AIM To stably correct tyrosinaemia in proliferating livers of fumarylacetoacetate-hydrolase knockout(Fah-/-)mice by homologous-recombination-mediated targeted addition of the Fah gene.METHODS C57 BL/6 Fah?exon5 mice s...AIM To stably correct tyrosinaemia in proliferating livers of fumarylacetoacetate-hydrolase knockout(Fah-/-)mice by homologous-recombination-mediated targeted addition of the Fah gene.METHODS C57 BL/6 Fah?exon5 mice served as an animal model for human tyrosinaemia type 1 in our study.The vector was created by amplifying human Fah c DNA including the TTR promoter from a lentivirus plasmid as described.The Fah expression cassette was flanked by homologous arms(620 bp and 749 bp long)of the Rosa26 gene locus.Mice were injected with 2.1×108 VP of this vector(r AAV8-ROSA26.HAL-TTR.FahROSA26.HAR)via the tail vein.Mice in the control group were injected with 2.1×108 VP of a similar vector but missing the homologous arms(r AAV8-TTR.Fah).Primary hepatocytes from Fah-/-recipient mice,treated with our vectors,were isolated and 1×106 hepatocytes were transplanted into secondary Fah-/-recipient mice by injection into the spleen.Upon either vector application or hepatocyte transplantation NTBC treatment was stopped in recipient mice.RESULTS Here,we report successful HR-mediated genome editing by integration of a Fah gene expression cassette into the"safe harbour locus"Rosa26 by recombinant AAV8.Both groups of mice showed long-term survival,weight gain and FAH positive clusters as determined by immunohistochemistry analysis of liver sections in the absence of NTBC treatment.In the group of C57 BL/6 Fah?exon5 mice,which have been transplanted with hepatocytes from a mouse injected with r AAV8-ROSA26.HAL-TTR.Fah-ROSA26.HAR 156 d before,6 out of 6 mice showed long-term survival,weight gain and FAH positive clusters without need for NTBC treatment.In contrast only 1 out 5 mice,who received hepatocytes from r AAV8-TTR.Fah treated mice,survived and showed few and smaller FAH positive clusters.These results demonstrate that homologous recombinationmediated Fah gene transfer corrects the phenotype in a mouse model of human tyrosinaemia type 1(Fah-/-mice)and is long lasting in a proliferating state of the liver as shown by withdrawal of NTBC treatment and serial transplantation of isolated hepatocytes from primary Fah-/-recipient mice into secondary Fah-/-recipient mice.This long term therapeutic efficacy is clearly superior to our control mice treated with episomal r AAV8 gene therapy approach.CONCLUSION HR-mediated r AAV8 gene therapy provides targeted transgene integration and phenotypic correction in Fah-/-mice with superior long-term efficacy compared to episomal r AAV8 therapy in proliferating livers.展开更多
Many newly emerging and re-emerging viruses have neuroinvasive potential,underscoring viral encephalitis as a global research priority.Upon entry of the virus into the CNS,severe neurological life-threatening conditio...Many newly emerging and re-emerging viruses have neuroinvasive potential,underscoring viral encephalitis as a global research priority.Upon entry of the virus into the CNS,severe neurological life-threatening conditions may manifest that are associated with high morbidity and mortality.The currently available therapeutic arsenal against viral encephalitis is rather limited,emphasizing the need to better understand the conditions of local antiviral immunity within the infected CNS.In this review,we discuss new insights into the pathophysiology of viral encephalitis,with a focus on myeloid cells and CD8^(+)T cells,which critically contribute to protection against viral CNS infection.By illuminating the prerequisites of myeloid and T cell activation,discussing new discoveries regarding their transcriptional signatures,and dissecting the mechanisms of their recruitment to sites of viral replication within the CNS,we aim to further delineate the complexity of antiviral responses within the infected CNS.Moreover,we summarize the current knowledge in the field of virus infection and neurodegeneration and discuss the potential links of some neurotropic viruses with certain pathological hallmarks observed in neurodegeneration.展开更多
