Percentage Of Lymphocyte Subpopulations Research Articles

Lymphocyte immunophenotyping and concentration of MMP-9 in transudates and exudates in horses

This study is the first to provide information on the lymphocyte subpopulations in peritoneal effusions in horses. Peritoneal transudates (n = 12), peritoneal exudates (n = 6) and a pleural exudate (n = 1) were analyzed. The total nucleated cell count (TNCC), total protein (TP) and matrix metalloproteinase-9 (MMP-9) concentration determined by ELISA were measured and routine cytological evaluation was performed. CD3, CD4, CD8 and CD21 positive cells were detected by flow cytometry. A higher percentage of neutrophils (P < 0.05) and higher MMP-9 (P < 0.01) levels were found in exudates. A higher percentage of macrophages (P < 0.05) and lymphocytes (P < 0.01) were found in transudates. CD4 + lymphocytes were the most common lymphocyte subpopulation in all samples. CD21 + lymphocytes were the least common in all samples. A large variability in the percentage of CD21 + lymphocytes was found in exudates. The percentage of CD21 + lymphocytes positively correlated with the level of total protein (r = 0.5704, P < 0.05). The correlation was even stronger in the group of exudates. The percentages of lymphocyte subpopulations did not correlate with the level of MMP-9 or with cytological findings. The level of MMP-9 positively correlated with the percentage of neutrophils (r = 0.4980, P < 0.05), the level of TP (r = 0.7855, P < 0.01) and TNCC (r = 0.6129, P < 0.01). A significantly higher level of MMP-9 was detected in euthanized horses than in horses that survived (P < 0.05). However, it was shown that the level of MMP-9 in the peritoneal fluid can change significantly in a short time. More studies on repeated abdominocentesis could contribute to elucidating the role of MMP-9 as a prognostic indicator.

Distinct Lymphocyte Immunophenotyping and Quantitative Anti-Interferon Gamma Autoantibodies in Taiwanese HIV-Negative Patients with Non-Tuberculous Mycobacterial Infections.

The presence of anti-interferon-γ autoantibodies (AutoAbs-IFN-γ) is not rare in patients suffering from persistent non-tuberculous mycobacterial (NTM) infections that are characteristic of adult-onset immunodeficiency syndrome. The immune disturbances in this distinct disorder remain to be elucidated. Patients with NTM infections but without effective response over 3months' treatment were referred to our institute to quantify their level of AutoAbs-IFN-γ after excluding defective IL12/23-IFN-γ circuit and reactive oxygen species production. The AutoAbs-IFN-γ and percentage of lymphocyte subpopulations most relevant to T and B cell pools were assessed and compared with age-matched healthy controls. A total of 31 patients were enrolled during the 15-year study period (2008-2022), 20 patients with > 50% suppression of IFN-γ detection at 1:100 serum dilution were classified into the Auto-NTM group. The remaining 11 with negligible suppression were assigned to the No Auto-NTM group. Mycobacterium chimaera-intracellulare group (MAC), M. kansasii, and M. abscessus were the most common pathogens. Pneumonia (19 vs 7), lymphadenitis (11 vs 5), Salmonellasepsis (6 vs 2), osteomyelitis (5 vs 1), and cutaneous herpes zoster (4 vs 4) were the main manifestations in both the Auto-NTM and No Auto-NTM groups who had similar onset-age (55.3 vs 53.6years; p = 0.73) and follow-up duration (71.9 vs 54.6months;p = 0.45). The Auto-NTM group had significantly higher transitional (IgM + + CD38 + +), CD19 + CD21-low, and plasmablast (IgM-CD38 + +) in the B cell pool, with higher effector memory (CD4 + /CD8 + CD45RO + CCR7 -), senescent CD8 + CD57 + , and Th17 cells, but lower naïve (CD4 + /CD8 + CD45RO - CCR7 +) and Treg cells in the T cell pool when compared to the No Auto-NTM and healthy groups. NTM patients with/without AutoAbs-IFN-γ had lower Th1-like Tfh (CD4 + CXCR5 + CXCR3 + CCR6 -) cells. All Auto-NTM patients still had non-remitted mycobacterial infections and higher AutoAbs-IFN-γ despite anti-CD20 therapy in 3 patients. In patients with suspected adult-onset immunodeficiency syndrome, two thirds (20/31) were recognized as having significantly inhibitory AutoAbs-IFN-γ with higher antibody-enhancing transitional, CD19 + CD21-low and plasmablast B cells; as well as higher effector memory, senescent CD8 + CD57 + and Th17 cells, but lower naïve T and Treg cells in contrast to those with negligible AutoAbs-IFN-γ. Such immunophenotyping disturbances might correlate with the presence of AutoAbs-IFN-γ. However, the mutual mechanisms need to be further clarified.

