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Multi-drug resistant Pseudomonas aeruginosa is a gram-negative bacillus responsible for nosocomial infections. Immunoglobulin Y (IgY) is a chicken immunoglobulin used for research, immunodiagnosis, and immunotherapy. IgY presents antimicrobial properties and it is under investigation for use as an adjunct to prophylactic therapies. The current study aimed to assess the synergistic action between anti-P aeruginosa IgY and the beta-lactams ceftazidime, imipenem, and meropenem. IgY antibodies were obtained from laying hens immunized with SPM-1 producing P. aeruginosa (Pa48

The objective of this study was to compare the antimicrobial activity of macrophages and serum in laying hen (MM, CC, and CCc) and broiler chicken lineages (TT and LL). Macrophages were evaluated for phagocytic and antimicrobial activity. Microbicidal serum activity was evaluated by the resistance test for serum and the agar test. The results showed that phagocytic activity was higher in males of the MM strain, with 13% of macrophages presenting phagocytosis, while the other lineages studied, and even female MM, presented a rate of 6% of phagocytic cells. However, antimicrobial activity in macrophages from males of CCc lineage and females of TT lineage were higher, eliminating more than 30% of the Salmonella enterica inoculum, while in the other strains, the results were similar, with inoculum reduction below 30%. In the serum resistance assay, female laying lines presented higher antibacterial activity than female broiler lines. In the trials to evaluate the microbicide activity of the serum, females of both broiler and laying lineages presented higher performance when compared with males of the same lineage. Females of laying hen lines (MM and CC) present a greater antibacterium activity than males. These results can contribute to a better understanding of the immune response in broiler chicken and laying hen lineages, to aid development of lineages of birds more resistant to pathogens Made available in DSpace on 2021-11-25T14:00:43Z (GMT). No. of bitstreams: 1 final9220.pdf: 375423 bytes, checksum: 93f1118fa449bc634140423360940b25 (MD5) Previous issue date: 2021

Rhythmic gymnastics has been an Olympic sport since 1984, however, there are relatively few studies about this sport. In order to understand whether genetic predisposition could play a role in defining the flexibility phenotype in rhythmic gymnastics, the purpose of this study was to review the current literature and systematically identify common polymorphisms linked to genes correlated with joint mobility in elite rhythmic gymnastics. Systematic computerized searches were performed from 1950 to 2017 in the following databases: Medline, Embase, Cinahl, Lilacs, SPORTDiscus, Web of Science, Scopus and the Cochrane Central. Although the search initially identified 9761 studies, after removing duplicates and excluding by title and abstract, only 10 studies demonstrated potential to be included. After reading of full-texts, 9 studies were entered in the qualitative synthesis, thus only 1 study was eligible for this systematic review. The results of Tringali's study demonstrated that the COL5A1 CT genotype was linked to high joint mobility and to the occurrence of genu recurvatum. From this systematic review, further investigations are suggested to confirm the results of involving genes related to physiological and anthropometric determinants of rhythmic gymnastics performance.

Background Considering that studies on the relationship between dental caries and trace elements present contradictory and inconclusive results, the purpose of this study was to determine the levels of salivary trace elements in saliva samples of preschool children and investigate their relationship with dental caries. Methods In total, 120 samples of unstimulated saliva were collected from children aged 36–72 months, of both sexes, who participate in the preventative educational program in oral health at the State University of Londrina, Brazil. The children were divided into two groups, caries (n = 60) and non-caries (n = 60). Levels of Al, Cu, Fe, Mn, and Zn were analyzed by total reflection X-ray fluorescence (TXRF). Descriptive statistics, the Student’s t-test, Mann-Whitney U test, and Pearson’s Chi-squared test were performed (P Results The concentrations of Mn and Fe were significantly higher in the caries group (Mn =0.015 mg/L [0.007−0.020]; Fe =0.080 mg/L [0.031−0.239] than the non-caries group (Mn =0.010 mg/L [0.001 – 0.017]; Fe =0.044 mg/L [0.023 – 0.107]). Conclusion The results suggest a relationship between trace elements and dental caries, indicating possible involvement of these elements in the metabolism of microorganisms involved in the carious process. In addition, the use of TXRF presented satisfactory results, with a simple and fast methodology for the detection of the studied elements.

