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Abstract: The mutated VAPBP56S (vesicle B associated membrane protein – P56S) protein has been described in a Brazilian family and classified as Amyotrophic Lateral Sclerosis type 8 (ALS8). We aimed to study altered biochemical and immunological parameters in cells from ALS8 patients to identify possible biomarkers or therapeutic targets. Wild-type VAPB, VAPBP56S, mTOR, pro-inflammatory cytokines, and oxidant/reducing ELISA, fluorimetry, and spectrophotometry measured balance levels in serum, leucocytes, and cellular lysate from ALS8 patients and Health Controls. Our results showed similar levels of mutant and wild-type VAPB in serum and intracellular lysate (p >0.05) when ALS8 patients and Controls were compared. IL-1β, IL-6, and IL-18 levels in patients and Controls showed no difference, suggesting an absence of peripheral inflammation (p > 0.05). Oxidative metabolic response, assessed by mitochondrial ROS production, and reductive response by MTT reduction, were higher in the ALS8 group compared to Controls (p < 0.05), although not characterizing typical oxidative stress in ALS8 patients. Total mTOR levels (phosphorylated or non-phosphorylated) of ALS8 patients were significantly lower in serum and higher in intracellular lysate than the mean equivalents in Controls (p < 0.05). A similar result was observed when we quantified the phosphorylated protein (p < 0.05). We demonstrate the possibility of using these biochemical and immunological parameters as potential therapeutic targets or biomarkers. Furthermore, by hypothesis,we suggest a hormetic response in which both VAPB forms could coexist in different proportions throughout life. The mutated VAPBP56S production would increase with aging and predominate over the wild-type VAPB levels, determining the onset of symptoms and aggravating the disease.

Background: Type 2 diabetes (DM2) and chronic kidney disease (CKD) are inflammatory pathologies. Diabetes is characterized by hyperglycemia and CKD by the gradual and irreversible loss of kidney function. Both diseases develop oxidative stress, and reactive oxygen species (ROS) play a pivotal role in the pathogenesis. This study aimed to determine ROS production by granulocytes from renal patients (CKD) with or without diabetes. Methods: Granulocytes from patients with DM2, CKD, CKD-DM2, and healthy controls were purified using the Ficoll-Hypaque gradient method. Granulocyte ROS generation in the absence or the presence of PDB (an activator of NADPH-oxidase) or Concanavalin A (Toll- receptor 3,9 activator) was evaluated in a luminol-dependent chemiluminescence method. The cell-free DNA in the serum of DM2, CKD, and CKD-DM2 patients was measured by the fluorescence method before and after hemodialysis. Results: Our results show a significant increase in ROS production by granulocytes from patients with CKD, DM2, and CKD-DM2 compared to healthy control (p

Background High mobility group box 1 (HMGB1) has been studied as a molecule associated with severe outcomes in sepsis and thrombomodulin (TM) seems to decrease HMGB1 activity. Aim To investigate the role of the thrombomodulin/high mobility group box 1 (T/H) ratio in patients with sepsis and their association with their clinic, testing the hypothesis that higher ratios are associated with better outcomes. Methods Twenty patients diagnosed with sepsis or septic shock, according to the 2016 criteria sepsis and septic shock (Sepsis-3), were studied. Patients were followed until they left the intensive care unit or until they achieved 28 d of hospitalization (D28). The following clinical outcomes were observed: Sequential Organ Failure Assessment (SOFA) score; Need for mechanical pulmonary ventilation; Presence of septic shock; Occurrence of sepsis-induced coagulopathy; Need for renal replacement therapy (RRT); and Death. Results The results showed that patients with SOFA scores greater than or equal to 12 points had higher serum levels of TM: 76.41 ± 29.21 pg/mL vs 37.41 ± 22.55 pg/mL among those whose SOFA scores were less than 12 points, P = 0.003. The T/H ratio was also higher in patients whose SOFA scores were greater than or equal to 12 points, P = 0.001. The T/H ratio was, on average, three times higher in patients in need of RRT (0.38 ± 0.14 vs 0.11 ± 0.09), P Conclusion Higher serum levels of TM and, therefore, higher T/H ratio in the first 24 h after the diagnosis of sepsis were associated with more severe disease and the need for renal replacement therapy, while those with better clinical outcomes and those who were discharged before D28 showed a tendency for lower T/H ratio values.

