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Biomedical subjects

Mark A Jutila

Publications and source records attributed to Mark A Jutila.

17 recordsLinked to original sources
PubMed2006

Natural products that reduce rotavirus infectivity identified by a cell-based moderate-throughput screening assay.

BACKGROUND: There is widespread interest in the use of innate immune modulators as a defense strategy against infectious pathogens. Using rotavirus as a model system, we developed a cell-based, moderate-throughput screening (MTS) assay to identify compounds that reduce rotavirus infectivity in vitro, toward a long-term goal of discovering immunomodulatory agents that enhance innate responses to viral infection. RESULTS: A natural product library consisting of 280 compounds was screened in the assay and 15 compounds that significantly reduced infectivity without cytotoxicity were identified. Time course analysis of four compounds with previously characterized effects on inflammatory gene expression inhibited replication with pre-treatment times as minimal as 2 hours. Two of these four compounds, alpha-mangostin and 18-beta-glycyrrhetinic acid, activated NFkappaB and induced IL-8 secretion. The assay is adaptable to other virus systems, and amenable to full automation and adaptation to a high-throughput format. CONCLUSION: Identification of several compounds with known effects on inflammatory and antiviral gene expression that confer resistance to rotavirus infection in vitro suggests the assay is an appropriate platform for discovery of compounds with potential to amplify innate antiviral responses.

Animals↗
PubMed2006

Use of early passage fetal intestinal epithelial cells in semi-high-throughput screening assays: an approach to identify new innate immune system adjuvants.

Innate immune system stimulants (innate adjuvants) offer complementary approaches to vaccines and antimicrobial compounds to increase host resistance to infection. The authors established fetal bovine intestinal epithelial cell (BIEC) cultures to screen natural product and synthetic compound libraries for novel mucosal adjuvants. They showed that BIECs from fetal intestine maintained an in vivo phenotype as reflected in cytokeratin expression, expression of antigens restricted to intestinal enterocytes, and induced interleukin-8 (IL-8) production. BIECs could be infected by and support replication of bovine rotavirus. A semi-high-throughput enzyme-linked immunosorbent assay-based assay that measured IL-8 production by BIECs was established and used to screen commercially available natural compounds for novel adjuvant activity. Five novel hits were identified, demonstrating the utility of the assay for selecting and screening new epithelial cell adjuvants. Although the identified compounds had not previously been shown to induce IL-8 production in epithelial cells, other known functions for 3 of the 5 were consistent with this activity. Statistical analysis of the throughput data demonstrated that the assay is adaptable to a high-throughput format for screening both synthetic and natural product derived compound libraries.

Adjuvants, Immunologic↗
PubMed2006

LTA recognition by bovine gammadelta T cells involves CD36.

CD36 has recently been shown to facilitate monocyte Toll-like receptor 2 (TLR2) recognition of lipoteichoic acid (LTA), much like CD14 in TLR4 recognition of lipopolysaccharide. We previously found that bovine gammadelta T cells express CD36 transcripts. Here, we tested whether bovine gammadelta T cells express CD36 protein and if so, whether it functions in a manner similar to the monocyte molecule. CD36 transcripts and internal and cell surface protein could be detected in resting, sorted gammadelta T cells. Phorbol 12-myristate 13-acetate (PMA)/ionomycin treatment increased CD36 transcript levels (detectable at 4 h) and protein expression (internal and cell surface). Increased surface antigen expression was detectable by 24 h and was maximal at 72 h following PMA/ionomycin stimulation. Anti-CD36 monoclonal antibody inhibited increased macrophage-inflammatory protein-1alpha gene expression in gammadelta T cells activated by LTA. In conclusion, gammadelta T cells express CD36, previously thought to be a myeloid and endothelial cell-restricted surface antigen, and it contributes to responses by these cells to microbial LTA.

Animals↗
PubMed2006

A comprehensive SAGE database for the analysis of gammadelta T cells.

