FixNCut: single-cell genomics through reversible tissue fixation and dissociation.

The use of single-cell technologies for clinical applications requires disconnecting sampling from downstream processing steps. Early sample preservation can further increase robustness and reproducibility by avoiding artifacts introduced during specimen handling. We present FixNCut, a methodology for the reversible fixation of tissue followed by dissociation that overcomes current limitations. We applied FixNCut to human and mouse tissues to demonstrate the preservation of RNA integrity, sequencing library complexity, and cellular composition, while diminishing stress-related artifacts. Besides single-cell RNA sequencing, FixNCut is compatible with multiple single-cell and spatial technologies, making it a versatile tool for robust and flexible study designs.

An atlas of cells in the human tonsil.

Palatine tonsils are secondary lymphoid organs (SLOs) representing the first line of immunological defense against inhaled or ingested pathogens. We generated an atlas of the human tonsil composed of >556,000 cells profiled across five different data modalities, including single-cell transcriptome, epigenome, proteome, and immune repertoire sequencing, as well as spatial transcriptomics. This census identified 121 cell types and states, defined developmental trajectories, and enabled an understanding of the functional units of the tonsil. Exemplarily, we stratified myeloid slan-like subtypes, established a BCL6 enhancer as locally active in follicle-associated T and B cells, and identified SIX5 as putative transcriptional regulator of plasma cell maturation. Analyses of a validation cohort confirmed the presence, annotation, and markers of tonsillar cell types and provided evidence of age-related compositional shifts. We demonstrate the value of this resource by annotating cells from B cell-derived mantle cell lymphomas, linking transcriptional heterogeneity to normal B cell differentiation states of the human tonsil.

Tumor heterogeneity and tumor-microglia interactions in primary and recurrent IDH1-mutant gliomas.

The isocitrate dehydrogenase (IDH) gene is recurrently mutated in adult diffuse gliomas. IDH-mutant gliomas are categorized into oligodendrogliomas and astrocytomas, each with unique pathological features. Here, we use single-nucleus RNA and ATAC sequencing to compare the molecular heterogeneity of these glioma subtypes. In addition to astrocyte-like, oligodendrocyte progenitor-like, and cycling tumor subpopulations, a tumor population enriched for ribosomal genes and translation elongation factors is primarily present in oligodendrogliomas. Longitudinal analysis of astrocytomas indicates that the proportion of tumor subpopulations remains stable in recurrent tumors. Analysis of tumor-associated microglia/macrophages (TAMs) reveals significant differences between oligodendrogliomas, with astrocytomas harboring inflammatory TAMs expressing phosphorylated STAT1, as confirmed by immunohistochemistry. Furthermore, inferred receptor-ligand interactions between tumor subpopulations and TAMs may contribute to TAM state diversity. Overall, our study sheds light on distinct tumor populations, TAM heterogeneity, TAM-tumor interactions in IDH-mutant glioma subtypes, and the relative stability of tumor subpopulations in recurrent astrocytomas.

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.

Phase angle by electrical bioimpedance is a predictive factor of hospitalisation, falls and mortality in patients with cirrhosis.

The phase angle is a versatile measurement to assess body composition, frailty and prognosis in patients with chronic diseases. In cirrhosis, patients often present alterations in body composition that are related to adverse outcomes. The phase angle could be useful to evaluate prognosis in these patients, but data are scarce. The aim was to analyse the prognostic value of the phase angle to predict clinically relevant events such as hospitalisation, falls, and mortality in patients with cirrhosis. Outpatients with cirrhosis were consecutively included and the phase angle was determined by electrical bioimpedance. Patients were prospectively followed to determine the incidence of hospitalisations, falls, and mortality. One hundred patients were included. Patients with phase angle ≤ 4.6° (n = 31) showed a higher probability of hospitalisation (35% vs 11%, p = 0.003), falls (41% vs 11%, p = 0.001) and mortality (26% vs 3%, p = 0.001) at 2-year follow-up than patients with PA > 4.6° (n = 69). In the multivariable analysis, the phase angle and MELD-Na were independent predictive factors of hospitalisation and mortality. Phase angle was the only predictive factor for falls. In conclusion, the phase angle showed to be a predictive marker for hospitalisation, falls, and mortality in outpatients with cirrhosis.

