IL-21 Enhances the Immune Protection Induced by the Vibrio vulnificus Hemolysin A Protein.

We previously reported that the Vibrio vulnificus hemolysin A (VvhA) protein elicited good immune protection and could effectively control V. vulnificus infection in mice. However, its molecular mechanism remains unknown. We hypothesized that hemolysin A induces an immunoprotective response via IL-21 regulation. To demonstrate this, IL-21 expression in mice was regulated by injecting either specific antibodies or rIL-21, and the immune response was evaluated by flow cytometry. Our results suggested that IL-21 enhances immune protection by inducing a T follicular helper cell and germinal center B cell response. We used RNA-seq to explore molecular mechanisms and identified 10 upregulated and 32 downregulated genes involved in IL-21-upregulated protection. Gene Ontology analysis and pathway analysis of the differentially expressed genes were also performed. Our findings indicate that IL-21 can enhance the immune protection effect of the VvhA protein and may serve as a novel strategy for enhancing the immune protection effect of protein vaccines.

Detection and Identification of Serum Peptides Biomarker in Papillary Thyroid Cancer.

BACKGROUND Papillary thyroid cancer (PTC) is currently the most commonly diagnosed endocrine malignancy. In addition, the sex- and age-adjusted incidence of PTC has exhibited a greater increase over the last 2 decades than in many other malignancies. Thus, discovering noninvasive specific serum biomarker to distinguish PTC from cancer-free controls in its early stages remains an important goal. MATERIAL AND METHODS Serum samples from 88 PTC patients and 80 cancer-free controls were randomly allocated into training or validation sets. Serum peptide profiling was performed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) after using weak cation exchange magnetic beads (WCX-MB), and the results were evaluated by use of ClinProTools™ Software. To distinguish PTC from cancer-free controls, quick classifier (QC), supervised neural network (SNN), and genetic algorithm (GA) models were established. The models were blindly validated to verify their diagnostic capabilities. The most discriminative peaks were subsequently identified with a nano-liquid chromatography-electrospray ionization-tandem mass spectrometry system. RESULTS Six peptide ions were identified as the most discriminative peaks between the PTC and cancer-free control samples. The QC model exhibited satisfactory sensitivity and specificity among the 3 models that were validated. Two peaks, at m/z 2671.17 and m/z 1464.68, were identified as fragments of the alpha chain of fibrinogen, while a peak at m/z 1738.92 was a fragment of complement component 4A/B. CONCLUSIONS MS combined with ClinProTools™ software was able to detect peptide biomarkers in PTC patients. In addition, the constructed classification models provided a serum peptidome pattern for distinguishing PTC from cancer-free controls. Both fibrinogen a and complement C4A/B were identified as potential markers for diagnosis of PTC.

Direct Detection of Vibrio vulnificus, Vibrio parahaemolyticus, and Vibrio alginolyticus from Clinical and Environmental Samples by a Multiplex Touchdown Polymerase Chain Reaction Assay.

BACKGROUND: Vibrio vulnificus, V. alginolyticus, and V. parahaemolyticus are commonly and opportunistically pathogenic to humans. METHODS: In this study, a novel multiple touchdown polymerase chain reaction method (MT-PCR) was developed to benefit rapid and simultaneous detection of the presence of the three Vibrio species from the enriched clinical and environmental samples. RESULTS: The method showed a sensitivity of 104 colony forming units (CFU)/mL for V. vulnificus, 103 CFU/mL for V. parahaemolyticus and V. alginolyticus, and a specificity of 100% for all the three Vibrio species. All strains of the three Vibrio species were detected in the spiked samples artificially contaminated with reference strains and were identified directly from the enriched clinical and environmental samples within three hours by this MT-PCR assay. All the corresponding bacteria were isolated from these enriched samples in 48 hours by standard microbiologic procedures. CONCLUSIONS: This MT-PCR method, which can detect V. vulnificus, V. parahaemolyticus, and V. alginolyticus directly and simultaneously, was rapid, sensitive, specific, and can be used in clinical diagnostics, food industry studies, and risk assessment of environment.

Vibrio vulnificus VvhA induces Th1 and Tfh cells to proliferate against Vibrio vulnificus in a mouse model of infection.

