Allelic loss mapping and physical delineation of a region harboring a putative thymic lymphoma suppressor gene on mouse chromosome 12.

Our previous allelic loss analysis of gamma-ray induced thymic lymphomas in F1 hybrid and backcross mice between BALB/c and MSM strains mapped the Tlsr4 region exhibiting a high frequency of allelic loss (62%) to a 2.9 cM interval between the markers D12Mit53 and D12Mit279 on mouse chromosome 12. To narrow further the interval harboring a putative tumor suppressor gene, a high-density scan has been carried out for informative 361 thymic lymphomas. Construction of a physical map of Tlsr4 with 3 YAC and 15 BAC clones and isolation of YAC- and BAC-derived polymorphic probes lead to fine allelic loss mapping. Three successive polymorphic sites within one BAC exhibit the retention of both alleles in seven, one and four lymphomas, suggesting that a common region of allelic loss for Tlsr4 exists within the BAC region. Pulsed-field gel electrophoresis of NotI digests of this and other clones determines that the commonly lost region is a 35 kb interval with a NotI site. NotI sites are frequently associated with coding regions, and our preliminary sequencing has identified ESTs in the region. Thus, the present study facilitates the identification of genes in the Tlsr4 region that would lead to isolation of a novel tumor suppressor gene.

Allelic loss analysis of gamma-ray-induced mouse thymic lymphomas: two candidate tumor suppressor gene loci on chromosomes 12 and 16.

A total of 429 gamma-ray-induced thymic lymphomas were obtained from F1 and backcross mice between BALB/c and MSM strains, about a half of which carried a p53-deficient allele. A genome-wide allelic loss analysis has revealed two loci exhibiting frequent allelic losses but no allelic preference, one is localized within a 2.9 cM region between D12Mit53 and D12Mit279 loci on chromosome 12, and the other is near the D16Mit122/D16Mit162 loci on chromosome 16. The frequency of allelic loss in the D12Mit279 region is 62% and does not differ in tumors between the presence and absence of the p53-deficient allele. In contrast, the loss frequency of D16Mit122 is raised by the existence of p53-deficient allele: 62% for p63(-/+) and 13% for p53(+/+), suggesting co-operative function of the two losses. The D12Mit279 and D16Mit122 regions probably harbor different types of tumor suppressor gene that play key roles in lymphoma development.

Homozygous deletions and point mutations of the Ikaros gene in gamma-ray-induced mouse thymic lymphomas.

Our previous genome-wide analysis of allelic loss for thymic lymphomas that were induced by gamma-irradiation in F1 hybrid mice between BALB/c and MSM strains suggested the centromeric region on chromosome 11 as a site harboring a tumor suppressor gene. Interestingly, to this region the mouse Ikaros gene was mapped which was postulated to participate in oncogenic process from the study of Ikaros knockout mice. Here we show fine allelic loss mapping in the vicinity of Ikaros in 191 lymphomas, indicating that the critical region of allelic loss was centered at the Ikaros locus. PCR analysis revealed that nine lymphomas failed to give PCR-amplification for either of two exon primer pairs, indicative of homozygous deletion. Six and five mutations were detected in the N-terminal zinc finger domain and the activation domain of Ikaros, respectively, and six of the eleven were frameshift or nonsense mutations that resulted in truncation of Ikaros protein. The results strongly suggest a direct role for Ikaros in development of mouse thymic lymphomas. This provides the experimental basis for further analysis of Ikaros mutations in human cancer.

Evidence for phase separation in the membrane of an osmotically stabilized fatty acid auxotroph of E. coli and its biological significance.

