Differential expression of the histone H1 zero gene in U937 and HL-60 leukemia cell lines. The expression of the human H1 zero histone gene and of a main type H1 gene was analyzed in two human leukemia cell lines. The main type, replication dependent H1 gene expression reflected the state of proliferation of both cell lines. No H1 zero mRNA was detected in the promyelocytic HL-60 line, whereas the monocytic U937 cells showed low steady-state levels of 1H zero mRNA. Stimulation of HL-60 with several known inducers of differentiation failed to induce any accumulation of H1 zero mRNA. Treatment of U937 with phorbol ester or butyrate, on the other hand, led to an increase of the H1 zero mRNA concentration.


Pericellular substrates of human mast cell tryptase: 72,000 dalton gelatinase and fibronectin. Migrating cells degrade pericellular matrices and basement membranes. For these purposes cells produce a number of proteolytic enzymes. Mast cells produce two major proteinases, chymase and tryptase, whose physiological functions are poorly known. In the present study we have analyzed the ability of purified human mast cell tryptase to digest pericellular matrices of human fibroblasts. Isolated matrices of human fibroblasts and fibroblast conditioned medium were treated with tryptase, and alterations in the radiolabeled polypeptides were observed in autoradiograms of sodium dodecyl sulphate polyacrylamide gels. It was found that an M(r) 72,000 protein was digested to an M(r) 62,000 form by human mast cell tryptase while the plasminogen activator inhibitor, PAI-1, was not affected. Cleavage of the M(r) 72,000 protein could be partially inhibited by known inhibitors of tryptase but not by aprotinin, soybean trypsin inhibitor, or EDTA. Fibroblastic cells secreted the M(r) 72,000 protein into their medium and it bound to gelatin as shown by analysis of the medium by affinity chromatography over gelatin-Sepharose. The soluble form of the M(r) 72,000 protein was also susceptible to cleavage by tryptase. Analysis using gelatin containing polyacrylamide gels showed that both the intact M(r) 72,000 and the M(r) 62,000 degraded form of the protein possess gelatinolytic activity after activation by sodium dodecyl sulphate. Immunoblotting analysis of the matrices revealed the cleavage of an immunoreactive protein of M(r) 72,000 indicating that the protein is related to type IV collagenase. Further analysis of the pericellular matrices indicated that the protease sensitive extracellular matrix protein fibronectin was removed from the matrix by tryptase in a dose-dependent manner. Fibronectin was also susceptible to proteolytic degradation by tryptase. The data suggest a role for mast cell tryptase in the degradation of pericellular matrices.


Changes in annexin (lipocortin) content in human amnion and chorion at parturition. Arachidonic acid is mobilized from fetal membrane phospholipids at parturition leading to increased production of oxytocic prostaglandins which may initiate or maintain myometrial contractions. Phospholipid mobilization requires activation of phospholipase A2 or C, both of which require calcium for activity. The annexins (lipocortins) are a superfamily of proteins which bind to calcium and phospholipids and thereby may alter phospholipase activity through two mechanisms: modulation of intracellular free Ca2+ concentrations or regulation of the accessibility of phospholipids to hydrolyzing enzymes. Using Western immunoblotting with monospecific polyclonal antibodies, annexins I-VI were identified in human amnion and chorion/decidua at term in tissues obtained from patients in labor or not in labor. Each annexin was present in two distinct pools: a pool which only associated with the membrane in the presence of calcium (calcium-dependent pool) and a calcium-independent pool that remained membrane bound in the presence of calcium chelators. Annexin I was present as two species, resolving at 36 kDa and 68 kDa. The total concentration of annexin I in both amnion and chorion/decidua was significantly decreased with labor, while the total concentration of annexin V in chorion significantly increased with labor. The size of individual pools of annexins also changed with labor: the calcium-dependent pool of annexins I and II in both amnion and chorion significantly decreased; the calcium-dependent pool of annexin V increased in chorion; and calcium-independent pools of annexin I in amnion and annexins I, II, and V in chorion significantly decreased with labor. The decrease in total annexin I concentration with labor in amnion reflects a substantial decrease (80-90%) in the pool tightly bound to the membrane in a calcium-independent manner. This striking change distinguishes annexin I as a potential candidate inhibitor which is specifically downregulated at parturition, potentially leading to increased access of phospholipases to substrate phospholipids and increased prostaglandin production at labor.


A human histone H2B.1 variant gene, located on chromosome 1, utilizes alternative 3' end processing. A variant human H2B histone gene (GL105), previously shown to encode a 2300 nt replication independent mRNA, has been cloned. We demonstrate this gene expresses alternative mRNAs regulated differentially during the HeLa S3 cell cycle. The H2B-Gl105 gene encodes both a 500 nt cell cycle dependent mRNA and a 2300 nt constitutively expressed mRNA. The 3' end of the cell cycle regulated mRNA terminates immediately following the region of hyphenated dyad symmetry typical of most histone mRNAs, whereas the constitutively expressed mRNA has a 1798 nt non-translated trailer that contains the same region of hyphenated dyad symmetry but is polyadenylated. The cap site for the H2B-GL105 mRNAs is located 42 nt upstream of the protein coding region. The H2B-GL105 histone gene was localized to chromosome region 1q21-1q23 by chromosomal in situ hybridization and by analysis of rodent-human somatic cell hybrids using an H2B-GL105 specific probe. The H2B-GL105 gene is paired with a functional H2A histone gene and this H2A/H2B gene pair is separated by a bidirectionally transcribed intergenic promoter region containing consensus TATA and CCAAT boxes and an OTF-1 element. These results demonstrate that cell cycle regulated and constitutively expressed histone mRNAs can be encoded by the same gene, and indicate that alternative 3' end processing may be an important mechanism for regulation of histone mRNA. Such control further increases the versatility by which cells can modulate the synthesis of replication-dependent as well as variant histone proteins during the cell cycle and at the onset of differentiation.


Post-transcriptional regulation of collagenase and stromelysin gene expression by epidermal growth factor and dexamethasone in cultured human fibroblasts. Epidermal growth factor (EGF) is a ubiquitous fibroblast mitogen which also stimulates the synthesis of the extracellular matrix degrading metalloproteinases, collagenase, and stromelysin. Using primary cultures of human skin fibroblast, we show that these metalloproteinase mRNAs are coordinately up-regulated by EGF; and that dexamethasone, a potent inhibitor of collagenase and stromelysin synthesis, coordinately down-regulates these EGF-induced mRNAs. Nuclear run-on assays showed that EGF increased transcription of collagenase and stromelysin approximately 2-fold over the untreated control, while repression by dexamethasone was difficult to detect. However, steady state mRNA levels were induced approximately 10-fold by EGF and co-treatment with dexamethasone decreased them to below control levels, suggesting modulation of mRNA stability. Thus, we measured the half-life of these mRNAs using "pulse-chase" methodology. Typically, the half-life of EGF-induced collagenase and stromelysin mRNAs was approximately 30 h, and co-treatment with dexamethasone decreased the half-life of these mRNAs by 30-50%. Additionally, we found that the transcription inhibitor DRB stabilized EGF-induced metalloproteinase mRNAs, suggesting an mRNA degradation pathway which requires transcription. Thus our data demonstrate that collagenase and stromelysin are coordinately regulated by EGF and by dexamethasone, primarily at the level of metalloproteinase mRNA stability.


Glucocorticoid regulation of alkaline phosphatase, osteocalcin, and proto-oncogenes in normal human osteoblast-like cells. In humans, glucocorticoids are known to have marked effects on bone metabolism and function, including the significant regulation of osteoblast cells. To aid in the understanding of the mechanism of glucocorticoid action on normal human osteoblasts (hOB), confluent cells were analyzed for the presence of glucocorticoid receptors (GR) as well as for the effects of the glucocorticoid dexamethasone (Dex) on the expression of both the rapid responding nuclear proto-oncogenes and the late responding structural genes for bone matrix proteins. The interactions between Dex and 1,25 dihydroxy vitamin D3 (1,25 D3) on the gene expression in these cells were also examined. Using a functional receptor assay, a mean of 11,600 functional nuclear bound glucocorticoid receptors (range 6,000-22,000) was measured in fifteen separate cell strains. Northern blot analysis with a cDNA probe to the human GR was used to demonstrate the presence of a 7Kb transcript which is a candidate mRNA for GR in these cells. In agreement with previous studies, treatment of the hOB cells with Dex increased the steady state mRNA levels for alkaline phosphatase (AP) but displayed little or no effect on the mRNA levels for osteocalcin (OC) and glyceraldehyde phosphate dehydrogenase (GAPDH). Interestingly, the 1,25 D3 inductions of mRNA levels for OC were blocked by Dex but enhanced for AP. The above effects of Dex on AP and OC gene expression, including the interaction with 1,25 D3, were also shown to occur at the level of protein. The effect of Dex on the mRNA levels of the nuclear proto-oncogenes c-myc, c-fos, and c-jun was also investigated, since the oncoproteins (Fos/Jun) appear to play a role in the delayed glucocorticoid regulation of structural genes. Interestingly, Dex increased the steady state levels of c-myc, c-fos, and c-jun mRNAs in nonproliferating (confluent) hOB cells by 3.5-, 10-, and 2.0-fold, respectively, over control (untreated cells) values within one h of steroid treatment. The Dex-induced mRNA levels were transient and returned to basal values within 24 h of the steroid treatment. A reduced but qualitatively similar pattern of response was found in proliferating hOB cells. The pattern of response of these genes to glucocorticoids in hOB cells mimics the response in avian liver cells but not in reproductive cells. These results support the theory that hOB cells are target cells for glucocorticoids, and that as a primary event glucocorticoids rapidly regulate the expression of the nuclear oncoproteins Fos/Jun in these cells.


