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M F Kagnoff

Publications and source records attributed to M F Kagnoff.

At least 55 records · Page 3Linked to original sources
PubMed1997

HIV-1 Tat protein can transactivate a heterologous TATAA element independent of viral promoter sequences and the trans-activation response element.

OBJECTIVE: To determine whether the HIV-1 transactivator protein Tat acts as a DNA sequence-specific transcription factor and activates transcription from a heterologous TATAA element in the absence of the trans-activation response (TAR) element and other sequences in the HIV-1 long terminal repeat (LTR). DESIGN: Activating protein-1 (AP-1) and Tat-induced transcription were assessed using Jun and hybrid Tat/Jun-expression plasmids and reporter gene constructs which contained AP-1 binding sites upstream of the rat prolactin TATAA element or an HIV-1 LTR construct in which AP-1 binding sites replaced the TAR element. METHODS: Tat-induced transcription was determined following transient transfection of colon epithelial cell lines with reporter gene constructs and Tat/Jun-expression plasmids in which Tat was fused to the DNA binding domain of Jun. Activation of prolactin (PL) and LTR reporter genes was assessed by luciferase (LUC) or chloramphenicol acetyltransferase (CAT) activity in cellular extracts. RESULTS: Cotransfection of cells with Tat/Jun and the AP-1 PL LUC or LTR AP-1 CAT reporter plasmid resulted in a marked increase in reporter gene activity which was comparable with that induced by transfection of cells with several different AP-1 expression plasmids (e.g., JunD, JunB, c-Fos), or that elicited by stimulation of the cells transfected with LTR AP-1 CAT plasmids with phorbol ester or tumor necrosis factor-alpha. Tat-induced transcription was DNA-mediated since both a Jun DNA binding domain fused to Tat as well as AP-1 binding sites within the promoter were required for the induction of CAT expression. CONCLUSIONS: Tat-activated transcriptor can occur strictly through a heterologous TATAA element independent of TAR and Sp1 binding sites or other HIV-1 LTR sequences. Tat appears to increase transcription initiated through the TATAA element by mechanisms similar to that of DNA sequence-specific transcription factors.

Animals↗
PubMed1997

Cryptosporidium parvum infection of human intestinal epithelial cells induces the polarized secretion of C-X-C chemokines.

Cryptosporidium parvum infects intestinal epithelial cells and does not invade deeper layers of the intestinal mucosa. Nonetheless, an inflammatory cell infiltrate that consists of neutrophils and mononuclear cells is often present in the lamina propria, which underlies the epithelium. This study investigated the host epithelial cell response to C. parvum by assessing in vitro and in vivo the expression and production of proinflammatory cytokines by intestinal epithelial cells after infection. The human colon epithelial cell lines HCT-8 and Caco-2 and human intestinal xenografts in SCID mice were infected with C. parvum. The expression and secretion of the C-X-C chemokines interleukin-8 (IL-8) and GROalpha were determined by reverse transcription-PCR analysis and enzyme-linked immunosorbent assay. Our results demonstrate that upregulated expression and secretion of IL-8 and GROalpha after C. parvum infection of intestinal epithelial cells first occurred 16 to 24 h after infection and increased over the ensuing 1 to 2 days. The kinetics of C-X-C chemokine production by C. parvum-infected epithelial cells contrast markedly with the rapid but transient expression of C-X-C chemokines by epithelial cells infected with invasive enteric bacteria. C-X-C chemokine secretion in C. parvum-infected epithelial cells occurred predominantly from the basolateral surface in polarized monolayers of Caco-2 cells grown in Transwell cultures, whereas cell lysis occurred at the apical surface. The basolateral secretion of IL-8 and GROalpha from C. parvum-infected epithelial cells suggests that C-X-C chemokines produced by those cells contribute to the mucosal inflammatory cell infiltrate in the underlying intestinal mucosa.

Animals↗
PubMed1997

Coccoid and spiral Helicobacter pylori differ in their abilities to adhere to gastric epithelial cells and induce interleukin-8 secretion.

