Question: A high microerythrocyte count raises vulnerability to severe anemia in homozygous alpha (+)- thalassemia trait subjects.

Title: Increased Microerythrocyte Count in Homozygous α+-Thalassaemia Contributes to Protection against Severe Malarial Anaemia Content: BACKGROUND The heritable haemoglobinopathy alpha(+)-thalassaemia is caused by the reduced synthesis of alpha-globin chains that form part of normal adult haemoglobin (Hb). Individuals homozygous for alpha(+)-thalassaemia have microcytosis and an increased erythrocyte count. Alpha(+)-thalassaemia homozygosity confers considerable protection against severe malaria, including severe malarial anaemia (SMA) (Hb concentration < 50 g/l), but does not influence parasite count. We tested the hypothesis that the erythrocyte indices associated with alpha(+)-thalassaemia homozygosity provide a haematological benefit during acute malaria. METHODS AND FINDINGS Data from children living on the north coast of Papua New Guinea who had participated in a case-control study of the protection afforded by alpha(+)-thalassaemia against severe malaria were reanalysed to assess the genotype-specific reduction in erythrocyte count and Hb levels associated with acute malarial disease. We observed a reduction in median erythrocyte count of approximately 1.5 x 10(12)/l in all children with acute falciparum malaria relative to values in community children (p < 0.001). We developed a simple mathematical model of the linear relationship between Hb concentration and erythrocyte count. This model predicted that children homozygous for alpha(+)-thalassaemia lose less Hb than children of normal genotype for a reduction in erythrocyte count of >1.1 x 10(12)/l as a result of the reduced mean cell Hb in homozygous alpha(+)-thalassaemia. In addition, children homozygous for alpha(+)-thalassaemia require a 10% greater reduction in erythrocyte count than children of normal genotype (p = 0.02) for Hb concentration to fall to 50 g/l, the cutoff for SMA. We estimated that the haematological profile in children homozygous for alpha(+)-thalassaemia reduces the risk of SMA during acute malaria compared to children of normal genotype (relative risk 0.52; 95% confidence interval [CI] 0.24-1.12, p = 0.09). CONCLUSIONS The increased erythrocyte count and microcytosis in children homozygous for alpha(+)-thalassaemia may contribute substantially to their protection against SMA. A lower concentration of Hb per erythrocyte and a larger population of erythrocytes may be a biologically advantageous strategy against the significant reduction in erythrocyte count that occurs during acute infection with the malaria parasite Plasmodium falciparum. This haematological profile may reduce the risk of anaemia by other Plasmodium species, as well as other causes of anaemia. Other host polymorphisms that induce an increased erythrocyte count and microcytosis may confer a similar advantage.

Title: GENOTYPING OF THALASSEMIA IN MICROCYTIC HYPOCHROMIC ANEMIA PATIENTS FROM SOUTHWEST REGION OF IRAN Content: Objective: To evaluate the frequency of α -gene, s-gene, and hemoglobin variant numbers in subjects with Microcytic hypochromic anemia. Methodology: In total out of 850, 340 subjects with microcytic hypochromic anemia [MCV<80fl; MCH<27pg] from Southwest part of Iran, were studied in Research Center of Thalassemia and Hemoglobinopathies (RCTH) which is the only center working on hematology and oncology in Southwest (Khuzestan) region of Iran. These include 325 individuals: 171 with Beta-thalassemia trait, 88 with Alpha-thalassemia trait, 13 with thalassemia major, 11 with hemoglobin variants (HbS, HbC, and HbD Punjab ) and 42 with iron-deficiency anemia. The rest 15 patients diagnosed with no definite etiology. Results: Genotyping for -α 3.7 , -α 4.2 , – α PA , - α 5NT and - - MED was done with gap-PCR. The overall frequency of - α 3.7 deletion in 325 individuals is 20%. Genotyping for 23 most known s-gene mutations was done with direct mutation analysis by Amplification Refractory Mutation System (ARMS). The most frequent mutations were CD 36/37, IVS II-I, and IVS I-110 with 9.7%, 11.7%, and 3.5% respected frequencies in 340 patients. There was statistically significant difference between Beta-thalassemia trait and Beta-thalassemia Major in case of MCV (p- value = 0.25) and MCH (P–value =0.23) indices, and also MCH index between Beta-thalassemia trait and Hb Variants (P-value = 0.04). Conclusion: The α -gene and s-gene mutation is quite common in the Southwest part of Iran. Molecular genotyping of α -thalassemia and s-thalassemia help to diagnose unexplained microcytosis, and thus prevent unnecessary iron supplementation.

Title: Microcytosis, iron deficiency and thalassaemia in a multi-ethnic community: a pilot study. Content: The high prevalence of microcytosis (defined here as mean cell haemoglobin<27 pg) with no other abnormality is a principal cause of confusion in screening for haemoglobin disorders. Here we report the results of a small pilot study aiming to resolve this confusion by routinely proceeding to plasma ferritin and HPLC assay, using the original sequestrene blood sample, when microcytosis is detected. Participants comprised a random sample of 1,302 people referred for a full blood count by their General Practitioner (GP) to the laboratory of a North London district general hospital serving a multi-ethnic inner-city population. Ethnicity was established by questionnaire. In North Europeans, microcytosis was present in 3% of males (half were iron-deficient) and 11% of females (most were iron-deficient). Among ethnic minorities, microcytosis was present in 35% of males (one tenth were iron-deficient), and 45% of females (less than half were iron-deficient): an exclusion diagnosis of "probable alpha thalassaemia" could be made in the remainder. We conclude that when microcytosis is present, routine further analysis of the original sequestrene sample by plasma ferritin assay and haemoglobinopathy screening could lead to a more efficient and cost-effective laboratory service for primary care and maternity services.

Title: Hyperhomocysteinemia and atherosclerotic vascular disease: pathophysiology, screening, and treatment. off. Content: Hyperhomocysteinemia has recently been identified as an important risk factor for atherosclerotic vascular disease. This article reviews homocysteine metabolism, causes of hyperhomocysteinemia, the pathophysiological findings of this disorder, and epidemiological studies of homocysteine and vascular disease. Screening for hyperhomocysteinemia should be considered for patients at high risk for vascular disease or abnormalities of homocysteine metabolism. For primary prevention of vascular disease, treatment of patients with homocysteine levels of 14 micromol/L or higher should be considered. For secondary prevention, treatment of patients with homocysteine levels of 11 micromol/L or higher should be considered. Treatment is most conveniently administered as a folic acid supplement (400-1000 microg) and a high-potency multivitamin that contains at least 400 microg of folate. Higher doses of folic acid and cyanocobalamin supplements may be required in some patients. Until prospective clinical trial data become available, these conservative recommendations provide a safe, effective, and evidence-based approach to the diagnosis, evaluation, and management of patients with hyperhomocysteinemia.

Title: Thalassemia and malaria: new insights into an old problem. Content: The hemoglobinopathies are probably the world's most common genetic diseases: The World Health Organization has estimated that at least 5% of the population are carriers for one or other of the most serious forms, the alpha- and beta-thalassemias and the structural variant hemoglobins S, C, and E, which are found at polymorphic frequencies in many countries. All these hemoglobinopathies are believed to provide protection against malaria, and it is thought that, in malarial regions of the world, natural selection has been responsible for elevating and maintaining their gene frequencies, an idea first proposed 50 years ago by J.B.S. Haldane. Epidemiological studies undertaken in the 1950s on hemoglobin S in Africa provided support for the "malaria hypothesis," but until recently it has proved extremely difficult to verify it for the thalassemias. The application of molecular methods has, however, provided new opportunities to address this old question. Population and molecular genetic analysis of thalassemia variants, and microepidemiological studies of the relationship between alpha-thalassemia and malaria in the southwest Pacific, have provided unequivocal evidence for protection. Surprisingly, some of this protection appears to derive from enhanced susceptibility in very young thalassemic children to both Plasmodium falciparum and, especially, P. vivax, and this early exposure appears to provide the basis for better protection in later life.

Title: Congenital leptin deficiency is associated with severe early-onset obesity in humans. Content: The extreme obesity of the obese (ob/ob) mouse is attributable to mutations in the gene encoding leptin, an adipocyte-specific secreted protein which has profound effects on appetite and energy expenditure. We know of no equivalent evidence regarding leptin's role in the control of fat mass in humans. We have examined two severely obese children who are members of the same highly consanguineous pedigree. Their serum leptin levels were very low despite their markedly elevated fat mass and, in both, a homozygous frame-shift mutation involving the deletion of a single guanine nucleotide in codon 133 of the gene for leptin was found. The severe obesity found in these congenitally leptin-deficient subjects provides the first genetic evidence that leptin is an important regulator of energy balance in humans.

Title: Homocysteine and the risk of ischemic stroke in a triethnic cohort: the NOrthern MAnhattan Study. Content: BACKGROUND AND PURPOSE The level of total homocysteine (tHcy) that confers a risk of ischemic stroke is unsettled, and no prospective cohort studies have included sufficient elderly minority subjects. We investigated the association between mild to moderate fasting tHcy level and the incidence of ischemic stroke, myocardial infarction, and vascular death in a multiethnic prospective study. METHODS A population-based cohort was followed for vascular events (stroke, myocardial infarction, and vascular death). Baseline values of tHcy and methylmalonic acid were measured among 2939 subjects (mean age, 69+/-10; 61% women, 53% Hispanics, 24% blacks, and 20% whites). Cox proportional models were used to calculate hazard ratios (HRs) and 95% CIs in tHcy categories after adjusting for age, race, education, renal insufficiency, B12 deficiency, and other risk factors. RESULTS The adjusted HR for a tHcy level > or =15 micromol/L compared with <10 micromol/L was greatest for vascular death (HR=6.04; 95% CI, 3.44 to 10.60), followed by combined vascular events (HR=2.27; 95% CI, 1.51 to 3.43), ischemic stroke (HR=2.01; 95% CI, 1.00 to 4.05), and nonvascular death (HR=2.02; 95% CI, 1.31 to 3.14). Mild to moderate elevations of tHcy of 10 to 15 micromol/L were not significantly predictive of ischemic stroke, but increased the risk of vascular death (2.27; 95% CI, 1.44 to 3.60) and combined vascular events (1.42; 95% CI, 1.06 to 1.88). The effect of tHcy was stronger among whites and Hispanics, but not a significant risk factor for blacks. CONCLUSIONS Total Hcy elevations above 15 micromol/L are an independent risk factor for ischemic stroke, whereas mild elevations of tHcy of 10 to 15 micromol/L are less predictive. The vascular effects of tHcy are greatest among whites and Hispanics, and less among blacks.

Title: Applicability of current staging/categorization of α-synuclein pathology and their clinical relevance Content: In Parkinson's disease (PD) and dementia with Lewy bodies (DLB) alpha-synuclein (alphaS) pathology is seen that displays a predictable topographic distribution. There are two staging/categorization systems, i.e. Braak's and McKeith's, currently in use for the assessment of alphaS pathology. The aim of these diagnostic strategies in pathology is, in addition to assess the stage/severity of pathology, to assess the probabilities of the related clinical symptomatology i.e. dementia and extrapyramidal symptoms (EPS). Herein, we assessed the applicability of these two staging/categorization systems and the frequency of dementia and EPS in a cohort of 226 alphaS-positive-subjects. These subject were selected from a large autopsy sample (n = 1,720), irrespective of the clinical presentation, based on the detection of alphaS-immunoreactivity (IR) in one of the most vulnerable nuclei; in the dorsal motor nucleus of vagus, substantia nigra and basal forebrain. The frequency of alphaS-IR lesions in this large cohort was 14% (248 out of 1,720). If applicable, each of the 226 subjects with all required material available was assigned a neuropathological stage/category of PD/DLB and finally the neuropathological data was analyzed in relation to dementia and EPS. 83% of subjects showed a distribution pattern of alphaS-IR that was compatible with the current staging/categorization systems. Around 55% of subjects with widespread alphaS pathology (Braak's PD stages 5-6) lacked clinical signs of dementia or EPS. Similarly, in respect to those subjects that fulfilled the McKeith criteria for diffuse neocortical category and displaying only mild concomitant Alzheimer's disease-related pathology, only 48% were demented and 54% displayed EPS. It is noteworthy that some subjects (17%) deviated from the suggested caudo-rostral propagation suggesting alternative routes of progression, perhaps due to concomitant diseases and genetic predisposition. In conclusion, our results do indeed confirm that current staging/categorization systems can readily be applied to most of the subjects with alphaS pathology. However, finding that around half of the subjects with abundant alphaS pathology remain neurologically intact is intriguing and raises the question whether we do assess the actual disease process.

Title: Ribosomal DNA copy loss and repeat instability in ATRX-mutated cancers Content: ATRX (alpha thalassemia/mental retardation X-linked) complexes with DAXX to deposit histone variant H3.3 into repetitive heterochromatin. Recent genome sequencing studies in cancers have revealed mutations in ATRX and their association with ALT (alternative lengthening of telomeres) activation. Here we report depletion of ATRX in mouse ES cells leads to selective loss in ribosomal RNA gene (rDNA) copy number. Supporting this, ATRX-mutated human ALT-positive tumors also show a substantially lower rDNA copy than ALT-negative tumors. Further investigation shows that the rDNA copy loss and repeat instability are caused by a disruption in H3.3 deposition and thus a failure in heterochromatin formation at rDNA repeats in the absence of ATRX. We also find that ATRX-depleted cells are reduced in ribosomal RNA transcription output and show increased sensitivity to RNA polymerase I (Pol I) transcription inhibitor CX5461. In addition, human ALT-positive cancer cell lines are also more sensitive to CX5461 treatment. Our study provides insights into the contribution of ATRX loss of function to tumorigenesis through the loss of rDNA stability and suggests the therapeutic potential of targeting Pol I transcription in ALT cancers.

Title: KLF1-null neonates display hydrops fetalis and a deranged erythroid transcriptome. Content: We describe a case of severe neonatal anemia with kernicterus caused by compound heterozygosity for null mutations in KLF1, each inherited from asymptomatic parents. One of the mutations is novel. This is the first described case of a KLF1-null human. The phenotype of severe nonspherocytic hemolytic anemia, jaundice, hepatosplenomegaly, and marked erythroblastosis is more severe than that present in congenital dyserythropoietic anemia type IV as a result of dominant mutations in the second zinc-finger of KLF1. There was a very high level of HbF expression into childhood (>70%), consistent with a key role for KLF1 in human hemoglobin switching. We performed RNA-seq on circulating erythroblasts and found that human KLF1 acts like mouse Klf1 to coordinate expression of many genes required to build a red cell including those encoding globins, cytoskeletal components, AHSP, heme synthesis enzymes, cell-cycle regulators, and blood group antigens. We identify novel KLF1 target genes including KIF23 and KIF11 which are required for proper cytokinesis. We also identify new roles for KLF1 in autophagy, global transcriptional control, and RNA splicing. We suggest loss of KLF1 should be considered in otherwise unexplained cases of severe neonatal NSHA or hydrops fetalis.

Title: British Journal of Nutrition (2003), 89, 295–301 q The Authors 2003 DOI: 10.1079/BJN2002776 Plasma homocysteine concentration is decreased by dietary intervention* Content: High plasma total homocysteine (tHcy) concentration is reported to be a risk factor for vascular diseases. We investigated the extent to which serum folate and plasma tHcy respond to a high intake of natural folate from food. Thirty-seven healthy females volunteered t o participate in a crossover dietary intervention. The study included a baseline period and two 5-week diet periods (low- and high-folate diets) with a 3-week washout in between. The low-folate diet contained one serving of both vegetables and fruit/d, while during the high-folate diet the subjects ate at least seven servings of vegetables, berries, and citrus fruit/d. Serum and erythrocyte (RBC) folate, serum vitamin B (12), and plasma tHcy concentrations were measured at the base-line and at the end of each diet period. The mean concentrations of serum and RBC folate were 11.0 (SD 3.0) nmol/l and 412 (SD 120) nmol/l at the end of the low-folate diet and 78 (95 % CI 62, 94) % and 14 (95 % CI 8, 20) % higher in response to the high-folate diet (P< 0.001). The serum concentration of vitamin B12 remained unchanged during the intervention. The mean plasma tHcy concentration was 8.0 pmol/ at the end of the low-folate diet and decreased by 13 (95% CI 9, 18) % in response to the high-folate diet (P<0.001). In conclusion, a diet high in fresh berries, citrus fruit, and vegetables effectively increases serum and RBC folate and decreases plasma homocysteine.

Title: A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function Content: Antigen stimulation of immune cells triggers Ca2+ entry through Ca2+ release-activated Ca2+ (CRAC) channels, promoting the immune response to pathogens by activating the transcription factor NFAT. We have previously shown that cells from patients with one form of hereditary severe combined immune deficiency (SCID) syndrome are defective in store-operated Ca2+ entry and CRAC channel function. Here we identify the genetic defect in these patients, using a combination of two unbiased genome-wide approaches: a modified linkage analysis with single-nucleotide polymorphism arrays, and a Drosophila RNA interference screen designed to identify regulators of store-operated Ca2+ entry and NFAT nuclear import. Both approaches converged on a novel protein that we call Orai1, which contains four putative transmembrane segments. The SCID patients are homozygous for a single missense mutation in ORAI1, and expression of wild-type Orai1 in SCID T cells restores store-operated Ca2+ influx and the CRAC current (ICRAC). We propose that Orai1 is an essential component or regulator of the CRAC channel complex.

