Phagocytotic activity and gene expression of leukocytes isolated from Astyanax lacustris.

The constant intensification of aquaculture has considerable increased the stress levels of farmed fish and, consequently, the number and intensity of diseases outbreaks. Thus, studies on fish immune response, especially regarding the interaction of fish leukocytes with potential pathogens and xenobiotics are of great importance in order to develop new prophylactic and curative strategies. We isolated leukocytes from the head kidney of Astyanax lacustris-an important Neotropical fish species for aquaculture and a potential model for Neotropical aquaculture research-using a Percoll centrifugation protocol. The isolated leukocytes were incubated with lipopolysaccharide (LPS), and the expression of genes IL-1ß, IL-8, LysC, and LysG were measured. We assessed the phagocytotic activity of leukocytes using Congo red-dyed yeast, a novel and cost-effective protocol that has been developed in this study. The isolated leukocytes responded to LPS induction, exhibiting strong IL-1ß and IL-8 upregulation, two of the most important pro-inflammatory interleukins for vertebrates immune reponse. The optimal concentration of yeast for the phagocytic assay was 106 cells mL-1, resulting in acceptable phagocytic capacity (PC) but without excess of yeasts during the counting process, ensuring a high precision and accuracy of the method. To the best of our knowledge, the present study is the first to investigate the in vitro gene expression and phagocytic activity of leukocytes isolated from A. lacustris. Our findings will serve as a reference for future studies on the immunology and toxicology of Neotropical fish.

Phagocytotic activity and gene expression of leukocytes isolated from Astyanax lacustris / Atividade fagocítica e expressão gênica de leucócitos isolados de Astyanax lacustris

The constant intensification of aquaculture has considerable increased the stress levels of farmed fish and, consequently, the number and intensity of diseases outbreaks. Thus, studies on fish immune response, especially regarding the interaction of fish leukocytes with potential pathogens and xenobiotics are of great importance in order to develop new prophylactic and curative strategies. We isolated leukocytes from the head kidney of Astyanax lacustris­an important Neotropical fish species for aquaculture and a potential model for Neotropical aquaculture research­using a Percoll centrifugation protocol. The isolated leukocytes were incubated with lipopolysaccharide (LPS), and the expression of genes IL-1ß, IL-8, LysC, and LysG were measured. We assessed the phagocytotic activity of leukocytes using Congo red-dyed yeast, a novel and cost-effective protocol that has been developed in this study. The isolated leukocytes responded to LPS induction, exhibiting strong IL-1ß and IL-8 upregulation, two of the most important pro-inflammatory interleukins for vertebrates immune reponse. The optimal concentration of yeast for the phagocytic assay was 106 cells mL-1, resulting in acceptable phagocytic capacity (PC) but without excess of yeasts during the counting process, ensuring a high precision and accuracy of the method. To the best of our knowledge, the present study is the first to investigate the in vitro gene expression and phagocytic activity of leukocytes isolated from A. lacustris. Our findings will serve as a reference for future studies on the immunology and toxicology of Neotropical fish.

A constante intensificação da aquicultura tem aumentado consideravelmente os níveis de estresse dos animais cultivados e, consequentemente, o número e a intensidade dos surtos de doenças. Logo, estudos sobre a resposta imune dos peixes, especialmente relacionados com a interação dos leucócitos de peixes com potenciais patógenos e xenobióticos, são de grande importância para o desenvolvimento de novas estratégias profiláticas e curativas. No presente trabalho, nós obtivemos sucesso ao isolar leucócitos oriundos do rim cranial de Astyanax lacustris ­ uma importante espécie de peixe Neotropical para a aquicultura e um modelo em potencial para pesquisas em aquicultura Neotropical ­ usando um protocolo de centrifugação com Percoll. Os leucócitos isolados foram incubados com lipossacarídeo (LPS) e, a expressão dos genes IL-1ß, IL-8, LysC, e LysG foi avaliada. Ainda, um novo protocolo para avaliação da atividade fagocítica dos leucócitos utilizando leveduras coradas com Vermelho Congo foi estabelecido. Os leucócitos isolados responderam à indução com LPS, exibindo up regulation dos genes IL-1ß e IL-8, duas das interleucinas pró-inflamatórias mais importantes para a resposta imune de vertebrados. Além do mais, a concentração ótima de leveduras para a avaliação da fagocitose foi de 106 células mL-1, resultando em uma capacidade fagocítica (PC) aceitável, mas sem excesso de leveduras durante o processo de contagem, garantindo maior precisão e eficácia do método. Até o presente momento, o presente estudo é o primeiro a investigar a expressão gênica e atividade fagocítica de leucócitos isolados de A. lacustris através da abordagem in vitro. Ainda, nossos resultados servirão de referência para futuros estudos em imunologia e toxicologia de peixes Neotropicais.

Lymphocyte subsets and Langerhans cells in the skin of kidney transplant recipients under three different immunosuppressive regimens.

