Whitney W. Stevens

Delayed clinical and ex vivo response to mammalian meat in patients with IgE to galactose-alpha-1,3-galactose

BACKGROUND In 2009, we reported a novel form of delayed anaphylaxis to red meat related to serum IgE antibodies to the oligosaccharide galactose-alpha-1,3-galactose (alpha-gal). Although patients were remarkably consistent in their description of a 3- to 6-hour delay between eating mammalian meat and the appearance of symptoms, this delay has not been demonstrated under observed studies. OBJECTIVES We sought to formally document the time course of clinical symptoms after the ingestion of mammalian meat in subjects with IgE to alpha-gal and to monitor ex vivo for the appearance of markers of an allergic reaction. METHODS Open food challenges were performed with mammalian meat in 12 subjects with a history of severe urticarial reactions 3 to 6 hours after eating beef, pork, or lamb, as well as in 13 control subjects. Blood samples were taken hourly during each challenge. RESULTS Ten of 12 subjects with IgE to alpha-gal had clinical evidence of a reaction during the food challenge (vs none of the control subjects, P < .001). The reactions occurred 3 to 7 hours after the initial ingestion of mammalian meat and ranged from urticaria to anaphylaxis. Tryptase levels were positive in 3 challenges. Basophil activation, as measured by increased expression of CD63, correlated with the appearance of clinical symptoms. CONCLUSION The results presented provide clear evidence of an IgE-mediated food allergy that occurs several hours after ingestion of the inciting allergen. Moreover, here we report that in vivo basophil activation during a food challenge occurs in the same time frame as clinical symptoms and likely reflects the appearance of the antigen in the bloodstream.

Chronic rhinosinusitis pathogenesis

There are a variety of medical conditions associated with chronic sinonasal inflammation, including chronic rhinosinusitis (CRS) and cystic fibrosis. In particular, CRS can be divided into 2 major subgroups based on whether nasal polyps are present or absent. Unfortunately, clinical treatment strategies for patients with chronic sinonasal inflammation are limited, in part because the underlying mechanisms contributing to disease pathology are heterogeneous and not entirely known. It is hypothesized that alterations in mucociliary clearance, abnormalities in the sinonasal epithelial cell barrier, and tissue remodeling all contribute to the chronic inflammatory and tissue-deforming processes characteristic of CRS. Additionally, the host innate and adaptive immune responses are also significantly activated and might be involved in pathogenesis. Recent advancements in the understanding of CRS pathogenesis are highlighted in this review, with special focus placed on the roles of epithelial cells and the host immune response in patients with cystic fibrosis, CRS without nasal polyps, or CRS with nasal polyps.

Cytokines in Chronic Rhinosinusitis. Role in Eosinophilia and Aspirin-exacerbated Respiratory Disease

RATIONALE The mechanisms that underlie the pathogenesis of chronic rhinosinusitis without nasal polyps (CRSsNP), chronic rhinosinusitis with nasal polyps (CRSwNP), and aspirin-exacerbated respiratory disease (AERD) are not clear. OBJECTIVES To first evaluate the inflammatory profiles of CRSsNP and CRSwNP tissues and then to investigate whether clinical differences observed between CRSwNP and AERD are in part secondary to differences in inflammatory mediator expression within nasal polyp (NP) tissues. METHODS Expression levels of numerous inflammatory mediators were determined by quantitative real-time polymerase chain reaction, ELISA, and multiplex immunoassay. MEASUREMENTS AND MAIN RESULTS CRSwNP NP had increased levels of type 2 mediators, including IL-5 (P < 0.001), IL-13 (P < 0.001), eotaxin-2 (P < 0.001), and monocyte chemoattractant protein (MCP)-4 (P < 0.01), compared with sinonasal tissue from subjects with CRSsNP and control subjects. Expression of IFN-γ messenger RNA or protein was low and not different among the chronic rhinosinusitis subtypes examined. Compared with CRSwNP, AERD NP had elevated protein levels of eosinophil cationic protein (ECP) (P < 0.001), granulocyte-macrophage colony-stimulating factor (GM-CSF) (P < 0.01), and MCP-1 (P = 0.01), as well as decreased gene expression of tissue plasminogen activator (tPA) (P = 0.02). Despite the higher eosinophilia in AERD, there was no associated increase in type 2 mediator protein levels observed. CONCLUSIONS CRSwNP was characterized by a predominant type 2 inflammatory environment, whereas CRSsNP did not reflect a classic type 1 milieu, as has been suggested previously. AERD can be distinguished from CRSwNP by elevated ECP levels, but this enhanced eosinophilia is not associated with elevations in traditional type 2 inflammatory mediators associated with eosinophil proliferation and recruitment. However, other factors, including GM-CSF, MCP-1, and tPA, may be important contributors to AERD pathogenesis.

