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PubMed1994

Flow cytometric immunophenotyping of lymphocyte subsets in samples that contain a high proportion of non-lymphoid cells.

Flow cytometric (FCM) immunophenotyping of peripheral blood from thalassemia patients presents technical difficulties because of the high proportion of immature red cells. The combination of forward scatter (FSC) and side scatter (SSC) with fluorescence associated with human leukocyte antigen/monocyte antigen (CD45/CD14) was unable to identify the lymphocyte population in thalassemia patients; therefore, it was necessary to exclude immature red cells to analyze lymphocyte subsets in these patients. A simultaneous three-color FCM method was developed, with the basis that transferrin receptor (CD71) or glycophorin A (glyco A) is present on all immature red cells, but is not expressed on CD45 positive leukocytes. In this study, the lymphocyte population was identified by gating out unwanted cell populations using the FSC/CD71-fluorescein isothiocyanate (FITC), FSC/glyco A-FITC, or FSC/CD45-peridinin chlorophyll protein (PerCP) profiles. The CD71-FITC negative cells, glyco A-FITC negative cells, or CD45-PerCP positive cells were identified, then analyzed on the basis of FSC/SSC to allow any remaining non-lymphocyte cells in FSC/SSC gate to be excluded. The cells in FSC/SSC gate were then analyzed using other irrelevant two-color antibodies. Of the three gating strategies, CD45-PerCP and glyco A-FITC methods are better than the CD71-FITC gating method. Both methods markedly increase the purity of lymphocytes in the analysis gate. Either method is easy, straightforward, requires a six-tube set of reagent tubes, and provides a reliable method for immunophenotyping lymphocyte subsets in preparations containing a large percentage of non-lymphoid cells.

Adult↗
PubMed1983

Lymphocyte subsets in Sjogren's syndrome: a quantitative analysis using monoclonal antibodies and the fluorescence-activated cell sorter.

Lymphocyte subsets in the peripheral blood of 18 patients with Sjogren's syndrome (SS) were studied using monoclonal antibodies and the fluorescence-activated cell sorter (FACS). The percentage of T cells was decreased when compared to normal controls. In primary SS, there was a proportional decrease in both suppressor/cytotoxic (anti-Leu-2a reactive) and helper/inducer (anti-Leu-3a reactive) T cells with an unchanged helper/suppressor ratio (1.8 vs. 1.7 for normals). In SS with an associated connective tissue disorder, there was a significant decrease only in the suppressor/cytotoxic subset. There was increase in B cells and null cells in primary SS compared to controls. Quantitative immunofluorescence allowed the calculation of determinant density per cell. Cells expressing low antigen density Leu-2a were increased in 8 patients (4 with primary SS and 4 with SS with an associated disorder). Thus, in addition to quantitative changes in lymphocyte subsets, we found changes in Leu-2a expression suggesting abnormal differentiation of the suppressor/cytotoxic subset. These changes may contribute to the immunoregulatory disturbance in Sjogren's syndrome.

Adult↗
PubMed1995

Efficacy of an analysis of lymphocyte subsets in predicting the clinical response to alpha-interferon therapy in thalassaemia patients with chronic infection by hepatitis C virus: a pilot study.

alpha-interferon (alpha-IFN) has been used to treat chronic non-A non-B hepatitis in thalassaemic patients with response rates from 45% to 83%. Unfortunately, treatment with alpha-IFN is associated with side-effects which have a negative effect on the quality of life of the patient. Therefore it would be useful if we could distinguish in advance those patients who would benefit from such therapy from those who would not. In the present study we found that the modification of lymphocyte subsets 20 h after the administration of the first dose of alpha-IFN revealed that relative numbers of T helper lymphocytes (CD4+) increased in three non-responding patients and decreased in five responding patients, whereas those of T suppressor lymphocytes (CD8+), and natural killer cells (CD57+, CD16+) decreased in non-responding patients and increased in responding patients. Therefore analysis of the lymphocyte subsets CD4, CD8, CD57 and CD16 before and 20 h after the administration of alpha-IFN can be used to predict the clinical response to treatment with alpha-IFN.

