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Biomedical subjects

D J Ferguson

Publications and source records attributed to D J Ferguson.

At least 73 records · Page 4Linked to original sources
PubMed1997

Electron microscopy findings on an intraocular lens in the uveitis, glaucoma, hyphaema syndrome.

PURPOSE: To report the electron microscopic findings on an explanted intraocular lens in a patient with the uveitis, glaucoma, hyphaema syndrome. METHODS: Scanning and transmission electron microscopy were undertaken on a coccoon of cellular material from the tip of the intraocular lens haptic. RESULTS: Scanning electron micrographs showed densely packed coccoid-like structures on the haptic surface. By transmission electron microscopy these structures proved to be melanosomes. CONCLUSIONS: The scanning electron micrographs described in this report are similar to those reported in patients with chronic post-operative uveitis, but to our knowledge have not been shown before in association with the uveitis, glaucoma, hyphaema syndrome. Transmission electron microscopy determined that the coccoid-like structures were melanosomes. The melanosomes are probably derived from damaged pigment epithelial cells or iris stromal melanocytes secondary to recurrent chafing of the haptic against the posterior surface of the iris.

Aged↗
PubMed1997

The interstitial response to renal injury: fibroblast-like cells show phenotypic changes and have reduced potential for erythropoietin gene expression.

To define the potential for erythropoietin gene expression in injured kidneys, marker gene expression was examined in transgenic mice bearing a homologously recombined erythropoietin--simian virus 40 T antigen (Epo-TAg) transgene. Three types of renal injury were studied: ureteric obstruction, global ischemia following clamping of the renal pedicle, and focal needlestick injury. All modes of injury were associated with an expansion of the interstitial space, which contained an increased number of cells. Alterations observed in the interstitial fibroblast-like cells included an increased number and complexity of cellular processes, enhanced expression of contractile elements, particularly of the intermediate filament desmin, and reduced expression of ecto-5'-nucleotidase. Following each type of injury there was a focal or general reduction in the proportion of such cells that could be stimulated to express Epo-TAg. However, some positively staining cells were present even in severely injured regions and more could be recruited to express Epo-TAg by severe anemic or hypoxic stimulation, indicating that cells with the potential for erythropoietin gene expression were neither absent nor completely refractory to stimulation in these regions. In all injured kidneys, Epo-TAg expression was limited to the fibroblast-like population. Double labeling experiments showed that cells expressing Epo-TAg also expressed increased amounts of desmin, demonstrating that the myofibroblast features which develop in response to injury and the capacity for erythropoietin gene expression are not mutually exclusive.

Animals↗
PubMed1997

Innate immunity to Toxoplasma gondii is influenced by gender and is associated with differences in interleukin-12 and gamma interferon production.

Given that differences between the sexes in relative susceptibility to parasitic infections have been noted, this study further elucidates the mechanisms responsible by demonstrating that male SCID mice are more resistant than female mice to infection with Toxoplasma gondii and that this difference correlates with enhanced innate immune responses in these animals. Male SCID mice exhibited longer survival times, lower parasite burdens, and less severe pathological changes postinfection. An immunological basis for these differences is demonstrated in that these animals produced interleukin-12 more rapidly and exhibited higher levels of gamma interferon earlier postinfection.

Animals↗
PubMed1997

Reconstitution of trimethylamine-dependent coenzyme M methylation with the trimethylamine corrinoid protein and the isozymes of methyltransferase II from Methanosarcina barkeri.

