[Cytogenetic and Clinical Characteristics of Newly Diagnosed Elderly Patients with Acute Myeloid Leukemia].

OBJECTIVE: To investigate the cytogenetic characteristics and prognostic risk factors for elderly patients with newly diagnosed elderly acute myeloid leukemia(AML). METHODS: Cytogenetic test results of 76 elderly patients with AML admitted to the First Affiliated Hospital of the University of Science and Technology of China (Anhui Provincial Hospital) from April 2015 to December 2021 were retrospectively analyzed, and analyzed clinical characteristics of patients and risk factors influencing prognosis. RESULTS: According to cytogenetic risk stratification, 76 newly treated elderly AML patients were divided into the favorable, intermediate, and unfavorable groups with 6(7.9%), 58(76.3%), and 12(15.8%) cases, respectively. There was no significant difference in the patient's clinical characteristics and prognosis with the cytogenetics-risk classification groups. Correlation analysis showed that patients' objective response rate (ORR) was related to the age of onset and the mutation status of the CEBPA gene. Logistic regression analysis found that age ≥70 years was an independent risk factor for patients' ORR (OR=0.110, P=0.005). Remission determined the 1-year OS rate (OR=0.049, P=0.005). CONCLUSION: There is no significant difference in clinical characteristics among aged AML patients treated at initial treatment in different cytogenetic risk groups. The age of onset ≥70 years is the determinant of whether patients can obtain ORR, and the rate of ORR is closely related to the 1-year OS rate.

Preventive effect of pidotimod on reactivated toxoplasmosis in mice.

As one of food-borne parasitic diseases, toxoplasmosis entails the risk of developing reactivation in immunocompromised patients. The synthetic dipeptide pidotimod is a potent immunostimulating agent that improves the immunodefenses in immunodepression. To investigate the efficacy of pidotimod as a preventive treatment, we used a murine model of reactivated toxoplasmosis with cyclophosphamide (CY)-induced immunosuppression. Pidotimod administration significantly restored the body weight and spleen organ index, increased survival time (from 70 to 90%), and decreased the parasitemia (from 80 to 35%) of CY-induced mice with reactivated toxoplasmosis. Cytokine profiles and CD4(+) T cells subpopulation analyses by Cytometric Bead Array and flow cytometry demonstrated that pidotimod treatment resulted in a significant upregulation of pro-inflammatory cytokines (IFN-γ, TNF-α, and IL-2) and Th1 cells (from 3.73 ± 0.39 to 5.88 ± 0.46%) after CY induction in infected mice. Additionally, histological findings and parasite DNA quantification revealed that mice administered with pidotimod had a remarkable reduction of parasite burden (two-log) and amelioration of histopathology in the brains. The in vitro studies showed that pidotimod significantly restored concanavalin A-induced splenocyte proliferation and pro-inflammatory cytokines in the supernatants of splenocyte culture. It could be concluded that the administration of pidotimod in immunocompromised mice significantly increases the Th1-biased immune response, prolongs survival time, and ameliorates the load of parasites in the blood. This is the first report of the preventive effect of pidotimod on reactivated toxoplasmosis.

Comparative studies of macrophage-biased responses in mice to infection with Toxoplasma gondii ToxoDB #9 strains of different virulence isolated from China.

