Reference ranges of lymphocyte subsets balanced for age and gender from a population of healthy adults in Chongqing District of China.

The enumeration of lymphocyte subsets plays an essential role in the monitoring of immunological disorders. Immunophenotyping values have been found to be influenced by race, age, gender, and environmental conditions. Therefore, it is important to establish reference ranges for healthy adults from the local population for clinical decision-making. The current study aimed to establish a normal reference range for peripheral blood lymphocyte subsets in healthy adults from the Chongqing District of China by using single-platform flow cytometry. Age- and gender-specific reference ranges were established in 268 healthy adult males and females between 21 and 60 years of age. The CD8+ cell counts decreased with age, CD4+ cell percentages and counts increased with age, and total T cell percentages were higher in the female population. Our results are similar to those reported from other parts of China but different from some results reported from other countries; this further stresses the need to establish local reference ranges by region. Our results will help in the management of patients with human immunodeficiency virus and other immunological disorders in Chongqing District. © 2015 International Clinical Cytometry Society.

Early secreted antigen ESAT-6 of Mycobacterium Tuberculosis promotes apoptosis of macrophages via targeting the microRNA155-SOCS1 interaction.

BACKGROUND: The early secreted antigenic target 6-kDa protein (ESAT-6) of Mycobacterium tuberculosis (Mtb) not only acts as a key player for virulence but also exhibits a strong immunotherapeutic potential against Mtb. However, little is known about the molecular basis for its potential in immunotherapy. The present study was designed to unravel the role of miRNA-155 in ESAT-6-mediated enhancement of host immunity and apoptosis in macrophages. METHODS: Lentivirus-mediated miR-155 sponge and miR-155 and SOCS1 overexpression vectors were developed in macrophages. TLR2- or p65-specific siRNA knockdown was employed to silence TLR2 or p65. Quantitative polymerase chain reaction and western blotting analyses were performed to determine mRNA and protein expression levels, respectively. Macrophage apoptosis was analyzed by flow cytometry. RESULTS: ESAT-6 significantly increased miR-155 expression, which was dependent on TLR2/NF-κB activation in macrophages. Induced expression of miRNA-155 was required for the ESAT-6-mediated protective immune response and macrophage apoptosis. ESAT-6 promoted macrophage apoptosis by targeting the miR-155-SOCS1 pathway. The differential expression levels of TLR2, BIC, and SOCS1 were involved in regulating the immune response in human peripheral blood mononuclear cells of patients with active tuberculosis (TB) and latent TB (LTB). CONCLUSION: ESAT-6 promotes apoptosis of macrophages via targeting the miRNA155-SOCS1 interaction.

Label-free and high-sensitive detection of human breast cancer cells by aptamer-based leaky surface acoustic wave biosensor array.

A label-free and high-sensitive sensing technology for tumor cell recognition and detection was developed based on a novel 2 × 3 model of leaky surface acoustic wave (LSAW) aptasensor array. In this methodology, every resonator crystal unit of the LSAW aptasensor array had an individual oscillator circuit to work without mutual interference, and could oscillate independently with the phase shift stability of ± 0.15° in air phase and ± 0.3° in liquid phase. The aptamer was firstly assembled to the gold electrode surface of 100 MHz LiTaO3 piezoelectric crystal, which could effectively captured target cells (MCF-7 cells) based on the specific interaction between aptamer and the overexpression of MUC1 protein on tumor cell surface. The aptamer-cell complexes increased the mass loading of LSAW aptasensor and led to phase shifts of LSAW. The plot of phase shift against the logarithm of concentration of MCF-7 cells was linear over the range from 1 × 10(2) cells mL(-1) to 1 × 10(7) cells mL(-1) with a correlation coefficient of 0.994. The detection limit as low as 32 cells mL(-1) was achieved for MCF-7 cells. The LSAW aptasensor also exhibited excellent specificity and stability. In addition, this aptasensor could be regenerated for ten times without irreversible loss of activity. Therefore, the LSAW aptasensor may offer a promising approach for tumor cell detection and have great potential in clinical applications.

Rapid detection of hepatitis B virus variants associated with lamivudine and adefovir resistance by multiplex ligation-dependent probe amplification combined with real-time PCR.