Reconstitution of trimethylamine-dependent coenzyme M (CoM) methylation was achieved with three purified polypeptides. Two of these polypeptides copurified as a trimethylamine methyl transfer (TMA-MT) activity detected by stimulation of the TMA:CoM methyl transfer reaction in cell extracts. The purified TMA-MT fraction stimulated the rate of methyl-CoM formation sevenfold, up to 1.7 micromol/min/mg of TMA-MT protein. The TMA-MT polypeptides had molecular masses of 52 and 26 kDa. Gel permeation of the TMA-MT fraction demonstrated that the 52-kDa polypeptide eluted with an apparent molecular mass of 280 kDa. The 26-kDa protein eluted primarily as a monomer, but some 26-kDa polypeptides also eluted with the 280-kDa peak, indicating that the two proteins weakly associate. The two polypeptides could be completely separated using gel permeation in the presence of sodium dodecyl sulfate. The corrinoid remained associated with the 26-kDa polypeptide at a molar ratio of 1.1 corrin/26-kDa polypeptide. This polypeptide was therefore designated the TMA corrinoid protein, or TCP. The TMA-MT polypeptides, when supplemented with purified methylcorrinoid:CoM methyltransferase (MT2), could effect the demethylation of TMA with the subsequent methylation of CoM and the production of dimethylamine at specific activities of up to 600 nmol/min/mg of TMA-MT protein. Neither dimethylamine nor monomethylamine served as the substrate, and the activity required Ti(III) citrate and methyl viologen. TMA-MT could interact with either isozyme of MT2 but had the greatest affinity for the A isozyme. These results suggest that TCP is uniquely involved in TMA-dependent methanogenesis, that this corrinoid protein is methylated by the substrate and demethylated by either isozyme of MT2, and that the predominant isozyme of MT2 found in TMA-grown cells is the favored participant in the TMA:CoM methyl transfer reaction.

Archaeal Proteins↗
PubMed1997

Biomaterial particle phagocytosis by bone-resorbing osteoclasts.

Abundant implant-derived biomaterial wear particles are generated in aseptic loosening and are deposited in periprosthetic tissues in which they are phagocytosed by mononuclear and multinucleated macrophage-like cells. It has been stated that the multinucleated cells which contain wear particles are not bone-resorbing osteoclasts. To investigate the validity of this claim we isolated human osteoclasts from giant-cell tumours of bone and rat osteoclasts from long bones. These were cultured on glass coverslips and on cortical bone slices in the presence of particles of latex, PMMA and titanium. Osteoclast phagocytosis of these particle types was shown by light microscopy, energy-dispersive X-ray analysis and SEM. Giant cells containing phagocytosed particles were seen to be associated with the formation of resorption lacunae. Osteoclasts containing particles were also calcitonin-receptor-positive and showed an inhibitory response to calcitonin. Our findings demonstrate that osteoclasts are capable of phagocytosing particles of a wide range of size, including particles of polymeric and metallic biomaterials found in periprosthetic tissues, and that after particle phagocytosis, they remain fully functional, hormone-responsive, bone-resorbing cells.

Bone Resorption↗
PubMed1997

The location of binding sites of pemphigus vulgaris and pemphigus foliaceus autoantibodies: a post-embedding immunoelectron microscopic study.

Pemphigus is a life-threatening autoimmune blistering disease of skin and mucous membranes that has two major subtypes based on clinical and histological features, pemphigus vulgaris (PV) and pemphigus foliaceus (PF). Autoantibodies against the PV antigen (desmoglein 3) and the PF antigen (desmoglein 1) are involved in the pathogenesis of blister formation. In the present study, the location of epitopes recognized by autoantibodies of patients with PV and PF was studied by postembedding immunogold electron microscopy. PV and PF autoantibodies were observed bound predominantly to the intercellular domains of desmosomes, but not to the non-desmosomal keratinocyte cell surface. The relationship between the location of PF antigen and other constitutive desmosomal proteins, desmocollin, desmoplakin and plakoglobin, in normal human skin was investigated using a double immunogold labelling technique. It was observed that PF antigen and desmocollin co-localize within the intercellular domain of the desmosomes. In contrast, the antibodies against desmoplakin and plakoglobin bound predominantly to the intracellular desmosomal attachment plaque with the binding site of the antibody against plakoglobin closer to the desmosomal cell membrane than that of the antibody to desmoplakin. We show that the LR White postembedded immunogold electronmicroscopy technique is convenient and easily applied to studies of autoimmune bullous skin diseases. We have used it to demonstrate the precise localization of the binding sites of PV and PF autoantibodies and their relationship with other constitutive desmosomal proteins.

Antigens↗
PubMed1996

Specific roles of methylcobamide:coenzyme M methyltransferase isozymes in metabolism of methanol and methylamines in Methanosarcina barkeri.

