PGE2-receptor subtype EP4-dependent adherence of mastocytoma P-815 cells to matrix components in subcutaneous tissues overlaying inside surface of air pouch cavity in CDF1 mouse.

OBJECTIVES: It remains to be fully clarified how adhesion of mast cells is regulated in vivo. We previously reported that PGE2-receptor EP4 stimulated the adhesion of mouse mastocytoma P-815 cells to plate-bound fibronectin. Our purpose in this study is to evaluate the adhesion using a system, which can mimic the in vivo adhesion. METHODS: P-815 cells were transplanted in an air pouch produced in the transplantable mice, CDF1. The number of cells that adhere to the subcutaneous tissues overlaying the inside cavity surface was determined. RESULTS: The number of adhered cells was decreased in mice administered with ibuprofen or an EP4 antagonist, ONO AE3-208. A local administration of PGE(2) or a phorbol ester, PMA, increased the number of adhered cells, which was also suppressed in the mice treated with ONO AE3-208. CONCLUSION: Our results suggest that PGE(2)-mediated adhesion of P-815 cells in the subcutaneous tissues of the air pouch is mediated by the EP4 subtype.

Isolation and structural analyses of positional isomers of 6(1),6(n)-di-O-(N-acetyl-beta-D-glucosaminyl)cyclomaltoheptaose (n=2, 3, and 4) and 6-O-[6-O-(N-acetyl-beta-D-glucosaminyl)-N-acetyl-beta-D-glucosaminyl]cyclomaltoheptaose.

6-O-[6-O-(N-acetyl-beta-D-glucosaminyl)-N-acetyl-beta-D-glucosaminyl]cyclomaltoheptaose (beta CD) and three positional isomers of 6(1),6(n)-di-O-(N-acetyl-beta-D-glucosaminyl)cyclomaltoheptaose (n=2, 3, and 4) in a mixture of products from beta CD and N-acetylglucosamine by the reversed reaction of beta-N-acetylhexosaminidase from jack bean were isolated and purified by HPLC. The structures of four isomers of di-N-acetylglucosaminyl-beta CDs were determined by FABMS and NMR spectroscopy. The degree of polymerization of the branched oligosaccharides produced by enzymatic degradation with bacterial saccharifying alpha-amylase (BSA) was established by LC-MS methods.

[Effects of stress on allergic reaction].

The purpose of the present study was to investigate the effects of stress on allergic reaction. We studied changes in the contact hypersensitivity reaction (CHR) of mice exposed to foot shock (FS) stress or psychological (PSY) stress induced by the communication box. CHR was elicited by applying antigen (2,4-dinitrofluorobenzene, DNFB) to the ear of the mice at 4 days after DNFB sensitization. In acute stress experiments, DNFB-sensitized mice were exposed for 2 h to FS or PSY stress after contact challenge with DNFB. Acute FS stress significantly inhibited CHR immediately after the end of the stress period (3 h after DNFB-challenge), and a significant enhancement of the CHR was observed at 5 h after DNFB-challenge. The concentration of the serum corticosterone level of the mice exposed to acute FS stress significantly increased compared to the control mice, immediately after stress loading. Both CHR and serum corticosterone levels after acute PSY stress loading were almost the same as those of the control groups. The temporary decrease of the inflammatory reaction at CHR caused by acute FS stress loading may have been correlated with serum corticosterone produced by the stress, and the increase of corticosterone may act as a trigger of enhancement of the CHR (delayed-type hypersensitivity). Chronic stress experiments were designed to expose mice to FS or PSY stress 2 h daily after sensitization with DNFB. These chronic stresses caused a significant reduction in the body weight of mice. The temporary decrease effect on the CHR of chronic FS stress-loaded mice was similar to the acute FS stress-loaded mice. In contrast, no significant enhancement of the CHR (late phase) at 24 h and 48 h after challenge was observed. Although the changes in body weight suggested that mice were influenced by chronic PSY stress, no significant difference from the control group for the CHR of mice exposed to chronic PSY stress was found, as in the case of acute PSY stress loading. The correlation between chronic PSY stress and CHR remains to be ascertained.

