Interplay of leucocyte-platelet complexes and clinical response to eltrombopag in immune thrombocytopenia patients.

Eltrombopag (ELT) is a thrombopoietin-receptor agonist that stimulates platelet (PLT) production in patients with primary immune thrombocytopenia (ITP). One potential mechanism of ELT is modulating the inflammatory response by increasing PLTs binding to leucocytes. This study examined the effect of ELT on leucocyte-PLTs complexes in 38 ITP patients. Patients, predominantly females with a mean age of 59 years, underwent treatments like corticosteroids, intravenous immunoglobulin and splenectomy. Compared to healthy donors, ITP patients exhibited lower percentages of lymphocyte with bound PLTs, but similar monocyte- or neutrophil with bound PLTs. ELT treatment increased PLTs counts and all types of leucocyte with bound PLTs. Network analysis showed dynamic changes in leucocyte with bound PLTs relationships due to ELT. Machine learning indicated that higher percentages of monocytes with bound PLTs were linked to a better clinical response to ELT. A possible mechanism was an increased IL-10 production in monocytes with bound PLTs from responder patients. This study provides insights into the immunological changes in ITP patients undergoing ELT and suggests potential predictive biomarkers for treatment response and disease monitoring.

Levels of Lysozyme and SLPI in Bronchoalveolar Lavage: Exploring Their Role in Interstitial Lung Disease.

Interstitial lung diseases (ILDs) are characterized by inflammation or fibrosis of the pulmonary parenchyma. Despite the involvement of immune cells and soluble mediators in pulmonary fibrosis, the influence of antimicrobial peptides (AMPs) remains underexplored. These effector molecules display a range of activities, which include immunomodulation and wound repair. Here, we investigate the role of AMPs in the development of fibrosis in ILD. We compare the concentration of different AMPs and different cytokines in 46 fibrotic (F-ILD) and 17 non-fibrotic (NF-ILD) patients by ELISA and using peripheral blood mononuclear cells from in vitro stimulation in the presence of lysozyme or secretory leukocyte protease inhibitor (SLPI) from 10 healthy donors. We observed that bronchoalveolar lavage (BAL) levels of AMPs were decreased in F-ILD patients (lysozyme: p < 0.001; SLPI: p < 0.001; LL-37: p < 0.001; lactoferrin: p = 0.47) and were negatively correlated with levels of TGF-ß (lysozyme: p = 0.02; SLPI: p < 0.001) and IL-17 (lysozyme: p < 0.001; SLPI: p < 0.001). We observed that lysozyme increased the percentage of CD86+ macrophages (p < 0.001) and the production of TNF-α (p < 0.001). We showed that lysozyme and SLPI were associated with clinical parameters (lysozyme: p < 0.001; SLPI: p < 0.001) and disease progression (lysozyme: p < 0.001; SLPI: p = 0.01). These results suggest that AMPs may play an important role in the anti-fibrotic response, regulating the effect of pro-fibrotic cytokines. In addition, levels of lysozyme in BAL may be a potential biomarker to predict the progression in F-ILD patients.

Immune Response and Risk of Decompensation following SARS-CoV-2 Infection in Outpatients with Advanced Chronic Liver Disease.

Advanced chronic liver disease (ACLD) is associated with a wide spectrum of immune dysfunction. The clinical impact of SARS-CoV-2 on the development of decompensation and immune response in unvaccinated outpatients has not as yet been clearly defined. This study aimed to evaluate the clinical and immunological impact of SARS-CoV-2 on outpatients with ACLD. This is an observational case-control study, in which ACLD outpatients were included prospectively and consecutively and classified into two groups: SARS-CoV-2 infected and non-infected. Patients' baseline characteristics and infection data were collected and analyzed. Immunoglobulin G (IgG) levels against Spike 1 were evaluated. The primary endpoint was risk of liver decompensation during follow-up, assessed after propensity score matching and adjusted by Cox regression. Between October 2020 and July 2021, ACLD outpatients (n = 580) were identified, and 174 patients with clinical follow-up were included. SARS-CoV-2 infection incidence was 7.6% (n = 44). Risk of liver decompensation was significantly higher after infection (HR = 2.43 [1.01-5.86], p = 0.048) vs. non-infection. The time of IgG evaluation was similar in all patients (n = 74); IgG concentrations were significantly higher in compensated vs. decompensated patients (1.02 ± 0.35 pg/mL vs. 0.34 ± 0.16 pg/mL, p < 0.0001) and correlated with hemoglobin levels. The dysregulation of the innate immune response in patients with decompensated liver disease increased the risk of further decompensation following SARS-CoV-2, mainly due to a worsening of ascites.

