Short Communication: Simultaneous Identification of Five κ-Casein (CSN3) Alleles in Domestic Goat by Polymerase Chain Reaction-Single Strand Conformation Polymorphism

Until now, a total of nine polymorphic sites corresponding to six different alleles have been described at the kappa-casein (CSN3) locus in the domestic goat (Capra hircus). A protocol for the rapid and simultaneous genotyping of five goat CSN3 alleles by using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique was developed. Moreover, the developed test was validated by screening the CSN3 variability in four Italian breeds, Garganica, Jonica, Maltese, and Camosciata. Seven different patterns were readily identifiable. These corresponded to five known alleles and two newly identified variants. The G/A substitution at nucleotide position 471, which is not identifiable at the protein level but was found to be very frequent in the typed breeds, is easily detectable by the protocol developed. The PCR-SSCP analysis is a powerful tool for the genetic study of CSN3 variability in domestic goats, allowing both the simultaneous identification of different alleles, and the detection of new variants

Chromosomal assignment of the ovine hairless (hr) gene by fluorescence in situ hybridization.

Congenital hypotrichosis in mammalian species consists of partial or complete absence of a hair coat at birth. Affected individuals having a partial hair coat at birth may loose it subsequently.The aim of this paper was to physically map the ovine hr gene using fluorescence in situ hybridization (FISH).A preliminary study on an internet data bank (http://www.informatics.jax.org) showed that the regions of the hr gene on murine chromosome 14 and human chromosome 8 present homology with ovine chromosome 2

Casein haplotype structure in five Italian goat breeds

The aim of this work was to investigate the genetic structure of the casein gene cluster in 5 Italian goat breeds and to evaluate the haplotype variability within and among populations. A total of 430 goats from Vallesana, Roccaverano, Jonica, Garganica, and Maltese breeds were genotyped at alphas1-casein (CSN1S1), alphas2-casein, (CSN1S2), beta-casein (CSN2), and kappa-casein (CSN3) loci using several genomic techniques and milk protein analysis. Casein haplotype frequencies were estimated for each breed. Principal component analysis was carried out to highlight the relationship among breeds. Allele and haplotype distributions indicated considerable differences among breeds. The haplotype CSN1S1*F- CSN1S2*F-CSN3*D occurred in all breeds with frequencies >0.100 and was the most common haplotype in the Southern breeds. A high frequency of CSN1S1*0-CSN1S2*C-CSN3*A haplotype was found in Vallesana population (0.162). Principal component analysis clearly separated the Northern and Southern breeds by the first component. The variability of the caprine casein loci and variety of resulting haplotypes should be exploited in the future using specific breeding programs aiming to preserve biodiversity and to select goat genetic lines for specific protein production

PULSATILE FLOW OF VISCOELASTIC FLUIDS IN TUBES

In this study, we present an experimental investigation of the flow structure changes in non-Newtonian fluids subjected to periodic variable fluxes inside rigid-walled tubes. We employ a liquid mixture composed of water and polyacrylamide to account for various rheological properties. We obtain the velocity fields for each experimental case using the Particle Image Velocimetry (PIV) technique and we analyze their variations based on different properties of the pulsatile input signal. Reynolds numbers between 200 and 350 are considered, with a 10 % variation in amplitude

Process development and validation of expanded regulatory T cells for prospective applications: an example of manufacturing a personalized advanced therapy medicinal product

