Identification of an ion channel-forming motif in the primary structure of CFTR, the cystic fibrosis chloride channel.

Synthetic peptides with sequences representingputative transmembrane (M) segments of CFTR (the cystic fibrosis transmembraneconductance regulator) were used as tools to identify the involvement of suchsegments in forming the ionic pore of the CFTR Cl- channel. Peptides withsequences corresponding to M2 and M6 form anion-selective channels afterreconstitution in lipid bilayers. In contrast, peptides with the sequences ofM1, M3, M4, and M5, or peptides of the same amino acid composition as M2 and M6but with scrambled sequences, do not form channels. Conductive heterooligomersof M2 and M6 exhibit a single channel conductance of 8 pS (in 0.15 M KCl) and a95% selectivity for anions over cations, properties that emulate both theconductance and the selectivity of the authentic CFTR channel. Theidentification of sequence-specific motifs that account for key functionalattributes of the CFTR channel suggests that such modules may representfundamental units of function and are plausible constituents of the pore-formingstructure of the CFTR Cl- channel.     

The relation between EEG and mental development following cardiac surgery performed under simple deep hypothermia in children

In order to assess the accuracy of electroencephalography (EEG), in children who have undergone cardiac surgery under simple deep hypothermia, the relation between IQ or schoolwork achievement and the duration of circulatory arrest was investigated in 75 such children. Abnormal preoperative EEG's were found in 16 per cent of the children while abnormal postoperative EEG's were found in 17 per cent. The children were divided into 4 groups, according to pre- and postoperative EEG results. Schoolwork achievement scores ranged between 3.0 and 3.2, the difference among the groups being insignificant. Moreover, no significant shift in IQ was found among the groups. Finally, regarding the number of children who were able to go on to a higher level of education, including high school the college or university, again no significant differences were found among the 4 groups. In a comparison with the number of such children in neighboring Nagasaki prefecture able to continue on to a higher level of education, no significant differences were seen either. The findings and statistics of this investigation therefore indicate that pre- and postoperative EEG's are not always a reliable reference for assessing the prognosis of cerebral activity.     

Congenital pseudarthrosis of the tibia: analysis of the histology and the <Emphasis Type="Italic">NF1</Emphasis> gene

Background Congenital pseudarthrosis of the tibia (CPT) is frequently, but not always, associated with neurofibromatosis type 1 (NF1). Double inactivation of the NF1 gene has been reported to be the pathogenesis of CPT in NF1 cases. Methods We analyzed the loss of heterozygosity (LOH) of the NF1 gene in cases of CPT with NF1 to examine whether double inactivation was seen in the case. In addition to morphological analysis, immunoexpression of differentiation markers was examined. Results and discussion The tibia tapered with the zone phenomenon from mature to immature bone with osteoblastic rimming, resembling osteofibrous dysplasia. Osteosclerotic bowed bone with a small number of osteoclasts suggested dysfunction of bone remodeling. Fibrous tissue at the site of pseudarthrosis was associated with the periosteum and demonstrated myofibroblastic differentiation accompanied by massive cartilage formation, suggesting some misdirection during the differentiation of periosteum to myofibroblasts or chondrocytes. LOH of the NF1 gene locus was not seen in fibrous tissue. This result suggests that CPT is not accompanied by double inactivation in every NF1 case.     

Point mutation in the band 4.2 gene associated with autosomal recessively inherited erythrocyte band 4.2 deficiency

Abstract: A patient who represented acute hemolytic crisis was studied. Analysis of the erythrocyte membrane proteins by SDS-PAGE revealed a deficiency of band 4.2. In the family, the sister of the patient who had been clinically normal was also shown to be deficient in band 4.2. Binding studies showed that the propositus' membranes were able to bind normal band 4.2 protein as much as control. It was suggested that the binding sites for the protein were prepared on the membrane. We analyzed the band 4.2 cDNA of the propositus and detected a mutation that changes a codon for alanine to one for threonine at residue 142. Band 4.2 exon III of genomic DNA which included the mutation site was amplified and sequenced directly in the family members, and it was revealed that only the homozygotes of the mutation allele manifested band 4.2 deficiency and the parents, who were heterozygotes, showed normal amounts of band 4.2. Recently, the same mutation was reported as Protein 4.2^NIPPON in another 4 cases (Bouhassira et al. Blood 1992: 79: 1846–1854). This study supports the hypothesis that this mutation is the pathogenetic cause of band 4.2 deficiency and not a polymorphism.     

