Optimization of immunomagnetic separation for cord blood-derived hematopoietic stem cells

BACKGROUND: There is a growing interest in cord blood as a source of primitive stem cells with the capacity for multilineage differentiation. Pure cell fractions are needed for the characterization and in vitro expansion of stem cells as well as for their use in preclinical research. However, enrichment of stem cells is challenging due to the lack of stem cell-specific markers and gentle protocols for the isolation of highly pure stem cell fractions. Protocols developed for the enrichment of peripheral blood-derived stem cells have been found to be suboptimal for cord blood. RESULTS: In this study, immunomagnetic cell sorting protocols to purify CD34+, CD133+ and Lin- cells from fresh and cryopreserved cord blood were optimized. Reproducible purities of up to 97% were reached. The selected cells were highly viable having substantial colony-forming potential. CONCLUSION: The optimized protocols enable rapid enrichment of highly pure hematopoietic stem cells from both fresh and cryopreserved cord blood

Porcine circovirus 2 (PCV-2) genetic variability under natural infection scenario reveals a complex network of viral quasispecies

Porcine circovirus 2 (PCV-2) is a virus characterized by a high evolutionary rate, promoting the potential emergence of different genotypes and strains. Despite the likely relevance in the emergence of new PCV-2 variants, the subtle evolutionary patterns of PCV-2 at the individual-host level or over short transmission chains are still largely unknown. This study aimed to analyze the within-host genetic variability of PCV-2 subpopulations to unravel the forces driving PCV-2 evolution. A longitudinal weekly sampling was conducted on individual animals located in three farms after the first PCV-2 detection. The analysis of polymorphisms evaluated throughout the full PCV-2 genome demonstrated the presence of several single nucleotide polymorphisms (SNPs) especially in the genome region encoding for the capsid gene. The global haplotype reconstruction allowed inferring the virus transmission network over time, suggesting a relevant within-farm circulation. Evidences of co-infection and recombination involving multiple PCV-2 genotypes were found after mixing with pigs originating from other sources. The present study demonstrates the remarkable within-host genetic variability of PCV-2 quasispecies, suggesting the role of the natural selection induced by the host immune response in driving PCV-2 evolution. Moreover, the effect of pig management in multiple genotype coinfections occurrence and recombination likelihood was demonstrated

Porcine circovirus 2 (PCV-2) genetic variability under natural infection scenario reveals a complex network of viral quasispecies

Porcine circovirus 2 (PCV-2) is a virus characterized by a high evolutionary rate, promoting the potential emergence of diferent genotypes and strains. Despite the likely relevance in the emergence of new PCV-2 variants, the subtle evolutionary patterns of PCV-2 at the individual-host level or over short transmission chains are still largely unknown. This study aimed to analyze the within-host genetic variability of PCV-2 subpopulations to unravel the forces driving PCV-2 evolution. A longitudinal weekly sampling was conducted on individual animals located in three farms after the frst PCV-2 detection. The analysis of polymorphisms evaluated throughout the full PCV-2 genome demonstrated the presence of several single nucleotide polymorphisms (SNPs) especially in the genome region encoding for the capsid gene. The global haplotype reconstruction allowed inferring the virus transmission network over time, suggesting a relevant within-farm circulation. Evidences of co-infection and recombination involving multiple PCV-2 genotypes were found after mixing with pigs originating from other sources. The present study demonstrates the remarkable within-host genetic variability of PCV-2 quasispecies, suggesting the role of the natural selection induced by the host immune response in driving PCV-2 evolution. Moreover, the efect of pig management in multiple genotype coinfections occurrence and recombination likelihood was demonstrated.info:eu-repo/semantics/publishedVersio

Cytokines impact natural killer cell phenotype and functionality against glioblastoma in vitro

ObjectiveNatural killer (NK) cells are a part of the innate immune system and first-line defense against cancer. Since they possess natural mechanisms to recognize and kill tumor cells, NK cells are considered as a potential option for an off-the-shelf allogeneic cell-based immunotherapy. Here, our objective was to identify the optimal cytokine-based, feeder-free, activation and expansion protocol for cytotoxic NK cells against glioblastoma in vitro.MethodsNK cells were enriched from human peripheral blood and expanded for 16 days with different activation and cytokine combinations. The expansion conditions were evaluated based on NK cell viability, functionality, expansion rate and purity. The cytotoxicity and degranulation of the expanded NK cells were measured in vitro from co‑cultures with the glioma cell lines U‑87 MG, U‑87 MG EGFR vIII, LN-229, U-118 and DK-MG. The best expansion protocols were selected from ultimately 39 different conditions: three magnetic cell‑selection steps (Depletion of CD3+ cells, enrichment of CD56+ cells, and depletion of CD3+ cells followed by enrichment of CD56+ cells); four activation protocols (continuous, pre-activation, re-activation, and boost); and four cytokine combinations (IL-2/15, IL‑21/15, IL‑27/18/15 and IL-12/18/15).ResultsThe expansion rates varied between 2-50-fold, depending on the donor and the expansion conditions. The best expansion rate and purity were gained with sequential selection (Depletion of CD3+ cells and enrichment of CD56+ cells) from the starting material and pre-activation with IL‑12/18/15 cytokines, which are known to produce cytokine-induced memory-like NK cells. The cytotoxicity of these memory-like NK cells was enhanced with re-activation, diminishing the donor variation. The most cytotoxic NK cells were produced when cells were boosted at the end of the expansion with IL-12/18/15 or IL-21/15.ConclusionAccording to our findings the ex vivo proliferation capacity and functionality of NK cells is affected by multiple factors, such as the donor, composition of starting material, cytokine combination and the activation protocol. The cytokines modified the NK cells' phenotype and functionality, which was evident in their reactivity against the glioma cell lines. To our knowledge, this is the first comprehensive comparative study performed to this extent, and these findings could be used for upscaling clinical NK cell manufacturing