1. An unsaturated fatty acid auxotroph of Escherichia coli accumulated a high content of saturated fatty acids in its membrane when it was cultured under osmotically stabilized conditions. The physicochemical properties of the phospholipid extracts and of the membrane fraction from the cells were investigated by means of proton magnetic resonance, infrared spectroscopy and differential scanning calorimetry. 2. Physicochemical studies indicate that the phospholipid bilayers in the membranes exhibit at least two phase transitions, a minor one at approx. 19 degrees C and a major one at approx. 43 degrees C. Between the two temperatures, gel and liquid crystalline domains co-exist. Moreover, even in the gel state, phospholipids seem to segregated into domains containing different proportions of unsaturated fatty acids. 3. The Arrhenius plot of beta-galactoside transport rates is biphasic. The inflection point is at 22 degrees C. This means that the appearance of the fluid region in the bilayer at approx. 19 degrees C is important in the activation of membrane transport.

Separation of aromatic isomers on cyclophane-bonded stationary phases.

A cyclophane (CP66)-bonded silica gel stationary phase (CP66-SP) was prepared and the retention of water-insoluble hydrophobic compounds on it was investigated in comparison with that on the CP44-bonded stationary phase (CP44-SP) reported previously. Like CP44-SP, it retained aromatic compounds more strongly than the corresponding alicyclic compounds, as was expected by the cavity size of the cyclophane. The CP66-SP also showed isomer-selectivity for monosubstituted and disubstituted naphthalenes, but its selectivity was perfectly reversed to that of the CP44-SP. On the CP66-SP, isomers having methyl and ethyl groups at beta-position were eluted prior to those having groups at alpha-position, whereas on the CP44-SP beta-substituted naphthalenes were retained more strongly than alpha-substituted ones. Isomers of three- and four-ring aromatic compounds were also separated on these cyclophane-bonded stationary phases. The retention order on the CP66-SP was almost opposite to that on the CP44-SP; on the CP66-SP, the retention order was phenanthrene > anthracene, and chrysene > 1,2-benzanthracene > 2,3-benzanthracene, whereas on the CP44-SP, anthracene > phenanthrene, and 2,3-benzanthracene > chrysene > 1,2-benzanthracene. The retention mechanism of aromatic compounds is discussed on the basis of the structure of the cyclophane-involved complex.

[Character of a human cholangiocarcinoma CHGS, serially transplanted to nude mice].

With the progress in surgical technique, remarkable improvement has been noted in the treatment of bile duct carcinoma. However, in the cholangiocarcinoma at porta hepatis or in the progressive carcinoma, many cases have been reported, for which radical surgery is not achievable. In recent years, discussion has been concentrated on the necessity of multidisciplinary treatment for the bile duct carcinoma, but fundamental research has not been done enough. In the present paper, the process for obtaining CHGS strain implantable to the nude mouse derived from a human cholangiocarcinoma as achieved in our department was discussed, and its biological characteristics-above all, the sensitivity to carcinostatic agents and to radiation-were evaluated. The doubling time of CHGS strain is 6.2 days, and nude mice showed stable proliferation with 100% viability. Histologically, it was tubular adenocarcinoma similar to the primary tumor. It has high mucin producing ability, and necrosis hardly occurs. The search for DNA ploidy by flow cytometry revealed the presence of two types of cells: The cells of diploid pattern and aneuploid pattern. In the tests to determine the sensitivity of CHGS strains to carcinostatic in MMC, ADR, 5-FU and CDDP groups, and to radiation according to the Battele Columbus Laboratories Protocol, the regression of tumor was observed in MMC, ADR, CDDP groups. Particularly, in MMC group, some of the tumors had disappeared. Recurrence was also noted in this case, but the survival, was still recognized nearly four years after the operation through the postoperative auxiliary therapy. This was regarded as the case, where the sensitivity test using the nude mouse implantable tumor strain was reflected well in clinical application.

Effect of hyperthermia on proliferation and deviation of carcinoembryonic antigen and squamous cell carcinoma-related antigen of human esophageal carcinoma cells in culture.

As a basic study of hyperthermia on malignant tumors, we investigated the kinetics of proliferative activity and the values of tumor markers carcinoembryonic antigen (CEA) and squamous cell carcinoma-related antigen (SCC) in a human esophageal carcinoma cell line, SGF-4, following a change of culture temperature. The temperature range allowing cultured SGF-4 cells to proliferate was from 37 degrees C to 40 degrees C. In an experiment examining the recovery of proliferative activity, no proliferative activity was observed after the cultured cells were exposed to 42 degrees C for 72 hours. The values of CEA and SCC as tumor markers were found to be increased in association with the cell damage due to the change of temperature. These markers could thus be useful as indicators for evaluations of hyperthermia therapy effectiveness.