Detection of anaphylactic reaction in the percutaneously sensitized mouse using the AW method.

Anaphylactic reactions of mice sensitized percutaneously with 2,4-dinitrofluorobenzene (DNFB) were investigated by the AW method assay, which is a mouse anaphylactic model using the abdominal wall as the site for induction with either 2,4-dinitrophenyl (DNP)-human serum albumin or anti-mouse IgE antibody and then estimation of the response. DNP-specific and IgE-dependent anaphylactic reaction after contact sensitization with DNFB could be induced and detected by the abdominal wall (AW) method assay in both groups with and without previous ear challenge with DNFB. Thus, the anaphylactic reaction in the group of twice-contact with 0.5% DNFB was observed on the 9th day from the sensitization (5th day from the ear challenge), and the reaction in the group of a single contact with 0.5% DNFB was observed 10 d after sensitization. The DNP-specific anaphylactic reaction was observed earlier than the 10th day with higher doses of DNFB. As for the mice of the former twice-contact group, the first and second characteristic ear swelling responses appeared within 1-6 h and 2 d of the ear challenge, respectively, and small swelling was observed 7 d after the challenge. It is suggested that Th1 and Th2 cells are activated at the almost same time, in other words, the preparation for both cell-mediated and humoral immunity could be accomplished to function, in vivo by a single percutaneous sensitization with DNFB.

Suppression and enhancement of the Freund's incomplete adjuvant-induced writhing reaction by sodium ascorbate in mice.

We noticed that an intraperitoneal injection of Freund's incomplete adjuvant (FIA) into mice could stimulate the induction of a writhing reaction. The FIA emulsion-induced writhing reaction was found to be remarkably inhibited by preadministration of oral indomethacin, a non-steroidal anti-inflammatory and analgesic drug. The induction of the writhing reaction was also inhibited by intravenous preadministration of sodium ascorbate (SAs) in saline. In the experiments where SAs was added to FIA, it was demonstrated that SAs had dual activity of suppression and enhancement. At lower concentrations SAs functioned as a suppressor of the writhing reaction, while at concentrations higher than about 1 mg/50 microl/mouse it acted as an enhancer of the reaction. Furthermore, this writhing reaction induced by FIA+SAs emulsion was also inhibited by preadministraion of SAs itself as well as indomethacin. These results suggested that the mechanism of the writhing reaction induced by FIA was concerned with the production of prostaglandins (PGs), and SAs might be involved in regulation of the writhing reaction. In this paper, we propose a mouse writhing model induced by FIA or FIA+SAs emulsion as a novel pain model useful for assessment of analgesic and anti-inflammatory agents.

Enzymic method for the determination of nitrite in meat and fish products.

A simple spectrophotometric method for the determination of nitrite is described. Nitrite is measured enzymically through its reaction with nitrite reductase coupled with NADH. The entire enzymic procedure required 15 min to complete. The calibration curve was linear in the range 0.1-10 micrograms cm-3 nitrite with a slope of 6.25. The relative standard deviation at 5 microgram g-1 was 1.7% (n = 5). The method greatly simplifies the procedure of nitrite determination and enables the routine inspection of a number of samples with very little laboratory equipment. A comparison study showed that the method was superior to the GC method for samples containing large amounts of reducing substances while good agreement was achieved between both methods for other foods.

A novel method for induction and detection of anaphylactic reaction using the mouse abdominal wall (AW method).

We found that an antigen-specific anaphylaxis was induced by antigen challenge to the abdominal wall, ear auricle, or subcutaneous tissue in mice sensitized 9 days previously with antigen and adjuvant. The anaphylactic reaction was detected by vascular permeability at the injected site 7 minutes after challenge, which was the best time for estimation. A novel method (AW method) for induction and detection of the anaphylactic reaction in mice was established using the abdominal wall as the challenge site. This method could detect the anaphylactic response in mice 1 to 3 weeks after sensitization. The increase in vascular permeability was completely inhibited by administration of diphenhydramine.

