Enhancement of CD4+ T Cell Function as a Strategy for Improving Antibiotic Therapy Efficacy in Tuberculosis: Does It Work?

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) remains a major public health problem worldwide due in part to the lack of an effective vaccine and to the lengthy course of antibiotic treatment required for successful cure. Combined immuno/chemotherapeutic intervention represents a major strategy for developing more effective therapies against this important pathogen. Because of the major role of CD4+ T cells in containing Mtb infection, augmentation of bacterial specific CD4+ T cell responses has been considered as an approach in achieving this aim. Here we present new data from our own research aimed at determining whether boosting CD4+ T cell responses can promote antibiotic clearance. In these studies, we first characterized the impact of antibiotic treatment of infected mice on Th1 responses to major Mtb antigens and then performed experiments aimed at sustaining CD4+ T cell responsiveness during antibiotic treatment. These included IL-12 infusion, immunization with ESAT-6 and Ag85B immunodominant peptides and adoptive transfer of Th1-polarized CD4+ T cells specific for ESAT-6 or Ag85B during the initial month of chemotherapy. These approaches failed to enhance antibiotic clearance of Mtb, indicating that boosting Th1 responses to immunogenic Mtb antigens highly expressed by actively dividing bacteria is not an effective strategy to be used in the initial phase of antibiotic treatment, perhaps because replicating organisms are the first to be eliminated by the drugs. These results are discussed in the context of previously published findings addressing this concept along with possible alternate approaches for harnessing Th1 immunity as an adjunct to chemotherapy

Lysosomal Cathepsin Release Is Required for NLRP3-Inflammasome Activation by Mycobacterium tuberculosis in Infected Macrophages

Lysosomal cathepsin B (CTSB) has been proposed to play a role in the induction of acute inflammation. We hypothesised that the presence of active CTSB in the cytosol is crucial for NLRP3-inflammasome assembly and, consequently, for mature IL-1β generation after mycobacterial infection in vitro. Elevated levels of CTSB was observed in the lungs of mice and rabbits following infection with Mycobacterium tuberculosis (Mtb) H37Rv as well as in plasma from acute tuberculosis patients. H37Rv-infected murine bone marrow-derived macrophages (BMDMs) displayed both lysosomal leakage, with release of CTSB into the cytosol, as well as increased levels of mature IL-1β. These responses were diminished in BMDM infected with a mutant H37Rv deficient in ESAT-6 expression. Pharmacological inhibition of cathepsin activity with CA074-Me resulted in a substantial reduction of both mature IL-1β production and caspase-1 activation in infected macrophages. Moreover, cathepsin inhibition abolished the interaction between NLRP3 and ASC, measured by immunofluorescence imaging in H37Rv-infected macrophages, demonstrating a critical role of the enzyme in NLRP3-inflammasome activation. These observations suggest that during Mtb infection, lysosomal release of activated CTSB and possibly other cathepsins inhibitable by CA07-Me is critical for the induction of inflammasome-mediated IL-1β processing by regulating NLRP3-inflammasome assembly in the cytosol

Remote sensing-based actual evapotranspiration assessment in a data-scarce area of Brazil : a case study of the Urucuia Aquifer System

The large groundwater reserves of the Urucuia Aquifer System (UAS) enabled agricultural development and economic growth in the western Bahia State, in northeastern Brazil. Over the last several years, concern has grown around the aquifer’s diminishing water levels, and water balance (WB) studies are in demand. Considering the lack of measured actual evapotranspiration (ETa), a major component of the water cycle, this work uses the Operational Simplified Surface Energy Balance (SSEBop) model to estimate ETa, and compares it to basin-scale estimates from the Soil Moisture Accounting Procedure (SMAP) monthly model and from an annual WB closure method, based on gridded meteorological data and the Gravity Recovery and Climate Experiment (GRACE) product. Additionally, a comparative assessment of different versions of the SSEBop parameterization was per-formed. Moderate Resolution Imaging Spectroradiometer (MODIS) imagery was used to implement eight different versions of the SSEBop algorithm over the UAS between 2000 and 2013. SSEBop and SMAP ETa yielded similar seasonal patterns, with correlation coefficient (r) up to 0.65, mean difference (MD) of 0.8 mm/month and mean absolute difference (MAD) of 18.5 mm/month. Comparison of SSEBop annual ETa estimates to annual SMAP and WB closure estimates yielded low MD (12.1 and 7.3 mm/year, respectively) and MAD (82.5 and 82.8 mm/year, respectively), but also low r values (0.00 and 0.37, respectively). The comparison of the different SSEBop versions indicated the need to incorporate a calibration step of the aerodynamic heat resistance (rah) parameter. SSEBop results were also used for land cover and drought monitoring. Analysis indicates that agri-culture, associated with an increasing trend of atmospheric evaporative demand, is responsible for the decrease in groundwater levels and streamflow in the studied time period

Influence of dietary pattern on anti-tuberculosis treatment outcomes in persons with dysglycemia: a Peruvian prospective cohort study

