Clonal analysis of bcr-abl rearrangement in T lymphocytes from patients with chronic myelogenous leukemia.

The cytogenetic hallmark of chronic myelogenous leukemia (CML) is the Philadelphia chromosome (Ph1), which reflects a chromosomal translocation t(9;22) and a rearrangement of the ABL and bcr genes. This marker is found in all cells arising from the same malignant precursor cell and can be detected in CML cells of the myeloid, monocytic, erythroid, and B-lymphocyte lineage. It is, however, controversial as to whether T lymphocytes of CML patients carry this gene rearrangement. An answer to this question would clarify whether the translocation in CML occurs in a pluripotent hematopoietic stem cell or in a precursor cell already committed to certain lineages, but not the T-cell lineage. To address this question, we established T-cell clones from peripheral venous blood cells of four patients with CML and screened these clones for bcr-abl fusion transcripts by means of polymerase chain reaction and Southern blot analysis. In four T-cell clones of three of these patients, the bcr-abl transcript could be detected. None of 12 T-cell clones of the fourth patient disclosed detectable bcr-abl amplification product. Both CD4+ as well as CD8+ clones displayed fused bcr-abl sequences. These data imply that in CML some but not all T lymphocytes may originate from the Ph1-positive stem cell.


Stem cell factor increases colony-forming unit-spleen number in vitro in synergy with interleukin-6, and in vivo in Sl/Sld mice as a single factor.

Hematopoiesis is thought to be modulated by interactions of progenitor cells with hematopoietic growth factors. We have shown that colony-forming units-spleen (CFU-S) and repopulating stem cells require interleukin-3 (IL-3) to survive in vitro, and that CFU-S number and long-term repopulating ability can be increased by culture in the combination of IL-3 and IL-6. In this report, we describe the effects of stem cell factor (SCF) on CFU-S and repopulating stem cells. Injection of SCF into anemic Sl/Sld mice caused a twofold and 20-fold increase in CFU-S number in the bone marrow and spleen of treated animals, respectively. After 6 days in suspension culture, CFU-S number increased threefold in cultures supplemented with SCF and IL-6, or SCF, IL-3, and IL-6 relative to the number at day 0. The long-term repopulating ability of cells cultured in SCF, IL-3, and IL-6 was approximately sevenfold better than that of cells cultured in IL-3 or SCF. Similar experiments were performed on populations of bone marrow cells enriched for, or depleted of, CFU-S by elutriation and lineage subtraction. The combination of SCF and IL-6 increased CFU-S number approximately fourfold to eightfold in the CFU-S-enriched fraction, but had no effect on the CFU-S-depleted cells. These results show that SCF alone can increase CFU-S number in vivo, and in combination with other growth factors increases CFU-S numbers in vitro.


The c-kit receptor ligand functions as a mast cell chemoattractant.

Mast cells accumulate at sites of neovascularization, solid tumors, and many immune reactions. Such accumulation requires directed migration of mature mast cells or their precursors. The nature of the chemoattractants that regulate mast cell motility and the identity of the receptors that mediate the chemotactic response are poorly understood. We have tested the ability of stem cell factor (SCF), a mast cell growth factor, to stimulate mast cell migration. Our results show that SCF is a potent mast cell attractant that stimulates directional motility of both mucosal and connective tissue-type mast cells. The activity is potentiated by costimulation with interleukin-3 (IL-3), another mast cell chemoattractant. SCF, a known ligand for the c-kit tyrosine kinase receptor, was unable to stimulate motility in W42 mutant mast cells, which have a defective c-kit tyrosine kinase. However, W42 mast cells were still able to migrate in response to IL-3. These results show that SCF is a chemotactic factor as well as a growth factor and that the c-kit receptor can transduce signals leading to both cell proliferation and increased directional cell motility.


Polyclonal hematopoiesis in interferon-induced cytogenetic remissions of chronic myelogenous leukemia.

Interferon (IFN) therapy of early chronic myelogenous leukemia (CML) frequently produces partial or complete cytogenetic remission of the disease. Patients with complete cytogenetic remission often continue on therapy for several years with bone marrow showing only diploid (normal) metaphases. We studied hematopoiesis in five female patients with major cytogenetic remissions from CML during IFN therapy. Clonality analysis using the BstXI PGK gene polymorphism showed that granulocytes were nonclonal in all patients during cytogenetic remission. BCR region studies showed rearrangement only in the one patient whose remission was incomplete at the time of sampling. Granulopoiesis is nonclonal in IFN-induced remissions of CML and may be derived from normal hematopoietic stem cells.


Treatment of Paget's disease by weekly infusions of 3-aminohydroxypropylidene-1,1-bisphosphonate (APD).

Thirty patients with symptomatic Paget's disease of bone were treated with weekly infusions of 30 mg APD for 6 weeks and followed for up to 3 years (mean 2 years). Bone pain diminished or disappeared in 83%. Six months following treatment the serum alkaline phosphatase (sAP) had normalized in 53% and had fallen by an average of 68% in the remainder. The average fall in sAP was 65% at 6 months, 59% at 1 year and 51% at 2 years. Urine hydroxyproline/creatinine ratios (OHp/Cr) fell by an average of 72% over the 6 weeks of treatment whilst serum bone gla protein (BGP) showed no significant change. However, there was a significant fall in the mean BGP of 25% by 6 months following treatment. Radionuclide bone scan abnormalities improved in all patients and showed complete resolution in two. The same regimen was used to retreat eight patients with a persistence or recurrence of symptoms after 1 year. At 6 months following retreatment the sAP had fallen to normal in four and in the remainder by an average of 39%. Two patients had a third course after a further year and by 6 months the sAP had fallen by an average of 50%. Normalization of sAP occurred in 86% of patients with a pretreatment sAP less than 900 iu/l (normal less than 300) and 89% of patients with a sAP less than 600 iu/l immediately following the course of treatment. Therefore, knowledge of the pretreatment and post-treatment sAP should enable prediction of the need for further therapy in most cases. We confirm that APD is an effective and well tolerated treatment for the management of Paget's disease, giving long-term suppression.(ABSTRACT TRUNCATED AT 250 WORDS)


Multiple myeloma.

Patients with multiple myeloma may present with insidious or acute clinical problems to wide variety of medical specialties. It is important for doctors to know how to diagnose myeloma accurately, to be aware of the clinical complications and to have some knowledge of the newer therapeutic approaches to chemotherapy and supportive treatment.


Presenting a talk at an academic meeting.

Presenting a paper at an academic meeting or giving a lecture is a small but significant part of a doctor's work. Nearly all doctors initially dread such a prospect, but with practice and experience it need not be such an ordeal.


Eosinophilic prostatitis and prostatic specific antigen.

Eosinophilic prostatitis is a rare form of abacterial prostatitis with uncertain aetiology. Its clinical presentation, like other types of abacterial prostatitis, commonly mimics carcinoma of the prostate. Transrectal ultrasound may be helpful in the diagnosis of prostatitis but histological confirmation is necessary. Prostatic specific antigen has been widely used in the diagnosis and follow-up of patients with prostatic carcinoma. High levels of this antigen (greater than 30 micrograms/l) have been claimed to be highly specific for prostate cancer, although lesser elevations may also occur in patients with large benign prostate glands and in bacterial prostatitis. We report 3 patients with histologically proven eosinophilic prostatitis and high levels of prostatic specific antigen. This diagnosis may closely mimic carcinoma of the prostate and must be excluded by histological examination of biopsy material before treatment for presumed prostate carcinoma is initiated.


Surgical palliation for pancreatic cancer: developments during the past two decades.

Improvements in pancreatic imaging over the past 20 years have revolutionized the preoperative diagnosis and assessment of resectability in patients with suspected pancreatic cancer. This review highlights the resultant trends in the surgical treatment of ductal carcinoma of the pancreas, comparing series reported between 1981 and 1990 with those from the previous decade. Small but worthwhile gains have been achieved both in overall resection rate and in the survival rate from such resections. Nevertheless, 80 per cent or more of affected patients are still unsuitable for resection because of the extent of their disease. Laparotomy retains a crucial role in the management of carcinoma of the pancreatic head, although percutaneous and endoscopic stents provide a useful alternative for palliation of malignant obstructive jaundice in elderly patients or those with carcinomatosis. Operation provides the chance to confirm the nature and full extent of the tumour, to circumvent duodenal obstruction and to abolish pain, besides relieving jaundice without the need for tubes (with their potential to block). By contrast, operative treatment generally has much less to offer in patients with carcinoma of the pancreatic body, unless diagnosis and irresectability remain in doubt. In combination, radiotherapy and 5-fluorouracil may achieve more as adjuncts to palliative surgery than either agent alone. The increasing safety of pancreaticoduodenectomy raises the possibility of palliative resection in younger patients with limited but incurable disease.


Tumor promoters induce basic fibroblast growth factor gene expression in human dermal fibroblasts.

Tumor-promoting phorbol esters have been shown previously to either induce or repress the expression of numerous cellular genes, and this property is likely to be important for the in vitro and in vivo biological effects of these compounds. In this report, we demonstrate that phorbol 12-myristate 13-acetate induces the accumulation of basic fibroblast growth factor mRNA and protein in human dermal fibroblasts. In contrast, acidic fibroblast growth factor expression was unaffected by this compound. The enhancement of basic fibroblast growth factor gene expression by phorbol 12-myristate 13-acetate was blocked by the isoquinolinesulfonamide derivative H7, a potent inhibitor of protein kinase C. Two additional tumor promoters that bind to and activate protein kinase C, phorbol 12,13-didecanoate and mezerein, also increased basic fibroblast growth factor mRNA levels. Basic fibroblast growth factor is a mitogen for many cell types and can stimulate angiogenesis; thus, some tumor promoter-induced cellular responses may be mediated by this polypeptide.


Role of neovasculature and vascular permeability on the tumor retention of photodynamic agents.

A variety of photodynamic sensitizers (chloroaluminum sulfonated phthalocyanine, tetraphenyl porphine sulfonate, mono-L-aspartyl chlorin e6, Photofrin, chlorin e6, and Uroporphyrin dihydrochloride I) were characterized by their ability to be retained in EMT-6 tumors growing in BALB/c mice. Two properties uniquely associated with tumors, proliferating neovasculature and vascular permeability, were tested for their relative importance in retaining the photosensitizer. A chick embryo model was used to compare photosensitizer uptake/retention in proliferating and nonproliferating neovasculature with retention in proliferating nonvascular tissue. Our results provide evidence that photosensitizers which are preferentially retained by tumors have a selective affinity for proliferating neovasculature. The chloroaluminum sulfonated phthalocyanine and tetraphenyl porphine sulfonate compounds possess the greatest affinity for proliferating neovasculature relative to nonvascular tissue, while the phthalocyanine has the largest tumor/normal differential in vivo of all the photosensitizers tested. Chlorin e6 and uroporphyrin dihydrochloride I were the only photosensitizers which were not retained in greater amounts by tumor tissues relative to normal tissues. Using a delayed-type hypersensitivity reaction, extended and constant vascular permeability was induced in BALB/c mice. Vascular permeability was quantitated by Evans blue extraction from the delayed-type hypersensitivity sites. Interestingly, leaky vessels alone did not result in photosensitizer retention, as seen with tumors. These data demonstrate that tumor-retained photosensitizers possess a selective affinity for proliferating neovasculature and that vascular permeability alone is not sufficient to retain these sensitizers.


