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N 6 -Methyladenosine (m 6 A) is a prevalent and highly regulated RNA modification essential for RNA metabolism and normal brain function. It is particularly important in the hippocampus, where m 6 A is implicated in neurogenesis and learning. Although extensively studied, its presence in specific cell types remains poorly understood. We investigated m 6 A in the hippocampus at a single-cell resolution, revealing a comprehensive landscape of m 6 A modifications within individual cells. Through our analysis, we uncovered transcripts exhibiting a dense m 6 A profile, notably linked to neurological disorders such as Alzheimer's disease. Our findings suggest a pivotal role of m 6 A-containing transcripts, particularly in the context of CAMK2A neurons. Overall, this work provides new insights into the molecular mechanisms underlying hippocampal physiology and lays the foundation for future studies investigating the dynamic nature of m 6 A RNA methylation in the healthy and diseased brain., (© 2024 Feng et al.; Published by Cold Spring Harbor Laboratory Press.)

Background: Rehabilitation of the anterior area when the mesio-distal space is reduced is a challenge for the clinician, due to the patient's anatomical limitations and aesthetic requirements. Narrow Diameter Implants (NDI) are an option of treatment when the standard diameter implant is not possible, but the evidence is scarce. This prospective clinical study aims to analyze the formation of the tooth-implant papilla between the implant and the adjacent natural tooth in the maxillary lateral incisors and mandibular incisors., Methods: A total of 40 patients treated with NDI, of titanium-zirconium (Ti-Zr) alloy i.e., 2.9 mm Test Group (TG) and 3.3 mm Control Group (CG), were included. The mesiodistal distance between the adjacent natural teeth was used for implant selection, maintaining 1.5 mm between the fixation and the adjacent tooth. Clinical assessment was performed by a clinical examiner at 6 and 12 months after the final prosthesis. The primary variable was the Jemt Papillary Index. Also, implant survival rate (SR), complications, Implant Stability Quotient (ISQ), and patient-reported outcomes measures (PROMs) such as aesthetics, chewing, phonation, comfort, and self-esteem were analyzed., Results: A significant amount of papilla filling was observed concerning the baseline, with a trend towards more formation of the papilla in the TG, with a JPI score of 3. No significant differences were observed between the two groups regarding implant SR, clinical parameters, and complications. In terms of PROMs, a higher satisfaction in the TG was observed, with significant intergroup differences for aesthetics, comfort, self-esteem, and primary stability ISQ (TG: 59.05 (SD: 5.4) vs. CG: 51.55 (SD: 5.7))., Conclusions: The 2.9 mm diameter Ti-Zr implants achieved a formation of papilla similar to 3.3 mm implants in the anterior region at 12 months of follow-up after the final prosthetic restoration. The use of Ti-Zr implants with a diameter of 2.9 mm to rehabilitate single teeth in areas of the anterior region, where the mesiodistal distance is limited, showed favorable clinical results and a high degree of satisfaction during 1 year of observation similar to 3.3 mm dental implants., Trial Registration: This study was retrospectively registered in ClinicalTrials.gov with the number NCT05642520, dated 18/11/2022., (© 2024. The Author(s).)

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.)

The lack of safe and cost-effective treatments against leishmaniasis highlights the urgent need to develop improved leishmanicidal agents. Antimicrobial peptides (AMPs) are an emerging category of therapeutics exerting a wide range of biological activities such as anti-bacterial, anti-fungal, anti-parasitic and anti-tumoral. In the present study, the approach of repurposing AMPs as antileishmanial drugs was applied. The leishmanicidal activity of two synthetic anti-lipopolysaccharide peptides (SALPs), so-called 19-2.5 and 19-4LF was characterized in Leishmania major . In vitro, both peptides were highly active against intracellular Leishmania major in mouse macrophages without exerting toxicity in host cells. Then, q-PCR-based gene profiling, revealed that this activity was related to the downregulation of several genes involved in drug resistance ( yip1 ), virulence ( gp63 ) and parasite proliferation ( Cyclin 1 and Cyclin 6 ). Importantly, the treatment of BALB/c mice with any of the two AMPs caused a significant reduction in L. major infective burden. This effect was associated with an increase in Th1 cytokine levels ( IL-12p35 , TNF-α , and iNOS ) in the skin lesion and spleen of the L. major infected mice while the Th2-associated genes were downregulated ( IL-4 and IL-6 ). Lastly, we investigated the effect of both peptides in the gene expression profile of the P2X7 purinergic receptor, which has been reported as a therapeutic target in several diseases. The results showed significant repression of P2X7R by both peptides in the skin lesion of L. major infected mice to an extent comparable to that of a common anti-leishmanial drug, Paromomycin. Our in vitro and in vivo studies suggest that the synthetic AMPs 19-2.5 and 19-4LF are promising candidates for leishmaniasis treatment and present P2X7R as a potential therapeutic target in cutaneous leishmaniasis (CL).

Around 15% of cancer cases are attributable to infectious agents. Epidemiological studies suggest that an association between leishmaniasis and cancer does exist. Recently, the homologue of PES1 in Leishmania major (LmjPES) was described to be involved in parasite infectivity. Mammalian PES1 protein has been implicated in cellular processes like cell cycle regulation. Its BRCT domain has been identified as a key factor in DNA damage-responsive checkpoints. This work aimed to elucidate the hypothetical oncogenic implication of BRCT domain from LmjPES in host cells. We generated a lentivirus carrying this BRCT domain sequence (lentiBRCT) and a lentivirus expressing the luciferase protein (lentiLuc), as control. Then, HEK293T and NIH/3T3 mammalian cells were infected with these lentiviruses. We observed that the expression of BRCT domain from LmjPES conferred to mammal cells in vitro a greater replication rate and higher survival. In in vivo experiments, we observed faster tumor growth in mice inoculated with lentiBRCT respect to lentiLuc HEK293T infected cells. Moreover, the lentiBRCT infected cells were less sensitive to the genotoxic drugs. Accordingly, gene expression profiling analysis revealed that BRCT domain from LmjPES protein altered the expression of proliferation- ( DTX3L , CPA4 , BHLHE41 , BMP2 , DHRS2, S100A1 and PARP9 ), survival- ( BMP2 and CARD9 ) and chemoresistance-related genes ( DPYD, Dok3 , DTX3L , PARP9 and DHRS2 ). Altogether, our results reinforced the idea that in eukaryotes, horizontal gene transfer might be also achieved by parasitism like Leishmania infection driving therefore to some crucial biological changes such as proliferation and drug resistance.

