The Role of Molecular or Cytogenetic Response as a Favorable Prognostic Factor Before Hematopoietic Stem Cell Transplantation for Chronic Myeloid Leukemia.

Tyrosine kinase inhibitors (TKIs) have revolutionized therapy for patients with chronic myeloid leukemia (CML) over the last two decades. However, some patients still do not achieve an adequate response to these drugs, and hematopoietic stem cell transplantation (HSCT) is indicated in this scenario. We present the results of a 20-year follow-up study of 70 patients who underwent transplantation after TKI failure. The primary objective of this study was to evaluate overall survival (OS) and the secondary objective was to evaluate the outcomes of relapse-free survival (RFS), GVHD-free, relapse-free survival (GFRS) and the incidences of relapse (RI), non-relapse mortality (NRM), acute and chronic GVHD. Median survival was 11 years, with a 1-year OS of 70% (57.8 to 79.3) and a 5-year OS of 57.7% (45.1 to 68.5). The estimated 5-year OS was not different for CP1 (60%) versus advanced stages (45%); P = .60. The degree of response immediately before transplantation was directly associated with worse outcomes [HR 5.89 (1.19-29.16); P = .03] for patients with only a hematological response compared with patients with a cytogenetic or molecular response. This study corroborates the potential of HSCT in the scenario of therapeutic failure and highlights the role of molecular or cytogenetic response as a potential target to be achieved before transplantation.

Correction: Molecular modelling of the HCMV IL-10 protein isoforms and analysis of their interaction with the human IL-10 receptor.

[This corrects the article DOI: 10.1371/journal.pone.0277953.].

Molecular modelling of the HCMV IL-10 protein isoforms and analysis of their interaction with the human IL-10 receptor.

The human cytomegalovirus (HCMV) UL111A gene encodes several homologs of the cellular interleukin 10 (cIL-10). Alternative splicing in the UL111A region produces two relatively well-characterized transcripts designated cmvIL-10 (isoform A) and LAcmvIL-10 (isoform B). The cmvIL-10 protein is the best characterized, both structurally and functionally, and has many immunosuppressive activities similar to cIL-10, while LAcmvIL-10 has more restricted biological activities. Alternative splicing also results in five less studied UL111A transcripts encoding additional proteins homologous to cIL-10 (isoforms C to G). These transcripts were identified during productive HCMV infection of MRC-5 cells with the high passage laboratory adapted AD169 strain, and the structure and properties of the corresponding proteins are largely unknown. Moreover, it is unclear whether these protein isoforms are able to bind the cellular IL-10 receptor and induce signalling. In the present study, we investigated the expression spectrum of UL111A transcripts in fully permissive MRC-5 cells and semi permissive U251 cells infected with the low passage HCMV strain TB40E. We identified a new spliced transcript (H) expressed during productive infection. Using computational methods, we carried out molecular modelling studies on the three-dimensional structures of the HCMV IL-10 proteins encoded by the transcripts detected in our work (cmvIL-10 (A), LAcmvIL-10 (B), E, F and H) and on their interaction with the human IL-10 receptor (IL-10R1). The modelling predicts clear differences between the isoform structures. Furthermore, the in silico simulations (molecular dynamics simulation and normal-mode analyses) allowed us to evaluate regions that contain potential receptor binding sites in each isoform. The analyses demonstrate that the complexes between the isoforms and IL-10R1 present different types of molecular interactions and consequently different affinities and stabilities. The knowledge about structure and expression of specific viral IL-10 isoforms has implications for understanding of their properties and role in HCMV immune evasion and pathogenesis.

Antibiotic resistance profiles and activity of clove essential oil (Syzygium aromaticum) against Pseudomonas aeruginosa isolated of canine otitis.

