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Biomedical subjects

M C Thomas

Publications and source records attributed to M C Thomas.

At least 19 recordsLinked to original sources
PubMed1998

Single amino acid substitutions disrupt tetramer formation in the dihydroneopterin aldolase enzyme of Pneumocystis carinii.

In the opportunistic pathogen Pneumocystis carinii, dihydroneopterin aldolase function is expressed as the N-terminal portion of the multifunctional folic acid synthesis protein (Fas). This region encompasses two domains, FasA and FasB, which are 27% amino acid identical. FasA and FasB also share significant amino acid sequence similarity with bacterial dihydroneopterin aldolases. In the present study, this enzyme function has been overproduced as an independent monofunctional activity in Escherichia coli. Recombinant FasAB-Met23 (amino acids 23-290 of the predicted open reading frame) was purified and shown to contain dihydroneopterin aldolase activity. The native FasAB-Met23 is a tetramer of the 30-kDa subunit, demonstrating characteristics of an associating-dissociating equilibrium system in which only the multimeric form of the enzyme is active. Multiple sequence alignment of FasA and FasB with other dihydroneopterin aldolases highlights only three positions where the amino acid is invariable between all the predicted proteins. The role of these conserved amino acid residues in enzyme function was investigated using site-directed mutagenesis. Mutant FasAB-Met23 species were overproduced and purified to near homogeneity. Three FasA domain mutants and two FasB domain mutants had little or no detectable dihydroneopterin aldolase activity, implicating both FasA and FasB in the catalytic mechanism. We show that each mutant protein containing an inactivating amino acid substitution has lost its ability to form stable tetramers.

Aldehyde-Lyases↗
PubMed1998

The impact of reference pricing on clinical lipid control.

AIM: Reference pricing has recently been introduced into New Zealand in an attempt to curb rising pharmaceutical costs. Although budget savings may be significant, the resulting alteration of established drug prescriptions has the potential to cause harm. We undertook to assess the impact of these changes in patients switching from simvastatin to fluvastatin following the introduction of reference-based pricing in New Zealand. METHODS: The fasting lipid profiles of 262 patients in a defined geographic region were obtained after at least six weeks of fluvastatin therapy. These were compared to mean lipid levels obtained from laboratory databases for the patients while previously receiving simvastatin. RESULTS: There was a significant increase in total cholesterol, LDL cholesterol and triglyceride levels (p < 0.01). The elevation was less pronounced where higher incremental doses of fluvastatin were used, although still significant for LDL cholesterol and total cholesterol (p < 0.01). Those receiving maximal therapy with fluvastatin experienced similar elevations in lipid as did those on lower doses. CONCLUSION: The lipid elevations seen in this audit relate both to the lesser potency of fluvastatin and underdosing. In this high risk population, significant lipid elevations may conceivably produce an excess of vascular events. The responsibility to the taxpayer should be weighed carefully against the ethical responsibility to the individual patient and the potential to do harm. Subtherapeutic treatment may prove more costly than all the savings from reference pricing.

Drug Utilization↗
PubMed1998

Enhanced tumor protection by granulocyte-macrophage colony-stimulating factor expression at the site of an allogeneic vaccine.

Murine tumor models have demonstrated that whole tumor cell vaccines engineered to secrete certain cytokines in a paracrine fashion elicit systemic immune responses capable of eliminating small amounts of established tumor. In particular, autologous tumors that express the cytokine GM-CSF induce potent systemic immune responses against poorly immunogenic murine tumors. However, phase I clinical trials have demonstrated the technical difficulty of routinely expanding primary autologous human tumor cells to the numbers required for vaccination, making the generalization of autologous vaccines impractical. Dissection of the mechanism by which antitumor immunity is generated has demonstrated that GM-CSF recruits professional antigen-presenting cells that act as intermediates in presenting tumor antigen to and activating effector T cells. Furthermore, the identification of commonly recognized murine and human tumor antigens indicates that many are shared rather than unique. These findings would suggest that allogeneic as well as autologous tumor cells can be used as the vaccinating cells for activating antitumor immunity. A major concern in the application of allogeneic vaccines relates to the potential interference of allogeneic MHC expression at the vaccine site with priming of tumor-specific T cell responses. Here we describe a series of experiments that directly examines the effects of allogeneic MHC molecules on the immune-priming capabilities of a whole cell tumor vaccine engineered to secrete GM-CSF. The results demonstrate that the expression of an allogeneic MHC molecule by a vaccine cell can actually enhance the induction of systemic antitumor immunity. In addition, allogeneic MHC expression has no inhibitory effect on the ability of GM-CSF-transduced vaccines to induce systemic antitumor immunity. These findings support the design of clinical trials for testing this more feasible and generalizable allogeneic whole tumor cell vaccine approach.