Gammadelta T cells have been conserved since the adaptive immune system arose, yet their importance is still unclear. In an attempt to compensate for the lack of a broad knowledge base of gammadelta T cells across species, global analyses of gammadelta T cell transcriptomes have been performed using serial analysis of gene expression (SAGE). Twelve new SAGE libraries were generated from the following bovine lymphocyte populations: magnetic bead-sorted blood gammadelta T cells, spleen gammadelta T cells and enriched spleen alphabeta T cells from a single calf, both rested and Con A/IL2 stimulated, and flow cytometry-sorted blood gammadelta and alphabeta T cells each either rested, Con A/IL2, or phorbol 12 myristate 13-acetate/ionomycin stimulated. These new libraries complement two earlier SAGE libraries of circulating gammadelta T cell subsets. These databases were analyzed using new web-based bioinformatic tools, which allow the user to rapidly compare gene expression patterns within these and other SAGE and standard expressed sequence tag libraries generated from different cell types and different species. These analyses revealed striking differences between blood and spleen gammadelta T cells and how these cells respond to mitogenic stimulation. These analyses also confirm previous studies that suggested that global gene expression in gammadelta and alphabeta T cells is quite similar; however, a 5-fold increase in gammadelta T cell-specific transcripts could be induced by Con A/IL2 stimulation. These new public databases provide additional resources for the annotation/analysis of global gene expression in gammadelta T cells, which will facilitate studies of the biology of this enigmatic lymphoid cell.

Animals↗
PubMed2006

Distinct gene expression in human Vdelta1 and Vdelta2 gammadelta T cells following non-TCR agonist stimulation.

The two major human gammadelta T cell subsets, Vdelta1 and Vdelta2, display differences in tissue tropism and agonist responses, but we have little insight into global differences that may exist at the gene expression level. This is due to the small numbers of these cells that can be obtained from healthy donors, which limit comprehensive, comparative gene expression analyses. We established a culture method that expands Vdelta1 and Vdelta2 cells from the same PBL preparation to levels sufficient for sorting and microarray analysis. Although the subsets were expanded identically (anti-TCR mAb, plus IL-15), 392 and 614 genes were identified, which were differentially expressed in the two subsets, from two donors, respectively. Approximately 4500 genes changed in both subsets following PMA/ionomycin treatment; about 50% of these genes were subset-specific. Both subsets responded to a crude LPS preparation, but only 6% of the responsive genes were the same. The differentially expressed genes were consistent with Vdelta2 cells being more inflammatory and Vdelta1 cells having more of a regulatory phenotype. Both subsets expressed transcripts encoding an array of innate and NK cell receptors, supporting the relationship of gammadeltaT cells to the innate immune system. Our results indicate that circulating Vdelta1 and Vdelta2 subsets in humans have considerable inherent differences in gene expression following treatment with the same agonist. The patterns of differentially expressed genes are consistent with unique functional roles for these cells in vivo.

Cell Culture Techniques↗
PubMed2006

Comparative gene expression by WC1+ gammadelta and CD4+ alphabeta T lymphocytes, which respond to Anaplasma marginale, demonstrates higher expression of chemokines and other myeloid cell-associated genes by WC1+ gammadelta T cells.

