Evaluation of the RT-LAMP and LAMP methods for detection of Mycobacterium tuberculosis.

BACKGROUND: The current methods for detecting Mycobacterium tuberculosis (Mtb) are not clinically optimal. Standard culture methods (SCMs) are slow, costly, or unreliable, and loop-mediated isothermal amplification (LAMP) cannot differentiate live Mtb. METHODS: This study compared reverse transcription (RT)-LAMP, LAMP, and an SCM for detecting Mtb. A first experiment tested the sensitivity and specificity of primers for 9 species of Mycobacterium (H37Rv, M. intracellulare, M. marinum, M. kansasii, M. avium, M. flavescens, M. smegmatis, M. fortuitum, and M. chelonae); and 3 non-Mycobacterium species (Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae). A second experiment tested sputum specimens for the presence of Mtb, from 100 patients with tuberculosis (clinical) and 22 from patients without tuberculosis (control), using Roche solid culture (SCM), LAMP, and RT-LAMP. In the clinical samples. RESULTS: The rates of positivity for Mtb of the SCM, LAMP, and RT-LAMP methods were 88%, 92%, and 100%, respectively. The difference in detection rate was significant between RT-LAMP and SCM, but RT-LAMP and LAMP were comparable. In the control group, the detection rates were nil for all three methods. CONCLUSION: The specificities of the methods were similar. The sensitivity of RT-LAMP was ~10-fold higher than that of LAMP for detecting Mtb. Unlike LAMP, RT-LAMP could identify viable bacteria, and was able to detect a single copy of Mtb. Among SCM, LAMP, and RT-LAMP, the latter is the most suitable for wide use in the lower-level hospitals and clinics of China for detecting Mtb in sputum samples.

Multiparametric diffusion-weighted imaging in breast lesions: Association with pathologic diagnosis and prognostic factors.

PURPOSE: To determine the utility of multiparametric diffusion-weighted imaging (DWI) including monoexponential (apparent diffusion coefficient [ADC]), biexponential (Df , Ds , and f), stretched-exponential (distributed diffusion coefficient [DDC] and α), and kurtosis (mean diffusivity [MD] and mean kurtosis [MK]) models in the differentiation and characterization of breast lesions, and assess their associations with prognostic factors in invasive breast cancer. MATERIALS AND METHODS: This study included 101 patients (44 benign and 57 malignant lesions) who underwent 3T breast multi-b-value DWI. Diffusion model selection was investigated in benign and malignant lesions using the Akaike information criteria (AIC). Mann-Whitney U-test and receiver operating characteristic (ROC) curves were used for statistical analysis. RESULTS: Goodness-of-fit analysis showed that most benign lesion voxels (50.5%) were preferred by the kurtosis model, and most malignant lesion voxels (51.2%) by the stretched-exponential model. All diffusion measures showed significant differences between benign and malignant lesions (P < 0.05), and between in situ and invasive cancers (P < 0.05) except MD (P = 0.103). There were no significant differences in areas under the ROC curves (AUCs) between ADC and non-monoexponential diffusion parameters (P > 0.05), except Df and α, whose AUCs were significantly lower than AUC of ADC for differentiating benign from malignant lesions (P = 0.03 and P < 0.01, respectively). In patients with invasive breast cancer, α was significantly correlated with tumor size (P = 0.007) and Ki-67 expression (P = 0.012), Df was significantly correlated with lymph node metastasis (P = 0.021) and Ki-67 expression (P = 0.042), and ADC, Ds , f, DDC, and MD were significantly correlated with estrogen receptor status (all P < 0.05). CONCLUSION: Multiparametric DWI shows relationships with pathologic outcomes and prognostic factors of breast lesions. LEVEL OF EVIDENCE: 3 Technical Efficacy: Stage 2 J. MAGN. RESON. IMAGING 2017;46:740-750.

Combination of CpG Oligodeoxynucleotide and Anti-4-1BB Antibody in the Treatment of Multiple Hepatocellular Carcinoma in Mice.

