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Lymphocyte subsets in patients with oestrogen deficiency.

We have previously shown that in patients with idiopathic premature ovarian failure there were significant changes in lymphocyte subsets. To test our hypothesis that these changes were due to oestrogen deficiency we studied lymphocyte subsets in patients with oestrogen deficiency due to other causes. Blood was taken for serum oestradiol, lymphocyte counts and lymphocyte subset counts (CD2+, CD4+, CD8+ and B cells) before oestrogen replacement in 19 patients with gonadal dysgenesis, 22 patients with hypothalamic-pituitary failure and 24 healthy female control subjects. The CD4:CD8 ratio in both groups of patients was significantly lower than that in the normal control subjects while the percentages and counts of lymphocytes and CD8+ cells were significantly higher. There was a significant positive correlation between the serum oestradiol level and the CD4:CD8 ratio. These findings support the hypothesis that the changes in lymphocyte subsets are due to oestrogen deficiency.

Antigens, CD↗

Lymphocyte subset numbers depend on the bacterial origin of sepsis.

OBJECTIVE: To determine the quantitative variances in peripheral blood lymphocyte subsets during sepsis, and their clinical significance. METHODS: Peripheral blood lymphocyte subsets were enumerated in 32 non-surgical septic patients during the first 14 days of hospitalization; results from septic patients were compared with those from 34 healthy controls. Influences of the severity and the bacterial etiology of sepsis on changes in lymphocyte subsets were also assessed. RESULTS: Significant decreases (P < 0.05) from normal values of CD4+, CD8+ and total T-lymphocytes were observed in septic patients, but the decline persisted only for CD4+ T-lymphocytes and natural killer (NK) cells for 3 and 7 days, respectively. In addition, the numbers of CD3+/DR+ lymphocytes were significantly elevated on day 14. There were no correlations between these alterations and the severity of sepsis. Gram-positive sepsis (n = 10), which was mainly due to Streptococcus pneumoniae and Staphylococcus aureus, caused prolonged decreases in CD4+, CD8+ and total T-lymphocytes, and a reduction in NK cells, that lasted for >or=14 days. Conversely, patients with sepsis due to Gram-negative pathogens (Neisseria meningitidis, n = 8; enterobacteria, n = 2) achieved full recovery of the subsets within 3 days. Moreover, the patients with Gram-negative sepsis demonstrated a significant increase in B-lymphocytes, and a rise in the numbers of CD3+/DR+ and CD4+ T-lymphocytes, which were more rapid than in patients with Gram-positive sepsis. CONCLUSION: Our results indicate that Gram-positive sepsis causes stronger suppression of peripheral blood lymphocyte subsets in comparison to sepsis due to Gram-negative pathogens.

Adolescent↗

Lymphocyte subsets in lung tissues of interstitial pneumonia associated with untreated polymyositis/dermatomyositis.

This study was designed to evaluate the distribution of lymphocyte subsets in lung specimens obtained by surgical lung biopsy from 12 patients with interstitial pneumonia associated with untreated polymyositis/dermatomyositis (PM/DM). Differences of histological findings and distributions of lymphocyte subsets between PM and DM were also evaluated. Distributions of B lymphocytes, CD4-positive T lymphocytes, and CD8-positive T lymphocytes were evaluated immunohistochemically. Interstitial pneumonia was pathologically classified as basically nonspecific interstitial pneumonia (NSIP) in all patients. Immunohistochemically, the distribution of B lymphocytes was mostly restricted to inside and/or around lymphoid follicles. The CD4-positive T lymphocytes were distributed diffusely in fibrotic areas and unrelated to lymphoid follicles. Most CD8-positive T lymphocytes were diffusely distributed, especially in relatively normal alveoli. There were no significant differences in the distribution of lymphocyte subsets between PM and DM. Although the distribution of B lymphocytes and CD4- and CD8-positive T lymphocytes in the lung were different, there were no significant differences in distributions of lymphocyte subsets between PM and DM.

Aged↗

Circulating lymphocyte subsets in human acute pancreatitis.

