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Metabolic state does not influence lymphocyte subsets in type 1 diabetic patients.

In most studies the distribution of peripheral lymphocyte subsets at diagnosis of type 1 diabetes has been found to be altered. Lymphocyte subpopulations were therefore studied during longitudinal changes in the glycaemic control of 11 type 1 diabetics to investigate whether poor metabolic status affects these results. To avoid any influence of the etiopathogenetic mechanisms, the patients studied had a disease duration of 10 +/- 2 (SEM) years and all but one had no residual beta-cell function. The patients were selected randomly amongst those with a long record of poor glycaemic control and at the first examination they had a mean fasting blood glucose of 15 +/- 1 mmol/l and a mean glucosuria of 67 +/- 11 g/24 h. They were then hospitalized and strictly regulated using pump treatment, resulting in a massive reduction in glucosuria (0 +/- 0 g/24 h) and fasting blood glucose (6 +/- 1 mmol/l) at a second examination a week later. Five of the patients were tested for a third time 35 +/- 4 days later and were still in very good glycaemic control. Peripheral lymphocytes were labelled with monoclonal antibodies and examined by flow cytometry (FACS). Neither CD3+ (pan) T-lymphocytes, CD4+ (helper) T-cells, CD8+ (suppressor/cytotoxic) T-cells, the relation between CD4+ and CD8+ T-cells, nor the total amount of lymphocytes, changed significantly between the first, second, and third examination. None of the results were significantly different from those of healthy controls. There was no correlation between any of the immunological and metabolic parameters. It is concluded that metabolic influence on the distribution of lymphocyte subsets is unlikely.

Adult↗

T-lymphocyte subsets (CD4/CD8 ratio) in breast cancer patients.

T-lymphocyte subsets (CD4/CD8 antigen positive cells) were determined in peripheral lymphocytes from 48 patients with breast cancer of different stages by flow immunocytometry with the aid of anti-CD4 and CD8 monoclonal antibodies. A broad individual variability of the CD4/CD8 ratio among both healthy donors and breast cancer patients was observed. The average value of CD4/CD8 ratio decreased in groups as follows: Healthy donors and Stage I patients, Stage IIA, IIB and Stage IV breast cancer patients. These differences were generally statistically not significant. The difference between healthy donors and Stage IV breast cancer patients was statistically significant (p < 0.01), if one exceedingly elevated value of the CD4/CD8 ratio was excluded from statistical evaluation. The average CD4/CD8 value in the group of breast cancer patients with lymph node or distant metastases was lower than that of patients without metastases, but their difference was not statistically significant either.

Breast Neoplasms↗

Lymphocyte subsets in tumour of patients with undifferentiated nasopharyngeal carcinoma: presence of lymphocytes with the phenotype of activated T cells.

We have analyzed lymphocytes infiltrating nasopharyngeal carcinomas, using a combination of immunoperoxidase staining of frozen and paraffin-embedded sections, and immunofluorescence on lymphocyte suspensions recovered from teased tumours. A panel of monoclonal antibodies was used to define lymphocytic subsets on frozen sections of 14 different tumours. The vast majority of peri- and intra-tumoral lymphocytes were stained by OKT3 antibody. In 8 sections, T4 positive cells were largely predominant, while T8 positive cells were the majority in three sections. Twenty-nine paraffin-embedded sections from other NPC patients stained with HNK-1 antibody showed a variable percentage of positive cells reaching 6 to 15% in nine patients. Most HNK-1 positive cells had the morphology of large granular lymphocytes typical of natural killer cells. Double staining experiments on lymphocytes isolated from 7 tumours revealed a constant presence of T3 positive, HLA-DR positive lymphocytes (from 6 to 29% of mononuclear cells), and of lymphocytes coexpressing the T3 and the Tac (IL-2 receptor) antigens (from 5 to 12% of mononuclear cells). Lymphocytes with a phenotype of activated T-cells are thus constantly found in NPC tumours.

