Effect of Mycobacterium cell wall fraction on endometrial histomorphometry of mares resistant and susceptible to persistent breeding-induced endometritis.

Mycobacterium cell wall fraction (MCWF) is a biological component made up of molecules with immunostimulant properties, which is therapeutically used to modulate persistent breeding-induced endometritis (PBIE). The aim of this study was to analyze the effect of this immunomodulator on the endometrial histological structure during the diestrus of PBIE-resistant and -susceptible mares that either received treatment with MCWF or not. The experiment was conducted with 10 resistant mares (RM) and 9 susceptible mares (SM). In the first estrous cycle of the trial, all mares were inseminated with dead semen as an inflammatory stimulus (Group A); at the next cycle, all mares were inseminated with dead semen and treated with a MCWF commercial immunomodulator (Group B). In both groups, endometrial biopsies were taken on day 7 post-ovulation (diestrus). Endometrial biopsies of untreated-RM (UTRM, n = 6), untreated-SM (UTSM, n = 7) MCWF-treated-RM (TRM, n = 6) and MCWF-treated-SM (TSM, n = 6) were evaluated. They were randomly chosen as representative mares of Group A and B, respectively. The height of lining and glandular epithelia, glandular diameter, glandular density and glandular area were evaluated. The histological structure revealed lymphocytic infiltration and dilated, tortuous glands with some glandular nests, particularly in UTSM. The histomorphometrical results showed no differences (ρ > 0.05) between the analyzed groups. This would indicate that post-service treatment with the MCWF immunomodulator does not modify the endometrial histoarchitecture but, apparently, its action would be mainly based on the stimulation of the cellular and humoral immune responses.

Observaciones sobre la Morfología del Oviducto del Coipo (Myocastor coypus) / Observations on Oviduct Morphology of the Coypu (Myocastor coypus)

Se efectuaron los análisis anatómico e histoarquitectónico del oviducto del coipo (Myocastor coypus) en la madurez sexual. Se trabajó con 34 oviductos que fueron segmentados en infundíbulo, ampolla, unión ámpulo-ístmica, istmo cefálico, medio y caudal y procesados con técnicas histológicas de rutina. Los oviductos se observaron como órganos tubulares y tortuosos, con amplia superficie infundibular, marcada flexura en la unión con los cuernos uterinos y el desarrollo de un colículo en la unión útero-tubárica. La mucosa presentó plegamientos que disminuyeron en número, grado de ramificación y altura desde la ampolla hasta los comienzos de la unión útero-tubárica. El epitelio de revestimiento estuvo compuesto por células ciliadas y células secretoras. En el infundíbulo y el istmo fue cilíndrico simple, en tanto que en la ampolla fue seudoestratificado cilíndrico. En las regiones caudales del istmo se observaron criptas revestidas por un epitelio cilíndrico de aspecto secretor. La lámina propia fue de tejido conectivo laxo y muy vascularizado. La túnica muscular incrementó su grosor y complejidad desde el infundíbulo a la unión útero-tubárica.

Anatomical and histoachitectonic analyses of coypu (Myocastor coypus) oviduct were performed at sexual maturity. Thirty-four oviducts were segmented into: infundibulum, ampulla, cephalic istmus, middle and caudal. Routine histological techniques were used. Oviducts were observed as tubular and tortuous organs with a wide infundibular surface, a pronounce flexure at junction of the uterine horns and with a colliculus at the utero-tubaric junction. The oviductal wall was composed of the mucosal, muscular and serosal tunics. The mucosal tunic showed foldings that diminished in number, ramification and height from the ampulla to the beginning of the utero-tubaric junction. The lining epithelium was composed of ciliated cells as well as secretory cells. In the infundibulum and isthmus, it was simple and cylindrical, while in the ampoule it was cylindrical pseudo-stratified. In the caudal regions of the isthmus, crypts covered by a cylindrical epithelium with secretroy aspect were observed. The lamina propria was composed of highly vascularized lax connective tissue. The muscular tunic increased its thickness and complexity from the infundibulum to the utero-tubaric junction.

Comparative plasma exposure and lung distribution of two human use commercial azithromycin formulations assessed in murine model: a preclinical study.

Azithromycin (AZM) therapeutic failure and relapses of patients treated with generic formulations have been observed in clinical practice. The main goal of this research was to compare in a preclinical study the serum exposure and lung tissue concentration of two commercial formulations AZM-based in murine model. The current study involved 264 healthy Balb-C. Mice were divided into two groups (n = 44): animals of Group A (reference formulation -R-) were orally treated with AZM suspension at 10 mg/kg of b.w. Experimental animals of Group B (generic formulation -G-) received identical treatment than Group A with a generic formulation AZM-based. The study was repeated twice as Phase II and III. Serum and lung tissue samples were taken 24 h post treatment. Validated microbiological assay was used to determine the serum pharmacokinetic and lung distribution of AZM. After the pharmacokinetic analysis was observed, a similar serum exposure for both formulations of AZM assayed. In contrast, statistical differences (P < 0.001) were obtained after comparing the concentrations of both formulations in lung tissue, being the values obtained for AUC and Cmax (AZM-R-) +1586 and 122%, respectively, than those obtained for AZM-G- in lung. These differences may indicate large differences on the distribution process of both formulations, which may explain the lack of efficacy/therapeutic failure observed on clinical practice.

