Biomedical subjects

James O'Rourke

Publications and source records attributed to James O'Rourke.

13 recordsLinked to original sources

PubMed2006

The suppression of delayed-type hypersensitivity by CD8+ regulatory T cells requires interferon-gamma.

CD8(+) regulatory (suppressor) T cells are induced by complex cellular pathways in the spleens of mice that have received an injection of antigen into the anterior chamber (AC) of an eye, an immune-privileged site. Although these CD8(+) regulatory T cells perform an antigen-specific regulatory function for an immune response to self and non-self antigens, the mechanisms of the activation or function of these regulatory cells are not clear. Here, we describe a novel mechanism for the activation of splenic CD8(+) regulatory T cells induced by injection of antigen into the AC. Immunization of mice with trinitrophenyl and bovine serum albumin (TNP-BSA) amplified AC-induced splenic CD8(+) regulatory T cells that suppressed the initiation of contact sensitivity when transferred to immunized, challenged mice. These CD8(+) regulatory T cells were produced independently of perforin, indicating that they are not canonical cytotoxic T cells. Fas ligand (FasL)-deficient CD8(+) regulatory T-cell function was rescued by inclusion of exogenous interferon-gamma (IFN-gamma), demonstrating that the expression of FasL by CD8(+) regulatory T cells was dispensable, but IFN-gamma was not. Ultimately, we demonstrated that the generation of these CD8(+) regulatory T cells occurred independently of IFN-gamma, but their suppressor function required IFN-gamma receptor stimulation.

Animals↗

PubMed2006

New transgenic evidence for a system of sympathetic axons able to express tissue plasminogen activator (t-PA) within arterial/arteriolar walls.

Sympathetic axons embedded in a few arterioles and vasa vasora were recently shown to store tissue plasminogen activator (t-PA) in vesicles. But the extension of such t-PA axons to arteries and arterioles throughout the organism has not been verified. Confirmation of this anatomy would identify a second significant source of vessel wall t-PA. To visualize fine embedded axons independent of endothelium, we created a transgenic mouse whose expressions of the t-PA promoter and enhanced green fluorescent protein are confined to sympathetic neurons and other neural crest derivatives. Confocal images reveal the extension of t-PA axons to arterioles serving heart, brain, kidney, lung, mesentery, and skin; plus aortic, carotid, and mesenteric artery walls. Ganglion neurons and adrenal chromaffin cells also show strong expressions. These new sightings confirm the existence of a system of t-PA axons that is prominent in arterioles, and compatible with the release of neural t-PA into their walls.

Animals↗

PubMed2006

Preparation of the Dräger Primus anesthetic machine for malignant hyperthermia-susceptible patients.

PURPOSE: Preparation of anesthesia machines for patients who are susceptible to malignant hyperthermia includes flushing the machine with vapour-free fresh gas to washout residual anesthetic agents. To establish guidelines for the preparation of the Dräger Primus machine, we compared the washout profiles for isoflurane and sevoflurane in the Dräger Primus and Ohmeda Excel 210 anesthesia machines. TECHNICAL FEATURES: The machines were primed with 1.5% isoflurane or 2.5% sevoflurane. Fresh gas flow (FGF) was set at 10 L.min(-1) during the early washout phase, and subsequently reduced to 3 L.min(-1) during the late washout phase. A Miran ambient air analyzer measured the anesthetic concentration every minute during washout until a concentration of 5 ppm was achieved in the inspiratory limb of the circle circuit. We found that at a FGF of 10 L.min(-1), maximum washout times for isoflurane and sevoflurane in the Primus, 70 and 74 min, respectively, were approximately tenfold greater than for isoflurane in the Excel 210 (7.0 min). Increasing the FGF to 18 L.min(-1) decreased the washout time for isoflurane in the Primus, only moderately, to 52 min. We observed a threefold increase in anesthetic concentration in the Primus during the late washout phase. CONCLUSION: We conclude that the Primus must be flushed for at least 70 min to decrease the anesthetic concentration to 5 ppm when using a FGF of 10 L.min(-1). We recommend maintaining a FGF of 10 L.min(-1) for the duration of anesthesia in order to prevent the rebound increase in anesthetic concentration in the FGF.