Objectives: More effective topical treatments remain an unmet need for the localized forms of cutaneous leishmaniasis (CL). The aim of this study was to evaluate the efficacy and safety of a topical berberine cream in BALB/c mice infected with Leishmania major parasites., Methods: A cream containing 0.5% berberine-β-glycerophosphate salt and 2.5% menthol was prepared. Its physicochemical and stability properties were determined. The cream was evaluated for its capacity to reduce lesion size and parasitic load as well as to promote wound healing after twice-a-day administration for 35 days. Clinical biochemical profile was used for estimating off-target effects. In vitro time-to-kill curves in L. major-infected macrophages and skin and plasma pharmacokinetics were determined, aiming to establish pharmacokinetic/pharmacodynamic relationships., Results: The cream was stable at 40°C for 3 months and at 4°C for at least 8 months. It was able to halt lesion progression in all treated mice. At the end of treatment, parasite load in the skin was reduced by 99.9% (4 log) and genes involved in the wound healing process were up-regulated compared with untreated mice.The observed effects were higher than expected from in vitro time-to-kill kinetic and plasma berberine concentrations, which ranged between 0.07 and 0.22 μM., Conclusions: The twice-a-day administration of a topical berberine cream was safe, able to stop parasite progression and improved the appearance of skin CL lesions. The relationship between drug plasma levels and in vivo effect was unclear., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Leishmaniasis is a neglected tropical disease caused by Leishmania spp. The improvement of existing treatments and the discovery of new drugs remain ones of the major goals in control and eradication of this disease. From the parasite genome, we have identified the homologue of the human oncogene PES1 in Leishmania major ( LmjPES ). It has been demonstrated that PES1 is involved in several processes such as ribosome biogenesis, cell proliferation and genetic transcription. Our phylogenetic studies showed that LmjPES encodes a highly conserved protein containing three main domains: PES N-terminus (shared with proteins involved in ribosomal biogenesis), BRCT (found in proteins related to DNA repair processes) and MAEBL-type domain (C-terminus, related to erythrocyte invasion in apicomplexan). This gene showed its highest expression level in metacyclic promastigotes, the infective forms; by fluorescence microscopy assay, we demonstrated the nuclear localization of LmjPES protein. After generating mutant parasites overexpressing LmjPES , we observed that these clones displayed a dramatic increase in the ratio of cell infection within macrophages. Furthermore, BALB/c mice infected with these transgenic parasites exhibited higher footpad inflammation compared to those inoculated with non-overexpressing parasites.

This study investigates if visceral leishmaniasis (VL) infection has some effects on the organ and cellular uptake and distribution of 100-200 nm near-infrared fluorescently labelled non-biodegradable polystyrene latex beads (PS NPs) or biodegradable polylactic- co -glycolic nanoparticles (PLGA NPs), as this parasitic infection produces morphological alterations in liver, spleen and bone marrow, organs highly involved in NP sequestration. The results showed that the magnitude of the effect was specific for each organ and type of NP. With the exception of the liver, the general trend was a decrease in NP organ and cellular uptake, mostly due to immune cell mobilization and/or weight organ gain, as vascular permeability was increased. Moreover, NPs redistributed among different phagocytic cells to adapt infection associated changes and cellular alterations. In the liver, it is noteworthy that only isolated Kuffer cells (KCs) captured NPs, whereas they were not taken up by KC forming granulomas. In the spleen, NPs redistributed from macrophages and dendritic cells towards B cells and inflammatory monocytes although they maintained their preferential accumulation in the marginal zone and red pulp. Comparatively, the infection rarely affected the NP cellular distribution in the bone marrow. NP cellular target changes in VL infection could affect their therapeutic efficacy and should be considered for more efficient drug delivery.

The human endometrium is receptive to the embryo for a specific period of time known as the window of implantation (WOI). During this period, the endometrium shows a specific gene expression profile suitable for endometrial function evaluation. ER Map is a molecular tool able to accurately predict endometrial receptivity status by transcriptomic analysis. In this retrospective study, including 2256 subfertile patients undergoing ART treatment, the clinical value of precise WOI determination is studied in detail. Results obtained when single embryo transfers (sET) were scheduled either within the WOI timeframe as established by ER Map, or deviating from this WOI, are assessed and compared. Data obtained showed that 34.18% (771/2256) of patients had a displaced WOI. Analysis of ART outcomes showed significantly higher pregnancy rates in transfers scheduled within the WOI predicted compared to transfers that deviated more than 12h from this WOI (44.35% vs 23.08%, p < 0.001). The deviation from the WOI had also an impact on the progression of pregnancy, with a significant increase in pregnancy loss (~ twofold) observed in transfers that deviated more than 12h from the WOI predicted. These results indicate that the precise determination of the WOI and personalised embryo transfer can significantly improve clinical outcomes.

Follicular lymphoma (FL) is a common indolent B-cell lymphoma that can transform into the more aggressive transformed FL (tFL). However, the molecular process driving this transformation is uncertain. In this work, we aimed to identify microRNA (miRNA)-binding sites recurrently mutated in follicular lymphoma patients, as well as in transformed FL patients. Using whole-genome sequencing data from FL tumors, we discovered 544 mutations located in bioinformatically predicted microRNA-binding sites. We then studied these specific regions using targeted sequencing in a cohort of 55 FL patients, found 16 recurrent mutations, and identified a further 69 variants. After filtering for QC, we identified 21 genes with mutated miRNA-binding sites that were also enriched for B-cell-associated genes by Gene Ontology. Over 40% of mutations identified in these genes were present exclusively in tFL patients. We validated the predicted miRNA-binding sites of five of the genes by luciferase assay and demonstrated that the identified mutations in BCL2 and EZH2 genes impaired the binding efficiency of miR-5008 and miR-144 and regulated the endogenous levels of messenger RNA (mRNA).

A novel serine/threonine protein kinase, LmjF.22.0810, was recently described in Leishmania major . After generating an L. major cell line overexpressing LmjF.22.0810 (named LmJ3OE), the ability of this novel protein to modulate the Th2-type immune response was analyzed. Our results suggest that the protein kinase LmjF.22.0810 might be involved in leishmaniasis outcomes. Indeed, our study outlined the LmJ3OE parasites infectivity in vitro and in vivo. Transgenic parasites displayed lower phagocytosis rates in vitro, and their promastigote forms exhibited lower expression levels of virulence factors compared to their counterparts in control parasites. In addition, LmJ3OE parasites developed significantly smaller footpad swelling in susceptible BALB/c mice. Hematoxylin-eosin staining allowed the observation of a lower inflammatory infiltrate in the footpad from LmJ3OE-infected mice compared to animals inoculated with control parasites. Gene expression of Th2-associated cytokines and effectors revealed a dramatically lower induction in interleukin (IL)-4 , IL-10 , and arginase 1 (ARG1) mRNA levels at the beginning of the swelling; no expression change was found in Th1-associated cytokines except for IL-12 . Accordingly, such results were validated by immunohistochemistry studies, illustrating a weaker expression of ARG1 and a similar induction for inducible NO synthase (iNOS) in footpads from LmJ3OE-infected mice compared to control L. major infected animals. Furthermore, the parasite burden was lower in footpads from LmJ3OE-infected mice. Our analysis indicated that such significant smaller footpad swellings might be due to an impairment of the Th2 immune response that subsequently benefits Th1 prevalence. Altogether, these studies depict LmjF.22.0810 as a potential modulator of host immune responses to Leishmania . Finally, this promising target might be involved in the modulation of infection outcome.

