Biomedical subjects
Publications and source records attributed to A L Smith.
The application of a novel solid-phase synthesis of 2,3-disubstituted indoles utilizing a carbamate indole linker is described resulting in the identification of the novel, high-affinity, selective h5-HT2A antagonist 19.
BACKGROUND: Historically, panel reactive antibody (PRA) analysis to detect HLA antibodies has been performed using cell-based complement-dependent cytotoxicity (CDC) techniques. Recently, a flow cytometric procedure (FlowPRA) was introduced as an alternative approach to detect HLA antibodies. The flow methodology, using a solid phase matrix to which soluble HLA class I or class II antigens are attached is significantly more sensitive than CDC assays. However, the clinical relevance of antibodies detected exclusively by FlowPRAhas not been established. In this study of cardiac allograft recipients, FlowPRA was performed on pretransplant sera with no detectable PRA activity as assessed by CDC assays. FlowPRA antibody activity was then correlated with clinical outcome. METHODS: PRA analysis by anti-human globulin enhanced (AHG) CDC and FlowPRA was performed on sera corresponding to final cross-match specimens from 219 cardiac allograft recipients. In addition, sera collected 3-6 months posttransplant from 91 patients were evaluated. The presence or absence of antibodies was correlated with episodes of rejection and patient survival. A rejection episode was considered to have occurred based on treatment with antirejection medication and/or histology. RESULTS: By CDC, 12 patients (5.5%) had pretransplant PRA >10%. In contrast, 72 patients (32.9%) had pretransplant anti-HLA antibodies detectable by FlowPRA (34 patients with only class I antibodies; 7 patients with only class II antibodies; 31 patients with both class I and class II antibodies). A highly significant association (P<0.001) was observed between pretransplant HLA antibodies detected by FlowPRA and episodes of rejection that occurred during the first posttransplant year. Fifteen patients died within the first year posttransplant. Of nine retrospective flow cytometric cross-matches that were performed, two were in recipients who had no pretransplant antibodies detectable by FlowPRA. Both of these cross-matches were negative. In contrast, five of seven cross-matches were positive among recipients who had FlowPRA detectable pretransplant antibodies. Posttransplant serum specimens from 91 patients were also assessed for antibodies by FlowPRA. Among this group, 58 patients had FlowPRA antibodies and there was a trend (although not statistically significant) for a biopsy documented episode of rejection to have occurred among patients with these antibodies. CONCLUSIONS: Collectively, our data suggest that pre- and posttransplant HLA antibodies detectable by FlowPRA and not AHG-CDC identify cardiac allograft recipients at risk for rejection. Furthermore, a positive donor reactive flow cytometric cross-match is significantly associated with graft loss. Thus, we believe that detection and identification of HLA-specific antibodies can be used to stratify patients into high and low risk categories. An important observation of this study is that in the majority of donor:recipient pairs, pretransplant HLA antibodies were not directed against donor antigens. We speculate that these non-donor-directed antibodies are surrogate markers that correspond to previous T cell activation. Thus, the rejection episodes that occur in these patients are in response to donor-derived MHC peptides that share cryptic determinants with the HLA antigens that initially sensitized the patient.
Progressive cerebral amyloid beta-protein (A beta) deposition is believed to play a central role in the pathogenesis of Alzheimer's disease (AD). Elevated levels of A beta(42) peptide formation have been linked to early-onset familial AD-causing gene mutations in the amyloid beta-protein precursor (A beta PP) and the presenilins. Sequential cleavage of A beta PP by the beta- and gamma-secretases generates the N- and C-termini of the A beta peptide, making both the beta- and gamma-secretase enzymes potential therapeutic targets for AD. The identity of the A beta PP gamma-secretase and the mechanism by which the C-termini of A beta are formed remain uncertain, although it has been suggested that the presenilins themselves are novel intramembrane-cleaving gamma-secretases of the aspartyl protease class [Wolfe, M. S., Xia, W., Ostaszewski, B. L., Diehl, T. S., Kimberly, W. T., and Selkoe, D. J. (1999) Nature 398, 513-517]. In this study we report the identification of L-685,458 as a structurally novel inhibitor of A beta PP gamma-secretase activity, with a similar potency for inhibition of A beta(42) and A beta(40) peptides. This compound contains an hydroxyethylene dipeptide isostere which suggests that it could function as a transition state analogue mimic of an aspartyl protease. The preferred stereochemistry of the hydroxyethylene dipeptide isostere was found to be the opposite to that required for inhibition of the HIV-1 aspartyl protease, a factor which may contribute to the observed specificity of this compound. Specific and potent inhibitors of A beta PP gamma-secretase activity such as L-685,458 will enable important advances toward the identification and elucidation of the mechanism of action of this enigmatic protease.
