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The serum levels of C-reactive protein (CRP) produced as an inflammatory response in dogs with various disorders and surgical traumas were measured by enzyme-linked immunoabsorbent assay and slide reversed passive latex agglutination test (RPLA). The CRP levels were greatly increased 1-2 days after surgery in most of the dogs (n = 29) subjected to surgery. These levels had markedly decreased by the time the sutures were removed. In dogs with various disorders (n = 58), the serum CRP levels at first diagnosis were high in infectious diseases. In dogs from which paired serum samples were examined, the serum CRP usually showed a decrease with improvement in the condition (n = 11) or a terminal increase (n = 4) but, conversely, some showed an increase with improvement in the condition (n = 3).
We have examined biochemical and functional characteristics of dispersed sinus mast cells and compared them with those of mast cells dispersed from other tissues. This experiment yielded the following results. 1) Although no difference was observed in histamine content, tryptase content in sinus mast cells was significantly lower than that of skin and lung mast cells. 2) In contrast with the situation in foreskin mast cells, anti-IgE-induced histamine release from sinus and lung mast cells was potentiated with lower concentrations of adenosine. 3) Similar to lung mast cells, sinus mast cells did not respond to compound 48/80 or substance P, whereas skin mast cells were stimulated to release histamine with either 10 micrograms/ml of compound 48/80 (14.0%) or 10(-4) M of substance P (23.4%). 4) Sinus mast cells are similar to lung mast cells in terms of release of arachidonic acid metabolites. Anti-IgE challenge of sinus mast cells caused the generation of both prostaglandin D2 (89.5 +/- 33.7 ng/10(6) mast cells, n = 14) and i-leukotriene D4 (78.7 +/- 46.8 ng/10(6) mast cells, n = 10).
We measured the airborne and floor dust allergen levels of the cat (Fel d I), dog (Can f I), and mite (Der I and Der II) allergens in 13 houses. Airborne allergens were sampled with a low-noise air sampler for 5 to 7 days in the living rooms where the inhabitants were living as usual. The mean levels of airborne Fel d I and Can f I in houses with cats or dogs were 5960 and 2880 pg/m3, respectively, which were about 160 and 100 times higher than levels of airborne Der I. In floor dust the mean levels of Fel d I and Can f I were 322 and 236 micrograms/gm fine dust, respectively, which were 59 and 10 times higher than the levels of Der I. These results suggest that the airborne cat and dog allergens might be important sources of allergens for persons who live in a house with those animals, because the absolute allergen levels in both the air and dust are significantly higher than those of mite.
The canine C-reactive protein (CRP) fraction isolated from canine acute-phase serum on a phosphorylcholine-Sepharose 4B column was further subjected to Sephacryl S-300 gel filtration. A canine CRP fraction not containing IgM was then obtained. The antisera, obtained after several immunizations with this canine CRP fraction, contained nonspecific antibodies that reacted with albumin, transferrin and IgG in addition to CRP. This antiserum could be easily changed to monospecific canine CRP serum, when it was subjected to absorption for only 15 min using glutaraldehyde-insolubilized normal canine serum protein containing 3.5 micrograms ml-1 of CRP. Pure canine CRP was isolated with a recovery rate of 95% from canine acute-phase serum by affinity chromatography using specific anti-canine CRP antibody.
Expression of muscarinic receptor mRNA subtypes (m1-5) and nicotinic receptor subunits (alpha 2-4, and beta 2) was examined in the rat submandibular gland by in situ hybridization histochemistry, using oligonucleotide probes for the muscarinic receptor and RNA probes for the nicotinic receptors. m2, alpha 3, and beta 2 mRNA were strongly expressed in the submandibular ganglion, and m3, alpha 2, alpha 3, alpha 4, and beta 2 were expressed in the striated and interlobular duct cells. Both muscarinic and nicotinic receptors were coexpressed in the same ganglion neurons, while none of these mRNA were detected in the terminal secretory units.