Lymphocyte Subpopulations Associated with Neutralizing Antibody Levels of SARS-CoV-2 for COVID-19 Vaccination.

The comprehensive knowledge regarding the immune response during coronavirus disease 2019 (COVID-19) vaccination is limited. The aim of this study was to longitudinally investigate not only the dynamic changes of peripheral lymphocyte subpopulations and cytokine levels but parallel changes of antibody levels against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Blood samples of 20 healthcare workers with two doses of COVID-19 vaccine were prospectively collected. The percentages of lymphocyte subpopulations from peripheral blood and cytokine production in lymphocytes with in vitro stimulation were assessed using eight-color flow cytometry. SARS-CoV-2 spike antibodies (anti-S Abs) and functional neutralizing antibodies (nAbs) were also measured. The relation between pre- and post-vaccination immunity was analyzed. There are 7 men and 13 women with a median age of 44.0 years (range: 25.7–59.5 years). The individuals had an increased percentage of lymphocytes at post-vaccination with statistical significance post first dose (p = 0.031). The levels of transitional cells (p = 0.001), such as plasmablasts (p < 0.001) and plasma cells (p = 0.031), were increased compared with pre-vaccination. Recent thymic emigrants of CD4+ T cells subsets were significantly higher at post-vaccination than those at pre-vaccination (p = 0.029). Intracellular levels of tumor necrosis factor-alpha, interferon-γ, interleukin (IL)-2, IL-21, transforming growth factor-beta and IL-17 produced by CD4+ T, CD8+ T, and natural killer cells were increased. All individual samples showed reactivity to anti-S Abs and the levels of nAbs were elevated after vaccination. The magnitude of adaptive immunity was associated with vaccine types and doses. Alterations of total memory B cells (p < 0.001), non-switched memory B cells (p = 0.016), and memory Treg cells (p < 0.001) were independent predictors for nAb levels. These findings might be helpful in elucidating the immune response of COVID-19 vaccination and in developing new strategies for immunization.

Assessment of anti-desmoglein antibodies levels and other laboratory indexes as objective comprehensive indicators of patients with pemphigus vulgaris of different severity: a single-center retrospective study.

The aim of this study is to assess the utility of anti-desmoglein (Dsg) antibodies levels, hematological biomarkers levels, the albumin to globulin (A/G) ratio, blood lipids levels, and lymphocyte subpopulation percentages as objective laboratory indicators of disease severity of pemphigus vulgaris (PV). A retrospective study of 187 PV patients with 256 medical records between January 2013 and December 2020. PV patients were divided into three groups by disease severity according to the pemphigus disease area index (PDAI) score: mild (0-8), moderate (9-24), and severe (≥ 25). The levels of anti-Dsg antibodies, hematological biomarkers, A/G ratio, blood lipids, and the percentage of lymphocyte subpopulations were measured. We assessed the correlations of quantitative variables by Pearson correlation (r). Multivariable linear regression was used to identify the variables associated with the disease severity of PV (PDAI score). The results show that the levels of Dsg1 (r = 0.294, P < 0.001) and Dsg3 (r = 0.206, P = 0.011), monocyte count (r = 0.210, P = 0.001), neutrophil-to-lymphocyte ratio (NLR) (r = 0.123, P = 0.049), and platelet-to-lymphocyte ratio (PLR) (r = 0.170, P = 0.006) were positively correlated with the PDAI score. However, the A/G ratio (r = -0.399, P < 0.001), and the levels of total serum cholesterol (r = -0.140, P = 0.026) and HDL (r = -0.143, P = 0.023) were negatively correlated with the PDAI score. Multiple linear regression showed that the factors associated with the PDAI score were higher level of anti-Dsg1 antibody (P = 0.001), a higher NLR (P = 0.005), and a lower A/G ratio (P < 0.001). The linear regression equation was Y(PDAI) = 32.798 + 0.058X(Dsg1) + 0.846 X(NLR)-16.472 X(A/G) (R2 = 0.586). Therefore, high levels of anti-Dsg1 antibody and NLR combined with a low A/G ratio could explain the PDAI score. These findings might provide a more comprehensive and objective evaluation system for reflecting the disease severity of PV based on laboratory indicators.

