Austrian tricentric real-life analysis of molecular profiles of metastatic biliary tract cancer patients.

Introduction: Metastatic biliary tract cancer (BTC) is a rare and aggressive entity associated with poor prognosis. It represents a major challenge for adequate treatment strategies. In recent years, BTC has become a model for precision medicine in gastrointestinal oncology. Therefore, the analysis of the individual molecular profile in BTC patients may lead to targeted therapies for the benefit of patients. Methods: In this Austrian, tricentric, real-world, retrospective analysis, we investigated patients diagnosed with metastatic BTC who underwent molecular profiling between 2013 and 2022. Results: In total, 92 patients were identified in this tricentric analysis and 205 molecular aberrations, including 198 mutations affecting 89 different genes in 61 patients were found. The predominant mutations were in KRAS (n=17; 22.4%), TP53 (n=17; 22.4%), PIK3CA (n=7; 9.2%), FGFR2 (n=7; 9.2%), DNMT3A (n=7; 9.2%), IDH1 (n=7; 9.2%), IDH2 (n=6; 7.9%), CDKN2A (n=6; 7.9%), BAP1 (n=4; 5.3%), NF1 (n=4; 5.3%), and NF2 (n=4; 5.3%). Three patients had HER2 amplification. MSI-H status and FGFR2 fusion genes were each observed in two different patients. One patient had a BRAF V600E mutation. Eventually, 10 patients received targeted therapy, of whom one-half derived clinical benefit. Conclusions: Molecular profiling of BTC patients is implementable in routine clinical practice and should be regularly employed to detect and exploit molecular vulnerabilities.

Evaluation of Antibody Responses in Patients with B-Cell Malignancies after Two and Three Doses of Anti-SARS-CoV-2 S Vaccination-A Retrospective Cohort Study.

Patients with B-cell malignancies are at a higher risk of severe SARS-CoV-2 infections. Nevertheless, extensive data on the immune responses of hematological patients and the efficacy of the third dose of the vaccine are scarce. The goal of this study was to determine standardized anti-SARS-CoV-2 S antibody levels and to evaluate differences between treatment modalities in response to the second and third vaccines among patients with B-cell malignancies treated at the University Hospital Krems and the University Hospital of Vienna. The antibody levels of a total of 80 patients were retrospectively analyzed. The results indicate a significant increase in antibody production in response to the third vaccination. The highest increases could be observed in patients in a "watchful-waiting" and "off-therapy" setting. Encouragingly, approximately one-third of patients who did not develop antibodies in response to two vaccinations achieved seroconversion after the third vaccination. "Watchful-waiting", "off-therapy" and treatment with BTK inhibitors were indicative for increased antibody response after the third dose compared to anti-CD19 CAR T-cell and anti-CD-20 antibody treatment. In summary, the results of this study underline the pre-eminent role of the need for complete vaccination with three doses for the development of protective immunity in patients with B-cell malignancies.

Can Mindfulness-Based Stress Reduction Influence the Quality of Life, Anxiety, and Depression of Women Diagnosed with Breast Cancer? -A Review.

INTRODUCTION: Breast cancer is the most common form of cancerous disease worldwide. Its treatment leads to a variety of physiological and psychological side effects. This review investigates the question of how mindfulness-based stress reduction (MBSR), a stress management program, can influence the quality of life, anxiety, and depression of women diagnosed with breast cancer. METHODS: A systematic literature search was conducted in PubMed/MEDLINE and Cochrane Library. Screening by title, abstract and full text was performed, whereby only those articles were included that fit the inclusion criteria. A risk of bias assessment was performed for each included study. RESULTS: Overall, six studies were included, but not every study investigated all three outcomes. Two studies found positive impacts on quality of life, whereas three did not find a positive correlation between the intervention and quality of life. Four out of six studies found a positive relation between MBSR and anxiety scores, but only half of the included studies found positive results for the interaction between MBSR and depression scores. CONCLUSION: Published data suggest that anxiety can be positively influenced by MBSR, which can be used to improve the psychological care of breast cancer patients, both during and after treatment. However, further studies with larger patient numbers and longer observation periods should be conducted in order to elucidate the full potential of MSBR on important areas such as depression and quality of life.

Evaluation of Ki-67 as a Prognostic Marker in Diffuse Large B-Cell Lymphoma-A Single-Center Retrospective Cohort Study.

