Assessment of platelet-derived thrombogenicity with the total thrombus-formation analysis system in coronary artery disease patients receiving antiplatelet therapy.

BACKGROUND: Accurate evaluation of thrombogenicity helps to prevent thrombosis and excessive bleeding. The total thrombus-formation analysis system (T-TAS) was developed for quantitative analysis of platelet thrombus formation by the use of microchips with thrombogenic surfaces (collagen, platelet chip [PL-chip]; collagen plus tissue factor, atherome chip [AR-chip]). We examined the utility of the T-TAS in the assessment of the efficacy of antiplatelet therapy in patients with coronary artery disease (CAD). METHODS AND RESULTS: In this cross-sectional study, 372 consecutive patients admitted to the cardiovascular department were divided into three groups: patients not receiving any antiplatelet therapy (control, n = 56), patients receiving aspirin only (n = 69), and patients receiving aspirin and clopidogrel (n = 149). Blood samples were used for the T-TAS to measure the platelet thrombus-formation area under the curve (AUC) at various shear rates (1500 s(-1) [PL18 -AUC10 ] and 2000 s(-1) [PL24 -AUC10 ] for the PL-chip; 300 s(-1) [AR10 -AUC30 ] for the AR-chip). The on-clopidogrel platelet aggregation was measured by the use of P2Y12 reaction units (PRUs) with the VerifyNow system. The mean PL24 -AUC10 levels were 358 ± 111 (± standard deviation) (95% confidence interval [CI] 328.9-387.1) in the control group, 256 ± 108 (95% CI 230.5-281.5) in the aspirin group, and 113 ± 91 (95% CI 98.4-127.6) in the aspirin/clopidogrel group. In the aspirin/clopidogrel group, the PL24 -AUC10 was higher in poor metabolizers (PMs) with cytochrome P450 2C19(CYP2C19) polymorphisms (152 ± 112, 95% CI 103.4-200.6) than in the non-PM group (87 ± 74, 95% CI 73.8-100.2). CONCLUSIONS: Our findings suggest that the PL24 -AUC10 level measured by the T-TAS is a potentially suitable index for the assessment of antiplatelet therapy in CAD patients.

Class A scavenger receptor (CD204) attenuates hyperoxia-induced lung injury by reducing oxidative stress.

To clarify the role of macrophage class A scavenger receptors (SR-A, CD204) in oxidative lung injury, we examined lung tissue of SR-A deficient (SR-A(-/-)) and wild-type (SR-A(+/+)) mice in response to hyperoxic treatment. Protein levels of bronchoalveolar lavage fluid (BALF) and pulmonary oedema (wet : dry weight ratios) were higher in SR-A(-/-) mice than those in SR-A(+/+) mice. Cumulative survival was significantly decreased in SR-A(-/-) mice. However, there were no differences in BALF macrophage and neutrophil count between the two groups. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that messenger RNA (mRNA) levels of the inducible nitric oxide synthase (iNOS) were increased during hyperoxic injury, and this increase was more prominent in SR-A(-/-) mice. Expression levels of iNOS in alveolar macrophages after hyperoxia in vivo and in vitro were higher in SR-A(-/-) macrophages compared with SR-A(+/+) macrophages. Immunohistochemistry using anti-nitrotyrosine antibodies revealed distinctive oxidative stress in the injured lung in both groups, but it was more remarkable in the SR-A(-/-) mice. After hyperoxic treatment, pulmonary mRNA levels of tumour necrosis factor-alpha(TNF-alpha) were elevated more rapidly in SR-A(-/-) mice than in SR-A(+/+) mice. Together these results suggest that SR-A expression attenuates hyperoxia-induced lung injury by reducing macrophage activation.

Cardiac amyloidosis presenting as microvascular angina--a case report.

Cardiac amyloidosis usually presents with heart failure, but rarely leads to coronary insufficiency. The authors report a case of a 69-year-old Japanese woman with cardiac amyloidosis presenting as microvascular angina. She had exertional angina with positive exercise test and normal coronary angiograms. However, heart failure developed, and she died 3 years after symptom onset. On autopsy, coronary arteries were patent. In contrast to that of the epicardial coronary arteries, histologic examination of the heart revealed severe obstructive alterations of the intramural coronary arteries with amyloid. Furthermore, amyloid was present mainly in the endocardium and the intramural coronary arteries, but there was little present in the myocardium. This case was a rare AL amyloidosis. There have been only 4 reported cases of cardiac amyloidosis that presented with exertional angina, a positive exercise test, and normal coronary angiograms and histologic examination.

Various AGC repeat numbers in the coding region of the human transcription factor gene E2F-4.

