Semaphorin 7A impairs barrier function in cultured human corneal epithelial cells in a manner dependent on nuclear factor-kappa B

●AIM:To evaluate the role of semaphorin 7A(Sema7A)and its associated regulatory mechanisms in modulating the barrier function of cultured human corneal epithelial cells(HCEs).●METHODS:Barrier models of HCEs were treated with recombinant human Sema7A at concentrations of 0,125,250,or 500 ng/mL for 24,48,or 72h in vitro.Transepithelial electrical resistance(TEER)as well as Dextran-fluorescein isothiocyanate(FITC)permeability assays were conducted to assess barrier function.To quantify tight junctions(TJs)such as occludin and zonula occludens-1(ZO-1)at the mRNA level,reverse transcriptionpolymerase chain reaction(RT-PCR)analysis was performed.Immunoblotting was used to examine the activity of the nuclear factor-kappa B(NF-κB)signaling pathway and the production of TJs proteins.Immunofluorescence analyses were employed to localize the TJs.Enzyme-linked immunosorbent assay(ELISA)and RT-PCR were utilized to observe changes in interleukin(IL)-1βlevels.To investigate the role of NF-κB signaling activation and IL^(-1)βin Sema7A’s anti-barrier mechanism,we employed 0.1μmol/L IκB kinase 2(IKK2)inhibitor IV or 500 ng/mL IL^(-1)receptor(IL-1R)antagonist.●RESULTS:Treatment with Sema7A resulted in decreased TEER and increased permeability of Dextran-FITC in HCEs through down-regulating mRNA and protein levels of TJs in a time-and dose-dependent manner,as well as altering the localization of TJs.Furthermore,Sema7A stimulated the activation of inhibitor of kappa B alpha(IκBα)and expression of IL-1β.The anti-barrier function of Sema7A was significantly suppressed by treatment with IKK2 inhibitor IV or IL-1R antagonists.●CONCLUSION:Sema7A disrupts barrier function through its influence on NF-κB-mediated expression of TJ proteins,as well as the expression of IL-1β.These findings suggest that Sema7A could be a potential therapeutic target for the diseases in corneal epithelium.

Outcomes of ABO-incompatible liver transplantation in end-stage liver disease patients co-infected with hepatitis B and human immunodeficiency virus

BACKGROUND Human immunodeficiency virus(HIV)-positive patients coinfected with hepatitis B virus(HBV)are eligible for liver transplantation(LT)in Africa and Southeast Asia,particularly China.However,the outcome of HIV-HBV coinfected patients referred for ABO-incompatible LT(ABOi-LT)is unknown.AIM To clarify the outcome of ABOi-LT for HIV-HBV coinfected patients with endstage liver disease(ESLD).METHODS We report on two Chinese HIV-HBV coinfected patients with ESLD who underwent A to O brain-dead donor LT and reviewed the literature on HIV-HBV coinfected patients treated with ABO-compatible LT.The pretransplantation HIV viral load was undetectable,with no active opportunistic infections.Induction therapy consisted of two sessions of plasmapheresis and a single dose of rituximab in two split doses,followed by an intraoperative regimen of intravenous immunoglobulin,methylprednisolone,and basiliximab.Post-transplant maintenance immunosuppressive agents consisted of tacrolimus and mycophenolate mofetil,and prednisone.RESULTS At the intermediate-term follow-up,patients showed undetectable HIV viral load,CD4(+)T cell counts greater than 150 cells/μL,no HBV recurrence,and stable liver function.A liver allograft biopsy showed no evidence of acute cellular rejection.Both patients survived at 36-42 mo of follow-up.CONCLUSION This is the first report of ABOi-LT in HIV-HBV recipients with good intermediate-term outcomes,suggesting that ABOi-LT may be feasible and safe for HIV-HBV coinfected patients with ESLD.

