Prevention of autoimmune symptoms in autoimmune-prone mice by elimination of B-1 cells

Our recent studies on an autoantibody-transgenic mouse linedemonstrated that peritoneal B-1 cells are responsible for autoimmunesymptoms. However, whether B-1 cells in the peritoneum are generallyinvolved in the pathogenesis of autoimmune disease remains controversial.To test the possible involvement of peritoneal B-1 cells inautoimmune symptoms of autoimmune-prone NZB mice, we eliminatedthe peritoneal cells by hypotonic shock with repeated I.p. injectionof distilled water every 7 days into neonatal or 8-week-oldNZB mice. By this treatment, B-1 cells, which self- renew withinthe peritoneal cavity, are expected to be preferentially eliminated,while other peritoneal cells can be easily supplied from bonemarrows after this treatment indeed, in distilled water-treatedold NZB mice, the number of B-1 cells decreased in spleen aswell as in lamina propria of the gut but the numbers of conventionalB cells and T cells did not change. Moreover, the productionof autoantibodies against erythrocytes significantly decreasedand the occurrence of autoimmune hemolytic anemia was reducedin 12-month-old treated NZB mice. Similarly, the eliminationof peritoneal cells of NZB/NZW (NZB/W) F₁; mice by water injectiondecreased anti-DNA IgG antibodies in the sera and reduced thepathological changes of the kidney. These results suggest thatperitoneal B-1 cells may be a source of autoantibody-producingcells in autoimmune diseases of NZB and NZB/W F₁; mice.     

Antigen-induced elevation of immunoreactive endothelin-1 (ET-1) levels in ovalbumin-sensitized guinea pig airway tissue

Changes in the immunoreactive ET-1 levels during the anaphylactic reaction of airway tissue from ovalbumin-sensitized guinea pigs were investigated. ET-1-immunoreactivity (ET-IR) was detected in the epithelial and smooth muscle layers of tracheal sections from normal guinea pigs and it was enhanced slightly by phosphoramidon (1 microM) treatment. The ET-IR level of the epithelial layer of ovalbumin-treated tissue from actively sensitized animals was slightly higher than that from normal animals, but it was enhanced markedly by phosphoramidon (1 microM) treatment. Furthermore, the mean ET-IR level of homogenates of antigen-treated tracheal tissues from sensitized guinea pigs (22.8 +/- 1.55 fmol mg-1 protein, n = 5) was significantly higher than the corresponding normal level (12.3 +/- 1.21 fmol mg-1 protein, n = 5). These results suggest that increased epithelial airway ET-1 levels contribute to the anaphylactic reaction of guinea pig airways.     

Increased peripheral blood Ia positive T cells and their effect on autologous mixed lymphocyte reaction in chronic active liver disease.

We measured Ia antigen bearing peripheral blood T cells, as an index of immunological stimulation, of patients with chronic active liver diseases (CALD) by the rosette assay method. We also examined the role of Ia antigen which represents the products of the genes of the major histocompatibility complex on the autologous mixed lymphocyte reaction (AMLR) since this reaction may reflect self regulation of immune responses. The percentages of Ia positive T cells of 29 patients with CALD (17.1 +/- 4.3%, P less than 0.001) and of 12 patients with other liver diseases (12.9 +/- 2.4%, P less than 0.05) were increased when compared with that of normal individuals (10.7 +/- 2.0%). However, levels of Ia positive T cells activated by phytohaemagglutinin-P in patients with CALD and other liver diseases did not differ from normal subjects. Ia positive cells in OKT8 positive cells were markedly elevated (P less than 0.001), whereas those in OKT4 positive cells were decreased (P less than 0.01) in CALD. The impaired values for the AMLR correlated inversely (P less than 0.01) with the increased percentages of Ia positive T cells in patients with CALD. Further analysis showed that there was no suppression of the proliferation of Ia and OKT4 positive cells by Ia and OKT8 positive cells although the culture of increasing numbers of Ia and OKT8 positive cells and decreasing numbers of Ia and OKT4 positive cells gave a lesser AMLR value. These data suggest that the increase in Ia positive T cells and the alteration of Ia positive cells in the T cell subsets reflect an activation of immune system and provide further evidence in favour of an abnormality of the immunoregulatory system in CALD.     