Adolescent↗
PubMed2000

Lymphocyte subset proliferative responses of Mycobacterium bovis-infected cattle to purified protein derivative.

Despite highly successful eradication efforts in several countries, Mycobacterium bovis infection of cattle remains a significant health concern worldwide. Immune mechanisms of resistance to and/or clearance of M. bovis infection of cattle, however, are unclear. Recent studies have provided evidence supporting a role for CD4(+), CD8(+), and gammadelta TCR(+) T cells in the response of cattle to M. bovis. In the present study, we utilized a flow cytometric-based proliferation assay to determine the relative contribution of individual lymphocyte subsets in the response to M. bovis infection and/or sensitization with mycobacterial purified protein derivative (PPD). Peripheral blood mononuclear cells (PBMC) from M. bovis-infected cattle proliferated in response to in vitro stimulation with M. bovis PPD. CD4(+) T cells and gammadelta TCR(+) cells were the predominate subsets of lymphocytes responding to PPD. gammadelta TCR(+) cells also proliferated in non-stimulated cultures; however, the gammadelta TCR(+) cell proliferative response of infected cattle was significantly (p<0.05) greater in PPD-stimulated cultures as compared to non-stimulated cultures. Intradermal injection of PPD for comparative cervical testing (CCT) induced a boost in the in vitro proliferative response of CD4(+) but not gammadelta TCR(+) cells of infected cattle. Administration of PPD for CCT also boosted interferon-gamma (IFN-gamma) production by PBMC of infected cattle following in vitro stimulation with M. bovis PPD. Injection of PPD for CCT did not, however, elicit a proliferative or IFN-gamma response in cells isolated from non-infected cattle. These data indicate that CD4(+) and gammadelta TCR(+) cells of M. bovis-infected cattle proliferate in a recall response to M. bovis PPD and that the CD4(+) cell response is boosted by intradermal injection with PPD for CCT.

Animals↗
PubMed2004

[Effect of integrative Chinese and western medicine on T-lymphocyte subsets in treating patients with severe acute respiratory syndrome].

OBJECTIVE: To investigate the therapeutic effect of integrative Chinese and western medicine (ICWM) in treating severe acute respiratory syndrome (SARS) and its influence on T-lymphocyte subsets. METHODS: Comparative study was conducted in 133 SARS inpatients in Beijing Ditan Hospital, who were divided into 3 groups according to the treatment applied, the basic treated group, the low dose steroid group and the high dose steroid group, and all the 3 groups were subdivided into two groups, Chinese herbs and non-Chinese herbs added, respectively. Chinese drugs for clearing-up heat, dispelling dampness, detoxication, removing stasis, supplementing Qi and nourishing Yin were selected according to patients' syndrome and given additionally to all the three ICWM groups. Retrospective analysis for significance test on changes of T-lymphocyte subsets before and after treatment were carried out. RESULTS: T-lymphocyte counts, including CD3+, CD4+, CD8+, lowered in all patients before treatment, but increased significantly after treated for 3 weeks, the increment in all the low dose steroid treated groups was higher than that in the basic treated groups, and that in ICWM groups was higher than that in non-ICWM groups, respectively. CONCLUSION: Glycocorticosteroid and Chinese herbal medicine treatments could promote the recovery of T-lymphocyte profile, rationally use of them is the effective therapeutic method.

Adolescent↗
PubMed1992

[An analysis of lymphocyte subsets and purified protein derivative-antibody in pulmonary tuberculosis].