Title: Translocation of sickle cell erythrocyte microRNAs into Plasmodium falciparum inhibits parasite translation and contributes to malaria resistance. Content: Erythrocytes carrying a variant hemoglobin allele (HbS), which causes sickle cell disease and resists infection by the malaria parasite Plasmodium falciparum. The molecular basis of this resistance, which has long been recognized as multifactorial, remains incompletely understood. Here we show that the dysregulated microRNA (miRNA) composition, of either heterozygous HbAS or homozygous HbSS erythrocytes, contributes to resistance against P. falciparum. During the intraerythrocytic life cycle of P. falciparum, a subset of erythrocyte miRNAs translocate into the parasite. Two miRNAs, miR-451 and let-7i, were highly enriched in HbAS and HbSS erythrocytes, and these miRNAs, along with miR-223, negatively regulated parasite growth. Surprisingly, we found that miR-451 and let-7i integrated into essential parasite messenger RNAs and, via impaired ribosomal loading, resulted in translational inhibition. Hence, sickle cell erythrocytes exhibit cell-intrinsic resistance to malaria in part through an atypical miRNA activity, which may represent a unique host defense strategy against complex eukaryotic pathogens.

Title: A tumor necrosis factor-α–mediated pathway promoting autosomal dominant polycystic kidney disease Content: Autosomal dominant polycystic kidney disease (ADPKD) is caused by heterozygous mutations in either PKD1 or PKD2, genes that encode polycystin-1 and polycystin-2, respectively. We show here that tumor necrosis factor-α (TNF-α), an inflammatory cytokine present in the cystic fluid of humans with ADPKD, disrupts the localization of polycystin-2 to the plasma membrane and primary cilia through a scaffold protein, FIP2, which is induced by TNF-α. Treatment of mouse embryonic kidney organ cultures with TNF-α resulted in formation of cysts, and this effect was exacerbated in the Pkd2+/− kidneys. TNF-α also stimulated cyst formation in vivo in Pkd2+/− mice. In contrast, treatment of Pkd2+/− mice with the TNF-α inhibitor etanercept prevented cyst formation. These data reveal a pathway connecting TNF-α signaling, polycystins and cystogenesis, the activation of which may reduce functional polycystin-2 below a critical threshold, precipitating the ADPKD cellular phenotype.

Title: The ATM missense mutation p.Ser49Cys (c.146C>G) and the risk of breast cancer. Content: Homozygous mutation in the ATM gene causes ataxia telangiectasia and heterozygous mutation carriers may be at increased risk of breast cancer. We studied a total of 22 ATM variants; 18 variants were analyzed in one of two large population-based studies from the U.S. and Poland, and four variants were analyzed in all 2,856 breast cancer cases and 3,344 controls from the two studies. The missense mutation Ser49Cys (c.146C>G, p. S49C), carried by approximately 2% of subjects, was more common in cases than controls in both study populations, combined odds ratio (OR) 1.69 (95% CI, 1.19-2.40; P=0.004). Another missense mutation at approximately 2% frequency, Phe858Leu (c.2572T>C, p. F858L), was associated with a significant increased risk in the U.S. study but not in Poland, and had a combined OR of 1.44 (95% CI, 0.98-2.11; P=0.06). These analyses provide the most convincing evidence thus far that missense mutations in ATM, particularly p. S49C, may be breast cancer susceptibility alleles. Because of their low frequency, even larger sample sizes are required to more firmly establish these associations.

Title: Rps14 haploinsufficiency causes a block in erythroid differentiation mediated by S100A8 and S100A9 Content: Impaired erythropoiesis in the deletion 5q (del(5q)) subtype of myelodysplastic syndrome (MDS) has been linked to heterozygous deletion of RPS14, which encodes the ribosomal protein small subunit 14. We generated mice with conditional inactivation of Rps14 and demonstrated an erythroid differentiation defect that is dependent on the tumor suppressor protein p53 (encoded by Trp53 in mice) and is characterized by apoptosis at the transition from polychromatic to orthochromatic erythroblasts. This defect resulted in age-dependent progressive anemia, megakaryocyte dysplasia and loss of hematopoietic stem cell (HSC) quiescence. As assessed by quantitative proteomics, mutant erythroblasts expressed higher levels of proteins involved in innate immune signaling, notably the heterodimeric S100 calcium-binding proteins S100a8 and S100a9. S100a8—whose expression was increased in mutant erythroblasts, monocytes and macrophages—is functionally involved in the erythroid defect caused by the Rps14 deletion, as addition of recombinant S100a8 was sufficient to induce a differentiation defect in wild-type erythroid cells, and genetic inactivation of S100a8 expression rescued the erythroid differentiation defect of Rps14-haploinsufficient HSCs. Our data link Rps14 haploinsufficiency in del(5q) MDS to activation of the innate immune system and induction of S100A8-S100A9 expression, leading to a p53-dependent erythroid differentiation defect.

Title: Homocysteine Induces Trophoblast Cell Death with Apoptotic Features1 Content: Abstract Hyperhomocysteinemia has been suggested as a possible risk factor in women suffering from habitual abortions, placental abruption or infarcts, preeclampsia, and/or intrauterine growth retardation. However, little is known about the pathogenic mechanisms underlying the action of homocysteine. The present study investigated the in vitro ability of homocysteine to affect trophoblast gonadotropin secretion and to induce cell death. In primary human trophoblast cells, homocysteine treatment (20 μmol/L) resulted in cellular flattening and enlargement, extension of pseudopodia, and cellular vacuolization. Cellular detachment, apoptosis, and necrosis were favored. With in situ nick end labeling, we investigated DNA degradation, and we used M30 CytoDEATH to selectively stain the cytoplasm of apoptotic cells. Cytochrome c release from mitochondria to the cytosol and DNA cleavage in agarose gel have been investigated. Homocysteine, but not cysteine, induced trophoblast apoptosis and significantly reduced human chorionic gonadotropin secretion. These findings suggest that trophoblast cell death might represent a pathogenic mechanism by which homocysteine may cause pregnancy complications related to placental diseases.

Title: Compound heterozygous ZMPSTE24 mutations reduce prelamin A processing and result in a severe progeroid phenotype. Content: Hutchinson–Gilford progeria syndrome (HGPS; OMIM 176670) is an extremely rare but devastating disorder that mimics premature aging.1–3 Affected children appear normal at birth but typically develop failure to thrive in the first two years. Other features include alopecia, micrognathia, loss of subcutaneous fat with prominent veins, abnormal dentition, sclerodermatous skin changes, and osteolysis of the clavicles and distal phalanges. The mean age of death is at age 13 years, most commonly due to atherosclerosis. HGPS is mainly sporadic in occurrence, but a genetic cause has now been implicated following the identification of de novo heterozygous mutations in the LMNA gene in the majority of HGPS patients.4,5 A single family showing autosomal recessive inheritance of homozygous LMNA mutations has also been reported.6 LMNA encodes lamins A and C, components of the nuclear lamina, a meshwork underlying the nuclear envelope that serves as a structural support and is also thought to contribute to chromatin organisation and the regulation of gene expression.7,8 Interestingly, mutations in LMNA have recently been associated with at least eight inherited disorders, known as laminopathies, with differential dystrophic effects on a variety of tissues including muscle, neurones, skin, bone, and adipose tissue (reviewed in Mounkes et al 9). However, the realisation that these disorders share common genetic defects has led to clinical re-evaluation, with emerging evidence of significant phenotypic overlap.10 Hence the laminopathies might reasonably be considered as a spectrum of related diseases. HGPS has phenotypic similarities to several other laminopathies, in particular the atypical Werner’s syndrome11 and mandibuloacral dysplasia (MAD; OMIM 248370 and 608612).12 These diseases are associated with lipodystrophy,3,13 which is the most prominent feature of another laminopathy, familial partial lipodystrophy of the Dunnigan variety (OMIM 151660).14 MAD has been further classified as two …

Title: Effect of homocysteine lowering on mortality and vascular disease in advanced chronic kidney disease and end-stage renal disease: a randomized controlled trial. Content: CONTEXT High plasma homocysteine levels are a risk factor for mortality and vascular disease in observational studies of patients with chronic kidney disease. Folic acid and B vitamins decrease homocysteine levels in this population but whether they lower mortality is unknown. OBJECTIVE To determine whether high doses of folic acid and B vitamins administered daily reduce mortality in patients with chronic kidney disease. DESIGN, SETTING, AND PARTICIPANTS Double-blind randomized controlled trial (2001-2006) in 36 US Department of Veterans Affairs medical centers. Median follow-up was 3.2 years for 2056 participants aged 21 years or older with advanced chronic kidney disease (estimated creatinine clearance < or =30 mL/min) (n = 1305) or end-stage renal disease (n = 751) and high homocysteine levels (> or = 15 micromol/L). INTERVENTION Participants received a daily capsule containing 40 mg of folic acid, 100 mg of pyridoxine hydrochloride (vitamin B6), and 2 mg of cyanocobalamin (vitamin B12) or a placebo. MAIN OUTCOME MEASURES The primary outcome was all-cause mortality. Secondary outcomes included myocardial infarction (MI), stroke, amputation of all or part of a lower extremity, a composite of these 3 plus all-cause mortality, time to initiation of dialysis, and time to thrombosis of arteriovenous access in hemodialysis patients. RESULTS Mean baseline homocysteine level was 24.0 micromol/L in the vitamin group and 24.2 micromol/L in the placebo group. It was lowered 6.3 micromol/L (25.8%; P < .001) in the vitamin group and 0.4 micromol/L (1.7%; P = .14) in the placebo group at 3 months, but there was no significant effect on mortality (448 vitamin group deaths vs 436 placebo group deaths) (hazard ratio [HR], 1.04; 95% CI, 0.91-1.18). No significant effects were demonstrated for secondary outcomes or adverse events: there were 129 MIs in the vitamin group vs 150 for placebo (HR, 0.86; 95% CI, 0.67-1.08), 37 strokes in the vitamin group vs 41 for placebo (HR, 0.90; 95% CI, 0.58-1.40), and 60 amputations in the vitamin group vs 53 for placebo (HR, 1.14; 95% CI, 0.79-1.64). In addition, the composite of MI, stroke, and amputations plus mortality (P = .85), time to dialysis (P = .38), and time to thrombosis in hemodialysis patients (P = .97) did not differ between the vitamin and placebo groups. CONCLUSION Treatment with high doses of folic acid and B vitamins did not improve survival or reduce the incidence of vascular disease in patients with advanced chronic kidney disease or end-stage renal disease. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00032435.

Title: The effect of folic acid supplementation on plasma homocysteine in an elderly population. Content: BACKGROUND Increased plasma homocysteine is associated with coronary artery disease, peripheral vascular disease and venous thrombosis. Folic acid is the most effective therapy for reducing homocysteine levels. The lowest effective supplement of folic acid is not known, particularly for the elderly who have the highest prevalence of these conditions. AIM To explore the effects of daily supplements of 0, 50, 100, 200, 400 and 600 microg folic acid on plasma homocysteine in an elderly population. DESIGN Randomized double-blind placebo-controlled trial. METHODS Participants (n=368) aged 65-75 years were randomly allocated to receive one of the treatments for 6 weeks. Plasma homocysteine was recorded after 3 weeks and 6 weeks of supplementation. RESULTS Only the 400 microg and 600 microg groups had significantly lower homocysteine levels compared to placebo (p=0.038 and p<0.001, respectively). Using multiple linear regression and each individual's total folic acid intake (diet plus supplement), a total daily folic acid intake of 926 microg per day would be required to ensure that 95% of the elderly population would be without cardiovascular risk from folate deficiency. DISCUSSION A daily folic acid intake of 926 microg is unlikely to be achieved by diet alone. Individual supplementation or fortification of food with folic acid will be required to reach this target.

Title: Gene--nutrition interactions in coronary artery disease: correlation between the MTHFR C677T polymorphism and folate and homocysteine status in a Korean population. Content: INTRODUCTION Elevated plasma total homocysteine is a major risk for coronary artery disease (CAD). Methyltetrahydrofolate reductase (MTHFR) is a main regulatory enzyme in homocysteine metabolism; a common C677T mutation in the MTHFR gene results in decreased enzyme activity, and contributes to increased homocysteine levels and decreased folate levels. We investigated the frequency of MTHFR C677T alleles in a Korean population, determined the genotype-specific threshold levels of folate or vitamin B12, and investigated the relationship between the TT genotype and the risk of CAD. MATERIALS AND METHODS We enrolled a study population of 163 CAD patients and 50 control subjects, and screened the MTHFR C677T polymorphism using real-time PCR with melting point analysis. Levels of plasma homocysteine, folate and vitamin B12 were also determined. We then defined the genotype-specific threshold values of folate and vitamin B12 required to keep homocysteine levels in a normal range for individuals of each MTHFR C677T genotype. RESULTS The frequency of the TT genotype was 18% in control subjects and 26% in patients group (P>0.05). Individuals homozygous for the TT genotype had significantly elevated homocysteine levels (P<0.05). The genotype-specific folate threshold level was significantly higher in TT individuals than in the CC or CT genotypes. The OR of individuals with low folate status and the TT genotype to estimate the relative risk of CAD was 2.2 and the OR of those with high folate status and the TT genotype was 1.5 (95% CI, 0.5-9.6 and 0.7-3.2, respectively). CONCLUSION We were able to define a gene-nutrient interaction that shows a higher risk for CAD based on specific threshold folate levels required by different MTHFR C677T genotypes in a Korean population.

Title: Prediagnostic levels of C-peptide, IGF-I, IGFBP -1, -2 and -3 and risk of endometrial cancer. Content: Conditions related to chronic hyperinsulinemia, such as obesity, noninsulin dependent diabetes mellitus and polycystic ovary syndrome, are associated with an increased risk of endometrial cancer. Elevated plasma IGF-I and decreased levels of IGF-binding proteins have been shown to be associated with increased risk of several cancer types that are frequent in affluent societies. We investigated for the first time in a prospective study the association of pre-diagnostic blood concentrations of C-peptide (a marker of pancreatic insulin production), IGF-I, IGFBP-1, -2 and -3 with endometrial cancer risk. A case-control study was nested within 3 cohorts in New York (USA), Umeå (Sweden) and Milan (Italy). It included 166 women with primary invasive endometrial cancer and 315 matched controls, of which 44 case and 78 control subjects were premenopausal at recruitment. Endometrial cancer risk increased with increasing levels of C-peptide (ptrend = 0.0002), up to an odds ratio (OR) of 4.76 [95% confidence interval (CI) = 1.91-11.8] for the highest quintile. This association remained after adjustment for BMI and other confounders [OR for the top quintile = 4.40 (1.65-11.7)]. IGFBP-1 levels were inversely related to endometrial cancer [ptrend = 0.002; OR in the upper quintile = 0.30 (0.15-0.62)], but the association was weakened and lost statistical significance after adjustment for confounders [ptrend = 0.06; OR in the upper quintile = 0.49 (0.22-1.07)]. Risk was unrelated to levels of IGF-I, IGFBP-2 and IGFBP-3. Chronic hyperinsulinemia, as reflected by increased circulating C-peptide, is associated with increased endometrial cancer risk. Decrease in the prevalence of chronic hyperinsulinemia, through changes in lifestyle or medication, is expected to prevent endometrial cancer.

Title: Tumor necrosis factor alpha mediates apoptosis of brown adipocytes and defective brown adipocyte function in obesity. Content: Severe quantitative and qualitative brown adipocyte defects are common in obesity. To investigate whether aberrant expression of tumor necrosis factor alpha (TNF-alpha) in obesity is involved in functional brown fat atrophy, we have studied genetically obese (ob/ob) mice with targeted null mutations in the genes encoding the two TNF receptors. The absence of both TNF receptors or p55 receptor alone resulted in a significant reduction in brown adipocyte apoptosis and an increase in beta(3)-adrenoreceptor and uncoupling protein-1 expression in obese mice. Increased numbers of multilocular functionally active brown adipocytes, and improved thermoregulation was also observed in obese animals lacking TNF-alpha function. These results indicate that TNF-alpha plays an important role in multiple aspects of brown adipose tissue biology and mediates the abnormalities that occur at this site in obesity.

Title: Atm-deficient mice exhibit increased sensitivity to dextran sulfate sodium-induced colitis characterized by elevated DNA damage and persistent immune activation. Content: The role of ataxia telangiectasia mutated (ATM), a DNA double-strand break recognition and response protein, in inflammation and inflammatory diseases is unclear. We have previously shown that high levels of systemic DNA damage are induced by intestinal inflammation in wild-type mice. To determine the effect of Atm deficiency in inflammation, we induced experimental colitis in Atm(-/-), Atm(+/-), and wild-type mice via dextran sulfate sodium (DSS) administration. Atm(-/-) mice had higher disease activity indices and rates of mortality compared with heterozygous and wild-type mice. Systemic DNA damage and immune response were characterized in peripheral blood throughout and after three cycles of treatment. Atm(-/-) mice showed increased sensitivity to levels of DNA strand breaks in peripheral leukocytes, as well as micronucleus formation in erythroblasts, compared with heterozygous and wild-type mice, especially during remission periods and after the end of treatment. Markers of reactive oxygen and nitrogen species-mediated damage, including 8-oxoguanine and nitrotyrosine, were present both in the distal colon and in peripheral leukocytes, with Atm(-/-) mice manifesting more 8-oxoguanine formation than wild-type mice. Atm(-/-) mice showed greater upregulation of inflammatory cytokines and significantly higher percentages of activated CD69+ and CD44+ T cells in the peripheral blood throughout treatment. ATM, therefore, may be a critical immunoregulatory factor dampening the deleterious effects of chronic DSS-induced inflammation, necessary for systemic genomic stability and homeostasis of the gut epithelial barrier.