Antigens, Surface↗

The effect of short-course high-dose methylprednisolone on peripheral blood lymphocyte subsets in children with acute leukemia during remission induction treatment.

We have previously demonstrated a favorable effect of high-dose steroid in the treatment of children with acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML). This study was performed to determine the effect of short-course high-dose methylprednisolone (HDMP) treatment on the peripheral blood (PB) T lymphocyte subsets, and blast cells, during remission induction treatment in 23 children with newly diagnosed acute leukemia (16 with ALL, seven with AML). All patients were administered HDMP as a single daily oral dose of 30mg/kg for the first 4 days of induction therapy. The number of PB lymphocyte subsets (CD3, CD4, CD8, CD16+56, CD45RA, and CD45RO) were determined by flow cytometry before and after 4 days of HDMP treatment. While the number of PB blast cells significantly decreased, the absolute number of T lymphocytes expressing CD3, CD4, CD8, CD45RA and the absolute number of CD16+56 (natural killer) cells increased in all patients. We suggest that the beneficial effects of HDMP in the induction treatment of acute leukemia may occur partly due to an increase in the number of PB T lymphocyte subsets. A study randomly assigning patients to treatment with either conventional therapy or HDMP may provide further information.

Acute Disease↗

Reference ranges for lymphocyte subsets in a defined Malaysian population.

The aim of this study was to establish the lymphocyte subset reference ranges in a defined Malaysian population as well as to determine inter-racial differences for these values. Normal blood obtained from 152 subjects (55.9% Malay, 26.3% Chinese and 17.7% Indian) was immunophenotyped. Results obtained (expressed as mean +/- SD %), absolute count (x 10(6) cells/mm3) were as follows: CD3:66.5 +/- 8.6%, 2,066; CD4:33.2 +/- 8.5%, 1,028; CD831.6 +/- 8.9%, 982; CD19:12.0 +/- 0%, 5,374, and CD56+CD16:20.9 +/- 9%, 1,638. There were no significant differences between the percent lymphocyte subsets of the three racial groups. However, the absolute number of CD4 cells and CD19 cells in Chinese was significantly lower (p < 0.05) compared to the Indian and the Indian and Malay groups respectively. Comparison of our results with other reports showed that the percentage of Natural Killer cells in this population is higher than that reported for Caucasian population.

Adolescent↗

Quantification of apoptosis in lymphocyte subsets and effect of apoptosis on apparent expression of membrane antigens.

Annexin V binding to phosphatidylserine was evaluated by flow cytometry to examine apoptosis in different lymphocyte subsets of peripheral blood mononuclear cells after a 24 h in vitro culture period. We also applied a 2 Gy dose gamma-irradiation prior to incubation to evaluate the additional apoptogenic effect of radiation on the lymphocyte subsets. Overall, B lymphocytes showed the highest number of apoptotic cells, followed by T lymphocytes. Within the T lymphocytes, CD4-positive and CD45RA-negative cells were more prone to apoptosis than the CD8-positive and CD45RA-positive cells. Natural killer cells turned out to be most apoptosis-resistant. In the irradiated samples about twice as many apoptotic cells were found and the differences between lymphocyte subpopulations remained. Backgating of the annexin V-positive cells showed that these cells had a clearly decreased forward scatter signal. The antibody binding capacity (ABC) of lymphocyte membrane antigens was determined with CD3-fluorescein isothiocyanate (FITC), CD45RA-FITC, CD4-phycoerythrin (PE), CD8-PE, CD56-PE, and CD20-PE in viable and apoptotic cells. In the apoptotic cells a decrease of ABC was found for all antigens, except for CD20. There was no significant cell loss in the cultures. We conclude that the change in scatter and in ABC must be considered in immunophenotyping experiments on cells kept in culture for 24 h. If these changes are taken into account, percentages of subpopulations or the numbers of cells that stain positive for the studied markers do not significantly change.

Animals↗

[Abnormality of T lymphocyte subsets in aged persons with kidney deficiency syndrome and its influence on immunologic regulation].

