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Lorea Manterola,1 Marta Fernandez-Mercado,1 Erika Larrea,1 Ibai Goicoechea,1 María Arestin,1 María Armesto,1 Luiza Hernandez,1 Charles H Lawrie1,2,3 1Oncology Area, Biodonostia Research Institute, San Sebastián, Spain; 2Nuffield Department of Clinical Laboratory Sciences, University of Oxford, Oxford, UK; 3Ikerbasque, Basque Foundation for Science, Bilbao, Spain Abstract: B-cell lymphomas represent a group of more than 35 recognized mature B-cell neoplasms differentiated largely on the basis of immunohistochemical staining patterns that are often challenging to accurately diagnose. Despite having been only formally recognized just over 10 years ago, microRNAs (miRNAs) have become one of the trendiest topics in biology. Dysregulation of miRNAs is a ubiquitous feature of cancer in general, including B-cell lymphomas. Many of the miRNAs aberrantly expressed in B-cell lymphomas also play a crucial regulatory role in normal hematopoietic function. MiRNAs show great potential as novel biomarkers of cancer, as they can differentiate cancers according to diagnosis and developmental stage, even discriminating between cancers that are poorly separated histologically. Furthermore, they can be robustly measured from routinely prepared formalin-fixed paraffin-embedded biopsy material and biological fluids such as blood. Here, we consider the identity, function, and biomarker potential of miRNAs in B-cell lymphomas and, most importantly, the hurdles that remain to be overcome if they are really to become part of future clinical practice. Keywords: microRNA, lymphoma, biomarker, B-cell

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.

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.

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.