Effects of chicken thymic stromal cells on the growth and differentiation of thymocytes in vitro

We examined contact-mediated effects of chicken thymic stromal cells (TSC) on thymocyte differentiation by co-cultivation of these cell populations. The primary cultures of TSC isolated from thymus mainly have consisted of epithelial cells which were polygonal in shape, possessed long processes and expressed MHC class II antigen. When thymocytes were co-cultured with TSC, 60% to 70% of thymocytes attached to TSC and some of them engulfed underneath TSC. These attached thymocytes were CD4-CD8- and CD4+CD8+ subsets and expressed alpha/beta TCRhigh or gamma/delta TCRlow. Some of the thymocytes attaching to TSC showed an increase of intracellular and nuclear density, fragmentation of cytoplasm and nuclei, and DNA fragmentation. And also, thymocytes attaching to TSC contained a higher percentage of cycling (S and G2 + M phase) cells than nonattaching cells. These results indicate that specific subsets in thymocytes selectively bind to TSC and undergo apoptotic death or proliferation because of interaction with TSC. Chicken TSC may play an important role in thymic differentiation by direct contact within the thymus as in mammals.     

Effects of cytokines from thymocytes and thymic stromal cells on chicken intrathymic T cell development

We have studied the ability of thymic stromal cells (TSC) and thymocytes to produce cytokines and the involvement of cytokines in intrathymic T cell development. When thymocytes were co-cultured with thymic stromal cells in absence of direct contact and mitogenic stimulation, induction of thymocyte proliferation was observed. Supernatants of cultured stromal cells (TSC-CS) promoted a high proliferative response on CD3- thymocytes but had little effect on CD3+ thymocytes. These results indicate that stromal cells have produced a cytokine which can induce immature thymocyte proliferation. Moreover, stromal cells express the MRNA for stem cell factor (SCF) and c-kit (the receptor for SCF) was detected on CD3- thymocytes but not on CD3+ thymocytes. Since SCF can enhance the proliferation of immature thymocytes in synergy with IL-7 in mammals, there is a possibility that chicken stromal cells may produce a IL-7-like factor. Thymocytes have clearly expressed interferon (IFN)-gamma. In contrast, thymic stromal cells showed no detectable expression of IFN-gamma. CD3+ thymocytes express IFN-gamma MRNA more strongly than CD3 thymocytes, suggesting that IFN-gamma from thymocytes may operate on stromal cells and then may indirectly induce clonal elimination of CD3+ cells on stromal cells. The expression of these cytokines and receptors by thymic stromal cells and thymocyte subpopulations suggests that these cytokines participate in paracrine interactions between these cell populations during thymocyte differentiation.     

Extracellular matrix proteoglycan decorin-mediated myogenic satellite cell responsiveness to transforming growth factor-beta1 during cell proliferation and differentiation Decorin and transforming growth factor-beta1 in satellite cells

Transforming growth factor-beta1 (TGF-beta1) is a potent inhibitor of muscle cell proliferation and differentiation. Decorin, a small proteoglycan in the extracellular matrix, binds to TGF-beta1 and modulates the activity of TGF-beta1 during muscle cell growth and development. However, its interaction with TGF-beta1 and involvement in myogenesis is not well characterized. In the present study, chicken myogenic satellite cells, myogenic precursors for muscle growth and repair, were isolated from the pectoralis major muscle and used to investigate the biological function of TGF-beta1 and decorin during myogenesis. The over-expression of decorin in satellite cells significantly increased cell proliferation, compared to the control cells. Consistent with this result, reducing decorin expression decreased cell proliferation, which suggests a decorin-mediated mechanism is involved in the regulation of myogenic satellite cell proliferation. Satellite cells over-expressing decorin were less sensitive to TGF-beta1 during proliferation, which indicates that decorin may sequester TGF-beta1 leading to increased proliferation. During satellite cell differentiation, the over-expression of decorin induced differentiation by increasing the muscle specific creatine kinase concentration. However, the addition of TGF-beta1 diminished decorin-mediated cell responsiveness to TGF-beta1 during differentiation. Taken together, these results suggest that decorin induces myogenic satellite cell proliferation and differentiation by regulating cellular responsiveness to TGF-beta1. An alternative TGF-beta1-independent pathway may be involved in the regulation of satellite cells by decorin.     

