Biomedical subjects

P Alard

Publications and source records attributed to P Alard.

At least 19 recordsLinked to original sources

PubMed2001

Endogenous oocyte antigens are required for rapid induction and progression of autoimmune ovarian disease following day-3 thymectomy.

Female (C57BL/6xA/J)F(1) mice undergoing thymectomy on day 3 after birth (d3tx) developed autoimmune ovarian disease (AOD) and autoimmune disease of the lacrimal gland. As both were prevented by normal adult CD25(+) T cells, regulatory T cell depletion is responsible for d3tx diseases. AOD began as oophoritis at 3 wk. By 4 wk, AOD progressed to ovarian atrophy with autoantibody response against multiple oocyte Ag of early ontogeny. The requirement for immunogenic endogenous ovarian Ag was investigated in d3tx female mice, d3tx male mice, and d3tx neonatally ovariectomized (OX) females. At 8 wk, all mice had comparable lacrimalitis but only those with endogenous ovaries developed AOD in ovarian grafts. The duration of Ag exposure required to initiate AOD was evaluated in d3tx mice OX at 2, 3, or 4 wk and engrafted with an ovary at 4, 5, or 6 wk, respectively. The mice OX at 2 wk did not have oophoritis whereas approximately 80% of mice OX at 3 or 4 wk had maximal AOD, thus Ag stimulus for 2.5 wk following d3tx is sufficient. AOD progression requires additional endogenous Ag stimulation from the ovarian graft. In mice OX at 3 wk, ovaries engrafted at 5 wk had more severe oophoritis than ovaries engrafted at 6 or 12 wk; moreover, only mice engrafted at 5 wk developed ovarian atrophy and oocyte autoantibodies. Similar results were obtained in mice OX at 4 wk. Thus endogenous tissue Ag are critical in autoimmune disease induction and progression that occur spontaneously upon regulatory T cell depletion.

Animals↗

PubMed2001

Hapten-specific tolerance induced by acute, low-dose ultraviolet B radiation of skin requires mast cell degranulation.

The deleterious effects of ultraviolet B radiation (UVR) on cutaneous immunity are mediated in part by cytokines released from cutaneous cells following radiation exposure. On the one hand, TNF-alpha has been advocated as the primary mediator of failed contact hypersensitivity induction, and, on the other hand, IL-10 has been held responsible for tolerance. While keratinocytes exposed to UVR have been found to produce both TNF-alpha and IL-10, there is reason to question whether these major cellular constituents of the epidermis are the relevant source of immunomodulatory cytokines after UVR. Dermal mast cells also produce TNF-alpha and IL-10, and we have recently reported that mast cell-derived TNF-alpha is required for UVR-induced impairment of CH induction. In this study, we have examined whether mast cells are also a relevant source of IL-10 in UVR-dependent tolerance. We found that (a) UVR fails to induce tolerance in mast cell-deficient mice, and (b) that tolerance occurs if mast cells are triggered to degranulate after ligation of the IgE receptor. Both types of tolerance were neutralized with anti-IL-10 antibodies, are hapten specific, and are associated with regulatory lymphoid cells. We conclude that mast cells are required in UVR-induced tolerance and may be one of the major sources of IL-10 that mediates the tolerance induced by acute, low-dose UVR.

Adjuvants, Immunologic↗

PubMed2001

Evidence that ultraviolet B radiation transiently inhibits emigration of Langerhans cells from exposed epidermis, thwarting contact hypersensitivity induction.

