Two closely related antigens expressed on granulocytes, macrophages and some reticular elements in rat lymphoid tissues: characterization by monoclonal antibodies

Two distinct novel antigen systems preferentially expressed in rat granulocytes and macrophages were detected using two different monoclonal antibodies (R2-1A6 and R2-2B1). These two antibodies reacted with approximately 50% of rat bone marrow cells, most granulocytes, blood monocytes, alveolar macrophages and peptone-elicited peritoneal macrophages, but not with red blood cells, platelets, thymocytes and T lymphocytes. In addition, R2-2B1 but not R2-1A6 antibody cross-reacted weakly with rat B cells. These two monoclonals also reacted with some reticular elements in rat lymphoid organs including epithelial reticular cells in the thymic medulla and follicular dendritic cells in the lymphoid germinal centre, as well as with the specialized endothelium in the marginal sinuses of the spleen and the post-capillary venules of the lymph node, where lymphocyte recirculation takes place. These antibodies, however, did not label so-called 'dendritic cells' bearing Ia antigens on their cell surfaces, which were found to be located in the thymic medulla, thymus-dependent areas of rat lymphoid tissues and the interstitium of various non-lymphoid organs, suggesting that these dendritic cells, presumably ascribed to those associated with accesory cell function, are separable from the mononuclear phagocyte system in rats by their different reactivities with R2-1A6 and R2-2B1 antibodies.     

Hepatic Campylobacter jejuni infection in patients with Castleman‐Kojima disease (idiopathic multicentric Castleman disease with thrombocytopenia,anasarca, fever,reticulin fibrosis,and organomegaly (TAFRO) syndrome)

Castleman‐Kojima disease, also known as idiopathic multicentric Castleman disease with TAFRO syndrome (iMCD‐TAFRO), is a recently recognized systemic inflammatory disorder with a characteristic series of clinical symptoms, including thrombocytopenia (T), anasarca (A), fever (F), reticulin fibrosis (R), and organomegaly (O). Patients with iMCD‐TAFRO often develop severe abdominal pain, elevated alkaline phosphatase levels, and systemic inflammation, but the etiological factors are unknown. To investigate the potential role of bacterial infection in the pathogenesis of iMCD‐TAFRO, we performed polymerase chain reaction (PCR) for the bacterial 16S rRNA gene with DNA extracted from liver specimens of three patients with iMCD‐TAFRO, four patients with amyotrophic lateral sclerosis, and seven patients with inflammatory conditions. Sequencing of the PCR product showed 99% DNA sequence identity with Campylobacter jejuni in all three patients with iMCD‐TAFRO and in two patients with inflammatory conditions. Immunohistochemical and electron microscopy analyses could not identify C. jejuni in patients with iMCD‐TAFRO. The findings indicated that C. jejuni infection is not the pathological cause of iMCD‐TAFRO; however, this ubiquitous bacterium may play a role in uncontrolled systemic hypercytokinemia, possibly through the development of cross‐reactive autoantibodies.     

Purification of a Brucella canis cell wall antigen by using immunosorbent columns and use of the antigen in enzyme-linked immunosorbent assay for specific diagnosis of canine brucellosis.

A cell wall antigen of Brucella canis was purified by immunosorbent columns. The antigen contained two proteins of 30 and 28 kilodaltons and a polysaccharide exhibiting a 12-kilodalton band upon 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibody to the purified antigen, which specifically reacted with the polysaccharide, was used as the first coating antibody in an enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of canine brucellosis. Dogs inoculated orally with live B. canis were positive and dogs from B. canis-free colonies were negative in the ELISA. Of 199 dogs from a brucellosis-contaminated area, 116 with negative titers in the tube agglutination test (TAT), using heat-inactivated whole B. canis cells as the antigen, were also negative in the ELISA. Seventy-eight of the dogs with questionable titers in the TAT were divided into two groups: 20 dogs that were positive in the ELISA and 58 that were negative. Of five dogs with positive titers in the TAT, three were positive in the ELISA and the gel immunodiffusion test (GD) with crude B. canis extract as the antigen and were also culture positive for B. canis. One dog was positive in the ELISA and GD but gave a negative culture result. Serum from the remaining dog, which was positive with high titer in the TAT but negative in the ELISA and in culture for B. canis, formed a spur precipitate with a homologous precipitate in the GD. These results indicate that the ELISA is a specific serological test for B. canis infection in dogs.     

