Biomedical subjects

A D Levine

Publications and source records attributed to A D Levine.

At least 19 recordsLinked to original sources

PubMed2001

A mucosal IgA-mediated excretory immune system in vivo.

The capacity of mucosal IgA Abs to serve as an excretory immune system in vivo was investigated. Mice expressing a transgenic TCR were immunized intragastrically with the cognate Ag to elicit a vigorous mucosal IgA Ab response. Soon after i.v. challenge, Ag was detected within the epithelial cells of the small intestinal crypts and to a lesser degree within the epithelial cells higher up the villi, paralleling the gradient in expression of the polymeric Ig receptor and the transport of its ligand, oligomeric IgA. Uptake of Ag into the epithelial cells occurred only from the basolateral aspect and only when Ag complexed to IgA Ab could be present in the lamina propria. The results support the concept that local IgA Abs can excrete Ags from the body by transporting them directly through mucosal epithelial cells, using the same mechanism that transports free IgA into the mucosal secretions.

Animals↗

PubMed2001

Reversal of established delayed type hypersensitivity reactions following therapy with IL-4 or antigen-specific Th2 cells.

Delayed-type hypersensitivity reactions (DTHR) are mediated by IFN-gamma-producing CD4+ (Th1) or CD8+ T cells (Tc1) and can be prevented by steering T cells toward an IL-4-producing Th2 or Tc2 phenotype. It is currently accepted that T cells can be directed toward a Th2 or Tc2 phenotype only during the initiation of an immune response. Once established, the cytokine pattern of immune reactions is believed to be stable. Therefore, inhibition of DTHR by the induction of Th2/Tc2 responses, termed immune deviation, is considered only as a prevention but not as a therapy of harmful DTHR. Here we demonstrate that therapeutic immune deviation can reverse established contact hypersensitivity (CHS), a Th1/Tc1-mediated DTHR. One or two weeks after induction of CHS, mice received either a single cycle of IL-4 therapy or adoptive transfer of antigen-specific Th2 cells. This treatment generated a novel state of immunity that provided long-lasting protection against tissue destruction and neutrophil recruitment during subsequent antigen exposures. Therapeutic immune deviation of established CHS was dependent on CD4+ T cells and the induction of endogenous IL-4 synthesis. Thus, a population of immunoregulatory Th2 cells persists during advanced inflammatory responses that can be used for therapeutic deviation of established DTHR.

Adoptive Transfer↗

PubMed2001

Regulation of life and death in lamina propria T cells.

T cells are essential to initiation, amplification, and regulation of an immune response. This response is terminated when T cells undergo apoptosis, a physiological process of cell death triggered by various mechanisms and regulated by signaling pathways leading to enzymatic degradation of chromatin. An effective immune response depends on the proper balance between proliferation and death of activated T cells. This is particularly important in the intestine, where mucosal T cells are subjected to the high antigenic pressure of lumenal antigens and apoptosis is required to induce tolerance and maintain a state of 'physiological' inflammation. Insufficient apoptosis may result in excessive T cell retention and chronic intestinal inflammation, as seen in conditions associated with defective apoptosis of lamina propria T cells.

Animals↗

PubMed2001

IL-4 instructs TH1 responses and resistance to Leishmania major in susceptible BALB/c mice.

Immunity to infection with intracellular pathogens is regulated by interleukin 12 (IL-12), which mediates protective T helper type 1 (TH1) responses, or IL-4, which induces TH2 cells and susceptibility. Paradoxically, we show here that when present during the initial activation of dendritic cells (DCs) by infectious agents, IL-4 instructed DCs to produce IL-12 and promote TH1 development. This TH1 response established resistance to Leishmania major in susceptible BALB/c mice. When present later, during the period of T cell priming, IL-4 induced TH2 differentiation and progressive leishmaniasis in resistant mice. Because immune responses developed via the consecutive activation of DCs and then T cells, the contrasting effects of IL-4 on DC development and T cell differentiation led to immune responses that had opposing functional phenotypes.

Animals↗

PubMed2001

Hierarchical cleavage of focal adhesion kinase by caspases alters signal transduction during apoptosis of intestinal epithelial cells.

