Elevation of matrix metalloproteinases and interleukin-6 in the culprit coronary artery of myocardial infarction.

BACKGROUND: Interleukin-6 (IL-6) and metalloproteinases (MMPs) are involved in the instability of vulnerable plaque associated with the induction of acute myocardial infarction (AMI). We examined the regional changes of cytokines, MMPs and adhesion molecules in patients with AMI to elucidate how these factors are involved in the onset of AMI. MATERIALS AND METHODS: One hundred and twenty-two patients with AMI were included. Blood was aspirated from the culprit coronary artery with a thrombectomy catheter, and was also sampled from peripheral veins during the coronary intervention. Control samples were obtained from the peripheral blood of age-matched patients. RESULTS: The serum levels of IL-6 (P < 0.05), tumour necrosis factor-alpha (P < 0.005), MMP-1 (P < 0.001), MMP-13 (P < 0.001), soluble intercellular adhesion molecule-1 (P < 0.005), and soluble vascular cellular adhesion molecule-1 (P < 0.05) in peripheral blood were significantly higher in the AMI group than in the controls. Aspirated serum contained significantly higher levels of IL-6 (P < 0.001), MMP-1 (P < 0.001), and MMP-13 (P < 0.05) compared to the peripheral blood of AMI. Serum IL-6 levels were significantly higher in the aspirated than in the peripheral blood in the patients hospitalized within 6 h and 6-12 h, but were similar in the aspirated and peripheral blood of the patients hospitalized 12-24 h after the onset of AMI. There were no differences between the aspirated serum and peripheral blood in the levels of interleukin-1beta and MMP-2. CONCLUSIONS: The levels of MMP-1, MMP-13 and IL-6 were higher in the culprit coronary artery than in the peripheral blood. These factors appear to be involved in the early stage of AMI.

Novel factor Xa and plasma kallikrein inhibitory-activities of the second Kunitz-type inhibitory domain of urinary trypsin inhibitor.

Urinary trypsin inhibitor is a glycoprotein with a structure in which two Kunitz-type inhibitory domains are linked in a row. We isolated two genes encoding the 70 amino acid sequence from the 78th amino acid (Thr) to the C-terminal and the 68 amino acid sequence from the 80th (Ala) to the C-terminal of human urinary trypsin inhibitor, both which correspond to the second Kunitz-type inhibitory domain, and then constructed expression plasmids by ligating it to the E. coli alkaline phosphatase signal peptide gene. These plasmids under the control of the tryptophan promoter expressed the second domain in E. coli strain JE5505 which lacks the membrane lipoprotein. The recombinant second domain purified from the culture supernatant of the transformant inhibited trypsin, plasmin, leukocyte elastase and chymotrypsin which are known to be inhibited by urinary trypsin inhibitor. In addition it inhibited blood coagulation factor Xa and plasma kallikrein in a concentration dependent and competitive manner, and significantly prolonged the plasma-based activated partial thromboplastin time (APTT). The truncated natural counterpart obtained by a limited degradation of human urinary trypsin inhibitor also revealed the identical inhibitory activities.

High sensitivity of human melanoma cell lines to the growth inhibitory activity of mycoplasmal arginine deiminase in vitro.

Arginine deiminase (AD) is a potent growth inhibitor for some but not all tumour cell lines in vitro. As AD catalyses the direct conversion of L-arginine to L-citrulline, the AD-sensitivity of various tumour cells might be attributed to the levels of urea cycle enzymes involved in L-arginine biosynthesis. This study demonstrated that human melanoma cells were highly sensitive to the growth inhibitory activity of AD. Five melanoma cell lines tested also exhibited reduced argininosuccinate synthetase (ASS) gene expression--being almost absent in four cell lines and at low level in one cell line. This resulted in an inability of the cells to utilize L-citrulline for growth. Based on the tissue-specific regulation of ASS gene, the feature of melanomas suggests that it might be possible to develop agents with strong AD activity for chemotherapeutic use for human melanomas in vivo.

Analysis of vocal fold vibration by x-ray stroboscopy with multiple markers.

