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J O'Rourke

Publications and source records attributed to J O'Rourke.

At least 37 records · Page 2Linked to original sources
PubMed1997

Direct thymic involvement in anterior chamber-associated immune deviation: evidence for a nondeletional mechanism of centrally induced tolerance to extrathymic antigens in adult mice.

Recent reports have suggested that the dichotomy between central (thymic) and peripheral T cell tolerance is not absolute and that self-tolerance in perinatal animals may also involve the intrathymic generation and release to the periphery of Ag-specific immunoregulatory T cells. We have expanded this concept to include tolerance to non self Ags administered extrathymically to adult animals. In this study, we use the anterior chamber-associated immune deviation (ACAID) to demonstrate that central regulation of acquired peripheral tolerance can be induced in adult mice by the intraocular administration of low doses of nonself Ag. The results show that adult thymectomy prevents the inhibition of trinitrophenol (TNP)-specific delayed-type hypersensitivity, which normally occurs after injection of TNP-BSA into the anterior chamber (AC) of the eye. Thymocytes obtained from mice 1 to 3 days, but not 5 to 7 days, after AC injection of TNP-BSA or BSA alone specifically transfer inhibition of delayed-type hypersensitivity to mice primed with the homologous Ag. The latter observation, when correlated with the time of onset of ACAID, suggests that immunoregulatory T cells are formed in the thymus within 24 h and are exported to the peripheral lymphoid tissues between 2 and 5 days after AC injection of Ag. Immunomagnetic separation of thymocytes revealed that the immunoregulatory activity resides within the minor subset of CD4-, CD8-, TCR-alphabeta+ cells, previously postulated to induce fas ligand-mediated apoptosis and Th1 to Th2 immune deviation. Hence, the present study identifies ACAID as a prototypical model of centrally induced, nondeletional tolerance to extrathymic nonself Ags.

Adoptive Transfer↗
PubMed1997

Morphologic evidence for a preferential storage of tissue plasminogen activator (t-PA) in perivascular axons of the rat uvea.

The uveal layer is thought to hold the largest stores of tissue plasminogen activator (t-PA) within the eye. However, the uveal cell types that contain and could release t-PA to contiguous tissues and fluids have not been clearly identified. In the present study the general distribution pattern of t-PA antigen in fresh rat iris and choroid tissue was determined by immunofluorescence in preliminary light microscopic (LM) cryosections. Transmission electron microscopic (TEM) immunogold localization was then used to detect specific cellular and subcellular sites of t-PA antigen. The primary antibody was rabbit anti-mouse t-PA IgG. The immunofluorescence in preliminary LM cryosections of both tissues was most intense over discrete linear and cross-sectioned structures that resembled the contours of axon bundles. This impression was strengthened when silver impregnation highlighted similar structures in separate sections of the same tissue samples. TEM immunogold labeling of thin sections then confirmed that the t-PA antigen was confined to the axoplasm of both myelinated and unmyelinated perivascular nerve fibers in both the iris and choroid. Gold particles were not observed over axonal membranes, myelin sheaths, Schwann cells, retinal pigment epithelium or vascular endothelial cells. Ultrathin TEM cryosections of the iris showed a localization of some particles over structures that resembled tubules and vesicles within the axoplasm, but not over mitochondria. The axonal location of t-PA was shown by the co-localization of t-PA with an antibody against rat neurofilaments. The typical axon morphology that enclosed the t-PA particle markers in all TEM sections also indicated an axonal location. Separate TEM sections were processed with conventional fixatives and stains to highlight the typical uveal axon morphology, which also confirmed the identity of perivascular axons as the sites of t-PA localization. Affinity of the primary antibody for rat t-PA was shown by an inhibition ELISA against rat uveal tissue extracts and by the inhibition of t-PA activity in aqueous humor. An amidolytic assay was used to quantify t-PA activity. Possible explanations for the preferential immunolocalization of t-PA antigen to the axoplasm of uveal nerve terminals and the need for additional functional studies to confirm a putative neural t-PA synthesis are discussed.

