Characteristics of Helicobacter pylori Heteroresistance in Gastric Biopsies and Its Clinical Relevance.

BACKGROUND: Antimicrobial susceptibility testing (AST) plays a vital role in anti-Helicobacter pylori treatment, but the traditional AST method has difficulty detecting heteroresistance, which may cause an increased prevalence of resistant strains and eradication failure. AIMS: To investigate the characteristics of heteroresistance in H. pylori in gastric biopsies and investigate its clinical relevance. METHOD: A total of 704 gastric biopsies were selected for 23S rRNA and gyrA gene sequencing, 470 H. pylori isolates from these biopsies were selected for AST, and the clinical characteristics of the patients were reviewed. RESULT: For the 699 biopsies that were positive for 23S rRNA gene, 98 (14.0%) showed a heteroresistance genotype, and a wild type (WT) combined with A2143G (86.7%) genotype was found in most samples. For the 694 biopsies that were positive for gyrA gene, 99 (14.3%) showed a heteroresistance genotype, and a WT combined with 87K (26.3%) or WT combined with 91N (23.2%) genotype was predominant. According to the E-test results, the resistance rates of heteroresistance genotype samples for clarithromycin and levofloxacin were 36.2% and 68.1%, respectively. When dividing the heteroresistance samples into different groups according to the sequencing profile peaks of the mutation position, the resistance rates were higher along with mutation peaks at the mutation position. In addition, patients infected with mutated or heteroresistant strains showed lower peptic ulcer detection rates than those infected with the WT strain (p < 0.05). CONCLUSION: Heteroresistance genotypes for clarithromycin and levofloxacin were not rare in H. pylori. Most cases with a heteroresistance genotype showed a susceptible phenotype for clarithromycin and a resistance phenotype for levofloxacin. Patients infected with heteroresistance genotype strains showed a lower peptic ulcer detection rate than those infected with the WT strain.

Reduction of Human DNA Contamination in Clinical Cerebrospinal Fluid Specimens Improves the Sensitivity of Metagenomic Next-Generation Sequencing.

Metagenomics next-generation sequencing (mNGS) is increasingly available for the detection of obscure infectious diseases of the central nervous system. However, human DNA contamination from elevated white cells, one of the characteristic cerebrospinal fluid (CSF) features in meningitis patients, greatly reduces the sensitivity of mNGS in the pathogen detection. Currently, effective approaches to selectively reduce host DNA contamination from clinical CSF samples are still lacking. In this study, a total of 20 meningitis patients were enrolled, including 10 definitively diagnosed tuberculous meningitis (TBM) and 10 definite cryptococcal meningitis (CM) cases. To evaluate the effect of reduced human DNA in the sensitivity of mNGS detection, three specimen-processing protocols were performed: (i) To remove human DNA, saponin, a nonionic surfactant, was used to selectively lyse white cells in CSF followed by DNase treatment prior to the extraction of DNA; (ii) to reduce host DNA, CSF was centrifuged to remove human cells, and the supernatant was collected for DNA extraction; and (iii) DNA extraction from the unprocessed specimens was set as the control. We found that saponin processing significantly elevated the NGS unique reads for Cryptococcus (P < 0.01) compared with the control but had no effects for Mycobacterium tuberculosis (P > 0.05). However, detection of centrifuged supernatants improved the NGS unique reads for both TBM and CM compared with controls (P < 0.01). Our results demonstrate that the use of mNGS of centrifuged supernatants from clinical CSF samples in patients with TBM and CM is a simple and effective method to improve the sensitivity of pathogen detection.

Fine particulate matter (PM2.5): The culprit for chronic lung diseases in China.

Air pollution is a world public health problem. Particulate matter (PM), a mix of solid and liquid particles in the air, becomes an increasing concern in the social and economic development of China. For decades, epidemiological studies have confirmed the association between fine particle pollutants and respiratory diseases. It has been reported in different populations that increased Fine particulate matter (PM2.5) concentrations cause elevated susceptibility to respiratory diseases, including acute respiratory distress, asthma, chronic obstructive pulmonary disease, and lung cancer. This review will discuss the pathophysiology of PM2.5 in respiratory diseases, which are helpful for the prevention of air pollution and treatment of respiratory tract inflammatory diseases.

MAPK/JNK signalling: a potential autophagy regulation pathway.