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma and prognostic information is essential in finding the right treatment. This study evaluated the prognostic significance of Ki-67 in patients with DLBCL. METHODS: Patients with DLBCL, treated with first-line R-CHOP, were retrospectively analyzed in groups of high (>70%) and low (≤70%) Ki-67. Parameters of interest were the international prognostic index (IPI), treatment response, progression-free survival (PFS) and overall survival (OS). A chi-squared test or Fisher's exact test was conducted to analyze categorical variables. Kaplan-Meier and log-rank tests were applied for survival analyses. Finally, a multivariate linear regression analysis was performed, including gender, Ki-67 ≤ 70% or >70%, IPI and presence of B symptoms. RESULTS: Overall, 58 patients were included. No significant association was found between Ki-67 status and IPI (p = 0.148) or treatment response (p = 0.373). Survival in patients with high Ki-67 was significantly inferior with respect to OS (p = 0.047) but not PFS (p = 0.138). Multivariate linear regression, however, yielded only IPI as a risk factor for OS. CONCLUSION: Future studies with larger patient cohorts are needed in order to elucidate the prognostic role of Ki-67 in patients with DLBCL treated with R-CHOP.

Evaluation of Antibody Responses to COVID-19 Vaccines among Solid Tumor and Hematologic Patients.

Vaccination is the primary public health strategy to cope with the COVID-19 pandemic. Although solid tumor and hematologic patients are at higher risk of serious COVID-19-related complications, data on immune responses to COVID-19 vaccines in this patient cohort are particularly scarce. The present study, therefore, aimed at the standardized determination of anti-SARS-CoV-2 spike protein antibody titers among non-vaccinated versus vaccinated solid tumor and hematologic patients who are under clinical observation or under treatment at the University Hospital Krems. Standardized anti-SARS-CoV-2 S antibody titers of a total of 441 patients were retrospectively analyzed. Our results show that antibody titers against the SARS-CoV-2 spike protein are significantly higher in solid tumor versus hematologic patients. While SARS-CoV-2 antibody titers were equal among sexes, an age-dependent decrease was observed. Of note, our studies additionally show that complete vaccination represents a valuable predictor for high anti-SARS-CoV-2 antibody responses in solid tumor and hematologic patients. In summary, to date, this is one of the largest studies to comprehensively evaluate the impact of various COVID-19 vaccines on anti-SARS-CoV-2 S antibody production in solid tumor and hematologic patients. Our findings aim to support future vaccination strategies in these highly vulnerable patients, including vaccination booster programs and alternative protective approaches.

Evaluation of personalized cancer therapies based on comprehensive genomic profiling in a middle-sized oncologic center in Austria, the University Clinic Krems.

BACKGROUND AND AIM: To successfully apply personalized cancer therapies, thorough understanding of the patient's tumor is needed. In-depth, comprehensive genomic profiling systems allow gathering this knowledge by testing hundreds of cancer-related genes. Several large institutions have established precision oncology programs in recent years with promising results for patients. However, especially middle-sized oncologic institutions face challenges to implement such programs. This study aims to retrospectively analyze the effects of comprehensive genomic tumor profiling with respect to feasibility and effectiveness in a middle-sized oncologic center in Austria. METHODS: From May 1st, 2016 to December 31st, 2019 patients at the University Clinic Krems, who suffered from CUP-syndromes plus patients, who were resistant to conventional therapy or have progressed after all available therapy lines, were offered to get their tumors analyzed by comprehensive genomic profiling in order to establish a customized therapy. RESULTS: Of 69 considered patients, 64 patients' samples could be profiled. The median number of detected alterations was 4 (minimum 0; maximum 23). Most frequent alterations were reported for TP53, KRAS and CDKN2A/B. In 13 patients (20% of 64 successful profiles), personalized therapies could be initiated. 22 patients were treated with another line of chemotherapy as no actionable alteration could be detected. Effectiveness, determined by a PFS of the newly initiated therapy longer than 130% of the last conventional therapy line, could be seen in 8 of 13 patients (61,5%) of the precision oncology cohort compared to only 3 of 22 patients (13,5%) in the chemotherapy group. Additionally, Kaplan-Meier curves of PFS demonstrate a significant benefit for personalized treated patients (p = 0,0165 with a median PFS of 151 days, compared to 83 days in the chemotherapy group). CONCLUSION: In summary, personalized cancer therapy based on comprehensive genomic profiling is effective and feasible also in the setting of a middle-sized oncologic center.

