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Background: SARS-CoV2 virus, responsible for the COVID-19 pandemic, has four structural proteins and 16 nonstructural proteins. S-protein is one of the structural proteins exposed on the virus surface and is the main target for producing neutralizing antibodies and vaccines. The S-protein forms a trimer that can bind the angiotensin-converting enzyme 2 (ACE2) through its receptor binding domain (RBD) for cell entry., Aims: The goal of this study was to express in HEK293 cells a new RBD recombinant protein in a constitutive and stable manner in order to use it as an alternative immunogen and diagnostic tool for COVID-19., Materials & Methods: The protein was designed to contain an immunoglobulin signal sequence, an explanded C-terminal section of the RBD, a region responsible for the bacteriophage T4 trimerization inducer, and six histidines in the pCDNA-3.1 plasmid. Following transformation, the cells were selected with geneticin-G418 and purified from serum-fre culture supernatants using Ni2+-agarand size exclusion chromatography. The protein was structurally identified by cross-linking and circular dichroism experiments, and utilized to immunize mice in conjuction with AS03 or alum adjuvants. The mice sera were examined for antibody recognition, receptor-binding inhibition, and virus neutralization, while spleens were evaluated for γ-interferon production in the presence of RBD., Results: The protein released in the culture supernatant of cells, and exhibited a molecular mass of 135 kDa with a secondary structure like the monomeric and trimeric RBD. After purification, it formed a multimeric structure comprising trimers and hexamers, which were able to bind the ACE2 receptor. It generated high antibody titers in mice when combined with AS03 adjuvant (up to 1:50,000). The sera were capable of inhibiting binding of biotin-labeled ACE2 to the virus S1 subunit and could neutralize the entry of the Wuhan virus strain into cells at dilutions up to 1:2000. It produced specific IFN-γ producing cells in immunized mouse splenocytes., Discussion: Our data describe a new RBD containing protein, forming trimers and hexamers, which are able to induce a protective humoral and cellular response against SARS-CoV2., Conclusion: These results add a new arsenal to combat COVID-19, as an alternative immunogen or antigen for diagnosis., (© 2024 The Author(s). Immunity, Inflammation and Disease published by John Wiley & Sons Ltd.)

Chagas disease (CD), caused by the protozoan parasite Trypanosoma cruzi , is a neglected disease affecting around 6 million people. About 30% of CD patients develop chronic Chagas disease cardiomyopathy (CCC), an inflammatory cardiomyopathy that occurs decades after the initial infection, while most infected patients (60%) remain asymptomatic in the so-called indeterminate form (IF). Death results from heart failure or arrhythmia in a subset of CCC patients. Myocardial fibrosis, inflammation, and mitochondrial dysfunction are involved in the arrhythmia substrate and triggering events. Survival in CCC is worse than in other cardiomyopathies, which may be linked to a Th1-T cell rich myocarditis with abundant interferon (IFN)-γ and tumor necrosis factor (TNF)-α, selectively lower levels of mitochondrial energy metabolism enzymes in the heart, and reduced levels of high-energy phosphate, indicating poor adenosine triphosphate (ATP) production. IFN-γ and TNF-α signaling, which are constitutively upregulated in CD patients, negatively affect mitochondrial function in cardiomyocytes, recapitulating findings in CCC heart tissue. Genetic studies such as whole-exome sequencing (WES) in nuclear families with multiple CCC/IF cases has disclosed rare heterozygous pathogenic variants in mitochondrial and inflammatory genes segregating in CCC cases. In this minireview, we summarized studies showing how IFN-γ and TNF-α affect cell energy generation, mitochondrial health, and redox homeostasis in cardiomyocytes, in addition to human CD and mitochondria. We hypothesize that cytokine-induced mitochondrial dysfunction in genetically predisposed patients may be the underlying cause of CCC severity and we believe this mechanism may have a bearing on other inflammatory cardiomyopathies., Competing Interests: Declaration Of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Chagas disease is a parasitic disease from South America, affecting around 7 million people worldwide. Decades after the infection, 30% of people develop chronic forms, including Chronic Chagas Cardiomyopathy (CCC), for which no treatment exists. Two stages characterized this form: the moderate form, characterized by a heart ejection fraction (EF) ≥ 0.4, and the severe form, associated to an EF < 0.4. We propose two sets of DNA methylation biomarkers which can predict in blood CCC occurrence, and CCC stage. This analysis, based on machine learning algorithms, makes predictions with more than 95% accuracy in a test cohort. Beyond their predictive capacity, these CpGs are located near genes involved in the immune response, the nervous system, ion transport or ATP synthesis, pathways known to be deregulated in CCCs. Among these genes, some are also differentially expressed in heart tissues. Interestingly, the CpGs of interest are tagged to genes mainly involved in nervous and ionic processes. Given the close link between methylation and gene expression, these lists of CpGs promise to be not only good biomarkers, but also good indicators of key elements in the development of this pathology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Brochet, Ianni, Nunes, Frade, Teixeira, Mady, Ferreira, Kuramoto, Pissetti, Saba, Cândido, Dias, Sampaio, Marin-Neto, Fragata, Zaniratto, Siqueira, Peixoto, Rigaud, Buck, Almeida, Lin-Wang, Schmidt, Martinelli, Hirata, Donadi, Rodrigues Junior, Pereira, Kalil, Spinelli, Cunha-Neto and Chevillard.)