T lymphocyte subsets were investigated in 57 aged persons with Kidney Deficiency Syndrome as well as in 30 normal controls with application of the monoclonal antibody (OKT3, OKT4, OKT8). The results showed that the percentage of OKT3, OKT4 in the aged persons was significantly lower than that of control (P < 0.01), while OKT8 was significantly higher (P < 0.01). The OKT4/OKT8 ratio was significantly lower than that of control (P < 0.01). However, the above-mentioned changes were more evident in the Kidney Yang Deficiency Syndrome than those in Kidney Yin Deficiency Syndrome (P < 0.01). The further analysis indicated that the aged persons with Kidney Yang Deficiency appeared as marked positive correlation (r = 0.64, P < 0.001) between the OKT4/OKT8 ratio and IgA, and they also appeared as marked negative correlation (r = -0.89, P < 0.001 and r = -0.665, P < 0.001 respectively) between the OKT4/OKT8 ratio and C3 or CIC. The results suggested that the imbalance of the cell mediated immunity expressed by the T lymphocyte subsets in aged persons with Kidney Deficiency might be one of the essences of senility.

Aged↗

Lymphocyte subsets in human tonsils: the effect of age and infection.

The aim of the present study was to determine the effect of repeated tonsillitis on the development of lymphocyte subsets in the tonsils and among peripheral blood lymphocytes (PBL) of children. Subsets of T- and B cells were analyzed in the tonsils and in PBL of patients undergoing tonsillectomy for idiopathic tonsillar hypertrophy, recurrent tonsillitis, or tonsillar hypertrophy and tonsillitis. The majority of the CD4+ cells in the tonsils displayed the CD45RO+ phenotype, while the majority of those in the PBL displayed the CD45RA+ phenotype. Likewise, the proportion of CD45RO+CD8+ cells was higher in the tonsils than among PBL. The proportion of CD4 cells expressing the CD45RO marker increased with age among PBL, but not in the tonsils. B cells, detected by their CD19, CD20, and CD21 markers, were three times more abundant in the tonsils than in the PBL. The proportion of CD38+ cells showed a negative correlation with age, both in the tonsils and among PBL. Among PBL a striking age-related reduction was seen in the proportion of CD19+, CD21+ and CD38+CD21+ B cells. In contrast, in the tonsils age-related changes could be detected only in the proportion of CD21+CD38+ cells. No difference among patients with various clinical diagnoses was detectable in any of the T- and B cell subsets in the tonsils and PBL. Thus, lymphocyte subsets evolve independently in the tonsils and peripheral blood, with the repeated antigenic challenge of tonsillar lymphocytes not influencing circulating memory cells.

ADP-ribosyl Cyclase↗

Modulatory effects of chitosan adipate on the T and B lymphocyte subsets in mice.

This study examined the subsets of T lymphocytes in the thymus, spleen and mesenteric lymph nodes as well as the subsets of B lymphocytes in the spleen and mesenteric lymph nodes in mice administered chitosan adipate (20 mg/kg) intraperitoneally once or four times at 24 h intervals. The results showed that chitosan adipate decreased the percentage of immature CD4+CD8+ thymic T cells and increased the percentage of mature CD4+ and CD8+ thymocytes. The most significant stimulating effect was observed after four injections. A single exposure to chitosan adipate increased the percentage of CD4+ mesenteric lymph node cells, but four injections of the drug increased the percentage of CD4+ and CD8+ mesenteric lymph node cells. Chitosan adipate had no effect on the subset of splenic T cells. In contrast, chitosan adipate administered either once or four times increased the percentage of CD19+ splenocytes but had no effect on the percentage of CD19+ mesenteric lymph node cells. Overall, chitosan adipate induces the maturation and differentiation of thymocytes, and regulates the number of B splenic cells and lymph node T cells irrespective of the number of doses.

Animals↗

Changes in lymphocyte subsets, mitogen responsiveness, and interleukin-2 production after cardiac operations.

