A. Ahmed-Ansari

Induction of major histocompatibility complex antigens within the myocardium of patients with active myocarditis: A nonhistologic marker of myocarditis

The histologic diagnosis of active myocarditis is frequently difficult to establish. A nonhistologic marker of immune activation would be clinically useful in identifying cases of immune-mediated myocarditis. A viral etiology with subsequent autoimmunity to cardiac antigens has been implicated in human myocarditis. Because autoimmunity and viral disease are commonly associated with increased expression of major histocompatibility complex (MHC) antigens on targeted tissue, we examined endomyocardial biopsy samples from patients with active myocarditis for abnormal levels of MHC antigen expression. Thirteen patients with active myocarditis and eight control patients with other well-defined cardiac diagnoses (coronary disease, amyloidosis or neoplasm) were studied. A sensitive radioimmunoassay was developed that utilized monoclonal antibodies to human MHC class I and class II antigens in order to quantitate the expression of both of these antigens within each biopsy. Abnormal MHC class I and class II antigen expression was present in 11 of 13 myocarditis specimens and 1 of 8 control samples (specificity 88%, sensitivity 84.6%). Active myocarditis samples had approximately a 10-fold increase in MHC class I and class II expression. Immunoperoxidase staining localized abnormal MHC expression primarily within microvascular endothelium and along myocyte surfaces (11 of 13). This study is the first to demonstrate a marked increase in major histocompatibility complex antigen expression within the myocardium of patients with active myocarditis. The identification of abnormal histocompatibility antigen expression within an endomyocardial biopsy may prove a useful adjunct to the histologic diagnosis of myocarditis.

Establishment of a human fetal cardiac myocyte cell line

SummaryHuman cardiac myocytes undergo degeneration, cytolysis, and necrosis in a number of clinical disease conditions such as myocarditis, dilated cardiomyopathy, and during episodes of cardiac allograft rejection. The precise cellular, biochemical, and molecular mechanisms that lead to such abnormalities in myocytes have been difficult to investigate because at present it is not possible to obtain and maintain viable cell cultures of human adult cardiac myocytes in vitro. However, human fetal cardiac myocytes are relatively easy to maintain and culture in vitro, but their limited availability and growth, variability from one preparation to another, and varying degrees of contamination with endothelial and epithelial cell types have made it difficult to obtain reliable data on the effect of cardiotropic viruses and cardiotoxic drugs on such myocytes. These thoughts prompted us to attempt to derive a cell line of human cardiac origin. Highly enriched human fetal cardiac myocytes were transfected with the plasmids pSV2Neo and pRSVTAg and gave rise to a cell line (W1) which has been maintained in culture for 1 yr. Morphologic and phenotypic analyses of W1 cells by flow microfluorometry and immunoperoxidase techniques indicate that the W1 cell line shares many properties of human fetal cardiac myocytes, but appears not to react with specific antibodies known to react with markers unique to human endothelial, epithelial, skeletal muscle, and dendritic cells. These preliminary data suggest that the W1 cells may provide a unique source of an established cell line that shares many properties ascribed to human cardiac myocytes.

Major histocompatibility complex class I and class II expression by myocytes in cardiac biopsies posttransplantation.

A total of 85 cardiac biopsies from patients 23–265 days posttransplant were studied for the correlation of the rejection grade score with the level of major histocompatibility complex (MHC) class I and class II expression on cardiac myocytes and endothelial cells, the quantitative level of leukocytic infiltrate, and the immunophenotype of the leukocytes. Results indicate a lack of absolute correlation between rejection grade scores and levels of MHC antigen expression. Further, a lack of absolute correlation was also seen with quantitation of leukocytic infiltrates and relative levels of MHC antigen expression. Of great interest was our preliminary finding that as early as 4 weeks prior to a rejection episode scored by routine histological criteria as grade 4, cardiac biopsy from the patient demonstrated high levels of MHC class I and class II expression. Similar increases of MHC antigen expression prior to an increase in histological rejection score grades were also noted in serial biopsies of 2 other patients. These data suggest that it may be quite useful to examine levels of MHC antigens on cardiac biopsies posttransplantation as an additional parameter for monitoring of cardiac rejection episodes.

