Endogenous carbon monoxide (CO) as a modulating factor of molecular biological clock in the hypothalamic structures involved in light transmission in pig and wild boar hybrid during long and short day season.

Mature males of a wild boar-pig crossbreed, during the long and short day season, were used for the study which demonstrates that the chemical light carrier CO regulates the expression of biological clock genes in the hypothalamus via humoral pathways. Autologous blood with experimentally elevated concentrations of endogenous CO (using lamps with white light-emitting diodes) was infused into the ophthalmic venous sinus via the right dorsal nasal vein. Molecular biology methods: qPCR and Western Blot were used to determine the expression of genes and biological clock proteins. The results showed that elevated endogenous CO levels, through blood irradiation, induces changes in genes expression involved in the functioning of the main biological clock located in suprachiasmatic nuclei. Changes in the expression of the transcription factors Bmal1, Clock and Npas2 have a similar pattern in both structures, where a very large decrease in gene expression was shown after exposure to elevated endogenous CO levels. The changes in the gene expression of PER 1-2, CRY 1-2, and REV-ERB α-ß and ROR ß are not the same for both POA and DH hypothalamic structures, indicating that both structures respond differently to the humoral signal received. The results indicate that CO is a chemical light molecule whose production in an organism depends on the amount of light. An adequate amount of light is an essential factor for the proper functioning of the main biological clock.

Effects of the phytoestrogen, genistein, and protein tyrosine kinase inhibitor-dependent mechanisms on steroidogenesis and estrogen receptor expression in porcine granulosa cells of medium follicles.

The use of soy-based products in pig diets had raised concerns regarding the reproductive toxicity of genistein, the predominant isoflavone in soybeans. Genistein was reported to exhibit weak estrogenic activity but its mechanism of action is not fully recognized. The aim of the study was to examine the in vitro effects of genistein on (1) progesterone (P(4)) and estradiol (E(2)) secretion by porcine granulosa cells harvested from medium follicles, (2) the viability of cultured granulosa cells, and (3) the mRNA and protein expression of estrogen receptors α and ß (ERα and ERß) in these cells. In addition, to verify the role of protein tyrosine kinase (PTK)-dependent mechanisms possibly involved in genistein biological action, we tested the effects of lavendustin C, the nonsteroidal PTK inhibitor, on granulosa cell steroidogenesis. We found that genistein inhibited (P < 0.05) basal P(4) secretion by granulosa cells harvested from medium follicles of pigs. In contrast, lavendustin C did not affect basal P(4) secretion by the cells. Moreover, genistein increased (P < 0.05) basal granulosal secretion of E(2). In contrast, lavendustin C did not alter basal E(2) secretion by porcine granulosa cells. In addition, we demonstrated that genistein increased mRNA and protein expression of ERß (P < 0.05) in the examined cells. The expression of ERα mRNA was not affected by genistein and ERα protein was not detected in the cultured granulosa cells of pigs. In summary, the genistein action on follicular steroidogenesis in pigs involved changes in the granulosal expression of ERß. However, the genistein action on P(4) and E(2) production by granulosa cells harvested from medium follicles did not seem to be associated with PTK.

The influence of steroids on noradrenaline-mediated contractile reactivity of the superficial nasal and facial veins in cycling gilts.

The nasal venous blood may be directed through the facial vein into the systemic circulation or through the frontal vein into the venous cavernous sinus of the perihypophyseal vascular complex, where hormones and pheromones permeate from the venous blood into the arterial blood supplying the brain and hypophysis. The present study was designed to determine the effect of noradrenaline (NA) on the tension of the nasal, frontal and facial veins of cycling gilts, and influence of ovarian steroid hormones on NA-mediated contractile reactivity. Additionally, the enzyme dopamine-beta-hydroxylase catalysing the conversion of dopamine to noradrenaline (DbetaH) was immunolocalized in these vessels. Among three studied veins, the frontal proximal vein, that fulfill a key role in the supply of the nasal venous blood into the venous cavernous sinus, reacted to NA most strongly (P < 0.001) and this reaction was weaker in the periestrous period than in luteal phase (P < 0.001). Inversely, the reaction to NA of the facial proximal vein, that carry blood to the peripheral circulation, was stronger in the periestrous period than in luteal phase (P < 0.05). P4, E2 and T significantly lowered NA-mediated tension of the frontal proximal vein during the periestrous period (P < 0.001), while in the luteal phase P4 might antagonize relaxing effect of E2 to this vessel. The result suggests that supply of the nasal venous blood into the venous cavernous sinus is greater during the periestrous period than during the luteal phase. DbetaH was clearly expressed in the muscular layer of the isolated superficial nasal and facial veins of gilts in both studied stages of the estrous cycle. We suggest that the reactivity of the superficial veins of the nose and face to NA combined with the previously demonstrated reactivity of these veins to steroid ovarian hormones and male steroid pheromones may regulate the access of priming pheromone androstenol (resorebed in the nasal cavity) to the brain of gilts during periestrous period via humoral local destination transfer.

