Group B Streptococcus DNA Copy Numbers Measured by Digital PCR Correlates with Perinatal Outcomes.

Group B Streptococcus (GBS) is a one of the main causes of perinatal disease, yet the method for GBS detection, broth-enriched culture, is time-consuming and has low sensitivity and accuracy. We aimed to develop a GBS digital PCR (GBS-dPCR) assay for detecting GBS colonization. More rapid and accurate detection of GBS colonization could increase GBS diagnosis and treatment closer to delivery. A single-center, retrospective, case-controlled study was performed. A total of 182 rectovaginal swabs from pregnant women, who were undergoing prenatal screening by broth-enriched culture, were evaluated using GBS-dPCR targeting the cfb gene of GBS. Pregnant women with GBS colonization were followed up for correlation analysis between GBS DNA copy numbers and perinatal outcomes. The results of the GBS-dPCR assay were compared to those from the broth-enriched culture, which is the gold standard for GBS detection. The sensitivity and specificity of GBS-dPCR were 98% and 92.5%, respectively. By discrepant result analysis, the specificity of GBS-dPCR was raised to 97.4%. The incidence of premature rupture of membrane (PROM) and neonatal infection were statistically significantly positively correlated with GBS DNA copy numbers. GBS-dPCR has the advantage of directly detecting GBS colonization from swabs with high specificity and sensitivity, while reducing turnaround time (<4 h). Analysis of clinical samples with GBS-dPCR shows that GBS DNA copy numbers are positively correlated with the incidence of PROM and neonatal infection, suggesting that dPCR is a promising method for detection of GBS colonization during pregnancy.

Elevated DLL3 in stomach cancer by tumor-associated macrophages enhances cancer-cell proliferation and cytokine secretion of macrophages.

Background: The notch signal pathway is important in the development of both tumor-associated macrophages (TAMs) and stomach cancer, but how Notch signaling affects TAMs in stomach cancer is barely understood. Methods: The expressions of Notch1, Notch2, Notch3, Notch4, hes family bHLH transcription factor 1 (Hes1), and delta-like canonical Notch ligand 3 (DLL3) were detected by Western blot and the expressions of interleukin (IL)-10, IL-12, and IL1-ß were detected using enzyme-linked immunosorbent assay after the co-culture of macrophages and stomach-cancer cells. The proliferation and migration of cancer cells were detected using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and scratch assay, respectively, and the cell cycle was detected using Annexin V/propidium iodide assay. The protein interactions with DLL3 were detected using co-immunoprecipitation and mass spectrometry. Results: The co-culture of macrophages and stomach-cancer cells MKN45 and BGC823 could enhance cell proliferation accompanied by the activation of Notch1/Notch2 signaling and upregulation of DLL3. Notch signaling gamma-secretase inhibitor (DAPT) blocked this process. The overexpression of DLL3 in stomach-cancer cells could promote the proliferation of cancer cells, enhance the activation of Notch1/Notch2 signaling, induce the expression of IL-33, lead to the degradation of galectin-3-binding protein (LG3BP) and heat shock cognate 71 kDa protein (HSPA8), and result in elevated IL-1ß, IL-12, and IL-10 secretion by macrophages. Higher expression of DLL3 or IL-33 could lead to a lower survival rate based on University of California, Santa Cruz Xena Functional Genomics Explorer and The Cancer Genome Atlas data set. Conclusions: This is evidence that DLL3 regulates macrophages in stomach cancer, suggesting that DLL3 may be a novel and potential target for stomach-cancer therapy.

The association between neutrophil-to-lymphocyte count ratio and mortality in septic patients: a retrospective analysis of the MIMIC-III database.

BACKGROUND: Neutrophil-to-lymphocyte count ratio (NLCR) has been shown as a feasible parameter associated with outcomes of tumor patients and an accessible predictor of bacteremia. However, only a handful of research shed the light on the association between NLCR and outcomes of septic patients. This study is aimed to evaluate the association between NLCR and all-cause mortality in a population of adult septic patients. METHODS: We extracted clinical data from Medical Information Mart for Intensive Care (MIMIC)-III V1.4, a free, large-scale, single-center database. NLCR was computed individually. Patients were categorized by quartiles of NLCR. The associations between NLCR quartiles and 28-day all-cause mortality in septic patients were assessed using Cox proportional hazards models and subgroup analyzes. To evaluate the accuracy of NLCR in predicting 28-day mortality of sepsis, receiver operator characteristic curves (ROC), areas under the curve (AUC), and the Youden's J Index were calculated. Other outcomes included 7-day all-cause mortality, mortality in the intensive care units (ICU), in-hospital mortality and length of ICU stay. RESULTS: A total of 3,043 eligible patients were included in the study, of which, 760, 759, 766 and 758 patients were fallen in the first quartile (≤5.89), the second quartile (>5.89, ≤10.69), the third quartile (>10.69, ≤20.25) and the fourth quartile (>20.25) of NLCR, respectively. The 7-day mortality (13.4%, 9.9%, 13.6% and 14.2%; P=0.064) showed no difference in the four quartiles. In multivariate analysis, after adjusting for confounding factors, the highest NLCR quartile (>20.25) was associated with increased 28-day all-cause mortality [hazard ratio (HR) 1.22, 95% Cl: 1.01-1.49; P=0.046]. The areas under the receiver operating characteristic curves (AUROCs) for NLCR was 0.553 (95% CI: 0.529-0.576) for 28-day mortality. CONCLUSIONS: High NLCR (>20.25) is independently related to increased 28-day all-cause mortality in adult septic patients of a limited sensibility and specificity. Further large multi-center prospective studies are needed to confirm such relationship and to validate whose clinical significance.