Adriamycin-Lipiodol suspension for i. a. chemotherapy of hepatocellular carcinoma

Summary Physicochemical properties of two types of adriamycin preparation, suspensions and emulsions prepared for i.a. chemotherapy of hepatocellular cacinoma, were investigated. A suspension was prepared by dispersing adriamycin directly into the lipid contrast medium, Lipiodol, whereas an emulsion was obtained by emulsifying an aqueous solution of adriamycin into Lipiodol. The dispersibility of the drug in each preparation was examined microscopically. The chemical stability of and drug release from the preparation were determined by high-performance liquid chromatography and spectrophotometry, respectively. The suspension was then given to ten patients with primary hepatocellular carcinoma. The suspension maintained good dispersibility without coagulation of drug particles, whereas coalescence of aqueous droplets and the resultant phase separation occurred 4 h after preparation of the emulsion. Both preparations maintained the initial drug content for at least 1 week at room temperature. The release of adriamycin was more prolonged in the suspension than in the emulsion. After i.a. administration of the suspension, a selective accumulation of Lipiodol in the tumor and decrease in serum -fetoprotein (AFP) levels were found in most patients. A significant amount of adriamycin was still detected in hepatic speciments resected from two patients 1 and 2 months after treatment. These findings suggest that the adriamycin-Lipiodol suspension may be a useful preparation for targeting chemotherapy to hepatocellular carcinoma.     

WIDESPREAD ECZEMA VACCINATUM ACQUIRED BY CONTACTS A Report of an Autopsy Case

A4-moth-old male infant predisposed to allergic dermatitis acquired widespread eczema vaccinatum by contacts with a recently vaccinated sibling. He died of acute purulent peritonitis following a perforation of multiple duodenal ulcers. Fluorescence immunocytochemical and electron microscopic studies on the skin lesions revealed the presence of viral antigens and numerous virus particles compatible morphologically with those of the mature form from the same batch of smallpox vaccine given to the sibling. A large number of virus particles in the developmental form were also predominantly scattered in the cytoplasm of cells at the stratum malpighii of the epidermis as well as in neutrophils and macrophages in the skin lesions. The virus isolation from the skin lesions was done by using the HeLa cells and the human embryonic lung fibroblasts. No abnormal laboratory data were noted in immunoglobulins. On the basis of atrophy of the thymus and other lymphatic tissues and an appearance of large pyroninophilic cells in association with blastoid transformation, the authors discussed a possible participation of the disturbance of cellular immunity secondary to the virus infection in the development of the disease. ACTA PATH. JAP. 29 : 435–455, 1979.     

Abnormal IgG galactosylation in MRL-lpr/lpr mice: pathogenic role in the development of arthritis

MRL-lpr/lpr (MRL/lpr) mice spontaneously develop arthritis by an increase in the incidence of agalactosylated oligosaccharides in serum IgG, similar to rheumatoid arthritis patients. However, whether this association has a pathogenic significance is still unknown. In this study, we analyzed the oligosaccharide structure of serum IgG in various MRL mice with or without arthritis, to clarify the relationship between the oligosaccharide abnormality and the development of arthritis. The level of agalactosylation in serum IgG was comparable in both arthritis-free MRL/lpr and MRL-+/+ (MRL/+) mice at 6 weeks of age. In contrast, the incidence of IgG lacking galactose markedly increased in MRL/lpr mice at 6 months of age (the age at which arthritis occurred), compared with that from age-matched MRL/+ mice without arthritis. However, the proportion of agalactosylated IgG increased similarly in anti-CD4 monoclonal antibody-treated MRL/lpr mice at 6 months of age, despite the absence of the development of arthritis, because of depletion of CD4+ T cells. These results suggest that the abnormality in IgG galactosylation of MRL/lpr mice developed in an age-dependent manner, but it did so independently of CD4+ T cell-dependent B-cell activation and is not a consequence of the development of arthritis.     

Genes predisposing to autoimmunity augment constitutive major histocompatibility complex class II-associated presentation of the self-antigen IgG2a in vivo

The self-antigen IgG2ab is poorly presented to a gamma2ab 435-451-reactive I-Ad-restricted T-cell hybridoma unless available in high concentrations or targeted to Fcgamma- or complement receptors. Environmental factors, probably the extent of microbial challenge, profoundly influence the constitutive gamma2ab/I-Ad presentation in IgCHb, H-2d mice. Here we report also a strong genetic impact. Constitutive presentation was highly efficient in spleen and thymus of (NZB x BXSB)F1 mice, which inherit a predisposition to develop lupus. Presentation correlated with disease progression and the serum levels of IgG2ab and IgG2ab complement factor 3 complexes. The finding that constitutive presentation was by far most efficient in males indicated that it was augmented by the Y chromosome-linked autoimmune acceleration Yaa gene. In line with previous data for healthy mice, constitutive gamma2ab/I-Ad presentation was most pronounced in the adherent spleen cell fraction and improved by further enrichment for dendritic cells. Notably, however, whereas in normal mice the gamma2ab determinant was undetectable on B cells lacking surface IgG2ab, such B cells contributed considerably to constitutive presentation in (NZB x BXSB)F1 hybrids. Presumably this resulted from complement receptor-mediated internalization of IgG2ab-containing immune complexes formed in lupus. These data add to the evidence that B cells with self-reactive receptors, known to exist in the mature repertoire, may present non-cognate foreign antigen to anti-foreign helper T lymphocytes and thus differentiate into autoantibody-secreting cells, and might likewise account for the polyclonal B-cell activation characteristic of several autoimmune syndromes.     

