Anti-inflammatory effect of Piper longum L. fruit methanolic extract on lipopolysaccharide-treated RAW 264.7 murine macrophages.

Context: The Piper species was studied several potential properties such as anti-tumor, anti-inflammatory and antioxidant activity. However, the specific anti-inflammatory activity of the extract from the fruits of P. longum L. has not been investigated. Objectives: Our study want to examine the anti-inflammatory effects of P. longum L. fruit methanolic extracts (PLE) on lipopolysachharide (LPS)-stimulated RAW 264.7 murine macrophages to understand the mechanism of this effect. Method: This study examined the chemical profiling of PLE by LC-HRMS analysis and measured the presence of nitric oxide (NO), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) in the supernatant using the Griess reagent assay and enzyme-linked immunosorbent assay (ELISA), respectively. The mRNA expression of IL-6, TNF-α, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) were evaluated by using real-time quantitative polymerase chain reaction (RT-qPCR). Furthermore, the protein expression of COX-2, iNOS and the phosphorylation of MAPK family, c-Jun N-terminal kinase (JNK), p38 in protein level were observed by western blotting. Result: PLE have detected 66 compounds which belong to different classes such as alkaloids, flavonoids, terpenoids, phenolics, lactones, and organic acids inhibited nitric oxide products with the IC50 = 28.5 ± 0.91 µg/mL. Moreover, PLE at 10-100 µg/mL up-regulate HO-1 protein expression from 3 to 10 folds at 3 h. It also downregulated the mRNA and protein expression of iNOS, COX-2, decreased IL-6 and TNF-α secretion by modulating the mitogen-activated protein kinase (MAPK) signaling pathway, specifically by decreasing the phosphorylation of p38 and JNK. Conclusion: These results shown chemical profiling of PLE and demonstrated that PLE exhibits anti-inflammatory effects by regulating the MAPK family and could be a potential candidate for the treatment of inflammatory diseases.

Characterization of Shigella flexneri in northern Vietnam in 2012-2016.

Introduction: Shigellosis remains a considerable public health concern in developing countries. Shigella flexneri and Shigella sonnei are prevalent worldwide and S. sonnei has been replacing S. flexneri . Gap Statement: S. flexneri still causes outbreaks of shigellosis in northern Vietnam but limited information is available on its genetic characteristics. Aim: This study aimed to characterize the genetic characteristics of S. flexneri strains from northern Vietnam. Methodology: This study used 17 isolates from eight incidents, collected in northern Vietnam between 2012 and 2016. The samples were subjected to whole genome sequencing, molecular serotyping, cluster analysis and identification of antimicrobial resistance genes. Additionally, phylogenetic analysis was performed including isolates from previous studies. Results: Clusters were identified according to spatiotemporal backgrounds. The results suggested that two incidents in Yen Bai province in 2015 and 2016 were derived from a very recent common ancestor. All isolates belonged to phylogroup (PG) 3, which was divided into two sub-lineages. Thirteen of 17 isolates, including those from the Yen Bai incidents, belonged to sub-lineage Sub-1 and were serotyped as 1a. The remaining four isolates belonged to sub-lineage Sub-2 and were the globally predominant serotype 2a. The Sub-1 S. flexneri isolates possessed the gtrI gene, which encodes the glycosyl transferase that determines serotype 1a, with bacteriophage elements in the vicinity. Conclusion: This study revealed two PG3 sub-lineages of S. flexneri in northern Vietnam, of which Sub-1 might be specific to the region.

Genomic characterization of endemic diarrheagenic Escherichia coli and Escherichia albertii from infants with diarrhea in Vietnam.

