Diagnostic value of orbicularis oculi muscle electromyography in functional epiphora.

BACKGROUND: Functional epiphora is a clinical condition which is not due to an anatomic defect. Most studies agree that it involves the action of the orbicularis oculi muscle, particularly its deeper segment (Horner's muscle), but the exact mechanism is not clear. AIM: To evaluate the orbicularis oculi muscle in functional epiphora patients using electromyography (EMG). METHODS: A total of 8 Chinese patients (16 eyes) with functional epiphora were enrolled in this study, and ten volunteers (10 eyes) were included as normal controls. Five epiphora patients (five eyes) with facial palsy served as positive controls. Quantitative EMG was performed in the deeper segment of orbicularis oculi muscle. The average duration of each EMG waveform was measured. RESULTS: The average duration of EMG waveforms in the normal control group, the functional epiphora group, and the facial palsy group were 6.39 ± 0.73 ms, 9.39 ± 1.32 ms and 11.2 ± 1.42 ms, respectively. The duration of EMG waveforms was significantly longer in the functional epiphora group than in the normal control group (P < 0.05), and shorter than that in the facial palsy group (P < 0.05). CONCLUSION: These data indicate the presence of neurogenic orbicularis oculi muscle damage in epiphora patients, which may be the cause of functional epiphora. The etiology of neurogenic damage in the orbicularis oculi muscle requires further investigation.

Role of Tim-3 in hepatitis B virus infection: An overview.

Hepatitis B virus (HBV) infection has received increasing public attention. HBV is the prototypical member of hepadnaviruses, which naturally infect only humans and great apes and induce the acute and persistent chronic infection of hepatocytes. A large body of evidence has demonstrated that dysfunction of the host anti-viral immune response is responsible for persistent HBV replication, unresolved inflammation and disease progression. Many regulatory factors are involved in immune dysfunction. Among these, T cell immunoglobulin domain and mucin domain-3 (Tim-3), one of the immune checkpoint proteins, has attracted increasing attention due to its critical role in regulating both adaptive and innate immune cells. In chronic HBV infection, Tim-3 expression is elevated in many types of immune cells, such as T helper cells, cytotoxic T lymphocytes, dendritic cells, macrophages and natural killer cells. Tim-3 over-expression is often accompanied by impaired function of the above-mentioned immunocytes, and Tim-3 inhibition can at least partially rescue impaired immune function and thus promote viral clearance. A better understanding of the regulatory role of Tim-3 in host immunity during HBV infection will shed new light on the mechanisms of HBV-related liver disease and suggest new therapeutic methods for intervention.

Tim-4 Inhibits NO Generation by Murine Macrophages.

OBJECTIVE: T cell immunoglobulin- and mucin-domain-containing molecule-4 (Tim-4) receives much attention as a potentially negative regulator of immune responses. However, its modulation on macrophages has not been fully elucidated so far. This study aimed to identify the role of Tim-4 in nitric oxide (NO) modulation. METHODS: Macrophages were stimulated with 100 ng/ml LPS or 100 U/ml IFN-γ. RT-PCR was performed to detect TIM-4 mRNA expression. Tim-4 blocking antibody and NF-κB inhibitory ligand were involved in the study. NO levels were assayed by Griess reaction. Phosphorylation of NF-κB, Jak2 or Stat1 was verified by western blot. RESULTS: Tim-4 was up-regulated in murine macrophages after interferon-gamma (IFN-γ) stimulation. Tim-4 over-expression decreased NO production and inducible nitric oxide synthase (iNOS) expression in lipopolysaccharide (LPS) or IFN-γ-stimulated macrophages. Consistently, Tim-4 blockade promoted LPS or IFN-γ-induced NO secretion and iNOS expression. Tim-4 over-expression decreased LPS-induced nuclear factor kappa B (NF-κB) p65 phosphorylation in macrophages, which was abrogated by NF-κB inhibitory ligand. On the contrary, Tim-4 blocking increased LPS-induced NF-κB signaling, which was also abrogated by NF-κB inhibition. In addition, Tim-4 blockade promoted Jak2 and Stat1 phosphorylation in IFN-γ stimulated macrophages. CONCLUSION: These results indicate that Tim-4 is involved in negative regulation of NO production in macrophages, suggesting the critical role of Tim-4 in immune related diseases.

[Early production of interleukin-17 in airway upon Chlamydia trachomatis infection increases the local secretion of IL-6 and MIP-2].

