The Capacity of Lymphokine Production by Peripheral Blood Lymphocytes from Aged Humans

The functional capability to produce interleukin 2 (IL2) and interferon gamma (IFNγ) by peripheral blood lymphocytes (PBL) from elderly humans was evaluated. Thirty-nine samples of PBL derived from donors aged from 56 to 79 yr were cultured with phytohaemagglutinin (PHA) as stimulus at 37°C for 48 hours. The IL2 activity in the supernatants was measured by its ability to sustain the IL2-dependent CTLL growth. Cultures of elder human T cells produced low level of IL2 activity, that is on the average of 9.7±9.8 μ/ml, equivalent to 28.1% the IL2 activity in parallel cultures of PBL derived from young donors (34.2±8.3 μ/ml, assessed on the basis of 145 samples). In the elder humans, the IL2 production by PBL decreased progressively as the increase of the age of the donors.

In contrast to the impairment of IL2 production, the amount of IFNγ secretion by elder human T cells was almost at the same level as that by young human T cells. The average IFNγ activity of 39 samples of elder PBL cultures and 128 samples of young PBL cultures was 2,961±736 μ/ml and 3133±950 μ/ml, respectively.

There was no significantly positive correlation between the level of IL2 and IFNγ when comparison was made based on each individual samples. This implies that it may exist an alternative way of IFNγ production which does not require the induction by IL2, and the relatively high level of IFNγ may not impose adverse effect on the IL2 production.     

Impaired Functions of Peripheral Blood Monocyte Subpopulations in Aged Humans

Aging is associated with increased susceptibility to microbial infections, and monocytes play an important role in microbial defense. In this study, we have identified and compared four subpopulations of monocytes (CD14^++(high)CD16⁻, CD14^+(low)CD16⁻, CD14^++(high)CD16⁺, and CD14^+(low)CD16⁺) in the peripheral blood of young and aged subjects with regard to their numbers, cytokine production, TLR expression, and phosphorylation of ERK1/2 in response to pam3Cys a TLR-1/2 ligand. Proportions and numbers of CD14^++(high)CD16⁺ and CD14^+(low)CD16⁺ monocytes were significantly increased, whereas proportions of CD14^+(low)CD16⁻ monocytes were decreased in aged subjects as compared to young subjects. In aged subjects, IL-6 production by all four subsets of monocytes was significantly decreased, whereas TNF-α production was decreased in monocyte subsets, except the CD14^+(low)CD16⁻ subset. A significantly reduced expression of TLR1 was observed in CD14^++(high)CD16⁺ and CD14^+(low)CD16⁺ monocyte subsets in aged subjects. Furthermore, following pam3Cys stimulation, ERK1/2 phosphorylation was significantly lower in CD14^+(low)CD16⁺, CD14^++(high)CD16⁺, and CD14^+(low)CD16⁻ subsets of monocytes from aged subjects. This is the first study of four subpopulations of monocytes in aging, which demonstrates that their functions are differentially impaired with regard to the production of cytokines, expression of TLR, and signaling via the ERK–MAPK pathway. Finally, changes in the number of monocyte subsets, and impairment of TLR1 expression, TNF-α production, and EK1/2 phosphorylation was more consistent in CD16⁺ monocyte subsets regardless of expression of CD14^high or CD14^+low, therefore highlighting the significance of further subdivision of monocytes into four subpopulations.     

从人外周血B淋巴细胞中应用PCR扩增人抗体基因

本文用常规ＰＣＲ法和半套式ＰＣＲ法，以一组人抗体重链和轻链引物，直接从人外周血淋巴细胞中扩增出抗体重链Ｆｄ基因和轻链基因。一些常规ＰＣＲ法不能直接扩增的人抗体基因，用半套式ＰＣＲ法扩增却得到了阳性结果。扩增的抗体基因的分子量与国内外同类报道一致。本文结果提示，在建立抗体基因文库时，半套式ＰＣＲ法能进一步丰富扩增的抗体基因的多样性     

外周血B淋巴细胞半套式PCR法扩增人抗体基因

目的用半套式PCR法,以一组人抗体重链和轻链引物直接从人外周血淋巴细胞中扩增人全套抗体基因片段.方法从不同人群外周血淋巴细胞中提取总RNA,经反转录后,以免疫球蛋白信号肽序列引物和家族特异性免疫球蛋白可变区基因引物,进行半套式PCR扩增人全套抗体基因片段.结果采用不同的引物进行重链、轻链Kappa和Lambda链的半套式PCR扩增,均能获得相应大小的PCR产物,其结果扩增率达到100%.结论在建立抗体基因文库时,半套式PCR法能进一步丰富扩增的抗体基因的多样性,可弥补由于转化效率不高而降低抗体库多样性的不足.     

