[Primary arterial switch operation for complete transposition of the great arteries (type I) of a neonate weighing 1,378 g].

A 2-day-old female baby, delivered by emergent cesarean section at 35 weeks of gestational age with a birth weight of 1,378 g, was referred to our institute for intensive care of heart failure. By echocardiography and cardiac catheterization, the patient was diagnosed with isolated complete transposition of the great arteries. Primary arterial switch operation was performed at 13 days of age. No technical difficulty arose, imposed by the small size of cardiovascular structure. On the 5th postoperative day, surgical repair of intestinal perforation was performed. Convalescence thereafter was uneventful. She returned home on the 64th postoperative day with the body weight of 2,310 g. We conclude that primary arterial switch operation can be a feasible surgical option even in a neonate with very low birth weight.

Th1/Th2 balance in childhood idiopathic nephrotic syndrome.

AIMS: In view of the conflicting evidence of helper T cell type 1 (Th1) or type 2 (Th2) pattern of cytokine synthesis in childhood idiopathic nephrotic syndrome (INS) this study examined the balance of Th1 and Th2 which are characterized by intracellular cytokine production of interferon-gamma (IFNgamma) and interleukin-4 (IL-4), respectively. SUBJECTS AND METHODS: Sixteen children with steroid-sensitive INS (mean age 9.0 years) were included in this study, together with 15 healthy normal children (mean age 7.9 years) for the control group. Intracellular production of both IFNgamma and IL-4 in helper T cell (CD4+ cell) was investigated by a 3-color flow cytometry. RESULTS: The cross-sectional data showed no significant differences of percentages of Th0 (IFNgamma+ IL-4+ CD4+ cell), Th1 (IFNgamma+ lL-4- CD4+ cell) and Th2 (IFNgamma- IL-4+ CD4+ cell) in CD4+ cells (p > 0.05). The Th1/Th2 ratio during nephrotic relapse did not differ from those during nephrotic remission and in normal healthy children (p > 0.05). CONCLUSION: We conclude that there is no significant skew of Th1/Th2 balance in childhood INS and that the cardinal immunological abnormality does not lie in helper T cells but in other cells, such as suppressor/cytotoxic T cells, natural killer cells or monocytes/macrophage. To clarify the pathogenesis of INS, comprehensive studies for these cells would be worthwhile.

Molecular cloning and expression of the cDNA encoding feline granulocyte colony-stimulating factor.

Both genomic DNA and cDNA of the feline granulocyte colony-stimulating factor (G-CSF) gene were cloned from CRFK cells. Southern blot analysis showed that the haploid genome contains a single copy of the G-CSF gene. The RT-PCR analysis of several feline cell lines revealed expression of G-CSF mRNA in response to lipopolysaccharide stimulation. Sequence analysis of genomic and cDNA clones indicated that the intron-exon junction structure is conserved between the human and the feline G-CSF genes. The G-CSF coding region encodes a predicted protein of 195 amino acids including a signal sequence of 21 amino acids. The feline G-CSF amino acid sequence shares a high degree of identity with the canine (90.8%), human (87.4%), ovine (83.9%), bovine (82.8%), porcine (80.5%), murine (70.7%) and rat (66.8%) G-CSF. The feline G-CSF expressed in insect cells using recombinant baculovirus vector was biologically active as measured in a proliferation assay using NFS-60 cells and an induction assay of leukocytes in cats.

Construction of canine herpesvirus vector expressing foreign genes using a lacZ-TK gene cassette as a double selectional marker.

An improved method for constructing canine herpesvirus (CHV) recombinants expressing foreign genes by using the lacZ-TK gene cassette as a double selectional marker was developed. A recombinant CHV carrying the lacZ-TK gene at a targeted gene locus was constructed and used as a parental virus for generating new recombinants. The parental virus formed blue plaques and was sensitive to TK-specific drugs, while newly generated recombinants, in which the lacZ-TK gene was replaced with the desired foreign gene, become both resistant to the TK-specific drugs and formed white plaques. Recombinants were isolated by using the combination of drug selection and color selection. This improved method allows construction of recombinant CHV with great ease, because the drug selection can enrich the frequency of recombinant CHV from 0.01-0.1% to 10-80%. This method was employed to construct a recombinant CHV that expressed rabies virus (RV) glycoprotein (G protein).

Biological and immunogenic properties of rabies virus glycoprotein expressed by canine herpesvirus vector.