BACKGROUND: Different from three clonal lineages of Toxoplasma gondii in North America and Europe, the genotype China 1 is predominantly prevalent in China. However, there are different virulent isolates within China 1, such as virulent TgCtwh3 and avirulent TgCtwh6, and little is known about differences in macrophage activation between them. The objective of this study focused on cytokine production, phenotype and markers of activated macrophages, and correlated signaling pathway induced by the two isolates. METHODS: Adherent peritoneal macrophages (termed Wh3-Mφ and Wh6-Mφ, respectively) harvested from infected mice were cultured for detection of Nitric Oxide and arginase activity, and activated markers on Wh3-Mφ/Wh6-Mφ were determined by flow cytometry. In in vitro experiments, the levels of IL-12p40 and TNF-α were measured using ELISA kits, and mRNA expressions of IL-12p40, TNF-α, iNOS, Arg-1 and Ym1 were assayed by real-time PCR. To confirm the activation state of NF-kB p65 in infected cells stained by IF, protein levels of iNOS, Arg-1, Ym1, nuclear NF-κB p65, and phosphorylation of STAT6/STAT3/IκBα were evaluated by Western Blotting. A one-way ANOVA test was used to compare differences among multiple groups. RESULTS: The result revealed that contrary to the virulent TgCtwh3, the less virulent TgCtwh6 isolate induced a significant increase in IL-12p40 and TNF-α. Although both isolates down-regulated CD80, CD86 and MHCII molecule expression on macrophages, TgCtwh3 promoted up-regulation of PD-L2 and CD206. Wh6-Mφ generated a high level of NO whereas Wh3-Mφ up-regulated Ym1 and arginase expression at transcriptional and protein levels. In terms of signaling pathway, TgCtwh3 induced phospho-STAT6, conversely, TgCtWh6 led to NF-κB p65 activation. CONCLUSIONS: The virulent TgCtwh3 isolate induced macrophages to polarize toward alternatively activated cells with STAT6 phosphorylation, whereas the less virulent TgCtwh6 elicited the development of classically activated macrophages with nuclear translocation of NF-κB p65. This discrepancy suggests that it is necessary to thoroughly analyze the genotype of TgCtwh3 and TgCtwh6, and to further study other effector molecules that contribute to the macrophage polarization in T. gondii.

Changes of Treg-associated molecules on CD4+CD25 +Treg cells in myasthenia gravis and effects of immunosuppressants.

OBJECTIVE: Myasthenia gravis (MG) is a CD4(+) T cell-dependent autoimmune disease, and close attention has been paid to the role of CD4(+)CD25(+)Treg cells (Tregs). Previous results regarding Tregs in MG patients have been conflicting. The discrepancy was partly ascribed to selecting different Treg-associated molecules in defining Tregs. Therefore, we considered it necessary to find a reliable index for assessing the immunologic state in MG patients and explore the effect of IS on them. METHODS: We adopted flow cytometric techniques to measure the numbers and frequencies of Tregs in peripheral blood taken from 57 patients and 91 age-matched healthy donors, and we also analyzed FOXP3 mean fluorescence intensity on Tregs. RESULTS: The number and frequency of Tregs in peripheral blood of MG patients significantly decreased, together with down-regulation of FOXP3 expression. There was dynamic change of Treg cell level and the inverse relationship with clinical symptom, suggesting that the immunologic disorder in MG patients was related to peripheral Tregs population. Meanwhile, CD4(+)CD25(+)FOXP3(+)Helios(+)T cells might be activated Tregs, rather than nTregs. Moreover, the number and frequency of CD4(+)CD25(+)FOXP3(+)Helios(+)T cells significantly decreased in MG patients, indicating that the reduction of the activated Tregs population might be a critical contributor to the pathogenesis of MG. CONCLUSIONS: The significant reduction of the peripheral Tregs population in MG patients might be responsible for the immunologic disorders in MG patients. IS such as GC took its effect possible by increasing the population size, and the underlying mechanism should be further investigated.

Promoter methylation of p16, Runx3, DAPK and CHFR genes is frequent in gastric carcinoma.