Glucocorticoids promote development of the osteoblast phenotype by selectively modulating expression of cell growth and differentiation associated genes. To understand the mechanisms by which glucocorticoids promote differentiation of fetal rat calvaria derived osteoblasts to produce bone-like mineralized nodules in vitro, a panel of osteoblast growth and differentiation related genes that characterize development of the osteoblast phenotype has been quantitated in glucocorticoid-treated cultures. We compared the mRNA levels of osteoblast expressed genes in control cultures of subcultivated cells where nodule formation is diminished, to cells continuously (35 days) exposed to 10(-7) M dexamethasone, a synthetic glucocorticoid, which promotes nodule formation to levels usually the extent observed in primary cultures. Tritiated thymidine labelling revealed a selective inhibition of internodule cell proliferation and promotion of proliferation and differentiation of cells forming bone nodules. Fibronectin, osteopontin, and c-fos expression were increased in the nodule forming period. Alkaline phosphatase and type I collagen expression were initially inhibited in proliferating cells, then increased after nodule formation to support further growth and mineralization of the nodule. Expression of osteocalcin was 1,000-fold elevated in glucocorticoid-differentiated cultures in relation to nodule formation. Collagenase gene expression was also greater than controls (fivefold) with the highest levels observed in mature cultures (day 35). At this time, a rise in collagen and TGF beta was also observed suggesting turnover of the matrix. Short term (48 h) effects of glucocorticoid on histone H4 (reflecting cell proliferation), alkaline phosphatase, osteopontin, and osteocalcin mRNA levels reveal both up or down regulation as a function of the developmental stage of the osteoblast phenotype. A comparison of transcriptional levels of these genes by nuclear run-on assays to mRNA levels indicates that glucocorticoids exert both transcriptional and post-transcriptional effects. Further, the presence of glucocorticoids enhances the vitamin D3 effect on gene expression. Those genes which are upregulated by 1,25(OH)2D3 are transcribed at an increased rate by dexamethasone, while those genes which are inhibited by vitamin D3 remain inhibited in the presence of dexamethasone and D3. We propose that the glucocorticoids promote changes in gene expression involved in cell-cell and cell-extracellular matrix signaling mechanisms that support the growth and differentiation of cells capable of osteoblast phenotype development and bone tissue-like organization, while inhibiting the growth of cells that cannot progress to the mature osteoblast phenotype in fetal rat calvarial cultures.


Localization of the cleavage sites on fibronectin following digestion by urokinase. Urokinase (u-PA) proteolytically cleaves both human plasma (pFn) and cellular (cFn) dimeric fibronectin (M(r) 440,000) into four major polypeptides of approximately M(r) 210,000, 200,000, 25,000, and 6,000. Amino acid sequence analysis of the polypeptide fragments indicated that the enzymatic cleavage of Fn occurs at two sites: 1) between an arginine/alanine peptide bond located C-terminal to residue 259; this cleavage liberates the N-terminal M(r) 25,000 fragment and the M(r) 210,000 and M(r) 200,000 polypeptides derived from the A and B chains of Fn, respectively; and 2) between an arginine/threonine peptide bond located C-terminal to residue 2,299, thereby yielding an M(r) 6,000 dimeric fragment containing the C-terminal interchain disulfide bonds. Predigestion of Fn with u-PA increased the molecule's vulnerability to further attack by the enzymes plasmin and cathepsin D. These data provide further biochemical evidence for the proteolytic cleavage of fibronectin by plasminogen activators and substantiate that u-PA digestion of Fn may be an initial event in the local degradation of the extracellular matrix by malignant cells, possessing elevated levels of these enzymes.


Specific changes in lymphocyte subpopulations: a potential mechanism for stress-induced immunomodulation. The mechanisms by which stressors alter immune function are not well understood. One hypothesis for stress-induced immunomodulation is that since immune responses require cooperation of different cell types, stress-induced shifts in cell populations might affect an organism's ability to mount an immune response. We sought to determine if inescapable shock (IS) could alter lymphocyte subpopulations and if so, whether this could be a mechanism for shock-induced immunomodulation. Our results suggest that IS produces changes in lymphocyte subpopulations and that these shifts could be responsible for modulation of in vivo antibody production. Exposure to IS resulted in an increase in the percent of CD4+ mesenteric lymphocytes and a decrease in the percent of CD8+ mesenteric lymphocytes when examined immediately after the cessation of IS. The stressor reduced antibody production to antigen processed at the altered mesenteric nodes, but did not alter antibody production to antigen processed at other sites. No measurable shifts were found in other compartments examined. The changes in CD4+ and CD8+ mesenteric lymphocytes resulted in an increased CD4+/CD8+ ratio that persisted for 1-24 h after stressor termination, becoming absent 48 h after IS termination. The stress-induced reduction in antibody production occurred only when antigen was given immediately prior to but not when antigen was given 48 h post stress. These findings suggest that the effects of a stressor could be specific to the manner in which the antigen enters the body, and that the stress-induced decrease in antibody production could be due to altered lymphocyte subpopulations as reflected by an increased CD4+/CD8+ ratio.


Chronic inflammatory effects of interleukin-1 on the blood-retina barrier. The chronic effects of human recombinant IL-1 (hrIL-1) on the specialized vasculature of the central nervous system (CNS) and on the CNS itself have been examined over a 35-day period in the rabbit retina. A single intraocular injection of physiological levels of hrIL-1 (300 units) induced a biphasic inflammatory reaction with well-defined acute and chronic phases in the challenged eye. Quantitative histopathological examination of the vascularized portion of the retina in the IL-1-challenged eye documented a persistent mononuclear (MN) cell response that peaked 7-14 days post-challenge. Included in the MN cell count were perivascular plasma cells. Elevated protein levels in the vitreous persisted throughout the time points studied and alterations in vascular permeability of the epiretinal vessels were demonstrated by tracer leakage at 2 weeks post-challenge. The results show that exposure of the CNS-vasculature to IL-1 induces long-lasting inflammatory changes typical of a chronic inflammatory reaction.


Inhibition of chronic relapsing experimental allergic encephalomyelitis in the Biozzi AB/H mouse. Chronic relapsing experimental allergic encephalomyelitis (CREAE) can be reproducibly induced in Biozzi AB/H mice following injection of spinal cord homogenate (SCH) emulsified in complete Freund's adjuvant (CFA). Active clinical disease is associated with mononuclear cell infiltration of the central nervous system (CNS), mainly the spinal cord. Whole brain homogenate (BH), however, failed to induce clinical or histological disease. In contrast, substituting sciatic nerve homogenate in the inoculum induced experimental allergic neuritis (EAN). Clinical disease was manifest earlier (13.1 +/- 0.3 days) than CREAE (16.2 +/- 1.4) and was accompanied by mononuclear infiltration of the peripheral nervous system (PNS). In comparison to CREAE induction, pretreating mice with SCH or BH in incomplete Freund's adjuvant (IFA) suppressed the development of SCH-induced disease. The BH was more tolerogenic than the SCH and this hyporesponsiveness was CNS antigen-specific as PNS tissue failed to inhibit the course of CREAE. Tolerance induced by pretreatment with SCH or BH in IFA was reversed by a single injection of 200 mg/kg cyclophosphamide, 2 days prior to CREAE induction. This suggests that IFA-induced hyporesponsiveness is actively regulated, possibly via the action of suppressor cells. In addition, treatment with neuroantigens in IFA appears to be mainly afferent acting as it serves to prevent initial disease induction. This treatment after immunization for CREAE, however, fails to prevent disease progression. Furthermore, treatment with CNS antigens emulsified in IFA during the post-acute remission stage appeared to synchronize and induce (32 +/- 1 days) the onset of clinical relapse, compared with untreated controls (41 +/- 5 days). This indicates that such IFA treatment has minimal value in controlling an ongoing immune disease of the CNS.


Ig-secreting cells pass the blood-brain barrier: studies on kappa and lambda light chain secreting cells in plasma cell dyscrasia. To study if immunoglobulin (Ig)-secreting cells actively pass the blood-brain barrier (BBB), 15 patients with monoclonal gammopathy underwent bonemarrow (BM) iliac crest aspiration biopsy, peripheral blood (PB) sampling and cerebrospinal fluid (CSF) analysis. With an enzyme-linked immunospot assay we investigated the number and ratio of mononuclear cells secreting Ig with kappa and lambda light chains in the three different compartments. A statistically significant (P < 0.05) correlation between the ratio of Ig kappa/lambda-secreting cells in CSF, PB and BM was found. The frequency of kappa and lambda (i.e. Ig in total) secreting mononuclear cells, of the same Ig class as the paraprotein, per 10(4) mononuclear cells was higher in BM (median 2.16%, range 0.43-9.28%) compared to CSF (median 0.44%, range 0.05-9.25%) and in CSF compared to PB (median 0.12%, range 0.02-0.96%). The proportion of all mononuclear cells with Ig kappa and lambda light chain (i.e. Ig) secretion was on average 5-fold greater in CSF compared to PB and 11-fold greater in BM compared to PB. The present study indicates that paraprotein-secreting cells preferentially pass from PB to CSF.


Afferent and efferent arms of the humoral immune response to CSF-administered albumins in a rat model with normal blood-brain barrier permeability. Cerebrospinal fluid (CSF) and serum antibody responses to albumin administered into CSF or muscle have been compared with respect to titer, isotype profile and complement-fixing activity in a rat model with normal brain barrier function. CSF/serum titer ratios and the ratio of IgG subclasses, IgG1/IgG2, were both elevated following CSF immunization. In contrast, there was no difference in complement-fixing activity between antibodies elicited by the two routes of immunization. It is suggested that intrathecal antibody synthesis accounts for the elevated CSF antibody titers in CSF-immunized rats, providing the first example of central nervous system antibody synthesis in an animal with normal brain barrier permeability.


Phenotypes and alloantigen-presenting activity of individual clones of microglia derived from the mouse brain. To clarify the origin and function of the microglia residing in the central nervous system, we cloned brain cells from newborn and adult mice in soft agar containing the macrophage-specific growth factor, colony-stimulating factor-1 and expanded the cells from individual colonies in liquid culture medium. The results of molecular, immunophenotypic and functional analyses showed that the clones consisted of microglia derived from the macrophage family of cells. For instance, the microglia contain mRNA transcripts for the receptor for colony-stimulating factor-1 and truncated CD4 transcripts similar to those found in mouse macrophages but not T helper cells. About a third of the microglial progenitors gave rise to progeny that constitutively induced the selective proliferation of naive allogeneic CD8+ T cells in a CD4+ T cell-independent manner, a response that was inhibited by monoclonal antibodies to major histocompatibility complex (MHC) class I molecules on the microglia. Since all microglia expressed similar levels of MHC class I molecules, the basis for the alloantigen presentation likely resides in the ability of some clones of microglia to synthesize co-stimulator molecules that are required for CD8+ T cell proliferation. Thus, at least some microglia in mouse brain arise from endogenous progenitors and appear capable of specialized functions.


Differential expression of heat shock proteins by human glial cells. Heat shock proteins (HSP) have been implicated in the interactions between the gamma delta T lymphocyte population and target tissues. gamma delta T cells are found in increased numbers in multiple sclerosis (MS) plaques compared to their proportion in peripheral blood, co-localizing with oligodendrocytes (OGC) expressing HSP. We have demonstrated that such gamma delta T cells can induce in vitro lysis of human adult-derived OGC. Using immunohistochemical and flow cytometry techniques, we examined the constitutive and/or inducible expression of HSP in or on adult human-derived glial cell cultures in vitro. HSP70 was expressed in OGC maintained at basal temperature, but the expression of the inducible HSP70 protein was upregulated by a prior 43 degrees C heat exposure. HSP70 could not be detected within astrocytes (GFAP+ cells), whether heat stress was applied or not. Constitutive expression of HSP60 could be discerned on the surface of all OGC under non-stressed culture conditions. Only some astrocytes demonstrated minor punctate surface HSP60 staining, whereas the remainder did not express HSP60 constitutively. These observations raise the possibility that OGC, by virtue of their differential expression of HSP compared to other glial cells, may be particularly prone to interaction with HSP-reactive gamma delta T cells. Such findings may further implicate gamma delta T cells in the pathogenesis of MS, a putative autoimmune disease in which immune-mediated injury is directed specifically against the oligodendrocyte-myelin unit within the central nervous system.