We studied 104 patients after cardiac operations to search for changes in peripheral blood lymphocyte subsets, mitogen responsiveness, and interleukin-2 production. Mononuclear cells obtained from 10 patients with cholecystectomy were examined as control samples. Cultures for phytohemagglutinin and interleukin-2 production were incubated in microtiter plates for 24 hours with cells labeled with tritium thymidine used as target cells. Lymphocyte subsets were measured by using monoclonal antibodies. The results obtained with preoperative blood samples were compared with those from the first, third, and seventh days after operation. In patients having cardiac operations, mitogen responsiveness was low on both the first and the seventh days after operation and interleukin-2 production was markedly depressed (greater than 90% depressed) on the first postoperative day. However, in control samples from patients having cholecystectomy, lymphocyte subsets, mitogen responsiveness, and interleukin-2 production showed no significant changes. Improvement in interleukin-2 production occurred immediately in patients without blood transfusion from random donors and reached normal levels by postoperative day 3. However, interleukin-2 production remained depressed on day 3 in all patients with transfusion from random donors and remained significantly diminished even on day 7 in such patients in New York Heart Association classes III and IV. We conclude that heart operations for which cardiopulmonary bypass is used are associated with depression of cellular immunity, including interleukin-2 production. Furthermore, it appears that both blood transfusion from random donors and the general preoperative condition of the patients contribute to suppression of measures of cell-mediated immunity.

Adult↗

Circulating lymphocyte subsets in patients with alopecia areata.

Lymphocyte subsets in peripheral blood of fourteen patients with patchy alopecia areata or alopecia universalis were estimated using monoclonal antibodies and immunofluorescence. The median percentage of circulating Leu 2a, 3a, 4 and 7 positive cells ("T-suppressor/cytotoxic", "T-helper/effector", total T-cells and killer and natural killer cells) were normal.

Adolescent↗

Lymphocyte subsets in whole blood and isolated mononuclear leucocytes of healthy infants and children.

The determination of lymphocyte subsets utilizing monoclonal antibodies and flow cytometry has become essential in the evaluation of immunological status. Using a standardized method it was found that in healthy children the percentage of CD 8+ (Leu 2+) positive cells increases significantly (P less than 0.01) during infancy, whereas the percentage of CD 4+ (Leu 3+) positive cells decreases with age (P less than 0.01). The percentage of CD 3+ (Leu 4+) cells remains constant. The ratio of CD 4/CD 8 positive cells is significantly (P less than 0.001) higher in infants than in older children. Other subpopulations (HLA DR+, Leu 7) were found to be constant in all age groups. For the comparison of data on lymphocyte subsets obtained by flow cytometry a standardized test procedure is important.

Adolescent↗

Shifts in circulating lymphocyte subsets in cats with feline infectious peritonitis (FIP): pathogenic role and diagnostic relevance.

Cats with feline infectious peritonitis (FIP) are usually lymphopenic and have lymphoid depletion evident in spleen and lymph nodes. In particular, the number of CD4+ lymphocytes in tissues decreases during the evolution of FIP lesions. This decrease is most likely due to increased lymphocyte apoptotic rate. In contrast, cats infected with the Feline Coronavirus (FCoV) develop a follicular hyperplasia in the peripheral lymph nodes. The current study was devised to evaluate the possible pathogenic role of shifts in circulating lymphocyte subsets in FIP. Peripheral blood from cats with FIP was evaluated and compared with peripheral blood from clinically healthy cats living in both FCoV-free and FCoV-endemic catteries. Blood from cats with diseases other than FIP was also examined in order to define the diagnostic relevance of the changes. Lymphocyte subsets were analysed by flow cytometry, using a whole blood indirect immunofluorescence technique and mAbs specific for feline CD5, CD4, CD8, CD21. The results of the current study suggest that cats recently infected with FCoV that do not develop the disease have a transient increase in T cells; cats from groups with high prevalence of FIP have a moderate but persistent decrease in T cell subsets; cats with FIP have a very severe decrease in all the subsets of lymphocytes. Moreover, during FIP many lymphocytes do not express any membrane antigen, most likely due to early apoptosis. Cats with diseases other than FIP also had decreased number of lymphocytes: as a consequence, the diagnostic relevance of these findings is very low. Nevertheless, the lack of flow cytometric changes had a high negative predictive value (NPV), thus allowing to exclude FIP from the list of possible diagnoses in cats with normal cytograms.

