New chemical modalities for leishmaniasis drug discovery

Leishmaniasis is one of the major parasitic diseases among neglected tropical diseases with a high rate of morbidity and mortality. Human migration and climate change have spread the disease from limited endemic areas all over the world, also reaching regions in Southern Europe, and causing significant health and economic burden. The currently available treatments are far from ideal due to host toxicity, elevated cost, and increasing rates of drug resistance. Safer and more effective drugs are thus urgently required. Nevertheless, the identification of new chemical entities for leishmaniasis has proven to be incredibly hard and exacerbated by the scarcity of well-validated targets. Trypanothione reductase (TR) represents one robustly validated target in Leishmania that fulfils most of the requirements for a good drug target. However, due to the large and featureless active site, TR is considered extremely challenging and almost undruggable by small molecules. This scenario advocates the development of new chemical entities by unlocking new modalities for leishmaniasis drug discovery. The classical toolbox for drug discovery has enormously expanded in the last decade, and medicinal chemists can now strategize across a variety of new chemical modalities and a vast chemical space, to efficiently modulate challenging targets and provide effective treatments. Beyond others, Targeted p Protein Degradation (TPD) is an emerging strategy that uses small molecules to hijack endogenous proteolysis systems to degrade disease-relevant proteins and thus reduce their abundance in the cell. Based on these considerations, this thesis aimed to develop new strategies for leishmaniasis drug discovery while embracing novel chemical modalities and navigating the chemical space by chasing unprecedented chemotypes. This has been achieved by four complementary projects. We believe that these next-generation chemical modalities for leishmaniasis will play an important role in what was previously thought to be a drug discovery landscape dominated by small molecules.

Bright-field multiplex immunohistochemistry for tumor. Microenvironment evaluation in mucosal melanoma of the head and neck region: technical aspects, results, and correlation with clinicopathologic features and molecular landscape

Mucosal melanoma of the head and neck region (MM-H&N) is a rare disease, characterized by a poor prognosis and limited therapeutic strategies, especially regarding targeted therapy (lower rate of targetable mutations compared to cutaneous melanoma) and immunotherapy (lack of diagnostic tools able to predict the response). Meanwhile, bright-field multiplex immunohistochemistry (BF-mIHC) is emerging as a promising tool for characterizing tumor microenvironment (TME) and predicting response to immunotherapy in several tumors, including melanoma. This PhD project aims to develop a BF-mIHC protocol to evaluate the TME in MM-H&N, analyze the correlation between immune markers/immune profiles and MM-H&N features (clinicopathologic and molecular), and find new biomarkers useful for prognostic-therapeutic stratification of these patients. Specific aims are: (I) describe the clinicopathological features of MM-H&N; (II) analyze the molecular status of MM-H&N and correlate it with the clinicopathological features; (III) analyze the molecular status of multiple specimens from the same patient to verify whether molecular heterogeneity of MM-H&N could affect the results with relevant prognostic-therapeutic implications; (IV) develop a BF-mIHC protocol to study TME in MM-H&N; (V) analyze the correlation between immune markers/immune profiles and MM-H&N features (clinicopathologic and molecular) to test whether BF-mIHC could be a promising tool for prognostic-therapeutic characterization of these patients.

S-nitrosoglutathione (gsno) Is Cytotoxic To Intracellular Amastigotes And Promotes Healing Of Topically Treated Leishmania Major Or Leishmania Braziliensis Skin Lesions-authors' Response.

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Anatomical Study Of Minor Alterations In Neonate Vocal Folds.

Minor structural alterations of the vocal fold cover are frequent causes of voice abnormalities. They may be difficult to diagnose, and are expressed in different manners. Cases of intracordal cysts, sulcus vocalis, mucosal bridge, and laryngeal micro-diaphragm form the group of minor structural alterations of the vocal fold cover investigated in the present study. The etiopathogenesis and epidemiology of these alterations are poorly known. To evaluate the existence and anatomical characterization of minor structural alterations in the vocal folds of newborns. 56 larynxes excised from neonates of both genders were studied. They were examined fresh, or defrosted after conservation via freezing, under a microscope at magnifications of 25× and 40×. The vocal folds were inspected and palpated by two examiners, with the aim of finding minor structural alterations similar to those described classically, and other undetermined minor structural alterations. Larynges presenting abnormalities were submitted to histological examination. Six cases of abnormalities were found in different larynges: one (1.79%) compatible with a sulcus vocalis and five (8.93%) compatible with a laryngeal micro-diaphragm. No cases of cysts or mucosal bridges were found. The observed abnormalities had characteristics similar to those described in other age groups. Abnormalities similar to sulcus vocalis or micro-diaphragm may be present at birth.

