These results suggest this hormone may be a determinant regulator of the beneficial effects of CR.eNOS activation resulting in mitochondrial biogenesis is believed to play a central role in life span extension promoted by calorie restriction (CR). We investigated the mechanism of this activation by treating vascular cells with serum from CR rats and found increased Akt and eNOS phosphorylation, in addition to enhanced nitrite release. Inhibiting Akt phosphorylation or immunoprecipitating adiponectin (found in high quantities in CR serum) completely prevented the increment in nitrite release and eNOS activation. Overall, we demonstrate that adiponectin in the serum from CR animals increases NO Mammals submitted to CR present lower insulin levels • signaling in cultured vascular cells, and uncover the signaling pathways involved.The mechanism which leads to NOAfter 26 weeks of CR, the average body weight of rats was lower than control AL rats, an effect accompanied by lower visceral fat deposits, serum glucose, insulin, and increased adiponectin levels , alterat2−, indicative of higher levels of NO• production compared to cells maintained in media containing serum from animals fed AL . Furthermore, active, phosphorylated, eNOS increased . This concentration of NTD completely prevented the accumulation of NO2− promoted by CR serum, but did not affect the release in cells grown in AL serum . Male, 8-week-old Sprague-Dawley rats were separated into 2 groups: AL, fed ad libitum with an AIN-93-M diet prepared by Rhoster  and CR, fed at levels 60% of AL ingested amounts a diet supplemented with micronutrients to reach the vitamin and mineral levels consumed by AL animals ad libitum. At 34 weeks (26 weeks on the diet), rats were sacrificed after 12 hours fasting and the serum was obtained as described in g. The supernatant was collected and stored (−20°C). Sera were thawed and heat-inactivated at 56°C for 30 min prior to use.All experiments were conducted in agreement with National Institutes of Health guidelines for humane treatment of animals and were approved (unnumbered) the local Animal Care and Use Committee . Adiponectin was detected by Western Blots.Insulin, glucose, triglycerides, HDL, total cholesterol and adiponectin levels from AL or CR sera were evaluated . Periphe2. Cells were passaged every 3 days. After the 8th passage, cells were cultured in medium where fetal bovine serum was substituted for AL rat serum. After 2 further passages, cells from a 70% confluent flask were washed and cultured in DMEM with CR or AL rat sera. Where used, naphthyridinone 17 (NTD) was pre-incubated with the cultures for 24 hours, while the control group was incubated with the same quantity of the solvent DMSO.Rat vascular smooth muscle cells (VSMC) were purchased from ATCC (CRL-2797™) and cultured in 25 mM glucose DMEM supplemented with 18 mM sodium bicarbonate, 4 mM glutamine, 0.3 mM geneticin, 100 µg/mL streptomycin, 100 U/mL penicillin and 10% v/v fetal bovine serum, at 37°C and 5% CO2− measurements. Cells were washed, detached and counted in a Newbauer chamber. The cells were then centrifuged  and homogenized in 50 mM Tris-HCl buffer, pH 7.4, supplemented with 1% glycerol, 10% protease inhibitor cocktail (Sigma), 1% octyl phenol ethoxylate, 10 mM sodium orthovanadate, 10 mM sodium fluoride and 10 mM sodium pyrophosphate. After 30 min over ice, cell lysates were centrifuged  and the resulting supernatants were collected.After 6, 12 or 24 hours, cell culture media were removed and stored at −80°C for NO2−, a marker of NO• levels • analyzer  according to manufacturer protocols through the detection of chemiluminescence in the presence of potassium iodide and acetic acid 2− levels from the media were subtracted.NOSer1177 ; Akt , phospho-AktSer473 ; and γ-actin . Chemiluminescent detection was performed using a secondary peroxidase-linked anti-rabbit  or anti-sheep IgG  and a detection system from Pierce KLP . The specificity of anti-NOS antibodies Total proteins from cell lysates or serum were diluted in Laemmli sample buffer  containing 100 mM dithiothreitol, with the exception of eNOS and phospho-eNOS Western Blots, which were performed without dithiothreitol. After heating at 90°C for 5 min, proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were blocked with 5% BSA and detection was carried out using specific primary antibodies against: Adiponectin ; eNOS ; phospho-eNOS7 cells were plated over 75 cm2 and cultured with AL or CR sera for 24 hours. Cells were homogenized in lysis buffer . After 20 min over ice, tissues lysates were centrifuged , and the resulting supernatants were collected. Solubilized proteins (1 mg/mL) were incubated overnight with 4 µg⋅mL−1 anti-IR beta subunit antibody at 4°C. Protein A-agarose (Sigma) beads (50%) were added (80 µL⋅mL−1), and the incubation was continued at 4°C for 2 hours. The beads were centrifuged , washed five times in lysis buffer and suspended in Laemmli sample buffer containing 5% 2-mercaptoethanol. Immunoprecipitation specificity was verified through SDS-PAGE separation followed by silver staining.10−1. After a 12 hour incubation period, protein A-agarose beads  were added (200 µL⋅mL−1), and the incubation was continued at 4°C for 2 hours. The beads were centrifuged  and the serum was analyzed by Western Blot to confirm efficiency.Adiponectin immunoprecipitation from the serum followed the same steps described above, except the serum was not diluted. The polyclonal adiponectin antibody was used at 50 µg⋅mLData shown are representative blots or averages ± SEM of at least three identical repetitions. Data were analyzed using GraphPad Prism and compared using t-tests (for data pairs) or two-tailed ANOVA followed by Tukey tests (multiple comparisons).


Using a murine model of parainfluenza virus infection , we compared the inflammatory responses to lethal and sub-lethal infections in inbred DBA/2 mice.3 or 1.6×105 infectious units (IU) of SeV or diluent control. Clinical data including daily weights, oxygen saturation, and lung function via whole body plethysmography were collected on days 0, 3–7, and 9–14. Clarified whole lung homogenates were evaluated for inflammatory markers by enzyme-linked immunoassay (ELISA). Data were analyzed using ANOVA or Student t-tests, as appropriate.Mice were intranasally inoculated with either 1.6×105 IU of SeV developed a lethal infection with 100% mortality by day 7, while mice inoculated with 1.6×103 IU developed a clinically significant infection, with universal weight loss but only 32% mortality. Interestingly, peak virus recovery from the lungs of mice inoculated with 1.6×105 IU of SeV did not differ substantially from that detected in mice that received the 100-fold lower inoculum. In contrast, concentrations of CCL5 (RANTES), CCL11 (eotaxin), interferon-γ, CXCL10 (IP-10), and CCL3 (MIP-1α) were significantly higher in lung tissue homogenates from mice inoculated with 1.6×105 IU (p < 0.05). In the lethal infection, levels of CCL11, interferon- γ and CCL3 all correlated strongly with disease severity.Mice inoculated with 1.6×10We observed that severity of SeV-infection in DBA/2 mice was not associated with virus recovery but rather with the levels of proinflammatory cytokines, specifically CCL11, interferon- γ and CCL3, detected in lung tissue in response to SeV infection. Paramyxovirus family, cause a spectrum of illness from mild upper respiratory infection and otitis media to severe laryngotracheobronchitis and bronchiolitis [Human parainfluenza viruses (hPIV), of the hiolitis -4. HPIV hiolitis -4. Pathohiolitis -7.Airway epithelium infected with hPIV produces a variety of cytokines and chemokines that act as immune mediators in response to infection . We haveSendai virus (SeV), murine PIV, induces acute bronchiolitis and interstitial pneumonia in rodents, and has been used to model severe human hPIV infection -12. DiffUnderstanding host inflammatory responses that contribute to illness severity offers the potential to identify future therapeutic targets. Toward this end, we compared the inflammatory responses to both sub-lethal and lethal SeV infection in mice.3 IU of SeV developed a clinically significant infection with 32% mortality in the 14-day period . Similarly, by day 5, oxygen saturations were as low as 74% and Penh values were at 7.5-fold over baseline, although these clinical parameters were not statistically different from the other infected cohort , interferon-γ, CXCL10 (interferon gamma-induced