Helicobacter pylori exists as an actively dividing spiral form and a nonculturable, but viable, metabolizing coccoid form. Both forms are present in the stomach, but their relative pathophysiologic significances are unknown. Here we show that the coccoid form of H. pylori, in contrast to the spiral form, binds poorly to gastric epithelial cells and induces little, if any, interleukin-8 secretion by these cells.

Bacterial Adhesion↗
PubMed1997

Human hepatocytes express an array of proinflammatory cytokines after agonist stimulation or bacterial invasion.

Inflammatory cells infiltrate the liver in response to microbial infection or hepatic injury. To assess the potential role hepatocytes may play in initiating or amplifying the acute inflammatory response in the liver, we used three human hepatocyte cell lines and primary human hepatocyte cultures to characterize the repertoire of cytokines that can be expressed and regulated in hepatocytes in response to agonist stimulation or bacterial infection. As reported herein, a proinflammatory cytokine gene program that includes C-X-C and C-C chemokines [interleukin-8(IL-8), growth related (GRO)-alpha, GRO-beta, GRO-gamma, epithelial neutrophil activating peptide-78 (ENA-78), and RANTES] and the cytokines tumor necrosis factor-alpha (TNF-alpha) and macrophage colony stimulating factor was upregulated in human hepatocytes after stimulation with IL-1 alpha or TNF-alpha or bacterial invasion. In contrast, expression of hematopoietic/ lymphoid growth factors by the same cells was either down-regulated (erythropoietin and stem cell factor) or unchanged (IL-7 and IL-15) in response to the identical stimuli. Hepatocytes did not express cytokines that often are associated with the regulation of antigen-specific immune responses (IL-2, IL-4, IL-5, IL-10, IL-12p40, IL-13, and interferon-gamma) or genes for several other proinflammatory cytokines [IL-1 alpha, IL-6, monocyte chemotactic protein-1 (MCP-1), and MCP-3] or hematopoietic growth factors (granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, IL-3, and IL-11). Together, these studies suggest that hepatocytes can both initiate and amplify acute inflammatory responses in the liver through the regulated expression and secretion of a specific array of proinflammatory cytokines.

Bacterial Infections↗
PubMed1996

Infection of human intestinal epithelial cells with invasive bacteria upregulates apical intercellular adhesion molecule-1 (ICAM)-1) expression and neutrophil adhesion.

The acute host response to gastrointestinal infection with invasive bacteria is characterized by an accumulation of neutrophils in the lamina propria, and neutrophil transmigration to the luminal side of the crypts. Intestinal epithelial cells play an important role in the recruitment of inflammatory cells to the site of infection through the secretion of chemokines. However, little is known regarding the expression, by epithelial cells, of molecules that are involved in interactions between the epithelium and neutrophils following bacterial invasion. We report herein that expression of ICAM-1 on human colon epithelial cell lines, and on human enterocytes in an in vivo model system, is upregulated following infection with invasive bacteria. Increased ICAM-1 expression in the early period (4-9 h) after infection appeared to result mainly from a direct interaction between invaded bacteria and host epithelial cells since it co-localized to cells invaded by bacteria, and the release of soluble factors by epithelial cells played only a minor role in mediating increased ICAM-1 expression. Furthermore, ICAM-1 was expressed on the apical side of polarized intestinal epithelial cells, and increased expression was accompanied by increased neutrophil adhesion to these cells. ICAM-1 expression by intestinal epithelial cells following infection with invasive bacteria may function to maintain neutrophils that have transmigrated through the epithelium in close contact with the intestinal epithelium, thereby reducing further invasion of the mucosa by invading pathogens.

Animals↗
PubMed1996

Genetically resistant (Ityr) and susceptible (Itys) congenic mouse strains show similar cytokine responses following infection with Salmonella dublin.

