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Biomedical subjects

N A Williams

Publications and source records attributed to N A Williams.

At least 19 recordsLinked to original sources
PubMed2003

Increased NF-kappaB DNA binding but not transcriptional activity during apoptosis induced by the COX-2-selective inhibitor NS-398 in colorectal carcinoma cells.

Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit colorectal neoplasia, an effect that is associated with their ability to induce apoptosis. Although NSAIDs have been reported to inhibit NF-kappaB, more recent studies show activation of NF-kappaB by NSAIDs. NF-kappaB commonly shows antiapoptotic activity and is implicated in the therapeutic resistance of cancer cells. The effects of highly COX-2-selective NSAIDs such as NS-398 on NF-kappaB in colorectal tumour cells have not been reported. Therefore, we addressed whether NF-kappaB has a role in NS-398-induced apoptosis of colorectal cancer cells. Treatment of HT-29 colorectal carcinoma cells with doses of NS-398 (50-75 microM) known to induce apoptosis had no effect on NF-kappaB for up to 48 h. However after 72 and 96 h NF-kappaB DNA-binding activity was increased by NS-398, in parallel with apoptosis induction. NS-398-treated HT-29 cells showed increased p50 homodimer binding and an induction of p50/p65 heterodimers, as demonstrated by supershift assay. However, although NS-398 increased NF-kappaB DNA binding it did not increase NF-kappaB-dependent reporter activity and inhibition of NF-kappaB DNA binding did not enhance NS-398-induced apoptosis. This indicates that NF-kappaB activated by NS-398 is transcriptionally inactive and is an encouraging result for the use of COX-2-selective NSAIDs not only in chemoprevention but also as novel therapies for colon cancer.

Apoptosis↗
PubMed2003

Protection against recurrent ocular herpes simplex virus type 1 disease after therapeutic vaccination of latently infected mice.

The potential of therapeutic vaccination of animals latently infected with herpes simplex virus type 1 (HSV-1) to enhance protective immunity to the virus and thereby reduce the incidence and severity of recurrent ocular disease was assessed in a mouse model. Mice latently infected with HSV-1 were vaccinated intranasally with a mixture of HSV-1 glycoproteins and recombinant Escherichia coli heat-labile enterotoxin B subunit (rEtxB) as an adjuvant. The systemic immune response induced was characterized by high levels of virus-specific immunoglobulin G1 (IgG1) in serum and very low levels of IgG2a. Mucosal immunity was demonstrated by high levels of IgA in eye and vaginal secretions. Proliferating T cells from lymph nodes of vaccinated animals produced higher levels of interleukin-10 (IL-10) than were produced by such cells from mock-vaccinated animals. This profile suggests that vaccination of latently infected mice modulates the Th1-dominated proinflammatory response usually induced upon infection. After reactivation of latent virus by UV irradiation, vaccinated mice showed reduced viral shedding in tears as well as a reduction in the incidence of recurrent herpetic corneal epithelial disease and stromal disease compared with mock-vaccinated mice. Moreover, vaccinated mice developing recurrent ocular disease showed less severe signs and a quicker recovery rate. Spread of virus to other areas close to the eye, such as the eyelid, was also significantly reduced. Encephalitis occurred in a small percentage (11%) of mock-vaccinated mice, but vaccinated animals were completely protected from such disease. The possible immune mechanisms involved in protection against recurrent ocular herpetic disease in therapeutically vaccinated animals are discussed.

Animals↗
PubMed2001

MHC matching and mechanisms of alloactivation in corneal transplantation.

BACKGROUND: In human corneal transplantation the value of matching, particularly for MHC class II, is unclear and controversial. The contribution of the direct pathway to T cell activation is also uncertain. We have determined the relative contribution of class I, II and non-MHC antigens to graft rejection and of the direct and indirect pathways to T cell activation in a rat model mimicking human incompatibilities. METHODS: DA (RT1a) strain recipients received fully mismatched PVG (RT1c) strain grafts or grafts from one of three recombinant strains bearing DA MHC genes on a PVG background. Graft survival was assessed and the specificity of T cells generated in the draining lymph nodes was determined in mixed lymphocyte (MLR) proliferation assays. To assess the contribution of the direct pathway, fully mismatched graft were performed and allospecific proliferation was measured after depletion of recipient APC from the MLR reaction. RESULTS: There was no significant difference in survival of grafts between the four grades of mismatch, which ranged from a full mismatch to non-MHC mismatches alone (median survival 12.5, 11, 13 and 12.5 days respectively). In conformity with clinical results, strong secondary responses were generated against targets matched for MHC with the recipient. Depletion of recipient APC from a fully allogeneic secondary MLR did not fully abrogate donor-specific proliferation. CONCLUSIONS: Class II matching is of no benefit in this model. Strong indirect responses to non-MHC mismatches are sufficient to induce the rapid rejection, but the small numbers of class II+ cells in the donor appear sufficient to generate a direct response.

