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Background: Vitamin K antagonists (VKA) are the most widely used anticoagulants for the prevention of thrombotic events. Several renal adverse effects have been associated with the use of VKA. The main aim of our study was to explore the association between international normalized ratio (INR) levels and microscopic hematuria in patients with VKA., Methods: We performed a cross-sectional study of patients treated with VKA that attended the outpatient clinic for routine INR control. A simple urinalysis was performed on the day of the INR control and the precise number of red cells in the urine sediment was quantified. Demographic data, kidney function tests, comorbidities, anticoagulant dose and concomitant treatment were registered., Results: A total of 337 patients were included with median INR levels of 2.6 (IQR 2.1-3.3). 11.9% of the patients presented microscopic hematuria (≥14 RBCs/µl). There was a significant correlation between INR levels and the number of red blood cells in the urine sediment (r = 0.201, p = 0.024). In the univariate analysis, microscopic hematuria was associated with having an INR >3.5 (19% vs. 10.2%, p = 0.046), bacteriuria (15.2% vs. 3.6%, p = 0.015), leukocyturia (14.8% vs. 6.6%, p =  0.026), hypertension (16.2% vs. 9.5%, p = 0.053), and the use of renin-angiotensin system (RAS) blockers (6.9% vs. 17.2%, p = 0.004). Multivariate logistic regression showed an association between microscopic hematuria and RAS blockade (OR 0.38, CI 95% 0.163-0.886, p = 0.025), independent from INR levels, hypertension, leukocyturia or bacteriuria., Conclusions: INR overdose was significantly associated with the presence of microscopic hematuria. RAS blockade is an independent protective factor for the presence of microscopic hematuria in anticoagulated patients., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest to disclose., (Copyright © 2022 Southern Society for Clinical Investigation. Published by Elsevier Inc. All rights reserved.)

Background: Cutaneous leishmaniasis (CL) skin lesions are the result of a deregulated immune response, which is unable to eliminate Leishmania parasites. The control of both, parasites and host immune response, is critical to prevent tissue destruction. The skin ulceration has been correlated with high TNF-α level., Objective: Because human anti-TNF-α antibodies (Ab) have been successfully assayed in several mice inflammatory diseases, we hypothesized that their anti-inflammatory effect could optimize the healing of CL lesions achieved after topical application of paromomycin (PM), the current chemotherapy against CL., Methods and Results: We first compared the in vitro efficacy of PM and Ab alone and the drug given in combination with Ab to assess if the Ab could interfere with PM leishmanicidal activity in L. major-infected bone marrow-derived macrophages. The combination therapy had similar antileishmanial activity to the drug alone and showed no influence on NO production, which allows macrophage-mediated parasite killing. Next, we demonstrated in an in vivo model of Imiquimod®-induced inflammation that topical Ab and PM inhibit the infiltration of inflammatory cells in the skin. In the efficacy studies in L. major-infected BALB/c mice, PM combined with Ab led to a sharp infection reduction and showed a stronger anti-inflammatory activity than PM alone. This was confirmed by the down-regulation of TNF-α, IL-1β, iNOS, IL-17, and CCL3 as well as by a decrease of the neutrophilic infiltrate during infection upon treatment with the Ab., Conclusions: In terms of parasite elimination and inflammation reduction, topical application of Ab in combination with PM was more effective than the drug alone., (Copyright © 2018 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

