Progesterone suppresses interferon signaling by repressing TLR-7 and MxA expression in peripheral blood mononuclear cells of patients infected with hepatitis C virus

This study aimed at investigating the effect of progesterone on interferon signaling pathways in peripheral blood mononuclear cells (PBMCs) of patients infected with hepatitis C virus (HCV). PBMCs were isolated from peripheral blood of 38 treatment-naïve HCV-infected patients, pooled, and stimulated with progesterone in the presence and absence of its receptor antagonist, mifepristone, along with interferon alpha (IFN-α) or imiquimod. Toll-like receptor (TLR) 7 and myxovirus resistance protein A (MxA) were quantified in PBMCs using RT-qPCR. Imiquimod alone or combined with progesterone did not change MxA expression in HCV-infected PBMCs. Progesterone decreased the inducing effect of IFN-α on TLR-7 expression in both males and females. Moreover, progesterone stimulation prior to IFN-α treatment attenuated the Jak/STAT pathway, which was reflected by decreased expression of MxA in females. Progesterone showed a negative impact on the IFN signaling pathway in HCV-infected PBMCs as it decreased the expression of TLR-7 in both genders, while MxA expression was decreased only in females.     

Prominent proliferative response of peripheral blood mononuclear cells to a recombinant non-structural (NS3) protein of hepatitis C virus in patients with chronic hepatitis C.

The proliferative response of peripheral blood mononuclear cells (PBMC) to a recombinant non-structural (NS3) protein of hepatitis C virus (HCV) was studied in 41 patients with chronic hepatitis C. Of them, 28 had chronic persistent hepatitis (CPH) and 13 chronic active hepatitis (CAH). The positive proliferation rate of PBMC to the recombinant NS3 protein, T9Ag, was 66% in the 41 patients (77% in CAH versus 61% in CPH; P > 0.05) when stimulation index (SI) = 4 was set as the cut-off value. However, mean SI of CAH patients was significantly higher than that of CPH patients (8.3 +/- 5.2 versus 5.1 +/- 3.6; P < 0.05). Six other chronic hepatitis patients who were repeatedly negative for anti-HCV antibody but positive for serum HCV RNA also had an SI of > or = 4.0. The frequency of cellular immune response to the T9Ag is among the highest results obtained by using HCV antigens tested so far. Our studies thus indicate that NS3 is an immunologically important region of HCV for T cells. Moreover, the proliferative response to T9Ag may help to establish hepatitis C etiology in chronic hepatitis patients who are seronegative with currently available anti-HCV assays.     

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.     

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.     

Interferon-gamma mRNA expression from peripheral blood mononuclear cells in hepatitis C virus infection: relation to viremia and combined peginterferon ribavirin response

It would be most helpful to identify serological markers associated with poor treatment outcome at baseline or in the early phase of therapy in order to spare unnecessary side effects of an expensive antiviral therapy (ribavirin and peginterferon). We hypothesized that pretreatment gamma-interferon gene expression level in peripheral blood mononuclear cells (PBMCs) and its protein could be used to predict treatment outcome (responders and nonresponders) in Egyptian HCV patients. This study involved 29 HCV subjects; they were classified after the 24 weeks of a treatment regimen (ribavirin and peginterferon) into two groups (16 patients with nondetectable HCV classified as responders and 13 HCV patients who had detectable HCV classified as nonresponders). Baseline interferon-gamma (IFN-γ) gene expression and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a housekeeping gene were measured by real-time polymerase chain reaction (RT-PCR) methods using Syber Green method. Serum interferon-gamma level was determined by an ELISA (Enzyme-Linked Immunosorbent Assay). It is shown that 7/13 (54%) of nonresponders and 2/16 (13%) of responders showed elevated blood IFN-gamma mRNA levels prior to the therapy (p?<?0.05). Serum interferon-gamma level in the nonresponder group was undetectable compared with the responder group (p?<?0.01). There was no correlation between IFN-gamma expression levels, stage of fibrosis, viral load level nor serum interferon-gamma level. An interferon-gamma gene expression cutoff level of 0.54 at baseline (before starting the treatment regimen) can discriminate patients with response from patients with failure of response. IFN-gamma was higher in PBMCs of nonresponders when compared to responders and that measuring IFN-gamma can be used in patients infected with chronic hepatitis C to predict treatment failure.     

