A possible role of two hydrophobic amino acids in antigen recognition by synovial T cells in rheumatoid arthritis

Synovial T cells play a crucial role in the pathogenesis of rheumatoid arthritis (RA) synovitis. We have quantitatively analyzed the T cell receptor (TcR) variable (V) region gene repertoire of freshly isolated synovial fluid (SF) T cells, comparing it with that of peripheral blood (PB) T cells in RA. The TcR V gene repertoire of PB and SF T cells in RA and osteoarthritis was heterogeneous. In contrast, Vail in SF was expressed to a greater degree in three of five RA patients, and increased levels of Vp6, 1-3 were found in the SF of four of six RA, compared with paired PB. Of note, Vβ6, 1–3 was universally used in four RA patients with a disease duration of less than 10 years, irrespective of their HLA-DR types. This was in contrast to two other RA patients, suffering for more than 20 years, who showed different Vα and Vβ usages. β-chain sequence analysis in RA patients with a preference for Vβ6, 1–3 has shown that a few clones dominated in SF, whereas polyclonality was observed in PB. These findings suggest oligoclonal expansion of T cells in response to specific antigen(s) in the SF of these patients with RA of relatively short duration. Concomitant use of two hydrophobic amino acids, leucine and valine, in the Dβ region was noticeable among the predominant SF clones. These two amino acids might directly contact a peptide specific for the induction of synovitis in RA patients. TcR-directed therapy may, therefore, be useful for the treatment of early RA synovitis.     

Synergistic effect of gelatin microspheres incorporating TGF-beta1 and a physical barrier for fibrous tissue infiltration on skull bone formation

The objective of this study is to examine whether or not bone formation at a skull bone defect induced by gelatin microspheres incorporating transforming growth factor (TGF)-beta1 is promoted by prevention of fibrous tissues into the defect. The 6-mm diameter bone defect of rabbit skulls was applied with gelatin microspheres incorporating TGF-beta1 or free TGF-beta1 and physically covered by a barrier membrane. When the bone formation at the defect was assessed 6 weeks postoperatively, combinational application of gelatin microspheres incorporating 0.1 microg of TGF-beta1 with the barrier membrane induced bone formation at the skull defect, in marked contrast to that of 0.1 microg of free TGF-beta1 and empty gelatin microspheres. Complete defect closure was histologically observed by the newly formed bone tissue. Without the barrier membrane, gelatin microspheres incorporating TGF-beta1 were less effective in inducing bone formation, whereas free TGF-beta1 and empty gelatin microspheres were ineffective. The skull defect was occupied by fibrous tissue infiltrated in place of bone tissue. The bone mineral density at the skull defect applied with gelatin microspheres incorporating TGF-beta1 plus the membrane was significantly higher than that of gelatin microspheres incorporating TGF-beta1 alone. The present data indicated that physical protection from the soft tissue infiltration enabled gelatin microspheres incorporating TGF-beta1 to synergistically enhance the osteoinductive ability at the skull defect.     

Non-adhesive cyanoacrylate as an embolic material for endovascular neurosurgery

Endovascular neurosurgery is now becoming available as one of strategies for the treatment of cerebro-spinal arterio-venous malformations and aneurysms. For this treatment, a microcatheter is advanced into or close to a lesion and then an embolic material is administered through it to obliterate the lesion. N-butyl-2-cyanoacrylate (NBCA) has preferentially been used as an embolic material in Europe and America. However, its exceptionally strong adhesive force sometimes causes adhesion between the tip of the microcatheter and the artery. In this study, a new non-adhesive cyanoacrylate, isostearyl-2-cyanoacrylate (ISCA), was developed. It carries a long hydrophobic side isostearyl group with lower reactivity and adhesion than other cyanoacrylates. Its polymerization rate is, however, too low to obliterate a vascular lesion with a rapid blood flow. To increase the polymerization rate. ISCA was mixed with NBCA. As a result, the adhesive force of the mixture became extremely low, compared with that of NBCA. The viscosity of the mixture was low enough to allow its' use as an embolic material. Tissue reactions against the mixture was milder than those against NBCA. Radio-angiography became possible by mixing further with Lipiodol. The evaluation of this new embolic material with a rabbit renal artery showed that the obliteration effect of the mixture of ISCA and NBCA was excellent to use as an embolic material for clinical applications.     

A possible pathogenic role of CD8+ T cells and their derived cytokine, IL-16, in SLE

Current investigations into the role of CD8+ T cells and their derived cytokine, interleukin (IL)-16, in the induction of CD4+ T cell abnormalities in systemic lupus erythematosus (SLE) were reviewed and discussed on the basis of results mainly obtained in our laboratory.     

