Intravenous Injection Of Antigen Research Articles

Rapid desensitization of humanized mice with anti-human FcεRIα monoclonal antibodies

Anaphylaxis is classically mediated by allergen cross-linking of IgE bound to the α chain of FcεRI, the mast cell/basophil high affinity IgE receptor. Allergen cross-linking of the IgE/FcεRI complex activates these cells, inducing release of disease-causing mediators, cytokines, and enzymes. We previously demonstrated that IgE-mediated anaphylaxis could be safely prevented in wild-type BALB/c mice by rapid desensitization with anti-mouse FcεRIα mAb. This study sought to use humanized mice to extend these results to humans. We actively immunized huFcεRIα/F709 mice, which express human (hu) instead of mouse FcεRIα and a mutant IL-4 receptor that lacks inhibitory function. We passively immunized huFcεRIα mice, as well as human cord blood-reconstituted reNSGS mice, which are immune-deficient, produce mast cell-stimulating human cytokines, and develop numerous human mast cells. For desensitization, we used anti-huFcεRIα mAbs that bind FcεRIα regardless of its association with IgE (noncompeting mAbs), and/or mAbs that compete with IgE for huFcεRIα binding (competing mAbs). Anaphylaxis was induced by intravenous injection of antigen or anti-huIgE mAb. Anti-huFcεRIα mAb rapid desensitization was safer and more effective than allergen rapid desensitization and suppressed anaphylaxis more rapidly than omalizumab or ligelizumab. Rapid desensitization of naïve, IgE-sensitized huFcεRIα mice and huFcεRIα/F709 mice that were egg-allergic with anti-FcεRIα mAbs safely removed >98% of IgE from peritoneal mast cells and completely suppressed IgE-mediated anaphylaxis. Rapid desensitization of reNSGS mice with anti-FcεRIα mAbs also safely removed ∼98% of mast cell IgE and prevented IgE-mediated anaphylaxis. Rapid desensitization with anti-FcεRIα mAbs may be a safe, effective, and practical way to prevent IgE-mediated anaphylaxis.

A TLR2 Ligand Suppresses Allergic Inflammatory Reactions by Acting Directly on Mast Cells

Background: Although much attention has been focused on the anti-allergic effects of probiotics, their mode of action is not fully understood. Mast cells, which play a central role in inducing allergic inflammation, are potential targets of probiotics given the recent discovery that they express Toll-like receptors (TLRs), the pattern recognition receptors for microbial components. In this study, we examined whether allergic reactions of mast cells are modulated by stimulation through TLR2. Methods: The effects on mast cells of the synthetic TLR2 ligand Pam3CSK4 and Bifidobacterium pseudocatenulatum JCM 7041 were evaluated in vitro. Furthermore, the effects of Pam3CSK4 on mast cell-induced increase in vascular permeability in vivo were investigated by employing mast cell-deficient W/W^v mice into which IgE-sensitized mouse bone marrow-derived mast cells were transferred. Results: Pam3CSK4 and Bifidobacterium pseudocatenulatum JCM 7041 suppressed degranulation of IgE-sensitized mast cells upon antigen stimulation in vitro. Pam3CSK4 also suppressed leukotriene C₄ production triggered by engagement of the high-affinity IgE receptor, FcεRI. Intracellular Ca²⁺ mobilization and phosphorylation of Erk were suppressed by pretreatment with Pam3CSK4, suggesting that the TLR2 ligand suppresses activation of mast cells by interrupting FcεRI-mediated intracellular signaling. Pam3CSK4 treatment of bone marrow-derived mast cells reduced the increase in vascular permeability in recipient W/W^v mice upon intravenous injection of antigen; the decrease was by about half, in a TLR-dependent manner. Conclusion: Collectively, these results demonstrate that the FcεRI-mediated inflammatory responses of mast cells are suppressed by stimulation through TLR2, suggesting that probiotics exert potential anti-allergic effects, at least in part, through direct effects on mast cells.

