PubMed Health⌕ Search

Biomedical subjects

R A Flavell

Publications and source records attributed to R A Flavell.

At least 181 records · Page 10Linked to original sources
PubMed1997

Cleavage of FLICE (caspase-8) by granzyme B during cytotoxic T lymphocyte-induced apoptosis.

Cytotoxic T lymphocytes induce apoptosis in target cells through the CD95(APO-1/Fas) and the perforin/granzyme B (GrB) pathway. The exact substrate of GrB in vivo is still unknown, but to induce apoptosis GrB requires the activity of caspases in target cells. We show here that in HeLa target cells induction of apoptosis through the perforin/GrB pathway resulted in minor direct cleavage of CPP32 (caspase-3) by GrB. Most caspase-3 cleavage resulted from activation of an upstream caspase. Moreover, target cells derived from caspase-3(-/-) mice displayed GrB-induced poly(ADP-ribose) polymerase (PARP) cleavage with only partially reduced efficiency compared to wild-type target cells. This indicates that other PARP-cleaving caspases can be activated during perforin/GrB-induced cell death. In contrast to caspase-3, FLICE (caspase-8) was directly cleaved by GrB in HeLa cells. We therefore conclude that FLICE not only plays a central role in CD95(APO-1/Fas)-induced apoptosis but can also be directly activated during perforin/GrB-induced apoptosis.

Animals↗
PubMed1997

The CD40 ligand. At the center of the immune universe?

For several years, the primary function of CD40 ligand (CD40L) has been believed to be in regulation of contact-dependent, CD40-CD40L-mediated signals between B- and T-cells, which are essential for the regulation of thymus-dependent (TD) humoral immune responses. Recently, a flurry of reports indicate that CD40 is expressed by variety of cell types other than B-cells that include dendritic cells, follicular dendritic cells, monocytes, macrophages, fibroblasts, and endothelial cells. These studies show that CD40-CD40L interactions are important in inflammatory process. For the past few years, through the availability of CD40L-knockout mice, new data have emerged to support the belief that CD40L has many more functions than its role in TD humoral immunity. CD40L-deficient mice have provided significant information towards our understanding of the in vivo role of CD40L. The current picture that emerges indicates that CD40-CD40L interactions mediate many cell-mediated immune responses and T-cell-mediated effector functions that are required for proper functioning of the host defense system. This article focuses on the in vivo role of the CD40L in regulation of cell-mediated effector functions.

Animals↗
PubMed1997

The CD40-CD154 system in anti-infective host defense.

Research in the past few years has documented significant advances in our understanding of the CD40-CD40 ligand (CD154) system in diverse immune functions. This system influences many T cell mediated inflammatory immune responses and effector functions, unmasking a previously unexpected role for CD40-CD154 in cell mediated immunity. Manipulation of CD154 in animal models of infection by the use of CD154-deficient mice or anti-CD154 antibodies has shown the importance of this system in the initiation of the inflammatory response, in the activation of antigen-presenting cells and in resistance to infections.

Animals↗
PubMed1997

Distinct roles in lymphoid organogenesis for lymphotoxins alpha and beta revealed in lymphotoxin beta-deficient mice.

Lymphotoxin alpha (LT alpha)-deficient mice revealed critical roles for LT alpha in lymphoid organogenesis, but it is not clear whether LT alpha functions through an LT alpha homotrimer (LT alpha3) or LT alpha/beta heterotrimers. We generated LTbeta-deficient mice and found them to lack Peyer's patches, peripheral lymph nodes, splenic germinal centers, and follicular dendritic cells. Unlike LT alpha-deficient mice, LT beta-deficient mice had cervical and mesenteric lymph nodes. Furthermore, the mesenteric lymph nodes had germinal center-like regions, although these structures appeared to lack follicular dendritic cells. The absence of cervical and mesenteric lymph nodes in LT alpha-deficient mice, and yet their presence in LT beta-deficient mice and in mice deficient in tumor necrosis factor receptor types I and II, suggest that LT alpha3 may signal via an as yet unidentified receptor.

