Clinical Implications of a Six-Protein Signature in Bone Metastasis of Renal Cell Carcinoma.

Bone metastases is prevalent from renal cell carcinoma (RCC) with poor quality of life and prognosis. Our previous proteomics analysis identified dysregulated proteins in the bone-tropism RCC cells. In this study, we further examined the clinical implications of these proteins using multiple clinical cohorts. We identified 6 proteins with significant upregulation in RCC tumor tissue in comparing to tumor adjacent normal tissue (p<0.05). High expression of these 6 protein-encoding genes significantly correlates with a poor survival in the TCGA-KIRC (Kidney renal clear cell carcinoma) cohort (log-rank test p=2.7e-05), and they all individually had a reverse-correlation with the gene expression of VHL and PBRM1 (p<0.001), and positive-correlation with the expression of VEGFA (p<0.001). Further gene set variation analysis (GSVA) revealed positive correlation with Th17 cells enrichment and negative CD8 T cell infiltration in the RCC tumor microenvironment. High expression of these 6 genes in pretreatment tumors favors longer overall survival (OS)(p=0.027) in anti-PDL1 treated patients (n=428). We treated one humeral metastases RCC patient with the anti-PDL1 antibody drug atezolizumab after examined the elevated expression of the 6 proteins in his nephrectomy tumor tissue, the tumor at the fracture site shrunk remarkably after four courses of treatment. These results altogether suggest a clinical implication of the 6-protein signature in RCC bone metastasis prognosis and response to immune-checkpoint inhibitor treatment.

Demethylating therapy increases cytotoxicity of CD44v6 CAR-T cells against acute myeloid leukemia.

Background: CD44v6 chimeric antigen receptor T (CD44v6 CAR-T) cells demonstrate strong anti-tumor ability and safety in acute myeloid leukemia (AML). However, the expression of CD44v6 on T cells leads to transient fratricide and exhaustion of CD44v6 CAR-T cells, which affect the application of CD44v6 CAR-T. The exhaustion and function of T cells and CD44v6 expression of AML cells are associated with DNA methylation. Hypomethylating agents (HAMs) decitabine (Dec) and azacitidine (Aza) have been widely used to treat AML. Therefore, there may be synergy between CD44v6 CAR-T cells and HAMs in the treatment of AML. Methods: CD44v6 CAR-T cells pretreated with Dec or Aza were co-cultured with CD44v6+ AML cells. Dec or aza pretreated AML cells were co-cultured with CD44v6 CAR-T cells. The cytotoxicity, exhaustion, differentiation and transduction efficiency of CAR-T cells, and CD44v6 expression and apoptosis in AML cells were detected by flow cytometry. The subcutaneous tumor models were used to evaluate the anti-tumor effect of CD44v6 CAR-T cells combined with Dec in vivo. The effects of Dec or Aza on gene expression profile of CD44v6 CAR-T cells were analyzed by RNA-seq. Results: Our results revealed that Dec and Aza improved the function of CD44v6 CAR-T cells through increasing the absolute output of CAR+ cells and persistence, promoting activation and memory phenotype of CD44v6 CAR-T cells, and Dec had a more pronounced effect. Dec and Aza promoted the apoptosis of AML cells, particularly with DNA methyltransferase 3A (DNMT3A) mutation. Dec and Aza also enhanced the CD44v6 CAR-T response to AML by upregulating CD44v6 expression of AML cells regardless of FMS-like tyrosine kinase 3 (FLT3) or DNMT3A mutations. The combination of Dec or Aza pretreated CD44v6 CAR-T with pretreated AML cells demonstrated the most potent anti-tumor ability against AML. Conclusion: Dec or Aza in combination with CD44v6 CAR-T cells is a promising combination therapy for AML patients.

[The Levels of Inflammatory Regulatory Factors in Acute Leukemia Complicated by Infection and Its Clinical Significance].