Drug-resistant mutations of hepatitis B virus (HBV) are the major obstacles to successful therapy for chronic hepatitis B infection. Although there are many methods for detecting the antiviral drug-resistant mutations of HBV, their applications are restricted because of their shortcomings, such as low sensitivity, the time required, and the high cost. For this study, a multiplex ligation-dependent probe real-time PCR (MLP-RT-PCR) method was developed to simultaneously detect lamivudine (LAM)- and adefovir (ADV)-resistant HBV mutants (those with the mutations rtM204V/I, rtA181V/T, and rtN236T). The new method combined the high-throughput nature of multiplex ligation-dependent probe amplification (MLPA) with the rapid and sensitive detection of real-time PCR. In this report, MLP-RT-PCR was evaluated by detecting drug-resistant mutants in 116 patients with chronic hepatitis B infection. By MLP-RT-PCR analysis, LAM-resistant mutations were detected in 41 patients (35.3%), ADV-resistant mutations were detected in 17 patients (14.7%), and LAM- and-ADV-resistant mutations were detected in 5 patients (4.3%). Based on the results of MLP-RT-PCR, the mutations rtM204V, rtM204I, rtA181T, rtA181V, and rtN236T were 95.7% (111/116 patients), 98.3% (114/116 patients), 99.1% (115/116 patients), 98.3% (114/116 patients), and 99.1% (115/116 patients) concordant, respectively, with those of direct sequencing. The MLP-RT-PCR assay was more sensitive than direct sequencing for detecting mutations with low frequencies. Four samples containing the low-frequency (<10%) mutants were identified by MLP-RT-PCR and further confirmed by clonal sequencing. MLP-RT-PCR is a rapid and sensitive method that enables the detection of multidrug-resistant HBV mutations in clinical practice.

Development and validation of a novel leaky surface acoustic wave immunosensor array for label-free and high-sensitive detection of cyclosporin A in whole-blood samples.

This manuscript described a novel 2×3 model of leaky surface acoustic wave (LSAW) immunosensor array for label-free and high-sensitive detection of Cyclosporin A (CsA) in whole-blood samples. In this technique, every resonator crystal unit of the LSAW immunosensor array had an individual oscillator circuit to work without mutual interference. The LSAW immunosensor was first immobilized with protein A from Staphylococcus aureus and monoclonal anti-CsA antibody on the gold electrode surface of 100 MHz LiTaO3 piezoelectric crystals, which then captured the CsA. The CsA increased the mass loading of LSAW immunosensor and leaded to phase shifts of LSAW. Consequently, under optimal conditions, the designed LSAW immunosensor exhibited a detection limit of 0.89 ng/mL, quantification limit of 2.96 ng/mL, and wide dynamic linear range from 1 ng/mL to 1000 ng/mL for CsA detection. Application of the LSAW immunosensor array to clinical sample revealed that consistency and comparability between LSAW immunosensor and the enzyme multiplied immunoassay method were good. Moreover, the immunosensor could be regenerated for ten times without appreciable loss of activity. Therefore, the self-designed LSAW immunosensor array provided a rapid, accurate, label-free, easy handling, and dynamic real-time method for the detection of immunosuppressive drugs in clinical laboratory.

Association between CD209 -336A/G and -871A/G polymorphisms and susceptibility of tuberculosis: a meta-analysis.

BACKGROUND: The association between CD209 promoter polymorphisms (-336A/G, -871A/G) and tuberculosis (TB) risk has been widely reported, but results of previous studies remain controversial and ambiguous. To assess the association between CD209 polymorphisms and TB risk, a meta-analysis was performed. METHODS: Based on comprehensive searches of the PubMed, Embase, Web of Science, Weipu, and CBM databases, we identified outcome data from all articles estimating the association between CD209 polymorphisms and TB risk. The pooled odds ratio (OR) with 95% confidence intervals (CIs) were calculated. RESULTS: A total of 14 studies with 3,610 cases and 3,539 controls were identified. There was no significant association between CD209 -336A/G polymorphism and TB risk (OR = 1.04, 95% CI = 0.91-1.19 for G vs. A; OR = 1.13, 95% CI = 0.84-1.53 for GG vs. AA; OR = 1.04, 95% CI = 0.87-1.24 for GG+AG vs. AA; OR = 1.11, 95% CI = 0.88-1.39 for GG vs. AG+AA). However, the significant association was revealed for Asians in GG vs. AA (OR = 2.48, 95% CI = 1.46-4.22, P = 0.0008) and GG vs. AG+AA (OR = 2.10, 95% CI = 1.33-3.32, P = 0.001). For the CD209 -871A/G polymorphism, lack of an association was also found (OR = 0.81, 95% CI = 0.70-0.95 for G vs. A; OR = 1.00, 95% CI = 0.52-1.93 for GG vs. AA; OR = 0.73, 95% CI = 0.60-0.89 for GG+AG vs. AA; OR = 1.09, 95% CI = 0.57-2.10 for GG vs. AG+AA). CONCLUSION: The present meta-analysis suggested that CD209 promoter polymorphisms (-336A/G, -871A/G) were unlikely to substantially contribute to TB susceptibility. However, the GG genotype of CD209 -336A/G polymorphism might be a genetic risk factor that increases TB susceptibility for Asians in GG vs. AA and GG vs. AG+AA.

