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D J Ferguson

Publications and source records attributed to D J Ferguson.

At least 91 records · Page 5Linked to original sources
PubMed1995

Conventional patterns of human intestinal proliferation in a severe-combined immunodeficient xenograft model.

The present work describes the pattern of human intestinal proliferation in an immunodeficient murine xenograft model, which we have shown to closely mimic cell division in normal paediatric gut. Cellular proliferation was measured using a double-label technique combining MIB-1 immunohistochemistry and [3H]thymidine autoradiography, to critically compare values for the tissue growth fraction (G1, G2, S- and M-phase cells) and DNA synthesizing (S-phase) cells in xenograft epithelium, lamina propria, muscularis externa and intraepithelial lymphocytes. The MIB-1 monoclonal antibody (which recognises the cell-cycle dependent nuclear antigen Ki-67) specifically labelled proliferating human cells within the xenografts and did not cross-react with dividing murine cells. This was confirmed using ultrastructural in situ hybridisation with human- and mouse-specific DNA probes to identify the genetic origin of proliferating cells. In general, we found a good tissue correlation between MIB-1 and [3H]thymidine labelling, the only exception being an apparent dysregulation of Ki-67 antigen expression in regenerating xenograft epithelium. In developed xenograft intestine, the highest levels of proliferation were consistently recorded within the crypt epithelium, where 15.7%-26.7% of cells were actively cycling and S-phase occupied approximately half of the cell cycle. The frequency distribution of proliferating epithelial cells within small and large intestinal xenograft crypts was clearly tissue-specific, showing typical patterns of cell division. Therefore, the presence of functional pluripotent epithelial stem cells and conventional spatio-temporal patterns in cellular proliferation, migration, de-cycling, lineage commitment and cytodifferentiation now makes this an attractive experimental model with which to study human intestinal crypt responses to various types of tissue manipulation, e.g. cytotoxic, radiotherapeutic, dietary, endocrine and gene-targeting therapy.

Animals↗
PubMed1995

Opc- and pilus-dependent interactions of meningococci with human endothelial cells: molecular mechanisms and modulation by surface polysaccharides.

The interplay between four surface-expressed virulence factors of Neisseria meningitidis (pili, Opc, capsule and lipopolysaccharide (LPS)) in host cell adhesion and invasion was examined using derivatives of a serogroup B strain, MC58, created by mutation (capsule, Opc) and selection of variants. To examine the role of Opc and of additional expression of pili, bacteria lacking the expression of Opa proteins were used. The effects of different LPS structures were examined in variants expressing either sialylated (L3 immunotype) or truncated non-sialylated (L8 immunotype) LPS. Studies showed that (i) pili were essential for meningococcal interactions with host cells in both capsulate and acapsulate bacteria with the sialylated L3 LPS immunotype, (ii) the Opc-mediated invasion of host cells by piliated and non-piliated bacteria was observed only in acapsulate organisms with L8 LPS immunotype, and (iii) expression of pili in Opc-expressing bacteria resulted in increased invasion. Investigations on the mechanisms of cellular invasion indicated that the Opc-mediated invasion was dependent on the presence of serum in the incubation medium and was mediated by serum proteins with arginine-glycine-aspartic acid (RGD) sequence. Cellular invasion in piliated Opc+ phenotype also required bridging molecules containing the RGD recognition sequence and appeared to involve the integrin alpha v beta 3 as a target receptor on endothelial cells. These studies extend the previous observations on variants of a serogroup A strain (C751) and show that Opc mediates cellular invasion in distinct meningococcal strains and provide confirmation of its mechanism of action. This is the first investigation that evaluates, using derivatives of a single strain, the interplay between four meningococcal surface virulence factors in host cell invasion.

Antigens, Bacterial↗
PubMed1995

Functional implications of the expression of PilC proteins in meningococci.