Current status of liver transplantation for human immunodeficiency virus-infected patients in China's Mainland

According to the report from the Chinese Center for Disease Control and Prevention,the prevalence of human immunodeficiency virus(HIV)infection exceeded 1.2 million individuals by the year 2022,with an annual increase of about 80000 cases.The overall prevalence of hepatitis B surface antigen among individuals co-infected with HIV reached 13.7%,almost twice the rate of the general population in China.In addition to the well-documented susceptibility to opportunistic infections and new malignancies,HIV infected patients frequently experience liver-related organ damage,with the liver and kidneys being the most commonly affected.This often leads to the development of end-stage liver and kidney diseases.Therefore,organ transplantation has emerged as an important part of active treatment for HIV infected patients.However,the curative effect is not satisfactory.HIV infection has been considered a contraindication for organ transplantation.Until the emergence of highly active anti-retroviral therapy in 1996,the once intractable replication of retrovirus was effectively inhibited.With prolonged survival,the failure of important organs has become the main cause of death among HIV patients.Therefore,transplant centers worldwide have resu-med exploration of organ transplantation for HIV-infected individuals and reached a positive conclusion.This study provides an overview of the current landscape of HIV-positive patients receiving liver transplantation(LT)in main-land China.To date,our transplant center has conducted LT for eight end-stage liver disease patients co-infected with HIV,and all but one,who died two months postoperatively due to sepsis and progressive multi-organ failure,have survived.Comparative analysis with hepatitis B virus-infected patients during the same period revealed no statistically significant differences in acute rejection reactions,cytomegalovirus infection,bacteremia,pulmonary infections,acute kidney injury,new-onset cancers,or vascular and biliary complications.

Effects of hepatitis B virus infection on human sperm chromosomes

AIM: To evaluate the level of sperm chromosome aberrations in male patients with hepatitis B, and to directly detect whether there are HBV DNA integrations in sperm chromosomes of hepatitis B patients.METHODS: Sperm chromosomes of 14 tested subjects (5healthy controls, 9 patients with HBV infection, including 1with acute hepatitis B, 2 with chronic active hepatitis B, 4with chronic persistent hepatitis B, 2 chronic HBsAg carriers with no clinical symptoms) were prepared using interspecific in vitro fertilization between zona-free golden hamster ova and human spermatozoa, and the frequencies of aberration spermatozoa were compared between subjects of HBV infection and controls. Fluorescence in situ hybridization (FISH) to sperm chromosome spreads was carried out with biotin-labeled full length HBV DNA probe to detect the specific HBV DNA sequences in the sperm chromosomes.RESULTS: The total frequency of sperm chromosome aberrations in HBV infection group (14.8%, 33/223) was significantly higher than that in the control group (4.3%,5/116). Moreover, the sperm chromosomes in HBV infection patients commonly presented stickiness, clumping, failure to staining, etc, which would affect the analysis of sperm chromosomes. Specific fluorescent signal spots for HBV DNA were seen in sperm chromosomes of one patient with chronic persistent hepatitis. In 9 (9/42) sperm chromosome complements containing fluorescent signal spots, one presented 5 obvious FISH spots, others presented 2 to 4signals. There was significant difference of fluorescence intensity among the signal spots. The distribution of signal sites among chromosomes was random.CONCLUSION: HBV infection can bring about mutagenic effects on sperm chromosomes. Integrations of viral DNA into sperm chromosomes which are multisites and nonspecific, can further increase the instability of sperm chromosomes. This study suggested that HBV infection can create extensively hereditary effects by alteration genetic constituent and/or induction chromosome aberrations, as well as the possibility of vertical transmission of HBV via the germ line to the next generation.

