Unraveling the differential mechanisms of revascularization promoted by MSCs & ECFCs from adipose tissue or umbilical cord in a murine model of critical limb-threatening ischemia.

BACKGROUND: Critical limb-threatening ischemia (CLTI) constitutes the most severe manifestation of peripheral artery disease, usually induced by atherosclerosis. CLTI patients suffer from high risk of amputation of the lower extremities and elevated mortality rates, while they have low options for surgical revascularization due to associated comorbidities. Alternatively, cell-based therapeutic strategies represent an effective and safe approach to promote revascularization. However, the variability seen in several factors such as cell combinations or doses applied, have limited their success in clinical trials, being necessary to reach a consensus regarding the optimal "cellular-cocktail" prior further application into the clinic. To achieve so, it is essential to understand the mechanisms by which these cells exert their regenerative properties. Herein, we have evaluated, for the first time, the regenerative and vasculogenic potential of a combination of endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) isolated from adipose-tissue (AT), compared with ECFCs from umbilical cord blood (CB-ECFCs) and AT-MSCs, in a murine model of CLTI. METHODS: Balb-c nude mice (n:32) were distributed in four different groups (n:8/group): control shams, and ischemic mice (after femoral ligation) that received 50 µl of physiological serum alone or a cellular combination of AT-MSCs with either CB-ECFCs or AT-ECFCs. Follow-up of blood flow reperfusion and ischemic symptoms was carried out for 21 days, when mice were sacrificed to evaluate vascular density formation. Moreover, the long-term molecular changes in response to CLTI and both cell combinations were analyzed in a proteomic quantitative approach. RESULTS: AT-MSCs with either AT- or CB-ECFCs, promoted a significant recovery of blood flow in CLTI mice 21 days post-ischemia. Besides, they modulated the inflammatory and necrotic related processes, although the CB group presented the slowest ischemic progression along the assay. Moreover, many proteins involved in the repairing mechanisms promoted by cell treatments were identified. CONCLUSIONS: The combination of AT-MSCs with AT-ECFCs or with CB-ECFCs promoted similar revascularization in CLTI mice, by restoring blood flow levels, together with the modulation of the inflammatory and necrotic processes, and reduction of muscle damage. The protein changes identified are representative of the molecular mechanisms involved in ECFCs and MSCs-induced revascularization (immune response, vascular repair, muscle regeneration, etc.).

Endothelial progenitor cells as biomarkers of diabetes-related cardiovascular complications.

Diabetes mellitus (DM) constitutes a chronic metabolic disease characterized by elevated levels of blood glucose which can also lead to the so-called diabetic vascular complications (DVCs), responsible for most of the morbidity, hospitalizations and death registered in these patients. Currently, different approaches to prevent or reduce DM and its DVCs have focused on reducing blood sugar levels, cholesterol management or even changes in lifestyle habits. However, even the strictest glycaemic control strategies are not always sufficient to prevent the development of DVCs, which reflects the need to identify reliable biomarkers capable of predicting further vascular complications in diabetic patients. Endothelial progenitor cells (EPCs), widely known for their potential applications in cell therapy due to their regenerative properties, may be used as differential markers in DVCs, considering that the number and functionality of these cells are affected under the pathological environments related to DM. Besides, drugs commonly used with DM patients may influence the level or behaviour of EPCs as a pleiotropic effect that could finally be decisive in the prognosis of the disease. In the current review, we have analysed the relationship between diabetes and DVCs, focusing on the potential use of EPCs as biomarkers of diabetes progression towards the development of major vascular complications. Moreover, the effects of different drugs on the number and function of EPCs have been also addressed.

Cardiovascular-related proteomic changes in ECFCs exposed to the serum of COVID-19 patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection significantly affects the cardiovascular system, causing vascular damage and thromboembolic events in critical patients. Endothelial dysfunction represents one of the first steps in response to COVID-19 that might lead to cardiovascular complications and long-term sequelae. However, despite the enormous efforts in the last two years, the molecular mechanisms involved in such processes remain poorly understood. Herein, we analyzed the protein changes taking place in endothelial colony forming cells (ECFCs) after the incubation with the serum from individuals infected with COVID-19, whether asymptomatic or critical patients, by application of a label free-quantitative proteomics approach. Specifically, ECFCs from healthy individuals were incubated ex-vivo with the serum of either COVID-19 negative donors (PCR-/IgG-, n:8), COVID-19 asymptomatic donors at different infective stages (PCR+/ IgG-, n:8and PCR-/IgG+, n:8), or hospitalized critical COVID-19 patients (n:8), followed by proteomics analysis. In total, 590 proteins were differentially expressed in ECFCs in response to all infected serums. Predictive analysis highlighted several proteins like CAPN5, SURF4, LAMP2 or MT-ND1, as highly discriminating features between the groups compared. Protein changes correlated with viral infection, RNA metabolism or autophagy, among others. Remarkably, the angiogenic potential of ECFCs in response to the infected serums was impaired, and many of the protein alterations in response to the serum of critical patients were associated with cardiovascular-related pathologies.