Multiple forms of PilC were found in Neisseria meningitidis (Nm) strains isolated from the oropharynx, blood or cerebrospinal fluid expressing either Class I or Class II pili. PilC expression was observed less frequently in case as opposed to carrier isolates. Moreover, PilC and pili were not always co-expressed. Several heavily piliated strains had no detectable PilC protein as determined by Western blotting using an antiserum previously used to detect such proteins in adhesive variants (Nassif et al., 1994). Serogroup B strain MC58 produced large numbers of pili, but expressed barely detectable amounts of PilC. A clonal variant of this strain with increased expression of PilC concurrently exhibited increased adherence to Chang conjunctival epithelial cells and human umbilical vein endothelial cells (Huvecs), but with more rapid binding to the former. No alteration in pilin sequence occurred in this variant, suggesting the involvement of PilC in increased adhesion. A Pil- backswitcher isolated from the hyper-adherent variant was PilC+ but was non-adherent, indicating that any PilC adherence function requires pilus expression. Parental variant (low PilC) produced pili in bundles that were easily detached from the bacterial surface and were frequently associated with Huvec surfaces after bacteria had been sheared off, but pili infrequently replaced bacteria during infection with the PilC-expressing variant. The hyper-adherent variant, which appeared to produce morphologically distinct pilus bundles, was able to withstand considerable shearing force and remained firmly attached to Huvecs. This raises the possibility that the observed hyper-adherence may arise from better anchorage of pili to the bacterial surface in addition to increased adhesion to some host cell surfaces.

Bacterial Adhesion↗
PubMed1995

Cloning and characterization of a bradyzoite-specifically expressed gene (hsp30/bag1) of Toxoplasma gondii, related to genes encoding small heat-shock proteins of plants.

Stage conversion between the tachyzoite and bradyzoite forms of the protozoan parasite Toxoplasma gondii is an important aspect in the pathogenesis of toxoplasmosis. In an initial investigation of molecular regulation of stage conversion in T. gondii, we describe the cloning and characterization of a bradyzoite-specifically expressed gene (hsp30/bag1). Bradyzoite formation was induced in cell culture by alkaline pH, and this was followed by purification of this parasitic stage using magnetic cell sorting. A bradyzoite cDNA library was constructed by random amplification using the polymerase chain reaction. Screening with a bradyzoite-specific monoclonal antibody identified a reactive clone. The amino acid sequence derived from the 687 bp open reading frame showed similarity to the conserved C-terminal region of small heat-shock proteins from plants. Stage-specific expression of the naturally occurring 30 kDa antigen in bradyzoites was confirmed by polyclonal antisera generated against the recombinant antigen. Immunoelectron microscopy indicated a cytosolic location of this antigen in bradyzoites. The expression of HSP30/BAG1 seems to be regulated at the mRNA level, since reverse polymerase chain reaction using bradyzoite-specific primers amplified transcripts in bradyzoites only, not in tachyzoites.

Amino Acid Sequence↗
PubMed1995

Golf tee hairs in Netherton disease.

We present a case of Netherton disease, where the hairs lacked the characteristic microscopic feature of trichorrhexis invaginata. In its place were certain hairs with a golf tee morphology. These represent the proximal half of the invaginate node seen in typical Netherton disease. Scanning electron microscopy demonstrated the three-dimensional quality of this abnormality, which is subtle when assessed by light microscopy alone. We describe this sign so that it might be recognized when seen in isolation, as here, and allow the diagnosis of Netherton disease to be confirmed.

Child, Preschool↗
PubMed1995

Monoclonal rat antibodies directed against Toxoplasma gondii suitable for studying tachyzoite-bradyzoite interconversion in vivo.

We previously reported the in vitro analysis of stage differentiation of Toxoplasma gondii in murine bone marrow-derived macrophages. The purpose of this study was to generate monoclonal rat antibodies that might be suitable for investigating tachyzoite-bradyzoite interconversion in vivo with the murine model. Immunization of Fischer rats with cysts of T. gondii NTE resulted in the generation of seven monoclonal antibodies of the immunoglobulin G2a, G2b, or M isotype, which were further characterized by the immunoblot technique, immunofluorescence assay, immunohistology, and immunoelectron microscopy. Immunoblots demonstrated specific reactivity of five monoclonal antibodies with proteins with molecular masses of 40, 52, 55, 60, 64, 65, and 115 kDa. One antibody (CC2) appeared to recognize a differently expressed antigen depending on the parasite stage, reacting with a 40-kDa molecule in tachyzoites and a 115-kDa antigen in bradyzoites and oocysts. Several other monoclonal antibodies were shown to be stage specific and to react in immunofluorescence assays or in immunoblots with either tachyzoites or bradyzoites. Kinetics of stage conversion in vitro could be monitored by immunofluorescence with two of these monoclonal antibodies. Preliminary immunohistological investigations of tissue sections from infected mice demonstrated the possible usefulness of these monoclonal antibodies for future in vivo studies on stage differentiation of T. gondii in the murine system.

