Vitrification protocol for immature Brycon orbignyanus ovarian tissue as an extinction escape strategy.

Piracanjunba (Brycon orbignyanus) is an endangered South American fish, and ovarian tissue cryopreservation is an alternative method for preserving maternal germplasm and genetic diversity. Therefore, our aim was to test a vitrification protocol for ovarian tissue containing primary growth (PG) oocytes of B. orbignyanus as a strategy to avoid the threat of extinction. Two vitrification solutions were evaluated (VS1: 1.5 M methanol + 4.5 M propylene glycol and VS2: 1.5 M methanol + 5.5 M Me2SO) and compared using control/fresh ovarian tissue. After vitrification, the following factors were analyzed: membrane integrity using trypan blue, morphology using a histological assessment, oxidative stress (total reactive antioxidant potential (TRAP) and reduced thiol [-SH]), mitochondrial activity using MTT, and DNA damage using a comet assay. The vitrified oocytes (VS1 = 24.3 ± 0.49% and VS2 = 24.8 ± 0.69%) showed higher DNA damage than the control group (control = 20.7 ± 1.03%) (P = 0.004). In contrast, in most evaluations (membrane integrity, membrane damage, oxidative stress, and mitochondrial activity), there were no discernible differences between the control group and the vitrified samples. In addition, oocyte (P = 0.883) and nuclear diameter (P = 0.118) did not change after vitrification. VS2 treatment resulted in higher nuclear damage (15.7 ± 1.45%) than in the control treatment (3.5 ± 1.19%); however, VS1 treatment did not result in significantly more damage (9.5 ± 3.01%) than in the control (P = 0.015). Therefore, the protocol for ovarian tissue vitrification tested in this study resulted in high maintenance of PG oocyte cell integrity, making it a promising alternative for B. orbignyanus maternal genome preservation.

Genotype by environment interaction for Holstein cattle populations using autoregressive and within- and across-country multi-trait reaction norms test-day models.

The progenies of international bulls in diverse climatic conditions and management levels may lead to different expressions of their genetic potential resulting in a re-ranking of these bulls. Therefore, evaluate the presence of genotype by environment interaction (G×E) within and across countries is important to guide the decision-making on alternative selection strategies. Thus, a two-step reaction norm (RN) approach was used to investigate the presence of G×E in Portuguese and Brazilian Holstein cattle. In step 1, we performed a within-country genetic evaluation using an autoregressive model to obtain precorrected phenotypes and environmental gradients (herd test-day solutions, HTD levels). In step 2, the precorrected phenotypes were considered as two distinct traits in a bi-trait RN model to estimate variance components across HTD levels, genetic correlation between HTD levels in Portugal and Brazil, and RN of the estimated breeding values. Additionally, the genetic correlation between countries using a bi-trait random regression (RR) sire model was obtained. In step 1, genetic additive variance for milk yield (MY) in Portugal was 14.1% higher than in Brazil. For somatic cell score (SCS), the genetic additive variance in Portugal was 12.7% lower than in Brazil. Although similar heritability estimates for SCS were observed in both countries, MY heritabilities were 0.31 for Portugal and 0.23 for Brazil. Genetic correlations (SD) between both countries obtained using RR sire model were 0.78 (0.051) for MY and 0.75 (0.062) for SCS. In step 2, MY genetic correlations among HTD levels within countries were higher than 0.94 for Portugal and 0.98 for Brazil. Somatic cell score genetic correlations among HTD levels ranged from 0.70 to 0.99 for Portugal and from 0.84 to 0.99 for Brazil. The average (SD) of genetic correlation estimates between Portuguese and Brazilian HTD levels were 0.74 (0.009) for MY and 0.57 (0.060) for SCS. These results suggest the presence of G×E for MY and SCS of Holstein cattle between both countries. Although there was no indication of G×E between Brazilian herd environments, the low genetic correlation for SCS indicates potential re-ranking of bulls between extreme environmental gradient in Portugal. Overall, the results of this study evidence the importance of national and international genetic evaluation systems to assist dairy farmers in the selection of the best genotypes to obtain the expected returns from investments in imported semen and to realize genetic progress in dairy populations under local environmental conditions.

Genetic evaluation for latent variables derived from factor analysis in broilers.

