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Biomedical subjects

J O'Rourke

Publications and source records attributed to J O'Rourke.

At least 19 recordsLinked to original sources
PubMed1992

A cytotonic, cholera toxin-like protein produced by Campylobacter jejuni.

1. Campylobacter jejuni is a major cause of gastroenteric infection. 2. This organism appears to produce both cytotonic and cytotoxic virulence factors. 3. We report here that culture filtrates of some clinical isolates of C. jejuni induce elongation of Chinese hamster ovary (CHO) cells in vitro but do not cause inhibition of fluid absorption in the rat ileum. 4. These culture filtrates contain low levels of a protein which cross-reacts immunologically with the cholera toxin. 5. The cholera toxin-like protein of C. jejuni behaved identically to cholera toxin on non-denaturing polyacrylamide gel electrophoresis. 6. Under denaturing conditions, however, this protein displayed no subunit structure and a molecular weight of approximately 50 kDa with many higher molecular weight aggregates. 7. In conclusion, isolates of C. jejuni produced small amounts of enterotoxin when grown in vitro. 8. The toxin cross-reacted immunologically with cholera toxin and has a similar native structure, but does not appear to possess subunits.

Animals↗
PubMed1992

An ultrastructural study of Helicobacter mustelae and evidence of a specific association with gastric mucosa.

The ultrastructure of Helicobacter mustelae, a natural inhabitant of the ferret stomach, has been studied and compared with the human gastroduodenal pathogen H. pylori. H. mustelae is a short, slightly curved rod, 2 microns x 0.5 micron, with four or more sheathed flagella. The most common flagellar configuration is a single flagellum at one terminus, bipolar arrangement at the other end and a lateral flagellum. Dense inclusion bodies were observed below the flagellar insertion sites. It is suggested that this configuration is a specialised adaptation to motility in a viscous environment. On examination of the ferret gastric mucosa, similarities to H. pylori were observed such as adherence to gastric tissue and the formation of adhesion pedestals. However, unlike H. pylori, significant numbers of bacteria were intracellular. Furthermore, a much greater proportion of H. mustelae were attached to the mucosa with few bacteria lying free in the mucus, as is seen with H. pylori. It is concluded that the ferret is an important model for the study of adherence of bacteria to gastric mucosa and their possible role in peptic ulceration.

Animals↗
PubMed1991

Cytokine regulation of C3 and C5 production by human corneal fibroblasts.

Recent investigations have suggested that cytokines play important roles during inflammation and host defense, primarily by regulating the diverse functions of immunologic cells (e.g. lymphocytes and monocytes). However, much less is known about the capacity of cytokines to also regulate the functions of resident tissue cells. We hypothesize that during inflammation, cytokines (e.g. monokines and lymphokines) directly regulate the expression of inflammatory precursors and mediators, such as the third and fifth complement components, by resident ocular cells and are therefore important in the local regulation of ocular inflammation. To test this hypothesis we developed an in vitro culture system utilizing isolated human corneal fibroblasts and examined the effects of specific cytokines, i.e. interleukins and interferons, on the production of the third and fifth components of the complement system. Human corneal fibroblasts were cultured in the presence of varying concentrations (1-500 U ml-1) of interleukin 1 alpha, interleukin 1 beta, interleukin 2, interferon alpha and interferon gamma for 48 hr at 37 degrees C, 5% CO2. The supernatants were then evaluated for antigen levels for the third and fifth components of complement using specific enzyme-linked immunospecific assays. These studies revealed that both interleukin 1 alpha and interleukin 1 beta induced seven to tenfold increases in the levels of the third component. Similarly interferon alpha and interferon gamma stimulated an approximate four and ninefold dose-dependent increase, respectively, in the production of the third component. Analysis of the effect of interleukin 2 on third component production demonstrated that higher concentrations (100 U ml-1) were required to induce a fivefold increase in the production of the third component.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗
PubMed1991

An aqueous paracentesis pipet.

