Comparative study of antioxidants as quenchers or scavengers of reactive oxygen species based on quenching of MCLA-dependent chemiluminescence.

The quenching or scavenging effect of non-enzymatic antioxidants against reactive oxygen species (ROS) was studied by comparing the degree of suppression of chemiluminescence (CL) caused by the oxidation of MCLA (methoxylated Cypridina luciferin analogue) by ROS. MCLA-dependent CL caused by O2- was effectively quenched by ascorbic acid, beta-carotene, lycopene and astaxanthin, while it was enhanced by alpha-tocopherol. The CL by 1O2 was quenched effectively by beta-carotene, lycopene and astaxanthin, moderately by ascorbic acid, and slightly by alpha-tocopherol. beta-Carotene and alpha-tocopherol remarkably suppressed the CL when ROS was HO*. The present study revealed that MCLA-dependent CL assay provides a simple and rapid method for the evaluation of antioxidants as a quencher or scavenger against any kind of ROS.

Effect of Helicobacter pylori eradication on ongoing mutation of immunoglobulin genes in gastric MALT lymphoma.

Gastric low-grade mucosa-associated lymphoid tissue (low-grade MALT) lymphomas has been associated with Helicobacter pylori (H. pylori) infection. Although infiltrating T cells with specificity for H. pylori are known to stimulate the development of MALT lymphomas, the effect of H. pylori eradication on rearranged immunoglobulin heavy chain (IgH) genes of low-grade gastric MALT lymphomas is unclear. Gastric biopsies from five cases were investigated by cloning and sequence analysis of rearranged IgH genes before and after the treatment for H. pylori. In all cases, IgH genes were mutated from their germline counterpart. The frequency of intraclonal sequence heterogeneity before the eradication of H. pylori varied from 0.25 to 0.49%. Clones obtained from the tumours before the eradication of H. pylori in cases 1 and 2 showed a tendency to display a mutation pattern by positive antigen selection and their monoclonarity disappeared after the eradication. The frequency of intraclonal sequence heterogeneity of the clones obtained from cases 3, 4 and 5 (0.12% in case 3, 0.10% in 4 and 0.18% in 5) after the eradication of H. pylori was lower than that in tumours before the eradication (0.30% in case 3, 0.49% in 4 and not determined in 5). These findings suggest that low-grade gastric MALT lymphomas expand due to the persistent presence of H. pylori in vivo. The characteristic feature of tumour clones obtained from the tumours after the eradication of H. pylori is a very low intraclonal heterogeneity, which may potentially be independent of H. pylori.

[A new formula to calculate perfusion rate in advanced hemodilution and tepid cardiopulmonary bypass].

BACKGROUND: During cardiopulmonary bypass, perfusion flow rate is generally calculated only with the patient body surface. Recently, far advanced hemodilution during cardiopulmonary bypass and tepid bypass circulation are common. PURPOSE: We have arrived at an appropriate flow rate formula, in which factors like temperature, hemoglobin concentration, the target mixed venous oxygen saturation (SvO2), and the amount of oxygen consumption are included. Our formula was compared with the conventional one. MATERIAL AND METHOD: Seventy-four points of cardiopulmonary bypass data under total cardiopulmonary bypass in 33 patients were studied. Our formula's validity was re-evaluated. Then, the SvO2 values were predicted by applying the flow rate value as per conventional calculation in to our formula. RESULTS: The flow rate of our formula and the actual flow rate are well correlated (r = 0.9212). In the prediction of the SvO2 by the conventional method, 36.5% were calculated to have a SvO2 of less than 60%. Furthermore, with a hemoglobin concentration of 7 g/dl, 73.3% were calculated to have a SvO2 of less than 60%. With a body temperature of 34 degrees centigrade, 53.8% were calculated to have a SvO2 of less than 60%. On the other hand, to maintain SvO2 level at 70% by the conventional method, if the patient hemoglobin concentration was 10 g/dl, temperature should be maintained at 36 degrees centigrade, and when hemoglobin concentration is 7 g/dl, the temperature should be maintained at 33 degrees centigrade. CONCLUSION: In advanced hemodilution or tepid cardiopulmonary bypass, use of appropriate flow rate formula is recommended, which takes into account the indispensable factors such as hemoglobin levels, temperature, and the target SvO2.