Interaction of the Pseudomonas aeruginosa secretory products pyocyanin and pyochelin generates hydroxyl radical and causes synergistic damage to endothelial cells. Implications for Pseudomonas-associated tissue injury. Pyocyanin, a secretory product of Pseudomonas aeruginosa, has the capacity to undergo redox cycling under aerobic conditions with resulting generation of superoxide and hydrogen peroxide. By using spin trapping techniques in conjunction with electron paramagnetic resonance spectrometry (EPR), superoxide was detected during the aerobic reduction of pyocyanin by NADH or porcine endothelial cells. No evidence of hydroxyl radical formation was detected. Chromium oxalate eliminated the EPR spectrum of the superoxide-derived spin adduct resulting from endothelial cell exposure to pyocyanin, suggesting superoxide formation close to the endothelial cell plasma membrane. We have previously reported that iron bound to the P. aeruginosa siderophore pyochelin (ferripyochelin) catalyzes the formation of hydroxyl free radical from superoxide and hydrogen peroxide via the Haber-Weiss reaction. In the present study, spin trap evidence of hydroxyl radical formation was detected when NADH and pyocyanin were allowed to react in the presence of ferripyochelin. Similarly, endothelial cell exposure to pyocyanin and ferripyochelin also resulted in hydroxyl radical production which appeared to occur in close proximity to the cell surface. As assessed by 51Cr release, endothelial cells which were treated with pyocyanin or ferripyochelin alone demonstrated minimal injury. However, endothelial cell exposure to the combination of pyochelin and pyocyanin resulted in 55% specific 51Cr release. Injury was not observed with the substitution of iron-free pyochelin and was diminished by the presence of catalase or dimethyl thiourea. These data suggest the possibility that the P. aeruginosa secretory products pyocyanin and pyochelin may act synergistically via the generation of hydroxyl radical to damage local tissues at sites of pseudomonas infection.


Insulin-like growth factor-I improves glucose and lipid metabolism in type 2 diabetes mellitus. Hyperglycemia, hyperinsulinemia, and insulin resistance cause vascular disease in type 2 diabetes mellitus. Dietary treatment alone often fails and oral drugs or insulin enhance hyperinsulinemia. In previous studies, an intravenous bolus of recombinant human insulin-like growth factor-I (rhIGF-I) caused normoglycemia in insulin-resistant diabetics whereas rhIGF-I infusions lowered insulin and lipid levels in healthy humans, suggesting that rhIGF-I is effective in insulin-resistant states. Thus, eight type 2 diabetics on a diet received on five treatment days subcutaneous rhIGF-I (2 x 120 micrograms/kg) after five control days. Fasting and postprandial glucose, insulin, C-peptide, proinsulin, glucagon, triglyceride, insulin-like growth factor-I and -II, and growth hormone levels were determined. RhIGF-I administration increased total IGF-I serum levels 5.3-fold above control. During the control period mean (+/- SD) fasting glucose, insulin, C-peptide, and total triglyceride levels were 11.0 +/- 4.3 mmol/liter, 108 +/- 50 pmol/liter, 793 +/- 250 pmol/liter, and 3.1 +/- 2.7 mmol/liter, respectively, and decreased during treatment to a nadir of 6.6 +/- 2.5 mmol/liter, 47 +/- 18 pmol/liter, 311 +/- 165 pmol/liter, and 1.6 +/- 0.8 mmol/liter (P < 0.01), respectively. Postprandial areas under the glucose, insulin, and C-peptide curve decreased to 77 +/- 13 (P < 0.02), 52 +/- 11, and 60 +/- 9% (P < 0.01) of control, respectively. RhIGF-I decreased the proinsulin/insulin ratio whereas glucagon levels remained unchanged. The magnitude of the effects of rhIGF-I correlated with the respective control levels. Since rhIGF-I appears to improve insulin sensitivity directly and/or indirectly, it may become an interesting tool in type 2 diabetes and other states associated with insulin resistance.


Age-dependent HLA genetic heterogeneity of type 1 insulin-dependent diabetes mellitus. The association of insulin-dependent diabetes mellitus (IDDM) with certain HLA alleles is well documented in pediatric patients. Whether a similar association is found in adult-on-set IDDM is not clear, although the disease occurs after the age of 20 in 50% of cases. HLA class II DRB1, DQA1, and DQB1 alleles were studied in 402 type I diabetics and 405 healthy controls (all Caucasian) using oligonucleotide typing after gene amplification. Alleles DRB1*03, DRB1*04, DQB1*0201, DQB1*0302, DQA1*0301, and DQA1*0501 were indeed enriched in diabetics and the highest relative risk was observed in patients carrying both the DRB1*03-DQB1*0201 and the DRB1*0402 or DRB1*0405-DQB1*0302 haplotypes. However none of these alleles, or specific residues, could alone account for the susceptibility to IDDM. Furthermore, there were major differences in HLA class II gene profiles according to the age of onset. Patients with onset after 15 yr (n = 290) showed a significantly higher percentage of non-DR3/non-DR4 genotypes than those with childhood onset (n = 112) and a lower percentage of DR3/4 genotypes. These non-DR3/non-DR4 patients, although presenting clinically as IDDM type 1 patients, showed a lower frequency of islet cell antibodies at diagnosis and a significantly milder initial insulin deficiency. These subjects probably represent a particular subset of IDDM patients in whom frequency increases with age. The data confirm the genetic heterogeneity of IDDM and call for caution in extrapolating to adult patients the genetic concepts derived from childhood IDDM.


Entactin stimulates neutrophil adhesion and chemotaxis through interactions between its Arg-Gly-Asp (RGD) domain and the leukocyte response integrin. Entactin is an integral component of basement membranes that plays a major role in basement membrane assembly through its ability to bind avidly to both laminin and type IV collagen. Because neutrophil (PMN) interactions with entactin have not been examined, we investigated the ability of natural and recombinant entactin to mediate PMN adhesion and chemotaxis. With both forms of entactin, we observed that entactin-coated surfaces promoted PMN adhesion and that entactin stimulated PMN chemotaxis. The increase in adhesion to entactin over control was two to threefold whereas the chemotactic response to 15 ng/ml (1 x 10(-10) M) entactin was equivalent to the chemotactic response elicited with 1 x 10(-8) M formyl-methionyl-leucyl-phenylalanine (fMLP). HL-60 cells, after differentiation with dimethylsulfoxide, also demonstrated adhesion and chemotaxis to entactin. A synthetic peptide of the Arg-Gly-Asp (RGD) domain in entactin, SIGFRGDGQTC (S-RGD), mediated PMN adhesion and chemotaxis, and preexposure of PMN to S-RGD blocked PMN adhesion and chemotaxis induced by entactin without diminishing the adhesive and chemotactic activities of fMLP. In contrast, preexposure to peptides SIGFRGEGQTCA or SIGFKGDGQTCA had no effect. The findings with synthetic peptides were confirmed with a recombinant entactin mutant in which aspartic acid at residue 674 was replaced with glutamic acid, thus converting the RGD sequence of entactin to RGE. RGE-entactin was neither adhesive nor chemotactic for neutrophils. Monoclonal antibodies to the leukocyte response integrin (LRI) and the integrin-associated protein blocked entactin-mediated adhesion and chemotaxis whereas monoclonal antibodies to beta 1 and beta 2 integrins had no effect and PMN from an individual with leukocyte-adhesion deficiency adhered normally to entactin-coated surfaces. These data demonstrate that entactin mediates biologically and pathologically important functions of PMN through its RGD domain and that LRI, which has been shown previously to mediate RGD-stimulated phagocytosis, is also capable of mediating RGD-stimulated PMN adhesion and chemotaxis.


O-linked carbohydrate of recombinant von Willebrand factor influences ristocetin-induced binding to platelet glycoprotein 1b. By transfecting the full-length cDNA for human von Willebrand factor (vWf) into a line of Chinese hamster ovary cells with a defect in carbohydrate metabolism, we have prepared recombinant vWf specifically lacking O-linked carbohydrates. We have compared this under-glycosylated protein to fully glycosylated recombinant vWf with respect to several structural and binding properties. vWf deficient in O-linked glycans was synthesized, assembled into multimers, and secreted in an apparently normal manner and was not prone to degradation in the extracellular milieu. It did not differ from fully glycosylated vWf in ability to bind to heparin or to collagen type I but did interact less well with glycoprotein 1b on formalin-fixed platelets. This decreased interaction was evidenced in both a lessened overall binding to platelets and in diminished capacity to promote platelet agglutination, in the presence of ristocetin. In contrast, no difference was seen in platelet binding in the presence of botrocetin. These data indicate a possible role for O-linked carbohydrates in the vWf-glycoprotein 1b interaction promoted by ristocetin and suggest that abnormalities in carbohydrate modification might contribute to the altered ristocetin-dependent reactivity between vWf and platelets described for some variant forms of von Willebrand disease.


Human pancreatitis-associated protein. Messenger RNA cloning and expression in pancreatic diseases. A human pancreatic cDNA library was screened with the cDNA encoding rat "pancreatitis-associated protein" (PAP). The selected clone encoded a secretory protein structurally related to rat PAP. The protein had the same size as rat PAP and showed 71% amino acid identity, the six half-cystines being in identical positions. Domains of the proteins showing homologies with calcium-dependent lectins were also conserved. In addition, expression in pancreas of the genes encoding the human protein and rat PAP showed similar characteristics: both were expressed at very low levels in control tissue and overexpressed during the acute phase of pancreatitis, contrary to most secretory products. The human protein was therefore named human pancreatitis-associated protein (PAP-H). Antibodies raised to a synthetic peptide of PAP-H detected a single band with an M(r) compatible with PAP-H in Western blot analysis of proteins extracted from a pancreas presenting with acute pancreatitis. In that tissue, the protein could be immunolocalized to the apical regions of acinar cells. An immunoassay was also constructed to quantify the protein in serum. Elevated PAP-H levels were observed in patients with acute pancreatitis and in some patients with chronic pancreatitis. Values were close to background in healthy subjects and in patients with other abdominal diseases. These results confirm that PAP-H synthesis increases during inflammation and suggest a possible use of the protein as biological marker of acute pancreatitis.