The Putative Leishmania Telomerase Rna (leishter) Undergoes Trans-splicing And Contains A Conserved Template Sequence.

Telomerase RNAs (TERs) are highly divergent between species, varying in size and sequence composition. Here, we identify a candidate for the telomerase RNA component of Leishmania genus, which includes species that cause leishmaniasis, a neglected tropical disease. Merging a thorough computational screening combined with RNA-seq evidence, we mapped a non-coding RNA gene localized in a syntenic locus on chromosome 25 of five Leishmania species that shares partial synteny with both Trypanosoma brucei TER locus and a putative TER candidate-containing locus of Crithidia fasciculata. Using target-driven molecular biology approaches, we detected a ∼2,100 nt transcript (LeishTER) that contains a 5' spliced leader (SL) cap, a putative 3' polyA tail and a predicted C/D box snoRNA domain. LeishTER is expressed at similar levels in the logarithmic and stationary growth phases of promastigote forms. A 5'SL capped LeishTER co-immunoprecipitated and co-localized with the telomerase protein component (TERT) in a cell cycle-dependent manner. Prediction of its secondary structure strongly suggests the existence of a bona fide single-stranded template sequence and a conserved C[U/C]GUCA motif-containing helix II, representing the template boundary element. This study paves the way for further investigations on the biogenesis of parasite TERT ribonucleoproteins (RNPs) and its role in parasite telomere biology.

S-nitrosoglutathione Accelerates Recovery From 5-fluorouracil-induced Oral Mucositis.

Mucositis induced by anti-neoplastic drugs is an important, dose-limiting and costly side-effect of cancer therapy. To evaluate the effect of the topical application of S-nitrosoglutathione (GSNO), a nitric oxide donor, on 5-fluorouracil (5-FU)-induced oral mucositis in hamsters. Oral mucositis was induced in male hamsters by two intraperitoneal administrations of 5-FU on the first and second days of the experiment (60 and 40 mg/kg, respectively) followed by mechanical trauma on the fourth day. Animals received saline, HPMC or HPMC/GSNO (0.1, 0.5 or 2.0 mM) 1 h prior to the 5-FU injection and twice a day for 10 or 14 days. Samples of cheek pouches were harvested for: histopathological analysis, TNF-α and IL-1β levels, immunohistochemical staining for iNOS, TNF-α, IL-1β, Ki67 and TGF-β RII and a TUNEL assay. The presence and levels of 39 bacterial taxa were analyzed using the Checkerboard DNA-DNA hybridization method. The profiles of NO released from the HPMC/GSNO formulations were characterized using chemiluminescence. The HPMC/GSNO formulations were found to provide sustained release of NO for more than 4 h at concentration-dependent rates of 14 to 80 nmol/mL/h. Treatment with HPMC/GSNO (0.5 mM) significantly reduced mucosal damage, inflammatory alterations and cell death associated with 5-FU-induced oral mucositis on day 14 but not on day 10. HPMC/GSNO administration also reversed the inhibitory effect of 5-FU on cell proliferation on day 14. In addition, we observed that the chemotherapy significantly increased the levels and/or prevalence of several bacterial species. Topical HPMC/GSNO accelerates mucosal recovery, reduces inflammatory parameters, speeds up re-epithelization and decreases levels of periodontopathic species in mucosal ulcers.

Distribution Of Lutzomyia Longipalpis Chemotype Populations In São Paulo State, Brazil.