Relationship between contractions of the uterus and concentration of PGF2α in uterine venous blood after luteolysis in gilts.

The origin and physiological significance of high pulses of prostaglandin F2α (PGF2α) in uterine venous blood that occur 2-3 days after luteolysis are not well understood. We studied the relationship between contractions of the uterus evoked by exogenous oxytocin (OT) and PGF2α concentration in uterine venous blood on day 17 of the porcine oestrous cycle. The infusion of OT into the uterine artery produced an immediate increase in the uterine intraluminal pressure (UIP) (p < 0.001) and a simultaneous elevation in PGF2α concentration in uterine venous blood (p < 0.0001). The infusion of indomethacin (IND) into the uterine artery slightly decreased PGF2α concentration in uterine venous blood, but it did not suppress uterine contraction or the rapid increase in PGF2α concentration in uterine venous blood just after OT infusion (p < 0.0001), which was lower that in gilts not treated with IND. We conclude that the spikes of PGF2α concentration in uterine venous blood occurring after OT infusion on day 17 of the porcine oestrous cycle are mainly caused by the excretion with venous blood from the remodelled uterus and that PGF2α synthesis may contribute to this. These results suggest that the high spikes in PGF2α concentration that occur 2-3 days after luteolysis in pigs, sheep, cows and mares all have a similar origin.

In vitro effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on ovarian, pituitary, and pineal function in pigs.

The aims of the study were: (1) to examine 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and/or prolactin (PRL) effects on in vitro secretion of progesterone (P(4)) and estradiol (E(2)) by luteinized granulosa and theca cells from porcine preovulatory follicles; and (2) to determine the effects of TCDD on PRL, luteinizing hormone (LH), and melatonin luteal phase in pigs. We found that TCDD itself did not affect progesterone secretion, but it abolished the stimulatory effect of PRL in the follicular cells. TCDD stimulated PRL secretion during the luteal phase and inhibited during the follicular phase. Moreover, TCDD increased luteinizing hormone secretion by pituitary cells during the follicular phase. In contrast to protein and steroid hormones, melatonin secretion in vitro was not affected by TCDD. In conclusion, it was found that the pituitary-ovarian axis in pigs is sensitive to TCDD, and the dioxin exhibited a profound ability to disrupt the ovarian actions of prolactin.

Local effect of progesterone infusion into ovarian artery on activin A and inhibin alpha-subunit secretion during the middle luteal phase in gilts.

The present study was undertaken to elucidate whether an increased, but physiological, amount of progesterone (P4) supplied to the porcine corpus luteum affects luteal secretion of activin A and inhibin alpha-subunit (Inhalpha) in freely moving gilts. On day 9 of the estrous cycle (EC), both ovarian arteries and both ovarian veins of gilts (n = 5) were cannulated. Progesterone was infused into the right ovarian arteries in gilts on days 10, 11 and 12 of the EC at a rate adequate to its physiological retrograde transfer found during the middle luteal phase of the EC. The P4 infusion rate was 0.62 microg/min (day 10), 2 x 0.62 microg/min (day 11) and 3 x 0.62 microg/min (day 12). The left ovarian arteries were infused with saline (control). Blood samples were collected from both ovarian veins on days 10-12 of the EC before and after P4 or saline infusion. The mean plasma activin A level in the ovarian vein ipsilateral to the P4-infused ovary was higher (P < 0.0001) on days 10-12 of the EC than this found in the contralateral ovarian vein. The level of activin A in the ovarian vein ipsilataral to the infusion of P4 was higher on days 11 (P < 0.01) and 12 (P < 0.0001) and tended to be higher (P < 0.07) on day 10 of the EC than this in contralateral ovarian vein. The level of Inhalpha in the ovarian vein ipsilateral to the P4-infused ovary on days 10-12 of the EC was not significantly different (P > 0.05) than this found in the contralateral ovarian vein. The results of the present study indicate that a local elevation of P4 concentration in blood supplying the ovary during the middle luteal phase of the porcine EC affects ovarian secretion of activin A. The effect of P4 on the secretion of activin A suggested the existence of a short regulatory loop of a positive feedback between P4 being retrogradely transferred into the ovary and the secretion of this peptide.

