Intrauterine infusion of a pathogenic bacterial cocktail is associated with the development of clinical metritis in postpartum multiparous Holstein cows.

Metritis is a uterine disorder common in dairy cattle caused by bacterial infection, with greater incidence in the early postpartum period. The disease causes delayed uterine involution, with a fetid, watery, red-brown discharge, with animals presenting different clinical signs including fever, dullness, inappetence, decreased milk yield, and dehydration. We developed an in vivo model of clinical metritis in Holstein multiparous cows using a pathogenic cocktail of Escherichia coli, Fusobacterium necrophorum, and Trueperella pyogenes. A total of 36 multiparous cows were randomly allocated to 1 of 3 treatment groups of 12 animals each. Cows assigned to the control group received an intrauterine administration of sterile saline solution, those in the low-dose group received a bacterial inoculum containing 106 cfu of Escherichia coli, Trueperella pyogenes, and Fusobacterium necrophorum; and those in the high-dose group received 109 cfu of these same organisms, all within 24 h of parturition. Clinical signs; milk yield; dry matter intake; serum concentration of acute phase proteins, metabolites, and cytokines; and bacterial counts in vaginal discharge were measured from parturition until 14 d in milk. Based on clinical signs, cows in the low-dose group had a greater incidence of metritis (83.3%) compared with the control (9%) or high-dose (25%) group. The low-dose group also had lesser dry matter intake compared with controls and an acute phase protein profile that typified metritis. The low-dose group presented greater relative abundance of bacteria from the genus Fusobacterium in the vaginal discharge compared with control cows, and the high-dose group was similar to the control group. Unexpectedly, intrauterine infusion of cows with a lower dose of bacteria was more effective than a higher dose, indicating that the bacterial load may affect metritis incidence in a nonlinear manner. These findings demonstrate that clinical metritis can be experimentally induced in postpartum multiparous Holstein cows, creating a relevant model for the study of uterine diseases.

Effects of different doses of estradiol benzoate used in a cervical relaxation protocol on the success of non-surgical embryo recovery and luteal function in superovulated ewes.

This study investigated the effectiveness of different doses of estradiol benzoate (EB) to promote cervical relaxation and their effects on luteal function and outcomes of non-surgical embryo recovery (NSER) in sheep. Multiparous (MULT) and nulliparous (NULL) crossbred Lacaune X Santa Inês ewes were superovulated and naturally bred. Seven days after progesterone withdrawal, females were randomly assigned to one of three distinct cervical relaxation protocols, consisting of i.m. treatment with 37.5 µg d-cloprostenol and different doses of EB: 0.0 mg (0.0EB group; n = 3 NULL and 14 MULT); 0.5 mg (0.5EB group; n = 4 NULL and 12 MULT) or 1.0 mg (1.0EB group, n = 6 NULL and 11 MULT) 16 h before NSER. All ewes received 50 IU of oxytocin 20 min before NSER (D17). Blood samples were collected and ultrasound exams (B-mode and color Doppler) were performed at two timepoints: immediately before d-cloprostenol and EB treatments and prior to NSER. Estrous behavior, corpora lutea count and NSER success outcomes were not affected by EB treatments nor parity (P > 0.05). Embryo recovery rate was greater for ewes in the 0.5EB group and in the NULL ewes (P < 0.05). Ovarian biometrics differed between the two evaluation timepoints in all groups (P < 0.05). Plasma estradiol increased over time, reaching a significant greater level in 1.0EB ewes compared to controls on D17 (P < 0.05), whereas progesterone concentrations decreased over time in all groups (P > 0.05). In conclusion, treatments did not affect NSER success but they did affect luteal function by altering P4 and E2 concentrations. Therefore, the NSER technique can be successfully performed in ewes with or without prior treatment with EB.

Anatomy of the mastoid triangle and morphometric sex differences.

