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Estrogens have been shown to regulate the immune system and modulate multiple autoimmune diseases. 17α-ethinyl estradiol (EE), a synthetic analog of 17β-estradiol, is prescribed commonly and found in oral contraceptives and hormone replacement therapies. Surprisingly, few studies have investigated the immunoregulatory effects of exposure to EE, especially in autoimmunity. In this study, we exposed autoimmune-prone female MRL/lpr mice to a human-relevant dose of EE through the oral route of exposure. Since lupus patients are prone to infections, groups of mice were injected with viral (Imiquimod, a TLR7 agonist) or bacterial (ODN 2395, a TLR9 agonist) surrogates. We then evaluated autoimmune disease parameters, kidney disease, and response to in vivo TLR7/9 pathogenic signals. EE-exposed mice had increased proteinuria as early as 7 weeks of age. Proteinuria, blood urea nitrogen, and glomerular immune complex deposition were also exacerbated when compared to controls. Production of cytokines by splenic leukocytes were altered in EE-exposed mice. Our study shows that oral exposure to EE, even at a very low dose, can exacerbate azotemia, increase clinical markers of renal disease, enhance glomerular immune complex deposition, and modulate TLR7/9 cytokine production in female MRL/lpr mice. This study may have implications for EE-exposure risk for genetically lupus-prone individuals.

Prevalence of immune-mediated glomerulonephritis has increased in preclinical toxicity studies, with more frequent use of biotherapeutic agents (especially antigenic humanized molecules) and antisense oligonucleotide (ASO) therapies. Immune complex disease affects a small number of study monkeys, often correlates with antidrug antibody (ADA) titers, and occurs at a dose that favors immune complex formation or impedes clearance. While preclinical glomerulonephritis often fails to correlate with evidence of glomerular or vascular injury in human clinical trials and is not considered predictive, additional animal investigative immunohistochemical work may be performed to substantiate evidence for immune complex pathogenesis. While ADA is most commonly encountered as a predisposing factor with biotherapeutic agents, complement activation may occur without circulating complexes, and other mechanisms of non-ADA immune-mediated glomerulonephritis have been observed including nonendogenous immune aggregates and immunoregulatory pharmacology. Although glomerulonephritis associated with oligonucleotide therapies has been noted occasionally in preclinical studies and more rarely with human patients, pathophysiologic mechanisms involved appear to be different between species and preclinical cases are not considered predictive for humans. ADA is not involved in oligonucleotide-associated cases, and complement fixation plays a more important role in monkeys. Recent screening of ASOs for proinflammatory activity appears to have decreased glomerulonephritis incidence preclinically.

This article summarizes a continuing education presentation on immunogenicity that was part of a continuing education course entitled, "Clinical Pathology of Biotherapeutics." Immunogenicity of a biotherapeutic can have diverse impacts including altered systemic exposure and pharmacologic responses and, in a fraction of the cases, safety concerns including cross-reactive neutralization of endogenous proteins or sequela related to immune complex disease (ICD). In most cases, immune complexes are readily cleared from circulation; however, based on physiochemical properties, insoluble complexes form, activate complement, and deposit in tissues. Using published information and personal experience, a set of repeat-dose monkey toxicity studies with manifestations suggestive of ICD was reviewed to summarize the spectrum of clinical and pathology findings. The most common live-phase observation linked to ICD was an acute postdosing reaction following multiple dose administrations characterized by generalized collapse and attributed to acute complement activation. Less common live-phase observations were related to syndromes such as a consumptive coagulopathy or a protein losing nephropathy. The most common histologic change attributed to ICD was multi-organ vascular/perivascular inflammation followed by glomerulonephritis. The presentation concluded with a description of the challenges in assessing the relevance of immunogenicity-related reaction in monkey to human clinical use.

Aim: Interactions between the co-stimulatory molecule programmed death 1 (PD-1) and its ligands, PD-L1 and PD-L2, constrain T-cell responses and help maintain peripheral tolerance. Glomerulonephritis can result from a variety of antigens, both self and foreign, and from humoural and cellular effector responses. These studies aimed to define the role of PD1 and its ligands in circulating immune complex glomerulonephritis induced by immunity to a foreign antigen., Methods: Immune complex glomerulonephritis was initiated by injecting BALB/c mice with horse spleen apoferritin intraperitoneally daily for 14 days. Inhibitory anti-mouse PD-1, anti-PD-L1 or anti-PD-L2 antibodies were administered every other day. Renal disease and immune responses were studied., Results: Daily injection of horse spleen apoferritin-induced proliferative immune complex glomerulonephritis in control antibody-treated mice, but inhibiting PD-1 did not augment renal injury. Specifically, blocking PD-1 did not increase serum antigen-specific antibodies or increase glomerular immunoglobulin G deposition, the hallmark of injury in this model. Furthermore, C3 deposition was unaffected and glomerular macrophages were reduced after anti-PD-1 antibodies. However, anti-PD-1 administration did increase splenocyte proliferation and cytokine production including interferon-γ, interleukin (IL)-4, and IL-17, but not IL-10. Neutralizing either PD-L1 or PD-L2 alone did not result in major alterations in renal injury., Conclusion: The endogenous PD-1/PD-L pathway does not limit acute experimental foreign antigen-induced circulating immune complex glomerulonephritis., (© 2015 Asian Pacific Society of Nephrology.)

Contagious bovine pleuropneumonia (CBPP) is a serious respiratory disease of cattle caused by Mycoplasma mycoides subsp. mycoides. Current vaccines against CBPP induce short-lived immunity and can cause severe postvaccine reactions. Previous studies have identified the N terminus of the transmembrane lipoprotein Q (LppQ-N') of M. mycoides subsp. mycoides as the major antigen and a possible virulence factor. We therefore immunized cattle with purified recombinant LppQ-N' formulated in Freund's adjuvant and challenged them with M. mycoides subsp. mycoides. Vaccinated animals showed a strong seroconversion to LppQ, but they exhibited significantly enhanced postchallenge glomerulonephritis compared to the placebo group (P = 0.021). Glomerulonephritis was characterized by features that suggested the development of antigen-antibody immune complexes. Clinical signs and gross pathological scores did not significantly differ between vaccinated and placebo groups. These findings reveal for the first time the pathogenesis of enhanced disease as a result of antibodies against LppQ during challenge and also argue against inclusion of LppQ-N' in a future subunit vaccine for CBPP., (Copyright © 2015 Mulongo et al.)