Animals↗
PubMed1997

A standardized mouse model of Helicobacter pylori infection: introducing the Sydney strain.

BACKGROUND & AIMS: Currently available Helicobacter pylori models show variable and, in some instances, poor colonization. There is a need for a strain with high colonizing ability to act as a standard for animal studies. METHODS: After screening a range of fresh clinical isolates and long-term adaptation in mice, a strain of H. pylon has been isolated with a very good colonizing ability. RESULTS: This strain, named the Sydney strain of H. pylori (strain SS1), is cagA and vacA positive. High levels of colonization (10(6)-10(7) colony-forming units/g tissue) were achieved consistently in C57BL/6 mice. Colonization levels varied depending on the mouse strain used with BALB/c, DBA/2, and C3H/He, all being colonized but in lower numbers. In all strains of mice, bacteria were clearly visible at the junctional zone between the antrum and the body. The phenotype was stable with colonizing ability remaining after 20 subcultures in vitro. The bacterium attached firmly to gastric epithelium. During 8 months, a chronic active gastritis slowly developed, progressing to severe atrophy in both C57BL/6 and BALB/c mice. CONCLUSIONS: The Sydney strain of H. pylori is available to all and will provide a standardized mouse model for vaccine development, compound screening, and studies in pathogenesis.

Animals↗
PubMed1997

Serum TABM produced during anterior chamber-associated immune deviation passively transfers suppression of delayed-type hypersensitivity to primed mice.

Injection of soluble protein antigen into the anterior chamber of the eye of primed mice induces anterior chamber-associated immune deviation (ACAID) which is manifested by suppression of delayed-type hypersensitivity (DTH) to the antigen. Recently, we found that ACAID induced in primed mice also results in a rapid rise in serum of soluble T lymphocyte-derived proteins specific for nominal antigen (TABM). Here, we demonstrate that serum TABM induced in primed mice during ACAID will transfer the suppression of DTH to mice primed to the same antigen. Sera from TNP-BSA-primed mice that received an anterior chamber injection of TNP-BSA, but not BSA alone, suppressed the DTH response to TNP when injected into other TNP-BSA-primed mice. Sera absorbed with Sepharose beads conjugated with either anti-TCR C(alpha), anti-TCR C(beta), anti-TABM or TNP-BSA did not contain TNP-specific TABM and did not transfer suppression of DTH. These results suggest that the antigen-specific, TCR C(alphabeta)+ TABM that appear in serum during ACAID are able to confer on or amplify the capacity of sensitized T cells to suppress DTH. We believe this to be the first demonstration of an in vivo immunologic function that is specifically associated with TABM produced in vivo.

Absorption↗
PubMed1997

The endotoxin of Helicobacter pylori is a modulator of host-dependent gastritis.

Atrophic gastritis caused by Helicobacter pylori is the precursor lesion in the development of intestinal-type gastric adenocarcinoma. In animal models, atrophic gastritis induced by Helicobacter felis has been shown to be host dependent, developing in some mouse strains and not in others. The lipopolysaccharide (LPS) of H. pylori has been suggested to play a role in the induction of gastritis. The goal of this study was to compare the inflammation induced by long-term infection of the C3H/He and the C3H/HeJ strains of mice with H. felis. C3H/HeJ mice are unresponsive to LPS. Six months after infection, severe atrophic gastritis had developed in the body mucosae of all infected C3H/He mice, with replacement of parietal and chief cells. Atrophy was associated with a loss of the H. felis from the antral mucosa. In contrast, no atrophy was seen in the infected C3H/HeJ non-LPS responder animals, and heavy colonization of the antrum remained. There were no significant differences between both the quantitative and qualitative serum immunoglobulin G (IgG) and salivary IgA levels in both strains of mice. The main difference between the two strains of long-term-infected mice was a lack of macrophage infiltration in the lamina propria. Immunization induced good protective immunity to challenge with viable H. felis. Helicobacter-induced, host-dependent gastritis is likely to be cell mediated. The C3H/He and C3H/HeJ mouse model provides an excellent opportunity to investigate the cellular basis of atrophic gastritis.

