[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.

Predictive factors for inadequate colon preparation before colonoscopy.

BACKGROUND: It could be helpful to ascertain which patients are at risk of poor bowel preparation prior to performing sedated colonoscopy. The aim of the present study was to identify the predictive factors for poor colon preparation prior to colonoscopy. METHODS: A prospective study was performed at Kaohsiung Chang Gung Memorial Hospital, Taiwan, from September 2011 to May 2013. Patient characteristics, food consumed within 2 days of colonoscopy, volume of polyethylene glycol (PEG) solution, interval between completing PEG and examination, number of bowel movements, and character of the last stool were evaluated. RESULTS: Seven hundred and three patients were enrolled (mean age 50.3 ± 11.6 years, 43 % female). In univariate analysis, character of the last stool (<0.001), body weight (p = 0.007), body mass index (p = 0.047), waist circumference (p = 0.008), buttock girth (p = 0.016), meal residue score (<0.001), and interval between end of PEG and colonoscopy (p = 0.01) were related to inadequate colon preparation. In multivariate analysis, waist circumference (p < 0.001), meal residue score (p < 0.001), and characteristics of last stool (p < 0.001) were variables that predicted poor colon preparation. CONCLUSIONS: Patients who have consumed a high residue diet and/or who report that their last stool is semisolid are likely to have poor bowel preparation, and consideration could be given to rescheduling the examination.

Insights into osteoarthritis progression revealed by analyses of both knee tibiofemoral compartments.

OBJECTIVE: To identify disease relevant genes and pathways associated with knee Osteoarthritis (OA) progression in human subjects using medial and lateral compartment dominant OA knee tissue. DESIGN: Gene expression of knee cartilage was comprehensively assessed for three regions of interest from human medial dominant OA (n = 10) and non-OA (n = 6) specimens. Histology and gene expression were compared for the regions with minimal degeneration, moderate degeneration and significant degeneration. Agilent whole-genome microarray was performed and data were analyzed using Agilent GeneSpring GX11.5. Significant differentially regulated genes were further investigated by Ingenuity Pathway Analysis (IPA) to identify functional categories. To confirm their association with disease severity as opposed to site within the knee, 30 differentially expressed genes, identified by microarray, were analyzed by quantitative reverse-transcription polymerase chain reaction on additional medial (n = 16) and lateral (n = 10) compartment dominant knee OA samples. RESULTS: A total of 767 genes were differentially expressed ≥ two-fold (P ≤ 0.05) in lesion compared to relatively intact regions. Analysis of these data by IPA predicted biological functions related to an imbalance of anabolism and catabolism of cartilage matrix components. Up-regulated expression of IL11, POSTN, TNFAIP6, and down-regulated expression of CHRDL2, MATN4, SPOCK3, VIT, PDE3B were significantly associated with OA progression and validated in both medial and lateral compartment dominant OA samples. CONCLUSIONS: Our study provides a strategy for identifying targets whose modification may have the potential to ameliorate pathological alternations and progression of disease in cartilage and to serve as biomarkers for identifying individuals susceptible to progression.

Comparison of clinical outcomes of open, laparoscopic and single port appendicectomies.

INTRODUCTION: Appendicectomy is the most common surgical procedure performed in general surgery. This study aimed to compare the outcomes of open appendicectomy (OA), laparoscopic appendicectomy (LA) and single port laparoscopic appendicectomy (SPLA). METHODS: Fifty consecutive patients with suspected acute appendicitis were studied (OA: n=20, LA: n=20, SPLA: n=10). Clinical outcomes were compared between the three groups in terms of operative time, blood loss, postoperative complications, length of hospital stay and cost. RESULTS: Patient demographics were similar among groups (p>0.05). SPLA was characterised by longer operative time (88.1 minutes vs 35.6 minutes in OA and 33.4 minutes in LA) and higher costs (12.84 thousand Chinese yuan [RMB] vs 8.41 thousand RMB in LA and 4.99 thousand RMB in OA). OA was characterised by more blood loss (9.8ml vs 7.5ml in SPLA and 6.8ml in LA), longer hospital stay (7.5 days vs 3.5 days in LA and 3.4 days in SPLA) and lower costs. The total number of complications was higher for OA (n=2) than for LA and SPLA (n=0) although this was not statistically significant. CONCLUSIONS: Where feasible, LA should be undertaken as the initial treatment of choice for most cases of suspected appendicitis.