Regulation of CD23 in the chronic inflammatory response in asthma: a role for interferon-gamma and heat shock protein 70 in the TH2 environment.

BACKGROUND: Monocytic cells and alveolar macrophages (AMs) are activated in patients with asthma, producing inflammatory cytokines. This occurs despite a TH2 environment that consists of the cytokines interleukin (IL) 4, IL-10, and IL-13. The mechanism by which this occurs may involve cross-linking of the low-alphaffinity IgE receptor CD23. OBJECTIVE: To determine the effect of the TH2 environment with interferon-gamma (IFN-gamma) and heat shock protein 70 (HSP 70) on CD23 receptor expression and tumor necrosis factor alpha (TNF-alpha) production. METHODS: We examined the effect of IL-4 and IL-13 in culture with IFN-gamma and HSP 70 on CD23 expression in both THP-1 cells and AMs from healthy controls via flow cytometry. AMs from mild asthmatic patients and THP-1 cells were evaluated for TNF-alpha production after cross-linking CD23 with immune complexes. RESULTS: Asthmatic AMs stimulated with anti-IgE exhibited a 5.7- +/- 1.9-fold increase in TNF-alpha protein. AMs from healthy controls increased the geometric mean +/- SD of CD23 2.00- +/- 0.50-fold in IL-4 and 2.14- +/- 0.50-fold in IL-13. THP-1 cells cultured with IL-4 and IL-13 then stimulated with IFN-gamma or HSP 70 increased CD23 expression above baseline as follows: IL-4, 2.16- +/- 0.31-fold; IL-13, 2.66- +/- 0.43-fold; IFN-gamma, 2.03- +/- 0.34-fold; IL-4/IFN-gamma, 9.14- to 4.02-fold; IL-13/IFN-gamma, 11.51- +/- 5.51-fold; IL-4/HSP, 5.20- +/- 0.61-fold; and IL-13/HSP, 5.60- +/- 0.79-fold. Stimulating the CD23 receptor with immune complexes significantly increased TNF-alpha production by THP-1 cells stimulated with IFN-gamma, IL-4, IL-13, or a combination of these. CONCLUSIONS: Both IFN-gamma and HSP 70, in the TH2 environment, up-regulate CD23 expression and thus may play an important role in maintaining the chronic inflammatory state in asthma.     

Characteristics of an anti-eosinophil monoclonal antibody that recognizes granulocytes from patients with blood eosinophilia but not from subjects without eosinophilia.

To investigate cell surface antigens of activated human eosinophils using monoclonal antibodies, we established a murine anti-human eosinophil monoclonal antibody AE500 by immunizing with blood eosinophils from patients with idiopathic hypereosinophilic syndrome (HES) and characterized the reactivity to a variety of human leucocytes by a fluorescence-activated cell sorter. AE500 reacted with blood eosinophils and neutrophils in nine out of 11 patients with marked eosinophilia (greater than or equal to 2500/microliters) (seven with idiopathic eosinophilia including HES and two with asthma), but not with those in asthmatic patients with mild eosinophilia (n = 10) or in healthy subjects (n = 8). AE500 did not react with blood lymphocytes, monocytes or platelets. AE500 did not react with human myeloid or lymphoid cell lines, including eosinophilic leukemia cell lines EOL-1 and EOL-3. The reactivity of AE500 to blood eosinophils and neutrophils in patients with marked eosinophilia changed in relation to blood eosinophil counts and prednisolone therapy. In addition, the reactivity of AE500 to blood eosinophils was increased in three out of four AE500-positive eosinophils by the incubation of the cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) at 37 degrees C for 30 min, but not with interleukin 3 or interleukin-5. These results suggest that the anti-eosinophil antibody AE500 detects a cell surface antigen expressed on blood granulocytes in a hypereosinophilic state. This anti-eosinophil antibody would be useful for analysing the mechanism of eosinophilia.