Title: Preventing coronary heart disease: B vitamins and homocysteine. Content: The list of preventable and reversible risk factors for atherosclerotic cardiovascular disease continues to grow. Cigarette smoking, high blood pressure, physical inactivity, elevated cholesterol, underlying lipoprotein abnormalities, lipoprotein(a), diabetes, overweight, male gender, and age are well-established risk factors. During the 1990s, there have been many reports associating elevated plasma homocysteine levels with arteriosclerotic cardiovascular disease and consistent evidence that dietary and supplemental folic acid can reduce homocysteine levels.1 2 The article by Robinson and colleagues3 in this issue of Circulation presents further evidence of the importance of homocysteine and suggestive evidence that plasma folate and plasma pyrixodal-l-phosphate (vitamin B6) are protective factors. Their study is part of the European Concerted Action Project,4 which examined 750 patients younger than age 60 with diagnoses within the previous 12 months of coronary, cerebrovascular, or peripheral vascular disease and 800 healthy control subjects. The patient groups were young (47 years for cases and 44 years for control subjects) and heterogeneous, with nonfatal clinical events or symptoms of arteriosclerotic cardiovascular disease supported by ECG, angiographic, or Doppler evidence; the study involved 19 centers in nine European countries. Men in the highest quintile for fasting total homocysteine (tHcy), compared with the remainder of the population, had an estimated relative risk of 2.2 (95% confidence interval [CI], 1.6 to 2.9), with a striking dose-response relationship and a more-than-multiplicative interaction with cigarette smoking and high blood pressure on vascular disease risk4 ; the corresponding estimated relative risk for coronary heart disease was similar (2.0; 95% CI 1.6 to 2.8). (tHcy is the sum of homocysteine and homocysteinyl moieties of oxidized disulfides, homocystine, and cysteine- homocysteine. ) Robinson and colleagues3 examined three B vitamins in detail to determine their effects on fasting and post–methionine-loading tHcy levels and any independent effects on cardiovascular disease …

Title: Macrosomia and Hyperinsulinaemic Hypoglycaemia in Patients with Heterozygous Mutations in the HNF4A Gene Content: Background Macrosomia is associated with considerable neonatal and maternal morbidity. Factors that predict macrosomia are poorly understood. The increased rate of macrosomia in the offspring of pregnant women with diabetes and in congenital hyperinsulinaemia is mediated by increased foetal insulin secretion. We assessed the in utero and neonatal role of two key regulators of pancreatic insulin secretion by studying birthweight and the incidence of neonatal hypoglycaemia in patients with heterozygous mutations in the maturity-onset diabetes of the young (MODY) genes HNF4A (encoding HNF-4α) and HNF1A/TCF1 (encoding HNF-1α), and the effect of pancreatic deletion of Hnf4a on foetal and neonatal insulin secretion in mice.

Title: A prospective study of plasma homocyst(e)ine and risk of myocardial infarction in US physicians. Content: OBJECTIVE To assess prospectively the risk of coronary heart disease associated with elevated plasma levels of homocyst(e)ine. DESIGN Nested case-control study using prospectively collected blood samples. SETTING Participants in the Physicians' Health Study. PARTICIPANTS A total of 14,916 male physicians, aged 40 to 84 years, with no prior myocardial infarction (MI) or stroke provided plasma samples at baseline and were followed up for 5 years. Samples from 271 men who subsequently developed MI were analyzed for homocyst(e)ine levels together with paired controls, matched by age and smoking. MAIN OUTCOME MEASURE Acute MI or death due to coronary disease. RESULTS Levels of homocyst(e)ine were higher in cases than in controls (11.1 +/- 4.0 [SD] vs 10.5 +/- 2.8 nmol/mL; P = .03). The difference was attributable to an excess of high values among men who later had MIs. The relative risk for the highest 5% vs the bottom 90% of homocyst(e)ine levels was 3.1 (95% confidence interval, 1.4 to 6.9; P = .005). After additional adjustment for diabetes, hypertension, aspirin assignment, Quetelet's Index, and total/high-density lipoprotein cholesterol, this relative risk was 3.4 (95% confidence interval, 1.3 to 8.8) (P = .01). Thirteen controls and 31 cases (11%) had values above the 95th percentile of the controls. CONCLUSIONS Moderately high levels of plasma homocyst(e)ine are associated with subsequent risk of MI independent of other coronary risk factors. Because high levels can often be easily treated with vitamin supplements, homocyst(e)ine may be an independent, modifiable risk factor.

Title: Original Article Content: UNLABELLED Fruit and vegetable consumption has been inversely associated with the risk of chronic diseases including cancer and cardiovascular disease, with the beneficial effects attributed to a variety of protective antioxidants, carotenoids and phytonutrients. The objective of the present study was to determine the effect of supplementation with dehydrated concentrates from mixed fruit and vegetable juices (Juice Plus+R) on serum antioxidant and folate status, plasma homocysteine levels and markers for oxidative stress and DNA damage. Japanese subjects (n=60; age 27.8 yrs; BMI 22.1) were recruited to participate in a double-blind placebo controlled study and were randomized into 2 groups of 30, matched for sex, age, BMI and smoking status (39 males, 22 smokers; 21 females, 13 smokers). Subjects were given encapsulated supplements containing mixed fruit and vegetable juice concentrates or a matching placebo for 28 days, with blood and urine samples collected at baseline, day 14 and day 28 for analytical testing. Compared with the placebo, 28 day supplementation significantly increased the concentration of serum beta-carotene 528% (p<0.0001), lycopene 80.2% (p<0.0005), and alpha tocopherol 39.5% (p<0.0001). Serum folate increased 174.3% (p<0.0001) and correlated with a decrease in plasma homocysteine of -19.9% (p<0.03). Compared with baseline, measures of oxidative stress decreased with serum lipid peroxides declining -10.5% (p<0.02) and urine 8OHdG decreasing -21.1% (p<0.02). Evaluation of data from smokers only (n=17) after 28 days of active supplementation showed comparable changes. CONCLUSION In the absence of dietary modification, supplementation with the fruit and vegetable juice concentrate capsules proved to be a highly bioavailable source of phytonutrients. Important antioxidants were elevated to desirable levels associated with decreased risk of disease while markers of oxidative stress were reduced, and folate status improved with a concomitant decrease in homocysteine, and these benefits occurred to a similar extent in smokers when compared to non-smokers.

Title: Long-term survival of beta thalassemia major patients treated with hematopoietic stem cell transplantation compared with survival with conventional treatment. Content: Allogeneic hematopoietic stem cell transplantation (HSCT) in thalassemia remains a challenge. We reported a single-centre case-control study of a large cohort of 516 children and adult patients treated with HSCT or blood transfusion support and iron chelation therapy; 258 patients (median age 12, range 1-45) underwent sibling (67%) or unrelated (33%) HSCT; 97 patients were adults (age ≥ 16 years). The median follow-up after HSCT was 11 years (range 1-30). The conditioning regimen was busulfan (80.6%) or treosulfan-based (19.4%). A cohort of 258 age-sex matched conventionally treated (CT) patients was randomly selected. In transplanted patients the 30-year overall survival (OS) and thalassemia-free survival (TFS) were 82.6 ± 2.7% and 77.8 ± 2.9%, compared to the OS of 85.3 ± 2.7% in CT patients (P = NS); The incidence of grade II-IV acute and chronic graft versus host disease (GvHD) was 23.6% and 12.9% respectively. The probability of rejection was 6.9%. Transplant-related mortality (TRM) (13.8%) was similar to the probability of dying of cardiovascular events in CT patients (12.2%). High-risk Pesaro score (class 3) was associated with lower OS (OR = 1.99, 95% C.I.=1.31-3.03) and TFS (OR = 1.54, 95% C.I.=1.12-2.12). In adult patients, the 23-years OS and TFS after HSCT were 70 ± 5% and 67.3 ± 5%, compared to 71.2 ± 5% of OS in CT (P = NS). Finally, treosulfan was associated with lower risk of acute GvHD (P = .004; OR = 0.28, 95% C.I.=0.12-0.67). In conclusion, the 30-year survival rate of ex-thalassemia patients after HSCT was similar to that expected in CT thalassemia patients, with the vast majority of HSCT survivors cured from thalassemia.

Title: Association of Toll-Like Receptor 4 on Human Monocyte Subsets and Vulnerability Characteristics of Coronary Plaque as Assessed by 64-Slice Multidetector Computed Tomography. Content: BACKGROUND Although Toll-like receptor 4 (TLR-4) is involved in monocyte activation in patients with accelerated forms of atherosclerosis, the relationship between the expression of TLR-4 on circulating monocytes and coronary plaque vulnerability has not previously been evaluated. We investigated this relationship using 64-slice multidetector computed tomography (MDCT) in patients with stable angina pectoris (SAP).Methods and Results:We enrolled 65 patients with SAP who underwent MDCT. Three monocyte subsets (CD14++CD16-, CD14++CD16+, and CD14+CD16+) and expression of TLR-4 were measured by flow cytometry. Intracoronary plaques were assessed by 64-slice MDCT. We defined vulnerability of intracoronary plaques according to the presence of positive remodeling (remodeling index >1.05) and/or low CT attenuation (<35 HU). The circulating CD14++CD16+monocytes more frequently expressed TLR-4 than CD14++CD16-and CD14+CD16+monocytes (P<0.001). The relative proportion of the expression of TLR-4 on CD14++CD16+monocytes was significantly greater in patients with vulnerable plaque compared with those without (10.4 [4.1-14.5] % vs. 4.5 [2.8-7.8] %, P=0.012). In addition, the relative proportion of TLR-4 expression on CD14++CD16+monocytes positively correlated with the remodeling index (r=0.28, P=0.025) and negatively correlated with CT attenuation value (r=-0.31, P=0.013). CONCLUSIONS Upregulation of TLR-4 on CD14++CD16+monocytes might be associated with coronary plaque vulnerability in patients with SAP.

Title: Randomized trial of folic acid supplementation and serum homocysteine levels. Content: BACKGROUND Lowering serum homocysteine levels with folic acid is expected to reduce mortality from ischemic heart disease. Homocysteine reduction is known to be maximal at a folic acid dosage of 1 mg/d, but the effect of lower doses (relevant to food fortification) is unclear. METHODS We randomized 151 patients with ischemic heart disease to 1 of 5 dosages of folic acid (0.2, 0.4, 0.6, 0.8, and 1.0 mg/d) or placebo. Fasting blood samples for serum homocysteine and serum folate analysis were taken initially, after 3 months of supplementation, and 3 months after folic acid use was discontinued. RESULTS Median serum homocysteine level decreased with increasing folic acid dosage, to a maximum at 0.8 mg of folic acid per day, when the homocysteine reduction (placebo adjusted) was 2.7 micromol/L (23%), similar to the known effect of folic acid dosages of 1 mg/d and above. The higher a person's initial serum homocysteine level, the greater was the response to folic acid, but there were statistically significant reductions regardless of the initial level. Serum folate level increased approximately linearly (5.5 nmol/L for every 0.1 mg of folic acid). Within-person fluctuations over time in serum homocysteine levels, measured in the placebo group, were large compared with the effect of folic acid, indicating that monitoring of the reduction in an individual is impractical. CONCLUSIONS A dosage of folic acid of 0.8 mg/d appears necessary to achieve the maximum reduction in serum homocysteine level across the range of homocysteine levels in the population. Current US food fortification levels will achieve only a small proportion of the achievable homocysteine reduction.

Title: Changes in white blood cells and platelets in children with falciparum malaria: relationship to disease outcome. Content: Little is known about the changes in white blood cells and platelets in children with falciparum malaria in endemic areas. We measured the white cell count (WCC) and platelets of 230 healthy children from the community, 1369 children admitted to hospital with symptomatic malaria, and 1461 children with other medical conditions. Children with malaria had a higher WCC compared with community controls, and leucocytosis was strongly associated with younger age, deep breathing, severe anaemia, thrombocytopenia and death. The WCC was not associated with a positive blood culture. In children with malaria, high lymphocyte and low monocyte counts were independently associated with mortality. A platelet count of less than 150 x 109/l was found in 56.7% of children with malaria, and was associated with age, prostration and parasite density, but not with bleeding problems or mortality. The mean platelet volume was also higher in children with malaria compared with other medical conditions. This may reflect early release from the bone marrow in response to peripheral platelet destruction. Thus, leucocytosis was associated with both severity and mortality in children with falciparum malaria, irrespective of bacteraemia, whereas thrombocytopenia, although very common, was not associated with adverse outcome.

Title: No net renal extraction of homocysteine in fasting humans. Content: BACKGROUND The pathophysiological mechanism of hyperhomocysteinemia in chronic renal failure in humans is unknown. The loss of a putative renal homocysteine extraction in chronic renal failure has been hypothesized as significant homocysteine uptake has been demonstrated in the normal rat kidney. We studied homocysteine extraction in the normal human kidney. METHODS We measured plasma total (free and protein-bound) and free homocysteine (tHcy and fHcy, respectively) in arterial and renal venous blood sampled from the aorta and right-side renal vein during cardiac catheterization in 20 fasting patients with normal renal function. Renal homocysteine extraction was calculated as the arteriovenous difference divided by the arterial levels times 100%. RESULTS No significant renal extraction was demonstrated either for tHcy: 0.9% (SD 5.8; 95% CI -1.8 to +3.6) or for fHcy: -0.2% (11.0; -5.4 to +4.9). CONCLUSIONS We conclude that no significant net renal uptake of homocysteine occurs in fasting humans with normal renal function. The loss of such uptake, therefore, cannot cause hyperhomocysteinemia in patients with renal failure.

Title: Hemoglobin concentration in white, black, and Oriental children: is there a need for separate criteria in screening for anemia? Content: The concentration of hemoglobin in blacks was found to be 0.5 to 1.0 g/dl lower than that of income-matched whites in several large surveys. This difference could be a racial characteristic of blacks, or it might be due to a higher frequency of genetic traits such as thalassemia minor and hemoglobinopathies, or to environmental factors such as iron deficiency. To help in making this distinction, we analyzed the data from multiphasic examinations (1973 to 1975) on 1718 white, 741 black, and 315 Oriental healthy, nonindigent children between 5 and 14 years of age. In the entire population, the median hemoglobin concentration averaged 0.5 g/dl lower in blacks than in whites of both sexes (t test, P less than 0.001). The differences still averaged 0.5 g/dl (P less than 0.001) after exclusion of all those with abnormal hemoglobin by electrophoresis (Hgb S and C) and those whose mean corpuscular volume was more than 5% below the normal mean for age (to exclude iron deficiency or thalassemia minor). The data strengthen the impression that blacks normally have a concentration of hemoglobin averaging about 0.5 g/dl less than in whites. If this is the case, about 10% of normal blacks will be mistakenly designated anemic, if the same norms are applied.

Title: Association of cystic fibrosis with abnormalities in fatty acid metabolism. Content: BACKGROUND Patients with cystic fibrosis have altered levels of plasma fatty acids. We previously demonstrated that arachidonic acid levels are increased and docosahexaenoic acid levels are decreased in affected tissues from cystic fibrosis-knockout mice. In this study we determined whether humans with mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have a similar fatty acid defect in tissues expressing CFTR. METHODS Fatty acids from nasal- and rectal-biopsy specimens, nasal epithelial scrapings, and plasma were analyzed from 38 subjects with cystic fibrosis and compared with results in 13 obligate heterozygotes, 24 healthy controls, 11 subjects with inflammatory bowel disease, 9 subjects with upper respiratory tract infection, and 16 subjects with asthma. RESULTS The ratio of arachidonic to docosahexaenoic acid was increased in mucosal and submucosal nasal-biopsy specimens (P<0.001) and rectal-biopsy specimens (P=0.009) from subjects with cystic fibrosis and pancreatic sufficiency and subjects with cystic fibrosis and pancreatic insufficiency, as compared with values in healthy control subjects. In nasal tissue, this change reflected an increase in arachidonic acid levels and a decrease in docosahexaenoic acid levels. In cells from nasal mucosa, the ratio of arachidonic to docosahexaenoic acid was increased in subjects with cystic fibrosis (P<0.001), as compared with healthy controls, with values in obligate heterozygotes intermediate between these two groups (P<0.001). The ratio was not increased in subjects with inflammatory bowel disease. Subjects with asthma and those with upper respiratory tract infection had values intermediate between those in subjects with cystic fibrosis and those in healthy control subjects. CONCLUSIONS These data indicate that alterations in fatty acids similar to those in cystic fibrosis-knockout mice are present in CFTR-expressing tissue from subjects with cystic fibrosis.

Title: Age as a risk factor for thrombocytopenia and anaemia in children treated for acute uncomplicated falciparum malaria. Content: BACKGROUND & OBJECTIVES Anaemia is commonly observed in children with malaria, but reports on leucocyte and platelet count abnormalities associated with malaria are inconsistent. This study examined the effect of age, gender, parasite density and temperature on haematological parameters in children with acute uncomplicated malaria. METHODS Haematological parameters were determined in children with acute uncomplicated malaria, and these were correlated with age, sex, temperature and parasite density. Statistical analysis was done using SAS 9.1. RESULTS Six hundred and ninety five children with acute uncomplicated malaria participated in the study. The mean age was 51.7 months +/- 33.8. At presentation, anaemia occurred in 43.8% of the patients and children <5 yr had a significantly lower haematocrit (28.4% +/- 4.8) than that of older children (32.8% +/- 4.8) (p <0.001), but the haematocrit was not significantly different by days 14 and 28. There was no difference between both sexes. Leucocytosis was more frequently seen than leucopenia (9.5% vs 3%). Thrombocytopenia was found in 59.3% of enrolled patients. More than half of the patients with thrombocytopenia had recovered by Day 28. Baseline platelet count was related to Day 14 (r = 0.6, p < 0.0001) and Day 28 (r = 0.2, p = 0.0015) and the haematocrit on Day 28 (r = 0.12, p = 0.00197). Platelet count showed no correlation with temperature, parasite density and leucocyte count. Haematocrit correlated with age (r = 0.4, p < 0.0001); but not with parasite density or temperature. Leucocyte count showed no correlation with age or parasite density. CONCLUSION While thrombocytopenia was the most common haematological finding and may be of diagnostic importance, anaemia and leucocytosis were more common in the under fives.

Title: Th2 cytokine response in Major Depressive Disorder patients before treatment Content: In Major Depressive Disorder (MDD), the neuroendocrine and immune systems interactions are impaired. We investigated the pro/anti-inflammatory Th1/Th2 cytokine balance in MDD patients and in non-depressed control group. The MDD subjects showed higher levels of cortisol and TNF-alpha, increased CD3+CD8+ and NK percentages, diminished B cell counts and no significant variations in CD3+CD4+ lymphocyte. Moreover, higher levels of IL-4 and IL-13 (Th2) and significantly lower measurements of IL-2 and IFN-gamma (Th1) cytokines were also observed in the MDD group. Overall, we propose that all these changes could be related to the elevated cortisol levels seen in the MDD patients. Further studies are necessary to explore these findings and its implication in future therapeutic approach of MDD patients.