A case of primary low grade mucosa associated lymphoid tissue (MALT) lymphoma of the oesophagus.

We report a very rare case of primary low grade mucosa associated lymphoid tissue (MALT) lymphoma of the oesophagus. An 83 year old woman was referred to our hospital in June 1999 for further examination and treatment of oesophageal tumour. Although a physical examination and laboratory data showed no significant abnormalities, endoscopic observation revealed two slightly elevated submucosal tumour-like lesions of the oesophagus. Tissue specimens were obtained by endoscopic mucosal resection of the oesophagus using a cap fitted panendoscope. The lesions were composed of diffuse small atypical lymphoid cells--that is, centrocyte-like cells--which were stained with CD20, L26, BCL-2, and kappa, but not with CD3, CD5, CD10, or cyclin D1. Monoclonality was detected by polymerase chain reaction analysis using the primer for CDR-3 of immunoglobulin H and diagnosed as low grade MALT lymphoma of the oesophagus. The tumours were considered to be completely resected and therefore additional treatment was not administered. The patient is alive and well 22 months after treatment and diagnosis.

Potential tumor-targeting peptide vector of histidylated oligolysine conjugated to a tumor-homing RGD motif.

We have developed a potential tumor-targeting peptide vector (cRGD-hK) that is intended to be systemically and repeatedly administered to patients with advanced solid tumors. The peptide vector of 36 l-amino acid residues, CRGDCF(K[H-]KKK)6, comprises a tumor-homing RGD motif, a DNA-binding oligolysine, and histidyl residues to facilitate the delivery into the cytosol. Using cytomegalovirus-driven luciferase expression plasmids as a reporter, we tested the transfection efficiency of cRGD-hK in hepatoma and pancreatic cancer cell lines. Transfection with the cRGD-hK/plasmid complexes (molar ratio 4000:1) was inhibited by 50 nM bafilomycin A1, an inhibitor of the vacuolar ATPase endosomal proton pump, or 10 microM cycloRGDfV, an integrin alphavbeta3 antagonist, indicating that the three elements of cRGD-hK could function as expected, at least in vitro. In nude mice bearing tumors created by subcutaneous inoculation, luciferase activity in the tumor tissues 48 hours after the injection of the cRGD-hK/plasmid complexes through the tail vein (20 microg plasmids per mouse) was significantly higher than that in the lung, kidney, and spleen, but only slightly higher than that in the liver. Although the latter difference was small, we propose a potential nonviral gene therapy for advanced solid tumors through use of the tumor-targeting peptide vector.

Hemorrhagic shock due to intrathoracic rupture of an osteosarcoma of the rib.

A 13-year-old girl presented with dyspnea and chest pain. Chest radiography showed a massive left pleural effusion. Computed tomography revealed a tumor of the fourth rib. A large bloody effusion was drained. Her anemia worsened (hemoglobin: 4.8 g/dl), and hemorrhagic shock ensued. An emergency thoracotomy was performed. Bleeding from the ruptured tumor was identified. The fourth rib, the tumor, and the adjacent tissues were resected. Histopathologic examination revealed a ruptured primary osteosarcoma of the rib with pleural dissemination.

[Surgical treatment of chronic pulmonary thromboembolism caused by a cavernous hemangioma at the lower limb].

A 48-years old man complained of dyspnea and was admitted to the hospital. Chest enhanced CT confirmed the presence of the thrombus in the pulmonary artery. Cardiac catheterization showed severe pulmonary hypertension (mean PAP 75 mmHg). ATIII level, protein C and S antigen were within normal range. Anticardiolipin antibody and lupus anti-coagulant determination were negative. He was diagnosed as chronic pulmonary thromboembolism, and underwent pulmonary thromboendarterectomy via median sternotomy under deep hypothermic intermittent circulatory arrest. At the same time IVC filter was inserted. The origin of the thrombus was not detected before operation, but after surgery, MR angiography of total body showed a cavernous hemangioma at left lower limb. We speculated this lesion was the origin of pulmonary embolism.

