Immunophenotyping analysis of peripheral blood, splenic, and thymic lymphocytes in male and female rats (original) (raw)
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Blood and spleen lymphocytes as targets for immunotoxic effects in the rat—a comparison
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Traditionally, immunotoxicological studies in the rat have been performed by measuring the effect of chemical substances on spleen lymphocytes in vivo and in vitro. However, rat blood lymphocytes may be more relevant than spleen cells for comparison with human blood lymphocytes. Further, lymphocytes in blood may be a more sensitive indicator of immunotoxic effects than spleen lymphocytes. Finally, in longitudinal studies peripheral blood specimens can be collected repeatedly from the same animals, thereby reducing the number of animals sacrificed and, possibly, experimental variation. We compared blood and spleen lymphocyte parameters in rats treated with a single dose of the immunosuppressant cyclophosphamide (CY), monitoring effects on blood and spleen lymphocytes by immunophenotyping. We also performed repeated bleedings to demonstrate the feasibility of following the time course of induced changes in the same animals. Immunophenotyping as well as total mononuclear cell counts consistently showed as large or lager effects of CY in blood lymphocytes than in spleen cells. Further, the measured effects in blood lymphocytes became statistically significant at an earlier time point, compared to spleen cells. Repeated bleedings of the same animals illustrated that blood specimens drawn from a peripheral vein give sufficient numbers of cells to perform immunotoxicological tests.
Functional informativeness of lymphocytes' cytomorphometric analysis of laboratory rats' blood
Journal of Advanced Biotechnology and Experimental Therapeutics, 2021
Immunological methods that objectively reflect the lymphogenetic processes in the examined organism are needed to assess the effectiveness of prophylactic or therapeutic agents that modulate regenerative processes in the immune system. Such requirements are met by the cytomorphometric method of analysis of circulating blood lymphocytes, the size (small, medium and large) populations of which reflect their proliferative processes in the lymphoid organs. However, the cytomorphometric characteristics of lymphocytes have species features that have to be determined experimentally. In the model of fecal peritonitis in white non-linear sexually mature male laboratory rats, the limits of statistical size classes of lymphocytes were determined, which were the same in control and experimental animals, namely: small-with a diameter of 8.5 μm and less, medium-with a diameter of more than 8.5 μm and less 11.0 μm, large-with a diameter of 11.0 μm and more. Acute infectious process significantly changed the levels of cytomorphometric classes of lymphocytes according to their functional activity in immunogenesis: small, medium and large size of lymphocytes as 42.5 to 7.0%, 45.0 to 54.0%, 10.5 to 39.0%, respectively, in the control and experimental groups of animals, which demonstrates the informativeness of the method. In this case, small and large lymphocytes belong to the activated lymphocytes, which determine the state of the immune system, while medium lymphocytes mainly make up the pool of memory cells. Based on the ratio of these size classes in the peripheral blood, a conclusion about the proliferative reaction of lymphocytes in the experiment is made, followed by the extrapolation of the results and in clinical practice.
Blood B, T, CD4+ and CD8+ lymphocytes in female Wistar rats
Annals of Hematology, 1993
We have established reference values of peripheral blood lymphocyte subsets in healthy female Wistar rats under highly standardized conditions. Using monoclonal antibodies and flow cytometry, T lymphocytes (OX19+), B lymphocytes (OX6 + and anti-Ig+), T-helper/inducer (W3/25 +), and T-suppressor/ cytotoxic subsets (OX8 +) were determined, from week 11 to week 21 after birth. The mean percentages of T and B lymphocytes with respect to total lymphocytes were 78.5% and 18%, respectively; the mean percentages of T-helper/inducer and T-suppressor/cytotoxic cells in relation to T lymphocytes were 59% and 25%, respectively (n =48). No difference in total leukocyte count, differential leukocyte analysis, or lymphocyte subsets was observed during the 10 weeks the rats were studied under standard housing conditions. Therefore, the period considered seems the most appropriate in which to carry out experiments that could involve lymphocyte subset disturbances.
