[Use of the ETV6/RUNX1 probe to verify the performance of the fluorescence in situ hybridization probe before clinical detection].

Objective: To explore the standardized performance of a FISH probe before clinical detection. Methods: The probe sensitivity and specificity of ETV6/RUNX1 were analyzed via interphase and metaphase FISH in 20 discarded healthy bone marrow samples. The threshold system of the probe was established using an inverse beta distribution, and an interpretation standard was established. Finally, a parallel-controlled polymerase chain reaction detection study was conducted on 286 bone marrow samples from patients at our hospital. The clinical sensitivity, specificity, and diagnostic coincidence rate of ETV6/RUNX1 FISH detection were analyzed, and the diagnostic consistency of the two methods was analyzed by the kappa test. Results: The probe sensitivity and specificity of the ETV6/RUNX1 probe were 98.47% and 100%, respectively. When 50, 100, and 200 cells were counted, the typical positive signal pattern cutoffs were 5.81%, 2.95%, and 1.49%, respectively, and the atypical positive signal pattern cutoffs were 13.98%, 9.75%, and 6.26%, respectively. The clinical sensitivity of FISH was 96.1%, clinical specificity was 99.6%, diagnostic coincidence rate was 99.00%, diagnostic consistency test kappa value was 0.964, and P value was <0.001. Conclusion: For FISH probes without a national medical device registration certificate, standardized performance verification and methodology performance verification can be performed using laboratory developed test verification standards to ensure a reliable and accurate reference basis for clinical diagnosis and treatment.

[Clinical characteristics of 11 patients with chronic lymphocytic leukemia with t (14;19) (q32;q13)].

Objective: To analyze the clinicopathological characteristics of 11 cases of chronic lymphocytic leukemia (CLL) with t (14;19) (q32;q13) . Methods: The case data of 11 patients with CLL with t (14;19) (q32;q13) in the chromosome karyotype analysis results of the Blood Diseases Hospital, Chinese Academy of Medical Sciences from January 1, 2018, to July 30, 2022, were retrospectively analyzed. Results: In all 11 patients, t (14;19) (q32;q13) involved IGH::BCL3 gene rearrangement, and most of them were accompanied by +12 or complex karyotype. An immunophenotypic score of 4-5 was found in 7 patients and 3 in 4 cases. We demonstrated that CLLs with t (14;19) (q32;q13) had a mutational pattern with recurrent mutations in NOTCH1 (3/7), FBXW7 (3/7), and KMT2D (2/7). The very-high-risk, high-risk, intermediate-risk, and low-risk groups consisted of 1, 1, 6, and 3 cases, respectively. Two patients died, 8 survived, and 2 were lost in follow-up. Four patients had disease progression or relapse during treatment. The median time to the first therapy was 1 month. Conclusion: t (14;19) (q32;q13), involving IGH::BCL3 gene rearrangement, is a rare recurrent cytogenetic abnormality in CLL, which is associated with a poor prognosis.

Identification of two novel anti-HCV E2 412-423 epitope antibodies by screening a Chinese-specific phage library.

The hepatitis C virus (HCV) E2 412-423 linear epitope has been found to be highly conserved across multiple HCV genotypes. The antibodies against this epitope have broadly neutralizing activity. Considering the poor immunogenicity of the epitope in humans and significant diversity in the global distribution of HCV genotypes, the aim of this study was to construct an anti-HCV phage library by using a series of optimal strategies to screen novel broadly neutralizing antibodies from Chinese donors. mRNA was isolated from peripheral blood samples of 39 patients who were anti-HCV positive. A phage library was constructed by inserting a single-chain variable fragment (scFv) gene repertoire into the T7Select10-3b vector. A synthetic peptide representing the HCV E2 N-terminal 412-423 region was used as "bait" for bio-panning. The binding affinities of phage clones to the synthetic peptide were evaluated through peptide-ELISA. Two scFv clones (R3-19 and R4-85) showing the strongest binding affinities were selected. The complementarity-determining regions (CDRs) of these clones were aligned with those of other previously reported broadly neutralizing anti-HCV antibodies, and multiple conserved amino acid sites were found. The optimized procedures ensured that two novel scFv antibodies were isolated from a constructed phage library and showed specific binding to the poorly immunogenic HCV E2 412-423 linear epitope. Keywords: phage antibody library; hepatitis C virus; broadly neutralizing antibody; synthetic peptide.