Reliability of iCare ic100 Rebound Tonometry and Agreement With Goldmann Applanation Tonometry in Healthy and Post-myopic LASIK Patients.

PRECIS: Rebound tonometry offers excellent reliability for obtaining intraocular pressure (IOP) measurements in healthy and post-myopic laser-assisted in situ keratomileusis (LASIK) patients regardless of corneal parameters and axial length and regardless of the instillation of ocular topical anesthesia. PURPOSE: The purpose of this study was to assess the reliability of rebound tonometry, its agreement with Goldmann applanation tonometry (GAT) and its concordance after topical anesthesia in a group of nonoperated healthy patients and a group of post-myopic LASIK patients. PATIENTS AND METHODS: Fifty-four patients were included in this prospective observational, case-control study. Patients were divided into 2 groups: control (n=32, 26.7±6.1 y) and LASIK (n=22, 35.8±7.6 y). Measurements of IOP were carried out using the iCare ic100 tonometer and GAT. The repeatability, intersession and interobserver reproducibility, GAT interchangeability and concordance after topical anesthesia of the iCare ic100 tonometer were assessed. RESULTS: Clinical reliability of the iCare ic100 was excellent in terms of repeatability [intraclass correlation coefficient (ICC)=0.909 and 0.951 in control and ICC=0.938 and 0.961 in LASIK] as well as interobserver reproducibility (ICC=0.896 in control and 0.916 in LASIK) and concordance after anesthesia (ICC=0.910 in control and 0.972 in LASIK) in both groups. Intersession reproducibility was clinically good in nonoperated patients (ICC=0.758 and 0.849) and excellent in LASIK patients (ICC=0.878 and 0.904), although statistically significant differences were obtained in the control group for both examiners (P=0.003 for examiner 1 and P=0.04 for examiner 2). Agreement with GAT was good in both groups (ICC=0.805 in control and 0.790 in LASIK), although statistically different (P<0.001 and P=0.02, respectively). Finally, the iCare ic100 reliability was independent of corneal parameters and axial length in both groups (P>0.05 for all parameters). CONCLUSIONS: Rebound tonometry offers good or excellent clinical reliability in both healthy and post-myopic LASIK patients. This article highlights the efficacy of iCare ic100 rebound tonometry as a reliable tool for the measurement of IOP and the management of glaucoma in healthy patients and patients undergoing myopic LASIK surgery.

Immunological and genetic kinetics from diagnosis to clinical progression in chronic lymphocytic leukemia.

BACKGROUND: Mechanisms driving the progression of chronic lymphocytic leukemia (CLL) from its early stages are not fully understood. The acquisition of molecular changes at the time of progression has been observed in a small fraction of patients, suggesting that CLL progression is not mainly driven by dynamic clonal evolution. In order to shed light on mechanisms that lead to CLL progression, we investigated longitudinal changes in both the genetic and immunological scenarios. METHODS: We performed genetic and immunological longitudinal analysis using paired primary samples from untreated CLL patients that underwent clinical progression (sampling at diagnosis and progression) and from patients with stable disease (sampling at diagnosis and at long-term asymptomatic follow-up). RESULTS: Molecular analysis showed limited and non-recurrent molecular changes at progression, indicating that clonal evolution is not the main driver of clinical progression. Our analysis of the immune kinetics found an increasingly dysfunctional CD8+ T cell compartment in progressing patients that was not observed in those patients that remained asymptomatic. Specifically, terminally exhausted effector CD8+ T cells (T-betdim/-EomeshiPD1hi) accumulated, while the the co-expression of inhibitory receptors (PD1, CD244 and CD160) increased, along with an altered gene expression profile in T cells only in those patients that progressed. In addition, malignant cells from patients at clinical progression showed enhanced capacity to induce exhaustion-related markers in CD8+ T cells ex vivo mainly through a mechanism dependent on soluble factors including IL-10. CONCLUSIONS: Altogether, we demonstrate that the interaction with the immune microenvironment plays a key role in clinical progression in CLL, thereby providing a rationale for the use of early immunotherapeutic intervention.