Efficacy and safety of immune checkpoint inhibitors in patients with advanced non-small cell lung cancer (NSCLC): a systematic literature review.

Background: Therapeutic strategies with immune checkpoint inhibitors (ICIs) counteract the immunosuppressive effects of programmed cell death protein-1 (PD-1) and ligand-1 (PD-L1). ICI treatment has emerged in first- and second-line therapy of non-small cell lung cancer (NSCLC). As immunotherapeutic treatment with ICIs is a dynamic field where new drugs and combinations are constantly evaluated, we conducted an up-to-date systematic review on comparative efficacy and safety in patients with advanced NSCLC. Methods: We searched PubMed up to February 2020 and Embase, CENTRAL, and clinical trial registries up to August 2018. Additionally, we checked reference lists. We dually screened titles, abstracts and, subsequently, full-texts for eligibility. Two reviewers assessed the risk of bias and graded the certainty of evidence following GRADE (Grading of Recommendations Assessment, Development and Evaluation). For second-line therapy, we performed random-effects meta-analyses. Due to considerable clinical heterogeneity, we reported first-line results narratively. Results: Of 1497 references, we identified 22 relevant publications of 16 studies. For first-line therapy, a combination of an ICI with chemotherapy improved progression-free survival and overall survival compared to chemotherapy but increased the risk of serious adverse events. Single-agent pembrolizumab increased overall and progression-free survival in patients with PD-L1 expression of ≥50% and resulted in less TRAE than chemotherapy. Compared to placebo, maintenance therapy with durvalumab increased overall and progression-free survival at the downside of higher risk of TRAE. For second-line therapy, a random-effects meta-analysis yielded a statistically significantly improved overall survival (OS) and progression-free survival (PFS) for ICIs compared to docetaxel (HR 0.69; 95% CI: 0.63-0.75 for OS; HR 0.85; 95% CI: 0.77 - 0.93 for PFS; 6 studies, 3478 patients; median OS benefit in months: 2.4 to 4.2). In meta-analysis, risk of any treatment-related adverse events of any grade was lower for ICI than docetaxel as second-line therapy (RR 0.76, 95% CI: 0.73-0.79; 6 studies, 3763 patients). Conclusion: In first-line therapy of patients with advanced NSCLC, ICI is effective when combined with chemotherapy not depending on PD-L1 expression, or as monotherapy in high PD-L1 expressing tumors. For second-line therapy, single-agent ICI improves efficacy and safety compared to docetaxel.

Precision medicine in clinical oncology: the journey from IgG antibody to IgE.

PURPOSE OF REVIEW: Cancer is one of the leading causes of death and the incidence rates are constantly rising. The heterogeneity of tumors poses a big challenge for the treatment of the disease and natural antibodies additionally affect disease progression. The introduction of engineered mAbs for anticancer immunotherapies has substantially improved progression-free and overall survival of cancer patients, but little efforts have been made to exploit other antibody isotypes than IgG. RECENT FINDINGS: In order to improve these therapies, 'next-generation antibodies' were engineered to enhance a specific feature of classical antibodies and form a group of highly effective and precise therapy compounds. Advanced antibody approaches include among others antibody-drug conjugates, glyco-engineered and Fc-engineered antibodies, antibody fragments, radioimmunotherapy compounds, bispecific antibodies and alternative (non-IgG) immunoglobulin classes, especially IgE. SUMMARY: The current review describes solutions for the needs of next-generation antibody therapies through different approaches. Careful selection of the best-suited engineering methodology is a key factor in developing personalized, more specific and more efficient mAbs against cancer to improve the outcomes of cancer patients. We highlight here the large evidence of IgE exploiting a highly cytotoxic effector arm as potential next-generation anticancer immunotherapy.

AllergoOncology: High innate IgE levels are decisive for the survival of cancer-bearing mice.