Henoch-Schönlein purpura (HSP) is a common systemic vasculitis in children. Animal models of HSP are needed to better understand the mechanism of HSP. Here, we investigated hematologic and immunologic profiles in HSP rat and rabbit models. Models were established with ovalbumin (OVA) based on type III hypersensitivity. During the acute phase, the models exhibited varying degrees of cutaneous purpura, joint inflammatory response, gastrointestinal bleeding, glomerular capsule protein exudation, vascular dilatation, and increased IgA expression and immune complex deposition. Twenty four hours after antigen challenge, compared with the controls, the models showed a significantly increased white blood cell count and granulocytes count and percentage, decreased number and percentage of lymphocytes, no change in platelet concentration, significantly increased serum IL-4 and TNF-α levels, and decreased CD4(+) T cell, CD4/CD8 ratio, and C3 and C4 levels. Compared with the hematologic and immunologic profiles in pediatric HSP patients, the rat and rabbit HSP models can mimic pediatric HSP characteristics. Our studies provide two useful animal models for further investigations of the pathogenesis, diagnosis, drug screening and treatment of HSP.

There were performed the studies of genotoxic stress and the ways of immunocompetent cells death (apoptosis and necrosis) in the modeling of immune system damage by immunization of CBA mice with the bovine serum albumin. Immunofluorescence studies of immunized mice were established the fixation of immune complexes in liver tissue, spleen, kidney and the aorta. Histological studies of these organs showed vascular system affection and, to a lesser extent, parenchyma. It has been shown that DNA comets index increases in 1,4 time in the lymph node cells and in 1,5 time in the thymus cells in the presence of BSA immunization. We also observed an increase in the number of cells with maximum damage DNA thymus preparations (3.4 fold) and lymph nodes (3.3-fold), respectively, indicating strong genotoxic stress. There were shown the reduce of live ICC number and their death increase, including the pro-inflammatory and immunogenic necrotic way. In that way, data which were obtained on the experimental model is evidenced that generalized immunecomplex pathologic process leads to DNA damage and ICC death both central and peripheral organs of the immune system. ICC genotoxic stress and their death amplification by the necrotic way may play a significant role in the immunecomplex deseases development. These factors of peripheral blood lymphocytes can serve as a prospective test system for assessing the severity of autoimmune and immune complex diseases and their treatment effectiveness.

Advanced glycation end products (AGEs) are thought to contribute to the abnormal lipoprotein profiles and increased risk of cardiovascular disease in patients with diabetes and renal failure. D-ribose is one of the naturally occurring pentose monosaccharide present in all living cells and is a key component of numerous biomolecules involved in many important metabolic pathways. Formation of D-ribose derived glycated low density lipoprotein (LDL) has been previously demonstrated but no studies have been performed to assess the immune complex deposition in the kidney of rabbits immunized with glycated LDL. In this study, LDL was glycated with D-ribose, and it was further used as an immunogen for immunizing NZW female rabbits. The results showed that female rabbits immunized with D-ribose modified LDL induced antibodies as detected by direct binding and competitive ELISA. The modified LDL was found to be highly immunogenic eliciting high titer immunogen-specific antibodies, while the native forms were moderately immunogenic. The induced antibodies from modified LDL exhibited wide range of heterogeneity in recognizing various proteins and amino acids conformers. Furthermore, our histopathological results illustrated the deposits of immune complex in glomerular basement membrane in rabbits immunized with D-ribose-LDL.

Two 6-month repeat-dose toxicity studies in cynomolgus monkeys illustrated immune complex-mediated adverse findings in individual monkeys and identified parameters that potentially signal the onset of immune complex-mediated reactions following administration of RN6G, a monoclonal antibody (mAb). In the first study, 3 monkeys exhibited nondose-dependent severe clinical signs accompanied by decreased erythrocytes with increased reticulocytes, neutrophilia, monocytosis, thrombocytopenia, coagulopathy, decreased albumin, azotemia, and increased serum levels of activated complement products, prompting unscheduled euthanasia. Histologically, immunohistochemical localization of RN6G was associated with monkey immunoglobulin and complement components in glomeruli and other tissues, attributable to immune complex disease (ICD). All 3 animals also had anti-RN6G antibodies and decreased plasma levels of RN6G. Subsequently, an investigational study was designed and conducted with regulatory agency input to detect early onset of ICD and assess reversibility to support further clinical development. Dosing of individual animals ceased when biomarkers of ICD indicated adverse findings. Of the 12 monkeys, 1 developed anti-RN6G antibodies and decreased RN6G exposure that preceded elevations in complement products, interleukin-6, and coagulation parameters and decreases in albumin and fibrinogen. All findings in this monkey, except for antidrug antibody (ADA), reversed after cessation of dosing without progressing to adverse sequelae typically associated with ICD., (© 2014 by The Author(s).)

Objective: To describe 2 cases of vasculitis that were attributed to a type III hypersensitivity reaction in critically ill dogs occurring 8-16 days postadministration of human serum albumin (HSA)., Case or Series Summary: Skin biopsies were obtained from 3 different sites in 2 critically ill dogs that developed vasculitis 8-16 days following treatment with HSA. Histopathological findings from both dogs indicated epidermal pallor, widespread edema and hemorrhage, degenerative neutrophilic perivascular infiltrates, and multifocal areas of neutrophilic or leukocytoclastic vasculitis. Immunohistochemical staining using an anti-human serum albumin rabbit antibody suggested that the antigen-antibody complexes seen in the dermis were associated with the administration of HSA., New or Unique Information Provided: In this case series, we documented a leukocytoclastic vasculitis and probable antigen-antibody complexes to human albumin in the dermis of 2 critically ill dogs after administration of HSA. Previously, type III hypersensitivity reactions had only been reported in healthy dogs that had received HSA. This report also describes the potential use of immunohistochemical staining to detect the HSA antigen in tissue sections through the use of specifically labeled antibodies., (© Veterinary Emergency and Critical Care Society 2013.)

