Showing papers in "Journal of Investigative Medicine in 1995"

Bronchial epithelial cell-cytokine interactions in airway inflammation.

Abstract: A variety of cytokine bronchial cell interactions may play an important role in normal host defense as well as in the pathogenesis of inflammatory airway disorders such as asthma, cystic fibrosis, acute and chronic bronchitis, and bronchiectasis. First, airway epithelial cells may participate in local cytokine networks by synthesizing interleukins, chemokines, colony stimulating factors and growth factors in response to inflammatory mediators. Bronchial epithelial cell derived cytokines may thereby amplify ongoing inflammatory processes via the recruitment and activation of specific subsets of inflammatory cells, as well as by prolonging their survival in the airway microenvironment. Second, airway epithelial cells can initiate inflammatory cascades by generating cytokines in direct response to viral and bacterial products, noxious gases, and sensitizing chemicals. Third, airway epithelial cells represent targets for paracrine acting cytokines, which may then modulate bronchial epithelial cell functions. Finally, airway epithelial cells may modulate ongoing inflammatory events in the airway microenvironment via the shedding of soluble TNF receptors. Cytokine-bronchial epithelial cell interactions represent an important mechanism by which inflammatory events in the airway microenvironment can be regulated and represent potential targets for novel anti-inflammatory therapies in airway disorders.

Regulation of endothelial cell gap formation and paracellular permeability.

Abstract: Investigation of the regulation of permeability properties of the endothelium has yielded evidence to support the concept of a dual regulation of EC gap formation and barrier function. In this model, the primary determinants of EC permeability are tethering/adhesive properties (Figure 1) and tensile centripetal force generation (Figure 2). The importance of actin-myosin interactions and active cellular contraction and force generation has been reviewed. In the model of thrombin-induced EC barrier dysfunction, there is a strong shift in the MLC species from the unphosphorylated to the diphosphorylated form, indicating activation of MLCK, a key enzyme whose importance in EC contraction has been well established. Although important differences between EC and SMC exist, endothelial cell gap formation involves actomyosin-dependent contractile mechanisms similar to SMC, a cellular system in which MLC phosphorylation correlates with the initial rate of tension development. The increase in MLC phosphorylation and isometric tension is consistent with the hypothesis that activation of signal transduction mediates an increase in isometric tension to a new level of "latch state" through the cytoskeleton. Thus, the available evidence implicates a strong role for cellular force generation and contraction in the evolution of thrombin-induced barrier dysfunction. Accumulating evidence also indicates that modulation of tethering properties, primarily those involving cell-matrix and cell-cell adhesion, is also a key determinant of basal EC barrier properties as well as agonist-mediated barrier dysfunction. Because each of these focal adhesion constituents may be involved in establishing tethering properties in endothelium, they each may be involved in determining barrier permeability and may be involved in the evolution of agonist-mediated barrier dysfunction. Therefore, in addition to MLCK-dependent active tensile force generation, agonist-induced barrier dysfunction may occur via MLCK-independent pathways that rely on basal levels of MLC phosphorylation or by affecting proteins involved in tethering properties of endothelium that contribute to barrier function. Further examination of tethering force properties, combined with elucidation of EC relaxation via MLC dephosphorylation may yield clues as to how this important vascular barrier is maintained and restored after vascular insult.

Cholesterol and Coronary Heart Disease: Predicting Risks in Men by Changes in Levels and Ratios

Abstract: BACKGROUND Little is known about the relative ability of different measures of change in cholesterol to discriminate coronary heart disease risk. We evaluated this ability for changes in low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, LDL/HDL ratios, and total cholesterol/HDL ratios. METHODS We predicted risks of coronary heart disease using data from 3641 men in the Lipid Research Clinics Coronary Primary Prevention Trial. Treating these patients as a cohort, we estimated risks associated with changes in cholesterol levels independent of the patients' randomization group. RESULTS Changes in LDL and HDL cholesterol when used in combination were each significant predictors of coronary heart disease risk (odds ratios [OR] for 10% increases, 1.15 and 0.84, respectively; P < 0.001). Changes in LDL/HDL and total cholesterol/HDL ratios had similar discriminating ability (OR for 10% increases, 1.17 and 1.21, respectively; P < 0.0001). In the best discriminating models, changes in ratios added information about risks to changes in LDL cholesterol, although changes in LDL cholesterol levels failed to add information to changes in ratios. CONCLUSIONS Changes in total cholesterol/HDL and LDL/HDL ratios were better predictors of risk for coronary heart disease than were changes in LDL cholesterol levels alone. When assessed as percentage changes averaged during the first two months of intervention, they were among the best discriminators of risk. Clinicians selecting treatments for intervention should include among their considerations the treatment's effect on both LDL and HDL cholesterol rather than their effects on LDL cholesterol levels alone.