Banff '09 meeting report: antibody mediated graft deterioration and implementation of Banff working groups.

The 10th Banff Conference on Allograft Pathology was held in Banff, Canada from August 9 to 14, 2009. A total of 263 transplant clinicians, pathologists, surgeons, immunologists and researchers discussed several aspects of solid organ transplants with a special focus on antibody mediated graft injury. The willingness of the Banff process to adapt continuously in response to new research and improve potential weaknesses, led to the implementation of six working groups on the following areas: isolated v-lesion, fibrosis scoring, glomerular lesions, molecular pathology, polyomavirus nephropathy and quality assurance. Banff working groups will conduct multicenter trials to evaluate the clinical relevance, practical feasibility and reproducibility of potential changes to the Banff classification. There were also sessions on quality improvement in biopsy reading and utilization of virtual microscopy for maintaining competence in transplant biopsy interpretation. In addition, compelling molecular research data led to the discussion of incorporation of omics-technologies and discovery of new tissue markers with the goal of combining histopathology and molecular parameters within the Banff working classification in the near future.

Retrograde transfer of 125I-radiolabelled activin and inhibin in the periovarian vascular complex in the sow.

This study investigated whether activin A and an inhibin-alpha subunit fragment (INHalpha) could permeate in a periovarian vascular complex from ovarian effluent into the ovarian artery and be retrograde transferred into the ovary. Radiolabelled activin A (125I-activin A) and INHalpha (125I-INHalpha) were injected (2.7 x 10(7) dpm) into follicles or corpora lutea (CL). It was demonstrated that 125I-activin A and 125I-INHalpha were released into the ovarian effluent and permeated into the arterial blood supplying the ovary in both phases of the cycle. The concentration of 125I-activin A in ovarian arterial blood was higher in the luteal phase (LP) than in the follicular phase (FP) (P<0.0001) in contrast to 125I-INHalpha which was higher in the FP (P<0.0001). The concentration of 125I-activin A in uterine tissues generally did not differ between the phases of the estrous cycle, but the concentration of 125I-INHalpha was higher (P<0.05) in the FP than in the LP. The concentration of 125I-activin A was higher in the LP in samples of endometrium and myometrium (P<0.05), as well as mesometrium (P<0.01), and higher in the FP in samples of mesometrium (P<0.05) close to the ovary than in the samples adjoining the uterine body. In the FP, the concentration of 125I-INHalpha was higher in endometrium and mesometrium close to the ovary than in samples adjoining the uterine body (P<0.05). In conclusion, the study demonstrated that it was possible for INHalpha and activin A to be retrograde transferred to the ovary. Thus this transfer could elevate their concentration in arterial blood supplied to the ovarian follicles or CL and may influence production of these peptides in the ovary, modulating ovarian function.

Synergistic deposition of C4d by complement-activating and non-activating antibodies in cardiac transplants.

The role of non-complement-activating alloantibodies in humoral graft rejection is unclear. We hypothesized that the non-complement-activating alloantibodies synergistically activate complement in combination with complement-activating antibodies. B10.A hearts were transplanted into immunoglobulin knock out (Ig-KO) mice reconstituted with monoclonal antibodies to MHC class I antigens. In allografts of unreconstituted Ig-KO recipients, no C4d was detected. Similarly, reconstitution with IgG1 or low dose IgG2b alloantibodies did not induce C4d deposition. However, mice administered with a low dose of IgG2b combined with IgG1 had heavy linear deposits of C4d on vascular endothelium. C4d deposits correlated with decreased graft survival. To replicate this synergy in vitro, mononuclear cells from B10.A mice were incubated with antibodies to MHC class I antigens followed by incubation in normal mouse serum. Flow cytometry revealed that both IgG2a and IgG2b synergized with IgG1 to deposit C4d. This synergy was significantly decreased in mouse serum deficient in mannose binding lectin (MBL) and in serum deficient in C1q. Reconstitution of MBL-A/C knock out (MBL-KO) serum with C1q-knock out (C1q-KO) serum reestablished the synergistic activity. This suggests a novel role for non-complement-activating alloantibodies and MBL in humoral rejection.