In order to evaluate whether canine herpesvirus (CHV) could be used as a live vector for the expression of heterologous immunogenes, we constructed a recombinant canine herpesvirus (CHV) expressing glycoprotein (G protein) of rabies virus (RV). The gene of G protein was inserted within the thymidine kinase gene of CHV YP11mu strain under the control of the human cytomegalovirus immediate early promoter. The G protein expressed by the recombinant CHV was processed and transported to the cell surface as in RV infected cells, and showed the same biological activities such as low pH dependent cell fusion and hemadsorption. The antigenic authenticity of the recombinant G protein was confirmed by a panel of monoclonal antibodies specific for G protein. Dogs inoculated intransally with the recombinant CHV produced higher titres of virus neutralizing antibodies against RV than those inoculated with a commercial, inactivated rabies vaccine. These results suggest that the CHV recombinant expressing G protein can be used as a vaccine to control canine rabies and that CHV may be useful as a vector to develop live recombinant against other infectious diseases in dogs.

[An autopsied case of purulent meningitis associated with ocular flutter].

We report a 72-year-old autopsied case of purulent meningitis associated with ocular flutter. She was admitted to our hospital because of disturbances of consciousness and fever. Physical examination revealed fever, tachycardia, and tachypnea. Neurological examination showed disturbance of consciousness (Japan Coma Scale 30), agitated state, anisocoria, sluggish and fixed reaction of pupils to light, and nuchal stiffness. Routine blood examination showed leukocytosis, thrombocytopenia, positive CRP, and elevated myocardial enzymes. Cerebrospinal fluid revealed pleocytosis with predominant leukocytes, elevated protein, and decreased glucose (22% of blood glucose), and Streptococcus pneumoniae was proved in culture. Brain CT scan revealed no abnormal findings. Electrocardiography showed tachycardia, left axis deviation, and elevated ST segment in aVF, and V3-V6. Ultrasonic echocardiography revealed slight hypokinesis of the left anterior wall, septum, and apex. She was diagnosed as having purulent meningitis, myocarditis, probable encephalitis. Thus, antibiotics, acycrovir, glycerol, and aspirin were administrated. But her respiration deteriorated and ocular flutter was observed for 15 minutes. After that, She required artificial ventilation and eventually died after 29 hours the admission to our hospital. Pathological examination revealed leukocyte accumulation in the arachnoid space of the derebral surface, especially frontal and parietal lobes. Uncal herniation was not observed. The brainstem and cerebellum were histologically within normal limits. These findings suggest that ocular flutter observed in this patient was caused by functional damage of the brainstem.

Development of an enzyme-linked immunosorbent assay using recombinant chicken anemia virus proteins expressed in a baculovirus vector system.

Recombinant baculoviruses were constructed to express the putative proteins VP1, VP2 or VP3 of the chicken anemia virus (CAV). The recombinant VP1, VP2 or VP3 were detected by SDS-PAGE, and their molecular weights were 50, 30/27 and 16 kDa, respectively. The VP2 and VP3 reacted with sera from CAV-infected chickens in Western blot analysis and when used as an enzyme-linked immunosorbent assay (ELISA) antigen, but VP1 did not. Antibodies to CAV were detected, by ELISA using crude insect cell lysates containing VP2 or VP3, from 2 to 20 weeks or 2 to 7 weeks after CAV infection, respectively. These findings indicate that recombinant VP2 and VP3 expressed in the baculovirus vector system can be used as antigens to detect anti-CAV antibodies in ELISA.

Cloning of the enomycin structural gene from Streptomyces mauvecolor and production of recombinant enomycin in Escherichia coli.

A genomic DNA library from the enomycin (ENM) producer, Streptomyces mauvecolor, was screened for the ENM structural gene (enm) by use of a segment of the phenomycin gene (phm) as the probe, and a plasmid, pENI, was constructed. By primer-walking along the insert, a 573 bp DNA sequence that contain an ORF corresponding to preENM was determined. The deduced amino acid composition of ENM was close to that previously reported (MIZUNO, S.; K. NITTA & H. UMEZAWA: Mode of action of enomycin, an antitumor antibiotic of high molecular weight. I. Inhibition of protein synthesis. J. Biochem. 61: 373 approximately 381, 1967). The producer cells expressed enm during an ENM-productive fermentation. An enm-expression plasmid, pENE 1, was constructed, with which E. coli AD202 was transformed. The transformant produced a fusion protein consisting of glutathione-S-transferase (GST) and ENM. The genetically engineered ENM (rENM) inhibited the growth of Hela cells in vitro. Comparison of the base sequence spanning enm with that spanning phm showed that the structural genes were conserved more extensively than were the flanking regions, though the genes were unlikely to be essential to the lives of the producers.

Prostaglandin E2 increases the calcium concentration in rat brown adipocytes and their consumption of oxygen.

