Staining Of Thymocytes Research Articles

Coxsackievirus B4 infection of murine foetal thymus organ cultures

The infection of foetal thymus with coxsackievirus B4 (CV-B4) E2 has been studied ex vivo by using CD-1 mice on foetal day 14, as a ready source of organs for experimentation to investigate the hypothesis of the role of thymic viral infections in the pathogenesis of type 1 diabetes. The replication of CV-B4 E2 in murine foetal thymus organ cultures has been demonstrated by evaluating the levels of positive- and negative-stranded viral RNA in cells by using a real-time quantitative RT-PCR method and by determining titres of infectious viral particles in culture supernatants for 7 days post-infection (p.i.). Staining of tissue sections with an anti-cytokeratin antibody and haematoxylin-eosin showed that CV-B4 infection had no visible effect on cell survival and organ integrity. Cell counts in mock- and virus-infected foetal thymus organ cultures increased from day 1 through day 7, and live cell numbers were comparable in both conditions as shown by Trypan blue exclusion test and 7-amino-actinomycin D staining of thymocytes. Compared with controls on day 7 p.i., cytofluorometric analyses on cells from CV-B4 E2-infected foetal thymus organ cultures displayed a marked increase in the percentage of the most immature CD3(-)CD4(-)CD8(-) thymocytes, and a decrease in the percentage of immature CD3(-)CD4(+)CD8(+) cells, together with an increase in the percentage of mature CD3(+)CD4(+) and CD3(+)CD8(+) cells. These data show that CV-B4 E2 disturbs T-cell maturation and differentiation processes in infected murine foetal thymus organ cultures and provide evidence of a suitable system to investigate the effect of viruses in T-cell differentiation.

Assessment of Apoptosis in Thymocytes and Splenocytes from Mice Exposed to Arsenate in Drinking Water: Cytotoxic Effects of Arsenate on the Cells In Vitro

This study provides an assessment of the level of apoptosis in thymocytes and splenocytes from mice exposed to arsenate in drinking water. To simulate the naturally occurring exposure conditions of humans, the animals were exposed to arsenate at the concentrations of 0.5, 5, and 50 mgAs/L. TUNEL method for staining of thymocytes and splenocytes isolated from the mice after 8 and 12 weeks revealed increased percentage of apoptotic cells in the exposed groups. Although statistically significant increases were observed only for the highest concentration of arsenate, the increases showed linear trend as a function of arsenate concentration in drinking water. {In vitro} experiments performed on isolated cells incubated for 24 hours with arsenate at 6.7–2000 μM showed very similar concentration-viability relationships for both cell populations (IC50 was 442 ± 15 μM and 427 ± 18 μM for thymocytes and splenocytes, respectively). Arsenate induced a concentration-dependent increase in the percentage of the cells undergoing apoptosis. At higher concentrations, apoptosis was the predominant mode of cell death. It can be speculated that proapoptotic effects of arsenate as observed in our {in vivo} study may contribute to some immunotoxic symptoms observed in people chronically exposed to arsenic in drinking water.

Membrane molecules in induction of apoptosis of thymocytes by mouse thymic dendritic cells which express Fas ligands

Apoptosis of thymocytes is involved in the negative selection of thymus, but it remains unclear how the cell death of thymocytes is regulated by the thymic stromal cells. By using immunohistochemistry, TdT-mediated dUTP nick end labeling (TUNEL) and flow cytometry methods, it was found that Fas ligand was expressed in the thymic medulla and a mouse thymic medullary type dendritic cell line (MTSC4). The DNA fragmentation and TUNEL positive staining of thymocytes were detected in 6 h of cocultures with MTSC4. 97 % of the thymocytes which bound to MTSC4 were Fas antigen positive cells. The DNA fragmentation of thymocytes induced by MTSC4 was inhibited by the addition of 20 mmol/L N-acetyl galactosamin monosaccharide and the pretreatment of a monoclonal antibody (PF-18-3) which recognized a putative antigen on MTSC4. These results suggested that the mechanisms of induction of cell death by stromal cells may include the interactions of multiple cell surface molecules, in addition to the Fas/Fas ligand system.

Fetal hematopoietic alterations after maternal exposure to benzo[a]pyrene: a cytometric evaluation.

In utero exposure to the environmental contaminant benzo[a]pyrene (BaP) was found to alter expression of murine thymocyte and liver fetal cell-surface markers. Pregnant mice were treated (via gavage) with 0, 50, 100, or 150 mg BaP/kg/d on gestational days (gd) 13-17, and offspring were examined on gd 18. Severe thymic atrophy and cellular depletion were found in BaP-exposed fetal mice. Flow cytometric analysis indicated that the BaP treatment resulted in a significant decrease in the percentage of CD4+8+ fetal thymocytes, as well as significantly increased CD4-8- and CD4-8+ thymocytes. Staining of thymocytes with anti-mouse heat-stable antigen (HSA) and CD8 monoclonal antibodies produced similar results. These data suggest that BaP, in addition to producing thymic hypocellularity, inhibits normal thymocyte maturation processes. The BaP treatment was also found to decrease total fetal liver cellularity including numbers of cells within resident hematopoietic subpopulations. In particular, prolymphocytic cells, identified by CD44 and CD45R antigen expression and by presence of nuclear terminal deoxynucleotidyl transferase (TdT), were significantly decreased in animals gestationally exposed to BaP. These data, taken together, indicate that postnatal suppression of cell and humoral-mediated immune function following in utero exposure to BaP may result from multiple targeting of immune cells at different hematopoietic levels. Furthermore, results of the present study identify both qualitative and quantitative changes in fetal immune cell antigen expression that correlate well with the postnatal immunosuppression that occurs in experimental animals exposed to this carcinogenic polycyclic aromatic hydrocarbon.

Flow Cytometric and Immunohistochemical Evaluation of Ethanol‐Induced Changes in Splenic and Thymic Lymphoid Cell Populations

Ethanol-induced alterations in the immune system are thought to play a major role in increasing the susceptibility of alcoholics to infections and tumors. One important change in the immune system is the noted loss of lymphoid cells from the thymus and spleen. To examine these alterations we used a model system where C57Bl/6 mice were pair-fed either a Leiber-DeCarli diet containing 7% (v/v) ethanol or an isocaloric control diet. Mice receiving ETOH for 7 days showed a loss of cells from the spleen and thymus; this loss was even more severe after withdrawal for 1 day. The most profound changes were seen after 2 weeks of ETOH. Spleen and thymus cell numbers were reduced to 36% and 6.2%, respectively compared to control mice. Staining of thymocytes with monoclonal antibodies to lymphocyte surface markers and evaluation with flow cytometry revealed that immature thymocytes (PNA+, CD4+/CD8+) were most reduced. Mature thymocytes (CD4+/CD8- or CD4-/CD8+) were depleted, and the CD4+ to CD8+ ratio was increased. Sections of thymus stained with hematoxylin and eosin or with immunohistochemical methods showed atrophy and lymphoid cell depletion. No cortex was histologically identifiable after 2 weeks of ETOH. The spleen cells most affected by ETOH were the B cells. They were reduced to 8.2 x 10(6) cells/spleen (31.5% of the lymphocytes), as compared to 38.5 x 10(6) cells/spleen (50.3% of the lymphocytes) in the control mice. The spleen was atrophic, but the immunoarchitecture was preserved. Ethanol causes a depletion of lymphocytes from the spleen and thymus with alterations in lymphocyte subpopulations.(ABSTRACT TRUNCATED AT 250 WORDS)