Gene expression analysis of primary normal human hepatocytes infected with human hepatitis B virus

AIM： To find the relationship between hepatitis B virus （HBV） and hepatocytes during the initial state of infection by cDNA microarray. METHODS： Primary normal human hepatocytes （PNHHs） were isolated and infected with HBV. From the PNHHs, RNA was isolated and inverted into complement DNA （cDNA） with Cy3- or Cy5- labeled dUTP for microarray analysis. The labeled cDNA was hybridized with microarray chip, including 4224 cDNAs. From the image of the microarray, expression profiles were produced and some of them were confirmed by RT-PCR, immunoblot analysis, and NF-κB luciferase reporter assay. RESULTS： From the cDNA microarray, we obtained 98 differentially regulated genes. Of the 98 genes, 53 were up regulated and 45 down regulated. Interestingly, in the up regulated genes, we found the TNF signaling pathway-related genes： LT-α, TRAF2, and NIK. By using RT-PCR, we confirmed the up-regulation of these genes in HepG2, HuhT, and Chang liver cells, which were transfected with pHBV1.2x, a plasmid encoding all HBV messages. Moreover, these three genes participated in HBV- mediated NF-κB activation. CONCLUSION： During the initial state of HBV infection, hepatocytes facilitate the activation of NF-κB through up regulation of LT-α, TRAF2, and NIK.

Stem cells from human exfoliated deciduous teeth ameliorate concanavalin A-induced autoimmune hepatitis by protecting hepatocytes from apoptosis

BACKGROUND Autoimmune hepatitis is a serious autoimmune liver disease that threatens human health worldwide,which emphasizes the urgent need to identify novel treatments.Stem cells from human exfoliated deciduous teeth(SHED),which are easy to obtain in a non-invasive manner,show pronounced proliferative and immunomodulatory capacities.AIM To investigate the protective effects of SHED on concanavalin A(ConA)-induced hepatitis in mice,and to elucidate the associated regulatory mechanisms.METHODS We used a ConA-induced acute hepatitis mouse model and an in vitro co-culture system to study the protective effects of SHED on ConA-induced autoimmune hepatitis,as well as the associated underlying mechanisms.RESULTS SHED infusion could prevent aberrant histopathological liver architecture caused by ConA-induced infiltration of CD3+,CD4+,tumor necrosis-alpha+,and interferon-gamma+inflammatory cells.Alanine aminotransferase and aspartate aminotransferase were significantly elevated in hepatitis mice.SHED infusion could therefore block ConA-induced alanine aminotransferase and aspartate aminotransferase elevations.Mechanistically,ConA upregulated tumor necrosisalpha and interferon-gamma expression,which was activated by the nuclear factor-kappa B pathway to induce hepatocyte apoptosis,resulting in acute liver injury.SHED administration protected hepatocytes from ConA-induced apoptosis.CONCLUSION SHED alleviates ConA-induced acute liver injury via inhibition of hepatocyte apoptosis mediated by the nuclear factor-kappa B pathway.Our findings could provide a potential treatment strategy for hepatitis.

Hepatitis C virus in human B lymphocytes transformed by Epstein-Barr virus in vitro by in situ reverse transcriptase-polymerase chain reaction

AIM: To study persistence and replication of hepatitis C virus (HCV) in patients' peripheral blood mononuclear cells (PBMC) cultured in vitro. METHODS: Epstein Barr virus (EBV) was used to transform the hepatitis C virus from a HCV positive patient to permanent lymphoblastoid cell lines (LCL). Positive and negative HCV RNA strands of the cultured cells and growth media were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) each month. Core and NS5 proteins of HCV were further tested using immunohistochemical SP method and in situ RT-PCR. RESULTS: HCV RNA positive strands were consistently detected the cultured cells for one year. The negative-strand RNA in LCL cells and the positive-strand RNA in supernatants were observed intermittently. Immunohistochemical results medicated expression of HCV NS3 and C proteins in LCL cytoplasm mostly. The positive signal of PCR product was dark blue and mainly localized to the LCL cytoplasm. The RT-PCR signal was eliminated by overnight RNase digestion but not DNase digestion. CONCLUSION: HCV may exist and remain functional in a cultured cell line for a long period.