Direct assessment of articular cartilage and underlying subchondral bone reveals a progressive gene expression change in human osteoarthritic knees.

OBJECTIVE: To evaluate the interaction of articular cartilage (AC) and subchondral bone (SB) through analysis of osteoarthritis (OA)-related genes of site-matched tissue. DESIGN: We developed a novel method for isolating site-matched overlying AC and underlying SB from three and four regions of interest respectively from the human knee tibial plateau (n = 50). For each site, the severity of cartilage changes of OA were assessed histologically, and the severity of bone abnormalities were assessed by microcomputed tomography. An RNA isolation procedure was optimized that yielded high quality RNA from site-matched AC and SB tibial regions. Quantitative polymerase chain reaction (Q-PCR) analysis was performed to evaluate gene expression of 61 OA-associated genes for correlation with cartilage integrity and bone structure parameters. RESULTS: A total of 27 (44%) genes were coordinately up- or down-regulated in both tissues. The expression levels of 19 genes were statistically significantly correlated with the severity of AC degeneration and changes of SB structure; these included: ADAMTS1, ASPN, BMP6, BMPER, CCL2, CCL8, COL5A1, COL6A3, COL7A1, COL16A1, FRZB, GDF10, MMP3, OGN, OMD, POSTN, PTGES, TNFSF11 and WNT1. CONCLUSIONS: These results provide a strategy for identifying targets whose modification may have the potential to ameliorate pathological alterations and progression of disease in both AC and SB simultaneously. In addition, this is the first study, to our knowledge, to overcome the major difficulties related to isolation of high quality RNA from site-matched joint tissues. We expect this method to facilitate advances in our understanding of the coordinated molecular responses of the whole joint organ.

Elevated expression of both MDR1 and MMP-2 genes in metastasized lymph node of invasive ductal breast cancer.

BACKGROUND: There is emerging evidence that matrix metalloproteinase (MMP)-2 plays a crucial role in cancer invasion/metastasis. However, little evidence is available about the connections of multidrug resistance protein 1 (MDR1) and cancer invasion/metastasis so far. AIM: To investigate the expression of MDR1 and MMP2 in primary breast tumors and their corresponding metastasized lymph nodes. MATERIALS AND METHODS: Only lymph nodes which were pathologically identified as metastases were included in this study to compare with the corresponding primary tumor. We determined the expression of MDR1 and MMP2 in primary breast tumor and its metastasized lymph node specimens of 21 patients. The quantitative real-time polymerase chain reaction (Q-RT-PCR) technique was used to assess the MDR1 and MMP2 RNA expression levels in primary breast tumor and lymph nodal specimens. Target gene copies were normalized using beta-actin (beta-actin) gene copies. Tumor characteristics and number of metastatic lymph nodes were gathered from the pathology reports. RESULTS: The Q-RT-PCR data showed that MDR1 expression in metastasized lymph node was higher than that of their corresponding primary tumors (p < 0.05), MMP2 expression in metastasized lymph nodes was also even higher compared with their matched primary tumors (p < 0.01). But SPSS bivariate correlation analysis revealed that MDR1 expression in lymph node was not correlated with MMP-2 expression in lymph node, number of metastasized lymph nodes and tumor size (p > 0.05). MDR1 expression in primary tumors was highly correlated with in corresponding lymph node metastases (p < 0.01 r = 0.795). CONCLUSIONS: All those indicated that MMP-2 should play an important role in the lymph node metastasis. However, further clinical studies with larger sample size need to be performed to verify these findings.

Prospective study of warfarin dosage requirements based on CYP2C9 and VKORC1 genotypes.