Background: Galanin (GAL) constitutes a family of neuropeptides composed of four peptides: (i) galanin (GAL), (ii) galanin-message associated peptide (GAMP), (iii) galanin-like peptide (GALP), and (iv) alarin. GAL contains 29/30 amino acids, and its biological action occurs through the interactions with its various receptors (GALR1, GALR2, and GALR3). The neuropeptide GAL regulates several physiological and pathophysiological functions in the central nervous system, the peripheral nervous system, and the peripheral organs. GAL is secreted mainly by oligodendrocytes, astrocytes, and the gastrointestinal tract, and its effect depends on the interaction with its different receptors. These receptors are expressed mainly in the central, peripheral nervous systems and the intestines. Objective: The present review evaluates the role of GAL family in inflammatory diseases. An overview is given of the signaling and pharmacological effects due to the interaction between GAL and GALR in different cell types. The potential use of GAL as a therapeutic resource is critically discussed. Conclusion: GAL is suggested to have an anti-inflammatory function in some situations and a proinflammatory function in others. The literature on GAL is controversial and currently not conclusive. This could be due to the complexity of the metabolic network signaling induced by the interactions between GAL and GALR. In the next future, GAL might be a promising therapeutic resource for several diseases, but its practical use for disease control is presently not advisable.

Background: Paracoccidioidomycosis (PCM) is a systemic mycosis caused by dimorphic fungi Paracoccidioides brasiliensis and Paracoccidioides lutzii. It is prevalent in Latin American, mainly in Brazil. Therefore, PCM has fundamental impact on the Brazilian global economy, especially in public health system, since it is affecting economical active population in different country regions. Objective: The present study aimed to standardize the Real Time-Polymerase Chain Reaction (rt-PCR) for an efficient and safe PCM diagnosis amplifying the recombinant protein PB27 gene, only expressed by specimens of Paracoccidioides genus. Methods: To standardize a methodology of rt-PCR using species-specific primers and probe designed for annealing in this specific region of the fungi´s genome, amplifying the recombinant protein PB27 gene, only expressed by specimens of Paracoccidioides genus. Followed by design in silico, experiments were performed in vitro to determine rt-PCR specificity, efficiency and genome detection limit. Results: The primers and probe sequences were deposited in Brazilian Coordination of Technological Innovation and Transfer (CTIT), under patent reference number BR1020160078830. The present study demonstrated the rt-PCR applicability for support on diagnosis of paracoccidioidomycosis, presenting low cost, which makes it affordable for public health services in developing countries as Brazil. It is noteworthy that it is necessary to validate this methodology using clinical samples before to use as a safe method of diagnosis. A review of all patents related to this topic was performed and it was shown that, to date, there are no records of patent on kits for paracoccidioidomycosis´s diagnostic. Indeed, there is still a lot to go to reach this goal. Conclusion: The reaction developed was standardized and patented, opening perspectives to molecular diagnosis development for paracoccidioidomycosis, since rt-PCR can be applied to a broad spectrum of infectious diseases. It would need to be tested in biological samples in order to validate this method and then generate a diagnostic kit for Paracoccidioidomycosis.

