Enhancing Lung Cancer Care in Portugal: Bridging Gaps for Improved Patient Outcomes.

Lung cancer has the highest incidence and cancer-related mortality worldwide. In Portugal, it ranks as the fourth most common cancer, with nearly 6000 new cases being diagnosed every year. Lung cancer is the main cause of cancer-related death among males and the third cause of cancer-related death in females. Despite the globally accepted guidelines and recommendations for what would be the ideal path for a lung cancer patient, several challenges occur in real clinical management across the world. The recommendations emphasize the importance of adequate screening of high-risk individuals, a precise tumour biopsy, and an accurate final diagnosis to confirm the neoplastic nature of the nodule. A detailed histological classification of the lung tumour type and a comprehensive molecular characterization are of utmost importance for the selection of an efficacious and patient-directed therapeutic approach. However, in the context of the Portuguese clinical organization and the national healthcare system, there are still several gaps in the ideal pathway for a lung cancer patient, involving aspects ranging from the absence of a national lung cancer screening programme through difficulties in histological diagnosis and molecular characterization to challenges in therapeutic approaches. In this manuscript, we address the most relevant weaknesses, presenting several proposals for potential solutions to improve the management of lung cancer patients, helping to decisively improve their overall survival and quality of life.

Unravelling the triad of neuroinvasion, neurodissemination, and neuroinflammation of human immunodeficiency virus type 1 in the central nervous system.

Since the identification of human immunodeficiency virus type 1 (HIV-1) in 1983, many improvements have been made to control viral replication in the peripheral blood and to treat opportunistic infections. This has increased life expectancy but also the incidence of age-related central nervous system (CNS) disorders and HIV-associated neurodegeneration/neurocognitive impairment and depression collectively referred to as HIV-associated neurocognitive disorders (HAND). HAND encompasses a spectrum of different clinical presentations ranging from milder forms such as asymptomatic neurocognitive impairment or mild neurocognitive disorder to a severe HIV-associated dementia (HAD). Although control of viral replication and suppression of plasma viral load with combination antiretroviral therapy has reduced the incidence of HAD, it has not reversed milder forms of HAND. The objective of this review, is to describe the mechanisms by which HIV-1 invades and disseminates in the CNS, a crucial event leading to HAND. The review will present the evidence that underlies the relationship between HIV infection and HAND. Additionally, recent findings explaining the role of neuroinflammation in the pathogenesis of HAND will be discussed, along with prospects for treatment and control.

Human immunodeficiency virus transmission-Mechanisms underlying the cell-to-cell spread of human immunodeficiency virus.

Despite the success of combined antiretroviral therapy in controlling viral load and reducing the risk of human immunodeficiency virus (HIV) transmission, an estimated 1.5 million new infections occurred worldwide in 2021. These new infections are mainly the result of sexual intercourse and thus involve cells present on the genital mucosa, such as dendritic cells (DCs), macrophages (Mø) and CD4+ T lymphocytes. Understanding the mechanisms by which HIV interacts with these cells and how HIV exploits these interactions to establish infection in a new human host is critical to the development of strategies to prevent and control HIV transmission. In this review, we explore how HIV has evolved to manipulate some of the physiological roles of these cells, thereby gaining access to strategic cellular niches that are critical for the spread and pathogenesis of HIV infection. The interaction of HIV with DCs, Mø and CD4+ T lymphocytes, and the role of the intercellular transfer of viral particles through the establishment of the infectious or virological synapses, but also through membrane protrusions such as filopodia and tunnelling nanotubes (TNTs), and cell fusion or cell engulfment processes are presented and discussed.