BACKGROUND: Renal transplant recipients (RTRs) are at increased risk of developing skin cancer; however, the role of immunosuppression is not yet fully understood. In this study, we evaluated the immunohistochemical changes in the skin of RTRs under three different immunosuppression regimens: mTOR inhibitors (mTORi), sirolimus or everolimus, mycophenolic acid (MPA) precursors such as mycophenolate sodium or mofetil, or azathioprine (AZA). METHODS: We evaluated biopsies of sun-exposed and sun-protected skin for immunohistochemical quantification of B lymphocytes (CD20+ ), T lymphocytes (CD3+ , CD4+ , and CD8+ ), and Langerhans cells (LCs) (CD1a+ ) in 30 RTRs and 10 healthy controls. The RTRs were divided into three groups: mTORi (n = 10), MPA (n = 10), and AZA (n = 10). RESULTS: No differences were observed in the number of B lymphocytes. However, a significant decrease in the number of T lymphocytes and LCs was observed in both sun-protected and sun-exposed skin in the AZA and MPA groups, although to a lesser degree in the latter group. The skin of the mTORi group did not differ from that of the control group in terms of the number of B and T lymphocytes and LCs. CONCLUSIONS: Patients treated with mTORi exhibit preserved cellular elements related to cutaneous immune surveillance. The use of AZA induced a greater degree of skin immunosuppression than in the control group, as demonstrated by the decrease in T lymphocytes and LCs.

Increased expression of Filaggrin and Claudin-1 in the ocular surface of patients with atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is an itchy, chronic and inflammatory skin condition, with dysfunctional immune response and skin barrier defects. Reduction of filaggrin (FLG) and tight junctions (TJ) proteins, such as claudin-1 (CLDN-1), expression in cutaneous epithelial barrier is remarkable in AD pathogenesis. Ocular involvement occurs in approximately 40% of AD patients leading to changes in the structure of the conjunctiva. OBJECTIVES: We aimed to evaluate the expression of FLG and CLDN-1 in the ocular surface of adults with AD, analysing bulbar conjunctival cells collected by a novel non-invasive cellular imprint. METHODS: Bulbar conjunctival epithelial cells were collected by cellular imprint technique, and FLG and CLDN-1 expression were assessed by immunofluorescence (IF) and real-time polymerase chain reaction (RT-PCR). RESULTS: We detected increased expression of FLG and CLDN-1, as well as their transcript levels in AD patients compared with healthy controls (HC). There was a positive correlation between tear film break-up time (TBUT) and FLG expression. Fluorescein staining was inversely associated with FLG expression. CONCLUSIONS: Our results may reflect a reactive response of the ocular surface to AD-related ocular inflammation and associated dry eye disease. Further investigations focusing on the role of FLG and TJ expression in the ocular surface of AD patients may increment the understanding of the pathophysiology of extracutaneous AD and developing future targeted therapies.

Domestication strategies for the endangered catfish species Pseudopimelodus mangurus Valenciennes, 1835 (Siluriformes: Pseudopimelodidae) / Estratégias de domesticação das espécies ameaçadas de peixe-gato Pseudopimelodus mangurus Valenciennes, 1835 (Siluriformes: Pseudopimelodidae)

Wild fish domestication can be considered a strategic approach to endangered species conservation, supporting studies and reducing economic and environmental costs. Three of the most important strategies in the domestication processes of fish are the adaptation of wild fish to captivity, the reproduction of the adapted fish and the production and maintenance of the young individuals. That being said, the present study is divided in three experiments: the 1st aimed to adapt wild Pseudopimelodus mangurus to captivity environment using different feeding approaches and a prophylactic strategie; the 2nd aimed to reproduce the adapted individuals from the 1st experiment; and the 3rd aimed to train the P. mangurus juveniles to accept commercial diets. The 1st and 2nd experiments were successful at the maintenance and artificial reproduction of P. mangurus kept in tanks between the reproductive seasons. The results suggest that the reproductive performance of animals kept in captivity (initial relative fertility-IRF = 609.25 ± 36.6 eggs/g) was similar (p > 0,05) to the performance found in wild individuals (IRF = 679.21 ± 45.66 eggs/g). Feed training of P. mangurus juveniles (3rd experiment) was also conducted, evaluating three feeding treatments with different concentrations of bovine heart and ration. At the end of the experiment, the treatment containing half bovine heart and half commercial feeding resulted in the highest values of weight gain (0.10 ± 0.16 g), specific growth rate (0.37 ± 0.11 mm), length (47.78 ± 2.35 mm) and growth (2.15 ± 2.27 mm), suggesting reasonable acceptability to artificial diets in the cultivation of this species. As conclusion, the present study contributes with the development of techniques for the domestication of fresh water fish species with commercial value or andangered of extinction, showing the domestication and reproduction of wild P. mangurus in captivity. However, more studies have to be conducted in order to improve the acceptance of artificial feeding by juveniles and to increase their survival rate.(AU)