A simplified courtship conditioning protocol to test learning and memory in Drosophila.

In Drosophila, a male that has previously been sexually rejected reduces its courtship behavior when confronted again with an unreceptive female. This reduced courting time reflects a memory formation process. Here, we describe a simplified protocol to perform the courtship conditioning assay for assessing the reduced courting time, using regular lab equipment and handmade tools. Every step of the procedure, from raising flies and training to testing and quantification of this memory-related behavior, can be implemented in any practice laboratory.

Relationship of persistent lymphocytosis, antibody titers, and proviral load with expression of interleukin-12, interferon-γ, interleukin-2, interleukin-4, interleukin-10, and transforming growth factor-ß in cows infected with bovine leukemia virus from a high-prevalence dairy complex.

Bovine leukemia virus (BLV) subclinical infection promotes persistent lymphocytosis (PL), which is related to susceptibility and progression to lymphoma. Moreover, lymphocyte counts directly correlate with BLV antibody titers and proviral load, and cell immune responses are considered atypical due to immune suppression. In order to determine the relationship of PL, antibody titers, and proviral load with interleukin (IL)-12, interferon (IFN)-γ, IL-2, IL-4, IL-10, and transforming growth factor (TGF)-ß expression in a 3-month interval, 58 cows were selected (30 BLV+ and 28 BLV-) from a high-prevalence dairy herd to complete 3 monthly blood samplings for the assessment of PL, BLV antibody titers, BLV proviral load, and IL-12, IFN-γ, IL-2, IL-4, IL-10, and TGF-ß expression. At sampling conclusion, the BLV-infected cows were grouped according to PL, BLV proviral load, and BLV antibody titers as follows: BLV+PL+ (n = 16) and BLV+PL- (n = 14); high proviral load (HPL) (n = 18) and low proviral load (LPL) (n = 13); high antibody titers (HAT) (n = 17) and low antibody titers (LAT) (n = 14). The BLV+PL+ cows showed significantly higher proviral load and antibody titers than the BLV+PL- group; however, the former suggested spread presumably unrelated to lymphoma outcome, because HPL was observed in PL- cows in the last sampling. Consistent with the data, a higher antibody response strongly indicated BLV susceptibility since it was linked to PL+ occurrence and a cytokine profile compatible with immune suppression. Furthermore, a reversion to lower antibody titers was observed in cows with HPL far ahead of time, most likely due to long-term immune suppression. In addition, high expression of IL-10 and TGF-ß was associated with reduced IL-12, IFN-γ, IL-2, and IL-4 expression alongside PL, HAT, and HPL in BLV-infected cows, suggesting an IL-10- and TGF-ß-induced immune suppression. The IL-10 expression was increasing throughout, implying disease progression, as described. In conclusion, the proliferative expansion of lymphocytes known as PL might enhance a regulatory-rich cell population (Bregs and/or Tregs) that secretes IL-10 and TGF-ß, leading to immune suppression. Further studies must be conducted regarding the types of regulatory cells involved in BLV-induced immune suppression.