Animals↗
PubMed1997

Accessory bone or unusual congenital anomaly.

This case study presents a congenital anomaly, diagnosed in a 23-year-old male, not found previously described in the medical literature. It also reviews the current classification systems used to describe congenital anomalies.

Adult↗
PubMed1996

Atrophic gastric changes in both Helicobacter felis and Helicobacter pylori infected mice are host dependent and separate from antral gastritis.

BACKGROUND/AIMS: The role of host factors has been neglected in studies of the pathogenesis of Helicobacter associated disease. The aim of this study was to assess the response of different mouse strains to infection with a single strain of Helicobacter felis. METHOD: Six strains of inbred mice were infected with the identical H felis culture and were killed at one month, two months, and six months after infection to assess histopathological changes. In addition, two strains of mice were infected with a mouse adapted strain of H pylori and examined at six months after infection. RESULTS: In SJL, C3H/He, DBA/2, and C57BL/6 infected mice, severe to moderate chronic active gastritis was observed only in the body of the stomach, which increased in severity over time with specialised cells in the body glands being replaced. As the severity of this damage in the body increased and atrophic changes were seen, the level of bacterial colonisation of the antrum decreased. In contrast, in BALB/c and CBA mice, there was only mild gastritis in the antrum, no remarkable changes were detected in their body mucosa, and no atrophy was seen over time. In both these strains of mice, heavy bacterial colonisation was seen, which tended to increase over the period of the experiment. Of particular importance in this experiment was that bacterial colonisation was mainly restricted to the antrum yet the atrophy, when present, was only observed in the body of the stomach. H pylori infected C3H/He mice showed moderate colonisation of the antrum, which persisted up to six months with little development of atrophy. In contrast, H pylori in C57BL/6 mice showed excellent colonisation of the antrum at two months but six months after infection there was moderate to severe body atrophy, which was associated with a loss of bacteria from the antrum. CONCLUSIONS: These findings challenge current concepts of the development of Helicobacter induced atrophy in that active chronic gastritis of antrum or the body mucosa, or both, is not a prerequisite. They also suggest an autoimmune basis for the pathology although no autoantibody or antibody to the H+/K+ ATPase was detected. Loss of infecting helicobacters from the stomach together with development of an atrophic gastritis in the body of the stomach is similar to the pattern found in certain H pylori infected human subjects.

Animals↗
PubMed1995

High-dose cyclophosphamide inhibits anterior chamber-associated immune deviation (ACAID) and the production of extracellular antigen-specific T cell proteins induced by trinitrophenylated (TNP) spleen cells.

Injection of antigen into the anterior chamber (AC) of the eye induces the production of IgM and IgG1 antibodies and potentiates the appearance in serum of extracellular antigen-specific T cell proteins (TABM) that are specific for the AC-injected antigen. In contrast, delayed-type hypersensitivity (DTH) to the injected antigen is suppressed. This manifestation of anterior chamber-associated immune deviation (ACAID) is believed to be a key part of the basis of the immune privilege of the eye. Because cyclophosphamide (CY) exerts selective effects on immunoregulatory T cells and macrophages, we sought to determine its effects on the appearance in serum of TNP-specific TABM in mice following intracameral injection with TNP murine spleen cells followed by epicutaneous sensitization and challenge with picrylchloride. Injection of 200 mg/kg CY 2 days before AC injection of TNP spleen cells, sensitization, and challenge prevented the suppression of DTH and the production of TNP-specific TABM. The production of TNP-specific immunoglobulins was not affected. Injection of a low dose of 20 mg/kg CY enhanced DTH 100-300% in control animals, but did not prevent either ACAID or the production of TABM. These results provide further evidence that serum TABM may be a serologic indicator of T lymphocyte activity in ACAID and that ACAID is mediated by cells sensitive to high-dose CY.

Animals↗
PubMed1995

Extravascular secretion of t-PA by the intact superfused choroid.

