Newborn Screening for the Detection of the TP53 R337H Variant and Surveillance for Early Diagnosis of Pediatric Adrenocortical Tumors: Lessons Learned and Way Forward.

The incidence of pediatric adrenocortical tumors (ACT) is high in southern Brazil due to the founder TP53 R337H variant. Neonatal screening/surveillance (NSS) for this variant resulted in early ACT detection and improved outcomes. The medical records of children with ACT who did not participate in newborn screening (non-NSS) were reviewed (2012-2018). We compared known prognostic factors between the NSS and non-NSS cohorts and estimated surveillance and treatment costs. Of the 16 non-NSS children with ACT carrying the R337H variant, the disease stages I, II, III, and IV were observed in five, five, one, and five children, respectively. The tumor weight ranged from 22 to 608 g. The 11 NSS children with ACT all had disease stage I and were alive. The median tumor weight, age of diagnosis, and interval between symptoms and diagnosis were 21 g, 1.9 years, and two weeks, respectively, for the NSS cohort and 210 g, 5.2 years, and 15 weeks, respectively, for the non-NSS cohort. The estimated surveillance/screening cost per year of life saved is US$623/patient. NSS is critical for improving the outcome of pediatric ACT in this region. Hence, we strongly advocate for the inclusion of R337H in the state-mandated universal screening and surveillance.

Penetrance of the TP53 R337H Mutation and Pediatric Adrenocortical Carcinoma Incidence Associated with Environmental Influences in a 12-Year Observational Cohort in Southern Brazil.

The TP53 R337H mutation is associated with increased incidence of pediatric adrenocortical tumor (ACT). The different environmental conditions where R337H carriers live have not been systematically analyzed. Here, the R337H frequencies, ACT incidences, and R337H penetrance for ACT were calculated using the 2006 cohort with 4165 R337H carriers living in Paraná state (PR) subregions. The effectiveness of a second surveillance for R337H probands selected from 42,438 tested newborns in PR (2016 cohort) was tested to detect early stage I tumor among educated families without periodical exams. Estimation of R337H frequencies and ACT incidence in Santa Catarina state (SC) used data from 50,115 tested newborns without surveillance, ACT cases from a SC hospital, and a public cancer registry. R337H carrier frequencies in the population were 0.245% (SC) and 0.306% (PR), and 87% and 95% in ACTs, respectively. The ACT incidence was calculated as ~6.4/million children younger than 10 years per year in PR (95% CI: 5.28; 7.65) and 4.15/million in SC (CI 95%: 2.95; 5.67). The ACT penetrance in PR for probands followed from birth to 12 years was 3.9%. R337H carriers living in an agricultural subregion (C1) had a lower risk of developing pediatric ACT than those living in industrial and large urban subregion (relative risk = 2.4). One small ACT (21g) without recurrence (1/112) was detected by the parents in the 2016 cohort. ACT incidence follows R337H frequency in each population, but remarkably environmental factors modify these rates.

The Prognostic Role of CD8+ T Lymphocytes in Childhood Adrenocortical Carcinomas Compared to Ki-67, PD-1, PD-L1, and the Weiss Score.

Adrenocortical carcinoma (ACC) is a rare disease among children. Our goal was to identify prognostic biomarkers in 48 primary ACCs from children (2.83 ± 2.3 y; mean age ± SD) by evaluating the tumor stage and outcome for an age of diagnosis before or after 3 years, and association with ACC cluster of differentiation 8 positive (CD8+) cytotoxic T lymphocytes (CD8+-CTL) and Ki-67 immunohistochemical expression (IHC). Programmed death 1(PD-1)/Programmed death-ligand 1 (PD-L1) immunohistochemistry (IHC) in ACC was analyzed in a second, partially overlapping cohort (N = 19) with a similar mean age. All patients and control children were carriers of the germline TP53 R337H mutation. Survival without recurrence for less than 3 years and death unrelated to disease were excluded. Higher counts of CD8+-CTL were associated with patients diagnosed with ACC at a younger age and stage I, whereas a higher percentage of the Ki-67 labeling index (LI) and Weiss scores did not differentiate disease free survival (DFS) in children younger than 3 years old. No PD-1 staining was observed, whereas weakly PD-L1-positive immune cells were found in 4/19 (21%) of the ACC samples studied. A high CD8+-CTL count in ACC of surviving children is compelling evidence of an immune response against the disease. A better understanding of the options for enhancement of targets for CD8+ T cell recognition may provide insights for future pre-clinical studies.