Animals↗
PubMed2001

A mutant cholera toxin B subunit that binds GM1- ganglioside but lacks immunomodulatory or toxic activity.

GM1-ganglioside receptor binding by the B subunit of cholera toxin (CtxB) is widely accepted to initiate toxin action by triggering uptake and delivery of the toxin A subunit into cells. More recently, GM1 binding by isolated CtxB, or the related B subunit of Escherichia coli heat-labile enterotoxin (EtxB), has been found to modulate leukocyte function, resulting in the down-regulation of proinflammatory immune responses that cause autoimmune disorders such as rheumatoid arthritis and diabetes. Here, we demonstrate that GM1 binding, contrary to expectation, is not sufficient to initiate toxin action. We report the engineering and crystallographic structure of a mutant cholera toxin, with a His to Ala substitution in the B subunit at position 57. Whereas the mutant retained pentameric stability and high affinity binding to GM1-ganglioside, it had lost its immunomodulatory activity and, when part of the holotoxin complex, exhibited ablated toxicity. The implications of these findings on the mode of action of cholera toxin are discussed.

Adjuvants, Immunologic↗
PubMed2001

The genetic and immunopathological processes underlying collagen-induced arthritis.

Animal models of rheumatoid arthritis (RA) have provided substantial insights into basic pathogenic mechanisms of chronic inflammatory arthritis and autoimmune disease in general. Of the variety of models reported, collagen-induced arthritis (CIA) has been the most characterized in terms of both its pathogenesis and its underlying immunological basis. Collagen-induced arthritis has also been the model of choice in terms of testing potential new therapeutic agents for the treatment of human RA. Nevertheless, the complex nature of the balance between T-cell cytokines and the chronic inflammatory processes is only recently becoming clear. This review focuses on these developments, highlighting their implications for our understanding of RA and for the use of CIA as a suitable animal model.

Animals↗
PubMed2001

Immunomodulation using bacterial enterotoxins.

Immunologic unresponsiveness (tolerance) is a key feature of the mucosal immune system, and deliberate vaccination by a mucosal route can effectively induce immune suppression. However, some bacterial-derived proteins, e.g. cholera toxin and the heat labile toxin of Escherichia coli, are immunogenic and immunomodulatory at mucosal surfaces and can effectively adjuvant immune responses to codelivered bystander antigens. This review summarizes some of the structural and biological characteristics of these toxins and provides examples of how these properties have been exploited for tolerance induction and mucosal vaccine development.

Adjuvants, Immunologic↗
PubMed2001

Antigen-receptor cross-linking and lipopolysaccharide trigger distinct phosphoinositide 3-kinase-dependent pathways to NF-kappa B activation in primary B cells.

The NF-kappaB/Rel transcription factors play an important role in the expression of genes involved in B cell development, differentiation and function. Nuclear NF-kappaB is induced in B cells by engagement of either the BCR or CD40 or by stimulation with lipopolysaccharide (LPS). Despite the importance of NF-kappaB to B cell function, little is known about the signaling pathways leading to NF-kappaB activation. In this report we address the role of phosphoinositide 3'-kinase (PI 3-kinase) in BCR- and LPS-induced NF-kappaB activation using populations of primary murine resting B cells. Using the specific pharmacological inhibitors of PI 3-kinase, Wortmannin and LY294002, we demonstrate that PI 3-kinase activity is vital for BCR-induced NF-kappaB DNA-binding activity. Furthermore, we show that this is achieved via protein kinase C-dependent degradation of IkappaBalpha. Similar analyses reveal that PI 3-kinase is also critical in triggering NF-kappaB DNA-binding activity and IkappaBalpha degradation following LPS stimulation. Interestingly, a PKC inhibitor which blocked the BCR-induced IkappaBalpha degradation had no effect on the degradation of IkappaBalpha after LPS stimulation. Taken together, our results indicate the involvement of PI 3-kinase in at least two distinct signaling pathways leading to activation of NF-kappaB in B cells.

Androstadienes↗
PubMed2001

Escherichia coli enterotoxin B subunit triggers apoptosis of CD8(+) T cells by activating transcription factor c-myc.