We describe a glass pipet tipped with a short, sharp, 30-gauge needle for performing aqueous paracentesis. The design eliminates the possibility of needle bending and minimizes fractional resistance during limbus penetration; it also allows quicker recovery of aqueous samples. A small plastic bulb at the upper end facilitates removal of aqueous samples from the pipet.

Aqueous Humor↗
PubMed1989

Use of the mouse for the isolation and investigation of stomach-associated, spiral-helical shaped bacteria from man and other animals.

Spiral-helical shaped bacteria other than Campylobacter pylori have been shown to infect the human stomach. The characteristic helical morphology of these bacteria appears to be similar to that of bacteria found in the stomachs of many other animal species. Early reports on gastric bacteria suggested that rodents may be useful for investigation and isolation of stomach-associated bacteria. Therefore, anaesthetised mice were given, through a stomach tube, a heavy suspension of a spiral-helical bacterium from a cat, scrapings of gastric mucus from primates, or a homogenised whole-antral biopsy from a human patient. At intervals after inoculation, gastric biopsies were examined by lightmicroscopy and electronmicroscopy for the presence of spiral-helical bacteria. Significant colonisation was observed in 40% of mice 1 week, and in 80% of mice 11 weeks, after inoculation with suspensions of the cat isolate. Mice were also successfully colonised by spiral bacteria present in homogenised human biopsy material and by other spiral bacteria from a monkey. These observations suggest that mice may prove to be useful animals for the study of gastric bacteria that are, as yet, non-cultivable and for analysis of some of the attributes commonly thought to be involved in colonisation.

Animals↗
PubMed1989

Aqueous humor composition in Fuchs' dystrophy.

Several studies have suggested differences in aqueous humor composition in Fuchs' dystrophy, including elevations in fibrinogen-related factors, compared with controls. In the current study, aqueous humor was obtained at surgery from 10 uninflamed eyes with advanced Fuchs' dystrophy and cataracts and 11 control eyes with cataracts alone. Total fibrinogen-related antigen was measured in a masked manner using an enzyme-linked immunosorbant assay (ELISA). There was no statistically significant difference between the means of the two groups (0.281 +/- 0.292 (SD) and 0.176 +/- 0.090 mg/ml, respectively). Similarly, there was no difference in ELISA-determined aqueous humor small molecular weight fibrinogen-derived metabolites between the two groups, 623 +/- 141 and 550 +/- 55 fibrinogen equivalents, respectively. Also, no statistically significant difference was detected between Fuchs' dystrophy and control eyes in aqueous humor ascorbate, glucose, carbon dioxide, bicarbonate, and pH. Therefore, this study found no evidence of alterations in aqueous humor composition in Fuchs' dystrophy and supports the hypothesis that the disease is a primary disorder of the corneal endothelium.

Antigens↗
PubMed1988

Isolation of a spiral-shaped bacterium from the cat stomach.

A spiral- or helix-shaped bacterium that colonizes the stomachs of cats has been isolated in pure culture for the first time. The organism is tightly coiled with tufts of 10 to 17 polar flagella positioned slightly off center at the end of the cell. The body of the cell is entwined with unique periplasmic fibrils that usually occur in pairs, although groupings of one and three fibrils were also seen. The organism is strongly urease, catalase, and oxidase positive and is likely to belong to an as yet unclassified group of bacteria that are specifically adapted to the ecological niche provided by gastrointestinal mucus. Isolation of this organism will allow study of the factors influencing colonization of gastric mucosae, information relevant to the association of another mucus colonizer, Campylobacter pylori, with the human stomach. Recent reports of the isolation of other bacteria with the characteristic periplasmic surface structures suggests that the group may be more widespread than was hitherto thought. Bacteria with the morphology of the organisms seen in the cat stomach have been seen in gastric biopsies from humans. The organism whose isolation is reported here has been used in previous serological studies to support the hypothesis that spiral bacteria from animals can colonize the human stomach.

