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Flow cytometry description of a novel CD3-/CD7+ intraepithelial lymphocyte subset in human duodenal biopsies: potential diagnostic value in coeliac disease.

Intraepithelial lymphocytes (IEL) represent a heterogeneous cellular compartment of unknown functions and controversial ontogeny. Previous observations in humans indicate that the majority of IEL subsets express the CD3 complex associated with either the alphabeta or the gammadelta T-cell receptor components, and describe the characteristic increase of CD3+TCRgammadelta+ IEL in coeliac disease. In the present work, we analyze the surface antigen expression of intraepithelial lymphocytes isolated from duodenal biopsies of control subjects and coeliac disease patients. We describe a CD3-CD7 + IEL subset frequently found in control subjects (41.41+/-21.8), with the following features: 1) most of these cells are CD45R0+ CD103+ and CD44- CD28- CD5-; 2) a significant percentage express CD56 (44.7%+/-21.3), CD2 (55.1%+/-16.2), and CD94 (16.2%+/-7.3). Furthermore, they are CD122+ and CD25-; 3) this CD3- IEL subset exhibit an activated phenotype expressing higher levels of CD69, CD103, and CD38 than the CD3+ subset. Interestingly, this CD3- subset is drastically reduced in CD patients (2.2+/-2.9 in active disease, 6.3+/-4.6 in treated patients versus 41.4+/-21.8 in control subjects). The imbalanced ratio "increased TCRgammadelta versus decreased CD3- CD7+" is a permanent finding in CD patients following clinical and histological remission. This parameter might provide helpful diagnostic information (easily obtained by 3-color FCM from diagnostic biopsies), and suggest a potential implication in the pathogenesis of coeliac disease.

Antigens, CD7↗

Differential requirement of CD28 costimulation for activation of murine CD8+ intestinal intraepithelial lymphocyte subsets and lymph node cells.

The CD8+CD4- (CD8+) murine small intestinal intraepithelial lymphocytes (IELs) contain two subpopulations, one expressing alpha alpha-CD8 homodimers and another alpha beta-CD8 heterodimers. In this study, plate-bound anti-TCR beta-chain (TCR-beta) mAb alone or combined with anti-CD28 mAb is used as a model system to study activation requirement of these two CD8+ IEL subsets. In contrast to CD8+ lymph node (LN) cells that require both TCR and CD28 triggering for activation, alpha beta-CD8+ IELs proliferate and produce IL-2 and IFN-gamma when stimulated with anti-TCR-beta mAb alone, and soluble CTLA-4 Ig has no effect on their responses. On the other hand, alpha alpha-CD8+ IELs neither make IL-2 or IFN-gamma nor proliferate even when both stimuli are provided. However, alpha alpha-CD8+ IELs are capable of proliferation in both CD8+ IEL subsets is lower than in CD8+ LN cells, which contributes to the weaker and delayed response of CD8+ IELs.

Animals↗

The effect of intranasal and inhaled corticosteroids in healthy volunteers on the number of circulating lymphocytes and lymphocyte subsets.

BACKGROUND: There has been an increasing interest in the potential systemic effects of inhaled corticosteroids. METHODS: The effect of locally inhaled corticosteroids in the nose and lung on blood lymphocytes was measured in two studies. In the first study, budesonide (BUD) (200 and 800 microg), fluticasone propionate (FP) (200 and 800 microg), and placebo were administered in the nose, and BUD (1600 microg) and FP (1500 microg) were inhaled into the lungs in a blinded, randomized fashion by 12 healthy volunteers. Blood samples were taken before and 4 h after the administration of the drug, and total lymphocyte count and different subpopulations were determined. In the second study, 15 healthy volunteers were randomized to BUD (1600 microg), FP (1600 microg), or placebo inhaled into the lungs. Blood samples were taken before and 4, 8, 24, 48, and 148 h (=7 days) after inhalation of the medication. RESULTS: Neither the nasal applications nor the inhalation of FP (1500 microg/1600 microg) showed significant differences in total lymphocyte count or different subpopulations between baseline and 4 h after the administration. In both studies, a significant reduction was found in the total lymphocyte count, B cells, T cells, and the CD4+ and the CD8+ fractions 4 h after application of BUD 1600 microg. CONCLUSIONS: Nasal application of BUD or FP in doses up to 800 microg do not induce lymphopenia. BUD 1600 microg inhalation in the lung reduces lymphocytes and their subfractions. Further studies have to be done to determine whether the results obtained in this study in healthy volunteers will also be found in patients with diseased mucosa and whether there is any correlation with adverse effects such as growth inhibition or osteoporosis.