The combination of gamma-interferon and tumor necrosis factor causes a rapid and extensive differentiation of human neuroblastoma cells.

Neuroblastoma (NB), a tumor originating from the sympathetic nervous system, is the most common extracranial neurological tumor of childhood. Human NB cells may differentiate in vitro under treatment with biological agents, as gamma-interferon (IFN-gamma) and tumor necrosis factor (TNF). Unfortunately, NB cell lines resistant to the differentiation-inducing effects of both drugs have been observed. Here we demonstrate that a combination of IFN-gamma plus TNF causes extensive and generalized differentiation of NB cells toward a neuronal phenotype. Both IFN-gamma and TNF, given alone, moderately reduced cell growth and induced partial morphological maturation. Their combination further reduced cell proliferation. The combined treatment gave a synergistic rather than additive cytostatic effect, documented also by a dramatically enhanced differentiation toward a neuronal morphology. Membrane immunofluorescence showed a homologous and heterologous up-regulation of IFN-gamma receptor, as well as a marked induction of HLA Class I antigens and, to a lesser extent, of Class II antigens on NB cells induced to differentiate. Treatment of NB cell lines with IFN-gamma/TNF results in the induction of a differentiated phenotype, as indicated by the increased expression of the Mr 160,000 and 200,000 neurofilament proteins and that of microtubule-associated proteins. Evaluation of biochemical markers of neuronal differentiation confirmed the ability of the combined treatment to induce neuroblast maturation. These results suggest that the combination of IFN-gamma and TNF should be considered for experimental clinical trials in neuroblastoma.


Introduction of the ras oncogene transforms a simian virus 40-immortalized hepatocyte cell line without loss of expression of albumin and other liver-specific genes.

Activated c-Ha-ras DNA sequences were introduced by transfection into a low passage simian virus 40 (SV40)-immortalized rat hepatocyte cell line, CWSV1, and stable ras transfectant cell lines were established to determine the effect of the addition of the activated c-Ha-ras oncogene on growth properties and differentiation. Control transfectant cell lines were generated by transfection with neo alone. CWSV1 cells at low passage and the control transfectants were not tumorigenic. The ras transfectants demonstrated anchorage-independent growth and were highly tumorigenic in syngeneic hosts. CWSV1 cells produce liver-like levels of albumin and express other liver-specific genes. The ras transfectants expressed RNA for albumin, transferrin, and the transcription factor HNF-1 at similar levels to the parental CWSV1 cells, indicating that the alterations in growth properties and tumorigenic potential of these cells did not decrease the ability of the cells to express several genes that are associated with hepatocyte differentiation. The addition of the ras oncogene did not induce the expression of alpha-fetoprotein and had no specific effect on expression of glutathione S-transferase-P. The tumors produced by the ras transfectants were not well differentiated; however, the cells in the tumors and tumor cell lines derived from the tumors continued to produce albumin and did not produce alpha-fetoprotein. We conclude that the addition of the activated c-Ha-ras oncogene to immortalized CWSV1 cells transformed these cells as measured by morphology, growth properties, and tumorigenicity without reducing their ability to express albumin and other significant liver-specific genes.


Successful treatment of human acute T-cell leukemia in SCID mice using the anti-CD7-deglycosylated ricin A-chain immunotoxin DA7.

The study of new therapeutic approaches for refractory human leukemia has been hampered by the lack of relevant in vivo models with disseminated disease, particularly T acute lymphoblastic leukemia (T-ALL). In the present study we evaluated methods for establishing and therapy of a human T-ALL cell line (MT-ALL) in 73 SCID mice. MT-ALL is a T-cell receptor alpha/beta +, CD3+, and CD7+ leukemia cell line, derived from a patient with refractory disease and early death. Injection of 5 x 10(7) MT-ALL cells i.v. caused disseminated human leukemia in hematopoietic and nonhematopoietic organs in 100% of SCID mice (n = 9) leading to death or terminal disease at 65 to 70 days after a uniform clinical course. To study possible therapeutic approaches for disseminated leukemia we utilized an immunotoxin, DA7, constructed by chemically linking the mouse IgG2b anti-CD7(3A1E) monoclonal antibody which recognizes a pan-T-cell marker expressed on almost all T-cell leukemias to deglycosylated ricin A-chain, a catalytic plant toxin and inhibitor of protein synthesis. Administration of DA7 led to greater than 5 log kill of clonogenic MT-ALL cells in vitro and selectively inhibited protein synthesis. DA7 was administered to mice at a dose of 10 micrograms/mouse/day for 5 consecutive days starting 8 days after i.v. inoculation of leukemia. The immunotoxin therapy resulted in significant long term survival over 348 days compared to untreated or control mice treated with anti-CD7 antibody and deglycosylated ricin A-chain which were all dead by day 70 (P less than 0.001). Even after more than 11 months there was no evidence of disease in 82% of the DA7 treated animals. SCID mice given i.p. injections (n = 9) developed an i.p. tumor mass but demonstrated metastasis outside the peritoneum with disseminated leukemia in hematopoietic and nonhematopoietic organs, a finding different from most conventional nude mouse models. The leukemia was fatal in 100% and killed the animals at 68-95 days. SCID mice given i.p. injections of MT-ALL completely responded to therapy with DA7, resulting in survival of 100% of the animals (n = 10) at 216 days (P less than 0.001 compared to untreated animals). Anti-CD7 antibody, deglycosylated ricin A-chain, and a control anti-melanoma immunotoxin (IND1-RTA) showed no therapeutic effect. We conclude that DA7 is an effective in vivo therapeutic agent against human MT-ALL in the SCID mouse system, suggesting potential usefulness for therapy of humans with poor prognosis T-cell leukemia.


Is hypomagnesemia arrhythmogenic?

An understanding of the role of magnesium in cardiac conduction is complicated by the multiplicity of intracellular events coordinated by the magnesium ion. Several reports have cited magnesium deficiency as the cause of a variety of ventricular and supraventricular arrhythmias. On further inspection, the circumstances of each report strongly suggest the coexistence of significant potassium depletion; isolated hypomagnesemia as a cause of arrhythmia is not reported. This discussion brings together new data from basic science with that of clinical research to refute the suggestion that isolated hypomagnesemia is arrhythmogenic. However, there is sufficient evidence to indicate that hypomagnesemia will significantly exacerbate the proarrhythmic effect of hypokalemia, particularly if occurring in the presence of digoxin toxicity. Potassium and magnesium depletion are commonly concomitant, and simultaneous repletion of both ions in the presence of hypokalemia-induced arrhythmia would be both logical and effective. The beneficial effects of intravenous magnesium in the acute control of ventricular tachyarrhythmia are concluded to occur as a result of a separate antiarrhythmic action, quite independent of underlying magnesium balance.


Chemotherapy-induced myocardial infarction in a young man with Hodgkin's disease.

A 32-year-old male with stage IIIA nodular sclerosing Hodgkin's disease and no cardiac risk factors presented with chest pain after receiving chemotherapy consisting of multiple drugs, including vinca alkaloids. He completed an uncomplicated anterior wall myocardial infarction. Coronary angiography documented the absence of significant coronary artery disease. Exercise stress testing with gated scan confirmed loss of anterior wall motion and a decreased left ventricular ejection fraction. Vascular toxicity, including, rarely, myocardial infarction, has been reported following antineoplastic regimens containing vinca alkaloids. Hypercoagulable states, cardiac invasion by tumor, and coronary artery spasm are possible etiologies. Of these, coronary artery spasm appears most likely. Management should include discontinuation of the offending drug and supportive care.


Patterns of cytokines, plasma endotoxin, plasminogen activator inhibitor, and acute-phase proteins during the treatment of severe sepsis in humans.

To study the patterns of plasma concentrations of endotoxin, tumor necrosis factor-alpha (TNF), interleukin-6 (IL-6), plasminogen activator inhibitor-1, C-reactive protein, and serum amyloid A during the treatment of human sepsis. A prospective case series study. ICU of the Department of Internal Medicine, University Hospital Groningen, The Netherlands. Twenty consecutive patients (11 female, 9 male, mean age 67 yrs) with clinically defined sepsis. Eighteen patients were admitted from the outpatient emergency ward; two patients were already inpatients. The control group (n = 7) comprised patients with nonseptic shock. Ten (50%) septic patients had detectable endotoxemia (greater than 5 (ng/L). TNF concentrations on admission were increased in 94% of the septic patients, whereas IL-6 and plasminogen activator inhibitor plasma concentrations were increased in all septic patients. The septic group showed significantly (p less than .05) higher concentrations of TNF, IL-6, plasminogen activator inhibitor-1, C-reactive protein, and serum amyloid A compared with the nonseptic patients. In the septic group, we found a correlation of both IL-6 and plasminogen activator inhibitor concentrations with severity of illness (r2 = .33, p less than .05; r2 = .22, p less than .05, respectively). After the start of antibiotic treatment, high concentrations of TNF and plasminogen activator inhibitor persisted in the nonsurvivors in contrast to decreasing concentrations in most of the survivors. After an initial increase in seven patients, IL-6 concentrations decreased in all septic patients and also in nonsurvivors. This study confirms previous findings that: a) TNF is a major mediator involved in the pathogenesis of septic shock; b) plasminogen activator inhibitor activity is significantly increased in septic patients and might be involved in the pathogenesis of disseminated intravascular coagulation associated with sepsis; and c) IL-6 is involved in the pathophysiology of septic shock, although further studies are needed to determine whether IL-6 is directly involved in mediating the lethal complications of septic shock or whether it should be considered an "alarm hormone" that reflects endothelial cell injury. Our findings also suggest that the concentrations and trends of these mediators during treatment are valuable for monitoring septic patients.


Resuscitation of intraoperative hypovolemia: a comparison of normal saline and hyperosmotic/hyperoncotic solutions in swine.

We compared a hypertonic saline-dextran solution (7.5% NaCl/6% dextran-70) with 0.9% NaCl (normal saline) for treatment of intraoperative hypovolemia. Fourteen anesthetized pigs (mean weight 36.3 +/- 2.1 kg) underwent thoracotomy, followed by hemorrhage for 1 hr to reduce mean arterial pressure to 45 mm Hg. A continuous infusion of either solution was then initiated and the flow rate was adjusted to restore and maintain aortic blood flow at baseline levels for 2 hrs. Full resuscitation to initial values of aortic blood flow was achieved with both regimens, but the normal saline group required substantially larger volumes and sodium loads to maintain stable hemodynamic values. Normal saline resuscitation produced increases in right ventricular preload (central venous pressure) and afterload (pulmonary arterial pressure and pulmonary vascular resistance), resulting in increased right ventricular work. Hypertonic saline-dextran solution resuscitation of intraoperative hypovolemia is performed effectively with smaller fluid and sodium loads, and is devoid of the deleterious effects associated with fluid accumulation induced by a conventional isotonic solution regimen.