Prevalence and significance of hepatitis B virus antigens: expression in peripheral blood mononuclear cells in chronic active hepatitis

One-hundred four chronic active hepatitis (CAH) patients were investigated for the expression of the hepatitis B virus (HBV) surface and core gene products (HBs Ag, HBc/HBe Ags) in peripheral blood mononuclear cells (PBMC). Two-thirds of 59 HBs antigenemic patients expressed HBs Ag in PBMC but 26% of cases positive for both anti-HBs and anti-HBc also expressed HBs Ag while none of the controls reacted. Among HBs antigenemic patients, only those who replicated HBV express the core gene products (HBc and/or HBe Ag) in PBMC, and high replicators did so more often than low replicators (P less than 0.05). The HBs Ag prevalence in PBMC, although slightly higher among HBe Ag/DNAp-positive cases could not be correlated with the intensity of HBV replication. In 16 cases (8 replicants and 8 nonreplicants) HBV DNA was detected by DNA hybridization spot test, while 8 controls devoid of HBV markers were negative. Both T and non-T cells reacted similarly for antigenic or genomic HBV markers. When the expression of HBV gene products in PBMC among 43 cases with HBs antigenemia was compared with that in the liver, a good correlation was found in 70% of cases for HBs Ag but in only 40% for HBc and HBe Ags. By contrast, among 38 cases lacking HBs Ag in the serum but positive for anti-HBc with or without anti-HBs, concordance between liver and PBMC expression of core gene products (69%) was better than for HBs antigenemic patients (40%). These data suggest that PBMC including T lymphocytes may represent the second-best HBV target and may mimic the steps of HBV cycle within hepatocytes.     

MxA and PKR expression in chronic hepatitis C.

The effectiveness of therapy for chronic hepatitis C (CHC) patients has greatly improved in the last few years, and the gold standard is currently held to be pegylated interferon (IFN) in combination with ribavirin. Overall, however, the percentage of patients achieving a sustained virologic response (SVR) is only around 50%,and it is not possible to predict those patients who will benefit from therapy. The molecular mechanisms underlying lack of therapeutic response remain unknown. In this study, we investigated the tissue expression of MxA and RNA-dependent protein kinase (PKR), two antiviral proteins modulated by IFN, in biopsy samples from hepatitis C patients before the beginning of therapy. Our results show that expression of MxA, but not of PKR, is significantly lower in responders compared with nonresponders. No differences were observed regarding the hepatitis C virus (HCV) genotype and the viral load. These results suggest that expression of the MxA protein could play a role among the mechanisms underlying responsiveness to therapy.     

Detection of hepatitis C virus RNA in serum and peripheral blood mononuclear cells in patients with chronic hepatitis C treated with interferon alpha

PCR was used to detect hepatitis C virus (HCV) RNA in serum and peripheral blood mononuclear cells (PBMCs) for evaluation of a six-month course of Interferon therapy in 18 patients with histologically confirmed chronic hepatitis C. At follow-up six months after the end of therapy positive-stranded (genomic) and negative-stranded (anti-genomic, presumptive replicative intermediate) HCV RNA could be detected in PBMCs of all ten patients who either did not respond to therapy or suffered a relapse; genomic strand RNA was detected in five patients who responded but then relapsed. The study confirms that interferon therapy leads to inhibition of HCV replication but not eradication of the virus. Persistence of the virus at extrahepatic sites may explain its reactivation after cessation of interferon therapy.     

