Nitric oxide stimulates progesterone and prostaglandin E2 secretion as well as angiogenic activity in the equine corpus luteum

Cytokines and nitric oxide (NO) are potential mediators of luteal development and maintenance, angiogenesis, and blood flow. The aim of this study was to evaluate (i) the localization and protein expression of endothelial and inducible nitric oxide synthases (eNOS and iNOS) in equine corpora lutea (CL) throughout the luteal phase and (ii) the effect of a nitric oxide donor (spermine NONOate, NONOate) on the production of progesterone (P4) and prostaglandin (PG) E(2) and factor(s) that stimulate endothelial cell proliferation using equine luteal explants. Luteal tissue was classified as corpora hemorrhagica (CH; n = 5), midluteal phase CL (mid-CL; n = 5) or late luteal phase CL (late CL; n = 5). Both eNOS and iNOS were localized in large luteal cells and endothelial cells throughout the luteal phase. The expression of eNOS was the lowest in mid-CL (P < 0.05) and the highest in late CL (P < 0.05). However, no change was found for iNOS expression. Luteal explants were cultured with no hormone added or with NONOate (10(-5) M), tumor necrosis factor-α (TNFα; 10 ng/mL; positive control), or equine LH (100 ng/mL; positive control). Conditioned media by luteal tissues were assayed for P4 and PGE(2) and for their ability to stimulate proliferation of bovine aortic endothelial cells (BAEC). All treatments stimulated release of P4 in CH, but not in mid-CL. TNFα and NONOate treatments also increased PGE(2) levels and BAEC proliferation in CH (P < 0.05). However, in mid-CL, no changes were observed, regardless of the treatments used. These data suggest that NO and TNFα stimulate equine CH secretory functions and the production of angiogenic factor(s). Furthermore, in mares, NO may play a role in CL growth during early luteal development, when vascular development is more intense.     

Vascular and immune regulation of corpus luteum development, maintenance, and regression in the cow

The bovine corpus luteum (CL) is a unique, transient organ with well-coordinated mechanisms by which its development, maintenance, and regression are effectively controlled. Angiogenic factors, such as vascular endothelial growth factor A and basic fibroblast growth factor, play an essential role in promoting progesterone secretion, cell proliferation, and angiogenesis. These processes are critically regulated, through both angiogenic and immune systems, by the specific immune cells, including macrophages, eosinophils, and neutrophils, that are recruited into the developing CL. The bovine luteolytic cascade appears to be similar to that of general acute inflammation in terms of time-dependent infiltration by immune cells (neutrophils, macrophages, and T lymphocytes) and drastic changes in vascular tonus and blood flow, which are regulated by luteal nitric oxide and the vasoconstrictive factors endothelin-1 and angiotensin II. Over the period of maternal recognition of pregnancy, the maternal immune system should be well controlled to accept the semiallograft fetus. The information on the presence of the developing embryo in the genital tract is suggested to be transmitted to the ovary by both the endocrine system and the circulating immune cells. In the bovine CL, the lymphatic system, but not the blood vascular system, is reconstituted during early pregnancy, and interferon tau from the embryo could trigger this novel phenomenon. Collectively, the angiogenic and vasoactive factors produced by luteal cells and the time-dependently recruited immune cells within the CL and their interactions appear to play critical roles in regulating luteal functions throughout the life span of the CL.     

Status of autophagy,lysosome activity and apoptosis during corpus luteum regression in cattle