Bronchus-associated lymphoid tissue(BALT)develops at unpredictable locations around lung bronchi following pulmonary inflammation.The formation and composition of BALT have primarily been investigated by immunohistolo...Bronchus-associated lymphoid tissue(BALT)develops at unpredictable locations around lung bronchi following pulmonary inflammation.The formation and composition of BALT have primarily been investigated by immunohistology that,due to the size of the invested organ,is usually restricted to a limited number of histological sections.To assess the entire BALT of the lung,other approaches are urgently needed.Here,we introduce a novel light sheet microscopy-based approach for assessing lymphoid tissue in the lung.Using antibody staining of whole lung lobes and optical clearing by organic solvents,we present a method that allows in-depth visualization of the entire bronchial tree,the lymphatic vasculature and the immune cell composition of the induced BALT.Furthermore,three-dimensional analysis of the entire lung allows the qualitative and quantitative enumeration of the induced BALT.Using this approach,we show that a single intranasal application of the replication-deficient poxvirus MVA induces BALT that constitutes up to 8%of the entire lung volume in mice deficient in CCR7,in contrast to wild type mice(WT).Furthermore,BALT induced by heat-inactivated E.coli is dominated by a pronounced T cell infiltration in Cxcr5-deficient mice,in contrast to WT mice.展开更多
Background:Post-Soviet Kyrgyzstan has experienced a major surge in blood-borne infections,but data from adequately powered,up-to-date studies are lacking.We thus examined a)the seroprevalences of hepatitis B virus sur...Background:Post-Soviet Kyrgyzstan has experienced a major surge in blood-borne infections,but data from adequately powered,up-to-date studies are lacking.We thus examined a)the seroprevalences of hepatitis B virus surface antigen(HBsAg),HIV-1 p24 antigen and antibodies against hepatitis C virus(anti-HCV),human immunodeficiency viruses(anti-HIV-1/2,HIV-1 group O),and Treponema pallidum among blood donors in Kyrgyzstan and assess their distribution according to sex,age,and provinces of residence;b)trends in the respective seroprevalences;and c)co-infection rates among the pathogens studied.Methods:Serological screening was performed on 37165 blood donors at the Republican Blood Centre in Bishkek,Kyrgyzstan,between January 2013 and December 2015.We applied poststratification weights to control for sampling bias and used logistic regression analyses to examine the association of seropositivity and co-infections with sex,age,provinces of residence,and year of blood donation.Results:Twenty nine thousand and one hundred forty-five(78%)donors were males and 8020(22%)were females.The median age was 27 years(range:18-64).The prevalences of HBsAg,anti-HCV,HIV(p24 Ag and anti-HIV),and anti-T.pallidum were 3.6%(95%CI:3.4-3.8%),3.1%(3.0-3.3%),0.78%(0.69-0.87%),and 3.3%(3.1-3.5%),respectively.Males were more likely to be seropositive for HBsAg than females(OR:1.63;95%CI:1.40-1.90),but less likely to be seropositive for anti-HCV(0.85;0.74-0.98)and HIV(0.65;0.49-0.85).Prevalences were lower in the capital than in the other provinces.There was a decreasing trend in the seroprevalences of HBsAg,anti-HCV,and anti-T.pallidum from 2012 to 2015(P-value for trend,P=0.01,P<0.0001,P<0.0001,respectively),while the seroprevalence of HIV increased(P=0.049).One hundred eighty donors(0.48%)were seropositive for multiple infections.The highest co-infection rate was observed between anti-T.pallidum and HBsAg(6.0%),followed by anti-HCV and anti-T.pallidum(5.2%),and HIV and anti-HCV(4.9%).Conclusions:The data suggest that Kyrgyzstan can be reclassified from high to lower-intermediate HBsAg endemicity,whereas the high HIV prevalence with a rising trend is an alarming finding that needs to be urgently addressed by public health authorities.The observed co-infections suggest common risk factors but also common preventive interventions.展开更多