We investigated peripheral lymphocyte subsets in 34 consecutive acute pancreatitis patients (21 males, 13 females; mean age, 57 years; range, 16-85 years) studied within 48 h of pain onset and for 5 consecutive days to understand better the immunological response during the course of the disease. The diagnosis was based on characteristic abdominal pain associated with a twofold increase in serum lipase and confirmed by imaging techniques in all patients. Acute pancreatitis was of biliary origin in 25 patients, due to alcohol abuse in 5, due to pancreas divisum in 1, and of unknown origin in 3. Fifteen patients had severe illness and 19 had mild disease. In all patients, total lymphocyte and lymphocyte subset counts were carried out on admission, as well as on the third and fifth day of hospitalization, using a flow cytometric analysis. Twenty-three patients (13 with severe illness and 10 with mild disease) also had a repeat count 1 month after recovery. Twenty-five healthy subjects and 27 patients with nonpancreatic acute abdomen comparable for sex and age were studied as controls. On the first day of the study, the leukocyte number was significantly higher in patients with acute pancreatitis and in those with nonpancreatic acute abdomen with respect to healthy subjects, whereas the number of total and CD4+, CD8+, CD3+ DR-, and CD3- DR+ lymphocytes was significantly lower in acute pancreatitis patients than in healthy subjects or in patients with nonpancreatic acute abdomen. These subject counts persisted on the third and fifth days of the study.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗

Effect of blood donation on the establishment of normal ranges of lymphocyte subsets.

BACKGROUND: Absolute counts of CD4+ T-lymphocytes are used in the management of patients with human immunodeficiency virus infection. Low absolute counts of CD3+CD4+ cells have also been observed in healthy people--a phenomenon called idiopathic CD4 lymphocytopenia. It is common practice for normal ranges for lymphocyte subsets to be derived from samples taken from blood donors. STUDY DESIGN AND METHODS: A sample of EDTA blood was taken through the donation line tubing, after donation from 565 blood donors in Sydney, Australia, who were selected from a range of age groups. An additional 12 donors provided a predonation sample as well as a postdonation sample. Hematologic assays were performed on two analyzers. Samples were stained for CD3, CD4, CD8, CD19, and CD56 and analyzed on a flow cytometer. RESULTS: Three donors were found to have absolute CD3+CD4+ counts < 300 cells per microL. The percentage of CD3+CD4+ cells was found to increase with age. Both the percentage and the absolute count of CD3+CD8+ cells decreased with age, which resulted in an increased CD4:CD8 ratio with age. Men had consistently higher absolute counts of CD3-CD56+ cells than women. The 12 additional donors all had greater percentages of CD3+CD4+ cells and lower absolute counts for CD3+, CD3+CD4+, CD3+CD8+, CD19+ and CD3-CD56+ cells after donation than they had before donation (p < 0.001). CONCLUSION: It is not satisfactory to base normal ranges for lymphocyte subsets on donor blood, from which the blood sample has been obtained after donation.

Adolescent↗

Lymphocyte subsets and natural killer cell responses during cytomegalovirus mononucleosis.

Cell-mediated immunity in seven patients with cytomegalovirus mononucleosis was assessed by monoclonal antibody to lymphocyte subsets, lymphocyte blastogenesis, and natural killer cell activity. The patients had decreased ratios of helper (Leu 3a) to suppressor-cytotoxic (Leu 2a) T cells as compared with normal donors. Leu 2a+ T cells from the patients were uniquely sensitive to overnight culturing, which resulted in a 2.6-fold increase in the helper/suppressor cell ratio and the appearance of an unusual third Leu 1+ 2a- 3a- T cell subset. This correlated directly with enhanced blastogenesis to concanavalin A after preculture. Base-line and interferon-boosted natural killer cell responses versus K562 cell targets during cytomegalovirus mononucleosis did not differ from those of normal donors. This suggests that certain cellular immune functions are depressed during cytomegalovirus mononucleosis in correlation with the emergence of a culture-sensitive subset of suppressor T cells. In contrast, cytotoxic lymphocytes appear to function normally and may aid in the resolution of the illness.

Adult↗

Changes of lymphocyte subsets in leukemia patients who received allogenic bone marrow transplantation.