IntroductionDietary patterns (DPs) are associated with overall nutritional status and may alter the clinical prognosis of tuberculosis. This interaction can be further intricated by dysglycemia (i.e., diabetes or prediabetes). Here, we identified DPs that are more common with tuberculosis–dysglycemia and depicted their association with tuberculosis treatment outcomes.MethodsA prospective cohort study of persons with tuberculosis and their contacts was conducted in Peru. A food frequency questionnaire and a multidimensional systems biology-based analytical approach were employed to identify DPs associated with these clinical groups. Potential independent associations between clinical features and DPs were analyzed.ResultsThree major DPs were identified. TB–dysglycemia cases more often had a high intake of carbohydrates (DP1). Furthermore, DP1 was found to be associated with an increased risk of unfavorable TB outcomes independent of other factors, including dysglycemia.ConclusionOur findings suggest that the evaluation of nutritional status through DPs in comorbidities such as dysglycemia is a fundamental action to predict TB treatment outcomes. The mechanisms underlying the association between high intake of carbohydrates, dysglycemia, and unfavorable tuberculosis treatment outcomes warrant further investigation

In Vitro CRISPR/Cas9 Transfection and Gene-Editing Mediated by Multivalent Cationic LiposomeDNA Complexes

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease 9 (Cas9) gene-editing offers exciting new therapeutic possibilities for disease treatment with a genetic etiology such as cancer, cardiovascular, neuronal, and immune disorders. However, its clinical translation is being hampered by the lack of safe, versatile, and effective nonviral delivery systems. Herein we report on the preparation and application of two cationic liposome–DNA systems (i.e., lipoplexes) for CRISPR/Cas9 gene delivery. For that purpose, two types of cationic lipids are used (DOTAP, monovalent, and MVL5, multivalent with +5e nominal charge), along with three types of helper lipids (DOPC, DOPE, and monoolein (GMO)). We demonstrated that plasmids encoding Cas9 and single-guide RNA (sgRNA), which are typically hard to transfect due to their large size (>9 kb), can be successfully transfected into HEK 293T cells via MVL5-based lipoplexes. In contrast, DOTAP-based lipoplexes resulted in very low transfection rates. MVL5-based lipoplexes presented the ability to escape from lysosomes, which may explain the superior transfection efficiency. Regarding gene editing, MVL5-based lipoplexes achieved promising GFP knockout levels, reaching rates of knockout superior to 35% for charge ratios (+/−) of 10. Despite the knockout efficiency being comparable to that of Lipofectamine 3000® commercial reagent, the non-specific gene knockout is more pronounced in MVL5-based formulations, probably resulting from the considerable cytotoxicity of these formulations. Altogether, these results show that multivalent lipid-based lipoplexes are promising CRISPR/Cas9 plasmid delivery vehicles, which by further optimization and functionalization may become suitable in vivo delivery systems.This research was funded by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte and the Project FCOMP-01– 0124-FEDER-021053 (PTDC/SAU-BMA/121028/2010). This research was also supported by the Microfluidic Layer-by-layer Assembly of Cationic Liposome—Nucleic Acid Nanoparticles for Gene Delivery project (032520) co-funded by FCT and the ERDF through COMPETE2020. Diana A. Sousa (D.A.S) and Celso J.O. Ferreira (C.J.O.F) acknowledge FCT for the grants PD/BD/139083/2018 and SFRH/BD/149199/2019, respectively.info:eu-repo/semantics/publishedVersio

Assessment of the risk of burnout and its associated factors in healthcare professionals during the COVID-19 pandemic: A prospective cohort study

IntroductionThe COVID-19 pandemic resulted in tremendous physical and psychological pressure on healthcare professionals, especially on those working in intensive care units (ICUs) and Emergency Departments (EDs). The present study intended to characterize the profile of these professionals which is associated with burnout and determine the potential predictors of such condition.MethodsA Prospective cohort study was carried out in a tertiary hospital between March 2020 and March 2021, in Salvador, Brazil. A standardized and validated version of the Oldenburg Burnout inventory (OLBI) was applied to assess risk of burnout together with data forms designed to collect information on sociodemographic characteristics and religious beliefs. ICU and ED healthcare professionals were evaluated during off-hours at two distinct periods of the COVID-19 pandemic, in 2020 and in 2021. Differences in the results obtained from each study participant between the timepoints were compared. A binary logistic regression analysis was performed to identify the predictors of burnout development independent of other confounding factors.ResultsSeventy-seven healthcare professionals with a median age of 33 (interquartile range [IQR]: 31–37.5) years and predominantly female (72.7%; n = 56) were enrolled. There were 62 professionals at risk of developing burnout through the OLBI. Those had a median age of 33 (IQR: 31–37) and female predominance (71%, n = 44). Disengagement and burnout were the only features which frequencies significantly changed over time, with increasing detection at the latest timepoint. Alcohol consumption was found to be an important risk factor for burnout development [adjusted odds ratio (aOR): 10.8 (95% CI: 1.8–64.2)]. Importantly, working in the ICU [aOR: 0.04 (95%CI: 0.01–0.32)] and the habit of praying daily [aOR: 0.07 (95%CI: 0.01–0.41)] were characteristics linked to reduced odds of burnout.DiscussionDisengagement substantially increased during the COVID-19 pandemic in healthcare professionals. Alcohol consumption favors the onset of burnout whereas habit of praying daily and working in the ICU are protective against such outcome. Institutional policies aimed at minimizing etilism may positively impact mental health of these professionals

