PREVALENCE AND PREDICTORS OF VITAMIN A DEFICIENCY AMONG INFANTS IN WESTERN KENYA USING A CROSS-SECTIONAL ANALYSIS

Vitamin A (VA) deficiency (VAD) is a major nutritional public health problem among children under-5-years-old in the developing world including Kenya. A community-based cross-sectional survey among 1,630 children (aged 6-23 mos) was undertaken in Western Kenya. A questionnaire was administered to collect demographic, socio-economic and dietary intake information. Prevalence of low retinol-binding protein (RBP) concentrations was assessed using Dried Blood Spot (DBS) methodology. Analysis of RBP was carried out using rapid enzyme immunoassay (EIA) and C-reactive protein (CRP) was carried out using enzyme linked immunosorbent assay (ELISA) to estimate VA and sub-clinical inflammation statuses, respectively. Values were adjusted for influence of inflammation using CRP (CRP >5 mg/L) and population prevalence of VAD (RBP <0.825 μmol/L, biologically equivalent to 0.70 μmol/L retinol) estimated. Anthropometric data gave three indices: stunting, wasting and underweight—all of which took age and sex into consideration. Mean (geometric± SD) concentration of RBP was adequate (1.56±0.79μmol/L) but the inflammation-adjusted mean (±SE) prevalence of VAD was high (20.1±1.1%) in this population. The level of CRP was within normal range (1.06±4.95 mg/L) whilst 18.4±0.9% of the children had subclinical inflammation (CRP>5 mg/L). Intake of VA capsule (VAC) by a child was a predictor of VAD with children who have not taken VA during the past 1 year prior to the survey having a 30% increased risk of VAD (OR (CI): 1.3 (1.1-1.7); p=0.025. Additionally, age of the child was a predictor with older children (18-23 mos) having a 30 % increased risk of VAD (OR (CI): 1.3 (1.1-1.9); p=0.035); the caretaker’s knowledge on VA and nutrition was also a predictor of VAD with children whose caretaker’s had poor knowledge having a 40 % increased risk of VAD (OR (CI): 1.4 (1.0-1.9); p=0.027. A child’s district of residence was also a significant predictor of VAD. Prevalence of VAD in this sample of infants was high. Predictors of VAD included child intake of VAC in the last 1 year before the survey, older children, children whose caretakers had poor VA and nutritional knowledge and a child’s district of residence. There is a need to improve knowledge on nutrition and VA of caretakers; undertake a targeted VAC distribution, particularly in children older than 1 year and above and use a sustainable food-based intervention in the areas with severe VAD.

Efeitos da terapia periodontal na contagem de leucócitos e níveis do fator de crescimento transformador beta no soro de indivíduos com periodontite severa

Tese (doutorado)—Universidade de Brasília, Faculdade de Ciências da Saúde, Programa de Pós-Graduação em Ciências da Saúde, 2015.

Original Research Article Humoral and cellular immune responses to modified hepatitis B plasmid DNA vaccine in mice

Purpose: To evaluate the immunogenicity and types of immune response of a quality-controlled modified recombinant hepatitis B surface antigen (HBsAg) plasmid encoding HBsAg in mice. Methods: The characterized plasmid DNA was used in the immunization of Balb/c mice. Three groups of mice were intramuscularly injected with three different concentrations (50, 25 and 10 μg/100 μL) of the modified plasmid. Humoral immune response was monitored by enzyme-linked immunosorbent assay (ELISA), while cellular immune response was investigated by analysis of spleen cytokine profile (TNFα, IFN γ and IL2) as well as CD69 expression level in CD4 and CD8 positive cells. Results: In general, the activated CD4 cells showing intracellular cytokines were higher than CD8 positive population of cells (p < 0.05). These findings indicate that the vaccine induced both a humoral and cellular immunity. Cytokine profile also showed high levels of TNFα, IFN γ and IL2 and CD69 expression in the group of animals immunized at a dose of 10 μg when compared to control group (p < 0.05). Conclusion: A 10 μg dose intramuscular injection of the modified DNA-based vaccine encoding HBsAg in mice induces both high humoral and cellular immune responses.