Title: Effect modification by population dietary folate on the association between MTHFR genotype, homocysteine, and stroke risk: a meta-analysis of genetic studies and randomised trials Content: BACKGROUND The MTHFR 677C→T polymorphism has been associated with raised homocysteine concentration and increased risk of stroke. A previous overview showed that the effects were greatest in regions with low dietary folate consumption, but differentiation between the effect of folate and small-study bias was difficult. A meta-analysis of randomised trials of homocysteine-lowering interventions showed no reduction in coronary heart disease events or stroke, but the trials were generally set in populations with high folate consumption. We aimed to reduce the effect of small-study bias and investigate whether folate status modifies the association between MTHFR 677C→T and stroke in a genetic analysis and meta-analysis of randomised controlled trials. METHODS We established a collaboration of genetic studies consisting of 237 datasets including 59,995 individuals with data for homocysteine and 20,885 stroke events. We compared the genetic findings with a meta-analysis of 13 randomised trials of homocysteine-lowering treatments and stroke risk (45,549 individuals, 2314 stroke events, 269 transient ischaemic attacks). FINDINGS The effect of the MTHFR 677C→T variant on homocysteine concentration was larger in low folate regions (Asia; difference between individuals with TT versus CC genotype, 3·12 μmol/L, 95% CI 2·23 to 4·01) than in areas with folate fortification (America, Australia, and New Zealand, high; 0·13 μmol/L, -0·85 to 1·11). The odds ratio (OR) for stroke was also higher in Asia (1·68, 95% CI 1·44 to 1·97) than in America, Australia, and New Zealand, high (1·03, 0·84 to 1·25). Most randomised trials took place in regions with high or increasing population folate concentrations. The summary relative risk (RR) of stroke in trials of homocysteine-lowering interventions (0·94, 95% CI 0·85 to 1·04) was similar to that predicted for the same extent of homocysteine reduction in large genetic studies in populations with similar folate status (predicted RR 1·00, 95% CI 0·90 to 1·11). Although the predicted effect of homocysteine reduction from large genetic studies in low folate regions (Asia) was larger (RR 0·78, 95% CI 0·68 to 0·90), no trial has evaluated the effect of lowering of homocysteine on stroke risk exclusively in a low folate region. INTERPRETATION In regions with increasing levels or established policies of population folate supplementation, evidence from genetic studies and randomised trials is concordant in suggesting an absence of benefit from lowering of homocysteine for prevention of stroke. Further large-scale genetic studies of the association between MTHFR 677C→T and stroke in low folate settings are needed to distinguish effect modification by folate from small-study bias. If future randomised trials of homocysteine-lowering interventions for stroke prevention are undertaken, they should take place in regions with low folate consumption. FUNDING Full funding sources listed at end of paper (see Acknowledgments).

Title: High frequency of p16 (CDKN2/MTS-1/INK4A) inactivation in head and neck squamous cell carcinoma. Content: The tumor suppressor gene p16 (CDKN2/MTS-1/INK4A) can be inactivated by multiple genetic mechanisms. We analyzed 29 invasive primary head and neck squamous cell carcinomas (HNSCC) for p16 inactivation with immunohistochemistry utilizing a new monoclonal antibody (mAb), DCS-50. p16 staining of the primary lesions was correlated with genetic analysis including: (a) detailed microsatellite analysis of markers at the p16 locus to detect homozygous deletion; (b) sequence analysis of p16; and (c) Southern blot analysis to determine the methylation status of the 5' CpG island of p16. Twenty-four of 29 (83%) head and neck squamous cell carcinoma tumors displayed an absence of p16 nuclear staining using immunohistochemistry. Of these 24 tumors, we found that 16 (67%) harbored homozygous deletions, 5 (21%) were methylated, 1 displayed a rearrangement at the p16 locus, and 1 displayed a frameshift mutation in exon 1. These data suggest that: (a) inactivation of the p16 tumor suppressor gene is a frequent event in squamous cell carcinomas of the head and neck; (b) p16 is inactivated by several distinct and exclusive events including homozygous deletion, point mutation, and promoter methylation; and (c) immunohistochemical analysis for expression of the p16 gene product is an accurate and relatively simple method for evaluating p16 gene inactivation.

Title: A common variant in BRCA2 is associated with both breast cancer risk and prenatal viability Content: Inherited mutations in the gene BRCA2 predispose carriers to early onset breast cancer, but such mutations account for fewer than 2% of all cases in East Anglia. It is likely that low penetrance alleles explain the greater part of inherited susceptibility to breast cancer; polymorphic variants in strongly predisposing genes, such as BRCA2, are candidates for this role. BRCA2 is thought to be involved in DNA double strand break-repair. Few mice in which Brca2 is truncated survive to birth; of those that do, most are male, smaller than their normal littermates and have high cancer incidence. Here we show that a common human polymorphism (N372H) in exon 10 of BRCA2 confers an increased risk of breast cancer: the HH homozygotes have a 1.31-fold (95% CI, 1.07–1.61) greater risk than the NN group. Moreover, in normal female controls of all ages there is a significant deficiency of homozygotes compared with that expected from Hardy-Weinberg equilibrium, whereas in males there is an excess of homozygotes: the HH group has an estimated fitness of 0.82 in females and 1.38 in males. Therefore, this variant of BRCA2 appears also to affect fetal survival in a sex-dependent manner.

Title: Angiopoietin-2 is increased in sepsis and inversely associated with nitric oxide-dependent microvascular reactivity Content: INTRODUCTION Angiopoietin-2 (ang-2), an angiogenic peptide released by endothelial cell Weibel-Palade bodies (WPBs), increases endothelial activation and vascular permeability. Ang-2 is raised in severe sepsis but the mechanisms underlying this are not known. Nitric oxide (NO) inhibits WPB exocytosis, and bioavailability of endothelial NO is decreased in sepsis. We hypothesized that endothelial NO bioavailability would be inversely correlated with ang-2 concentrations in sepsis. METHODS Plasma ang-2, vascular endothelial growth factor (VEGF) and endothelial-active cytokines were assessed in 83 patients with early sepsis and 41 hospital controls, and related to reactive hyperaemia-peripheral arterial tonometry, RH-PAT, a measure of endothelial NO bioavailability. RESULTS Plasma Ang-2 was elevated in sepsis (median [interquartile range (IQR)], ng/ml: severe sepsis 12.4 [8.5-33.4], sepsis without organ failure 6.1 [5.0-10.4], controls 2.7 [2.2-3.6], P < 0.0001). It correlated inversely with RH-PAT (r = -0.38, P < 0.0001) and positively with IL-6 (r = 0.57, P < 0.0001) and degree of organ failure (sequential organ function assessment score) (r = 0.58, P < 0.0001). The correlation of ang-2 with RH-PAT persisted after controlling for sepsis severity. In a longitudinal mixed-effects model, recovery of RH-PAT over time was associated with decline in ang-2. CONCLUSIONS Ang-2 is elevated in proportion to sepsis severity, and inversely correlated with NO-dependent microvascular reactivity. Impaired endothelial NO bioavailability may contribute to increased endothelial cell release of ang-2, endothelial activation and capillary leak. Agents that increase endothelial NO bioavailability or inhibit WPB exocytosis and/or Ang-2 activity may have therapeutic potential in sepsis.

Title: Mutation of the fumarase gene in two siblings with progressive encephalopathy and fumarase deficiency. Content: We report an inborn error of the tricarboxylic acid cycle, fumarase deficiency, in two siblings born to first cousin parents. They presented with progressive encephalopathy, dystonia, leucopenia, and neutropenia. Elevation of lactate in the cerebrospinal fluid and high fumarate excretion in the urine led us to investigate the activities of the respiratory chain and of the Krebs cycle, and to finally identify fumarase deficiency in these two children. The deficiency was profound and present in all tissues investigated, affecting the cytosolic and the mitochondrial fumarase isoenzymes to the same degree. Analysis of fumarase cDNA demonstrated that both patients were homozygous for a missense mutation, a G-955-->C transversion, predicting a Glu-319-->Gln substitution. This substitution occurred in a highly conserved region of the fumarase cDNA. Both parents exhibited half the expected fumarase activity in their lymphocytes and were found to be heterozygous for this substitution. The present study is to our knowledge the first molecular characterization of tricarboxylic acid deficiency, a rare inherited inborn error of metabolism in childhood.

Title: Oxymetholone Therapy of Fanconi Anemia Suppresses Osteopontin Transcription and Induces Hematopoietic Stem Cell Cycling Content: Androgens are widely used for treating Fanconi anemia (FA) and other human bone marrow failure syndromes, but their mode of action remains incompletely understood. Aged Fancd2(-/-) mice were used to assess the therapeutic efficacy of oxymetholone (OXM) and its mechanism of action. Eighteen-month-old Fancd2(-/-) mice recapitulated key human FA phenotypes, including reduced bone marrow cellularity, red cell macrocytosis, and peripheral pancytopenia. As in humans, chronic OXM treatment significantly improved these hematological parameters and stimulated the proliferation of hematopoietic stem and progenitor cells. RNA-Seq analysis implicated downregulation of osteopontin as an important potential mechanism for the drug's action. Consistent with the increased stem cell proliferation, competitive repopulation assays demonstrated that chronic OXM therapy eventually resulted in stem cell exhaustion. These results expand our knowledge of the regulation of hematopoietic stem cell proliferation and have direct clinical implications for the treatment of bone marrow failure.

Title: Ribosomal protein S17 gene (RPS17) is mutated in Diamond-Blackfan anemia. Content: Diamond-Blackfan anemia (DBA) is a congenital erythroid aplasia characterized as a normochromic macrocytic anemia with a selective deficiency in red blood cell precursors in otherwise normocellular bone marrow. In 40% of DBA patients, various physical anomalies are also present. Currently two genes are associated with the DBA phenotype--the ribosomal protein (RP) S19 mutated in 25% of DBA patients and RPS24 mutated in approximately 1.4% of DBA patients. Here we report the identification of a mutation in yet another ribosomal protein, RPS17. The mutation affects the translation initiation start codon, changing T to G (c.2T>G), thus eliminating the natural start of RPS17 protein biosynthesis. RNA analysis revealed that the mutated allele was expressed, and the next downstream start codon located at position +158 should give rise to a short peptide of only four amino acids (Met-Ser-Arg-Ile). The mutation arose de novo, since all healthy family members carry the wild-type alleles. The identification of a mutation in the third RP of the small ribosomal subunit in DBA patients further supports the theory that impaired translation may be the main cause of DBA pathogenesis.

Title: IDH1 mutations alter citric acid cycle metabolism and increase dependence on oxidative mitochondrial metabolism. Content: Oncogenic mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in several types of cancer, but the metabolic consequences of these genetic changes are not fully understood. In this study, we performed (13)C metabolic flux analysis on a panel of isogenic cell lines containing heterozygous IDH1/2 mutations. We observed that under hypoxic conditions, IDH1-mutant cells exhibited increased oxidative tricarboxylic acid metabolism along with decreased reductive glutamine metabolism, but not IDH2-mutant cells. However, selective inhibition of mutant IDH1 enzyme function could not reverse the defect in reductive carboxylation activity. Furthermore, this metabolic reprogramming increased the sensitivity of IDH1-mutant cells to hypoxia or electron transport chain inhibition in vitro. Lastly, IDH1-mutant cells also grew poorly as subcutaneous xenografts within a hypoxic in vivo microenvironment. Together, our results suggest therapeutic opportunities to exploit the metabolic vulnerabilities specific to IDH1 mutation.

Title: Activation of AMPKα2 in adipocytes is essential for nicotine-induced insulin resistance in vivo Content: Cigarette smoking promotes body weight reduction in humans while paradoxically also promoting insulin resistance (IR) and hyperinsulinemia. However, the mechanisms behind these effects are unclear. Here we show that nicotine, a major constituent of cigarette smoke, selectively activates AMP-activated protein kinase α2 (AMPKα2) in adipocytes, which in turn phosphorylates MAP kinase phosphatase-1 (MKP1) at serine 334, initiating its proteasome-dependent degradation. The nicotine-dependent reduction of MKP1 induces the aberrant activation of both p38 mitogen-activated protein kinase and c-Jun N-terminal kinase, leading to increased phosphorylation of insulin receptor substrate 1 (IRS1) at serine 307. Phosphorylation of IRS1 leads to its degradation, protein kinase B inhibition, and the loss of insulin-mediated inhibition of lipolysis. Consequently, nicotine increases lipolysis, which results in body weight reduction, but this increase also elevates the levels of circulating free fatty acids and thus causes IR in insulin-sensitive tissues. These results establish AMPKα2 as an essential mediator of nicotine-induced whole-body IR in spite of reductions in adiposity.

Title: Plasma homocysteine is a risk factor for recurrent vascular events in young patients with an ischaemic stroke or TIA Content: Young patients with an ischaemic stroke or transient ischaemic attack (TIA) often have no vascular risk factors. Hyperhomocysteinaemia is an established risk factor for stroke in elderly patients but it is uncertain whether it is also important for the prognosis of young ischaemic stroke and TIA patients. We examined the possible effect of the plasma homocysteine level on the risk of recurrent vascular events in patients between 18 and 45 years of age. The study population consisted of 161 consecutive patients with a recent cerebral infarction or TIA. Data on the primary event and the homocysteine level were collected retrospectively from hospital records. General practitioners and patients were contacted by telephone to record vascular events and the type of medication used during the follow–up period. Vascular events included cerebral infarction, TIA, pulmonary embolism, venous thrombosis, myocardial infarction and peripheral arterial disease. A Kaplan- Meier curve showed a dose effect relationship between event-free survival time and tertiles of the homocysteine level (Log rank statistic 5.91; p = 0.05). The Cox hazard ratio, after adjustment for homocysteine lowering treatment, was 1.7 (95 % CI, 1.1 to 2.8) for any vascular outcome event, 1.9 (95% CI, 1.1 to 3.0) for arterial outcome events and 1.8 (95 % CI, 1.1 to 2.9) for cerebral outcome events. In spite of our small number of outcome events we found a significant association at the 95% confidence level between homocysteine level and the risk of recurrent vascular events in young patients with an ischaemic stroke or TIA. The association is of the same magnitude as in elderly people.

Title: Specific deficit of the ON response in visual transmission by targeted disruption of the mGIuR6 gene Content: Taking advantage of the restricted expression of metabotropic glutamate receptor subtype 6 (mGluR6) in retinal ON bipolar cells, we generated knockout mice lacking mGluR6 expression. The homozygous mutant mice showed a loss of ON responses but unchanged OFF responses to light. The mutant mice displayed no obvious changes in retinal cell organization nor in the projection of optic fibers to the brain. Furthermore, the mGluR6-deficient mice showed visual behavioral responses to light stimulation as examined by shuttle box avoidance behavior experiments using light exposure as a conditioned stimulus. The results demonstrate that mGluR6 is essential in synaptic transmission to the ON bipolar cell and that the OFF response provides an important means for transmitting visual information.

Title: Murine embryonic stem cell differentiation is promoted by SOCS-3 and inhibited by the zinc finger transcription factor Klf4. Content: Embryonic stem (ES) cells homozygous for a Shp-2 mutation (Shp-2(Delta46-110)) demonstrate leukemia inhibitory factor (LIF) hypersensitivity and increased LIF-stimulated phosphorylation of signal transducer and activator of transcription (STAT3). We hypothesized that LIF-responsive genes in Shp-2(Delta46-110) cells would represent potential candidates for molecules vital for ES cell self-renewal. Using microarray analysis, we detected 41 genes whose expression was modified by LIF in Shp-2(Delta46-110) ES cells. Induction of 2 significantly up-regulated genes, suppressor of cytokine signaling-3 (SOCS-3) and Kruppel-like factor 4 (Klf4), was verified using Northern blotting. ES cells overexpressing SOCS-3 had an increased capacity to differentiate to hematopoietic progenitors, rather than to self-renew. In contrast, ES cells overexpressing Klf4 had a greater capacity to self-renew based on secondary embryoid body (EB) formation. Klf4-transduced d6 EBs expressed higher levels of Oct-4, consistent with the notion that Klf4 promotes ES cell self-renewal. These findings verify the negative role of SOCS-3 on LIF signaling and provide a novel role for Klf4 in ES cell function.

Title: The effect of folate fortification on folic acid-based homocysteine-lowering intervention and stroke risk: a meta-analysis. Content: OBJECTIVE Folate and vitamin B12 are two vital regulators in the metabolic process of homocysteine, which is a risk factor of atherothrombotic events. Low folate intake or low plasma folate concentration is associated with increased stroke risk. Previous randomized controlled trials presented discordant findings in the effect of folic acid supplementation-based homocysteine lowering on stroke risk. The aim of the present review was to perform a meta-analysis of relevant randomized controlled trials to check the how different folate fortification status might affect the effects of folic acid supplementation in lowering homocysteine and reducing stroke risk. DESIGN Relevant randomized controlled trials were identified through formal literature search. Homocysteine reduction was compared in subgroups stratified by folate fortification status. Relative risks with 95 % confidence intervals were used as a measure to assess the association between folic acid supplementation and stroke risk. SETTING The meta-analysis included fourteen randomized controlled trials, SUBJECTS A total of 39 420 patients. RESULTS Homocysteine reductions were 26·99 (sd 1·91) %, 18·38 (sd 3·82) % and 21·30 (sd 1·98) %, respectively, in the subgroups without folate fortification, with folate fortification and with partial folate fortification. Significant difference was observed between the subgroups with folate fortification and without folate fortification (P=0·05). The relative risk of stroke was 0·88 (95 % CI 0·77, 1·00, P=0·05) in the subgroup without folate fortification, 0·94 (95 % CI 0·58, 1·54, P=0·82) in the subgroup with folate fortification and 0·91 (95 % CI 0·82, 1·01, P=0·09) in the subgroup with partial folate fortification. CONCLUSIONS Folic acid supplementation might have a modest benefit on stroke prevention in regions without folate fortification.

