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Epiphyseal growth cartilage of the femoral head obtained from Wistar rats was investigated after fixation by a rapid-freezing and freeze-substitution. Liquid helium was used in order to achieve a fast cooling rate without ice-crystal damage during the rapid freezing. Use of the rapid-freezing and freeze-substitution procedure provided better ultrastructural preservation of the chondrocyte than conventional chemical fixation methods. This procedure allowed a more reliable approach to electron probe analysis. X-ray microanalysis of the specimens confirmed that calcium is not detected in the initial matrix vesicles as a result of the freezing process. The results suggest that calcium release from precipitates occurs in the free state without any detectable formation of hydroxyapatite at the initial stage of calcification and that calcium is not tightly bound to the matrix vesicles.

A total of 124 lesions from 1 to 6cm in diameter, including 31 cavernous hemangiomas, 32 metastases and 61 hepatocellular carcinomas (HCC) were analyzed to study the usefulness of magnetic resonance imaging (MRI) at 0.5 Tesla to differentiate focal hepatic lesions on the basis of qualitative criteria. Each focal hepatic lesion was assessed for shape, internal architecture and signal intensity relative to normal liver parenchyma. While all cavernous hemangiomas and metastases except one lesion could be detected, detection rate of HCC was significantly inferior to that of the other two diseases. A tumor capsule and a hyperintense focus on T1-weighted images were demonstrated in only HCC lesions in strong contrast with the other two diseases; however, metastases with slow-growing characteristics or subacute hematoma may appear as similar images. Cavernous hemangiomas appeared markedly hyperintense on T2-weighted images in 23 of 31 lesions, but one metastasis and one HCC had similar images. A multivariate analysis of several MRI resulted in the following mean discriminant scores: cavernous hemangioma, -1.2652; metastasis, 0.1830; and HCC, 0.7138. It appeared to be possible to differentiate the three diseases with 84.4 percent accuracy.

We report second malignant neoplasms which developed between 7 and 19 years after treatment in 3 pediatric patients with osteosarcoma. Two patients had been treated with only surgery, and another patient had been treated with a combination of surgery with chemotherapy and radiation therapy for primary lesions. Pediatric patients with osteosarcoma, in particular, require careful long-term follow-up to monitor not only metastases but also development of second malignant neoplasms.

The aim of this study is to obtain data for improving a training program for patients with diabetes mellitus. One hundred eighty-seven patients with non-insulin dependent diabetes mellitus were tested with 20 questions about their knowledge for self-management of diabetes mellitus. Then to draw out factors in their personal backgrounds relating to their correct answers, multiple regression analyses were conducted. As a result, four factors showed significant differences in the following order: Educational careers > ages > duration of disease > socioeconomic strata. The results of the present study have shown for the first time, that these four factors closely concern patients to acquire the necessary knowledge for their self-management of the disease. In addition, this study has raised some fundamental problems regarding the training program for patients: how education should be given to patients.

To confirm the formation of Charcot-Leyden crystals (CLC) in basophils, we observed basophils in sputum and peripheral blood. Sealed slide and suspension culture methods were used to observe the process of CLC formation in peripheral blood basophils and eosinophils under electron microscopy. CLC formation was observed in basophils and eosinophils, and was found to be augmented by sealed slide method. A temperature of 4 degrees C was better than 37 degrees C for promoting the formation of crystals. There was no correlation between the degranulation of these cells and the formation of CLC after stimulation with anti-IgE or anti-IgG antibodies. CLC were initially detected in the cytoplasmic granules of basophils where they continued to enlarge. No CLC were identified in mast cells under any conditions studied. These findings confirm that CLC in sputum are not exclusive to eosinophils and that CLC appear to be present in basophil-rich sites under the cell damage.