AIM: To characterize the roles of VvhA in host's acquired immune response to Vibrio vulnificus infection. MATERIALS & METHODS: The recombinant VvhA fusion protein was used to immunize mice and the anti-VvhA polyclonal antibody was produced in vivo for prophylactic and therapeutic efficacy assay. The roles of VvhA in T helper (Th) cells differentiation were analyzed by vvhA-deleted mutant during the early phase of infection, while the ratio of Th2 and T follicular helper (Tfh) cells were examined in VvhA immunization. RESULTS: Anti-VvhA antibody exhibited neutralization activity against V. vulnificus. Wild-type strain induced higher level of Th1 cells than the mutant, and the concentrations of IgG2a and IFN-γ were increased during the early phase of infection. The spontaneous development of Tfh was observed in immunized model, and the serum IL-21 was increased. CONCLUSION: V. vulnificus VvhA elicited cellular and humoral immune responses by Th1 and Tfh cells to provide protection against VvhA.

Diagnostic value of KLK6 as an ovarian cancer biomarker: A meta-analysis.

Kallikrein-related peptidase 6 (KLK6) is a new potential serum biomarker of ovarian cancer. The aim of the present study was to assess the diagnostic value of KLK6 systematically for ovarian cancer. All the selected studies regarding the changes of KLK6 in ovarian cancer were published prior to April 2015. Five studies involving 485 patients with ovarian cancer, 420 benign cysts and 245 healthy controls met the inclusion criteria. The value of sensitivity, specificity, positive-likelihood ratio (LR+), negative-likelihood ratio (LR-) and area under the receiver operating characteristic curve (ROC) were obtained. All these indices were used to evaluate the diagnostic value of KLK6 for ovarian cancer. The values of sensitivity, specificity, LR+ and LR- (95% confidence interval) of KLK6 were 0.50 (0.47-0.54), 0.91 (0.89-0.93), 7.20 (3.34-15.52) and 0.51 (0.43-0.62), respectively. The area under the summary ROC of KLK6 was 0.86. The index of Q* was 0.79. In conclusion, KLK6 showed high specificity for the diagnosis of ovarian cancer. It can improve the diagnostic accuracy of cancer antigen 125 (CA125). A combined panel of CA125 and KL K6 shows a high diagnostic efficiency for advanced ovarian cancer. Owing to the small number of studies and lack of samples, additional studies meeting the inclusion criteria are required to further analyze the diagnostic value of KLK6 for ovarian cancer.

Identification of Novel Inflammatory Cytokines and Contribution of Keratinocyte-Derived Chemokine to Inflammation in Response to Vibrio vulnificus Infection in Mice.

Currently, only tumor necrosis factor alpha (TNF-α) and interleukin family cytokines have been found to be elicited in Vibrio vulnificus (V. vulnificus)-infected animal models and humans. However, multiple other cytokines are also involved in the immune and inflammatory responses to foreign microorganism infection. Antibody array technology, unlike traditional enzyme-linked immunosorbent assay (ELISA), is able to detect multiple cytokines at one time. Therefore, in this study, we examined the proinflammatory cytokine profile in the serum and liver homogenate samples of bacterial-infected mice using antibody array technology. We identified nine novel cytokines in response to V. vulnificus infection in mice. We found that keratinocyte-derived chemokine (KC) was the most elevated cytokine and demonstrated that KC played a very important role in the V. vulnificus infection-elicited inflammatory response in mice, as evidenced by the fact that the blocking of KC by anti-KC antibody reduced hepatic injury in vivo and that KC induced by V. vulnificus infection in AML-12 cells chemoattracted neutrophils. Our findings implicate that KC may serve as a novel diagnostic biomarker and a possible therapeutic target for V. vulnificus infection.

Complete Genome Sequence of Vibrio alginolyticus ATCC 17749T.

Vibrio alginolyticus is a Gram-negative halophilic bacterium and has been recognized as an opportunistic pathogen in both humans and marine animals. It is the causative agent of food-borne diseases, such as gastroenteritis, and it invades through wounds in predisposed individuals. In this study, we present the completed genome of V. alginolyticus ATCC 17749(T) through high-throughput sequencing.

Serum peptide pattern that differentially diagnoses hepatitis B virus-related hepatocellular carcinoma from liver cirrhosis.