Effect of hydroxyethyl starch solutions on blood glucose concentrations in diabetic and nondiabetic rats.

The effects of iv infusions of 6% hetastarch or 10% pentastarch on blood glucose concentrations were tested in 12 nondiabetic and 12 streptozotocin-induced diabetic pentobarbital-anesthetized Sprague-Dawley rats weighing 313 to 473 g. All rats were paralyzed and mechanically ventilated. After control measurements of blood glucose concentrations were taken, rats were divided into four groups. Groups 1 (n = 6) and 2 (n = 6) comprised nondiabetic rats that received iv infusions of hetastarch or pentastarch, respectively, at a rate of 2.0 mL over 30 mins. Groups 3 (n = 6) and 4 (n = 6) comprised diabetic rats that also received iv hetastarch or pentastarch, respectively, at the same rate and duration of infusion. Neither hetastarch nor pentastarch infusions significantly altered blood glucose values over the 3-hr study period, regardless of whether the rats were diabetic or nondiabetic. Assuming these data are transferable to humans, the authors conclude that hydroxyethyl starch solutions do not produce or exacerbate hyperglycemia, and furthermore, that their use is not contraindicated in subjects having hyperglycemia from diabetes mellitus or iatrogenic causes.


[The palliative therapy of malignant esophageal obstruction with self-expanding metal endoprostheses].

A total of 23 self-expanding metal stents were implanted in 17 patients (12 men, 5 women; mean age 66 [44-83] years) with inoperable malignant obstruction of the oesophagus or the oesophago-gastric junction. A primary success was achieved in all, a good functional result in 16 (94%). There were no complications. In the follow-up period (mean of 15.2 +/- 13 weeks) re-obstruction by the tumour process occurred in three patients. Twelve patients died after a mean survival time of 15.8 +/- 14 weeks. In ten of these the stent was still patent at death, while two had again developed dysphagia. The cumulative patency rate of the stents was 79%. These observations indicate that self-expanding metal stents can achieve satisfactory palliation in dysphagia due to a malignancy. The mortality and morbidity rates of the method seem to be less than those of other palliative measures.


Flow-induced release of adenosine 5'-triphosphate from endothelial cells of the rat mesenteric arterial bed.

Adenosine 5'-triphosphate (ATP) was released into the perfusate of rat isolated mesenteric arterial beds during each of two consecutive increases in flow. There was no significant difference between the amounts of ATP released on each occasion. Substance P was also released into the perfusate by increased flow, although its release was more variable. Removal of the endothelium of the mesenteric vessels with sodium deoxycholate led to a significant reduction (74%) in the amount of ATP released compared with the release before the endothelium had been removed. This suggests that the ATP released into the mesenteric arterial perfusate during increased flow arises from endothelial cells.


An improved thioflavine S method for staining neurofibrillary tangles and senile plaques in Alzheimer's disease.

Large differences are usually observed when standard staining methods for a number of pathological lesions in neurodegenerative disorders are compared. With the modified thioflavine S method presented here (easy and cheap to perform), the morphological appearance of the stained neurofibrillary tangles (NFT) and senile plaques (SP) is greatly improved. Furthermore, the intense contrast between stained lesions and background obtained with this technique permits an accurate automatic quantification of NFT and SP using a computer-assisted image analysis system.


Correlation between first disease-free interval from mastectomy to second disease-free interval from chest wall resection.

Between 24 November 1977 and 16 September 1988, 18 consecutive chest wall resections for recurrent breast cancer after failure of radiotherapy, were evaluated as of 1 January 1990. Chest wall involvement was the only site of recurrence in 14 patients (Group I), and the most painful of the multiple recurrences in the remaining four (Group II). Of Group I, chest wall recurrence was local in eight patients (four with necrosis after radiotherapy), regional in four, and distant in two. Chest wall reconstruction was effected by contralateral breast flap in six, by random cutaneous flap in seven and by myocutaneous flap in the remaining five. Cosmetic results were better if both marlex mesh and myocutaneous flap were used. Of Group I, at surgical/pathological staging, one recurrence with sarcomatous findings, two multiple recurrences and residual cancer in all necrosed local recurrences were found: in three of these cases radionecrosis was prominent. Mortality was 0% and surgical morbidity 5%. For Group I, median disease-free interval from mastectomy was extended from 1611 days to 3220 by recurrence resection, and disease-free interval from chest wall resection was 28% cancer-free at 1657 days, without any difference between the local vs regional-distant recurrence. Correlation factor between first and second disease interval was 0.99 and R2 was 0.98. For Group II, survival was 0% at 635 days. Chest wall resection must be considered as an important part of palliative treatment in breast cancer, but the results reflects the biology of the disease more than the chest wall surgery.


Adenomatoid tumours: a mucin histochemical and immunohistochemical study.

We have examined 16 cases of adenomatoid tumour using a panel of monoclonal and polyclonal antibodies and also stained them for the presence of hyaluronidase-sensitive alcianophilic material. Fourteen cases expressed cytokeratins and a proportion of these also expressed S-100 protein, neuron-specific enolase, vimentin, and human milk fat globule protein 2. The same 14 cases also showed hyaluronidase-sensitive staining with alcian blue. No expression of factor VIII-related antigen was seen in these cases. We conclude that this provides further evidence of a mesothelial origin of these tumours. The remaining two cases did not express cytokeratins and no hyaluronidase-sensitive staining with alcian blue was seen. They did however express factor VIII-related antigen. Although they were morphologically indistinguishable from the other 14 cases, we suggest that they should be more properly regarded as angiomas.


Specificity and protective activity of murine monoclonal antibodies directed against the capsular polysaccharide of type III group B streptococci.

We have obtained 41 monoclonal antibodies directed against type III group B streptococci by immunizing Balb/c mice with formalin-killed bacteria. All of these antibodies reacted with purified type-specific carbohydrate by enzyme-linked immunosorbent assay and immunoprecipitation tests. The epitope recognized by all of these antibodies was associated with terminal sialic acid residues, as indicated by abrogation of immune reactions by treatment of the type-specific carbohydrate with neuraminidase. Two purified monoclonal antibodies (the IgM P9D8 and the IgG3 P4F12) were further characterized for their protective activity in a neonatal rat model of infection. P9D8 and P4F12 antibodies were significantly protective when administered in a dose of 0.5 and 2.5 mg/kg, respectively, at the same time as 3 x 10(5) colony forming units of type III streptococci. Protection was still observed when the antibodies were given up to 9 h after challenge. No protection was afforded against infections with type Ia/c and II streptococci. Similarly, both antibodies effectively opsonized type III, but not Ia, Ib or II bacteria, in an in vitro assay. These and similar, previously described, monoclonal antibodies may be useful, possibly after "humanization" by genetic engineering, for the therapy of neonatal group B streptococcal infections.


Immunohistochemical study on the pattern of 50 kd cytokeratin in psoriasis.

Three biopsies of normal skin and 15 biopsies collected from patients with psoriasis vulgaris were analyzed for the expression of the 50 kd cytokeratin using direct immunofluorescence and ABC technique before and after local treatment with anthralin 0.1% in a petrolatum base, with 0.05% betamethasone dipropionate cream, and finally, after PUVA treatment. Antiserum against the 50 kd anti-cytokeratin reacted with tissue sections of normal skin, staining cells in the basal layer, while the psoriatic skin sections before the various treatments showed a staining concerning the whole thickness of the epidermis. After the various therapies, the 50 kd cytokeratin immunoreactivity was observed only in the basal layer of those psoriatic skin sections that showed complete clinical clearing, while it was observed in the whole thickness of psoriatic patches that did not clear. These data suggest that the normalization of the 50 kd cytokeratin expression pattern can be considered as a marker of clinical remission of psoriasis.


The Hsp56 component of steroid receptor complexes binds to immobilized FK506 and shows homology to FKBP-12 and FKBP-13.

Extracts from human Jurkat cells or from calf thymus contain a 60-kDa protein that is bound to immobilized FK506. As expected, the NH2-terminal sequences of the 60-kDa protein from these two species were found to be nearly the same. We were surprised to discover, however, that the sequence of the human protein was identical to that of Hsp56, a heat shock protein of unknown function that has been shown to be a component of several steroid receptor complexes. Further analysis of the calf thymus protein revealed a peptide with homology to a region near the COOH terminus of both FKBP-12 and FKBP-13. It would appear, therefore, that this 60-kDa protein, or as we refer to it provisionally, "Hsp56," could have the capacity to bind FK506 directly. These observations lead us to speculate that "Hsp56" may mediate immunosuppression and inhibition of T-cell proliferation by FK506 and may do so via a cytosolic signal transduction pathway separate, but not necessarily exclusive, from that of FKBP-12 and FKBP-13.


Lithium inhibits hepatic gluconeogenesis and phosphoenolpyruvate carboxykinase gene expression.

Incubation of isolated hepatocytes from fasted rats with 20 mM LiCl for 1 h decreased glucose production from lactate, pyruvate, and alanine. In addition, phosphoenolpyruvate carboxykinase (PEPCK) gene expression in FTO-2B rat hepatoma cells was inhibited by treatment with LiCl. Lithium was also able to counteract the increased PEPCK mRNA levels caused by both Bt2cAMP and dexamethasone, in a concentration-dependent manner. A chimeric gene containing the PEPCK promoter (-550 to +73) linked to the amino-3-glycosyl phosphotransferase (neo) structural gene was transduced into FTO-2B cells using a Moloney murine leukemia virus-based retrovirus. In these infected cells, 20 mM LiCl decreased both the concentration of neo mRNA transcribed from the PEPCK-neo chimeric gene and mRNA from the endogenous PEPCK gene. Lithium also inhibited the stimulatory effect of Bt2cAMP and dexamethasone on both genes. The stability of neo mRNA was not altered by lithium, since in cells infected with retrovirus containing only the neo gene transcribed via the retroviral 5'-LTR and treated with 20 mM LiCl, no change in neo mRNA levels was observed. The intraperitoneal administration of LiCl to rats caused a decrease in hepatic PEPCK mRNA, indicating that lithium could also modify gene expression in vivo. The effects of lithium were not due to an increase in the concentration of insulin in the blood but were correlated with an increase in hepatic glycogen and fructose 2,6-bisphosphate levels. These results indicate that lithium ions, at concentrations normally used therapeutically for depression in humans, can inhibit glucose synthesis in the liver by a mechanism which can selectively modify the expression of hepatic phosphoenolpyruvate carboxykinase.