Animals↗
PubMed1997

Borrelia burgdorferi P35 and P37 proteins, expressed in vivo, elicit protective immunity.

p35 and p37 are Borrelia burgdorferi genes encoding 35 and 37 kDa proteins. The gene products were identified by differential screening of a B. burgdorferi expression library with sera from B. burgdorferi infected- and B. burgdorferi-hyperimmunized mice. Northern blot and RT-PCR analyses confirmed that these genes were selectively expressed in vivo. ELISA, using P35 and P37, showed that infected mice (5 of 5, 100%) and patients (31 of 43, 72%) with Lyme borreliosis developed P35 or P37 antibodies. Mice developed peak IgG titers to P35 and P37 within 30 days, followed by decline. Mice given both P35 and P37 antisera were protected from challenge with 10(2) B. burgdorferi, and P35 and P37 antisera also afforded protection when administered 24 hr after spirochete challenge. The use of in vivo-expressed antigens such as P35 and P37 represents a new approach for Lyme disease serodiagnosis and for understanding the role of B. burgdorferi-specific immune responses in host immunity.

Amino Acid Sequence↗
PubMed1997

Immunological tolerance to a pancreatic antigen as a result of local expression of TNFalpha by islet beta cells.

Recent experiments have suggested that tumor necrosis factor alpha (TNFalpha) can down-regulate islet-specific T cells and prevent the development of autoimmune diabetes. Here we demonstrate that transgenic mice expressing both TNFalpha and the Leishmania major LACK antigen in the pancreas (RIP-TNFalpha/RIP-LACK) exhibit an impaired ability to mount a CD4+ T cell response against LACK. In addition, peripheral CD4+ T cells from TCR transgenic mice (TCR-LACK/RIP-TNFalpha/RIP-LACK) produced reduced interleukin-2 but elevated levels of T helper 2 cytokines in response to LACK peptide in vitro. Taken together, our data suggest that TNFalpha may act in vivo to modulate a potentially damaging self-reactive T cell response by inducing tolerance to pancreatic antigens.

Animals↗
PubMed1997

Reprogramming the signalling requirement for AP-1 (activator protein-1) activation during differentiation of precursor CD4+ T-cells into effector Th1 and Th2 cells.

Upon antigenic stimulation, precursor CD4+ helper T-cells differentiate into two subsets of effector cells, Th1 and Th2. These two subpopulations are defined by the pattern of cytokine expression that distinguishes these differentiated cells from their precursors. We have used reporter transgenic mice here to show that, during differentiation of precursor T-cells into effector Th1 or Th2 cells, high levels of preformed activator protein (AP)-1 complexes are accumulated. However, upon stimulation, the preformed AP-1 complexes in effector Th2 cells, but not in Th1 cells, are able to induce high levels of AP-1 transcriptional activity. Furthermore, in contrast to precursor T-cells, the induction of AP-1 transcriptional activity is independent of calcium and co-stimulatory signals in effector Th2 cells. This AP-1 transcriptional activity appears to correlate with the presence of JunB complexes, which accumulate differentially in effector Th2 cells, but not in precursor CD4+ T-cells or effector Th1 cells. Unlike precursor cells, the activation of AP-1 does not appear to be mediated by c-Jun N-terminal kinase (JNK) in effector Th2 cells. These results indicate that during differentiation of T-cells, and probably other cell types, the signal requirements for the AP-1 transcription machinery are reprogrammed to enable the differentiated cells to perform their specialized functions.

Amino Acid Sequence↗
PubMed1997

Lyme borreliosis in transgenic mice tolerant to OspA from Borrelia burgdorferi 25015.

Lyme borreliosis in transgenic mice tolerant to Borrelia burgdorferi outer surface protein A (OspA) from the weakly virulent B. burgdorferi 25015 was assessed because the response to OspA has been implicated in protective immunity and is suspected in disease pathogenesis. B. burgdorferi 25015 is infectious but does not cause significant arthritis in C3H/HeN mice. Tolerance of mice to B. burgdorferi N40 OspA had no effect on disease. Since B. burgdorferi N40 is more virulent than and genetically distinct from B. burgdorferi 25015, the generality of these findings was investigated. OspA-25015-transgenic mice were tolerant to OspA-25015 and did not develop OspA antibodies or cellular responses following immunization with OspA-25015. The development and resolution of Lyme arthritis was similar in transgenic and nontransgenic littermates, suggesting that ablation of the OspA-25015 response does not cause B. burgdorferi 25015 to become more arthritogenic.