Cleavage of amyloid precursor protein (APP) by the beta- and gamma-secretases generates the amino and carboxy termini, respectively, of the A beta amyloidogenic peptides A beta40 and A beta42--the major constituents of the amyloid plaques in the brain parenchyma of Alzheimer's disease patients. There is evidence that the polytopic membrane-spanning proteins, presenilin 1 and 2 (PS1 and PS2), are important determinants of gamma-secretase activity: mutations in PS1 and PS2 that are associated with early-onset familial Alzheimer's disease increase the production of A beta42 (refs 4-6), the more amyloidogenic peptide; gamma-secretase activity is reduced in neuronal cultures derived from PS1-deficient mouse embryos; and directed mutagenesis of two conserved aspartates in transmembrane segments of PS1 inactivates the ability of gamma-secretase to catalyse processing of APP within its transmembrane domain. It is unknown, however, whether PS1 (which has little or no homology to any known aspartyl protease) is itself a transmembrane aspartyl protease or a gamma-secretase cofactor, or helps to colocalize gamma-secretase and APP. Here we report photoaffinity labelling of PS1 (and PS2) by potent gamma-secretase inhibitors that were designed to function as transition state analogue inhibitors directed to the active site of an aspartyl protease. This observation indicates that PS1 (and PS2) may contain the active site of gamma-secretase. Interestingly, the intact, single-chain form of wild-type PS1 is not labelled by an active-site-directed photoaffinity probe, suggesting that intact wild-type PS1 may be an aspartyl protease zymogen.
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In vivo antigenic competition of naive CD4+ TCR transgenic T cells was visualized by tracking cell division. Competition reduced both recruitment into cell division and burst size per recruited precursor cell, minimizing the effect of differences in precursorfrequency while maintaining the dose-response relationship with antigen. Competition was restricted to T cells of the same specificity, indicating that cells were competing for access to Ag-MHC complexes rather than for Ag nonspecific factors. Moreover, the qualitative distinction between the responses to i.v. peptide and s.c. peptide/CFA was unaffected by precursor frequency. These data explain the paradoxical ability of the immune system to tailor responses to the type and dose of Ag even in individuals with large differences in initial precursor frequency.
We studied the seroprevalence of three viruses (mouse cytomegalovirus (MCMV), minute virus of mice (MVM), and mouse parvovirus (MPV)) in house mice (Mus domesticus) in 1995 7. In the first year average mouse density was less than 1 mouse/ha. From November 1995 to May 1996 the population increased at an average rate of 7% per week, a doubling time of about 10 weeks. From August 1996 to May 1997 the population increased at an average rate of 10% per week, a doubling time of about 7.5 weeks. From a peak around 250 mice/ha in May 1997, the mouse population fell 19% per week to 5 mice/ha in October 1997. The seroprevalence for all three viruses varied dramatically over time. MCMV had the highest seroprevalence (61.7%), followed by MVM (8.5%) and MPV (18.4%). Time series data indicated that MCMV spread rapidly through the population of mice once trap success was greater than 14% (40-100 mice/ha). By contrast MVM and MPV seroprevalence occurred with a 2-3 month and 3-4 month time lag, respectively. The current study supports the contention that MCMV would be a good carrier for an immunocontraceptive vaccine for controlling field populations of mice.
Although gammadelta cells are commonly hypothesized to provide a 'first line of defence', gammadelta-cell-deficient mice are generally only marginally more susceptible to pathogens. Because gammadelta cells are enriched within epithelia, it is important to resolve whether immunoprotective capacity towards epithelial-tropic pathogens is absent from the gammadelta-cell compartment, or whether such activity is present but simply redundant with that of alphabeta T cells. In this work, following infection of the intestinal epithelium of alphabeta T-cell-deficient mice with the coccidian parasite, Eimeria vermiformis, gammadelta cells were shown to support the rapid activation of other lymphoid cells and to confer a transferable antipathogen effect that could be eradicated by neutralization of interferon-gamma. However, unlike alphabeta T cells, these effects of gammadelta cells showed no evidence of functional immunological memory. These results are directly relevant to coccidiosis, an economically significant disease of livestock, and should have general relevance to infections involving alphabeta T-cell deficiencies, e.g. cryptosporidiosis in patients with acquired immune deficiency syndrome (AIDS).