Langerhans cells (LC) play a critical role in the induction of contact hypersensitivity (CH), and ultraviolet B radiation (UVR) impairs CH induction in UVB-susceptible (UVB-S) mice via a TNF-alpha-dependent mechanism. A possible explanation of this effect is that UVR impairs CH in UVB-S mice by immobilizing LC transiently in the epidermis and upper dermis, thereby preventing their timely migration to draining lymph nodes. To test this hypothesis we examined in vitro and in vivo the effects of in vivo UVR on migration of Ia(+) cells from skin of UVB-S and UVB-resistant (UVB-R) mice. Dorsal surfaces of ears of mice were irradiated with 400 J/cm(2) UVB and either explanted in vitro or transplanted orthotopically to the thoracic wall of syngeneic recipients. After 24, 48, and 72 h the epidermis was recovered from these explants/grafts and the number of Ia(+) cells determined by immunohistochemistry. Culture medium obtained after explants were removed was also evaluated for content of Ia(+) cells. The density of Ia(+)-bearing cells in the epidermis of cultured untreated skin explants and of grafted skin fell progressively for both UVB-S and UVB-R skin during the observation period. The rate of decline in Ia(+) cells density during this interval was greatly impaired if the skin was exposed to UVR prior to excision; this effect was seen equally in UVB-S and UVB-R skin. Recovery of Ia(+) cells in the medium after removal of cultured untreated skin explants was maximum after 24 h and comparable in UVB-S and UVB-R skin. However, the number of Ia(+) cells recovered in the medium from UVB-exposed skin was significantly reduced only if the skin donor was UVB-S. We conclude that the ability of UVR to impede LC migration from epidermis is significantly greater for UVB-S mice, accounting in part for the failure of these mice after UVR to acquire CH.

Animals↗

PubMed2001

Regulatory T-cell, endogenous antigen and neonatal environment in the prevention and induction of autoimmune disease.

Recent studies on autoimmune ovarian disease (AOD) induced by thymectomy on d3 (d3tx), and AOD induced by immunization with the ovary-specific zona pellucida 3 peptide (pZP3), have yielded the following results. First, female tolerance to pZP3 depends on the persistence of endogenous antigen (Ag). Second, following regulatory T-cell depletion, endogenous Ag in prepubertal d3tx mice triggers AOD and drives disease progression. Third, endogenous ZP3 from ovaries without AOD stimulates a diversified IgG autoantibody (autoAb) response that rapidly follows pZP3 T epitope immunization. Fourth, induction of AOD and autoimmune memory in neonatal female mice by pZP3 in incomplete Freund's adjuvant depends on endogenous Ag stimulation within the neonatal week. Fifth, in a rodent pinworm-positive environment, neonatal but not adult female mice injected with pZP3 in water develop Th2-mediated AOD and Th2 memory. Sixth, neonatal T cells transfer AOD to syngeneic athymic recipients, whereas adult T cells are non-pathogenic and in fact suppress AOD conferred by neonatal T cells. Therefore: 1) the continuous presence of physiologically-expressed autoAg is critical for both tolerance maintenance and autoimmune disease pathogenesis; the outcome is determined by the integrity of regulatory T cells; and 2) the neonatal mice, deficient in the regulatory T-cell function, are more responsive than adults to Ag and environmental stimuli that promote autoimmune disease and memory.

Animals↗

PubMed2000

Retargeting T cell-mediated inflammation: a new perspective on autoantibody action.

To understand the pathogenesis of organ-specific autoimmune disease requires an appreciation of how the T cell-mediated inflammation is targeted, and how the organ function is compromised. In this study, autoantibody was documented to influence both of these parameters by modulating the distribution of T cell-mediated inflammation. The murine autoimmune ovarian disease is induced by immunization with the ZP3330-342 peptide of the ovarian zona pellucida 3 glycoprotein, ZP3. Passively transferred or actively induced Ab to ZP3335-342 bound to the zona pellucida in the functional and degenerative ovarian follicles, and the ovaries remained histologically normal. Transfer of ZP3330-342 peptide-specific T cells targeted the degenerative follicles and spared the functional follicles, and the resultant interstitial oophoritis was associated with unimpaired ovarian function. Unexpectedly, the coexistence of ZP3330-342 peptide-specific T cells and zona-bound autoantibody led to a dramatic translocation of the ovarian inflammation to the growing and mature ovarian follicles, with destruction of the ovarian functional unit. Ab retargeted both Th1-induced mononuclear inflammation and Th2-induced eosinophilic inflammation, and retargeting was induced by murine and rat polyclonal Abs to multiple distinct native B cell determinants of the zona pellucida. Therefore, by reacting with the native determinants in tissue Ag, Ab alters the distribution of T cell-mediated inflammation, and results in destruction of the functional units of the target organ. We propose that this is a clinically important and previously unappreciated element of Ab action in autoimmune disease.

Amino Acid Sequence↗

PubMed1999

Neural influences on induction of contact hypersensitivity.

