Sex and Urbanicity Contribute to Variation in Lymphocyte Distribution across Ugandan Populations

Management of patient care and interpretation of research data require evaluation of laboratory results in the context of reference data from populations with known health status to adequately diagnose disease or make a physiological assessment. Few studies have addressed the diversity of lymphocyte subsets in rural and urban Ugandan populations. Here, 663 healthy blood bank donors from semi-urban centers of Kampala consented to participate in a study to define lymphocyte reference ranges. Whole blood immunophenotyping was performed to determine the frequency and absolute counts of T, B, and NK cells using clinical flow cytometry. Results from blood bank donors were compared to a rural cohort from the district of Kayunga and more urban clinical trial participants from the capital city, Kampala. Relationships between hematological and lymphocyte parameters were also explored. In the semi-urban blood donors, females were significantly different from males in all parameters except the frequency of CD8 T and B cells. Females had higher absolute counts of CD4 T, CD8 T and B cells, whereas males had higher NK cell counts. NK cell frequency and counts were significantly higher in semi-urban blood donors, regardless of sex, compared to more urban study participants. CD8 T cell frequency and counts were significantly higher in the blood donors compared to the rural participants, irrespective of sex. Interestingly, basophil counts were positively associated with overall T cell counts. These findings suggest that both sex and level of cohort urbanicity may influence lymphocyte subset distributions in Ugandans.     

Daily variation in circulating lymphocyte counts, T and B proportions and responsiveness to phytohemagglutinin.

Peripheral blood was obtained from six healthy individuals over five consecutive days under uniform conditions and the total lymphocyte counts, T and B proportions, and response to phythohemagglutinin (PHA) were determined. The daily variation in T lymphocytes as measured by the spontaneous sheep erythrocyte (SRBC) assay was much greater when total T concentrations rather than T percentages were compared. There was considerable daily variation in PHA responsiveness and in the percentages of cells bearing Fc and C3 receptors and surface immunoglobulin (SIg). Cryopreservation did not affect the proportions of T and B lymphocytes although it resulted in a significant enhancement of PHA responsiveness following the freeze-thaw procedure. The significance of these results is discussed.     

Sustained impairment of IFN-gamma secretion in suppressed HIV-infected patients despite mature NK cell recovery: evidence for a defective reconstitution of innate immunity

The impairment of NK cell functions in the course of HIV infection contributes to a decreased resistance against HIV and other pathogens. We analyzed the proportion of mature and immature NK cell subsets, and measured subsets of IFN-gamma and TNF-alpha-producing NK and T cells in viremic or therapy-suppressed HIV-infected subjects, and noninfected control donors. Viremic HIV(+) individuals had significantly lower proportions of mature CD3(-)/CD161(+)/CD56(+) NK cells and of IFN-gamma-producing NK cells compared with noninfected donors, independent of CD4(+) T cell counts. HIV-infected subjects with undetectable viral load recovered mature CD3(-)/CD161(+)/CD56(+) NK cells and cytotoxicity against tumor (K562) and HSV-infected target cells to percentages comparable with those of uninfected individuals, but their NK cells remained impaired in their ability to produce IFN-gamma. In parallel to these ex vivo findings, in vitro NK cell differentiation of CD34-positive cord blood precursors in the presence of R5 or X4 HIV-1 resulted in the production of NK cells with a normal mature phenotype, but lacking the ability to produce IFN-gamma, whereas coculture of uninfected PBMC with HIV failed to affect mature NK cell properties or IFN-gamma secretion. Altogether, our findings support the hypothesis that mature NK cell phenotype may be uncoupled from some mature functions following highly active antiretroviral therapy-mediated suppression of HIV-1, and indicate that relevant innate immune functions of NK cell subsets may remain altered despite effective viral suppression following antiretroviral treatment.     