Animals↗
PubMed1997

Serologic diagnosis of canine and equine borreliosis: use of recombinant antigens in enzyme-linked immunosorbent assays.

Serum samples from dogs and equids suspected of having canine or equine borreliosis, respectively, were analyzed in polyvalent enzyme-linked immunosorbent assays (ELISAs) with whole-cell or recombinant antigens of Borrelia burgdorferi sensu stricto. Purified preparations of recombinant antigens included outer surface protein A (OspA), OspB, OspC, OspE, OspF, and p41-G (a fragment of flagellin). Of the 36 dog sera that reacted positively to whole-cell antigen, 32 (88.9%) contained antibodies to one or more recombinant antigens. Reactivities to OspF (88.9% positive) and p41-G (75% positive) were most prevalent. In analyses of 30 equid sera positive in an ELISA with whole cells, 24 (80%) contained antibodies to one or more recombinant antigens. Seropositivities in ELISAs with p41-G (50% positive) and OspF (46.7% positive) were more than twofold greater than in ELISAs with OspA, OspB, or OspC (10 to 20% positive). In parallel tests of eight canine and three equine sera, there was good agreement in results of Western blot (immunoblot) analyses and ELISAs. Although dog and equid sera with antibodies to whole-cell B. burgdorferi frequently reacted positively to one or more recombinant antigens, the inclusion of OspF and p41-G antigens in ELISAs was most useful in the serologic diagnosis of canine and equine borreliosis.

Animals↗
PubMed1997

Differential transcription directed by discrete gamma interferon promoter elements in naive and memory (effector) CD4 T cells and CD8 T cells.

Acquisition of the ability to produce gamma interferon (IFN-gamma) is a fundamental property of memory T cells and enables one subset (T helper 1 [TH1]) to deliver its effector functions. To examine regulation of IFN-gamma gene expression in a model system which recapitulates TH1 differentiation, we prepared reporter transgenic mice which express the luciferase gene under the control of proximal and distal regulatory elements (prox.IFN gamma and dist.IFN gamma) from the IFN-gamma promoter. Memory T cells, but not naive T cells, secreted IFN-gamma and expressed both prox.IFN gamma and dist.IFN gamma transcriptional activities. Naive T cells required priming to become producers of IFN-gamma and to direct transcription by these elements. While both CD4+ and CD8+ T cells produced IFN-gamma, only CD4+ T cells expressed prox.IFN gamma transcriptional activity. Induction of transcriptional activity was inhibited by known antagonists of effector T-cell populations. Cyclosporin A inhibited transcriptional activity directed by both elements in effector T cells. Elevated cyclic AMP inhibited transcriptional activity directed by prox.IFN gamma in primed CD4+ T cells but enhanced transcriptional activity directed by dist.IFN gamma in primed CD8+ T cells. Taken together, these data show that prox.IFN gamma and dist.IFN gamma transcriptional activities mirror IFN-gamma gene expression in naive and memory CD4+ T cells but suggest that differences exist in regulation of IFN-gamma gene expression in CD4+ and CD8+ T-cell subsets.

Animals↗
PubMed1997

Transcription mediated by NFAT is highly inducible in effector CD4+ T helper 2 (Th2) cells but not in Th1 cells.

Transcriptional factors of the NFAT family play an important role in regulating the expression of several cytokine genes during the immune response, such as the genes for interleukin 2 (IL-2) and IL-4, among others. Upon antigen stimulation, precursor CD4+ T helper (pTh) cells proliferate and differentiate into two populations of effector cells (eTh1 and eTh2), each one expressing a specific pattern of cytokines that distinguishes them from their precursors. eTh2 cells are the major source of IL-4, while gamma interferon is produced by eTh1 cells. Here we have used reporter transgenic mice to show that DNA binding and transcriptional activities of NFAT are transiently induced during the differentiation of pTh cells into either eTh1 or eTh2 cells to mediate the expression of IL-2 as a common growth factor in both pathways. However, although NFAT DNA binding is similarly induced in both eTh1 and eTh2 cells upon antigen stimulation, only the NFAT complexes present in eTh2 cells are able to mediate high-level transcription, and relatively little NFAT transcriptional activity was induced in eTh1 cells. In contrast to activated pTh cells, neither eTh1 nor eTh2 cells produced significant IL-2 upon stimulation, but the high levels of NFAT transcriptional activities directly correlate with the IL-4 production induced in response to antigen stimulation in eTh2 cells. These data suggest that activated NFAT is involved in the effector function of eTh2 cells and that the failure of eTh1 cells to produce IL-4 in response to an antigen is due, at least partially, to a failure to induce high-level transcription of the IL-4 gene by NFAT. Regulation of NFAT could be therefore a critical element in the polarization to eTh1 or eTh2.

