Functional polymorphisms of TLR8 are associated with hepatitis C virus infection.

Chronic hepatitis C virus (HCV) infection is a worldwide threat to public health. Toll-like receptor 8 (TLR8) is critical for eliminating RNA viruses, and variation within the TLR8 gene may alter the function of TLR8 in response to HCV infection. Our previous study demonstrated that the TLR8-129G>C (rs3764879) and TLR8+1G>A (rs3764880) variants were in complete linkage disequilibrium, and that the frequency of TLR8-129C/+1A was significantly higher in male patients with HCV infection compared with the healthy controls. In the present study, we found that the promoter activity of TLR8-129G was higher than that of TLR8-129C in THP-1 cells. Moreover, TLR8-129G mRNA stability and competitive DNA-binding ability were significantly lower than that of TLR8-129C. To investigate the functional effects of TLR8 polymorphisms, we compared the nuclear factor-κB (NF-κB)-driven luciferase activity in HEK293 cells transfected with the TLR8 variants. TLR8+1A plasmids induced less NF-κB signalling than did those transfected with TLR8+1G after 20 µm CL075 (P = 0.011) stimulation. We also analysed the mRNA expression and cytokine production in whole blood and monocytes from people of various genotypes stimulated ex vivo by the interferon-γ and TLR7/8 agonist CL075, R848. TLR8 expression in CD14⁺ cells derived from volunteers with TLR8-129G/+1G was significantly higher than that derived from TLR8-129C/+1A, and interleukin-12p40 production was higher in volunteers with TLR8-129G/+1G after stimulation. The data indicate that variations in TLR8 genes may modulate immune responses during HCV infection.

Increased toll-like receptor 2 expression in peptidoglycan-treated blood monocytes is associated with insulin resistance in patients with nondiabetic rheumatoid arthritis.

The close relationship between increased TLR-2 expression in blood monocytes and insulin resistance in RA patients is shown in this study. Traditional risk factors for metabolic disorders, including the waist circumstance, body mass index (BMI), triglyceride (TG), and ratio of TG to high density lipoprotein (HDL) cholesterol, were closely correlated with HOMA (homoeostasis model assessment) index in patients with nondiabetic RA. Expressions of TLR2 in peripheral blood monocytes, following stimulation with peptidoglycan which is known as a TLR2 agonist, were closely correlated with the HOMA index, TNF-α, and IL-6 concentrations. Accordingly, TLR-2 receptor and its related inflammatory cytokines could be potential therapeutic targets in managing insulin resistance in RA patients.

Experimental phage therapy in treating Klebsiella pneumoniae-mediated liver abscesses and bacteremia in mice.

Intragastric inoculation of mice with Klebsiella pneumoniae can cause liver abscesses, necrosis of liver tissues, and bacteremia. A newly isolated phage (φNK5) with lytic activity for K. pneumoniae was used to treat K. pneumoniae infection in an intragastric model. Both intraperitoneal and intragastric administration of a single dose of φNK5 lower than 2 × 10(8) PFU at 30 min after K. pneumoniae infection was able to protect mice from death in a dose-dependent manner, but the efficacy achieved with a low dose of φNK5 by intragastric treatment provided the more significant protection. Phage φNK5 administered as late as 24 h after K. pneumoniae inoculation was still protective, while intraperitoneal treatment with phage was more efficient than intragastric treatment as a result of the dissemination of bacteria into the circulation at 24 h postinfection. Surveys of bacterial counts for mice treated with φNK5 by the intraperitoneal route revealed that the bacteria were eliminated effectively from both blood and liver tissue. K. pneumoniae-induced liver injury, such as liver necrosis, as well as blood levels of aspartate aminotransferase and alanine aminotransferase and inflammatory cytokine production, was significantly inhibited by φNK5 treatment. These data suggest that a low dose of φNK5 is a potential therapeutic agent for K. pneumoniae-induced liver infection.

Paraneoplastic Leukemoid Reaction after Primary Tumor Resection in Patients with Urothelial Carcinoma: A Report of 2 Cases.