1. The aim of this study was to investigate the associations between several carcass, performance and meat quality traits in broilers through factor analysis and use the latent variables (i.e. factors) as pseudo-phenotypes in genetic evaluations.2. Factors were extracted using the principal components method and varimax rotation algorithm. Genetic parameters were estimated via Bayesian inference under a multiple-trait animal model.3. All factors taken together explained 71% of the original variance of the data. The first factor, denominated as 'weight', was associated with carcass and body weight traits; and the second factor, defined as 'tenderness', represented traits related to water-holding capacity and shear force. The third factor, 'colour', was associated with traits related to meat colour, whereas the fourth, referenced as 'viscera', was related to heart, liver and abdominal fat.4. The four biological factors presented moderate to high heritability (ranging from 0.35 to 0.75), which may confer genetic gains in this population.5. In conclusion, it seems possible to reduce the number of traits in the genetic evaluation of broilers using latent variables derived from factor analysis.

A note on transgenerational epigenetics affecting egg quality traits in meat-type quail.

1. The aim of the following experiment was to estimate transgenerational epigenetic variance for egg quality traits using genealogical and phenotypic information in meat-type quail. Measured traits included egg length (EL) and width (EWD), albumen weight (AW), shell weight (SW), yolk weight (YW) and egg weight (EW). 2. A total of 391 birds were evaluated for egg quality by collecting a sample of one egg per bird, during three consecutive days, starting on the 14th d of production. Analyses were performed using mixed models including the random epigenetic effect. Variance components were estimated by the restricted maximum likelihood method. A grid-search for values for the auto-recursive parameter (λ) was used in the variance components estimation. This parameter is directly related to the reset (v) and epigenetic transmissibility (1 - v) coefficients. 3. The epigenetic effect was not significant for any of the egg quality traits evaluated. Direct heritability estimates for egg quality traits ranged in magnitude from 0.06 to 0.33, whereby the higher estimates were found for AW and SW. Epigenetic heritability estimates were low and close to zero (ranging from 0.00 to 0.07) for all evaluated traits. 4. The current breeding strategies accounting for additive genetic effect seem to be suitable for egg quality traits in meat-type quail.

Transgenerational epigenetic variance for body weight in meat quails.

We aimed to estimate transgenerational epigenetic variance for body weight using genealogical and phenotypic information in meat quails. Animals were individually weighted from 1 week after hatching, with weight records at 7, 14, 21, 28, 35 and 42 days of age (BW7, BW14, BW21, BW28, BW35 and BW42, respectively). Single-trait genetic analyses were performed using mixed models with random epigenetic effects. Variance components were estimated by the restricted maximum likelihood method. A grid search for values of autorecursive parameter (λ) ranging from 0 to 0.5 was used in the variance component estimation. This parameter is directly related to the reset coefficient (ν) and the epigenetic coefficient of transmissibility (1-ν). The epigenetic effect was only significant for BW7. Direct heritability estimates for body weight ranged in magnitude (from 0.15 to 0.26), with the highest estimate for BW7. Epigenetic heritability was 0.10 for BW7, and close to zero for the other body weights. The inclusion of the epigenetic effect in the model helped to explain the residual and non-Mendelian variability of initial body weight in meat quails.

Avaliação da imobilidade tônica em codornas de corte via análise de sobrevivência' / Tonic immobility evaluation in meat quail via survival analysis

Tonic immobility (TI) is a way to measure fear, as characterized by the reduced capacity to respond to external stimuli. The time spent in TI indicates the level of fear. Since TI is measured as the time until event occurrence, survival analysis stands out as a suitable statistical method to treat these data. We aimed to investigate the influence of possible factors (lines, sex and age) on fear behavior in meat quail (Coturnix coturnix) measured through TI by using survival analysis (non parametric Kaplan-Meyer method via logrank test). The dataset was composed by TI information provided by 50 animals from each line (UFV1 and UFV2) in each age (14 and 28 days of age), totalizing 200 records. Despite the slight difference between the two evaluated ages, there was no significance for this factor between each studied line, UFV1 (P= 0.1493) and UFV2 (P= 0.2583). The logrank test indicated significant difference (P= 0.0407) between levels of line/sex groups at 14 days of age. We noted that males from UFV2 line presented higher fear behavior in relation to males from UFV1 line. No significant differences were observed for this factor when considering 28 days of age.(AU)

