Potential predictive role of gut microbiota to immunotherapy in HCC patients: a brief review.

The recent evolution of immunotherapy has revolutionised the treatment of hepatocellular carcinoma (HCC) and has led to new therapeutic standards. The advances in immunotherapy have been accompanied by the recognition of the role of the gut-liver axis in the progression of HCC but also of the clinical relevance of the gut microbiota, which influences host homeostasis but also cancer development and the response to treatment. Dysbiosis, by altering the tumour microenvironment, favours the activation of intracellular signalling pathways and promotes carcinogenesis. The gut microbiota, through their composition and immunomodulatory role, are thus strong predictors of the response to immune checkpoint inhibitor (ICI) treatment as well as an available target to improve ICI efficacy and reduce drug toxicities. In this review we examine the novel role of the gut microbiota as biomarkers in both the diagnosis of HCC and the clinical response to immunotherapy as well as its potential impact on clinical practice in the future.

Lichenysin-like Polypeptide Production by Bacillus licheniformis B3-15 and Its Antiadhesive and Antibiofilm Properties.

We report the ability of the crude biosurfactant (BS B3-15), produced by the marine, thermotolerant Bacillus licheniformis B3-15, to hinder the adhesion and biofilm formation of Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213 to polystyrene and human cells. First, we attempted to increase the BS yield, optimizing the culture conditions, and evaluated the surface-active properties of cell-free supernatants. Under phosphate deprivation (0.06 mM) and 5% saccharose, the yield of BS (1.5 g/L) increased by 37%, which could be explained by the earlier (12 h) increase in lchAA expression compared to the non-optimized condition (48 h). Without exerting any anti-bacterial activity, BS (300 µg/mL) prevented the adhesion of P. aeruginosa and S. aureus to polystyrene (47% and 36%, respectively) and disrupted the preformed biofilms, being more efficient against S. aureus (47%) than P. aeruginosa (26%). When added to human cells, the BS reduced the adhesion of P. aeruginosa and S. aureus (10× and 100,000× CFU/mL, respectively) without altering the epithelial cells' viability. As it is not cytotoxic, BS B3-15 could be useful to prevent or remove bacterial biofilms in several medical and non-medical applications.

Anti-Severe Acute Respiratory Syndrome Coronavirus 2 IgG Is Present in the Amniotic Fluid of both Infected and Vaccinated Women at Second Trimester of Pregnancy: A Cohort Study.

INTRODUCTION: Pregnant women are protected from the complications of COVID-19 infection, thanks to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination. The benefit of this vaccination to prevent morbidity and mortality in the fetus has not yet been completely elucidated. Our aim was to test the presence of anti-SARS-CoV-2 antibodies in the amniotic fluid during the second trimester of pregnancy and then to compare them to the antibody levels in maternal serum to evaluate their correlation and to improve amniotic fluid immunological characteristics knowledge. METHODS: This cohort study took place at the Policlinico G. Martino of Messina from September 2021 to February 2022; 22 pregnant women had amniocentesis: we analyzed serum and amniotic fluid samples of women who contracted the SARS-CoV-2 infection or vaccinated against the same virus within 1 year, and women never infected or vaccinated against it. Amniotic fluids and peripheral blood were collected to evaluate IgG anti-SARS-CoV-2 nucleocapsid and spike S1 protein antibodies. RESULTS: Patients vaccinated had higher S1 receptor-binding domain antibody levels both in amniotic fluid (p < 0.006; mean 68.70; standard deviation [SD] 85.46) and maternal blood (p < 0.005; mean 1,989.86; SD 3,777.15) than unvaccinated women. Anti-nucleocapsid antibodies were present in women who developed COVID infection both in amniotic fluid and maternal blood but not in unvaccinated women. There was a high correlation between the concentrations of anti-spike antibody levels in serum and amniotic fluid of vaccinated women (p < 0.001; R = 1.0) and of anti-nucleocapsid antibody levels in serum and amniotic fluid of women who developed COVID infection (p < 0.001; R = 0.93). CONCLUSION: Recent studies have shown that SARS-CoV-2 vaccination during pregnancy is safe. Moreover, we can assume that there is an early transplacental antibody transfer after anti-SARS-CoV-2 immunization to protect the fetus, and there is also a high correlation between levels of anti-nucleocapsid antibodies in blood and amniotic fluid of pregnant women previously infected.