Effects of labour and medication on major lymphocyte subsets in cord

It is envisaged that flow cytometric analysis of lymphocyte subsets in cord blood may be used as a biomarker for effects on the immune system of exposure to environmental factors. In order to investigate the possible application of this parameter, we first studied the effects of other factors that may influence the outcome of subset analysis in cord blood. FACS analysis was performed in 112 pairs of umbilical cord blood and of peripheral maternal blood sampled at labour. Whereas in maternal blood no statistically significant effects of medication during labour on T lymphocyte numbers and NK cells were found, in oxytocin and in oxytocin and prostaglandin treated mothers B cell numbers showed a statistically significant increase. In cord blood, the course of labour and or medication during labour were identified as the most important factors determining distribution of major lymphocyte subsets. In cord blood after deliveries without medication or after neuroplegic analgesia NPA, the mean percentage of cord blood T lymphocytes CD3 was highest 59 and that of NK lymphocytes CD3- CD16 56 lowest 20 . The mean percentage of T lymphocytes was significantly lower 52 and that of NK lymphocytes higher 28 in cord blood where deliveries were done under NPA in combination with infusion of oxytocin. The combination of NPA with oxytocin and induction of labour by prostaglandin E2 led to a further reduction of T lymphocytes and an increase of NK cells 39 and 38 respectively. The changes in ratio of T and NK lymphocytes were due both to decreasing absolute counts of T lymphocytes and increasing counts of NK lymphocytes. Thus, the effects of labour and or medication during labour must be taken into account when this parameter is applied as a potential biomarker of effects of environmental factors on the immune system.     

Influence of gender and age on T-cell responses in a murine model of trauma-hemorrhage: differences between circulating and tissue-fixed cells.

Clinical studies indicate that peripheral blood lymphocyte functions are depressed following trauma; however, it is unclear whether tissue-fixed lymphocyte functions are also altered under those conditions. Moreover, the impact of gender and age on peripheral T-cell responses following trauma-hemorrhage (TH) are unknown. To study this, immature (approximately 3 wk of age), mature (approximately 7 wk of age), and aged (approximately 23 mo of age) male and proestrus female C3H/HeN mice were sham operated or subjected to trauma (i.e., midline laparotomy) and hemorrhagic shock (30+/-5 mmHg for 90 min). Twenty-four hours after resuscitation, blood and splenocytes were harvested and T-cell functions assessed. In immature animals, TH induced an enhanced immune response in the splenic compartment and a suppressed response in the peripheral blood mononuclear cells (PBMC) that was independent of gender. Differential responses were observed in cells from mature mice. Splenic responses were enhanced following TH, independent of gender, whereas PBMC displayed gender dimorphism with suppressed proliferation and T-cell helper 1 responses in males but not in females. A similar pattern was observed in cells from aged mice. Splenic T cells from male mice displayed a suppressed CD4-to-CD8 ratio after TH, whereas no such change was observed in cells from proestrus females. In contrast, only PBMC from mature males displayed a suppressed CD4-to-CD8 ratio after TH. Thus gender differences exist in PBMC responses after TH that do not necessarily correlate with changes in the tissue-fixed compartment. Age is also an important factor in the immune responses after TH. In view of this, both gender and age should be taken into consideration in evaluating the immune status and in treatment of TH shock.     

Effects of labour and medication on major lymphocyte subsets in cord

It is envisaged that flow cytometric analysis of lymphocyte subsets in cord blood may be used as a biomarker for effects on the immune system of exposure to environmental factors. In order to investigate the possible application of this parameter, we first studied the effects of other factors that may influence the outcome of subset analysis in cord blood. FACS analysis was performed in 112 pairs of umbilical cord blood and of peripheral maternal blood sampled at labour. Whereas in maternal blood no statistically significant effects of medication during labour on T lymphocyte numbers and NK cells were found, in oxytocin and in oxytocin and prostaglandin treated mothers B cell numbers showed a statistically significant increase. In cord blood, the course of labour and or medication during labour were identified as the most important factors determining distribution of major lymphocyte subsets. In cord blood after deliveries without medication or after neuroplegic analgesia NPA , the mean percentage of cord blood T lymphocytes CD3 was highest 59 and that of NK lymphocytes CD3- CD16 56 lowest 20 . The mean percentage of T lymphocytes was significantly lower 52 and that of NK lymphocytes higher 28 in cord blood where deliveries were done under NPA in combination with infusion of oxytocin. The combination of NPA with oxytocin and induction of labour by prostaglandin E2 led to a further reduction of T lymphocytes and an increase of NK cells 39 and 38 respectively . The changes in ratio of T and NK lymphocytes were due both to decreasing absolute counts of T lymphocytes and increasing counts of NK lymphocytes. Thus, the effects of labour and or medication during labour must be taken into account when this parameter is applied as a potential biomarker of effects of environmental factors on the immune system.     