Title: Review article Content: Ribosomopathies compose a collection of disorders in which genetic abnormalities cause impaired ribosome biogenesis and function, resulting in specific clinical phenotypes. Congenital mutations in RPS19 and other genes encoding ribosomal proteins cause Diamond-Blackfan anemia, a disorder characterized by hypoplastic, macrocytic anemia. Mutations in other genes required for normal ribosome biogenesis have been implicated in other rare congenital syndromes, Schwachman-Diamond syndrome, dyskeratosis congenita, cartilage hair hypoplasia, and Treacher Collins syndrome. In addition, the 5q- syndrome, a subtype of myelodysplastic syndrome, is caused by a somatically acquired deletion of chromosome 5q, which leads to haploinsufficiency of the ribosomal protein RPS14 and an erythroid phenotype highly similar to Diamond-Blackfan anemia. Acquired abnormalities in ribosome function have been implicated more broadly in human malignancies. The p53 pathway provides a surveillance mechanism for protein translation as well as genome integrity and is activated by defects in ribosome biogenesis; this pathway appears to be a critical mediator of many of the clinical features of ribosomopathies. Elucidation of the mechanisms whereby selective abnormalities in ribosome biogenesis cause specific clinical syndromes will hopefully lead to novel therapeutic strategies for these diseases.

Title: Hyperglycemia is associated with increased bone mineral density and decreased trabecular bone score in elderly Japanese men: The Fujiwara-kyo osteoporosis risk in men (FORMEN) study. Content: PURPOSE Patients with type 2 diabetes mellitus (T2DM) have an increased fracture risk despite having higher areal bone mineral density (aBMD). This study aimed to clarify the association between glycemic and insulin resistance status and bone microarchitecture, and whether pentosidine and bone turnover markers play any roles in the association. METHODS A total of 2012 community-dwelling men aged ≥65years completed baseline measurements of spine aBMD, fasting plasma glucose (FPG) and serum insulin, hemoglobin A1c (HbA1c), osteocalcin, type I procollagen N-terminal propeptide, type I collagen C-terminal crosslinking telopeptide, tartrate-resistant acid phosphatase isoenzyme 5b, pentosidine, height and weight and an interview regarding past disease history. Homeostasis model assessment-insulin resistance (HOMA-IR) was also calculated. T2DM was defined as physician-diagnosed middle age or elderly-onset diabetes mellitus, or according to biochemical test results. To evaluate bone microarchitecture, trabecular bone score (TBS) was calculated at the same vertebrae as those used for aBMD measurement. RESULTS After excluding participants who had a disease history and/or were taking medications affecting bone metabolism, 1683 men (age, 72.9±5.2years) were analyzed. Men with T2DM had significantly higher aBMD compared to those without T2DM. There was no significant difference in TBS. However, FPG, HbA1c and HOMA-IR levels were significantly inversely correlated with TBS after adjusting for age, BMI and aBMD. Multivariate linear regression analyses revealed that glycemic indices (FPG and HbA1c) were significantly associated with increased aBMD and decreased TBS, and that HOMA-IR was associated only with TBS. These associations did not change after further adjusting for bone turnover makers and pentosidine levels. CONCLUSIONS Hyperglycemia and elevated insulin-resistance were associated with low TBS independently of bone turnover and pentosidine levels.

Title: Increased lipid peroxidation as a mechanism of methionine-induced atherosclerosis in rabbits. Content: Methionine is converted by the transmethylation/transsulfuration pathway to homocysteine which may exert atherogenic effects by several mechanisms, including lipid peroxidation. Therefore, the excessive dietary methionine may induce the development of atherosclerosis. To test this hypothesis, plasma and aortic thiobarbituric acid reactive substances (TBARS), as well as activities of aortic and erythrocyte superoxide dismutase (SOD), catalase and selenium-dependent glutathione peroxidase (GPX) were measured in rabbits fed a diet enriched with 0.3% methionine for 6 or 9 months. Histological examinations of aortas also were performed. Feeding rabbits a methionine-enriched diet for 6 or 9 months resulted in significant increases in plasma and aortic TBARS levels and aortic antioxidant enzyme activities. However, a decrease in plasma antioxidant activity (AOA) was observed. In erythrocytes, SOD activity increased, catalase remained normal and GPX decreased in the treated animals. Histological examination of aortas showed typical atherosclerotic changes, such as intimal thickening, deposition of cholesterol, and calcification in methionine-fed rabbits. These results confirm that high-methionine diet may induce atherosclerosis in rabbits and indicate disturbances in lipid peroxidation and antioxidant processes as possible mechanisms of its atherogenic influence.

Title: A paediatric case of sideroblastic anaemia. Ultrastructural studies of erythroblasts cultured from marrow BFU-E in a methylcellulose micromethod. Content: We examined the morphological and functional characteristics of erythroblasts derived from marrow erythroid progenitor cells grown in a methylcellulose microculture, which were taken from a female child with rare atypical sideroblastic anaemia (SA) partially responsive to pyridoxine. Colony formation was within the normal range in three successive cultures (median values: 82.25 CFU-E and 16.4 BFU-E derived colonies/6.6 X 10(4) cells) compared to growth by normal cells (65-315 CFU-E and 9-40 BFU-E). We evaluated in vitro differentiation by biochemical microassay of a cytosol enzyme involved in the haem pathway: uroporphyrinogen I synthase (UROS). The UROS values in the erythroid colonies from SA marrow were at the lowere end of the normal range (median values: 6.7 +/- 0.3 and 14.4 +/- 3.8 pmol uroporphyrinogen/h in CFU-E and BFU-E-derived colonies respectively versus 17.4 +/- 7.3 and 25 +/- 7.2 pmol/h in CFU-E and BFU-E colonies from normal subjects. Ultrastructural examination of the SA erythroblasts from non-cultured bone marrow or derived from cultured BFU-E revealed the characteristic deposition of iron in mitochondria around the nucleus of most cells (ringed sideroblasts). However, the majority of cultured cells had marked dyserythropoietic features, with a large number of bilobulated or trilobulated erythroblasts, multiple cytoplasmic vacuoles, numerous abnormalities of the nucleus, and excessive membrane material beneath the plasma membrane, all features difficult to observe in non-cultured marrows.

Title: Role of beta adrenergic receptor polymorphisms in heart failure: systematic review and meta-analysis. Content: Heart Failure (HF) is a common disorder associated with substantial morbidity and mortality. beta adrenergic receptors (betaAR) are the primary pathway through which cardiac function is influenced. Chronic beta(1)AR activation is implicated in the pathogenesis of HF and betaAR blockade improves survival in left ventricular systolic dysfunction. Common functional polymorphisms in beta adrenergic receptor genes (ADRB) have been associated with HF phenotypes, and with pharmacogenetic interaction with beta adrenergic receptor blockers (beta blockers). However, these associations have not been consistently replicated. The evidence for ADRB variant involvement in pathogenesis, progression and response to beta blockers in HF is reviewed. In addition, a meta-analysis of three studies analysing the effect of ADRB1 Arg389Gly polymorphism on left ventricular remodelling with the use of beta blockers, demonstrating a 5% improvement in left ventricular ejection fraction in Arg389 homozygotes, is presented. There is now accumulating molecular evidence for a different functional response to beta blockers associated with this polymorphism. In the future, confirmed genotypic associations may enable patients to be identified who are either at greater risk of developing HF, whose HF may rapidly progress, or who are unlikely to benefit from beta blockers, and such patients may benefit from targeted aggressive therapy.

Title: β-Adrenergic receptor antagonism prevents anxiety-like behavior and microglial reactivity induced by repeated social defeat. Content: Psychosocial stress is associated with altered immune function and development of psychological disorders including anxiety and depression. Here we show that repeated social defeat in mice increased c-Fos staining in brain regions associated with fear and threat appraisal and promoted anxiety-like behavior in a β-adrenergic receptor-dependent manner. Repeated social defeat also significantly increased the number of CD11b(+)/CD45(high)/Ly6C(high) macrophages that trafficked to the brain. In addition, several inflammatory markers were increased on the surface of microglia (CD14, CD86, and TLR4) and macrophages (CD14 and CD86) after social defeat. Repeated social defeat also increased the presence of deramified microglia in the medial amygdala, prefrontal cortex, and hippocampus. Moreover, mRNA analysis of microglia indicated that repeated social defeat increased levels of interleukin (IL)-1β and reduced levels of glucocorticoid responsive genes [glucocorticoid-induced leucine zipper (GILZ) and FK506 binding protein-51 (FKBP51)]. The stress-dependent changes in microglia and macrophages were prevented by propranolol, a β-adrenergic receptor antagonist. Microglia isolated from socially defeated mice and cultured ex vivo produced markedly higher levels of IL-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1 after stimulation with lipopolysaccharide compared with microglia from control mice. Last, repeated social defeat increased c-Fos activation in IL-1 receptor type-1-deficient mice, but did not promote anxiety-like behavior or microglia activation in the absence of functional IL-1 receptor type-1. These findings indicate that repeated social defeat-induced anxiety-like behavior and enhanced reactivity of microglia was dependent on activation of β-adrenergic and IL-1 receptors.

Title: Increased susceptibility to UV-induced skin carcinogenesis in polymerase eta-deficient mice. Content: Xeroderma pigmentosum variant (XPV) patients with mutations in the DNA polymerase eta (pol eta) gene are hypersensitive to sunlight and have greatly increased susceptibility to sunlight-induced skin cancer. Consistent with the ability of Pol eta to efficiently bypass UV light-induced cyclobutane pyrimidine dimers, XPV cells lacking Pol eta have diminished capacity to replicate UV-damaged DNA and are sensitive to UV light-induced killing and mutagenesis. To better understand these and other Pol eta functions, we generated Pol eta-deficient mice. Mice homozygous for a null mutation in pol eta are viable, fertile, and do not show any obvious spontaneous defects during the first year of life. However, fibroblasts derived from these mutant mice are sensitive to killing by exposure to UV light, and all Pol eta-deficient mice develop skin tumors after UV irradiation, in contrast to the wild-type littermate controls that did not develop such tumors. These results and biochemical studies of translesion synthesis by mouse Pol eta indicate that Pol eta-dependent bypass of cyclobutane pyrimidine dimers suppresses UV light-induced skin cancer in mice. Moreover, 37.5% of pol eta heterozygous mice also developed skin cancer during 5 months after a 5-month exposure to UV light, suggesting that humans who are heterozygous for mutations in pol eta may also have an increased risk of skin cancer.

Title: Enhanced monocyte Fc phagocytosis by a homologue of interleukin-10 encoded by human cytomegalovirus. Content: Human cytomegalovirus (HCMV) expresses several homologues of human interleukin 10 (hIL-10) possessing immunomodulatory properties which may promote viral infection by modulating the function of myeloid cells. We examined the phenotype and phagocytic capability of human monocytes exposed to hIL-10, an HCMV-encoded hIL-10 homologue expressed during the productive phase of infection (cmvIL-10), and a differentially spliced form of cmvIL-10 expressed during latent and productive phases of infection, (LAcmvIL-10). hIL-10 and cmvIL-10 upregulated expression of Fcgamma receptors, stimulated phagocytosis of IgG-opsonised erythrocytes and decreased MHC class II (HLA-DR) expression on purified monocytes within 24 h. In contrast, LAcmvIL-10 decreased HLA-DR expression at later times (48 h and 72 h) but did not increase Fcgamma receptor expression. We conclude that cmvIL-10 promotes differentiation of monocytes towards a pro-phagocytic phenotype and that LAcmvIL-10 does not affect monocytes by the same mechanism as cmvIL-10. The significance of these properties to cytomegalovirus pathogenesis is discussed.

Title: CD11b+ monocytes abrogate Th17 CD4+ T cell-mediated experimental autoimmune myocarditis. Content: Experimental autoimmune myocarditis (EAM) represents a Th17 T cell-mediated mouse model of postinflammatory heart disease. In BALB/c wild-type mice, EAM is a self-limiting disease, peaking 21 days after alpha-myosin H chain peptide (MyHC-alpha)/CFA immunization and largely resolving thereafter. In IFN-gammaR(-/-) mice, however, EAM is exacerbated and shows a chronic progressive disease course. We found that this progressive disease course paralleled persistently elevated IL-17 release from T cells infiltrating the hearts of IFN-gammaR(-/-) mice 30 days after immunization. In fact, IL-17 promoted the recruitment of CD11b(+) monocytes, the major heart-infiltrating cells in EAM. In turn, CD11b(+) monocytes suppressed MyHC-alpha-specific Th17 T cell responses IFN-gamma-dependently in vitro. In vivo, injection of IFN-gammaR(+/+)CD11b(+), but not IFN-gammaR(-/-)CD11b(+), monocytes, suppressed MyHC-alpha-specific T cells, and abrogated the progressive disease course in IFN-gammaR(-/-) mice. Finally, coinjection of MyHC-alpha-specific, but not OVA-transgenic, IFN-gamma-releasing CD4(+) Th1 T cell lines, together with MyHC-alpha-specific Th17 T cells protected RAG2(-/-) mice from EAM. In conclusion, CD11b(+) monocytes play a dual role in EAM: as a major cellular substrate of IL-17-induced inflammation and as mediators of an IFN-gamma-dependent negative feedback loop confining disease progression.

Title: Folic acid improves endothelial function in coronary artery disease via mechanisms largely independent of homocysteine lowering. Content: BACKGROUND Homocysteine is a risk factor for coronary artery disease (CAD), although a causal relation remains to be proven. The importance of determining direct causality rests in the fact that plasma homocysteine can be safely and inexpensively reduced by 25% with folic acid. This reduction is maximally achieved by doses of 0.4 mg/d. High-dose folic acid (5 mg/d) improves endothelial function in CAD, although the mechanism is controversial. It has been proposed that improvement occurs through reduction in total (tHcy) or free (non-protein bound) homocysteine (fHcy). We investigated the effects of folic acid on endothelial function before a change in homocysteine in patients with CAD. METHODS AND RESULTS A randomized, placebo-controlled study of folic acid (5 mg/d) for 6 weeks was undertaken in 33 patients. Endothelial function, assessed by flow-mediated dilatation (FMD), was measured before, at 2 and 4 hours after the first dose of folic acid, and after 6 weeks of treatment. Plasma folate increased markedly by 1 hour (200 compared with 25.8 nmol/L; P<0.001). FMD improved at 2 hours (83 compared with 47 microm; P<0.001) and was largely complete by 4 hours (101 compared with 51 microm; P<0.001). tHcy did not significantly differ acutely (4-hour tHcy, 9.56 compared with 9.79 micromol/L; P=NS). fHcy did not differ at 3 hours but was slightly reduced at 4 hours (1.55 compared with 1.78 micromol/L; P=0.02). FMD improvement did not correlate with reductions in either fHcy or tHcy at any time. CONCLUSIONS These data suggest that folic acid improves endothelial function in CAD acutely by a mechanism largely independent of homocysteine.

Title: Circulating microRNAs as biomarkers for hepatocellular carcinoma. Content: GOALS We investigated whether measurement of serum levels of the microRNAs (miRNAs) miR-16, miR-195, and miR-199a, alone or in combination with conventional serum markers, can help to differentiate hepatocellular carcinoma (HCC) from chronic liver diseases (CLDs). BACKGROUND Recent reports suggest a link between aberrant expression of miRNA, and HCC. STUDY This retrospective analysis was conducted using sera from 105 HCC patients, 107 CLD patients, and 71 normal control subjects. The miRNAs were measured using real-time reverse transcription-polymerase chain reaction. The conventional HCC markers α-fetoprotein (AFP), lens culinaris agglutinin-reactive AFP (AFP-L3%), and des-γ-carboxyprothrombin (DCP) were measured with commercial kits. RESULTS Serum levels of miR-16 and miR-199a were significantly lower in HCC than in CLD patients or control subjects (P<0.01). As a single marker, miR-16 had the highest sensitivity for HCC, followed by miR-199a, AFP, DCP, AFP-L3%, and miR-195. The combination of miR-16, AFP, AFP-L3%, and DCP yielded the optimal combination of sensitivity (92.4%) and specificity (78.5%) for HCC, overall and when analysis was restricted to patients with tumors size smaller than 3 cm. As a second-line HCC marker, miR-16 yielded positive HCC predictions in 18 of the 26 (69.2%) HCC patients with negative results on all 3 conventional markers, most of whom had tumors size smaller than 3 cm; miR-16 was falsely positive in only 12 of 96 (12.5%) CLD patients. CONCLUSIONS The addition of miR-16 to conventional serum markers improved sensitivity and specificity for HCC. Use of miR-16 for second-line testing in cases considered negative on the basis of conventional HCC markers should be explored in larger, prospective studies.

Title: Disruption of tumor necrosis factor-alpha gene diminishes the development of atherosclerosis in ApoE-deficient mice. Content: Inflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha) have been implicated in atherogenesis. However, the precise role of TNF-alpha in atherogenesis is still unclear. To examine the effect of TNF-alpha on atherogenesis, we generated compound-deficient mice in apolipoprotein E (apoE) and TNF-alpha (apoE-/-/TNF-alpha-/-) and compared them with apoE-/- mice. Although serum total cholesterol levels were markedly elevated in both apoE-/-/TNF-alpha-/- and apoE-/- mice compared to wild-type mice, no differences were observed between apoE-/-/TNF-alpha-/- and apoE-/- mice. The atherosclerotic plaque area in the aortic luminal surface of apoE-/-/TNF-alpha-/- mice (n=8, 3.1+/-0.4%) was significantly smaller than that of apoE-/- mice (n=7, 4.7+/-0.4%, p<0.001) despite the lack of difference in serum cholesterol levels. The atherosclerotic lesion size in the aortic sinus of apoE-/-/TNF-alpha-/- mice (n=10, 5.1+/-0.3 x 10(5)microm(2)) was also significantly smaller than that of apoE-/- mice (n=11, 7.0+/-0.3 x 10(5)microm(2), p<0.0001). RT-PCR analysis indicated that the expression levels of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1) were significantly higher in apoE-/- than apoE-/-/TNF-alpha-/- mice. Macrophages from apoE(-/-) mice showed higher uptake level of oxidized LDL and increased expression level of scavenger receptor class A (SRA) compared to those from apoE-/-/TNF-alpha-/- mice. These results indicate that TNF-alpha plays an atherogenic role by upregulating the expressions of ICAM-1, VCAM-1 and MCP-1 in the vascular wall, and by inducing SRA expression and oxidized LDL uptake in macrophages.

