Nocardia Rubra Cell Wall Skeleton Up-Regulates T Cell Subsets and Inhibits PD-1/PD-L1 Pathway to Promote Local Immune Status of Patients With High-Risk Human Papillomavirus Infection and Cervical Intraepithelial Neoplasia.

The Nocardia rubra cell wall skeleton (Nr-CWS) for external use is an immune enhancer, which has been widely used in human cervix diseases such as cervical erosion, but the mechanism of Nr-CWS enhancing immunity is still unclear. The purpose of this study was to explore the effect and mechanism of Nr-CWS on the local immune status of cervical tissue in patients with high-risk human papillomavirus (HR-HPV) infection and cervical precancerous lesion, cervical intraepithelial neoplasia (CIN). The recruited patients with HR-HPV infection and CIN were treated with Nr-CWS. The specimens were taken from these patients before and after local application of Nr-CWS respectively. The normal control specimens were tested simultaneously. Serial section analysis of immunohistochemistry and co-expression analysis were performed to characterize populations of T cells and the expressions of programmed cell death-1 (PD-1) and programmed cell death-ligand 1 (PD-L1). The levels of cytokines in local cervical tissue were also detected. Nr-CWS significantly increased T cells including CD4+, CD8+ T cells, and reduced the expression of PD-L1 in the patients' local cervical tissues. Co-expression analyses showed that the proportions of PD-1+CD4+ cells in CD4+ T cells and PD-1+CD8+ cells in CD8+ T cells decreased after Nr-CWS application. Furthermore, the increase in the number of immune cells was accompanied by increased pro-inflammatory cytokines interleukin-12 (IL-12), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and decreased suppressive cytokine IL-10. The results indicate that Nr-CWS, as an immunotherapeutic agent for HR-HPV infection and CIN, plays an immune promoting role related to the upregulation of T cell subsets and the inhibition of PD-1/PD-L1 pathway.

Hepcidin links gluco-toxicity to pancreatic beta cell dysfunction by inhibiting Pdx-1 expression.

OBJECTIVE: Gluco-toxicity is a term used to convey the detrimental effect of hyperglycemia on ß-cell function through impaired insulin synthesis. Although it is known that the expression and activity of several key insulin transcription regulators is inhibited, other molecular mechanisms that mediate gluco-toxicity are poorly defined. Our objective was to explore the role of hepcidin in ß-cell gluco-toxicity. DESIGN: We first confirmed that high glucose levels inhibited hepcidin expression in the mouse insulinoma cell line, MIN6. The downregulation of hepcidin decreased Pdx-1 expression, which reduced insulin synthesis. METHODS: MIN6 cells were exposed to high glucose concentrations (33.3 mmol/L). Glucose-stimulated insulin secretion (GSIS) and serum hepcidin levels were measured by ELISA. The mRNA levels of insulin1, insulin2, Pdx-1 and hepcidin were measured by real-time polymerase chain reaction. Western blot analysis was used to detect the changes in PDX-1 expression. Transient overexpression with hepcidin was used to reverse the downregulation of Pdx-1 and insulin synthesis induced by gluco-toxicity. RESULTS: Exposure of MIN6 cells to high glucose significantly decreased GSIS and inhibited insulin synthesis as well as Pdx-1 transcriptional activity and expression at both the mRNA and protein levels. High glucose also decreased hepcidin expression and secretion. Hepcidin overexpression in MIN6 cells partially reversed the gluco-toxicity-induced downregulation of Pdx-1 and insulin expression and improved GSIS. The restoration of insulin synthesis by transfection of a hepcidin overexpression plasmid confirmed the role of hepcidin in mediating the gluco-toxic inhibition of insulin synthesis. CONCLUSIONS: Our observations suggest that hepcidin is associated with gluco-toxicity-reduced pancreatic ß-cell insulin synthesis in type 2 diabetes by inhibiting Pdx-1 expression.

