Paracoccidioides brasiliensis Induces α3 Integrin Lysosomal Degradation in Lung Epithelial Cells.

Studies on the pathogen-host interaction are crucial for the understanding of the mechanisms involved in the establishment, maintenance, and spread of infection. In recent years, our research group has observed that the P. brasiliensis species interact with integrin family receptors and increase the expression of α3 integrin in lung epithelial cells within 5 h of infection. Interestingly, α3 integrin levels were reduced by approximately 99% after 24 h of infection with P. brasiliensis compared to non-infected cells. In this work, we show that, during infection with this fungus, α3 integrin is increased in the late endosomes of A549 lung epithelial cells. We also observed that the inhibitor of the lysosomal activity bafilomycin A1 was able to inhibit the decrease in α3 integrin levels. In addition, the silencing of the charged multivesicular body protein 3 (CHMP3) inhibited the reduction in α3 integrin levels induced by P. brasiliensis in A549 cells. Thus, together, these results indicate that this fungus induces the degradation of α3 integrin in A549 lung epithelial cells by hijacking the host cell endolysosomal pathway.

Alternative Methods for Pulmonary-Administered Drugs Metabolism: A Breath of Change.

Prediction of pulmonary metabolites following inhalation of a locally acting pulmonary drug is essential to the successful development of novel inhaled medicines. The lungs present metabolic enzymes, therefore they influence drug disposal and toxicity. The present review provides an overview of alternative methods to evaluate the pulmonary metabolism for the safety and efficacy of pulmonary delivery systems. In vitro approaches for investigating pulmonary drug metabolism were described, including subcellular fractions, cell culture models and lung slices as the main available in vitro methods. In addition, in silico studies are promising alternatives that use specific software to predict pulmonary drug metabolism, determine whether a molecule will react with a metabolic enzyme, the site of metabolism (SoM) and the result of this interaction. They can be used in an integrated approach to delineate the major cytochrome P450 (CYP) isoforms to rationalize the use of in vivo methods. A case study about a combination of experimental and computational approaches was done using fluticasone propionate as an example. The results of three tested software, RSWebPredictor, SMARTCyp and XenoSite, demonstrated greater probability of the fluticasone propionate being metabolized by CYPs 3A4 at the S1 atom of 5-S-fluoromethyl carbothioate group. As the in vitro studies were not able to directly detect pulmonary metabolites, those alternatives in silico methods may reduce animal testing efforts, following the principle of 3Rs (Replacement, Reduction and Refinement), and contribute to the evaluation of pharmacological efficacy and safety profiles of new drugs in development.

Shared Nearest Neighbors Approach and Interactive Browser for Network Analysis of a Comprehensive Non-Small-Cell Lung Cancer Data Set.

PURPOSE: Advances in biological measurement technologies are enabling large-scale studies of patient cohorts across multiple omics platforms. Holistic analysis of these data can generate actionable insights for translational research and necessitate new approaches for data integration and mining. METHODS: We present a novel approach for integrating data across platforms on the basis of the shared nearest neighbors algorithm and use it to create a network of multiplatform data from the immunogenomic profiling of non-small-cell lung cancer project. RESULTS: Benchmarking demonstrates that the shared nearest neighbors-based network approach outperforms a traditional gene-gene network in capturing established interactions while providing new ones on the basis of the interplay between measurements from different platforms. When used to examine patient characteristics of interest, our approach provided signatures associated with and new leads related to recurrence and TP53 oncogenotype. CONCLUSION: The network developed offers an unprecedented, holistic view into immunogenomic profiling of non-small-cell lung cancer, which can be explored through the accompanying interactive browser that we built.

Respiratory Epithelial Cells: More Than Just a Physical Barrier to Fungal Infections.

The respiratory epithelium is highly complex, and its composition varies along the conducting airways and alveoli. In addition to their primary function in maintaining the respiratory barrier and lung homeostasis for gas exchange, epithelial cells interact with inhaled pathogens, which can manipulate cell signaling pathways, promoting adhesion to these cells or hosting tissue invasion. Moreover, pathogens (or their products) can induce the secretion of chemokines and cytokines by epithelial cells, and in this way, these host cells communicate with the immune system, modulating host defenses and inflammatory outcomes. This review will focus on the response of respiratory epithelial cells to two human fungal pathogens that cause systemic mycoses: Aspergillus and Paracoccidioides. Some of the host epithelial cell receptors and signaling pathways, in addition to fungal adhesins or other molecules that are responsible for fungal adhesion, invasion, or induction of cytokine secretion will be addressed in this review.