Corpus luteum (CL) regression is required during the estrous cycle. During CL regression, luteal cells stop producing progesterone and are degraded by apoptosis. However, the detailed mechanism of CL regression in cattle has not been fully elucidated. The aim of this study was to evaluate autophagy, lysosome activity, and apoptosis during CL regression in cattle. The expression of autophagy-related genes (LC3α, LC3β, Atg3, and Atg7) and the protein LC3-II was significantly higher in the late CL than in the mid CL. In addition, autophagy activity was significantly increased in the late CL. Moreover, gene expression of the autophagy inhibitor mammalian target of rapamycin (mTOR) was significantly lower in the late CL than in the mid CL. Lysosome activation and expression of cathepsin-related genes (CTSB, CTSD, and CTSZ) showed significant increases in the late CL and were associated with an increase in cathepsin B protein. In addition, mRNA expression and activity of caspase 3 (CASP3), an apoptotic enzyme, were significantly higher in the late CL than in the mid CL. These results suggest simultaneous upregulation of autophagy-related factors, lysosomal enzymes and apoptotic mediators, which are involved in regression of the bovine CL.     

Inhibition of lipopolysaccharide-induced suppression of luteal function in isolated perfused bovine ovaries

Recently, we observed that lipopolysaccharide (LPS) suppresses corpus luteum (CL) function in isolated perfused ovaries. It remained unclear if this suppression was due to increased luteal PGF_2α secretion or LPS-induced apoptosis. Therefore, possible impacts of PGF_2α and LPS were inhibited by a non-steroidal anti-inflammatory drug (flunixin) and an endotoxin-binding agent (polymyxin B), respectively. Bovine ovaries with a mid-cycle CL were collected immediately after slaughter and perfused for 240 min. After 50 min of equilibration, either flunixin or polymyxin B (5 μg/ml of each) were added to the perfusion medium of six ovaries, respectively. All ovaries (n = 12) were treated with E. coli LPS (0.5 μg/ml) 60 min after the onset of perfusion, and received 500 I.U. of hCG after 210 min of perfusion. Progesterone and PGF_2α were measured in the effluent perfusate every 10 and 30 min, respectively. Biopsies of the CL were collected every 60 min to determine the mRNA expression of the cytokine TNFA and factors of apoptosis (CASP3, -8). Flunixin-treatment inhibited the increase of PGF_2α after LPS-challenge that was observed in the polymyxin B-treated (PX-LPS) ovaries. After hCG-stimulation, progesterone secretion increased (P < 0.05) in group PX-LPS but not in the flunixin-treated (F-LPS) ovaries. Compared to initial values before LPS-challenge, luteal mRNA expression of TNFA and CASP3 was increased (P < 0.05) in group F-LPS at 120 and 180 min, respectively, and those of CASP8 was decreased (P < 0.05) in PX-LPS at 60 and 120 min after LPS-treatment. In conclusion, although flunixin managed to inhibit PGF_2α, it did not suffice to successfully prevent LPS-induced apoptosis. However, endotoxin-binding polymyxin B resulted in luteal responsiveness to hCG after LPS-challenge.     

Local mechanisms for luteolysis in the cow: Novel roles of vasoactive substances in the luteolytic cascade within the corpus luteum

The corpus luteum (CL) in the estrous cycle in the cow is a dynamic organ which has a lifespan of approximately 17–18 days. As the CL matures, the steroidogenic cells establish contact with many capillary vessels and the CL is composed of a large number of vascular endothelial cells that can account for up to 50% of the bovine CL. Furthermore, luteal cells and endothelial cells secrete several vasoactive substances such as prostaglandin F_2α (PGF_2α), endothelin‐1 and angiotensin II. These vasoactive substances also function in regulating progesterone secretion in an autocrine/paracrine manner in the CL. The blood vessels and endothelial cells in the CL therefore have an essential role in the luteal function in the cow. Endometrial PGF_2α, the primary luteolysin in the cow, stimulates luteal vasoactive substances during luteolysis. Moreover, luteal vasoactive substances may have key roles in the regulation of luteolysis to induce vasodilatation, vasoconstriction and angiolysis. This review describes the current concept for possible roles of vasoactive substances in the luteolytic cascade within the bovine CL.     

