Risk factors for reactions and adverse effects during oral immunotherapy.

Oral immunotherapy (OIT) involves the potential for a variety of adverse events, which range from serious systemic reactions that require epinephrine to minimal oral reactions that require no treatment. This chapter describes common types of reactions seen in the course of OIT, reviews the frequency of and risk factors for different types of events as reported in recent literature (with a focus on real-world reports from private practice), and discusses treatment strategies for these adverse events. As the availability of OIT expands, it is paramount to ensure that allergists who offer OIT have a robust understanding of these reactions and mechanisms, with the overarching goal being the safety and tolerability of the therapy for the individual patient.

Validation of inducible basophil biomarkers: Time, temperature and transportation.

BACKGROUND: The short stability window of several hours from blood collection to measuring basophil activation has limited the use of flow cytometry-based basophil activation assays in clinical settings. We examine if it is possible to extend this window to 1 day allowing for shipment of samples between laboratories. Several options exist for reporting the results including reporting all the measured values directly, calculating ratios and reporting a single value covering all measured results. Each of these options have different stability and value to the physician. METHODS: Whole blood samples from peanut allergic patients were stimulated with four different peanut concentrations at Day 0, Day 1, and Day 2. Samples were stored under temperature-controlled conditions. Flow cytometry was used to analyze the samples. The basophil activation and degranulation were measured as percentage of positive CD63 basophils and CD203c MFI fold change. Shipped samples were transported under ambient conditions. RESULTS: The results show that CD63 is a stable marker at Day 1. The CD203c ratio decreases significantly at Day 1. Calculating the CD63/IgE ratio proves to be more stable than CD63 alone. The most stable readouts are the semi-quantitative results and the trajectory of the dose response curve. Finally, we confirmed that the stability can be extended to samples shipped overnight to the laboratory. CONCLUSIONS: It is possible to extend the stability of the basophil activation assay to 1 day for samples stored at 18-25°C as well as samples shipped under ambient conditions as long as the temperature is within the 2-37°C range.

Should Epinephrine Autoinjectors Be Prescribed to All Patients on Subcutaneous Immunotherapy?

Subcutaneous allergen immunotherapy (SCIT) clearly benefits appropriately selected patients with allergic rhinitis, asthma and anaphylaxis to stinging insects. Since inception of SCIT, systemic allergic reactions (SRs) and severe anaphylaxis have been risk management challenges facing the practicing allergist. Recently it has estimated that 14% of reported SRs begin at least 30 minutes after injection administration or after the 30 minute recommended clinic observation period. Faced with the possibility that SRs could occur after the patient leaves the clinic, some practicing allergists routinely prescribe epinephrine auto-injectors to all injection patients. This article summarizes the key arguments for and against routine prescription of epinephrine auto-injectors for all allergen injection patients, discussed in a PRO/CON debate at the 2015 AAAAI meeting. Currently, there is insufficient clinical evidence to make a strong recommendation for or against this practice.

G Protein Coupled Receptor Kinase 3 Regulates Breast Cancer Migration, Invasion, and Metastasis.

Triple negative breast cancer (TNBC) is a heterogeneous disease that has a poor prognosis and limited treatment options. Chemokine receptor interactions are important modulators of breast cancer metastasis; however, it is now recognized that quantitative surface expression of one important chemokine receptor, CXCR4, may not directly correlate with metastasis and that its functional activity in breast cancer may better inform tumor pathogenicity. G protein coupled receptor kinase 3 (GRK3) is a negative regulator of CXCR4 activity, and we show that GRK expression correlates with tumorigenicity, molecular subtype, and metastatic potential in human tumor microarray analysis. Using established human breast cancer cell lines and an immunocompetent in vivo mouse model, we further demonstrate that alterations in GRK3 expression levels in tumor cells directly affect migration and invasion in vitro and the establishment of distant metastasis in vivo. The effects of GRK3 modulation appear to be specific to chemokine-mediated migration behaviors without influencing tumor cell proliferation or survival. These data demonstrate that GRK3 dysregulation may play an important part in TNBC metastasis.

