IL-2预孵育影响G-CSF动员的小鼠脾淋巴细胞增殖及极化观察

目的 探讨IL-2预孵育对粒细胞集落刺激因子(G-CSF)动员后的小鼠脾淋巴细胞功能的影响,从而为减轻异基因造血干细胞移植时急性移植物抗宿主病(GVHD)的发生提供一种新途径.方法 IL-2(50 U/ml)预孵育rhG-CSF(每天0.25 μg/g)体内动员的C57BL/6N小鼠脾细胞和na(i)ve CD4+T细胞6 h,然后在体外以BALB/c小鼠异基因抗原分别对其进行刺激,测定动员后的C57BL/6N小鼠脾细胞和na(i)ve CD4+T细胞的增殖能力以及上清液IL-4、IFN-γ的浓度,实验分为G-CSF+IL-2组、G-CSF组、IL-2组和对照组.结果 G-CSF+IL-2组较列照组A值降低(P＜0.01);G-CSF+IL-2组IL-4水平高于对照组(P＜0.01),而IFN-γ水平低于对照组(P＜0.01).结论 rhG-CSF动员的C57BL/6N小鼠脾细胞和na(i)ve CD4+T细胞经IL-2预孵育后对同种异基因抗原免疫增殖能力明显减弱并向Th2方向分化,两者合用对诱导T淋巴细胞免疫耐受有协同效应.

Abstract：

Objective To explore the influence of IL-2 pretreatment on splenic lymphocyte following mobilization with G-CSF, which may provide a new approach to attenuate acute graft-versus-host disease (GVHD). Methods Splenic cells and na(i)ve CD4+T cells from C57BL/6N mice receiving G-CSF-mobilized were pretreated with IL-2(50 U/ml) , and cocultured with the allogeneic antigens from BALB/c mice. The proliferation responses and the polarization of T cells were determined. C57BL/6N mice were randomly divided into 4 groups: G-CSF + IL-2 group, G-CSF group, IL-2 group , control group. Results Compared with the control group, IL-4 increased obviously while IFN-γ decreased significantly in the group of G-CSF + IL-2. The proliferation responses were also suppressed in vitro. Conclusion The proliferation responses of splenic cells and naive CD4 + T cells from C57BI/6N mice receiving G-CSF-mobilized to the allogeneic antigens were significantly abrogated by the pretreatment with IL-2, T cells were polarized toward the production of type-2 cytokines. The combination of G-CSF and IL-2 is potentially synergetic in the induction of T lymphocyte immune tolerance.     

湖南艾滋病患者Th17和Tr细胞及IL-17在抗病毒治疗过程中的变化

目的 观察HIV感染者和AIDS患者血IL-17、CD3+CD4+IL-17+细胞(Th17细胞)和CD4+CD25+Foxp3+T细胞(Tr细胞)的平衡状态及其在1年高效抗反转录病毒治疗(HAART)中的变化.方法 选取经HAART治疗的HIV/AIDS患者33例,同时选取33例健康志愿者为对照,分别于治疗0、6、12个月采集静脉血,检测血清IL-17水平、Th17细胞及Tr细胞百分比,并对比分析其相互关系.结果 HIV/AIDS在HAART治疗前、治疗6个月、12个月及健康对照外周血的Th17细胞在CD4+T细胞中的比例分别为(1.20±0.37)%、(2.50±1.03)%、(3.70±1.56)%和(4.70±1.43)%;Tr细胞在CD4+T细胞中的比例分别为(9.16±3.33)%、(7.19±2.91)%、(5.53±1.88)%和(4.43±0.97)%;血清IL-17水平分别为(5.3±2.5) pg/ml、(7.7±2.4) pg/ml、(10.4±3.1) pg/ml和(17.7±6.6) pg/ml.Th17细胞水平与CD4+T细胞计数正相关,与病毒载量负相关;Tr细胞水平与CD4+T细胞计数负相关,与病毒载量正相关.结论 HIV感染导致IL-17、Th17细胞和Tr细胞的失平衡,而HAART治疗可能逐渐恢复二者的免疫平衡状态.提示三者可能在艾滋病发病机制中起作用,并有可能成为观察艾滋病进展和HAART治疗效果的有效指标.