Pathogenesis of nasal polyposis

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a complex inflammatory condition that affects a large proportion of the population world‐wide and is associated with high cost of management and significant morbidity. Yet, there is a lack of population‐based epidemiologic studies using current definitions of CRSwNP, and the mechanisms that drive pathogenesis in this disease remain unclear. In this review, we summarize the current evidence for the plethora of factors that likely contribute to CRSwNP pathogenesis. Defects in the innate function of the airway epithelial barrier, including diminished expression of antimicrobial products and loss of barrier integrity, combined with colonization by fungi and bacteria likely play a critical role in the development of chronic inflammation in CRSwNP. This chronic inflammation is characterized by elevated expression of many key inflammatory cytokines and chemokines, including IL‐5, thymic stromal lymphopoietin and CCL11, that help to initiate and perpetuate this chronic inflammatory response. Together, these factors likely combine to drive the influx of a variety of immune cells, including eosinophils, mast cells, group 2 innate lymphoid cells and lymphocytes, which participate in the chronic inflammatory response within the nasal polyps. Importantly, however, future studies are needed to demonstrate the necessity and sufficiency of these potential drivers of disease in CRSwNP. In addition to the development of new tools and models to aid mechanistic studies, the field of CRSwNP research also needs the type of robust epidemiologic data that has served the asthma community so well. Given the high prevalence, costs and morbidity, there is a great need for continued research into CRS that could facilitate the development of novel therapeutic strategies to improve treatment for patients who suffer from this disease.

Basophils are elevated in nasal polyps of patients with chronic rhinosinusitis without aspirin sensitivity