A simple method for purification of basophils from a relatively small volume of blood has been developed, which enables us to collect basophils with a purity of over 80%. Basophils were partially purified from 10-20 ml of citrated whole blood using the discontinuous Percoll density gradient centrifugation technique and then contaminant cells were removed using monoclonal antibodies against CD2, CD19, CD14 and CD16. At the end of the procedure, basophils were > 80% pure with lymphocytes accounting for most of the contaminating cells. When stimulated with anti-IgE or fMLP, histamine release from purified basophils was similar to that from mixed leukocytes. When highly purified basophils were challenged with calcium ionophore A23187, generation of leukotriene C4 (LTC4) was not significantly different between asthmatic patients and normal subjects (45.6 +/- 22.6 vs 52.7 +/- 25.6 ng/10(6) cells). Basophils were capable of generating LTC4 in approximately the same quantities as eosinophils (46.5 +/- 11.7 ng/10(6) cells, n = 3). Furthermore, it has been shown that incubation of basophils and eosinophils with calcium ionophore generates only small quantities of thromboxane B2 (TXB2).
A new set of allergens from Dermatophagoides pteronyssinus and D. farinae (provisionally named DP5 and DF5, respectively) was isolated from the whole culture of mites. The apparent molecular weights of both allergens were shown to be 25,000 on SDS-PAGE under a reducing condition and 27,000 on Sephadex G-75 gel filtration chromatography. Both DP5 and DF5, as well as Der f III, possessed proteolytic activity. The results of substrate specificity and susceptibility to various protease inhibitors of DP5 and DF5 strongly suggested that they belonged to the chymotrypsin-like serine protease family. In sera from 88 mite-allergic patients, specific IgE antibodies to DP5 and/or DF5 were detected in only 41% of the sera by radio-allergosorbent test, while 90% and 93% had specific IgE antibodies to Der p I and/or Der f I and Der p II and/or Der f II, respectively.
EoL-1 cells differentiated into mature eosinophil-like cells when incubated with butyric acid (BA). The differentiated cells possessed many granules that stained with Luxol fast blue and acquired the ability to produce leukotriene C4 and eosinophil cationic protein (ECP), and migrated toward some eosinophillotactic attractants, including PAF, IL-5, IL-3 and GM-CSF. Monoclonal antibody to GM-CSF and IL-5, especially GM-CSF, strongly decreased the ability of BA to induce differentiation, whereas antibody to IL-3 did not. Recombinant human (rh) IL-3, rhGM-CSF and recombinant mouse (rm) IL-5, by themselves, did not have the ability to induce differentiation. On the other hand, the presence of these cytokines significantly (p < 0.01) augmented BA-induced cell differentiation. Other cytokines including rhIL-1 alpha/beta, rhIL-2, rhIL-4, rhIL-6, rhIL-8, rhTNF-alpha and rhIFN-gamma had no effect on differentiation. Expression of GM-CSF and GM-CSF receptor mRNA was detected in cellular RNA obtained from BA-stimulated EoL-1 cells. Moreover, about 5-10 pg/ml of GM-CSF was detected in the culture supernatant of BA-stimulated EoL-1 cells. These findings suggested that BA-induced EoL-1 cell differentiation was elicited under the influence of GM-CSF endogenously produced in these cells. GM-CSF also seemed to be an autocrine differentiating factor in EoL-1 cells.
Since increased levels of serum soluble CD23/Fc epsilon RII (sCD23) were evidently demonstrated in patients with autoimmune diseases such as rheumatoid arthritis (RA), the possible mechanisms responsible for the elevation of serum sCD23 were investigated in RA patients. In keeping with increased serum sCD23, high proportion of CD23+ B cells was detected in the patients; this was associated with the enhanced expression of only Fc epsilon RIIa mRNA. Upon incubation at 37 degrees C, peripheral blood mononuclear cells of the patients spontaneously released high levels of sCD23 into the culture supernatant, while the CD23 expression on their B cells was considerably maintained even after the culture. Dot blot analysis further revealed that in contrast to normal subjects, RA patients showed no complete disappearance of Fc epsilon RIIa mRNA after the spontaneous culture. In addition, sCD23 release was significantly reduced in the patients by the addition of cycloheximide. It was also found that cycloheximide exerted the inhibitory influence on the spontaneous culture-mediated expression of CD23 on CD5+ but not CD5- B cells of the patients. However, the disappearance of CD23 from CD5+ as well as CD5- B cells of cord blood samples was unaffected by the agent. These results strongly suggest that CD5+ B cells of RA patients may be specifically activated by some mechanisms responsible for the persistent expression of Fc epsilon RIIa mRNA leading to the accelerated turnover of CD23 and in turn the increased release of sCD23.