Administration, Inhalation↗

Quantitation of lymphocyte subsets in cerebrospinal fluid and blood during the clinical course of aseptic and bacterial meningitis.

Mononuclear cell subsets in cerebrospinal fluid (CSF) and peripheral blood (PB) were monitored during the clinical course in 23 patients with acute meningitis using 6 monoclonal antibodies. Significant differences between aseptic and bacterial meningitis mainly consisted of a higher percentage of OKT4-positive cells in PB in the acute phase of bacterial meningitis. Significant differences between CSF and PB are found in the amount of most cell subtypes at all times except the acute phase of bacterial meningitis. The OKT4/OKT8 ratio was always significantly higher in CSF and correlated with the acuity of inflammation in bacterial meningitis.

Aged↗

Effects of Cordyceps sinensis on murine T lymphocyte subsets.

It was shown by flow cytometry analysis that crystalized preparation of Cordyceps sinensis (Cs-Cr) caused significant elevation of the number of T helper cells and Lyt-1/Lyt-2 (T helper to T suppressor cell) ratio both in peripheral blood and the treated mice spleen. The spleen weight, phagocyte counts and phagocytic activity were also elevated in the treated group. In addition, Cs-Cr could protect T helper cells from the immunosuppressive effects of prednisolone acetate and cyclophosphamide. These results further substantiate the fact that Cs-Cr is an immunoregulator/biological response modifier of cellular immunity and may be potentially useful in handling immunodeficient or immunosuppressed patients.

Animals↗

Macrophages interact with enriched populations of distinct T lymphocyte subsets for the induction of severe destructive Lyme arthritis.

Severe destructive Lyme arthritis was detected in the hind paws of hamsters infused with enriched populations of either CD4+ or CD4- T lymphocytes along with macrophages exposed in vitro to formalin-inactivated Borrelia burgdorferi and then infected with the Lyme spirochete. Swelling was detected 4 days after infection, increased rapidly, peaked on day 8 of infection, and gradually decreased. Similarly, severe destructive arthritis was induced in hamsters infused with enriched populations of unfractionated T lymphocytes and macrophages exposed to spirochetes after infection with B. burgdorferi. Histopathological examination affirmed that hamsters infused with CD4+, CD4-, or unfractionated T lymphocytes and macrophages exposed to B. burgdorferi-induced arthritis. In addition, macrophages exposed in vitro to B. burgdorferi demonstrated both conventional and coiling phagocytosis, suggesting a mechanism by which CD4+ and CD4- T lymphocytes induce arthritis, respectively. These findings demonstrate that both CD4+ and CD4- subpopulations of T lymphocytes are capable of interacting with macrophages for the induction of severe destructive Lyme arthritis.

Animals↗

Effect of zinc or zinc plus arginine supplementation on antibody titre and lymphocyte subsets after influenza vaccination in elderly subjects: a randomized controlled trial.

OBJECTIVE: to evaluate whether oral supplementation with zinc or zinc/arginine increases the antibody response to influenza vaccine or modulates the lymphocyte phenotype in elderly subjects. DESIGN: a randomized controlled trial with two supplemented groups and one control group. SETTING: a community nursing home. PARTICIPANTS: 384 subjects aged 64-100 (mean age 82 years) examined in three separate studies. INTERVENTION: oral supplementation with zinc (400 mg/day) or zinc plus arginine (4 g/day) for 60 days starting 15 days before influenza vaccination. The control groups received vaccine only. MEASUREMENTS: haematological and nutritional indices, antibody titre against influenza viral antigens, lymphocyte phenotype. RESULTS: supplementation with zinc or zinc plus arginine increased zinc plasma concentrations restoring the age-related impairment in zinc concentrations to values found in younger people. The antibody titre against influenza viral antigens was not increased in zinc or zinc/arginine supplemented groups in comparison with subjects receiving vaccine alone. The number of CD3, CD4 or CD8 lymphocytes was not affected by zinc or zinc/arginine supplementation. CONCLUSION: prolonged supplementation with zinc or zinc/arginine restores zinc plasma concentrations but is ineffective in inducing or ameliorating the antibody response after influenza vaccination in elderly subjects.