BACKGROUND: Although alpha-fetoprotein (AFP) is a useful serologic marker of hepatocellular carcinoma (HCC), it is not sufficiently sensitive to differentiate HCC and liver cirrhosis (LC) caused by hepatitis B virus (HBV) infection. AIMS: The aim is to discover novel noninvasive specific serum biomarkers for the differential diagnosis of HBV-related HCC and LC. METHODS: With a highly optimized peptide extraction and matrix-assisted laser desorption/ionization time of flight/time of flight mass spectrometric approach, we investigated serum peptide profiles of 80 HCC and 67 LC patients. Three supervised machine learning methods were employed to construct classifiers. Receiver operator curves were plotted to evaluate the performance of classifiers. RESULTS: With a support vector machine-based strategy, we picked nine peaks with m/z ratios of 819.49, 1076.14, 1341.72, 2551.44, 3156.44, 3812.88, 4184.26, 4465.92, and 4776.41 to construct the classifier. We proposed a novel method for distinguishing HCC from cirrhosis, based on a multilayer perceptron (MLP) method. We obtained a sensitivity of 90.0%, specificity of 79.4%, and overall accuracy of 85.1% on an independent test set. The combination of the MLP model and serum AFP level outperformed serum AFP marker alone in distinguishing HCC patients from LC patients. In this experience, sensitivity increased from 62.5% to 87.5%, and specificity increased from 79.4% to 88.2%. CONCLUSIONS: Our results indicate that the MLP model is a novel and useful serum peptide pattern for distinguishing HCC and LC. The peptidome signature alone or together with serum AFP determination may be a more effective method for early diagnosis of HCC in patients with HBV-related LC.

Diagnostic accuracy of osteopontin for malignant pleural mesothelioma: a systematic review and meta-analysis.

BACKGROUND: Osteopontin has been viewed as a promising biomarker for malignant pleural mesothelioma (MPM); however, the conclusions of various studies on diagnostic accuracy of osteopontin have not been consistent. The aim of this study was to conduct a systematic review and meta-analysis to evaluate the diagnostic accuracy of circulating osteopontin for MPM. METHODS: Using appropriate key words, scientific literature that evaluated circulating levels of osteopontin for the diagnosis of MPM was retrieved from electronic databases. Only articles published in English till March 26, 2013 were included in this study. The quality of the studies was assessed using the revised Quality Assessment for Studies of Diagnostic Accuracy (QUADAS-2) tools. The random-effects models were applied for analysing the performance of pooled characteristics. RESULTS: Six studies were included in the analysis. The overall diagnostic sensitivity and specificity were 0.65 (95% CI: 0.60-0.70) and 0.81 (95% CI: 0.78-0.85), respectively. The area under summary receiver operating characteristic (sROC) curves (AUC) was 0.83. The diagnostic accuracy of serum and plasma osteopontin was comparable. CONCLUSIONS: Osteopontin is an effective marker for MPM diagnosis. However, more studies with a larger sample size and better design are needed to rigorously assess the diagnostic power of osteopontin.

[Characteristics of complex chromosomal rearrangement in Chinese male carriers and its impact on male fertility].

OBJECTIVE: To analyze the characteristics of complex chromosomal rearrangement (CCR) in Chinese male carriers and its influence on male fertility. METHODS: Using the G band technique, we conducted karyotype analysis on the peripheral blood lymphocytes of 1,625 Chinese males with reproductive problems. We also searched CNKI and Wanfang database for CCR-related literature published between January 1984 and November 2013, followed by statistical analysis on the CCR characteristics and reproduction-related data of the CCR carriers. RESULTS: Two CCR carriers were found among the 1,625 males and another 47 cases identified from the databases. Among the 49 CCR carriers, there were 17 three-way exchange cases (34.7%), 17 double two-way exchange cases (34.7%), and 15 exceptional cases (30.6%), with no statistically significant differences in the incidence of the three types (P > 0.05). Azoospermia- or oligospermia-induced infertility was found in 19 (38.8% ) of the CCR carriers. A total of 87 pregnancies were achieved in the other 30 (61.2%), among which spontaneous abortion occurred in 75.9% (66/87), dead fetus and malformed infant death in 9.2% (8/87), and phenotypically normal offspring in 14.9% (13/87). Recurrent abortion was associated frequently with breakpoints on CCR-involved chromosomes 6, 7, 8, 11, and 16, while dyszoospermia mostly with breakpoints on CCR-involved chromosomes 10 and 14. The breaking occurred more than 3 times at 1p22, 1q25, 2q31, 5p13, 5q35, 6q23, 8q13, and 20p13. Moreo- ver, the breakpoints at 2q31, 5q35, and 8q13 were particularly related to recurrent abortion, while that at 1p22 only to dyszoospermia. CONCLUSION: CCR is extremely rare. Male CCR carriers are often identified through reproductive problems and have high risks of infertility and abnormal pregnancy and a very low rate of normal newborns. In addition, chromosomes and breakpoints involved in CCR may affect the fertility of male CCR carriers, and some particular chromosomal breakpoints may play a key role in gametogenesis.

Diagnostic accuracy of serum glypican-3 for hepatocellular carcinoma: a systematic review and meta-analysis.