L'infection subclinique par le virus de la leucémie bovine (BLV) favorise une lymphocytose persistante (PL), qui est liée à la susceptibilité et à la progression vers le lymphome. De plus, le nombre de lymphocytes est directement corrélé aux titres d'anticorps BLV et à la charge provirale, et les réponses immunitaires cellulaires sont considérées comme atypiques en raison de la suppression immunitaire. Afin de déterminer la relation entre PL, les titres d'anticorps et la charge provirale avec l'interleukine (IL)-12, l'interféron (IFN)-γ, l'IL-2, l'IL-4, l'IL-10 et l'expression du facteur de croissance transformant (TGF)-ß dans un intervalle de 3 mois, 58 vaches ont été sélectionnées (30 BLV+ et 28 BLV−) à partir d'un troupeau laitier à forte prévalence pour compléter trois prélèvements sanguins mensuels pour l'évaluation de PL, des titres d'anticorps BLV, de la charge provirale BLV et l'expression d'IL-12, IFN-γ, d'IL-2, d'IL-4, d'IL-10 et TGF-ß. À la fin de l'échantillonnage, les vaches infectées par le BLV ont été regroupées en fonction du PL, de la charge provirale du BLV et des titres d'anticorps du BLV comme suit : BLV+PL+ (n = 16) et BLV+PL− (n = 14); charge provirale élevée (HPL) (n = 18) et charge provirale faible (LPL) (n = 13); titres d'anticorps élevés (HAT) (n = 17) et titres d'anticorps faibles (LAT) (n = 14). Les vaches BLV+PL+ ont montré une charge provirale et des titres d'anticorps significativement plus élevés que le groupe BLV+PL−; cependant, le premier suggère une propagation vraisemblablement sans rapport avec l'issue du lymphome, car HPL a été observé chez les vaches PL− lors du dernier échantillonnage. Conformément aux données, une réponse anticorps plus élevée indiquait fortement une sensibilité au BLV puisqu'elle était liée à l'apparition de PL+ et à un profil de cytokines compatible avec la suppression immunitaire. De plus, un retour à des titres d'anticorps plus faibles a été observé chez les vaches atteintes de HPL bien avant le temps, probablement en raison d'une immunosuppression à long terme. De plus, une expression élevée d'IL-10 et de TGF-ß était associée à une expression réduite d'IL-12, d'IFN-γ, d'IL-2 et d'IL-4 aux côtés de PL, HAT et HPL chez les vaches infectées par le BLV, suggérant une immunosuppression induite par IL-10 et le TGF-ß. L'expression d'IL-10 augmentait tout au long, impliquant une progression de la maladie, comme décrit. En conclusion, l'expansion proliférative des lymphocytes connus sous le nom de PL pourrait renforcer une population de cellules riches en régulation (Bregs et/ou Tregs) qui sécrète d'IL-10 et du TGF-ß, conduisant à une suppression immunitaire. D'autres études doivent être menées sur les types de cellules régulatrices impliquées dans la suppression immunitaire induite par le BLV.(Traduit par Docteur Serge Messier).

Assessment of endothelial colony forming cells delivery routes in a murine model of critical limb threatening ischemia using an optimized cell tracking approach.

BACKGROUND: Endothelial colony forming cells (ECFCs), alone or in combination with mesenchymal stem cells, have been selected as potential therapeutic candidates for critical limb-threatening ischemia (CLTI), mainly for those patients considered as "no-option," due to their capability to enhance revascularization and perfusion recovery of ischemic tissues. Nevertheless, prior to translating cell therapy to the clinic, biodistribution assays are required by regulatory guidelines to ensure biosafety as well as to discard undesired systemic translocations. Different approaches, from imaging technologies to qPCR-based methods, are currently applied. METHODS: In the current study, we have optimized a cell-tracking assay based on DiR fluorescent cell labeling and near-infrared detection for in vivo and ex vivo assays. Briefly, an improved protocol for DiR staining was set up, by incubation of ECFCs with 6.67 µM DiR and intensive washing steps prior cell administration. The minimal signal detected for the residual DiR, remaining after these washes, was considered as a baseline signal to estimate cell amounts correlated to the DiR intensity values registered in vivo. Besides, several assays were also performed to determine any potential effect of DiR over ECFCs functionality. Furthermore, the optimized protocol was applied in combination with qPCR amplification of specific human Alu sequences to assess the final distribution of ECFCs after intramuscular or intravenous administration to a murine model of CLTI. RESULTS: The optimized DiR labeling protocol indicated that ECFCs administered intramuscularly remained mainly within the hind limb muscle while cells injected intravenously were found in the spleen, liver and lungs. CONCLUSION: Overall, the combination of DiR labeling and qPCR analysis in biodistribution assays constitutes a highly sensitive approach to systemically track cells in vivo. Thereby, human ECFCs administered intramuscularly to CLTI mice remained locally within the ischemic tissues, while intravenously injected cells were found in several organs. Our data corroborate the need to perform biodistribution assays in order to define specific parameters such as the optimal delivery route for ECFCs before their application into the clinic.

