Injection Of Autologous Serum Research Articles

Effects of complement inactivation and IgG depletion on skin reactivity to autologous serum in chronic idiopathic urticaria

Background: Intradermal injection of autologous serum elicits a wheal-and-flare response in about 60% of patients with chronic idiopathic urticaria (CIU). This reactivity has been attributed to the presence of IgG autoantibodies directed against IgE or the α-chain of the high-affinity IgE receptor (FcϵRIα) expressed on basophils and mast cells, leading to the hypothesis that at least some forms of CIU could be sustained by an autoimmune process. Objectives: The aim of this study was to investigate the relationship between the presence of anti-IgE or anti-FcϵRI antibodies and the ability to induce wheal-and-flare responses in CIU sera selected for the capacity to give a positive skin test response. Methods: Fifteen patients with CIU and a positive skin test response to autologous serum were injected intradermally with native serum and with serum heated at 56°C for 30 minutes and then adsorbed on Sepharose-protein G to obtain IgG depletion. Serum levels of anti-IgE and anti-FcϵRIα antibodies were measured by ELISA by using purified IgE and recombinant RIα-soluble double-fusion protein RIα-human serum albumin-RIα, respectively. The histamine-releasing activity of sera was tested by using ELISA with whole human blood from a healthy donor. Results: All patients had positive cutaneous responses to native serum injection. Anti-FcϵRIα antibodies were present in 14 of 15 native sera, only two of which were able to induce in vitro basophil degranulation. On the contrary, detectable amounts of anti-IgE antibodies were not found in any serum. IgG depletion by protein G resulted in complete (10/14 samples) or considerable (4/14 samples) removal of anti-FcϵRIα antibodies. The two sera endowed with functional activity lost their capacity to trigger histamine release from basophils after heating and protein G adsorption. Nonetheless, heat-decomplemented/IgG-depleted sera elicited wheal-and-flare reactions comparable with those observed with untreated sera. Conclusions: These results strongly suggest that skin reactivity to autologous serum could be due to as yet unidentified non–Ig reactants present in the sera of patients with CIU. (J Allergy Clin Immunol 2000;106:567-72.)

The relationship between Helicobacter pylori IgG antibody and autologous serum test in chronic urticaria.

In chronic urticaria, suspicious factors are Helicobacter pylori (HP) infection and autoimmunity, as well as other etiologic agents. Autologous serum injection is the first step to identify autoimmune urticaria. Our study was performed to determine the prevalence of HP infection in patients with chronic urticaria, and to evaluate the results of autologous serum testing in chronic urticaria patients who had HP antibodies. HP immunoglobulin G (IgG) antibody was defined by an immunochromatographic method in 61 chronic urticaria patients and 15 healthy persons. Autologous serum testing was performed in 32 chronic urticaria patients. HP IgG antibody was found to be positive in 41% of chronic urticaria patients and 26% of healthy controls (chi2= 7.82, P= 0.005). Autologous serum testing was positive in 40% of chronic urticaria patients who had HP IgG antibody. This ratio was 14.3% in chronic urticaria patients who did not have HP IgG antibody (chi2 = 9.23, P= 0.002). In this study, autologous serum testing was found to highly positive in chronic urticaria patients with HP IgG antibody, but the relationship between autoimmunity and HP infection requires further investigations.

Chronic generalized pruritus caused by nitrate intolerance

Chronic generalized pruritus caused by nitrate intolerance

Inhibitory effect of heparin on skin reactivity to autologous serum in chronic idiopathic urticaria

