Investigation of the combination of anti-PD-L1 mAb with HER2/neu-loaded dendritic cells and QS-21 saponin adjuvant: effect against HER2 positive breast cancer in mice.

BACKGROUND: Human epidermal growth factor receptor 2 (HER2) is overexpressed in a subset of cancers including 25% of breast cancers. Since combination therapy consisting of multiple therapeutic approaches is considered a promising regimen, we examined combination treatment modalities in a xenograft model in Balb/c mice injected with 4T1-HER2 cells. We used HER2/neu-loaded bone marrow-derived dendritic cells (BM-DC's) along with anti-PD-L1 monoclonal antibody in a new combination immunotherapy model. METHODS: The combination was composed of an active immunotherapy (i.e. BM-DC-based vaccine) designed to boost the immune response against target antigen and was augmented by using anti-PD-L1 mAb to prevent immune evasion by the xenografted tumors. The vaccine combination was further supported using a QS-21 saponin adjuvant and the immune response was evaluated. RESULTS: Mice treated with HER2/neu-loaded BM-DCs, combined with QS-21 and anti-PD-L1 mAb had significantly decreased tumor sizes and their splenocytes had enhanced cytotoxic activity, based on the lactate dehydrogenase (LDH) assay, compared to vaccine and adjuvant groups alone. The same vaccination group demonstrated a remarkable increase in IFN-γ secreting CD8+ T-cells analyzed by flow cytometry. ELISA data also revealed a significant increase in the serum anti-HER2 IgG1 response; in addition, there was significant splenocyte proliferation upon stimulation with antigen compared to vaccine and adjuvant groups. Consistently, a significant infiltration of CD4+, CD8+ immune cells in and around the tumors was observed. CONCLUSIONS: Our data suggest that the BM-DC + HER2/neu + QS-21 + anti-PD-L1 vaccine combination paradigm synergistically generates anti-tumor activity and immune responses against HER2 overexpressing breast cancer in mice.

Characteristics and genetic diversity of bioluminescent Shewanella woodyi strains isolated from the Gulf of Izmir, Turkey.

The purpose of this study was to isolate bioluminescent strains and to phenotypically and biochemically identify them based on the 16S rRNA gene sequence. 16S rRNA gene sequence analysis of the 11 isolates revealed that they belonged to Shewanella woodyi. Nevertheless, they were determined to exhibit various growth characteristics, enzymatic activities, assimilation of carbon and nitrogen sources, and different characteristics in antibiotic resistance profiles, and also, it was determined that different growth conditions affect the amount of biofilm. Pulsed-field gel electrophoresis (PFGE) analysis of S. woodyi strains performed with SmaI and NotI restriction enzymes revealed that they exhibited restriction fragment pattern homology ranging from 56 to 89 % and from 82 to 94 %, respectively. As a result, PFGE analysis of the genome S. woodyi (as the first record) revealed that although these strains inhabiting the Gulf of Izmir exhibit common characteristics, they also have high levels of genomic polymorphism.

Production of laccase from Trametes trogii TEM H2: a newly isolated white-rot fungus by air sampling.

This work represents the first report of isolation of potential laccase producers by air sampling using media supplemented with 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonate) and guaiacol for laccase production and secretion indicators. Nine fungal isolates showed positive reactions with 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonate) and guaiacol. The isolate named TEM H2 exhibited the largest and intensive oxidation zones with 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonate) (85 mm) and guaiacol (66 mm) and therefore it was selected for detailed investigations. The strain was identified as Trametes trogii TEM H2 due to the morphological characteristics and the comparison of internal transcribed spacer ribosomal DNA gene sequences. The laccase production was screened in different liquid cultures. The best laccase production medium was determined as soluble starch yeast extract medium in which laccase production was reached to a maximum level (989.6 U l(-1) ) on the 8(th) day of cultivation. Effects of different initial pH values on laccase production were tested. Optimum pH value for laccase production in soluble starch yeast extract medium was determined as pH 3.0 with 15425.0 U l(-1) laccase production at 12(th) day of cultivation. In addition, effects of eight inducers (veratryl alcohol, ferulic acid, 1-Hydroxybenzotriazole, syringic acid, 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonate), 1 mmol l(-1) CuSO(4) , 3% ethanol, guaiacol) were examined. Only cultures with 2,5-xylidine exhibited 1.9 fold increase in laccase activity reaching to 28890.0 U l(-1).

Production of monoclonal antibody against Salmonella H: g,m flagellar antigen and potential diagnostic application.

In this study, the flagella antigen was detached at high speed by shaking vigorously with glass pearl beads, from Salmonella enteritidis in a yield of 3.9 mg/mL(-1) after being concentrated with polyethylene glycol (PEG). A monoclonal antibody (MAb), designated D7 clone, was generated from Balb/c mice immunized with Salmonella enteritidis flagella using the conventional hybridoma method. The D7 clone secreting MAb was classified as IgG2a isotype. ELISA analyses of D7 MAb to Salmonella-specific flagella demonstrated that the antibody reacted with H: m. However, Western immunoblot analyses of D7 clone appear to be secreting heavy chain of IgG2a antibody, which was eligible for the diagnosis of Salmonella enteritidis.

Production of monoclonal antibodies in a mouse model via lipopolysaccharide conjugates with synthetic polymers specific to Salmonella Enteritidis O antigen.

Monoclonal antibodies (Mabs) specific for lipopolysaccharide (LPS) of Salmonella Enteritidis were evaluated in a model of LPS conjugated synthetic polymer immunization of Balb/c mice by conventional hybridoma method. Nine hybridoma cell lines were determined as antibody positive against LPS. The clone 5A8 secreting the highest antibody was selected for further characterization. Evaluated results indicate that the synthetic polymer can be used as an effective adjuvant in immunization with LPS, because the 5A8 Mab were obtained using synthetic polymer as an adjuvant. 5A8 Mab was classified as IgG2a isotype by antibody capture enzyme-linked immunosorbent assay. The reactivity of the Mab against lipid A and different LPS of Salmonella were investigated using an indirect enzyme-linked immunosorbent assay. Mab presented a wide spectrum of reactivity, coupling with antigens against Salmonella. The hybridoma 5A8 determined in this study has a great potential to be used in the development of diagnostic, prophylactic, and therapeutic agents specific for Salmonella Enteritidis and Salmonella LPS.