OBJECTIVE OF THE STUDY: This study aimed to quantify the area of the mastoid triangle (MT) and assess potential morphometric differences between males and females. PATIENTS: The sample consisted of 244 dry human skulls, with biological sex known based on genetic analysis, collected from a medicolegal osteological database from Central-Western Brazil. MATERIALS AND METHODS: The study was observational, analytical, and cross-sectional. The skulls were analyzed using Heron's equation to calculate the area of the MT. The landmarks connecting each of the sides of the triangle were: Porion (Po)>Mastoidale (Ma)>Asterion (Ast). Morphometric references were calculated and compared based on sex. RESULTS: The area of the MT was nearly 14% larger in males compared to females (p<0.05). The mean MT area for the right and left sides of males were 684.11±93.25mm2 and 668.94±111.95mm2, respectively. In females, the mean MT for the right and left sides were 588.93±91.09mm2 and 582.88±102.98mm2, respectively. Right and left side measurements were significantly different (p<0.05), except for Po-Ast (p=0.232). CONCLUSION: Morphometric features regarding the MT were slightly different between males and females. Application of the MT as a dimorphic tool should be adjuvant. Moreover, this tool should be considered carefully, especially because the sex-based differences were statistically significant, but discrete between males and females.

Single dose of 300 IU hCG in the early luteal phase in superovulated ewes: Effects on corpora lutea, progesterone profile, and embryo recovery.

This study aimed to evaluate the effects of hCG treatment during the early luteal phase on ovarian function, progesterone profile, and embryo yield in superovulated ewes. Superovulated sheep were randomly assigned to receive 300 IU hCG i.m. (GhCG, n = 24) or not (GControl, n = 25) at 96 h after the removal of the progesterone (P4) device (D13). Non-surgical embryo recovery (NSER) was performed eight days after P4 withdrawal. Ultrasound evaluations were performed on D13, D14, D16, and D17. Blood samples were collected on D14, D16, and D17. Superovulation scores were recorded based on the number of corpora lutea (CL) as follows: 1 (≤ 2), 2 (3-5), 3 (6-8), and 4 (≥ 9). NSER efficiency, superovulation response, and luteal tissue area were similar in both groups (P > 0.05). Structural luteolysis tended to be higher in GControl (P = 0.07; 47.0 %) while functional luteolysis was similar in both groups (P > 0.05; 0.0 % and 5.9 %). The recovery rate was greater (P < 0.05) in GhCG (89.8 %) compared with GControl (71.0 %), with similar overall ova/embryo numbers observed for both groups (P > 0.05). GhCG showed a higher concentration of animals with a superovulatory response score of 4 (54.5 %; P < 0.05) compared with the lowest scores. Plasma progesterone on D16 was higher (P < 0.05) in GhCG ewes (11.1 ± 1.5 vs 6.9 ± 1.5 ng/mL). In conclusion, the hCG treatment improved circulating P4 and embryo recovery rate, tended to maintain luteal functionality, and thus constitutes an additional tool for improving embryo yield in superovulated ewes.

Chronic Aerobic Training at Different Volumes in the Modulation of Macrophage Function and in vivo Infection of BALB/c Mice by Leishmania major.

Physical inactivity is one of the main causes of chronic diseases; however, strenuous exercise can induce immunosuppression. Several studies suggest that moderate amounts of exercise lead to a Th1 response, favoring the resolution of infections caused by intracellular microorganisms, while high volumes of exercise tend to direct the response to Th2, favoring infection by them. Leishmaniasis is a parasitic disease promoted by parasites of the Leishmania genus, with clinical manifestations that vary according to the species of the parasite and the immune response of the host. The experimental Leishmania major-BALB/C mouse model provides a good model for the resistance (Th1 response) or susceptibility (Th2 response) that determines the progression of this infection. The aim of this study was to evaluate the effect of aerobic training at different volumes on modulation of in vitro macrophage infection by L. major, as well as to assess the effect of high volume (HV) aerobic training on the development of L. major in vivo in BALB/c mice. Uninfected animals were submitted to various exercise volumes: none (SED), light (LV), moderate (MV), high (HV), very high (VHV), and tapering (TAP). The macrophages of these animals were infected by L. major and the LV and MV groups showed a decrease in the infection factor, while the VHV showed an increase in the infection factor, when treated with LPS. The cytokine concentration pattern measured in the supernatants of these macrophages suggested a predominant Th1 response profile in the LV and MV groups, while the Th2 profile predominated in the VHV and TAP groups. Groups of BALB/C mice infected with L. major were subjected to high volume (iHV) or non-periodized high volume (iNPHV) exercise or kept sedentary (iSED). The exercised animals suffered a significant increase in injuries caused by the parasites. The animals in the group submitted to high volume exercise (iHV) showed visceralization of the infection. These data strongly suggest that a very high volume of aerobic training increased the susceptibility of BALB/C mice to L. major infection, while moderate distribution of training loads promoted immunological balance, better controlling the infection by this parasite.

