Different cytokine profiles of peripheral blood mononuclear cells from patients with persistent and self-limited hepatitis C virus infection

An imbalance between T helper cell Th1 and Th2 like cytokines has been described in several chronic infectious diseases. In an attempt to characterise the mechanism responsible for viral persistence in hepatitis C virus (HCV)-related chronic infection, we analyzed Th1 cytokines (IL-2, IL-12, IFN-gamma) and Th2 cytokines (IL-4, IL-10) production by phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) derived from ten patients with viremic chronic hepatitis C, five healthy HCV seropositive individuals and four HCV seronegative individuals. Cytokine production was determined by enzyme-linked immunosorbent assay (ELISA) after 72 h of stimulation. The results showed that the production of IFN-gamma by PHA-stimulated PBMC was decreased in patients with hepatitis C infection (P=0.05). IL-4 production was not detected in both patients and controls, while no difference was observed for IL-2, IL-10 and IL-12 production between patients and controls. Furthermore, IL-12 and IFN-gamma production was weaker in patients with viremic chronic hepatitis C than in subjects who were able to clear the virus (P=0.01; P=0.03, respectively). These results clearly indicate that a defect both in IL-12 and IFN-gamma production may contribute to the persistence of HCV infection.     

Cytokine mRNA levels in antigen-stimulated peripheral blood mononuclear cells

Levels of cytokine mRNA coding for granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-gamma (IFN-gamma), interleukin (IL) 2, IL 1 beta, IL 4 or IL 6 have been measured by Northern blot analysis after antigen stimulation. As source for RNA we used peripheral blood mononuclear cells (PBMC) from donors which showed a proliferative response after tetanus toxoid or Candida albicans stimulation. For comparison PBMC were also stimulated with lectins and anti-CD3 antibody. With some variations among donors, antigens clearly induced measurable levels of IFN-gamma, GM-CSF and IL 2 mRNA. Increased levels for IL 6 were also detected after antigen stimulation. In contrast to polyclonal T cell stimuli, antigens showed delayed kinetics of mRNA steady-state levels and resembled in this respect more closely the stimulation with pokeweed mitogen. Thus, cytokine mRNA levels may be assessed in unfractionated PBMC after antigen stimulation. The two tested antigens also clearly show a cytokine pattern distinct from that induced in polyclonal stimulations such as anti-CD3.     

慢性乙型肝炎患者树突状细胞表型的研究

目的探讨慢性乙型肝炎患者外周血来源树突状细胞（Dendritic cell，DC）数量及表型的改变，并对其与肝功能、乙肝病毒复制水平的关系进行研究。方法检测37例慢性乙肝患者和21例健康人肝功能及血清HBV DNA水平，并提取外周血单个核细胞（Peripheral blood mononuclear cell，PBMC）进行体外诱导培养，促使其发育成DC，计数其数量并检测膜表面分子的变化。分析DC数量及表型与肝功能、乙肝病毒复制水平的关系。结果与正常对照组比较，慢性乙肝患者的树突状细胞数量明显减少（P〈0．05），且其膜表面分子CD83、CD86的表达均明显降低（P〈0．05）。在慢性乙肝患者中，DC数量、DC膜表面分子CD83和CD86与血清HBV DNA之间呈负相关关系，而与肝功能之间无明显相关关系。结论慢性乙肝患者体内存在DC数量减少及成熟障碍，这种改变与肝内炎症反应程度不相关，但与乙肝病毒（Hepatitis Bvirus，HBV）的复制水平呈负相关，提示DC参与慢性乙肝患者体内HBV的清除。     

Cytokine profiles in peripheral blood mononuclear cells and lymph node cells from piglets infected in utero with porcine reproductive and respiratory syndrome virus