Respiratory epithelial cells: more than just a physical barrier to fungal infections

The respiratory epithelium is highly complex, and its composition varies along the conducting airways and alveoli. In addition to their primary function in maintaining the respiratory barrier and lung homeostasis for gas exchange, epithelial cells interact with inhaled pathogens, which can manipulate cell signaling pathways, promoting adhesion to these cells or hosting tissue invasion. Moreover, pathogens (or their products) can induce the secretion of chemokines and cytokines by epithelial cells, and in this way, these host cells communicate with the immune system, modulating host defenses and inflammatory outcomes. This review will focus on the response of respiratory epithelial cells to two human fungal pathogens that cause systemic mycoses: Aspergillus and Paracoccidioides. Some of the host epithelial cell receptors and signaling pathways, in addition to fungal adhesins or other molecules that are responsible for fungal adhesion, invasion, or induction of cytokine secretion will be addressed in this review.

PRMT1-dependent regulation of RNA metabolism and DNA damage response sustains pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer that has remained clinically challenging to manage. Here we employ an RNAi-based in vivo functional genomics platform to determine epigenetic vulnerabilities across a panel of patient-derived PDAC models. Through this, we identify protein arginine methyltransferase 1 (PRMT1) as a critical dependency required for PDAC maintenance. Genetic and pharmacological studies validate the role of PRMT1 in maintaining PDAC growth. Mechanistically, using proteomic and transcriptomic analyses, we demonstrate that global inhibition of asymmetric arginine methylation impairs RNA metabolism, which includes RNA splicing, alternative polyadenylation, and transcription termination. This triggers a robust downregulation of multiple pathways involved in the DNA damage response, thereby promoting genomic instability and inhibiting tumor growth. Taken together, our data support PRMT1 as a compelling target in PDAC and informs a mechanism-based translational strategy for future therapeutic development.Statement of significancePDAC is a highly lethal cancer with limited therapeutic options. This study identified and characterized PRMT1-dependent regulation of RNA metabolism and coordination of key cellular processes required for PDAC tumor growth, defining a mechanism-based translational hypothesis for PRMT1 inhibitors.

Bovine serum promotes the formation and phenotype memory retention of persister cells in Escherichia coli liquid cultures.

Persister cells, or persisters, are a fraction of bacterial cells that have become temporarily tolerant to antibiotics despite their lack of typical antibiotic-resistant genes. In a previous study, we found that colony-biofilm culture (i.e., biofilm formed at an air-solid interface) promoted the formation and phenotype memory retention of persisters of Escherichia coli and other bacteria. To assess whether these same effects are caused by other types of stimuli that bacterial cells encounter in the environment, we examined the effects of bovine serum on the formation and phenotype retention of ampicillin-tolerant persisters in E. coli liquid culture. Bovine serum did indeed exert these effects significantly, and its effects were negated by heat treatment. Similar effects were observed with bovine serum albumin, albeit weaker than those of BS. Given that serum is a component of blood and lymph and is thus a general substance within animal and human bodies, our findings suggest that bacteria encountering these body fluids may enhance their abilities for persister formation and phenotype memory retention to allow their longer survival in antibiotic-containing environments.

Paracoccidioides brasiliensis downmodulates α3 integrin levels in human lung epithelial cells in a TLR2-dependent manner.