Animals↗
PubMed1998

Interleukin-6 and small intestinal luminal immunoglobulins.

Our aim was to determine the relationships between interleukin-6 and immunoglobulin levels within small intestinal luminal secretions. Twenty adult subjects with small intestinal bacterial overgrowth (N = 13), irritable bowel syndrome (N = 4), and nonulcer dyspepsia (N = 3) underwent endoscopic aspiration of secretions from the small intestinal mucosal surface for assessment of IL-6, IgA1, IgA2, IgM, IgG1, IgG2, IgG3, and IgG4 concentrations. Serum immunoglobulin concentrations and small intestinal histology were also determined. IgA2 and IgG3 were the predominant IgA and IgG subclasses in luminal secretions in 19/20 (95%) and 20/20 (100%) subjects, respectively. IgA1 and IgG1 predominated in serum in all subjects. No subject had villous atrophy. Luminal IL-6 concentrations correlated significantly with luminal IgA2, IgM, and IgG3 concentrations but not with IgA1 or any other IgG subclass levels. Conversely, luminal IL-6 or immunoglobulin concentrations did not correlate significantly with levels of any immunoglobulin isotype in serum. These observations suggest that important relationships exist between local IL-6 and IgA2, IgM, and IgG3 responses in human small intestinal luminal secretions. Local investigation is mandatory when assessing intestinal immune activity.

Adult↗
PubMed1998

Immune responses to Plasmodium falciparum antigens during a malaria vaccine trial in Tanzanian children.

Among Tanzanian children living in an area of intense and perennial malaria transmission, prevalence of naturally acquired IgG antibodies that recognize SPf66, NANP, p190 and a 19 kDa fragment of the merozoite surface protein-1 (MSP-1) is high and increases with age. This possibly reflects the high level of natural exposure of the children to P. falciparum. The prevalences of IgG antibodies that recognize the three putative merozoite derived sequences contained in the malaria vaccine SPf66 (83.1, 55.1 and 35.1) is low but also show some age dependence. Three doses of the SPf66 vaccine induce a strong IgG antibody response against both the SPf66 construct, NANP and the three individual peptides. Vaccination with SPf66 did not result in an increase of anti19 kDa fragment antibodies. This reflects the specificity of the humoral immune response induced by the SPf66 construct. Among vaccinated children, antibody titres against SPf66 decreased over time following the third dose. However, 18 months after the third dose, SPf66 recipients still had significantly higher IgG titres and stimulation indices of peripheral blood mononuclear cells (PBMC) than placebo recipients. Within the vaccine group, there is a trend for increasing anti-SPf66 IgG titre to be associated with decreasing risk of clinical malaria but this was not statistically significant. Results also show the difficulties of establishing whether antibody responses are related to protection in field trials in endemic areas.

Animals↗
PubMed1997

A transformation vector for stage-specific expression of heterologous genes in Trypanosoma cruzi epimastigotes.

