Targeting NAD metabolism regulates extracellular adenosine levels to improve the cytotoxicity of CD8+ effector T cells in the tumor microenvironment of gastric cancer.

PURPOSE: Nicotinamide adenine dinucleotide (NAD+) is closely related to the pathogenesis of tumors. However, the effect of NAD+ metabolism of gastric cancer (GC) cells on immune cells remains unexplained. We targeted nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting enzyme in the NAD+ synthesis salvage pathway, to observe its effect in the immune microenvironment. METHODS: NAMPT of GC cell lines was inhibited by using the small molecule inhibitor (FK866) and short hairpin RNA (shRNA). CCK-8 test and flow cytometry were performed to detect cell viability and apoptosis. Immunofluorescence was used to observe changes in mitochondrial membrane potential (MMP).The transfected GC cells (AGS) and patient-derived organoids (PDOs) were cocultured with activated PBMCs, followed by flow cytometric analysis (FCA) for cytokines and inhibitory marker. The level of NAD and ATP of GC cells (AGS & MKN45) was tested combined with NMN and CD39 inhibitor. RESULTS: Targeting NAD+ by FK866 obviously reduced MMP, which ultimately inhibited proliferation and increased the apoptosis of GC cells. NAMPT silencing reduced intracellular NAD and ATP，further decreased extracellular adenosine. Meawhile, the cytokines of CD8+T cells were significantly increased after cocultured with transfected AGS, and the expression of PD-1 was distinctly decreased. NMN reversed the effect of shNAMPT and enhanced the immunosuppression. Consistent results were obtained by coculturing PBMCs with PDOs. CONCLUSION: Restraining the function of NAMPT resulted in the functional improvement of effector CD8+ T cells by decreasing extracellular adenosine levels and inducing apoptosis of GC cells simultaneously. Therefore, this study demonstrates that NAMPT can be an effective target for gastric cancer immunotherapy.

Direct Macromolecular Drug Delivery to Cerebral Ischemia Area using Neutrophil-Mediated Nanoparticles.

Delivery of macromolecular drugs to the brain is impeded by the blood brain barrier. The recruitment of leukocytes to lesions in the brain, a typical feature of neuroinflammation response which occurs in cerebral ischemia, offers a unique opportunity to deliver drugs to inflammation sites in the brain. In the present study, cross-linked dendrigraft poly-L-lysine (DGL) nanoparticles containing cis-aconitic anhydride-modified catalase and modified with PGP, an endogenous tripeptide that acts as a ligand with high affinity to neutrophils, were developed to form the cl PGP-PEG-DGL/CAT-Aco system. Significant binding efficiency to neutrophils, efficient protection of catalase enzymatic activity from degradation and effective transport to receiver cells were revealed in the delivery system. Delivery of catalase to ischemic subregions and cerebral neurocytes in MCAO mice was significantly enhanced, which obviously reducing infarct volume in MCAO mice. Thus, the therapeutic outcome of cerebral ischemia was greatly improved. The underlying mechanism was found to be related to the inhibition of ROS-mediated apoptosis. Considering that neuroinflammation occurs in many neurological disorders, the strategy developed here is not only promising for treatment of cerebral ischemia but also an effective approach for various CNS diseases related to inflammation.

[Cell-penetrating chimeric apoptotic peptide AVPI-LMWP/DNA co-delivery system for cancer therapy].

To develop a cell-penetrating chimeric apoptotic peptide AVPI-LMWP/DNA co-delivery system for cancer therapy, we prepared the AVPI-LMWP/pTRAIL self-assembled complexes containing a therapeutic combination of peptide drug AVPI and DNA drug TRAIL. The chimeric apoptotic peptide AVPI-LMWP was synthesized using the standard solid-phase synthesis. The cationic AVPI-LMWP could condense pTRAIL by electrostatic interaction. The physical-chemical properties of the AVPI-LMWP/pTRAIL complexes were characterized. The cellular uptake efficiency and the inhibitory activity of the AVPI-LMWP/pTRAIL complexes on tumor cell were also performed. The results showed that the AVPI-LMWP/pTRAIL complexes were successfully prepared by co-incubation. With the increase of mass ratio (AVPI-LMWP/DNA), the particle size was decreased and the zeta potential had few change. Agarose gel electrophoresis showed that AVPI-LMWP could fully bind and condense pTRAIL at a mass ratio above 15:1. Cellular uptake efficiency was improved along with the increased ratio of W(AVPI-LMWP)/WpTRAIL. The in vitro cytotoxicity experiments demonstrated that the AVPI-LMWP/pTRAIL (W:W = 20:1) complexes was significantly more effective than the pTRAIL, AVPI-LMWP alone or LMWP/pTRAIL complexes on inhibition of HeLa cell growth. Our studies indicated that the AVPI-LMWP/pTRAIL co-delivery system could deliver plasmid into HeLa cell and induce tumor cell apoptosis efficiently, which showed its potential in cancer therapy using combination of apoptoic peptide and gene drugs.

