CD137 is a Useful Marker for Identifying CD4+ T Cell Responses to Mycobacterium tuberculosis.

Upregulation of CD137 on recently activated CD8+ T cells has been used to identify rare viral and tumour antigen-specific T cells from the peripheral blood. We aimed to evaluate the accuracy of CD137 for identifying Mycobacterium tuberculosis (Mtb)-reactive CD4+ T cells in the peripheral blood of infected individuals by flow cytometry and to investigate the characteristics of these CD137+ CD4+ T cells. We initially enrolled 31 active tuberculosis (TB) patients, 31 individuals with latent TB infection (LTBI) and 25 healthy donors. The intracellular CD137 and interferon-γ (IFN-γ) production by CD4+ T cells was simultaneously detected under unstimulated and CFP10-stimulated (culture filtrate protein 10, a Mtb-specific antigen) conditions. In unstimulated CD4+ T cells, we found that the CD137 expression in the TB group was significantly higher than that in the LTBI group. Stimulation with CFP10 largely increased the CD4+ T cell CD137 expression in both the TB and LTBI groups. After CFP10 stimulation, the frequency of CD137+ CD4+ T cells was higher than that of IFN-γ+ CD4+ T cells in both the TB and LTBI groups. Most of the CFP10-activated IFN-γ-secreting cells were CD137-positive, but only a small fraction of the CD137-positive cells expressed IFN-γ. An additional 20 patients with TB were enrolled to characterize the CD45RO+ CCR7+ , CD45RO+ CCR7- and CD45RO- subsets in the CD137+ CD4+ T cell populations. The Mtb-specific CD137+ CD4+ T cells were mainly identified as having an effector memory phenotype. In conclusion, CD137 is a useful marker that can be used for identifying Mtb-reactive CD4+ T cells by flow cytometry.

Expression level and clinical significance of IL-2, IL-6 and TGF-ß in elderly patients with goiter and hyperthyroidism.

OBJECTIVE: To investigate the level of expression and the clinical significance of IL-2 (interleukin-2), IL-6 (interleukin-6) and TGF-ß (transforming growth factor-ß) in elderly patients with goiter and hyperthyroidism. PATIENTS AND METHODS: Gender, age, course of disease, BMI (Body Mass Index), serum FT3 (Free triiodothyronine-3), FT4 (Free triiodothyronine-4), TT3 (Total triiodothyronine-3), TT4 (Total triiodothyronine-4), TSH (Thyroid Stimulating Hormone) and clinical manifestations on admission and other general clinical data and laboratory examination results were collected and statistically analyzed as case group in 128 elderly patients with goiter and hyperthyroidism. Additional 128 over 60-year-old patients with hyperthyroidism were selected as control group. The thyroid tissue of these patients and the control group were examined by fine needle aspiration biopsy. The expressions of IL-2, IL-6, TGF-ß of the thyroid tissue in all patients were detected by immunohistochemistry, qRT-PCR (Real-time Quantitative Polymerase Chain Reaction) and Western blot method respectively, and the statistical analysis was carried out. p < 0.05 indicated that the difference had statistical significance. RESULTS: Compared with the control group, the expressions of IL-2, IL-6 and TGF-ß in the group of patients were significantly higher (p < 0.05). The significantly higher expression of IL-2, IL-6, and TGF-ß was mainly concentrated in the thyroid follicular cells of patients with hyperthyroidism and thyroid enlargement (p < 0.05). In the patients with goiter, hyperthyroidism, and symptoms of exophthalmos, the level of expression of IL-6 was significantly higher than that of patients without exophthalmos (p < 0.05). In the patients with goiter, hyperthyroidism and symptoms of exophthalmos, and the patients with goiter, hyperthyroidism without symptoms of exophthalmos, IL-2 and TGF-ß expression level were not different (p > 0.05). CONCLUSIONS: The expression levels of IL-2, IL-6, and TGF-ß were significantly increased in the patients with senile goiter and hyperthyroidism, but in the senile patients with goiter, hyperthyroidism and exophthalmos symptoms, IL-6 levels were significantly higher than those without exophthalmos. The use of IL-2, IL-6, and TGF-ß is of great significance in the diagnosis of goiter with hyperthyroidism, especially for elderly patients with atypical clinical symptoms of hyperthyroidism.

MiR-370 promotes cell death of liver cancer cells by Akt/FoxO3a signalling pathway.

OBJECTIVE: MicroRNAs have emerged as key regulators in cancer cell biology. In the present study, we investigate the role and the involving mechanism of miR-370 in the progression of liver cancers. MATERIALS AND METHODS: MiR-370 levels were detected by real-time PCR assay. Cell proliferation of HepG2, MHCC-97H and SMMC-7721 was determined by MTT assay. PI staining was detected by FACS analysis. Colony formation was used to test liver cancer cell growth. FoxO3a and Akt expression was determined by western blotting analysis. RESULTS: MiR-370 level was significantly down-regulated in liver cancer cells. Functional analysis revealed that miR-370 mimics suppressed cell proliferation of liver cancer cells, while transfection with miR-370 inhibitor increased cell proliferation of liver cancer cells. Moreover, miR-370 mimics induced cell death of HepG2. Furthermore, Western blotting analysis results demonstrated that miR-370 inhibited the proliferation of liver cancer cells by activating FoxO3a. CONCLUSIONS: MiR-370 inhibited cell proliferation of liver cancer cells by PI3K/Akt signaling pathway. It worked as a tumor suppressor to suppress the progression of human liver cancers.