Paracoccidioidomycosis (PCM) is the most prevalent systemic mycosis in Latin America and may be caused by the species Paracoccidioides brasiliensis. In the lungs, this fungus interacts with epithelial cells, activating host cell signalling pathways, resulting in the production of inflammatory mediators. This event may be initiated through the activation of Pattern-Recognition Receptors such as Toll-like Receptors (TLRs). By interacting with cell wall components, TLR2 is frequently related to fungal infections. In this work, we show that, after 24 h post-infection with P. brasiliensis, A549 lung epithelial cells presented higher TLR2 levels, which is important for IL-8 secretion. Besides, integrins may also participate in pathogen recognition by host cells. We verified that P. brasiliensis increased α3 integrin levels in A549 cells after 5 h of infection and promoted interaction between this receptor and TLR2. However, after 24 h, surprisingly, we verified a decrease of α3 integrin levels, which was dependent on direct contact between fungi and epithelial cells. Likewise, we observed that TLR2 is important to downmodulate α3 integrin levels after 24 h of infection. Thus, P. brasiliensis can modulate the host inflammatory response by exploiting host cell receptors and cell signalling pathways.

Allosteric SHP2 Inhibitor, IACS-13909, Overcomes EGFR-Dependent and EGFR-Independent Resistance Mechanisms toward Osimertinib.

Src homology 2 domain-containing phosphatase (SHP2) is a phosphatase that mediates signaling downstream of multiple receptor tyrosine kinases (RTK) and is required for full activation of the MAPK pathway. SHP2 inhibition has demonstrated tumor growth inhibition in RTK-activated cancers in preclinical studies. The long-term effectiveness of tyrosine kinase inhibitors such as the EGFR inhibitor (EGFRi), osimertinib, in non-small cell lung cancer (NSCLC) is limited by acquired resistance. Multiple clinically identified mechanisms underlie resistance to osimertinib, including mutations in EGFR that preclude drug binding as well as EGFR-independent activation of the MAPK pathway through alternate RTK (RTK-bypass). It has also been noted that frequently a tumor from a single patient harbors more than one resistance mechanism, and the plasticity between multiple resistance mechanisms could restrict the effectiveness of therapies targeting a single node of the oncogenic signaling network. Here, we report the discovery of IACS-13909, a specific and potent allosteric inhibitor of SHP2, that suppresses signaling through the MAPK pathway. IACS-13909 potently impeded proliferation of tumors harboring a broad spectrum of activated RTKs as the oncogenic driver. In EGFR-mutant osimertinib-resistant NSCLC models with EGFR-dependent and EGFR-independent resistance mechanisms, IACS-13909, administered as a single agent or in combination with osimertinib, potently suppressed tumor cell proliferation in vitro and caused tumor regression in vivo. Together, our findings provide preclinical evidence for using a SHP2 inhibitor as a therapeutic strategy in acquired EGFRi-resistant NSCLC. SIGNIFICANCE: These findings highlight the discovery of IACS-13909 as a potent, selective inhibitor of SHP2 with drug-like properties, and targeting SHP2 may serve as a therapeutic strategy to overcome tumor resistance to osimertinib.

[Effects of Medium Chain Triglycerides Intake on Lipid Metabolism and Intestinal Disaccharidase Activities in Rats].

It has been reported that medium-chain triglyceride (MCT) have various physiological functions, such as anti-obesity and hypolipidemic effects. They can also elicit increased disaccharidase activity and intestinal cell proliferation. However, a meta-analysis of randomized controlled trials, comparing the effects of MCT on weight loss and body composition, detected commercial bias. Additional research on the physiological functions is needed in order to have conclusive evidence. Thus, we sought to evaluate the various functions of MCT by conducting a feeding study in rats. Rats fed a diet containing 15% (w/w) MCT, had significantly lower visceral fat weight, plasma and liver lipid concentrations; they had significantly higher intestinal maltase and glucoamylase activities; and they had a greater number of Ki-67 positive cells/crypt, compared to the rats fed a diet containing 15% (w/w) lard. The effects of a diet containing 5% (w/w) MCT was observed only for plasma cholesterol levels and the number of Ki-67 positive cells/crypt; in which some results were found to be inconsistent with previous reports. These results indicate that physiological functions of MCT are numerous and need to be confirmed by additional research.

Effects of a novel roflumilast and formoterol fumarate dry powder inhaler formulation in experimental allergic asthma.