Berberine (BER)-an anti-inflammatory quaternary isoquinoline alkaloid extracted from plants-has been reported to have a variety of biologic properties, including antileishmanial activity. This work addresses the preparation of BER-loaded liposomes with the aim to prevent its rapid liver metabolism and improve the drug selective delivery to the infected organs in visceral leishmaniasis (VL). BER liposomes (LP-BER) displayed a mean size of 120 nm, negative Z-potential of -38 mV and loaded 6 nmol/μmol lipid. In vitro, the loading of BER in liposomes enhanced its selectivity index more than 7-fold by decreasing its cytotoxicity to macrophages. In mice, LP-BER enhanced drug accumulation in the liver and the spleen. Consequently, the liposomal delivery of the drug reduced parasite burden in the liver and spleen by three and one logarithms (99.2 and 93.5%), whereas the free drug only decreased the infection in the liver by 1-log. The organ drug concentrations-far from IC 50 values- indicate that BER immunomodulatory activity or drug metabolites also contribute to the efficacy. Although LP-BER decreased 10-fold-an extremely rapid clearance of the free drug in mice-the value remains very high. Moreover, LP-BER reduced plasma triglycerides levels.

The identification and clarification of the mechanisms of action of drugs used against leishmaniasis may improve their administration regimens and prevent the development of resistant strains. Herein, for the first time, we describe the structure of the putatively essential Ser/Thr kinase LmjF.22.0810 from Leishmania major . Molecular dynamics simulations were performed to assess the stability of the kinase model. The analysis of its sequence and structure revealed two druggable sites on the protein. Furthermore, in silico docking of small molecules showed that aminoglycosides preferentially bind to the phosphorylation site of the protein. Given that transgenic LmjF.22.0810-overexpressing parasites displayed less sensitivity to aminoglycosides such as paromomycin, our predicted models support the idea that the mechanism of drug resistance observed in those transgenic parasites is the tight binding of such compounds to LmjF.22.0810 associated with its overexpression. These results may be helpful to understand the complex machinery of drug response in Leishmania ., Competing Interests: The authors declare no conflict of interest.

Ionothermal synthesis is a little used method for the preparation of coordination polymers. By this method, two cadmium compounds were synthesized, 1 , with formula Cd 3 (ox)F 2 (Ina) 2 (Ina = isonicotinate) and 2 , Cd(NO 3 ) 2 (4,4'-Bpy) (4,4'-Bpy = 4,4'-Bipyridine). The modification of the reaction conditions has allowed to obtain 2 as a pure phase. The structure of both compounds was determined by a single-crystal X-ray diffraction. Compound 1 is isostructural to the previously reported Cd 2 Zn(ox)(OH) 2 (Ina) 2 . It crystallizes in the monoclinic space group P 2 1 /n and present a three-dimensional (3D) network, built-up from [Cd 3 (ox)F 2 ] n 2n+ layers, linked by isonicotinate ligands. Crystals of 2 are formed by twins of two components which are rotated ca. 180° to each other. This compound crystallizes in the triclinic P -1 space group and its structure can be describe as a two-dimensional (2D) 4 connected 'sql' net. The layers are composed by [Cd(NO 3 ) 2 ] n chains linked through 4,4'-Bpy ligands, and are pillared along the [011] direction. The thermal decomposition of 2 was studied by thermogravimetric and thermodiffractiometric techniques. The compound decomposes gradually starting from 160 °C, and due to heating, the structure suffers slight reversible changes in the bond distances and angles.

Conventional chemotherapy against leishmaniasis includes agents exhibiting considerable toxicity. In addition, reports of drug resistance are not uncommon. Thus, safe and effective therapies are urgently needed. Isoselenocyanate compounds have recently been identified with potential antitumor activity. It is well known that some antitumor agents demonstrate effects against Leishmania In this study, the in vitro leishmanicidal activities of several organo-selenium and organo-sulfur compounds were tested against Leishmania major and Leishmania amazonensis parasites, using promastigotes and intracellular amastigote forms. The cytotoxicity of these agents was measured in murine peritoneal macrophages and their selectivity indexes were calculated. One of the tested compounds, the isoselenocyanate derivative NISC-6, showed selectivity indexes 2- and 10-fold higher than those of the reference drug amphotericin B when evaluated in L. amazonensis and L. major , respectively. The American strain ( L. amazonensis ) was less sensitive to NISC-6 than L. major , showing a trend similar to that observed previously for amphotericin B. In addition, we also observed that NISC-6 significantly reduced the number of amastigotes per infected macrophage. On the other hand, we showed that NISC-6 decreases expression levels of Leishmania genes involved in the cell cycle, such as topoisomerase-2 ( TOP-2 ), PCNA , and MCM4 , therefore contributing to its leishmanicidal activity. The effect of this compound on cell cycle progression was confirmed by flow cytometry. We observed a significant increase of cells in the G 1 phase and a dramatic reduction of cells in the S phase compared to untreated cells. Altogether, our data suggest that the isoselenocyanate NISC-6 may be a promising candidate for new drug development against leishmaniasis., (Copyright © 2019 American Society for Microbiology.)

The innate immune system provides a primary line of defense against pathogens. Stimulator of IFN genes (STING), encoded by the TMEM173 gene, is a critical protein involved in IFN-β induction in response to infection by different pathogens. In this study, we describe the expression of three different alternative-spliced human (h) TMEM173 mRNAs producing STING truncated isoforms 1, 2, and 3 in addition to the full-length wild-type (wt) hSTING. All of the truncated isoforms lack exon 7 and share the N-terminal transmembrane region with wt hSTING. Overexpression of the three STING truncated isoforms failed to induce IFN-β, and they acted as selective pathway inhibitors of wt hSTING even in combination with upstream inducer cyclic-di-GMP-AMP synthase. Truncated isoforms alter the stability of wt hSTING, reducing protein t 1/2 to some extent by the induction of proteasome-dependent degradation. Knocking down expression of truncated isoforms increased production of IFN-β by THP1 monocytes in response to intracellular cytosolic DNA or HSV-1 infection. At early stages of infection, viruses like HSV-1 or vesicular stomatitis virus reduced the ratio of full-length wt hSTING/truncated STING isoforms, suggesting the skewing of alternative splicing of STING toward truncated forms as a tactic to evade antiviral responses. Finally, in silico analysis revealed that the human intron-exon gene architecture of TMEM173 (splice sites included) is preserved in other mammal species, predominantly primates, stressing the relevance of alternative splicing in regulating STING antiviral biology., (Copyright © 2018 The Authors.)

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.)