The production of antiserum against Myrobalan latent ringspot virus for detection of the virus using ELISA

<I>Myrobalan latent ringspot virus</I> (MLRSV) was purified from the extract of infected <I>Chenopodiumquinoa</I> Willd., using <I>n</I>-octanol, differential centrifugation, and saccharose gradient centrifugation. Electron microscopy showed 28 nm large isometric particles in a sample of purified virus. Antiserum to MLRSV was prepared by immunisation of rabbits with intravenous injections of antigen in combination with intramuscular injections of antigen with Freund’s adjuvant. The antibody titer of the obtained antiserum was determined by drop-precipitation method to be 1:1024. Immunoglobulins against MLRSV were isolated from antiserum with ammonium acetate, caprylic acid, and precipitation with ammonium sulphate. The isolated immunoglobulins (IgG) were conjugated with alkaline phosphatase. The optimal dilution of IgG for detection of MLRSV using ELISA was 1×10<sup>–3</sup>μg/ml which was also the optimal dilution of conjugated IgG. Using this dilution of antibodies, the absorbance of samples from MLRSV-infected plants of <I> C. quinoa</I> varied between 0.71 and 1.45, while absorbance of samples from healthy plants (control) was 0.01 to 0.07.

Head-Down Tilt Posture Attenuates Anaphylactic Hypotension in Mice and Rats

A head-down tilt posture, the Trendelenburg position, which could facilitate venous return from the splanchnic organs and lower extremities, is recommended for the treatment of anaphylactic shock. However, few data of animal studies support its effectiveness. We examined the effects of a head-down tilt maneuver on anaphylactic hypotension in BALB/c mice and Sprague-Dawley rats. We measured systemic arterial pressure (Sap) and portal venous pressure (Pvp) in spontaneously breathing anesthetized animals sensitized with ovalbumin. At either supine (control) or a 30-degree head-down tilt position, anaphylactic hypotension was induced by an intravenous injection of antigen. In the control rats, an increase in Sap by 66 mmHg and a decrease in Pvp by 11.5 cmH(2)O were observed at 2.5 and 6 min, respectively, after antigen. In contrast, in control mice injected with antigen, Sap decreased similarly, but Pvp increased by only 4 cmH(2)O. A head-down tilt maneuver in mice substantially attenuated the antigen-induced decrease in Sap throughout the 60 min measurements, though it aggravated slightly, but significantly, only at the late phase of after 25 min in rats. We conclude that a head-down tilt maneuver attenuates anaphylactic hypotension in anesthetized mice and rats. These beneficial effects were smaller in rats than in mice probably because of substantial portal hypertension, which might prevent the head-down tilt-induced increase in venous return from the splanchnic vascular bed.

A role for TGFbeta and B cells in immunologic tolerance after intravenous injection of soluble antigen.

Intravenous injection of soluble antigen has been reported to induce immunologic tolerance through a variety of mechanisms including T-cell deletion, anergy, and suppression. To clarify the reported discrepancies, we studied mechanisms of intravenous tolerance to a defined transgenic minor transplantation antigen in mice. Wild-type C57BL/6 (B6) mice or congenic B6 B-cell knockout mice were made tolerant to beta-galactosidase (beta-gal). Clinical tolerance was assessed by placement of B6 beta-gal transgenic (tg) and third-party skin grafts. In vitro analysis of T- and B-cell immunity and in vivo treatment with anti-TGFbeta antibodies were used to define mechanisms of induced tolerance. Intravenous injection of beta-gal induced true immunologic tolerance to beta-gal tg skin in wild-type but not in B-cell-deficient recipients, suggesting that antigen presentation by B cells was required for the effect. The tolerogenic manipulation primed a population of CD4+, beta-gal-specific, TGFbeta-producing T cells. Although evidence for both anergy and suppression were observed, subsequent data demonstrated that TGFbeta was a critical immunoregulatory mediator of the tolerant state: neutralizing anti-TGFbeta antibodies fully prevented the induction of tolerance to B6 beta-gal tg skin grafts. Second male beta-gal tg grafts placed onto female recipients that were previously made tolerant to female beta-gal tg skin were rapidly rejected, however, suggesting that this TGFbeta-induced tolerance could not be linked to additional antigenic determinants. The studies demonstrate a critical role for TGFbeta in mediating tolerance after intravenous injection of antigen but additionally raise concerns about the stability of this tolerant state.

Method for systemic anaphylactic challenge in the rat using subplantar route

Intravenous injection of antigen is the fastest and most effective way of eliciting anaphylacticc shock in previously sensitized rats. When intravenous injection is difficult or undesirable, subplantar challenge is a preferable alternative to the intraperitoneal route.

