Multiple Drug Hypersensitivity in a Small Child with DRESS Syndrome - A Case Report.

Drug reaction with eosinophilia and systemic symptoms (DRESS) belongs to the group of severe cutaneous adverse reactions. Here we report a case of drug hypersensitivity against multiple antibiotics with DRESS in a young child with necrotizing pneumonia.

Vaccination status of resident pediatricians and the potential risk for their patients - a cross-sectional questionnaire study in pediatric practices in Vienna.

BACKGROUND: Pediatricians are advised by the Austrian ministry of health to be vaccinated against diphtheria, tetanus, pertussis, poliomyelitis, measles, mumps, rubella, varicella, hepatitis A, hepatitis B, meningococcus ACWY and meningococcus B, pneumococcus and seasonal influenza. As they take care of a vulnerable patient group including newborns and infants, who have not been vaccinated yet, it is important that they have a positive immunization status in order to protect their patients. This cross-sectional study aims to investigate the vaccination status of pediatricians and their assistants in practices in Vienna. METHODS: All 196 resident pediatricians in Vienna were invited to participate in this cross-sectional, questionnaire-based study. They had to specify their sex, medical profession, self-reported vaccination status for the respective vaccine preventable diseases and the type of practice they are working in (private versus government funded practice). RESULTS: High vaccination rates above 90% were found for measles, poliomyelitis, pertussis and hepatitis B, whereas seasonal influenza, meningococcus and pneumococcus were the least accepted vaccinations in this cohort. No significant differences were observed for male and female vaccination habits. Influenza and pneumococcus vaccines were more frequently received by pediatricians than their assistants. Health care workers (HCW) of private practices had significantly lower hepatitis B vaccination rates compared to those working in practices covered by the Vienna health insurance fund. CONCLUSION: Resident pediatricians in Vienna reveal rather high vaccination rates for some but not all of the recommended immunizations, which puts their pediatric patients at risk. Measures for higher vaccination rates are needed especially for this medical professional group.

Quality of life in children and adolescents with migraine: an Austrian monocentric, cross-sectional questionnaire study.

BACKGROUND: Migraine is a disabling primary headache disorder that occurs in about 10 % of children and might lead to a lower quality of life. There are several possible migraine triggers in a patient's environment, which should be avoided where possible. The objective of this Austrian monocentric study was to identify migraine triggers and the areas, in which children and adolescents with migraine have a lower quality of life than healthy, headache-free children. METHODS: In this cross-sectional, questionnaire study, 76 children from ages 8 to 17 years were included. Thirty-seven were classified as migraineurs, 39 as non-migraineurs. Participants filled in a questionnaire surveying the areas of physical, socio-economic and school functioning. Migraineurs further answered migraine-specific questions. RESULTS: The study included 33 (43.4%) males and 43 (56.6%) females. Median age was 13.00 (10.00-16.00) years. Average age of onset for migraine was 9.22 ± 3.34 years. Non-migraineurs skipped trendwise fewer meals (p.adjust = 0.108) and exercised more often (p.adjust = 0.108). In socio-economic functioning, the father's nationality being Austrian might be related to migraine (p.adjust = 0.108). Children with migraine had a significantly lower quality of life in school functioning (PedsQL 4.0 questionnaire, p.adjust = 0.04) and had significantly less often "good" grades than children without migraine (p.adjust = 0.048). CONCLUSION: Children with migraine show a reduced quality of life in the areas of physical, socio-economic and school functioning.

Characterization of Vibrio cholerae neuraminidase as an immunomodulator for novel formulation of oral allergy immunotherapy.

To improve current mucosal allergen immunotherapy Vibrio cholerae neuraminidase (NA) was evaluated as a novel epithelial targeting molecule for functionalization of allergen-loaded, poly(D,L-lactide-co-glycolide) (PLGA) microparticles (MPs) and compared to the previously described epithelial targeting lectins wheat germ agglutinin (WGA) and Aleuria aurantia lectin (AAL). All targeters revealed binding to Caco-2 cells, but only NA had high binding specificity to α-L fucose and monosialoganglioside-1. An increased transepithelial uptake was found for NA-MPs in a M-cell co-culture model. NA and NA-MPs induced high levels of IFN-γ and IL10 in naive mouse splenocytes and CCL20 expression in Caco-2. Repeated oral gavage of NA-MPs resulted in a modulated, allergen-specific immune response. In conclusion, NA has enhanced M-cell specificity compared to the other targeters. NA functionalized MPs induce a Th1 and T-regulatory driven immune response and avoid allergy effector cell activation. Therefore, it is a promising novel, orally applied formula for allergy therapy.