A domesticação de peixes selvagens pode ser considerada uma abordagem estratégica para a conservação de espécies ameaçadas, apoiando estudos e reduzindo custos econômicos e ambientais. Três das estratégias mais importantes para o processo de domesticação de peixes são a adaptação dos peixes ao cativeiro, a reprodução dos peixes adaptados e a produção e manutenção dos indivíduos jovens. O presente estudo está dividido em três partes: a 1ª objetivou adaptar Pseudopimelodus mangurus selvagens ao ambiente de cativeiro usando diferentes abordagens alimentares e uma estratégia profilática; o 2º objetivou reproduzir os indivíduos adaptados do 1º experimento; e o 3º teve como objetivo treinar os juvenis de P. mangurus para aceitar dietas comerciais. O 1o e 2o experimento obteveram sucesso na manutenção e reprodução artificial de P. mangurus mantidos em tanques entre as estações reprodutivas. Os resultados sugerem que o desempenho reprodutivo dos animais mantidos em cativeiro (fertilidade inicial relativa-FIR = 609,25 ± 36,6 ovos / g) foi similar (p> 0,05) ao dos indivíduos selvagens (FIR = 679,21 ± 45,66 ovos / g). O treinamento alimentar de P. mangurus juvenis (3º experimento) também foi realizado avaliando-se 3 tratamentos alimentares com diferentes concentrações de coração bovino e ração. Ao final do experimento, o tratamento contendo metade coração bovino e metade ração gerou os maiores valores de ganho de peso (0,10 ± 0,16 g), taxa de crescimento específico (0,37 ± 0,11 mm), comprimento (47,78 ± 2,35 mm) e crescimento (2,15 ± 2,27 mm), sugerindo razoável aceitabilidade para dietas artificiais no cultivo desta espécie. Como conclusão, o presente estudo contribui com o desenvolvimento de técnicas para a domesticação espécies de peixes de água doce, de interesse comercial ou ameaçados de extinção, mostrando a domesticação e reproduçao de em cativeiro P. Mangurus selvagens. No entanto, mais estudos devem ser conduzidos no intuito de aumentar a aceitação de dietas comerciais pelos juvenis e melhorar sua taxa sobrevivência.(AU)

Differential Age-Dependent Mitochondrial Dysfunction, Oxidative Stress, and Apoptosis Induced by Neonatal Hypoxia-Ischemia in the Immature Rat Brain.

Neonatal hypoxia-ischemia (HI) is among the main causes of mortality and morbidity in newborns. Experimental studies show that the immature rat brain is less susceptible to HI injury, suggesting that changes that occur during the first days of life drastically alter its susceptibility. Among the main developmental changes observed is the mitochondrial function, namely, the tricarboxylic acid (TCA) cycle and respiratory complex (RC) activities. Therefore, in the present study, we investigated the influence of neonatal HI on mitochondrial functions, redox homeostasis, and cell damage at different postnatal ages in the hippocampus of neonate rats. For this purpose, animals were divided into four groups: sham postnatal day 3 (ShP3), HIP3, ShP11, and HIP11. We initially observed increased apoptosis in the HIP11 group only, indicating a higher susceptibility of these animals to brain injury. Mitochondrial damage, as determined by flow cytometry showing mitochondrial swelling and loss of mitochondrial membrane potential, was also demonstrated only in the HIP11 group. This was consistent with the decreased mitochondrial oxygen consumption, reduced TCA cycle enzymes, and RC activities and induction of oxidative stress in this group of animals. Considering that HIP3 and the sham animals showed no alteration of mitochondrial functions, redox homeostasis, and showed no apoptosis, our data suggest an age-dependent vulnerability of the hippocampus to hypoxia-ischemia. The present results highlight age-dependent metabolic differences in the brain of neonate rats submitted to HI indicating that different treatments might be needed for HI newborns with different gestational ages.

Morphophysiological analysis of the salivary glands of Rhipicephalus sanguineus sensu lato (Acari: Ixodidae) exposed to ozonated water: A control strategy.

The tick Rhipicephalus sanguineus sensu lato has great medical and veterinary importance, mainly because the ability to transmit many diseases, causing harm to pets but also risks to public health. The blood spoliation and transmission of pathogens occur because of the immunosuppressive action of these ticks' saliva, a potent mixture of bioactive substances that is secreted by the salivary glands, one of the organs responsible for their biological success, and hence the target of studies for their control. Ozone has promise for use as an alternative acaricide, due to its proven efficiency in controlling agricultural and food pests, besides posing no risk of environmental contamination or to animal and human health. Therefore, this study evaluated the acaricidal potential of exposure of females of R. sanguineus s.l. to ozonated water at many concentrations and analysed the morphophysiological alterations of the salivary glands, employing histological and light microscopic techniques. The results demonstrated that the ozonated water at the concentrations investigated caused severe alterations in the salivary glands, bringing a new perspective for control of R. sanguineus s.l., through an ecologically correct method due to the absence of harm to non-target organisms and the environment.

Domestication strategies for the endangered catfish species Pseudopimelodus mangurus Valenciennes, 1835 (Siluriformes: Pseudopimelodidae).

Wild fish domestication can be considered a strategic approach to endangered species conservation, supporting studies and reducing economic and environmental costs. Three of the most important strategies in the domestication processes of fish are the adaptation of wild fish to captivity, the reproduction of the adapted fish and the production and maintenance of the young individuals. That being said, the present study is divided in three experiments: the 1st aimed to adapt wild Pseudopimelodus mangurus to captivity environment using different feeding approaches and a prophylactic strategie; the 2nd aimed to reproduce the adapted individuals from the 1st experiment; and the 3rd aimed to train the P. mangurus juveniles to accept commercial diets. The 1st and 2nd experiments were successful at the maintenance and artificial reproduction of P. mangurus kept in tanks between the reproductive seasons. The results suggest that the reproductive performance of animals kept in captivity (initial relative fertility-IRF = 609.25 ± 36.6 eggs/g) was similar (p > 0,05) to the performance found in wild individuals (IRF = 679.21 ± 45.66 eggs/g). Feed training of P. mangurus juveniles (3rd experiment) was also conducted, evaluating three feeding treatments with different concentrations of bovine heart and ration. At the end of the experiment, the treatment containing half bovine heart and half commercial feeding resulted in the highest values of weight gain (0.10 ± 0.16 g), specific growth rate (0.37 ± 0.11 mm), length (47.78 ± 2.35 mm) and growth (2.15 ± 2.27 mm), suggesting reasonable acceptability to artificial diets in the cultivation of this species. As conclusion, the present study contributes with the development of techniques for the domestication of fresh water fish species with commercial value or andangered of extinction, showing the domestication and reproduction of wild P. mangurus in captivity. However, more studies have to be conducted in order to improve the acceptance of artificial feeding by juveniles and to increase their survival rate.

