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Biomedical subjects

A M Lew

Publications and source records attributed to A M Lew.

At least 37 records · Page 2Linked to original sources
PubMed2000

Transgenic anti-CD4 monoclonal antibody secretion by mouse segmental pancreas allografts promotes long term survival.

To compare the effectiveness of transgenic and systemic monoclonal antibody therapy for pancreas transplantation, vascularised segmental pancreas allografts from wild-type or transgenic pancreatic tissue that secreted monoclonal anti-CD4 were placed in CBA recipients in which diabetes had been induced chemically by streptozotocin (STZ, non-autoimmune diabetes). In untreated CBA recipients, wild-type BALB/c or C57BL/6 bml pancreas transplants were rejected in a mean survival time (MST) of 27 and 30 days, respectively. BALB/c and C57BL/6 graft survival improved when recipients were given a short course of T cell depleting monoclonal anti-CD4 antibody, (GK 1.5, 2 mg total on days -1, 0, 1, 2 with grafting on day 0) with MST +/- S.D. of 71 +/- 29 and 44 +/- 36 days, respectively. Thus, transient depletion of CD4 was effective in delaying pancreas allograft rejection in these strain combinations. The use of C57BL/6 bml mice transgenic for a rat anti-CD4 antibody (GK5 mice) as pancreas donors provided allografts that secreted sufficient anti-CD4 antibody to cause CD4 T cell depletion in the recipients (CD4 cells decreased from 30 to < 5% of small lymphocytes). This degree of depletion was not sustained and the CD4 recovery inversely correlated with graft survival. Mice with > 20% CD4 cells in the splenic lymphocyte population 4 weeks post-transplant rejected their grafts (3 of 10 mice). However, in 7 of 10 mice CD4 cells remained low (< 15%) and allografts survived for > 80 days. The GK5 allografts survived significantly longer than those from non-transgenic bml controls (MST 83 +/- 32 days, compared with 30 days, P < 0.0005). This survival time was similar to that of BALB/c allografts in CBA recipients treated with a high dose of anti-CD4 antibody. Thus, transgenic secretion of anti-CD4 antibody by the pancreas allograft was very effective in prolonging its survival.

Animals↗
PubMed2000

Delayed rejection of fetal pig pancreas in CD4 cell deficient mice was correlated with residual helper activity.

CD4 cells have been shown to play a dominant role in the rejection of xenografts. Depletion of murine CD4 cells by injecting anti-CD4 antibody prolongs the graft survival, but does not prevent its rejection. For a more stable phenotype, we used genetically modified mice. To test whether the delayed rejection is caused by incomplete depletion of CD4 cells, we evaluated the response to fetal pig pancreas (FPP) xenografts in three types of CD4 cell deficient mice. They are MHC class II deficient mice (MHC II(o/o), CD4 deficient mice (CD4(o/o)) and a novel type of CD4 cell deficient mice (designated GK). GK mice were rendered permanently and completely CD4 deficient by transgenic expression of anti-CD4 antibody, whereas both MHC II(o/o) and CD4(o/o) mice have a residual helper cell population. FPP grafts in wild type mice were rejected within a week, whereas FPP grafts survived up to 4 weeks in MHC II(o/o) and CD4(o/o) mice. Survival of grafts in GK mice was even longer (8 weeks). Differences in histology were also noted. Rejecting grafts in MHC II(o/o) and wild-type mice were infiltrated with both eosinophils and mononuclear cells, whereas the infiltrates in CD4(o/o) and GK mice were exclusively mononuclear cells. Immunohistochemistry showed that they were primarily CD8 cells. The immune response to FPP was clearly different in the three types of CD4 cell deficient mice. Splenocytes of MHC II(o/o) 3 weeks post-transplant with FPP produced substantial amounts of IFN-gamma and IL-5, whereas splenocytes of CD4(o/o) mice produced low levels of IFN-gamma but no detectable IL-5. At similar times, these cytokines were not detected in GK mice. Furthermore, CD4(o/o) mice were capable of mounting helper dependent, although reduced, IgG responses to FPP antigens, while GK mice were not. The above results indicate that residual helper activity in some types of CD4 cell deficient mice could still contribute to xenograft rejection. Caution needs to be exercised where such mice are used as models of CD4 cell deficiency. Also, because there is eventual rejection of xenograft FPP in GK mice which lack detectable helper activity, we argue that these mice are a better model to investigate the involvement of CD4-independent rejection mechanisms.

