Electron microscopic specimen preparation from low concentration of cell suspension using cytospin technique.

Electron microscopic examinations are sometimes limited due to the small number of cells available for analysis. The purpose of this study was to determine the limit of cell concentration for a successful transmission electron microscopic preparation. Various concentrations of monocyte cell suspension were fixed in glutaraldehyde and osmium tetroxide according to the standard methods. Cell preparations were made on silane-coated glass slides in a cytospin centrifuge. The attached cells to the glass slides were dehydrated, and embedded in epoxy resin by routine electron microscopic technique. By this method, cell suspensions containing as low as 2x10(3) cells could show approximately 5 to 10 cells in each hole of the 150-mesh grids which was designated as the lowest limit for the successful preparation with detectable cells for evaluation. The fine structure of cells was clearly evident and the preparations were uniformly free from artifacts, similar or superior to those of cell pellet preparations. This method is useful whenever dealing with the samples containing a low number of cells, particularly those of clinical samples.

Identification of an antigenic epitope in Helicobacter pylori urease that induces neutralizing antibody production.

We previously reported a mouse monoclonal antibody (MAb), termed L2, specific for Helicobacter pylori urease strongly inhibited its enzymatic activity. Here, to gain insight into how this antibody affects urease activity, the epitope that was recognized by the antibody was determined. By screening a panel of overlapping synthetic peptides covering the entire sequence of the two subunits (UreA and UreB), we identified a stretch of UreB-derived 19 amino acid (aa) residues (UB-33; aa 321 to 339, CHHLDKSIKEDVQFADSRI) that was specifically recognized by the L2 antibody. Further sequential amino acid deletion of the 19-mer peptide from either end allowed us to determine the minimal epitope as 8 amino acid residues (F8; SIKEDVQF) for L2 reactivity. This epitope appears to lie exactly on a short sequence which formed a flap over the active site of urease, suggesting that binding of the L2 antibody sterically inhibits access of urea, the substrate of urease. Finally, immunization of rabbits with either the 19-mer peptide or the 8-mer minimal epitope resulted in generation of antiurease antibodies that were capable of inhibiting the enzymatic activity. Since urease is critical for virulence of H. pylori, antigenic peptides that induce production of antibodies to inhibit its enzymatic activity may potentially be a useful tool as a vaccine for prevention and treatment of H. pylori infection.

Role of the liver in T cell differentiation--generation of CD3-CD4+/CD8+TCRbeta- cells and CD3-4-8-TCRbeta+ cells from CD4-8-TCRbeta- athymic nude bone marrow cells by culture with parenchymal liver cells.

To investigate the influence of the liver on differentiation of hematopoietic stem cells/pro-T cells, TN-NWP-BMC (athymic nude bone marrow cells that were treated with anti-TCRbeta, anti-CD4, and anti-CD8 Abs plus complement and then passed through a nylon wool column) were cultured on parenchymal liver cells. After culture for 2.5 days, CD3-4-8-TCRbeta+ cells and CD3-CD4+/CD8+TCRbeta- cells were developed from TN-NWP-BMC. TCRVbeta8+ cells comprised 19.9% of CD3-4-8-TCRbeta+ cells, and Vbeta8 mRNA was detected in the CD3-4-8-TCRbeta+ cells by reverse transcriptase-polymerase chain reaction. The CD3-CD4+/CD8+TCRbeta- cells contained not only single-positive cells but also CD4+8+ double-positive cells. The CD8 protein consisted of 88.9% CD8alpha+beta-, 10.1% CD8alpha+beta+, and 1% CD8alpha-beta+ molecules. From these results and the finding of co-expressed antigens, CD3-4-8-TCRbeta+ cells and CD3-CD4+/CD8+TCRbeta- cells appear to be immature cells not committed to a certain cell lineage.

Systemic and local immune responses against Helicobacter pylori urease in patients with chronic gastritis: distinct IgA and IgG productive sites.