Title: EIF2AK3, encoding translation initiation factor 2-α kinase 3, is mutated in patients with Wolcott-Rallison syndrome Content: Wolcott-Rallison syndrome (WRS) is a rare, autosomal recessive disorder characterized by permanent neonatal or early infancy insulin-dependent diabetes. Epiphyseal dysplasia, osteoporosis and growth retardation occur at a later age. Other frequent multisystemic manifestations include hepatic and renal dysfunction, mental retardation and cardiovascular abnormalities. On the basis of two consanguineous families, we mapped WRS to a region of less than 3 cM on chromosome 2p12, with maximal evidence of linkage and homozygosity at 4 microsatellite markers within an interval of approximately 1 cM. The gene encoding the eukaryotic translation initiation factor 2-α kinase 3 (EIF2AK3) resides in this interval; thus we explored it as a candidate. We identified distinct mutations of EIF2AK3 that segregated with the disorder in each of the families. The first mutation produces a truncated protein in which the entire catalytic domain is missing. The other changes an amino acid, located in the catalytic domain of the protein, that is highly conserved among kinases from the same subfamily. Our results provide evidence for the role of EIF2AK3 in WRS. The identification of this gene may provide insight into the understanding of the more common forms of diabetes and other pathologic manifestations of WRS.

Title: Autosomal dominant immune dysregulation syndrome in humans with CTLA4 mutations Content: The protein cytotoxic T lymphocyte antigen-4 (CTLA-4) is an essential negative regulator of immune responses, and its loss causes fatal autoimmunity in mice. We studied a large family in which five individuals presented with a complex, autosomal dominant immune dysregulation syndrome characterized by hypogammaglobulinemia, recurrent infections and multiple autoimmune clinical features. We identified a heterozygous nonsense mutation in exon 1 of CTLA4. Screening of 71 unrelated patients with comparable clinical phenotypes identified five additional families (nine individuals) with previously undescribed splice site and missense mutations in CTLA4. Clinical penetrance was incomplete (eight adults of a total of 19 genetically proven CTLA4 mutation carriers were considered unaffected). However, CTLA-4 protein expression was decreased in regulatory T cells (Treg cells) in both patients and carriers with CTLA4 mutations. Whereas Treg cells were generally present at elevated numbers in these individuals, their suppressive function, CTLA-4 ligand binding and transendocytosis of CD80 were impaired. Mutations in CTLA4 were also associated with decreased circulating B cell numbers. Taken together, mutations in CTLA4 resulting in CTLA-4 haploinsufficiency or impaired ligand binding result in disrupted T and B cell homeostasis and a complex immune dysregulation syndrome.

Title: Direct in vivo assessment of microcirculatory dysfunction in severe falciparum malaria. Content: BACKGROUND This study sought to describe and quantify microcirculatory changes in the mucosal surfaces of patients with severe malaria, by direct in vivo observation using orthogonal polarization spectral (OPS) imaging. METHODS The microcirculation in the rectal mucosa of adult patients with severe malaria was assessed by use of OPS imaging, at admission and then daily. Comparison groups comprised patients with uncomplicated falciparum malaria, patients with bacterial sepsis, and healthy individuals. RESULTS Erythrocyte velocities were measured directly in 43 adult patients with severe falciparum malaria, of whom 20 died. Microcirculatory blood flow was markedly disturbed, with heterogeneous obstruction that was proportional to severity of disease. Blocked capillaries were found in 29 patients (67%) and were associated with concurrent hyperdynamic blood flow (erythrocyte velocity, >750 mm/s) in adjacent vessels in 27 patients (93%). The proportion of blocked capillaries correlated with the base deficit in plasma and with the concentration of lactate. Abnormalities disappeared when the patients recovered. In healthy individuals and in patients with uncomplicated malaria or sepsis, no stagnant erythrocytes were detected, and, in patients with sepsis, hyperdynamic blood flow was prominent. CONCLUSION Patients with severe falciparum malaria show extensive microvascular obstruction that is proportional to the severity of the disease. This finding underscores the prominent role that microvascular obstruction plays in the pathophysiology of severe malaria and illustrates the fundamental difference between the microvascular pathophysiology of malaria and that of bacterial sepsis.

Title: Lipoatrophy and severe metabolic disturbance in mice with fat-specific deletion of PPARγ. Content: Adipose tissue is an important metabolic organ, the dysfunction of which is associated with the development of obesity, diabetes mellitus, and cardiovascular disease. The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) is considered the master regulator of adipocyte differentiation and function. Although its cell-autonomous role in adipogenesis has been clearly demonstrated in cell culture, previous fat-specific knockouts of the murine PPARγ gene did not demonstrate a dramatic phenotype in vivo. Here, using Adipoq-Cre mice to drive adipose-specific recombination, we report a unique fat-specific PPARγ knockout (PPARγ FKO) mouse model with almost no visible brown and white adipose tissue at age 3 mo. As a consequence, PPARγ FKO mice had hugely enlarged pancreatic islets, massive fatty livers, and dramatically elevated levels of blood glucose and serum insulin accompanied by extreme insulin resistance. PPARγ FKO mice also exhibited delayed hair coat formation associated with absence of dermal fat, disrupted mammary gland development with loss of mammary fat pads, and high bone mass with loss of bone marrow fat, indicating the critical roles of adipose PPARγ in these tissues. Together, our data reveal the necessity of fat PPARγ in adipose formation, whole-body metabolic homeostasis, and normal development of fat-containing tissues.

Title: Disruption of the Hepcidin/Ferroportin Regulatory System Causes Pulmonary Iron Overload and Restrictive Lung Disease Content: Emerging evidence suggests that pulmonary iron accumulation is implicated in a spectrum of chronic lung diseases. However, the mechanism(s) involved in pulmonary iron deposition and its role in the in vivo pathogenesis of lung diseases remains unknown. Here we show that a point mutation in the murine ferroportin gene, which causes hereditary hemochromatosis type 4 (Slc40a1C326S), increases iron levels in alveolar macrophages, epithelial cells lining the conducting airways and lung parenchyma, and in vascular smooth muscle cells. Pulmonary iron overload is associated with oxidative stress, restrictive lung disease with decreased total lung capacity and reduced blood oxygen saturation in homozygous Slc40a1C326S/C326S mice compared to wild-type controls. These findings implicate iron in lung pathology, which is so far not considered a classical iron-related disorder.

Title: Nephrin mutations cause childhood- and adult-onset focal segmental glomerulosclerosis. Content: Mutations in the NPHS1 gene cause congenital nephrotic syndrome of the Finnish type presenting before the first 3 months of life. Recently, NPHS1 mutations have also been identified in childhood-onset steroid-resistant nephrotic syndrome and milder courses of disease, but their role in adults with focal segmental glomerulosclerosis remains unknown. Here we developed an in silico scoring matrix to evaluate the pathogenicity of amino-acid substitutions using the biophysical and biochemical difference between wild-type and mutant amino acid, the evolutionary conservation of the amino-acid residue in orthologs, and defined domains, with the addition of contextual information. Mutation analysis was performed in 97 patients from 89 unrelated families, of which 52 presented with steroid-resistant nephrotic syndrome after 18 years of age. Compound heterozygous or homozygous NPHS1 mutations were identified in five familial and seven sporadic cases, including one patient 27 years old at onset of the disease. Substitutions were classified as 'severe' or 'mild' using this in silico approach. Our results suggest an earlier onset of the disease in patients with two 'severe' mutations compared to patients with at least one 'mild' mutation. The finding of mutations in a patient with adult-onset focal segmental glomerulosclerosis indicates that NPHS1 analysis could be considered in patients with later onset of the disease.

Title: The VirB type IV secretion system of Bartonella henselae mediates invasion, proinflammatory activation and antiapoptotic protection of endothelial cells. Content: Bartonella henselae is an arthropod-borne zoonotic pathogen causing intraerythrocytic bacteraemia in the feline reservoir host and a broad range of clinical manifestations in incidentally infected humans. Remarkably, B. henselae can specifically colonize the human vascular endothelium, resulting in inflammation and the formation of vasoproliferative lesions known as bacillary angiomatosis and bacillary peliosis. Cultured human endothelial cells provide an in vitro system to study this intimate interaction of B. henselae with the vascular endothelium. However, little is known about the bacterial virulence factors required for this pathogenic process. Recently, we identified the type IV secretion system (T4SS) VirB as an essential pathogenicity factor in Bartonella, required to establish intraerythrocytic infection in the mammalian reservoir. Here, we demonstrate that the VirB T4SS also mediates most of the virulence attributes associated with the interaction of B. henselae during the interaction with human endothelial cells. These include: (i) massive rearrangements of the actin cytoskeleton, resulting in the formation of bacterial aggregates and their internalization by the invasome structure; (ii) nuclear factor kappaB-dependent proinflammatory activation, leading to cell adhesion molecule expression and chemokine secretion, and (iii) inhibition of apoptotic cell death, resulting in enhanced endothelial cell survival. Moreover, we show that the VirB system mediates cytostatic and cytotoxic effects at high bacterial titres, which interfere with a potent VirB-independent mitogenic activity. We conclude that the VirB T4SS is a major virulence determinant of B. henselae, required for targeting multiple endothelial cell functions exploited by this vasculotropic pathogen.

Title: Enzymatically active lysosomal proteases are associated with amyloid deposits in Alzheimer brain. Content: The formation of beta-amyloid in the brains of individuals with Alzheimer disease requires the proteolytic cleavage of a membrane-associated precursor protein. The proteases that may be involved in this process have not yet been identified. Cathepsins are normally intracellular proteolytic enzymes associated with lysosomes; however, when sections from Alzheimer brains were stained by antisera to cathepsin D and cathepsin B, high levels of immunoreactivity were also detected in senile plaques. Extracellular sites of cathepsin immunoreactivity were not seen in control brains from age-matched individuals without neurologic disease or from patients with Huntington disease or Parkinson disease. In situ enzyme histochemistry of cathepsin D and cathepsin B on sections of neocortex using synthetic peptides and protein substrates showed that senile plaques contained the highest levels of enzymatically active cathepsin. At the ultrastructural level, cathepsin immunoreactivity in senile plaques was localized principally to lysosomal dense bodies and lipofuscin granules, which were extracellular. Similar structures were abundant in degenerating neurons of Alzheimer neocortex, and cathepsin-laden neuronal perikarya in various stages of disintegration could be seen within some senile plaques. The high levels of enzymatically competent lysosomal proteases abnormally localized in senile plaques represent evidence for candidate enzymes that may mediate the proteolytic formation of amyloid. We propose that amyloid precursor protein within senile plaques is processed by lysosomal proteases principally derived from degenerating neurons. Escape of cathepsins from the stringently regulated intracellular milieu provides a basis for an abnormal sequence of proteolytic cleavages of accumulating amyloid precursor protein.

Title: Ataxia, dementia, and hypogonadotropism caused by disordered ubiquitination. Content: BACKGROUND The combination of ataxia and hypogonadism was first described more than a century ago, but its genetic basis has remained elusive. METHODS We performed whole-exome sequencing in a patient with ataxia and hypogonadotropic hypogonadism, followed by targeted sequencing of candidate genes in similarly affected patients. Neurologic and reproductive endocrine phenotypes were characterized in detail. The effects of sequence variants and the presence of an epistatic interaction were tested in a zebrafish model. RESULTS Digenic homozygous mutations in RNF216 and OTUD4, which encode a ubiquitin E3 ligase and a deubiquitinase, respectively, were found in three affected siblings in a consanguineous family. Additional screening identified compound heterozygous truncating mutations in RNF216 in an unrelated patient and single heterozygous deleterious mutations in four other patients. Knockdown of rnf216 or otud4 in zebrafish embryos induced defects in the eye, optic tectum, and cerebellum; combinatorial suppression of both genes exacerbated these phenotypes, which were rescued by nonmutant, but not mutant, human RNF216 or OTUD4 messenger RNA. All patients had progressive ataxia and dementia. Neuronal loss was observed in cerebellar pathways and the hippocampus; surviving hippocampal neurons contained ubiquitin-immunoreactive intranuclear inclusions. Defects were detected at the hypothalamic and pituitary levels of the reproductive endocrine axis. CONCLUSIONS The syndrome of hypogonadotropic hypogonadism, ataxia, and dementia can be caused by inactivating mutations in RNF216 or by the combination of mutations in RNF216 and OTUD4. These findings link disordered ubiquitination to neurodegeneration and reproductive dysfunction and highlight the power of whole-exome sequencing in combination with functional studies to unveil genetic interactions that cause disease. (Funded by the National Institutes of Health and others.).

Title: Effect of exercise-induced ischemia on QT interval dispersion. Content: An increased spatial dispersion of ventricular repolarization duration (QT dispersion) is associated with an increased vulnerability to arrhythmias. This study was designed to examine the effect of exercise on QT dispersion in ischemic heart disease (IHD). QT dispersion, corrected QT dispersion, and percentage change in uncorrected and corrected QT dispersion between rest and peak exercise were examined in 14 members of a control group, 17 patients with IHD, and 14 patients with IHD who were receiving beta-blockers (IHD-B). All subjects had undergone a standard Bruce protocol exercise test, and QT intervals were measured at rest and peak exercise with a digitizing tablet interfaced to a personal computer. QT dispersion at rest was markedly increased in the IHD group compared with that in the control and IHD-B groups, respectively (corrected QT dispersion in milliseconds), 74 +/- 7, 40 +/- 4, 49 +/- 5, p < 0.03). The corrected QT dispersion at peak exercise was greater in the IHD group compared with that in the control group (57 +/- 5 vs 26 +/- 3 msec, p < 0.03). The percentage change in QT dispersion with exercise was significantly higher in the IHD group (52% +/- 5%) compared with that in both the control group (28% +/- 4%, p < 0.002) and the IHD-B group (30% +/- 3%, p < 0.01). A larger mean QT dispersion at peak exercise and an increased percentage change in QT dispersion with exercise may help explain the increased susceptibility of the IHD group for arrhythmias. The cardioprotective action of beta-blockers may be explained by their blunting effect on exercise-related changes in QT dispersion.

Title: Early lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice Content: Interleukin 7 (IL-7) stimulates the proliferation of B cell progenitors, thymocytes, and mature T cells through an interaction with a high affinity receptor (IL-7R) belonging to the hematopoietin receptor superfamily. We have further addressed the role of IL-7 and its receptor during B and T cell development by generating mice genetically deficient in IL-7R. Mutant mice display a profound reduction in thymic and peripheral lymphoid cellularity. Analyses of lymphoid progenitor populations in IL-7R-deficient mice define precisely those developmental stages affected by the mutation and reveal a critical role for IL-7R during early lymphoid development. Significantly, these studies indicate that the phase of thymocyte expansion occurring before the onset of T cell receptor gene rearrangement is critically dependent upon, and mediated by the high affinity receptor for IL-7.

Title: Laminin-based cell adhesion anchors microtubule plus ends to the epithelial cell basal cortex through LL5α/β Content: LL5beta has been identified as a microtubule-anchoring factor that attaches EB1/CLIP-associating protein (CLASP)-bound microtubule plus ends to the cell cortex. In this study, we show that LL5beta and its homologue LL5alpha (LL5s) colocalize with autocrine laminin-5 and its receptors, integrins alpha3beta1 and alpha6beta4, at the basal side of fully polarized epithelial sheets. Depletion of both laminin receptor integrins abolishes the cortical localization of LL5s, whereas LL5 depletion reduces the amount of integrin alpha3 at the basal cell cortex. Activation of integrin alpha3 is sufficient to initiate LL5 accumulation at the cell cortex. LL5s form a complex with the cytoplasmic tails of these integrins, but their interaction might be indirect. Analysis of the three-dimensional distribution of microtubule growth by visualizing EB1-GFP in epithelial sheets in combination with RNA interference reveals that LL5s are required to maintain the density of growing microtubules selectively at the basal cortex. These findings reveal that signaling from laminin-integrin associations attaches microtubule plus ends to the epithelial basal cell cortex.

Title: Conditional Kif3a ablation causes abnormal hedgehog signaling topography, growth plate dysfunction, and excessive bone and cartilage formation during mouse skeletogenesis. Content: The motor protein Kif3a and primary cilia regulate important developmental processes, but their roles in skeletogenesis remain ill-defined. Here we created mice deficient in Kif3a in cartilage and focused on the cranial base and synchondroses. Kif3a deficiency caused cranial base growth retardation and dysmorphogenesis, which were evident in neonatal animals by anatomical and micro-computed tomography (microCT) inspection. Kif3a deficiency also changed synchondrosis growth plate organization and function, and the severity of these changes increased over time. By postnatal day (P)7, mutant growth plates lacked typical zones of chondrocyte proliferation and hypertrophy, and were instead composed of chondrocytes with an unusual phenotype characterized by strong collagen II (Col2a1) gene expression but barely detectable expression of Indian hedgehog (Ihh), collagen X (Col10a1), Vegf (Vegfa), MMP-13 (Mmp13) and osterix (Sp7). Concurrently, unexpected developmental events occurred in perichondrial tissues, including excessive intramembranous ossification all along the perichondrial border and the formation of ectopic cartilage masses. Looking for possible culprits for these latter processes, we analyzed hedgehog signalling topography and intensity by monitoring the expression of the hedgehog effectors Patched 1 and Gli1, and of the hedgehog-binding cell-surface component syndecan 3. Compared with controls, hedgehog signaling was quite feeble within mutant growth plates as early as P0, but was actually higher and was widespread all along mutant perichondrial tissues. Lastly, we studied postnatal mice deficient in Ihh in cartilage; their cranial base defects only minimally resembled those in Kif3a-deficient mice. In summary, Kif3a and primary cilia make unique contributions to cranial base development and synchondrosis growth plate function. Their deficiency causes abnormal topography of hedgehog signaling, growth plate dysfunction, and un-physiologic responses and processes in perichondrial tissues, including ectopic cartilage formation and excessive intramembranous ossification.