Pneumatosis cystoides intestinalis after chemotherapy for hematological malignancies: report of 4 cases.

Pneumatosis cystoides intestinalis (PCI) is known to be a relatively rare condition which is characterized by gas cysts in the gastrointestinal mucosa. We treated four cases of PCI accompanied by hematological malignancies during chemotherapy treatment. All cases suffered from abdominal discomfort. Abdominal X-ray films revealed gas cysts in the intestine. PCI was observed during leukocytopenic states, and three cases had septicemia. Etoposide was administered to three cases, and prednisolone to all cases. It is considered that PCI sometimes occurs in patients with hematological malignancies during a period of leukocytopenia, and may be caused by intestinal mucosal damage due to myelosuppressive agents and immunosuppression from prednisolone.

Estimate of gas transfer rates of an intravascular membrane oxygenator.

An asymmetric hollow fiber membrane was prepared from a newly synthesized fluorinated aromatic polyimide (6FDA-6FAP) by using a dry/wet phase inversion process. The membrane was used in a membrane oxygenator over a long period of time. In this study, the potential of the membrane for intravascular membrane oxygenation (IVOX) was studied in respect to oxygen transfer. The gas permeance of the membrane and three commercially available hollow fiber membranes for membrane oxygenators was measured in a gas-gas system and a gas-liquid system and discussed relative to the membrane structures. The oxygen transfer rates of the IVOX devices using these four membranes were estimated by a mathematical kinetic model, with the oxygen permeance measured in the gas-liquid system. The results showed that the device using the 6FDA-6FAP hollow fiber membrane has the highest oxygen transfer rate and is believed to be applicable to IVOX. The methods to determine oxygen transfer rate of a hollow fiber membrane and the mathematical kinetic model, are useful for developing a hollow fiber membrane and a device for oxygenation.

Development of a novel polyimide hollow fiber for an intravascular oxygenator.

The authors have synthesized a novel fluorinated polyimide to develop a membrane material for oxygenators and fabricated polyimide hollow fibers for use in an intravascular oxygenator. A dry/wet phase inversion process has been applied to a spinning process to prepare an asymmetric polyimide hollow fiber. The outer surface of the hollow fiber consists of an ultrathin, dense skin layer, with a calculated apparent thickness of approximately 60 nm. The fiber diameter was 800 microns with a wall thickness of 130 microns. The asymmetric hollow fiber has two advantages because (a) the hollow fiber does not produce plasma leakage due to the dense skin layer of the surface and (b) O2 and CO2 transfer rates through the hollow fiber are enhanced due to the ultrathin skin layer and are significantly larger than those of presently available membrane oxygenators. The blood compatibility of the polyimide hollow fiber without heparinization has been evaluated in vitro. Deformation and aggregation of platelets adherent to the fibers were not observed, and the polyimide suppressed platelet activation. The polyimide significantly reduced the production of anaphylatoxin and also suppressed complement activation.

System design of a bioartificial liver with a high performance hemodialyzer as an immunoisolator using a mathematical kinetic model.

We propose a new bioartificial liver (BAL) system equipped with a high performance hemodialyzer to act as an immunoisolation device. We discuss the design of the BAL system using a mathematical kinetic model with the experimentally obtained mass-transfer performances of various hemodialyzers. The mass transfer resistances of the hemodialyzers did not adversely influence the ammonia-removal and bioactive-substance-supply performances of the BAL system. A suitable hemodialyzer for the BAL system is available even at present using an engineering design. The remaining problems to be overcome before realizing clinical use of the BAL system are to increase the rate constant of the first order reaction of the BAL for ammonia metabolism and to develop a new method of blood access that can be used safely with long term reliability at a high blood flow rate (ca. 556 ml/min).