Tubulocystic renal cell carcinoma (TC-RCC) is a rare recently described renal neoplasm characterized by gross, microscopic, and immunohistochemical differences from other renal tumor types and was recently classified as a distinct entity. However, this distinction remains controversial particularly because some genetic studies suggest a close relationship with papillary RCC (PRCC). The molecular basis of this disease remains largely unexplored. We therefore performed noncoding (nc) RNA/miRNA expression analysis and targeted next-generation sequencing mutational profiling on 13 TC-RCC cases (11 pure, two mixed TC-RCC/PRCC) and compared with other renal neoplasms. The expression profile of miRNAs and other ncRNAs in TC-RCC was distinct and validated 10 differentially expressed miRNAs by quantitative RT-PCR, including miR-155 and miR-34a, that were significantly down-regulated compared with PRCC cases (n = 22). With the use of targeted next-generation sequencing we identified mutations in 14 different genes, most frequently (>60% of TC-RCC cases) in ABL1 and PDFGRA genes. These mutations were present in <5% of clear cell RCC, PRCC, or chromophobe RCC cases (n > 600) of The Cancer Genome Atlas database. In summary, this study is by far the largest molecular study of TC-RCC cases and the first to investigate either ncRNA expression or their genomic profile. These results add molecular evidence that TC-RCC is indeed a distinct entity from PRCC and other renal neoplasms., (Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Patients affected by cutaneous leishmaniasis need a topical treatment which cures lesions without leaving scars. Lesions are produced not only by the parasite but also by an uncontrolled and persistent inflammatory immune response. In this study, we proposed the loading of β-lapachone (β-LP) in lecithin-chitosan nanoparticles (NP) for targeting the drug to the dermis, where infected macrophages reside, and promote wound healing. Although the loading of β-LP in NP did not influence the drug antileishmanial activity it was critical to achieve important drug accumulation in the dermis and permeation through the skin. When topically applied in Leishmania major infected BALB/c mice, β-LP NP achieved no parasite reduction but they stopped the lesion progression. Immuno-histopathological assays in CL lesions and quantitative mRNA studies in draining lymph nodes confirmed that β-LP exhibited anti-inflammatory activity leading to the down-regulation of IL-1β and COX-2 expression and a decrease of neutrophils infiltrate., From the Clinical Editor: Cutaneous leishmaniasis often leaves patients with unsightly scars due to the body's inflammatory response to the infection. The authors in this paper described topical treatment using β-lapachone (β- LP) loaded in lecithin-chitosan nanoparticles (NP) in an animal model. Results confirmed the reduction of inflammatory response without affecting the parasite killing efficacy. These findings would pave way for further clinical testing in the near future., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background & Aims: Oncostatin M (OSM) is an inflammatory cytokine which interacts with a heterodimeric receptor formed by gp130 and either OSMRβ or LIFR. Here we have analysed OSM and its receptors in livers with chronic hepatitis C (CHC) and studied the factors that regulate this system., Methods: OSM, OSM receptors and OSM-target molecules were studied by immunohistochemistry and/or qPCR analysis in livers from CHC patients and controls. We determined the production of OSM by CD40L-stimulated antigen presenting cells (APC) and its biological effects on HuH7 cells containing HCV replicon (HuH7 Core-3')., Results: OSM was upregulated in livers with CHC and its production was mapped to CD11c+ cells. OSM levels correlated directly with inflammatory activity and CD40L expression. In vitro studies showed that OSM is released by APC upon interaction with activated CD4+ T cells in a CD40L-dependent manner. Culture of HuH7 Core-3' cells with supernatant from CD40L-stimulated APC repressed HCV replication and induced IL-7 and IL-15Rα. These effects were dampened by antibodies blocking OSM or gp130 and by silencing OSMRβ. In CHC livers OSMRβ and LIFR were significantly downregulated and their values correlated with those of OSM-induced molecules. Experiments in HuH7 cells showed that impaired STAT3 signaling and exposure to TGFβ1, two findings in CHC, are factors involved in repressing OSMRβ and LIFR, respectively., Conclusions: OSM is a cytokine possessing vigorous antiviral and immunostimulatory properties which is released by APC upon interaction with CD40L present on activated CD4+ T cells. In livers with CHC, OSM is overexpressed but its biological activity appears to be hampered because of downregulation of its receptor subunits., (Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background & Aims: Tremelimumab is a monoclonal antibody that blocks cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), an inhibitory co-receptor that interferes with T cell activation and proliferation. The purpose of this pilot clinical trial was to test the antitumor and antiviral effect of tremelimumab in patients with hepatocellular carcinoma (HCC) and chronic hepatitis C virus (HCV) infection; and to study the safety of its administration to cirrhotic patients., Methods: Tremelimumab at a dose of 15 mg/kg IV every 90 days was administered until tumor progression or severe toxicity. Twenty patients were assessable for toxicity and viral response and 17 were assessable for tumor response. Most patients were in the advanced stage and 43% had an altered liver function (Child-Pugh class B)., Results: A good safety profile was recorded and no patient needed steroids because of severe immune-mediated adverse events. Some patients had a transient albeit intense elevation of transaminases after the first dose, but not following subsequent cycles. Partial response rate was 17.6% and disease control rate was 76.4%. Time to progression was 6.48 months (95% CI 3.95-9.14). A significant drop in viral load was observed while new emerging variants of the hypervariable region 1 of HCV replaced the predominant variants present before therapy, particularly in those patients with a more prominent drop in viral load. This antiviral effect was associated with an enhanced specific anti-HCV immune response., Conclusions: Tremelimumab safety profile and antitumor and antiviral activity, in patients with advanced HCC developed on HCV-induced liver cirrhosis, support further investigation., (Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background & Aims: The mechanisms by which Foxp3+ T regulatory cells (Treg) accumulate in HCV infected livers are not known. Here, we studied the role of chemokines CCL17 and CCL22 in this process., Methods: Chemokine mRNA levels were determined by qPCR in liver biopsies from 26 HCV chronically infected patients (CHC), 11 patients with treatment-induced sustained virological response (SVR), 16 patients with other liver diseases unrelated to HCV, and 24 normal livers. Double-immunofluorescence Foxp3/CD3 or CD11c/CCL22 was performed in liver sections. Chemokine production by monocyte-derived dendritic cells (MDDC) co-cultured with uninfected or HCV-JFH1 infected Huh7 cells was measured by qPCR and ELISA. Chemotactic activity of culture supernatants was also tested., Results: Foxp3+ Treg were increased in CHC livers as compared to controls. Patients with CHC showed elevated intrahepatic levels of CCL17 mRNA compared to normal livers or livers from subjects with SVR or other forms of liver disease. Intrahepatic CCL22 expression was also higher in CHC than in healthy subjects or SVR patients but similar to that observed in other liver diseases. Dendritic cells producing CCL22 could be found inside the hepatic lobule in CHC patients. Contact between MDDC and HCV-JFH1-infected Huh7 cells induced the expression of CCL17 and CCL22 in a process partially dependent on ICAM-1. Transwell experiments showed that upregulation of these chemokines enhanced Treg migration., Conclusions: Contact of HCV-infected cells with dendritic cells induces the production of Treg-attracting chemokines, an effect which may favour liver accumulation of Treg in CHC. Our findings contribute to explain the mechanism by which HCV escapes the immune response and thus reveals novel therapeutic targets., (Copyright © 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background/aims: Hepatocellular carcinoma (HCC) often lacks curative treatment; therefore new efficient therapies are needed. In this work we aimed at evaluating the antitumor effect of interleukin-12 (IL-12)-based gene therapy on HCC occurring spontaneously in mice., Methods: A plasmid-vector expressing IL-12 in a liver-specific and doxycycline (Dox)-inducible manner was transferred by hydrodynamic injection to the liver of L-PK/c-myc mice with HCC. IL-12 expression was induced by administering Dox (3 cycles of 1 month duration separated by 1 month rest)., Results: Dox administration increased serum IL-12 and IFN-gamma and induced tumor lymphocytic infiltration in all treated mice which was accompanied by tumor stabilization or regression in 40% of animals. The antitumor effect did not correlate with levels of IL-12 or IFN-gamma nor with the intensity of tumor mononuclear infiltration. However, tumors from non-responder mice showed more abundance of Foxp3+ regulatory T cells and higher expression of the immunosuppressive molecules PD-1, PD-L1, VEGF, CTLA-4, IDO, and IL-10 than those that responded to therapy., Conclusions: Although long-term induction of IL-12 expression in the liver can inhibit HCC growth, the efficacy of the treatment appears to be limited by the activation of immunosuppressive mechanisms.