Porcine circovirus 2 immunology and viral evolution

Porcine circovirus 2 (PCV2) has and is still causing important economic losses to pig industry. This is due to PCV2-systemic disease (PCV2-SD), formerly known as postweaning multi-systemic wasting syndrome (PMWS), which increases mortality rates and slows down the growth of the animals, as well as other conditions collectively included within the so-called porcine circovirus diseases (PCVD). PCV2-SD affected pigs are considered to be immunosuppressed, with severe lymphocyte depletion and evidence of secondary infections. However, PCV2-infected pigs not developing the disease are able to mount humoral and cellular immune responses and clear the virus or limit the infection. On the contrary, insufficient amounts of neutralizing antibodies have been linked to increased PCV2 replication, severe lymphoid lesions and development of PCV2-SD. Central role in controlling PCV2 infection are played by the antigen specific memory T cells. These cells persist long term post-infection or vaccination and are able to expand rapidly after recall antigen recognition. Most farms in the main pig producing countries are applying vaccination against PCV2 to prevent the disease and improve the farm performance. Vaccines do not induce sterilizing immunity and PCV2 keeps on circulating even in farms applying vaccination. This, together with the high mutation rate of PCV2, world-wide fluctuations in the genotype dominance and emergence of novel genetic variants, warrant close molecular survey of the virus in the field

Optimization of immunomagnetic separation for cord blood-derived hematopoietic stem cells

Abstract Background There is a growing interest in cord blood as a source of primitive stem cells with the capacity for multilineage differentiation. Pure cell fractions are needed for the characterization and in vitro expansion of stem cells as well as for their use in preclinical research. However, enrichment of stem cells is challenging due to the lack of stem cell-specific markers and gentle protocols for the isolation of highly pure stem cell fractions. Protocols developed for the enrichment of peripheral blood-derived stem cells have been found to be suboptimal for cord blood. Results In this study, immunomagnetic cell sorting protocols to purify CD34+, CD133+ and Lin- cells from fresh and cryopreserved cord blood were optimized. Reproducible purities of up to 97% were reached. The selected cells were highly viable having substantial colony-forming potential. Conclusion The optimized protocols enable rapid enrichment of highly pure hematopoietic stem cells from both fresh and cryopreserved cord blood.</p

Optimization of immunomagnetic separation for cord blood-derived hematopoietic stem cells

Background: There is a growing interest in cord blood as a source of primitive stem cells with the capacity for multilineage differentiation. Pure cell fractions are needed for the characterization and in vitro expansion of stem cells as well as for their use in preclinical research. However, enrichment of stem cells is challenging due to the lack of stem cell-specific markers and gentle protocols for the isolation of highly pure stem cell fractions. Protocols developed for the enrichment of peripheral blood-derived stem cells have been found to be suboptimal for cord blood. Results: In this study, immunomagnetic cell sorting protocols to purify CD34+, CD133+ and Lin- cells from fresh and cryopreserved cord blood were optimized. Reproducible purities of up to 97% were reached. The selected cells were highly viable having substantial colony-forming potential. Conclusion: The optimized protocols enable rapid enrichment of highly pure hematopoietic stem cells from both fresh and cryopreserved cord blood

A novel rolling circle amplification assay to detect members of the family Anelloviridae in pigs and humans

The study describes a novel rolling circle amplification (RCA) assay to detect members of the family Anelloviridae from swine and human serum samples. The RCA was carried out using short anellovirus (AV)-specific primers based on a highly conserved region among available AV full-length genomes. Then, RCA products were used as templates to amplify full-length genomes with an AV-specific PCR. Amplification products were separated by agarose gel electrophoresis and full-length genomes were isolated based on the known size. With this novel AV-RCA/PCR approach it was possible to detect Torque teno sus virus 1 (TTSuV1) and Torque teno sus virus 2 (TTSuV2) in swine and many species of Torque teno virus (TTV) in human sera, which were previously tested negative by conventional PCRs. \uc2\ua9 2011 Elsevier B.V

Torque teno virus (TTV) is highly prevalent in the European wild boar (Sus scrofa)

The present study represents the first survey of Torque teno virus (TTV) prevalence in European wild boar (Sus scrofa). The prevalence of two distinct TTV genogroups in 178 Spanish wild boar sera from different geographic regions, management conditions, gender and age was determined by a nested PCR method. The overall prevalence of TTV genogroups was 84% (58% for genogroup 1 and 66% for genogroup 2), and differences between genogroup prevalence were observed depending on the geographical region analysed. Significantly higher prevalence for TTV genogroup 2 was found in fenced managed wild boar, juvenile animals and females. No other significant differences in TTV genogroup prevalence were observed. The phylogenetic analysis of nucleotide sequences obtained from the untranslated region of selected samples revealed that the same TTV genogroups are infecting wild boar and domestic pig. The results indicate that TTV is apparently ubiquitous in European wild boar populations.This work was partly funded by the Project no. 513928 from the Sixth Framework Programme of the European Commission, and by OAPN-MMA and SDGSA-MAPA, as well as Ministerio de Educación, Plan Nacional de I + D, grant AGL2005-07401-GAN. Francisco Ruiz-Fons is an FPU-MEC grantee.Peer reviewe