The genetic landscape of mitochondrial diseases in the next-generation sequencing era: a Portuguese cohort study.

Introduction: Rare disorders that are genetically and clinically heterogeneous, such as mitochondrial diseases (MDs), have a challenging diagnosis. Nuclear genes codify most proteins involved in mitochondrial biogenesis, despite all mitochondria having their own DNA. The development of next-generation sequencing (NGS) technologies has revolutionized the understanding of many genes involved in the pathogenesis of MDs. In this new genetic era, using the NGS approach, we aimed to identify the genetic etiology for a suspected MD in a cohort of 450 Portuguese patients. Methods: We examined 450 patients using a combined NGS strategy, starting with the analysis of a targeted mitochondrial panel of 213 nuclear genes, and then proceeding to analyze the whole mitochondrial DNA. Results and Discussion: In this study, we identified disease-related variants in 134 (30%) analyzed patients, 88 with nuclear DNA (nDNA) and 46 with mitochondrial DNA (mtDNA) variants, most of them being pediatric patients (66%), of which 77% were identified in nDNA and 23% in mtDNA. The molecular analysis of this cohort revealed 72 already described pathogenic and 20 novel, probably pathogenic, variants, as well as 62 variants of unknown significance. For this cohort of patients with suspected MDs, the use of a customized gene panel provided a molecular diagnosis in a timely and cost-effective manner. Patients who cannot be diagnosed after this initial approach will be further selected for whole-exome sequencing. Conclusion: As a national laboratory for the study and research of MDs, we demonstrated the power of NGS to achieve a molecular etiology, expanding the mutational spectrum and proposing accurate genetic counseling in this group of heterogeneous diseases without therapeutic options.

Toxigenic characterization, spoilage potential, and antimicrobial susceptibility of coagulase-positive Staphylococcus species isolated from Minas Frescal cheese.

This study aimed to identify coagulase-positive staphylococci (CPS) species from 21 samples of clandestine Minas Frescal cheese, investigate the potential for deterioration in psychrotrophic and mesophilic conditions, verify the toxigenic potential of Staphylococcus aureus, and determine the antimicrobial susceptibility profile of toxigenic S. aureus. Species determination was performed based on the detection of ß-hemolysis in 5% ovine blood agar; fermentation of mannitol, maltose, and trehalose sugars; and production of acetoin. After species determination, DNA extraction and analysis was performed for S. aureus colonies for genes encoding staphylococcal toxins (eta, etb, tst, sea, seb, sec, sed, and see) using 2 multiplex PCR assays. Isolates identified as toxigenic S. aureus were tested for antimicrobial susceptibility to tetracycline, erythromycin, clindamycin, gentamicin, ciprofloxacin, sulfazotrim, trimethoprim, streptomycin, cefoxitin, vancomycin and enrofloxacin. Elevated CPS counts were observed with an average of >6 log cfu/g. Of the 355 isolates, 177 (49.86%) were identified as S. aureus. Staphylococcus hyicus, Staphylococcus intermedius, Staphylococcus delphini, and Staphylococcus coagulans were identified in 3 (0.84%), 2 (0.56%), 2 (0.56%), and 1 (0.28%) isolates, respectively. Of the total number of S. aureus, 25 (52.08%) were positive for the gene that encodes for toxic shock toxin (TSST-1). Another 16 (33.33%) were positive for the sea gene, and 4 isolates (8.33%) were positive for see and one isolate each was positive for seb (2.08%), sec (2.08%), and etb (2.08%) genes. All isolates demonstrated lipolytic activity under mesophilic and psychrotrophic conditions. S. intermedius and S. hyicus had the most prominent proteolytic potential. Multidrug resistance was observed in most of the potentially toxigenic isolates, with clindamycin having the lowest efficiency (40%), whereas the aminoglycosides (gentamicin and streptomycin) had the highest effectiveness demonstrating inhibition in all evaluated isolates. Methicillin-resistant S. aureus (MRSA) was detected. Minas Frescal cheeses, marketed in the north of Tocantins in the Brazilian Amazon region, do not comply with legal quality standards and pose a public health risk due to the enterotoxigenic potential of multiresistant isolates, in addition to low shelf life of the samples given the high spoilage potential of this microbiota.

Sustained PGC-1α2 or PGC-1α3 expression induces astrocyte dysfunction and degeneration.

Peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) transcriptional coactivators are key regulators of energy metabolism-related genes and are expressed in energy-demanding tissues. There are several PGC-1α variants with different biological functions in different tissues. The brain is one of the tissues where the role of PGC-1α isoforms remains less explored. Here, we used a toxin-based mouse model of Parkinson's disease (PD) and observed that the expression levels of variants PGC-1α2 and PGC-1α3 in the nigrostriatal pathway increases at the onset of dopaminergic cell degeneration. This increase occurs concomitant with an increase in glial fibrillary acidic protein levels. Since PGC-1α coactivators regulate cellular adaptive responses, we hypothesized that they could be involved in the modulation of astrogliosis induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Therefore, we analysed the transcriptome of astrocytes transduced with expression vectors encoding PGC-1α1 to 1α4 by massively parallel sequencing (RNA-seq) and identified the main cellular pathways controlled by these isoforms. Interestingly, in reactive astrocytes the inflammatory and antioxidant responses, adhesion, migration, and viability were altered by PGC-1α2 and PGC-1α3, showing that sustained expression of these isoforms induces astrocyte dysfunction and degeneration. This work highlights PGC-1α isoforms as modulators of astrocyte reactivity and as potential therapeutic targets for the treatment of PD and other neurodegenerative disorders.

Cholesterol Exacerbates the Pathophysiology of Non-Alcoholic Steatohepatitis by Upregulating Hypoxia-Inducible Factor 1 and Modulating Microcirculatory Dysfunction.

Cholesterol is a pivotal lipotoxic molecule that contributes to the progression of Non-Alcoholic Steatohepatitis NASH). Additionally, microcirculatory changes are critical components of Non-Alcoholic Fatty Liver Disease (NAFLD) pathogenesis. This study aimed to investigate the role of cholesterol as an insult that modulates microcirculatory damage in NAFLD and the underlying mechanisms. The experimental model was established in male C57BL/6 mice fed a high-fat high-carbohydrate (HFHC) diet for 39 weeks. Between weeks 31-39, 2% cholesterol was added to the HFHC diet in a subgroup of mice. Leukocyte recruitment and hepatic stellate cells (HSC) activation in microcirculation were assessed using intravital microscopy. The hepatic microvascular blood flow (HMBF) was measured using laser speckle flowmetry. High cholesterol levels exacerbated hepatomegaly, hepatic steatosis, inflammation, fibrosis, and leukocyte recruitment compared to the HFHC group. In addition, cholesterol decreased the HMBF-cholesterol-induced activation of HSC and increased HIF1A expression in the liver. Furthermore, cholesterol promoted a pro-inflammatory cytokine profile with a Th1-type immune response (IFN-γ/IL-4). These findings suggest cholesterol exacerbates NAFLD progression through microcirculatory dysfunction and HIF1A upregulation through hypoxia and inflammation. This study highlights the importance of cholesterol-induced lipotoxicity, which causes microcirculatory dysfunction associated with NAFLD pathology, thus reinforcing the potential of lipotoxicity and microcirculation as therapeutic targets for NAFLD.

Pro-resolving role of glucagon in lipopolysaccharide-induced mice lung neutrophilia.

Prior research demonstrated that glucagon has protective roles against inflammation, but its effect on the resolution of inflammation remains elusive. Using in vitro and in vivo approaches, this study aimed to investigate the pro-resolving potential of glucagon on pulmonary neutrophilic inflammation caused by lipopolysaccharide. Lipopolysaccharide induced an increase in the proportions of neutrophils positives to glucagon receptor (GcgR) in vitro. In addition, lipopolysaccharide induced an increase in the neutrophil accumulation and expression of GcgR by the inflammatory cells in the lungs, however, without altering glucagon levels. Intranasal treatment with glucagon, at the peak of neutrophilic inflammation, reduced the neutrophil number in the bronchoalveolar lavage (BAL), and lung tissue within 24 h. The reduction of neutrophilic inflammation provoked by glucagon was accompanied by neutrophilia in the blood, an increase in the apoptosis rate of neutrophils in the BAL, enhance in the pro-apoptotic Bax protein expression, and decrease in the anti-apoptotic Bcl-2 protein levels in the lung. Glucagon also induced a rise in the cleavage of caspase-3 in the lungs; however, it was not significant. Glucagon inhibited the levels of IL-1ß and TNF-α while increasing the content of pro-resolving mediators transforming growth factor (TGF-ß1) and PGE2 in the BAL and lung. Finally, glucagon inhibited lipopolysaccharide-induced airway hyper-reactivity, as evidenced by the reduction in lung elastance values in response to methacholine. In conclusion, glucagon-induced resolution of neutrophilic inflammation by promoting cessation of neutrophil migration and a rise of neutrophil apoptosis and the levels of pro-resolving mediators TGF-ß1 and PGE2.