OBJECTIVE: To derive a more precise description of vocal fold vibration, experimental phonation of excised canine larynxes was studied. STUDY DESIGN AND SETTING: Multiple X-ray-positive markers were inserted, and their vibratory movement was observed with x-ray stroboscopy with change of pitch and intensity. A histologic study was also carried out. RESULT: Regular waves were observed just above the lowest point of the lamina propria of the mucous membrane, which shifted upward at high pitch, but downward in high intensity. CONCLUSIONS: The starting point of the mucosal wave was confirmed on the lower surface of the vocal fold, histologically just above the lowest point of the lamina propria of the mucous membrane and shifted upward at high pitch, but downward in high intensity. SIGNIFICANCE: This is the first study investigating the starting point of mucosal wave in vocal fold vibration in a frontal plane using x-ray stroboscopy, providing the evidence for the body-cover theory.

Primary small cell carcinoma of the larynx.

Small cell carcinoma is a rare tumor of the larynx. We present such a case in a 78-year-old female. The histopathological diagnosis at the time of laryngomicroscopic biopsy was squamous cell carcinoma, upon which basis we initially chose surgical treatment. The surgical specimen, however, revealed small cell carcinoma. Despite the administration of radiotherapy and chemotherapy, the patient died 9 months after initial presentation. We believe that this case illustrates the need for a sufficiently large biopsy specimen in order to arrive at the correct histopathological diagnosis when small cell carcinoma of the larynx is present, and that immunohistochemistry and electron microscopy should be performed to aid the diagnosis.

[Fundamental evaluation of the subrenal capsule assay as an in vivo chemosensitivity test].

Histological analyses of the subrenal capsule assay (SRCA) carried out with modification of Bogden's original method revealed that the major portion of cancer tissue implanted were almost replaced by the host immune cells and reactive tissue by day 6. For the purpose to find a more improved condition, the following experiments were performed using human cancer lines xenografted in nude mice. No prominent differences in the host reaction to xenograft were found between 3 kinds of immunocompetent mice. Subrenal space of the athymic nude mice allowed cancer tissue to grow well until 15 days after inoculation, and then chemosensitivity test to judge the growth inhibition rates. The similar condition as in nude mice was achieved in CDF1 mice by daily injection of 60 mg/kg of cyclosporin A.

[Subrenal capsule (SRC) assay as a chemosensitivity test (IV)--Evaluation of the SRC assay method as a system for growing implanted tumor-xenografts].

Using tumor specimens of human cancer serially transplanted in nude mice, we examined fundamentally the subrenal capsule assay method in order to improve the subrenal space as an area where implanted tumor-xenografts persistently grow. Host reaction in immunocompetent mice treated with cyclosporin A (CsA) (60 mg/kg subcutaneously, daily) was suppressed almost completely, and tumor-xenografts persistently grew similarly to those implanted under the renal capsule of BALB/c-nu/nu mice. CsA treatment, 30 mg/kg given subcutaneously; daily, or 60 mg/kg subcutaneously; every other day, could not suppress the host reaction. Bredinin treatment, 200 mg/kg subcutaneously; every other day, or 100 mg/kg subcutaneously; daily, could not suppress the host reaction also, and tumor-xenografts implanted under the renal capsule were rejected.

[Subrenal capsule assay as a chemosensitivity test (V)--Experimental chemotherapy of cyclosporin A-treated mice and nude mice].

We studied fundamentally subrenal capsule assay, using human tumor specimens (breast, gastric and colon cancers) serially transplanted in nude mice. Mitomycin C, 5-fluorouracil, adriamycin, cisplatinum or cyclophosphamide was injected into immunocompetent CDF1 mice treated with cyclosporin A (CsA) after tumor implantation. On day 6 and day 9 after inoculation, the chemosensitivity profiles of tumor xenografts were similar in CsA-treated mice and nude mice, macroscopically and microscopically. It is suggested that CsA-treated mice were an appropriate model as hosts for chemosensitivity testing. When we examine chemosensitivity effect macroscopically, a method of comparing chemotherapy groups with control groups; i.e. inhibition rate by measurement of tumor volume, was induced, in addition to the tumor size measurement. High toxicity due to cancer chemotherapeutic agents was found in CsA-treated mice, so that careful examination on treatment schedules with CsA and chemotherapeutic agents will be required.

[Experimental studies on subrenal capsule assay using cyclosporin A treated mice--the optimal treatment schedules of CsA and anticancer agents (mitomycin C and 5-fluorouracil)].

