Desenvolvimento de novas abordagens terapêuticas para o tratamento da mucopolissacaridose tipo I

A Mucopolissacaridose I (MPS I) é uma doença autossômica recessiva causada por mutações no gene da alpha-L-iduronidase (IDUA), o que leva a uma atividade enzimática baixa ou nula. O principal tratamento é a terapia de reposição enzimática (TRE), que tem limitações como o alto custo, a necessidade de infusões repetidas e a ineficácia do tratamento dos sintomas neurológicos dos pacientes com a forma grave da doença. Novas estratégias terapêuticas são necessárias para superar esses obstáculos. A tradução alternativa tem potencial para aumentar a atividade enzimática, uma vez que há supressão de mutações sem sentido, permitindo que a proteína seja totalmente traduzida. Outra possibilidade é a aplicação de estratégias de nanoestruturação, as quais podem aumentar a eficiência enzimática e a biodistribuição, podendo representar uma redução da dose. Neste trabalho, abordamos ambas as estratégias em dois estudos. No primeiro, fibroblastos de três pacientes com MPS I foram tratados com geneticina, um conhecido indutor de misreading, e cloranfenicol, um inibidor da peptidil transferase que também pode atuar da mesma forma. A geneticina não foi capaz de aumentar a atividade ou expressão de IDUA, enquanto o cloranfenicol aumentou 100 vezes apenas nos fibroblastos heterozigotos compostos. O sequenciamento de cDNA sugere que os alelos com mutação sem sentido são alvo do nonsense mediated mRNA decay, o que indica que o cloranfenicol atua através de um mecanismo que não a tradução alternativa. Este fármaco é capaz de ultrapassar a barreira hematoencefálica, podendo ser utilizado como adjuvante à TRE. No segundo estudo, utilizamos uma nanocápsula de núcleo lipídico e paredes múltiplas revestidas com quitosana e íons de Fe2+que permitem a ligação química com a Laronidase. A enzima nanoestruturada apresentou maior Vmax e Kcat e foi capaz de produzir maior quantidade de produto. Ela não apresentou citotoxicidade na dose utilizada in vivo e foi internalizada por transporte ativo não apenas mediado por receptores de manose-6-fosfato. Além disso, o perfil de clearance foi melhor, apresentando maior área sob a curva e tempo de meia vida. A análise da biodistribuição mostrou maior atividade da enzima nanoestruturada em diferentes órgãos em 4 h e 24 h. Neste trabalho, ambos os estudos procuraram solucionar diferentes limitações da TRE. É importante ressaltar que estes dois estudos foram baseados em fármacos aprovados para uso, o que viabiliza sua aplicabilidade terapêutica.Mucopolysaccharidosis I (MPS I) is an autosomal recessive disorder caused by mutations on alpha-L-iduronidase (IDUA) gene, leading to low or null enzyme activity. The treatment of choice is enzyme replacement therapy (ERT), which has limitations such as the high cost, the need of repeated infusions and the inefficacy to treat the patient’s neurological symptoms. New treatment strategies are necessary to overcome these hurdles. Stop codon read through (SCRT) is a potential alternative to achieve enhanced enzyme activity as there is suppression of premature stop codon mutations by the addition of a random amino acid in that position, allowing the protein to be fully translated. Other possibility is the application of nanostructuration strategies that may enhance enzyme efficiency and biodistribution, thus leading to reduced dose. In this work, we accessed both strategies in two studies. In the first one fibroblasts from three MPS I patients were treated with geneticin, a known misreading inducer, and chloramphenicol, a peptidyl transferase inhibitor that has been suggested to induce misreading. Geneticin was not able to enhance IDUA activity or expression and chloramphenicol enhanced IDUA activity 100-fold only on compound heterozygous fibroblasts. cDNA sequencing suggested that the nonsense alleles were being target to nonsense mediated mRNA decay and that chloramphenicol acts through a mechanism other than SCRT. As chloramphenicol is able to cross the blood brain barrier, it could be used as an adjuvant to ERT. In the second study we used a multiple wall lipid core nanocapsule coated with chitosan and Fe2+ ions that allows the chemical linkage of Laronidase. The nanostructured enzyme has an enhanced Vmax and Kcat and is able to produce higher amounts of product. The modified enzyme is not cytotoxic at the dose used in vivo and is uptaken by active transport not only mediated by mannose-6- phosphate receptors. Moreover, the clearance profile is better, showing a higher area under the curve and half-life time. The biodistribution analysis showed enhanced enzyme activity of nanostructured enzyme in different organs in 4 h and 24 h. In this work, both studies access different limitations of ERT with some benefits. Importantly, they are based on drugs approved for use in patient care; therefore, its therapeutic applicability becomes more feasible