A newly introduced compound, EPC-K1, represents a phosphate diester linkage of vitamin E and vitamin C. The effect of EPC-K1 on the reperfusion injury was evaluated in a heterotopic cardiac transplantation model using syngenic combination rats. Prior to the warm ischemia, 12mg EPC-K1/kg was administered intravenously to donor rats. After 15 min of warm ischemic time, hearts were harvested and perfused with 4 degrees C saline. After completion of the transplantation, recipient rats were also treated with intravenous 12 mg EPC-K1/kg, before reperfusion. Saline was used instead of EPC-K1 for both donors and recipients in the control group. On the 7th post-transplantation day, graft survival was 7 out of 8 in EPC-K1 group, versus 1 out of 9 in the control group (p < 0.001). Thiobarbituric acid-reactive substance levels in the recipient serum, three hours after reperfusion, were significantly limited, in the group in which EPC-K1 was administered only to donors. But it was not possible to clarify whether the effect of EPC-K1 is primarily at the donor or recipient levels at this time. These results indicate that EPC-K1 may reduce reperfusion injury after cardiac transplantation. This beneficial effect may be mediated by the hydroxyl radical scavenging properties of EPC-K1.

A sustained release system for interleukin-2 (IL-2), and IL-2 mini-pellet (IL-2 mp), was developed by fusing IL-2 into a needle shaped collagen. Serum concentration of IL-2 after a single subcutaneous injection of the IL-2 mp into C57BL/6 mice remained elevated longer than after an injection of aqueous IL-2. IL-2 in the serum became undetectable by 6h after a subcutaneous injection of 1 x 10(6) unit of IL-2 in phosphate-buffered saline (PBS). In contrast, after a single subcutaneous injection of IL-2 mp containing the same amount of IL-2, the concentration of IL-2 increased to its maximum at 6h after injection, then began to decrease gradually. IL-2 was detected even on the third day after a single subcutaneous injection of one IL-2 mp. Augmentation of NK activity and generation of IL-2 activated killer cells were observed in the spleen from day 1--day 3 after a single subcutaneous injection of IL-2 mp into C57BL/6 mice. This activation was not observed following a single subcutaneous injection of the same amount of IL-2 in PBS. Adoptive immunotherapy by a single subcutaneous injection of IL-2 mp followed by intravenous injections of in vitro cultured IL-2 activated killer cells showed better results in decreasing the number of metastases of Lewis lung carcinoma in C57BL/6 mice than immunotherapy using IL-2 solution.(ABSTRACT TRUNCATED AT 250 WORDS)

A 56 year-old rectal cancer patient who developed a chronic rectoabdominocutaneous fistula postoperatively was treated with fibrin clot, and the fistula healed completely. Occlusion of chronic postoperative fistulas with fibrin clot appears to be a useful technique.

Doppler left ventricular (LV) inflow is reportedly affected by LV diastolic properties. We evaluated 48 subjects consisting of 27 patients with chronic mitral regurgitation (MR) and 21 patients with noncardiac disorders who received echocardiographic examinations. The deceleration rate divided by diastolic dimension (DR/Dd) derived from Doppler early diastolic LV inflow was correlated with the peak diastolic velocity divided by diastolic dimension (peak DV/Dd), a conventional index of LV diastolic function derived from the M-mode echocardiogram in the 48 patients, regardless of the presence of normal sinus rhythm or atrial fibrillation. LV diastolic function was then estimated by comparing perioperative echocardiographic examination and LV micro-and ultrastructural findings of biopsy specimens from 12 patients with MR who received mitral valve replacement. Fiber diameter, volume fraction of interstitial fibrosis (int. % Fib), and volume fractions of three intracellular components; the myofibrils (% MF), the sarcoplasmic reticulum (% SR) and the mitochondria (% MT), were measured in LV transmural biopsy specimens. DR/Dd was significantly correlated with peak DV/Dd before and after operation. Peak DV/Dd and DR/Dd were inversely correlated with int. % Fib and % SR, and were positively correlated with % MF. We subdivided the 12 MR patients according to their postoperative DR/Dd values as "recovered", and "non-recovered" based on their postoperative LV diastolic function. % MF was significantly lower in the 'non-recovered' group. Thus, DR/Dd can serve as an index of LV diastolic function. A decrease in % MF may inhibit the recovery of postoperative LV diastolic function.

