Introduction:Tuberculosis (TB) continues to be one of the leading infectious causes of mortality world-wide, while the Bacillus Calmette-Guérin (BCG) vaccine provides variable protection. To address this limitation, our group developed a recombinant BCG strain expressing a detoxified Escherichia coli heat-labile toxin subunit (rBCG-LTAK63), which confers superior protection against Mycobacterium tuberculosis (Mtb). However, the mechanisms underlying this enhanced efficacy remain to be better characterized. Here, we investigated the capacity of rBCG-LTAK63 to enhance inflammasome-associated innate immune responses and its impact on T cell activation and protection against Mtb. Methods:Bone marrow-derived macrophages (BMDMs) from wild-type (C57Bl/6), Casp-1-/-, Nlrp3-/-, Aim-2-/- and phenotypically selected AIRmax and AIRminTT mice were used to evaluate inflammasome-associated responses using IL-1β as a primary readout. A co-culture system of inflammasome-activated BMDMs and splenocytes was employed to assess CD4+ T cell activation and polarization. Additionally, immunization of AIRmax and AIRminTT mice with BCG or rBCG-LTAK63 was performed to evaluate protection against Mtb. Results:rBCG-LTAK63 induced significantly higher IL-1β production than parental BCG. IL-1β production was largely ASC-associated and predominantly dependent on the NLRP3/caspase-1 axis; however, residual IL-1β production was still detected in Casp-/- and Nlrp3-/- BMDMs, indicating the contribution of additional processing pathways. Co-culture of inflammasome-activated BMDMs and splenocytes showed that rBCG-LTAK63-primed macrophages strongly promoted CD4+ T cell activation and polarization toward Th1/Th17 responses. In vivo, BCG only induced protection in AIRmax mice, while rBCG-LTAK63 induced protection in both AIRmax and AIRminTT genotypes. Conclusion:This demonstrates that protection is achieved in a diminished innate inflammatory response scenario, but its full engagement can further reduce pulmonary bacterial loads. Together, these findings demonstrate that rBCG-LTAK63 enhances protection against TB through a broad innate activation including inflammasome-associated mechanisms.

A scientific study begins with a central question, and search engines like PubMed are the first tools for retrieving knowledge and understanding the current state of the art. Large Language Models (LLMs) have been used in research, promising acceleration and deeper results. However, besides caution, they demand rigorous validation. Assessing complex biological relationships remains challenging for SQL-based tools and LLM models. Here, we introduce the Digital Pathway Curation (DPC) pipeline to evaluate the reproducibility and accuracy of the Gemini models against PubMed search and human expert curation. Using two omics experiments, we created a large dataset (Ensemble) based on determining pathway-disease associations. With the Ensemble dataset, we demonstrate that Gemini achieves high run-to-run reproducibility of approximately 99 calculate the crowdsourced consensus using a smaller dataset. The CSC allows us to calculate accuracies, and the Gemini multi-model consensus reached a significant accuracy of about 87 reproducible, reliable, and valuable tools for navigating complex biomedical knowledge.

The incorporation of drugs into nanostructured silica has proven to be an effective strategy for delaying drug release, protecting against enzymatic degradation, and enhancing therapeutic efficacy. Specifically, crotoxin, a component derived from the venom of Crotalus durissus terrificus, exhibits notable analgesic and immunomodulatory properties. Previous studies have demonstrated that encapsulating crotoxin within SBA-15 nanostructured mesoporous silica not only reduces its toxicity and enhances its analgesic effects but also enables effective oral administration. Given its promising efficacy and the expanding interest in its application across various experimental models and potential therapeutic uses, this study aimed to conduct a detailed analysis of the physicochemical properties of crotoxin when incorporated into SBA-15 silica. Following characterization, the crotoxin-SBA-15 complex was orally administered to mice in an experimental model of ulcerative colitis (UC). The most widely adopted experimental model for studying UC involves the administration of dextran sodium sulfate (DSS) in drinking water to induce colonic inflammation in susceptible animals. In this study, we hypothesized that crotoxin incorporated into ordered mesoporous silica (SBA-15) could modulate DSS-induced UC. Crotoxin was successfully incorporated into SBA-15 and administered orally, as its physicochemical properties supported this route of delivery. Mice received the crotoxin-SBA-15 complex either at the onset of UC induction or on days 1 and 4 after DSS exposure. Seven days after the start of DSS administration, we observed a substantial reduction (approximately 50%) in Disease Activity Index (DAI) scores, accompanied by marked improvements in the histopathological features of the colon. These findings indicate for the first time that crotoxin incorporated into SBA-15 exhibits significant therapeutic potential in the treatment of experimentally induced ulcerative colitis.

