Assessment of the inflammatory process by endomyocardial biopsy in patients with dilated cardiomyopathy based on pathological and immunohistochemical methods.

INTRODUCTION: Myocarditis may lead to dilated cardiomyopathy (DCM) in immunogenetically predisposed individuals. The diagnosis of myocardial inflammation is currently based on histopathological and immunohistochemical methods. Previous studies indicate that inflammatory cardiomyopathy occurs in approximately 50% of patients with DCM. AIM: The goal of the study was to assess the inflammatory process in patients with DCM by endomyocardial biopsy using histopathological and immunohistochemical methods. METHODS: Endomyocardial biopsy specimens was examined using routine histopathological methods and immunochemical staining for T lymphocytes (CD3(+), n=84), major histocompatibility complex I (HLA ABC, n=48) and II (HLA DPQR, n=84) antigens and the adhesion molecules ICAM-1 (n=51) and VCAM-1 (n=48) in 84 patients (69 male, 15 female; mean age 35.0+/-10.5 years) with angiographically-confirmed DCM. Familial disease occurrence was noted in 14 (16.7%) patients. Cardiac samples obtained from 18 patients who died of non-cardiovascular causes were used as a control group. RESULTS: Myocarditis was diagnosed, according to the Dallas criteria, in 8 (9.5%) patients. The frequency of inflammatory cardiomyopathy, defined as the presence of >2 CD3(+) T lymphocytes per high-power field (hpf) in myocardial biopsy, was 14.3%. When broader criteria were applied (presence of >2.0 CD3(+) lymphocytes/hpf and/or 1.5 CD3(+) lymphocytes/hpf in multiple foci and increased expression of class I/II HLA), inflammatory cardiomyopathy was diagnosed in 32.1% of patients. Inflammatory activation of the endothelium, indicated by increased expression of at least three adhesion molecules (class I and II HLA, ICAM-1, VCAM-1), was present in 22 (45.8%) patients. The expression of HLA DPQR, HLA ABC and ICAM-1 was observed on the endothelium of capillaries and larger vessels, interstitial cells, and the surface of activated lymphocytes; immunohistochemical reactions were diffuse. In patients with markedly elevated expression of the aforementioned adhesion molecules, the expression was also present on cardiomyocyte cell membranes. VCAM-1 was restricted to the endothelium of individual small veins. The control group did not demonstrate any signs of myocarditis, inflammatory cardiomyopathy or inflammatory endothelial activation. CONCLUSIONS: The application of immunohistochemical methods to myocardial biopsy in order to identify the inflammatory cell phenotype and the presence of adhesion molecules permits the diagnosis of inflammatory cardiomyopathy in 14% or 32% of patients, depending on the criteria used, while conventional pathology allows for this diagnosis in 9% of patients. The observed frequency of inflammatory cardiomyopathy, defined as the presence of >2 CD3(+) T lymphocytes/hpf in the myocardium, was lower (14%) than in previous studies, while the frequency of inflammatory endothelial activation was similar (45%).

Quantitative assessment of human leukocyte antigen-G protein in amniotic fluid by a double-determinant enzyme-linked immunosorbent assay using anti-human leukocyte antigen-G-specific antibody '87G'.

PROBLEM: The objective of this study was to establish an enzyme-linked immunosorbent assay (ELISA) system, in an attempt to quantify the amount of human leukocyte antigen (HLA)-G protein in amniotic fluid. METHOD OF STUDY: We established a double-determinant ELISA system using the anti-HLA-G specific mouse monoclonal antibody '87G' as a capture antibody and the horseradish-peroxidase labeled rabbit anti-human beta2-microglobulin antibody as a detection antibody. We then measured the concentration of HLA-G protein in amniotic fluid samples from nine normal second-trimester pregnant women and in serum samples from eight normal males. RESULTS AND CONCLUSION: HLA-G protein was detected in amniotic fluid at a concentration of 275 ng/ml (197-343 ng/ml) (median value and 95% confident range), whereas the concentration of HLA-G protein in male serum was below the minimum detection level.