We studied fundamentally subrenal capsule assay, using human tumor specimens (breast, gastric and colon cancers) serially transplanted in nude mice. When cancer anticancer agents such as mitomycin C (MMC) and 5-fluorouracil (5-FU) were injected into immunocompetent mice treated with various dosages of cyclosporin A (CsA) after tumor implantation, optimal schedule of each drug was examined on the points of effects and toxicity against host mice. The following results were obtained. Control groups were set up as immunocompetent mice which treated daily with 60 mg/kg CsA from day 1 after tumor implantation. Optimal treatment schedule was judged as MMC 3 mg/kg i.v. injection on day 1 following by daily 60 mg/kg CsA treatment, and 5-FU was injected 25 mg/kg subcutaneous injection every day from day 1 without CsA treatment, each schedule showed an appropriate anti-tumor activity profiles against implanted tumor xenografts, and had less toxicity to the hosts.

[Subrenal capsule assay for chemosensitivity test (II)--Sequential changes in the implanted tumor tissue and histological findings].

We carried out fundamental subrenal capsule assay methodology, using tumor specimens of human cancer xenografts (breast cancer and colon cancer) serially transplanted into nude mice. With regard to sequential changes in the tumor grafts implanted under the renal space of immunocompetent mice, tumor size was largest macroscopically around day 6 after inoculation, and later involuted gradually. Histological findings showed that implanted tumor tissues were preserved to a moderate extent until day 4 after inoculation, but leukocyte infiltration by host reaction had begun by day 4, and tumor tissues were almost replaced by host reactive tissues on day 6. Labeling index scoring did not indicate growth of implanted tumor cells. We found that macroscopic tumor size was largest around day 6 because we measured tumor size with involved leukocyte infiltration, and the macroscopic tumor did not represent the true extent of the tumor tissue.

[Application to chemosensitivity tests of the human tumor-nude mouse system and subrenal capsule assay system].

Since 1974, approximately 200 fresh cancer tissues obtained from various types of cancer patients were inoculated into athymic BALB/c nude mice, of which 30 percent were taken and grown in the subcutaneous space of mice. Among them 15 lines of gastrointestinal and breast cancer xenografts were selected for experimental single agent chemotherapy. The response rates of 14 drugs examined in this xenograft system were compared with the cumulative clinical response rate of each drug in the same type of cancer. Drugs which were clinically effective against one type of tumor were found to be also effective against the corresponding xenograft in nude mice. Thus the human cancer-nude mouse system was considered useful as a predictive secondary screening method for new drugs. This evidence suggested to us the feasibility of utilizing the system as a chemosensitivity test for determining the drugs effective for an individual human malignancy. In our present study, the responses to 15 experimental chemotherapies with single agent or drug combination of 11 lines of cancer xenografts in nude mice were directly compared with the clinical response in each donor patient to the corresponding chemotherapy. Good correlation was obtained between these respective results, the overall predictive accuracy of the experimental results being 93%. Therefore, if the rate of transplantability to nude mice were to be improved, this nude mouse system would become a promising tool for the individual chemosensitivity test. The subrenal capsule (SRC) assay recently introduced by Bogden and his colleagues has excited much attention among Japanese clinical oncologists. In our study, cancer tissues implanted under the renal capsule 6 days after inoculation, did not show marked proliferation and a high percentage of implants was almost replaced by host reactive tissue. It therefore seems necessary to solve some fundamental problems before we can apply this assay method to clinical chemosensitivity trial.

[Subrenal capsule assay for chemosensitivity testing].

The subrenal capsule (SRC) assay for cancer chemotherapy was tested according to Bogden's methodology. Of 37 patients providing tumor tissue for assay, 29 cases were considered suitable for evaluable assays. Fourteen patients had clinically evaluable diseases and 10 cases were evaluable for SRC assays. Correspondence between sensitive assay and clinical sensitivity was seen in 2 cases, and that between resistant assay and clinical resistance was seen in 4 cases. Discordance between sensitive assay and clinical resistance was seen in 4 cases. In histological studies, cancer tissues implanted in the subrenal space in immunocompetent mice did not show marked proliferation and were replaced by prominent leukocyte infiltration and fibrosis on day 6 after inoculation. The degree of leukocyte infiltration in the xenografts in the mice administered some anti-cancer drugs was slight in comparison with that in untreated control mice, which showed a remarkable trend in xenografts treated with 5-fluorouracil and cyclophosphamide, respectively. Our study suggests that there are many problems involved in the SRC assay methodology of Bogden, and that careful examination of this aspect will be required.

[Subrenal capsule assay as a chemosensitivity test (III)--Comparison of host reaction, experimental chemotherapy and use of nude mice].

