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D J Ferguson

Publications and source records attributed to D J Ferguson.

At least 55 records · Page 3Linked to original sources
PubMed1999

Potent immunostimulatory dendritic cells can be cultured in bulk from progenitors in normal infant and adult myasthenic human thymus.

Low density cells can readily be enriched from thymus tissue both of children undergoing cardiac surgery and of older patients with myasthenia gravis, and can be cryostored in bulk. When fresh or thawed cells are cultured with granulocyte-macrophage colony-stimulating factor and stem cell factor with or without tumour necrosis factor-alpha (TNF-alpha), they generate numerous cells with the characteristic ultrastructural, phenotypic and functional properties of dendritic cells. These proved to be very potent, both as stimulators of primary mixed leucocyte responses and as costimulators in oxidative mitogenesis. Especially after exposure to TNF-alpha, these dendritic cells also processed a natural epitope from a 437-residue polypeptide and presented it efficiently to an autoimmune T-cell clone (of T helper type 0 phenotype). Thus, immunostimulatory dendritic cells can be cultured in relative abundance from progenitors in infant and adult human thymus. Both are convenient sources of potent antigen-presenting cells of identifiable origins, e.g. for use in selecting human T-cell lines.

Adolescent↗
PubMed1999

Cell surface protease PRT1 identified in the fungal pathogen Pneumocystis carinii.

The subtelomeric regions of the chromosomes of many organisms contain gene families that allow adaptation to a changing environment. In a number of parasites, these subtelomeric gene families encode cell surface proteins that undergo antigenic variation. Proteases are another important virulence determinant in pathogenic microorganisms. We report the localization of the PRT1 protease of the pathogenic fungus Pneumocystis carinii sp. f. carinii, encoded by a subtelomeric gene family, to the cell surface of both the trophozoite and the cyst phase of the organism. Using anti-PRT1 antiserum, we demonstrated specificity to P. carinii sp. f. carinii in sections of infected rat lungs and, using immunofluorescence, we showed that the PRT1 protease has the characteristic distribution of a surface protein. The anti-PRT1 antiserum showed cross-reactivity with a number of P. carinii sp. f. carinii proteins migrating between 185 kDa and 28 kDa, the majority migrating between 42 kDa and 52 kDa, a region that has been shown by serological studies to contain important immunodominant P. carinii proteins. Cross-reactivity was also observed with P. carinii sp. f. hominis proteins. We have also cloned a portion of the catalytic domain of PRT1 from P. carinii sp. f. hominis, P. carinii sp. f. muris and P. carinii sp. f. rattus. Our data suggest that the PRT1 protease plays an important role in the pathogenicity of P. carinii.

Amino Acid Sequence↗
PubMed1998

The relationship between nm23, angiogenesis, and the metastatic proclivity of node-negative breast cancer.

Distant metastases are the major cause of morbidity and mortality in women with breast cancer. The prediction of this metastatic proclivity is essential in determining prognosis and should allow an appropriate choice of therapy. A critical look at the metastatic process and its phenotypic expression offers an opportunity to identify some of the important events in the process that may relate to prognosis, with the goal of identifying those patients with occult metastases and also sparing systemic treatment in those patients whose tumors have not developed the capacity for distant spread. To evaluate the significance of nm23 and angiogenesis in the metastatic cascade, we used archival material from 163 node-negative breast cancer patients who had a median follow-up of 14 years. All patients underwent mastectomy and received no adjuvant chemotherapy or hormone or radiation therapy. Immunohistochemistry was used to detect nm23-H1 expression, whereas angiogenesis was determined by microvessel count (MVC). We found the 15-year disease-free survival (DFS) to be significantly better in patients with high nm23 compared with low nm23 (91% compared with 70%, P = 0.008). Low MVC is associated with excellent (92%) long-term DFS. In those patients with high MVC, high nm23 allows the identification of a subgroup with significantly higher DFS (90% compared with 66%, P = 0.02). Among high nuclear grade tumors, if nm23 is high, the DFS is significantly better (89% compared with 68%, P = 0.03). Thus, nm23 is still associated with excellent survival, even when there is unfavorable angiogenesis or nuclear grade. Multivariate analysis confirms that nm23 and MVC are important prognostic factors. High MVC appears necessary but not sufficient for metastasis to occur, whereas low nm23 may further contribute to metastatic progression. Both nm23 and MVC contribute valuable information in characterizing the malignant phenotype.

