Toll-like receptor-guided therapeutic intervention of human cancers: molecular and immunological perspectives.

Toll-like receptors (TLRs) serve as the body's first line of defense, recognizing both pathogen-expressed molecules and host-derived molecules released from damaged or dying cells. The wide distribution of different cell types, ranging from epithelial to immune cells, highlights the crucial roles of TLRs in linking innate and adaptive immunity. Upon stimulation, TLRs binding mediates the expression of several adapter proteins and downstream kinases, that lead to the induction of several other signaling molecules such as key pro-inflammatory mediators. Indeed, extraordinary progress in immunobiological research has suggested that TLRs could represent promising targets for the therapeutic intervention of inflammation-associated diseases, autoimmune diseases, microbial infections as well as human cancers. So far, for the prevention and possible treatment of inflammatory diseases, various TLR antagonists/inhibitors have shown to be efficacious at several stages from pre-clinical evaluation to clinical trials. Therefore, the fascinating role of TLRs in modulating the human immune responses at innate as well as adaptive levels directed the scientists to opt for these immune sensor proteins as suitable targets for developing chemotherapeutics and immunotherapeutics against cancer. Hitherto, several TLR-targeting small molecules (e.g., Pam3CSK4, Poly (I:C), Poly (A:U)), chemical compounds, phytocompounds (e.g., Curcumin), peptides, and antibodies have been found to confer protection against several types of cancers. However, administration of inappropriate doses of such TLR-modulating therapeutics or a wrong infusion administration is reported to induce detrimental outcomes. This review summarizes the current findings on the molecular and structural biology of TLRs and gives an overview of the potency and promises of TLR-directed therapeutic strategies against cancers by discussing the findings from established and pipeline discoveries.

The Shigella flexneri virulence factor apyrase is released inside eukaryotic cells to hijack host cell fate.

IMPORTANCE: In this paper, we demonstrated that apyrase is released within the host cell cytoplasm during infection to target the intracellular ATP pool. By degrading intracellular ATP, apyrase contributes to prevent caspases activation, thereby inhibiting the activation of pyroptosis in infected cells. Our results show, for the first time, that apyrase is involved in the modulation of host cell survival, thereby aiding this pathogen to dampen the inflammatory response. This work adds a further piece to the puzzle of Shigella pathogenesis. Due to its increased spread worldwide, prevention and controlling strategies are urgently needed. Overall, this study highlighted apyrase as a suitable target for an anti-virulence therapy to tackle this pathogen.

Insightful Improvement in the Design of Potent Uropathogenic E. coli FimH Antagonists.

Selective antiadhesion antagonists of Uropathogenic Escherichia coli (UPEC) type-1 Fimbrial adhesin (FimH) are attractive alternatives for antibiotic therapies and prophylaxes against acute or recurrent urinary tract infections (UTIs) caused by UPECs. A rational small library of FimH antagonists based on previously described C-linked allyl α-D-mannopyranoside was synthesized using Heck cross-coupling reaction using a series of iodoaryl derivatives. This work reports two new members of FimH antagonist amongst the above family with sub nanomolar affinity. The resulting hydrophobic aglycones, including constrained alkene and aryl groups, were designed to provide additional favorable binding interactions with the so-called FimH "tyrosine gate". The newly synthesized C-linked glycomimetic antagonists, having a hydrolytically stable anomeric linkage, exhibited improved binding when compared to previously published analogs, as demonstrated by affinity measurement through interactions by FimH lectin. The crystal structure of FimH co-crystallized with one of the nanomolar antagonists revealed the binding mode of this inhibitor into the active site of the tyrosine gate. In addition, selected mannopyranoside constructs neither affected bacterial growth or cell viability nor interfered with antibiotic activity. C-linked mannoside antagonists were effective in decreasing bacterial adhesion to human bladder epithelial cells (HTB-9). Therefore, these molecules constituted additional therapeutic candidates' worth further development in the search for potent anti-adhesive drugs against infections caused by UPEC.

Inflammatory Response in Caco-2 Cells Stimulated with Anisakis Messengers of Pathogenicity.