Leukemoid reaction (LR), which is defined as leukocytosis with a white blood cell (WBC) count above 50 000/µL, can be caused by various conditions, while paraneoplastic leukemoid reaction (PLR), a rare type of paraneoplastic syndrome, occurs in cases of solid tumors. Here we report 2 cases of high-grade urothelial carcinoma (HGUC) with PLR accompanied by rapid tumor progression after complete resection of the primary tumor. We reviewed the patient's clinical history, histopathology, and the results of laboratory tests to rule out LR induced by non-tumor causes. In both cases, PLR appeared after primary tumor resection, and the patients died of disease at the peak of PLR at 6 and 8 weeks. Immunohistochemistry for granulocyte colony-stimulating factor and its receptor was performed on tumor tissues. Patients with HGUC and PLR are rare and have an extremely poor prognosis. The mechanism by which solid tumors are associated with PLR and rapid tumor progression after surgical resection of the primary tumor is incompletely understood and will be discussed here.

Endotoxin and CD14 in the progression of biliary atresia.

BACKGROUND: Biliary atresia (BA) is a typical cholestatic neonatal disease, characterized by obliteration of intra- and/or extra-hepatic bile ducts. However, the mechanisms contributing to the pathogenesis of BA remain uncertain. Because of decreased bile flow, infectious complications and damaging endotoxemia occur frequently in patients with BA. The aim of this study was to investigate endotoxin levels in patients with BA and the relation of these levels with the expression of the endotoxin receptor, CD14. METHODS: The plasma levels of endotoxin and soluble CD14 were measured with a pyrochrome Limulus amebocyte lysate assay and enzyme-linked immunosorbent assay in patients with early-stage BA when they received the Kasai procedure (KP), in patients who were jaundice-free post-KP and followed-up at the outpatient department, in patients with late-stage BA when they received liver transplantation, and in patients with choledochal cysts. The correlation of CD14 expression with endotoxin levels in rats following common bile duct ligation was investigated. RESULTS: The results demonstrated a significantly higher hepatic CD14 mRNA and soluble CD14 plasma levels in patients with early-stage BA relative to those with late-stage BA. However, plasma endotoxin levels were significantly higher in both the early and late stages of BA relative to controls. In rat model, the results demonstrated that both endotoxin and CD14 levels were significantly increased in liver tissues of rats following bile duct ligation. CONCLUSIONS: The significant increase in plasma endotoxin and soluble CD14 levels during BA implies a possible involvement of endotoxin stimulated CD14 production by hepatocytes in the early stage of BA for removal of endotoxin; whereas, endotoxin signaling likely induced liver injury and impaired soluble CD14 synthesis in the late stages of BA.

Faster waning of the rubella-specific immune response in young pregnant women immunized with MMR at 15 months.

PROBLEM: Vaccination is the best protection against rubella and congenital rubella infection. Although a high rate of immunization coverage is achieved in Taiwan, it is unknown if the vaccine-induced immunity persists from the age of vaccination to childbearing age. METHODS OF STUDY: A total of 5,988 prenatal rubella IgG test results of young pregnant women aged 19-23 years old from six hospitals during January 2001 to December 2008 and January 2013 to December 2017 were analyzed. We compared the rubella seropositivity rates and titers in these women who were vaccinated with MMR vaccine in four different vaccination age cohorts. RESULTS: The overall rubella seropositivity rate was 87.4% (95% CI: 86.6%-88.3%), and the mean rubella IgG level was 39 IU/mL among young pregnant women aged 19-23 years. Women in the elementary cohort had the highest rubella positivity of 90.8% (95% CI: 89.6%-91.9%), and levels gradually decrease to 84.6% (95% CI: 82.4%-86.7%) in 15-month plus cohort. The average rubella IgG was only 25 IU/mL for the 15-month plus cohort. Women in cohorts immunized at younger age exhibited significantly lower chances of being seropositive relative to women in older cohort after adjusting other factors (all P < .01). CONCLUSION: The rubella seropositivity rate and rubella IgG levels were low among young women aged 19-23 years, especially in cohorts immunized at younger age. As rubella immunity wanes over time, a third dose of MMR may be a protective strategy for women who conceive later in life.