Lymphocyte abnormalities in the BB rat
Metabolism, 1983
The BB rat has a marked T cell lymphocytopenia, with a near abso,lco of peripheral "helper" T coils recognized by monoclonal antibody W3/25 (W3/25+ T cells}. The lymphocytes of the BB rat's spleen and thymus were examined for the presence of W3/25+ T cells, which were found to be absent in the spleen but present in normal amounts in the thymus. Concanavalin A (Con A) re,~ponsiveness was absent in the BB's peripheral blood and spleen but present in the thymus. Thus, in these three lymphoid compartments, Con A responsiveness directly correlated with the presence or absence of W3/25+ T cells. These lymphocyte abnormallt;~es in the BB rat are notably different from lymphocyte changes present in human type i diabetes.
Distribution of T-lymphocyte subsets in porcine lymphoid tissues
Immunology, 1987
The distribution of the functional subsets of porcine T cells, the cytolytic/suppressor (Tc/s) and the helper/inducer (Th/i) cells was studied in cryostat sections of thymus, lymph nodes, tonsils, Peyer's patches, spleen and liver using the indirect immunoperoxidase technique. Three murine monoclonal antibodies (mAb) were used. The mAb 8/1 reacts with an antigen present on all T cells and on cells of the myeloid lineage; the antigen has not yet been characterized biochemically. The mAb 295/33 (anti-T8) binds to the porcine T8 antigen and defines the Tc/s subset, while mAb PT-4 (anti-T4) detects the porcine T4 antigen and defines the Th/i subset. Practically all thymocytes were stained by mAb 8/1. The majority of cortical thymocytes apparently co-expressed T8 and T4, whereas distinct fractions of medullary cells were labelled by either anti-T8 or anti-T4. In peripheral lymphoid organs all three mAb reacted with cells in the thymus-dependent areas and with cells scattered in the l...
Experimental Gerontology, 2000
We previously demonstrated that the rat thymus undergoes a progressive remodelling long before the appearance of typical signs of involution . Age-dependent remodelling of rat thymus. Morphological and cytofluorimetric analysis from birth up to one year of age. Eur. J. Cell. Biol. 76,[156][157][158][159][160][161][162][163][164][165][166]. To focus better on the complex remodelling that occurs in the rat immune system during the first year of life, we analysed the phenotype profile of thymocytes, and T lymphocytes from mesenteric lymph nodes and peripheral blood of the same animals by flow cytometry. Two experimental sets were performed simultaneously using the same animal strain, but starting and ending the study at different ages (15 days up to 300 days in the first experimental set, and 90 days up to 360 days of life in the second). In the rat these ages appear to be crucial not only for developmental, maturative and early involutional processes of the thymus, but also of the entire immune system. The main findings were the following: (1) in the thymus, CD8 Ϫ CD4 Ϫ cells increased, CD5 ϩ ab TCR Ϫ and CD8 ϩ CD4 ϩ thymocytes decreased, while the most mature cell subset appeared well preserved with ageing; (2) in the lymph nodes, T helper and T cytotoxic lymphocytes decreased in the most aged animals. Memory/activated CD4 ϩ CD45RC Ϫ T cells decreased, while naive/resting M. Capri et al. / Experimental Gerontology 35 (2000) 613-625 613 Experimental Gerontology 35 (2000) 613-625 www.elsevier.nl/locate/expgero 0531-5565/00/$ -see front matter ᭧ (M. Capri).