The E2F family of transcription factors regulates the expression of genes required for DNA synthesis and cell cycle control. The AGC triplet repeat in the coding region of the E2F-4 gene, a member of the family, has been reported to be mutated in colorectal cancers with a microsatellite instability (MSI) phenotype. We found a wider range variation of the repeat number in DNAs from tumors, the corresponding normal mucosa, and healthy individuals. A total of 5 repeat variants, ranging from 8 to 17 AGC repeats, was detected in 6 (9.7%) of the 62 healthy individuals and 8 (8.9%) of the 90 normal DNAs of the patients. The wild-type 13 repeat was present in all of these individuals. The variation of the AGC repeat number may be a polymorphism. Further, loss of heterozygosity (LOH) at the E2F-4 locus in the tumor tissues of 2 (25%) of the 8 informative cases was detected. The variation may be a useful marker for detection of LOH in primary tumors.

[Cooperative study of intraarterial preventive chemotherapy after resection of hepatic metastasis from colorectal cancer].

This is a compilation of the results of preventive intraarterial infusion following resection of hepatic metastasis from colorectal cancer at four surgical centers. The cases studied included two groups: A) 76 patients who underwent normal liver resection only, and B) 78 patients who underwent resection with adjuvant chemotherapy. Methods included: 1) WHF, 50 cases; 2) other methods using 5-FU, 18 cases; and 3) intraarterial infusions other than 5-FU, 10 (2 cases, outcome unknown). Survival rates for groups A and B for 1 and 5 years were 71.2, 18.9% and 91.5, 56.2%, respectively, with the rates for the intraarterial infusion group showing far better results. The 1- and 5-year survival rates in terms of infusion methods were: 1) 90.7% and 64.6%; 2) 94.4% and 39.3%; and 3) 90% and 60%, respectively, showing no remarkable differences between methods. Total doses of 5-FU were (a) less than 5 g, 7 patients (b) 5-15 g, 16 patients (c) 15-30 g, 22 patients (d) greater than 30 g, 23 patients. A comparison of 1- and 5-year survival rates shows (a) 85.7% and 17.1%; (b) 66.5% and 44.3%; (c) 100% and 62.7%; (d) 100% and 66.5%, respectively, with doses (c) and (d) showing markedly better results than the (a) dosage. From this we conclude that the group undergoing intraarterial hepatic infusion had a markedly improved prognosis compared to the group not undergoing any type of adjuvant therapy. Also, groups receiving a dosage of 15 g or greater of 5-FU showed prolonged survival rates.

Highly scattering intralipid-10% assisted lasing from microdroplets with acridine orange dye.

One order or greater of magnitude enhancement of lasing emission is confirmed experimentally from liquid microdroplets of Acridine Orange dye mixed with the fat emulsion Intralipid-10% suspension as highly scattering media, compared with pure dye-doped droplets. This novel method that makes use of a high-gain laser dye soft scatter microsystem allows for a wide range of lasing of microdropslets. Originally without lasing pure dye droplets enabled one to lase with a well-defined threshold and an appreciably increased emission intensity in suitable conditions. Spectral characteristics and emission peak intensities from these microdroplets are measured quantitatively as a function of volume content of Intralipid-10% solutions.

Undectable expression of hMLH1 protein in sporadic colorectal cancer with replication error phenotype.

PURPOSE: Four DNA mismatch repair genes have been identified as being susceptible genes for hereditary nonpolyposis colorectal cancer. Deficiency of one of the mismatch repair genes causes the replication error phenotype in more than 80 percent of patients with hereditary nonpolyposis colorectal cancer and in 10 to 30 percent of patients with sporadic colorectal cancer. To determine which mismatch repair gene is lacking the function in patients with replication error-positive colorectal cancer, several approaches have been used at the nucleic acid and protein levels. We studied replication error in 40 samples of randomly selected colorectal cancers and expression of hMSH2 and hMLH1 proteins analyzed by immunoblot in the tumor and normal tissues of the replication error-positive and replication error-negative samples. MATERIALS AND METHODS: Frozen tumor and normal tissues were obtained from 40 Japanese patients who had colorectal cancer. According to the Amsterdam criteria, those patients were classified as having 39 sporadic and 1 unknown colorectal cancers. Genomic DNA was extracted from tumor and normal tissues for determining replication error with eight microsatellite markers. Expression of hMSH2 and hMLH1 proteins in cell lysates of tumor and normal tissues of 16 patients was analyzed by immunoblot. RESULTS: The replication error phenotype was found in 6 (15 percent) of the 39 sporadic cases. hMLH1 protein was not detected in two of the six replication error-positive tumor tissues and not in the normal tissues, indicating that the tumor cells of the two patients had severe mutations in both alleles of the hMLH1 gene. Another four replication error-positive and ten replication error-negative tumors and normal tissues expressed hMLH1 protein. hMSH2 protein was detected in all samples. CONCLUSION: hMLH1 protein was undetectable in the two tumor tissues of the six replication error-positive samples of sporadic colorectal cancer. The detection procedure used here may have potential use for determining a dysfunctional mismatch repair gene product.