American visceral leishmaniasis (AVL) is an emerging disease in the state of São Paulo, Brazil. Its geographical expansion and the increase in the number of human cases has been linked to dispersion of Lutzomyia longipalpis into urban areas. To produce more accurate risk maps we investigated the geographic distribution and routes of expansion of the disease as well as chemotype populations of the vector. A database, containing the annual records of municipalities which had notified human and canine AVL cases as well as the presence of the vector, was compiled. The chemotypes of L. longipalpis populations from municipalities in different regions of São Paulo State were determined by Coupled Gas Chromatography - Mass Spectrometry. From 1997 to June 2014, L. longipalpis has been reported in 166 municipalities, 148 of them in the Western region. A total of 106 municipalities were identified with transmission and 99 were located in the Western region, where all 2,204 autochthonous human cases occurred. Both the vector and the occurrence of human cases have expanded in a South-easterly direction, from the Western to central region, and from there, a further expansion to the North and the South. The (S)-9-methylgermacrene-B population of L. longipalpis is widely distributed in the Western region and the cembrene-1 population is restricted to the Eastern region. The maps in the present study show that there are two distinct epidemiological patterns of AVL in São Paulo State and that the expansion of human and canine AVL cases through the Western region has followed the same dispersion route of only one of the two species of the L. longipalpis complex, (S)-9-methylgermacrene-B. Entomological vigilance based on the routes of dispersion and identification of the chemotype population could be used to identify at-risk areas and consequently define the priorities for control measures.

Resultados da cirurgia de reservatórios ileais em pacientes com doença de Crohn

BACKGROUND: Total rectocolectomy and ileal pouch-anal anastomosis is the choice surgical procedure for patients with ulcerative colitis. In cases of Crohn's disease post-operative diagnosis, it can be followed by pouch failure. AIM: To evaluate ileal pouch-anal anastomosis long-term outcome in patients with Crohn's disease. METHODS: Between February 1983 and March 2007, 151 patients were submitted to ileal pouch-anal anastomosis by Campinas State University Colorectal Unit, Campinas, SP, Brazil, 76 had pre-operative ulcerative colitis diagnosis and 11 had post-operative Crohn's disease diagnosis. Crohn's disease diagnosis was made by histopathological biopsies in nine cases, being one in surgical specimen, two cases in rectal stump, small bowel in two cases, ileal pouch in three and in perianal abscess in one of them. The median age was 30.6 years and eight (72.7%) were female. RESULTS: All patients had previous ulcerative colitis diagnosis and in five cases emergency colectomy was done by toxic megacolon. The mean time until of Crohn's disease diagnosis was 30.6 (6-80) months after ileal pouch-anal anastomosis. Ileostomy closure was possible in 10 cases except in one that had ileal pouch fistula, perianal disease and small bowel involvement. In the long-term follow-up, three patients had perineal fistulas and one had also a pouch-vaginal fistula. All of them were submitted to a new ileostomy and one had the pouch excised. Another patient presented pouch-vaginal fistula which was successfully treated by mucosal flap. Three patients had small bowel involvement and three others, pouch involvement. All improved with medical treatment. Presently, the mean follow-up is 76.5 months and all patients are in clinical remission, and four have fecal diversion. The remaining patients have good functional results with 6-10 bowel movements/day. CONCLUSION: Crohn's disease diagnosis after ileal pouch-anal anastomosis for ulcerative colitis may be usual and later complications such fistulas and stenosis are common. However, when left in situ ileal pouch is associated with good function.

Reviewed evidence about the safety of the daily use of alcohol-based mouthrinses

Current scientific knowledge provides clear evidence that alcohol-based mouthwashes can be beneficial in a daily oral health routine, including dental hygiene and plaque control. Several issues are worth discussing, in spite of the wealth of supporting evidence. Despite some undesirable effects to some people, like burning sensation, and some contraindications, like the use by infants, alcohol addicts and patients with mucosal injuries, there is no reason to avoid the use of alcohol-containing mouthwashes as long as they are used following proper guidance by dental professionals and the manufacturers' instructions. The alleged correlation between oral cancer and alcohol-based mouthrinses presents so little, weak, inconsistent and even contradictory evidence in the literature that any kind of risk warning to patients would be uncalled for. Antimicrobial mouthrinses are safe and effective in reducing plaque and gingivitis, and should be part of a comprehensive oral health care regimen that includes brushing, flossing and rinsing to prevent or minimize periodontal disease.