Short communication: Does previous superovulation affect fertility in dairy heifers?

The aim of this study was to evaluate the potential negative effects of superovulation on subsequent fertility of dairy heifers. Holstein heifers (n = 1,783), 312 to 387 d of age, and 273 to 307 kg of body weight (BW) from 2 commercial farms were enrolled. These animals were first selected to be donors (446) or nondonors (CON, n = 1,327) according to their genomic values. Nondonor heifers (CON) were artificially inseminated (AI) according to standard procedures of each farm after reaching 320 kg of BW. Donor heifers were superovulated using a fixed FSH dose (180 mg NIH-FSH-P1; Folltropin-V, Vetoquinol Brasil, Mairiporã, SP, Brazil) and embryos were collected following standard procedures. Heifers that produced fewer than 8 viable embryos after first superovulation (SOV1, n = 337) were no longer used as donors, whereas the remaining heifers (SOV2, n = 109) were superovulated a second time within an interval of 48 to 54 d. Donor heifers (SOV1 and SOV2) were AI once they reached 320 kg of BW, at least 15 d after the last embryo collection. Data on age at first AI, at conception, and at parturition, as well as the number of services per conception, were analyzed by ANOVA, using the PROC MIXED procedure of SAS (SAS Institute Inc., Cary, NC) procedure. Binomial variables (pregnancy per AI, overall pregnancy rate, open heifers at 500 d age, and late pregnancy loss) were analyzed using the GLIMMIX procedure of SAS. The heifers selected to undergo superovulation twice (SOV2) yielded more total (12.6 ± 5.3 vs. 6.8 ± 4.6; respectively) and viable embryos (8.5 ± 3.8 vs. 3.9 ± 2.8; respectively) than those superovulated only once (SOV1). Age at first AI, conception, and at parturition was greater in SOV2, but not in SOV1 compared with nondonor controls. In addition, pregnancy per first AI, overall pregnancy rate, services per conception, open heifers at 500 d of age, and occurrence of pregnancy losses after 60 d of gestation were similar among CON, SOV1, and SOV2 heifers. In summary, a single superovulation performed before heifers reach a minimum weight for breeding did not affect age at conception, calving or other indicators of reproductive efficiency. On the other hand, heifers superovulated twice were first inseminated at a later age than their birth cohorts, but had similar reproductive performance.

Differential expression of LHCGR and its isoforms is associated to the variability in superovulation responses of Gir cattle.

The aim of this study was to evaluate the pattern of expression of LHCGR isoforms in Gir heifers characterized as good (10.3 ±â€¯1.2 ova/embryos per flush, n = 5) or poor responders (1.1 ±â€¯0.3 ova/embryos per flush, n = 5) to superovulation protocols. In both groups, an adapted ultrasound-guided follicular aspiration system was used to collect granulosa cells from 8 mm follicles formed either during a synchronized, non-stimulated follicular wave (no stimulation control, NS) or on the fourth day of a superovulation protocol (SOV) induced with 200 IU of pFSH. The recovered follicular fluid was centrifuged and granulosa cells were washed with NaCl 0.9% and kept in RNAlater®. RNA extraction was performed using a commercial RNeasy Micro Kit and eluted samples were quantified and reverse transcribed using the commercial Superscript III kit. cDNA samples were amplified by real-time PCR using a primer to target LH/hCG receptor gene - not selective for LHCGR isoforms (total LHCGR) - and four sets of isoforms selective primers (S1, S10, S10 + 11, and S11). Analyses were performed using the REST software and expression levels are shown as mean ± SEM. Under physiological conditions (NS), poor responders had a higher expression of total LHCGR (4.9 ± 1.7 fold-change, P < 0.01) as well as isoforms S10, S11 and S10 + 11, compared to good responders. In both phenotypes, superovulation down-regulated total LHCGR expression (-0.5 ± 0.2 and -0.9 ± 0.0 for good and poor responders, respectively; P < 0.05). However, in poor responders the exogenous FSH treatment up-regulated the S10 (2.4 ± 2.0; P < 0.05), S10 + 11 (3.8 ± 3.2; P < 0.01), and S1 isoforms (1.8 ± 1.3; P < 0.05), compared to good responders We conclude that down-regulation of total LHCGR, associated to up-regulation of their inactive isoforms, may have compromised follicle development and thus contributed to the low efficiency of superovulation in heifers with a poor responder phenotype.