This study explores the direct effects of amphibian skin secretions on human cells involved in joint diseases, aiming to identify species with potential for inflammatory modulation. Secretions were obtained from sixteen species distributed across Brazilian biomes and one European species. Following biochemical characterization, human chondrocytes, synoviocytes, and macrophages were treated with secretions for 24 h. The cytotoxicity and modulation of the IL-6, IL-8, TNF-α, and IL-1β release were assessed. Synoviocytes showed the greatest resistance to cytotoxic effects, though sensitivity varied by species. Secretions from Trachycephalus mesophaeus, Pipa carvalhoi, and Phyllomedusa bahiana exhibited the highest cytotoxicity. At non-cytotoxic concentrations, P. carvalhoi and Leptodactylus fuscus strongly induced IL-6 and IL-8 in chondrocytes and synoviocytes, with P. carvalhoi also stimulating IL-1β and TNF-α release in macrophages. Among Bufonidae species, particularly Rhinella jimi and Bufo bufo, were potent inducers of TNF-α and IL-1β in macrophages. Secretions lacking pro-inflammatory effects were further tested for anti-inflammatory activity. P. bahiana reduced TNF-α production in stimulated macrophages and IL-6 in synoviocytes, while Siphonops annulatus and T. mesophaeus reduced LPS-induced TNF-α in macrophages. Our data underscore the rich biodiversity of amphibians, supporting the bioprospecting of their cutaneous secretions. These data reveal substantial potential for uncovering bioactive compounds with pharmacological applications.

Ulcerative Colitis (UC) is an inflammatory disease characterized by colonic mucosal lesions associated with an increased risk of carcinogenesis. UC pathogenesis involves environmental and genetic factors. Genetic studies have indicated the association of gene variants coding for the divalent metal ion transporter SLC11A1 protein (formerly NRAMP1) with UC susceptibility in several animal species. Two mouse lines were genetically selected for high (AIRmax) or low (AIRmin) acute inflammatory responses (AIR). AIRmax is susceptible, and AIRmin is resistant to DSS-induced colitis and colon carcinogenesis. Furthermore, AIRmin mice present polymorphism of the Slc11a1 gene. Here we investigated the possible modulating effect of the Slc11a1 R and S variants in DSS-induced colitis by using AIRmin mice homozygous for Slc11a1 R (AIRmin RR ) or S (AIRmin SS ) alleles. We evaluated UC by the disease activity index (DAI), considering weight loss, diarrhea, blood in the anus or feces, cytokines, histopathology, and cell populations in the distal colon epithelium. AIRmin SS mice have become susceptible to DSS effects, with higher DAI, IL6, G-CSF, and MCP-1 production and morphological and colon histopathological alterations than AIRmin RR mice. The results point to a role of the Slc11a1 S allele in DSS colitis induction in the genetic background of AIRmin mice.

Bothrops jararaca venom (BjV) can induce mast cell degranulation. In order to investigate the role of mast cells and the interference of the host genetic background in the inflammation induced by BjV, we have used mouse strains selected for maximal (AIRmax) or minimal (AIRmin) acute inflammatory response (AIR). Mice were pretreated with an inhibitor of mast cell degranulation, cromolyn (CROM), and injected in footpads or intraperitoneally (i.p.) with BjV. Pain was measured with von Frey hairs, cell migration in the peritoneum by flow cytometry, and reactive oxygen species (ROS) production by chemiluminescence assays. The nociceptive response to BjV was higher in AIRmax than AIRmin mice; however, this difference was abolished by pretreatment with CROM. BjV induced peritoneal neutrophil (CD11b + GR-1 + ) infiltration and ROS secretion in AIRmax mice only, which were partially inhibited by CROM. Our findings evidence a role for mast cells in pain, neutrophil migration, and ROS production triggered by BjV in AIRmax mice that are more susceptible to the action of BjV.