Proportional changes of lymphocyte subsets in the peripheral blood were monitored by two-color flow-cytometry in seven leukemia patients who had received allogenic bone marrow transplantation (BMT). Lymphocyte counts, and proportions of T and B-cells returned to normal ranges between the 2nd and 12th months after BMT. Activated T-cells prominently increased after BMT, and the values gradually returned toward normal. As to lymphocyte subsets, the proportions of CD 4+ cells had remained low, while those of CD 8+ cells high for a whole observation period after BMT. The changes of CD 4+ cells were caused by the decrease of suppressor-inducer T-cells (CD 4+ Leu 8+). High proportion of CD 8+ cells was mainly associated with increased suppressor T-cells (CD 8+ CD 11+). Among natural killer (NK) cells, highly active NK cells (CD 16+ CD 57-) markedly increased shortly after BMT, and gradually returned to normal. CD 16 -CD 57+ NK cells increased beyond normal ranges after the 2nd month. The incidence or degree of acute and chronic graft-versus-host diseases (GVHD) did not correlate with the changes of any lymphocyte subsets. The present results suggest that the increase of activated T-cells shortly after BMT reflects lymphocyte reconstitution. The prolonged immune deficiency after BMT might be related to either deficient expression of homing receptor (Leu 8 antigen) on CD 4+ cells or increased suppressor T-cells (CD 8+ CD 11+). In addition, the early increase of NK cells after BMT may compensate for the immune deficiency in BMT patients.

Adolescent↗

Lymphocyte subsets and serum immunoglobulins in patients with premature ovarian failure before and after oestrogen replacement.

We have previously demonstrated that in patients with oestrogen deficiency, there was a significant reduction in the ratio of CD4+ to CD8+ lymphocytes. To test our hypothesis that oestrogen deficiency was the cause of the changes in lymphocyte subsets, we have studied the lymphocyte subsets and serum immunoglobulin levels in 35 patients with idiopathic premature ovarian failure before and after oestrogen replacement therapy. Before oestrogen replacement the lymphocyte subsets in peripheral blood were enumerated with indirect immunofluorescence techniques using monoclonal antibodies. The serum immunoglobulins (Ig) G, A and M were measured with nephelometry. After oestrogen replacement therapy, the tests were repeated around day 7-11 of the 2nd, 4th and 24th month. There was no significant change in any of these parameters by the 2nd or 4th months. After 2 years of oestrogen replacement, there was a significant decrease in the percentage of CD8+ cells, a significant increase in the CD4:CD8 ratio and a significant increase in the mean serum IgG concentration. There was no significant change in the other parameters. Our results support the hypothesis that oestrogen deficiency is the cause for the changes in the lymphocyte subsets.

Adult↗

T-lymphocyte subsets in acute illness.

OBJECTIVES: To determine the range of T-lymphocyte subsets (CD4, CD8, and CD4/CD8 ratios) in acutely ill, hospitalized patients and to determine whether these concentrations correlate with illness severity, survival rate, or immunodepression. DESIGN: Cross-sectional study, comparing Acute Physiology and Chronic Health Evaluation II (APACHE II) scores and the calculated, disease-specific, predicted mortality rate with T-lymphocyte subsets. SETTING: Urban county hospital intensive care unit (ICU), serving as the designated trauma center. PATIENTS: One hundred two consecutively admitted ICU patients (72 medical and 30 surgical). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Patient clinical data, APACHE II scores, and their associated predicted mortality rate were recorded. Blinded human immunodeficiency virus (HIV) and lymphocyte testing was performed on samples from all patients on ICU admission. Despite only three (2.9%) of 102 patients testing positive for HIV antibodies, 41% (42/102) of patients had CD4 concentrations of < 400 cells/microL, and 29% (29/102) had CD4 concentrations of < 300 cells/microL. Mean CD8 concentrations were even lower, compared with normal laboratory values, resulting in a slight increase in CD4/CD8 ratios, although 16% (16/102) of patients had a CD4/CD8 ratio of < 1. CD4 counts were linearly related to total lymphocyte concentrations (Pearson correlation coefficient = 0.948), but no relationship was found between total lymphocyte or lymphocyte subset counts and APACHE II score, predicted mortality rate, or survival rate. CONCLUSIONS: Acute illness alone, in the absence of HIV infection, can be associated with profound decreases of T-lymphocyte populations. This problem is unpredictable and does not correlate with severity of illness, predicted mortality rate, or actual mortality rate. No conclusions regarding HIV serostatus or survival can be made based on single measurements of T-cell concentrations in acutely ill hospitalized patients.