Crosstalk between ILC3s and Microbiota: Implications for Colon Cancer Development and Treatment with Immune Check Point Inhibitors.

Type 3 innate lymphoid cells (ILC3s) are primarily tissue-resident cells strategically localized at the intestinal barrier that exhibit the fast-acting responsiveness of classic innate immune cells. Populations of these lymphocytes depend on the transcription factor RAR-related orphan receptor and play a key role in maintaining intestinal homeostasis, keeping host-microbial mutualism in check. Current evidence has indicated a bidirectional relationship between microbiota and ILC3s. While ILC3 function and maintenance in the gut are influenced by commensal microbiota, ILC3s themselves can control immune responses to intestinal microbiota by providing host defense against extracellular bacteria, helping to maintain a diverse microbiota and inducing immune tolerance for commensal bacteria. Thus, ILC3s have been linked to host-microbiota interactions and the loss of their normal activity promotes dysbiosis, chronic inflammation and colon cancer. Furthermore, recent evidence has suggested that a healthy dialog between ILC3s and gut microbes is necessary to support antitumor immunity and response to immune checkpoint inhibitor (ICI) therapy. In this review, we summarize the functional interactions occurring between microbiota and ILC3s in homeostasis, providing an overview of the molecular mechanisms orchestrating these interactions. We focus on how alterations in this interplay promote gut inflammation, colorectal cancer and resistance to therapies with immune check point inhibitors.

Anti-Bacterial Adhesion on Abiotic and Biotic Surfaces of the Exopolysaccharide from the Marine Bacillus licheniformis B3-15.

The eradication of bacterial biofilm represents a crucial strategy to prevent a clinical problem associated with microbial persistent infection. In this study we evaluated the ability of the exopolysaccharide (EPS) B3-15, produced by the marine Bacillus licheniformis B3-15, to prevent the adhesion and biofilm formation of Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213 on polystyrene and polyvinyl chloride surfaces. The EPS was added at different times (0, 2, 4 and 8 h), corresponding to the initial, reversible and irreversible attachment, and after the biofilm development (24 or 48 h). The EPS (300 µg/mL) impaired the initial phase, preventing bacterial adhesion even when added after 2 h of incubation, but had no effects on mature biofilms. Without exerting any antibiotic activity, the antibiofilm mechanisms of the EPS were related to the modification of the (i) abiotic surface properties, (ii) cell-surface charges and hydrophobicity, and iii) cell-to-cell aggregation. The addition of EPS downregulated the expression of genes (lecA and pslA of P. aeruginosa and clfA of S. aureus) involved in the bacterial adhesion. Moreover, the EPS reduced the adhesion of P. aeruginosa (five logs-scale) and S. aureus (one log) on human nasal epithelial cells. The EPS could represent a promising tool for the prevention of biofilm-related infections.

Mechanical bacterial lysate enhances antimicrobial barrier mechanisms in human airway epithelial cells.

Polyvalent mechanical bacterial lysate is effective in the prevention of respiratory tract infections, although its mechanism of action is not entirely elucidated. Because epithelial cells constitute the frontline defense against infections, we investigated the molecular mechanisms of innate response exerted by bronchial epithelial cells in the presence of polyvalent mechanical bacterial lysate. By using primary human bronchial epithelial cells, we observed that polyvalent mechanical bacterial lysate was able to increase the expression of cellular adhesion molecules such as ICAM-1 and E-cadherin, as well as the expression of amphiregulin, a growth factor able to support human bronchial epithelial cell proliferation. Remarkably, polyvalent mechanical bacterial lysate promoted in human bronchial epithelial cells the de novo expression of human ß-defensin-2, a major antimicrobial peptide, conferring them a direct antimicrobial activity. Moreover, polyvalent mechanical bacterial lysate-stimulated human bronchial epithelial cells provided signals for increased IL-22 production by innate lymphoid cells via IL-23, which could further contribute to the release of antimicrobial peptides by epithelial cells. In agreement with these in vitro data, the concentration of both IL-23 and antimicrobial peptides (human ß-defensin-2 and LL-37) increased in the saliva of healthy volunteers after sublingual administration of polyvalent mechanical bacterial lysate. Altogether, these results indicate that polyvalent mechanical bacterial lysate administration might support mucosal barrier integrity and promote mechanisms of antimicrobial activity in airway epithelial cells.