Contact hypersensitivity (CH)-induction begins when cutaneous antigen-presenting cells (APC) capture hapten that has been applied epicutaneously, and the process prepares hapten for presentation to T-cells. APCs are functionally plastic, are influenced by the microenvironment in which they reside, and their functional properties have a profound effect on the phenotype of the hapten-specific T-cells that they activate. Ultraviolet B radiation (UVR) distorts the cutaneous microenvironment, thereby altering local APC function, and changing the immune outcome from sensitization to unresponsiveness. Although UVR induces keratinocytes to produce TNF alpha and IL-10 (cytokines that have been implicated in failed CH-induction and tolerance, respectively, after UVR), dermal mast cells turn out to be the source of these immunomodulatory cytokines. Mast cell degranulation is triggered by CGRP released from UVR-exposed cutaneous nerve termini. Even in normal skin, cutaneous nerves influence the immune response to haptens. Substance P released from cutaneous nerves acts as an adjuvant, raising the immunogenicity of epicutaneously applied haptens. Thus, the nerves and the neuropeptides that these processes release contribute to the cutaneous microenvironment. By altering APC function, cutaneous nerves can dictate the quality and the quantity of immune responses to antigens of the skin.

Animals↗

PubMed1999

Anterior chamber-associated immune deviation-inducing cells activate T cells, and rescue them from antigen-induced apoptosis.

Immune responses to antigens injected into the anterior chamber of the eye are devoid of T helper 1 (Th1)-type responses of the delayed hypersensitivity type, which has been termed anterior chamber-associated immune deviation (ACAID). Recently, it has been found that peritoneal exudate cells (PEC) from normal mice can be made to acquire the capacity to induce ACAID in vivo when the cells are pulsed with antigen in vitro in the presence of transforming growth factor-beta2 (TGF-beta2), a major cytokine in the ocular microenvironment. We now report that when ovalbumin (OVA)-specific T cells from DO11.10 transgenic mice, or from OVA-primed normal mice, were activated in vitro by normal (untreated) PEC pulsed with OVA, the responding T cells were induced to undergo apoptosis. However, when PEC were first treated with TGF-beta2 and then used to stimulate DO11.10 T cells in the presence of OVA, T-cell proliferation occurred without evidence of increased apoptosis. The ability of TGF-beta2 to rescue responding T cells from apoptosis rested with the capacity of this cytokine to inhibit interleukin-12 (IL-12) production by PEC. Untreated PEC produced large amounts of IL-12 upon interaction with responding T cells. Under these conditions, tumour necrosis factor-alpha (TNF-alpha) production was up-regulated, and this cytokine, in turn, triggered apoptosis among T cells stimulated with OVA-pulsed PEC. From these results, we conclude that TGF-beta2-treated APC promote ACAID by rescuing antigen-activated T cells from apoptosis, and by conferring upon these cells the capacity to down-regulate delayed hypersensitivity.

Animals↗

PubMed1999

Local ultraviolet B irradiation impairs contact hypersensitivity induction by triggering release of tumor necrosis factor-alpha from mast cells. Involvement of mast cells and Langerhans cells in susceptibility to ultraviolet B.

Our laboratory has previously demonstrated that ultraviolet B radiation impairs contact hypersensitivity induction in ultraviolet B susceptible mice through a tumor necrosis factor-alpha-dependent mechanism, involving calcitonin gene related peptide and cutaneous mast cells. This study was designed to test directly whether mast cells are the source of tumor necrosis factor-alpha, to account for the ultra-violet B-susceptible phenotype. As dermal mast cells seem to release tumor necrosis factor-alpha following exposure to ultraviolet B, we investigated whether tumor necrosis factor-alpha released by mast cells could mediate impairment of contact hypersensitivity in a manner similar to that found with ultraviolet B radiation treatment. First, we loaded Fcepsilon receptors of mast cells of ultraviolet B-susceptible (C3H/HeN), ultraviolet B-resistant (C3H/HeJ), and mast-cell deficient (Sl/Sld) mice by intradermal injections of anti-dinitrophenyl immunoglobulin E antibodies. Twenty-four hours later, dinitrophenyl was injected intravenously, and within 30 min oxazolone was painted on injected skin sites. Contact hypersensitivity induction was impaired in ultraviolet B-susceptible mice, but not in ultraviolet B-resistant or Sl/Sld mice, and treatment with anti-tumor necrosis factor-alpha antibodies was able to reverse this impairment of contact hypersensitivity. Second, we have found that ultraviolet B radiation did not impair contact hypersensitivity induction when haptens were painted on irradiated skin of mast cell deficient mice. As ultraviolet B radiation impairs contact hypersensitivity induction through a tumor necrosis factor-alpha-dependent mechanism, we conclude that ultraviolet B radiation triggers the prompt release of tumor necrosis factor-alpha from dermal mast cells, and that mast cell-derived tumor necrosis factor-alpha interferes with generation of the hapten-specific signal required for contact hypersensitivity induction. In addition, we are providing data that indicate that tumor necrosis factor-alpha levels released from mast cells as well as sensitivity of Langerhans cells to tumor necrosis factor-alpha contribute in defining the phenotypes of resistance versus sensitivity to ultra-violet B radiation.