Background: Anisakis spp. third-stage larvae (L3) are the causative agents of human zoonosis called anisakiasis. The accidental ingestion of L3 can cause acute and chronic inflammation at the gastric, intestinal, or ectopic levels. Despite its relevance in public health, studies on pathogenetic mechanisms and parasite-human interplay are scarce. The aim of this study was to investigate the human inflammatory response to different Anisakis vehicles of pathogenicity. Methods: Human colorectal adenocarcinoma (Caco-2) cells were exposed to Anisakis L3 (the initial contact with the host), extracellular vesicles (EVs, Anisakis-host communication), and crude extract (CE, the larval dying). The protein quantity and gene expression of two pro-inflammatory cytokines (IL-6 and IL-8) were investigated using an ELISA test (6 h and 24 h) and a qReal-Time PCR (1 h, 6 h, and 24 h), respectively. Results: The L3 and EVs induced a downregulation in both the Il-6 and Il-8 gene expression and protein quantity. On the contrary, the CE stimulated IL-6 gene expression and its protein release, not affecting IL-8. Conclusions: The Caco-2 cells seemed to not react to the exposure to the L3 and EVs, suggesting a parasite's immunomodulating action to remain alive in an inhospitable niche. Conversely, the dying larva (CE) could induce strong activation of the immune strategy of the host that, in vivo, would lead to parasite expulsion, eosinophilia, and/or granuloma formation.

Optimization of 3D islet-like cluster derived from human pluripotent stem cells: An efficient in vitro differentiation protocol.

Development of an optimized protocol to produce sufficient functional human insulin-producing islet-like cluster is important as a potential therapeutic strategy for diabetes as well as in vitro studies. Here, we described a stepwise protocol for differentiation of the human induced pluripotent stem cell line (R1-hiPSC1) into the islet-like cluster using several growth factors and small molecules. Therefore, various differentiation steps have been adopted to maximize mimicking of developmental processes in order to form functional islet like cluster. The differentiation protocol enables us to generate 3D islet-like clusters with highly viable cells, which are insulin producer and glucose responsive. Transcriptome analysis of transcription factors and functional genes revealed high coordination between gene expressions and resembling to those reported during natural development of islet cell. This coordination was further confirmed by hierarchical clustering of genes during differentiation. Furthermore, the islet-like clusters were enriched with insulin producing cells and formed glucose responsiveness behavior upon stimulation with glucose. Our protocol provides a robust platform and well-behaved model for additional developmental studies and shed light our clusters as a good candidate for in vitro model. Further studies are needed to assess the hormonal content of this cluster as well as transplantation into the animal model.

Adaptive strategies of uropathogenic Escherichia coli CFT073: from growth in lab media to virulence during host cell adhesion / Estrategias adaptativas de Escherichia coli CFT073 uropatógena: desde el crecimiento en medios de laboratorio hasta la virulencia durante la adhesión de la célula huésped

Urinary tract infections (UTIs) are a major concern in public health. The prevalent uropathogenic bacterium in healthcare settings is Escherichia coli. The increasing rate of antibiotic-resistant strains demands studies to understand E. coli pathogenesis to drive the development of new therapeutic approaches. This study compared the gene expression profile of selected target genes in the prototype uropathogenic E. coli (UPEC) strain CFT073 grown in Luria Bertani (LB), artificial urine (AU), and during adhesion to host bladder cells by semi-quantitative real-time PCR (RT-PCR) assays. AU effectively supported the growth of strain CFT073 as well as other E. coli strains with different lifestyles, thereby confirming the appropriateness of this medium for in vitro models. Unexpectedly, gene expression of strain CFT073 in LB and AU was quite similar; conversely, during the adhesion assay, adhesins and porins were upregulated, while key global regulators were downregulated with respect to lab media. Interestingly, fimH and papGII genes were significantly expressed in all tested conditions. Taken together, these results provide for the first time insights of the metabolic and pathogenic profile of strain CFT073 during the essential phase of host cell adhesion.(AU)

Improving the Diagnostic Potential of Extracellular miRNAs Coupled to Multiomics Data by Exploiting the Power of Artificial Intelligence.