Dendritic cells(DCs)are indispensable for defense against pathogens but may also contribute to immunopathology.Activation of DCs upon the sensing of pathogens by Toll-like receptors(TLRs)is largely mediated by pattern...Dendritic cells(DCs)are indispensable for defense against pathogens but may also contribute to immunopathology.Activation of DCs upon the sensing of pathogens by Toll-like receptors(TLRs)is largely mediated by pattern recognition receptor/nuclear factor-κB(NF-κB)signaling and depends on the appropriate ubiquitination of the respective signaling molecules.However,the ubiquitinating and deubiquitinating enzymes involved and their interactions are only incompletely understood.Here,we reveal that the deubiquitinase OTU domain,ubiquitin aldehyde binding 1(OTUB1)is upregulated in DCs upon murine Toxoplasma gondii infection and lipopolysaccharide challenge.Stimulation of DCs with the TLR11/12 ligand T.gondii profilin and the TLR4 ligand lipopolysaccharide induced an increase in NF-κB activation in OTUB1-competent cells,resulting in elevated interleukin-6(IL-6),IL-12,and tumor necrosis factor(TNF)production,which was also observed upon the specific stimulation of TLR2,TLR3,TLR7,and TLR9.Mechanistically,OTUB1 promoted NF-κB activity in DCs by K48-linked deubiquitination and stabilization of the E2-conjugating enzyme UBC13,resulting in increased K63-linked ubiquitination of IRAK1(IL-1 receptor-associated kinase 1)and TRAF6(TNF receptor-associated factor 6).Consequently,DC-specific deletion of OTUB1 impaired the production of cytokines,in particular IL-12,by DCs over the first 2 days of T.gondii infection,resulting in the diminished production of protective interferon-γ(IFN-γ)by natural killer cells,impaired control of parasite replication,and,finally,death from chronic T.encephalitis,all of which could be prevented by low-dose IL-12 treatment in the first 3 days of infection.In contrast,impaired OTUB1-deficient DC activation and cytokine production by OTUB1-deficient DCs protected mice from lipopolysaccharide-induced immunopathology.Collectively,these findings identify OTUB1 as a potent novel regulator of DCs during infectious and inflammatory diseases.展开更多
Egypt has the highest prevalence of chronic hepatitis C virus (HCV) infection and seropositivity worldwide, and it has been proposed that this enhanced susceptibility to HCV is related to coinfection with schistosomia...Egypt has the highest prevalence of chronic hepatitis C virus (HCV) infection and seropositivity worldwide, and it has been proposed that this enhanced susceptibility to HCV is related to coinfection with schistosomiasis. Although currently, there are no studies regarding the actual prevalence of both human schistosomiasis and schistosomiasis/HCV coinfection evi-dences strongly support that eliminating human schistoso-miasis from Egypt is necessary to reduce both HCV prevalence and liver pathology. The present review highlights the significant impact of the neglected tropical disease human schistosomiasis on both susceptibility of Egyptians to HCV coinfection, severity of the resulting liver pathology, and poor response to antiviral therapy. The immune evasion mechan-isms exerted by the HCV-NS3/4A protease domain, and the possible impact of immune evasion mechanisms exerted by proteases of larval, worm and egg stages of the parasite Schistosoma on human susceptibility to HCV infection are discussed. In addition, schistosome immune evasion mechanisms may include immunosuppression that in turn prevents clearance of HCV viremia and leads to relapsing HCV infection and severe liver pathology. I propose the generation of a replicon system from the most prevailing genotype (HCV-4a) in Egypt and establishing its replication on hepatoplas-toma or immune cells in presence of bilharzial antigens. Finally, the use of a humanized small animal model that can acquire both HCV and S. mansoni infections will be important to further understand in real time the impact of coinfection on both the immune system and liver pathology.展开更多
基金SFB 738the"Deutsche Forschungsgemeinschaft"(Gerok-Grant)for financial support
