European expert consensus statement on the systemic treatment of alopecia areata.

Alopecia areata is an autoimmune form of non-scarring hair loss. It is usually characterized by limited areas of hair loss. However, the disease may progress to complete scalp and body hair loss (alopecia totalis, alopecia universalis). In patients with alopecia areata hair loss significantly impacts the quality of life. Children and adolescents with alopecia areata often experience bullying, including physical aggression. The disease severity evaluation tools used in clinical practice are: the Severity of Alopecia Tool (SALT) score and the Alopecia Areata Scale (AAS). A SALT score equal to or greater than 20 constitutes a commonly accepted indication for systemic therapy in alopecia areata. When using the AAS, moderate to severe alopecia areata should be considered a medical indication for systemic treatment. Currently, the only two EMA-approved medications for alopecia areata are baricitinib (JAK 1/2 inhibitor) for adults and ritlecitinib (JAK 3/TEC inhibitor) for individuals aged 12 and older. Both are EMA-approved for patients with severe alopecia areata. Other systemic medications used off-label in alopecia areata include glucocorticosteroids, cyclosporine, methotrexate and azathioprine. Oral minoxidil is considered an adjuvant therapy with limited data confirming its possible efficacy. This consensus statement is to outline a systemic treatment algorithm for alopecia areata, indications for systemic treatment, available therapeutic options, their efficacy and safety, as well as the duration of the therapy.

Guidelines for clinical trials of frontal fibrosing alopecia: consensus recommendations from the International FFA Cooperative Group (IFFACG).

BACKGROUND: Frontal fibrosing alopecia (FFA) has become one of the most common causes of cicatricial alopecia worldwide. However, there is a lack of clear aetiology and robust clinical trial evidence for the efficacy and safety of agents currently used for treatment. OBJECTIVES: To enable data to be collected worldwide on FFA using common criteria and assessment methods. METHODS: A multicentre, international group of experts in hair loss was convened by email to create consensus recommendations for clinical trials. Consensus was defined at > 90% agreement on each recommended part of these guidelines. RESULTS: Standardized diagnostic criteria, severity rating, staging, and investigator and patient assessment of scalp hair loss and other clinical features of FFA were created. CONCLUSIONS: These guidelines should allow the collection of reliable aggregate data on FFA and advance efforts in both clinical and basic research to close knowledge gaps in this condition.

Cell and platelet composition assays by flow cytometry: basis for new platelet-rich fibrin methodologies.

Platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) are selective blood fractions obtained by cen¬trifugation. They act locally on inflammation and immunity as adjuvant homeostatic modulators during tissue regeneration. In recent years, many methods for achieving these blood concentrates have emerged, whose parameters of time and force of centrifugation presented themselves as critical, conflicting, and poorly understood points. Thus, the present study aimed to evaluate the effect of different centrifugal experimental parameters on the concentration of cells and platelets in samples of anticoagulated blood. Blood samples were centrifuged by forces of 200, 400 and 800 x g for 5, 10 and 15 minutes of centrifugation times to obtain three fractions: a) platelet-poor plasma (PPP), b) leukocyte-rich plasma (L- PRP) and c) red blood cell sed¬iment (RBC). The leukocyte and platelet content of each centrifuged fraction was measured by automated flow cytometry associated with the peroxidase reaction for differential leukocyte count. The application of 200 x g generated a more significant dispersive content of leukocytes and platelets in the supernatant fraction of PPP when compared to the other two strength ranges. However, it presented the highest concentration of platelets in the sediment (P <0.05 ANOVA), representing a loss of total mass during processing. The 400 and 800 x g forces showed leukocytes and platelets condensed in the L-PRP fraction and lower levels in the sedi¬ment, demonstrating the greater effectiveness of buoyancy in the resuspension of these sedimented elements. Our experimental data showed that the concentration and organization of leukocytes and platelets in the centrifuged blood matrices are very sensitive to variations in g force and centrifugation time, thus generating products with different biological composition and characteristics, and with specific potential therapeutic effects. The present study did not focus on comparing authoring methods, but on presenting the impact of methodological variations on the biological nature of centrifuged blood matrices. Further in vivo studies are needed to assess the specific clinical effect of each methodological change.

Stereochemical features of the envelope protein Domain III of dengue virus reveals putative antigenic site in the five-fold symmetry axis.