Biofilm Development and Approaches to Biofilm Inhibition by Exopolysaccharides.

Bacteria biofilm consists of microorganisms, accounting for 5-35% of the biofilm volume, and of the extracellular matrix (65-95%), made of water (97%), proteins (2%), polysaccharides (1-2%) and nucleic acids (DNA/RNA, both <1%). The physiology of bacteria in the biofilms entails adaptive changes with expression of genes which are different from those translated in the planktonic state. While most of our applied knowledge on bacterial biology stems from the study in the planktonic state, an increasing interest is currently paid to bacterial behaviour as biofilm generators, as it is estimated that 65% of all bacterial infections are associated with bacterial biofilms. Infections of both upper and lower airways, bacterial endocarditis, chronic otitis media, urinary tract infections, periodontitis, ocular infections and chronic wound infections (including diabetic foot ulcer) are all associated with biofilm formation. The role of biofilm is also relevant in case of infections taking place on abiotic surfaces, as in the case of infections occurring on prostheses and several other medical devices. Here, we review current knowledge on biofilm formation and its impact on human infections, discussing recent means for its inhibition, with particular emphasis on an interesting anti-biofilm activity exerted by exopolysaccharides derived from marine strains of Bacillus licheniformis.

Cutting Edge: Hyperinflammatory Monocytes Expressing CD56 Abound in Severe COVID-19 Patients.

Proinflammatory monocytes play a preponderant role in the development of a cytokine storm causing fatal consequences in coronavirus disease 2019 (COVID-19) patients, highlighting the importance of analyzing in more detail monocyte distribution in these patients. In this study, we identified an atypical monocyte subpopulation expressing CD56 molecules that showed a low level of HLA-DR and high level of l-selectin. They released higher amounts of TNF-α and IL-6 and expressed genes associated with an excessive inflammatory process. Remarkably, the frequency of CD56+ monocytes inversely correlated with that of CD16+ monocytes and a high CD56+/CD16+monocyte ratio was associated with both disease severity and mortality, as well as with serum concentration of type I IFN, a factor able to induce the appearance of CD56+ monocytes. In conclusion, severe COVID-19 is characterized by the abundance of hyperinflammatory CD56+ monocytes, which could represent a novel marker with prognostic significance and, possibly, a therapeutic target for controlling the inflammatory process occurring during COVID-19.

Chitosan-Hyaluronan Nanoparticles for Vinblastine Sulfate Delivery: Characterization and Internalization Studies on K-562 Cells.

In the present study, we developed chitosan/hyaluronan nanoparticles (CS/HY NPs) for tumor targeting with vinblastine sulfate (VBL), that can be directed to the CD44 transmembrane receptor, over-expressed in cancer cells. NPs were prepared by coating with HY-preformed chitosan/tripolyphosphate (CS/TPP) NPs, or by polyelectrolyte complexation of CS with HY. NPs with a mean hydrodynamic radius (RH) of 110 nm, 12% polydispersity index and negative zeta potential values were obtained by a direct complexation process. Transmission Electron Microscopy (TEM) images showed spherical NPs with a non-homogeneous matrix, probably due to a random localization of CS and HY interacting chains. The intermolecular interactions occurring between CS and HY upon NPs formation were experimentally evidenced by micro-Raman (µ-Raman) spectroscopy, through the analysis of the spectral changes of characteristic vibrational bands of HY during NP formation, in order to reveal the involvement of specific chemical groups in the process. Optimized NP formulation efficiently encapsulated VBL, producing a drug sustained release for 20 h. In vitro studies demonstrated a fast internalization of labeled CS/HY NPs (within 6 h) on K-562 human myeloid leukemia cells. Pre-saturation of CD44 by free HY produced a slowing-down of NP uptake over 24 h, demonstrating the need of CD44 for the internalization of HY-based NPs.