Effect of prostaglandin F2alpha on ovarian, adrenal, and pituitary hormones and on luteal blood flow in mares

The effect of a single injection of prostaglandin F2alpha (PGF) during mid-diestrus on systemic concentrations of progesterone, LH, FSH, estradiol, and cortisol and on blood flow to the corpus luteum was studied in 10 controls and 10 PGF-treated mares. Blood flow was assessed by estimating the percentage of corpus luteum with color-Doppler signals of blood flow during real-time scanning of the entire structure and by the diameter of the vascular pedicle near its attachment to the ovary. Treatment was done 8 days after ovulation and 0 h was immediately before the treatment. Examinations and collection of blood samples were done at 0 h, every 5 min until 1h, and then at 1.5, 2, 4, 8, 12, 24, 48, and 72 h. The concentrations of estradiol did not change, but progesterone, LH, FSH, and cortisol increased significantly within 5 min. Concentrations of LH and FSH in the PGF group remained elevated until a temporarily lower concentration at 8 or 4h, respectively, rebounded to 12h, and then slowly decreased. Cortisol remained elevated, until a decrease between 1 and 4h. Progesterone in the PGF group increased significantly until 10 min after 0 h and then decreased by 40 min to below the concentrations in controls. Within the PGF group, progesterone decreased significantly by 45 min to below the concentrations at 0 h. The values for each of the two indicators of blood flow did not differ significantly between the PGF and control groups until a decrease at 24h in the PGF group. Results did not support the hypothesis that the immediate transient post-PGF increase in progesterone was associated with an increase in luteal blood flow. Luteolysis, as indicated by decreasing progesterone, began well before the beginning of a decrease in luteal blood flow.     

Endocrine Changes after Mating in Pregnant and Non-Pregnant Llamas and Alpacas

Plasma concentrations of oestradiol-17ß, progesterone, 15-keto–dihydro–PGF_2α and luteinizing hormone (LH) were monitored in llamas and alpacas after mating with an intact male. Concentrations of LH and PGF_2α metabolite were high immediately after copulation. Ovulation occurred in 92% of the animals. The first significant increases in progesterone were recorded on day 4 after mating. In non-pregnant animals the lifespan of the corpus luteum was estimated to be 8–9 days. Luteolysis occurred in association with the release of PGF_2α. In pregnant animals, a transient decrease in progesterone concentrations was observed between days 8 and 18 in both species. No significant changes in PGF_2α secretion were registered during this period. Oes– tradiol–17ß concentrations were high on the day of mating, declined to low values on day 4, and started to increase again on day 8. Peak values after luteolysis in non-pregnant animals were significantly higher than those registered in pregnant ones. Furthermore, concentrations of oestradiol-17ß were elevated for a longer period in non–pregnant than in pregnant animals. The results suggest that progesterone from the corpus luteum exerts a negative influence on follicular activity in pregnant animals by reducing oes– tradiol-17ß secretion.     

Intraluminal Pressure Variations in the Isthmus of the Porcine Oviduct after Intrauterine Insemination with Saline,Oestrogen Solution or Boar Seminal Plasma

The purpose of this study was to investigate if an intrauterine deposition of saline, boar seminal plasma, or an oestrogen solution containing 11.5 µg oestrogens affected the intraluminal pressure in the isthmus of the oviduct in unrestrained gilts. In order to monitor variations in intraluminal pressure, 2 ultra-miniature pressure sensors, located at 2 different points along the same isthmus were used. After an initial recording period, either saline, boar seminal plasma, or the oestrogen solution was deposited directly into the uterus. Intraluminal pressure recordings were conducted up to 6 h after insemination, and blood samples, for monitoring oestradiol-17B and 15-ketodihydroprostaglandin F_2α levels, were collected. None of the inseminates had any consistent effect on the intraluminal pressure in the porcine oviduct. After deposition of the oestrogen solution, increases in the circulating levels of both oestradiol-17B and 15-ketodihydropro- staglandin F_2α were seen.     