Animals↗

Lymphocyte subsets in inline filtered packed red blood cell units: comparison between low and high spin procedures.

Lymphocyte subsets were determined in 20 packed red blood cell units (PRC) before and after filtration (FPRC) with the Pall Leukotrap RC inline filter system; 10 units were prepared by low spin and platelet rich plasma (PRP) removal (Group A) and 10 with high spin, plasma and buffy-coat (BC) removal (Group B). Flow cytometry was employed for white blood cell (WBC) enumeration and phenotype analysis. Median WBCs in prefiltered units was 2.08 x 10(9) (Group A) vs. 0.8 x 10(9) (Group B) (p < 0.0001). Five Group A and three Group B filtered units had WBC counts above the limit of detection (LD), median values being 25.59 and 3.08 x 10(3), respectively. Whereas CD3+, CD3+CD4+ and CD3+CD8+ lymphocyte subsets were assessable in 20-40% of Group A units, inline filtration of Group B units lowered lymphocytes below the LD of the present study. Post-filtration CD19+ lymphocytes were below the LD in all the 20 units.

Antigens, CD↗

Study of the diurnal variation of human lymphocyte subsets.

The diurnal variation of peripheral white cells and in particular of different lymphocyte subsets has been investigated in 11 normal subjects. Monoclonal antibodies (Mab) phenotyping total T lymphocytes (UCHT1), cytotoxic/suppressor (UCHT4), helper T cells (Leu 3a), B lymphocytes (231) and K/NK cells (H25) have been used. In addition to the traditional parametric statistics, the chronobiological approach of cosinor was adopted in order to inferentially obtain the waveform covering the 24-hr period. Cosinor analysis validated the existence of a significant rhythm for leucocytes (p = 0.04, lymphocytes (p = 0.03), and Leu 3a positive cells (p = 0.04). The best fitting period was equal to 28 hr. No circadian rhythm was documented for lymphocyte subsets positive for Mab UCHT4, 231, H25, by testing the variability with a frequency ranging from 1 cycle every 12hr to 1 cycle every 28 hr. By the non-inferential analysis of temporal curves, a significant peak of Leu 3a positive cells at 20.00 hr (p less than 0.001) was observed. The acrophase has been recorded by cosinor analysis at 20.36 hr. We conclude that the circadian variability of Leu 3a positive cell profile must be taken into account when the helper/suppressor ratio is calculated. The time-dependence of this lymphocyte subpopulation suggests that when therapy with corticosteroids is considered in autoimmunity, it should be planned by keeping the peak of Leu 3a positive cells as a marker time.

Adult↗

Immunohistochemical investigation of hepatitis B virus associated antigens, HLA antigens and lymphocyte subsets in type B chronic hepatitis.

HLA antigens, hepatitis B virus (HBV)-associated antigens and lymphocyte subsets in liver tissue from 35 patients with HBs antigenemia were studied using an immunoperoxidase double staining method and immunoelectron microscopy in order to clarify the immune mechanism of hepatocyte lysis in type B hepatitis. Immune light and electron microscopy using monoclonal antibodies to lymphocyte subsets revealed that infiltrating lymphocytes in the areas of piecemeal necrosis and focal necrosis were predominantly CD8-positive, showing direct contact with hepatocytes. In contrast, CD4(+) cells were infrequently observed in necrotizing inflammatory lesions. HLA-A,B,C antigens were mainly found on hepatocytes in areas of piecemeal necrosis and focal necrosis, in association with CD8(+) lymphocyte infiltration. HLA-DR antigens were demonstrated on a few hepatocytes in the same lesions. In cases of CAH with serum HBeAg positive, HLA-A,B,C, antigens and HBV antigens simultaneously demonstrated on the same hepatocytes. Especially, hepatocytes expressing both HLA-A,B,C antigen and HBsAg on the plasma membrane showed direct contact with CD8(+)lymphocytes. This finding fulfilled the morphological requirements for HBsAg as a target antigen. On the other hand, HBcAg was hardly demonstrated in the liver cell membrane but was demonstrated mainly in the cytoplasm. Compared with the nuclear localization of HBcAg in cases of NSR, cytoplasmic localization of this antigen may be associated with membranous expression of new antigens induced by HBV infection.