AIMS AND BACKGROUND: Transcriptional silencing induced by hypermethylation of CpG islands in the promoter regions of genes is believed to be an important mechanism of carcinogenesis in human cancers including gastric cancer. A number of reports on methylation of various genes in gastric cancer have been published, but most of these studies focused on cancer tissues or only a single gene. In this study, we determined the promoter hypermethylation status and mRNA expression of 4 genes: p16, Runx3, DAPK and CHFR. METHODS: Methylation-specific polymerase chain reaction (MSP) was used to determine the methylation status of p16, Runx3, DAPK and CHFR gene promoters in cancer and adjacent normal gastric mucosa specimens from 70 patients with gastric cancer, as well as normal gastric biopsy samples from 30 people without cancer serving as controls. In addition, the mRNA expression of p16, Runx3, DAPK and CHFR was investigated in 34 gastric cancer patients by RT-PCR. Bisulfite DNA sequence analysis was applied to check the positive samples detected by MSP. RESULTS: When carcinoma specimens were compared with adjacent normal gastric mucosa samples, a significant increase in promoter methylation of p16, Runx3, DAPK and CHFR was observed, while all 30 histologically normal gastric specimens were methylation free for all 4 genes. The methylation rate of the 4 genes increased from normal stomach tissue to tumor-adjacent gastric mucosa to gastric cancer tissue. Concurrent methylation in 2 or more genes was found in 22.9% of tumor-adjacent normal gastric mucosa and 75.7% of cancer tissues. No correlation was found between hypermethylation and other clinicopathological parameters such as sex, age, and tumor location. However, the frequency of DAPK and CHFR methylation in cancer tissues was significantly associated with the extent of differentiation and lymph node metastasis (P < 0.05) and the frequency of Runx3 methylation was significantly associated with tumor size (P < 0.05). Weak expression and loss of expression of the 4 genes was observed in cancer tissues and was significantly associated with promoter hypermethylation (P < 0.05). CONCLUSIONS: Promoter hypermethylation of p16, Runx3, DAPK and CHFR is frequent in gastric cancer. DAPK and CHFR promoter hypermethylation may be an important help in evaluating the differentiation grade and lymph node status of gastric cancer. Weak gene expression and loss of gene expression due to promoter hypermethylation may be a cancer-specific event.

[Clinical significance of detection of T-cell subgroups in patients with aplastic anemia].

The study was aimed to investigate the changes of T-cell subgroups in the peripheral blood (PB) of patients with aplastic anemia (AA) and the relationships between these changes and the pathogenesis of AA and the immunosuppressive therapeutic effects in AA, in order to provide a basis for selecting rational therapy of AA patients. T-cell subtype and the ratio of CD4+/CD8+ cell in the PB of 88 AA patients which had been diagnosed clearly and given conventional therapy or conventional therapy combined with immunotherapy were analyzed by tri-colour fluorescence-labeled monoclonal antibody and using multiparameter flow cytometry. The patients with AA were divided into normal type of ratio, inverted type of ratio, hypernormal type of ratio according to the ratio of CD4+/CD8+ cell in normal group, and then the relations of these subtype with patients' conditions and therapeutic effects were investigated. The results showed that the percentage of normal type of ratio in all patients was 39.8%, the percentage of inverted type of ratio in all patients was 44.3%, The percentage of hypernormal type of ratio in all patients was 15.9%. In the conventional therapy alone, there was no significant difference on therapeutic effects among these three immunological subtypes. In combined immunotherapy, total therapeutic efficacy of AA patients with inverted type of ratio and AA patients with immunologic abnormality (inverted type + hypernormal type) was 84.2% and 82.6% respectively, which were more than that in conventional therapy (45.5% and 42.8%) (p < 0.05). Total therapeutic efficacy in these patients was better than that in AA patients with normal type. It is concluded that significant abnormal ratios of CD4+/CD8+ exist in the majority of AA patients, abnormal ratios of CD4+/CD8+ both may be showed as increase or decrease, immunologic abnormality may play a role in pathogenesis of the patients with AA. The detection of PB T-cell subtype in patients with aplastic anemia contributes to evaluation of patients' condition and choice of rational treatment prescription, and enhancement of diagnostic level and therapeutic efficacy significantly, which is an important indicator for therapeutic strategy also.

[Identification of a novel mutation of F (13) A gene in a pedigree with factor XIII deficiency].

OBJECTIVE: To explore F (13) A gene mutation in a pedigree with hereditary coagulation factor XIII (FXIII) deficiency. METHODS: The FXIII deficiency was diagnosed by clot solubility test and other standard laboratory clotting tests. All exons, exon-intron boundary sequences of F(13) A gene were amplified by PCR and the products were sequenced directly. Any mutation identified by direct sequencing was confirmed by reverse sequencing. The mutation identified in the proband was screened in the family members. RESULTS: The assays of PT, Qiulan, fibrinogen leveling, platelet counts, bleeding time were normal and the clot solubility test was positive in the proband. The homozygous deletion of 33 nucleotides (127067de133) in exon 10 of F(13) A gene which resulted in deletion of 11 amino acids in FXIIII A protein with 720aa residues was identified in the proband. Family studies showed that the mutation was inherited from the parents both of whom carried the heterozygous deletion mutation. CONCLUSION: The homozygous 127067de133 mutation of F(13) A gene is responsible for the disorder of the pedigree.