A comparison of performance of mathematical predictive methods for medical diagnosis: identifying acute cardiac ischemia among emergency department patients.

Abstract: Background There is increasing interest in mathematical methods for the prediction of medical outcomes. Three methods have attracted particular attention: logistic regression, classification trees (such as ID3 and CART), and neural networks. To compare their relative performance, we used a large clinical database to develop and compare models using these methods. Methods Each modeling method was used to generate predictive instruments for acute cardiac ischemia (which includes acute myocardial infarction and unstable angina pectoris), using prospectivel-collected clinical data on 5773 patients, who presented over a two year period to six hospitals' emergency departments with chest pain or symptoms suggesting acute ischemia. This data set was then split into training (n = 3453) and test (n = 2320) sets. Of 200 available variables, modeling was restricted to those available within the first 10 minutes of emergency department care (history, physical exam, and electrocardiogram). Results When the number of variables was limited to eight, representing a practical number for input in the real-time clinical setting, the logistic regression's receiver-operating characteristic (ROC) curve area, as a measure of diagnostic performance, was 0.887; the classification tree model's ROC curve area was 0.858, and the neural network's ROC curve area was 0.902. When the number of variables used by a model was not limited, the logistic regression's ROC area was 0.905, the classification tree model's 0.861, and the neural network's 0.923. Among these models the neural networks had noticeably poorer calibration. When the outputs from each of these unrestricted models were presented to each of the other methods as an additional independent variable, the ROC areas of the new "hybrid" models were not significantly better than the original unlimited models (ROC areas 0.858 to 0.920). Conclusions Logistic regression, classification tree, and neural network models all can provide excellent predictive performance of medical outcomes for clinical decision aids and policy models. Their ultimate limitations seem due to the availability of the information in data (a "data barrier") rather than their respective intrinsic properties. Choices between these methods would seem to be most appropriately based on the needs of the specific application, rather than on the premise that any one of these methods is intrinsically more powerful.

Abnormal vascular function following ischemia-reperfusion injury.

Abstract: Ischemia-reperfusion injury as a general rule is accompanied by dramatic changes in basal and reactive vascular function in most organs. There are similarities in altered organ vascular function, particularly in the first 24 to 48 hours, with decreased basal organ blood flow, hypersensitivity to vasoconstrictor stimuli, attenuated responses to vasodilators, and increased vascular permeability. The reduced responsiveness to endothelium-dependent vasodilators may be due to reduced endothelial NOS activity or to spontaneous maximal activation of NOS/NO activity, which cannot be stimulated further by endothelium-dependent agents. There are also notable quantitative and qualitative differences in ischemia-reperfusion injury vasoreactive response in organs such as kidney, heart, and brain, the basis of which is unexplored, but may reflect regional differences in endothelium and/or organ parenchyma. Further examination of both the mechanisms and consequences of ischemia-reperfusion injury to the vasculature, as well as the clinical implications, should be a rewarding pursuit in organ pathophysiology.

Angiotensin-I converting enzyme genotype DD is a risk factor for coronary artery disease.