Emerging Evidence and Treatment Perspectives from Randomized Clinical Trials in Systemic Sclerosis: Focus on Interstitial Lung Disease.

Systemic sclerosis (SSc) is a complex rare autoimmune disease with heterogeneous clinical manifestations. Currently, interstitial lung disease (ILD) and cardiac involvement (including pulmonary arterial hypertension) are recognized as the leading causes of SSc-associated mortality. New molecular targets have been discovered and phase II and phase III clinical trials published in the last 5 years on SSc-ILD will be discussed in this review. Details on the study design; the drug tested and its dose; the inclusion and exclusion criteria of the study; the concomitant immunosuppression; the outcomes and the duration of the study were reviewed. The two most common drugs used for the treatment of SSc-ILD are cyclophosphamide and mycophenolate mofetil, both supported by randomized controlled trials. Additional drugs, such as nintedanib and tocilizumab, have been approved to slow pulmonary function decline in SSc-ILD. In this review, we discuss the therapeutic alternatives for SSc management, offering the option to customize the design of future studies to stratify SSc patients and provide a patient-specific treatment according to the new emerging pathogenic features of SSc-ILD.

Correlation between Hyperkalemia and the Duration of Several Hospitalizations in Patients with Chronic Kidney Disease.

(1) Background: This observational study aimed to verify the association between serum potassium levels and hospitalization days in patients with chronic kidney disease in a follow up of nine months. (2) Methods: Patients with chronic kidney disease were divided into group A (180 patients, potassium ≤ 5.1 mEq/L) and B (90 patients, potassium > 5.1 mEq/L). Student's t-test, Mann-Whitney test, Pearson's Chi-Square test, Pearson/Spearman's correlation test and linear regression test were performed in the entire sample and in stage-G4/5 subsample. (3) Results: Groups A and B differed for estimated glomerular filtration rate (eGFR) (34.89 (IQR, 16.24-57.98) vs. 19.8 (IQR, 10.50-32.50) mL/min/1.73 m2; p < 0.0001), hemoglobin (11.64 ± 2.20 vs. 10.97 ± 2.19 g/dL, p = 0.048), sum of hospitalization days (8 (IQR, 6-10) vs. 11 (IQR, 7-15) days; p < 0.0001) and use of angiotensin II receptor blockers (40.2% vs. 53.3%; p = 0.010). Considering patients with eGFR 6-30 mL/min/1.73 m2, differences in the sum of hospitalization days were confirmed. Multivariable regression analysis showed that hyperkalemia is an independent risk factor of increased hospital length. In stage G4-G5, regression analysis showed that hyperkalemia is the only independent risk factor (ß = 2.93, 95% confidence interval, 0.077-5.794, p = 0.044). (4) Conclusions: We observed significantly greater odds of increased length of hospital stay among patients with higher potassium, mostly in stages G4-G5 chronic kidney disease.

Monocyte to HDL ratio: a novel marker of resistant hypertension in CKD patients.

