Objective To investigate the antitumor immunity induced by tumor derived mixed heat shock protein/peptide(mHSPs),interleukin-12(IL-12)and Cyclophosphamide(Cy).MethodsPurified mixed HSP was prepared from tumor by S180 protein extraction and purification,SDS-PAGE,Western blot and animal experiment were applied for mixed HSPs analysis.ResultsThe proliferation of cytotoxic T lymphocyte(CTL)cocultured in the mHSPs+Cy+IL-12 group was significantly remarkable and the content of CD8+ CTLs was significantly more in comparison with the other groups(P<0.01).To the tumor bearing mice,mHSPs+Cy+IL-12 group showed partial therapeutic efficacy,the averaged survival period was over 60 d,and 90% of the mice in this group got long period tumor free survival(>90d),obvious difference(P<0.05)from the other groups.ConclusionTumor derived mixed HSPs can induce powerful antitumor immune efficacy and show favorable therapeutic efficacy.

Objective: To evaluate the anti-tumor activity of mouse multi-subtype heat shock protein/peptide (mHSP/P) vaccine in combination with a programmed death ligand 1 (PD-L1) inhibitor in mouse sarcoma. Methods: Immunohistochemical staining and en-zyme-linked immunosorbent assay (Elisa) was used to quantitatively identify the expression of heat shock proteins (HSP70, HSP90, Grp94) in the sarcoma cell line MCA207. From the protein suspension prepared, mHSP/P and Grp94/peptide (Grp94/P) sarcoma vac-cines were isolated using chromatography and were identified by Western blot (WB). Flow cytometry was used to determine their cy-totoxic effects. The levels of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) produced upon mHSP/P and Grp94/P stimulation were measured by Elisa. The effect of sarcoma vaccines on the growth and survival of sarcoma was evaluated in mice. The expression of PD-L1 on the surface of MCA207 sarcoma cells was evaluated by immunofluorescent staining. The effect of IFN-γ treatment on the expression of PD-L1 was determined by WB. Animal experiments explored the effects of PD-L1 inhibitor in combination with mHSP/P treatment on tumors. Results: Tumor tissue carries a variety of HSP subtypes (HSP70, HSP90, Grp94). We successfully isolated sarco-ma tissue-derived mHSP/P and Grp94/P tumor vaccines, which were identified by WB; flow cytometry analysis demonstrated their cy-totoxicity. The levels of IFN-γ and TNF-α cytokines upon mHSP/P stimulation were significantly higher than that observed upon Grp94/P stimulation (P<0.05). The expression of PD-L1 on the surface of sarcoma cells increased with IFN-γ treatment. Animal experiments demonstrated that PD-L1 inhibitor in combination with mHSP/P significantly increased the immune response against tumor (P<0.05). Conclusions: Tumor-derived mHSP/P and Grp94/P can be used as tumor vaccines in animal models. The mHSP/P can elicit a stronger anti-tumor immune response than Grp94/P. IFN-γ stimulates the expression of PD-L1 in sarcoma cells, which results in immune eva-sion. The PD-L1 inhibitor in combination with mHSP/P increased the anti-tumor effect in the tumor microenvironment.

OBJECTIVETo investigate the relationship of the mutational status of the ND4 gene and the clinical features of acute myelogenous leukemia (AML) patients with ND4 mutations.

METHODSUsing PCR combined with directly sequencing, we identified somatic mutations of ND4 in 121 primary AML patients to couple with their clinical features.

RESULTSThere were 58 male patients and 63 female patients (median age 49 years, 10-86 years). Eight of 121 patients (6.6%) with de novo AML were found harboring missense mutation of ND4 gene, including 3 patients with A131V (3/8, 37.5%), 2 patients with A404T (2/8, 25%), 1 patient with F149L (1/8, 12.5%), 1 patient with G242D (1/8, 12.5%) and 1 patient with Y409H (1/8, 12.5%), respectively. Patients with ND4 mutations were associated with good karyotype (P=0.049), regardless of gender, age, white blood cell, hemoglobin, platelet, blast cells of bone marrow or immunophenotype (P>0.05). There were no statistical significance in mutations of FLT3-ITD, NPM1, CEBPA, c-KIT and DNMT3A between patients with ND4 mutation and wild-type (wt) ND4 (P>0.05). The median overall survival of patients with ND4 mutations and wt ND4 were all not reached. The median relapse-free survival were not reached and 29(2-53) months, respectively (P>0.05). There was no significance in the ratio of CR and RR patients between wt ND4 and ND4 mutated groups (P>0.05).

CONCLUSIONIt was concluded that novel ND4 mutations could be found in de novo AML patients, especially in patients with good karyotype. Thus, ND4 mutations might play an important role in AML prognosis. However, whether the mitochondria dysfunction contribute to leukemogenesis needs to be further investigated.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Child ; Female ; Humans ; Leukemia, Myeloid, Acute ; drug therapy ; genetics ; Male ; Middle Aged ; Mutation ; NADH Dehydrogenase ; genetics ; Prognosis ; Young Adult