A distinct microbiota composition is associated with protection from food allergy in an oral mouse immunization model.

In our mouse model, gastric acid-suppression is associated with antigen-specific IgE and anaphylaxis development. We repeatedly observed non-responder animals protected from food allergy. Here, we aimed to analyse reasons for this protection. Ten out of 64 mice, subjected to oral ovalbumin (OVA) immunizations under gastric acid-suppression, were non-responders without OVA-specific IgE or IgG1 elevation, indicating protection from allergy. In these non-responders, allergen challenges confirmed reduced antigen uptake and lack of anaphylactic symptoms, while in allergic mice high levels of mouse mast-cell protease-1 and a body temperature reduction, indicative for anaphylaxis, were determined. Upon OVA stimulation, significantly lower IL-4, IL-5, IL-10 and IL-13 levels were detected in non-responders, while IL-22 was significantly higher. Comparison of fecal microbiota revealed differences of bacterial communities on single bacterial Operational-Taxonomic-Unit level between the groups, indicating protection from food allergy being associated with a distinct microbiota composition in a non-responding phenotype in this mouse model.

Interleukin-10: an anti-inflammatory marker to target atherosclerotic lesions via PEGylated liposomes.

Atherosclerosis (AS) causes cardiovascular disease, which leads to fatal clinical end points like myocardial infarction or stroke, the most prevalent causes of death in developed countries. An early, noninvasive method of detection and diagnosis of atherosclerotic lesions is necessary to prevent and treat these clinical end points. Working toward this goal, we examined recombinant interleukin-10 (IL-10), stealth liposomes with nanocargo potency for NMRI relevant contrast agents, and IL-10 coupled to stealth liposomes in an ApoE-deficient mouse model using confocal laser-scanning microscopy (CLSM). Through ex vivo incubation and imaging with CLSM, we showed that fluorescently labeled IL-10 is internalized by AS plaques, and a low signal is detected in both the less injured aortic surfaces and the arteries of wild-type mice. In vivo experiments included intravenous injections of (i) fluorescent IL-10, (ii) IL-10 targeted carboxyfluorescin (CF-) labeled stealth liposomes, and (iii) untargeted CF-labeled stealth liposomes. Twenty-four hours after injection the arteries were dissected and imaged ex vivo. Compared to free IL-10, we observed a markedly stronger fluorescence intensity with IL-10 targeted liposomes at AS plaque regions. Moreover, untargeted CF-labeled liposomes showed only weak, unspecific binding. Neither free IL-10 nor IL-10 targeted liposomes showed significant immune reaction when injected into wild-type mice. Thus, the combined use of specific anti-inflammatory proteins, high payloads of contrast agents, and liposome particles should enable current imaging techniques to better recognize and visualize AS plaques for research and prospective therapeutic strategies.

Perspectives on immunomodulation early in life.

The immune system early in life is characterized by immature activation and function of immune cells and a preponderance of Th2 cytokines. Together with other factors such as genetics and epigenetics, these immature immune responses might prone newborns susceptible to severe infections as well as allergic diseases. Immunomodulation therapy may have potential as therapeutic strategy against those disorders and might have implication in early-life interventions in the future. In this review, we will focus on two immunomodulatory substance classes, Toll-like receptor (TLR) ligands and sphingolipids, which are the focus of extensive research to date. Both TLRs and sphingolipid receptors have a very distinct distribution pattern and function on immune cells. Therefore, they can potentially modulate and balance immune responses, which might be in particular beneficial for the immaturity of the immune response early in life.

[Mechanisms and risk factors for type 1 food allergies: the role of gastric digestion]. / Mechanismen und Risikofaktoren für Typ 1 Nahrungsmittelallergien: Die Rolle der gastrischen Verdauung.

