Biologic Therapy in Rare Eosinophil-Associated Disorders: Remaining Questions and Translational Research Opportunities.

Rare eosinophil-associated disorders (EADs), including hypereosinophilic syndrome, eosinophilic granulomatosis with polyangiitis and eosinophilic gastrointestinal disorders, are a heterogeneous group of conditions characterized by blood and/or tissue hypereosinophilia and eosinophil-related clinical manifestations. Although the recent availability of biologic therapies that directly and indirectly target eosinophils has the potential to dramatically improve treatment options for all EADs, clinical trials addressing their safety and efficacy in rare EADs have been relatively few. Consequently, patient access to therapy is limited for many biologics, and the establishment of evidence-based treatment guidelines has been extremely difficult. In this regard, multicenter retrospective collaborative studies focusing on disease manifestations and treatment responses in rare EADs have provided invaluable data for physicians managing patients with these conditions and helped identify important questions for future translational research. During the Clinical Pre-Meeting Workshop held in association with the July 2023 biennial meeting of the International Eosinophil Society in Hamilton, Ontario, Canada, the successes and limitations of pivotal multicenter retrospective studies in EADs were summarized, and unmet needs regarding the establishment of guidelines for use of biologics in rare EADs were discussed. Key topics of interest included: 1) clinical outcome measures, 2) minimally invasive biomarkers of disease activity, 3) predictors of response to biologic agents, and 4) long-term safety of eosinophil depletion. Herein, we report a summary of these discussions, presenting a state-of-the-art overview of data currently available for each of these topics, the limitations of the data, and avenues for future data generation through implementation of multidisciplinary and multicenter studies.

Standardized Quantification of Mast Cells in the Gastrointestinal Tract in Adults.

BACKGROUND: Current data on the normal quantity of mast cells throughout the adult gastrointestinal tract are limited in several domains. These include microanatomic localization of mast cells, standardization of staining and counting methods, and reporting of microscope field of view. OBJECTIVE: To address this lack of reliable reference ranges to facilitate the study of and diagnosis of emerging mast cell-mediated diseases. METHODS: We examined biopsies obtained from the esophagus, stomach, duodenum, and colon from an unselected cohort. Mean and peak mast cell density were determined on slides stained for tryptase and CD117, and were expressed per high power field (hpf) and surface area (mm2), thus deriving reference ranges (average ± 2 SDs). RESULTS: For the most common hpf surface area (0.238 mm2), upper limits of the derived reference ranges for average/peak mast cells were 0.15/3.67 (esophagus, tryptase), 0.70/5.98 (esophagus, CD117), 22.56/35.30 (stomach, tryptase), 31.32/53.10 (stomach, CD117), 30.28/49.77 (duodenal crypts, tryptase), 41.96/65.26 (duodenal crypts, CD117), 4.98/11.56 (duodenal villi, tryptase), 8.38/14.17 (duodenal villi, CD117), 26.58/41.08 (colon, tryptase), and 35.57/57.92 (colon, CD117). Interobserver variability was moderate to good. There was significant correlation between average and peak mast cell counts. CONCLUSIONS: These data help standardize mast cell reference ranges throughout the gastrointestinal tract in adults, which can be used to determine whether abnormal levels of mast cells are present in patients with suspected mast cell-mediated disease. Our data show that the commonly used cutoff of 20 mast cells per hpf irrespective of the gastrointestinal tract segment is an underestimate of an appropriate cutoff in stomach, duodenum (crypt area), and colon.

Breakthroughs in understanding and treating eosinophilic gastrointestinal diseases presented at the CEGIR/TIGERs Symposium at the 2022 American Academy of Allergy, Asthma & Immunology Meeting.

The Consortium of Eosinophilic Gastrointestinal Diseases and The International Gastrointestinal Eosinophil Researchers organized a day-long symposium at the 2022 Annual Meeting of the American Academy of Allergy, Asthma & Immunology. The symposium featured a review of recent discoveries in the basic biology and pathogenesis of eosinophilic gastrointestinal diseases (EGIDs) in addition to advances in our understanding of the clinical features of EGIDs. Diagnostic and management approaches were reviewed and debated, and clinical trials of emerging therapies were highlighted. Herein, we briefly summarize the breakthrough discoveries in EGIDs.