Abstract: BACKGROUND Coronary artery disease (CAD) is a polygenic disease whose phenotypic manifestation is due to interaction of a number of environmental factors with an underlying genetic background A number of genes, including the angiotensin-I converting enzyme (ACE) gene, have been implicated in the pathogenesis of CAD ACE can affect oxidation of LDL, endothelial cell function, and smooth muscle cell migration and proliferation: all important components of atherosclerosis A variant of ACE gene, genotype DD is associated with a higher plasma level of ACE and an increased risk of myocardial infarction, and cardiomyopathies In this study, we sought to determine the distribution of ACE genotypes and the frequency of allele D in patients with CAD undergoing coronary angioplasty METHODS DNA from 182 white patients undergoing coronary angioplasty and 338 apparently healthy white individuals was amplified by polymerase chain reaction (PCR) in the region of the polymorphism using the previously published protocol RESULTS PCR amplification of alleles I and D resulted in 490 bp and 190 bp products, respectively ACE genotype DD was present in 47% of patients with CAD as compared to 30% in the general population (p = 00002, Odds ratio 27) The frequency of allele D was 068 in patients with CAD and 055 in general population, respectively (p < 00001) Genotype DD was associated with CAD only in males (54% vs 30%, p = 00001, Odds ratio 20), but not in female patients There was no association between the frequency of ACE genotype DD and the prior history of myocardial infarction, or the extent of CAD The frequency of ACE genotype DD was the highest among patients with restenosis following angioplasty (55%), however, the difference was not significantly changed as compared to those without restenosis (40%) CONCLUSIONS ACE genotype DD is more common in patients with CAD as compared to the general population, indicating that genotype DD is a genetic risk factor for CAD

Pulmonary responses to purified zinc oxide fume.

Abstract: BACKGROUND Metal fume fever is a flu-like illness caused by zinc oxide fume inhalation and mediated by unknown mechanisms. It is one of a group of work-related febrile inhalational syndromes. We studied bronchoalveolar lavage (BAL) obtained from cigarette smoking and nonsmoking human volunteers after controlled exposure to purified zinc oxide fume to explore the possible roles of proinflammatory cytokines in this condition. METHODS We studied 14 volunteers after inhalation exposure to purified zinc oxide fume and after sham exposure to air. The mean cumulative exposure was 537 +/- 232 mg min per cubic meter elemental zinc. Twenty hours after exposure we performed BAL. We analyzed BAL cells and studied BAL supernatant for cytokines including tumor necrosis factor-alpha (TNF alpha), interleukin(IL)-8, and IL-1 by enzyme-linked immunosorbant assay (ELISA). RESULTS Polymorphonuclear leukocytes (PMNs) were significantly increased in the BAL fluid obtained post-exposure compared to sham (mean difference = 41.3 +/- 16.8 x 10(3) per mL; p < 0.05). Cumulative zinc exposure positively correlated with exposure-sham differences in BAL supernatant concentrations of both TNF (r2 = 0.58; p = .002) and IL-8 (r2 = 0.44, p = 0.01). Exposure-sham concentration differences in BAL supernatant IL-8 and BAL PMNs were also positively correlated (r2 = 0.60; p < 0.001). Cigarette smoking was not associated with exposure-sham differences in BAL TNF or IL-8, but did demonstrate a packs-per-day dependent increase in BAL supernatant IL-1 (t = 2.3, p = 0.04) post-exposure compared to sham, after taking into account the zinc exposure response. CONCLUSIONS Purified zinc oxide fume inhalation causes an exposure-dependent increase in proinflammatory cytokines and PMNs in the lung. This supports a role for cytokine networking in mediating metal fume fever.

Interferon gamma modulates the expression of neutrophil-derived chemokines.

Abstract: Specific cell recruitment to a site of acute inflammation is a crucial event characterized by the elicitation of mainly polymorphonuclear neutrophils (PMNs). Recently, it has been reported that PMNs can express and secrete chemotactic cytokines or chemokines, including IL-8, MIP-1 alpha, and MIP-1 beta. Moreover, PMN-derived chemokines are regulated by various soluble mediators, such as dexamethasone, prostaglandin E, classic chemoattractant factors (e.g., fMLP, C5a, leukotriene B4), IL-4, and IL-10. In this article we demonstrate that PMNs treated with IFN-gamma, a Th1-derived cytokine, can inhibit early mRNA expression for MIP-1 alpha, MIP-1 beta, and IL-8 (up to 8 hours post IFN-gamma addition), while augmenting their production at 24 hours post IFN-gamma addition. Furthermore, our studies demonstrate that one of the mechanisms for the activity of IFN-gamma in this system is via the autocrine activity of TNF-alpha. These data imply that PMN-derived chemokines are regulated by not only proinflammatory cytokines, including IL-1 beta and TNF-alpha, but also Th1- and Th2-derived cytokines, including IL-4, IL-10, and IFN-gamma. The role of these cytokine networks in regulating PMN-derived chemokines may play an important role in leukocyte elicitation during the initiation and maintenance of an inflammatory response.