Ischaemic heart disease deaths in a Japanese rural area evaluated by clinical records.

A community-based survey to examine the actual number of ischaemic heart disease (IHD) deaths was undertaken in the western area of Toyama, a rural area with a population of 209,000. IHD deaths (International Classification of Diseases [ICD], ninth revision, codes 410-414) and heart failure (HF) deaths (ICD code 428) according to the death certificate aged 15 to 74 in 1987 to 1990 were reevaluated from medical records using the criteria of the multinational monitoring of trends and determinants in cardiovascular disease (MONICA). Of 97 subjects with IHD and of 170 subjects with HF according to the death certificate, 85 cases (87.6%) and 143 cases (84.1%) could be examined, respectively. Using the MONICA criteria, of 85 subjects with IHD, 18 (21.2%) were reevaluated as "definite acute myocardial infarction (AMI)" and 30 (35.3%) as "possible AMI". On the other hand, of 143 subjects with HF, 1 (0.7%) were reevaluated as "definite AMI" and 12 (8.4%) as "possible AMI". From these results, the number of deaths as reevaluated IHD was estimated 104.5 in the study period, and it was 7.7% increase at least compared with the number of deaths judged from the death certificate.

Enzymic method for the spectrophotometric determination of benzoic acid in soy sauce and pickles.

A simple spectrophotometric method for the determination of benzoic acid is described. Benzoic acid is measured enzymically through its reaction with benzoate 4-hydroxylase coupled with NADPH and O2. The entire enzymic procedure required 20 min to complete. The method greatly simplifies the procedure for benzoic acid determination and permits the routine inspection of a number of samples with very little laboratory equipment. The method was compared with HPLC and satisfactory agreement was achieved.

Enzymic method for the amperometric determination of nicotinic acid in meat products.

An enzymic method for the determination of nicotinic acid is described, based on the indirect electrochemical monitoring of nicotinic acid via its reaction with oxygen in the presence of nicotinic acid hydroxylase. Derivative amperometric signals due to oxygen depletion enable a one-point kinetic analysis to be carried out. Nicotinic acid hydroxylase catalyses the hydroxylation of nicotinic acid to produce 6-hydroxynicotinic acid, which is accompanied by a stoichiometric consumption of oxygen. The method was applied successfully to the analysis of real samples.

Comparative study on mortality patterns in Japan and China.

On the basis of the 1990 Chinese death notice data and 1990 Japanese populations vital statistical materials, the mortality patterns and proportions of individual causes of death in the two countries were compared. In both sexes, the mortality rates were the highest in almost all age groups in Chinese rural areas followed by urban areas and Japan. In Japan and in Chinese urban areas, malignant neoplasms cardiovascular diseases and cerebrovascular diseases were the major causes of death, with these three making up about 60% of the total. In Chinese rural areas, these three conditions were responsible for 43% of all deaths, a considerably lower figure as compared to those in the other two areas, and the mortality rates for infectious diseases and accident/suicide were higher than those in the other two areas. Of the three major adult diseases, cerebrovascular diseases were found to be especially frequent in both Chinese urban and rural areas. On the other hand, ischemic heart disease was found at comparable levels in Japan and China and the rate was lower than in other developed countries. Site-specific mortality rates for malignant neoplasms were characterized by high rates for lung, liver and esophageal cancers in China. The mortality rates for bronchitis in both Chinese urban and rural areas were markedly higher than those in Japan These differences in mortality rates and proportions of individual causes of death between China and Japan are thought to be attributable to the differences in medical services and the level of risk factors for each disease.

Participation of nitric oxide in mouse anaphylactic hypotension.