Fluorescent "in situ" hybridization of hepatitis C virus RNA in peripheral blood mononuclear cells from patients with chronic hepatitis C

Although the liver is the main target for hepatitis C virus (HCV) infection, HCV RNA of positive and negative polarity has also been detected in peripheral blood mononuclear cells (PBMCs) by polymerase chain reaction. However, no data have been published on the relationship between the number of HCV-infected PBMCs and serum viremia levels. To address this issue, PBMC samples from 20 patients with chronic hepatitis C were examined by fluorescent "in situ" hybridization. Serum viremia levels and viral load in infected PBMC were measured using the Amplicor Monitor test. HCV was detected in all PBMC samples corresponding to the HCV-positive patients. Fluorescent signals were found mainly in the cytoplasm of the cell. The percentage of positive cells ranged from 0.08% to 4%, with a statistical correlation with the viral load in PBMC (r = 0.69; p =. 001) but not with the serum viremia levels (r = 0.23). It was demonstrated that HCV infection of PBMCs is a common feature of HCV chronic carriers. The results suggest that HCV infection of PBMCs does not contribute significantly to HCV viremia.     

慢性乙型肝炎患者外周血单个核细胞Toll样受体3的表达降低

目的 探讨慢性乙型肝炎患者外周血单个核细胞Toll样受体3(TLR3)的表达及其临床意义.方法 分别采集慢性乙型肝炎患者和健康志愿者外周血,荧光定量PCR法检测血清HBV DNA复制水平;使用RT-PCR、流式细胞术以及免疫印迹技术分别检测外周血单个核细胞TLR3的mRNA、蛋白的表达;使用ELISA法检测血清中肿瘤坏死因子α(TNF-α)和干扰素β(IFN-p)水平.结果 慢性乙型肝炎患者外周血单个核细胞中的TLR3表达显著低于健康志愿者,且降低水平与血清HBV DNA复制水平相关;慢性乙型肝炎患者外周血TNF-α、IFN-β浓度显著低于健康志愿者,且降低的水平与血清HBV DNA复制水平相关.结论 慢性乙型肝炎患者外周血单个核细胞TLR3的表达与乙肝病毒的复制水平相关.     

MxA gene expression in juvenile dermatomyositis peripheral blood mononuclear cells: association with muscle involvement

Juvenile dermatomyositis (JDM), a systemic vasculopathy, is characterized by inflammation of skin and muscle. Muscle biopsies from untreated JDM patients show upregulation of type I interferon (IFN)-inducible genes, including myxovirus resistance protein A (MxA). The present study examines whether MxA mRNA expression in peripheral blood mononuclear cells (PBMC) from JDM patients: (1) is elevated compared to healthy controls, (2) reflects disease activity, and (3) changes with the onset of clinically effective treatment. MxA mRNA expression in JDM PBMC obtained at the initial clinic visit was elevated compared to controls and was positively correlated with Disease Activity Score (DAS) for muscle, but not with DAS for skin, suggesting that damage to skin and muscle in JDM may each have a discrete pathophysiology. During the course of clinically effective treatment, decrease in muscle symptoms was associated with a decrease in PBMC MxA mRNA expression.     

CpG-ODN对慢性乙型肝炎患者外周血单个核细胞T-bet表达的影响

目的 探讨T-bet在乙型肝炎病毒(HBV)感染中的作用以CpD-ODN对其表达的调节作用。方法应用半定量RT-PCR，对22例慢性乙型肝炎活动期患者、10例HBsAg阳性无症状携带者和12例正常对照外周血单个核细胞(PBMC)T-bet的表达进行检测；同时用CpD-ODN体外刺激以上3组实验对象的PBMC，RT-PCR观察T-bet的表达情况。结果无症状携带者T-bet表达最低，慢性乙型肝炎活动期患者T-bet表达最高；经cpG-ODN刺激后，无症状携带者和正常对照的T-bet表达明显上调，结论T-bet在HBV感染的不同状态中具有差异表达，提示T-bet可能参与了机体抗HBV的免疫应答，具体的机制有待进一步研究。CpG-ODN作为一种新型的免疫调节剂，可在一定程度上恢复HBV感染者特别是无症状携带者的Th1型细胞免疫应答．     

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。