Research advances in the molecular diagnosis of spontaneous bacterial peritonitis / 临床肝胆病杂志

Spontaneous bacterial peritonitis (SBP) is the most common type of infection in end-stage liver disease, and the diagnosis and treatment of SBP are facing great difficulties and challenges. In recent years, great achievements have been made in molecular diagnostic techniques, but they have not been widely used in clinical practice. Based on the current status of the diagnosis of SBP, this article reviews the advances in molecular microbiological methods in the diagnosis of SBP. Bacterial qualitative analysis alone cannot clarify the association between bacterial DNA and clinical manifestations, and the combination of bacterial quantitative analysis and bacterial type can more accurately describe the biological characteristics of SBP, which may help with the diagnosis of SBP and its special types and the application of antimicrobial agents.

Estructura genética de cepas de Mycobacterium tuberculosis farmacorresistentes en Perú con base en haplotipos obtenidos de un ensayo de sonda lineal / Genetic structure of drug-resistant strains of Mycobacterium tuberculosis in Peru based on haplotypes obtained from a linear probe assay

RESUMEN Objetivo. Determinar la estructura genética de las cepas drogorresistentes de Mycobacterium tuberculosis que circularon en todo el Perú durante los años 2011-2015 a través de haplotipos obtenidos de un ensayo con sondas en línea. Materiales y métodos. Se analizaron 6589 muestras que ingresaron al Instituto Nacional de Salud para el diagnóstico rutinario mediante el ensayo GenoType® MTBDRplus v2, durante el periodo de estudio. Se crearon haplotipos resistentes mediante la concatenación de 21 sitios polimórficos de los genes evaluados por el ensayo con sondas en línea, y se realizó el análisis de asociación con fenotipos obtenidos por el método de proporciones agar 7H10. Resultados. Las mutaciones de mayores frecuencias fueron: rpoB S531L (55,4%) y rpoB D516V (18,5%) para la resistencia a rifampicina, y katG S315T (59,5%) e inhA c-15t (25,7%) para la resistencia a isoniacida. Se obtuvieron 13 haplotipos representativos (87,8% de muestras analizadas) de los cuales seis correspondieron al genotipo multidrogorresistente, cuatro al genotipo monorresistente a isoniacida y tres al genotipo monorresistente a rifampicina. Dieciocho departamentos, y la provincia del Callao, presentaron una alta diversidad haplotípica; cuatro presentaron moderada diversidad y dos presentaron baja diversidad. Conclusiones. Existe una alta diversidad haplotípica en la mayoría de los departamentos, además de una concentración de las cepas de Mycobacterium tuberculosis drogorresistentes en las ciudades de Lima y Callao. Asimismo, las cepas de Mycobacterium tuberculosis con perfil drogorresistente que circulan en el Perú contienen principalmente los marcadores genéticos de mayor prevalencia a nivel mundial asociados con la resistencia frente a rifampicina e isoniacida.

ABSTRACT Objective. To determine the genetic structure of drug-resistant strains of Mycobacterium tuberculosis that circulated throughout Peru during the years 2011-2015, by using haplotypes obtained from a line probe assay. Materials and methods. A total of 6589 samples that were admitted to the Instituto Nacional de Salud for routine diagnosis using the GenoType® MTBDRplus v2 assay were analyzed during the study period. Resistant haplotypes were created by concatenating 21 polymorphic sites of the evaluated genes using the line probe assay; and the association analysis was carried out with phenotypes obtained by the 7H10 agar ratio method. Results. The most frequent mutations were: rpoB S531L (55.4%) and rpoB D516V (18.5%) for rifampicin resistance, and katG S315T (59.5%) and inhA c-15t (25.7%) for isoniazid resistance. We obtained 13 representative haplotypes (87.8% of analyzed samples), 6 corresponded to the multidrug-resistant genotype, 4 to the isoniazid mono-resistant genotype and 3 to the rifampicin mono-resistant genotype. Eighteen regions and the province of Callao showed high haplotype diversity; four showed moderate diversity and two showed low diversity. Conclusions. Most regions showed high haplotype diversity; in addition, most drug-resistant strains of Mycobacterium tuberculosis were concentrated in the cities of Lima and Callao. Likewise, drug-resistant Mycobacterium tuberculosis strains circulating in Peru mainly contain the genetic markers with the highest prevalence worldwide, which are associated with resistance to rifampicin and isoniazid.