The association of C (-260)-->T polymorphism in CD14 promoter and Chlamydia pneumoniae infection in ischemic stroke patients.

The C (-260) --> T polymorphism has been reported to regulate CD14 gene expression. It has also been implicated in atherosclerotic diseases, and in addition, it could be a genetic factor responsible for interindividual differences in the susceptibility to Chlamydia pneumoniae infection. This case-control study is aimed at evaluating the association between CD14 promoter polymorphisms, frequency of persistent C pneumoniae infection, and anti-chlamydial heat shock protein 60 (cHsp60) induction in stroke patients. Persistent C pneumoniae infection was observed in 43.3% of control subjects and 53.3% of patients (P = .005). The odds ratio of persistent infection was 3.25 for the CC genotype in stroke patients (P = .018). However, the serologic positive responses to cHsp60 in people with the TT genotype were significantly lower in stroke patients (9.3% vs 38.6%; P < .001). Our findings suggest that persistent C pneumoniae infection is independently associated with stroke in CC genotypes and the lowering of cHsp60 antibody levels in TT genotypes.

Functional polymorphisms of the TLR7 and TLR8 genes contribute to Mycobacterium tuberculosis infection.

Tuberculosis (TB) has recently re-emerged as a major global public health threat and Mycobacterium tuberculosis (MTB) is a highly successful pathogen that evolved remarkable strategies to establish persistent infection. There is strong evidence that host genetic factors influence individual susceptibility to TB. In this study, we evaluated the associations between the TLR7 and TLR8 genetic polymorphisms and TB susceptibility in Chinese individuals. The results demonstrated that the frequency of the TLR8-129C allele was higher in male patients with pulmonary TB than in healthy controls (22.9% vs. 6.8%, p < 0.001). Based on haplotype analysis, the frequency of the TLR7 IVS2-151A/TLR8 -129C haplotype increased the risk for TB infection compared to the wild-type allele (TLR7 IVS2-151A/TLR8 -129G), with OR = 3.23 (95% CI = 1.58-6.61; p = 0.001). An ex vivo phagocytosis assay that examined the functional effects of these polymorphisms on the defense against MTB revealed higher phagocytosis in monocytes from males with the TLR7 IVS2-151A/TLR8 -129C genotype than in those with the wild-type allele (73.0 ± 20.3% versus 34.6 ± 8.1%; p = 0.03). In addition, mRNA expression and cytokine production were analyzed in the whole blood of male healthy volunteers stimulated with inactivated MTB ex vivo. TNFα production was lower in TLR7 IVS2-151A/TLR8 -129C subjects than in those with the wild-type allele (578.4 ± 90.3 pg/ml versus 1043 ± 136 pg/ml; p = 0.03), and the expression of TLR7 was significantly impaired (0.8 ± 0.1 folds, p = 0.05) after MTB stimulation. In conclusion, these findings provide evidence that TLR7 and TLR8 genetic polymorphisms are associated with susceptibility to MTB infection, and the link is shaped by less effective MTB phagocytosis and impaired TLR signaling.

Down-regulation of Toll-like receptor 7 expression in hepatitis-virus-related human hepatocellular carcinoma.

Toll-like receptors (TLRs) play a pivotal role in innate immunity, controlling inflammatory responses, and further development of adaptive immunity. Hepatitis virus can establish chronic infection, and the associated inflammatory responses are important determinants of virus-associated liver damage. However, the contributions of the host immune system to chronic presence of virus are not clear in patients with hepatitis virus infection. Chronic inflammatory conditions caused by persistent hepatitis virus infections and interferon (IFN)-γ-related immunopathology are known to be related to carcinogenesis. To gain insight into the role of immune modulation in the pathogenesis of hepatocellular carcinoma (HCC), we studied the expression of TLR7 in cancerous and non-cancerous liver tissue from 87 patients with HCC. Our results showed that TLR7 is significantly down-regulated in neoplastic hepatocytes (P < .001), especially in the patients with hepatitis B (n = 52) or C (n = 24) virus infection. We confirmed this decreased TLR7 expression by quantitative analysis of mRNA using real-time reverse transcription-polymerase chain reaction in 26 liver specimens of HCC patients. Using serial deletion analysis of the TLR7 promoter, a hepatocyte-specific regulatory region was found at nucleotides -156 to -98 in the TLR7 promoter. Furthermore, the effects of IFN-γ on TLR7 expression in a hepatoma cell line (HepG2) were investigated in vitro. We demonstrated that IFN-γ significantly decreased TLR7 promoter activity and expression in a dose-dependent manner. We thus propose that hepatitis virus induces down-regulation of TLR7 gene expression through IFN-γ, thereby modulating inflammatory signaling in hepatoma cells.