We studied fundamentally subrenal capsule assay, using human tumor specimens (gastric, breast and pancreas cancers) serially transplanted in nude mice. Any prominent difference of host reaction was not found between the host of BALB/c-nu/+, BALB/c-+/+ and CDF1 mice. Using immunocompetent BALB/c-nu/+ mice, experimental chemotherapy with mitomycin C (MMC) and 5-fluorouracil (5-FU) was carried out. On day 6, macroscopic and histological findings corresponded relatively well with 5-FU effect but not with MMC. Using BALB/c-nu/nu mice, we tried 15-day SRC assay. When the sensitivity of anti-cancer drugs was compared between early and intermediate phase after inoculation, no obvious difference was found macroscopically and histologically. BALB/c-nu/nu mouse will be useful as a host of SRC assay, and could be applicable to clinical fresh cases.

[Evaluation of predictability of in vitro SDI assay in comparison with in vivo nude mouse assay].

Twenty lines of human gastro intestinal and breast cancer xenografts, in which chemosensitivity spectra by the in vivo nude mouse assay had been clarified. were subjected to the in vitro SDI (succinate dehydrogenase inhibition) assay using MTT dye to assess the accuracy of this drug sensitivity test against 4 drugs i.e., mitomycin C (MMC), adriamycin (ADM) 5 fluorouracil (5-FU), and cisplatin (CDDP). After 3 days incubation, the suspension of every tumor cells including small fragments showed a marked decrease of SD activity even when no anticancer drug was added to the assay medium. Among these 4 drugs evaluated MMC exhibited a statistically significant correlation between chemosensitivity values of the in vitro SDI assay and those of the nude mouse assay. However, the other 3 drugs demonstrated no correlation between the values of these two methods. Since the primary cultured fibroblasts revealed, in general, lower sensitivity to these drugs, contamination of fibroblast may decrease the SDI values when materials from solid tumors with rich stroma such as a type of stomach cancer were subjected. It is considered that the prediction of chemosensitivity to every drug will be impossible by a in vitro SDI assay.

[Accordance of the chemosensitivity between clinical specimens and their xenografts in nude mice by SDI test and the value of in vivo chemosensitivity test using nude mice].

We assessed the sensitivity to anticancer drugs by SDI (succinate dehydrogenase inhibition) test with 11 clinical specimens obtained from operated patients. The results were compared with those of xenografted specimen in nude mice, using the adjuvant part of the specimens assayed. Chemosensitivity of clinical specimens against 3 drugs is mitomycin (MMC), adriamycin (ADM) and cisplatin (CDDP), showed a good accordance (73%) with those of xenografted tumors. The drug sensitivity was considered to be one of the features of original tumors and to be well preserved in the xenografts in nude mice. We also studied in vivo experimental chemotherapy on 19 tumor lines in nude mice to compare the chemosensitivity with clinical response to the same chemotherapy observed in each donor patient. A close correlation (83% on responder, 100% on non-responder and the overall predicting accuracy rate were 95%) was shown, suggesting in vivo nude mouse assay having an excellent predictability for clinical results.

[The persistence and proliferation of tumor-xenografts implanted under the renal capsule of immunocompetent mice, cyclosporin A-treated mice and nude mice].

The subrenal capsule assay for cancer chemotherapy was performed, using tumor-specimens of 19 patients' cancers. Twelve tumor-specimens were implanted simultaneously under the renal space of immunocompetent CDF1 mice, cyclosporin A (CsA) 60 mg/kg treated mice, and BALB/c-nu/nu (nude) mice. The persistence and growth of implanted tumor-xenografts of each mouse, was evaluated, on day 6 and 9 after inoculation. The tumor-xenografts implanted under the renal space of immunocompetent mice, grew larger on days 6 in 9 cases, but histological evaluation showed tumor tissues were in various degree replaced by host reactive tissues. Host reaction in CsA-treated mice or nude mice was suppressed almost completely, but the persistence and proliferation of tumor-xenografts of both mice was varied, depending on the nature of original tumors. The judgment for cancer chemotherapy on our modified SRC assay was almost similar between CsA-treated mice and nude mice, but there were some cases in which macroscopical judgment didn't correspond with histological effect. The DNA synthesis of tumor-xenografts of 7 patients, was examined by using sequential changes of BrdU labeling index (LI) in the renal space of CsA-treated mice. It was showed LI rather indicated the nature of original tumors itself.