Anesthesiology↗

PubMed2006

Phenotypic and immunoregulatory characteristics of monocytic iris cells.

The introduction of antigen into the anterior chamber of an eye induces the antigen-specific suppression of cell-mediated immunity and the antigen-induced production of immunoglobulin G2 antibodies. To define further the role of iris monocytic cells in the systemic suppression of cell-mediated immunity that follows the entry of foreign antigen into the anterior chamber, murine iris wholemounts or cell suspensions of iris cells were stained with fluorescent anti-F4/80 and/or anti-CD11c, anti-CD11b antibodies and examined by confocal microscopy or flow cytometry, respectively. Monocytic cells in iris cell suspensions were recovered from mice receiving an injection of trinitrophenylated bovine serum albumin (TNP-BSA) into an anterior chamber and Percoll-enriched iris cells separated into cells expressing F4/80 or CD11c were injected intravenously into TNP-BSA-immunized or naive recipients. The recipients were challenged to induce delayed-type hypersensitivity (DTH) or were provided with splenocytes or thymocytes that transfer the suppression of DTH. The homing of monocytic bone marrow cells to the iris was determined by the intravenous injection of bone marrow cells from green fluorescent protein (GFP)-transgenic donors into C57 mice, and the staining of recipient iris wholemounts with anti-F4/80 antibodies. Iris cells with a dendritic morphology expressing both F4/80 and/or CD11c and CD11b, some cells expressing only F4/80 or CD11c, were detected. The irides of irradiated GFP- mice that received intravenous GFP+ bone marrow cells contained GFP+ F4/80+ cells. F4/80+ and CD11c+ cells from the irides of donors that received intracameral TNP-BSA transferred the suppression of DTH when injected intravenously into TNP-BSA-immunized recipients, activated immunoregulatory thymocytes and activated antigen-specific splenic regulatory effector cells. These results support the hypothesis that iris monocytic cells may participate in the systemic induction of regulatory T cells.

Animals↗

PubMed2005

Distribution of sympathetic tissue plasminogen activator (tPA) to a distant microvasculature.

Tissue plasminogen activator (tPA) is the predominant plasminogen activator present in the vascular and nervous systems. Prior studies of the two have emphasized different tPA sources; respectively, endothelium and neurons. A closer relationship is now suggested by evidence that the peripheral sympathetic nervous system synthesizes and infuses enzymatically active tPA into small artery walls and the microcirculation. TPA may thus be the only known neural product able to effect degradation of the artery wall extracellular matrix. This brief review considers historical and current indications for the existence of such an autonomically controlled system and some physiologic implications. Immunohistochemical tPA expression in small arteries and arterioles is more prominent in the outer wall sympathetic axon plexus than in endothelium. Its presence in nerve filaments beneath the seldom-studied adventitia was obscured in earlier localizations. The systemic impact of a neural distribution is suggested by a 60% reduction of blood tPA activity after chemical sympathectomy. TPA-bearing axons extend outward from ganglion neuron cell bodies to reach even thin-walled vasa vasora and uveal microvessels. Ganglion cell bodies synthesize and package tPA in vesicles for the long axoplasmic transport. Densely innervated intact vessels release much greater amounts of tPA in vitro than do larger vessels, indicating a high neuron tPA production capacity and a large storage reservoir available within axon networks. The influence of an autonomically controlled plasmin production within small artery walls on regulation of blood pressure and capillary perfusion awaits further investigation. Its possible role in the pathogenesis of vessel wall matrix degradations in aging, hypertension, and diabetes may also merit further consideration.

Animals↗

PubMed2005

Stimulated release of tissue plasminogen activator from artery wall sympathetic nerves: implications for stress-associated wall damage.