Cellular localization of inflammatory cytokines in human glomerulonephritis

We evaluated the expression of inflammatory cytokines in renal tissues obtained from 45 patients with several types of glomerulonephritis. Immunofluorescence studies with specific antibodies to interleukin (IL)-1, IL-1, IL-6, tumour necrosis factor (TNF)-, and TNF- showed intense cytoplasmic staining in the glomeruli and interstitium. Cells positive for these cytokines were found frequently in tissue from patients with lupus nephritis (WHO Class IV) and membranoproliferative glomerulonephritis, and, to a lesser extent, in tissue from patients with mesangial proliferative glomerulonephritis, Henoch-Schönlein purpura nephritis, and minimal change nephrotic syndrome. Most of these cells were dual-stained with a monoclonal antibody to monocytes-macrophages. In situ hybridization for cytokine mRNA, combined with immunoperoxidase staining for monocytes-macrophages, detected IL-1, IL-6, and TNF- mRNA in monocytes-macrophages infiltrating the glomeruli and interstitium. Occasionally, there was weak or moderate immunostaining for IL-1, IL-6, and TNF- in the glomerular mesangial and epithelial cells, but in situ hybridization signals were rarely found in these loci. These findings suggest that infiltrating monocytes-macrophages, rather than resident glomerular cells, are the major source of inflammatory cytokines in human glomerulonephritis.     

Lectin binding patterns in normal liver, chronic active hepatitis, liver cirrhosis, and hepatocellular carcinoma. An immunohistochemical and immunoelectron microscopic study.

We studied the binding patterns of 14 lectins in human normal and cirrhotic liver (LC) tissues and hepatocellular carcinomas (HCC) using the ABC method. Lectins were divided into 4 groups according to their binding patterns in normal tissues: (A) PHA, MPA, LcH, RCA-I, and WGA, which bound to hepatocytes and all three types of sinusoidal cells; (B) BPA, GS-I, PNA, and SBA, which bound to Kupffer cells and endothelia of interlobular arteries and veins and bile duct epithelia in the portal tract, but not to hepatocytes; (C) UEA-I, which bound only to endothelia of interlobular arteries and veins and bile duct epithelia in the portal tract; (D) LBA, Lotus, LPA, and SJA, which showed no binding. Thus group B lectins may be useful markers of Kupffer cells. Only electron microscopic examination revealed the precise binding sites of lectins in sinusoidal cells and hepatocytes. Hepatocyte cell surface polarities demonstrated by lectin binding in LC and HCC were different from those in the normal liver. The binding pattern of PHA to LC hepatocytes changed from a membranous to both a membranous and a cytoplasmic pattern, and that of LcH to HCC cells changed to dot-like staining in the cytoplasm. These changes of polarities in LC and HCC might be caused by changes in the distribution of lectin-binding carbohydrates or by the altered glycosylation of glycoconjugates.     

Efficacy of early use of continuous isoproterenol inhalation therapy for severe asthma attacks in children]

The aim of this study was to evaluate the efficacy and safety of continuous isoproterenol inhalation therapy for asthma attacks in children. We used l-body isoproterenol (Proternol L) in 22 children with 32 episodes of severe attacks. One of them did not respond to this therapy, and two had complications (atelectasis and pneumothorax). Twenty-nine cases were divided into three subgroups according to their clinical scores; A) scores less than or equal to 4, which meant that they were in the early stage of severe attack (n = 9), B) scores 5-6, which meant impending respiratory failure (n = 17), C) scores greater than or equal to 7, which meant respiratory failure (n = 3). The values of SpO2 at the start of this therapy were 94.8, 91.5, 82.0%, respectively. The more severe their attacks were, the lower their SpO2 levels were. The periods until their scores became zero were 0.78, 6.3, 17.2 hours, respectively. There were significant differences between each period respectively (p less than 0.001, p less than 0.01). Heart rates decreased when their symptoms improved, and other adverse effects were not detected. These results suggest that this therapy is effective and safe for children with severe asthma attacks, especially in the early stage.     