Clinical impact of confinement due to the COVID-19 pandemic on patients with fibromyalgia: a cohort study.

OBJECTIVES: To our knowledge, the impact of the COVID-19 pandemic on fibromyalgia (FM) patients has not been studied before. FM patients often experience clinical impairment with stress. The aim of this study was to determine whether severity of FM increases because of confinement by the COVID-19 pandemic. METHODS: This prospective study includes patients from the Combined Index of Severity of Fibromyalgia (ICAF) cohort who met the 2010 ACR FM criteria. In this cohort, all patients have a periodical evaluation of their quality of life through two questionnaires, the ICAF, which assesses the ability to perform daily living activities, anxiety and depression, and through the Patient Global Impression of Change (PGIC), which assesses overall change after a therapeutical intervention. Pre- and post-confinement measurements were analysed. Inferential statistical analysis and ANOVA for repeated measurements were used. RESULTS: A total of 93 patients received a phone consultation, (95.5% females), mean (SD) age of 48.23 (8.38) years. Four patients were excluded as presenting COVID-19 and 51 (57%) completed the post-confinement ICAF. Following confinement, 25 (49%) patients got worse (group-worse) and 26 (51%) patients experienced no change or improved (group-stable). Comparisons between pre- and post-confinement ICAF did not show significant differences in both groups. Passive coping was significantly different in group-worse in pre-confinement evaluation. In the 80% of patients with passive coping predominance there were no changes in coping strategy. CONCLUSIONS: No clinical impairment due to COVID-19 confinement occurred. The perceived worsening among FM patients relies primarily on how patients cope with their disease, without a real impact on clinical manifestations.

Bacteriophages immunomodulate the response of monocytes.

Bacteriophages are present in fluids from cirrhosis patients. However, their effect on the immune response is unknown. In this work, we explore the role of phages in the phenotype, function, and cytokine production of monocytes. We stimulated healthy monocytes with five different butanol-purified phage suspensions infective for Gram-negative and Gram-positive bacteria. We studied the expression of the monocyte markers involved in lipopolysaccharide recognition (LPS; CD14), antigen presentation (HLA-DR) and co-stimulation (CD86), and the concentration of induced cytokines (TNF-α, IFN-α, and IL-10) by phages. To confirm the direct role of phages without the interference of contaminating soluble LPS in phage suspensions, polymyxin B was added to the cell cultures. Phagocytosis experiments were assessed by flow cytometry using labeled phage suspensions. We observed that butanol-purified phages reduced the surface levels of CD14 and CD86 in monocytes and increased the secreted levels of TNF-α and IL-10 compared with the control sample containing only butanol buffer. All phage suspensions showed downregulation of HLA-DR expression but only Staphylococcus aureus phage contaminated with Escherichia coli reached statistical significance. The addition of polymyxin B did not restore the monocytic response induced by phages, suggesting that the effect was not caused by the presence of LPS. Monocytes were able to phagocyte phages in a dose- and time-dependent manner. To conclude, the phagocytosis of butanol-purified phages altered the phenotype and cytokine production of monocytes suggesting they become tolerogenic.

Adherence to subcutaneous biological therapies in patients with inflammatory rheumatic diseases and inflammatory bowel disease: a systematic review.