BACKGROUND & AIMS: Purified intestinal epithelial cells die of detachment-induced apoptosis due to loss of cell anchorage during isolation. Anchorage-dependent cells form focal adhesions, sites of enhanced cell-matrix attachment that confer survival signals. Focal adhesion kinase (FAK), a component of the focal adhesion signaling complex, transduces these antiapoptotic signals. In this report, the molecular events leading to cleavage of FAK by caspases during apoptosis and its functional implications are defined. METHODS: Cytosolic extracts of human intestinal epithelial cells undergoing detachment-induced apoptosis were analyzed by Western blotting, immunoprecipitation, and kinase assay. RESULTS: FAK is cleaved by the ordered proteolytic activity of 2 different members of the caspase-3 family. The first cleavage is mediated by caspase-3, generating a 94/92-kilodalton-terminal fragment, which is processed by caspase-6 to an 84-kilodalton fragment. After apoptosis is initiated, the level of FAK phosphorylation is rapidly decreased, and the phosphorylation pattern of FAK-associated proteins is dramatically modified, showing significant yet divergent changes in signal transduction. CONCLUSIONS: Cleavage of FAK during apoptosis of normal human cells is an example of the sequential, highly regulated, and coordinate action of caspases that not only dismantle a cell by proteolysis, but also alter the cell's signaling machinery.

Amino Acid Chloromethyl Ketones↗

PubMed2001

Decreased Bax expression by mucosal T cells favours resistance to apoptosis in Crohn's disease.

BACKGROUND: Activated T cells are more susceptible to apoptosis than resting T cells. As intestinal T cells normally exhibit a higher state of activation, increased apoptosis may be necessary to maintain immune homeostasis in the specialised microenvironment of the mucosa. On the other hand, in Crohn's disease (CD) mucosal T cells are resistant to apoptosis, suggesting abnormal regulation of cell death mechanisms. AIMS: To investigate differences in expression of anti- and proapoptotic Bcl-2 family proteins, key regulators of apoptosis, between circulating and mucosal T cells, and possible alterations in CD. PATIENTS AND METHODS: Lamina propria T cells (LPT) were isolated from 10 control, seven CD, and eight ulcerative colitis (UC) patients, and peripheral blood T cells (PBT) from healthy volunteers. Purified T cells were stained intracellularly for Bcl-2, Bcl-x(L), and Bax, and mean fluorescence intensity measured by flow cytometry. RESULTS: Compared with PBT, the expression level of Bcl-2 and Bax, but not Bcl-x(L), was significantly greater in LPT, resulting in lower Bcl-x(L)/Bax ratios. In PBT, Bax expression was highly and significantly correlated with both Bcl-2 and Bcl-x(L), but correlation with Bcl-2 was absent in LPT. Bax expression in CD, but not UC, LPT was significantly lower than in control LPT, resulting in a significantly higher Bcl-x(L)/Bax ratio. The significant correlation of Bcl-x(L) to Bax was preserved in CD, but not UC, LPT. CONCLUSIONS: Regulation of Bcl-2 family protein expression differs between circulating and mucosal T cells, probably underlying diverse survival potentials. In CD LPT, a low Bax expression and a high Bcl-x(L)/Bax ratio favour resistance to apoptosis and may contribute to the chronicity of inflammation.

Adolescent↗

PubMed2000

Immunology of inflammatory bowel disease.

The immunology of inflammatory bowel disease continues to be an intense area of investigation for clues to the pathogenesis of Crohn disease and ulcerative colitis. As typical with complex diseases, inflammatory bowel disease research is continuously evolving. Without abandoning traditional areas of study, such as humoral and cellular immunity and cytokines, investigation is broadening to explore new molecules and biologic phenomena. Novel cytokines and cell adhesion molecules appear to be involved in inflammation, while the role of nitric oxide is being clarified. Leukocyte resistance to apoptosis appears to be a major contributing factor to Crohn disease. Epithelial cell-derived defensins and receptors are arising as key molecules mediating the interaction of innate and acquired mucosal immunity with the enteric flora, and explaining how the latter participates in gut inflammation. The results of these combined studies are opening novel therapeutic horizons whose implementation offers better forms of treatment.

Journal Article↗

PubMed2000

Apoptosis: implications for inflammatory bowel disease.

Cells of the intestinal mucosa live in a harsh environment and therefore rely heavily on the highly regulated process of cell death, apoptosis, to maintain tissue integrity. Imbalance in the intracellular events that modulate apoptosis may contribute to the pathogenesis of inflammatory bowel disease.

Apoptosis↗

PubMed1999

The differentiated state of intestinal lamina propria CD4+ T cells results in altered cytokine production, activation threshold, and costimulatory requirements.