Analysis of difference in oral bacteria flora in saliva among elderly people in longevity zone of Guangxi / 中华老年医学杂志

Objective To investigate the bacterial species,characteristics and differences of oral bacteria flora of saliva in the longevous elderly between in Bama county and in Debao county in Guangxi,in order to explore the relationship between longevity and oral salivary bacteria flora in the elderly.Methods The saliva was taken from the longevous elderly in Bama county(BM group)and people aged over 60 years in Debao county(BS group)separately,and the total DNA was extracted.The 16S rDNA-V4 region was amplified by PCR and analyzed by sequencing.The main species and diversity of bacterial colonies were recorded for difference analysis.Results A total of 14 saliva samples were collected from 7 cases in BM group and 7 cases in BS group.A total of 369 OTUs were generated by cluster analysis of 14 samples.At the genus level,the dominant salivary bacteria flora were Ctinomyces,Ca pnoc ytophaga,Chryseobacterium,Fusobacterium,Haemophilus,Lactobacillus,Leptotrichia,Neisseria,Porphyromonas,Prevotella,Rothia,Streptococcus,Veillonella in both BM group and BS group.The OTU PCA analysis showed that some evidence for indeterminate differences was found,but statistically significant differences did not exist in the dominant components of oral flora between the two groups(P＞0.05).Also,the same tendency toward the diversity(P＞0.05)was presented.Similarly,the species annotation analysis and the heat map showed that there were no significant differences (P ＞ 0.05) in oral salivary flora composition between the two groups.Lactobacillu was always the prevailing flora in the Phylume,Class,Order,Family and Genus,but the abundance ratio was different between the two groups as following:Lactobacillus abundance in salivary bacteria flora was higher in BM Group than in the BS group,while Mycoplasma abundance was lower in BM Group than in the BS group(P＜0.05).Conclusions The dominant salivary bacteria flora is Lactobacillus in both BM and BS group,while,the abundance of Lactobacillus is higher in the BM group than in the BS group,which indicates that the longevity of population in Bama county may be related to Lactobacillus.

Analysis of difference in oral bacteria flora in saliva among elderly people in longevity zone of Guangxi / 中华老年医学杂志

Objective@#To investigate the bacterial species, characteristics and differences of oral bacteria flora of saliva in the longevous elderly between in Bama county and in Debao county in Guangxi, in order to explore the relationship between longevity and oral salivary bacteria flora in the elderly.@*Methods@#The saliva was taken from the longevous elderly in Bama county(BM group)and people aged over 60 years in Debao county(BS group)separately, and the total DNA was extracted.The 16S rDNA-V4 region was amplified by PCR and analyzed by sequencing.The main species and diversity of bacterial colonies were recorded for difference analysis.@*Results@#A total of 14 saliva samples were collected from 7 cases in BM group and 7 cases in BS group.A total of 369 OTUs were generated by cluster analysis of 14 samples.At the genus level, the dominant salivary bacteria flora were Ctinomyces, Capnocytophaga, Chryseobacterium, Fusobacterium, Haemophilus, Lactobacillus, Leptotrichia, Neisseria, Porphyromonas, Prevotella, Rothia, Streptococcus, Veillonella in both BM group and BS group.The OTU PCA analysis showed that some evidence for indeterminate differences was found, but statistically significant differences did not exist in the dominant components of oral flora between the two groups(P>0.05). Also, the same tendency toward the diversity(P>0.05)was presented.Similarly, the species annotation analysis and the heat map showed that there were no significant differences(P>0.05)in oral salivary flora composition between the two groups.Lactobacillu was always the prevailing flora in the Phylume, Class, Order, Family and Genus, but the abundance ratio was different between the two groups as following: Lactobacillus abundance in salivary bacteria flora was higher in BM Group than in the BS group, while Mycoplasma abundance was lower in BM Group than in the BS group(P<0.05).@*Conclusions@#The dominant salivary bacteria flora is Lactobacillus in both BM and BS group, while, the abundance of Lactobacillus is higher in the BM group than in the BS group, which indicates that the longevity of population in Bama county may be related to Lactobacillus.

