Investigation of the immunological mechanisms involved in the response to fungi and characterization of Brazilian patients with susceptibility to mycoses

As micoses sistêmicas e micoses oportunistas que cursam de forma disseminada são uma preocupação mundial devido às suas elevadas taxas de morbimortalidade. Dentre os componentes associados à resposta imunológica frente a infecções fúngicas, destacam-se na imunidade inata os receptores de padrão molecular, tais quais receptores do tipo Toll e receptores do tipo lectina C, as células do sistema fagocítico monócitos/macrófagos e os neutrófilos e seus mecanismos efetores, como a produção de citocinas pró-inflamatórias, o burst oxidativo e a geração de armadilhas extracelulares de neutrófilos. Tal conhecimento é ratificado pelo estudo de pacientes com Erros Inatos da Imunidade (EII), conjunto de doenças causadas por alterações genéticas em componentes do sistema imune que tornam indivíduos afetados propensos a infecções graves e recorrentes causadas por diversas classes de microrganismos, dentre os quais os fungos. Complementarmente a abordagem de genômica amplamente utilizada em estudos relacionados aos EII, técnicas modernas tais quais sequenciamento de transcriptoma e análises de single-cell permitem analisar não somente as alterações em nível celular, mas também o impacto sistêmico destas. Assim, o presente trabalho teve duas linhas principais de estudo: a investigação dos mecanismos imunológicos na resposta a fungos por meio de uma análise integrativa realizada com dados públicos e a caracterização de pacientes brasileiros com suscetibilidade a micoses. Para isso, com o auxílio de ferramentas de bioinformática, oito diferentes dados públicos de transcriptoma humano infectados com Candida spp. foram avaliados. Identificamos uma rede de interação conectando as vias de receptores do tipo Toll e cascata de sinalização por interferon tipo I/II moduladas em resposta a Candida spp., reforçando estudos anteriores e demonstrando uma interessante sobreposição com a resposta imune antiviral. Em relação ao estudo com enfoque nos EII, realizamos a triagem de pacientes a partir da avaliação do Eixo IL-12/IFN-&#947 e do Burst Oxidativo , além da caracterização genética por Whole Exome Sequencing e Sanger . Foram incluídos no estudo oito pacientes, dos quais quatro apresentaram alterações nos exames de triagem; entretanto considerando histórico clínico e familiar, todos foram encaminhados para investigação genética. De acordo com análises de predição in silico , encontramos variantes provavelmente patogênicas em três destes pacientes, sendo que para a paciente P3 estas apresentaram-se como de significado incerto. Identificamos uma família (de P1 e P2) afetada pela variante patogênica conhecida como Santiago de Cuba no gene G6PD cujos indivíduos acometidos apresentaram baixa produção de ROS e suscetibilidade à histoplasmose disseminada, variante ainda não identificada em indivíduos brasileiros e associação até o momento não descrita na literatura. O estudo da relação entre suscetibilidade a micoses sistêmicas e EII torna-se fundamental na busca da compreensão e caracterização destas doenças, auxiliando no seu diagnóstico, nas estratégias terapêuticas aplicadas e, consequentemente, na qualidade de vida de pacientes afetadosSystemic mycoses and opportunistic mycoses that occur in a disseminated way are a worldwide concern due to their high rates of morbidity and mortality. Among the components associated with the immune response to fungal infections, are highlighted in innate immunity the molecular pattern receptors, such as Toll-like receptors and C-type lectin receptors, cells of the phagocytic system, monocytes/macrophages and neutrophils as well as their effector mechanisms, including the production of pro-inflammatory cytokines, oxidative burst and also the generation of extracellular neutrophil traps. Such knowledge is confirmed by the study of patients with Inborn Errors of Immunity (IEI), diseases caused by genetic alterations in immune systems components that make affected individuals susceptible to severe and recurrent infections caused by several classes of microorganisms, among which the fungi. In addition to the genomics approach widely used in studies related to IEI, modern techniques such as transcriptome sequencing and single-cell analysis allow analyzing not only alterations at the cellular level, but also their systemic impact. Thus, the present study had two main research lines: investigation of immunological mechanisms in the response to fungi through an integrative analysis performed with public datasets and the characterization of Brazilian patients with susceptibility to mycoses. To this end, approaching by bioinformatics tools, eight different public data of human transcriptome infected with Candida spp. were evaluated. We identified an interaction network connecting the Toll-like receptor pathways and the type I/II interferon signaling cascade modulated in response to Candida spp., reinforcing previous studies and demonstrating an interesting overlap with the antiviral immune response. Regarding the study focusing on IEI, we performed a screening of patients based on the evaluation of IL-12/IFN-&#947 axis and Oxidative Burst, in addition to the genetic characterization by Whole Exome Sequencing and Sanger. Eight patients were included in the study, four of which had alterations in the screening tests; however, considering clinical and family history, all of them were referred for genetic investigation. According to in silico prediction analyses, we found likely pathogenic variants in three of these patients, and for patient P3 they were of uncertain significance. We identified a family (of patients P1 and P2) affected by the pathogenic variant known as Santiago de Cuba in the G6PD gene, whose affected individuals had low ROS production and susceptibility to disseminated histoplasmosis, a variant not yet identified in Brazilian individuals and an association not yet described in the literature. The study of the relationship between susceptibility to systemic mycoses and IEI becomes fundamental in the search for understanding and characterization of these diseases, helping in their diagnosis, in the therapeutic strategies applied and, consequently, in the quality of life of patients