We bring to attention a characteristic parasitic pattern present in the dengue virus: it undergoes several intensive thermodynamic variations due to host environmental changes, from a vector's digestive tract, through the human bloodstream and intracellular medium. Comparatively, among the known dengue serotypes, we evaluate the effects that these medium variations may induce to the overall structural characteristics of the Domain III of the envelope (E) protein, checking for stereochemical congruences that could lead to the identification of immunologic relevant regions. We used molecular dynamics and principal component analysis to study the protein in solution, for all four dengue serotypes, under distinct pH and temperature. We stated that, while the core of Domain III is remarkably rigid and effectively unaffected by most of the mentioned intensive variations, the loops account for major and distinguishable flexibilities. Therefore, the rigidity of the Domain III core provides a foothold that projects specifically two of these high flexible loop regions towards the inner face of the envelope pores, which are found at every five-fold symmetry axis of the icosahedron-shaped mature virus. These loops bear a remarkable low identity though with high occurrence of ionizable residues, including histidines. Such stereochemical properties can provide very particular serotype-specific electrostatic surface patterns, suggesting a viral fingerprint region, on which other specific molecules and ions can establish chemical interactions in an induced fit mechanism. We assert that the proposed regions share enough relevant features to qualify for further immunologic and pharmacologic essays, such as target peptide synthesis and phage display using dengue patients' sera.

Neolignans from plants in northeastern Brazil (Lauraceae) with activity against Trypanosoma cruzi.

Trypanosoma cruzi is the ethiological agent for Chagas disease in Latin America. This study aimed to test the trypanocidal effect of licarin A and burchellin isolated from plants in northeastern Brazil. These neolignans were tested on T. cruzi and on peritoneal macrophages, to evaluate drug toxicity. Epimastigote growth was inhibited in 45% with licarin A and 20% with burchellin with an IC(50)/96 h of 462.7 microM and 756 microM, respectively. Epimastigotes treated with licarin A presented swollen mitochondria and disorganized mitochondrial cristae, kDNA and Golgi complex. When treated with burchellin, they presented enormous autophagosomes and chromatin disorganization. Licarin A and burchellin were able to induce trypomastigote death with IC(50)/24 h of 960 microM and 520 microM, respectively. Although licarin A presented an IC(50) for trypomastigotes higher than for epimastigotes, both substances acted as therapeutic trypanocidal agents, because they were able to kill parasites without affecting macrophages. Due to our results, burchellin and licarin A need to be further analysed to observe if they may be used as alternative blood additive prophylaxis against Chagas disease, since it has been established that blood transfusion is an important mechanism in the transmission process.

Insights into the Interactions of Fasciola hepatica Cathepsin L3 with a Substrate and Potential Novel Inhibitors through In Silico Approaches.

BACKGROUND: Fasciola hepatica is the causative agent of fascioliasis, a disease affecting grazing animals, causing economic losses in global agriculture and currently being an important human zoonosis. Overuse of chemotherapeutics against fascioliasis has increased the populations of drug resistant parasites. F. hepatica cathepsin L3 is a protease that plays important roles during the life cycle of fluke. Due to its particular collagenolytic activity it is considered an attractive target against the infective phase of F. hepatica. METHODOLOGY/PRINCIPAL FINDINGS: Starting with a three dimensional model of FhCL3 we performed a structure-based design of novel inhibitors through a computational study that combined virtual screening, molecular dynamics simulations, and binding free energy (ΔGbind) calculations. Virtual screening was carried out by docking inhibitors obtained from the MYBRIDGE-HitFinder database inside FhCL3 and human cathepsin L substrate-binding sites. On the basis of dock-scores, five compounds were predicted as selective inhibitors of FhCL3. Molecular dynamic simulations were performed and, subsequently, an end-point method was employed to predict ΔGbind values. Two compounds with the best ΔGbind values (-10.68 kcal/mol and -7.16 kcal/mol), comparable to that of the positive control (-10.55 kcal/mol), were identified. A similar approach was followed to structurally and energetically characterize the interface of FhCL3 in complex with a peptidic substrate. Finally, through pair-wise and per-residue free energy decomposition we identified residues that are critical for the substrate/ligand binding and for the enzyme specificity. CONCLUSIONS/SIGNIFICANCE: The present study is the first computer-aided drug design approach against F. hepatica cathepsins. Here we predict the principal determinants of binding of FhCL3 in complex with a natural substrate by detailed energetic characterization of protease interaction surface. We also propose novel compounds as FhCL3 inhibitors. Overall, these results will foster the future rational design of new inhibitors against FhCL3, as well as other F. hepatica cathepsins.