Background and Aim: Pseudomonas aeruginosa is often isolated from acute and chronic otitis and deep pyoderma in dogs. The increase in bacterial resistance to antibiotics induced the need for alternative therapies to treat infections, with an emphasis on essential oils (EOs). This study aimed to investigate clove oil's in vitro bactericidal action as a therapeutic alternative against strains of P. aeruginosa isolated from canine otitis. Materials and Methods: The antibacterial activity of clove oil was evaluated by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) using the broth microdilution technique in 96-well plates. Serial concentrations of 10-0.31% of the oil were used, equivalent to 104.5-3.26 mg/mL. The susceptibility of isolates to different classes of antibiotics was determined by the disk diffusion technique using 20 antibiotics belonging to eight classes. Isolates resistant to at least one antibiotic of three different classes were considered multidrug-resistant (MDR). Results: A high occurrence of resistance was observed for three antibiotics belonging to the cephalosporin classes (cefadroxil, cephalexin, and ceftriaxone), namely, sulfamethoxazole + trimethoprime, doxycycline, and enrofloxacin. The lowest resistance rates were observed for meropenem (4.88%), amikacin (12.20%), and tobramycin (12.2%). All isolates were susceptible to clove oil with an equivalent MIC and MBC from 3.26 to 6.53 mg/mL. Eugenol was the major component of the oil. Conclusion: Clove EO was effective against MDR strains of P. aeruginosa, indicating an alternative for developing an efficient and low-cost antimicrobial agent to treat canine otitis.

Immunoproteomic and Immunopeptidomic Analyses of Histoplasma capsulatum Reveal Promiscuous and Conserved Epitopes Among Fungi With Vaccine Potential.

As there are more than 6 million human deaths due to mycoses each year, there is an urgent need to develop fungal vaccines. Moreover, given the similarities among pathogenic fungi, it may be possible to create a multi-fungi vaccine. In this study, we combined immunoproteomic and immunopeptidomic methods, for which we have adapted a technique based on co-immunoprecipitation (Co-IP) that made it possible to map Histoplasma capsulatum epitopes for the first time in a natural context using murine dendritic cells (DCs) and macrophages (Mφ). Although polysaccharide epitopes exist, this research focused on mapping protein epitopes as these are more immunogenic. We used different algorithms to screen proteins and peptides identified by two-dimensional electrophoresis (2-D) and Co-IP. Seventeen proteins were revealed by 2-D gels, and 45 and 24 peptides from distinct proteins were presented by DCs and Mφ, respectively. We then determined which epitopes were restricted to MHC-I and II from humans and mice and showed high promiscuity, but lacked identity with human proteins. The 4 most promising peptides were synthesized, and the peptides with and without incorporation into glucan particles induced CD4+ and CD8+ T cell proliferation and produced a Th1 and Th17 response marked by the secretion of high levels of IFN-γ, IL-17 and IL-2. These epitopes were from heat shock protein 60, enolase, and the ATP-dependent molecular chaperone HSC82, and they each have a high degree of identity with proteins expressed by other medically important pathogenic fungi. Thus, the epitopes described in this study have the potential for use in the development of vaccines that could result in cross-protection among fungal species.

Multidrug-resistant mcr-1 gene-positive Klebsiella pneumoniae ST307 causing urinary tract infection in a cat.

RESUMO: The Klebsiella pneumoniae (K. pneumoniae) bacterium is responsible for many opportunistic infections such as sepsis, and a multidrug-resistant (MDR) clone sequence type (ST) 307 has recently begun to spread. The objective of this study was to report the first occurrence of this virulent genotype, which was found in the context of a urinary infection in a domestic feline in Brazil. The K. pneumoniae isolate was identified from the urine of a 6-month-old male crossbreed cat using 16S rRNA sequencing. It was then subjected to antimicrobial susceptibility testing, followed by multilocus sequence typing analysis, and PCR detection of virulence and resistance genes. The antimicrobial susceptibility profile demonstrated that the isolate was MDR and associated with the presence of the colistin resistance gene (mcr-1). Genotyping allowed us to classify the isolate as K. pneumoniae ST307 with the presence of wabG, uge, and entB genes. MDR K. pneumoniae is important in human and veterinary medicine because it causes many types of infections. Clonal propagation of virulent or MDR genotypes such as K. pneumoniae ST307 is a global concern. This report of ST307 isolation from a urine sample in a domestic feline is the first in Brazil.