Heat-labile enterotoxin from enterotoxinogenic Escherichia coli is not only an important cause of diarrhea in humans and domestic animals but also possesses potent immunomodulatory properties. Recently, the nontoxic, receptor-binding B subunit of heat-labile enterotoxin (EtxB) was found to induce the selective death of CD8(+) T cells, suggesting that EtxB may trigger activation of proapoptotic signaling pathways. Here we show that EtxB treatment of CD8(+) T cells but not of CD4(+) T cells triggers the specific up-regulation of the transcription factor c-myc, implicated in the control of cell proliferation, differentiation, and death. A concomitant elevation in Myc protein levels was also evident, with peak expression occurring 4 h posttreatment. Preincubation with c-myc antisense oligodeoxynucleotides demonstrated that Myc expression was necessary for EtxB-mediated apoptosis. Myc activation was also associated with an increase of IkappaBalpha turnover, suggesting that elevated Myc expression may be dependent on NF-kappaB. When CD8(+) T cells were pretreated with inhibitors of IkappaBalpha turnover and NF-kappaB translocation, this resulted in a marked reduction in both EtxB-induced apoptosis and Myc expression. Further, a non-receptor-binding mutant of EtxB, EtxB(G33D), was shown to lack the capacity to activate Myc transcription. These findings provide further evidence that EtxB is a signaling molecule that triggers activation of transcription factors involved in cell survival.

Animals↗
PubMed2001

Protective mucosal immunity to ocular herpes simplex virus type 1 infection in mice by using Escherichia coli heat-labile enterotoxin B subunit as an adjuvant.

The potential of nontoxic recombinant B subunits of cholera toxin (rCtxB) and its close relative Escherichia coli heat-labile enterotoxin (rEtxB) to act as mucosal adjuvants for intranasal immunization with herpes simplex virus type 1 (HSV-1) glycoproteins was assessed. Doses of 10 microg of rEtxB or above with 10 microg of HSV-1 glycoproteins elicited high serum and mucosal anti-HSV-1 titers comparable with that obtained using CtxB (10 microg) with a trace (0.5 microg) of whole toxin (Ctx-CtxB). By contrast, doses of rCtxB up to 100 microg elicited only meager anti-HSV-1 responses. As for Ctx-CtxB, rEtxB resulted in a Th2-biased immune response with high immunoglobulin G1 (IgG1)/IgG2a antibody ratios and production of interleukin 4 (IL-4) and IL-10 as well as gamma interferon by proliferating T cells. The protective efficacy of the immune response induced using rEtxB as an adjuvant was assessed following ocular challenge of immunized and mock-immunized mice. Epithelial disease was observed in both groups, but the immunized mice recovered by day 6 whereas mock-immunized mice developed more severe corneal disease leading to stromal keratitis. In addition, a significant reduction in the incidence of lid disease and zosteriform spread was observed in immunized animals and there was no encephalitis compared with 95% encephalitis in mock-immunized mice. The potential of such mucosal adjuvants for use in human vaccines against pathogens such as HSV-1 is discussed.

Adjuvants, Immunologic↗
PubMed2000

Immune modulation by the cholera-like enterotoxin B-subunits: from adjuvant to immunotherapeutic.

Cholera toxin (Ctx) and its close relative, Escherichia coli heat-labile enterotoxin (Etx) have long been established as potent mucosal and systemic adjuvants. Problems arising from their inherent toxicity have, however, precluded human use. Here we describe findings which demonstrate that contrary to the established dogma the non-toxic B-subunit of Etx (EtxB) is a highly potent mucosal adjuvant capable of potentiating protective immunity to viral infection. The mechanisms which underlie this activity arise from an ability to trigger specific signaling processes in lymphocyte populations which modulate differentially their activation, differentiation and survival. The elucidation of these properties has led to the further use of EtxB as an agent capable of preventing the establishment of autoimmune diseases. The basis for these activities and their potential applicability to human therapies are discussed.

Adjuvants, Immunologic↗
PubMed2000

Effect of tibial bone resection on the development of fast- and slow-twitch skeletal muscles in foetal sheep.

To determine if longitudinal bone growth affects the differentiation of fast- and slow-twitch muscles, the tibial bone was sectioned at 90 days gestation in foetal sheep so that the lower leg was permanently without structural support. At 140 days (term is approximately 147 days) the contractile properties of whole muscles, activation profiles of single fibres and ultrastructure of fast- and slow-twitch muscles from the hindlimbs were studied. The contractile and activation profiles of the slow-twitch soleus muscles were significantly affected by tibial bone resection (TIBX). The soleus muscles from the TIBX hindlimbs showed: (1) a decrease in the time to peak of the twitch responses from 106.2 +/- 10.7 ms (control, n = 4) to 65.1 +/- 2.48 ms (TIBX, n = 5); (2) fatigue profiles more characteristic of those observed in the fast-twitch muscles: and (3) Ca2+ - and Sr2+ -activation profiles of skinned fibres similar to those from intact hindlimbs at earlier stages of gestation. In the FDL, TIBX did not significantly change whole muscle twitch contraction time, the fatigue profile or the Ca2+ - and Sr2+ -activation profiles of skinned fibres. Electron microscopy showed an increased deposition of glycogen in both soleus and FDL muscles. This study shows that the development of the slow-twitch phenotype is impeded in the absence of the physical support normally provided by the tibial bone. We suggest that longitudinal stretch is an important factor in allowing full expression of the slow-twitch phenotype.