Adult↗
PubMed1998

Evidence for the shikimate pathway in apicomplexan parasites.

Parasites of the phylum Apicomplexa cause substantial morbidity, mortality and economic losses, and new medicines to treat them are needed urgently. The shikimate pathway is an attractive target for herbicides and antimicrobial agents because it is essential in algae, higher plants, bacteria and fungi, but absent from mammals. Here we present biochemical, genetic and chemotherapeutic evidence for the presence of enzymes of the shikimate pathway in apicomplexan parasites. In vitro growth of Toxoplasma gondii, Plasmodium falciparum (malaria) and Cryptosporidium parvum was inhibited by the herbicide glyphosate, a well-characterized inhibitor of the shikimate pathway enzyme 5-enolpyruvyl shikimate 3-phosphate synthase. This effect on T. gondii and P. falciparum was reversed by treatment with p-aminobenzoate, which suggests that the shikimate pathway supplies folate precursors for their growth. Glyphosate in combination with pyrimethamine limited T. gondii infection in mice. Four shikimate pathway enzymes were detected in extracts of T. gondii and glyphosate inhibited 5-enolpyruvyl shikimate 3-phosphate synthase activity. Genes encoding chorismate synthase, the final shikimate pathway enzyme, were cloned from T. gondii and P. falciparum. This discovery of a functional shikimate pathway in apicomplexan parasites provides several targets for the development of new antiparasite agents.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1998

Targeted disruption of the bradyzoite-specific gene BAG1 does not prevent tissue cyst formation in Toxoplasma gondii.

Expression of the 30 kDa small heat shock protein BAG1 is restricted to the latent bradyzoite 'tissue cyst' form of Toxoplasma gondii, first appearing approximately 2-3 days after the initiation of bradyzoite differentiation. Although developmental expression of small heat shock proteins has been described for many species, their precise function is unclear. In order to examine the function of BAG1 in T. gondii bradyzoites and its role during parasite differentiation, we have used homologous recombination to produce a knock-out mutant in the cyst-forming strain P(LK), a clonal derivative of ME49. Under tissue culture conditions that stimulate bradyzoite differentiation (alkaline pH), the mutant was found to express several bradyzoite-specific markers with the same kinetics and frequency as the parental strain. Neither enhanced nor decreased susceptibility to stress was observed for the BAG1-deficient mutant. In vivo studies revealed that tachyzoites of the bag1 knock-out mutant were fully able to establish a chronic infection in C57BL/6 mice, producing brain cysts of a size, morphology and frequency indistinguishable from cysts formed by the parental control strain. Brain cysts of the bag1 knock-out mutant contained viable parasites capable of establishing an acute infection after oral administration, demonstrating that conversion of bradyzoites to tachyzoites is also unimpaired. We conclude that BAG1 is not essential for normal function of bradyzoite containing tissue cysts, at least in intermediate host species. This clone of P(LK) was found to be unable to produce oocysts and is therefore unsuitable for studies in cats.

Amino Acid Sequence↗
PubMed1998

Electron microscopy study of explanted intraocular lenses from clinically noninfected eyes.

PURPOSE: To determine whether intraocular lenses (IOLs) in clinically noninfected eyes are coated with a significant, bacteria-containing biofilm. SETTING: The Oxford Eye Hospital, Oxford, United Kingdom. METHODS: Twenty-six IOLs, removed for reasons other than endophthalmitis from 26 patients attending the Oxford Eye Hospital over a 3 year period, were examined by electron microscopy. Immediately following explantation, the IOL was placed in glutaraldehyde 4% in 0.1 M phosphate buffer solution and processed for scanning electron microscopy (SEM). Areas of interest were reprocessed for transmission electron microscopy (TEM). RESULTS: There was no evidence of a bacterial biofilm on any IOL. In 5 IOLs, significant host cellular debris was seen at the tip of the haptic or at the optic-haptic junction. In 4 of them, clusters of coccoid-shaped structures were seen at the optic-haptic junction on SEM, but examination by TEM showed these structures to be melanosomes, not bacteria. CONCLUSION: We found no evidence to suggest that a significant number of IOLs are coated with a bacterial biofilm in clinically noninfected cases. We advocate the use of TEM to distinguish between coccoid bacteria and melanosomes.