BACKGROUND: To investigate the effect of topical application of CpG oligodeoxynucleotide (CpG-ODN) combined with anti-4-1BB antibodies on mouse HCC multiple tumor-bearing models and the degree of improvement of anti-tumor immune response in mice. MATERIALS AND METHODS: We inoculated each BALB/c male mouse subcutaneously with one tumor in the axillae of the four limbs and divided them into four groups. We only selected the tumor-bearing part of the left lower limb for drug treatment. We measured the tumor-bearing volume of mice in each group. Then, we tested the organ coefficients of mice, the concentrations of IL-12 and IFN-γ in peripheral blood, the ratio of spleen Tregs and CD8+T cells, the spleen CTL killing activity, and the survival time of mice. RESULTS: We found that the tumor-bearing volume decreased significantly after the combination of CpG-ODN and anti-4-1BB antibody (P<0.001). The organ coefficients of treated mice were not significantly different from normal mice (P>0.05). The concentration of IL-12 and IFN-in serum and the ratio of CD8+T cells in spleen were increased, while the ratio of spleen Tregs was decreased. CTL activity of spleen was increased. The survival time of mice was significantly prolonged (P<0.001). CONCLUSION: The treatment programme combining CpG-ODN with an anti-4-1BB antibody can significantly reduce tumor growth at the treatment site, slow the growth rate of metastases and improve host prognosis.

Changes in aortic arch geometry and the risk for Stanford B dissection.

BACKGROUND: The increase in aortic diameter is not closely associated with type B aortic dissection (TBAD); morphological risk factors other than aortic diameter may help to better identify patients at risk for TBAD. The purpose of this study was to investigate possible morphological factors associated with the occurrence of TBAD. METHODS: This study was a retrospective, multicenter, cross-sectional study. We collected 94 patients with TBAD as the TBAD group and 534 patients with healthy aortas as the healthy control group. Morphometric data were collected on three-dimensional models of the thoracic aorta. A propensity score matching (PSM) analysis was conducted to reduce the potential for confounding by baseline factors. RESULTS: The number of patients in the TBAD group was 75 after PSM. Longer lengths of the aortic arch (28.00±7.42 vs. 25.14±7.11 cm) were observed in patients with TBAD. The width (80.04±17.27 vs. 71.73±15.55 mm) and height (24.92±11.39 vs. 19.37±10.10 mm) of the aortic arch in patients with TBAD were both larger than those of healthy controls. The morphological changes associated with the occurrence of type B acute dissection were most pronounced in the geometry of the aortic arch. CONCLUSIONS: This study demonstrates that TBAD was associated with longer lengths of aortic arch and with larger arch height and width.

Specific hepatic stellate cell-penetrating peptide targeted delivery of a KLA peptide reduces collagen accumulation by inducing apoptosis.

Liver fibrosis is an aberrant wound-healing process to chronic hepatic inflammation and is characterized by excessive accumulation of extracellular matrix (ECM) that is produced by activated hepatic stellate cells (HSCs). Thus, activated HSCs play a key role in the pathogenesis of liver fibrosis and are a potential target for the treatment of liver fibrosis. Herein, we report that a specific HSC-penetrating peptide reduced collagen accumulation by inducing the apoptosis of HSC-T6 cells. We first screened HSC-specific transduction peptides and identified a novel HSC-targeted cell-penetrating peptide (HTP) that specifically interacted with HSC-T6 cells. A chimeric peptide termed HTPK25 was consequently generated by coupling HTP with the antimicrobial peptide KLA, which is capable of initiating cell apoptosis in mammalian cells. HTPK25 entered cells in a dose-dependent manner, reduced the cell viability and induced apoptosis via the caspase 3 pathway in HSC-T6 cells. Furthermore, HTPK25 inhibited the α-smooth muscle actin and collagen I expression in HSC-T6 cells. Our results demonstrated that the HTP was able to specifically and efficiently deliver the KLA peptide into HSC-T6 cells to induce apoptosis, indicating that HTP-delivered functional agents may present a promising approach for liver fibrosis therapy.

B7-H4 overexpression is essential for early hepatocellular carcinoma progression and recurrence.

B7-H4, another member of costimulatory molecule, has been shown to be overexpressed in multiple types of tumors, including hepatocellular carcinoma (HCC). However, the specific biological role of B7-H4 in HCC still needs to be further explored. In this study, we observed that B7-H4 was highly overexpressed in HCC tissues and cells, and its overexpression strongly correlated with patient's TNM stage, overall survival and early recurrence. Downregulation of B7-H4 significantly suppressed cell growth, invasion, and stemness of HCC by inducing apoptosis in the in vitro experiment. In addition, depletion of B7-H4 could help restore CD8+ T anti-tumor immunity by elevating the expression and secretion levels of CD107a, granzyme A, granzyme B, perforin and IFN-γ. In a xenografted mouse model of HCC, stable depletion of B7-H4 resulted in significantly smaller mean tumor volume and less mean tumor weight after 30 days of growth, compared to the control group. Together, our results provide insights into the diverse functions of B7-H4 involved in the pathogenesis, recurrence and anti-tumor immunity of HCC, indicating B7-H4 as a novel and effective approach for future treatment strategies that benefits anticancer therapy.