We identified Pycard and BC017158 genes as putative effectors of the Quantitative Trait locus (QTL) that we mapped at distal chromosome 7 named Irm1 for Inflammatory response modulator 1, controlling acute inflammatory response (AIR) and the production of IL-1β, dependent on the activation of the NLRP3 inflammasome. We obtained the mapping through genome-wide linkage analysis of Single Nucleotide Polymorphisms (SNPs) in a cross between High (AIRmax) and Low (AIRmin) responder mouse lines that we produced by several generations of bidirectional selection for Acute Inflammatory Response. A highly significant linkage signal (LOD score peak of 72) for ex vivo IL-1β production limited a 4 Mbp interval to chromosome 7. Sequencing of the locus region revealed 14 SNPs between “High” and “Low” responders that narrowed the locus to a 420 Kb interval. Variants were detected in non-coding regions of Itgam, Rgs10 and BC017158 genes and at the first exon of Pycard gene, resulting in an E19K substitution in the protein ASC (apoptosis associated speck-like protein containing a CARD) an adaptor molecule in the inflammasome complex. Silencing of BC017158 inhibited IL1-β production by stimulated macrophages and the E19K ASC mutation carried by AIRmin mice impaired the ex vivo IL-1β response and the formation of ASC specks in stimulated cells. IL-1β and ASC specks play major roles in inflammatory reactions and in inflammation-related diseases. Our results delineate a novel genetic factor and a molecular mechanism affecting the acute inflammatory response.

Two non-inbred mouse lines, phenotypically selected for maximal (AIRmin) and minimal (AIRmax) acute inflammatory response, show differential susceptibility/resistance to the development of several chemically-induced tumor types. An intercross pedigree of these mice was generated and treated with the chemical carcinogen dimethylhydrazine, which induces lung and intestinal tumors. Genome wide high-density genotyping with the Restriction Site-Associated DNA genotyping (2B-RAD) technique was used to map genetic loci modulating individual genetic susceptibility to both lung and intestinal cancer. Our results evidence new common quantitative trait loci (QTL) for those phenotypes and provide an improved understanding of the relationship between genomic variation and individual genetic predisposition to tumorigenesis in different organs.

Apoptosis-associated speck-like protein containing a caspase recruit domain (ASC), encoded by PYCARD gene, is a 22 kDa small molecule, which aggregates into ASC specks during inflammasome activation. ASC protein is an adaptor protein present in several inflammasome complexes that performs several intra- and extracellular functions, in monomeric form or as ASC specks, during physiological and pathological processes related to inflammation and adaptive immunity. Extracellular ASC specks (eASC specks) released during cell death by pyroptosis can contribute as a danger signal to the propagation of inflammation via phagocytosis and activation of surrounding cells. ASC specks are found in the circulation of patients with chronic inflammatory diseases and have been considered as relevant blood biomarkers of inflammation. eASC amplifies the inflammatory signal, may induce the production of autoantibodies, transports molecules that bind to this complex, contributing to the generation of antibodies, and can induce the maturation of cytokines promoting the modelling of the adaptive immunity. Although several advances have been registered in the last 21 years, there are numerous unknown or enigmatic gaps in the understanding of the role of eASC specks in the organism. Here, we provide an overview about the ASC protein focusing on the probable roles of eASC specks in several diseases, up to the most recent studies concerning COVID-19.