Characterization of the proteases in the crude mite extract

Characterization of the proteases was performed in the crude mite extract fractionated by Sephacryl S-200 gel filtration. Three peaks of protease activities were detected in the fractions. From the results of substrate specificity and susceptibility to the inhibitors, PK.1 protease (about 60 kD) is suggested to be a trypsin-like protease of mites. From the results of susceptibility to various agents, PK.2 (about 30 kD) and PK.3 (about 20 kD) proteases may be cysteine proteases, e.g., papain and cathepsin B. PK.3 protease existed in the precipitate of 60% ammonium sulfate fractionation. The data in the present study suggest the possibility that Dermatophagoides farinae I allergen might be a cysteine protease probably derived from the gastrointestinal tract of the house dust mite.     

Early events in liver allograft rejection. Delineation of sites of simultaneous intragraft and recipient lymphoid tissue sensitization.

The early events of liver allograft rejection in untreated rats were studied in the DA to BN rejection strain combination and compared with DA and BN liver isograft recipients. In the liver allografts, T-cell infiltration first occurred at 2 days after transplantation and localized to the portal triads and subjacent to the terminal hepatic venules (THV), regions rich in intensely Ia + spindle and dendritic-shaped interstitial cells. Double staining showed distinct 'clustering' between donor Ia-positive dendritic-shaped cells and W3/25+ infiltrating lymphocytes, or to a lesser extent, OX8+ cells. The infiltrating mononuclear cells underwent blastogenesis and proliferated in both the triads and THV regions at 3 and 4 days. Donor Ia-positive cells were also noted in the W3/25+ periarterial lymphatic sheath and marginal zone of the recipient spleen 1 day after transplantation. The number of these cells in the spleen peaked at 3 to 4 days, but were no longer detectable by 10 to 12 days. Mitotic activity became evident in these same regions by days 3 and 4. Paracortical blastogenesis (day 2) and proliferation (days 3 and 4) were also noted in the regional lymph nodes of liver allograft recipients, but no donor Ia+ cells were found in the mesenteric nodes or thymus of the allograft recipients. These results demonstrate that sensitization of the recipient lymphoid tissue to liver allografts can occur both peripherally (intragraft) and centrally (spleen and lymph nodes). Passenger leukocytes (donor dendritic cells) are likely the primary stimulators of the rejection reaction. Still, it is probable that other pathways of sensitization exist.     

Safety and efficacy of water-in-oil-in-water emulsion vaccines containing Newcastle disease virus haemagglutinin-neuraminidase glycoprotein

Subunit vaccines containing haemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus (NDV), formulated as water-in-oil-in-water (W/O/W) emulsions, were prepared. First, the suitable constituents of a W/O/W emulsion adjuvant were investigated with polyvalent vaccines using NDV, infectious bronchitis virus and Haemophilus paragallinarum. The W/O/W emulsion adjuvant, composed of the antigen in phosphate-buffered saline (PBS), liquid paraffin, squalene, diglyceryl monooleate, polysorbate 80 and PBS in a 30:25:10:5:2:28 ratio, induced a good antibody response with less adverse local reactions. HN protein of NDV was expressed by an improved baculovirus expression vector, a hybrid nucleopolyhedrovirus (HyNPV) between Autographa californica NPV and Bombyx mori NPV,and was prepared from silkworm pupae infected with the recombinant baculovirus, HyNPV-HN. Then, the W/O/W emulsion vaccine containing HN protein was prepared using the aforementioned constituents. Chickens showed 100, 100 and 80% protection against challenge exposure to virulent NDV at 4 weeks after vaccination with W/O/W emulsion vaccines containing 30, 6 and 3% of HyHPV-HN-infected pupae, respectively. The vaccines containing HN protein did not induce adverse local reactions at the site of injection. The subunit vaccine for NDV containing HN protein expressed in the recombinant baculovirus-infected pupae, formulated as a W/O/W emulsion vaccine composed of the antigen in PBS, liquid paraffin, squalene, diglyceryl monooleate, polysorbate 80 and PBS in a 30:25:10:5:2:28 ratio, was therefore found to be safe and effective.     