Animals↗
PubMed2000

An amphioxus Emx homeobox gene reveals duplication during vertebrate evolution.

Members of the Emx homeobox gene class are expressed during embryogenesis in the brain and/or other head structures of phylogenetically diverse phyla. Here, we describe sequence, genomic structure, and molecular phylogenetic analysis of a cephalochordate (amphioxus) Emx class gene termed AmphiEmxA. The genomic structure of AmphiEmxA is very similar to that of vertebrate Emx genes, with two conserved intron sites. The Drosophila homolog empty spiracles (ems) has just one intron, which may be shared with chordates; the other has been secondarily lost in this Drosophila gene and in a cnidarian Emx-related gene. We identify a highly conserved peptide motif close to the amino terminus of Emx proteins, demonstrate its similarity to a sequence found in a variety of transcription factors, and argue that it arose through convergent evolution in homeobox and forkhead genes. Finally, our molecular phylogenetic analysis strongly supports the presence of a single Emx gene in the ancestor of chordates and gene duplication along the vertebrate lineage.

Amino Acid Motifs↗
PubMed1999

Immune modulation by the cholera-like enterotoxins: from adjuvant to therapeutic.

Cholera toxin and its close relative, Escherichia coli heat-labile enterotoxin, are potent immunogens and mucosal adjuvants. The recent findings that their B subunits can promote tolerance highlights the complexity of their interactions with the immune system. Here, Neil Williams and colleagues review the mechanisms by which these molecules modulate leukocyte populations and seek to explain the paradox.

Adjuvants, Immunologic↗
PubMed1999

Biofilm formation by Helicobacter pylori.

Helicobacter pylori NCTC 11637 produces a water-insoluble biofilm when grown under defined conditions with a high carbon:nitrogen ratio in continuous culture and in 10% strength Brucella broth supplemented with 3 g l-1 glucose. Biofilm accumulated at the air/liquid interface of the culture. Light microscopy of frozen sections of the biofilm material showed few bacterial cells in the mass of the biofilm. The material stained with periodic acid Schiff's reagent. Fucose, glucose, galactose, and glycero-manno-heptose, N-acetylglucosamine and N-acetylmuramic acid were identified in partially purified and in crude material, using gas chromatography and mass spectrometry. The sugar composition strongly indicates the presence of a polysaccharide as a component of the biofilm material. Antibodies (IgG) to partially purified material were found in both sero-positive and sero-negative individuals. Treatment of the biofilm material with periodic acid reduced or abolished immunoreactivity. Treatment with 5 mol l-1 urea at 100 degrees C and with phenol did not remove antigenic recognition by patient sera. The production of a water-insoluble biofilm by H. pylori may be important in enhancing resistance to host defence factors and antibiotics, and in microenvironmental pH homeostasis facilitating the growth and survival of H. pylori in vivo.

Amino Acids↗
PubMed1998

Effects of freeze-dry processing conditions on the crystallization of pentamidine isethionate.

The results of this study show that pentamidine isethionate (PI) can exist in at least four crystalline forms-three anhydrates designated as forms A, B, and C, and a trihydrate. Form C is the high-temperature modification, produced by heating forms A, B, and the trihydrate above 130 degrees C and cannot be produced under actual lyophilization conditions. The crystal forms of PI present after freeze-drying depend on the initial solution concentration and the thermal history of freezing. At low concentrations of PI (4% and less), form A is observed regardless of freezing method. At a higher concentration (10%), the crystal forms observed are a function of the freezing method. Three freezing methods were used to effect different cooling rates: (1) cooling on the shelf to 2 degrees C and holding for 3 h prior to decreasing the temperature to -45 degrees C, (2) directly cooling on the shelf from room temperature to -45 degrees C, and (3) dipping the vials in liquid nitrogen. The results show that form A, form B, or a mixture of both forms are present in the freeze-dried solid depending upon whether the trihydrate crystallizes during freezing or not. Since form B can only be produced by dehydration of the trihydrate at low temperature, the presence of this form in the freeze-dried powders depends on the nucleation and growth of the trihydrate during freezing. Photostability studies have demonstrated marked differences between freeze-dried solids frozen under different conditions. The results underscore the importance of recognizing that seemingly subtle differences in processing conditions can have a significant impact on critical quality attributes of freeze-dried products.

Antiprotozoal Agents↗