Difference Analysis Based on 16S rRNA Sequencing of Different Soil Bacterial Communities / 法医学杂志

Objective To study the structure and differences of bacterial communities in different soils, and to explore the effectiveness of 16S rRNA sequencing in identification of different soil. Methods Soil samples from 7 places in Shanghai were collected, then bacterial genomic DNA were extracted from them. The fragments of hypervariable region from 16S rRNA sequences were sequenced with high-throughput sequencing techniques. The results were quantified or visualized with bioinformatics software. The differences in diversity and abundance among the three kinds of bacterial communities in soil samples from grassland, forests and beaches were compared and analyzed. Results The statistical differences that existed among the alpha diversity indexes of bacterial communities in soil samples of grassland, forests and beaches had statistical significance. The relative abundance and diversity of bacterial communities in these three kinds of soil were significantly different. Grassland soil had higher Acidobacteria abundance, forest soil had higher Proteobacteria abundance, beach soil had higher Actinobacteria abundance. However, the differences in soil bacterial communities in artificial grasslands, natural grasslands and industrial district grasslands did not have statistical significance. Conclusion 16S rRNA sequencing can effectively distinguish different soils. This method may be able to provide clues for first crime scene inference in criminal cases.

Detecção de patógenos em ponta de cateter venoso central por reação em cadeia da polimerase / Detecção de patógenos em ponta de cateter venoso central por reação em cadeia da polimerase

As infecções relacionadas à assistência à saúde (IRAS) são eventos adversos preocupantes em saúde pública, que se configuram como importante causa de morbidade e mortalidade em unidades de terapia intensiva. Dispositivos invasivos como o cateter venoso central (CVC) favorecem um tipo de IRAS, a infecção da corrente sanguínea. Esse evento é comumente diagnosticado por hemocultura e ou cultura da ponta do cateter, entretanto nem sempre o tempo de resposta dos exames ou os achados contribuem com o adequado tratamento. Os avanços em biotecnologia apontam ferramentas capazes de contribuir com diagnósticos de infecção. O objetivo da presente tese foi testar a técnica de reação em cadeia da polimerase (PCR) como ferramenta para detecção de bactérias potencialmente patogênicas em ponta de CVC de pacientes com suspeita de infecção da corrente sanguínea relacionada ao cateter, internados na Unidade de Terapia Intensiva de Adultos de um hospital filantrópico e de ensino no interior de Minas Gerais. Foram abordados os temas extração de DNA e rastreamento molecular em CVC. Tratou-se de um estudo molecular, transversal, descritivo e exploratório. Testes laboratoriais de comparação entre métodos de extração de DNA foram realizados com cepa da bactéria Staphylococcus aureus para posterior aplicação em cateteres coletados de pacientes. Durante um período de seis meses, uma amostra de conveniência com trinta e quarto cateteres removidos de pacientes internados na Unidade de Terapia Intensiva de Adultos, sob suspeita de infecção da corrente sanguínea, foram submetidos à extração de DNA do material biológico contido na parede externa e no interior dos lúmens dos mesmos. Procedeu-se a identificação de bactérias por PCR utilizando um padrão de reagentes e temperaturas. Os resultados encontrados na análise por biologia molecular foram comparados com os resultados das culturas desses pacientes, realizadas pelo hospital. Houve ainda, o levantamento em prontuário de dados dos pacientes: sexo, idade, uso de outros dispositivos invasivos, tempo de permanência do CVC e local de inserção do cateter; e presença de sinais flogísticos no local de inserção do dispositivo. Testes estatísticos com auxílio do programa Stata, versão 15, foram utilizados. A prevalência das bactérias no CVC por teste de PCR foi: Staphylococcus aureaus (50%), Enterococcus faecalis (41,2%), Klebsiella pneumoniae (32,4%), Pseudomonas aeruginosa (20,6%), Acinetobacter baumannii (38,2%) e Escherichia coli (2,9%). Todas as hemoculturas realizadas tiveram ausência de bactérias como resultado do exame. A cultura de ponta de cateter revelou bactérias em 21 (61,8%) dispositivos, enquanto a PCR apresentou positividade em 31 (91,2%). Os patógenos mais detectados são comumente encontrados no ambiente e no microbioma humano, transmitidos aos pacientes inclusive pelas mãos dos profissionais de saúde. Estes achados são relevantes ao se programar medidas de prevenção de infecção da corrente sanguínea relacionada ao CVC. O método de extração do material genômico, o painel de primers e protocolo de amplificação deste estudo identificaram os principais bactérias comumente prevalentes nas infecções da corrente sanguínea. Desta forma, a identificação molecular de bactérias poderá auxiliar na detecção de infecção da corrente sanguínea e a tomada de decisão relativa à escolha da melhor terapia.