Wilson's disease: a new gene and an animal model for an old disease

Abstract: Wilson's disease is an autosomal recessive, inherited disorder of copper metabolism. In normal individuals, copper homeostasis is controlled by the balance between intestinal absorption of dietary copper and hepatic excretion of excess copper in bile. In Wilson's disease, hepatic copper is neither excreted in bile nor incorporated into ceruloplasmin and copper accumulates to toxic levels. The Wilson's disease gene (WND) encodes a putative copper-transporting protein that is expressed almost exclusively in the liver. The predicted structure of the protein product is that of a P-type ATPase with striking homology to bacterial copper transporters and the gene product of another inherited disorder of copper metabolism, Menkes' disease. A rat model of Wilson's disease has recently been identified. The Long-Evans Cinnamon (LEC) rat manifests elevated hepatic copper, defective incorporation of copper into ceruloplasmin, and reduced biliary excretion of copper. The rat homologue of the WND is abnormal in LEC rats. Clinical manifestations of Wilson's disease arise directly from copper-induced damage to hepatocytes (hepatic presentation) or indirectly after the release of copper from the liver with subsequent damage to the brain (neuropsychiatric presentation) and other organs. Genetic heterogeneity (different mutations in a single gene) may account for some of the variability in Wilsonian presentations. The diagnosis of Wilson's disease depends on the demonstration of disordered copper metabolism, manifested as elevated urinary and hepatic copper and low ceruloplasmin levels. However, none of the abnormal findings in Wilson's disease is pathognomonic. Genetic diagnosis, in the absence of family studies, is likely to be difficult since many different mutations result in the disease. Management of Wilson's disease involves decreasing excess levels of copper accumulated in the liver, brain, and other organs. Copper chelation therapy, to increase urinary excretion of copper, is the mainstay of treatment. In addition, oral zinc therapy may be useful at decreasing absorption of dietary copper and rendering tissue copper nontoxic, by increasing the formation of complexes with copper-binding proteins. Liver transplantation can be necessary for individuals with acute hepatic failure or complications of cirrhosis. Gene therapy may evolve in the future; however, medical management is effective in most patients.

Informed consent for research: a study to evaluate readability and processability to effect change.

Abstract: Background Ninety million Americans are reported to have low literacy skills, and the problems associated with illiteracy permeate all areas of our society, including medical research. The purpose of this study was to determine the readability and comprehensibility of human research informed consent forms using established reading comprehension processes. Further, if comprehension problems were present in the forms, the study sought to identify the exact nature of these difficulties and identify specific areas where the forms could be rewritten to increase readability and understanding. Methods All human research informed consents approved by Hartford Hospital in 1993 were evaluated by a Readability and Processability Form (RPF) based on reading research, including the Fry Scale which yields an approximate grade reading level. The RPF assigned points to each of the 20 areas of analysis according to strict scoring criteria, and target scores were established by the authors in consultation with the hospital. Results Seventy-six informed consent forms were evaluated, and neither the Fry score or the RPF score was in the target range. Ninety-six percent of the forms were found to have readability levels higher than the target level (8th grade). The mean readability and processability score was 46, resulting in the classification, Minimally Adequate/Needs Improvement. (The target range was Good, 61-100.) A question by question analysis of each of the 20 checklist items on the RPF identified important aspects of text writing style that were scored as Unacceptable or Poor. Conclusions The descriptive data indicates that there were problems with the readability of the informed consent documents studied. The prescriptive portion of this study provides researchers with information on specific areas where their forms need to be studied and rewritten. The comprehension problems found in this study may alert others to similar problems and may provide the basis for other institutional review boards (IRBs) to study their consent forms in order to ensure that the interests of both the patient and the researcher are protected and that the obtained consent is truly informed.

The effects of corticosteroids on lymphocyte recirculation in humans: analysis of the mechanism of impaired lymphocyte migration to lymph node following methylprednisolone administration.