Animals↗
PubMed1995

Natural history of node-negative breast cancer: a study of 826 patients with long-term follow-up.

PURPOSE: We were interested in examining the long-term outcome of patients with node-negative breast cancer to address the following questions: (1) Is node-negative breast cancer a disease that is curable by local modalities? (2) Are there predictors of disseminated disease in node-negative breast cancer? (3) Are there subgroups of tumors that have different times to recurrence? METHODS: From 1927 to 1984, 826 women with node-negative breast cancer were treated at the University of Chicago. Patients underwent either a radical or extended radical mastectomy (83%) or a modified radical mastectomy (13%). RESULTS: Follow-up evaluation ranged from 9 to 523 months (43.6 years); the mean follow-up period of survivors is 162 months (13.5 years). On multivariate analysis, the strongest predictor of outcome and time to relapse was pathologic tumor size. Patients with tumors less than 2 cm had a 20-year disease-free survival (DFS) rate of 79% and a median time to recurrence of 48 months as compared with patients with tumors greater than 2 cm, who had a survival rate of 64% (P < .001) and a median time to recurrence of 37 months (P = .01). CONCLUSION: With extended follow-up evaluation, node-negative breast cancer is a curable disease. Size is the strongest predictor of dissemination and rate of relapse. These data suggest that given the natural history of node-negative breast cancer, analysis of clinical trials with short follow-up periods can be misleading, since it may identify those patients whose tumors have a greater virulence but not necessarily a greater likelihood to metastasize.

Actuarial Analysis↗
PubMed1995

Inactivation of Cdc2 increases the level of apoptosis induced by DNA damage.

A number of lines of evidence have suggested a possible involvement of the mitosis-promoting protein kinase Cdc2 in the process of apoptotic cell death, and one recent study concluded that premature activation of Cdc2 is required for apoptosis. Here we have used a temperature-sensitive murine Cdc2 mutant cell line and Cdc2 inhibitor compounds to study the effect of inhibition of this protein kinase on apoptosis induced by DNA-damaging drugs. Inhibition of Cdc2 activity before or during exposure to DNA strand break-inducing drugs had the effect of increasing the level of subsequent apoptosis, as assessed by electron microscopy and flow cytometry. We conclude that, far from being required for cell death, a form of mammalian Cdc2 suppresses apoptosis induced by DNA damage. This form of Cdc2 appears to be active in G2-arrested cells and is therefore presumably distinct from the mitosis-promoting Cdc2-cyclin B heterodimer.

Animals↗
PubMed1994

Immediate destruction of xenogeneic islets in a primate model.

Transplantation of pancreatic islets from other species to man has the potential to cure diabetes, but whether such islet grafts will be subject to damage due to natural antibody-mediated hyperacute rejection is unknown. We have examined the fate of islet xenografts in a recipient with direct relevance to man, the cynomolgus monkey. Rabbit islets were prepared by an intraductal collagenase technique and incubated in neat rabbit, human, or cynomolgus serum, with and without heat inactivation, for up to 6 days. Islets were analyzed by flow cytometry for IGG and IGM binding, and scored for viability by supravital staining. For in vivo studies, isolated islets were prepared from 4 New Zealand White rabbits (15-34 x 10(3) islets 70-85% purity) and transplanted beneath the kidney capsule of normal cynomolgus monkeys after aggregation in either a rabbit or monkey blood clot. The tissue was retrieved at various times up to 4 days after transplantation and processed for light and electron microscopy. The results showed that rabbit islets bind heterophile antibody of both IGG and IGM subtypes. There was slow loss of islet viability in vitro over 3 days of culture in neat human or cynomolgus serum. Destruction of islets in vivo was more rapid with visible damage within 6 hr associated with neutrophil infiltration. Subsequently, there was heavy mononuclear cell infiltration leading to total destruction within 4 days. The results suggest that immediate mechanisms of graft rejection, possibly compliment and neutrophil mediated, represent a major barrier to islet xenotransplantation in humans.