Domestication strategies for the endangered catfish species Pseudopimelodus mangurus Valenciennes, 1835 (Siluriformes: Pseudopimelodidae) / Estratégias de domesticação das espécies ameaçadas de peixe-gato Pseudopimelodus mangurus Valenciennes, 1835 (Siluriformes: Pseudopimelodidae)

Wild fish domestication can be considered a strategic approach to endangered species conservation, supporting studies and reducing economic and environmental costs. Three of the most important strategies in the domestication processes of fish are the adaptation of wild fish to captivity, the reproduction of the adapted fish and the production and maintenance of the young individuals. That being said, the present study is divided in three experiments: the 1st aimed to adapt wild Pseudopimelodus mangurus to captivity environment using different feeding approaches and a prophylactic strategie; the 2nd aimed to reproduce the adapted individuals from the 1st experiment; and the 3rd aimed to train the P. mangurus juveniles to accept commercial diets. The 1st and 2nd experiments were successful at the maintenance and artificial reproduction of P. mangurus kept in tanks between the reproductive seasons. The results suggest that the reproductive performance of animals kept in captivity (initial relative fertility-IRF = 609.25 ± 36.6 eggs/g) was similar (p > 0,05) to the performance found in wild individuals (IRF = 679.21 ± 45.66 eggs/g). Feed training of P. mangurus juveniles (3rd experiment) was also conducted, evaluating three feeding treatments with different concentrations of bovine heart and ration. At the end of the experiment, the treatment containing half bovine heart and half commercial feeding resulted in the highest values of weight gain (0.10 ± 0.16 g), specific growth rate (0.37 ± 0.11 mm), length (47.78 ± 2.35 mm) and growth (2.15 ± 2.27 mm), suggesting reasonable acceptability to artificial diets in the cultivation of this species. As conclusion, the present study contributes with the development of techniques for the domestication of fresh water fish species with commercial value or andangered of extinction, showing the domestication and reproduction of wild P. mangurus in captivity. However, more studies have to be conducted in order to improve the acceptance of artificial feeding by juveniles and to increase their survival rate.

A domesticação de peixes selvagens pode ser considerada uma abordagem estratégica para a conservação de espécies ameaçadas, apoiando estudos e reduzindo custos econômicos e ambientais. Três das estratégias mais importantes para o processo de domesticação de peixes são a adaptação dos peixes ao cativeiro, a reprodução dos peixes adaptados e a produção e manutenção dos indivíduos jovens. O presente estudo está dividido em três partes: a 1ª objetivou adaptar Pseudopimelodus mangurus selvagens ao ambiente de cativeiro usando diferentes abordagens alimentares e uma estratégia profilática; o 2º objetivou reproduzir os indivíduos adaptados do 1º experimento; e o 3º teve como objetivo treinar os juvenis de P. mangurus para aceitar dietas comerciais. O 1o e 2o experimento obteveram sucesso na manutenção e reprodução artificial de P. mangurus mantidos em tanques entre as estações reprodutivas. Os resultados sugerem que o desempenho reprodutivo dos animais mantidos em cativeiro (fertilidade inicial relativa-FIR = 609,25 ± 36,6 ovos / g) foi similar (p> 0,05) ao dos indivíduos selvagens (FIR = 679,21 ± 45,66 ovos / g). O treinamento alimentar de P. mangurus juvenis (3º experimento) também foi realizado avaliando-se 3 tratamentos alimentares com diferentes concentrações de coração bovino e ração. Ao final do experimento, o tratamento contendo metade coração bovino e metade ração gerou os maiores valores de ganho de peso (0,10 ± 0,16 g), taxa de crescimento específico (0,37 ± 0,11 mm), comprimento (47,78 ± 2,35 mm) e crescimento (2,15 ± 2,27 mm), sugerindo razoável aceitabilidade para dietas artificiais no cultivo desta espécie. Como conclusão, o presente estudo contribui com o desenvolvimento de técnicas para a domesticação espécies de peixes de água doce, de interesse comercial ou ameaçados de extinção, mostrando a domesticação e reproduçao de em cativeiro P. Mangurus selvagens. No entanto, mais estudos devem ser conduzidos no intuito de aumentar a aceitação de dietas comerciais pelos juvenis e melhorar sua taxa sobrevivência.

Difficulty in detecting low levels of polymyxin resistance in clinical Klebsiella pneumoniae isolates: evaluation of Rapid Polymyxin NP test, Colispot Test and SuperPolymyxin medium.