文摘AIM To stably correct tyrosinaemia in proliferating livers of fumarylacetoacetate-hydrolase knockout(Fah-/-)mice by homologous-recombination-mediated targeted addition of the Fah gene.METHODS C57 BL/6 Fah?exon5 mice served as an animal model for human tyrosinaemia type 1 in our study.The vector was created by amplifying human Fah c DNA including the TTR promoter from a lentivirus plasmid as described.The Fah expression cassette was flanked by homologous arms(620 bp and 749 bp long)of the Rosa26 gene locus.Mice were injected with 2.1×108 VP of this vector(r AAV8-ROSA26.HAL-TTR.FahROSA26.HAR)via the tail vein.Mice in the control group were injected with 2.1×108 VP of a similar vector but missing the homologous arms(r AAV8-TTR.Fah).Primary hepatocytes from Fah-/-recipient mice,treated with our vectors,were isolated and 1×106 hepatocytes were transplanted into secondary Fah-/-recipient mice by injection into the spleen.Upon either vector application or hepatocyte transplantation NTBC treatment was stopped in recipient mice.RESULTS Here,we report successful HR-mediated genome editing by integration of a Fah gene expression cassette into the"safe harbour locus"Rosa26 by recombinant AAV8.Both groups of mice showed long-term survival,weight gain and FAH positive clusters as determined by immunohistochemistry analysis of liver sections in the absence of NTBC treatment.In the group of C57 BL/6 Fah?exon5 mice,which have been transplanted with hepatocytes from a mouse injected with r AAV8-ROSA26.HAL-TTR.Fah-ROSA26.HAR 156 d before,6 out of 6 mice showed long-term survival,weight gain and FAH positive clusters without need for NTBC treatment.In contrast only 1 out 5 mice,who received hepatocytes from r AAV8-TTR.Fah treated mice,survived and showed few and smaller FAH positive clusters.These results demonstrate that homologous recombinationmediated Fah gene transfer corrects the phenotype in a mouse model of human tyrosinaemia type 1(Fah-/-mice)and is long lasting in a proliferating state of the liver as shown by withdrawal of NTBC treatment and serial transplantation of isolated hepatocytes from primary Fah-/-recipient mice into secondary Fah-/-recipient mice.This long term therapeutic efficacy is clearly superior to our control mice treated with episomal r AAV8 gene therapy approach.CONCLUSION HR-mediated r AAV8 gene therapy provides targeted transgene integration and phenotypic correction in Fah-/-mice with superior long-term efficacy compared to episomal r AAV8 therapy in proliferating livers.
基金supported by funding from the Deutsche Forschungsgemeinschaft(DFGGerman Research Foundation)—398066876/GRK 2485/1 to UK,the DFG under Germany’s Excellence Strategy—EXC 2155“RESIST”—Project ID 39087428 to UKby the Helmholtz Association(Zukunftsthema“Immunology&Inflammation”(ZT-0027))to UK.Figures were created using Biorender.com.We thank Elisabeth Janecek-Erfurth for her help with the submission of the manuscript.
文摘Many newly emerging and re-emerging viruses have neuroinvasive potential,underscoring viral encephalitis as a global research priority.Upon entry of the virus into the CNS,severe neurological life-threatening conditions may manifest that are associated with high morbidity and mortality.The currently available therapeutic arsenal against viral encephalitis is rather limited,emphasizing the need to better understand the conditions of local antiviral immunity within the infected CNS.In this review,we discuss new insights into the pathophysiology of viral encephalitis,with a focus on myeloid cells and CD8^(+)T cells,which critically contribute to protection against viral CNS infection.By illuminating the prerequisites of myeloid and T cell activation,discussing new discoveries regarding their transcriptional signatures,and dissecting the mechanisms of their recruitment to sites of viral replication within the CNS,we aim to further delineate the complexity of antiviral responses within the infected CNS.Moreover,we summarize the current knowledge in the field of virus infection and neurodegeneration and discuss the potential links of some neurotropic viruses with certain pathological hallmarks observed in neurodegeneration.