APACHE↗

[Quantitative flow cytometric determination of cytoplasmic bcl-2 oncoprotein in peripheral blood lymphocyte subsets].

We measured serial changes of cytoplasmic bcl-2 oncoprotein in peripheral blood lymphocyte subsets after preparation of cell samples or cultivation with mitogen or interleukin-2 (IL-2) by two-color flow cytometry using monoclonal antibodies to lymphocyte subsets and human bcl-2. The bcl-2 levels determined by mean fluorescence intensity were diminished in most lymphocyte subsets when the samples were preserved at 4 degrees C for 6 hours or more without 1% paraformaldehyde (PFA) fixation. However, the bcl-2 levels were kept constant for a week or more in the samples preserved at 4 degrees C after immediate staining of cell surface markers and subsequent fixation with PFA. The percentages of CD4+ cells, CD8+, CD19+ cells and Fas+ cells became high when lymphocytes were cultured with concanavalin-A, and bcl-2 levels were also enhanced in these subsets. In addition, the bcl-2 levels were compared between lymphocytes cultured with and those without recombinant IL-2. IL-2 inhibited spontaneous decrease of bcl-2 in cultured lymphocytes. Apoptosis of the cultured lymphocytes was also determined by a flow cytometric method. IL-2 seemed to diminish apoptosis of cultured lymphocytes.

Adult↗

[Lymphocyte subsets analysis and glycosylphosphatidylinositol determination in patients with PNH].

OBJECTIVE: Absolute number of lymphocyte subsets and the proportion of glycosylphosphatidylinositol (GPI)-deficient clones among T, B and NK cells were determined. METHODS: By using multicolor flow cytometry with two and three color labelled monoclonal antibodies, absolute number of lymphocyte subsets and the proportion of CD(48), CD(55), CD(59)-deficient T, B and NK cells were analyzed. RESULTS: Lymphocyte subsets were found abnormal in all patients, low absolute number of natural killer cells and B cells were obvious. In most patients, GPI-deficient T, B and NK cells were detectable. The proportion of GPI-deficient clones in T, B and NK cells were 78.7%, 53.4% and 30.5%, respectively. There was significant difference among T, B and NK cells (P < 0.01). In two continuous remission cases, no GPI-deficient clones were found in erythrocytes, but there were GPI-deficient T and B cells, and in a 3 year remission there were still GPI-deficient NK cells. CONCLUSIONS: In PNH, similar to other blood cells, lymphocytes also had GPI deficiency, and GPI-deficient T and B cells could exist for a long time. NK cells of PNH patients are the predominant, GPI-deficiency cells with a high frequency (93.3%) and high proportion (78.7%).

Adult↗

Radioiodine-induced changes in lymphocyte subsets in patients with differentiated thyroid carcinoma.

This study evaluated changes in lymphocyte subsets in patients with thyroid carcinoma who received iodine-131 for diagnostic and therapeutic purposes. Twenty thyroid cancer patients were entered in the study after total thyroidectomy: ten patients (group A) underwent whole-body scintigraphy with 185 MBq of (131)I and the other ten (group B) received 3700 MBq of (131)I therapy. All patients were in a hypothyroid state at the time of administration of (131)I and started L-thyroxine 150 microg/day 3 days after (131)I administration. Free and bound triiodothyronine and thyroxine, thyroid-stimulating hormone, thyroglobulin, thyroglobulin antibodies, thyroid peroxidase/microsomal antibodies, white blood cell, lymphocyte counts and lymphocyte subsets were serially determined at baseline and at days 2, 7, 15, 30 and 60 after (131)I administration. Twenty healthy age- and sex-matched individuals were used as a reference population for lymphocyte subset values. In group A only a reduction in NK cells at days 7 (P=0.043) and 15 (P=0.037) was observed. In group B, patients showed a delayed reduction in the total lymphocyte count at days 15, 30 and 60 (P=0.008, 0.004 and 0. 018, respectively), and a decrease in B cells throughout the study (at days 7, 15, 30 and 60: P=0.006, 0.0017, 0.0017 and 0.0017 respectively). A transient decrease in NK cells was observed at days 15 (P=0.025) and 30 (P=0.008). Among T cells, the helper phenotype (CD4+) was mainly affected, resulting in a reduction in the CD4+/CD8+ ratio at day 60 (P=0.046). Comparing the two groups, the numbers of B lymphocytes at day 30 (P=0.023) and NK cells at days 2 (P=0.037) and 30 (P=0.023) were significantly lower in group B. Neither group showed any clinical sign of immunosuppression during the follow-up period. In patients with thyroid cancer the sensitivity of lymphocytes to the effects of (131)I administered for diagnostic or therapeutic purposes depends upon lymphocyte phenotype and (131)I activity. NK cells are the most radiosensitive cells, being reduced even by low (131)I activity. At higher activity all subtypes show a reduction, which is more marked and prolonged for B lymphocytes and, to a lesser extent, for T-helper lymphocytes. These changes do not result in clinically relevant immunosuppression.

