[An interlaboratory comparison study on the detection of RUNX1-RUNX1T1 fusion transcript levels and WT1 transcript levels].

Objective: To investigate the current status and real performance of the detection of RUNX1-RUNX1T1 fusion transcript levels and WT1 transcript levels in China through interlaboratory comparison. Methods: Peking University People's Hospital (PKUPH) prepared the samples for comparison. That is, the fresh RUNX1-RUNX1T1 positive (+) bone morrow nucleated cells were serially diluted with RUNX1-RUNX1T1 negative (-) nucleated cells from different patients. Totally 23 sets with 14 different samples per set were prepared. TRIzol reagent was added in each tube and thoroughly mixed with cells for homogenization. Each laboratory simultaneously tested RUNX1-RUNX1T1 and WT1 transcript levels of one set of samples by real-time quantitative PCR method. All transcript levels were reported as the percentage of RUNX1-RUNX1T1 or WT1 transcript copies/ABL copies. Spearman correlation coefficient between the reported transcript levels of each participated laboratory and those of PKUPH was calculated. Results: ①RUNX1-RUNX1T1 comparison: 9 samples were (+) and 5 were (-) , the false negative and positive rates of the 20 participated laboratories were 0 (0/180) and 5% (5/100) , respectively. The reported transcript levels of all 9 positive samples were different among laboratories. The median reported transcript levels of 9 positive samples were from 0.060% to 176.7%, which covered 3.5-log. The ratios of each sample's highest to the lowest reported transcript levels were from 5.5 to 12.3 (one result which obviously deviated from other laboratories' results was not included) , 85% (17/20) of the laboratories had correlation coefficient ≥0.98. ②WT1 comparison: The median reported transcript levels of all 14 samples were from 0.17% to 67.6%, which covered 2.6-log. The ratios of each sample's highest to the lowest reported transcript levels were from 5.3-13.7, 62% (13/21) of the laboratories had correlation coefficient ≥0.98. ③ The relative relationship of the reported RUNX1-RUNX1T1 transcript levels between the participants and PKUPH was not always consistent with that of WT1 transcript levels. Both RUNX1-RUNX1T1 and WT1 transcript levels from 2 and 7 laboratories were individually lower than and higher than those of PKUPH, whereas for the rest 11 laboratories, one transcript level was higher than and the other was lower than that of PKUPH. Conclusion: The reported RUNX1-RUNX1T1 and WT1 transcript levels were different among laboratories for the same sample. Most of the participated laboratories reported highly consistent result with that of PKUPH. The relationship between laboratories of the different transcript levels may not be the same.

Mice vaccinated with enteropathogenic Escherichia coli ghosts show significant protection against lethal challenges.

AIM: To prepare enteropathogenic Escherichia coli (EPEC) E2348/69 ghosts and investigate whether immunization with EPEC bacterial ghosts can elicit protective immune responses. METHODS AND RESULTS: A recombinant plasmid with double λPL/PR-cI857 temperature-sensitive regulatory cassettes was constructed. The lysis gene E and/or the staphylococcal nuclease A (SNA) gene were separately inserted downstream of the two regulatory cassettes to construct the lysis plasmids pBV220::E and pBV220::E::CI-P-SNA. An EPEC reference strain E2348/69 (serotype O127:H6) was transformed with the lysis plasmids to produce EPEC ghosts. Mice injected with bacterial ghosts EGE (EPEC ghosts produced using lysis protein E) or EGES (EPEC ghosts produced using a combination of lysis protein E and SNA) gained weight normally and showed no clinical signs of disease. Vaccination trials showed that mice immunized with EGE or EGES were significantly protected against subsequent challenge with the wild-type virulent parent strain, EPEC E2348/69 (42/50 and 45/50 survival, respectively); in contrast, none of the 30 control mice survived. CONCLUSIONS: Immunization with EPEC ghosts can elicit protective immune responses in BALB/c mice. SIGNIFICANCE AND IMPACT OF THE STUDY: EPEC ghosts may represent a promising new approach for vaccination against EPEC infection.

Surfactant and inhaled nitric oxide in rats alleviate acute lung injury induced by intestinal ischemia and reperfusion.

