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Biomedical subjects

H E Thomas

Publications and source records attributed to H E Thomas.

At least 19 recordsLinked to original sources
PubMed2001

Lack of expression of Gp-130 makes pancreatic beta cell lines unresponsive to the IL-6 family of cytokines.

Cytokine receptors from the IL-6 receptor family are comprised of ligand specific alpha chains and a common signalling chain, gp-130, which is also required for high affinity binding. A cDNA library generated from the beta-TC3 SV40 T-antigen transformed insulinoma cell line was screened for members of this receptor family potentially relevant to both beta cell development and autoimmunity. Degenerate oligonucleotide primers to a consensus region of these receptors were used and the IL-11 receptor alpha chain was identified. Despite confirmation of IL-11 receptor mRNA expression, iodinated bioactive IL-11 did not bind specifically to beta-TC3 cells and gp-130-dependent cytokines did not elicit signalling events in beta cell lines. This was explained by absence of gp-130 protein or mRNA in the beta cell lines tested and in primary islets. We conclude from these results that the previously recognised effects of IL-6 family member cytokines on pancreatic islets must be indirect via other non-beta cells within the islet, rather than due to direct effects on beta cells themselves.

Animals↗
PubMed2001

gamma-Interferon signaling in pancreatic beta-cells is persistent but can be terminated by overexpression of suppressor of cytokine signaling-1.

Proinflammatory cytokines, including gamma-interferon (IFN-gamma), have been implicated in the destruction of beta-cells in autoimmune diabetes. IFN-gamma signaling is transient in some cell types, but there is indirect evidence that it may be prolonged in beta-cells. In this study, we have shown that IFN-gamma signaling, measured by signal transducer and activator of transcription-1 (STAT1) activation and the expression of IFN-gamma-responsive genes, is persistent in beta-cells for as long as the cytokine is present. Because members of the suppressor of cytokine signaling (SOCS) family may regulate the duration of IFN-gamma signaling, their expression was investigated in beta-cells. We found that cytokine-inducible SH2-containing protein, SOCS-1, and SOCS-2 are expressed in primary islets and NIT-1 insulinoma cells, both at the mRNA and protein levels, after treatment with IFN-gamma and other proinflammatory cytokines. Transfected SOCS-1 was found to inhibit responses to IFN-gamma in NIT-1 insulinoma cells, including STAT1 activation, class I major histocompatibility complex upregulation, and IFN-gamma-induced cell death, but only when expressed at levels higher than those found in untransfected cells. Consistent with this, IFN-gamma signaling was not affected in SOCS-1-deficient beta-cells. Therefore, persistent IFN-gamma signaling in beta-cells is associated with SOCS-1 expression that is not sufficient to terminate signaling. Because overexpression of SOCS-1 can suppress responses to IFN-gamma, this may be a useful strategy for protecting beta-cells from cytotoxicity mediated by IFN-gamma and possibly other proinflammatory cytokines.

Animals↗
PubMed2000

Protective effect of CTLA4Ig secreted by transgenic fetal pancreas allografts.

BACKGROUND: Pancreas allotransplantation offers a cure for insulin-dependent diabetes mellitus. Systemic immunosuppression used to prevent immune destruction of the graft has side-effects, including increased susceptibility to infection and neoplasia. These unwanted effects may be limited by engineering the graft to secrete immunomodulatory molecules, to achieve local immunosuppression. Several studies have shown that transient local CTLA4Ig results in partial protection of allogeneic grafts. Our intent has been to determine whether sustained secretion of transgenic CTLA4Ig from pancreatic islets is able to protect against allograft rejection. METHODS AND RESULTS: Mouse CTLA4 (test=CTLA4Ig) or CD5 leader sequence (control=CD5LIg) was fused to the Fc of mouse IgG2c, and expressed transgenically under the control of the rat insulin promoter in C57BL/6 mice carrying the bml mutation of H-2K(b) (B6.C-H-2(bm1)). This resulted in expression in pancreatic islets. We used ELISA quantification of transgene products secreted into the supernatants of cultured fetal pancreata to select high (CTLA4Ig(hi)) and low (CTLA4Ig(lo)) expresser transgenic mice. Cultured fetal pancreata were transplanted under the kidney capsule of wholly allogeneic CBA recipient mice. CTLA4Ig(hi) but not CTLA4Ig(lo) expresser grafts showed enhanced survival compared with control CD5LIg grafts at 6 weeks posttransplant, provided the recipient mice were transiently depleted of CD4 T cells (by a single low-dose injection of GK1.5) before transplantation. CONCLUSIONS: Sustained local secretion of CTLA4Ig from transgenic grafts in combination with transient systemic CD4 T-cell depletion can enhance allograft acceptance.

