Project description:We analyzed the transcriptional profile of small-intestinal lamina propria (SI-LP) CD4+ T cells isolated from germ-free and mice monocolonized with Bifidobacterium adolescentis, SFB, and Nexabiotic (a 23-strain, Th17-inducing, probiotic mix).
Project description:Varicella Zoster Virus (VZV) is a skin-tropic virus that infects epidermal keratinocytes and causes chickenpox. Although common, VZV infection can be life-threatening particularly in the immunocompromised. Therefore, understanding VZV-keratinocyte interactions is important to find new treatments beyond vaccination and anti-viral drugs. In VZV- infected skin, Kallikrein 6 (KLK6), and the ubiquitin-ligase MDM2 are up-regulated concomitant with Keratin 10 (K10) down-regulation. MDM2 binds to K10 targeting it for degradation via the ubiquitin-proteasome pathway. Preventing K10 degradation reduced VZV propagation in culture and prevented epidermal disruption in skin explants. K10 knockdown induced expression of the nuclear receptor subfamily 4, group A, member 1 (NR4A1) and enhanced viral propagation in culture. NR4A1 knockdown prevented viral propagation in culture, reduced LC3 levels and increased LAMP2 expression. We therefore describe a novel drug-able pathway whereby MDM2 ubiquitinates and degrades K10 increasing NR4A1 expression allowing VZV replication and propagation.
Project description:Background: Smallpox was eradicated by a global program of inoculation with Vaccinia virus (VV). Robust VV-specific CD4 T-cell responses during primary infection are likely essential to controlling VV replication. Although there is increasing interest in cytolytic CD4 T-cells across many viral infections, the importance of these cells during acute VV infection is unclear. Methods: We undertook a detailed functional and genetic characterization of CD4 T-cells during acute VV-infection of humans. VV-specific T-cells were identified by up-regulation of activation markers directly ex vivo and through cytokine and co-timulatory molecule expression. At day-13-post primary inoculation with VV, CD38highCD45RO+ CD4 T-cells were purified by cell sorting, RNA isolated and analysed by microarray. Differential expression of up-regulated genes in activated CD4 T-cells was confirmed at the mRNA and protein levels. We compared analyses of VV-specific CD4 T-cells to studies on 12 subjects with primary HIV infection (PHI). VV-specific T-cells lines were established from PBMCs collected post vaccination and checked for cytotoxicity potential. Results: A median 11.9% CD4 T-cells were CD38highCD45RO+ at day-13 post-VV inoculation, compared to 3.0% prior and 10.4% during PHI. Activated CD4 T-cells had an up-regulation of genes related to cytolytic function, including granzymes K and A, perforin, granulysin, TIA-1, and Rab27a. No difference was seen between CD4 T-cell expression of perforin or TIA-1 to VV and PHI, however granzyme k was more dominant in the VV response. At 25:1 effector to target ratio, two VV-specific T-cell lines exhibited 62% and 30% cytotoxicity respectively and CD107a degranulation. Conclusions: We show for the first time that CD4 CTL are prominent in the early response to VV. Understanding the role of CD4 CTL in the generation of an effective anti-viral memory may help develop more effective vaccines for diseases such as HIV.
Project description:Smallpox is a highly communicable, often fatal diseae. There is currently no licensed treatment for smallpox and vaccinia virus (VV) is currently used for immunization. While immunization with VV can provide good protection against exposure to the smallpox virus, the current vaccine is far from optimal. Complications occur in 1/1,000-1/10,000 vaccinees, with at least one death per million vaccinees. We have constructed recombinant VV strains which are less pathogenic, yet provide a protective immune response. These viruses contain various mutations in the E3L which is known to block the host antiviral response. Identifying the host genes involved in producing a strong protective immunological response to these attenuated viruses would not only increase our understanding of the proteins and pathways involved in effective smallpox vaccination, but aid in the development of alternative vaccine strains which enhance these specific immune responses. We will determine gene expression patterns in HeLa cells at various times following infection with wtVV and several VV constructs containing mutations in the E3L gene. The VV E3L gene product blocks the host antiviral response by sequestering viral danger signals, including double-stranded RNA and Z-DNA. VV constructs containing mutations in E3L which allow host cell recognition of either of these danger signals leads to a decrease in viral pathogensis. In this project we will dissect the cellular inflammatory response to infection with wtVV in comparison to VV containing mutations in the E3L gene. By understanding why certain strains of VV are non-pathogenic, yet highly immunogenic, it is possible to gain a better understanding on the mechanisms of poxvirus pathogenesis and the host response. We will examine three times points following infection with VV: 2 HPI, 6 HPI and 9 HPI. These times points represent keys points in the virus replication cycle. Several VV constructs will be used which contain mutations in the E3L gene. These constructs alter the ability of E3L to sequester double-stranded RNA and/or Z-DNA and therefore have a direct effect on viral pathogenesis. Fourteen constructs will be used including: mock, wtVV, VVdelE3L, VVE3Ldel83N, VVE3Ldel37N, VVE3Ldel26C, VVE3Ldel7C, VVE3L Y48A, VVE3L P63A, VVE3L K167T, VV-ATV, VV-ADAR/E3L, VVdelK3L, VVdelK3L-E3Ldel37N. Cells will be infected at an MOI of 5 to allow infection of all cells. At each time point, cells will be harvested by scraping. RNA will be isolated using a Trizol RNA extraction protocol (Invitrogen) followed by RNA purification using the RNeasy cleanup kit available from Qiagen. Keywords: time-course
Project description:microRNAs(miRNAs) play critical regulatory roles mainly through cleaving targeted mRNAs or repressing gene translation during plant developments. Grapevine is amongst the most economically important fruit crops with whole genome available, and the study on grapevine miRNAs (Vv-miRNAs) have also been emphasized. However, the regulation mode of Vv-miRNAs on their target mRNAs during grapevine development has not been studied well, especially on a transcriptome-wide level. Here, six small RNA (sRNA) and mRNA libraries from various grapevine tissues were constructed for Illumina and Degradome sequencing. Subsequently, the spatiotemporal variation in the Vv-miRNAs’ regulation on their target genes was systematically analyzed. Totally, 242 known and 132 novel Vv-miRNAs were identified, and 193 target mRNAs including 103 for known and 90 for novel miRNAs were validated in one or more of tissues examined. The interesting finding was that over 50% of novel miRNAs were expressed exclusively in flowers or berries where they had tissue-specific cleavage roles on their target genes, especially, the breadth of their cleavage sites in flower tissues. Moreover, six novel miRNAs in berries were found to response to exogenous gibberellin (GA) and/or ethylene by real time RT-PCR (qRT-PCR) analysis, confirming their regulatory functions during berry development. Other finding was that about 93.6% of the known miRNAs possessed the high conservation in various tissues where their expression levels exhibited some dynamic variations during grapevine development. Significantly, it was found the phenomena that some Vv-miRNA families exist one key member that act as the main regulator of their target genes during grapevine development.