The inflammatory and autoimmune events preceding clinical symptoms in rheumatoid arthritis (RA) and other autoimmune diseases are difficult to study in human patients. Therefore, animal models that share immunologic and clinical features with human RA, such as pristane-induced arthritis (PIA), are valuable tools for assessing the primordial events related to arthritis susceptibility. PIA-resistant HIII and susceptible LIII mice were injected i.p. with pristane, and peritoneal lavage fluid was harvested in the early (7 days) and late (35 days) preclinical phases of PIA. Chemokine and cytokine levels were measured in lavage supernatant with ELISA, peritoneal inflammatory leukocytes were immunophenotyped by flow cytometry, and gene expression was determined by qRT-PCR. Leukocyte recruitment was quantitatively and qualitatively divergent in the peritoneum of HIII and LIII mice, with an early increase of CC chemokines (CCL2/CCL3/CCL5/CCL12/CCL22) in the susceptible LIII strain. Also, cytokines such as IL-12p40, IL-23, and IL-18 were elevated in LIII mice while IL-6 was increased in HIII animals. The results show that an early peritoneal CC chemokine response is an important feature of arthritis susceptibility and defines potential biomarkers in this model.

AIRmax and AIRmin mouse strains phenotypically selected for high and low acute inflammatory responsiveness (AIR) are, respectively, susceptible or resistant to developing hepatocellular carcinoma (HCC) induced by the chemical carcinogens urethane and diethylnitrosamine (DEN). Early production of TNF-α, IL-1β, and IL-6 in the liver after DEN treatment correlated with tumor development in AIRmax mice. Transcriptome analysis of livers from untreated AIRmax and AIRmin mice showed specific gene expression profiles in each line, which might play a role in their differential susceptibility to HCC. Linkage analysis with SNP markers in F2 (AIRmax×AIRmin) intercross mice revealed two quantitative trait loci (QTL) in chromosomes 2 and 9, which are significantly associated with the number and progression of urethane-induced liver tumors. An independent linkage analysis with an intercross population from A/J and C57BL/6J inbred mice mapped regions in chromosomes 1 and 7 associated with the progression of urethane-induced liver tumors, evidencing the heterogeneity of HCC genetic control.