Aim: To assess adherence to subcutaneous biologicals in adults with inflammatory rheumatic diseases or inflammatory bowel disease and evaluate factors possibly associated with adherence. Materials & methods: Systematic searches were conducted of main databases from January 2000 to June 2019. Results: 41 articles (32 full papers and nine abstracts) were included in the review. Among studies which used a medication possession ratio threshold of ≥80% as the end point, adherence varied from 28.8 to 89.4%. Possible predictors of adherence were older age, professional or family member support, belief in medication necessity, lower concerns about medication and monthly versus weekly administration. Conclusion: Considerable variability in adherence rates across published studies reflects study heterogeneity and the absence of a 'gold standard' to measure adherence.

Cell free circulating tumor DNA in cerebrospinal fluid detects and monitors central nervous system involvement of B-cell lymphomas.

The levels of cell free circulating tumor DNA (ctDNA) in plasma correlated with treatment response and outcome in systemic lymphomas. Notably, in brain tumors, the levels of ctDNA in the cerebrospinal fluid (CSF) are higher than in plasma. Nevertheless, their role in central nervous system (CNS) lymphomas remains elusive. We evaluated the CSF and plasma from 19 patients: 6 restricted CNS lymphomas, 1 systemic and CNS lymphoma, and 12 systemic lymphomas. We performed whole exome sequencing or targeted sequencing to identify somatic mutations of the primary tumor, then variant-specific droplet digital PCR was designed for each mutation. At time of enrolment, we found ctDNA in the CSF of all patients with restricted CNS lymphoma but not in patients with systemic lymphoma without CNS involvement. Conversely, plasma ctDNA was detected in only 2/6 patients with restricted CNS lymphoma with lower variant allele frequencies than CSF ctDNA. Moreover, we detected CSF ctDNA in 1 patient with CNS lymphoma in complete remission and in 1 patient with systemic lymphoma, 3 and 8 months before CNS relapse was confirmed; indicating CSF ctDNA might detect CNS relapse earlier than conventional methods. Finally, in 2 cases with CNS lymphoma, CSF ctDNA was still detected after treatment even though a complete decrease in CSF tumor cells was observed by flow cytometry (FC), indicating CSF ctDNA better detected residual disease than FC. In conclusion, CSF ctDNA can better detect CNS lesions than plasma ctDNA and FC. In addition, CSF ctDNA predicted CNS relapse in CNS and systemic lymphomas.

Frailty in outpatients with cirrhosis: A prospective observational study.

BACKGROUND AND AIM: Frailty is increasingly recognized as a major prognostic factor in cirrhosis in addition to conventional liver insufficiency scores. The aim was to compare the prevalence and characteristics of frailty between patients with cirrhosis and controls, and to analyse its prognostic value. METHODS: We included outpatients with cirrhosis and age- and gender-matched non-cirrhotic controls. Frailty was defined according to the Fried frailty criteria. In patients with cirrhosis, we analysed the ability of the degree of frailty to predict a composite endpoint, consisting of hospitalization, admission to a long-term care centre, falls or death. RESULTS: We included 135 patients with cirrhosis and 135 controls. The prevalence of frailty was higher among patients with cirrhosis: 35 (25.9%) frail, 74 (54.8%) pre-frail and 26 (19.2%) robust vs 14 (10.4%) frail, 67 (49.6%) pre-frail and 54 (40%) robust (P < .001) in controls. This difference was mainly as a result of decreased muscle strength in patients with cirrhosis. During follow-up, frail patients with cirrhosis showed a higher probability of composite endpoint, hospitalization and falls than pre-frail and robust cirrhotic patients but mortality was similar. MELD-Na score and frailty were independent predictive factors for hospitalization, frailty for falls, and MELD-Na score and albumin for survival. Vitamin D deficiency and increased cystatin C were associated with frailty. CONCLUSIONS: Frailty was more frequent in outpatients with cirrhosis than in controls, mainly because of a decrease in muscle strength, and it could be a predictive factor for hospitalization and falls in these patients.

Experimental supporting data on the influence of platelet-derived factors of malignant pleural effusions on T cell effector functions and their relevance in predicting prognosis of lung adenocarcinoma patients with pleural metastasis.