曲马多、吗啡术后自控镇痛对食管癌患者细胞免疫功能的影响

目的：比较曲马多、吗啡对食管癌术后患者的镇痛疗效以及对机体免疫功能的影响。方法：将本院50 例行食管癌手术患者随 机分成对A 组和B 组,A 组接受硬膜外吗啡镇痛,B 组接受硬膜外曲马多镇痛,常规监测心电图（ECG）、无创血压（BP）、心率 （HR）、血氧饱和度（SPO2）、呼气末二氧化碳分压(PETCO2）。记录两组患者不同时间点视觉模拟疼痛（VAS）评分、不良反应发生 率、外周血T 淋巴细胞亚群(CD3+、CD4+、CD8+)、NK 细胞的变化。结果：两组患者均得到满意的术后镇痛效果,术后1d,A、B两组 患者外周血T淋巴细胞亚群、NK 细胞的水平较术前降低（P<0.05）,且A 组降低幅度明显大于B 组（P<0.05）;术后2d,A 组外周 血T 淋巴细胞亚群、NK 细胞的水平虽有所升高,但仍较术前降低（P<0.05）,B 组CD3+、CD4+、CD8+及NK细胞水平恢复至术前水 平;术后3d,A 组上述指标恢复至麻醉前水平。结论：曲马多在镇痛的同时对机体外周血T 淋巴细胞亚群和NK细胞水平影响较 小,减轻了麻醉对细胞免疫功能的抑制效应。     

基于流式细胞术快速检测人鼻黏膜组织淋巴细胞亚群的实验研究

目的：基于流式细胞术检测正常人的鼻黏膜组织中的淋巴细胞亚群(CD3+T、CD4+T、CD8+T、NK、CD19+B)的比例,初步探讨 鼻局部黏膜免疫功能的意义,为鼻局部黏膜免疫性疾病的研究提供更多的参考。方法：用鼻黏膜刮匙获取21 例正常人的鼻黏膜 组织,并抽取其外周血2 mL。采用流式细胞术分别检测其鼻黏膜和外周血中的淋巴细胞亚群(包括CD3+T、CD4+T、CD8+T、NK、 CD19+B)分别所占的比例。结果：鼻黏膜和外周血的淋巴细胞亚群存在很大差异,与外周血相比,CD3+T 比例增加（t=15.34,P<0. 0001),CD4+T 比例降低(t=5.952,P<0.0001)、CD8+T 比例增加(t=12.44,P<0.0001)、NK 比例降低(t=4.865,P<0.0001)、CD19+B 比例降 低(t=15.56,P<0.0001),CD4+T/CD8+T 降低。结论：流式细胞术可以用来检测鼻黏膜的淋巴细胞亚群,鼻黏膜的淋巴细胞亚群和外 周血的淋巴细胞亚群存在很大差异,这种差异体现鼻黏膜组织独特的局部黏膜免疫功能,本方法为变应性鼻炎的研究提供了新 的研究途径。     

Cellular immunosuppression in children with acute lymphoblastic leukemia: Effect of consolidation chemotherapy