Early diagnosis of laryngeal squamous cell carcinoma (LSCC) at the stage of dysplasia could greatly improve the outcome of affected patients. For the first time we compared the mutational landscape of non-progressing dysplasia (NPD; n = 42) with progressing dysplasia (PD; n = 24), along with patient-matched LSCC biopsies; a total of 90 samples. Using targeted next-generation sequencing identified non-synonymous mutations in six genes (PIK3CA, FGFR3, TP53, JAK3, MET, FBXW7), and mutations were validated by Sanger sequencing and/or qPCR. Analysis was extended in silico to 530 head and neck (HNSCC) cases using TCGA data. Mutations in PIK3CA and FGFR3 were detected in PD and LSCC cases, as well as other HNSCC cases, but absent in NPD cases. In contrast, mutations in JAK3, MET and FBXW7 were found in NPD cases but not PD, LSCC or other HNSCC cases. TP53 was the most frequently mutated gene in both PD and NPD cases. With the exception of R248W, mutations were mutually exclusive. Moreover, five of seven PD mutations were located in motif H2 of p53, whereas none of the NPD mutations were. In summary, we propose that the mutational profile of laryngeal dysplasia has utility for the early detection of patients at risk of progression.

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.)

Vaccine delivery using microneedles (MNs) represents a safe, easily disposable and painless alternative to traditional needle immunizations. The MN delivery of DNA vaccines to the dermis may result in a superior immune response and/or an equivalent immune response at a lower vaccine dose (dose-sparing). This could be of special interest for immunization programs against neglected tropical diseases such as leishmaniasis. In this work, we loaded a MN device with 60μg of a plasmid DNA cocktail encoding the Leishmania infantum nucleosomal histones H2A, H2B, H3 and H4 and compared its immunogenicity and protective capacity against conventional s.c. or i.d. injection of the plasmid. Mice immunized with MNs showed increased ratios of IFN-γ/IL-10, IFN-γ/IL-13, IFN-γ/IL-4, and IFN-γ/TGF-β in the spleens and lymph nodes compared with mice immunized by s.c. and i.d. routes. Furthermore, CCXCL9, CXCL10 and CCL2 levels were also higher. These data suggest that the nucleic acid immunization using MNs produced a better bias towards a Th1 response. However, none of the immunizations strategies were able to control Leishmania major infection in BALB/c mice, as illustrated by an increase in lesion size and parasite burden., (Copyright © 2017 Elsevier B.V. All rights reserved.)

MicroRNAs (miRNAs) are small non-coding RNAs that regulate many human genes including those involved in normal B-cell development. When these miRNAs are aberrantly expressed in B-cells they play key pathogenetic roles in the development and maintenance of B-cell lymphomas and by association may serve as useful biomarkers. In this review, we provide an overview of the importance of miRNAs to B-cell lymphomagenesis, as well as considering their use as biomarkers, and their potential usefulness for the clinic., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background : The outcome for oestrogen receptor positive (ER+) breast cancer patients has improved greatly in recent years largely due to targeted therapy. However, the presence of involved multiple synchronous lymph nodes remains associated with a poor outcome. Consequently, these patients would benefit from the identification of new prognostic biomarkers and therapeutic targets. The expression of G-protein-coupled receptor kinase-interacting protein 1 (GIT1) has recently been shown to be an indicator of advanced stage breast cancer. Therefore, we investigated its expression and prognostic value of GIT1 in a cohort of 140 ER+ breast cancer with synchronous lymph node involvement. Methods : Immunohistochemistry was employed to assess GIT1 expression in a tissue microarray (TMA) containing duplicate non-adjacent cores with matched primary tumour and lymph node tissue (n=140). GIT1 expression in tumour cells was scored and statistical correlation analyses were carried out. Results : The results revealed a sub-group of patients that displayed discordant expression of GIT1 between the primary tumour and the lymph nodes (i.e. spatial intratumoural heterogeneity). We observed that loss of GIT1 expression in the tumour cells of the metastasis was associated with a shorter time to recurrence, poorer overall survival, and a shorter median survival time. Moreover, multivariate analysis demonstrated that GIT1 expression was an independent prognostic indicator. Conclusions : GIT1 expression enabled the identification of a sub-class of ER+ patients with lymph node metastasis that have a particularly poor prognostic outcome. We propose that this biomarker could be used to further stratify ER+ breast cancer patients with synchronous lymph node involvement and therefore facilitate adjuvant therapy decision making., Competing Interests: Competing interests: No competing interests were disclosed.

Background : The outcome for oestrogen receptor positive (ER+) breast cancer patients has improved greatly in recent years largely due to targeted therapy. However, the presence of involved multiple synchronous lymph nodes remains associated with a poor outcome. Consequently, these patients would benefit from the identification of new prognostic biomarkers and therapeutic targets. The expression of G-protein-coupled receptor kinase-interacting protein 1 (GIT1) has recently been shown to be an indicator of advanced stage breast cancer. Therefore, we investigated its expression and prognostic value of GIT1 in a cohort of 140 ER+ breast cancer with synchronous lymph node involvement. Methods : Immunohistochemistry was employed to assess GIT1 expression in a tissue microarray (TMA) containing duplicate non-adjacent cores with matched primary tumour and lymph node tissue (n=140). GIT1 expression in tumour cells was scored and statistical correlation analyses were carried out. Results : The results revealed a sub-group of patients that displayed discordant expression of GIT1 between the primary tumour and the lymph nodes (i.e. spatial intratumoural heterogeneity). We observed that loss of GIT1 expression in the metastasis was associated with a shorter time to recurrence, poorer overall survival, and a shorter median survival time. Moreover, multivariate analysis demonstrated that GIT1 expression was an independent prognostic indicator. Conclusions : GIT1 expression enabled the identification of a sub-class of ER+ patients with lymph node metastasis that have a particularly poor prognostic outcome. We propose that this biomarker could be used to further stratify ER+ breast cancer patients with synchronous lymph node involvement and therefore facilitate adjuvant therapy decision making., Competing Interests: Competing interests: No competing interests were disclosed.

Leishmania is the causative agent of leishmaniasis, a neglected tropical disease that affects more than 12 million people around the world. Current treatments are toxic and poorly effective due to the acquisition of resistance within Leishmania populations. Thus, the pursuit for new antileishmanial drugs is a priority. The available methods for drug screening based on colorimetric assays using vital dyes are time-consuming. Currently, the use of fluorescent reporter proteins is replacing the use of viability indicator dyes. We have constructed two plasmids expressing the red fluorescent protein mCherry with multiple cloning sites (MCS), adequate for N- and C-terminal fusion protein constructs. Our results also show that the improved pXG-mCherry plasmid can be employed for drug screening in vitro . The use of the red fluorescent protein, mCherry, is an easier tool for numerous assays, not only to test pharmacological compounds, but also to determine the subcellular localization of proteins., Competing Interests: The authors declare that they have no competing interests.

B-cell lymphomas represent a heterogeneous collection of more than twenty-five different malignancies. Classification is often challenging as primarily based upon, sometimes subjective, histopathological criteria and misdiagnosis can result in inappropriate treatment decisions. MicroRNAs (miRNAs) hold great promise as novel biomarkers (diagnostic, prognostic and predictive) of B-cell lymphoma in addition to being potential therapeutic targets. The most promising of these miRNAs more often than not play key regulatory roles in lymphopoiesis (development of lymphocytes) when under physiological conditions, and in the pathology of lymphoid malignancies when aberrantly expressed. In this review we consider the identity and functional role of miRNAs in the most common forms of B-cell lymphomas, their role in lymphopoiesis and their potential as biomarkers for these malignancies.