Gasoline discrimination using infrared spectroscopy and virtual samples based on measurement uncertainty.

In a previous work, we proposed a methodology for pair-wise discrimination of gasoline samples by creating virtual samples based on physicochemical assays or distillation curves. Satisfactory results were achieved, although specialist and specific apparatus (not commonly available at police laboratories) were required. The present study goes a step further and for the first time investigates the possibility of infrared (IR) spectroscopy to enable a virtual samples-based methodology for comparison of gasoline samples in pairs. IR spectroscopy feasibility for in situ applications is attractive for forensic investigations. The performances of one handheld NIR device and one dual-range (FT-NIR and FT-IR) benchtop spectrometer were evaluated. The estimation of uncertainty in infrared spectral measurement (needed to generate virtual samples) is barely discussed in literature. So far, there are no literature reports describing quantification and comparison of measurement uncertainties for the spectral acquisitions evaluated here, especially regarding their use for generating virtual samples. A stepwise procedure to quantify uncertainties associated with IR spectral acquisition, at each wavenumber, is described. This method can be useful for understanding both the sources of variability in IR measurements and the system under investigation. Uncertainty estimation was based on experimental data and considered intermediate precision, repeatability and variations in sample temperature as sources of variability. Virtual samples were employed in a discrimination approach using SIMCA models. Results for portable NIR, FT-NIR and FT-IR data sets showed complete discrimination for 96.3%, 93.4% and 93.7% of the 1431 pairs of gasoline samples evaluated, respectively. These results were comparable and similar to those obtained for the physicochemical properties data set (95.7%), although slightly inferior to the result obtained for distillation curves (99.2%). Using IR non-destructive methods in this case could enable faster investigations and simpler analysis, especially for the low-cost handheld spectrometer. In a screening approach, atmospheric distillation assays can be employed only if infrared techniques are not capable of distinguishing the samples subject to comparison. In this work, a pair of samples was considered to be completely discriminated only when a null false positive error (FPR) was achieved, although a more flexible criterium may be acceptable in practice. Finally, the methodology could be extended to other applications where sample comparison is important.

Microbiota and Nod-like receptors balance inflammation and metabolism during obesity and diabetes.

Gut microbiota influence host immunity and metabolism during obesity. Bacterial sensors of the innate immune system relay signals from specific bacterial components (i.e., postbiotics) that can have opposing outcomes on host metabolic inflammation. NOD-like receptors (NLRs) such as Nod1 and Nod2 both recruit receptor-interacting protein kinase 2 (RIPK2) but have opposite effects on blood glucose control. Nod1 connects bacterial cell wall-derived signals to metabolic inflammation and insulin resistance, whereas Nod2 can promote immune tolerance, insulin sensitivity, and better blood glucose control during obesity. NLR family pyrin domain containing (NLRP) inflammasomes can also generate divergent metabolic outcomes. NLRP1 protects against obesity and metabolic inflammation potentially because of a bias toward IL-18 regulation, whereas NLRP3 appears to have a bias toward IL-1ß-mediated metabolic inflammation and insulin resistance. Targeting specific postbiotics that improve immunometabolism is a key goal. The Nod2 ligand, muramyl dipeptide (MDP) is a short-acting insulin sensitizer during obesity or during inflammatory lipopolysaccharide (LPS) stress. LPS with underacylated lipid-A antagonizes TLR4 and counteracts the metabolic effects of inflammatory LPS. Providing underacylated LPS derived from Rhodobacter sphaeroides improved insulin sensitivity in obese mice. Therefore, certain types of LPS can generate metabolically beneficial metabolic endotoxemia. Engaging protective adaptive immunoglobulin immune responses can also improve blood glucose during obesity. A bacterial vaccine approach using an extract of the entire bacterial community in the upper gut promotes protective adaptive immune response and long-lasting improvements in blood glucose control. A key future goal is to identify and combine postbiotics that cooperate to improve blood glucose control.

