Hylan G-F 20 induces delayed foreign body inflammation in Guinea pigs and rabbits

Recent clinical evidence suggests that hylan, a modified hyaluronan, and related products potentially elicit foreign body granulomatous inflammation in human soft tissue. We investigated the biocompatibility of hylan G-F 20 (Synvisc) for up to 28 days after intradermal injection in guinea pigs and intramuscular injection in rabbits. Compared to saline and unmodified hyaluronan, hylan induced definitive macroscopic changes in guinea pigs by day 14 or later and in rabbits by 28 days after injection. Histologically, at the injection sites, there was severe granulomatous inflammation in guinea pigs and acute inflammation with minimal infiltration of macrophages and foreign body giant cells in rabbits. Furthermore, specific antibodies against hylan were demonstrated in guinea pigs by passive cutaneous anaphylaxis, and substantial deposits of IgG on hylan were evident by immunohistochemistry. The present results contradict previous reports on biocompatibility of hylan and suggest that hylan may potentially induce similar unfavorable reactions in humans.     

Immunological studies on delayed hypersensitivity to phenytoin--I. A new method for preparing phenytoin antiserum

The present study was initiated to produce an antiserum to phenytoin with high specificity and sensitivity which would be suitable not only for determination of blood phenytoin concentration but also for induction of a hypersensitivity reaction to phenytoin in experimental animals. p-Aminophenytoin was synthesized and identified by means of IR, 1H-NMR and mass spectroscopy. BSA-phenytoin conjugate was prepared by using p-aminophenytoin, BSA and, as a coupling reagent, glutaraldehyde. Satisfactory response to immunization was achieved at a 9.8:1 molar ratio of p-aminophenytoin to BSA. The antiserum obtained from rabbits immunized with BSA-phenytoin conjugate exhibited practically no cross-reactivity with either phenytoin metabolites or other anti-epileptic drugs, indicating that this antiserum provides sufficiently high specificity. In our experiments, the lower limit for detecting phenytoin was 2 ng using RIA, whereas 200 ng was the minimum amount detectable by HPLC. Thus, by a difference of two orders of magnitude, the present RIA method shows a much higher sensitivity than that of HPLC, though we found a good correlation of simultaneous determinations of serum phenytoin between the two methods. Reproducibility of phenytoin determination in plasma was confirmed by calculating the coefficient of variance. The values were less than 10%.     

Transfer of delayed Hypersensitivity by Lymph-Node Cells in Testis Autosensitization

Guinea pigs sensitized by an extract of homologous testis and Freund's adjuvant developed delayed skin hypersensitivity towards a purified testis antigen. When lymph-node cells from sensitized animals were transferred into normal guinea pigs by intravenous, intraperitoneal or intracutaneous injection the recipients also developed delayed skin hypersensitivity.

Maximum reactions in recipients were obtainable after the transfer of cells from donors which had been sensitized for only 6 days. The recipients became sensitized immediately after cell transfer but their sensitization lasted only a few days.

It can be concluded from this and earlier work that sensitized cells as well as free circulating antibody play a part in testis autosensitization.

     

Specific immunological and bronchopulmonary responses following intradermal sensitization to free trimellitic anhydride in guinea pigs

We have developed a guinea pig model of trimellitic anhydride-induced airway hypersensitivity responses. In one group of guinea pigs, injected intradermally with 0.1 ml 30% trimellitic anhydride (TMA), we examined the specificity of the bronchopulmonary response to TMA comparing the effect of intravenous TMA conjugated to guinea pig serum albumin (GPSA) with a control hapten (procion dye) protein conjugate (PD-GPSA). A significant increase in pulmonary inflation pressure (PIP) was provoked in sensitized animals following intravenous injection with TMA-GPSA (20%; 0-400, median; range) as compared to intravenous injection of PD-GPSA. In the second group we compared three different methods of sensitization: single injection of 0.1 ml of 0.3% TMA; four injections of 0.1 ml of 0.1% TMA; and a single high dose injection of 30% TMA. Following intravenous TMA-GPSA guinea pigs sensitized with a single injection 0.3% TMA had an increase in PIP of 395%; 220-600, while those given four repeat injections of 0.1% TMA had an increase in PIP of 343%; 315-490. These results were significantly higher than the increase in PIP (160%; 0-220) which occurred in guinea pigs sensitized with a single dose of 30% TMA. Four of 11 guinea pigs given low dose injections of TMA had bronchopulmonary responses to inhaled TMA-GPSA. All sensitized guinea pigs had specific IgG1 antibodies demonstrated by enzyme linked immunosorbent assay (ELISA) and confirmed by ELISA inhibition. Four guinea pigs sensitized by low dose injections of TMA had IgE antibodies demonstrated by passive cutaneous anaphylaxis.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Prevention of Delayed Hypersensitivity to Homologous Serum and Transplantation Antigens in Guinea Pigs