The lymphocyte subsets and PPD-antibody in peripheral blood were determined in 72 cases of bacteriological positive pulmonary tuberculosis using monoclonal antibody to anti-T lymphocyte and ELISA assay. The results showed that the percentage of T3 was 52.44 +/- 11.46% and the OD of PPD-antibody was 0.47 +/- 0.27 in initially treated patient group; 40.27 +/- 7.81%, 0.81 +/- 0.17 in chronic carrier group respectively. There were statistically significant difference between the two groups (P < 0.01). The percentage of T3 was significantly larger in 30-59 yr group than in more than 60 yr group among non chronic carriers (55.9 +/- 11.3% vs 44.4 +/- 2.4, P < 0.05). PPD-antibody was strongly associated with extent of lesion in lung.

Adolescent↗
PubMed2001

Clinical prognosis of patients with early-stage human immunodeficiency virus (HIV) disease: contribution of HIV-1 RNA and T lymphocyte subset quantitation.

Systems for the staging of individuals with human immunodeficiency virus type 1 (HIV-1) infection were developed 15 years ago. Subsequently, assays for quantitating HIV-1 RNA and immunophenotyping of lymphocyte subsets have been developed and validated. The utility of these assays for improved staging in early disease was evaluated in 256 HIV-infected adults (52% minority) with CD4 counts > or = 400 cells/microL followed in U.S. military medical centers before the highly active anti-retroviral therapy era. HIV viral load (RNA) was quantitated; the frequencies of select CD4+ immunophenotypes were determined in 112 subjects. The results were analyzed in relation to three outcome measures: death, first acquired immunodeficiency syndrome-defining opportunistic infection, and CD4 count < or = 200 cells/microL. Serum RNA level and CD4 count were each found to be predictive of all three outcomes. In addition, increases in the T-cell subsets CD28-CD4+ and CD29+CD26-CD4+ were found to be independently predictive of more rapid progression. The classification of early-stage HIV patients is improved by the quantitation of both viral RNA and T-lymphocyte subsets.

Adult↗
PubMed2004

Changes in lymphocyte subsets after short-term pharmacotherapy in patients with panic disorder.

Panic disorder is associated with a high frequency of comorbid immunological diseases, such as allergies and asthma, although the psychoneuroimmunology of panic disorder is relatively unexplored. The objective of this study was to determine whether panic patients have different immunological findings compared with normal healthy subjects and whether changes in immune function are associated with short-term pharmacotherapy. We also examined whether immunological variables were associated with clinical severity and serum catecholamine levels. Patients with panic disorder (n=26) and healthy control subjects (n=26) were recruited for this study. All patients were treated with paroxetine for 3 months. We measured the lymphocyte subsets, psychopathological characteristics and serum catecholamine (norepinephrine and epinephrine) levels. Panic patients did not differ initially from control subjects in peripheral lymphocyte phenotypic markers. After drug therapy, however, percentages of circulating CD3+, CD4+ and CD8+ T lymphocytes were significantly increased, while the percentage of CD19+ B lymphocytes was significantly decreased in the patients. The difference in the percentage of CD8+ T lymphocytes before and after treatment was negatively correlated with pretreatment Global Clinical Impression scores. The lymphocyte subsets were not significantly associated with serum catecholamine levels in panic patients. In conclusion, panic patients showed increased CD3+, CD4+ and CD8+ T lymphocyte proportions and a decreased B lymphocyte proportion after 3 months of drug therapy. This finding suggests that pharmacological treatment may affect immune function in panic patients.

Adult↗
PubMed1995

The effect of 2-acetyl-4-tetrahydroxybutylimidazole on lymphocyte subsets during a contact hypersensitivity response in the NOD mouse.