Objective: To observe the histopathological and ultrastructural features of coronary artery vasculitis in rabbits caused by repeated intravenous injections of bovine serum albumin (BSA), mimicking Kawasaki disease., Materials and Methods: Twenty weanling rabbits were randomly and equally divided into treatment groups for BSA or normal saline (NS), and administered the respective treatment by intravenous injection once every 12 days for two cycles. Six weeks after the first treatment, rabbits underwent coronary angiography and coronary arteries were removed within 1 h. Histopathological examination was performed by light, scanning electron, and transmission electron microscopy., Results: Coronary arteriography revealed that 3 rabbits (3/10) in the BSA group had various levels of dilation and narrowing of the left coronary arteries, while histological examination showed that 10 rabbits (10/10) had infiltration of the coronary arteries by inflammatory cells. Incomplete endothelium, breakage of elastic fiber, and intimal thickening were also observed in 8 rabbits (8/10) from the BSA group. Ultrastructurally, in 3 rabbits (3/10) in the BSA group, leukocyte migration, shedding of endothelial microparticles from the plasma membranes, incomplete endothelium, abscission of endothelial cells, breakage of the internal elastic lamina (IEL), degeneration of smooth muscle cells in the medial membrane, and swollen mitochondria were detected. By contrast, the IEL of the NS control group was continuous and of uniform thickness., Conclusion: This rabbit model of coronary arteritis displayed histopathological and ultrastructural features similar to those of Kawasaki disease in humans. Breakage of the IEL, a key factor in aneurysm formation, was observed in the coronary arteries. Therefore weanling rabbits may serve as an experimental model for immune complex vasculitis involving coronary arteries that mimics Kawasaki disease.

Background: In this study, the effect of intravitreal injection of bevacizumab on choroidal blood vessels was examined in primate eyes., Methods: Four Cynomolgus monkeys received an intravitreal injection of 1.25 mg bevacizumab. The eyes were enucleated on days 1, 4, 7 and 14. For each animal, one eye was embedded in paraffin whereas the other eye was embedded for electron microscopy. Seven untreated or PBS (phosphate buffered saline)-injected monkeys were used as controls., Results: Thrombotic microangiopathy was found in the choriocapillaris and choroidal vessels of all eight injected eyes. Acute microangiopathy was characterized ultrastructurally as swelling of the endothelium, loss of fenestrations and complete collapse of the capillaries, and was commonly observed in bevacizumab-treated eyes. Quantitative analysis showed reduction of the lumina of the choriocapillaris in the eyes of three of the monkeys. Bevacizumab was frequently localized inside the blood vessels, often filling the entire breadth of the vessels, and formed clusters with blood cells. Death of photoreceptors occurred in two monkeys., Conclusions: This study indicate that intravitreal injection of bevacizumab in monkeys induces activation of platelets, degranulation of thrombocytes and neutrophils, formation of immune complexes, thrombotic microangiopathy and alteration of the blood flow in choroidal vessels.

Aim: Renal expression of matrix metalloproteinases (MMP) and tissue inhibitors of MMP (TIMP) contribute to the development of tubulointerstitial fibrosis characteristic of progressive forms of primary glomerulonephritis (GN). The aim of this study was to investigate the therapeutic effect of MMP inhibitor, doxycycline, administration in an experimental rat model of immune-complex nephritis (ICN)., Methods: The induction of immune-complex glomerulonephritis was carried out by the administration of an i.v. dose of 2 mg bovine serum albumin (BSA) daily for 28 days after 8 weeks of s.c. immunization with 1 mg of BSA in complete Freund's adjuvant. Doxycycline (30 mg/kg) was given daily (in groups 2 and 4) by gavage for 28 days., Results: Animals treated with doxycycline showed significant reduction in glomerular area and cell proliferation than non-treated controls. Glomerular deposition of immunoglobulin (Ig)G and C3 was less intense in treated rats than non-treated controls. Although not statistically significant, interstitial inflammation was less intense in treated rats than non-treated controls. Glomerular expression of MMP-9 by immunoflourescence was significantly inhibited in the treated group. In addition pro-MMP-2 on gelatin zymography was importantly suppressed by doxycycline in ICN., Conclusion: Doxycycline, in addition to its antibiotic property, may, following further investigation, provide a possible survival benefit in proliferative glomerulonephritis.

The extensive use of therapeutic monoclonal antibodies in clinic has shown that toxic effects may occur in patients. Examples such as muromonab (anti-CD3) and recently TGN1412 (anti-CD28) clearly establish that prediction of toxic effects for these targeted therapies is still complex. These undesirable effects can be classified in four categories: cytokine release syndrome, auto-immune diseases, organ toxicity and opportunistic infections. Immunogenicity, which is highly variable depending on the degree of humanization, could also potentially lead to adverse effects due to immune-complexes formation. The recent accident observed with the anti-CD28 TGN1412 has led to the conclusion that the relative confidence in the safety of monoclonal antibodies should be revised. In addition, the prediction of non-clinical models is rather limited and extrapolation of animal results to the human situation should be performed with caution. A better knowledge of the cellular target of the antibodies and of their mechanisms of action and the identification of risk factors (immune cell activation, wide expression of the target...) should improve the clinical safety of these products. .

Background: The kidney is one of the organs affected in patients with propylthiouracil (PTU)-associated antineutrophil cytoplasmic antibody (ANCA)-positive vasculitis. We present a series of Chinese patients with renal involvement in PTU-associated ANCA-positive vasculitis and describe their clinical and pathological characteristics., Methods: Clinical and pathological data from patients with PTU-associated ANCA-positive vasculitis with renal involvement, diagnosed in Peking University First Hospital, were collected and analyzed retrospectively., Results: Nineteen patients with PTU-associated ANCA-positive vasculitis were treated at Peking University First Hospital between December 1999 and December 2005, and 15 of them had renal involvement. Of the 15 patients, 13 were female and 2 were male, with an average age of 26.3 +/- 11.8 years. All 15 patients were perinuclear ANCA positive with specificities to myeloperoxidase (15 of 15), cathepsin G (9 of 15), human leukocyte elastase (8 of 15), lactoferrin (7 of 15), azurocidin (5 of 15), and proteinase 3 (4 of 15). Duration of PTU administration was 43.0 +/- 31.2 months. All 15 patients had clinical markers of renal involvement, including hematuria (100%), proteinuria (100%), and renal function abnormality (47%). All 15 patients underwent percutaneous renal biopsy. Ten patients had necrotizing crescentic glomerulonephritis, and 7 of these 10 patients had immune complex deposition. Three patients had minimal involvement, 2 patients had immunoglobulin A nephropathy, and 2 patients had membranous nephropathy. PTU treatment was discontinued in all 15 patients. All except 2 patients with minimal renal involvement received immunosuppressive treatment. Eleven of 15 patients achieved complete clinical remission., Conclusion: Renal involvement in our case series of patients with PTU-associated ANCA-positive vasculitis was heterogeneous, and nearly half our patients had renal immune complex deposition.

