Characteristics and advantages of adeno-associated virus vector-mediated gene therapy for neurodegenerative diseases

Common neurodegenerative diseases of the central nervous system are characterized by progressive damage to the function of neurons, even leading to the permanent loss of function. Gene therapy via gene replacement or gene correction provides the potential for transformative therapies to delay or possibly stop further progression of the neurodegenerative disease in affected patients. Adeno-associated virus has been the vector of choice in recent clinical trials of therapies for neurodegenerative diseases due to its safety and efficiency in mediating gene transfer to the central nervous system. This review aims to discuss and summarize the progress and clinical applications of adeno-associated virus in neurodegenerative disease in central nervous system. Results from some clinical trials and successful cases of central neurodegenerative diseases deserve further study and exploration.

Hemophagocytic lymphohistiocytosis caused by primary Epstein-Barr virus in patient with Crohn's disease

We present a case of a 19-year-old man with a 6-year history of Crohn's disease(CD), previously treated with 6-mercaptopurine, who was admitted to our department for Epstein-Barr virus(EBV) infection and subsequently developed a hemophagocytic lymphohistiocytosis(HLH). HLH is a rare disease which causes phagocytosis of all bone marrow derived cells. It can be a primary form as a autosomic recessive disease, or a secondary form associated with a variety of infections; EBV is the most common, the one with poorer prognosis. The incidence of lymphoproliferative disorders was increased in patients with inflammatory bowel disease(IBD) treated with thiopurines. Specific EBV-related clinical and virological management should be considered when treating a patient with IBD with immunosuppressive therapy. Moreover EBV infection in immunosuppressed patient can occur with more aggressive forms such as encephalitis and diffuse large B cell lymphoma. Our case confirms what is described in the literature; patients with IBD, particularly patients with CD receiving thiopurine therapy, who present 5 d of fever and cervical lymphadenopathy or previous evidence of lymphopenia should be screened for HLH.

Contributions of neurotropic human herpesviruses herpes simplex virus 1 and human herpesvirus 6 to neurodegenerative disease pathology

Human herpesviruses （HVs） have developed ingenious mechanisms that enable them to traverse the defenses of the central nervous system （CNS）. The ability of HVs to enter a state of latency, a defining char- acteristic of this viral family, allows them to persist in the human host indefinitely. As such, HVs represent the most frequently detected pathogens in the brain. Under constant immune pressure, these infections are largely asymptomatic in healthy hosts. However, many neurotropic HVs have been directly connected with CNS pathology in the context of other stressors and genetic risk factors. In this review, we discuss the potential mechanisms by which neurotropic HVs contribute to neurodegenerative disease （NDD） patholo- gy by highlighting two prominent members of the HV family, herpes simplex virus 1 （HSV-1） and human herpesvirus 6 （HHV-6）. We （i） introduce the infectious pathways and replicative cycles of HSV-1 and HHV-6 and then （ii） review the clinical evidence supporting associations between these viruses and the NDDs Alzheimer＇s disease （AD） and multiple sclerosis （MS）, respectively. We then （iii） highlight and dis- cuss potential mechanisms by which these viruses exert negative effects on neurons and glia. Finally, we （iv） discuss how these viruses could interact with other disease-modifying factors to contribute to the initiation and/or progression of NDDs.

Hepatitis A virus-associated acute acalculous cholecystitis in an adult-onset Still’s disease patient:A case report and review of the literature

BACKGROUND Acute acalculous cholecystitis(AAC)is inflammation of the gallbladder without evidence of calculi.Although rarely reported,its etiologies include hepatitis virus infection(e.g.,hepatitis A virus,HAV)and adult-onset Still’s disease(AOSD).There are no reports of HAV-associated AAC in an AOSD patient.CASE SUMMARY Here we report a rare case of HAV infection-associated AAC in a 39-year-old woman who had a history of AOSD.The patient presented with an acute abdomen and hypotension.Elevated hepatobiliary enzymes and a thickened and distended gallbladder without gallstones on ultrasonography suggested AAC,but there were no signs of anemia nor thrombocytopenia.Serological screening revealed anti-HAV IgM antibodies.Steroid treatment did not alleviate her symptoms,and she was referred for laparoscopic cholecystectomy.The resected gallbladder was hydropic without perforation,and her clinical signs gradually improved after surgery.CONCLUSION AAC can be caused by HAV in AOSD patients.It is crucial to search for the underlying etiology for AAC,especially uncommon viral causes.