Advances in the fields of glycobiology and immunology have provided many insights into the role of carbohydrate-protein interactions in the immune system. Jacalin of Artocarpus integrifolia (JCA) and structural mannoprotein of Saccharomyces uvarum (MPS) are molecules with immunomodulatory properties. JCA is an IgA human lectin binding molecule that causes the mitogenic stimulation of immune cells, production of cytokines, chemotaxis, and activation of leukocytes. Studies on the immunomodulatory properties of JCA and MPS in mammals and fish suggest that they have an action on antibody production. The aim of this study was to investigate the possible action of JCA and MPS on the production of specific antibodies in laying hens. For this, laying hens were inoculated with an intra abdominal injection of sheep red blood cells (SRBC) with either JCA (0.075 µg, 0.75 µg, and 7.5 µg) or MPS (20 µg and 100 µg). Levels of anti-SRBC antibodies of the IgY, IgM, and IgA classes were evaluated by ELISA. Results showed that JCA and MPS have immunomodulatory effects on levels of anti-SRBC IgM, IgA, and IgY. An immunostimulatory effect of JCA was observed in primary immune response on anti-SRBC IgY, while an inhibitory effect of JCA and MPS was observed in secondary immune response on the production of IgM and IgA anti-SRBC. These results suggested that MPS and JCA have immunomodulatory effects on antibody production and could be used in future studies on humoral immune response in poultry. RESUMO: Avanços nos campos glicobiologia e imunologia forneceram muitas informações sobre o papel das interações da proteína-carboidrato na modulação do sistema imunológico. A jacalina extraída de Artocarpus integrifolia (JCA) e a manoproteína da parede celular de Saccharomyces uvarum (MPS) são moléculas com propriedades imunomoduladoras. JCA é uma lectina com afinidade pela IgA humana e tem ação mitogênica sobre células do sistema imunológico estimulando a produção de citocinas, a quimiotaxia e a ativação de leucócitos. Estudos sobre as propriedades imunomoduladoras de JCA e MPS em mamíferos e peixes sugerem que essas moléculas podem ter um efeito sobre a produção de anticorpos. O objetivo deste estudo foi investigar a ação da JCA e MPS sobre a produção de anticorpos específicos em galinhas poedeiras. Para isso, galinhas poedeiras foram inoculadas por via intra-abdominal com eritrócitos de carneiro (SRBC) em associação com o JCA (0,075 µg, 0,75 µg, e 7,5 µg) ou MPS (20 µg e 100 µg). Os níveis de anticorpos anti-SRBC das classes IgY, IgM, e IgA foram avaliados por ELISA. Os resultados mostraram que a JCA e a MPS têm um efeito imunomodulador sobre a produção IgY, IgM, ou IgA anti-SRBC. Um efeito imunoestimulador da JCA foi observado sobre a produção de anticorpos IgY na resposta imune primária, enquanto um efeito imuno inibitório da JCA e da MPS sobre a produção de IgM e IgA anti-SRBC na resposta imune secundária. Estes resultados sugerem que o MPS e JCA tem efeito modulador sobre a produção de anticorpos e podem ser utilizados em estudos futuros sobre a imunidade humoral em aves comerciais.

Influenza A virus (IAV) causes an important respiratory disease in mammals and birds leading to concerns in animal production industry and public health. Usually, antibodies produced in mammals are employed in diagnostic tests. However, due to animal welfare concerns, technical advantages and the high cost of production, alternatives to the production of antibodies in mammals have been investigated. The aim of this study was to produce egg yolk immunoglobulin (IgY) in laying hens against a highly conserved protein (nucleoprotein- NP) of IAV and to evaluate the application of anti-NP IgY antibodies in virus detection by immunocytochemistry (ICC) and immunohistochemistry (IHC). Three laying hens of the White Leghorn line were inoculated seven times with a recombinant NP protein and their eggs collected seven days after the 3rd, 5th and 7th inoculations. Immunoglobulin Y antibodies were purified from egg yolk through precipitation with ammonium sulfate. The titers and specificity of the purified antibodies were determined by ELISA, western blotting, ICC and IHC. High levels of specific anti-NP antibodies were detected by ELISA after the 5th inoculation, reaching a peak after the 7th inoculation. The mean yield of total protein in yolk after the 7th inoculation was 13.5 mg/mL. The use of western blotting and ICC demonstrated that anti-NP IgY binds specifically to NP protein. Moreover, the use of anti-NP IgY antibody in ICC test revealed positive staining of MDCK cells infected with IAV of the three subtypes circulating in swine (H1N1, H1N2, and H3N2). However, no staining was observed in lung tissues through the IHC test. The data obtained showed that anti-NP IgY antibodies bound specifically to influenza virus NP protein, detecting the main virus subtypes circulating in swine, reinforcing their usefulness in the influenza diagnosis.