Visceral leishmaniasis (VL) is a zoonosis caused by the protozoa of the genus Leishmania. Among the species, L. infantum and/or L. infantum (chagasi) are the most important species affecting the Americas. Domestic dogs are the main reservoir of the parasite and participate effectively in the parasite' transmission cycle. The Canine Visceral Leishmaniasis Control Program (PCLV) adopted in Brazil present as strategies the vector control, health education and serological diagnosis of CVL in dogs followed by culling of the seropositive ones. The resolution to eliminate seropositive dogs by euthanasia, when necessary, are the most controversial and least accepted by society. The diagnostic methods for canine visceral leishmaniasis, currently indicated and approved in Brazil by the Ministry of Health from Brazil are the Dual Path Platform (DPP)® as a screening test and the Enzyme immunoassay test (ELISA®). This study aimed to verify the presence of Leishmania spp. DNA in peripheral blood samples of dogs presenting positive serological results byDPP® and ELISA® tests,throughreal-time polymerase chain reaction (rt-PCR), using the pair of primers 150–152 already described. For this purpose, were collected blood samples from 185 seropositive dogs among them, 41 (22%) exhibited some clinical signal of disease, whereas 144 (78%) was asymptomatic. The animals were also analyzed according to gender, race and hair size. According to the results of rt-PCR, it was observed that among the185 seropositive dogs analyzed, only 132 (71%) presented positive results for CVL and 53 (29%) presented negative results. From this, 41/41 symptomatic dogs were positive (100%), while among the asymptomatic dogs, 91/144 were positive (63, 2%) and 53/144 were negative (36, 8%). Concerning the hair size of seropositive dogs, we found that 41 (22%) had long hair, while 144 (78%) had short hair. No statistical significance occurred between the results of rt-PCR, ELISA and DPP tests and the profile of the animals (gender, size of the dogs and hair size), probably due to the small number of samples and the sampling differences of each profile. But statistical significance occurred between the results of rt-PCR and the clinical evaluation, since the rt-PCR was positive in all symptomatic dogs. Thus, through these results, we reached at the following question, which may contribute to an important current debate: the dogs presenting CVL seropositive diagnosis confirmed by tests distributed by the Ministry of Health were in reality ill or were they seropositive by living in an endemic area of the disease? Would these asymptomatic seropositive dogs spread the disease to the inhabitants even presenting a low parasite charge circulating in the blood.

Leishmaniasis is considered a neglected tropical disease having a worldwide distribution. The disease is caused by protozoa belonging to the genus Leishmania, being clinically divided into visceral leishmaniasis (VL), cutaneous leishmaniasis (CL) and mucocutaneous leishmaniasis (MCL). Domestic dogs are the main parasite reservoir and effectively participate in the protozoa transmission. The human leishmaniasis treatment is based on a selection of therapeutic compounds, but the available drugs are toxic, presenting adverse side effects. The decision to treat or not to treat seropositive dogs is under discussion in some countries, because treatment is even more toxic to animals and, also, can generate drug resistant strains. Therefore, very few treatment choices have reached the clinic for this disease and there is an urgent need of new chemotherapy for both humans and animals. This study presents a review of new patents related to treatment and prevention of Leishmania infections. Some of these patents are related to new vaccine formulations for combating leishmaniasis. Likewise, the inventions related to the cream formulations for the treatment of cutaneous leishmaniasis are very important, avoiding the side effects of drugs during the treatment. Furthermore, anti leishmaniasis products that are extracted from nature has increasingly been patented each year, demonstrating the importance of bioprospection studies to improve the armamentarium of anti leishmaniasis drugs.

Paracoccidioidomycosis (PCM) is a chronic mycosis caused by the saprobic and dimorphic species Paracoccidioides brasiliensis and P. lutzii. This disease is prevalent in Latin American countries. PCM appears as a relevant concern and challenge for the mycologists, since until now there is no a methodology suitable for an efficient and safe diagnosis and species identification. Thus, the present study aimed to validate a methodology for PCM´s diagnosis, using quantitative Polymerase Chain Reaction (qPCR) through target amplification of the gene encoding the recombinant protein Pb27, a common protein to the both species Paracoccidioides brasiliensis and P. lutzii. The experiments were performed in vitro to determine the specificity, efficiency and detection limit of qPCR assay, using specific primers and probe, which sequences were subject to a patent deposited in Brazilian CTIT, under the registration number: BR1020160078830. According to the results the technique showed sensitivity of 94% and specificity of 100%, demonstrating that it will be possible to develop a new fast and safe diagnostic PCM and can be standardized in order to present a low cost, accessible to the patient served by the public health system in Brazil and Latin America.