Colorimetric B cell proliferation assay based on alkaline phosphatase activity. Selective measurement of B cell proliferation in the presence of other cell types

In this study we show that a rapid colorimetric determination of alkaline phosphatase (APase) activity can be used in conjunction with a multiwell scanning photometer to quantitatively measure lymphokine-dependent B cell proliferation. Despite the fact that only activated but not resting B cells exhibit APase activity, i.e., that APase expression in B cells is variable, the results obtained by monitoring the enzyme activity were very similar to those obtained by measuring thymidine incorporation in two murine B cell systems. With regard to a potential application to the screening of the effects of hybridoma supernatants on B cells, it is relevant that HAT medium has practically no influence on the colorimetric B cell proliferation assay. Another advantage of the method is that since T cells and macrophages lack APase activity, one can detect the proliferation of B cells even in the presence of a high number of such cells, for example, in studying a T cell-dependent response.     

HLA-DR Antigens in Pemphigus among Japanese

The frequency of HLA-DR4 was significantly increased at P < 0.02 in 37 unrelated pemphigus patients (62.2%), when compared with unrelated 73 healthy controls (30.1%). This antigen was more frequently found in pemphigus foliaceus (70.6%) than pemphigus vulgaris (55.8%).     

Effect of dextran sulphates on thrombin activity.

To clarify the action of dextran sulphate, a heparin analogue, in the clotting of fibrinogen by thrombin, determinations were carried out on the clotting activity, the release of fibrinopeptides from fibrinogen, and the hydrolytic activity of thrombin against a peptide chromogenic substrate in the absence or presence of antithrombin III (heparin cofactor). It was shown that dextran sulphate itself inhibited thrombin activity, and its inhibition was dependent on the molecular weight and the sulphur content of the dextran sulphate. Although heparin markedly enhanced the antithrombin activity of antithrombin III, dextran sulphate did not activate antithrombin III.     

Augmentation of cholesterol esterification in the microsomal membrane by the removal of membrane-bound ribosomes

Acyl-CoA: cholesterol O-acyltransferase (ACAT) activity in rough microsomes was enhanced (2-fold) by the removal (40%) of ribosomes from the microsomal membrane with RNase. Although EDTA was as efficient as RNase in the removal of ribosomes the stimulation (3.2-fold) of ACAT activity was even more, suggesting that additional effects were induced by EDTA. Reconstitution of EDTA-treated microsomes with ribosomes decreased the cholesterol-esterifying activity (40%) of the degranulated microsomes. Alternate possibilities were considered for the enhancement of ACAT activity by EDTA, namely, suppression of the hydrolysis of added palmitoyl-CoA substrate, the hydrolysis of cholesteryl ester, and the removal of metal suppressor of ACAT activity. Neither acyl-CoA hydrolase nor cholesteryl ester hydrolase activity was decreased after degranulation of microsomes. ACAT activity of EDTA-treated microsomes compared to the control was enhanced rather than suppressed after the addition of Ca2+, Mg2+, and Ba2+ ions whereas other metal ions (Co2+, Cu2+, Zn2+) almost completely suppressed ACAT activity in both the EDTA-treated and buffer-treated microsomes. It is concluded that the enhanced ACAT activity was largely due to the removal of ribosomes from the microsomal membrane.     

Relation between yawning behavior and central serotonergic neuronal system in rats

Summary Subcutaneously (s.c.) administered apomorphine (0.0125–0.4 mg/kg) or physostigmine (0.025–0.4 mg/kg) to rats elicited yawning. The dose-response curves were bellshaped. The peak effects of apomorphine and physostigmine were observed with a dose of 0.1 mg/kg of each drug. Yawning elicited by apomorphine (0.1 mg/kg) or physostigmine (0.1 mg/kg) was reduced by intraperitoneally (i.p.) administered 5-hydroxytryptophan (5-HTP, 50–200 mg/kg, given 30 min before). Yawning elicited by apomorphine but not by physostigmine was enhanced by p-chlorophenylalanine (p-CPA, 25–400 mg/kg i.p., given 24 h before). Apomorphine elicited but not physostigmine-elicited yawning was enhanced by pretreatment with 5,7-dihydroxytryptamine (5,7-DHT, 8 g/rat, given 14 days before into the dorsal raphe). This treatment led to a 35% depletion of serotonin (5-HT) in the striatum. 5-HTP, p-CPA or 5,7-DHT given alone did not elicit yawning. Bilateral, intrastriatal microinjection of apomorphine (1.5 –50 g/site) but not physostigmine (5–50 g/site) elicited yawning. The dose-response curve was also bell-shaped. These results indicate that central serotonergic pathways play an important role in modulating drug-elicited yawning in rats. Send offprint requests to S. Okuyama at the above address