In recent years, the clinical use of extracellular miRNAs as potential biomarkers of disease has increasingly emerged as a new and powerful tool. Serum, urine, saliva and stool contain miRNAs that can exert regulatory effects not only in surrounding epithelial cells but can also modulate bacterial gene expression, thus acting as a "master regulator" of many biological processes. We think that in order to have a holistic picture of the health status of an individual, we have to consider comprehensively many "omics" data, such as miRNAs profiling form different parts of the body and their interactions with cells and bacteria. Moreover, Artificial Intelligence (AI) and Machine Learning (ML) algorithms coupled to other multiomics data (i.e., big data) could help researchers to classify better the patient's molecular characteristics and drive clinicians to identify personalized therapeutic strategies. Here, we highlight how the integration of "multiomic" data (i.e., miRNAs profiling and microbiota signature) with other omics (i.e., metabolomics, exposomics) analyzed by AI algorithms could improve the diagnostic and prognostic potential of specific biomarkers of disease.

Biomarkers to Monitor Adherence to Gluten-Free Diet by Celiac Disease Patients: Gluten Immunogenic Peptides and Urinary miRNAs.

Celiac disease (CD) is a multifactorial autoimmune enteropathy with a prevalence greater than 1% in the pediatric population. The only therapy for CD patients is a strict gluten-free diet (GFD). Gluten-free food contamination by other cereals during packaging and cooking or accidental ingestion of gluten may cause several intestinal and extraintestinal symptoms in CD patients. Therefore, the monitoring of gluten contamination in food and assessing the level of ingested gluten by analytical biomarkers has been of great interest in recent years. To this aim, small gluten immunogenic peptides (GIPs) obtained by the hydrolysis of gluten and present in urine and feces have been studied as biomarkers of gluten intake and to monitor adherence to GFD by CD patients. More recently, the use of circulating, fecal and urinary miRNAs has emerged as a novel diagnostic tool that can be potentially applied to assess adherence to GFD. Moreover, the presence of GIPs and miRNAs in both feces and urine suggests a similar excretion modality and the possibility of using urinary miRNAs, similarly to GIPs, as potential biomarkers of GFD in CD patients.

The Interleukin-1 (IL-1) Superfamily Cytokines and Their Single Nucleotide Polymorphisms (SNPs).

Interleukins (ILs)-which are important members of cytokines-consist of a vast group of molecules, including a wide range of immune mediators that contribute to the immunological responses of many cells and tissues. ILs are immune-glycoproteins, which directly contribute to the growth, activation, adhesion, differentiation, migration, proliferation, and maturation of immune cells; and subsequently, they are involved in the pro and anti-inflammatory responses of the body, by their interaction with a wide range of receptors. Due to the importance of immune system in different organisms, the genes belonging to immune elements, such as ILs, have been studied vigorously. The results of recent investigations showed that the genes pertaining to the immune system undergo progressive evolution with a constant rate. The occurrence of any mutation or polymorphism in IL genes may result in substantial changes in their biology and function and may be associated with a wide range of diseases and disorders. Among these abnormalities, single nucleotide polymorphisms (SNPs) can represent as important disruptive factors. The present review aims at concisely summarizing the current knowledge available on the occurrence, properties, role, and biological consequences of SNPs within the IL-1 family members.

Genetic Diversity of Antimicrobial Resistance and Key Virulence Features in Two Extensively Drug-Resistant Acinetobacter baumannii Isolates.

