Blockade of CD40/CD40 ligand interactions attenuates skin fibrosis and autoimmunity in the tight-skin mouse.

OBJECTIVE: To assess the association of CD40/CD40 ligand (CD40L) interactions with the development of skin fibrosis and autoimmunity in tight-skin (TSK/+) mouse, which is a mouse model for human systemic sclerosis. METHODS: Newly born TSK/+ mice were treated with murine anti-CD40L monoclonal antibody (100 microg intraperitoneally weekly). Hypodermal thickness of 8-week-old female mice (defined as the thickness of a subcutaneous loose connective tissue layer beneath the panniculus carnosus) was measured under a light microscope. All skin sections were taken from the para-midline, upper back region. Serum anti-topoisomerase I autoantibody levels, serum immunoglobulin levels and plasma soluble CD40L levels were determined by enzyme-linked immunosorbent assay. For analysis of lymphocyte surface molecules, single cell suspensions of lymphocytes were stained by monoclonal antibodies. Proliferation of TSK/+ B cells and fibroblasts to anti-CD40 antibodies was assessed by the uptake of [3H]-labelled thymidine and bromodeoxyuridine, respectively. RESULTS: The blockade of CD40/CD40L interactions by anti-CD40L monoclonal antibody significantly reduced cutaneous fibrosis (65%) and anti-topoisomerase I autoantibody in TSK/+ mice. Anti-CD40L monoclonal antibody also normalised B lymphocyte abnormal activation in TSK/+ mice, demonstrated by hyper-gamma-globulinaemia. Furthermore, augmented CD40/CD40L interactions in TSK/+ mice were suggested by upregulated expression of CD40L on CD4(+) T cells, elevated plasma soluble CD40L levels. The hyperresponsiveness to CD40 stimulation was also observed in TSK/+ B cells and fibroblasts. CONCLUSIONS: Cutaneous fibrosis and autoimmunity in TSK/+ mice are closely correlated with CD40/CD40L interactions.

Arteriopathy after transarterial chemo-lipiodolization for hepatocellular carcinoma.

OBJECTIVES: The purpose of this study was to investigate the incidence of and the risk factors for arteriopathy in hepatic arteries after transarterial chemo-lipiodolization in patients with hepatocellular carcinoma and the subsequent treatment strategy changes due to arteriopathy. PATIENTS AND METHODS: A total of 365 arteries in 167 patients (126 men and 41 women; mean age, 60.4±15.0 [SD] years [range: 18-87 years]) were evaluated for the development of arteriopathy after chemo-lipiodolization with epirubicin- or doxorubicin-Lipiodol® emulsion. The development of arteriopathy after chemo-lipiodolization was assessed on arteriograms performed during subsequent transarterial treatments. The treatment strategy changes due to arteriopathy, including change in the chemo-lipiodolization method and the application of alternative therapies was also investigated. Univariate and multivariate binary logistic regression models were used to identify risk factors for arteriopathy and subsequent treatment strategy change. RESULTS: One hundred two (27.9%) arteriopathies were detected in 62/167 (37.1%) patients (45 men, 17 women) with a mean age of 63.3±7.1 [SD] years (age range, 50-86 years). The incidence of arteriopathy was highly patient dependent, demonstrating significant correlation in a fully-adjusted multivariate regression model (P<0.0001). Multivariate-adjusted regression analysis with adjustment for the patient effect showed a statistically significant association of super-selective chemo-lipiodolization (P=0.003) with the incidence of arteriopathy. Thirty of the 102 arteriopathies (29.4%) caused a change in treatment strategy. No factors were found to be significantly associated with the treatment strategy change. CONCLUSION: The incidence of arteriopathy after chemo-lipiodolization is 27.9%. Among them, 29.4% result in a change in treatment strategy.

Mechanisms of Günther Tulip filter tilting during transfemoral placement.

OBJECTIVE: The purpose of this study was to characterize the mechanisms of Günther Tulip filter (GTF) tilting during transfemoral placement in an experimental model with further validation in a clinical series. MATERIALS AND METHODS: In an experimental study, 120 GTF placements in an inferior vena cava (IVC) model were performed using 6 configurations of pre-deployment filter position. The angle between the pre-deployment filter axis and IVC axis, and the proximity of the constrained filter legs to IVC wall prior to deployment were evaluated. The association of those pre-deployment factors with post-deployment filter tilting was analyzed. The association noted in the experimental study was then evaluated in a retrospective clinical series of 21 patients. RESULTS: In the experimental study, there was a significant association between the pre-deployment angle and post-deployment filter tilting (P<0.0001). With a low pre-deployment angle (≤5°), a significant association was noted between filter tilting and the proximity of the constrained filter legs to the far IVC wall (P=0.001). In a retrospective clinical study, a significant association between the pre-deployment angle and post-deployment filter tilting was also noted with a linear regression model (P=0.026). CONCLUSION: Significant association of the pre-deployment angle with post-deployment GTF tilting was shown in both the experimental and clinical studies. The experimental study also showed that proximity of filter legs is relevant when pre-deployment angle is small. Addressing these factors may result in a lower incidence of filter tilting.

