Novel enhanced green fluorescent protein-expressing NOG mouse for analyzing the microenvironment of xenograft tissues.

The interaction between transplanted cells and host tissues is important for the growth and maintenance of transplanted cells. To analyze the mechanisms of these interactions, a systemic fluorescent protein-expressing mouse is a useful recipient. In this study, we generated a novel NOG strain, which strongly expresses enhanced green fluorescent protein (EGFP; PgkEGFP-NOG), especially in the liver, kidney, gastrointestinal tract, and testis. Because the host tissues expressed EGFP, xenotransplanted human cancer cells were clearly identified as EGFP-negative colonies in PgkEGFP-NOG mice. Immunohistochemical analysis revealed that EGFP-expressing stromal tissues formed a complicated tumor microenvironment within xenograft tissues. Moreover, a similar microenvironment was observed in human iPS cell-derived teratomas. Collectively, these results indicated that a suitable microenvironment is essential for the growth and maintenance of xenotransplanted cells and that PgkEGFP-NOG mice represent a useful animal model for analyzing the mechanisms of microenvironment formation.

Significant relationship between platelet activation and intra-media thickness of the carotid artery in patients with ischemic cerebrovascular disease.

INTRODUCTION: We have studied the relationship between the ratio of activated platelets and the thickness of intima and media of the carotid artery in ischemic CVD patients in the chronic stage. METHODS: Platelet activation was assessed by means of flow cytometry of whole blood using activation-dependent monoclonal antibodies (MoAb). Forty-one MRI-proven normative subjects and 55 patients with a history of ischemic CVD were examined. The intima-media thickness of the carotid artery was measured by using B-mode ultrasound in all subjects. RESULTS: The appearance rates of PAC-1-positive and CD62P-positive platelets (%) were increased in ischemic CVD patients compared with those in controls (p<0.0001, p<0.001, respectively) The patients and controls were divided into those with atherosclerosis (Ath+), defined as intima-media thickness 1.1 mm, and those without (Ath-). There was no significant difference of PAC-1-positive platelets between the Ath- and Ath+ subgroups in either group, but there was increase in Ath- ischemic CVD patients versus Ath- control subjects (p<0.01), and in Ath+ patients versus Ath+ controls (p<0.05). CD62-positive platelets in the Ath+ subgroup were significantly increased versus the Ath- subgroup in both the controls (p<0.001) and ischemic CVD patients (p<0.05), and there was also an increase in Ath- patients versus Ath- controls (p<0.05). CONCLUSION: Platelet activation markers were increased in patients with ischemic CVD compared with controls. A significant relationship was found between increased CD62-P-positive platelets and carotid artery abnormalities in both controls and ischemic CVD patients, suggesting that platelet activation may be a potential marker for atherosclerosis.

Large diversity in transport-mediated methotrexate resistance in human leukemia cell line CCRF-CEM established in a high concentration of leucovorin.

To elucidate the mechanism(s) of methotrexate (MTX) resistance as a possible reason underlying treatment failure in high-dose MTX regimens combined with leucovorin (LV) rescue, we established MTX-resistant human T-cell leukemia cell line CCRF-CEM cells in the presence of excess LV, and characterized their properties. Continuous exposure of the cells to escalating concentrations of MTX up to 20 microM in the presence of 1000 nM LV resulted in establishment of three MTX-resistant sublines with a wide disparity of resistance degree over a 4 logarithmic range (approximately 40-, 900- and 44,000-fold, respectively). Transmembrane transport of MTX in these sublines was diminished to 52%, 35% and 12%, respectively. Intracellular retention of MTX in these sublines was not different from that of the parent cells. A cell growth study in various concentrations of LV showed that cells with higher resistance to MTX required more LV for optimal growth. In parallel with the resistance levels, there was an increase in mRNA expression of dihydrofolate reductase gene and a decrease in that of thymidylate synthase gene, but no change in that of reduced folate carrier (RFC1) gene, as assessed by northern blot analysis. Sequencing of the RFC1 gene in all 3 sublines revealed a point mutation in codon 47 (TCC-->TTC) resulting in substitution of Phe for Ser residue, and additional deletion of CTG of codon 112 in the subline with the highest resistance. In summary, MTX exposure to CCRF-CEM cells in the presence of 1000 nM LV resulted in the establishment of heterogeneous cell populations with a wide range of transport-mediated MTX resistance, which was associated with differential alterations of RFC gene. These cell lines may serve as models for investigation of the molecular mechanism(s) underlying refractory tumors in high-dose MTX regimens with LV rescue.