Notch 1 Is Involved in CD4+ T Cell Differentiation Into Th1 Subtype During Helicobacter pylori Infection.

Helicobacter pylori infection induces CD4+ T differentiation cells into IFN-γ-producing Th1 cells. However, the details of mechanism underlying this process remain unclear. Notch signal pathway has been reported to regulate the differentiation of CD4+ T cells into Th1 subtype in many Th1-mediated inflammatory disorders but not yet in H. pylori infection. In the present study, the mRNA expression pattern of CD4+ T cells in H. pylori-infected patients differed from that of healthy control using Human Signal Transduction Pathway Finder RT2 Profiler PCR Array, and this alteration was associated with Notch signal pathway, as analyzed by Bioinformation. Quantitative real-time PCR showed that the mRNA expression of Notch1 and its target gene Hes-1 in CD4+ T cells of H. pylori-infected individuals increased compared with the healthy controls. In addition, the mRNA expression of Th1 master transcription factor T-bet and Th1 signature cytokine IFN-γ was both upregulated in H. pylori-infected individuals and positively correlated with Notch1 expression. The increased protein level of Notch1 and IFN-γ were also observed in H. pylori-infected individuals confirmed by flow cytometry and ELISA. In vitro, inhibition of Notch signaling decreased the mRNA expression of Notch1, Hes-1, T-bet, and IFN-γ, and reduced the protein levels of Notch1 and IFN-γ and the secretion of IFN-γ in CD4+ T cells stimulated by H. pylori. Collectively, this is the first evidence that Notch1 is upregulated and involved in the differentiation of Th1 cells during H. pylori infection, which will facilitate exploiting Notch1 as a therapeutic target for the control of H. pylori infection.

[Over-expression of human Notch ligand Delta-like 3 promotes proliferation of human gastric cancer cells in vitro].

OBJECTIVE: To construct a eukaryotic expression plasmid carrying human full-length Notch ligand Delta-like 3 (DLL3) gene and study the effect of DLL3 knockdown and overexpression on the proliferation of gastric cancer cells in vitro. METHODS: Human full-length DLL3 gene was amplified by PCR and cloned into the eukaryotic expression vector pCMV-Tag4. After verification by restriction enzymes and sequencing, the recombinant DLL3/pCMV-Tag4 vector was transiently transfected into HEK293T cells, in which the expressions of human DLL3 mRNA and protein were detected using real-time quantitative PCR and Western blotting, respectively. The expression of DLL3 in normal gastric epithelial cells and gastric cancer cell lines was detected by qRT-PCR and Western blotting. DLL3/pCMV-Tag4 was transfected into 3 gastric cancer cell lines, and their proliferation was assessed with MTT assay. Human gastric cancer cells MGC803 and MKN45 were also transfected with a specific human DLL3-siRNA to assess the effect of DLL3 down-expression on the cell proliferation. RESULTS: The recombinant eukaryotic expression vector DLL3/pCMV-Tag4 was successfully constructed and human full-length DLL3 was expressed in HEK293T cells. MTT assay showed that DLL3 over-expression obviously promoted the proliferation and down-regulation of DLL3 inhibited the proliferation of the gastric cancer cells. CONCLUSIONS: DLL3 overexpression can promote the proliferation of gastric cancer cells in vitro, and down-regulation of DLL3 inhibits the proliferation of gastrc cancer cells, which provides a novel strategy for targeted thrapy of gastric cancer.

Identification of Two Lpp20 CD4+ T Cell Epitopes in Helicobacter pylori-Infected Subjects.

