Detection of myeloma cell-derived microvesicles: a tool to monitor multiple myeloma load.

The persistence of tumor load in multiple myeloma (MM) lead to relapse in patients achieving complete remission (CR). Appropriate and effective methods of myeloma tumor load monitoring are important for guiding clinical management. This study aimed to clarify the value of microvesicles in monitoring MM tumor load. Microvesicles in bone marrow and peripheral blood were isolated by differential ultracentrifugation and detected by flow cytometry. Western blotting was applied to assess myosin light chain phosphorylation levels. Flow cytometry to detect Ps+CD41a-, Ps+CD41a-CD138+, Ps+CD41a-BCMA+ microvesicles from bone marrow can be used to predict myeloma burden, furthermore, Ps+CD41a- microvesicles may as a potential index to MRD test. Mechanistically, the releasing of microvesicles from MM cell was regulated by Pim-2 Kinase via Phosphorylation of MLC-2 protein.

[Expression and Significance of PD-1 and ICOS in Patients with Primary Immune Thrombocytopenia].

OBJECTIVE: To investigate the expression of programmed death receptor-1 (PD-1) and inducible costimulator (ICOS) on the surface of CD8+ T cells in peripheral blood of patients with primary immune thrombocytopenia (ITP), and explore the roles of PD-1 and ICOS in the occurrence and development of ITP. METHODS: A total of 28 ITP patients treated in Tianjin Medical University General Hospital from September to December 2020 were selected, including 13 patients with newly diagnosed ITP, 15 patients with chronic ITP, and 22 healthy volunteers were recruited as control group. Flow cytometry was used to detect the expression levels of PD-1 and ICOS, and evaluate their correlation with clinical indicators. RESULTS: The percentage of CD8 + T cells in ITP patients of chronic group was higher than that of the newly diagnosed group and the control group (P<0.05). The expression level of PD-1 on CD8+ T cells in ITP patients of newly diagnosed group and chronic group were significantly lower than that of the control group (P<0.05), while the expression level of ICOS were significantly higher (P<0.05). In ITP patients, PD-1 was negatively correlated with platelet count (r=-0.4942, P<0.01), but positively with ICOS (r=0.4342). PD-1 and ICOS were both negatively correlated with lymphocyte count (rPD-1=-0.4374; rICOS=-0.4492). CONCLUSION: In ITP patients, the unbalanced expression of PD-1 and ICOS may interfere with the immune homeostasis of the body, which can be used as a therapeutic target for ITP patients.

Mutant Allele Frequency-Based Intra-Tumoral Genetic Heterogeneity Related to the Tumor Shrinkage Mode After Neoadjuvant Chemotherapy in Breast Cancer Patients.

The shrinkage mode of tumor extent after neoadjuvant chemotherapy (NAC) is an important index to evaluate the odds of breast-conserving surgery. However, there is no sufficient measurement to predict the shrinkage mode after NAC. In this study, we analyzed 24 patients' formalin-fixed, paraffin-embedded samples before and after treatment and analyzed 456 cancer-related genes panel by using target next-generation sequencing. Meanwhile, the pathological shrinkage mode was reconstructed in three dimensions after surgery, and the genetic heterogeneity level was estimated by mutant-allele tumor heterogeneity (MATH). We measured the genetic intra-tumor heterogeneity and explored its correlation with the shrinkage mode after NAC. A total of 17 matched pair samples of primary tumor tissue and residual tumor tissue were successfully accessed. It was found that the most common mutated genes were TP53 and PIK3CA in both samples before and after NAC, and no recurrent mutations were significantly associated with the shrinkage mode. Besides, the MATH value of formalin-fixed, paraffin-embedded samples before and after NAC was analyzed by the area under the curve of the receiver operating characteristic, and it is feasible to classify patients into concentric shrinkage mode and non-concentric shrinkage mode in NAC based on the MATH threshold of 58. Our findings indicate that the MATH value was associated with the shrinkage mode of breast cancer in a non-linear model. Patients with the MATH value below the threshold of 58 before and after NAC displayed a concentric shrinkage mode. The area under the curve was 0.89, with a sensitivity of 0.69 and specificity of 1. Our study might provide a promising application of intra-tumor heterogeneity that is measured by MATH to make a choice of surgery.