Immune Sensing and Potential Immunotherapeutic Approaches to Control Chromoblastomycosis.

Chromoblastomycosis (CBM) is a neglected, chronic, and progressive subcutaneous mycosis caused by different species of fungi from the Herpotrichiellaceae family. CBM disease is usually associated with agricultural activities, and its infection is characterized by verrucous, erythematous papules, and atrophic lesions on the upper and lower limbs, leading to social stigma and impacts on patients' welfare. The economic aspect of disease treatment is another relevant issue. There is no specific treatment for CBM, and different anti-fungal drug associations are used to treat the patients. However, the long period of the disease and the high cost of the treatment lead to treatment interruption and, consequently, relapse of the disease. In previous years, great progress had been made in the comprehension of the CBM pathophysiology. In this review, we discuss the differences in the cell wall composition of conidia, hyphae, and muriform cells, with a particular focus on the activation of the host immune response. We also highlight the importance of studies about the host skin immunology in CBM. Finally, we explore different immunotherapeutic studies, highlighting the importance of these approaches for future treatment strategies for CBM.

Fonsecaeapedrosoi Conidia and Hyphae Activate Neutrophils Distinctly: Requirement of TLR-2 and TLR-4 in Neutrophil Effector Functions.

Chromoblastomycosis is a chronic and progressive subcutaneous mycosis caused mainly by the fungus Fonsecaea pedrosoi. The infection is characterized by erythematous papules and histological sections demonstrating an external layer of fibrous tissue and an internal layer of thick granulomatous inflammatory tissue containing mainly macrophages and neutrophils. Several groups are studying the roles of the innate and adaptive immune systems in F. pedrosoi infection; however, few studies have focused on the role of neutrophils in this infection. In the current study, we verify the importance of murine neutrophils in the killing of F. pedrosoi conidia and hyphae. We demonstrate that phagocytosis and reactive oxygen species during infection with conidia are TLR-2- and TLR-4-dependent and are essential for conidial killing. Meanwhile, hyphal killing occurs by NET formation in a TLR-2-, TLR-4-, and ROS-independent manner. In vivo experiments show that TLR-2 and TLR-4 are also important in chromoblastomycosis infection. TLR-2KO and TLR-4KO animals had lower levels of CCL3 and CXCL1 chemokines and impaired neutrophil migration to the infected site. These animals also had higher fungal loads during infection with F. pedrosoi conidia, confirming that TLR-2 and TLR-4 are essential receptors for F. pedrosoi recognition and immune system activation. Therefore, this study demonstrates for the first time that neutrophil activation during F. pedrosoi is conidial or hyphal-specific with TLR-2 and TLR-4 being essential during conidial infection but unnecessary for hyphal killing by neutrophils.

Vancomycin susceptibility profiles of Staphylococcus spp. isolates from domestic and wild animals / Perfil de suscetibilidade à Vancomicina de isolados de Staphylococcus spp. de animais domésticos e silvestres

ABSTRACT: Staphylococcus spp. are bacteria involved in human and animal infections. They are resistant to antimicrobials and have become a major public health concern. In recent years, there has been a significant increase in methicillin-resistant Staphylococcus strains and vancomycin is the drug of choice for the treatment of such isolates. However, the minimum inhibitory concentration (MIC) of vancomycin ​​necessary to combat this microorganism has been showing an increase. The aim of the present study was to determine the susceptibility profile of the Staphylococcus spp. of domestic and wild animals to vancomycin, using the microdilution in broth and E-test® techniques, as well as comparing the results of both tests. Of the 50 isolates tested, 47 (94 %) were sensitive to vancomycin in the microdilution and 43 (86 %) were sensitive to vancomycin in the E-test®. Seven (14 %) isolates had an intermediate result showing a risk to public health since the detection of these isolates may precede the occurrence of isolates resistant to vancomycin. In addition, the mecA gene was detected in 78 % of the tested samples. Six of the seven isolates with intermediate resistance to vancomycin were carriers of the mecA gene, showing that these isolates had a potential risk of becoming resistant. Thus, control measures must be taken to prevent the spread of these isolates with intermediate resistance and preserve the effectiveness of this antimicrobial for the treatment of infections caused by multiresistant Staphylococcus spp.