Healthcare-associated infections (HAIs) are worrying adverse events in public health. They are an important cause of morbidity and mortality in intensive care units. Invasive devices such as the central venous catheter (CVC) favors a type of HAIs, the bloodstream infection. This event is commonly diagnosed by blood culture and/or culture of the catheter tip, however, the response time of these tests or their results not always contribute to the appropriate treatment. Advances in biotechnology provide tools capable of contributing to diagnoses of infection. The aim of the present thesis was to detect potentially pathogenic bacteria at the tip of a central venous catheter through polymerase chain reaction (PCR). Subjects were treated with DNA extraction and molecular tracing in CVC. It is a cross-sectional molecular study. Laboratory tests comparing DNA extraction methods were performed with the Staphylococcus aureus bacterium for subsequent application to catheters collected from patients. Over a period of 6 months, in an Adult Intensive Care Unit of a philanthropic and training hospital, (n=34) catheters were removed from patients under suspicion of bloodstream infection. All the thirty-four catheters were subjected to DNA extraction from the biological material contained in their wall and inside their lumens. The bacteria were identified by PCR using a standard set of reagents and temperatures. The results found in the analysis by molecular biology were compared with the results of the cultures of these patients, performed by the hospital. Collection of patients' data was also carried out: sex, age, use of other invasive devices, CVC insertion location and period of catheters use; and presence of phlogistic signs in the insertion site of the device. Statistical tests were used with the help of the Stata software, version 15. The prevalence of bacteria in CVCs was: Staphylococcus aureaus (50%), Enterococcus faecalis (41,2%), Klebsiella pneumoniae (32,4%), Pseudomonas aeruginosa (20,6%), Acinetobacter baumannii (38,2%) and Escherichia coli (2,9%). All blood cultures performed had no bacteria as a result of the examination. Catheter-tip culture revealed microorganisms in 21 (61.8%) devices, whereas PCR showed positivity in 31 (91.2%). The most commonly detected pathogens are usually found in the environment and in the microbioma of the skin and they are possibly transmitted to patients by the hands of health professionals. These findings are relevant when programming CVC-related bloodstream infection prevention measures. The genomic material extraction method, primers panel and amplification protocol of this study identified the major pathogens prevalent in bloodstream infections. In this way, molecular identification of bacteria may assist in the detection of bloodstream infection and decision-making regarding the choice of the best therapy.

Impact of postnatal exposure to antibiotics on intestinal microbiome in preterm infants / 中华围产医学杂志

Objective To assess the impact of postnatal exposure to antibiotics on intestinal microbiome in preterm infants with 16S rDNA sequencing technology.Methods This study was conducted on 19 preterm infants admitted to the neonatal intensive care unit (NICU) at Tongji Hospital immediately after birth from September 2015 to February 2016.Two groups were set up according to the duration of antibiotic exposure (＜3 d,n=10;＞7 d,n=9).Fecal samples were collected from each infant within the first day and 2 or 3 weeks after bitrth.High-throughput sequencer (Hiseq 2500) was used for sequencing,from which information on composition and abundance of species,phylogenetic evolution and bacterial community diversity was obtained.Intergroup differences was analyzed with independent samples t-test or Fisher's exact test.Results (1) No statistically significant difference was found in general information about the infants between the two groups.(2) The intestinal flora in preterm infants was mainly composed of Lactococcus,Enterococcus and Bacillus for both groups before antibiotic treatment (36.41％,23.40％ and 14.98％).The proportions of Lactococcus and Bacillus were decreased significantly (1.73％ and 1.25％,P＜0.01) with Enterococcus becoming the predomiant genus (16.73％) after antibiotic treatment,while the relative proportions of Staphylococcus,Clostridium and Bifidobacterium were raised.(3) The Shannon index was decreased after antibiotic exposure [(2.34±0.84) vs (1.06±0.96) in ＜3 d group,and (2.64± 1.04) vs (0.35±0.36) in ＞7 d group,both P＜0.05],and the other three Alpha diversity indexes,including observed species,Chaol and PD whole tree indexes,were also decreased within each group (all P＜0.05).(4) Bacterial assemblages showed high beta diversity in both groups before the usage of antibiotics,but antibiotic therapy reduced the diversity.(5) Anoism analysis showed significant differences in the composition of intestinal flora within each group before and after antibiotic exposure (R=0.555and 0.733,both P=0.001),but no difference was found between the two groups after antibiotic exposure (R=0.060,P=0.138).Conclusions Antibiotic exposure,even short-term (＜3 d) administration,may significantly change the distribution of intestinal microbiota in preterm infants.Prolonged usage of antibiotics could have detrimental influence on intestinal flora.Therefore,for preterm babies,prescription of antibiotics should be cautious,even short-term empirical usage.