In recent decades, Acinetobacter baumannii emerged as a major infective menace in healthcare settings due to scarce therapeutic options to treat infections. Therefore, undertaking genome comparison analyses of multi-resistant A. baumannii strains could aid the identification of key bacterial determinants to develop innovative anti-virulence approaches. Following genome sequencing, we performed a molecular characterization of key genes and genomic comparison of two A. baumannii strains, #36 and #150, with selected reference genomes. Despite a different antibiotic resistance gene content, the analyzed strains showed a very similar antibiogram profile. Interestingly, the lack of some important virulence determinants (i.e., bap, ata and omp33-36) did not abrogate their adhesive abilities to abiotic and biotic surfaces, as reported before; indeed, strains retained these capacities, although to a different extent, suggesting the presence of distinct vicarious genes. Conversely, secretion systems, lipopolysaccharide (LPS), capsule and iron acquisition systems were highly similar to A. baumannii reference strains. Overall, our analyses increased our knowledge on A. baumannii genomic content and organization as well as the genomic events occurring in nosocomial isolates to better fit into changing healthcare environments.

Adaptive strategies of uropathogenic Escherichia coli CFT073: from growth in lab media to virulence during host cell adhesion.

Urinary tract infections (UTIs) are a major concern in public health. The prevalent uropathogenic bacterium in healthcare settings is Escherichia coli. The increasing rate of antibiotic-resistant strains demands studies to understand E. coli pathogenesis to drive the development of new therapeutic approaches. This study compared the gene expression profile of selected target genes in the prototype uropathogenic E. coli (UPEC) strain CFT073 grown in Luria Bertani (LB), artificial urine (AU), and during adhesion to host bladder cells by semi-quantitative real-time PCR (RT-PCR) assays. AU effectively supported the growth of strain CFT073 as well as other E. coli strains with different lifestyles, thereby confirming the appropriateness of this medium for in vitro models. Unexpectedly, gene expression of strain CFT073 in LB and AU was quite similar; conversely, during the adhesion assay, adhesins and porins were upregulated, while key global regulators were downregulated with respect to lab media. Interestingly, fimH and papGII genes were significantly expressed in all tested conditions. Taken together, these results provide for the first time insights of the metabolic and pathogenic profile of strain CFT073 during the essential phase of host cell adhesion.

The Acinetobacter baumannii model can explain the role of small non-coding RNAs as potential mediators of host-pathogen interactions.

Bacterial small RNAs (sRNAs) research has accelerated over the past decade, boosted by advances in RNA-seq technologies and methodologies for capturing both protein-RNA and RNA-RNA interactions. The emerging picture is that these regulatory sRNAs play important roles in controlling complex physiological processes and are required to survive the antimicrobial challenge. In recent years, the RNA content of OMVs/EVs has also gained increasing attention, particularly in the context of infection. Secreted RNAs from several bacterial pathogens have been characterized but the exact mechanisms promoting pathogenicity remain elusive. In this review, we briefly discuss how secreted sRNAs interact with targets in infected cells, thus representing a novel perspective of host cell manipulation during bacterial infection. During the last decade, Acinetobacter baumannii became clinically relevant emerging pathogens responsible for nosocomial and community-acquired infections. Therefore, we also summarize recent findings of regulation by sRNAs in A. baumannii and discuss how this emerging bacterium utilizes many of these sRNAs to adapt to its niche and become successful human pathogen.

Gram-Negative Bacteria Holding Together in a Biofilm: The Acinetobacter baumannii Way.

Bacterial biofilms are a serious public-health problem worldwide. In recent years, the rates of antibiotic-resistant Gram-negative bacteria associated with biofilm-forming activity have increased worrisomely, particularly among healthcare-associated pathogens. Acinetobacter baumannii is a critically opportunistic pathogen, due to the high rates of antibiotic resistant strains causing healthcare-acquired infections (HAIs). The clinical isolates of A. baumannii can form biofilms on both biotic and abiotic surfaces; hospital settings and medical devices are the ideal environments for A. baumannii biofilms, thereby representing the main source of patient infections. However, the paucity of therapeutic options poses major concerns for human health infections caused by A. baumannii strains. The increasing number of multidrug-resistant A. baumannii biofilm-forming isolates in association with the limited number of biofilm-eradicating treatments intensify the need for effective antibiofilm approaches. This review discusses the mechanisms used by this opportunistic pathogen to form biofilms, describes their clinical impact, and summarizes the current and emerging treatment options available, both to prevent their formation and to disrupt preformed A. baumannii biofilms.