Hydrometra in dairy goats: Ultrasonic variables and therapeutic protocols evaluated during the reproductive season.

Hydrometra is characterized by the accumulation of fluid within the uterus due to the persistence of corpus luteum. The diagnosis of this disorder occurs with an ultrasonic exam. This study evaluated uterine drainage and fertility rates in goats after the use of d-cloprostenol in association or not with Gonadotropin-releasing hormone (GnRH) treatment. Twenty Saanen goats, diagnosed with hydrometra, received three 37.5-µg doses of d-cloprostenol laterovulvarly at 10-day intervals. On D5, the goats were assigned into two groups receiving 1 mL of GnRH or saline solution intramuscularly. Ultrasonography (US) was performed from D0 to D25. An US approach was used to rank hydrometra in scores. The pregnancy rate was assessed 45 and 90 days after the end of treatment. The uterine fluid was totally drained after the first and second administration of d-cloprostenol in 50% and 95% of the goats, respectively. In one female, full emptying of the uterus occurred only after D20. US performed at 45 and 90 days after the end of treatment indicated there was a pregnancy rate of 45.0% and 55.0%, respectively. Fertility did not differ between the GnRH-treated and control goats. Those goats not pregnant at 45 days had a follicular cyst, hydrosalpinx or hydrometra. At 90 days, no change was observed in the hydrosalpinx, and four goats had hydrometra. The use of three doses of d-cloprostenol 10 days apart was efficient for induction of draining the contents of the uterus, resulting in a relatively acceptable pregnancy rate. This treatment associated with the US approach can be important when applied in the field.

Identification of potential embryokines in the bovine reproductive tract.

Knowledge of the molecules used by the maternal reproductive tract to regulate development of the preimplantation embryo is largely incomplete. The goal of the present experiment was to identify candidates for this function. The approach was to assess expression patterns in the endometrium and oviduct of 93 genes encoding for hormones, growth factors, chemokines, cytokines, and WNT-related molecules. Results show that all of the genes were expressed in the reproductive tract. Expression in oviduct was affected by day of the estrous cycle for 21 genes with 11 genes having highest expression at estrus (CCL21, CTGF, CXCL10, CXCL16, DKK3, FGF10, IL18, IL33, IL34, PGF, and SFRP2), 1 gene at d 3 (WNT4), 8 at d 5 (BMP7, HGF, IL6, SFRP1, TGFB1, WIF1, WNT2, and WNT5A), and 1 at d 7 (IK). For endometrium, expression of 34 genes was affected by day of the estrous cycle with 11 having highest expression at d 0 (BMP7, CCL14, CCL21, CCL26, CTGF, CXCL12, IGF2, IL16, IL33, SFRP2, and WIF1), 2 at d 3 (HDGF, IL15), 14 at d 5 (CSF2, CX3CL1, CXCL3, FGF1, FGF2, GRO1, HGF, IGF1, IL1B, IL8, SFRP1, SFRP4, WNT5A, and WNT16), and 7 at d 7 (CXCL16, FGF13, HDGFRP2, TDGF1, VEGFB, WNT7A, and WNT11). Results are consistent with a set of genes regulated by estradiol early in the estrous cycle and another set regulated by progesterone later in the cycle. The cell-signaling genes identified here as being expressed in the oviduct and endometrium could serve to regulate early embryonic development in a stage-of-pregnancy-specific manner.

Potential drug-drug interactions and polypharmacy in institutionalized elderly patients in a public hospital in Brazil.