Polymyxins are important therapeutic options for treating infections, mainly those caused by carbapenem-resistant Klebsiella pneumoniae. Specific chemical characteristics of polymyxins make it difficult to perform antimicrobial susceptibility testing, especially within the clinical laboratory. Here we aimed to evaluate the performance of three phenotypic methods: Rapid NP Polymyxin Test, ColiSpot test and the SuperPolymyxin medium. To accomplish this, 170 non-duplicate clinical K. pneumoniae isolates were analysed (123 colistin-resistant and 47 susceptible). The sensitivity and specificity obtained for Rapid Polymyxin NP Test, Colispot and SuperPolymyxin medium were, respectively, 90% and 94%, 74% and 100%, and 82% and 85%. Very major errors occurred more frequently in low-level colistin-resistant isolates (MICs 4 and 8 µg/mL). Rapid Polymyxin NP proved to be a method capable of identifying colistin-resistant strains in acceptable categorical agreement. However, major errors and very major errors of this method were considered unacceptable for colistin-resistance screening. Although the Colispot test is promising and easy to perform and interpret, the results did not reproduce well in the isolates tested. The colistin-containing selective medium (SuperPolymyxin) showed limitations, including quantification of mucoid colonies and poor stability. Nevertheless, Colispot and SuperPolymyxin medium methods did not present acceptable sensitivity, specificity and categorical agreement. It is essential to use analytical tools that faithfully reproduce bacterial resistance in vitro, especially in last-line drugs, such as polymyxins, when misinterpretation of a test can result in therapeutic ineffectiveness.

In vivo phagocytosis and hematology in Astyanax altiparanae, a potential model for surrogate technology / Fagocitose in vivo e hematologia em Astyanax altiparanae, um potencial modelo para tecnologia de propagação mediada

Abstract Although the potential of surrogate propagation technology for aquaculture and conservation of Neotropical fish, the poor understanding of the host immune system may results in rejection and destruction of the donor material. Thus, it is necessary to study and to develop methods to evaluate the effects of immunosuppressive drugs employment and to evaluate the immunocompatibility between donor and receptor. Thus, the present study aimed to optimize a methodology to assess in vivo phagocytosis in Astyanax altiparanae using Saccharomyces cerevisiae and to evaluate their hematological response resultant from the inflammatory induction. To this, S. cerevisiae were labeled with Congo red and injected in the coelomic cavity of A. altiparanae at the concentration of 2.5 x 106 cells mL-1. A PBS solution and a non-injected group were kept as control. Fish blood was sampled and the phagocytic capacity and index were determined at 1, 2, 3 and 6 h post-injection (hpi). The yeast injection successfully stimulated phagocytosis, with the best result for phagocytosis assessment after 2 hpi. Moreover, it was achieved a high traceability of phagocytized and non-phagocytized yeast under optic microscopy analysis due to the Congo red labeling. The hematological profile was similar to usually observed in early infections, indicating lymphocyte migration to inflammatory site and increase in number of circulating phagocytes due to natural response to inflammatory stimulus. In conclusion, our method was efficient to assess in vivo phagocytosis in A. altiparanae and will be an important tool to evaluate the efficacy of immunosuppressive drugs in this species. Additionally, these results may serve as support for further studies in fish immunocompetence, both in laboratory and in field conditions.

Resumo Apesar do potencial apresentado pela tecnologia de propagação mediada para a aquicultura e conservação de peixes Neotropicais, o pobre entendimento do sistema imune do hospedeiro pode resultar na rejeição e destruição do material do doador. Com isso, se fazem necessários o estudo e o desenvolvimento de métodos para análise tanto dos efeitos de drogas imunossupressoras quanto para a avaliação da imunocompatibilidade entre doadores e receptores. Logo, o presente estudo teve como objetivo aperfeiçoar um método para analisar a fagocitose in vivo em Astyanax altiparanae usando Saccharomyces cerevisiae marcado e avaliar seu perfil hematológico resultante da indução inflamatória. Para isso, S. cerevisiae foram marcados com vermelho Congo e injetados na cavidade celomática dos A. altiparanae na concentração de 2,5 x 106 células.mL-1. Peixes injetados com PBS e peixes não injetados foram mantidos como controle. Sangue foi colhido e a capacidade fagocítica e o índice fagocítico foram determinados após 1, 2, 3 e 6 horas após à injeção (hpi). A injeção de levedura estimulou a fagocitose com sucesso, com o melhor resultado atingido após 2 hpi. Ainda, foi observada uma alta rastreabilidade das leveduras fagocitadas e não fagocitadas sob microscopia óptica devido à marcação com vermelho Congo. O perfil hematológico foi similar ao observado usualmente em infecções recém-induzidas, indicando migração de linfócitos ao sítio inflamatório e aumento no número de fagócitos circulantes devido à resposta natural ao estímulo inflamatório. Como conclusão, nosso método foi eficiente para analisar a fagocitose in vivo em A. altiparanae e será uma ferramenta importante para a avaliação de eficácia de drogas imunossopressoras para esta espécie. Em adição, estes resultados podem contribuir para futuros estudos em imunocompetência em peixes, tanto em âmbito laboratorial quanto a campo.

In vivo phagocytosis and hematology in Astyanax altiparanae, a potential model for surrogate technology.