Platelet-activating factor (PAF)-induced augmentation of spontaneous differentiation into eosinophil-lineage cells from hematopoietic precursor cells was found among human umbilical cord blood mononuclear cells (CBMCs). This ability of PAF to augment eosinophil production was significantly (p < 0.01) inhibited by the addition of the anti-PAF agent WEB 2086. This enhancing effect of PAF was significantly (p < 0.01) diminished by the presence of antibodies to interleukin (IL)-1 alpha, -beta and IL-3, whereas antibodies to IL-5 or granulocyte-macrophage colony-stimulating factor (GM-CSF) did not diminish it. IL-1 beta, IL-3 or IL-5 gene expression was detected in cellular RNA isolated from both medium- and PAF-stimulated CBMCs using the reverse transcription polymerase chain reaction (RT-PCR) method; however, no expression of GM-CSF was detected. Moreover, 5 times as much IL-3 was detected in the PAF-stimulated CBMC culture supernatant than in that of cells treated with the medium. In the case of IL-1 beta, there was no difference between the 2 cell preparations. On the other hand, no IL-5 or GM-CSF was detected in the supernatant after stimulation with medium or PAF. Depletion of CD2+, CD16+ or CD19+ cells, but not CD14+ cells, from CBMCs resulted in a marked decrease in PAF-induced augmentation of eosinophil production. These results suggest that PAF induces the augmentation of IL-3 production in CBMCs which in turn enhances differentiation into eosinophil-lineage cells.
To elucidate the conformational properties of DNA with a 2-deoxyribosylformamide residue (dF), an oligodeoxyribonucleotide containing this abasic residue in a specific position of the nucleotide sequence was synthesized by the standard solid-phase phosphotriester method. Deprotection of the synthesized oligonucleotide was performed under routine alkaline and acidic conditions. The presence of a dF residue in the oligomer was confirmed by ion-spray mass spectrometry. A dF residue was found to affect considerably the stability of the DNA duplex, as determined from the melting behavior of the dF-containing duplex.
The effect of IPD-1151T, a new dimethylsulfonium compound, on the IgE response was investigated in the mouse system. The oral administration of IPD-1151T to immunized BALB/c mice suppressed the primary IgE antibody response and depressed the elevation of serum IgE levels, whereas the same treatment did not affect the IgG antibody response. The enhanced expression of low-affinity IgE receptor (Fc epsilon RII/CD23) on the spleen cells of immunized mice was also inhibited by IPD-1151T administration. It was further demonstrated from the adoptive transfer experiment that IPD-1151T, administered to hapten-primed B cell donors, but not to carrier-primed T cell donors, exerted its suppressive influence on the hapten-specific secondary IgE antibody response in irradiated syngeneic recipients. Interestingly, IPD-1151T concentration-dependently inhibited the production of interleukin 4 (IL-4) by D10G4.1, known to be a typical Th2 clone. However, IPD-1151T did not suppress the production of IgE and IgG1 by normal splenic B cells stimulated with lipopolysaccharide and IL-4. Moreover, IL-4-induced expression of Fc epsilon RII on normal spleen cells was not inhibited by the agent. These results strongly suggest that the IgE-suppressive activity of IPD-1151T is most likely due to the inhibition of IL-4 production at the T cell level.