基金This work was supported by a Lichtenberg-stipend to DTM through the University of Veterinary Medicine,Hannover,the German Excellence Initiative grant EXC62-Rebirth(to RF)DFG grant SFB 900/B1(to RF).
文摘Bronchus-associated lymphoid tissue(BALT)develops at unpredictable locations around lung bronchi following pulmonary inflammation.The formation and composition of BALT have primarily been investigated by immunohistology that,due to the size of the invested organ,is usually restricted to a limited number of histological sections.To assess the entire BALT of the lung,other approaches are urgently needed.Here,we introduce a novel light sheet microscopy-based approach for assessing lymphoid tissue in the lung.Using antibody staining of whole lung lobes and optical clearing by organic solvents,we present a method that allows in-depth visualization of the entire bronchial tree,the lymphatic vasculature and the immune cell composition of the induced BALT.Furthermore,three-dimensional analysis of the entire lung allows the qualitative and quantitative enumeration of the induced BALT.Using this approach,we show that a single intranasal application of the replication-deficient poxvirus MVA induces BALT that constitutes up to 8%of the entire lung volume in mice deficient in CCR7,in contrast to wild type mice(WT).Furthermore,BALT induced by heat-inactivated E.coli is dominated by a pronounced T cell infiltration in Cxcr5-deficient mice,in contrast to WT mice.
基金The study was funded by internal funds of the Helmholtz Centre for Infection Research,Braunschweig,Germany and by iMed-the Helmholtz Association’s Cross-Program Initiative on Personalized Medicine.
文摘Background:Post-Soviet Kyrgyzstan has experienced a major surge in blood-borne infections,but data from adequately powered,up-to-date studies are lacking.We thus examined a)the seroprevalences of hepatitis B virus surface antigen(HBsAg),HIV-1 p24 antigen and antibodies against hepatitis C virus(anti-HCV),human immunodeficiency viruses(anti-HIV-1/2,HIV-1 group O),and Treponema pallidum among blood donors in Kyrgyzstan and assess their distribution according to sex,age,and provinces of residence;b)trends in the respective seroprevalences;and c)co-infection rates among the pathogens studied.Methods:Serological screening was performed on 37165 blood donors at the Republican Blood Centre in Bishkek,Kyrgyzstan,between January 2013 and December 2015.We applied poststratification weights to control for sampling bias and used logistic regression analyses to examine the association of seropositivity and co-infections with sex,age,provinces of residence,and year of blood donation.Results:Twenty nine thousand and one hundred forty-five(78%)donors were males and 8020(22%)were females.The median age was 27 years(range:18-64).The prevalences of HBsAg,anti-HCV,HIV(p24 Ag and anti-HIV),and anti-T.pallidum were 3.6%(95%CI:3.4-3.8%),3.1%(3.0-3.3%),0.78%(0.69-0.87%),and 3.3%(3.1-3.5%),respectively.Males were more likely to be seropositive for HBsAg than females(OR:1.63;95%CI:1.40-1.90),but less likely to be seropositive for anti-HCV(0.85;0.74-0.98)and HIV(0.65;0.49-0.85).Prevalences were lower in the capital than in the other provinces.There was a decreasing trend in the seroprevalences of HBsAg,anti-HCV,and anti-T.pallidum from 2012 to 2015(P-value for trend,P=0.01,P<0.0001,P<0.0001,respectively),while the seroprevalence of HIV increased(P=0.049).One hundred eighty donors(0.48%)were seropositive for multiple infections.The highest co-infection rate was observed between anti-T.pallidum and HBsAg(6.0%),followed by anti-HCV and anti-T.pallidum(5.2%),and HIV and anti-HCV(4.9%).Conclusions:The data suggest that Kyrgyzstan can be reclassified from high to lower-intermediate HBsAg endemicity,whereas the high HIV prevalence with a rising trend is an alarming finding that needs to be urgently addressed by public health authorities.The observed co-infections suggest common risk factors but also common preventive interventions.