Clinicopathological Manifestations and Immune Phenotypes in Adult-Onset Immunodeficiency with Anti-interferon-γ Autoantibodies.

Anti-interferon (IFN)-γ autoantibodies (anti-IFN-γ Abs) is an emerging adult-onset immunodeficiency syndrome. Immune dysfunction in this distinct disorder remains to be clarified. We prospectively collected blood samples of 20 patients with anti-IFN-γ Abs and 40 healthy normal subjects. The percentages of lymphocyte subpopulations, most relevant to T, B, and NK cells, and the percentages of stimulated lymphocytes with cytokine production were assessed using eight-color flow cytometry. The results were adjusted to age and absolute lymphocyte counts. Most (85%) patients presented nontuberculous mycobacterial infection. Skin lesions were predominantly manifested by neutrophilic dermatoses. The involved lymph nodes had granulomatous inflammation, except 22.2% showing atypical lymphoid hyperplasia without granuloma formation. The percentages of CD4 + T cells and nonactivated subpopulations (recent thymic emigrants and naïve subtypes) decreased significantly with increased expression of activation markers and polarization to differentiated cells. The percentage of NK cells increased, but that of two major NK subpopulations, CD161 + CD56bright and CD161 + CD56 + CD16 + subsets, decreased. Increased CD161dim, CD161 + CD56 - CD16 + , and CD57 + NK cell subsets coupled with the decreased expression of NKp30 and NKp46 indicate reconfiguration of the NK cell population and acquisition of adaptive features. Intracellular cytokine production of the lymphocyte subpopulations was significantly low in the patients compared with the control group. We conclude that the immune system in patients with anti-IFN-γ Abs could be exhausted in T cells and be adaptive in NK cells, contributing to the distinct clinicopathologic features.

Immunosenescence profile and expression of the aging biomarker (p16INK4a) in testicular cancer survivors treated with chemotherapy

BackgroundCytotoxic chemotherapy can cure advanced germ cell tumors. Nevertheless, cancer treatment may induce cellular senescence and accelerate molecular aging. The aging process implies an increase of cells expressing p16INK4a and changes in lymphocyte subpopulations. Our aim was to study the potential induction of premature immunosenescence in testicular cancer survivors (TCS) exposed to chemotherapy.MethodsCase-control exploratory study of TCS treated with chemotherapy (≥3 BEP cycles, disease-free ≥3 months) compared with age matched healthy controls. Peripheral blood mononuclear cells were isolated, and lymphocyte subpopulations were analyzed by flow cytometry. CDKN2A/p16INK4a expression in T cells was measured using qPCR. The percentage of lymphocyte subpopulations and the CDKN2A/p16INK4a expression in TCS were compared with the control group using the Wilcoxon signed-rank test.ResultsWe included 16 cases and 16 controls. The median age was 27 years (minimum 24, maximum 54) and the median time on surveillance was 26.5 months (minimum 3, maximum192). TCS had a lower percentage of total T cells and CD4+ T cells in total lymphocytes. Among the CD4+ T lymphocytes, TCS had less naïve CD4+ and increased memory CD4+ cells. Within the CD8+ T lymphocytes, TCS exhibited a decrease in the percentage of naïve cells and an increase in CD8 + CD45RA + CD57+ cells. TCS also exhibited decreased memory CD19+ B cells compared to the controls. The relative expression of CDKN2A/p16INK4a in T cells was increased in TCS (mean 1.54; 95% CI of the mean: 1.074–2.005; p = 0.048).ConclusionIn this exploratory study, TCS showed increased expression of CDKN2A/p16INK4a and a lymphocyte phenotype that has been associated with immunosenescence. Further studies are warranted to define the clinical implications of these alterations in TCS.