Group formation of female pigtail macaques (Macaca nemestrina)

Human epidemiological studies have suggested that social variables can modulate the effects of stress on the immune system, and this concept has been gaining increasing attention with positive results emerging from empirical studies using nonhuman primates over the last two decades. Results from a previous study in rhesus monkeys suggested that receiving grooming positively affected recovery of T‐helper and T‐suppressor cells following the initial stress associated with group formation, and this co‐varied with high dominance rank. Thus, the present study was undertaken in order to determine: (1) if the stress effect of formation could be replicated in another species and (2) if social behaviors or dominance rank, given that formation is a stressor, might independently correlate with physiological recovery from the stressor. Eight adult female pigtail macaques were moved from individual cages and simultaneously introduced into an outdoor enclosure along with an adult male, while eight weight‐matched controls remained in individual caging. Behavioral data were collected during the introduction and over 4 weeks thereafter. Blood samples were collected prior to and at intervals for 4 weeks following formation. Compared to control subjects, the test subjects showed an increase in basal cortisol secretion (+28.9%) and a significant decrease in T‐helper cells (‐33.6%), T‐suppressor cells (‐30.8%), and B cells (‐22.5%), while there was a significant increase in white blood cells (+29.5%) 24 hr following formation. When dominance rank and seven behavioral categories were analyzed, only the frequency of receiving grooming significantly predicted change, with animals who received a greater frequency of grooms showing a lesser negative percent change from baseline in the absolute number of T‐helper cells 1 week following formation. The establishment of a dominance hierarchy, apparent within 1 week, was accomplished with no serious fighting and a complete absence of wounding or trauma, suggesting that psychosocial stress was responsible for the physiological changes observed.

Phenotypic and functional differences in NK and LAK cells in the peripheral blood of sooty mangabeys and rhesus macaques

Greater than 75% of the sooty mangabey monkeys at the Yerkes Regional Primate Research Center are naturally infected with SIV without any apparent clinical symptomology. On the other hand, experimental infection of rhesus macaques with SIV results in a clinical syndrome similar to human AIDS. These differences with regard to SIV infection prompted us to examine the natural immunosurveillance system of peripheral blood mononuclear cells (PBMC) from SIV-infected and uninfected monkeys of these two species. Phenotypic and functional studies of precursor and effector NK and LAK cells in the PBMC from these two species were carried out using monoclonal reagents, flow microfluorometry (FMF), and the standard in vitro 51Cr release assay against prototype K562 (NK sensitive) and RAJI (NK resistant, LAK susceptible) target cell lines. Data indicate that both NK and LAK cell activities in the PBMC of sooty mangabeys were significantly (P less than 0.01) greater than those in rhesus macaques. The predominant NK effector cells and LAK cell precursors were shown to be Leu 19-CD8+ in the PBMC of sooty mangabeys and Leu19+ CD8- in the PBMC of rhesus macaques as determined by panning depletion techniques and FMF analysis. On the other hand, the predominant LAK effector cells were found to be dual marked Leu 19+ CD8+ in rhesus macaques and Leu 19- CD8+ in sooty mangabeys. These qualitative and quantitative differences were not due to SIV infection of these two species since PBMC from both SIV-seropositive and virus-positive and SIV-sero-negative and virus-negative monkeys gave similar results. Moreover, of importance is the finding that the functional NK and LAK precursor cells are CD8+ and CD8- in sooty mangabeys and rhesus macaques, respectively. These data may have implications for the natural SIV/SMM virus-positive asymptomatic state of sooty mangabeys and may provide useful tools for tracing the ontogeny and lineage derivation of NK and LAK cells.

Interferon-γ-treated K562 target cells distinguish functional NK cells from lymphokine-activated killer (LAK) cells