Anti-amyloid beta single-chain Fv ameliorates behavioral impairment in Alzheimer's disease mice via adeno-associated virus delivery

Intracranial delivery of human Fc-deleted antibody specific to amyloid-β peptide （Aβ, anti-Aβ single-chain Fv, scFv） via adeno-associated virus （AAV） inhibits amyloid deposition in transgenic mice. However, the effects of AAV-mediated Fc-deleted antibody on animal behavior remain unclear. In this study, the anti-Aβ scFv antibody gone, isolated from phage display, was fused to the 5＇ end of the scFv antibody gone for antibody secretion by 2 rounds of polymerase chain reaction amplification. The fused antibody cDNA was cloned into a pSNAV2 plasmid under the control of the cytomegalovirus promoter. The sequence verified expression vector pSNAV2/scFv was transferred to BHK-21 ceils, and stable transfected BHK-21/scFv cells were established by G418 selection and infected with the recombinant herpes simplex virus rHSV/repcap for AAV production. Recombinant AAV was injected into the left quadriceps femoris of PDAPP transgenic mice. After 3 months, Morris water-maze results confirmed significantly improved cognitive function in a mouse model of Alzheimer＇s disease. Key Words： Alzheimer＇s disease; adeno-associated virus; amyloid-β peptide; single-chain antibody; neurodegenerative diseases; neural regeneration

Dynamic Pathology and Antigen Location Study on Broiler Breeders with Coinfection of Marek's Disease Virus and Reticuloendotheliosis Virus

To further understand the generation and development of coinfection of Marek＇s disease virus （MDV） and reticuloendotheliosis virus （REV） in broiler breeders, and then find the method and optimal time of differential diagnosis for complex clinic multiple infection, the authors studied the pathohistological changes, apoptosis, immunohistochemistry （immunofluorescence）, and ultrastructure of tumor tissues of broiler breeders inoculated with MDV and REV. The study showed that proliferation of small lymphocytes was seen in the main organs at the age of 1 week, then immature lymphocytes, all kinds of lymphocytes, primitive reticulum cells, and Marek＇s disease cells （MDCs） were observed at 2-9 weeks. Apoptosis of lymphocytes could not be seen until the age of 10 weeks in the immune system. Immunohistochemistry detection showed that the positive signs of MDV and REV antigen were observed in the main organs at 2 weeks of age. Multi-morphology lymphocytes, MDV, and REV, mitotic figures and apoptosis of lymphocytes were observed with the help of transmission electron microscopy. MDV cooperating with REV promotes the course of disease of coinfection. Differential diagnosis can be done by immunohistochemistry in the early stage （before 2 weeks）, and histopathology in the late stage （post 4 weeks）. MDCs, primitive reticulum cells, immature lymphocytes, and two kinds of virions can serve as a basis for bistopathology differential diagnosis.

Positive correlation between latent Epstein-Barr virus infection and severity of illness in inflammatory bowel disease patients

BACKGROUND Emerging studies indicate the critical involvement of microorganisms,such as Epstein-Barr virus(EBV),in the pathogenesis of inflammatory bowel disease(IBD).Immunosuppressive therapies for IBD can reactivate latent EBV,complicating the clinical course of IBD.Moreover,the clinical significance of EBV expression in B lymphocytes derived from IBD patients’intestinal tissues has not been explored in detail.AIM To explore the clinical significance of latent EBV infection in IBD patients.METHODS Latent EBV infection was determined by double staining for EBV encoded RNA and CD20 in colon specimens of 43 IBD patients who underwent bowel resection.Based on the staining results,the patients were divided into two groups,according to their latent EBV infection states-negative(n=33)and positive(n=10).Illness severity of IBD were assigned according to Crohn’s disease activity index(ulcerative colitis)and Mayo staging system(Crohn’s disease).The clinicpathological data were analyzed between the two different latent EBV groups and also between the mild-to-moderate and severe disease groups.RESULTS Systolic pressure(P=0.005),variety of disease(P=0.005),the severity of illness(P=0.002),and pre-op corticosteroids(P=0.025)were significantly different between the EBV-negative and EBV-positive groups.Systolic pressure(P=0.001),variety of disease(P=0.000),pre-op corticosteroids(P=0.011)and EBV infection(P=0.003)were significantly different between the mild-to-moderate and severe disease groups.CONCLUSION IBD patients with latent EBV infection may manifest more severe illnesses.It is suggested that the role of EBV in IBD development should be further investigated,latent EBV infection in patients with serious IBD should be closely monitored,and therapeutic course should be optimized.

