Flow cytometric detection of human telomerase reverse transcriptase (hTERT) expression in a subpopulation of bone marrow cells.

Telomerase activity has been found in most common cancers, thus indicating that telomerase detection may be a useful marker in cancer diagnosis. The telomeric amplification protocol (TRAP) assay and RT-PCR are customarily used to detect telomerase activity and the expression of the associated genes in cells. However, these methods do not provide any information about telomerase activation at an individual cell level. To analyze cells separately, those cells have to be isolated by sometimes complicated method. The immunohistochemical detection of human telomerase reverse transcriptase (hTERT) is useful to detect telomerase positive cells in a background of non-cancerous cells. A method has been developed for the detection of intranuclear hTERT protein, in a subpopulation of hematopoietic cells, using concurrent staining of a cell surface antigen and multicolor flow cytometry. Only mouse monoclonal anti-hTERT antibody demonstrated the specific positivity in immunocytochemistry and immunofluorescent flow cytometry. Human leukemia and myeloma cell lines showed 100% positivity, whereas normal neutrophils showed 0% positivity. hTERT expression was analyzed in hematopoietic precursor cells of bone marrow samples using concurrent staining of surface CD34 antigen and intracellular hTERT protein and multi-parameter flow cytometry. CD34 positive cells demonstrated higher expression of hTERT than CD34 negative cells. A quick, easy and sensitive assay for determining the hTERT protein expression has been developed. Using this method and the multi-parameter nature of flow cytometry and its ability to identify cellular subpopulations will provide a better understanding of the mechanisms regarding the activation of telomerase.

Clinical significance of neutrophil apoptosis in peripheral blood of patients with type 2 diabetes mellitus.

UNLABELLED: Although neutrophils are essential components of the natural immune system, they have also been implicated in the pathogenesis of tissue injuries. We assessed the clinical significance of neutrophil apoptosis in the peripheral blood of patients with type 2 diabetes mellitus (T2DM). PATIENTS AND METHODS: The study included 52 patients with T2DM (30 men, 22 women). Control subjects were 16 healthy volunteers without diabetes (7 men, 9 women). Neutrophil apoptosis levels were measured active caspase-3 positive rate by flow cytometry. RESULTS: The mean rate of neutrophil apoptosis in patients with T2DM was 15.0% (95% confidence interval [CI]: 9.5% approximately 20.5%), while that in the control group was 5.8% (95% CI: 1.6% approximately 10.0%). There were significant negative correlations between neutrophil apoptosis rate and hemoglobin (Hb) A1c levels (r = -0.352, P < .01). The mean rate of neutrophil apoptosis in the patient group with the 3 major complications (diabetic retinopathy, nephropathy, and neuropathy) was 11.1% (95% CI: 5.5%-16.7%, n = 36) and that of another group without complications was 23.8% (95% CI: 11.4%-36.2%, n = 16). There was a significant difference between these 2 groups (P < .05). CONCLUSIONS: The neutrophil apoptosis rate in patients with T2DM was significantly correlated with HbA1C levels. The mean rate of neutrophil apoptosis in the patient group with 3 major diabetic complications remained lower than that in another patient group without complications. The inhibition of neutrophil apoptosis by chronic hyperglycemia is thought to promote tissue injury and to enhance the risk of microangiopathy.

High Th1/Th2 ratio in patients with multiple myeloma.

To determine the clinical significance of the T helper 1(Th1)/T helper 2 (Th2) ratio in multiple myeloma, the intracellular IFN-gamma and IL-4 were analyzed by flow cytometry in 56 patients with multiple myeloma. The mean Th1/Th2 ratio of cases in the initial diagnosis phase and the refractory phase were higher than that of the control group. The mean serum beta-2-microglobulin in the high Th1/Th2 subgroup was significantly higher than that in the normal Th1/Th2 subgroup (P < 0.05). In conclusion, the Th1/Th2 ratio was closely related to the disease status of multiple myeloma.

[Urinary 8-hydroxy-2'-deoxyguanosin (8-OHdG) in patients with chronic liver diseases].

Urinary 8-hydroxy-2'-deoxyguanosin(8-OHdG) has been reported as sensitive biomarker of oxidative DNA damage and also of oxidative stress. We measured the urinary 8-OHdG in patients with chronic liver diseases by competitive ELISA, and analyzed the relationship with clinical characteristics. Fifty patients (male/female: 22/28) with chronic liver disease were enrolled this study. The mean concentration of urinary 8-OHdG in healthy control and patients with liver cirrhosis, chronic hepatitis C, chronic hepatitis B, and autoimmune hepatitis were 10.40+/-3.14, 10.14+/-4.19, 11.79+/-5.58, 14.99+/-4.46, and 13.64+/-3.84 microg/gCr, respectively. There were no significant differences among the five group. The mean concentration of urinary 8-OHdG in inveterate drinker was significantly higher than that in non-drinker (16.67+/-4.29 vs. 11.19+/-4.80 microg/gCr, p<0.05). The smoking enhanced the elevation of urinary 8-OHdG in drinkers. In clinical characteristics, serum y-GTP, a marker of alcoholic liver disease, had significant positive correlation with urinary 8-OHdG on the drinker with chronic hepatitis. In addition, there was a positive correlation between serum ferritin levels and urinary 8-OHdG levels. Iron in the liver suggested oxidative damage of hepatocytes through the fenton reaction in patients with chronic liver disease. In conclusion, drinking and smoking induced liver damage by oxidative stress, and urinary 8-OHdG may be reliable marker of oxidative stress in patients with chronic liver disease.