Scavenger receptor class B type I (SR-B1) binds pathogen-associated molecular patterns participating in the regulation of the inflammatory reaction but there is no information regarding potential interactions between SR-B1 and the interferon system. Herein, we report that SR-B1 ligands strongly regulate the transcriptional response to interferon α (IFNα) and enhance its antiviral and antitumor activity. This effect was mediated by the activation of TLR2 and TLR4 as it was annulled by the addition of anti-TLR2 or anti-TLR4 blocking antibodies. In vivo, we maximized the antitumor activity of IFNα co-expressing in the liver a SR-B1 ligand and IFNα by adeno-associated viruses. This gene therapy strategy eradicated liver metastases from colon cancer with reduced toxicity. On the other hand, genetic and pharmacological inhibition of SR-B1 blocks the clathrin-dependent interferon receptor recycling pathway with a concomitant reduction in IFNα signaling and bioactivity. This effect can be applied to enhance cancer immunotherapy with oncolytic viruses. Indeed, SR-B1 antagonists facilitate replication of oncolytic viruses amplifying their tumoricidal potential. In conclusion, SR-B1 agonists behave as IFNα enhancers while SR-B1 inhibitors dampen IFNα activity. These results demonstrate that SR-B1 is a suitable pharmacology target to enhance cancer immunotherapy based on IFNα and oncolytic viruses.

Viral clearance during acute hepatitis C virus (HCV) infection is associated with the induction of potent antiviral T-cell responses. Since dendritic cells (DC) are essential in the activation of primary T-cell responses, gene expression was analyzed in DC from patients during acute HCV infection. By using microarrays, gene expression was compared in resting and activated peripheral blood plasmacytoid (pDC) and myeloid (mDC) DC from acute HCV resolving patients (AR) and from patients who become chronically infected (ANR), as well as in healthy individuals (CTRL) and chronically-infected patients (CHR). For pDC, a high number of upregulated genes was found in AR patients, irrespective of DC stimulation. However, for mDC, most evident differences were detected after DC stimulation, again corresponding to upregulated genes in AR patients. Divergent behavior of ANR was also observed when analyzing DC from CTRL and CHR, with ANR patients clustering again apart from these groups. These differences corresponded to metabolism-associated genes and genes belonging to pathways relevant for DC activation and cytokine responses. Thus, upregulation of relevant genes in DC during acute HCV infection may determine viral clearance, suggesting that dysfunctional DC may be responsible for the lack of efficient T-cell responses which lead to chronic HCV infection., (© 2015 Wiley Periodicals, Inc.)

The effective and efficient management of cancer patients relies upon early diagnosis and/or the monitoring of treatment, something that is often difficult to achieve using standard tissue biopsy techniques. Biological fluids such as blood hold great possibilities as a source of non-invasive cancer biomarkers that can act as surrogate markers to biopsy-based sampling. The non-invasive nature of these "liquid biopsies" ultimately means that cancer detection may be earlier and that the ability to monitor disease progression and/or treatment response represents a paradigm shift in the treatment of cancer patients. Below, we review one of the most promising classes of circulating cancer biomarkers: microRNAs (miRNAs). In particular, we will consider their history, the controversy surrounding their origin and biology, and, most importantly, the hurdles that remain to be overcome if they are really to become part of future clinical practice.

Development of reporter systems for in vivo examination of IFN-β induction or signaling of type I interferon (IFN-I) pathways is of great interest in order to characterize biological responses to different inducers such as viral infections. Several reporter mice have been developed to monitor the induction of both pathways in response to different agonists. However, alternative strategies that do not require transgenic mice breeding have to date not been reported. In addition, detection of these pathways in vivo in animal species other than mice has not yet been addressed. Herein we describe a simple method based on the use of an adeno-associated viral vector (AAV8-3xIRF-ISRE-Luc) containing an IFN-β induction and signaling-sensitive promoter sequence controlling the expression of the reporter gene luciferase. This vector is valid for monitoring IFN-I responses in vivo elicited by diverse stimuli in different organs. Intravenous administration of the vector in C57BL/6 mice and Syrian hamsters was able to detect activation of the IFN pathway in the liver upon systemic treatment with different pro-inflammatory agents and infection with Newcastle disease virus (NDV). In addition, intranasal instillation of AAV8-3xIRF-ISRE-Luc showed a rapid and transient IFN-I response in the respiratory tract of mice infected with the influenza A/PR8/34 virus lacking the NS1 protein. In comparison, this response was delayed and exacerbated in mice infected with influenza A/PR/8 wild type virus. In conclusion, the AAV8-3xIRF-ISRE-Luc vector offers the possibility of detecting IFN-I activation in response to different stimuli and in different animal models with no need for reporter transgenic animals.

Background: The limited efficacy of current treatments against pancreatic cancer has prompted the search of new alternatives such as virotherapy. Activation of the immune response against cancer cells is emerging as one of the main mechanisms of action of oncolytic viruses (OV). Direct oncolysis releases tumor antigens, and viral replication within the tumor microenvironment is a potent danger signal. Arming OV with immunostimulatory transgenes further enhances their therapeutic effect. However, standard virotherapy protocols do not take full advantage of OV as cancer vaccines because repeated viral administrations may polarize immune responses against strong viral antigens, and the rapid onset of neutralizing antibodies limits the efficacy of redosing. An alternative paradigm based on sequential combination of antigenically distinct OV has been recently proposed., Methods: We have developed a protocol consisting of sequential intratumor administrations of new Adenovirus (Ad) and Newcastle Disease Virus (NDV)-based OV encoding the immunostimulatory cytokine oncostatin M (OSM). Transgene expression, toxicity and antitumor effect were evaluated using an aggressive orthotopic pancreatic cancer model in Syrian hamsters, which are sensitive to OSM and permissive for replication of both OVs., Results: NDV-OSM was more cytolytic, whereas Ad-OSM caused higher OSM expression in vivo. Both viruses achieved only a marginal antitumor effect in monotherapy. In addition, strong secretion of OSM in serum limited the maximal tolerated dose of Ad-OSM. In contrast, moderate doses of Ad-OSM followed one week later by NDV-OSM were safe, showed a significant antitumor effect and stimulated immune responses against cancer cells. Similar efficacy was observed when the order of virus administrations was reversed., Conclusion: Sequential administration of oncolytic Ad and NDV encoding OSM is a promising approach against pancreatic cancer.

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.)