Modulation of the mitochondrial megachannel by divalent cations and protons.

In patch-clamp experiments on rat liver mitoplasts, the 1.3 nanosiemens (in 150 mM KCl) mitochondrial megachannel was activated by Ca2+ and competitively inhibited by Mg2+, Mn2+, Ba2+, and Sr2+. Cyclosporin A, which inhibits the megachannel, also showed a competitive behavior versus Ca2+. The pore is regulated by pH in the physiological range; lower pH values cause its closure in a Ca(2+)-reversible manner. The modulating sites involved in these effects are located on the matrix side of the membrane. As illustrated in the companion paper (Bernardi, P., Vassanelli, S., Veronese, P., Colonna, R., Szabó, I., and Zoratti, M. (1992) J. Biol. Chem. 267, 2934-2939), the calcium-induced permeability transition of mitochondria is affected by these various agents in a similar manner. The results support the identification of the megachannel with the pore believed to be involved in the permeabilization process. The kinetic characteristics of the single channel events support the idea that the megachannel is composed of cooperating subunits.


Isolation and characterization of a cDNA clone encoding a cognate 70-kDa heat shock protein of the chloroplast envelope.

The translocation of proteins into the endoplasmic reticulum, the mitochondrion, and the chloroplast has recently been shown to involve homologues of the highly conserved 70-kDa heat shock protein (HSP70) family. In this study, we have isolated and sequenced a full-length cDNA clone encoding a cognate 70-kDa heat shock protein of the spinach chloroplast envelope (SCE70). The cDNA insert is 2,535 base pairs long and codes for 653 amino acid residues of a protein with a predicted molecular mass of 71,731 daltons. The deduced amino acid sequence shows a high degree of homology with HSP70 proteins from other organisms. Southern genomic and RNA analyses reveal different hybridization patterns than that observed for a heat-inducible 70-kDa protein gene. The protein synthesized from the SCE70 cDNA insert co-migrates with a 70-kDa polypeptide of the chloroplast envelope following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blot analysis and import studies indicate that SCE70 is associated with the chloroplast outer envelope. The import data suggest that SCE70 is targeted to the envelope membrane via a pathway different from other plastidic precursors but similar to that recently reported for outer envelope proteins SOE1 and OM14.


Anion uniport in plant mitochondria is mediated by a Mg(2+)-insensitive inner membrane anion channel.

It has long been established that the inner membrane of plant mitochondria is permeable to Cl-. Evidence has also accumulated which suggests that a number of other anions such as Pi and dicarboxylates can also be transported electrophoretically. In this paper, we present evidence that anion uniport in plant mitochondria is mediated via a pH-regulated channel related to the so-called inner membrane anion channel (IMAC) of animal mitochondria. Like IMAC, the channel in potato mitochondria transports a wide variety of anions including NO3-, Cl-, ferrocyanide, 1,2,3-benzene-tricarboxylate, malonate, Pi, alpha-ketoglutarate, malate, adipate, and glucuronate. In the presence of nigericin, anion uniport is sensitive to the medium pH (pIC50 = 7.60, Hill coefficient = 2). In the absence of nigericin, transport rates are much lower and much less sensitive to pH, suggesting that matrix H+ inhibit anion uniport. This conclusion is supported by measurements of H+ flux which reveal that "activation" of anion transport at high pH by nigericin and at low pH by respiration is associated with an efflux of matrix H+. Other inhibitors of IMAC which are found to block anion uniport in potato mitochondria include propranolol (IC50 = 14 microM, Hill coefficient = 1.28), tributyltin (IC50 = 4 nmol/mg, Hill coefficient = 2.0), and the nucleotide analogs Erythrosin B and Cibacron Blue 3GA. The channel in plant mitochondria differs from IMAC in that it is not inhibited by matrix Mg2+, mercurials, or N,N'-dicyclohexylcarbodiimide. The lack of inhibition by Mg2+ suggests that the physiological regulation of the plant channel may differ from IMAC and that the plant IMAC may have functions such as a role in the malate/oxaloacetate shuttle in addition to its proposed role in volume homeostasis.


Ion channels are likely to be involved in the two steps of phage T5 DNA penetration into Escherichia coli cells.

Phage T5 injects its DNA into Escherichia coli cells in two steps; 8% of the chromosome is first injected, and then there is a pause during which proteins encoded by this DNA fragment are synthesized allowing the remaining DNA to be injected. Using a potassium-selective electrode, we show that the injection of the two DNA fragments is associated with an efflux in two steps, of cytoplasmic potassium. The rate of efflux is linearly related to the number of added phages suggesting that each phage induces the formation of at least one channel in the inner membrane. The first efflux occurs even in depolarized cells suggesting that the insertion and the opening of the channel can take place in the absence of the electrochemical gradient of protons (delta mu H+). The channel is in a closed configuration during the time required for the synthesis of the phage-encoded proteins; this closing and the second efflux are prevented by the depolarization of the cell. The insertion of the channel in the inner membrane requires a fluid membrane. The results obtained suggest that the function of this channel is to translocate phage T5 DNA.


Identification of the transcriptionally active genes of the chorionic gonadotropin beta gene cluster in vivo.

The chorionic gonadotropin beta (CG beta) subunit is encoded by a multigene cluster composed of six homologous sequences (genes or pseudogenes). They are primarily distinguished by sequences in the 5' nontranslated region of the first exon. To determine which CG beta genes are active in vivo, we employed the reverse transcription-polymerase chain reaction technique. DNA complementary to RNA from placenta tissue and a choriocarcinoma cell line was subjected to polymerase chain reaction with CG beta-specific primers. The amplified DNA was cloned into M13 and sequenced. Most of steady-state CG beta mRNAs are transcribed from CG beta genes, 5, 3, and 8. The level of expression is beta 5 greater than beta 3 = beta 8 greater than beta 7, beta 1/2. Transcripts from the CG beta 1 and CG beta 2 genes, which were previously considered pseudogenes because of their noncanonical splice site, were detected. These CG beta transcripts arising from alternative splicing sites in the CG beta 1/2 genes were also detected in polysomes which suggests they are translation-competent. Northern blotting with a CG beta 1/2 probe revealed low amounts of CG beta 1/2 transcripts which were smaller than the transcripts of the other genes. The results indicate that at least five, and possibly all six, CG beta genes are transcribed in vivo, and at least one of them (CG beta 1 and/or CG beta 2) undergoes alternative splicing.


Human lymphocytes transcribe the cystic fibrosis transmembrane conductance regulator gene and exhibit CF-defective cAMP-regulated chloride current.

Cystic fibrosis (CF) is the most common lethal genetic disease among Caucasians, primarily affecting epithelial tissues of the lung and gut. Mutations in a single gene, the cystic fibrosis transmembrane conductance regulator (CFTR), are responsible for this disease. Whether a physiological defect exists in the immune system of CF patients has remained controversial. A chloride ion transport defect has been described in human CF-derived lymphocytes; however, it has not been possible to detect CFTR mRNA in lymphocytes. We report here that normal human B-lymphoblasts display whole cell Cl- conductances induced by calcium-mediated pathways, volume regulation, and cAMP which are equivalent to currents described in epithelial cells. B-lymphoblasts from CF-affected humans demonstrated defective Cl- conductance regulation by cAMP but preserved regulation by calcium-mediated and volume regulation mechanisms. CFTR involvement in cAMP regulation of Cl- conductance in lymphocytes is further supported by our demonstration of the presence of appropriately spliced CFTR mRNA segments in human B and T lymphocytes as detected by an optimized reverse-transcription and polymerase chain reaction approach. The identity of the amplified products was confirmed by hybridization to CFTR-specific probes and DNA sequencing. Furthermore, the 3'-end of the gene was found in a T cell cDNA library. We conclude that CFTR mRNA is expressed in lymphocytes, consistent with the cAMP regulation of chloride transport present in normal lymphocytes but defective in CF-derived lymphocytes.


Characterization of mouse thrombospondin 2 sequence and expression during cell growth and development.

Thrombospondin (TSP) is an extracellular matrix glycoprotein whose expression has been associated with a variety of cellular processes including growth and embryogenesis. The recent discovery of the existence of a second mouse TSP gene necessitates careful examination of the discrete biochemical and functional properties associated with each molecule. In this report, the primary structures of human TSP, mouse TSP1 (mTSP1), mouse TSP2 (mTSP2), and chicken TSP are compared; and the expression of mTSP1 and mTSP2 during embryogenesis and growth factor-mediated cell proliferation is examined. The cloning and sequencing of the entire coding regions of mTSP1 and mTSP2 revealed considerable conservation of residues critical for TSP structure and function; these data suggest that TSP2 is capable of trimer formation and many of the same cell-surface and ligand interactions that mediate TSP function. Comparison of the various TSP sequences also allowed the assignment based on sequence homology of previously reported human TSP as TSP1 and chicken TSP as TSP2. mTSP2, like mTSP1, was shown to be a primary response gene when quiescent Swiss 3T3 cells were stimulated with serum, platelet-derived growth factor BB, basic fibroblast growth factor, or interleukin-1 beta. Interestingly, TSP1 and TSP2 exhibited markedly different tissue- and stage-specific patterns of mRNA expression during mouse embryogenesis, implying that the two TSP molecules possess discrete functional properties important for development. Additionally, the TSP genes (Thbs1 and Thbs2) were mapped to single loci on mouse chromosomes 2 and 17, respectively.


Simultaneous deficiency of sphingolipid activator proteins 1 and 2 is caused by a mutation in the initiation codon of their common gene.

Sphingolipid activator proteins (SAPs) are small, nonenzymic glycoproteins that stimulate lysosomal degradation of various sphingolipids. SAP-1, SAP-2, and two additional potential activator proteins are derived from a common precursor by proteolytic processing. A severe case of sphingolipid storage disease that led to death within 16 weeks was attributed to a possible total deficiency of the SAPs generated by this gene (Harzer, K., Paton, B. C., Poulos, A., Kustermann-Kuhn, B., Roggendorf, W., Grisar, T., and Popp, M. (1989) Eur. J. Pediatr. 149, 31-39). Analysis of the SAP precursor cDNA from the patient and his fetal sibling showed an A to T transversion in the initiation codon. Allele-specific oligonucleotide hybridization revealed that both parents are heterozygous carriers for this mutation. In pulse-chase experiments using antisera raised against SAP-1 or SAP-2, no cross-reacting material could be detected in the patients' fibroblasts.


PPIase catalysis by human FK506-binding protein proceeds through a conformational twist mechanism.