Quantitative assessment of feline epidermal Langerhans cells.

The densities of feline epidermal dendritic cells expressing CD18, MHC class II and CD1a antigens were determined for four anatomical locations in 19 cats of European breed in blind conditions. The densities (+/- SD) of CD1a+ Langerhans cells in the skin of the abdominal wall (269 +/- 68 cells/mm2), the back (363 +/- 19), the internal side of the ear (572 +/- 30) and the external side of the ear (502 +/- 32) were significantly different, with young and old animals displaying less stained cells than adults. No significant differences in the mean densities were found with regard to sex, colour or antibody used.

Assessment of cytotoxic T-lymphocyte phenotype using the specific markers granzyme B and TIA-1 in cervical neoplastic lesions.

Cervical carcinomas are closely associated with high-risk human papillomavirus (HPV) types and are preceded by cervical intraepithelial neoplasia (CIN). Most CIN lesions regress spontaneously and will not evolve to invasive carcinoma. The cellular immune system mediated by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are thought to play an important role in the ultimate decline of CIN lesions. Although TIA-1 is constitutively expressed in the majority of circulating T cells and defines a subpopulation of CD8+ T cells with cytotoxic potential, granzyme B is only expressed in CTLs upon activation. In the present study we have evaluated the expression of these proteins by lymphocytes present in 24 randomly chosen CIN lesions with increasing degree of atypia and in 14 cervical squamous cell carcinomas. As major histocompatibility complex (MHC) class I expression is frequently down-regulated in HPV-induced lesions, thus possibly frustrating tumour cell recognition by infiltrating CTLs, these lesions were also analysed for MHC class I expression. The results indicated that in most CIN lesions only a minority of CTLs are activated, whereas in some carcinomas a massive infiltration of activated, i.e. granzyme B-positive, CTLs were observed. The percentage of activated CTLs was not related to expression of MHC class I on neoplastic cells. These results suggest that in some carcinomas proper activation of CTLs occurs but that most likely local factors or immunoselection of resistant neoplastic cells inhibit a proper response of CTLs to these neoplastic cells.

Assessment of CD4 expression by early T precursor cells and by dendritic cells in the human thymus.

The adult mouse thymus contains a minute population of early lymphoid precursor cells that express moderate levels of CD4. We searched for a corresponding population of early T precursors in the infant human thymus, by first depleting the majority of more mature thymocytes, then using immunofluorescence and flow cytometry to analyze cells bearing a range of early T lineage markers. No discrete population of early T precursors expressing CD4 was observed, in contrast to the murine thymus. Most putative very early human thymocytes were CD4-8-3-1-2lo44+34+7hi class I MHChi class II MHC-. However, a distinct population of human thymic dendritic cells expressing high levels of CD4 was isolated. These were CD4hi8-3-1-2-44+34-7- class I MHChi class II MHChi, and lacked markers of B cells, NK cells, or myeloid cells. They were large cells that exhibited dendritic morphology after brief periods of culture, and they were efficient stimulators of allogeneic T cells. The biologic implications of CD4 expression by thymic dendritic cells are discussed.

The peripheral nerve allograft in the primate immunosuppressed with Cyclosporin A: I. Histologic and electrophysiologic assessment.

Nerve regeneration across peripheral nerve allografts and control autografts in primates immunosuppressed with Cyclosporin A was quantitatively evaluated by electrophysiologic and histologic methods. Twelve cynomolgus monkeys received 3-cm autografts and allografts in contralateral ulnar nerves. They were immunosuppressed with Cyclosporin A at 25 mg/kg per day or placebo vehicle. Morphometric analysis of nerve graft and distal nerve segments was assessed at 1 year after engraftment. Quantitative electrophysiologic studies were performed percutaneously at 6 and 12 months, and compound action potentials were measured directly across the nerve grafts at 1 year. Excellent regeneration was seen across autografts and allografts in Cyclosporin A-treated and placebo-treated recipients.