The functions of gammadelta T cells are enigmatic, and these cells are often considered as evolutionary remnants of well-characterized alphabeta T cells. However, their conservation throughout evolution suggests that gammadelta T cells are biologically unique. In ruminants, gammadelta T cells expressing the workshop cluster 1 (WC1) scavenger receptor comprise a large proportion of circulating lymphocytes, suggesting these cells are biologically relevant and functionally different from alphabeta T cells. In fact, bovine WC1(+) gammadelta T cells can act as APC for alphabeta T cells, indicating they may express genes encoding proteins associated with innate immunity. The present study was designed to compare immune function gene expression profiles of clonal populations of WC1(+) gammadelta and CD4(+) alphabeta T cells derived from the same animal, which respond to major surface protein 2 (MSP2) of the intraerythrocytic rickettsial pathogen of cattle, Anaplasma marginale. Gene expression profiles of activated T cell clones were compared using a microarray format, and differential gene expression was confirmed by real-time RT-PCR and protein analyses. We demonstrate that although MSP2-specific alphabeta and gammadelta T cell clones express many of the same genes, gammadelta T cell clones express high levels of genes associated with myeloid cells, including chemokines CCL2, CXCL1, CXCL2, CXCL6, and surface receptors CD68, CD11b, macrophage scavenger receptor 1, macrophage mannose receptor, and galectin-3. It is important that many of these genes were also expressed at higher levels in polyclonal WC1(+) gammadelta T cells when compared with CD4(+) alphabeta T cells selected from peripheral blood.

Anaplasma marginale↗
PubMed2005

Gamma delta T cells respond directly to pathogen-associated molecular patterns.

Gammadelta T cells recognize unprocessed or non-peptide Ags, respond rapidly to infection, and localize to mucosal surfaces. We have hypothesized that the innate functions of gammadelta T cells may be more similar to those of cells of the myeloid lineage than to other T cells. To begin to test this assumption, we have analyzed the direct response of cultured human and peripheral blood bovine gammadelta T cells to pathogen associated molecular patterns (PAMPs) in the absence of APCs using microarray, real-time RT-PCR, proteome array, and chemotaxis assays. Our results indicate that purified gammadelta T cells respond directly to PAMPs by increasing expression of chemokine and activation-related genes. The response was distinct from that to known gammadelta T cell Ags and different from the response of myeloid cells to PAMPs. In addition, we have analyzed the expression of a variety of PAMP receptors in gammadelta T cells. Freshly purified bovine gammadelta T cells responded more robustly to PAMPs than did cultured human cells and expressed measurable mRNA encoding a variety of PAMP receptors. Our results suggest that rapid response to PAMPs through the expression of PAMP receptors may be another innate role of gammadelta T cells.

Adult↗
PubMed2004

Impact of polyunsaturated fatty acids on cytoskeletal linkage of L-selectin.

Polyunsaturated fatty acid [omega-3 polyunsaturated fatty acids (omega-3PUFAs)] incorporation into cell membranes has been shown to have potent anti-inflammatory activity, though the mechanisms involved are only partially characterized. Here, we show that PUFA enrichment of T cell membranes decreased the overall expression of L-selectin as well as a highly conserved epitope on L-selectin that may serve as a marker for optimal protein function. Additionally, PUFA enrichment inhibited L-selectin cytoskeletal association, which is thought to be important for optimal functional activity. In support of this, PUFA enrichment of gammadelta T cell membranes reduced L-selectin-dependent rolling interactions under conditions mimicking physiological flow. Taken together, these data suggest that the anti-inflammatory activity of omega-3 polyunsaturated fatty acids may be due, in part, to a novel effect on L-selectin, namely PUFA reduction or prevention of cytoskeletal association of L-selectin.

Animals↗
PubMed2003

Serial analysis of gene expression in circulating gamma delta T cell subsets defines distinct immunoregulatory phenotypes and unexpected gene expression profiles.