RESUMO: Staphylococcus spp. são bactérias envolvidas em infecções de humanos e animais, resistentes a antimicrobianos e tem se tornado uma grande preocupação em saúde pública. Nos últimos anos houve um aumento significativo de Staphylococcus resistentes à meticilina e a vancomicina é a droga de escolha para o tratamento desses isolados, porém vem apresentando elevação nos valores de Concentração Inibitória Mínima (CIM) necessários para combater este microrganismo. O objetivo do presente trabalho foi determinar o perfil de suscetibilidade à vancomicina para isolados de Staphylococcus spp. de animais domésticos e silvestres pelas técnicas de Microdiluição em caldo e E-test®, bem como comparar os resultados de ambos os testes. Dos 50 isolados testados 47 (94%) foram sensíveis à vancomicina na Microdiluição e 43 (86%) foram sensíveis à vancomicina no E-test®. Sete (14%) isolados tiveram resultado intermediário demonstrando um risco à saúde pública visto que a detecção destes isolados pode preceder a ocorrência de isolados resistentes à vancomicina. Ademais o gene mecA foi detectado em 78% das amostras testadas, sendo que dos sete isolados com resistência intermediária à vancomicina, seis eram portadores do gene mecA, evidenciando que esses isolados possuem potencial risco de se tornarem resistentes. Dessa forma medidas de controle devem ser tomadas para evitar a propagação destes isolados com resistência intermediária e preservar a eficácia deste antimicrobiano para o tratamento de infecções causadas por Staphylococcus multirresitentes.

Genetic Diversity of the Cryptococcus gattii Species Complex in Mato Grosso State, Brazil.

Cryptococcosis is caused by fungi of the genus Cryptococcus. Owing to its importance, this study aimed to analyze the genetic diversity of C. gattii isolates from animals, humans, and the environment in Mato Grosso State (MT), Brazil, during November 2010-December 2017. All isolates of the C. gattii species complex were subjected to molecular genotyping via Restriction Fragment Length Polymorphism (PCR-RFLP) and Multi-locus Sequence Typing (MLST). PCR-RFLP analysis revealed that 21 isolates presented the genotype VGII, which is considered the most common and virulent genotype globally among. MLST analysis revealed the presence of 14 sequence types (STs), of which 5 are considered new genotypes. Clonal Complex (CC) CC182 (n = 5; 23,80%) and CC309 (n = 3; 14,28%) were the most frequent. CC distribution in relation to origin revealed that three CCs were found in animals with a predominance of CC182 (66,66%), while nine were found in humans, and two CCs were found in the environment. Extensive genetic variability was observed among the isolates in the State of Mato Grosso. STs belonging to the already described clonal complexes (CC) indicate the global expansion and adaptation of isolates in several other countries. Therefore, detection of clonal complexes and STs already described in other regions and the occurrence of new STs in the present study help further the current understanding of the geographic dispersion and genetic origin of the C. gattii species complex.

Impact of human cytomegalovirus on glioblastoma cell viability and chemotherapy treatment.