Lymphocyte Subsets in a Population of Nonfrail Elderly Individuals

Age-related frailty is characterized by increased vulnerability to stress due to decline in homeostatic reserve, which results in increased risk of adverse health outcomes including disability, hospitalization, and death. The relationship between frailty and immunological system alterations is well established. Thus, analysis of immunological changes, such as alterations in lymphocyte subsets, during senescence may provide useful markers for frailty and associated pathologies. Since reference ranges currently used for lymphocyte subsets do not specifically differentiate the elderly group, the aim of this study was to (1) establish reference ranges in nonfrail elderly individuals and (2) assess the evolution of these parameters with age. Further, the influence of other physiological and lifestyle factors was also evaluated. The study was performed on 144 elderly individuals (aged 65–95) from Galicia (in northwestern Spain). Percentages of lymphocyte subpopulations (CD3+ T lymphocytes, CD4+ T-helper lymphocytes, CD8+ T-cytotoxic lymphocytes, CD19+ B lymphocytes, and CD56+16+ natural killer cells) were analyzed in peripheral blood by flow cytometry, and reference ranges were calculated. The individual status as nonfrail or prefrail did not markedly affect the immunological parameters, but an apparent influence of age was obtained for %CD3+, %CD4+, and %CD19+ cells, all of which fell with increasing age. Women showed higher levels of %CD19+ lymphocytes. No significant influence of smoking habits, physical activity, or drinking alcohol or caffeine beverages was observed. The results obtained may serve as a basis to establish comparisons between frail and nonfrail elderly individuals, in order to determine the usefulness of lymphocyte subsets as immunological biomarkers of frailty.

Value of lymphocyte subgroups jointly detected in assessing poor prognosis of patients with sepsis

Objective To investigate the predictive value of T, B, and NK lymphocyte subsets jointly detected in poor prognosis in patients with sepsis. Methods Totally 101 patients with sepsis were enrolled for prospective study. The percentages of lymphocyte sub-populations in peripheral blood were detected within 48 h after admission. The outcomes of patients during hospitalization were observed by follow-up study. The predictive value of lymphocyte sub-populations jointly detected in death risk was assessed. Results The percentages of CD3, CD3 +/CD4 + in patients with septic shock were lower than those in patients with mild/moderate sepsis and severe sepsis. The percentages of CD3 +/CD8 + , CD3 -/CD19 + , CD4 +/CD8 + in patients with severe sepsis and septic shock were lower than those in patients with mild/moderate sepsis. The percentage of CD (16 +56) + in severe sepsis and septic shock was higher than that in mild/moderate sepsis group (P <0.05) . The total number of monocytes in the deaths was higher than that in survivals. The percentages of CD3, CD3 -/CD19 + , CD3 +/CD4 + , CD4 +/CD8 + were lower in deaths than those in survivals (P <0.05) . The CD3 -/CD19 + , CD3 +/CD8 and CD4 +/CD8 + were protective factors against sepsis with their OR values of 0.235, 0.006 and 0.108, respectively. The ROC curve of lymphocyte subsets jointly detected, APACHEⅡ score and SOFA score to predict the risk of death were 0.993, 0.877 and 0.848, respectively. The misjudgment rate of discriminating disease severity function of lymphocyte subgroups jointly detected was 19.8% , and that of outcomes was 4%. Conclusion The percentages of lymphocyte sub-populations in patients with sepsis is abnormal, and therefore, the lymphocyte subgroups jointly detected has value in the judgment of disease severity and prognosis in patients with sepsis. Key words: Sepsis; Lymphocyte subsets; Prognosis; Perspective study

Effect of Diet and Exercise on the Peripheral Immune System in Young Balb/c Mice.

Although diet and exercise clearly have an influence on immune function, studies are scarce on the effect caused by exercise and the consumption of a carbohydrate-rich or fat-rich diet on the peripheral immune system. The aim of the present study was to evaluate the effect of exercise and the two aforementioned unbalanced diets on young Balb/c mice, especially in relation to BMI, the level of glucose, and the percentage of lymphocyte subpopulations in peripheral blood. The changes found were then related to the synthesis of leptin and adiponectin as well as the production of oxidative stress. The increase in BMI found with the carbohydrate-rich and fat-rich diets showed correlation with the levels of leptin and adiponectin. An increase in leptin and a decrease in adiponectin directly correlated with an increase in total lymphocytes and CD4+ cells and with a decrease in B cells. The increase in leptin also correlated with an increase in CD8+ cells. Glycemia and oxidative stress increased with the two unbalanced diets, negatively affecting the proliferation of total lymphocytes and the percentage of B cells, apparently by causing alterations in proteins through carbonylation. These alterations caused by an unbalanced diet were not modified by moderate exercise.