Effect of Lycii fructus polysaccharides on ovulation failure in rats

Purpose: To investigate the effect of Lycii fructus polysaccharides (LFPS) on ovulation failure. Methods: A rat model of ovulation failure was established by intragastric administration of hydroxyurea (300 mg/kg). Rats with ovulation failure then received LFPS via oral administration at doses of 100, 200, or 400 mg/kg. The body, uterus and ovary of each rat were weighed using electronic scales. The hypothalamic-pituitary-ovarian (HPO) axis hormones, including estradiol (E2) level, follicle-stimulating hormone (FSH) activity, and luteinizing hormone (LH) activity in the serum of each rat were determined by enzyme-linked immunosorbent assay (ELISA). The levels of pro-apoptotic proteins (Fas, FasL, FADD, c-caspase-8, c-caspase-10, c-caspase-3, c-caspase-6, and c-caspase-7) in the ovarian tissue of each rat were detected by western blot. Results: Hydroxyurea reduced significantly (p < 0.01) uterus and ovary indices (uterus or ovary weight/body weight) (0.119 and 0.026 %), E2 level (3.42 pmol/L), and FSH and LH activities (2.28 and 2.76 U/L), compared with those in the normal group (0.169 and 0.039 %; 6.72 pmol/L; 2.76 and 3.75 U/L). Hydroxyurea increased significantly (p < 0.01) the levels of the above-mentioned pro-apoptotic proteins relative to those in the normal group. LFPS (100, 200, or 400 mg/kg) reversed significantly (p < 0.05 or 0.01) the effect of hydroxyurea on all of the above indices. Conclusion: LFPS exhibits a protective effect on hydroxyurea-induced ovulation failure by regulating the HPO axis hormones and death receptor-mediated apoptotic pathway.

Effects of coagulation factors and inflammatory cytokines on development of acute myocardial infarction in patients younger than 60 years

Purpose: To investigate the effects of coagulation factors and inflammatory cytokines on acute myocardial infarction (AMI) development in patients younger than 60 years. Methods: In this study, 60 patients admitted to The First Affiliated Hospital of Dalian Medical University (Dalian, China) with AMI and 30 other subjects matched with the patients for age and ethnicity but without AMI were enrolled. Blood samples were collected from the AMI patients and the control subjects after a 12-h fast. Subsequently, the levels of coagulation factors (F) II (FII), VII (FVII), VIII (FVIII), fibrinogen (Fg) and von Willebrand factor (vWF) in plasma were analyzed by enzyme-linked immunosorbent assay (ELISA). The protein expression levels of these coagulation factors were determined by Western blot analysis. Inflammatory factors including C-reactive protein (CRP), tumor necrosis factor-α (TNF-α) and interleukin- 6 (IL-6) were also measured by ELISA. Results: FII, FVII, FVIII, Fg and vWF levels in plasm were significantly higher in AMI patients compared with control subjects (p < 0.01). Furthermore, the protein expression levels of FII, FVII, FVIII, Fg and vWF were also significantly up-regulated in AMI patients compared with those in control subjects. Additionally, no significant difference was observed in CRP between AMI patients and control subjects (p > 0.05). However, TNF-α and IL-6 levels in the plasma of AMI patients were significantly higher than those in control subjects (p < 0.05). Conclusion: The results reveal that the pathogenesis of AMI in patients younger than 60 years might be closely related to the high levels of coagulation factors and inflammatory cytokines in the blood.

Cardioprotective effects of Dan-Yang-Fu-Xin decoction on chronic heart failure in rats

Purpose: To evaluate the cardioprotective effects and possible mechanisms of Dan-Yang-Fu-Xin decoction (DYFX) in a rat chronic heart failure (CHF). Methods: A CHF rat model induced by ligation of the left anterior descending coronary artery was used to investigate the cardioprotective effects of DYFX. After intragastric administration for 8 weeks, several functional cardiac indices, including fractional shortening (FS), ejection fraction (EF), heart rate (HR) and cardiac output (CO) were assessed by ultrasound examination. Subsequently, inflammatory markers, viz, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), myocardial enzymes, namely, lactate dehydrogenase (LDH) and creatine kinase (CK), were also assessed by enzyme-linked immunosorbent assay (ELISA). Results: Intragastric administration of DYFX (200, 400 and 600 mg/kg) significantly reversed the decrease in body weight and increase in cardiac weight (p < 0.05) induced by CHF. Treatment with DYFX also significantly reversed EF, FS, HR, and CO changes in CHF rats. In addition, DYFX inhibited the two inflammatory cytokines (TNF-α and IL-6) and myocardial enzymes (CK and LDH), suggesting that these effects may include the mechanisms of cardioprotectiion involved in attenuation of CHF. Conclusion: DYFX possesses cardioprotective effects involving CHF. The protective mechanisms may include the suppression of expression of inflammatory mediators and myocardial enzymes.