Background: Most patients with chronic idiopathic urticaria (CIU) show cutaneous reactivity to intradermal injection of autologous serum. In some cases this reactivity is associated with the presence of autoantibodies directed against IgE or IgE receptors expressed on mast cells, whereas in others no autoimmune mechanisms can be documented. Objectives: The aims of this study were to compare the cutaneous reactivity to serum and plasma samples in a series of patients with active CIU and to address the mechanisms of the inhibitory effect exerted by heparin on the cutaneous responsiveness to the histamine-releasing factors (HRFs) present in CIU serum. Methods: Fourteen patients with CIU were injected intradermally with autologous serum, plasma (anticoagulated by either heparin or EDTA), or serum samples to which heparin had been added. The effects of heparin injection on cutaneous responsiveness to allergens was tested in 5 atopic patients. Moreover, in a set of experiments sera were also adsorbed with Sepharose-conjugated heparin. Results: All the patients had positive cutaneous reactions to autologous serum injection. When heparinized plasma was injected, negative reactions were observed in 12 of 14 patients, and a sizable reduction in the wheal-and-flare reactions was recorded in the remaining 2. Compared with results obtained with serum, no substantial change was observed in 6 of 8 patients injected with EDTA-anticoagulated plasma. When heparin was added to serum, abrogation of skin reactivity was seen; nonetheless, no change in the cutaneous response to allergens was associated with locally administered heparin in 5 atopic patients with no history of CIU. Finally, adsorption of CIU sera with solid-phase heparin abrogated the ability to induce cutaneous reactions in 5 of 7 patients, whereas in the remaining 2 a sizable reduction was observed. Conclusions: These data indicate that heparin is able to profoundly inhibit the cutaneous response to HRFs present in the sera of patients with CIU. Although the precise level of action of this heparin-mediated effect is unclear from present data, preliminary evidence seems to indicate that heparin could directly interfere with HRFs present in CIU sera. (J Allergy Clin Immunol 1999;103:1143-7.)

Human leucocyte antigen class II associations in chronic idiopathic urticaria.

The major histocompatibility complex (MHC) acts as a marker for self during T-cell ontogeny and is associated with the pathogenesis of many autoimmune diseases. Recent investigations have shown about 30% of patients with chronic idiopathic urticaria (CIU) have IgG autoantibodies against the high-affinity IgE receptor, FcepsilonRI, or IgE. A link between MHC class II alleles and CIU has not been reported previously. DNA was extracted from blood of 100 Caucasian patients with CIU, and the MHC class II type determined using the polymerase chain reaction with sequence-specific primers, testing for DRB and DQB1 alleles. The frequency of alleles in CIU patients was compared with that found in 603 controls. Further human leucocyte antigen (HLA) typing on patient subsets, classified by the patients' responses to intradermal injection of autologous serum and their serum-induced histamine release from basophil leucocytes of healthy donors, was undertaken. HLA DRB1*04 (DR4) and its associated allele, DQB1*0302 (DQ8), are raised in CIU patients compared with a control population (P = 2 x 10-5 and P = 2 x 10-4, respectively). HLA DRB1*15 (DR15) and its associated allele, DQB1*06 (DQ6), are significantly less frequently associated with CIU. The HLA DRB1*04 association is particularly strong (corrected P = 3.6 x 10-6) for patients whose serum has in vivo and in vitro histamine-releasing activity. HLA class II typing is consistent with the concept that CIU is a heterogeneous disease, and supports an autoimmune pathogenesis in a subset of patients.

The autologous serum skin test: a screening test for autoantibodies in chronic idiopathic urticaria.

One-third of patients with chronic idiopathic urticaria (CIU) have circulating functional autoantibodies against the high affinity IgE receptor FcepsilonRI, or IgE. The intradermal injection of autologous serum causes a weal and flare reaction in many patients with CIU, and this reaction forms the basis of the autologous serum skin test (ASST). We have determined the parameters of the ASST which define the optimal sensitivity and specificity for the identification of patients with autoantibodies. Two physicians (R.A. S. and C.E.H.G.) performed ASSTs in a total of 155 patients with CIU, 40 healthy control subjects, 15 patients with dermographism, nine with cholinergic urticaria and 10 with atopic eczema. Patients were classified as having functional autoantibodies by demonstrating in vitro serum-evoked histamine release from the basophils of two healthy donors. There were significant differences (P < 0.001) in the mean weal diameter, weal volume, weal redness and flare area of the intradermal serum-induced cutaneous responses at 30 min between patients with CIU with autoantibodies and either those without autoantibodies or control subjects. The optimum combined sensitivity and specificity of the ASST was obtained if a positive test was defined as a red serum-induced weal with a diameter of >/= 1.5 mm than the saline-induced response at 30 min. For R.A.S. and C.E.H.G., the ASST sensitivity was 65% and 71% and specificity was 81% and 78%, respectively. Using these criteria, the following subjects had positive ASSTs: none of 15 dermographics, none of 10 atopics, one of nine cholinergics and one of 40 controls.