protein 10 (IP-10)), and CCL3 (MIP-1α) detected in lung homogenates in response to SeV-infection were significantly higher in the mice inoculated with 1.6×102 = 0.86) and less so with diminished oxygen saturation (R2 = 0.54).In lethal SeV infection, CCL3, CCL11, and interferon-Υ each strongly correlated with severity of clinical symptoms, including weight loss, diminished oxygen saturation, and Penh Figures , 6 and 73 IU of SeV, there was no correlation between proinflammatory mediators and clinical parameters. Correlation coefficient (R2) for CCL11 and Penh, oxygen saturations, and weight loss were 0.02, 0.006, and 0.09, respectively. R2 for IFN-Υ and Penh, oxygen saturations, and weight loss were 0.19, 0.26, and 0.30, respectively. R2 for CXCL10 and Penh, oxygen saturations, and weight loss were 0.27, 0.41, and 0.21, respectively. R2 for CCL3 and Penh, oxygen saturations, and weight loss were 0.33, 0.44, and 0.59, respectively. R2 for CCL5 and Penh, oxygen saturations, and weight loss were 0.13, 0.24, and 0.10, respectively.On the other hand, in mice inoculated with 1.6×102 of 0.57, 0.58, and 0.25 for Penh, oxygen saturation, and weight loss and inoculum of 1.6×105 IU of SeV, respectively, and R2 of 0.12, 0.19, and 0.44 for Penh, oxygen saturation, and weight loss and inoculum of 1.6×103 IU of SeV, respectively (data not in figure).Similarly, virus titers did not correlate with clinical parameters at either dose inoculum, with R5 IU of SeV) sustained greater weight loss and mortality than the mice that received the lower inoculum (1.6x103 IU of SeV). Despite the increased severity of disease, virus recovery from mice receiving a lethal dose was not substantially different from that determined for mice receiving the lower virus inoculum. Interestingly, on day 3 post-inoculation, the mice receiving the lower inoculum had statistically higher BAL fluid cellularity when compared to the mice receiving the higher inoculum. The combination of these findings support the observation that severity of illness is not determined by virus recovery alone, and likely results from the combination of direct virus cytotoxicity and the subsequent, host inflammatory response [In this study, we evaluated the clinical impact, virus recovery, and inflammatory responses to both lethal and sublethal SeV infections in inbred DBA/2 mice. As has been previously described, DBA/2 mice are highly susceptible to lethal infection with SeV ,13-15. Sresponse ,13-15.5 IU of SeV developed a more robust inflammatory response, eliciting higher local levels of CCL11, interferon-γ, CCL3, CCL5, and CXCL10 when compared to mice inoculated with 1.6×103 IU of SeV, despite similar peak virus titers. It is possible that significant differences in early virus kinetics led to the differences in the chemokine response. While this observation is unusual, it is not unique to this study. There are several well-documented parallels demonstrating that there are host- and virus-specific contributions to virus kinetics and resulting chemokine response. Neonatal and adult mice inoculated with the same dose of pneumovirus of mice (PVM), similar to mice of the same strain and age inoculated with either PVM strain 15 or PVM strain J3666, develop differing chemokine responses despite similar virus replication [The inflammatory response to SeV in the mice differed based on the inoculum. The DBA/2 mice inoculated with 1.6×10lication ,17.In the lethal infection, CCL11, interferon-γ, and CCL3 each strongly correlated with worsening clinical symptoms, including weight loss, diminished oxygen saturation, and increasing Penh. While interferon- γ and CCL3 are among the mediators identified previously in response to SeV infection ,18, hereCCL11, CCL3, and interferon- γ, are also produced in response to other paramyxovirus infections, including PVM and human RSV infections -26. FurtIdentifying mediators which correlate with disease severity provide targets for therapeutic interventions. In RSV-challenged mice, for example, the use of blocking antibody to CCL11, an eosinophil chemoattractant, resulted in reduced pulmonary eosinophilia and disease severity . SimilarWe have described here the inflammatory responses to lethal and sublethal inocula in DBA/2 mice and have shown that CCL11, CCL3, and interferon- γ each correlate with disease severity. Further understanding of these responses in the murine model of severe PIV lower respiratory tract infection will aid in the development of therapeutic agents for human disease.3 IU of SeV over 7 experiments and 52 DBA/2 mice were inoculated with 1.6×105 IU of SeV over 3 experiments to collect the following data.Six to eight week old DBA/2 mice  were intra-nasally inoculated with SeV strain 52, obtained from American Type Culture Collection . Infected mouse lungs were harvested 5 days post-inoculation, homogenized, pooled, clarified via centrifugation, aliquoted, and stored in liquid nitrogen. All procedures were reviewed an approved by the institution’s Committee for the Humane Use of Animals (CHUA 191). In total, 128 DBA/2 mice were inoculated with 1.6x10Lungs from uninfected 6–8 week old DBA/2 mice purchased from Charles River  were harvested 5 days post-inoculation, homogenized, pooled, clarified via centrifugation, aliquoted, and stored in liquid nitrogen and used as sham stock control.3 and 1.6×105 infectious units (IU)/50 μl. Under light isofluorane anesthesia, the DBA/2 mice were intranasally inoculated with either 1.6×103 or 1.6×105 IU in 50 μl. Control mice received either an intra-nasal dose of PBS diluent or sham stock (50 μl).Specific pathogen-free, 5–8 week old DBA/2 mice were purchased from Charles River . Virus stocks of mouse passaged SeV stored in liquid nitrogen were diluted in phosphate buffered saline (PBS) to final concentrations of 1.6×10Clinical symptoms, as per scoring system previously described, together with body weights, and pulse oximetry were recorded daily . Whole b260/A280). cDNA was prepared from 2 μg DNase I-treated RNA from each lung sample and subjected to quantitative RT-PCR using primers and probe targeting the virus C’ gene: probe: 5′-56FAM-ACTTCTCCTTCGCCCTCACTT 36TAMSp-3′, primer 1: 5′-AACACCAATCAACACTCCCC-3′, primer 2: 5′-CCTGATCGATTATCTTGGGTC-3′, with reference to a standard curve generated using serial ten-fold dilutions of the C’ gene as target. The cDNAs were then subjected to a second set of quantitative PCR assays, with the commercial rodent GAPDH primers and probe  and serial ten-fold dilutions of the 905 bp rodent GAPDH fragment  to generate a standard curve. Controls include reactions with no reverse transcriptase and no template. Reactions were performed in an ABI 7500 Sequence detector, with cycling parameters including: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 65°C for 1 min. Data are expressed as copies of C’ per 105 copies GAPDH.Virus titers were determined on days 0, 3, 5, 7, 10, and 14 after intranasal inoculation with SeV by a quantitative reverse transcriptase PCR assay that targets the SeV C’ gene. Lung tissue was excised from SeV-infected mice, homogenized in RNazol B and RNA prepared per manufacturer’s instructions. Integrity of the RNA was evaluated on formaldehyde agarose gels and quantified spectrophotometrically , and bronchoalveolar lavage (BAL) fluid was harvested by transtracheal instillation of pre-chilled PBS with 2% EDTA, with recovery of approximately 700 μl of BAL fluid. Leukocyte counts were obtained by visual inspection and quantitative analysis cytospin preparations that were stained following Fisher Scientific Hema 3 protocol.Concentrations of proinflammatory mediators, including CCL5, CCL11, interferon-γ, CXCL10, and CCL3 were determined on days 0, 1, 2, 3, 4, 5, 7, 10, and 14 on total lung homogenates from SeV- , sham stock-, and diluent control-inoculated mice (four mice each per time point) using commercially-available ELISA kits .Data were analyzed using ANOVA or Student t-test’s as appropriate.The authors have no competing interests to declare.MS participated in the design of the study, performing experiments, data analysis and interpretation, and drafted the manuscript. CB participated in the design of the study and performing experiments. HR participated in the design of the study, interpretation of the data, and critical review of the manuscript. JD participated in the design of the study, data analysis and interpretation, and critical review of the manuscript. All authors have read and approved the final manuscript.


