A Network of MicroRNAs and mRNAs Involved in Melanosome Maturation and Trafficking Defines the Lower Response of Pigmentable Melanoma Cells to Targeted Therapy

Simple Summary Selective inhibitors of mutant BRAFV600E (BRAFi) have revolutionized the treatment of metastatic melanoma patients and represent a powerful example of the efficacy of targeted therapy. However, one of the main limitations of BRAFi is that treated cells put in place several adaptive response mechanisms, which initially confer drug tolerance and later provide a gateway for the insurgence of genetically acquired resistance mechanisms. We previously discovered that pigmentation is one of these adaptive response mechanisms. Upon BRAFi treatment, those cells that increase their pigmentation level are more resistant to BRAFi than those that do not. Here, we demonstrate that pigmentation limits BRAFi activity through an increase in the number of intracellular mature melanosomes. We also show that this increase derives from increased maturation and/or trafficking. In addition, we identify the miRNAs and mRNAs that are involved in these biological processes. Finally, we provide the rationale for testing a new combinatorial therapeutic strategy that aims at increasing BRAFi efficacy by blocking the adaptive responses that they elicit. This strategy is based on the combined use of BRAFi with inhibitors of pigmentation, specifically inhibitors of melanosome maturation and/or trafficking. Background: The ability to increase their degree of pigmentation is an adaptive response that confers pigmentable melanoma cells higher resistance to BRAF inhibitors (BRAFi) compared to non-pigmentable melanoma cells. Methods: Here, we compared the miRNome and the transcriptome profile of pigmentable 501Mel and SK-Mel-5 melanoma cells vs. non-pigmentable A375 melanoma cells, following treatment with the BRAFi vemurafenib (vem). In depth bioinformatic analyses (clusterProfiler, WGCNA and SWIMmeR) allowed us to identify the miRNAs, mRNAs and biological processes (BPs) that specifically characterize the response of pigmentable melanoma cells to the drug. Such BPs were studied using appropriate assays in vitro and in vivo (xenograft in zebrafish embryos). Results: Upon vem treatment, miR-192-5p, miR-211-5p, miR-374a-5p, miR-486-5p, miR-582-5p, miR-1260a and miR-7977, as well as GPR143, OCA2, RAB27A, RAB32 and TYRP1 mRNAs, are differentially expressed only in pigmentable cells. These miRNAs and mRNAs belong to BPs related to pigmentation, specifically melanosome maturation and trafficking. In fact, an increase in the number of intracellular melanosomes-due to increased maturation and/or trafficking-confers resistance to vem. Conclusion: We demonstrated that the ability of pigmentable cells to increase the number of intracellular melanosomes fully accounts for their higher resistance to vem compared to non-pigmentable cells. In addition, we identified a network of miRNAs and mRNAs that are involved in melanosome maturation and/or trafficking. Finally, we provide the rationale for testing BRAFi in combination with inhibitors of these biological processes, so that pigmentable melanoma cells can be turned into more sensitive non-pigmentable cells

The natural compound climacostol as a prodrug strategy based on pH activation for efficient delivery of cytotoxic small agents

We synthesized and characterized MOMO as a new small molecule analog of the cytotoxic natural product climacostol efficiently activated in mild extracellular acidosis. The synthesis of MOMO had a key step in the Wittig olefination for the construction of the carbon-carbon double bond in the alkenyl moiety of climacostol. The possibility of obtaining the target (Z)-alkenyl MOMO derivative in very good yield and without presence of the less active (E)-diastereomer was favored from the methoxymethyl ether (MOM)-protecting group of hydroxyl functions in aromatic ring of climacostol aldehyde intermediate. Of interest, the easy removal of MOM-protecting group in a weakly acidic environment allowed us to obtain a great quantity of climacostol in biologically active (Z)-configuration. Results obtained in free-living ciliates that share the same micro-environment of the climacostol natural producer Climacostomum virens demonstrated that MOMO is well-tolerated in a physiological environment, while its cytotoxicity is rapidly and efficiently triggered at pH 6.3. In addition, the cytostatic vs. cytotoxic effects of acidified-MOMO can be modulated in a dose-dependent manner. In mouse melanoma cells, MOMO displayed a marked pH-sensitivity since its cytotoxic and apoptotic effects become evident only in mild extracellular acidosis. Data also suggested MOMO being preferentially activated in the unique extra-acidic microenvironment that characterizes tumoural cells. Finally, the use of the model organism Drosophila melanogaster fed with an acidic diet supported the efficient activity and oral delivery of MOMO molecule in vivo. MOMO affected oviposition of mating adults and larvae eclosion. Reduced survival of flies was due to lethality during the larval stages while emerging larvae retained their ability to develop into adults. Interestingly, the gut of eclosed larvae exhibited an extended damage (cell death by apoptosis) and the brain tissue was also affected (reduced mitosis), demonstrating that orally activated MOMO efficiently targets different tissues of the developing fly. These results provided a proof-of-concept study on the pH-dependence of MOMO effects. In this respect, MOM-protection emerges as a potential prodrug strategy which deserves to be further investigated for the generation of efficient pH-sensitive small organic molecules as pharmacologically active cytotoxic compounds