OBJECTIVE: Many individual studies have evaluated the diagnostic efficiency of serum glypican-3 (GPC-3) for diagnosing hepatocellular carcinoma (HCC), but the results have been inconsistent. The aim of present study was to meta-analyze the overall diagnostic accuracy of serum GPC-3 for diagnosing HCC. DESIGN AND METHODS: English language studies which evaluated the diagnostic performance of GPC-3 and published before March 22, 2013 were retrieved. The quality of the studies was assessed by revised QUADAS tools. The performance characteristics were pooled and determined by random-effects models. RESULTS: Twelve studies with a total of 898 HCC patients and 835 non-HCC patients were included. For the studies in which the majority of reference participants had HBV or HCV infections, the overall diagnostic sensitivity and specificity were 0.53 (95% CI: 0.49-0.57) and 0.77 (95% CI: 0.74-0.81), respectively. The area under summary receiver operating characteristic (sROC) curves (AUC) was 0.82. The major design deficiencies of included studies were differential verification bias, and a lack of clear exclusion and inclusion criteria. CONCLUSIONS: GPC-3 has moderate diagnostic accuracy for HCC. Due to the design limitations, results in published studies should be carefully interpreted. In addition, more well-designed studies with large sample sizes should be performed to rigorously evaluate the diagnostic value of the GPC-3.

An MLP classifier for prediction of HBV-induced liver cirrhosis using routinely available clinical parameters.

BACKGROUND: Liver cirrhosis (LC) is the final stage of most of chronic liver diseases and is almost caused by chronic hepatitis B (CHB) in China. Liver biopsy is the reference method for the evaluation of liver cirrhosis. However, it is an invasive procedure with inherent risk. The aim of this study was to construct a new classifier based on the routine clinical markers for the prediction of HBV-induced LC. SUBJECTS AND METHODS: We collected routine clinical parameters from 124 LC patients with CHB and 115 with CHB. Training set (n = 120) and test set (n = 119) were built for model construction and evaluation, respectively. RESULTS: We describe a new classifier, MLP, for prediction of LC with CHB. MLP was built with seven routinely available clinical parameters, including age, ALT, AST, PT, PLT, HGB, and RDW. With optimal cutoff, we obtained a sensitivity of 95.2%, a specificity of 84.2%, and an overall accuracy of 89.9% on an independent test set, which were superior to those of FIB-4 and APRI. CONCLUSIONS: Our study suggests that the MLP classifier can be implemented for discriminating LC and non-LC cohorts by using machine learning method based on the routine available clinical parameters. It could be used for clinical practice in HBV-induced LC assessment.

Draft Genome Sequence of the Human-Pathogenic Bacterium Vibrio alginolyticus E0666.

Vibrio alginolyticus is a Gram-negative halophilic bacterium with worldwide distribution. In this work, we report the draft genome sequence of a V. alginolyticus strain (E0666) isolated from Epinephelus coioides ascites in the Shantou city of Guangdong Province, China.

Expression and biological significance of human kallikrein 6 in gastric cancer tissues.

AIM OF THE STUDY: This study aims to investigate the expression of human kallikrein 6 (hK6) in gastric cancer, gastric ulcer and normal gastric mucosa tissues and its biological significance. MATERIAL AND METHODS: The expression of hK6 in 15 normal gastric mucosa (NGM) tissues, 15 gastric ulcer (GU) tissues and 55 gastric carcinoma (GC) tissues was respectively detected by immunohistochemistry. The correlations between the expression of hK6 and the clinical pathological parameters of gastric cancer were also analyzed. RESULTS: Human kallikrein 6 was mainly expressed in cytoplasm. The positive rate of hK6 was significantly higher in gastric cancer tissues than that in gastric ulcer or normal gastric mucosa tissues (70.9%, 40% and 20%, respectively, p < 0.01). With the increase of the invasion depth of gastric cancer cells, aggravation of TNM stage and development of lymph node metastasis, the expression of hK6 increased significantly (p < 0.05 and p < 0.01). There was no obvious correlation between the expression of hK6 and sex, age, tumor diameter, histodifferentiation degree or primary pathological location of gastric cancer (p > 0.05). CONCLUSIONS: The overexpression of hK6 is related to the depth of invasion, lymph node metastasis and clinical stage of gastric carcinoma, which suggests that hK6 may act as a new marker of gastric cancer biological behavior.

Two classifiers based on serum peptide pattern for prediction of HBV-induced liver cirrhosis using MALDI-TOF MS.