Summary The present study was designed to evaluate the chemotherapy-induced cellular immunosuppression in 20 children with acute lymphoblastic leukemia (ALL) in remission and receiving maintenance chemotherapy. Peripheral blood was serially obtained from leukemic children during vincristine/cyclophosphamide/6-mercaptopurine/prednisone combined consolidation chemotherapy. The mean absolute number of peripheral blood lymphocytes as well as the mean absolute numbers of lymphocyte subsets (T cells, T cell subsets, B cells, and natural killer cells) from leukemic children before consolidation chemotherapy were all significantly lower than in control subjects; however, the percentages of lymphocyte subsets were similar in both groups. After consolidation chemotherapy, the percentages of CD4⁺ T lymphocytes and natural killer (NK) cells were significantly decreased and the percentages of monocytes and CD8⁺ T lymphocytes were significantly increased. Phytohemagglutinin- and 12-O-tetradecanoylphorbol-13-acetate-induced production of interleukin-2 (IL-2) and NK-cell-mediated cytotoxic activity by peripheral blood mononuclear cells (PBMC) were also substantially decreased in the post-therapy groups. NK activity correlated with the percentage of NK cells in PBMC. In contrast, OK432-induced production of tumor necrosis factor (TNF) and killer activity against NK-resistant target cells were significantly increased after therapy as compared with the pre-therapy and control groups. TNF production correlated with the percentage of monocytes in PBMC. These results demonstrate that substantial quantitative and qualitative chemotherapy-induced abnormalities of the cellular immune system are present in the majority of patients treated with ALL. It is also suggested that the increased TNF production by monocytes and the appearance of potent killing activity against NK-resistant targets might compensate for the defects of IL-2 production and NK activity during intensive consolidation chemotherapy.This work was supported by a grant-in-aid for cancer research from the Ministry of Health and Welfare, Japan, and a grant-in-aid from the Association for the Support of Children with Cancer     

Possible role of peripheral CD14low monocytes in the development of collagen-induced arthritis in cynomolgus monkeys (Macaca fascicularis).

The changes in levels of peripheral major lymphocyte subsets were monitored with 10 adult cynomolgus monkeys (5 females and 5 males) during the 9 weeks after immunization with chick type-II collagen in Freund's complete adjuvant. Three females and 3 males developed overt arthritis determined by swelling of small joints and increase of plasma alkaline phosphatase as well as C-reactive protein. An increase of CD16+ NK cells was observed in four non-arthritis-developed monkeys (two females and two males). There was no significant difference in the fluctuation pattern of CD4+ T cell, CD8+ T cell and CD20+ B cell levels between arthritis-developed monkeys and non-developed ones. In addition, the percentages of CD45RA+ CD4+ T cells to total CD4+ T cells, CD28- CD8+ T cells to total CD8+ T cells, and IgD- B cells to total B cells did not significantly differ between them. On the other hand, a significant increase was demonstrated in CD14-positive cells at 3 weeks after immunization in only arthritis-developed monkeys regardless of sex. The expression of CD14 antigen on the surface of increased cells was low in comparison with those appearing in blood obtained before immunization. In addition, increased CD14low cells showed no response to LPS stimulation. However, there was no significant difference in antibody titer to both chick type-II and monkey type-II collagen between arthritis-developed monkeys and non-developed ones. These results suggest that an increase in number of CD14low monocytes with immature function might be a part of the autoimmune response, and that the appearance of these cells is of pathogenic importance in the arthritic process in cynomolgus monkeys regardless of the production of autoantibody.     

Ontogeny of con A and PHA responses of chicken blood cells in MHC-compatible lines 6(3) and 7(2)