Title: Suppression of Myc oncogenic activity by ribosomal protein haploinsufficiency Content: The Myc oncogene regulates the expression of several components of the protein synthetic machinery, including ribosomal proteins, initiation factors of translation, RNA polymerase III and ribosomal DNA. Whether and how increasing the cellular protein synthesis capacity affects the multistep process leading to cancer remains to be addressed. Here we use ribosomal protein heterozygote mice as a genetic tool to restore increased protein synthesis in Emu-Myc/+ transgenic mice to normal levels, and show that the oncogenic potential of Myc in this context is suppressed. Our findings demonstrate that the ability of Myc to increase protein synthesis directly augments cell size and is sufficient to accelerate cell cycle progression independently of known cell cycle targets transcriptionally regulated by Myc. In addition, when protein synthesis is restored to normal levels, Myc-overexpressing precancerous cells are more efficiently eliminated by programmed cell death. Our findings reveal a new mechanism that links increases in general protein synthesis rates downstream of an oncogenic signal to a specific molecular impairment in the modality of translation initiation used to regulate the expression of selective messenger RNAs. We show that an aberrant increase in cap-dependent translation downstream of Myc hyperactivation specifically impairs the translational switch to internal ribosomal entry site (IRES)-dependent translation that is required for accurate mitotic progression. Failure of this translational switch results in reduced mitotic-specific expression of the endogenous IRES-dependent form of Cdk11 (also known as Cdc2l and PITSLRE), which leads to cytokinesis defects and is associated with increased centrosome numbers and genome instability in Emu-Myc/+ mice. When accurate translational control is re-established in Emu-Myc/+ mice, genome instability is suppressed. Our findings demonstrate how perturbations in translational control provide a highly specific outcome for gene expression, genome stability and cancer initiation that have important implications for understanding the molecular mechanism of cancer formation at the post-genomic level.

Title: Human monocyte characteristics are altered in hypercholesterolaemia. Content: Peripheral blood monocytes are involved during atherogenesis in adhering to endothelium, migrating into the subendothelial space and taking-up lipoproteins to become macrophage/foam cells. We have assessed whether peripheral blood monocyte characteristics are altered in human hyperlipidaemia in age/sex/smoking status matched pairs of patients and controls. Monocytes from the hypercholesterolaemic patients, as opposed to the controls, were more sensitive to stimulation by the agonist, N-formyl-methionyl-leucyl-phenylalanine, with respect to chemokinesis (stimulation index 1.48 +/- 0.17 vs. 1.10 +/- 0.14), chemotaxis (4.05 +/- 0.55 vs. 2.72 +/- 0.24) and adhesion to porcine aortic endothelial monolayers (1.26 +/- 0.05 vs. 1.17 +/- 0.06). The patients' monocyte total surface expression of the adhesion glycoprotein CD11b/CD18 (37.5 +/- 7.1 vs. 36.0 +/- 7.1), but not CD11c/CD18 (31.6 +/- 7.2 vs. 31.4 +/- 6.8), was increased; however, the monocytes in hyperlipidaemia were larger (9.15 +/- 0.11 microns vs. 8.98 +/- 0.11 microns) such that the surface density of CD11b/CD18 was not altered (0.144 +/- 0.029 vs. 0.142 +/- 0.029). The data suggest that circulating monocytes are functionally different in hypercholesterolaemia. This may explain the increased involvement by monocytes in hypercholesterolaemia-related atherogenesis.

Title: Myelodysplasia and leukemia of Fanconi anemia are associated with a specific pattern of genomic abnormalities that includes cryptic RUNX1/AML1 lesions. Content: Fanconi anemia (FA) is a genetic condition associated with bone marrow (BM) failure, myelodysplasia (MDS), and acute myeloid leukemia (AML). We studied 57 FA patients with hypoplastic or aplastic anemia (n = 20), MDS (n = 18), AML (n = 11), or no BM abnormality (n = 8). BM samples were analyzed by karyotype, high-density DNA arrays with respect to paired fibroblasts, and by selected oncogene sequencing. A specific pattern of chromosomal abnormalities was found in MDS/AML, which included 1q+ (44.8%), 3q+ (41.4%), -7/7q (17.2%), and 11q- (13.8%). Moreover, cryptic RUNX1/AML1 lesions (translocations, deletions, or mutations) were observed for the first time in FA (20.7%). Rare mutations of NRAS, FLT3-ITD, MLL-PTD, ERG amplification, and ZFP36L2-PRDM16 translocation, but no TP53, TET2, CBL, NPM1, and CEBPα mutations were found. Frequent homozygosity regions were related not to somatic copy-neutral loss of heterozygosity but to consanguinity, suggesting that homologous recombination is not a common progression mechanism in FA. Importantly, the RUNX1 and other chromosomal/genomic lesions were found at the MDS/AML stages, except for 1q+, which was found at all stages. These data have implications for staging and therapeutic managing in FA patients, and also to analyze the mechanisms of clonal evolution and oncogenesis in a background of genomic instability and BM failure.

Title: Differential assembly of inwardly rectifying K+ channel subunits, Kir4.1 and Kir5.1, in brain astrocytes. Content: The inwardly rectifying K+ channel subunit Kir5.1 is expressed abundantly in the brain, but its precise distribution and function are still largely unknown. Because Kir5.1 is co-expressed with Kir4.1 in retinal glial Muller cells, we have compared the biochemical and immunological properties of Kir5.1 and Kir4.1 in the mouse brain. Immunoprecipitation experiments suggested that brain expressed at least two subsets of Kir channels, heteromeric Kir4.1/5.1 and homomeric Kir4.1. Immunolabeling using specific antibodies showed that channels comprising Kir4.1 and Kir5.1 subunits were assembled in a region-specific fashion. Heteromeric Kir4.1/5.1 was identified in the neocortex and in the glomeruli of the olfactory bulb. Homomeric Kir4.1 was confined to the hippocampus and the thalamus. Homomeric Kir5.1 was not identified. Kir4.1/5.1 and Kir4.1 expression appeared to occur only in astrocytes, specifically in the membrane domains facing the pia mater and blood vessels or in the processes surrounding synapses. Both Kir4.1/5.1 and Kir4.1 could be associated with PDZ domain-containing syntrophins, which might be involved in the subcellular targeting of these astrocyte Kir channels. Because heteromeric Kir4.1/5.1 and homomeric Kir4.1 have distinct ion channel properties (Tanemoto, M., Kittaka, N., Inanobe, A., and Kurachi, Y. (2000) J. Physiol. (Lond.) 525, 587-592 and Tucker, S. J., Imbrici, P., Salvatore, L., D'Adamo, M. C., and Pessia, M. (2000) J. Biol. Chem. 275, 16404-16407), it is plausible that these channels play differential physiological roles in the K+ -buffering action of brain astrocytes in a region-specific manner.

Title: Downloaded from Content: Physical activity induces a subclinical inflammatory response, mediated in part by leukocytes, and manifested by elevated concentrations of circulating proinflammatory cytokines, including interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α). However, the source of the cytokines that appear during exercise remains unknown. In this study, we examined exercise-induced changes in plasma cytokine concentrations and their corresponding mRNA expression in peripheral blood mononuclear cells. Ten healthy [peak oxygen uptake = 48.8 ± 6.5 (SD) ml · kg−1 · min−1] but untrained men [age = 25 ± 5 (SD) yr] undertook 3 h of exercise (cycling and inclined walking) at 60–65% peak oxygen uptake. Circulating leukocyte subset counts were elevated during and 2 h postexercise but returned to normal within 24 h. Plasma concentrations of IL-1β, IL-6, and TNF-α peaked at the end of exercise and remained elevated at 2 h (IL-6) and up to 24 h (IL-1β and TNF-α) postexercise. Cytokine gene expression in circulating mononucl...

Title: Enhanced pathological angiogenesis in mice lacking β3 integrin or β3 and β5 integrins Content: Inhibition of αvβ3 or αvβ5 integrin function has been reported to suppress neovascularization and tumor growth, suggesting that these integrins are critical modulators of angiogenesis. Here we report that mice lacking β3 integrins or both β3 and β5 integrins not only support tumorigenesis, but have enhanced tumor growth as well. Moreover, the tumors in these integrin-deficient mice display enhanced angiogenesis, strongly suggesting that neither β3 nor β5 integrins are essential for neovascularization. We also observed that angiogenic responses to hypoxia and vascular endothelial growth factor (VEGF) are augmented significantly in the absence of β3 integrins. We found no evidence that the expression or functions of other integrins were altered as a consequence of the β3 deficiency, but we did observe elevated levels of VEGF receptor-2 (also called Flk-1) in β3-null endothelial cells. These data indicate that αvβ3 and αvβ5 integrins are not essential for vascular development or pathological angiogenesis and highlight the need for further evaluation of the mechanisms of action of αv-integrin antagonists in anti-angiogenic therapeutics.

Title: Increased Adipocyte O2 Consumption Triggers HIF-1α, Causing Inflammation and Insulin Resistance in Obesity Content: Adipose tissue hypoxia and inflammation have been causally implicated in obesity-induced insulin resistance. Here, we report that, early in the course of high-fat diet (HFD) feeding and obesity, adipocyte respiration becomes uncoupled, leading to increased oxygen consumption and a state of relative adipocyte hypoxia. These events are sufficient to trigger HIF-1α induction, setting off the chronic adipose tissue inflammatory response characteristic of obesity. At the molecular level, these events involve saturated fatty acid stimulation of the adenine nucleotide translocase 2 (ANT2), an inner mitochondrial membrane protein, which leads to the uncoupled respiratory state. Genetic or pharmacologic inhibition of either ANT2 or HIF-1α can prevent or reverse these pathophysiologic events, restoring a state of insulin sensitivity and glucose tolerance. These results reveal the sequential series of events in obesity-induced inflammation and insulin resistance.

Title: Fibroblast Growth Factor 21 Prevents Atherosclerosis by Suppression of Hepatic Sterol Regulatory Element-Binding Protein-2 and Induction of Adiponectin in Mice Content: BACKGROUND Fibroblast growth factor 21 (FGF21) is a metabolic hormone with pleiotropic effects on glucose and lipid metabolism and insulin sensitivity. It acts as a key downstream target of both peroxisome proliferator-activated receptor α and γ, the agonists of which have been used for lipid lowering and insulin sensitization, respectively. However, the role of FGF21 in the cardiovascular system remains elusive. METHODS AND RESULTS The roles of FGF21 in atherosclerosis were investigated by evaluating the impact of FGF21 deficiency and replenishment with recombinant FGF21 in apolipoprotein E(-/-) mice. FGF21 deficiency causes a marked exacerbation of atherosclerotic plaque formation and premature death in apolipoprotein E(-/-) mice, which is accompanied by hypoadiponectinemia and severe hypercholesterolemia. Replenishment of FGF21 protects against atherosclerosis in apolipoprotein E(-/-)mice via 2 independent mechanisms, inducing the adipocyte production of adiponectin, which in turn acts on the blood vessels to inhibit neointima formation and macrophage inflammation, and suppressing the hepatic expression of the transcription factor sterol regulatory element-binding protein-2, thereby leading to reduced cholesterol synthesis and attenuation of hypercholesterolemia. Chronic treatment with adiponectin partially reverses atherosclerosis without obvious effects on hypercholesterolemia in FGF21-deficient apolipoprotein E(-/-) mice. By contrast, the cholesterol-lowering effects of FGF21 are abrogated by hepatic expression of sterol regulatory element-binding protein-2. CONCLUSIONS FGF21 protects against atherosclerosis via fine tuning the multiorgan crosstalk among liver, adipose tissue, and blood vessels.

Title: Distinct and essential roles of transcription factors IRF-3 and IRF-7 in response to viruses for IFN-alpha/beta gene induction. Content: Induction of the interferon (IFN)-alpha/beta gene transcription in virus-infected cells is an event central to innate immunity. Mice lacking the transcription factor IRF-3 are more vulnerable to virus infection. In embryonic fibroblasts, virus-induced IFN-alpha/beta gene expression levels are reduced and the spectrum of the IFN-alpha mRNA subspecies altered. Furthermore, cells additionally defective in IRF-7 expression totally fail to induce these genes in response to infections by any of the virus types tested. In these cells, a normal profile of IFN-alpha/beta mRNA induction can be achieved by coexpressing both IRF-3 and IRF-7. These results demonstrate the essential and distinct roles of thetwo factors, which together ensure the transcriptional efficiency and diversity of IFN-alpha/beta genes for the antiviral response.

Title: Interferon alpha and zidovudine therapy in adult T-cell leukaemia lymphoma: response and outcome in 15 patients. Content: Adult T-cell leukaemia lymphoma (ATLL) is an aggressive disease caused by the human T-lymphotropic virus 1 (HTLV-I) with a short survival. Responses to interferon alpha (IFN-alpha) and zidovudine (AZT) have been documented but not with long-term follow-up. We treated 15 ATLL patients with IFN and AZT. Eleven patients had acute ATLL, two had lymphoma and two smouldering ATLL, with progression. The main features were: organomegaly (14), skin lesions (10), high white blood cell (WBC) count (11) and hypercalcaemia (9). Eleven patients had previously received chemotherapy and one had received an autograft. At the time of the study, seven patients had progressive disease and eight were in partial or complete clinical remission. Responses (PR) lasting 2+ to 44+ months were seen in 67%; 26% did not respond (NR) and one patient was not evaluable. Hypercalcaemia predicted a poor outcome but differences were not significant. Eight of the 15 patients have died 3-41 months from diagnosis. Median survival for the 15 patients was 18 months. Survival of the NR ranged from 4 to 20 months; six PR patients are alive 8-82 months from diagnosis. The differences in survival between NR (median: 6 months) and PR (55% of patients alive at 4 years) were statistically significant (P = 0.002). In conclusion, IFN and AZT improves the outcome of ATLL patients and helps maintain responses.

Title: Alcohol Intake and Blood Pressure: A Systematic Review Implementing a Mendelian Randomization Approach Content: BACKGROUND Alcohol has been reported to be a common and modifiable risk factor for hypertension. However, observational studies are subject to confounding by other behavioural and sociodemographic factors, while clinical trials are difficult to implement and have limited follow-up time. Mendelian randomization can provide robust evidence on the nature of this association by use of a common polymorphism in aldehyde dehydrogenase 2 (ALDH2) as a surrogate for measuring alcohol consumption. ALDH2 encodes a major enzyme involved in alcohol metabolism. Individuals homozygous for the null variant (*2*2) experience adverse symptoms when drinking alcohol and consequently drink considerably less alcohol than wild-type homozygotes (*1*1) or heterozygotes. We hypothesise that this polymorphism may influence the risk of hypertension by affecting alcohol drinking behaviour. METHODS AND FINDINGS We carried out fixed effect meta-analyses of the ALDH2 genotype with blood pressure (five studies, n = 7,658) and hypertension (three studies, n = 4,219) using studies identified via systematic review. In males, we obtained an overall odds ratio of 2.42 (95% confidence interval [CI] 1.66-3.55, p = 4.8 x 10(-6)) for hypertension comparing *1*1 with *2*2 homozygotes and an odds ratio of 1.72 (95% CI 1.17-2.52, p = 0.006) comparing heterozygotes (surrogate for moderate drinkers) with *2*2 homozygotes. Systolic blood pressure was 7.44 mmHg (95% CI 5.39-9.49, p = 1.1 x 10(-12)) greater among *1*1 than among *2*2 homozygotes, and 4.24 mmHg (95% CI 2.18-6.31, p = 0.00005) greater among heterozygotes than among *2*2 homozygotes. CONCLUSIONS These findings support the hypothesis that alcohol intake has a marked effect on blood pressure and the risk of hypertension.

Title: Alzheimer’s Disease Risk Gene CD33 Inhibits Microglial Uptake of Amyloid Beta Content: The transmembrane protein CD33 is a sialic acid-binding immunoglobulin-like lectin that regulates innate immunity but has no known functions in the brain. We have previously shown that the CD33 gene is a risk factor for Alzheimer's disease (AD). Here, we observed increased expression of CD33 in microglial cells in AD brain. The minor allele of the CD33 SNP rs3865444, which confers protection against AD, was associated with reductions in both CD33 expression and insoluble amyloid beta 42 (Aβ42) levels in AD brain. Furthermore, the numbers of CD33-immunoreactive microglia were positively correlated with insoluble Aβ42 levels and plaque burden in AD brain. CD33 inhibited uptake and clearance of Aβ42 in microglial cell cultures. Finally, brain levels of insoluble Aβ42 as well as amyloid plaque burden were markedly reduced in APP(Swe)/PS1(ΔE9)/CD33(-/-) mice. Therefore, CD33 inactivation mitigates Aβ pathology and CD33 inhibition could represent a novel therapy for AD.