Abatacept↗
PubMed2000

Virus-induced autoimmune diabetes: most beta-cells die through inflammatory cytokines and not perforin from autoreactive (anti-viral) cytotoxic T-lymphocytes.

Autoimmune diabetes is caused by selective loss of insulin-producing pancreatic beta-cells. The main factors directly implicated in beta-cell death are autoreactive, cytotoxic (islet-antigen specific) T-lymphocytes (CTL), and inflammatory cytokines. In this study, we have used an antigen-specific model of virally induced autoimmune diabetes to demonstrate that even high numbers of autoreactive CTL are unable to lyse beta-cells by perforin unless major histocompatibility complex class I is upregulated on islets. This requires the presence of inflammatory cytokines induced by viral infection of the exocrine pancreas but not of the beta-cells. Unexpectedly, we found that the resulting perforin-mediated killing of beta-cells by autoreactive CTL is not sufficient to lead to clinically overt diabetes in vivo, and it is not an absolute prerequisite for the development of insulitis, as shown by studies in perforin-deficient transgenic mice. In turn, destruction of beta-cells also requires a direct effect of gamma-interferon (IFN-gamma), which is likely to be in synergy with other cytokines, as shown in double transgenic mice that express a mutated IFN-gamma receptor on their beta-cells in addition to the viral (target) antigen and do not develop diabetes. Thus, destruction of most beta-cells occurs as cytokine-mediated death and requires IFN-gama in addition to perforin. Understanding these kinetics could be of high conceptual importance for the design of suitable interventions in prediabetic individuals at risk to develop type 1 diabetes.

Animals↗
PubMed1999

Evidence that beta cell death in the nonobese diabetic mouse is Fas independent.

Recent studies suggest that Fas expression on pancreatic beta cells may be important in the development of autoimmune diabetes in the nonobese diabetic (NOD) mouse. To address this, pancreatic islets from NOD mice were analyzed by flow cytometry to directly identify which cells express Fas and Fas ligand (FasL) ex vivo and after in vitro culture with cytokines. Fas expression was not detected on beta cells isolated from young (35 days) NOD mice. In vitro, incubation of NOD mouse islets with both IL-1 and IFN-gamma was required to achieve sufficient Fas expression and sensitivity for islets to be susceptible to lysis by soluble FasL. In islets isolated from older (>/=125 days) NOD mice, Fas expression was detected on a limited number of beta cells (1-5%). FasL was not detected on beta cells from either NOD or Fas-deficient MRLlpr/lpr islets. Also, both NOD and MRLlpr/lpr islets were equally susceptible to cytokine-induced cell death. This eliminates the possibility that cytokine-treated murine islet cells commit "suicide" due to simultaneous expression of Fas and FasL. Last, we show that NO is not required for cytokine-induced Fas expression and Fas-mediated apoptosis of islet cells. These findings indicate that beta cells can be killed by Fas-dependent cytotoxicity; however, our results raise further doubts about the clinical significance of Fas-mediated beta cell destruction because few Fas-positive cells were isolated immediately before the development of diabetes.

Animals↗
PubMed1999

Tumor necrosis factor-alpha-activated cell death pathways in NIT-1 insulinoma cells and primary pancreatic beta cells.

