Biomedical subjects
Publications and source records attributed to A Mayne.
Children and adults can have recurrent infection with invasive encapsulated bacterial pathogens such as Streptococcus pneumoniae as a result of a selective inability to respond to polysaccharide antigens. We have identified a mutation in the gene encoding Bruton's tyrosine kinase in a male patient with selective anti-polysaccharide antibody deficiency.
As a close cousin to Asian macaques, the African sooty mangabey monkey, a species naturally infected with SIV without ever developing disease, represents an intriguing and thought provoking model for the study of lentiviral infection and disease development. Pursuant to our previous findings that documented the presence of high frequencies of CD8+ T-cells capable of inhibiting lentiviral replication in vitro via a soluble factor, the potential contribution of beta-chemokines and their receptor was evaluated in samples from sooty mangabeys and disease susceptible macaques. Both mangabey and Rhesus macaque PBMC were found to secrete comparable levels of MIP-1alpha, MIP-1beta and RANTES after stimulation in vitro. Constitutive expression of CCR-5 appeared lower in mangabey PBMC but the vast majority of T-cells from mangabeys or macaques were found to express CCR-5 after in vitro activation. Sequence analysis of macaque and mangabey MIP-1alpha and RANTES showed complete homology to the human counterpart. MIP-1beta on the other hand while highly conserved among both monkey species, showed only 93% homology to human MIP-1beta. In addition, CCR-5, CCR-2b and CXCR-4 presented no consistent differences between rhesus and mangabey sequences. Thus, based on current data, differences in disease susceptibility between macaques and mangabeys do not appear to rely on differences in chemokine pathways. However, analyses of the ontogeny of CD8+ T-cell-mediated inhibition of SIV replication showed that macaque PBMC acquire this function shortly after infection, and show a progressive loss thereafter, correlated with progression towards disease. Mangabeys, on the other hand, appear to acquire the CD8+ T-cell inhibitory function long before any evidence of seroconversion to SIV and maintain this function for the lifetime of the animal. In vitro analyses of induction of the CD8+ inhibitory function showed that rhesus CD8+ T-cells have the potential to secrete the inhibitory factor(s) prior to SIV infection.
Previous studies have shown that in vitro culture of human CD4+ T cells with antibodies to CD3 and CD28 immobilized on beads induced an antiviral effect to HIV-1 infection. Herein, we have used CD4+ T cells from nonhuman primates to address issues critical for use of such cells for therapy and immune reconstitution of humans and nonhuman primates infected with HIV and simian immunovirus (SIV). These studies include definition of the kinetics of the antiviral effect, the relative stability of the acquired phenotype, and whether such activated and expanded CD4+ T cells retain their immune function. Results of our studies show that antiviral effect is induced rapidly following activation with anti-CD3/CD28-coated beads. Additionally, the antiviral effect is not stable in these cells and requires continuous culture with anti-CD3/CD28 beads. Removal of CD4+ T cells from anti-CD3/CD28 stimulation renders these cells susceptible to infection, demonstrating that the resistant phenotype is not stable in these cultures. However, anti-CD3/CD28 expanded CD4+ T cells do retain immune function. Thus, although these findings imply a note of caution for therapeutic strategies aimed at providing patients with virus-resistant CD4+ T cells, the present study suggests that transfusion of such cells with retained immune function may have immune restoration capability.