The compound 2-acetyl-4-tetrahydroxybutylimidazole (THI) is known to suppress a contact hypersensitivity (CH) response. The effect of THI on lymphocyte subsets during treatment and in a CH response has not been shown in mice. To further define the immunosuppressive potential of THI, a time-course study during the CH response to oxazolone (OX) was performed. While THI can prevent the induction of CH, if treatment is started before sensitization, it has a low therapeutic capability since it could not significantly inhibit the response when continuous oral treatment was commenced during the course of CH. We report that during this response continuous oral treatment of non-obese diabetic (NOD) mice with THI reduced the number of CD4+ and CD8+ T cells in the peripheral blood. In the draining lymph nodes THI treatment had no effect on lymphocyte subsets prior to contact sensitization, but subsequent sensitization and elicitation with OX could not stimulate a significant increase in the number of CD4+ T cells in the treated mice, whereas untreated control mice showed elevated numbers of these lymphocytes. These findings suggest that THI can inhibit an CH response by preventing the recruitment of CD4+ T cells in the draining lymph nodes through an unknown mechanism.

Animals↗
PubMed1990

Nonrandom migration of CD4+, CD8+, and gamma delta+T19+ lymphocyte subsets following in vivo stimulation with antigen.

We report here the results of experiments in which the migration of three T cell subsets (CD4+, CD8+, and gamma delta+T19+ cells) through antigen-stimulated lymph nodes and subcutaneous granulomas has been compared with that through normal skin and resting lymph nodes. The percentage of gamma delta+T19+ lymphocytes was halved and the percentage of CD8+ lymphocytes was doubled in lymph draining stimulated compared with control tissues, and all lymphocyte subsets except gamma delta+T19+ lymphocytes had higher hourly outputs in lymph draining antigen-stimulated compared with control tissues. Antigen also resulted in a higher percentage of CD8+ lymphoblasts and a lower percentage of gamma delta+T19+ lymphoblasts in efferent lymph draining antigen-stimulated lymph nodes. The data indicate that lymphocyte subsets leave the blood with differing efficiencies in different vascular beds and raise the possibility that antigen can influence the rate at which tissues extract individual T cell subsets from the blood.

Animals↗
PubMedDate not supplied

Inhibition of mixed lymphocyte response by monoclonal antibody specific for a rat T lymphocyte subset.

Cell-surface differentiation antigens which are associated with particular lymphocyte subsets are likely to be molecules which mediate specific functions of the cells which display them. One way to investigate this hypothesis is to see whether antibodies directed against such antigens will inhibit functional systems. This approach can be attempted with confidence if monoclonal antibodies are used, as these react with one antigenic determinant only and can be used in purified form. In this report we show that a monoclonal antibody which is specific for a subset of rat tT lymphocytes inhibits the mixed lymphocyte response (MLR). The antibody acts on the responder cells without killing them.

Animals↗
PubMed2001

The distribution of major lymphocyte subsets in cord blood is associated with its pH.

OBJECTIVES: To assess in venous cord blood the distribution of major lymphocyte subsets according to pH and medications used during labor. DESIGN AND METHODS: Venous cord blood was sampled immediately after labor from 70 newborns (35 males and 35 females) delivered vaginally. Lymphocytes were immunophenotyped by flow cytometry and pH was measured using the AVL 900 automated blood gas analysis system. Data on birth weight, gestational age at delivery, length of labor, presence of stained amniotic fluid, medications used during labor, maternal risk factors, age and parity were collected. RESULTS: The percentage of T lymphocytes decreased while the percentage of NK lymphocytes increased with decreasing pH over the whole range of pH values. The proportions of T and NK lymphocytes were associated with the administration of neuroplegics, spasmolytics or dihydroergotoxin in the first stage of labor. CONCLUSIONS: Cord blood pH and labor-associated variables should be taken into account to adequately interpret the profile of major lymphocyte subsets as a marker of the effect of different prenatal factors on the immune system of neonates.

Adolescent↗
PubMedDate not supplied

Lymphocyte subsets in lung tissues of non-specific interstitial pneumonia and pulmonary fibrosis associated with collagen vascular disorders: correlation with CD4/CD8 ratio in bronchoalveolar lavage.