True food allergens are considered as digestion stable proteins, which are absorbed through the gastrointestinal epithelium in an intact form leading to sensitization and causing systemic symptoms. According to classifications, allergens, which are digestion-labile, cause local symptoms by their cross-reactivity towards inhalative allergens. Our recent studies revealed that digestion labile allergens can also have sensitizing capacity if gastric digestion is hindered. The increase of gastric pH via acid-suppression by proton pump inhibitors, sucralfate or antacids, interferes with protein digestion, and leads to sensitization and allergic reaction in mouse models as well as in human patients. Furthermore, the inhibition of digestion increases the risk for anaphylactic responses in sensitized individuals.Even though also other factors, such as sphingolipid metabolites, are associated with the development of food allergies, it is without any doubt that the stomach has an important gate keeping function against food allergies.

Food allergy: only a pediatric disease?

Epidemiologic studies report an increase in food allergies in industrialized countries, but mainly focus on children and young adults. This leads to the impression that food allergies do not occur in the older population. However, age-related changes dramatically affect both the innate as well as the adaptive immune system - a phenomenon known as immunosenescence. Deficiencies in micronutrients, especially zinc and iron, as well as vitamin D, in the elderly may also contribute to the development of allergies. A further risk factor of the elderly in developing food allergies could also be the decreased digestive ability of the stomach due to atrophic gastritis or anti-ulcer medication. In these settings, undigested proteins may persist and become allergenic. In fact, mouse models indicate that these pharmaceuticals support the induction of Th2 responses not only in young adult, but also in aged animals. Previous reports have already suggested that allergies are underdiagnosed among the elderly. Based on our own recent study conducted in a geriatric nursing home, we also suggest that food allergies may be underestimated.

Plasma Levels of the Bioactive Sphingolipid Metabolite S1P in Adult Cystic Fibrosis Patients: Potential Target for Immunonutrition?

Recent research has linked sphingolipid (SL) metabolism with cystic fibrosis transmembrane conductance regulator (CFTR) activity, affecting bioactive lipid mediator sphingosine-1-phosphate (S1P). We hypothesize that loss of CFTR function in cystic fibrosis (CF) patients influenced plasma S1P levels. Total and unbound plasma S1P levels were measured in 20 lung-transplanted adult CF patients and 20 healthy controls by mass spectrometry and enzyme-linked immunosorbent assay (ELISA). S1P levels were correlated with CFTR genotype, routine laboratory parameters, lung function and pathogen colonization, and clinical symptoms. Compared to controls, CF patients showed lower unbound plasma S1P, whereas total S1P levels did not differ. A positive correlation of total and unbound S1P levels was found in healthy controls, but not in CF patients. Higher unbound S1P levels were measured in ΔF508-homozygous compared to ΔF508-heterozygous CF patients (p = 0.038), accompanied by higher levels of HDL in ΔF508-heterozygous patients. Gastrointestinal symptoms were more common in ΔF508 heterozygotes compared to ΔF508 homozygotes. This is the first clinical study linking plasma S1P levels with CFTR function and clinical presentation in adult CF patients. Given the emerging role of immunonutrition in CF, our study might pave the way for using S1P as a novel biomarker and nutritional target in CF.

Risk of infection in the first year of life in preterm children: An Austrian observational study.

Newborns, especially preterm infants, have an immature immune system, which, in combination with the required medical interventions necessary for keeping the neonate alive may lead to an increased risk of infection. Even after reaching stability and adapting to the environment, preterm infants have adverse prognoses regarding infections and long-term outcomes compared to their full-term counterparts. The objective of this study was to research differences in the number and severity of infections between preterm and full-term infants during their first year of life. To answer this question, a monocentric prospective study was conducted in a pediatric practice in Vienna, including 71 full-term infants and 72 preterm infants who were observed during their first year of life regarding occurring infections. In respective samples, there was a significantly higher total number of infections in preterm (mean 6.01 ± 3.90) compared to full-term infants (3.85 ± 1.72) during the observation period of one year. Particularly the count of respiratory and severe infections was considerably higher in preterm infants. Otorhinolaryngeal infections were the most frequent of all types of infections in both groups. The pregnancy period, number of siblings, and length of the postnatal hospital stay, were observed as significantly influencing factors which affected the total number of infections. The group of early term infants (37+0 weeks to 38+6) was not significantly different to late term babies (>39+0). The acquired knowledge about the increased risk of infections should lead to a more extensive care for preterm infants, with the objective of reducing the rates of complications, morbidity and mortality in this vulnerable age group in the future.