Image_1_Immunological signatures unveiled by integrative systems vaccinology characterization of dengue vaccination trials and natural infection.pdf

IntroductionDengue virus infection is a global health problem lacking specific therapy, requiring an improved understanding of DENV immunity and vaccine responses. Considering the recent emerging of new dengue vaccines, here we performed an integrative systems vaccinology characterization of molecular signatures triggered by the natural DENV infection (NDI) and attenuated dengue virus infection models (DVTs).Methods and resultsWe analyzed 955 samples of transcriptomic datasets of patients with NDI and attenuated dengue virus infection trials (DVT1, DVT2, and DVT3) using a systems vaccinology approach. Differential expression analysis identified 237 common differentially expressed genes (DEGs) between DVTs and NDI. Among them, 28 and 60 DEGs were up or downregulated by dengue vaccination during DVT2 and DVT3, respectively, with 20 DEGs intersecting across all three DVTs. Enriched biological processes of these genes included type I/II interferon signaling, cytokine regulation, apoptosis, and T-cell differentiation. Principal component analysis based on 20 common DEGs (overlapping between DVTs and our NDI validation dataset) distinguished dengue patients by disease severity, particularly in the late acute phase. Machine learning analysis ranked the ten most critical predictors of disease severity in NDI, crucial for the anti-viral immune response. ConclusionThis work provides insights into the NDI and vaccine-induced overlapping immune response and suggests molecular markers (e.g., IFIT5, ISG15, and HERC5) for anti-dengue-specific therapies and effective vaccination development. </p

Table_1_Immunological signatures unveiled by integrative systems vaccinology characterization of dengue vaccination trials and natural infection.xlsx

IntroductionDengue virus infection is a global health problem lacking specific therapy, requiring an improved understanding of DENV immunity and vaccine responses. Considering the recent emerging of new dengue vaccines, here we performed an integrative systems vaccinology characterization of molecular signatures triggered by the natural DENV infection (NDI) and attenuated dengue virus infection models (DVTs).Methods and resultsWe analyzed 955 samples of transcriptomic datasets of patients with NDI and attenuated dengue virus infection trials (DVT1, DVT2, and DVT3) using a systems vaccinology approach. Differential expression analysis identified 237 common differentially expressed genes (DEGs) between DVTs and NDI. Among them, 28 and 60 DEGs were up or downregulated by dengue vaccination during DVT2 and DVT3, respectively, with 20 DEGs intersecting across all three DVTs. Enriched biological processes of these genes included type I/II interferon signaling, cytokine regulation, apoptosis, and T-cell differentiation. Principal component analysis based on 20 common DEGs (overlapping between DVTs and our NDI validation dataset) distinguished dengue patients by disease severity, particularly in the late acute phase. Machine learning analysis ranked the ten most critical predictors of disease severity in NDI, crucial for the anti-viral immune response. ConclusionThis work provides insights into the NDI and vaccine-induced overlapping immune response and suggests molecular markers (e.g., IFIT5, ISG15, and HERC5) for anti-dengue-specific therapies and effective vaccination development. </p

Severe COVID-19 Shares a Common Neutrophil Activation Signature with Other Acute Inflammatory States

