The Predictive Value of Changes in the Absolute Counts of Peripheral Lymphocyte Subsets for Progression and Prognosis in Breast Cancer Patients.

Background: As the number and proportion of lymphocyte subsets are an important indicator of the immune function, an in depth understanding of the immune function of patients with malignant tumor has important clinical values for the treatment, prognosis, and evaluation of the disease. This retrospective study was to evaluate the clinical value of the absolute counts of lymphocyte subsets as potential blood biomarkers for progression and prognosis in breast cancer patients. Methods: A total of 237 BC patients and 55 age-matched female normal healthy donors were included in this study. Flow cytometry was used to determine the absolute counts and the percentages of CD3+, CD4+, CD8+, B, and NK cells. The receiver operating characteristic curve (ROC) was used to evaluate the accuracy of absolute count of lymphocyte subsets in the curative efficacy assessment. The clinicopathological parameters influencing the disease progression were determined by Cox proportional hazards regression. Progression-free survival (PFS) was estimated using the Kaplan-Meier method with the log-rank test. Results: Compared with the healthy donors, the absolute counts of lymphocyte subsets in patients decreased significantly. ROC analysis showed that the area under the curve of the CD4+ absolute count was 90% (95% confidence interval 0.859-0.940), and the sensitivity and specificity were 80.9% and 85.3%, respectively. The analysis of Cox regression showed that the cutoff value of the CD4+ absolute count ≥451 cells/µL might be a favorable prognostic factor. Multivariate analysis of prognostic factors of PFS showed that the CD4+ and CD8+ absolute count were independent factors for predicting PFS. Conclusions: The remarkably impaired absolute counts of the CD3+, CD4+, CD8+, B, and NK cells in patients with breast cancer can be used as potential susceptible biomarkers to evaluate the patient's immune status. The higher level of CD4+ and CD8+ absolute counts probably contributed to the longer PFS and favorable outcome of BC patients.

Reference range of naïve T and T memory lymphocyte subsets in peripheral blood of healthy adult.

Naïve T and T memory cell subsets are closely related to immune response and can provide important information for the diagnosis and treatment of immunological and hematological disorders. Lymphocyte compartment undergoes dramatic changes during adulthood; age-related reference values derived from healthy individuals are crucial. However, extensively detailed reference values of peripheral blood lymphocytes in the whole spectrum of adulthood detected by multi-color flow cytometry on a single platform are rare. Three hundred and nine healthy adult volunteers were recruited from Tianjin in China. The absolute counts and percentages of CD3+CD4+ T cells, CD3+CD8+ T cells, naïve T cells (Tn), T memory stem cells (Tscm), central memory T cells (Tcm), effector memory T cells (Tem), and terminal effector T cells (Tte) were detected by flow cytometry with single platform technologies. Reference range of absolute counts and percentage of T lymphocyte subsets were formulated by different age and gender. The results showed that Tn and Tscm cells, which had stem cell properties, decreased with aging; while, Tcm and Tem increased with aging, which increased from 18 to 64 years old but presented no significant change over the 65 years old. Gender had an influence on the fluctuation of lymphocyte subsets, the absolute count of CD3+CD8+, CD8+Tcm, CD8+Tem in males were higher than those in females. The reference values of percentages and absolute numbers of naïve T and T memory cell subsets can help doctors to understand the immune state of patients and evaluate conditions of prognosis then adjust the treatment for patients. (Chinese Clinic Trial Registry number: ChiCTR-IOR-17014139.).

The clinical value of the changes of peripheral lymphocyte subsets absolute counts in patients with non-small cell lung cancer.

INTRODUCTION: Immune function strongly influences the outcome of patients with non-small cell lung cancer (NSCLC). It's vital to understand the immune state of patients through detecting the percentage and number of lymphocyte subsets accurately, and helpful to evaluate conditions of prognosis and adjust treatment for patients. METHODS: We conducted a retrospective cohort study in First Teaching Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin, China. The absolute counts and percentages of CD3+, CD3 + CD4+, CD3 + CD8+, B and NK cells were determined by single platform technologies. 172 patients received treatment including surgery or chemotherapy after surgery. The factors affecting disease progression were analyzed by Binary Logistic regression. Progression free survival (PFS) calculating survivals were with the method of Kaplan-Meier. The log-rank test and cox's proportional hazard regression (enter method) were used for univariable and multivariable analyses respectively. RESULTS: Relative to normal controls, patients with NSCLC at different stages showed decreased absolute lymphocyte count obviously, rather than lymphocyte percentages. Different treatments had unlike influence on the homeostasis of lymphocytes and the effects last for a long time. Logistic regression showed CD3 + CD4+ and CD3 + CD8+ could contribute to favorable prognosis. Multivariate analysis of prognostic factors of PFS showed CD3 + CD4+ cell was independent factor for predicting PFS. CONCLUSIONS: The absolute count of CD3+, CD3 + CD4+, CD3 + CD8+, B and NK cells were better indication of the patient's immune state than percentages of each lymphocyte subsets. Immune function was impaired in patients with non-small cell lung. The high level of baseline absolute CD3 + CD4+ cells count contributed to longer progression free survival. Chinese Clinic Trial Registry number: ChiCTR-IOR-17014139; Registry date: 2017/12/25.

