The role of lipoprotein­associated phospholipase A2 in inflammatory response and macrophage infiltration in sepsis and the regulatory mechanisms.

Lipoproteinassociated phospholipase A2 (Lp-PLA2), encoded by the phospholipase A2 group VII (Pla2g7) gene, has been pertinent to inflammatory responses. This study investigates the correlation between Lp-PLA2 and inflammatory injury in septic mice and explores its regulatory mechanism. Lp-PLA2 was found to be upregulated in the serum of septic mice induced by cecal ligation and puncture and in the culture supernatant of RAW264.7 cells following lipopolysaccharide and adenosine triphosphate treatments. The contents of Lp-PLA2 were positively correlated with increased concentrations of proinflammatory cytokines in patients with sepsis. Both animal and cellular models showed increased concentrations of proinflammatory cytokines. Spi-1 proto-oncogene (Spi1), highly expressed in these models, was found to activate Pla2g7 transcription. Knockdown of Pla2g7 or Spi1 reduced the proinflammatory cytokine production, mitigated organ damage in mice, and suppressed macrophage migration in vitro. Retinoblastoma binding protein 6 (Rbbp6), poorly expressed in both models, was found to reduce Spi1 protein stability through ubiquitination modification. Rbbp6 overexpression similarly suppressed inflammatory activation of RAW264.7 cells, which was counteracted by Pla2g7 or Spi1 upregulation. In summary, this study demonstrates that the Pla2g7 loss and Spi1 upregulation participate in inflammatory responses in sepsis by elevating the Lp-PLA2 levels.

Optical Control of the GTP Affinity of K-Ras(G12C) by a Photoswitchable Inhibitor.

Photocontrol of protein activity is an emerging field in biomedicine. For optical control of a mutant small GTPase K-Ras(G12C), we developed small-molecule inhibitors with photoswitchable efficacy, where one configuration binds the target protein and exert different pharmacological effects upon light irradiation. The compound design was based on the structure feature of a previously identified allosteric pocket of K-Ras(G12C) and the chemical structure of covalent inhibitors, and resulted in the synthesis and characterization of two representative azobenzene-containing compounds. Nucleotide exchange assays demonstrated the different efficacy to control the GTP affinity by photoswitching of one potent compound PS-C2, which would be a useful tool to probe the conformation of mutational K-Ras. Our study demonstrated the feasibility of designing photoswitchable modulators from allosteric covalent inhibitor of small GTPases.

Antagonism by Ganoderma lucidum polysaccharides against the suppression by culture supernatants of B16F10 melanoma cells on macrophage.

It is well-documented that macrophages have the functions to regulate antitumor immune response. Antitumor response can be launched by a series of events, starting with inflammation mediated by monocyte/macrophages, which stimulates natural killer and dendritic cells and finally activates the cytotoxic lymphoid system. Monocytes/macrophages may be the first line of defense in tumors. However, specific and nonspecific immunotherapy for human cancer has shown no success or limited success in clinical trials. Part of the reasons attribute to tumor-derived soluble factors that suppress functions of immune cells or induce apoptosis of these cells, including macrophages. Therefore, antagonism of the suppression on the macrophages is an important goal for tumor immunotherapy. To achieve this purpose, Ganoderma lucidum polysaccharides (Gl-PS) with multiple bioactivities were used on mouse peritoneal macrophages incubating with culture supernatants of B16F10 melanoma cells (B16F10-CS). It was shown that the viability, phagocytic activity, NO production, TNF-α production and activity in peritoneal macrophages after activation by lipopolysaccharide were suppressed by B16F10-CS, while the suppressions were fully or partially antagonized by Gl-PS. In conclusion, B16F10-CS is suppressive to the viability, phagocytic activity, NO production, TNF-α production and activity in peritoneal macrophages while Gl-PS had the antagonistic effects against this suppression, suggesting this potential of Gl-PS to facilitate cancer immunotherapy.

A case report of Vibrio vulnificus sepsis in a diabetic patient.

Vibrio vulnificus is a facultative anaerobic, alkalophilic, halophilic, mesophilic, Gram-negative bacterium that can cause severe wound infection, sepsis and diarrhea. This paper reported a case of 85-year-old male patient infected with Vibrio vulnificus due to being stabbed by a sea shrimp. This patient also had diabetes with a long history of alcoholism. Due to bacterial pathogenicity and the patient's underlying diseases, his condition deteriorated rapidly. Based on the rapid diagnosis of Vibrio vulnificus using the next-generation sequencing（NGS）technology and blood culture method, as well as the selection of the most effective antibiotics via drug sensitivity test, this patient underwent precise antimicrobial treatment, thorough debridement and drainage within the shortest possible time, and thus the prognosis of this patient was greatly improved. In this paper, we have systematically explored the epidemiology, clinical features, diagnosis and treatment of Vibrio vulnificus infection, thus providing a practical reference for the clinicians to quickly identify and treat possible Vibrio vulnificus infection in diabetic patients after contacting with sea water or seafood.