The data described in this article are supplementary to our primary article "Platelet factor 4 regulates T cell effector functions in malignant pleural effusions". Malignant pleural effusion (MPE) is a common complication of advanced lung adenocarcinoma (LAC) associated with a poor life expectancy [1]. Several challenges need to be addressed to identify non-invasive molecular biomarkers that help to predict the prognosis of LAC patients with MPE [2]. In the primary publication, we proposed that platelet-derived factors, especially platelet factor 4 (PF4), can negatively regulate T lymphocyte activation and granzyme B expression in pleural metastasis and its levels were associated with a worse prognosis. Here, we provide data on the influence of other platelet-derived factors, including transforming growth factor ß (TGF-ß), vascular endothelial factor (VEGF), and P-selectin on T lymphocyte response in MPE and their relevance as prognostic factors in lung cancer patients with pleural metastasis. Pleural fluids from 35 lung adenocarcinoma (LAC) and 20 heart failure (HF) patients were collected by thoracentesis and its platelet-derived factors' content was measured by specific enzyme-linked immunosorbent assay (ELISAs). Correlations between pleural levels of platelet-derived factors and T cell functions were analyzed by Pearson coefficients. Kaplan-Meier curves were used to estimate the effect of pleural concentrations of platelet-derived factors on overall survival of LAC patients with pleural metastasis. These analyses showed that the concentration of platelet-derived factors was not associated with T cell proliferation and cytotoxicity. Furthermore, their levels do not predict the survival of LAC with MPE.

Platelet factor 4 regulates T cell effector functions in malignant pleural effusions.

Malignant pleural effusion (MPE) is defined as the presence of tumor cells in pleural fluid and it is a fatal complication of advanced lung adenocarcinoma (LAC). To understand the immune response to the tumor in MPE, we compared the concentration of immunomodulatory factors in MPE of LAC and pleural effusion of heart failure (HF) patients by ELISA, and the proliferation and cytotoxic phenotype of T cells stimulated in the presence of LAC and HF pleural fluids by cytometry. Platelet factor 4 (PF4), vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF-ß) and P-selectin levels were higher in LAC than in HF pleural fluids. However, plasmatic PF4 and P-selectin levels were similar in LAC and HF. VEGF positively correlated with TGF-ß and sPD-L1 in LAC but not in HF pleural fluids. LAC pleural fluids also inhibited T lymphocyte proliferation and cytotoxicity and reduced IL-17 production. PF4 levels inversely correlated with T cell function. The high content of PF4 in MPE was associated with poor prognosis. Our findings suggest that an impaired response of T lymphocytes induced by PF4 provides a significant advantage for tumor progression.

Repolarization of tumor infiltrating macrophages and increased survival in mouse primary CNS lymphomas after XPO1 and BTK inhibition.

BACKGROUND: Patients diagnosed with primary central nervous system lymphoma (PCNSL) often face dismal outcomes due to the limited availability of therapeutic options. PCNSL cells frequently have deregulated B-cell receptor (BCR) signaling, but clinical responses to its inhibition using ibrutinib have been brief. In this regard, blocking nuclear export by using selinexor, which covalently binds to XPO1, can also inhibit BCR signaling. Selinexor crosses the blood-brain barrier and was recently shown to have clinical activity in a patient with refractory diffuse large B-cell lymphoma in the CNS. We studied selinexor alone or in combination with ibrutinib in pre-clinical mouse models of PCNSL. METHODS: Orthotopic xenograft models were established by injecting lymphoma cells into the brain parenchyma of athymic mice. Tumor growth was monitored by bioluminescence. Malignant cells and macrophages were studied by immunohistochemistry and flow cytometry. RESULTS: Selinexor blocked tumor growth and prolonged survival in a bioluminescent mouse model, while its combination with ibrutinib further increased survival. CNS lymphoma in mice was infiltrated by tumor-promoting M2-like macrophages expressing PD-1 and SIRPα. Interestingly, treatment with selinexor and ibrutinib favored an anti-tumoral immune response by shifting polarization toward inflammatory M1-like and diminishing PD-1 and SIRPα expression in the remaining tumor-promoting M2-like macrophages. CONCLUSIONS: These data highlight the pathogenic role of the innate immune microenvironment in PCNSL and provide pre-clinical evidence for the development of selinexor and ibrutinib as a new promising therapeutic option with cytotoxic and immunomodulatory potential.