Microspectrophotometry of nitric oxide-dependent changes in hemoglobin in single red blood cells incubated with stimulated macrophages.

A highly sensitive microspectrophotometer was developed to measure spectral changes of oxyhemoglobin (oxy Hb) in single red blood cells (RBC) incubated with stimulated macrophages as a model of nitric oxide (NO) dependent cytotoxicity. Our microspectrophotometer, using a modified acousto-optic tunable filter (AOTF) and a 2-dimensional CCD array, allows fast spectrophotometric data acquisition. Human RBC treated with various concentrations of NO showed spectral changes due to the conversion of oxy Hb to methemoglobin (met Hb), in which the change in absorption differences at alpha (557 590 nm) and beta (542-525 nm) bands showed a linear relationship with the concentration of NO up to 100 microM. In contrast to highly diffusible NO, nitrite ions (NO2-) seem to enter RBC very slowly, resulting in negligible formation of met Hb in the presence of 5 mM glucose even during a prolonged incubation period. RBC were incubated with murine macrophages with and without lipopolysaccharide (LPS) in the presence of glucose for 24 and 40 h and subjected to the microspectrophotometric assay. The RBC incubated with LPS-stimulated macrophages showed significant changes in the spectrum due to NO-dependent conversion of oxy Hb to met Hb, which corresponded to the spectral changes of RBC treated with a several times higher concentration of NO than that in the culture medium. The trapping efficiency was calculated from the amounts of the NO released from macrophages and of the met Hb formed in the RBC, which gave a high efficiency (43%). The results suggest that RBC trap NO directly by cell cell interaction with macrophages. This spectrophotometric system is available for use with just a few drops of samples to study NO-specific cytotoxicity as a model of RBC without the use of any chemical reagent, in parallel with microscopic observations on changes of the cellular morphology under physiological conditions, such as membrane damage leading to hemolysis, adherence, and phagocytosis.

[An immunohistochemical study of the relationship between p53 expression and the DNA ploidy pattern using image cytometry in colorectal carcinoma].

We studied p53 expression in 46 colorectal carcinomas by immunohistochemistry. We examined the relationship between p53 expression and the pathological findings, the prognosis and tumor proliferative activity by Ki-67 staining or DNA ploidy pattern using an image cytometer. Expression of p53 was observed in 41.3% of carcinomas. No correlation was found between p53 expression and the depth of invasion, lymph node metastasis or Dukes' stage. No difference in survival was found between p53 positive and negative patients after curative surgery. We also studied the DNA ploidy pattern using an image cytometer in 28 carcinomas. Aneuploidy was found more frequently in the p53 positive areas than in the negative ones, but there was no significant difference. The DNA index showed no difference between the p53 positive and negative areas. We examined proliferating activity using Ki-67. There was no difference in the Ki-67 labeling index between p53 positive and negative areas. In the present study, there was no correlation between p53 expression and pathological findings, prognosis, tumor proliferative activity using Ki-67 staining or DNA ploidy pattern. Thus, p53 expression was suggested to have little relationship to grade of malignancy.

[Study on relationship between intratumoral DNA heterogeneity and clinicopathological findings in colorectal cancer].

We studied the relationship between intratumoral DNA heterogeneity and clinicopathological findings in 85 colorectal cancers. DNA ploidy was analyzed in 5 specimens from different sites of each tumor. When the difference in the DNA index (D.I) between several peaks in the same tumor was more than 0.1, the tumor was considered to consist of heterogeneous subpopulations with different DNA clones. DNA heterogeneity was found in 34 cases (40.0%). There was no relationship between DNA heterogeneity and depth of tumor invasion, lymph node metastasis or stage. The incidence of heterogeneity was significantly higher in the tumor with lower differentiation, liver metastasis or vessel invasion.

[Studies on the antigenicity of MY-5116].

Antigenicity of MY-5116 was studied in the experimental animals and the following results were obtained. MY-5116 was shown to be non-immunogenic in rabbits, guinea pigs and mice when given alone as immunogen. Protein conjugate of MY-5116 induced antibody of hapten specific antibody when using guinea pigs and mice as immunizing species. However, MY-5116 was shown to be not capable of reaction with anti MY-5116-OVA antibodies. Guinea pigs had no skin contact sensitivity against MY-5116 examined by means of maximization test. In conclusion, MY-5116 lacks immunogenicity and eliciting antigenicity in this experimental conditions and this suggests that drug allergic response would either not occur or be minimal, if any, when MY-5116 is administered clinically.