Autophagy refers to a lysosomal degradative pathway or a process of self-cannibalization. This pathway maintains nutrients levels for vital cellular functions during periods of starvation and it provides cells with survival advantages under various stress situations. However, the mechanisms responsible for the induction and regulation of autophagy are poorly understood. The c-Jun NH2-terminal kinase (JNK) signal transduction pathway functions to induce defence mechanisms that protect organisms against acute oxidative and xenobiotic insults. This pathway has also been repeatedly linked to the molecular events involved in autophagy regulation. The present review will focus on recent advances in understanding of the relationship between mitogen-activated protein kinase (MAPK)/JNK signalling and autophagic cell death.

Effect of Golgi phosphoprotein 2 (GOLPH2/GP73) on autophagy in human hepatocellular carcinoma HepG2 cells.

This study aims to investigate the effect of Golgi Protein 73 (GP73) on autophagy in human hepatoma line cells HepG2. We investigated the functional effects of GP73 on autophagy in hepatoma cell line HepG2 using immunofluoscence staining, Western blotting and real-time PCR. Our data showed that specific small interference RNA (siRNA) notably induced formation of autophagic vacuoles. In addition, upregulation of GP73 significantly inhibited formation of starvation-induced LC3-positive structures. We provide the first experimental evidence to show that GP73 may play an important role in the inhibitory regulation of autophagy. Therefore, our data suggest a new molecular mechanism for GP73-related hepatoma progression.

Large scale preparation of midkine antisense oligonucleotides nanoliposomes by a cross-flow injection technique combined with ultrafiltration and high-pressure extrusion procedures.

The midkine antisense oligonucleotide (MK-ASODN, 5'-CCC CGG GCC GCC CTT CTT CA-3') nanoliposomes have been identified to suppress hepatocellular carcinoma (HCC) growth effectively, and have a great potential to be an effective target drug for HCC. In this study, a facile and reproducible method for large-scale preparation of MK-ASODN nanoliposomes followed by lyophilization has been developed successfully. Meanwhile, the MK-ASODN nanoliposomes characteristics, storage stability and their antitumor efficiency were studied. The mean particle size of MK-ASODN nanoliposomes were 229.43±15.11 nm, and the zeta potential were 29.7±1.1 mV. High entrapment efficiency values were achieved around 90%. Transmission electron microscopy images revealed spherical shaped nanoliposomes. Nanoliposomes allowed sustained MK-ASODN release for as long as 14 days. During 180 days of storage, freeze-dried nanoliposomes showed no significant change in the mean size, zeta potential, entrapment efficiency and drug release ratio. Regarding their antitumor efficiency, the in vitro proliferation of human liver cancer cells were significantly inhibited by the MK-ASODN nanoliposomes. Furthermore, the MK-ASOND nanoliposomes also significantly inhibited the growth of HCC in the mouse model. In summary, the results confirmed that this large-scale preparation of MK-ASOND nanoliposomes was facile and reproducible, and potentially, could speed up the application process of our MK-ASOND nanoliposomes for HCC therapy.

[Flexible bronchoscopic intervention for endobronchial hamartoma].

OBJECTIVE: To evaluate the effectiveness and safety of interventional treatment in the removal of endobronchial hamartoma by flexible bronchoscopy. METHODS: A retrospective analysis was conducted in 8 inpatients with histologically confirmed endobronchial hamartoma , diagnosed between May 2009 to January 2012 in the First Affiliated Hospital of Nanjing Medical University. The clinical, radiological and bronchoscopic features of hamartoma, and the clinical outcomes after bronchoscopic intervention were described. The endoscopic interventional treatments included resection by electrosurgical snare, electrocautery, argon plasma coagulation (APC) and cryotherapy. Thoracic computed tomography(CT)and bronchoscopy were used to evaluate the airway stenosis during follow-up. RESULTS: The 8 patients, 7 males and 1 female, aged (62 ± 8) years, underwent 13 times of interventional treatment for endobronchial hamartoma. Four patients were cured after receiving a single endoscopic treatment, while 3 patients had recurrence after initial interventional treatment but were cured after the second treatment. Three times of interventional treatment was carried out in 1 patient who had two relapses but later became stable with a 40% stenosis of the airway lumen. The rates of cure and effectiveness were 87.5% and 100%, respectively. Following interventional treatment, pneumothorax occurred in 1 patient who was cured after oxygen therapy. There were no serious complications such as massive haemorrhage, airway perforation, airway ignition and suffocation. CONCLUSION: Interventional treatments through flexible bronchoscopy appear to be safe and effective for removing endobronchial hamartoma.

[Advances on Golgi glycoprotein 73 and its association with diseases].