The development of T cell responsiveness to Con A and PHA was examined in two MHC-compatible inbred chicken lines, RPRL 6(3) and 7(2), at ages 2 to 118 days posthatching. These lines are respectively resistant or susceptible to Marek's disease, a naturally occurring, virally induced T cell lymphoma. Between-line comparisons were made of optimal in vitro responses of diluted serum-free blood cells to each mitogen in two groups of chicks tested over ages 2 to 63 and 41 to 118 days. Over 2 to 63 days, Con A responses increased with age at the same rate in each line, but 7(2) responses averaged 2.3 times higher than 6(3). The increase with age was dependent on blood lymphocyte counts, which also increased with age in parallel in both lines. In contrast, the between-line difference in responsiveness was dependent on intrinsic reactivity of cells as well as lymphocyte counts. Covariance analysis was used to estimate that line 7(2) was 1.4 times higher than 6(3) in intrinsic cell reactivity, after accounting for the effect of the twofold higher blood lymphocyte counts in 7(2), and that this intrinsic difference contributed almost one-half the total difference. Over 41 to 118 days Con A responses no longer increased with age, although lymphocyte counts were still increasing, and the line difference (2.6 times) was now almost entirely contributed by a 2.3-fold superiority of 7(2) blood cells in intrinsic reactivity. The line difference in PHA responses was the reverse of the above in young chicks, with 6(3) responses greater than 7(2) in spite of lower lymphocyte counts. In additional chicks tested over 5 to 26 days, intrinsic reactivity of 6(3) cells to PHA averaged 4.5 times higher than 7(2). There was an abrupt decline in intrinsic reactivity of line 6(3) blood cells between 26 and 41 days to a level equal with 7(2). After this age, line 7(2) responses were 1.8 times greater than those of 6(3), and this difference was dependent solely on lymphocyte count differences. The results suggest that different gene systems mediate blood cell responses to PHA as compared with Con A. The pattern of developmental differences between inbred lines indicates the existence of distinct or partly overlapping T cell subsets with different reactivities to PHA or Con A, and of higher suppressor activity of adherent cells in line 6(3) blood. Both these differences may be related to line 6(3) inherited resistance to Marek's disease.     

Purine nucleoside modulation of functions of human lymphocytes.

The accumulation of endogenous substrates in patients with adenosine deaminase deficiency or purine nucleoside phosphorylase deficiency is believed to be responsible for the immunodeficiency observed in these patients. To identify the lymphocyte populations that are most susceptible to these substrates, we investigated the effect of their nucleoside analogs on a number of T and B cell functions of human lymphocytes. We found that tubercidin (Tub), 2-chloro 2'deoxyadenosine (2CldA), 2-fluoro adenine arabinoside-5'phosphate (FaraAMP), and 9-beta-D-arabinosyl guanine (AraGua) inhibited the proliferative responses of human peripheral blood mononuclear cells (PBMC) to polyclonal activators (PHA, OKT3 mab) or to allogeneic PBMC in mixed lymphocyte cultures (MLC). Addition of recombinant IL-2 from the beginning of the culture did not alter the inhibition by Tub of the proliferative responses of PBMC. These purine nucleoside analogs also inhibited the proliferative responses of purified human peripheral blood CD4+ and CD8+ T cells to PHA and of purified B cells to SAC. The concentrations of these nucleosides required to achieve a given degree of inhibition of proliferative responses of T lymphocyte subpopulations or B cells was similar, suggesting that these analogs do not exhibit any selectivity for these purified lymphocyte populations. Tub and FaraAMP, respectively, inhibited and enhanced, at the effector phase, both NK cytotoxicity and specific T cell-mediated cytotoxicity. In contrast to these findings, LAK cytotoxicity at the effector phase was not significantly inhibited by Tub, and was not enhanced by FaraAMP. Both analogs inhibited rIL-2-induced proliferative responses of PBMC, but did not affect the generation of LAK cytotoxicity (induction phase) against the K562 targets when added at the beginning of the culture. This suggests that DNA synthesis is not required for LAK cell induction. Both Tub and FaraAMP inhibited immunoglobulin production (IgG and IgM) by PBMC in the PWM-induced system. These results demonstrate that purine nucleoside analogs significantly inhibited a number of functions of human lymphocytes. Although selectivity for T lymphocyte subpopulations and B cells was not observed, a differential effect of Tub and FaraAMP on LAK cytotoxicity versus NK cytotoxicity and specific T cell cytotoxicity was found.     

Sex- and age-specific clinical and immunological features of coronavirus disease 2019