Serum microRNAs targeting ACE2 and RAB14 genes distinguish asymptomatic from critical COVID-19 patients.

Despite the extraordinary advances achieved to beat COVID-19 disease, many questions remain unsolved, including the mechanisms of action of SARS-CoV-2 and which factors determine why individuals respond so differently to the viral infection. Herein, we performed an in silico analysis to identify host microRNA targeting ACE2, TMPRSS2, and/or RAB14, all genes known to participate in viral entry and replication. Next, the levels of six microRNA candidates previously linked to viral and respiratory-related pathologies were measured in the serum of COVID-19-negative controls (n = 16), IgG-positive COVID-19 asymptomatic individuals (n = 16), and critical COVID-19 patients (n = 17). Four of the peripheral microRNAs analyzed (hsa-miR-32-5p, hsa-miR-98-3p, hsa-miR-423-3p, and hsa-miR-1246) were upregulated in COVID-19 critical patients compared with COVID-19-negative controls. Moreover, hsa-miR-32-5p and hsa-miR-1246 levels were also altered in critical versus asymptomatic individuals. Furthermore, these microRNA target genes were related to viral infection, inflammatory response, and coagulation-related processes. In conclusion, SARS-CoV-2 promotes the alteration of microRNAs targeting the expression of key proteins for viral entry and replication, and these changes are associated with disease severity. The microRNAs identified could be taken as potential biomarkers of COVID-19 progression as well as candidates for future therapeutic approaches against this disease.

The serum of COVID-19 asymptomatic patients up-regulates proteins related to endothelial dysfunction and viral response in circulating angiogenic cells ex-vivo.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has already caused 6 million deaths worldwide. While asymptomatic individuals are responsible of many potential transmissions, the difficulty to identify and isolate them at the high peak of infection constitutes still a real challenge. Moreover, SARS-CoV-2 provokes severe vascular damage and thromboembolic events in critical COVID-19 patients, deriving in many related deaths and long-hauler symptoms. Understanding how these processes are triggered as well as the potential long-term sequelae, even in asymptomatic individuals, becomes essential. METHODS: We have evaluated, by application of a proteomics-based quantitative approach, the effect of serum from COVID-19 asymptomatic individuals over circulating angiogenic cells (CACs). Healthy CACs were incubated ex-vivo with the serum of either COVID-19 negative (PCR -/IgG -, n:8) or COVID-19 positive asymptomatic donors, at different infective stages: PCR +/IgG - (n:8) and PCR -/IgG + (n:8). Also, a label free quantitative approach was applied to identify and quantify protein differences between these serums. Finally, machine learning algorithms were applied to validate the differential protein patterns in CACs. RESULTS: Our results confirmed that SARS-CoV-2 promotes changes at the protein level in the serum of infected asymptomatic individuals, mainly correlated with altered coagulation and inflammatory processes (Fibrinogen, Von Willebrand Factor, Thrombospondin-1). At the cellular level, proteins like ICAM-1, TLR2 or Ezrin/Radixin were only up-regulated in CACs treated with the serum of asymptomatic patients at the highest peak of infection (PCR + /IgG -), but not with the serum of PCR -/IgG + individuals. Several proteins stood out as significantly discriminating markers in CACs in response to PCR or IgG + serums. Many of these proteins particiArticle title: Kindly check and confirm the edit made in the article title.pate in the initial endothelial response against the virus. CONCLUSIONS: The ex vivo incubation of CACs with the serum of asymptomatic COVID-19 donors at different stages of infection promoted protein changes representative of the endothelial dysfunction and inflammatory response after viral infection, together with activation of the coagulation process. The current approach constitutes an optimal model to study the response of vascular cells to SARS-CoV-2 infection, and an alternative platform to test potential inhibitors targeting either the virus entry pathway or the immune responses following SARS-CoV-2 infection.

Long Term Response to Circulating Angiogenic Cells, Unstimulated or Atherosclerotic Pre-Conditioned, in Critical Limb Ischemic Mice.