Tumor necrosis factor-alpha (TNFalpha) is a potential mediator of beta cell destruction in insulin-dependent diabetes mellitus. We have studied TNF-responsive pathways leading to apoptosis in beta cells. Primary beta cells express low levels of the type I TNF receptor (TNFR1) but do not express the type 2 receptor (TNFR2). Evidence for TNFR1 expression on beta cells came from flow cytometry using monoclonal antibodies specific for TNFR1 and TNFR2 and from RT-PCR of beta cell RNA. NIT-1 insulinoma cells similarly expressed TNFR1 (at higher levels than primary beta cells) as detected by flow cytometry and radio-binding studies. TNF induced NF-kappaB activation in both primary islet cells and NIT-1 cells. Apoptosis in response to TNFalpha was observed in NIT-1 cells whereas apoptosis of primary beta cells required both TNFalpha and interferon-gamma (IFNgamma). Apoptosis could be prevented in NIT-1 cells by expression of dominant negative Fas-associating protein with death domain (dnFADD). Apoptosis in NIT-1 cells was increased by coincubation with IFNgamma, which also increased caspase 1 expression. These data show that TNF-activated pathways capable of inducing apoptotic cell death are present in beta cells. Caspase activation is the dominant pathway of TNF-induced cell death in NIT-1 cells and may be an important mechanism of beta cell damage in insulin-dependent diabetes mellitus.

Animals↗
PubMed1998

IFN-gamma action on pancreatic beta cells causes class I MHC upregulation but not diabetes.

We have generated transgenic nonobese diabetic (NOD) mice expressing dominant negative mutant IFN-gamma receptors on pancreatic beta cells to investigate whether the direct effects of IFN-gamma on beta cells contribute to autoimmune diabetes. We have also quantitated by flow cytometry the rise in class I MHC on beta cells of NOD mice with increasing age and degree of islet inflammatory infiltrate. Class I MHC expression increases gradually with age in wild-type NOD mice; however, no such increase is observed in the transgenic beta cells. The transgenic mice develop diabetes at a similar rate to that of wild-type animals. This study dissociates class I MHC upregulation from progression to diabetes, shows that the rise in class I MHC is due to local IFN-gamma action, and eliminates beta cells as the targets of IFN-gamma in autoimmune diabetes.

Adoptive Transfer↗
PubMed1997

Protection of NIT-1 pancreatic beta-cells from immune attack by inhibition of NF-kappaB.

We have recently observed that inhibition of NF-kappaB in NIT-1 insulinoma cells protects them from tumour necrosis factor (TNF)-induced cell death in vitro, possibly because expression of interleukin-1 (IL-1)beta-converting enzyme (ICE), a member of the cysteine protease pathway of cell death, is decreased. In the current study we have examined the effect of the same inhibitor of NF-kappaB on class I major histocompatibility complex (MHC) protein expression in NIT-1 cells and shown that inhibition of NF-kappaB activation decreased basal and TNF-induced class I MHC levels. Although inducible nitric oxide synthase (iNOS) may also be inhibited by inhibition of NF-kappaB, this could not be demonstrated in NIT-1/delta sp cells because wild-type NIT-1 cells express very little iNOS. When NIT-1/delta sp12 cells, expressing high levels of the NF-kappaB inhibitor, are transplanted into immunodeficient NOD/scid mice, tumorigenesis and death by hypoglycemia proceed similarly to untransfected NIT-1 cells. Untransfected NIT-1 cells were killed by co-transfer of splenic T cells from diabetic but not non-diabetic NOD mice. NIT-1/delta sp12 cells were protected from killing in vivo by T cells from diabetic mice, in that tumours developed in four out of five mice and the kinetics of tumour development were not significantly delayed. NIT-1/delta sp12 cells were not protected from killing by T cells from mice previously primed with NIT-1 cells. In conclusion, inhibition of NF-kappaB is likely to suppress several different pathways of immune-mediated cell death in beta-cells and protects NIT-1 cells from immune attack by diabetogenic T cells in vivo. Inhibition of NF-kappaB is a potentially effective strategy for protection of pancreatic beta-cells in autoimmune diabetes.

Adoptive Transfer↗
PubMed1997

Interferon regulatory factor 1 is induced by interferon-gamma equally in neurons and glial cells.