BACKGROUND: Diarrheagenic Escherichia coli (DEC) is a group of bacterial pathogens that causes life-threatening diarrhea in children in developing countries. However, there is limited information on the characteristics of DEC isolated from patients in these countries. A detailed genomic analysis of 61 DEC-like isolates from infants with diarrhea was performed to clarify and share the characteristics of DEC prevalent in Vietnam. PRINCIPAL FINDINGS: DEC was classified into 57 strains, including 33 enteroaggregative E. coli (EAEC) (54.1%), 20 enteropathogenic E. coli (EPEC) (32.8%), two enteroinvasive E. coli (EIEC) (3.3%), one enterotoxigenic E. coli (ETEC), and one ETEC/EIEC hybrid (1.6% each), and surprisingly into four Escherichia albertii strains (6.6%). Furthermore, several epidemic DEC clones showed an uncommon combination of pathotypes and serotypes, such as EAEC Og130:Hg27, EAEC OgGp9:Hg18, EAEC OgX13:H27, EPEC OgGp7:Hg16, and E. albertii EAOg1:HgUT. Genomic analysis also revealed the presence of various genes and mutations associated with antibiotic resistance in many isolates. Strains that demonstrate potential resistance to ciprofloxacin and ceftriaxone, drugs recommended for treating childhood diarrhea, accounted for 65.6% and 41%, respectively. SIGNIFICANCE: Our finding indicate that the routine use of these antibiotics has selected resistant DECs, resulting in a situation where these drugs do not provide in therapeutic effects for some patients. Bridging this gap requires continuous investigations and information sharing regarding the type and distribution of endemic DEC and E. albertii and their antibiotic resistance in different countries.

3-Hydroxyolean-12-en-27-oic Acids Inhibit RANKL-Induced Osteoclastogenesis in Vitro and Inflammation-Induced Bone Loss in Vivo.

Olean-12-en-27-oic acids possess a variety of pharmacological effects. However, their effects and underlying mechanisms on osteoclastogenesis remain unclear. This study aimed to investigate the anti-osteoclastogenic effects of five olean-12-en-27-oic acid derivatives including 3α,23-isopropylidenedioxyolean-12-en-27-oic acid (AR-1), 3-oxoolean-12-en-27-oic acid (AR-2), 3α-hydroxyolean-12-en-27-oic acid (AR-3), 23-hydroxy-3-oxoolean-12-en-27-oic acid (AR-4), and aceriphyllic acid A (AR-5). Among the five olean-12-en-27-oic acid derivatives, 3-hydroxyolean-12-en-27-oic acid derivatives, AR-3 and AR-5, significantly inhibited receptor activator of nuclear factor-κB ligand (RANKL)-induced mature osteoclast formation by reducing the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts, F-actin ring formation, and mineral resorption activity. AR-3 and AR-5 decreased RANKL-induced expression levels of osteoclast-specific marker genes such as c-Src, TRAP, and cathepsin K (CtsK) as well as c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1). Mice treated with either AR-3 or AR-5 showed significant protection of the mice from lipopolysaccharide (LPS)-induced bone destruction and osteoclast formation. In particular, AR-5 suppressed RANKL-induced phosphorylation of JNK and ERK mitogen-activated protein kinases (MAPKs). The results suggest that AR-3 and AR-5 attenuate osteoclast formation in vitro and in vivo by suppressing RANKL-mediated MAPKs and NFATc1 signaling pathways and could potentially be lead compounds for the prevention or treatment of osteolytic bone diseases.

Analysis of the interactions between environmental and food contaminants, cadmium and deoxynivalenol, in different target organs.

Cadmium (Cd), a common and widespread toxic heavy metal, and mycotoxins such as deoxynivalenol (DON) are frequent contaminants of the food supply. Most of the data on their toxicity concern their effects when present alone. However, consumers can be exposed to a cocktail of DON and Cd. To improve the understanding of their combined toxicity, the effects of DON and Cd alone or in combination were investigated in different human cell lines from the kidney (HEK-293), intestine (Caco-2), blood (HL-60) and liver (HepG2). Cytotoxicity was assessed through ATP measurement and types of interactions determined by the Isobologram-Combination index method. HEK-293 cells were exposed to increasing doses of DON, Cd and their combination at different ratios (DON/Cd of 2/1; 1/1; 1/2 and 1/8). Regardless of the ratio, the type of interaction observed in HEK-293 cells ranged from moderate antagonism to nearly additive with increasing cytotoxicity. In Caco-2 cells, the interactions ranged from nearly additive to antagonism whatever the ratio. At ratio 1/1, in HL-60 and HepG2 cells, interactions ranged from synergy to antagonism depending on the cytotoxicity level. Using human cells lines, this study indicates that the consequences of combined exposure to environmental and food contaminants are specific to the target organ. Further studies are needed to confirm these data in vivo.

