Down-Regulated Expression of Perforin-Positive/CD16+ Cells in the Peripheral Blood Lymphocytes in the First Trimester of Pregnancy and Up-Regulation at the End of Pregnancy

PROBLEM: Immunophenotypic profiles of perforin (P)-positive peripheral blood lymphocytes in the first trimester and at the term of human pregnancy were analyzed. METHOD OF STUDY: Perforin expression in peripheral blood lymphocyte subsets was measured by simultaneous detection of P (intracellular antigen) and cell-surface antigens (CD3, CD4, CD8, CD16, and CD56) by flow cytometry in nonpregnant (NP) and pregnant women in the first trimester (FTP) and at the time of parturition (TP). RESULTS: The percentage of total P⁺ cells in peripheral blood compared to nonpregnant women was slightly lower in the FTP but significantly higher at TP. Perforin-positive cells were significantly elevated in T lymphocyte subsets (CD3⁺P⁺, CD4⁺P\ CD8⁺P⁺) in both the FTP and TP groups, as was the percentage of CD56⁺P⁺ cells. Profound changes in the CD16⁺ subpopulation were found in the FTP group compared to both the NP and TP groups (a drastic decrease of CD16⁺P⁺ cells; CD16⁺ cells among P⁺ cells; P⁺ cells among CD16⁺ cells). A considerable part of CD3⁺ cells in both the FTP and TP groups are CD3⁺CD56⁺P⁺. The average fluorescence intensity (AFI) for P (a measure of P content per cell) was significantly decreased in FTP and increased in TP groups. CONCLUSIONS: The CD16 molecule is FcγRIIIA which is the only Fc receptor responsible for antibody dependent cell-cytotoxicity (ADCC) of NK and T-cells. In the first-trimester human pregnancy this mechanism is severely down-regulated compared to both the NP and TP groups.     

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．     

Characterization of NKT Cells in Human Peripheral Blood and Decidual Lymphocytes

PROBLEM: To examine whether natural killer (NKT) cells are present in human pregnancy decidua. METHOD OF STUDY: We calculated the percentage of CD3+CD161+Valpha 24+-NKT cells in peripheral blood and early pregnancy decidua, and analyzed intracellular cytokines, interleukin (IL)-4 and interferon (IFN)gamma in NKT cells using flow cytometry. RESULTS: A distinct subset of CD3+ CD161+ lymphocytes expressing an invariant antigen receptor encoded by the Valpha24 and Vbeta11 segment was accumulated in the decidua. In pregnant subjects the percentages of NKT cells were significantly increased in the decidua compared with peripheral blood. Both NKT cells in the decidua and the peripheral blood had an ability to rapidly produce cytokine associated with Th1 (IFNgamma) and Th2 (IL-4). Interestingly, the percentages of IL-4 and IFNgamma producing NKT cells were significantly higher in the decidua compared with the peripheral blood. CONCLUSIONS: These findings suggest that NKT cells might control the Th1/Th2 balance by producing IL-4 and IFNgamma at the feto-maternal interface.     

Studying the Proliferation of Human Peripheral Blood T Lymphocytes in Serum-Free Medium

We compared the cultivation of human peripheral blood lymphocytes in serum-free medium Hybris-2 and RPMI 1640 medium with 10% fetal bovine serum in the presence of phytohemagglutinin and interleukin-2. The optimal concentration of phytohemagglutinin significantly differed in serum-free and serum-containing media (0.5 and 5 mg/ml, respectively). Both mitogens were more potent in stimulating the proliferation of lymphocytes in serum-free medium than in serum-containing medium. Strong proliferation of CD3⁺ and CD4⁺ T lymphocytes was observed in both media. The dynamics of other markers was similar in serum-free and serum-containing media. However, significant differences were revealed between individual donors. Our results indicate that the developed serum-free medium may be used in lymphocyte cultivation for scientific, diagnostic, and therapeutic purposes. Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 1, pp. 10-15, 2009 An erratum to this article can be found at     