To express heterologous genes in a stage-specific manner, we constructed a transformation vector for Trypanosoma cruzi containing a selection gene (hyg) and a reporter gene (luc) flanked by sequences of the multicopy 1f8 gene arranged so as to provide a trans-splicing acceptor site to hyg and a putative polyadenylation signal to luc. The intergenic region of the T. cruzi genes 294 and KAP was placed between hyg and luc, contributing the polyadenylation signal of 294 to hyg and the KAP trans-splicing acceptor site to luc. Transformation was carried out by electroporation, and transformed epimastigotes were selected in medium containing hygromycin B. Through double homologous recombination of the 1f8 sequences with their chromosomal counterparts, the construction is inserted into the 1f8 locus, substituting probably one and no more than a few copies of the 1f8 gene without having apparent deleterious effects on the parasite. cDNA analysis demonstrated that the introduced signals were correctly processed, resulting in translatable hyg and luc mRNAs. Whereas epimastigotes express luciferase, no expression is found in the trypomastigote stage.

Animals↗
PubMed1997

Identification of a Trypanosoma cruzi antigenic epitope implicated in the infectivity of fibroblast LLC-MK2 cells.

In the present paper we describe the identification of an antigenic epitope that appears to be associated with the surface membrane of the Trypanosoma cruzi parasite, probably implicated in infectivity. Anti-TcMe antibodies inhibited the infectivity of fibroblast LLC-MK2 cells by 34% relative to a preimmune serum. The epitope was specifically recognized by 55% of the sera from 80 chagasic patients. The anti-TcMe antibody immunoprecipitated proteins of about 60 and 40 kDa from epimastigote and cell-culture trypomastigote forms, respectively. It is likely that the 60- and 40-kDa proteins are processed from higher-molecular-weight precursors, since the antibody immunoprecipitated protein fractions in the range of 115-150 kDa from in vitro translation products of poly A+ RNA.

Amino Acid Sequence↗
PubMed1997

Reverse transcriptase-like activity in Trypanosoma cruzi.

The use of specific synthetic RNA homopolymers as templates and short oligonucleotides as primers has allowed evidence of the existence of a reverse transcriptase-like activity in Trypanosoma cruzi, to be revealed. The RNA:DNA products derived from this reaction are of approximately 110 nucleotides in length. The enzyme has greater affinity for poly(rA)/ oligo(dT) templates than for poly(rC)/oligo(dG) having a 20 mM Mg+2 ion requirement. The detected reverse transcriptase-like activity is not affected by aphidicolin and ddTTP but is inhibited by actinomycin D. novobiocin, rifamycin SV and AZT.

Animals↗
PubMed1997

Local and systemic complement activity in small intestinal bacterial overgrowth.

It is unknown whether bacteriolysis due to luminal complement activation contributes to local defense mechanisms against small intestinal bacterial overgrowth, particularly with gram-negative bacteria. This study addressed this issue. Thirty adult subjects were investigated with culture of luminal secretions adherent to proximal small intestinal mucosa. Luminal and plasma concentrations of C3 and C3d and C3d/C3 ratios were determined. Activated terminal complement complex was sought in surface epithelium to which aspirated secretions had been adherent. Small intestinal bacterial overgrowth with gram-negative bacteria was present in 12/30 (40.0%) subjects. C3, C3d, and C3d/C3 profile indicated that increased local but not systemic C3 activation occurs in this group. Conversely, no activation of terminal complement complex was evident in this circumstance. Thus, complement-mediated bacteriolysis is unlike to contribute to local defense mechanisms against small intestinal bacterial overgrowth, even when overgrowth flora includes gram-negative bacteria. Factors preventing full local activation of the complement cascade in this circumstance require investigation.

Adult↗
PubMed1997

Luminal bacteria and small-intestinal permeability.