In this study we aimed to develop a roflumilast (R) and formoterol fumarate (F) dry powder inhaler formulation (DPI) incorporating HPßCD by spray drying and evaluated if it attenuates the inflammatory process and improves lung function in a murine model of ovalbumin induced allergic asthma. The DPI was characterized by powder X-ray diffraction, thermal analysis, scanning electron microscopy, particle size, density, specific surface area and dynamic vapor sorption analyses. In vitro deposition studies were performed using a NGI, while transepithelial permeability and in vivo effects on lung mechanics and inflammation in a model of allergic asthma were also assessed. The R:F formulation was amorphous with high glass transition temperatures, comprised of wrinkled particles, had low bulk and tapped densities, high surface area, suitable particle size for pulmonary delivery and exhibited no recrystallization even at high relative humidities. MMAD were statistically similar of 4.22 ± 0.19 and 4.32 ± 0.13 µm for F and R, respectively. Fine particle fractions (<5 µm) were of more than 50% of the emitted dose. The R:F formulation led to reduced eosinophil infiltration and airway collagen fiber content, yielding decreased airway hyperresponsiveness. In the current asthma model, the R:F formulation combination decreased inflammation and remodeling, thus improving lung mechanics.

Discovery of IACS-9439, a Potent, Exquisitely Selective, and Orally Bioavailable Inhibitor of CSF1R.

Tumor-associated macrophages (TAMs) have a significant presence in the tumor stroma across multiple human malignancies and are believed to be beneficial to tumor growth. Targeting CSF1R has been proposed as a potential therapy to reduce TAMs, especially the protumor, immune-suppressive M2 TAMs. Additionally, the high expression of CSF1R on tumor cells has been associated with poor survival in certain cancers, suggesting tumor dependency and therefore a potential therapeutic target. The CSF1-CSF1R signaling pathway modulates the production, differentiation, and function of TAMs; however, the discovery of selective CSF1R inhibitors devoid of type III kinase activity has proven to be challenging. We discovered a potent, highly selective, and orally bioavailable CSF1R inhibitor, IACS-9439 (1). Treatment with 1 led to a dose-dependent reduction in macrophages, promoted macrophage polarization toward the M1 phenotype, and led to tumor growth inhibition in MC38 and PANC02 syngeneic tumor models.

Histoplasma capsulatum chemotypes I and II induce IL-8 secretion in lung epithelial cells in distinct manners.

The cell wall is one of the most important structures of pathogenic fungi, enabling initial interaction with the host and consequent modulation of immunological responses. Over the years, some researchers have shown that cell wall components of Histoplasma capsulatum vary among fungal isolates, and one of the major differences is the presence or absence of α-(1,3)-glucan, classifying wild-type fungi as chemotypes II or I, respectively. The present work shows that an isolate of H. capsulatum chemotype I induced lower levels of interleukin (IL)-8 secretion by the lung epithelial cell line A549, when compared to chemotype II yeasts. Thus, we expected that the absence of α-glucan in spontaneous variant yeasts, which were isolated from chemotype II cultures, would modify IL-8 secretion by A549 cells, but surprisingly, these fungi promoted similar levels of IL-8 secretion as their wild-type counterpart. Furthermore, when using a specific inhibitor for Syk activation, we observed that this inhibitor reduced IL-8 levels in A549 cell cultures infected with wild type chemotype I fungi. This inhibitor failed to reduce this cytokine levels in A549 cell cultures infected with chemotype II and their spontaneous variant yeasts, which also do not present α-glucan on their surface. The importance of SFKs and PKC δ in this event was also analyzed. Our results show that different isolates of H. capsulatum modulate distinct cell signaling pathways to promote cytokine secretion in host epithelial cells, emphasizing the existence of various mechanisms for Histoplasma pathogenicity.

Paracoccidioides species present distinct fungal adherence to epithelial lung cells and promote different IL-8 secretion levels.