Golgi glycoprotein 73(GP73) is a transmembrane glycoprotein residing in the cis-Golgi complex, which is strongly expressed in hepatocellular carcinoma (HCC) and secreted into the blood. It has been regarded as a promising serum tumor marker for the detection of HCC with higher sensitivity and specificity than AFP. GP73 is also significantly elevated in kidney cancer, prostate cancer, lung adenocarcinoma, esophageal cancer and seminomas; therefore, it would be helpful for the diagnosis of these diseases. However, the function of GP73 and the regulatory mechanism for its expression are unclear. In this article, the physical-chemical properties, the regulation of its expression, the relation with various cancers and the clinical applications of GP73 are reviewed.

Unusual life-threatening Rosai-Dorfman disease of the trachea: role of NF-κB.

Rosai-Dorfman disease (RDD) is a rare non-neoplastic histioproliferative disorder characterised by painless lymphadenopathy, low fever, high erythrocyte sedimentation rate, leucocytosis and hypergammaglobulinaemia. Overactivity of nuclear factor κB (NF-κB) is linked with inflammatory, cancerous and autoimmune diseases. The first case is described of an unusual life-threatening RDD of the trachea with no lymphadenopathy at risk of suffocation in a 39-year-old Chinese woman. A diagnosis of RDD was made following CT scans, thoracotomy and histological examination. Gel shift assay revealed an essential role for NF-κB overactivity in RDD. The patient remains well with no evidence of progression without treatment. Histological confirmation should be sought in all cases as the clinical manifestation of RDD is similar to asthma or lung carcinoma.

IQGAP1 is overexpressed in hepatocellular carcinoma and promotes cell proliferation by Akt activation.

The scaffold protein IQGAP1 shows elevated levels in several cancer types, but its expression in hepatocellular carcinoma is unknown. We found that 58% of human hepatocellular carcinoma tissue samples had increased IQGAP1 expression compared to adjacent normal tissue. Overexpressing IQGAP1 raised the in vivo tumorigenicity of hepatocellular carcinoma cells, and forced overexpression of IQGAP1 in vitro stimulated cell proliferation. Cell growth was reduced by knockdown or mutation of IQGAP1, or by treatment of cells with a phosphotidylinositol 3-kinase inhibitor. To determine the mechanism by which IQGAP1 overexpression affected hepatocellular carcinoma cells, we confirmed its interaction in these cells with mammalian target of rapamycin (mTOR), a serine/threonine kinase that integrates signals about nutrient and energy status with downstream effectors that influence cell division. In addition, we discovered a new interaction involving IQGAP1, mTOR and Akt, which is a downstream target of mTOR. Akt phosphorylation on Ser-473, which is catalyzed by mTOR and required for Akt activation, increased with increasing amounts of IQGAP1, and decreased with IQGAP1 mutation. We hypothesize that IQGAP1 is a scaffold that facilitates mTOR and Akt interaction.

Inhibition of c-FLIP expression by miR-512-3p contributes to taxol-induced apoptosis in hepatocellular carcinoma cells.

Dysregulation of the antiapoptotic protein cellular FLICE-like inhibitory protein (c-FLIP) has been proven to be associated with tumorigenesis and progress of most human cancers. However, its aberrant expression is poorly elucidated. MicroRNAs (miRNAs) are small non-coding RNAs that are involved in tumorigenesis through negatively regulating gene expression. Our study disclosed that c-FLIP was overexpressed in HepG2 hepatocellular carcinoma cells and down-regulation of c-FLIP enhanced taxol-induced apoptosis. Taxol induction significantly decreased the protein level of c-FLIP. While no decrease in c-FLIP mRNA level was observed, indicating taxol decreased c-FLIP expression through a post-transcriptional mechanism. miR-512-3p was a predicted suppressor of c-FLIP and exhibited an opposite expression manner to c-FLIP before and after taxol induction. Luciferase report assay demonstrated miR-512-3p negatively regulated c-FLIP expression via a conserved miRNA-binding site in 3' untranslated region (3'UTR) of c-FLIP. The decrease of c-FLIP protein due to transfection of miR-512-3p further validated the inhibitory effect of miR-512-3p on c-FLIP. Additional transfection of miR-512-3p remarkably promoted taxol-induced apoptosis, confirming its involvement in apoptosis. In summary, our study disclosed a novel regulatory mechanism that down-regulation of c-FLIP by miR-512-3p contributed to taxol-induced apoptosis. Importantly, the pivotal role of miR-512-3p in determining c-FLIP abundance helps to broaden the implications for cancer therapy by developing small molecules to directly target c-FLIP at mRNA level.