Effects of topical FK506 on experimental allergic conjunctivitis

In vitro studies of FK506 demonstrated that the agent inhibited the T-cell receptormediated signal transduction that results in the transcription of interleukin 2. Recent in vitro studies have demonstrated an identical spectrum of activities of FK506 on IgE receptor-mediated signal transduction that results in exocytosis of secretory granules from basophils and mast cells. The effects of topical FK506 on allergic conjunctivitis were therefore studied in the rat. Passive anaphylaxis was induced in Wistar rats by injecting anti-ovalbumin IgE at the conjunctiva followed by intravenous injection of antigen (ovalbumin) and Evans blue. Topical FK506 (0.03-0.1%) significantly suppressed the passive anaphylaxis in the rat conjunctiva when given at 15 and five minutes, or six, four and two hours before the antigen challenge. The efficacy was more intense than that of 0.1% betamethasone or 2% disodium cromoglycate. These data thus suggest that topical FK506 might be beneficial for the therapy of severe allergic diseases in the eye.

Antiasthmatic Activity of a Novel Thromboxane A<sub>2</sub> 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)

Generation of leukotrienes in guinea-pigs in vivo during antigen-induced bronchospasm.

Anaphylactic bronchoconstriction in passively sensitized guinea-pigs was induced by intravenous injection of antigen (ovalbumin). Five min after injection of the antigen, when the increase in airways resistance to overflow (bronchospasm) was maximum, the plasma levels of leukotrienes LTC4 and LTD4 had increased from undetectable levels of 0.280 and 1.423 ng/ml, respectively. Administration of 5-lipoxygenase inhibitors clearly suppressed the increase in the leukotriene levels concomitant with the suppression of bronchospasm.

IgA nephropathy is not induced in mice by oral administration of TNP-conjugated ovalbumin.

An IgA nephropathy model based on long-term oral administration of protein antigens was evaluated in three mouse strains using trinitrophenyl (TNP)-conjugated ovalbumin. Administration of the antigen for 14 weeks did not induce a significant IgA response nor deposition of IgA in the mesangium in any of the mouse strains. If, however, serum IgA anti-TNP antibodies were induced by intraperitoneal injection of anti-TNP producing MOPC-315 tumor cells, subsequent intravenous injection of antigen resulted in the deposition of IgA immune complexes in the mouse kidneys. Hematuria did not occur. In conclusion, previous data showing that long-term oral administration of protein antigens induces mesangial IgA deposits could not be confirmed with TNP ovalbumin. However, mesangial IgA deposits were obtained in animals treated with MOPC-315 tumor cells as described by Rifai et al. [J. exp. Med. 150: 1161-1173, 1979].

Antigen-induced bronchial anaphylaxis in actively sensitized SD rats. Effects of immunization and provocation doses of antigen and of pretreatment with DSCG, theophylline, terbutaline and a new anti-allergic xanthine derivative, D4026.

Bronchial anaphylactic reactions, estimated as increase in intratracheal pressure, were precipitated by intravenous injections of antigen into actively sensitized SD rats. The degree of bronchial reactivity was found to depend on both the challenge dose and the immunization dose of antigen; therefore the course of the capacity to respond was recorded as a function of these variables. The degree of the bronchial anaphylactic response could be reduced by pretreatment with disodium cromoglycate, terbutaline or a new anti-allergic xanthine derivative, D 4026, in some groups of animals. The efficacy of each agent was found to depend on the dose of antigen used for sensitization and for provocation of the bronchial reaction rather than on the strength of the response. Taken together, the data suggest that more than one type of homocytotropic antibody mediates bronchial anaphylactic reactivity in the SD rat.

Antigen-specific densensitization in a rabbit model of acute hypersensitivity pneumonitis

Rabbits that had been prepared to develop acute alveolitis after aerosol challenge with simple protein antigens did not develop chronic alveolitis but rather gradually recovered despite continued challenge. Immunologic accompaniments of waning disease were compared in this model to those associated with intravenous injections of antigen causing “desensitization”. We also studied the effects of aerosol challenge prior to systemic immunization, antigen specificity, and the duration of desensitization by aerosolized and intravenous antigen. We found that repeated aerosol or intravenous challenges produced antigen-specific desensitization in this model, and the effect lasted several weeks. Prior exposure to aerosolized antigen was not protective. Neither aerosol nor intravenous desensitization maneuvers abrogated antigen-specific lymphocyte blastogenesis, although an early transient fall did occur. Humoral responses were boosted. These findings suggest that chronic alveolitis is prevented in this model by specific desensitization, without the induction of true tolerance or of nonspecific anergy. Such immunoregulation may result from development of antigen-specific blockade or blocking factors (e.g., lymphokines), antigen-antibody complexes, or suppressor cells affecting specific effector cells. Evaluation of these mechanisms may have implications for diagnosis and prognosis in human hypersensitivity pneumonitis.