The relationship between human cytomegalovirus (HCMV) and tumours has been extensively investigated, mainly in glioblastoma multiforme (GBM), a malignant tumour of the central nervous system with low overall survival rates. Several reports have demonstrated the presence of HCMV in GBM, although typically restricted to a low number of cells, and studies have indicated that viral proteins have the ability to dysregulate cellular processes and increase tumour malignancy. Treatment of GBM involves the use of the chemotherapeutic agents temozolomide (TMZ) and carmustine (bis-chloroethylnitrosourea, BCNU), which lead to the attachment of adducts to the DNA backbone, causing errors during replication and consequent cell death. It is known that HCMV infection can modulate DNA repair pathways, but what effects the virus may exhibit during chemotherapy are unknown. Here we approach this question by analysing HCMV infection and viral protein accumulation in GBM cell lines with different genotypes and their response to TMZ and BCNU in the presence of the virus. We demonstrate that A172, TP365MG and U251MG GBM cells are efficiently infected by both low-passage (TB40E) and high-passage (AD169) HCMV strains. However, the GBM cell lines vary widely in their permissiveness to viral gene expression and exhibit very different patterns of immediate early, early and late protein accumulation. HCMV reduces the viability of permissive GBM cells in a multiplicity-dependent manner in both the absence and presence of TMZ or BNCU. In sum, we demonstrate that GBM cell lines are equally susceptible but differentially permissive to infection by both low- and high-passage strains of HCMV. This observation not only indicates that viral replication is largely controlled by cellular factors in this system, but also provides a possible explanation for why viral gene products are only found in a subset of cells in GBM tumours. Furthermore, we conclude that the virus does not confer increased resistance to chemotherapeutic drugs in various GBM cell lines, but instead reduces tumour cell viability. These results highlight that the oncomodulatory potential of HCMV is not limited to cancer-promoting activities, but also includes adverse effects on tumour cell proliferation or survival.

Qualidade microbiológica do leite cru produzido no Norte de Minas Gerais* / Microbiological quality of raw milk produced in northern of Minas Gerais

Objetivou-se verificar a contaminação do leite cru por micro-organismos mesófilos, psicrotróficos, coliformes a 45°C e Enterococcus spp., produzido por rebanhos mestiços, em 30 unidades agrícolas familiares produtoras de leite em Juramento e Montes Claros, na região Norte de Minas gerais. Houve quatro coletas de amostras durante um ano, sendo duas na época de chuva e duas na época da seca. Coletaram-se amostras de leite cru e cru refrigerado, observando-se a contagem total de micro-organismos mesófilos, psicrotróficos, Enterococcus spp. e a contagem de coliformes a 45°C. Coletaram-se também, amostras de Swabs de equipamentos utilizados em ordenha (baldes e latões) utilizando-se a técnica do esfregaço em superfície, a fim de avaliar a contagem total bacteriana presente nesses utensílios para verificar a sua higienização. As amostras de leite apresentaram contaminação elevada por micro-organismos aeróbios mesófilos, psicrotróficos, Enterococcus spp.,coliformes a 45°C, comprometendo a qualidade higiênico-sanitária do produto, bem como as amostras de Swabs, que apresentaram níveis de contaminação elevados, quanto à presença de aeróbios mesófilos. Existiu deficiência quanto às práticas de antissepsias adotadas nos equipamentos utilizados na ordenha, levando a contaminações acima do permitido por mesófilos aeróbios, tornando-se evidente a necessidade de adoção de boas práticas agropecuárias e maiores investimentos em treinamentos e assistência técnica de qualidade com o intuito de provocar melhorias na qualidade microbiológica do leite produzido nessa região.

This study aimed to investigate the contaminationby mesophilic, psychrotrophic, coliform microorganisms at 45°C and Enterococcus spp., produced by crossbred herds, in raw milk produced in 30 family dairy farms in Juramento and Montes Claros, Northern Minas Gerais. Samples were collected 4 times, 2 during the rainy season and 2 during the dry season. Raw milk samples and milk samples refrigerated were collected, observing the total count of mesophilic, psychrotrophic microorganisms, Enterococcus sppand coliform at 45°C. Was collected too, Swabs samples from milking materials (buckets, cans) were collected using the surface scrubbing technique, in order to assess the total bacterial count of those utensils to verify their hygiene. The milk samples showed high contamination by micro-organisms aerobic mesophilic, psychrotrophic, Enterococcus spp., Coliforms at 45°C, compromising the sanitary quality of the product as well as Swab samples that hight contamination levels, with aerobic mesophilic. What compromised the sanitary quality of the product. There are shortcomings regarding antiseptic practices adopted on the milking equipment, leading to aerobic mesophilic contamination higher than allowed. Becoming evident the need for adoption of good agricultural practices, greater investments in training and quality technical assistance in order to stimulate improvements in the microbiological quality of the milk produced in the region.