Intestinal lamina propria (LP) CD4+ T cells are memory-like effector cells that proliferate at relatively low levels and require high levels of TCR signaling and costimulation for full activation in vitro. To study LP CD4+ T cell functional potential we used DO11.10 TCR transgenic (Tg) mice specific for the class II MHC-restricted OVA323-339 peptide and nontransgenic BALB/c mice. Activation of LP Tg+ T cells with Ag using mucosal explants induced high levels of IL-2, IL-4, and IFN-gamma. Culturing isolated LP cells with IL-12 enhanced IFN-gamma production and down-regulated IL-4 and IL-2, whereas addition of IL-4 maintained IL-4 production without inhibiting IFN-gamma production. Systemic administration of relatively high dose (HD; 100 nM) OVA323-339 peptide induced similar levels of bromodeoxyuridine (BrdU) incorporation by LP and splenic Tg+ T cells in vivo, whereas low dose (LD; 4.5 nM) peptide injections induced 4-fold greater levels of BrdU incorporation for LP compared with splenic Tg+ T cells. Coadministration of CTLA-4Ig reduced BrdU incorporation for splenic cells by 70% with HD and LD stimulation, but had little effect on LP responses to HD stimulation. Results of in vivo studies were confirmed in nontransgenic BALB/c mice using HD (200 microg) and LD (10 microg) anti-CD3 mAb+/- CTLA-4Ig. These results suggest that LP T cells are differentiated effector cells that respond at high levels when activated with relatively low levels of Ag- and B7-mediated costimulation in vivo. The reduced activation threshold of LP T cells may facilitate responses to low levels of Ag derived from mucosal pathogens.

Animals↗

PubMed1999

Resistance of Crohn's disease T cells to multiple apoptotic signals is associated with a Bcl-2/Bax mucosal imbalance.

Crohn's disease (CD) is a condition characterized by excessive numbers of activated T cells in the mucosa. We investigated whether a defect in apoptosis could prolong T cell survival and contribute to their accumulation in the mucosa. Apoptotic, Bcl-2+, and Bax+ cells in tissue sections were detected by the TUNEL method and immunohistochemistry. T cell apoptosis was induced by IL-2 deprivation, Fas Ag ligation, and exposure to TNF-alpha and nitric oxide. TUNEL+ leukocytes were few in control, CD, and ulcerative colitis (UC) mucosa, with occasional CD68+ and myeloperoxidase+, but no CD45RO+, apoptotic cells. Compared with control and UC, CD T cells grew remarkably more in response to IL-2 and were significantly more resistant to IL-2 deprivation-induced apoptosis. CD T cells were also more resistant to Fas- and nitric oxide-mediated apoptosis, whereas TNF-alpha failed to induce cell death in all groups. Compared with control, CD mucosa contained similar numbers of Bcl-2+, but fewer Bax+, cells, while UC mucosa contained fewer Bcl-2+, but more Bax+, cells. Hence, the Bcl-2/Bax ratio was significantly higher in CD and lower in UC. These results indicate that CD may represent a disorder where the rate of T cell proliferation exceeds that of cell death. Insufficient T cell apoptosis may interfere with clonal deletion and maintenance of tolerance, and result in inappropriate T cell accumulation contributing to chronic inflammation.

Abatacept↗

PubMed1999

Regulation of ICAM-1-mediated fibroblast-T cell reciprocal interaction: implications for modulation of gut inflammation.

BACKGROUND & AIMS: Immune-nonimmune cell interactions modulate mucosal immunity. We investigated the expression of adhesion molecules by intestinal fibroblasts, the effect of immune cell-derived factor on fibroblast binding of T cells, and the consequences of interfering with adhesion molecule expression on fibroblast-T cell interaction. METHODS: Expression of fibroblast intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 surface and messenger RNA (mRNA) was measured before and after exposure to immune cell-derived supernatants. Fibroblasts were treated with antibodies to ICAM-1 or VCAM-1, or ICAM-1 antisense oligonucleotide Isis 2302, before a T-cell adhesion assay. RESULTS: Fibroblast activation by immune cell-derived cytokines enhanced ICAM-1 and VCAM-1 surface expression and mRNA as well as adhesiveness for T cells. Blockade with neutralizing antibodies showed that binding was almost exclusively dependent on ICAM-1. Isis 2302 specifically reduced fibroblast ICAM-1 mRNA and dose-dependently inhibited ICAM-1 surface expression and T-cell binding. CONCLUSIONS: ICAM-1 is essential for intestinal fibroblast binding of T cells, a phenomenon that is efficiently and specifically disrupted by ICAM-1 antisense oligonucleotides. These observations emphasize the crucial regulatory role of fibroblasts in mucosal immunity and their potential as targets for therapeutic intervention in intestinal inflammation.