Title: Exosomal miR-99a-5p is elevated in sera of ovarian cancer patients and promotes cancer cell invasion by increasing fibronectin and vitronectin expression in neighboring peritoneal mesothelial cells Content: BACKGROUND microRNAs (miRNAs) stably exist in circulating blood and are encapsulated in extracellular vesicles such as exosomes. The aims of this study were to identify which exosomal miRNAs are highly produced from epithelial ovarian cancer (EOC) cells, to analyze whether serum miRNA can be used to discriminate patients with EOC from healthy volunteers, and to investigate the functional role of exosomal miRNAs in ovarian cancer progression. METHODS Exosomes were collected from the culture media of serous ovarian cancer cell lines, namely TYK-nu and HeyA8 cells. An exosomal miRNA microarray revealed that several miRNAs including miR-99a-5p were specifically elevated in EOC-derived exosomes. Expression levels of serum miR-99a-5p in 62 patients with EOC, 26 patients with benign ovarian tumors, and 20 healthy volunteers were determined by miRNA quantitative reverse transcription-polymerase chain reaction. To investigate the role of exosomal miR-99a-5p in peritoneal dissemination, neighboring human peritoneal mesothelial cells (HPMCs) were treated with EOC-derived exosomes and then expression levels of miR-99a-5p were examined. Furthermore, mimics of miR-99a-5p were transfected into HPMCs and the effect of miR-99a-5p on cancer invasion was analyzed using a 3D culture model. Proteomic analysis with the tandem mass tag method was performed on HPMCs transfected with miR-99a-5p and then potential target genes of miR-99a-5p were examined. RESULTS The serum miR-99a-5p levels were significantly increased in patients with EOC, compared with those in benign tumor patients and healthy volunteers (1.7-fold and 2.8-fold, respectively). A receiver operating characteristic curve analysis showed with a cut-off of 1.41 showed sensitivity and specificity of 0.85 and 0.75, respectively, for detecting EOC (area under the curve = 0.88). Serum miR-99a-5p expression levels were significantly decreased after EOC surgeries (1.8 to 1.3, p = 0.002), indicating that miR-99a-5p reflects tumor burden. Treatment with EOC-derived exosomes significantly increased miR-99a-5p expression in HPMCs. HPMCs transfected with miR-99a-5p promoted ovarian cancer invasion and exhibited increased expression levels of fibronectin and vitronectin. CONCLUSIONS Serum miR-99a-5p is significantly elevated in ovarian cancer patients. Exosomal miR-99a-5p from EOC cells promotes cell invasion by affecting HPMCs through fibronectin and vitronectin upregulation and may serve as a target for inhibiting ovarian cancer progression.

Title: Percentile curves for hemoglobin and red cell volume in infancy and childhood. Content: Percentile curves were calculated for hemoglobin and mean corpuscular volume in children between 0.5 and 16 years of age. The curves were derived from several populations of non-indigent white children who lived near sea level. Subjects were excluded from the reference population if they had laboratory evidence of iron deficiency, thalassemia minor, and/or hemoglobinopathy. The final reference populations included 9,946 children for the derivation of the hemoglobin curves and 2,314 for the MCV curves. The percentile curves should be particularly applicable to the diagnosis and screening of iron deficiency and thalassemia minor.

Title: High-resolution peripheral quantitative computed tomographic imaging of cortical and trabecular bone microarchitecture in patients with type 2 diabetes mellitus. Content: CONTEXT Cross-sectional epidemiological studies have found that patients with type 2 diabetes mellitus (T2DM) have a higher incidence of certain fragility fractures despite normal or elevated bone mineral density (BMD). OBJECTIVE In this study, high-resolution peripheral quantitative computed tomography was applied to characterize cortical and trabecular microarchitecture and biomechanics in the peripheral skeleton of female patients with T2DM. DESIGN AND SETTING A cross-sectional study was conducted in patients with T2DM recruited from a diabetic outpatient clinic. PARTICIPANTS Elderly female patients (age, 62.9 ± 7.7 yr) with a history of T2DM (n = 19) and age- and height-matched controls (n = 19) were recruited. OUTCOME MEASURES Subjects were imaged using high-resolution peripheral quantitative computed tomography at the distal radius and tibia. Quantitative measures of volumetric (BMD), cross-sectional geometry, trabecular and cortical microarchitecture were calculated. Additionally, compressive mechanical properties were determined by micro-finite element analysis. RESULTS Compared to the controls, the T2DM cohort had 10% higher trabecular volumetric BMD (P < 0.05) adjacent to the cortex and higher trabecular thickness in the tibia (13.8%; P < 0.05). Cortical porosity differences alone were consistent with impaired bone strength and were significant in the radius (>+50%; P < 0.05), whereas pore volume approached significance in the tibia (+118%; P = 0.1). CONCLUSION The results of this pilot investigation provide a potential explanation for the inability of standard BMD measures to explain the elevated fracture incidence in patients with T2DM. The findings suggest that T2DM may be associated with impaired resistance to bending loads due to inefficient redistribution of bone mass, characterized by loss of intracortical bone offset by an elevation in trabecular bone density.

Title: Urban malaria and anaemia in children: a cross-sectional survey in two cities of Ghana. Content: OBJECTIVE To describe the epidemiology of urban malaria, an emerging problem in sub-Saharan Africa. METHOD Cross-sectional surveys of communities in Accra and Kumasi, Ghana, determining risk factors for malaria infection and anaemia in children aged 6-60 months. RESULTS Malaria prevalence rates ranged from 2% to 33% between urban communities. 47.1% of children were anaemic (Hb<11.0 g/dl). Factors associated with malaria prevalence were low socio-economic status, age and anaemia. The attributable risks of anaemia and severe anaemia (Hb<8.0 g/dl) caused by malaria were 5% and 23% respectively. CONCLUSIONS Malaria in urban areas displayed a heterogeneity and complexity that differed from the rural environment, which has important implications for malaria control. Marked intra-city variation indicates the importance of targeting specific areas or districts. The most vulnerable group, the urban poor, should be prioritized when designing control measures. This would require careful assessment of the malaria risk pattern in any city to guide an integrated control program.

Title: Transcriptome analysis reveals human cytomegalovirus reprograms monocyte differentiation toward an M1 macrophage. Content: Monocytes are primary targets for human CMV (HCMV) infection and are proposed to be responsible for hematogenous dissemination of the virus. Monocytes acquire different functional traits during polarization to the classical proinflammatory M1 macrophage or the alternative antiinflammatory M2 macrophage. We hypothesized that HCMV induced a proinflammatory M1 macrophage following infection to promote viral dissemination because, biologically, a proinflammatory state provides the tools to drive infected monocytes from the blood into the tissue. To test this hypothesis of monocyte conversion from a normal quiescent phenotype to an inflammatory phenotype, we used Affymetrix Microarray to acquire a transcriptional profile of infected monocytes at a time point our data emphasized is a key temporal regulatory point following infection. We found that HCMV significantly up-regulated 583 (5.2%) of the total genes and down-regulated 621 (5.5%) of the total genes>or=1.5-fold at 4 h postinfection. Further ontology analysis revealed that genes implicated in classical M1 macrophage activation were stimulated by HCMV infection. We found that 65% of genes strictly associated with M1 polarization were up-regulated, while only 4% of genes solely associated with M2 polarization were up-regulated. Analysis of the monocyte chemokinome at the transcriptional level showed that 44% of M1 and 33% of M2 macrophage chemokines were up-regulated. Proteomic analysis using chemokine Ab arrays confirmed the secretion of these chemotactic proteins from HCMV-infected monocytes. Overall, the results identify that the HCMV-infected monocyte transcriptome displayed a unique M1/M2 polarization signature that was skewed toward the classical M1 activation phenotype.

Title: Disturbed sleep in bipolar disorder is related to an elevation of IL-6 in peripheral monocytes. Content: Bipolar disorder is a severe psychiatric disorder that is associated with persistent changes in the quality, duration and architecture of sleep. Currently there is no unifying hypothesis explaining the alterations in sleep observable in patients with bipolar disorder and management is often difficult though vital. Sleep is modified by various cytokines including IL-6. Elevated levels of IL-6 are associated with a poorer quality of sleep and changes in the architecture of sleep similar to those observed in bipolar disorder. Therapeutic administration of Interferon causes elevations of intrathecal IL-6 concentrations and appears to provoke a deteriorating quality of sleep. The blockade of IL-6 with tocilizumab in rheumatoid arthritis is associated with improvements in the quality of sleep. Bipolar disorder is associated with elevated levels of IL-6 and in particular elevated levels of mRNA coding for IL-6 in peripheral monocytes. We propose that the changes observed in the sleep of patients with bipolar disorder are related to the elevation of IL-6 and that this correlates with an elevated expression of mRNA coding for IL-6 expression in peripheral monocytes.

Title: Obesity due to melanocortin 4 receptor (MC4R) deficiency is associated with increased linear growth and final height, fasting hyperinsulinemia, and incompletely suppressed growth hormone secretion. Content: CONTEXT Melanocortin receptor 4 (MC4R) deficiency is characterized by increased linear growth greater than expected for the degree of obesity. OBJECTIVE The objective of the investigation was to study the somatotroph axis in obese MC4R-deficient patients and equally obese controls. PATIENTS AND METHODS We obtained anthropometric measurements and insulin concentrations in 153 MC4R-deficient subjects and 1392 controls matched for age and severity of obesity. We measured fasting IGF-I, IGF-II, IGF binding protein (IGFBP)-1, IGFBP-3, and acid-labile subunit levels in a subset of 33 MC4R-deficient patients and 36 control subjects. We examined pulsatile GH secretion in six adult MC4R-deficient subjects and six obese controls. RESULTS Height sd score was significantly greater in MC4R-deficient children under 5 yr of age compared with controls (mean ± SEM: 2.3 ± 0.06 vs. 1.8 ± 0.04, P < 0.001), an effect that persisted throughout childhood. Final height (cm) was greater in MC4R-deficient men (mean ± SEM 173 ± 2.5 vs. 168 ± 2.1, P < 0.001) and women (mean 165 ± 2.1 vs. 158 ± 1.9, P < 0.001). Fasting IGF-I, IGF-II, acid-labile subunit, and IGFBP-3 concentrations were similar in the two groups. GH levels were markedly suppressed in obese controls, but pulsatile GH secretion was retained in MC4R deficiency. The mean maximal GH secretion rate per burst (P < 0.05) and mass per burst (P < 0.05) were increased in MC4R deficiency, consistent with increased pulsatile and total GH secretion. Fasting insulin levels were markedly elevated in MC4R-deficient children. CONCLUSIONS In MC4R deficiency, increased linear growth in childhood leads to increased adult final height, greater than predicted by obesity alone. GH pulsatility is maintained in MC4R deficiency, a finding consistent with animal studies, suggesting a role for MC4R in controlling hypothalamic somatostatinergic tone. Fasting insulin levels are significantly higher in children carrying MC4R mutations. Both of these factors may contribute to the accelerated growth phenotype characteristic of MC4R deficiency.

Title: CCL2/monocyte chemoattractant protein-1 mediates enhanced transmigration of human immunodeficiency virus (HIV)-infected leukocytes across the blood-brain barrier: a potential mechanism of HIV-CNS invasion and NeuroAIDS. Content: Encephalitis and dementia associated with acquired immunodeficiency syndrome (AIDS) are characterized by leukocyte infiltration into the CNS, microglia activation, aberrant chemokine expression, blood-brain barrier (BBB) disruption, and eventual loss of neurons. Little is known about whether human immunodeficiency virus 1 (HIV-1) infection of leukocytes affects their ability to transmigrate in response to chemokines and to alter BBB integrity. We now demonstrate that HIV infection of human leukocytes results in their increased transmigration across our tissue culture model of the human BBB in response to the chemokine CCL2, as well as in disruption of the BBB, as evidenced by enhanced permeability, reduction of tight junction proteins, and expression of matrix metalloproteinases (MMP)-2 and MMP-9. HIV-infected cells added to our model did not transmigrate in the absence of CCL2, nor did this condition alter BBB integrity. The chemokines CXCL10/interferon-gamma-inducible protein of 10 kDa, CCL3/macrophage inflammatory protein-1alpha, or CCL5/RANTES (regulated on activation normal T-cell expressed and secreted) did not enhance HIV-infected leukocyte transmigration or BBB permeability. The increased capacity of HIV-infected leukocytes to transmigrate in response to CCL2 correlated with their increased expression of CCR2, the chemokine receptor for CCL2. These data suggest that CCL2, but not other chemokines, plays a key role in infiltration of HIV-infected leukocytes into the CNS and the subsequent pathology characteristic of NeuroAIDS.

Title: Redefining the Genetic Hierarchies Controlling Skeletal Myogenesis: Pax-3 and Myf-5 Act Upstream of MyoD Content: We analyzed Pax-3 (splotch), Myf-5 (targeted with nlacZ), and splotch/Myf-5 homozygous mutant mice to investigate the roles that these genes play in programming skeletal myogenesis. In splotch and Myf-5 homozygous embryos, myogenic progenitor cell perturbations and early muscle defects are distinct. Remarkably, splotch/Myf-5 double homozygotes have a dramatic phenotype not seen in the individual mutants: body muscles are absent. MyoD does not rescue this double mutant phenotype since activation of this gene proves to be dependent on either Pax-3 or Myf-5. Therefore, Pax-3 and Myf-5 define two distinct myogenic pathways, and MyoD acts genetically downstream of these genes for myogenesis in the body. This genetic hierarchy does not appear to operate for head muscle formation.

Title: A point mutation in KINDLIN3 ablates activation of three integrin subfamilies in humans Content: Monogenic deficiency diseases provide unique opportunities to define the contributions of individual molecules to human physiology and to identify pathologies arising from their dysfunction. Here we describe a deficiency disease in two human siblings that presented with severe bleeding, frequent infections and osteopetrosis at an early age. These symptoms are consistent with but more severe than those reported for people with leukocyte adhesion deficiency III (LAD-III). Mechanistically, these symptoms arose from an inability to activate the integrins expressed on hematopoietic cells, including platelets and leukocytes. Immortalized lymphocyte cell lines isolated from the two individuals showed integrin activation defects. Several proteins previously implicated in integrin activation, including Ras-associated protein-1 (RAP1) and calcium and diacylglycerol-regulated guanine nucleotide exchange factor-1 (CALDAG-GEF1), were present and functional in these cell lines. The genetic basis for this disease was traced to a point mutation in the coding region of the KINDLIN3 (official gene symbol FERMT3) gene. When wild-type KINDLIN-3 was expressed in the immortalized lymphocytes, their integrins became responsive to activation signals. These results identify a genetic disease that severely compromises the health of the affected individuals and establish an essential role of KINDLIN-3 in integrin activation in humans. Furthermore, allogeneic bone marrow transplantation was shown to alleviate the symptoms of the disease.

Title: Genetic Interaction between Mutations in c-Myb and the KIX Domains of CBP and p300 Affects Multiple Blood Cell Lineages and Influences Both Gene Activation and Repression Content: Adult blood cell production or definitive hematopoiesis requires the transcription factor c-Myb. The closely related KAT3 histone acetyltransferases CBP (CREBBP) and p300 (EP300) bind c-Myb through their KIX domains and mice homozygous for a p300 KIX domain mutation exhibit multiple blood defects. Perplexingly, mice homozygous for the same KIX domain mutation in CBP have normal blood. Here we test the hypothesis that the CBP KIX domain contributes subordinately to hematopoiesis via a genetic interaction with c-Myb. We assessed hematopoiesis in mice bearing compound mutations of c-Myb and/or the KIX domains of CBP and p300, and measured the effect of KIX domain mutations on c-Myb-dependent gene expression. We found that in the context of a p300 KIX mutation, the CBP KIX domain mutation affects platelets, B cells, T cells, and red cells. Gene interaction (epistasis) analysis provides mechanistic evidence that blood defects in KIX mutant mice are consistent with reduced c-Myb and KIX interaction. Lastly, we demonstrated that the CBP and p300 KIX domains contribute to both c-Myb-dependent gene activation and repression. Together these results suggest that the KIX domains of CBP, and especially p300, are principal mediators of c-Myb-dependent gene activation and repression that is required for definitive hematopoiesis.

Title: The Ratio of Monocytes to Lymphocytes in Peripheral Blood Correlates with Increased Susceptibility to Clinical Malaria in Kenyan Children Content: BACKGROUND Plasmodium falciparum malaria remains a major cause of illness and death in sub-Saharan Africa. Young children bear the brunt of the disease and though older children and adults suffer relatively fewer clinical attacks, they remain susceptible to asymptomatic P. falciparum infection. A better understanding of the host factors associated with immunity to clinical malaria and the ability to sustain asymptomatic P. falciparum infection will aid the development of improved strategies for disease prevention. METHODS AND FINDINGS Here we investigate whether full differential blood counts can predict susceptibility to clinical malaria among Kenyan children sampled at five annual cross-sectional surveys. We find that the ratio of monocytes to lymphocytes, measured in peripheral blood at the time of survey, directly correlates with risk of clinical malaria during follow-up. This association is evident among children with asymptomatic P. falciparum infection at the time the cell counts are measured (Hazard ratio (HR) = 2.7 (95% CI 1.42, 5.01, P = 0.002) but not in those without detectable parasitaemia (HR = 1.0 (95% CI 0.74, 1.42, P = 0.9). CONCLUSIONS We propose that the monocyte to lymphocyte ratio, which is easily derived from routine full differential blood counts, reflects an individual's capacity to mount an effective immune response to P. falciparum infection.

Title: Pdgfrβ+ Mural Preadipocytes Contribute to Adipocyte Hyperplasia Induced by High-Fat-Diet Feeding and Prolonged Cold Exposure in Adult Mice. Content: The expansion of white adipose tissue (WAT) in obesity involves de novo differentiation of new adipocytes; however, the cellular origin of these cells remains unclear. Here, we utilize Zfp423(GFP) reporter mice to characterize adipose mural (Pdgfrβ(+)) cells with varying levels of the preadipocyte commitment factor Zfp423. We find that adipose tissue contains distinct mural populations, with levels of Zfp423 distinguishing adipogenic from inflammatory-like mural cells. Using our "MuralChaser" lineage tracking system, we uncover adipose perivascular cells as developmental precursors of adipocytes formed in obesity, with adipogenesis and precursor abundance regulated in a depot-dependent manner. Interestingly, Pdgfrβ(+) cells do not significantly contribute to the initial cold-induced recruitment of beige adipocytes in WAT; it is only after prolonged cold exposure that these cells differentiate into beige adipocytes. These results provide genetic evidence for a mural cell origin of white adipocytes in obesity and suggest that beige adipogenesis may originate from multiple sources.