Baccharis dracunculifolia (Asteraceae) essential oil toxicity to Culex quinquefasciatus (Culicidae).

The control of mosquitoes by means of chemical insecticides has been a problem, mainly due to the possibility of resistance developed by insects to xenobiotics. For this reason, demand for botanical insecticides has increased. In this sense, the present work aims to verify the susceptibility and morphological and biochemical alterations of Culex quinquefasciatus larvae after exposure to essential oil (EO) of leaves of Baccharis dracunculifolia. To observe the larvicidal action, larvae were exposed to EO at concentrations of 25, 50, 100, and 200 mg/L, until their emergence to adults. The control group was exposed to deionized water and dimethyl sulfoxide. Morphological analyses were also carried out using hematoxylin and eosin, mercury bromophenol blue, Nile blue, and periodic acid Schiff. Biochemical analyses of total glucose, triacylglyceride (TAG), protein, and acetylcholinesterase levels were performed. The phytochemical analysis of the EO showed (E)-nerolidol as the major compound (30.62%). Larvae susceptibility results showed a LC50 of 34.45 mg/L for EO. Morphological analysis showed that there were histological changes in midgut. For biochemical analyses, the glucose level in the larvae exposed to EO for 24 h decreased significantly, unlike the TAG levels, which increased. The total protein level of the larvae also increased after exposure for 24 h, and acetylcholinesterase levels decreased significantly. Taking all our data into account, we can conclude that EO causes destabilization in larva, leading to histological changes, metabolic deregulation and, consequently, their death.

The role of tannins as antiulcer agents: a fluorescence-imaging based study

Abstract Condensed tannins have been used for many years in folk medicine to treat gastric problems. The mechanism of action that explains why tannins improve gastritis symptoms is based on their ability to chelate metals, antioxidant activity, and their complexation power with other molecules. Even though these uses are well-known, the requirements to become an herbal medicine are much more complex. Herein, we analyzed Stryphnodendron rotundifolium Mart., Fabaceae, extract using MALDI for tannin characterization and carried out a fluorescence-imaging study to prove the gastroprotective effects of tannins as coating agents. Through these methods we show that condensed tannins form a gastroprotective layer. Moreover, we revise and discuss other possible mechanisms of action for phenolic-rich plant extracts and their potential in the development of herbal medicines to treat ulcers and gastritis.

Artepillin C Induces Selective Oxidative Stress and Inhibits Migration and Invasion in a Comprehensive Panel of Human Cervical Cancer Cell Lines.

BACKGROUND: Artepillin C (3,5-diprenyl-4-hydroxycinnamic acid) is the main bioactive component of Brazilian green propolis, and possesses, among other things, anticancer properties. However, to the best of our knowledge, there are no studies of artepillin C in cervical cancer. METHOD: To explore a new therapeutic candidate for cervical cancer, we have evaluated the effects of artepillin C on cellular viability in a comprehensive panel of human cervical cancer-derived cell lines including HeLa (human papillomavirus/HPV 18-positive), SiHa (HPV 16-positive), CaSki (HPV 16- and 18-positive) and C33A (HPV-negative) cells compared to a spontaneously immortalized human epithelial cell line (HaCaT). RESULTS: Our results demonstrated that artepillin C had a selective effect on cellular viability and could induce apoptosis possibly by intrinsic pathway, likely a result of oxidative stress, in all cancer-derived cell lines but not in HaCaT. Additionally, artepillin C was able to inhibit the migration and invasion of cancer cells. CONCLUSION: Thus, artepillin C appears to be a promising new candidate as an anticancer drug for cervical cancer induced by different HPV types.

Leaf and Root Extracts from Campomanesia adamantium (Myrtaceae) Promote Apoptotic Death of Leukemic Cells via Activation of Intracellular Calcium and Caspase-3.