Project description:microRNAs(miRNAs) play critical regulatory roles mainly through cleaving targeted mRNAs or repressing gene translation during plant developments. Grapevine is amongst the most economically important fruit crops with whole genome available, and the study on grapevine miRNAs (Vv-miRNAs) have also been emphasized. However, the regulation mode of Vv-miRNAs on their target mRNAs during grapevine development has not been studied well, especially on a transcriptome-wide level. Here, six small RNA (sRNA) and mRNA libraries from various grapevine tissues were constructed for Illumina and Degradome sequencing. Subsequently, the spatiotemporal variation in the Vv-miRNAs’ regulation on their target genes was systematically analyzed. Totally, 242 known and 132 novel Vv-miRNAs were identified, and 193 target mRNAs including 103 for known and 90 for novel miRNAs were validated in one or more of tissues examined. The interesting finding was that over 50% of novel miRNAs were expressed exclusively in flowers or berries where they had tissue-specific cleavage roles on their target genes, especially, the breadth of their cleavage sites in flower tissues. Moreover, six novel miRNAs in berries were found to response to exogenous gibberellin (GA) and/or ethylene by real time RT-PCR (qRT-PCR) analysis, confirming their regulatory functions during berry development. Other finding was that about 93.6% of the known miRNAs possessed the high conservation in various tissues where their expression levels exhibited some dynamic variations during grapevine development. Significantly, it was found the phenomena that some Vv-miRNA families exist one key member that act as the main regulator of their target genes during grapevine development.
Project description:Smallpox is a highly communicable, often fatal diseae. There is currently no licensed treatment for smallpox and vaccinia virus (VV) is currently used for immunization. While immunization with VV can provide good protection against exposure to the smallpox virus, the current vaccine is far from optimal. Complications occur in 1/1,000-1/10,000 vaccinees, with at least one death per million vaccinees. We have constructed recombinant VV strains which are less pathogenic, yet provide a protective immune response. These viruses contain various mutations in the E3L which is known to block the host antiviral response. Identifying the host genes involved in producing a strong protective immunological response to these attenuated viruses would not only increase our understanding of the proteins and pathways involved in effective smallpox vaccination, but aid in the development of alternative vaccine strains which enhance these specific immune responses. We will determine gene expression patterns in HeLa cells at various times following infection with wtVV and several VV constructs containing mutations in the E3L gene. The VV E3L gene product blocks the host antiviral response by sequestering viral danger signals, including double-stranded RNA and Z-DNA. VV constructs containing mutations in E3L which allow host cell recognition of either of these danger signals leads to a decrease in viral pathogensis. In this project we will dissect the cellular inflammatory response to infection with wtVV in comparison to VV containing mutations in the E3L gene. By understanding why certain strains of VV are non-pathogenic, yet highly immunogenic, it is possible to gain a better understanding on the mechanisms of poxvirus pathogenesis and the host response. We will examine three times points following infection with VV: 2 HPI, 6 HPI and 9 HPI. These times points represent keys points in the virus replication cycle. Several VV constructs will be used which contain mutations in the E3L gene. These constructs alter the ability of E3L to sequester double-stranded RNA and/or Z-DNA and therefore have a direct effect on viral pathogenesis. Fourteen constructs will be used including: mock, wtVV, VVdelE3L, VVE3Ldel83N, VVE3Ldel37N, VVE3Ldel26C, VVE3Ldel7C, VVE3L Y48A, VVE3L P63A, VVE3L K167T, VV-ATV, VV-ADAR/E3L, VVdelK3L, VVdelK3L-E3Ldel37N. Cells will be infected at an MOI of 5 to allow infection of all cells. At each time point, cells will be harvested by scraping. RNA will be isolated using a Trizol RNA extraction protocol (Invitrogen) followed by RNA purification using the RNeasy cleanup kit available from Qiagen.