Evaluation of bone repair with platelet-rich fibrin following the extraction of impacted third molars - randomized clinical trial.

BACKGROUND: The aim of the present study was to evaluate postoperative effects of platelet-rich fibrin (PRF) in wound and bone healing, pain, swelling and periodontal complications outcomes after impacted third molars extraction. MATERIAL AND METHODS: A prospective, randomized, split-mouth, double-blind clinical trial was conducted. PRF was placed within sockets following tooth removal and before suturing mucoperiosteal flap while no treatment was performed on control group's sockets. Patients were evaluated considering bone volume which was obtained in the 90-day postoperative period. Other variables included trabecular thickness, trabecular distance and grey values, pain, swelling, and wound healing. A Wilcoxon test and a t-Student test were used at a 5% significance level and a Friedman test was used to multiple comparisons. RESULTS: Forty-four surgeries were performed in the present study. The patients' mean age was 22.41 (± 2.75 years) and 72.73% were women. PRF was associated to increased trabecular thickness and bone volume means (p < 0.001). The experimental group had significantly lower pain scores at 4h, 6h, 8h, 16h, 24h, and 72h (p ˂ 0.05). Mean swelling was lower on the experimental group (p < 0.001). The PRF group showed significant higher wound healing (p ˂ 0.001). CONCLUSIONS: Alveolar filling with PRF improves wound and bone healing after extractions while also decreasing pain and swelling in the postoperative period.

Effect of the renin-angiotensin system on the exacerbation of adrenal glucocorticoid steroidogenesis in diabetic mice: Role of angiotensin-II type 2 receptor.

Prior investigation shows an increase in the activity of both hypothalamus-pituitary-adrenal (HPA) axis and the renin-angiotensin system (RAS) in diabetic patients. Moreover, activation of angiotensin-II type 1 receptor (AT1) has been associated with adrenal steroidogenesis. This study investigates the role of RAS on the overproduction of corticosterone in diabetic mice. Diabetes was induced by intravenous injection of alloxan into fasted Swiss-webster mice. Captopril (angiotensin-converting enzyme inhibitor), Olmesartan (AT1 receptor antagonist), CGP42112A (AT2 receptor agonist) or PD123319 (AT2 receptor antagonist) were administered daily for 14 consecutive days, starting 7 days post-alloxan. Plasma corticosterone was evaluated by ELISA, while adrenal gland expressions of AT1 receptor, AT2 receptor, adrenocorticotropic hormone receptor MC2R, pro-steroidogenic enzymes steroidogenic acute regulatory protein (StAR), and 11ß-hydroxysteroid dehydrogenase type 1 (11ßHSD1) were assessed using immunohistochemistry or western blot. Diabetic mice showed adrenal gland overexpression of AT1 receptor, MC2R, StAR, and 11ßHSD1 without altering AT2 receptor levels, all of which were sensitive to Captopril or Olmesartan treatment. In addition, PD123319 blocked the ability of Olmesartan to reduce plasma corticosterone levels in diabetic mice. Furthermore, CGP42112A significantly decreased circulating corticosterone levels in diabetic mice, without altering the overexpression of MC2R and StAR in the adrenal glands. Our findings revealed that inhibition of both angiotensin synthesis and AT1 receptor activity reduced the high production of corticosterone in diabetic mice via the reduction of MC2R signaling expression in the adrenal gland. Furthermore, the protective effect of Olmesartan on the overproduction of corticosterone by adrenals in diabetic mice depends on both AT1 receptor blockade and AT2 receptor activation.

Records of Human Deaths from Echinococcosis in Brazil, 1995-2016.

Echinococcosis is a zoonotic disease relevant to public health in many countries, on all continents except Antarctica. The objective of the study is to describe the registered cases and mortality from echinococcosis in Brazil, from 1995 to 2016. The records of two national databases, the Hospital Information System (HIS) and the Mortality Information System (MIS), were accessed during the period of 1995-2016. Demographic, epidemiological, and health care data related to the occurrence of disease and deaths attributed to echinococcosis in Brazil are described. The results showed that 7955 records of hospitalizations were documented in the HIS, during the study period, with 185 deaths from echinococcosis, and 113 records of deaths were documented in the MIS Deaths in every state of Brazil in the period. When comparing between states, the HIS showed great variability in mortality rates, possibly indicating heterogeneity in diagnosis and in the quality of health care received by patients. Less severe cases that do not require specialized care are not recorded by the information systems, thus the true burden of the disease could be underrepresented in the country. A change in the coding of disease records in the HIS in the late 1990s, (the integration of echinococcosis cases with other pathologies), led to the loss of specificity of the records. The records showed a wide geographic distribution of deaths from echinococcosis, reinforcing the need to expand the notification of the disease in Brazil. Currently, notification of cases is compulsory in the state of Rio Grande do Sul.