Anomalous rearrangements of the immunoglobulin heavy chain genes in human leukemias support the loop-out mechanism of class switch. Discrete rearrangements of immunoglobulin genes are characteristic of lymphoproliferative diseases of B cells and provide direct evidence of their clonal nature. In addition, because leukemic transformation and growth may amplify B cell clones regardless of selection by antigen, analysis of rearranged Ig genes in leukemic clones may give insight into molecular events taking place during the ontogenesis of normal B cells. We have tested DNA samples from patients with chronic B cell leukemias in search for abnormal rearrangements of the Ig heavy chain gene region. By Southern blot analysis we found an unexpected break in the JH-C mu region in 7 out of 118 cases. Two of these cases were investigated in detail by constructing from each a phage genomic library and isolating the phage clones containing the break points. In both cases the JH-C mu separation was confirmed. Further analysis demonstrated that in both cases the abnormality was an inversion of the Ig heavy chain gene between C mu and one of the C gamma segments. This inversion structure strongly suggests that, as has been demonstrated in murine cell lines and in splenocytes stimulated in vitro, class switching in human B lymphocytes occurs in vivo via a loop-out deletion mechanism. The frequency of abnormal events may be as high as 15%. Our data indicate that a proportion of cases of chronic B cell leukemia arise from a cell which has attempted an Ig class switch.


Adenosine triphosphate-dependent taurocholate transport in human liver plasma membranes. Transport systems involved in uptake and biliary secretion of bile salts have been extensively studied in rat liver; however, little is known about these systems in the human liver. In this study, we investigated taurocholate (TC) transport in canalicular and basolateral plasma membrane vesicles isolated from 15 human livers (donor age 6-64 yr). ATP stimulated the uptake of TC into both canalicular and basolateral human liver plasma membrane vesicles (cLPM and blLPM, respectively). Considerable interindividual variations in the transport velocity were observed in the different membrane preparations used: 9.0 +/- 1.3 (mean +/- SEM, n = 17; range 1.6-18.0) and 9.3 +/- 2.0 (range 1.1-29.8) pmol TC.mg protein-1.min-1 at 1.0 microM TC for cLPM and blLPM, respectively. TC transport was temperature sensitive and showed saturation kinetics with a high affinity for TC (Km 4.2 +/- 0.7 microM and 3.7 +/- 0.5 microM for cLPM and blLPM, respectively). Transport was dependent on the ATP concentration and saturable (Km 0.25 +/- 0.03 mM, n = 3). Neither nitrate, which reduces membrane potential, nor the protonophore FCCP strongly inhibited ATP-dependent TC transport, indicating that membrane potential and proton gradient are not involved in this process. TC transport was significantly inhibited by the classical anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonate (250 microM) and the glutathione conjugate S-(2,4-dinitrophenyl)glutathione (100 microM). In conclusion, high affinity ATP-dependent TC transport is present in human liver at both the canalicular and the basolateral sides of the hepatocyte.


Detection, characterization, and bioavailability of membrane-associated iron in the intact sickle red cell. It is hypothesized that membrane-associated iron in the sickle red cell is of pathophysiologic importance, but the actual existence of such iron in the intact cell has been questioned. Using a strategy whereby membrane iron can be detected through its bioavailability for catalyzing peroxidation, we used phospholipid exchange protein to load membranes of intact erythrocytes (RBC) with approximately 2% phosphatidylethanolamine hydroperoxide (PEOOH) and monitored the development of peroxidation by-products during subsequent incubation. Normal RBC loaded with PEOOH developed very little peroxidation, but vitamin E-replete sickle RBC showed an exuberant peroxidation response that was not seen in cells loaded with control nonoxidized phosphatidylethanolamine. Ancillary studies of sickle RBC revealed that the catalytic iron included both heme iron and free iron located at the bilayer inner leaflet. Significantly, these studies also revealed that peroxidation after PEOOH loading is promoted by cellular dehydration and inhibited by hydration, thus identifying a dynamic interaction between hemoglobin (sickle >> normal) and membrane lipid. High-reticulocyte control RBC and sickle trait RBC behaved exactly like normal RBC, while HbCC RBC and RBC having membranes gilded with hemoglobin iron because of prior exposure to acetylphenylhydrazine showed an abnormal peroxidation response like that of sickle RBC. Indeed, the peroxidation response of RBC loaded with PEOOH paralleled amounts of iron measured on inside-out membranes prepared from them (r = 0.783, P < 0.01). These studies corroborate existence of membrane-associated heme and free iron in the intact sickle cell, and they document its bioavailability for participation in injurious peroxidative processes. That association of cytosolic sickle hemoglobin with membrane lipid is modulated by cell hydration status provides a mechanism that may help explain increased development of oxidative membrane lesions in abnormally dehydrated sickle RBC regardless of the mechanism underlying their formation.


Insulin sensitivity of protein and glucose metabolism in human forearm skeletal muscle. Physiologic increases of insulin promote net amino acid uptake and protein anabolism in forearm skeletal muscle by restraining protein degradation. The sensitivity of this process to insulin is not known. Using the forearm perfusion method, we infused insulin locally in the brachial artery at rates of 0.00 (saline control), 0.01, 0.02, 0.035, or 0.05 mU/min per kg for 150 min to increase local forearm plasma insulin concentration by 0, approximately 20, approximately 35, approximately 60, and approximately 120 microU/ml (n = 35). L-[ring-2,6-3H]phenylalanine and L-[1-14C]leucine were infused systemically, and the net forearm balance, rate of appearance (Ra) and rate of disposal (R(d)) of phenylalanine and leucine, and forearm glucose balance were measured basally and in response to insulin infusion. Compared to saline, increasing rates of insulin infusion progressively increased net forearm glucose uptake from 0.9 mumol/min per 100 ml (saline) to 1.0, 1.8, 2.4, and 4.7 mumol/min per 100 ml forearm, respectively. Net forearm balance for phenylalanine and leucine was significantly less negative than basal (P < 0.01 for each) in response to the lowest dose insulin infusion, 0.01 mU/min per kg, and all higher rates of insulin infusion. Phenylalanine and leucine R(a) declined by approximately 38 and 40% with the lowest dose insulin infusion. Higher doses of insulin produced no greater effect (decline in R(a) varied between 26 and 42% for phenylalanine and 30-50% for leucine). In contrast, R(d) for phenylalanine and leucine did not change with insulin. We conclude that even modest increases of plasma insulin can markedly suppress proteolysis, measured by phenylalanine R(a), in human forearm skeletal muscle. Further increments of insulin within the physiologic range augment glucose uptake but have little additional effect on phenylalanine R(a) or balance. These results suggest that proteolysis in human skeletal muscle is more sensitive than glucose uptake to physiologic increments in insulin.


The HLA-DRB1 locus as a genetic component in giant cell arteritis. Mapping of a disease-linked sequence motif to the antigen binding site of the HLA-DR molecule. Giant cell arteritis (GCA) is a granulomatous vasculitis affecting persons over 50 years of age. The inflammatory infiltrate, which is targeted at the aorta and its proximal branches, includes activated CD4+ helper T cells, histiocytes, and giant cells. To investigate whether the genetic polymorphism of the HLA-DRB1 genes contributes to the local accumulation of activated T cells, we have analyzed both HLA-DRB1 alleles in a cohort of 42 patients with biopsy-proven GCA. The majority of patients (60%) expressed the B1*0401 or B1*0404/8 variant of the HLA-DR4 haplotype, both of which also represent the major genetic factors underlying the disease association in RA. GCA patients negative for the disease-linked HLA-DR4 alleles were characterized by a nonrandom distribution of HLA-DRB1 alleles. Sequence comparison among the allelic products identified in the GCA cohort demonstrated heterogeneity for the sequence polymorphism of the third hypervariable region (HVR), but homology for the polymorphic residues within the HVR2 of the HLA-DRB1 gene. The GCA patients shared a sequence motif spanning amino acid positions 28-31 of the HLA-DR beta 1 chain. In the structural model for HLA-DR molecules, this sequence motif can be mapped to the antigen-binding site of the HLA complex, suggesting a crucial role of antigen selection and presentation in GCA. In contrast, the sequence polymorphism linked to RA has been mapped to the HVR3 of the HLA-DRB1 gene and translates into a distinct domain of the HLA-DR molecule, the alpha-helical loop surrounding the antigen-binding groove. A consecutive case series study demonstrated that GCA and RA rarely co-occurred, supporting the interpretation that distinct functional domains of the HLA-DR molecule are implicated in the pathomechanisms of these two autoimmune diseases.


Gas exchange by intratracheal insufflation in a ventilatory failure dog model. Respiratory insufficiency patients who need only partial ventilatory support are, nevertheless, intubated and connected to a respirator. In search of a partial respiratory assistance method we evaluated the gas exchange, mechanisms, and hemodynamic effects of intratracheal insufflation (ITI) via a narrow (0.2-cm) catheter. The effects of flow rate (0.05-0.2 liter/min per kg), catheter tip position (carina, bronchus, and trachea), and superimposed chest vibration at 22 Hz were studied in seven anesthetized and partially paralyzed dogs. ITI in the carina induced CO2 removal (VCO2) of 48 +/- 16 ml/min in the periods between breaths, which was 39% of the control VCO2. CO2 removal rates between breaths with ITI in a bronchus and in the trachea were 63 and 28% of control, respectively (P < 0.05). ITI at 0.15-0.2 liter/min per kg augmented total VCO2 by > 50% over control (P < 0.05) and decreased PaCO2 by 10% (P < 0.05) despite a 28% fall in VE and 32% lower work of breathing (P < 0.05). Adding vibration to ITI at 0.15 liter/min per kg induced VCO2 of 162 +/- 34 ml/min, which was significantly greater than control, while PaCO2 fell from 69 +/- 24 to 47 +/- 6 mmHg (P < 0.05), despite complete cessation of spontaneous breathing. ITI with or without vibration did not cause any hemodynamic changes, except for a fall in the shunt fraction from 14.6 +/- 9.9% to 5.8 +/- 2.8% with vibration. Thus, ITI at low flow rates can support respiration with no hemodynamic side effects. Adding chest vibration further enhances gas exchange and can provide total ventilation.


Pleiotropic effects of antithrombin strand 1C substitution mutations. Six different substitution mutations were identified in four different amino acid residues of antithrombin strand 1C and the polypeptide leading into strand 4B (F402S, F402C, F402L, A404T, N405K, and P407T), and are responsible for functional antithrombin deficiency in seven independently ascertained kindreds (Rosny, Torino, Maisons-Laffitte, Paris 3, La Rochelle, Budapest 5, and Oslo) affected by venous thromboembolic disease. In all seven families, variant antithrombins with heparin-binding abnormalities were detected by crossed immunoelectrophoresis, and in six of the kindreds there was a reduced antigen concentration of plasma antithrombin. Two of the variant antithrombins, Rosny and Torino, were purified by heparin-Sepharose and immunoaffinity chromatography, and shown to have greatly reduced heparin cofactor and progressive inhibitor activities in vitro. The defective interactions of these mutants with thrombin may result from proximity of s1C to the reactive site, while reduced circulating levels may be related to s1C proximity to highly conserved internal beta strands, which contain elements proposed to influence serpin turnover and intracellular degradation. In contrast, s1C is spatially distant to the positively charged surface which forms the heparin binding site of antithrombin; altered heparin binding properties of s1C variants may therefore reflect conformational linkage between the reactive site and heparin binding regions of the molecule. This work demonstrates that point mutations in and immediately adjacent to strand 1C have multiple, or pleiotropic, effects on this serpin, leading ultimately to failure of its regulatory function.