The effect of in vivo exposure to zearalenone on cytokine secretion by Th1 and Th2 lymphocytes in porcine Peyer's patches after in vitro stimulation with LPS.

Most research studies investigating the estrogenic effects of zearalenone (ZEN) focus on the mycotoxin's effect on the reproductive system. Since estrogen receptors are present on various types of immunocompetent cells, ZEN can also modify diverse immune functions. This study analyzed immunocompetent cells isolated from Peyer's patches in the ileum of pigs administered ZEN in the estimated daily dose of 8 μg kg(-1) BW (equivalent of 100 μg kg(-1) feed per day(-1)). The objective of the study was to determine whether long-term exposure to low ZEN doses below the NOEL threshold leads to changes in the percentages of lymphocyte subpopulations and cytokine secretion by Th1 (IL-2, IFN-γ) and Th2 (IL-4 and IL-10) lymphocytes in Peyer's patches of the ileum after in vitro stimulation with lipopolysaccharides (LPS). Immunocompetent cells isolated from Payer's patches on experimental days 0, 14, 28 and 42 were cultured in vitro and stimulated with LPS. The presence of IL-2, IFN-γ, IL-4 and IL-10 in culture media was determined by the ELISA method. The results of the study indicate that ZEN inhibits IL-2 and IFN-γ secretion and stimulates IL-4 and IL-10 produc- tion by Th1 and Th2 lymphocytes by shifting the Th1/Th2 balance towards the humoral immune response. The above can promote allergic responses, as demonstrated by the increase in the size of B1 cell populations producing more autoantibodies. ZEN can also lower resistance to viruses and tumors by inhibiting the proliferation of NK cells and IFN-γ secretion.

Evaluation of lymphocyte subpopulations in cord blood of Bulgarian newborns.

Umbilical cord blood (UCB) is a clinically useful source of hematopoietic stem and progenitor cells for treatment of a wide variety of malignant and non-malignant disorders. An important way to completing infor- mation on the quality and composition of units for transplantation is more extensive immunophenotyping of UCB. Moreover, phenotyping of lymphocyte subpopulations is essential for the diagnosis and follow-up of children with immunodeficiencies and other immune disorders and therefore, establishment of age-matched reference values of lymphocyte subsets is a necessity for each population. The aim of this study was to determine the normal range of T and B lymphocytes, and NK cells as well as the CD4 and CD8 subpopulations of T cells in cord blood collected from healthy term infants. The relative and absolute number distributions (median, 5th and 95th percentile) of lymphocyte subsets in cord blood samples from 72 healthy newborns were examined by multi-colour flow cytometry with a view to obtaining reference values for Bulgarian neonates at birth. Mean percentages of lymphocyte subpopulations were: CD3 (62.27 ± 9.64), CD19 (17.47 ± 5.46), CD3- CD16/CD56+ (17.27 ± 8.4). Our results show the prevalence of helper-inducer CD3+CD4+ (44.88-8.21) compared to the suppressor-cytotoxic CD3+CD8+ (16.65 ± 4.54) T-cell subpopulation, which determines the positive CD4/CD8 ratio (2.86 ± 0.82; 1.4-4.8). Also, the absolute numbers of studied populations varied widely due to differences of the absolute number of lymphocytes in the samples. This study on distribution of lymphocyte subpopulations in UCB helps to enhance our knowledge about cell phenotypes in cord blood and improve characterization of products for cellular therapy, as well as contributes to the correct interpretation of laboratory results for infants with possible immune disorders. Our data can be used as normal intervals for lymphocyte subsets in Bulgarian neonates.