Background: A growing number of clinical trials have shown that regulatory T (Treg) cell transfer may have a favorable effect on the maintenance of self-tolerance and immune homeostasis in different conditions such as graft-versus-host disease (GvHD), solid organ transplantation, type 1 diabetes, and others. In this context, the availability of a robust manufacturing protocol that is able to produce a sufficient number of functional Treg cells represents a fundamental prerequisite for the success of a cell therapy clinical protocol. However, extended workflow guidelines for nonprofit manufacturers are currently lacking. Despite the fact that different successful manufacturing procedures and cell products with excellent safety profiles have been reported from early clinical trials, the selection and expansion protocols for Treg cells vary a lot. The objective of this study was to validate a Good Manufacturing Practice (GMP)-compliant protocol for the production of Treg cells that approaches the whole process with a risk-management methodology, from process design to completion of final product development. High emphasis was given to the description of the quality control (QC) methodologies used for the in-process and release tests (sterility, endotoxin test, mycoplasma, and immunophenotype). Results: The GMP-compliant protocol defined in this work allows at least 4.11 7 109 Treg cells to be obtained with an average purity of 95.75 \ub1 4.38% and can be used in different clinical settings to exploit Treg cell immunomodulatory function. Conclusions: These results could be of great use for facilities implementing GMP-compliant cell therapy protocols of these cells for different conditions aimed at restoring the Treg cell number and function, which may slow the progression of certain diseases

Regulatory T cells from patients with end-stage organ disease can be isolated, expanded and cryopreserved according good manufacturing practice improving their function

Background Here, we isolated, expanded and functionally characterized regulatory T cells (Tregs) from patients with end stage kidney and liver disease, waiting for kidney/liver transplantation (KT/LT), with the aim to establish a suitable method to obtain large numbers of immunomodulatory cells for adoptive immunotherapy post-transplantation. Methods We first established a preclinical protocol for expansion/isolation of Tregs from peripheral blood of LT/KT patients. We then scaled up and optimized such protocol according to good manufacturing practice (GMP) to obtain high numbers of purified Tregs which were phenotypically and functionally characterized in vitro and in vivo in a xenogeneic acute graft-versus-host disease (aGVHD) mouse model. Specifically, immunodepressed mice (NOD-SCID-gamma KO mice) received human effector T cells with or without GMP-produced Tregs to prevent the onset of xenogeneic GVHD. Results Our small scale Treg isolation/expansion protocol generated functional Tregs. Interestingly, cryopreservation/thawing did not impair phenotype/function and DNA methylation pattern of FOXP3 gene of the expanded Tregs. Fully functional Tregs were also isolated/expanded from KT and LT patients according to GMP. In the mouse model, GMP Tregs from LT or KT patient proved to be safe and show a trend toward reduced lethality of acute GVHD. Conclusions These data demonstrate that expanded/thawed GMP-Tregs from patients with end-stage organ disease are fully functional in vitro. Moreover, their infusion is safe and results in a trend toward reduced lethality of acute GVHD in vivo, further supporting Tregs-based adoptive immunotherapy in solid organ transplantation

Finding a new therapeutic approach for no-option Parkinsonisms : Mesenchymal stromal cells for progressive supranuclear palsy

Background: The trophic, anti-apoptotic and regenerative effects of bone marrow mesenchymal stromal cells (MSC) may reduce neuronal cell loss in neurodegenerative disorders. Methods: We used MSC as a novel candidate therapeutic tool in a pilot phase-I study for patients affected by progressive supranuclear palsy (PSP), a rare, severe and no-option form of Parkinsonism. Five patients received the cells by infusion into the cerebral arteries. Effects were assessed using the best available motor function rating scales (UPDRS, Hoehn and Yahr, PSP rating scale), as well as neuropsychological assessments, gait analysis and brain imaging before and after cell administration. Results: One year after cell infusion, all treated patients were alive, except one, who died 9 months after the infusion for reasons not related to cell administration or to disease progression (accidental fall). In all treated patients motor function rating scales remained stable for at least six-months during the one-year follow-up. Conclusions: We have demonstrated for the first time that MSC administration is feasible in subjects with PSP. In these patients, in whom deterioration of motor function is invariably rapid, we recorded clinical stabilization for at least 6 months. These encouraging results pave the way to the next randomized, placebo-controlled phase-II study that will definitively provide information on the efficacy of this innovative approach. Trial registration ClinicalTrials.gov NCT0182412