Fungi that belong to the genus Paracoccidioides are the etiologic agents of paracoccidioidomycosis, a human systemic mycosis, which occurs in Latin America. Epithelial cell is one of the first cells that interact with these fungi and responds by secreting inflammatory mediators such as cytokines. In the present study, we demonstrate that yeasts of different isolates of Paracoccidioides brasiliensis (Pb18 and Pb03) and Paracoccidioides lutzii (Pb01) distinctly promoted interleukin (IL)-8 secretion by the lung epithelial cell line A549. Depending on the isolate, this cytokine release may rely on the epithelial cell interaction with fungal secreted components or direct contact with the pathogen. In addition, adhesion of yeasts to the pulmonary epithelial cells was also different among Paracoccidioides isolates, and the highest percentage of A549 cells with adhered fungi was observed with P. lutzii. All Paracoccidioides isolates induced an expression increase of α3 and α5 integrins in A549 cells and, using small interfering RNA, we observed that the integrin silencing promoted a reduction of P. lutzii adhesion, which suggests the involvement of integrins in this event. Together, these results indicate that host epithelial cell response may depend on the isolate of Paracoccidioides.

Secretion of high amounts of hepatocyte growth factor is a characteristic feature of cancer-associated fibroblasts with EGFR-TKI resistance-promoting phenotype: A study of 18 cases of cancer-associated fibroblasts.

Humoral factors from cancer-associated fibroblasts (CAFs) reportedly affect epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) resistance in cancer cells with EGFR mutations. The aim of this study was to identify the robust humoral factors secreted from CAFs that induce the primary resistance to EGFR-TKI. We evaluated the EGFR-TKI sensitivity of EGFR-mutant lung adenocarcinoma cell line (PC-9) treated with condition media (CM) from 18 cases of CAFs and matched non-cancerous-tissue-associated fibroblasts (NCAFs). We measured the expression levels of hepatocyte growth factor (HGF), interleukin-6, fibroblast growth factor-2, insulin-like growth factor-1, and vascular endothelial growth factor-A in CAFs and NCAFs. We examined whether HGF neutralizing antibody could annul the EGFR-TKI resistance induced by CM from CAFs. Compared to CM from NCAFs, CM from CAFs increased the resistance of PC-9 cells to EGFR-TKI in five out of 18 cases. Relative expression ratio of HGF messenger RNA was significantly higher in these five CAFs compared to others (P = 0.0013), whereas other cytokines were not. In four of these five cases, the addition of HGF neutralizing antibody significantly decreased the survival ratio of PC-9 cells. This study suggests that the secretion of higher amounts of HGF is the robust feature of EGFR-TKI resistance-promoting CAFs.

Horizontal Plasmid Transfer by Transformation in Escherichia coli: Environmental Factors and Possible Mechanisms.

Transformation is one mode of horizontal gene transfer (HGT) in bacteria, wherein extracellular naked DNA is taken up by cells that have developed genetic competence. Sensitivity to DNase, which degrades naked DNA, is the key to distinguishing transformation from the DNase-resistant HGT mechanisms. In general, Escherichia coli is not believed to be naturally transformable; it develops high competence only under artificial conditions, including exposure to high Ca2+ concentrations. However, E. coli can reportedly express modest competence under certain conditions that are feasible in natural environments outside laboratory. In addition, recent data suggest that environmental factors influence multiple routes of transformation. In this mini review, we (1) summarize our studies on transformation-based HGT using E. coli experimental systems and (2) discuss the possible occurrence of transformation via multiple mechanisms in the environment and its possible impact on the spread of antibiotic resistance genes.

Development of a new formulation of roflumilast for pulmonary drug delivery to treat inflammatory lung conditions.

The aim of this study was to develop roflumilast dry powder inhaler (DPI) formulations by spray drying using hydroxypropyl-ß-cyclodextrin (HPßCD) and to determine their suitability for pulmonary delivery. Different feed solution concentrations, solvent systems and spray drying parameters were used to obtain the formulations which were characterized using X-ray powder diffraction, thermal analysis, scanning electron microscopy, particle size distribution, bulk and tapped density, specific surface area, dynamic vapour sorption, in vitro deposition properties using a Next Generation Impactor (NGI) and transepithelial permeability. Microparticles spray dried from ethanol were wrinkled and amorphous, exhibiting high glass transition temperatures while those from methanol:n-butyl acetate consisted of irregularly shaped porous particles partially crystalline. All formulations presented low density, particle size and residual solvent content exhibiting high depositon in the lower stages of the NGI. Mass median aerodynamic diameters (MMADs) were in the range of 3.32-4.49 µm, with high fine particle fractions (FPF < 5 µm). Stability studies demonstrated no significant modifications in the solid-state nature and in the aerolisation performance of the selected formulation which presented a Papp of 8.73 × 10-6 ±â€¯4.70 × 10-7 cm/s. The developed roflumilast DPI formulations have potential therapeutic applications in the treatment of lung diseases.