Chalcones with potential antibacterial and antibiofilm activities against periodontopathogenic bacteria.

OBJECTIVES: Periodontitis is a pathology resulting from complex interaction of microorganisms in the dental biofilm with the host's immune system. Increased use of antibiotics associated with their inappropriate use has increased resistance levels in anaerobic bacteria. Therefore, identifying new antimicrobial compounds, such as chalcones, is urgent. This study evaluates the antibacterial activity and the antibiofilm activity of 15 chalcones against the periodontopathogenic bacteria Prevotella nigrescens (ATCC 33563), P. oralis (ATCC 33269), Peptostreptococcus anaerobius (ATCC 27337), Actinomyces viscosus (ATCC 43146), Porphyromonas asaccharolytica (ATCC 25260), and Fusobacterium nucleatum (ATCC 25586). METHODS: The compounds were evaluated by minimum inhibitory concentration (MIC) and minimum biofilm inhibitory concentration (MBIC) tests. RESULTS: Compounds 1-6 showed good antibacterial and antibiofilm activities against most of the evaluated bacteria: MIC was lower than or equal to 6.25 µg/mL, biofilm biomass was reduced by 95%, and the compounds at concentrations between 0.78 and 100 µg/mL totally inhibited cell viability. Among the tested chalcones, 3 stood out: it was effective against all the bacteria, as revealed by the MIC and MBIC results. CONCLUSIONS: Our results have consolidated a base for the development of new studies on the effects of the tested chalcones as agents to combat and to prevent periodontitis.

Spurious infection by Calodium hepaticum (Bancroft, 1983) Moravec, 1982 and intestinal parasites in forest reserve dwellers in Western Brazilian Amazon.

Subsistence hunting is the main source of protein for forest reserve dwellers, contributing to the development of spurious infections by Calodium hepaticum, frequently associated with the consumption of the liver from wild mammals. The prevalence of infections by soil-transmitted helminths (STHs) and intestinal protozoa is considered an indicator of the social vulnerability of a country, besides providing information on habits, customs and quality of life of a given population. Intestinal parasites mostly affect poor rural communities with limited access to clean water and adequate sanitation. This study reports the results of a parasitological survey carried out in 2017 and 2019, in two municipalities (Xapuri and Sena Madureira) in Acre State. Stool samples were collected from 276 inhabitants. Upon receipt, each sample was divided into two aliquots. Fresh samples without preservative were processed and examined by the Kato-Katz technique. Samples fixed in 10% formalin were processed by the spontaneous sedimentation and the centrifugal sedimentation techniques. Calodium hepaticum eggs were found in three stool samples. The overall STH prevalence was 44.9%. The hookworm prevalence (19.2%) was higher than that of Ascaris lumbricoides (2.5%) and Trichuris trichiura (0.7%), an unexpected finding for municipalities belonging to the Western Brazilian Amazon. When considering parasites transmitted via the fecal-oral route, Endolimax nana and Entamoeba coli showed the highest positivity rates, of 13% and 10.9%, respectively. This study is the first report of spurious infection by C. hepaticum among forest reserve dwellers that consume undercooked liver of lowland pacas. Additionally, this is the first report of Blastocystis sp. in Acre State.

Toxocara canis 30-35 kDa excretory-secretory antigen is an important marker in mice challenged by inocula containing different parasite load levels.

The Western-blotting technique was applied to identify antigenic fractions of excretory-secretory Toxocara canis antigen recognized by IgG antibodies throughout an experimental infection in mice challenged by different inocula. Mice were inoculated with 5, 50 and 500 embryonated eggs and serum samples were collected 15, 30, 60, 90 and 120 days post-infection. Serum samples were analyzed using an excretory-secretory Toxocara antigen. Antibodies recognized antigenic fractions from 30 to 90 kDa. The protein fraction of 30-35 kDa was the most frequently recognized regardless of the size of inoculum and the stage of infection represented by the different collection times, but the antigenic recognition was more evident in groups infected with 50 and 500 eggs. This study presents an antigenic panel of the excretory-secretory antigen of T. canis and suggests that the 30-35 kDa antigenic fraction is a promising marker of the infection and should be further explored in future studies on experimental toxocariasis.