WHAT IS KNOWN ON THE SUBJECT?: Older individuals constitute an increasing proportion of the population, and therefore, are the major consumers of drugs. The elderly, especially those with mental disabilities, frequently develop chronic diseases and start using numerous drugs. Drug-drug interactions (DDIs) are a major clinical problem in the elderly population, and previous studies have focused only on antidepressants and others types of drugs used to treat mental health conditions. WHAT THIS ARTICLE ADDS TO EXISTING KNOWLEDGE?: This study shows that in hospitalized elderly patients with mental disorders (aged 60-69 years), polypharmacy (≥5 drugs) and the use of drugs that act on the cardiovascular, respiratory and nervous systems can lead to potential drug-drug interactions. Moreover, it was reported that the prevalence of drug-drug interactions in elderly patients with mental disorders was high during their hospitalization in a public hospital in Brazil. WHAT ARE THE IMPLICATIONS FOR PRACTICE?: Nurses should know the factors associated with drug-drug interactions in hospitalized elderly patients with mental disorders to choose appropriate strategies for avoiding treatment failure and adverse events in patients. ABSTRACT: Introduction Despite the impact on patient safety and the fact that prevalence is higher in older patients, previous research did not analyse drug-drug interactions (DDIs) in view of nursing care of elderly psychiatric patients. Aim To identify potential drug-drug interactions and polypharmacy in prescriptions of aged inpatients with psychiatric disorders and analyse associated factors. Methods In this retrospective cross-sectional study, we analysed the medical records of institutionalized patients diagnosed with psychiatric disorders (n = 94), aged >60 years, and prescribed multiple medications. Drug prescriptions were checked at admission, midway through and the last prescription. Factors associated with DDI occurrence were assessed using multivariable logistic regression analysis. Results A DDI prevalence potential of 67.0%, 74.5% and 80.8% occurred in patients at admission, midway through hospitalization and the last prescription, respectively. Most of the prescribed drugs were nervous system agents. A high percentage of serious and contraindicated potential DDIs occurred. Age between 60 and 69 years, use of cardiovascular and respiratory system drugs, and the number of medications contributed significantly to DDI. Implications for mental health nursing Knowledge on the factors associated with DDIs in patients with mental disorders can contribute to the improvement of effectiveness and safety of nursing care.

Intrafollicular oestradiol production, expression of the LH receptor (LHR) gene and its isoforms, and early follicular deviation in Bos indicus.

The aim of the present study was to characterise the roles of intrafollicular oestradiol production and granulosa cell (GC) expression of the LH receptor (LHR) gene and its isoforms during follicular deviation in Bos indicus. Follicular wave emergence was synchronised in heifers from a Bos taurus dairy (Holstein; n=10) and a B. indicus dairy breed (Gir; n=10). Follicles were aspirated individually at sizes corresponding to the periods of predeviation, deviation and postdeviation. Intrafollicular oestradiol (IF-E2) and progesterone (IF-P4) concentrations were determined in the follicular fluid (FF) by radioimmunoassay, and relative expression of P450 aromatase (CYP19A1) and LHR forms was evaluated in GC using real-time quantitative-polymerase chain reaction. Despite differences in the size of the dominant follicle at deviation, changes in CYP19A1 expression and IF-E2 concentrations were similar in follicles of the same diameter in both breeds. A peak in total LHR expression occurred after follicular deviation in association with low expression of LHR isoforms. The results suggest that regulation of LHR function by sequential changes in the expression pattern of LHR isoforms may play a role in the early deviation of the dominant follicle, as observed in B. indicus breeds.

Associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) in colorectal liver metastasis: the radiologist's perspective.