To detect the expression and clinical significances of Lewis y antigen and integrin αv, β3 in epithelial ovarian tumors, and to explore the expression correlation between Lewis y antigen and integrin αv, β3.Immunohistochemical staining was performed in 95 cases of epithelial ovarian cancer, 37 cases of borderline tumors, 20 cases of benign tumors, and 20 cases of normal ovarian tissue, for the detection of Lewis y antigen and integrin αv, β3 expressions, and to analyze the relationship between Lewis y antigen and integrin, and the relationship between clinical and pathological parameters of ovarian cancer. In addition, immunofluorescence double labeling was utilized to detect the expression correlation between Lewis y antigen and integrin αv, β3 in ovarian cancer.P < 0.05) and benign (25%) (P < 0.01) tumors, and normal ovarian tissues (0) (P < 0.01). The expression rate of integrin αv, β3 in malignant epithelial ovarian tumors was 78.95% and 82.11%, respectively, significantly higher than that of the borderline  (both P < 0.05), benign group  (both P < 0.01) and normal ovary group  (both P < 0.01).In epithelial ovarian tumors, the expression rate of Lewis y antigen was 81.05%, significantly higher than that of borderline (51.53%) (Lewis y and integrins αv, β3 are relevant to pelvic and abdominal diffusion and metastasis of ovarian cancer cells, suggesting that these two molecules mediate a boosting function for tumor metastasis. Among these 95 cases of ovarian cancer, 60 cases showed spread and metastasis in pelvic and abdominal cavities . Seventy-five (75) cases were positive for CA125 expression.Rabbit anti-human integrin αv, β3 polyclonal antibody was purchased from Wuhan Boster Company, mouse anti-human Lewis y monoclonal antibody was purchased from Abcam Inc., biotinylated goat anti-mouse IgM, biotinylated goat anti-mouse IgG were purchased from Zhongshan Biotechnology Co., Ltd. Serum albumin (BSA) and DAB kit were purchased from Zhongshan Biotechnology Co., Ltd. Goat anti-rabbit IgG fluorescein isothiocyanate (FITC) and goat anti-mouse IgG tetraethyl rhodamine isothiocyanate (TRITC) were purchased from Fujian Wanxin company, and DAPI was purchased from Shenyang Baoxin company.Streptavidin-biotin-peroxidase (SP) immunohistochemistry was performed. Tissues were fixed in 4% formaldehyde and embedded in paraffin, and 4 mm thick serial sections were prepared at the same organizational part. The working concentrations of Lewis y antibody and integrin αv, β3 antibody were 1:200 and 1:100, respectively. The staining procedure was performed according to SP kit manual. The group with PBS instead of primary antibody was used as a negative control. A colon cancer sample served as positive control for Lewis y antigen, and a breast cancer sample was a positive control for integrin αv, β3.The sample slices of strong expression for immunohistochemistry were selected to performed immunofluorescence double labeling method. Primary antibody combinations were anti-integrin αv with anti-Lewis y, or anti-integrin β3 with anti-Lewis y, with the PBS instead of primary antibody as the negative control. The working concentrations of rabbit anti-human integrin αv, β3 and mouse anti-human Lewis y antibody were all 1:100. The working concentrations of goat anti-rabbit IgM FITC and goat anti-mouse IgG TRITC were 1:100. The working concentration of nuclear dye DAPI was 1:100. The staining was performed according to the instructions of immunofluorescence kit.The presence of brown colored granules on the cell membrane or in the cytoplasm was taken as a positive signal, and was divided by color intensity into not colored, light yellow, brown, tan and is recorded as 0, 1, 2, and 3, respectively. We choose five high-power fields in series from each slice, then score them and take the average percentage of chromatosis cells. A positive cell rate of less than 5% was a score of 0, a positive cell rate of 5~25% was a score of 1, a positive cell rate of 26~50% was a score of 2, positive cell rate of 51~75% was a score of 3, positive cell rate of more than 75% was a score of 4. The final score was determined by multiplying positive cell rate and score values: 0~2 was equal to negative expression (−), 3~4 was equal to weakly positive (+), 5~8 was equal to moderate positive (++), 9~12 was equal to strong positive (+++). The results were read by two independent observers to control for variability.Microscopic red fluorescence indicated Lewis y antigen labeled by TRITC, green fluorescence indicated integrin αv, β3 labeled by FITC, while blue fluorescence indicated DAPI-stained nucleus. Pictures of the three individual fluorescence channels were superimposed using image analysis software, with a yellow fluorescence indicated co-localization of Lewis y antigen and integrin αv, β3.2) test was applied to analyze the expression of Lewis y antigen, integrin αv, β3 and clinicopathological parameters. The Spearman correlation analysis method was applied to calculate the coefficient R of indexes and to analyze its correlation, A P value <0.05 was considered statistically significant.Statistical analyses were performed using the SPSS software Version 11.5. Data expressed as mean ± SD was applied for statistical analysis. The Student’s t test was applied to compare data between the two groups, and analysis of variance was applied to compare data among multiple groups. The Chi-square (χIn summary, Lewis y and integrins αv, β3 are relevant to pelvic and abdominal diffusion and metastasis of ovarian cancer cells, suggesting that these two molecules mediate a boosting function for tumor metastasis.


Escherichia coli sfa+ strains isolated from poultry were serotyped and characterized by polymerase chain reaction (PCR) and amplified fragment length polymorphism (AFLP). Isolates collected from 12 Brazilian poultry farms mostly belonged to serogroup O6, followed by serogroups O2, O8, O21, O46, O78, O88, O106, O111, and O143. Virulence genes associated were: iuc 90%, fim 86% neuS 60%, hly 34%, tsh 28%, crl/csg 26%, iss 26%, pap 18%, and 14% cnf. Strains from the same farm presented more than one genotypic pattern belonging to different profiles in AFLP. AFLP showed a clonal relation between Escherichia coli sfa+ serogroup O6. The virulence genes found in these strains reveal some similarity with extraintestinal E. coli (ExPEC), thus alerting for potential zoonotic risk. Escherichia coli (APEC) is considered an outstanding pathogen for the poultry industry, due to several economic losses associated with chronic respiratory disease, septicemia, salpingitis, omphalitis, and embrionary death. Serogroups O2 and O78 are preferentially associated with colibacillosis outbreaks in poultry and represent 80% of disease cases worldwide . AμL) contained PCR buffer (1X), MgCl2 (1.5 Mm), 200 mM of each deoxyribonucleotide , 50 pmol of each oligonucleotide, 1.0 U of Taq DNA polymerase, autoclaved ultrapure water, and 5 μL of DNA template. Primers for specific amplification of the iuc, neuS, hly, tsh, crl/csg, iss, pap, and cnf genes, annealing temperatures, fragment size, and references are described in The polymerase chain reaction (PCR) procedure was applied after DNA extraction, according to the protocol described by Boom et al. 1990) [90 [19]. μL of the DNA extracted, 24 U of Hind III  were added along with ultrapure water, to a final volume of 20 μL, and this reaction was incubated overnight at 37°C. An aliquot of digested DNA (5 μL) was added to 0.2 μg of each adapter ADH1 and ADH2 oligonucleotides, 1 U of T4 DNA ligase  along with ultrapure water, to a final volume of 20 μL, and this reaction was incubated at room temperature for 3 hours. Ligated DNA was heated to 80°C for 10 min, and 5 μL were used for each PCR reaction. PCR reactions were performed in 50 μL of the final volume, containing 5 μL of ligated DNA, 2.5 mM of MgCl2, 30 pmol of primer , and 1 U of Taq polymerase, in a 1 X PCR buffer. This mixture was subjected to an initial denaturing step at 94°C for 4 min, followed by 35 cycles of 1 min at 94°C, 1 min at 60°C, and 2.5 min at 72°C. The base sequences of adapter and selective primers were: ADH1—5′ACGGTATGCGACAG 3′, ADH2—5′AGCTCTGTCGCATACCGTGAG 3′, HI-A—5′GGTATGCGACAGAGCTTA 3′, HI-G—5′GGTATGCGACAGAGCTTG 3′, and HI-T—5′GGTATGCGACAGAGCTTT 3′. Electrophoresis was conducted on 1.5% agarose gel at 22 V for 24 hours. The amplified products were visualized with ethidium bromide staining and then were compared to a 100 bp DNA ladder . Restriction endonuclease digestion and ligation was performed using a modified method described by McLauchiln et al. (2000) . To 10 μ The levels of relatedness of the isolates were determined by comprehensive pairwise comparison of restriction fragment sizes, using Dice coefficient. Mean values obtained from Dice coefficients were employed in UPGMA, using the NTSYS pc 2.0 version software to generate the dendrogram.Escherichia