Upregulation of endogenous heparin-binding EGF-like growth factor and its role as a survival factor in skeletal myotubes.

To investigate the role of heparin-binding EGF-like growth factor (HB-EGF) in skeletal muscle, we studied its function in skeletal myotubes in vitro using mouse C2C12 cells. Expression levels of membrane-anchored HB-EGF (proHB-EGF) protein were increased specifically during their differentiation among epidermal growth factor receptor (EGFR) ligands. Production levels of EGFR on the cell surface were constant. Tyrosine phosphorylation of EGFR, however, was constitutively increased during differentiation. Quenching of endogenous HB-EGF significantly rendered myotubes sensitive to apoptotic cell death induced by hypoxic stress, suggesting that proHB-EGF in the skeletal muscle is specifically upregulated to function as a survival factor.

The enhanced immunosuppressive efficacy of newly developed liposomal FK506 in canine liver transplantation.

Local delivery of immunosuppressants to the graft and lymphatic tissue is a potential appraoch to enhance the immunosuppressive efficacy and to alleviate systemic adverse effects simultaneously. By taking advantage of this method, we developed liposomal FK506. Previous pharmacokinetic study of liposomal FK506 indicated increased FK506 levels in the liver and spleen. Because the liver is the site of the allograft in liver transplantation and the spleen is a major lymphoid tissue, we hypothesized that liposomal FK506 would increase immunosuppressive efficacy in liver transplantation. We evaluated this hypothesis in a canine model. Orthotopic liver transplantation was performed using beagle dogs, and the recipients were divided into the following groups: group I, no immunosuppression (n = 5); group II, 0.05 mg/kg/day of FK506 i.v. in a commercially available i.v. formulation for 14 days (n = 5); and group III, 0.05 mg/kg/day of FK506 i.v. in a liposomal formulation for 14 days (n = 5). All recipients in group I died within 2 weeks. Recipients in group II died within 33 days. In contrast, three recipients in group III survived for more than 200 days (P < 0.05 versus group I or group II). In DNA analysis, splenocyte proliferation activity in group III was significantly suppressed in comparison with group II. These results suggest that liposomal FK506 markedly increase the immunosuppressive efficacy of FK506 in liver transplantation. A local immunosuppressive effect in the grafted liver and significant suppression of splenocyte proliferation might contribute to enhancement of the immunosuppressive efficacy of liposomal FK506.

Effect of stirring during fixation upon immunofluorescence. Results with distribution of albumin-producing cells in liver.

When the immunofluroscent study on the distribution and the incidence of albumin-producing hepatocytes in the rat liver was performed by the method of Sainte-Marie, the number of positive cells showed various values (10-60%). It was surmised that when the permeability of the fixative was delayed, albumin had flowed out from the cytoplasm of the unfixed hepatocytes. By the simple means of constant stirring of the fixative using a magnetic stirrer, we accomplished rapid fixation and achieved results in which positive cells attained 100%. On the other hand, the incidence of positive cells decreased markedly when rats were fed a protein-free diet.

Lung cancer mortality among a cohort of male chromate pigment workers in Japan.

In 1975, five manufacturers of chromate pigment in Japan were examined in a study of the carcinogenicity of chromates. These companies were producing lead chromate, zinc chromate, molybdate orange and/or strontium chromate. The current study covers a cohort of 666 workers involved in the manufacture of chromate pigment for at least 1 year between 1950 and 1975. The workers were followed up for 15-40 years, until 1989. Many previous reports have found an excess lung cancer risk among workers involved in the manufacture of chromate pigments and chromate chemicals. In the current study, subjects were classified on the basis of years worked, years of observation, characteristics of company, type of work engaged in for the longest period of time, and involvement in the manufacture of zinc chromate. Mortality was compared with that of all Japanese males by means of the person-year method. The route of exposure was primarily inhalation through the respiratory system. None of the results showed statistically significant differences that would suggest an excess risk for malignant neoplasms, particularly lung cancer, among workers engaged in the manufacture of chromate pigment in Japan.