Chagas disease (CD), caused by the protozoan parasite Trypanosoma cruzi, affects 8 million people, and around 1/3 develop chronic cardiac (CCC) or digestive disease (megaesophagus/megacolon), while the majority remain asymptomatic, in the indeterminate form of Chagas disease (ASY). Most CCC cases in families with multiple Chagas disease patients carry damaging mutations in mitochondrial genes. We searched for exonic mutations associated to chagasic megaesophagus (CME) in genes essential to mitochondrial processes. We performed whole exome sequencing of 13 CME and 45 ASY patients. We found the damaging variant MRPS18B 688C > G P230A, in five out of the 13 CME patients (one of them being homozygous; 38.4%), while the variant appeared in one out of 45 ASY patients (2.2%). We analyzed the interferon (IFN)-γ-induced nitro-oxidative stress and mitochondrial function of EBV-transformed lymphoblastoid cell lines. We found the CME carriers of the mutation displayed increased levels of nitrite and nitrated proteins; in addition, the homozygous (G/G) CME patient also showed increased mitochondrial superoxide and reduced levels of ATP production. The results suggest that pathogenic mitochondrial mutations may contribute to cytokine-induced nitro-oxidative stress and mitochondrial dysfunction. We hypothesize that, in mutation carriers, IFN-γ produced in the esophageal myenteric plexus might cause nitro-oxidative stress and mitochondrial dysfunction in neurons, contributing to megaesophagus.

CoronaVac is an inactivated SARS-CoV-2 vaccine that has been rolled out in several low and middle-income countries including Brazil, where it was the mainstay of the first wave of immunization of healthcare workers and the elderly population. We aimed to assess the T cell and antibody responses of vaccinated individuals as compared to convalescent patients. We detected IgG against SARS-CoV-2 antigens, neutralizing antibodies against the reference Wuhan SARS-CoV-2 strain and used SARS-CoV-2 peptides to detect IFN-g and IL-2 specific T cell responses in a group of CoronaVac vaccinated individuals (N = 101) and convalescent (N = 72) individuals. The frequency among vaccinated individuals, of whom 96% displayed T cell and/or antibody responses to SARS-CoV-2, is comparable to 98.5% responses of convalescent individuals. We observed that among vaccinated individuals, men and individuals 55 years or older developed significantly lower anti-RBD, anti-NP and neutralization titers against the Wuhan strain and antigen-induced IL-2 production by T cells. Neutralizing antibody responses for Gamma variant were even lower than for the Wuhan strain. Even though some studies indicated CoronaVac helped reduce mortality among elderly people, considering the appearance of novel variants of concern, CoronaVac vaccinated individuals above 55 years old are likely to benefit from a heterologous third dose/booster vaccine to increase immune response and likely protection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Medeiros, Sasahara, Magawa, Nunes, Bruno, Kuramoto, Almeida, Ferreira, Scagion, Candido, Leal, Oliveira, Durigon, Silva, Rosa, Boscardin, Coelho, Kalil, Santos and Cunha-Neto.)