The literature presents several instances of interaction between the nervous system (NS) and the immune system (IS). These interactions are promoted by several molecules, such as cytokines and hormones, with modulating action for both the NS and IS. In this sense, the two systems may influence each other: changes in behavior may be accompanied by alterations in the IS (e.g., immunosuppression) and immunological disorders, such as infections, may modulate behavior (e.g., anxiety and depression). Considering that chronic stress, in addition to affecting behavior, also modulates the IS and that there is evidence that moderate intensity physical exercise (PE) protects physical and mental health, the objective of this review is to explore the influence of moderate-intensity PE on behavior and immunity. Level of Evidence V; Expert opinion.

Scorpionism is a relevant public health problem in several countries in tropical and subtropical regions. In Brazil, Tityus serrulatus sting can induce acute lung injury in part as a consequence of inflammation. Despite the occurrence of other scorpions of Tityus genus in Brazilian scorpiofauna, the knowledge regarding pulmonary alterations is related to T. serrulatus venom (Tsv). Here we studied, comparatively, the pathophysiological changes in the rat airways envenomed by Tsv or T. bahiensis venom (Tbv), since both scorpions are involved in human accidents but with severe envenomations occurring when victims are stung by T. serrulatus. After intravenous injection of the venoms (200 μg/kg), both were able to induce Evans blue extravasation (in 30 min) into airways and increased protein extravasation into lungs at 4 and 24 h, but the magnitude of such events was higher in Tsv group. Hemorrhage (in 60 min) in the lungs was higher in Tbv group, while IL-1β (at 1 h) and IL-6 (at 1 and 4 h) in lung homogenates were detected only in Tsv group. Four and 24 h after envenomation, myeloperoxidase activity in lung was equally augmented in the envenomed groups, as well as an increased in polymorphonuclear cell numbers in bronchoalveolar lavage fluid. At 4 h blood leukogram showed increased leukocyte values with the highest neutrophilia in Tsv group. The numbers of leukocytes and neutrophils remained higher than control at 24 h in Tsv and Tbv groups, and it was accompanied by lympho (envenomed groups) and monocytosis (Tsv group). In conclusion, although Tbv was capable of inducing acute lung injury and blood leukocyte mobilization, most of the evaluated parameters were more affected by the Tsv. These results could help to explain the pathophysiology of the scorpionism and the clinical data arguing toward the greatest severity associated with T. serrulatus stings.

The aim of this study was to investigate the effects of route of inoculation and dose on production and avidity of IgY antibodies in chickens. The animals were inoculated with 20 µg of human IgG by intramuscular, intradermal, or subcutaneous routes, or with 2, 20, or 200 µg of human IgG via IM to evaluate the effect of dose. All routes led to significant levels of specific antibodies after the fifth inoculation. There was a significant difference in the levels of antibodies produced with 2 and 200 µg of antigen after the third and seventh inoculation. It was observed that there is no influence of route of inoculation or dose on the avidity of the antibodies produced. Furthermore, the index of avidity is influenced directly by the number of inoculations. These results are important to help future studies where antibody production of IgY from laying hens is necessary.

It was evaluated the effects of maternal treatment with the Trichilia catigua (ExTc) crude extract on the antibodies’ production by their offspring. Female rats received ExTc or saline from the first day of pregnancy until the twenty-first day after the birth of the pups, when the pups were weaned. All pups were inoculated with two doses of 50 μg of IgY diluted in aluminium hydroxide/PBS on days 26 and 40 of life. Antibody levels were analysed by ELISA. Our results show an increase in levels of IgG1 and IgG2a anti-IgY in female offspring of mothers treated with ExTc compared to female offspring of untreated mothers. Furthermore, ExTc treatment suppressed the production of IgG2a anti-IgY antibodies in males. The data show that maternal exposure to ExTc can modulate the production of antibodies in the offspring.

RESUMO A adicao de oleos na dieta de frangos de corte proporciona muitas vantagens, visto que, dependendo do perfil de acidos graxos, pode melhorar o desempenho e atuar como estimulante do sistema imune. Assim, este estudo teve como objetivo avaliar as caracteristicas produtivas, o rendimento de carcaca, os cortes e a resposta imune humoral de frangos de corte alimentados com diferentes fontes de oleos e vitamina E. Foram utilizados 312 pintainhos de corte machos da linhagem Cobb com um dia de idade, distribuidos em delineamento inteiramente ao acaso, com oito repeticoes compostas de 13 aves por parcela experimental. Os tratamentos experimentais consistiram em oleo de soja, oleo de canola e oleo de canola mais adicao de vitamina E. As variaveis analisadas foram ganho de peso, consumo de racao, conversao alimentar, rendimento de carcaca, cortes comerciais e resposta imune humoral. Os resultados obtidos mostram que houve diferenca significativa no desempenho somente na fase pre-inicial, quando as aves que receberam o tratamento com oleo de canola e vitamina E apresentaram piores ganhos de peso. Nao foram observadas diferencas significativas para as outras variaveis analisadas. Conclui-se que a utilizacao de diferentes fontes lipidicas associadas ou nao a vitamina E nao afeta as caracteristicas produtivas de carcaca, cortes e resposta imune humoral em frangos de corte em relacao ao uso de oleo de soja. Palavras-chave: aves, oleo de canola, oleo de soja, tocoferol ABSTRACT