Upregulation of transforming growth factor-beta and interleukin-10 in cows with clinical Johne's disease

Johne's disease progresses through distinct stages including a protracted subclinical stage in which the infection appears to be controlled; followed by a more acute stage in which the host animal demonstrates clinical signs such as diarrhea and weight loss. Little is known about the dynamics of the host immune response during these two phases of disease, however, it is possible that immune modulation in the early stages of disease may play an important role in disease progression. We hypothesized that the clinical stage of Johne's disease is mediated by the expression of cytokines such as transforming growth factor-beta (TGF-beta) and interleukin-10 (IL-10) that may be accompanied by the downregulation of IFN-gamma gene expression. In the present study, tissue samples were collected from the ileum, ileocecal junction, ileocecal lymph node, and mesenteric lymph nodes of healthy, subclinically or clinically infected cows. The expression of TGF-beta, IL-10, and IFN-gamma genes in these tissues was determined by quantitative competitive RT-PCR. The results demonstrate that TGF-beta and IL-10 mRNA levels are higher in cows that have progressed to the clinical stage of disease compared to subclinically infected or healthy cows. In contrast, IFN-gamma gene expression was significantly higher in subclinically infected cows. These results suggest that a change in the balance of cytokines at the site of infection may contribute to the ability of the host to control Mycobacterium avium subsp. paratuberculosis infection.     

Comparison of hematologic values and transforming growth factor-beta and insulin-like growth factor concentrations in platelet concentrates obtained by use of buffy coat and apheresis methods from equine blood

OBJECTIVE: To evaluate the buffy coat and apheresis methods for preparation of platelet concentrates from equine blood by comparing platelet and growth factor concentrations. ANIMALS: 15 mature mixed-breed geldings. PROCEDURE: Whole blood samples were collected and processed by use of a buffy coat or apheresis method to obtain platelet poor and platelet concentrated fractions. The PCV, WBC count, and platelet count were compared among whole blood samples, platelet poor fractions, concentrates obtained by use of the apheresis method (ie, apheresis platelet concentrates), and concentrates obtained by use of the buffy coat method (ie, buffy coat platelet concentrates). Concentrations of transforming growth factor-beta (ie,TGF-beta1 andTGF-beta2) and insulin-like growth factor were compared between buffy coat and apheresis platelet concentrates. RESULTS: Platelet concentrations were 8.9-fold and 5.2-fold greater in buffy coat and apheresis platelet concentrates, respectively, compared with whole blood. Platelet concentrations were 13.1-fold greater in filtered apheresis platelet concentrates, compared with whole blood. TGF-beta1 concentrations were 2.8-fold and 3.1-fold greater in buffy coat and apheresis platelet concentrates, respectively, and TGF-beta1 concentrations were 10.5-fold greater in filtered apheresis platelet concentrates, compared with whole blood. TGF-beta2 concentrations were 3.6-fold greater in apheresis platelet concentrates, compared with whole blood. Platelet concentrations correlated with growth factor concentrations across all blood and platelet fractions. White blood cell counts had a significant positive correlation with TGF-beta1 concentration in buffy coat platelet concentrates. CONCLUSIONS AND CLINICAL RELEVANCE: Platelets and TGF-beta1 can be concentrated reliably from equine blood by use of buffy coat or apheresis methods, without modification of the protocols used for humans.     

