Is Malassezia nana the main species in horses' ear canal microbiome?

The objective of this study was to characterize genotypically Malassezia spp. isolated from the external ear canal of healthy horses. Fifty-five horses, 39 (70.9%) males and 16 (29.1%) females, from different breeds and adults were studied. External ear canals were cleaned and a sterile cotton swab was introduced to collect cerumen. A total of 110 samples were cultured into Dixon medium and were incubated at 32 degrees C for up to 15 days. Macro- and micromorphology and phenotypic identification were performed. DNA was extracted, strains were submitted to polymerase chain reaction technique, and the products obtained were submitted to Restriction Fragment Length Polymorphism using the restriction enzymes BstCI and HhaI. Strains were sent off to genetic sequencing of the regions 26S rDNA D1/D2 and ITS1-5.85-ITS2 rDNA. Malassezia spp. were isolated from 33/55 (60%) animals and 52/110 (47%) ear canals. No growth on Sabouraud dextrose agar was observed, confirming the lipid dependence of all strains. Polymerase chain reaction-Restriction fragment length polymorphism permitted the molecular identification of Malassezia nana-42/52 (81%) and Malassezia slooffiae- 10/52 (19%). Sequencing confirmed RFLP identification. It was surprising that M. nana represented over 80% of the strains and no Malassezia equina was isolated in this study, differing from what was expected. (C) 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND licenseCapes-PROSUP fellowship, Federal Government, BrazilUniv Paulista, Mol & Cellular Biol Lab, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Microbiol Immunol & Parasitol, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Microbiol Immunol & Parasitol, Sao Paulo, BrazilWeb of Scienc

Targeting the Immune System With Mesenchymal Stromal Cell-Derived Extracellular Vesicles: What Is the Cargo's Mechanism of Action?

[EN]The potent immunomodulatory activities displayed bymesenchymal stromal cells (MSCs) have motivated their application in hundreds of clinical trials to date. In some countries, they have subsequently been approved for the treatment of immune disorders such as Crohn’s disease and graft-versus-host disease. Increasing evidence suggests that their main mechanism of action in vivo relies on paracrine signaling and extracellular vesicles. Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) play a prominent role in intercellular communication by allowing the horizontal transfer of microRNAs, mRNAs, proteins, lipids and other bioactive molecules between MSCs and their targets. However, despite the considerable momentum gained by MSC-EV research, the precise mechanismby whichMSC-EVs interact with the immune systemis still debated. Available evidence is highly context-dependent and fragmentary, with a limited number of reports trying to link their efficacy to specific active components shuttled within them. In this concise review, currently available evidence on the molecular mechanisms underlying the effects of MSC-EV cargo on the immune system is analyzed. Studies that pinpoint specific MSC-EV-borne mediators of immunomodulation are highlighted, with a focus on the signaling events triggered by MSC-EVs in target immune cells. Reports that study the effects of preconditioning or “licensing” inMSC-EV-mediated immunomodulation are also presented. The need for further studies that dissect the mechanisms of MSC-EV cargo in the adaptive immune system is emphasized. Finally, the major challenges that need to be addressed to harness the full potential of these signaling vehicles are discussed, with the ultimate goal of effectively translating MSC-EV treatments into the clinic.Instituto de Salud Carlos III ; Consejería de Sanidad de Castilla y León ; Consejería de Educación de Castilla y León ; Fundación Científica de la Asociación Española contra el Cáncer ; Sociedad Española de Hematología y Hemoterapia

Could a B-1 Cell Derived Phagocyte “Be One” of the Peritoneal Macrophages during LPS-Driven Inflammation?