外周血B淋巴细胞半套式PCR法扩增人抗体基因

目的用半套式PCR法，以一组人抗体重链和轻链引物直接从人外周血淋巴细胞中扩增人全套抗体基因片段．方法从不同人群外周血淋巴细胞中提取总RNA，经反转录后，以免疫球蛋白信号肽序列引物和家族特异性免疫球蛋白可变区基因引物，进行半套式PCR扩增人全套抗体基因片段．结果采用不同的引物进行重链、轻链Kappa和Lambda链的半套式PCR扩增，均能获得相应大小的PCR产物，其结果扩增率达到100％．结论在建立抗体基因文库时，半套式PCR法能进一步丰富扩增的抗体基因的多样性，可弥补由于转化效率不高而降低抗体库多样性的不足．     

螺旋藻对人外周血淋巴细胞增殖的作用

目的:探讨螺旋藻水提物对人外周血淋巴细胞体外增殖的影响和机制。方法:利用MTT法检测不同剂量组螺旋藻水提膜内、膜外物对人外周血淋巴细胞体外增殖的影响,同时设刀豆蛋白A(ConA)和植物血凝素(PHA)进行对照分析。结果:螺旋藻水提物对人外周血淋巴细胞具有增殖作用,并且高浓度螺旋藻水提膜内物增殖作用优于PHA。结论:螺旋藻水提物对人外周血淋巴细胞具有明显的增殖作用。     

Stimulation of Peripheral Blood Lymphocytes by Herpes Simplex Virus in Vitro

A method for stimulating sensitized lymphocytes by inactivated herpes simplex virus was established by using cultures of washed whole blood cells. The development of the immune response of herpes simplex virus-infected guinea pigs was examined at different times in the 4-month period after infection. Humoral and cellular immune responses were compared in individual animals by measuring lymphocyte stimulation ratios and neutralizing serum antibodies.     

Thymus-Dependent Lymphocytes of Peripheral Blood in Leprosy Patients

Study of the numbers of thymus-derived lymphocytes by the rosette assay (T-RFC) in patients with leprosy reveals that lower than normal numbers of T-RFC are regularly seen in those patients with the active lepromatous form of this disease. Essentially normal numbers of T-RFC were found in inactive lepromatous, borderline, and indeterminate types of leprosy. The lowest percentages and lowest absolute numbers of T-RFC were encountered in patients with lepromatous leprosy resistant to chemotherapy. Patients with lepromatous leprosy complicated by erythema nodosum leprosum show numbers of T-RFC that are more nearly normal than the numbers of T-RFC in patients with uncomplicated lepromatous leprosy. These findings are discussed with respect to the pathogenesis of lepromatous leprosy and the T-RFC deficiency demonstrated in this disease. The possibility that transient defects in T-RFC numbers or function may predispose to lepromatous leprosy is proposed.     

Immunophenotyping of Peripheral Blood Lymphocytes in Saudi Men

Flow cytometry is an important tool for the diagnosis and follow-up of immunodeficiency patients, as well as for pateints with leukemia and lymphoma. Lymphocytes and their subsets show variations with race. The aim of this study was to establish reference ranges for lymphocytes and their subsets in an Saudi adult population by using flow cytometry. Blood samples obtained from 209 healthy Saudi men were used for this study. All blood donors were between 18 and 44 years old. Lymphocytes and their subsets were analyzed by flow cytometry, and the absolute and percentage values were calculated. We investigated the expression of T-cell markers (CD3, CD4, and CD8), B cells (CD19), and natural killer cells (CD16 and CD56). The absolute and percent values of each cell subset were compared with published data from different populations by using the Student t test. Reference ranges, each expressed as the mean ± the standard deviation, were as follows: leukocytes (6,335 ± 1759), total lymphocytes (2,224 ± 717), CD3 cells (1,618 ± 547), CD4 cells (869 ± 310), CD8 cells (615 ± 278), CD19 cells (230 ± 130), and CD3-CD16⁺/CD56+ cells (262 ± 178). The CD4/CD8 ratio was 1.6 ± 0.7. Our results for B cells, CD4 cells, and CD8 cells and for the CD4/CD8 ratio fell in between the reported results for Ethiopian and Dutch subjects. Our results were also different from previously reported findings in an Saudi adult population that showed no increase in CD8 T cells. We thus establish here the reference ranges for lymphocytes and their subsets in a large cohort of Saudi men. The CD8 cell count was not abnormally high, as previously reported, and fell in between previous results obtained for African and European populations.     

