Expression properties, structural features and functional analysis of CALR E381A in MPN patients.

OBJECTIVE: To investigate the expression properties, structural features and function of CALR E381A in myeloproliferative neoplasms (MPN) patients. METHODS: In this retrospective study, 435 MPN patients admitted to the Department of Hematology, Ningbo First Hospital from July 2015 to July 2021 were selected as the study subjects. Mutations in CALR exon 9 from genomic DNA samples were identified by PCR, followed by Sanger sequencing. The physicochemical properties of the wild-type calreticulin and the p.E381A variant, and the structural information of the p.E381A variant were analyzed by using the bioinformatics databases. Growth assay of UT-7/mpl cells with CALR E381A was used for the functional analysis of CALR E381A. RESULTS: The predominant types of CALR variants were identified as follows: p.L367fs*46 (38.1%), p.K385fs*47 (25.8%) and p.E381A (19.6%). Notably, the frequency of the p.E381A variant (c.1142A >C) in polycythemia vera or essential thrombocythemia was significantly higher than the frequency of that as a single nucleotide polymorphism (SNP) in the East Asian population. Furthermore, CALR E381A coexisted with other genetic variants, of which JAK2 V617F was more common. Bioinformatics analysis confirmed that CALR E381A did not change the physicochemical properties of the calreticulin protein, but did change the electrical charge, energy state and steric hindrance of amino acid residues at site 381. UT-7/mpl cells harboring CALR E381A overexpression did not exhibit altered cell growth, which is distinctly different from the stereotypical frameshift mutation. CONCLUSION: CALR E381A is not a driver mutation for the development of MPN but may be a risk SNP implying an inherited predisposition for MPN disease in East Asian populations.

The gut microbiota correlate with the disease characteristics and immune status of patients with untreated diffuse large B-cell lymphoma.

Background: Gut microbiota characteristics in patients with diffuse large B-cell lymphoma (DLBCL) are reportedly different when compared with the healthy population and it remains unclear if the gut microbiota affects host immunity and clinical disease features. This research investigated the gut microbiota in patients with untreated DLBCL and analyzed its correlation with patient clinical characteristics, humoral, and cell immune status. Methods: Thirty-five patients with untreated DLBCL and 20 healthy controls (HCs) were recruited to this study and microbiota differences in stool samples were analyzed by 16S rDNA sequencing. Absolute ratios of immune cell subset counts in peripheral blood were detected by flow cytometry and peripheral blood cytokine levels were detected by enzyme-linked immunosorbent assay. Relationships between changes in patient microbiomes and clinical characteristics, such as clinical stage, international prognostic index (IPI) risk stratification, cell origin, organ involved and treatment responses were investigated and correlations between differential microbiota and host immune indices were analyzed. Results: The alpha-diversity index of intestinal microecology in DLBCL patients was not significantly different when compared with HCs (P>0.05), nonetheless beta-diversity was significantly decreased (P=0.001). p_Proteobacteria were dominant in DLBCL, while p_Bacteroidetes abundance was significantly decreased when compared with HCs (P<0.05). Gut microbiota characteristics were identified that were associated with clinical features, such as tumor load, risk stratification and cell origin, and correlation analyses were performed between differential flora abundance associated with these clinical features and host immune status. The p_Firmicutes was positively correlated with absolute lymphocyte values, g_Prevotella_2 and s_un_g_Prevotella_2 were negatively correlated with absolute lymphocyte values, T cell counts and CD4 cell counts, while g_Pyramidobacter, s_un_g_Pyramidobacter, and f_Peptostreptococcaceae were negatively correlated with IgA. Conclusions: Dominant gut microbiota, abundance, diversity, and structure in DLBCL were influenced by the disease, correlated with patient immune status and this suggested that the microecology-immune axis may be involved in regulating lymphoma development. In the future, it may be possible to improve immune function in patients with DLBCL by regulating the gut microbiota, improve treatment response rates and increase patient survival rates.

Gastrointestinal Bleeding Secondary to High-Dose Melphalan Pretreatment in Patients with Multiple Myeloma Was Associated with the Mode of Melphalan Administration.