The inflammatory response is driven by signals that recruit and elicit immune cells to areas of tissue damage or infection. The concept of a mononuclear phagocyte system postulates that monocytes circulating in the bloodstream are recruited to inflamed tissues where they give rise to macrophages. A recent publication demonstrated that the large increase in the macrophages observed during infection was the result of the multiplication of these cells rather than the recruitment of blood monocytes. We demonstrated previously that B-1 cells undergo differentiation to acquire a mononuclear phagocyte phenotype in vitro (B-1CDP), and we propose that B-1 cells could be an alternative origin for peritoneal macrophages. A number of recent studies that describe the phagocytic and microbicidal activity of B-1 cells in vitro and in vivo support this hypothesis. Based on these findings, we further investigated the differentiation of B-1 cells into phagocytes in vivo in response to LPS-induced inflammation. Therefore, we investigated the role of B-1 cells in the composition of the peritoneal macrophage population after LPS stimulation using osteopetrotic mice, BALB/Xid mice and the depletion of monocytes/macrophages by clodronate treatment. We show that peritoneal macrophages appear in op/op(−/−) mice after LPS stimulation and exhibit the same Ig gene rearrangement (VH11) that is often found in B-1 cells. These results strongly suggest that op/op(−/−) peritoneal “macrophages” are B-1CDP. Similarly, the LPS-induced increase in the macrophage population was observed even following monocyte/macrophage depletion by clodronate. After monocyte/macrophage depletion by clodronate, LPS-elicited macrophages were observed in BALB/Xid mice only following the transfer of B-1 cells. Based on these data, we confirmed that B-1 cell differentiation into phagocytes also occurs in vivo. In conclusion, the results strongly suggest that B-1 cell derived phagocytes are a component of the LPS-elicited peritoneal macrophage population

Co-administration of human MSC overexpressing HIF-1α increases human CD34+ cell engraftment in vivo

Background: Poor graft function or graft failure after allogeneic stem cell transplantation is an unmet medical need, in which mesenchymal stromal cells (MSC) constitute an attractive potential therapeutic approach. Hypoxia-inducible factor-1α (HIF-1α) overexpression in MSC (HIF-MSC) potentiates the angiogenic and immunomodulatory properties of these cells, so we hypothesized that co-transplantation of MSC-HIF with CD34+ human cord blood cells would also enhance hematopoietic stem cell engraftment and function both in vitro and in vivo. Methods: Human MSC were obtained from dental pulp. Lentiviral overexpression of HIF-1α was performed transducing cells with pWPI-green fluorescent protein (GFP) (MSC WT) or pWPI-HIF-1α-GFP (HIF-MSC) expression vectors. Human cord blood CD34+ cells were co-cultured with MSC WT or HIF-MSC (4:1) for 72 h. Then, viability (Annexin V and 7-AAD), cell cycle, ROS expression and immunophenotyping of key molecules involved in engraftment (CXCR4, CD34, ITGA4, c-KIT) were evaluated by flow cytometry in CD34+ cells. In addition, CD34+ cells clonal expansion was analyzed by clonogenic assays. Finally, in vivo engraftment was measured by flow cytometry 4-weeks after CD34+ cell transplantation with or without intrabone MSC WT or HIF-MSC in NOD/SCID mice. Results: We did not observe significant differences in viability, cell cycle and ROS expression between CD34+ cells co-cultured with MSC WT or HIF-MSC. Nevertheless, a significant increase in CD34, CXCR4 and ITGA4 expression (p = 0.009; p = 0.001; p = 0.013, respectively) was observed in CD34+ cells co-cultured with HIF-MSC compared to MSC WT. In addition, CD34+ cells cultured with HIF-MSC displayed a higher CFU-GM clonogenic potential than those cultured with MSC WT (p = 0.048). We also observed a significant increase in CD34+ cells engraftment ability when they were co-transplanted with HIF-MSC compared to CD34+ co-transplanted with MSC WT (p = 0.016) or alone (p = 0.015) in both the injected and contralateral femurs (p = 0.024, p = 0.008 respectively). Conclusions: Co-transplantation of human CD34+ cells with HIF-MSC enhances cell engraftment in vivo. This is probably due to the ability of HIF-MSC to increase clonogenic capacity of hematopoietic cells and to induce the expression of adhesion molecules involved in graft survival in the hematopoietic niche