单次高剂量率辐射和持续低剂量率辐射对外周血淋巴细胞培养的对比研究

目的:通过外周血淋巴细胞培养,对比研究单次高剂量率外放射和持续低剂量率内放射的辐射生物效应。方法:应用直线加速器对外周血淋巴细胞分别进行1、2、4、8、16、32Gy的单次辐射;应用P-32胶体0.01、0.1、0.5、2.7MBq,分别进行持续的外周血淋巴细胞辐射,培养3d后进行制片染色,对比分析研究细胞核的形态大小和染色体形成率。结果:单次1、2、4、8、16、32Gy的外放射后,外周血淋巴细胞染色体形成率分别为4.7%、5.5%、3.6%、1.7%、0.1%、0.02%;P-32胶体0.01、0.1、0.5、2.7MBq,连续3d的内辐射后,染色体形成率分别为3.3%、2.6%、1.7%、0.004%,未辐射对照组淋巴细胞形成率为3.5%。结论:单次小剂量(1、2、4Gy)辐射对外周血淋巴细胞培养可能有促进作用,单次高剂量(8、16、32Gy)辐射有明显抑制作用,高活度的P-32胶体(0.5、2.7MBq)对外周血淋巴细胞生长有明显抑制作用,对比认为,P-32胶体0.5MBq持续3d辐射的生物效应近似单次8Gy的外放射生物效应。     

In Vitro Humoral Immune Response of Human Peripheral Blood Lymphocytes to Tetanus Toxoid Sepharose 4B

The in vitro immune response of unfractionated human peripheral blood lymphocytes (PBL) from immune donors who had not been re-immunized with tetanus toxoid (TT) prior to donation was investigated. In this study we were able to stimulate PBL with tetanus toxoid coupled to Sepharose 4B (STT) for production of anti-tetanus toxoid antibody (Ab). Soluble tetanus toxoid or STT alone did not stimulate production of specific Ab. Pokeweed mitogen (PWM) and STT were required for optimal production of IgG and IgM antibodies specific to tetanus toxoid. Specific Ab responses were reduced in low and high concentrations of STT. Depletion of monocytes had no effect on either total IgG or specific IgG synthesis, but decreased the synthesis of both total and specific IgM. Depletion of E-rosette-forming cells decreased the production of specific Ab, suggesting T-dependency of the immune response to STT. Simultaneous production of total immunoglobulin and specific Ab by Sepharose 4B was negligible in the absence of PWM. In the presence of PWM, total immunoglobulin production was optimal, and specific anti-TT Ab production was undetectable. The specificity of the antiTT Ab was studied by absorption of the culture supernates with an STT column which removed all measurable specific Ab.     

Role of Bacterial Lipopolysaccharides in the Development of Natural Antibacterial Activity Mediated by Human Peripheral Blood T Lymphocytes

The role of bacterial lipopolysaccharides (LPS) has been evaluated for their influence on the human T cell-mediated anti-Salmonella typhi activity. In nonendemic areas for salmonellosis this activity is exerted by CD4⁺ lymphocytes armed by IgA, whereas in endemic zones besides these cells also CD8⁺ lymphocytes armed by IgG display an elevated anti bacterial activity. These results suggest that, in endemic regions, continuous antigenic challenge and, in particular, that exerted by lipid A (the active moiety of LPS) may play a role in triggering this activity.

In other series of experiments, pretreatment of endemic peripheral blood lymphocytes (PBL) with smooth and rough (Rb and Re) forms of Salmonella LPS leads to the inhibition of antibacterial activity. In this respect, Re-LPS, which contains lipid A covalently linked to the core-chetodeoxyoctonate, gives rise to the maximum of inhibition.

Finally, fractionation of PBL by means of S. minnesota R345 (Rb) cytoadherence has led to the conclusion that anti bacterial activity is present in the Rb-unbound population, thus indicating that bacterial adherence to PBL is a distinct phenomenon from natural anti-S. typhi activity.

The overall results suggest that lipid A is able to modulate the expression of antibacterial activity exerted by human peripheral blood T cells.     

老年鼠外周血淋巴细胞凋亡及基因调控的研究

本文采用TUNEL法及流式细胞仪观察不同鼠龄外周血淋巴细胞凋亡及凋亡发生率 ,并用免疫组织化学检测Bcl 2、Bcl xl及Bax基因表达变化。结果发现老年鼠组外周血淋巴细胞群中存有典型的凋亡细胞 ,凋亡细胞发生率明显高于青年组。老年鼠PBL的Bcl 2 ,Bcl xl表达下调 ,而Bax的表达量明显增加。提示在衰老过程中存在着免疫细胞凋亡 ,其发生与相关调控基因的表达一致。     