Abstract: Background Steroid therapy profoundly inhibits lymphocyte migration into lymph nodes (LN), thereby disrupting lymphocyte recirculation. This study was undertaken in human subjects to examine two nonmutually exclusive hypotheses proposed to explain this steroid effect: (1) steroids decrease the capacity of LN high endothelial venules (HEV) to support lymphocyte adherence; and/or (2) steroids alter the expression of lymphocyte adhesion molecules specific for entry into LNs. Methods Human LNs were obtained from cadaveric organ donors before and after methylprednisolone infusion and examined for their capacity to support lymphocyte adherence using an in vitro lymphocyte-HEV adherence assay. Additionally, six healthy volunteers underwent infusions of saline (placebo) and methylprednisolone (3 mg/kg in saline), and peripheral blood lymphocytes were isolated before, immediately after, and at 1, 2, 4, 8, 24, and 48 hours following infusions. At each timepoint, flow cytometric analysis of lymphocyte L-selectin, CD44, and LFA-1 expression was assessed. Short-term tissue culture studies were also performed to examine the direct effects of steroids on the expression of these lymphocyte proteins. Results LNs obtained from organ donors before and after steroid infusion displayed no differences in the capacity of HEV to support lymphocyte adherence. In controlled steroid infusion studies, lymphopenia was observed within 1 hour and persisted for 8 hours. Cells isolated during the period of lymphopenia did not adhere to LN HEV, and there was a marked decrease in both the percentage and mean fluorescence intensity level of L-selectin+ lymphocytes; minor changes were observed in the percentage of CD44+ and LFA-1+ cells, while the mean fluorescence intensity level decreased for CD44+ cells and increased for LFA-1+ cells. All such changes in adhesion protein levels among circulating cells reversed with the resolution of lymphopenia. However, in tissue culture experiments, steroids did not alter the expression of lymphocyte adhesion proteins. Conclusions The decreased migration of lymphocytes to LN following steroid administration is not due to changes in the capacity of HEV to bind lymphocytes, but results from decreases in the level of L-selectin expressed among circulating cells. Culture studies suggest that steroids do not directly alter lymphocyte adhesion protein expression. The possibility that these agents act indirectly by stimulating the release and/or promoting the activity of relevant biologic mediators affecting lymphocyte adhesion protein expression needs further exploration.

Increased rate of thrombin generation in hepatitis C virus cirrhotic patients. Relationship to venous thrombosis.

Abstract: BACKGROUND Venous thrombosis may complicate the clinical course of liver cirrhosis (LC), but the pathogenesis is still uncertain. We have previously demonstrated that antiphospholipid (aPL) antibodies were a risk factor for thrombosis. In the same study it was also evident that there was a higher prevalence of hepatitis C virus (HCV) infection in patients with thrombosis, and an association between anti-HCV positivity and aPL. METHODS In a case-control study, 18 consecutive patients with LC, who had suffered from splanchnic venous thrombosis (n = 12), or thrombophlebitis (n = 6), were matched for age, sex, and degree of liver failure with 36 LC patients without thrombosis. RESULTS In comparison to patients without thrombosis, patients with thrombosis had higher prevalence of HCV infection (p = 0.0027), positivity for aPL antibodies (p = 0.0003), and higher values of fragment F1+2, a marker of thrombin generation (p = 0.0083). While F1+2 values were similar in aPL (+) and aPL (-) patients, HCV patients had significantly higher values of F1+2 than patients with hepatitis B virus (HBV) infection and/or alcoholism (2.6 +/- 0.94 vs 1.5 +/- 0.66, p = 0.0001). CONCLUSIONS These data suggest that in LC, HCV infection may contribute to clotting system activation, thus predisposing patients to venous thrombosis.

Regulation of calcium-activated potassium channels by S-nitrosothiol compounds and cyclic guanosine monophosphate in rabbit coronary artery myocytes.