The injection of normal guinea-pig serum in Freund's complete adjuvant into guinea pigs other than the serum donor led to the development of a long-lasting, delayed hypersensitivity. Serum alone, without adjuvant, had no sensitizing capacity. Circulating antibodies to the allotypic antigens could not be detected.

Injection of as much as 8 ml. of serum into sensitized animals did not achieve desensitization. However, the intravenous injection of the same amount of serum prevented normal guinea pigs from becoming sensitized to the same antigen for over 100 days. This unresponsiveness was interpreted to be due to an interference by the serum with the process of sensitization.

This method of producing unresponsiveness was applied to the homograft reaction: guinea pigs, given a series of intravenous injections of spleen extracts containing transplantation antigens, could not be subsequently sensitized by the injection of spleen cells from the same donors. However, immunization provided by skin grafting could probably break through this unresponsiveness: guinea pigs, judged to be unresponsive by the intradermal injection of spleen cells before skin grafting, all developed an intense cutaneous hypersensitivity after they had rejected the graft.

     

Decreased sensitivity and response of isolated tracheal muscle to methacholine and histamine without changing the activity of pulmonary muscarinic receptors in the egg albumin-sensitized guinea pig

Guinea pigs were sensitized by daily intraperitoneal injections of 100 mg of egg albumin for 3 consecutive days and sacrificed at 30-35 days after the last injection. The in vitro sensitivity and response of the sensitized tracheal muscle to methacholine and histamine were significantly less than those of littermate controls. However, there was no difference between the dissociation constants and concentrations of pulmonary muscarinic receptor binding sites of the control and sensitized guinea pigs. We conclude that subsensitivity and hyporesponsiveness to methacholine and histamine occur in the airway muscle of this guinea pig model of experimental asthma and suggest that the decreased reactivity to methacholine might be due to defects beyond the receptor.     

Delayed hypersensitivity to hapten-skin protein conjugates in guinea pigs sensitized to benzo(a)pyrene

Guinea pigs were sensitized to 3,4-benzo(a)pyrene, by epicutaneous application or by footbad injection in Freund's complete adjuvant. Conjugates of benzo(a)pyrene and guinea pig skin protein formed by ultraviolet radiation could elicit cutaneous delayed hypersensitivity and could inhibit the migration of macrophages obtained from guinea pigs sensitized to the carcinogen. Extracts of benzo(a)pyrene-treated guinea pig skin and conjugates formed in vitro with benzo(a)pyrene isocyanate were unable to consistently elicit delayed hypersensitivity reactions in vivo or in vitro. The results indicate a high degree of hapten-carrier specificity to contact sensitivity to benzo(a)pyrene.     

Immunologic Events in Experimental Hypersensitivity Granulomas

Polyacrylamide beads were covalently linked to proteins and injected intravenously into normal or immune guinea pigs. The beads trapped in the lung produced very mild foreign body granulomas in nonimmune guinea pigs. In immune guinea pigs, severe granulomas developed which progressed through the several characteristic histologic stages with time. Severe granulomatous reactions developed only upon recognition of carrier determinants of hapten-protein conjugates; thus guinea pigs immune to dinitrophenyl hapten (DNP)-hemocyanin developed characteristic granulomas only upon injection of beads coated with hemocyanin or DNP-hemocyanin, but not with DNP on another unrelated antigen. Granulomatous reactivity was passively transferred into normal animals by lymph node cells but not by serum antibody from sensitized guinea pigs.     