Animals↗

PubMed1999

A new concept of skin-associated lymphoid tissue (SALT): UVB light impaired cutaneous immunity reveals a prominent role for cutaneous nerves.

More than 20 years have passed since the concept that the skin has its own associated immune system was first proposed by Streilein. This proposal was advanced in part on evidence that cutaneous contact hypersensitivity (CH) reactions are closely correlated with Langerhans cells (LC). Recent reports have demonstrated that LC have neural connectivity with cutaneous nerve termini containing calcitonin gene-related peptide (CGRP), suggesting that a link exists between innervation and immune responses in the skin. Here we discuss the neural components which have recently been found to be participants in skin-associated lymphoid tissue (SALT). In part, discovery of a functional link between the nervous system and SALT is based on studies in which cutaneous immunity was impaired by ultraviolet-B radiation (UVR). The deleterious effects of UVR on cutaneous immunity include failed CH induction and promotion of hapten-specific tolerance, effects that are mediated by tumor necrosis factor-alpha and interleukin-10, respectively. The source of these cytokines after UVR appears to be dermal mast cells. Evidence indicates that mast cells are triggered to release these cytokines in response to CGRP, which is released from UVR-damaged cutaneous nerve endings. Moreover, a substance P agonist was able to reverse the deleterious effects of UVR on CH induction, rendering the mice able to develop intense CH. These observations indicate that two cell types not originally included in the SALT concept are critical to the functional integrity of cutaneous immunity: mast cells and cutaneous nerves. We propose that cutaneous nerves dictate whether antigen applied to or arising within skin will lead to sensitivity or tolerance.

Animals↗

PubMed1998

Alterations in cytokine production following intraocular injection of soluble protein antigen: impairment in IFN-gamma and induction of TGF-beta and IL-4 production.

Immune deviation induced by intraocular injection of soluble protein Ag, referred to as anterior chamber-associated immune deviation (ACAID), is characterized by impairment of delayed hypersensitivity (DH). Two populations of splenic regulatory cells that impair the induction and expression phases of DH are involved in the ACAID response and may mediate their effects through cytokines. The purpose of the present study was to evaluate the role that cytokines play in ACAID. IFN-gamma production in draining lymph nodes induced by conventional immunization with protein Ag and adjuvant was suppressed after intraocular injection of protein Ag administered either before or after sensitization; IL-12 production in these mice was not decreased, suggesting that suppression of IL-12 may not be the mechanism involved in the impairment in IFN-gamma production. Surprisingly, although significant amounts of IL-4 (but not IL-10) were produced by spleen and lymph node cells from several different strains of mice, experiments in IL-4 knockout mice showed that impairment of neither DH nor IFN-gamma production required IL-4. Interestingly, significant levels of TGF-beta were detected in cultures of spleen cells from mice with ACAID. As determined by quantitative RT-PCR, TGF-beta was produced primarily by the splenic CD4 and non-T cells and was of the TGF-beta1 type. These results suggest that the Th1 response is impaired in ACAID by a mechanism(s) that does not require Th2-type cytokines, but may involve TGF-beta at several different (including the effector) phases during the response.

Animals↗

PubMed1998

TGF-beta promotes immune deviation by altering accessory signals of antigen-presenting cells.