Severe Plasmodium vivax malaria exhibits marked inflammatory imbalance

<p>Abstract</p> <p>Background</p> <p>Despite clinical descriptions of severe vivax malaria cases having been reported, data regarding immunological and inflammatory patterns are scarce. In this report, the inflammatory and immunological status of both mild and severe vivax malaria cases are compared in order to explore immunopathological events in this disease.</p> <p>Methods and Results</p> <p>Active and passive malaria case detections were performed during 2007 in Buritis, Rondônia, in the Brazilian Amazon. A total of 219 participants enrolled the study. Study individuals were classified according to the presence of <it>Plasmodium vivax </it>infection within four groups: non-infected (n = 90), asymptomatic (n = 60), mild (n = 50) and severe vivax infection (n = 19). A diagnosis of malaria was made by microscopy and molecular assays. Since at present no clear criteria define severe vivax malaria, this study adapted the consensual criteria from falciparum malaria. Patients with severe <it>P. vivax </it>infection were younger, had lived for shorter time in the endemic area, and recalled having experienced less previous malaria episodes than individuals with no malaria infection and with mild or asymptomatic infection. Strong linear trends were identified regarding increasing plasma levels of C reactive protein (CRP), serum creatinine, bilirubins and the graduation of disease severity. Plasma levels of tumour necrosis factor (TNF), interferon-gamma(IFN-gamma) and also IFN-gamma/interleukin-10 ratios were increased and exhibited a linear trend with gradual augmentation of disease severity. Both laboratory parameters of organ dysfunction and inflammatory cytokines were reduced during anti-parasite therapy in those patients with severe disease.</p> <p>Conclusion</p> <p>Different clinical presentations of vivax malaria infection present strong association with activation of pro-inflammatory responses and cytokine imbalance. These findings are of utmost importance to improve current knowledge about physiopathological concepts of this serious widespread disease.</p

Immunization of Experimental Dogs With Salivary Proteins From Lutzomyia longipalpis, Using DNA and Recombinant Canarypox Virus Induces Immune Responses Consistent With Protection Against Leishmania infantum

Metacyclic Leishmania promastigotes are transmitted by sand flies that inject parasites and saliva into the host's skin. Previous studies have demonstrated that DNA plasmids encoding Lutzomyia longipalpis salivary proteins LJM17 and LJL143, when used to immunize dogs, resulted in a systemic and local Th1 cell-mediated immunity that interfered in parasite survival in vitro. Here we evaluated the ability of these same salivary antigens to induce anti-Leishmania immunity and to confer protection by immunizing dogs using a novel vaccination strategy more suitable for use in the field. The strategy consisted of a single dose of plasmid followed by two doses of recombinant Canarypoxvirus (rCanarypoxvirus) expressing L. longipalpis salivary proteins (LJM17 or LJL143). Thirty days after the final immunization, dogs were intradermally challenged with 107Leishmania infantum promastigotes in the presence of L. longipalpis saliva. We followed the experimentally infected dogs for 10 months to characterize clinical, parasitological, and immunological parameters. Upon vaccination, all immunized dogs presented strong and specific humoral responses with increased serum concentrations of IFN-γ, TNF, IL-7, and IL-15. The serum of dogs immunized with LJM17 also exhibited high levels of IL-2, IL-6, and IL-18. L. infantum infection was established in all experimental groups as evidenced by the presence of anti-Leishmania IgG, and by parasite detection in the spleen and skin. Dogs immunized with LJM17-based vaccines presented higher circulating levels of IFN-γ, IL-2, IL-6, IL-7, IL-15, IL-18, TNF, CXCL10, and GM-CSF post-infection when compared with controls. Results demonstrated that relevant Leishmania-specific immune responses were induced following vaccination of dogs with L. longipalpis salivary antigen LJM17 administered in a single priming dose of plasmid DNA, followed by two booster doses of recombinant Canarypox vector. Importantly, a significant increase in pro-inflammatory cytokines and chemokines known to be relevant for protection against leishmaniasis was evidenced after challenging LJM17-vaccinated dogs as compared to controls. Although similar results were observed following immunization with LJL143, the pro-inflammatory response observed after immunization was attenuated following infection. Collectively, these data suggest that the LJM17-based vaccine induced an immune profile consistent with the expected protective immunity against canine leishmaniosis. These results clearly support the need for further evaluation of the LJM17 antigen, using a heterologous prime-boost vaccination strategy against canine visceral leishmaniosis (CVL)