Cold-adaptation of human rotavirus

A human rotavirus strain was cold-adapted for possible future use as a live vaccine. The original strain was isolated in 1980 in primary cynomolgus monkey kidney cells and has a serotype I and subgroup II antigenicity. The virus was serially passaged in African green monkey kidney cells; it was cultivated at 37 degrees C at the first stage of passages, and the cultivation temperature was then shifted down stepwise by 3 degrees C per each 10 passages. Finally the virus was passaged 10 times at 25 degrees C (total passage number of 55). The virus formed small-size plaques with irregular shaped borders at 31 degrees C. Growth at 25 degrees C of the cold-adapted virus was higher than that of the original virus. There was no difference between the migration patterns of 11 dsRNA segments in polyacrylamide gel electrophoresis of the original and the cold-adapted viruses.     

Germ cell transplantation: a review and progress report on ICSI from spermatozoa generated in xenogeneic testes

Results from the transplantation of donor male germ cells into xenogeneic recipient seminiferous tubules indicate that donor spermatogonia are capable of differentiating to form spermatozoa morphologically characteristic of the donor species. Germ cell transplantation procedures combined with developments in freezing, culturing or enriching germ cell populations have applications of paramount importance in medicine, basic sciences and animal reproduction. Additionally, these techniques can serve as an alternative approach for gonadal protection and fertility preservation in patients with cancer. This article is a chronological critical review of the technological advances that followed the initial successful transplantation of mouse germ cells into recipient mice. Furthermore, the factors responsible for the immunological privilege properties of the testis and the parameters influencing the potential of mammalian germ cells to undergo mitosis and meiosis within a xenogeneic testis are described. Finally, the role of human germ cell transplantation procedures in the therapeutic management of non-obstructive azoospermia is discussed.     

Expression and characterization of mouse angiotensin II type 1a receptor tagging hemagglutinin epitope in cultured cells

The octapeptide angiotensin II mediates the physiological actions of the renin-angiotensin system through activation of several angiotensin II receptor (AT) subtypes, in particular AT1 (AT1a and AT1b in the case of rodents). Although we and others have generated mutant mice in which the AT1a gene was disrupted, the function of mouse AT1 remains to be fully elucidated, due to the lack of effective tools involving antibodies against AT1 for detecting biological responses in cellular conditions. To avoid these problems, we constructed the hemagglutinin (HA)-tagged mouse AT1a, and stably introduced this recombinant receptor into human embryonic kidney 293-T cells. Radioligand binding of [(125)I] angiotensin II to AT1a was specific, saturable, and reversible. Scatchard analysis demonstrated that the transfected receptor had a dissociation constant of 1.7 nM with a density of 1.2 x 10(5) sites/cells. Angiotensin II stimulated a rapid increase in cytosolic free calcium, and angiotensin II-induced phosphorylation of extracellular signal-regulated kinases (Erk) was found in a dose-dependent manner. After solubilization, Western blot analysis showed specific interactions between an anti-HA antibody and HA-tagged mouse AT1a. Furthermore, a significant proportion of HA-tagged mouse AT1a was specifically immunoprecipitated with this antibody. In the immunocytochemical and electronmicroscopic studies, treatment of this cell line with angiotensin II resulted in decrease in signals of the surface receptors. Based on these results, the cell line established here provides an excellent tool for studying angiotensin II actions mediated through mouse AT1a, at sub-nanomolar concentrations.