The effect of presynchronization with prostaglandin F2α and gonadotropin-releasing hormone simultaneously, 7 d before Ovsynch, compared with Presynch-10/Ovsynch on luteal function and first-service pregnancies per artificial insemination.

Pregnancy per artificial insemination (P/AI) following Ovsynch is optimized when cows ovulate to the first GnRH of Ovsynch. Fertility programs are designed to presynchronize cows to d 6 or 7 of the estrous cycle to increase the chances of ovulation of a first-wave dominant follicle to the first GnRH of Ovsynch. The hypothesis of this experiment was that simplification of a presynchronization program through the combination of PGF2α and GnRH on the same day, 7 d before Ovsynch, would allow for similar P/AI compared with Presynch-10. Lactating dairy cows (n = 432) 41 to 47 d in milk (DIM) were randomly assigned to 2 treatments within parities for first service. Control cows received Presynch-10/Ovsynch consisting of the following: PGF2α-14 d-PGF2α-10 d-GnRH-7 d-PGF2α-56 h-GnRH-16 h-AI. Treated cows received PGF2α and GnRH-7 d-GnRH-7 d-PGF2α-56 h-GnRH-16 h-AI. All cows received a supplemental injection of PGF2α 24 h after the PGF2α of Ovsynch to enhance complete luteolysis. All cows received timed AI between 75 and 81 DIM. Blood was collected to assess circulating concentrations of progesterone (P4), and the number and size of corpora lutea (CL) were recorded using ultrasonography on day of PGF2α of Ovsynch. The administration of PGF2α simultaneously with GnRH and 7 d before Ovsynch (PG+G) had similar P/AI at 28 (46 vs. 48%), 35 (43 vs. 43%), 49 (39 vs. 39%), and 77 d post-AI (38 vs. 39%) compared with Presynch-10. No differences were observed in P/AI in primiparous versus multiparous cows at 28 (52 vs. 45%), 35 (48 vs. 41%), 49 (45 vs. 37%), and 77 d post-AI (43 vs. 36%). No difference existed between treatments in percentage of cows with functional CL at PGF2α of Ovsynch, total luteal area (mm2), or serum concentrations of P4 at time of PGF2α of Ovsynch, regardless of parity. Number of CL had a tendency to be greater for multiparous PG+G vs. Presynch-10 cows (2.34 ± 0.09 vs. 2.15 ± 0.08) but not in primiparous cows (1.95 ± 0.10 vs. 1.98 ± 0.11). In summary, administering both PGF2α and GnRH on the same day, 7 d before the start of Ovsynch, appears to be a simple and effective alternative to Presynch-10 Ovsynch.

Influence of donor specific HLA antibodies detected by Luminex in kidney graft survival: a multivariate analysis.

Some studies have demonstrated the clinical relevance of a positive virtual crossmatch in graft survival; nevertheless, other donor and recipient variables influence the outcome of the transplant. The aim of this study was to investigate the relevance of a positive virtual crossmatch in the graft survival performing a multivariate analysis including other pretransplant variables. A total of 879 deceased kidney transplantations were included. Univariate and multivariate analyses were performed using Cox regression model. After performing the multivariate analysis, a positive virtual crossmatch against class I (adjusted HR 6.613; 95% CI 3.222-13.573), class II (adjusted HR 2.419; 95% CI 1.170-5.002) and class I+II (adjusted HR 5.717; 95% CI 1.925-16.975) detected by single antigen Luminex was the variable conferring the greatest relative risk of graft loss. A positive virtual crossmatch predicts a worse kidney graft survival even after correction by other variables and therefore, transplantation of patients with positive virtual crossmatches should be avoided.

Efficiency of gene transfection reagents in NG108-15, SH-SY5Y and CHO-K1 cell lines.

Several gene delivery reagents were analyzed for their transfection efficiency. Genes studied belonged to the class of mammalian proteins termed regulators of G-protein signaling (RGS), ranged in size up to 2.2 Kb long and were transfected into the NG108-15, SH-SY5Y and CHO-K1 cell lines. Prior to transfection, genes were cloned into a nonviral vector pcDNA 6.2/EmGFP, so as to express a green fluorescent protein tag at the 3' end. Flow cytometry was used to analyze cell fluorescent activity and thereby transfection efficiency. Gene delivery reagents Lipofectamine 2000 and ExGen 500 produced more effective transfection in NG108-15 cells whereas Lipofectamine 2000, ExGen 500 and TurboFectin 8.0 were more effective in CHO-K1 cells. In both these cell lines, transfection efficiency reached 60-80%. In SH-SY5Y cells, TurboFectin 8.0 produced the best transfection result; however efficiency level was only 5%. Gene size had no effect on transfection efficiency. Unlike Lipofectamine 2000, cells transfected using ExGen 500 showed morphological deformation. Our results suggest that Lipofectamine 2000 is the most suitable transfection medium for gene delivery to NG108-15 and CHO-K1 cells.

Donor-specific antibody detection: comparison of single antigen assay and Luminex crossmatches.

Luminex bead-based assays are routinely used in the study of anti-human leukocyte antigen (HLA) donor-specific antibodies (DSA). Single antigen (SA) assays use beads coated with recombinant antigens whereas Luminex crossmatch (Xm-DSA) tests consist of beads coated with isolated donor-specific HLA molecules. The aim of this study was to compare these techniques used to detect DSA. A total of 24 sera recognizing different HLA class I (seven anti-HLA-A and seven anti-HLA-B) as well as class II (seven anti-HLA-DR and three anti-HLA DQ) specificities by complement dependent cytotoxicity were included in the study. These sera were used undiluted and in serial dilutions to perform both class I and II SA and Xm-DSA assays. In the case of Xm-DSA the same serum was checked with different lysates. A total of 42 lysates were used to perform a total of 61 crossmatches: 42 to detect anti-class I and 19 to detect anti-class II antibodies. The maximum positive dilution was higher for SA in 76% of the class I and in 90% of the class II crossmatches. Those cases with a higher sensitivity of the Xm-DSA could not be explained by a larger number of antigen targets.