Morphine improved the antitumor effects on MCF-7 cells in combination with 5-Fluorouracil.

BACKGROUND: The most frequently used opioid in cancer pain management is morphine which remains a cornerstone for the management of cancer pain, due to the largest experience existing among physicians and widely availability in a variety of formulation. Considering that analgesics on cancer pain is often under the condition of chemotherapy and 5-Fluorouracil (5-FU) is widely used today as a potent drug for the treatment of advanced cancers, whether analgesics such as morphine, interferes the chemotherapy such as 5-FU, arose as a considerable problem. METHODS: In this study, the MCF-7 breast cancer cells were used to determine the antitumor effects of the 5-FU in combination with morphine. The cell proliferation was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and the apoptosis was determined by the Annexin V/PI staining and flow cytometry. The immunocytochemistry and western blot was used to determine the Bcl-2 and Bax expression. RESULTS: It was shown that in MCF-7 cells, the proliferation was inhibited, the apoptosis was promoted, the Bcl-2 expression was suppressed and the Bax expression was promoted by both 5-FU alone and morphine alone, while the superior effects were achieved in combination with the two drugs. CONCLUSION: These results suggest that the morphine may have the beneficial effects on the antitumor chemotherapy with 5-FU, in stead of interferential effects.

Effects of T-cadherin expression on B16F10 melanoma cells.

Melanoma is one of the most deadly skin cancers. T-cadherin is an atypical member of the cadherin superfamily as it lacks the transmembrane and cytoplasmic domains and is anchored to cell membranes through glycosylphosphatidylinositol (GPI) anchors. T-cadherin downregulation is associated with a poorer prognosis in various carcinomas, such as lung, ovarian, cervical and prostate cancer, while in the majority of cancer cell lines, T-cadherin re-expression inhibits cell proliferation and invasiveness, increases susceptibility in apoptosis and reduces tumor growth in in vivo models. The functional relevance of T-cadherin gene expression in melanoma progression remains to be clarified. The present study was designed for this purpose. The T-cadherin gene was transfected into B16F10 melanoma cells to express T-cadherin in the cells which were originally deficient in T-cadherin expression. The proliferation, invasiveness, apoptosis and cell cycle of the transfected B16F10 melanoma cells were analyzed. The present study showed that the expression of T-cadherin in B16F10 melanoma cells markedly reduced cell proliferation and permeation through Matrigel-coated membranes, representing invasiveness. The percentage of early apoptotic cells and cells in the G2/M phase of the cell cycle was markedly increased compared with either parental B16F10 (without transfection) or empty pEGFP-N1 (without T-cadherin gene)-transfected B16F10 cells, suggesting G2/M arrest, with similarity between the parental and empty pEGFP-N1-transfected B16F10 cells. T-cadherin is important in melanoma progression and may be a possible target for therapy in melanoma and certain other types of cancer.

Ganoderma lucidum polysaccharides counteract inhibition on CD71 and FasL expression by culture supernatant of B16F10 cells upon lymphocyte activation.

Immune responses to tumor-associated antigens are often detectable in tumor-bearing hosts, but they fail to eliminate malignant cells or prevent development of metastases. Tumor cells produce factors such as interleukin-10, transforming growth factor-ß1 and vascular endothelial growth factor (VEGF) that suppress the function of immune cells or induce apoptosis of immune cells. Culture supernatant of tumor cells may contain these immunosuppressive factors which suppress lymphocyte activation. CD71 and FasL are two important molecules that are expressed upon lymphocyte activation. Counteraction against suppression CD71 and FasL expression upon lymphocyte activation may benefit tumor control. A potential component with this effect is Ganoderma lucidum polysaccharides (Gl-PS). In this study, Gl-PS was used on lymphocytes incubating with culture supernatant of B16F10 melanoma cells (B16F10-CS) in the presence of phytohemagglutinin. Following induction with phytohemagglutinin, B16F10-CS suppressed CD71 expression in lymphocytes (as detected by immunofluorescence and flow cytometry), proliferation in lymphocytes (as detected by MTT assay), and FasL expression in lymphocytes (as detected by immunocytochemistry and western blot analysis), while Gl-PS fully or partially counteracted these suppressions. Gl-PS showed counteractive effects against suppression induced by B16F10-CS on CD71 and FasL expression upon lymphocyte activation, suggesting the potential of Gl-PS to facilitate cancer immunotherapy.