[Biochemistry of platelet membrane glycoproteins].

Platelet membranes are rich in glycoproteins which mediate the key functions of platelets, adhesion and aggregation, by their binding of specific adhesive proteins. The membrane glycoproteins were initially identified by surface labeling with 125I or 3H followed by SDS-polyacrylamide gel electrophoresis. Many of these glycoproteins are now cloned and sequenced and have been found to be members of major gene families, such as integrin, leucine rich glycoprotein (LRG), selectin, etc. In this chapter, the structure and the functions of membrane glycoproteins are briefly reviewed.

Simple, rapid, and automated method for detection of hyperaggregability of platelets using a hematology analyzer.

Estimation of hyperaggregability of platelets is important for diagnosis and prevention of vascular events. We have developed and evaluated a simple and rapid method for detection of a hyperaggregable state of platelets by using an Abbott CELL-DYN(R) 4000 hematology analyzer. Citrated blood samples were collected from 62 patients with chronic cerebral infarction (CCI), of whom 19 patients were treated with ticlopidine, and from 9 healthy subjects. Platelet clumps were detected in the scatter plots for white blood cell populations with the hematology analyzer. Platelet clumps were positive in 20 of 43 (46.5%) CCI patients who were not treated with anti-platelet agents but not at all in 9 healthy subjects and in 19 CCI patients treated with ticlopidine. The detection of platelet clumps in citrated blood by the hematology analyzer was proved useful in detecting a platelet hyperaggregability in CCI patients. This method is simple, rapid, and automated and thus should be suitable for routine clinical use for monitoring indications and the efficacy of anti-platelet drugs.

Quantitative assay of hepatitis C virus RNA using an automated extraction system for specific capture with probes and paramagnetic particle separation.

A commercially available automated specimen preparation instrument for specific probe capture and paramagnetic separation has been developed (AmpliCap/GT-12; Roche Molecular Systems). We evaluated assay performance of the AmpliCap/GT-12 in the quantitative assay for hepatitis C virus (HCV) RNA with the AMPLICOR HCV MONITOR Test (version 2.0). Assay linearity using serial dilutions from a serum panel was observed in the range of 500 to 850000 IU/ml, with a slightly compromised slope in the higher viral titers. The overall within-run and between-run reproducibility of the entire detection process for 3 and 5 log(10) (IU/ml) of HCV RNA in samples had a standard deviation of <0.2, which was comparable to a manual method based on organic extraction and isopropanol precipitation (Roche Molecular Systems). Comparison of the test results with those obtained by the manual method showed a good correlation (R(2) = 0.972, n = 86). Using heparin (3, 6.5, and 13 U/ml), dextran sulfate (0.1, 1, and 5 mM), hemoglobin (1.13, 2.25, and 4.5 g/liter), conjugated or unconjugated bilirubin (7.5, 15, and 30 mg/dl), and ATP (1.25, 2.5, and 5.0 mM) as known inhibitors, inhibition was only detected at a dextran sulfate concentration of 1 mM with the manual method but not with the AmpliCap/GT-12 extraction. In summary, the AmpliCap/GT-12 system was shown to permit a stable extraction process and accurate results for the quantitative assay of HCV RNA, successfully eliminating the inhibitory effect of dextran sulfate. This automated extraction system provides reliable and reproducible test results and saves labor; thus, it is suitable for routine diagnostic PCR.

Extensive wall thickening in intestinal Burkitt lymphoma.

OBJECTIVE: To evaluate intestinal lesions in Burkitt lymphoma. METHODS: Ultrasonography was used in the initial evaluation of 6 Japanese patients with intestinal Burkitt lymphoma. RESULTS: Ultrasonography revealed marked wall thickening of the colon from the cecum through either the ascending or the transverse colon, which led to a target sign (4 cases) or a pseudokidney sign (2 cases). The target sign histopathologically corresponded to invagination of an occult tumor of the cecum into the ascending colon. Wall thickening of the colon when associated with a target or pseudokidney sign corresponded to marked lymph edema or a diffuse infiltration of Burkitt lymphoma cells into the intramural layers, respectively CONCLUSIONS: Ultrasonography provides useful information in the initial evaluation of intestinal lesions with distinctive histopathologic characteristics in Burkitt lymphoma.

Smoldering myeloma associated with zonisamide treatment.