Aged↗

Early events in cell-mediated recognition of vascularized xenografts: cooperative interactions between selected lymphocyte subsets and natural antibodies.

Cell-mediated early immune recognition of xenogeneic vascularized discordant grafts is poorly characterized. It has been the purpose of our studies to elucidate the role of lymphocytes in the recognition and rejection phenomena. To this end, we have utilized both ex vivo and in vitro model systems. We demonstrate that selected human lymphocyte subpopulations (mainly NK cells) rapidly and specifically adhere to xenogeneic endothelia. Efficient lysis of endothelial cells is subsequently mediated by such a population. Adhesion and cytotoxicity occur via at least two pathways, one dependent on and the other independent of the presence of human natural antibodies of the G class. Both IgG-dependent and IgG-independent adhesion and cytotoxicity can be partly inhibited by the use of anti-leukocyte integrin monoclonal antibodies. IgG-dependent adhesion and cytotoxicity can be also partly inhibited by carbohydrate structures that contain the alpha-galactosyl epitope. A possible role of these events in the eventual outcome of discordant vascularized xenogeneic transplants can be postulated.

Animals↗

Lymphocyte subset reference ranges in Romanian adult Caucasians.

The paper reports the distribution of lymphocyte subpopulations in the Caucasian population of Romania. Investigations were carried out in cells bearing the following antigens: CD3 (T cells), CD19 (B cells), CD4 (T helper/inducer cells), CD8 (T suppressor/cytotoxic and some NK cells), and CD16 CD56 (NK cells). Reference values for the lymphocyte subpopulation were obtained from over 100 healthy Caucasian adult volunteers. Blood from these donors was analyzed using FACScan flow cytometer, Leuco-GATE, Simultest and FACS Lysing Solution, and SimulSET software. As an internal quality control, it was verified that %T+%B+%NK approximates 100% in all samples. The results presented here, obtained on healthy donors (51 males and 49 females), showed that there are no statistically significant variations of CD4/CD8 ratio related to sex or age, the mean value of this ratio (2.0 +/- 0.02) being similar to that reported by the West-European countries. Additional similarities were found when the relative percentage, mean +/- standard deviation (CD3 = 74.2 +/- 2.8; CD19 = 10.8 +/- 1.6; CD4 = 42.0 +/- 2.5; CD8 = 28.9 +/- 5.7; CD56 = 15.2 +/- 0.4) and the absolute cell number of the major peripheral blood mononuclear subsets established in our study were compared with other published results. This study was entirely supported by Becton Dickinson--Europe (Division Heidelberg, Germany).

Adolescent↗

Direct impact of inactivated HIV-1 virions on B lymphocyte subsets.

Although there is no convincing evidence that HIV infects primary B cells, marked changes in B cell responses have been described in HIV-1-infected subjects, including B cell repertoire perturbations, depression of B cell memory and paucity of CD5(+) B cells. As it is hard to assess the consequences of these in vitro and ex vivo observations in patients, the pathogenic mechanisms responsible for the B cell deficit are unclear, and direct and indirect effects of HIV-1 remain possible. To gain further insight into the impact of HIV-1 on the B cell compartment in vivo, we used XenoMouse mice, mice genetically engineered to express human antibodies with an absence of mouse antibody expression. In these transgenic animals, B cells expressing a virtually full human Ig repertoire develop, which allows investigation of the in vivo consequences of confronting B cells expressing human immunoglobulins with HIV-1. We found that soluble gp120 induced an inversion in the B-1a/B-1b cell ratios, without impacting B-2 cells or affecting substantially the T cell compartment. Virion treatment specifically and dramatically depressed B-1a cells, which represent the majority of B-1 cells in normal mice. The observed B cell changes were associated with a functional alteration of the humoral response to tetanus toxoid. Thus, the results reveal a capacity of HIV-1 to specifically impact a highly specialized B cell subpopulation. Because there is evidence that human IgM memory B cells are functionally equivalent to murine B-1a cells, our findings suggest that gp120 may have a direct deleting activity on B cell memory.

Animals↗

Characterization of peripheral blood lymphocyte subsets in patients with acute Plasmodium falciparum and P. vivax malaria infections at Wonji Sugar Estate, Ethiopia.

