Tim-3 Is Upregulated in NK Cells during Early Pregnancy and Inhibits NK Cytotoxicity toward Trophoblast in Galectin-9 Dependent Pathway

NK cells accumulate at the maternal-fetal interface (MFI) and play essential roles in maintaining immune tolerance during pregnancy. The mechanisms that facilitate NK cells tolerance to fetal tissue are largely unknown. T cell Ig and mucin domain-containing protein 3 (Tim-3) is a newly defined molecule with essential immunological function in many physiological and pathological processes. Recent study showed that Tim-3 was involved in the regulation of immune tolerance at MFI. However, whether Tim-3 regulates NK cells cytotoxicity toward trophoblasts is unclear. Here, we showed Tim-3 was mainly expressed by decidual NK cells (dNK) and Tim-3 level in dNK was higher than peripheral NK cells (pNK). Tim-3⁺ dNK expressed more levels of mature markers CD94 and CD69 than Tim-3^- dNK cells and blocking Tim-3 significantly inhibited dNK IFN-γ and TNF-α secretion. Furthermore, we found TGF-β1 may contribute to such up-regulation of Tim-3 in NK cells. Interestingly, blocking Tim-3 enhanced NK cytotoxicity toward trophoblast cell line HTR-8 but not K562. We found HTR-8 expressed Tim-3 ligand Galectin-9, in contrast K562 did not. Small interfering RNA-mediated silencing of Galectin-9 expression enhanced NK cytotoxicity toward HTR-8. We further showed Tim-3/Galecin-9 inhibited NK cytotoxicity toward trophoblast partially via impairing the degranulation process. In addition, clinical data showed that abnormal Tim-3 level on pNK might be associated with recurrent spontaneous abortion (RSA). Thus, our data demonstrate Tim-3/Galectin-9 pathway maintains local tolerance by suppressing NK cytotoxicity toward trophoblasts which may represent a new immunologic tolerance mechanism at MFI.     

Diagnostic Evaluation of Des-Gamma-Carboxy Prothrombin versus α-Fetoprotein for Hepatitis B Virus-Related Hepatocellular Carcinoma in China: A Large-Scale,Multicentre Study

An efficient serum marker for hepatocellular carcinoma (HCC) is currently lacking and requires intensive exploration. We aimed to evaluate the performance of des-gamma-carboxy prothrombin (DCP) for identifying hepatitis B virus-related HCC in a large, multicentre study in China. A total of 1034 subjects in three cohorts (A, B, and C) including HCC and various non-HCC controls were enrolled from 4 academic medical centers in China from January 2011 to February 2014. Blind parallel detections were conducted for DCP and AFP. The area under the receiver operating characteristic curve (AUC) was used to evaluate the diagnostic efficacies. In cohort A, which comprised 521 subjects, including patients with HCC, liver metastasis, liver cirrhosis (LC), and liver hemangiomas as well as healthy controls (HCs), the accuracy of DCP for distinguishing HCC from various controls was 6.2–9.7% higher than that of AFP. In cohort B, which comprised 447 subjects, including patients with HCC, LC, and chronic hepatitis B as well as HC, the accuracy of DCP was further elevated (12.3–20.67% higher than that of AFP). The superiority of DCP to AFP was more profound in the surveillance of early HCC [AUC 0.837 (95% CI: 0.771–0.903) vs. 0.650 (0.555–0.745)] and AFP-negative HCC [AUC: 0.856 (0.798–0.914)] and in discriminating HCC from LC (accuracy: 92.9% vs.64.71%). Higher DCP levels were associated with worse clinical behaviors and shorter disease-free survival. DCP not only is complementary to AFP in identifying AFP-negative HCC and in excluding AFP-positive non-HCC (liver cirrhosis), but also demonstrates improved performance in HCC surveillance, early diagnosis, treatment response and recurrence monitoring in the HBV-related population.     

Biomechanical Characteristics of Osteoporotic Fracture Healing in Ovariectomized Rats: A Systematic Review

Biomechanical tests are widely used in animal studies on osteoporotic fracture healing. However, the biomechanical recovery process is still unknown, leading to difficulty in choosing time points for biomechanical tests and in correctly assessing osteoporotic fracture healing. To determine the biomechanical recovery process during osteoporotic fracture healing, studies on osteoporotic femur fracture healing with biomechanical tests in ovariectomized rat (OVX) models were collected from PUBMED, EMBASE, and Chinese databases. Quadratic curves of fracture healing time and maximum load were fitted with data from the analyzed studies. In the fitted curve for normal fractures, the predicted maximum load was 145.56 N, and the fracture healing time was 88.0 d. In the fitted curve for osteoporotic fractures, the predicted maximum load was 122.30 N, and the fracture healing time was 95.2 d. The maximum load of fractured femurs in OVX rats was also lower than that in sham rats at day 84 post-fracture (D84 PF). The fracture healing time was prolonged and maximum load at D84 PF decreased in OVX rats with closed fractures. The maximum load of Wister rats was higher than that of Sprague-Dawley (SD) rats, but the fracture healing time of SD and Wister rats was similar. Osteoporotic fracture healing was delayed in rats that were < = 12 weeks old when ovariectomized, and at D84 PF, the maximum load of rats < = 12 weeks old at ovariectomy was lower than that of rats >12 weeks old at ovariectomy. There was no significant difference in maximum load at D84 PF between rats with an osteoporosis modeling time <12 weeks and > = 12 weeks. In conclusion, fracture healing was delayed and biomechanical property decreased by osteoporosis. Time points around D95.2 PF should be considered for biomechanical tests of osteoporotic femur fracture healing in OVX rat models. Osteoporotic fracture healing in OVX rats was affected by the fracture type but not by the strain of the rat.     