Safety concerns about di-(2-ethylhexyl)phthalate (DEHP), a plasticizer and a probable endocrine disruptor, have attracted considerable public attention, but there are few studies about long-term exposure to DEHP. DEHP toxicity is thought to involve peroxisome proliferator-activated receptor alpha (PPARalpha), but this contention remains controversial. For investigation of the long-term toxicity of DEHP and determination of whether PPARalpha mediates toxicity, wild-type and PPARalpha-null mice were fed a diet that contained 0.05 or 0.01% DEHP for 22 mo. PPARalpha-null mice that were exposed to DEHP exhibited prominent immune complex glomerulonephritis, most likely related to elevated glomerular oxidative stress. Elevated NADPH oxidase, low antioxidant enzymes, and absence of the PPARalpha-dependent anti-inflammatory effects that normally antagonize the NFkappaB signaling pathway accompanied the glomerulonephritis in PPARalpha-null mice. The results reported here indicate that PPARalpha protects against the nephrotoxic effects of long-term exposure to DEHP.

Problem: This study was undertaken to evaluate whether the anti-GnRH antibodies and immune complexes (IC) generated by immunization with GnRH-TT cause cellular damage within the animal., Method of Study: Chronic immunization of rats with GnRH-TT injected i.m. was followed by tissue/organ analysis for immune complex deposition by immunofluorescence microscopy. Two groups were studied: (1) those immunized throughout the experiment until their ultimate demise, and (2) those given a chance to recover from the effects of chronic immunization before final analysis., Results: GnRH-TT was effective in stopping spermatogenesis, which resumed after withdrawal of the immunogen. Most tissues from chronically immunized animals were not significantly different than controls, however the kidneys of treated animals exhibited a higher accumulation of IC. Despite increased IC deposition, pathologic effects were not detected at the cellular level., Conclusions: GnRH-TT is an effective immunocontraceptive although the accumulation of glomerular IC represents a potential deleterious side effect.

The purpose of this report was to present 2 cases of similar erythematous lesions in patients using Listerine Cool Mint PocketPaks Oral Care Strips. The first case, a 44-year-old female presented with an erythematous, well-defined, macular lesion centrally located on the midhard palatal gingiva. The second case involved a healthy 7-year-old Hispanic female who presented with a similar lesion and the same clinical presentation. Both presented as solitary lesions involving the height of the palate, with bilateral congruency to the mid-palatal suture line. The lesions were square in shape, with 3 sides being distinctly demarcated. The fourth side feathered anteriorly, gradually disappearing as it approached the anterior third of the palate. On verbal questioning in both cases, no discomfort or irritation was indicated in the medical history or by the patient. Both patients indicated regular use of Listerine Cool Mint PocketPaks Oral Care Strips. Treatment consisted of a: (1) recommendation that the strips be discontinued; and (2) re-evaluation of the lesions at the follow-up appointments to make assessments for any possible changes in lesion color, size, or shape. Following discontinuance of the Oral Care Strips, the lesions disappeared. Follow-up appointments suggest an allergic contact focal erythema caused by prolonged contact with the irritant, in this case due to use of Listerine Cool Mint PocketPaks Oral Care Strips.

The incidence of antiplatelet factor-4/heparin antibody formation in patients who receive contemporary doses of unfractionated heparin in the setting of percutaneous coronary revascularization is unknown. Also unknown is the ability of these antibodies to activate platelets or adversely affect clinical outcome in the absence of clinically recognized heparin-induced thrombocytopenia. To address these questions, we serially measured antiplatelet factor-4/heparin antibody levels and performed serotonin release assays in patients who underwent percutaneous coronary intervention. Correlations were then made across antibody induction, heparin exposure, and clinical outcome at 6 months.

In previous studies we have found that FcgammaRI determines chondrocyte death and matrix metalloproteinase (MMP)-mediated cartilage destruction during IFN-gamma-regulated immune complex arthritis (ICA). Binding of immune complexes (ICs) to FcgammaRI leads to the prominent production of oxygen radicals. In the present study we investigated the contribution of NADPH-oxidase-driven oxygen radicals to cartilage destruction by using p47phox-/- mice lacking a functional NADPH oxidase complex. Induction of a passive ICA in the knee joints of p47phox-/- mice resulted in a significant elevation of joint inflammation at day 3 when compared with wild-type (WT) controls as studied by histology. However, when IFN-gamma was overexpressed by injection of adenoviral IFN-gamma in the knee joint before ICA induction, a similar influx of inflammatory cells was found at days 3 and 7, comprising mainly macrophages in both mouse strains. Proteoglycan depletion from the cartilage layers of the knee joints in both groups was similar at days 3 and 7. Aggrecan breakdown in cartilage caused by MMPs was further studied by immunolocalisation of MMP-mediated neoepitopes (VDIPEN). VDIPEN expression in the cartilage layers of arthritic knee joints was markedly lower (between 30 and 60%) in IFN-gamma-stimulated arthritic p47phox-/- mice at day 7 than in WT controls, despite significant upregulation of mRNA levels of various MMPs such as MMP-3, MMP-9, MMP-12 and MMP-13 in synovia and MMP-13 in cartilage layers as measured with quantitative RT-PCR. The latter observation suggests that oxygen radicals are involved in the activation of latent MMPs. Chondrocyte death, determined as the percentage of empty lacunae in articular cartilage, ranged between 20 and 60% at day 3 and between 30 and 80% at day 7 in WT mice, and was completely blocked in p47phox-/- mice at both time points. FcgammaRI mRNA expression was significantly lower, and FcgammaRII and FcgammaRIII were higher, in p47phox-/- mice than in controls. NADPH-oxidase-driven oxygen radical production determines chondrocyte death and aggravates MMP-mediated cartilage destruction during IFN-gamma-stimulated IC-mediated arthritis. Upregulation of FcgammaRI by oxygen radicals may contribute to cartilage destruction.

After introduction of recombinant human insulin, immunological reactions have become rare, but symptomatic immune reactions to insulin still occur. We present four insulin-treated diabetic patients who developed hypersensitivity to insulin or additives to insulin preparations. We show that symptomatic immunological reactions to insulin have not been eliminated by the introduction of recombinant human insulin. Furthermore it demonstrates the importance of skin-biopsy-verified diagnoses and the relevance of differentiation between different types of insulin allergy.