Urinary tract infections: Can we prevent uropathogenic Escherichia coli infection with dietary intervention?

Urinary tract infections (UTIs) are among the most common causes of infections in women. Via the fecal-perineal-urethral route, uropathogenic Escherichia coli (UPEC) can cause ascending urinary tract infections, including cystitis and pyelonephritis. These infections re-occur within six months or they account for, at least, three episodes within a year of recurrent UTIs (rUTIs). Long term and continuous antibiotic treatment or prophylaxis should be considered as the last options in rUTIs. Conversely, updated European Association of Urology guidelines recommend non-antimicrobial approaches to prevent rUTIs. Accordingly, several studies reported the efficacy of number of natural molecules in inhibiting UPEC adhesion to bladder cells, restraining bacterial growth, as well as stimulating the host innate immune defenses, and protecting the bladder and the kidney mucosa. Therefore, we propose an "anti-UPEC" diet enriched of foods containing natural compounds that were proven effective against UPEC, such as D-mannose, cranberry extracts and medicinal plants. Being a valuable and safe clinical approach to reduce UTI recurrence and limiting the detrimental effects of long and continuous antibiotic prophylaxis, dietary interventions should be evaluated in future clinical trials.

Acinetobacter baumannii: An Ancient Commensal with Weapons of a Pathogen.

Acinetobacter baumannii is regarded as a life-threatening pathogen associated with community-acquired and nosocomial infections, mainly pneumonia. The rise in the number of A. baumannii antibiotic-resistant strains reduces effective therapies and increases mortality. Bacterial comparative genomic studies have unraveled the innate and acquired virulence factors of A. baumannii. These virulence factors are involved in antibiotic resistance, environmental persistence, host-pathogen interactions, and immune evasion. Studies on host-pathogen interactions revealed that A. baumannii evolved different mechanisms to adhere to in order to invade host respiratory cells as well as evade the host immune system. In this review, we discuss current data on A. baumannii genetic features and virulence factors. An emphasis is given to the players in host-pathogen interaction in the respiratory tract. In addition, we report recent investigations into host defense systems using in vitro and in vivo models, providing new insights into the innate immune response to A. baumannii infections. Increasing our knowledge of A. baumannii pathogenesis may help the development of novel therapeutic strategies based on anti-adhesive, anti-virulence, and anti-cell to cell signaling pathways drugs.

Protective Effect of HLA-E∗0101∕∗0103 Genotype in Survival of Patients After Allogeneic Hematopoietic Stem Cell Transplant.

OBJECTIVES: HLA-E is located on the nonclassical major histocompatibility complex class I and acts as the ligand for natural killer cells. Consequently, it has a main role in the regulation of innate immune responses by involving cell identification by natural killer cells. Differences in expression levels among HLA-E alleles have been suggested to affect transplant outcomes. In this study, we evaluated the effects of different HLA-E genotypes on allogeneic hematopoietic stem cell transplant in southern Iran. MATERIALS AND METHODS: We investigated 200 patients (donors and recipients) who underwent allogeneic hematopoietic stem-cell transplant and 100 normal participants (control group) in a case-control study. Detection of HLA-E polymorphisms was performed using a sequence-specific primer polymerase chain reaction method. RESULTS: Statistical analyses indicated that genotypes in the transplant group were not distributed in accordance with Hardy-Weinberg equilibrium (χ²=76.56; P < .001), whereas genotypes in the control group were distributed in accordance with Hardy-Weinberg equilibrium (χ²=0.39; P = .53). No significant differences were observed in cumulative incidence of acute (P = .76; hazard ratio = 0.80; 95% confidence interval, 0.19-3.31) and chronic (P = .75, hazard ratio = 0.048; 95% confidence interval, 0.00) graft-versus-host disease in recipients harboring HLA-E∗0103 allele compared with those homozygous for the HLA-E∗0101 allele. The HLA-E∗0103 allele showed a trend toward lower cumulative incidence of relapse compared with the homozygous HLA-E∗0101 genotype (8% vs 21.5%; P = .37; hazard ratio = 2.50; 95% confidence interval, 0.32-19.20). CONCLUSIONS: Genotypes of the HLA-E molecule did not correlate with acute and chronic graft-versus-host disease in hematopoietic stem cell transplant recipients except for the HLA-E∗0101∗∕∗0103 genotype, which was protective in survival of our study patients.