Molecular epidemiology of Vibrio cholerae O1 in northern Vietnam (2007-2009), using multilocus variable-number tandem repeat analysis.

Cholera is an infectious disease of major concern in Vietnam and other Asian countries. In 2009, there was a large outbreak of cholera in northern Vietnam. To investigate relationships among isolates of the causative pathogen Vibrio cholerae in this region since 2007, we carried out a multilocus variable-number tandem repeat analysis (MLVA) of 170 isolates collected between 2007 and 2009. A total of 24 MLVA types were identified using seven loci. Five clones (1-5) were identified using five loci of the large V. cholerae chromosome; clones 1 and 2 were major, and the others were minor. Clone 1 isolates were responsible for the 2009 outbreak. A shift in the predominant clone occurred between 2007 and 2009, with clone 1 likely derived from clone 2. Moreover, the former was less diverse than the latter, suggesting a single source of cholera dissemination. Epidemiological data indicated a wavelet prior to the large outbreak, suggesting that drinking water source or food chain became contaminated during dissemination. Our results reveal the utility of MLVA for analysis of V. cholerae isolates within a relatively short period and broaden our understanding of its transmission and response to cholera.

Characterization of Vibrio cholerae O139 of an Aquatic Isolate in Northern Vietnam.

During the cholera survey in Namdinh province (northern Vietnam) in July, 2010, one strain of Vibrio cholerae O139 was isolated from 7 environmental water samples positive for ctxA, toxR,VCO139 genes and named as V. cholerae O139, ND1 strain. This strain was lysogenic harbouring a genome similar to the filamentous phage fs1. The replicative form DNA of this phage (named as ND1-fs1, 6856 bp) was sequenced and compared with the other filamentous phages. The filamentous phage ND1-fs1 integrates into the region between ctxB and rtxA genes. The genetic organization of the CTXϕ of V. cholerae O139, strain ND1 was determined and the schematic representation of the genetic organization was shown together with the ND1-fs1 prophage.

Filamentous vibriophage fs2 encoding the rstC gene integrates into the same chromosomal region as the CTX phage [corrected].

The genome of the filamentous phage of Vibrio cholerae fs2 was found to contain rstC and rstB1 (truncated) genes downstream of ORF500. att-fs2-dir and att-fs2-rev sequences homologous to that of att-CTXphi were found between orf500 and rstC of the fs2 genome. This prompted us to search for the integration site of fs2 in the genomes of V. cholerae O1 and O139. The genome of fs2 was found to integrate downstream of attRS of the CTXphi phage, which integrated into chromosome I of V. cholerae O1 and O139. When infected with fs2, a fimbriate strain of V. cholerae O1 appeared to reduce fimbrial production in an adult rabbit ileal loop assay.

Application of Pulsed field gel electrophoresis (PFGE) for typing sudy base on Haemophilus influenzae molecular

The study was conducted to compare the molecular differences of Haemophilus influenzae type b (Hib) strains causing meningitis in young children with H. influenzae strains causing acute upper respiratory tract infection, 44 Hib strains and 37 H. influenzae strains were analysed by pulsed field gel electrophoresis (PFGE) with SmaI restriction enzyme. The results: 44 Hib strains were mainly distributed into 4 DNA restriction patterns (PFGE patterns) whereas 37 H. influenzae strains were distributed into 22 patterns and 81% of them were not encapsulated. The genome of Hib strains causing bacterial meningitis showed less variation than that of H. influenzae causing acute upper respiratory infection. PFGE is a useful tool for epidemiological research.