Induction of K562 lysis in autologous LGL and dense T cell cocultures

Human large granular lymphocytes (LGL) were cultured with autologous dense T lymphocytes for 0-8 days. The mixed LGL dense T cell cultures had enhanced lytic activity against K562 on day 4, as compared to LGL cultured alone. Dense T cells cultured alone had no K562 lytic activity. The mixed LGL dense T cell cultures were reseparated on day 4 into low dense SRBC- and SRBC+ and into high dense subsets. Both low dense subsets did lyse K562 whereas the high dense subset did not lyse K562. Since the LGL population was the low dense SRBC- cells, the results suggested that the day 4 low dense SRBC+ cytotoxic population in the mixed LGL dense T cell culture originated from noncytotoxic dense T cells. Dense T cells were also separated into OKT8- and OKT8+ subsets and cultured together with LGL for 0-8 days. The LGL dense OKT8- T cell coculture had enhanced K562 lytic activity while the LGL dense OKT8+ T cell coculture had no enhanced lytic activity, as compared to LGL cultured alone. These results indicated that LGL interacted with the dense OKT8- T cells to produce the enhanced K562 lysis.     

A Cross‐Talk of Decidual Stromal Cells,Trophoblast, and Immune Cells: A Prerequisite for the Success of Pregnancy

Embryo implantation and formation of a functional placenta are complex processes that require a plethora of regulatory mechanisms involving both mother and embryo cells. Recently, an important role in this complicated cells and factors network was assigned to the decidual stromal cells (DSC) and trophoblast cells. Decidualization includes biochemical changes that trigger DSC to produce a number of factors required for the implantation and induction of immunotolerance in maternal immune system. Immunotolerance is achieved by a cascade of strictly controlled events starting with selective homing of immune cells to the feto‐maternal site, regulated proliferation, and predominant differentiation into a regulatory type of immune cells. Furthermore, cytotoxic effector functions are reduced owing to the influence of steroid hormones, factors, cytokines, and inhibitory receptors. Altogether the entire immune system of the mother is switched to tolerogenic functional state which is a prerequisite for the successful maintenance of pregnancy.     

SEB活化的人外周血T细胞CD25,CD69的表达

本文用超抗原葡萄球菌肠毒素B(SEB)诱导外周血淋巴细胞增殖。结果显示出微量超抗原能诱导外周血淋巴细胞的增殖,大量的增殖反应发生在SEB刺激后的第5天,增殖的细胞是CD4~+T细胞,它们由刺激前的27％增加到42％。外周血活化的T细胞不依赖外源性IL-2:大量内源性IL-2的存在抑制T细胞的增殖反应。伴随CD4~+T细胞的增殖,CD25和CD69分子表达明显增加。提示SEB能改变外周血T细胞表面的分子表达。     

淋巴瘤患者外周血大颗粒淋巴细胞及淋巴细胞微核的分析

目的　探讨淋巴瘤患者外周血中大颗粒淋巴细胞 (LGL)及淋巴细胞微核的数量变化。方法　对 15例淋巴瘤患者及 2 0例输血员外周血做血涂片 ,瑞氏染色 ,显微镜观察。结果　淋巴瘤患者外周血中LGL百分率为 2 4 0 1± 7 5 1,对照组LGL百分率为 5 6 5± 2 6 0 ,差异有显著性 (p <0 0 0 1)。淋巴瘤患者外周血中淋巴细胞微核率 0 87‰ ,对照组为 0 6 5‰ ,差异无显著性 (p± 0 0 5 )。 结论　淋巴瘤患者外周血中LGL的变化对病人机体的免疫状态及预后等有一定的临床意义     

Peripheral Blood T Lymphocytes from Systemic Sclerosis Patients Show Both Th1 and Th2 Activation

Our objective was to investigate the phenotype of helper T cells in the peripheral blood of patients with systemic sclerosis (SSc). PBMC from 15 patients with SSc and 15 sex- and age-matched controls were investigated for lymphocyte subpopulations (CD3, CD4, CD8, CD19, CD16/CD56, CD3-DR); IL-2, IL-4, and IFN- mRNAs; and the relative cytokines in their cytoplasm. The last assay was carried out both in unstimulated and in PMA-activated PBMC. SSc patients presented a higher percentage of activated T cells, CD3+ DR+ (19.7 ± 9.9 vs 5.1 ± 2.5%; P < 0.0001); 12 of them presented IFN- mRNA-positive cells; and none IL-2 or IL-4 mRNAs. Under basal conditions, PBMC from six SSc patients contained IL-2, IL-4, and IFN- (i.e., they showed both Th1 and Th2 activation), and 1 IFN- only. PMA-stimulated PBMC of patients differed from those of controls only in the increased percentage of IFN- positive cells (52 ± 12 vs 37 ± 11%; P < 0.01). Our study demonstrates that Th1 activation occurs in the peripheral blood of SSc patients. This evidence must be faced with from both a pathogenetic and a therapeutical point of view.     