Abstract：

Objective To observe the Th17, IL-17 and Tr cells equilibrium state as well as their changes of HIV infected or AIDS suffered patients in one-year HAART treatment. Methods Select 33 HIV/AIDS patients received HAART treatment while 33 healthy volunteers as controls. Flow cytometry was used to analyze Th17 and Tr cells in venous blood at the time of pre-therapy, 6th, 12th month when IL-17 levels in serum are tested by ELISA. Results The ratio of Th17 cells in CD4 cells in HIV/AIDS patients and volunteers were (1.20±0.37)%, (2.50±1.03)%, (3.70±1.56)%, (4.70±1.43)%, respectively; The ratio of Tr cells were (9.16±3.33)%, (7.19±2.91)%, (5.53±1.88)%, (4.43±0.97)%, respectively; The levels of IL-17 in serum were (5.3±2.5) pg/ml, (7.7±2.4) pg/ml, (10.4±3.1) pg/ml, (17.7±6.6) pg/ml respectively. The Th17 cells' level was positively correlative with the amount of CD4 cells, negatively correlate with the count of viral load. However, the Tr cells level is positively correlative with the count of viral load, negatively relate to the quantity of CD4 cells. Conclusion HIV could make IL-17, Th17 cells and Tr cells lost their balance, but the immune equilibrium state may gradually recover after HAART treatment. Which indicates the IL-17, Th17 cells and Treg cells may play an important role in the pathogenesis of AIDS, and they are likely to be the effective indexes to observe the progress of AIDS and the treatment effect of highly active antiretroviral therapy(HAART).     

禽流感H5N1亚型病毒感染BALB/c小鼠的免疫应答

目的 研究禽流感H5N1病毒感染BALB/c小鼠后对宿主细胞免疫功能和细胞因子水平变化的影响,探讨禽流感H5N1病毒感染哺乳动物的免疫发病机制.方法 选用鹅源禽流感H5N1病毒感染BALB/c小鼠,采用流式细胞仪检测血液和脾脏T淋巴细胞及其亚群的变化,采用ELISA检测血液中细胞岗子(IFN-γ、TNF-α、IL-4、IL-18、IL-10、IL-2)及禽流感H5N1病毒特异性抗体的变化.结果 禽流感H5N1病毒感染可引起对宿主短暂的、可恢复的细胞免疫功能损伤:血液CD3+、CD4+、CD8+ T淋巴细胞数量于染毒后第2～4天下降(第4天为最低值),脾脏T淋巴细胞数最于染毒后第5～8天下降(第6天为最低值),然后均逐渐恢复到正常水平.染毒后血液细胞因子变化表现为:血清IFN-γ、TNF-α水平下降,IL-4、IL-18、IL-10水平上升,IL-2水平无明显变化.从感染第7天开始检测H5N1禽流感特异性抗体为阳性,抗体水平逐渐升高至实验结束的感染第14天.结论 H5N1禽流感病毒感染可引起宿主T细胞免疫功能低下是其主要的免疫病理改变之一,细胞因子表达失平衡或过多的表达都可能对宿主产生免疫病理损伤.

Abstract：

Objective To study the cell immunity and eytokines responses to avian influenza A H5N1 virus infections in a BALB/c model to better understand the pathogenesis of H5N1 avian influenza disease. Methods Two hundred and twenty BALB/c mice of the infected group were inoculated with 0.1 ml (10-4.875 TCID50) of A/Goose/Guangdong/NH/2003 ( H5N1 ) virus intra-nasally. Fifty control mice received noninfectious allantoic fluid and another fifty control mice received normal sodium. Blood and spleen samples were collected from the live mice every 24 h during the 14 d post-infection. The changes of CD3 + T cells , CD4 + T cells, CD8 + T cells for cell immunity in blood circulation and spleen were detected by flow cytometry. And the cytokines and antibody responses in blood circulation were detected by ELISA. Necropsy was performed on mice that died during the experiment and those euthanized at end of study. Results Avian influenza A( H5N1) virus infections can make damages to the cell immune system transiently. The CD3 + T cells, CD4 + T cells, CDS + T cells declined at 24 days post infection in blood circulation and declined at 5-8 days in spleen, then recovered to the normal level gradually. The eytokines responses to the infections can be detected: the level of IFN-γ,TNF-α declined, IL-4, IL-18, IL-10 increased, and IL-2 changed little. The antibody increased rapidly from day 7 post infection until the end of the study (day 14 post infection). Conclusion Collectively, avian influenza A(H5N1) virus can cause cell immunity deficiency and an imbalance in the level of eytokines, which may contribute to the unusual severity of disease caused by the H5N1 avian influenza virus.     