Effects of prostaglandin E2 (PGE2) were examined on the oxygen consumption and intracellular calcium concentration of rat brown adipose tissue (BAT). PGE2 0.1 nM-1 microM increased oxygen consumption of the tissue blocks of BAT, with a maximum 2-13 min after PGE2 administration. PGE2 was most effective at 1 and 10 nM, and the oxygen consumption was elevated for over 40 min. Pretreatment of BAT with indomethacin, a prostaglandin synthesis inhibitor, did not affect the increase in oxygen consumption induced by noradrenaline. PGE2 at 1-10 nM gradually increased the intracellular calcium concentration of freshly dispersed single brown adipocytes by 3-4 times in 30 min. PGF2 also increased the intracellular calcium concentration of brown adipocytes in calcium-free medium. These results raise the possibility that PGE2 and noradrenaline affect heat genesis and metabolism of BAT independently.

Characterization of pseudorabies virus glycoprotein gII expressed by recombinant baculovirus.

The gene encoding the complete glycoprotein gII (homologue of gB of herpes simplex virus) of pseudorabies virus (PrV) was inserted into a baculovirus transfer vector, and a recombinant virus expressing gII was isolated. Three gII-related recombinant baculovirus-expressed peptides of 100, 60, and 45 to 50 kDa were detected with a polyclonal antibody against gII; these correspond to the authentic subunits gIIa and its cleavage products gIIb and gIIc, respectively. These proteins were subjected to N-terminal sequencing, and the results showed that the protease cleavage sites were identical to those of authentic gII. The expressed gII was shown to be transported to the surface of infected cells as judged by an indirect immunofluorescence test. Antibodies raised in mice immunized with the recombinant gII neutralized the infection of PrV in vitro. Mice inoculated with the recombinant gII were completely protected from lethal challenge with PrV.

Characterization of rabies virus glycoprotein expressed by recombinant baculovirus.

A cDNA of the glycoprotein (G protein) gene of rabies virus Nishigahara strain was cloned and inserted into a baculovirus genome under the control of the polyhedrin promoter. Infection of Spodoptera frugiperda cells with this recombinant virus produced a large quantity of new protein instead of the parental polyhedrin protein. By immunofluorescent and immunoblotting analyses, the recombinant protein was antigenically similar to the authentic G protein. Its molecular mass estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, however, was slightly smaller than that of the authentic one, and this observation was suggested to be due to the difference in glycosylation level between the two G proteins. The recombinant G protein expressed on the cell surface of the insect cells showed a fusion activity at low pH. The fusion activity was inhibited by antiserum against either whole virions or G protein of rabies virus.

Reconstitution of influenza virus RNA polymerase from three subunits expressed using recombinant baculovirus system.

Influenza virus RNA polymerase catalyzes multiple step reactions in transcription and replication of the genome RNA. The core enzyme is composed of each one of the three P proteins, PB1, PB2 and PA (Honda et al. (1990) J. Biochem. 107, 624-628). For detailed analysis of the role of each P protein and of the functional domains on each P polypeptide, we expressed individual P proteins in cultured insect cells after infection with recombinant baculoviruses. PB1 and PB2 accumulated in cell nuclei whereas PA stayed in cytoplasm. Both the PB1 and PB2 proteins were purified from aggregates in the respective nuclear extract, and the PA was partially purified from the cytoplasm. RNA polymerase was reconstituted by mixing the three P proteins in a urea solution and then dialyzing against a reconstitution buffer. The reconstituted enzyme was able to transcribe model RNA templates. Minus-sense RNA was a better template than plus-sense RNA.

Wire-directed detachable balloon. Work in progress.

A new silicone detachable balloon has two self-sealing valves. The proximal valve grips the catheter tip, and the distal valve allows a guide wire to pass through. The balloon is advanced over the guide wire. Detachment is performed after the wire is withdrawn. Five balloons were successfully placed in the intended arteries and veins of three dogs. This wire-directed detachable balloon is placed more easily and accurately than the conventional detachable balloons that are placed with the flow-directed method.

[Study on argyrophilic inclusions of multisystem atrophy (Oppenheimer)].