Immune parameters in mares resistant and susceptible to persistent post-breeding endometritis: effects of immunomodulation.

Our objective was to characterize immune parameters in susceptible (SM) and resistant (RM) mares, with and without artificial insemination (AI) and immunomodulation. Eight RM and eight SM were selected based on their reproductive history and functional tests. Both groups of mares were evaluated during three consecutive cycles: Cycle 1, untreated cycle (control); Cycle 2, AI with dead semen; Cycle 3, AI with dead semen and immunomodulation. Endometrial biopsies were taken during the three cycles as follows: Cycle 1--at estrus, when follicles > or =35mm and at diestrus (7+/-1 days after ovulation); Cycle 2--at estrus 24h post-AI, and at diestrus; Cycle 3--at estrus 24h after treatment with a Mycobacterium phlei cell-wall extract (MCWE) and AI, and at diestrus. The mRNA transcription (mRNAT) of IL-8 and IL-10 were determined by real-time PCR. Image analysis of immunohistochemistry slides was performed using digital software (Image-Pro Plus v 5.0; Media Cybernetics); the percentage of stained area was determined for Major Histocompatibility Complex II (MHC-II), polymorphonuclear leukocytes (PMN) and T lymphocytes (TL) on each tissue section. In Cycle 1, SM had significantly higher MHC-II, TL, PMN and IL-8 than RM during estrus (P<0.006, P<0.0005, P<0.05, respectively), while transcription of IL-10 was significantly lower than in RM (P<0.0001). During diestrus, SM had higher levels of TL, PMN and IL-8 than RM (P<0.0001). After AI (Cycle 2), SM had higher levels of IL-8 and lower levels of IL-10 than RM at estrus and no differences were detected for MHC-II, TL and PMN positive cells. During diestrus in the same cycle, all the immune parameters were higher in SM mares (P<0.005, P<0.0004, P<0.0001, P<0.02, respectively). When MCWE was applied at the time of AI (Cycle 3), SM expressed significant higher levels of IL-10 24h after treatment (P<0.005), which were also higher than in the control Cycle 2 or after AI (Cycle 2). However, no significant differences were detected for MHC-II, lymphocytes-PMN or IL-8 between SM and RM during diestrus in Cycle 3. This study showed that SM had higher levels of all immune parameters except IL-10 than RM during Cycle 1. After AI (Cycle 2), the inflammatory condition persisted in SM but not RM mares until day 7 post-ovulation. Following treatment with MCWE at the time of AI (Cycle 3) uterine immunological changes in SM resulted in an endometrial immune environment similar to that found in normal RM.

Endometrial IL-1beta, IL-6 and TNF-alpha, mRNA expression in mares resistant or susceptible to post-breeding endometritis. Effects of estrous cycle, artificial insemination and immunomodulation.

Endometrial mRNA expression of the pro-inflammatory cytokines interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-alpha) was assessed in mares resistant (RM) or susceptible (SM) to persistent post-breeding endometritis (PPBE). Eight RM and eight SM, were selected based on reproductive records and functional tests out of a herd of 2,000 light cross-type mares. Three experiments were done to study transcription patterns in (i) basal conditions; (ii) after artificial insemination (AI); and (iii) after administration of an immunomodulator at time of artificial insemination. Endometrial biopsies were taken during consecutive cycles: (i) at estrus, when follicles reached 35 mm and at diestrus (7 +/- 1 days after ovulation); (ii) at 24 h post-AI, with dead semen (estrus) and in diestrus; (iii) at 24 h after treatment with a Mycobacterium phlei cell-wall extract (MCWE) preparation and AI (with dead semen), and at diestrus. mRNA expression was quantitated by real time PCR. Under basal conditions, SM had significantly higher mRNA expression of all cytokines in estrus and of IL-1beta and TNF-alpha in diestrus, compared to RM. After AI, there were no differences between RM and SM in estrus; however, mRNA expression for all three pro-inflammatory cytokines was higher than under basal conditions. In diestrus, RM showed significantly lower IL-1beta and TNF-alpha mRNA expression than SM. When MCWE was administered at time of AI, no differences between cytokine induction from RM and SM were found. Globally, mRNA expression for all three cytokines correlated well among themselves when expression was high. The present study showed that (i) in basal conditions RM had lower mRNA expression of pro-inflammatory cytokines than SM with no effect of estrous cycle; (ii) AI upregulated mRNA expression for all three cytokines in both RM and SM, with persistance in diestrus in the latter; (iii) treatment with MCWE at time of AI down-regulated mRNA expression of IL-1 with significant effects in SM which behaved like RM. Immunomodulation with MCWE could be of help in restoring homeostatic local inflammatory mechanisms, thus assisting in the prophylaxis of post-breeding endometritis in mares.