Identification of "active" T lymphocytes among effector cells in guinea pigs
Immunopharmacology, 1982
Guinea pig T lymphocytes have receptors of different affinity for rabbit red blood cells (RRBC): those binding RRBC immediately are termed "active" T cells; the remainder, which bind RRBC only after longer incubation times, are "non-active" or "late-rosetting" T cells. We have found that these two subpopulations have different functional characteristics. Active T cells could not be stimulated effectively with phytohemagglutinin (PHA), and stimulation with concanavalin A (ConA) increased their DNA synthesis only at high concentrations. The non-active subpopulation responded better to PHA but poorly to ConA. Unseparated (total) T cells, however, responded well to both mitogens, suggesting a helper effect by the active T cells. The presence of monocytes in T-cell cultures further enhanced mitogen-induced DNA synthesis. Active T cells were not present in guinea pig bone marrow, whereas they constituted 10% of all lymphocytes in the thymus, 13% in the spleen...
Distribution of T -Iymphocyte subsets in porcine lymphoid tissues
1987
Tbe distribution ofthe functional subsets ofporcine T cells, the cytolytic/suppressor (Tc/s) and the helper/inducer (Tb/i) cells was studied in cryostat sections ofthymus, Iymph nodes, tonsils, Peyer's patches, spleen and liver using the indirect immunoperoxidase technique. Tbree murine monoclonal antibodies (mAb) were used. Tbe mAb 8/1 reacts with an antigen present on all T cells and on cells of the myeloid Iineage; the antigen has not yet been characterized biochemically. Tbe mAb 295/33 (antiT8) binds to the porcine T8 antigen and defines the Tc/s subset, while mAb PT -4 (anti-T4) detects the porcine T4 antigen and defines the Tb/i subset. Practically all thymocytes were stained by mAb 8/1. Tbe majority of corticaI thymocytes apparently co-expressed T8 and T 4, whereas distinct fractions of medullary cells were labelIed by either anti-T8 or anti-T4. In peripherallymphoid organs all three mAb reacted with cells in the thymus-dependent areas and with cells scattered in the lymp...
Lymphocyte Subsets in Normal Human Lymphoid Tissues
American Journal of Clinical Pathology, 1983
A series of B, T, natural killer (NK) cell, and monocyte-specific monoclonal antibodies was used to determine the localization of lymphocyte subpopulations in frozen tissue sections of human lymph node, spleen, tonsil, and thymus by means of an immunohistochemical technic. In thymus, most cortical thymocytes reacted with Leu 1, Leu 2a, Leu 3a, Leu 4, Lyt 3, OKT3, and OKT6 antibodies. Except for OKT6, Leu 2a, and Leu 3a, these antibodies also reacted with medullary thymocytes. The majority (70-80%) of medullary thymocytes reacted with Leu 3a and a smaller fraction (20-30%) with Leu 2a antibody. The staining pattern of thymic medulla approximates the staining pattern of peripheral T cells. In peripheral lymphoid tissues, the majority of cells in the paracortical region of lymph node and in the periarteriolar sheath of spleen stained with Leu 1, Leu 4, OKT3, and Lyt 3 antibodies. Staining with Leu 3a and Leu 2a identified 60-80% and 20-40% of total T cells, respectively, as defined by Lyt 3 positivity. In addition, a substantial number of Leu 3a + and Leu 7 + cells were found in the germinal centers of secondary follicles. This finding supports the importance of these subsets of lymphocytes in regulation of the human immune response. Leu 2a + cells were rare in tissues with prominent follicular hyperplasia, but appeared in considerable number in the red pulp of spleen. In the mantle zone of lymphoid follicles, the majority of lymphocytes were positive for IgM, IgD, and Bl. Approximately 60-70% of these cells bore K chain and 30-40% X chain immunoglobulin. The extracellular substance in germinal centers was positive for Bl, IgG, IgM, x, and X. The majority of germinal center cells appeared to contain no surface or cytoplasmic immunoglobulins. Small mononuclear cells bearing OKM I marker were abundant in the marginal zone of white pulp and in the red pulp of spleen but, rarely were observed in other portions of lymphoid tissues. OKM1 also reacted with granulocytes. Leu 7 + (NK) cells were rare in the thymus, but frequent in the GC of secondary follicles. The distribution of Leu 7 + cells did not correspond to staining with Lyt 3 and Leu 2a.