Lymphocyte subpopulations in Chinese women with Turner syndrome

Turner syndrome (TS) is associated with deficiency of cellular and humoral immunity. However, the characteristics of lymphocyte subpopulations in Chinese women with TS have not been reported. In this study, the percentage of lymphocyte subpopulations and the mRNA expression of some transcription factors were determined in patients with TS. The effect of the hormone substitution on lymphocyte subpopulations was also analyzed. Thirteen Chinese TS women and eight age and sex-matched healthy volunteers were studied. The percentage and mean fluorescence intensity (MFI) of lymphocyte subpopulations including CD3+CD4+, CD3+CD8+, CD19-CD138+, CD4+CD25+FoxP3+ and CD4+CD8-IL17A+ cells were determined by flow cytometry. The mRNA expression of some transcription factors were detected by RT-PCR. Compared to control, the percentage of CD3+CD4+ cells was significantly reduced (p < 0.05), while the percentage of CD19-CD138+, CD4+CD25+FoxP3+ and CD4+CD8-IL17A+ cells was significantly increased in TS patients. No difference was observed in the percentage of CD3+CD8+, CD19+ B cells between TS patients and healthy volunteers, with the similar changes in the mean fluorescence intensity of these cells. The mRNA expression of some transcription factors slightly enhanced in TS patients. Estrogen therapy did not affect the percentage of lymphocyte subpopulations. These findings suggested that Turner syndrome might be associated with changes of lymphocyte subpopulations.

Altered time structure of neuro-endocrine-immune system function in lung cancer patients

BackgroundThe onset and the development of neoplastic disease may be influenced by many physiological, biological and immunological factors. The nervous, endocrine and immune system might act as an integrated unit to mantain body defense against this pathological process and reciprocal influences have been evidenced among hypothalamus, pituitary, thyroid, adrenal, pineal gland and immune system. In this study we evaluated differences among healthy subjects and subjects suffering from lung cancer in the 24-hour secretory profile of melatonin, cortisol, TRH, TSH, FT4, GH, IGF-1 and IL-2 and circadian variations of lymphocyte subpopulations. MethodsIn ten healthy male volunteers (age range 45-66) and ten male patients with untreated non small cell lung cancer (age range 46-65) we measured melatonin, cortisol, TRH, TSH, FT4, GH, IGF-1 and IL-2 serum levels and percentages of lymphocyte subpopulations on blood samples collected every four hours for 24 hours. One-way ANOVA between the timepoints for each variable and each group was performed to look for a time-effect, the presence of circadian rhythmicity was evaluated, MESOR, amplitude and acrophase values, mean diurnal levels and mean nocturnal levels were compared.ResultsA clear circadian rhythm was validated in the control group for hormone serum level and for lymphocyte subsets variation. Melatonin, TRH, TSH, GH, CD3, CD4, HLA-DR, CD20 and CD25 expressing cells presented circadian rhythmicity with acrophase during the night. Cortisol, CD8, CD8bright, CD8dim, CD16, TcRδ1 and δTcS1 presented circadian rhythmicity with acrophase in the morning/at noon. FT4, IGF-1 and IL-2 variation did not show circadian rhythmicity. In lung cancer patients cortisol, TRH, TSH and GH serum level and all the lymphocyte subsubsets variation (except for CD4) showed loss of circadian rhythmicity. MESOR of cortisol, TRH, GH, IL-2 and CD16 was increased, whereas MESOR of TSH, IGF-1, CD8, CD8bright, TcRδ1 and δTcS1 was decreased in cancer patients. The melatonin/cortisol mean nocturnal level ratio was decreased in cancer patients.ConclusionThe altered secretion and loss of circadian rhythmicity of many studied factors observed in the subjects suffering from neoplastic disease may be expression of gradual alteration of the integrated function of the neuro-immune-endocrine system

Adenoid as a source of lymphocytes in the surface secretions of nasopharynx

The significance and protective efficacy of surface secretions on mucosal membranes in the upper airways are well recognized. The aim of our study was to reveal the role of the adenoids as a source of cellular components in the mucosal secretion. The adenoid removed because of its hypertrophy and the samples of surface secretions taken by "imprint method" described by Ebenfelt et al. from the group of 38 children were examined by flow cytometry. The lymphocyte subsets with following antigens: CD3, CD4, CD8, CD19, CD23, CD16+56, CD45RA, CD45RO, HLA-DR were analyzed. Two groups were studied: (1) AH, children with adenoid hyperplasia without otitis media; (2) OME, children with adenoid hyperplasia and persistent otitis media with effusion. Age-related changes in percentages of lymphocyte subpopulations were analyzed. The percentage of lymphocyte subsets in adenoid and secretion were similar, however, in the surface secretion there was higher proportion of lymphocytes B and NK and lower of T. There was no correlation between percentages of lymphocyte in adenoid and surface secretion except for lymphocytes Th with memory phenotype and lymphocytes B with marker CD23(+). In adenoid the percentage of T and Th lymphocytes increased with age and the percentage of B lymphocytes decreased. In the secretion age-related changes were less pronounced and only the percentage of lymphocytes B CD23(+) showed positive correlation with age. In cases of otitis media with effusion there was higher proportion of lymphocytes B (CD19(+) and CD19(+)CD23(+)) and lower of lymphocytes Ts and Th with naive phenotype in the secretion. In adenoid however, the cases of OME revealed lower percentage of lymphocytes B and higher of lymphocytes Ts. Similar proportions of lymphocyte subpopulations in surface secretion and in adenoid imply that adenoid is the source of those cells. However, lack of correlation between the percentages of lymphocytes in secretion and in adenoid suggest that the process of lymphocyte migration has active and selective character.