C4d deposition and clearance in cardiac transplants correlates with alloantibody levels and rejection in rats.

Antibody-mediated rejection of human cardiac transplants is correlated with C4d deposits and macrophage infiltrates in capillaries of endomyocardial biopsies. We produced an antibody to rat C4d to study C4d deposition and clearance in Lewis rats that were sensitized with a blood transfusion from DA rats 7, 14 or 21 days before cardiac transplantation. Cyclosporin A (CsA) immunosuppression was initiated after transplantation at a dose that inhibited graft rejection, antibody production and C4d deposition in unsensitized recipients. Blood transfusion elicited high levels of circulating IgG alloantibodies, predominantly of the complement-activating IgG2b subclass, that peaked 14 days after transplantation. At this time, macrophages accumulated in capillaries, and C4d deposits were diffuse and intense on arteries, capillaries and veins. Grafts that survived 90 days in sensitized recipients still had deposits of C4d that were associated with increased interstitial fibrosis and vasculopathy in arteries. Clearance of C4d was determined by retransplanting DA cardiac allografts from Lewis recipients back to DA recipients. C4d deposits were decreased to minimal levels within 5 days after retransplantation. Thus, C4d deposition is not limited to the capillaries, but extends throughout the arterial tree, and despite formation of a covalent bond, C4d is cleared within days.

The effect of intramuscular injections of boar pheromone 5alpha-androstenol on the hormonal regulation of the estrous cycle in hypoosmatic gilts.

Until 1999 it was accepted that pheromones act exclusively by stimulating the dendritic receptors present in olfactory epithelium. Cycling gilts with an experimentally-disrupted neural olfactory pathway were used to test the hypothesis that boar pheromone 5alpha-androstenol may affect the secretion of hormones involved in the regulation of the estrous cycle by the humoral pathway. On day 12 of the estrous cycle the nasal cavity of gilts (n=15) was irrigated with zink sulfate solution. From day 16 to 20, the experimental group (n=10) was injected intramuscularly with 5alpha-androstenol (20 microg) twice a day. Blood samples were collected from the jugular vein at 4 h intervals on days 17-21 to estimate plasma concentration of LH, oxytocin, estradiol-17beta, testosterone and progesterone. The experimental group displayed a significantly lower mean concentration of LH than the control animals (P<0.0001). The decrease in concentration of LH was accompanied by the reduction of oxytocin (P<0.001), estradiol-17beta (P<0.001) and testosterone (P<0.01) secretion. These results demonstrated that 5alpha-androstenol influenced hormonal regulation by humoral pathway and might be considered to be the priming pheromone in gilts.

Expression of CR1/2 receptor on alloantigen-stimulated mouse T cells.

Antibodies can mediate injury of organ transplants by several mechanisms, including complement activation and interaction with Fc receptors on cells. We tested the hypothesis that antibodies could also cause up-regulation of complement receptors on cells to increase the responses to complement activation by interaction with split products of C3. In our experimental model, B10.A (H-2(a)) cardiac transplants survive significantly longer in C57BL/6 (H-2(b)) immunoglobulin knockout recipients (IgKO) than in their wild-type counterparts. Passive transfer of specific antibodies to donor MHC class I to IgKO recipients of cardiac allografts at the time coinciding with a vigorous cellular infiltration reconstituted acute rejection. We tested the effects of alloantibodies on CR1/2 expression by alloantigen-stimulated T cells. Both CD4(+)/CR1/2(+) and CD8(+)/CR1/2(+) populations of T cells were expanded in C57BL/6 splenocytes stimulated by B10.A alloantigen in 7-day MLR after coculture with endothelial cells sensitized with IgG1 and IgG2b mAb specific to MHC. Endothelial cells sensitized with antibodies also caused an expansion of CD8(+) T cells expressing CR1/2 in lymph node lymphocytes harvested from a C57BL/6 recipient of a B10.A cardiac allograft. These data suggest that antibodies can augment the cellular rejection process through expanding the population of T cells interacting with complement split products.

A rat model of pregnancy-induced sensitization to transplants.