Animals↗
PubMed1994

Expression of a homologously recombined erythopoietin-SV40 T antigen fusion gene in mouse liver: evidence for erythropoietin production by Ito cells.

We have obtained transgenic mice in which an erythropoietin-SV40 virus T antigen fusion gene is homologously recombined into the native Epo locus. This gene is expressed in a tissue-specific manner closely resembling that of the native Epo gene. Immunohistochemical detection of SV40 T antigen has been used to characterize the hepatic cell populations expressing the transgene. In mice stimulated by anaemia or hypobaric hypoxia, SV40 T antigen was demonstrated in two liver cell populations: a subset of hepatocytes and a nonparenchymal cell type. Immunohistochemical and ultrastructural characterization of these cells by light and electron microscopy showed the nonparenchymal cell type to be the Ito cells, which lie in a persinusoidal position within the space of Disse. We therefore conclude that Ito cells are the nonhepatocytic source of liver Epo production. These cells show many similarities to the Epo-producing fibroblastoid interstitial cells of the kidney.

Anemia↗
PubMed1994

The chromatin-associated protein H-NS alters DNA topology in vitro.

H-NS is one of the two most abundant proteins in the bacterial nucleoid and influences the expression of a number of genes. We have studied the interaction of H-NS with DNA; purified H-NS was demonstrated to constrain negative DNA supercoils in vitro. This provides support for the hypothesis that H-NS influences transcription via changes in DNA topology, and is evidence of a structural role for H-NS in bacterial chromatin. The effects of H-NS on topology were only observed at sub-saturating concentrations of the protein. In addition, a preferred binding site on DNA was identified by DNase I footprinting at sub-saturating H-NS concentrations. This site corresponded to a curved sequence element which we previously showed, by in vivo studies, to be a site at which H-NS influences transcription of the proU operon. When present in saturating concentrations, H-NS did not constrain supercoils and bound to DNA in a sequence-independent fashion, covering all DNA molecules from end to end, suggesting that H-NS may form distinct complexes with DNA at different H-NS:DNA ratios. The data presented here provide direct support for the hypothesis that H-NS acts at specific sites to influence DNA topology and, hence, transcription.

Bacterial Proteins↗
PubMed1994

A morphological study of chronic cerebral toxoplasmosis in mice: comparison of four different strains of Toxoplasma gondii.

The pathological changes, host-parasite relationship and structure of the tissue cysts in the brains of mice chronically infected with four different strains of Toxoplasma gondii were examined by light and electron microscopy. In mice infected with the mouse-adapted ME49 strain for 4, 8, 12, 16 and 25 weeks, the pathological changes consisted of moderate to severe meningitis and cuffing of blood vessels by inflammatory cells. At 4 weeks post-infection (p.i.), lymphocytes were the major cell type, but at later time points, plasma cells predominated. Large numbers of cysts were observed at between 4 and 12 weeks p.i., with a decrease being seen at 16 weeks p.i. Microglial nodules, many containing tachyzoites or bradyzoites, were present at all time points. In contrast, the three strains isolated from patients with the acquired immune deficiency syndrome (AIDS) resulted in no meningitis in two cases (DEY, DAG) and in mild meningitis in one case (WIL), although all three showed some cuffing of blood vessels. In addition, only very low numbers of cysts and nodules were observed. Ultrastructurally, the cysts of all four strains were seen to be located within host cells. The cysts of the ME49 strain differed from those of the other strains in that a proportion contained immature and dividing bradyzoites at all time points, whereas those of the other strains contained only mature bradyzoites. From the observation of nodules with parasites and cysts with immature zoites, it would appear that the ME49 strain may result in an unstable chronic infection with a continuous turnover of cysts, a feature that should be taken into consideration when this strain is used as an experimental model of chronic toxoplasmosis.

Acquired Immunodeficiency Syndrome↗