Bronchial hyperresponsiveness to histamine following antigen challenges in actively sensitized guinea pigs

Guinea pigs were actively sensitized by intraperitoneally administered ovalbumin and challenged by intravenous injections of ovalbumin. Respiratory resistance and dynamic compliance were measured by pulmonary mechanics analyzer Buxco Model 6. Changes in resistance (delta R) and compliance (delta C) induced by intravenously administered histamine were investigated before and after the challenge. Responses to the histamine in sensitized guinea pigs were also compared with those in naive guinea pigs. The following results were obtained. 1) A significant increase in delta R by histamine administration was observed following the antigen challenge. delta C by histamine administration showed a tendency to increase after the challenge. 2) A significant increase of delta R by 7.2 micrograms/kg histamine administration was observed in actively sensitized guinea pigs. However, delta C by the histamine administration did not change in the sensitized animals.     

Histamine-releasing activity of lymphocyte supernatants of guinea pig spleen cell cultures

We demonstrated the production of a histamine releasing factor (HRF) by 24-h cultures of guinea pig spleen cells which were stimulated or not with specific antigen (ovalbumin, OA) or mitogen (phytohemagglutinins or concanavalin A). HRF induced the release of histamine from homologous mesenteric mast cells in a dose-dependent fashion. The HRF-induced histamine release was not high compared to the release induced by calcium ionophore A23187, but higher than that induced by compound 48/80, polymyxin B and con canavalin A. The mast cells from sensitized guinea pigs released histamine when challenged with OA. We found that HRF-induced histamine release was additive to that induced by antigen, when both agents were added simultaneously to sensitized mast cells. The phenomenon was most significant when a suboptimal dose of antigen was used. Moreover, we did not observe any differences in the magnitude of HRF-induced histamine release between the mast cells from nonsensitized and sensitized guinea pigs. The time course of histamine release induced by HRF was significantly slower than that with specific antigen (10 min and 45 sec, respectively). Our results may suggest that HRF acts on mast cells through a different not immunological mechanism.     

Effect of amlexanox on experimental allergic rhinitis in actively sensitized guinea pigs

The effect of amlexanox given orally for 3 weeks was studied on the IgE-mediated experimental allergic rhinitis in the actively sensitized guinea pigs. The intranasal instillation of antigen (egg albumin) induced the increase of nasal vascular permeability (dye leakage), histamine content in nasal perfusate and nasal resistance in sensitized guinea pig. Amlexanox, 20 and 60 mg/kg/day given orally for 3 weeks significantly inhibited the increase of dye leakage into the nasal cavity, histamine content and nasal resistance in a dose-dependent manner. These results suggest that amlexanox given orally may be useful therapeutic agent for human allergic rhinitis.     

Physiological and immunological effects of chronic antigen exposure in immunized guinea pigs

Antigenic tolerance was induced in previously sensitized guinea pigs by challenging with ovalbumin (OA) aerosol 1 h/day, 5 days/week for 6 weeks. Reactivity was assessed visually and by lung mechanics. Sera from tolerant and sensitized animals showed comparable titers of antigen-specific antibody by passive cutaneous anaphylaxis with 6-hour, 4-day (heated serum) and 7-day sensitizations. In vitro contractile responses of airway smooth muscle revealed comparable histamine responses in sensitized and tolerant guinea pigs but decreased OA sensitivity in smooth muscle from tolerant animals. Although lung histamine content was equivalent in the two groups, antigen-induced histamine release from chopped lung preparations was significantly less in tolerant animals at a low antigen concentration. We conclude that antigen-induced histamine release is impaired in tolerant animals.     

Death of adherent spleen cells (macrophages)in vitro in hypersensitivity of delayed type to microbial antigens

The cytotoxic action of immune lymphocytes on adherent spleen cells obtained from unsensitized guinea pigs or guinea pigs sensitized with BCG was studied in autologous and allogeneic systems. The low cytotoxic effect found during culture of a suspension of spleen cells of sensitized guinea pigs with tuberculin was greatly increased after the addition of lymph node cells obtained from the same animal. Determination of death of adherent spleen cells, as also of adherent lymph node cells, can be used as a sensitive method for the detection of hypersensitivity of delayed type. The use of spleen cells as target cells is more convenient, for there are many more adherent cells in the spleen than in a suspension of lymph node cells.Laboratory of Streptococcal Infections, N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR P. A. Vershilova.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 85, No. 5, pp. 577–579, May, 1978.     