Animals↗
PubMed2000

Predominant transgene expression in exocrine pancreas directed by the CMV promoter.

The enhancer/promoter of the human cytomegalovirus gene encoding the major immediate-early protein (CMVp) is reputed to be one of the strongest and most promiscuous regulatory elements for directing transcription of heterologous genes in vitro. However, transgene expression under the promoter in adult transgenic mice is often more restricted. We selected a CMVp segment from position -350 to +59 to control expression of transgenes for two secretory fusion proteins. Expression was analyzed by immunohistology staining and quantified by Northern blot, Western blot, and ELISA of secretions from explanted tissues. In all six lines of transgenic mice, the highest expression of transgenes at the mRNA and protein level was observed in the exocrine tissue of the pancreas, although the levels of expression varied among the lines. The results indicate not only that CMVp is not a universal promoter in vivo but indeed that it can be relatively specific for the exocrine pancreas, where expression of the gene it controlled was consistently very high.

Animals↗
PubMed2000

Transgenic overexpression of human Bcl-2 in islet beta cells inhibits apoptosis but does not prevent autoimmune destruction.

Insulin-dependent diabetes mellitus results when > 90% of the insulin-producing beta cells in the pancreatic islets are killed as a result of autoimmune attack by T cells. During the progression to diabetes, islet beta cells die as a result of different insults from the immune system. Agents such as perforin and granzymes, CD95 ligand and tumor necrosis factor-alpha, or cytokines and free-radicals have all been shown to cause beta cell apoptosis. The anti-apoptotic protein, Bcl-2, might protect against some of these stimuli. We have therefore generated transgenic mice expressing human Bcl-2 in their islet beta cells. Although Bcl-2 was able to prevent apoptosis induced by cytotoxic agents against beta cells in vitro, Bcl-2 alone could not prevent or ameliorate cytotoxic or autoimmune beta cell damage in vivo.

Age of Onset↗
PubMed1999

BAC-VAC, a novel generation of (DNA) vaccines: A bacterial artificial chromosome (BAC) containing a replication-competent, packaging-defective virus genome induces protective immunity against herpes simplex virus 1.

This study aimed to exploit bacterial artificial chromosomes (BAC) as large antigen-capacity DNA vaccines (BAC-VAC) against complex pathogens, such as herpes simplex virus 1 (HSV-1). The 152-kbp HSV-1 genome recently has been cloned as an F-plasmid-based BAC in Escherichia coli (fHSV), which can efficiently produce infectious virus progeny upon transfection into mammalian cells. A safe modification of fHSV, fHSVDeltapac, does not give rise to progeny virus because the signals necessary to package DNA into virions have been excluded. However, in mammalian cells fHSVDeltapac DNA can still replicate, express the HSV-1 genes, cause cytotoxic effects, and produce virus-like particles. Because these functions mimic the lytic cycle of the HSV-1 infection, fHSVDeltapac was expected to stimulate the immune system as efficiently as a modified live virus vaccine. To test this hypothesis, mice were immunized with fHSVDeltapac DNA applied intradermally by gold-particle bombardment, and the immune responses were compared with those induced by infection with disabled infectious single cycle HSV-1. Immunization with either fHSVDeltapac or disabled infectious single cycle HSV-1 induced the priming of HSV-1-specific cytotoxic T cells and the production of virus-specific antibodies and conferred protection against intracerebral injection of wild-type HSV-1 at a dose of 200 LD(50). Protection probably was cell-mediated, as transfer of serum from immunized mice did not protect naive animals. We conclude that BAC-VACs per se, or in combination with genetic elements that support replicative amplification of the DNA in the cell nucleus, represent a useful new generation of DNA-based vaccination strategies for many viral and nonviral antigens.