Mouse hen egg-white lysozyme-specific anaphylaxis was estimated by monitoring changes in blood pressure by using a tail-cuff method. Stimulation of histamine H1 receptors of the vascular endothelium was suggested to be critical for mouse anaphylactic hypotension, because pretreatment with diphenhydramine but not with cimetidine completely inhibited the hypotension. Nitric oxide (NO) was indicated to play an important role in mouse anaphylaxis, because NG-nitro-L-arginine methyl ester, a NO synthase inhibitor, significantly blocked the hypotension while a large amount of L-arginine, a precursor of NO synthesis, restored the hypotension.

Activation of idiotype-specific CD4+ T-cell line: cellular processing of exogenous self-immunoglobulin.

An idiotype (Id)-specific long-term cultured T-cell line has been generated from BALB/c mice immunized with M315 (alpha, lambda 2). The cell line comprises both CD3+ and CD4+ but CD8- cells. The T-cell line is stimulated in a class II major histocompatibility complex (MHC)-restricted manner, and is capable of producing interleukin-2 (IL-2) in response to the Id along with Iad-bearing antigen-presenting cells (APC). Fine Id specificity analysis has shown that changes in amino acid residues, Phe-94, Arg-95 and Asn-96, located on the VL-315, resulting from a somatic mutation mechanism of the mouse V lambda 2 gene, contribute to the T-cell activation. Pretreatment of APC with either glutaraldehyde or paraformaldehyde prevented both Fv-315 and VL-315 from triggering the T cells. This suggested that further processing of VL is required for T-cell activation. To clarify this point, we have generated a synthetic peptide, designated P18, which spans residues 91-108 of VL-315. In sharp contrast to VL, prefixed APC were capable of presenting P18 to stimulate the T-cell line to induce IL-2.

Immunomodulating activities of staphylococcal enterotoxins. I. Effects on in vivo antibody responses and contact sensitivity reaction.

The immunomodulating effects of staphylococcal enterotoxins on in vivo immune responses in C57BL/6 mice were examined. Of the five serological types A (SEA), B, C, D, and E (SEE), only SEA and SEE markedly suppressed the antibody response to sheep red blood cells (SRBC) when injected 1 day before or on the day of immunization with SRBC. Further study of SEA revealed that it did not affect the antibody response to a thymus-independent antigen, salmonella flagella, but did affect the T-cell-mediated immune response. Contact sensitivity to dinitrofluorobenzene (DNFB) was suppressed when SEA was injected before sensitization or before challenge with DNFB, indicating that SEA affected both the afferent and efferent phases of DNFB contact sensitivity. As the suppression of DNFB contact sensitivity could be transferred by anti-Thy-1.2 antibody-sensitive spleen cells of SEA injected donors into normal or DNFB-sensitized recipients, the suppression was thought to be an active one. However, SEA could augment the DNFB contact sensitivity when injected on the third day after sensitization with DNFB. These results indicate that the immunomodulating effects of SEA can be mediated by the T-cell function.

Induction of suppressor T cells to dinitrofluorobenzene contact sensitivity by application of sensitizer through Langerhans cell-deficient skin. II. Kinetics of the induction of suppressor T cells.

Tolerance to dinitrofluorobenzene (DNFB) contact sensitivity (DNFB CS) appeared in C3H/HeN mice within 2 days after application of DNFB on Langerhans cell-deficient tail skin. A single painting on tail skin led to complete prevention of DNFB CS for at least 14 days. Spleen cells from tolerant mice could transfer the suppression to syngeneic recipients, while the cells treated in vitro with anti-Thy 1.2 lost their suppressive activities completely. From these facts, this tolerance might be due to active suppression caused by suppressor T cells. These suppressor T cells were antigen-specific, because recipients exhibited normal CS to picryl chloride. Furthermore, the suppressor T cells were demonstrated to be afferent blockers in nature and their precursors were sensitive to cyclophosphamide. Therefore, the suppressor T cells induced by DNFB percutaneously administered through epidermal Langerhans cell-deficient region were thought to be generated in the same or in a very similar suppressor pathway as the intravenously administered dinitrobenzene sulfonate (DNBSO3), a soluble reactive dinitrophenyl congener.