Recurrence of COVID-19 in recovered patients has been increasingly reported. However, the immune mechanisms behind the recurrence have not been thoroughly investigated. The presence of neutralizing antibodies (nAbs) in recurrence/reinfection cases suggests that other types of immune response are involved in protection against recurrence. Here, we investigated the innate type I/III interferon (IFN) response, binding and nAb assays and T-cell responses to severe acute respiratory distress syndrome coronavirus 2 (SARS-CoV-2) with IFN gamma (IFN γ ) enzyme-linked spot assay (ELISPOT) in three pairs of young adult monozygotic (MZ) twins with previous confirmed COVID-19, one of them presenting a severe recurrence four months after the initial infection. Twin studies have been of paramount importance to comprehend the immunogenetics of infectious diseases. Each MZ twin pair was previously exposed to SARS-CoV-2, as seen by clinical reports. The six individuals presented similar overall recovered immune responses except for the recurrence case, who presented a drastically reduced number of recognized SARS-CoV-2 T-cell epitopes on ELISPOT as compared to her twin sister and the other twin pairs. Our results suggest that the lack of a broad T-cell response to initial infection may have led to recurrence, emphasizing that an effective SARS-CoV-2-specific T-cell immune response is key for complete viral control and avoidance of clinical recurrence of COVID-19.

Mastitis is one of the most important causes of loss of cattle production, burdening producers due to the increased cost of milk production and decreased herd productivity. The development of alternative methods for the treatment and prevention of mastitis other than traditional chemical antibiotic therapy needs to be implemented to meet international pressures to reduce the use of these drugs and promote the elimination of multiresistant microbial strains from the environment. Treatment with probiotic bacteria or yeast strains offers a possible strategy for the control of mastitis. The objective of this work was to isolate, identify, and characterize lactic bacteria from milk and the intramammary duct of Gyr, Guzerat, Girolando 1/2, and Holstein cattle breeds from Brazil. Samples of 115 cows were taken, a total of 192 bacteria isolates belonging to 30 species were obtained, and 81 were selected to evaluate their probiotic potential in in vitro characterization tests. In general, bacteria isolated from the mammary gland have low autoaggregation, cell surface hydrophobicity, and co-aggregation with mastitis etiological bacteria Staphylococcus aureus and Escherichia coli. Also, they have biofilm assembly capacity, inability to produce exopolysaccharides, high production of H 2 O 2 , and strong antagonism against mastitis pathogens. Ten lactic bacteria isolates were used in co-culture with human MDA-MB-231 breast epithelial cells to assess their adhesion capacity and impairment of the S. aureus invasion. Our results, therefore, contribute to the future production of new prevention and treatment tools for bovine mastitis., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Infection by the protozoan Trypanosoma cruzi causes Chagas disease cardiomyopathy (CCC) and can lead to arrhythmia, heart failure and death. Chagas disease affects 8 million people worldwide, and chronic production of the cytokines IFN-γ and TNF-α by T cells together with mitochondrial dysfunction are important players for the poor prognosis of the disease. Mitochondria occupy 40% of the cardiomyocytes volume and produce 95% of cellular ATP that sustain the life-long cycles of heart contraction. As IFN-γ and TNF-α have been described to affect mitochondrial function, we hypothesized that IFN-γ and TNF-α are involved in the myocardial mitochondrial dysfunction observed in CCC patients. In this study, we quantified markers of mitochondrial dysfunction and nitro-oxidative stress in CCC heart tissue and in IFN-γ/TNF-α-stimulated AC-16 human cardiomyocytes. We found that CCC myocardium displayed increased levels of nitro-oxidative stress and reduced mitochondrial DNA as compared with myocardial tissue from patients with dilated cardiomyopathy (DCM). IFN-γ/TNF-α treatment of AC-16 cardiomyocytes induced increased nitro-oxidative stress and decreased the mitochondrial membrane potential (ΔΨm). We found that the STAT1/NF-κB/NOS2 axis is involved in the IFN-γ/TNF-α-induced decrease of ΔΨm in AC-16 cardiomyocytes. Furthermore, treatment with mitochondria-sparing agonists of AMPK, NRF2 and SIRT1 rescues ΔΨm in IFN-γ/TNF-α-stimulated cells. Proteomic and gene expression analyses revealed that IFN-γ/TNF-α-treated cells corroborate mitochondrial dysfunction, transmembrane potential of mitochondria, altered fatty acid metabolism and cardiac necrosis/cell death. Functional assays conducted on Seahorse respirometer showed that cytokine-stimulated cells display decreased glycolytic and mitochondrial ATP production, dependency of fatty acid oxidation as well as increased proton leak and non-mitochondrial oxygen consumption. Together, our results suggest that IFN-γ and TNF-α cause direct damage to cardiomyocytes' mitochondria by promoting oxidative and nitrosative stress and impairing energy production pathways. We hypothesize that treatment with agonists of AMPK, NRF2 and SIRT1 might be an approach to ameliorate the progression of Chagas disease cardiomyopathy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Nunes, Andrieux, Brochet, Almeida, Kitano, Honda, Iwai, Andrade-Silva, Goudenège, Alcântara Silva, Vieira, Levy, Bydlowski, Gallardo, Torres, Bocchi, Mano, Santos, Bacal, Pomerantzeff, Laurindo, Teixeira, Nakaya, Kalil, Procaccio, Chevillard and Cunha-Neto.)