coli sfa+ were isolated from twelve poultry farms located in five Brazilian states. sfa+ strains. Three strains could not be identified with the serum tested.11 serogroups were identified: O2, O6, O8, O21, O25, O46, O78, O88, O106, O111, and O143. Serogroup O6 was the most frequent, representing 62% of the total number of strains. Serogroups O2, O21, and O78, commonly found in poultry affected by colibacillosis, comprised only 10% of the fim operon revealed 86% of positive strains. Results from PCR revealed that 45 (90%) strains were positive to the aerobactin gene—iucD; 30 (60%) to the K1 gene—neuS; 17 (34%) to haemolysin—hlyA; 14 (28%) to temperature-sensitive hemagglutinin—tsh; 13 (26%) to serum resistance—iss; 9 (18%) to P fimbriae—papEF, 7 (14%) to the cytotoxic necrotizing factor—cnf1. Forty-seven isolates (94%) presented the gene that regulates the curli operon, but only 13 strains (26%) possessed the structural csgA gene. None of the strains were positive to nonfimbrial adhesion—afaBC. According to the data presented in Colony hybridization with Analysis of the samples with AFLP revealed the presence of 20 profiles. Each profile produced from 4 to 10 fragments (bands), with approximately 600 to 3200 bp . Thirty-The upper dendogram region showed greater homogeneity, and it is composed by 29 strains classified in profiles A and B, which are derived from subgroup VIIa with higher than 82% similarity. Twenty-eight strains belonging to serogroup O6, and only one from O8, were classified as profile A.In profile A, 10 out of 11 strains belonging to genotype G13 were grouped, besides 3 strains belonging to G4, and 2 belonging to genotypes G16, G17, and G21. Strains with identical genotypes G9, G12, and G23 patterns, however, were placed in distant dendogram regions.sfa+ were characterized phenotypically and genotypically. Eleven serogroups were identified , which detected 4.2% of E. coli sfa+, after analyzing 1601 isolates from European poultry farms [sfa gene-positive strains were identified at 12 poultry farms, localized in five Brazilian states: SP, PR, SC, RS, and GO. Paraná state presented the highest percentile of positive strains (52%). These results differ from data obtained by Delicato et al. (2003), who report none positive strains for sfaDE and sfaS, in a study with 200 strains from different farms located in Paraná state [ The occurrence of avian extraintestinal ry farms . Here, sná state .sfa gene. The results showed that the frequency of some virulence genes reported here was different from those previously reported [tsh gene was detected in 85.3% of the APEC analyzed by Janben et al. (2001), in 39.5% of the strains analyzed by Delicato et al. (2003), and in 53.3% of the strains analyzed by Ewers et al. (2004). In this study, the tsh+ strains frequency remained at 28%. Similarly, Ewers et al. (2004) detected 82.7% of strains positive to the iss gene, Delicato et al. (2003) reported 38.5%, and this study detected only 26%. Many epidemiological studies on avian colibacillosis were based on the comparative frequency of virulence genes, in fecal strains and organs obtained from diseased poultry. In this study, we compared the frequency of virulence genes between traditional APEC strains (reported in the literature) and those positive to the reported –28. The pap gene also was observed at a lower frequency (18%), when compared to the data obtained by Janben et. al. (2001), who found 30% of APEC pap+. However, some researchers, such as Delicato et al. (2003), obtained a lower percentage, 18.5% in Brazil, Ewers et al. (2004), 22.7% in Germany, thus suggesting geographical variation for pap+ strains prevalence [ The evalence –28.tsh, iss, and pap genes have been detected at a lower percentage in the literature, the detection of other genes goes beyond the estimated prevalence. Thirty (60%) strains positive to the gene that encoded for the K1 capsule were identified, while the literature reports a percentage ranging from 8 to 20% for APEC [ Although for APEC .cnf1+ gene. Although genes encoding the presence of cytotoxic necrotizing factors in avian strains have been investigated by various authors, the occurrence of positive strains has not yet been notified [hly+, and the occurrence of hly+ APEC was also uncommon [hly and cnf genes act as a virulence marker of E.coli associated to mammals, and their presence in birds should be monitored as an indicator of interspecies barrier transposition. Seven strains 14%) were positive to the notified , 29, 30.uncommon . There auncommon , 29. Dat4% were psfa, crl, iuc, fim, and neuS genes, comprising 22% of the isolates. This profile was identified in four poultry farms in São Paulo, Paraná, and Rio Grande do Sul states. Other frequent profile was the G12 profile, representing 12% of the total number of strains, with the crl+iuc+fim+neuS+tsh+csgA+iss+ gene combination, and serogroups O2, O78, O88, O111, O143 detected in 3 farms in São Paulo state and 1 farm in Paraná. According to  According to the data exhibited in E. coli [ A study involving the genomic subtraction in the APEC serogroup O2, aiming at the identification of new virulence genes, revealed high homology among six of the nine genomic sequences identified with the genes described in human strains, associated with meningitis and urinary tract infection. These observations suggest a clonal relation between avian and human E. coli .Escherichia coli has been widely employed in epidemiological surveys. Many molecular approaches have been indicated for the discrimination of E. coli from avian origin: profiles for plasmid and isoenzymes, ribotyping, restriction endonuclease analysis (REA), pulsed field gel electroforesis (PFGE), random fragment length polymorphism (RFLP), and ERIC PCR [ Clonal analysis of ERIC PCR .E. coli isolates from broilers carcass, obtaining a higher discriminatory index. However, they observed an excessive division, with 41 distinct patterns and lower similarity, thus suggesting the absence of clonal relation between carcass and fecal strains [ AFLP has been employed for differentiation of important bacterial species, because this technique can identify disperse mutations in genomic, with the same sensitivity of RAPD or PFGE . AFLP wi strains . Dendogram analysis  shows th Ewers et al. (2004) employed PFGE for the analysis of 150 APEC strains and reported great difficulty in associating the serogroup and the profiles obtained with virulence genes distribution . Similar To the A profile, 90.3% of serogroup O6 strains were allocated. The C profile grouped two strains of serogroup O88. Serogroup O78 was allocated to D and E profiles, with 80% similarity, while two O2 isolates with the same origin and genotypic pattern were grouped in M profiles, with 96% similarity. Serogroup O143 strains were allocated to the Q and R profiles, with 94% similarity.E. coli sfa+ APEC. The existence of these strains in Brazilian poultry farms must be investigated by epidemiological surveys, with particular attention to the geographical distribution of serogroup O6 in recent isolates (2002 to 2011). There is a consensus in the literature as to the existence of limited APEC clones , 34, 37.


To investigate the correlations between aqueous concentrations of interleukin 1β, 6, 8, 10, 12p , and tumor necrosis factor α (TNF-α) and the parameters of macular edema acquired by optical coherence tomography (OCT) in patients with choroidal neovascularization.IL-1β, IL-6, IL-8, IL-10, IL-12p, and TNF-α in the aqueous humor samples of 17 patients with exudative age-related macular degeneration (AMD), ten patients with pathological myopia (PM), seven patients with idiopathic choroidal neovascularization (CNV), and 14 patients with cataract and idiopathic epiretinal membrane or macular hole in the control group were measured with cytometric bead array. The maximum macular thickness and macular volume within 1 mm, 3 mm, and 6 mm were measured with OCT.In the CNV groups, the aqueous levels of IL-6 and IL-8 were significantly associated with macular volume within 6 mm , while IL-1β, IL-10, IL-12p, and TNF-α showed no significant correlation with either the maximum macular thickness or the macular volume. By further selecting patients with CNV who had accepted their last intravitreal injection of bevacizumab within 3 months, the level of IL-6 still significantly correlated with the maximum macular thickness (p=0.019) and macular volume within 1 mm (p=0.018), 3 mm (p=0.018), and 6 mm (p=0.022). In patients with exudative AMD, the level of IL-6 was significantly associated with the maximum macular thickness (p=0.025) and macular volume within 1 mm (p=0.025), 3 mm (p=0.006), and 6 mm (p=0.002). The aqueous level of all cytokines did not vary significantly between the CNV patients who had accepted their last intravitreal injection of bevacizumab within 3 months and the other patients, nor was a difference