Severe COVID-19 patients present a clinical and laboratory overlap with other hyperinflammatory conditions such as hemophagocytic lymphohistiocytosis (HLH). However, the underlying mechanisms of these conditions remain to be explored. Here, we investigated the transcriptome of 1596 individuals, including patients with COVID-19 in comparison to healthy controls, other acute inflammatory states (HLH, multisystem inflammatory syndrome in children [MIS-C], Kawasaki disease [KD]), and different respiratory infections (seasonal coronavirus, influenza, bacterial pneumonia). We observed that COVID-19 and HLH share immunological pathways (cytokine/chemokine signaling and neutrophil-mediated immune responses), including gene signatures that stratify COVID-19 patients admitted to the intensive care unit (ICU) and COVID-19_nonICU patients. Of note, among the common differentially expressed genes (DEG), there is a cluster of neutrophil-associated genes that reflects a generalized hyperinflammatory state since it is also dysregulated in patients with KD and bacterial pneumonia. These genes are dysregulated at the protein level across several COVID-19 studies and form an interconnected network with differentially expressed plasma proteins that point to neutrophil hyperactivation in COVID-19 patients admitted to the intensive care unit. scRNAseq analysis indicated that these genes are specifically upregulated across different leukocyte populations, including lymphocyte subsets and immature neutrophils. Artificial intelligence modeling confirmed the strong association of these genes with COVID-19 severity. Thus, our work indicates putative therapeutic pathways for intervention

Protein Design: Toward Functional Metalloenzymes

The scope of this Review is to discuss the construction of metal sites in designed protein scaffolds. We categorize the effort of designing proteins into redesign, which is to rationally engineer desired functionality into an existing protein scaffold,(1-9) and de novo design, which is to build a peptidic or protein system that is not directly related to any sequence found in nature yet folds into a predicted structure and/or carries out desired reactions.(10-12) We will analyze and interpret the significance of designed protein systems from a coordination chemistry and biochemistry perspective, with an emphasis on those containing constructed metal sites as mimics for metalloenzymes

Candida bloodstream infections in intensive care units: analysis of the extended prevalence of infection in intensive care unit study

Item does not contain fulltextOBJECTIVES: To provide a global, up-to-date picture of the prevalence, treatment, and outcomes of Candida bloodstream infections in intensive care unit patients and compare Candida with bacterial bloodstream infection. DESIGN: A retrospective analysis of the Extended Prevalence of Infection in the ICU Study (EPIC II). Demographic, physiological, infection-related and therapeutic data were collected. Patients were grouped as having Candida, Gram-positive, Gram-negative, and combined Candida/bacterial bloodstream infection. Outcome data were assessed at intensive care unit and hospital discharge. SETTING: EPIC II included 1265 intensive care units in 76 countries. PATIENTS: Patients in participating intensive care units on study day. INTERVENTIONS: None. MEASUREMENT AND MAIN RESULTS: Of the 14,414 patients in EPIC II, 99 patients had Candida bloodstream infections for a prevalence of 6.9 per 1000 patients. Sixty-one patients had candidemia alone and 38 patients had combined bloodstream infections. Candida albicans (n = 70) was the predominant species. Primary therapy included monotherapy with fluconazole (n = 39), caspofungin (n = 16), and a polyene-based product (n = 12). Combination therapy was infrequently used (n = 10). Compared with patients with Gram-positive (n = 420) and Gram-negative (n = 264) bloodstream infections, patients with candidemia were more likely to have solid tumors (p < .05) and appeared to have been in an intensive care unit longer (14 days [range, 5-25 days], 8 days [range, 3-20 days], and 10 days [range, 2-23 days], respectively), but this difference was not statistically significant. Severity of illness and organ dysfunction scores were similar between groups. Patients with Candida bloodstream infections, compared with patients with Gram-positive and Gram-negative bloodstream infections, had the greatest crude intensive care unit mortality rates (42.6%, 25.3%, and 29.1%, respectively) and longer intensive care unit lengths of stay (median [interquartile range]) (33 days [18-44], 20 days [9-43], and 21 days [8-46], respectively); however, these differences were not statistically significant. CONCLUSION: Candidemia remains a significant problem in intensive care units patients. In the EPIC II population, Candida albicans was the most common organism and fluconazole remained the predominant antifungal agent used. Candida bloodstream infections are associated with high intensive care unit and hospital mortality rates and resource use

Candida bloodstream infections in intensive care units: analysis of the extended prevalence of infection in intensive care unit study