Development of novel-nanobody-based lateral-flow immunochromatographic strip test for rapid detection of recombinant human interferon a2b

Recombinant human interferon a2b(rhIFNa2b)is widely used as an antiviral therapy agent for the treatment of hepatitis B and hepatitis C.The current identification test for rhIFNa2b is complex.In this study,an anti-rhIFNa2b nanobody was discovered and used for the development of a rapid lateral flow strip for the identification of rhIFNa2b.RhIFNa2b was used to immunize an alpaca,which established a phage nanobody library.After five steps of enrichment,the nanobody I22,which specifically bound rhIFNa2b,was isolated and inserted into the prokaryotic expression vector pET28a.After subsequent purification,the physicochemical properties of the nanobody were determined.A semiquantitative detection and rapid identification assay of rhIFNa2b was developed using this novel nanobody.To develop a rapid test,the nanobody I22 was coupled with a colloidal gold to produce lateral-flow test strips.The developed rhIFNa2b detection assay had a limit of detection of 1 mg/mL.The isolation of I22 and successful construction of a lateral-flow immunochromatographic test strip demonstrated the feasibility of performing ligand-binding assays on a lateral-flow test strip using recombinant protein products.The principle of this novel assay is generally applicable for the rapid testing of other commercial products,with a great potential for routine use in detecting counterfeit recombinant protein products.

Detection of Human Parvovirus B19 Nonstrutural Protein DNA by Nested-Polymerase Chain Reaction in Gravida Serum and Pregnant Tissues

A new nested-polymerase chain reaction （nested-PCR） assay was developed to detect human parvovirus B19 DNA corresponding to the nonstructural protein in clinical specimens in a routine diagnostic laboratory. The sensitivity of this highly specific assay was up to 0. 005 fg of B19 DNA. Parvovirus B19 was identified in sera of 20 pregnant women with abnormal pregnant outcome. Among these 20 cases, intrauterine parvovirus infection did exist in 7 pregnant women because parvovirus B19 DNA was detected in the pregnant tissues of them such as placenta tissues, chorionic villi, amniotic fluid, fetal spleen, liver and abdominal fluids.

EXPERIMENTAL PRIMARY LIVER CANCER IN TREE SHREWS EXPOSED TO HUMAN HEPATITIS B VIRUS AND AFLATOXIN B_(1)

On the basis of the successful establishment of an animal model in tree shrews experimentally in fected with human hepatitis B virus (HHBV), a study on the hepatocarcinogenic effects of HHBV and aflatoxin B1 (AFB1) by using this animal model was conducted through a lifelong experiment. Among 41 tree shrews exposed to AFB1, 17 were experimentally infected by HHBV and 24 were uninfected. After 158 weeks, significant difference of primary liver cancer (PLC) incidence was present between the HHBV infected (52.94%) and uninfected (12.5%) groups (p<0.05). No difference was found between these two groups in the amount of AFB4 ingestion. Moreover, 1/9 of the tree shrews infected only by HHBV but not exposed to AFB4 developed PLC. No PLC was found in 6 tree shrews that had neither been infected with HHBV nor been exposed to AFB4. These results suggest the possible etiologic relationship between HHBV infection and PLC, as well as the synergetic effects of HHBV and AFB4 during PLC development.

Translating transitions-how to decipher peripheral human B cell development

During the last two decades our understanding of human B cell differentiation has developed considerably. Our understanding of the human B cell compartment has advanced from a point where essentially all assays were based on the presence or not of class-switched antibodies to a level where a substantial diversity is appreciated among the cells involved. Several consecutive transitional stages that newly formed IgM expressing B cells go through after they leave the bone marrow, but before they are fully mature, have been described, and a significant complexity is also acknowledged within the IgM expressing and class-switched memory B cell compartments. It is possible to isolate plasma blasts in blood to follow the formation of plasma cells during immune responses, and the importance and uniqueness of the mucosal IgA system is now much more appreciated. Current data suggest the presence of at least one lineage of human innate-like B cells akin to B 1 and/or marginal zone B cells in mice. In addition, regu- latory B cells with the ability to produce IL-10 have been identified. Clinically, B cell depletion therapy is used for a broad range of conditions. The ability to define different human B cell subtypes using flow cytometry has therefore started to come into clinical use, but as our understanding of human B cell development further progresses, B cell subtype analysis will be of increasing importance in diagnosis, to measure the effect of immune therapy and to understand the underlying causes for diseases. In this review the diversity of human B cells will be discussed, with special focus on current data regarding their phenotypes and functions.