Avaliação da imobilidade tônica em codornas de corte via análise de sobrevivência' / Tonic immobility evaluation in meat quail via survival analysis

Tonic immobility (TI) is a way to measure fear, as characterized by the reduced capacity to respond to external stimuli. The time spent in TI indicates the level of fear. Since TI is measured as the time until event occurrence, survival analysis stands out as a suitable statistical method to treat these data. We aimed to investigate the influence of possible factors (lines, sex and age) on fear behavior in meat quail (Coturnix coturnix) measured through TI by using survival analysis (non parametric Kaplan-Meyer method via logrank test). The dataset was composed by TI information provided by 50 animals from each line (UFV1 and UFV2) in each age (14 and 28 days of age), totalizing 200 records. Despite the slight difference between the two evaluated ages, there was no significance for this factor between each studied line, UFV1 (P= 0.1493) and UFV2 (P= 0.2583). The logrank test indicated significant difference (P= 0.0407) between levels of line/sex groups at 14 days of age. We noted that males from UFV2 line presented higher fear behavior in relation to males from UFV1 line. No significant differences were observed for this factor when considering 28 days of age.(AU)

Tolerability of up to 200 days of prophylaxis with valganciclovir oral solution and/or film-coated tablets in pediatric kidney transplant recipients at risk of cytomegalovirus disease.

This multicenter, open-label study evaluated the tolerability of extended prophylaxis with valganciclovir in pediatric kidney transplant recipients at risk of CMV disease. Fifty-six patients aged 4 months to 16 years received once-daily valganciclovir oral solution and/or tablets, dosed by BSA and renal function, for up to 200 days. The most common AEs on treatment were upper respiratory tract infection (33.9%), urinary tract infection (33.9%), diarrhea (32.1%), leukopenia (25.0%), neutropenia (23.2%), and headache (21.4%). There were fewer AEs during days 101-228 vs days 1-100. Twenty-seven patients (48.2%) had treatment-related AEs during valganciclovir treatment, most commonly leukopenia (21.4%), neutropenia (19.6%), anemia (7.1%), and tremor (5.4%). Treatment-related serious AEs were reported for nine patients (16.1%) and six withdrew due to AEs. Viremia was centrally confirmed in 10 patients; there was no confirmed CMV disease. One patient tested positive for a resistance mutation (UL97 L595F). Biopsy-proven acute rejection occurred in six patients (10.7%), but no graft loss or deaths occurred. In conclusion, up to 200 days of valganciclovir prophylaxis in pediatric kidney allograft recipients showed a safety profile consistent with that established in adult transplant patients.

Planned randomized conversion from tacrolimus to sirolimus-based immunosuppressive regimen in de novo kidney transplant recipients.

Planned conversion from tacrolimus to sirolimus was evaluated in de novo kidney transplant recipients. In this multicenter, randomized, open-label study, 297 patients were initially treated with tacrolimus, mycophenolate sodium and prednisone. Of the 283 patients reaching 3 months, 97 were converted to sirolimus (SRL), 107 were maintained on tacrolimus (TAC) and 79 were patients receiving TAC without criteria to undergo intervention at month 3 (TACex). The primary objective was to show superior estimated glomerular filtration rate (eGFR) in the SRL group at month 24. Of the 258 patients who completed 24 months, 91 (94%) were in the SRL group, 101 (94%) in the TAC group and 66 (84%) in the TACex group. In the intention-to-treat population there were no differences in eGFR (66.2 ± 25.3 vs. 70.7 ± 25.1, p = 0.817) or in the severity of chronic sclerosing lesions scores in 24-month protocol biopsies. Higher mean urinary protein-to-creatinine ratio (0.36 ± 0.69 vs. 0.15 ± 0.53, p = 0.03) and higher incidence of treated acute rejection between months 3-24 (13.4% vs. 4.7%, p = 0.047) were observed in SRL compared to TAC group. In this population planned conversion from TAC to SRL 3 months after kidney transplantation was not associated with improved renal function at 24 months.

Medical image registration using TSallis Entropy in Statistical Parametric Mapping (SPM).