To simultaneously determine clinical and immunological responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in young and old females and males, 681 coronavirus disease 2019 (COVID-19) patients and 369 normal controls (NCs) were analyzed based on age and sex classifications using multiple linear regression analysis. Compared to the age-matched NCs, both young and old male and female non-comorbid COVID-19 patients had lower lymphocyte counts and alanine aminotransferase (ALT) concentration, and only young male and female patients had lower neutrophil counts. Compared to young patients, both old males and females had significantly higher plasma ALT and AST concentrations. Compared to young and old females, age-matched males had higher plasma ALT and AST concentrations, but only young males had higher C-reactive protein (CRP) concentration. Compared to females, old males, but not young males, showed higher incidence of critical illness. Compared to young patients, old females had more leukocyte and neutrophil counts above the normal upper limit and B cell count below the normal lower limit (NLL), while old males had more lymphocyte and natural killer (NK) cell counts below the NLL. No sex or age associations with B cell and NK cell counts were observed. However, there were age-dependent decreases in CD8⁺ T-cell counts in both male and female COVID-19 patients. Age was negatively associated with CD8⁺ T cell counts but positively associated with neutrophil count, CRP, ALT, and AST concentrations, and sex (females) was negatively associated with neutrophil count, CRP, ALT, and AST concentrations. The present study suggests that SARS-CoV-2 infection mainly induced 1) beneficial sex (female)-related differences regarding reduced COVID-19 disease severity and negative associations with inflammatory responses and liver damage, and 2) harmful age-related differences relating to negative associations with CD8⁺ T cell count and positive associations with inflammatory responses and liver damage. Thus, sex and age are biological variables that should be considered in the prevention and treatment of COVID-19.     

多发性骨髓瘤患者外周血淋巴细胞亚群分析及与预后因子的相关性

目的通过对多发性骨髓瘤（MM）患者外周血淋巴细胞亚群的检测以评价MM患者机体的免疫功能状态。方法采用流式细胞术检测36例MM患者和25例健康志愿者外周血T、B淋巴细胞、NK细胞及CD4＋CD25＋T细胞的表达。结果与正常对照组相比,MM患者外周血的CD4、CD19细胞的表达显著下调,CD8细胞的表达显著上调,CD4/CD8比值则显著降低（P〈0.05或〈0.01）;MM患者外周血的CD4＋CD25＋T细胞占CD3＋T细胞的比例明显增高（P〈0.01）,且与血清中的β2-MG浓度成正相关（γ=0.56,P〈0.05）。结论 MM患者体内存在淋巴细胞亚群的异常表达、CD4＋CD25＋Treg细胞的异常扩增,可能是MM患者体内广泛存在免疫缺陷的一个主要原因。     

NK cell function in severe combined immunodeficiency (SCID): evidence of a common T and NK cell defect in some but not all SCID patients

The immunologic work-up of eight infants with the clinical diagnosis of severe combined immunodeficiency (SCID) was performed with special emphasis on natural killer (NK) cell function and ontogeny. Contrary to previous reports, our study shows that not all SCID patients lack NK activity; some may even express very high NK- and antibody-dependent cellular cytotoxicity (ADCC). The present group of eight SCID infants was homogeneous with respect to normal levels of the purine metabolism enzymes adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP). They all had low serum Ig levels and were defective for specific antibody formation against BSA and diphtheria toxin (DiT). None of the infants' peripheral blood mononuclear cells (PBMC) proliferated significantly upon in vitro stimulation with PHA, concanavalin A (Con A), pokeweed mitogen (PWM), and irradiated allogeneic lymphocytes. Seven of eight patients, however, responded significantly to mitogenic factors present in a lectin-free interleukin 2 (IL 2) preparation, and two exhibited a positive costimulation as well with simultaneous exposure to IL 2 + Con A. The lymphocyte marker analysis revealed high percentages of OKT10+ cells in seven of eight infants, whereas peripheral T cells (OKT3+) with suppressor/killer (OKT8+) or helper/inducer (OKT4+) phenotypes were abnormally low in all infants with one exception. The PBMC of two patients formed low to normal percentages of E rosettes but expressed no B cell markers (B-/SCID). The six other infants had high percentages of B cells (B+/SCID) but lacked E rosette-forming cells. High NK and ADCC activity was found in the two B-/SCID patients. The B+/SCID infants either totally lacked NK and ADCC function (four of six) or expressed low to normal NK activity together with some T cell markers as revealed by monoclonal antibody staining but not by E rosette formation (two of six). From the data presented, an ontogenic model is proposed that assumes the status of an independent cell lineage in between T cells and monocytes for human NK cells, or that places these cells in close proximity to early differentiation steps of the T cell lineage. In any case, NK cell function clearly constitutes an additional parameter of heterogeneity in the immunologic analysis of SCID.     