FK506-binding protein (FKBP) catalyzes the cis-trans isomerization of the peptidyl-prolyl amide bond (the PPIase reaction) and is the major intracellular receptor for the immunosuppressive drugs FK506 and rapamycin. One mechanism proposed for catalysis of the PPIase reaction requires attack of an enzyme nucleophile on the carbonyl carbon of the isomerized peptide bond. An alternative mechanism requires conformational distortion of the peptide bond with or without assistance by an enzyme hydrogen bond donor. We have determined the kinetic parameters of the human FKBP-catalyzed PPIase reaction. At 5 degrees C, the isomerization of Suc-Ala-Leu-Pro-Phe-pNA proceeds in 2.5% trifluorethanol with kcat = 600 s-1, Km = 0.5 mM and kcat/Km = 1.2 x 10(6) M-1s-1. The kcat/Km shows little pH dependence between 5 and 10. A normal secondary deuterium isotope effect is observed on both kcat and kcat/Km. To investigate dependence on enzyme nucleophiles and proton donors, we have replaced eight potential catalytic residues with alanine by site-directed mutagenesis. Each FKBP variant efficiently catalyzes the PPIase reaction. Taken together, these data support an unassisted conformational twist mechanism with rate enhancement due in part to desolvation of the peptide bond at the active site. Fluorescence quenching of the buried tryptophan 59 residue by peptide substrate suggests that isomerization occurs in a hydrophobic environment.


Positive and negative control of the skeletal alpha-actin promoter in cardiac muscle. A proximal serum response element is sufficient for induction by basic fibroblast growth factor (FGF) but not for inhibition by acidic FGF.

Like mechanical load in vivo, basic fibroblast growth factor (bFGF) selectively provokes cardiac expression of "fetal" genes including skeletal alpha-actin (SkA). Antithetically, acidic FGF (aFGF) suppresses SkA transcription. To define sites controlling SkA transcription in cardiac muscle cells, rat cardiac myocytes were transfected with internal-deletion and block-substitution mutations in the SkA promoter, including three motifs resembling the fos serum response element (SRE). The upstream, central, and proximal SREs each contributed to basal expression in cardiac myocytes. To determine whether identical elements mediate induction by bFGF versus inhibition by aFGF, the proximal SRE (SRE1) and fos SRE were positioned upstream from a neutral promoter. In cardiac myocytes, both the SRE1 and fos SRE were expressed at levels up to one-third that of the SkA promoter (nucleotides -202 to -11). Neither was expressed in quiescent cardiac fibroblasts. bFGF augmented SRE1-CAT activity, whereas aFGF produced no change; the fos SRE was induced by both. The transcriptional and mitogenic actions of aFGF were contingent on the presence of a putative nuclear translocation motif. Thus 1) the SkA SRE1 and fos SRE each suffice for tissue specificity in cardiac myocytes; 2) unlike the c-fos SRE, the SkA SRE1 is induced selectively by bFGF yet not aFGF; 3) sequences alternative or in addition to the SRE1 are obligatory for aFGF to suppress the SkA promoter; and 4) possible differences in intracellular localization are one basis for divergent actions of aFGF and bFGF in cardiac muscle cells.


Structural and functional characterization of an inositol polyphosphate receptor from cerebellum.

An inositol polyphosphate receptor has been purified from bovine cerebellum which consists of three different polypeptides with Mr of 111,000, 102,000, and 52,000. Negative staining electron microscopy reveals globular-like structures 10-13 nm in diameter. The receptor has a Stokes radius of 400,000 daltons as determined by molecular sieve high performance liquid chromatography. The receptor preparation binds inositol 1,3,4,5-tetrakisphosphate, inositol hexaphosphate (or phytol), and inositol 1,4,5-trisphosphate (IP4, IP6, and IP3, respectively) with submicromolar affinity (0.19, 0.15, and 0.54 microM, respectively) at conditions approximating physiological ionic strength and pH. The purified receptor preparation, when reconstituted into planar bilayers, displays ion channel activity, preferentially permeable to K+. Permeability ratios of the channel are PK+/PNa+ approximately 5 and PK+/PCl approximately 19. In symmetrical 100 mM KCl, the channel is characterized by long open times (minutes) with a conductance of 7.2 picosiemens. The channel is selectively modulated by IP4. That is, at 1 microM IP4, the mean open time decreased substantially to rapid flicker behavior and the channel is completely closed at 10 microM IP4. IP6 and IP3 did not modulate the channel under similar conditions. Thus, the channel appears to be an IP4-modulated K+ channel.


Hormone withdrawal triggers a premature and sustained gene activation from delayed secondary glucocorticoid response elements.

Glucocorticoid regulatory elements, denoted GREs and delayed secondary GREs (sGREs), bind the purified glucocorticoid receptors via distinctive sequence motifs and confer a primary and delayed secondary hormone inducibility, respectively, upon a linked reporter construct in stably transfected mammalian cells. The delayed secondary responses, but not the primary responses, are preceded by a time lag of several hours and blocked by protein synthesis inhibitors. In this report, we further characterized and distinguished these hormonal inductions. A 206-base pair DNA fragment from the hepatic rat alpha 2u-globulin (RUG) gene, containing at least two delayed sGREs, was specifically activated by glucocorticoids in a dose-dependent manner via a process which is sensitive to receptor antagonist RU486. Delayed sGRE-stimulated production of correctly initiated transcripts was preceded by a time lag of 2 h, a time when the GRE-mediated induction had reached maximal levels. A pulse of glucocorticoids sustained maximal activation of the delayed secondary response but not the primary response. In fact, hormone withdrawal triggered a premature induction of this delayed secondary response, suggesting that delayed sGREs are under both negative and positive control of the hormone receptor. Two separable elements of the 206-base pair fragment, including the 29-base pair sequence of a single receptor binding site, activated the reporter expression as effectively with transient, pulsatile exposure to hormone as with continuous exposure. Our results suggest that the information content of a hormonal pulse is retained, or "memorized," more persistently by a receptor binding site of delayed sGREs than those of the prototypical GREs.


Ligand-regulated internalization and recycling of human beta 2-adrenergic receptors between the plasma membrane and endosomes containing transferrin receptors.

Agonist-regulated redistribution of human beta 2-adrenergic receptors was examined in 293 cells. A specific antiserum recognizing the carboxyl-terminal hydrophilic domain of the receptor was developed, characterized, and used for immunocytochemical localization of receptors in fixed cells by conventional fluorescence and confocal fluorescence microscopy. The beta-adrenergic agonist isoproterenol induced redistribution of receptors from the surface of cells into small (less than 1 micron diameter) punctuate accumulations which were detected in cells within 2 min of agonist addition. The time course of receptor redistribution paralleled that of receptor sequestration measured by ligand binding, and receptor redistribution was reversible in the presence of the beta-adrenergic antagonist alprenolol. Optical sections imaged through cells by confocal microscopy localized receptor accumulations within the cytoplasm. To address the question of receptor internalization further, a mutant receptor possessing an engineered antigenic epitope in the amino-terminal hydrophilic domain was constructed, transfected into cells, and localized using both a monoclonal antibody recognizing the epitope tag (receptor ectodomain) and an antiserum recognizing the carboxyl terminus (receptor endodomain). In untreated cells most receptor antigen was detected at the cell surface, as assessed by accessibility to ectodomain antibodies in unpermeabilized specimens. In isoproterenol-treated cells, however, little receptor antigen was detected at the cell surface. Punctate receptor accumulations present in isoproterenol-treated cells were labeled by antibodies only following permeabilization of cells, as expected if these receptor accumulations were intracellular. Finally, internalized beta-adrenergic receptors colocalized with transferrin receptors, which are markers of endosomal membranes. These data provide several lines of evidence establishing that beta-adrenergic receptors undergo ligand-regulated internalization, they suggest that internalized receptors may be recycled back to the cell surface, and they provide the first direct indication that these processes involve the same endosomal membrane system passaged by constitutively recycling receptors.


Pediatric psychopharmacotherapy: a review of recent research.

In the past 5 years, we have witnessed the continuation of important trends in clinical research that began earlier in the decade. With regard to the treatment of specific disorders in children and adolescents, the most significant developments have been the examination of the tricyclics for the treatment of depression and the initiation of controlled studies for the treatment of Tourette syndrome. Unfortunately, the findings from the depression studies have been uniformly negative, and the results of research on both depression and tic disorders show a relatively high rate of placebo responsivity, which raises nagging questions about the role of case reports and open trials. Another important trend in pediatric psychopharmacotherapy is the search for substitutes for the neuroleptics. Potential candidates include agents such as lithium, naltrexone, fenfluramine, clonidine, and carbamazepine. The most underresearched disorders are a combination of the least common (e.g. schizophrenia, mania) and those that are apparently perceived as less serious (e.g. sleep disorders, certain anxiety disorders). Not surprisingly, the most studied disorder and treatment is hyperactivity and stimulant medication, respectively. Considerable progress has been made in understanding the social implications of the associated symptoms and their response to stimulant drugs, aided greatly by the use of direct observation procedures. Researchers are beginning to attend to the implications of comorbidity for assessing response to medication. There has been additional confirmation of efficacy of stimulant treatment for preschoolers and adolescents. Dose-response issues remain to some extent unresolved, the primary impediments being interpretive misconceptions associated with trend analysis, an overreliance on the syndromal perspective and too little attention to target behaviors and their clinical implications, and the failure to operationalize the minimal effective dose with regard to the normalization and supranormalization of target and collateral behaviors. Disagreement over whether hyperactivity is a learning or a behavior disorder (or both) and what academic underproductivity means clinically and socially is also impeding progress. With regard to developmental disorders, controlled studies indicate that fenfluramine and naltrexone are effective for managing associated symptoms in some individuals. However, given the limited amount of research on these agents, their status as clinically useful palliatives must be considered tentative.(ABSTRACT TRUNCATED AT 400 WORDS)


Physicochemical and physiological properties of cholylsarcosine. A potential replacement detergent for bile acid deficiency states in the small intestine.

The properties of cholylsarcosine (the synthetic N-acyl conjugate of cholic acid with sarcosine [N-methylglycine]) were examined to determine its suitability as a bile acid replacement agent for conditions of bile acid deficiency in the small intestine, which causes fat malabsorption. Previous studies in rodents had shown that the compound was well transported by the liver and ileum and underwent neither deconjugation nor dehydroxylation during enterohepatic cycling. By 1H-nuclear magnetic resonance, cholylsarcosine was found to exist in dilute aqueous solution as an almost equimolar mixture of two geometric isomers--cis and trans (around the amide bond)--in contrast to cholylglycine, which was present entirely in the trans form. The critical micellization concentration was 11 mmol/liter, similar to that of cholylglycine (10 mmol/liter). By nonaqueous titrimetry, the pKa' of cholylsarcosine was 3.7, only slightly lower than that of cholylglycine (3.9). Cholylsarcosine was poorly soluble below pH 3.7, but highly soluble above pH 4. In vitro, cholylsarcosine behaved as cholylglycine with respect to promoting lipolysis by lipase/colipase. There was little difference between cholylsarcosine and cholylglycine in their solubilization of an equimolar mixture of oleic acid, oleate, and monoolein (designed to simulate digestive products of triglyceride) or in their solubilization of monooleyl-glycerol alone. When a [3H]triolein emulsion with either cholylsarcosine or cholyltaurine was infused intraduodenally in biliary fistula rats, recovery of 3H in lymph was 52 +/- 10% (mean +/- SD) for cholylsarcosine and 52 +/- 11% for cholyltaurine. When perfused into the colon of the anesthetized rabbit, cholylsarcosine (5 mmol/liter) did not influence water absorption or permeability to erythritol, in contrast to chenodeoxycholate, which induced vigorous water secretion and caused erythritol loss. We conclude that cholylsarcosine possesses the physicochemical and physiological properties required for a suitable bile acid replacement in deficiency states.