Purpose: Rheumatoid Arthritis (RA) is an autoimmune and chronic inflammatory disease that affects all articulations, mainly extremity joints. The NLRP3 inflammasome activation pathway seems to play a key role in several diseases related to inflammation, including RA. Outbred mouse lines, named AIRmax (maximal inflammatory reactivity) and AIRmin (minimal inflammatory reactivity), established after phenotypic selection for acute inflammation induced by polyacrylamide beads (Biogel), diverge widely in Pristane Induced Arthritis (PIA) susceptibility, with an incidence of 65% in AIRmax and only 7% in AIRmin at 200 days post-induction. Bioinformatics analysis comparing transcriptome datasets from synovial tissue of rheumatoid arthritis patients and from paws of pristane-treated and control AIRmax and AIRmin mice revealed that common relevant pathways are enriched in human RA and the PIA model. Linkage analysis performed in (AIRmax X AIRmin) F2 intercrosses mapped a major locus, Inflammatory response modulator 1 (Irm1) at distal chr 7 controlling acute inflammation intensity and quantitative IL-1β production through inflammasome NLRP3 activation. One single nucleotide polymorphism at this locus with alternative C/T alleles is situated at exon 1 of Pycard gene encoding PYCARD or ASC (apoptosis associated speck-like protein containing a caspase recruitment domain) the adaptor protein of inflammasome complex which forms aggregates (ASC specks) leading to cell death by pyroptosis. The C/T mutation causes a substitution of glutamic acid by lysine at position 19 of the protein (E19K). AIRmax mice present the WT allele (E19) whereas the majority of AIRmin mice present the K19 mutation. Methods: In order to evaluate the role of inflammation during PIA, AIRmax/AIRmin groups were given two ip injections of 0.5 mL pristane at 60 days intervals. After the first signal of arthritis, susceptible AIRmax were randomly separated into control and Prednisolone treated groups (20mg/Kg bw po/day) up to 200 days. In vivo X-ray images were acquired during PIA and bone remodeling was confirmed by fluorescent biphosponate probe. The cytokine levels in the serum were evaluated by Luminex assay. AIRmin mice bearing the 3 genotypes at Pycard (AIRminCC, AIRminCT and AIRminTT) were produced by genotype assisted mating to evaluate the effect of Pycard genotype in inflammatory phenotypes. AIRmax are homozygous for the CC allele. IL-1β secretion by circulating leukocytes was evaluated by ELISA and ASC-Speck formation in BMDM from AIRmax, AIRminCC and AIRminTT after NLRP3 inflammasome activation using LPS (1ug/ml) 3h and ATP (5mM) 30 minutes was visualized by confocal microscopy. Results: X -ray images indicates that AIRmax and AIRmin differ in arthritis development in the PIA model. AIRmax present edema and bone erosion that were reverted by prednisolone treatment. In addition, this treatment down modulated IL17A, MCP-1, MIP-1alpha and TNF-alpha levels in the serum of arthritic mice (Figure 1). IL-1β production by circulating leukocytes after inflammasome activation was higher in AIRmax CC and AIRmin CC mice whereas AIRmin CT and AIRmin TT were non responsive. (Figure 2). Moreover, in the same LPS/ATP assay we have observed large ASC-SPECK formation in AIRmax, small ASC specks in AIRmin CC but not in AIRminTT (Figure 3). Conclusions: All data suggest that E19K mutation in Pycard gene from AIRmin mice interfere in the IL-1β secretion via ASC-SPECK inhibition. Several works have shown that PYCARD is involved in Rheumatoid arthritis in humans. The E19K mutation in Pycard gene carried by AIRmin mice could contribute to their resistance to develop PIA. IL-17A, MCP-1, MIP-1alpha and TNF-alpha are potential biomarkers for RA in PIA model.View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Mouse lines selected for maximal (AIRmax) or minimal acute inflammatory reaction (AIRmin) were used to characterize the immune response and the influence of genetic background during pristane-induced arthritis (PIA). Susceptible AIRmax mice demonstrated exacerbated cellular profiles during PIA, with intense infiltration of lymphocytes, as well as monocytes/macrophages and neutrophils, producing higher levels of IL-1β, IFN-γ, TNF-α, IL-10, total IgG3, and chemokines. Resistant AIRmin mice controlled cell activation more efficiently than the AIRmax during arthritis progression. The weight alterations of the spleen and thymus in the course of PIA were observed. Our data suggest that selected AIRmax cellular and genetic immune mechanisms contribute to cartilage damage and arthritis severity, evidencing many targets for therapeutic actions.

Pristane-induced arthritis (PIA) in mice is an experimental model that resembles human rheumatoid arthritis, a chronic autoimmune disease that affects joints and is characterized by synovial inflammation and articular cartilage and bone destruction. AIRmax and AIRmin mouse lines differ in their susceptibility to PIA, and linkage analysis in this model mapped arthritis severity QTLs in chromosomes 5 and 8. miRNAs are a class of small RNA molecules that have been extensively studied in the development of arthritis. We analyzed miRNA and gene expression profiles in peritoneal cells of AIRmax and AIRmin lines, in order to evaluate the genetic architecture in this model. Susceptible AIRmax mice showed higher gene (2025 vs 1043) and miRNA (240 vs 59) modulation than resistant AIRmin mice at the onset of disease symptoms. miR-132-3p/212-3p, miR-106-5p, miR-27b-3p, and miR-25-3p were among the miRNAs with the highest expression in susceptible animals, showing a negative correlation with the expression of predicted target genes (Il10, Cd69, and Sp1r1). Our study showed that global gene and miRNA expression profiles in peritoneal cells of susceptible AIRmax and resistant AIRmin lines during pristane-induced arthritis are distinct, evidencing interesting targets for further validation.