BACKGROUND: Atopics have a lower risk for malignancies, and IgE targeted to tumors is superior to IgG in fighting cancer. Whether IgE-mediated innate or adaptive immune surveillance can confer protection against tumors remains unclear. OBJECTIVE: We aimed to investigate the effects of active and passive immunotherapy to the tumor-associated antigen HER-2 in three murine models differing in Epsilon-B-cell-receptor expression affecting the levels of expressed IgE. METHODS: We compared the levels of several serum specific anti-HER-2 antibodies (IgE, IgG1, IgG2a, IgG2b, IgA) and the survival rates in low-IgE ΔM1M2 mice lacking the transmembrane/cytoplasmic domain of Epsilon-B-cell-receptors expressing reduced IgE levels, high-IgE KN1 mice expressing chimeric Epsilon-Gamma1-B-cell receptors with 4-6-fold elevated serum IgE levels, and wild type (WT) BALB/c. Prior engrafting mice with D2F2/E2 mammary tumors overexpressing HER-2, mice were vaccinated with HER-2 or vehicle control PBS using the Th2-adjuvant Al(OH)3 (active immunotherapy), or treated with the murine anti-HER-2 IgG1 antibody 4D5 (passive immunotherapy). RESULTS: Overall, among the three strains of mice, HER-2 vaccination induced significantly higher levels of HER-2 specific IgE and IgG1 in high-IgE KN1, while low-IgE ΔM1M2 mice had higher IgG2a levels. HER-2 vaccination and passive immunotherapy prolonged the survival in tumor-grafted WT and low-IgE ΔM1M2 strains compared with treatment controls; active vaccination provided the highest benefit. Notably, untreated high-IgE KN1 mice displayed the longest survival of all strains, which could not be further extended by active or passive immunotherapy. CONCLUSION: Active and passive immunotherapies prolong survival in wild type and low-IgE ΔM1M2 mice engrafted with mammary tumors. High-IgE KN1 mice have an innate survival benefit following tumor challenge.

Tolerability of obinutuzumab therapy in patients with rituximab-relapsed/refractory B-cell malignancies - a retrospective single center cohort study.

BACKGROUND & AIM: In randomised clinical trials, the type II anti-CD20 antibody obinutuzumab has been shown to be more effective than rituximab for therapy of chronic lymphocytic leukemia (CLL) and follicular lymphoma (FL). However, this enhanced efficacy was linked with elevated rates of high-grade adverse events. The aim of this study was to assess the tolerability and toxicity profile of obinutuzumab treatment in routine patients with CLL and FL, of whom the majority had experienced toxicity or resistance to rituximab. METHODS: This retrospective cohort study investigated fifteen obinutuzumab-treated patients, eight with CLL and seven with FL. The course of the disease, comorbidities and treatment-related toxicities were recorded. All patients with CLL and all but three FL patients had any form of pre-treatment with rituximab. RESULTS: Between October 2014 and August 2017, 15 patients were treated with obinutuzumab at the University Hospital Krems. In the CLL-cohort, 1 patient (12,5%) developed pneumonia, 2 (25%) febrile neutropenia, 6 (75%) anemia and 7 (87,5%) thrombocytopenia, respectively. One patient exhibited an infusion-related allergic reaction. In the FL-cohort, 6 patients (85,7%) presented with thrombocytopenia, 3 (42,9%) with anemia and one patient with neutropenia. No sepsis or consecutive solid tumors were seen in any of the patients. CONCLUSION: Obinutuzumab was mostly well tolerated in mild to heavily pre-treated patients with CLL and therapy-naïve or pre-treated patients with FL. The frequency and profile of adverse events and toxicity was comparable to data from previous clinical studies and could be managed adequately in the setting of a University Clinic.

Evaluation of vascular events in patients with myeloproliferative syndromes and mutations of either the januskinase-2 or calreticulin gene at the university hospital Krems from 2008 to 2015.