Induction of oncogenic transformation by low doses of X rays and dose-rate effect.

Golden hamster embryo cells were used to study morphological transformation induced by low doses of X rays at different dose rates. X irradiation at lower dose rates was less effective in cell killing and induction of transformation than at higher dose rates. The general shapes of induction curves of transformants were almost the same at all dose rates. At shoulder doses (1 to 150 R), the transformation frequencies increased steeply with increasing dose at all dose rates. At doses higher than 150 R survivals declined exponentially, but the frequencies of transformants increased only slightly. However, irradiation at higher dose rates was more effective in induction of transformants than that at lower dose rates. We conclude that transformational damage introduced at a low dose rate, as well as sublethal damage, may be repaired during low-dose-rate irradiation, but transformational damage may be different from sublethal damage.

A novel brain-derived member of the epidermal growth factor family that interacts with ErbB3 and ErbB4.

A novel member of the epidermal growth factor (EGF) family, the neural- and thymus-derived activator for ErbB kinases (NTAK), has been purified and cloned. Five alternative spliced isoforms have been detected in the rat adrenal pheochromocytoma cell line, PC-12 cells. The rat NTAK alpha2a isoform exhibits 94% identity in its primary sequence with the human NTAK alpha isoform. In vivo, NTAK is only expressed in the brain of rat E11.5 embryos, and in the brain and thymus of adult rats. The soluble 46 kDa form binds directly to ErbB3 and B4, but not to ErbB1 or B2. NTAK, however, transactivates ErbB1 and B2 via heterodimerization with ErbB3 or B4. NTAK stimulates the differentiation of MDA-MB-453 cells and competitively inhibits the binding of [125I]neuregulin to these cells. In addition to these neuregulin-like properties, NTAK exhibits limited structural homology to neuregulins in the immunoglobulin (Ig)-like, EGF-like, and hydrophobic domains. Thus, NTAK appears to be a new member of the EGF family displaying neuregulin properties.

Comparison of clinical and laboratory findings of intrathoracic sarcoidosis between children and adults.

Children were different from adults in the following findings of intrathoracic sarcoidosis: All of the 69 pediatric patients studied had no complaints and were found by chest x-ray examinations, in contrast with 71% of the 62 adult patients studied. At the ages 10-14 yr there were far more boys than girls. In pediatric patients eye involvements were rare, blood eosinophilia was more frequent, biopsy of conjunctival follicles was more frequently positive, cold serum hemagglutinins were higher at first and decreased later, and the prognosis was far better than in adults.

The use of zona-free aged unfertilized human oocytes as a predictor for successful subzonal insemination.

OBJECTIVE: To examine the reliability of the zona-free aged (1 day old) human unfertilized oocyte sperm penetration assay for assessing sperm fertilizing ability and to determine the predictive value of this assay for subsequent subzonal insemination (SUZI) outcomes. DESIGN: A total of 253 unfertilized oocytes from total fertilization failure patients and from good fertilization rate (> 70%) patients in standard IVF were inseminated with donors' spermatozoa, and penetration rates were compared in each group. Two hundred seventy-two unfertilized oocytes from total fertilization failure, poor fertilization (< 30%), and normal fertilization (> 30%) were inseminated with husbands' spermatozoa, and penetration rates were compared between the three groups. In 29 patients, the results of the zona-free assay performed in previous IVF were compared with the fertilization rates of subsequent SUZI. RESULTS: In the zona-free assay using donors' spermatozoa, there was no difference in penetration rates between the two groups (109/122, 89.3% versus 114/131, 87.0%). Penetration rates using partners' spermatozoa were positively correlated with fertilization rates in standard IVF (total fertilization failure 34/75, 45.3%; poor fertilization 56/77, 72.7%; normal fertilization 108/120, 90.0%). There was a significant difference in fertilization rates after SUZI between the patients with negative and positive penetration in zona-free assay (4/53, 7.5%, versus 54/174, 31.0%). CONCLUSION: The zona-free human oocyte assay may primarily reflect sperm fertilizing ability. This asssay also could be a reliable predictor for subsequent SUZI outcome.

Hyperthermo-chemo-hypoxic isolated liver perfusion for hepatic metastases: a possible adjuvant approach.