We studied whether laboratory confirmation of heparin-induced thrombocytopenia (HIT) can be improved after antigen clearance by determining free antibody and combining the results of an antigenic and a biologic assay. Blood samples taken over 40 days in 14 patients with HIT with thromboembolism underwent fluorescence-linked immunofiltration and the carbon 14-serotonin release assays. Of the 14 patients, 11 showed positive results in both assays at day 1 after stopping heparin. The 3 patients with negative results seroconverted in one or both assays during the subsequent 7 days. Combining the positive results of the assays increased the sensitivity from 85% at day 1 to 100% at day 7. Assay results became negative in all patients within 40 days. The platelet count normalized between days 2 and 9 after withdrawal of heparin. It is assumed that the free antibody can be detected after withdrawal of heparin and after clearance of the platelet factor 4-heparin complex in patients with HIT.

A 71-year-old man developed an exfoliative dermatitis of the palms of the hands and soles of the feet, and a generalized itch, during treatment with intravesical instillations of mitomycin C for an undifferentiated carcinoma of the bladder. Patch tests with mitomycin C 0.03%, 0.1% and 0.3% aq. were positive. Because of the serious consequences of this finding, the patient was retested with mitomycin C in pet. (same concentrations), a more stable preparation. This showed clear positive reactions. During this last series of patch tests, he developed palpable purpura on the legs. We postulated that this reaction was an immune-complex-mediated reaction, caused by the 2nd series of patch tests with mitomycin C. To prove this, we performed histopathological and immunofluorescence investigations, and these showed the reaction to be consistent with Henoch-Schonlein-type purpura. We therefore conclude that this patient developed systemic reactions to mitomycin C, characterized by an eczematous dermatitis as well as purpuric reactions. The intravesical installations with mitomycin C have been stopped. The patient's skin problems (the purpura as well as the eczema) have completely resolved and have not recurred.

The Spanish Societies of Allergology and Anesthesiology have established a protocol for action for the performance of allergy tests for anesthesia. The protocol was published by the General Directorate of INSALUD, along with an informed consent form for general and loco-regional anesthesia, on 30 June 1994. Despite this, demands for care and requests for allergologic and anesthetic studies have increased. This review examines the diagnostic, preventive, and therapeutic criteria for adverse reactions during general anesthesia according to the most relevant international literature. Pseudoallergic reactions are one of the most frequent and important adverse reactions that occur during general anesthesia. These reactions are caused by the release of histamine and, probably, other mediators. The histamine-releasing effect depends on the dose of the medication. The most potent medications are morphine and almost all muscular relaxants, in the following proportions (succinylcholine: 43%; vecuronium: 37%; pancuronium: 13%; alcuronium: 76%; atracurion: 6.8%; galamine: 56%). Somewhat less than 50% of all severe intraoperative allergic reactions are really anaphylactoid reactions. The mediators involved in anaphylactic/anaphylactoid responses are: IgE in type I hypersensitivity mechanisms, IgA, immunocomplexes, complement activated by an alternative pathway, tryptase, and histamine. Numerous and varied agents are involved. Table III lists general intravenous anesthetics; table IV shows muscular relaxants. According to national studies, intraoperative latex sensitization occurs in 1% to 5% of health-care personnel and 40% of children with spina bifida. More than 6% of blood donors have IgE against latex. Cross-reactions between latex and fruit proteins, such as banana and kiwi, have been reported. As many as 50% of patients with latex allergy have such cross-reactions, according to some authors. Diagnostic methods include skin tests, challenge, histamine release test, RIA, human basophil optical degranulation test, and ImmunoCAP. Skin tests are the technique most often used in Allergology Units in Spain. Risk factors are debated. Although the female sex is accepted as predominant, atopy is controversial. The only known negative factor is the insertion of an endotracheal tube in asthmatic patients. Prevention should begin with the selection of less potent drugs, such as histamine releasing agents. Slow administration of drugs, as opposed to bolus administration, has been demonstrated to be more effective. The use of combined H1 and H2 antihistamines as a preanesthetic medication can significantly reduce tachycardia/bradycardia, hypotension, skin response, and even gastric pH changes induced by histamine release.

Hypersensitivity reactions to the commonly used denture base resins are infrequently reported. When they have been reported, most acrylic hypersensitivity reactions have been described as local contact reactions with few reports identifying any significant systemic symptoms. This paper reports a case where the patient suffered extensive systemic symptoms which were strongly linked to denture wear. A variety of alternative dentures of different resin content were constructed over time with varying reactions. The patient was patch-tested and responded with positive reactions to pure dye samples supplied by manufacturers of the resins. She also failed to react to dentures made in a clear acrylic with no dye components. These factors strongly support the hypothesis that the reactions experienced by this patient to some denture resins was the result of the incorporated colouring agents. It is therefore suggested that in cases where a hypersensitivity reaction with systemic manifestations to a denture base resin is suspected, questioning with regard to other reactions to colourants and patch testing for dyestuffs should be considered in addition to the use of a resin with no colouring agents in construction of replacement prostheses.

Intraperitoneal injection of pristane induces a lupus-like disease in BALB/c and other non-autoimmune mice characterized by autoantibody production and the development of immune complex disease closely resembling lupus nephritis. Two subsets of autoantibodies are induced by pristane: IgG anti-DNA DNA and -chromatin autoantibodies are strongly IL-6-dependent, whereas IgG anti-nRNP/Sm and -Su antibodies are not. The present studies were carried out to examine the role of T cells in establishing this dichotomy between the production of anti-nRNP/Sm/Su versus anti-DNA/chromatin autoantibodies. Autoantibody production and renal disease were evaluated in athymic (nude) mice treated with pristane. BALB/c nu/nu mice spontaneously developed IgM and IgG anti-single-stranded (ss)DNA and -chromatin, but not anti-nRNP/Sm or -Su, autoantibodies. Pristane treatment increased the levels of IgG anti-chromatin antibodies in nu/nu mice, but did not induce production of anti-nRNP/Sm or -Su antibodies. In contrast, BALB/c nu/+ and +/+ control mice did not spontaneously produce autoantibodies, whereas anti-nRNP/Sm and -Su autoantibodies were induced by pristane in approx. 50% of nu/+ and +/+ mice and anti-DNA/chromatin antibodies at lower frequencies. Nude mice spontaneously developed mild renal lesions that were marginally affected by pristane, but were generally milder than the lesions developing in pristane-treated nu/+ and +/+ mice. The data provide further evidence that two distinct pathways with different cytokine and T cell requirements are involved in autoantibody formation in pristane-induced lupus. This dichotomy may be relevant to understanding differences in the regulation of anti-DNA versus anti-nRNP/Sm autoantibodies in systemic lupus erythematosus, as well as the association of anti-DNA, but not anti-nRNP/Sm, with lupus nephritis.