Eltrombopag increases the hematopoietic supporting ability of mesenchymal stem/stromal cells

[Background]: Eltrombopag (EP) is a small molecule that acts directly on hematopoietic stem cells (HSCs) and megakaryocytes to stimulate the hematopoietic process. Mesenchymal stem/stromal cells (MSCs) are key hematopoietic niche regulators. [Objectives]: We aimed to determine whether EP has any effect on MSC function and properties (especially on their hematopoietic-supporting ability) and if so, what changes (e.g. genome-wide transcriptomic alterations) are induced in MSC after EP treatment. [Design/Methods]: MSCs were isolated from 12 healthy donors and treated with 15 µM and 50 µM of EP for 24 h. The toxicity of the drug on MSCs and their differentiation ability were analyzed, as well as the transcriptomic profile, reactive oxygen species (ROS) and DNA damage and the changes induced in the clonogenic capacity of HSCs.[Results]: The results show that EP also modifies MSC functions, decreasing their adipogenic differentiation, increasing the expression of genes involved in hypoxia and other pathways related to oxygen homeostasis, and enhancing their ability to support hematopoiesis in vitro.[Conclusion]: Our findings support the use of EP in cases where hematopoiesis is defective, despite its well-known direct effects on hematopoietic cells. Our findings suggest that further studies on the effects of EP on MSCs from patients with aplastic anemia are warranted.This study was supported by research funding from Novartis Pharmaceuticals to FS-G. SP is supported by Fundación Española de Hematología y Hemoterapia (FEHH). SM is supported by RETIC and RICORS programs of ISCIII European Regional Development Fund (RD16/0011/0015, RD21/0017/0006), ‘A way to make Europe’ and NextGenerationEU. GJMC and ESL are supported by the Spanish Ministerio de Ciencia e Innovación (FPU18/03533 and PFIS/19/00272 respectively).Peer reviewe

Characterization of ExPEC (\"Extraintestinal Pathogenic Escherichia coli\") isolated from dogs and cats with uinary tract infections (UTI).

As ITU são as mais freqüentes infecções ocasionadas por ExPEC. Entre os fatores de virulência (FV) encontram-se nestas cepas adesinas, invasinas, toxinas, sideróforos, e evasinas, localizados em plasmídios ou ilhas de patogenecidade. O objetivo deste estudo foi caracterizar 45 cepas de E. coli isoladas de 33 cães e 7 gatos com ITU, quanto aos sorotipos, FV e grupos filogenéticos. Dos sorogrupos relacionados às ITU foram encontrados O6 (20%), O2 (16%), O25 (4%), O4 e O11 (4% cada um). Entre os genes pesquisados, foram encontrados fimH (100%), pap (47%), sfa (33%) e iha (4%); ibeA (29%); cnf1 (31%), hlyA (27%); fyuA (80%), iucD (22%); traT (51%); cvaC (20%) e malX (67%). Os isolados felinos foram agrupados em B2 (89%) e D (11%), enquanto os caninos em A (5,5%), B1 (19,5%), B2 (55,5%) e D (19,5%). Estes resultados sugerem que as ExPEC isoladas de cães e gatos apresentam potencial patogênico para ocasionar doenças mais graves que as ITU, assim como ocorre em humanos. Além disso, a similitude com as amostras humanas reforça a hipótese acerca de seu potencial zoonótico.The ability of ExPEC to cause extraintestinal infections in humans, dogs, and cats is associated with the expression of a variety of virulence factors (VF). The aim of this study was to evaluate the frequency of VF related to ExPEC, serotypes, and phylogenetic groups in 45 strains isolated from 33 dogs and 7 cats with UTI. These strains presented serogroups related with extraintestinal infections, e.g. O6 (20%), O2 (16%), O25 (4%), O4 e O11 (each one) and the following genes: fimH (100%), pap (47%), sfa (33%) e iha (4%); ibeA (29%); cnf1 (31%), hlyA (27%); fyuA (80%), iucD (22%); traT (51%); cvaC (20%) e malX (67%), cvaC (20%), and malX (67%). All feline strains were concentrated in B2 (89%) and D (11%) phylogenetic groups, whereas the canine ones were distributed in the four groups, A (5,5%), B1 (19,5%), B2 (55,5%) and D (19,5%). These findings suggesting that ExPEC isolated from dog and cat contain virulence markers to cause diseases, more severe than UTI, likewise in humans. Besides, these the close similarity between human and animal ExPEC supports the hypotesis of zoonotic potencial of them