Characterization of intrinsic and extrinsic risk factors for celery allergy in immunosenescence.

Recent studies indicated an underestimation of allergies in elderly. In our experimental food allergy model of protein feeding under acid-suppression we aimed to assess whether food allergy can be induced in immunosenescent mice. Furthermore, the impact of gastric digestion on celery allergenicity was evaluated in aged patients. Measurements of serum zinc and iron levels in senescent and adult BALB/c mice for definition of the nutritional status indicated a possible alteration of the immune response in the aged animals due to reduced zinc and iron levels. Feedings of mice with digestion-sensitive celery proteins under physiological gastric conditions induced IgG1 and IgG2a in the aged and preferentially IgG1 in the adult animals. In contrast, incomplete digestion due to acid-suppression rendered celery-specific IgE, positive skin tests and elevated IL-5 levels in both age groups. Also in aged celery allergic patients (mean age 72 years) properly digested celery showed decreased capacity to bind and crosslink IgE as evaluated by skin tests and IgE immunoblot. Thus, in the geriatric murine model, celery allergy was induced only if gastric digestion was hindered. Accordingly, gastric proteolysis decreased in vitro and in vivo IgE-reactivity against celery proteins in aged allergic patients.

GM-CSF Down-Regulates TLR Expression via the Transcription Factor PU.1 in Human Monocytes.

Toll-like receptors (TLR) are crucial sensors of microbial agents such as bacterial or viral compounds. These receptors constitute key players in the induction of inflammation, e.g. in septic or chronic inflammatory diseases. Colony-stimulating factors (CSFs) such as granulocyte-macrophage-CSF (GM-CSF) or granulocyte-CSF (G-CSF) have been extensively investigated in their capacity to promote myelopoiesis in febrile neutropenia or to overcome immunosuppression in patients suffering from sepsis-associated neutropenia or from monocytic immunoincompetence. We report here that GM-CSF, downregulates TLR1, TLR2 and TLR4 in a time- and dose-dependent fashion in human monocytes. Diminished pathogen recognition receptor expression was accompanied by reduced downstream p38 and extracellular-signal-regulated kinase (ERK) signaling upon lipoteichoic acid (LTA) and lipopolysaccharide (LPS) binding-and accordingly led to impaired proinflammatory cytokine production. Knockdown experiments of the transcription factors PU.1 and VentX showed that GM-CSF driven effects on TLR regulation is entirely PU.1 but not VentX dependent. We further analysed monocyte TLR and CD14 expression upon exposure to the IMID® immunomodulatory drug Pomalidomide (CC-4047), a Thalidomide analogue known to downregulate PU.1. Indeed, Pomalidomide in part reversed the GM-CSF-mediated effects. Our data indicate a critical role of PU.1 in the regulation of TLR1, 2, 4 and of CD14, thus targeting PU.1 ultimately results in TLR modulation. The PU.1 mediated immunomodulatory properties of GM-CSF should be taken into consideration upon usage of GM-CSF in inflammatory or infection-related conditions.

Surgical Elimination of the Gastric Digestion by Roux-en-Y Gastric Bypass Impacts on Food Sensitisation-a Pilot Study.

BACKGROUND: Impairment of gastric digestion due to pH elevation increases the risk for food allergy induction. As patients after Roux-en-Y gastric bypass (RYGB) surgery have lower gastric acidity and less gastric gland secretion, we aimed to analyse in a prospective study the effect of limiting gastric digestion capacity by surgical intervention on the immune response towards allergens. METHODS: Nine patients undergoing RYGB surgery for morbid obesity and one control patient having undergone surgery for treatment of an incisional hernia were enrolled in the study. Before and 1, 3, 6, 9 and 12 months after surgery, blood was collected for analysis of specific IgE antibodies, and patients were subjected to skin prick testing with 16 food and 18 aeroallergens. RESULTS: Skin prick test results revealed an increase of positive reactions indicating sensitisations towards the tested food and aeroallergens in 77.8 and 88.9 % of the patients, respectively, after surgical elimination of gastric digestion. These results were in line with elevated titers of food- and aeroallergen-specific IgE antibodies in 7 out of 9 (7/9) and 5/9 patients, respectively, after RYGB surgery. Serum cytokine levels revealed a mixed response for IFN-γ and were mostly beneath detection limit for IL-4. CONCLUSION: A change of IgE reactivity pattern occurred after impairment of gastric digestion due to surgical elimination underlining the important gastric gatekeeping function during oral sensitisation. Even though this study indicates an increased allergy risk for gastric bypass patients, further studies are needed to investigate in-depth the immunological changes associated with RYGB surgery.