The gold standard for cancer diagnosis remains the histological examination of affected tissue, obtained either by surgical excision, or radiologically guided biopsy. Such procedures however are expensive, not without risk to the patient, and require consistent evaluation by expert pathologists. Consequently, the search for non-invasive tools for the diagnosis and management of cancer has led to great interest in the field of circulating nucleic acids in plasma and serum. An additional benefit of blood-based testing is the ability to carry out screening and repeat sampling on patients undergoing therapy, or monitoring disease progression allowing for the development of a personalized approach to cancer patient management. Despite having been discovered over 60 years ago, the clear clinical potential of circulating nucleic acids, with the notable exception of prenatal diagnostic testing, has yet to translate into the clinic. The recent discovery of non-coding (nc) RNA (in particular micro(mi)RNAs) in the blood has provided fresh impetuous for the field. In this review, we discuss the potential of the circulating transcriptome (coding and ncRNA), as novel cancer biomarkers, the controversy surrounding their origin and biology, and most importantly the hurdles that remain to be overcome if they are really to become part of future clinical practice., (© 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

The lack of antiviral cellular immune responses in patients with chronic hepatitis C virus (HCV) infection suggests that T-cell vaccines may provide therapeutic benefit. Due to the central role that dendritic cells (DC) play in the activation of T-cell responses, our aim was to carry out a therapeutic vaccination clinical trial in HCV patients using DC. Five patients with chronic HCV infection were vaccinated with three doses of 5 × 10(6) or 10(7) autologous DC transduced with a recombinant adenovirus encoding NS3 using the adapter protein CFh40L, which facilitates DC transduction and maturation. No significant adverse effects were recorded after vaccination. Treatment caused no changes in serum liver enzymes nor in viral load. Vaccination induced weak but consistent expansion of T-cell responses against NS3 and adenoviral antigens. Patients' DC, as opposed to murine DC or DC from healthy subjects, secreted high IL-10 levels after transduction, inducing the activation of IL-10-producing T cells. IL-10 blockade during vaccine preparation restored its ability to stimulate anti-NS3 Th1 responses. Thus, vaccination with adenovirus-transduced DC is safe and induces weak antiviral immune responses. IL-10 associated with vaccine preparation may be partly responsible for these effects, suggesting that future vaccines should consider concomitant inhibition of this cytokine.

RIG-I-like receptors (RLRs) are cellular sensor proteins that detect certain RNA species produced during viral infections. RLRs activate a signaling cascade that results in the production of IFN-β as well as several other cytokines with antiviral and proinflammatory activities. We explored the potential of different constructs based on RLRs to induce the IFN-β pathway and create an antiviral state in type I IFN-unresponsive models. A chimeric construct composed of RIG-I 2CARD and the first 200 amino acids of MAVS (2CARD-MAVS200) showed an enhanced ability to induce IFN-β when compared to other stimulatory constructs. Furthermore, this human chimeric construct showed a superior ability to activate IFN-β expression in cells from various species. This construct was found to overcome the restrictions of blocking IFN-β induction or signaling by a number of viral IFN-antagonist proteins. Additionally, the antiviral activity of this chimera was demonstrated in influenza virus and HBV infection mouse models using adeno-associated virus (AAV) vectors as a delivery vehicle. We propose that AAV vectors expressing 2CARD-MAVS200 chimeric protein can reconstitute IFN-β induction and recover a partial antiviral state in different models that do not respond to recombinant IFN-β treatment., (© 2015 S. Karger AG, Basel.)

We report a new method to generate high-expressing mammalian cell lines in a quick and efficient way. For that purpose, we developed a master cell line (MCL) containing an inducible alphavirus vector expressing GFP integrated into the genome. In the MCL, recombinant RNA levels increased >4,600-fold after induction, due to a doxycycline-dependent RNA amplification loop. The MCL maintained inducibility and expression during 50 passages, being more efficient for protein expression than a conventional cell line. To generate new cell lines, mutant LoxP sites were inserted into the MCL, allowing transgene and selection gene exchange by Cre-directed recombination, leading to quick generation of inducible cell lines expressing proteins of therapeutic interest, like human cardiotrophin-1 and oncostatin-M at several mg/l/24 h. These proteins contained posttranslational modifications, showed bioactivity, and were efficiently purified. Remarkably, this system allowed production of toxic proteins, like oncostatin-M, since cells able to express it could be grown to the desired amount before induction. These cell lines were easily adapted to growth in suspension, making this methodology very attractive for therapeutic protein production.

Di-ammonium hexa-phosphito-penta-ferrate(II), (NH4)2[Fe5(HPO3)6], was synthesized under mild hydro-thermal conditions and autogeneous pressure, yielding twinned crystals. The crystal structure exhibits an [Fe(II) 5(HPO3)6](2-) open framework with NH4 (+) groups as counter-cations. The anionic skeleton is based on (001) sheets of [FeO6] octa-hedra (one with point-group symmetry 3.. and one with .2.) linked along [001] through [HPO3](2-) oxoanions. Each sheet is constructed from 12-membered rings of edge-sharing [FeO6] octa-hedra, giving rise to channels with a radius of ca 3.1 Å in which the disordered NH4 (+) cations are located. The IR spectrum shows vibrational bands typical for phosphite and ammonium groups.

Background: IL-7 and IL-15 are produced by hepatocytes and are critical for the expansion and function of CD8 T cells. IL-15 needs to be presented by IL-15Rα for efficient stimulation of CD8 T cells., Methods: We analysed the hepatic levels of IL-7, IL-15, IL-15Rα and interferon regulatory factors (IRF) in patients with chronic hepatitis C (CHC) (78% genotype 1) and the role of IRF1 and IRF2 on IL-7 and IL-15Rα expression in Huh7 cells with or without hepatitis C virus (HCV) replicon., Results: Hepatic expression of both IL-7 and IL-15Rα, but not of IL-15, was reduced in CHC. These patients exhibited decreased hepatic IRF2 messenger RNA levels and diminished IRF2 staining in hepatocyte nuclei. We found that IRF2 controls basal expression of both IL-7 and IL-15Rα in Huh7 cells. IRF2, but not IRF1, is downregulated in cells with HCV genotype 1b replicon and this was accompanied by decreased expression of IL-7 and IL-15Rα, a defect reversed by overexpressing IRF2. Treating Huh7 cells with IFNα plus oncostatin M increased IL-7 and IL-15Rα mRNA more intensely than either cytokine alone. This effect was mediated by strong upregulation of IRF1 triggered by the combined treatment. Induction of IRF1, IL-7 and IL-15Rα by IFNα plus oncostatin M was dampened in replicon cells but the combination was more effective than either cytokine alone., Conclusions: HCV genotype 1 infection downregulates IRF2 in hepatocytes attenuating hepatocellular expression of IL-7 and IL-15Rα. Our data reveal a new mechanism by which HCV abrogates specific T-cell responses and point to a novel therapeutic approach to stimulate anti-HCV immunity.

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.)