Long-term restoration of alpha-L-iduronidase activity in fibroblasts from patients with mucopolysaccharidosis type I after non-viral gene transfer

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disorder due to deficiency of alpha-L-iduronidase (IDUA). Limitations such as need of weekly injection, high morbidity and mortality  and high cost of the current treatments show the need for new approaches to treat this disease. In this work we aimed to correct fibroblasts from a MPS I patient using non-viral gene therapy. Using a plasmid encoding the human IDUA cDNA, we achieved stable high IDUA levels in transfected fibroblasts up to 6 months of treatment. These results serve as proof-of concept that a non-viral approach can correct the enzyme deficiency in cells from lysosomal storage disorders patients, which can be used as a tool for research a series of disease aspects. Future studies will focus on verify if this approach can be useful in small animals and clinical trials

Genomic analysis of Enterococcus durans LAB18S, a potential probiotic strain isolated from cheese

Gut microbiota exerts a fundamental role in human health and increased evidence supports the beneficial role of probiotic microorganisms in the maintenance of intestinal health. Enterococcus durans LAB18S was previously isolated from soft cheese and showed some desirable in vitro probiotic properties, for that reason its genome was sequenced and evaluated for genes that can be relevant for probiotic activity and are involved in selenium metabolism. Genome sequencing was performed using the Illumina MiSeq System. A variety of genes potentially associated with probiotic properties, including adhesion capability, viability at low pH, bile salt resistance, antimicrobial activity, and utilization of prebiotic fructooligosaccharides (FOS) were identified. The strain showed tolerance to acid pH and bile salts, exhibited antimicrobial activity and thrived on prebiotic oligosaccharides. Six genes involved in selenium metabolism were predicted. Analysis of the SECIS element showed twelve known selenoprotein candidates. E. durans LAB18S was the only food isolate showing absence of plasmids, virulence and antimicrobial resistance genes, when compared with other 30 E. durans genomes. The results of this study provide evidence supporting the potential of E. durans LAB18S as alternative for probiotic formulations