Formalin-fixed, paraffin-embedded tissue sections of resected tumors from 90 patients were immunohistochemically studied to assess the prognostic value of proliferating cell nuclear antigen (PCNA) expression in non-small cell lung cancer. The individual tumors were classified into groups of high, moderate or low proliferative grade, and 38 (42.2%) patients had a high grade of proliferation. No statistically significant correlations were observed between PCNA grade and TNM status, pathological stage, resectability, histological type, degree of histological differentiation. Only vascular invasion significantly correlated with proliferative grade (p < 0.05). Survival analysis showed that patients with low proliferative grade tumors survived significantly longer (a 5-year survival rate of 83.3%) than those with high proliferative grade tumors (39.4%, p < 0.005). Cox's multivariate analysis revealed that PCNA grade was a significant prognostic determinant of survival. These results suggest that PCNA expression provides an independent prognostic variable for patients with non-small cell lung cancer and that it may be useful to consider this factor in treatment planning.

The relationship between MR configuration and pathological grade was studied in 41 histologically verified supratentorial astrocytic gliomas with a 0.5T superconductive MR system. The gliomas included 13 low-grade astrocytomas (LGAs), 14 anaplastic astrocytomas (AAs) and 14 glioblastoma multiformes (GBMs). MRI configurations were classified into nine criteria which were scored and statistically analyzed. The mean values of LGAs, AAs and GBMs were 0.45 +/- 0.31, 1.18 +/- 0.20 and 1.47 +/- 0.22. In each grade, MRI score increased as pathological grades increased (p < 0.01-0.001). LGAs had significantly lower values than AAs in five of the nine criteria (55.6%); heterogeneity, cyst or necrosis, edema or mass effect, border definition, and the degree of contrast enhancement, and lower values than GBMs in eight criteria (88.9%) except for hemorrhage. Three criteria (33.3%), heterogeneity, cyst or necrosis, and flow void sign were significantly higher in GBMs than AAs. The four variables, heterogeneity, cyst or necrosis, edema or mass effect and border definition, proved to be important factors related to the pathological grade in a multiple regression analysis.

Stump problems in amputations resulting from employment related injuries were investigated in 397 cases in the Chugoku and Shikoku districts of Japan between 1987 and 1991. Ninety-seven patients (24%) had stump problems which interfered the prosthetic fitting. Stump problems of the upper extremity were seen in about 9% (17 amputees), two thirds of which were skin troubles. Stump problems of the lower extremity were seen in about 37% (80 amputees). Certain complaints were associated with specific methods of amputation; abnormal keratosis in Syme's amputation, equinus deformity in Chopart's amputation, reduced muscle power in above the knee (A/K) amputation and joint dysfunction in below the knee (B/K) amputation. Adequate prosthetic fitting was achieved by the modification of the socket and alignment in almost all amputees with stump problems. In only two cases, Chopart's amputation required subsequent Syme's amputation due to equinus deformity with abnormal keratosis. In almost every case, stump problems are avoidable by means of surgeons' deliberate evaluation of the affected limb and adequate choice of the amputation level.

Newly designed oligonucleotide primers, KI-7 and KI-8 for the human T cell lymphotropic virus type I (HTLV-I) pX gene were synthesized using an automated DNA synthesizer. Previously known HTLV-I-infected cell lines, MT-1 and MT-2, were used as positive controls and HTLV-I-uninfected cell lines, Molt-4, SBC-3, ABC-1, and EBC-1, as negative controls. Peripheral blood mononuclear cells from 17 patients with anti-HTLV-I antibody and 10 healthy individuals without anti-HTLV-I antibody were studied by polymerase chain reaction (PCR) with KI-7 and KI-8. All DNA samples from HTLV-I-infected cell lines and 17 patients with anti-HTLV-I antibodies showed positive signals of the HTLV-I pX gene. None of the DNA samples from HTLV-I-uninfected cell lines or 10 healthy individuals showed positive signals. When serially diluted DNA of MT-2 cells were amplified by 35 cycles of PCR, the detection limit of the pX gene by using the primer pairs was DNA from about 1.5 MT-2 cells. Specificity and detectable capacity of primer pairs, KI-7 and KI-8 were confirmed to be enough to use for the diagnosis of HTLV-I infection.