Adult↗
PubMed1998

The localization of target antigens and autoantibodies in linear IgA disease is variable: correlation of immunogold electron microscopy and immunoblotting.

Linear IgA disease (LAD) of adults and children is a dapsone-responsive, autoimmune subepidermal blistering disease characterized by linear IgA deposits at the basement membrane zone (BMZ) of the skin and mucosa. Circulating IgA antibodies to BMZ components are often present. In this study we investigated the ultrastructural localization of the antigens and autoantibodies in six patients with LAD (five adults and one child). Using a direct postembedding immunogold electron microscopy (EM) technique, three different patterns of IgA antibody deposition were seen in the skin of four patients with LAD. The IgA deposits localized within the uppermost part of the lamina lucida and to the basal surface of the hemidesmosome in two patients, to the lamina lucida in one, and to the lamina densa in the fourth patient. Using an indirect immunogold EM technique and serum or purified blister fluid from two additional LAD patients, we showed that the serum autoantibodies of one patient bound to the hemidesmosome of the BMZ, while the autoantibodies in the blister fluid of the other patient bound to the lamina densa and sublamina densa including the anchoring fibrils in a labelling pattern similar to that of the monoclonal antibody (LH7.2) to collagen VII. All the autoantibodies binding to the hemidesmosome or lamina lucida recognized a protein in epidermal extracts of molecular weight 180 kDa or its breakdown product of 97 kDa, 200 kDa or 230 kDa. The antibodies binding to the lamina densa recognized proteins of 180 and 285 kDa. The antibodies that bound to the lamina densa and anchoring fibrils recognized collagen VII. In this immunogold EM study we have shown four patterns of IgA labelling in six patients with LAD, associated with five different antigens as recognized by immunoblotting. These results, together with our previous immunofluorescence and immunoblotting findings add support to the contention that LAD is a heterogeneous disease as regards both the target antigens and epitopes.

Adult↗
PubMed1998

A protective role for IL-6 during early infection with Toxoplasma gondii.

IL-6 deficient mice were found to be significantly more susceptible to peroral infection with Toxoplasma gondii than their wild-type counterparts as measured by survival, brain cyst burdens and brain pathology at 28 days post infection. The physical manifestations of disease, such as weight loss, were not observed in IL-6 deficient animals until at least seven days later than such changes occurred in wildtype mice. During this early stage of infection IL-6+/+ but not IL-6-/- mice mounted a peripheral blood neutrophilia. Furthermore, between 6-8 days post-infection there was a significant increase in plasma IFN-gamma levels in wild-type but not IL-6 deficient mice. Not until days 18-23 post-infection, concurrent with the majority of deaths in IL-6-/- mice, were plasma IFN-gamma levels substantially and significantly raised in IL-6-/- mice. At this time not only were these plasma IFN-gamma levels 20-fold higher than background but eight-fold greater than peak (6-8 days post-infection) IFN-gamma levels in IL-6+/+ mice. IFN-gamma dependent parasite specific IgG2a levels were also significantly higher in IL-6-/- mice over this period and thereafter. Overall the evidence suggests that in the absence of IL-6 mice are unable to initiate a rapid proinflammatory response against T. gondii, which allows increased parasite growth. Increased mortality in IL-6-/- mice may be directly due to this increased parasite burden and the excessive inflammatory response this induces three weeks post-infection.

Animals↗
PubMed1998

Partially protective vaccination permits the development of latency in a normally virulent strain of Toxoplasma gondii.

The virulent RH strain of Toxoplasma gondii is acutely lethal in mice and fails to establish chronic infection. Vaccination of BALB/c mice with a soluble tachyzoite antigen preparation, STAg, in combination with the immunostimulatory cytokine interleukin-12 results in partial protection against RH lethal challenge. Nevertheless, brain tissue obtained from surviving, vaccinated mice as late as 1 year after RH infection contained latent parasite forms as demonstrated by subinoculation into naive recipients. The tachyzoites arising in the subinoculated animals were genetically indistinguishable from the original RH inoculum. Microscopic examination revealed that the persistent parasite forms present in the brains of vaccinated and challenged mice have a tissue cyst-like morphology and express the bradyzoite antigen BAG-1 but not the tachyzoite-specific antigen SAG-2 but are different from the cysts formed by avirulent T. gondii strains in that the internal parasite stages display ultrastructural features intermediate between tachyzoites and bradyzoites. Moreover, the zoites within the RH tissue cysts are clearly distinct from conventional bradyzoites in their sensitivity to pepsin-HCl digestion. In contrast to the observations made with partially resistant STAg/interleukin-12-vaccinated animals, no latent forms could be detected in brain tissue after RH challenge of mice immunized with a live attenuated tachyzoite vaccine which confers total protection against this parasite isolate. The above findings demonstrate the potential of a virulent T. gondii strain to generate latent parasite stages, a process which may be promoted under conditions of incomplete vaccination.