Recurrent stress is clinically associated with early onset hypertension and coronary artery disease. A mechanism linking emotion to pathogenic remodeling of the artery wall has not been identified. Stress stimulates acute regulated release of tissue plasminogen activator (t-PA) into the circulation, which is presently attributed to the vascular endothelium. Sympathetic neurons also synthesize t-PA and axonally transport it to the arterial smooth muscle. Unlike release by the endothelium, a stress-stimulated sympathetic discharge would potentially accelerate degradation of the wall matrix by plasmin. To assess whether sympathetic axons are the principal source of acute stress-induced arterial release of t-PA, we compared the output from small densely innervated and large sparsely innervated isolated artery segments before and after sympathetic stimulation, and after ablations. Following phenylephrine infusion densely-innervated microvessels in uveal eyecups were released over 60-fold greater amounts of active t-PA per milligram than the sparsely innervated aorta; and ten-fold more than carotid artery segments. Mesenteric artery release was 4.8-fold greater than release by the carotid artery. In vivo, uveal release of t-PA increased more than three-fold within one minute following superior cervical sympathetic ganglion electrical stimulation, and after phenylephrine, or nicotine infusions of the anterior chamber. Circulating levels of t-PA fell 70% following chemical sympathectomy. We propose that sympathetic nerves are the primary source of stress-induced release of t-PA into and from the densely innervated resistance arteries and arterioles, where dysregulated plasmin-induced proteolysis could damage the wall matrix.

Animals↗

PubMed2004

The induction of splenic suppressor T cells through an immune-privileged site requires an intact sympathetic nervous system.

Antigen injection into the eye's anterior chamber (AC) induces the antigen-specific suppression of delayed-type hypersensitivity (DTH) that is mediated by NKT cells and splenic CD8+ suppressor T cells. Because the AC, uveal tissues, the thymus and spleen required to induce anterior chamber-associated immune deviation (ACAID) have dense sympathetic innervations, we examined the effects of chemical sympathectomy of mice by 6-hydroxydopamine (6-OHDA) on the induction of the suppression of contact sensitivity to trinitrophenol (TNP) induced by the injection of TNP-bovine serum albumin (BSA) into the anterior chamber. DTH measured as contact sensitivity to picrylchloride was not induced in mice that received 6-OHDA before immunization with TNP-BSA. Although spleen cells from 6-OHDA-treated TNP-BSA-immunized mice produced IFN-gamma when stimulated by TNP-BSA, the number of DTH-initiating hepatic NKT cells was reduced markedly in 6-OHDA-treated mice. Chemically denervated mice did not produce splenic suppressor T cells or thymic NKT cells that activate splenic suppressor T cells. We suggest that an intact sympathetic nervous system (SNS) is required to maintain cellular immunoregulation.

Animals↗

PubMed2004

Thymocytes induced by antigen injection into the anterior chamber activate splenic CD8+ suppressor cells and enhance the antigen-induced production of immunoglobulin G1 antibodies.

Injection of antigen into the ocular anterior chamber (AC) of a mouse eye (an immunologically privileged site) induces the activation of immunoregulatory NK1.1+, CD4- CD8-, T-cell receptor (TCR) alphabeta+ thymocytes. These thymocytes transfer the suppression of delayed-type hypersensitivity (DTH) when injected into mice sensitized to the same antigen but do not effect the suppression of DTH. On the other hand, the immunized recipients of these transferred thymocytes produce splenic CD8+ T cells that effect the suppression of DTH. However, it is unclear whether the thymocytes transferred from the AC-injected donor differentiate into and/or activate CD8+ T-splenic suppressor cells. We therefore sought to determine the origin of splenic suppressor cells produced in the recipients of immunoregulatory thymocytes transferred from donors that receive an injection of antigen into the AC. CD45.1+ thymocytes from mice that received an AC injection of 2,4,6-trinitrobenzene sulphonic acid (TNP)-bovine serum albumin (BSA) were transferred to congenic CD45.2+ TNP-BSA-immune recipients. Spleen cells from the recipients were then sorted based on anti-CD45.1 or -CD45.2 antibody binding and assayed for suppressor cells. This was done by the injection of separated spleen cells into the footpad of TNP-BSA-immunized mice, concurrent with the induction of footpad swelling (contact sensitivity) of the footpad elicited by an epicutaneous application of picryl chloride. The systemic distribution of antigen after the injection of antigen into the AC was demonstrated by the injection of fluorescein or 125I-labelled TNP-BSA into the AC. The results demonstrate that (i) splenic CD8+ T-suppressor cells produced in the immunized recipients of immunoregulatory thymocytes are derived from the CD45.2 recipient of the CD45.1+ thymocytes; (ii) the induction of recipient splenic suppressor T cells by the transferred immunoregulatory thymocytes requires that the recipient be immunized to the same antigen as that used to induce immunoregulatory thymocytes; (iii) antigen is introduced to the thymus after an injection of antigen into the AC; (iv) although the transfer of the suppression of DTH by regulatory thymocytes was not dependent on interleukin-4 (IL-4), CD4+ NK1.1- regulatory thymocytes from AC-injected donors enhanced the production of immunoglobulin G1 antibodies to TNP-BSA by an IL-4-dependent mechanism. These observations suggest that the adult thymus plays an active role in the induction and maintenance of anterior chamber-associated immune deviation as manifested by the generation of the suppression of cell-mediated immunity to exogenous antigen and the antigen-induced production of IgG1 antibodies.