The aim of the present study was to investigate at 2, 4, and 6 weeks after birth cytokine expression by peripheral blood mononuclear cells and bronchial lymph node cells from piglets infected in utero with porcine reproductive and respiratory syndrome virus (PRRSV). Technically, by flow cytometry we were able to measure gamma interferon (gamma-IFN), tumor necrosis factor alpha (TNF-alpha), interleukin-4 (IL-4), and IL-8 levels. In general, we found increases in the percentages of IL-4-, gamma-IFN-, and TNF-alpha-producing lymphocytes in the infected piglets compared to the percentages in the uninfected control animals, while there was a decrease in the percentage of IL-8-producing monocytes. We believe that these findings reflect a general lymphocyte activation stage that is created due to the infection and that occurs in combination with impairment of the monocyte function, possibly due to the ongoing viral replication in these cells. Single-cell bronchial lymph node preparations exhibited very much the same cytokine profiles as peripheral blood mononuclear cells except for a lack of IL-8 production. When the levels of the individual cytokines in the three groups of PRRSV-infected piglets were compared, the levels of cytokine expression at 4 weeks diverged from those at 2 and 6 weeks, in that there was a significant decrease in the numbers of lymphocytes producing gamma-IFN and TNF-alpha. This tendency was also observed among blood monocytes and lymph node macrophages. Possible reasons for this temporary immunosuppression in the piglets at 4 weeks are discussed.     

Cytokine mRNA profiles in mononuclear cells in acute aseptic meningoencephalitis.

Cytokines are important modulators of inflammation and immune responses. Using in situ hybridization with radiolabelled cDNA oligonucleotide probes, we studied the expression of mRNA encoding the cytokines gamma interferon (IFN-gamma), interleukin 4 (IL-4), IL-6, IL-10, transforming growth factor beta (TGF-beta), tumor necrosis factor alpha (TNF-alpha), lymphotoxin, and perforin in mononuclear cells (MNC) from blood and cerebrospinal fluid (CSF) of patients with acute aseptic meningoencephalitis (AM) and from blood of healthy controls. Patients in the acute phase of AM had elevated numbers of IFN-gamma mRNA-expressing cells in the blood compared with that of controls and higher numbers of IFN-gamma mRNA-expressing cells in their CSF compared with that of convalescent-phase patients, which is in accordance with the antiviral effects of this cytokine. Upregulation of IL-4, IL-6, and IL-10 was found in convalescent-phase patients, which is consistent with the longstanding B-cell response found in AM. TGF-beta and perforin were upregulated in both stages of AM, while the numbers of blood and CSF MNC expressing cytokine mRNA of the TNF family (TNF-alpha and lymphotoxin) did not differ between patients with AM and controls. An even higher elevation in CSF was noticed for MNC expressing most of the cytokines, particularly IL-4 and TGF-beta, reflecting the autonomy of the immune response in the CSF. The definition of cytokine profiles in AM, a self-limiting and benign disease, provides a foundation for future comparisons with other infectious and inflammatory nervous system diseases.     

Cytokine gene expression in peripheral blood mononuclear cells (PBMC) from patients with pharmacologically treated cystic echinococcosis

The aim of the study was to assess the role of the complement system in Staphylococcus aureus arthritis and septicaemia. The murine model of haematogenously acquired septic arthritis was used, injecting intravenously toxic shock syndrome toxin-1 (TSST-1), producing S. aureus LS-1. Complement was depleted using cobra venom factor (CVF). Evaluation of arthritis was performed clinically and histopathologically. In addition, the effect of complement depletion on the phagocytic activity of leucocytes was assessed in vivo and in vitro. Six days after inoculation of S. aureus the prevalence of arthritis in decomplemented mice was three-fold higher than that in controls (91% versus 25%). The clinical severity of arthritis at the end of the experiment, expressed as arthritic index, was 7.3 and 1.9, respectively. These findings were confirmed by histological index of synovitis as well as of cartilage and/or bone destruction being significantly higher in decomplemented mice than in controls (9.8 ± 1.7 versus 4.9 ± 1.2, P < 0.05; and 7.9 ± 1.7 versus 3.0 ± 0.9, P < 0.05, respectively). Also, the septicaemia-induced mortality was clearly higher in decomplemented mice compared with the controls. CVF treatment significantly reduced in vivo polymorphonuclear cell-dependent inflammation induced by subcutaneous injection of olive oil and mirroring the capacity of polymorphonuclear cells (PMNC) to migrate and/or extravasate. Besides, the decomplementation procedure significantly impaired phagocytic activity of peripheral blood leucocytes in vitro, since the number of phagocytes being able to ingest bacteria decreased by 50% when the cells were maintained in decomplemented serum compared with those in intact serum. The conclusion is that complement depletion aggravates the clinical course of S. aureus arthritis and septicaemia, possibly by a combination of decreased migration/extravasation of PMNC and an impairment of phagocytosis.     