Gene expression profiles were compared in circulating bovine GD3.5+ (CD8-) and GD3.5- (predominantly CD8+) gammadelta T cells using serial analysis of gene expression (SAGE). Approximately 20,000 SAGE tags were generated from each library. A comparison of the two libraries demonstrated 297 and 173 tags representing genes with 5-fold differential expression in GD3.5+ and GD3.5- gammadelta T cells, respectively. Consistent with their localization into sites of inflammation, GD3.5+ gammadelta T cells appeared transcriptionally and translationally more active than GD3.5- gammadelta cells. GD3.5- gammadelta T cells demonstrated higher expression of the cell proliferation inhibitor BAP 37, which was associated with their less activated gene expression phenotype. The immune regulatory and apoptosis-inducing molecule, galectin-1, was identified as a highly abundant molecule and was higher in GD3.5+gammadelta T cells. Surface molecules attributed to myeloid cells, such as CD14, CD68, and scavenger receptor-1, were identified in both populations. Furthermore, expression of B lymphocyte-induced maturation protein, a master regulator of B cell and myeloid cell differentiation, was identified by SAGE analysis and was confirmed at the RNA level to be selectively expressed in gammadelta T cells vs alphabeta T cells. These results provide new insights into the inherent differences between circulating gammadelta T cell subsets.

Animals↗
PubMed2003

Differential mRNA expression in circulating gammadelta T lymphocyte subsets defines unique tissue-specific functions.

To elucidate the functions of circulating gammadelta T cells, in the absence of antigen stimulation, the differential gene expression of two circulating gammadelta T cell subsets was analyzed. The two subsets, with distinct trafficking phenotypes in young calves, were GD3.5(+), CD8(-), WC1(+) or GD3.5(-), CD2(+), WC1(-), and 90-100% CD8(+) and were sorted based on GD3.5 and gammadelta T cell receptor expression. Results from two different human arrays probed with cDNA from these gammadelta T cell subsets indicated that they have markedly different tissue-specific functions. The genes preferentially expressed by GD3.5(+) (CD8(-)) gammadelta T cells demonstrated that they were highly activated, proliferative, and inflammatory, whereas those expressed by GD3.5(-) (primarily CD8(+)) gammadelta T cells were involved in promoting quiescence, consistent with a role for gammadelta T cells as sentinel mucosal cells, and several were interferon-regulated genes. Gene expression and phenotypic assays indicated that CD8(+) gammadelta T cells were apoptotic, whereas CD8(-) gammadelta T cells were apoptosis-resistant. Differential expression of multiple genes was confirmed in both arrays: That of 14 genes was confirmed by quantitative reverse transcriptase-polymerase chain reaction and that of seven proteins was confirmed by flow cytometry. This novel, genomic analysis of circulating gammadelta T cell subsets, without confounding effects of the tissue microenvironment, offers new insight into the biology and development of neonatal gammadelta T cells.

Animals↗
PubMed2002

Bovine gamma delta T cell subsets express distinct patterns of chemokine responsiveness and adhesion molecules: a mechanism for tissue-specific gamma delta T cell subset accumulation.

Subsets of gammadelta T cells localize to distinct tissue sites in the absence of exogenous Ag stimulation or development of effector/memory cells. Selective lymphocyte homing from the blood into tissues is controlled by a multistep process involving vascular and lymphocyte adhesion molecules, and G protein-linked chemokine receptors. The role of these mechanisms in the tissue tropism of gammadelta T cells is still poorly understood. In this study, we demonstrate that a subset of gammadelta T cells, most of which express an antigenically distinct TCR and are characterized by coexpression of CD8, selectively accumulated in tissues that expressed high levels of the mucosal vascular addressin, mucosal addressin cell adhesion molecule 1. These cells expressed higher levels of alpha(4)beta(7) integrins than other gammadelta T cell subsets and selectively migrated to the CCR7 ligand secondary lymphoid-tissue chemokine (CCL21). Integrin activation by CCL21 selectively increased CD8(+)gammadelta T cell binding to recombinant mucosal addressin cell adhesion molecule 1. These results suggest that the tropism of circulating CD8(+)gammadelta T cells for mucosal tissues is due, at least in part, to selective developmental expression of adhesion molecules and chemokine receptors.

Animals↗
PubMed2002

L-selectin serves as an E-selectin ligand on cultured human T lymphoblasts.