Adoptive Transfer↗

PubMed2003

Enhanced tissue plasminogen activator synthesis by the sympathetic neurons that innervate aging vessels.

We investigated the source of the increased release of tissue plasminogen activator (t-PA) into the circulation that occurs during natural aging. Both the basal release and the acute stress-associated release induced by sympathetic stimulations are greater in older subjects. It is widely assumed that the source of these increases is vascular endothelium. However, the sympathetic neurons that densely innervate resistance vessel walls were recently shown to synthesize and transport active t-PA to axon terminals in vascular smooth muscle, suggesting an alternative source. These fine t-PA-bearing axons lie in the seldom-studied deep adventitia of vessel walls, where they are less visible than endothelium in tissue sections. Using Northern blot analysis, we observed that t-PAmRNA synthesis is increased 54% in the ganglion parent neuron cell bodies that innervate aged vessels. The t-PA release from isolated, aged ganglia in cultures was twofold greater than that from younger controls. In addition, aged whole-artery explants showed a 20% greater basal and a 50% greater acute release of stored t-PA in vitro. In vivo levels of active t-PA were 33% greater in the blood and 40% greater in the aqueous humor. These results are consistent with an increased infusion of the active t-PA protease from sympathetic axon terminals into the vessel wall extracellular matrix and the blood during natural aging, in addition to the basal endothelial release. We suggest that the cumulative impact of an accelerated plasmin production and matrix degradation within vessel walls, especially during repetitive stress, may play an unrecognized role in the pathogenesis of vascular aging. The possibility that increased sympathetic nervous system plasminogenesis influences the aging process in nonvascular tissues also deserves further investigation.

Adrenergic Fibers↗

PubMed2003

Splenic T cells from mice receiving intracameral antigen suppress in-vitro antigen-induced proliferation and interferon-gamma production by sensitized lymph node cells.

PURPOSE: To characterize the immunoregulatory mechanisms in vitro of spleen cells that are activated by intracameral injection of antigen (AC-spleen cells). METHODS: AC-spleen cell regulation of in-vitro antigen-induced proliferation and interferon-gamma production by lymph node cells from TNP-BSA-immunized mice was quantified by co-culture of the lymph node cells with TNP-BSA and AC-spleen cells induced by intracameral TNP-BSA. Cytokine production was quantified by ELISA. RESULTS: AC-spleen cells produced significantly more IL-4 and IL-10 than spleen cells from TNP-BSA-immunized mice or naive spleen cells; unlike spleen cells from immunized mice, AC-spleen cells did not produce IFN-gamma. AC-splenic CD4(+), CD8(+), CD4(-)/CD8( -) (DN) T cells differentially suppressed antigen-induced proliferation and IFN-gamma production by immunized lymph node cells by a mechanism dependent on IL-10 and antigen. Cultures of lymph node cells, antigen, and AC-splenic T cells contained increased amounts of IL-10 and/or TGFbeta2. CONCLUSIONS: The differential, cytokine-dependent immunoregulatory effects of CD4( +) and CD8(+) AC-spleen cells observed in vitro parallel their effects in vivo. We suggest that the suppression of antigen-induced lymphocyte proliferation and IFN-gamma production by AC-spleen cells provides a useful in-vitro assay of the immunoregulatory activity of cell populations that are induced by the injection of antigen into the anterior chamber.