Chronic infection with hepatitis B virus (HBV) is associated with the majority of cases of liver cirrhosis (LC) in China. Although liver biopsy is the reference method for evaluation of cirrhosis, it is an invasive procedure with inherent risk. The aim of this study is to discover novel noninvasive specific serum biomarkers for the diagnosis of HBV-induced LC. We performed bead fractionation/MALDI-TOF MS analysis on sera from patients with LC. Thirteen feature peaks which had optimal discriminatory performance were obtained by using support-vector-machine-(SVM-) based strategy. Based on the previous results, five supervised machine learning methods were employed to construct classifiers that discriminated proteomic spectra of patients with HBV-induced LC from those of controls. Here, we describe two novel methods for prediction of HBV-induced LC, termed LC-NB and LC-MLP, respectively. We obtained a sensitivity of 90.9%, a specificity of 94.9%, and overall accuracy of 93.8% on an independent test set. Comparisons with the existing methods showed that LC-NB and LC-MLP held better accuracy. Our study suggests that potential serum biomarkers can be determined for discriminating LC and non-LC cohorts by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. These two classifiers could be used for clinical practice in HBV-induced LC assessment.

RNA interference-mediated silencing of UBCH10 gene inhibits colorectal cancer cell growth in vitro and in vivo.

1. UbcH10 is the cancer-related E2 ubiquitin-conjugating enzyme, and its overexpression has been demonstrated in a variety of malignancies. The aim of the present study is to silence UbcH10 gene by RNA interference (RNAi) and to observe its inhibitory effect on the colorectal cancer cell growth in vitro and in vivo. 2. We constructed the expression vector pGPU6/GFP/Neo/UbcH10-RNAi (pUbcH10-RNAi), which contained a UbcH10 short hairpin RNA expression cassette. Then the UbcH10 gene silencing cell lines LoVo/UbcH10-RNAi and HT-29/UbcH10-RNAi were established. Reverse transcription-polymerase chain reaction and western blot analysis were used to evaluate the expression of the UbcH10 gene. Cell Counting Kit-8 was used to assess properties of tumour cell growth in vitro. Flow cytometry was used to detect the effect of pUbcH10-RNAi on the cell cycle of colorectal cancer cells. Furthermore, the anti-tumour effects of pUbcH10-RNAi were evaluated in vivo in a nude mouse xenografts model. 3. Results demonstrated that UbcH10 gene expression was significantly decreased in pUbcH10-RNAi treated cells. Colorectal cancer cells growth was markedly suppressed in the pUbcH10-RNAi group compared with control conditions and colorectal cancer cells were arrested in the G2-M phase. In vivo, the downregulation of UbcH10 gene expression by pUbcH10-RNAi also inhibited tumour growth in a nude mice xenograft model. 4. Our study suggests that RNA interference-mediated silencing of UbcH10 gene has anti-tumour activity on colorectal cancer and might have therapeutic potential for the treatment of colorectal cancer.

[Correlation of hK6 expression with clinicopathological features and prognosis in epithelial ovarian cancer].

OBJECTIVE: To approach the relationship between the expression of hK6 in ovarian neoplasm and clinicopathological variables and prognosis in ovarian cancer patients for finding a new tumor marker of the ovarian cancer. METHODS: The expression of hK6 was detected by immunohistochemistry in 19 cases of benign, 11 cases of borderline and 45 cases of malignant ovarian neoplasms and statistically analyzed whether its expression correlate with clinicopathological variables and prognosis in patients with ovarian cancer. RESULTS: The expression of hK6 in ovarian cancer tissues (60.0%) was significantly higher than that in the benign (15.8%) and borderline (27.3%) ovarian neoplasm tissues (P < 0.01). The expression of hK6 in higher-grade ovarian cancer tissues (68.4% ) was higher than that in low-grade ones (14.3%, P < 0.05). The expression of hK6 in late-stage (stage III, 76.7%) was significantly higher than that in early-stage (stage I or II, 26.7%, P < 0.01). The expression of hK6 was significantly higher in patients with lymph node metastasis (77.8%) than that in patients without (33.3%, P < 0.01). The expression of hK6 in the cancer tissues in the patients died, or with reccurence or metastasis within 3 years after surgery was higher (75.0%) than that in the patients with stable disease (42.9%, P < 0.05). CONCLUSION: The expression of hK6 in ovarian cancer was higher than that in benign and borderline ovarian neoplasms. The expression of hK6 is higher in the ovarian cancer of late stage, higher-grade, with lymph node metastasis and is associated with a poorer prognosis. hK6 may become a new markers in prediction of prognosis of the patients with ovarian tumors.