Anti-HBV effects of CpG oligodeoxynucleotide-activated peripheral blood mononuclear cells from patients with chronic hepatitis B

Unmethylated CpG dinucleotides in bacterial DNA or synthetic oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG ODN) are known as a potent Th1-like immune enhancer in vertebrates. Chronic hepatitis B is the immunocompromising condition. We therefore investigated the effects of CpG ODN on cultured cells from chronic hepatitis B patients and healthy controls. The inhibitory effects of CpG ODN on hepatitis B virus (HBV) were also studied. The secretion of IFN-alpha by CpG ODN-activated peripheral blood mononuclear cells (PBMCs) from chronic hepatitis B patients and healthy controls was significantly increased when compared with PBMCs alone or GpC ODN-stimulated PBMCs. After activation with CpG ODN, the IFN-alpha secretion by chronically HBV-infected patient PBMCs is less than that by healthy control PBMCs. Treatment of HepG2 2.2.15 cells with culture supernatants of PBMCs activated by CpG ODN can significantly suppress the secretion of HBsAg, HBeAg and HBV DNA as compared with that of PBMCs without CpG ODN activation under the same conditions. No inhibitory effect on the replication of HBV was found for CpG ODN treatment alone. Our results indicated that CpG ODN could efficiently enhance the immune response of chronic hepatitis B patients. Moreover, the CpG ODN-activated PBMCs from chronic hepatitis B patients were able to significantly inhibit HBV replication in vitro, suggesting that CpG ODN may be a potential immunoregulator against HBV infection in the future.     

Correlation of AIM2 expression in peripheral blood mononuclear cells from humans with acute and chronic hepatitis B

The AIM2 (absent in melanoma 2) protein promotes host defenses against invading viruses and pathogenic bacteria through corresponding adapter molecules leading to the initiation of innate immune responses. We investigated the expression of AIM2 in peripheral blood mononuclear cells (PBMCs) from patients with acute hepatitis B (AHB) and chronic hepatitis B (CHB) during different clinical phases, and analyzed the correlation between AIM2 and clinical profiles in these groups. This study indicated that there is higher expression of AIM2, IL-1β, and IL-18 in AHB compared with expression in CHB. The expression of AIM2 mRNA was significantly negatively correlated with serum hepatitis B virus (HBV) load, HBeAg, and significantly positively correlated with IL-1β and IL-18 in AHB patients and CHB patients with immune clearance, which suggests that AIM2 expression is correlated with the immune clearance of HBV in the host. We summarized that there is a higher immune status in AHB, and a lower immune response in CHB. This suggests that the down-regulation of AIM2 may be associated with the chronic development of HB.     

外周血单个核细胞中乙型肝炎病毒核酸与细胞因子含量变化

目的：探讨慢性乙型肝炎患者外周血单个核细胞中乙型肝炎病毒核酸与血清细胞因子变化关系。方法：采用荧光实时PCR技术定量检测76例慢性乙型肝炎患者和15例健康者外周血单个核细胞中HBVDNA含量，用ELISA法定量检测血浆细胞因子(IFN-γ、TNF—α、IL-4、IL-6、TGF-β1、sIL-2R)水平。结果：①慢性乙型病毒性肝炎中细胞因子(IFN-γ、TNF-α、IL-4、IL-6、TGF-β1、sIL-2R)水平明显高于健康对照组；②单个核细胞HBVDNA阳性组IFN-γ、TNF-α含量明显低于阴性组，而sIL-2R含量则高于阴性组但无统计学差异，IL-4、IL-6、TGF-β1含量无差异；③随着PBMCs中HBVDNA含量升高，TNF—α含量逐渐降低，IL-4和sIL-2R含量逐渐升高，而IFN-γ，IL-6、TGF-β1含量无显著性变化。结论：外周血单个核细胞HBVDNA持续存在及含量变化与细胞因子相对异常有关，进而导致肝细胞损伤。     

Cytokine production by human milk cells and peripheral blood mononuclear cells from the same mothers