Cell-penetrating peptides: Possible transduction mechanisms and therapeutic applications.

Cell-penetrating peptides (CPPs), also known as protein transduction domains, are a class of diverse peptides with 5-30 amino acids. CPPs are divided into cationic, amphipathic and hydrophobic CPPs. They are able to carry small molecules, plasmid DNA, small interfering RNA, proteins, viruses, imaging agents and other various nanoparticles across the cellular membrane, resulting in internalization of the intact cargos. However, the mechanisms of CPP internalization remain to be elucidated. Recently, CPPs have received considerable attention due to their high transduction efficiency and low cytotoxicity. These peptides have a significant potential for diagnostic and therapeutic applications, such as delivery of fluorescent or radioactive compounds for imaging, delivery of peptides and proteins for therapeutic application, and delivery of molecules into induced pluripotent stem cells for directing differentiation. The present study reviews the classifications and transduction mechanisms of CPPs, as well as their potential applications.

Suppression of hepatitis B virus antigen production and replication by wild-type HBV dependently replicating HBV shRNA vectors in vitro and in vivo.

Chronic infection with hepatitis B virus (HBV), a small DNA virus that replicates by reverse transcription of a pregenomic (pg) RNA precursor, greatly increases the risk for terminal liver disease. RNA interference (RNAi) based therapy approaches have shown potential to overcome the limited efficacy of current treatments. However, synthetic siRNAs as well as small hairpin (sh) RNAs expressed from non-integrating vectors require repeated applications; integrating vectors suffer from safety concerns. We pursue a new concept by which HBV itself is engineered into a conditionally replicating, wild-type HBV dependent anti-HBV shRNA vector. Beyond sharing HBV's hepatocyte tropism, such a vector would be self-renewing, but only as long as wild-type HBV is present. Here, we realized several important aspects of this concept. We identified two distinct regions in the 3.2 kb HBV genome which tolerate replacement by shRNA expression cassettes without compromising reverse transcription when complemented in vitro by HBV helper constructs or by wild-type HBV; a representative HBV shRNA vector was infectious in cell culture. The vector-encoded shRNAs were active, including on HBV as target. A dual anti-HBV shRNA vector delivered into HBV transgenic mice, which are not susceptible to HBV infection, by a chimeric adenovirus-HBV shuttle reduced serum hepatitis B surface antigen (HBsAg) up to ∼4-fold, and virus particles up to ∼20-fold. Importantly, a fraction of the circulating particles contained vector-derived DNA, indicating successful complementation in vivo. These data encourage further investigations to prove antiviral efficacy and the predicted self-limiting vector spread in a small animal HBV infection model.

Stable Human Hepatoma Cell Lines for Efficient Regulated Expression of Nucleoside/Nucleotide Analog Resistant and Vaccine Escape Hepatitis B Virus Variants and Woolly Monkey Hepatitis B Virus.

Hepatitis B virus (HBV) causes acute and chronic hepatitis B (CHB). Due to its error-prone replication via reverse transcription, HBV can rapidly evolve variants that escape vaccination and/or become resistant to CHB treatment with nucleoside/nucleotide analogs (NAs). This is particularly problematic for the first generation NAs lamivudine and adefovir. Though now superseded by more potent NAs, both are still widely used. Furthermore, resistance against the older NAs can contribute to cross-resistance against more advanced NAs. For lack of feasible HBV infection systems, the biology of such variants is not well understood. From the recent discovery of Na+-taurocholate cotransporting polypeptide (NTCP) as an HBV receptor new in vitro infection systems are emerging, yet access to the required large amounts of virions, in particular variants, remains a limiting factor. Stably HBV producing cell lines address both issues by allowing to study intracellular viral replication and as a permanent source of defined virions. Accordingly, we generated a panel of new tetracycline regulated TetOFF HepG2 hepatoma cell lines which produce six lamivudine and adefovir resistance-associated and two vaccine escape variants of HBV as well as the model virus woolly monkey HBV (WMHBV). The cell line-borne viruses reproduced the expected NA resistance profiles and all were equally sensitive against a non-NA drug. The new cell lines should be valuable to investigate under standardized conditions HBV resistance and cross-resistance. With titers of secreted virions reaching >3 x 10(7) viral genome equivalents per ml they should also facilitate exploitation of the new in vitro infection systems.