Clinical Relevance of Tumour-Infiltrating Immune Cells in HER2-Negative Breast Cancer Treated with Neoadjuvant Therapy.

Currently, therapy response cannot be accurately predicted in HER2-negative breast cancer (BC). Measuring stromal tumour-infiltrating lymphocytes (sTILs) and mediators of the tumour microenvironment and characterizing tumour-infiltrating immune cells (TIICs) may improve treatment response in the neoadjuvant setting. Tumour tissue and peripheral blood samples were retrospectively collected from 118 patients, and sTILs were evaluated. Circulating exosomes and myeloid-derived suppressor cells were determined by flow cytometry. TIICs markers (CD4, CD8, CD20, CD1a, and CD68) were assessed immunohistochemically. High sTILs were significantly associated with pathological complete response (pCR; p = 0.048) and event-free survival (EFS; p = 0.027). High-CD68 cells were significantly associated with pCR in triple-negative (TN, p = 0.027) and high-CD1a cells with EFS in luminal-B (p = 0.012) BC. Cluster analyses of TIICs revealed two groups of tumours (C1 and C2) that had different immune patterns and clinical outcomes. An immunoscore based on clinicopathological variables was developed to identify high risk (C1) or low-risk (C2) patients. Additionally, cluster analyses revealed two groups of tumours for both luminal-B and TNBC. Our findings support the association of sTILs with pCR and show an immunological component in a subset of patients with HER2-negative BC. Our immunoscore may be useful for future escalation or de-escalation treatments.

The immunoregulatory role of IL-35 in patients with interstitial lung disease.

Pulmonary fibrosis involves various types of immune cells and soluble mediators, including TGF-ß and IL-35, a recently identified heterodimeric cytokine that belongs to the IL-12 cytokine family. However, the effect of regulatory IL-35 may play an important role in fibrotic diseases. The aim of this paper is to explore the immunoregulatory role of IL-35 in the development of fibrosis in interstitial lung disease (ILD). To gain a better understanding of this issue, the concentrations of IL-35 and different profibrotic cytokines in fibrotic (F-ILD) and non-fibrotic (NF-ILD) patients by ELISA were compared to that of intracellular IL-35 and IL-17 on CD4+ T cells stimulated in the presence of BAL or with different ratios of recombinant IL-35 (rIL-35) and TGF-ß (rTGF-ß), which were evaluated by flow cytometry. We observed that BAL concentration of IL-35 was lower in F patients (p < 0.001) and was negatively correlated with concentrations of TGF-ß (p < 0.001) and IL-17 (p < 0.001). In supplemented cell cultures, BAL from NF but not F patients enhanced the percentage of IL-35 + CD4+ T (p < 0.001) cells and decreased the percentage of IL-17 + CD4+ T cells (p < 0.001). The percentage of IL-35 + CD4+ T cells correlated positively with BAL concentration of IL-35 (p = 0.02), but correlated negatively with BAL concentrations of IL-17 (p = 0.007) and TGF-ß (p = 0.01). After adjusting the concentrations of recombinant cytokines to establish a TGF-ß: IL-35 ratio of 1:4, an enhanced percentage of IL-35 + CD4+ T cells (p < 0.001) but a decreased percentage of IL-17 + CD4+ T cells (p < 0.001) was observed. After adding recombinant IL-35 to the BAL from F patients until a 1:4 ratio of TGF-ß: IL-35 was reached, a significantly increased percentage of IL-35 + CD4+ T cells (p < 0.001) and a decreased percentage of IL-17 + CD4+ T cells (p = 0.003) was found. These results suggest that IL-35 may induce an anti-fibrotic response, regulating the effect of TGF-ß and the inflammatory response on CD4+ T cells. In addition, the TGF-ß: IL-35 ratio in BAL has been shown to be a potential biomarker to predict the outcome of F patients with ILD.

Dysregulated neutrophil extracellular traps formation in sepsis.

The migration and antimicrobial functions of neutrophils seem to be impaired during sepsis and contribute to the dysregulation of immune responses and disease pathogenesis. However, the role of neutrophil extracellular traps (NETs) remains to be clarified. The study aimed to analyse sequential phenotypic and functional changes of neutrophils during the time following the diagnosis of sepsis. We prospectively enrolled 49 septic and 18 non-septic patients from the intensive care unit (ICU) and emergency room (ER) and 20 healthy volunteers (HV). Baseline blood samples from septic and non-septic patients were collected within 12 h of admission to the hospital. Additional septic samples were drawn at 24, 48 and 72 h after baseline. Neutrophil phenotype and degranulation capacity were assessed by flow cytometry and NET formation was quantified by fluorescence. Neutrophils from septic patients exhibited increased CD66b, CD11b and CD177 expression but displayed reduced NET formation at baseline compared with non-septic patients and HV controls. Neutrophils expressing CD177 interacted less with platelets, were related to reduced NETosis and tended to indicate a worse sepsis outcome. In vitro experiments revealed that neutrophil function is compromised by the origin of sepsis, including the pathogen type and the affected organ. Assessing a decision tree model, our study showed that CD11b expression and NETosis values are useful variables to discriminate septic from non-septic patients. We conclude that sepsis induces changes in neutrophil phenotype and function that may compromise the effective capacity of the host to eliminate pathogens.