Endocardite vegetativa associada à Helcococcus ovis em uma vaca no Sul do Brasil

A 4-year-old cow with a history of breathing difficulty, progressive weight loss, and muffled heart sound was treated for a period of two weeks, but died and underwent necropsy examination. Macroscopic examination revealed heart with vegetative proliferative lesion firmly adhered to pulmonary valve, lungs with marked consolidation, and the presence of thrombus in vessels. There were grade II ulcers in abomasal mucosa associated to blood clots. Microscopic examination revealed marked thickening of the pulmonary valve due to the proliferation of fibrous connective tissue, inflammatory neutrophil infiltration, fibrin deposition, and a significant number of coccoid basophilic bacteria. Septic thromboemboli were present in the large and small pulmonary blood vessels suggestive of embolic pneumonia. The bacterial culture of the valve showed growth of small, nonhemolytic colonies that demonstrated satellitism to coagulase-negative staphylococci contaminating colonies, which were subjected to 16S gene sequencing and were compatible with Helcococcus ovis in GenBank. This was the first report of H. ovis endocarditis in cattle in South America.Um bovino, fêmea, 4 anos com histórico de dificuldade respiratória, perda de peso progressiva e som cardíaco abafado, foi tratado por um período de duas semanas, porém veio a óbito e foi submetido a exame de necropsia. No exame macroscópico, notou-se coração com lesão proliferativa vegetativa firmemente aderida em valva pulmonar, pulmões com consolidação acentuada, e presença de trombos no interior de vasos. Havia ainda úlcera abomasal grau II em mucosa associada a coágulo sanguíneo. No exame microscópico notou-se acentuado espessamento da valva pulmonar por proliferação de tecido conjuntivo fibroso, infiltrado inflamatório de neutrófilos, deposição de fibrina e acentuado número de miríades bacterianas basofílicas cocoides. Tromboembolia séptica foi vista no interior de vasos pulmonares de pequeno e médio calibre, sugestivo de pneumonia embólica. Em cultivo bacteriano da valva notou-se crescimento de colônias pequenas, não hemolíticas que demonstravam satelitismo a colônias contaminantes de estafilococos coagulase negativa, essas foram submetidas ao sequenciamento do gene 16S e foram compatíveis com Helcococcus ovis no GenBank. Este foi o primeiro relato de endocardite por H. ovis em bovino na América do Sul

Surto de tuberculose em suínos de criação intensiva no Sul do Brasil

Mycobacterium tuberculosis var. bovis is the etiologic agent of animal tuberculosis (aTB), a neglected zoonotic disease. Animal tuberculosis can affect many species, including swine. aTB-consistent granulomas in these animals lead to carcass disposal, generating economic losses and posing risks to human health. In the present study, an aTB outbreak was identified at an intensive swine farming operation in Southern Brazil. Inspection during swine slaughter revealed aTB-suspected lesions, which were collected for diagnosis by histology, PCR, and bacterial isolation. The animals had no clinical signs of tuberculosis. Granulomatous lesions were identified in 0.73% (59/8,071) of the slaughtered swine, and were confirmed by histology. Nine samples were further examined by PCR and bacterial isolation, with 44.4% and 55.5% positive results, respectively. Data from abattoirs subjected to federal surveillance show an aTB prevalence in Brazil of <0.001%. The present data thus indicate a swine aTB outbreak in intensive breeding. Swine infection can be related to exposure to infected animals or to contaminated food or environment. Biosecurity measures must be taken to avoid aTB transmission. Although certified swine breeding farms adopt such measures, this report indicates that constant monitoring is crucial, and greater control in swine breeding and finishing units is required to prevent outbreaks and spread of tuberculosis.Mycobacterium bovis é o agente etiológico da tuberculose animal (aTB), uma doença zoonótica negligenciada. A tuberculose animal pode afetar muitas espécies, incluindo suínos. Os granulomas compatíveis com a aTB nesses animais levam ao descarte de carcaças, gerando perdas econômicas e trazendo riscos à saúde humana. No presente estudo, um surto de aTB foi identificado em um sistema de criação intensiva de suínos na região Sul do Brasil. A inspeção durante um abate de suínos revelou lesões suspeitas de tuberculose, as quais foram coletadas para diagnóstico por histologia, PCR e isolamento bacteriano. Os animais não apresentavam sinais clínicos de tuberculose. Lesões granulomatosas foram identificadas em 0,73% (59/8.071) dos suínos abatidos, e foram confirmadas pela histologia. Nove amostras foram posteriormente examinadas por PCR e isolamento bacteriano, com 44,4% e 55,5% de resultados positivos, respectivamente. Dados de frigoríficos submetidos à vigilância federal demostram prevalência da aTB de < 0,001% no Brasil. Os dados presentes indicam, portanto, um surto de aTB em suínos de criação intensiva. A infecção em suínos pode estar relacionada à exposição a animais infectados, alimentos ou ambientes contaminados. Medidas de biossegurança devem ser tomadas para evitar a transmissão da aTB. Embora as granjas suinícolas certificadas adotem tais medidas, este relato indica que o monitoramento constante é crucial, e maior controle nas unidades de criação e terminação de suínos é necessário para prevenir surtos e disseminação da tuberculose