Identification of pathgenic bacteria in aqueous and vitreous of endophthalmitis by 16S rDNA sequencing technique / 中华实验眼科杂志

Background Endophthalmitis is a serious complication of intraocular surgery.Conventional identification methods for bacteria are becterial culture and smear method,but these laboratory tests spend a long time and have low positive rates.16S rDNA is the bacterial chromosome encoding ribosomal RNA sequences,and it is determined that 16S rDNA sequencing has a high specificity for the identification of bacteria.Objective This study was to identify the infectious bacteria from aqueous humor or vitreous body in the eyes with endophthalmitis by 16S rDNA sequencing technique,and to investigate the diagnosis efficency of 16S rDNA sequencing technique on bacterial endophthalmitis.Methods Anterior chamber fluid (0.1-0.2 ml) or vitreous humor (0.5-1.0 ml) specimens were collected from 5 eyes of 5 patients with endophthalmitis in Qingdao Eye Hospital from June to December 2015 and used for high throughput sequencing,bacterial culture and smear,respectively.Bacteria DNA was extracted from the specimen with D3096-01 trace DNA kit for the amplification of V3-V4 region of 16S rRNA gene and sequencing of hypervariable region of all microbes in the samples by MiSeq Illumina Sequencing Platform.Then the bioinformatic analysis including analysis of taxonomy,abundance and alpha diversity were performed.Nucleasefree water of 50 μl in the centrifuge tube was used as control.Results Five aqueous humor or vitreous body samples were collected,and the positive results were exhibited by smear examination,with the Gram positive bacilli in the trumatic endophthalmitis eye and Gram negative bacilli in the filtering bleb infectious endophthalmitis eye,and all culture results were negative.16S rDNA squencing showed the positive outcomes in all the 5 samples.The high abundent nacteral genuses were Staphylococcus (65.28％),Streptococcus (18.90％) and Pseudomonas (12.76％) in the trumatic endophthalmitis eye;the major components of sample were Pseudomonas (53.68％),Acinetobacter (8.62％) and Limnobacter (5.96％) in the eye with acute endophthalmitis occurring at 2 days following cataract surgery;the major components in the filtering bleb infectious endophthalmitis eye were Moraxella (88.89％) and Pseudomonas (9.52％);the Pseudomonas was major components in the later-onset endophthalmitis eye (84.63％) and the eye with acute endophthalmitis occurring at 1 day after cataract surgery (97.89％).Conclusions A distinct advantage is found in 16S rDNA sequencing technique for the indentification of the pathogenic bacteria in endophthalmitis eyes due to its high positive rate in comparison with bacterial culture and smear method.

Early diagnosis of colonic anastomotic leakage by detecting bacterial DNAs in rots with PCR / 中国医师杂志

Objective To asses the value of detecting bacterial DNAs in rat's blood with PCR for early diagnosis of colonic anastemotic leakage.Methods 48 healthy female Wistar rats were random divided into three groups: Group A(n=8,sham operation group),Group B(n=20,colonic anastomosis group),Group C(n=20,colonic anastomotic leakage group).Group B and C rats underwent standardized colon resection 3cm away from the ileocecal junction 10cm,Group B rats were done with a complete anastomosis(end-to-end single layer anastomosis with 0# silk sutures) while Group C rats were done with an anastomosis leaving a 5mm opening in colonic anterior wall.lml and 3ml venous blood samples were collected from Group A,B and C.DNAs were extracted from these blood samples and PCR techniques were used to amplify lacZ genes from Escherichia coli and 16S ribosomal RNA genes(16SrRNA genes) 3 days after operation.The data were analyzed by chi square test.Specimens of the experimental intestine were HE stained for pathological studies.Results The positive ratios of expressing lacZ genes in peripheral blood(PB) with PCR in Group C were significantly higher than that in Group B(P<0.05),hut there were no differences between the two groups in expressing 16SrRNA genes(P>0.05).Conclusions It could be a useful way to detect lacZ genes of Escherichia coli from PB by PCR but not 16SrRNA genes for diagnosis of colonic anastomotic leakages.