CLONING AND SEQUENCING OF MATURE FRAGMENT OF HUMAN BMP4 GENE

Objective To study the cloning and sequencing of mature fragment of human bone morphogenetic protein 4 gene. Methods The template DNA was obtained from the human osteosarcoma cell line U2OS. By using RT PCR method, the cDNA coding for the mature fragment of BMP 4 was amplified, cloned into the vector pUC19, and sequenced by Sanger Dideoxy mediated Chain Termination method. Results The mature fragment of BMP4 cDNA was obtained by RT PCR and determined by sequencing. Through the computer search on Genebank, the analysis showed that the homology of nucleotides and amino acids between cDNA of rhBMP4 mature fragment of this study and the published sequence was 99%. Sequence analysis showed that there were two differences, one was at base 1154(201): G→C, which had no influence on the corresponding amino acids(Val). Another was at base1222(269):C→T, the mutation at the base 1222 had the change of Ala to Val. Conclusion The mature fragment of BMP4 gene has been cloned. The results will be of great significance in treatment of skeletal injuries and diseases.

Genetic Correction and Hepatic Differentiation of Hemophilia B-specific Human Induced Pluripotent Stem Cells

Objective To genetically correct a disease-causing point mutation in human induced pluripotent stem cells （iPSCs） derived from a hemophilia B patient. Methods First, the disease-causing mutation was detected by sequencing the encoding area of human coagulation factor IX （F IX） gene. Genomic DNA was extracted from the iPSCs, and the primers were designed to amplify the eight exons of F IX. Next, the point mutation in those iPSCs was genetically corrected using CRISPR/Cas9 technology in the presence of a 129-nucleotide homologous repair template that contained two synonymous mutations. Then, top 8 potential off-target sites were subsequently analyzed using Sanger sequencing. Finally, the corrected clones were differentiated into hepatocyte-like cells, and the secretion of F IX was validated by immunocytochemistry and ELISA assay.Results The cell line bore a missense mutation in the 6th coding exon （c.676 C〉T） of F IX gene. Correction of the point mutation was achieved via CRISPR/Cas9 technology in situ with a high efficacy at about 22% （10/45） and no off-target effects detected in the corrected iPSC clones. F IX secretion, which was further visualized by immunocytochemistry and quantified by ELISA in vitro, reached about 6 ng/ml on day 21 of differentiation procedure. Conclusions Mutations in human disease-specific iPSCs could be precisely corrected by CRISPR/Cas9 technology, and corrected cells still maintained hepatic differentiation capability. Our findings might throw a light on iPSC-based personalized therapies in the clinical application, especially for hemophilia B.

Expression of hepatitis B virus genes in early embryonic cells originated from hamster ova and human spermatozoa transfected with the complete viral genome

Aim： To detect the expression of hepatitis B virus （HBV） genes （HB S and C genes） in early embryonic cells after introducing motile human sperm carrying HBV DNA into zona-free hamster oocytes via the in vitro fertilization （IVF） technique. Methods： Human sperm-mediated HBV genes were delivered into zona-free hamster oocytes by the IVF method. Polymerase chain reaction （PCR） was used to detect HB S and pre-Core/Core （pre-C/C） coding genes both in one- and two-cell embryos. Reverse transcription-PCR （RT-PCR） analysis was used to study the expression of the two genes. Fluorescence in situ hybridization （FISH） analysis using the full-length HBV DNA as the hybridization probe was performed to confirm the integration of viral DNA in the host embryonic genome. Results： Both HB S and pre-C/C coding genes are present and transcribed in one- and two-cell embryos originated from hamster ova IVF with human spermatozoa carrying HBV DNA sequences. Conclusion： Sperm-mediated HBV genes are able to replicate and express themselves in early embryonic cells. These results provide direct evidence that HBV DNA could transmit vertically to the next generation via the male germ line.