The hematologic abnormalities of SIV and HIV are well described, although the mechanisms that lead to hematopoietic dysfunction are yet to be fully defined. A number of growth factors and cytokines have been used to induce the differentiation, maturation, and proliferation of appropriate lineages, with the aim that such therapy will lead to functional hematopoietic reconstitution. Within this context, some cytokines have been shown to influence HIV and SIV replication in vitro and, in selected cases, in vivo. However, few studies detail the effects of hematopoietic cytokines such as IL-3, Flt-3 ligand, G-CSF, Tpo, and Epo or correlate the effects on virus replication. In an effort to address this issue, we infected 12 rhesus macaques with 500 TCID50 of SIVmac239 and intensively evaluated hematologic, virologic, and immunologic parameters during administration of cytokines. When all animals had lymphadenopathy, hepatosplenomegaly, and CD4+ cell counts > or =1000/microl, subgroups of three rhesus macaques were administered either rhFlt-3; rrIL-3a; combination of rhG-CSF, rhTpo, and rhEpo (rhGET); or rrIL-12. Fourteen days of rhFlt-3 administration induced expansion of the bone marrow CD34+ cells and granulocyte-macrophage colony-forming units (GM-CFUs) and increased absolute peripheral blood CD34+ cells and total CFUs. Following rrIL-3 and rhGET administration absolute peripheral blood CD34+ cells and total CFUs increased. rhGET also increased granulocyte, platelet, and reticulocyte counts by day 14 of administration. Branched DNA and coculture assays did not demonstrate any significant change in viral load with any of the cytokines administered. These data suggest that SIV-infected rhesus macaques have the hematopoietic capability to expand and mobilize CD34+ and GM-CFU progenitors and formed elements at 6-8 months postinfection in response to various cytokines, without increasing viral load.
A number of rhesus macaques experimentally infected with SIV isolates such as SIVmac251, fail to seroconvert, develop high plasma viremia and die rapidly (within 6-7 months p.i.). We hypothesized that such rapid progression is a result of a state of hyperimmune activation and concomitant immune suppression of these animals at the time of virus challenge. In efforts to test the hypothesis that immune activation leads to rapid progression of lentivirus-induced disease, adult rhesus macaques were infected with SIV mac251 and received an alternate monthly schedule of repeated immunization with allogeneic cells, keyhole limpet hemocyanin and tetanus toxoid (group I). For purposes of controls, a group of monkeys was infected with the same pool and dose of virus but were not immunized (group II) and a group was immunized with the same schedule of multiple antigens as group I but were not infected with SIV (group III). All the animals in group I (n = 3) either failed to seroconvert or developed very low levels of SIV antibodies, had high plasma p27 defined antigenemia, and died within 8 months (2/3 died within 4 months). Of the animals in group II (n = 8), two patterns emerged as we had noted before. One subgroup (3 animals), displayed the same profile as group I (failure to fully seroconvert, high p27 levels and death by 8 months), whereas the other subgroup (5 animals) seroconverted, had low plasma p27 levels, and survived past 11 months (2/5 still alive past 22 months). All 3 animals in group III remained healthy. The data provided herein suggest that either experimental or natural (due to factors not clear at present) immune stimulation may lead to accelerated lentivirus induced disease progression most likely due to immune suppression and has implications for the understanding of the mechanisms for the rate of disease progression in human HIV-1 infection.
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Nonhuman primates naturally infected with simian immunodeficiency virus (SIV), while maintaining chronic viremia, do not develop any disease associated with lentiviral infection. Thus they provide a unique model to define the mechanism(s) by which they remain infected but disease-resistant. The purpose of this article is to summarize our current knowledge of the virological and immunological studies that have been performed in sooty mangabeys naturally infected with SIVsmm and in disease-susceptible rhesus macaques experimentally infected with SIVsmm. Data on virological studies demonstrate that the naturally infected sooty mangabeys are infected predominantly with SIV that have nef sequences distinct from those shown to cause disease in the inappropriate host, a factor which may contribute to disease resistance. Hyperimmunization with a variety of antigens or chronic infection contributes to accelerated disease and death in rhesus macaques if hyperimmunizations are initiated at the time of SIV infection, whereas similar hyperimmunization and chronic infection do not lead to disease in naturally infected seropositive sooty mangabeys. However, in both species infected with SIV, hyperimmunization leads to increased