Myeloproliferative neoplasms (MPN), classified as polycythemia vera (PV), essential thrombocytosis (ET) and myelofibrosis (MF) are stem-cell derived disorders. Mutations in either the januskinase-2 (JAK-2) or the calreticulin (CALR) gene are characteristic for MPN and may result in enhanced proliferation of red blood cells, white blood cells and platelets, and thus increase the risk for vascular events. This study is a retrospective and descriptive analysis of records of patients, who underwent treatment for myeloproliferative syndromes at the Department of Hemato-Oncology of the University hospital Krems from 2008 to the end of 2015. Out of 250 patients, who were suspected for MPN, 51 patients displayed a JAK-2 V617F mutation. These were analyzed with regard to their blood values, gender, age at diagnosis, therapy and vascular events before and after diagnosis (during therapy). Of the 51 patients diagnosed with MPN and a JAK-2 V617F mutation, 33 suffered from PV, 15 from ET and 3 from MF. More men than women were diagnosed with MPN and the median age at diagnosis was 72 years. Acetylsalicylic acid, phlebotomy and Hydroxyurea were the most frequent therapies applied. In our study cohort, the most common vascular events were acute coronary syndrome and transitory ischemic attack. Thromboembolic events were effectively reduced by MPN therapy while no elevation in bleeding events could be observed.

Development of a radiolabeled caninized anti-EGFR antibody for comparative oncology trials.

Due to large homology of human and canine EGFR, dogs suffering from spontaneous EGFR+ cancer can be considered as ideal translational models. Thereby, novel immunotherapeutic compounds can be developed for both human and veterinary patients. This study describes the radiolabeling of a canine anti-EGFR IgG antibody (can225IgG) with potential diagnostic and therapeutic value in comparative clinical settings. Can225IgG was functionalized with DTPA for subsequent chelation with the radionuclide 99mTc. Successful coupling of 10 DTPA molecules per antibody on average was proven by significant mass increase in MALDI-TOF spectroscopy, gel electrophoresis and immunoblots. Following functionalization and radiolabeling, 99mTc-DTPA-can225IgG fully retained its binding capacity towards human and canine EGFR in flow cytometry, immuno- and radioblots, and autoradiography. The affinity of radiolabeled can225IgG was determined to KD 0.8 ±0.0031 nM in a real-time kinetics assay on canine carcinoma cells by a competition binding technique. Stability tests of the radiolabeled compound identified TRIS buffered saline as the ideal formulation for short-term storage with 87.11 ±6.04% intact compound being still detected 60 minutes post radiolabeling. High stability, specificity and EGFR binding affinity pinpoint towards 99mTc-radiolabeled can225IgG antibody as an ideal lead compound for the first proof-of-concept diagnostic and therapeutic applications in canine cancer patients.

A case report of septic shock syndrome caused by S. pneumoniae in an immunocompromised patient despite of vaccination.

BACKGROUND AND CASE PRESENTATION: We report a case of septic shock syndrome caused by Streptococcus pneumoniae in a patient who had undergone splenectomy due to an autoimmune lymphoproliferative syndrome (ALPS), which is characterized as a dysfunction of immunoregulation. Although the patient was vaccinated with a conjugated polysaccharide vaccine after the splenectomy, he was still susceptible to S. pneumoniae infection, because the isolated serovar (24F), a serovar long thought to be apathogenic, is not covered by any vaccine currently approved, neither a conjugated nor an unconjugated polysaccharide one. CONCLUSIONS: This case demonstrates that, due to presence of different serovars, also infections with bacteria against which patients are vaccinated have to be considered as differential diagnosis. Although vaccine development has extended the coverage of S. pneumoniae from 7 to 23 serovars within recent years, there is still demand for novel vaccines which can provide broader protection also against so-thought "apathogenic" strains, especially for groups at high risk.

Long term storage in liquid nitrogen leads to only minor phenotypic and gene expression changes in the mammary carcinoma model cell line BT474.

BACKGROUND/AIM: Cancer cell lines are indispensible surrogate models in cancer research, as they can be used off-the-shelf, expanded to the desired extent, easily modified and exchanged between research groups for affirmation, reproduction or follow-up experiments.As malignant cells are prone to genomic instability, phenotypical changes may occur after certain passages in culture. Thus, cell lines have to be regularly authenticated to ensure data quality. In between experiments these cell lines are often stored in liquid nitrogen for extended time periods.Although freezing of cells is a necessary evil, little research is performed on how long-term storage affects cancer cell lines. Therefore, this study investigated the effects of a 28-year long liquid nitrogen storage period on BT474 cells with regard to phenotypical changes, differences in cell-surface receptor expression as well as cytokine and gene expressional variations. METHODS: Two batches of BT474 cells, one frozen in 1986, the other directly purchased from ATCC were investigated by light microscopy, cell growth analysis, flow cytometry and cytokine as well as whole-transcriptome expression profiling. RESULTS: The cell lines were morphologically indifferent and showed similar growth rates and similar cell-surface receptor expression. Transcriptome analysis revealed significant differences in only 26 of 40,716 investigated RefSeq transcripts with 4 of them being up-regulated and 22 down-regulated. CONCLUSION: This study demonstrates that even after very long periods of storage in liquid nitrogen, cancer cell lines display only minimal changes in their gene expression profiles. However, also such minor changes should be carefully assessed before continuation of experiments, especially if phenotypic alterations can be additionally observed.