Evaluation of anti-inflammatory-related activity of essential oils from the leaves and resin of species of Protium.

The resins and leaves of species of Protium are commonly used by folk medicine. In the present study, we analyse the pharmacological effects of essential oils obtained by steam distillation (leaves and resin) from Protium species. Analysis by gas chromatography (GC) coupled to mass spectrometry and retention indices calculations demonstrate that the resin oil is constituted mainly of monoterpenes and phenylpropanoids: alpha-terpinolene (22%), p-cymene (11%), p-cimen-8-ol (11%), limonene (5%) and dillapiol (16%), whereas sesquiterpenes predominate as the volatile constituents of the leaves. The resin of Protium heptaphyllum (PHP) and leaves of P. strumosum (PS), P. grandifolium (PG), P. lewellyni (PL) and P. hebetatum (PHT) were screened for anti-inflammatory activity by the use of mouse pleurisy model induced by zymosan (500 microg/cavity) and lipopolysaccharide (LPS) (250 ng/cavity), for antinociceptive effect (by means of preventing mice abdominal writhings), as well as NO production from stimulated macrophages and proliferation of neoplasic cell lines: Neuro-2a (mouse neuroblastoma), SP2/0 (mouse plasmocytoma) and J774 (mouse monocytic cell line). The oils from PHP, PS and PL were able to inhibit protein extravasation but no sample inhibited total or differential leucocyte counts after administrating p.o. (100 mg/kg) 1 h before stimulation with zymosan. The oils from PG, PL and PHT inhibited neutrophil accumulation whereas PHP and specially PL inhibited LPS-induced eosinophil accumulation in mouse pleural cavity. PHT was also able to inhibit mononuclear cells accumulation. Antinociceptive effect was not observed, when animals received oral administration of the essential oils (100 mg/kg). In vitro treatment with essential oils (100 microg/well) changed the NO production from stimulated mouse macrophages. PHP inhibited in 74% and PS in 46% the LPS-induced NO production. In contrast, treatment with PL was able to increase in 49% the NO production. Cell lines proliferation was affected by the oils assayed in the range of 60-100% for Neuro-2a, 65-95% for SP2/0 and 70-90% for J774. Taken together these results showed that essential oils could be useful as efficient pharmacological tools.

[Crohn disease with unusual cutaneous expression]. / Doença de Crohn com expressão dermatológica invulgar.

Perianal fistula is a frequent complication of Crohn's disease. The diagnosis of the later may be difficult if cutaneous disease is dominant, and sometimes an important clinical challenge. The authors report a case of a 34 year old male complaining of painful perianal nodules for the three last years. Cutaneous histopathological examination was inespecific and colonoscopy normal. Pelvic CT scan and fistulography showed an intercommunicated fistular system invading both isquiorectal fossae, without intestinal communication. Repetition of colonoscopy showed normal mucosa, but in two of the biopsy samples noncaseous granulomas were found at the submucosa. The authors explain their main difficulties to reach the diagnosis of Crohn's disease and discuss the investigation and the evolution of the patient.

Novel nitroimidazoles with trypanocidal and cell growth inhibition activities.

Chagas' disease, caused by the protozoan parasite Trypanosoma cruzi, affects 18 million people in Latin America and is an important cause of heart disease. Although transmission has been reduced, an effective therapy for the infected population is lacking. New nitroimidazoles were designed and synthesized aimed at the development of a trypanocidal drug. The coupling of nitroimidazoles with heterocyclic N-trifluoromethyltriazolyl and pyrazolyl groups, 5-[N-(3-(5-trifluoromethyl)-1H-1,2,4-triazolyl)]amino-1-methyl-4-nitroimidazole (compound 4) and 5-N-(1-pyrazolyl)-1-methyl-4-nitroimidazole (compound 5). The in vitro trypanocidal effects of compounds 4 and 5 were evaluated. The results demonstrated that compound 5 was the most active compound, killing about 100% and 64% of the parasites in 0.3 mg/ml and 0.003 mg/ml concentrations, respectively. Interestingly, compound 4 also inhibited myeloma cell growth in a dose-dependent manner.