Although the potential of surrogate propagation technology for aquaculture and conservation of Neotropical fish, the poor understanding of the host immune system may results in rejection and destruction of the donor material. Thus, it is necessary to study and to develop methods to evaluate the effects of immunosuppressive drugs employment and to evaluate the immunocompatibility between donor and receptor. Thus, the present study aimed to optimize a methodology to assess in vivo phagocytosis in Astyanax altiparanae using Saccharomyces cerevisiae and to evaluate their hematological response resultant from the inflammatory induction. To this, S. cerevisiae were labeled with Congo red and injected in the coelomic cavity of A. altiparanae at the concentration of 2.5 x 106 cells mL-1. A PBS solution and a non-injected group were kept as control. Fish blood was sampled and the phagocytic capacity and index were determined at 1, 2, 3 and 6 h post-injection (hpi). The yeast injection successfully stimulated phagocytosis, with the best result for phagocytosis assessment after 2 hpi. Moreover, it was achieved a high traceability of phagocytized and non-phagocytized yeast under optic microscopy analysis due to the Congo red labeling. The hematological profile was similar to usually observed in early infections, indicating lymphocyte migration to inflammatory site and increase in number of circulating phagocytes due to natural response to inflammatory stimulus. In conclusion, our method was efficient to assess in vivo phagocytosis in A. altiparanae and will be an important tool to evaluate the efficacy of immunosuppressive drugs in this species. Additionally, these results may serve as support for further studies in fish immunocompetence, both in laboratory and in field conditions.

In vivo phagocytosis and hematology in Astyanax altiparanae, a potential model for surrogate technology

Although the potential of surrogate propagation technology for aquaculture and conservation of Neotropical fish, the poor understanding of the host immune system may results in rejection and destruction of the donor material. Thus, it is necessary to study and to develop methods to evaluate the effects of immunosuppressive drugs employment and to evaluate the immunocompatibility between donor and receptor. Thus, the present study aimed to optimize a methodology to assess in vivo phagocytosis in Astyanax altiparanae using Saccharomyces cerevisiae and to evaluate their hematological response resultant from the inflammatory induction. To this, S. cerevisiae were labeled with Congo red and injected in the coelomic cavity of A. altiparanae at the concentration of 2.5 x 106 cells mL-1. A PBS solution and a non-injected group were kept as control. Fish blood was sampled and the phagocytic capacity and index were determined at 1, 2, 3 and 6 h post-injection (hpi). The yeast injection successfully stimulated phagocytosis, with the best result for phagocytosis assessment after 2 hpi. Moreover, it was achieved a high traceability of phagocytized and non-phagocytized yeast under optic microscopy analysis due to the Congo red labeling. The hematological profile was similar to usually observed in early infections, indicating lymphocyte migration to inflammatory site and increase in number of circulating phagocytes due to natural response to inflammatory stimulus. In conclusion, our method was efficient to assess in vivo phagocytosis in A. altiparanae and will be an important tool to evaluate the efficacy of immunosuppressive drugs in this species. Additionally, these results may serve as support for further studies in fish immunocompetence, both in laboratory and in field conditions.(AU)

Apesar do potencial apresentado pela tecnologia de propagação mediada para a aquicultura e conservação de peixes Neotropicais, o pobre entendimento do sistema imune do hospedeiro pode resultar na rejeição e destruição do material do doador. Com isso, se fazem necessários o estudo e o desenvolvimento de métodos para análise tanto dos efeitos de drogas imunossupressoras quanto para a avaliação da imunocompatibilidade entre doadores e receptores. Logo, o presente estudo teve como objetivo aperfeiçoar um método para analisar a fagocitose in vivo em Astyanax altiparanae usando Saccharomyces cerevisiae marcado e avaliar seu perfil hematológico resultante da indução inflamatória. Para isso, S. cerevisiae foram marcados com vermelho Congo e injetados na cavidade celomática dos A. altiparanae na concentração de 2,5 x 106 células.mL-1. Peixes injetados com PBS e peixes não injetados foram mantidos como controle. Sangue foi colhido e a capacidade fagocítica e o índice fagocítico foram determinados após 1, 2, 3 e 6 horas após à injeção (hpi). A injeção de levedura estimulou a fagocitose com sucesso, com o melhor resultado atingido após 2 hpi. Ainda, foi observada uma alta rastreabilidade das leveduras fagocitadas e não fagocitadas sob microscopia óptica devido à marcação com vermelho Congo. O perfil hematológico foi similar ao observado usualmente em infecções recém-induzidas, indicando migração de linfócitos ao sítio inflamatório e aumento no número de fagócitos circulantes devido à resposta natural ao estímulo inflamatório. Como conclusão, nosso método foi eficiente para analisar a fagocitose in vivo em A. altiparanae e será uma ferramenta importante para a avaliação de eficácia de drogas imunossopressoras para esta espécie. Em adição, estes resultados podem contribuir para futuros estudos em imunocompetência em peixes, tanto em âmbito laboratorial quanto a campo.(AU)

Domestication strategies for the endangered catfish species Pseudopimelodus mangurus Valenciennes, 1835 (Siluriformes: Pseudopimelodidae)