Comparative analysis of the upper respiratory bacterial communities of pigs with or without respiratory clinical signs : from weaning to finishing phase

A prospective study was conducted to identify bacterial communities in the nasal and laryngeal cavities of pigs with or without clinical signs of respiratory disease in a longitudinal fashion, from weaning to the finishing phase. Nasal and laryngeal swabs were collected from asymptomatic pigs (n = 30), as well as from pigs with clinical signs of respiratory disease (n = 30) at the end of the weaning (T1—33 days) phase, end of the nursery phase (T2—71 days), and finishing (T3—173 days). Total DNA was extracted from each sample, and the V4 hypervariable region of the 16S rRNA gene was amplified and sequenced with the Illumina MiSeq platform. Principal coordinates analysis indicated no significant differences between the nasal and laryngeal bacterial communities. Nevertheless, the microbiota composition in the upper respiratory tract (URT) was clearly distinct between animals, with or without signs of respiratory disease, particularly at post-weaning and the end of nursery. In pigs with clinical signs of respiratory disease, Actinobacillus, Streptococcus Porphyromonas, Veillonella, and an unclassified genus of Pasteurellaceae were more abundant than in pigs with no signs. Metabolic prediction identified 28 differentially abundant pathways, mainly related to carbohydrate, energy, amino acid, anaerobic, and nucleotide metabolism in symptomatic pigs (especially in T2). These findings provide evidence that the composition of the URT bacterial microbiota differs significantly when comparing pigs with or without respiratory clinical signs after weaning, and this difference is maintained in the nursery phase; such differences, however, were not evident at the finishing phase

Evaluation of enterotoxins and antimicrobial resistance in microorganisms isolated from raw sheep milk and cheese : ensuring the microbiological safety of these products in southern Brazil

This study emphasizes the importance of monitoring the microbiological quality of animal products, such as raw sheep’s milk and cheese, to ensure food safety. In Brazil, there is currently no legislation governing the quality of sheep’s milk and its derivatives. Therefore, this study aimed to evaluate: (i) the hygienic-sanitary quality of raw sheep’s milk and cheese produced in southern Brazil; (ii) the presence of enterotoxins and Staphylococcus spp. in these products; and (iii) the susceptibility of the isolated Staphylococcus spp. to antimicrobial drugs and the presence of resistance genes. A total of 35 samples of sheep’s milk and cheese were examined. The microbiological quality and presence of enterotoxins were accessed using Petrifilm and VIDAS SET2 methods, respectively. Antimicrobial susceptibility tests were conducted using VITEK 2 equipment and the disc diffusion method. The presence of resistance genes tet(L), sul1, sul2, ermB, tetM, AAC(6)’, tetW, and strA were evaluated through PCR. In total, 39 Staphylococcus spp. were obtained. The resistance genes tetM, ermB, strA, tetL, sul1, AAC(6)’, and sul2 were detected in 82%, 59%, 36%, 28%, 23%, 3%, and 3% of isolates, respectively. The findings revealed that both raw sheep’s milk and cheese contained Staphylococcus spp. that exhibited resistance to antimicrobial drugs and harbored resistance genes. These results underscore the immediate need for specific legislation in Brazil to regulate the production and sale of these product