This study was designed to evaluate the distribution of lymphocyte subsets in lung specimens that were obtained by open-lung biopsy from 8 patients with idiopathic nonspecific interstitial pneumonia/fibrosis (NSIP) and 10 patients with pulmonary fibrosis associated with collagen vascular disorders (PF-CVD). Distributions of B lymphocytes, CD4-positive T lymphocytes, and CD8-positive T lymphocytes were evaluated immunohistochemically and compared with the cell composition in BALF. Correlation between CD4/CD8 ratios in bronchoalveolar lavage fluids (BALF) and CD4/CD8 ratios in lung tissues was also examined. B lymphocytes were mostly restricted in lymphoid follicles. CD4-positive T lymphocytes were observed inside and around lymphoid follicles and in the thick fibrotic wall of reconstructed alveoli with fibrosis. In contrast, CD8-positive T lymphocytes were diffusely distributed, especially in relatively thin alveoli. Correlation was weak between CD4/CD8 ratios in lung tissue and CD4/CD8 ratios in BALF. However, even in patients with very low CD4/CD8 ratios in BALF, many CD4 lymphocytes were observed in lung tissues, suggesting that CD8-positive lymphocytes diffusely distributed in thin alveolar architecture were more easily recovered in BALF than CD4-positive lymphocytes. Therefore, a low CD4/CD8 ratio in BALF may indicate that the alveolar structure was not severely reconstructed by fibrosis. This is the first report that compared lymphocyte subsets in lung tissues and in BALF.

Aged↗
PubMed1992

Activated lymphocyte subsets in adult periodontitis.

The activation state of T and B lymphocytes in the peripheral blood of periodontitis patients may be a reflection of disease activity. We have utilized 2- and 3-color flow cytometric analyses using a new chromophore, peridinin chlorophyll A protein, and conventional dyes, fluorescein isothiocyanate and phycoerythrin, conjugated to monoclonal antibodies against activated lymphocyte surface markers to measure blood lymphocyte subsets from 18 periodontitis patients and 16 periodontally healthy control subjects. Two-color flow cytometric analysis demonstrated that the frequency of CD4+ and CD5+ T cells, CD20+ B cells, and CD16+ NK (natural killer) cells were increased in periodontitis patients. Of particular interest, CD4+ activated "memory" T cells, CD5+ B cells, and CD56+ NK effector cells were increased significantly in periodontitis patients (p less than 0.05). While the relationship of lymphocyte activation to periodontal disease activity remains unclear, there may be potential for using 2- and 3-color flow cytometry to subcategorize periodontitis patients into high- and moderate-risk groups.

Adult↗
PubMed1998

Premedication with clonidine does not attenuate suppression of certain lymphocyte subsets after surgery.

UNLABELLED: Sixty-four patients undergoing elective major surgery were randomly assigned into a double-blinded, placebo-controlled, clinical trial to test the hypothesis that premedication with clonidine would attenuate postoperative reductions in circulating lymphocytes. The treatment group (n = 28) received a clonidine skin patch (0.3 mg/d) and a 0.6-mg oral loading dose 60-90 min before surgery. The control group (n = 36) received placebo patches and pills. Absolute blood levels of the following lymphocyte subsets were measured before induction of a standardized general anesthetic (baseline) and the morning after surgery: CD2, CD3, CD4, CD8, CD20, CD56, and the CD4:CD8 ratio. Significant decreases in lymphocyte subsets CD2, CD3, and CD4 were found in both groups; CD56 was significantly decreased only in the placebo group. However, the extent of lymphocyte depletion from baseline to Postoperative Day 1 between the clonidine and placebo groups was not different. Plasma concentrations of epinephrine, norepinephrine, and cortisol were measured from blood samples drawn at 8:00 AM on Postoperative Day 1. Plasma norepinephrine levels were significantly lower among patients who received clonidine. However, no significant differences were found in plasma epinephrine or cortisol levels between the clonidine and placebo groups. With a clinical dose, clonidine did not prevent postoperative lymphocyte depletion. alpha2-Agonists may not suppress adrenocortical stress responses sufficiently to prevent postoperative immune suppression. IMPLICATIONS: Lymphocyte (white blood cell) counts often decrease after major surgery. We hypothesized that clonidine would reduce hormonal stress and blunt reductions in lymphocytes after major surgery. In a randomized trial, we found no differences from placebo in cortisol levels or lymphocyte changes. Lymphocyte levels did not predict infectious complications.