Binding of Pseudomonas cepacia to normal human intestinal mucin and respiratory mucin from patients with cystic fibrosis.

Although not as prevalent as Pseudomonas aeruginosa, Pseudomonas cepacia is another opportunistic pathogen which colonizes the lungs of at least some patients with cystic fibrosis. A subgroup of these patients exhibits the "cepacia syndrome", i.e., a rapid clinical deterioration and death within one year. To investigate potential early sites of bacterial attachment, we have measured the specific binding of P. cepacia isolates from cystic fibrosis (CF) sputa to both CF and non-CF mucins purified from respiratory and intestinal secretions, respectively. As shown in microtiter binding assays, clinical isolates from 19/22 patients were found to bind to both mucins, with the highest specific binding exhibited by isolates from eight patients, seven of whom later died with the cepacia syndrome. No differences were observed in the binding capacity of the two (CF versus non-CF) mucins. Binding was specific, saturable, and not influenced by tetramethylurea, a disruptor of hydrophobic associations. Individual sugars were ineffective as hapten inhibitors, as were several lectins. Mucins treated by reduction/alkylation or chloroform/methanol extraction showed enhanced bacterial binding, findings which were attributed to exposure of underlying binding sites. Deglycosylation procedures indicated that mucin receptors for P. cepacia include N-acetylglucosamine and N-acetylgalactosamine, probably linked together as part of core oligosaccharide structures. P. cepacia isolates also bound to buccal epithelial cells, and mucin partially inhibited the binding of those isolates of P. cepacia that also had the ability to bind to mucin. We speculate that specific binding of P. cepacia to secreted mucins may be an early step in the pathogenesis of the cepacia syndrome.


Basic fibroblast growth factor enhances the coupling of intimal hyperplasia and proliferation of vasa vasorum in injured rat arteries.

Basic fibroblast growth factor (bFGF) is mitogenic for smooth muscle cells (SMC) and angiogenic. We examined the in vivo effects of bFGF in balloon denuded carotid arteries of laboratory rats. bFGF was administered continuously from polymer-based devices at 34 ng/d into the periadventitial space of rat carotid arteries for 2 wk. Intimal hyperplasia was not observed in the absence of injury or with lipopolysaccharide induced endothelial dysfunction. Different degrees of vascular injury produced proportionally more intimal hyperplasia. bFGF increased the intimal hyperplastic response 1.3-fold with severe vascular injury, and 2.4-fold with more mild injury. Increased cell proliferation, not extracellular matrix production, accounted for these effects. Cell density was unchanged for the control and bFGF-treated groups, and the number of proliferating intimal cells at 2 wk rose to an amount equivalent to the increase in mass; 1.9- and 4.0-fold for severe and lesser injury, respectively. The relative ability of heparin to reduce SMC proliferation was not altered by the presence of bFGF.bFGF also induced profound angiogenesis within and surrounding the polymeric releasing device, and in the vasa vasorum immediately around the injured arteries. bFGF's effect on vasa was linearly related to the amount of SMC proliferation within the blood vessel. Thus, the in vivo mitogenic and angiogenic potential of bFGF are coupled, and may be similarly modulated by the products of local injury and/or factors in the vessel wall.


Selective induction of rheumatoid factors by superantigens and human helper T cells.

Production of autoantibodies specific for the Fc region of autologous IgG, called rheumatoid factors (RF), is a characteristic finding in patients with rheumatoid arthritis (RA). To study the requirements regulating the synthesis of these autoantibodies, we have cloned human helper T cells and co-cultured them with purified B cells. To mimic cognate T-B cell interaction, we have used bacterial superantigens that function by cross-linking HLA molecules on the B cell with selected T cell receptor (TCR) molecules expressing a particular polymorphism of the V beta gene segment. Data presented here demonstrate that the staphylococcal enterotoxin D (SE D), but not other bacterial superantigens, exhibits an ability to induce IgM, IgG, and especially RF production, in B cells from RA patients and normal individuals. Comparison with the polyclonal antibody production in B cell cultures driven by anti-CD3-stimulated T cell clones confirmed that SE D shifted the repertoire of secreted antibodies toward immunoglobulins with Fc binding specificity, suggesting that SE D preferentially stimulates RF+ B lymphocytes. B cells with the potential to secrete RF were highly frequent in RA patients, requiring as few as 150 peripheral B cells/culture to detect RF in the culture supernatants. SE D-induced RF synthesis was strictly dependent on the presence of selected CD4+T helper cells and required a direct membrane contact between B cells and T helper cells. Here, we propose a model that SE D selectively induces RF production depending on the availability of SE D responsive T cells in the TCR repertoire of the responder.


Human postnatal thymocytes generate phenotypically immature CD3dim, CD5dim, CD1abright progeny in organ culture.

We previously described an organ culture system that supports the in vitro development of human fetal thymocytes in murine alymphoid thymic rudiments without addition of exogenous factors. This approach to study human T cell precursors is limited by the requirement for fetal tissue. We show that human thymocytes from pediatric sources can be expanded under similar conditions. Organ cultures generated predominantly CD4 CD8 double positive cells, most of which maintained the phenotype CD3dim, CD5dim, and CD1abright, characteristic for double positive thymocytes ex vivo. This culture system should facilitate in vitro studies on human thymocyte development and repertoire selection.


Cyclosporin A and FK506 mediate differential effects on T cell activation in vivo.

Modified limiting dilution analysis techniques were used to evaluate the effects of the immunosuppressants cyclosporin A (CsA) and FK506 on alloantigen-induced T cell activation in vivo. Treatment of sponge matrix allograft recipients with either CsA or FK506 inhibited lymphocytic infiltration of the allograft, a process thought to be dependent on local lymphokine production. In addition, both immunosuppressants markedly reduced the absolute number of lymphocytes recovered from the draining lymph nodes (LN) and prevented CTL activation in the LN. However, Ag-primed helper T lymphocytes (HTL) were present in the draining LN of sponge allograft recipients treated with CsA, but not in recipients treated with FK506. T cell depletion experiments were performed to determine the phenotype of primed HTL in the LN of untreated and CsA-treated sponge allograft recipients. In untreated sponge allograft recipients, CD4+ and CD8+ Ag-primed HTL were present in the draining LN in equivalent numbers. In contrast, the majority of primed HTL in the LN of CsA-treated sponge allograft recipients were CD8+, rather than CD4+ T cells. These observations indicate that CsA and FK506 exert distinct in vivo effects at the level of HTL priming, and CD4+ and CD8+ HTL exhibit differential sensitivity to CsA in vivo.


Antibody against the Leu-CAM beta-chain (CD18) promotes both LFA-1- and CR3-dependent adhesion events.

The Leukocytic cell-adhesion molecule (beta 2 integrin) family of adhesion molecules play a key role in the intercellular adhesive interactions necessary for normal immune cell function. In this study, we report an antibody that recognizes an epitope on the Leukocytic cell-adhesion molecule common beta-chain (CD18) and promotes both lymphocyte function-associated Ag-1- and CR3-dependent adhesion events. The antibody recognizes a temperature-sensitive epitope that is not dependent on the presence of divalent cations. It is proposed that antibody binding promotes a conformational change in both lymphocyte function-associated Ag-1 and CR3, which may mimic a natural activation mechanism, resulting in increased cellular adhesion.


IL-4 induces adherence of human eosinophils and basophils but not neutrophils to endothelium. Association with expression of VCAM-1.

The present studies were performed to explore potentially selective mechanisms of leukocyte adhesion in an attempt to understand how preferential recruitment of eosinophils and basophils might occur during allergic and other inflammatory reactions. Stimulation of human vascular endothelial cells for 24 h with IL-4 (30 to 1,000 U/ml) induced adhesion for eosinophils (up to approximately four-fold of control) and basophils (up to approximately twofold of control) but not neutrophils (less than 125% of control). Analysis of endothelial expression of adhesion molecules by flow cytometry revealed that IL-4 treatment induced vascular cell adhesion molecule-1 (VCAM-1) expression without significantly affecting the expression of other adhesion molecules, namely endothelial-leukocyte adhesion molecule-1 (ELAM-1) or intercellular adhesion molecule-1 (ICAM-1). The concentration-response curve for IL-4-induced VCAM-1 expression paralleled that for adhesion. Endothelial cells stimulated with IL-4 expressed adhesive properties for eosinophils by 3 h; the response increased steadily during a 24-h time course study. Eosinophils and basophils adhered to plates coated with a recombinant form of VCAM-1. This adhesion was blocked with antibodies to VCAM-1 but not ELAM-1. mAb directed against either VCAM-1 or VLA-4 inhibited (by approximately 75%) the binding of eosinophils and basophils to IL-4-stimulated endothelial cells. Because VLA-4 and VCAM-1 have been demonstrated to bind to each other in other adhesion systems, these results suggest that IL-4 stimulates eosinophil and basophil adhesion by inducing endothelial cell expression of VCAM-1 which binds to eosinophil and basophil VLA-4. The lack of expression of VLA-4 on neutrophils and the failure of IL-4 to stimulate neutrophil adherence support this conclusion. It is proposed that local release of IL-4 in vivo in allergic diseases or after experimental allergen challenge may partly explain the enrichment of eosinophils and basophils (vs neutrophils) observed in these situations.


Role of LFA-1 and VLA-4 in the adhesion of cloned normal and LFA-1 (CD11/CD18)-deficient T cells to cultured endothelial cells. Indication for a new adhesion pathway.