To provide a global, up-to-date picture of the prevalence, treatment, and outcomes of Candida bloodstream infections in intensive care unit patients and compare Candida with bacterial bloodstream infection. DESIGN: A retrospective analysis of the Extended Prevalence of Infection in the ICU Study (EPIC II). Demographic, physiological, infection-related and therapeutic data were collected. Patients were grouped as having Candida, Gram-positive, Gram-negative, and combined Candida/bacterial bloodstream infection. Outcome data were assessed at intensive care unit and hospital discharge. SETTING: EPIC II included 1265 intensive care units in 76 countries. PATIENTS: Patients in participating intensive care units on study day. INTERVENTIONS: None. MEASUREMENT AND MAIN RESULTS: Of the 14,414 patients in EPIC II, 99 patients had Candida bloodstream infections for a prevalence of 6.9 per 1000 patients. Sixty-one patients had candidemia alone and 38 patients had combined bloodstream infections. Candida albicans (n = 70) was the predominant species. Primary therapy included monotherapy with fluconazole (n = 39), caspofungin (n = 16), and a polyene-based product (n = 12). Combination therapy was infrequently used (n = 10). Compared with patients with Gram-positive (n = 420) and Gram-negative (n = 264) bloodstream infections, patients with candidemia were more likely to have solid tumors (p < .05) and appeared to have been in an intensive care unit longer (14 days [range, 5-25 days], 8 days [range, 3-20 days], and 10 days [range, 2-23 days], respectively), but this difference was not statistically significant. Severity of illness and organ dysfunction scores were similar between groups. Patients with Candida bloodstream infections, compared with patients with Gram-positive and Gram-negative bloodstream infections, had the greatest crude intensive care unit mortality rates (42.6%, 25.3%, and 29.1%, respectively) and longer intensive care unit lengths of stay (median [interquartile range]) (33 days [18-44], 20 days [9-43], and 21 days [8-46], respectively); however, these differences were not statistically significant. CONCLUSION: Candidemia remains a significant problem in intensive care units patients. In the EPIC II population, Candida albicans was the most common organism and fluconazole remained the predominant antifungal agent used. Candida bloodstream infections are associated with high intensive care unit and hospital mortality rates and resource use

Evaluation of a quality improvement intervention to reduce anastomotic leak following right colectomy (EAGLE): pragmatic, batched stepped-wedge, cluster-randomized trial in 64 countries

Background Anastomotic leak affects 8 per cent of patients after right colectomy with a 10-fold increased risk of postoperative death. The EAGLE study aimed to develop and test whether an international, standardized quality improvement intervention could reduce anastomotic leaks. Methods The internationally intended protocol, iteratively co-developed by a multistage Delphi process, comprised an online educational module introducing risk stratification, an intraoperative checklist, and harmonized surgical techniques. Clusters (hospital teams) were randomized to one of three arms with varied sequences of intervention/data collection by a derived stepped-wedge batch design (at least 18 hospital teams per batch). Patients were blinded to the study allocation. Low- and middle-income country enrolment was encouraged. The primary outcome (assessed by intention to treat) was anastomotic leak rate, and subgroup analyses by module completion (at least 80 per cent of surgeons, high engagement; less than 50 per cent, low engagement) were preplanned. Results A total 355 hospital teams registered, with 332 from 64 countries (39.2 per cent low and middle income) included in the final analysis. The online modules were completed by half of the surgeons (2143 of 4411). The primary analysis included 3039 of the 3268 patients recruited (206 patients had no anastomosis and 23 were lost to follow-up), with anastomotic leaks arising before and after the intervention in 10.1 and 9.6 per cent respectively (adjusted OR 0.87, 95 per cent c.i. 0.59 to 1.30; P = 0.498). The proportion of surgeons completing the educational modules was an influence: the leak rate decreased from 12.2 per cent (61 of 500) before intervention to 5.1 per cent (24 of 473) after intervention in high-engagement centres (adjusted OR 0.36, 0.20 to 0.64; P &lt; 0.001), but this was not observed in low-engagement hospitals (8.3 per cent (59 of 714) and 13.8 per cent (61 of 443) respectively; adjusted OR 2.09, 1.31 to 3.31). Conclusion Completion of globally available digital training by engaged teams can alter anastomotic leak rates. Registration number: NCT04270721 (http://www.clinicaltrials.gov)