BACKGROUND: Helicobacter pylori urease is a major target for immune responses among various bacterial components in H pylori infected patients. AIMS: To analyse the relation between systemic and local humoral immune responses to H pylori urease and grades of chronic gastritis. PATIENTS: Seventy five patients with chronic gastritis associated with H pylori infection were classified into three groups (grade I, superficial gastritis; II, atrophic gastritis, quiescent; or III, atrophic gastritis, active). METHODS: Anti-H pylori urease specific antibodies in the serum, gastric juice, and biopsy specimens were determined by ELISA or western blotting analysis. The sites for H pylori urease and its specific antibody producing B lymphocytes were confirmed by immunohistochemical analysis. RESULTS: In the sera of patients with grade I gastritis, weak IgG but relatively strong IgG responses to H pylori urease were observed; dominant strong IgG responses were detected in grade II gastritis. In grade III gastritis, significant IgG and IgA responses were obtained. A similar pattern of IgA and IgG responses was detected in gastric juice and tissue. H pylori urease specific, antibody producing B cells were not found in the gastric mucosa of patients with grade I gastritis despite the presence of such B cells in the duodenal bulb. Specific B cells were observed in the gastric mucosa of patients with grade II and III gastritis with atrophy. CONCLUSIONS: Purified H pylori urease, together with localisation of its specific antibody producing B cells, are useful for serological testing and histopathological analysis for determining the stage of chronic gastritis and studying the pathogenesis of H pylori infection.

Changes in M-CSF-like activity during chicken embryonic development.

We have established a method for separation of chicken bone-marrow cells using Percoll density gradient centrifugation, and have developed a new method for determining chicken M-CSF-like activity employing a liquid culture. Using this method, we determined M-CSF-like activities in egg yolk, chorioallantoic fluid (CAF) and amniotic fluid (AmF), and studied the effects of M-CSF on development of chicken embryos. M-CSF-like activity in egg yolk was at a high level before the incubation of the egg; it began to decrease on the third day of incubation and rapidly decreased on the fourth day, and no significant activity was detected after the tenth day of incubation. M-CSF-like activity in CAF was very low, and it exhibited almost no change during development. No M-CSF-like activity was detected in AmF throughout the experimental period.

[Analysis of immune response to Helicobacter pylori--identification of the protein recognized by anti-Helicobacter pylori antibodies from sera of patients with gastroduodenal diseases].

HP-specific antibodies were measured in each collected fraction obtained from gel-filtrated separation method with selected patients sera by enzyme-linked immunosorbent assay (ELISA). Most of the fractions reacted with the patients sera also responded to HP urease-specific monoclonal antibodies. In addition, the fractions which showed urease activity strongly correlated with the HP antibody positive ones. Also, we could not detect any cross-reactivity to ureases of other species. Moreover, most patient sera strongly responded to the purified HP urease B subunit separated with SDS/PAGE by western blotting analysis. These findings suggest that the major target for HP-specific antibodies appears to be HP-urease, in particular around 68kd large molecule, urease B subunit and such urease-specific antibodies are isolate-species specific.

Modification of T-lymphocyte subsets in peripheral blood, the liver, and the spleen during liver regeneration after partial hepatectomy of mice.

Flow cytometry analysis was performed to investigate the modifications to the T lymphocytes in peripheral blood, the spleen, and two intrahepatic lymphocyte fractions--the intrahepatic lymphocyte fraction 1 (IHL Fr.1), which is easily washed out from the liver by perfusion of collagenase solution, and the intrahepatic lymphocyte fraction 2 (IHL Fr.2), which remains in the liver after the perfusion--occurring in the liver regeneration process after partial hepatectomy of mice. The following findings were obtained: 1) The nontreated murine liver contains Thy 1.2+ cells, alpha beta TCR+ cells, gamma delta TCR+ cells, CD4+ cells, and CD8+ cells. 2) The percentage of each T-cell subset in IHL Fr.1 shows an intermediate value between that in the peripheral blood lymphocytes (PBL) and that in the IHL Fr.2. 3) One day after partial hepatectomy, the Thy 1.2+ cells, alpha beta TCR+ whole cells, and CD4+ cells in IHL Fr.2 all showed a transient, yet significant, decrease, but did not reveal any major change in the other fractions. 4) Both the alpha beta TCR dull+ cells and the CD8+ cells showed practically no change after partial hepatectomy in any of the fractions. This indicates that the alpha beta TCR dull+ cell/alpha beta TCR+ whole cell and the CD8+ cell/CD4+ cell ratios show a significant increase 1 day after partial hepatectomy only in the IHL Fr.2. Double-positive cells, which were scarcely found in the spleen of nontreated mice, appeared in the spleen after partial hepatectomy.(ABSTRACT TRUNCATED AT 250 WORDS)

The liver as a potential hematolymphoid organ examined from modifications occurring in the systemic and intrahepatic hematolymphoid system during liver regeneration after partial hepatectomy.