The impact of nintedanib and pirfenidone on lung function and survival in patients with idiopathic pulmonary fibrosis in real-life setting.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic, fibrosing interstitial pneumonia of unknown cause that is associated with radiological and/or histological features of usual interstitial pneumonia (UIP). A mean survival of 2-5 years was reported previously to the advent of antifibrotics. According to clinical trials, nintedanib and pirfenidone induce a significant delay in functional decline, with a favorable impact on survival. METHODS: A real-life retrospective and longitudinal study was conducted to assess the efficacy and tolerability of antifibrotics in IPF patients, between January 2014 and December 2020. Two groups (under nintedanib or pirfenidone) were analyzed at diagnosis through their clinical features and radiological patterns. Lung function was assessed at diagnosis (time 0) and after 6, 12 and 24 months of treatment. We also compared this antifibrotic cohort with an older naïve antifibrotic cohort, mainly treated with immunosuppressive drugs and/or N- acetylcysteine. Survival was analyzed and prognostic features were also studied. Statistical analysis was performed with IBM® SPSS®. RESULTS: A cohort of 108 patients under antifibrotics (nintedanib n = 54; pirfenidone n = 54) was assessed. Lung function analysis showed an overall stabilization in FVC and DLCO mean predicted percentages at 6, 12 and 24 months of treatment. The mean decline in FVC and DLCO, at 12 months, was -40.95 ± 438.26 mL and -0.626 ± 1.31 mL/min/mmHg, respectively. However, during this period, 34.2% of the patients died mostly due to acute exacerbation associated with a poorer lung function at diagnosis. Mean survival in the naïve antifibrotic cohort was significantly lower than in the antifibrotic cohort (39.9 months versus 58.2 months; p < 0.005). Regarding lung function evolution and survival, we found no differences between definitive or probable UIP radiological patterns, both on patients under nintedanib and pirfenidone (p = 0.656). CONCLUSIONS: In this real-life observational study, the positive impact of antifibrotic therapy on the IPF clinical course and on survival was corroborated. Regarding efficacy, there was no difference between patients taking nintedanib or pirfenidone. The need for an early treatment was also demonstrated, since a worse outcome is clearly associated with lower lung volumes and lower diffusing capacity at diagnosis.

Modulation of Cystatin F in Human Macrophages Impacts Cathepsin-Driven Killing of Multidrug-Resistant Mycobacterium tuberculosis.

Tuberculosis (TB) treatment relies primarily on 70-year-old drugs, and prophylaxis suffers from the lack of an effective vaccine. Among the 10 million people exhibiting disease symptoms yearly, 450,000 have multidrug or extensively drug-resistant (MDR or XDR) TB. A greater understanding of host and pathogen interactions will lead to new therapeutic interventions for TB eradication. One of the strategies will be to target the host for better immune bactericidal responses against the TB causative agent Mycobacterium tuberculosis (Mtb). Cathepsins are promising targets due to their manipulation of Mtb with consequences such as decreased proteolytic activity and improved pathogen survival in macrophages. We recently demonstrated that we could overcome this enzymatic blockade by manipulating protease inhibitors such as cystatins. Here, we investigate the role of cystatin F, an inhibitor that we showed previously to be strongly upregulated during Mtb infection. Our results indicate that the silencing of cystatin F using siRNA increase the proteolytic activity of cathepsins S, L, and B, significantly impacting pathogen intracellular killing in macrophages. Taken together, these indicate the targeting of cystatin F as a potential adjuvant therapy for TB, including MDR and XDR-TB.

ESAT-6 a Major Virulence Factor of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis (TB), is one of the most successfully adapted human pathogens. Human-to-human transmission occurs at high rates through aerosols containing bacteria, but the pathogen evolved prior to the establishment of crowded populations. Mtb has developed a particular strategy to ensure persistence in the host until an opportunity for transmission arises. It has refined its lifestyle to obviate the need for virulence factors such as capsules, flagella, pili, or toxins to circumvent mucosal barriers. Instead, the pathogen uses host macrophages, where it establishes intracellular niches for its migration into the lung parenchyma and other tissues and for the induction of long-lived latency in granulomas. Finally, at the end of the infection cycle, Mtb induces necrotic cell death in macrophages to escape to the extracellular milieu and instructs a strong inflammatory response that is required for the progression from latency to disease and transmission. Common to all these events is ESAT-6, one of the major virulence factors secreted by the pathogen. This narrative review highlights the recent advances in understanding the role of ESAT-6 in hijacking macrophage function to establish successful infection and transmission and its use as a target for the development of diagnostic tools and vaccines.