The ability of IPD-1151T to suppress the induction of human IgE synthesis was investigated with an in vitro model of IgE production mediated by an allergen-specific helper T cell line (SN-4) from a patient allergic to Japanese cedar pollen. IPD-1151T induced a concentration-dependent suppression of purified allergen (Cry j 1)-dependent IgE synthesis in autologous B cell cultures mediated by SN-4, without significantly affecting the IgG synthesis. In addition, the production of interleukin 4 (IL-4) by Cry j 1-activated SN-4 as well as that by phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) of normal donors was inhibited in a concentration-dependent manner by the agent. Interestingly, IPD-1151T clearly depressed PHA-induced expression of IL-4 mRNA in normal PBMC, indicating that this agent inhibits IL-4 gene transcription. However, IPD-1151T had no antagonistic action on IL-4, since neither IL-4-induced expression of low-affinity IgE receptor (Fc epsilon RII/CD23) on normal B cells nor soluble Fc epsilon RII release from IL-4-stimulated B cells was affected by the agent. On the other hand, IPD-1151T had no effect on the production of interferon-gamma by both Cry j 1-stimulated SN-4 and anti-CD3 monoclonal antibody-activated T cells of normal donors. These results suggest that the selective suppression of IgE synthesis by IPD-1151T results from the inhibition of IL-4 production by T cells at the gene level.
We examined the clinical usefulness of three diagnostic tests including the histamine release test, RAST and prick test in determining which of a total of 349 allergens were responsible for individual cases of food hypersensitivity. The subjects were 42 children (mean 3.3 years old). When compared with the results of the offending allergen confirmed by elimination and provocation test, each diagnostic test showed a fairly good correlation in percentage agreement and negative agreement, but there was poor correlation in positive agreement (histamine release test 58.1%, RAST 62.8%, prick test 48.8%). False positive results were more frequently observed in RAST (12.6%) as compared with the histamine release test (4.0%) and prick test (2.3%). McNemar's analysis demonstrated that the histamine release test, but not RAST or the prick test, was matched the diagnosis by elimination and provocation test (alpha = 0.30). These results suggest that the histamine release test is more clinically useful than RAST or the prick test in the diagnosis of food hypersensitivity.
Nine cases were diagnosed to be eosinophilic cystitis on the basis of histological criteria and clinical observations were made on these cases. In all cases, a past history of allergic or collagen diseases were revealed. Eosinophils in urine were present in 8 out of 9 cases and allergic tests were then performed. Histamine release from biopsied bladder tissue, incubated with anti-IgE, were first examined and a higher level than control was found. Next, intravesical provocation test (IgE RAST positive allergen was introduced into vesical cavity) revealed the appearance of bladder irritabilities within 30 minutes and release of urinary histamine within 3 hours. In 3 cases, provocation tests by eating food were performed and 2 out of 3 cases showed a positive reaction. These results show that allergy may be related to the development of eosinophilic cystitis in part.
In order to study the structural properties of Holliday junctions, we designed and synthesized oligodeoxyribonucleotides to construct the models of the Holliday junctions with no sequence symmetry at the sites of branching called immobile junctions. Electrophoretic and spectroscopic analyses of the immobile junctions indicated that the Holliday junctions constructed from four deoxy 24-mers and four 18-mers were stable in 0.1 M NaCl at approximately 5 x 10(-6) M strand concentration at room temperature. Addition of MgCl2 to the solution of HJs resulted significantly in an increase in melting temperature of HJs.
The species of Candida albicans (C. albicans) are known to colonize mucocutaneous regions as part of the endogenous flora. C. albicans is also known as an important allergen in human allergic diseases. However, the major allergen of C. albicans has not been identified yet. Acid protease released from C. albicans (CAAP) cleaves IgA or the secretory component to enable C. albicans to colonize on the mucous membrane. In this study, using CAAP and crude Candida antigen as challenging antigens, we carried out conjunctival provocation tests (CPT) on 68 adult bronchial asthmatic patients who showed immediate positive skin reactions to crude Candida antigen. In several patients we performed serum IgE antibody (RAST), histamine release from peripheral leukocytes (HRT) and bronchial provocation tests (BPT) to CAAP. Six of the 8 patients who showed positive CPT to crude Candida antigen (75%) showed positive CPT responses to CAAP. On the other hand, only 1 of the 59 patients who showed negative CPT to crude Candida antigen (1.7%) showed positive CPT reactions to CAAP. Two female patients showed positive RAST and HRT to both crude Candida and CAAP. They also showed positive CPT and BPT. One patient had positive RAST results but negative HRT, CPT and BPT results. These results show that CAAP is an important allergen for atopic patients allergic to C. albicans.