OBJECTIVE: To examine the clinical spectrum of hepatitis C virus (HCV) infected children in our care by determining presentation, mode of acquisition, degree of co-infection, biochemical evidence of persisting hepatitis and treatment outcome. METHODOLOGY: A retrospective review of the medical records of all children attending the Royal Children's Hospital, Melbourne, between 1990 and 1998, who had antibodies to HCV infection detected. Detailed clinical information, investigations and the results of treatment were extracted from the clinical notes. RESULTS: A total of 94 children (age range 2 weeks to 19.7 years) were identified, of whom nine had passive transfer of maternal antibodies from HCV-positive mothers and were excluded from analysis. Sixty-seven children (79%) were infected by transfusion of blood or blood products. Perinatal transmission occurred in 11 children (13%), and six children (7%) had a history of i.v. drug abuse. The majority of children were asymptomatic at presentation. Of the 65 patients tested for HCV-ribonucleic acid, 43 (66%) were positive. Fifty-seven cases had serial alanine aminotransaminase (ALT) measurements over a mean of 28 months. Of these, 38 (67%) had an abnormal ALT. Ten cases (12%) were co-infected with hepatitis B virus, HIV or both. Of 12 patients treated with interferon, four responded with normalisation of ALT from 3 to 12 months post-commencement of therapy. CONCLUSIONS: Although HCV was largely an asymptomatic condition in our clinic population, more than half the patients had biochemical evidence of ongoing liver damage. Given the chronicity of this infection in the majority of patients and the long-term risks of cirrhosis and hepatocellular carcinoma, children with HCV infection represent a high-risk group worthy of regular follow up.
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This essay surveys four recent theological works on the issue of personhood: John F. Crosby's The Selfhood of the Human Person, Stanley Rudman's Concepts of Person and Christian Ethics, Metropolitan Hierotheos Vlachos' The Person in the Orthodox Tradition, and James Walter's What is a Person? An Ethical Exploration.
In August 1996, a patient in Kansas developed an Enterobacter cloacae bloodstream infection (BSI) shortly after receiving Albuminar, a brand of human albumin. Albuminar contamination was suspected. A case-control study of patients with primary gram-negative bacterial BSIs showed that patients with E. cloacae BSIs were significantly more likely than patients with non-E. cloacae gram-negative BSIs to have received Albuminar within 3 days of developing their BSIs (3 of 5 vs. 0 of 9; OR, undefined; P=.03). The E. cloacae isolate from the Kansas patient was found by pulsed-field gel electrophoresis to be identical to the isolate from the patient's Albuminar vial, to isolates from 2 previously unopened Albuminar vials, and to an isolate from a Wisconsin patient who had received Albuminar. A worldwide recall of approximately 116,000 Albuminar vials took place. This multistate outbreak was detected because of clinical astuteness and prompt reporting. Combined epidemiological and laboratory approaches are valuable when investigating potentially contaminated blood components and plasma derivatives.
Because most pathogens initially challenge the body at epithelial surfaces, it is important to dissect the mechanisms that underlie T-cell responses to infected epithelial cells in vivo. The coccidian parasites of the genus Eimeria are protozoan gut pathogens that elicit a potent, protective immune response in a wide range of host species. CD4+ alpha beta T cells and gamma interferon (IFN-gamma) are centrally implicated in the primary immunoprotective response. To define any additional requirements for the primary response and to develop a comparison between the primary and the secondary response, we have studied Eimeria infections of a broad range of genetically altered mice. We find that a full-strength primary response depends on beta(2)-microglobulin (class I major histocompatibility complex [MHC] and class II MHC and on IFN-gamma and interleukin-6 (IL-6) but not on TAP1, perforin, IL-4, Fas ligand, or inducible nitric oxide synthetase. Indeed, MHC class II-deficient and IFN-gamma-deficient mice are as susceptible to primary infection as mice deficient in all alpha beta T cells. Strikingly, the requirements for a highly effective alpha beta-T-cell-driven memory response are less stringent, requiring neither IFN-gamma nor IL-6 nor class I MHC. The class II MHC dependence was also reduced, with adoptively transferable immunity developing in MHC class II(-/-) mice. Besides the improved depiction of an immune response to a natural gut pathogen, the finding that effective memory can be elicited in the absence of primary effector responses appears to create latitude in the design of vaccine strategies.