Clear cell tubulopapillary renal cell carcinoma (CCPRCC) is a recently described rare renal malignancy that displays characteristic gross, microscopic and immunohistochemical differences from other renal tumour types. However, CCPRCC remains a very poorly understood entity. We therefore sought to elucidate some of the molecular mechanisms involved in this neoplasm by carrying out targeted next-generation sequencing (NGS) to identify associated mutations, and in addition examined the expression of non-coding (nc) RNAs. We identified multiple somatic mutations in CCPRCC cases, including a recurrent [3/14 cases (21%)] non-synonymous T992I mutation in the MET proto-oncogene, a gene associated with epithelial-to-mesenchymal transition (EMT). Using a microarray approach, we found that the expression of mature (n = 1105) and pre-miRNAs (n = 1105), as well as snoRNA and scaRNAs (n = 2214), in CCPRCC cases differed from that of clear cell renal cell carcinoma (CCRCC) or papillary renal cell carcinoma (PRCC) tumours. Surprisingly, and unlike other renal tumour subtypes, we found that all five members of the miR-200 family were over-expressed in CCPRCC cases. As these miRNAs are intimately involved with EMT, we stained CCPRCC cases for E-cadherin, vimentin and β-catenin and found that the tumour cells of all cases were positive for all three markers, a combination rarely reported in other renal tumours that could have diagnostic implications. Taken together with the mutational analysis, these data suggest that EMT in CCPRCC tumour cells is incomplete or blocked, consistent with the indolent clinical course typical of this malignancy. In summary, as well as describing a novel pathological mechanism in renal carcinomas, this study adds to the mounting evidence that CCPRCC should be formally considered a distinct entity. Microarray data have been deposited in the GEO database [GEO accession number (GSE51554)]., (Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

It has been shown that the liver of immunodeficient mice can be efficiently repopulated with human hepatocytes when subjected to chronic hepatocellular damage. Mice with such chimeric livers represent useful reagents for medical and clinical studies. However all previously reported models of humanized livers are difficult to implement as they involve cross-breeding of immunodeficient mice with mice exhibiting genetic alterations causing sustained hepatic injury. In this paper we attempted to create chimeric livers by inducing persistent hepatocellular damage in immunodeficient Rag2(-/-) γc(-/-) mice using an adenovirus encoding herpes virus thymidine kinase (AdTk) and two consecutive doses of ganciclovir (GCV). We found that this treatment resulted in hepatocellular damage persisting for at least 10 weeks and enabled efficient engraftment and proliferation within the liver of either human or allogenic hepatocytes. Interestingly, while the nodules generated from the transplanted mouse hepatocytes were well vascularized, the human hepatocytes experienced progressive depolarization and exhibited reduced numbers of murine endothelial cells inside the nodules. In conclusion, AdTk/GCV-induced liver damage licenses the liver of immunodeficient mice for allogenic and xenogenic hepatocyte repopulation. This approach represents a simple alternative strategy for chimeric liver generation using immunodeficient mice without additional genetic manipulation of the germ line.

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: IFN-α5 has been demonstrated to induce stronger signaling and higher expression of antiviral genes than IFN-α2, which is the current treatment in chronic viral hepatitis. However, there is no specific and validated quantification method in order to conduct kinetic studies as part of the preclinical and clinical evaluation for regulatory purposes., Results: A novel integration of an antiviral assay against the cytopathic effect of the encephalomyocarditis virus in HeLa cells with a very sensitive method for assay processing - the Vialight(®) Plus assay - is presented for IFN-α5 activity quantification. The bioassay has been validated in macaque and human serum and it has been demonstrated to be selective, precise and accurate., Conclusion: The validated bioassay meets suitable acceptance criteria for these types of biological assays.

Previous mouse and human studies have demonstrated that direct IFN-α/β signaling on naive CD8 T cells is critical to support their expansion and acquisition of effector functions. In this study, we show that human naive CD8 T cells primed in the presence of IFN-α possess a heightened ability to respond to homeostatic cytokines and to secondary Ag stimulation, but rather than differentiating to effector or memory CTLs, they preserve nature-like phenotypic features. These are qualities associated with greater efficacy in adoptive immunotherapy. In a mouse model of adoptive transfer, CD8 T cells primed in the presence of IFN-α are able to persist and to mediate a robust recall response even after a long period of naturally driven homeostatic maintenance. The long-lasting persistence of IFN-α-primed CD8 T cells is favored by their enhanced responsiveness to IL-15 and IL-7, as demonstrated in IL-15(-/-) and IL-7(-/-) recipient mice. In humans, exposure to IFN-α during in vitro priming of naive HLA-A2(+) CD8 T cells with autologous dendritic cells loaded with MART1(26-35) peptide renders CD8 T cells with an improved capacity to respond to homeostatic cytokines and to specifically lyse MART1-expressing melanoma cells. Furthermore, in a mouse model of melanoma, adoptive transfer of tumor-specific CD8 T cells primed ex vivo in the presence of IFN-α exhibits an improved ability to contain tumor progression. Therefore, exposure to IFN-α during priming of naive CD8 T cells imprints decisive information on the expanded cells that can be exploited to improve the efficacy of adoptive T cell therapy.

Unlabelled: Regulatory T cells (Treg) play a critical role in the modulation of immune responses to viral antigens in chronic viral hepatitis. Woodchucks (Marmota monax) infected with the woodchuck hepatitis virus (WHV) represent the best animal model for chronic hepatitis B virus (HBV) infection. Examination of intrahepatic and peripheral Treg in uninfected and WHV chronically infected woodchucks showed a significant increase of intrahepatic Treg numbers in chronically infected animals, whereas no differences were found in peripheral blood. In agreement with these data, higher expression levels of Forkhead box P3 (Foxp3), interleukin (IL)-10, transforming growth factor beta (TGF-β) were detected in the liver of chronic WHV carriers in comparison to uninfected animals. Furthermore, treatment of WHV-infected animals with an adenovirus encoding IL-12 failed to reduce viral load, a finding that was associated with lymphocyte unresponsiveness to IL-12 stimulation in vitro. We observed that TGF-β and Treg play a major role in the lack of lymphocyte response to IL-12 stimulation, as TGF-β inhibition and Treg depletion allowed recovery of T-cell responsiveness to this cytokine. Based on these results, woodchucks were treated with IL-12 in combination with a TGF-β inhibitory peptide or Treg depletion. However, no antiviral effect was achieved and, instead, an enhancement of the intrahepatic tolerogenic environment was observed., Conclusion: Our data show that TGF-β inhibition or Treg depletion had no added benefit over IL-12 therapy in chronic WHV infection. IL-12 immunostimulation induces a strong immunosuppressive reaction in the liver of chronic WHV carriers that counteracts the antiviral effect of the treatment., (Copyright © 2012 American Association for the Study of Liver Diseases.)