基金funded by the Deutsche Forschungsgemeinschaft(DFG,German Research Foundation)-Project-ID 97850925-SFB 854(project TP5 to M.N.and DS),Project-ID 406922110-Joint French-German Project cGASVAC(to U.K.)Project-ID 158989968-SFB900(project B2 to U.K.)。
文摘Dendritic cells(DCs)are indispensable for defense against pathogens but may also contribute to immunopathology.Activation of DCs upon the sensing of pathogens by Toll-like receptors(TLRs)is largely mediated by pattern recognition receptor/nuclear factor-κB(NF-κB)signaling and depends on the appropriate ubiquitination of the respective signaling molecules.However,the ubiquitinating and deubiquitinating enzymes involved and their interactions are only incompletely understood.Here,we reveal that the deubiquitinase OTU domain,ubiquitin aldehyde binding 1(OTUB1)is upregulated in DCs upon murine Toxoplasma gondii infection and lipopolysaccharide challenge.Stimulation of DCs with the TLR11/12 ligand T.gondii profilin and the TLR4 ligand lipopolysaccharide induced an increase in NF-κB activation in OTUB1-competent cells,resulting in elevated interleukin-6(IL-6),IL-12,and tumor necrosis factor(TNF)production,which was also observed upon the specific stimulation of TLR2,TLR3,TLR7,and TLR9.Mechanistically,OTUB1 promoted NF-κB activity in DCs by K48-linked deubiquitination and stabilization of the E2-conjugating enzyme UBC13,resulting in increased K63-linked ubiquitination of IRAK1(IL-1 receptor-associated kinase 1)and TRAF6(TNF receptor-associated factor 6).Consequently,DC-specific deletion of OTUB1 impaired the production of cytokines,in particular IL-12,by DCs over the first 2 days of T.gondii infection,resulting in the diminished production of protective interferon-γ(IFN-γ)by natural killer cells,impaired control of parasite replication,and,finally,death from chronic T.encephalitis,all of which could be prevented by low-dose IL-12 treatment in the first 3 days of infection.In contrast,impaired OTUB1-deficient DC activation and cytokine production by OTUB1-deficient DCs protected mice from lipopolysaccharide-induced immunopathology.Collectively,these findings identify OTUB1 as a potent novel regulator of DCs during infectious and inflammatory diseases.
文摘Egypt has the highest prevalence of chronic hepatitis C virus (HCV) infection and seropositivity worldwide, and it has been proposed that this enhanced susceptibility to HCV is related to coinfection with schistosomiasis. Although currently, there are no studies regarding the actual prevalence of both human schistosomiasis and schistosomiasis/HCV coinfection evi-dences strongly support that eliminating human schistoso-miasis from Egypt is necessary to reduce both HCV prevalence and liver pathology. The present review highlights the significant impact of the neglected tropical disease human schistosomiasis on both susceptibility of Egyptians to HCV coinfection, severity of the resulting liver pathology, and poor response to antiviral therapy. The immune evasion mechan-isms exerted by the HCV-NS3/4A protease domain, and the possible impact of immune evasion mechanisms exerted by proteases of larval, worm and egg stages of the parasite Schistosoma on human susceptibility to HCV infection are discussed. In addition, schistosome immune evasion mechanisms may include immunosuppression that in turn prevents clearance of HCV viremia and leads to relapsing HCV infection and severe liver pathology. I propose the generation of a replicon system from the most prevailing genotype (HCV-4a) in Egypt and establishing its replication on hepatoplas-toma or immune cells in presence of bilharzial antigens. Finally, the use of a humanized small animal model that can acquire both HCV and S. mansoni infections will be important to further understand in real time the impact of coinfection on both the immune system and liver pathology.