Macrophages incubated with OVA in the presence of TGF-beta2 induce immune deviation in vivo (impaired delayed hypersensitivity and IgG2a Ab production) when injected into naive, syngeneic mice. OVA-specific TCR transgenic naive T cells (DO11.10 T cells) produce Th1-type cytokines when stimulated in vitro with OVA-pulsed peritoneal exudate cells (PEC), but if PEC are first treated with TGF-beta2 and then pulsed with OVA, the T cells secrete Th2-type cytokines instead. In this study, we investigated the mechanisms that are involved in the modified Ag-presenting functions of macrophages by TGF-beta2 pretreatment. We have found that: 1) TGF-beta2 impaired the capacity of PEC to produce IL-12 and to express CD40; 2) reduced CD40 expression on TGF-beta2-treated PEC impaired IL-12 production when the cells were cocultured with DO11.10 T cells; 3) the failure of TGF-beta2-treated PEC to stimulate DO11.10 T cells to secrete IFN-gamma was due to their impaired IL-12 production. From these results, we conclude that TGF-beta2 treatment impairs the ability of macrophages to produce IL-12 and to express CD40. As a consequence, TGF-beta2-treated PEC fail to promote development of pT cells toward the Th1 phenotype.

Animals↗

PubMed1998

Interleukin 1 receptor antagonist suppresses allosensitization in corneal transplantation.

OBJECTIVE: To delineate the mechanisms by which topical interleukin 1 receptor antagonist (IL-1RA) treatment promotes orthotopic corneal allograft survival. METHODS: Corneal buttons were prepared from eyes of C57BL/6 mice and placed orthotopically in normal or neovascularized (high-risk) eyes of BALB/c mouse recipients. Topical IL-1RA (or vehicle alone) was applied to grafts 3 times daily until the grafted eyes were enucleated. Corneal specimens were evaluated for content of Langerhans cells. A week after enucleation, 1 group of recipients was tested for allospecific delayed-type hypersensitivity elicited by intrapinnae injections of donor splenocytes. In companion experiments, a second group of mice that underwent transplantation, IL-1RA treatment, and enucleation was challenged with orthotopic skin grafts from B10.D2 donor mice (sharing minor H antigens with C57BL/6 mice) to determine whether the second group of mice could reject grafts bearing corneal donor minor H alloantigens in an accelerated fashion. RESULTS: Mice whose orthotopic corneal allografts were treated topically with IL-1RA acquired neither donor-specific delayed-type hypersensitivity (P<.001) nor the capacity to reject orthotopic donor-type skin allografts in an accelerated manner (P<.05), whereas controls treated with vehicle alone developed delayed-type hypersensitivity and rejected B10.D2 grafts in an accelerated manner. Moreover, IL-1RA-treated grafts placed in both high-risk (P = .01) and normal-risk (P = .004) eyes displayed significantly reduced levels of infiltrating Langerhans cells compared with vehicle-treated controls. CONCLUSIONS: Topical IL-1RA promotes corneal allograft survival in large part by preventing activity of recipient Langerhans cells, and thereby preventing these cells from inducing systemic allosensitization. These data suggest that IL-1 plays a key role in promoting allosensitization when corneal allografts are placed orthotopically. CLINICAL RELEVANCE: Suppression of allosensitization by topical IL-1RA may prove a clinically useful method for enhancing corneal transplant survival.

Animals↗

PubMed1997

Calcitonin gene-related peptide is necessary for ultraviolet B-impaired induction of contact hypersensitivity.

Since nerve termini on Langerhans cells (LC) contain calcitonin gene-related peptide (CGRP), and since ultraviolet B radiation (UVR) causes CGRP to be released from cutaneous nerve endings, we examined whether CGRP participates in the immune aberrations caused in skin by UVR. First, intradermally injected CGRP, in a dose-dependent manner, reduced LC density and impaired CH induction when hapten was painted on the injected site. Second, CGRP antagonist restored CH induction after UVR. Third, anti-TNF-alpha Abs injected before CGRP prevented the loss of LC density and restored CH induction. Fourth, CGRP failed to impair CH induction in mast cell-deficient mice. Fifth, CGRP induced mast cells to release TNF-alpha. We conclude that CGRP plays an essential role in the loss of CH induction after UVR. These data indicate that UVR, by causing the release of CGRP from cutaneous nerve endings, triggers mast cell release of TNF-alpha, which impairs CH induction.