Prevention of surgical site infection by antibiotic spraying in the operative field during cardiac surgery.

OBJECTIVE: Despite the many procedures introduced to prevent surgical site infection during cardiothoracic surgery, serious infections still occur. We attempted to reduce surgical site infection by spraying antibiotic solution in the operative field--a procedure since introduced at 4 other Japanese institutions. METHODS: In the latter half of 1990, we began spraying an antibiotic solution of cefazolin (1g) and gentamicin (40 mg)/40 ml of saline placed in a 50 ml syringe and dispensed through an 18 G needle bent at 60 to 80 degrees to clean the wound during surgery. RESULT: No deep surgical site infections or deaths due to infection have occurred among the 502 patients undergoing cardiothoracic surgery under cardiopulmonary bypass at our hospital. This method was used in over 2,100 cases of similar procedures at 4 other institutions. There were 3 deaths due to severe surgical site infection (0.11%). At one institution treating over 1,000 cases a year, the incidence of death due to surgical site infection decreased significantly after this method was introduced. CONCLUSION: These preliminary experiences show that spraying antibiotic solution in the operative field reduces the risk of surgical site infection in cardiothoracic surgery.

Hepatocarcinogenesis inhibition by caffeine in ACI rats treated with 2-acetylaminofluorene.

The inhibitory effects of caffeine have been demonstrated on the development of various organs in animals. The purpose of the present study was to examine the inhibitory effect of caffeine on hepatocarcinogenesis and to determine the responsive dose of caffeine on hepatocarcinogenesis in young male ACI rats. Animals given a diet containing 2-acetylaminofluorene (2-AAF) for 12 weeks and then a basal diet and tap water containing caffeine for 18 weeks showed statistically significant decreases in the incidence, multiplicity (the number of hepatic tumors per rat) and histological grade compared with rats fed a diet containing carcinogen for 12 weeks followed by tap water alone. Dose-dependent inhibition of hepatocarcinogenesis by caffeine was also seen. The inhibitory effect of caffeine on hepatocarcinogenesis in rats was found when caffeine was administered during the initiation phase.

Soluble E-selectin induces monocyte chemotaxis through Src family tyrosine kinases.

Cellular adhesion molecules such as E-selectin function to recruit leukocytes into the inflammatory lesions of diseases such as rheumatoid arthritis (RA) and atherosclerosis. Monocytes are the key components of the cellular infiltrates present in these disorders. We hypothesized that soluble E-selectin (sE-selectin) might mediate the chemotaxis of monocytes. In this report, we show that sE-selectin induced normal human peripheral blood monocyte migration in the nanomolar range in a concentration-dependent manner. Neutralization studies using RA human joint synovial fluids and anti-E-selectin antibody showed a mean 31% reduction in RA synovial fluid-mediated monocyte chemotaxis (p < 0.05), indicating that sE-selectin is a major monocyte recruiter in RA. Next, we investigated the role of tyrosine phosphorylation pathways in sE-selectin-induced monocyte chemotaxis. Human peripheral blood monocytes stimulated with sE-selectin showed a time-dependent increase in the tyrosine phosphorylation of a broad range of cellular proteins, predominantly in the molecular size range of Src family kinases (50-60 kDa) and mitogen-activated protein kinases (MAPKs). Western blot analysis of Src family kinases showed a time-dependent increase in Src, Hck, and Lyn phosphorylation. The pretreatment of monocytes with the Src inhibitor AG1879: 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine (PP2) prior to stimulation with sE-selectin markedly inhibited Hck and Lyn phosphorylation, whereas the phosphorylation of Src was partially inhibited. In addition, the sE-selectin stimulation of monocytes resulted in the increased phosphorylation of extracellular signal-related kinase (ERK1/2) and p38 MAPK. The pretreatment of monocytes with PP2 showed 89 and 83% inhibition of ERK1/2 and p38 MAPK phosphorylation, respectively. sE-selectin also showed a time-dependent activation of Ras kinase. Furthermore, the pretreatment of monocytes with PP2 completely inhibited sE-selectin-mediated monocyte chemotaxis. Taken together, our data demonstrate a novel function for sE-selectin as a monocyte chemotactic agent and suggest that sE-selectin might be mediating its biological functions through the Src-MAPK pathway.

