Optimization of Lyophilized Hyperacute Serum (HAS) as a Regenerative Therapeutic in Osteoarthritis

Hyperacute serum (HAS) is a blood derivative product that promotes the proliferation of various cell types and controls inflammation in vitro. The aim of this study is to investigate the regenerative potential of different formulations of HAS, including lyophilized and hyaluronic acid combined versions, to obtain a stable and standardized therapeutic in osteoarthritis (OA), which may be able to overcome the variability limitations of platelet-rich plasma (PRP). Primary human osteoarthritic chondrocytes were used for testing cellular viability and gene expression of OA-related genes. Moreover, a co-culture of human explants of cartilage, bone and synovium under inflammatory conditions was used for investigating the inflammatory control capacities of the different therapeutics. In this study, one formulation of lyophilized HAS achieved the high cell viability rates of liquid HAS and PRP. Gene expression analysis showed that HAS induced higher Col1a1 expression than PRP. Cytokine quantification from supernatant fluids revealed that HAS treatment of inflamed co-cultures significantly reduced levels of IL-5, IL-15, IL-2, TNFα, IL-7 and IL-12. To conclude, lyophilized HAS is a stable and standardized therapeutic with high potential in joint regeneration.

Scrapie Resistance Gene Identification using Optimized Taqman Test qPCR Method in Sheep on the Territory of the Republic of Serbia

Scrapie is an infectious neurodegenerative disease affecting the central nervous system of sheep and goats that belongs to transmissible spongiform encephalopathies. The disease is caused by the accumulation of proteinase-resistant isoform of the prion protein. The sheep predisposition to scrapie is associated with polymorphisms of the PrP gene. Genetic susceptibility to scrapie is mainly related to codons 136, 154, and 171. ARR sheep are strongly scrapie resistant and VRQ genotype is the most susceptible. Many countries have scrapie eradication programs based on using rams with resistant genotype. The eradication program has not yet been implemented in the Republic of Serbia. To examine the genetic makeup of sheep in Serbia related to scrapie, we optimized TaqMan probes of real-time polymerase chain reaction (qPCR) technique for three codons. Blood samples from 100 sheep were analyzed by qPCR and the majority of the examined sheep were AA homozygous for the 136 codon. For codon 154 the most frequent genotype was RR and for codon 171 the most frequent genotype was QQ.

Two distinct conformers of PrPD type 1 of sporadic Creutzfeldt–Jakob disease with codon 129VV genotype faithfully propagate in vivo

Current classifications of sporadic Creutzfeldt–Jakob disease (sCJD) identify five subtypes associated with different disease phenotypes. Most of these histopathological phenotypes (histotypes) co-distribute with distinct pairings of methionine (M)/valine (V) genotypes at codon 129 of the prion protein (PrP) gene and the type (1 or 2) of the disease-associated PrP (PrPD). Types 1 and 2 are defined by the molecular mass (~ 21 kDa and ~ 19 kDa, respectively) of the unglycosylated isoform of the proteinase K-resistant PrPD (resPrPD). We recently reported that the sCJDVV1 subtype (129VV homozygosity paired with PrPD type 1, T1) shows an electrophoretic profile where the resPrPD unglycosylated isoform is characterized by either one of two single bands of ~ 20 kDa (T120) and ~ 21 kDa (T121), or a doublet of ~ 21–20 kDa (T121−20). We also showed that T120 and T121 in sCJDVV have different conformational features but are associated with indistinguishable histotypes. The presence of three distinct molecular profiles of T1 is unique and raises the issue as to whether T120 and T121 represent distinct prion strains. To answer this question, brain homogenates from sCJDVV cases harboring each of the three resPrPD profiles, were inoculated to transgenic (Tg) mice expressing the human PrP-129M or PrP-129V genotypes. We found that T120 and T121 were faithfully replicated in Tg129V mice. Electrophoretic profile and incubation period of mice challenged with T121−20 resembled those of mice inoculated with T121 and T120, respectively. As in sCJDVV1, Tg129V mice challenged with T121 and T120 generated virtually undistinguishable histotypes. In Tg129M mice, T121 was not replicated while T120 and T121−20 generated a ~ 21–20 kDa doublet after lengthier incubation periods. On second passage, Tg129M mice incubation periods and regional PrP accumulation significantly differed in T120 and T121−20 challenged mice. Combined, these data indicate that T121 and T120 resPrPD represent distinct human prion strains associated with partially overlapping histotypes.

PrP gene polymorphism and its influence on some productive traits of sheepbreeds reared in Bulgaria

The rapiddissemination of scrapie over the past few decades led to development of aspecific eradication programme, based on the polymorphisms within the prionprotein gene (PRNP). Current approach encourages the selection ofanimals carrying the resistant ARR/ARR genotype, while other genotypes areconsidered less preferable. Although the strategy seems to be working quitewell, farmers are concerned whether this will affect sheep productivity andsubsequently decrease net profits. The current study was aimed to elucidatethe linkage between the PrP gene polymorphism (based on codons 136,154 and 171) and some productive traits (live weight, reproduction, milk andfleece yield) of three sheep breeds reared in Bulgaria – Assaf, NortheastBulgarian Merino and Blackhead Pleven. The total number of detectedgenotypes was six – ARR/ARR, ARR/ARQ, ARR/ARH, ARQ/ARQ, AHQ/ARQ and ARR/VRQ,with different prevalence within each breed. The observed lack ofsignificant differences in the studied performance traits between the PRNP genotypes suggests that PRNP polymorphisms did not influencethe sheep productive performance. Therefore, selection of animals on theresistant genotype (ARR/ARR) would not worsen their productivity. Theobtained results should help the better understanding of scrapie selectionand the positive effect that it would have to both health care and industry.

Molecular Detection of Cellular Prion Protein in Brain Tissues of Black Bengal Goats in Bangladesh

The cellular prion protein (PrPC) is a membrane-bound glycoprotein mainlypresent in the central nervous system which is necessary for the establishmentand further evolution of prion disease in human and animals. The aim of thepresent study was to investigate the PrPC protein in brain tissues of black Bengalgoat. Fifteen brain tissues were collected from different slaughter houses ofthree districts (Mymensingh, Manikgonj and Netrokona) of Bangladesh duringJanuary to February, 2011. The PrPC protein was detected in the brain tissuesof black Bengal goats using polymerase chain reaction. The result showed allpositive (100%) of the amplified samples. The standardized PCR could be usedfor detection of PrPC protein in different tissues of animals and humans.Sequencing of PrP gene in the black Bengal goats for the risk assessment ofscrapie is needed for further study. To our knowledge, detection of PrPC proteinin the brain tissues of indigenous goats is the first time in Bangladesh.