IFN-α治疗慢性乙型肝炎疗效与记忆性T细胞的相关性

目的 研究IFN-α在治疗慢性乙型肝炎患者中对记忆性T细胞亚群变化的影响,及其临床治疗效果与记忆性T细胞亚群变化的相关性.方法 57例接受IFN-α治疗的慢性乙型肝炎(CHB)患者,分别在治疗前(O周),及疗程期内不同时间点(12周和24周)抽取外周血,分离外周血单个核细胞(PBMCs)后加入抗体染色,流式细胞仪检测,统计学分析.结果 非活动性HBsAg携带者CD8效应记忆性T细胞(CD8+TEM)的比例显著高于未经IFN-α治疗的CHB患者;相反,CD8中心记忆性T细胞(CD8+TCM)的百分比则显著低于未经IFN-α治疗的CHB患者(P<0.05);有效组CD8+TEM百分比在IFN-α治疗前和治疗24周时均高于无效组,CD8+TCM百分比在IFN-α治疗前和治疗24周时则低于无效组(P<0.05);有效组IFN-α的剂量明显高于无效组.结论 病毒的清除与效应记忆性T细胞亚群比例可能有关,TEM亚群在数量上占优势可能对IFN-α治疗效果有益.

Abstract：

Objective To investigate the influence of interferon-a therapy on CD8 T memory subsets in patients with chronic hepatitis B(CHB) and correlation between the effect of IFN-α and CD8 T memory subsets. Methods Blood samples from 57 patients with CHB were collected before treatment (0 week), at 12 weeks and 24 weeks of treatment with pegylated IFN-α. Assays were performed on freshly isolated peripheral blood mononuclear cells ( PBMCs). For phenotype analysis, All data were acquired on a flow cytometer instrument and prepared for analysis. Results A significantly higher frequency of CD8+ TEM and lower frequency of CD8+ TCM in inactive HBsAg carriers than that in CHB patients prior to treatment was observed (P <0.05). The proportion of CD8 + TCM was higher in group nonresponders than in group respond-ers, and the proportion of CD8 + TEM was lower in group nonresponders than in group responders (P < 0.05 ). The average dosage of IFN-α applied to patients with response was significantly higher than nonresponders. Conclusion The dominance of circulating effector memory T cells may be associated with elimination of viral infection, and possibly benefit for response to therapy with IFN-α.     

Effects of lysedEnterococcus faecalis FK-23 on experimental allergic rhinitis in a murine model

In the current study,we sought to investigate whether lysed Enterococcus faecalis FK-23(LFK),a heat-killed probiotic preparation,attenuated eosinophil influx into the upper airway and had immunomodulatory activity in a murine allergic rhinitis model.Eighteen BALB/c mice were divided into three groups;the ovalbumin(OVA)-sensitized/challenged group,which received saline orally for 6 weeks(OVA group),the OVA-sensitized/challenged group,which received LFK orally for 6 weeks(LFK-fed group),and the non-sensitized group,which received saline for 6 weeks(saline control group).Nasal rubbing and sneezing were monitored during the study.After the final challenge,interleukin(IL)-4,interferon(IFN)-γ,and OVA-specific IgE levels in the sera and splenocyte culture supernatants were determined,eosinophilic infiltrate into the upper airway was quantified,and splenic CD4+CD25+ regulatory T cells(Tregs) were examined by flow cytometry.We found that nasal rubbing was significantly reduced in LFK-fed mice compared to the OVA group on d 27 and 35,and sneezing was significantly inhibited by LFK administration for 35 d.LFK-fed mice had significantly less eosinophil influx into the nasal mucosa than the OVA group.There were no significant differences between the LFK-fed group and OVA group in the serum and splenocyte culture supernatant levels of IL-4,IFN-γ,and OVA-specific IgE.Interestingly,the LFK-fed mice had a significantly greater percentage of splenic CD4+CD25+ Tregs than OVA group.Our results indicate that oral administration of LFK may alleviate nasal symptoms,reduce nasal eosinophilia,and increase the percentage of CD4+CD25+ Tregs in experimental allergic rhinitis.     