A case of glossopharyngeal zoster diagnosed by detecting viral specific antigen in the pharyngeal mucous membrane.

Glossopharyngeal nerve paralysis caused by varicella zoster virus reactivation is rare. We present a case of glossopharyngeal zoster confirmed by direct immunofluorescence staining for virus antigens. A 35-year-old man presented with right-sided, severe swallowing pain and dysgeusia. Physical examination showed a loss of ipsilateral gag reflex. White spots on the posterior wall of the right pyriform sinus were seen by laryngofibroscopy, and a loss of taste on the right posterior part of the tongue was confirmed by gustometry using the filter paper disc method. The varicella zoster virus antigen was revealed by direct immunofluorescence staining by fluorescein isothiocyanate labelled mouse monoclonal antibody specific for varicella zoster virus glycoprotein, using samples obtained from the mucosal lesion by abrasion with a cotton swab. The patient was treated by intravenous administration of acyclovir. His throat pain and dysgeusia completely resolved. We discuss the advantages of direct immunofluorescence staining for varicella zoster virus antigen for the diagnosis of glossopharyngeal zoster.

Microbubble destruction with ultrasound augments neovascularisation by bone marrow cell transplantation in rat hind limb ischaemia.

OBJECTIVE: To examine the effects of microbubble destruction with ultrasound (MB) combined with bone marrow derived mononuclear cell transplantation (BMT) into ischaemic tissues in rat hind limb ischaemia. METHODS AND RESULTS: Unilateral hind limb ischaemia was surgically induced in Lewis rats. At postoperative day 7, rats were randomly divided into three groups: a vehicle treated group, an ultrasound treated group, and an MB treated group. MB treatment increased vascular endothelial growth factor mRNA as assessed by real time polymerase chain reaction (3.0-fold, p < 0.05). At four weeks, the MB group had increases in laser Doppler blood flow index (LDBFI; 1.2-fold, p < 0.05), angiographically detectable collateral vessels (angiographic score: 1.4-fold, p < 0.01), and capillary to muscle fibre ratio (1.4-fold, p < 0.01) in ischaemic limbs compared with the vehicle treated group. No differences were seen between the vehicle and ultrasound treated groups. Secondly, rats were allocated to vehicle treatment, BMT (5 x 10(6) cells/rat), or a combination of MB and BMT (MB+BMT) at seven days after hind limb ischaemia. BMT treatment significantly increased LDBFI, angiographic score, and capillary to muscle fibre ratio compared with vehicle treatment. Interestingly, MB+BMT treatment produced significantly greater LDBFI (1.2-fold, p < 0.01), angiographic score (1.5-fold, p < 0.01), and capillary to muscle fibre ratio (1.5-fold, p < 0.05) than BMT treatment alone. CONCLUSIONS: MB may be a useful technique to enhance BMT induced neovascularisation.

Effects of eplerenone on transcriptional factors and mRNA expression related to cardiac remodelling after myocardial infarction.

OBJECTIVE: To examine the effects of eplerenone, a selective aldosterone blocker, on cardiac function after myocardial infarction (MI) and myocardial remodelling related transcriptional factors and mRNA expression in non-infarcted myocardium. METHODS: MI was induced by ligation of the coronary artery in Wistar rats. Rats were randomly assigned to a vehicle treated group or an eplerenone treated group (100 mg/kg/day). RESULTS: At four weeks after MI, left ventricular (LV) end diastolic pressure, LV weight, and LV end diastolic dimension were increased in MI rats. Eplerenone significantly reduced the increase in LV end diastolic pressure, LV weight, and LV end diastolic dimension. In the MI rats the decreased ejection fraction indicated systolic dysfunction and the increased E wave to A wave ratio and E deceleration rate indicated diastolic dysfunction. Eplerenone significantly attenuated this systolic and diastolic dysfunction. Myocardial interstitial fibrosis, transcriptional activities of activator protein 1 and nuclear factor kappaB, and mRNA expression of monocyte chemoattractant protein 1, plasminogen activator inhibitor 1, atrial natriuretic peptide, brain natriuretic peptide, and collagen types I and III were significantly increased at four weeks after MI. Eplerenone significantly attenuated interstitial fibrosis and suppressed transcriptional activity and mRNA expression of these genes. CONCLUSIONS: When administered after MI, eplerenone prevents cardiac remodelling accompanied by systolic and diastolic dysfunction and inhibits abnormal myocardial transcriptional activities and gene expression.