PURPOSE: Hepatic resection is the only potentially curative treatment for patients with colorectal liver metastasis (CRLM). Many multidisciplinary approaches, including the associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) procedure, have been proposed to increase the resectability rate in these patients. ALPPS is the most recently described staged liver resection technique, representing an advantageous strategy to induce a rapid and marked increase in the future liver remnant (FLR) volume. The aim of this article is to describe the radiological evaluation of this procedure and its variation. METHODS: This retrospective study included 9 patients with CRLM who underwent the ALPPS procedure. Abdominal imaging studies were reviewed, with an emphasis on a rational radiological approach. The number of liver metastases, the FLR volume (pre- and postportal vein ligation), anatomical variations, potential pitfalls related to disease progression, and postoperative complications were evaluated. RESULTS: The types of hepatic resection included 4 classical ALPPS cases, 3 right ALPPS variations, and 2 left ALPPS variations. The mean FLR volume calculated in the initial evaluation was 453 mL (213-790 mL). Following the first surgery, the mean FLR volume increased to 634 mL (410-957 mL), which indicated a mean volume increase of 181.1 mL (95% CI 149.7-212.5 mL; p < 0.001) and a mean absolute volume increase of 48% (19%-88%). CONCLUSION: The ALPPS procedure is an emerging form of two-stage hepatectomy. In this context, radiologists should provide crucial preoperative and perioperative information that may change surgical planning and contribute to an improvement in the oncologic outcome.

Efficacy of induction of luteolysis in superovulated cows is dependent on time of prostaglandin F2alpha analog treatment: effects on plasma progesterone and luteinizing hormone profiles.

The objectives were to (1) evaluate the effectiveness of induction of luteolysis in superovulated (SOV) cows at two distinct time points after embryo flushing; and (2) compare the pattern of LH release after treatment with PGF in cows with single vs. multiple ovulations. In the first experiment, Holstein cows were SOV with 400 IU of FSH following standard procedures. Uterine flushing for embryo recovery was performed 7 days after artificial insemination (Day 0), and cows were randomly allocated into two groups to receive PGF (0.5-mg sodium cloprostenol, intramascular) either immediately after flushing (Day 7 group, N = 19) or 4 days later (Day 11 group, N = 20). Time of luteolysis was determined on the basis of plasma progesterone (P4) concentrations. There was no difference (P > 0.05) in plasma P4 before treatment between Day 7 and Day 11 groups. A decline in plasma P4 was observed 48 hours after PGF treatment in both the groups (P < 0.0001). In Day 11 cows, P4 continued to decrease thereafter, whereas Day 7 animals had no further reduction in plasma P4. Luteolysis (P4 < 1 ng/mL) occurred in all Day 11 cows. In the Day 7 group, however, luteolysis failure was observed for 11 of 19 cows (57.9%). In cows without luteolysis, plasma P4 increased after the initial PGF-induced decline. The second experiment compared luteolysis in (SOV, N = 6) vs. non-SOV (control, N = 8) cows. Both groups received a single PGF treatment on Day 11 after estrus, and luteolysis was monitored daily by ovarian ultrasonography and plasma P4 measurements. In addition, plasma LH was measured in blood samples taken every 20 minutes for 1 hour during five consecutive days after treatment. A similar percentage of reduction in P4 was observed in both groups 24 hours after treatment; however, SOV cows only reached plasma P4 values similar (P > 0.05) to controls 96 hours after treatment. There was no difference in initial LH values between SOV and controls (P > 0.05). The slower decrease in plasma P4 in the SOV group prevented an increase in LH for up to 96 hours after luteolysis induction, whereas LH values increased (P < 0.05) in controls 24 hours after treatment. In conclusion, (1) luteolysis may fail or be incomplete when PGF treatment is given on the day of uterine flushing (Day 7) in SOV cows; (2) induction of luteolysis 4 days later (Day 11) is effective, but the initial high-plasma P4 concentrations result in a slower slope of P4 decline to basal levels, and consequently, delayed increase in LH pulses.

Influence of the addition of ß-TCP on the morphology, thermal properties and cell viability of poly (lactic acid) fibers obtained by electrospinning.