Rapid urinary trypsinogen-2 test in the early diagnosis of acute pancreatitis: a meta-analysis.

OBJECTIVES: Urinary trypsinogen-2 has been implicated as a promising biomarker for the early diagnosis of acute pancreatitis (AP). The meta-analysis was used to establish the overall accuracy of urinary trypsinogen-2 test for diagnosing AP. METHODS: Based on comprehensive searches of the PubMed and Embase databases, we identified and abstracted outcome data from all articles evaluating the diagnostic value of urinary trypsinogen-2. A summary estimate for sensitivity, specificity, 95% confidence region and 95% prediction region was calculated using the bivariate random-effects approach. RESULTS: The meta-analysis included 13 studies (2342 patients, the proportion of severe AP from 13.21% to 30.00%). Overall, the pooled sensitivity was 82.3% (95%CI 79.3%-85.1%) and specificity was 93.5% (95%CI 92.2%-94.6%). The diagnostic odds ratios (DOR) was 85.23 (95%CI 40.14-180.99). The area under the summary ROC curve (AUC) was 0.9673. CONCLUSION: The urinary trypsinogen-2 test is a reliable and rapid method for the early diagnosis of AP.

Rapid and effective diagnosis of tuberculosis and rifampicin resistance with Xpert MTB/RIF assay: a meta-analysis.

OBJECTIVES: Xpert MTB/RIF (Cepheid) assay has been introduced for the diagnosis of tuberculosis (TB) and RIF-resistance. The meta-analysis was used to establish the overall accuracy of Xpert MTB/RIF assay for diagnosing TB and RIF-resistance. METHODS: Based on comprehensive searches of the Pubmed and Embase, we identified outcome data from all articles estimating diagnostic accuracy with Xpert MTB/RIF assay. A summary estimation for sensitivity, specificity, diagnostic odds ratios (DOR) and the area under the summary ROC curve (AUC) was calculated by using the bivariate random-effects approach. RESULTS: The meta-analysis included 18 studies (10,224 suspected specimens). The summary estimate was 90.4% (95%CI 89.2%-91.4%) for sensitivity, 98.4% (95%CI 98.0%-98.7%) for specificity and 328.3/0.9822 for DOR/AUC in pulmonary tuberculosis (PTB). The sensitivity, specificity and DOR/AUC of detecting RIF-resistance were 94.1%, 97.0% and 177.8/0.9832, respectively. For extrapulmonary tuberculosis, the overall pooled sensitivity was 80.4% and specificity was 86.1%. The findings in subgroup analysis were as follows: the accuracy of Xpert MTB/RIF assay is higher in smear-positive specimens and the sensitivity of diagnosing PTB in adults was higher than that in children (90.8% versus 74.3%). CONCLUSIONS: TB and RIF-resistance can be rapidly and effectively diagnosed with Xpert MTB/RIF assay.

Detection of single-nucleotide polymorphisms with novel leaky surface acoustic wave biosensors, DNA ligation and enzymatic signal amplification.

This manuscript describes a new technique for detecting single-nucleotide polymorphisms (SNPs) by integrating a leaky surface acoustic wave (LSAW) biosensor, enzymatic DNA ligation and enzymatic signal amplification. In this technique, the DNA target is hybridized with a capture probe immobilized on the surface of a LSAW biosensor. Then, the hybridized sequence is ligated to biotinylated allele-specific detection probe using Taq DNA ligase. The ligation does not take place if there is a single-nucleotide mismatch between the target and the capture probe. The ligated detection probe is transformed into a streptavidin-horseradish peroxidase (SA-HRP) terminal group via a biotin-streptavidin complex. Then, the SA-HRP group catalyzes the polymerization of 3,3-diaminobenzidine (DAB) to form a surface precipitate, thus effectively increasing the sensitivity of detecting surface mass changes and allowing detection of SNPs. Optimal detection conditions were found to be: 0.3 mol/L sodium ion concentration in PBS, pH 7.6, capture probe concentration 0.5 µmol/L and target sequence concentration 1.0 µmol/L. The detection limit was found to be 1 × 10(-12)mol/L. Using this technique, we were able to detect a single-point mutation at nucleotide A2293G in Japanese encephalitis virus.