Crohn’s disease in human immunodeficiency virus-infected patient:A case report

BACKGROUND Inflammatory bowel disease(IBD)is an autoimmune condition treated with immunosuppressive drugs.However,the need for immune system suppression becomes questionable when infection with the human immunodeficiency virus(HIV)occurs simultaneously and impacts the course of IBD.Our reported case represents the clinical course,prescribed treatment and its effect,as well as clinical challenges faced by physicians in a combination of such diseases.We also present a comprehensive literature review of similar cases.CASE SUMMARY A 49-year-old woman suffering from a newly diagnosed Crohn’s disease was hospitalized due to exacerbated symptoms(abdominal pain,fever,and weight loss).During her hospital stay,she tested positive for HIV.With conservative treatment,the patient improved and was discharged.In the outpatient clinic,her HIV infection was confirmed as stage C3,and antiretroviral treatment was initiated immediately.That notwithstanding,soon the patient was rehospitalized with pulmonary embolism and developed a series of complications because of the subsequent coexistence of IBD and HIV.After intensive and meticulous treatment,the patient’s condition has improved and she remains in remission.CONCLUSION The paucity of studies and data on the coexistence of HIV and IBD leaves clinicians doubting the optimal treatment options.

Varicella zoster meningitis complicating combined anti-tumor necrosis factor and corticosteroid therapy in Crohn's disease

Opportunistic viral infections are a well-recognized complication of anti-tumor necrosis factor (TNF) therapy for inflammatory bowel disease (IBD). Cases of severe or atypical varicella zoster virus infection, both primary and latent reactivation, have been described in association with immunosuppression of Crohn's disease (CD) patients. However, central nervous system varicella zoster virus infections have been rarely described, and there are no previous reports of varicella zoster virus meningitis associated with anti-TNF therapy among the CD population. Here, we present the case of a 40-year-old male with severe ileocecal-CD who developed a reactivation of dermatomal herpes zoster after treatment with prednisone and adalimumab. The reactivation presented as debilitating varicella zoster virus meningitis, which was not completely resolved despite aggressive antiviral therapy with prolonged intravenous acyclovir and subsequent oral valacyclovir. This is the first reported case of opportunistic central nervous system varicella zoster infection complicating anti-TNF therapy in the CD population. This paper also reviews the literature on varicella zoster virus infections of immunosuppressed IBD patients and the importance of vaccination prior to initiation of anti-TNF therapy.

A study on pathogenicity of hepatitis G virus

AIM To study the pathogenicity of hepatitis G virus (HGV) and observe the genesis and pathological process of hepatitis G.METHODS HGV-RNA in serum was detected by RT-PCR assay. The immunohistochemical assays of liver tissue were performed with HGV monocoloned antibody (McAb)expressed from the region of HGV NS5 nucleic acid sequence. The clinical and pathological data of 52 patients with hepatitis G were discussed. In animal experiment,the Chinese Rhesus monkeys were infected with the serum of a patient with HGV infection. And the dynamic changes in serology and liver histology of animals were observed.RESULTS One hundred and fifty-four patients with HGVRNA positive were selected from 1552 patients with various kinds of hepatitis. Of 154 patients with HGV infection, 52 were infected with HGV only, which accounted for 33.8% (52/154) and 102 with positive HGVRNA were super-infected with other hepatitis viruses,which accounted for 66.2% (102/154). The clinical and pathological observation showed that the acute and chronic hepatitis could be induced by HGV. The slight abnormality of transaminases ALT and AST in serum of monkeys lasted nearly 12 months and histological results showed a series of pathological changes.CONCLUSION HGV is a hepatotropic virus and has pathogenicty.