氯前列烯醇对鸡等级前卵泡颗粒细胞增殖的促进作用

探讨了前列腺素PGF2α类似物氯前列烯醇（Cloprostenol,CLO）对鸡等级前小黄卵泡颗粒细胞增殖的作用及其信号转导途径。颗粒细胞经16 h预培养后用CLO处理24 h,测定细胞的增殖情况。结果显示：0.1～10μg/L的CLO促进了颗粒细胞的增殖,10μg/L时刺激效果最为明显。蛋白激酶A（PKA）激活后颗粒细胞数量增加,PKA抑制剂H89抑制了CLO的促增殖作用。PKC的激活或抑制对CLO的促增殖作用无显著影响。这表明,CLO可通过PKA途径促进鸡等级前小黄卵泡颗粒细胞的增殖。     

Oral ingestion of colostrum alters intestinal transforming growth factor-beta receptor intensity in newborn pigs

Mammary gland secretion contains numerous bioactive compounds including transforming growth factor-beta (TGF-β). The concentrations of such bioactive compounds are usually much higher in colostrum compared with those in mature milk. To investigate possible effects of colostrum-borne TGF-β on the suckling animal, newborn piglets were naturally suckled or bottle-fed with porcine colostrum, bovine colostrum, porcine milk, infant formula or water for 24 h and intestinal TGF-β receptor intensity was assessed using an immunohistochemical staining technique in combination with computerized image analysis. The intestinal structure was also analyzed by morphometric analysis technique. It was observed that newborn pigs naturally suckled or bottle-fed with porcine or bovine colostrum had significantly greater intestinal villous height and crypt depth when compared with those fed with porcine milk, infant formula or water (p < 0.05). The immunostaining intensity for TGF-β receptors in the intestinal epithelium, particularly on the apical membrane of the villous epithelium, was significantly lower in naturally suckled or colostrum fed piglets compared with that in piglets fed with milk, infant formula or water (p < 0.05). Such decline in receptor intensity is likely the result of receptor internalization and degradation following exposure to colostrum-borne TGF-β. These findings suggest that colostrum-borne TGF-β can modulate intestinal TGF-β signalling pathways and may play a role in postnatal adaptation of the gut in newborn pigs.     

Temporal expression of transforming growth factor-beta2 and myostatin mRNA during embryonic myogenesis in Indian broilers

TGF-beta2 and myostatin, the members of TGF family, act through both autocrine and paracrine mechanisms to regulate the growth and differentiation at various developmental stages in chicken. The kinetics and expression profile of these two growth factors were investigated by semi-quantitative RT-PCR, during the myogenesis of Indian broiler chickens. Total RNA was isolated from whole embryos on each of embryonic days (E) 0-6 (n=3 per day) and from the biceps femoris muscle at E7-E18 (n=3 per day). The expression of TGF-beta2 was noticed on E2 that remained at the same level until E6. In biceps femoris muscle, higher level of TGF-beta2 expression was observed during E7-E12, which decreased gradually thereafter. These findings suggested that TGF-beta2 might be a regulatory factor participating in the myogenesis of chicken embryos. Initial myostatin expression was noticed on E1, even before the myogenic lineage is established in embryo. This finding suggested an additional role of myostatin in early chicken embryo development, other than myogenesis. Furthermore, myostatin expression was significantly higher on E3 as compared to earlier studies, where initial higher level was observed at E2, suggesting the differential expression of myostatin among breeds. Higher and almost static myostatin expression was noticed in biceps femoris muscle during the entire period of myogenesis (E7-E18). In the present study, the ontogeny of myostatin expression coincided with myogenesis of chicken. Therefore, it may be hypothesized that myostatin is not only a major determinant of muscle mass, but also involved in early embryogenesis in chickens.     

Regulation of secondary follicle growth by theca cells and insulin-like growth factor 1

Ovaries contain follicles at various stages of development, including primordial, primary, secondary, antral and Graafian follicles. Although the growth of these follicles is controlled to maintain regular ovulation, the mechanism through which this occurs remains unclear. In our study, we found that the growth rate of cultured secondary follicles separated from mice ovaries differed between follicles. After 4 days of culture, the size of some secondary follicles was markedly increased, while that of others had either slightly increased, remained unchanged or shrunk. We compared the expression levels of growth factors between these secondary follicles and found that the growth rate of cultured secondary follicles correlated with the expression level of insulin-like growth factor 1 (Igf1) mRNA. Igf1 mRNA expression level in secondary follicles containing theca cells was higher than that in secondary follicles without theca cells, and the granulosa cell proliferation around follicles containing theca cells was increased. Furthermore, an IGF1 inhibitor also inhibited the granulosa cell proliferation, and administration of IGF1 to secondary follicles without growth promoted granulosa cell proliferation. These results indicated that the theca cells of secondary follicles induced the expression of IGF1 and promoted the follicle growth.     