Estrogen receptors and biologic response in rat parathyroid tissue and C cells. The expression of the PTH and calcitonin genes is dramatically decreased by 1,25(OH)2D3 in vivo, and the PTH gene expression is increased by hypocalcemia. We have now studied the effect of estrogens on the expression of these genes in vivo. 17 beta-Estradiol, given to ovariectomized rats, led to a fourfold increase in PTH mRNA and calcitonin mRNA levels. These effects occurred 24 h after single injections of 37-145 nmol estradiol, or after constant infusions of 12 pmol/d for 1 or 2 wk, where there was no effect on serum calcium levels. The estrogen receptor mRNA was demonstrated in the thyroparathyroid tissue by polymerase chain reaction. The estrogen binding was localized to the parathyroid and C cells by immunohistochemistry. Uterus weight was increased by repeated larger doses (73 nmol/d x 7) of estradiol, but not by the small doses (12 pmol/d for 1 or 2 wk) which were effective on the PTH and calcitonin genes, suggesting a sensitive endocrine effect. These results confirm that the parathyroid and C cells are target organs for estrogen, leading to an increased expression of PTH and calcitonin, which by their combined anabolic effect on bone would help prevent osteoporosis.


Impaired secretion of the elongated mutant of protein C (protein C-Nagoya). Molecular and cellular basis for hereditary protein C deficiency. Genetic analysis of a heterozygous protein C-deficient patient revealed a novel deletion of a single guanine residue (8857G) among four consecutive guanine nucleotides [380Trp(TGG)-381Gly(GGT)] in exon IX, which encodes the carboxyl-terminal region of protein C. This deletion results in a frameshift mutation and substitution of the last 39 amino acids (381Gly-419Pro) with 81 abnormal amino acid residues, and we have designated this elongated variant as Protein C-Nagoya. A mutagenic primer was designed which replaced the third guanine residue upstream from the deletion with cytosine, thereby creating a new AvaI site in an otherwise normal allele. Analysis of the polymerase chain reaction products derived from this mutagenic primer showed that the abnormal allele has been inherited in this family. To elucidate how this molecular abnormality leads to protein C deficiency, an expression plasmid containing this mutation was transfected into COS 7, BHK, and psi-2 cells, and the secretory process of the expressed Protein C-Nagoya was analyzed. ELISA and immunoprecipitation analysis with [35S]methionine labeling indicated that the mutant protein C, which was larger in size than normal, was mostly retained within the cells, and only a small portion of it was secreted into the medium. These results suggest that most of Protein C-Nagoya undergoes degradation within the producing cells, and this frameshift mutation apparently leads to protein C deficiency by impairment of secretion of the elongated protein C into plasma.


Chronic metabolic acidosis increases the serum concentration of 1,25-dihydroxyvitamin D in humans by stimulating its production rate. Critical role of acidosis-induced renal hypophosphatemia. Chronic metabolic acidosis results in metabolic bone disease, calcium nephrolithiasis, and growth retardation. The pathogenesis of each of these sequelae is poorly understood in humans. We therefore investigated the effects of chronic extrarenal metabolic acidosis on the regulation of 1,25-(OH)2D, parathyroid hormone, calcium, and phosphate metabolism in normal humans. Chronic extrarenal metabolic acidosis was induced by administering two different doses of NH4Cl [2.1 (low dose) and 4.2 (high dose) mmol/kg body wt per d, respectively] to four male volunteers each during metabolic balance conditions. Plasma [HCO3-] decreased by 4.5 +/- 0.4 mmol/liter in the low dose and by 9.1 +/- 0.3 mmol/liter (P < 0.001) in the high dose group. Metabolic acidosis induced renal hypophosphatemia, which strongly correlated with the severity of acidosis (Plasma [PO4] on plasma [HCO3-]; r = 0.721, P < 0.001). Both metabolic clearance and production rates of 1,25-(OH)2D increased in both groups. In the high dose group, the percentage increase in production rate was much greater than the percentage increase in metabolic clearance rate, resulting in a significantly increased serum 1,25-(OH)2D concentration. A strong inverse correlation was observed for serum 1,25-(OH)2D concentration on both plasma [PO4] (r = -0.711, P < 0.001) and plasma [HCO3-] (r = -0.725, P < 0.001). Plasma ionized calcium concentration did not change in either group whereas intact serum parathyroid hormone concentration decreased significantly in the high dose group. In conclusion, metabolic acidosis results in graded increases in serum 1,25-(OH)2D concentration by stimulating its production rate in humans. The increased production rate is explained by acidosis-induced hypophosphatemia/cellular phosphate depletion resulting at least in part from decreased renal tubular phosphate reabsorption. The decreased serum intact parathyroid hormone levels in more severe acidosis may be the consequence of hypophosphatemia and/or increased serum 1,25-(OH)2D concentrations.


Estrogen status and heredity are major determinants of premenopausal bone mass. To analyze their relative effects on premenopausal bone mass, we have studied the impact of lifelong estrogen exposure, assessed by an estrogen score (ES; computed on age at menarche, average length of menstrual cycles since menarche, and use of birth control pills), heredity, and some environmental factors on vertebral bone density (VBD), of 63 premenopausal women (age, 19-40 yr). Compared with women with normal bone density (Z score > -1), subjects with low VBD (Z score < -1) had significantly lower ES (15.1 +/- 3.9 vs. 18.7 +/- 2.4, P = 0.001), higher age at menarche (13.8 +/- 1.7 vs. 12.6 +/- 1.4 yr, P = 0.005), and lower serum estradiol (46.9 +/- 37 vs. 86.6 +/- 57 pg/ml, P = 0.023) and estrone levels (107.4 +/- 60 vs. 178.8 +/- 9.0 pg/ml, P = 0.05). Likewise, women in the lowest quartile for VBD had significantly lower ES (15.3 +/- 4.5 vs. 18.1 +/- 2.7, P = 0.006) and higher age at menarche (13.9 +/- 1.9 vs. 12.8 +/- .4, P = 0.02) than those in the upper three quartiles. A higher proportion of subjects with irregular menses (52 vs. 23%, P = 0.03) and a positive family history of osteoporosis (86 vs. 61%, P = 0.04) was found in the low VBD group compared with subjects with normal VBD. VBD correlated positively with ES (r = 0.44, P = < 0.001) and negatively with age at menarche (r = -0.30, P = 0.03) by simple linear regression, whereas no correlation was found between VBD and age, body mass index, parity, lactation, physical activity, sunlight exposure, and dietary calcium and vitamin D intakes. The correlation between VBD and ES improved after correcting for the effect of all the other variables by partial correlation analysis (Pearson partial r = 0.57, P = < 0.01), which also disclosed a significant contribution of dietary calcium to VBD. However, ES was the only significant independent determinant of VBD, by stepwise multiple regression analysis (R2 = 0.24). Therefore, premenopausal estrogen exposure, and possibly genetic predisposition, rather than environmental factors, are the major determinants for the development of peak bone mass before menopause.


Germline variable region gene segment derivation of human monoclonal anti-Rh(D) antibodies. Evidence for affinity maturation by somatic hypermutation and repertoire shift. To date, there has been no systematic study of the process of affinity maturation of human antibodies. We therefore sequenced the variable region genes (V genes) of 14 human monoclonal antibodies specific for the erythrocyte Rh(D) alloantigen and determined the germline gene segments of origin and extent of somatic hypermutation. These data were correlated with determinations of antibody affinity. The four IgM antibodies (low affinity) appear to be derived from two germline heavy chain variable region gene segments and one or two germline light chain variable region gene segments and were not extensively mutated. The 10 IgG antibodies (higher affinity) appear to be derived from somatic hypermutation of these V gene segments and by use of new V gene segments or V gene segment combinations (repertoire shift). Affinity generally increased with increasing somatic hypermutation; on average, there were 8.9 point mutations in the V gene segments of the four IgM antibodies (Ka = 1-4 x 10(7)/M-1) compared with 19 point mutations in the V gene segments of the 10 IgG antibodies. The four highest affinity antibodies (Ka = 0.9-3 x 10(9)/M-1) averaged 25.5 point mutations. The use of repertoire shift and somatic hypermutation in affinity maturation of human alloantibodies is similar to data obtained in inbred mice immunized with haptens.


Prepubertal increases in gonadotropin-releasing hormone mRNA, gonadotropin-releasing hormone precursor, and subsequent maturation of precursor processing in male rats. Changes in gonadotropins and gonadal steroids during sexual maturation in rats and humans are well documented but little is known about hypothalamic gonadotropin-releasing hormone (GnRH) gene expression in relation to these events. This study measured hypothalamic proGnRH mRNA, GnRH precursor, and fully processed GnRH from postnatal day 8 until day 62 in male rats. GnRH precursor increased on day 22, reached a peak on day 24, declined on day 25 and returned to infantile levels by day 28. A secondary rise in precursor occurred at about day 40 when testosterone levels increased. GnRH mRNA increased on day 22 and remained elevated over the study period to day 26. GnRH increased on day 24 and remained at this level until a secondary rise occurred coincident with the testosterone rise at about day 40. The ratio of GnRH precursor to GnRH was high until day 24 and was low from day 26 onwards, reflecting a maturation of the processing enzyme system between these 2 d. Thus, an abrupt increase in GnRH gene transcription (mRNA) occurs early in juvenile male rats (day 22), well before the onset of puberty. An increase in GnRH precursor accompanies these early changes and this is followed by the maturation of processing as evidenced by the rapid decline of precursor and increase in GnRH from day 24 onward.