As a possible intraoperative adjuvant approach to treating hepatic metastases we developed a method of hyperthermo-chemo-hypoxic isolated liver perfusion in combination with hepatic resection. This method was applied to 11 patients with colorectal hepatic metastases between 1992 and 1995. One patient died on postoperative day 14 of hepatic failure (9% mortality), the cause of which was live temperature that reached 42.9 degree C, which seems to be the maximum limit for thermal toxic effect on the human liver. The other 10 patients tolerated the perfusion well, with mild hepatic and non systemic toxicity after minor or even major hepatic resection; the serum aminotransferase and total bilirubin levels returned to normal levels by postoperative day 14. Only one of eight patients (13%) for whom cytotoxic drugs were added to the perfusate (mitomycin C 10 micrograms/ml or cisplatin 2 micrograms/ml) had hepatic recurrence by 19 months after the perfusion (mean follow-up 25.8 months; median 23 months; range 8-57 months). Two patients were alive with no evidence of disease at 13 and 57 months, respectively after the perfusion; the other five patients had postperfusion extrahepatic recurrences (median: 19 months; range 7-20 months). In contrast, hepatic metastases recurred 7 and 20 months after the perfusion, respectively, in the two patients not given a cytotoxic drug.

Differential activation of ERK 1/2 and JNK in normal human fibroblast-like cells in response to UVC radiation under different oxygen tensions.

The mechanisms by which mitogen-activated protein kinases (MAPK) respond to the input of UV-induced signal transduction pathways and the resulting biological functions are not well understood. We investigated whether the level of oxygen tension of culture was responsible for the differential activation of MAPK and different cellular outcomes in UVC-irradiated cells. The intracellular oxidative level of normal human fibroblast-like cells in a normal atmosphere (normoxic, 20% O2) was increased within 30 min after UVC irradiation. When cells were cultured at lower oxygen tension in the presence of an antioxidant N-acetyl-L-cysteine (NAC) or under physiologically hypoxic (5% O2) conditions, the elevation of the oxidative level by UV-irradiation was significantly reduced. Among MAPK, extracellular-signal related kinase (ERK) 1/2 was activated by UV regardless of the oxidative level, while c-Jun N-terminal kinase (JNK) activation was inhibited in NAC-treated and in hypoxic cultures. In addition, in cultures at lower oxygen tension, there was less apoptosis and cell survival was enhanced. These results suggest that UV-induced oxidative stress was responsible for intracellular signaling through the JNK pathway. Furthermore, the balance between ERK1/2 and JNK activities after UV irradiation under different oxygen tensions possibly modified cellular outcome in response to UV.

Analyses of differential sensitivities of synchronized HeLa S3 cells to radiations and chemical carcinogens during the cell cycle. Part IV. X-rays.

Radiation-induction and rejoining of single-strand breaks (SSBs) in the DNA of synchronized HeLa S3 cells were investigated by alkaline sucrose density gradients. We could not find any significant differences in the extent of SSBs induced in cellular DNA and in the extent of their rejoining throughout the cell cycle, including mitosis. The cyclic variation curve of the content of non-protein sylfhydryls (NPSH) during the cell cycle is similar to that of X-ray survivals except in mitosis, although there was no close correlation between the content of apparent total sulfhydryls (APSH) and X-ray survivals. Radiation-induced mutants resistant to 8-azaguanine (8AG) occurred in higher frequency in the radio-sensitive G1-S boundary phase than in the radio-resistant G1, S and early G2 phases. Further, the pre-irradiation treatment with 50 mM cysteamine prevented reproductive death and induction of 8AG-resistant mutants by X-rays throughout the cell cycle. These findings seem to indicate that there is a close correlation between the extent of lethal radiation damage to the cells and their mutability, and that sulfhydryls may play an important role as a factor governing cellular radio-sensitivity.

Analyses of differential sensitivities of synchronized HeLa S3 cells to radiations and chemical carcinogens during the cell cycle. III. 4-Nitroquinoline 1-oxide and its derivatives.

Sensitivity to the chemical carcinogens 4-nitroquinoline 1-oxide (4-NQO) and 4-hydroxyaminoquinoline I-oxide (4-HAQO), during the cell cycle of synchronized HeLa S3 cells, decreases from the late S to the early G2 phases. Cells in other phases are relatively sensitive to both carcinogens. [3-H]4-NQO and [ 3-H]4-HAQO seem to be bound preferably more with cellular DNA of the mitotic phase to the middle of the S phase than with that of the late S phase in which the cells are rather insensitive to these carcinogens. However, we found no significant difference in the excision rates of these carcinogens from the DNA of HeLa S3 cells through the cell cycle. These findings indicate that the cyclic variation of 4-NQO and 4-HAQO cell survivals during the cell cycle may be due to the differences in the amounts of 4-NQO and 4-HAQO bound with cellular DNA.