Detection of periodental pathogens in atherosclerotic lesions in patients / 北京大学学报(医学版)

Objective:To detect periodontopathic bacterial DNA in atherosclerotic vascular lesions in a group of Chinese patients by using polymerase chain reaction(PCR) techniques.Methods: Ten human specimens of atherosclerotic plaque were obtained sterilely.The sclerotic lesions were blade into fine pieces and DNA was isolated from the samples.To detect Porphyromonas gingivalis(Pg),Tannerella forsythia(Tf),Aggregatibacter actinomycetemcomitans(Aa),Prevotella intermedia(Pi),Prevotella nigre-scens(Pn),Treponema denticola(Td),Campylobacter rectus(Cr),PCR amplification of bacterial 16S ribosomal DNA(rDNA) was carried out.Results: PCR assays for bacterial 16S rDNA indicated the presence of periodontal pathogens in 3 out of 10 surgical specimens.DNA of Pg were found in 3 samples,Tf was found only in one among the 3 samples and Pn was found in another sample among the 3 samples.Conclusion: The data confirm that DNA of periodontal pathogens present in atherosclerotic plaques.Pg,Tf,Pn may play a role in the development and progression of atherosclerosis in these Chinese patients.Further studies with large size samples are needed.

Comparison of sequence between adenoviral DNA and Escherichia coli DNA / 解放军医学杂志

Objective To identify the putative CpG-N ODNs in adenovisus 2 DNA (Adv2 DNA) and Adv5 DNA by comparing the sequence difference among Adv2, 5, 12 DNA and E.coli (EC) DNA. Methods Sequences of Adv2, 5, 12 DNA and EC DNA were obtained from the Entrez Nucleotides database at NCBI. The specific CpG motifs of Adv2 DNA and Adv5 DNA were identified after above sequences were analyzed and compared by softwares such as DNATools, BioEdit, and so on. All the 12-ODNs with specific CpG motif core were searched from Adv2 DNA and Adv5 DNA. Results 19 specific CpG motifs were ascertained and 504 12-ODNs were detected in Adv2 DNA and Adv5 DNA. Conclusion 504 12-ODNs were putative CpG-N ODNs in Adv2 DNA and Adv5 DNA.

Real-time quantitative PCR in detection of neonatal sepsis / 中华围产医学杂志

Objective To explore a rapid diagnostic method in neonatal sepsis and bacterial me- ningitis. Methods The primers and TaqMan probes were designed and synthesized based on the sequences of bacterial 16S rRNA gene. Nineteen bacterial strains, 3 different viruses, fungus and human genomic DNA were tested by FQ-PCR assay. Blood specimens and CSF from 195 cases of suspected septicemia were detected with both TaqMan PCR assay and blood or/and CSF culture. Results The FQ-PCR showed very high sensitivity and specificity and was able to detect at least 10 copies of 16S rRNA gene equivalent to 1~2 copies bacterium. No cross-reaction was found with human genomic DNA, other fungus and viruses. Among the 195 cases, the positive rate by FQ-PCR was 12.8%(25 cases) and 7.1%(15 cases) by blood culture ( P

Molecular mechanism of immune cells activated by bacterial DNA / 中国病理生理杂志

Bacterial DNA taken up by immune cells in a CpG motif-independent manner is translocated into endosome. Endosomal maturation is essential for subsequent bacterial DNA-mediated signal transduction. TLR9 is recruited into endosome to recognize bacterial DNA and initiate the TLR/IL-1R signal transduction pathway. As a result , transcription factors NF-?B and AP-1 are activated, which, in turn, leads to proinflammatory cytokine expression and induces a strong acute Th1-like inflammatory response.