The superposition of medical images, technically known as co-registration, can take a major role in determining the topographic and morphological changes in functional diagnostic and therapeutic purposes. This paper describes a study focused on to find an alternative cost function method for medical images co-registration through the study of performance and robustness of the TSallis Entropy in Statistical Parametric Mapping package (SPM). Images of Magnetic Resonance (MR) and Single Photon Emission Computed Tomography (SPECT) of 3 patients morphologically normal were used for the construction of anatomic phantoms containing predetermined geometric variations. The simulated images were co-registered with the original images using traditional techniques and the proposed method. The comparative analysis of the Root Mean Square (RMS) error showed that the Tsallis Entropy was more efficient in the intramodality alignment, while the Shannon Entropy in the intermodality one; revealing therefore the importance of the implementation of the Tsallis Entropy in SPM for applications in neurology and neuropsychiatric evaluation.

One-year results with extended-release tacrolimus/MMF, tacrolimus/MMF and cyclosporine/MMF in de novo kidney transplant recipients.

Once-daily tacrolimus extended-release formulation (Prograf XL, formerly referred to as MR or MR4) was compared with the twice-a-day tacrolimus formulation (TAC) and cyclosporine microemulsion (CsA), all administered in combination with mycophenolate mofetil (MMF), corticosteroids and basiliximab induction, in a phase 3, randomized (1:1:1), open-label trial in 638 de novo kidney transplant recipients. In combination with MMF and corticosteroids, XL had an efficacy profile comparable to TAC and CsA. XL/MMF and TAC/MMF were statistically noninferior at 1-year posttransplantation to CsA/MMF for the primary efficacy endpoint, efficacy failure (death, graft loss, biopsy-confirmed acute rejection (BCAR) or lost to follow-up). One-year patient and graft survival were 98.6% and 96.7% in the XL/MMF group, 95.7% and 92.9% in TAC/MMF group and 97.6% and 95.7% in CsA/MMF group. The safety profile of XL in comparison with CsA was similar to that observed with TAC in this study and consistent with previously published reports of TAC in comparison with CsA. The results support the safety and efficacy of tacrolimus in combination with MMF, corticosteroids and basiliximab induction, as well as XL as a safe and effective once-daily dosing alternative.

FTY720 impairs necrosis development after ischemia-reperfusion injury.

Ischemia-reperfusion (IR) injury is a common early feature that contributes to graft damage by impairing resident cell function. Our previous results showed that IR injury impaired renal function, by causing extensive tubular necrosis and increasing MHC class II and ICAM-1 molecule expression by mesangial cells (MC). MCs are likely candidates to come into close contact with immune cells such as monocytes or lymphocytes. It has been suggested that under inflammatory circumstances, there is increased MC expression of MHC class II, of adhesion molecules (such as ICAM-1), of cytokines receptors, and of molecules associated with cellular death (apoptosis). The immunosuppressive properties of FTY720 have been shown in clinical and experimental situations. It has also been shown to be protective against IR injury in rats. We sought to evaluate the role of FTY720 in a murine IR model by measuring renal function, tubular necrosis, and surface molecule expression by cultured mesangial cells. Intravenous administration of FTY720 (1 mg/kg) immediately before IR induction did not improve the short-term (24 hours) outcome of renal function or reduced MHC class II and ICAM-1 surface molecule expression. However, there was a decreased percentage of tubular necrosis in mice treated with FTY720 (51.3% +/- 1.6%) compared with vehicle-treated mice (66% +/- 5.5%). These results suggest a protective role of FTY720 in an IR injury model. More studies are required to identify the mechanisms involved in the protective activity of FTY720 in the IR injury model.

FTY720 treatment prolongs skin graft survival in a completely incompatible strain combination.

