Low-dose tolerance is mediated by the microfold cell ligand, reovirus protein sigma1.

Mucosal tolerance induction generally requires multiple or large Ag doses. Because microfold (M) cells have been implicated as being important for mucosal tolerance induction and because reovirus attachment protein sigma1 (psigma1) is capable of binding M cells, we postulated that targeting a model Ag to M cells via psigma1 could induce a state of unresponsiveness. Accordingly, a genetic fusion between OVA and the M cell ligand, reovirus psigma1, termed OVA-psigma1, was developed to enhance tolerogen uptake. When applied nasally, not parenterally, as little as a single dose of OVA-psigma1 failed to induce OVA-specific Abs even in the presence of adjuvant. Moreover, the mice remained unresponsive to peripheral OVA challenge, unlike mice given multiple nasal OVA doses that rendered them responsive to OVA. The observed unresponsiveness to OVA-psigma1 could be adoptively transferred using cervical lymph node CD4(+) T cells, which failed to undergo proliferative or delayed-type hypersensitivity responses in recipients. To discern the cytokines responsible as a mechanism for this unresponsiveness, restimulation assays revealed increased production of regulatory cytokines, IL-4, IL-10, and TGF-beta1, with greatly reduced IL-17 and IFN-gamma. The induced IL-10 was derived predominantly from FoxP3(+)CD25(+)CD4(+) T cells. No FoxP3(+)CD25(+)CD4(+) T cells were induced in OVA-psigma1-dosed IL-10-deficient (IL-10(-/-)) mice, and despite showing increased TGF-beta1 synthesis, these mice were responsive to OVA. These data demonstrate the feasibility of using psigma1 as a mucosal delivery platform specifically for low-dose tolerance induction.

Infusion of exogenous tumor necrosis factor dose dependently alters the length of the luteal phase in cattle: differential responses to treatment with indomethacin and L-NAME, a nitric oxide synthase inhibitor.

We examined whether prostaglandins (PGs) and nitric oxide (NO) mediate tumor necrosis factor (TNF) actions in the estrus cycle. On Day 14 of the cycle, the following solutions were infused into the aorta abdominalis of a total of 51 heifers (Experiments 1 and 2): saline; 1 or 10 microg of TNF; 480 mg indomethacin (INDO), an inhibitor of prostaglandin H synthase; 800 mg L-NAME, an inhibitor of NO synthase; and TNF (1 or 10 microg) in combination with INDO or L-NAME. TNF at 1 microg infused directly into aorta abdominalis increased the level of PGF(2alpha) and decreased the level of progesterone (P4) in the peripheral blood and shortened the estrus cycle. The high TNF dose stimulated P4 and PGE(2) and prolonged the corpus luteum (CL) lifespan. INDO blocked the effects of both TNF doses on the CL lifespan and hormone output. L-NAME completely blocked the effects of the luteolytic TNF dose, whereas the effects of the luteotropic TNF dose were not inhibited. In Experiment 3 (Day 14), saline or different TNF doses were infused into the jugular vein (n = 9) or into the uterine lumen (n = 18). The CL lifespans of the different groups were not different when TNF was infused into the jugular vein. Although high TNF doses (1 and 10 microg) infused into the uterine lumen prolonged the CL lifespan, low doses (0.01 and 0.1 microg) induced premature luteolysis. We suggest that the actions of exogenous TNF on the CL lifespan depend on PG synthesis stimulated by TNF in the uterus. TNF at low concentrations initiates a positive cascade between uterine PGF(2alpha) and various luteolytic factors, including NO, to complete premature luteolysis. PGE(2) is a good candidate mediator of the luteotropic actions of exogenous TNF action.

The distribution and influence of calcitonin gene-related peptide (CGRP) on the perfusion pressure in the isolated ovarian artery in the pig.

A moderate number of delicate CGRP-immunoreactive (CGRP-IR) varicose nerve terminals were found at the adventitia-media border of the sexually mature porcine ovarian artery by means of a routine single immunolabelling technique. Additionally, a pharmacological analysis was performed of the function fulfilled by alpha-CGRP and its C-terminal fragment (Tyr27) alpha-CGRP(27-37) in the porcine isolated ovarian artery, collected on days 8-13 of the oestrous cycle. It was shown that alpha-CGRP (10(-8) M) caused a decrease in the perfusion pressure of the porcine isolated ovarian artery of 16% (p < 0.05), while (Tyr27) alpha-CGRP(27-37), added at the same concentration, reduced perfusion pressure by 13% (p > 0.05). Thus, we concluded that alpha-CGRP released from perivascular terminals may cause relaxation of the ovarian artery and, furthermore, that the potency of this action is dependent on the length of the chain of this peptide produced during the deactivation of the molecule by tissue proteases.