A small hypervariable segment in the variable domain of an immunoglobulin light chain stimulates formation of anti-idiotypic suppressor T cells.

The induction in BALB/c mice of suppressor T cells that block a delayed-type hypersensitivity (DTH) response to the idiotype of M315, a myeloma protein of BALB/c origin, was examined with a variety of immunoglobulin chains and fragments whose amino acid sequences are known. Normal BALB/c mice receiving either the light chain of M315 (L315, lambda 2 isotype) or the variable (V) domain of this chain prior to sensitization with M315 showed marked suppression of DTH to the M315 idiotype. In contrast, neither the heavy chain nor the variable domain of the heavy chain of M315 affected the DTH response. Two other lambda 2 chains were tested and they also failed to suppress DTH to M315. Comparison of amino acid sequences in the three lambda 2 chains indicates that in L315 at most four V region amino acid substitutions (each resulting from a somatic mutation in the V lambda 2 germ-line gene) determine the specificity of the T-cell suppressor pathway. One of the four is in the framework and probably of negligible importance; the other three, however, are all clustered in the third hypervariable loop of the L315 V domain. The tertiary structure of L315 may also be essential, because disruption of intrachain disulfide bonds abolished the ability of the chain to induce suppression.

Regulation of delayed-type hypersensitivity (DTH) response to hen egg-white lysozyme (HEL).

Delayed-type hypersensitivity (DTH) can be demonstrated in A/J mice immunized with hen egg-white lysozyme (HEL) in complete Freund's adjuvant (CFA) by challenging primed animals in the ear with aqueous HEL. Normal A/J mice receiving soluble HEL derivative peptide, P-Ib, sequence 29-54 and 109-123 linked by a single S-S bond, 7 days prior to immunization with HEL showed much lower DTH response specific to the protein. The inhibition of DTH reactivity is due to active suppression and involves the generation of suppressor T-cells (Ts). Thus, the suppression induced with a single i.v. injection of P-Ib solution was able to be transferred into syngeneic recipients by the spleen cells from mice pretreated with P-Ib. These suppressor cells are T-cells, since their ability to suppress DTH is completely abrogated by treatment wit anti-Thy 1.2 and complement. Amongst HEL derivative peptides tested in the present study, only P-Ib could induce the tolerance.

Idiotypic analysis of antibodies to hen egg-white lysozyme (HEL). III. Further studies on the idiotypic cross-reactivity among anti-HEL antibodies in mice.

An isolated antibody preparation directed to the native hen egg-white lysozyme (HEL) from a single A/J mouse (#a-11), termed IDHELa-11, was inoculated into rabbits to produce anti-idiotypic sera (anti-Id). The antisera were extensively absorbed with normal A/J Ig to render then idiotype specific. Radioimmunoassay utilizing 125I-IdHELa-11 and the anti-ID sera (R103 and R104) was performed to examine the idiotypic cross-reactivity of the humoral immune response to HEL in various mouse strains and other animal species. Idiotypes shared by IdHELa-11 were detected in the sera of five mouse strains tested, but not in any of the examined sera of other animal species such as rats, goats, guinea pigs, or sheep, indicating the occurrence of species-specific cross-reactive idiotypes (CRI) of antibodies to HEL. Our experiments also suggested the presence of intrastrain CRI. The present experiments, along with our earlier results (15), suggest that idiotypic cross-reactivity among murine antibodies to HEL appears to be weak. Thus when R103 and R104 were the anti-Id sera used, the frequency of occurrence of CRI shared by IdHELa-11 in 5 micrograms of anti-HEL antibody in strain A mice was 74 ng and 111 ng; in other strains it was 25-44 ng and 60-98 ng, respectively.