In Vivo Biodistribution and Anti-Tumor Efficacy Evaluation of Doxorubicin and Paclitaxel-Loaded Pluronic Micelles Decorated with c(RGDyK) Peptide

The treatment of squamous carcinoma, especially multidrug resistance (MDR) tumors, represents one of the most formidable challenges in oncology. In this study, integrin-mediated Pluronic-based micellar system (c(RGDyK)-FP-DP) was proposed as a drug delivery system to enhance the in vivo anti-tumor efficacy in MDR human squamous carcinoma (KBv)-bearing. Following the recognition by integrin proteins express on the cell surface, cellular uptake and in vitro anti-tumor efficacy of c(RGDyK)-FP-DP were better than conventional PF-DP in KBv cells. The tumor homing specificity and further in vivo anticancer efficacy of c(RGDyK)-FP-DP were performed using subcutaneous KBv tumor-bearing mice model, respectively. Compared with PF-DP, c(RGDyK)-FP-DP demonstrated more drug accumulation in tumor and relatively less drug accumulation in heart, and an extended median survival time in the KBv tumor-bearing mice model. Furthermore, preliminary in vivo subacute toxicity evaluation was also conducted by the measurement of histopathology, blood cell counts and clinical biochemistry parameters. Results showed that no obvious toxicity was observed to the hematological system or heart after a series of intravenous administration of c(RGDyK)-FP-DP. In conclusion, our results suggested that c(RGDyK) peptide conjugated Pluronic micelles could be a promising vehicle for enhancing the treatment of MDR human squamous carcinoma.     

Factors Associated with Knowledge of and Willingness for Adult Male Circumcision in Changsha,China

Background

Male circumcision (MC) has been shown to reduce the risk of male genital diseases. MC is not commonly practiced among Chinese males and little is known about the factors associated with their knowledge of and willingness for MC. This study was to explore the knowledge regarding the foreskin among Chinese males and to identify factors associated with their willingness to undergo circumcision.

Methods

A total of 237 patients with redundant prepuce/phimosis were interviewed through face-to-face interviews. The items on the questionnaire included: demographics, an objective scale assessing knowledge about the foreskin, willingness to have MC, the attitudes of sexual partners and doctors toward redundant prepuce/phimosis, and the approaches that patients used to acquire knowledge regarding the prepuce. Univariate analysis and multiple logistic regression analysis were performed to identify factors that are associated with willingness to be circumcised (WTC).

Results

A total of 212 patients completed the interview. Multivariable logistic regression showed that three factors were significantly associated with WTC: being married (OR = 0.43), perceiving redundant prepuce/phimosis as a disease (OR = 1.93), and if a patient’s partner supported MC (OR = 1.39). 58% (n = 122) had received information about the foreskin from another party: 18% (n = 37) from school, 8% (n = 17) from family, 17% (n = 36) from friends, 27% (n = 57) from health care providers. About 4% (n = 8) believed that their partners disliked their redundant prepuce/phimosis. 20% (n = 42) had received doctors’ advice to undergo circumcision.

Conclusion

Knowledge about the foreskin was low among Chinese males. Our study elucidates the factors associated with WTC and suggests that more education of the population about the foreskin can help improve the recognition of a correctible abnormality and help patients assess the potential role of MC in their health.     

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 °C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in ≥50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.     

Intensified plant N and C pool with more available nitrogen under experimental warming in an alpine meadow ecosystem

Nitrogen (N) availability is projected to increase in a warming climate. But whether the more available N is immobilized by microbes (thus stimulates soil carbon (C) decomposition), or is absorbed by plants (thus intensifies C uptake) remains unknown in the alpine meadow ecosystem. Infrared heaters were used to simulate climate warming with a paired experimental design. Soil ammonification, nitrification, and net mineralization were obtained by in situ incubation in a permafrost region of the Qinghai‐Tibet Plateau (QTP). Available N significantly increased due to the stimulation of net nitrification and mineralization in 0–30 cm soil layer. Microbes immobilized N in the end of growing season in both warming and control plots. The magnitude of immobilized N was lower in the warming plots. The root N concentration significantly reduced, but root N pool intensified due to the significant increase in root biomass in the warming treatment. Our results suggest that a warming‐induced increase in biomass is the major N sink and will continue to stimulate plant growth until plant N saturation, which could sustain the positive warming effect on ecosystem productivity.     

Rapid identification of ubiquitination and SUMOylation target sites by microfluidic peptide array

SUMOylation and ubiquitination are two essential post translational modifications (PTMs) involved in the regulation of important biological processes in eukaryotic cells. Identification of ubiquitin (Ub) and small ubiquitin-related modifier (SUMO)-conjugated lysine residues in proteins is critical for understanding the role of ubiquitination and SUMOylation, but remains experimentally challenging. We have developed a powerful in vitro Ub/SUMO assay using a novel high density peptide array incorporated within a microfluidic device that allows rapid identification of ubiquitination and SUMOylation sites on target proteins. We performed the assay with a panel of human proteins and a microbial effector with known target sites for Ub or SUMO modifications, and determined that 80% of these proteins were modified by Ub or specific SUMO isoforms at the sites previously determined using conventional methods. Our results confirm the specificity for both SUMO isoform and individual target proteins at the peptide level. In summary, this microfluidic high density peptide array approach is a rapid screening assay to determine sites of Ub and SUMO modification of target substrates, which will provide new insights into the composition, selectivity and specificity of these PTM target sites.