Animals↗
PubMed1988

Differences in the gastrointestinal microbiota of specific pathogen free mice: an often unknown variable in biomedical research.

Large differences were found in the numbers of facultatively anaerobic Gram-negative bacteria in the gastrointestinal tract of mice from 3 major specific pathogen free (SPF) units in Australia. The species isolated also differed between mouse colonies. In one unit the presence of Enterobacter cloacae was found to dramatically influence the survival of mice following total body irradiation. This finding conforms with previous studies which have shown the influence of variation in gastrointestinal microbiota on the immune system and on susceptibility to infection. Given that the presence or absence of Enterobacteriaceae in the intestines of mice under investigation may influence experimental results, researchers using SPF rodents are encouraged to determine the baseline loading of these bacteria in their animals. Where results of immunological or irradiation studies from different colonies are likely to be compared, the enterobacterial status of the colony being used should be reported.

Animals↗
PubMed1988

Morphology of the fibrinogen exudate during evolution of a mycobacterial-induced murine eye granuloma.

The intent of this study was to visualize changes in the density and location of fibrinogen-related antigen (FRA) depositions within the murine vitreous space during the formation of a chronic mycobacterial-induced uveitis (CMIU) granuloma. Concurrent changes in cellular morphology of the granuloma were also examined. Fibrinogen derivatives within the exudates of granulomatous cell-mediated inflammations may induce physical induration and numerous other phlogistic effects. However, technical limitations of conventional FRA staining methods have tended to underestimate the extent of their presence within this category of inflammatory lesions. Conventional H and E sections of the CMIU granuloma confirmed the classic progression-early PMN influx, monocyte maturation and final macrophage and epithelioid cell dominance-described for such lesions. Avidin-biotin-complex staining utilizing a polyclonal mouse antifibrinogen then revealed a progressive increase in amorphous extracellular fibrinogen-FRA-positive staining material as the granuloma evolved. Thus, on day one the PMN influx showed no evidence of fibrinogen-FRA staining; at one week heavy staining was evident in the anterior chamber and in consolidated (i.e. macrophage) regions of the granuloma; at one month a heavy uniform staining appeared throughout the indurated granuloma where macrophages and epithelioid cells predominated. Patterns of heavy deposition on macrophage surfaces were suggested. The likelihood that bulky accumulations of FRA in mature granulomas are not solely fibrin, and may account for granulomatous induration and persistence, is discussed.

Animals↗
PubMed1987

Inflammatory mediators in alkali-burned corneas: preliminary characterization.

Leukocyte chemotactic factors (LCF) are important inflammatory mediators which activate and recruit leukocytes from the circulation into sites of tissue damage. These factors were recently detected in the tear fluid of inflamed eyes induced by alkali burn. It remains unclear, however, whether the detected LCF are released from injured corneal tissues or leaked from the circulation. Using a corneal cup model developed in our laboratory, we began examining the capability of corneal tissues to produce LCF in response to alkali injury. We also evaluated the influence of citric acid on the production of LCF from corneas preinjured by the alkali sodium hydroxide (NaOH). For these studies, the epithelial surfaces of corneas isolated from bovine and human eyes were exposed to 1N NaOH for 35 seconds at room temperature. The NaOH was then removed and the epithelial surfaces washed once with buffer and incubated with culture medium for 1, 2, 4, and 6 hours at 37 degrees C/5% CO2 atmosphere. Our results showed that (1) NaOH2 induced corneal epithelial cell injury ranging from cell discoloration and moderate damage of the upper half of the epithelium (1-4 hrs) to total destruction of the epithelium (6 hrs); (2) NaOH-injured corneas (2 hr incubation post injury) produced significant levels of chemotactic activity (via checkerboard analysis) specific for neutrophils (115% maximum chemotactic response [MCR]) and mononuclear cells (94% MCR); (3) preliminary characterization of these factors revealed that they are protease and heat sensitive, extractable by organic solvents, and possess molecular weight values greater than 100,000 daltons; and (4) incubation of NaOH-pretreated corneas with 0.01% citric acid for 2 hours markedly inhibited the production of LCF for both neutrophils (98% inhibition) and mononuclear cells (91% inhibition). Results of these studies indicate that alkali-burned bovine and human corneas generate leukocyte chemoattractants which differ in their biochemical characteristics from previously known low molecular weight chemotactic factors such as C5a, interleukin-1, or leukotriene B4.