found among patients with exudative AMD, PM, and idiopathic CNV, and the control group.Intraocular concentrations of IL-6 and IL-8 (particularly IL-6) are significantly associated with the volume of macular edema in patients with CNV. However, intravitreal injection of antivascular endothelial growth factor drugs did not change the intraocular level of these inflammation cytokines. Choroidal neovascularization (CNV), either idiopathic CNV , exudatiVascular endothelial growth factor (VEGF), first discovered as a vasopermeability factor , has beeIn this study, we explored the relationship between levels of inflammatory cytokines in aqueous humor of patients with CNV after and not after recent anti-VEGF therapy and the parameters of the macula acquired with optical coherence tomography (OCT).This study included 17 patients (17 eyes) with exudative AMD, ten patients (ten eyes) with pathological myopia (PM), seven patients (seven eyes) with idiopathic choroidal neovascularization (CNV) who underwent intravitreal injection of bevacizumab  as the study group, and 14 patients (14 eyes) with cataract and idiopathic epiretinal membrane or a macular hole who underwent combined cataract and vitrectomy surgery (CCVS) as the control group. The patients were consecutive in each group. The inclusion criterion in the study group was the presence of active CNV. Exclusion criteria included glaucoma, previous photodynamic therapy, and other retinal diseases, such as diabetic retinopathy and retinal vascular occlusion.All patients underwent an ophthalmic examination including best-corrected visual acuity (BCVA) recording using manifest refraction and the logMAR visual acuity chart, non-contact tonometry, slit lamp assisted biomicroscopy of the anterior segment and posterior segment of the eye, fundus fluorescein angiography (FFA), and OCT . All patients in the study group showed leakage on fluorescein angiographies (active CNV), and they were further divided into two subgroups according to the interval between the last intravitreal injection of bevacizumab (IVB) and this time . OCT was analyzed by one investigator, and the maximum macular thickness and macular volume within 1 mm, 3 mm, and 6 mm were measured using built-in software. The OCT measurements were converted from millimeters to proportionally corresponding micrometers.Aqueous humor was collected during intravitreal injection or CCVS. All procedures conformed to the Declaration of Helsinki for research involving human subjects. Informed consent was obtained from all participants. Undiluted aqueous humor samples (100–200 µl) were obtained through anterior chamber paracentesis. All injections and sample collections were performed using a standard sterilization procedure that included the use of topical povidone-iodine and levofloxacin drops. Samples were stored in a sterilized plastic Corning  at −70 °C until use.The concentration of aqueous interleukin 1β, 6, 8, 10, 12p  and TNF-α were measured with cytometric bead array (CBA), a method for capturing a soluble analyte or set of analytes with beads of known size and fluorescence, making it possible to detect analytes using flow cytometry. Each capture bead has been conjugated with a specified antibody. The detection reagent is a mixture of phycoerythrin (PE)-conjugated antibodies, which provides a fluorescent signal in proportion to the amount of bound analyte. When capture beads and detector reagent are incubated with an unknown sample containing recognized analytes, sandwich complexes  are formed that can be measured using flow cytometry  to identify particles with the fluorescence characteristics of the bead and detector. Six bead population with distinct fluorescence intensities coated with specific capture antibodies for IL-1β, IL-6, IL-8, IL-10, IL-12p, and TNF-α were mixed together to form the bead array. Thus, the concentration of each cytokine in one unknown sample can be calculated from regressive analysis of the standard curve simultaneously. In this study, 50 μl of undiluted aqueous humor sample was used for each test. Quality control procedures were performed according to the CBA kit instructions.t test were used to compare noncontinuous variables. Values were reported as mean±standard deviation (SD). Statistical significance was set at p<0.05 for two-tail analysis.The Pearson correlation test was used to explore the relationship between the aqueous level of a cytokine and the OCT parameters . Parameters were compared using the Mann–Whitney U test between two groups and the Kruskal–Wallis H test to compare variables among various groups. The chi-square test and the Fisher exact A total of 48 aqueous humor samples from 48 patients were collected . The ageIL-6=0.011, R2=0.176, PIL-8=0.008, R2=0.187; 2=0.382), 3 mm , and 6 mm  and macular volume within 1 mm , 3 mm , and 6 mm .In exudative AMD , the levThe aqueous level of all cytokines investigated in this study showed no significant difference between the patients with CNV who had accepted their last IVB within the last 3 months (<3 months), patients with CNV who had accepted their last IVB more than 3 months before or never (>3 months), and the control group  of the IL-8 promoter is more prevalent in AMD cases than controls. Izumi-Nagai et al. [Our results were in agreement with several previous studies. Higgins  found thi et al.  found thThere were limitations in this study. First, the selection of the control group may have caused selection bias, since the aqueous level of all cytokines investigated in this study did not vary significantly between the CNV group and the control group . Patients with idiopathic epiretinal membrane or a macular hole -35 were A previous study investigated the relationship of recurrent CNV and cytokines, but this study may be the first one that states the association between cytokines and CNV volume, suggesting their importance. In conclusion, the aqueous level of IL-6 is positively related to the maximum macular thickness and macular volume within 1 mm, 3 mm, and 6 mm in patients with CNV who had undergone their last IVB within the last 3 months. Intravitreal injection of anti-VEGF drugs did not change the intraocular level of inflammation cytokines, including IL-1β, IL-6, IL-8, IL-10, IL-12p, and TNF-α. Furthermore, IL-6 is positively related to these OCT parameters in exudative AMD. IL-6 and IL-8, especially the former, may be additional target molecules in therapy for CNV.


To the Editor: Because methicillin-resistant Staphylococcus aureus (MRSA) has been identified in retail meat worldwide , mecA identification, staphylococcal cassette chromosome (SCC) mec typing, Panton-Valentine leukocidin identification, agr typing, MLST, and spa typing as described  from 30 grocery stores in Detroit, Michigan, USA, during August 2009–January 2010. Up to 3 presumptive S. aureus: 32 beef (20.5%), 19 chicken (25.0%), and 14 turkey (24.6%) samples. Six samples, consisting of 2 beef (1.3%), 3 chickens (3.9%), and 1 turkey (1.7%), were positive for MRSA as evidenced by the presence of mecA. The overall lower prevalence of S. aureus and MRSA than that found in a previous study in the United States   samples yielded tively) ,.mec typing, agr typing, and pvl detection showed all strains to be positive for ST8, SCCmec IVa, agr I, and Panton-Valentine leukocidin, which are typical characteristics of USA300 clones. However, spa typing identified 2 distinct spa types, t008 (11–19–12–21–17–34–24–34–22–25) and t2031 (11–19–12–12–34–34–24–34–22–25) (repeat variants in boldface), which differed by 5 nucleotides. t008, the most common spa type of USA300, was identified in 6 isolates of beef, chicken, and turkey origin, whereas t2031 was recovered from MRSA4a, 4b, and 4c from a chicken sample. The nucleotide variation in t2031 caused amino acid changes from glycine-asparagine in t008 to asparagine-lysine. The single nucleotide difference between repeats 12 (GGT) and 21 (GGC) and repeats 34 (AAA) and 17 (AAG) resulted in no amino acid change, with glycine and lysine encoded, respectively.Although an extra band was generated in MRSA 2a, 2b, 3, 5, and 6 by PFGE, all 9 MRSA isolates belonged to USA300 . MultiplUnlike studies in Europe, where researchers have reported the animal MRSA clone ST398 from various meat products . These include whether t2031 is more adaptable to chicken production because of the 2 amino acid difference from t008, or whether t2031 is linked with specific antimicrobial drug resistance phenotypes other than β-lactam resistance.Despite the recovery of MRSA from retail chicken and t2031 that has an antibiogram distinct from t008, except for β-lactam resistance, several questions remain about whether more