Previous pregnancy is a known risk factor for alloantibody production and graft rejection in clinical transplantation. However, in previous rat models, immune responses to RT1.A antigens induced by allogeneic pregnancy resulted in prolonged survival of subsequent allografts. This study was designed to investigate the effects of a previous pregnancy on alloantibody response, complement activation, and allograft survival in a highly immunogenic rat strain combination. C6-sufficient and -deficient female PVG.1U (RT1.A(u)B(u)) rats were mated with allogeneic PVG.R8 (RT1.A(a)B(u)) males or control isogeneic PVG.1U (RT1.A(u)B(u)) males. Three weeks after parturition, experimental and control females received cardiac allografts from female PVG.R8 donors. A low dose of cyclosporine (CsA, 5 mg/kg on alternate days) was used for immunosuppression after transplantation. Allogeneic, but not control isogeneic, pregnancy elicited a weak, transient IgG alloantibody response that declined before transplantation. Experimental female recipients produced a rapid, vigorous IgM and IgG alloantibody response to the transplant despite CsA treatment. C6-sufficient recipients rejected their transplants at an accelerated rate (5 days, n = 6) compared with control animals (7 days, n = 5). In contrast, allografts to C6-deficient recipients functioned until sacrifice at 90 days in both the experimental group (n = 7) and control group (n = 4). Most experimental C6-deficient recipients continued to produce strong IgG alloantibodies for 90 days. Complement activation resulting from the alloantibody response was evidenced by the diffuse deposition of C3d on the vascular endothelium of the grafts. In summary, previous pregnancy leads to memory alloantibody responses that accelerate allograft rejection even with immunosuppression. Membrane attack complex is required for accelerated rejection induced by previous pregnancy.

Retrograde transfer of steroid hormones to the ovary in luteal and follicular phases of porcine oestrous cycle in vivo.

The efficiency of the retrograde transfer of steroid ovarian hormones from the ovarian effluent into blood supplying the ovary and the rate of its back transport to the ovary were determined for the first time in in vivo conditions. Sexually mature gilts (n = 25) were used in the physiological study. The concentration of oestradiol and progesterone in blood collected from the ovarian artery was higher in both the follicular phase (by 87.9 +/- 2.9% and 150.0 +/- 4.8%, respectively, P < 0.001) and the luteal phase (by 82.1 +/- 3.9% and 77.7 +/- 2.7%, respectively, P < 0.001) than in systemic blood reaching the initial part of the ovarian artery. The high efficiency of the retrograde transfer was not dependent on the concentration of hormones in the ovarian venous blood. However, the efficiency and rate of the retrograde transfer differed between phases of the oestrous cycle. We suggest that such effective retrograde transfer of ovarian hormones must affect the secretory function of the ovary.

Boar pheromone androstenol may affect the ovarian morphology in cycling gilts by humoral pathway.

Up to 1999 it was accepted that pheromones act exclusively by stimulation of dendritic receptors of olfactory neurons massed in the olfactory epithelium, but in 1999-2000, the presence of local humoral pathway for transfer of boar pheromone androstenol from the nasal cavity to the hypophysis and brain was demonstrated in gilts. The aim of the present study was to ascertain whether boar pheromone androstenol may affect by humoral pathway the ovarian morphology in gilts. This study demonstrated that intramuscular injections of androstenol in the follicular phase (17-20 day) of the estrous cycle in anosmatic gilts, in which the neural pathway for olfactory function was experimentally blocked, produced lack of the ovulation and changes in the morphology of ovaries. Histological analysis of the ovaries, collected seven days after androstenol injections, revealed the absence of corpora lutea and healthy follicles of a diameter over 6 mm as well as a significant decrease in the number of the follicles up to I mm in diameter (P<0.01). In androstenol-treated gilts, the number of atretic follicles from 1 mm to 6 mm in size was increased (P<0.01-P<0.001) and in one gilt cysts were found. The obtained results provided some evidence that in gilts in addition to acting by standard neural pathway, androstenol as a priming pheromone may affect the ovarian morphology by a humoral pathway.

Retrograde transfer of ovarian steroid hormones to the ovary in the porcine periovarian vascular complex.