Posttransplant cyclophosphamide after allogeneic hematopoietic cell transplantation mitigates the immune activation induced by previous nivolumab therapy.

Patients receiving an allogeneic hematopoietic cell transplantation (allo-HCT) after the use of PD-1 inhibitors seem to be at a higher risk of developing acute graft-versus-host disease (aGHVD) through etiopathogenetic mechanisms not fully elucidated. Herein, we investigated the effect of nivolumab administered prior to allo-HCT on the following early T-cell reconstitution and its modulation by the GVHD prophylaxis (tacrolimus/sirolimus vs. posttransplant cyclophosphamide [PTCY]). In all nivolumab-exposed patients we detected circulating nivolumab in plasma for up to 56 days after allo-HCT. This residual nivolumab was able to bind and block PD-1 on T-cells at day 21 after allo-HCT, inducing a T cell activation that was differentially modulated depending on the GVHD prophylactic regimen. Among patients receiving tacrolimus/sirolimus, nivolumab-exposed patients had a higher incidence of severe aGVHD and a more effector T-cell profile compared with anti-PD-1-naïve patients. Conversely, patients receiving PTCY-based prophylaxis showed a similar risk of aGVHD and T-cell profile irrespective of the previous nivolumab exposure. In conclusion, nivolumab persists in plasma after transplantation, binds to allogeneic T cells and generates an increased T-cell activation. This T-cell activation status can be mitigated with the use of PTCY, thus reducing the risk of aGVHD.

Development of semiquantitative ultrasound scoring system to assess cartilage in rheumatoid arthritis.

OBJECTIVES: To develop and test the reliability of a new semiquantitative scoring system for the assessment of cartilage changes by ultrasound in a web-based exercise as well as a patient exercise of patients with RA. METHODS: A taskforce of the Outcome Measures in Rheumatology Ultrasound Working Group performed a systematic literature review on the US assessment of cartilage in RA, followed by a Delphi survey on cartilage changes and a new semiquantitative US scoring system, and finally a web-based exercise as well as a patient exercise. For the web-based exercise, taskforce members scored a dataset of anonymized static images of MCP joints in RA patients and healthy controls, which also contained duplicate images. Subsequently, 12 taskforce members used the same US to score cartilage in MCP and proximal interphalangeal joints of six patients with RA in in a patient reliability exercise. Percentage agreement and prevalence of lesions were calculated, as intrareader reliability was assessed by weighted kappa and interreader reliability by Light's kappa. RESULTS: The three-grade semiquantitative scoring system demonstrated excellent intrareader reliability (kappa: 0.87 and 0.83) in the web-based exercise and the patient exercise, respectively. Interreader reliability was good in the web-based exercise (kappa: 0.64) and moderate (kappa: 0.48) in the patient exercise. CONCLUSION: Our study demonstrates that ultrasound is a reliable tool for evaluating cartilage changes in the MCP joints of patients with RA and supports further development of a new reliable semiquantitative ultrasound scoring system for evaluating cartilage involvement in RA.

Migrated T lymphocytes into malignant pleural effusions: an indicator of good prognosis in lung adenocarcinoma patients.