Samples of milk (n = 80) and venous blood were collected at 5 weeks postpartum from 82 lactating mothers. Human milk cells and peripheral blood mononuclear cells were isolated and the production of interleukin-1, interleukin-6, and tumor necrosis factor- in the absence and presence of lipopolysaccharide was determined by enzyme-linked immunosorbent assay. Human milk cells spontaneously produced significantly less interleukin-1, interleukin-6, and tumor necrosis factor- than peripheral blood mononuclear cells in the absence of stimulation. In vitro stimulation of human milk cells with lipopolysaccharide (500 ng/ml) for 24 hr increased cytokine production by approximately 40–50%, whereas peripheral blood mononuclear cells responded to lipopolysaccharide (200 ng/ml) with increased cytokine production of up to 350%. These observations suggest that cells in milk are capable of active involvement in the production of the interleukin-1, interleukin-6, and tumor necrosis factor- in the mammary gland and have the capacity to respond to further stimulation after leaving the breast.     

Cytokine production by peripheral blood mononuclear cells following birch-pollen immunotherapy

We studied the Th2/Th1 balance by short-term stimulation of peripheral blood mononuclear cells (PBMC) isolated during the pollen season from seven allergic patients treated with conventional birch-pollen immunotherapy (IT) for 18 months, eight matched allergic control patients and 10 non-atopic individuals. The PBMC were cultured for 7 days with birch-pollen extract (BPE) or tetanus toxoid (TT), and then restimulated with PHA and PMA to induce high IL-5, IL-10 and IFN-gamma production. The serum levels of birch-pollen-specific IgG and IgG4 were significantly elevated after IT treatment. The proliferative response to BPE was significantly enhanced in the allergic control group, but not in the IT-treated group, compared to the non-atopic group (P<0.05). Birch-pollen-specific IL-5 production was significantly enhanced in both the IT-treated group and the allergic control group (P<0.01-0. 05). Furthermore, both the IT-treated group and the allergic control group had a cytokine profile to BPE significantly more Th2 polarized (high IL-5/IFN-gamma ratio) than to TT (P<0.05 and P<0.01, respectively). No differences in IL-10 production between the three study groups were observed. The Th2/Th1 balance in vitro correlated with the serum concentrations of birch-pollen-specific IgE (r=0.60, P<0.05), and in the IT-treated group, also with the IgG and IgG4 levels (r=0.79, P<0.05 and r=0.86, P<0.05, respectively). We conclude that conventional birch-pollen IT does not lead to changes in the cytokine profile of the circulating pool of allergen-specific T cells during birch-pollen season. However, induction of peripheral T-cell tolerance and increased production of specific IgG and IgG4 might be part of the mechanisms of IT.     

Cytokine gene expression and protein production in peripheral blood mononuclear cells of children with acute dengue virus infections

Plasma leakage in dengue hemorrhagic fever (DHF) is associated with elevated plasma levels of cytokines. To define further the contribution of immune activation to DHF and the source of cytokines, we analyzed the production of cytokines in peripheral blood mononuclear cells (PBMC) obtained from children with dengue, using RT-PCR and immunostaining. Tumor necrosis factor-alpha (TNF-alpha) and TNF-beta expression was detected in all samples by PCR and in < 50% of samples by immunostaining. Interferon-gamma (IFN-gamma) expression was detected in < 50% of samples by either method. Interleukin-2 (IL-2) and IL-4 expression was detected in a few samples by immunostaining but was not detectable by PCR. We found greater expression of TNF-alpha and IL-4 in DHF than in dengue fever or other (non-dengue) febrile illnesses. These results support the model of immunopathogenesis of DHF. However, low levels of cytokine expression in PBMC suggest that cellular activation in tissues may contribute to high serum cytokine levels in DHF.     