Cell-to-Cell Transmission of HIV-1 and HIV-2 from Infected Macrophages and Dendritic Cells to CD4+ T Lymphocytes.

Macrophages (Mø) and dendritic cells (DCs) are key players in human immunodeficiency virus (HIV) infection and pathogenesis. They are essential for the spread of HIV to CD4+ T lymphocytes (TCD4+) during acute infection. In addition, they constitute a persistently infected reservoir in which viral production is maintained for long periods of time during chronic infection. Defining how HIV interacts with these cells remains a critical area of research to elucidate the pathogenic mechanisms of acute spread and sustained chronic infection and transmission. To address this issue, we analyzed a panel of phenotypically distinct HIV-1 and HIV-2 primary isolates for the efficiency with which they are transferred from infected DCs or Mø to TCD4+. Our results show that infected Mø and DCs spread the virus to TCD4+ via cell-free viral particles in addition to other alternative pathways. We demonstrate that the production of infectious viral particles is induced by the co-culture of different cell populations, indicating that the contribution of cell signaling driven by cell-to-cell contact is a trigger for viral replication. The results obtained do not correlate with the phenotypic characteristics of the HIV isolates, namely their co-receptor usage, nor do we find significant differences between HIV-1 and HIV-2 in terms of cis- or trans-infection. The data presented here may help to further elucidate the cell-to-cell spread of HIV and its importance in HIV pathogenesis. Ultimately, this knowledge is critical for new therapeutic and vaccine approaches.

HIV/Mtb Co-Infection: From the Amplification of Disease Pathogenesis to an "Emerging Syndemic".

Human immunodeficiency virus (HIV) and Mycobacterium tuberculosis (Mtb) are pathogens responsible for millions of new infections each year; together, they cause high morbidity and mortality worldwide. In addition, late-stage HIV infection increases the risk of developing tuberculosis (TB) by a factor of 20 in latently infected people, and even patients with controlled HIV infection on antiretroviral therapy (ART) have a fourfold increased risk of developing TB. Conversely, Mtb infection exacerbates HIV pathogenesis and increases the rate of AIDS progression. In this review, we discuss this reciprocal amplification of HIV/Mtb coinfection and how they influence each other's pathogenesis. Elucidating the infectious cofactors that impact on pathogenesis may open doors for the design of new potential therapeutic strategies to control disease progression, especially in contexts where vaccines or the sterile clearance of pathogens are not effectively available.

Development of Chitosan Particles Loaded with siRNA for Cystatin C to Control Intracellular Drug-Resistant Mycobacterium tuberculosis.

The golden age of antibiotics for tuberculosis (TB) is marked by its success in the 1950s of the last century. However, TB is not under control, and the rise in antibiotic resistance worldwide is a major threat to global health care. Understanding the complex interactions between TB bacilli and their host can inform the rational design of better TB therapeutics, including vaccines, new antibiotics, and host-directed therapies. We recently demonstrated that the modulation of cystatin C in human macrophages via RNA silencing improved the anti-mycobacterial immune responses to Mycobacterium tuberculosis infection. Available in vitro transfection methods are not suitable for the clinical translation of host-cell RNA silencing. To overcome this limitation, we developed different RNA delivery systems (DSs) that target human macrophages. Human peripheral blood-derived macrophages and THP1 cells are difficult to transfect using available methods. In this work, a new potential nanomedicine based on chitosan (CS-DS) was efficiently developed to carry a siRNA-targeting cystatin C to the infected macrophage models. Consequently, an effective impact on the intracellular survival/replication of TB bacilli, including drug-resistant clinical strains, was observed. Altogether, these results suggest the potential use of CS-DS in adjunctive therapy for TB in combination or not with antibiotics.

