Uvants. There are little data however, that address whether these compounds have similar activity when taken orally, and whether purified compounds or crude extracts commonly used as dietary supplements affect host defense responses through this route of administration. In this study, potential mechanisms of immune system modulating activity of orally administered GRA were investigated. Analysis of cytokine gene expression in small intestinal tissue following administration of GRA revealed a specific pattern of chemokine and chemokine receptor gene expression that was predictive of B cell recruitment to the gut mucosa. Increases in CD19+ B cells in the small intestinal lamina propria were observed in GRA-treated mice, and histological analyses identified B220+ B cell 
clusters with morphology and cell content consistent with structures of isolated lymphoid follicles (ILFs). The ability of GRA to induce lymphoid tissue 1326631 maturation independently of ectopic antigenic stimulus suggests GRA affects immune cell responses in the gut and activates signaling pathways favorable to modulation of mucosal B cell populations. Using the adult mouse model of rotavirus infection, we further show that GRA shortened the duration of viral antigen shedding, suggesting the changes in gene expression and lymphocyte recruitment to the intestine induced by GRA likely is functionally relevant in enteric virus infection.(Qiagen) at 4uC for a minimum of 18 hrs. All sections were devoid of Peyer’s Patches. RNA was extracted with the RNeasy system (Qiagen) and quantified with a Nanodrop 1000 (Fisher Scientific). Cytokine transcripts were measured with the SABiosciences Mouse MedChemExpress Madrasin Inflammatory Cytokine Array (PAMM-011A) or Custom Mouse RT2 ProfilerTM. Custom arrays included Cxcr5, Ccl19, Ccl21b, Cxcl13, Lta, Ltb, Ccr6, Ccr7, Ccr9, Ifng, and Il10. One mg of RNA was reverse transcribed with RT2 First Strand kit (SABiosciences) following the Licochalcone A web manufacturer’s instructions. PCR reactions were performed on an Realplex 4 s (Eppendorf ). Reaction conditions consisted of 95uC for 10 minutes, followed by 40 cycles of 95uC for 15 seconds, and 60uC for one minute. Data from a minimum of three mice per group were combined and are expressed as fold-change over vehicle-treated animals. Foldchanges .2 were scored as significant.Harvesting and Analysis of Intestinal Cell PopulationsAt the indicated times post-dosing and/or post-infection, cells from the Peyer’s Patches (PPs), mesenteric lymph nodes (MLNs), and small intestinal lamina propria (LP) were isolated as previously described [21]. Antibodies used for staining and analysis by flow cytometry included: anti-CD4 A488, anti-CD8 PE, anti-CD19 PE-cy7, anti-CD69 eF605, anti-CD127 PE-cy5, anti-CD185 PE, and anti-CD8a AF700, all from eBiosciences. Anti-CD138 PE and anti-CD11c APC were from BD Biosciences. Flow cytometry was performed on a BD LSR flow cytometer using FacsDIVA software and data were analyzed with FlowJo software.Materials and Methods Ethics StatementAll animal experiments were performed according to the NIH Guidelines for Care and Use of Animals, with approval from the Montana State University Institutional Animal Care and Use Committee (Protocol number 2011-44).ELISAs for Fecal Rotavirus Antigen Shedding and Antirotavirus Serum AntibodyELISA for fecal rotavirus antigen detection was performed as previously described [22]. Fecal samples were diluted 10-fold w/v in TNC (50 mM Tris, 150 mM NaCl, 5 mM CaCl2) containing 0.05 Tween-.Uvants. There are little data however, that address whether these compounds have similar activity when taken orally, and whether purified compounds or crude extracts commonly used as dietary supplements affect host defense responses through this route of administration. In this study, potential mechanisms of immune system modulating activity of orally administered GRA were investigated. Analysis of cytokine gene expression in small intestinal tissue following administration of GRA revealed a specific pattern of chemokine and chemokine receptor gene expression that was predictive of B cell recruitment to the gut mucosa. Increases in CD19+ B cells in the small intestinal lamina propria were observed in GRA-treated mice, and histological analyses identified B220+ B cell clusters with morphology and cell content consistent with structures of isolated lymphoid follicles (ILFs). The ability of GRA to induce lymphoid tissue 1326631 maturation independently of ectopic antigenic stimulus suggests GRA affects immune cell responses in 
the gut and activates signaling pathways favorable to modulation of mucosal B cell populations. Using the adult mouse model of rotavirus infection, we further show that GRA shortened the duration of viral antigen shedding, suggesting the changes in gene expression and lymphocyte recruitment to the intestine induced by GRA likely is functionally relevant in enteric virus infection.(Qiagen) at 4uC for a minimum of 18 hrs. All sections were devoid of Peyer’s Patches. RNA was extracted with the RNeasy system (Qiagen) and quantified with a Nanodrop 1000 (Fisher Scientific). Cytokine transcripts were measured with the SABiosciences Mouse Inflammatory Cytokine Array (PAMM-011A) or Custom Mouse RT2 ProfilerTM. Custom arrays included Cxcr5, Ccl19, Ccl21b, Cxcl13, Lta, Ltb, Ccr6, Ccr7, Ccr9, Ifng, and Il10. One mg of RNA was reverse transcribed with RT2 First Strand kit (SABiosciences) following the manufacturer’s instructions. PCR reactions were performed on an Realplex 4 s (Eppendorf ). Reaction conditions consisted of 95uC for 10 minutes, followed by 40 cycles of 95uC for 15 seconds, and 60uC for one minute. Data from a minimum of three mice per group were combined and are expressed as fold-change over vehicle-treated animals. Foldchanges .2 were scored as significant.Harvesting and Analysis of Intestinal Cell PopulationsAt the indicated times post-dosing and/or post-infection, cells from the Peyer’s Patches (PPs), mesenteric lymph nodes (MLNs), and small intestinal lamina propria (LP) were isolated as previously described [21]. Antibodies used for staining and analysis by flow cytometry included: anti-CD4 A488, anti-CD8 PE, anti-CD19 PE-cy7, anti-CD69 eF605, anti-CD127 PE-cy5, anti-CD185 PE, and anti-CD8a AF700, all from eBiosciences. Anti-CD138 PE and anti-CD11c APC were from BD Biosciences. Flow cytometry was performed on a BD LSR flow cytometer using FacsDIVA software and data were analyzed with FlowJo software.Materials and Methods Ethics StatementAll animal experiments were performed according to the NIH Guidelines for Care and Use of Animals, with approval from the Montana State University Institutional Animal Care and Use Committee (Protocol number 2011-44).ELISAs for Fecal Rotavirus Antigen Shedding and Antirotavirus Serum AntibodyELISA for fecal rotavirus antigen detection was performed as previously described [22]. Fecal samples were diluted 10-fold w/v in TNC (50 mM Tris, 150 mM NaCl, 5 mM CaCl2) containing 0.05 Tween-.