Phytochemical studies are seeking new alternatives to prevent or treat cancer, including different types of leukemias. Campomanesia adamantium, commonly known as guavira or guabiroba, exhibits pharmacological properties including antioxidant, antimicrobial, and antiproliferative activities. Considering the anticancer potential of this plant species, the aim of this study was to evaluate the antileukemic activity and the chemical composition of aqueous extracts from the leaves (AECL) and roots (AECR) of C. adamantium and their possible mechanisms of action. The extracts were analyzed by LC-DAD-MS, and their constituents were identified based on the UV, MS, and MS/MS data. The AECL and AECR showed different chemical compositions, which were identified as main compounds glycosylated flavonols from AECL and ellagic acid and their derivatives from AECR. The cytotoxicity promoted by these extracts were evaluated using human peripheral blood mononuclear cells and Jurkat leukemic cell line. The cell death profile was evaluated using annexin-V-FITC and propidium iodide labeling. Changes in the mitochondrial membrane potential, the activity of caspases, and intracellular calcium levels were assessed. The cell cycle profile was evaluated using propidium iodide. Both extracts caused concentration-dependent cytotoxicity only in Jurkat cells via late apoptosis. This activity was associated with loss of the mitochondrial membrane potential, activation of caspases-9 and -3, changes in intracellular calcium levels, and cell cycle arrest in S-phase. Therefore, the antileukemic activity of the AECL and AECR is mediated by mitochondrial dysfunction and intracellular messengers, which activate the intrinsic apoptotic pathway. Hence, aqueous extracts of the leaves and roots of C. adamantium show therapeutic potential for use in the prevention and treatment of diseases associated the proliferation of tumor cell.

Antibacterial activity of Limonium brasiliense (Baicuru) against multidrug-resistant bacteria using a statistical mixture design.

ETHNOPHARMACOLOGICAL RELEVANCE: Limonium brasiliense (Boiss.) Kuntze (Plumbaginaceae) is commonly known as "baicuru" or "guaicuru" and preparations of its dried rhizomes have been popularly used in the treatment of premenstrual syndrome and menstrual disorder, and as an antiseptic in genito-urinary infections. This study evaluated the potential antibacterial activity of rhizome extracts against multidrug-resistant bacterial strains using statistical mixture design. MATERIALS AND METHODS: The statistical design of four components (water, methanol, acetone, and ethanol) produced 15 different extracts and also a confirmatory experiment, which was performed using water:acetone (3:7, v/v). The crude extracts and their ethyl-acetate fractions were tested against vancomycin-resistant Enterococcus faecium (VREfm), methicillin-resistant Staphylococcus aureus (MRSA) and Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae, all of which have been implicated in hospital and community-acquired infections. The dry residue, total polyphenol, gallocatechin and epigallocatechin contents of the extracts were also tested and statistical analysis was applied in order to define the fit models to predict the result of each parameter for any mixture of components. The principal component and hierarchical clustering analyses (PCA and HCA) of chromatographic data, as well as mass spectrometry (MS) analysis were performanced to determine the main compounds present in the extracts. RESULTS: The Gram-positive bacteria were susceptible to inhibition of bacterial growth, in special the ethyl-acetate fraction of ternary extracts from water:acetone:ethanol and methanol:acetone:ethanol against, respectively, VREfm (MIC=19µg/mL) and MRSA (MIC=39µg/mL). On the other hand, moderate activity of the ethyl-acetate fractions from primary (except water), secondary and ternary extracts (MIC=625µg/mL) was noted against KPC. The quadratic and special cubic models were significant for polyphenols and gallocatechin contents, respectively. Fit models to dry residue and epigallocatechin contents were not possible. PCA and HCA of the chromatographic fingerprints were disturbed by displacement retention time of some peaks, but the ultraviolet spectra indicated the homogeneous presence of flavan-3-ols characteristic of tannins. The MS confirmed the presence of gallic acid, gallocatechin, and epigallocatechin in extracts, and suggested the presence of monomers and dimers of B- and A-type prodelphinidins gallate, as well as a methyl gallate. CONCLUSION: Our results showed the antibacterial potential of L. brasiliense extracts against multidrug-resistant Gram-positive bacteria, such as VREfm and MRSA. The statistical design was a important tool to evaluate the biological activity by optimized form. The presence of some phenolic compounds was also demonstrated in extracts.

Natural Green coating inhibits adhesion of clinically important bacteria.

Despite many advances, biomaterial-associated infections continue to be a major clinical problem. In order to minimize bacterial adhesion, material surface modifications are currently being investigated and natural products possess large potential for the design of innovative surface coatings. We report the bioguided phytochemical investigation of Pityrocarpa moniliformis and the characterization of tannins by mass spectrometry. It was demonstrated that B-type linked proanthocyanidins-coated surfaces, here termed Green coatings, reduced Gram-positive bacterial adhesion and supported mammalian cell spreading. The proposed mechanism of bacterial attachment inhibition is based on electrostatic repulsion, high hydrophilicity and the steric hindrance provided by the coating that blocks bacterium-substratum interactions. This work shows the applicability of a prototype Green-coated surface that aims to promote necessary mammalian tissue compatibility, while reducing bacterial colonization.