Pregnancy recognition signaling mechanisms in ruminants and pigs

Maternal recognition of pregnancy refers to the requirement for the conceptus (embryo and its associated extra-embryonic membranes) to produce a hormone that acts on the uterus and/or corpus luteum (CL) to ensure maintenance of a functional CL for production of progesterone; the hormone required for pregnancy in most mammals. The pregnancy recognition signal in primates is chorionic gonadotrophin which acts directly on the CL via luteinizing hormone receptors to ensure maintenance of functional CL during pregnancy. In ruminants, interferon tau (IFNT) is the pregnancy recognition signal. IFNT is secreted during the peri-implantation period of pregnancy and acts on uterine epithelia to silence expression of estrogen receptor alpha and oxytocin receptor which abrogates the oxytocin-dependent release of luteolytic pulses of prostaglandin F2-alpha (PGF) by uterine epithelia; therefore, the CL continues to produce progesterone required for pregnancy. Pig conceptuses secrete interferon delta and interferon gamma during the peri-implantation period of pregnancy, but there is no evidence that they are involved in pregnancy recognition signaling. Rather, pig conceptuses secrete abundant amounts of estrogens between Days 11 to 15 of pregnancy required for maternal recognition of pregnancy. Estrogen, likely in concert with prolactin, prevents secretion of PGF into the uterine venous drainage (endocrine secretion), but maintains secretion of PGF into the uterine lumen (exocrine secretion) where it is metabolized to a form that is not luteolytic. Since PGF is sequestered within the uterine lumen and unavailable to induce luteolysis, functional CL are maintained for production of progesterone. In addition to effects of chorionic gonadotrophin, IFNT and estrogens to signal pregnancy recognition, these hormones act on uterine epithelia to enhance expression of genes critical for growth and development of the conceptus.     

Protein kinase C isoforms in the porcine corpus luteum: Temporal and spatial expression patterns

Porcine corpora lutea (CL) fail to show a luteolytic response to prostaglandin-F-2α (PGF-2α) (ie, luteolytic sensitivity, or LS) until ∼day 13 of the estrous cycle. In view of the importance of protein kinase C (PRKC) in PGF-2α signal transduction, it was hypothesized that limiting levels of 1 or more PRKC isoforms may explain the lack of LS before day 13. This hypothesis was tested by examining expression of mRNA and protein, and the cellular localization patterns of the 11 PRKC isoforms throughout the porcine estrous cycle, to determine whether PRKC expression correlates with and thus may be associated with the control of the acquisition of LS in the pig. The expression patterns show that for most PRKC isoforms (ie, PRKC alpha, beta 1, beta 2, delta, epsilon, theta, iota, and zeta), mRNA was maximally expressed on day 7 or day 10 (protein kinase D1 only) of the cycle, whereas PRKCs gamma, eta, and lambda were unchanged. At the protein level, only PRKC epsilon (PRKCE) significantly changed during the estrous cycle and was elevated on day 13 (versus days 4, 7, and 15; P < 0.05). By immunofluoresence, most PRKC isoforms, including PRKCE, were localized to steroidogenic large luteal cells (LLC) and small (nonendothelial cell) luteal cell subtypes (SLC). In conclusion, since the increase in PRKCE protein expression (day 13) occurred coincidentally with the onset of LS (≥day 12), these results support a potential role for PRKCE in control of the acquisition of LS in the pig.     

The Effect of Induced Standing Reflex,Cervical Stimulation and Insemination on Intraluminal Pressure Variations in the Isthmus of the Oviduct in Unrestrained Gilts

Four gilts were each equipped with 2 ultra-miniature pressure sensors, placed at 2 different points along the same isthmus of the oviduct, on the morning of the first day of standing oestrus (Day 1). Intraluminal pressure recordings were started the same afternoon. After an initial recording period, intraluminal pressure was recorded while the gilts showed standing oestrus and during cervical stimulation followed by insemination with either 100 ml saline or 100 ml boar semen. Monitoring of the pressure variations in the isthmus was continued for up to 6.5 h after the last insemination. Blood samples for monitoring oestradiol-17B, progesterone and 15-ketodihydroprostaglandin F_2α were collected before and after each manipulation of the gilt and every 30 min during the rest of the test period. None of the above manipulations had any consistent effect on the intraluminal pressure in the porcine isthmus, although, a clear 15-ketodihydroprostaglandin F_2α peak could be seen after insemination with boar semen.     