Establishment of transgenic mouse harboring hepatitis B virus (adr subtype) genomes

INTRODUCTIONHepatitis B virus (HBV) belongs to the group ofhepatovirus, a major pathogen of human acute andchronic hepatitis B[1 4], which has a very closeassociation with human hepatocellular carcinoma(HCC)[5-8], For example, a statistical data from ahospital in Shanghai showed that 80% of HCCpatients were positive for HBsAg ( personalcommunication).

Follow up of infection of chacma baboons with inoculum containing a and non-a genotypes of hepatitis B virus

AIM: To determine whether one genotype (A or non-A genotypes of HBV) predominated over the other during the course of HBV infection.METHODS: Four baboons were inoculated with HBV. DNA was extracted from serum obtained at monthly intervals postinoculation for 52 weeks and HBV DNA was amplified using primers specific for the core region containing an insert characteristic of genotype A (nt 2 354-2 359, numbering from the EcoRI site). The amplicons were cloned into PCRScriptTM and a minimum of 15 clones per time point were sequenced in both directions.RESULTS: Both genotypes persisted for the entire followup period of 52 weeks. Genotype non-A predominated in two baboons and genotype A in one baboon. Neither genotype predominated in the fourth baboon, as shown at a 5 % level of testing.CONCLUSION: No conclusions concerning the dominance of either genotype or the natural progression or replication rates of HBV could be drawn because the pattern of the genotypes found may have been caused by sampling fluctuations at the time of DNA extraction and cloning as a result of the very low viral loads in the baboon sera.

Inducing agent tamoxifen of CreER^(T2) to reduce the side effects of gene therapy for Parkinson’s disease

BACKGROUND：Gene therapy for Parkinson＇s disease is being explored as an effective strategy to restore and protect the function of neuronal cells in the substantia nigra. Regulation of gene expression is necessary for gene therapy to avoid adverse effects due to excessive synthesis of transgene products.OBJECTIVE：Here we developed recombinant adeno-associated virus （AAV） as a viral vector-mediated gene regulation system based on Cre recombinase fused to the mutated ligand-binding domain of the estrogen receptor （CreERT2） ＋ inducing agent tamoxifen. Inducible Cre recombinase was used to reduce tyrosine hydroxylase gene expression and to prevent the excessive increase in dopamine.DESIGN, TIME AND SETTING：A genetic engineering in vitro comparative study and randomized controlled animal experiment. This study was conducted at the Gene Therapy Center, Jichi Medical School, Japan from June 2002 to June 2004.METHODS：To construct a recombinant AAV vector carrying a dopamine synthase gene. The tyrosine hydroxylase gene was inserted using a IoxP fragment that could be regulated by Cre recombinase. The recombinant AAV vector carrying the CreERT2 gene was co-transduced with HEK293 cells and the corpus striatum in a rat model of Parkinson＇s disease, with inducing agent tamoxifen to regulate gene expression.MAIN OUTCOME MEASURES：The levels of dopamine and aromatic L-amino acid decarboxylase （AADC） activity were detected in HEK293 cell medium and in the corpus striatum in a rat model of Parkinson＇s disease using high-performance liquid chromatography. Immunofluorescence double staining was used to observe tyrosine hydroxylase and Cre or AADC co-expression in HEK293 cell medium. Immunohistochemical staining was employed to observe tyrosine hydroxylase and AADC expression and behavioral changes were measured in Parkinson＇s rats.RESULTS：Transfected AAV-CreERT2 and AAV expressing dopamine synthesis enzymes could increase the synthesis of dopamine in HEK293 medium and Parkinson＇s rat striatum （P 〈 0.01） and improve the rotational behavior of Parkinson＇s rats. While tamoxifen markedly reduced overproduction of dopamine caused by cotransfection of viral vectors （P 〈 0.01）, but did not affect the expression and activity of AADC.CONCLUSION：The application of AAV vector-encoded tyrosine hydroxylase gene under the gene regulation system of Cre-ERT2〉, after tamoxifen treatment, can effectively control the generation of genetically modified products to reduce the production of excessive dopamine in vivo and in vitro. Therefore, this method can increase the safety of gene therapy.

