48-h Supernatants Research Articles

Evaluation of Anti-Tumor Potential of Lactobacillus acidophilus ATCC4356 Culture Supernatants in MCF-7 Breast Cancer.

The anti-cancer activity of some lactic acid bacterial strains is well documented in several kinds of literatures. Lactobacillus strains have received considerable attention as a beneficial microbiota. The aim of this study is to evaluate the effects of anti-tumor activities of L. acidophilus ATCC4356 culture supernatants on the MCF-7 human breast cancer cells. The anti-cancer effects of 24h and 48h culture supernatants at various concentrations (1.25, 2.5, 5, 10 and 20 μg/ml) were determined by various in vitro and in vivo assays including MTT, tumor volume measurement as well as 99mTc-MIBI biodistribution in MCF-7 tumor bearing nude mice and histopathology test. For evaluation of the related mechanism of action, quantitative PCR was conducted. The 48h culture supernatants at 10 and 20 μg/ml exhibited significant in vitro inhibition of MCF-7 cell proliferation. However, this inhibition was not observed for HUVEC human endothelial normal cells. Q-PCR indicated that treatment by the supernatant led to a significant downregulation of VEGFR (~ 0.009 fold) and Bcl- 2 (~ 0.5 fold) and upregulation of p53 (~ 1.3 fold). In vivo study using MCF-7 xenograft mouse models demonstrated a reduction in tumor weight and volume by both 24h and 48h supernatants (2 mg/kg) after 15 days. According to the 99mTc-MIBI biodistribution result, treatment of MCF-7 bearing nude mice with both 24h and 48h supernatant (2mg/kg) led to a significant decrease in tumor uptake compared with the control group. These results suggest that the culture supernatants of L. acidophilus ATCC4356 at suitable concentrations can be considered as a good alternative nutraceutical with promising therapeutic indexes for breast cancer.

Abstract P2-04-17: Directing NK cells to Trop-2-expressing breast and other cancers, with chimeric antigen receptors

Abstract Adoptive immunotherapy with chimeric antigen receptor (CAR)-engineered T or NK cells is in active clinical and preclinical development, with recent interest increasingly focused on advancing the availability of off-the-shelf allogeneic clonal NK cell lines capable of targeting tumors with appropriately designed CARs. Among the human NK cell lines established, the IL-2-dependent NK-92 is the most studied and has been transduced with human IL-2 to generate NK-92MI, which exhibits cytotoxicity similar to NK-92, and is independent of IL-2 for its cytotoxicity, viability and proliferation. We report the assembly of E1.BB.3z-92MI, a novel CAR-NK for potential therapy of diverse Trop-2-expressing breast, lung, bladder, ovarian, and other cancers. The Trop-2-specific CAR, referred to as hRS7-CAR, consists of the CD8α signal peptide, the VK and VH of hRS7 (a humanized anti-human Trop-2 mAb used in the antibody-drug conjugate, IMMU-132), the hinge region and transmembrane domain of CD8α, the intracellular domains (ICD) of 4-1BB, and the ICD of CD3ξ. The construct of hRS7-CAR is introduced into NK-92MI either by electroporation of the mRNA synthesized in vitro, or by inoculation with the lentiviral particles harvested from the 48-h supernatants of Lenti-X 293T cells transduced with pLVX-puro-hRS7-CAR. The presence of hRS7-CAR in E1.BB.3z-92MI was probed by Western blot using an HRP conjugate of a rat anti-id antibody against hRS7, which detected a distinct band of about 50 kDa from the cell lysates of NK-92MI transfected with hRS7-CAR mRNA, but not from the mock-transfected NK-92MI. As the calculated molecular weight of hRS7-CAR is about 51 kDa, these results support that hRS7-CAR is produced in NK-92MI cells transfected with hRS7-CAR mRNA. Additional evidence by flow cytometry shows about 41% of NK-92MI cells transfected with hRS7-CAR mRNA are alive at the time of analysis and 25% of this subpopulation expresses hRS7. The cytotoxicity of E1.BB.3z-92MI was evaluated against the Trop-2-expressing, human breast cancer cell line, HCC1806, in 96-well plates (4,500 cells/well) at 3 different effector-to-target (E/T) ratios (1:1, 2:1, or 4:1). As shown in the Table, the results of the MTS assay indicate that NK-92MI cells transfected with hRS7-CAR mRNA significantly killed more HCC1806 cells at the E/T ratio of 2:1 or 4:1, in comparison to mock-transfected NK-92MI. Another study performed by flow cytometry using dye-labeled HCC1806 at the E/T ratio of 3:1 indicates that the specific lysis of HCC1806 cells by NK-92MI cells transfected with hRS7-CAR mRNA was about 2-fold higher than that observed for mock-transfected NK-92MI. Finally, the initial results obtained for NK-92MI cells transduced with the lentiviral pLVX-puro-hRS7-CAR show about 70% viability and greater than 30-fold higher MFI in the live population than the control transduced with pLVX-puro or not transduced. Together, these results demonstrate the feasibility of developing E1.BB.3z-92MI as a novel CAR-NK for targeting Trop-2-expressing cancers, including breast cancer. Table. Cytotoxicity of Trop-2-targeting NKcellsE/TViability (mean ± SD)Mock vs. hRS7-CAR MockhRS7-CARΔP-value1:188.5 ± 9.980.3 ± 11.18.20.392:189.9 ± 10.667.4 ± 4.422.50.0274:184.8 ± 4.072.4± 2.512.40.010 Citation Format: Chang C-H, Liu D, Goldenberg DM. Directing NK cells to Trop-2-expressing breast and other cancers, with chimeric antigen receptors [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-04-17.