Lysine-specific demethylase 1 in breast cancer cells contributes to the production of endogenous formaldehyde in the metastatic bone cancer pain model of rats.

BACKGROUND: Bone cancer pain seriously affects the quality of life of cancer patients. Our previous study found that endogenous formaldehyde was produced by cancer cells metastasized into bone marrows and played an important role in bone cancer pain. However, the mechanism of production of this endogenous formaldehyde by metastatic cancer cells was unknown in bone cancer pain rats. Lysine-specific demethylase 1 (LSD1) is one of the major enzymes catalyzing the production of formaldehyde. The expression of LSD1 and the concentration of formaldehyde were up-regulated in many high-risk tumors. OBJECTIVE: This study aimed to investigate whether LSD1 in metastasized MRMT-1 breast cancer cells in bone marrows participated in the production of endogenous formaldehyde in bone cancer pain rats. METHODOLOGY/PRINCIPAL FINDINGS: Concentration of the endogenous formaldehyde was measured by high performance liquid chromatography (HPLC). Endogenous formaldehyde dramatically increased in cultured MRMT-1 breast cancer cells in vitro, in bone marrows and sera of bone cancer pain rats, in tumor tissues and sera of MRMT-1 subcutaneous vaccination model rats in vivo. Formaldehyde at a concentration as low as the above measured (3 mM) induced pain behaviors in normal rats. The expression of LSD1 which mainly located in nuclei of cancer cells significantly increased in bone marrows of bone cancer pain rats from 14 d to 21 d after inoculation. Furthermore, inhibition of LSD1 decreased the production of formaldehyde in MRMT-1 cells in vitro. Intraperitoneal injection of LSD1 inhibitor pargyline from 3 d to 14 d after inoculation of MRMT-1 cancer cells reduced bone cancer pain behaviors. CONCLUSION: Our data in the present study, combing our previous report, suggested that in the endogenous formaldehyde-induced pain in bone cancer pain rats, LSD1 in metastasized cancer cells contributed to the production of the endogenous formaldehyde.

[Study on functions and mechanism of curcumin in inducing gastric carcinoma BGC apoptosis].

AIM: discuss the biological function and regulation mechanism of curcumin in promoting human gastric carcinoma BGC-823 apoptosis. METHODS: Conventional in virto culture in logarithmic phase gastric carcinoma BGC-823 cells; cells are divided into four groups: control group, low treatment group, middle treatment group and high treatment group, with curcumin concentration being 0 mg/L, 5 mg/L, 10 mg/L, and 20 mg/L, respectively. 24 hours after curcumin is treated, cell proliferation level and apoptosis rate are measured with MTT colorimetry and flow cytometry, Bax, Bcl-2 protein expression is measured with immunohistochemistry; mRNA of Caspase-3 is tested by means of PCR. RESULTS: MTT test indicates that curcumin can inhibit human gastric carcinoma BGC-823 cell proliferation, showing concentration dependency; flow cytometry shows that curcumin can effectively induce apoptosis, showing concentration dependency, where the apoptosis rate is 48.3% 24 hours after 20 mg/L curcumin is treated; immunohistochemistry test shows that curcumin treatment enables Bax expression level in human gastric carcinoma BGC-823 cells to go up, meanwhile, the Bcl-2 protein expression level to go down, besides, the mRNA expression level of Caspase-3 in cells increases through induction of curcumin. CONCLUSION: Curcumin has obvious inhibitory effect on human gastric carcinoma BGC-823 cell proliferation, showing concentration dependency to promote apoptosis. Such biological effect may be associated with activating Caspase-3 signal channel by activating Bax protein expression and inhibiting Bcl-2 protein. This study lays an important foundation for further discussing the mechanism of curcumin in inducing human gastric carcinoma BGC-823 apoptosis.

[Potential application of human amniotic mesenchymal cells for seeding cells in bone tissue engineering].