Abstract Wild fish domestication can be considered a strategic approach to endangered species conservation, supporting studies and reducing economic and environmental costs. Three of the most important strategies in the domestication processes of fish are the adaptation of wild fish to captivity, the reproduction of the adapted fish and the production and maintenance of the young individuals. That being said, the present study is divided in three experiments: the 1st aimed to adapt wild Pseudopimelodus mangurus to captivity environment using different feeding approaches and a prophylactic strategie; the 2nd aimed to reproduce the adapted individuals from the 1st experiment; and the 3rd aimed to train the P. mangurus juveniles to accept commercial diets. The 1st and 2nd experiments were successful at the maintenance and artificial reproduction of P. mangurus kept in tanks between the reproductive seasons. The results suggest that the reproductive performance of animals kept in captivity (initial relative fertility-IRF = 609.25 ± 36.6 eggs/g) was similar (p > 0,05) to the performance found in wild individuals (IRF = 679.21 ± 45.66 eggs/g). Feed training of P. mangurus juveniles (3rd experiment) was also conducted, evaluating three feeding treatments with different concentrations of bovine heart and ration. At the end of the experiment, the treatment containing half bovine heart and half commercial feeding resulted in the highest values of weight gain (0.10 ± 0.16 g), specific growth rate (0.37 ± 0.11 mm), length (47.78 ± 2.35 mm) and growth (2.15 ± 2.27 mm), suggesting reasonable acceptability to artificial diets in the cultivation of this species. As conclusion, the present study contributes with the development of techniques for the domestication of fresh water fish species with commercial value or andangered of extinction, showing the domestication and reproduction of wild P. mangurus in captivity. However, more studies have to be conducted in order to improve the acceptance of artificial feeding by juveniles and to increase their survival rate.

Resumo A domesticação de peixes selvagens pode ser considerada uma abordagem estratégica para a conservação de espécies ameaçadas, apoiando estudos e reduzindo custos econômicos e ambientais. Três das estratégias mais importantes para o processo de domesticação de peixes são a adaptação dos peixes ao cativeiro, a reprodução dos peixes adaptados e a produção e manutenção dos indivíduos jovens. O presente estudo está dividido em três partes: a 1ª objetivou adaptar Pseudopimelodus mangurus selvagens ao ambiente de cativeiro usando diferentes abordagens alimentares e uma estratégia profilática; o 2º objetivou reproduzir os indivíduos adaptados do 1º experimento; e o 3º teve como objetivo treinar os juvenis de P. mangurus para aceitar dietas comerciais. O 1o e 2o experimento obteveram sucesso na manutenção e reprodução artificial de P. mangurus mantidos em tanques entre as estações reprodutivas. Os resultados sugerem que o desempenho reprodutivo dos animais mantidos em cativeiro (fertilidade inicial relativa-FIR = 609,25 ± 36,6 ovos / g) foi similar (p> 0,05) ao dos indivíduos selvagens (FIR = 679,21 ± 45,66 ovos / g). O treinamento alimentar de P. mangurus juvenis (3º experimento) também foi realizado avaliando-se 3 tratamentos alimentares com diferentes concentrações de coração bovino e ração. Ao final do experimento, o tratamento contendo metade coração bovino e metade ração gerou os maiores valores de ganho de peso (0,10 ± 0,16 g), taxa de crescimento específico (0,37 ± 0,11 mm), comprimento (47,78 ± 2,35 mm) e crescimento (2,15 ± 2,27 mm), sugerindo razoável aceitabilidade para dietas artificiais no cultivo desta espécie. Como conclusão, o presente estudo contribui com o desenvolvimento de técnicas para a domesticação espécies de peixes de água doce, de interesse comercial ou ameaçados de extinção, mostrando a domesticação e reproduçao de em cativeiro P. Mangurus selvagens. No entanto, mais estudos devem ser conduzidos no intuito de aumentar a aceitação de dietas comerciais pelos juvenis e melhorar sua taxa sobrevivência.

Lichen planus: altered AIM2 and NLRP1 expression in skin lesions and defective activation in peripheral blood mononuclear cells.

BACKGROUND: Lichen planus (LP) is an inflammatory skin disease with unknown aetiology. Activation by pathogen-associated molecular patterns or environmental stimuli may activate some components of inflammasomes that contribute to the inflammatory process in LP lesions. AIM: To characterize the inflammasomes in skin lesions and peripheral blood mononuclear cells (PBMCs) of patients with LP under Toll-like receptor (TLR) activation. METHODS: In total, 15 patients with LP and 14 healthy controls (HCs) were enrolled in the study. Inflammasome expression in skin was evaluated by real-time PCR and immunohistochemistry, while ELISA was used to assess the production of interleukin (IL)-1ß by PBMCs under stimulation with TLR4 and TLR7/TLR8 agonists and adenosine triphosphate (ATP). RESULTS: Compared with the levels in HC samples, increased expression of the inflammasome AIM2 was verified in both epidermal and dermal sections of LP skin lesions, whereas NLRP1 and IL-ß expression levels were enhanced in the dermis. LP skin lesion samples exhibited higher AIM2 transcript levels, similar NLRP1 levels and lower pro-IL-1ß mRNA levels compared with HC samples. We verified that, compared with PBMCs from HC subjects, PBMCs from patients with LP produced similar amounts of IL-1ß after induction by TLR4 agonists but lower IL-1ß levels after induction by TLR7/TLR8 agonists, regardless of the addition of ATP. CONCLUSION: Alterations in innate immunity, such as inflammasome component expression in skin lesions and PBMCs, were observed in patients with LP. Further investigations of dysfunctional inflammasome activation and the chronic inflammatory status of LP are required.