Pathogenesis of Crohn's disease: Bug or no bug

The possibility of an infectious origin in inflammatory bowel disease(IBD)has been postulated since the first description of Crohn’s disease(CD).Many observations implicate bacteria as a trigger for the development of CD:lesions occur in regions with higher bacterial concentrations;aphthous ulcers occur in Peyer’s patches;inflammation resolves when the fecal stream is diverted and is reactivated following reinfusion ofbowel contents;severity of the disease is correlated with bacterial density in the mucosa;granulomas can contain bacteria;and susceptible mice raised in germfree conditions develop inflammation when bacteria are introduced in the 1990’s,several studies sought to establish a relationship with viral infections and the onset of IBD,finally concluding that no direct link had been demonstrated.In the past fifteen years,evidence relating IBD pathogenesis to Mycobacterium avium paratuberculosis,salmonella,campylobacter,etc.,has been found.The tendency now under discussion to regard microbiota as the primary catalyst has led to the latest studies on microbiota as pathogens,focusing on Escherichia coli,mainly in ileal CD.The present review discusses the literature available on these"bugs".

Growth differentiation factor 5:a neurotrophic factor with neuroprotective potential in Parkinson’s disease

Parkinson’s disease is the most common movement disorder worldwide,affecting over 6 million people.It is an age-related disease,occurring in 1%of people over the age of 60,and 3%of the population over 80 years.The disease is characterized by the progressive loss of midbrain dopaminergic neurons from the substantia nigra,and their axons,which innervate the striatum,resulting in the characteristic motor and non-motor symptoms of Parkinson’s disease.This is paralleled by the intracellular accumulation ofα-synuclein in several regions of the nervous system.Current therapies are solely symptomatic and do not stop or slow disease progression.One promising disease-modifying strategy to arrest the loss of dopaminergic neurons is the targeted delivery of neurotrophic factors to the substantia nigra or striatum,to protect the remaining dopaminergic neurons of the nigrostriatal pathway.However,clinical trials of two well-established neurotrophic factors,glial cell line-derived neurotrophic factor and neurturin,have failed to meet their primary end-points.This failure is thought to be at least partly due to the downregulation byα-synuclein of Ret,the common co-receptor of glial cell line-derived neurorophic factor and neurturin.Growth/differentiation factor 5 is a member of the bone morphogenetic protein family of neurotrophic factors,that signals through the Ret-independent canonical Smad signaling pathway.Here,we review the evidence for the neurotrophic potential of growth/differentiation factor 5 in in vitro and in vivo models of Parkinson’s disease.We discuss new work on growth/differentiation factor 5’s mechanisms of action,as well as data showing that viral delivery of growth/differentiation factor 5 to the substantia nigra is neuroprotective in theα-synuclein rat model of Parkinson’s disease.These data highlight the potential for growth/differentiation factor 5 as a disease-modifying therapy for Parkinson’s disease.

AAV mediated carboxyl terminus of Hsp70 interacting protein overexpression mitigates the cognitive and pathological phenotypes of APP/PS1 mice

The E3 ubiquitin ligase,carboxyl terminus of heat shock protein 70(Hsp70)interacting protein(CHIP),also functions as a co-chaperone and plays a crucial role in the protein quality control system.In this study,we aimed to investigate the neuroprotective effect of overexpressed CHIP on Alzheimer’s disease.We used an adeno-associated virus vector that can cross the blood-brain barrier to mediate CHIP overexpression in APP/PS1 mouse brain.CHIP overexpression significantly ameliorated the performance of APP/PS1 mice in the Morris water maze and nest building tests,reduced amyloid-βplaques,and decreased the expression of both amyloid-βand phosphorylated tau.CHIP also alleviated the concentration of microglia and astrocytes around plaques.In APP/PS1 mice of a younger age,CHIP overexpression promoted an increase in ADAM10 expression and inhibitedβ-site APP cleaving enzyme 1,insulin degrading enzyme,and neprilysin expression.Levels of HSP70 and HSP40,which have functional relevance to CHIP,were also increased.Single nuclei transcriptome sequencing in the hippocampus of CHIP overexpressed mice showed that the lysosomal pathway and oligodendrocyte-related biological processes were up-regulated,which may also reflect a potential mechanism for the neuroprotective effect of CHIP.Our research shows that CHIP effectively reduces the behavior and pathological manifestations of APP/PS1 mice.Indeed,overexpression of CHIP could be a beneficial approach for the treatment of Alzheimer’s disease.