Animals↗
PubMed1997

Activation of the STAT signaling pathway can cause expression of caspase 1 and apoptosis.

Protein tyrosine kinases activate the STAT (signal transducer and activator of transcription) signaling pathway, which can play essential roles in cell differentiation, cell cycle control, and development. However, the potential role of the STAT signaling pathway in the induction of apoptosis remains unexplored. Here we show that gamma interferon (IFN-gamma) activated STAT1 and induced apoptosis in both A431 and HeLa cells, whereas epidermal growth factor (EGF) activated STAT proteins and induced apoptosis in A431 but not in HeLa cells. EGF receptor autophosphorylation and mitogen-activated protein kinase activation in response to EGF were similar in both cell lines. The breast cancer cell line MDA-MB-468 exhibited a similar response to A431 cells, i.e., STAT activation and apoptosis correlatively resulted from EGF or IFN-gamma treatment. In addition, in a mutant A431 cell line in which STAT activation was abolished, no apoptosis was induced by either EGF or IFN-gamma. We further demonstrated that both EGF and IFN-gamma induced caspase 1 (interleukin-1beta converting enzyme [ICE]) gene expression in a STAT-dependent manner. IFN-gamma was unable to induce ICE gene expression and apoptosis in either JAK1-deficient HeLa cells (E2A4) or STAT1-deficient cells (U3A). However, ICE gene expression and apoptosis were induced by IFN-gamma in U3A cells into which STAT1 had been reintroduced. Moreover, both EGF-induced apoptosis and IFN-gamma-induced apoptosis were effectively blocked by Z-Val-Ala-Asp-fluoromethylketone (ZVAD) in all the cells tested, and studies from ICE-deficient cells indicated that ICE gene expression was necessary for IFN-gamma-induced apoptosis. We conclude that activation of the STAT signaling pathway can induce apoptosis through the induction of ICE gene expression.

Apoptosis↗
PubMed1997

A novel role for murine IL-4 in vivo: induction of MUC5AC gene expression and mucin hypersecretion.

Mucus hypersecretion and plugging of lower respiratory tract airways contributes to the morbidity and mortality associated with asthma. Interleukin (IL)-4 plays a putative role in some forms of asthma. Thus, transgenic mice that overexpress murine IL-4 selectively within the lung were used to study the effect of IL-4 on mucus glycoprotein gene expression and mucin release. Histologic examination of lung sections from IL-4 mice revealed that nonciliated epithelial cells from conducting airways were hypertrophic, due at least in part to the accumulation of mucus glycoprotein. The cytoplasm of these cells stained positively for glycoproteins using mucicarmine, alcian blue (AB), and periodic acid-Schiff (PAS). Ciliated cells were also enlarged but did not show any mucin-specific staining. Inclusion granules typically found in nonciliated (Clara) cells of control mice were absent in the IL-4 transgenic mice. Northern blot analysis of total RNA from lung tissue revealed that the expression of the MUC5AC, but not MUC2, mucin gene was distinctly upgraded in IL-4 transgenic mice compared to transgene-negative controls. In addition, a 5- to 10-fold increase in AB- and PAS-positive material was found in lavage fluid from IL-4 overexpressing mice compared to transgene-negative controls. Thus, the overexpression of IL-4 locally within the lung enhances mucus glycoprotein synthesis by altering gene expression, results in the accumulation of mucus glycoprotein in nonciliated epithelial cells, and induces the release of mucus into the airway lumen. We therefore hypothesize that the overproduction of mucus seen in some patients with asthma may be a direct result of the action of IL-4 within the inflamed lung.

Alcian Blue↗
PubMed1997

Placenta-specific expression of the rat growth hormone-releasing hormone gene promoter in transgenic mice.