Abstract: BACKGROUND: The patch-clamp technique was used to study a large conductance, calcium-activated potassium channel (IK(Ca) in coronary arterial smooth muscle cells from rabbits. The properties of this channel are similar to those of IK(Ca) found in many types of vascular tissue. A brief single channel characterization of IK(Ca) in this tissue type has been completed for this study. METHODS: The effects of S-nitrosothiol compounds on IK(Ca) were studied in cell-attached patches. RESULTS: The probability of opening for IK(Ca) increased from 0.008 +/- 0.004 to 0.780 +/- 0.07 following application of S-nitroso-L-cysteine. S-nitroso-N-acetylpenicillamine (SNAP) also increased the probability of opening for IK(Ca) from 0.022 +/- 0.01 to 0.601 +/- 0.05. The probability of opening for IK(Ca) also increased from 0.026 +/- 0.01 to 0.809 +/- 0.02 following application of membrane-permeable analogs of cyclic guanosine monophosphate (cGMP) to the bath of cell-attached patches, suggesting that IK(Ca) in coronary artery smooth muscle cells is regulated by a cGMP-dependent mechanism. Rp-8-pCPT-cGMP, a protein kinase G inhibitor, blocked the effect of SNAP, an S-nitrosothiol compound. CONCLUSIONS: These findings suggest that one of the effects of nitrosothiol compounds is the activation of IK(Ca) through a cGMP-dependent mechanism in coronary artery smooth muscle cells.

High prevalence of colorectal cancer in HLA-B27 transgenic F344 rats with chronic inflammatory bowel disease.

Abstract: Background/aims Transgenic rats expressing the human major histocompatibility class I molecule HLA-B27 develop a spontaneous multisystem disease that includes a chronic colitis resembling ulcerative colitis. The availability of this phenotype in B27 transgenic rats of 2 different inbred strains provided the opportunity to inquire whether colorectal neoplasia, which occurs with increased frequency in humans with inflammatory bowel disease (IBD), would develop in either or both rat genetic backgrounds. Methods Clinical and histologic evaluation of B27 transgenic rats with chronic inflammatory bowel disease (IBD) on the F344 and LEW inbred backgrounds. Results In B27 transgenic rats on an inbred F344 background, hyperplastic lesions evolved in the setting of chronic colitis, with a high frequency of colorectal polyp formation and frequent histologic progression from adenoma to adenocarcinoma. In contrast, no neoplasia occurred in B27 transgenic rats on an inbred LEW background, despite similar colitis. Conclusion A high incidence of spontaneous colorectal neoplasia occurs in a line of B27 F344 rats that shares some features of both sporadic and inflammatory bowel disease-associated human colorectal cancer. This represents a novel example of spontaneous colorectal neoplasia in rodents that is not based on germline modification of one or more already-identified cancer-related genes.

Immunization with vesicular stomatitis virus nucleocapsid protein induces autoantibodies to the 60 kD Ro ribonucleoprotein particle.

Abstract: BACKGROUND Systemic lupus erythematosus (SLE) is an autoimmune disorder of unknown etiology that is characterized by antibodies binding ribonucleoprotein complexes. The epitopes of SLE associated 60 kd Ro (or SSA) autoantigen share sequence with vesicular stomatitis virus (VSV) nucleocapsid (N) protein and SLE patients with anti-Ro are more likely to bind the N-protein than anti-Ro negative patients. METHOD We immunized New Zealand white (NZW) rabbits with purified VSV N-protein to examine the relationship between the immune response to N-protein and anti-Ro. RESULTS After multiple immunizations with VSV N-protein, NZW rabbits produced not only IgG antibodies to VSV N-protein but also an autoimmune response to Ro antigen. The IgG fraction of the rabbit immune serum precipitates 60 kd Ro protein as well as its associated Y RNAs. Antibodies elicited by 10 immunizations of VSV N-protein bind to at least 20 linear epitope groups on VSV N-protein and over 25 linear epitope groups on 60 kd Ro protein. These antibodies also bound 5 of the 6 shared sequences between 60 kd Ro protein and VSV N-protein. CONCLUSIONS Our data demonstrate that an immune response initiated towards a foreign antigen, selected on the basis of sharing short sequence similarity with the autoantigenic epitopes of an autoantigen, can lead to an autoimmune response.

Method for mass retrieval, morphologic, and functional characterization of adult porcine islets of Langerhans: a potential nonhuman pancreatic tissue resource for xenotransplantation in insulin-dependent diabetes mellitus.

