Homocytotropic Antibody Research Articles

The spleen dictates platelet destruction, anti-platelet antibody production, and lymphocyte distribution patterns in a murine model of immune thrombocytopenia.

For many years, splenectomy has been used to treat immune thrombocytopenia (ITP), and this procedure benefits approximately two-thirds of the treated patients. Although splenectomy may raise platelet counts, antibody-coated platelets and cytotoxic T lymphocytes appear to persist or can change over time. To better understand how the spleen may affect anti-platelet immune responses, we used a murine model of ITP demonstrating both antibody-mediated and T lymphocyte-mediated thrombocytopenia. Mice with severe combined immunodeficiency (SCID) were either splenectomized or not and transfused with splenocytes from CD61 (GPIIIa) knockout mice immunized against CD61(+) platelets. Platelet counts and anti-platelet antibody levels were performed weekly. After 4weeks, the mice were sacrificed, and lymphoid organs were harvested and examined by flow cytometry to quantify CD4(+)CD25(+)FoxP3(+) Tregs and conventional cross-presenting XCR1(+) and tolerizing SIRPα+ dendritic cells. The results indicate that compared with control non-splenectomized mice, thrombocytopenia was improved and anti-platelet antibody production was significantly diminished in all splenectomized mice that received immune splenocytes. Splenectomized SCID mice also had a marked reduction in Tregs in the thymus together with an increased proportion of both thymic dendritic cell subsets that correlated with increased platelet counts. Of interest, although splenectomy diminished anti-platelet antibody production and raised platelet counts, marrow megakaryocyte densities were still significantly reduced in mice that received immune splenocytes. These results suggest that the spleen in murine ITP not only is the primary site responsible for platelet destruction, but it also controls, to a significant extent, the antibody response against platelets and the migration patterns of lymphocyte subsets.

Lack of homocytotropic antibody potentiation by Ascaris suum infection in pigs.

Summary Ascaris suum adults were transplanted into the small intestine of pigs with homocytotropic antibodies to equine serum. Ascaris suum infection did not potentiate the homocytotropic response to equine serum. Zusammenfassung Keine Potenzierung der Bildung homocytotroper Antikörper nach Ascaris suum Infektionen des Shweines Schweine, die im Serum homocytotrope Antikörper gegenüber Pferdeserum besaßen, wurden mit Ascaris suum in den Dünndarm infiziert. Die Ascaris suum Infektion potenzierte nicht die homocytotropen Antikörper gegenüber Pferdeserum. Résumé Aucune potentialisation de la formation des anticorps homocytotropes après des infections du porc avec Ascaris suum Des pous possédant des anticorps homocytotropes sériques anti-sérum de cheval ont été infectés dans l'intestin grêle avec Ascaris suum. L'infection à Ascaris suum n'a pas potentialisé les anticorps homocytotropes anti-sérum de cheval. Resumen Ninguna potencialización de la formación de anticuerpos homocitotropos tras infestaciones por Ascaris suum en cerdos Se infestaron cerdos, que tenían en el suero sanguíneo anticuerpos homocitotropos frente a suero sanguíneo de caballo, con Ascaris suum en el intestino delgado. La infestación por Ascaris suum no potenció los anticuerpos homocitotropos frente al suero equino.

Potentiation of allergic bronchoconstriction by repeated exposure to formaldehyde in guinea-pigs in vivo.

Indoor formaldehyde (FA) might worsen allergies and be an underlying factor for the increasing incidence and severity of asthma; the exact mechanism, however, remains unclear. The present study examined the effects of repeated exposure to FA on methacholine- and antigen-induced bronchoconstriction in guinea-pigs in vivo. First, non-sensitized guinea-pigs were transnasally treated with 0.1 or 1.0% FA or saline three times a week for 6 weeks, and increasing concentrations of methacholine (50, 100, and 200 microg/mL) were inhaled at 5-min intervals. Second, guinea-pigs pre-treated with transnasal administration of FA or saline using the same protocol were passively sensitized with anti-ovalbumin (OA) serum 7 days before antigen challenge. Third, guinea-pigs were actively sensitized with OA and pre-treated with transnasal administration of FA or saline using the same protocol. The lateral pressure of the tracheal tube (Pao) was measured under anesthesia and artificial ventilation. The antigen-induced increase in Pao in actively sensitized guinea-pigs was significantly potentiated by FA exposure in a dose-dependent manner. The dose-response curve of the methacholine-induced increase in Pao in non-sensitized guinea-pigs or of the antigen-induced increase in Pao in passively sensitized guinea-pigs was not altered by FA exposure. Transnasal administration of FA significantly increased the serum anti-OA homocytotropic antibody titre (IgG) as measured by the passive cutaneous anaphylaxis reaction in actively sensitized guinea-pigs. The results suggest that repeated exposure to FA worsens allergic bronchoconstriction through enhancing antigen sensitization.