A novel A2 allele found in Leishmania (Leishmania) infantum chagasi

Visceral leishmaniasis (VL) is a widely spread zoonotic disease. In Brazil the disease is caused by Leishmania (Leishmania) infantum chagasi. Peridomestic sandflies acquire the etiological agent by feeding on blood of infected reservoir animals, such as dogs or wildlife. The disease is endemic in Brazil and epidemic foci have been reported in densely populated cities all over the country. Many clinical features of Leishmania infection are related to the host-parasite relationship, and many candidate virulence factors in parasites that cause VL have been studied such as A2 genes. The A2 gene was first isolated in 1994 and then in 2005 three new alleles were described in Leishmania (Leishmania) infantum. In the present study we amplified by polymerase chain reaction (PCR) and sequenced the A2 gene from the genome of a clonal population of L. (L.) infantum chagasi VL parasites. The L. (L.) infantum chagasi A2 gene was amplified, cloned, and sequenced in. The amplified fragment showed approximately 90% similarity with another A2 allele amplified in Leishmania (Leishmania) donovani and in L.(L.) infantum described in literature. However, nucleotide translation shows differences in protein amino acid sequence, which may be essential to determine the variability of A2 genes in the species of the L. (L.) donovani complex and represents an additional tool to help understanding the role this gene family may have in establishing virulence and immunity in visceral leishmaniasis. This knowledge is important for the development of more accurate diagnostic tests and effective tools for disease control.

Anti-oxidative and anti-ulcerogenic activity of Ipomoea imperati

Ipomoea imperati (Vahl) Griseb., Convolvulaceae, is used in traditional medicine for the treatment of inflammation, swelling and wounds, as well as to treat pains and stomach problems. This work evaluates the anti-oxidative activity by ESR (Electron Spin Resonance spectroscopy) and the preventive and curative actions of I. imperati in gastric ulcer animal model. Ipomoea imperati (200 mg/kg, p.o.) prevented the formation of gastric lesions in 78% (p<0.05) when compared with the negative control tween 80. Lanzoprazole, prevented in 85% the gastric lesions formation induced by ethanol (p<0.05). Therefore, the oral administration of I. imperati one hour before the ulcerogenic agent prevented the ulcer formation, conserving the citoprotection characteristics of the gastric mucosa and assuring the integrity of gastric glands and gastric fossets. The healing activity of I. imperati (200 mg/kg, p.o.) evaluated in chronic ulcer experiments induced by the acetic acid, was 72% (p<0.05). The positive control, ranitidine, healed 78% of the gastric lesions (p<0.05). The histological analysis confirmed the recovery of the mucosal layer and the muscle mucosal layer harmed by the acetic acid. Experiments in vitro with DPPH (2.2-diphenyl-1-picrylhydrazyl) of anti-oxidative activity demonstrated that I. imperati presents an IC50 of 0.73±0.01 mg/mL.

Leishmania spp. parasite isolation through inoculation of patient biopsy macerates in interferon gamma knockout mice

Isolation of Leishmania parasite and species identification are important for confirmation and to help define the epidemiology of the leishmaniasis. Mice are often used to isolate pathogens, but the most common mouse strains are resistant to infection with parasites from the Leishmania (Viannia) subgenus. In this study we tested the inoculation of interferon gamma knockout (IFNγ KO) mice with biopsy macerates from Leishmania-infected patients to increase the possibility of isolating parasites. Biopsies from twenty five patients with clinical signs of leishmaniasis were taken and tested for the presence of parasites. Immunohistochemical assay (IHC) and conventional histopathology detected the parasite in 88% and 83% of the patients, respectively. Leishmania sp. were isolated in biopsy macerates from 52% of the patients by culture in Grace's insect medium, but 13% of isolates were lost due to contamination. Inoculation of macerates in IFNγ KO mice provides isolation of parasites in 31.8% of the biopsies. Most isolates belong to L. (Viannia) subgenus, as confirmed by PCR, except one that belongs to L. (Leishmania) subgenus. Our preliminary results support the use of IFNγ KO mice to improve the possibility to isolate New World Leishmania species.