Abstract: Background We aimed to implement and expand an original technique for mass procurement, maintenance, and study of adult porcine pancreatic islets (PPIs), which had been previously developed in our laboratory. Methods By screening through specific criteria for pig donor strain selection, we acquired new leads for separation of large, intact islets from the whole pancreas. Accomplishment of this goal also was permitted by adjusting, and or replacing physical/chemical parameters of the pancreatic digestion process. Results High yield isolation and purification of large and intact PPIs was thoroughly associated with full retention of islet morphologic integrity, as proven by accurate assessment of islet cell structure and ultra-structure, as well as viability and functional competence, in vitro and in vivo. In particular, we observed that isolated PPIs may retain capability of synthesizing insulin "de novo," and regulate this property on glucose stimulation. We also began to explore new approaches for in vitro, long-term PPI culture maintenance. Conclusion We found that a graft of PPI containing algin/polyaminoacidic microcapsules, in nonimmunosuppressed diabetic rodents, was associated preliminary with remission of hyperglycemia, providing further insight into the physiologic competence of these islets.

Effect of H2-receptor antagonists on bile acid metabolism.

Abstract: Background Several reports have been presented concerning pronounced overgrowth of bacteria in gastric juices of patients treated with H2-receptor antagonists. However, there has been no report concerning influence of H2-receptor antagonists on jejunal flora. Thus, to investigate the influence and its effect on bile acid metabolism, this study was performed: 1) to examine whether patients with gastric ulcers who have been treated with H2-receptor antagonists have positive bile acid breath tests due to bacterial overgrowth in their jejuna; 2) to verify that these bacteria, isolated and identified, have deconjugation ability; and 3) to determine whether the changes in the gastric pH are related to bacterial overgrowth. Methods The methods used were detection of deconjugation of bile acids in early phase by a bile acid breath test using 5 muCi of oral glycine-1-14C labeled glycocholate, aspiration of jejunal fluids by a double lumen tube with a rubber cover on the tip, and examination of deconjugation ability by thin layer chromatography. Results Expired breath samples from all 18 patients after administration of H2-receptor antagonists showed a significant increase in 14CO2 specific activity compared with those before administration of H2-receptor antagonist and the normal controls, and bacterial overgrowth was found in the jejunal fluid of the patients after administration of H2-receptor antagonist. The administration of tetracycline to the 18 patients reduced the 14CO2 specific activity significantly. The following species were identified in the jejunal fluid samples obtained from the patients: Escherichia coli, Candida albicans, Pseudomonas aeruginosa, enterococcus, Lactobacillus bifidus, Bacteroides vulgatus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Eubacterium lentum, and Eubacterium parvum. All of the species identified except for Escherichia coli, Pseudomonas aeruginosa, and Candida albicans deconjugated bile acids. There were significant correlations between the 14CO2 activity and gastric pH before and after administration of H2-receptor antagonist, respectively. Conclusions Patients with gastric ulcers who were treated with H2-receptor antagonists have increased bile acid deconjugation due to bacterial overgrowth in their jejuna containing species that can deconjugate bile acids. The bacterial overgrowth is probably associated with a shift to neutral pH in the gastric juice caused by the H2-receptor antagonists.

A diagnostic assay for the Wiskott-Aldrich syndrome and its variant forms.

Abstract: BACKGROUND: The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disease characterized by severe thrombocytopenia, eczema, and impaired immunity. While the diagnosis is usually straightforward, the syndrome may be expressed in an attenuated form, a phenotype which is difficult to distinguish from other types of congenital thrombocytopenia. Although a molecular-based assay for diagnosis of the spectrum of WAS patients has not been available, recent data indicate that WAS is associated with a specific profile of impaired mitogen responsiveness and suggest that detection of this abnormality may provide a diagnostic marker for all forms of the disease. To address this issue, we have studied patients with classical and atypical WAS for their lymphocyte proliferative responses to four T cell mitogenic stimuli and compared their response patterns to those detected in unaffected children. METHODS: Clinical histories and informed consent were obtained from 23 patients with either classical or putative (ie, atypical) WAS, 16 subjects with other disorders, and 12 healthy children. Peripheral blood mononuclear cells (PBMCs) collected from patients and controls were resuspended in culture medium, stimulated with the T cell mitogens phytohemagglutinin (PHA), concanavalin A (Con A), neuraminidase/galactose oxidase (NAGO), or periodate, and cultured for 60 h in 0.2 mL aliquots. Following a 20 h pulse with 3H-thymidine, cultures were harvested and the 3H-thymidine uptake was evaluated by liquid scintillation counting. RESULTS: The most striking observation involved response to periodate. While lymphocytes from all healthy control children proliferated in response to periodate treatment, cells from both classical as well as atypical WAS patients consistently failed to proliferate in response to this mitogen. By contrast, lymphocyte proliferative responses to PHA, Con A, and NAGO were detected in all patients and controls, although responses generally were lower in cells from classical WAS patients compared to other children. In two WAS patients, bone marrow transplantation and clinical improvement were associated with a change from no periodate response (pre-transplant) to periodate responsiveness (post-transplant). In contrast to the WAS patients, cells from patients with other hematologic and primary immune deficiency diseases responded uniformly to all four mitogens, including periodate. CONCLUSIONS: The data presented here indicate that T cells from patients with either classical or attenuated WAS fail to undergo proliferation in response to periodate, an agent that induced extensive T cell mitogenesis of cells from all healthy controls as well as patients with diseases other than WAS. As the WAS patients' cells did proliferate in response to treatment with other T cell mitogens, it appears that periodate induced T cell proliferation is selectively impaired in WAS and that detection of this defect may be of value in the distinction of both classical and attenuated WAS from other thrombocytopenic conditions.