Ochratoxin A (OTA), an immunosuppressive mycotoxin, can increase the risk of many infectious diseases and contribute to economic losses to the poultry industry. The immunosuppressive effect has mainly been investigated through oral exposure, however, birds may also be contaminated through skin absorption. The present study investigated the influence of OTA exposure on the defense system of broiler chicks through the subcutaneous route and including low doses. Groups of broiler chicks (Cobb), 05 days old, were exposed to subcutaneous inoculation of OTA at concentrations of 0.1, 0.5, 0.9, 1.3, and 1.7 mg OTA/kg body weight. The size of the lymphoid organs, circulating immune cells, and total IgY and IgA levels were evaluated 21 days post inoculation. Subcutaneous OTA exposure decreased the weight of the thymus, spleen, and bursa of Fabricius, and leukocytopenia (p &lt, 0.05) was detected in chicks of the OTA treated groups. In a dose-dependent way, decreased levels of circulating lymphocytes and heterophils (p &lt, 0.05), and increased levels of monocytes (p &lt, 0.05) were detected. Decreased IgY and IgA serum concentrations were noted in the OTA treated groups (p &lt, 0.05). In conclusion, subcutaneous OTA exposure induces immunosuppression even at low levels.

Ochratoxin A (OTA) is a mycotoxin produced by species of Penicillium and Aspergillus that can contaminate products of plant origin that are used as animal feed. Through oral exposure, this mycotoxin primarily affects the chicken gastrointestinal system. The present study evaluated the intestinal toxic effects of OTA and the introduction of L-tryptophan to alleviate these effects in chickens. One-day-old chicks were exposed to a single OTA dose (1.4 mg/kg body weight&mdash, b.w.) and treated with or without four daily doses of L-tryptophan (100 mg/kg b.w.). Duodenal villus height/crypt depth, fecal immunoglobulin A/immunoglobulin Y (IgA/IgY) levels, and duodenal positive immunoglobulin A cells (IgA+) were evaluated by histology, ELISA, and immunohistochemistry, respectively, on the 14th day. There were significant changes in the duodenal villus height, crypt depth, and levels of fecal IgA/IgY and duodenal IgA+ cells (p &lt, 0.05) in groups exposed to OTA. On the other hand, groups exposed to OTA and treated with L-tryptophan showed similar levels of villus height, IgA/IgY levels, and duodenal IgA+ cells to those of the control group (p &gt, 0.05). In conclusion, exposure to a single dose of OTA orally induces changes in intestinal morphology, levels of IgA/IgY antibodies, and IgA+ cells. Thus, treatment with L-tryptophan may be a valid alternative means to reduce the harmful effects of OTA on the intestinal mucosa, which requires further study.

The mycotoxin, ochratoxin-A (OTA), produced by some fungi, and is a natural contaminant of many foods and animal feeds worldwide. Due to its toxic effects, the recommended maximum daily intake of OTA for poultry feeds is 0.1 mg OTA/kg (ECR2006/575/EC), this dose does not induce changes in hepatic/renal parameters, but decreases thymus size and serum globulin concentrations. Accordingly, in this study, we assessed quantitatively the total circulating IgY and IgA serum levels, in chicks consuming a 0.1 mg OTA/kg diet (limit) and higher doses (0.3&ndash, 1.1 mg OTA/kg diet) for 14 or 21 days. We also evaluated other immunological parameters (thymus, bursa of Fabricius, and spleen weights and leukocyte profiles) at day 21. Decreased IgY serum levels were observed in all OTA-treated groups (p &lt, 0.05). In the low-dose group, IgA levels were decreased on day 21, but not on day 14. The size of the thymus and the bursa of Fabricius was decreased in all OTA-treated groups (p &lt, 0.05), whereas reduced spleen size and altered leukocyte profiles were detected only in the high-dose group (p &lt, 0.05). We concluded that chronic exposure to OTA, even at the recommended highest dose, affected IgY and IgA production in chicks.