IgE antibody in the serum--detection and diagnostic significance.

IgE antibody in the serum--detection and diagnostic significance.

Tick modulation of host immunity: an important factor in pathogen transmission

Immunological interactions at the tick–host interface involve innate and specific acquired host immune defenses and immunomodulatory countermeasures by the tick. Tick feeding stimulates host immune response pathways involving antigen-presenting cells, cytokines, B-cells, T-cells, circulating and homocytotropic antibodies, granulocytes, and an array of biologically active molecules. In response to host immune defenses, tick-mediated host immunosuppressive countermeasures inhibit: host antibody responses; complement activation; T-cell proliferation; and cytokine elaboration by macrophages and Th1-lymphocytes. Immunosuppressive proteins identified in tick salivary glands and saliva have been partially characterised. Tick-induced host immunosuppression facilitates blood meal acquisition and is an important factor in the transmission/establishment of the tick-borne disease-causing agent, Borrelia burgdorferi. A novel strategy for control of tick-borne pathogens is proposed.

Production and characterisation of polyclonal antisera against feline IgE

Cats, naturally or experimentally infected with Toxocara cati were immunised with dinitrophenylated ascaris antigen (DNP-Asc). All cats developed immediate skin reactivity to DNP coupled to bovine serum albumin (DNP–BSA) and the sera of the nine cats had a heat labile homocytotropic antibody detectable by homologous Prausnitz–Küstner (PK) tests. Reagin-rich fractions were prepared from these sera and used for the preparation of polyclonal antisera in rabbits. Resultant antisera were passed through a immunoabsorbent column of Sepharose 4B coupled to heated normal cat serum. An immunoabsorbent column prepared with the resultant antisera removed the PK reactivity from the cat sera, and the activity was recovered following acid elution. The antiserum failed to detect any recognised immunoglobulin in cat sera, but precipitated with a heat labile protein with γ-1 electrophoretic mobility in the sera of parasited cats. These findings support the contention that the antisera are specific for feline IgE.

Detection of homocytotropic antibody in lambs infested with the louse, Bovicola ovis, using a basophil histamine-release assay

The utility of a basophil histamine-release assay using washed whole blood cells was examined in lambs and was used to determine if homocytotropic antibody with specificity for Bovicola ovis was produced in response to infestation with the louse. Maximal histamine release in the assay in response to Concanavalin A, anti-ovine IgE monoclonal antibody and, in sensitized lambs, to B. ovis antigen ranged from 18 to 48%. Histamine release from blood cells in response to B. ovis antigen was significantly higher in louse-infested lambs than in louse-naive lambs and was significantly correlated with louse and cockle scores. Passive cutaneous anaphylaxis (PCA) tests were negative with sera obtained from the lambs at the same time as blood for the basophil histamine-release assay. Serum histamine levels also were significantly higher in the louse-infested lambs than in louse-naive lambs and were significantly correlated with louse and cockle scores. The present results support a role for B. ovis-specific homocytotropic antibody in the development of cockle and indicate that the basophil histamine-release assay is more sensitive than the PCA test.

A guinea pig model of hypersensitivity to allergenic fractions of natural rubber latex.