virus load, suggesting that virus load per se cannot account for disease, at least in naturally infected nonhuman primates. Immunological studies concerning changes in subsets of T cells, based on cytokine profile (TH0/TH1/TH2), showed that whereas rhesus macaques early post SIV infection show a dominant TH1 profile, this profile rapidly changes to TH0. On the other hand, mangabeys continuously demonstrate a TH2-like profile. Studies also showed a high frequency of in vivo-activated cells in the peripheral blood of SIV-infected rhesus macaques and mangabeys. Of interest, however, is the finding of a similar level of in vivo-activated cells from ELISA seronegative mangabeys. Although cells from SIV-infected mangabeys fail to show increased levels of apoptotic cells following incubation with immobilized anti-CD3, PBMC from rhesus macaques at varying time intervals do show increased levels of apoptotic cells, an increase which is predominantly seen in CD8+ T cells and is unrelated to levels of viremia. Sooty mangabeys maintain a high frequency of CD8+ T cells that regulate virus replication throughout their lifetime, a frequency that develops prior to ELISA-based seroconversion, whereas rhesus macaques only show a frequency of CD8+ T cells high enough to regulate virus replication shortly post infection, and this regulatory function is gradually lost prior to CD8+ cell loss and death. HIV and SIV infection do have profound effects on the expression of a number of costimulatory and adhesion molecules. There appear to be differences in the nature of the intracellular phosphorylated proteins in cells from activated rhesus macaques and mangabeys. We believe that careful studies of the detailed mechanisms of the issues described above may provide an understanding of the constellation of virological and immunological mechanisms responsible for the disease-resistant state of naturally infected sooty mangabeys. These findings can be employed for evaluating a nonvirus sterilizing form of SIV/HIV vaccines.
The pathogenesis of AIDS in simian immunodeficiency virus (SIV)-infected rhesus macaques models the pathogenesis of human AIDS. We investigated whether certain immunological abnormalities associated with HIV infection and implicated in disease progression also occur in SIV infection. We observed striking parallels between the rhesus macaque humoral immune response to SIV and unusual features of the human humoral immune response to HIV infection. Anti-SIV envelope antibodies from SIV-infected macaques exhibited skewed kappa/lambda L chain usage relative to the ratio of kappa/lambda L chain usage detected on total plasma Ig. In addition, the same idiotope conserved on human anti-HIV gag, pol and env antibodies was detected with a similar distribution pattern on macaque anti-SIV env, gag and pol antibodies. Skewed Ig L chain representation and selection of a highly conserved possible dominant regulatory idiotope on antibodies against key neutralizing antigens raise the possibility that the progressive failure of the humoral immune response to SIV and HIV involves skewed oligoclonal humoral immunity. We also found that SIV-infected macaques, like HIV-infected humans, have elevated levels of CD8+ lymphocyte-mediated cytotoxicity. Cytotoxicity against human B cells was higher in CD8+ effector lymphocytes from SIV-infected macaques than in those from uninfected macaques (P < 0.05). Cell-mediated cytotoxicity against human NK cell targets was not elevated in effector lymphocytes from the SIV-infected macaques, suggesting that CTL activity was selectively elevated. Two out of seven SIV-infected macaques tested had CD8+ cytotoxic lymphocytes that selectively killed activated uninfected CD4+ macaque lymphocytes. Oligoclonal B cell responses and persistently elevated CD8+ lymphocyte-mediated cytotoxicity are consistent features of primate retroviral infections leading to AIDS. Understanding immune predispositions to these features triggered by HIV and SIV may advance new prophylactic and therapeutic strategies.
One of the manifestations of human HIV-1 and nonhuman primate SIV infection that lead to disease is reasoned to be secondary to generalized T-cell dysfunction. The molecular mechanisms associated with the T-cell dysfunction remain to be elucidated. To address this issue, we sought to utilize the nonhuman primate model to study intracellular signaling events in cells from disease-susceptible rhesus macaques and disease-resistant sooty mangabeys. Because relatively little is known about these events in nonhuman primates, our laboratory defined optimal conditions, reagents, and assays for the study of signal transduction events in cells from nonhuman primates. The protein phosphorylation patterns in the two monkeys exhibited quantitative, qualitative, and kinetic differences. Antibodies to Stat6 detected a unique band in macaque cell lysates. This band is markedly decreased human cell lysates and never seen in mangabey cell lysates. Detection of various other intracellular signaling proteins is also described.