Adenocarcinoma, Follicular↗

Peripheral blood lymphocyte subsets in multiple myeloma and monoclonal gammopathy of undetermined significance.

Peripheral blood lymphocyte subsets were analysed by flow cytometry and compared among 43 patients with untreated multiple myeloma (MM), 16 patients with monoclonal gammopathy of undetermined significance (MGUS) and 26 controls. The age and sex distributions of the patients and controls were comparable, which is important, since in the controls there was a significant effect of age and/or sex on the number of CD3+, CD57+, CD8+57+, CD16+ and CD3-56+ lymphocyte subsets, and on the CD4+/CD8+ and CD4+Leu-8+/CD4+ ratios. In MM, the number of CD8+ and CD57+ cells and the CD4+/CD8+ ratio were related to the clinical stage. The number of CD20+, CD3+, CD4+, CD16+ and CD3-56+ cells and the CD3+/CD20+ ratio were significantly different in MM patients compared to age- and sex-matched controls as was the number of CD3+ and CD4+ cells of MGUS patients compared to controls. Further, there were significant differences in the CD3+/CD20+ ratio between MM and MGUS patients and between stage I MM and MGUS. The role of peripheral blood lymphocyte subsets in differentiating monoclonal gammopathies merits further study.

Age Factors↗

Lymphocyte subset analysis on frozen whole blood.

An approach to perform lymphocyte subset analysis on frozen-thawed whole blood (F/T WB) is described. WB from 24 human immunodeficiency virus type 1 (HIV-1) seropositive individuals and 21 controls was analyzed fresh and after frozen storage (with or without dimethyl sulfoxide) at -80 degrees C, in liquid nitrogen (LN2), and at -20 degrees C. Analysis of F/T WB utilized 3-color flow cytometry with CD45 and right angle light scatter gating. Absolute cell counts were obtained for 30 samples by using staining tubes containing internal bead standards [TruCount, Becton Dickinson Immunocytometry Systems (BDIS), San Jose, CA]. The mean difference between CD3+4+ percentages for F/T (-80 degrees C storage for up to 1 year) and fresh WB was less than -0.2% (95% limits +/-3%, P = 0.5) with 39 of 45 (87%) results falling within 2% of the fresh values (P = 0.74). Absolute CD3+4+ cell counts for F/T WB were generally lower than corresponding results for fresh aliquots (median difference was 33 cells/microl, P < 0.0001), but the results were highly correlated (r2 = 0.975, P < 0.0001). Results were more variable, although still highly correlated, for CD3+8+ cells, and with other freezing and storage conditions. It is concluded that lymphocyte subset analysis using F/T WB yields comparable results to fresh samples, which should prove useful for a number of practical applications.

Blood Preservation↗

Changes in T-lymphocyte subsets during childhood Epstein-Barr virus infectious mononucleosis.

Lymphocyte subsets were measured using monoclonal antibodies in 11 children with Epstein-Barr virus-induced infectious mononucleosis and compared with those of 10 normal children. In acute infectious mononucleosis the percentage of T8+ lymphocytes was greater while the percentage of T4+ lymphocytes and the T4+ to T8+ ratio were less than those measured in normal children. The percentage and absolute number of T lymphocytes, as enumerated by E rosetting, did not differ from the values for normal children. The children with acute infectious mononucleosis had a somewhat lower T8+ response than that observed in four adult infectious mononucleosis patients. With clinical recovery, the T lymphocyte-subset values returned toward normal. T8+ lymphocytes, a phenotype subset with predominantly suppressor activity, presumably reduce normal cellular immune functions transiently and may limit the continued proliferation of Epstein-Barr virus-infected B lymphocytes.

