Minoru Harada

Antiasthmatic Activity of a Novel Thromboxane A2 Antagonist, S-1452, in Guinea Pigs

We examined the effect of a potent thromboxane (Tx) A2 receptor antagonist, calcium (1R, 2S, 3S, 4S)-(5Z)-7-(((phenylsulfonyl)amino)bicyclo[2.2.1] hept-2-yl)-5-heptenoate dihydrate (S-1452), on antigen- and various allergic-spasmogen-induced contractions of guinea pig lung parenchymal strips and on the increase in insufflation pressure, an index of bronchoconstriction, in anesthetized guinea pigs. In isolated guinea pig lung parenchymal strips, S-1452 showed competitive antagonism of the contractile activity of U-46619, a TxA2 mimetic, with a pA2 value of 8.9. The compound also inhibited the contraction induced by prostaglandin (PG) D2 and PGF2 alpha, but a TxA2 synthetase inhibitor, OKY-046, did not. In contrast, both drugs inhibited not only leukotriene (LT) D4-induced contraction but also antigen-induced contraction in the presence of a histamine antagonist. In anesthetized guinea pigs, oral administration of S-1452 markedly inhibited the bronchoconstrictions induced by intravenous injection of U-46619, PGD2, PGF2 alpha, LTD4 and platelet-activating factor (PAF) with ED50 values of 0.006, 0.031, 0.112, 0.033 and 0.115 mg/kg, respectively, but OKY-046 inhibited only that by LTD4 and PAF. Additionally, bronchoconstriction following intravenous injection of antigen was almost completely suppressed by S-1452 (0.1 mg/kg) and partially by OKY-046 (300 mg/kg) in passively sensitized guinea pigs which were treated with diphenydramine and propranolol. The inhibitory effect of S-1452 against U-46619-induced broncho-constriction persisted up to 7 h after oral administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of thromboxane A2 in bronchial hyperresponsiveness but not lung inflammation induced by bacterial lipopolysaccharide in guinea pigs.

We examined both a possible association of bronchial hyperresponsiveness with lung inflammatory responses and the role of thromboxane (Tx) A2 in these responses after lipopolysaccharide (LPS) exposure in guinea pigs treated with metyrapone, a cortisol synthesis inhibitor. The increase in bronchial responsiveness to i.v. acetylcholine was transient, with a peak at 2 h after LPS exposure, which was associated with increases in TxB2 and tumor necrosis factor in bronchoalveolar lavage (BAL) fluid. However, the levels of 6-keto-prostaglandin (PG) F1 alpha, interleukin-1 and interleukin-6 in BAL fluid, and the influx of leukocytes in airway and pulmonary edema were not associated with bronchial hyperresponsiveness. Oral administration of S-1452, a selective TxA2 receptor antagonist, markedly suppressed bronchial hyperresponsiveness without affecting cellular responses, pulmonary edema and production of PGs and cytokines. These findings suggest that LPS-induced bronchial hyperresponsiveness is dependent on secondarily generated TxA2, which appears to be independent of lung inflammation.

Impaired cell-mediated immunity in lipoid nephrosis.

Cell-mediated immunity (CMI) was evaluated in 26 patients with lipoid nephrosis (LN), 50 patients suffering from chronic diffuse proliferative glomerulonephritis (CGN) without renal sufficiency and 24 healthy controls. The following parameters were measured: delayed hypersensitivity skin test responses to purified protein derivative (PPD) and candida, circulating lymphocytes. T lymphocytes and T lymphocytes with receptors for the Fc portion of IgG (T gamma cells) or IgM (Tmu cells). Patients with LN in relapse had less mean induration of skin reactivity and a smaller proportion reacting to both antigens as compared with the control subjects. In contrast, the intensity of skin reactivity and the frequency of negative reactions in patients with LN in remission and CGN were similar to those of the control subjects. It was also found that the LN patients in relapse had a significant T lymphocytopenia as well as a significant decrease in absolute numbers of Tmu and T gamma cells, whereas the patients with LN in remission and CGN did not differ significantly from the control population. Thus, the majority of patients with LN in relapse demonstrated an impaired response in a CMI assay system. The disturbed CMI may be secondary to hypoproteinemia and other nutritional factors induced by the nephrotic syndrome.

Impaired Cell-Mediated Immunity in Focal Glomerular Sclerosis

Cell-mediated immunity (CMI) was evaluated in 8 patients with focal glomerular sclerosis (FGS), 50 patients suffering from chronic mesangial proliferative glomerulonephritis without renal insufficiency and 24 healthy controls. The following parameters were measured: delayed skin reactivity to purified protein derivative, circulating lymphocytes, lymphocyte cell-surface markers (neuraminidase-treated sheep erythrocyte and erythrocyte-antibody-complement rosettes) and functional markers (mitogenic responses to concanavalin A and phytohemagglutinin). The FGS patients with nephrotic syndrome (NS) had a significant depression in CMI, characterized by decreased responses of the lymphocytes to both concanavalin A and phytohemagglutinin, impaired delayed hypersensitivity to purified protein derivative and a decreased proportion of T lymphocytes as compared with normal subjects. In contrast, the levels of all CMI parameters studied in FGS patients in remission and in patients with chronic glomerulonephritis with or without NS did not differ from normal subjects. Thus, the majority of FGS patients with NS demonstrated an impaired response in a CMI assay system. The possible significance of these phenomena in the pathophysiology of FGS is discussed.