Objective: To analyze the characteristics of gastrointestinal bleeding secondary to high-dose melphalan pretreatment in patients with multiple myeloma. Methods: Between 1 January 2016 and 31 October 2021, 26 patients with multiple myeloma after autologous peripheral blood hematopoietic stem cell transplantation with high-dose melphalan pretreatment were recruited. They were assigned to either the oral administration group or the intravenous administration group according to the administration modes, or to either the gastrointestinal bleeding group or the nongastrointestinal bleeding group. Analyses were performed based on the differences in general gastrointestinal symptoms and hemorrhage between different administration modes and on the differences in the general gastrointestinal symptoms, platelet alterations, and the intestinal microecology before pretreatment between patients with and without gastrointestinal bleeding. Results: Of the 26 included patients, heavy secondary gastrointestinal bleeding was found in 6 patients with intravenous pretreatment. In patients given intravenous melphalan, those with gastrointestinal bleeding showed more pronounced general symptoms such as nausea and vomiting versus those without. There was no significant difference in platelet alterations between the two groups. Gastrointestinal bleeding was associated with more significant microecological disturbances than no bleeding. Conclusion: Gastrointestinal bleeding secondary to high-dose melphalan pretreatment in patients with multiple myeloma was associated with the mode of melphalan administration, adverse symptoms at pretreatment, and the intestinal microecology prior to pretreatment. Thus, improvement of the intestinal microecology prior to pretreatment and mitigation of adverse gastrointestinal symptoms during pretreatment may contribute to a lower incidence of secondary gastrointestinal bleeding and enhanced transplantation safety.

The Differential Expression of CD47 may be Related to the Pathogenesis From Myelodysplastic Syndromes to Acute Myeloid Leukemia.

Myelodysplastic syndrome (MDS) can lead to the development of peripheral blood cytopenia and abnormal cell morphology. MDS has the potential to evolve into AML and can lead to reduced survival. CD47, a member of the immunoglobulin family, is one molecule that is overexpressed in a variety of cancer cells and is associated with clinical features and poor prognosis in a variety of malignancies. In this study, we analyzed the expression and function of CD47 in MDS and AML, and further analyzed its role in other tumors. Our analysis revealed significantly low CD47 expression in MDS and significantly high expression in AML. Further analysis of the function or pathway of CD47 from different perspectives identified a relationship to the immune response, cell growth, and other related functions or pathways. The relationship between CD47 and other tumors was analyzed from four aspects: DNA methyltransferase, TMB, MSI, and tumor cell stemness. Changes in gene expression levels have a known association with aberrant DNA methylation, and this methylation is the main mechanism of tumor suppressor gene silencing and clonal variation during the evolution of MDS to AML. Taken together, our findings support the hypothesis that the differential expression of CD47 might be related to the transformation of MDS to AML.

[Clinical Features of Pregnant Women with Thalassemia in Non Endemic Area].

OBJECTIVE: To investigate the clinical features of pregnant women with thalassemia in non endemic area, and to prevent the births of babies with intermedia or major thalassemia. METHODS: Two hundred and thirty-five pregnants women with thalassemia diagnosed from March 2015 to April 2016 in our hospital were enrolled and retrospectively analysed. The blood routine and hemoglobin electrophoresis were performed respectively by XN-9000 automatic blood cell analyzer and HYDRASYS hemoglobin electrophoresis apparatus. The three commonest deletion of α-thalassemia, the three non-deletion α-thalassemia and 21 known ß-thalassemia mutation were all detected by fluorescence melting curve analysis. RESULTS: Among 235 pregnant women of thalassemia, the majority were ß-thalassemia, which were followed by α-thalassemia and composite thalassemia. Most pregnant women showed a mild anemia, and suffered from microcytic anemia, but less suffered from iron deficiency anemia. The ratio of second-child pregnant women was increased, and the ratio was close to one third both in α-thalassemia and ß-thalassemia patients, and 75% patients were composite thalassemia. HbF was found to be more in native pregnant women with ß-thalassemia. Hemoglobin isomer was easy to found in the pregnant with α-thalassemia, and they were all non native. The genotype of --sea were found majority in both native and non native pregnant women with thalassemia. The genotype of IVS-II-654 made up a large majority(55.38%) in native pregnant with ß-thalassemia, as well as one of whose parents was native pregnant women. The genotypes of CD41-42,IVS-II-654 and CD17 were found to be a large majority in non native pregnant women, each of them accounted for 30%. CONCLUSION: More pregnant women with thalassemia are founded to be in non endemic area, and shows their own unique clinical features. It is certainly to detect thalassemia mutation in their spouse and their babies, to prevent the births of babies with intermedia or major thalassemia.

Effects of arsenic trioxide combined with bortezomib on apoptosis of multiple myeloma cell line KM3 and its mechanisms.