Leishmanicidal evaluation of tetrahydroprotoberberine and spirocyclic erythrina-alkaloids.

Leishmaniasis is one of the World's most problematic diseases in developing countries. Traditional medicines to treat leishmaniasis have serious side effects, as well as significant parasite resistance problems. In this work, two alkaloids 1 and 2 were obtained from Corydalis govaniana Wall and seven alkaloids 3-9, were obtained from Erythrina verna. The structures of the compounds were confirmed by mass spectrometry and 1D- and 2D-NMR spectroscopy. The leishmanicidal activity of compounds 1-9 against Leishmania amazonensis was tested on promastigote forms and cytotoxicity against J774 (macrophage cell line) was assessed in vitro. Compound 1 showed potent activity (IC50 = 0.18 µg/mL), compared with the standard amphotericin B (IC50 = 0.20 µg/mL). The spirocyclic erythrina-alkaloids showed lower leishmanicidal activity than dibenzoquinolizine type alkaloids.

Acute lymphoblastic leukemia in infants: 20 years of experience.

OBJECTIVE: To analyze patients younger than 2 years with acute lymphoblastic leukemia, treated in the period between 1990 and 2010 in a state reference center. METHODS: This was a clinical-epidemiological, cross-sectional, observational, and descriptive study. It included patients younger than 2 years with acute lymphoblastic leukemia, treated in the period of 1990 to 2010 in a pediatric oncology unit of a state reference center, totaling 41 cases. RESULTS: All patients were white ethnicity, and 60.9% were females. Regarding age, 24.38% were younger than 6 months, 17.07% were between 6 months and 1 year, and 58.53% were older than 1 year. The age of 6 months was statistically significant for the outcome of death. Predominant signs and symptoms were fever, bruising, and petechiae. A leukocyte count > 100,000 was found in 34.14% of cases, hemoglobin count < 11 in 95.13%, and platelet count < 100,000 in 75.61. Infiltration of central nervous system was present in 12.91% of patients. According to the lineage, B-cell lineage predominated (73%), but the T-cell line was statistically significant for death. 39% of patients had disease recurrence. In relation to vital status, 70.73% of the patients died; septic shock was the main cause. CONCLUSIONS: Acute lymphoblastic leukemia in infants has a high mortality rate, especially in children under 1 year and those with T-cell derived lineage.

Leucemia linfoblástica aguda em lactentes: 20 anos de experiência / Acute lymphoblastic leukemia in infants: 20 years of experience

OBJETIVO: Analisar pacientes com menos de dois anos de idade com leucemia linfoblástica aguda atendidos no período de 1990 a 2010, em um centro de referência estadual. MÉTODOS: Estudo clínico, epidemiológico, transversal, descritivo e observacional. Pacientes incluídos tinham menos de dois anos de idade, com leucemia linfoblástica aguda, tratados no período de 1990 a 2010 na unidade de oncologia pediátrica de um centro de referência estadual, totalizando 41 casos. RESULTADOS: Todos os pacientes eram Caucasianos e 60,9% eram do sexo feminino. Com relação à idade, 24,38% tinham menos de seis meses, 17,07% tinham entre seis meses e um ano e 58,53% mais do que um ano de idade. A idade de seis meses foi estatisticamente significante para o desfecho de óbito. Os sinais e sintomas predominantes foram febre, hematomas e petéquias. Uma contagem de leucócitos superior a 100.000 foi observada em 34,14% dos casos; hemoglobina inferior a 11 em 95,13% e contagem de plaquetas inferior a 100.000, em 75,61% dos casos. Infiltração do sistema nervoso central estava presente em 12,91% dos pacientes. Em relação à linhagem, a linhagem B predominou (73%), mas a linhagem de células T foi estatisticamente significativa para o óbito. Trinta e nove por cento dos pacientes tiveram recorrência da doença. Em relação ao estado vital, 70,73% dos pacientes morreram, sendo choque séptico a principal causa. CONCLUSÕES: leucemia linfoblástica aguda em crianças tem uma alta taxa de mortalidade, principalmente em crianças menores de um ano e linhagem derivada de células T.