Multiple intranigral unilateral LPS infusion protocol generates a persistent cognitive impairment without cumulative dopaminergic impairment.

Inflammation in Parkinson's disease (PD) is a continuous process and might be implicated in the progression of neuronal degeneration. Taking this into account, we proposed a new protocol with multiple and consecutive intranigral lipopolysaccharide (LPS) administration in order to analyze its effects on cognitive behavior. Additionally, striatal concentrations of the neurotransmitters dopamine (DA) and serotonin and their respective metabolites were assessed in three different time-points with the purpose of identifying the consecutive and cumulative effects of LPS infusions. We demonstrated that with a minimum administered dose there was stabilization of neuronal damage as revealed by absence of synergic effect on DA concentration. Although the DA decrease (-43%) generates an animal model of early phase of PD, without apparent motor impairment, the LPS group exhibited deficit in episodic-like memory behavior from the first time-point until the last one, indicating persisted disturbances in memory-recognition responses. These findings provide evidence that multiple intranigral LPS infusions are not sufficient to cause cumulative and progressive damage to dopaminergic neurons, but confirm that the LPS model can be adopted as a useful tool providing insight about the cognitive impairment observed in pre-motor phase of PD.

Eficiência da compostagem sobre a qualidade do biossólido de esgoto doméstico e resíduos agrícolas, utilizado para cultivo do cogumelo do sol (Agaricus blazei Murril) / Efficiency of composting biosolids on the quality of domestic sewage and agricultural waste, used for cultivation of the mushroom of the sun (Agaricus blazei Murrill)

A eficácia da compostagem foi estudada para remoção de contaminantes presentes em substratos alternativos contendo lodo de esgoto a serem utilizados para cultivo do cogumelo do sol (Agaricus blazei Murril). Substratos contendo cinco relações de carbono/nitrogênio: 25, 30, 35,40 e 45 eram compostos por cinco combinações diferentes de resíduos agrícolas. O material foi preparado e submetido ao processo de compostagem. Para avaliar a eficiência da remoção de coliformes fecais e aeróbios mesófilos, realizaram-se análises microbiológicas de amostras de cada ingrediente a ser utilizado para preparo dos compostos, de amostras de cada biossólido referentes aos 5 tratamentos com as cinco diferentes relações C/N, antes e após da pasteurização. Amostras dos frutos cultivados nos diferentes substratos também foram analisadas. Ao serem analisados os resultados, observou- se que houve redução na presençade aeróbios mesófilos como de coliforme, em 80% das amostras analisadas. Essa ocorrência de microorganismos permitiu a classificação como lodo A, sendo de uso irrestrito. Não houve contaminação dos frutos cultivados em nenhum dos substratos avaliados. Concluiu-se que o tratamento adotado promoveu a higienização dos substratos tomando os aptos para aplicação no cultivo do cogumelo do sol e demais hortaliças.

The effectiveness of composting was studied in order to remove the contaminants in alternative substratescontaining sludge, used in the cultivation of Sun Mushroom (Agaricus blazei Murril).Substrates containing five relations of carbon/ nitrogen: 25, 30, 35, 40 and 45 were composed of five different agricultural waste combinations. The material was prepared and submitted to the composting process. With the purpose of evaluating the efficiency of the removal of fecal coliforms and aerobic mesophiles, were conducted microbiological analysis of samples of each ingredient used in the compost preparation and samples consisting of each biosolid relating to the five different relations C/N, before and after the pasteurization. Samples of fruit grown on different substrates were also analyzed. According to the results analyzed, it was observed the decrease of aerobic mesophiles and coliforms, in 80% of the samples. The sludge was classified as "A ", because of the occurrence of microorganisms. There was no contaminationof fruits grown in any of the substrates evaluated. It was concluded that the treatment promoted the substrates hygiene, making them suitable for application in the cultivation of Sun Mushroom and other vegetables.