PURPOSE: Demonstrate the continuous extravascular secretion of enzymatically active t-PA by the intact choroid. METHODS: Enucleated rat eyes were dissected under microscopic control to prepare eyecups for superfusion in a small organ culture system. Individual preparations provided access to intact retinal, retinal pigment epithelial, or choroidal surfaces in situ. Cups were superfused with oxygenated Kreb's buffer (100 microliters/minute) at 37 degrees C and pH 7.4 while suspended in a microincubator. Released t-PA activity was measured by an amidolytic assay in 1-minute overflow samples and in cup media after flow interruptions. RESULTS: Minimal basal secretion was detected in the overflow with the retina in place. Interruption of flow (10 minutes) produced a static accumulation of 0.10 +/- 0.01 IU/ml. With the retinal pigment epithelium in place after retinal removal, t-PA release was moderately accelerated. Removal of the retinal pigment epithelium to expose the Bruch's-choroidal surface produced the greatest acceleration. The 10-minute static level was 0.48 +/- 0.13 IU/ml. Release from the scleral shell after choroid removal was negligible. An acute burst release of t-PA activity (1.3 +/- 0.18 IU/ml) followed the infusion of bradykinin (5 to 20 microM) into choroidal cups. CONCLUSION: Choroid appears to be the dominant source of a continuously secreted t-PA in superfused rat eyecup organ cultures. This enzymatically active t-PA is able to pass through an apparently intact Bruch's membrane but is largely impeded by an intact retinal pigment epithelium. It is also possible that retinal pigment epithelium contains inhibitors that could reduce the released t-PA activity. The authors propose that this secretory function of the uveal tissue, which has thus far not been recognized, may be a principal mechanism for the regulation of intraocular fluid circulation and matrix remodeling by plasmin in vivo.

Animals↗
PubMed1995

Intracameral injection of antigen potentiates the production of antigen-specific T cell proteins in serum after the induction of delayed-type hypersensitivity.

PURPOSE: To determine whether the introduction of antigen into the anterior chamber induces the production of extracellular antigen-specific T cell proteins (T cell antigen-binding molecules [TABM] specific for the antigen. METHODS: Balb/c mice received an intracameral or subconjunctival injection of trinitrophenylated spleen cells (TNP spleen cells) before skin sensitization and challenge with picrychloride. The production of TNP-specific TABM in serum was quantified by enzyme-linked immunosorbent assay-based antigen binding and immunoblotting using a rabbit antiserum raised against a monoclonal antigen-specific T cell protein that induces suppressor T cells. RESULTS: Intracameral, but not subconjunctival, injection of TNP spleen cells before contact sensitization increased the level of TNP-specific TABM in serum. At 2 days, TABM levels began to rise and peaked at 5 days after contact sensitization. Anterior chamber injection of TNP spleen cells alone into nonimmunized mice did not induce a detectable increase of TNP-specific TABM in serum. Mice that did not receive an intracameral TNP spleen cell injection but were made contact sensitive to TNP showed a diminished TNP-specific TABM response. Specificity of the TABM induced by intracameral-injection and sensitization for the TNP hapten was documented by showing that the induced TABM binds TNP-bovine serum antigen (BSA) but does not bind either BSA alone or azobenzenearsonate-ovalbumin and that immune sera raised against ovalbumin did not contain an increase in TNP-specific TABM. TNP-specific TABM in the sera of mice receiving an intracameral injection of TNP spleen cells, followed by contact sensitization with picrylchloride (PCl), were purified by affinity for TNP and were resolved by polyacrylamide gel electrophoresis and immunoblotting as M(r) 110,000 polypeptides. CONCLUSIONS: Serum levels of TNP-specific TABM induced by contact sensitization alone are enhanced fourfold to fivefold when sensitization is preceded by the intracameral, but not the subconjunctival, injection of TNP spleen cells. The authors propose that besides suppression of systemic delayed type hypersensitivity, the intracameral injection of antigen into nonimmune mice may prime an increased production of TABM, which may indicate systemic activation of the immunoregulatory T cell circuit.