Formation of novel non-cyclooxygenase-derived prostanoids (F2-isoprostanes) in carbon tetrachloride hepatotoxicity. An animal model of lipid peroxidation. These studies examine the in vivo formation of a unique series of PGF2-like compounds (F2-isoprostanes) derived from free radical-catalyzed nonenzymatic peroxidation of arachidonic acid. We have previously shown that levels of these compounds increase up to 50-fold in rats administered CCl4. To understand further the formation of these compounds in vivo, we carried out a series of experiments assessing factors influencing their generation. After CCl4 (2 ml/kg) was administered to rats, plasma F2-isoprostanes increased 55-fold by 4 h. Levels declined thereafter, but at 24 h, they were still elevated 21-fold, indicating continued lipid peroxidation. Pretreatment of rats with isonicotinic acid hydrazide and phenobarbital to induce cytochrome P-450 enhanced the production of F2-isoprostanes after CCl4 administration eightfold and fivefold, respectively, whereas inhibition of the cytochrome P-450 system with SKF-525A and 4-methylpyrazole decreased formation of F2-isoprostanes after CCl4 by 55 and 82%, respectively. Further, the glutathione-depleting agents buthionine sulfoximine and phorone augmented the F2-isoprostane response to CCl4 by 22- and 11-fold, respectively. F2-isoprostanes are formed in situ esterified to lipids and, in addition to increases in levels of free F2-isoprostanes in the circulation, levels of F2-isoprostanes esterified to lipids in various organs and plasma also increase sharply during CCl4 poisoning. The measurement of F2-isoprostanes may facilitate investigation of the role of lipid peroxidation in human diseases.


CD10/neutral endopeptidase 24.11 in developing human fetal lung. Patterns of expression and modulation of peptide-mediated proliferation. The cell membrane-associated enzyme CD10/neutral endopeptidase 24.11 (CD10/NEP) functions in multiple organ systems to downregulate responses to peptide hormones. Recently, CD10/NEP was found to hydrolyze bombesin-like peptides (BLP), which are mitogens for normal bronchial epithelial cells and small cell lung carcinomas. Growth of BLP-responsive small cell lung carcinomas was potentiated by CD10/NEP inhibition, implicating CD10/NEP in regulation of BLP-mediated tumor growth. BLP are also likely to participate in normal lung development because high BLP levels are found in fetal lung, and bombesin induces proliferation and maturation of human fetal lung in organ cultures and murine fetal lung in utero. To explore potential roles for CD10/NEP in regulating peptide-mediated human fetal lung development, we have characterized temporal and cellular patterns of CD10/NEP expression and effects of CD10/NEP inhibition in organ cultures. Peak CD10/NEP transcript levels are identified at 11-13 wk gestation by Northern blots and localized to epithelial cells and mesenchyme of developing airways by in situ hybridization. CD10/NEP immunostaining is most intense in undifferentiated airway epithelium. In human fetal lung organ cultures, inhibition of CD10/NEP with either phosphoramidon or SCH32615 increases thymidine incorporation by 166-182% (P < 0.025). The specific BLP receptor antagonist, [Leu13-psi(CH2NH)Leu14]bombesin abolishes these effects on fetal lung growth, suggesting that CD10/NEP modulates BLP-mediated proliferation. CD10/NEP expression in the growing front of airway epithelium and the effects of CD10/NEP inhibitors in lung explants implicate the enzyme in the regulation of peptide-mediated fetal lung growth.


Inhibition and stimulation of nitric oxide synthesis in the human forearm arterial bed of patients with insulin-dependent diabetes. Patients with insulin-dependent diabetes mellitus have an increased mortality and morbidity due to vascular complications. Nitric oxide from the vascular endothelium contributes to the control of normal vascular tone, and endothelial dysfunction has been implicated in the pathogenesis of diabetic vascular disease. In this study we have examined basal and stimulated nitric oxide-mediated vasodilatation in insulin-dependent diabetics and age- and sex-matched healthy controls. Drugs were infused locally into the brachial artery and forearm blood flow measured using venous occlusion plethysmography. Noradrenaline and NG-monomethyl-L-arginine produced similar reductions in resting forearm blood flow in healthy controls. However, in the diabetics, NG-monomethyl-L-arginine was significantly less effective than noradrenaline. Comparing between groups, the response to NG-monomethyl-L-arginine was also significantly less in the diabetics compared with the healthy controls. The response to sodium nitroprusside was significantly less in the diabetics compared with the healthy controls, whereas the responses to both acetylcholine and verapamil were the same in the two groups. The results provide evidence for an abnormality of basal nitric oxide-mediated dilatation in the forearm arterial bed of patients with insulin-dependent diabetes mellitus, and suggest that the vascular smooth muscle is less sensitive to nitric oxide.


Human Toxoplasma gondii-specific secretory immunoglobulin A reduces T. gondii infection of enterocytes in vitro. Whey from 17 women (four acutely infected with Toxoplasma gondii, eight chronically infected, and five uninfected) was studied. T. gondii-specific secretory IgA antibodies were demonstrated by ELISA in whey from acutely infected and one of eight chronically infected women. Such antibodies to tachyzoite proteins of < or = 14, 22, 26-28, 30, 46, 60, 70-80, and > 100 kD (eliminated by protease but not periodate or neuraminidase treatment) were demonstrated in whey from acutely infected subjects when Western blots were probed with their whey and antibodies to human secretory IgA or IgA or secretory piece. Secretory IgA from four of eight chronically infected women recognized the 46- and 69-kD epitopes. Other whey samples were negative. Incubation of T. gondii tachyzoites with whey or purified secretory IgA from acutely infected (but not seronegative) women caused 50-75% reduction in infection of enterocytes in vitro. Whey reactive with the 46-kD epitope from three of six chronically infected women caused less (> or = 40%) inhibition. Whey and purified secretory IgA from two of three acutely infected women agglutinated tachyzoites. Whey did not result in complement-dependent lysis of T. gondii. These results indicate that it may be possible to produce human secretory IgA to T. gondii capable of reducing initial infection of enterocytes, as such IgA is present during natural infection. They also demonstrate candidate epitopes for such protection.


60- and 72-month follow-up of children prenatally exposed to marijuana, cigarettes, and alcohol: cognitive and language assessment. Cognitive and receptive language development were examined in 135 60-month-old and 137 72-month-old children for whom prenatal exposure to marijuana, cigarettes, and alcohol had been ascertained. Discriminant Function analysis revealed an association between prenatal cigarette exposure and lower cognitive and receptive language scores at 60 and 72 months. This paralleled and extended observations made with this sample at annual assessments at 12 to 48 months of age. Unlike observations made at 48 months, prenatal exposure to marijuana was not associated with the cognitive and verbal outcomes. Relatively low levels of maternal alcohol consumption did not have significant relationships with the outcome variables. The importance of assessing subtle components rather than global cognitive and language skills to detect potential behavioral teratogenic effects of the drugs being examined is discussed.


Medical illness in school-age Kenyans in relation to nutrition, cognition, and playground behaviors. Because medical illness is associated with malnutrition, it might contribute to the poor outcomes seen in malnourished children. This study explored relations between frequency of mild illness and development in 133 mild-to-moderately malnourished school-age Kenyan children. Morbidity information was collected for one year. Concurrent assessments of cognitive status and playground behaviors were obtained. Nutritional and environmental variables were also assessed. Girls with more reported days of mild illness performed less well on developmental measures than did their healthier female peers. They were less cognitively advanced, and, on the playground, girls with more mild illness were less active, happy, and social. Furthermore, relations between frequency of mild illness and playground behaviors (but not cognitive score) in the girls remained statistically significant when other variables, such as socioeconomic status, parental literacy, food intake, and anthropometry, were considered. For the boys, frequency of mild illness was not related to cognition or play behaviors.


Developmental reading disorder: predictors of outcome in adolescents who received early diagnosis and treatment. In this study of the course of developmental reading disorder, 40 Caucasian children, who were diagnosed and began reading therapy at 7-years-old were followed-up at 14-years-old. A variety of methodological concerns were addressed by careful screening of the subjects, by attempting to include a representative population, by comparing standardized measures statistically at initial and follow-up testing, and by using a homogeneously delayed population. It was found that 40% of the subjects were reading appropriately at follow-up. Improvement and recovery were significantly related to parents' educational status, especially to IQ, and not to gender of subject or to having a speech articulation or hyperactivity problem. It was concluded that for those who receive early diagnosis and treatment of developmental reading disorder (1) IQ may be a good predictor of recovery potential and (2) remedial efforts for the low IQ dyslexic child may be optimal if cognitive therapy is included in addition to reading therapy.


Medical stress, appraised stress, and the psychological adjustment of mothers of children with myelomeningocele. This study investigated the relationship between medical severity, stress appraisals, and psychological symptoms in a representative sample of mothers of children with myelomeningocele. Although these mothers may be at risk for the development of depression and anxiety problems, substantial variability in their psychological adjustment exists, perhaps due to differences in illness severity and stress appraisals. A sample of 66 mothers answered several questionnaires, including the Stress Questionnaire and SCL-90-R. Medical indexes reflecting severity were obtained from medical charts. Correlational analyses indicated no relationship between medical indexes and maternal adjustment. Appraised illness stress was not related to medical indexes but was significantly related to adjustment. Regression analyses including demographic, medical, and appraised stress variables accounted for 32% of the variance in adjustment. The findings suggest the importance of cognitive appraisal of stress as opposed to objective medical severity in the explanation of maternal psychological adjustment to myelomeningocele.


The effect of very low birth weight and social risk on neurocognitive abilities at school age. We tested the hypothesis that very low birth weight (VLBW < 1.5 kg) children would have significantly poorer neurocognitive abilities at school age than would normal birth weight full-term age mates, that differences would persist after control for neurologic impairment and social risk, and that VLBW would interact with social risk. Two hundred forty-nine VLBW children and a randomly selected sample of 363 normal birth weight age mates born 1977 through 1979 were tested at 8 years. A neurologic examination and tests of intelligence, language, speech, reading, mathematics, spelling, visual and fine motor abilities, and behavior were performed. Twenty-four (10%) VLBW had a major neurologic abnormality compared with none of the controls. VLBW had significantly poorer scores on all tests, with the exception of speech and the total behavior score. These differences persisted among VLBW children without major neurologic abnormality, with the exception of social competence, reading, and spelling. Even normal IQ, neurologically normal VLBW had significantly poorer scores than did controls in expressive language, memory, visuomotor, and fine motor function, and measures of hyperactivity. When social risk was controlled in multiple regression analyses, VLBW still had an adverse effect on all outcome measures with the exception of speech. Social risk was, however, the major determinant of outcome. We found an interaction between VLBW and social risk only in verbal IQ and in the opposite direction than hypothesized.


The neurobehavioral organization of Efe (pygmy) infants. The neurobehavioral organization of 16 Efe Mbuti (pygmy) infants was examined over the first weeks of life using the Brazelton Neonatal Behavioral Assessment Scale. Mbuti infants are the smallest well-grown, full-term infants in the world, i.e., length and weight below the 10th percentile but ponderal index above the 50th percentile. The Efe infants are a relatively isolated population that allows for an evaluation of ethnic differences in newborn behavior; their small stature permits the examination of the relations among size, neurobehavioral organization, and morbid processes. The neurobehavioral organization of the Efe was compared with three groups of full-term infants: two full-statured, well-grown groups of infants, one African, one United States; and a group of symmetrically growth-retarded infants whose weight and length were proportionally compromised in utero. The behavioral organization of the Efe infants was found to be similar to the neurobehavior of the well-grown infants and superior to the group of growth-retarded infants. No unique neurobehavioral features were found compared with the other groups. These findings suggest that neurobehavioral organization has a universal form and that expected small size per se does not compromise infant behavior.