Aborto bovino por uma cepa vacinal de Bacillus anthracis

This paper reports the abortion of a male Aberdeen Angus bovine by a vaccine strain of Bacillus anthracis, describing the pathological and microbiological findings and the genome sequence. Necropsy findings included multifocal areas of hemorrhage in different organs. Histologically, various organs showed hemorrhage, fibrin exudation, necrosis associated with countless bacillary bacterial clumps and severe neutrophilic inflammatory infiltrate. In the microbiological examination, numerous rough, nonhemolytic, gray and dry colonies with irregular edges were isolated from liver, lung and abomasum content samples. Gram staining revealed square-ended Gram-positive rods arranged in chains. B. anthracis identification was confirmed by detection of the molecular chromosomal marker Ba813. The genomes from the isolated B. anthracis (named SPV842_15) and from the isolated vaccinal strain (Brazilian vaccinal strain), which was recovered from a commercial vaccine used in the pregnant cow, were sequenced. Genomic comparisons displayed a high level of nucleotide identity in the comparisons between B. anthracis SPV842_15 and the B. anthracis Brazilian vaccinal strain (98,2%). Furthermore, in both strains, only the plasmid pX01 sequence was detected. Although, vaccination against anthrax is characterized by an elevated protective profile and very low residual virulence, immunization with Sterne strains can cause abortion in cattle, presumably by the plasmid pX01 toxins in rare or special situations.Este trabalho relata um aborto de um bovino, macho, Aberdeen Angus, por uma cepa vacinal de Bacillus anthracis, descreve os achados patológicos, microbiológicos e o sequenciamento do genoma. Os achados de necropsia incluíram áreas multifocais de hemorragias em diferentes órgãos. Histologicamente, órgãos afetados apresentaram hemorragia, exsudação de fibrina, necrose associada a miríades bacterianas bacilares e intenso infiltrado inflamatório neutrofílico. No exame microbiológico, foram isoladas numerosas colônias rugosas, não hemolíticas, cinzas e secas, com bordas irregulares a partir de amostras de fígado, pulmão e conteúdo do abomaso. A coloração de Gram revelou bastonetes Gram-positivos dispostos em cadeias. A identificação do B. anthracis foi confirmada pela detecção do marcador cromossômico molecular Ba813. Os genomas do isolado B. anthracis (SPV842_15) e do isolado vacinal (cepa vacinal brasileira), recuperado de uma vacina comercial utilizada na vaca prenhe, foram sequenciados. Comparações genômicas mostraram um elevado nível de identidade de nucleotídeos entre B. anthracis SPV842_15 e cepa vacinal brasileira (98,2%). Além disso, em ambas as estirpes foi detectada apenas a sequência do plasmídeo pX01. Embora a vacinação contra o antraz seja caracterizada por um perfil protetor elevado e uma virulência residual muito baixa, a imunização com estirpes de Sterne pode causar aborto em bovinos, presumivelmente pelas toxinas do plasmídeo pX01 em situações raras ou específicas

The genetic diversity of “papillomavirome” in bovine teat papilloma lesions

Background: Papillomaviruses are small nonenveloped, circular double-stranded DNA viruses that belong to the Papillomaviridae family. To date, 29 Bos taurus papillomavirus (BPV) types have been described. Studies involving mixed BPV infections have rarely been reported in contrast to human papillomavirus (HPV), which is commonly described in numerous studies showing coinfections. Moreover, previous studies had shown that HPV coinfections increase the risk of carcinogenesis. In the present study, we used rolling-circle amplification followed by a high-throughput sequencing (RCA-HTS) approach in 23 teat papillomas from southern Brazil. Results: Eleven well-characterized BPV types and 14 putative new BPV types were genetically characterized into the Xi, Epsilon and Dyoxipapillomavirus genera according to phylogenetic analysis of the L1 gene, which expands the previous 29 BPV types to 43. Moreover, BPV coinfections were detected in the majority (56.3%) of the papilloma lesions analyzed, suggesting a genetic diverse “papillomavirome” in bovine teat warts. Conclusions: The data generated in this study support the possibility that a wide range of BPV is probably underdetected by conventional molecular detection tools, and that BPV coinfections are underestimated and probably genetic diverse. Additionally, 14 new BPV types were characterized, increasing the knowledge regarding BPV genetic diversity