Ochratoxicosis is one of the most common mycotoxicoses in poultry, specially commercial poultry. It is caused by most dangerous mycotoxin because it causes oncogenic effects in embryos, that is, ochratoxin A. The presence of ochratoxin-A in poultry feed contributes significantly to health disorders and decreases production. This is one of the causes of economic losses in poultry industry due to increased mortality, reduced body weight gain, reduction of carcass quality, greater feed conversion rate and immunosuppression. The risk associated with ochratoxin residues in poultry meat represents a public health concern. The present article reviews most significant scientific literature on ochratoxin and their possible detrimental effects on poultry birds and subsequent public health hazards. Recent studies have revealed that embryos, new born chicks and young poultry are more sensitive to ochratoxin A than adults. Ochratoxin-A has a high affinity for liver, kidneys, bursa of Fabricius and thymus. It causes an appreciable increase in the size of liver and kidneys where as the size of bursa and thymus is reduced. It also causes nephrotoxicity and hepatotoxicity with carcinogenic effect. In embryo, it causes teratologic defects in the form of anophthalmia followed by mandibular hypoplasia, micropthalmia, maxillary retrognathism, reduced body size, everted viscera, spina bifida and exencephaly. Biochemically it causes hypoproteinemia, hypoalbuminemia, hypoglobulinemia and hypoglycaemia. Similarly, it also causes increased levels of blood urea nitrogen (BUN), serum creatinine, uric acid, alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and serum triglycerides. In order to prevent and reduce implications of these mycotoxins in poultry feed, there is needs for both global and national strategic programs to reduce the residual accumulation of mycotoxins in grain, to use advanced analytic techniques and to establish new limits concerning the maximum amount of mycotoxins allowed in poultry feed and products from poultry for human consumptions. Key words: Ochratoxin, toxicity, teratologic defects, immunoglobulins.

Phospholipase is an important virulence factor for pathogenic fungi. In this study, we demonstrate the following: (i) the Paracoccidioides brasiliensis pld gene is preferentially expressed in mycelium cells, (ii) the plb1 gene is mostly up-regulated by infection after 6 h of co-infection of MH-S cells or during BALB/c mice lung infection, (iii) during lung infection, plb1, plc and pld gene expression are significantly increased 6-48 h post-infection compared to 56 days after infection, strongly suggesting that phospholipases play a role in the early events of infection, but not during the chronic stages of pulmonary infection by P. brasiliensis.

MicroRNAs (miRNAs) are small single stranded RNA sequences involved in post-transcriptional regulation of different biological and physiological processes. Paracoccidioidomycosis (PCM) is an infection caused by Paracoccidioides brasiliensis, and it is a major cause of mortality due to systemic mycoses in Brazil. To date, there have been few reports on the role of miRNAs in the immune response against fungi, especially PCM. The objective of this study was to evaluate the differential expression of miRNAs related to the inflammatory response associated with pulmonary infection by P. brasiliensis. For this purpose, lungs from BALB/c mice, intravenously infected with P. brasiliensis (2.7×107 yeast cells/ml, n = 12) and noninfected BALB/c mice (n = 8), were collected at the 28 and 56 day after infection. The lung parenchyma presented a great number of yeast cells, granulomas, and edema at 28 days and a framework of resolution of the inflammatory process after 56 days. The mRNAs gata-3, ror-γt, foxp3, and IL-6 were positively regulated at the moment at the 56 day, while the TGF-β1 mRNA was positively regulated at both moments. The miRNAs 126a-5p, 340-5p, 30b-5p, 19b-3p, 221-3p, 20a-5p, 130a-3p, and 301a-3p, 466k presented the greatest increase in expression levels 28 days after infection, and the miRNAs let-7f-5p, let-7a-5p, 5p-26b, let-7e-5p and 369-3p, 466k presented a greater increase in levels of expression 56 days after infection. This study shows a set of differentially expressed miRNAs possibly involved in the immune response in mice during pulmonary infection by P. brasiliensis.

No presente trabalho, os efeitos comportamentais e imunomoduladores da fluoxetina foram avaliados num modelo animal de depressão, o nado forçado. Nesse, o comportamento de flutuar é sensível a antidepressivos e é usado como índice de desespero comportamental. Foram utilizados dois grupos experimentais, sendo um grupo controle tratado com salina e outro grupo teste tratado com a fluoxetina, ambos administrados por via intra-peritoneal. Os animais foram tratados três vezes ao dia por 12 dias. Adicionalmente, a resposta imune humoral a uma imunoestimulação (hemácias de carneiro) foi avaliada. A fluoxetina provocou aumento no tempo em flutuação, queda na massa corporal e menor produção total de anticorpos. Os resultados indicam uma modulação simultânea do comportamento e do sistema imunológico pela fluoxetina. In the present study, behavioral and immunomodulatory effects of fluoxetine were evaluated with the forced swim test, an animal model of depression. In this model, floating behavior is responsive to antidepressants and is used as a behavioral index of despair. During 12 days, three times a day, two groups were treated intraperitoneally with saline or fluoxetine. Additionally, the humoral immune response to immunostimulation (sheep red blood cells) was evaluated. The animals treated with fluoxetine floated longer, showed decreases in body mass and had lower total production of antibodies. The results indicate simultaneous modulation of behavior and antibody production by fluoxetine.