To the Editor: Chronic rhinosinusitis (CRS) is a common chronic diseasewith significant burden and effect on the quality of life of affected individuals, characterized by inflammation of the nasal mucosa and paranasal sinuses. Patients can be subdivided into CRS with nasal polyps (CRSwNP) and CRS without nasal polyps (CRSsNP). In addition, aspirin exacerbated respiratory disease (AERD) is a subgroup of CRSwNP characterized by the triad of CRSwNP, asthma, and hypersensitivity to aspirin or nonsteroidal antiinflammatory drugs. Eosinophilia is evident in the nasal mucosa and submucosa of nasal polyps (NPs) from patients in Europe and the United States, but less so in Asian countries such as China or Japan. In this study we attempted, for the first time, to evaluate the role of another important inflammatory leukocyte, the basophil, in CRS. Basophils are granulocytes found mainly in the circulation and are known to have a role in allergic diseases, parasite expulsion, and immunity against ectoparasites. Basophil numbers are elevated in the bronchial mucosa and submucosa of asthmatic patients, the nasal submucosa in patients with allergic rhinitis, and in the skin of those with multiple inflammatory dermatologic conditions, including eczema and urticaria. AlthoughCRSwNP is a highlyTH2-biaseddisease,making basophil involvement likely, there are no reports of basophils in CRS. In the current study we used 2D7, a mouse mAb against a basophilspecific intermediate form of major basic protein, to detect basophils in uncinate tissue (UT)or polyp tissue obtained during surgery from patients with CRS and control subjects as described previously. This antibody is shown to be a specific marker for basophils and does not recognize any other cell type. We used the immunohistochemistry protocol detailed in previous studies using 2D7. Mucosa and submucosa in the tissue biopsies were delineated, and the total number of positively stained cells in each field was counted in 50 random hpf with 1003magnification. Results are reported as the number of positive cells per square millimeter. A tissue section was stained with hematoxylin and eosin. Eosinophils were identified on the basis of their cellular features and distinct cytoplasmic eosinophilic granules. For all polyp samples, a serial section was stained with a tryptase mAB, a specificmarker for mast cells as described previously. Cells were counted and data expressed as described above for basophils. Data are presented as mean 6 SEM. Comparisons between groups were assessed by using ANOVA and Newman-Keuls multiple comparison tests. Correlations were measured using a Spearman rank correlation test. All statistical analyses were performed using GraphPad Prism version 6.00 for Windows (GraphPad Software, La Jolla, Calif; www.graphpad.com). A P value of less than .05 was considered statistically significant. A total of 73 sections were labeled with 2D7, including polyps from 27 patients with and without AERD (10 with AERD and 17 without AERD) and UT from 16 patients with CRSwNP, 15 patients with CRSsNP, and 15 control cases with no evidence of CRS. Cells were identified as basophils on the basis of a brown cytoplasmic granular staining pattern (Fig 1, A). Negative control slides were labeled with an isotype-matched nonimmune antibody and showed no staining. As detailed in Table I, there were no 2D7 basophils detected within the uncinate mucosa and submucosa of control subjects. A small number of these cells were detected in the UTof patients with CRSsNP, and a greater number was detected in the UTof patients with CRSwNP, though the quantities detected were not significantly greater than in control UT. The 2D7-positive cells per square millimeter of tissue were significantly higher in the NP tissue of patients with CRSwNP than in the UT of control subjects or patients with CRSsNP and CRSwNP (Fig 1, B). This count was also significantly higher when compared with the corresponding UT of the same patients with CRSwNP, paired sets of the polyp tissue and the UT having been evaluated from 12 patients with CRSwNP. Interestingly, the number of basophils detected in NPs from subjects with AERD was not higher than in normal or CRS UT and was significantly lower than in NPs from patients without AERD. Eosinophil counts in the UT of control patients with no evidence of sinusitis were significantly lower than in the UT of patients with CRSwNP (Table I). Polyp tissue had a significantly higher number of eosinophils than did the UT of all groups, including controls and patients with CRSsNP and CRSwNP. Polyps from subjects with AERD had a significantly higher eosinophil count than did the UT of all groups, with a trend toward higher levels than in non-AERD polyps. Basophil and eosinophil counts in all tissues (excluding AERD polyps) significantly correlated with one another (P < .001; Spearman r 5 0.67) (Fig 1, D). But the basophil and mast cell counts did not correlate. Basophils and eosinophils utilize similar pathways for recruitment and might be expected to be recruited to an inflammatory site in parallel.We therefore calculated the ratio of eosinophils to basophils by dividing the total number of eosinophils per hpf by the number of basophils per hpf in a fixed area in serially cut slides. The eosinophil to basophil ratio in normal UT had a mean value of 3.0; this value was 13.1 in CRSsNP UT and 49.0 in CRSwNPUT. Furthermore, the magnitude of the ratio of eosinophils to basophils was highest in NPs, with a mean of 89.2 in non-AERD polyps and 287.3 in AERD polyps. Values of this ratio were significantly higher in polyps than in the UT of CRSwNP, CRSsNP, and control cases. The blood basophil counts within 6 months of the time of surgery were available for 40 cases: 13 with analysis of polyp tissue and 27 with analysis of the UT. There was no correlation between the blood basophil count and the number of basophils in either the polyp tissue or the UT. This appears to exclude systemic basophilia as a likely cause of the observed increase in the number of basophils in the polyp tissue. We also correlated the basophil numbers in polyps with total serum IgE (available in 13 cases) and the presence of anosmia or hyposmia as a marker for disease severity and positive skin test result. None of these comparisons was significant (both including and excluding AERD polyps). Potentially other patient-related factors, such as treatment, might have affected the number of basophils. We compared the number of basophils in the UT and the polyp tissue of patients who were on oral steroids at the time of surgery compared with those who

Increased noneosinophilic nasal polyps in chronic rhinosinusitis in US second-generation Asians suggest genetic regulation of eosinophilia

In this study we found a significantly lower eosinophilia in nasal polyps surgically removed from second-generation Asian patients, similar to studies of native-born patients in Asian countries, suggesting the hypothesis that there may be genetic regulation of eosinophilia.

Differences in both prevalence and titre of specific immunoglobulin E among children with asthma in affluent and poor communities within a large town in Ghana

Background Reports from several African countries have noted an increasing prevalence of asthma in areas of extensive urbanization.