Ameliorative effect and potential mechanism of Ermiao san on adjuvant-induced arthritis in rats

Purpose: To investigate the effect and mechanism of action of Ermiao san (EMS), a traditional Chinese herbal formula, on inflammation development and production of inflammatory mediators in adjuvantinduced arthritis (AIA). Methods: AIA was induced by injection of 0.1 ml Freund’s complete adjuvant (FCA, 10 mg/ml) in the left hind footpad of the rats. AIA rats were intragastricly treated with 0.5, 1, 2 g/kg EMS or 0.1 g/kg methotrexate from day 7 to 28 after FCA challenge. Foot volume and histological score were measured. Osteoclast number was calculated by tartrate-resistant acid phosphatase (TRAP) staining assay. Levels of prostaglandin (PG) E2, tumor necrosis factor (TNF) -α and interleukin (IL)-1β in serum were determined by enzyme-linked immunosorbent assay (ELISA) while the level of nitric oxide (NO) in serum was analyzed by Griess reaction method. Results: Foot volume, histological score, osteoclast number and serum levels of TNF-α, IL-1β, PGE2 and NO were all increased in AIA group rats on day 28 after FCA challenge (all p < 0.01) compared with control. EMS (1 and 2 g/kg) significantly decreased the foot volume of AIA rats by 10 % (p < 0.05) and 19 % (p < 0.01), respectively, compared with AIA group. Furthermore, 1 and 2 g/kg EMS significantly reduced histological score by about 28 % (p < 0.05) and 46 % (p < 0.01), respectively, as well as osteoclast number by 12 % (p < 0.05) and 15 % (p < 0.05), respectively, compared with AIA group. In addition, 1 and 2 g/kg EMS significantly decreased the serum levels of TNF-α about 23 % (p < 0.05) and 43 % (p < 0.01), IL-1β by15 % (p < 0.05) and 26 % (p < 0.01), NO 13 % (p < 0.05) and 26 % (p < 0.01) as well as PGE2 by 11 % (p < 0.05) and 15 % (p < 0.01), respectively, compared with AIA group. Conclusion: These results suggest that EMS probably alleviates arthritis development and joint destruction by decreasing the production of inflammatory mediators in AIA rats.

In vitro antioxidative activity of moss extract, and effect of moss on serum lipid level of mice fed with high-fat diet

Purpose: To evaluate the potential of active compounds derived from moss in the prevention and treatment of various diseases. Methods: Three species of moss were extracted with deionized water at 95 °C, and with 70.5 % ethanol at 85 °C. Analysis of total phenolic contents (TPC) of the extracts were performed by FolinCiocalteu (FC) method. The antioxidant activity of the extracts were determined using three methods, namely, by 2,2\'-azino-bis(3-ethylbenzothiazoline-6-sulphonic) acid (ABTS), 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP). In vivo effects were evaluated in mice fed high fat diet (HFD) supplemented with 20 % ground moss. Cholesterol levels in HFD were evaluated by ophthalaldehyde method. Serum triglyceride levels were measured using triglyceride (TG) kit, while blood insulin level and leptin concentration were measured by enzyme-linked immunosorbent assay (ELISA) kit. Results: The moss extracts exhibited antioxidative effects, as evidenced of . TPC of 47.20 ± 11.20 to 119.87 ± 11.51 mg GAE/mg, respectively. ABTS scavenging activity was 1078.11 ± 18.95 to 2587.33 ± 46.19 μmol Trolox/mg, DPPH scavenging activity of were 42.11 ± 8.22 to 298.78 ± 20.02 μmol Trolox/mg, and FRAP value of 393.19 ± 24.64 to 1070.14 ± 17.92 μmol Trolox/mg, respectively. Mice fed with 20 % ground moss did not show any significant effect (p < 0.05) on visceral weight and blood lipid levels of HFD, while leptin concentrations reduced significantly to 4.74 ± 0.00 and 0.20 ± 0.00 ng/dL) relative to HFD alone (26.72 ± 6.53 ng/dL). Conclusion: Moss can potentially be used as an antioxidative ingredient, for the improvement of overall human health, suggesting that important medical benefits associated with moss consumption. However, further investigations are required to ascertain this.