Papel da via wnt no desenvolvimento de células b-1

Hematopoietic stem cells (HSC) are capable to generate multipotent progenitors that produce all blood cell lineages, and exhibit high proliferative potential and are able to self-renewal. Several molecules has been described to participate in the embryo cells development and commitment. In this context, Wnt proteins have an important role in many biological processes, included participation in different stages of the lymphocyte development and HSC self-renewal. Although B-1 cells are considered mature cells, they express two different programs simultaneously, the lymphoid and myeloid ones, and are self-renewing cells. In order to evaluate the role that Wnt pathway plays in this permissiveness and the self-renewal property, B-1 cells were purified and the Wnt receptors expression were verified by qPCR, showing the Fzd6 are the most expressed Wnt receptor. In co-culture experiments of B-1 and OP9 cells, stimulated or not with Wnt3a or Wnt5a, increased amount of viable and proliferative cells were observed in the Wnt3a treated group. The canonical Wnt signaling activation was assessed by Axin2 expression that was detected in the both treated groups. When the transcription factors related to lymphoid and myeloid lineage were analyzed, the Flt3 gene was upregulated after the both stimuli and IL- 7R was upregulated only when Wnt3a was added to the cultures. Despite of in vitro results indicated the role of canonical Wnt signaling in the B-1 self-renewal, the in vivo assays involving adoptive Wnt-reporter mice (Axin2+/lacZ) derived-B-1 cell transference to RAG-1 mice did not demonstrate increase number of the Wnt activated B-1 cells. Interestingly, B-1 progenitors (B-1P) showed an expressive response when cultivated onto Wnt5a-transduced OP9 cells, expanding their pool 40 times more in relation to the input cell number. Moreover, in the presence of Wnt3a- transduced OP9 cells, B-1P originated a significant population of CD5+ cells. Altogether, these data suggest that mature B-1 cells, and mainly B-1 progenitors are able to respond to Wnt ligands and this signaling could be important to maintenance of this B cell subtype. The pronounced response to Wnt ligands observed in the progenitors suggests that this Wnt pathway could be involved in the B-1 cell development and differentiation process.Células-tronco hematopoéticas (HSC) são capazes de originar progenitores de múltiplas linhagens que compõe o sangue, possuem alto potencial de proliferação, além de se autorrenovarem. Dentre as moléculas envolvidas no desenvolvimento e comprometimento de células embrionárias, as proteínas Wnt se destacam pelo papel que exercem em diversos processos biológicos, sendo relacionadas a vários estágios de desenvolvimento de linfócitos e autorrenovação de HSC. Células B-1, embora sejam células já diferenciadas, são capazes de expressar os programas linfoide e mieloide simultaneamente e também são mantidas por autorrenovação. Para verificar o envolvimento da via Wnt na permissividade da coexistência destes dois programas, assim como na manutenção e proliferação de linfócitos B-1, estas células foram purificadas e a expressão dos receptores da via foi avaliada, sendo o receptor Fzd6 o mais expresso. Nas coculturas de células B-1 com células OP9, estimuladas ou não com Wnt3a e Wnt5a recombinantes, foi observado aumento de células viáveis e maior quantidade de células em proliferação no grupo tratado com Wnt3a. A ativação da via foi avaliada pela qPCR sendo constatada a expressão de Axin2 após o tratamento com os dois ligantes. A avaliação dos genes relacionados aos programas linfomieloide revelou aumento da expressão de Flt3 após estímulo com Wnt3a e Wnt5a e IL-7R, somente após a adição do primeiro ligante. Embora os resultados in vitro indicassem a atuação da via clássica na autorrenovação de células B-1, os ensaios in vivo de transferência adotiva de células provenientes de animais repórter para Axin2 (Axin2+/lacZ) não mostraram aumento de células B-1 ativadas para a via Wnt. Interessantemente, observou-se uma resposta expressiva dos progenitores de células B-1 à sinalização mediada por Wnt5a. Foi observada expansão de 40 vezes em relação ao número inicial de células, quando progenitores de células B-1 foram cultivados com células OP9 transduzida com Wnt5a. Ainda, o cultivo destes progenitores com células OP9 transduzidas com Wnt3a induziu aumento nas células CD5+. Os dados obtidos até o momento sugerem que células B-1 adultas e, principalmente, as precursoras, são responsivas aos ligantes da via Wnt, indicando a atuação desta via na manutenção desta população de células B. A expressiva resposta aos ligantes da via pelos progenitores sugere o papel da mesma no processo de desenvolvimento e diferenciação de células B-1.Dados abertos - Sucupira - Teses e dissertações (2013 a 2016