Class I MHC protein is induced in glia but not mature neurons by IFN-gamma. We have compared IFN-gamma signal transduction in these populations. There were identical levels of STAT1 homodimers and IRF-1 by gel-shift and IRF-1 mRNA was induced equally. However class I MHC, beta2-microglobulin and interleukin 1-beta converting enzyme mRNA levels were greatly reduced in neurons. These experiments show that there is no defect in expression of IRF-1 in response to IFN-gamma in mature mouse neurons but that insufficient class I MHC gene expression is induced for detectable cell surface protein expression.

Animals↗
PubMed1997

Transgenic expression of mouse proinsulin II prevents diabetes in nonobese diabetic mice.

IDDM in humans and in nonobese diabetic (NOD) mice is a T-cell-dependent autoimmune disease in which the beta-cells of the pancreatic islets are destroyed. Several putative beta-cell autoantigens have been identified, but insulin and its precursor, proinsulin, are the only ones that are beta-cell specific. (Pro)insulin may be a key autoantigen in IDDM. To address the role of proinsulin in the development of IDDM, we generated NOD mice transgenic for the mouse proinsulin II gene driven off a major histocompatibility complex (MHC) class II promoter to direct expression of the transgene to MHC class II bearing cells, including those in the thymus, with the aim of deleting proinsulin-reactive T-cells. The mononuclear cell infiltration of the islets (insulitis) is almost completely absent, and diabetes is prevented in these transgenic NOD mice. The mononuclear cell infiltration of the salivary glands (sialitis) and immune responses to ovalbumin (OVA) are not altered, indicating that the protective effect of the transgene is specific for islet pathology and not due to general immunosuppression. We conclude that autoimmunity to proinsulin plays a pivotal role in the development of IDDM.

Aging↗
PubMed1996

RIP-beta 2-microglobulin transgene expression restores insulitis, but not diabetes, in beta 2-microglobulin null nonobese diabetic mice.

Beta2m-deficient nonobese diabetic (NODbeta2mnull) do not develop insulitis or diabetes. Expression of a beta2m transgene controlled by the rat insulin promoter (RIP-beta2m) in NODbeta2mnull mice resulted in reconstitution of IFN-gamma-inducible cell surface MHC class I protein on pancreatic beta-cells. These mice developed insulitis, but did not develop diabetes. Transfer of T cells from diabetic NOD mice to NODbeta2mnull recipients resulted in insulitis, which took several months to progress to diabetes. In contrast, transgenic RIP-beta2m/NODbeta2mnull mice with islet MHC class I reconstitution developed diabetes rapidly after transfer of diabetic NOD spleen cells. Administration of cyclophosphamide, which accelerates diabetes in NOD mice, resulted in 43% of RIPbeta2m/NODbeta2mnull mice becoming diabetic compared with 75% of wild-type mice and 0% of NODbeta2mnull mice. Acceleration of diabetes by cyclophosphamide was prevented by anti-CD8 mAb treatment. FACS analysis of peripheral blood and lymphoid organs from transgene-bearing animals did not show an increase in the number of CD8+ T cells compared with that in NODbeta2mnull mice. In summary, beta-cell expression of beta2m in NODbeta2mnull mice resulted in a return of insulitis, but not spontaneous diabetes. These studies demonstrate that beta2m and cell surface MHC class I expression on beta-cells are essential for the initiation of diabetes in the NOD mouse and further confirm that efficient progression to diabetes requires both CD4+ and CD8+ T cells.

Animals↗
PubMed1995

Experiencing a shame response as a precursor to violence.

The shame response is a primitive physiological response to a rejection of oneself by another. The discomfort of this response may vary from intense physical pain to one that is barely noticeable, if at all. When this pain is sufficient, it causes anger that may be directed outward against another or inward against oneself. The intensity of the shame response, hence the intensity of the pain and anger, is related to the significance of the other, the significance of witnesses to the rejection, one's vulnerability, whether or not the rejection is of oneself or an aspect of oneself, and if the rejection comes as a surprise. When most intense (i.e., most painful), the shame response may include a tightness of the throat, nausea, stomach pain, and a sense that the contents of one's chest and abdomen are collapsing, exploding, or imploding. In reviewing what preceded an act of violence, it is necessary to determine whether the assailant had experienced a shame response and how intense it was. Understanding that a shame response can lead to anger and violence allows for the prevention of violence. This requires that individuals do not experience rejections that are so painful as to lead to violence.