Detection of Eosinophils in Tissue Sections by Immunohistochemistry.

Eosinophils are bone marrow-derived hematopoietic cells which represent a small subset in the peripheral blood, and under homeostatic conditions predominantly reside in certain organs, such as the gastrointestinal tract. However, eosinophil numbers increase both in the peripheral blood and tissues during allergic inflammation, parasitic infestation, drug reactions, vasculitides, as well as certain hematopoietic neoplasms. Their presence in tissues can be detected by hematoxylin and eosin staining; however, this may be challenging particularly at times of activation and/or degranulation, e.g., during allergic lung inflammation. Thus, detection of eosinophils and/or their released granule proteins is significantly enhanced by immunohistochemistry. This chapter describes methods for the detection of mouse or human eosinophils by using granule protein-specific antibodies in formalin-fixed paraffin-embedded tissue.

Targeting Eosinophils in Mouse Models of Asthma.

Eosinophils are bone marrow-derived hematopoietic cells that accumulate significantly in the lungs and bronchoalveolar lavage fluid in patients with asthma and models of allergic airway inflammation. Their role in the pathophysiology of asthma and other diseases can be studied using mouse models in which eosinophils are depleted. This review article focuses on two main approaches for depleting eosinophils in vivo, antibody-mediated and genetic models. Specific antibodies and genetic models are reviewed, along with their strengths and weaknesses.

Desmoplakin and periplakin genetically and functionally contribute to eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is a chronic allergic inflammatory disease with a complex underlying genetic etiology. Herein, we conduct whole-exome sequencing of a multigeneration EoE pedigree (discovery set) and 61 additional multiplex families with EoE (replication set). A series of rare, heterozygous, missense variants are identified in the genes encoding the desmosome-associated proteins DSP and PPL in 21% of the multiplex families. Esophageal biopsies from patients with these variants retain dilated intercellular spaces and decrease DSP and PPL expression even during disease remission. These variants affect barrier integrity, cell motility and RhoGTPase activity in esophageal epithelial cells and have increased susceptibility to calpain-14-mediated degradation. An acquired loss of esophageal DSP and PPL is present in non-familial EoE. Taken together, herein, we uncover a pathogenic role for desmosomal dysfunction in EoE, providing a deeper mechanistic understanding of tissue-specific allergic responses.

Developing a standardized approach for assessing mast cells and eosinophils on tissue biopsies: A Work Group Report of the AAAAI Allergic Skin Diseases Committee.

Mast cells and eosinophils are commonly found, expectedly or unexpectedly, in human tissue biopsies. Although the clinical significance of their presence, absence, quantity, and quality continues to be investigated in homeostasis and disease, there are currently gaps in knowledge related to what constitutes quantitatively relevant increases in mast cell and eosinophil number in tissue specimens for several clinical conditions. Diagnostically relevant thresholds of mast cell and eosinophil numbers have been proposed and generally accepted by the medical community for a few conditions, such as systemic mastocytosis and eosinophilic esophagitis. However, for other mast cell- and eosinophil-associated disorders, broad discrepancies remain regarding diagnostic thresholds and how samples are processed, routinely and/or specially stained, and interpreted and/or reported by pathologists. These discrepancies can obfuscate or delay a patient's correct diagnosis. Therefore, a work group was assembled to review the literature and develop a standardized consensus for assessing the presence of mast cells and eosinophils for a spectrum of clinical conditions, including systemic mastocytosis and cutaneous mastocytosis, mast cell activation syndrome, eosinophilic esophagitis, eosinophilic gastritis/enteritis, and hypereosinophilia/hypereosinophilic syndrome. The intent of this work group is to build a consensus among pathology, allergy, dermatology, hematology/oncology, and gastroenterology stakeholders for qualitatively and quantitatively assessing mast cells and eosinophils in skin, gastrointestinal, and bone marrow pathologic specimens for the benefit of clinical practice and patients.

Myeloid neoplasm with a novel cryptic PDGFRB rearrangement detected by next-generation sequencing.