IFN-gamma, TNF-alpha, IL-1, and granulocyte-macrophage CSF (GM-CSF) play an important role in host resistance to infection with nontyphoid Salmonella. In mice, resistance to Salmonella is determined by alleles of the susceptibility gene, Nramp, which maps to the Ity/Lsh/Bcg locus and is expressed in macrophages. In vitro studies suggested that macrophages from Salmonella-susceptible mice (Itys phenotype) are impaired functionally in their ability to produce, or stimulate the production of, cytokines such as TNF-alpha and IFN-gamma. BALB/c and BALB/c.DBA2 Idh-lb-Ityr-Pep-3b mice are congenic strains that differ at the Ity/Lsh/Bcg locus and in their susceptibility to Salmonella infection. These strains were used to question whether differences in the host cytokine response determine the outcome of Salmonella infection in genetically susceptible and resistant mice. As reported in this work, the in vivo response to Salmonella dublin infection in both Itys and Ityr mice was characterized by increased expression of IFN-gamma, TNF-alpha, GM-CSF, IL-1 alpha, IL-2, IL-6, IL-10, and IL-12 p40. In contrast, expression of IL-4, IL-5, and TGF-beta 1 was not altered, or decreased, during the course of infection. Moreover, the kinetics and magnitude of the cytokine response following S. dublin infection were similar in susceptible Itys and resistant Ityr mice, even though the former group died while the latter survived the infection. Thus, in vivo cytokine responses that are associated with survival of Ityr mice following S. dublin infection do not confer protection in mice of the Itys phenotype.

Animals↗
PubMed1996

Mucosal immunology: new frontiers.

Mucosal immunology is one of the most rapidly developing and exciting fields of immunological research. A recent meeting discussed advances in the fundamental knowledge of this area, and reviewed possibilities for its clinical application.

Animals↗
PubMed1996

Oral tolerance: mechanisms and possible role in inflammatory joint diseases.

Decreased systemic immune responsiveness to a specific antigen following exposure to that antigen by the enteric route is termed 'oral tolerance.' Oral tolerance is revealed when attempts are made to parenterally immunize the host to the same antigen that was previously administered orally or intragastrically. A similar phenomenon is also seen following antigen exposure via the nasal mucosa and a related phenomenon is seen following antigen exposure in the upper respiratory tract. There has been a marked renewal of interest in the mechanisms that underlie oral tolerance because of its potential role for preventing and treating autoimmune and inflammatory diseases and IgE-mediated allergic disorders. The specific factors that determine whether or not the host develops mucosal tolerance to an antigen administered by the mucosal route are also of substantial importance for those involved in mucosal vaccine development. Furthermore, putative abnormalities in the ability of the host to develop mucosal tolerance may play a pathogenetic role in certain autoimmune and allergic diseases and disorders. Several well-defined immunological mechanisms mediate oral tolerance. These include the induction, following mucosal antigen exposure, of regulatory populations of T-cells that can down-regulate specific immune responses (e.g. DTH) via the production of specific cytokines (e.g. TGF-beta 1, IL-10 and IL-4). In addition, clonal anergy, clonal deletion and antibody-mediated suppression can be shown to play a role in the induction and maintenance of mucosal tolerance in several experimental systems. In animal studies, the onset of collagen-induced, adjuvant-induced, antigen-induced and pristane-induced arthritis has been delayed and the severity of ongoing disease diminished following feeding collagen type II. Mucosal tolerance has been clearly demonstrated in humans and clinical studies have been undertaken to treat rheumatoid arthritis using a similar approach. Results of initial clinical studies in rheumatoid arthritis indicated a modest improvement and further studies are ongoing in this and other autoimmune diseases (e.g. multiple sclerosis, autoimmune uveitis and insulin-dependent diabetes). This approach, if successful, could offer a new and novel therapeutic modality for preventing autoimmune and allergic disorders, and modulating ongoing disease.

Administration, Oral↗
PubMed1996

Activating protein-1 cooperates with phorbol ester activation signals to increase HIV-1 expression.