Adult↗

Flow cytometric study of lymphocyte subsets in patients at different stages of colorectal carcinoma.

PURPOSE: The evaluation of lymphocyte subsets by using monoclonal antibodies in neoplastic patients has provided different results, partly in relation to the stage of the disease. Therefore, as a preliminary study of cancer patients treated with immunomodulating drugs, an analysis of lymphocyte subsets was performed in colorectal carcinoma patients. METHODS: In this study, a flow cytometric evaluation of lymphocyte subsets was performed in 33 patients affected by colorectal carcinoma, with or without metastases. RESULTS: A significant reduction of hemoglobin concentrations and hematocrit was observed in all of these subjects, associated with an evident increase of white blood cells, platelets, and HLA DR-positive T lymphocytes, whereas CD 3-CD 4-positive and CD 20-positive lymphocyte concentrations were decreased. Subjects without metastases showed an evident decrease of hemoglobin concentrations and an increase of white blood cells, platelets and CD 3-HLA DR-positive lymphocytes, while patients with disseminated disease also had reduced mean values of hematocrit, red blood cells, CD 3-CD 4-positive, and CD 20-positive lymphocytes. CONCLUSIONS: The main differences between colorectal carcinoma patients with or without metastases were represented by a decrease of red blood cells, CD 3-CD 4-positive, and CD 20-positive lymphocyte concentrations in the latter group.

Blood Cell Count↗

Studies on lymphocyte subsets in thymic tissue and functions of peripheral lymphocytes in patients with tumorous thymus with special reference to myasthenia gravis.

In order to obtain a better understanding of the immunological abnormalities present in myasthenia gravis (MG), which is often accompanied by thymoma or thymic hyperplasia, we investigated lymphocyte subsets and their functions using samples of thymoma or thymic hyperplasia tissues from 11 patients (6 cases with MG), and peripheral blood from 6 patients (4 cases with MG). In most thymic tissues from patients with MG, a maturating tendency of lymphocytes was generally observed. Especially in the medulla of thymic hyperplasia, an entirely peripheral blood type of T-lymphocytes, which were Leu-6- and either Leu-2a+ or 3a + 3b+, were encountered abundantly. Therefore, the presence of abnormal maturation of lymphocytes in the thymus or destruction of the barrier between the thymus and the peripheral blood in MG cases was indicated. In cases without MG, no such tendency was noted. As to the peripheral blood in patients with MG, concanavalin A-induced suppressor cells were significantly decreased (p less than 0.01). All of these changes were considered to be intimately related to the appearance of MG.

Adult↗

Lymphocyte subsets and their proliferation in a model for a delayed-type hypersensitivity reaction in the skin.

A delayed-type hypersensitivity (DTH) reaction was induced in the skin of young pigs, by local injection of phytohaemagglutinin, and evaluation was carried out on the resulting accumulation of lymphocyte subsets and lymphocyte production by incorporation of bromodeoxyuridine in the skin and the draining lymph node. There was a rapid increase in mononuclear cells, which were found in clusters around venules. These included very few B lymphocytes, and CD8+ lymphocytes far outnumbered CD4+ cells. Underlining the importance of determining absolute numbers, the relative and absolute numbers of lymphocyte subsets showed quite different patterns during the development of the skin reaction. Lymphocytes in the normal skin incorporated the DNA precursor bromodeoxyuridine at higher rates than have been found for peripheral lymphoid organs. After intradermal phytohaemagglutinin injections, all subsets showed high proliferation rates in the skin, with kinetics which differed from the reaction in the draining lymph node. The labelling indexes of cells labelled with bromodeoxyuridine in vitro and in vivo were comparable. The phytohaemagglutinin injections also caused a marked and rapid increase in the proliferation of the cells in the basal layer of the epidermis. This model DTH-like reaction in skin with major CD8+ T-cell accumulation and proliferation locally and in the lymph nodes provides a reliable model for study of such reactions and for investigation of the regulatory role of cytokines.

Animals↗