Blotting, Northern↗

PubMed1998

New isolation technique to study apoptosis in human intestinal epithelial cells.

Intestinal epithelial cells derive from stem cells at the base of the crypt and migrate along the crypt-lumen axis. Their life is terminated as they reach the luminal surface where they detach and are shed. Intestinal epithelial cells show evidence of apoptosis in the region of shedding, and cell death is thought to resemble a form of apoptosis called detachment-induced cell death, or anoikis. Human intestinal epithelial cells die rapidly in vitro due to loss of anchorage during isolation, making primary culture of these cells a goal that has not yet been reached. However, the molecular mechanisms underlying this process of anoikis are largely unknown. In this study, a novel protocol for the rapid, temperature-controlled isolation of highly purified human colonic epithelial cells from surgical specimens is described. Using this method, early molecular events of anoikis in nontransformed epithelial cells were studied. Intestinal epithelial cells were isolated at the beginning of the apoptotic cascade, before the activation of caspase 3 family members and cleavage of poly(ADP-ribose) polymerase and DNA fragmentation. Elucidating the molecular mechanisms of detachment-induced cell death may facilitate the establishment of long-term primary cultures of human intestinal epithelial cells and enhance our understanding of homeostasis in the intestinal epithelium.

Apoptosis↗

PubMed1998

Sequential and rapid activation of select caspases during apoptosis of normal intestinal epithelial cells.

Detachment-induced cell death (DICD) is considered to be one of the means by which intestinal epithelial cells (IEC) die of apoptosis as they reach the lumen and are shed. Caspases, a family of cysteine proteases, play a central role in initiating, amplifying, and executing apoptosis; however, the pattern of caspase activation in response to distinct apoptotic stimuli remains unknown. We investigated the kinetics of caspase activation during DICD in freshly isolated human IEC. DNA fragmentation is observed 90 min after detachment and is preceded by the sequential activation of preformed members of the CPP32 family of caspases. Activation of caspase 6 and cleavage of the endogenous caspase substrate poly(ADP-ribose) polymerase (EC 2.4.2.30) are detected within 15 min of detachment, 30-45 min before caspase 3 activation. Caspase 1 and caspase 10 are present as proenzymes, yet they remain inactive in response to this trigger of apoptosis. Human IEC are primed to rapidly undergo detachment-induced apoptosis involving the selective and sequential activation of preformed caspases. This study may enhance our understanding of physiological events occurring as IEC are shed. Their rapid apoptotic response to detachment may facilitate the high turnover of cells and ensure homeostasis in the intestinal epithelium.

Apoptosis↗

PubMed1997

Biogeochemical assessment of natural attenuation of JP-4-contaminated ground water in the presence of fluorinated surfactants.

The biogeochemistry of the natural attenuation of petroleum-contaminated ground water was investigated in a field study. The focus of the study was a fire training site located on Tyndall Air Force Base in Florida. The site has been used by the Air Force for approximately 11 years in fire fighting exercises. An on-site above-ground tank of JP-4 provided fuel for setting controlled fires for the exercises. Various amounts of water and aqueous film forming foams (AFFF) were applied to extinguish the fires. The sources of contamination included leaks from pipelines transporting the fuel, leaks from an oil/water separator and runoff and percolation from the fire fighting activities. Previous investigations had identified jet fuel contamination at the site, however, no active remediation efforts have been conducted to date. The goal of this study was to use biogeochemical monitoring data to delineate redox zones within the site and to identify evidence of natural attenuation of JP-4 contamination. In addition to identifying several hydrocarbon metabolites, fluorinated surfactants (AFFF) were detected down-gradient of the hydrocarbon plume.

Environmental Exposure↗

PubMed1997

Crohn's disease and ulcerative colitis mucosal T cells are stimulated by intestinal epithelial cells: implications for immunosuppressive therapy.

BACKGROUND: Crohn's disease (CD) and ulcerative colitis (UC) are chronic inflammatory diseases, and their pathogenesis is attributed, in part, to alterations of the mucosal immune system. This study was designed to define the possible contribution of epithelial cells to the activation of lamina propria T lymphocytes (LPTs) in CD and UC. METHODS: LPTs isolated from CD, UC, and control surgical specimens were cocultured with freshly isolated allogeneic or autologous epithelial cells or epithelial cell lines. Resulting T-cell proliferation was evaluated by tritiated thymidine incorporation on day 5. RESULTS: When intestinal epithelial cells were used to stimulate mucosal T-cell proliferation, CD and UC LPTs were less responsive than control LPTs (p < 0.05 and p < 0.03, respectively). This difference between inflamed and control T cells was consistently observed by using a variety of different intestinal epithelial cell types. CONCLUSIONS: CD and UC mucosal T cells are hyporesponsive to activation by intestinal epithelial cells when compared with control LPTs. Elucidating the mechanism underlying the differential activation of CD and UC LPTs may help to better understand the immunopathogenesis of these conditions.