Why man's best friend, the dog, could also benefit from an anti-HER-2 vaccine.

Human epidermal growth factor receptor-2 (HER-2) is a well-established target for anticancer anticancerprecision medicine in humans. A HER-2 homologue with 92% amino acid identity has been described in canine mammary tumors, which whichis termed here as 'dog epidermal growth factor receptor-2 (DER-2)', with similar biological implications as those in human breast cancer. Both antigens can principally be immunologically targeted by anti-HER-2 antibodies, such as trastuzumab; however, the in vivo application applicationof humanized antibodies to other species would lead to specific hypersensitivity reactions. Therefore, HER-2 mimotope vaccines that actively induce autologous trastuzumab-like immunoglobulins represent a novel and economic treatment option to overcome species-specific limitations. Thus, the present review proposes the implementation of clinical trials with HER-2 vaccines in canine cancer model modelpatients with spontaneous DER-2 positive mammary gland carcinomas in order to assess their safety and efficacy. This approach would not only pave the way into the veterinary oncology market, but would also similarly generate robust data for human trials and facilitate the testing of novel combinatorial treatments.

Proof of concept study with an HER-2 mimotope anticancer vaccine deduced from a novel AAV-mimotope library platform.

BACKGROUND: Anticancer vaccines could represent a valuable complementary strategy to established therapies, especially in settings of early stage and minimal residual disease. HER-2 is an important target for immunotherapy and addressed by the monoclonal antibody trastuzumab. We have previously generated HER-2 mimotope peptides from phage display libraries. The synthesized peptides were coupled to carriers and applied for epitope-specific induction of trastuzumab-like IgG. For simplification and to avoid methodological limitations of synthesis and coupling chemistry, we herewith present a novel and optimized approach by using adeno-associated viruses (AAV) as effective and high-density mimotope-display system, which can be directly used for vaccination. METHODS: An AAV capsid display library was constructed by genetically incorporating random peptides in a plasmid encoding the wild-type AAV2 capsid protein. AAV clones, expressing peptides specifically reactive to trastuzumab, were employed to immunize BALB/c mice. Antibody titers against human HER-2 were determined, and the isotype composition and functional properties of these were tested. Finally, prophylactically immunized mice were challenged with human HER-2 transfected mouse D2F2/E2 cells. RESULTS: HER-2 mimotope AAV-vaccines induced antibodies specific to human HER-2. Two clones were selected for immunization of mice, which were subsequently grafted D2F2/E2 cells. Both mimotope AAV clones delayed the growth of tumors significantly, as compared to controls. CONCLUSION: In this study, a novel mimotope AAV-based platform was created allowing the isolation of mimotopes, which can be directly used as anticancer vaccines. The example of trastuzumab AAV-mimotopes demonstrates that this vaccine strategy could help to establish active immunotherapy for breast-cancer patients.

Translating clinical trials from human to veterinary oncology and back.

In human medicine clinical trials are legally required for drug development and approval. In contrast, clinical trials in small animal cancer patients are less common and legally perceived as animal experiments. Comparative oncology has been recognized as a method to speed up the development of medications by introducing animal patients with naturally developing tumours. In such cases, using animal patients would generate more robust data, as their spontaneous disease resembles the "real life" situation and thus could be more likely to predict the situation in human disease. This would not only provide veterinary oncology access to the latest developments in medicine before they are available for clinical use in animals, but could also lead to generation of clinical data in animal patients that could be translated to humans. Nevertheless, there are several limitations to practical conduct of clinical trials in veterinary medicine. In this review, the possible application of similar standards of Good Clinical Practice as in human clinical drug development will be discussed in detail, with special consideration of legal and ethical aspects in Europe and the US.

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.