OBJECTIVE: To evaluate the early interleukin-17 (IL-17) production in airway upon Chlamydia trachomatis infection and its relationship with the secretion of interleukin-6 (IL-6) and macrophage inflammatory protein 2 (MIP-2) in local site. METHODS: In vivo, a murine model of pneumonia induced by intranasal inoculation with Chlamydia trachomatis mouse pneumonitis (MoPn, now classified as a new species C. muridarum) was used for the study. Chlamydial growth in the lung was assessed by inoculating HeLa cell monolayer with lung homogenates followed by enzyme-linked immunosorbent assay (IFA). IL-17, IL-6 and MIP-2 were measured by enzyme-linked immunosorbent assay (ELISA). Mice without infection acted as the control group. In vitro, L929 cells were pretreated with recombinant murine IL-17 (rmIL-17) at a dose ranging from 20, 100 to 500 µg/L for 24 h then infected with MoPn for 24 h. The supernatants were harvested and tested for IL-6 and MIP-2 concentration using ELISA. The cells were assayed for the number of inclusion-forming unit (IFU) by IFA. L929 cells without pretreatment with rmIL-17 but infected with MoPn was the control group. RESULTS: The study showed that in vivo, Chlamydial growth in the lung was found on day 1 after infection, and reached its peak at day 8 (6.49±0.19, lg IFU/lung) with subsequent decline in quantity. IL-17 peaked at 48 h (83.0 ng/L±35.8 ng/L) while IL-6 peaked on day 3 [(3.98±0.04) µg/L], MIP-2 peaked on day 8 [(2.19±0.71) µg/L]. The study showed that in vitro, compared with control group [(55.10±16.54) ng/L for IL-6 production and (13.71±0.84) ng/L for MIP-2], L929 cells pretreated with rmIL-17 at the different concentrations of 20, 100 and 500 µg/L for 24 h then infected with MoPn for 24 h, could significantly increase IL-6 (P <0.01) and MIP-2 secretion (P <0.05). The productions of IL-6 in the supernatants were (531.65±24.40), (629.95±7.71), and (646.51±35.92) ng/L. Meanwhile, the productions of MIP-2 were (107.21±28.40), (181.95±25.51), and (221.90±17.32) ng/L, respectively. RmIL-17 alone had no effect on IL-6 and MIP-2 secretion, and no direct effect on growth of chlamydial inclusion body was demonstrated either. CONCLUSION: IL-17 was produced early in airway upon Chlamydia trachomatis, and rmIL-17could induce IL-6 and MIP-2 production in L929 cells after infection with MoPn. These suggest that an early IL-17 response may play an important role by inducing the secretion of IL-6 and MIP-2 in initiating host defense against infection with Chlamydia trachomatis in the airway.

[Survey on host animal and molecular epidemiology of hantavirus in Chuxiong prefecture, Yunnan province].

OBJECTIVE: To determine the hosts of hantavirus (HV) and its molecular epidemiological characteristics, to provide evidence for prevention and control on hemorrhagic fever with renal syndrome (HFRS). METHODS: Rodents were captured by a special trap within the residential area. The antigens of HV in lung tissues were detected by direct immuno-fluorescence assay (DFA). Nucleotide sequences of HV were amplified by RT-PCR with HV genotype-specific primer. The amplified genes were then sequenced. Phylogenetic tree were built on nucleotide sequence with ClustalX 1.83 software. RESULTS: 1421 rodents were captured and classified into 8 species of 4 Genera in the epidemic area within 10 counties of Chuxiong prefecture, Yunnan province, between 2005 and 2006. Out of the 1421 rodents, 1056 (74.31% ) of them were Rattus norvegicus and 280 (19.70%) belonged to Rattus flavipectus. The antigens of HV were detected by DFA in lung tissues and the total positive rate of HV was 5.15% (53/ 1029). After applying the sequencing nucleotide method to the 53 positive specimens, data showed that 21 specimens were positive and all of them belonged to Seoul type (15 samples were from Rattus norvegicus, 4 samples Rattus flavipectus, 2 samples Rattus nitidus). The partial S segments from 12 specimens were sequenced which appeared homologic with R22, L99 and HLD65 from GenBank in relatively high level (87.1% -99.7%). When compared to 76-118 strain of Hantaan type, their homologic degree was only 64.4%-69.1%. Results from Phylogenetic analysis showed that 12 specimens belonged to Seoul type. As for their homology, they were significantly similar to Seoul type and could be tentatively divided into two subtypes S1 and S3. CONCLUSION: It was confirmed that the Seoul type virus, as HFRS's pathogenetic agent mainly carried by rats, prevailed widely in Chuxiong prefecture. Owing to the local ecological environment, we also noticed the characteristics of different HV subtypes among Seoul type.