An In Vitro Evaluation of the Potential Neuroprotective Effects of Intranasal Lipid Nanoparticles Containing Astaxanthin Obtained from Different Sources: Comparative Studies.

The intranasal route has been suggested as a promising alternative to improve the direct transport of molecules to the brain, avoiding the need to cross the blood-brain barrier (BBB). In this area, the use of lipid nanoparticles, namely solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC), has been highlighted as a promising strategy to improve the treatment of neurodegenerative diseases. In this work, formulations containing SLN and NLC that were loaded with astaxanthin that was obtained from different sources (astaxanthin extract (AE) from the algae Haematococcus pluvialis and pure astaxanthin (PA) from the fungi Blakeslea trispora) were prepared for nose-to-brain administration, and comparative in vitro experiments were performed to evaluate the biocompatibility of the formulations with nasal (RPMI 2650) and neuronal (SH-SY5Y) cells. Afterwards, the antioxidant activity of the formulations was evaluated for its potential neuroprotective effects, using different chemical aggressors. Finally, the cellular uptake of the astaxanthin was evaluated for the formulations that showed the greatest neuroprotection of the neuronal cells against chemical-induced damage. On the production day, all the formulations showed a particle size, a high encapsulation efficiency (EE), the presence of nanoparticles with a typical spherical shape, and a polydispersity index (PDI) and zeta potential (ZP) that are suitable for nose-to-brain administration. After three months of storage at room temperature, no significant changes were observed in the characterization parameters, predicting a good long-term stability. Furthermore, these formulations were shown to be safe with concentrations of up to 100 µg/mL in differentiated SH-SY5Y and RPMI 2650 cells. Regarding neuroprotection studies, the PA-loaded SLN and NLC formulations showed an ability to counteract some mechanisms of neurodegeneration, including oxidative stress. Moreover, when compared with the PA-loaded SLN, the PA-loaded NLC showed greater neuroprotective effects against the cytotoxicity induced by aggressors. In contrast, the AE-loaded SLN and NLC formulations showed no significant neuroprotective effects. Although further studies are needed to confirm these neuroprotective effects, the results of this study suggest that the intranasal administration of PA-loaded NLC may be a promising alternative to improve the treatment of neurodegenerative diseases.

Liposomal Delivery of Saquinavir to Macrophages Overcomes Cathepsin Blockade by Mycobacterium tuberculosis and Helps Control the Phagosomal Replicative Niches.

Mycobacterium tuberculosis is able to establish a chronic colonization of lung macrophages in a controlled replication manner, giving rise to a so-called latent infection. Conversely, when intracellular bacteria undergo actively uncontrolled replication rates, they provide the switch for the active infection called tuberculosis to occur. Our group found that the pathogen is able to manipulate the activity of endolysosomal enzymes, cathepsins, directly at the level of gene expression or indirectly by regulating their natural inhibitors, cystatins. To provide evidence for the crucial role of cathepsin manipulation for the success of tuberculosis bacilli in their intracellular survival, we used liposomal delivery of saquinavir. This protease inhibitor was previously found to be able to increase cathepsin proteolytic activity, overcoming the pathogen induced blockade. In this study, we demonstrate that incorporation in liposomes was able to increase the efficiency of saquinavir internalization in macrophages, reducing cytotoxicity at higher concentrations. Consequently, our results show a significant impact on the intracellular killing not only to reference and clinical strains susceptible to current antibiotic therapy but also to multidrug- and extensively drug-resistant (XDR) Mtb strains. Altogether, this indicates the manipulation of cathepsins as a fine-tuning strategy used by the pathogen to survive and replicate in host cells.

Mortality risk prediction with ILD-GAP index in a fibrotic hypersensitivity pneumonitis cohort.