We expressed mouse cytochrome P1-450 and P3-450 using recombinant vaccinia virus gene expression system in HeLa cells that were devoid of significant basal levels of P-450. HeLa cells were infected with the recombinant vaccinia virus containing either mouse cytochrome P1-450 or P3-450 cDNA, and the cell lysates were analyzed for the kinetics of P-450 enzyme activity and protein expression at the same time. 7-Ethoxycoumarin O-deethylase and ethoxyresorufin O-deethylase activities were measured as an expression of the cytochrome P-450 enzyme activities. Both cell lines began to express these enzyme activities as early as 12h after infection. The activities increased linearly up to the 24 h time point, and were kept for 36 h. Western immunoblot analysis showed that these cytochrome P-450 proteins were detected at 16 h and reached maximum quantity at 24 h after infection. These data showed a good correlation between cytochrome P-450 enzyme activity and protein concentration throughout the process of P-450 gene expression by vaccinia virus vector, suggesting a complete formation of cytochrome P-450 holoenzyme from the early stage of the protein expression.

Experiments were undertaken to determine the relationship between evoked spinal cord potential (ESP) and the partial pressure of oxygen in tissue in the epidural space (E-pO2) during aortic clamping. Eighteen adult mongrel dogs were studied as follows. In group I (n = 6), the descending thoracic aorta was clamped partially at the proximal site for 15 min to maintain the distal arterial pressure at 60, 40, and 20 mmHg consecutively at 15 min intervals. In group II (n = 6), the descending thoracic aorta was clamped proximally for 30 min. In group III (n = 6), the descending thoracic aorta was cross-clamped at proximal and distal sites for 30 min. Postoperative complete paraplegia was observed in 4 of 6 dogs in group III, but none in group II. The change in ESP with aorta cross-clamping was very mild in groups I and II. Transient increases and decreases in the ESP amplitude were observed in group III. The decrease of E-pO2 correlated well with the distal arterial pressure, and a rapid return to baseline of the E-pO2 was observed after declamping. The E-pO2 changed in response to spinal ischemia more rapidly than did ESP in all groups. The critical level of E-pO2 was 50 mmHg or a 40% decrease from baseline. Because the ESP reflects spinal function and the E-pO2 reflects spinal blood pressure, we propose that combined recording of ESP and E-pO2 would improve spinal monitoring during thoracic aortic surgery.

To determine whether a relationship exists between DNA ploidy and the prognosis of hepatocellular carcinoma (HCC), flow cytometric DNA analysis was performed in paraffin-embedded specimens obtained from 44 patients with HCC who underwent hepatectomy. There were 26 diploid (59%) and 18 aneuploid (41%) tumors. No correlation was shown between DNA ploidy pattern and patient age, sex, liver cirrhosis, hepatitis B virus antigen and serum alpha-fetoprotein level. The ploidy pattern had no significant correlation with the presence of vascular invasion or intrahepatic metastasis. Only Edmondson's grade was well correlated with the ploidy pattern. We noted a significant correlation between survival rates and the presence of vascular invasion or intrahepatic metastasis (p < 0.05). In contrast, no significant correlation was found between DNA ploidy pattern and the prognosis of HCC. The results of this study indicate that DNA ploidy pattern may not be a useful indicator for the prognosis of HCCs after hepatic resection, unlike the results of gastric and colon cancers.