[Expression and Significance of IL-9 and IL-6 in Patients with BCR-ABL- MPN].

OBJECTIVE: To investigate the expression of IL-9 and IL-6 in patients with BCR-ABL- bone marrow proli- ferative tumor (MPN), and to explore its role in the occurrence and development of MPN. METHODS: A total of 71 newly diagnosis MPN patients treated in Tianjin Medical University General Hospital from 2018 to 2019 were selected, including 32 patients with polycythemia vera (PV) and 22 patients with primary thrombocytosis (ET), and 17 patients with primary myelofibrosis (PMF). Then 58 patients who retestine after treatment were selected as therapy group，and 20 healthy volunteers were recruited as control group. ELISA was used to detect the expression level of IL-6 and IL-9 in bone marrow supernatant, and the relative expression level of IL-6 and IL-9 mRNA in BMMNC was detected by real-time PCR. The proportion of Th9 cells in peripheral blood were detected by flow cytometry (FCM). The expression level of IL-6 mRNA and IL-9 mRNA of BMMNC and clinical indicators were analyzed, and the correlation between JAK2 gene mutation load and IL-9 level was further analyzed. RESULT: The level of IL-6 in bone marrow supernatant and the expression of IL-6 mRNA in BMMNC were higher in the newly diagnosed group as compared with those in the treated group and the control group (P<0.001). The expression level of IL-9 in bone marrow supernatant and the expression of IL-9 mRNA in BMMNC were lower in the newly diagnosed group as compared with those in the treated group and the control group (P<0.05). The proportion of Th9 cells in peripheral blood was lower in the newly diagnosed group as compared with that in the treated group and the control group (P<0.001). The level of IL-6 in bone marrow supernatant and the expression of IL-6 mRNA in BMMNC in JAK2+ group were higher than those in JAK2- group (P<0.05). The expression level of IL-9 in bone marrow supernatant and the expression of IL-9 mRNA in BMMNC were lower in JAK2+ group as compared with those in JAK2- group (P<0.05). The expression of IL-6 and IL-9 in the patient group showed correlation with the number of lymphocytes (IL-6: r=-0.49, P<0.01; IL-9: r=0.53, P<0.001), and also related with Hb in PV patients (IL-6: r= 0.87, P<0.001; IL-9: r=-0.54, P<0.01), and platelets in ET patients (IL-6: r=0.64, P<0.05; IL-9: r=-0.46, P<0.05). CONCLUSION: The increased expression of IL-6 in MPN and hyperfunction may promote the progression of BCR-ABL- MPN disease. The expression of IL-9 in MPN decreases, and it negatively correlates with the mutation load of JAK2 gene, which may be related with the decrease of tumor environmental antitumor immune effect.

[Relationship of Peripheral Blood IL-37 Expression with T Lymphocytes Subsets and NK Cells in Patients with Primary Immune Thrombocytopenia].

OBJECTIVE: To study the correlation of IL-37 with T lymphocytes subsets and NK cells in ITP patients, and to explore its possible mechanisms involved in the pathogenesis of ITP. METHODS: Forty-five patients with newly diagnosed ITP(newly diagnosed group), 32 patients of complete remission (remission group) and 22 healthy persons(control group) were selected. The serum level of IL-37 in 3 groups was determined by enzyme linked immunosorbent assay (ELISA). The mRNA expression of IL-37, IL-17 and IL-18 in peripheral blood mononuclear cells（PBMNC） in 3 groups was measured by real-time fluorescence quantitative polymerase chain reaction (PCR). The number of IL-18Rα+CD4+ T cells and Tim-3+NK cells in the peripheral blood in 3 groups was detected by flow cytometry (FCM). RESULTS: The serum level of IL-37 in the peripheral blood of ITP patients in the newly diagnosed group was significantly higher than that in the control group and the remission group(P＜0.01) . The expression level of IL-37 in PBMNC of the ITP patients in newly diagnosed group was higher than that in the control group and the remission group(P＜0. 05). The expression level of IL-17 and IL-18 in PBMNC of the ITP patients in newly diagnosed group was higher than that in the control group and the remission group(P＜0. 01); the expression of IL-18Rα in CD4+ T cells in newly diagnosed group was significantly higher than that in both the control and the remission group(P＜0.01).The expression of Tim-3 in NK cells in ITP patients was significantly lower than that in the control group (P＜0. 01). In ITP patients, the serum IL-37 level and IL-18Rα+CD4+T cells ratio both negatively correlated with Plt count (r=-0.58, r=-0.48) moreo-ver the serum IL-37 level also negatively correlated with amount of CD4+ T cells and NK cells (r=-0.29, r=-0.28), but positively correlated with amount of CD8+ T cells (r=0.329). CONCLUSION: The IL-37 and its receptors may play an immunoregulatory role in CD4+ T cells and NK cells, the IL-37 may be a therapeutic target for ITP patients.