Effects of α2-adrenergic drugs on small intestinal motility in the horse: An in vitro study

The effects of selective α₂-agonists (xylazine, detomidine and medetomidine) and antagonists (yohimbine and atipamezole) on in vitro small intestine motility in the horse were evaluated. Samples of equine jejunum were placed in isolated organ baths and drug-induced modifications of motility were measured by means of an isotonic transducer. All tested α₂-agonists dose-dependently reduced both spontaneous and electrically-evoked phasic contractions. Conversely, α₂-antagonists were ineffective when tested alone, and showed a heterogeneous and dose-independent ability to inhibit agonist activity. In particular, the antagonism exerted by higher concentrations of both yohimbine and atipamezole against α₂-agonists was weaker than when lower concentrations were used. The data are indicative of the presence of both pre- and post-synaptic α₂-adrenoceptors with inhibitory activity on equine jejunum motility, and support a possible therapeutic utility of these drugs in horse intestinal disorders associated with hypermotility.     

Effects of GnRH in combination with PGF2α on the dynamics of follicular and luteal cells in post-pubertal Holstein heifers

Holstein heifers were randomly allotted by weight, age and body condition score to one of three treatments to test the hypothesis that GnRH administration concurrent with PGF_2α injection would advance follicle or corpus luteum (CL) development parallel to an induced luteolysis of the pre-existing CL. Heifers in the control group (n = 14) received two treatments of PGF_2α(25 mg, im) given 10 days apart. Groups 2 (n = 14) and 3 (n = 14) received an additional treatment of GnRH (100 μg, im) after the first and second PGF_2α respectively. Estrus detection began immediately after PGF_2α and continued for 80 h. Blood sampling was initiated 7 days prior to the first PGF_2α (day − 7) and continued on days 0, 7, 10 (prior to the second PGF_2α), 17 and 24. Heifers were artificially inseminated after the second PGF_2α and pregnancy diagnosed at 60 days. There was a trend (P < .10) toward a lower estrus response in group 3 when compared to the other groups. Pregnant heifers in group 2 had lower progesterone (0.44 ± 0.09 vs. 1.72 ± 0.56 ng/ml) a week after the second PGF_2α than the non-pregnant animals in that group (P < .05). Similar results were observed in the control group but only within the responding heifers (0.61 ± 0.08 vs. 0.93 ± 0.03 ng/ml; P < .05). Progesterone in heifers in group 2 remained high on day 0, 7, and 10 (1.48 ± 0.37, 1.23 ± 0.39, 1.96 ± 0.36 ng/ml) in spite of the treatment with PGF_2α. This data suggest that administration of GnRH following PGF_2α alters bovine luteal and/or follicular cell function.     

Effects of hyper‐ and hypothyroidism on the development and proliferation of testicular cells in prepubertal rats

Thyroid hormones are important in the development and regulation of testes. This study was conducted to determine the effects of hyper‐ and hypothyroidism on testicular development in prepubertal rats aged 20–70 days. Weaning male rats (20 days old) until day 70 age were randomly divided into four groups: control, hyperthyroid (hyper‐T), hypothyroid (hypo‐T) and hypothyroid treated with thyroxine (T4) (hypo‐T+T4). The results indicated that thyroid hormones caused a significant effect in body and testis weights, and food and water consumption. In addition there were changes in serum concentrations of tri‐iodothyronine, T4, thyroid stimulating hormone (TSH) and testosterone. Histomorphology showed a significant decrease in seminiferous tubule diameter in hyper‐T compared to the other groups. Leydig cell numbers showed a significant elevation in hyper‐T but not in hypo‐T groups. Immunostaining indicated that TSH receptor (TSHR), thyroid hormone receptors α/β (TRαβ) and proliferating cell nuclear antigen (PCNA) have the roles in testicular development. Our findings suggest that hyper‐ and hypo‐thyroidism regulate testicular cell proliferation and spermatogenesis in prepubertal rats, indicating that expression of TSHR, TRαβ and PCNA may be regulated by thyroid hormones that are involved in testicular development; and that the administration of T4 to the hypo‐T+T4 group leads to an improvement in the testicular condition.     

