Sensitivity of activated human lymphocytes to cyclosporine and its metabolites

Alloreactive T cells generated as clones from mixed lymphocyte cultures, or propagated from heart or liver transplant biopsies, were tested for secondary proliferation measured in the primed lymphocyte test in the presence of Cyclosporine A and metabolites fractionated from human bile. Significant differences were observed in Cyclosporine A sensitivity between various cell cultures ranging as high as 100-fold. The liver is the primary site of Cyclosporine A metabolism, which yields a number of hydroxylated and N-dimethylated derivatives that are eventually secreted into the bile. Bile was collected from adult liver transplant patients on Cyclosporine A therapy and following extraction with diethyl ether, separated by high pressure liquid chromatography. Thirteen fractions were tested for their effect on lymphocyte proliferation in concanavalin A activation, mixed lymphocyte cultures and primed lymphocyte test assays. The strongest immunosuppressive effect was found with fraction 8, which contained metabolite M17, which has a single hydroxylation in position 1. Only three other fractions 9, 10, and 13, which contained metabolites M1, M18, and M21, respectively, exhibited immunosuppressive activity, albeit much lower than that of Cyclosporine A. Differences in Cyclosporine A sensitivity among alloreactive T cells followed similar patterns with Cyclosporius A metabolites. Thus, the assessment of the Cyclosporine A effect must consider differences in drug sensitivity of lymphocytes involved in transplant immunity and the generation of metabolites with immunosuppressive activity. © 1988

Uji Aktivitas Imunostimulator Fraksi Etil Asetat Ekstrak Etanol Kelopak Bunga Rosella (Hibiscus Sabdariffa L.) Terhadap Proliferasi Sel Limfosit Mencit Galur Swiss Secara in Vitro Beserta Identifikasi Kandungan Senyawa Kimianya

Immunostimulatory agent has an important role in the treatment of infectious disease caused by pathogen. Roselle calyx (Hibiscus sabdariffa L.) used by community as a health drink to enhance body's immune system. The aims of this research was to know immunostimulatory activity of ethyl acetate fraction of ethanolic extract of roselle calyx on lymphocyte cell proliferation Swiss strain mice and to identified the chemical compounds of the fraction. Roselle calyx was extracted by maceration method using ethanol 96% as a solvent. The ethanolic extract was partitioned gradually by n-hexan and ethyl acetate. Series of ethyl acetate fraction were made to concentration 10, 20, 50, 100, 200, 400 μg/mL and positive control PHA 10 μg/mL for tested to lymphocyte cell culture. Immunostimulatory activity test was done according to MTT Assay method. Lymphocyte proliferation activity was analyzed statistically to the value of Optical Density (OD) result of the Enzyme Linked Immunosorbent Assay (ELISA) reader using Kruskal Wallis Test continued by Mann Whitney Test (p<0,05). Identification of the contents phenolic compounds and flavonoids performed by Thin Layer Chromatography (TLC). The result of the research showed that ethyl acetate fraction had lymphocyte cell proliferation activity at the concentration of 50, 100, 200, and 400 μg/mL. The ethyl acetate fraction of roselle calyx contained phenolic compounds and flavonoids

Spatial Organization and Molecular Correlation of Tumor-Infiltrating Lymphocytes Using Deep Learning on Pathology Images

Beyond sample curation and basic pathologic characterization, the digitized H&E-stained images of TCGA samples remain underutilized. To highlight this resource, we present mappings of tumorinfiltrating lymphocytes (TILs) based on H&E images from 13 TCGA tumor types. These TIL maps are derived through computational staining using a convolutional neural network trained to classify patches of images. Affinity propagation revealed local spatial structure in TIL patterns and correlation with overall survival. TIL map structural patterns were grouped using standard histopathological parameters. These patterns are enriched in particular T cell subpopulations derived from molecular measures. TIL densities and spatial structure were differentially enriched among tumor types, immune subtypes, and tumor molecular subtypes, implying that spatial infiltrate state could reflect particular tumor cell aberration states. Obtaining spatial lymphocytic patterns linked to the rich genomic characterization of TCGA samples demonstrates one use for the TCGA image archives with insights into the tumor-immune microenvironment

Uji Aktivitas Imunostimulator Fraksi Etil Asetat Ekstrak Etanol Kelopak Bunga Rosella (Hibiscus Sabdariffa L.) Terhadap Proliferasi Sel Limfosit Mencit Galur Swiss Secara in Vitro Beserta Identifikasi Kandungan Senyawa Kimianya