Contribution of thromboxane A2 to the antigen-induced immediate asthmatic response mediated by IgG1 antibody by augmentation of bronchial responsiveness in guinea-pigs.

1 IgG1‐mediated anaphylactic bronchoconstriction was elicited by intravenous administration of antigen to guinea‐pig 2 days after passive sensitization with IgG1‐rich serum, and this response was not affected by heating the serum (at 56°C, for 4 h). IgE‐mediated bronchoconstriction, provoked 14 days after passive sensitization with IgE‐rich serum, was completely abolished by the heating of the serum. 2 S‐1452 (10 mg kg−1, p.o.), a selective thromboxane (Tx) A2 antagonist, significantly but incompletely suppressed the IgG1‐mediated bronchoconstriction, but did not affect the IgE‐mediated one, while diphenhydramine (5 mg kg−1, i.v.), a histamine antagonist, almost completely inhibited both IgG1‐ and IgE‐mediated bronchoconstriction. 3 Pretreatment with propranolol (1 mg kg−1, i.v.), a β‐adrenergic blocker, in addition to diphenhydramine, caused a long‐lasting bronchoconstriction following antigen challenge in both animal models. This histamine‐independent bronchoconstriction was markedly suppressed by S‐1452 at a low dose of 0.1 mg kg−1. 4 A significant increase in bronchial responsiveness to i.v. acetylcholine (ACh), compared to the prechallenge value, occurred as early as 3 min and persisted for 24 h after antigen challenge in the IgG1 model, but was not observed in the IgE model. S‐1452 (10 mg kg−1, p.o.) inhibited the IgG1‐mediated bronchial hyperresponsiveness, as assessed 60 min after antigen challenge. 5 A marked elevation of TxB2 levels was observed in bronchoalveolar lavage fluid (BALF) 3 min after antigen challenge in the IgG1 model, while levels were not changed in the IgE model. In contrast, the plasma TxB2 level assessed 1 min after antigen challenge was increased in both the IgG1 and IgE models. 6 The results indicate that the inhibition of IgG1‐ but not IgE‐mediated bronchoconstriction by higher doses of S‐1452 may result from the suppression of increased bronchial responsiveness to allergic mediators such as histamine, which is probably due to TxA2 generated in the airway lumen rather than in plasma. In both the IgG1 and IgE models, plasma TxA2 appeared to contribute directly to the bronchoconstriction, its action being almost completely masked by histamine‐mediated bronchoconstriction.

Alteration of T-lymphocyte subpopulations in patients with primary renal diseases and systemic lupus erythematosus.

Forty‐eight patients with a variety of primary renal diseases and systemic lupus erythematosus (SLE) were examined for the proportion of circulating T lymphocytes bearing receptors for IgM Tμ cells) or IgG (Tγ cells). Although the control group showed strikingly similar mean values for both Tμ and Tγ cells, the whole group of patients with primary renal diseases and SLE showed a wide scatter of values. Sixteen patients with primary renal diseases and SLE had higher proportions of Tγ cells than the control group, whereas seven patients with chronic glomerulonephritis (CGN), membranoproliferative glomerulonephritis (MPGN), lipoid nephrosis (LN), and SLE showed very marked decrease in the proportions of Tγ cells in the peripheral blood. On the other hand, six out of the total group of patients had low proportions of Tμ cells in the peripheral blood. However, no consistent relationship between the proportion of Tμ and Tγ cells was found in our study. These findings indicate that there exists a heterogeneity of T‐lymphocyte subpopulation distribution in some patients with primary renal diseases and SLE. The possible significance of these phenomena in the pathophysiology of renal diseases is discussed.

Cell-Mediated Immunity in Idiopathic Membranous Nephropathy

Cell-mediated immunity (CMI) was evaluated in 11 patients with idiopathic membranous nephropathy (MN), 50 patients suffering from chronic proliferative glomerulonephritis (CGN) without renal insufficiency and 24 healthy controls. The following parameters were measured: delayed skin reactivity to purified protein derivative (PPD), circulating lymphocytes, lymphocyte cell-surface markers (En and EAC rosettes) and functional markers (mitogenic responses to Con A and PHA). The MN patients with nephrotic syndrome (NS) had less mean induration of skin reactivity and a smaller proportion reacting to the PPD antigen as compared with the control subjects. In contrast, the intensity of skin reactivity and the frequency of negative reactions in MN patients in remission and CGN were similar to those of the control subjects. During the nephrotic stage of MN the proportion of T lymphocytes decreased with simultaneous increase of the proportion of B lymphocytes. It was also found that the MN patients with NS showed impaired lymphocyte reactivity with lower Con A and PHA responses compared to the normal controls. Conversely, the mean mitogenic responses to the antigens in patients with MN in remission and CGN were similar to those of the control subjects. Thus, the majority of MN patients with NS demonstrated an impaired response in a CMI assay system. The possible significance of these phenomena in the pathophysiology of MN is discussed.