Some Reproductive and Clinical Aspects of Endotoxins in Cows with Special Emphasis on The Role of Prostaglandins

Endotoxin from Salmonella typhimurium was administered intravenously to 3 cows. It produced an immediate increase in blood prostaglandin metabolite levels causing either luteolysis or a temporary drop in progesterone concentrations depending on the degree of prostaglandin synthesis. The clinical effects on heart rate, respiratory rate, ruminai contraction rate, body temperature and other general signs were studied. The cows showed a transient increase in heart and respiratory rate and a decrease in ruminai activity. The temperature responded by a small initial decrease and a secondary increase. Other symptoms included muscular weakness and shivering, excessive salivation and diarrhoea. Plasma Ca levels showed a direct decrease and then slowly increased to initial levels in the course of several days. The role of endotoxins in Gram-negative infections and their mode of action on the body are discussed with special emphasis on the role of prostaglandins on reproductive function.     

细胞自噬相关信号通路分子在猪卵巢黄体形成与退化过程中的表达

为了研究细胞自噬相关信号通路分子在猪卵巢黄体发育与退化过程中的表达,本试验以猪卵巢为对象,分别采集卵泡期卵巢(F)、早期黄体(E)、中期黄体(M)和晚期黄体(L),应用Western blot检测LC3-B、p-Akt、Akt、p-JAK2、JAK2、p-STAT3及STAT3的表达,应用免疫组织化学技术检测STAT3在卵巢中的细胞定位。Western blot结果显示,LC3-B在早期及中期黄体表达量较高(P0.01);p-Akt在卵泡期卵巢的表达量高于黄体期(P0.05),且黄体发育的不同时期差异不显著(P0.05);p-JAK2在早期黄体表达量较高(P0.05);p-STAT3在晚期黄体表达量显著升高(P0.01)。Akt、JAK2与STAT3的表达量在整个卵巢周期无显著差异(P0.05)。免疫组化结果显示,STAT3主要定位在猪卵巢卵泡的颗粒细胞、膜细胞和黄体的类固醇生成细胞中。结果表明,猪黄体细胞的自噬可能受多种信号通路的协同调控,为进一步研究黄体形成和退化的分子机制提供依据。     

Changes in the C-reactive protein and 13,14-dihydro-15-keto-prostaglandin F2α concentrations of uterine lavage samples after the administration of povidone-iodine in cows

Changes in the C-reactive protein (CRP) and 13,14-dihydro-15-keto-prostaglandin F_2α (PGFM) concentrations of uterine lavage fluid were examined in cows given an intrauterine povidone-iodine (PI) infusion. The mean polymorphonuclear leukocyte (PMN) ratios (the ratio of PMN to total cells) and CRP concentration of uterine lavage fluid on the day after the treatment were significantly (P<0.05) greater in the PI infusion group (PMN: 53.0 ± 32.7%, CRP: 50.2 ± 32.3 ng/mL) than in the non-treatment control group (PMN: 7.9 ± 21.9%, CRP: 17.2 ± 5.9 ng/mL), whereas there was no significant difference in the mean PGFM concentration between the two groups. The present findings suggest that the uterine CRP level is a useful biomarker of local uterine inflammation in cows.     