BACKGROUND: Inflammation, oxidative stress (OS), atherosclerosis and resistant hypertension (RH) are common features of chronic kidney disease (CKD) leading to a higher risk of death from cardiovascular disease. These effects seem to be modulated by impaired anti-oxidant, anti-inflammatory and reverse cholesterol transport actions of high-density lipoprotein cholesterol (HDL). HDL prevents and reverses monocyte recruitment and activation into the arterial wall and impairs endothelial adhesion molecule expression. Recently, monocyte count to HDL-cholesterol ratio (MHR) has emerged as a potential marker of inflammation and OS, demonstrating to be relevant in CKD. Our research was aimed to assess, for the first time, its reliability in RH. METHODS: We performed a retrospective study on 214 patients with CKD and arterial hypertension who were admitted between January and June 2019 to our Department, 72 of whom were diagnosed with RH. RESULTS: MHR appeared inversely related to eGFR (ρ = - 0.163; P = 0.0172). MHR was significantly higher among RH patients compared to non-RH ones (12.39 [IQR 10.67-16.05] versus 7.30 [5.49-9.06]; P < 0.0001). Moreover, MHR was significantly different according to the number of anti-hypertensive drugs per patient in the whole study cohort (F = 46.723; P < 0.001) as well as in the non-RH group (F = 14.191; P < 0.001). Moreover, MHR positively correlates with diabetes mellitus (ρ = 0.253; P = 0.0002), white blood cells (ρ = 0.664; P < 0.0001) and C-reactive protein (ρ = 0.563; P < 0.0001). CONCLUSIONS: MHR may be a reliable biomarker due to the connection between HDL and monocytes. Our study suggests that MHR is linked with the use of multiple anti-hypertensive therapy and resistant hypertension in CKD patients, and can be a useful ratio to implement appropriate treatment strategies.

A multivariate analysis of Multiple Myeloma subtype plasma cells.

Trusted methods for identifying different Multiple Myeloma (MM) cells and their biological diversity due to their immunophenotypic variety are often little detailed and difficult to find in literature. In this work, we show that micro-Raman spectroscopy can be used to highlight if there is a certain degree of distinction or correlation between the MM subtype plasmacells in relation to the cluster of differentiation (CD45+/CD38+/CD138-) and (CD45-/CD38+/CD138+). After taking samples from the bone marrow of patients with Multiple Myeloma, the PCs were sorted by flow cytometry, selecting the most common CD of the disease, i.e. CD 45, CD38 and CD138. Some spectral differences are observed comparing the Raman spectra of the two set of samples investigated. To better define in which spectral regions there are greater differences and, therefore, to which biological contributions the changes refers, we also explored the principal component analysis (PCA) of the collected Raman data. The spectral variations between the different sorted cells have been highlighted by plotting loading vectors PC1 and PC2, which shows a net differentiation between the two set of cells. Ultimately, the differences shown by PCA have been associated with the spectral variations observed and explained in terms of changes of proteins and lipid contributions. Thus, the differentiation of Multiple Myeloma subtype plasma cells by confocal micro-Raman spectroscopy can be proposed as a diagnostic tool in the speeding up of cell identification, assessing the intracellular biochemical changes that take place in cancer cells.

Circulating ILC precursors expressing CD62L exhibit a type 2 signature distinctly decreased in psoriatic patients.

Human CD117+ CRTH2neg innate lymphoid cells (ILC) comprise multipotent precursors (ILCp), which are able to differentiate into subtypes in response to different signals received in peripheral tissues. NKp46+ ILCp have been reported to associate with ILC3 whereas KLRG1+ ILCp with ILC2, although the latter can also generate other ILC subsets, thus, maintaining a substantial plasticity. We here showed that CD62L is expressed by ILCp exclusively within KLRG1+ population and its expression marks a loss of their broad differentiation potential. Analysis of cytokine production and relevant markers demonstrated that CD62L+ ILCp mainly differentiate into ILC2 whereas CD62Lneg counterpart can also differentiate into other ILC subsets depending on the signals they receive. Remarkably, in peripheral blood of psoriatic patients, where ILC3 are usually enriched, CD62L+ ILC were drastically reduced, whereas CD62Lneg ILC2 upregulated both RORγt and NKp46, thus, suggesting an ongoing conversion to ILC3. Therefore, CD62L now emerges as a potential marker to identify a skewing toward type 2 among ILCp.

ILC in chronic inflammation, cancer and targeting with biologicals.