Allergy to natural rubber latex is a growing medical problem with life-threatening aspects. The aim of this study was to learn if guinea pigs could serve as a suitable model for immediate-type hypersensitivity to latex. Guinea pigs were immunized either with whole non-ammoniated latex extract, or with one of nine SDS-PAGE-separated components. Other animals were injected with electroeluted latex components localized on gel at 14, 24 and a cluster around 45 kD. Before and after immunization, sera from the animals were examined by ELISA, immunoblots, passive cutaneous anaphylaxis (PCA) and passive systemic anaphylaxis. Latex-specific antibodies were detected by ELISA and immunoblots in sera from all immunized animals. PCA assays showed that the guinea pigs had homocytotropic antibodies dilutable to 1:250. PCA was abolished when sera from animals immunized with allergens in alum were heated at 56 degrees C for 30 min indicating the antibodies were of the E isotype. Passive systemic anaphylaxis was induced in 4 of 10 guinea pigs. The results show that guinea pigs are capable of making antibodies to latex protein components that mediate dermal and systemic anaphylaxis, paralleling the spectrum of clinical and laboratory findings of humans with immediate-type clinical latex hypersensitivity.

Validation of a non‐invasive technique to assess immediate or delayed onset of airway hypersensitivity in guinea‐pigs

Conscious Hartley guinea-pigs were sensitized with trimellitic anhydride (TMA) or trimeric hexamethylene diisocyanate biuret (Des-N) by repeated intradermal injections or by inhalation exposure. Immediate- and delayed-onset pulmonary reactions were recorded during/after challenges with the hapten or protein conjugate of the hapten, respectively. The positive control was sensitized and challenged with ovalbumin (OA) by inhalation. Homocytotropic antibodies of the IgG1 type were determined to correlate antibody titres and pulmonary responses. Immediate-onset pulmonary reactions were apparent in guinea-pigs sensitized by inhalation of TMA and OA. Animals sensitized intradermally with TMA demonstrated more vigorous immediate-onset reactions than animals sensitized by inhalation. The measurement of breathing parameters demonstrated that the ability to detect immediate-onset airway hyperreactivity was best when the breathing rate and tidal and minute volumes were measured. None of the measurements for delayed-onset reactions presented conclusive results, since sensitized as well as naive guinea-pigs demonstrated a delayed increase of breathing frequency. Antigen-specific IgG1 antibodies were several orders of magnitude higher in intradermally sensitized animals compared with animals sensitized by inhalation. Although Des-N-sensitized guinea-pigs experienced high IgG1-antibody titres, no response of pulmonary hypersensitivity could be elicited after hapten or conjugate challenge. In summary, it would appear that pulmonary hypersensitivity depends on factors other than IgG1 antibody titres. For the screening of chemicals, the dermal induction and inhalation challenge protocol was revealed to be more sensitive than the inhalation induction and inhalation challenge protocol.

Production and isolation of guinea pig IgE antibody

Immunoglobulins of the IgE and IgG classes have been causally associated with hypersensitivity reactions in man and in numerous animal species including mice, rats and guinea pigs. The use of the guinea pig as an animal model for both pulmonary and dermal hypersensitivity reactions, and the recent recognition of the importance of IgE antibodies in both early- and late-onset hypersensitivity responses, has heightened interest in production, separation, and isolation of this immunoglobulin class from the guinea pig. IgE antibodies were produced by treatment of strain 13 guinea pigs with cyclophosphamide followed by injection with S. aureus enterotoxin. Serum was obtained and the globulin fraction isolated by addition of caprylic acid then ammonium sulfate. Immunoglobulins were separated into classes using fast protein liquid chromatography (FPLC) employing a Mono Q column and a linear gradient of 0.01–0.3 M Na,K phosphate buffer, pH 7.5 (buffer B). IgG eluted in two major peaks. IgG2 was not retained on the column and emerged with the starting buffer; IgG1 was eluted with 15–20% buffer B. IgE, detected as heat labile homocytotropic antibody, was found in the fraction eluting with 30–35% buffer B. The elution profile of the guinea pig immunoglobulins was predicted from the pattern obtained with immunoglobulin classes from other species. This chromatographic procedure enabled rapid isolation of immunoglobulin classes from guinea pig sera and effectively separated IgG1 from IgE, the two classes associated with hypersensitivity reactions.