BACKGROUND: The influence of luminal bacteria on small-intestinal permeability has not been fully assessed. This study addressed this issue. METHODS: Thirty-four subjects (mean age 64 years; range 22-95 years) were investigated for possible small-intestinal bacterial overgrowth (SIBO) with culture of a small-intestinal aspirate. A lactulose/mannitol small-intestinal permeability test was performed, small-intestinal histology assessed and serum vitamin B12 concentrations measured in all subjects. Permeability was also assessed in a control group of 34 asymptomatic volunteers. RESULTS: Urinary lactulose/mannitol ratios were significantly increased in subjects with SIBO with colonic-type flora (P < 0.0005), even in the absence of villous atrophy. Urinary lactulose/mannitol ratios were increased in this group due to significantly increased urinary lactulose concentrations (P < 0.0005) rather than reduced urinary mannitol levels, after correcting for inter-subject variations in renal function. Counts of intraepithelial lymphocytes of CD8 phenotype were significantly increased in this group (P = 0.003). Although a significant correlation was found between intraepithelial lymphocyte counts and small-intestinal permeability overall (P < 0.002), these counts were not significantly different in subjects with SIBO with colonic-type flora whose permeability values were < or = > 0.028, the upper limit of normal in asymptomatic controls. Serum vitamin B12 concentrations did not differ significantly between groups (P > 0.5). Ageing did not independently influence small-intestinal permeability (P > 0.5). CONCLUSIONS: Small-intestinal permeability is increased in subjects with SIBO with colonic-type bacteria. This effect is independent of ageing and not mediated by vitamin B12 deficiency. Although counts of intraepithelial lymphocytes of CD8 phenotype are increased in this disorder, it is also unlikely that these cells play an important causative role in this process. Routine light microscopic assessment underestimates the prevalence of small-intestinal functional disturbance in this disorder.

Bacterial Infections↗
PubMed1997

Small intestinal bacterial overgrowth in the symptomatic elderly.

OBJECTIVE: 1) To determine the prevalence of small intestinal overgrowth with colonic-type bacteria in symptomatic elderly subjects, particularly those without important "clues" such as clinically apparent predisposition or vitamin B12 deficiency, and 2) to investigate defense mechanisms such as gastric acidity, small intestinal motility, and luminal IgA in this setting. METHODS: Fifty-two symptomatic subjects without vitamin B12 deficiency or clinically apparent predisposition to bacterial overgrowth or disturbed mucosal immunity, including 22 subjects > or = 75 yr old, underwent culture of small intestinal luminal secretions. Indicator paper was used to measure fasting gastric pH. The presence of bacteria of confirmed nonsalivary origin in small intestinal secretions served as an index of small intestinal dysmotility. Small intestinal luminal IgA concentrations were measured by radial immunodiffusion. RESULTS: Small intestinal overgrowth with colonic-type flora was not present in any subject investigated for dyspepsia, irrespective of age. In subjects with chronic diarrhea, anorexia, or nausea, overgrowth with colonic-type flora (Enterobacteriaceae) was present in 0/12 (0%), 1/10 (10.0%), and 9/14 (64.3%) subjects aged < 50 yr, 50-74 yr, and > or = 75 yr, respectively. Enterobacteriaceae were not concurrently recovered from saliva of any subject > or = 75 yr old with small intestinal overgrowth with these bacteria. Fasting hypochlorhydria was present in only 1/9 (11.1%) such subjects. Luminal IgA concentrations were significantly greater in subjects > or = 75 yr old with bacterial overgrowth than in culture-negative subjects (p < or = 0.003). CONCLUSIONS: Small intestinal overgrowth with colonic-type bacterial should be considered in subjects > or = 75 yr old with chronic diarrhea, anorexia, or nausea, even in the absence of clues such as clinically apparent predisposition or vitamin B12 deficiency. Small intestinal dysmotility, rather than fasting hypochlorhydria or mucosal immunosenescence, probably is responsible for the prevalence of bacterial overgrowth in this group.

Aged↗
PubMed1997

Luminal antigliadin antibodies in small intestinal bacterial overgrowth.