Enhanced MHC class I and costimulatory molecules on B16F10 cells by Ganoderma lucidum polysaccharides.

PURPOSE: It is obvious that malignant cells evade from immune system in patients with manifest malignancy. Deficient major histocompatibility complex (MHC) class I and costimulatory molecules on malignant cells partially consist of evasion strategy since antigen bond MHC and costimulatory molecules provide two signals necessary for T cell activation. Therefore, enhancement of MHC-I and costimulatory molecules may favor restraint of the evasion. For this purpose, Ganoderma lucidum Polysaccharides (Gl-PS) was used on B16F10 melanoma cells in this study. METHODS: Immunocytochemistry and flowcytometry were used to determine the H-2K(b) and H-2D(b) (two prominent MHC class I molecules in C57BL mouse) as well as B7-1 and B7-2 (two prominent costimulatory molecules) expression on B16F10 cells after incubation with Gl-PS, while messenger ribonucleic acid (mRNA) of these molecules was detected by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: The H-2K(b) and H-2D(b), and B7-1 and B7-2 on B16F10 cells and mRNAs of these molecules were enhanced by Gl-PS, and more efficient antitumor cytotoxicity was induced by the Gl-PS treated cells. CONCLUSIONS: The MHC class I molecules and costimulatory molecules may be enhanced by Gl-PS, and more efficient immune cell mediated cytotoxicity against these B16F10 cells may be induced, which may favor cancer therapy.

Promoting effects of Ganoderma lucidum polysaccharides on B16F10 cells to activate lymphocytes.

The immune system in patients with cancer often fails to control tumour growth because of deficient immunogenicity of tumour cells. Ganoderma lucidum polysaccharides (Gl-PS) are believed to have anti-tumour effects by boosting host immune function. Additionally, Gl-PS may have some direct effects on tumour cells in the activation of lymphocytes, thus enhancing the immunogenicity of tumour cells. We tested the effects of Gl-PS in lymphocyte activation by incubating Gl-PS with a tumour cell line deficient in antigen presentation. Our study showed that Gl-PS can promote B16F10 melanoma cells to induce lymphocyte proliferation, CD69 and FasL expression and IFN-γ production, indicating that Gl-PS can improve the nature of B16F10 cells to activate lymphocytes. Furthermore, H-2D(b) [a major histocompatibility (MHC) class I molecule], and B7-1 and B7-2 (two prominent co-stimulatory molecules expressed on B16F10 cells) were enhanced by Gl-PS, suggesting that these molecules may at least partially be involved in the process of Gl-PS on B16F10 cells to activate lymphocytes.

Ganoderma lucidum polysaccharides antagonize the suppression on lymphocytes induced by culture supernatants of B16F10 melanoma cells.

OBJECTIVES: Tumour cells produce factors such as interleukin 10 (IL-10), transforming growth factor ß1 (TGF-ß1) and vascular endothelial growth factor (VEGF) that suppress the function of immune cells or induce apoptosis of immune cells. One of the most important goals of tumour immunotherapy is to antagonize this suppression on immune cells. Ganoderma lucidum polysaccharides (Gl-PS) may have this potential. The purpose of this study was to determine the antagonistic effects of Gl-PS on the suppression induced by B16F10 melanoma cell culture supernatant (B16F10-CS) on lymphocytes. METHODS: Gl-PS was used on lymphocytes incubated with B16F10-CS. Enzyme-linked immunosorbent assay was used to determine the levels of IL-10, TGF-ß1 and VEGF in B16F10-CS. The MTT assay was used to determine the proliferation of lymphocytes. Immunocytochemistry and Western blot assay were used to determine perforin and granzyme B production in lymphocytes. KEY FINDINGS: There were elevated levels of IL-10, TGF-ß1 and VEGF in B16F10-CS. The lymphocyte proliferation, and perforin and granzyme B production in lymphocytes after induction with phytohemagglutinin, as well as lymphocyte proliferation in the mixed lymphocyte reaction, were suppressed by B16F10-CS. This suppression was fully or partially antagonized by Gl-PS. CONCLUSIONS: B16F10-CS suppressed lymphocyte proliferation and perforin and granzyme B production in lymphocytes after induction with phytohemagglutinin, as well as lymphocyte proliferation in the mixed lymphocyte reaction. This suppression may be associated with elevated levels of immunosuppressive IL-10, TGF-ß1 and VEGF in B16F10-CS. Gl-PS had antagonistic effects on the immunosuppression induced by B16F10-CS, suggesting the potential for Gl-PS in cancer immunotherapy.