Animals↗
PubMed1999

CD8 T cell ignorance or tolerance to islet antigens depends on antigen dose.

There are two major mechanisms reported to prevent the autoreactivity of islet-specific CD8(+) T cells: ignorance and tolerance. When ignorance is operative, naïve autoreactive CD8(+) T cells ignore islet antigens and recirculate without causing damage, unless activated by an external stimulus. In the case of tolerance, CD8(+) T cells are deleted. Which factor(s) contributes to each particular outcome was previously unknown. Here, we demonstrate that the concentration of self antigen determines which mechanism operates. When ovalbumin (OVA) was expressed at a relatively low concentration in the pancreatic islets of transgenic mice, there was no detectable cross-presentation, and the CD8(+) T cell compartment remained ignorant of OVA. In mice expressing higher doses of OVA, cross-presentation was detectable and led to peripheral deletion of OVA-specific CD8(+) T cells. When cross-presentation was prevented by reconstituting the bone marrow compartment with cells incapable of presenting OVA, deletional tolerance was converted to ignorance. Thus, the immune system uses two strategies to avoid CD8(+) T cell-mediated autoimmunity: for high dose antigens, it deletes autoreactive T cells, whereas for lower dose antigens, it relies on ignorance.

Adoptive Transfer↗
PubMed1999

Specific inhibition of skeletal alpha-actin gene transcription by applied mechanical forces through integrins and actin.

Skeletal alpha-actin (skA), a prominent fetal actin isoform that is re-expressed by adult cardiac myocytes after chronic overload in vivo, provides a model for studying cytoskeletal gene regulation by mechanical forces in vitro. We have determined the mechanisms by which perpendicular applied forces acting through integrins and the actin cytoskeleton regulate the expression of skA. Rat-2 fibroblasts were transiently transfected with plasmids containing 5'-regulatory regions of the skA gene fused to luciferase coding sequences. A constant, perpendicular force (0.2 pN/micrometer(2)) was applied by using a collagen-magnetic bead model; a 25% deformation was obtained on the dorsal cell surface. In this system, force is applied through focal adhesion integrins and strongly induces actin assembly [Glogauer, Arora, Yao, Sokholov, Ferrier and McCulloch (1997) J. Cell Sci. 110, 11-21]. skA promoter activity was inhibited by 68% in cells subjected to 4 h of applied force, whereas Rous sarcoma virus promoter activity was unaffected. In cells transiently transfected with a skA expression vector there was also a parallel 40% decrease in skA protein levels by force, as shown by Western blotting. In L8 cells, constitutive skA expression was decreased by more than 50%. Analyses of specific motifs in the skA promoter revealed that transcriptional enhancer factor 1 and Yin and Yang 1 sites, but not serum response factor and Sp1 sites, mediated inhibitory responses to force. In cells treated with cycloheximide the force-induced inhibition was abrogated, indicating a dependence on new protein synthesis. Inhibition of actin filament assembly with either cytochalasin D or Ca(2+)-depleted medium blocked the inhibitory effect induced by the applied force, suggesting that actin filaments are required for the regulation of skA promoter activity. Western blot analysis showed that p38 kinase, but not Jun N-terminal kinase or extracellular signal-regulated protein kinase 1/2, was activated by force; indeed, the p38 kinase inhibitor SB203580 relieved the force-induced inhibition of skA. We conclude that the force-induced inhibition of skA promoter activity requires an intact actin cytoskeleton and can be mapped to two different response elements. This inhibition might be mediated through the p38 kinase.

Actins↗
PubMed1999

Inhibition of naïve class I-restricted T cells by altered peptide ligands.