OBJECTIVE: To determine whether Jun and Fos, components of the activating protein-1 (AP-1) transcription factor, transactivate HIV-1 proviral expression. DESIGN: The effects of phorbol myristate acetate (PMA) and Jun or Fos transcription factors on HIV-1 expression were investigated using a provirus clone and long terminal repeat (LTR)-reporter gene constructs. The influence of PMA stimulation on AP-1 binding activity was determined with antibodies in gel mobility shift assays. METHODS: Activation of HIV-1 provirus and transcription of HIV-1 LTR sequences in response to cotransfection of Jun or Fos expression plasmids into a permissive colon cancer cell line, SW480, were assessed by p24 core antigen capture and reporter gene assays, respectively. The effect of protein kinase C activation was evaluated by comparing cells grown in the presence or absence of PMA (20 ng/ml). RESULTS: Cotransfection of HIV-1 provirus and expression plasmids for c-Jun or JunB into SW480 cells resulted in increased p24 core antigen and this response was markedly increased following PMA stimulation of cells. c-Fos or JunD alone did not increase p24 production but markedly increased p24 production in PMA-stimulated cells. PMA increased c-Fos and JunD binding activity on an AP-1 binding site within the U5 region of the LTR, as shown in gel mobility shift assays. Functional analysis of this site by transient transfections demonstrated it was required to mediate c-Fos and JunD transactivation of the HIV-1 LTR. CONCLUSIONS: Specific Jun and Fos transcription factors can transactivate the HIV-1 provirus and this response is markedly increased in cooperation with cellular activation signals elicited by PMA. Taken together, the data indicate that AP-1 binding sites downstream of the transcriptional start site in the HIV-1 LTR are capable of binding c-Fos and JunD and may contribute to transactivation of HIV-1 provirus.

Colonic Neoplasms↗
PubMed1996

Expression of Salmonella typhimurium rpoS and rpoS-dependent genes in the intracellular environment of eukaryotic cells.

Adaptation to the intracellular environment of host cells is crucial for the pathogenesis of Salmonella infections. The alternative sigma factor RpoS is a global regulator of gene expression during starvation and stress conditions and is required for virulence in Salmonella spp. We have used lacZ reporter fusions to rpoS and rpoS-dependent genes to study rpoS regulation after entry of Salmonella typhimurium into macrophages and epithelial cells. The results demonstrate that expression of an rpoS::lacZ translational fusion increases rapidly in S. typhimurium after phagocytosis. Activity of RpoS also increases after bacterial entry into both macrophages and epithelial cells, as demonstrated by the induction of the rpoS-regulated genes katE and spvB. A control rpoS-independent promoter for neomycin resistance does not show significant induction after cell entry. These results demonstrate that the regulatory system mediated by RpoS in S. typhimurium is activated by the intracellular environment of eukaryotic cells.

Animals↗
PubMed1995

Inverse PCR amplification of rare T cell receptor delta messages from mucosal biopsy specimens.

Reverse transcription (RT) PCR is an important tool in studies of the repertoire of T cell antigen receptors (TCR). In combination with inverse PCR, it can be used to amplify TCR transcripts whose V regions are unknown. Little is currently known regarding the delta TCR repertoire in human intestine. Here, RT inverse PCR was adapted to efficiently amplify low abundance delta TCR sequences from human colonic mucosal biopsy specimens. In this protocol, high yields of PCR products were obtained by optimizing the conditions at which cDNA autoligation was performed.

Base Sequence↗
PubMed1995

Intestinal epithelial cells as watchdogs for the natural immune system.

Intestinal epithelial cells secrete a spectrum of chemoattractant and proinflammatory cytokines after invasion by bacteria. We suggest the novel concept that epithelial cells not only act as a mechanical barrier to invasive bacteria, but that they also signal the presence of invasive pathogens to the mucosal immune and inflammatory cells.

Animals↗
PubMed1995

A distinct array of proinflammatory cytokines is expressed in human colon epithelial cells in response to bacterial invasion.