Characterization of ExPEC (\"Extraintestinal Pathogenic Escherichia coli\") isolated from dogs and cats with uinary tract infections (UTI).

As ITU são as mais freqüentes infecções ocasionadas por ExPEC. Entre os fatores de virulência (FV) encontram-se nestas cepas adesinas, invasinas, toxinas, sideróforos, e evasinas, localizados em plasmídios ou ilhas de patogenecidade. O objetivo deste estudo foi caracterizar 45 cepas de E. coli isoladas de 33 cães e 7 gatos com ITU, quanto aos sorotipos, FV e grupos filogenéticos. Dos sorogrupos relacionados às ITU foram encontrados O6 (20%), O2 (16%), O25 (4%), O4 e O11 (4% cada um). Entre os genes pesquisados, foram encontrados fimH (100%), pap (47%), sfa (33%) e iha (4%); ibeA (29%); cnf1 (31%), hlyA (27%); fyuA (80%), iucD (22%); traT (51%); cvaC (20%) e malX (67%). Os isolados felinos foram agrupados em B2 (89%) e D (11%), enquanto os caninos em A (5,5%), B1 (19,5%), B2 (55,5%) e D (19,5%). Estes resultados sugerem que as ExPEC isoladas de cães e gatos apresentam potencial patogênico para ocasionar doenças mais graves que as ITU, assim como ocorre em humanos. Além disso, a similitude com as amostras humanas reforça a hipótese acerca de seu potencial zoonótico.The ability of ExPEC to cause extraintestinal infections in humans, dogs, and cats is associated with the expression of a variety of virulence factors (VF). The aim of this study was to evaluate the frequency of VF related to ExPEC, serotypes, and phylogenetic groups in 45 strains isolated from 33 dogs and 7 cats with UTI. These strains presented serogroups related with extraintestinal infections, e.g. O6 (20%), O2 (16%), O25 (4%), O4 e O11 (each one) and the following genes: fimH (100%), pap (47%), sfa (33%) e iha (4%); ibeA (29%); cnf1 (31%), hlyA (27%); fyuA (80%), iucD (22%); traT (51%); cvaC (20%) e malX (67%), cvaC (20%), and malX (67%). All feline strains were concentrated in B2 (89%) and D (11%) phylogenetic groups, whereas the canine ones were distributed in the four groups, A (5,5%), B1 (19,5%), B2 (55,5%) and D (19,5%). These findings suggesting that ExPEC isolated from dog and cat contain virulence markers to cause diseases, more severe than UTI, likewise in humans. Besides, these the close similarity between human and animal ExPEC supports the hypotesis of zoonotic potencial of them