Propolis is a honeybee product showing several biological properties that enhance the immune response, depending on the concentration and intake period. Because propolis possesses an immunomodulatory action on mammals, the objective of our study was to investigate the effects of propolis on the humoral immune response of laying hens by evaluating antibody production. Laying hens (ISA Brown) were divided into 5 groups with 7 birds each. Group 1 was a nonimmunized control, whereas birds in group 2 were immunized intravenously with SRBC, and those in groups 3, 4, and 5 were treated intraperitoneally with propolis (2, 10, and 50 mg/kg, respectively) on 3 consecutive days and then inoculated intravenously with SRBC. Hematological and serological analyses were carried out on d 0, 3, and 38. Natural and specific antibody levels were determined by hemagglutination with rabbit red blood cells and SRBC, respectively. Propolis-treated birds (50 mg/kg) showed a significant decline in heterophils and in the heterophil:lymphocyte ratio. After SRBC immunization, significant increases in levels of IgG were observed in groups 4 and 5. Furthermore, higher levels of natural antibodies were observed in propolis-treated laying hens. The administration of propolis to laying hens increased the production of IgG specific to SRBC and natural antibodies, and could be used to increase antigen-specific antibody responses to vaccines.

This study investigated the major soluble antigens produced by Paracoccidioides brasiliensis (Pb339) cultured in solid Sabouraud (pH 5.6 and 8.5), Sabouraud plus brain heart infusion and liquid tomato juice-enriched complex medium media at intervals of 3 days over 30 days by immunoblotting and concluded that, to optimize the source of each antigen, both time and growth conditions should be considered.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by the fungus Paracoccidioides brasiliensis (Pb). The cyclosporin A (CsA) is an immunosuppressant drug that inhibits calcineurin and has been described as a potential antifungal drug. The present study investigated the effect of CsA on the immune response, fungal load/antigenemia in experimental murine PCM. It was used four groups of BALB/c mice: (a) infected with 1 x 10⁵ Pb18 yeast cells (Pb), (b) infected and treated with CsA every other day 10 mg/kg of CsA (s.c.) during 30 days (Pb/CsA), (c) treated with CsA (CsA) and (d) no infected/treated (PBS). The immune response was evaluated by lymphocyte proliferation, DTH assays to exoAgs, ELISA for IgG anti-gp43 (specific immune responses) and cytokine serum levels (IFN-γ, TNF-α, IL-4 and IL-10). Fungal load was determined by lung colony-forming units (CFU) counts, lung and liver histopathology analysis and antigenemia determined by inhibition-ELISA. As expected, CsA was able to inhibit the specific cellular and humoral immune response (P < 0.05), with decrease in serum IFN-γ, TNF-α and IL-4 levels (P < 0.05). Cyclosporin A treatment also resulted in significantly decreased lung Pb CFU (P < 0.05) as well as a lower number of yeasts in the lung and liver (P < 0.05) by histopathology. In concordance, the decreased antigenemia was observed in Pb/CsA group (P < 0.05). In conclusion, even with immunosuppressive action, treatment with CsA results in decreased lung fungal load/antigenemia in experimental PCM in BALB/c mice. Further study is required to determine whether this represents less severe disease or protection by CsA.

Introduction Herein, we report a one-tube, semi-nested-polymerase chain reaction (OTsn-PCR) assay for the detection of Paracoccidioides brasiliensis. Methods We developed the OTsn-PCR assay for the detection of P. brasiliensis in clinical specimens and compared it with other PCR methods. Results The OTsn-PCR assay was positive for all clinical samples, and the detection limit was better or equivalent to the other nested or semi-nested PCR methods for P. brasiliensis detection. Conclusions The OTsn-PCR assay described in this paper has a detection limit similar to other reactions for the molecular detection of P. brasiliensis, but this approach is faster and less prone to contamination than other conventional nested or semi-nested PCR assays.