Pathogenic bacteria that penetrate the intestinal epithelial barrier stimulate an inflammatory response in the adjacent intestinal mucosa. The present studies asked whether colon epithelial cells can provide signals that are important for the initiation and amplification of an acute mucosal inflammatory response. Infection of monolayers of human colon epithelial cell lines (T84, HT29, Caco-2) with invasive strains of bacteria (Salmonella dublin, Shigella dysenteriae, Yersinia enterocolitica, Listeria monocytogenes, enteroinvasive Escherichia coli) resulted in the coordinate expression and upregulation of a specific array of four proinflammatory cytokines, IL-8, monocyte chemotactic protein-1, GM-CSF, and TNF alpha, as assessed by mRNA levels and cytokine secretion. Expression of the same cytokines was upregulated after TNF alpha or IL-1 stimulation of these cells. In contrast, cytokine gene expression was not altered after infection of colon epithelial cells with noninvasive bacteria or the noninvasive protozoan parasite, G. lamblia. Notably, none of the cell lines expressed mRNA for IL-2, IL-4, IL-5, IL-6, IL-12p40, IFN-gamma, or significant levels of IL-1 or IL-10 in response to the identical stimuli. The coordinate expression of IL-8, MCP-1, GM-CSF and TNF alpha appears to be a general property of human colon epithelial cells since an identical array of cytokines, as well as IL-6, also was expressed by freshly isolated human colon epithelial cells. Since the cytokines expressed in response to bacterial invasion or other proinflammatory agonists have a well documented role in chemotaxis and activation of inflammatory cells, colon epithelial cells appear to be programmed to provide a set of signals for the activation of the mucosal inflammatory response in the earliest phases after microbial invasion.

Animals↗
PubMed1995

The delta T cell receptor repertoire in human colon and peripheral blood is oligoclonal irrespective of V region usage.

The majority of gamma/delta T cell receptors (TCR) in the human intestinal mucosa are thought to use the TCRDV1 (V delta 1) variable region gene segment, whereas gamma/delta T cells in the circulation predominantly express the TCRDV2 (V delta 2) gene segment. delta T cell receptors that use the TCRDV2 variable region gene segment generally have been regarded as highly diverse, whereas those that use the TCRDV1 gene segment are oligoclonal, whether present in the intestinal tract or in peripheral blood. We report herein that oligoclonality is a general feature of the peripheral delta T cell receptor repertoire in healthy human adults, irrespective of the variable region used and regardless of whether gamma/delta T cells reside in the intestinal mucosa or in peripheral blood. In addition, the delta T cell receptor repertoire is shown to be highly compartmentalized between such sites as the colon and peripheral blood, relatively stable over at least a 10-16-mo period, and unique in each individual. Further, the spectrum of variable region genes used by delta T cell receptor transcripts in the human colon is greater than previously recognized. Thus, in addition to the TCRDV1 and TCRDV2 variable region gene segments, delta T cell receptors in normal intestinal mucosa can use TCRDV3 (V delta 3) and TCRAV (V alpha) gene segments which, in some individuals, comprise a significant component of the mucosal delta T cell receptor repertoire. Our studies indicate that the potential of delta T cell receptors for extensive diversity is not reflected in the mature human repertoire. Moreover, these findings suggest a model wherein the delta T cell receptor repertoire in the colon and peripheral blood is shaped by selection and clonal expansion of gamma/delta T cells that ultimately seed throughout the length of the colon mucosa and populate the circulation.

Aged↗
PubMed1995

Entamoeba histolytica trophozoites induce an inflammatory cytokine response by cultured human cells through the paracrine action of cytolytically released interleukin-1 alpha.