Effects of hepatocyte CD14 upregulation during cholestasis on endotoxin sensitivity.

Cholestasis is frequently related to endotoxemia and inflammatory response. Our previous investigation revealed a significant increase in plasma endotoxin and CD14 levels during biliary atresia. We therefore propose that lipopolysacharides (LPS) may stimulate CD14 production in liver cells and promote the removal of endotoxins. The aims of this study are to test the hypothesis that CD14 is upregulated by LPS and investigate the pathophysiological role of CD14 production during cholestasis. Using Western blotting, qRT-PCR, and promoter activity assay, we demonstrated that LPS was associated with a significant increase in CD14 and MD2 protein and mRNA expression and CD14 promoter activity in C9 rat hepatocytes but not in the HSC-T6 hepatic stellate cell line in vitro. To correlate CD14 expression and endotoxin sensitivity, in vivo biliary LPS administration was performed on rats two weeks after they were subjected to bile duct ligation (BDL) or a sham operation. CD14 expression and endotoxin levels were found to significantly increase after LPS administration in BDL rats. These returned to basal levels after 24 h. In contrast, although endotoxin levels were increased in sham-operated rats given LPS, no increase in CD14 expression was observed. However, mortality within 24 h was more frequent in the BDL animals than in the sham-operated group. In conclusion, cholestasis and LPS stimulation were here found to upregulate hepatic CD14 expression, which may have led to increased endotoxin sensitivity and host proinflammatory reactions, causing organ failure and death in BDL rats.

TLR7 and TLR8 gene variations and susceptibility to hepatitis C virus infection.

Toll-like receptors (TLRs) play pivotal roles in the innate immune system and control inflammatory responses and adaptive immunity. We previously evaluated associations between TLR7 and TLR8 gene SNPs and susceptibility to hepatitis C virus (HCV) infection. Our results suggested that TLR7IVS2-151G and TLR8-129G alleles were present at higher frequency in males of an HCV-infected group as compared to a control group (24.1% vs. 14.4%, p = 0.028; 17.6% vs. 6.8%, p = 0.004, respectively). Based upon their recognition of single stranded viral RNA, this suggested that TLR7 and TLR8 played a significant role in anti-HCV immune responses. Here, we studied the functional effects of these polymorphisms by analyzing the mRNA expressions of TLR7 and TLR8 and cytokine production induced ex vivo by TLR7- and TLR8-specific agonists using whole blood of subjects with different genotypes. The percentage of CD14+ cells from those with an AG haplotype that expressed TLR7 and TLR8 was significantly lower, but higher in intensity compared to cells from those with GG and AC haplotypes. Cells from those with an AG haplotype produced more IFN-α and less amounts of pro-inflammatory cytokines upon stimulation. This suggests that variations in TLR7 and TLR8 genes might impair immune responses during HCV infection.

Concanavalin A protects mice from a lethal inoculation of intragastric Klebsiella pneumoniae and reduces the induced liver damage.