Decreased Th17 and antigen-specific humoral responses in CX3 CR1-deficient mice in the collagen-induced arthritis model.

OBJECTIVE: CX(3) CR1 is a chemokine receptor that uniquely binds to its ligand fractalkine (CX(3) CL1) and has been shown to be important in inflammatory arthritis responses, largely due to its effects on cellular migration. This study was undertaken to test the hypothesis that genetic deficiency of CX(3) CR1 is protective in the chronic inflammatory arthritis model collagen-induced arthritis (CIA). Because CX(3) CR1 is expressed on T cells and antigen-presenting cells, we also examined adaptive immune functions in this model. METHODS: Autoantibody formation, clinical, histologic, T cell proliferative, and cytokine responses were evaluated in wild-type and CX(3) CR1-deficient DBA/1J mice after immunization with heterologous type II collagen (CII). RESULTS: CX(3) CR1(-/-) mice had an ∼30% reduction in arthritis severity compared to wild-type mice, as determined by 2 independent measures, paw swelling (P < 0.01) and clinical disease score (P < 0.0001). Additionally, compared to wild-type mice, CX(3) CR1(-/-) mice had an ∼50% decrease in anti-CII autoantibody formation (P < 0.05), decreased Th17 intraarticular cytokine expression (P < 0.01 for interleukin-17 [IL-17] and P < 0.001 for IL-23), and decreased total numbers of Th17 cells in inflamed joints (P < 0.05). CONCLUSION: Our findings indicate that CX(3) CR1 deficiency is protective in inflammatory arthritis and may have effects that extend beyond migration that involve adaptive immune responses in autoimmune disease.

Allergen immunotherapy: a history of the first 100 years.

PURPOSE OF REVIEW: To provide a historical perspective on the development of allergen immunotherapy and to describe the progress that has been made in both the clinical application and the scientific understanding of this therapeutic technique in the 100 years since its inception. RECENT FINDINGS: Although allergen immunotherapy has been part of allergy practice for a century, it is only in relatively recent years that the cellular and molecular mechanisms which underlie its clinical efficacy have been elucidated. Most recent studies implicate the T-regulatory cell response as central to the development of a tolerogenic state in response to allergen immunotherapy, with both IL-10 and TGF-ß playing crucial roles in the development of this cell subset. The clinical application of immunotherapy continues to advance, with promising contemporary studies noting improved safety and efficacy with pretreatment using omalizumab prior to an immunotherapy program as well as the potential for innate immune system modulation with allergen conjugates which can stimulate pattern recognition receptors such as the toll-like receptors. SUMMARY: After 100 years of clinical application, allergen immunotherapy remains the only treatment modality with the potential for long-term immunologic amelioration of atopic diseases. Future treatment advances in allergen immunotherapy will likely harness the increasing power of molecular and genomic medicine to achieve greater allergen specificity, while improving overall efficacy and minimizing the potential for systemic reactions.

History of immunotherapy: the first 100 years.

Allergen-specific immunotherapy (SIT) defines and distinguishes the modern practice of clinical allergy and immunology as the 100th anniversary of this pioneering technique is celebrated. Despite the tremendous advancements made in therapeutics, pharmacology, and the basic science of allergy, SIT remains the only treatment modality that offers a potential cure for atopic diseases rather than simply an amelioration of symptoms. A historical perspective not only offers an opportunity to tell some of the fascinating stories that led to the conception of SIT but also gives an occasion to recognize, remember, and honor those individuals who have contributed to its development.

Bromelain treatment decreases neutrophil migration to sites of inflammation.