In vitro incubation of the erythroleukemic cell line K562 with interferon-gamma (IFN-gamma) renders these cells relatively resistant to natural killer (NK) cell lysis. However, such treatment does not alter their sensitivity to LAK cell lysis. Thus, the lytic susceptibility of interferon-gamma-treated K562 (I-K562) cells to LAK cells as opposed to its relative resistance to NK cell lysis provides a functional assay to help distinguish these two types of effector cells. The relative resistance of I-K562 for NK cell-mediated lysis was not secondary to the release of soluble factors or the frequency of Leu-19+, CD3+ T cells, residual IFN-gamma, or expression of MHC Class I molecules. Coincubation of I-K562 cells with NK or LAK cells overnight did not appreciably change the pattern of lytic responses against K562 and I-K562 target cells. However, incubation of PBMC in vitro with I-K562 but not native K562 in the presence of r-IL-2 leads to a marked decrease in the generation of LAK cells. The inhibition of LAK cell generation was not secondary to differences in the consumption of bioactive levels of IL-2. Differences in the lytic capability of NK and LAK effector cells suggest heterogeneity among cells that mediate such non-MHC-restricted lysis. Use was made of cells from a patient with a large granular lymphocyte lymphoproliferative disease (greater than 85% Leu-19+) to determine if such cells could be used to distinguish clonal population of cells which would represent NK or LAK cell function. Of interest was the finding that such cells, even after incubation in vitro with IL-2, showed lytic function representative of NK cells but not LAK cells. Data concerning the inhibition of LAK cell generation by I-K562 cells have important implications for future therapeutic trials of IFN-gamma and IL-2 in the treatment of human malignancies.

Influence of cytokines and immunosuppressive drugs on major histocompatibility complex class I/II expression by human cardiac myocytes in vitro

Human cardiac myocytes do not express detectable levels of major histocompatibility complex (MHC) class II antigens and express low levels, if any, of MHC class I antigens. During rejection episodes, cardiac biopsies show massive increases of MHC antigens, which are thought to be induced by cytokines released by donor-sensitized recipient mononuclear cells. In efforts to determine the nature of the cytokines that induce MHC expression on cardiac myocytes, human fetal cardiac myocyte cultures were established. Interferon-alpha (IFN-alpha), interferon-gamma (IFN-gamma), interleukin (IL)-1, IL-2, IL-3, IL-4, and tumor necrosis factor (TNF)-alpha were added to these cultures and dose/kinetics of MHC class I/II induction quantitated. Data show that IFN-gamma induces both MHC class I and II expression, and all the other cytokines (except IL-2) induce only MHC class I but not class II. Cytokines used in combination showed that IFN-alpha with TNF-alpha was the only combination that induced MHC class II expression. Addition of immunosuppressive drugs such as cytoxan, azathioprine, cyclosporine-A, and FK-506, even when added at the initiation of the cultures, did not appreciably affect the ability of the appropriate cytokines to induce MHC expression by the myocytes in vitro.

Simian immunodeficiency virus-specific T-cell-mediated proliferative response of infected rhesus macaques

While cell-mediated immunity is known to play an important role in controlling viral infections, its role in human and experimental animal models of human AIDS has not been established. To address this issue, four juvenile rhesus macaques were infected with simian immunodeficiency virus SIVMAC. Freshly isolated peripheral blood mononuclear cells from these SIVMAC-infected macaques and four uninfected control macaques were assessed for T-cell proliferative activity to SIV, monthly, for 10 consecutive months. T cells from SIV-infected monkeys failed to proliferate in response to SIV added directly to the culture. However, when SIV was processed by autologous antigen-presenting cells prior to culture with purified T cells, proliferative responses were uniformly demonstrated in SIV-infected monkeys, but not in uninfected controls. Proliferation in response to heat-inactivated SIV was mediated by CD4+ T cells and was shown to be MHC class II-restricted. However, the proliferative response to infectious SIV was mediated by both CD4+ and CD8+ T cells and was MHC class-restricted. As disease progressed, a decline in the T-cell proliferative response was observed.

Requirements for simian immunodeficiency virus antigen-specific in vitro proliferation of T cells from infected rhesus macaques and sooty mangabeys

The measurement of cell-mediated immunity against the etiologic agent of human AIDS (HIV) in the non-human primate model of AIDS (simian immunodeficiency virus, SIV) has been difficult. In general, culture of peripheral blood mononuclear cells from HIV-1− and SIV-infected humans and monkeys, respectively, with purified inactivated HIV and SIV virus preparations has given inconsistent or negative proliferative responses. However, we describe herein an assay which consists of coculturing monocytes that have been pulsed with inactivated SIVsmm with nylon-wool-purified autologous T cells, leading to antigen-specific T-cell proliferation. The proliferative response, which predominantly occurs in CD4+ T cells, is major histocompatibility complex (MHC) class II-restricted and requires antigen processing. This assay will greatly facilitate the identification of the immunodominant epitopes recognized by T cells in sooty mangabeys, which are naturally infected but remain clinically asymptomatic, and in rhesus macaques, in which experimental infection leads to clinical symptomatology similar to human AIDS, eventually resulting in death.