Difference in Cytokine Production and Cell Activation between Adenoidal Lymphocytes and Peripheral Blood Lymphocytes of Children with Otitis Media

We evaluated the immunological potential of adenoidal lymphocytes from children with recurrent otitis media. Interleukin-4 release and CD69 expression were lower in adenoidal lymphocytes than in peripheral blood lymphocytes (PBL). Our results suggest that there may be a difference between the immunological potential of adenoidal lymphocytes and that of PBL in children with otitis.     

Expression of a Novel Protein by Regenerating Hepatocytes and Peripheral Blood Lymphocytes

Regeneration and tolerance factor (RTF) is a protein with immunosuppressive activity and is normally present in the thymus and placenta. RTF was measured in the livers of patients with regenerating nodules due to alcoholic cirrhosis and hepatitis C. RTF was expressed in the regenerating nodules of 26 patients with alcoholic cirrhosis. All patients with chronic hepatitis C without cirrhosis failed to express RTF. Flow cytometry revealed upregulation of RTF on the lymphocytes from alcoholic cirrhosis and downregulation in hepatitis C disease.     

Blastogenesis and Lymphokine Synthesis by T and B Lymphocytes from Patients with Periodontal Disease

Thymus-derived (T) and bone marrow-derived (B) lymphocytes were isolated from human peripheral blood and cultured with various mitogens and antigens. Purified protein derivative of tuberculin stimulated both purified T and B cells from patients with positive skin reactivity to purified protein derivative but did not stimulate nonimmune lymphocytes. Similarly, both T and B lymphocytes from patients with periodontal disease were stimulated to proliferate when incubated with dental plaque, whereas cells from normal individuals without gingivitis were unresponsive. In contrast, one component of plaque, bacterial endotoxins (lipopolysaccharide), minimally stimulated B lymphocytes from both normal or gingivitis patients. T lymphocytes from patients with periodontal disease were also stimulated by plaque antigen to produce chemotactic lymphokine activity (CTX) for human monocytes. B cells purified by the EAC rosetting method nonspecifically produced CTX without concomitant blastogenesis; however, after dissociation of adherent EAC these immune B cells did not spontaneously produce CTX. Lymphokine synthesis by B cells was not dependent on concomitant blastogenesis. Dissociated B cells from periodontitis patients also produced CTX activity after stimulation with dental plaque antigen. Therefore, both T and B lymphocytes, after stimulation with nonendotoxin antigenic components of plaque, proliferated and produced lymphokines, which are presumed to contribute to the pathogenesis of periodontal disease.     

Modulation of Mitogen-Induced Proliferation of Autologous Peripheral Blood Lymphocytes by Human Alveolar Macrophages

Experiments were carried out to determine the effect of cocultivation of T-cell-enriched human peripheral blood lymphocytes with autologous alveolar macrophages on mitogen-induced proliferation as determined by [³H]thymidine uptake. Cells obtained by fiberoptic bronchoscopy and saline bronchial lavage from 14 normal volunteers were enriched for macrophages by adherence in plastic dishes for 1 h in RPMI 1640 medium supplemented with 10% fetal calf serum. Nonadherent mononuclear cells were prepared from heparinized venous blood after Ficoll-Hypaque sedimentation by passage over nylon wool columns. T-cell-enriched populations were incubated with and without alveolar macrophages, either in the presence or absence of phytohemagglutinin. In these experiments, the number of lymphocytes was held constant (10⁵ per well), while the number of alveolar macrophages was varied (0.1 × 10⁵ to 4.0 × 10⁵ per well). Alveolar macrophages generally tended to stimulate phytohemagglutinin-induced lymphoproliferation at lymphocyte/macrophage ratios of 10:1 but consistently and significantly suppressed proliferation at ratios which approach those usually observed in recovered human bronchial lavage fluid, namely, 1:4. The suppressive effect of alveolar macrophages was observed as early as 48 h after culture initiation, while the magnitude of suppression increased with time. Suppression did not appear to be due to alteration in lymphocyte viability, nor was it sensitive to indomethacin. These results indicate that human alveolar macrophages can modulate the in vitro proliferative response of autologous peripheral blood lymphocytes. This observation may have relevance to interactions between alveolar macrophages and bronchial lymphocytes in the human lung in vivo.     