Polymorphisms in CYP2C9 and VKORC1 have been shown to be associated with warfarin dose requirements and could be used to predict warfarin dose. We conducted a prospective study in which warfarin dose was prescribed based on CYP2C9 and VKORC1 polymorphisms in 108 Han-Chinese patients without prior warfarin treatments. Using the genotype-based dosing, 83% of patients reached stable, therapeutic international normalized ratio (INR) within 2 weeks of treatment initiation and none of the patients developed clinical bleeding or thromboembolic event. Ten percent (11) of patients with INR > 4 and no clinical bleeding were detected during this study. At 12 weeks, 69% of the patients' maintenance doses matched the prediction. Dosing algorithms incorporating genetic factors, age, and body surface area were developed, which could explain up to 62% of the total variation (R(2) of 0.62). This study demonstrated that pharmacogenetics-based dosing could improve time to stable, therapeutic INR, reduce adverse events, and achieve high sensitivity.

CD70 (TNFSF7) is expressed at high prevalence in renal cell carcinomas and is rapidly internalised on antibody binding.

In order to identify potential markers of renal cancer, the plasma membrane protein content of renal cell carcinoma (RCC)-derived cell lines was annotated using a proteomics process. One unusual protein identified at high levels in A498 and 786-O cells was CD70 (TNFSF7), a type II transmembrane receptor normally expressed on a subset of B, T and NK cells, where it plays a costimulatory role in immune cell activation. Immunohistochemical analysis of CD70 expression in multiple carcinoma types demonstrated strong CD70 staining in RCC tissues. Metastatic tissues from eight of 11 patients with clear cell RCC were positive for CD70 expression. Immunocytochemical analysis demonstrated that binding of an anti-CD70 antibody to CD70 endogenously expressed on the surface of A498 and 786-O cell lines resulted in the rapid internalisation of the antibody-receptor complex. Coincubation of the internalising anti-CD70 antibody with a saporin-conjugated secondary antibody before addition to A498 cells resulted in 50% cell killing. These data indicate that CD70 represents a potential target antigen for toxin-conjugated therapeutic antibody treatment of RCC.

[Study on the improvement of process technology of L(+)-tartaric acid fermentation].

This work proposes an improved process technology of L(+)-tartaric acid produced by using cis-epoxysuccinates as the substrate for fermentation. The key to the question is to apply dipotassium cis-epoxysuccinate as the substrate instead of disodium cis-epoxysuccinate. As compared with the original process technology, the improved one has prominent advantages: 1. High yield of acid, increased by 20%-30% over the old one; 2. High rate of recovery, from about 60% to 80%; 3. One of the raw materials is cheaper, the KOH is expensive than NaOH, but half of the K kions could be used cyclically, and the varied products could be obtained easily; 4. The tough working procedure of filtration of fermented liquor could be evaded, the total working procedures might increase to some extent, but the cost of production will be reduced obviously, it is advantageous to industrial production.

Characterization and differentiation of an early murine yolk sac-derived IL-7-independent pre-pro-B cell line.

We describe a unique, stable pre-pro-B cell line (YS-PPB) derived from AA4.1+ yolk sac cells from day 10 mouse embryos. This cell line, discovered fortuitously during the course of studies of in vitro B cell differentiation, is independent of IL-7 supplementation for long term expansion in vitro. YS-PPB cells as well as clonal sublines expressed AA4.1, CD43, B220, Sca-1, CD19, heat stable antigen, MHC class I, IL-7R, and FcyR, but did not express cytoplasmic mu-chain, surface IgM (sIgM), or MHC class II molecules. PCR analysis showed that the cells expressed TdT, lambda5, and RAG-1 genes, but that their Ig genes were still in germline configuration. The cell line was dependent on direct contact with S17 stromal cells for growth, but, in contrast to bone marrow stem cells, required no additional growth factors for maintenance and expansion. When stimulated with IL-7 and LPS, YS-PPB cells and cells from all tested clonal sublines differentiated into sIgM+ B cells in vitro. Irradiated mice reconstituted with YS-PPB cells yielded spleens containing 38% sIgM+ donor-derived B cells, demonstrating that YS-PPB cells, although stably arrested in development at the boundary between pre-pro-B and pro-B stages of B cell differentiation, still retain their competence to differentiate into mature, Ig-producing B cells when transferred to a normal host environment. Thus, this new cell line can provide a reproducible source of B cell precursors arrested at that critical time in B cell differentiation when the machinery for Ig gene rearrangement is in place but rearrangement has not yet occurred.