Pharmacological studies on 6-amidino-2-naphthyl[4-(4,5-dihydro-1H-imidazol-2-yl)amino] benzoate dimethane sulfonate (FUT-187). I: Inhibitory activities on various kinds of enzymes in vitro and anticomplement activity in vivo.

FUT-187, a newly synthesized compound, was studied on its inhibitory activities mainly on proteolytic enzymes, in comparison with those of FUT-175 and FOY-305, known serine protease inhibitors. FUT-187, as well as FUT-175 and FOY-305, had selective inhibitory activities on serine proteases including Clr, Cls, kallikrein, trypsin, plasmin and thrombin; its activities on these enzymes except Clr and pancreatic kallikrein were relatively lower than those of FUT-175 and FOY-305. Further studies were conducted focusing on complement-mediated reactions. In spite of its lower activities against Clr and Cls, inhibitions by FUT-187 on the complement-mediated hemolysis in vitro and in vivo were only a little weaker than or equivalent to that of FUT-175. FOY-305 was ineffective in these tests. Forssman shock in guinea pigs is known to be initiated by the activation of the complement system. The protective effect of intravenous or oral FUT-187 against this shock was definitely superior to that of FUT-175. Furthermore, FUT-187 inhibited changes accompanied with Forssman shock, such as increase in lung weight, the decrease in platelet counts and CH50, and histopathological changes. These results suggested that FUT-187 should be a more potent oral therapeutic agent than FUT-175 for various inflammatory diseases attributed to the excessive activation of the complement system followed by platelet aggregation.     

Inhibitory effect of nafamostat mesilate (FUT-175) on O2- production in rat polymorphonuclear leucocytes

Effect of nafamostat mesilate (FUT-175), a serine protease inhibitor, having anti-inflammatory effects was studied on superoxide (O2-) production in rat polymorphonuclear leucocytes (PMN) and compared with those of other serine protease inhibitors and typical anti-inflammatory agents. 1) O2- productions in rat PMN stimulated with concanavalin A (Con A) and cytochalasin B (Cyt B) were too weak to observe. With NADH, however, strong O2- production was induced by Con A and Cyt B. 2) FUT-175 at 10(-6) and 10(-5) M inhibited O2- production in rat PMN induced by Con A and Cyt B with NADH in a concentration-dependent manner. 3) The serine protease inhibitor L-tosylamido-2-phenylethyl-chloromethyl ketone (TPCK) and soybean trypsin inhibitor (SBTI) inhibited O2- production at 10(-5) M and 10(-4) M, respectively, while aprotinin, chymostatin and leupeptin did not. 4) Neither indomethacin nor dexamethasone, typical anti-inflammatory agents, inhibited O2- production. Mepacrine, a phospholipase A2 inhibitor, strongly inhibited it. 5) O2- production in PMN prepared from the rat administered FUT-175, 200 mg/kg, p.o., was significantly decreased in comparison with that of the control rat. 6) FUT-175 had no effect on O2- production by hypoxanthine-xanthine oxidase. These results showed FUT-175 had a strong inhibitory effect on O2- production in rat PMN which other typical anti-inflammatory agents did not have.     

Transdermal absorption of bupranolol in rabbit skin in vitro and in vivo

This study was designed to clarify the percutaneous penetration of bupranolol (BP), a beta-adrenoceptor antagonist, through rabbit skin and to compare the in vitro penetration with the in vivo absorption. BP penetrated across the skin slowly in the absence of enhancers in vitro. Isopropyl myristate and N-methyl-2-pyrrolidone enhanced the in vitro penetration, with a 3.6 times higher flux compared with that without enhancers. However, in the in vivo percutaneous absorption, the maximal penetration was obtained with the formulation added dlimonene, with a 3.0 times higher area under the concentration-time curve (AUC) than that for the formulation without enhancers. The plasma levels of BP determined, however, were extremely lower than the theoretical plasma steady-state concentrations predicted. The plasma levels of BP after application of these formulations were maintained in the range of 7-22 ng/ml for 30 h, of which concentrations were above the therapeutically effective concentration (1.5-4 ng/ml). Therefore, the transdermal systems will offer an efficient drug delivery system for the treatment of angina pectoris and tachycardia.