Antigen-specific CD4+ T cells play an essential role in effective immunity against Helicobacter pylori (H. pylori) infection. Lpp20, a conserved lipoprotein of H. pylori, has been investigated as one of major protective antigens for vaccination strategies. Our previous study identified two H-2d-restricted CD4+ T cell epitopes within Lpp20 and an epitope vaccine based on these epitopes was constructed, which protected mice in prophylactic and therapeutic vaccination against H. pylori infection. Immunodominant CD4+ T cell response is an important feature of antiviral, antibacterial, and antitumor cellular immunity. However, while many immunodominant HLA-restricted CD4+ T cell epitopes of H. pylori protective antigens have been identified, immunodominant HLA-restricted Lpp20 CD4+ T cell epitope has not been elucidated. In this study, a systematic method was used to comprehensively evaluate the immunodominant Lpp20-specific CD4+ T cell response in H. pylori-infected patients. Using in vitro recombinant Lpp20 (rLpp20)-specific expanded T cell lines from H. pylori-infected subjects and 27 18mer overlapping synthetic peptides spanned the whole Lpp20 protein, we have shown that L55-72 and L79-96 harbored dominant epitopes for CD4+ T cell responses. Then the core sequence within these two 18mer dominant epitopes was screened by various extended or truncated 13mer peptides. The immunodominant epitope was mapped to L57-69 and L83-95. Various Epstein-Barr virus (EBV) transformed B lymphoblastoid cell lines (B-LCLs) with different HLA alleles were used as antigen presenting cell (APC) to present peptides to CD4+ T cells. The restriction molecules were determined by HLA class-antibody blocking. L57-69 was restricted by DRB1-1501 and L83-95 by DRB1-1602. The epitopes were recognized on autologous dendritic cells (DCs) loaded with rLpp20 but also those pulsed with whole cell lysates of H. pylori (HP-WCL), suggesting that these epitopes are naturally processed and presented by APC. CD4+ T cells were isolated from H. pylori-infected patients and stimulated with L57-69 and L83-95. These two epitopes were able to stimulate CD4+ T cell proliferation. This study may be of value for the future development of potential H. pylori vaccine.

Annonaceous acetogenins mediated up-regulation of Notch2 exerts growth inhibition in human gastric cancer cells in vitro.

BACKGROUND: Gastric cancer (GC) is a global health problem because of limited treatments and poor prognosis. Annonaceous acetogenins (ACGs) has been reported to exert anti-tumorigenic effects in cancer, yet the mechanism underlying its effects on GC remains largely unknown. Notch signaling plays a critical role in cell proliferation, differentiation and apoptosis. Therefore, it may contribute to the development of GC. This study aims to explore the role of Notch2 in ACGs' activities in GC cells. RESULTS: ACGs inhibited GC cells' viability in a dose dependent manner and led to cell apoptosis and cell cycle arrest in G0/G1 phase with an increased Notch2 expression. Additionally, Notch2 siRNA reduced ACGs-induced cell growth inhibition while Notch2 cDNA transfection did the opposite. MATERIALS AND METHODS: ACGs were administrated in GC cells and cell proliferation was assayed by MTS, cell apoptosis and cell cycle were detected by flow cytometry. Additionally, the expression of Notch2 and the downstream target Hes1 were identified by Western blot. Furthermore, Notch2-siRNA transfection and Notch2-cDNA were performed to investigate the role of Notch2 in the antitumor effect of ACGs. CONCLUSIONS: Up-regulation of Notch2 by ACGs is a potential therapeutic strategy for GC.

Clinical pharmacology and pharmacokinetics of once-daily hydromorphone hydrochloride extended-release capsules.

Hydromorphone hydrochloride extended release (HHER) is a multiparticulate melt-extrusion pellet capsule formulation administered q24h. Study 1 investigated the bioavailability of 24-mg HHER fed, as well as 24-mg and 12-mg HHER and 8-mg hydromorphone hydrochloride immediate-release (HHIR) tablets fasting. No clinically significant food effect was observed on hydromorphone C(max) or AUC for the 24-mg HHER, and dose proportionality (AUC) was demonstrated between 12- and 24-mg HHER. Study 2 demonstrated dose strength proportionality for 3 x 12-mg HHER versus 1 x 32-mg HHER. Study 3 evaluated 12-mg HHER q24h versus 3-mg HHIR q6h at steady state. HHER produced relatively constant steady-state concentrations over 24 hours. HHER and HHIR were equivalent for AUC(ss). C(ssmax) was 26% lower for HHER than HHIR, C(ssmin) was 43% higher for HHER, and peak-to-trough fluctuation was 126% for HHER versus 328% for HHIR, which are ideal attributes of a once-daily oral extended-release dosage form. HHER administration resulted in fewer adverse events than HHIR in study 3.

Quantification of small molecules in plasma with direct analysis in real time tandem mass spectrometry, without sample preparation and liquid chromatographic separation.