Comparison of Immune Effect of Four Commercial Marek's Disease Vaccines in Wenchang Chicken

Marek＇s disease（MD） is a lymphoproliferative disease of domestic chickens caused by a highly infectious,oncogenic alpha-herpesvirus known as Marek＇s disease virus（MDV）.The aim of this study was to compare the efficacy of four commercial MDV vaccines in Wenchang chicken.The 1-day old Wenchang chickens tested were injected with one of four different vaccines or not unvaccinated as control;five days later,they were then challenged by virulent MDV strain MD5.The results showed that,in comparison with HVT vaccines,the CVI988 vaccine gave the immunized chickens more potent immunities against challenges of MDV strain MD5.

MDV-1 VP22 conjugated VP2 enhancing immune response against infectious bursal disease virus by DNA vaccination in mice

VP22 of Marek’s disease virus serotype 1 (MDV-1) could function in protein transduction. In this study, an infectious bursal disease virus VP2 gene was fused to the carboxyl termini of VP22. It showed that the fusion protein did not spread into the bystander cells from the cells transfected with pVP22-VP2, as the VP22 alone could. The VP22 proteins were found to be translocated into all the nuclei in the neighboring COS-1 cells, as analyzed by a fluorescence assay. Although mice were immunized with the recombinant DNAs mixed with polyethylenimine (PEI) at a dose of 1:2, it failed to enhance the antibody response against IBDV VP2, as measured by the indirect ELISA assay, yet the cell mediated immune response was significantly increased. The ratio of CD8+/CD4+ T cells was significantly increased in the immunized group with the fusion genes, compared with the group immunized with VP2 (P<0.05). Our results demonstrated that VP22 indeed enhances the cell-mediated response in the fused VP2 in a mice model system, possibly due to the fact that the IBDV VP2 could be carried into the surrounding cells at a limited level under pressure from MDV VP22.

Expression and intercellular trafficking of the VP22 protein of CVI988/Rispens vaccine strain of Marek’s disease virus

The viral protein 22 (VP22) in the tegument of Marek’s disease virus serotype 1 (MDV-1) plays an im-portant role in cell-to-cell spread and viral propagation. Antiserum against the carboxyl terminus of VP22 was prepared by immunizing mice with recombinant VP22 expressed in E. coli, and used to in-vestigate its expression in chicken embryo fibroblast (CEF) cells infected with different MDV-1 strains. At an infection dose of PFU=50, intercellular trafficking of the VP22 into the nuclei of the surrounding receipt cells was detected as early as 3 hours post infection. By 6 hours after infection (before viral plague formation), the protein was detected in the whole nuclei of the recipient cells with no difference among MDV-1 strains CVI988/Rispens, GA and RB1B. Intra-nuclear accumulation of the VP22 protein was further increased when the viral plagues started to form. These results indicate that, albeit the ex-istence of the 201TKSERT206 deletion, the VP22 of the CVI988/Rispens vaccine strain has also intercel-lular-trafficking function, which might serve as a potential alternative delivering protein instead of virulent strains VP22.

Study on the structure of heteropolymer pp38/pp24 and its enhancement on the bi-directional promoter upstream of pp38 gene in Marek’s disease virus

In the latest report, Chloramphenicol acetyltransferase (CAT) gene was used as a reporter to investi-gate the influence of pp38 on its upstream bi-directional promoter, and it was found that the co-expression of pp38 and pp24 can significantly enhance the transactivity of the bi-directional pro-moter between pp38 gene and 1.8-kb mRNA transcript in genome of Marek’s disease virus (MDV). In this study, enhanced green fluorescence protein (EGFP) gene was used as another reporter to further investigate the promoter activity. The transfection shows the promoter has the complete activity under the condition of co-expression of pp38 and pp24 in the same cells. Immunoprecipitation test was used to verify the structure of pp38/pp24 heteropolymer. The pp38-specific monoclonal antibody H19 was used in this test, and pp38, pp24 or both were prepared from the pcDNA-pp38, pcDNA-pp24 or pBud-pp38-pp24 transfected chicken embryonic fibroblast (CEF), respectively. Immunoprecipitation indicates that pp24 could be co-precipitated with pp38 by MabH19, implying that pp24 and pp38 were able to form a heteropolymer in the natural condition. The two separated tests clarify that pp38 and pp24 form a heteropolymer, which enhances the activity of the promoter.