The presence of the HTLV-I gene in peripheral blood mononuclear cells was studied by polymerase chain reaction in 42 patients including 16 with lung cancer, 12 with diffuse panbronchiolitis (DPB), 11 with idiopathic interstitial pneumonia (IIP), and 3 with pneumoconiosis and hematological malignancy. Sequences equal to a part of the pX gene were found in 44% of the lung cancer cases, 50% of the DPB cases, 55% of the IIP cases, and 100% of the cases of pneumoconiosis and leukemia. In the lung cancer cases, detection of the pX gene was frequently associated with the existence of diffuse interstitial pulmonary shadows. The pX gene was detected in 100% of patients with anti-HTLV-I antibody, 50% of patients with HTLV-I-related reaction and 14% of patients who tested seronegative. It may be inferred from the results that respiratory diseases that produce diffuse interstitial pulmonary shadows are closely associated with HTLV-I infection and that the HTLV-I-related reaction to the immunofluorescent test might reflect the latent infection state of HTLV-I.

Early diagnosis of rejection and timely immunosuppression are absolutely important in clinical lung transplantation. We studied surface markers of peripheral blood lymphocytes (PBL), graft infiltrating lymphocytes (GIF) and bronchoalveolar lavage fluid (BALF) in a rat using flow cytometric monitoring to diagnose rejection. Left lung transplantation was performed on Brown Norway (BN) rats and Lewis (LEW) rats in the following groups; Group 1: LEW-LEW (isograft), Group 2: BN-LEW (allograft; no immunosuppression), Group 3: BN-LEW (allograft; treated with Cyclosporine A at a dose of 15 mg/kg/day i.m.). In each group, rats were killed 3, 5, 7 days postoperatively (n = 6 on each day). Monoclonal antibodies investigated in this study were W3/25 (anti-helper T lymphocyte), OX8 (anti-suppressor/cytotoxic T lymphocyte), and OX39 (anti-interleukin 2 receptor). Histological classification of rejection in Group 2 showed vascular phase at 3 days, alveolar phase at 5 days, and destructive phase at 7 days, respectively. No evidence of rejection was found in Group 1 or 3. In Group 2, W3/25 positive cell proportion in GIL and BALF significantly decreased as the rejection progressed, but OX8 positive and OX39 positive cell proportion increases were significantly greater than in Groups 1 and 3 as the rejection progressed. These results lead us to speculate that the studies of T cell subsets in GIL and BALF lymphocytes are useful for diagnosis of rejection in lung transplantation.

To determine how interleukin-7 (IL-7) affects the proliferation of T cells in patients with rheumatoid arthritis (RA), we evaluated the response of mononuclear cells (MNC) obtained from their peripheral blood (PB), synovial fluid (SF) and synovial tissue (ST) to stimulation by recombinant IL-7 and interleukin-2 (IL-2). Each cytokine was administered alone or combined with phytohemagglutinin (PHA). Cellular DNA synthesis was assayed by the [3H]-thymidine incorporation method. The stimulatory effect of 500 u/ml IL-7 on PBMNC obtained from 19 patients with RA was significantly lower than on PBMNC from 19 healthy controls. However, the same degree of stimulatory activity of 500 u/ml IL-2 was observed on the PBMNC from both RA patients and control subjects. The response of PBMNC to a suboptimal dose of PHA (0.2 micrograms/ml) was enhanced by adding either IL-7 or IL-2 (100 or 500 u/ml) to the cultures. The enhanced synthesis of DNA by both RA and control PBMNC on exposure to IL-7 following stimulation by a suboptimal dose of PHA was higher than that of IL-2. The effect of IL-7 on RA PBMNC was significantly greater than that of IL-2 at the concentration of 100 u/ml on PBMNC from the same RA patients. The stimulatory activity of IL-2 at the concentrations of 100 and 500 u/ml on SF MNC and ST MNC exceeded that of IL-7. In particular, an IL-2 dose of 500 u/ml had a marked effect on SF MNC. The PHA response of SF MNC was the lowest seen among the MNC from three different compartments.(ABSTRACT TRUNCATED AT 250 WORDS)