An immunochemical approach was employed as a direct test for functional activities of isozymes of methylcobamide:coenzyme M methyltransferase (MT2-M and MT2-A) in the metabolic pathways of methane formation from: methanol, acetate, monomethylamine, dimethylamine, and trimethylamine. Specific removal of the MT2 isozymes from buffer soluble cell extracts of Methanosarcina barkeri was accomplished by use of immobilized, affinity-purified, ovine polyclonal antibodies. Extracts of methanol-grown cells depleted of MT2-M lost entirely the ability to carry out conversion of methanol to 2-(methylthio)ethanesulfonate (methyl-CoM). Methanol:CoM methyl transfer activity was completely restored by addition of purified MT2-M, but no activity was recovered by addition of MT2-A. In contrast, the activity of trimethylamine-grown cell extracts to convert monomethylamine and dimethylamine to methyl-CoM was lost almost entirely by immunosorptive removal of MT2-A. Addition of purified MT2-A, but not MT2-M, to the MT2-A-depleted extract fully reconstituted methyl-CoM formation from both mono- and dimethylamine. Interestingly, in extracts resolved of MT2-A, trimethylamine-dependent methylation of coenzyme M was observed at approximately 20% of the rate of controls not treated with antibody. Furthermore, both isozymes were effective in full restoration of trimethylamine conversion. Tests indicated that neither of the two MT2 isozymes are involved in methane formation from acetate. The results establish that MT2-A plays a specific role in metabolism of methylated amine substrates, whereas, MT2-M functions in methane formation from trimethylamine and methanol.

Cell-Free System↗
PubMed1996

Carcinoembryonic antigens (CD66) on epithelial cells and neutrophils are receptors for Opa proteins of pathogenic neisseriae.

Opa protein-expressing pathogenic neisseriae interact with CD66a-transfected COS (African green monkey kidney) and CHO (Chinese hamster ovary) cells. CD66a (BGP) is a member of carcinoembryonic antigen (CEA, CD66) family. The interactions occur at the N-terminal domain of CD66a, a region that is highly conserved between members of the CEA subgroup of the CD66 family. In this study, we have investigated the roles of CD66 expressed on human epithelial cells and polymorphonuclear phagocytes (PMNs) in adhesion mediated via Opa proteins. Using human colonic (HT29) and lung (A549) epithelial cell lines known to express CD66 molecules, we show that these receptors are used by meningococci. A monoclonal antibody, YTH71.3, against the N-terminal domain of CD66, but not 3B10 directed against domains, A1/ B1, inhibited meningococcal adhesion to host cells. When acapsulate bacteria expressing Opa proteins were used, large numbers of bacteria adhered to HT29 and A549 cells. In addition, both CD66a-transfected CHO cells and human epithelial cells were invaded by Opa-expressing meningococci, suggesting that epithelial cell invasion may occur via Opa-CD66 interactions. In previous studies we have shown that serogroup A strain C751 expresses three Opa proteins, all of which mediate non-opsonic interactions with neutrophils. We have examined the mechanisms of these interactions using antibodies and soluble chimeric receptors. The results indicate that the nature of their interactions with purified CD66a molecules and with CD66 on neutrophils is alike and that these interactions occur at the N-terminal domain of CD66. Thus, the Opa family of neisserial ligands may interact with several members of the CD66 family via their largely conserved N-terminal domains.

Animals↗
PubMed1996

Structural determinants within the subunit protein of Ty1 virus-like particles.

The Ty virus-like particles (VLPs) are functionally analogous to retroviral particles. They package the enzymes and the RNA necessary for retrotransposition, and mediate the integration of the reverse-transcription product into the genome of the host cell. Here we map three structural determinants of particle assembly in the subunit protein. We have also identified key residues in these regions that seem to be involved in subunit interaction and particle morphology. In particular, two point mutations in putative amphipathic helices have remarkable effects on VLP morphology, increasing the diameter as much as eightfold.

Amino Acid Sequence↗
PubMed1996

Different roles for interleukin-4 during the course of Toxoplasma gondii infection.