Electrospinning is a simple and low-cost way to fabricate fibers. Among the various polymers used in electrospinning process, the poly (lactic acid) (PLA) stands out due to its excellent biodegradability and biocompatibility. Calcium phosphate ceramics has been recognized as an attractive biomaterial because their chemical composition is similar to the mineral component of the hard tissue in the body. Furthermore, they are bioactive and osteoinductive and some are even quite biodegradable. The beta-tricalcium phosphate (ß-TCP) particles were synthesized by solid state reaction. Different contents of ß-TCP particles were incorporated in polymer matrices to form fibers of PLA/ß-TCP composites by electrospinning aiming a possible application as a scaffold for tissue engineering. The fibers were characterized by scanning electron microscopy (SEM), infrared (FTIR), differential scanning calorimetry (DSC) and thermal gravimetric analysis (TGA). The average diameter of the fibers varied in the range of 260-519.6 ± 50 nm. The presence of ß-TCP particles promoted changes on thermal properties of the fibers. The composite with 8 wt-% of ß-TCP showed a low degree of crystallinity and can be used for application in tissue engineering. The cell viability was analyzed by reduction of the methyl tetrazolium salt by the pyruvate dehydrogenase enzymatic complex present in the matrix of mitochondria (MTT test). All PLA fiber groups, with different contents of ß-TCP, showed cytocompatibility ability with non-cytotoxicity effect and bioactive properties using SBF assay.

Allergic reaction in a cohort of haemophilia A patients using plasma-derived factor VIII (FVIII) concentrate is rare and not necessarily triggered by FVIII.

In contrast to haemophilia B, allergic manifestations are rare complications in haemophilia A (HA) patients treated with factor VIII (FVIII) concentrates. Nevertheless, it can be serious and hamper replacement therapy in these cases. The aims of this study were to evaluate the frequency of allergic reaction in a cohort of HA patients treated only with plasma-derived FVIII (pdFVIII) concentrates, and assess the possible immune mechanisms involved. History of allergic reaction was retrospectively assessed. Patients with allergic manifestations were followed, and had plasma samples collected in different timepoints in relation to the allergic episode. These samples were analysed for the presence of inhibitor and anti-FVIII immunoglobulins subclasses. Three of 322 HA patients (0.9%) developed allergic reaction after exposure to pdFVIII products during the last 15 years in our centre. The first patient, with severe HA, without inhibitor, had anti-pdFVIII IgE and IgG4, but no anti-recombinant FVIII (rFVIII) IgE. The second patient, with severe HA, and high-responding inhibitor, presented allergic manifestation with both, pdFVIII concentrate and activated prothrombin complex concentrate. Although anti-pdFVIII and anti-rFVIII IgG4 were detected, no anti-FVIII IgE was present. The third patient, with moderate HA without inhibitor, atopic, had no anti-FVIII immunoglobulin detected, and allergic symptoms disappeared after switching to rFVIII concentrate. This study corroborates the low incidence of allergic reactions in HA patients. In the three cases presented, the anti-FVIII immunoglobulin profile demonstrated that the allergic manifestation was triggered by other proteins contained in pdFVIII products, and not directed to FVIII.

A longitudinal evaluation of anti-FVIII antibodies demonstrated IgG4 subclass is mainly correlated with high-titre inhibitor in haemophilia A patients.

The development of inhibitory antibodies against factor VIII (FVIII) (inhibitor) is the major complication in haemophilia A patients. The FVIII-binding antibodies development comprises a polyclonal immunoglobulin (Ig) G response. Recent studies showed strong correlation between the presence of neutralizing anti-FVIII antibodies (inhibitors) and IgG4 subclass. The aim of this study was to evaluate anti-FVIII IgG subclasses in haemophilia A patients with inhibitor both in a cross-sectional and in a longitudinal analysis. Inhibitors were determined by Nijmegen-Bethesda assay. Anti-FVIII IgG subclasses were performed by ELISA, and samples from 20 healthy individuals were used to validate the test. We studied 25 haemophilia A patients with inhibitor, previously treated exclusively with plasma-derived FVIII concentrates or bypassing agents. The IgG subclasses distributions were evaluated in two groups of patients classified according to inhibitor response. IgG1 and IgG4 antibodies were most prominent in haemophilia A patients with inhibitors when compared with IgG2 and IgG3. This study reports for the first time the behaviour of FVIII-binding IgG1 and IgG4 subclasses in a longitudinal analysis, in a clinical setting, of high-response inhibitor haemophilia A patients, showing the correlation of IgG4 and the inhibitor titres. In spite of being considered a non-pathologic antibody subclass with anti-inflammatory properties in other situations, IgG4 is correlated with the presence of high-titre inhibitor in the haemophilia setting. The comprehension of the IgG4 role in immune response may be crucial to establish the process for designing specific tolerance to FVIII.