[A novel mutation in antithrombin gene results in hereditary antithrombin deficiency].

OBJECTIVE: To investigate the antithrombin (AT) activity (AT: A) and AT antigen (AT: Ag) level in a Chinese family with type I antithrombin (AT) deficiency, and to explore the molecular mechanism of AT deficiency. METHODS: Immuno-nephelometry and chromogenic assay were used to detect the plasma level of AT: A and AT: Ag, respectively. Genomic DNA was isolated from the peripheral blood, and all the seven exons and exon-intron boundaries of AT gene were amplified by PCR and direct sequencing. RESULTS: The plasma levels of AT: A and AT: Ag of the proband were 45% and 97 mg/L, respectively, which led to a type I AT deficiency. A heterozygous T to A mutation was found at nucleotide 9833 in exon 5 resulting in a Tyr363Stop nonsense mutation. The sequencing results from the pedigree indicated that four other members also had this mutation. CONCLUSION: This heterozygous nonsense mutation of T9833A in exon 5 resulting in venous thrombosis is a novel genetic defect of hereditary AT deficiency, which has not been described before.

[Severe hereditary coagulation factor V deficiency caused by two novel heterozygous mutations].

OBJECTIVE: To identify gene mutations of a pedigree with inherited factor V (FV) deficiency. METHODS: The activated partial thromboplastin time (APTT), prothrombin time (PT), FV activity (FV:C) and FV antigen (FV:Ag) tests were performed for phenotypic diagnosis. The genomic DNA was extracted from the peripheral blood of the proband and all the 25 exons and their flanks of FV gene were amplified by polymerase chain reaction (PCR). The PCR products were screened by direct sequencing and the mutations were further confirmed by restriction enzyme digestion. RESULTS: APTT, PT, TT, FV:C, FV:Ag of the proband were 249.2 s, 46.6 s, 17.9 s, 0.1% and 1.5%, respectively. FII, FVII, FVIII, FIX, FX activities, vWF and Fg were within normal ranges. Taking the GenBank Z99572 sequence as the reference, four mutations were identified in FV gene of the proband. They were a heterozygous two bases deletion in exon 13 (2238 approximately 2239delAG) introducing a frameshift and a premature stop at codon 689, and a heterozygous missense mutation in exon 23 (G6410T) resulting in the substitution of Gly for Val at codon 2079, respectively. The proband's father and mother were heterozygous for G6410T and for 2238 approximately 2239delAG, respectively. CONCLUSION: The severe FV deficiency of the proband is caused by a frameshift mutation of 2238 approximately 2239delAG and a missense mutation of G6410T, which haven't been identified before.

[Fibrinogen beta chain gene mutation contributes to one congenital afibrinogenemia].

OBJECTIVE: To identify the fibrinogen (Fg) gene mutations in a Chinese pedigree of congenital afibrinogenemia. METHODS: The plasma Fg activity and protein of the proband and his family members were detected. Genomic DNA was isolated from the peripheral blood mononuclear cells. All the exons and exon-intron boundaries of fibrinogen gene were amplified by PCR and sequenced thereafter. RESULTS: Two mutations, 7972 del G in FGB and T2543A in FGG, were found in the proband. CONCLUSIONS: FGG2543 is a polymorphism site, which lead to the polymorphism of gamma144 I/K. The G deletion at base 7972 of FGB contributes to the frameshift mutation after amino acid 419, resulting in the truncated beta chain without the terminal 27 amino acids. The latter may contributes to the pathogenetic mechanisms in Chinese congenital afibrinogenemia patients. The G deletion at base 7972 of FGB is identified for the first time.