Amino acid variants of an antigenic peptide or altered peptide ligands have previously been investigated with CD4+ and CD8+ T cells. However, for CD8+ T cells, only clones (which are continually restimulated in vitro) have been assessed. Using TCR transgenic mice specific for a class I Kb-restricted OVA peptide (OVAp; OT-I mice) as a source of naïve CD8+ T cells, single amino acid variants of the OVAp were analysed in vitro for their ability to antagonize the proliferative and cytotoxic function of naïve OT-I cells. Peptides with substitutions at TCR contact residues were found to be the most potent antagonists of OT-I cell function. Those peptides that inhibited activation of cells to proliferate also inhibited activation of cells to become killers. Inhibition was inversely correlated with interferon (IFN)-gamma production. It was found that levels of antagonist peptide required for inhibition were higher than that described for T cell clones, presumably due to affinity differences.

Amino Acid Sequence↗
PubMed1999

Targeting improves the efficacy of a DNA vaccine against Corynebacterium pseudotuberculosis in sheep.

A large-scale DNA vaccination trial was performed with sheep to investigate whether an antigen targeted by CTLA-4 enhanced and accelerated the humoral immune response. Vaccination with genetically detoxified phospholipase D (DeltaPLD) has been shown to be effective, at least partially, against Corynebacterium pseudotuberculosis, the causal agent of caseous lymphadenitis in sheep. CTLA-4 binds to B7 on antigen-presenting cells and thus was used to direct the fusion antigens to sites of immune induction. Here we demonstrated that targeting DeltaPLD as a CTLA-4 fusion protein significantly enhanced the speed, magnitude, and longevity of the antibody response compared to that obtained with DNA encoding DeltaPLD. While all groups of sheep vaccinated with DNA encoding DeltaPLD were afforded better protection against an experimental challenge with C. pseudotuberculosis than those immunized with an irrelevant plasmid or those left unimmunized, the best protection was provided by the targeted DNA vaccine. We propose that targeting antigens to antigen-presenting cells offers a generic strategy for enhancing the efficacy of DNA vaccines.

Abatacept↗
PubMed1998

Enhanced responses to a DNA vaccine encoding a fusion antigen that is directed to sites of immune induction.

Viral infection and vaccination with DNA both induce similar immune responses to encoded antigens that are produced by the host. The availability of antigens in lymphoid organs is important in generating an immune response to viral challenge. Antigen availability may also be important in the response to DNA vaccines, because immune responses are stronger when antigen is secreted from DNA-transfected cells. We directed antigen to lymphoid organs by vaccination with DNA encoding antigen-ligand fusion proteins. The two ligands examined bind to receptors that are present on high endothelial venule cells of lymph nodes or on antigen-presenting cells. Here we show that both the humoral and the cellular immune responses to a model DNA vaccine were enhanced using either antigen-targeting strategy. Moreover, directing antigen to antigen-presenting cells speeded up, and altered the form of, the immune response. Directing antigen to sites of immune-response induction may represent a generic means of tailoring a potent and effective immune response to a DNA vaccine.

Abatacept↗
PubMed1998

The need for IgG2c specific antiserum when isotyping antibodies from C57BL/6 and NOD mice.

Isotyping and quantitation of murine IgG2a antibodies are widely performed with commercial monoclonal and polyclonal antisera raised against BALB/c IgG2a myeloma proteins. Recently it became evident that inbred mouse strains with the Igh1-b allele do not have the gene for IgG2a and instead express the IgG2c isotype. We show that commercial anti-IgG2a sera cross-react inadequately against IgG2c in immunoblot and ELISA and hence, are not suitable to detect and measure this subclass in mouse strains such as C57BL/6, C57BL/10 and NOD. We have used DNA immunization to generate polyclonal anti-IgG2c serum and demonstrated that it is essential to use IgG2c-specific antiserum to quantify accurately isotypic responses in mouse strains with the Igh1-b allele.

Alleles↗
PubMed1998

Inhibitory effect of lipopolysaccharide on immune response after DNA immunization is route dependent.