Background: Several occurrences of immune complex-mediated, cephalosporin-induced intravascular hemolysis have been reported. This report describes the first case of hemolytic anemia caused by an immune-complex mechanism associated with ceftizoxime and delineates the epitope responsible for hemolysis., Case Report: The patient's serum was tested for antibody that reacted with five penicillins and 30 cephems (all types of cephalosporins) by using protocols to detect drug-adsorption and immune-complex mechanisms. The patient's antibody that formed immune complexes with ceftizoxime reacted with 10 of 30 cephems. These 10 drugs were classified as oxime-type cephalosporins, which have a common structural formula consisting of [(Z)-2-(2-amino-4-thiazolyl)-2-methoxyiminoacetoamido] at the C7 position on 7-aminocephalosporinic acid with or without substitution at the C3 position., Conclusion: The patient's antibody recognized a common structure in 10 oxime-type cephalosporins, and immune complexes formed by the antibody specifically or nonspecifically bound to red cell membranes. Therefore, when intermittent antibiotic therapy is required, as in this case, care should be taken in antibiotic selection to avoid drug-induced hemolytic anemia. In addition, when this type of hemolysis is observed, tests for antibody that reacts by adsorption and immune-complex mechanisms should be performed against penicillins and cephems to select antibiotics not showing a cross-reaction.

The awareness of the effects of metallic mercury vapour on the human immune system has increased only in the last decade. The regulatory guidelines relating to testing for immunotoxicity of metals are not standardized so far. A full understanding of the relevance of the tests to man is still incomplete. Immunotoxicity investigation of metals in rodents, with subsequent extrapolation to man, forms the basis of human risk assessment. Human contact with mercury vapour is mainly in chloralkali plants and in factories producing controlling and measuring devices. When the immune system acts as a target of xenobiotic insults, the result can be a decreased resistance to infection, cancers, or immune disregulation that can induce the development of allergy, or autoimmunity (Fig. 1). This article reviews literature data and our studies concerning the immunotoxicity of metallic mercury vapour. A number of data shows that mercury exerts a suppressing effect but another data suggest stimulating effects on the human immune system. The results of immunological monitoring of individuals exposed to mercury vapour were either positive or negative as well as borderline and uncertain as to the influence of mercury vapour on human immune system. The positive data had no influence on the resistance of workers to infections and neoplasms. Skin and mucosa hypersensitivity to metallic mercury is rare. No positive report that mercury vapour could be carcinogenic in man has appeared up to now.

Dental amalgam restorations are a significant source of mercury exposure in the human population, but their potential to cause systemic health effects is highly disputed. We examined effects on the immune system by giving genetically mercury-susceptible Brown Norway (BN) rats and mercury-resistant Lewis (LE) rats silver amalgam restorations in 4 molars of the upper jaw, causing a body burden similar to that described in human amalgam-bearers (from 250 to 375 mg amalgam/kg body weight). BN rats with amalgam restorations, compared with control rats given composite resinous restorations, developed a rapid activation of the immune system, with a maximum 12-fold increase of the plasma IgE concentration after 3 wks (p < 0.001; Mann-Whitney's test). LE rats receiving amalgam restorations showed no significant increase of plasma IgE (p > 0.05). After 12 wks, BN rats with amalgam restorations showed significantly increased (p < 0.05) titers of immune-complex (IC) deposits in the renal glomeruli and in the vessel walls of internal organs. These rats also showed a significant (p < 0.05), from six- to 130-fold, increase in tissue mercury concentration in the concentration order kidney > spleen > cerebrum occipital lobe > cerebellum > liver > thymus, and the tissue silver concentration was significantly (p < 0.05) increased from three- to 11-fold. Amalgam-implanted BN rats showed a significant (p < 0.05) increase in copper concentration in the kidney and spleen, and in kidney selenium concentration. We conclude that dental amalgam restorations release substantial amounts of their elements, which accumulate in the organs and which, in genetically susceptible rats, give rise to activation of the immune system and systemic IC deposits.

Contact with many drugs and chemicals can produce glomerular injury. A common sign of such injury is proteinuria. Chemicals and drugs act through diverse mechanisms to produce injury, including direct damage to cellular and membranous glomerular components, as well as to renal vasculature. Several basic pathophysiologic mechanisms, including the "intact nephron hypothesis" and the "hyperfiltration hypothesis," help to explain the mode of toxicity of many chemicals. Furthermore, they provide a means to understand the basis for renal damage and the progression of renal disease once injury has occurred.

P-selectin is one of the key early mediators of leukocyte adhesion in inflammatory conditions. This report examines the role of P-selectin in a neutrophil- and platelet-mediated model of glomerulonephritis (the concanavalin A [con A] model). The administration of neutralizing anti-P-selectin antibody (PB 1.3) reduced the platelet influx at 10 min (P < 0.05) and was associated with a 60% reduction in the neutrophil infiltrate and a 50% reduction in the number of oxidant-producing cells at 3 h within glomeruli. No effect on glomerular monocyte-macrophage accumulation was observed, and proteinuria was reduced by 20% but did not reach significance. It is concluded that P-selectin plays an important role in mediating the neutrophil and platelet accumulation in this model and likely has a role in mediating the glomerular injury.