Previous studies reported that L-selectin (CD62L) on human peripheral blood neutrophils serves as an E-selectin ligand. This study shows that CD62L acquired E-selectin-binding activity following phorbol ester (PMA) treatment of the Jurkat T cell line and anti-CD3/IL-2-driven proliferation of human T lymphocytes in vitro. The recombinant porcine E-selectin/human Ig chimera P11.4 showed neuraminidase-sensitive and calcium-dependent attachment to PMA-stimulated human Jurkat T cells in a flow cytometry assay. The anti-CD62L mAb (DREG 56) blocked this binding interaction by approximately 60% and P11.4 precipitated CD62L from detergent lysates of PMA-activated Jurkat cells. In contrast, P11.4 precipitated minimal amounts of CD62L from detergent lysates of nonactivated human PBL. As reported previously, P-selectin glycoprotein ligand 1 and a distinct 130-kDa glycoprotein were the major species in these precipitates. However, T cell activation on plate-immobilized anti-CD3 and growth in low-dose IL-2 increased the percentage of CD62L molecules with E-selectin-binding activity. After two cycles of activation and culture, approximately 60-70% of the CD62L was precipitated with the P11.4 chimera. These cultured T lymphoblasts rolled avidly on both E-selectin and P-selectin at physiologic levels of linear shear stress. The DREG 56 Ab partially blocked rolling on the E-selectin substrate, whereas no effect was seen on P-selectin. Thus, CD62L on human cultured T lymphoblasts is one of several glycoproteins that interacts directly with E-selectin and contributes to rolling under flow.

Antibodies, Monoclonal↗
PubMed2002

Mucosal addressin expression and binding-interactions with naive lymphocytes vary among the cranial, oral, and nasal-associated lymphoid tissues.

The head and neck lymph nodes (LN)--or cranial, oral, and nasal-associated lymphoid tissue (CONALT)--help disseminate activated lymphocytes to produce salivary immune responses, especially after intranasal immunization. To elucidate the mechanisms that induce immunity at these sites, we investigated the interactions between addressins and homing-receptors that allow for lymphocyte binding to high endothelial venules (HEV) of CONALT. In vivo lymphocyte trafficking to CONALT was mediated primarily through interactions between peripheral node addressin (PNAd) and L-selectin, whereas interactions between mucosal addressin cell adhesion molecule-1 (MAdCAM-1) and alpha 4 beta 7 played a role in retention in cervical LN (CLN). Upon immunofluorescent staining for PNAd and MAdCAM-1, nearly all HEV in CONALT expressed PNAd, with varying MAdCAM-1 expression among these LN. The parotid gland LN (PRLN) and submaxillary gland LN (SMLN) rely exclusively upon PNAd-L-selectin interactions for naive-lymphocyte binding, whereas the CLN utilize PNAd-L-selectin interactions and MAdCAM-1-alpha 4 beta 7 interactions for binding. Intense staining of non-HEV-expressed vascular cell adhesion molecule-1 (VCAM-1) was observed in PRLN, whereas SMLN and CLN displayed less VCAM-1 but showed intense staining for diffuse MAdCAM-1. This study suggests that though PNAd-L-selectin interactions play an important role in the trafficking of lymphocytes throughout CONALT, varying MAdCAM-1 and VCAM-1 addressin expression and usage impart important differences among the PRLN, SMLN, and CLN.

Animals↗
PubMed2002

Ultrastructural examination of cytoskeletal linkage of L-selectin and comparison of L-selectin cytoskeletal association to that of other human and bovine lymphocyte surface antigens.