Effect of transforming growth factor-beta1, insulin-like growth factor-I,and hepatocyte growth factor on proteoglycan production and regulation in canine melanoma cell lines

OBJECTIVE:To identify extracellular proteoglycans produced by canine melanoma cell lines and analyze the effect of transforming growth factor-beta1 (TGF-beta1), insulin-like growth factor-I (IGF-I), and hepatocyte growth factor (HGF) on these proteoglycans. SAMPLE POPULATION: 3 canine melanoma cell lines (ie, CML-1, CML-6M, and CML-10c2). PROCEDURE: Extracellular proteoglycans were analyzed by use of metabolic labeling and western immunoblot analysis. The effect of TGF-beta1 on cell proliferation was determined by incorporation of 5-bromo-2'-deoxyuridine. RESULTS: The CML-1 and CML-6M melanoma cell lines produced 2 main extracellular proteoglycans. One of them was identified as versican, a proteoglycan found in undifferentiated human melanoma cell lines. The CML-10c2 cells produced a small amount of extracellular proteoglycans. Addition of TGF-beta1 (1.25 to 6.25 ng/ml) increased the release of sulfated proteoglycans into the medium. The TGF-beta1 had mainly a posttranslational effect, because it increased the molecular mass of the sulfated bands. Addition of IGF-I (50 ng/ml) slightly increased production of proteoglycans in the CML-6M cell line, whereas HGF (50 ng/ml) did not have any effect on proteoglycan production. CONCLUSIONS AND CLINICAL RELEVANCE: The proteoglycan content and response toTGF-beta1 treatment for CML-1 and CML-6M canine melanoma cell lines are similar to that for undifferentiated human melanoma cell lines. In contrast, CML-10c2 cells produced a low amount of proteoglycans with high molecular weight. Because these extracellular proteoglycans are involved in the control of cell adhesion, proliferation, and migration, they may play an important role in the progression of melanomas in dogs.     

Relationship among growth, steroid production and immunolocalization of transforming growth factor-beta 1 in the normally developing mouse follicles cultured in vitro

This study examined the relationship among growth, steroid production and transforming growth factor-beta 1 (TGF-beta 1) immunolocalization in the mouse follicles cultured in vitro to evaluate the hypothesis that normally developing follicles should express TGF-beta 1 in the granulosa cells around the time of antrum formation. Preantral follicles with 151-175 microns (large category) and 125-150 microns (small category) of initial diameters were used as models for normal and retarded follicles, respectively. Growth rate and timing of antrum formation in both categories were comparable to those of in-vivo grown follicles. At the time of antrum formation, follicular diameters were similar between the two follicle categories; however, antral follicles from the large category showed larger number of granulosa cells, higher estradiol production and proportion of follicles with TGF-beta 1 positive granulosa cells. Two days after antrum formation, there were no differences in the number of granulosa cells and the proportions of follicles with TGF-beta 1 positive granulosa or theca cells between the two categories. Temporal association in large follicles between the increase in estradiol production and proportion of follicles with TGF-beta 1 positive granulosa cells at the time of antrum formation supports our hypothesis. Furthermore, this study demonstrated the usefulness of the follicle culture system in the investigations of follicular physiology.     