Endopleura uchi is a typical Amazonian tree and its bark is popularly employed in the preparation of teas against myomas, arthritis, influenza, diarrhea and cancer. In this study, the antioxidant activity, cytotoxicity and antimicrobial activity of five different extracts of the bark, selected by their total tannin content, were assessed. The potential antioxidant activity of the extracts was determined by 2.2-diphenyl-1-picrylhydrazyl radical scavenging assay and the values found were very similar among the extracts and to the standards antioxidants used in the tests. Cytotoxicity analysis in mammalian cells indicated that all the tested extracts exhibited IC50 values higher than the highest concentration used, showing that they do not present a risk when consumed under these conditions. Extract tested against five bacterial strains and one yeast strain did not show satisfactory growth inhibitory activity, and even the extracts that showed some antimicrobial activity were not effective at any dilution to determine the minimum inhibitory concentration. The results may serve as a reference for subsequent works, since such reference values described in the literature for the bark of E. uchi. Approximately 25% of the medicines prescribed in the industrialized countries originate from plants and about 120 compounds of natural origin, obtained from approximately 90 species of plants, are used in modern therapy ,4.The abusive and indiscriminate use of antimicrobial compounds over many years is the main factor responsible for the appearance of the phenomenon of bacterial resistance to such compounds . SeveralEndopleura uchi (Huber) Cuatrec. (Humiriaceae) is a native tree of the Brazilian Amazon rainforest and is found scattered over the whole Amazonian Basin. The family Humiriaceae was described by Antoine Laurent de Jussieu and includes 50 species classified in eight genera, spread in tropical areas of America, and one specie in tropical West Africa. The plant is known in the Amazon as “uchi”, “uxi-amarelo”, “cumatê”, “axuá”, “pururu”, “uxi-liso”, “uxi-ordinário” or “uchi-pucu” /[1000 × m], NAP (%) = [15625 × Abs]/[1000 × m], TT (%) = TP − NAP, where TP = total polyphenolics (%); NAP = non-adsorvent polyphenolics (%); Abs = Absorbance; m = mass (g) of samples; TT = total tannins (%).Briefly, the samples were dissolved in 250 mL water to give the mother liquor (ML). A 5 mL aliquot of ML was diluted in water to 25 mL and 2 mL of this solution, were transferred to a 25 mL vial with Folin-Ciocalteau phenol reagent 2 N (1 mL) and Milli-Q3.4.Bacillus subtilis (ATCC 9372), Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 27853), Escherichia coli (ATCC 25922), Shigella sonnei  and Candida albicans (ATCC 64548). A colony of each bacterial strain used, or 150 μL of a previously prepared bacterial suspension was inoculated in Brain Heart Infusion (BHI) broth and incubated at 37 °C for 24 hours. The turbidity of each bacterial cell suspension was then adjusted with saline to match the 0.5 McFarland scale (∼1.5 × 108 CFU/mL). Five colonies of Candida albicans were inoculated in Sabouraud broth and incubated at 35 °C for 24–48 hours. The turbidity of the yeast cell suspension was then adjusted to 0.5 on the McFarland scale (1× 106–5 × 106 CFU/mL) with sterile saline. Ampicillin (50 μg/mL) was used as a positive control for the bacterial strains and amphotericin B for the Candida albicans strain. The later was prepared by dissolving 16 mg of amphotericin B in 10 mL of dimethylsulfoxide (DMSO) and diluting this, twice, in the proportion 1:5 (w/v), obtaining the stock solution 64 μg/mL.The agar disk diffusion technique (both with “templates” and “paper discs”) was employed to test for antimicrobial activity against strains of E. uchi extract  and 50 μL of DMSO:BHI solution (1:1 v/v) as a negative control were added. Sterilized filter paper discs (Whatman® grade No. 1) were individually impregnated with 40 μL of each 10 mg/mL extracts solution (resuspended in DMSO), 40 μL of DMSO:BHI  as negative control and 40 μL of ampicillin or amphotericin B as positive controls. The plates were incubated at 37 °C for 24–48 hours. The inhibition of the bacterial and/or fungal growth was determined by measuring the haloes around of the wells and discs with the aid of a digital calliper, and expressed as the average of three independent experimental determinations.The agar diffusion tests were performed as in the approved standards M2-A8 and M44-A of the Clinical and Laboratory Standards Institute, with modifications ,34. Bact3.5.The minimum inhibitory concentration (MIC) of the extracts that presented some activity against the tested microbial strains was determined by the broth micro-dilution method, as described in the M7-A6 reference guideline of the Clinical and Laboratory Standards Institute, with modifications .N-morpholino) propanesulfonic acid (MOPS) buffer, 0.165 mol/L) for the yeast in 96-well flat-bottomed microtitration plates, containing 0.1 mL medium in each well. The extracts were prepared in DMSO at an initial concentration of 200 mg/mL and diluted in DMSO:BHI  to obtain 10 mg/mL in test solutions. Sample solutions (0.1 mL) were two-fold serially diluted in the plates with liquid medium. The working inoculum suspension (0.1 mL) was added to give a final inoculum concentration of 1 × 105–5 × 105 and 0.55 × 103–2.5 × 103 CFU/mL, for bacteria and yeast assays, respectively. Ampicillin final dilutions ranging from 12.5 μg/mL to 0.012 μg/mL and amphotericin B final dilutions ranging from 16 μg/mL to 0.015 μg/mL were used for bacteria and yeast, respectively, as positive control. Negative contamination controls using only medium (BHI or RPMI), and with or without extract were used in the tests. The plates were incubated at 37 °C for 24 and 48 hours for bacteria and yeast, respectively. No inhibitory effects were observed in the presence of DMSO at the highest concentration used. To perform the visual reading, 20 μL of 0.01% (w/v) resazurin (Sigma®) was added to each well. The plates were incubated for a further 30 minutes, and estimated visually for any change in color from blue to pink, indicating reduction of the dye due to microbial growth. The lowest concentration that remained blue was taken as the MIC. Experiments were performed in duplicate.The test was carried out in BHI broth for bacterial strains or RPMI-1640 (adjusted to pH 7.0 with 3-(3.6.2 in Eagle medium (pH 7) supplemented with 15% fetal bovine serum, without sodium bicarbonate. The extract samples were prepared by dissolving 10 mg of 20% decoction, 10% infusion, 5% maceration (EtOH 50%), 10% percolation (EtOH 50%) and 20% turboextraction (EtOH 50%) in 1 mL of DMSO. These initial stock solutions were diluted  in Eagle medium, to obtain the test solutions.Cytotoxicity was tested on rabbit corneal fibroblasts . These cells were maintained in culture bottles incubated at 37 °C with 5% CO5 cells/mL in Eagle medium. The cells were incubated in 96-well flat-bottomed microtitration plates at 37 °C in an atmosphere of 5% CO2 for 72 h. E. uchi samples, diluted in DMSO:Eagle , were added. The extracts were serially diluted  across the plate resulting in an initial concentration of 200 μg/mL to a final concentration of 39 μg/mL. The plate was incubated for 24 hours at 37 °C in a humid atmosphere with 5% CO2 [2 at 37 °C. The wells were read visually after 3 hours by distinguishing the original blue color (absence of living cells) from pink (presence of living cells) and with a microplate fluorescence reader , with filters of 530 and 590 nm [For cytotoxicity evaluation, the cells were collected, centrifuged  counted and adjusted to the concentration 1 × 10h 5% CO2 –38. Afte3.7.Ginkgo biloba L. extract (Santosflora®) was also used in this test, at the concentration of 250 μg/mL (in methanol), for its recognized antioxidant activity [Abscontrol − Abssample)/Abscontrol × 100, where Abs = absorbance.The assay of antioxidant activity of the extracts was based on the scavenging activity of 2.2-diphenyl-1-picrylhydrazyl solution (DPPH) . Gallic activity . The tes3.8.®. When necessary, analysis of variance (ANOVA) was performed, with P < 0.05. The Spearman and Kendall-Tau tests (α = 0.05) were performed with Stat Plus Professional 2009® to verify correlation coefficient among total tannins content, total phenols content and antioxidant activity.The results were expressed as the average of three determinations ± standard deviation, using Microsoft Office Excel 20074.Although satisfactory antimicrobial activity was not observed for any extract used, new tests with more sensitive techniques should be performed, since high levels of tannins were found in the samples, which are well recognized for antimicrobial properties. Moreover, the absence of cytotoxicity and high antioxidant activity and the previously reported lack of acute oral toxicity, confirm that the bark used in the preparation of teas by the population can be consumed safely. The results presented here, in association with others in the literature, should guide future work in the search for a possible anti-cancer activity.