Discovery and characteristics of B cell-like T cells: A potential novel tumor immune marker?

BACKGROUND: CD3 and CD19 are the characteristic surface markers of mature T lymphocytes and B lymphocytes of human respectively. A special subset of immune cells that characteristically expressed the surface markers CD19+ of B lymphocytes and CD3+ of T lymphocytes simultaneously (CD19+CD3+ cells, hereinafter referred to as B-T cells) was found in the peripheral blood of human, yet it has not been reported in cancer research before. Our aims were to characterize the expression and possible value of B-T cells in cancer patients. METHODS: Flow cytometry was applied to analyse the CD19+CD3+ cells, and laser scanning confocal microscope was utilized to prove co-expressing CD19+ of B lymphocytes and CD3+ of T lymphocytes simultaneously on the surface of the cells. Then a total of 523 patients with malignant tumor were enrolled in this study, and 177 healthy donors were recruited as the control group. The levels of CD19+CD3+ cells in peripheral blood were measured by flow cytometry, and the differences between the two groups were compared. RESULTS: The healthy donors and cancer patients all had B-T cells in their peripheral blood, but the percentage of B-T cells was 0.16 % ± 0.11 % and 0.58 % ± 0.38 % respectively, showing statistically significant (P < 0.0001). There was no significant correlation between the percentage of B-T cells and lymphocyte subsets (P > 0.05). The percentages of B-T cells in different tumor species were different. The proportion of B-T cells was high in esophageal cancer, non-Hodgkin's lymphoma and lung cancer, but it was low in pancreatic cancer, ovarian cancer and kidney cancer. Meanwhile, there was significant difference between esophageal cancer and kidney cancer (P < 0.001). The distribution of B-T cells in pancreatic cancer and kidney cancer was more concentrated, yet more dispersed in other cancers. Although there was a trend of increase in clinical stage Ⅲ+Ⅳ and a trend of decrease in age above 60 years for breast cancer, gastric cancer and liver cancer, there was no significant difference in the percentage of B-T cells in age, gender, different clinical stages, tumor metastasis, lymph node metastasis, and splenomegaly (P > 0.05). CONCLUSION: The percentage of B-T cells in cancer patients was significantly higher than that of healthy donors. B-T cells maybe play a very complicated role in tumor, whether it could be a potential tumor immune marker or not and what are the specific phenotypes and functions of it to need be further verified.

[Effects of "Tiaoyi Sanjiao" acupuncture and moxibustion on cancer-induced fatigue and immune function in patients with advanced non-small cell lung cancer].

OBJECTIVE: To observe the therapeutic effect of "Tiaoyi Sanjiao" （regulating and tonifying the triple energizer）acupuncture and moxibustion in the treatment of cancer-related fatigue of advanced non-small cell lung cancer and observe its influence on lymphocyte count. METHODS: The cancer-related fatigue patients with advanced non-small cell lung cancer who met the inclusive criteria were randomly divided into 2 groups, with 77 cases in each group. Patients in the control group were treated with conventional Chinese and Western medicine. In the treatment group, on the base of the treatment as the control group, "Tiaoyi Sanjiao" acupuncture and moxibustion was applied（mild moxibustion at Danzhong ［CV17］, Zhongwan［CV12］, Qihai［CV6］, bilateral Zusanli［ST36］, and acupuncture at bilateral Xuehai［SP10］, Waiguan［SJ5］, Taichong［LR3］）. KPS score, Piper scale, percentage and absolute count of lymphocyte subsets of the two groups before and after treatment were observed. RESULTS: Compared with pre-treatment, the KPS scores of both groups were significantly increased after treatment (P<0.05); the behavioral dimension score of the Piper scale, and the percentage of B cells of the control group decreased significantly(P<0.05); the behavioral dimension, emotional dimension, sensory dimension scores, and total score of the Piper scale in the treatment group were significantly reduced(P<0.05), while the percentage of CD3+T cells and absolute counts of CD3+T cells, CD4+T cells and CD8+T cells of the treatment group were significantly increased (P<0.05). After treatment, in comparison with the control group, behavioral dimension score of the Piper scale in the treatment group decreased significantly(P<0.05); the percentage and absolute counts of B cells and CD4+T cells, the absolute count of CD3+T cells in the treatment group were up-regulated (P<0.05). CONCLUSION: "Tiaoyi Sanjiao" acupuncture and moxibustion can significantly alleviate the fatigue state and improve the quality of life in patients with advanced non-small cell lung cancer, which may be achieved by regulating the number of lymphocytes and improving immune function.