The presence of leukocyte subpopulations in malignant pleural effusions (MPEs) can have a different impact on tumor cell proliferation and vascular leakiness, their analysis can help to understand the metastatic microenvironment. We analyzed the relationship between the leukocyte subpopulation counts per ml of pleural fluid and the tumor cell count, molecular phenotype of lung adenocarcinoma (LAC), time from cancer diagnosis and previous oncologic therapy. We also evaluated the leukocyte composition of MPEs as a biomarker of prognosis. We determined CD4+ T, CD8+ T and CD20+ B cells, monocytes and neutrophils per ml in pleural effusions of 22 LAC and 10 heart failure (HF) patients by flow cytometry. Tumor cells were identified by morphology and CD326 expression. IFNγ, IL-10 and IL-17, and chemokines were determined by ELISAs and migratory response to pleural fluids by transwell assays. MPEs from LAC patients had more CD8+ T lymphocytes and a tendency to more CD4+ T and CD20+ B lymphocytes than HF-related fluids. However, no correlation was found between lymphocytes and tumor cells. In those MPEs which were detected >1 month from LAC diagnosis, there was a negative correlation between pleural tumor cells and CD8+ T lymphocytes. CXCL10 was responsible for the attraction of CD20+ B, CD4+ T and CD8+ T lymphocytes in malignant fluids. Concentrations of IL-17 were higher in MPEs than in HF-related effusions. Survival after MPE diagnosis correlated positively with CD4+ T and CD8+ T lymphocytes, but negatively with neutrophils and IL-17 levels. In conclusion, lymphocyte enrichment in MPEs from LAC patients is mostly due to local migration and increases patient survival.

Ascitic fluid regulates the local innate immune response of patients with cirrhosis.

Ascitic neutrophils from cirrhotic patients with spontaneous bacterial peritonitis (SBP) exhibit an impaired oxidative burst that could facilitate bacterial infection. However, the influence of the cell-free ascitic fluid of these patients on neutrophil function has not been investigated. To analyze this influence, we determined the ascitic levels of cytokines, resistin, and lactoferrin and their association with neutrophil function, disease severity score, and SBP resolution. We analyzed NETosis induction by microscopy and oxidative burst by the flow cytometry of healthy neutrophils cultured in ascitic fluid from cirrhotic patients with sterile ascites (SA) and with SBP before and after antibiotic treatment. Resistin, IL-6, IL-1 receptor antagonist, IL-1ß, and lactoferrin levels were measured in ascitic fluids and supernatants of cultured neutrophils and PBMCs by ELISA. Upon stimulation, healthy neutrophils cultured in SBP ascitic fluid produced lower NETosis and oxidative burst than those cultured in SA. Ascitic resistin levels were negatively correlated with NETosis, oxidative burst, and ascitic glucose levels; and positively correlated with the model for end-stage liver disease score. After an E. coli or TNF-α stimulus, neutrophils were the major resistin producers. Resistin indirectly reduced the oxidative burst of neutrophils and directly reduced the inflammatory phenotype of monocytes and TNF-α production. Bacterial-induced resistin production can down-regulate the inflammatory response of macrophages and neutrophil function in ascitic fluid. Consequently, this down-regulation may jeopardize the elimination of bacteria that translocate to ascitic fluid in patients with cirrhosis.

Teeth infection may "shunt" through Fontan in high-altitude conditions.

The Fontan surgery involves the creation a conduit between the inferior vena cava and the right pulmonary artery. This conduit has a small fenestration that shunts the blood from right to left in case the pulmonary blood flow is limited; namely, if the pulmonary vascular resistance (PVR) is increased then the shunt is increased. Bacteria may bypass the pulmonary circulation and easily get access to the systemic circulation (bacteremia). We report the case of a patient that underwent Fontan surgery in 2010 and remained in a high-altitude city for 7 years, during this time he was asymptomatic until 2017 when he developed a brain abscess due to Streptococcus gordonii, a pathogen of dental plaque. Since high-altitude may raise PVR in response to reduction in the partial pressure of oxygen, we conclude that the long-term outcome of increased altitude on Fontan hemodynamics can lead to the shunt of teeth flora and consequently leading to severe infections.