Partial left ventriculectomy in a 3-year-old boy with dilated cardiomyopathy.

A 3-year-old boy suffered severe heart failure 2 months after ventricular septal defect repair. The cardiothoracic ratio was 67% and the ejection fraction 13%. Echocardiography showed a dilated left ventricle and thin myocardium. After thorough study, we made a diagnosis of dilated cardiomyopathy. Because conventional therapy was unsuccessful, we conducted partial left ventriculectomy with Alfieri repair of the mitral valve. The postoperative cardiothoracic ratio was 57% at 1 year of follow-up and the ejection fraction 40%. The New York Heart Association functional class improved from IV to I. In conclusion, the role of partial left ventriculectomy is both as a bridge to transplantation and as a definitive repair in dilated cardiomyopathy during childhood.

[Whole-body oxygen consumption at different temperatures during cardiopulmonary bypass].

For evaluation of the adequacy of tissue perfusion during cardiopulmonary bypass (CPB), whole-body oxygen consumption rates at different body weights and temperatures are basic and important data. But few studies have analyzed the oxygen consumption estimated from real-time data during CPB in clinical situations. We analyzed retrospectively the oxygen consumption at different body weights (BW) during normothermia (VO2 37) and the oxygen consumption ratio at different body temperatures (BT; %VO2X; X = BT) from the CPB charts of 189 cases. From these charts, 877 data points for oxygen consumption during total CPB were calculated by Fick's law. By statistical analysis of these data, we derived the following formulae: VO2 37 (ml/kg/min) = 7.6481 x BW0.0679, R2 = 1.0 (BW < 8 kg) VO2 37 (ml/kg/min) = 32.394 x BW-0.625, R2 = 0.92 (BW > or = 8 kg) %VO2X (%) = 4 x 10(-5) x BT4.0777, R2 = 0.42. These formulae indicate that (1) the whole-body oxygen consumption during total CPB can be measured by subtracting the oxygen consumption associated with circulatory and respiratory processes from the oxygen consumption derived from the basal metabolism; and (2) although the change in oxygen consumption induced by hypothermia is variable depending on body weight, the rate of change shows no difference regardless of the patient's weight.

Reassessment of K-ras mutations at codon 12 by direct PCR and sequencing from tissue microdissection in human pancreatic adenocarcinomas.

K-ras mutations at codon 12 have been detected in almost all pancreatic adenocarcinomas by highly sensitive assays. We reassessed the K-ras mutation status by direct polymerase chain reaction (PCR) and sequencing from tissue microdissection without DNA extraction in 10 pancreatic adenocarcinomas, and also assessed the K-ras and DPC4 genes in nine pancreatic cancer cell lines. Eight pancreatic adenocarcinomas were found to harbor K-ras mutations at codon 12 of either GTT or GAT, five of which were inferred to harbor amplified mutant alleles. Mutations at the sites other than codon 12 were found in seven of 70 clones (seven of 9,380 bases) by the TA cloning analysis, suggesting that artifactual mutations at the first or second base of codon 12 before and during PCR could occur at a frequency of approximately 10(-3), enough for highly sensitive assays to detect. Two cell lines without K-ras mutations at codon 12 were found to have homozygous deletions at the DPC4 gene. Thus the K-ras mutation status was demonstrated to be correctly determined by just direct sequencing from tissue microdissection. All possible mutations or multiple mutations at K-ras codon 12 that have been reported in pancreatic adenocarcinomas might include artifacts or mutations without a selective advantage. In addition, we must be very cautious about contamination.

Co-occurrence of mucoepidermoid carcinoma and squamous cell carcinoma of the esophagus: report of a case.