Critical limb ischemia (CLI), the most severe form of peripheral artery disease, results from the blockade of peripheral vessels, usually correlated to atherosclerosis. Currently, endovascular and surgical revascularization strategies cannot be applied to all patients due to related comorbidities, and even so, most patients require re-intervention or amputation within a year. Circulating angiogenic cells (CACs) constitute a good alternative as CLI cell therapy due to their vascular regenerative potential, although the mechanisms of action of these cells, as well as their response to pathological conditions, remain unclear. Previously, we have shown that CACs enhance angiogenesis/arteriogenesis from the first days of administration in CLI mice. Also, the incubation ex vivo of these cells with factors secreted by atherosclerotic plaques promotes their activation and mobilization. Herein, we have evaluated the long-term effect of CACs administration in CLI mice, whether pre-stimulated or not with atherosclerotic factors. Remarkably, mice receiving CACs and moreover, pre-stimulated CACs, presented the highest blood flow recovery, lower progression of ischemic symptoms, and decrease of immune cells recruitment. In addition, many proteins potentially involved, like CD44 or matrix metalloproteinase 9 (MMP9), up-regulated in response to ischemia and decreased after CACs administration, were identified by a quantitative proteomics approach. Overall, our data suggest that pre-stimulation of CACs with atherosclerotic factors might potentiate the regenerative properties of these cells in vivo.

Current Status of Angiogenic Cell Therapy and Related Strategies Applied in Critical Limb Ischemia.

Critical limb ischemia (CLI) constitutes the most severe form of peripheral arterial disease (PAD), it is characterized by progressive blockade of arterial vessels, commonly correlated to atherosclerosis. Currently, revascularization strategies (bypass grafting, angioplasty) remain the first option for CLI patients, although less than 45% of them are eligible for surgical intervention mainly due to associated comorbidities. Moreover, patients usually require amputation in the short-term. Angiogenic cell therapy has arisen as a promising alternative for these "no-option" patients, with many studies demonstrating the potential of stem cells to enhance revascularization by promoting vessel formation and blood flow recovery in ischemic tissues. Herein, we provide an overview of studies focused on the use of angiogenic cell therapies in CLI in the last years, from approaches testing different cell types in animal/pre-clinical models of CLI, to the clinical trials currently under evaluation. Furthermore, recent alternatives related to stem cell therapies such as the use of secretomes, exosomes, or even microRNA, will be also described.

REX-001, a BM-MNC Enriched Solution, Induces Revascularization of Ischemic Tissues in a Murine Model of Chronic Limb-Threatening Ischemia.

Background: Bone Marrow Mononuclear Cells (BM-MNC) constitute a promising alternative for the treatment of Chronic Limb-Threatening ischemia (CLTI), a disease characterized by extensive blockade of peripheral arteries, clinically presenting as excruciating pain at rest and ischemic ulcers which may lead to gangrene and amputation. BM-MNC implantation has shown to be efficient in promoting angiogenesis and ameliorating ischemic symptoms in CLTI patients. However, the variability seen between clinical trials makes necessary a further understanding of the mechanisms of action of BM-MNC, and moreover, to improve trial characteristics such as endpoints, inclusion/exclusion criteria or drug product compositions, in order to implement their use as stem-cell therapy. Materials: Herein, the effect of REX-001, a human-BM derived cell suspension enriched for mononuclear cells, granulocytes and CD34+ cells, has been assessed in a murine model of CLTI. In addition, a REX-001 placebo solution containing BM-derived red blood cells (BM-RBCs) was also tested. Thus, 24 h after double ligation of the femoral artery, REX-001 and placebo were administrated intramuscularly to Balb-c nude mice (n:51) and follow-up of ischemic symptoms (blood flow perfusion, motility, ulceration and necrosis) was carried out for 21 days. The number of vessels and vascular diameter sizes were measured within the ischemic tissues to evaluate neovascularization and arteriogenesis. Finally, several cell-tracking assays were performed to evaluate potential biodistribution of these cells. Results: REX-001 induced a significant recovery of blood flow by increasing vascular density within the ischemic limbs, with no cell translocation to other organs. Moreover, cell tracking assays confirmed a decrease in the number of infused cells after 2 weeks post-injection despite on-going revascularization, suggesting a paracrine mechanism of action. Conclusion: Overall, our data supported the role of REX-001 product to improve revascularization and ischemic reperfusion in CLTI.

Atherosclerotic Pre-Conditioning Affects the Paracrine Role of Circulating Angiogenic Cells Ex-Vivo.