Distribution and structure of identified tonic and phasic synapses between L-neurones in the locust ocellar tract. The ultrastructures and distributions of the discrete anatomical synapses which constitute two distinct types of output connections made by individual ocellar L-neurons, L1-3, are described. Outputs to neurones L4-5 are excitatory and transmit tonically, whereas reciprocal connections among the three L1-3 neurones are inhibitory and incapable of transmission for longer than a few milliseconds. The tonically transmitting synapses are located in the lateral ocellar tract and are made between the axons of L1-3, which do not receive inputs, and short branches of L4-5, which make no outputs. Each excitatory connection is composed of a few hundred discrete anatomical synapses, each characterised by a bar-shaped presynaptic density which is 0.15-1.5 microns in length and associated with a large number of round synaptic vesicles. Two postsynaptic profiles are apposed to each presynaptic density. Associated with tonic synapses are abundant invaginations of the presynaptic membrane. Synapses of the reciprocal, inhibitory, phasic connections occur in the protocerebral arbors of L1-3, among numerous output synapses of these neurones. Each phasic connection is composed of a few tens of discrete anatomical synapses. Each bar-shaped presynaptic density is associated with two postsynaptic profiles, and is 0.1-1.0 microns long. Compared with the tonic, excitatory connection, there are fewer vesicles and fewer invaginations of the presynaptic membrane associated with each synapse.


Development of commissural neurons in the embryonic rat spinal cord. Little is known about the development of the various populations of interneurons in the mammalian spinal cord. We have utilized the lipid-soluble tracer DiI in fixed tissue to study the migration and dendritic arborization of spinal neurons with axons in the ventral commissure in embryonic rats. Crystals of DiI were placed in various locations in the thoracic spinal cord in order to label commissural neurons within the dorsal horn, intermediate zone, and ventral horn at E13.5, E15, E17, and E19. Seven different groups of commissural interneurons are present in the spinal cord by E13.5. Migration is relatively simple with groups occupying a position along the dorsoventral axis roughly corresponding to their position of origin along the neuroepithelium. By E15, commissural cells are near their final locations and exhibit characteristic morphology. One striking feature is the tendency of cells with similar morphology to cluster in distinct groups. By E19, at least 18 different types of commissural interneurons can be identified on morphological grounds. Although the situation is complex, some generalities about dendritic morphology are apparent. Commissural neurons located in the dorsal horn are small and have highly branched dendrites oriented along the dorsoventral axis. In more ventral regions, commissural neurons are larger and possess dendritic arbors oriented obliquely or parallel to the mediolateral axis with long dendrites extending toward the lateral and ventral funiculi. The number of primary dendrites of most groups is set by E15 and dendritic growth occurs in the transverse plane by lengthening and branching of these primary processes. This study demonstrates that a large number of classes of commissural interneurons can be recognized on the basis of characteristic morphologies and locations within the dorsal horn, intermediate zone and ventral horn of the embryonic rat spinal cord. This finding is consistent with the fact that commissural neurons project to many different targets and mediate a variety of different functions. The demonstration that dendritic arbors of spinal interneurons with characteristic morphologies can be conveniently labelled with DiI should prove useful in future studies on the development of specific circuits in the mammalian spinal cord.


Cellular localization of the neural cell adhesion molecule L1 in adult rat neuroendocrine and endocrine tissues: comparisons with NCAM. The tissue distribution and cellular localization of the neural cell adhesion molecule L1 was determined by immunocytochemistry at the optical and ultrastructural levels in adult rat neuroendocrine tissues and pancreatic endocrine cells. L1 was found to be abundant in the neurohypophysis but undetectable in the rest of the pituitary gland. It was barely detectable in the normal rat endocrine pancreas, but a rat pancreatic insulinoma cell line was found by immunofluorescence to express low levels of L1. In the adrenal medulla, it was present on a sub-population of chromaffin cells and its density appeared to be lower on surfaces exposed to the extracellular matrix. Double immunolabelling showed this sub-population to consist of noradrenergic chromaffin cells. Adrenergic chromaffin cells were found not to express L1. In addition, the tissue distribution and cellular localization of NCAM mRNAs was determined by in situ hybridization, extending our previous studies on the cellular expression of NCAM proteins in endocrine and neuroendocrine tissues. This confirmed that the NCAM message has a wider cellular distribution than L1 within the hypophysis and the adrenal gland. In addition to secretory cells, L1 immunoreactivity was detected in glial cells, in particular in the pituicytes of the neurohypophysis, which further distinguishes them from astrocytes, their counterparts in the central nervous system. These data are discussed in terms of the different embryological origins of the various endocrine tissues examined and also in terms of the specific design constraints imposed on these tissues during their development.


Biphasic retinal neurogenesis in the brush-tailed possum, Trichosurus vulpecula: further evidence for the mechanisms involved in formation of ganglion cell density gradients. We investigated cell generation in the retina of the brush-tailed possum (Trichosurus vulpecula) by using tritiated (3H)-thymidine labelling of newly generated cells. Animals aged between postnatal day (P) 5 and 85 each received a single injection of 3H-thymidine. Following autoradiographic processing, maps of labelled cells were constructed from retinal sections. Retinal cell generation takes place in two phases, the first is concluding in the retinal periphery at P53 as the second is seen to commence in midtemporal retina. In the first phase, cells in central retina are generated earlier than those in peripheral regions. In the second phase, cells complete their final division in midtemporal retina first and in the periphery last. Cells generated in the first phase comprise virtually all cells in the ganglion cell layer, amacrine cells, horizontal cells, and cones. Ganglion cells are produced at a slightly earlier stage than displaced amacrine cells, horizontal cells, or cones. Amacrine cells in the inner nuclear layer are the final cells produced in the first phase. When ganglion cells and amacrine cells are pooled, their combined rate of production matches that of the other cell types. These data indicate that the ratio of displaced amacrine cells: horizontal cells: cones: combined ganglion cells and amacrine cells does not change throughout development. However, the ratio of ganglion cells:macrines changes steadily as development proceeds to favour amacrine cells. In the second phase, sparse numbers of nonganglion cells in the ganglion cell layer and large numbers of bipolar and Müller cells are produced along with all rods. The two phases in the possum are similar to those seen in the wallaby, the quokka. However, fewer cells are added in central retina in the possum than in the quokka and cell addition continues for a more extended period in the periphery in the possum. We suggest that this difference in cell addition could account for the development of a more pronounced visual streak of retinal ganglion cells in the possum than in the quokka. A comparison of possum retinal cell generation with that of other marsupials adds support for the "homochrony theory."


Nerve growth factor-induced sprouting of mature, uninjured sympathetic axons. The infusion of nerve growth factor (NGF) into the lateral ventricle of the mature rat brain elicits a sprouting response from axons associated with the intradural segment of the internal carotid artery. Using electron microscopic techniques, we observed a three-fold increase in the total number of perivascular axons. This NGF-elicited response is characterized by a dramatic reduction in glial cell ensheathment similar to that observed during development and by the presence of profiles devoid of organelles that may represent newly formed sprouts. In spite of the increase in axon number, no significant changes in the percentage of small, medium, or large axons were observed. The three-fold increase in the total number of axons was accompanied by an increase in the number of axons/fascicle but no change in the number of fascicles. This, along with the observation that a majority of sprouted axons were associated with other axons, supports the idea that the sprouted axons tend to associate preferentially with other axons. Bilateral superior cervical ganglionectomies following cytochrome C infusion indicate that approximately 60% of the axons associated with the internal carotid artery arise from the superior cervical ganglion and that the majority of axons contacting the smooth muscle layer arise from this ganglion. Sympathectomy following NGF infusion resulted in a 79% reduction in the total number of perivascular axons, demonstrating overwhelmingly that the majority of sprouted axons are sympathetic fibers. These results demonstrate that infusion of NGF into the mature rat brain results in the preferential sprouting of sympathetic axons associated with the internal carotid artery. These findings are consistent with the hypothesis that NGF normally plays a role in the regulation of autonomic cerebrovascular innervation in the adult animal and that mature, uninjured sympathetic neurons remain responsive to NGF.


Selective retrograde transport of nipecotic acid, a GABA analog, labels a subpopulation of gerbil olivocochlear neurons. Perfusion of the gerbil cochlea with micromolar quantities of 3H-gamma-aminobutyric acid (GABA) results in rapid, selective labeling of 50-60% of the olivocochlear (OC) efferent terminals on afferent dendrites beneath the inner hair cells, and all of the efferent terminals beneath the outer hair cells. In order to identify the neurons from which these GABA-accumulating terminals originate, the cell bodies were localized by using retrograde transport of 3H-nipecotic acid, a metabolically inert GABA analog. With survival times of 6-30 hours after cochlear injection, myelinated OC efferent fibers and cell bodies were well labeled, with the greatest number being labeled at 12-18 hours. All of the labeled neurons belonged to the medial OC system, and no lateral OC neurons were labeled. It is concluded that the GABA-accumulating endings in the gerbil cochlea arise from medial OC neurons, and therefore that medial OC efferent neurons in this species project to both inner and outer hair cell regions.


Distribution of nerve growth factor-like immunoreactive neurons in the adult rat brain following colchicine treatment. Using immunohistochemical techniques, we have previously localized nerve growth factor (NGF)-like immunoreactivity in the normal adult rat central nervous system (CNS) exclusively in the hippocampal mossy fiber region and within basal forebrain cholinergic neurons--a cell population believed to be primary NGF consumers within the CNS. In the present investigation, we have attempted to identify potential producers of NGF by pretreating animals with colchicine. Such a treatment would be expected to block microtubule-assisted neuritic transport mechanisms, thus preventing the accumulation of antigens normally obtained by retrograde transport and forcing the accumulation of cell products normally exported anterogradely. Forty-eight hours after colchicine administration within their innervation territories, basal forebrain cholinergic neurons showed a marked loss of NGF-like immunoreactivity. Conversely, following colchicine treatment, many new populations of NGF-like immunoreactive cells were detected, several of which have been previously observed with in situ hybridization techniques for NGF mRNA. Many NGF-like immunoreactive populations, however, were not previously recognized by in situ hybridization methods, including cells of the striatum, reticular thalamic nucleus, paraventricular hypothalamic nucleus, supraoptic nucleus, lateral and medial septum, substantia innominata, and nucleus basalis. Furthermore, evidence is provided that colchicine-blocked, NGF-like immunoreactive neurons within the basal forebrain are not cholinergic, thus reinforcing the hypothesis that trophic support for these NGF-dependent neurons may be derived from distant and local sources. The distinctive distribution of NGF-like immunoreactive cells observed in this study strongly correlates with the reported distribution of NGF mRNA in CNS neurons, thus suggesting that our antibodies are uniquely recognizing NGF and not other related neurotrophins.