Bacterial Memory of Persisters: Bacterial Persister Cells Can Retain Their Phenotype for Days or Weeks After Withdrawal From Colony-Biofilm Culture.

Persister cells, or persisters, are a specific subpopulation of bacterial cells that have acquired temporary antibiotic-resistant phenotypes. In this study, we showed that Escherichia coli produces many more persister cells in colony-biofilm culture than in the usual liquid culture and that these persisters can be maintained in higher numbers than those from liquid culture for up to 4 weeks at 37°C in a fresh, nutrient-rich, antibiotic-containing medium, even after complete withdrawal from the colony-biofilm culture. This suggests the presence of a long-retention effect, or "memory effect", in the persister cell state of E. coli cells. We also discovered that such increases in persisters during colony-biofilm culture and their memory effects are common, to a greater or lesser degree, in other bacterial species. This is true not only for gram-negative bacteria (Acinetobacter and Salmonella) but also for gram-positive bacteria (Staphylococcus and Bacillus). This is the first report to suggest the presence of a common memory mechanism for the persister cell state, which is inscribed during colony-biofilm culture, in a wide variety of bacteria.

Candida albicans: The Ability to Invade Epithelial Cells and Survive under Oxidative Stress Is Unlinked to Hyphal Length.

In its hyphal form, Candida albicans invades epithelial and endothelial cells by two distinct mechanisms: active penetration and induced endocytosis. The latter is dependent on a reorganization of the host cytoskeleton (actin/cortactin recruitment), whilst active penetration does not rely on the host's cellular machinery. The first obstacle for the fungus to reach deep tissues is the epithelial barrier and this interaction is crucial for commensal growth, fungal pathogenicity and host defense. This study aimed to characterize in vitro epithelial HeLa cell invasion by four different isolates of C. albicans with distinct clinical backgrounds, including a C. albicans SC5314 reference strain. All isolates invaded HeLa cells, recruited actin and cortactin, and induced the phosphorylation of both Src-family kinases (SFK) and cortactin. Curiously, L3881 isolated from blood culture of a patient exhibited the highest resistance to oxidative stress, although this isolate showed reduced hyphal length and displayed the lowest cell damage and invasion rates. Collectively, these data suggest that the ability of C. albicans to invade HeLa cells, and to reach and adapt to the host's blood, including resistance to oxidative stress, may be independent of hyphal length.

Paracoccidioides brasiliensis induces cytokine secretion in epithelial cells in a protease-activated receptor-dependent (PAR) manner.

Paracoccidioides brasiliensis is one of the etiological agents of the human systemic mycosis paracoccidioidomycosis. Protease-activated receptors (PARs) are expressed in many cell types and comprise a family of G protein-coupled receptors (PAR-1, PAR-2, and PAR-4), which may be activated by proteases secreted by several pathogens. In the present study, we showed that the pathogenic fungus P. brasiliensis secretes components that promote interleukin (IL)-6 and IL-8 secretion by the lung epithelial cell line A549. Cytokine secretion was reduced by antagonistic peptides for PAR-1 and PAR-2, but not for PAR-4. P. brasiliensis proteases were isolated from fungal culture supernatants in a p-aminomethylbenzamidine-Sepharose column. The obtained fractions were tested for enzymatic activity against fluorescence resonance energy transfer (FRET) peptides derived from sequences that spanned the activation sites of human PARs. The eluted fraction, termed PbP, contained protease activities that were able to hydrolyze the FRET peptides. PbP also induced IL-6 and IL-8 secretion in A549 epithelial cells, which was reduced upon heat inactivation of PbP, incubation with antagonistic peptides for PAR-1 and PAR-2, and the protease inhibitors aprotinin, leupeptin, and E-64. Together, these results show for the first time that P. brasiliensis yeasts secrete proteases that activate PARs in lung epithelial cells, leading to cytokine secretion.