The potential diagnostic power of CD138+ microparticles from the plasma analysis for multiple myeloma clinical monitoring.

Multiple myeloma (MM) is malignant tumor with abnormal proliferation of bone marrow plasma cells. The existing clinical tools used to determine treatment response and tumor relapse are limited in sensitivity. We investigated the CD138+ microparticles (MPs) of MM patients to find out whether MPs could provide a novel means to monitor the malignant cells in MM patients. Our study showed that the levels of MPs were significantly elevated in MM patients. The MP counts in peripheral blood from new diagnosed MM patients were significantly higher than patients in CR and HD. Consist with the total MPs, the number of the PC-derived MPs (CD138+) increased in BM from MM patients compared with CR and HD. The ratio of the PC-derived MPs (CD138+) in BM increased in MM patients compared with CR and HD. The correlation test revealed that the CD138+ MPs in BM and PB were all positively correlated with the plasmacyte ratio in bone marrow (BMPC) and the ß2 -MG. New diagnosed MM patients and controls were compared, and ROC curves were used to identify cutoff points with optimal sensitivity and specificity concerning the ratios and counts of CD138+ MPs in BM and PB. The AUC of the CD138+ MP counts in BM was 0.767, and in PB was 0.680. The AUC of the CD138+ MP ratios in BM was 0.714, and in PB was 0.666. According to this, the counts of CD138+ MPs in BM showed to be a powerful marker of diagnosis. We demonstrated that CD138+ MPs from the plasma provide support for a potential monitoring biomarker of MM.

[Number and Function of Myeloid-Derived Suppressor Cells in Patients with Adult Primary Immune Thrombocytopenia].

OBJECTIVE: To analyze the number of myeloid-derived suppressor cells(MDSC) and the level of prostaglandin E2(PGE2) in the bone marrow of adult ITP patients, and to explore their possible mechanisms involved in the pathogenesis of this disease. METHODS: Twenty-five patients of newly diagnosed ITP, 25 patients of complete remission group and 15 patients of control group were selected. The number of MDSC in the bone marrow between 3 groups was detect by flow cytometry (FCM). The serum level of prostaglandin E2 (PGE2) in 3 groups was determined by enzyme linked immunosorbent assay (ELISA). The relative expression of IFN-γ mRNA in bone marrow mononuclear cells was measured by real time fluorescence quantitative polymerase chain reaction (RT-qPCR) in each groups. RESULTS: The number of MDSC in the complete remission group was significantly higher than that in the control group (P<0.05); the number of MDSC in the newly diagnosed group was higher than that in the control group; the number of MDSC in the complete remission group was higher than that in the newly diagnosed group. The serum level of PGE2 in bone marrow of ITP patients in the newly diagnosed group was higher than that of the control group（P<0.05）. The serum level of PGE2 in the bone marrow of ITP patients of the complete remission group was higher than that of the control group (P<0.05）. The level of PGE2 in bone marrow serum of ITP patients of the newly diagnosed group was lower than that in the complete remission group(P<0.05）. The relative expression level of IFN-gamma in bone marrow mononuclear cells of the ITP patients in newly diagnosed group was higher than that in the control group and the complete remission group（P<0.001）. The relative quantification (RQ) of IFN-γ in bone marrow mononuclear cells was 2.60 between the newly diagnosed group and the complete remission group. CONCLUSION: When adult ITP disease is remitted, the number of MDSC rises and correlates with the therapeutic response and PGE2 level in the bone marrow.

Protective effects of microRNA-330 on amyloid ß-protein production, oxidative stress, and mitochondrial dysfunction in Alzheimer's disease by targeting VAV1 via the MAPK signaling pathway.