OBJECTIVE: Elevated antigliadin antibody levels in small intestinal luminal secretions of subjects with normal or only mildly abnormal small intestinal histology are considered indicative of "latent" or "potential" celiac disease. The purpose of this study was to determine whether small intestinal bacterial overgrowth (SIBO) might provide an alternative explanation for positive luminal antigliadin antibodies in such subjects. METHODS: Twenty-six adult subjects without predisposition to disturbed mucosal immunity were investigated with culture of small intestinal luminal secretions. Luminal total IgA and IgA-antigliadin antibody concentrations were measured by radial immunodiffusion and indirect enzyme immunoassay, respectively. Local mucosal counts of IgA-plasma cells were determined by immunohistochemistry. Small intestinal histology and intraepithelial lymphocyte counts were assessed by light microscopy. Corresponding serum antigliadin antibody concentrations were determined. RESULTS: SIBO was present in 17/26 (65.4%) subjects. No subject with SIBO had villous atrophy. Luminal total IgA concentrations (p < 0.0005), mucosal IgA-plasma cell counts (p < 0.01), and intraepithelial lymphocyte counts (p < 0.01) were significantly increased in subjects with SIBO. Luminal IgA-antigliadin antibodies were detected in 6/17 (35.3%) subjects with SIBO and 0/9 (0%) subjects without SIBO. Luminal IgA-antigliadin antibody concentrations correlated significantly with luminal total IgA levels (p < 0.01) but not with serum values (p < 0.1). Serum IgG-antigliadin antibody concentrations were elevated in 2/6 (33.3%) subjects with SIBO and positive luminal antigliadin antibodies. CONCLUSIONS: SIBO may be an alternative explanation to "latent" or "potential" celiac disease for positive luminal antigliadin antibodies in subjects with either normal or only mildly abnormal small intestinal histology, even when serum antigliadin antibody concentrations are elevated. Positive luminal antigliadin antibodies in SIBO probably occur as epiphenomena in the context of a graded mucosal immune response to local bacterial antigens.

Adult↗
PubMed1996

The immunodominant major histocompatibility complex class I-restricted antigen of a murine colon tumor derives from an endogenous retroviral gene product.

Tumors express peptide antigens capable of being recognized by tumor-specific cytotoxic T lymphocytes (CTL). Immunization of mice with a carcinogen-induced colorectal tumor, CT26, engineered to secrete granulocyte/macrophage colony-stimulating factor, routinely generated both short-term and long-term CTL lines that not only lysed the parental tumor in vitro, but also cured mice of established tumor following adoptive transfer in vivo. When either short-term or long-term CTL lines were used to screen peptides isolated from CT26, one reverse-phase high performance liquid chromatography peptide fraction consistently sensitized a surrogate target for specific lysis. The bioactivity remained localized within one fraction following multiple purification procedures, indicating that virtually all of the CT26-specific CTL recognized a single peptide. This result contrasts with other tumor systems, where multiple bioactive peptide fractions have been detected. The bioactive peptide was identified as a nonmutated nonamer derived from the envelope protein (gp70) of an endogenous ecotropic murine leukemia provirus. Adoptive transfer with CTL lines specific for this antigen demonstrated that this epitope represents a potent tumor rejection antigen. The selective expression of this antigen in multiple non-viral-induced tumors provides evidence for a unique class of shared immunodominant tumor associated antigens as targets for antitumor immunity.

Animals↗
PubMed1996

Enhanced immune priming with spatial distribution of paracrine cytokine vaccines.

In preclinical models, tumor cells genetically modified to express cytokines or other costimulatory molecules can generate systemic antitumor immunity. In some cases, these tumor vaccines have been shown to eradicate micrometastases. These results have led to the initiation of numerous phase I clinical trials employing either autologous or allogeneic tumor vaccines genetically modified to express cytokines and other genes. In this report, we use our murine model to identify a number of parameters that may be critical for enhancing vaccine efficacy. In addition to antigen dose and cytokine level, the distribution of vaccine inoculation was found to have a significant impact on vaccine potency. These results require consideration in early clinical trials designed to evaluate cellular vaccine therapy.

Animals↗
PubMed1996

Systemic and local paracrine cytokine therapies using transduced tumor cells are synergistic in treating intracranial tumors.