A case of co-occurrence of a mucoepidermoid carcinoma (MEC) and a squamous cell carcinoma (SCC) in the esophagus is described. The present patient was a 61-year-old man who underwent a curative esophagectomy with a regional lymph node dissection for a MEC in the lower esophagus and a SCC near the esophagogastric junction. The two lesions were endoscopically and histologically divided by a normal esophageal mucosa. The MEC of the esophagus consisted of SCC cells and signet-ring cells, and a mucin product and carcinoembryonic antigen, which were found at high levels in the blood serum before surgery, were detected histochemically in the signet-ring cells. The follow-up survey of the patients with esophageal MEC previously reported in Japan showed that most of the patients died of either local recurrence or widespread metastasis after treatment; the overall 5-year survival rate was 24.4% in the total 25 cases, and 27.7% in the 22 resected cases. However, 6 patients who died of therapeutic complications were included among these patients; furthermore, the 5-year survival rate after surgery was 29.2% in the patients treated over the last decade (1989-1998). We expect that the clinical outcome of patients treated for esophageal MEC will therefore improve in the future.

Soluble vascular cell adhesion molecule 1 mediation of monocyte chemotaxis in rheumatoid arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is characterized by infiltration of leukocytes, including monocyte/ macrophages, into synovial tissue (ST), but factors mediating the ingress of these cells are poorly understood. Vascular cell adhesion molecule 1 (VCAM-1) plays an important role in adhesion of leukocytes to the vasculature. This study was undertaken to test the hypothesis that soluble VCAM-1 (sVCAM-1) might mediate chemotaxis of monocytes in RA. METHODS: Chemotaxis assays were performed using a modified Boyden chamber to determine the effects of sVCAM-1 on and the role of very late activation antigen 4 (VLA-4) in peripheral blood (PB) monocyte migration. Synovial fluids (SF) were immunodepleted of sVCAM-1 to identify a role for sVCAM-1 in RA. Immunohistochemistry and flow cytometry analyses were performed to show the expression of VLA-4 in ST, SF, and PB. Tyrosine phosphorylation was studied by Western blot analysis on PB monocyte lysates in the presence of signaling inhibitors. RESULTS: Soluble VCAM-1 induced monocyte migration in the nM range, in a concentration-dependent manner. Anti-VLA-4 significantly inhibited sVCAM-1-induced monocyte migration, suggesting that sVCAM-1 acts in part via a VLA-4-dependent mechanism. In RA SF, incubation with anti-VCAM-1 resulted in a reduction in the ability to induce monocyte migration (mean 28%). VLA-4 immunolocalized to RA ST, SF, or PB, monocytes, macrophages, and lymphocytes. Soluble VCAM-1 stimulated tyrosine phosphorylation in monocytes, and pertussis toxin, chelerythrine chloride, and staurosporine significantly reduced sVCAM-1-mediated monocyte chemotaxis, suggesting that signaling pathways via G proteins and protein kinase C are required for sVCAM-1-mediated monocyte migration. CONCLUSION: These results demonstrate a novel function for sVCAM-1 as a monocyte chemotactic agent in RA and suggest a new potential target for modulating monocyte ingress into inflamed RA ST.

API2-MALT1 chimeric transcripts involved in mucosa-associated lymphoid tissue type lymphoma predict heterogeneous products.

Recent progress in molecular analysis of low-grade B cell lymphoma has revealed that API2 at 11q21 and a novel gene, MALT1 at 18q21, are involved in t(11;18)(q21;q21), a characteristic chromosome aberration for mucosa-associated lymphoid tissue (MALT) type lymphoma. We describe here the establishment of a reverse transcription-polymerase chain reaction (RT-PCR) assay that we used to analyze 22 cases of MALT lymphoma. All five cases that were shown to possess t(11;18)(q21;q21) showed the specific amplification of API2-MALT1 chimeric transcripts. Of the remaining 17 cases for which cytogenetic data were not available, three cases demonstrated the presence of fusion transcripts, indicating that a significant percentage of MALT lymphoma cases of the present series appeared to possess t(11;18). A single fragment was observed in each of these cases, but the size varied from case to case. Sequencing analysis revealed that there are two breakpoints in API2 and three in MALT1, and that all of the fusion transcripts are in-frame. On the basis of these results, four kinds of chimeric proteins can be predicted for the present series. Thus, the RT-PCR assay used here should serve as an effective molecular tool for understanding molecular pathogenesis and the clinical significance of API2-MALT1 for MALT lymphomas.