UNLABELLED: Non-hereditary olivo-ponto-cerebellar atrophy (OPCA) and striato-nigral degeneration (SND) have been looked upon as a single disease entity called multisystem atrophy (MSA) by Oppenheimer. This study revealed that both intracytoplasmic argyrophilic inclusions (AI) in pontine neurons and glial (argyrophilic) cytoplasmic inclusions (GCIs) widely distributed in the CNS are characteristics of MSA. MATERIALS: a) 12 cases with MSA, b) 16 cases with autosomal dominant (AD) form of spinocerebellar degeneration (SCD): AD form of OPCA 5 cases, Joseph disease 4 cases, AD-dentatorubropallidoluysian atrophy (Naitoh & Oyanagi's form) 6 cases, AD-spastic ataxia (Brown) 1 case, c) 4 cases with autosomal recessive (AR) form of SCD: AR form of OPCA 1 case, myoclonic epilepsy with ragged-red fibers (MERRF) 1 case, complicated form of spastic paraplegia 2 cases, d) 6 cases with non-hereditary SCD including intoxications: late cortical cerebellar atrophy 1 case, alcoholic cerebellar degeneration 2 cases, phenytoin-induced cerebellar degeneration 1 case, neuroleptic malignant syndrome 1 case, and e) 27 cases with other neuropsychiatric diseases: Alzheimer disease 20 cases, progressive supranuclear palsy 5 cases, schizophrenia 2 cases. METHOD: We examined 10 mu-thick paraffin sections stained with HE, Klüver-Barrera, Bodian, Holzer, Gallyas, and Bielschowski methods. RESULTS: AI in pontine neurons were found only in two cases of MSA. Interestingly no AI could be detected even in cases with AD form of OPCA showing mild degeneration in the pontocerebellar system. On the other hand, GCIs were found in all cases with MSA irrespective of the degree of degeneration in the olivo-ponto-cerebellar or striato-nigral system. However, there was no GCIs in cases with other form of SCD and other neuropsychiatric diseases. Gallyas stain was the best method for detecting GCIs. GCIs were widely distributed in the CNS except for superficial layers of the cerebral cortex, the cerebellar cortex, and the dorsal column of the spinal cord. There were also many GCIs in the putamen, pontine base, and cerebellar white matter, even though these sites were well preserved.

Enzyme-linked immunosorbent assay for the detection of canine coronavirus and its antibody in dogs.

Two methods of enzyme-linked immunosorbent assay (ELISA) were developed for the diagnosis of canine coronavirus (CCV) infection in dogs. One ELISA, in which CCV-infected CRFK cell lysate is used as antigen, is for the detection and titration of antibody against CCV, and the other ELISA uses the double antibody sandwich method for the detection of CCV antigen. The first ELISA procedure demonstrated antibody responses in dogs inoculated with CCV, as did the virus neutralization test; the second ELISA detected specific CCV antigen in feces and organ homogenates of inoculated dogs.

Serodiagnosis of canine herpesvirus infection--development of an enzyme-linked immunosorbent assay and its comparison with two improved methods of serum neutralization test.

An enzyme-linked immunosorbent assay (ELISA) for the diagnosis of canine herpesvirus (CHV) infection using antigen prepared by solubilizing infected cells was developed. The ELISA and two improved methods of serum neutralization test, the microplate serum neutralization test (MSNT) with complement and the 50% plaque reduction (PR) assay with complement, were compared for the results of antibody detection from a total of 557 field canine sera. Of 529 sample sera that were negative in the MSNT with complement, 119 were ELISA positive, and this result together with time course of serum antibody detection in a dog experimentally infected with CHV strongly suggested that the MSNT with complement is less sensitive for the detection of antibody in CHV infected dogs, especially those in early stages of infection. A correlation was found between the titers measured by the ELISA and 50% PR assay with complement, however, for field use, the ELISA is recommended as a highly sensitive test method of serodiagnosis of CHV infection adequate for dealing with a large number of samples with less demand on time and effort.

[Can radiologic features of community-acquired pneumonia presume etiologic agents?].

During a one-year and nine months (from June 1987 to February 1989) survey of community-acquired pneumonia, we investigated in 130 patients if radiologic features presume etiologic agents. Incidences of etiologic agents are 21 (16%) pneumococcus, 18 (14%) mycoplasma, 14 (11%) tuberculosis, 12 (9%) hemophilus, and 54 (42%) unknown agents, respectively. In correlates of radiologic features and etiologic agents, alveolar shadows spreading bilateral lungs presume tuberculosis and pneumococcal pneumonia. Lobar distributing alveolar shadows presume pneumococcal, mycoplasmal tuberculous diseases and other agents, equally. Segmentally distributing shadows presume pneumococcal and mycoplasma pneumonia. Radiologic subgrouping features of alveolar shadows composed of acinar, lobular, and lobar shadows did not presume specific agents. Centrilobular (peribronchiolar) shadows suspect hemophilus infections. Pleural fluid accumulations suspect tuberculosis and anaerobic infections and cavitary shadows, tuberculosis, respectively. Radiologic features can presume etiologic agents.