FTY720 has shown potent immunomodulatory activity in a variety of animal organ transplant models. However, the in vivo immunosuppressive mechanism of FTY720 is still not fully understood. It has been suggested that the marked decrease in the number of peripheral blood lymphocytes during FTY720 administration could be responsible for its immunosuppressive effects. Our aims were: (1) to study the effects of FTY720 treatment on skin graft survival using a fully mismatched strain combination and (2) to evaluate lymphocyte numbers in different sites at 5 days after skin transplant. C57BL/6 mice and BALB/c mice were the donors and recipients respectively. BALB/c mice received FTY720 (1 mg/kg/d) orally for 4 consecutive days. Drug administration started 1 day before skin transplants. A small segment of tail skin was affixed on the right dorsal side of the mouse via sutures. The administration of FTY720 (4 mg/kg) prolonged skin graft survival from 12.6 +/- 2.2 days (no treatment) to 16.6 +/- 4.2 days. The histologic findings of rejection were similar for all groups. Five days after transplant, lymphocyte numbers were significantly increased in lymph nodes compared with nontransplanted or isogenic graft mice. FTY720 decreased lymphocyte numbers only in the spleen. In conclusion, FTY720 prolonged skin graft survival in a fully mismatched strain combination when administered for 4 days (day -1 to day +2) at a dose of 1 mg/kg/d. The decreased number of lymphocytes in the spleen suggests that the spleen may be a target of FTY720 activity, during the early posttransplant period.

Post-transplant lymphoproliferative disorders (PTLD) after renal transplantation: management and evolution of seven cases among 1002 renal transplants in São Paulo, Brazil.

We reported seven cases (0.7%) of PTLD among 1002 renal transplants performed at Renal Transplant Service from Hospital São Paulo-Universidade Federal de São Paulo/Escola Paulista de Medicina, São Paulo, Brazil, between 1976 and 1997. There were three male and four female patients with median age of 37 year-old. According to Ann Arbor staging system there were four localized extra-nodal intermediate-grade NHL, one disseminated low-grade NHL and two polyclonal lymphoid hyperplasia. Four patients received cadaveric, two received related and one received unrelated renal transplant. PTLD occurred after a median latency period of 36 months (ranging from 5 to 84 months). In situ hybridization for EBER1 was performed in five patients and molecular evidence of EBV was found in 3 cases (two DLCL and one lymphoplasmocytoid lymphoma). All patients were treated with immunosuppression withdrawal, four patients received anthracyclin-based chemotherapy for control of localized or systemic clonal disease and three were treated with resection of primary PTLD. Four of seven patients (57%) are in complete remission 11, 20, 25 and 79 months after PTLD onset. One patient lost follow-up and two patients died due to lymphoma relapse, respectively 4 and 10 months after completion of treatment. In conclusion, our experience with this small group of patients showed that: 1) immunosuppression withdrawal is not necessarily associated with loss of renal transplant and can be used as the only treatment for polyclonal PTLD; 2) chemotherapy can simultaneously lead to clonal PTLD remission and periodic immunosuppression, avoiding graft rejection after immunosuppression withdrawal.

Leflunomide and malononitriloamides.

Leflunomide is a new immunomodulatory drug effective in experimental models of autoimmune diseases and allo- or xenotransplantation. In a Phase II clinical trial leflunomide has shown high tolerability and efficacy in patients with advanced rheumatoid arthritis. The immunomodulatory activity of leflunomide is attributed to its primary metabolite, A77 1726, a malononitriloamide. The in vitro and in vivo mechanisms of action of this class of compounds remain to be completely defined. A77 1726 and several malononitriloamide analogues inhibit T- and B-cell proliferation, suppress immunoglobulin production, and interfere with cell adhesion. While no one central molecular mechanism of action has been proposed to explain all the effects of the malononitriloamides, inhibition of de novo pyrimidine biosynthesis and inhibition of cytokine- and growth factor-receptor associated tyrosine kinase activity are leading hypotheses for the effects of A77 1726 on T- and B-cell proliferation and function. Leflunomide is effective when administered at daily doses of 10 and 25 mg to patients with active rheumatoid arthritis. The improved efficacy at the 25 mg dose is associated with a higher incidence of adverse effects (gastrointestinal symptoms, weight loss, allergic reactions, skin rash, and reversible alopecia). Due to the long plasma half-life of A77 1726 (11-16 days), loading doses are required to achieve steady-state concentrations. Phase III randomised, placebo-controlled trials using daily doses of 10 or 20 mg are underway in the US and Europe to confirm and extend the results of the Phase II study. Malononitriloamide analogues of A77 1726 are being evaluated for immunosuppressive efficacy in preclinical models of transplantation, because these compounds have a shorter half-life in animals than A77 1726. If these analogues show efficacies similar to leflunomide in these models and have shorter half-lives than A77 1726 in Phase I trials, the preclinical and Phase I data will be used to select the analogues for Phase II trials in organ transplant recipients.