Rearrangements of PDGFRB are defining cytogenetic abnormalities seen in "Myeloid/lymphoid neoplasms with eosinophilia and rearrangement of PDGFRB" and are generally evident by common cytogenetic methods. Here we present an unique case in which karyotyping and fluorescence in situ hybridization (FISH) analysis were negative, and the PDGFRB rearrangement was detected by next-generation sequencing (NGS) analysis. The patient presented with approximately one-year history of leukocytosis including neutrophilia, eosinophilia, basophilia and granulocytic left shift. Bone marrow biopsy revealed a hypercellular marrow with panmyelosis, eosinophilia and mast cell hyperplasia. Blasts were not increased. Ancillary studies revealed a normal karyotype and absence of BCR-ABL1 fusion gene. NGS identified AFAP1L1-PDGFRB fusion, which was confirmed by polymerase chain reaction amplification followed by direct Sanger sequencing. The patient was treated with imatinib and showed normalization of peripheral blood leukocytosis, which lasted for at least six months. This case highlights that cytogenetics/FISH study alone may be insufficient to detect all PDGFRB rearrangement, which is critical for the patient's management. We suggest that molecular analysis capable of detecting fusion genes should be performed in all similar cases.

Heart disease in a mutant mouse model of spontaneous eosinophilic myocarditis maps to three loci.

BACKGROUND: Heart disease (HD) is the major cause of morbidity and mortality in patients with hypereosinophilic diseases. Due to a lack of adequate animal models, our understanding of the pathophysiology of eosinophil-mediated diseases with heart complications is limited. We have discovered a mouse mutant, now maintained on an A/J inbred background, that spontaneously develops hypereosinophilia in multiple organs. Cellular infiltration into the heart causes an eosinophilic myocarditis, with affected mice of the mutant line (i.e., A/JHD) demonstrating extensive myocardial damage and remodeling that leads to HD and premature death, usually by 15-weeks old. RESULTS: Maintaining the A/JHD line for many generations established that the HD trait was heritable and implied the mode of inheritance was not too complex. Backcross and intercross populations generated from mating A/JHD males with females from four different inbred strains produced recombinant populations with highly variable rates of affected offspring, ranging from none in C57BL/6 J intercrosses, to a few mice with HD using 129S1/SvImJ intercrosses and C57BL/6 J backcrosses, but nearly 8% of intercrosses and > 17% of backcrosses from SJL/J related populations developed HD. Linkage analyses of these SJL/J derived recombinants identified three highly significant loci: a recessive locus mapping to distal chromosome 5 (LOD = 4.88; named Emhd1 for eosinophilic myocarditis to heart disease-1); and two dominant variants mapping to chromosome 17, one (Emhd2; LOD = 7.51) proximal to the major histocompatibility complex, and a second (Emhd3; LOD = 6.89) that includes the major histocompatibility region. Haplotype analysis identified the specific crossovers that defined the Emhd1 (2.65 Mb), Emhd2 (8.46 Mb) and Emhd3 (14.59 Mb) intervals. CONCLUSIONS: These results indicate the HD trait in this mutant mouse model of eosinophilic myocarditis is oligogenic with variable penetrance, due to multiple segregating variants and possibly additional genetic or nongenetic factors. The A/JHD mouse model represents a unique and valuable resource to understand the interplay of causal factors that underlie the pathology of this newly discovered eosinophil-associated disease with cardiac complications.

Characterization of a mouse model of hypereosinophilia-associated heart disease.