A novel tumor suppressor SPINK5 targets Wnt/ß-catenin signaling pathway in esophageal cancer.

Esophageal cancer is one of the most common tumor in the world, and the morbidity rate is as high as 100/100 000 in some parts of China. Therefore, it is important and urgent to explore the pathogenesis of esophageal cancer and find new therapeutic targets for esophageal cancer. In this study, we found that a novel tumor suppressor SPINK5 is significantly reduced in the development of esophageal cancer, and is closely related to the pathological differentiation and lymph node metastasis of esophageal cancer via bioinformatics analysis and esophageal cancer tissue array. Further studies have found that SPINK5 is closely related to Wnt/ß-catenin signaling pathway by bioinformatics analysis and western blot. In esophageal cancer cells, SPINK5 overexpression can inhibit Wnt/ß-catenin signaling pathway. Combined with LiCl or MG-132 treatment, SPINK5 can inhibit GSK3ß phosphorylation and promote ß-catenin protein degradation, thus inhibit Wnt/ß-catenin signaling pathway. In vivo study, SPINK5 overexpression can significantly inhibit the growth of esophageal cancer cells. Our study shows that SPINK5 can inhibit the proliferation, migration, and invasion of esophageal cancer cells by inhibiting Wnt/ß-catenin signaling pathway, and thus plays an important role in the development of esophageal cancer, and may serve as a treatment target of esophageal cancer.

Wenyang Huazhuo Tongluo formula inhibits fibrosis via suppressing Wnt/ß-catenin signaling pathway in a Bleomycin-induced systemic sclerosis mouse model.

BACKGROUND: Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of the skin and internal organs. So far, no Western medicine treatment can completely inhibit or reverse the progress of SSc, while at the same time, our previous series of studies have shown that the treatment of SSc by the Wenyang Huazhuo Tongluo formula (WYHZTL), a Chinese herbal decoction, shows a delightful prospect. The aim of this study is to further investigate the mechanism of anti-fibrosis of WYHZTL formula in SSc mouse model. METHODS: The Bleomycin-induced SSc mouse model was treated with saline (BLM), high-dosage of WYHZTL formula (WYHZTL-H), medium-dosage of WYHZTL formula (WYHZTL-M), low-dosage of WYHZTL formula (WYHZTL-L) and XAV-939, a small molecule inhibitor of Wnt/ß-catenin signaling pathway, by the intragastric administration and intraperitoneal injection, respectively. The mRNA and protein levels of Wnt/ß-catenin signaling pathway associated genes, fibrosis markers and histopathology were detected by reverse transcription-quantitative polymerase chain reaction, Western blotting and hematoxylin/eosin-staining. The levels of Wnt1, CTGF and DKK1 protein in serum were detected by enzyme-linked immunosorbent assay. RESULTS: Compared with BLM group, the WYHZTL formula and XAV-939 could significantly inhibit the thickness of the skin tissue of the SSc mouse model. The mRNA expression levels of GSK3ß and DKK1 in the WYHZTL formula and XAV-939-treated group were significantly higher than those in the BLM group, while Wnt1, ß-catenin, TCF4, cyclin D1, survivin, VEGF, CTGF, FN1, collagen I/III were decreased. Compared with BLM group, the protein expression levels of GSK3ß and DKK1 in the WYHZTL formula and XAV-939-treated group were upregulated, while Wnt1, ß-catenin, cyclin D1, survivin, CTGF, FN1, collagen I/III were downregulated. WYHZTL formula and XAV-939 could inhibit expression of Wnt1 and CTGF, but promoted DKK1 in serum. Furthermore, WYHZTL-H seemed more effective than WYHZTL-M and/or XAV-939 on regulating Wnt1, ß-catenin, TCF4, GSK3ß, DKK1, cyclin D1, survivin, VEGF, CTGF, FN1 and collagen I/III. CONCLUSION: This present study demonstrates that WYHZTL formula has anti-fibrosis effect in Bleomycin-induced SSc mouse model in a dosage-dependent manner, and the molecular mechanism may be related to the inhibition of Wnt/ß-catenin signaling pathway.