This study aims to explore the effect of miR-330 targeting VAV1 on amyloid ß-protein (Aß) production, oxidative stress (OS), and mitochondrial dysfunction in Alzheimer's disease (AD) mice through the MAPK signaling pathway. Putative targeted gene of miR-330 was performed by a miRNA target prediction website and dual-luciferase reporter gene assay. AD mouse model was successfully established. Fourteen C57 mice were randomized into AD and control groups. The positive protein expression rate of VAV1 was measured by immunohistochemistry. Neuron cells were assigned into control, blank, negative control (NC), miR-330 mimics, miR-330 inhibitors, siRNA-VAV1, and miR-330 inhibitors + siRNA-VAV1 groups. Expression of miR-330, VAV1, ERK1, JNK1, P38MAPK, Aß, COX, and lipoprotein receptor-related protein-1 (LRP-1) were determined using RT-qPCR and Western blotting. Colorimetry was applied to measure the levels of OS parameters of superoxide dismutase (SOD) and malondialdehyde (MDA). Aß production in brain tissue was detected using ELISA, while that in neuron cell was measured by radioimmunoassay. MiR-330 was down-regulated in neuron cells of AD mice and VAV1 was negatively regulated by miR-330. Compared with the control group, the positive protein expression rate of VAV1 was significantly elevated in the AD group. Overexpression of miR-330 decreased the expression of VAV1, ERK1, JNK1, P38MAPK, and Aß, but increased the expression of COX and LRP-1. AD mice revealed elevated Aß production and MDA with decreased SOD level. The result indicates that overexpressed miR-330 targeting VAV1 through the MAPK signaling pathway reduces Aß production and alleviates OS and mitochondrial dysfunction in AD.

Prognostic significance of lymphatic vessel invasion diagnosed by D2-40 in Chinese invasive breast cancers.

Lymphatic vessel invasion (LVI) is promising in determining prognosis and treatment strategies, but the application of LVI as a histopathological criterion in breast cancer patients especially those of different subgroups is controversial. This research aims to evaluate the prognostic value of LVI assessed by D2-40 not only in patients with early invasive breast cancer but also in lymph node-negative, lymph node-positive, luminal A-like, luminal B-like, HER2-enriched, and triple-negative subgroups.The study cohort included 255 patients with a median follow-up of 101 months. Immunohistochemical staining for D2-40 was performed to identify LVI.LVI was present in 64 (25.1%), 15 (12.1%), 49 (37.4%), 19 (20.9%), 23 (27.7%), 13 (31.7%), and 9 (22.5%), respectively, in the whole cohort, lymph node-negative, lymph node-positive, luminal A-like, luminal B-like, HER2-enriched, and triple-negative patients. LVI was associated with large tumor size (P = .04), high histological grade (P = .004), involved lymph node (P < .001), and high expression of Ki-67 (P = .003). No significant difference was found among patients with different subtypes and LVI status. The presence of LVI was significantly associated with adverse disease-free survival in the whole cohort (P < .001), lymph node-negative (P < .001), lymph node-positive (P < .001), luminal A-like (P < .001), and luminal B-like patients (P < .001) in both of the univariate and multivariate survival analysis.This study indicated that the presence of LVI stained by D2-40 provided independent prognostic information not only in the whole cohort but also in the subgroup of patients with lymph node-negative, lymph node-positive, luminal A-like, and luminal B-like diseases, which may make a case for routine clinical assessment of LVI using D2-40.

Primary or metastatic hepatic carcinoma? A breast cancer patient after adjuvant chemotherapy and radiotherapy postoperatively with intrahepatic cholangiocarcinoma and review of the literature.

BACKGROUND: The liver is a common site of metastases, followed by the bone and lung in breast cancer. The symptoms of hepatic metastases are similar to intrahepatic cholangiocarcinoma (ICC). ICC is rare, with an overall incidence rate of 0.95 cases per 100,000 adults. The incidence of ICC for patients with breast cancer is very uncommon. Breast cancer patient with ICC is easily misdiagnosed as hepatic metastases. CASE PRESENTATION: We report a breast cancer patient postoperatively who was hospitalized because of having continuous irregular fever for 1 month. Antibiotics were given for 1 week without any significant effect. Her admission bloods revealed elevated levels of carcino-embryonic antigen. Magnetic resonance imaging diagnosis showed multiple liver metastases. We believed that the woman had hepatic metastases until biopsy guided by computed tomography. The liver biopsy pathology analysis considered the possibility of primary intrahepatic cholangiocarcinoma. CONCLUSIONS: Breast cancer patient with space-occupying lesions in the liver is easily considered to be progressed hepatic metastases. Image-guided biopsy is the best diagnostic method for breast cancer with liver mass to avoid misdiagnosis and classify the molecular subtypes to make appropriate treatment.