Animals↗

PubMed2002

Storage and release of tissue plasminogen activator by sympathetic axons in resistance vessel walls.

We studied the immunolocalization of tissue plasminogen activator (t-PA) in rat precapillary arteries, arterioles, and terminal arterioles. Lack of information about the precise location of t-PA within small vessel walls has contributed to uncertainty about its cellular source. The presumed origin has been an endothelial phenotype largely restricted to certain small vessels. However, vessel wall sympathetic axons were recently also shown to store significant amounts of a neuron-generated t-PA in secretory vesicles. Using immunolocalizations we determined the extension of t-PA-bearing axons into the resistance vasculature. Light and confocal images revealed the persistence of t-PA-bearing sympathetic nerve filaments down to the level of 15-microm-diameter terminal arterioles in vasa vasora and the choroidal microvasculature. Immunoelectron localizations confirmed the confinement of t-PA within individual nerve filaments in the deep adventitia. A complete plasminogen activator system (t-PA, plasminogen, and plasmin) was localized in the arteriolar wall matrix. Isolated iris-choroid and mesenteric artery explants from sympathectomized animals released 65 and 43% less t-PA, respectively, than controls. These data support the hypothesis that resistance vessel sympathetic axons release neural t-PA into the wall matrix and the microvascular plasma.

Animals↗

PubMed2002

Tissue plasminogen activator is released into cultured medium by cultured human uveal melanocytes.

Melanoma cells produce tissue plasminogen activator (t-PA) that plays an important role in tumor invasion and metastasis. The production of t-PA by normal human uveal melanocytes has not been reported previously. In order to explore this possibility, we studied the production of t-PA by cultured human uveal melanocytes and compared that with the production by cultured human uveal melanoma cells and epidermal melanocytes. Human adult uveal melanocytes were isolated and cultured from donor eyes. The cells were cultured in serum-free medium for 48 h and the conditioned medium then collected for the plasminogen activator (PA) activity assay. Free PA activity was tested in an amidolytic assay using a t-PA standard curve. PA type was identified by fibrinography and antihuman t-PA and urokinase plasminogen activator (u-PA) blocking antibodies. Free PA activity was found in the conditioned medium of normal melanocytes and melanoma cells. The predominant PA activity was t-PA. Normal uveal melanocytes produced more t-PA (3.23 +/- 0.73 IU/105 cells/24 h) than that of epidermal melanocytes (1.25 IU/105 cells/24 h) but much less than uveal melanoma cells (11.0 +/- 3.39 IU/105 cells/24 h). Western blot analysis revealed that most t-PA in conditioned media were one-chain t-PA with molecular weight of 69 kDa. Our study indicates that uveal melanocytes may contribute to the free t-PA activity previously found in aqueous humor and choroidal eye cup superfusions. Therefore, this function of uveal melanocytes may play a role in intraocular matrix remodeling, fibrinolysis and aqueous humor outflow.

Blotting, Western↗

PubMedDate not supplied

Compact bulb pipette simplifies paracentesis.

Anterior chamber paracentesis is typically done using a tuberculin syringe, hypodermic needle, fixation forceps, and lid speculum. A compact suction pipette design simplifies the tap by eliminating unneeded instrumentation and minimizing needle dead space. The short (1/4-inch) 30-gauge needle tip eases limbal penetration. The soft plastic suction bulb provides a prompt and controlled aspiration of small volumes. Compared with syringe-needle methods, the pipette refines paracentesis by reducing the complexity and duration of aqueous withdrawal.

Animals↗