Epidemiologia da leishmaniose tegumentar americana no Estado do Acre, Amazônia brasileira

Efetuou-se estudo estatístico descritivo em 8.516 casos de leishmaniose tegumentar americana no Estado do Acre, Brasil, no período de 2001 a 2006 (segundo período), comparando-se com os resultados de publicação anterior com dados no mesmo estado entre 1992 e 1997 (primeiro período). A prevalência no estado mais que dobrou entre os dois momentos (55,7/10 mil habitantes de 1992 a 1997 e 128,5/10 mil habitantes entre 2001 e 2006. O sexo masculino foi o mais acometido (68,8%, n = 5.860) no segundo período. A média das idades foi de 26,3 anos, valor discretamente menor do que o do primeiro período. Pacientes com baixa escolaridade foram os mais acometidos pela doença. As demais variáveis avaliadas foram zona de residência, ocupação, critério de diagnóstico, forma clínica, tratamento, recidiva, tempo para procurar tratamento e evolução da doença. Conclui-se que houve piora nos indicadores epidemiológicos da leishmaniose tegumentar americana avaliados no estado entre os períodos estudados.

Epidemiologia da Leishmaniose Tegumentar e descrição das populações de flebotomíneos no município de Acrelândia, Acre, Brasil

São apresentadas neste artigo a distribuição da leishmaniose tegumentar (LT) e descrição das populações de flebotomíneos em Acrelândia, Acre. Os dados epidemiológicos foram obtidos a partir de fichas de notificação de casos ocorridos entre 2001 e 2004, e os dados entomológicos são provenientes de capturas com armadilhas luminosas efetuadas entre 2004 e 2005 na zona rural de Acrelândia. Ocorreram 82 novos casos de LT, com idade entre 2 e 69 anos, sendo 75,6% em homens e 83,9% na zona rural. Predominou a LT com lesões únicas (78%). A microscopia direta da lesão, intradermorreação de Montenegro e biópsia apresentaram positividade de 100%, 98% e 79,5%, respectivamente. A resposta ao tratamento farmacológico foi bem sucedida em 71,6% dos casos; a falência terapêutica foi maior em pacientes com diagnóstico exclusivamente clínico (41,2%) e nos que receberam dose diária inadequada de antimonial pentavalente (64,3%). Foram coletados 40 espécimes de flebotomíneos em propriedades rurais com casos de LT (3 gêneros, 14 espécies), sendo 3 espécies conhecidas como vetoras ou possíveis vetoras de Leishmania: Nyssomyia antunesi predominou no peridomicílio (59,1%) e em margens de matas; Nyssomyia whitmani foi freqüente no peridomicílio (15%) e a única espécie encontrada no intradomicílio, e Trichophoromyia ubiquitalis foi capturada no peridomicílio. O uso de dados epidemiológicos existentes no serviço de saúde de Acrelândia, embora com várias limitações, permitiu avaliar a eficácia do diagnóstico e o tratamento empregados no município, enquanto os dados entomológicos coletados podem orientar estudos mais amplos visando identificar os vetores e espécies circulantes na região.

Nyssomyia intermedia (Lutz & Neiva) and Nyssomyia neivai (Pinto) (Diptera, Psychodidae, Phlebotominae) in a sympatric area: seasonal and nocturnal hourly rhythm in black and white modified Shannon traps

Captures with black and white Shannon traps were undertaken to identify aspects of the behavior of the two cryptic and sympatric species implicated as vectors of cutaneous leishmaniasis, Nyssomyia intermedia (Lutz & Neiva, 1912) and Nyssomyia neivai (Pinto, 1926). The traps were installed side by side, monthly, from July 2001 to June 2002, from 18 to 07 hours, in a peridomicile of Iporanga municipality, state of São Paulo, Brazil. A total of 2,142 specimens were captured, Ny. intermedia (47.4%) and Ny. neivai (50.5%). The white trap was more attractive to both sexes of both species. Males of Ny. neivai predominated (70%) over those of Ny. intermedia on the two traps; on the black trap, the females of Ny. intermedia predominated (63.3%) over those of Ny. neivai (36.7%). Seventy percent of the specimens of both species were captured between 18 and 24 h. Females of Ny. intermedia presented the highest peak at 19-20 h, and those of Ny. neivai at 20-21 h. The highest hourly average for females of Ny. intermedia on the black trap occurred in the winter and that for males in the summer. For Ny. neivai, both sexes predominated in the summer. The two species probably transmit the cutaneous leishmaniasis in the area due to their great predominance.