Blockade of Tim-3 pathway ameliorates interferon-gamma production from hepatic CD8+ T cells in a mouse model of hepatitis B virus infection.

T cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) has been reported to participate in the pathogenesis of inflammatory diseases. However, whether Tim-3 is involved in hepatitis B virus (HBV) infection remains unknown. Here, we studied the expression and function of Tim-3 in a hydrodynamics-based mouse model of HBV infection. A significant increase of Tim-3 expression on hepatic T lymphocytes, especially on CD8+ T cells, was demonstrated in HBV model mice from day 7 to day 18. After Tim-3 knockdown by specific shRNAs, significantly increased IFN-gamma production from hepatic CD8+ T cells in HBV model mice was observed. Very interestingly, we found Tim-3 expression on CD8+ T cells was higher in HBV model mice with higher serum anti-HBs production. Moreover, Tim-3 knockdown influenced anti-HBs production in vivo. Collectively, our data suggested that Tim-3 might act as a potent regulator of antiviral T-cell responses in HBV infection.

Blockade of TRAIL pathway ameliorates HBV-induced hepatocyte apoptosis in an acute hepatitis model.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) may play important roles during hepatitis B virus (HBV) infection. In this study, we used a recombinant human soluble death receptor 5 (sDR5) to explore its effect in a mouse model of HBV-induced acute hepatitis. By measuring blood transaminase activity and hepatocyte apoptosis, we found that sDR5 could alleviate liver damage by blocking TRAIL-induced apoptosis of HBV-transfected hepatocytes. sDR5 injection at 16 mg/kg 24h before HBV transfection was the most effective. Additionally, we showed that sDR5 was equally effective in protecting liver injury as the Stronger Neo-Minophagen C (SNMC), a commonly used drug for patients with liver diseases. Thus, sDR5 represents a potential novel therapeutic drug for patients with fulminant hepatitis.

Human endostatin gene transfer, either naked or with liposome, has the same inhibitory effect on growth of mouse liver tumor cells in vivo.

AIM: To explore a safe and efficient strategy of tumor therapy using anti-angiogenetic agents. METHODS: Endostatin gene with a signal sequence of human IgGgamma chain was amplified by PCR and cloned into pVAX1 plasmid which was the only vector authorized by FDA in clinical trial to construct a recombinant plasmid named as pVAX-sEN. The recombinant plasmid was detected with EcoRI/KpnI and DNA sequencing. BALB/c mice bearing hepatocarcinoma cell line H22 were treated with naked pVAX-sEN or liposome-DNA complex in which the dose of DNA and the ratio of DNA and liposome were different from each other. To compare the efficiency of gene transfection, expression of endostatin at the treated tumor site was assayed with ELISA. To investigate the effect of pVAX1-sEN on hepatocellular carcinoma, pVAX-sEN either naked or in liposome-DNA complex was injected into BALB/c mice bearing H22, then the diameter of tumors was measured, microvessel density was detected by immunohistochemistry, endostatin expression in vivo was assayed at different time points. RESULTS: DNA sequencing showed the endostatin gene with the signal peptide was correctly cloned. In situ gene expression assay indicated that both the ratio of DNA and liposome and the dose of DNA could affect the gene transfection efficiency. Interestingly, naked pVAX-sEN had a similar in situ endostatin expression to pVAX-sEN with liposome. Animal experiments showed that pVAX-sEN together with pVAX-sEN-liposome complex could efficiently suppress the growth of mouse hepatoma cells. CONCLUSION: Naked endostatin plasmid intratumoral injection can get a similar gene transfection efficiency to liposome-DNA complex when used in situ.

Establishment of mice model with human viral hepatitis B.

