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).

Gas transfer and blood compatibility of asymmetric polyimide hollow fiber.

We have fabricated an asymmetric polyimide hollow fiber for use as a membrane oxygenator. A dry/wet phase inversion process has been applied to a spinning process to prepare the hollow fiber. The fiber structure consisted of a complete defect-free skin layer and a porous substructure characterized by the presence of an open-cell structure and macrovoids. The outer diameter was 480 microm with a wall thickness of 50 microm. Transfer rates of O2 and CO2 in the asymmetric polyimide fiber were 2.3 x 10(-5) and 1.1 x 10(-4) (cm3 (STP)/(cm2 s cmHg)), respectively, which were four times higher than those measured in the polydimethylsiloxane (PDMS) fiber of the presently-available membrane oxygenator. The (QO2/QN2) selectivity of the polyimide fiber was 4.9, indicating that the surface skin layer is essentially defect-free. The blood compatibility of the polyimide hollow fiber has been evaluated in vitro and in vivo. The polyimide had an excellent blood compatibility when compared with PDMS.

[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.

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.

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.

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.

Accumulation of aberrant Y chromosomes in gamma-ray-induced thymic lymphomas lacking p53.

Male F(1) hybrids between MSM mice carrying a deficient p53 allele and BALB/c mice were irradiated with gamma-rays, and 80 thymic lymphomas were obtained, 46 of which developed in mice carrying the deficient p53 allele. Because the Y chromosome contributes little to cellular function, the stability of the Y chromosome in the tumors was assessed by polymerase chain reaction by examining three genes: Smcy and Sry on the short arm and Sts in the pseudoautosomal region of the long arm of the Y chromosome. Twenty-one lymphomas had lost one or two genes, probably as a result of mitotic recombination or interstitial deletion, whereas no lymphomas had lost all three genes. The p53 status of the lymphomas was determined by genotyping and allelic loss analysis; 34 had retained two wild-type p53 alleles, suggesting normal function; 34 had lost both alleles, indicating loss of function; and the other 12 had at least one wild-type p53 allele, so their p53 status was unclear. Compilation of these data revealed that changes in the Y chromosome were detected in only two of the 34 lymphomas retaining functional p53 but in 18 of the 34 lymphomas lacking p53 function, suggesting that p53 deficiency leads to an increase in the accumulation of radiation-induced aberrant chromosomes. This is consistent with our previous result from analysis of the inactive X chromosome. In contrast, a decrease in the fidelity of mitotic transmission in p53-deficient lymphomas was not noted for the Y chromosome.

Photoinactivation of vesicular stomatitis virus with fullerene conjugated with methoxy polyethylene glycol amine.

Fullerene is known to effectively produce mainly singlet oxygen by absorbing light energy, and therefore may be useful as a virucidal photosensitizer. This study was designed to investigate the virucidal activity of a water-soluble fullerene derivative, which is conjugated with methoxy polyethylene glycol amine to enhance its water solubility. Vesicular stomatitis virus (VSV) was inactivated upon illumination with this water-soluble fullerene, in a concentration- or dose-dependent manner. The titer of VSV was reduced by >5log 10 in the presence of 10 mg/ml (400 microM) fullerene derivative with 120 J/cm2 white light irradiation. VSV inactivation was inhibited by oxygen removal or by the addition of sodium azide, a known singlet oxygen scavenger. The substitution of H2O by D2O, which is known to prolong the lifetime of singlet oxygen, promoted the virucidal activity. These results indicate that singlet oxygen may play a major role in VSV photoinactivation by the water-soluble fullerene derivative. The concentration needed for virus inactivation is higher than that of other sensitizers such as methylene blue.

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.

Mutations in the p53 and scid genes do not cooperate in lymphomagenesis in doubly heterozygous mice.

Analysis of double mutant mice of the p53 and scid genes, which have a combination of cell cycle checkpoint/apoptosis and DNA repair defects, shows that the latter defect synergistically enhances lymphoma development with loss of the former function. These mice lack the ability to eliminate lymphocytes predisposed to neoplastic transformation resulting from faulty antigen receptor gene rearrangement. Here we examine the cooperativity in double heterozygotes of p53 and scid in which normal development of lymphocytes is not impaired. MSM mice carrying a p53-knockout allele were crossed with BALB/c mice heterozygous for the scid locus and 129 offspring were obtained. They were subjected to gamma-ray irradiation, 84 thymic lymphomas being generated. The tumors and host mice were genotyped of p53 and scid. Among 42 mice developing p53-deficient lymphomas, scid/+ and +/+ genotypes did not provide difference in onset and latency. Besides, allelic loss of the Scid gene occurred at a high frequency in those lymphomas but the loss exhibited no allelic bias. The results suggest that the scid/+ genotype is not a modifier of loss of p53 function in the double heterozygotes.

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.

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.

[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.

Genetic and physical delineation of the region of the mouse deafness mutation shaker-2.

A total of 951 backcross progeny have been obtained from a backcross segregating for the mouse deafness mutation, shaker-2(sh-2). Linkage analysis provides a detailed genetic map in the vicinity of sh-2 which comprises 40 backcross mice identified as recombinant within a 4 cM region. This allows construction of a contig consisting of 21 BAC clones across an approximately 700-kb region of sh-2. This covers the entire nonrecombinant region of sh-2 and is therefore useful to facilitate the identification of genes in the sh-2 region.

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.

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.

[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.

Temperature sensitivity on proliferation and morphologic alteration of human esophageal carcinoma cells in culture.

As basic studies of hyperthermia and hypothermia on malignant tumor, the kinetics of proliferative activity, the morphologic changes in the two cell lines, SGF-3 and SGF-5, established in our department after the change of culture temperature were examined. The results obtained were: a) A significant difference was found in the sensitivity to temperatures between the two cell lines originated from human esophageal squamous cell carcinoma. The temperature range allowing cultured cell to proliferate were from 31 degrees to 39 degrees C in SGF-3 and from 29 degrees to 41 degrees C in SGF-5. b) Minor difference occurred in the results between the two cell lines examined during the recovery of proliferative activity, but no proliferative activity was discovered after the cells were exposed to 42 degrees C for 72 h. Two cell lines resumed their proliferation after having been exposed to 27 degrees or 28 degrees C for 72 h. c) Morphologic changes of the cell lines cultured at high temperature were cytoplasmic vacuolation and cell aggregation by phase contrast microscope and the increase of heterochromatin, the decrease of granular formation in nucleoli, and nucleolar vacuolation by transmission electron microscopy (TEM). At low temperatures the changes observed included cytoplasmic ballooning and circumnuclear halo formation by phase contrast microscope, and the increase of heterochromatin, nucleolar segregation, swelling of mitochondria, and dilatation of rough endoplasmic reticulum (rER) by TEM.