Lactobacillus GG in inducing and maintaining remission of Crohn's disease

BACKGROUND: Experimental studies have shown that luminal antigens are involved in chronic intestinal inflammatory disorders such as Crohn's disease and ulcerative colitis. Alteration of the intestinal microflora by antibiotic or probiotic therapy may induce and maintain remission. The aim of this randomized, placebo-controlled trial was to determine the effect of oral Lactobacillus GG (L. GG) to induce or maintain medically induced remission. METHODS: Eleven patients with moderate to active Crohn's disease were enrolled in this trial to receive either L. GG (2 × 10(9 )CFU/day) or placebo for six months. All patients were started on a tapering steroid regime and received antibiotics for the week before the probiotic/placebo medication was initiated. The primary end point was sustained remission, defined as freedom from relapse at the 6 months follow-up visit. Relapse was defined as an increase in CDAI of >100 points. RESULTS: 5/11 patients finished the study, with 2 patients in each group in sustained remission. The median time to relapse was 16 ± 4 weeks in the L. GG group and 12 ± 4.3 weeks in the placebo group (p = 0.5). CONCLUSION: In this study we could not demonstrate a benefit of L. GG in inducing or maintaining medically induced remission in CD

Cost effectiveness of ulcerative colitis treatment in Germany: a comparison of two oral formulations of mesalazine

<p>Abstract</p> <p>Background</p> <p>The treatment of ulcerative colitis (UC) can place a substantial financial burden on healthcare systems. The anti-inflammatory compound 5-aminosalicylic acid (5-ASA; mesalazine) is the recommended first-line treatment for patients with UC. In this analysis, the incremental cost effectiveness ratio (ICER) of two oral formulations of 5-ASA (Mezavant^®and Asacol^®) is examined in the treatment of patients with mild-to-moderate, active UC in Germany.</p> <p>Methods</p> <p>A Markov cohort model was developed to assess the cost effectiveness of Mezavant compared with Asacol over a 5-year period in the German Statutory Health Insurance (SHI). Drug pricing details for 2009 were applied throughout the model, and overall resource use was determined and also fitted to 2009 from published results of a large cross sectional study of German SHI patients. Cost per quality adjusted life year (QALY) was the primary endpoint for this study. Remission rates were obtained using data from a randomised, phase III trial of Mezavant with an active Asacol reference arm and a long-term, open label, safety and tolerability trial of Mezavant. Uncertainty in the study model was assessed using one-way and probabilistic sensitivity analyses applying a Monte Carlo simulation.</p> <p>Results</p> <p>Over a 5-year period, healthcare costs for patients receiving Mezavant were 624 Euro lower than for patients receiving Asacol. Additionally, patients receiving Mezavant gained 0.011 QALYs or 18 more days in remission compared with Asacol. One-way sensitivity analyses suggest that these results are driven by both differences in the acquisition cost between mesalazine formulations and differences in treatment efficacy. Furthermore, sensitivity analyses suggest a probability of 76% for cost savings and higher QALYs with Mezavant compared with Asacol. If adherence and its influence on the remission rates and the risk of developing colorectal cancer were included in the model, the results might have even been more favorable to Mezavant due to its once daily dosing regimen.</p> <p>Conclusions</p> <p>This model suggests that patients treated with Mezavant may achieve increased time in remission and higher QALYs, with lower direct costs to the SHI when compared with Asacol. Mezavant may therefore be a suitable first-line option for the induction and maintenance of remission in UC.</p