Animals↗
PubMed1987

Extravascular plasminogen activator and inhibitor activities detected at the site of a chronic mycobacterial-induced inflammation.

Levels of extravascular tissue plasminogen activator activity (PA) and those of inhibitors of PA and of urokinase (UK) present within the anterior chamber of normal and inflamed feline eyes were assessed with the use of a direct PA assay of microsamples of aqueous humor. Purposes of the study were, first, to confirm prior indirect evidence that this extravascular space normally contains higher levels of uninhibited PA, but lower levels of inhibitor activity, than does plasma and, second, to determine patterns of change in these activities under in vivo conditions imposed by a chronic mycobacterial-induced uveitis (CMIU) disease model. The PA assay utilized a 125I-plasminogen substrate whose cleavage by PA contained in samples was both visualized during gel electrophoreis, and quantified by gamma counting. The results provided the first direct evidence that the higher fibrinolytic activity previously observed in normal aqueous in comparison with plasma is in fact associated with higher levels of available (uninhibited) PA (P less than 0.01) The data also indicated that normal aqueous contains a much higher level of PA inhibitor activity than previously suspected--roughly 40 times more than available PA levels. These normal values for PA and inhibitors occupied a relatively narrow, threefold range, in contrast to the wide scattering of individual values that appeared during 18-20 weeks of the chronic inflammation disease model. Despite this, however, the general pattern of observation for all individual eyes during CMIU was a significant increase in levels of both PA and inhibitors. The net effect of CMIU was thus to cause the 1:40 ratio noted above to be tilted more strongly in favor of inhibitor activity, ie, up to 1:80. Increases in local vasopermeability in this disease model were believed contributory to this change. However, local generations of PA and APA in vivo by inflammatory cells, especially monocyte-macrophages, must also be considered. Assays for UK inhibitor showed levels of activity and directions of change that closely followed those of PA inhibitor, which suggests the possibility that they may be identical. It is surmised that the above patterns, along with results of our prior studies, indicate an apparent need for a multistep, strict inhibitory control of plasmin generation and proteolysis in vivo within normal extravascular spaces such as the anterior chamber.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Characterization of neutrophil and monocyte specific chemotactic factors derived from the cornea in response to hydrogen peroxide injury.