Subcutaneous phaeohyphomycosis is a form of infection caused by dematiaceous fungi and frequently reported in the literature. It is considered as an emerging mycosis because it is increasingly reported among immunosuppressive patients. The most commonly cited etiologic agent is Exophiala jeanselmei, followed by Alternaria spp. We present a case of a 48-year-old woman, with a history of lepromatous leprosy, using corticosteroid in immunosuppressive doses due to a type 2 repetitive reaction leprosy outbreak, revealing subcutaneous phaeohyphomycosis lesions. The diagnosis was confirmed by fine-needle aspiration of the secretion, with subsequent molecular analysis, and culture and direct mycological analyses.

In this study, we report four cases of Histoplasma capsulatum infection in eight biologists who made an expedition to determine the prevalence of this fungus in a cave localized in the northwest of Minas Gerais state, Brazil. This case report demonstrates the importance of evaluating the H. capsulatum presence in Brazilian caves before opening to public visitations.

The diagnosis of visceral leishmaniasis (VL) is a challenging issue and several studies worldwide have evaluated the different tools to reach a diagnostic solution. The polymerase chain reaction (PCR) has proven to be effective in detecting the genome ofLeishmaniaspecies in different biological samples. In this study, we compared the conventional PCR and real-time PCR using the Sybr Green system and their application in molecular diagnosis of visceral leishmaniasis in peripheral blood as a biological sample. The genus-specific conserved region of kinetoplast DNA (kDNA) was the target of amplification. We studied 30 samples from patients with suspect of visceral leishmaniasis who were treated by the Medical Clinic of Santa Casa de Belo Horizonte Hospital, Brazil. Among the samples studied, 19 had a confirmed diagnosis for VL by serology and/or by clinical findings. Among these 19 samples, 63% (n=12) presented positive results for serology and 79% (n=15) positive results in both PCR methodologies. This fact suggests that the PCR technique can assist in the diagnosis of visceral leishmaniasis in patients who do not have detectable antibodies by serology but can present the genome of the parasite circulating in whole blood. Also, it was possible to observe that there was conformity between the results of the techniques of cPCR and qPCR using the Sybr Green system in 100% of samples analyzed. These data suggest that both PCR techniques were equally effective for detection of the genome of the parasite in the patient’s blood.

Introduction: invasive aspergillosis (AI) is a serious fungal infection caused by species of the genus Aspergillus that primarily affects leukemic and diabetic patients and those recipients of stem cell transplants, which have neutropenia. The fungi spores that colonize the lung epithelium may invade the endothelial cell lining and vascular access and thus, spread to other organs through the blood. The high mortality of the disease is related to severe immunosuppression, rapid infection progression, and especially lack of an early and efficient diagnosis. Therefore, the diagnosis in the initial infection phase is beneficial, providing a more effective therapy that can reduce the disease’s mortality rate. Objective: this study aimed at evaluating the applicability of the polymerase chain reaction (PCR) Diagnosis of invasive aspergillosis: Application of Polymerase Chain Reaction Techniques (PCR) and Enzyme-Linked Immunosorbent Assay for Detection of Galactomannan (EIA-GM) Alisson Brandao Ferreira1, Claudia Barbosa Assuncao2, Thamara Thascila Silva Silveira2, Raquel Acaiah2, Antonio Tarcisio Faria Freire3, Jorge Luiz Saliba3, Sonia Maria de Figueiredo4, Fabiana Rocha-Silva5, Rachel Basques Caligiorne6 1 Farmaceutico. Mestre em Biomedicina. Instituto de Ensino e Pesquisa da Santa Casa de Belo Horizonte – IEP/SCBH. Belo Horizonte, MG – Brasil. 2 Biologa. Bolsista de Iniciacao Cientifica. IEP/SCBH. Belo Horizonte, MG – Brasil. 3 Medico. Coordenador do Servico de Controle de Infeccao Hospitalar – SCBH. Belo Horizonte, MG – Brasil. 4 Nutricionista. Doutora pela Pos-Graduacao em Biomedicina do IEP/SCBH. Belo Horizonte, MG – Brasil. 5 Biologa. Mestre em Biomedicina. IEP/SCBH. Belo Horizonte, MG – Brasil. 6 Biologa. Doutora em Ciencias. IEP/SCBH. Belo Horizonte, MG – Brasil. Apoio: CNPq – Conselho Nacional de Pesquisa e Desenvolvimento Cientifico e FAPEMIG – Fundacao de Pesquisa do Estado de Minas Gerais. Diagnostico da aspergilose invasiva: Aplicacao das Tecnicas de Reacao em Cadeia da Polimerase (PCR) e Ensaio Imunoenzimatico de Deteccao da Galactomanana (EIA-GM®) DOI: 10.5935/2238-3182.20150076