Involvement of stathmin in proliferation and differentiation of immortalized human endometrial stromal cells

Uterine endometrial stromal cells differentiate into decidual cells during the late secretory phase of the menstrual cycle and pregnancy. However, the biochemical mechanisms of decidualization have yet to be definitively elucidated. In the present study, we transfected primary human endometrial stromal cell with a temperature-sensitive mutant of simian virus 40 large T antigen and thereby established an immortalized stromal cell line (EtsT) in order to examine the role of stathmin, a cytosolic phosphoprotein that regulates microtubule dynamics, in stromal cell differentiation. When treated with the decidual stimulus dibutyryl-cAMP (db-cAMP) or forskolin, the fibroblastic cell-shaped EtsT cells transformed into large- and round-shaped cells and secreted large amounts of the decidual markers prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1). Analysis of the stathmin protein levels in the db-cAMP- and forskolin-treated EtsT cells revealed that the total and phosphorylated protein levels dropped as decidualization progressed. Suppression of stathmin expression by transfection with small interfering RNA (siRNA) suppressed EtsT cell proliferation. It also abolished db-cAMP-induced PRL and IGFBP-1 mRNA expression and protein secretion. Thus, stathmin expression can be considered an integral factor regulating the initial stage of the process of human endometrial stromal cell differentiation.     

Effect of transforming growth factor-beta1 on bone regeneration in critical-sized bone defects after irradiation of host tissues

OBJECTIVE: To determine whether sustained release of transforming growth factor (TGF)-beta1 from a gelatin hydrogel would enhance bone regeneration in critical-sized long-bone defects and overcome inhibitory effects of preoperative irradiation. ANIMALS: 24 adult New Zealand White rabbits. PROCEDURE: Rabbits were allocated to 2 groups. Twelve rabbits received localized megavoltage radiation to the right ulna by use of a cobalt 60 teletherapy unit, and 12 rabbits received no irradiation. Then, a 1.5-cm defect was aseptically created in the right ulna of each rabbit. Gelatin hydrogel that contained 5 microg of adsorbed recombinant-human (rh)TGF-beta1 was placed in the defect of 12 rabbits (6 irradiated and 6 nonirradiated), and the other 12 rabbits received hydrogel without rhTGF-beta1. Rabbits were euthanatized 10 weeks after surgery. New bone formation within the defect was analyzed by use of nondecalcified histomorphometric methods. A 1-way ANOVA was used to compare differences among groups. RESULTS: New bone formation within the defect was significantly greater in TGF-beta1-treated rabbits than in rabbits treated with hydrogel carrier alone. Local delivery of rhTGF-beta1 via a hydrogel carrier in irradiated defects resulted in amounts of bone formation similar to those for nonirradiated defects treated by use of rhTGF-beta1. CONCLUSIONS AND CLINICAL RELEVANCE: Local delivery of TGF-beta1 by use of a hydrogel carrier appears to have therapeutic potential for enhancing bone formation in animals after radiation treatments. IMPACT FOR HUMAN MEDICINE: This technique may be of value for treating human patients at risk for delayed bone healing because of prior radiation therapy.     

Reduced incidence of insect-bite hypersensitivity in Icelandic horses is associated with a down-regulation of interleukin-4 by interleukin-10 and transforming growth factor-beta1

Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses caused by IgE-mediated reactions to bites of insects of the genus Culicoides. IBH does not occur in Iceland due to the absence of Culicoides. However, Icelandic horses exported to mainland Europe as adults (1st generation) have a > or =50% incidence of developing IBH. In contrast, their progeny (2nd generation) has a <10% incidence of IBH. Here we show that peripheral blood mononuclear cells (PBMC) from Icelandic horses born in mainland Europe and belonging either to the IBH or healthy subgroup produce less interleukin (IL)-4 after polyclonal or allergen-specific stimulation when compared with counterparts from horses born in Iceland. We examined a role of IL-10 and transforming growth factor (TGF)-beta1 in down-regulation of IL-4 in healthy 2nd generation Icelandic horses. Supernatants of PBMC from 2nd generation healthy horses down-regulated the proportion of IL-4-producing cells and IL-4 production in stimulated cultures of PBMC from 1st generation IBH. This inhibition was mimicked by a combination of IL-10 and TGF-beta1 but not by the single cytokines. Cultures of stimulated PBMC of healthy 2nd generation horses produced a low level of IL-4, but IL-4 production was increased by anti-equine IL-10 and anti-human TGF-beta1. This shows for the first time that in horses, IL-10 and TGF-beta1 combined regulate IL-4 production in vitro. It is suggested that in this naturally occurring IgE-mediated allergy, IL-10 and TGF-beta1 have a role in the down-regulation of IL-4-induced allergen-specific Th2 cells, thereby reducing the incidence of IBH.     