Influence of interferon-α therapy on CD8 T memory subsets in patients with chronic hepatitis B

Objective To investigate the influence of interferon-a therapy on CD8 T memory subsets in patients with chronic hepatitis B(CHB) and correlation between the effect of IFN-α and CD8 T memory subsets. Methods Blood samples from 57 patients with CHB were collected before treatment (0 week), at 12 weeks and 24 weeks of treatment with pegylated IFN-α. Assays were performed on freshly isolated peripheral blood mononuclear cells ( PBMCs). For phenotype analysis, All data were acquired on a flow cytometer instrument and prepared for analysis. Results A significantly higher frequency of CD8+ TEM and lower frequency of CD8+ TCM in inactive HBsAg carriers than that in CHB patients prior to treatment was observed (P <0.05). The proportion of CD8 + TCM was higher in group nonresponders than in group respond-ers, and the proportion of CD8 + TEM was lower in group nonresponders than in group responders (P < 0.05 ). The average dosage of IFN-α applied to patients with response was significantly higher than nonresponders. Conclusion The dominance of circulating effector memory T cells may be associated with elimination of viral infection, and possibly benefit for response to therapy with IFN-α.     

Influence of interferon-α therapy on CD8 T memory subsets in patients with chronic hepatitis B

Objective To investigate the influence of interferon-a therapy on CD8 T memory subsets in patients with chronic hepatitis B(CHB) and correlation between the effect of IFN-α and CD8 T memory subsets. Methods Blood samples from 57 patients with CHB were collected before treatment (0 week), at 12 weeks and 24 weeks of treatment with pegylated IFN-α. Assays were performed on freshly isolated peripheral blood mononuclear cells ( PBMCs). For phenotype analysis, All data were acquired on a flow cytometer instrument and prepared for analysis. Results A significantly higher frequency of CD8+ TEM and lower frequency of CD8+ TCM in inactive HBsAg carriers than that in CHB patients prior to treatment was observed (P <0.05). The proportion of CD8 + TCM was higher in group nonresponders than in group respond-ers, and the proportion of CD8 + TEM was lower in group nonresponders than in group responders (P < 0.05 ). The average dosage of IFN-α applied to patients with response was significantly higher than nonresponders. Conclusion The dominance of circulating effector memory T cells may be associated with elimination of viral infection, and possibly benefit for response to therapy with IFN-α.     

Increased serum IL-10 in lupus patients promotes apoptosis of T cell subsets via the caspase 8 pathway initiated by Fas signaling

We sought to investigate the expression of Fas and FasL on T cell surface and caspase 8 involvement in T cell apoptosis promoted by serum IL-10 in systemic lupus erythematosus(SLE) patients.Cells and sera were obtained from 35 SLE patients.Apoptosis of T cells in patients with SLE was increased and associated with the SLE disease activity index(SLEDAI).Elevated expression of Fas and FasL on T cell surface contributed to increased apoptosis of T cells.Increased IL-10 in the sera of SLE patients was capable of inducing Fas and FasL expression on CD4~+T cell surface,promoting apoptosis of this cell subset.Decreased IL-10 serum levels and low expression of Fas were found in 5 patients of the first follow-up group after 2-month treatment.In another group with one-year treatment,the SLEDAI declined to inactive scores.Serum IL-10 was decreased significantly,and expression of Fas and FasL on T cells was also reduced.Declined apoptosis was predominant only in CD4~+T cell subset.When sera with high level of IL-10 were used to culture PBMCs from healthy controls,activated caspase 8 was elevated in CD3~+T,CD4~+T and CD8~+T cells.The study showed that serum IL-10 induced apoptosis of T cell subsets via the caspase8 pathway initiated by Fas signaling.Increased apoptosis of T cells contributes to autoantigen burden,which is pathogenic in the development of SLE.     