BALB/c ByJ mice develop a lupus-like syndrome characterized by anti-nRNP/Sm and Su autoantibodies and immune complex glomerulonephritis after a single i.p. pristane injection. In contrast, mercuric chloride induces anti-fibrillarin Abs only in SJL and other H-2s mice, and not in BALB/c (H-2d) mice. In the present study, the specificities of autoantibodies induced by pristane and HgCl2 were compared in SJL and BALB/c mice to examine whether these strains are "programmed" to make different sets of autoantibodies in response to nonspecific immune stimulation. Unexpectedly, the predominant autoantibodies induced by pristane in SJL mice were neither those characteristic of HgCl2-treated SJL mice nor those associated with pristane-induced disease in BALB/c mice but, rather, anti-ribosomal P, another lupus-related specificity. The autoantibodies were strongly reactive with the C-terminal 22 amino acids of the ribosomal P2 protein, indicating that they exhibited similar fine specificities to anti-P Abs in human SLE and MRL/Ipr mice. Like BALB/c mice, pristane-treated SJL mice developed severe glomerulonephritis characterized by proteinuria, mesangial proliferation, and glomerular immune complex deposits. This is the first evidence that the induction of a lupus-like syndrome by pristane is not restricted to BALB/c mice. The predominance of anti-P Abs in SJL mice contrasts sharply with the predominance of anti-nRNP/Sm and Su, in pristane-treated BALB/c mice, even though the renal lesions were similar in both strains. The data suggest that H-2s does not program mice to produce anti-fibrillarin Abs in response to nonspecific immune stimulation, arguing that autoantibody induction by pristane involves Ag-specific mechanisms.

It is well established that in susceptible mouse strains, chronic treatment with subtoxic doses of mercuric chloride (HgCl2) induces a systemic autoimmune disease, which is characterized by increased serum levels of IgG1 and IgE antibodies, by the production of anti-nucleolar antibodies and by the development of immune complex-mediated glomerulonephritis. Susceptibility to mercury is partly under the control of major histocompatibility complex genes. To study the susceptibility to mercury further, we investigated the in vivo effects of mercury in young autoimmune disease prone (NZB x NZW)F1 (H-2d/z) mice prior to establishment of spontaneous autoimmune disease. Mercury-susceptible SJL (H-2s) mice and mercury low-responder BALB/c (H-2d) mice were used as positive and negative controls, respectively. In (NZB x NZW)F1 mice, treatment with mercury stimulated an intense antibody formation characterized by increased numbers of splenic IgG1 and IgG3 antibody-producing cells as well as by elevated serum IgE levels. Injection with mercury also induced an increased production of IgG1, IgG2b and IgE antibodies in SJL, but not in BALB/c mice. The mercury-induced IgG1 response in (NZB x NZW)F1 and SJL mice was found to be polyclonal and autoantibodies against double-stranded (ds)DNA, IgG, collagen, cardiolipin, phosphatidylethanolamine as well as antibodies against the hapten trinitrophenol were produced. In addition, SJL, but not (NZB x NZW)F1 or BALB/c mice, produced IgG1 anti-nucleolar antibodies after treatment with mercury. Further studies demonstrated that (NZB x NZW)F1 and SJL mice developed high titers of renal mesangial immune complex deposits containing IgG1 antibodies 3 weeks after injection with mercury. Thus, a mouse strain genetically prone to develop spontaneous autoimmune diseases is highly susceptible to mercury-induced immunopathological alterations.

Objective: To determine the regulating role of interleukin-1 alpha and beta (IL-1 alpha, beta) and tumor necrosis factor alpha (TNF-alpha) on inhibition of proteoglycan synthesis and proteoglycan degradation in early immune complex arthritis (ICA) in the mouse., Methods: In the early phases of arthritis, IL-1 and TNF were measured using cytokine specific bioassays, the NOB.1 EL-4 and L929 assay, respectively. The impact of IL-1 in proteoglycan synthesis was studied by neutralizing the formed IL-1 during early arthritis either by giving anti-IL-1 specific antibodies intravenously or IL-1 receptor antagonist (IL-1ra) intraperitoneally by osmotic pumps. TNF-alpha was neutralized by giving monoclonal antibodies directed against murine TNF-alpha. Synthesis of proteoglycans was measured ex vivo by uptake of 35S-sulfate by patellae derived from inflamed and control, noninflamed knee joints. In vivo formation of 35S-sulfate labeled proteoglycans was studied by autoradiography. Degradation of proteoglycans was measured by labeling patellae in vivo with 35S-sulfate before arthritis induction., Results: High levels of IL-1 are formed during the first phase of immune complex arthritis (ICA). Neutralization of either IL-1 alpha or beta with specific polyclonal antibodies resulted only in partial blocking, whereas a combination fully blocked inhibition of proteoglycan synthesis. Full blocking was also found after systemic treatment with high amounts of IL-1 receptor antagonist (1.2 mg/day during 3 days). Influx of cells was also significantly reduced both in the anti-IL-1 as well as in the IL-1ra treated groups. Whether infiltrating cells are involved in inhibition of proteoglycan synthesis was further investigated in neutropenic mice. Significantly higher levels of IL-1 were found in arthritic joints of neutropenic compared with control mice. Suppression of proteoglycan synthesis was similar in arthritic knee joints of normal and neutropenic mice. However, only minor proteoglycan degradation was found in the latter. TNF-alpha was undetectable in the bioassay in early ICA and neutralization of TNF-alpha did not change either swelling, cell influx, proteoglycan synthesis or proteoglycan degradation., Conclusion: Local production of IL-1 in ICA in knee joints seems directly responsible for inhibition of proteoglycan synthesis. A direct role of IL-1 in proteoglycan loss is unlikely, but indirectly IL-1 may be involved in proteoglycan breakdown by attracting inflammatory leukocytes and activating synovial cells. TNF-alpha seemed to have no effect on either cell influx, proteoglycan synthesis or proteoglycan degradation in this model.

A 45-year-old mechanic employed in blowing hot air (350 degrees-600 degrees C) onto the surface of a polyurethane methylene diphenyl diisocyanate (MDI) conveyer belt developed dyspnoea, rhinoconjunctivitis and fever. The illness was suggestive of an MDI-associated illness, compatible with both immediate hypersensitivity and a complement-mediated immune-complex reaction. In his serum there were specific IgG and IgE antibodies against MDI and other isocyanates, and high values of circulating immune complexes. The patient's blood and urine samples were analysed for the presence of 4,4'-methylenedianiline (MDA) in hydrolysed urine and plasma. MDA was derivatized to amides using pentafluoropropionic acid anhydride (PFPA). Gas chromatography-mass spectrometry determinations were made monitoring the (M-20; M = molecular mass) fragments from the MDA-PFPA and the [2H2]MDA-PFPA derivative. The first urine sample was obtained 22 h and the last sample 114 h after start of exposure. The urine concentrations of MDA were corrected for creatinine. The half-time of MDA was 70-80 h. The first serum sample was obtained 19 h and the last sample 1967 days after the start of exposure. The half-time was 21 days, which suggests the presence of MDI/MDA plasma protein adducts in the exposed worker.