Antiviral activity and mechanism of action of arbidol against Hantaan virus infection

Purpose: To investigate the activity and mechanism of action of arbidol against Hantaan virus (HTNV) activity by modulating inflammation via TLR-4 pathway. Methods: HUVEC cells infected with HTNV 76-118 were treated with serially diluted arbidol solutions at -2h (2 h before viral infection, pre-treatment mode), 0 h (at the same time as viral infection, simultaneous treatment mode) or 2 h (2 h after viral infection, post-treatment mode). The transcript levels of TLR4 were detected by semi-quantitative reverse transcription-PCR (RT-PCR) at 6, 12, 18, and 24 h later. The levels of iNOS and TNF-α were examined using enzyme-linked immunosorbent assay (ELISA). Results: Pre-treatment with arbidol, rather than simultaneous treatment or post-treatment, effectively inhibited up-regulation of cellular TLR4 expression (up to 40 ± 6.1 % inhibition) and activity of supernatant iNOS induced by HTNV infection(up to 44.1 ± 9.4 % inhibition), as well as in a LPSstimulated inflammatory endothelial cell. Arbidol decreased the elevated TNF-α levels induced by LPS stimulation. Conclusion: These results are the first evidence that arbidol modulates viral PRRs signaling and its consequential inflammatory cytokine/chemokine response during hantavirus infection.

Protective effect of rhamnopyranosyl vanilloyl isolated from Scrophularia ningpoensis Hemsl (Scrophulariaceae) root against acute liver injury in mice

Purpose: To investigate the protective effect of rhamnopyranosyl vanilloyl (RV) from Scrophularia ningpoensis root against tetrachloromethane (CCl4)-induced acute liver injury (ALI) in mice. Methods: RV was isolated from S. ningpoensis by column chromatography. ALI model of mice was established by intraperitoneal injection of CCl4. Liver index, liver function indices, as well as serum alanine transaminase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBIL) were evaluated. Lipid peroxidation (LPO)-related indices, including malonaldehyde (MDA), glutathione (GSH), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Apoptotic proteins (Bcl-2, Bax and caspase-3) in liver tissue were determined by enzyme-linked immunosorbent assay (ELISA) and Western blot. Results: After treatment with RV (10, 20 or 40 mg/kg), liver index (5.65 - 5.21 vs. 6.68 %), ALT (90.18 - 79.68 vs. 112.47 U/L), AST (64.44 - 57.63 vs. 75.41 U/L) and TBIL (2.68 - 1.95 vs. 3.21 U/L) activities, as well as MDA (3.58 - 2.88 vs. 4.13 μmol/g), Bax and caspase-3 levels significantly (p < 0.05 or 0.01) decreased, compared with those in control group. After treatment with RV (10, 20 or 40 mg/kg), GSH (16.58 - 22.14 vs. 12.34 μmol/g), Bcl-2, SOD (86.45 - 107.61 vs. 68.43 U/mg) and GSH-Px (295.64 - 329.47 vs. 268.49 U/mg) levels or activities significantly (p < 0.05 or 0.01) increased, compared with those in control group. Conclusion: RV has protective effect against CCl4-induced ALI in mice, and the mechanisms involve the inhibition of LPO and apoptosis in liver cells. Thus, RV is a potential drug for the treatment of liver injury

Genotype-phenotype correlation in trisomy 21

Down syndrome (DS) or trisomy 21 (T21) is the most common genetic cause of intellectual disability (ID). Subjects with DS are characterized by complex and variable clinical features including intellectual disability (ID) and craniofacial dysmorphisms. The aim of the thesis is to uncover genotype-phenotype relationships in DS possibly useful to devise therapies based on molecular and cellular mechanisms. In this work, we have investigated different aspects of DS: - we have collected clinical data of children with DS and we have evaluated the cognitive impairment through specific cognitive tests - we have analysed genomics of DS through the study of partial trisomy (PT21) cases. We have described new PT21 cases confirming the hypothesis of the highly restricted DS critical region (HR-DSCR) recently identified as the minimal region whose duplication is shared by all PT21 subjects diagnosed with DS, while it is absent in all PT21 non-DS subjects. Moreover, we have characterized new transcripts included in the HR-DSCR; - we have studied gene expression through RNAseq in blood cells of children with DS; -metabolic alterations in plasma of children with DS were identified through different methods: Nuclear Magnetic resonance, routine blood exams performed during the follow up of the subjects and enzyme-linked immunosorbent assay (ELISA); - to test possible correlations between specific Hsa21 regions and alterations in transcriptomics and metabolomics, we have used trisomic iPSCs and differentiated them into neuronal derivatives. Significant alterations in gene expression and metabolic profiles have been identified, as well as significant correlations with clinical and cognitive aspects. Specific genes and the HR-DSCR may play a role in these alterations: cell models need to be developed to investigate this role. Neural derivatives from trisomic iPSCs are a promising model to better understand genotype-phenotype correlations in DS.