Adult↗
PubMed1993

Heterodimerization of the Drosophila ecdysone receptor with retinoid X receptor and ultraspiracle.

Ecdysone in Drosophila has been a paradigm for steroid hormones since its ability to induce gene activity directly was demonstrated by its effects on moulting and polytene chromosome puffing. The ecdysone receptor (EcR) was recently confirmed as a member of the nuclear receptor superfamily by cloning and characterization in a Drosophila cell line. Here we show that EcR needs to heterodimerize with either the retinoid X receptor (RXR) or its Drosophila homologue, ultraspiracle (USP), for DNA binding and transactivation. These results place the ecdysone receptor in the heterodimerizing class of the nuclear receptor superfamily and demonstrate that the role of RXR/USP as a central and promiscuous partner in mediating the activity of these receptors is highly conserved. Whereas EcR-USP DNA-binding activity is unaffected by hormone, EcR-RXR DNA-binding activity is stimulated by either ecdysteroid or 9-cis-retinoic acid, demonstrating that hormone can play a role in heterodimer stabilization.

Animals↗
PubMed1991

The diagnosis of Duchenne and Becker muscular dystrophies: two years' experience in a comprehensive carrier screening and prenatal diagnostic laboratory.

This article describes the diagnostic algorithm being used for the management of the 148 families affected by Duchenne or Becker muscular dystrophy who are known to the Molecular Neurogenetics Laboratory in the Department of Neuropathology, Royal Perth Hospital. In 60 families from whom DNA has been obtained, 41 mutations (39 deletions and two duplications) of the Duchenne muscular dystrophy gene (DMD) have been identified by means of complementary DNA (cDNA) probes. DNA-based screening has clarified the carrier status of 45 at-risk women, and 13 pregnancies have been monitored. In addition, cDNA screening of all relevant patients with autosomal recessive muscular dystrophy, spinal muscular atrophy or limb-girdle muscular dystrophy facilitated the correct diagnosis of Becker muscular dystrophy in three patients.

Algorithms↗
PubMed1991

Risk factor changes after cessation of intervention in the Multiple Risk Factor Intervention Trial. The MRFIT Research Group.

BACKGROUND: The degree of maintenance of risk factor changes observed in post-trial follow-up of participants in the Multiple Risk Factor Intervention Trial (MRFIT) provides insights into long-term determinants of preventive behaviors, as well as post-trial mortality differentials. METHODS: Nine hundred eighty-nine former MRFIT participants at four clinical centers were invited to be reexamined 2-3.5 years after the end of this 6- to 8-year trial. Seventy percent came to the clinics for measurements of systolic and diastolic blood pressure, plasma lipids and lipoproteins, serum thiocyanate, and dietary food frequency. With the addition of phone interviews, 82% provided self-report data on cigarette smoking and use of antihypertensive drugs. RESULTS: Comparison of Special Intervention (SI) and Usual Care (UC) groups showed that differences persisted for total (6.5 mg/dl, P = 0.02) and low-density lipoprotein cholesterol (5.4 mg/dl, P = 0.04), and for diastolic blood pressure (1.6 mm Hg, P = 0.02). Differences were not significant for systolic blood pressure (1.8 mm Hg, P = 0.12), cigarette smoking, and serum thiocyanate. The plasma cholesterol difference represented 70% of the value at the end of intervention, and food frequency profiles were consistent with maintenance of an SI - UC difference. Loss of the SI - UC difference in smoking was attributable to both UC cessation and SI recidivism. The blood pressure difference was not attributable to different proportions of SI and UC men on medications, but possibly to differences in diuretic doses, use of multiple drugs, and adherence. There was a persisting contrast in use of specific diuretics, with more frequent use of chlorthalidone and less of hydrochlorothiazide in the SI group. CONCLUSION: These findings suggest that cholesterol-lowering dietary changes are self-sustaining, while smoking cessation is less so, and may require continued intervention. Finally, the SI - UC differences in use of specific diuretics remains one hypothesis for explaining a portion of post-trial mortality trends.

Adult↗