BACKGROUND: Fibrotic hypersensitivity pneumonitis (fHP) is associated with significant morbidity and mortality. Interstitial lung disease-gender-age-physiology (ILD-GAP) performance in fHP outside the initial cohort was never performed. AIM: To assess the ILD-GAP index's ability to predict mortality in a Portuguese cohort of patients with fHP and analyse whether other clinical variables add value. METHODS: Retrospective analysis of fHP cohort in two Portuguese ILD centres. The baseline ILD-GAP index was calculated. Survival was analysed in months; mortality was the primary outcome. Univariate and multivariate analyses to identify mortality risk factors were performed. RESULTS: A total of 141 patients were included. Fifty-three patients (37.6%) died during the follow-up. The usual interstitial pneumonia (UIP) pattern was found in 49.6%, and their survival was inferior to non-UIP [32 months (interquartile range, IQR = 19, 60) versus 52 months (IQR = 28, 98), p = 0.048]. Patients with an ILD-GAP index higher than three double their risk of mortality [hazard ratio (HR) = 6.48, 95% confidence interval (CI) = (3.03-13.96)] when compared with the patients with an index between 2 and 3 [HR = 3.04, 95% CI = (1.62-5.71)] adjusting for acute exacerbation history. Even though UIP patients had worse survival, it did not reach statistical significance when UIP pattern was added to this model. Acute exacerbation history was an independent risk factor for mortality; however, ILD-GAP still predicted mortality after adjusting for this factor. PaO2 and 6-minute walk test desaturation were not significant risk factors. CONCLUSION: ILD-GAP index is a good predictor for mortality in fHP, even after adjusting for other mortality risk factors.

Drought-heatwave nexus in Brazil and related impacts on health and fires: A comprehensive review.

Climate change is drastically altering the frequency, duration, and severity of compound drought-heatwave (CDHW) episodes, which present a new challenge in environmental and socioeconomic sectors. These threats are of particular importance in low-income regions with growing populations, fragile infrastructure, and threatened ecosystems. This review synthesizes emerging progress in the understanding of CDHW patterns in Brazil while providing insights about the impacts on fire occurrence and public health. Evidence is mounting that heatwaves are becoming increasingly linked with droughts in northeastern and southeastern Brazil, the Amazonia, and the Pantanal. In those regions, recent studies have begun to build a better understanding of the physical mechanisms behind CDHW events, such as the soil moisture-atmosphere coupling, promoted by exceptional atmospheric blocking conditions. Results hint at a synergy between CDHW events and high fire activity in the country over the last decades, with the most recent example being the catastrophic 2020 fires in the Pantanal. Moreover, we show that HWs were responsible for increasing mortality and preterm births during record-breaking droughts in southeastern Brazil. This work paves the way for a more in-depth understanding on CDHW events and their impacts, which is crucial to enhance the adaptive capacity of different Brazilian sectors.

Predictive value of common genetic variants in idiopathic pulmonary fibrosis survival.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrosing interstitial pneumonia of unknown etiology. The role of genetic risk factors has been the focus of numerous studies probing for associations of genetic variants with IPF. We aimed to determine whether single-nucleotide polymorphisms (SNPs) of four candidate genes are associated with IPF susceptibility and survival in a Portuguese population. A retrospective case-control study was performed with 64 IPF patients and 74 healthy controls. Ten single-nucleotide variants residing in the MUC5B, TOLLIP, SERPINB1, and PLAU genes were analyzed. Single- and multi-locus analyses were performed to investigate the predictive potential of specific variants in IPF susceptibility and survival. Multifactor dimensionality reduction (MDR) was employed to uncover predictive multi-locus interactions underlying IPF susceptibility. The MUC5B rs35705950 SNP was significantly associated with IPF: T allele carriers were significantly more frequent among IPF patients (75.0% vs 20.3%, P < 1.0 × 10-6). Genotypic and allelic distributions of TOLLIP, PLAU, and SERPINB1 SNPs did not differ significantly between groups. However, the MUC5B-TOLLIP T-C-T-C haplotype, defined by the rs35705950-rs111521887-rs5743894-rs5743854 block, emerged as an independent protective factor in IPF survival (HR = 0.37, 95% CI 0.17-0.78, P = 0.009, after adjustment for FVC). No significant multi-locus interactions correlating with disease susceptibility were detected. MUC5B rs35705950 was linked to an increased risk for IPF, as reported for other populations, but not to disease survival. A haplotype incorporating SNPs of the MUC5B-TOLLIP locus at 11p15.5 seems to predict better survival and could prove useful for prognostic purposes and IPF patient stratification. KEY MESSAGES : The MUC5B rs35705950 minor allele is associated with IPF risk in the Portuguese. No predictive multi-locus interactions of IPF susceptibility were identified by MDR. A haplotype defined by MUC5B and TOLLIP SNPs is a protective factor in IPF survival. The haplotype may be used as a prognostic tool for IPF patient stratification.