Paracoccidioidomycosis, despite being the most important deep mycosis in Latin America, still has many blindspots in terms of its approach, especially in relation to duration of treatment, cure control and prophylaxis. Depending on severity, the following can be used in the treatment: sulfonamides, azoles (itraconazole and ketoconazole), and amphotericin. The prognosis depends on severity, time between onset and diagnosis, and therapy instituted. In mild forms, prognosis is good; in moderate and severe forms, for which there is risk of developing sequelae and death, it is guarded.

The diagnosis of paracoccidioidomycosis requires epidemiological data to be available and for the presence of some more typical clinical manifestations.It requires complementary investigation with interventional methods, differential diagnosis of pathologies of great importance such as tuberculosis and lymphomas, and cure control. This update discusses the advances in these various areas, which include complementary investigation, differential diagnosis and cure control, pointing to development prospects that may help better define the best approach to this disease.

Background The topical application of mitomycin C has been evaluated as a complementary therapy for eosinophilic nasal polyposis (ENP). However, the mechanism underlying the additional benefits of mitomycin C for the control of eosinophilic inflammation and prevention of posttherapeutic relapse remains to be elucidated. In this work, the aim was to characterize the gene expression profile by quantitative real-time polymerase chain reaction (qPCR) of proinflammatory and regulatory biomarkers that are typically associated with ENP and to assess the impact of the topical application of mitomycin C on the nasal mucosal tissue immunologic milieu after ENP surgery. Methods We have selected 20 patients with ENP that were recommended to undergo surgical intervention. Normal mucosal tissue was obtained from healthy nasal mucosa from six patients with absence of eosinophilic infiltration. To test the effect of mitomycin C, one side of the maxillary sinus mucosa was selected for topical application of this drug and the other received no further treatment and acted as the control. The genes interleukin-4 (IL-4), IL-5, IL-10, IL-13, chemokine (C-C motif) ligand 5 (CCL5), CCL24, colony-stimulating factor 2 (CSF2), transforming growth factor beta 1 (TGFB1), tumor necrosis factor alpha (TNF-alpha), and beta actin (ACTB) were selected for gene expression analysis by qPCR. Results The data showed higher expression of proinflammatory biomarkers and lower levels of regulatory TGFB1 transcripts in ENP mucosal tissue. Surgery with topical application of mitomycin C induced a prominent transcriptional down-regulation of the immunologic biomarkers, CCL24, TNF-alpha, CSF2, and IL-5, in ENP mucosal tissue. Additionally, this treatment restored the levels of chemokines and cytokines to those observed in the nasal mucosal tissue of control subjects, except for TGFB1, which remained below the reference pattern. Moreover, CSF2 was identified as a putative biomarker with significant predictive value for complementary prophylactic purposes after surgery in ENP patients. Conclusion After the characterization of the expression signatures of immunologic biomarkers in ENP, we observed that the topical use of mitomycin C is important for the reestablishment of the immunologic microenvironment of a normal expression profile of biomarkers involved in ENP mucosal tissue.