[Association between A-kinase anchor proteins 12 methylation and recurrence of hepatocellular carcinoma].

Objective: To study the association between the AKAP12 promoter methylation and recurrence of hepatocellular carcinoma. Methods: A total of 142 primary liver cancer patients underwent surgery in department of Hepatobiliary surgery in Peking University Cancer Hospital from 2003 to 2009 were selected as subjects in the survey; with the inclusion criteria as hepatocellular carcinoma, no cancer cells were observed in the surgical margin(SM) samples. All patients had neither lymph nor distant metastasis at the time of surgery, and receiving complete follow-up data for at least 3 years. By the end of May 2014, a total of 75 patients had relapsed of whom 71 died and there were no lost. All samples were acquired from the frozen surgical tissues. Genomic DNA was extracted using phenol/chloroform method and performed bisulfite modification following with polymerase chain reaction (PCR). AKAP12 methylation in hepatoma and the corresponding SM samples from 142 patients was determined by denature high-performance liquid chromatography (DHPLC) and bisulfite clone sequencing. Kaplan-Meier and Cox proportion hazard regression model were used to identify the factors related to the survival time. Results: In 142 cases, 125 patients (88.0%) were male and 17 (12.0%) cases were female. The median age was 52.5 years, ranging from 34 years to 76 years. AKAP12 methylation-positive rate was significantly higher in hepatomas than SMs (54.9% vs. 10.2%, P<0.001). Patients with AKAP12 methylation-positive had less risk of the recurrence (HR=0.62, 95%CI: 0.39-0.99); with tumor diameter more than 5 cm (HR=1.53, 95%CI: 1.00-2.50),portal vein invasion(HR=4.53, 95% CI:2.69-7.64) increased the recurrence risk. Moreover, portal vein invasion had a higher risk of death (HR=2.98, 95% CI: 1.73-4.98). Conclusion: There was significant association between AKAP12 DNA methylation and low risk of recurrence and long progression-free survival of hepatocellular carcinoma patients.

MKL1-184C>T gene polymorphism is associated with coronary artery disease in the Chinese Han population.

We investigated genetic susceptibility to coronary artery disease (CAD) by studying the association of MKL1 gene polymorphisms with CAD in the Chinese Han population. We performed a case-control study with 476 unrelated CAD patients and 325 non-CAD controls. All SNPs were genotyped with a TaqMan SNP genotyping assay. The distribution of MKL1-184C>T gene polymorphism in each group was in Hardy-Weinberg equilibrium. The frequency of the MKL1 T allele in the CAD group was significantly higher than in the control group (38.6 vs 30.8%). After logistic regression models adjusted for CAD risk factors, the risk of CAD among CT genotypes was 1.765 times higher than among the CC genotypes [odds ration (OR) = 1.765, 95% confidence interval (CI) = 1.246-2.5], and for TT genotypes it was 1.806 times higher than for the CC genotypes (OR = 1.806, 95%CI = 1.203-2.71). In summary, genotypes with at least one T allele (CT or TT genotypes) had a significantly increased CAD risk than the CC genotypes, with a ratio of 1.78 to 1 (OR = 1.780, 95%CI = 1.311-2.418). There was a close association between -184 T allele and 3VD (OR = 1.614, 95%CI = 1.259-2.07, P < 0.05). We conclude that the -184C>T of MKL1 is an important susceptibility factor for CAD in the Han Chinese in Henan Province. Homozygosity for the T allele is not only associated with an increased risk for CAD, it is also correlated with severity of stenosis in the Chinese Han population.

Upregulated response to chemokines in oxidative metabolism of eosinophils in asthma and allergic rhinitis.