GH-releasing hormone (GHRH) is a hypothalamic peptide that plays a critical role in controlling the synthesis and secretion of GH in the anterior pituitary. Along with many other hypothalamic hormones, GHRH is also expressed in the placenta, although its physiological role in this tissue has not yet been determined. The placental prepro-GHRH is identical to that found in the hypothalamus. However, the placental and hypothalamic GHRH messenger RNAs differ in the region corresponding to the untranslated exon 1. A combined mechanism involving the use of tissue-specific promoters and the differential splicing of exon 1 generates the mature GHRH messenger RNAs in placenta and hypothalamus. As a first step toward the localization of the regulatory elements involved in the placenta-specific expression of the GHRH gene, we have generated transgenic mice containing constructs in which potential regulatory sequences of the rat GHRH gene were fused to the chloramphenicol acetyltransferase (CAT) reporter gene. Construct GHRH-CAT1, which contains 7.5 kilobases of flanking sequences upstream to the placental transcription start site, did not promote CAT expression in the transgenic animals. In contrast, construct GHRH-CAT2, which differs from construct GHRH-CAT1 in having additional sequences located downstream to placental exon 1, exhibited high levels of CAT expression in brain and placenta. Our results show that the sequences included in construct GHRH-CAT2 contain the cis-acting regulatory elements necessary to direct developmentally regulated and cell type-specific expression of the CAT gene in the placenta. Unexpectedly, the expression of the transgene in the brain was detected in glial cells of different areas, but not in the hypothalamus.

Animals↗
PubMed1996

Targeted expression of IL-11 in the murine airway causes lymphocytic inflammation, bronchial remodeling, and airways obstruction.

Interleukin-11 is a pleotropic cytokine produced by lung stromal cells in response to respiratory viruses, cytokines, and histamine. To further define its potential effector functions, the Clara cell 10-kD protein promoter was used to express IL-11 and the airways of the resulting transgene mice were characterized. In contrast to transgene (-) littermates, the airways of IL-11 transgene (+) animals manifest nodular peribronchiolar mononuclear cell infiltrates and impressive airways remodeling with subepithelial fibrosis. The inflammatory foci contained large numbers of B220(+) and MHC Class II(+) cells and lesser numbers of CD3(+), CD4(+), and CD8(+) cells. The fibrotic response contained increased amounts of types III and I collagen, increased numbers of alpha smooth muscle actin and desmin-containing cells and a spectrum of stromal elements including fibroblasts, myofibroblasts, and smooth muscle cells. Physiologic evaluation also demonstrated that 2-mo-old transgene (+) mice had increased airways resistance and non-specific airways hyperresponsiveness to methacholine when compared with their transgene (-) littermates. These studies demonstrate that the targeted expression of IL-11 in the mouse airway causes a B and T cell-predominant inflammatory response, airway remodeling with increased types III and I collagen, the local accumulation of fibroblasts, myofibroblasts, and myocytes, and obstructive physiologic dysregulation. IL-11 may play an important role in the inflammatory and fibrotic responses in viral and/or nonviral human airway disorders.

Airway Obstruction↗
PubMed1996

Identification of a Borrelia burgdorferi OspA T cell epitope that promotes anti-OspA IgG in mice.

Lyme disease, due to infection with the tick-borne spirochete Borrelia burgdorferi, is a multisystem disorder that can lead to chronic disabling symptoms. Abs to the outer surface protein A (OspA) of B. burgdorferi provide protection against infection, and OspA is now the basis of a first generation recombinant vaccine undergoing phase III efficacy studies. Recent studies have suggested that T cells reactive with N-terminal epitopes in OspA could contribute to the development of treatment-resistant Lyme arthritis. In the present studies, we use the murine model of Lyme borreliosis to define an OspA T cell epitope located in the carboxyl terminus that accelerates anti-OspA IgG production after infection. In addition, we show that this T cell epitope is elicited by immunization with rOspA or with a truncated form of OspA that contains the B cell epitope targeted by protective OspA mAb. Polyclonal antisera to the truncated OspA fragment can protect mice from challenge infection. These results are the first demonstration of a B. burgdorferi-specific peptide that elicits a biologically important T cell response in vivo and have implications for the design of a second generation OspA-based subunit vaccine.

Amino Acid Sequence↗