Mitochondria are the energy center of the cell. They are found in the cell cytoplasm as dynamic networks where they adapt energy production based on the cell's needs. They are also at the center of the proinflammatory response and have essential roles in the response against pathogenic infections. Mitochondria are a major site for production of Reactive Oxygen Species (ROS; or free radicals), which are essential to fight infection. However, excessive and uncontrolled production can become deleterious to the cell, leading to mitochondrial and tissue damage. Pathogens exploit the role of mitochondria during infection by affecting the oxidative phosphorylation mechanism (OXPHOS), mitochondrial network and disrupting the communication between the nucleus and the mitochondria. The role of mitochondria in these biological processes makes these organelle good targets for the development of therapeutic strategies. In this review, we presented a summary of the endosymbiotic origin of mitochondria and their involvement in the pathogen response, as well as the potential promising mitochondrial targets for the fight against infectious diseases and chronic inflammatory diseases.

Cardiomyopathies are an important cause of heart failure and sudden cardiac death. Little is known about the role of rare genetic variants in inflammatory cardiomyopathy. Chronic Chagas disease cardiomyopathy (CCC) is an inflammatory cardiomyopathy prevalent in Latin America, developing in 30% of the 6 million patients chronically infected by the protozoan Trypanosoma cruzi, while 60% remain free of heart disease (asymptomatic (ASY)). The cytokine interferon-γ and mitochondrial dysfunction are known to play a major pathogenetic role. Chagas disease provides a unique model to probe for genetic variants involved in inflammatory cardiomyopathy., Methods: We used whole exome sequencing to study nuclear families containing multiple cases of Chagas disease. We searched for rare pathogenic variants shared by all family members with CCC but absent in infected ASY siblings and in unrelated ASY., Results: We identified heterozygous, pathogenic variants linked to CCC in all tested families on 22 distinct genes, from which 20 were mitochondrial or inflammation-related - most of the latter involved in proinflammatory cytokine production. Significantly, incubation with IFN-γ on a human cardiomyocyte line treated with an inhibitor of dihydroorotate dehydrogenase brequinar (enzyme showing a loss-of-function variant in one family) markedly reduced mitochondrial membrane potential (ΔψM), indicating mitochondrial dysfunction., Conclusion: Mitochondrial dysfunction and inflammation may be genetically determined in CCC, driven by rare genetic variants. We hypothesize that CCC-linked genetic variants increase mitochondrial susceptibility to IFN-γ-induced damage in the myocardium, leading to the cardiomyopathy phenotype in Chagas disease. This mechanism may also be operative in other inflammatory cardiomyopathies.