Development of an effective immunotherapeutic approach for treatment of CNS tumors must take into account the unique anatomic and immunologic features of the brain. We explored the antitumor immune response in the brain elicited by nonreplicating melanoma cells genetically engineered to produce either granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-2 (IL-2) in a paracrine fashion. Using a new model of intracranial melanoma in C57BL/6 mice, the cytokine-producing cells were given either as a subcutaneous vaccine to induce systemic antitumor immunity or as a direct injection into the brain as local immunotherapy. We found that GM-CSF-transduced cells, as a subcutaneous vaccine but not as an intracranial injection, afforded some protection from intracranial challenge with the wild-type tumor. In contrast, direct intracranial injection of tumor cells secreting IL-2 was protective whereas flank vaccination with IL-2 transductants was not. Combination therapy with both the subcutaneous GM-CSF-transductants as a vaccine and local administration of IL-2-transductants in the brain achieved a synergistic response. These findings provide a basis for the application of paracrine cytokine delivery to brain cancer therapy both as a systemic vaccine and via local administration. The demonstration of synergy between paracrine cytokine therapies holds promise as a novel therapy for brain tumors.

Animals↗
PubMed1996

Multiprimer PCR system for differential identification of mycobacteria in clinical samples.

A novel multiprimer PCR method with the potential to identify mycobacteria in clinical samples is presented. The assay relies on the simultaneous amplification of three bacterial DNA genomic fragments by using different sets of oligonucleotide primers. The first set of primers amplifies a 506-bp fragment from the gene for the 32-kDa antigen of Mycobacterium tuberculosis, which is present in most of the species belonging to the genus Mycobacterium. The second set of primers amplifies a 984-bp fragment from the IS6110 insertion sequence of the bacteria belonging to the M. tuberculosis complex. The third set of primers, derived from an M. tuberculosis species-specific sequence named MTP40, amplifies a 396-bp genomic fragment. Thus, while the multiprimer system would render three amplification fragments from the M. tuberculosis genome and two fragments from the Mycobacterium bovis genome, a unique amplification fragment would be obtained from nontuberculous mycobacteria. The results obtained, using reference mycobacterial strains and typed clinical isolates, show that the multiprimer PCR method may be a rapid, sensitive, and specific tool for the differential identification of various mycobacterial strains in a single-step assay.

Base Sequence↗
PubMed1996

Mucosal cytokine production in small-intestinal bacterial overgrowth.

BACKGROUND: Mucosal production of interferon-gamma, interleukin-6, and tumour necrosis factor-alpha is increased in inflammatory bowel disease and parallels disease activity. Interferon-gamma production is also increased in coeliac disease. Conversely, local cytokine profiles have not been investigated in small-intestinal bacterial overgrowth. This study addressed this issue. METHODS: Eighteen adult subjects were studies with culture of proximal small-intestinal luminal secretion and measurement of luminal interferon-gamma, interleukin-6, and tumour necrosis factor-alpha concentrations by enzyme-linked immunosorbent assay. Small-intestinal histology was assessed by light microscopy. RESULTS: Interferon-gamma, interleukin-6, and tumour necrosis factor-alpha were measurable in proximal small-intestinal luminal secretions of all subjects, even in the absence of light microscopic evidence of enteropathy. Small-intestinal bacterial overgrowth was present in 12 of 18 (66.7%) subjects. Luminal concentrations of neither interferon-gamma nor tumour necrosis factor-alpha differed significantly in subjects with and without small-intestinal bacterial overgrowth (P + 0.06 and P = 1.0, respectively). Conversely, luminal interleukin-6 concentrations were significantly increased in subjects with this disorder (P = 0.02). Multivariate linear regression analysis suggested that colonic-type rather than salivary-type flora mediated this increased interleukin-6 response (P = 0.02 and P = 0.64, respectively). No correlation was found between luminal interleukin-6 and tumour necrosis factor-alpha concentrations, even after the confounding influence of colonic-type bacteria was excluded (P = 0.60). CONCLUSIONS: These findings suggest that increased mucosal production of interleukin-6 occurs in small-intestinal bacterial overgrowth, particularly when the overgrowth flora includes colonic-type bacteria. Conversely, luminal levels of neither interferon-gamma nor tumour necrosis factor-alpha are increased in the circumstance, distinguishing the local cytokine profile in this disorder from those that occur in coeliac disease and inflammatory bowel disease.

Adult↗