Effects of the Overall Alkaloid of a Traditional Chinese Medicine “Tongbiling” on the Cytokine Expression in Thl and Th2 Cells of Rheumatoid Arthritis and Their Signaling Pathway

To investigate the effects of overall alkali of a traditional Chinese medicine “Tongbiling” (brucine and strychnine alkaloids in main) on the cytokines expression in Th1 and Th2 cells in the synovial fluid of patients with rheumatism arthritis and their signal pathway, the mononuclear cells in the synovial fluid (SFMC) of patients were isolated by Ficoll-Hypaque gradient centrifugation, and the CD3^+ CD69^+ and CD3^+ HLA-DR antigen were analyzed by flow cytometry in comparison with those of the peripheral blood. The rest of cells were cultured after resuspension with RPMI 1640 culture medium. Phorbol 12, 13-dibutyrate (PDB) and ionomycin were added successively into the culture with various concentration of overall alkali Tongbiling (TBL). After 4 h of cultivation, the expression of IFN-γ and IL-4 in CD3^+ cells were analyzed by flow cytometry. The influence of overall alkali TBL ( 100 mg/I,) on the intracellular calcium was investigated after Fluo-3/AM labeling and stimulation with PDB and ionomycin at 1, 2, 4 and 10 min, and the influence of TBL on the expression of CD3^+ CD69^+ cells were determined with stimulation of PDB for 24 h in the whole blood lymphocytes culture. It was found that the percentage of T cells bearing CD69 was significantly up-regulated (77%), while that of T cells bearing HLA-DR was 44% in the synovial mononucleated cells. After PDB and ionomycin stimulation, the expression of IFN-7 in CD3 ~ cells were up-regulated, but there was no change on the expression of IL-4 in CD3^+ cells, indicating that ratio of Th1/Th2 was significantly increased and Th cells differentiate to Thl cells in mainly. Four concentrations of overall alkaloid of TBI, (200 mg/L, 100 mg/L, 50 mg/L, 25 mg/L) could down-regulated the expression of IFN-γ in CD3^+ cells and the Th1/Th2 ratio obviously, but all the concentrations of the overall alkaloids had no effect on the expression of IL-4 in CD3^+ cells. 100 mg/L concentration of the overall alkaloid did not down-regulate the intracellular calcium level. Each concentration of the overall alkaloid could down-regulated the expression CD69 obviously on the PDB-activated mouse T cells. It concluded from the above observations that the overall alkaloid of TBL could relieve the inflammatory and immune damages by suppressing the expression of Thl type cytokines and Th1 cell differen-tiation, regulating the imbalance of Th1/Th2 cells and inhibiting the early activation of the T lymphocytes bearing CD69. There was no remarkable influence on the intracellular calcium signaling transduction pathway. The inhibitory effected on T cells to express 1FN-γ might be due to the suppression of PKC-MAPK signaling pathway. From the standpoint of traditional Chinese medicine, this might be due to the regulation of “Yin” and “Yang” imbalance of joints to modify the pathological status in rheumatoid arthritis. This study provided an experimental basis for the application of overall alkaloids of TBL in the treatment of rheumatoid arthritis.     

I L- 15 trans-presentation regulates homeostasis of CD4^＋ T lymphocytes

Interleukin-15 （IL-15） is essential for the survival of memory CD8^＋ and CD4^＋ T cell subsets, and natural killer and natural killer T cells. Here, we describe a hitherto unreported role of IL-15 in regulating homoeostasis of naive CD4^＋ T cells. Adoptive transfer of splenocytes from non-obese diabetic （NOD） mice results in increased homeostatic expansion of T cells in lymphopenic NOD.scid.II15^-/- mice when compared to NOD.scid recipients. The increased accumulation of CD4^＋ T cells is also observed in NOD.II15^-/- mice, indicating that IL-15-dependent regulation also occurs in the absence of lymphopenia. NOD.scid mice lacking the I L- 15Ra chain, but not those lacking the common gamma chain, also show increased accumulation of CD4^＋ T cells. These findings indicate that the IL-15-mediated regulation occurs directly on CD4^＋ T cells and requires trans-presentation of IL-15. CD4^＋ T cells expanding in the absence of IL-15 signaling do not acquire the characteristics of classical regulatory T cells. Rather, CD4^＋ T cells expanding in the absence of IL-15 show impaired antigen-induced activation and IFN-7 production. Based on these findings, we propose that the IL-15-dependent regulation of the naive CD4^＋ T-cell compartment may represent an additional layer of control to thwart potentially autoreactive cells that escape central tolerance, while permitting the expansion of memory T cells.     