[Meningococcemia associated with C7 deficiency].

We report a 20-year-old woman who developed meningococcemia. The patient developed fever, vomiting and skin rash, then was sent to our hospital for shock. Physical and laboratory examination revealed septic shock and DIC. Her blood culture grew Neisseria meningitidis (W135). She recovered promptly with PCG, gabexate mesilate and intensive care for shock. Hemolytic activities of the patient's complement were less than 12/CH50 during the course. Screening for each component of the complements suggested that this patient had deficiency of C7. Meningococcal disease has seldom seen in Japan. Early recognition is essential so that appropriate antibiotic therapy and supportive care can be promptly started because shock and death may ensure within hours after onset of symptoms.

Effect of recombinant human granulocyte colony-stimulating factor on combination therapy with aztreonam and clindamycin for infections in neutropenic patients with hematologic diseases.

The present multicenter study was performed to evaluate the effect of recombinant human granulocyte-colony stimulating factor (rhG-CSF) on combination therapy using aztreonam (AZT) and clindamycin (CLDM) to treat severe infection in neutropenic patients with hematologic diseases. Forty-three neutropenic patients with infections (rhG-CSF group) were treated with AZT (2 g) and CLDM (600 mg) 2-3 times daily as well as rhG-CSF (Lenograstim or Filgrastim: 2-5 mu/kg/day). The clinical efficacy of this regimen was compared to that obtained in 44 febrile neutropenic patients, with hematologic diseases, who received only AZT and CLDM in a previous study (historical control group). The overall efficacy rate was 69.8% (30/43) in the rhG-CSF group and 65.9% (29/44) in the historical control group. Although the neutrophil count was significantly increased and C-reactive protein tended to be lower in the rhG-CSF group, the daily maximum body temperature profiles of the 2 groups were nearly the same. These results suggest that rhG-CSF is of little benefit in the treatment of single infectious episodes in neutropenic patients, and that appropriate antibiotic therapy is more important.

Experience of 26 acute renal failure cases.

Data from 26 patients in acute renal failure biopsy have been analysed to define the highest risk factors and to evaluated the reversibility of renal functions. The overall mortality rate was 38 per cent. The mortality (69%) in postsurgical and traumatic cases was significantly higher than that (8%) in cases of obstetrical and medical origin. Survivors and nonsurvivors did not differ in time of initiation of dialysis. There was an increased incidence of gastrointestinal hemorrhage in those who died. A serious underlying disease, infection and gastrointestinal hemorrhage were mainly responsible for deaths. Five of the 16 survivors were left with renal failure and two of them required chronic hemodialysis.

Clinical report on hemodialysis in the Department of Transplantation, Tokai University School of Medicine.

The Department of Transplantation, Tokai University School of Medicine was opened in April 1975. There were 286 patients treated by regular hemodialysis and 30 transplantation patients in the five year period from 1975 to 1980 in our Department. The total number of patients treated by regular hemodialysis alive in June 30, 1980 was 244 (85.3%). In the regular hemodialysis group, the peak age was 30-39 years. The youngest patient was nine years old and the oldest was 85 years old. The male : female ratio of chronic renal failure patients treated by regular hemodialysis was 67 : 33%. The actual five-year survival rate for patients treated by regular hemodialysis was 82%. The most prominent cause of chronic renal failure was glomerulonephritis (86.6%). The major cause of death in regular dialysis patients was gastrointestinal hemorrhage (22.6%). We had 81 cases of acute renal failure and 20 (24.7%) of these patients were treated by hemodialysis. Six cases with no acute or chronic renal failure were treated by hemodialysis.

IgA nephropathy associated with myasthenia gravis and scleritis.

A 29-year-old male patient with IgA nephropathy developed scleritis and myasthenia gravis during the follow-up period. No lymphocytic infiltration or atrophy in muscle fibers was observed in muscle biopsy specimens. However, an immunofluorescent study on the same specimens showed deposits of immunoglobulin A in muscular vessels. The development of scleritis and myasthenia gravis in a patient with IgA nephropathy suggests a common immunologic basis for these disorders.