Protein N-terminal acetylation (Nt-acetylation) is an important mediator of protein function, stability, sorting, and localization. Although the responsible enzymes are thought to be fairly well characterized, the lack of identified in vivo substrates, the occurrence of Nt-acetylation substrates displaying yet uncharacterized N-terminal acetyltransferase (NAT) specificities, and emerging evidence of posttranslational Nt-acetylation, necessitate the use of genetic models and quantitative proteomics. NatB, which targets Met-Glu-, Met-Asp-, and Met-Asn-starting protein N termini, is presumed to Nt-acetylate 15% of all yeast and 18% of all human proteins. We here report on the evolutionary traits of NatB from yeast to human and demonstrate that ectopically expressed hNatB in a yNatB-Δ yeast strain partially complements the natB-Δ phenotypes and partially restores the yNatB Nt-acetylome. Overall, combining quantitative N-terminomics with yeast studies and knockdown of hNatB in human cell lines, led to the unambiguous identification of 180 human and 110 yeast NatB substrates. Interestingly, these substrates included Met-Gln- N-termini, which are thus now classified as in vivo NatB substrates. We also demonstrate the requirement of hNatB activity for maintaining the structure and function of actomyosin fibers and for proper cellular migration. In addition, expression of tropomyosin-1 restored the altered focal adhesions and cellular migration defects observed in hNatB-depleted HeLa cells, indicative for the conserved link between NatB, tropomyosin, and actin cable function from yeast to human.

Rabbit hemorrhagic disease virus (RHDV) causes lethal fulminant hepatitis closely resembling acute liver failure (ALF) in humans. In this study, we investigated whether cardiotrophin-1 (CT-1), a cytokine with hepatoprotective properties, could attenuate liver damage and prolong survival in virus-induced ALF. Twenty-four rabbits were infected with 2 × 10(4) hemagglutination units of RHDV. Twelve received five doses of CT-1 (100 μg/kg) starting at 12 h postinfection (hpi) (the first three doses every 6 h and then two additional doses at 48 and 72 hpi), while the rest received saline. The animals were analyzed for survival, serum biochemistry, and viral load. Another cohort (n = 22) was infected and treated similarly, but animals were sacrificed at 30 and 36 hpi to analyze liver histology, viral load, and the expression of factors implicated in liver damage and repair. All infected rabbits that received saline died by 60 hpi, while 67% of the CT-1-treated animals survived until the end of the study. Treated animals showed improved liver function and histology, while the viral loads were similar. In the livers of CT-1-treated rabbits we observed reduction of oxidative stress, diminished PARP1/2 and JNK activation, and decreased inflammatory reaction, as reflected by reduced expression of tumor necrosis factor alpha, interleukin-1β, Toll-like receptor 4, VCAM-1, and MMP-9. In addition, CT-1-treated rabbits exhibited marked upregulation of TIMP-1 and increased expression of cytoprotective and proregenerative growth factors, including platelet-derived growth factor B, epidermal growth factor, platelet-derived growth factor receptor β, and c-Met. In conclusion, in a lethal form of acute viral hepatitis, CT-1 increases animal survival by attenuating inflammation and activating cytoprotective mechanisms, thus representing a promising therapy for ALF of viral origin.

Unlabelled: Interferon alpha (IFNα) is widely used for the treatment of viral hepatitis but substantial toxicity hampers its clinical use. In this work, we aimed at improving the efficacy of IFNα therapy by increasing the IFNα half-life and providing liver tropism. We selected apolipoprotein A-I (ApoA-I) as the stabilizing and targeting moiety. We generated plasmids encoding IFNα, albumin bound to IFNα (ALF), or IFNα linked to ApoA-I (IA) and mice were treated either by hydrodynamic administration of the plasmids or by injection of the corresponding recombinant proteins or high-density lipoproteins containing IA. The plasma half-life of IA was intermediate between IFNα and ALF. IA was targeted to the liver and induced higher hepatic expression of interferon-stimulated genes than IFNα or even ALF. IA exhibits stronger in vivo antiviral activity than IFNα and the hematologic cytopenic effects of IA are milder than those observed when using IFNα or ALF. In contrast to IFNα, IA does not cause activation-dependent cell death of lymphocytes in vitro. Accordingly, in vivo studies showed that IA boosts T-cell immune responses more efficiently than IFNα or ALF. The difference in immunostimulatory activity between IFNα and IA disappears in scavenger receptor class B type I (SR-BI) knockout mice, suggesting that crosstalk between SR-BI and IFNα receptor is essential for enhanced induction of cytotoxic T cells by IA., Conclusion: Anchoring IFNα to ApoA-I prolongs the half-life of IFNα and promotes targeting to the liver. Importantly, the fusion protein shows increased immunostimulatory properties and lower hematological toxicity., (Copyright © 2011 American Association for the Study of Liver Diseases.)

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.)

Unlabelled: The high levels of interleukin 10 (IL-10) present in chronic hepatitis C virus (HCV) infection have been suggested as responsible for the poor antiviral cellular immune responses found in these patients. To overcome the immunosuppressive effect of IL-10 on antigen-presenting cells such as dendritic cells (DCs), we developed peptide inhibitors of IL-10 to restore DC functions and concomitantly induce efficient antiviral immune responses. Two IL-10-binding peptides (p9 and p13) were selected using a phage-displayed library and their capacity to inhibit IL-10 was assessed in a bioassay and in STAT-3 (signal transducer and activator of transcription 3) phosphorylation experiments in vitro. In cultures of human leukocytes where HCV core protein induces the production of IL-10, p13 restored the ability of plasmacytoid DC to produce interferon alpha (IFN-α) after Toll-like receptor 9 (TLR9) stimulation. Similarly, when myeloid DCs were stimulated with CD40L in the presence of HCV core, p9 enhanced IL-12 production by inhibiting HCV core-induced as well as CD40L-induced IL-10. Moreover, in vitro, p13 potentiated the effect of maturation stimuli on human and murine DC, increasing their IL-12 production and stimulatory activity, which resulted in enhanced proliferation and IFN-γ production by responding T-cells. Finally, immunization with p13-treated murine DC induced stronger anti-HCV T-cell responses not only in wildtype mice but also in HCV transgenic mice and in mice transiently expressing HCV core in the liver., Conclusion: These results suggest that IL-10 inhibiting peptides may have important applications to enhance anti-HCV immune responses by restoring the immunostimulatory capabilities of DC., (Copyright © 2010 American Association for the Study of Liver Diseases.)

IFN-α/β link innate and adaptive immune responses by directly acting on naïve CD8(+) T cells. This concept unveiled in mice remains unexplored in humans. To investigate that, human CD8(+) CD45RO(-) cells were stimulated with beads coated with anti-CD3 and anti-CD28 mAb, mimicking Ag (type-1) and co-stimulatory (type-2) signals, in the presence or absence of IFN-α and their transcriptional profiles were defined by cDNA-microarrays. We show that IFN-α provides a strong third signal directly to human CD8(+) T cells resulting in regulation of critical genes for their overall activation. This transcriptional effect was substantiated at the protein level and verified by functional assays. Interestingly, the biological effects derived from this stimulation vary depending on the CD8(+) T-cell population. Thus, whereas IFN-α increases the proliferative capacity of naïve CD8(+) T cells, it inhibits or does not affect the proliferation of Ag-experienced cells, such as memory and effector CTL, including CMV-specific lymphocytes. Cytolysis and IFN-γ-secretion of all these populations are enhanced by IFN-α-derived signals, which are critical in naïve CD8(+) T cells for acquisition of effector functions. Our findings in human CD8(+) T cells are informative to understand and improve IFN-α-based therapies for viral and malignant diseases., (Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)