The aim of the present study was to investigate the mechanism of the retrograde transfer of ovarian steroid hormones from the ovarian lymphatic and venous effluent to the arterial blood supplying the ovary. In the first experiment, reproductive organs were collected from gilts in the luteal (n = 10) and follicular (n = 10) phase of the oestrous cycle. The ovary with the mesovarium was isolated and perfused through the ovarian artery with warmed, oxygenated autologous blood. The concentrations of progesterone and oestradiol in ovarian arterial blood increased on passing through the ovarian artery to the ovary, in the luteal phase, from 20.3 +/- 2.1 to 31.4 +/- 3.9 ng ml(-1) (P < 0.001) and from 6.2 +/- 0.8 to 11.4 +/- 1.4 pg ml(-1) (P < 0.001), respectively, and in the follicular phase, from 1.2 +/- 0.2 to 2.2 +/- 0.4 ng ml(-1) (P < 0.001) and from 8.2 +/- 1.8 to 13.2 +/- 2.3 pg ml(-1) (P < 0.001), respectively. Approximately 17.5 +/- 3.9 % of the progesterone and 12.6 +/- 1.7 % of the oestradiol found in the ovarian venous effluent was retrogradely transferred from the ovarian venous blood to the ovary in the luteal phase. In the follicular phase, these values were 10.1 +/- 2.0 % and 8.6 +/- 1.4 %, respectively. The efficiency of retrograde transfer of oestradiol and the rate of retrograde transfer of progesterone differed between phases of the oestrous cycle (P < 0.05 and P < 0.0001, respectively). A direct relationship between the concentration of the steroids in the venous effluent and the efficiency and rate of the retrograde transfer to the ovary was not found. In the second experiment (luteal phase, n = 10; follicular phase, n = 5), the concentration of progesterone and oestradiol increased in both ovarian arterial blood (P < 0.0001) and in the venous effluent (P < 0.0001) after administration of the steroids into the lymphatic vessels of the isolated mesovarium with separated ovary. In the third experiment (follicular phase, n = 5), with the mesovarium isolated after the ovary was removed and ovarian venous blood flowing out under the force of gravity (without the blood pressure in the ovarian vein), it was demonstrated that the veno-venous network covering the branches of the ovarian artery was supplied with the blood flowing out from the mesovarian tissue and that the filling of the veno-venous network was dependent on the blood pressure in the ovarian artery. We conclude that the effective retrograde transfer of steroid hormones from ovarian venous and lymphatic effluent to the ovary is accomplished not only by the classical counter-current exchange mechanism, but also as a result of complex processes that may be dependent on a specific part of the circulation of the blood and lymph in the periovarian vascular complex of the mesovarium.

Inducible nitric oxide synthase inhibition of weibel-palade body release in cardiac transplant rejection.

BACKGROUND: Inducible nitric oxide synthase (iNOS, or NOS2) reduces the severity of accelerated graft arteriosclerosis (AGA) in transplanted organs, although the precise mechanism is unclear. METHODS AND RESULTS: We transplanted wild-type murine hearts into either wild-type or NOS2-null recipient mice; we then measured cardiac allograft survival and analyzed tissue sections by immunohistochemistry. We have confirmed that NOS2 increases cardiac allograft survival. We now show that there is less inflammation of cardiac allografts in wild-type hosts than in NOS2-null hosts. Furthermore, staining for von Willebrand factor reveals that the presence of NOS2 is correlated with the presence of Weibel-Palade bodies inside endothelial cells, whereas the absence of NOS2 is correlated with the release of Weibel-Palade bodies. CONCLUSIONS: Weibel-Palade bodies contain mediators that promote thrombosis and inflammation. Therefore, nitric oxide (NO) may stabilize the vessel wall and prevent endothelial activation in part by inhibiting the release of the contents of Weibel-Palade bodies. Prevention of Weibel-Palade body release might be a mechanism by which NO protects the vessel wall from inflammatory disorders such as atherosclerosis or graft arteriosclerosis.

Apoptotic cell death in the porcine endometrium during the oestrous cycle.

It has been reported that apoptosis plays an essential role in controlling the physiological cell kinetics in the human and rodent endometrium but this type of death has never been studied in the porcine endometrium. The aim of this study was to investigate the apoptotic cell death in the porcine endometrium during the middle (Days 9-11) and late (Day 13) luteal phase, during the luteolysis (Day 15) and early follicular phase (Days 17-19) of the oestrous cycle. Apoptotic cells were identified by in situ DNA 3'-end labelling method. It was revealed that the greatest number of apoptotic cells in the luminal and glandular epithelium was found on Days 17-19 and on Day 15 of the oestrous cycle, respectively. In the stroma, the greatest number of these cells was found on Days 9-11. Our data have shown that in the porcine endometrium, both epithelial and stromal cells undergo apoptosis and that the number of apoptotic cells varies depending on the phase of the oestrous cycle.