Phenotypes of Human Large Granular Lymphocytes as Defined by Monoclonal Antibodies

Four monoclonal antibodies VEP8, VEP9, VIM-D5, VIB-C5 against antigens expressed on human mature myeloid cells (polymorphonuclear leukocytes [PMNL] and/or monocytes) as well as on immature cells in the bone marrow were tested for reactivity with cell preparations highly enriched for large granular lymphocytes (LGL). These cells are known to be the main effector cells responsible for natural killer (NK) cell activity in human peripheral blood. Using indirect membrane immunofluorescence (IMF), none of these antibodies showed any reactivity at all. In addition, LGL-enriched cell preparations were tested with the anti-lymphocyte monoclonal antibodies OKT6, anti-Leu1, anti-Leu2a, anti-Leu3a, and anti-human Lyt3, and also with OKM1 antibody. Significant reactivity was found with anti-Leu2a (59 ± 8 %), anti-Lyt3 (55 ± 4 %) and OKM1 (81 ± 11 %) antibodies, whereas T6, Leu1, and Leu3a antigens were less pronounced or missing on LGL. As a further approach, another monoclonal antibody, VEP13, which reacts with LGL, granulocytes but not monocytes and is therefore different in its specificity from OKM1 and OKT10, was used for identification of LGL. The coexpression of antigens as defined by the above-mentioned antibodies and OKT10 on VEP13⁺ cells was studied. Again, phenotypes similar to those observed on LGL enriched by Percoll^® gradient centrifugation were found: of VEP13⁺ cells 84 ± 6 % reacted with OKM1, 82 ± 5 % with OKT10, 52 ± 17 % with anti-human Lyt3, and 48 ± 14 % with anti-Leu2a, whereas VEP8, VEP9, VIM-D5, VIB-C5, T6, Leu1, Leu3a antigens were not expressed on VEP13⁺ cells. Taken together as an overall evaluation of phenotypic characteristics, our data indicate that LGL cannot be integrated into one of the known lymphocytic or myelomonocytic lineages. LGL show an intermediate phenotype depending possibly on varying differentiation or activation stages of haemopoietic cells. However, the possibility also exists that LGL belong to a separate, yet undefined cell lineage.     

人外周血淋巴细胞酶活性光镜细胞化学定位

目的　探讨人外周血淋巴细胞酶结构定位与活性。方法　应用光镜酶细胞化学方法对人外周血淋巴细胞ATP酶与G 6 P酶进行酶的定位与活性分析。结果　ATP酶阳性反应颗粒清晰 ,见于淋巴细胞膜内侧 ,G 6 P酶阳性反应颗粒见于淋巴细胞胞质内。结论　二种酶存在于人淋巴细胞内。     

Comparative Analysis of Peripheral Natural Killer Cells in the Two Phases of the Ovarian Cycle

Changes in endometrial Natural Killer (NK) cells during the luteal phase of the ovarian cycle are important in initiating/maintaining a subsequent pregnancy. In the present study it was investigated whether during the menstrual cycle changes occur also in peripheral blood (PB) NKs.

Method of study

Blood samples during the follicular and the luteal phase were collected from 30 women without fertility problems. Samples were analyzed by flow-cytometry for: (1) NK cells (CD3⁻CD16⁺CD56⁺) and (2) intracellular production of interferon-γ (IFN-γ) by NK cells. For the comparison and correlation of the two populations between the two phases, Wilcoxon signed-rank test and Spearman's Coefficient were used.

Results

The differences in percentages of CD3⁻CD16⁺CD56⁺ cells and that of CD3⁻CD16⁺CD56⁺/IFN-γ⁺ cells between the follicular and the luteal phase were not statistically significant (10.61 ± 5.11 versus 9.76 ± 4.57 and 6.48 ± 7.90 versus 7.30 ± 6.77, respectively, P  > 0.05). The correlation between the two variables (NK% and NK/IFN-γ%) was weakly positive ( P  = 0.07) only in the follicular phase.

Conclusion

The study did not reveal menstrual cycle-depended changes in PB NK cells. Thus, a suggestion to measure these cells in a specific phase of the cycle in order to predict the outcome of a subsequent pregnancy in women with fertility problems is objected.