The effects of conjugated linoleic acid isomers cis-9,trans-11 and trans-10,cis-12 on in vitro bovine embryo production and cryopreservation.

Conjugated linoleic acid (CLA) isomers can affect the lipid profile and signaling of cells and thereby alter their function. A total of 5,700 bovine oocytes were used in a structured series of experiments to test the effects of CLA cis-9,trans-11 and CLA trans-10,cis-12 in vitro. In experiment 1, high doses of each CLA isomer during in vitro maturation (IVM) were compared with high or low doses during the entire in vitro culture (IVC) of parthenogenetic embryos. High doses of the CLA isomers ranged from 50 to 200 µM and low doses were 15 and 25 µM. In experiment 2, the low doses of each CLA isomer were tested during IVM/IVC on embryos produced by in vitro fertilization (IVF). Experiment 3 compared the effects of 15 µM doses of each CLA isomer during IVM or IVC of IVF embryos. In experiment 4, post-rewarming survival rates and blastomere counts were assessed for embryos supplemented with each CLA isomer during IVM or for 36 h before vitrification. In experiment 1, when either CLA isomer was provided only during IVM, we observed no effects on overall rates of maturation, cleavage, or blastocysts (92.2 ± 1.6%, 78.3 ± 4.1%, and 28.9 ± 5.1%, respectively). However, high doses of each CLA isomer, but not low doses, during the entire embryo culture period decreased blastocyst rates (5-20%) in a dose-dependent manner. Cleavage rates improved with 15 or 50 µM CLA trans-10,cis-12. Progesterone concentrations in maturation media were significantly increased by high doses of each CLA isomer compared with control, but low doses of CLA isomers had no effect. In experiment 2 with IVF embryos, low doses of each CLA isomer did not alter cleavage rates (average 84.9 ± 1.9%) and only 25 µM CLA trans-10,cis-12 during IVC reduced blastocyst rates below those of controls (25.5 ± 2.1 vs. 38.2 ± 2.3%). The lipid content of embryos was increased and relative expression of the BIRC5 (baculoviral IAP repeat containing 5) gene was depressed by CLA trans-10,cis-12. In experiment 3, low doses (15µM) of each CLA isomer during IVC significantly reduced blastocyst rates (20.6 ± 2.4% and 27.7 ± 1.2% vs. 34.18 ± 1.2% for CLA trans-10,cis-12 and CLA cis-9,trans-11 compared with control, respectively) with less effect of each CLA during IVM. In experiment 4, adding 100 µM CLA cis-9,trans-11 during the final 36 h of culture resulted in a high survival rate after rewarming and culture, and the higher embryo blastomere count was comparable to that of control embryos not undergoing vitrification. In conclusion, supplementation with either CLA isomer did not improve embryo production, but inclusion of CLA cis-9,trans-11 before vitrification improved the quality of bovine IVF embryos after rewarming and culture.

Preventive effect of CuCl2 on behavioral alterations and mercury accumulation in central nervous system induced by HgCl2 in newborn rats.

This study investigated the benefits of Cu preexposition on Hg effects on behavioral tests, acetylcholinesterase (AChE) activity and Hg, and essential metal contents in the cerebrum and cerebellum of neonate rats. Wistar rats received (subcutaneous) saline or CuCl2 ·2H2O (6.9 mg/kg/day) when they were 3 to 7 days old and saline or HgCl2 (5.0 mg/kg/day) when they were 8 to 12 days old. Mercury exposure reduced the performance of rats in the negative geotaxis (3-13 days) and beaker test (17-20 days), inhibited cerebellum AChE activity (13 days), increased cerebrum and cerebellum Hg (13 days), cerebrum Cu (13 days), and cerebrum and cerebellum Zn levels (33 days). The performance of rats in the tail immersion and rotarod tests as well as Fe and Mg levels were not altered by treatments. Copper prevented all alterations induced by mercury. These results are important to open a new perspective of prevention and/or therapy for mercury exposure.