Chronic Disease↗

Lung and blood lymphocyte subsets in asbestosis and in mixed dust pneumoconiosis.

To study the local distribution of lymphocyte subsets in inorganic dust diseases, bronchoalveolar lavage (BAL) was performed in seven patients with asbestosis, and in 13 patients with mixed dust pneumoconiosis (anthracosidero-silicosis). Lymphocyte subsets in BAL and blood were determined by the monoclonal antibodies OKT3 (pan T), OKT4 (helper/inducer), OKT8 (suppressor/cytotoxic), B1 (B-cells), OKIa (HLA-DR antigens), and Leu-7 (natural killer). The BAL lymphocytes were moderately elevated to 15 +/- 8 percent (mean +/- SD) in mixed dust pneumoconiosis, and even more markedly increased to 28 +/- 21 percent in asbestosis. The OKT4/OKT8 ratio in BAL was significantly increased to 4.5 +/- 2.1 in asbestosis, and significantly reduced to 0.9 +/- 0.8 in mixed dust pneumoconiosis. In blood, the ratio of T-cell subsets remained unchanged, though total lymphocytes were decreased in asbestosis. These results suggest altered cellular immune processes in the lungs of patients with pneumoconiosis and may indicate different immunoregulatory changes depending on the nature of the inhaled dust.

Adolescent↗

Contribution of exertional hyperthermia to sympathoadrenal-mediated lymphocyte subset redistribution.

The contribution of hyperthermia to the differential leukocytosis of exercise remains obscure. This study examined changes in circulating sympathoadrenal hormone concentrations and patterns of leukocyte and lymphocyte subset (CD3(+), CD4(+), CD8(+), CD19(+), CD3(-)16(+)/56(+)) redistribution during exercise, with and without a significant rise of rectal temperature (T(re)). Ten healthy men [age 26.9 +/- 5.7 (SD) yr, body mass 76.0 +/- 10.9 kg, body fat 13.9 +/- 4.6%, peak O(2) consumption: 48.0 +/- 12.4 ml x kg(-1) x min(-1)] exercised for 40 min (65% peak O(2) consumption) during water immersion at 39 or 18 degrees C. T(re) increased from 37.2 to 39.3 degrees C (P < 0.0001) after 40 min of exercise in 39 degrees C water but was held constant to an increment of 0.5 degrees C during exercise in 18 degrees C water. Application of this thermal clamp reduced exercise-associated increments of plasma epinephrine (Epi) and norepinephrine (NE) by >50% (P < 0.05) and abolished the postexercise increase in cortisol. Thermal clamping also reduced the exercise-induced leukocytosis and lymphocytosis. Multiple regression demonstrated that T(re) had no direct association with lymphocyte subset mobilization but was significantly (P < 0.0001) correlated with hormone levels. Epi was an important determinant of total leukocytes, lymphocytes, and CD3(+), CD4(+), CD8(+), and CD3(-)CD16(+)/56(+) subset redistribution. The relationship between NE and lymphocyte subsets was weaker than that with Epi, with the exception of CD3(-)CD16(+)/56(+) counts, which were positively (P < 0.0001) related to NE. Cortisol was negatively associated with leukocytes, CD14(+) monocytes, and CD19(+) B- and CD4(+) T-cell subsets but was positively related to granulocytes. We conclude that hyperthermia mediates exercise-induced immune cell redistribution to the extent that it causes sympathoadrenal activation, with alterations in circulating Epi, NE, and cortisol.

Adrenal Glands↗