Hepatitis C virus (HCV) is remarkably efficient at establishing persistent infection. The current treatment with IFN-alpha given alone or in combination with ribavirin is ineffective in eliminating the virus in a large proportion of individuals with chronic hepatitis C. Recent data suggest that HCV blocks IFN-alpha signalling, an effect that facilitates viral persistence. We have used the HCV genomic and subgenomic replicon system to analyze the effect of structural and non-structural viral proteins on the activation of the Jak/STAT pathway and induction of antiviral activity by IFN-alpha. Our results show that IFN-alpha-mediated STAT activation (but not IFN-gamma-stimulated STAT phosphorylation) is blocked in Huh7 cell line containing the genomic replicon, while this is not observed in cells with the subgenomic replicon. In agreement with these findings, the transcriptional activity and the antiviral effect of IFN-alpha were significantly lower in cells harboring the genomic replicon than in cells with the subgenomic replicon. These results indicate that HCV structural proteins play an important role in the escape of HCV from the interferon system.

Background/aims: Oxidative stress could play a role in the pathogenesis of hepatitis C virus infection. We investigated the oxidant/antioxidant status in peripheral blood mononuclear cells from patients with chronic hepatitis C and controls., Methods/results: Lipid peroxidation products and superoxide dismutase activity in peripheral blood mononuclear cells were higher in chronic hepatitis C patients than in healthy subjects while glutathione S-transferase activity was reduced in patients as compared to controls. Catalase, glutathione peroxidase and glutathione reductase were similar in chronic hepatitis C and normal individuals. No statistically significant differences were found between patients and controls with regard to glutathione levels in peripheral blood mononuclear cells, but 35% of patients with chronic hepatitis C showed values of glutathione and oxidized glutathione which were below and above, respectively, the limits of normal controls. Finally, the glutathione synthetic capacity of the cytosol of peripheral blood mononuclear cells was significantly higher in patients than in controls, indicating increased glutathione turnover in lymphocytes from patients with chronic hepatitis C., Conclusions: Oxidative stress is observed in peripheral blood mononuclear cells from chronic hepatitis C patients. This process might alter lymphocyte function and facilitate the chronicity of the infection.

alpha-Interferon therapy normalizes aminotransferase levels in approximately 50% of the patients with chronic hepatitis C, but post-therapy relapses are common and predictive factors of sustained response remain largely unknown. We retrospectively assessed several parameters as predictors of sustained remission after a 12-month course of lymphoblastoid alpha-interferon: the Knodell histological activity index, serum levels of procollagen type III peptide, serum HCV-RNA, anti-alpha-interferon antibodies, and anti-HCV antibodies (C-100-3), all at month 12. Thirty-seven patients were studied. Fourteen patients were non-responders (38%), 15 patients experienced a sustained response (40.5%) and eight patients responded similarly but relapsed after alpha-interferon withdrawal (21.5%). A decrease in the histological activity index above 5, normalization of procollagen type III peptide levels (< 12 ng/ml) and the absence of viremia after treatment were all significantly associated with a sustained response (p = 0.008, p = 0.007 and p = 0.037, respectively). Anti-interferon antibodies were detected in only one non-responder patient. Anti-C-100-3 antibodies became undetectable at month 12 in 5 of the 15 sustained responders. The best prediction of sustained response was obtained from the three variables independent of multivariate analysis according to the following equation: F = 0.872 + 0.067 x K (decrease of histological index) -0.052 x P (procollagen type III peptide levels at month 12) -0.28 x R (HCV-RNA at month 12; R = 2 when present and R = 1 when absent). A score higher than 0 predicted sustained remission with a 100% sensitivity and specificity in this series of patients.(ABSTRACT TRUNCATED AT 250 WORDS)

The insertion of two lysine residues (cleavage sites of cathepsin B) at the boundary of a peptide recognized by B cells (BD) and a class-II- presentable sequence (TDh) enhanced the anti-BD antibody induction capacity of this type of peptide construct, as well as production of IL2. It is postulated that these lysines generate a neoprocessable site which helps in release of the TDh moiety from the construct, enabling its presentation to class II molecules, an essential step in clonal expansion of the antibody-producing B cell after internalization of the construct via the BD moiety.

Exposure of human peripheral blood mononuclear cells (PBMC) to the stilbene derivative DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) (60 microM and above) significantly increased the release of tumor necrosis factor-alpha (TNF-alpha), as determined by TNF-alpha activity in the incubation media. When the TNF-alpha message was analyzed in PBMC by a reverse transcription/polymerase chain reaction (RT/PCR)-based procedure, it was found that incubation with DIDS (60 microM) was followed by a time-dependent accumulation of TNF-alpha mRNA. Measurements of intracellular pH showed that the presence of increasing concentrations of DIDS resulted in a progressive intracellular alkalinization of PBMC. It is suggested that the known DIDS effect of inhibiting transmembrane anion exchange, i.e., chloride/bicarbonate exchange, might play a role in the stimulation of TNF-alpha production by PBMC exposed to DIDS.