Experimental Lung Injury Induced by Trimellitic Anhydride Inhalation on Guinea Pigs

Trimellitic anhydride (TMA) causes lung injury by inhalation exposure in humans. In order to investigate more precisely the mechanism of lung injury by TMA, an experimental animal model of TMA-induced lung injury was established. Guinea pigs were intramuscularly injected with trimellitylated bovine serum albumin (TM BSA) with complete Freund's adjuvant (CFA). Appreciable amounts of antibodies against TM epitopes were detected in the sera of these animals. Guinea pigs that were passively sensitized with anti-TM antisera as well as the actively immunized animals developed hemorrhagic pneumonitis after TMA inhalation. It is well recognized that hyperimmune antisera of guinea pigs contain two subclasses of IgG antibodies, IgG1 and IgG2, which are known as homocytotropic and heterocytotropic antibodies, respectively. To determine the role of these antibodies in the airway injury with alveolar hemorrhages, they were isolated by gel filtration and by ion exchange column chromatography, and the guinea pigs that were sensitized with each of these antibodies were exposed to TMA inhalation. The extent of lung injury in these animals was quantitatively determined by the measurements of lung extravasation of Evans blue dye injected intravenously after TMA inhalation. Significant increases in the extravasation of dye were observed in both animal groups sensitized with IgG1 and IgG2 antibodies. In addition, results obtained with heat-treated antisera and IgG1 antibody did not significantly differ from those obtained with untreated samples. These results suggest that the lung injury resulting from TMA inhalation exposure can be induced with humoral antibodies, not only IgG1 but also IgG2, and that at least two types of allergic reactions are involved in the pathogenesis of lung injury.

Characteristics of weekly pulmonary hypersensitivity responses elicited in the guinea pig by inhalation of ovalbumin aerosols

We have reported the development of a guinea pig animal model for pulmonary hypersensitivity to inhaled chemicals in which respirations of animals are monitored continuously for 24 hr permitting detection of immediate- (IAR) and late-onset (LAR) airway reactions. Additionally implanted temperature transmitters allow determination of accompanying febrile responses. The current study investigated the relationship between severity of IAR and occurrence of LAR in individual animals. To quantify severity, a grading system was devised which took into account time to response, increase in breathing rate, and occurrence of airway constriction (ACR). Guinea pigs were sensitized by ip injection with 1 mg ovalbumin (OA). On Day 14, inhalation challenge with 12 mg/m 3 OA resulted in severe IAR in all animals. No LAR were detected. Subsequent weekly inhalation challenges with OA resulted in less severe IARs and occasionally in absence of response. Febrile reactions were not detected. On one occasion a LAR was observed. It occurred in the animal demonstrating the most severe IARs and having the highest titer of specific homocytotropic antibody. These results are consistent with the mechanism of IAR involving release of spasmogenic mediators from mast cells as a result of antigen crosslinking of surface antibody. The mechanism of the LAR is addressed in the accompanying paper in which LARs are more consistently produced in OA-sensitized guinea pigs.

The induction of respiratory allergy in guinea-pigs following intradermal injection of trimellitic anhydride: a comparison with the response to 2,4-dinitrochlorobenzene

Guinea-pigs injected intradermally with the known respiratory sensitiser trimellitic anhydride (TMA) developed high-titre antigen-specific homocytotropic (IgG 1 and IgE) antibodies. Many of the sensitised animals responded to a challenge by inhalation with either free TMA or a TMA-protein conjugate with a change in respiratory rate, reflecting the onset of bronchoconstriction. Guinea-pigs were also injected intradermally with 2,4-dinitrochlorobenzene (DNCB), which is a potent skin sensitiser in man but which has not been reported to cause respiratory allergy. These animals developed only low-titre homocytotropic antibodies and were unresponsive to an inhalation challenge with either free or conjugated hapten. The animals were, however, contact-sensitised to the chemical.

Active immunization induces lung hyperresponsiveness in the guinea pig. Pharmacologic modulation and triggering role of the booster injection.