Since their discovery, Innate Lymphoid Cells (ILC) have emerged as important effector cells, serving multiple roles in maintaining tissue homeostasis and responding to tissue insults. As such, dysregulations of their function and distribution have been observed in a variety of immune-mediated diseases, suggesting a specific role for ILC in the pathophysiology of several disorders including chronic inflammation and cancer. Here, we provide an updated view on ILC biology dissecting their pathological or protective contribution in chronic inflammatory diseases such as multiple sclerosis, inflammatory bowel diseases, psoriasis, rheumatoid arthritis, asthma and COPD, atherosclerosis, also exploring ILC role in tumor surveillance and progression. Throughout the review, we will also highlight how the potential dual role of these cells for protective or pathogenic immunity in many inflammatory diseases makes them interesting targets for the development of novel therapeutic strategies, particularly promising.

Human Hepatitis B Virus Negatively Impacts the Protective Immune Crosstalk Between Natural Killer and Dendritic Cells.

BACKGROUND AND AIMS: Natural killer (NK) cells play a crucial role in the clearance of human viruses but their activity is significantly impaired in patients infected with chronic hepatitis B (CHB). Cooperation with dendritic cells (DCs) is pivotal for obtaining optimal NK cell antiviral function; thus, we investigated whether HBV might impact the ability of DCs to sustain NK cell functions. APPROACH AND RESULTS: Human DCs were poor stimulators of interferon-gamma (IFN-γ) production by NK cells when exposed to HBV, while maintaining the capability to trigger NK cell cytotoxicity. HBV prevented DC maturation but did not affect their expression of human leukocyte antigen class I, thus allowing DCs to evade NK cell lysis. Tolerogenic features of DCs exposed to HBV were further supported by their increased expression of IL-10 and the immunosuppressive enzyme indoleamine 2,3-dioxygenase, which contributed to the impairment of DC-mediated NK cell IFN-γ production and proliferation, respectively. HBV could also inhibit the expression of inducible immunoproteasome (iP) subunits on DCs. In fact, NK cells could induce iP subunit expression on DCs, but they failed in the presence of HBV. Remarkably, circulating blood DC antigen1 (BDCA1)+ DCs isolated from patients with CHB were functionally compromised, hence altering, in turn, NK cell responses. CONCLUSIONS: The abnormal NK-DC interplay caused by HBV may significantly impair the efficacy of antiviral immune response in patients with CHB.

Safety profile of immune checkpoint inhibitors: An analysis of the Italian spontaneous reporting system database.

AIMS: To provide an overview of immune checkpoint inhibitors (ICIs) safety profile using the Italian spontaneous adverse drug reaction (ADR) reporting system. METHODS: We selected all ADR reports attributed to ipilimumab (CTLA-4 inhibitor), nivolumab, pembrolizumab, atezolizumab (PD-1/PD-L1 inhibitors) from the Italian spontaneous reporting system (2011-2018). Descriptive analyses of reports for ICIs have been conducted. Time to onset of adverse effects was stratified by system organ class. Reporting odds ratio was used as measure of ADR reporting disproportionality. ICI-related ADR reports were compared with 2 reference groups, i.e. all other suspected drugs or all other antineoplastic agents. RESULTS: Overall, 2217 (0.7%) reports were related to ICIs (nivolumab: 72.2% of those reports; ipilimumab: 14.3%; pembrolizumab: 10.3%; and atezolizumab: 3.5%). ICI-related ADR reports mostly involved males (65%) and median age was 67 (interquartile range 59-73) years. Serious reports accounted for 48.8%. Frequencies of endocrine, general, hepatobiliary, metabolism, musculoskeletal, respiratory disorders, infections and neoplasms were significantly higher for ICIs than for all other drugs (P < .001). Except for infections, similar results emerged through comparison with other anticancer drugs. Colitis, hypophysitis and skin disorders were more frequently reported for anti-CTLA-4 drugs than PD-1/PD-L1 ICIs, and the opposite for musculoskeletal effects, pneumonia, and thyroid dysfunctions. ICIs were disproportionally associated also with less known risks, e.g. ischaemic heart disease, cardiac failure and optic nerve disorders. CONCLUSION: The most frequently reported safety issues were probably immune-related adverse events including general, gastrointestinal and respiratory disorders. Potentially emerging safety signals, such as ischaemic heart disease and cardiac failure, requiring further investigation were detected.