Risk Factors and Frequency of Tuberculosis-associated Immune Reconstitution Inflammatory Syndrome among HIV/Tuberculosis Co-infected Patients in Southern India

Immune reconstitution inflammatory syndrome (IRIS) continues to be a complication in HIV/tuberculosis (TB) co-infected patients initiating highly active antiretroviral therapy (HAART). The aim of this study was to evaluate the risk factors associated with developing IRIS to identify a possible biomarker to predict or diagnose IRIS in patients initiating HAART. A total of 175 HIV/TB co-infected patients initiating HAART were followed up longitudinally during September 2010 to May 2013 attending a HIV care clinic in Chennai. Patients were followed up longitudinally after HAART initiation and baseline demographic, laboratory parameters and treatment characteristics between patients with IRIS events and those without IRIS events were compared. Chi-square or Fisher’s exact test for categorical variables and a Wilcoxon rank-sum test for continuous variables were performed using SPSS, version 12.0 software. Patients with IRIS had a significantly lower median baseline CD4+ T-cell count (P = 0.0039). There were no differences in terms of sex, CD4 T-cell %, plasma viral load, time interval between initiating ATT and HAART between the IRIS and non-IRIS patients. Low CD4+ T-cell count (<100 cells/μL) could be used as a marker to screen and monitor patients initiating HAART.     

Distinctive in vitro effects of T-cell growth cytokines on cytomegalovirus-stimulated T-cell responses of HIV-infected HAART recipients

Functional immune reconstitution is limited after HAART, maintaining the interest in adjunctive immune-modulators. We compared in vitro the effects of the gamma-chain T-cell growth cytokines IL-2, IL-4, IL-7 and IL-15 on cytomegalovirus-stimulated cell-mediated immunity. IL-2 and IL-15 increased cytomegalovirus-specific lymphocyte proliferation in HAART recipients, whereas IL-4 and IL-7 did not. The boosting effect of IL-2 and IL-15 on proliferation correlated with their ability to prevent late apoptosis. However, IL-2 increased the frequency of cells in early apoptosis, whereas IL-15 increased the frequency of fully viable cells. Both IL-2 and IL-15 increased cytomegalovirus-induced CD4+ and CD8+ T-cell proliferation and the synthesis of Th1 and pro-inflammatory cytokines and chemokines. However, only IL-2 increased the frequency of regulatory T cells and Th2 cytokine production, both of which have the potential to attenuate antiviral immune responses. Overall, compared to other gamma-chain cytokines, IL-15 had the most favorable profile for boosting antiviral cell-mediated immunity.     

Cd38 expression on Cd8+ T cells in Human immunodeficiency virus 1-positive adults treated with HAART

The aim of this study was to assess whether the density of CD38 antigen expression on CD8+ T cells can be used as a marker of activation of the immune system in Human immunodeficiency virus 1 (HIV-1)-positive patients treated with highly active antiretroviral therapy (HAART). T cell subsets, expression of CD38 antigen on CD8+T cells, HIV-1 viral load and stage of the disease were analyzed at baseline and after 12 months of HAART in 24 HIV-1-infected patients. Our data showed that the use of HAART is effective in reducing plasma viral load and in achieving a stable CD4+ count and percentage of CD8+/CD38+ cells. The percentages of CD8/CD38+ cells in HIV-1-infected patients at baseline and after 12 months of HAART were significantly higher than those of controls. Analysis of the density of CD38 expression revealed that it was due to CD8+/CD38+ subsets with low and medium density of antigen expression. Absolute number of CD4+ T cells correlated negatively with the percentage of CD8+/CD38+ cells at baseline of the study. Persistent up-regulation of the CD38 expression on CD8+ T cells and its correlation with the decreased CD4+ count despite the reduction of plasma viral load may reflect residual replication of HIV-1 in reservoirs. Thus, this immunological parameter can serve as a biological marker of HIV-1 infection and might have utility in clinical management of HIV-1-infected persons.     