Role of endothelium in the control of mouse yolk sac stem cell differentiation.

Studies in our laboratory have shown that as early as day 8.5 of development, mouse yolk sac cells can generate T cells when placed in a thymic microenvironment. At this stage, yolk sac cells can also differentiate into myeloid cells in vitro. B cell differentiation in vitro was achieved with day 9 yolk sac by providing a bone marrow stromal feeder layer. We have now established endothelial cell lines and clones from yolk sacs of day 8-12 mouse embryos. These vary in their ability to support stem cell maintenance and differentiation. Our principal work has been carried out with day 12 cloned endothelial cell lines. One clone supported the > 100 fold expansion of yolk sac hematopoietic stem cells that subsequently could generate B cells, T cells and myeloid cells both in vitro and in vivo. Preliminary experiments with endothelial cells from younger embryos are also described.

In vitro and in vivo differentiation into B cells, T cells, and myeloid cells of primitive yolk sac hematopoietic precursor cells expanded > 100-fold by coculture with a clonal yolk sac endothelial cell line.

The yolk sac, first site of hematopoiesis during mammalian development, contains not only hematopoietic stem cells but also the earliest precursors of endothelial cells. We have previously shown that a nonadherent yolk sac cell population (WGA+, density < 1.077, AA4.1+) can give rise to B cells, T cells and myeloid cells both in vitro and in vivo. We now report on the ability of a yolk sac-derived cloned endothelial cell line (C166) to provide a suitable microenvironment for expansion of these early precursor cells. Single day 10 embryonic mouse yolk sac hematopoietic stem cells wer expanded > 100 fold within 8 days by coculture with irradiated C166 cells. Colony-forming ability was retained for at least three passages in vitro, with retention of the ability to differentiate into T-cell, B-cell, and myeloid lineages. Stem cell properties were maintained by a significant fraction of nonadherent cells in the third passage, although these stem cells expressed a somewhat more mature cell surface phenotype than the initial yolk sac stem cells. When reintroduced into adult allogeneic immunocompromised (scid) hosts, they were able to give rise to all of the leukocyte lineages, including T cells, B cells, and myeloid cells. We conclude that yolk sac endothelial cells can support the stable proliferation of multipotential hematopoietic stem cells, thus generating adequate numbers of cells for study of the mechanisms involved in their subsequent development and differentiation, for in vivo hematopoietic restitution, and for potential use as a vehicle for gene transfer.

[Functional roles of LFA-1 involved in signal transduction for thymocyte activation].

The biological significance of the signals triggered by the interaction of cell surface expressed LFA-1 and ICAM-1 has been investigated in Con A and immobilized anti-CD3 mAb stimulated cultures. When added at the beginning of activation in the presence of Con A, soluble anti-LFA-1 and anti-ICAM-1 mAbs could strongly inhibit cell proliferation. Such inhibitory effect was also exhibited in the proliferative response of thymocytes to immobilized anti-CD 3 mAb activation. However, the soluble anti-LFA-1 mAb was unable to inhibit the proliferation of primed thymocytes preactivated with Con A for 24 h or of IL-1 + IL-2 activated fresh thymocytes. Anti-LFA-1 mAb could profoundly inhibit Con A-induced thymocytes to produce IL-2 and IL-6 and to reduce IL-2 receptor expression. By contrast, anti-LFA-immobilized on plastic plates together with immobilized anti-CD 3 mAbs or CD 3 cross-linking with LFA-1 by secondary antibodies resulted in an enhanced activation signals for thymocytes to proliferate compared with that activated by anti-CD 3 alone. Thus, mAb to LFA-1 is a functional molecule for thymocyte activation, mediating signals contributed to very early phases of signal transduction through TCR/CD 3 pathway, and that LFA-1 might provide a costimulatory signal for expression of IL-2 R and IL-2 production.