Standardization of dot-ELISA for the serological diagnosis of toxocariasis and comparision of the assay with ELISA

The dot-enzyme-linked immunosorbent assay (dot-ELISA) was standardized using somatic (S) and excretory-secretory (ES) antigens of Toxocara-canis for the detection of specific antibodies in 22 serum samples from children aged 1 to 15 years, with clinical signs of toxocariasis. Fourteen serum samples from apparently normal individuals and 28 sera from patients with other pathologies were used as controls. All samples were used before and after absorption with Ascaris suum extract. When the results were evaluated in comparison with ELISA, the two tests were found to have similar sensitivity, but dot-ELISA was found to be more specific in the presence of the two antigens studied. Dot-ELISA proved to be effective for the diagnosis of human toxocariasis, presenting advantages in terms of yield, stability, time and ease of execution and low cost.

EVALUATION OF A RAPID SCREENING ASSAY FOR BACTERIAL IDENTIFICATION (DOT-ELISA) IN FECAL SAMPLES FROM CHILDREN

With the objective of standardizing a Dot Enzyme-Linked Immunosorbent Assay (Dot-ELISA) to detect antigens of fecal bacterial enteropathogens, 250 children, aged under 36 months and of both sexes, were studied; of which 162 had acute gastroenteritis. The efficacy of a rapid screening assay for bacterial enteropathogens (enteropathogenic Escherichia coli "EPEC", enteroinvasive Escherichia coli "EIEC", Salmonella spp. and Shigella spp.) was evaluated. The fecal samples were also submitted to a traditional method of stool culture for comparison. The concordance index between the two techniques, calculated using the Kappa (k) index for the above mentioned bacterial strains was 0.8859, 0.9055, 0.7932 and 0.7829 respectively. These values express an almost perfect degree of concordance for the first two and substantial concordance for the latter two, thus enabling this technique to be applied in the early diagnosis of diarrhea in infants. With a view to increasing the sensitivity and specificity of this immunological test, a study was made of the antigenic preparations obtained from two types of treatment: 1) deproteinization by heating; 2) precipitation and concentration of the lipopolysaccharide antigen (LPS) using an ethanol-acetone solution, which was then heated in the presence of sodium EDTA

A Putative Otu Domain-containing Protein 1 Deubiquitinating Enzyme Is Differentially Expressed In Thyroid Cancer And Identifies Less-aggressive Tumours.

This study aimed to identify novel biomarkers for thyroid carcinoma diagnosis and prognosis. We have constructed a human single-chain variable fragment (scFv) antibody library that was selected against tumour thyroid cells using the BRASIL method (biopanning and rapid analysis of selective interactive ligands) and phage display technology. One highly reactive clone, scFv-C1, with specific binding to papillary thyroid tumour proteins was confirmed by ELISA, which was further tested against a tissue microarray that comprised of 229 thyroid tissues, including: 110 carcinomas (38 papillary thyroid carcinomas (PTCs), 42 follicular carcinomas, 30 follicular variants of PTC), 18 normal thyroid tissues, 49 nodular goitres (NG) and 52 follicular adenomas. The scFv-C1 was able to distinguish carcinomas from benign lesions (P=0.0001) and reacted preferentially against T1 and T2 tumour stages (P=0.0108). We have further identified an OTU domain-containing protein 1, DUBA-7 deubiquitinating enzyme as the scFv-binding antigen using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. The strategy of screening and identifying a cell-surface-binding antibody against thyroid tissues was highly effective and resulted in a useful biomarker that recognises malignancy among thyroid nodules and may help identify lower-risk cases that can benefit from less-aggressive management.

DOT-ELISA for detection of anti-Cysticercus cellulosae antibodies in human cerebrospinal fluid using a new solid-phase (resin-treated polyester fabrics): preliminary report

A dot enzyme-linked immunosorbent assay (DOT-ELISA) was developed to detect specific antibodies in cerebrospinal fluid (CSF) for human neurocysticercosis immunodiagnosis, with Cysticercus cellulosae antigen dotted on a new solid-phase. This was represented by sheets of a synthetic polyester fabric impregnated with a polymerized resin (N-methylol-acrylamide). A very stable preparation was thus obtained, the antigen being covalently bound by cross-linking with free N-methylol groups on the resin. Since robust, no special care was necessary for handling the solid-phase. The test could be performed at room-temperature. From 30 CSF samples assayed, 14 were positive, from a group of 15 cases of neurocysticercosis, with titers from 1 to 128; 15 other samples, from normals or other neurological diseases, were all negative. Test characteristics seem to indicate it as adequate for epidemiological surveys. A more detailed study on sensitivity, specificity, reproducibility and the use in serum samples is being conducted.