Antigens, CD↗

PubMed1996

A strict requirement of interleukin-4 for interleukin-4 induction in antigen-stimulated human memory T cells.

The role of interleukin-4 (IL-4) in the induction of IL-4 in mouse T cells is well established, but conflicting results have been reported with anti-CD3-primed human T cells and T cell clones. Therefore, IL-4 regulation was investigated in short-term cultured human T cells primed in vitro with either a superantigen or a hapten, nickel sulfate (NiSO4), for 3 days and expanded with IL-2 for another 5 days. Under these conditions, antigen-specific IL-4 producing T cells were generated in 35/40 cultures. Priming for IL-4 production was abrogated in all cultures by anti-IL-4 antibody or soluble IL-4 receptor (sIL-4R). Primed T cells that were IL-4- when cultured with IL-2 only developed an IL-4 producing phenotype when primed and expanded in the presence of exogenous IL-4. T cells primed in the presence of either endogenous or exogenous IL-4 produced 10-200-fold more IL-4 than T cells primed in the presence of anti-IL-4 antibody or sIL-4R. While IL-4 induction was absolutely dependent on IL-4, neither endogenous nor exogenous IL-4 influenced IFN-gamma synthesis. Most importantly, IL-4 induced and sIL-4R abolished priming for IL-4 production even in NiSO4-specific memory T cells from sensitized individuals. Thus, IL-4 induction in antigen-specific human memory T cell populations absolutely required IL-4. The IL-4 pathway of memory T cells retained a remarkable plasticity in sensitized individuals.

Adult↗

PubMed1996

Interleukin 4 in inflammatory bowel disease and mucosal immune reactivity.

BACKGROUND & AIMS: Interleukin (IL) 4 has immunoregulatory and anti-inflammatory activities, but little is known about IL-4 in the human gut. We investigated production of IL-4 by isolated lamina propria mononuclear cells (LPMCs) from normal and inflamed intestine and its capacity to modulate local immune responses. METHODS: IL-4 levels were measured by enzyme-linked immunosorbent assay in cultures of control and inflammatory bowel disease LPMCs, and the effect of IL-4 on LPMC proliferation and interaction with IL-2, IL-1 beta, lipopolysaccharide, bacterial antigens, superantigen, and antibodies to various T-cell receptors was investigated. RESULTS: Various stimuli induced LPMCs to produce IL-4, but inflammatory bowel disease cells expressed IL-4 messenger RNA and secreted protein in significantly lower amounts than control cells. IL-4 failed to stimulate proliferation by fresh LPMCs, but a vigorous dose-dependent response was observed after preactivation by phytohemagglutinin, IL-2, or IL-4. When added to fresh LPMCs, IL-4 inhibited IL-2-induced proliferation. IL-4 amplified proliferation to IL-1 beta, lipopolysaccharide, peptidoglycan-polysaccharide complexes, staphylococcus enterotoxin A, and antibodies to the CD3 and CD28 receptors but not to tetanus toxoid. CONCLUSIONS: Decreased production of IL-4 in inflammatory bowel disease may cause defective immunosuppressive and anti-inflammatory mechanisms and may contribute to disease pathogenesis. The ability of IL-4 to differentially modulate LPMC reactivity probably influences mucosal immune homeostasis.

Adolescent↗

PubMed1995

Microbial biosorption of copper and lead from aqueous systems.

Biosorption of metal ions from aqueous systems was evaluated using a culture of acidic soil isolates grown in a completely mixed, aerobic, semi-batch culture reactor. The laboratory scale system was used to test single and bimetallic solutions of copper and lead with sulfates, chlorides, or nitrates. To elucidate the key factors influencing biosorption and to characterize metal uptake by cellular and extra cellular components of the microbial system, a dialysis testing procedure was developed. A direct contact technique was used to determine the rate of metal sorption on cellular surfaces. The effectiveness of biosorption was influenced by pH, initial metal concentrations, and anionic composition. Respirometric tests were carried out to identify potential inhibitory effects of metal accumulation on microbial oxygen uptake rates.

Absorption↗