Hypereosinophilic syndrome is characterized by sustained and marked eosinophilia leading to tissue damage and organ dysfunction. Morbidity and mortality occur primarily due to cardiac and thromboembolic complications. Understanding the cause and mechanism of disease would aid in the development of targeted therapies with greater efficacy and fewer side effects. We discovered a spontaneous mouse mutant in our colony with a hypereosinophilic phenotype. Mice develop peripheral blood eosinophilia; infiltration of lungs, spleen, and heart by eosinophils; and extensive myocardial damage and remodeling. This ultimately leads to heart failure and premature death. Histopathological assessment of the hearts revealed a robust inflammatory infiltrate composed primarily of eosinophils and B-lymphocytes, associated with myocardial damage and replacement fibrosis, consistent with eosinophilic myocarditis. In many cases, hearts showed dilatation and thinning of the right ventricular wall, suggestive of an inflammatory dilated cardiomyopathy. Most mice showed atrial thrombi, which often filled the chamber. Protein expression analysis revealed overexpression of chemokines and cytokines involved in innate and adaptive immunity including IL-4, eotaxin, and RANTES. Disease could be transferred to wild-type mice by adoptive transfer of splenocytes from affected mice, suggesting a role for the immune system. In summary, the pathologies observed in the mutant lines are reminiscent of those seen in patients with hypereosinophilia, where cardiac-related morbidities, like congestive heart failure and thrombi, are the most common causes of death. As such, our model provides an opportunity to test mechanistic hypotheses and develop targeted therapies.NEW & NOTEWORTHY This article describes a new model of heart disease in hypereosinophilia. The model developed as a spontaneous mouse mutant in the colony and is characterized by peripheral blood eosinophilia and infiltration of lungs, spleen, and heart by eosinophils. In the heart, there is extensive myocardial damage, remodeling, fibrosis, and thrombosis, leading to heart failure and death. The immune microenvironment is one of increased innate and adaptive immunity, including Th1 and Th2 cytokines/chemokines. Finally, adoptive transfer of splenocytes transfers disease to recipient mice. In summary, this model provides an opportunity to test mechanistic hypotheses and develop targeted therapies for this rare but devastating disease.

Sialic acid-binding immunoglobulin-like lectin (Siglec) 8 in patients with eosinophilic disorders: Receptor expression and targeting using chimeric antibodies.

BACKGROUND: Sialic acid-binding immunoglobulin-like lectin (Siglec) 8 is selectively expressed on eosinophils, mast cells, and basophils and, when engaged on eosinophils, can cause cell death. OBJECTIVE: We sought to characterize surface and soluble Siglec-8 (sSiglec-8) levels in normal donors (NDs) and eosinophilic donors (EOs) and assess the efficacy of anti-Siglec-8 antibodies in inducing eosinophil cell death in vitro. METHODS: Eosinophil expression of Siglec-8 was assessed by using flow cytometry and quantitative PCR. Serum sSiglec-8 levels were measured by means of ELISA. Induction of eosinophil death by IgG4 (chimeric 2E2 IgG4) and afucosylated IgG1 (chimeric 2E2 IgG1 [c2E2 IgG1]) anti-Siglec-8 antibodies was evaluated in vitro by using flow cytometry and in vivo in humanized mice. RESULTS: Siglec-8 was consistently expressed on eosinophils from NDs and EOs and did not correlate with absolute eosinophil count or disease activity. sSiglec-8 levels were measurable in sera from most donors unrelated to absolute eosinophil counts or Siglec-8 surface expression. c2E2 IgG1 and chimeric 2E2 IgG4 were equally effective at inducing cell death (Annexin-V positivity) of purified eosinophils from NDs and EOs after overnight IL-5 priming. In contrast, killing of purified eosinophils without IL-5 was only seen in EOs, and natural killer cell-mediated eosinophil killing was seen only with c2E2 IgG1. Finally, treatment of humanized mice with anti-Siglec antibody led to robust depletion of IL-5-induced eosinophilia in vivo. CONCLUSIONS: Siglec-8 is highly expressed on blood eosinophils from EOs and NDs and represents a potential therapeutic target for eosinophilic disorders. Enhanced killing of eosinophils in the presence of IL-5 might lead to increased efficacy in patients with IL-5-driven eosinophilia.

Previously Unreported Complication of Coronary Artery Bypass Grafting: Suture Rupture Resulting in Patient's Death.

Traditionally, the manner of death in most hospital autopsy cases is natural, in which death is due to the natural course of disease or reasonably anticipated outcomes of medical interventions. Some cases fall into a potential gray zone between natural and accident, including rare or unanticipated outcomes of medical interventions. We present a case of a patient postcoronary artery bypass graft. Autopsy revealed the proximal anastomosis of the aorta-to-first-diagonal-coronary-artery-to-second-obtuse-marginal-artery graft was detached from the aorta. A broken suture was present at the disconnected anastomosis, with intact knots but was broken along its length. In-hospital mortality rates of CABG range from 1% to 3%, with several autopsy studies identifying surgical complications as the cause of death in one-third of perioperative deaths. No publications were found that described suture rupture as directly relating to the cause of death. This case report describes a previously unreported complication of coronary artery bypass grafting.