Sympathetic nerves in adult rats regenerate normally and restore pilomotor function during an anti-NGF treatment that prevents their collateral sprouting. We have used anti-nerve growth factor (anti-NGF) [corrected] administration to study the NGF dependency of the reinnervation of denervated skin by sympathetic nerves in the adult rat. Sympathetic pilomotor fields were revealed by electrical stimulation of selected dorsal cutaneous nerves; the affected skin rapidly assumed a "gooseflesh" appearance, sharply demarcated from surrounding unstimulated skin. Examined 2-5 days after section of neighboring nerves, the "isolated" pilomotor field of the spared nerve was found to be coextensive with an area of amine-fluorescent fibers that were associated with pilomotor muscles and blood vessels. After its isolation, a pilomotor field begins to expand into the surrounding deprived territory, reaching a maximum size at approximately 40 days. Fluorescence studies confirmed that new sympathetic fiber growth had occurred into the expanded regions of such fields. Daily injections of polyclonal anti-NGF serum completely prevented these pilomotor field expansions. Following termination of the anti-NGF treatment, expansion proceeded normally. Finally, if the onset of anti-NGF treatment was delayed until pilomotor field expansion had already commenced, further expansion was halted. Regeneration of sympathetic fibers was evoked by crushing a selected nerve. Recovery of pilomotor function in the totally denervated skin was first detected at about 20 days postcrush, and the field progressively enlarged over the next 40 days. Although the imposed NGF deprivation is known to cause a demonstrable shrinkage, and presumably atrophy, of sympathetic ganglia, the anti-NGF treatment appeared to impair neither the restoration of a pilomotor field after nerve crush, nor its continued expansion into skin regions well beyond that originally supplied by the nerve, i.e., into territory whose invasion by collateral sprouts would have been totally prevented by the treatment. During such NGF deprivation, fluorescent regenerating fibers were visualized in the nerve trunk. We conclude that even though the regenerating and collaterally sprouting sympathetic fibers probably utilise the same degenerating dermal pathways to reach and functionally reinnervate the same denervated targets, only the collateral sprouting of the uninjured axons is dependent upon endogenous NGF. These findings extend the results described earlier for nociceptive fibers, and suggest that the contrasting dependencies upon growth factors of sprouting and regeneration might apply throughout the adult nervous system.


Thalamic connections of the vestibular cortical fields in the squirrel monkey (Saimiri sciureus). The afferent thalamic connections to cortical fields important for control of head movement in space were analysed by intracortical retrograde tracer injections. The proprioceptive/vestibular area 3aV, the neck-trunk region of area 3a, receives two thirds of its thalamic projections from the oral and superior ventroposterior nucleus (VPO/VPS), which is considered as the proprioceptive relay of the ventroposterior complex (Kaas et al., J. Comp. Neurol. 226:211-240, 1984). The parieto-insular vestibular cortex (PIVC, area retroinsularis, Ri) receives its main thalamic input from posterior parts of the ventroposterior complex and from the medial pulvinar. Anatomical evidence is presented that the posterior region of the ventroposterior complex is a special compartment within this principal somatosensory relay complex. The parietotemporal association area T3, mainly involved in visual-optokinetic signal processing, receives a substantial input from the medial, the lateral, and the inferior pulvinar. Dual tracer experiments revealed that about 5% of the thalamic neurons projecting to 3aV were spatially intermingled with neurons projecting to areas PIVC or T3. This spatial intermingling was distributed over small but numerous, circumscribed thalamic regions, called "common patches," which were found mainly in the intralaminar nuclei, the posterior group of thalamic nuclei, and the caudal parts of the ventroposterior complex. The "common patches" may indicate a functional coupling of area 3aV with the PIVC or area T3 on the thalamic level. In control experiments thalamic projections to the granular insula Ig and the anterior part of area 7, two cerebral structures connected with the vestibular cortical areas, were studied. Some overlap in the thalamic relay structures projecting to these areas with those projecting to the vestibular cortices was found. A quantitative evaluation of thalamic regions projecting to different cortical structures was performed by constructing so-called "thalamograms." A scheme was developed that describes the afferent thalamic connections by which vestibular, visual-optokinetic, and proprioceptive signals reach the vestibular cortical areas PIVC and 3aV.


Growth and regression of thalamic efferents in the song-control system of male zebra finches. A serial forebrain pathway in the songbird brain plays a critical role in vocal learning; Area X of the parolfactory lobe (X) projects to the medial portion of the dorsolateral nucleus of the anterior thalamus (DLM), which in turn projects to the lateral magnocellular nucleus of the anterior neostriatum (IMAN). Lesions of this pathway in juvenile birds disrupt vocal development, whereas identical lesions in adult birds do not influence the production of already learned song. During the course of vocal learning, IMAN undergoes a phase of massive neuronal loss, whereas the neuronal population of X more than doubles. In the present study, the development of neuron number in DLM was analyzed and found not to change during the course of vocal learning. Anterograde DiI labeling of DLM efferent fibers was then used to analyze the morphological development of this projection in relation to both the loss of neurons from lMAN and the loss of the ability of X-DLM-lMAN lesions to influence vocal production. We found that DLM axons arrive within lMAN by 15 days of age, prior to both the loss of neurons from lMAN and the onset of vocal production. The volume of anterograde DiI label over lMAN did not change between 15 and 20 days of age, but this volume more than doubled between 20 and 35 days of age. During this phase of exuberant growth, anterograde label matched the dorsal border of lMAN but extended beyond all other borders of lMAN into a surrounding "shell" of parvicellular neurons.(ABSTRACT TRUNCATED AT 250 WORDS)


Analysis of low affinity nerve growth factor receptor during pulpal healing and regeneration of myelinated and unmyelinated axons in replanted teeth. Nerve regeneration was examined in rat molars that were briefly extracted and then replanted in the socket for 1-90 days. Immunocytochemistry was used to evaluate neural and nonneural immunoreactivity (IR) for low affinity nerve growth factor receptor (p75-NGFR) and for laminin and calcitonin gene-related peptide (CGRP). Three different types of pulpal response to replantation were found. Type I: Some replanted teeth had mild injury and still contained coronal odontoblasts and associated fibroblasts that retained p75-NGFR-IR; they continued regular dentin formation and had excellent reinnervation. Type II: Teeth with intermediate injury lost most or all of the coronal pulp tissue, but they regenerated odontoblast-like cells that formed irregular dentin, they had numerous dispersed p75-NGFR-IR fibroblasts in crown pulp during early regeneration, and they had excellent reinnervation. Type III: Severely injured teeth lost their original pulp; they filled with dense connective tissue and bone and had poor reinnervation. After Type I or II injury the Schwann cells around degenerating myelinated and unmyelinated axons had increased expression of p75-NGFR by 1-3 days. By 7-10 days those Schwann cells had formed hollow tubes (bands of Bungner) along the degenerating axon tracks. They maintained their increased p75-NGFR-IR during and after regeneration of unmyelinated axons, whereas Schwann cells involved in remyelination lost p75-NGFR-IR at that stage. The number of CGRP-IR axons in the regenerating pulp increased from 7 to 90 days. Laminin-IR increased in all replanted teeth at 3-10 days and only returned to normal patterns in teeth with Type I or Type II response at 20-90 days. The special p75-NGFR-IR of pulpal fibroblasts of adult rat molars did not usually persist in regenerated, reinnervated pulp. The extensive depletion of fibroblast p75-NGFR-IR and the continuing enhanced p75-NGFR-IR in unmyelinated nerve fibers at 90 days show that altered growth factor conditions characterize regenerated pulp of replanted teeth.


Autonomic neurone degeneration in equine dysautonomia (grass sickness). Histological investigations were undertaken on four sympathetic autonomic ganglia and on the myenteric and sub-mucosal plexuses of the jejunum in healthy animals, in naturally occurring cases of acute, sub-acute and chronic equine dysautonomia and in ponies in which neuronal damage had been induced by the injection of acute grass sickness sera. The degree of neuronal damage is related to the type of dysautonomia. The coeliac-mesenteric ganglion reacts differently from other ganglia and is less severely damaged in cases of short duration. Extensive experimentally induced damage to the coeliac-mesenteric ganglion, even when jejunal damage is also present, is not associated with clinical illness. It is proposed that the rate of autonomic neurone loss and the extent of the damage may both influence the clinical manifestations of grass sickness.


Cooperioides antidorca from intestinal nodules in Gazella thomsoni and G. granti in Kenya. Examination of nodules from the intestine of Gazella thomsoni and G. granti in Kenya revealed the presence of the trichostrongylid nematode Cooperioides antidorca. Nodules from the two hosts were histologically similar and took the form of chronic inflammatory lesions extending through the wall of the gut from the submucosa into the muscular layers. There was some necrosis around the parasite located in the centre of the nodule, a varying population of inflammatory cells and a varying degree of fibrous tissue encapsulation. One nodule possessed an aperture into the lumen of the gut and there was also evidence of serosal proliferation, probably due to the parasitic lesion penetrating the deeper layers of the wall. The morphology of Cooperioides antidorca recovered from the nodules was examined by scanning electron microscopy and is briefly described.


Upper motor neurone and descending tract pathology in bovine spinal muscular atrophy. The extent of neuropathology in a recently reported disease of Brown Swiss cattle, spinal muscular atrophy (SMA), was investigated with light and electron microscopy. Many regions of the central nervous system were sampled from 10 SMA-affected and three normal Brown Swiss calves. In addition to extensive necrosis of lower motor neurones there was extensive upper motor neurone degeneration and descending tract pathology. Since these abnormalities are also hallmarks of amyotrophic lateral sclerosis (ALS), SMA may be an animal model for that disease. There was also considerable vacuolar degeneration, which is a feature of the wobbler mouse and murine type C RNA virus-induced paralytic disease models of ALS.


Histopathological and morphometric studies on the hooves of dairy and beef cattle in relation to overgrown sole and laminitis. In dairy cattle, histopathology of the outer hind claws with overgrown sole revealed arteriosclerosis of blood vessels at the ulcer site in the sole. The laminae did not show any hyperplasia of the epidermis and thrombi were not seen in the blood vessels. Partial or complete disappearance of onychogenic substance was observed in the sole as well as in the wall epidermis. In beef animals, typical changes suggestive of chronic laminitis were seen: these included hyperplasia of the epidermis of the laminae, thrombus formation, arteriosclerosis in the corium of the sole, the abaxial wall and the coronary corium. Morphometric analysis quantified measurements of hooves of normal, beef and dairy cattle. It may be concluded that the changes associated with overgrowth of the sole in dairy cattle are localized to the sole without involvement of the laminae and that the term laminitis is not appropriate in the condition seen in dairy cattle.