This study was purposed to investigate the effect of bortezomib (Bor) and arsenic trioxide (As(2)O(3)) combination on multiple myeloma cell line KM3 and its mechanisms. KM3 cells were cultured with different concentration of Bor or As(2)O(3) as well as both for a certain time. The cell proliferation was analysed by MTT assay and the concentration of 50% proliferation inhibition (IC(50)) was calculated. Early apoptosis and late apoptosis of KM3 cells were detected by Annexin-V-FITC Kit, and the change of transmembrane potential was measured by flow cytometry. mRNA of Caspase-3, Bim and Bcl-xL were detected by RT-PCR. The results showed that the proliferation inhibitory rate of KM3 cells treated by Bor plus As(2)O(3) was much higher than that of KM3 cells treated by Bor only for 72 h [ (27.64 ± 0.81)% vs (21.67 ± 2.20)%, P < 0.05]. There were more KM3 cells treated by Bor plus As(2)O(3) in early apoptosis at 48 h and late apoptosis at 72 h than that of KM3 cells treated only by Bor [ (53.20 ± 3.70)% vs (35.40 ± 2.58)%, P < 0.01; (63.96 ± 2.97)% vs (54.08 ± 3.76)%, P < 0.01]. Transmembrane potential (Δψm) of KM3 cells treated by Bor plus As(2)O(3) decreased more at 48 h, as compared with Bor alone. The expression levels of caspase-3 mRNA and Bim mRNA in KM3 cells treated with Bor plus As(2)O(3) were higher than that in KM3 cells treated with Bor alone. But the expression level of Bcl-xL mRNA was lower than that in KM3 cells treated with Bor alone. It is concluded that As(2)O(3) can enhance the apoptosis-inducing effect of Bor on multiple myeloma cell line KM3, which is associated with decreasing the expression of Bcl-xl mRNA and increasing the expression of Caspase-3 and Bim mRNA.

[Effect of P38MAPK signal transduction pathway on apoptosis of THP-1 induced by allicin].

OBJECTIVE: The objective of this paper was to study the change of P38MAPK and Fas in the apoptosis of THP-1 cells induced by allicin. METHOD: The proliferation inhibition rates of THP-1 cells after various treatments were examined by MTT assay. Apoptosis rate was determined with Annexin V- FITC/PI double staining by flow cytometry. The expression and distribution change of the phosphorylation p38MAPK (P-p38MAPK) were detected by immunohistochemical staining. The changes of P-p38 MAPK and Fas proteins were detected by Western blot. RESULT: The proliferations of leukemia cell line THP-1 are inhibited by allicin. MTT assay showed that allicin can inhibit the proliferation of the THP-1 cell, and the inhibition was dependent on both dose and time. The IC50 of 72 hours was 12.8 mg x L(-1). Apoptosis rate detected by Annexin V-FITC/PI was proportional to the concentration of the allicin. After the immunohistochemical staining test, the P-p38MAPK was located in the cell nucleus and plasma, showing deep brown, when adding allicin to THP-1 cell. Western blot test showed that the P-p38MAPK proteins expression was proportional to the concentration of Allicin and was also dose dependent. The levels of P-p38MAPK in negative control group, 1/2 IC50 of 72 hours group and IC50 of 72 hours group were 0.259 8 +/- 0.013 2, 0.61 2 +/- 0.008 3 and 0.505 6 +/- 0.005 5 respectively, and the levels of Fas proteins were 0.287 4 +/- 0.008 9, 0.426 8 +/- 0.007 9 and 0.597 1 +/- 0.010 9 respectively. The difference was statistically significant when compared with the negative control group (P < 0.01). CONCLUSION: Allicin can significantly induce THP-1 cells apoptosis, and its mechanism may be related to the activation of P38MAPK/Fas.

[Apoptosis of U937 cell line promoted by matrine through MAPK signal transduction pathway].

OBJECTIVE: To study the anti-cancer effect of matrine (Mat) on U937 cell line and its possible molecular mechanism. METHOD: The cells were cultured in medium containing either 0.1, 0.2, 0.3, 0.4, or 0.5 g x L(-1) of Mat. The morphological alteration was observed by inverted microscopy and electron microscopy. Cell proliferation was analyzed by Try pan blue staining and MTT. The method of Western Blot was used to detect phosphorylation activity of MAPK. RESULT: Matrine had a significant inhibitory effect on proliferation of U937 cell line at the concentration of 0.2 g x L(-1). Treated with matrine of 0.2 g x L(-1) for 48 h, U937 cells became smaller and appeared more round than previously. The number of U937 cells showing apoptosis increased with elevation of the concentration of the matrine. Matrine had an ability of inhibiting the activity of ERK and increasing the activities of p38 and JNK to some degree in U937 cells. CONCLUSION: Matrine can inhibit the proliferation of U937 cell line in vitro and induce its apoptosis possibly through inhibiting the activity of ERK and increasing the activities of p38 and JNK in U937 cells.