Nitration of ß-Lactoglobulin but Not of Ovomucoid Enhances Anaphylactic Responses in Food Allergic Mice.

BACKGROUND: We revealed in previous studies that nitration of food proteins reduces the risk of de novo sensitization in a murine food allergy model. In contrast, in situations with preformed specific IgE antibodies, in vitro experiments suggested an increased capacity of effector cell activation by nitrated food proteins. OBJECTIVE: The aim of this study was to investigate the influence of protein nitration on the effector phase of food allergy. DESIGN: BALB/c mice were immunized intraperitoneally (i.p.) with the milk allergen ß-lactoglobulin (BLG) or the egg allergen ovomucoid (OVM), followed by intragastric (i.g.) gavages to induce a strong local inflammatory response and allergen-specific antibodies. Subsequently, naïve and allergic mice were intravenously (i.v.) challenged with untreated, sham-nitrated or nitrated BLG or OVM. Anaphylaxis was monitored by measuring core body temperature and determination of mouse mast cell protease-1 (mMCP-1) levels in blood. RESULTS: A significant drop of body temperature accompanied with significantly elevated concentrations of the anaphylaxis marker mMCP-1 were only observed in BLG allergic animals challenged with nitrated BLG and not in OVM allergic mice challenged with nitrated OVM. SDS-PAGE and circular dichroism analysis of the differentially modified allergens revealed an effect of nitration on the secondary protein structure exclusively for BLG together with enhanced protein aggregation. CONCLUSION: Our data suggest that nitration affects differently the food allergens BLG and OVM. In the case of BLG, structural changes favored dimerization possibly explaining the increased anaphylactic reactivity in BLG allergic animals.

Protamine nanoparticles with CpG-oligodeoxynucleotide prevent an allergen-induced Th2-response in BALB/c mice.

The currently applied immunotherapy of type I allergy with aluminum hydroxide (alum) as adjuvant elicits - among other side effects - an initial IgE-boost. In contrast, CpG-oligodeoxynucleotides (ODNs) drive the immune response toward Th1. The biodegradable material protamine can spontaneously form nanoparticles together with such ODNs. Our aim was to investigate the immune response induced by protamine-based nanoparticles (proticles) with CpG-ODN as an allergen delivery system. Proticles complexed with Ara h 2 extracted from raw peanuts as model allergen were injected subcutaneously into naïve BALB/c mice. Ara h 2-specific antibodies were analyzed by ELISA and rat basophilic leukemia (RBL) cell assay. Cytokine levels were investigated in supernatants of stimulated splenocytes. The in vivo distribution after subcutaneous injection was examined via fluorescence imaging. BMDCs were stimulated with proticles, and expression of stimulation and maturation markers as well as cytokines in supernatants was investigated. A favorable increase in Ara h 2-specific IgG2a antibodies was found after immunization with proticles-Ara h 2, whereas Ara h 2-specific IgE was not detectable. Accordingly, the ratio of IL-5/IFN-gamma was low in this group. Granuloma formation was completely absent at injection sites of proticles. The distribution of Ara h 2 after subcutaneous injection was markedly decelerated when complexed to proticles. Stimulation of BMDCs with proticles-Ara h 2 caused upregulation of CD11c and CD80 as well as an increased IL-6 production. Our data suggest that biodegradable protamine-based nanoparticles with CpG-ODN counteract the Th2-dominated immune response induced by an allergen and therefore are suitable as novel carrier system for immunotherapy of allergy.

Use of lectin-functionalized particles for oral immunotherapy.

Immunotherapy, in recent times, has found its application in a variety of immunologically mediated diseases. Oral immunotherapy may not only increase patient compliance but may, in particular, also induce both systemic as well as mucosal immune responses, due to mucosal application of active agents. To improve the bioavailability and to trigger strong immunological responses, recent research projects focused on the encapsulation of drugs and antigens into polymer particles. These particles protect the loaded antigen from the harsh conditions in the GI tract. Furthermore, modification of the surface of particles by the use of lectins, such as Aleuria aurantia lectin, wheatgerm agglutinin or Ulex europaeus-I, enhances the binding to epithelial cells, in particular to membranous cells, of the mucosa-associated lymphoid tissue. Membranous cell-specific targeting leads to an improved transepithelial transport of the particle carriers. Thus, enhanced uptake and presentation of the encapsulated antigen by antigen-presenting cells favor strong systemic, but also local, mucosal immune responses.