In atherosclerosis, circulating angiogenic cells (CAC), also known as early endothelial progenitor cells (eEPC), are thought to participate mainly in a paracrine fashion by promoting the recruitment of other cell populations such as late EPC, or endothelial colony-forming cells (ECFC), to the injured areas. There, ECFC replace the damaged endothelium, promoting neovascularization. However, despite their regenerative role, the number and function of EPC are severely affected under pathological conditions, being essential to further understand how these cells react to such environments in order to implement their use in regenerative cell therapies. Herein, we evaluated the effect of direct incubation ex vivo of healthy CAC with the secretome of atherosclerotic arteries. By using a quantitative proteomics approach, 194 altered proteins were identified in the secretome of pre-conditioned CAC, many of them related to inhibition of angiogenesis (e.g., endostatin, thrombospondin-1, fibulins) and cell migration. Functional assays corroborated that healthy CAC released factors enhanced ECFC angiogenesis, but, after atherosclerotic pre-conditioning, the secretome of pre-stimulated CAC negatively affected ECFC migration, as well as their ability to form tubules on a basement membrane matrix assay. Overall, we have shown here, for the first time, the effect of atherosclerotic factors over the paracrine role of CAC ex vivo. The increased release of angiogenic inhibitors by CAC in response to atherosclerotic factors induced an angiogenic switch, by blocking ECFC ability to form tubules in response to pre-conditioned CAC. Thus, we confirmed here that the angiogenic role of CAC is highly affected by the atherosclerotic environment.

Identification of the initial molecular changes in response to circulating angiogenic cells-mediated therapy in critical limb ischemia.

BACKGROUND: Critical limb ischemia (CLI) constitutes the most aggressive form of peripheral arterial occlusive disease, characterized by the blockade of arteries supplying blood to the lower extremities, significantly diminishing oxygen and nutrient supply. CLI patients usually undergo amputation of fingers, feet, or extremities, with a high risk of mortality due to associated comorbidities. Circulating angiogenic cells (CACs), also known as early endothelial progenitor cells, constitute promising candidates for cell therapy in CLI due to their assigned vascular regenerative properties. Preclinical and clinical assays with CACs have shown promising results. A better understanding of how these cells participate in vascular regeneration would significantly help to potentiate their role in revascularization. Herein, we analyzed the initial molecular mechanisms triggered by human CACs after being administered to a murine model of CLI, in order to understand how these cells promote angiogenesis within the ischemic tissues. METHODS: Balb-c nude mice (n:24) were distributed in four different groups: healthy controls (C, n:4), shams (SH, n:4), and ischemic mice (after femoral ligation) that received either 50 µl physiological serum (SC, n:8) or 5 × 105 human CACs (SE, n:8). Ischemic mice were sacrificed on days 2 and 4 (n:4/group/day), and immunohistochemistry assays and qPCR amplification of Alu-human-specific sequences were carried out for cell detection and vascular density measurements. Additionally, a label-free MS-based quantitative approach was performed to identify protein changes related. RESULTS: Administration of CACs induced in the ischemic tissues an increase in the number of blood vessels as well as the diameter size compared to ischemic, non-treated mice, although the number of CACs decreased within time. The initial protein changes taking place in response to ischemia and more importantly, right after administration of CACs to CLI mice, are shown. CONCLUSIONS: Our results indicate that CACs migrate to the injured area; moreover, they trigger protein changes correlated with cell migration, cell death, angiogenesis, and arteriogenesis in the host. These changes indicate that CACs promote from the beginning an increase in the number of vessels as well as the development of an appropriate vascular network.

A retrospective study of DNA prevalence of porcine parvoviruses in Mexico and its relationship with porcine circovirus associated disease.

Worldwide, many emerging porcine parvoviruses (PPVs) have been linked to porcine circovirus-2 (PCV2) associated disease (PCVAD), which includes post-weaning multi-systemic wasting syndrome (PMWS), PCV2-related reproductive failure (PCV2-RF), as well as other syndromes. To determine the DNA prevalence of PPVs and their relationship with PMWS and PCV2-RF in Mexico, 170 formalin-fixed paraffin-embedded tissues were selected from archival collections to detect PPVs using a nested polymerase chain reaction. The tissues were composed of 50 PMWS cases, 20 age-matched tissues from healthy pigs, 56 PCV2-related reproductive failure (PCV2+ -RF) cases, and 44 PCV2- -RF cases. Overall, PPV2 and PPV6 were the most prevalent species (90.0% and 74.7%, respectively). In 8-11 week old pigs, the highest prevalence was for PPV6 and PPV3. Concerning reproductive failure, the PCV2-affected farms had a significantly higher prevalence for PPV6 (61.6%) and PPV5 (36.4%) than the PCV2-unaffected farms (35.0% and 5.0%, respectively). The concurrent infection rate was high, being significant for PPV2/PPV4 and PPV1/PPV5 within the PMWS cases and for PPV6/PPV5 among the PCV2+ -RF tissues. PPV5 showed a significant relationship with PMWS, whereas PPV5 and PPV6 were significant for PCVAD. The prevalence and coinfection rate of PPVs in Mexico were markedly higher than that described in other countries, denoting that PPV5 and PPV6 might have a potential role in PCVAD in Mexico. It is concluded that it is likely that the density population of pigs in Mexico is contributing to high PPV inter-species and PCV2 coinfections which might lead to a different pathogenic outcome.