A murine model of communicating hydrocephalus: Role of TGF-β1

Subarachnoid haemorrhage (SAH) often causes communicating hydrocephalus. We recently found that intrathecal injection of human recombinant transforming growth factor (hrTGF)-β1 induced communicating hydrocephalus in mice. In this experiment we examined whether hydrocephalus could be generated in mice by intrathecal injection of autologous serum, which would contain TGF-β1 in a similar situation to SAH. Mouse serum, plasma and a range of quantities of hrTGF-β1 were injected intrathecally into 10 day old C57BL/6 mice. The sizes of the lateral ventricles were measured some weeks after injection. The serum induced hydrocephalus which was prevented by additional injection of anti-TGF-β1 antibody. Immunoblot analysis showed a band of the active form of TGF-β1 in the serum which was not detected in the plasma. This study therefore suggests that active TGF-β1 in cerebrospinal fluid after SAH contributes to the generation of communicating hydrocephalus.

Dermal Mast Cell Activation by Autoantibodies Against the High Affinity IgE Receptor in Chronic Urticaria

Previous studies identified autoantibodies against the IgE high affinity receptor alpha-chain, Fc epsilon RI alpha, in sera of selected patients with severe chronic idiopathic urticaria. We have now determined the incidence of anti-Fc epsilon RI alpha autoantibodies in a group of 163 patients. Intradermal injection of autologous serum caused skin reactions indicative of mast cell degranulation in 98 (60%) patients. Based on histamine release from IgE-sensitized and nonsensitized basophil leukocytes of healthy donors, we detected anti-Fc epsilon RI alpha autoantibodies in sera from 38 (23%) urticaria patients and evidence for anti-IgE antibodies in a further nine patients. The sera that released histamine from basophils induced histamine release (4-34%, n = 12) from mast cells in incubated skin slices. Protein-G affinity chromatography of sera demonstrated that mast cell histamine release was IgG-mediated. Preincubation of sera or the IgG fraction with a recombinant alpha-chain of Fc epsilon RI inhibited histamine release from mast cells and basophils. Further studies with the mouse anti-human Fc epsilon RI alpha antibody 29C6 showed that mast cells and basophils were similarly sensitive to IgG-mediated direct cross-linking of Fc epsilon RI, with 0.01-1.0 micrograms/ml 29C6 evoking histamine release in each case. These studies demonstrate that circulating levels of anti-Fc epsilon RI alpha autoantibodies mediate histamine release from skin mast cells in vitro and, taken together with in vivo evidence of mast cell degranulation following intradermal injection of autologous serum, support the concept that anti-Fc epsilon RI alpha autoantibodies are relevant to the pathogenesis of severe chronic urticaria in about 25% of patients.

Autoantibodies against the High-Affinity IgE Receptor as a Cause of Histamine Release in Chronic Urticaria

Most urticarias are induced by vasoactive mediators such as histamine released from mast cells. Although mast cells are activated by allergens through cross-linking of cell-surface--bound IgE, this mechanism does not appear to explain most cases of chronic urticaria, which, when allergic, infectious, drug-induced, or physical causes cannot be identified, are classified as idiopathic. We recruited 26 patients with chronic idiopathic urticaria, in whom intradermal injection of autologous serum caused a wheal-and-flare response. Serum from four patients that induced marked histamine release from basophils from a donor with very low serum IgE levels was studied with respect to the IgE dependence of the histamine release, the activity of the IgG fractions, and the neutralizing effect of a recombinant preparation of the soluble extracellular domain of the alpha subunit of the high-affinity IgE receptor (sFc epsilon RI alpha). The histamine-releasing activity of the serum was abolished by passive sensitization of basophils with myeloma IgE, enhanced after dissociation of IgE by treatment with lactic acid, and induced by IgG fractions from the serum of all four patients. Preincubation of the serum and isolated IgG with sFc epsilon RI alpha resulted in almost complete neutralization. Histamine-releasing IgG autoantibodies against the alpha subunit of the high-affinity IgE receptor are present in the circulation of some patients with chronic urticaria. Autoantibody-induced cross-linking of IgE receptors may be an important mechanism in the pathogenesis of chronic urticaria and other diseases mediated by mast cells.