[Effects of pig manure and oxytetracycline on microbial number and activity in soils with different fertility levels].

An incubation test was conducted to study the effects of antibiotics oxytetracycline on the microbial number and activity in three soils varied with fertility level under the conditions with or without pig manure application. The soils were injected with six concentrations (0, 0.1, 1, 10, 100, and 1000 mg x kg(-1)) oxytetracycline, and incubated for 30 d at 25 degrees C. Soil samples were collected on the 1st, 4th, and 30th day of incubation to measure the bacterial number, respiratory activity, and enzyme activity. Under no pig manure application, oxytetracycline had little effects on the soil bacterial number and activity. The EC10 values of oxytetracycline based on a 10% decrement of bacterial number, respiratory activity, enzyme activity, and NO3(-)-N concentration in treatment without pig manure were 36-1000 mg x kg(-1), 20-1000 mg x kg(-1), and 4-1000 mg x kg(-1) for S1, S2 and S3, while those in treatment with pig manure application were 2-656 mg x kg(-1), 2-81 mg x kg(-1), and 1-42 mg x kg(-1) for S1, S2 and S3, respectively. Soil fertility level had obvious effects on the dose-response relations between oxytetracycline and soil microbial number and activity. The effects of oxytetracycline on soil microbial number and activity increased with increasing soil fertility level, and the effects on microbial number and respiratory activity were higher than those on enzyme activity and NO3(-)-N concentration. The effects of oxytetracycline on soil microbial number and activity were time-depending, being the greatest on the 4th day of incubation. Overall speaking, oxytetracycline could exert temporary inhibition on soil microbes.

Pioglitazone decreased CD40/CD40L expression on human umbilical vein endothelial cells induced by oxidized low-density lipoprotein.

BACKGROUND: The CD40/CD40 ligand pathway mediated inflammatory processes are important in atherogenesis and the formation of the intraplaque lipid pool. We tested the hypothesis that pioglitazone could decrease lectin-like oxLDL receptor-1 (LOX-1) and CD40/CD40L expression on human umbilical vein endothelial cells (HUVECs) induced by oxidized low-density lipoprotein (oxLDL). METHODS: HUVECs were incubated with oxLDL for 24h with or without pretreated by pioglitazone. Expression of CD40/CD40L on the cell surface was detected by flow cytometry. CD40/CD40L and LOX-1 mRNA expression were evaluated by RT-PCR. The expression of LOX-1 on HUVECs was determined by cell immunohistochemistry. RESULTS: OxLDL increased the expression of CD40 and CD40L in a dose- and time-dependent manner. Pretreatment of HUVECs with pioglitazone (1 and 10 micromol/l) for 60 min decreased the expression of CD40 mRNA induced by oxLDL by 16% and 52%, respectively (both P<0.05). Pretreatment of HUVECs with pioglitazone (1 and 10 micromol/l) for 60 min decreased the expression of CD40L mRNA induced by oxLDL by 16% and 43% (both P<0.05). Also, pretreatment of HUVECs with pioglitazone (1 and 10 micromol/l) for 60 min also significantly decreased CD40 and CD40L expression on HUVECs induced by oxLDL in a concentration-dependent manner. Pretreatment of HUVECs with pioglitazone (1 and 10 micromol/l) decreased oxLDL induced upregulation mRNA of LOX-1 by 11% and 28%, respectively. Furthermore, through immunohistochemistry, we found that pioglitazone could decrease the LOX-1 expression on HUVECs induced by oxLDL. CONCLUSION: Pioglitazone inhibited the upregulation of LOX-1 on HUVECs elicited by oxLDL and subsequently decreased HUVECs CD40/CD40L expression induced by oxLDL. These observations provided novel insight into a potential novel anti-inflammatory pathway of thiazolidinediones.