OBJECTIVE: To explore the expression levels and clinical significance of helper T cell 1/helper T cell 2 (Th1/Th2) cytokine and interleukin-6 (IL-6) in patients with acute leukemia (AL) complicated by infection. METHODS: 68 patients with AL complicated by infection admitted to Wuhan Fifth Hospital from May 2017 to January 2020 were enrolled as study group, 50 AL patients without infection were enrolled as AL group, and 30 healthy volunteers checked in physical examination center were enrolled as healthy control group. The levels of serum tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-10 (IL-10), and peripheral blood Th1/Th2 cells subsets were measured and compared among the three groups. The serum IL-6, IL-10, TNF-α and Th1/Th2 were compared between the patients with mild to moderate infection (n=52) and septic shock (n=16). The relationship between IL-6, IL-10, TNF-α, Th1/Th2 and AL infection was analyzed. RESULTS: The levels of IL-6, IL-10 , TNF-α, and the proportion of Th2 of the patients in study group and AL group were significantly higher than those in healthy control group (P<0.001), while the proportion of Th1 and Th1/Th2 were significantly lower than those in healthy control group (P<0.001). The levels of IL-6, IL-10 and TNF-α, and the proportion of Th2 the patients in study group were significantly higher than those in AL group (P<0.001), while the proportion of Th1 and Th1/Th2 were significantly lower than those in AL group (P<0.001). The serum IL-6, IL-10 and TNF-α level of the patients in septic shock group were significantly higher than those in mild-to-moderate infection group (P<0.001), while Th1/Th2 was lower than those in mild-to-moderate infection group (P<0.001). The results of ROC curve analysis showed that the area under the ROC curve (AUC) values of IL-6, IL-10, TNF-α and Th1/Th2 alone for the diagnosis of septic shock were 0.779, 0.761, 0.724 and 0.718, which were lower than that their combination (0.910) (P<0.05). CONCLUSION: The levels of serum IL-6, IL-10 and TNF-α are high in patients with AL complicated infection and septic shock, while Th1/Th2 cell subsets is low. The combined detection of serum IL-6, IL-10, TNF-α and Th1/Th2 is a good diagnostic value for predicting the occurrence of severe septic shock.

MiR-185-5p suppresses acute myeloid leukemia by inhibiting GPX1.

Acute myeloid leukemia (AML) has been characterized by the swift development of abnormal cells in the bone marrow. This research aimed to examine the impacts of the miR-185-5p-GPX1 axis on AML progression and differentiation. Findings indicated that miR-185-5p and GPX1 levels were significantly reduced and elevated, respectively. The upregulation of miR-185-5p was observed to restrict the proliferation and invasion abilities of AML cells, and promote differentiate and apoptosis. Moreover, the overexpression of GPX1 was noticed to enhance the growth of AML cells. In conclusion, this research suggested that by targeting GPX1, miR-185-5p inhibited AML progression and downregulated AML cells' proliferation and invasion.

[In vitro cytotoxic effects of CML28 specific T cells on acute leukemia cells].

OBJECTIVE: To explore the activation and proliferation of specific T cells induced by artificial antigen-presenting cells (aAPCs) simulated dendritic cells (DCs) and to observed the effect of these T cells on leukemic cell killing. METHODS: aAPCs were developed by coating a human leukocyte antigen-immunoglobulin fusion protein ( HLA-lg), which was connected each one of the four CML28 antigen epitopes (DLMSSTKGL, DLMSSTKGL, ALFCGVACA, VLTFALDSV), and CD28-specific antibody, to magnet-beads CML cell specific peptides (CML28) served as target peptides. Bone marrow (BM) or peripheral blood (PB) mononuclear cells (MNCs) were isolated from HLA-A2 healthy volunteers, and co-cultured with aAPCs. Specific T lymphocyte were detected by flow cytometry. The fresh acute leukemic cells were used as target cells. The specific T cells incubated with leukemic cells for 4 h at ratios of 5:1, 10:1, 20:1, 40:1, 80: 1, respectively. The effect of leukemic cells killing was detected by lactate dehydrogenase release test. RESULTS: The average ratio of CML-28 specific T lymphocyte in control group was (2.2 +/- 0.4)% and in experimental groups (DLMSSTKGL, DLMSSTKGL, ALFCGVACA, VLTFALDSV) were (13.5 +/- 1.6)%, (15.2 +/- 1.5)%, (14.7 +/- 1.8)% and (34.3 +/- 3.5)%, respectively, being significantly higher than that in control group (P < 0.01). Induction efficiencies of acute leukemic cells killing were significantly enhanced by increase of effector cells. The cytotoxic activity of specific T lymphocyte in one experimental group (VLTFALDSV) was much higher than that in other three experimental group (P < 0.05). CONCLUSION: This "prime and expand" regimen should be an alternative method for large scale amplification of rare tumor-specific CTLs and aAPCs might be a useful tool for leukemia immunotherapy.

[Activation of specific T lymphocyte induced by artificial antigen presenting cell complex in vitro].