In our previous study, we found that acute anterior uveitis (AAU) could be induced in wild-type mice (C3H/HeN), but it could not be induced in TLR4 gene-deficient mice (C3H/HeJ), we concluded that the translocation of transcription factor nuclear factor-κB (NF-κB) may play an important role. In this study, we examined the concentration of different cytokines in the aqueous humor of C3H/HeN mice and C3H/HeJ mice with the aim of exploring the role of different cytokines in the lipopolysaccharide (LPS) and TLR4-mediated signal transduction in the development of AAU. Acute anterior uveitis (AAU) is the most common form of uveitis. In the past few years, inflammation has been recognized as a major driving force of AAU. It is now well established that, starting from the initial lesion to the iris and the aqueous humor in the eye, numerous cellular and molecular inflammatory components participate in the disease process. Monocyte-derived macrophages and T-lymphocytes are the predominant invading immune cells found in evolving lesions. Both cell types produce a wide array of soluble inflammatory mediators (cytokines and chemokines) that are critically important in the initiation and perpetuation of the disease . ExtensiRecently, our research group have demonstrated a higher expression of TLR4 on uvea-resident tissue macrophages in endotoxin-induced uveitis (EIU) than is found in normal rats , and C3Hp < 0.001), then gradually decreased. Twelve hours after injection, the concentration reached the second peak. There was no significant difference between 3 h group and 12 h group (p = 0.410). After 48 h, TNF-α concentration in the aqueous humor returned to their original levels. The original concentration of TNF-α was higher in C3H/HeJ than that in C3H/HeN mice. After injection of LPS, the concentration gradually increased, reaching a peak 24 h after injection, which was found to be statistically significant in comparison with the control group (p < 0.001), then gradually decreased , reached a peak at 12 h after injection (p < 0.001), then gradually decreased. It had finally returned to its original concentration at 48 h (p = 0.293). There was no significant difference of original concentration of IL-1 between C3H/HeN mice and C3H/HeJ mice (p = 0.637). After injection of LPS, the concentration of IL-1 in C3H/HeJ mice increased, but its peak concentration was still much lower than the concentration of C3H/HeN mice . Inconceivably, the level of the concentration of IL-6 in C3H/HeJ mice was much higher than that in C3H/HeN mice , and returned to its original concentration 48 h after injection (p = 0.736). Surprisingly, in C3H/HeJ mice, the peak concentration was the concentration of the control group. After LPS injection, the concentration decreased gradually, but 24 h after injection, the concentration reached its second peak, then decreased , and returned to its original concentration at 48 h (p = 0.499). In contrast, in C3H/HeJ mice, the peak concentration was the original concentration, and the concentration decreased with LPS injection  was kindly provided by Lanzhou Institute of Biologic Products . Endotoxin-induced uveitis was induced as previously described [Lipopolysaccharide (escribed . Mice ren = 50) and a control group (n = 10). The experimental group was further randomly divided into five subgroups of 10 mice each, according to post-LPS injection time points . Forty C3H/HeJ male mice, 6 to 8 weeks old and weighing between 16 g and 25 g, were obtained from Model Animal Research Center  and housed in 12 h light/12 h dark cycles. The mice were randomly divided into an experimental group (n = 30) and a control group (n = 10). The experimental group was randomly divided into three subgroups of 10 mice each, according to post-LPS injection time points  and housed in 12 h light/12 h dark cycles. The mice were randomly divided into an experimental group . BD™ Cell Quest and CBA software  BD™ CBA Mouse TNF, IL-1, IL-6, IL-10, and IFN-γ Flex Set . The BD FACSCalibur used is an automated, multicolour, bench top flow cytometer. It uses BD software for data analysis.g, at 4 °C, for 3 min. The supernatant was collected and stored at −80 °C.The aqueous humor samples were collected using a microinjector. Ten samples were pooled together and replicated performed for analysis. Then samples were centrifuged at 705× The cytokine standard solutions included with the BD CBA Flex Sets were serially diluted to the following concentrations: 2500 μg/L, 1250 μg/L, 625 μg/L, 312 μg/L, 156 μg/L, 80 μg/L, 40 μg/L, 20 μg/L, and 0 μg/L. A control sample was also included in the measurements. Measurements for the different concentrations were obtained using the FACSCalibur, and standard curves were generated using the BD CBA software.A total of 50 μL of sample was mixed with 50 μL of capture beads and 50 μL of PE-labeled detection antibodies. After incubation in the dark at room temperature for 2 h, 1 mL of soluble protein master buffer lotion was added. The sample was then centrifuged at 200× g for 5 min and washed twice, before the addition of 300 μL buffer lotion. Results were obtained using BD CellQuest and BD CBA software.p value of 0.05 or less was interpreted as indicating statistical significance when comparing experimental and control groups.Quantitative data were analyzed with one-way analysis of variance (ANOVA) followed by Significant Difference Procedure (LSD) test for multiple comparisons among experimental groups with control groups. Data was performed by SPSS  statistical software. A The cytokine levels in the aqueous humor of C3H/HeN mice were consistent with what would be expected during the TLR4 signal transduction related inflammation process. Further investigation into the interaction and dynamic changes of cytokines may provide insights into the pathogenesis of AAU, potentially leading to more effective treatments.


In vitro activity against CQ-sensitive Plasmodium falciparum 3D7 and an evaluation of cytotoxicity in a mammalian cell line (HEK293T) are reported. Furthermore, two possible mechanisms of action of anti-malarial compounds have been evaluated: effects on mitochondrial membrane potential (ΔΨm) and inhibition of β-haematin formation.The discovery and development of anti-malarial compounds of plant origin and semisynthetic derivatives thereof, such as quinine (QN) and chloroquine (CQ), has highlighted the importance of these compounds in the treatment of malaria. Ursolic acid analogues bearing an acetyl group at C-3 have demonstrated significant anti-malarial activity. With this in mind, two new series of betulinic acid (BA) and ursolic acid (UA) derivatives with ester groups at C-3 were synthesized in an attempt to improve anti-malarial activity, reduce cytotoxicity, and search for new targets. P. falciparum 3D7, and were non-cytotoxic. These derivatives were three to five times more active than BA and UA. A DiOC6(3) ΔΨm assay showed that mitochondria are not involved in their mechanism of action. Inhibition of β-haematin formation by the active derivatives was weaker than with CQ. Compounds of the BA series were generally more active against P. falciparum 3D7 than those of the UA series.Among the 18 derivatives synthesized, those having shorter side chains were most effective against CQ-sensitive Three new anti-malarial prototypes were obtained from natural sources through an easy and relatively inexpensive synthesis. They represent an alternative for new lead compounds for anti-malarial chemotherapy. Plasmodium falciparum is a protozoan parasite and the causative agent of the most virulent form of malaria in humans. In endemic areas, malaria accounts for nearly one million deaths, primarily among children under the age of five [Malaria is among the most significant of infectious diseases, influencing the health of humankind to this day . Plasmod of five .Cinchona (Rubiaceae) species [Plasmodium strains, the therapeutic impact of these compounds has declined, urging development of new and effective anti-malarials [in vitro anti-plasmodial activity (IC50) of BA against chloroquine (CQ)-resistant (K1) and CQ-sensitive (T9-96) P. falciparum strains was found to be 19.6 μg/mL and 25.9 μg/mL, respectively. In a murine malaria model using Plasmodium berghei NK65 strain, BA exhibited some toxicity when a high dosage was employed (250 mg/kg/day), but no toxicity was observed at a dose of 125 mg/kg/day [Higher plants are commonly used as sources for the discovery of new drug leads. Quinine (QN) and artemisinin are examples of plant-derived products with anti-malarial activity . QN, one species . Many ot species . Neverthalarials . The lacalarials . The natalarials ,9. The ig/kg/day .P. berghei-infected mice and reduce in vitro proliferation of P. falciparum strains 3D7, W2, and K1 [et al.[Another triterpene of pharmacological importance is ursolic acid , which is known to have anti-inflammatory and antihyperlipidemic properties as well as antitumour-promoting effects . Moreove, and K1 ,12. The 1 [et al. describeP. falciparum 3D7) of two new series of BA and UA analogues possessing various ester substituents at C-3, as well as the in vitro cytotoxicity of the most active compounds in HEK293T mammalian cells and their effect on mitochondrial membrane potential (ΔΨm) and inhibition of β-haematin formation. These mechanisms of action are likely involved in the anti-malarial activity of atovaquone and CQ respectively [In this context, the present study report the synthesis and anti-malarial activity  peels obtained from a local juice factory. This compound was identified using full spectroscopy data, which consistent with those previously described [2Cl2 (2 mL) and refluxed for 24 hours (for cyclic anhydrides) or processed without refluxing for one hour (for acyclic anhydrides). Column chromatography of the crude residue was performed to give the expected pure compounds. Series BA and UA are illustrated in Figure All commercially available reagents were used without further purification unless otherwise stated. Solvents were distilled under a positive-pressure dry nitrogen atmosphere when necessary. Reactions requiring anhydrous conditions were performed under a nitrogen atmosphere. BA and UA derivatives were synthesized according to the general procedure described in the literature . Briefly254 plates (Merck) and spots visualized by spraying with anisaldehyde/sulphuric acid solution followed by heating at 100°C. Melting points were determined using a Kofler bench. High-resolution mass spectra (HR-EI-MS) were obtained on a Micromass-Waters Q-TOF Ultima spectrometer. Infrared spectra were recorded on a PerkinElmer FTIR BX spectrometer. 