Spatial localization of cathepsins: Implications in immune activation and resolution during infections.

Cathepsins were first described, as endolysosomal proteolytic enzymes in reference to the organelles where they degrade the bulk of endogenous and exogenous substrates in a slightly acidic environment. These substrates include pathogens internalized via endocytosis and/or marked for destruction by autophagy. However, the role of cathepsins during infection far exceeds that of direct digestion of the pathogen. Cathepsins have been extensively investigated in the context of tumour associated immune cells and chronic inflammation. Several cathepsin-dependent immune responses develop in the endocytic pathway while others take place in the cytosol, the nucleus, or in the extracellular space. In this review we highlight the spatial localization of cathepsins and their implications in immune activation and resolution pathways during infection.

Pleuroparenchymal fibroelastosis as a complication of chemotherapy: A case report.

Pleuroparenchymal fibroelastosis (PPFE) is a rare and recently described distinct pattern of lung apical fibrosis involving the upper lobe parenchyma and pleural dome. PPFE has definable and reproducible clinical, radiological and histopathological criteria, which allowed its classification as an independent interstitial lung disease. Several factors have been associated with PPFE, such as chemotherapy, especially with alkylating agents. The authors present a case of a 34-year-old female with previous history of Hodgkin lymphoma treated with first line chemotherapy (doxorubicin, bleomycin, vinblastine and dacarbazine). The patient had no other known comorbidities or relevant exposure to lung irritants. A total of 2 years after completing cancer treatment, the patient developed clinical and radiological features of PPFE. Given their previous history of malignancy, a biopsy of the lesion was obtained, which confirmed the diagnosis of PPFE. The authors present this case to raise awareness of this disease and to demonstrate that PPFE can develop months to years following chemotherapy treatment. Moreover, to date, none of these chemotherapy agents have been associated with the development of PPFE.

The Role of Ultrasonography in the Diagnosis and Decision Algorithm for the Management of Pneumothorax after Transbronchial Lung Cryobiopsy.

BACKGROUND: Pneumothorax is one of the main complications of transbronchial lung cryobiopsy (TBLC). Chest ultrasound (CUS) is a radiation-free alternative method for pneumothorax detection. OBJECTIVE: We tested CUS diagnostic accuracy for pneumothorax and assessed its role in the decision algorithm for pneumothorax management. Secondary objectives were to evaluate the post-procedure pneumothorax occurrence and risk factors. METHODS: Eligible patients underwent TBLC, followed by chest X-ray (CXR) evaluation 2 h after the procedure, as our standard protocol. Bedside CUS was performed within 30 min and 2 h after TBLC. Pneumothorax by CUS was defined by the absence of lung sliding and comet-tail artefacts and confirmed with the stratosphere sign on M-mode. Pneumothorax size was determined through lung point projection on CUS and interpleural distance on CXR and properly managed according to clinical status. RESULTS: Sixty-seven patients were included. Nineteen pneumothoraces were detected at 2 h after the procedure, of which 8 (42.1%) were already present at the first CUS evaluation. All CXR-detected pneumothoraces had a positive CUS detection. There were 3 discordant cases (κ = 0.88, 95% CI: 0.76-1.00, p < 0.001), which were detected by CUS but not by inspiration CXR. We calculated a specificity of 97.5% (95% CI: 86.8-99.9) and a sensitivity of 100% (95% CI: 87.2-100) for CUS. Pneumothorax rate was higher when biopsies were taken in 2 lobes and if histology had pleural representation. Final diagnosis was achieved in 79.1% of patients, with the most frequent diagnosis being hypersensitivity pneumonitis. Regarding patients with large-volume pneumothorax needing drainage, the rate of detection was similar between CUS and CRX. CONCLUSION: CUS can replace CXR in detecting the presence of pneumothorax after TBLC, and the lung point site can reliably indicate its size. This useful method optimizes time spent at the bronchology unit and allows immediate response in symptomatic patients, helping to choose optimal treatment strategies, while preventing ionizing radiation exposure.