Studies on the integration of hepatitis B virus DNA sequence in human sperm chromosomes

Aim: To study the integration of hepatitis B virus (HBV) DNA into sperm chromosomes in hepatitis B patients and the features of its integration. Methods: Sperm chromosomes of 14 subjects (5 healthy controls and 9 HB patients, including 1 acute hepatitis B, 2 chronic active hepatitis B, 4 chronic persistent hepatitis B, 2 HBsAg chronic carriers with no clinical symptoms) were prepared using interspecific in vitro fertilization between zona-free hamster oocytes and human spermatozoa. Fluorescence in situ hybridization (FISH) to sperm chromosome spreads was carried out with biotin-labeled full length HBV DNA probe to detect the specific HBV DNA sequences in the sperm chromosomes. Results: Specific fluorescent signal spots for HBV DNA were seen in sperm chromosomes of one patient with chronic persistent hepatitis B. In 9(9/42) sperm chromosome complements containing fluorescent signal spots, one presented 5 obvious FISH spots and the others 2 to 4 signals. The fluorescence intensity showed significant difference among the signal spots. The distribution of signal sites among chromosomes seems to be random. Conclusion: HBV could integrate into human sperm chromosomes. Results suggest that the possibility of vertical transmission of HBV via the germ line to the next generation is present.

Therapeutic and prevention strategies against human enterovirus 71 infection

Human enterovirus 71(HEV71) is the cause of hand,foot and mouth disease and associated neurological complications in children under five years of age.There has been an increase in HEV71 epidemic activity throughout the Asia-Pacific region in the past decade,and it is predicted to replace poliovirus as the extant neurotropic enterovirus of highest global public health significance. To date there is no effective antiviral treatment and no vaccine is available to prevent HEV71 infection. The increase in prevalence, virulence and geographic spread of HEV71 infection over the past decade provides increasing incentive for the development of new therapeutic and prevention strategies against this emerging viral infection. The current review focuses on the potential, advantages and disadvantages of these strategies. Since the explosion of outbreaks leading to large epidemics in China, research in natural therapeutic products has identified several groups of compounds with anti-HEV71 activities. Concurrently, the search for effective synthetic antivirals has produced promising results. Other therapeutic strategies including immunotherapy and the use of oligonucleotides have also been explored. A sound prevention strategy is crucial in order to control the spread of HEV71. To this end the ultimate goal is the rapid development, regulatory approval and widespread implementation of a safe and effective vaccine. The various forms of HEV71 vaccine designs are highlighted in this review. Given the rapid progress of research in this area, eradication of the virus is likely to be achieved.

Hepatitis B and human immunodeficiency virus co-infection

Hepatitis B and human immunodeficiency virus(HBV and HIV)infection share transmission patterns and risk factors,which explains high prevalence of chronic HBV infection in HIV infected patients.The natural course of HBV disease is altered by the HIV infection with less chance to clear acute HBV infection,faster progression to cirrhosis and higher risk of liver-related death in HIVHBV co-infected patients than in HBV mono-infected ones.HIV infected patients with chronic hepatitis B should be counseled for liver damage and surveillance of chronic hepatitis B should be performed to screen early hepatocellular carcinoma.Noninvasive tools are now available to evaluate liver fibrosis.Isolated hepatitis B core antibodies(anti-HBc)are a good predictive marker of occult HBV infection.Still the prevalence and significance of occult HBV infection is controversial,but its screening may be important in the management of antiretroviral therapy.Vaccination against HBV infection is recommended in non-immune HIV patients.The optimal treatment for almost all HIV-HBV co-infectedpatients should contain tenofovir plus lamivudine or emtricitabine and treatment should not be stopped to avoid HBV reactivation.Long term tenofovir therapy may lead to significant decline in hepatitis B surface Antigen.The emergence of resistant HBV strains may compromise the HBV therapy and vaccine therapy.