Molecular characterization of the ORF2 of Torque teno sus virus 1a and Torque teno sus virus 1b detected in cases of postweaning multisystemic wasting syndrome in Mexico.

Worldwide Torque teno sus virus (TTSuV, genus Iotatorquevirus) species have been regarded as possible agents associated with porcine circovirus-associated disease. Iotatorquevirus species possess high genomic variability, suggesting that diverse genotypes are widely geographically distributed. In this study, we validated the genomic variability of Iotaroquevirus species in pigs with postweaned multisystemic wasting syndrome. Genomic DNA from nine TTSuV1a-positive tissues and 15 TTSuV1b-positive tissues was used to amplify the complete ORF2 of each species by nested PCR to perform a molecular characterization. It was found that Mexican TTSuV1a sequences belong to genotype B, sharing phylogenetic origin, high nucleic acid and amino acid sequence similarity and dominant epitope conformation with commercially linked countries, such as the United States, Canada and China, whereas the Mexican TTSuV1b sequences belong to genotype A, being more divergent among each other and displaying low nucleotide identity with worldwide genotype A sequences. In both Iotatorquevirus species, a PTPase-like signature motif was identified in the predicted amino acid sequence, being more conserved for Mexican TTSuV1b sequences than for Mexican TTSuV1a sequences, in which several substitutions were observed. These changes may influence the conformation of dominant epitopes as different arrays were determined among TTSuV1a genotypes. ORF2 variability may account for pathogenic differences by modifying viral replication and immune response, as depicted for human TTV.

Retrospective study of the relationship of Torque teno sus virus 1a and Torque teno sus virus 1b with porcine circovirus associated disease.

Genus Iotatorquevirus consists of 2 species, Torque teno sus virus 1a and Torque teno sus virus 1b, which are ubiquitous in swine populations, and are widely reported in association with porcine circovirus associated disease (PCVAD). To evaluate the relationship with PCVAD, 100 formalin-fixed paraffin-embedded tissue samples were used to detect both Iotatorquevirus species by nested PCR and sequencing. Sixty-eight PCVAD cases were selected as well as 32 porcine circovirus type 2 (PCV2) non-affected cases. Overall, 33 of the 100 cases were positive for Torque teno sus virus 1a and 8 of 100 were positive for Torque teno sus virus 1b. Only 24 of 68 (35%) PCVAD cases were positive for Torque teno sus virus 1a; 39% (9/23) of post-weaning multisystemic wasting syndrome, and 33% (15/45) of PCV2-associated reproductive failure cases. Among PCV2 non-affected cases, 28% were positive for Torque teno sus virus 1a and 6% were positive for Torque teno sus virus 1b. Torque teno sus virus 1b was not detected in PCV2-associated reproductive failure cases. Regardless of the PCV2-status, a lower frequency of both Iotatorquevirus species was found than depicted in other reports and there was no statistical relationship with PCVAD (χ 2 < 0.01). Given the worldwide genomic variability of Iotatorquevirus species, it is feasible that species prevalent in Mexico share a lower nucleotide sequence identity, leading to different pathogenic potential.