SARS-CoV-2: Comparative analysis of different RNA extraction methods.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent of the COVID-19 pandemic. Although other diagnostic methods have been introduced, detection of viral genes on oro- and nasopharyngeal swabs by reverse-transcription real time-PCR (rRT-PCR) assays is still the gold standard. Efficient viral RNA extraction is a prerequisite for downstream performance of rRT-PCR assays. Currently, several automatic methods that include RNA extraction are available. However, due to the growing demand, a shortage in kit supplies could be experienced in several labs. For these reasons, the use of different commercial or in-house protocols for RNA extraction may increase the possibility to analyze high number of samples. Herein, we compared the efficiency of RNA extraction of three different commercial kits and an in-house extraction protocol using synthetic ssRNA standards of SARS-CoV-2 as well as in oro-nasopharyngeal swabs from six COVID-19-positive patients. It was concluded that tested commercial kits can be used with some modifications for the detection of the SARS-CoV-2 genome by rRT-PCR approaches, although with some differences in RNA yields. Conversely, EXTRAzol reagent was the less efficient due to the phase separation principle at the basis of RNA extraction. Overall, this study offers alternative suitable methods to manually extract RNA that can be taken into account for SARS-CoV-2 detection.

Acinetobacter baumannii Targets Human Carcinoembryonic Antigen-Related Cell Adhesion Molecules (CEACAMs) for Invasion of Pneumocytes.

Multidrug-resistant Acinetobacter baumannii is regarded as a life-threatening pathogen mainly associated with nosocomial and community-acquired pneumonia. Here, we show that A. baumannii can bind the human carcinoembryonic antigen-related cell adhesion molecule (CEACAM) receptors CEACAM1, CEACAM5, and CEACAM6. This specific interaction enhances A. baumannii internalization in membrane-bound vacuoles, promptly decorated with Rab5, Rab7, and lipidated microtubule-associated protein light chain 3 (LC3). Dissecting intracellular signaling pathways revealed that infected pneumocytes trigger interleukin-8 (IL-8) secretion via the extracellular signal-regulated kinase (ERK)1/2 and nuclear factor-kappa B (NF-κB) signaling pathways for A. baumannii clearance. However, in CEACAM1-L-expressing cells, IL-8 secretion lasts only 24 h, possibly due to an A. baumannii-dependent effect on the CEACAM1-L intracellular domain. Conversely, the glycosylphosphatidylinositol-anchored CEACAM5 and CEACAM6 activate the c-Jun NH2-terminal kinase (JNK)1/2-Rubicon-NOX2 pathway, suggestive of LC3-associated phagocytosis. Overall, our data show for the first time novel mechanisms of adhesion to and invasion of pneumocytes by A. baumannii via CEACAM-dependent signaling pathways that eventually lead to bacterial killing. These findings suggest that CEACAM upregulation could put patients at increased risk of lower respiratory tract infection by A. baumannii IMPORTANCE This work shows for the first time that Acinetobacter baumannii binds to carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), CEACAM5, and CEACAM6. This binding significantly enhances A. baumannii internalization within alveolar host cell epithelia. Intracellular trafficking involves typical Rab5 and Rab7 vacuolar proteins as well as light chain 3 (LC3) and slowly progresses to bacterial killing by endosome acidification. CEACAM engagement by A. baumannii leads to distinct and specific downstream signaling pathways. The CEACAM1 pathway finely tunes interleukin-8 (IL-8) secretion, whereas CEACAM5 and CEACAM6 mediate LC3-associated phagocytosis. The present study provides new insights into A. baumannii-host interactions and could represent a promising therapeutic strategy to reduce pulmonary infections caused by this pathogen.