T Cells Contribute to Stroke-Induced Lymphopenia in Rats

Stroke-induced immunodepression (SIID) results when T cell and non-T immune cells, such as B cells, NK cells and monocytes, are reduced in the peripheral blood and spleen after stroke. We investigated the hypothesis that T cells are required for the reductions in non-T cell subsets observed in SIID, and further examined a potential correlation between lymphopenia and High-mobility group protein B1 (HMGB1) release, a protein that regulates inflammation and immunodepression. Our results showed that focal ischemia resulted in similar cortical infarct sizes in both wild type (WT) Sprague Dawley (SD) rats and nude rats with a SD genetic background, which excludes the possibility of different infarct sizes affecting SIID. In addition, the numbers of CD68-positive macrophages in the ischemic brain did not differ between WT and nude rats. Numbers of total peripheral blood mononuclear cells (PBMCs) or splenocytes and lymphocyte subsets, including T cells, CD4⁺ or CD8⁺ T cells, B cells and monocytes in the blood and spleen, were decreased after stroke in WT rats. In nude rats, however, the total number of PBMCs and absolute numbers of NK cells, B cells and monocytes were increased in the peripheral blood after stroke; nude rats are athymic therefore they have few T cells present. Adoptive transfer of WT splenocytes into nude rats before stroke resulted in lymphopenia after stroke similar to WT rats. Moreover, in vitro T cell proliferation stimulated by Concanavalin A was significantly inhibited in WT rats as well as in nude rats receiving WT splenocyte adoptive transfer, suggesting that T cell function is indeed inhibited after stroke. Lastly, we demonstrated that stroke-induced lymphopenia is associated with a reduction in HMGB1 release in the peripheral blood. In conclusion, T cells are required for stroke-induced reductions in non-T immune cells and they are the most crucial lymphocytes for SIID.     

The distribution of the major peripheral blood T, B and NK cell subsets does not predict the clinical outcome of Graves' disease patients after methimazole therapy

Biological markers capable of predicting the clinical outcome of antithyroid drug therapy could be clinically useful in selecting the modality of treatment for Graves' disease, but at present they are unavailable. In the present study we prospectively explore the value of 22 different peripheral blood T, B and NK lymphocyte subsets to predict remission and relapse in a group of 42 Graves' disease patients. Eighteen patients were studied at diagnosis, before treatment, and 24 during antithyroid drug therapy. All cases were followed-up for at least one year after finishing an 18 month cycle of methimazole therapy. The combination of flow cytometry and 3- color immunofluorescence did not reveal significant differences in the distribution of the major peripheral blood T, B and NK cell subsets between the relapsed patients and those in remission, both in the groups studied at diagnosis and in those analyzed during the cycle of antithyroid drug therapy. In our search for a prognostic marker for relapse prediction we found that some lymphoid subpopulations such as total B cells, total NK and NK CD8+ cells showed high sensitivity (88-100%). In turn, other subsets such as TCD8+, total T and B cells expressing the CD25 antigen displayed high specificity (77-88%).     

Age and gender related changes in hematological parameters of thoroughbred foals

Abstract

Hematological and biochemical profiles commonly are required in equine medicine. We studied hematological parameters including red blood cells (RBC), white blood cells (WBC), hemoglobin (Hb), hematocrit (PCV), differential leukocyte counts, mean cell volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) in thoroughbred foals at different ages and for both sexes. Sixty healthy thoroughbred foals, 1 day, 3 days and 1 year old were used. Each age group consisted of 10 male and 10 female animals. We found significant differences related to age in RBC values of females, PCV, MCV values of males, WBC, neutrophil percentages, lymphocyte percentages, monocyte percentages of females, and eosinophil percentages and basophil percentages. Significant differences related to gender were found only with regard to PCV at 1 year and WBC at 1 day. The hematological parameters of thoroughbred foals up to one year old may be useful for evaluating and monitoring the health of these animals.     