Lymphocyte proliferation assays may underestimate antigen responsiveness in human immunodeficiency virus infection.

The objective of this study was to examine the relationships among lymphocyte proliferation, interferon-gamma (IFN-gamma) production, and apoptosis in peripheral blood mononuclear cells (PBMC) of HIV-1-infected patients and controls. PBMC were prepared from 19 HIV-1-infected patients and 16 healthy controls. Using tetanus toxoid (TT) as a recall antigen, we assessed lymphocyte proliferation using [(3)H]thymidine incorporation after 2, 4, 6, and 7 days' culture and IFN-gamma production in 48-h culture supernatants by ELISA. Apoptosis was measured using TdT-mediated dUTP nick-end labeling. Median stimulation indices (SIs) in HIV-1-infected patients were 2.8 and 3.7 as opposed to 24.9 and 25.1 in healthy controls after 6 and 7 days' culture, respectively (P < 0. 001). Among the controls, peak proliferation was seen after 7 days in culture whereas in patients, SIs peaked at 4 days and fell progressively by days 6 and 7. At 2 and 4 days of stimulation with tetanus, patients' T cells showed increased apoptosis (19 and 25%) vs 12 and 15% apoptosis seen in controls' cells, P < 0.05. Interferon-gamma in 48-h supernatants of TT-stimulated PBMC was comparable among patients and controls. Whereas in our system, 6 and 7 day assays of lymphocyte proliferation provide increasing responses to TT among healthy controls, these durations of culture may underestimate antigen responsiveness in HIV-1 infection. Cell death due to apoptosis may account for this phenomenon. Whether shorter term or longer term assays of lymphocyte responsiveness more accurately reflect in vivo immune competence is unknown. Nonetheless, shorter duration assays may provide a more realistic estimate of the frequency of antigen-reactive cells in persons with HIV-1 infection.

Peripheral blood monocytes in psoriatic patients overproduce cytokines

Interleukin-1 α (IL-1 α), interleukin-1 β (IL-1 β), tumor necrosis factor- α (TNF- α) and interleukin-8 (IL-8) production by peripheral monocytes in 15 patients with psoriasis vulgaris and 13 healthy controls were investigated using an enzyme-linked immunosorbent assay. Polymorphonuclear leukocytes, mononuclear leukocytes, monocytes, lymphocytes and serum were isolated and cultured for 1, 12, 24 and 48 h in RPMI 1640 medium without fetal calf serum to release the cytokines. All cytokine levels in serum were very low in both psoriatic patients and the controls. However, a significant increase in the IL-1 α levels in the monocyte culture supernatants was observed in the psoriatic patients compared with that of the controls. IL-1 β and IL-8 levels in the monocyte supernatants were also significantly increased in the psoriatic patients compared with the controls. TNF- α levels in the 12-h supernatants of the monocytes increased in some samples from psoriatic patients but not in the 48-h supernatants. Our study confirmed the overproduction of cytokines in the peripheral monocytes of the psoriatic patients and suggest the activation of monocytes in the patients with psoriasis.