Immunostimulatory agent has an important role in the treatment of infectious disease caused by pathogen. Roselle calyx (Hibiscus sabdariffa L.) used by community as a health drink to enhance body's immune system. The aims of this research was to know immunostimulatory activity of ethyl acetate fraction of ethanolic extract of roselle calyx on lymphocyte cell proliferation Swiss strain mice and to identified the chemical compounds of the fraction. Roselle calyx was extracted by maceration method using ethanol 96% as a solvent. The ethanolic extract was partitioned gradually by n-hexan and ethyl acetate. Series of ethyl acetate fraction were made to concentration 10, 20, 50, 100, 200, 400 μg/mL and positive control PHA 10 μg/mL for tested to lymphocyte cell culture. Immunostimulatory activity test was done according to MTT Assay method. Lymphocyte proliferation activity was analyzed statistically to the value of Optical Density (OD) result of the Enzyme Linked Immunosorbent Assay (ELISA) reader using Kruskal Wallis Test continued by Mann Whitney Test (p<0,05). Identification of the contents phenolic compounds and flavonoids performed by Thin Layer Chromatography (TLC). The result of the research showed that ethyl acetate fraction had lymphocyte cell proliferation activity at the concentration of 50, 100, 200, and 400 μg/mL. The ethyl acetate fraction of roselle calyx contained phenolic compounds and flavonoids

Antibody localization in horse, rabbit, and goat antilymphocyte sera

The localization of antibodies was studied in rabbit, goat, and horse ALS raised by weekly immunization with canine or human spleen cells for 4 to 12 weeks. A combination of analytic techniques was used including column chromatography, electrophoresis, immunoelectrophoresis, determination of protein concentration, and measurement of antibody titers. In the rabbit and goat ALS, virtually all of the leukoagglutinins and lymphocytotoxins were in the easily separable IgG; accidentally induced thromboagglutinins were in the same location. In the rabbit hemagglutinins were found in both the IgG and IgM, whereas in the goat these were almost exclusively in the IgM. The antiwhite cell antibodies were most widely distributed in the horse. The cytotoxins were primarily in the IgG, but the leukoagglutinins were most heavily concentrated in the T-equine globulin which consists mostly of IgA. By differential ammonium sulfate precipitation of a horse antidoglymphocyte serum, fractions were prepared that were rich in IgG and IgA. Both were able to delay the rejection of canine renal homografts, the IgA-rich preparation to a somewhat greater degree. The findings in this study have been discussed in relation to the refining techniques that have been used for the production of globulin from heterologous ALS. © 1970

A T-lymphoma transmembrane glycoprotein (gp180) is linked to the cytoskeletal protein, fodrin.

A major mouse T-lymphoma surface glycoprotein (gp180) has been identified by labeling cells with 125I and [3H]glucosamine. After ligand-induced receptor patching and/or capping, the amount of gp 180 in the membrane-associated cytoskeleton fraction increases in direct proportion to the percentage of patched/capped cells. There is a parallel increase in the amount of fodrin in the membrane-associated cytoskeleton fraction. Evidence is presented that gp180 is the same as or very similar to the T-lymphocyte-specific glycoprotein T-200. An immunobinding assay of Nonidet P-40-solubilized plasma membrane selectively co-isolates gp180 and fodrin. After induction of receptor rearrangement, double-label immunofluorescence reveals that fodrin accumulated directly beneath gp180 patches and caps. Membrane extraction with Triton X-114 followed by sucrose gradient centrifugation permits isolation of a gp180-fodrin complex with a 1:1 molar ratio and sedimentation coefficient(s) of approximately 20. This complex remains stable during isoelectric focusing and exhibits a pl in the range of 5.2-5.7. On the basis of our results we conclude that gp180, an integral membrane glycoprotein, and fodrin, a component of the membrane-associated cytoskeleton, are closely associated into a complex. Furthermore, we contend that, through fodrin's association with actin, this complex is of functional significance in ligand-induced patching and capping of gp180. We also propose that, through lateral interactions in the plane of the membrane, the gp180-fodrin complex might be responsible for linking other surface receptors to the intracellular microfilament network during lymphocyte patching and capping

Explicit kinetic heterogeneity: mechanistic models for interpretation of labeling data of heterogeneous cell populations