[Role of LFA-1/ICAM-1 in the activation of murine thymocytes stimulated by Con A].

The biological significance of the signals triggered by the interaction of cell surface expressed LFA-1 and ICAM-1 has been investigated in Con A stimulated murine thymocytes. Addition of anti-LFA-1 or/and anti-ICAM-1 McAb to the thymocyte culture in the presence of Con A, the cell proliferation was profoundly inhibited. Such inhibitory effect was more significant by the addition of anti-LFA-1 than by the addition of anti-ICAM-1 McAb. FACS analysis revealed that the proliferation of CD8+ T cells was more sensitive to the inhibitory effect of anti-LFA-1 McAb than did the proliferation of CD4+ T cells. The anti-LFA-1 McAb imposed inhibitory effect, however, could be reversed by the supplementation of IL-2 in the initiation of the culture. The proliferation of cultured Con A activated T blast cells could no longer be inhibited by the addition of anti-LFA-1 McAb. Furthermore, neither anti-LFA-1 nor anti-ICAM-1 McAb could block PMA+A23187 induced thymocyte proliferation. The results imply that the ligand-receptor pair of LFA-1-ICAM-1 molecules also takes part in the early events of Con A activated signal transduction pathway which leads to the proliferation of thymocytes.

[The production of IL-2, IL-6 in relation to the functional development of NK cells in human fetal spleens].

The production of IL-2, IL-6 by fetal splenic mononuclear cells (FSMC) and relationship between them and the ontogenetic development of natural killer cell function were studied in human fetal spleens. As a results, before 20 weeks of gestation, both IL-2 and NK cell activities were not measured, but IL-6 was done. It was found that IL-2, IL-6 and NK cell activities were increased with the gestational age, and shown that there were linear positive correlation between the activities of three ones above (r > 0.86). Before the birth, the induced IL-2 activity was the same as adult levels (p > 0.05), although both IL-6 production and NK activity in fetal spleens were significantly lower than that in adults (p < 0.01). Lastly, the production of IL-2, IL-6 in relation to the functional development of NK cells during the embryonic development was discussed.

[Experimental study on IL-6 activity against tumor: I. Regulation of IL-2-induced LAK cells].

The ability of human recombinant interleukin-6 (IL-6) to regulate the induction and function of lymphokine activated killer (LAK) cells from human fetal spleens was studied. The results showed that IL-6 alone was unable to induce fetal splenic mononuclear cells (FSMC) to develop functional LAK cells, nor was it able to affect the number of IL-2-induced LAK cells and to alter the lytic activity of the induced LAK cells in effector phase. However, when IL-6 was used in combination with IL-2 during the induction phase, the resulting LAK cells displayed considerably greater lytic activity than that with IL-2 alone (P less than 0.01), and this effect was IL-6 dose-dependent. The possible mechanism of the synergetic effect of IL-2 and IL-6 in LAK cell induction from human fetal spleens is discussed.

Carbon Assimilation and Leaf Water Status in Sugar Beet Leaves during a Simulated Natural Light Regimen.

Carbon assimilation and leaf water status were studied in sugar beet (Beta vulgaris L., Klein E-type multigerm) leaves during a light period in which illumination either increased rapidly to full irradiance or changed gradually in a sinusoidal manner as generally occurs during a natural day. A light regimen that simulated the light of a natural day was produced by adjusting irradiance with a neutral-density filter under the control of a computer. Under this light regimen, photosynthesis, transpiration, and stomatal conductance followed the irradiance pattern very closely and ribulose bisphosphate carboxylase was nearly fully activated. When illumination was increased rapidly at the beginning of a light period, transpiration also increased quickly, causing leaves to wilt to some extent. The activation state of ribulose bisphosphate carboxylase increased to only 52%, but ribulose bisphosphate level was nearly twice as high as during the simulated natural day. In spite of the differences in activation state and ribulose bisphosphate levels, photosynthesis rates were very similar under both regimens. Nevertheless, differences in parameters between leaves under the two irradiance regimens can affect how a plant responds to internal or external factors, and therefore, the rate at which irradiance increases at the beginning of a light period is an important consideration when interpreting data.