The DNA prepared from E. coli contained high levels of lipopolysaccharide (LPS). When antigen-encoding DNA was injected into mice, toxicity and increased IgM responses were observed. A method for purifying high yields of DNA (up to 12 mg/L of broth culture) with very low levels of LPS (0.05 ng/mg) was developed. When this purified DNA was used for immunization studies, the toxicity and increased IgM responses were abrogated. Thus, LPS was added to DNA in order to examine its influence on the IgG and cytotoxic T lymphocyte (CTL) response after intramuscular (i.m.) or intradermal (i.d.) DNA immunization. The IgG response to DNA-encoded antigen was inhibited in a dose-dependent manner by the i.d., but not the i.m., route of immunization. Surprisingly, no effect on the CTL response was observed. Therefore, the ability to produce high yields of plasmid DNA with very low levels of endotoxin contamination is advantageous for DNA immunization studies, not only for toxicologic but also for immunologic considerations. Furthermore, these results provide further evidence that immune induction occurs via different mechanisms after i.m. and i.d. DNA immunization.

Animals↗
PubMed1998

T cell unresponsiveness in vitro can be due to activation in vivo.

During investigations into the behaviour and fate of ovalbumin (OVA)-specific CD8+ T cells in a TCR transgenic system, cytotoxic T lymphocyte (CTL) activity (as measured by classical in vitro stimulation followed by 51Cr-release assay) was found to be reduced after OVA peptide administration. This paradoxically occurred even during the peak of activation and expansion of these T cells. The reduced responsiveness occurred for both classical CTL assays and in vitro proliferation assays, and would apparently be consistent with induction of anergic or suppressor T cells. Instead, we provide evidence that in vivo peptide treatment generated activated killers which consequently killed the stimulator cells during in vitro culture, thus resulting in the unresponsive phenotype. In co-culture experiments, the proliferation and classical CTL activity were severely reduced when naive OVA-specific CD8+ T cells (OT-I) cells were co-cultured with cells from OVA peptide-treated mice. Moreover, cells obtained after peptide injection did not require in vitro stimulation to be able to kill target cells. Therefore, activation of killers in vivo should be considered as one pathway whereby unresponsiveness is found in assays requiring in vitro stimulation.

Animals↗
PubMed1997

DNA immunization: induction of higher avidity antibody and effect of route on T cell cytotoxicity.

Immunizations of mice with plasmid DNAs encoding ovalbumin (OVA), human Ig, and hen egg lysozyme were compared with doses of soluble protein (without adjuvant) that induced similar IgG responses. The route of immunization influenced the magnitude of the antibody (Ab) response in that intradermal (i.d.) injection elicited higher IgG Ab levels than i.m. injection in both DNA- and protein-immunized mice. Although total IgG levels were similar to soluble protein controls, the avidity of the anti-OVA Abs generated by DNA immunization were 100- and 1,000-fold higher via the i.m. or i.d. route, respectively. However, despite the generation of high-avidity Ab in DNA-immunized mice, germinal centers could not be detected in either DNA- or protein-immunized mice. Examination of the IgG subclass response showed that IgG2a was induced by i.m. DNA immunization, coinciding with elevated interferon gamma production, whereas a dominant and elevated IgG1 response, coinciding with detectable interleukin 4 production, was generated after i.d. immunization with DNA or soluble OVA and hen egg lysozyme but not human Ig protein. As expected, cytotoxic T cell (CTL) responses could be detected only after DNA immunization. I.d. immunization produced the strongest CTL responses early (2 weeks) but was similar to i.m. later. Therefore, DNA immunization can differ from protein immunization by its ability to induce rapid CTL responses and higher avidity Ab, both of which are advantageous for vaccination.

Animals↗
PubMed1997

The nonpolymorphic MHC class II isotype, HLA-DQA2, is expressed on the surface of B lymphoblastoid cells.