Engineering a pharmacologically superior form of granulocyte-colony-stimulating factor by fusion with gelatin-like-protein polymer.

The plasma half-life of therapeutic proteins is a critical factor in many clinical applications. Therefore, new strategies to prolong plasma half-life of long-acting peptides and protein drugs are in high demand. Here, we designed an artificial gelatin-like protein (GLK) and fused this hydrophilic GLK polymer to granulocyte-colony-stimulating factor (G-CSF) to generate a chimeric GLK/G-CSF fusion protein. The genetically engineered recombinant GLK/G-CSF (rGLK/G-CSF) fusion protein was purified from Pichia pastoris. In vitro studies demonstrated that rGLK/G-CSF possessed an enlarged hydrodynamic radius, improved thermal stability and retained full bioactivity compared to unfused G-CSF. Following a single subcutaneous administration to rats, the rGLK/G-CSF fusion protein displayed a slower plasma clearance rate and stimulated greater and longer lasting increases in circulating white blood cells than G-CSF. Our findings indicate that fusion with this artificial, hydrophilic, GLK polymer provides many advantages in the construction of a potent hematopoietic factor with extended plasma half-life. This approach could be easily applied to other therapeutic proteins and have important clinical applications.

Targeted NF-kappaB inhibition of asthmatic serum-mediated human monocyte-derived dendritic cell differentiation in a transendothelial trafficking model.

Transendothelial trafficking model mimics in vivo differentiation of monocytes into dendritic cells (DC). The serum from patients with systemic lupus erythematosus promotes the differentiation of monocytes into mature DC. We have shown that selective inhibition of NF-kappaB by adenoviral gene transfer of a novel mutated IkappaBalpha (AdIkappaBalphaM) in DC contributes to T cell tolerance. Here we demonstrated for the first time that asthmatic serum facilitated human monocyte-derived DC (MDDC) maturation associated with increased NF-kappaB activation in this model. Furthermore, selective blockade of NF-kappaB by AdIkappaBalphaM in MDDC led to increased apoptosis, and decreased levels of CD80, CD83, CD86, and IL-12 p70 but not IL-10 in asthmatic serum-stimulated MDDC, accompanied by reduced proliferation of T cells. These results suggest that AdIkappaBalphaM-transferred MDDC are at a more immature stage which is beneficial to augment the immune tolerance in asthma.

In vitro and in vivo suppression of hepatocellular carcinoma growth by midkine-antisense oligonucleotide-loaded nanoparticles.

AIM: To synthesize antisense oligonucleotides (ASODNs) of midkine (MK), package the ASODNs with nanoparticles, and to inhibit hepatocellular carcinoma (HCC) growth using these nanoparticles. METHODS: HepG2 cell proliferation was analyzed in vitro using the 3-(4,5-dimethythiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium, inner salt assay. The in vivo activity of nanoparticles delivering the MK-ASODNs was analyzed by histopathological and immunohistochemical staining and quantitative real time polymerase chain reaction (PCR). RESULTS: The in vitro proliferation of HepG2 cells was significantly inhibited by the nanoparticles packaged with MK-ASODNs (NANO-ASODNs). Furthermore, the NANO-ASODNs significantly inhibited the growth of HCC in the mouse model. CONCLUSION: NANO-ASODNs can significantly suppress the growth of HCC in vitro and in vivo.

Expression of surface markers on peripheral CD4+CD25high T cells in patients with atopic asthma: role of inhaled corticosteroid.

BACKGROUND: CD4(+)CD25(+) regulatory T cells (Tregs) mediate immune suppression through cell-cell contact with surface molecules, particularly cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR), and transforming growth factor beta (TGF-beta), but little is known about the exact role of Tregs in the pathogenesis of asthma. This study sought to characterize the expression of surface markers on peripheral blood mononuclear cells-derived Tregs in patients with atopic asthma and healthy subjects, and to investigate the effect of inhaled corticosteroid on them. METHODS: The expression of surface molecules on CD4(+)CD25(high) Tregs was detected by flow cytometry. The effect of inhaled corticosteroid on expression of the surface molecules on Tregs was determined in vivo and in vitro. Total serum immunoglobulin E (IgE) and high-sensitivity C-reactive protein were measured by enzyme linked immunosorbent assay and latex enhanced immunoturbidimetric assay, respectively. RESULTS: Equivalent numbers of peripheral Tregs were found in patients with atopic asthma (stable and acute) and healthy subjects. Tregs preferentially expressed CTLA-4, GITR, toll-like receptor 4 (TLR4), latency-associated peptide (LAP/TGF-beta1), and forkhead box P3 (FOXP3). Patients with acute asthma had decreased numbers of CD4(+)CD25(high)LAP(+) T cells compared to healthy subjects and stable asthmatics. Inhaled corticosteroid enhanced the percentage of Tregs expressing LAP in vivo and in vitro dose-dependently. Furthermore, the percentages of Tregs expressing LAP were negatively correlated with total serum IgE levels and severity of asthma, but positively correlated with forced expiratory volume in one second percentage of the predicted value in patients with asthma. CONCLUSIONS: The results suggest that membrane-bound TGF-beta1 is a potential candidate for predicting the severity of asthma, and may contribute to the sustained remission of asthma. Strategies targeting Tregs on their surface markers, especially TGF-beta1, are promising for future therapy of asthma.