Animals↗
PubMed1994

Molecular analysis of urease genes from a newly identified uncultured species of Helicobacter.

"Gastrospirillum hominis" is an uncultured gastric spiral bacterium that has recently been shown by 16S rDNA sequence analysis to be a newly recognized species of Helicobacter that infects humans, and it has been provisionally designated "Helicobacter heilmannii." We used PCR to directly amplify the urease structural genes of "H. heilmannii" from infected gastric tissue. DNA sequence analysis identified two open reading frames, ureA and ureB, which code for polypeptides with predicted molecular weights of 25,729 and 61,831, respectively. The urease subunit genes from "H. heilmannii" were cloned and expressed in Escherichia coli. Western blot (immunoblot) analysis showed that antiserum directed against the ureA and ureB gene products from H. pylori was cross-reactive with the corresponding polypeptides from "H. heilmannii." Analysis of the derived amino acid sequences of "H. heilmannii" UreA and UreB demonstrated that "H. heilmannii" urease is more highly related to the urease from H. felis (found in the stomachs of cats and dogs) than to the urease from H. pylori. These data are consistent with 16S rDNA sequence analysis and suggest that "H. heilmannii" is phylogenetically most closely related to H. felis.

Amino Acid Sequence↗
PubMed1994

Increased basal levels of free plasminogen activator activity found in human aqueous humor.

PURPOSE: To quantify the basal free t-PA activity in human aqueous humor. METHODS: Human aqueous humor obtained by simplified pipette paracentesis at cataract surgery was tested for free t-PA activity in a revised 15-hour amidolytic assay using a t-PA standard curve (n = 15). Total antigenic levels of PAI-1, the principal PA inhibitor, were determined using an ELISA kit. The available PAI-1 activity was tested indirectly using anti-human PAI-1 antibody blocking before t-PA activity assay (n = 11). Plasminogen activator type was determined by anti-human t-PA and urokinase (u-PA) antibody blocking before activity assay (n = 7). RESULTS: Free PA activity ranged widely (0.072 to 0.47 IU/ml; mean, 0.20 +/- 0.10 IU/ml) and was almost completed inactivated (> or = 89%) by antibody against human t-PA but not by the u-PA antibody. PAI-1 total antigen also ranged widely between (0.25 to 8.0 ng/ml; mean, 2.25 +/- 2.54 ng/ml). However, pretreatment of samples with PAI-1 antibody or by acidification (pH 3.2) to inactivate inhibitors did not increase t-PA activity levels. CONCLUSIONS: A basal-free t-PA activity, which is predominantly t-PA, is present in human aqueous humor at approximately 20 times higher levels than previously found with an earlier assay. This activity is estimated to represent roughly 10% of total released t-PA antigen. PAI-1 is present in aqueous humor at levels considerably lower than reported values for plasma in a predominantly PA-complexed or inactive form.

Aged↗
PubMed1994

Tissue plasminogen activator activity in human aqueous humor.

PURPOSE: To determine the levels of free plasminogen activator activity in human aqueous humor and to identify the type of activity (i.e., tissue-type t-PA or urokinase-type u-PA) that is responsible. METHODS: Aqueous humor was obtained by a simplified pipette paracentesis before cataract surgery in 31 subjects, ages 57 to 93 years. Levels of plasminogen activator activity were determined using a modified 17-hour specific amidolytic assay. The type of plasminogen activator was investigated in selected samples based on its dependence on soluble fibrin, inhibition by amiloride, and specific antibody blocking. Activity-antigen ratios were compared in seven samples. RESULTS: Plasminogen activator (PA) activity was present in all samples tested. PA activity ranged widely between 0.54 and 26.7 mIU/ml, with a mean value of 10.8 +/- 8.1 mIU/ml. Soluble fibrin, a known stimulator of tissue-type plasminogen activator (t-PA), was required in the assay system. Its absence decreased the measured activity by more than 90%. Amiloride, a known inhibitor of urokinase-type PA, had little or no effect in selected samples tested. The activity was blocked by anti-human t-PA antibodies but not by antibodies against human u-PA, further defining the type of PA responsible for the detected activity. t-PA antigen levels showed less variation among individuals than did activity levels. Antigen-activity ratios ranged between 89 and 552. CONCLUSION: Plasminogen activator activity is present in the human aqueous humor in measurable quantities. The type of PA activity present is almost exclusively t-PA. t-PA activity varies more widely than antigen, as is the case in plasma.