黄体生成素促进鸡小黄卵泡膜细胞增殖的作用机理

利用产蛋鸡小黄卵泡膜细胞培养模型研究黄体生成素（LH）对等级前发育的小黄卵泡膜细胞增殖的影响以及LH作用的信号转导途径。单层培养的外层膜细胞用LH（0.1～100μg/L）、蛋白激酶A（PKA）激活剂forskolin（FRSK,0.1～10μmol/L）及抑制剂H89（0.01～1μmol/L）或蛋白激酶C（PKC）激活剂佛波酯（PMA,0.1～10nmol/L）及抑制剂H7（0.01～1μmol/L）单独或共同处理,36 h后测定膜细胞增殖的变化。结果显示,LH和FRSK促进小黄卵泡膜细胞的增殖,且LH的促增殖作用可被H89阻断,而PMA无显著的促进作用,且H7不能阻断LH的促增殖作用。由此推断LH的促小黄卵泡外层膜细胞增殖作用主要是通过PKA途径进行的。     

Follicular fluid concentration of transforming growth factor-beta1 is negatively correlated with estradiol and follicle size at the early stage of development of the first-wave cohort of bovine ovarian follicles

The objectives of this study were to characterize the relationship between estradiol and transforming growth factor-β1(TGF-β1) concentrations in follicular fluid of growing bovine ovarian follicles, and to examine the effect of TGF-β1 on FSH-stimulated estradiol secretion in cultured bovine granulosa cells. Follicular fluid was collected from individual follicles >5 mm in diameter by ultrasound-guided transvaginal puncture (n = 12 heifers). Follicles were sampled at four different stages of development of the first post-ovulatory wave during selection of the single dominant follicle. Estradiol, progesterone and total TGF-β1 were measured in follicular fluid of the three or four largest follicles sampled when the largest follicle (F1) had reached either 6.5, 7.5, 8.5 or 9.5 ± 0.5 mm stage of development. There was a significant negative relationship between follicular fluid TGF-β1 and estradiol concentrations (R² = 0.44; p < 0.002), and between TGF-β1 concentrations and follicle diameter (R² = 0.23; p < 0.01) in cohort follicles at the 6.5 mm stage, but not at any later stage of development of the follicle wave. There was no correlation between progesterone and TGF-β1 concentrations at any stage. To assess the causal relationship between TGF-β1 and estradiol, granulosa cells from follicles measuring 2–5 mm at dissection were placed in serum-free culture. TGF-β1 caused a dose-dependent decrease in FSH-stimulated estradiol secretion (P < 0.001). These findings suggest that TGF-β1 has an inhibitory effect on estradiol secretion in FSH-stimulated follicles and that a reduction in TGF-β1 inhibition may be part of the mechanism of selection of a single dominant follicle.     

外源谷氨酰胺对AA肉鸡肠道早期生长发育的影响

谷氨酰胺 (Ｇｌｕｔａｍｉｎｅ ,Ｇｌｎ)作为一种重要的能量来源 ,是动物血液及其他组织含量最丰富的一种氨基酸 ,是核苷酸和某些氨基酸合成的前体物质 ,也是快速分裂细胞的主要能源物质 (Ｗｕ和Ｋｎａｂｅ,1 994) ,参与体内许多生化反应 ,具有许多生理功能。由于动物能够内源合成Ｇｌｎ ,因而长期以来一直被人们视为非必需氨基酸。近年来许多研究显示 ,当动物处于某种特殊应激状态 (饥饿、小肠受损、感染疾病等 )时 ,内源性Ｇｌｎ不能满足机体需要 ,这时外源性Ｇｌｎ在促进受损肠道的修复以及维持正常的局部免疫功能中具有积极作用。因此 ,…     

Chicken PRDX3 is required for proliferation of chicken embryo fibroblast cells

ABSTRACT

1. This experiment investigated the influence of chicken PRDX3 on cell proliferation in chick embryo fibroblast cells using PRDX3 knockdown technology.