Two major issues severely limit the studies of human recombinant cytokines/growth factors in nonhuman primates. First, assays and reagents specific for the detection and quantitation of human cytokines do not all function when utilized to detect/quantitate the nonhuman primate cytokines. Second, although most of the human cytokines appear to induce similar, if not identical, biologic function when used with cells from nonhuman primates in vitro or in vivo, they invariably induce Ab responses in vivo, precluding their repeated and/or continued use in vivo. Our laboratory has thus initiated studies to clone, sequence, and prepare recombinant cytokines from nonhuman primates and to define assays and reagents for their detection and quantitation at the nucleic acid and protein level. The data that were derived from such studies show that the nonhuman primate cytokines IL-1 alpha, IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12 alpha, IL-12 beta, IL-15, IFN-alpha, IFN-gamma, and TNF-alpha share 93 to 99% homology at the nucleic acid and protein level with the human equivalents. The most prominent differences between human and nonhuman primate cytokine sequences were noted for IL-1 alpha/beta, IL-2, IL-8, IFN-alpha, IFN-gamma, and IL-12 beta. The aligned sequences of cytokines for human and several nonhuman primate species are provided herein, and a phylogenetic analysis of the published sequences of select cytokines from other species, along with those of the nonhuman primates, are described. In addition, comparative analysis of the relative bioactivity of our immunoaffinity-purified recombinant rhesus macaque IL-4, IL-15, and IFN-gamma with commercially available human recombinant cytokines is described herein.
BACKGROUND: Histological evaluation of serial endomyocardial biopsies performed at fixed time intervals after cardiac transplantation is the universal method used for the detection of cardiac rejection and assessment of the adequacy of antirejection therapy. No noninvasive methodology thus far investigated has achieved a high enough sensitivity and predictive accuracy to be considered as a potential replacement for endomyocardial biopsy in the detection of rejection in adults. The present study exploited the finding that the rate of spontaneous mutation in the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene is higher in proliferating human T cells than in resting cells. Thus, it was reasoned that in the posttransplantation setting, the frequency of HPRT- cells in peripheral blood may provide an indirect measure of alloactivated T lymphocytes. METHODS AND RESULTS: This study consisted of determining the clonal frequency of HPRT- mutant cells (FMC/10(6) peripheral blood mononuclear cells [PBMCs]) within a total of 293 peripheral blood samples representing various numbers of sequential samples from each of 27 transplant recipients. These sequential samples represented time periods when endomyocardial biopsy specimens showed either (1) no evidence of rejection (n = 5 patients), (2) a single initial episode after transplantation of early (< 1 year) or late (> 1 year) rejection (n = 12 patients), or (3) multiple rejection episodes (n = 10 patients). Statistical analyses were used to quantify the time profiles of FMC/10(6) PBMCs in serial samples among transplant recipients and to determine the association of these profiles with both the onset of first rejection episodes and, in appropriate patients, the recurrence of rejection episodes. Data showed that PBMCs from patients with no evidence of rejection uniformly gave low values of < 6 FMC/10(6) cells, a frequency similar to that seen in healthy nontransplanted volunteers. In contrast, 19 of the 22 PBMC samples that were obtained from patients whose corresponding biopsy sample was diagnosed with a histological rejection grade of > or = 3 gave values of > 6 FMC/10(6) cells, 11 of which gave values > 50/10(6) cells (range, 146 to 46,982 FMC/10(6) cells). A significant association between the onset of first rejection and an increased rate of FMC/10(6) values was noted (P = .0001). The ability of a rising trend in FMC/10(6) values to correctly identify the onset of rejection was 81.8% and to correctly identify no rejection, 100%. In addition, a significant association between recurrent rejection episodes and persistence of high FMC/10(6) values in the weeks after treated rejection episodes was noted (P = .0003). The ability of a persistently elevated trend in values of FMC/10(6) cells to correctly identify recurrent rejection was 90% and to correctly identify no rejection, 100%. CONCLUSIONS: Increasing frequencies of HPRT- mutant cells in peripheral blood correlated with the onset of first rejection, and persistently elevated HPRT- mutant cells in the weeks after a treated rejection episode correlated with recurrent rejection. This quantitative noninvasive assay may thus serve as a useful adjunct to endomyocardial biopsy for monitoring post-cardiac transplantation patients, and its use as a prospective diagnostic tool merits further study.