Carbohydrate Metabolism in PHA Stimulated Human Peripheral Blood Lymphocytes

Human peripheral blood lymphocytes (PBL) stimulated in vitro by phytohemoagglutinin (PHA) manifest augmented glycolysis and oxidation of glucose-1-¹⁴C, indicating an increased utilization of the pentose pathway. Lactic acid production, as index of increased glycolysis, follows the same kinetic of thymidine incorporation and can he easily quantitated by an enzymatic assay.     

Polyclonal Activation of Human Peripheral Blood B Lymphocytes by Formaldehyde-Fixed Salmonella paratyphi B

B-cell 'activation' in cultures stimulated with pokeweed mitogen (PWM), Staphylococcus aureus strain Cowan I, or formaldehyde-fixed Salmonella paratyphi B (SPB) was evaluated by enumeration of cells secreting immunoglobulin (Ig) and by quantitation of Ig released into culture supernatants. A dissociation between these two values was found after day 6 in cultures activated with PWM or SPB, suggesting that Ig-secreting cells (ISC) are heterogeneous in terms of Ig secretion rate. Generation of ISC in cultures activated with PWM or SPB was partially inhibited by hydroxyurea, but [g levels in culture supernatants were not affected. These results indicate that there are at least two subpopulations of ISC in stimulated peripheral blood lymphocyte cultures, one sensitive to, and the other resistant to, hydroxyurea. The hydroxyurea-resistant subpopulation appeared to be more mature and to release almost all of the Ig detected in culture supernatants. Furthermore, time-course studies of ISC numbers and Ig levels showed that each ISC in SPB-stimulted cultures (but not in PWM-stimulated cultures) was more active in Ig synthesis and secretion after day 8 than before day 6, indicating that after day 8 most of the ISC in cultures activated with SPB were hydroxyurea-resistant. These studies suggest that SPB is another useful polyclonal B-cell 'activator' for studies of human B-cell differentiation and function, and that SPB defines two distinct subsets of B cells.     

Blastogenic Response of Peripheral Blood Lymphocytes From Multiparous Pregnant Ewes

ABSTRACT: The immunocompetence of pregnant multiparous ewes was investigated with respect to the blastogenic response of peripheral blood lymphocytes of (PBL) to three mitogens: PHA, Con A, and PWM. The profile of PBL responses shows 1) progressive suppression at 36 and 66 days of gestation, 2) enhanced response at 97 days of gestation, which approaches the mean values observed at the premating period, and 3) a redecline of the response at 137 days of gestation to depressed values lower than observed at 36 and 66 days of gestation. The results suggest that mitogen-treated lymphocytes were depressed and that the immunodepressive factor(s), which can influence lymphocytes at the systemic level, may be involved in the maintenance of the ovine fetal semiallograft.     

苏丹红Ⅰ对人淋巴细胞基因表达谱的影响

目的利用基因芯片技术,研究苏丹红Ⅰ对外周血淋巴细胞基因表达谱的作用,着重探索其对人类肿瘤形成的影响。方法用10-5mol/L苏丹红Ⅰ处理人外周血淋巴细胞8h,提取实验组和对照组细胞mRNA,经逆转录和标记后与人类全基因组芯片(约35000个点)杂交。芯片扫描后提取并处理数据,筛选出与肿瘤发生相关的基因,进行生物信息学分析。结果306个表达上调的基因中有11个与肿瘤形成相关的基因;表达下调的181个基因中有16个与肿瘤形成相关的基因。结论苏丹红Ⅰ可引起淋巴细胞基因表达谱的变化,其中对肿瘤相关基因表达有影响,提示苏丹红Ⅰ有诱发肿瘤的倾向。