2. A methyl thiazolyl tetrazolium (MTT) assay was performed to assess the effect of chPRDX3 knockdown on fibroblast proliferation. The antioxidant effect was investigated to determine if it directly mediated fibroblast cell proliferation.

3. To determine the role of chPRDX3 on cell proliferation, an siRNA mediated knockdown was performed in chick fibroblast cells using an in vitro assay. The proliferation of fibroblast cells transfected with siPRDX3 #3 and siPRDX3 Mix was significantly decreased after 48 h (P < 0.01). In addition, the knockdown of chicken PRDX3 suppressed cell proliferation through an increase in oxidative stress.

4. The results demonstrated that chPRDX3 is required for cell proliferation in chicken fibroblast cells. Such findings have important implications for the maintenance of chicken fibroblast cells.     

Expression of transforming growth factor-beta 1 (TGF-beta1) in different types of granulomatous lesions in bovine and ovine paratuberculosis

Paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (Map), is manifested by a broad spectrum of clinical and lesional presentations. We have evaluated the expression of transforming growth factor-beta1 (TGF-beta1), a cytokine known to have immunosuppressor effects, by immunohistochemistry, in different paratuberculosis lesions in the intestine and lymph nodes from 20 sheep and 25 cattle. Peripheral immune responses were assessed by interferon-gamma (IFN-gamma) test and the presence of antibodies. Expression of TGF-beta1, observed in macrophages and giant cells forming the lesions, was closely related to the amount of Map. In focal and multifocal forms, usually positive to IFN-gamma test, bacilli were difficult to detect and TGF-beta1 expression was low or absent. Diffuse multibacillary lesions, negative to IFN-gamma, show large numbers of Map and the highest percentage of immunolabelled cells. Diffuse paucibacillary forms, positive to IFN-gamma, have low numbers of AFB and scant or no cells positive to TGF-beta1. The high expression of TGF-beta1 would be related to the inability of macrophages to limit the multiplication of Map.     

Fluorometric detection of platelet activating factor receptor in cultured oviductal epithelial and stromal cells and endometrial stromal cells from bovine at different stages of the oestrous cycle and early pregnancy.

During the oestrous cycle and early pregnancy, the oviduct and uterus undergo a variety of morphological and physiological modifications in which the platelet activating factor receptor (PAF-R) plays an important role. PAF-R levels were quantified in bovine oviductal epithelial and stromal cells and endometrial stromal cells at days 2 to 4, 12, and 20 of the estrous cycle and during early pregnancy. Cells were grown in vitro and their intracellular PAF-R concentration was measured by flow cytometry using a polyclonal anti-PAF-R antibody system. A significant increase (P < 0.05) in the portion of PAF-R-positive oviductal epithelial and stromal cells was detected in both non-pregnant and pregnant cattle on days 2 to 4 in comparison to day 12 and 20. In endometrial stromal cells derived from day 20 pregnant bovine, a significant increase (P < 0.05) in PAF-R staining was observed in comparison to the day 20 non-pregnant and days 2 to 4 or 12 pregnant and non-pregnant animals. The PAF-R was detected in oviductal cells by using immunoblotting and immuno-gold postembedding method. Positive binding of the anti-PAF-R antibody was found on the cell membrane and in the cytoplasm. We concluded that the increased PAF-R concentration measured in cultured oviductal epithelial and stromal cells of cyclic and pregnant heifers on days 2 to 4 was hormonally regulated. The increased PAF-R in endometrial stromal cells on day 20 of pregnant heifers was a pregnancy-specific effect and may mediate a local increase in endometrial vascular permeability known to precede the implantation.