Patients with the leukocyte adhesion deficiency (LAD) syndrome have a genetic defect in the common beta 2-chain (CD18) of the leukocyte integrins. This defect can result in the absence of cell surface expression of all three members of the leukocyte integrins. We investigated the capacity of T cell clones obtained from the blood of an LAD patient and of normal T cell clones to adhere to human umbilical vein endothelial cells (EC). Adhesion of the number of LAD T cells to unstimulated EC was approximately half of that of leukocyte function-associated antigen (LFA)-1+ T cells. Stimulation of EC with human rTNF-alpha resulted in an average 2- and 2.5-fold increase in adhesion of LFA-1+ and LFA-1- cells, respectively. This effect was maximal after 24 h and lasted for 48 to 72 h. The involvement of surface structures known to participate in cell adhesion (integrins, CD44) was tested by blocking studies with mAb directed against these structures. Adhesion of LFA-1+ T cells to unstimulated EC was inhibited (average inhibition of 58%) with mAb to CD11a or CD18. Considerably less inhibition of adhesion occurred with mAb to CD11a or CD18 (average inhibition, 20%) when LFA-1+ T cells were incubated with rTNF-alpha-stimulated EC. The adhesion of LFA-1- T cells to EC stimulated with rTNF-alpha, but not to unstimulated EC, was inhibited (average inhibition, 56%) by incubation with a mAb directed to very late antigen (VLA)-4 (CDw49d). In contrast to LAD T cell clones and the LFA-1+ T cell line Jurkat, mAb to VLA-4 did not inhibit adhesion of normal LFA-1+ T cell clones to EC, whether or not the EC had been stimulated with rTNF-alpha. We conclude that the adhesion molecule pair LFA-1/intercellular adhesion molecule (ICAM)-1 plays a major role in the adhesion of LFA-1+ T cell clones derived from normal individuals to unstimulated EC. Adhesion of LFA-1-T cells to TNF-alpha-stimulated EC is mediated by VLA-4/vascular cell adhesion molecule (VCAM)-1 interactions. Since we were unable to reduce significantly the adhesion of cultured normal LFA-1+ T cells to 24 h with TNF-alpha-stimulated endothelium with antibodies that block LFA-1/ICAM-1 or VLA-4/VCAM-1 interactions, and lectin adhesion molecule-1 and endothelial leukocyte adhesion molecule-1 appeared not to be implicated, other as yet undefined cell surface structures are likely to participate in T cell/EC interactions.


Immunization with antigen bound to bispecific antibody induces antibody that is restricted in epitope specificity and contains antiidiotype.

Mice can be efficiently immunized in the absence of adjuvant using chemically cross-linked bispecific antibody (biAb) that bind to both class II MHC molecules and a protein Ag of interest. In our experiments, mice were immunized with the protein Ag hen egg lysozyme (HEL) bound to several different biAb, each of which contained a different mAb specific for a distinct (nonoverlapping) epitope of HEL. Primary and secondary serum antibody responses of the immunized mice were analyzed for their specificity for different epitopes of HEL. The results show that immunization with each HEL-biAb complex produced a bias in the epitope specificity of the primary antibody response. This bias was determined by the individual specificity of the anti-HEL mAb used in each biAb. The primary response was dominated by antibody reacting with epitopes distinct from that bound by the mAb in the immunizing complex, and was deficient in antibody that recognized the epitope bound by the biAb during immunization. This bias in antibody specificity was maintained during the secondary antibody response that followed a single challenge with soluble HEL alone. However, an additional challenge with HEL induced a switch in the specificity pattern, with increased amounts of antibody against the epitope that was previously ignored. In addition, immunization with Ag bound to biAb resulted in a substantial primary anti-Id response, detected by serum antibody specific only for the Fab'2 fragment of the mAb used in the biAb. These studies illustrate two unique features of immunization using biAb that allow for fine manipulation of the epitope specificity and anti-Id repertoires of the antibody response to whole protein Ag.


Stimulation of human monocytes via CD45, CD44, and LFA-3 triggers macrophage-colony-stimulating factor production. Synergism with lipopolysaccharide and IL-1 beta.

Stimulation of human monocytes with immobilized mAb directed against the CD45, CD44, or LFA-3 Ag induced the production of macrophage-CSF (M-CSF). M-CSF-specific transcripts appeared at 3 h poststimulation, were further increased by 12 h, and were still detectable at 24 h. M-CSF gene expression was accompanied by the induction of small but detectable amounts of M-CSF protein. LPS and IL-1 beta, but not IL-6 or TNF-alpha, dramatically augmented the ability of anti-CD45, anti-CD44, and anti-LFA-3 antibodies to induce M-CSF secretion but failed to stimulate M-CSF secretion in the absence of antibody. M-CSF activity in the culture supernatants was first detectable at 8 h of culture, peaked at day 2, and declined thereafter. Purified F(ab)2 fragments of anti-CD45 antibody were also effective in inducing M-CSF message and secretion, indicating that the Fc gamma RII is not involved in this response. Stimulation of cells with antibodies to the monocyte surface Ag MAC-1, LFA-1, and ICAM-1 did not result in M-CSF secretion. Moreover, LPS and IL-1 beta failed to synergize with these Ag in inducing M-CSF production. Together, these results indicate that stimulation of monocytes via the cell surface Ag CD45, CD44, and LFA-3 can trigger M-CSF production. However, second signals that can be provided by IL-1 beta or LPS are required to regulate optimal M-CSF protein secretion.


Comparative study of the T cell response to two allelic forms of a malarial vaccine candidate protein.

T cell responses to two allelic forms of the merozoite surface Ag 2 (MSA2) of Plasmodium falciparum were mapped in mice using the rMSA2 proteins, Ag 1609 which has the sequence of the FCQ27/PNG strain and Ag 1615 which has the sequence of the Indochina 1 strain. Lymph node cells of BL/10 and B10.BR mice immunized with either Ag 1609 or Ag 1615 responded to both Ag in in vitro proliferation assays. Lymph node cells of BALB/c mice did not respond. The T cell determinants recognized by the responder strains were mapped to conserved and variant regions of these Ag using overlapping synthetic peptides. The determinants recognized by each mouse strain were distinct. Marked difference in sequence between the central regions of the two rMSA2 proteins did not affect antigenic processing of the conserved N and C terminal regions. Hence lymph node cells of BL/10 mice immunized with either Ag 1615 or Ag 1609 recognized an immunodominant T cell determinant at the highly conserved N terminal end within the sequence YSNTFINNAYNMSIR (peptide 3b) and B10.BR mice similarly immunized recognized an immunodominant determinant at the highly conserved C terminal within the sequence CTDGNKENCGAATSL (peptide 23). Several peptides identified as containing immunodominant T cell determinants specific to BL/10 mice induced peptide-specific T cells in both BL/10 and B10.BR mouse strains when used as immunogens. However, the ability of the peptide-primed T cells to proliferate in response to the rMSA2 proteins was confined to BL/10 mice. An example of this was observed with peptides 3b and N (KNESKYSNTFINNAYNMSIRRSMAN). Peptide N was able to prime B10.BR and BL/10 mice for an enhanced antibody response when these mice were subsequently immunized with Ag 1615 even though Ag 1615-specific T cell proliferation was not detected in B10.BR mice primed with N. The study concluded that 1) conserved sequences such as peptide N when used in vaccines may give rise to MSA2-specific memory Th cells amenable to boosting by subsequent exposure to all parasite strains and 2) peptide priming may be a useful pathway for inducing defined memory Th cells in a wider population and for preferentially inducing T dependent over T independent responses to some malarial Ag.


Activation of cytokine genes in HIV-1 infected myelomonoblastic cells by phorbol ester and tumor necrosis factor.

The differential production of inflammatory cytokines (IL-1 alpha, IL-1 beta, IL-6, and TNF-alpha) was analyzed in the PLB-985 myelomonoblastic cell line, chronically infected or not by the IIIB strain of HIV-1. After treatment with phorbol ester (PMA) or TNF-alpha, a 20- to 40-fold increase in the level of IL-1 beta mRNA was observed in the HIV-infected PLB-IIIB as compared with the parental PLB-985 cells. The majority of the IL-1 beta activity detected in both cell types remained cell associated. In contrast, TNF-alpha mRNA levels were increased in both infected and uninfected cells; the t1/2 of TNF RNA was 90 min in uninfected cells and 30 min in HIV-infected cells. Interestingly, about 14-fold more TNF activity was secreted from PLB-IIIB than from similarly stimulated PLB-985 cells, indicating an enhanced translational efficiency of TNF RNA in PLB-IIIB cells. The PMA- or TNF-induced levels of IL-1 alpha mRNA did not vary significantly between the two cell types whereas IL-6 was poorly inducible in both cells. These results illustrate a differential cytokine response to HIV-1 infection in myeloid cells and demonstrate that HIV-1 infection of myelomonoblastic cells may alter both transcriptional and translational mechanisms controlling cytokine expression.


The cDNA structure, expression, and chromosomal assignment of the mouse Fas antigen.

The cell surface Fas antigen is a membrane-associated polypeptide which can mediate apoptosis. cDNA clones encoding the Fas antigen were isolated from a cDNA library constructed with mRNA from the mouse macrophage cell line BAM3. The nucleotide sequence and the deduced amino acid sequence of the mouse Fas antigen were 58.5 and 49.3% identical, respectively, to the corresponding sequences of human Fas antigen cDNA. The mouse Fas antigen consists of 306 amino acids with a calculated Mr of 34,971 and contains a single transmembrane domain which divides the molecule into extracellular and cytoplasmic domains. A 2.1-kb mRNA coding for the Fas antigen was detected in the mouse thymus, heart, liver, and ovary but not in brain and spleen. The expression of the Fas antigen gene in mouse fibroblast L929 and macrophage BAM3 cell lines was significantly induced by treatment with IFN-gamma but not by IFN-alpha/beta. Interspecific backcross analysis indicated that the gene coding for the Fas antigen is in the distal region of mouse chromosome 19.


Peptide-presenting similarities among functionally distant HLA-B27 subtypes revealed by alloreactive T lymphocytes of unusual specificity.

Functional dissection of HLA-B27 subtypes using alloreactive or B27-restricted CTL has shown that the structurally related B*2704 and B*2706 are the most distant subtypes relative to the prototype B*2705. In particular, previous studies have failed to find anti-B*2705 CTL cross-reacting with B*2704 or B*2706. Such failure can be accounted for by the drastic effect on T cell recognition of the change at residue 152 in both subtypes relative to B*2705, as established with site-directed mutants. B*2704 and B*2706 are also related in ethnic distribution, as they are restricted to Orientals, jointly being the predominant HLA-B27 subtypes in this population. As far as it is known, there are no differences relative to B*2705 in their linkage to ankylosing spondylitis. In our study, 5 of 13 examined anti-B*2705 limiting dilution CTL lines from a particular HLA-B27- individual were shown to crossreact with B*2704, B*2706 or both. The monoclonal nature of this cross-reaction was established by cold target competition analysis. This result demonstrates that the apparent differences in T cell antigenicity among anti-B27 subtypes are strongly influenced by the responder individual, as the spectrum of clonal specificities in anti-B27 responses may show significant differences among unrelated responders. Fine specificity differences among the cross-reactive CTL allowed unambiguous functional distinction between B*2704 and B*2706. The molecular basis of such cross-reactivity was examined by correlating CTL reaction patterns with the structure of both subtypes, which differ only by two residues located in the beta-pleated sheet bottom of the peptide binding site, and with site-directed mutants mimicking HLA-B27 subtype polymorphism. The results suggest that: 1) distinct peptides are involved in the allospecific epitopes recognized by the various crossreactive CTL, and 2) B*2704, B*2706, and B*2705 differ in their peptide-presenting specificity, but can present some identical or structurally similar peptides.