The course of Toxoplasma gondii infection from initiation of disease perorally until day 28 postinfection was compared between interleukin-4 (IL-4) gene knockout (IL-4-/-) mice and their wild-type (IL-4+/+) counterparts on a disease-susceptible genetic background. The rate of mortality was significantly greater in mice deficient in Il-4 than in the immunocompetent controls. Although levels of T. gondii-specific spleen cell proliferation measured in vitro were similar between groups at all time points examined throughout infection, the quantities of cytokines released into the culture supernatant differed. Culture supernatants from spleen cells derived from IL-4-deficient mice contained significantly more gamma interferon than those derived from IL-4+/+ mice at day 7 postinfection. Conversely, IL-10 production was significantly greater from the spleen cells derived from wild-type mice at day 28 postinfection. Splenocytes from both groups of mice had a marked inhibition of proliferation in response to soluble tachyzoite antigen as well as reduced proliferation in response to concanavalin A between days 7 and 14 postinfection and marked proliferation on days 21 and 28 postinfection. At day 28 postinfection, histological examination of the brains indicated that IL-4+/+ mice had more severe pathological changes and more cysts than IL-4-/- mice. In addition, although many nonencysted single organisms were present in IL-4+/+ mice within both necrotic lesions and microglial nodules, few nonencysted parasites were found, and no necrotic lesions were present in IL-4-deficient animals. These results suggest that the observed reduction in mortality during the early acute phases of infection may be due to the down-regulatory effects of Il-4 or associated Th2-derived products on proinflammatory cytokines such as gamma interferon. However, the long-term effects of IL-4 are detrimental, possibly because of the ability of this cytokine to inhibit proinflammatory antiparasitic products. This may explain the increased parasite multiplication with cysts observed in the brains of IL-4+/+ mice.

Animals↗
PubMed1996

Natural history of node-positive breast cancer: the curability of small cancers with a limited number of positive nodes.

PURPOSE: The long-term outcome of node-positive breast cancer was analyzed to determine the risk of metastatic disease as a function of tumor size and number of positive nodes. METHODS: From 1927 to 1987, 501 women with node-positive breast cancer were treated at the University of Chicago Medical Center. Patients were treated with radical, extended radical, or modified radical mastectomy. Forty-eight patients received multiagent chemotherapy, and 118 were treated with hormonal therapy. The mean survival duration is 120 months, with a maximal follow-up time of 485 months (40 years). RESULTS: The number of nodes that contained metastatic disease and the pathologic size of the primary tumor were significant determinants of disease-free-survival (DFS) by multivariate analysis (P < .001). In patients with fewer than four positive nodes, tumor size was of prognostic importance, with small tumors more likely to be cured by local-regional therapy. The 20-year DFS rate for patients with one positive node was 69%; however, if the primary tumor was < or = 2 cm, the 20-year DFS rate was 81%, compared with 59% if the tumor was larger than 2 cm. Patients with two or three positive nodes had a 73% 20-year DFS rate if the tumor size was < or = 2 cm, compared with 53% 20-year DFS in patients with tumors larger than 2 cm. CONCLUSION: In patients with T1 lesions with less than four nodes positive, the long-term DFS rate is comparable to that for node-negative breast cancer of the same size. Four or more nodes positive is an indicator of likely systemic disease.

Adrenalectomy↗
PubMed1996

Molecular characterisation of ninein, a new coiled-coil protein of the centrosome.

We describe the cDNA cloning of ninein, a novel component of centrosomes. In the mouse, ninein is predicted to be an acidic protein (calculated pI of 4.8) with alternatively spliced forms of 245 kDa and 249 kDa that contain extensive regions of coiled-coil structure flanked by non-coiled ends. Other interesting features of this protein include an EF-hand-like domain, a potential GTP binding site and four leucine zipper domains. Specific polyclonal antisera were raised to two non-overlapping recombinant fragments of the protein and used to characterise the cellular distribution of ninein. Immunofluorescence and immunoelectron microscopy experiments with macrophage-like cells, Mm1, showed that ninein is localised specifically in the pericentriolar matrix of the centrosome. Studies with NIH3T3 fibroblasts demonstrated that ninein is associated with the centrosome throughout the cell cycle and can also be detected within nuclei at interphase. At mitosis ninein was also observed in association with the mitotic spindle. Immunocytochemical staining of mouse tissues showed that ninein was expressed in a heterogeneous fashion. Staining, if present, was always consistent with a centrosomal localisation and was never associated with nuclei. Ninein provides a new molecular tool for analysing the structure and function of the centrosome.