Le genre Iotatorquevirus consiste en deux espèces, le virus Torque teno sus 1a et le virus Torque teno sus 1b, qui sont ubiquitaires dans la population porcine, et couramment rapportés en association avec la maladie associée au circovirus porcin (MACVP). Afin d'évaluer la relation avec MACVP, 100 échantillons de tissus fixés dans la formaline et enrobés de paraffine ont été utilisés pour détecter les deux espèces de Iotorquevirus par réaction d'amplification en chaîne par la polymérase nichée et séquençage. Soixante-huit cas de MACVP ont été sélectionnés ainsi que 32 cas non-affectés d'infection par le circovirus porcin de type (CVP2). Globalement, 33 des 100 cas étaient positifs pour le virus Torque teno sus 1a et 8 des 100 étaient positifs pour le virus Torque tenos sus 1b. Seulement 24 des 68 (35 %) cas de MACVP étaient positifs pour le virus Torque tenos sus 1a; 39 % (9/23) du syndrome de dépérissement post-sevrage, et 33 % (15/45) des cas de problèmes reproducteurs associés au CVP2. Parmi les cas non-affectés de CVP2, 28 % étaient positifs pour le virus Torque teno sus 1a et 6 % étaient positifs pour le virus Torque tenos sus 1b. Le virus Torque tenos sus 1b n'a pas été détecté dans les cas de problèmes reproducteurs associés au CVP2. Indépendamment du statu vis-à-vis le CVP2, une fréquence plus basse des deux espèces d'Iotatorquevirus fut trouvée comparativement à ce qui est décrit dans d'autres études et il n'y avait pas de relation statistiquement significative avec MACVP (χ2 < 0,01). Étant donné la variabilité génomique mondiale des espèces d'Iotatorquevirus il est possible que les espèces prévalentes au Mexique partagent une plus faible identité de séquences nucléotidiques, entrainant ainsi un potentiel pathogène différent.(Traduit par Docteur Serge Messier).

Psychological and Personality Predictors of Weight Loss and Comorbid Metabolic Changes After Bariatric Surgery.

Bariatric surgery (BS) has proven to be the most effective treatment for weight loss and for improving comorbidities in severe obesity. A comprehensive psychological assessment prior to surgery is proposed to prepare patients for a successful post-surgical outcome. Therefore, the main aim of the present study was to assess psychological and personality predictors of BS outcome. The sample comprised 139 severely obese patients who underwent BS. Assessment measures included the Eating Disorders Inventory-2, the Symptom Checklist-Revised and the Temperament and Character Inventory-Revised. Our results show that favourable BS outcome, after 2 years follow up, was associated with younger age, less depression, moderate anxiety symptoms and high cooperativeness levels. Likewise, metabolic improvements were found to be linked to younger age and certain psychopathological factors. In conclusion, our findings suggest that age, baseline body mass index, psychopathological indexes and personality traits predict successful BS outcome.

Full HPV typing by a single restriction enzyme.

BACKGROUND: Restriction fragment length polymorphism (RFLP) methods for genotyping genital human papillomavirus (HPV) are considered labor consuming and constrained by the reduced set of restriction enzymes capable of detecting specific mutations. However, we think that these methods have not taken full advantage of the high diversity of the known restriction enzymes. OBJECTIVE: We have set out to find the best restriction enzyme for HPV typing. STUDY DESIGN: An extensive search for enzymes was carried out by combining statistical methods and database information. The search maximized the discrimination between high- and low-risk types by examining the sequence of the L1 gene flanked by primers MY09/11. Different electrophoretic resolutions and two variations of the RFLP method were considered. RESULTS: HpyCH4V is the best enzyme for discriminating between risk types. Moreover, HpyCH4V generates different patterns for virtually all the HPV types. The typical pattern consists of two or three fragments, which facilitates typing in mixed infections. The typing of a set of clinical samples confirmed the expectations. CONCLUSIONS: This result illustrates the possibilities of statistical methods to exploit the high diversity of restriction enzymes in order to classify samples in a pre-established hierarchy of types for which DNA sequences are known.

Early cell-mediated immune responses to Marek's disease virus in two chicken lines with defined major histocompatibility complex antigens.

N2a and P2a chickens, resistant and susceptible to Marek's disease (MD), respectively, were used to examine relationships between major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) and natural killer (NK)-like cell activity with resistance to infection with Marek's disease virus (MDV). Ten-day-old chickens were infected with MDV and euthanatized at selected times to evaluate for NK cell and MHC-restricted cytotoxicity. The N2a MDV-infected chickens had an early cell-mediated immune response characterized by a sustained NK-like cytotoxicity that coincided with a measurable MHC-cytotoxicity that was lower than controls. Although MHC-restricted and NK cell cytotoxicity was demonstrated in P2a MDV-infected chickens at 8 dpi, both abruptly decreased and remained low for the remainder of the 20-day experiment. The critical time point that may determine the resistance to MD appears to be within the first 2 weeks post-infection. Improvement of the chicken NK cell activity may be a good candidate for both selection and immunomodulation MD control programs.