The more centromeric of the two human MHC class II DQA genes, DQA2, has not previously been shown to express a protein product. However, its high degree of sequence similarity to DQA1, the fact that it is highly evolutionarily conserved, and the recent demonstration of its transcription in some cell lines suggest that a DQA2 alpha-chain may be expressed. Polyclonal anti-peptide antisera were generated and shown to be specific for DQA2 in Western blotting of Triton X-114 preparations of B lymphoblastoid cell lines. DQA2 expression is variable, although always at least threefold less than that of DQA1, and is found in all haplotypes examined. Immunoprecipitations of biotin-labeled cell surface proteins reveal that DQA2 appears at the cell surface. In keeping with others' findings, no evidence for an expressed DQB2 beta-chain was found, prompting investigation of the means by which DQA2 gains access to the cell surface. Antisera specific for allotypic and isotypic MHC class II beta-chains were used in sequential immunoprecipitation experiments to search for a beta-chain partner for DQA2, and a transfectant system was established in an attempt to promote pairing of DQA2 with a selected DQB1 (*0501) chain. In neither case was DQA2 found to dimerize with an MHC class II beta-chain. Despite this, DQA2 is capable of forming a complex with invariant chain.

Alleles↗
PubMed1997

Androgen-dependent cell cycle arrest and apoptotic death in PC-3 prostatic cell cultures expressing a full-length human androgen receptor.

To assess the function of androgen receptor in androgen-independent prostate cancer cells, human PC-3 prostate carcinoma cells, which lack androgen receptor (AR) expression, were transfected with a full length human AR cDNA sequence inserted into an episomal expression vector system. Several clonal lines of transfected cells expressing varying levels of a 110 kDa AR, as determined by immunoblotting and ligand binding assay, were isolated. The expressed ectopic receptors displayed nuclear binding following androgen treatment and mediated androgen inducibility of a mouse mammary tumor virus (MMTV)-luciferase reporter gene construct in a dose-dependent manner. 5 alpha-dihydrotestosterone (DHT) activation of luciferase activity was blocked by the AR antagonist hydroxyflutamide, and was promoter-specific based on the inability of the hormone-insensitive RSV promoter to respond to DHT. Treatment of AR-expressing PC-3 cells with physiological levels of DHT for 3 days resulted in paradoxical inhibition of cell growth. The growth-inhibitory effect was observed in clonal lines expressing low, moderate and high levels of AR, indicating that it was not the result of AR overexpression. To determine whether AR-expressing PC-3 cells had become androgen dependent, albeit with slowed growth, the effect of 1.0 nM DHT on the growth of two clonal lines expressing low and moderate receptor levels (PC-3(AR)13 and PC-3(AR)2, respectively) was examined on over an 18 day period. DHT removed after 3, 6, or 9 days and replaced with steroid-free medium. Surprisingly, after 6 days of DHT treatment, the number of PC-3(AR)2 cells began to decrease such that all cells were dead by 15 days after initiation of DHT treatment. A similar effect was observed in PC-3(AR)13 cells, but required a longer initial period of DHT exposure. PC-3(AR)2 cells were rescued from cell death if DHT was withdrawn 3 days but not 6 or 9 days after initiation of DHT treatment. As determined by DNA cell cycle analysis, the proportion of cells in the G1 phase was enhanced by DHT treatment, accompanied by a decrease in cells in the S and G2M phase of the cell cycle. After 6 days of DHT treatment, the proportion of cells in G1 decreased which was accompanied by an increase in cells in a subG1 population consistent with apoptosis. DNA fragmentation in PC-3(AR)2 cells after 3 or 6 days of DHT treatment was demonstrated by agarose gel electrophoresis, further indicating the cell death was apoptotic. Removal of DHT from PC-3(AR)2 cultures after 3 days, but not after 6 or 9 days, was followed by a large shift in cells from G1 to S and G2M. These data suggest that DHT blocks the progression of AR transfected PC-3 cells through the cell cycle, resulting in growth inhibition and apoptosis.

Androgens↗