Quantitation of hepatitis C virus in liver and peripheral blood mononuclear cells from patients with chronic hepatitis C virus infection

Since the natural history of hepatitis C virus-associated liver disease and the therapeutic responsiveness might vary according to liver and blood mononuclear cells viral levels, it may be important to quantitate viral RNA in liver, blood mononuclear cells and serum, and to compare these data with genotype, biochemical and histologic data. A polymerase chain reaction-based assay available for serum hepatitis C virus RNA quantitation has been optimized to quantitate viral genomes in liver and peripheral blood mononuclear cells from 47 chronic hepatitis C patients. The procedure permitted hepatitis C virus RNA quantitation in freshly isolated mononuclear cells and in total RNA extracted from frozen mononuclear cells and liver tissue. The intrahepatic viral amount (median: 2.6 × 10³ copies/μg RNA; range: 0 to 3.6 × 10⁴ copies/μg RNA) correlated significantly with the hepatitis C virus RNA concentration in serum (r = 0.76, P < .001) but not in mononuclear cells. Viral RNA concentrations in liver (P < .001), serum (P < 0.01) and PBMC (P < 0.05) were significantly higher in hepatitis C virus genotype 1 patients (essentially type 1b) than in non-1 type cases, but were unrelated to biochemical or histologic indexes of disease activity. In conclusion, the optimized assay permit HCV RNA quantitation in liver and peripheral blood mononuclear cells, suggesting that serum viral level is an accurate measurement of intrahepatic viral burden. J. Med. Virol. 54:265–270, 1998. © 1998 Wiley-Liss, Inc.     

乙型肝炎患者外周血单个核细胞凋亡的检测及意义

目的研究激活诱导细胞死亡 (AICD)现象在乙型肝炎慢性化和重型化机制中的意义。方法分离 2 0例慢性 /慢性重型乙型肝炎病人与 10例健康献血员外周血单个核细胞 (PBMC) ,在 PHA的刺激下培养 72 h后收集细胞 ,经流式细胞仪检测凋亡。结果 PBMC凋亡率乙型肝炎组明显高于正常对照组 [(2 5 .48± 14.0 7) % vs(11.45± 5 .2 7) % ,P<0 .0 1];慢性乙型肝炎组 [(30 .5 7± 13.43) % ]明显高于正常对照组 [(11.45± 5 .2 7) % ,P<0 .0 1]和慢性重型乙型肝炎组 [(13.5 9± 6 .44 ) % ,P<0 .0 1];PBMC凋亡率乙型肝炎 HBe Ag(+ )组明显高于正常对照组 [(2 9.5 0± 12 .5 4) % vs(11.45± 5 .2 7) % ,P<0 .0 1]。结论 AICD可能是形成 HBV慢性感染免疫耐受的一个重要机制。     

Expression of CD1 antigens by peripheral blood mononuclear cells from hepatitis B patients

We have studied the expression of CD1 antigens on peripheral blood mononuclear cells (PBMC) from acute hepatitis B patients in order to analyse a possible role for CD1 antigens in hepatitis B virus (HBV) infection. Using immunofluorescence and the monoclonal antibodies which recognized CD1a, CD1b and CD1c molecules, we have shown that CD1 antigens were expressed on PBMC from acute hepatitis B patients but not from other acute and chronic liver disease. Dot blot analysis on nitrocellulose sheets of the lysates of the cells confirmed these observations. Cell fractionation and double-labelling experiments clearly demonstrated the CD1 antigens were expressed only on non-T cells. Furthermore, CD1 antigens were coexpressed with hepatitis B surface antigen (HBsAg) on the surface of Ig-positive cells. These results could indicate that CD1 expression may be associated with the lymphotropic effect of HBV.     

Induction of suppressor cell activity in normal peripheral blood mononuclear cells by sera from predialytic uremic patients

Normal peripheral blood mononuclear cells (PBMC) were primed with sera from 22 predialytic uremic patients, normal sera, concanavalin A, or both. Their suppressive activity was subsequently tested on fresh phytohemagglutinin-stimulated allogeneic responders (i.e., genetically unrelated to either primed cell or sera donors). Uremic serum induced suppressor cell activity in the normal PBMC. No correlation was found between serum urea/creatinine levels and their effects on suppressor cell activity. The induced activity, expressed as percent suppression, was similar to that induced by concanavalin A. In PBMC primed with both concanavalin A and uremic serum, an additive suppressive effect was evident. The suppressor subset(s) induced by uremic serum proved to belong to adherent cell population. Addition of indomethacin or catalase to responder systems did not abolish the suppressive effects, thus suggesting a mechanism of action other than prostaglandin or hydrogen peroxide release.