Analgesic use in home hospice cancer patients.

Pain control in hospice patients in the home may be compromised by concerns about overuse of analgesics, particularly narcotics. A retrospective chart audit of analgesic type and amount was performed on the medical records of 100 cancer patients receiving hospice care in the home. Different types and amounts of analgesics were converted to a common standard, an oral morphine equivalent (OME) relative to 1 mg of oral morphine sulfate. Descriptive statistics were used to characterize patient analgesic use during the entire course of hospice care and the last 5 days of life. Associations between analgesic use and select patient characteristics (age, sex, cancer site, metastases, and pain intensity at admission) were explored. Ninety-one percent of the sample had used analgesics at some time during hospice care. The proportion of patients using analgesics increased as death approached. The mean and median daily analgesic use over the entire period were 114 and 82 OMEs and during the last 5 days 140 and 84 OMEs, respectively. The range of mean daily analgesic use was between 10 and 735 OMEs. Individual variability in analgesic use was demonstrated. Not all patients required analgesics, and among those who did there was remarkable variation in the amount used. Large and even enormous doses of analgesics may sometimes be required to control cancer pain.


Care of cancer patients in a home-based hospice program: a comparison of oncologists and primary care physicians.

The purpose of this study was to describe a group of patients cared for in a home-based hospice program and to determine if there was a difference in patients' experiences dependent on whether the attending physician was a primary care physician or an oncologist. Information about cancer patients admitted to the Burlington Visiting Nurse Association (VNA) Hospice program from January 1986 to December 1990 was reviewed to compare the experiences of the patients of the oncologists with those of the patients of the primary care physicians. There was no difference in average length of stay or overall ambulatory status between the patient groups. The patient group cared for by oncologists had more hospitalizations than the group cared for by primary care physicians though there was not a significant difference in the percentage of hospital vs home deaths. There was a significant difference between the groups in the use of controlled-release morphine, with oncologists using this approach more often than primary care physicians. Oncologists also had more patients on continuous parenteral morphine infusions during hospice care. Primary care physicians as well as oncologists provide effective cancer care and pain control in this home-based hospice program. The hospice interdisciplinary team can be a valuable resource for physicians in supplying information on appropriate narcotics dosages and routes of administration for their dying patients.


Use of latex agglutination testing in diagnosing pediatric meningitis.

Latex agglutination (LA) tests are ordered frequently on cerebral spinal fluid (CSF) specimens obtained from pediatric patients to identify pathogenic bacteria as early as possible in an acute infection. Six hundred ten LA tests were performed on 176 patients suspected of having meningitis. Five patients with meningitis had positive LA tests. We found that the CSF white blood cell (WBC) count, and differential were the best predictors of meningitis. By limiting the use of LA tests to those patients having CSF with abnormal WBC counts or with positive Gram stains, the number of tests ordered would have been reduced. This practice would greatly reduce laboratory expense.


Chronic pain beyond patienthood.

Forty nonconsumers (subjects with pain lasting for more than 1 year who had not consulted a doctor because of it during the preceding year) were compared with 46 chronic patients at a pain clinic. The mean duration of the nonconsumers' pain was twice as long as that of the patients'. Although their pain intensity, as assessed with pain diaries, did not differ, their estimated pain intensity was less than that of the pain patients; they took fewer analgesics, were less functionally impaired, made less use of coping strategies associated with poor adjustment, were less depressed, and expected less help from external resources. The "non-consumer attitude" appears to be independent of the duration of the pain complaints.


Target-dependent regulation of retinal nicotinic acetylcholine receptor and tubulin RNAs during optic nerve regeneration in goldfish.

A fundamental issue in central nervous system development regards the effect of target tissue on the differentiation of innervating neurons. We address this issue by characterizing the role the retinal ganglion cell target, i.e., the optic tectum, plays in regulating expression of tubulin and nicotinic acetylcholine receptor genes in regenerating retinal ganglion cells. Tubulins are involved in axonal growth, whereas nicotinic acetylcholine receptors mediate communication across synapses. Retinal ganglion cell axons were induced to regenerate by crushing the optic nerve. Following crush, there was a rapid increase in alpha-tubulin RNAs (3 days), which preceded the increase in nicotinic acetylcholine receptor RNAs (10-15 days). Both classes of RNAs approached control levels by the time retinotectal synapses and functional recovery were restored (4-6 weeks). If the optic nerve was repeatedly crushed or its target ablated, tubulin RNAs remained elevated, and the increase in receptor RNAs that would otherwise be seen 2 weeks after a single nerve crush did not occur. The interaction of retinal ganglion cell axons with their targets in the optic tectum appears, then, to exert a suppressive effect on the RNA encoding a cytoskeletal protein, tubulin, and an inductive effect on RNAs encoding nicotinic acetylcholine receptors involved in synaptic communication.


Steroid inhibition of neural microvessel morphogenesis in vitro: receptor mediation and astroglial dependence.

Steroid hormones alter several aspects of microvascular function within the CNS. Both microvessel formation and blood-brain barrier expression appear to be influenced by interactions between astrocytes and endothelial cells. To determine if steroids alter astrocyte-endothelial interactions, we studied their effects on astroglial-induced microvessel morphogenesis in vitro. C6 astroglial cells induce bovine retinal microvascular endothelial cells to differentiate into capillary-like structures. Dexamethasone, hydrocortisone, and progesterone at 10 nM inhibited C6-induced microvessel morphogenesis by 75, 35, and 30%, respectively. Inhibition by dexamethasone was both time and concentration dependent, reaching 80-100% at 1 microM. Tetrahydrocortisone and 17 alpha-hydroxyprogesterone had only marginal inhibitory effects. Cortexolone, a glucocorticoid receptor antagonist, blocked inhibition by dexamethasone. Progesterone receptors were expressed in C6 but not bovine retinal microvascular endothelial cells, identifying the astroglial cell as the likely effector of progesterone-mediated inhibition. Astroglial cells were further implicated as the effectors of steroid-mediated inhibition because none of the steroids inhibited astroglial-independent capillary-like structure formation in response to a reconstituted extracellular matrix, Matrigel. These findings are evidence that steroids modulate neural microvascular endothelial cell functions indirectly through perivascular astrocytes via a receptor-mediated mechanism.


Temporal expression of HNK-1-reactive sulfoglucuronyl glycolipid in cultured quail trunk neural crest cells: comparison with other developmentally regulated glycolipids.

Monoclonal antibody HNK-1 is an important marker for embryonic neural crest cells and some of their differentiated derivatives. We have identified 3-sulfoglucuronylneolactotetraosylceramide (SGGL-1) as one of the HNK-1 antigens present in cultures of trunk neural crest cells. This lipid was present at 2 days in vitro and increased in amount with time in culture. Other major HNK-1-reactive antigens present in the culture were glycoproteins of apparent molecular masses of 120, 180, and 200 kDa. The 180- and 200-kDa bands were present at 2, 7, and 17 days in vitro, whereas the 120-kDa band was present only at 17 days in vitro. Gangliosides GD3, LD1, and LM1 were also found in the cultures and exhibited distinct temporal patterns of expression. Ganglioside GD3 was present at all stages examined and its expression peaked at 7 days in vitro. In contrast, LD1 was present only at 2 days in vitro and was not detectable at later times. Ganglioside LM1 increased in amount with time in culture in a pattern similar to that seen for SGGL-1. Taken together, these results indicate that several HNK-1-reactive molecules are expressed in neural crest cultures in a temporally regulated manner along with several glycolipids that do not bear this epitope.


Biochemical characterization and localization of a non-N-methyl-D-aspartate glutamate receptor in rat brain.

The structure and distribution of non-N-methyl-D-aspartate glutamate receptors in the rat brain were studied using subunit-specific antibodies that recognize the receptor subunit GluR1. The GluR1 protein, a 106-kDa glycoprotein, appears predominantly in synaptic plasma membranes, where it is highly enriched in the postsynaptic densities. When synaptic plasma membranes are solubilized with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, high-affinity alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) binding and GluR1 immunoreactivity comigrate at a native Mr of 610,000. GluR1 is enriched in the hippocampus and cerebellar cortex but is present throughout the CNS. It is found on neuronal cell bodies and processes within most regions of the brain; within the cerebellum, however, it is localized to the Bergmann glia. These data suggest that the GluR1 protein is a subunit of multimeric AMPA-preferring glutamate receptors present on neurons and on specialized glia.


Expression of size-selected RNA encoding brain serotonin transporter in Xenopus laevis oocytes.

The mRNA that encodes a serotonin transporter was expressed using the Xenopus laevis oocyte expression system. Poly(A)+ RNA isolated from mouse brainstem was injected into Xenopus laevis oocytes, and the ability of oocytes to take up serotonin was measured 3 days postinjection. RNA-dependent serotonin uptake was sensitive to citalopram, a specific inhibitor of serotonin uptake, whereas background levels of serotonin uptake were not citalopram sensitive. Two RNA size fractions, 4.0 and 4.5 kb, were most efficient in stimulating uptake. Injection into Xenopus laevis oocytes of the 4.5-kb size fraction of mouse brainstem RNA resulted in threefold more serotonin uptake than did injection of unfractionated poly(A)+ RNA.


Characterization of substance P-like immunoreactivity and tachykinin-encoding mRNAs in rat medullary thyroid carcinoma cell lines.

Rat thyroid tissue and three rat medullary thyroid carcinoma cell lines, 6-23, WE4/2, and CA77, have been examined for substance P (SP) and SP-like peptide expression. Analysis by combined HPLC and radioimmunoassay revealed the presence of SP in thyroid and 6-23 cell extracts. The presence of SP-encoding mRNAs was also detected in 6-23 cells by solution hybridization-nuclease protection analysis. SP-encoding mRNA expression was increased (fourfold) by maintaining the 6-23 cells in low serum (2%) for 4 or 10 days. The 6-23 cells also expressed other SP-like immunoreactive species, which were chromatographically and immunologically distinct from established tachykinin peptides. WE4/2 cells did not contain SP but did display SP-like immunoreactivity (SPLI), which migrated like the unidentified SPLI in 6-23 cells. CA77 cells did not contain SP or SP-encoding mRNA but did contain SPLI that migrated identically to the unidentified SPLI in the other cell lines. This novel SPLI was detected with an antiserum directed against the SP carboxyl terminus and to a lesser extent with an antiserum directed against the neurokinin A carboxyl terminus, but it showed minimal cross-reactivity using an antiserum directed against the midportion of SP. Treatment with 50 mM KCl resulted in secretion of this SPLI from CA77 cells. Gel filtration analysis demonstrated that this novel SPLI had an apparent molecular weight of approximately 1,000. These results are discussed in terms of cell lines that express tachykinin peptides and in terms of the molecular nature of the new SPLI detected in CA77 cells.