BACKGROUND: Respiratory distress and acute lung injury (ALI) are severe complications of intestinal ischemia and reperfusion injury (I/R). The authors hypothesize that a combined surfactant and inhaled nitric oxide (iNO) may alleviate I/R-induced ALI. METHODS: Adult rats (body weight 285 to 315 g, n = 45) were allocated randomly to either a negative control group (N-Control, n = 9) with only sham laparotomy, or groups (n = 9 each) for induction of I/R by occlusion of superior mesenteric artery, followed by treatment with (1) surfactant at 100 mg/kg (Surf), (2) iNO at 20 ppm (NO), (3) both surfactant and iNO (SNO), or (4) no surfactant no iNO (a positive control, P-Control). Mechanical ventilation was provided for 120 minutes with variable peak insufflation pressure and FIO2 to achieve adequate arterial pH, PaO2, and PaCO2. Blood gas values, dynamic lung compliance (Cdyn), and airway resistance (Raw) were measured during the 2-hour treatment. Lung wet-to-dry weight ratio (W/D), histopathology, and morphometric analysis of alveolar expansion (V(V)) were determined at the end of the experiment. RESULTS: After 120 minutes of treatment, the SNO group had the highest PaO2 and Cdyn values, close to that of the N-Control group. The Surf, NO, and SNO groups had higher V(V) and lower CV (V(V)) values than the P-Control, but modest decrease of W/D values. The NO group had moderately improved PaO2 but Cdyn and V(V) were as low as that of the P-Control. The lungs of the SNO group had significantly alleviated edema and neutrophil infiltration compared with the P-Control. CONCLUSIONS: The combined surfactant and iNO treatment alleviated rat ALI induced by I/R, and exerted effects better than the use of surfactant or iNO alone.

Prevention of rabbit acute lung injury by surfactant, inhaled nitric oxide, and pressure support ventilation.

Improvement of pulmonary perfusion and blood oxygenation and prevention of acute lung injury (ALI) may rely on ventilation strategy. We hypothesized that application of a combined surfactant, inhaled nitric oxide (iNO), and pressure support ventilation (PSV) should more effectively protect the lungs from injury. Anesthetized and intubated adult rabbits weighing 2.8 +/- 0.3 kg were allowed to breathe room air while receiving oleic acid intravenously (60 microl/kg). Within 90 min this caused a reduction of Pa(O(2)) from 94 +/- 7 to 48 +/- 3 mm Hg and dynamic lung compliance (Cdyn) from 1.59 +/- 0.22 to 0.85 +/- 0.10 ml/cm H(2)O/kg (both p < 0.01), and increase of intrapulmonary shunting (Q S/Q T) from 9.4 +/- 1.2 to 27 +/- 5% (p < 0.05). PSV was subsequently applied with 3 cm H(2)O of continuous positive airway pressure and FI(O(2)) of 0.3, and the animals were randomly allocated to four groups, receiving: (1) PSV only (Control, n = 10); (2) iNO at 20 ppm (NO, n = 9); (3) surfactant phospholipids at 100 mg/kg (Surf, n = 8); and (4) surfactant at 100 mg/kg and iNO at 20 ppm (SNO, n = 8). PSV level was varied to maintain a tidal volume of 8 to 10 ml/kg for another 12 h or until early animal death. Five animals in the SNO, three each in the NO and Surf group, and one in the Control group survived 12 h (SNO versus Control, p < 0.05). The NO, Surf, and SNO groups had significantly improved mean Pa(O(2)) (> 70 mm Hg, p < 0.05), and reduced Q S/Q T (15, 19, and 17%, respectively, p < 0.05) at 6 and 12 h, but not in the Control group. The SNO group had the highest values of Cdyn at 12 h, alveolar aeration and disaturated phosphatidylcholine-to-total protein ratio in bronchoalveolar lavage fluid, and the lowest wet-to-dry lung weight ratio and lung injury score (p < 0.05). The results indicate that early alleviation of ALI by surfactant, iNO, and PSV is due to synergistic effects, and only PSV in this model had limited effects.

Effects of inhaled nitric oxide and high-frequency ventilation in rabbits with meconium aspiration.

AIM: To evaluate effects of inhaled nitric oxide (iNO) in experimental meconium aspiration treated with high-frequency (HFV) or conventional mechanical ventilation (CMV). Ventilated adult rabbits had meconium instilled intratracheally resulting in respiratory failure as evidenced by more than 50% reduction of dynamic lung compliance (Cdyn) and increase in mean oxygenation index (OI) from 1 to 16. The animals were then allocated to 2 groups treated without (control) or with iNO at 20 ppm (NO). In each group the animals were initially ventilated with CMV or HFV mode for 3 h and then in a crossover fashion with HFV or CMV for another 3 h (CMV-->HFV, HFV-->CMV), respectively. In the first 3 h of treatment, the animals subjected to HFV-CMV in the control, and those with both HFV-CMV and CMV-HFV in the NO group had significantly reduced OI. In the subsequent 3 h, the animals in the control group with CMV-HFV did not improve in OI and those with HFV-CMV had deteriorated. In the NO group with both CMV-HFV and HFV-CMV moderate improvement of OI was observed. Platelet aggregation capability and counts were significantly decreased and bleeding time prolonged in animals receiving iNO treatment. These results suggest that both HFV alone and a combined treatment of iNO with either CMV or HFV are more effective in improving blood oxygenation than that of CMV in this animal model. The influence of iNO on platelet aggregation should be considered.

Comparison of effects of surfactant and inhaled nitric oxide in rabbits with surfactant-depleted respiratory failure.