[Value of DAPK Gene Methylation Patterns in the Diagnosis of Leukemia].

OBJECTIVE: To screen the differential methylation patterns of tumor suppressor gene DAPK and evaluate its value as a biomarker for the diagnosis of leukemia. METHODS: The methylation status of DAPK gene promoter's CpG island was analyzed in the genomes of normal human white blood cells and HL-60, U937 and Jurkat cell lines by bisulfite sequencing PCR (BSP). The effectiveness of differential methylation patterns of DAPK gene for diagnosis of leukemia was verified in the leukemia cell lines and peripheral blood samples by methylation specific PCR (MSP). RESULTS: The methylation pattern of DAPK gene in different cell genomes displayed that the degree of unmethylation in normal cell genome was higher than that of leukemia cell lines. The differential CpG sites were found and could be used to differentiate HL-60 and the other 3 cell lines by MSP. Meanwhile, the differential methylation patterns in clinical specimens could distinguish acute non-lymphocytic leukemia (ANLL) and other types of leukemia by MSP. The diagnostic sensitivity, specificity and accuracy were 59.1%, 100% and 82.7% respectively. No relationship was found between MSP diagnosis results and clinical pathological typing. CONCLUSION: The differential methylation patterns of DAPK gene as potential tumor biomarker for diagnosis of leukemia can enrich the means of diagnosis of leukemia, provide idea and basis for finding all kinds of tumor's DNA methylation biomarkers in the future.

[Improvement and Evaluation on Bisulfite Modification of DNA for DNA Methylation Detection].

OBJECTIVE: To establish a rapid and convenient method of DNA modification by bisulfite sodium for the detection of DNA methylation. METHODS: Through increasing the bisulfite sodium concentration and the temperature of treatment, cutting down the modification time, besides using glassmilk to adsorb the DNA in the purification and recovery, to improve the methods of DNA modification. Efficiency of cytosine converted to thymine in MAGE-A3 gene and DAP-K gene fragments were analyzed by bisulfite sequencing PCR in order to evaluate the DNA modification effect among the improved method, traditional method and kit method. RESULTS: The operating time of test was shortened to about 3 hours by the improved method; conversion rate of unmethylated cytosine to thymine was over 99%; compared with the traditional method and kit method, there was no significant difference (χ(2) = 0.0564, P > 0.05); the improved method was only for the unmethylated cytosine conversion modification, and there was no significant difference in process of methylated cytosine converted to thymine comparing with the traditional method (χ(2) = 0.0149, P > 0.05). CONCLUSION: The improved method has high efficiency of DNA modification and has no significant effect on excessive modification;meanwhile, it has many advantages such as time-saving and easy to operate etc.

Paeonol enhances thrombus recanalization by inducing vascular endothelial growth factor 165 via ERK1/2 MAPK signaling pathway.

Paeonol (2'-hydroxy-4'-methoxyacetophenone) is the major active compound of Mautan cortex and has been demonstrated to inhibit platelet aggregation in previous studies. The current study aimed to elucidate the underlying molecular mechanism of paeonol in recanalizing thrombi. The presence of indicators of prothrombotic state (PTS) in the serum of the model animals were determined by enzyme­linked immunosorbent assay (ELISA) assay and the cytotoxicity of paeonol on human umbilical vein endothelial cell (HUVEC) cultures was estimated by 3­(4,5 dimethylthiazol­2­yl)-2,5-diphenyltetrazolium bromide assay. The possible underlying signaling pathway involved in the interaction between paeonol and vascular endothelial growth factor 165 (VEGF165) was investigated using western blotting. The levels of 6­keto­prostaglandin F1α, fibronectin, and VEGF165 in serum were significantly upregulated by the treatment of paeonol while the levels of fibrinogen, D­dimer, and thromboxane B2 were significantly downregulated (P<0.05). With increased paeonol concentration, the cell viability of HUVECs gradually decreased. The results of the western blot analysis demonstrated that paeonol increased the expression levels of phosphorylated­extracellular signal­regulated kinase (ERK1/2) and VEGF165 but had no marked effect on the expression level of ERK1/2. Paeonol has the potential to improve PTS and recanalize thrombi in animal models, which may be by the upregulation of VEGF165 via the ERK1/2 mitogen activated protein kinase signaling pathway. However, this positive effect depended on the concentration of paeonol used, an unsuitably high concentration of the compound exerted negative effects on the anti­thrombosis signaling pathways.