Sphingosine-kinase 1 and 2 contribute to oral sensitization and effector phase in a mouse model of food allergy.

BACKGROUND: Sphingosine-1-phosphate (S1P) influences activation, migration and death of immune cells. Further, S1P was proposed to play a major role in the induction and promotion of allergic diseases. However, to date only limited information is available on the role of S1P in food allergy. OBJECTIVE: We aimed to investigate the role of sphingosine-kinase (SphK) 1 and 2, the enzymes responsible for endogenous S1P production, on the induction of food allergy. METHODS AND RESULTS: Human epithelial colorectal CaCo2 cells stimulated in vitro with S1P revealed a decrease of transepithelial resistance and enhanced transport of FITC labeled OVA. We studied the effect of genetic deletion of the enzymes involved in S1P production on food allergy induction using a mouse model of food allergy based on intragastrically (i.g.) administered ovalbumin (OVA) with concomitant acid-suppression. Wild-type (WT), SphK1(-/-) and SphK2(-/-) mice immunized with OVA alone i.g. or intraperitoneally (i.p.) were used as negative or positive controls, respectively. SphK1- and SphK2-deficient mice fed with OVA under acid-suppression showed reduced induction of OVA specific IgE and IgG compared to WT mice, but had normal responses when immunized by the intraperitoneal route. Flow cytometric analysis of spleen cells revealed a significantly reduced proportion of CD4(+) effector T-cells in both SphK deficient animals after oral sensitization. This was accompanied by a reduced accumulation of mast cells in the gastric mucosa in SphK-deficient animals compared to WT mice. Furthermore, mouse mast cell protease-1 (mMCP-1) levels, an IgE-mediated anaphylaxis marker, were reliably elevated in allergic WT animals. CONCLUSION: Modulation of the S1P homeostasis by deletion of either SphK1 or SphK2 alters the sensitization and effector phase of food allergy.

Nitration of the egg-allergen ovalbumin enhances protein allergenicity but reduces the risk for oral sensitization in a murine model of food allergy.

BACKGROUND: Nitration of proteins on tyrosine residues, which can occur due to polluted air under "summer smog" conditions, has been shown to increase the allergic potential of allergens. Since nitration of tyrosine residues is also observed during inflammatory responses, this modification could directly influence protein immunogenicity and might therefore contribute to food allergy induction. In the current study we have analyzed the impact of protein nitration on sensitization via the oral route. METHODOLOGY/PRINCIPAL FINDINGS: BALB/c mice were immunized intragastrically by feeding untreated ovalbumin (OVA), sham-nitrated ovalbumin (snOVA) or nitrated ovalbumin (nOVA) with or without concomitant acid-suppression. To analyze the impact of the sensitization route, the allergens were also injected intraperitoneally. Animals being fed OVA or snOVA under acid-suppressive medication developed significantly elevated levels of IgE, and increased titers of specific IgG1 and IgG2a antibodies. Interestingly, oral immunizations of nOVA under anti-acid treatment did not result in IgG and IgE formation. In contrast, intraperitoneal immunization induced high levels of OVA specific IgE, which were significantly increased in the group that received nOVA by injection. Furthermore, nOVA triggered significantly enhanced mediator release from RBL cells passively sensitized with sera from allergic mice. Gastric digestion experiments demonstrated protein nitration to interfere with protein stability as nOVA was easily degraded, whereas OVA and snOVA remained stable up to 120 min. Additionally, HPLC-chip-MS/MS analysis showed that one tyrosine residue (Y(107)) being very efficiently nitrated is part of an ovalbumin epitope recognized exclusively after oral sensitization. CONCLUSIONS/SIGNIFICANCE: These data indicated that despite the enhanced triggering capacity in existing allergy, nitration of OVA may be associated with a reduced de novo sensitizing capability via the oral route due to enhanced protein digestibility and/or changes in antibody epitopes.