Bromelain, a mixture of proteases derived from pineapple stem, has been reported to have therapeutic benefits in a variety of inflammatory diseases, including murine inflammatory bowel disease. The purpose of this work was to understand potential mechanisms for this anti-inflammatory activity. Exposure to bromelain in vitro has been shown to remove a number of cell surface molecules that are vital to leukocyte trafficking, including CD128a/CXCR1 and CD128b/CXCR2 that serve as receptors for the neutrophil chemoattractant IL-8 and its murine homologues. We hypothesized that specific proteolytic removal of CD128 molecules by bromelain would inhibit neutrophil migration to IL-8 and thus decrease acute responses to inflammatory stimuli. Using an in vitro chemotaxis assay, we demonstrated a 40% reduction in migration of bromelain- vs. sham-treated human neutrophils in response to rhIL-8. Migration to the bacterial peptide analog fMLP was unaffected, indicating that bromelain does not induce a global defect in leukocyte migration. In vivo bromelain treatment generated a 50-85% reduction in neutrophil migration in 3 different murine models of leukocyte migration into the inflamed peritoneal cavity. Intravital microscopy demonstrated that although in vivo bromelain treatment transiently decreased leukocyte rolling, its primary long-term effect was abrogation of firm adhesion of leukocytes to blood vessels at the site of inflammation. These changes in adhesion were correlated with rapid re-expression of the bromelain-sensitive CD62L/L-selectin molecules that mediate rolling following in vivo bromelain treatment and minimal re-expression of CD128 over the time period studied. Taken together, these studies demonstrate that bromelain can effectively decrease neutrophil migration to sites of acute inflammation and support the specific removal of the CD128 chemokine receptor as a potential mechanism of action.

Oral immunogenicity of the plant proteinase bromelain.

Bromelain is a natural mixture of proteolytic enzymes derived from pineapple stem that has been shown to have anti-inflammatory activity when administered orally. Although most proteins given orally without adjuvant (e.g., food) result in tolerance, we previously reported that long-term oral exposure to bromelain stimulated the development of high serum anti-bromelain antibody titers. The purpose of these studies was to further investigate the mechanisms responsible for the immunogenicity of oral bromelain. Results showed that repeated exposure was required for development of anti-bromelain antibodies, with strong antibody responses in all mice that received at least 12 doses of bromelain either orally or intragastrically over 3-6 weeks. Proteolytic activity was required for strong oral immunogenicity in the absence of conventional adjuvant, with strong serum antibody responses generated against proteolytically active bromelain and trypsin, but not against ovalbumin, lysozyme, or inactivated bromelain. Significantly higher anti-bromelain antibody titers were seen in IL-10-deficient versus wild-type mice, suggesting that simultaneous treatments that decrease IL-10 activity may further enhance systemic antibody responses following oral exposure. The antibodies generated did not affect the proteolytic activity of bromelain. The data demonstrate that proteolytically active antigens such as bromelain can stimulate both systemic and mucosal immune responses following repeated oral exposure. Further studies of the mechanisms involved in generation of immune responses following oral exposure to proteolytically active antigens can lead to a better understanding of mechanisms of oral tolerance and to the development of novel adjuvants for oral vaccines.

Structure/function analysis of the Saccharomyces cerevisiae Trf4/Pol sigma DNA polymerase.

The Trf4p/Pol sigma DNA polymerase (formerly Trf4p/Pol kappa) couples DNA replication to the establishment of sister chromatid cohesion. The polymerase is encoded by two redundant homologs in Saccharomyces cerevisiae, TRF4 and TRF5, that together define a fourth essential nuclear DNA polymerase in yeast and probably in all eukaryotes. Here we present a thorough genetic analysis of the founding member of this novel family of DNA polymerases, TRF4. Analyses of mutants carrying 1 of 34 "surface-targeted" alanine scanning mutations in TRF4 have identified those regions required for Pol sigma's essential function, for its role in DNA double-strand break repair, and for its association with chromosomes. The data strongly support the importance of the regions of predicted structural similarity with the Pol beta superfamily as critical for Trf4p/Pol sigma's essential and repair functions. Surprisingly, five lethal mutations lie outside all polymerase homology in a C-terminal region. The protein possesses Mg2+-dependent 3' to 5' exonuclease activity. Cell cycle analysis reveals that Trf4p/Pol sigma associates with chromosomes in G1, S, and G2 phases, but that association is abolished coincident with dissolution of cohesion at the metaphase-to-anaphase transition.