Adjuvants, Anesthesia↗
PubMed1992

Imbalance of CD4+ lymphocyte subsets in patients with mixed connective tissue disease.

CD4+ (helper/inducer) T lymphocyte subsets were studied in the peripheral blood from patients with mixed connective tissue disease (MCTD) by double-labelling immunofluorescence. The proportion of CD4+CD45RA+ cells was higher (P less than 0.01) when compared with controls, whereas CD4+CD29+ cells were markedly diminished (P less than 0.001). CD4+CD29+ cells were lower than in patients with progressive systemic sclerosis who were studied in parallel. Upon stimulation with phytohaemagglutinin, CD4+ cells from MCTD patients showed a strong reactivity to acquire the CD29+ phenotype. Expression of high levels of CD29 and other adhesion molecules might lead to facilitated localization of CD4+ cells to inflamed tissue. It is suggested that an increased responsiveness of CD4+ cells to activation signals in vivo and accumulation of CD4+CD29+ cells at tissue sites could result in depletion of this cell subset in the peripheral blood of patients with MCTD.

Adult↗
PubMed1992

[Lymphocyte subsets of peripheral blood in patients with sarcoidosis].

Cytofluorometric analysis of lymphocyte subsets was performed in the peripheral blood samples from fifteen sarcoidosis patients with uveitis as well as normal controls. The percentage of T cells (CD3+) was significantly decreased, whereas that of B cells (CD19+) was markedly increased in patients with sarcoidosis compared to controls. Natural killer cells (CD8+CD57+) as well as activated T cells (CD3+HLA-DR+) were significantly increased in patients with sarcoidosis. The percentage of the Leu 1-B cells and the ratio of Leu 1-B/B were significantly higher in sarcoidosis patients with respect to controls. Moreover, the ratio of Leu 1-B/B was significantly correlated with the serum level of angiotensin converting enzyme (ACE) in sarcoidosis. Therefore, it is considered that the Leu 1-B/B value could be a useful indicator not only for the activity but also for the diagnosis of sarcoidosis. No significant difference was found in any subset between the patients with active uveitis and patients with inactive uveitis.

Adolescent↗
PubMed1999

Lymphocyte subsets in renal transplant recipients during growth hormone therapy.

OBJECTIVE: To evaluate the effect of growth hormone treatment on growth, levels of insulin-like growth factor I (IGF-I) and lymphocyte subsets in immunosuppressed renal allograft recipients. METHODS: 18 children (aged 8.0-16.6 years) received growth hormone 1 IU/Kg/week daily for two years. Height, IGF-I levels and in 11/18 patients, lymphocyte subsets were evaluated serially. RESULTS: Standardized growth velocity increased from -1.0+1.5 to +1.2+2.2 and standardized IGF-I levels from +0.8+1.5 to +3.1+1.1 (1 year) and to +1.4+1.7 (2 years). The total lymphocyte count and the number of T lymphocytes (CD3+) decreased. The decrease was more marked in CD8+ (from 1.5+0.3 x10(9)/L to 0.9+0.3 x10(9)/L, 1 year and to 0.8+0.1 x10(9)/L, 2 years) compared to CD4+ (from 1.5+0.3 x10(9)/L to 1.0+0.2 x10(9)/L, 1 year and to 1.3+0.2 x10(9)/L, 2 years), resulting in an increment of the CD4+/CD8+ index. CONCLUSIONS: The differential effect of growth hormone treatment on CD4+ and CD8+ lymphocytes might be explained by different expression of the IGF-I receptor in these distinct subsets.

Adolescent↗