A 39-year-old man was found to have hyperproteinemia after being treated with zonisamide for 10 years. Laboratory examination revealed a serum M-protein which consisted of IgG (lambda) and an increased number of plasma cells in the bone marrow, resulting in a diagnosis of smoldering myeloma. Considering his age, zonisamide was suspected to play an etiologic role in the occurrence of smoldering myeloma. Zonisamide was changed to sodium valproate. Subsequently the M-protein did not increase over 13 months. When zonisamide is used, the monitoring of serum levels of M-protein and patterns of gammaglobulin is warranted.

Platelet activation in patients with chronic hepatitis C.

OBJECTIVE: To elucidate the mechanisms of thrombocytopenia in chronic hepatitis C (CHC), we investigated platelet activation in patients with chronic viral liver diseases. METHODS: Platelet activation was evaluated with flow cytometry in twenty-five patients with chronic viral hepatitis and 11 patients with liver cirrhosis of viral etiology. Liver biopsies were carried out in all patients. RESULTS: The platelet counts decreased significantly in patients with CHC and in patients with liver cirrhosis compared to controls, but not in patients with chronic hepatitis B (CHB). Patients with CHC had a significantly higher percentage of platelets positive for activation-dependent monoclonal antibodies (MoAbs), and also had a higher percentage of platelet microparticles (PMP), a marker of platelet activation, than patients with CHB. There was a significant correlation between the percentage of PMP and the levels of liver fibrosis markers, such as serum hyaluronate and N-terminal propeptide of type III procollagen (P-III-P), in CHC, suggesting the relationship between platelet activation and liver fibrosis. Platelet activation was markedly enhanced in CHC patients with high histological scores of liver fibrosis. CONCLUSION: Patients with CHC have increased platelet activation, which may contribute to the occurrence of thrombocytopenia in CHC. Liver fibrosis may play a role in activation of platelets in CHC.

Platelet activation in patients with obstructive sleep apnea syndrome and effects of nasal-continuous positive airway pressure.

OBJECTIVE: Our study was undertaken to determine whether increased platelet activation occurs in patients with obstructive sleep apnea syndrome (OSAS) and whether a therapy with nasal-continuous positive airway pressure (N-CPAP) alters this activation. METHODS: We measured the positive rate of activated platelets using activation-dependent monoclonal antibodies (MoAb) and flow cytometry in whole blood from 94 patients with OSAS, and from 31 age-matched controls. Thrombotic vascular diseases were surveyed as a background of alternative of platelet activation. RESULTS: The positive rate for activated platelets was significantly higher in patients with OSAS ( PAC1 52.6 +/- 22.9 %, CD62P 6.8 +/- 7.1%, mean +/- SD), as compared with healthy control subjects ( PAC1 16.7 +/- 8.6 %, CD62P 0.7 +/- 0.5 %, p < 0.001). The activation indexes were significantly reduced after 1 month with N-CPAP treatment as a whole (PAC1; from 52.6 +/- 22.9 to 44.2 +/- 22.4, p < 0.05, CD62P; from 6.8 +/- 7.1 to 5.3 +/- 5.5, p < 0.05). In nearly 60 % of patients, platelets activation remained high despite significant improvement of sleep apnea-episodes after N-CPAP. These patients had significantly higher incidence of previous myocardial infarction and/or cerebral infarction and abnormalities of head MRI and carotid sonograpy; indicating that the platelet activation appears to be induced by existing atheroma plaque and not by sympathetic activity in OSAS. CONCLUSION: In conclusion, patients with OSAS have increased percentages of activated platelets as assessed by flow cytometrical analysis of activation dependent surface markers, and were divided into two groups, one group with response to N-CPAP treatment in the reduction of platelet activation and the other without. One possible reason of no response to N-CPAP treatment in the reduction of platelet activation was suggested to be thrombotic diseases.

ACQUISITION OF COMPETENCE FOR CULMINATION DURING FINGER FORMATION IN DICTYOSTELIUM DISCOIDEUM.

Finger-like structures of the cellular slime mold, Dictyostelium discoideum, were disrupted with a fine needle and the resulting cell masses were allowed to develop. When complete fingers formed under overhead lighting were disrupted, the cell masses rapidly became transformed into fruiting bodies. Development of similar cell masses from fingers reared in the dark was affected by the lighting conditions after disruption: under overhead lighting the cell masses rapidly culminated; under unilateral lighting, they formed fingers again and then migrating slugs. In contrast, the cell masses from mounds with tips formed fingers regardless of the lighting conditions. It is concluded from these findings that the cells become competent for culmination during finger formation under overhead lighting.