OBJECTIVE: To investigate the potential of human amniotic mesenchymal cells (hAMC) serving as seeding cells in bone tissue engineering. METHODS: hAMC were isolated and cultured. The third passage of hAMC was cultured in osteogenic induced media [DMEM supplemented with 10% (v/v) FBS, 0.1 mumol/L dexamethasone, 50 mg/L ascorbic acid and 10 mmol/L beta-glycerophosphate] for one week. Calcified nodules were shown by alizarin red staining and counted under light microscope. Immunofluorescence cytochemical staining was used to detect collagen I (COL I) and alkaline phosphatase (ALP). Expression of FasL was examined in the amnion and hAMC by immunohistochemistry or immunocytochemistry. RESULTS: After osteoblast differentiation, calcified nodules were formed, on the average 18 per well. hAMC in calcified nodules showed positive expression of COL I and ALP. FasL was detected positive both in cells contained in amnion and in hAMC. CONCLUSION: hAMC are potential ideal candidates for seeding cells in bone tissue engineering.

The spleen as a target in severe acute respiratory syndrome.

It has been proposed that immune injury is the central mechanism of pathogenesis of the infectious disease, severe acute respiratory syndrome (SARS). To gain a better understanding of immune injury in the spleen, we investigated the number and distribution of various immune cell types in the spleens of SARS patients. We performed autopsies on six confirmed SARS cases, with six normal subjects as controls; spleen samples from these autopsies were examined with hematoxylin and eosin (H&E) sections, in situ hybridization for SARS virus genomic sequences, and immunohistochemistry with seven monoclonal antibodies to five cell types. The number and distribution of these cells were measured and analyzed using an image analysis system. SARS genomic sequences were detected in all SARS spleens. The SARS spleens all had severe damage to the white pulp and showed an alteration of the normal distribution of various cell types. Immunocytes in the red pulp were decreased by 68.0-90.7% except for CD68+ macrophages and human leukocyte antigen (HLA)-DR positive antigen-presenting cells (APC), which were decreased to a lesser degree. On average, CD68+ macrophages were increased in size by 2.21-fold. We hypothesize that the collapse of the splenic immune system plays a key role in the clinical outcome of these patients.

Histological and ultrastructural changes induced by selenium in early experimental gastric carcinogenesis.

AIM: To investigate the effect and significance of selenium in early experimental gastric carcinogenesis. METHODS: Weaning male Wistar rats were divided randomly into normal control group, experiment control group, low selenium (2 mg/L) group and high selenium (4 mg/L) group. Wistar rat gastric carcinogenesis was induced by N-methyl-N-nitro-N-nitroso guanidine (MNNG) (20 mg/kg) gavage daily for 10 d. Na2SeO3 was given by piped drinking 1 wk prior to MNNG gavage. The rats were killed at the 43rd wk. The surface characteristics of gastric mucosa were observed with naked eyes. Histopathologic changes of rat gastric mucosa were observed by HE staining and AB-PAS methods. The changes of cellular ultrastructure were observed under transmission electron microscope. Statistical analysis was carried out by SPSS. RESULTS: The incidence rate of gastric mucosa erosion, hemorrhage and intestinal metaplasia was 0, 45.5%, 66.7%, and 92.9%, respectively (92.9% vs 45.5%, P < 0.05) in the normal control group, experiment control group, low selenium group, and high selenium group. Leiomyoma formed in the process of inducement of rat gastric carcinoma. Dietary Na2SeO3 (2 and 4 mg/L) slightly increased the incidence rate of leiomyoma (0, 23%, 46.6%, and 46.6%). gastric mucosa did not change in the course of rat gastric carcinogenesis. Dietary Na2SeO3 by pipe drinking could expand the intracellular secretory canaliculus of parietal cells and increase the number of endocrine cells and lysosomes. CONCLUSION: Dietary Na2SeO3 by pipe drinking aggravates gastric erosion, hemorrhage and promotes intestinal metaplasia of gastric mucosa. The mechanism may be related with the function of parietal cells.