AIM: To establish a mice model of hepatitis B by using HBV-transgenic mice, and to transfer HBV-specific cytotoxic T lymphocytes (CTL) induced from syngeneic BALB/c mice immunized by a eukaryotic expression vector containing HBV complete genome DNA. METHODS: HBV DNA was obtained from digested pBR322-2HBV and ligated with the vector pcDNA3. Recombinant pcDNA3-HBV was identified by restriction endonuclease assay and transfected into human hepatoma cell line HepG2 with lipofectin. ELISA was used to detect the expression of HBsAg in culture supernatant, and RT-PCR to determine the existence of HBV PreS1 mRNA. BALB/c mice were immunized with pcDNA3-HBV or pcDNA3 by intramuscular injection. ELISA was used to detect the expression of HBsAb in serum. MTT assay was used to measure non-specific or specific proliferation ability and specific killing activity of spleen lymphocytes. Lymphocytes from immunized mice were transferred into HBV-transgenic mice (2.5X10(7) per mouse). Forty-eight hours later, the level of serum protein and transaminase was detected with biochemical method, liver and kidney were sectioned and stained by HE to observe the pathological changes. RESULTS: By enzyme digestion with Eco RI, Xho I and Hind III, the recombinant pcDNA3-HBV was verified to contain a single copy of HBV genome, which was inserted in the positive direction. HepG2 cells transfected with the recombinant could stably express PreS1 mRNA and HBsAg. After immunized by pcDNA3-HBV for 4 weeks, HBsAb was detected in the serum of BALB/c mice. The potential of spleen lymphocytes for both non-specific and specific proliferation and the specific killing activity against target cells were enhanced. The transgenic mice in model group had no significant changes in the level of serum protein but had an obvious increase of ALT and AST. The liver had obvious pathological changes, while the kidney had no evident damage. CONCLUSION: A eukaryotic expression vector pcDNA3-HBV containing HBV complete genome is constructed successfully. HepG2 cells transfected with the recombinant can express PreS1 mRNA and HBsAg stably. Specific cellular immune response can be induced in mice immunized by pcDNA3-HBV. A mice model of acute hepatitis with HBV has been established.

A novel HBV antisense RNA gene delivery system targeting hepatocellular carcinoma.

AIM: To construct a novel HBV antisense RNA delivery system targeting hapatocellular carcinoma and study its inhibitory effect in vitro and in vivo. METHODS: GE7,a 16-peptide specific to EGFR, and HA20,a homologue of N-terminus of haemagglutinin of influenza viral envelope protein, were synthesized and conjugated with polylysin. The above conjugates were organized into the pEBAF-as-preS2, a hepatocarcinoma specific HBV antisense expression vector, to construct a novel HBV antisense RNA delivery system, named AFP-enhancing 4-element complex. Hepatocelluar carcinoma HepG2.2.15 cells was used to assay the in vitro inhibition of the complex on HBV. Expression of HBV antigen was assayed by ELISA. BALB/c nude mice bearing HepG2.2.15 cells were injected with AFP-enhancing 4-element complex. The expression of HBV antisense RNA was examined by RT-PCR and the size of tumor in nude mice were measured. RESULTS: The AFP-enhancing 4-element complex was constructed and DNA was completely trapped at the slot with no DNA migration when the ratio of polypeptide to plasmid was 1:1. The expression of HBsAg and HBeAg of HepG2.2.15 cells was greatly decreased after being transfected by AFP-enhancing 4-element complex. The inhibitory rates were 33.4 % and 58.5 % respectively. RT-PCR showed HBV antisense RNA expressed specifically in liver tumor cells of tumor-bearing nude mice. After 4 injections of AFP-enhancing 4-element complex containing 0.2 micro g DNA, the diameter of the tumor was 0.995 cm+/-0.35, which was significantly smaller than that of the control groups(2.215 cm+/-0.25, P<0.05). CONCLUSION: AFP-enhancing 4-element complex could deliver HBV antisense RNA targeting on hepatocarcinoma and inhibit both HBV and liver tumor cells in vitro and in vivo.

Detection of soluble TRAIL in HBV infected patients and its clinical implications.

AIM: To detect the expression of soluble TRAIL (TNF-related apoptosis inducing ligand, TRAIL) in the peripheral blood of HBV infected patients and try to elucidate whether the expression level of sTRAIL have any correlativity with the clinical staging, the expression level of HBV markers and the degree of liver damage. METHODS: 52 cases of HBV infected patients were investigated, including 8 HBV carriers, 30 chronic hepatitis B, 11 cirrhotics and 3 HBV infection related hepatocellular carcinoma. Expression of soluble TRAIL and markers of the hepatitis B were measured by enzyme-linked immunosorbent assay. RESULTS: The expression level of sTRAIL in the peripheral blood of the HBV infected patients was significantly higher than that of healthy controls (1378.35+/-540.23 pg/ml vs 613.75+/-175.80 pg/ml, P<0.001). In the group of chronic hepatitis, the expression level of sTRAIL was coincident with the status of the disease and was significantly correlated with the level of ALT. In the group of cirrhosis and liver cancer, its expression level was significantly higher than that of the healthy persons and HBV carriers, but lower than that of the hepatitis B patients; meanwhile, the expression of sTRAIL did not have any correlativity with the functional indexes of the liver. CONCLUSION: The soluble TRAIL in the HBV infected people may participate in the liver damage. Our results indicated that the expression level of soluble TRAIL may reflect the ravage of liver caused by host immune reaction to a certain degree.