Thermodissociation of staphylococcal immune complexes and detection of staphylococcal antigen in serum from patients with Staphylococcus aureus bacteremia.

We describe a simple and rapid method to dissociate antigen-antibody complexes to allow antigen detection by radioimmunoassay. Immunoglobulins and immune complexes were precipitated from serum in 50%-saturated ammonium sulfate. The precipitate was dissolved in distilled water and heated at 90 degrees C for 15 min to free the antigen and denature the antibody irreversibly. With this treatment, the sensitivity for detecting staphylococcal antigen added to human serum improved greater than or equal to 16-fold and was similar to the sensitivity in buffer. After intravenous injection of antigen into a rabbit with high levels of staphylococcal antibodies, antigen was detected only after thermodissociation. Also, in 4 of 26 patients with Staphylococcus aureus bacteremia, circulating antigen was detected after thermodissociation. Thermodissociation of immune complexes is a simple, rapid method and should be useful for detecting other heat-stable antigens.

Rabbit lymphocytes with receptors for IgM: I. The development of receptors on lymphocytes following intravenous injection of antigen

Under conditions optimal for detection of receptors for IgM (RFcμ) on human lymphocytes, RFcμ + lymphocytes were not found in any of the tissues of nonimmune rabbits. However, 1 day after iv immunization with alum-precipitated keyhole limpet hemocyanin (KLH) or polymerized human IgG (pol-Ig), rabbit peripheral blood (PB) and spleen (S) demonstrated a significant percentage of lymphocytes (10%) with RFcμ. At 3 days after immunization there was a further increase in RFcμ + lymphocytes (10–13%). By 6 days, however, the percentage of lymphocytes bearing receptors for IgM had returned nearly to zero. Pronase treatment was found to increase expression of RFcμ at 1 and 3 days in pol-Ig-primed tissues (up to 25%) but not in KLH-primed tissues. The percentage of RFcμ + lymphocytes in tissues remained low at 6 days even after Pronase treatment. This time sequence of development of RFcμ + cells after immunization seems interesting in the light of the findings of others that human lymphocytes with receptors for IgM are responsible for help in the PWM-induced differentiation of B cells to plasma cells.

An immunofluorescence study of the passive arthus reaction in rat sciatic nerve.

The Arthus reaction, passively induced in rat sciatic nerve by local injection of antibody and intravenous injection of antigen, was studied by immunofluorescence, using fluorescein and tetramethylrhodamine isothiocyanate. The reaction in nerve is similar to that occurring in skin. Antigen-antibody complexes formed at the site of the reaction activate complement and attract large numbers of polymorphonuclear leucocytes which then ingest the immune complexes. The significance of the Arthus reaction in relation to diseases of the peripheral nervous system is mentioned.

Immunochemical properties of rabbit antibodies in membranous glomerulonephritis.

Four of 15 rabbits, injected with hen's egg albumin (EA) intramuscularly once a week for a period in excess of 12 weeks, developed membranous glomerulonephritis (MGN). It was demonstrated that the quantities and qualities of antibodies produced in the MGN rabbits were different than those of non‐membranous glomerulonephritis (non‐MGN) rabbits. MGN rabbits produced small to moderate amounts of total antibodies and precipitating antibodies as measured by antigen‐binding capacity (ABC‐33) of Farr's method and precipitation with radioactive antigen (P‐80), particularly after 12 weeks of immunization. The precipitating efficiencies of the antibodies in their sera declines remarkably and their avidity were lower than those of non‐MGN rabbits late in the course of immunization. MGN rabbits formed relatively small antigen‐antibody complexes after intravenous injection of antigen, which were slowly removed from their circulation. From these results, it was concluded that the properties of antibodies correlated closely with the development of MGN.

Sensitization of mice by inhalation of antigen after infection with Bordetella pertussis.

Intranasal infection of mice with Bordetella pertussis or injection of pertussis vaccine previous to administration of an albumin aerosol augments sensitivity toward albumin. Sensitization was demonstrated by provocation of anaphylactic reactions following intravenous injection of antigen.

