Analysis of components of immune complexes in sera of patients with systemic lupus erythematosus]

The samples of immune complexes (IC) isolated through Clq tube from serum of SLE patients were analysed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting for their antigens and antibodies. The isolated IC contained gamma, mu and L chain of immunoglobulin and various sizes of DNA ranging from 16KD to 55KD. The isolated IC showed antibodies reacting to 16KD DNA. In the isolated IC, DNAs with molecules less than 300 base pairs were detected. These DNA were double-stranded and hybridized with nuclear DNAs originated from human but not from other species.     

Influence of acute-phase proteins on the activity of natural killer cells

The effects of ₁-antitrypsin ( ₁,-AT), ₁,-acid glycoprotein ( ₁AGP), and haptoglobin (Hp), the main constituents of-globulin and which belong to acute phase proteins, on NK activity were examined using K562 cells as the NK target cells. Among the three proteins, ₁,-AT and ₁AGP had inhibitory effects on NK activity for fast target K562 cells. The,-AT preparations having the same protein concentration and a different trypsin inhibitory capacity (TIC) had an equal effect. Although ₁AT and ₁,-AGP equally reduced the NK activity, the mechanism involved in the reduction differed, in that the effect of ₁,-AT directed toward NK cells reduced their binding capacity with the target cells, ₁,-AGP probably interacts with a cytotoxic factor secreted from NK cells following effector-target interaction. These studies suggest that each of the acute-phase proteins, which increase following inflammation, inhibits NK cell function by two distinct mechanisms.     

Production of human interferon-gamma in serum-free medium.

Interferon (IFN)-gamma was produced with a high yield in cultures of human peripheral mononuclear cells by combined stimulation with OK-432 and staphylococcal enterotoxin B. Human mononuclear cells cultured in serum-free medium produced several times as much IFN as those in RPMI-1640 medium containing 10% fetal bovine serum. A synergistic effect of OK-432 and staphylococcal enterotoxin B on the production of IFN-gamma was demonstrated. Ultrogel AcA54 column chromatography of crude IFN showed a single peak with an apparent molecular weight of 43,000. Our production system for human IFN-gamma offers a feasible approach to preparation of large quantities of purified IFN-gamma for structure studies, antibody production, and clinical applications.     

Characterization of diethylstilbestrol‐induced hypospadias in female mice

The urethral duct and vagina are formed from the urogenital sinus (UGS) during the early neonatal period in mice. Neonatal estrogen exposure results in hypospadias, or the malpositioning of vaginal and urethral openings, with wide cleft clitoris. We sought to characterize diethylstilbestrol (DES) influence on UGS morphogenesis and hypospadias formation. Newborn (day 0) and 1–4‐day‐old female mice (ICR/Jcl) were given (s.c.) oil or 3.0 μg DES. Animals were killed 24 hr later; then hypospadias formation and epithelial apoptosis and proliferation within the developing UGS were assessed. DES did not alter normal UGS morphogenesis by day 1, in comparison with controls. However, hypospadias formation was observed in DES‐treated mice by day 3. In these mice, the distal dorsal urethral duct appeared to fuse with and open into the lower vaginal solid cord region. Further, DES treatment produced a gradual significant increase in dorsal urethral epithelial apoptosis (P < 0.05) just prior to and during fusion and hypospadias formation. DES‐induced urethral epithelial and sinus cord proliferation appeared significantly increased (P < 0.05) and unchanged, respectively, just prior to fusion. By day 5, DES‐treated mice exhibited wide cleft clitoris. In addition, if DES was given on day 3 or 5, a gradual, distinct caudal shift in the vaginal‐urethral junction was observed compared to mice treated on days 0–2. Although hypospadias was not induced when neonates were given DES on day 7, these mice continued to display early vaginal opening. Dose‐response analysis indicated that 0.03 μg DES for 5 days is the lowest known critical dose for hypospadias induction. We have shown for the first time that DES‐induced hypospadias onset may primarily be the result of changes in developing dorsal urethral epithelial cell apoptotic and proliferative activity, and that the location of DES‐induced hypospadias formation is dependent on age at time of exposure. Anat Rec 266:43–50, 2002. © 2002 Wiley‐Liss, Inc.     

En(a-) phenotype in a Japanese blood donor

The first Japanese En(a-) individual (T.N.) was found by screening red cells from 250,000 Japanese blood donors with monoclonal anti-Ena. His serum contained no atypical antibodies and his partial red cell phenotype was M-N-S+s-, although a trypsin- resistant N antigen was detected. His red cells were En(a-) and Wr(b-), as determined by various human and mouse monoclonal antibodies. The absence of glycophorin A (GPA) and the presence of apparently normal glycophorin B (GPB) were demonstrated by immunoblotting with antibodies to the extracellular and cytoplasmic domain of GPA and to epitopes common to GPA and GPB. Sialic acid levels of T.N.'s intact red cells were substantially lower than those of control MN cells. Serologic tests suggested that both of T.N.'s parents were heterozygous for a recessive GPA deficiency gene.     

Possible involvement of keratinocyte growth factor and its receptor in enhanced epithelial-cell proliferation and acquired recurrence of middle-ear cholesteatoma

Middle-ear cholesteatoma is characterized by enhanced proliferation of epithelial cells and granular tissue formation. However, the molecular mechanism underlying these pathological changes is largely unknown. Keratinocyte growth factor (KGF) is a mesenchymal cell-derived paracrine growth factor that specifically stimulates epithelial cell proliferation. In the present study, we investigated the possible involvement of KGF and its receptor, KGFR, in the pathogenesis of cholesteatoma using in situ hybridization and immunohistochemistry, respectively. We examined 56 cholesteatoma specimens, and 8 normal skin areas as control. KGF and KGFR expression was examined by immunohistochemistry using rabbit anti-human KGF and anti-human KGFR polyclonal antisera raised in our laboratories against synthetic peptides corresponding to parts of human KGF and KGFR, respectively. KGF protein and mRNA were detected exclusively in stromal fibroblasts and infiltrating T lymphocytes in 80% of cholesteatoma cases, whereas KGFR protein and mRNA were localized in the epithelium in 72% of cases. Assessment of the proliferative activity of cholesteatoma using the labeling index for Ki-67 showed a significantly higher Ki-67 labeling index (66%) in KGF+/KGFR+ cases than other cases. There was a significant correlation between KGF+/KGFR+ expression and recurrence. Our results indicate the possible involvement of both KGF and KGFR in enhanced epithelial cell proliferative activity and recurrence of cholesteatoma.