Intrauterine insulin resistance in fetuses of overweight mothers.

AIM: To investigate the relationship between maternal overweight and fetal insulin resistance. MATERIAL AND METHODS: Nineteen overweight and 30 lean pregnant women were recruited in the present study. Maternal and fetal insulin resistance were determined by measuring sex hormone binding globulin (SHBG) concentrations in maternal venous or umbilical cord serum, respectively. Maternal age, gestational age, height, pre-gravidity weight, pre-partum weight, as well as fetal gender, birth weight, birth height, and head circumference were collected as clinical data. RESULTS: Fetuses of overweight mothers had larger birth weight (3.58±0.55kg vs 3.32±0.42, adjusted P=0.006) and lower SHBG concentrations (26.64±3.65 vs 34.36±7.84, adjusted P=0.007) than those of lean mothers after values were adjusted for potential cofactors. Fetal SHBG level was negatively correlated with pre-gravidity body mass index (R=-0.392, adjusted P=0.025) and weight gain during pregnancy (R=-0.332, adjusted P=0.026) even with adjustment for potential cofactors. Among the 29 pregnant women with gestational diabetes mellitus, the overweight mothers had higher H1AC levels than their lean counterparts (6.47±0.44 vs 5.74±0.52, adjusted P=0.004). CONCLUSION: Intrauterine insulin resistance is more prominent in fetuses of overweight mothers, an effect that is decreased by weight gain control during pregnancy.

[Relationship between P-glycoprotein function in peripheral blood cells and multidrug resistance in breast carcinoma].

OBJECTIVE: To analyze the relationship between P-glycoprotein function in peripheral blood cells and primary multidrug resistance in breast carcinoma. METHODS: P-gp function was investigated by flow cytometry in NK cells of 16 breast cancer patients treated with anthracyclines and taxanes. Among all the patients, 8 were in chemotherapy-sensitive group and 8 in chemotherapy-resistant group. P-gp function was determined by rhodamine 123 (Rh123)-ejection test. Mathematical model was established by a regression of the fluorescence-time curve. The efflux rate constants of the chemotherapy-sensitive and -resistant groups were compared. RESULTS: There was no significant difference of Rh123 accumulation, retention or efflux between the two groups. The mathematical model of F(t) = F(0) · e(-kt) was established. K was the efflux rate constant, which was significantly different between the chemotherapy-sensitive and -resistant groups (P = 0.025). When k > 3.9 was used as diagnostic criterium for primary resistance, the sensitivity, specificity and accuracy were 75.0%, 100% and 87.5%, respectively. CONCLUSION: P-glycoprotein function in peripheral blood cells is associated with primary multidrug resistance in breast carcinoma. The efflux rate constant may be a good predictor for chemotherapy sensitivity.

[Correlation of peripheral blood micrometastasis to distant metastasis of breast cancer].

BACKGROUND & OBJECTIVE: Breast cancer, a whole body disease, can metastasize at early stage. This study was to explore the correlation of peripheral blood cancer cell (PBCC) content to distant metastasis of breast cancer. METHODS: The PBCC content of 65 breast cancer patients and 8 healthy donors was detected by multi-parameter flow cytometry (FCM) with CD45 and cytokeratin staining. RESULTS: Cancer cells were detected in peripheral blood samples from 57 of the 65 patients; the positive rate was 87.7%. No cancer cell was found in peripheral blood samples from healthy donors. The positive rate of PBCCs was correlated to T stage (r=0.271,P=0.017) and N stage (r=0.393, P=0.002). The patients were followed for 5 years; 2 were lost. Distant metastasis was found in 25 patients with PBCCs. In contrast, no metastasis was found in 8 patients without PBCCs (P<0.05). CONCLUSION: Preoperative PBCC content is closely related to distant metastasis of breast cancer. The detection of PBCCs might be useful for individual treatment decision for breast cancer.

[Application of flow cytometric immunophenotypic analysis in the diagnosis of malignant lymphoma].

OBJECTIVE: To explore the value of flow cytometric immunophenotyping (FCI) in the diagnosis and differentiated diagnosis of lymphoma and explain the immunophenotypic features and differences of malignant lymphoma. METHODS: Seventy four fresh samples of suspicious lymphoma were collected from Nov. 2004 to Aug. 2006. Each sample was individually evaluated by FCI. The results were analyzed and compared with the histopathological diagnosis. RESULTS: Among the 74 cases, the FCI data consisted with the final morphological diagnosis in 61 cases (82.4%). For the diagnosis of B and T non-Hodgkin's lymphoma (NHL), thymoma, carcinoma and benign lesions of lymph node, the concordance between FCI data and morphological diagnosis were 93.5%, 100%, 100%, 100% and 81.3%, respectively. CONCLUSION: Multi-parameter FCI analysis can provide important information and help for diagnosis of lymphoma. It is an assistant but necessary approach for the diagnosis and differential diagnosis of lymphoma.