Predictable value of PCR in detecting bacterial DNAs for early diagnosis of jejunal anastomotic leakage and ileal anastomotic leakage in rats / 中国普通外科杂志

0.05),but the positive ratio of 16SrRNA genes expression in PB in Group C and Group E was significantly higher than that in group B and Group D respectively(P0.05).Conclusions(1)Detecting 16SrRNA genes from PB with PCR has certain significance for early diagnosis of jejunal anastomotic leakage and ileal anastomotic leakage,(2)PCR might be a useful tool for early diagnosis of jejunal anastomotic leakages and ileal anastomotic leakages by detecting lacZ genes or 16SrRNA genes from ascites.

Mechanism of systemic inflammatory response syndrome induced by EC DNA / 中国病理生理杂志

AIM: To investigate whether the bacterial DNA participates in SIRS and its possible mechanism. METHODS: Escherichia coli genomic DNA (EC DNA) was extracted and purified from Escherichia coli 25922 by alkaline lysis method. Mortality of mice challenged with EC DNA and the changes of TNF-? and IL-6 in rat serum were observed. ANA-1 cells were cultured in vitro, after the cells were stimulated by different concentrations of EC DNA and LPS, the level of TNF-? and IL-6 in supernatant were tested. Meanwhile,expression of TLR9 and TLR4 on cell surface was measured. Activation of NF-?B was also observed. RESULTS: The lethal effect of EC DNA on mice with an obvious dose-effect relationship was observed. The death happened within 24 hours. Calf thymus DNA and DNase I-treated EC DNA did not lead to mice to die. The changes of serum TNF-? and IL-6 in rats induced by EC DNA and LPS were similar, but TNF-? peak level of EC DNA group appeared 1 hour earlier than that of LPS group. In vitro, large amount of TNF-? and IL-6 were released from ANA-1 cells stimulated by EC DNA. High expression of TLR9 and TLR4 was observed on surfaces of THP-1 cells. In particularly, LPS induced strong activation of NF?B. The results suggested other pathway possibly took part in the signal transduction inducea by EC DNA. CONCLUSION: EC DNA has the abilities to lead to death of mice, and induces serum TNF-? and IL-6 level to increase in rats and ANA-1 cells to release cytokines in vitro. High expression of TLR9 and TLR4, strong activation of NF-?B may be its important molecular mechanism, but other pathway probably exists to play an important role.

Screening of bacterial DNA antagonizing CpG oligodeoxynucleotides / 解放军医学杂志

Objective To obtain a potent bacterial DNA antagonizing CpG oligonucleotides (CpG-N ODN) from the structures of Adv2, 5 DNA. Methods Ten putative CpG-N ODNs were synthesized and investigated. Their abilities to inhibit the TNF-? release from hPBMC were observed. Based on the above results, the putative CpG-N ODN was redesigned according to the relationship between the structure and free energy using RNA structure software (version 3.71). Eleven putative CpG-N ODNs were synthesized and screened. Results Out of the ten initial CpG ODNs, ODN101 was the only CpG-N ODN with weak activity to inhibit TNF-? release from hPBMC induced by CpG-N ODN. After redesign, five CpG-N ODNs with strong activity were confirmed. Conclusion Six CpG-N ODNs were obtained with activity to inhibit TNF-? release from hPBMC induced by CpG-N ODN.

INHIBITION OF CHLOROQUINE ON IL-6 RELEASE INDUCED SYNERGISTICALLY BY EC DNA AND ENDOTOXIN / 解放军医学杂志

The aim of this study was to explored the mechanism of inhibitory effect of chloroquine on IL-6 release induced synergistically by bacterial DNA and endotoxin. Before experiment mice were sensitized for 1 hour with D-GalN by I.P. injection. Mortality within seven days was observed after mice were challenged with CpG ODN or LPS with or without chloroquine treatment. ANA-1 cell line cells were cultivated in vitro. We investigated the influence of chloroquine on IL-6 release induced by both EC DNA and LPS. Expressions of TLR4 and TLR9 and activation of NF-?B p65 were investigated in cultured THP-1 cells pretreated with chloroquine and stimulated by EC DNA and LPS. The results showed that all mice died within 4 hours after challenged with CpG ODN and LPS. Only 4 or 5 mice pretreated with chloroquine (30mg/kg) died after challenged with CpG ODN and LPS (P