Passive transfer of alloantibodies restores acute cardiac rejection in IgKO mice.

BACKGROUND: Alloantibody is an intrinsic component of the immune response to organ transplants. Although alloantibodies have been correlated with decreased graft survival, the mechanisms of alloantibody-mediated injury remain largely undefined in vivo. In the present study, we have established a model of alloantibody-mediated graft injury using B10.A (H-2a) hearts transplanted to wild type (WT) or immunoglobulin knock out (IgKO) C57BL-Igh-6 (H-2b) mice. METHODS: Alloantibodies were measured in the circulation and graft by flow cytometry and in immunofluorescence staining, respectively. Intragraft cytokine mRNA expression was evaluated using a competitive template reverse transcriptase polymerase chain reaction (RT-PCR) technique. P-selectin and von Willebrand factor expression were localized by immunoperoxidase staining. The capacity of alloantibodies to restore acute cardiac allograft rejection was tested by passive transfer of monoclonal antibodies (mAbs) against donor major histocompatibility complex (MHC) class I antigens to IgKO recipients. RESULTS: B10.A cardiac allografts are rejected acutely by WT C57BL/6 recipients, but over 50% of the cardiac allografts survived more than 50 days after transplantation in IgKO mice. Competitive template RT-PCR on the cardiac transplants demonstrated similar levels of IL-1-alpha, IL-12 (p40), TNF-alpha, IL-2, IFN-gamma, IL-4, and IL-10 mRNA in WT and IgKO recipients 8-10 days after transplantation, indicating that macrophage- and T-cell-dependent immune responses were intact in IgKO recipients. The rejection of B10.A hearts in WT recipients was characterized by interstitial and perivascular cellular infiltration; IgG, IgM, and complement (C3) deposition; vascular cell injury and intravascular platelet aggregation; and release of von Willebrand factor and P-selectin. In IgKO recipients the lower degree of vascular injury in the absence of alloantibody responses was reflected by the lack of release of von Willebrand factor and P-selectin, which remained confined to cytoplasmic storage granules of endothelial cells and platelets. Acute rejection of cardiac allografts was restored to IgKO recipients by passive transfer of proinflammatory IgG2b mAbs against donor MHC; recipients injected with isotype-matched control mAbs did not reject. In contrast, passive transfer of IgG1 mAbs against donor MHC failed to restore acute rejection of cardiac allografts to IgKO recipients. Passive transfer of IgG2b, but not IgG1 mAbs was associated with endothelial cell activation and plate. let aggregation together with the release of preformed von Willebrand factor and P-selectin from storage granules. CONCLUSIONS: Acute rejection of cardiac allografts can be reconstituted in IgKO recipients by passive transfer of IgG2b, but not IgG1 antibody. This model allows the mechanism of alloantibody-mediate graft injury to be dissected in vivo.

Adjunctive rapamycin and CsA treatment inhibits monocyte/macrophage associated cytokines/chemokines in sensitized cardiac graft recipients.

BACKGROUND: Although treatment of LEW rats with Rapamycin (RPM) prolongs the survival of LBNF1 cardiac allografts to ca. 50 days, it fails to prevent late activation of macrophage/monocyte-associated chemokines. METHODS: A 7-day course with RPM or CsA was introduced at 1-week posttransplant in RPM-pretreated hosts. At day 35, intragraft mRNA expression of IL-2, IFN-gamma, TGF-beta, IL-12 (p40), MCP-1, and RANTES was evaluated. RESULTS: RPM as a sequential treatment markedly inhibited mRNA levels coding for IL-2/IFN-gamma, and MCP-1. However, RPM monotherapy failed to prevent the expression of IL-12 (p40) and RANTES. Adjunctive treatment with CsA markedly depressed IL-12 (p40) and RANTES, and to a lesser extent MCP-1 mRNA, as compared with RPM-treated groups. Significant increase of TGF-beta mRNA expression was revealed after sequential RPM and adjunctive CsA treatment. CONCLUSIONS: A combination of RPM and CsA is more effective in restraining mRNA expression than RPM alone; however, either therapy is associated with TGF-beta hyperexpression.