To unravel the contributions of corneal tissue in endocular inflammation, we examined the capability of corneal endothelial cells (CECs) to release leukocyte chemotactic factors (LCFs) following injury induced by the leukocyte product hydrogen peroxide. Hydrogen peroxide (H2O2) was chemically generated by the interactions of glucose and glucose oxidase prepared in serum-free minimal essential medium (MEM). For these studies, endothelial surfaces of isolated bovine corneas were incubated with glucose (1 mg/ml) and glucose oxidase (20 U/ml) for 4, 6, and 10 hours at 37 C/in a 5% CO2 atmosphere. Supernatants were then removed and assayed for bovine neutrophil or mononuclear cell chemotactic activity. C5 fragment was our positive control for 100% chemotactic response. Corneas were also fixed in buffered formalin for histopathologic evaluation. Results of these studies indicated that 1) 6-hour interactions of the glucose (G)/glucose oxidase (GO) mixture with endothelial surfaces resulted in both endothelial cell injury (cytoplasmic vacuolization and convoluted nuclei) and production of chemotactic factors (via checkerboard analysis) specific for both neutrophils (58% maximum chemotactic response [MCR]) and mononuclear cells (75% MCR); 2) control corneas treated with either G or GO for 4 and 6 hours produced low levels of LCFs (5-15% MCR); 3) preliminary molecular weight characterization of cornea-derived LCFs obtained from corneas incubated with G/GO for 6 hours revealed the detection of chemotactic activity specific for mononuclear cells in two major fractions, one near the void volume (greater than 130,000 daltons) and one near the elution volume (less than or equal to 10,000-15,000 daltons). Chemotactic activity specific for neutrophils was detected only in one major fraction near the elution volume (less than or equal to 10,000-15,000 daltons); and 4) the production of these LCFs by isolated corneas was significantly inhibited, in a dose-response fashion, when the enzyme catalase (1200-6000 U/ml) was added to corneas incubated with G/GO for 6 hours. To investigate whether isolated CECs were capable of producing LCFs in response to G/GO injury, the authors incubated cultured bovine CECs with G/GO for 3, 6, and 20 hours at 37 C in a 5% CO2 atmosphere. Similar to isolated corneas, cultured CECs incubated with G/GO for 6 hours produced significant levels of LCFs specific for neutrophils (67% MCR) and mononuclear cells (75% MCR). Furthermore, the addition of catalase (3000 U/ml) to corneas incubated with G/GO for 6 hours markedly reduced the production of LCFs. These in vitro studies suggest that corneal cells and/or corneal matrix may play important roles in the initiation and amplification of endocular inflammation in vivo by elaborating factors that control leukocyte influx to the anterior chamber region.

Animals↗
PubMed1985

An in vitro model of leukocyte mediated injury to the corneal epithelium.

To enhance efforts directed at unraveling the role and mechanisms of leukocytes in mediating injury to corneal epithelium, an isolated bovine corneal cup was developed and evaluated. Bovine peripheral leukocytes and lysates were added to the corneal epithelial surface of isolated cornea for various periods after which the degree of morphologic changes and cell damage were assessed using light and electron microscopy. Results of these studies indicate that leukocyte/epithelial cell interactions are characterized by five successive stages: (1) leukocyte adhesion to superficial layer of the epithelium, (2) leukocyte penetration beneath the superficial epithelium, (3) epithelial cell injury, (4) leukocyte phagocytosis of killed epithelial cells and (5) ulceration and total destruction of the full thickness of the epithelial layer. The above sequence appears to be both time and dose dependent; that is epithelial cells exposed to leukocytes for short periods (5-60 minutes) or to low dose levels (10(5) - 10(7) cells/ml) shows leukocyte adhesion and penetration beneath the superficial layer of the epithelium, (stage 1 and 2), while longer exposures (2-3 hours) or higher numbers of leukocytes (10(7) - 10(8) cells/ml), leads to deeper penetration of epithelium by leukocytes and epithelial injury (Stages 3,4,and 5). We also observed that direct contact of intact leukocytes with epithelial cells is apparently necessary to induce this type of injury. These findings demonstrate the ability of leukocytes to destroy corneal epithelial cells and the value of this new ocular model for studies of the basic immunology of ocular inflammation.

Animals↗
PubMed1985

Fibrin: mediator of in vivo and in vitro injury and inflammation.

We examined the role that fibrin deposition and fibrin-associated factors (FAF) play in acute anterior segment inflammatory responses in the rabbit eye. It was demonstrated by immunofluorescence that fibrin represented a major component of the exudative meshwork deposited within the anterior chamber and on leukocyte surfaces therein. Using our in vivo model of endocular inflammation we next demonstrated that fibrin and fibrinogen-derived peptides, but not thrombin, induced inflammatory responses characterized by both leukocyte influx and endothelial cell injury. Fibrin formation within the anterior chamber induced a leukocyte influx consisting primarily of PMN's. Fibrinogen-derived peptides induced primarily a monocyte influx. This dichotomy suggests that multiple inflammatory mediators are elaborated or released during endocular fibrinogenesis and fibrinolysis. To investigate direct effects of fibrin deposition on the corneal endothelial cells (CEC) an in vitro "corneal cup" organ culture model was next developed. Studies comparing various types of mediators demonstrated that only fibrin- derived preparations directly induced CEC injury. Fibrin deposition may thus play multiple roles in endocular inflammation, including the modulation of leukocyte influx, and the direct mediation of corneal endothelial cell injury.