Relationship of in vivo and ex vivo levels of TH1 and TH2 cytokines with viremia in HAART patients with and without opportunistic infections

TH1/TH2 cytokines' imbalance is critical to HIV-1 progression and pathogenesis. Opportunistic infections-related cytokine perturbations in the setting of highly active antiretroviral therapy (HAART) are unclear. The objective of this cross-sectional study was to identify the relationship between TH1/TH2 cytokines and viremia in HAART patients with/without opportunistic infections. Sera from 17 HAART patients with and 43 without opportunistic infections, and 20 HIV-seronegative controls were used to measure the levels of IL-2, IFN-gamma, IL-4, and IL-10 proteins and mRNAs by ELISA and RNase protection assays, respectively. Ex vivo cytokine production by the CD4+/CD8+ T cells from four low and four high viremia patients randomly selected from non-opportunistic infection group was also evaluated. Serum IL-2 and IFN-gamma levels were lower (P < 0.05) in patients than controls; this reduction was more pronounced for IFN-gamma in non-opportunistic infection patients. IL-4 and IL-10 were higher in patients than controls; this elevation was more remarkable in patients with opportunistic infections. Serum TH1/TH2 cytokine levels correlated with viremia. In vitro cytokine production assays showed that CD4+ T cells from low viremia patients mainly produced IL-2 and IFN-gamma, CD8+ T cells from high viremia patients produced IL-4, and both subsets comparably produced IL-10 in patients with similar viremia. Positive correlations between sera/supernatant proteins and cellular mRNAs were also found statistically significant (P < 0.05). It was therefore concluded that in vivo TH1/TH2 cytokine levels in HAART patients and their ex vivo production by the CD4+/CD8+ T cells correlated with viremia and were also modulated by the presence of opportunistic infections in these patients.     

Role of common gamma chain utilizing cytokines for immune reconstitution in HIV infection

Many cytokines that utilize the common gamma (Cγ) chain signaling pathway, viz Interleukin (IL)-2, IL-15, and IL-7 are known to be important for inducing T cell maturation, proliferation, or survival. Untreated chronic HIV infection is associated with profound quantitative and qualitative deficiency of CD4 T cells, which is partially reversed following highly active antiretroviral therapy (HAART). A subset of patients, however, fail to recover CD4 T cells despite virologic suppression. The role of Cγ chain cytokines in influencing immune reconstitution following potent antiretroviral therapy is discussed. Maturation markers (naïve, central memory, effector memory, and effector), cytokine receptors IL-2Rβ, Cγ chain, IL-7Rα, IL-15Rα, and cytokine-induced proliferative responses of T cells in a cohort of HIV-infected pediatric patients and adults classified on the basis of immunologic and virologic response to antiretroviral therapy were examined. The studies indicated that patients had increased percentages of effector memory CD8+ T cells in comparison to healthy volunteers. While patients with partially controlled viremia and poor CD4 T cell reconstitution manifested poor proliferative responses to anti-CD3 or HIV gag antigen stimulation, proliferative responses to Cγ chain utilizing cytokines IL-2, IL-7, and IL-15 were robust. Another Cγ chain utilizing cytokine, IL-21 had no influence on cellular proliferation but enhanced perforin expression in effector CD8 T cells. Thus, cytokine receptor deficiencies may contribute to immune deficiency in HIV-infected patients, and Cγ chain cytokines may play an important role in vivo in immune homeostasis in lymphopenic patients by maintaining the memory subsets of T cells in patients with CD4 T cell deficiency.     

Normalization of immune activation in lymphoid tissue following highly active antiretroviral therapy

Although significant progress has been made in understanding immune reconstitution in peripheral blood following highly active antiretroviral therapy (HAART), less is known about immune changes in lymphoid tissue. Here, the expression of cytokine proteins (interferon gamma [IFN-gamma], interleukin [IL]-2, IL-4, IL-10, IL-1alpha, and IL-1beta) and surface antigens (CD4, CD8, CD1a, CD68) as well as cellular proviral HIV-1 DNA were determined in sequential tonsil biopsies before and at 4, 12, and 48 to 56 weeks posttherapy by quantitative in situ image analysis and fluorescent in situ 5;-nuclease assay (FISNA). Despite plasma virus suppression, a fraction of tonsil cells harbored pro-viral DNA for up to 1 year. A fourfold to eightfold increase in CD8+ T cells in tissue compared with seronegative controls and an increased frequency of CD1a+ dendritic cells prior to HAART reached control levels at week 56. The frequency of IFN-gamma expressing cells was 10-to 15-fold higher than controls before therapy and was comparable with findings in seronegative controls by week 56. Elevated baseline expression of IL-1alpha and IL-1beta was reduced by week 4 but IL-1alpha levels remained elevated in 1 of 3 patients at week 56. These findings suggest that with effective viral suppression the immune system in tissue may return to a more resting state.     