Estimation of division and death rates of lymphocytes in different conditions is vital for quantitative understanding of the immune system. Deuterium, in the form of deuterated glucose or heavy water, can be used to measure rates of proliferation and death of lymphocytes in vivo. Inferring these rates from labeling and delabeling curves has been subject to considerable debate with different groups suggesting different mathematical models for that purpose. We show that the three models that are most commonly used are in fact mathematically identical and differ only in their interpretation of the estimated parameters. By extending these previous models, we here propose a more mechanistic approach for the analysis of data from deuterium labeling experiments. We construct a model of "kinetic heterogeneity" in which the total cell population consists of many sub-populations with different rates of cell turnover. In this model, for a given distribution of the rates of turnover, the predicted fraction of labeled DNA accumulated and lost can be calculated. Our model reproduces several previously made experimental observations, such as a negative correlation between the length of the labeling period and the rate at which labeled DNA is lost after label cessation. We demonstrate the reliability of the new explicit kinetic heterogeneity model by applying it to artificially generated datasets, and illustrate its usefulness by fitting experimental data. In contrast to previous models, the explicit kinetic heterogeneity model 1) provides a mechanistic way of interpreting labeling data; 2) allows for a non-exponential loss of labeled cells during delabeling, and 3) can be used to describe data with variable labeling length

Antigen-specific electrophoretic cell separation for immunological investigations

Preincubation of human blood lymphocytes with cell surface antigen specific antibodies under non-capping conditions reduces the electrophoretic mobility of the corresponding lymphocyte subpopulation. Antigen-positive and antigen-negative cells can be separated by free flow electrophoresis with high yield, purity and viability. The use of fluorescence-labelled second antibodies augments the induced decrease in net surface charge density, and allows rapid detection of antigen-positive cells in the fractions of electrophoresis. Carrier-free cell electrophoresis of human peripheral blood lymphocytes after reaction with anti-IgM-antibody or the monoclonal antibodies OKT4 or OKT8, and sandwich staining with tetrarhodamine isothiocyanate-labelled anti-IgG resulted in the large-scale separation of high pure human B and T lymphocyte subpopulations. Their functional integrity was shown in assays of lymphocyte transformation and of antigen-specific induction and regulation of antibody synthesis in vitro. These separate lymphocyte subpopulations are useful tools for immunological investigations. While, for instance, the effects of drugs on human lymphocytes are obscured by coincident changes in cell composition of the peripheral blood tested that do not by themselves reflect whole body immunocompetence, the cell separation and in vitro assays at a defined cell number and cell composition allow the recording of quantitative changes in the function of different cell subpopulations. We studied the influence of the anesthetic thiopental on separated human lymphocyte subsets. In both polyclonal lectin stimulation and in vitro antibody production, thiopental exhibited a noncytotoxic suppression of lymphocyte functions. B-Cells, T-helper and T-suppressor cells were equally affected and showed the same dose response.(ABSTRACT TRUNCATED AT 250 WORDS

Air Pollution and Lymphocyte Phenotype Proportions in Cord Blood

Effects of air pollution on morbidity and mortality may be mediated by alterations in immune competence. In this study we examined short-term associations of air pollution exposures with lymphocyte immunophenotypes in cord blood among 1,397 deliveries in two districts of the Czech Republic. We measured fine particulate matter < 2.5 μm in diameter (PM(2.5)) and 12 polycyclic aromatic hydrocarbons (PAHs) in 24-hr samples collected by versatile air pollution samplers. Cord blood samples were analyzed using a FACSort flow cytometer to determine phenotypes of CD3(+) T-lymphocytes and their subsets CD4(+) and CD8(+), CD19(+) B-lymphocytes, and natural killer cells. The mothers were interviewed regarding sociodemographic and lifestyle factors, and medical records were abstracted for obstetric, labor and delivery characteristics. During the period 1994 to 1998, the mean daily ambient concentration of PM(2.5) was 24.8 μg/m(3) and that of PAHs was 63.5 ng/m(3). In multiple linear regression models adjusted for temperature, season, and other covariates, average PAH or PM(2.5) levels during the 14 days before birth were associated with decreases in T-lymphocyte phenotype fractions (i.e., CD3(+) CD4(+), and CD8(+)), and a clear increase in the B-lymphocyte (CD19(+)) fraction. For a 100-ng/m(3) increase in PAHs, which represented approximately two standard deviations, the percentage decrease was −3.3% [95% confidence interval (CI), −5.6 to −1.0%] for CD3(+), −3.1% (95% CI, −4.9 to −1.3%) for CD4(+), and −1.0% (95% CI, −1.8 to −0.2%) for CD8(+) cells. The corresponding increase in the CD19(+) cell proportion was 1.7% (95% CI, 0.4 to 3.0%). Associations were similar but slightly weaker for PM(2.5). Ambient air pollution may influence the relative distribution of lymphocyte immunophenotypes of the fetus