Effect of oral lactulose on clinical and immunohistochemical parameters in patients with inflammatory bowel disease: a pilot study

<p>Abstract</p> <p>Background</p> <p>The prebiotic potential of lactulose is well established and preclinical studies demonstrated a protective effect of lactulose in murine models of colitis. The aim of the present study was to investigate the clinical and histological efficacy of lactulose in patients with inflammatory bowel disease (IBD), for which probiotic therapy yielded promising results.</p> <p>Methods</p> <p>Patients were treated with standard medication alone or combined with 10 g lactulose daily as adjuvant therapy for 4 months. Clinical efficacy of treatment was assessed using clinical activity indices, a quality of life index (IBDQ), endoscopic scores, defecation frequency and monitoring corticosteroid medication. Orsomucoid, alpha1-antitrypsin and other laboratory parameters were determined. In addition, in some participants colonic biopsies were analyzed with haematoxylin-eosin staining or with antibodies against HLA-DR, CD68, IgA and CD3, and evaluated systematically. All measurements were performed both at enrolment and at the end of the trial.</p> <p>Results</p> <p>14 patients presenting ulcerative colitis (UC) and 17 patients presenting Crohn's disease (CD), most of them in a clinically active state, were enrolled in this pilot study. After 4 month no significant improvement of clinical activity index, endoscopic score or immunohistochemical parameters was observed in CD or UC patients receiving lactulose in comparison to the control group. However, significant improvement of quality of life was observed in UC patients receiving lactulose compared to the control group (p = 0.04).</p> <p>Conclusion</p> <p>The findings of the present pilot study indicate that oral lactulose has no beneficial effects in IBD patients in particular with regard to clinical activity, endoscopic score or immunohistochemical parameters. The importance of the beneficial effect of lactulose in UC patients regarding the quality of life needs further evaluation in larger controlled clinical trials.</p> <p>Trial registration</p> <p>Current Controlled Trials ISRCTN92101486</p

One week of levofloxacin plus dexamethasone eye drops for cataract surgery: an innovative and rational therapeutic strategy

Background: Cataract surgery is the most common operation performed worldwide. A fixed topical corticosteroid-antibiotic combination is usually prescribed in clinical practice for 2 or more weeks to treat post surgical inflammation and prevent infection. However, this protracted schedule may increase the incidence of corticosteroid-related adverse events and notably promote antibiotic resistance. Methods: This International, multicentre, randomized, blinded-assessor, parallel-group clinical study evaluated the non-inferiority of 1-week levofloxacin/dexamethasone eye drops, followed by 1-week dexamethasone alone, vs. 2-week gold-standard tobramycin/dexamethasone (one drop QID for all schedules) to prevent and treat ocular inflammation and prevent infection after uncomplicated cataract surgery. Non-inferiority was defined as the lower limit of the 95% confidence interval (CI) around a treatment difference >\u201310%. The study randomized 808 patients enrolled in 53 centres (Italy, Germany, Spain and Russia). The primary endpoint was the proportion of patients without anterior chamber inflammation on day 15 defined as the end of treatment. Endophthalmitis was the key secondary endpoint. This study is registered with EudraCT code: 2018-000286-36. Results: After the end of treatment, 95.2% of the patients in the test arm vs. 94.9% of the control arm had no signs of inflammation in the anterior chamber (difference between proportions of patients = 0.028; 95% CI: 120.0275/0.0331). No case of endophthalmitis was reported. No statistically significant difference was evident in any of the other secondary endpoints. Both treatments were well tolerated. Conclusions: Non-inferiority of the new short pharmacological strategy was proven. One week of levofloxacin/dexamethasone prevents infection, ensures complete control of inflammation in almost all patients and may contain antibiotic resistance

The Intestinal Microbiota Plays a Role in Salmonella-Induced Colitis Independent of Pathogen Colonization