BACKGROUND: Wilson's disease is a rare but treatable condition that often presents diagnostic dilemmas. These dilemmas have for the most part not been resolved by the identification and cloning of the Wilson's disease gene. AIMS: To report our experience over three decades with patients with Wilson's disease in order to illustrate the diverse patterns of presentation and thereby broaden the approach to diagnosis. METHODS: Clinical and laboratory findings of 30 patients with Wilson's disease were reviewed. RESULTS: Twenty two patients presented with liver manifestations (eight with fulminant hepatic failure and 14 with chronic liver disease), three with neurological disease, and one with haemolysis; four were asymptomatic siblings of patients with Wilson's disease. Seventy per cent were diagnosed within six months of the onset of symptoms, but diagnosis was delayed for up to nine years. Age range at diagnosis was wide (7-58 years) and five patients were over 40. In patients presenting with non-fulminant disease, 18% had neither Kayser-Fleischer rings nor low caeruloplasmin concentrations. Increased liver copper concentrations were found in all but one patient who had undergone six years of penicillamine treatment. In fulminant hepatic failure (n=8) additional features helpful in the diagnosis included evidence of haemolysis, increased urinary copper (range 844-9375 microg/24 h), and a high non-caeruloplasmin copper (range 325-1743 microg/l). CONCLUSIONS: The diagnosis of Wilson's disease still depends primarily on the evaluation of clinical and laboratory evidence of abnormal copper metabolism. No one feature is reliable, but the diagnosis can usually be made provided that it is suspected. Wilson's disease should be considered in patients of any age with obscure hepatic or neurological abnormalities.
BACKGROUND: There are limited data on the use of percutaneous revascularization techniques for transplant coronary artery disease (CAD). METHODS: Medical records and angiographic results for cardiac transplant patients undergoing percutaneous revascularization at Emory University Hospital were reviewed. Procedural results, results of angiography 4Eth 6 months after intervention, and clinical follow-up were recorded. RESULTS: Nineteen patients underwent 51 interventions. Thirty-eight lesions (75%) were de novo and 13 (25%) were restenotic. All patients had hypertension, 37% had diabetes, 79% had elevated lipid levels, and 53% had at least one episode of moderate to severe allograft rejection (grade 3A or greater). The primary procedural success rate was 100% with no major complications. Six-month restenosis rate (defined as > 50%) was 49%. At 23+/-17 months follow-up, 6 patients were dead or retransplanted (31%). Thirteen patients were alive without retransplantation (9 New York Heart Association class I, 3 class II, 1 class III). CONCLUSION: Percutaneous revascularization is safe and has a high initial procedural success rate in patients with transplant CAD. However, the restenosis rate in this population remains higher than reported for atherosclerotic coronary disease and the long-term prognosis remains poor.
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BACKGROUND AND PURPOSE: The major goal was to determine whether variations in the method of CO2 euthanasia would induce significant immunologic differences. METHODS: Young adult C57BL/6 mice (n = 40) were euthanized, using four regimens: 70% CO2/30% O2; 70% CO2/30% O2-->100% CO2; 100% CO2-naïve chamber; and 100% CO2 pre-charged chamber. Time to recumbency and euthanasia and body, liver, lung, spleen, and thymus masses were determined. Blood and spleen were further evaluated for leukocyte, lymphocyte, and thrombocyte counts, erythrocyte characteristics, distribution of lymphocyte subpopulations, spontaneous and mitogen-induced blastogenesis, complement activity, and cytokine production. RESULTS: Time to euthanasia was five- to eightfold longer in mice exposed to 70% CO2/30% O2 than that for any other group. There were slight increases in mean erythrocyte volume (MCV) and mean erythrocyte hemoglobin (MCH) for all groups, compared with those for the 100% CO2 pre-charged group. Circulating cytotoxic T (CD8+) lymphocyte percentages and numbers, and spontaneous blastogenesis of leukocytes in blood and spleen, also were affected by euthanasia method. CONCLUSIONS: The method of CO2 euthanasia can result in significant differences in immunologic/hematologic variables. Thus, consistency in euthanasia procedures may be important in accurate interpretation of research data.