Impaired Regulation by IL-35 in Systemic Sclerosis.

This study investigated the role of IL-35 in systemic sclerosis (SSc) patients, focusing on CD4+ T cell response and immunomodulatory cytokine production. By comparing the cytokine levels in healthy donors (HD) and SSc patients using ELISAs, we found a significantly lower plasma IL-35 concentration in the SSc patients (52.1 ± 5.6 vs. 143 ± 11.1, p < 0.001). Notably, the IL-35 levels showed a negative correlation with TGF-ß (p < 0.001) and IL-17 (p = 0.04). Assessing the IL-35R expression across cell types in the SSc patients and HDs via flow cytometry, we found higher levels on monocytes (40.7 + 5.7 vs. 20.3 ± 1.9, p < 0.001) and lower levels on CD8+ T cells (61.8 ± 9.2 vs. 83.4 ± 0.8, p < 0.05) in the SSc patients. The addition of recombinant IL-35 to stimulated peripheral blood mononuclear cells reduced the IL-17+CD4+ T cell percentage (9.0 ± 1.5 vs. 4.8 ± 0.7, p < 0.05) and increased the IL-35+CD4+ T percentage (4.1 ± 2.3 vs. 10.2 ± 0.8, p < 0.001). In a Treg:Tresponder cell Sco-culture assay with HD and SSc samples, rIL35 decreased the cell proliferation and levels of IL-17A (178.2 ± 30.5 pg/mL vs. 37.4 ± 6.4 pg/mL, p < 0.001) and TGF-ß (4194 ± 777 pg/mL vs. 2413 ± 608 pg/mL, p < 0.01). Furthermore, we observed a positive correlation between the modified Rodnan skin score (mRSS) and TGF-ß (p < 0.001), while there was a negative correlation between mRSS and IL-35 (p = 0.004). Interestingly, higher levels of plasmatic IL-35 were detected in individuals with limited disease compared to those with diffuse disease (60.1 ± 8.0 vs. 832.3 ± 4.1, p < 0.05). These findings suggest that IL-35 exhibits anti-inflammatory properties in SSc and it may serve as a marker for disease severity and a therapeutic target.

The Effects of Interstitial Lung Diseases on Alveolar Extracellular Vesicles Profile: A Multicenter Study.

Diagnosis of interstitial lung diseases (ILD) is difficult to perform. Extracellular vesicles (EVs) facilitate cell-to-cell communication, and they are released by a variety of cells. Our goal aimed to investigate EV markers in bronchoalveolar lavage (BAL) from idiopathic pulmonary fibrosis (IPF), sarcoidosis and hypersensitivity pneumonitis (HP) cohorts. ILD patients followed at Siena, Barcelona and Foggia University Hospitals were enrolled. BAL supernatants were used to isolate the EVs. They were characterized by flow cytometry assay through MACSPlex Exsome KIT. The majority of alveolar EV markers were related to the fibrotic damage. CD56, CD105, CD142, CD31 and CD49e were exclusively expressed by alveolar samples from IPF patients, while HP showed only CD86 and CD24. Some EV markers were common between HP and sarcoidosis (CD11c, CD1c, CD209, CD4, CD40, CD44, CD8). Principal component analysis distinguished the three groups based on EV markers with total variance of 60.08%. This study has demonstrated the validity of the flow cytometric method to phenotype and characterize EV surface markers in BAL samples. The two granulomatous diseases, sarcoidosis and HP, cohorts shared alveolar EV markers not revealed in IPF patients. Our findings demonstrated the viability of the alveolar compartment allowing identification of lung-specific markers for IPF and HP.

Kinetics of IFNγ-Induced Cytokines and Development of Immune-Related Adverse Events in Patients Receiving PD-(L)1 Inhibitors.