Analyses of differential sensitivities of synchronized HeLa S3 cells to radiations and chemical carcinogens during the cell cycle. Par V. Radiation- and chemical carcinogen-induced mutagenesis.

8-Azaguanine(8AG)-resistant mutations induced by X-rays, ultraviolet radiation (UV) and a chemical carcinogen, 4-hydroxyaminoquinoline 1-oxide (4-HAQO) were examined during the cell cycle of synchronized HeLa S3 cells. Mutants induced by 400 R of X-rays occurred in a higher frequency in the X-ray-SENSITIVE Gl-S boundary phase than in the X-ray-resistant G1, S and early G2 phases. 8AG-resistant mutants induced by treatment with 10(-5) M 4-HAQO for 20 min appeared in a higher frequency in the early to middle S phases than in the other phases. In the case of UV, however, we found no significant difference in the induced mutation frequencies during the cell cycle, because the mutation frequencies induced by the UV doses (0-20 J/m2) used were too low for detection of the difference. These results suggest that there is a close correlation between the critical damage induced in DNA molecule(s) at the DNA-synthetic phase in the cell cycle and mutagenesis, because mitotic cells have a low mutability in spite of their high radio-sensitivity.

Abdominal cerebrospinal fluid pseudocyst in a severely handicapped patient with hydrocephalus.

We present a case of an abdominal cerebrospinal fluid pseudocyst as a rare complication of a ventriculoperitoneal shunt. The patient is a severely handicapped bedridden spastic quadriplegic with hydrocephalus. He underwent surgical reconstruction of a shunt tube because of shunt disconnection at the age of 12 years. Fever and frequent vomiting developed 2 months after surgery, and abdominal fullness gradually became severe. He was diagnosed as having an abdominal cerebrospinal fluid pseudocyst on abdominal ultrasonography. If a shunt dysfunction is suspected, this type of cyst should also be considered, and abdominal ultrasonography should be performed as one of the screening tests for determining the cause of the shunt dysfunction.

Long-term observation of the changing brainstem auditory evoked potentials in a case of infantile opsoclonus-polymyoclonia syndrome.

Brainstem auditory evoked potential (BAEP) studies were repeatedly carried out on a boy with infantile opsoclonus-polymyoclonia syndrome, from 15 months to 39 months of age, the following being revealed: the I-III interpeak latency (I-III IPL) was more or less in the upper normal range until 24 months and then remained at the same level throughout the rest of the observation period, not showing physiological shortening with maturation. It should be noted that the prolongation of I-III IPL continued in spite of the disappearance of the opsoclonus.

A case of Isaacs syndrome with high CSF protein and a large cisterna magna.

An 11-year-old boy exhibited continuous muscle fiber activity. He had suffered from stiffness of his hands, difficulty in relaxing his hands after gripping, and making skilled movements with his fingers. His clinical symptoms improved after treatment with carbamazepine. Electromyography (EMG) showed that he had continuous electrical discharges both at rest and during sleep. These discharges completely disappeared after the peripheral nerve was blocked with Lidocaine. An evoked electromyogram showed suppression of abnormal discharges after the F response. These electrophysiological findings indicated that the disorder originated in the spinal anterior horn cells. CT scanning showed a large cisterna magna in the posterior cranial fossa. Protein in the cerebrospinal fluid was elevated.

The Rett syndrome and CSF lactic acid patterns.

We investigated both blood and cerebrospinal fluid (CSF) lactate and pyruvate levels in seven girls with the Rett syndrome (RS) and evaluated the relationship between CSF lactate and pyruvate levels and the clinical manifestations, particularly seizures, anticonvulsant medication, and breathing dysfunction including breath holding, apnea and hyperventilation. Elevated lactate and pyruvate levels in CSF with normal serum lactate were found in two RS patients. Elevated CSF lactate correlated significantly with the clinical occurrence of hyperventilation (P0 = 0.048, Fisher exact probability). We measured native and dichloroacetate (DCA)-activated pyruvate dehydrogenase (PDH) complex activities in two patients (#1 and 2) using cultured lymphoblastoid cell lines which were transformed by EB virus and the results were normal. We also analyzed CSF citric acid intermediates from 7 RS patients including citric acid, cis-aconitate, alpha-ketoglutarate, succinate, fumarate, malate and oxaloacetate. These concentrations were not significantly different from those control patients (N = 21). An elevated lactate level may be a clue to clarify the etiology of RS.