Animals↗

PubMed1997

High and low doses of haptens dictate whether dermal or epidermal antigen-presenting cells promote contact hypersensitivity.

In the induction of contact hypersensitivity (CH) to an epicutaneously applied hapten, we have previously proposed that low doses of hapten sensitize primarily through epidermal Langerhans' cells (LC), whereas high doses rely largely on dermal antigen-presenting cells (APC). To examine this issue further, we applied either high or low doses of dinitrofluorobenzene (DNFB) epicutaneously to mice. We observed reduced LC density at the site after 12 h (nadir), which returned to normal levels at 24 h only after a low dose of hapten. When a low dose of an unrelated hapten, oxazolone, was painted on skin that had been painted 12 h previously with high dose of DNFB, oxazolone-specific CH was impaired. When grafts of whole skin, dermis alone, and epidermis alone prepared from skin painted 2 h previously with low or high doses of DNFB were placed onto naive, syngeneic mice, CH was induced by whole skin after both types of doses, by epidermis only after a low dose, and by dermis only after high dose. When epidermal cell suspensions were derivatized in vitro with low or high doses of DNFB, only cells exposed to a low dose induced proliferation of hapten-specific Tcells. Thus, only a low dose of hapten reveals the APC functions of LC without the participation of dermal APC.

Adjuvants, Immunologic↗

PubMed1997

On the mechanisms by which transforming growth factor-beta 2 alters antigen-presenting abilities of macrophages on T cell activation.

Peritoneal exudate cells (PEC) incubated with antigen in the presence of transforming growth factor-(TGF)-beta 2 selectively suppress delayed hypersensitivity and IgG2a antibody production when injected intravenously into naive syngeneic recipients. In this study, we have examined in vitro the effects of TGF-beta 2 on the antigen presenting abilities of PEC to activate DO11.10 T cells that express a transgenic T cell receptor that recognizes ovalbumin peptide fragment 323-339 in the context of I-Ad. PEC were pretreated overnight with TGF-beta 2, washed extensively, then co-cultured with DO11.10 T cells in the presence of native OVA or P323-339. We found that TGF-beta 2-treated PEC induced the production of the T helper type 2 (Th2) cytokine, interleukin-4 (IL-4), but unlike untreated PEC, were unable to stimulate the Th1 cytokines, IL-2 and interferon-gamma (IFN-gamma). Furthermore, TGF-beta 2 was produced in an autocrine fashion by TGF-beta 2-treated PEC and was responsible for this shift to a Th2 response. This conclusion was supported by the following results. First, TGF-beta 2-treated PEC were found to express much more TGF-beta 1 and TGF-beta 2 mRNA than untreated PEC. Second, TGF-beta 2-treated PEC secreted large amounts of TGF-beta including its mature form. Third, addition of neutralizing anti-TGF-beta 2 antibodies, but not neutralizing anti-TGF-beta 1 antibodies, restored the ability of antigen-pulsed, TGF-beta 2-pretreated PEC to stimulate DO11.10 T cells to secrete IL-2 and IFN-gamma. These results indicate that antigen-presenting cells that encounter antigen in a TGF-beta-enriched environment (e.g., in the eye) shift responding native T cells toward Th2 responses by producing TGF-beta during antigen presentation.

Animals↗

PubMed1997

Aqueous humor induces transforming growth factor-beta (TGF-beta)-producing regulatory T-cells.

PURPOSE: The intraocular microenvironment is an immune-privileged site where immunogenic inflammation is suppressed. Suppression of immunogenic inflammation has been associated with immunosuppressive factors found in aqueous humor produced by ocular tissues. To further understand the mechanisms suppressing immunogenic inflammation in the eye, we have examined the production of lymphokines by primed T-cells activated in the presence of aqueous humor. METHODS: Enriched in vivo primed T-cells were T-cell receptor-stimulated in the presence of fresh aqueous humor. The culture supernatant was assayed for IFN-gamma and IL-4 by sandwich ELISA. TGF-beta production by T-cells stimulated in the presence of aqueous humor was assayed by a TGF-beta bioassay of the culture supernatant and by quantitative RT-PCR for TGF-beta 1 mRNA expression. Aqueous humor-treated T-cells were assayed for their capacity to suppress IFN-gamma production by stimulated, primed T-cells. RESULTS: Aqueous humor-enhanced proliferation but irreversibly suppressed production of both IFN-gamma and IL-4 by in vitro-activated, in vivo-primed T-cells. Aqueous humor induced in vivo primed T-cells to produce TGF-beta in vitro, and these TGF-beta-producing T-cells suppressed IFN-gamma production by other T-cells activated in co-cultures. CONCLUSIONS: Aqueous humor alters the functional program of TCR-ligand-activated, primed T-cells, converting the cells to TGF-beta-producing regulatory cells.