Abstract A SNP based linkage study in mouse lines phenotypically selected for maximal (AIRmax) or minimal (AIRmin) acute inflammatory response (AIR), mapped a major locus, Inflammatory response modulator 1 (Irm1) at 128Mb (3.5Mb interval) in chr 7 controlling AIR, measured by leukocyte and IL-6 levels in exudates and IL-1β production by circulating leukocytes after Nlpr3 inflammasome activation. Sequencing of this region in mice with extreme high or low IL-1β levels revealed 14 SNPs between the two groups, narrowing the locus interval to 420Kb. Candidate genes at Irm1 include Pycard with an undescribed exon 3 (C/T) mutation leading to E19K substitution at the pyrin domain, and Itgam, Rgs10 and BC017158 with intronic SNPs. In Pycard, the C allele was fixed in high responder AIRmax mice whereas the C and T alleles frequencies were 39% and 61%, respectively in AIRmin. We then investigated the effect of this novel Pycard SNP in inflammation phenotypes. Methods AIRmin mice bearing the 3 genotypes at Pycard: CC, CT, TT were produced by genotype-assisted mating. Inflammatory response was measured in AIRmax and in the 3 AIRmin sublines by the number of infiltrating cells and IL-6 concentration in the 24h exudate induced by sc Biogel P-100 bead injection and ex vivo IL-1β production by circulating leukocytes after E coli LPS (1 ug) and ATP (5mM) activation. Results IL-1β levels were similar in AIRmaxCC (4.5-±0.4 ng/ml) and AIRminCC (3.4±2.4 ng/ml) whereas AIRminCT produced 0.3±0.5 and AIRminTT <0.05 ng/ml IL-1β. Leukocyte influx and IL-6 levels in inflammatory exudates were not affected. Conclusion The E19K substitution in Pycard causes a negative effect in inflammasome activation for IL-1β production, without interfering in other inflammation phenotypes.

Somatic KRAS mutations are common in human lung adenocarcinomas and are associated with worse prognosis. In mice, Kras is frequently mutated in both spontaneous and experimentally induced lung tumors, although the pattern of mutation varies among strains, suggesting that such mutations are not random events. We tested if the occurrence of Kras mutations is under genetic control in two mouse intercrosses. Codon 61 mutations were prevalent, but the patterns of nucleotide changes differed between the intercrosses. Whole genome analysis with SNPs in (A/J x C57BL/6)F4 mice revealed a significant linkage between a locus on chromosome 19 and 2 particular codon 61 variants (CTA and CGA). In (AIRmax × AIRmin) F2 mice, there was a significant linkage between SNPs located on distal chromosome 6 (around 135 Mbp) and the frequency of codon 61 mutation. These results reveal the presence of two loci, on chromosomes 6 and 19, that modulate Kras mutation frequency in different mouse intercrosses. These findings indicate that somatic mutation frequency and type are not simple random events, but are under genetic control.

Pristane-induced arthritis (PIA) in AIRmax mice homozygous for Slc11a1 R and S alleles was used to characterize the influence of Slc11a1 gene polymorphism on immune responses during disease manifestation. Previous reports demonstrated that the presence of the Slc11a1 S allele increased the incidence and severity of PIA in AIRmax SS , suggesting that this gene could interact with inflammatory loci to modulate PIA. We investigated the effects of Slc11a1 alleles on the activation of phagocytes during PIA.

OBJECTIVE AND DESIGN:AIRmax and AIRmin mice differ in their local acute inflammatory reactions to polyacrylamide beads (Biogel). These lines were developed to identify genes that affect the intensity of the acute inflammatory response (AIR) and to investigate the cellular and molecular mechanisms of acute inflammation. Although these lines are well established, differences in their responses to chronic inflammatory Biogel exposure have not yet been described. We investigated whether the selective process that modified the acute inflammatory responses in these animals also affected the development of their chronic inflammatory responses.RESULTS:Inflammatory exudate cell infiltration was more intense in AIRmax than AIRmin mice at both 48 h and 30 days. Genes involved in signal transduction and immune/inflammatory responses were differentially expressed in the treated skin of AIRmax and AIRmin mice, and divergent expression of some acute inflammatory response genes was detected up to 30 days post-Biogel. However, distinct expression of several pro and anti-inflammatory response genes in both periods was observed.CONCLUSION:These results indicate that the selective process for acute inflammation affected the development of chronic inflammatory responses to Biogel, suggesting common genetic control.