Expression of Aldo-keto Reductase 1C23 in the Equine Corpus Luteum in Different Luteal Phases

Regression of the corpus luteum (CL) is characterized by a decay in progesterone (P₄) production (functional luteolysis) and disappearance of luteal tissues (structural luteolysis). In mares, structural luteolysis is thought to be caused by apoptosis of luteal cells, but functional luteolysis is poorly understood. 20α-hydroxysteroid dehydrogenase (20α-HSD) catabolizes P₄ into its biologically inactive form, 20α-hydroxyprogesterone (20α-OHP). In mares, aldo-keto reductase (AKR) 1C23, which is a member of the AKR superfamily, has 20α-HSD activity. To clarify whether AKR1C23 is associated with functional luteolysis in mares, we investigated the expression of AKR1C23 in the CL in different luteal phases. The luteal P₄ concentration and levels of 3β-hydroxysteroid dehydrogenase (3β-HSD) mRNA were higher in the mid luteal phase than in the late and regressed luteal phases (P<0.05), but the level of 3β-HSD protein was higher in the late luteal phase than in the regressed luteal phase (P<0.05). The luteal 20α-OHP concentration and the level of AKR1C23 mRNA were higher in the late luteal phase than in the early and mid luteal phases (P<0.05), and the level of AKR1C23 protein was also highest in the late luteal phase. Taken together, these findings suggest that metabolism of P₄ by AKR1C23 is one of the processes contributing to functional luteolysis in mares.     

The innate immune response against Brucella in humans

Pathogens have developed different strategies to survive and multiply within their host. Among them is the ability to control phagocyte apoptosis while another is to affect the expression of cytokines which is necessary for a normal protective function of the immune response. To establish themselves and cause chronic disease in humans and animals, Brucella spp. invade and proliferate within monocytic phagocytes. We have established that in humans, Brucella suis impairs the apoptosis of monocytes and macrophages, thus preventing its host cell elimination. In mice, which are not naturally colonized by the bacteria, Brucella infection results in Type1 (Th1) cellular immune response which promotes a clearance of the bacterial organism. The development of this response is under the control of major cytokines like TNF-alpha, IFN-gamma and IL-12 produced at the onset of infection. We have observed that in humans, B. suis-infected macrophages which produce IL-1, IL-6, IL-10 and several chemokines including IL-8, do not secrete TNF-alpha. By constructing null mutants, we demonstrated that this inhibition involves the outer membrane protein Omp25 of Brucella, however the mechanism regulating the inhibition has not yet been clearly defined. It is likely that the Omp25-induced effect on TNF-alpha production assists bacterial evasion of antimicrobial defences at different levels. Firstly, by preventing the autocrine activation of macrophages thus inhibiting innate immunity and secondly by impairing the production of IL-12 and the development of a Th1 type specific immunity. In addition to the central role of the macrophage in Brucella infection, others cells of the innate immune response are recruited and influenced by the interactions between bacteria and host. For instance, human Vgamma9Vdelta2 T-cells play an important role in the early response to infection with intracellular pathogens. Evidence has been presented that their number dramatically increased in the peripheral blood of patients with acute brucellosis. We have shown that human Vgamma9Vdelta2 T-cells can be specifically activated by non-peptidic low molecular weight compound(s) from B. suis lysate or by soluble factors produced by B. suis-infected macrophages. Under these conditions, they produce TNF-alpha and IFN-gamma and reduce the bacterial multiplication inside infected autologous macrophages. This impairment of B. suis multiplication is due to both soluble factors released from activated gammadeltaT-cells (including TNF-alpha and IFN-gamma) and to a contact-dependent cytotoxicity directed against the infected cells. The interactions between the bacteria and these cells can counteract the intramacrophagic development of the bacteria and finally influence the further development of the host defense. We hypothesize that the chronicity or the elimination of the infection will depend on the balance between contradictory effects induced by the bacteria which favor either the host or the pathogen. Moreover, the interrelationship between the different cells must be taken into account in the analysis of the virulence of the bacteria and in the development of in vitro models of human macrophage infection.