This study was aimed to investigate the effect of artificial antigen-presenting cells (aAPCs) on inducing activation and proliferation of specific T-lymphocytes through stimulating biological function of dendritic cells with aAPCs in vitro. The specific antigen of chronic myeloid leukemia CML-28 was screened as objective antigen peptide by using magnetic microbeads as vector; the CML-28 epitope sequence (Vltfaldsv) was obtained by antigen epitope prediction software; this epitope was coupled with MHC molecule and used as first signal molecule, the B7-1 molecule was used as second signal molecule; these 2 molecules simultaneously were loaded onto surface of magnetic microbeads so as to contract aAPC complex. The bone marrow mononuclear cells were derived from HLA-A2(+) healthy bone marrow donors, CD8(+) T lymphocytes were screened and co-cultured with aAPC complex. During culture the 5, 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) was added and proliferation of T-lymphocytes was detected by CPSE and proliferation level of specific T lymphocytes was detected by flow cytometry. The results showed that the proliferation level of CML-28 specific T lymphocytes obviously increased in experimental group, average level was 17.34 +/- 0.65%, while average level in control was 2.25 +/- 0.43%, there was significant difference between them (p < 0.01). It is concluded that the aAPC complex can mimic human APCs in vitro, and stimulate activation and proliferation of CD8(+) specific T lymphocytes.

Selection of HLA-A2 restricted CML28 peptide by artificial antigen-presenting cells.

Chronic myelogenous leukemia (CML) 28 is a broadly immunogenic antigen that can have an antileukemia effect. We adopted the SYFPEITHI database to predict human leukocyte antigen-A2 restricted CML28 peptide. We designed artificial antigen-presenting cells (aAPCs) by coating micro beads with human leukocyte antigen-A2-immunoglobulin dimer and CD28-specific antibody. We used the selected peptides-pulsed aAPCs to induce cytotoxic T lymphocytes (CTLs), to choose the CML28 peptide, which was best for inducing CTLs. The result showed that the peptides-pulsed aAPCs could induce CTLs and that the peptide VLTFALDSV was the best choice for significantly inducing specific CTLs.

[Expression of plk-1 gene in acute leukemia patients and its significance].

To investigate expression of plk-1 gene and PLK-1 protein in acute leukemia patients and its clinical significance, and to observe the distribution of PLk-1 protein in acute leukemin cells, the mononuclear cells were separated from the bone marrow or peripheral blood of acute leukemia patients, bone marrow benign proliferation individuals and normal individuals. The expression of plk-1 gene and PLK-1 protein in those cells were detected with RT-PCR and flow cytometry respectively, the distribution pattern of PLK-1 was observed by fluorescent inverted microscope. The result showed that the expressions of plk-1 gene and PLK-1 protein in mononuclear cells of acute leukemia patients were much higher than that of bone marrow benign proliferation individuals and normal individuals. Fluorescent inverted microscopy revealed that PLK-1 was highly concentrated in cytoplasm of acute leukemia cells during interphase of mitosis, and it was found that PLK-1 was mainly distributed between sister chromatid during the mitosis in mononuclear cells of acute leukemia patients, but the expressions of plk-1 gene and PLK-1 protein almost were not observed in cells of benign proliferative bone marrow and normal bone marrow. It is concluded that increased plk-1 gene and protein perhaps play an important role in abnormal proliferation of acute leukemia cells and correlate with the malignamcy of leukemia. plk-1 gene or PLK-1 protein may be considered as a new target of therapy, and one of useful indicators in evaluation of curative efficiency and prognosis.

Strain-dependent structural variants of herpes simplex virus type 1 ICP34.5 determine viral plaque size, efficiency of glycoprotein processing, and viral release and neuroinvasive disease potential.

The ability of certain strains of herpes simplex virus type 1 (HSV-1) to cause encephalitis or neuroinvasive disease in the mouse upon peripheral infection is dependent on a combination of activities of specific forms of viral proteins. The importance of specific variants of ICP34.5 to neuroinvasive disease potential and its correlation with small-plaque production, inefficient glycoprotein processing, and virus release were suggested by comparison of ICP34.5 from the SP7 virus, originally obtained from the brain of a neonate with disseminated disease, and the tissue culture-passaged progeny of SP7 (SLP5 and SLP10) and the KOS321 virus. SLP5, SLP10, and KOS321 are attenuated and exhibit a large-plaque phenotype, including efficient glycoprotein processing and viral release. We show that expression of the KOS321 ICP34.5 protein in cells infected with SP7 or ICP34.5 deletion mutants promotes large plaque formation and efficient viral glycoprotein processing, while expression of the SP7 ICP34.5 protein decreases efficiency of viral glycoprotein processing. In addition, a recombinant virus, 4hS1, with the SP7 ICP34.5 gene replacing the KOS321-like ICP34.5 gene in the SLP10a background, rescues the small-plaque phenotype and neuroinvasive disease. The major difference in the ICP34.5 gene product is the number of Pro-Ala-Thr repeats in the middle region of the protein, with 18 for SP7 and 3 for KOS321. Strain-dependent differences in the ICP34.5 protein can therefore alter the tissue culture behavior and the virulence of HSV-1.