AIM: To compare effects of pulmonary surfactant and inhaled nitric oxide (iNO) in improvement of survival and blood oxygenation in ventilated rabbits with acute hypoxic respiratory failure induced by repeated bronchoalveolar lavage (BAL). METHODS: After BAL all the rabbits had more than 50% reduction of dynamic lung compliance (Cdya), 50% increment of resistance of respiratory system (Rrs), and an increase of mean oxygenation index (OI) from 1 to 22. The rabbits were then randomly allocated to groups receiving (1) mechanical ventilation only (Control), (2) iNO 0.8 mumol.L-1 (20 ppm) (NO), (3) intratracheal bolus surfactant phospholipids at 100 mg.kg-1 (Surf), and (4) combined surfactant at 100 mg.kg-1 with inhaled NO at 0.8 mumol.L-1 (Surf + NO). All the rabbits were ventilated with standardized tidal volume (8-10 mL.kg-1) for another 8 h or until early death. RESULTS: The rabbits in both control and NO groups had the lowest survival rate, deterioration of lung mechanics and OI, whereas those in the Surf and Surf + NO groups had modestly improved Cdyn, Rrs, and OI. Only rabbits in the Surf + NO group had significantly improved survival rate and alveolar expansion. CONCLUSION: Surfactant with or without iNO is more effective compared to the control and iNO groups in rabbit, suggesting that iNO is not effective unless a method to recruit alveoli is applied.

[DNA content analysis of breast cancer cells recognized by monoclonal antibody].

DNA content of breast cancer cells recognized by monoclonal antibody Ki-67 was assayed with flow cytometry in 50 cases. The relationships between sorted or unsorted assay data and metastasis in axillary lymph nodes were analyzed. The results showed that: (1) Aneuploidy rate of breast cancer determined by sorting with Ki-67 was increased and significantly correlated with metastasis in axillary lymph nodes. (2) S-phase fraction (SPF) determined by both sorting and unsorting assay were significantly correlated with axillary lymph node metastasis. (3) Proliferative index (PI) was significantly correlated with axillary lymph node metastasis. This correlation was more significant by sorting with Ki-67. (4) The range of growth fraction (GF) was 28.8%-83.4%, with a mean value of 52.6%. The authors concluded that (1) accuracy of DNA content analysis of breast cancer cells can be increased by sorting with monoclonal antibody Ki-67. (2) DNA ploidy and SPF are important prognostic factors of breast cancer. (3) PI is of great importance in the evaluation of biological behaviours of breast cancer.

[Immune function and abdominal surgery in blood stasis syndrome in patients with gastrointestinal diseases].

52 Patients with Blood Stasis Syndrome (BSS) in abdominal surgical diseases were divided into 3 types according to their symptoms, signs and natures of diseases. Peripheral blood T lymphocyte subsets of these BSS patients and 12 healthy persons were studied with Flow Cytometry and monoclonal antibodies. The immunoglobulins and complements of these cases were also studied. There were no difference in T cell subsets, immunoglobulins and complements between Qizhi-BSS group and normal control. The Shire-BSS group showed that CD8 cell, IgG, IgM, and C1, C3c were increased. These results showed that the immune response increased in this type of BSS patients. In the Qixu-BSS group, the CD3 was nearly normal, and the CD4+, CD4+/CD8+ ratio, and CD16+ were statistically decreased. However, the CD8+ cells markedly increased, the IgG, IgM and IgD were also lowered significantly in this type. The marked morphologic abnormal changes in ultrastructures of T lymphocyte were found in 6 patients with Qixu-BSS group. These results showed that the Qixu type of Blood Stasis Syndrome patients were in the immuno-suppressive status.

Detection of onco-fetal bladder antigen in urine of patients with transitional cell carcinoma.

Studies of antigens associated with transitional cell carcinoma were extended by using murine IgM monoclonal antibody E7, developed earlier by this laboratory. These antibodies react preferentially with human bladder tumors and transitional cell carcinoma (TCC) cell line 647V. We now report that monoclonal antibody E7 detected the presence of antigen in midgestational and third trimester amniotic fluids, and in urine of patients with advanced transitional cell carcinoma. Western blot analysis showed that the antigen present in amniotic fluids consists of a sharp band with molecular weight greater than 200 kdaltons. A similar molecular weight pattern was seen with the solubilized membrane of 647V. A sensitive and convenient sandwich ELISA was developed and the urine of patients with bladder cancer was assayed for the presence of the E7 antigen. Antigen was detected in the urine of patients with advanced transitional cell carcinoma but not in the urine of normal adults or in urine from patients with prostate cancer, renal cell carcinoma, or benign prostate hyperplasia. An inhibition enzyme immunoassay was developed with monomeric forms of the E7 antibody and confirmed the presence of antigen in the urine of patients with TCC. We conclude that the E7 antigen is an onco-fetal antigen expressed in patients with transitional cell carcinoma of the bladder.