To assess whether vomitoxin-induced dysregulation of IgA production and IgA nephropathy are reversible, relevant immunologic parameters were compared among experimental groups of B6C3F1 mice that were fed: (1) 25 ppm vomitoxin in AIN-76A semipurified diet for 24 weeks (treatment group), (2) 25 ppm vomitoxin for 8 weeks and then control diet for 16 weeks (withdrawal group), and (3) control diet for 24 weeks (control group). Levels of serum IgA and microhematuria index in the treatment group were elevated after 4 to 8 weeks and continued to increase with further vomitoxin exposure. IgA immune complexes and mesangial IgA deposition, as quantitated by interactive laser cytometer image analysis, were also increased with toxin exposure at Weeks 8, 16, and 24, whereas IgM, IgG, and complement component C3 deposition were unaffected or depressed. Serum IgA, microhematuria index, and mesangial IgA deposition in withdrawal mice remained elevated over those of the controls at Weeks 16 and 24 but were less than those of the treatment group. Cell recovery from Peyer's patches (PP) as well as the percentages of IgA+ and CD4+ cells in PP and spleen at Weeks 16 and 24 were greater in treatment mice than in controls, but only the percentage of IgA+ cells in PP was elevated in the withdrawal mice at these the same time points. When IgA secretion by unstimulated and LPS-stimulated splenic lymphocytes was used as the measure of systemic production, it was elevated in both treatment and withdrawal mice at Weeks 16 and 24. The results indicated that experimental dysregulation of IgA production and IgA nephropathy persisted up to 4 months after a discrete period of dietary vomitoxin exposure, but that the severity of these effects did not increase in a progressive fashion.

Female SJL/N mice were given either 5.0, 2.5, 1.25, or 0.625 mg mercuric chloride per liter drinking water (ppm HgCl2). Serum antinucleolar antibodies (ANuA) of the IgG class were seen in mice given at least 1.25 ppm HgCl2 for 10 weeks, a dose which corresponded to a mean renal mercury concentration, as measured with atomic absorption spectrophotometry, of 2.4 +/- 0.43 microgram Hg/g wet weight (ppm Hg; means +/- 1 SD). At a dose of 5.0 ppm HgCl2 all mice showed IgG ANuA with a mean titer of 1:846 and a mean renal mercury concentration of 14.8 +/- 3.9 ppm. Significantly increased titers of granular IgG deposits, corresponding to immune-complex (IC) deposits, developed in the renal mesangium of mice given 5.0 ppm HgCl2. Mice with heavy mesangial IgG deposits showed a mild glomerular endocapillary cell proliferation and widening of the mesangium. Renal vessel wall IgG deposits were found only in mice given 5.0 ppm HgCl2, whereas such deposits were seen in splenic and cardiac arteries of mice receiving 1.25 ppm or more of HgCl2. The renal and splenic mercury concentration was significantly increased in all groups of mercuric chloride-exposed mice and correlated with the dose. We conclude that 10 weeks peroral treatment with mercuric chloride in drinking water is able to elicit autoimmunity and IC disease in genetically homogeneous, mercury-sensitive mice at a body burden similar to that reported in some occupationally exposed humans.

Fatal hemolytic anemia developed in a 52-year-old woman who was treated with a cephalosporin, ceftriaxone. The patient's red cells (RBCs) were coated with C3, but no RBC-bound IgG, IgA, or IgM was detected. Her serum contained an antibody that did not react with cephalosporin-coated RBCs but reacted strongly with RBCs in vitro when her serum was added to drug and RBCs. This is the first case of immune hemolytic anemia associated with ceftriaxone, the first case of fatal cephalosporin-induced hemolytic anemia, and the second case in which a cephalosporin antibody showed in vitro and in vivo characteristics usually thought to be associated with the so-called immune complex mechanism.

In rodents, mercuric chloride (HgCl2) causes an autoimmune disorder with glomerulonephritis (GN), and represents an animal model for the pathogenesis of GN. We have tested the hypothesis that HgCl2 induces major histocompatibility complex (MHC) expression in renal parenchymal cells, and studied the kinetics of this induction and its temporal relation to the development of immune complex deposition in the glomeruli. Mice treated with doses of HgCl2 between 2 and 3.2 mg/kg three times for one week had increased renal expression of MHC class I and class II (at the mRNA and the product levels). Class I induction was observed in proximal tubule cells, endothelial cells and glomerular cells. Class II induction was seen mainly in interstitial cells and, to a lesser extent, in tubule cells. Renal MHC expression was maximal at one week, decreased progressively after the second week of HgCl2 administration, and reached basal levels by 23 weeks. In contrast, the amount of lymphocyte infiltration in the kidney increased from the first to the fifth week and was followed by the appearance of glomerular immune deposits from the third week on. Glomerular immune complex deposits were maximal at five weeks and, by 23 weeks, immune deposits in HgCl2-treated mice were only slightly increased over those observed in the sham group. Renal MHC induction by HgCl2 was significantly reduced by treatment with monoclonal antibody against interferon gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

Glomerulonephritis was induced in rats by daily intravenous cationised bovine serum albumin. The conjugate of horseradish peroxidase with poly-L-lysine was injected as a tracer of endocytosis by glomerular epithelial cells. The rats were sacrificed serially from 1 min to 24 h after the injection of the conjugate. The cytochemical localisation of peroxidase was observed by light- and electron-microscopy. Peroxidase was detectable in the GBM and immune deposits from 1 min after the injection but was cleared by 2 h. Cellular membrane staining by the conjugate and the membrane invaginations containing concentrated peroxidase were prominent adjacent to the subepithelial immune deposits. Peroxidase was initially localised intracellularly in membrane-bound vesicles adjacent to the subepithelial deposits. They subsequently moved towards the cell centre and became larger, some having the characteristics of lysosomes. Peroxidase activity significantly diminished within the cells by 16 h. The results show that glomerular epithelial-cell endocytosis in immune-complex glomerulonephritis is similar to that observed in the normal, but cellular membrane binding and invagination are more prominent adjacent to immune deposits. The trapping of the tracer in the deposits did not seem to alter its property of being taken up by the epithelial cells.