Mechanochemical solid-state polymerization (X): the influence of copolymer structure in copolymeric prodrugs on the nature of drug release.

From the standpoint of the mechanism of mechanochemical polymerization, two kinds of copolymeric prodrug, whose monomer sequence distribution (MSD) is different from each other, can be prepared by this polymerization under appropriate operational conditions: one is a random copolymer abundant in the longer block consisting of the same repeating units (multi-block copolymer), and the other is a block copolymer. To confirm the difference of MSD, the 13C-NMR spectra of poly(acrylamide-co-sodium acrylate) prepared by mechanochemical polymerization were measured and compared with the spectrum of that synthesized by a conventional radical-initiated solution polymerization, which produces the random copolymer normally. The results show that MSD in copolymers depends on the polymerization method (operational condition). We prepared three kinds of copolymeric prodrug consisting of acrylamide and vinyl monomer of 5-fluorouracil, whose MSD is different from one another. These copolymeric prodrugs had almost the same number average molecular weight, particle diameter and composition, and differed only in MSD. We compared the rate of drug release of these copolymeric prodrugs. The rate of drug release was the highest with the random copolymer, followed by the mechanochemically produced multi-block copolymer and the block copolymer. This result suggests that the rate of drug release depends on MSD of copolymeric prodrugs. These results are useful as they give a fundamental insight into the synthesis of copolymeric prodrugs having the desired rate of drug release.

Selectivity and sensitivity in the measurement of reactive oxygen species (ROS) using chemiluminescent microspheres prepared by the binding of acridinium ester or ABEI to polymer microspheres.

Two kinds of chemiluminescent microspheres were prepared as tools for measuring reactive oxygen species (ROS) released into phagosomes in phagocytizing cells, by chemically binding acridinium ester or ABEI (isoluminol derivative) to polymer microspheres, and were examined from the viewpoint of specificity and sensitivity to ROS. Acridinium ester-bound microspheres (AE-ms) were found to be a sensitive probe to superoxide anion and hydrogen peroxide under a neutral condition (pH 7.2). AE-ms emitted strong chemiluminescence (CL) by hypoxanthine (HPX)/xanthine oxidase (XOD) or hydrogen peroxide. The CL by HPX/XOD was initially inhibited by superoxide dismutase. At pH 5.6, the CL intensity from AE-ms in the presence of HPX/XOD was reduced to about one-eighth of that at pH 7.2. ABEI-bound microspheres (ABEI-ms) were found to be a selective probe for singlet oxygen although not highly sensitive. ABEI-ms emitted CL of moderate intensity with hydrogen peroxide/myeloperoxidase (MPO), but not with hydrogen peroxide alone or with hypochlorite/MPO at pH 5.6. The CL from ABEI-ms with hydrogen peroxide/MPO was completely inhibited by azide. ABEI-ms did not emit CL in the presence of HPX/XOD or by potassium superoxide at pH 5.6. The result of supplemental experiments using dissolved chemiluminescent probes and non-enzymatically generated ROS supported the above-described selectivity and sensitivity of chemiluminescent microspheres.

[Surgical treatment of 2 cases of irradiation induced constrictive pericarditis].

A 72-years-old man underwent radiation therapy (62 Gy) for esophageal carcinoma. Twelve months later, symptoms of heart failure such as syncope, cough and hepatomegaly manifested. On catheter study, a dip and plateau pattern of right ventricular pressure curve was evident. Pericardiectomy without extracorporeal circulation was performed. Operative findings and pathological results were compatible with radiation-induced constrictive pericarditis. He recovered from the heart failure, and has been doing well 3 months after the surgery. A 54-years-old man underwent thymectomy for malignant thymoma. He underwent a radiation therapy (52 Gy) postoperatively. After 12 months from the irradiation, syncope and dyspnea manifested. On catheter study, a dip and plateau pattern of right ventricular pressure curve was observed. Pericardiectomy with extracorporeal circulation was performed. He recovered from the heart failure after pericardiectomy, however he died of radiation-induced pneumonitis 6 months later.