Intragastric inoculation of Klebsiella pneumoniae can cause invasive diseases, including necrosis of liver tissues and bacteremia. The effect of concanavalin A (ConA) on K. pneumoniae was tested. Pretreatment with ConA was able to protect mice from K. pneumoniae infection in an intragastric model. K. pneumoniae-induced mouse death and liver injury such as liver necrosis, as well as blood levels of aspartate aminotransferase and alanine aminotransferase, were inhibited in a dose-dependent manner by ConA. ConA administered intravenously as late as 24 h after K. pneumoniae inoculation was still protective. In an in vitro assay, ConA was able to bind K. pneumoniae cells directly and further agglutinate them but had no effect on their in vitro growth. Surveys of bacterial counts of ConA-treated mice revealed that the bacteria were eliminated effectively in both blood and liver tissues. Furthermore, the bactericidal activity of macrophages against K. pneumoniae was also enhanced in a dose-dependent manner by ConA in an in vitro culture. These data suggest that ConA is a potentially therapeutic agent for K. pneumoniae-induced liver infection.

Streptococcal pyrogenic exotoxin B cleaves properdin and inhibits complement-mediated opsonophagocytosis.

Streptococcal pyrogenic exotoxin B (SPE B), a cysteine protease, is an important virulence factor in group A streptococcal (GAS) infection. The reduction of phagocytic activity by SPE B may help prevent bacteria from being ingested. In this study, we investigated the mechanism SPE B uses to enable bacteria to resist opsonophagocytosis. Using Western blotting and an affinity column immobilized with SPE B, we found that both SPE B and C192S, an SPE B mutant lacking protease activity, bound to serum properdin, and that SPE B, but not C192S, degraded serum properdin. Further study showed that SPE B-treated, but not C192S-treated, serum blocked the alternative complement pathway. Reconstitution of properdin into SPE B-treated serum unblocked the alternative pathway. GAS opsonized with SPE B-treated serum was more resistant to neutrophil killing than GAS opsonized with C192S-treated or normal serum. These results suggest that a novel SPE B mechanism, one which degrades serum properdin, enables GAS to resist opsonophagocytosis.

The HSP expression of passive repetitive plyometric trained skeletal muscle.

This study aims to understand the effect of ten-week passive repetitive plyometric (PRP) training on human skeletal muscle and the application of PRP training for performance. Vastus lateralis of nine candidates were aspirated before (pre) and after (post) PRP training. Histochemical approaches with regular hematoxylene-eosin (HE) and Mallory's phosphotungstic acid hematoxylin (PTAH) stains were used to demonstrate the changes of muscle fibers. Immunohistochemical studies with heat shock protein (anti-hsp72, Stressgen, Canada) were employed to display cellular activities. Each set of slides was quantitatively analyzed by using a modified morphometric method (Russ and Dehoff, 1999) on a Nikon ECLIPSE 80i microscope, equipped with an Evolution VF COOLED color video camera, and the Image-Pro Plus software (5.0 for Win; Media Cybernetics, USA). Finally, hsp72 mRNAs of both pre-PRP and post-PRP specimens were amplified through RT-PCR. Signal intensities were read by a densitometer and analyzed through the SPSS (11.0 for Win) statistically. Post-PRP muscle cells demonstrated hypertrophic change with increased cellular content and a narrowed inter-cellular space according to both HE and PTAH profiles. Post-PRP cellular hsp72 proteins were higher by up to five percent, as measured by a gray-scale reading. Further, after a training period of 10 weeks, hsp72 mRNA expression was several times higher.

Association of CD14 promoter gene polymorphism and Chlamydia pneumoniae infection.

Recent studies have suggested that Chlamydia pneumoniae infection is an important factor in the development of atherosclerosis. The C(-260)-->T polymorphism in the CD14 promoter gene has been reported to regulate the density of CD14 expression on monocytes for the activation of monocytes to secrete inflammatory cytokines by lipopolysaccharide. We investigated this genetic marker and its association with C. pneumoniae infection. Among 315 healthy subjects, the distribution of the C(-260)-->T polymorphism in the CD14 promoter gene was 14.9% for the CC genotype, 54.3% for the CT genotype, and 30.8% for the TT genotype. Among subjects with the 3 CD14 genotypes, 59.5%, 64.9%, and 78.3%, respectively, were seropositive for C. pneumoniae. With multiple logistic regression analysis, the odds ratio of C. pneumoniae infection was 2.08 for CD14 TT genotype (95% confidence interval, 1.18-3.69; P=.016). A significant association between the CD14 TT genotype and C. pneumoniae infection was found.