Paracoccidioides brasiliensis is a pathogenic fungus that undergoes a temperature-dependent cell morphology change from mycelium (22 degrees C) to yeast (36 degrees C). It is assumed that this morphological transition correlates with the infection of the human host. Our goal was to identify genes expressed in the mycelium (M) and yeast (Y) forms by EST sequencing in order to generate a partial map of the fungus transcriptome. Individual EST sequences were clustered by the CAP3 program and annotated using Blastx similarity analysis and InterPro Scan. Three different databases, GenBank nr, COG (clusters of orthologous groups) and GO (gene ontology) were used for annotation. A total of 3,938 (Y = 1,654 and M = 2,274) ESTs were sequenced and clustered into 597 contigs and 1,563 singlets, making up a total of 2,160 genes, which possibly represent one-quarter of the complete gene repertoire in P. brasiliensis. From this total, 1,040 were successfully annotated and 894 could be classified in 18 functional COG categories as follows: cellular metabolism (44%); information storage and processing (25%); cellular processes-cell division, posttranslational modifications, among others (19%); and genes of unknown functions (12%). Computer analysis enabled us to identify some genes potentially involved in the dimorphic transition and drug resistance. Furthermore, computer subtraction analysis revealed several genes possibly expressed in stage-specific forms of P. brasiliensis. Further analysis of these genes may provide new insights into the pathology and differentiation of P. brasiliensis.

Kexin-like protein is a component of the subtilase family of proteinases involved in the processing of proproteins to their active forms. Kexin-like proteins are also synthesized as a propeptide and this is involved in (auto)inhibition, correct folding and subcellular sorting of proteins. The kexin-like protein was described as the product of the kex2 gene for Aspergillus niger, Candida albicans, Saccharomyces cerevisiae, Yarrowia lipolytica and other fungi. Disruption of the kex2 gene in C. albicans and Y. lipolytica affects hyphae production and induces morphological cell defects, strongly suggesting a possible role of kexin-like proteins in dimorphism of human pathogenic fungi. In this work, we report the nucleotide sequence of the kex2 gene cloned from the dimorphic and human pathogenic fungus Paracoccidioides brasiliensis (Pbkex2). An open reading frame (ORF) of 2622 bp was identified in the complete sequence, interrupted by only one intron of 93 bp. The 5' non-coding region contains consensus sequences such as canonical TATA, CAAT boxes and putative motifs for transcriptional factors binding sites, such as HSE-like regulating genes involved in thermo-dependent processes; Xbp1, reported as a transcriptional factor that may control genes involved in cell morphology; and StuAp, which may regulate spore differentiation and pseudohyphal growth in fungi. In the 3' non-coding region were observed the canonical motifs necessary for correct mRNA processing and polyadenylation. The deduced protein sequence consists of 842 amino acid residues, showing identity to kexin-like proteinases from A. niger (55%), Emericella nidulans (53%) and C. albicans (48%). Comparative sequence analysis of P. brasiliensis kexin-like protein reveals the presence of homologous regions related to a signal peptide, a propeptide, a subtilisin-like catalytic domain, a P domain, a S/T rich region and a transmembrane domain. A putative Golgi retrieval signal (YEFEMI) has also been found in the cytoplasmic tail. The complete nucleotide sequence of Pbkex2 and its flanking regions have been submitted to GenBank database under Accession No. AF486805.

Tityus scorpion stings are an important public health problem in Brazil, where the incidence of such stings exceeds the incidence of the health problems caused by other venomous animals, including snakes. In this study, we have analysed specific enzymatic activities of the venom from the Brazilian scorpions of Tityus genus, i.e., Tityus serrulatus, Tityus bahiensis and Tityus stigmurus. The data presented here revealed that Tityus spp. venoms exhibited significant hyaluronidase activity but no phospholipase activity. All the venom samples exhibited the ability to hydrolyse Abz-FLRRV-EDDnp and dynorphin 1-13 substrates. These activities were inhibited by 1,10-phenanthroline but not by PMSF, indicating the presence of metalloproteinases in the Tityus spp. venoms. The venom peptidase activity on Abz-FLRRV-EDDnp and on dynorphin 1-13 was partially inhibited by therapeutic Brazilian anti-scorpion and anti-arachnidic antivenoms. Dynorphin 1-13 (YGGFLRRIRPKLK) contains two scissile bonds between the residues Leu-Arg and Arg-Arg that are susceptible to cleavage by the Tityus venom metallopeptidase(s). Their cleavage releases leu-enkephalin, an important bioactive peptide. The detection of metalloproteinase(s) with specificity for both dynorphin 1-13 degradation and leu-enkephalin releasing can be important for the mechanistic understanding of hypotension and bradycardia induction in cases of scorpion stings, whereas hyaluronidases might contribute to the diffusion of the toxins present in these venoms. Furthermore, the limited inhibition of the toxic enzymatic activities by commercial antivenoms illustrates the necessity of improvements in current antivenom preparation.