GENETIC CONTROL OF PERIPHERAL T-CELL DEFICIENCY IN THE CATARACT SHIONOGI (CTS) MOUSE LINKED TO CHROMOSOME 7

The cataract Shionogi (CTS) mouse characterized by cataract and microphthalmia has been established at Shionogi Aburahi Laboratories as an inbred strain from a population of outbred ICR mice (Ohotori et al. 1968). This is a strain related to the NOD mouse which is widely used as an animal model of human Type I diabetes mellitus, having the same class II major histocompatibility complex (MHC) and class I D-end as those of the NOD mouse (Makino et al. 1980; Ikegami et al. 1988). In previous studies on CTS mice, we demonstrated a marked decrease of T cells in blood and peripheral lymphatic tissues and a resultant deficiency of T-cell-mediated in vitro and in vivo immune responses (Yagi et al. 1990a, b). Our recent study revealed that numerous CD4 single positive T cells are accumulated in the CTS thymus, suggesting that peripheral T-cell deficiency can be attributed to the defect of migration of mature T cells from the thymus (Yagi et al. 1996). These findings led us to investigate the inheritance mode of this unique immunological abnormality and to determine the locus of the responsible gene(s). The CTS and C3H/He strains bred at Shionogi Aburahi Laboratories were used as parent strains and their reciprocal matings gave F1 hybrids. F1 hybrids were then backcrossed to CTS and C3H/He mice to produce the BC1 and BC2 generations, respectively. The F2 offspring were obtained by mating F1 hybrids. All the mice were bred under specific pathogen-free conditions and used for the following analysis at about 8 weeks of age. To examine the phenotype of T-cell deficiency, CD3+ cells in peripheral blood were analyzed by flow-cytometry. Approximately 0.3 ml of blood was removed from the ophthalmic plexus of each mouse. Blood lymphocytes were stained with FITC-labeled mouse CD3-specific monoclonal antibody (1:50 dilution of ascites of Clone 145-2C11; PharMingen, San Diego, Calif.) and analyzed by using FACScan (Becton Dickinson, Mountain View, Calif.). Mice showing CD3+ cell percentages 515% were taken as the T-cell-deficient (CTS) phenotype, and the remaining mice as the normal (C3H/He) phenotype (details are described below). The rates of phenotype segregation at the BC1 and F2 generations were statistically tested for matching with the Mendelian laws by the c2 test. The percentage of CD3+ cells in peripheral blood differed greatly between the parent strains, being low (510%) in CTS mice (n = 29) and high (70% to 90%) in C3H/He mice (n = 38). A high percentage (averaged 64.4%) was also obtained with F1 hybrids (n = 48), although it was distributed over a relatively wide range from 45% to 85%. Similarly, all the BC2 mice (n = 102) showed high percentages ranging from 55% to 95% (averaged 78.3%). In contrast, segregation of low and high percentages occurred in BC1 mice: 54 of 109 mice displayed low values (510%), and the remaining mice over 15% (averaged 43.3% and ranged from 15% to 85%). Segregation was also observed in F2 mice: 53 of 248 mice displayed low percentages (515%), and the remaining mice over 20% (averaged 64.5% and ranged from 20% to 90%). Thus, the incidence of T-cell deficiency showing the CD3+ cell percentage below the arbitrarily determined criterion, i.e., 15%, was 49.5% in BC1 mice and 21.4% in F2 mice. Statistical examination of these results by the c2 test revealed that the segregation rates matched well the value (50% and 25% in the BC1 and normal F2 generations, respectively) expected from the hypothesis of one-recessive gene regulation (Table 1), which also agreed well with our preliminary test results (Yagi et al. 1996). We decided to tentatively call the present locus ptcd (peripheral T-cell deficiency). Since the average percentage of the normal S. Kimura ( ) ? K. Nakamura ? M. Harada Developmental Research Laboratories, Shionogi & Co., Ltd., 3-1-1 Futaba-cho, Toyonaka, Osaka 561, Japan

Leukocyte Adherence Inhibition Test in Renal Diseases

Leukocyte adherence inhibition (LAI) test was examined in 35 patients with renal diseases and 14 normal controls, using collagenase-treated glomerular basement membrane, glycosidase-treated glomerular basement membrane and renal tubular epithelium as antigens. Although the control group showed strikingly similar mean LAI indices for all antigens tested, the whole group of patients with renal diseases showed a wide scatter of values. Two categories of patients had significantly increased LAI indices (p less than 0.01) when their mean values were compared with those of normal controls: (1) rapidly progressive glomerulonephritis (RPGN) and (2) lupus nephritis (SLE). In the serial studies of the RPGN and SLE cases, there were no significant changes in the pattern of LAI and they continued to give positive and very comparable results when re-examined at intervals of 1-6 months. Out of the 30 patients who were able to be evaluated with the three antigens, 15 cases exhibited positive LAI response to two or more antigens simultaneously. These in vitro findings suggest that there is an abnormal cellular response to certain antigen or widespread LAI reactivity to a variety of renal antigens in certain forms of human glomerulonephritis.