Detection of circulating histamine releasing autoantibodies with functional properties of anti‐IgE in chronic urticaria

Circulating histamine releasing factor(s) have been demonstrated previously in chronic urticaria by an immediate weal-and-flare response to intradermal autologous serum injection. We have studied 25 chronic urticaria patients by in vivo skin testing with autologous sera and an in vitro histamine release assay using mixed leukocytes of healthy donors, to define the nature and functional properties of the serum factor(s). Twenty showed a weal response to autologous serum (mean +/- s.e.m., 37.3 +/- 6.8 mm3). Weal formation was confined to ultrafiltered serum fractions greater than 100 kD in nine of nine patients. There was no response in 10 healthy controls of five patients with symptomatic dermographism. Fourteen chronic urticaria sera elicited histamine release greater than 10% (mean +/- s.e.m., 44.3% +/- 6.7) above basal levels from leukocytes of at least one of seven healthy donors. This in vitro response was also confined to ultrafiltered serum fractions greater than 100 kD in seven of seven sera and was present in IgG fractions of six of seven chronic urticaria sera that showed histamine releasing activity. Functional studies indicated that this histamine releasing autoantibody had the properties of anti-IgE: chronic urticaria sera 'desensitized' basophil leukocytes to subsequent challenge with other chronic urticaria sera and to goat anti-human IgE antibody; human myeloma IgE inhibited histamine release from leukocytes in response to chronic urticaria sera; removal of surface-bound IgE by lactic acid 'stripping' reduced histamine release in response to chronic urticaria sera and anti-IgE and subsequent passive sensitization with IgE myeloma serum partially restored it. Stainable peripheral blood basophils/mm3 in chronic urticaria patients were significantly reduced (mean +/- s.e.m, 7.9 +/- 2.0) when compared to healthy controls (39.6 +/- 4.4), P less than 0.001. These results suggest that histamine releasing autoantibodies are important in the pathogenesis of chronic urticaria by stimulating or facilitating degranulation of basophils and cutaneous mast cells through cross-linking cell surface IgE receptors.

Effect of indomethacin on generation of chemotactic activity in inflammatory exudates induced by carrageenan

Chemotactic activity of protein origin was demonstrated in carrageenan-induced pleural exudates by the chemotactic response of neutrophils in the modified Boyden chamber. The activity was partly neutralized by monospecific antisera to complement component 5 and was destroyed by trypsin and chymotrypsin treatment but it differed from that in rat serum or plasma in that it was stable for 30 min at 56°C. Indomethacin (5 mg/kg i.v.) reduced equally protein content (56%) and total chemotactic activity (58%); i.e., chemotactic activity/mg of exudate protein was unchanged. Intrapleural injection of autologous or homologous serum also induced an infiltration of neutrophils; the protein content of the pleural fluid decreased by 60–70% in 4 h, whereas with carrageenan there was a progressive increase in exudate protein. When serum was injected in two doses to maintain protein levels comparable to those found following carrageenan injection, the number of neutrophils in the exudates was also comparable. In contrast to carrageenan, the response to serum was not inhibited by indomethacin. From these and other data we suggest that the exudate chemotactic activity is generated from plasma protein and that indomethacin acts primarily to reduce extravasation of plasma and consequently generation of chemotactic activity.

Sensitization of rabbits with chlorhexidine-protein complexes

Rabbits were immunized weekly with subcutaneous injections of autologous serum exposed to chlorhexidine gluconate, or with injections of chlorhexidine in distilled water. After 6 weeks, the animals were tested for antibodies by Ovary's cutaneous anaphylaxis test. The titre of chlorhexidine-treated serum showing a positive test was 1: 5120 for immunized animals as compared with that of 1:320 for non-immunized animals. The results are discussed in relation to the introduction of chlorhexidine gluconate into oral hygiene.

Significance of reactions to intradermal injection of autologous granulocytes, mononuclear cells, and serum.

Significance of reactions to intradermal injection of autologous granulocytes, mononuclear cells, and serum.