Preparation and characterization of a novel exendin-4 human serum albumin fusion protein expressed in Pichia pastoris.

A novel recombinant exendin-4 human serum albumin fusion protein (rEx-4/HSA) expressed in Pichia pastoris was prepared and characterized. Ex-4 is a 39-amino acid peptide isolated from the salivary gland of the lizard Heloderma suspectum and is thought to be a novel therapeutic agent for type 2 diabetes. But to gain a continued effect, the peptide has to be injected twice a day owing to its short plasma half-life (t(1/2) = 2.4 h). To extend the half-life of Ex-4 molecule in vivo, we designed a genetically engineered Ex-4/HSA fusion protein. Between Ex-4 and HSA, a peptide linker GGGGS was inserted and the fusion protein was expressed in methylotrophic yeast P. pastoris with native HSA secretion signal sequence. The recombinant protein was secreted correctly and was obtained with high purity (typically > 98%) by a three-step purification procedure. cAMP assay demonstrated that the fusion protein had a bioactivity similar to Ex-4 for interaction with GLP-1 receptors in vitro. Results from oral glucose tolerance test indicated that rEx-4/HSA could effectively improve glucose tolerance in diabetic db/db mice. Pharmacokinetics studies in cynomologus monkeys also showed that rEx-4/HSA had a much longer plasma half-life. Therefore, rEx-4/HSA fusion protein could potentially be used as a new recombinant biodrug for type 2 diabetes therapy.

[Human papillomavirus infection in women with cervical lesion of Huzhou area of Zhejiang province].

OBJECTIVE: To observe human papillomavirus (HPV) infection in women with cervical lesions in Huzhou area of Zhejiang province. METHODS: 720 samples of cervical secretion or exfoliated cells were collected from women with cervical lesion in Huzhou area. Human papillomavirus was detected by suspension array technique. RESULTS: Positive HPV infection was detected in 25.42% cases (183/720), with 135 cases of single HPV type, 33 of dual HPV types and 15 of multiple HPV types. HPV16 and HPV58 were the most prevalent types in 183 HPV positive cases. CONCLUSION: The most prevalent high-risk types of HPV are HPV16 and HPV58 in Huzhou area of Zhejiang province.

Wilson disease: identification of two novel mutations and clinical correlation in Eastern Chinese patients.

AIM: To study mutations in the P-type ATPase (ATP7B) gene responsible for Wilson disease (WD) in the Eastern Chinese population, and the possible correlation of specific mutations with clinical characteristics. METHODS: Mutations of the ATP7B gene were sought by means of direct sequencing in 50 Eastern Chinese WD patients of Han ethnic origin. RESULTS: Two novel mutations, Asp96Gly and Asp196Glu, were first identified. We also compared the characterization of mutations in ATP7B with the clinical findings, and a significant correlation with hepatic manifestations between patients carrying the Arg778Leu mutation and those without was found. CONCLUSION: Gene sequencing analysis was shown to have a high detection rate and accuracy. It may become the first priority in screening of WD patients.

Surveillance of viral contamination of invasive medical instruments in dentistry.

OBJECTIVE: To investigate the viral contamination of invasive medical instruments in dentistry and to provide health administrative institutions with surveillance data. METHODS: Sterilized samples were randomly collected from the department of dentistry to detect HBV-DNA, HCV-RNA, HIV-RNA and HBsAg. RESULTS: Of the invasive medical instruments that were sterilized with 2% glutaraldehyde, one of the samples was positive for HBV-DNA, and another sample was positive for HBsAg. CONCLUSION: Though massive virus contamination of invasive medical instruments in dentistry has been reduced to a low level, the occurrence of contamination still remains.