Animals↗

PubMed1996

The attenuated V60 strain of channel catfish virus possesses a deletion in ORF50 coding for a potentially secreted glycoprotein.

A wild-type strain of channel catfish virus was compared at the genomic level with the attenuated strain V60. In addition to several minor differences, restriction mapping revealed one major deletion (approximately 1200 bp) in ORF50 of the V60 strain. Cloning and sequencing of part of this ORF confirmed the presence of a 1164-bp deletion. It should result in a protein of 282 amino acids instead of 670. The predicted truncated protein lacks most of a threonine-rich, highly repetitive region in its central part. Since the protein encoded by ORF50 possesses a hydrophobic N-terminal leader sequence and no membrane anchor sequence, we suggest that it could be a secreted glycoprotein. This protein might be N-glycosylated (35 potential sites) and, given the repetitive arrangement of its residues (mainly threonines), also heavily O-glycosylated like the mucin-type glycoproteins. The deletion observed in ORF50 of the V60 strain implies the loss of 24 potential N-glycosylation sites and should considerably reduce the extent of O-glycosylation.

Amino Acid Sequence↗

PubMed1996

MHC class II tolerant T cells undergo apoptosis upon re-exposure to tolerogen in vivo.

Tolerance of MHC class II alloantigens can be achieved by intravenous injection of semiallogeneic hematopoietic cells into neonatal mice. Lymphoid cells of tolerant mice fail to proliferate or secrete interleukins IL-2 or IL-4 when stimulated in vitro with tolerogen. Since the lymphoid organs of B10.T(6R) tolerant mice contain normal levels of I-E reactive (V beta 11+) CD4+ T cells, deletion of alloreactive T cells does not appear to be the mechanism involved in the tolerance induction. To test whether T cells from tolerant animals can become activated under conditions that do not involve alloantigen stimulation, we stimulated these cells with immobilized anti-V beta 11 antibodies. Spleen cells from grafted tolerant and rejector mice proliferated in response to anti-V beta 11+ antibodies, suggesting they were not inert. We then tested whether V beta 11+ T cells from grafted mice can be induced to proliferate following stimulation with alloantigen in vivo. We adoptively transferred T cells from grafted tolerant and rejector mice into irradiated (B10.AQR x B10.T(6R))F1 mice and harvested the lymphoid organs after 65 h. Cells from both grafted tolerant and rejector mice underwent blast transformation, but only cells from rejector mice proliferated when exposed to immobilized anti-V beta 11 antibodies. The failure of V beta 11+ cells from tolerant mice to proliferate after in vivo stimulation may be because they are apoptotic. To test this hypothesis, spleen cells from naive or neonatally tolerized (with (B10.AQR x B10.T(6R))F1 cells) B10.T(6R) mice were adoptively transferred into irradiated (B10.AQR x B10.T(6R))F1 mice and bcl-2 expression was analysed in harvested V beta 11+ cells. Large cells recovered from recipients of naive 6R cells expressed bcl-2 mRNA. By contrast, large cells harvested from recipients of tolerized 6R cells did not express bcl-2 mRNA, suggesting bcl-2 mRNA expression was downregulated in these mice. Moreover, in another experiment, large V beta 11+ cells from grafted tolerant animals recovered after transfer into irradiated (B10.AQR x B10.T(6R))F1 mice did not express the bcl-2 protein as determined by flow cytometry, and contained fragmented DNA as assessed by the TUNEL method. Taken together, these data suggest that MHC class II tolerant T cells undergo apoptosis upon re-exposure to tolerogen in vivo.

Animals↗