Thrombosis recanalization by paeoniflorin through the upregulation of urokinase­type plasminogen activator via the MAPK signaling pathway.

Paeoniflorin, the major component of Paeonia lactiflora pall, has previously been reported to prevent thrombosis. Plasminogen activator urokinase (uPA) is a serine protease that markedly facilitates normal thrombosis resolution. Paeoniflorin and uPA have been linked to the mitogen­activated protein kinase (MAPK) signaling pathway. In the current study, the influence of paeoniflorin on the expression of uPA was investigated and the underlying regulatory mechanism was preliminarily determined. The prothrombotic state of the model animals treated with paeoniflorin were assessed by enzyme­linked immunosorbent assay (ELISA). Additionally, the cytotoxicity of paeoniflorin on human umbilical vein endothelial cell (HUVEC) cultures was estimated using a methyl thiazolyl tetrazolium assay and the possible pathways involved in the interaction between paeoniflorin and uPA were evaluated using western blot analysis. The ELISA results demonstrated that the levels of 6­keto prostaglandin F1a, fibronectin and uPA were significantly upregulated by treatment with paeoniflorin compared with control (P<0.05). By contrast, the expression of fibrinogen, D­dimer and thromboxane B2 were inhibited. With an increase in the concentration of paeoniflorin the cell viability of HUVECs decreased gradually. The results of western blot analysis demonstrated that paeoniflorin increased the phosphorylation of MAPK 14 (p38) and MAPK 8 (JNK). The present study demonstrated that paeoniflorin has the potential to improve the prethrombotic state and recanalize thrombosis by increasing the expression of uPA, which may be mediated via regulation of the p38 and JNK MAPK signaling pathways. However, this treatment effect was dependent on the concentration of paeoniflorin used, an unsuitable concentration of the agent would result in a negative effect on the anti­thrombosis pathways.

Wenyang Huazhuo Tongluo formula, a Chinese herbal decoction, improves skin fibrosis by promoting apoptosis and inhibiting proliferation through down-regulation of survivin and cyclin D1 in systemic sclerosis.

BACKGROUND: Fibrosis is a major contributor to systemic sclerosis (SSc)-related morbidity, and rapid, progressive skin involvement predicts later mortality. Western medicine therapies for SSc cannot produce satisfactory effects currently, while Traditional Chinese Medicine (TCM), such as the Wenyang Huazhuo Tongluo (WYHZTL) formula, a Chinese herbal decoction, has shown amazing anti-fibrosis efficacy on SSc in clinical applications. This study is aiming to investigate the anti-fibrotic mechanism of WYHZTL formula for the treatment of SSc. METHODS: Fibroblasts from primary culture of skin lesions of SSc patients were exposed to rat medicated sera containing WYHZTL or XAV939, a small-molecule inhibitor of both tankyrase 1/2 and Wnt/ß-catenin pathway. Cell counting kit-8 assay and Annexin V FITC/PI apoptosis kit were used to analyze cell proliferation and apoptosis in fibroblasts, respectively. Reverse transcription-polymerase chain reaction (RT-PCR) and western blotting were used to detect the mRNA and protein levels of cyclin D1 and survivin. RESULTS: After 28, 48 and 72 h of incubation, the proliferative ability of the fibroblasts cells was obviously reduced by the sera containing WYHZTL compared with that in the control group; the percentage of apoptotic cell population in the sera containing WYHZTL treated fibroblasts cells was significantly higher than that in those treated with the control sera, and was about similar to that in those treated with XAV939. The sera containing WYHZTL could down-regulate both mRNA and protein levels of cyclin D1 and survivin, compared with the control group. CONCLUSIONS: The present study demonstrates the antiproliferative and pro-apoptotic actions of WYHZTL formula against fibroblasts and the effect may be related to the down-regulation of mRNA and protein levels of cyclin D1 and survivin in SSc.