In order to investigate whether bronchopulmonary hyperresponsiveness represents a unique property of sensitized lungs, we examined the responses of lungs from either actively sensitized, passively sensitized, or nonsensitized (control) guinea pigs to in vitro bronchoconstriction (BC) and release of thromboxane (TX) B2, 6-keto-PGF1 alpha, and histamine induced by platelet-activating factor (PAF-acether) or leukotriene (LT) D4. Guinea pigs were actively sensitized with 10 micrograms of either ovalbumin or Dermatophagoides farinae extract in AI(OH)3 injected intraperitoneally twice at a 2-wk interval. Seven days after the second injection (booster injection), the lungs were removed, ventilated, and perfused via the pulmonary artery with Krebs solution containing 2.5 g/L bovine serum albumin. In lungs from actively sensitized animals, BC was induced by significantly lower doses of PAF-acether and LTD4 than those required to elicit the same response in control preparations. In addition, sensitized lungs released more TxB2, 6-keto-PGF1 alpha, and histamine in response to PAF-acether and LTD4 than did control lungs. Increased mediator release was also observed upon challenge of lungs from actively sensitized animals with arachidonic acid and histamine. Lungs from guinea pigs passively sensitized with serum from actively sensitized animals did not exhibit increased responsiveness to PAF-acether as compared to control lungs. The hyperresponsiveness induced after booster injection of the antigen occurred concomitantly with an increase in the homocytotropic antibody titer (as measured by passive cutaneous anaphylaxis) and persisted for 3 months after sensitization, when the levels of circulating antibodies and lung response to antigen challenge returned to control values.(ABSTRACT TRUNCATED AT 250 WORDS)

Production of passive cutaneous anaphylaxis (PCA) and reversed PCA by rat IgE antibody in the mouse.

Although IgE antibody is generally characterized as a homocytotropic antibody, it has been well known for some time that mouse IgE antibody causes potent sensitization of rat skin for PCA. The present study clearly shows the reciprocal cross-sensitization of mouse skin with rat IgE molecules. PCA and RPCA were produced by rat IgE antibody in an inbred mouse strain. DS/Shi, though not in C3H/HeShi, C57BL/6JShi and BALB/cCrj strains. Sensitization of DS/Shi mouse skin for PCA with rat IgE antibody was comparable in sensitivity with that of rat skin, but lasted only for a short term in comparison with the long persistence in rat skin.

Sensitisation of guinea pigs by inhalation exposure to low molecular weight chemicals

Guinea pigs could be immunologically sensitised (as shown by the development of antigen-specific homocytotropic antibodies) to toluene diisocyanate by exposing them for 3 h a day for 5 consecutive days to atmospheres containing free chemical. Pulmonary reactions could be elicited in many of the sensitised animals by challenging them with atmospheres containing protein conjugates of the chemical and then measuring changes in respiratory rate. Successful elicitation of pulmonary reactions appeared to depend upon a number of factors, including the quality of the protein conjugate used for the challenge, but possibly also the development of IgE as well as IgGl antibodies. Antigen-specific homocytotropic antibodies were detected in guinea pigs similarly exposed by inhalation to two non-isocyanate respiratory allergens, trimellitic anhydride and a reactive dye. Although the animals were immunologically sensitised to the chemicals, challenge with atmospheres containing appropriate chemical-protein conjugates failed to stimulate changes in respiratory rate.

Host immune responsiveness to the chigger, Eutrombicula cinnabaris.

Chigger infestation is often associated with severe cutaneous reactions. Mice were given four infestations with the pest chigger Eutrombicula cinnabaris, and each exposure was separated by a 14-day mite-free period. Mean duration of engorgement was nine to ten days for a first exposure and four to five days for a fourth exposure. An initial exposure did not elicit macroscopic changes at chigger attachment sites, while all third and fourth exposure animals had marked reactions consisting of erythema, epidermal thickening and serous exudation. Approximately 20% of second exposure animals had macroscopic changes at chigger feeding sites, but these reactions were much less intense than the responses of third and fourth infestation hosts. Third and fourth exposure animals had infiltrates of lymphocytes, eosinophils, basophils and neutrophils at attachment sites, with eosinophil influx the most intense. Cutaneous reactivity to chigger feeding was adoptively transferred with lymphocytes from fourth exposure animals. Passive transfer of serum from fourth infestation donors resulted in heightened reactivity to a challenge infestation. Skin testing, after a fourth infestation, with an extract of whole E. cinnabaris larvae provided evidence for Arthus and delayed type hypersensitivity responses to chigger antigens. Chigger-reactive homocytotropic antibody was not detected by skin testing and active cutaneous anaphylaxis.

Attenuation of rat conjunctival response by repeated hapten applications.