Direct stimulation of lymphoid tissue of the chicken. II. Changes in lymphoid organs following intrathymic and intravenous injection of antigen.

AbstractLymphoid tissue from chickens immunized intrathymically or intravenously with BSA was examined histologically at various time intervals after challenge. Quantitative analyses of the thymus medulla and the periarterial area of the spleen were based on 4080 to 6942 counted cells. Three days after immunization, thymic lobes injected with BSA exhibited an increase in the number of blasts. This was followed by a rise in plasma cell population up to 5 days. Significant diminution of small lymphocytes and moderate increase of medium and large lymphocytes occurred after immunization. Contralateral noninjected thymic lobes of the same chickens showed similar cellular kinetics. In thymuses of birds immunized intravenously with BSA there was a moderate increase in plasma cells. Thymic lobes of intrathymically or intravenously immunized bursectomized chickens were normal.In the spleen, an augmented number of blasts was found 3 days after intrathymic or intravenous challenge. Intrathymic injection of BSA produced profound changes in the bursal follicles and intense plasmacytic reaction 3 and 9 days after immunization.The results are discussed in the light of the hypothesis that the chicken thymus is not only a primary lymphoid organ, but also an organ capable of acting as a secondary lymphoid tissue.

Direct stimulation of lymphoid tissue of the chicken. I. Antibody-producing, antibody-transferring and plaque forming capacity of the thymus, spleen and bursa of Fabricius following intrathymic and intravenous injection of antigen.

AbstractBovine serum albumin (BSA) and human 0 red blood cells (0 RBC) were injected into six thymic lobes or into the vein of 8‐week old normal and neonatally bursectomized New Hampshire chickens in order to study the antibody production. Thymus and spleen cells from donors immunized by the intrathymic or intravenous route with BSA were used in the transfer of antibody response to homologous bursaless nonimmunized recipients. The thymus, spleen and bursa of chickens immunized with 0 RBC were inspected for the presence of plaque‐forming cells.Intrathymic injection of BSA induced a prompt appearance of 2‐mercaptoethanol (ME)‐sensitive anti‐BSA antibodies in a number of chickens, and progressive increase of antibody titer. Bursectomized chickens showed very weak primary antibody response (only antibody of ME‐sensitive type) following intrathymic or intravenous stimulation with BSA.The amount of ME‐sensitive hemagglutinins increased 2 days after intrathymic or intravenous administration of 0 RBC, but there was no immediate rise of hemagglutinin titer in intrathymically injected birds. Intravenous injection of 0 RBC was superior to intrathymic injection in inducing ME‐sensitive and ME‐resist ant he magglutinins.Thymus cells from donors immunized intrathymically with BSA were capable of transferring antibody response to neonatally bursectomized recipients. The most effective were thymus cells from BSA‐injected lobes. Thymus and spleen cells from intravenously immunized chickens exhibited a lower capacity for transferring the production of antibody. On the other hand, thymus and spleen cells from bursaless donors immunized intrathymically with BSA failed completely to induce antibody production in nonimmunized bursectomized recipients.Plaque‐forming cells were found in the thymus, spleen and bursa of chickens immunized intrathymically or intravenously with human erythrocytes.The results were interpreted as further support for the concept that the thymus is actively involved in immune reactions, and that the chicken thymus shares properties of both primary and secondary lymphoid tissues.

The invivo and in vitro immunogenicity and antigenic specificity of lymphoid antigen fragment-oligoribonucleopeptide conjugates

A unique low molecular weight antigen fragment-oligoribonucleopeptide conjugates that was isolated from the spleens and lymph nodes at 5 hr after the intravenous injection of antigen, S 35-BSA, produced the anamnestic response 4 days after the injection into rabbits which were previously immunized with BSA. These lymphoid conjugates after incubation with the specific BSA antibodies formed radioactive precipitin arcs when subjected to radioimmunoelectrophoresis against goat anti-rabbit immunoglobulin G. These data suggest that the antigen fragments which conjugate with the oligoribonucleopeptides were the antigenic determinants derived from the BSA molecule. The immunogenicity of the conjugates was shown in normal rabbits 3 days after the injection: that is, detecting the antibodies to S-BSA in the concentrated immunoglobulin G fraction of sera by radioimmunodiffusion and the antibody-forming cells in spleens by the liquid plaque technique. In view of the chemical properties of these conjugates, their capacities for inducing antibody formation suggests that they may be the fundamental antibody inducer.