The aim of this retrospective study is to evaluate the advantage of thoracoscopy and the efficacy of talcage in the treatment of spontaneous pneumothorax (SP). Two hundred cases have been analyzed with a follow-up of 1 to 8 years after the occurrence of the disorder. The ratio man/woman is 4/1. One hundred and forty two pneumothorax are considered as being of idiopathic origin and 58 are associated to bronchopneumopathy, with a mean age of 33 and 56 years, respectively. The percentage of smokers is 69.5% with a mean smoking of 14 packets/years. The endoscopic aspect of pleura is either normal (30%) or shows adhesions (23.5%), blebs (17%) or bullaes (29.5%). Thoracoscopy allowed talc poudrage in 191 patients and allowed to indicate the need for surgery in nine patients. The immediate success rate of talcage is 93.7%. In the group of immediate failure (6.3%), unexpected bullous structures (8/12) are found at tomodensitometry (TDM), as well as during surgery. Late recurrence is reported in 2 cases (1%) at 20 and 25 months. Radiological sequelaes are minimum (9%). Lung function testing in patients with idiopathic pneumothorax (n = 64) shows, before talc poudrage, signs of pulmonary hyperdistension (total lung capacity (TLC) at 116% of predicted values), reflecting the illness pathology, 3 months after talcage a discrete restrictive syndrome (TLC 93%) and one year after the partial recovery of the lost volume (TLC 105%). Tomodensitometry revealed to be complementary to thoracoscopy in secondary SP and very instructive in idiopathic SP after immediate failure of talc poudrage.(ABSTRACT TRUNCATED AT 250 WORDS)
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The self-expandable Gianturco stent was used in eleven cases of tracheal stenosis treated from September 92 to July 94. The stent is placed through a 9 mm outside diameter bronchoscope under optical control after cross-section and dilation of the stenosis with the CO2 laser according to the Shapshay technique. The follow-up varies from 3 to 22 months with a mean of 12 +/- 6 months. Pulmonary function tests (PFTs) show a mean improvement of the PEF (peak expiratory flow). Complications have been corrected in three patients with a second endoscopic procedure: the first case for malposition, the second for granuloma at the superior end of the prosthesis, and the third for the development of a mucous membrane webbing between the two loops of the stent. Owing to the lack of long-term follow-up, we reserve this technique for the treatment of severe tracheal stenosis with cartilage impairment in patients contraindicated when external reconstructive surgery is contraindicated.
Sera from 29 of 48 patients with idiopathic dilated cardiomyopathy (IDCM) and six of six patients with dilated cardiomyopathy (DCM) secondary to suspected viral myocarditis were shown to react with the branched chain alpha-ketoacid dehydrogenase (BCKD) complex mitochondrial proteins. Whereas sera from only 1 of 26 patients with ischemic heart disease showed reactivity against the BCKD complex protein, 0 of 30 sera from normal human volunteers, 0 of 64 sera from patients with lupus, and 0 of 34 sera from patients with rheumatoid arthritis showed detectable reactivity, denoting an element of specificity for the reactivity of sera from IDCM patients. The major reactivity was localized to the dihydrolipoyl transacylase (E2) component of BCKD complex. By using recombinant techniques, the immunodominant BCKD-E2 epitope recognized by sera from IDCM patients was localized to amino acid (aa) sequences 116 to 134. Each of the IDCM sera that reacted with the native BCKD complex was shown to react with the immunodominant peptide, as defined by a peptide inhibition ELISA and by an ELISA using the reactive peptide conjugated to BSA. Sera from IDCM patients that reacted with the native BCKD complex and the reactive peptide also showed inhibition of BCKD enzyme activity. The possible mechanisms for the induction of the Abs and the implications of these findings for the pathogenesis of IDCM are discussed.