In order to investigate whether or not the adult murine liver can function as one of the hematolymphoid organs, we studied the alteration of cellular characteristics in the systemic and intrahepatic hematolymphoid systems during liver regeneration after partial hepatectomy. Liver regeneration affects the systemic hematolymphoid system, as can been seen from the transient increase in colony forming unit in culture (CFU-C) frequency observed in the bone marrow at 20 hr and its gradual increase in the spleen up to the 6th day after partial hepatectomy. CFU-C were found not only in peripheral blood lymphocyte-rich fraction (PBL), but also in intrahepatic lymphocyte-rich fraction (IHL) of the normal adult liver. CFU-C frequency showed a weak increase in PBL and a dramatic increase, up to the 6th post-hepatectomy day, in IHL subfraction cells, forming a very strong liver association. Colonies, which were generated from IHL in a fibrin clot culture system, were mainly composed of granulocytes, macrophages and mast cells. Wheat germ agglutinin (WGA) positive cells too were detected in IHL and found to increase after partial hepatectomy. IHL of the normal liver were proliferated not only by IL-3 and GM-CSF but also by IL-2. The proliferative responses of IHL to these cytokines were further augmented on day 6 after partial hepatectomy. Similar results were obtained in peripheral blood and splenic lymphocyte-rich fractions. These observations, therefore, suggest that the adult murine liver has hematolymphoid cells as its component and has the function of a hematolymphoid organ closely associated with the systemic hematolymphoid system.

Suppressive effect of bradykinin to cellular immune responses in vivo and in vitro.

Bradykinin, as well as histamine, one of the mediators in IgE mediated immediate type allergic reactions or acute inflammation, may affect the in vitro and in vivo cell-mediated immune reactions of the immunized animals. It was demonstrated in our present experiment that the appearance of delayed type hypersensitivity (DTH) skin reaction in the immunized guinea pig was remarkably suppressed by treatment of bradykinin or histamine and the suppression of cutaneous DTH by bradykinin was inhibited by H-2 antagonist (burimamide) but not by H-1 receptor blocker (chlorpheniramine). It was also clearly demonstrated that bradykinin suppressed the production of antigen-induced macrophage migration inhibitory factor (MIF) of the immune guinea pig peritoneal exudate cells (PECs) and the production of MIF was blocked by H-2 antagonist (burimamide) or H-2 agonist (tolazolin) but not by H-1 antagonist (chlorpheniramine). Antigen-induced lymphocyte proliferation of the immunized mice, one of the indicators of the cellular immune response, was also suppressed by treatment of bradykinin. These results indicate that bradykinin as well as histamine may have some role in the subsequent expression of cellular immune reactions.

Adjuvant activities in production of reaginic antibody by bacterial cell wall peptidoglycan or synthetic N-acetylmuramyl dipeptides in mice.

This paper is concerned with the adjuvant activity in stimulatory immunoglobulin E production against ovalbumin (OA) by bacterial cell walls, cell wall peptidoglycan (PG), and their PG fragments and synthetic N-acetylmuramyl (MurNAc) dipeptides in A/J mice. A PG isolated from Streptococcus pyogenes, PG subunit polymer and dimer obtained from Staphylococcus epidermidis, and water-soluble fragments of cell walls or PG prepared from Nocardia corynebacteriodes and Streptomyces gardneri were found to enhance both the primary and secondary responses of anti-OA immunoglobulin E antibody production. It was suggested that the PG portion, either intact or highly degraded, was capable of enhancing the immunoglobulin E antibody production, and there was no need for the non-PG moiety or intactness of PG structure for the adjuvant activity. This finding was confirmed and extended by the use of synthetic MurNAc dipeptides. Among eight MurNAc dipeptides tested, MurNAc-l-Ala-d-isoGln, MurNAc-l-Ala-d-Gln, MurNAc-l-Ala-d-Glu, and MurNAc-l-Ser-d-isoGln were found active as an adjuvant in the stimulation of the primary and secondary reaginic anti-OA antibody production in a similar way to the cell wall PG and their fragments. None of the synthetic MurNAc-l-Ala-l-isoGln, MurNAc-l-Ala-l-Gln, MurNAc-l-Ala-l-Glu, and MurNAc-l-Ala-d-isoAsn, on the other hand, stimulated the anti-OA immunoglobulin E antibody production in either primary or secondary response, indicating the importance for the adjuvancy in immunoglobulin E production of the configuration of the glutamic acid residues adjacent to the l-Ala (or l-Ser) in muramyl dipeptides.

Production of homocytotropic antibody against acid extracts isolated from group A streptococcal cell walls in intact and adrenalectomized rats.

Intact and adrenalectomized rats of male Sprague-Dawley were injected with acid-extracted materials isolated from group A streptococcal cell walls using aluminum gel as an adjuvant. We have clearly demonstrated the production of homocytotropic antibodies against this antigens in rats. However, the homocytotropic antibody titers in adrenalectomized rats were much higher than those in intact rats.