Localization of neuropeptide Y and norepinephrine in the porcine ovarian artery and their influence on the local blood pressure.

The aim of the present study was to determine: (i) the presence of dopamine-beta-hydroxylase (DbetaH)- and neuropeptide Y (NPY)-immunoreactive (IR) nerve fibres in the wall of the porcine ovarian artery, (ii) the influence of NPY and norepinephrine (NE) on the contractile activity of the pig ovarian arteries, and (iii) the pharmacological analysis of the interaction between NPY and NE in the isolated porcine ovarian arteries collected from immature pigs and from animals in different days of the estrous cycle. Ovarian arteries for immunohistochemistry and isolated arteries for pharmacological studies were excised from immature pigs and mature animals on days 1-5, 8-13 and 17-20 of the estrous cycle. The study showed that both DbetaH- and NPY-IR nerve fibres were present in the pig ovarian arteries in all periods examined, and, that in some fibres DbetaH and NPY were co-localized. Both NE (10(-6) M) and NPY (10(-7) M) increased blood pressure of examined preparations, however, NE caused stronger changes in the vessel wall tension (P<0.001), than did NPY. NE significantly increased (P<0.001) blood pressure of all isolated arteries, however, this response was stronger in vessels from days 1-5 of the cycle, when compared to days 8-13 (P<0.01), 17-20 and immature pigs (P<0.001). NPY increased significantly blood pressure in isolated arteries from days 8-13 and 17-20 of the cycle (P<0.001), while in preparations taken from immature pigs and animals on days 1-5 of the estrous cycle this response was somewhat weaker (P<0.01). A higher elevation (P<0.001) of blood pressure after NPY administration was observed in isolated arteries from days 17-20 of the cycle, when compared to vessels from days 1-5 and 8-13 and those from immature pigs. Moreover, NE significantly intensified (P<0.001) an increase in the blood pressure in isolated arteries pre-treated with NPY in all periods examined. NPY insignificantly (P>0.05) potentiated increase of blood pressure in NE pre-treated vessels of immature pigs and in isolated arteries from days 17-20 of the cycle and significantly (P<0.05) in vessels from days 1-5 and 8-13 of the estrous cycle. Our results indicate that DbetaH- and NPY-IR nerve fibres are present in the pig ovarian arteries. NE and NPY administered alone increased blood pressure in the pig isolated ovarian artery and simultaneous administration of both substances caused each other potentiation of vasocontractile effect, however, the strength of observed changes was dependent on the stage of the estrous cycle.

Administration of a nitric oxide synthase inhibitor counteracts prostaglandin F2-induced luteolysis in cattle.

The objective of this study was to determine whether nitric oxide (NO) is produced locally in the bovine corpus luteum (CL) and whether NO mediates prostaglandin F2alpha (PGF2alpha)-induced regression of the bovine CL in vivo. The local production of NO was determined in early I, early II, mid, late, and regressed stages of CL by determining NADPH-d activity and the presence of inducible and endothelial NO synthase immunolabeling. To determine whether inhibition of NO production counteracts the PGF2alpha-induced regression of the CL, saline (10 ml/h; n = 10) or a nonselective NOS inhibitor (Nomega-nitro-l-arginine methyl ester dihydrochloride [L-NAME]; 400 mg/h; n = 9) was infused for 2 h on Day 15 of the estrous cycle into the aorta abdominalis of Holstein/Polish Black and White heifers. After 30 min of infusion, saline or cloprostenol, an analogue of PGF2alpha (aPGF2alpha; 100 microg) was injected into the aorta abdominalis of animals infused with saline or L-NAME. NADPH-diaphorase activity was present in bovine CL, with the highest activity at mid and late luteal stages (P < 0.05). Inducible and endothelial NO synthases were observed with the strongest immunolabeling in the late CL (P < 0.05). Injection of aPGF2alpha increased nitrite/nitrate concentrations (P < 0.01) and inhibited P4 secretion (P < 0.05) in heifers that were infused with saline. Infusion of L-NAME stimulated P4 secretion (P < 0.05) and concomitantly inhibited plasma concentrations of nitrite/nitrate (P < 0.05). Concentrations of P4 in heifers infused with L-NAME and injected with aPGF2alpha were higher (P < 0.05) than in animals injected only with aPGF2alpha. The PGF2alpha analogue shortened the cycle length compared with that of saline (17.5 +/- 0.22 days vs. 21.5 +/- 0.65 days P < 0.05). L-NAME blocked the luteolytic action of the aPGF2alpha (22.6 +/- 1.07 days vs. 17.5 +/- 0.22 days, P < 0.05). These results suggest that NO is produced in the bovine CL. NO inhibits luteal steroidogenesis and it may be one of the components of an autocrine/paracrine luteolytic cascade induced by PGF2alpha.