We investigated the absolute counts of CD4+, CD8+, B, NK, and CD3+ cells and total lymphocytes in patients with acute Plasmodium falciparum and Plasmodium vivax malaria. Three-color flow cytometry was used for enumerating the immune cells. After slide smears were stained with 3% Giemsa stain, parasite species were detected using light microscopy. Data were analyzed using STATA and SPSS software. A total of 204 adults of both sexes (age, >15 years) were included in the study. One hundred fifty-eight were acute malaria patients, of whom 79 (50%) were infected with P. falciparum, 76 (48.1%) were infected with P. vivax, and 3 (1.9%) were infected with both malaria parasites. The remaining 46 subjects were healthy controls. The leukocyte count in P. falciparum patients was lower than that in controls (P=0.015). Absolute counts of CD4+, CD8+, B, and CD3+ cells and total lymphocytes were decreased very significantly during both P. falciparum (P<0.0001) and P. vivax (P<0.0001) infections. However, the NK cell count was an exception in that it was not affected by either P. falciparum or P. vivax malaria. No difference was found in the percentages of CD4, CD8, and CD3 cells in P. falciparum or P. vivax patients compared to controls. In summary, acute malaria infection causes a depletion of lymphocyte populations in the peripheral blood. Thus, special steps should be taken in dealing with malaria patients, including enumeration of peripheral lymphocyte cells for diagnostic purposes and research on peripheral blood to evaluate the immune status of patients.

Acute Disease↗

Activation of T lymphocyte subsets by synthetic TSH receptor peptides and recombinant glutamate decarboxylase in autoimmune thyroid disease and insulin-dependent diabetes.

We have postulated that a defect in specific antigenic induction of suppressor T lymphocytes may account for the immunoregulatory disorder in autoimmune thyroid disease. In this context, we have measured the proliferative responses of peripheral blood mononuclear cells (PBMC) to the synthetic peptides corresponding to the extracellular domain of the TSH receptor (TSHR) and recombinant glutamate decarboxylase (GAD65) by means of 3H thymidine incorporation. We have also studied the antigenic activation of CD4+ and CD8+ T lymphocytes by measuring human leukocyte antigen-DR (HLA-DR) expression on the cell surface by flow cytometric analysis. PBMC obtained from 47 patients with Graves' disease (GD) [including 19 hyperthyroid GD (hyper GD)], 18 with Hashimoto's thyroiditis (HT), 7 with nontoxic nodular goiter (NG), 18 with insulin-dependent diabetes (IDDM), and 20 normal controls (N), were cultured for 7 days in the presence or absence of the pool peptides representing 3 different segments of TSHR or GAD65 at final concentration of 30 micrograms/mL or 10 micrograms/mL. The proportion of subjects whose PBMC gave a positive proliferative response with a stimulation index (SI) of over 2.3 (i.e. above the mean +2 SD for N) to TSHR peptides was significantly higher in the hyper GD group than among euthyroid GD (eu GD), HT, IDDM, and N group. The corresponding differences in mean SI provided analogous results, showing significant responses above normal in only hyper GD. The CD4+ T lymphocytes from hyper GD group were significantly more activated by TSHR peptides compared to eu GD, HT, IDDM, and N, and this induction correlated to their thyroid hormone levels. Quite differently, the activation of CD8+ T lymphocytes from both hyper GD and eu GD group in response to TSHR peptides was impaired compared to HT, IDDM, and the N group; in contrast to the findings with CD4+ T lymphocytes, this was independent of thyroid hormone levels. On the other hand, while the CD8+ T lymphocytes from GD and N groups were activated equally by GAD65, the activation of CD8+ T lymphocytes from the IDDM group by GAD65 was impaired compared to the GD and N groups. In conclusion, the activation of CD8+ T lymphocytes from GD and IDDM by relevant antigens (i.e. TSHR peptides for GD and GAD65 for IDDM) was impaired, but not by irrelevant antigens (i.e. GAD65 for GD and TSHR peptides for IDDM). There was also a modest stimulation of CD8+ T cells from all groups by tetanus toxoid and cardiac myosin light chain peptide, both irrelevant antigens.(ABSTRACT TRUNCATED AT 400 WORDS)

Adolescent↗