OBJECTIVE: To analyze patients younger than 2 years with acute lymphoblastic leukemia, treated in the period between 1990 and 2010 in a state reference center. METHODS: This was a clinical-epidemiological, cross-sectional, observational, and descriptive study. It included patients younger than 2 years with acute lymphoblastic leukemia, treated in the period of 1990 to 2010 in a pediatric oncology unit of a state reference center, totaling 41 cases. RESULTS: All patients were white ethnicity, and 60.9% were females. Regarding age, 24.38% were younger than 6 months, 17.07% were between 6 months and 1 year, and 58.53% were older than 1 year. The age of 6 months was statistically significant for the outcome of death. Predominant signs and symptoms were fever, bruising, and petechiae. A leukocyte count > 100,000 was found in 34.14% of cases, hemoglobin count < 11 in 95.13%, and platelet count < 100,000 in 75.61. Infiltration of central nervous system was present in 12.91% of patients. According to the lineage, B-cell lineage predominated (73%), but the T-cell line was statistically significant for death. 39% of patients had disease recurrence. In relation to vital status, 70.73% of the patients died; septic shock was the main cause. CONCLUSIONS: Acute lymphoblastic leukemia in infants has a high mortality rate, especially in children under 1 year and those with T-cell derived lineage.

Variação química no óleo essencial das folhas de seis indivíduos de Duguetia furfuracea (Annonaceae) / Chemical variability of the essential oil of the leaves from six individuals of Duguetia furfuracea (Annonaceae)

Duguetia furfuracea, Annonaceae, uma invasora de pastagens, é também usada na medicina tradicional em diversos estados no Brasil. Devido à significante diferença de odor entre dois grupos de indivíduos desta planta em um remanescente de cerrado em Campo Grande, Mato Grosso do Sul, seis amostras foram selecionadas de acordo com a intensidade de odor em suas folhas: In-01, In-03 e In-05: odor pronunciado (Perfil A); In-02, In-04 e In-06: odor fraco ou ausência de odor (Perfil B). As folhas foram coletadas e submetidas a hidrodestilação para obtenção dos óleos essenciais, os quais foram analisados por CG/EM. Uma boa compatibilidade foi encontrada entre os resultados obtidos por CG/EM e a análise olfativa adotada para as amostras: espécimes odor pronunciado apresentaram uma alta porcentagem de monoterpenos e alguns sesquiterpenos: In-01 (β-felandreno, 42,2 por cento; mirceno, 6,8 por cento; α-felandreno, 4,6 por cento); In-03 (terpin-4-ol, 21,6 por cento; sabineno, 17,3 por cento; p-cimeno, 5,6 por cento); In-05 (sabineno, 25,1 por cento; terpin-4-ol, 16,2 por cento; p-cimeno, 8,3 por cento). Apenas sesquiterpenos foram encontrados nos espécimes que apresentaram odor fraco ou ausência do mesmo (com constituinte majoritário biciclogermacreno: 21,4 por cento, 24,0 por cento, and 29,1 por cento, respectivamente, para os In-02, In-04 e In-06).

Duguetia furfuracea, Annonaceae, a pasture weed, is also used in folk medicine in several Brazilian states. Because of the significant difference in odor between two groups of this plant in a remnant patch of savanna in Campo Grande county, Mato Grosso do Sul, six samples were selected according to the odor intensity in the leaves (In-01, In-03, and In-05: pronounced odor; In-02, In-04, and In-06: weak odor or its absence). The leaves were collected and subjected to steam distillation for extraction of essential oils, which were analyzed by GC-MS. A good agreement was found between CG/MS results and olfactory evaluation of the samples: specimens exhibiting leaf scent had a high percentage of monoterpenes and some sesquiterpenes, as follows: In-01 (β-phellandrene, 42.2 percent; myrcene, 6.8 percent; α-phellandrene, 4.6 percent); In-03 (terpin-4-ol, 21.6 percent; sabinene, 17.3 percent; p-cymene, 5.6 percent); In-05 (sabinene, 25.1 percent; terpin-4-ol, 16.2 percent; p-cymene, 8.3 percent). Only sesquiterpenes were found in the specimens having weakly scented or scentless leaves (main constituent bicyclogermacrene: 21.4 percent, 24.0 percent, and 29.1 percent, respectively, for In-02, In-04 and In-06).

Divisao em risco da leucemmia linfocitica aguda da infancia: criterios para classificacao conforme risco, seu valor pratico e prognostico / Division in risk of the acute lymphocytic leukemia in childhood: criteria for classification according to the risk, its practice value and prognosis

Este trabalho procura mostrar como o protocolo brasileiro para o tratamento da leucemia linfocitica aguda na infancia adota criterios para a classificacao da LLA, mostrando seu valor pratico, modo de tratamento e prognostico.