The use of flow cytometry to discriminate avian lymphocytes from contaminating thrombocytes

Evaluation of peripheral blood mononuclear cells (PBMC) in avian species by flow cytometry is complicated by the presence of large numbers of nucleated thrombocytes. With light scattering properties similar to those of lymphocytes, variations in the proportion of thrombocytes in PBMC can result in apparent shifts in percentages of lymphocyte subpopulations. We have applied a dual-labeling procedure for flow cytometric analyses to exclude thrombocytes from the analyzed population by labeling with K55 monoclonal antibody (MAb), which differentially labeled leukocyte and thrombocyte populations. Flow cytometric analyses with K55 labeled chicken PBMC differentiated leukocytes into three populations according to their intensity of fluorescence. Using the Kl MAb, the K55-low population was shown to consist of thrombocytes. Dual-labeling with K55 MAb and MAb specific for B lymphocyte, CD4 or CD8 markers indicated that the K55 intermediate population consisted of lymphocytes. Therefore, concentrations of CD4 + and CD8 + T lymphocytes could be determined from the lymphocyte fraction by gating specifically on the K55 intermediate cells. Selecting cross-reactive chicken MAbs that included K55, this protocol was shown to identify CD4 + and CD8 + T lymphocytes in PBMC of another avian species, the endangered Attwater's prairie chicken.

Circadian rhythms in surface molecules of rat blood lymphocytes.

The present article examines whether the expression of certain surface molecules that trigger immune responses shows a circadian rhythm. We also analyzed the rhythms in the number and percentage of lymphocyte subpopulations, in the leukocyte differential counts, and in the total red and white blood cell counts. Blood samples obtained from rats at 2-h intervals for 24 h were stained with several mouse monoclonal antibodies directed against lymphocyte surface molecules and processed by flow cytometry. The number of B, total T, Tgammadelta, Th, and Ts/c cells followed a 24-h rhythm with a peak in the first half of the resting period. The expression of CD45, CD5, CD3, and CD4 followed a circadian rhythm. Their acrophases suggested temporal association between CD45 and CD5 at the end of the active phase and between CD4 and CD3 at the beginning of this phase. This temporal organization could have an important role for immune cell function.

Quantitative Analysis of the Neuroendocrine–Immune Axis: Linear Modeling of the Effects of Exogenous Corticosterone and Restraint Stress on Lymphocyte Subpopulations in the Spleen and Thymus in Female B6C3F1 Mice

The effects of exogenous corticosterone and restraint stress on the number and percentage of lymphocyte subpopulations in the spleen and thymus were evaluated. The data were used to generate linear models that describe the relationship between these parameters and the area under the corticosterone concentration vs time curve (AUC). Comparison of the models revealed that the number of nucleated cells in the spleen was decreased similarly by exogenous corticosterone and restraint (at equivalent corticosterone AUC values). However, exogenous corticosterone caused a greater decrease in cell number in the thymus than it did in the spleen. Corticosterone preferentially depleted CD4+CD8+ cells in the thymus, whereas the same corticosterone exposure produced by restraint stress did not. In the spleen, cell number for all major cell types was decreased by both treatments, but there were minor differences in the change in percentage of some subpopulations induced by exogenous corticosterone as compared to restraint. The models derived here provide quantitative data that indicate the magnitude of corticosterone and stress-induced effects on lymphocyte populations in the spleen and thymus. These results have mechanistic implications, and they may be useful in future efforts to extrapolate from mouse to human by completing a risk assessment parallelogram.