Modified cytospin slide microscopy method for rapid diagnosis of smear-negative pulmonary tuberculosis.

OBJECTIVE: To evaluate a modified method of the Ziehl-Neelsen stain and determine whether it improves the detection rate of acid-fast bacilli (AFB) in bronchoalveolar lavage fluid (BALF) specimens. DESIGN: Bronchoscopy of patients with suspected smear-negative pulmonary tuberculosis (PTB) patients was conducted to collect BALF to assess the efficacy and accuracy of the modified method for PTB diagnosis. RESULTS: A total of 106 BALF specimens was collected from 74 PTB patients on the basis of BALF samples that were culture-positive for Mycobacterium tuberculosis. When analysed by patient, the sensitivity and specificity of our modified method were respectively 87.8% and 99.6%, while the positive predictive (PPV) and negative predictive values (NPV) were respectively 98.5% and 96.8%. Conversely, the sensitivity of direct smears and concentrated smears was respectively 16.2% and 37.8%, with 100% specificity. On analysing 106 samples, the culture positivity rate of the direct smear and the concentrated smear methods was respectively 76.4%, 13.2% and 34%, while it was 91.5% for the modified method. CONCLUSION: The sensitivity of our modified method was significantly higher than that of direct or concentrated smears. Overall, the modified method improved the detection rate of AFB in BALF specimens, and provided an efficient and accurate diagnosis of PTB in patients with suspected smear-negative PTB.

Inadequate values from an interferon-gamma release assay for smear-negative tuberculosis in a high-burden setting.

OBJECTIVE: To examine the usefulness of an interferon-gamma release assay (IGRA) for the diagnosis of smear-negative tuberculosis (TB) in China. DESIGN: A total of 624 patients with presumed pulmonary TB were enrolled prospectively and categorised as smear-negative TB, smear-positive TB or no TB. All patients were tested using T-SPOT.TB. RESULTS: Both the smear-negative and smear-positive TB groups had significantly more spot-forming cells (SFCs) than the no TB group (all P < 0.001), while the smear-negative group had fewer SFCs than the smear-positive TB group (P < 0.001). The specificity of T-SPOT.TB was 60.4% (95%CI 53.4-67.1). The sensitivities of T-SPOT.TB in the smear-negative and smear-positive TB groups were respectively 81.4% (95%CI 75.7-86.0) and 93.2% (95%CI 87.6-96.4). The sensitivity in the smear-negative TB group was much lower than that in the smear-positive TB (P < 0.05). CONCLUSIONS: The sensitivity of T-SPOT.TB was lower due the paucibacillary nature of the samples, and the specificity was lower due to the high prevalence of latent tuberculous infection in the smear-negative TB patients. The T-SPOT.TB test should only be used as a supplementary test and not as a single test to rule in or rule out smear-negative TB.

Pyrosequencing analysis of the gyrB gene to differentiate bacteria responsible for diarrheal diseases.

Pathogens causing acute diarrhea include a large variety of species from Enterobacteriaceae and Vibrionaceae. A method based on pyrosequencing was used here to differentiate bacteria commonly associated with diarrhea in China; the method is targeted to a partial amplicon of the gyrB gene, which encodes the B subunit of DNA gyrase. Twenty-eight specific polymorphic positions were identified from sequence alignment of a large sequence dataset and targeted using 17 sequencing primers. Of 95 isolates tested, belonging to 13 species within 7 genera, most could be identified to the species level; O157 type could be differentiated from other E. coli types; Salmonella enterica subsp. enterica could be identified at the serotype level; the genus Shigella, except for S. boydii and S. dysenteriae, could also be identified. All these isolates were also subjected to conventional sequencing of a relatively long ( approximately1.2 kb) region of gyrB DNA; these results confirmed those with pyrosequencing. Twenty-two fecal samples were surveyed, the results of which were concordant with culture-based bacterial identification, and the pathogen detection limit with simulated stool specimens was 10(4) CFU/ml. DNA from different pathogens was also mixed to simulate a case of multibacterial infection, and the generated signals correlated well with the mix ratio. In summary, the gyrB-based pyrosequencing approach proved to have significant reliability and discriminatory power for enteropathogenic bacterial identification and provided a fast and effective method for clinical diagnosis.

Observation of Multi-TeV Gamma Rays from the Crab Nebula using the Tibet Air Shower Array.

The Tibet experiment, operating at Yangbajing (4300 m above sea level), is the lowest energy air shower array, and the new high-density array constructed in 1996 is sensitive to gamma-ray air showers at energies as low as 3 TeV. With this new array, the Crab Nebula was observed in multi-TeV gamma-rays and a signal was detected at the 5.5 sigma level. We also obtained the energy spectrum of gamma-rays in the energy region above 3 TeV which partially overlaps those observed with imaging atmospheric Cerenkov telescopes. The Crab spectrum observed in this energy region can be represented by the power-law fit dJ&parl0;E&parr0;&solm0;dE=&parl0;4.61+/-0.90&parr0;x10-12&parl0;E&solm0;3 TeV&parr0;-2.62+/-0.17 cm-2 s-1 TeV-1. This is the first observation of gamma-ray signals from point sources with a conventional air shower array using scintillation detectors.