"Clara, esta sou eu!" Nome, acesso à saúde e sofrimento social entre pessoas transgênero / "Clara, that's me!" Name, access to health care and social suffering among transgender people / "¡Clara, esta soy yo!" Nombre, acceso a la salud y sufrimiento social entre personas transgénero

Esta pesquisa qualitativa objetivou compreender as relações entre acesso a serviços de saúde e experiências de sofrimento social entre pessoas trans. Foram conduzidas entrevistas semiestruturadas com cinco interlocutores e observações participantes. Os dados foram analisados por meio da codificação temática, originando duas categorias: "A negação do nome" e "Acesso à saúde e transfobia institucionalizada no Sistema Único de Saúde (SUS)". As narrativas das/os interlocutores permitiram localizar tais relações na sociogênese de experiências de sofrimento social. A negação do nome implica negação da humanidade da pessoa trans, bem como patologização de sua identidade e prejuízos no acesso à saúde, colocando a automedicação como uma possibilidade de agência. Concluiu-se que a transfobia institucionalizada no setor de saúde reproduz a precarização da cidadania de pessoas trans, destacando quanto a ação estatal com vistas a mitigar o sofrimento social pode, por vezes, intensificá-lo. (AU)

The aim of this qualitative study was to understand the relationships between access to health services and experiences of social suffering among trans people. We conducted semi-structured interviews with five participants and participant observation. The data were analyzed using thematic coding, giving rise to two categories: "Name denial" and "Access to health care and institutionalized transphobia in Brazilian National Health System". The participants' narratives allowed the researchers to situate these relationships within the sociogenesis of social suffering experiences. Name denial implies the negation of the humanity of trans people and pathologization of their identity, and compromises access to health care, making self-medication a possibility of agency. We conclude that institutionalized transphobia in the public health care system reproduces the precariousness of the citizenship of trans people, highlighting how government actions aimed at mitigating social suffering can sometimes intensify it. (AU)

Esta investigación cualitativa tuvo el objetivo de comprender las relaciones entre acceso a servicios de salud y experiencias de sufrimiento social entre personas trans. Se realizaron entrevistas semiestructuradas con cinco interlocutores y observaciones participantes. Los datos se analizaron por medio de la codificación temática originando dos categorías: "La negación del nombre" y "Acceso a la salud y transfobia institucionalizada en el Sistema Único de Salud". Las narrativas de los interlocutores permitieron localizar tales relaciones en la sociogénesis de experiencias de sufrimiento social. La negación del nombre implica la negación de la humanidad de la persona trans, así como la patologización de su identidad y prejuicios en el acceso a la salud, planteando la automedicación como una posibilidad de agencia. Se concluyó que la transfobia institucionalizada en el sector de la salud reproduce la precarización de la ciudadanía de personas trans, destacando hasta qué punto la acción estatal dirigida a mitigar el sufrimiento social puede, algunas veces, intensificarlo. (AU)

Toxocara canis 30-35 kDa excretory-secretory antigen is an important marker in mice challenged by inocula containing different parasite load levels

ABSTRACT The Western-blotting technique was applied to identify antigenic fractions of excretory-secretory Toxocara canis antigen recognized by IgG antibodies throughout an experimental infection in mice challenged by different inocula. Mice were inoculated with 5, 50 and 500 embryonated eggs and serum samples were collected 15, 30, 60, 90 and 120 days post-infection. Serum samples were analyzed using an excretory-secretory Toxocara antigen. Antibodies recognized antigenic fractions from 30 to 90 kDa. The protein fraction of 30-35 kDa was the most frequently recognized regardless of the size of inoculum and the stage of infection represented by the different collection times, but the antigenic recognition was more evident in groups infected with 50 and 500 eggs. This study presents an antigenic panel of the excretory-secretory antigen of T. canis and suggests that the 30-35 kDa antigenic fraction is a promising marker of the infection and should be further explored in future studies on experimental toxocariasis.