Animals↗
PubMed1985

Regulation of anterior chamber fibrinolysis.

Levels of plasminogen activator (PA), plasminogen (Plg), and antiplasmin activity (APli) were compared in feline aqueous humor obtained from normal eyes, eyes inflamed by chronic mycobacterial-induced uveitis (CMIU), opposite eyes, and in plasma. Fibrin-agar plate microassays were utilized to visually confirm the extent of differences in in vitro fibrinolysis, per se. Chromogenic peptide (S2251) microassays were utilized to quantify differences. Normal AH showed much more available PA than did plasma. However, when APli activity was first neutralized their levels of total PA were comparable. Available PA activity in both normal and CMIU AH was considerably amplified in the presence of trace amounts of free plasmin. Plasma failed to show this response. AH levels of circulating Plg and APli in normal eyes were far below plasma levels. During CMIU, total PA levels remained approximately normal while levels of Plg and APli were greatly increased. The net effect of these concurrent rises was that antiplasmin activity (APli) prevailed over free plasmin formation; i.e., in vitro fibrinolysis was suppressed. Plasma levels of PA, Plg and APli did not change during CMIU. Changes in the normal PA+Plg/APli balance induced by CMIU suggested a hypothetical model of AH fibrinolysis wherein exclusion of APli from the normal anterior chamber and the high levels attained during CMUI are posited as key determinants of AH fibrinolytic capability.

Animals↗
PubMed1984

Leukocyte-mediated injury to corneal endothelial cells. A model of tissue injury.

Although leukocyte-mediated cell injury has long been suggested as a major mechanism of general tissue injury in acute and chronic inflammation, only limited data have been developed to directly implicate leukocytes in ocular tissue injury. In an effort to unravel the role and mechanisms of leukocyte-mediated injury to the cornea, an isolated corneal cup model was developed. For this model, peripheral bovine leukocytes were added to isolated bovine corneal cultures (corneal cup) in vitro, after which endothelial cell damage and death were evaluated morphologically. In general, corneal endothelial cell swelling was observed within 5-10 minutes after leukocyte exposure, followed by detachment of the corneal endothelium at a 30-75-minute interval. Grossly, the detached endothelium appeared as a floating sheet. Using trypan blue dye exclusion, cells in the sheet were found to be nonviable. When examined ultrastructurally, the sheet was found to consist of leukocytes and endothelial cell debris. Light-microscopic examination of the corneal cup at 5-10 minutes after leukocyte action demonstrated extensive endothelial cell damage, as indicated by cell membrane thickening, cytoplasmic vacuolization, and changes in nuclear shape. These changes in the endothelial cells were confirmed by electron microscopy. Vacuolization and swelling of the endothelium appeared as the first changes, induced by leukocyte interactions with the endothelial cell layer. In general, the endothelial cell nucleus became convoluted, mitochondria swelled, and the endoplasmic reticulum became dilated. These changes become more pronounced as the endothelial cells become detached from the underlying Descemet's membrane. Leukocyte interaction with corneal epithelium, on the other hand, did not result in cell detachment, but only cell damage. These studies demonstrate the ability of leukocytes to denude Descemet's membrane of endothelial cells and destroy corneal endothelial cells in vitro. They also suggest that the corneal cup model system will enhance both morphologic and biochemical evaluation of the mechanisms and consequences of leukocyte-mediated injury to the cornea, and thereby provide new insights into the mechanisms of endocular inflammation and tissue injury in vivo.

Animals↗