Role of carbohydrate metabolism in hormonal regulation of spermatogenesis

In mammals the testis, the site of male germ cell development, can be divided morphologically in two cellular compartments, viz. the seminiferous tubules and the interstitial tiSsue between the tubules. The seminiferous tubules contain developing germ cells and Sertoli cells and are surrounded by a boundary layer of myoid cells. The interstitial tissue contains Leydig cells, blood.and lymph vessels, nerves and fibroblasts. In intact animals transfer between the two compartments is restricted (Setchell & Waites, 1975). A harrier to substances of widely varying molecular size is formed, because the Sertoli cells which line the seminiferous tubules are closely connected by elaborate tight junctions between the basal parts of the cells (blood-testis harrier). Insome species a second, less effective harrier is formed by myoid cells which surround the seminiferous tubules (Fawcett, 1975) (Figure I. 1). Development of gerrn cells starts with the spermatogonial stem cells which are located at the basis of the Sertoli cells in the seminiferous tubules but outside the blood-testis barrier (basal compartment). After several mitotic divisions of the spermatogonia, preleptotene spermatocytes develop which synthesize DNA) so that they finally contain twice the amount of DNA present in non-dividing somatic cells. In the propbase following the preleptotene stage, rearrangement of the chromosomal material takes place as a preparatien for the first meiotic division. The meiotic propbase is subdivided in the leptotene, zygotene, pachytene and diplotene. During the leptotene and zygotene tight junctions are formed between the Sertoli cells at the basal side of the germ cells and subsequently the tight junctions at the luminal side are dissolved (Russell, 1980). In this way the germ cells are transported through the blood-testis harrier to the lumen of the seminiferous tubules (adluminal compartment) without disruption of the blood-testis harrier. The propbase is foliowed by the first meiotic di vis ion, and the generated secondary spermatocytes_ go quickly through the Second meiotic division, without synthesis of DNA

Culture of graft-infiltrating cells from cryopreserved endomyocardial biopsies

Graft-infiltrating cells can be cultured from fresh endomyocardial biopsies (EMB) taken after heart transplantation to determine their growth patterns, phenotypic composition, and functional characteristics for clinical or scientific purposes. In this study we investigated whether graft-infiltrating cells can also be cultured successfully after cryopreservation of these EMB. Three different cryopreservation methods were used. One method gave successful growth in 100% of the cases (n = 6): The biopsy fragments were preincubated in 10% vol/vol dimethyl sulfoxide during 5 min at O°C, frozen to -70°C at approximately 1°C per minute, and subsequently immersed and stored in liquid nitrogen. Thawing was performed rapidly in water at 37°C. In addition, the effect of cryopreservation on cell surface phenotype and donor-specific cytotoxicity of these graft-infiltrating cells was analyzed. When compared to cultures of nonfrozen control biopsies, both qualities remained constant in most cases, although a variation in CD4+/CD8+ cell ratio was observed in 33% of these cultures. However, when nonfrozen fragments of size-matched biopsies were cultured separately, a similar variation in phenotype was noted, indicating that this phenomenon can be attributed to sampling variation and not to the cryopreservation procedure. The present findings suggest that it is no longer required to culture fresh (nonfrozen) post-transplant EMB to propagate graft-infiltrating cells: Culturing can be limited to cryopreserved EMB that are selected retrospectively, depending on actual clinical or scientific interests. Besides greatly facilitating the long-term monitoring of heart transplant recipients, this also means a substantial decrease in cost and work load for laboratories involved in heart transplantation

Peripheral monitoring of direct and indirect alloantigen presentation pathways in clinical heart transplant recipients

It has been reported that the response to alloantigens presented by the direct and indirect pathway may be of differential relevance after human kidney transplantation. Accordingly, we monitored these routes in peripheral blood mononuclear cells (PBMC) of heart transplant patients from before transplantation and up to 2 years thereafter in an attempt to find a correlation with the clinical status of the patients. Both before and after transplantation, comparable proportions of PBMC samples reacted in mixed lymphocyte culture to nondepleted donor spleen cells (direct route), but never to donor cells depleted for antigen-presenting cells (indirect route). In contrast, the latter route could easily be activated by a nominal antigen and persisted after transplantation, although the proportion of PBMC samples responding was significantly suppressed, irrespective of the occurrence of rejection. Consequently, complete removal of antigen-presenting cells from the stimulator population in a mixed lymphocyte culture with PBMC as responder is not a suitable tool for measuring indirect presentation of alloantigens, and therefore not relevant for monitoring the immunological status of heart transplant recipients

Characteristics of graft-infiltrating lymphocytes after human heart transplantation: HLA mismatches and the cellular immune response within the transplanted heart

The influence of HLA mismatches between donor and recipient on the phenotypes, function, and specificity of T-lymphocyte cultures derived from endomyocardial biopsies was studied in 118 heart transplant recipients. In case of HLA-DR mismatches, the majority of the EMB-derived cultures were dominated by CD4+ T cells while, in patients with HLA-A and -B mismatches but without DR mismatches, CD8+ T cells comprised the predominant T-cell subset. Cytotoxicity against donor antigens was observed in 75% of the cultures. A significantly (p < 0.005) lower proportion of the cultures showed cytotoxicity against HLA-A antigens (36%) when compared with HLA-B (53%) or HLA-DR (49%). An HLA-A2 mismatch elicited a cytotoxic response that was comparable to that found against HLA-B and -DR antigens: 62% of the cultures from HLA-A2 mismatched donor-recipient combinations was reactive against A2. A higher number of A, B, or DR mismatches resulted in a higher number of cytotoxic cultures directed against these antigens. A higher number of HLA-B and -DR mismatches was associated with a lower freedom from rejection. Our data indicate that, despite the use of adequate immunosuppressive therapy, the degree of HLA matching plays a crucial role in the immune response against a transplanted heart, resulting in a significant effect on freedom from rejection

Alloreactive lymphoid infiltrates in human heart transplants: Loss of class II-directed cytotoxicity more than 3 months after transplantation

Abstract From 535 endomyocardial biopsies (87 heart transplant recipients) 283 cell cultures could be generated. All cultures tested contained T lymphocytes and in most cases CD4 was the predominant phenotype at any time posttransplant. A significantly higher proportion of CD8-dominated cultures was found among cultures from biopsies without myocytolysis. In the first 3 months post transplant 57% of cultures showed cytotoxicity against both class I and class II mismatched donor major histocompatibility complex (MHC) antigens, changing to an incidence of 33% at > 90 days. This proved to be due to a significant decrease in the number of cultures with human leukoctye antigen class II-directed cytotoxicity. This study shows that early after transplantation a heart transplant is infiltrated with activated donor-specific cytotoxic T cells which recognize a broad spectrum of mismatched donor MHC antigens, and that in time this spectrum becomes more restricted