Humoral and Cellular Immune Response to Elastin in Patients with Systemic Sclerosis

The humoral immune response against elastin and collagen was studied in parallel with the delayed type hypersensitivity (DTH) reaction to elastin and the percentage of lymphocyte subpopulations in peripheral blood in 20 patients with systemic sclerosis (SSc). An increase of anti-elastin antibodies of all subclasses was found with a significant prevalence of IgE and IgA antibodies. The profile of anti-collagen type I and type IV antibodies showed an increase of IgE isotypes. In 25% of the patients (5 out of 20) positive DTH reactions to elastin were observed as compared to the negative skin reactions in all control individuals. At the same time a significant hyporeactivity to common bacterial and mould antigens was found in 40% of the patients (versus 16% in the control group) which could be an explanation for the low incidence of positive anti-elastin DTH reaction. The DTH hyporeactivity in SSc cases was in contrast with the increased percentage of CD4 T cells (58.4 vs. 42.0) and increased CD4/CD8 ratio (2.5 vs. 1.5) in the peripheral blood of the patients. This finding together with the increased IgE antibodies to elastin and collagen type I and type IV might suggest a possible shift of the immune balance towards the Th2 type of immune response. This is in line with the increased CD8+CD57+ cells which correlated with the highest number of other parameters studied - disease duration, total skin score, IgE anti-elastin antibodies, IgG anti-collagen type I antibodies, CD4/CD8 ratio and CD 19 B cells.The results of this study demonstrated the existence of both humoral and cell-mediated immune response against elastin in SSc patients. However, we could not define whether this was an essential part of pathogenetic mechanisms or a secondary phenomenon reflecting the extent of the damage of connective tissue.

An immunological and genetic study of patients with gestational diabetes mellitus.

The aim of the study was to evaluate the frequency of islet cell (ICA) and insulin (IAA) antibodies and of HLA antigen typing in a group of subjects diagnosed with gestational diabetes mellitus (GDM) in a screening-diagnostic program during pregnancy. ICA, complement-fixing (CF) ICA and other autoantibodies, absolute number and percentage of lymphocyte subpopulations, and HLA antigens were evaluated in 68 women with GDM and compared with those of matched controls. ICA were found in 2 (2.9%) and IAA in 1 (1.5%). Both ICA-positive women had CF-ICA; one of them was receiving insulin therapy. while the other was on a special diet. No correlations were found between ICA and IAA, nor between IAA and insulin treatment. As far as lymphocyte subsets were concerned, we found a significant increase in the absolute number of total and activated (CD3+HLA-DR+) T lymphocytes and a significant increase in the absolute number and percentage of suppressor/cytotoxic T lymphocytes (CD8) and NK lymphocytes (CD57) in GDM patients compared with normal pregnant controls. Concerning frequency for HLA A, B, C, DR antigens in the GDM population, only Cw7 was found to be significantly increased and A10 significantly decreased in comparison with controls. Our study suggests that GDM is a heterogeneous disorder in which few patients present with the immunologic and genetic markers of type 1 diabetes.

Propofol infusion anaesthesia and the immune response in elderly patients undergoing ophthalmic surgery

In our earlier studies, propofol infusion anaesthesia increased the percentage of T helper cells in middle-aged surgical patients undergoing minor or major surgery. In the present study we compared the effects of total intravenous propofol anaesthesia and combined isoflurane anaesthesia on the immune response to ophthalmic surgery in elderly patients. Twenty patients (median age 75 years, ASA 2-3) were randomly allocated to receive total intravenous propofol anaesthesia (median total dose of propofol 710 mg) or combined isoflurane anaesthesia (median end-expiratory concentration of isoflurane 0.45 vol %). The following were measured pre-operatively, at the end of operation and on the first postoperative morning: leucocyte and differential counts: percentages of lymphocyte subpopulations (CD3, CD4, CD8, CD20, CD16) and monocytes (CD14); phytohaemagglutinin-, concanavalin A- and pokeweed mitogen-induced and unstimulated lymphocyte proliferative responses: polyclonal immunoglobulin synthesis as well as serum cortisol concentrations. The immune response to ophthalmic surgery was basically similar in both anaesthetic groups. The percentage of T helper cells in the blood circulation increased in the propofol group (p < 0.05) but not in the isoflurane group. The difference in the time-response profile for T helper cell percentages between the groups was also statistically significant (p < 0.01).