Yeasts discarded in industrial processes can be used as a nutritional supplement and to extract cellular components with biotechnological aims. In this study, the humoral immune response of Swiss mice treated with mannoproteins (MP) from the yeast Saccharomyces uvarum was evaluated. The mice were treated with MPs at different doses and times and inoculated with 2% sheep red blood cells. An increase in total Ig in mice treated with 100 μg of MP at the time of immunization or 24 h before was observed in the primary immune response; in the secondary immune response, an increase was observed in total Ig values for all groups, and an increase of IgG was observed in the mice treated with MPs (100 μg) at the time of immunization or 24 h before. These results show that S. uvarum MPs present an immunostimulatory action on the humoral immune response in mice.

The term stress was introduced into the biomedical field by Hans Selye (1936) in reference to a General Adaptation Syndrome which would consist of all non-specific systemic reactions that occur during an intense and chronic exposure to a stressor (e.g., pressure at work and poor diet). This syndrome would be different from the specific adaptive reactions (such as muscle hypertrophy caused by exercise performed on a regular basis) and immune responses (Selye, 1936).

INTRODUCTION: Paracoccidioidomycosis is a systemic infection caused by Paracoccidioides brasiliensis. METHODS: In this study, a semi-nested PCR for paracoccidioidomycosis diagnosis was developed. The primers ITS1 and ITS4 were used in the first reaction, while the primers MJ03 and ITS1 primer were used in the second reaction. The semi-nested PCR was used to investigate biopsies of five patients with oral lesions that resembled paracoccidioidomycosis. RESULTS: The semi-nested PCR was positive for four samples and negative for a sample from a patient later diagnosed with leishmaniasis. CONCLUSIONS: The new semi-nested PCR describe is useful for aracoccidioidomycosis diagnosis. ________________________________________________________________________________________________________________ RESUMO INTRODUÇÃO: A paracoccidioidomicose é uma infecção sistêmica causada pelo Paracoccidioides brasiliensis. MÉTODOS: Neste estudo, uma semi-nested PCR foi desenvolvida para o diagnóstico da paracoccidioidomicose. Os oligonucleotídeos iniciadores ITS1 e ITS4 foram usados na primeira reação, enquanto os oligonucleotídeos iniciadores MJ03 e ITS1 foram usados na segunda reação. A semi-nested PCR foi usada para investigar biopsias de cinco pacientes com lesões orais que se assemelhavam a paracoccidioidomicose. RESULTADOS: A semi-nested PCR foi positiva para quatro amostras e negativa para a amostra de um paciente, posteriormente diagnosticado com leishmaniose. CONCLUSÕES: A semi-nested PCR descrita aqui é útil para o diagnóstico da paracoccidioidomicose.

The dimorphic fungus Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, a human granulomatous disease. Recently the first case of natural disease in dogs was reported. The complement system is an important effector component of humoral immunity against infectious agents. Therefore, the aim of this study was to evaluate the activation of the dog alternative complement pathway by P. brasiliensis. Initially, the ability of erythrocytes of guinea pig, rabbit, sheep, chicken and swine to activate the dog alternative pathway was evaluated. The guinea pig erythrocytes showed the greatest capacity to activate dog alternative pathway. The alternative (AH50) hemolytic activity was evaluated in 27 serum samples from healthy dogs and the mean values were 87.2 AH50/ml. No significant differences were observed in relation to sex and age. The alternative pathway activation by P. brasiliensis was higher in serum samples from adult dogs when compared to puppies and aged dogs (p ≤ 0.05). This is the first report of dog alternative complement pathway activation by P. brasiliensis and suggests that it may play a protective role in canine paracoccidioidomycosis.

In this work, two accurate and sensitive real-time polymerase chain reaction (PCR) assays to differentiate pathogenic Cryptococcus gattii sensu lato (s.l.) and C. neoformans sensu lato (s.l.) targeting the intergenic spacer 1 (IGS1) region from rDNA locus were developed. Specific primers were designed based on their IGS1 sequence analyses and the optimal real-time PCR assays showed that the dissociation curves generated two different melting peaks, at 82.8 and 84.2ºC for C. gattii s.l. and C. neoformans s.l., respectively. No amplifications were observed in the negative template control. The minimum limit of detection of both primers was 100 plasmid copies per reaction, and they were highly specific when tested with a range of fungal DNAs. Overall, the results showed that the designed primers completely differentiated C. gattii s.l. and C. neoformans s.l. from clinical and environmental sources with great accuracy when compared to phenotypic identification, with no cross-reactivity to other fungal DNA.