3T3 Cells↗
PubMed1995

Combined immunocytochemistry and non-isotopic in situ hybridization for the ultrastructural investigation of human parvovirus B19 infection.

Parvovirus B19 is a single-stranded DNA virus with a specific tropism for human erythroid precursor cells. The virus codes for two overlapping structural (capsid) proteins and one non-structural protein which is thought to perform essential functions in viral replication, transcription and packaging. The ultrastructural localization of these proteins was achieved in cultured haemopoietic cells derived from fetal liver which had been infected in vitro and subsequently embedded in LR White acrylic resin. Postembedding immunogold detection of B19 structural and non-structural proteins was combined with localization of viral nucleic acid by in situ hybridization using a digoxigenin-labelled probe and different sized gold labels. The majority of the B19 capsid protein and DNA present in cells harvested 48 hours post-infection co-localized within the centri-nuclear region of erythroid cells demonstrating characteristic chromatin margination. Relatively little DNA hybridization signal was present over paracrystalline inclusions strongly labelled with anti-capsid protein monoclonal antibody R92F6. Viral DNA and capsid protein were co-localized in apparent egress from the nucleus through nuclear pores. B19 non-structural protein was detected in association with both nuclear and cytoplasmic arrays of capsids, supporting the view that this protein plays an important role in viral packaging and remains associated with the complete viral particle until its release from the cell. Co-localization of viral nucleic acid and proteins at the ultrastructural level is a flexible, rapid and highly specific tool for examination of viral life-cycles within cells.

Capsid↗
PubMed1995

The effect of minoxidil analogues and metabolites on the contraction of collagen lattices by human skin fibroblasts.

Minoxidil, in addition to its effect on hypertension and hair growth, has been proposed as a potential antifibrotic agent. Minoxidil inhibits the contraction of collagen lattices by human fibroblasts in vitro. However, the mechanism of inhibition is unknown. As minoxidil is metabolised in the body to minoxidil glucuronide and minoxidil sulphate, we investigated the potencies of these metabolites to inhibit collagen lattice contraction. We also studied selected analogues of minoxidil to assess the influence of certain functional groups in the inhibition. The major metabolite, minoxidil glucuronide, proved to be inactive, whereas minoxidil sulphate was considerably more active than minoxidil. In terms of the structural analogues, the substitution of one amino group by a methyl group resulted in loss of the inhibitory activity; removal of the nitroxide oxygen led to stronger inhibition than with minoxidil. Further studies are planned to learn more about a possible role for minoxidil in wound contraction.

Animals↗
PubMed1995

Human intestinal development in a severe-combined immunodeficient xenograft model.

The present work describes a severe-combined immunodeficient murine xenograft model used to investigate human gastrointestinal ontogenesis. Specifically, the study has tested whether carefully selected regions of human fetal gut are able to undergo region-specific morphogenesis and epithelial cytodifferentiation when transplanted subcutaneously into immunodeficient mice. In addition, double-label in situ hybridisation techniques, utilising specific human and mouse DNA probes, have been adopted to characterise host and donor cell types and to investigate the potential developmental roles for non-epithelial cells in the regulation of epithelial differentiation pathways in vivo. Human fetal small and large bowel developed to form a characteristic mucosa 10 weeks after transplantation, which displayed clear region-specific structural and functional gradients. The initial phase of xenograft epithelialisation closely resembled the stratified type of epithelium which is present during early fetal gastrointestinal development. Idiosyncratic epithelial differentiation pathways were recorded during xenograft regeneration, with an absence of Paneth cells and an abundance of enteroendocrine cells when compared with developed xenograft and paediatric intestine. Such differences may, therefore, be important in ensuring rapid and region-specific development in the absence of conventional luminal stimuli and hormonal changes that occur normally during pregnancy. In situ hybridisation demonstrated an exclusively human origin for the intestinal xenograft epithelium and muscularis mucosa and externa. Although the submucosa and lamina propria were comprised of a chimeric mixture, murine cells were rarely seen to contact with the epithelium, which interacted primarily with human myofibroblasts and human intraepithelial lymphocytes. It is proposed that a 'selection' process operates to maintain species-specific cellular interactions, and this mechanism may subsequently play an important role in regulating epithelial cell differentiation, orchestrated in part by juxtaposed non-epithelial cell types.

Absorption↗