Background: Hepatobiliary surgery and hepatic trauma are frequent causes of bile leaks and this feared complication can be safely managed by endoscopic retrograde cholangiopancreatography (ERCP). The approach consists of sphincterotomy alone, biliary stenting or a combination of the two but the optimal form remains unclear., Objective: The aim of this study is to compare sphincterotomy alone versus sphincterotomy plus biliary stent placement in the treatment of post-surgical and traumatic bile leaks., Methods: We retrospectively analyzed 31 patients with the final ERCP diagnosis of "bile leak". Data collected included patient demographics, etiology of the leak and the procedure details. The treatment techniques were divided into two groups: sphincterotomy alone vs. sphincterotomy plus biliary stenting. We evaluated the volume of the abdominal surgical drain before and after each procedure and the number of days needed until cessation of drainage post ERCP., Results: A total of 31 patients (18 men and 3 women; mean age, 51 years) with bile leaks were evaluated. Laparoscopic cholecystectomy was the etiology of the leak in 14 (45%) cases, followed by conventional cholecystectomy in 9 (29%) patients, hepatic trauma in 5 (16%) patients, and hepatectomy secondary to neoplasia in 3 (9.7%) patients. The most frequent location of the leaks was the cystic duct stump with 12 (38.6%) cases, followed by hepatic common duct in 10 (32%) cases, common bile duct in 7 (22%) cases and the liver bed in 2 (6.5%) cases. 71% of the patients were treated with sphincterotomy plus biliary stenting, and 29% with sphincterotomy alone. There was significant difference between the volume drained before and after both procedures (P<0.05). However, when comparing sphincterotomy alone and sphincterotomy plus biliary stenting, regarding the volume drained and the days needed to cessation of drainage, there was no statistical difference in both cases (P>0.005)., Conclusion: ERCP remains the first line treatment for bile leaks with no difference between sphincterotomy alone vs sphincterotomy plus stent placement.

Chagas disease is caused by infection with the protozoan Trypanosoma cruzi and affects over 8 million people worldwide. In spite of a powerful innate and adaptive immune response in acute infection, the parasite evades eradication, leading to a chronic persistent infection with low parasitism. Chronically infected subjects display differential patterns of disease progression. While 30% develop chronic Chagas disease cardiomyopathy (CCC)-a severe inflammatory dilated cardiomyopathy-decades after infection, 60% of the patients remain disease-free, in the asymptomatic/indeterminate (ASY) form, and 10% develop gastrointestinal disease. Infection of genetically deficient mice provided a map of genes relevant for resistance to T. cruzi infection, leading to the identification of multiple genes linked to survival to infection. These include pathogen resistance genes (PRG) needed for intracellular parasite destruction, and genes involved in disease tolerance (protection against tissue damage and acute phase death-DTG). All identified DTGs were found to directly or indirectly inhibit IFN-γ production or Th1 differentiation. We hypothesize that the absolute need for DTG to control potentially lethal IFN-γ PRG activity leads to T. cruzi persistence and establishment of chronic infection. IFN-γ production is higher in CCC than ASY patients, and is the most highly expressed cytokine in CCC hearts. Key DTGs that downmodulate IFN-γ, like IL-10, and Ebi3/IL27p28, are higher in ASY patients. Polymorphisms in PRG and DTG are associated with differential disease progression. We thus hypothesize that ASY patients are disease tolerant, while an imbalance of DTG and IFN-γ PRG activity leads to the inflammatory heart damage of CCC.

Recent advances in biological imaging techniques and the enormous amount of data they generate call for the development of computational tools for efficient and reliable high-throughput analysis. Several software applications with this functionality are available, and one of the most commonly used is ImageJ. Here, we present two independent macros (WH&#95;NJ and SA&#95;NJ) for automating and facilitating the analysis of images acquired from two in vitro assays frequently used in cancer studies and drug screening: the wound-healing and soft-agar assays. These two algorithms combine, in a single command, the steps required for the individual analysis of each image using ImageJ. WH&#95;NJ and SA&#95;NJ allow fast, reproducible data analysis without the experimental bias inherent in manual analyses, thus guaranteeing the robustness and reliability of the results.