Acute irreversible rejection after withdrawal of azathioprine in renal allograft recipients.

Azathioprine was withdrawn from five recipients after renal transplantation because of hepatic damage. In three out of five cases, clinical courses were uneventful for more than 1 year after withdrawal (Group A). In the other two cases, rejection episodes occurred 3 and 5 months after withdrawal (Group B). Differences in histocompatibility, the interval between the operation and withdrawal, and the maintenance dosis of prednisolone were not practically striking in either group, but rejection episodes occurred early in group B. We evaluated the results and considered the mechanisms of tolerance after withdrawal of azathioprine because of hepatic damage in renal allograft recipients.

[Myelofibrosis with marked subcapsular bleeding of the spleen--a case report].

A 22-year old male was admitted to Ashikaga Red Cross Hospital because of the progressing abdominal distension in May 1988. The physical examinations disclosed marked hepato-splenomegaly, lymphadenopathy and cachexic state. In the laboratory examinations, anemia, leukocytosis with left shift of nuclei and severe inflammatory reactions were found. Bone marrow aspirations were dry taps, and the biopsy showed myelofibrotic changes. Abdominal computed tomography and ultrasonography revealed a marked hepatomegaly and a giant splenomegaly with subcapsular hematoma. Philadelphia chromosome was negative and neutrophilic alkaline phosphatase score was slightly high. A little amount of peritoneal effusions was obtained and yielded S. aureus. A diagnosis of myelofibrosis with subcapsular hematoma due to spontaneous rupture of spleen and peritonitis of unknown cause was made. Operative indication was considered, but his family did not agree with it. Then conservative therapies for general conditions, those are, administrations of antibiotics and diuretics, and blood transfusions were taken. On the enlarged spleen, total irradiation was done. After the therapies, his spleen got smaller and hematoma was going to be absorbed. A rare case of myelofibrosis with splenic hematoma is reported and the conservative therapies were effective to this case.

[A case of idiopathic spinal epidural hematoma with a fructuation of the symptoms].

We reported a 9-year-old girl with idiopathic spinal epidural hematoma. She complained of her back and neck pain only during night for a few days, followed by days with no symptoms (a few days remission of her symptoms). This episode repeated, and she was suspected to have a psychogenic reaction. However, 28 days later after the first symptoms appeared, paralysis of lower extremities, anesthesia and urinary bladder dysfunction (neurogenic bladder) appeared. Spinal MRI showed a spinal epidural hematoma at the C 5-Th 2 level. An emergency operation of laminectomy and evacuation of the hematoma was done, and she has recovered gradually. We diagnosed her as having idiopathic spinal epidural hematoma. Excerbations of symptoms during nights were thought to be due to an elevation of venous pressure by lying, and transient remissions were due to intermittent small hemorrhages.

[Effect of thermotherapy on human esophageal carcinoma in culture and clinical cases].

Multidisciplinary treatment for advanced esophageal carcinoma, which is not resectable in many cases, has become an important subject to study. We have reported the effectiveness of thermotherapy in treatment of esophageal carcinoma by basic experiments using three cell strains of human esophageal carcinoma designated as SGF-3,4,5. Based on the results of the basic experiments, we have performed thermotherapy combined with radiotherapy, chemotherapy and immunotherapy on cases of advanced esophageal carcinoma since October 1985. As for the basic experiment, proliferation of SGF-3 was inhibited at more than 40 degrees C, SGF-4 at more than 41 degrees C and SGF-5 at more than 42 degrees C. We did not observe the recovery of the growth of SGF-3 after heating at 41 degrees C for 72 hours and 42 degrees C for more than 24 hours. And the growth of SGF-4 after heating at 42 degrees C for more than 48 hours did not recover either. No recovery of the growth of SGF-5 after heating at 42 degrees C for 72 hours was noted. Based on the data, combined therapy was performed in 12 patients with advanced esophageal carcinoma. The evaluation of the local response to the combined thermotherapy revealed CR in five cases, PR in five and NC in two.