Attenuation of the rat conjunctival response by repeated topical challenge with dinitrophenyl (DNP) hapten was demonstrated in our study. Adult rats were immunized by intraperitoneal injections of dinitrophenylated Ascaris suum extract (DNP-Asc) and alum. Serum levels of anti-DNP homocytotropic antibody were determined by passive cutaneous anaphylaxis in rats prepared with antibody 48 hours earlier. In other animals, topical challenge was performed by applying N,N'-di-2,4-DNP-L-lysine (di-DNP-lysine) in phosphate-buffered saline (PBS) to one eye; PBS alone was applied to the fellow eye. The degree of conjunctival reaction was assessed clinically, and ocular tissues were processed for histological evaluation. The intensity of the conjunctival reaction and extent of mast cell degranulation were significantly greater after one challenge with di-DNP-lysine than after multiple challenges. In the multiple-challenge group, the contralateral eye remained responsive to a single challenge with di-DNP-lysine. These results may have implications for therapeutic interventions in ocular anaphylaxis.

Hypersensitivity in fishes: a review

Immune hypersensitivity reactions, whereby secondary exposure to antigen elicits a heightened, accelerated immune response of sufficient intensity to cause tissue damage, are characteristic of well‐developed immune systems. Four (or five) types of hypersensitivity are usually recognized in Mammalia. In fishes, several hypersensitivity reactions have been recorded, particularly in Teleostei, but their mechanisms may sometimes indicate analogies rather than strict homologies with mammalian hypersensitivities. Type I anaphylactic hypersensitivity reactions have been described frequently in fishes, but the involvement of antigen as an initiating factor is problematical, as are the roles of homocytotropic antibody (if any), the vasoactive amine and other humoral factors implicated, and types of leucocyte. It is often difficult to distinguish between ‘classical’ anaphylactic hypersensitivity reactions and non‐specific inflammatory responses to invasive insults. The presence of a viable complement system in fishes (including the faculty to activate both the classical and the alternative complement pathways) together with NK cell‐mediated cytotoxicity and opsonizing antibody suggest that the feasibility of Type II cytotoxic hypersensitivity reactions can be entertained. Type III complex‐mediated hypersensitivity has not been described as such, although ‘neutrophil’ presence in inflammatory reactions suggests elements in fishes similar to an Arthus reaction. There are few reports of a piscine Type IV cell‐mediated, delayed hypersensitivity reaction; the existence of Type V stimulatory hypersensitivity has not yet been described. Tissue‐damaging immune hypersensitivity phenomena are associated with immunological sophistication, the ‘cost’ of effective immunity. Delayed hypersensitivity reactions in Mammalia are linked with expression of class II region genes of the MHC. In Teleostei, the existence of immune hypersensitivities might not be unexpected and would be concomitant with other features suggesting an advanced immune system, perhaps associated with the independent evolution or uncovering in these Osteichthyes of an MHC.

A review of in-vitro assays for IgG and IgG4 antibodies: concept and potential applications.

In-vitro assays for the diagnosis of allergy presently are quite common, having been demonstrated by a number of investigators to possess a high degree of specificity and sensitivity. These assays are for the most part rapid, flexible, and when utilized properly, cost effective. It has long been known that antibodies, other than the homocytotropic antibodies, are involved in allergy. The concept of "blocking antibodies" was first introduced by Cooke in 1935. He demonstrated that these antibodies could neutralize the effects of reagins present in the sera of allergic individuals. Since that time investigators have developed a series of techniques to assess and define blocking antibodies. These techniques include inhibition of antigen-induced histamine release from peripheral blood leukocytes, hemagglutination with allergen coated erythrocytes, radio-allergo-sorbent test (RAST), define antigen substrate sphere (DASS), antigen binding radioimmuno assay, and more recently, enzyme-linked immunosorbent assays (ELISA). These methods have been utilized to show that "blocking antibodies" are predominantly of the IgG class. Recently, several assays have been introduced to the clinical laboratory for the purpose of monitoring the production of blocking antibodies (i.e., IgG) in response to immunotherapy. These assays, while adding an important tool to the arsenal of the clinical practitioner, may have limited utility because of a lack of specificity and sensitivity. Recently, 3M Diagnostic Systems (3MDS) has introduced an assay via its Reference Laboratory designed for the measurement of what many investigators feel is the appropriate marker for monitoring immunotherapy . . . the IgG4 subclass.(ABSTRACT TRUNCATED AT 250 WORDS)