Immune function and phenotype before and after highly active antiretroviral therapy.

Immune functions represented by equal CD4 counts before and after highly active antiretroviral therapy (i.e., pre- and post-HAART) in the same HIV-infected patients, were examined. Twelve HIV-infected patients were included. Patients had equal CD4 counts pre- and post-HAART and were studied on average 30 months pre-HAART and 17 months post-HAART. Post-HAART, CD8+ T cells expressed greater amounts of CD28 (p < .02), smaller amounts of CD38 (p < .02), and a reduced proportion of CD4+CD28+ T cells expressed CD38+ (p < .01). Proliferation increased (p < 10) in lymphocyte cell cultures stimulated with pokeweed mitogens or Candida, and was correlated to expression of CD28 on T cells (p < .02). The proportion of CD3-CD16-CD56+ natural killer (NK) cells increased (p < .05) and CD3-CD16+CD56- NK cells declined (p < .01). Production of interferon-gamma increased (p < .10). The number of naive and memory T cells, the non-major histocompatibility complex (non-MHC)-restricted and HIV-specific MHC-restricted cytotoxicity and the production of macrophage inflammatory protein-1gamma were unchanged. The finding of increased expression of CD28, correlating to increased proliferation capacity, and diminished expression of CD38 on T cells, indicates that following long-term HAART, repopulation occurs with less activated cells with increased proliferative capacity. This finding may be of clinical importance in considering risk and vulnerability for progression of opportunistic infections post-HAART.     

Influence of CD4+ T cell counts on viral evolution in HIV-infected individuals undergoing suppressive HAART

We analyzed the viral C2-V4 envelope diversity, glycosylation patterns, and dS/dN ratios of plasma HIV-1 in an attempt to better understand the complex interaction between viral quasispecies and the host-selective pressures pre- and post-HAART. Phylogenetic analysis of the envelope gene of five patients revealed monophyletic clustering in patients with higher CD4+ T cell counts and sequence intermingling in those with lower CD4+ T cells in relation to the stage of HAART. Our analyses also showed clear shifts in N-linked glycosylation patterns in patients with higher CD4+ T cells, suggesting possible distinct immunological pressures pre- and post-HAART. The relative preponderance of synonymous/nonsynonymous changes in the envelope region suggested a positive selection in patients with higher CD4+ T cells, whereas lack of evidence for positive selection was found in the patients with lower CD4+ T cells. An exception to the last analysis occurred in the only patient who reached complete viral suppression, maybe due to drug pressure exerted over the pol gene that may obscure the immune pressure/selection at the envelope in this analysis. All these indications may suggest that even when HAART generates viral suppression, quasispecies evolve in the envelope gene probably resulting from host-selective pressure.     

Interleukin-18 regulates T helper 1 or 2 immune responses of human cord blood CD4+ V alpha 24+V beta 11+ natural killer T cells

Natural killer T (NKT) cells, exhibiting both T-cell and NK-cell markers, are known to regulate immune responses by secreting T-helper (Th) 1 and Th2 cytokines. We analyzed NKT cells in cord blood (CB) for phenotypical and functional characteristics and regulatory mechanisms that control Th1 and Th2 determination. Human CB V alpha 24+V beta 11+ NKT cells were predominantly the CD4+ single positive (SP) phenotype (approximately 96%), in contrast to adult peripheral blood V alpha 24+V beta 11+ NKT cells which are composed of a dominant population of the CD4-CD8-double negative (DN) phenotype and a minor population of the CD4+ SP phenotype. The CB CD4+ V alpha 24+ NKT cells, following stimulation with the primary culture, gained the capacity to secrete interferon (IFN)-gamma, a Th1 cytokine, and interleukin (IL)-13, a Th2 cytokine. The combination of IL-18 and IL-12 induced IFN-gamma production in CB CD4+ V alpha 24+ NKT cells, while IL-18 in combination with IL-2 induced IL-13 production in these cells. Thus, IL-18 regulates the determination of the Th1 or Th2 immune response by human CD4+ V alpha 24+ NKT cells through different cytokine combinations.