Pulmonary Eosinophilia Is Attenuated by Early Responding CD8+ Memory T Cells in a Murine Model of RSV Vaccine-Enhanced Disease

Vaccination with formalin-inactivated respiratory syncytial virus (RSV) vaccine results in enhanced respiratory tract inflammation and injury following subsequent RSV infection. RSV vaccine-enhanced disease can also be produced in mice by prior vaccination with a vaccinia virus vector containing the RSV G protein, followed by intranasal infectious RSV challenge, a process characterized by induction of a potent memory CD4(+) T-cell response to challenge infection with some features characteristic of Th-2 CD4(+) T-cell responses, including increased eosinophil accumulation in pulmonary inflammatory infiltrates. The adaptive immune response to the RSV G protein in immunized BALB/c mice is characterized by a weak or absent primary and secondary recall CD8(+) T-cell response. These and related results have led to the hypothesis that the failure of the infected animals to mount an effective CD8(+) memory T-cell (CD8(+) Tm) response in this model could account for the pulmonary eosinophilia associated with the development of enhanced disease, and that CD8(+) T cells may control the development of eosinophilia. In this study, we investigated how and when the generation of a CD8(+) Tm response to RSV infection might affect the development of pulmonary eosinophilia in this model of vaccine-enhanced disease. By defining the CD8(+) T-cell response kinetics and monitoring lung parenchymal eosinophil accumulation, we show that the establishment of an RSV-specific CD8(+) Tm response in the infected lungs early after challenge infection (i.e., within the first 3 d of RSV infection) is necessary and sufficient to control pulmonary eosinophilia development. Additionally, our work suggests that the mechanism by which CD8(+) T cells regulate this process is not by modulating the differentiation or development of the CD4(+) Tm response. Rather, we demonstrate that IL-10 produced by early responding CD8(+) Tm cells may regulate the pulmonary eosinophilia development observed in RSV vaccine-enhanced disease.

Accuracy of phenotyping chronic rhinosinusitis in the electronic health record

Background Chronic rhinosinusitis (CRS) is prevalent, morbid, and poorly understood. Extraction of electronic health record (EHR) data of patients with CRS may facilitate research on CRS. However, the accuracy of using structured billing codes for EHR-driven phenotyping of CRS is unknown. We sought to accurately identify CRS cases and controls using EHR data and to determine the accuracy of structured billing codes for identifying patients with CRS. Methods We developed and validated distinct algorithms to identify patients with CRS and controls using International Classification of Diseases, Ninth Revision (ICD-9) and Current Procedural Terminology codes. We used blinded clinician chart review as the reference standard to evaluate algorithm and billing code accuracy. Results Our initial control algorithm achieved a control positive predictive value (PPV) of 100% (i.e., negative predictive value of 100% for CRS). Our initial algorithm for CRS cases relied exclusively on billing codes and had a low case PPV (54%). Notably, ICD-9 code 471.x was associated with a case PPV of 85%, whereas the case PPV of ICD-9 code 473.x was only 34%. After multiple algorithm iterations, we increased the case PPV of our final algorithm to 91% by adding several requirements, e.g., that ICD-9 codes occur with 1 or more evaluations by a CRS specialist to enhance availability of objective clinical data for accurately phenotyping CRS. Conclusion These algorithms are an important first step to identify patients with CRS, and may facilitate EHR-based research on CRS pathogenesis, morbidity, and management. Exclusive use of coded data for phenotyping CRS has limited accuracy, especially because CRS symptomatology overlaps with that of other illnesses. Incorporating natural language processing (e.g., to evaluate results of nasal endoscopy or sinus computed tomography) into future work may increase algorithm accuracy and identify patients whose disease status may not be ascertained by only using billing codes.

Immunodeficiency in chronic sinusitis: recognition and treatment.

Chronic rhinosinusitis (CRS) is estimated to affect over 35 million people. However, not all patients with the diagnosis respond to standard medical and surgical treatments. Although there are a variety of reasons a patient may be refractory to therapy, one possible etiology is the presence of an underlying immunodeficiency. This review will focus on the description, recognition, and treatment of several antibody deficiencies associated with CRS, including common variable immunodeficiency (CVID), selective IgA deficiency, IgG subclass deficiency, and specific antibody deficiency (SAD). The diagnosis of antibody deficiency in patients with CRS is important because of the large clinical implications it can have on sinus disease management. CVID is treated with immunoglobulin replacement, whereas SAD may be managed symptomatically and sometimes with prophylactic antibiotics and/or immunoglobulin replacement.