Spurious infection by Calodium hepaticum (Bancroft, 1983) Moravec, 1982 and intestinal parasites in forest reserve dwellers in Western Brazilian Amazon

ABSTRACT Subsistence hunting is the main source of protein for forest reserve dwellers, contributing to the development of spurious infections by Calodium hepaticum, frequently associated with the consumption of the liver from wild mammals. The prevalence of infections by soil-transmitted helminths (STHs) and intestinal protozoa is considered an indicator of the social vulnerability of a country, besides providing information on habits, customs and quality of life of a given population. Intestinal parasites mostly affect poor rural communities with limited access to clean water and adequate sanitation. This study reports the results of a parasitological survey carried out in 2017 and 2019, in two municipalities (Xapuri and Sena Madureira) in Acre State. Stool samples were collected from 276 inhabitants. Upon receipt, each sample was divided into two aliquots. Fresh samples without preservative were processed and examined by the Kato-Katz technique. Samples fixed in 10% formalin were processed by the spontaneous sedimentation and the centrifugal sedimentation techniques. Calodium hepaticum eggs were found in three stool samples. The overall STH prevalence was 44.9%. The hookworm prevalence (19.2%) was higher than that of Ascaris lumbricoides (2.5%) and Trichuris trichiura (0.7%), an unexpected finding for municipalities belonging to the Western Brazilian Amazon. When considering parasites transmitted via the fecal-oral route, Endolimax nana and Entamoeba coli showed the highest positivity rates, of 13% and 10.9%, respectively. This study is the first report of spurious infection by C. hepaticum among forest reserve dwellers that consume undercooked liver of lowland pacas. Additionally, this is the first report of Blastocystis sp. in Acre State.

Occurrence of potential wild hosts of Echinococcus vogeli in the forests of southwestern Brazilian Amazonia / Ocorrência de potenciais hospedeiros silvestres de Echinococcus vogeli nas florestas do sudoeste da Amazônia brasileira

Abstract The helminth Echinococcus vogeliRausch & Bernstein, 1972 is a causative agent of Neotropical Echinococcosis, a chronic zoonotic disease which is endemic to the Neotropical region. This parasite is transmitted from bush dogs (Speothos venaticus) to their prey, which include lowland pacas (Cuniculus paca) and agoutis (Dasyprocta spp.). In Brazil, most human cases of Neotropical Echinococcosis have been recorded in the Amazonian states of Acre and Pará, although few data are available on the occurrence of the potential definitive or intermediate hosts of E. vogeli in the Amazon region. In the present study, we surveyed the forests surrounding 46 human communities located within and around of outside six sustainable-use protected areas in the southwestern Amazon basin of Brazil. The forests were surveyed using camera traps to determine the local presence of potential wild hosts of E. vogeli, and the exploitation of these hosts for game meat was evaluated through interviews with 136 subsistence hunters resident in the local communities. We recorded pacas, agoutis, and bush dogs, as well as domestic dogs (Canis familiaris), all potential reservoirs of Neotropical Echinococcosis, using the same habitats. We also confirmed the frequent consumption of paca and agouti meat by subsistence hunters and their families in the study communities. Our data contribute to the understanding of the occurrence of E. vogeli in Brazilian ecosystems.

Resumo O helminto Echinococcus vogeliRausch & Bernstein, 1972 é o agente causador da Equinococose Neotropical, uma doença zoonótica crônica e endêmica da região Neotropical. Este parasito é transmitido entre o cachorro-vinagre (Speothos venaticus) e suas presas, como pacas (Cuniculus paca) e cutias (Dasyprocta spp.). No Brasil, a maioria dos casos humanos de Equinococose Neotropical é registrada nos estados do Acre e Pará, embora existam poucos dados disponíveis sobre a ocorrência de potenciais hospedeiros definitivos e intermediários de E. vogeli na Amazônia. No presente estudo, foram investigadas áreas de floresta ao redor de 46 comunidades humanas localizadas no interior e entorno de seis unidades de conservação de uso sustentável no sudoeste da bacia amazônica brasileira e, por meio de armadilhas fotográficas, foram avaliadas as presenças de potenciais hospedeiros silvestres de E. vogeli. Adicionalmente, foram avaliados o padrão de consumo da carne dos hospedeiros silvestres por meio de entrevistas com 136 moradores dessas comunidades. Foram registradas pacas, cutias e cachorros-vinagre, bem como cães domésticos (Canis familiaris) utilizando os mesmos habitats, todos potenciais reservatórios da Equinococose Neotropical. Além disto, confirmamos a alto consumo de paca e cutia nas comunidades. Os dados do presente trabalho contribuem para pesquisas em andamento sobre a presença dos potenciais reservatórios de E. vogeli em ambientes brasileiros.

Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular Consensus on genetically modified cells. Special Article: Advanced therapy medicinal products in Brazil: regulatory panorama.

Advanced therapy medicinal products, considered special medications, requires Anvisa approval for use and commercialization in Brazil. They include the advanced cellular therapy products, tissue engineering products and gene therapy products, which due to their complexity involve innovation and risks, optimized regulatory channels for their development and life cycle monitoring. The scientific elements and the compliance with applicable regulatory aspects are fundamental pillars for the advancement of clinical trials, the positive evidence of the benefit-risk profile and the definition of the critical quality attributes, from the perspective of making safe, effective and high-quality products available to the population. The approval models of these products in Brazil adapt to the specificities and characteristics of the technology and the patient target population, with accelerated regulatory analyses, use in emergency situations by risk controls and specific monitoring mechanisms, principally those related to rare diseases without other therapeutic alternatives. The opportune access to the advance therapy product with safety, efficacy and quality involves innovative normative elements that include the long-term follow-up of the safety and efficacy and of the adaptive pharmacovigilance requisites, as well as the traceability mechanisms for the start-off materials, products and patients.

Toxocara DNA amplification in serum and tissue samples in BALB/c mice.

Toxocariasis is still a neglected parasitic disease worldwide and much about its biology and diagnosis has yet to be understood. The migration of third stage larvae via bloodstream suggests a potential use of molecular tools in diagnosis as well to deepen the knowledge about its migration behaviors. Conventional PCR was applied in serum and tissue samples from BALB/c mice infected with 5 and 500 embryonated eggs. Blood samples were collected at 15, 30, 60, 90 and 120 days post-infection. Organs were excised at 170 days post infection. There was no DNA amplification in serum samples in any group or day post-infection; contrarily, tissue samples showed DNA amplification. These results also support a continuous larval migration after and/or simultaneously with the neurotropic-myotropic phase. Thus, molecular tools might be useful as a differential diagnosis method, but do not replace immunodiagnostics techniques.

Behavioral alterations in long-term Toxoplasma gondii infection of C57BL/6 mice are associated with neuroinflammation and disruption of the blood brain barrier.

The Apicomplexa protozoan Toxoplasma gondii is a mandatory intracellular parasite and the causative agent of toxoplasmosis. This illness is of medical importance due to its high prevalence worldwide and may cause neurological alterations in immunocompromised persons. In chronically infected immunocompetent individuals, this parasite forms tissue cysts mainly in the brain. In addition, T. gondii infection has been related to mental illnesses such as schizophrenia, bipolar disorder, depression, obsessive-compulsive disorder, as well as mood, personality, and other behavioral changes. In the present study, we evaluated the kinetics of behavioral alterations in a model of chronic infection, assessing anxiety, depression and exploratory behavior, and their relationship with neuroinflammation and parasite cysts in brain tissue areas, blood-brain-barrier (BBB) integrity, and cytokine status in the brain and serum. Adult female C57BL/6 mice were infected by gavage with 5 cysts of the ME-49 type II T. gondii strain, and analyzed as independent groups at 30, 60 and 90 days postinfection (dpi). Anxiety, depressive-like behavior, and hyperactivity were detected in the early (30 dpi) and long-term (60 and 90 dpi) chronic T. gondii infection, in a direct association with the presence of parasite cysts and neuroinflammation, independently of the brain tissue areas, and linked to BBB disruption. These behavioral alterations paralleled the upregulation of expression of tumor necrosis factor (TNF) and CC-chemokines (CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1ß and CCL5/RANTES) in the brain tissue. In addition, increased levels of interferon-gamma (IFNγ), TNF and CCL2/MCP-1 were detected in the peripheral blood, at 30 and 60 dpi. Our data suggest that the persistence of parasite cysts induces sustained neuroinflammation, and BBB disruption, thus allowing leakage of cytokines of circulating plasma into the brain tissue. Therefore, all these factors may contribute to behavioral changes (anxiety, depressive-like behavior, and hyperactivity) in chronic T. gondii infection.