气道速激肽在卵蛋白致敏豚鼠模型咳嗽反应中的作用

目的：研究气道速激肽在卵蛋白致敏和激发豚鼠咳嗽发生机制中的作用。 方法： 正常和卵蛋白致敏豚鼠各20只，卵蛋白雾化吸入激发。24 h后，正常组和致敏组豚鼠随机各分为2组，每组10只，分别依次腹腔注射生理盐水，0.1 mg/kg、0.3 mg/kg和1.0 mg/kg的NK1受体拮抗剂SR140333 或NK2受体拮抗剂SR48968，观察吸入10-4 mol/L的辣椒素溶液诱导的咳嗽反应。用非侵入性方法测量正常和卵蛋白致敏豚鼠注射SR140333或SR48968前后吸入辣椒素溶液所产生的特异性气道阻力。 结果： 致敏豚鼠咳嗽反应明显高于正常对照组［(9.3±1.2)times/3 min vs (19.5±5.7)times/3 min，P<0.05］。SR140333不影响正常对照组豚鼠的咳嗽反应，而SR48968则可降低咳嗽频率达30% (P<0.05)，两者均抑制吸入辣椒素后增加的气道阻力。而在卵蛋白致敏豚鼠，SR140333或SR48968均抑制吸入辣椒素溶液诱导的咳嗽频率［(9.9±4.7)times/3 min，(8.0±1.6)times/3 min，P<0.05］和增高的气道阻力。 结论： NK受体拮抗剂抑制致敏豚鼠卵蛋白激发后增高的咳嗽反应。因此，气道速激肽可能是嗜酸性粒细胞性气道炎症所致咳嗽的重要介质。     

Measurement of MPO activity as model for detection of granulocyte infiltration in different tissues

Activity of myeloperoxidase (MPO) was determined in different tissues to detect granulocyte infiltration. MPO was measured in the mouse ear after injection of interleukin-1β, in the rat paw after carrageenan-induced edema and in the lung of sensitized guinea pigs after ovalbumin inhalation. Pretreatment of the animals with antiinflammatory drugs abolished the increase of MPO activity in tissues induced by this different stimuli.

     

Elicitation of homologous passive cutaneous anaphylactic reactions by a benzylpenicillin preparation

Four out of five commercially available benzylpenicillin preparations elicited homologous passive cutaneous anaphylactic (PCA) reaction in sensitized guinea pigs with anti-benzylpenicilloyl (anti-BPO) reaginic sera. The same preparations could not evoke PCA reaction in sensitized guinea pigs with anti-BPO γ₁ homocytotropic antibodies. The PCA reactions were completely inhibited by a prior injection of BPO--aminocaproic acid (BPO-EACA). Chromatographic analysis of one of the benzylpenicillin preparations on Sephadex G 10 revealed that the reagin-mediated PCA reaction was not evoked with the fractions from the main peak of the benzylpenicillin but with fractions eluted earlier. None of the fractions gave positive γ₁-mediated PCA reactions. These results indicated that some commercial benzylpenicillin preparations contained minute amounts of the impurities that could elicit the homologous PCA reaction in guinea pigs sensitized with anti-BPO reaginic sera. It was also indicated that the PCA elicitation activity of the benzylpenicillin preparation in the system of reagin-mediated PCA differed from that of γ₁-mediated PCA.     

Eosinophil infiltration and enhancement of airway reactivity by leukocyte chemotactic factor, formyl-methionyl-leucyl-phenylalanine (fMLP), in guinea pigs.