1H and 13C Nuclear Magnetic Resonance (NMR) spectra were recorded on a Bruker AC 400 spectrometer operating at 300 and 75 MHz, respectively, using tetramethylsilane as the internal standard and chloroform-d. The chemical shifts (δ) were expressed in parts per million (ppm).Column chromatography was carried out using silica gel 60 (Merck). Analytical thin layer chromatography was performed on silica gel 60 FPlasmodium falciparum (3D7 strain) was cultured in RPMI 1640 supplemented with 37.5 mM HEPES, 7 mM D-glucose, 6 mM NaOH, 25 μg/mL gentamicin sulfate, 2 mM L-glutamine and 10% human serum and maintained in human erythrocytes under a gas mixture of 5% O2, 5% CO2, and 90% N2[50) was obtained from the drug concentration response curve. The DMSO concentration never exceeded 0.1% and did not inhibit parasite growth. The definition of anti-malarial activity used herein was: IC50 ≤1 μM, potent activity; IC50 1-10 μM, good activity; IC50 10-30 μM, moderate activity; IC50 ≥30 μM, inactive [nd 90% N2. Culturend 90% N2. Infecteinactive . Reagent2, in Dulbecco’s modified essential medium (Gibco BRL) supplemented with 10% (v/v) foetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells (5.0 x 104/well) were seeded into 48-well plates. Twenty-four hours after plating, cells were treated with decreasing concentrations  of compounds 1e, 1f and 2e and evaluated after 24 and 48 hours. After incubation, cells were harvested with trypsin, gently centrifuged and resuspended in phosphate buffer saline. The cells were stained with dihydroethidium solution , gently vortexed, and incubated for 40 min at 37°C in the dark. Ten thousand gated events were acquired for each sample . The percentage of cell viability calculated as intrinsic cytotoxicity is represented by the concentration leading to 50% of cell death (IC50) on flow cytometry.Cytotoxicity was estimated using HEK293T (human embryonic kidney) cells. This cell line was cultured in 75 sq cm vented tissue culture flasks at 37°C in a humidified atmosphere containing 5% COet al.[′-dihexyloxacarbocyanine iodide (DiOC6(3)) in mitochondria was assessed in the presence of the active compounds 1e, 1f and 2e and in their absence, in comparison with cells treated with the mitochondrial respiratory chain uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP). In this manner, iRBC in RPMI 1640 medium containing 10% human serum and 10% parasitaemia were incubated with a 2 nM final concentration of DiOC6(3) for 30 min at 37°C. Various concentrations of the compounds were then added to the culture and incubated for an additional 30 min at 37°C. At the end of the incubation period, samples were centrifuged for 5 min at 3,700 rpm and the supernatant discarded. Samples were then resuspended in phosphate buffer saline and 105 events analyzed by flow cytometry.Changes in mitochondrial membrane potential were assessed as per Srivastava et al.. Accumulet al.[1e, 1f and 2e or water for CQ). The final concentration of compounds in wells ranged from 0.5 to 20 mM. After 24 hours of incubation while gently shaking at 37°C, samples were centrifuged for 15 min at 3,300 g, the supernatant discarded and the pellet washed with 200 μL DMSO. This latter step was repeated once more and the pellet was finally washed with water. The pellet was then dissolved in 200 μL of 0.1 M NaOH. After a further 1:8 dilution, absorption at 405 nm was measured using a FlexStation plate reader. Experiments were carried out at least in triplicate. Results are expressed as percentage of inhibition of β-haematin formation in comparison to the negative control result, as calculated with the equationInhibition of β-haematin formation was determined as described by Baelmens et al.. CQ was t test was chosen for between-group comparisons, whereas repeated measures ANOVA was used for comparisons amongst more than two groups. Statistical significance was defined as p < 0.05. Analyses were carried out in GraphPad Prism version 4.00 for Windows .All results are expressed as mean ± standard error or mean ± standard deviation of at least three individual experiments. Student’s The structures of the isolated compounds are shown in Figure P. acerifolia bark, and UA from M. domestica peel; both sources were highly advantageous due to their low cost and good yields . After semisynthesis of the eighteen compounds in series BA and UA, their structural elucidation could be deduced from IR, 1H, 13C NMR, HR-EI-MS and elemental analysis data . The IC50 for BA and UA were 18 and 36 μM respectively, which is consistent with previously published data [1e, 1f, and 2e showed good activity and BA, 1b, and 1h showed moderate activity, whereas the other compounds, including UA, were considered inactive.Table hed data ,22. Comp50 of compounds 1e, 1f and 2e for HEK293T was > 100 μM; therefore, none was cytotoxic in the tested conditions.The IC1e, 1f and 2e . This suggests that the tested derivatives exert their anti-malarial effects via a mechanism of action distinct from that of CQ in the haemin detoxification pathway.Inhibition percentages for the most active derivatives are reported in Table In this study, semisynthetic compounds were obtained, derived from natural sources, with potential utility as anti-malarial agents, using a simple and inexpensive strategy.in vitro[P. falciparum 3D7, with three compounds exhibiting good activity . These three derivatives possessed a four-carbon side-chain and had anti-malarial activity three to five times greater than that of BA and UA. Some reports have reported a change in activity after modification on the triterpene carbon 3. The 3-O-acetyl ester of ursolic acid exhibited enhanced activity against CQ-resistant P. falciparum FcB1 (IC50 = 24.93 μM) as compared with UA (IC50 = 52.93 μM) [P. falciparum K1 strain [P. falciparum FCR-3 [1e, 1f and 2e) had improved anti-malarial activity . It was also obtained information on the presence of a second carboxylic acid group, as in derivatives 1d (IC50 = 70 μM), 1g (IC50 = 66 μM) and 2g (IC50 = 58 μM); and the presence of a halogen, such as chlorine and fluorine, as in derivatives 1h (IC50 = 22 μM), 1i (IC50 >100 μM), 2h (IC50 = 58 μM) and 2i (IC50 >100 μM). These functional groups did not potentiate anti-malarial action.The triterpenes BA and UA exhibited moderate anti-malarial activity in vitro,22. Both2.93 μM) . Interes1 strain . In anot1 IC50 = .93 μM asP. falciparum 3D7 strain than UA derivatives. The most active derivatives synthesized, 1e, 1f and 2e, did not show any cytotoxicity against HEK293T cells at the tested concentration of 100 μM. This excellent result justifies potential in vivo experiments with these compounds in future. Follow-up studies with non-cytotoxic compounds are important, as adverse effects such as hypoglycaemia, cardiotoxicity, and gastrointestinal discomfort have been described with the anti-malarials: QN, halofantrine, and CQ/proguanil [Comparison of derivatives with the same substituents derived from series BA and series UA demonstrated that BA derivatives are generally more active against roguanil .ΔΨm. The ΔΨm consists of chemical and electrical components generated by electron transport chain enzymes, and its determination is widely used to characterize cellular metabolism, viability and apoptosis [ΔΨm assay was performed for the more active derivatives, 1e, 1f and 2e, by accumulation of the lipophilic cationic fluorescent probe DiOC6(3). When DiOC6(3) is incubated with iRBC, it diffuses into cells and concentrates several orders of magnitude into negative-inside mitochondria. Probe accumulation into parasite mitochondria is dependent on the presence of a membrane potential, collapse of which will result in diffusion of the probe out of the mitochondria, resulting in signal dissipation. The tested derivatives did not exhibit histogram displacement, as observed after incubation with CCCP, indicating dissipation of the membrane potential and abolition of probe accumulation. This experiment showed that, unlike CCCP, the tested compounds were not able to collapse ΔΨm , whereas 1f displayed less inhibitory activity (14%) than the other tested derivatives. Thus, it was observed that modification of carbon 3 plays an important role in the inhibition of β-haematin formation.Derivatives aemozoin . DerivatP. falciparum 3D7. Derivatives 1e, 1f and 2e exhibited good anti-malarial activity in the micromolar range. Derivatives of series BA were generally more active against P. falciparum 3D7 than compounds derived from series UA, with a three- to five-fold difference in activity in relation to their respective aglycones. Furthermore, 1e, 1f and 2e did not exhibit any cytotoxicity against a HEK293T cell line at 100 μM. Regarding mechanism of action, these triterpene derivatives was weaker inhibit β-haematin formation than CQ and did not act on ΔΨm. Further assays are required to elucidate the pathways whereby these anti-malarial compounds exert their effects. This study can contribute to the design and development of potent anti-malarial compounds derived from natural sources.In this study, 18 new derivatives of betulinic acid (series BA) and ursolic acid (series UA) were synthesized with the purpose of investigating the importance of substituents at carbon 3 for anti-malarial activity against BA: Betulinic acid; CQ: Chloroquine; OD: Optical density; UA: Ursolic acidThe authors declare that they have no competing interests.Ψm and inhibition of β-haematin formation. GNSS and LNC drafted the manuscript. SCBG, GG and CRSG corrected the manuscript. All authors approved the final manuscript.SCBG, GG, CRSG and GNSS designed and coordinated the research. GNSS and NRGM obtained the betulinic and ursolic acids, and corresponding derivatives. GNSS and MN performed the experiments for the anti-malarial activity. GNSS and MSM performed the cytotoxicity assays. GNSS, DCS and LNC were responsible for the assay on ΔExtraction of BA and UA. Description: The data provided represent the extraction of betulinic and ursolic acids.Click here for fileIdentification of synthesized derivatives. Description: The data provided represent the identification of the synthesized derivatives.Click here for file