Familial factor in contagiousness of Haemophilus influenzae type B meningitis in children

Seventy-nice pediatric patient - family pairs were detected for Hib or Hib-antigen. The study also examined 22 pairs of H. influenzae strains isolated from CSF of patients and from upper respiratory tract of the family members of the patients to determine serotype, biotype, presence of Beta-lactamase, PCR with Hib-specified primers, and PFGE with SmaI restriction enzyme. The results showed that 44.4% of the patients` families had Hib carriers; all of 22 H. influenzae isolated pairs from patients and their families had serotype b; 17 of 22 pairs had characteristics of biotype II; 16 of 22 pairs had Beta-lactamase; and 9 of 22 pairs had both the PCR pattern and PFGE pattern. Close contact with Hib carriers was a factor of Hib meningitis contagiousness in children under 5 years of age

Haemophilus influenzae type b (Hib) molecular relationship of Hib pathogenous strains isolated in Hanoi

34 Haemophilus influenzae type b (Hib) strains isolated from cerebrospinal fluids (CSFs) of meningitis patients at National Hospital of Pediatrics, Hanoi, between 11/2002 and 12/2003 were biotyped, serotyped and analyzed by using PCR with specific primers, Pulsed field gel electrophoresis (PFGE) with Smal restriction enzyme. The results showed that 24 of 34 Hib strains (70.6%) belong to biotype II; 8 of 34 (23.5%) belong to biotype I. PCR patterns with capsular polysaccharide type b specified primers of the 34 Hib strains were identical. DNA restriction patterns generated by PFGE (so-called PFGE patterns) of the 34 Hib strains were mainly distributed into 2 PFGE patterns. DNA restriction patterns had close relationship to biotype. The presence of PFGE patterns of Hib strains was not related to season

Use of mycobacterium vaccae as immuno-stimulant in the preparation of H.influenzae type b (Hib) antisera

In order to enhance the capability of antiserum production rabbits, Mycobacterium vaccae was used to stimulate rabbit’s immune system unspecifically before immunization with Haemophilus influenzae type b (Hib). The results showed that only single immunization with Hib antigen: the number of rabbits had response fulfill depended-time requirements (harvested after 1 or 6 months) varied from 30% to 70%, harvesting time for antiserum with fulfilled quantity and quality was 24 weeks (6 months) (2 courses induced immunization). Stimulating rabbit’s immune system by M. vaccae, then inducing rabbit’s immunization with Hib antigen: the number of rabbits had response fulfiling requirements varied from 71.4% to 100%; harvesting time for antiserum with fulfilled quantity and quality was 5 weeks (only after 1 course induced immunization). Sensitivity and specificity of antiserum were unchanged

Some techniques of detection of type B Haemophilus influenza in the diagnosis of meningitis due to type B H. influenza

Some techniques of detection of Hib were compared, including classic techniques such as bacterial culture and modern techniques such as PCR method. The results were as follows: 21% (+) culture with Hib compared to the total of clinically diagnosed cases, Hib-latex agglutination with 95% sensitivity and 99.6% specificity, counter immunophoresis assay to detect Hib by 97% sensitivity and 100% specificity. PCR increased the number of positive cases of Hib meningitis by 42.8% compared to culture alone. Blood culture gave a highly significant value to diagnose (51% Hib-possitive culture)

Early diagnosis of meningitis due to haemophilus influenzae type b in children under 5 years of age

Clinical characteristics and cerebrospinal fluid abnormalities in 100 cases of confirmed B type of H.influenzae (Hib) meningititis were analysed in comparing with 105 cases of meningitis caused by other bacterial pathogens. The basic criteria for early diagnosis of Hib meningitis in under five year old children were found out. Results showed that 48.8% of cases of meningititis were due to Hib; 96% of CSF samples due to Hib had physiological change in comparing with 72.4% in meningitis due to other bacterial pathogens, in 92% there were clinical manifestations and biochemical/cytological typical changes in CSF, in 100% of cases of Hib meningitis(+), there were typical changes; 51% of cases there were positive blood culture and 47% there were Hib positive CSF and blood culture concurrently