Immune checkpoint inhibitors (ICI) have the potential to trigger unpredictable immune-related adverse events (irAEs), which can be severe. The underlying mechanisms of these events are not fully understood. As PD-L1 is upregulated by IFN, the heightened immune activation resulting from PD-1/PD-L1 inhibition may enhance the IFN response, triggering the expression of IFN-inducible genes and contributing to irAE development and its severity. In this study, we investigated the interplay between irAEs and the expression of IFN-inducible chemokines and cytokines in 134 consecutive patients with solid tumours treated with PD-(L)1 inhibitors as monotherapy or in combination with chemotherapy or other immunotherapy agents. We compared the plasma levels of IFN-associated cytokines (CXCL9/10/11, IL-18, IL-10, IL-6 and TGFß) at various time points (at baseline, at the onset of irAE and previous to irAE onset) in three patient groups categorized by irAE development and severity: patients with serious irAEs, mild irAEs and without irAEs after PD-(L)1 inhibitors. No differences were observed between groups at baseline. However, patients with serious irAEs exhibited significant increases in CXCL9/10/11, IL-18 and IL-10 levels at the onset of the irAE compared to baseline. A network analysis and correlation patterns highlighted a robust relationship among these chemokines and cytokines at serious-irAE onset. Combining all of the analysed proteins in a cluster analysis, we identified a subgroup of patients with a higher incidence of serious irAEs affecting different organs or systems. Finally, an ROC analysis and a decision tree model proposed IL-18 levels ≥ 807 pg/mL and TGFß levels ≤ 114 pg/mL as predictors for serious irAEs in 90% of cases. In conclusion, our study elucidates the dynamic changes in cytokine profiles associated with serious irAE development during treatment with PD-(L)1 inhibitors. The study's findings offer valuable insights into the intricate IFN-induced immune responses associated with irAEs and propose potential predictive markers for their severity.

Potential Role of Circulating PD-L1+ Leukocytes as a Predictor of Response to Anti-PD-(L)1 Therapy in NSCLC Patients.

PD-(L)1 inhibitors are part of the treatment strategy for non-small cell lung cancer (NSCLC) although its efficacy is limited to certain patients. Our study aimed to identify patients who might benefit from anti-PD-(L)1 inhibitors by analyzing the PD-L1 expression on circulating leukocytes and its evolution during treatment. One hundred thirteen NSCLC patients, according to their radiological response after 10-12 weeks of treatment, were classified into responders, stable, and progressive disease. Percentages of circulating PD-L1+ leukocytes, PD-L1+ platelets (PLTs), and leukocyte-PLT complexes were assessed using flow cytometry, and plasma concentrations of soluble immunomodulatory factors were quantified by ELISA. Responders exhibited significantly higher pre-treatment percentages of PD-L1+ neutrophils, PD-L1+ CD14+ cells, and PD-L1+ PLTs than progressors. The percentages of these populations decreased in responders post-treatment, contrasting with stables and progressors. PLTs notably contributed to PD-L1 expression in CD14+ cells and neutrophils. Plasma cytokine analysis revealed baseline differences only in IL-17 concentration among groups, whereas network analyses highlighted distinct association patterns between plasma molecules and PD-L1+ leukocytes after 10-12 weeks of treatment. Our findings suggest that pre-treatment assessment of circulating PD-L1+ neutrophils, PD-L1+ CD14+ cells, and PD-L1+ PLTs may be helpful in identifying NSCLC patients who are potential candidates for anti-PD-(L)1 therapy.

Binge drinking at time of bariatric surgery is associated with liver disease, suicides, and increases long-term mortality.

BACKGROUND AND AIMS: Alcohol use disorder has been reported in patients undergoing bariatric procedures, but the pattern of alcohol consumption has not been evaluated. We investigated the prevalence, risk factors, and impact of binge drinking (BD) at the time of surgery and during follow-up. METHODS: A prospective, longitudinal study of subjects undergoing bariatric surgery was included in the LABS-2 registry between 2006 and 2009. Participants with AUDIT questionnaire at the time of surgery and a minimum of 12 months follow-up were included. BD was defined as consuming ≥5 drinks on at least 1 occasion in the previous month. Liver biopsies were obtained during bariatric procedures in not all cases. Survival analysis was performed with the adjusted Cox regression model and competing risk. RESULTS: A total of 2257 subjects were included, with a median follow-up of 79 months. The prevalence of BD at time of surgery was 12%, and it raised up to 23% during follow-up. Patients with BD predominantly had a binge eating disorder (OR=1.35 [95% CI: 1.04-1.76]), regularly consumed fast food [OR=1.4 (95% CI: 1.07-1.85)] and used other drugs (OR=2.65 [95% CI: 1.74-4.04]). Within liver biopsies evaluation, BD showed higher hepatic iron deposits (OR=3.00 [95% CI: 1.25-7.21]). BD at the time of surgery was associated with a higher risk of BD during follow-up (OR=10.49 [95% CI: 7.86-14.00]) and long-term mortality (HR: 3.21 [95% CI: 1.67-6.18]). Specific causes of death in these patients with BD were liver disease (p=0.020), suicide (p=0.015), neoplasms (p=0.034), and respiratory (p=0.025). CONCLUSIONS: The prevalence of BD in patients undergoing bariatric surgery is high and increases the risk of postoperative liver disease, suicides, and long-term mortality.