Active but not inactive granulomatosis with polyangiitis is associated with decreased and phenotypically and functionally altered CD56dim natural killer cells

BackgroundThe role of natural killer (NK) cells in granulomatosis with polyangiitis (GPA) is poorly understood. We recently reported that peripheral blood NK cell percentages correlate with the suppression of GPA activity (cohort I). The purpose of the current study was to further characterize NK cell subsets, phenotype and function in a second GPA cohort (cohort II).MethodsPeripheral blood lymphocyte subsets were analyzed at a clinical diagnostic laboratory. Clinical data were extracted from medical records and patients were grouped according to their activity state (remission vs. active/non-remission). Separate analysis (cohort II, n = 22) and combined analysis (cohorts I and II, n = 34/57) of NK cell counts/percentages was performed. NK cell subsets and phenotypes were analyzed by multicolor flow cytometry. Cytotoxicity assays were performed using ⁵¹Cr-labeled K562 target cells.ResultsIn cohort II, NK cell counts were lower than the lower limit of normal in active GPA, despite normal percentages due to lymphopenia. NK cell counts, but not other lymphocyte counts, were significantly higher in remission. Combined analysis of cohorts I and II confirmed decreased NK cell counts in active GPA and increased percentages in long-term remission. Follow-up measurements of six patients revealed increasing NK cell percentages during successful induction therapy. Multicolor analysis from cohort II revealed that in active GPA, the CD56^dim subset was responsible for decreased NK cell counts, expressed more frequently CD69, downregulated the Fc-receptor CD16 and upregulated the adhesion molecule CD54, the chemokine receptor CCR5 and the activating receptor NKG2C. In remission, these markers were unaltered or marginally altered. All other receptors investigated (NKp30, NKp44, NKp46, NKG2D, DNAM1, 2B4, CRACC, 41BB) remained unchanged. Natural cytotoxicity was not detectable in most patients with active GPA, but was restored in remission.ConclusionsNK cell numbers correlate inversely with GPA activity. Reduced CD56^dim NK cells in active GPA have an activated phenotype, which intriguingly is associated with profound deficiency in cytotoxicity. These data suggest a function for NK cells in the pathogenesis and/or modulation of inflammation in GPA. NK cell numbers, phenotype (CD16, CD69, NKG2C) or overall natural cytotoxicity are promising candidates to serve as clinical biomarkers to determine GPA activity.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-016-1098-7) contains supplementary material, which is available to authorized users.     

Phenotypic and Functional Characterization of Lymphocytes from Different Age Groups of Bolivian Squirrel Monkeys (Saimiri boliviensis boliviensis)

Due to many physiological and genetic characteristic similarities to humans, squirrel monkeys provide an ideal animal model specifically for studying malaria, and transmissible spongiform encephalopathies (Creutzfeldt-Jacob disease). While squirrel monkeys three years and older are generally considered adult subjects suitable for use in medical research studies, little is known about the functional properties of lymphocytes in relation to the age of these animals, which could significantly impact the quality and quantity of innate and adaptive immune responses. In this study, we investigated differences in the phenotype and function of lymphocytes subsets of young (3–4 years), adult (8–10 years) and aged (16–19 years) squirrel monkeys. In general, animals in all three age groups exhibited comparable numbers of different lymphocyte subsets except for CD20+ B cells that were significantly lower in aged relative to young animals and T cells subsets expressing both CD4 and CD8 (double positive) were significantly higher in aged relative to young animals. With increasing age, phenotypic differences in central and effector memory T cells subsets were observed, that were more pronounced for the CD8+ T cells. Despite equal proportions of CD3+ T cells among the three age groups, responses of peripheral blood mononuclear cells to T cell mitogens PHA and Con A showed lower IFN-γ producing cells in the aged group than that in the young group. Furthermore, aged animals showed significantly higher plasma levels of inflammatory cytokines IL-6, IFN-γ, TNF-α, IL-10 and IL-12. These findings suggest that while the squirrel monkeys in general share phenotypic and functional similarities of lymphocyte subsets with humans in relation to age, specific differences exist in immune function of lymphocytes between young and old animals that could potentially impact experimental outcomes for which the measurement of immunologic endpoints are critical.