Infection with the protozoan parasite Entamoeba histolytica results in a high mortality worldwide. To initiate infection, E. histolytica trophozoites in the bowel lumen penetrate the epithelium, and cause extensive lysis of host cells. The acute amebic lesions in animal models are characterized by infiltration with inflammatory cells, particularly neutrophils. The acute host response is likely important for determining whether the infection will spread systemically, but little is known regarding the signals which initiate an acute inflammatory response to E. histolytica. In the studies reported herein, we used an in vitro model system to define the proinflammatory signals produced by epithelial and other host cells in response to infection with E. histolytica trophozoites. Coculture of human epithelial and stromal cells and cell lines with trophozoites is shown to increase expression and secretion of an array of chemoattractant and proinflammatory cytokines, including IL-8, GRO alpha, GM-CSF, IL-1 alpha, and IL-6. Moreover, high-level secretion of those cytokines is regulated by the paracrine action of cytolytically released IL-1 alpha. A second mechanism for trophozoite-induced IL-8 production involves trophozoite-target cell contact via a galactose-inhibitable amebic adherence protein, and appears to be mediated through increased intracellular calcium levels. These studies define novel mechanisms through which acute inflammation can be initiated in the host in response to a cytolytic pathogen, such as E. histolytica.

Animals↗
PubMed1994

Increased transcription and coordinate stabilization of mRNAs for secreted immunoglobulin alpha heavy chain and kappa light chain following stimulation of immunoglobulin A expressing B cells.

Immunoglobulin A (IgA) plays a key role in host protection at mucosal surfaces, yet little is known regarding the molecular mechanisms that govern the expression of this isotype. The studies herein investigated mechanisms that control IgA secretion in response to stimulation with interleukin-4 and interleukin-5, cytokines which are known to regulate IgA responses, and to bacterial lipopolysaccharide. Two mechanisms were shown to govern agonist induced IgA expression in a murine IgA expressing B cell line. In the initial period after stimulation, increased mRNA levels for the secreted form of alpha heavy chain (alpha s), and kappa light chain were paralleled by a transient increase in transcription rates of the corresponding genes. However, with prolonged agonist stimulation, gene transcription rates decreased to near control levels, and increased mRNA levels were associated with a coordinate increase in alpha s and kappa mRNA stability. In striking contrast, these agonists did not affect levels or stability of mRNA for the membrane form of alpha heavy chain (alpha m). Alpha s mRNAs contained multiple poly(A) addition sites located 13-32 nucleotides downstream of a single AAUAAA sequence. Nonetheless, the differential usage of these sites as a mechanism for controlling alpha s mRNA stability could be excluded, since the relative abundance of these different alpha s mRNAs did not differ significantly after agonist stimulation. These data, taken together, suggest that sequences within 107 nucleotides of the 3' end of alpha s mRNA, which are absent in alpha M mRNA, are required for the regulation of alpha s mRNA stability, possibly by acting as targets for regulated trans-acting cellular factors.

Animals↗
PubMed1994

The V delta 1 T cell receptor repertoire in human small intestine and colon.

V delta 1 bearing T cells comprise the major population of gamma/delta T cells in the human intestinal tract. To gain insight into mechanisms involved in the generation of these cells and the diversity of their repertoire, we have characterized the junctional sequences of V delta 1 T cell receptor transcripts in the human small intestine and colon. Mucosal biopsies obtained from defined regions along the length of the small intestine or colon contained a high frequency of either one or a few identical in frame V delta 1 sequences. Less abundant sequences were also detected repeatedly throughout the length of small intestine or colon. Moreover, the intestinal V delta 1 repertoire in the small intestine and colon appeared compartmentalized and showed no overlap with the V delta 1 repertoire in peripheral blood. Dominant V delta 1 transcripts in each subject differed between the small intestine and colon, and the dominant transcripts within these sites differed among individuals. Analysis of small intestinal transcripts obtained at a 1-yr interval revealed that the V delta 1 repertoire was stable over time. The fact that the majority of V delta 1 transcripts, both dominant and rare, are distributed throughout a several meter length of the adult intestinal tract and are stable over time suggests they are not generated by an ongoing process of in situ VDJ gene rearrangement. Our results favor a model in which the repertoire of V delta 1 T cells in the intestinal tract is shaped by positive selection in response to a limited array of ligands before the migration of V delta 1 cells throughout the small intestine or colon.

Adult↗