L-selectin is constitutively expressed on most leukocytes and is responsible for the initial events in cell trafficking termed tethering and rolling. Recently, L-selectin has been shown to associate with the actin-based cytoskeleton under a variety of conditions. In an effort to better understand L-selectin cytoskeletal association and the ultrastructural nature of the cytoskeleton itself, we provide a comparison of the cytoskeletal association of various human and bovine surface proteins in relation to L-selectin. Electron microscopic examination of the cytoskeleton provided further data on the ultrastructure of freshly isolated peripheral lymphocytes as well as demonstrated L-selectin localization to the periphery of the cytoskeleton following low dose detergent treatment of the cell. Clusters of colloidal-gold-stained L-selectin were found on the surface of the detergent-treated lymphocytes, even though these particles completely lacked microvilli. By flow cytometry, we have defined three distinct patterns of cytoskeletal association; constitutive, inductive, and mAb crosslink-induced, and assigned human and bovine CD2, CD3, CD4, CD5, CD8, CD18, CD19, CD44, CD45RA, CD45RO, alphabeta TCR, gammadelta TCR, E-selectin ligands, and L-selectin surface antigens to one of these respective patterns. SDS-PAGE analyses confirmed most of the flow cytometry results. Depending upon its conformation, L-selectin fell into the inductive or mAb crosslink-induced pattern of association, similar to E-selectin ligand(s). Our data provide additional insight into the functional role of L-selectin and the cytoskeleton in immunological events.

Animals↗
PubMed2002

Bluetongue virus-induced activation of primary bovine lung microvascular endothelial cells.

Bluetongue is an insect-transmitted viral disease of sheep and some species of wild ruminants. Infection of lung microvascular endothelial cells (ECs) is central to the pathogenesis of bluetongue virus (BTV) infection of ruminants, but it is uncertain as to why cattle are resistant to BTV-induced microvascular injury and bluetongue disease. Thus, in order to better understand the pathogenesis of BTV infection of cattle, mRNAs encoding a variety of inflammatory mediators were quantitated by real-time polymerase chain reaction in primary bovine lung microvascular ECs (BLmVECs) exposed to BTV and/or EC-derived mediators. BTV infection of BLmVECs significantly increased the transcription of genes encoding interleukin-1 (IL-1), IL-6, IL-8, cyclooxygenase-2, and inducible nitric oxide synthase. Treatment of BLmVECs with EC-lysates that contained BTV as well as cytokines increased both the incidence of apoptosis and expression of cellular adhesion molecules, as compared to infection of BLmVECs with BTV alone. Thus, BTV infection caused activation of BLmVECs with production of inflammatory mediators that alter the mechanism of cell death of BLmVECs and exert potentially potent effects on blood coagulation. The activities of BTV-induced-EC-derived inflammatory mediators likely contribute to the resistance of cattle to BTV-induced microvascular injury and bluetongue disease.

Animals↗
PubMed2001

Infection kinetics, prostacyclin release and cytokine-mediated modulation of the mechanism of cell death during bluetongue virus infection of cultured ovine and bovine pulmonary artery and lung microvascular endothelial cells.

Bluetongue virus (BTV) infection causes a haemorrhagic disease in sheep, whereas BTV infection typically is asymptomatic in cattle. Injury to the endothelium of small blood vessels is responsible for the manifestations of disease in BTV-infected sheep. The lungs are central to the pathogenesis of BTV infection of ruminants; thus endothelial cells (ECs) cultured from the pulmonary artery and lung microvasculature of sheep and cattle were used to investigate the basis for the disparate expression of bluetongue disease in the two species. Ovine and bovine microvascular ECs infected at low multiplicity with partially purified BTV were equally susceptible to BTV-induced cell death, yet ovine microvascular ECs had a lower incidence of infection and produced significantly less virus than did bovine microvascular ECs. Importantly, the relative proportions of apoptotic and necrotic cells were significantly different in BTV-infected EC cultures depending on the species of EC origin and the presence of inflammatory mediators in the virus inoculum. Furthermore, BTV-infected ovine lung microvascular ECs released markedly less prostacyclin than the other types of ECs. Results of these in vitro studies are consistent with the marked pulmonary oedema and microvascular thrombosis that characterize bluetongue disease of sheep but which rarely, if ever, occur in BTV-infected cattle.

Animals↗