Influence of Malignant Pleural Fluid from Lung Adenocarcinoma Patients on Neutrophil Response.

Malignant pleural effusion (MPE) is a common severe complication of advanced lung adenocarcinoma (LAC). Neutrophils, an essential component of tumor infiltrates, contribute to tumor progression and their counts in MPE have been associated with worse outcome in LAC. This study aimed to evaluate phenotypical and functional changes of neutrophils induced by MPE to determine the influence of MPE immunomodulatory factors in neutrophil response and to find a possible association between neutrophil functions and clinical outcomes. Pleural fluid samples were collected from 47 LAC and 25 heart failure (HF) patients. We measured neutrophil degranulation products by ELISA, oxidative burst capacity and apoptosis by flow cytometry, and NETosis by fluorescence. The concentration of degranulation products was higher in MPE-LAC than in PE-HF. Functionally, neutrophils cultured with MPE-LAC had enhanced survival and neutrophil extracellular trap (NET) formation but had reduced oxidative burst capacity. In MPE, NETosis was positively associated with MMP-9, P-selectin, and sPD-L1 and clinically related to a worse outcome. This is the first study associating NETs with a worse outcome in MPE. Neutrophils likely contribute to tumor progression through the release of NETs, suggesting that they are a potential therapeutic target in LAC.

Identification of Two Eosinophil Subsets in Induced Sputum from Patients with Allergic Asthma According to CD15 and CD66b Expression.

Two subsets of eosinophils have been described: resident eosinophils with homeostatic functions (rEOS) in healthy subjects and in patients with nonallergic eosinophilic asthma, and inflammatory eosinophils (iEOS) in blood and lung samples from patients with allergic asthma. We explored if it would be possible to identify different subsets of eosinophils using flow cytometry and the gating strategy applied to induced sputum. We conducted an observational cross-sectional single-center study of 62 patients with persistent allergic asthma. Inflammatory cells from induced sputum samples were counted by light microscopy and flow cytometry, and cytokine levels in the supernatant were determined. Two subsets of eosinophils were defined that we call E1 (CD66b-high and CD15-high) and E2 (CD66b-low and CD15-low). Of the 62 patients, 24 were eosinophilic, 18 mixed, 10 paucigranulocytic, and 10 neutrophilic. E1 predominated over E2 in the eosinophilic and mixed patients (20.86% vs. 6.27% and 14.42% vs. 4.31%, respectively), while E1 and E2 were similar for neutrophilic and paucigranulocytic patients. E1 correlated with IL-5, fractional exhaled nitric oxide, and blood eosinophils. While eosinophil subsets have been identified for asthma in blood, we have shown that they can also be identified in induced sputum.

Effector Mechanisms of CD8+ HLA-DR+ T Cells in Breast Cancer Patients Who Respond to Neoadjuvant Chemotherapy.

Cytotoxic T lymphocyte (CTLs) activation is an independent predictor of response to neoadjuvant chemotherapy (NACT) in breast cancer (BC) patients. Here, we go deeper into the function of CD8+ HLA-DR+ T cells from NACT treated HER2 negative BC patients. Flow cytometry analysis revealed that CD8+ HLA-DR+ T cell percentage was increased in NACT responder (R) compared to non-responder (NR) patients. R patients with ER-/PR- hormone receptors had the highest CD8+ HLA-DR+ T cell frequencies, while no differences were found when patients were classified according to cancer stage or menopause status. Interestingly, the cytotoxicity and production of anti-tumor cytokines were enhanced when CD8+ HLA-DR+ T cells from healthy donors were cultured with plasma from R, but not from NR patients. The induced anti-tumor profile of CD8+ HLA-DR+ T cells was associated with plasmatic IL-12 and IFN-γ levels, increased cytokines in R patients. IL-12 or IFN-γ neutralization decreased cytotoxic activity and TNF-α production by cultured CD8+ HLA-DR+ T cells in R plasma presence. All these data suggest that an effective response to NACT in BC patients is associated with increased IL-12 or IFN-γ levels involved in the induction of cytotoxic and pro-inflammatory mechanisms in CD8+ HLA-DR+ T cells.