Reactive oxygen species (ROS) from eosinophils are known to cause tissue damage in allergic inflammation. CC chemokines, especially eotaxin and regulated on activation, normal T-cell expressed and secreted (RANTES), are involved not only in chemotaxis but also in eosinophil activation, such as ROS production. It has been shown that eosinophils from allergic patients are not functionally equivalent to those from normal subjects. In the present study, the characteristics of chemokine-primed ROS production in eosinophils from allergic patients and normal controls were compared. After pretreatment with chemokines, eosinophils were stimulated with calcium ionophore A23187. ROS production by eosinophils was measured using luminol-dependent chemiluminescence. Both RANTES and eotaxin exhibited a priming effect on calcium ionophore-induced ROS production from eosinophils. Despite there being no difference in expression of CC chemokine receptor 3, the priming effect of RANTES and eotaxin was significantly enhanced in eosinophils from the patients. Interleukin-5 further enhanced the priming effect of chemokines in eosinophils from normal subjects, but not those from allergic subjects. The present results suggest an upregulated response to chemokines in eosinophils from allergic patients, and that interleukin-5 can induce a similar phenotype to that found in vivo in allergic patients.

Expression of VLA-4 on eosinophils decreases in patients with eosinophilia.

BACKGROUND: It is assumed that the very late antigen-4 (VLA-4) plays a key role in selective migration and accumulation of eosinophils to the allergic inflammatory focus. The regulatory mechanism for VLA-4 expression is poorly understood, as is its relationship between other adhesion molecules. OBJECTIVE: The aim of the study was to elucidate the relationship between VLA-4 expression and the activation of eosinophils. METHODS: The surface expression of VLA-4, Mac-1, ICAM-1, CD4, CD25, CD69, CD89, IL-5 receptor and GM-CSF receptor on eosinophils isolated from the peripheral blood of 15 patients with eosinophilia and 16 healthy volunteers was measured. RESULTS: The surface expression of VLA-4 presented in mean fluorescent intensity by flow-cytometric analysis showed a significant decrease in the patients with eosinophilia (>700 eosinophils/microl) compared to that of the subjects without eosinophilia. On the other hand, the surface expression of Mac-1 was significantly increased in the patients with eosinophilia. There was an inverse correlation between the expression of VLA-4 and that of Mac-1 (r = -0.81) on the eosinophils obtained from the patients with eosinophilia. CONCLUSION: The changes on the surface expressions of Mac-1 and VLA-4 may be indicating the activation of eosinophils in the patients with eosinophilia and may contribute to their migration to the allergic inflammatory focus.

Effect of roxithromycin on eotaxin-primed reactive oxygen species from eosinophils.

BACKGROUND: The CC chemokine eotaxin not only attracts eosinophils to inflamed sites but also promotes adhesion, degranulation and reactive oxygen species production of eosinophils. Reactive oxygen species released from eosinophils are believed to injure epithelial cells at inflamed sites, resulting in airway hyperresponsiveness. Roxithromycin has been reported to have antiasthmatic effects, although its mechanism of action is not thoroughly understood. Therefore, the effect of roxithromycin on eotaxin-primed reactive oxygen species production from eosinophils was studied. METHODS: Reactive oxygen species production by eosinophils cultured with or without roxithromycin was evaluated using luminol-dependent chemiluminescence. RESULTS: Roxithromycin inhibited the release of reactive oxygen species from eosinophils evoked with the calcium ionophore A23187, regardless of pretreatment with or without eotaxin. CONCLUSION: Roxithromycin may protect epithelial cells at inflamed sites, at least partly by inhibiting the release of reactive oxygen species from eosinophils.

Studies of the effect on tumor and normal cells in vitro with bioactive materials isolated from algae by a microcalorimetric method.

Recently much attention has been focused on the human physiological actions with bioactive materials from algae to enhance immunocompetence and to strengthen antineoplastic activity. In the study reported in this paper an MS 80 standard Calvet microcalorimeter was used for measuring the thermogram of HeLa, human breast carcinoma (Bcap-37) and diploid fibroblasts from human feral lung (2BS) under conditions with or without the presence of bioactive materials (Sp) from algae. At the same time, the cell number was counted, the inhibition rate of growth and the death rate were obtained. It has been shown that the Sp (100 micrograms/ml) was the growth inhibitor and lethal to tumor cells (Bcap-37 and HeLa), but had no influence on the normal (2BS) cells under the same conditions in vitro.