In vivo phagocytosis and hematology in Astyanax altiparanae, a potential model for surrogate technology

Abstract Although the potential of surrogate propagation technology for aquaculture and conservation of Neotropical fish, the poor understanding of the host immune system may results in rejection and destruction of the donor material. Thus, it is necessary to study and to develop methods to evaluate the effects of immunosuppressive drugs employment and to evaluate the immunocompatibility between donor and receptor. Thus, the present study aimed to optimize a methodology to assess in vivo phagocytosis in Astyanax altiparanae using Saccharomyces cerevisiae and to evaluate their hematological response resultant from the inflammatory induction. To this, S. cerevisiae were labeled with Congo red and injected in the coelomic cavity of A. altiparanae at the concentration of 2.5 x 106 cells mL-1. A PBS solution and a non-injected group were kept as control. Fish blood was sampled and the phagocytic capacity and index were determined at 1, 2, 3 and 6 h post-injection (hpi). The yeast injection successfully stimulated phagocytosis, with the best result for phagocytosis assessment after 2 hpi. Moreover, it was achieved a high traceability of phagocytized and non-phagocytized yeast under optic microscopy analysis due to the Congo red labeling. The hematological profile was similar to usually observed in early infections, indicating lymphocyte migration to inflammatory site and increase in number of circulating phagocytes due to natural response to inflammatory stimulus. In conclusion, our method was efficient to assess in vivo phagocytosis in A. altiparanae and will be an important tool to evaluate the efficacy of immunosuppressive drugs in this species. Additionally, these results may serve as support for further studies in fish immunocompetence, both in laboratory and in field conditions.

Resumo Apesar do potencial apresentado pela tecnologia de propagação mediada para a aquicultura e conservação de peixes Neotropicais, o pobre entendimento do sistema imune do hospedeiro pode resultar na rejeição e destruição do material do doador. Com isso, se fazem necessários o estudo e o desenvolvimento de métodos para análise tanto dos efeitos de drogas imunossupressoras quanto para a avaliação da imunocompatibilidade entre doadores e receptores. Logo, o presente estudo teve como objetivo aperfeiçoar um método para analisar a fagocitose in vivo em Astyanax altiparanae usando Saccharomyces cerevisiae marcado e avaliar seu perfil hematológico resultante da indução inflamatória. Para isso, S. cerevisiae foram marcados com vermelho Congo e injetados na cavidade celomática dos A. altiparanae na concentração de 2,5 x 106 células.mL-1. Peixes injetados com PBS e peixes não injetados foram mantidos como controle. Sangue foi colhido e a capacidade fagocítica e o índice fagocítico foram determinados após 1, 2, 3 e 6 horas após à injeção (hpi). A injeção de levedura estimulou a fagocitose com sucesso, com o melhor resultado atingido após 2 hpi. Ainda, foi observada uma alta rastreabilidade das leveduras fagocitadas e não fagocitadas sob microscopia óptica devido à marcação com vermelho Congo. O perfil hematológico foi similar ao observado usualmente em infecções recém-induzidas, indicando migração de linfócitos ao sítio inflamatório e aumento no número de fagócitos circulantes devido à resposta natural ao estímulo inflamatório. Como conclusão, nosso método foi eficiente para analisar a fagocitose in vivo em A. altiparanae e será uma ferramenta importante para a avaliação de eficácia de drogas imunossopressoras para esta espécie. Em adição, estes resultados podem contribuir para futuros estudos em imunocompetência em peixes, tanto em âmbito laboratorial quanto a campo.

Energy drinks and their component modulate attention, memory, and antioxidant defences in rats.

PURPOSE: This study aimed to evaluate the effects of the subchronic consumption of energy drinks and their constituents (caffeine and taurine) in male Wistar rats using behavioural and oxidative measures. METHODS: Energy drinks (ED 5, 7.5, and 10 mL/kg) or their constituents, caffeine (3.2 mg/kg) and taurine (40 mg/kg), either separately or in combination, were administered orally to animals for 28 days. Attention was measured though the ox-maze apparatus and the object recognition memory test. Following behavioural analyses, markers of oxidative stress, including SOD, CAT, GPx, thiol content, and free radicals, were measured in the prefrontal cortex, hippocampus, and striatum. RESULTS: The latency time to find the first reward was lower in animals that received caffeine, taurine, or a combination of both (P = 0.003; ANOVA/Bonferroni). In addition, these animals took less time to complete the ox-maze task (P = 0.0001; ANOVA/Bonferroni), and had better short-term memory (P < 0.01, Kruskal-Wallis). The ED 10 group showed improvement in the attention task, but did not differ on other measures. In addition, there was an imbalance in enzymatic markers of oxidative stress in the prefrontal cortex, the hippocampus, and the striatum. In the group that received both caffeine and taurine, there was a significant increase in the production of free radicals in the prefrontal cortex and in the hippocampus (P < 0.0001; ANOVA/Bonferroni). CONCLUSIONS: Exposure to a combination of caffeine and taurine improved memory and attention, and led to an imbalance in the antioxidant defence system. These results differed from those of the group that was exposed to the energy drink. This might be related to other components contained in the energy drink, such as vitamins and minerals, which may have altered the ability of caffeine and taurine to modulate memory and attention.