Aged↗
PubMed1993

Adenosine diphosphate stimulates the endothelial release of tissue-type plasminogen activator but not von Willebrand factor from isolated-perfused rat hind limbs.

The acute release of tissue-type plasminogen activator (t-PA) activity and of von Willebrand Factor (vWF) antigen from vascular endothelium was studied ex vivo using the rat hindquarter isolated perfusion model. The release of these proteins has been reported to occur simultaneously in response to a variety of endothelial cell agonists including bradykinin, thrombin and platelet activating factor. It has therefore been suggested that similar endothelial cell pathways and mechanisms are involved in the release of t-PA and vWF in vivo. This paper shows that the releases of t-PA and of vWF are not always closely linked and may depend on the agonist utilized to effect release. In our hands, the bradykinin-induced release of these proteins appears to be essentially identical, but this is not true for adenosine diphosphate (ADP). Rather, ADP is capable of causing the acute release of t-PA without the simultaneous release of vWF in the ex vivo rat hindquarter model. This indicates that the bradykinin- and ADP-induced pathways for t-PA release are probably distinct and that the releases of t-PA and vWF are not as closely linked as previously believed.

Adenosine Diphosphate↗
PubMed1993

Long term infection of the gastric mucosa with Helicobacter species does induce atrophic gastritis in an animal model of Helicobacter pylori infection.

Gastric atrophy is a precursor lesion in the development of gastric cancer. It has been proposed that atrophy is part of a natural progression of inflammatory changes that result from long term infection with the bacterium Helicobacter pylori. The aim of this study was to test this hypothesis using an animal model of human Helicobacter infection. Conventional mice were infected with either a cat isolate of Helicobacter felis or a human isolate of "Gastrospirillum hominis". All infected mice showed a slowly progressive chronic gastritis with increasing numbers of infiltrating mononuclear cells and polymorphonuclear leucocytes. After a year and a half, the inflammatory reaction was so severe that atrophic changes were seen in both the antral and fundic mucosa. Control animals initially showed no inflammatory changes however as the animals aged, the gastric mucosa of some animals became infected with a bacterium Helicobacter muridarum that normally inhabits the small and large bowel of the rodent. The presence of this bacterium was also associated with gastritis and atrophic changes. This is the first report of experimentally induced atrophic changes induced by a gastric bacterium and opens the way for important experiments that will help better understand the induction of gastric cancer.

Animals↗
PubMed1993

An uncultured gastric spiral organism is a newly identified Helicobacter in humans.

"Gastrospirillum hominis" is an uncultivated spiral bacterium in human gastric mucosa that is larger and more tightly coiled than Helicobacter pylori. In an attempt to determine if this organism is a new species of Helicobacter, its 16S rRNA gene was cloned and sequenced. Gastric mucosa from 2 patients infected with "Gastrospirillum hominis" was fed to specific pathogen-free mice. Electron microscopy of gastric tissue confirmed that the mice became colonized with "Gastrospirillum hominis." The 16S rRNA gene from bacterial target sequences was amplified directly from mouse stomach tissue by the polymerase chain reaction (PCR), cloned into Escherichia coli, and sequenced. Both fragments were 16S rRNA genes from the Helicobacter genus that were most closely related to Helicobacter felis. "Gastrospirillum hominis" is probably a newly recognized Helicobacter infection in humans. Because this is the only Helicobacter organism that infects both humans and small animals, it may be particularly suited for studies of pathogenesis.

Adult↗