N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) is a bacterial-derived chemotactic factor for eosinophils and neutrophils. This study is aimed to examine whether or not eosinophil infiltration induced by intra-airway administration of fMLP causes the damage of the bronchial epithelium and results in airway hyperresponsiveness in normal non-sensitized guinea pigs. In normal guinea pigs fMLP administered by aerosol inhalation or intratracheal injection caused significant infiltration of eosinophils in the tracheal mucosa and enhanced bronchial reactivity to inhaled histamine 6 and 24 hours after exposure. Electron microscopic examination showed damage of the alignment of the epithelial cells in the bronchial mucosa in fMLP-treated guinea pigs. PAF antagonists CV3988 and WEB2086 and a 5-lipoxygenase inhibitor (AA-861) did not prevent fMLP induced eosinophil infiltration, which suggests that fMLP caused eosinophil infiltration mainly by its chemotactic activity, not by the release of platelet activating factor (PAF) or leukotrienes in this experimental condition. These results showed that in normal guinea pigs a bacteria-derived chemoattractant of fMLP could reproduce a sequence of eosinophil infiltration and airway hyperresponsiveness, similar to the inflammatory pathophysiology after antigen challenge in sensitized animals. We concluded that eosinophil infiltration induced by either immunological or non-immunological mechanisms can cause airway damage and airway hyperresponsiveness.     

IgG2 antibodies block IgE antibody-induced asthma in guinea pigs

In order to examine the blocking activity of IgG2 antibodies to guinea pig for IgE antibodies-induced guinea pig asthma, experiments were carried out as follows. Guinea pigs were passively sensitized intravenously with guinea pig serum containing IgE antibodies to ovalbumin (OA). 8 days after sensitization, IgG2 purified from guinea pigs hyperimmunized with OA was intravenously injected. One hour later, the guinea pigs were challenged by inhalation of OA solution. Asthma attacks were not observed in the guinea pigs, whereas the attacks were observed in guinea pigs passively sensitized with the IgE antibodies but injected IgG2 fraction from normal guinea pigs 1 h before inhalation. These observations suggested that IgG antibodies that increased after immunotherapy might block asthma caused by inhalation of allergens in humans.     

Effect of Y-20811, a long-lasting thromboxane A2 synthetase inhibitor, on antigen-induced airway hyperresponsiveness in guinea pigs.

Effect of Y-20811 on airway hyperresponsiveness was studied in sensitized guinea pigs. Airway hyperresponsiveness to acetylcholine (ACh) reached maximum 7 h after antigen challenge in guinea pigs sensitized actively. Y-20811 (0.3-3 mg/kg) administered orally 3 h prior to challenge inhibited this airway hyperresponsiveness in a dose-dependent manner. Y-20811 (3 mg/kg) administered orally 4 h after antigen challenge also decreased the airway hyperresponsiveness. On the other hand, Y-20811 did not affect the bronchoconstriction induced by ACh, serotonin and histamine in nonsensitized guinea pigs. The number of eosinophils in bronchoalveolar lavage fluid in the guinea pig reached the peak 7 h after antigen challenge. Y-20811 had a tendency to decrease the number of total cells, macrophages and eosinophils in a dose-dependent manner. These results suggest that Y-20811 suppress the asthmatic mechanism which causes antigen-induced airway hyperresponsiveness.     

Attenuation of antigen-induced bronchoconstriction in guinea pigs by a new xanthine derivative (HWA448).

We examined the effect of a new xanthine derivative, HWA448, on antigen-induced bronchoconstriction in actively sensitized guinea pigs. Guinea pigs were sensitized by intraperitoneal injection of bovine serum albumin (BSA) on two occasions, separated by 10 days. Two weeks after the second injection, the animal was placed in a two-chambered whole body plethysmograph and specific airway resistance (SRaw) was monitored for 10 min after an aerosol inhalation of BSA. HWA448 prevented the increase in SRaw after challenge (at 5 and 20 mg/kg i.p.). Aminophylline also prevented the increase in SRaw at 20 mg/kg, but not at a 5-mg/kg dose. The concentration of HWA448, which produced 50% relaxation of the tracheal rings constricted with 0.1 mM of histamine, was 49.9 microM as compared with 18.2 microM in aminophylline. HWA448 has a protective effect on antigen-induced bronchoconstriction in guinea pigs and may be a useful agent in the therapy of bronchial asthma.