Animals↗
PubMed1997

Osteoclasts are capable of particle phagocytosis and bone resorption.

Osteoclasts are multinucleated cells specialized for the function of lacunar bone resorption. Although they are known to be capable of phagocytosis of inert particles, it is not known whether this abolishes their ability to respond to hormones or to form resorption lacunae. Human and rat osteoclasts were isolated from giant cell tumours of bone and rat long bones, respectively, and cultured on coverslips and cortical bone slices, both in the presence and in the absence of particles of latex (1 micron diameter) and polymethylmethacrylate (PMMA) (< 50 microns). By light microscopy, it was evident that osteoclasts which had phagocytosed both latex and PMMA particles remained responsive to calcitonin. Osteoclast phagocytosis of particles was also evident on scanning electron microscopy, where it could also be seen that these cells were associated with the formation of resorption lacunae. These findings underline the fact that the osteoclast is a true member of the mononuclear phagocyte system and that phagocytosis does not abrogate either its hormonal response to calcitonin or its highly specialized function of bone resorption. That osteoclasts which have phagocytosed biomaterial particles such as PMMA are still able to carry out lacunar bone resorption is of interest in clinical conditions such as aseptic loosening, where a heavy foreign body particle load is often associated with extensive bone resorption.

Animals↗
PubMed1997

Effect of desferrioxamine cardioplegia on ischemia-reperfusion injury in isolated rat heart.

BACKGROUND: The protective effect of desferrioxamine against myocardial ischemia-reperfusion injury remains uncertain. In this study we examined a broad range of ischemia-reperfusion indices to determine the effect of desferrioxamine cardioplegia in a model that reflects surgical practice. METHODS: Isolated rat hearts were subjected to 90 minutes of ischemia with cold cardioplegia, with or without 0.5-mmol/L desferrioxamine. Left ventricular mechanical function and the levels of thiobarbituric acid-reactive substances and nonprotein thiol compounds were measured after reperfusion. Electron microscopic analysis of mitochondria was performed using diaminobenzidine staining, together with histochemical staining for glycogen and marker enzymes in left ventricular muscle and the atrioventricular node. RESULTS: The desferrioxamine group showed better preservation of diastolic function (chamber stiffness coefficient at 15 minutes and maximum rate of decrease of left ventricular pressure at 45 minutes of reperfusion). Histochemical analysis showed that mitochondria-specific succinate dehydrogenase and the nonspecific esterase of the atrioventricular node were better preserved in the desferrioxamine group. CONCLUSIONS: The findings from this study indicate that there is added protection against ischemia-reperfusion injury when desferrioxamine is added to the cardioplegic solution; however, the study also highlighted that, in this clinically applicable model, desferrioxamine is not universally protective against all aspects of ischemia-reperfusion injury.

Animals↗
PubMed1997

Loss of oral infectivity of tissue cysts of Toxoplasma gondii RH strain to outbred Swiss Webster mice.

We have developed a method for obtaining cysts of Toxoplasma gondii RH strain. Outbred Swiss Webster mice were infected subcutaneously (s.c.) or intraperitoneally (i.p) with 10(5) tachyzoites and given sulfadiazine 400 mg l-1 + NaHCO3 10 g l-1 in drinking water from day 1 to day 15 post-infection (p.i.). None of the mice infected i.p. survived, compared with 50% of the mice infected s.c. Cysts were detectable in the brain on day 45 p.i., and had ultrastructural features consistent with those of bradyzoites. However, these cysts were incapable of infecting mice via the oral route. In addition, immunofluorescence studies showed the persistence of P36 protein expression, indicating that the conversion to bradyzoites was incomplete.

Animals↗