Recently, a new ion source, Direct Analysis in Real Time (DART), has been introduced which allows direct biological sample introduction into a mass spectrometry (MS) system. The elimination of conventionally required sample preparation and separation by high-performance liquid chromatography (HPLC) prior to MS analysis represents a remarkable opportunity to reduce assay turn-around time, environmental impact and capital/manpower investment. This new technology initially was used in various qualitative applications to directly detect chemicals on solid surfaces, in liquids and gases. In this study, a DART source operating under ambient pressure with ground potential was installed onto a Sciex 4000 tandem mass spectrometer and employed in the sample analysis of plasma based on direct introduction into the DART-MS/MS system. Reasonable precision and accuracy (%CV and %Error, both <10%) were achieved of a significant number of compounds tested in biological fluids. In addition, the limit of detection for 80% of the tested compounds reached 5 ng/mL or lower which is sufficient for pharmaceutical drug discovery support. Finally, experimental conditions that significantly impacted assay performance were investigated with respect to optimization and limitation. Because of its simplicity, fast data acquisition (3-5 s) and low cost, DART has the potential to significantly impact quantitative pharmaceutical analysis in biological matrices.

The dose response and effects of dexamethasone on bupivacaine microcapsules for intercostal blockade (T9 to T11) in healthy volunteers.

Biodegradable microcapsules containing bupivacaine/dexamethasone produce an anesthetic duration of 7-11 days in animal models. In this investigation, we explored the effect of increasing doses (Part 1) and the effect of including dexamethasone (Part 2) on the onset, density, and duration of analgesia and anesthesia produced by bupivacaine microcapsules. Concentrations ranging from 0.3125% to 5.0% in microcapsules were compared with 0.25% aqueous bupivacaine (bilateral injection, three intercostal nerves, 2 mL per nerve) (Part 1). Part 2 compared 2.5% microcapsules with or without the inclusion of dexamethasone by unilateral blockade. Sensory block was assessed by pinprick, temperature sensation, and subjective numbness (0, not numb; 10, totally numb). Pharmacodynamic assessments and plasma drug concentrations of bupivacaine and dexamethasone were measured for 96 h. The onset time was reduced and the duration of analgesia increased over the 0.3125%-5.0% dose range (P < 0.02). Onset with 2.5% microcapsules approximated that of 0.25% aqueous bupivacaine. Microcapsule block duration increased to at least 96 h and was significantly longer than aqueous bupivacaine (P < 0.001). Inclusion of dexamethasone increased the duration of pinprick anesthesia in 2.5% microcapsules (P = 0.03). We conclude that bupivacaine/dexamethasone microcapsules are well tolerated and demonstrate a dose-related effect in onset and duration of intercostal blockade. Inclusion of dexamethasone increases intercostal block anesthesia.

A model to evaluate the pharmacokinetic and pharmacodynamic variables of extended-release products using in vivo tissue microdialysis in humans: bupivacaine-loaded microcapsules.

UNLABELLED: Biodegradable microcapsules produce an ultra-long duration of local anesthesia. We hypothesized that this duration is caused by the sustained-release of bupivacaine from the microcapsules into the surrounding tissue. Previous studies investigated the pharmacokinetics (PKs) of bupivacaine after release from microcapsules and absorption into the systemic circulation. Microdialysis sampling can determine the PKs of any drug at its site of injection. This study was performed to characterize the PKs of bupivacaine and dexamethasone released from microcapsules at a subcutaneous injection site over a 96-h period in volunteers. Bupivacaine concentrations were compared with clinical variables of local anesthetic blockade. This study demonstrates that bupivacaine is released in a sustained manner from microcapsules, that bupivacaine concentrations increase for 24-34 h after microcapsule injection, and that analgesia parallels the tissue bupivacaine concentration obtained by microdialysis. Analgesia was equally rapid in onset with aqueous and microcapsule bupivacaine (P = 0.23). Analgesia was still present at 78% of microcapsule-injected sites after 96 h, significantly longer than for aqueous bupivacaine (P < 0.001). Mild pruritus was the most common side effect, occurring with 56% of the microcapsule injections. Dexamethasone-containing bupivacaine microcapsules are well tolerated and produce a prolonged duration of skin analgesia. Systemic absorption of bupivacaine produces higher peak plasma levels after aqueous injection than after microcapsule injection, despite the injection of a threefold larger load of bupivacaine in the latter. IMPLICATIONS: Microcapsules loaded with bupivacaine and dexamethasone and administered by subcutaneous injection produce prolonged cutaneous anesthesia and analgesia. Determination of local tissue pharmacokinetic variables of bupivacaine by microdialysis confirms that the prolonged duration of anesthesia is caused by the extended release characteristics of the microcapsules.