The intestinal microbiota is composed of hundreds of species of bacteria, fungi and protozoa and is critical for numerous biological processes, such as nutrient acquisition, vitamin production, and colonization resistance against bacterial pathogens. We studied the role of the intestinal microbiota on host resistance to Salmonella enterica serovar Typhimurium-induced colitis. Using multiple antibiotic treatments in 129S1/SvImJ mice, we showed that disruption of the intestinal microbiota alters host susceptibility to infection. Although all antibiotic treatments caused similar increases in pathogen colonization, the development of enterocolitis was seen only when streptomycin or vancomycin was used; no significant pathology was observed with the use of metronidazole. Interestingly, metronidazole-treated and infected C57BL/6 mice developed severe pathology. We hypothesized that the intestinal microbiota confers resistance to infectious colitis without affecting the ability of S. Typhimurium to colonize the intestine. Indeed, different antibiotic treatments caused distinct shifts in the intestinal microbiota prior to infection. Through fluorescence in situ hybridization, terminal restriction fragment length polymorphism, and real-time PCR, we showed that there is a strong correlation between the intestinal microbiota composition before infection and susceptibility to Salmonella-induced colitis. Members of the Bacteroidetes phylum were present at significantly higher levels in mice resistant to colitis. Further analysis revealed that Porphyromonadaceae levels were also increased in these mice. Conversely, there was a positive correlation between the abundance of Lactobacillus sp. and predisposition to colitis. Our data suggests that different members of the microbiota might be associated with S. Typhimurium colonization and colitis. Dissecting the mechanisms involved in resistance to infection and inflammation will be critical for the development of therapeutic and preventative measures against enteric pathogens

Chromosome-wide DNA methylation analysis predicts human tissue-specific X inactivation

X-chromosome inactivation (XCI) results in the differential marking of the active and inactive X with epigenetic modifications including DNA methylation. Consistent with the previous studies showing that CpG island-containing promoters of genes subject to XCI are approximately 50% methylated in females and unmethylated in males while genes which escape XCI are unmethylated in both sexes; our chromosome-wide (Methylated DNA ImmunoPrecipitation) and promoter-targeted methylation analyses (Illumina Infinium HumanMethylation27 array) showed the largest methylation difference (D = 0.12, p < 2.2 E−16) between male and female blood at X-linked CpG islands promoters. We used the methylation differences between males and females to predict XCI statuses in blood and found that 81% had the same XCI status as previously determined using expression data. Most genes (83%) showed the same XCI status across tissues (blood, fetal: muscle, kidney and nerual); however, the methylation of a subset of genes predicted different XCI statuses in different tissues. Using previously published expression data the effect of transcription on gene-body methylation was investigated and while X-linked introns of highly expressed genes were more methylated than the introns of lowly expressed genes, exonic methylation did not differ based on expression level. We conclude that the XCI status predicted using methylation of X-linked promoters with CpG islands was usually the same as determined by expression analysis and that 12% of X-linked genes examined show tissue-specific XCI whereby a gene has a different XCI status in at least one of the four tissues examined

ESPEN Guideline: Clinical Nutrition in inflammatory bowel disease

Introduction: The ESPEN guideline presents a multidisciplinary focus on clinical nutrition in inflammatory bowel disease (IBD). Methodology: The guideline is based on extensive systematic review of the literature, but relies on expert opinion when objective data were lacking or inconclusive. The conclusions and 64 recommendations have been subject to full peer review and a Delphi process in which uniformly positive responses (agree or strongly agree) were required. Results: IBD is increasingly common and potential dietary factors in its aetiology are briefly reviewed. Malnutrition is highly prevalent in IBD – especially in Crohn's disease. Increased energy and protein requirements are observed in some patients. The management of malnu-trition in IBD is considered within the general context of support for malnourished patients. Treatment of iron deficiency (parenterally if necessary) is strongly recommended. Routine provision of a special diet in IBD is not however supported. Parenteral nutrition is indicated only when enteral nutrition has failed or is impossible. The recommended perioperative man-agement of patients with IBD undergoing surgery accords with general ESPEN guidance for patients having abdominal surgery. Probiotics may be helpful in UC but not Crohn's disease. Primary therapy using nutrition to treat IBD is not supported in ulcerative colitis, but is mod-erately well supported in Crohn's disease, especially in children where the adverse conse-quences of steroid therapy are proportionally greater. However, exclusion diets are generally not recommended and there is little evidence to support any particular formula feed when nutritional regimens are constructed. Conclusions: Available objective data to guide nutritional support and primary nutritional therapy in IBD are presented as 64 recommendations, of which 9 are very strong recom-mendations (grade A), 22 are strong recommendations (grade B) and 12 are based only on sparse evidence (grade 0); 21 recommendations are good practice points (GPP)