Correlates and in-hospital outcome of painless presentation of acute myocardial infarction: a prospective study of a consecutive series of patients admitted to the coronary care unit.

Abstract: BACKGROUND The past literature has long ago identified painless myocardial infarction as a clinical entity; however, the term has been applied, often loosely, to denote diagnosis of infarction made at autopsy, or during routine electrocardiography, or at presentation with atypical symptoms. Many of the old studies were retrospective or included patients who could not contribute a reliable history. METHODS Systematic interviews of 517 consecutive patients with an acute myocardial infarction admitted to the Coronary Care Unit were carried out; a large array of data was collected prospectively while the patients were taken care of in the hospital. RESULTS A reliable history of symptoms at the inception of the clinical episode could be provided by 501 patients; 40 patients (8.0%) presented with painless (not silent) infarction, while the remaining 461 had pain. Multivariate analysis revealed that painless presentation of myocardial infarction correlated positively with age (OR 1.05, CI 1.02-1.08) and admission Killip class (OR 2.58, CI 2.16-2.97), and negatively with history of prior angina (OR 0.16, CI 0.15-064); also it was not a predictor of increased rate of mortality or life-threatening arrhythmias. CONCLUSIONS Presentation with painless myocardial infarction occurs in older patients with increased degrees of pulmonary congestion on admission, and less frequent rate of prior angina than the ones encountered in patients with pain; however such presentation is not an independent predictor of worse than expected in-hospital outcome.

The sensitivity of leukemic bone marrow to simvastatin is lost at remission: a potential purging agent for autologous bone marrow transplantation.

Abstract: BACKGROUND Autologous bone marrow transplantation (ABMT) is frequently used in the treatment of malignant disease but carries the risk of reintroducing tumor cells into the patient. Methods are required for removing malignant cells from harvested bone marrow (BM) without impairing hematopoietic reconstitution. We have shown that simvastatin is toxic to leukemic progenitor cells at a concentration that conserves normal BM progenitors and may be of use clinically as a novel BM purging agent. METHODS A two-stage culture system was used to compare the effects of simvastatin on both normal BM progenitor and primary acute myeloid leukemia (AML) cells. AML cells and normal BM mononuclear cells were incubated for 18 hours in suspension culture with 10 micrograms per mL simvastatin and the numbers of surviving clonogenic progenitor cells assayed in semisolid agar culture. RESULTS Following simvastatin treatment of 18 AML cell populations, the mean surviving fraction of progenitor cells was 21.3 +/- 4.8% ( +/- standard error of the mean [SEM]). In contrast, the mean survival of normal BM progenitors from 16 donors was 89.6 +/- 8.6% ( +/- SEM). Samples were taken from 6 AML patients before treatment and after remission of disease had been induced by chemotherapy. In 5 of these cases the AML sample was significantly more sensitive to simvastatin than the remission sample, 4 of the 5 showed > 80% difference in progenitor cell survival. CONCLUSIONS AML progenitor cells are sensitive to a short-term exposure to simvastatin that spares normal BM hematopoietic progenitor cells. We conclude that simvastatin may be an effective in vitro purging agent in ABMT for AML.