Modulation of Cystatin C in Human Macrophages Improves Anti-Mycobacterial Immune Responses to Mycobacterium tuberculosis Infection and Coinfection With HIV.

Tuberculosis owes its resurgence as a major global health threat mostly to the emergence of drug resistance and coinfection with HIV. The synergy between HIV and Mycobacterium tuberculosis (Mtb) modifies the host immune environment to enhance both viral and bacterial replication and spread. In the lung immune context, both pathogens infect macrophages, establishing favorable intracellular niches. Both manipulate the endocytic pathway in order to avoid destruction. Relevant players of the endocytic pathway to control pathogens include endolysosomal proteases, cathepsins, and their natural inhibitors, cystatins. Here, a mapping of the human macrophage transcriptome for type I and II cystatins during Mtb, HIV, or Mtb-HIV infection displayed different profiles of gene expression, revealing cystatin C as a potential target to control mycobacterial infection as well as HIV coinfection. We found that cystatin C silencing in macrophages significantly improves the intracellular killing of Mtb, which was concomitant with an increased general proteolytic activity of cathepsins. In addition, downmodulation of cystatin C led to an improved expression of the human leukocyte antigen (HLA) class II in macrophages and an increased CD4+ T-lymphocyte proliferation along with enhanced IFN-γ secretion. Overall, our results suggest that the targeting of cystatin C in human macrophages represents a promising approach to improve the control of mycobacterial infections including multidrug-resistant (MDR) TB.

Origin, phylogeny, variability and epitope conservation of SARS-CoV-2 worldwide.

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses innumerous challenges, like understanding what triggered the emergence of this new human virus, how this RNA virus is evolving or how the variability of viral genome may impact the primary structure of proteins that are targets for vaccine. We analyzed 19471 SARS-CoV-2 genomes available at the GISAID database from all over the world and 3335 genomes of other Coronoviridae family members available at GenBank, collecting SARS-CoV-2 high-quality genomes and distinct Coronoviridae family genomes. Additionally, we analyzed 199,984 spike glycoprotein sequences. Here, we identify a SARS-CoV-2 emerging cluster containing 13 closely related genomes isolated from bat and pangolin that showed evidence of recombination, which may have contributed to the emergence of SARS-CoV-2. The analyzed SARS-CoV-2 genomes presented 9632 single nucleotide variants (SNVs) corresponding to a variant density of 0.3 over the genome, and a clear geographic distribution. SNVs are unevenly distributed throughout the genome and hotspots for mutations were found for the spike gene and ORF 1ab. We describe a set of predicted spike protein epitopes whose variability is negligible. Additionally, all predicted epitopes for the structural E, M and N proteins are highly conserved. The amino acid changes present in the spike glycoprotein of variables of concern (VOCs) comprise between 3.4% and 20.7% of the predicted epitopes of this protein. These results favors the continuous efficacy of the available vaccines targeting the spike protein, and other structural proteins. Multiple epitopes vaccines should sustain vaccine efficacy since at least some of the epitopes present in variability regions of VOCs are conserved and thus recognizable by antibodies.