Eosinophilic oesophagitis endotype classification by molecular, clinical, and histopathological analyses: a cross-sectional study.

BACKGROUND: Eosinophilic oesophagitis is understood in terms of quantifiable histological, endoscopic, and molecular features. Data are scant for inter-relations of these features and their potential to identify distinct disease endotypes. We aimed to identify clinical-pathological correlations between endoscopic and histological disease variables by transcription profiling of the oesophagus of patients with eosinophilic oesophagitis of varying severity and disease activity states. METHODS: We did a cross-sectional study across ten hospital sites in the USA associated with the Consortium of Eosinophilic Gastrointestinal Disease Researchers. We analysed oesophageal biopsy specimens taken from paediatric and adult patients with eosinophilic oesophagitis (discovery cohort), using the eosinophilic oesophagitis diagnostic panel (EDP), a set of 96 informative transcripts. Histological and endoscopic features were assessed by quantification of oesophageal eosinophils and use of the eosinophilic oesophagitis histology scoring system (HSS) and the eosinophilic oesophagitis endoscopic reference score (EREFS). Associations among the various histological, endoscopic, and molecular features were analysed by Spearman correlation. Results were replicated in a biologically independent, single-centre, validation cohort of patients with active eosinophilic oesophagitis. FINDINGS: The discovery cohort contained 185 samples and the validation cohort comprised 100 specimens. In the discovery cohort, EDP showed intersite consistency, significant correlation with oesophageal eosinophils (p<0·0001), and similar findings between paediatric and adult patients. Of eight HSS domains, basal zone hyperplasia correlated with the EDP (median Spearman ρ 0·47 [IQR 0·36-0·60]). Of five EREFS features, distal furrows correlated with the EDP (median Spearman ρ 0·42 [0·32-0·50]). By analysing active eosinophilic oesophagitis in the discovery cohort, the EDP identified three clusters associated with distinct endotypes (termed EoEe1-3) despite similar eosinophil levels. EoEe1 was associated with a normal-appearing oesophagus (risk ratio [RR] 3·27, 95% CI 1·04-10·27; p=0·0443), an inverse association with a history of oesophageal dilation (0·27, 0·09-0·82; p=0·0105) and showed relatively mild histological, endoscopic, and molecular changes. EoEe2 showed an inflammatory and steroid-refractory phenotype (RR 2·77, 95% CI 1·11-6·95; p=0·0376) and had the highest expression of inflammatory cytokines and steroid-responding genes. EoEe3 was associated with a narrow-calibre oesophagus (RR 7·98, 95% CI 1·84-34·64; p=0·0013) and adult onset (2·22, 1·19-4·12; p=0·0155), and showed the highest degree of endoscopic and histological severity and the lowest expression of epithelial differentiation genes. These endotypes were replicated in the validation cohort by clustering and with an eosinophilic oesophagitis endotype-prediction algorithm. INTERPRETATION: Our new disease classification stratifies patients with eosinophilic oesophagitis into subgroups with potential clinical and therapeutic significance and provides a framework for a precision medicine approach to eosinophilic oesophagitis. FUNDING: National Institutes of Health.

Hypereosinophilic syndrome in the differential diagnosis of pulmonary infiltrates with eosinophilia.

OBJECTIVE: To describe key diagnostic considerations in patients with pulmonary infiltrates with eosinophilia, with a special emphasis on raising awareness of hypereosinophilic syndrome (HES), a disease that often involves the lungs and prompts investigation for clonal neoplastic processes that determine prognosis and treatment. DATA SOURCES: Studies and review articles were selected from PubMed and Scopus for relevance to pertinent topics. STUDY SELECTIONS: The literature was screened for studies that described lung eosinophilia and HES. Studies relevant to the topic were included in this review. RESULTS: Pulmonary eosinophil infiltrates in lung biopsy specimens present a broad differential diagnosis, including eosinophilic pneumonia; hypersensitivity reactions, such as allergic bronchopulmonary fungal disease; and pulmonary manifestations of systemic diseases, such as eosinophilic granulomatosis with polyangiitis. An additional important consideration in the differential diagnosis is pulmonary involvement by HES. HES is a rare syndrome that comprises a heterogeneous group of conditions characterized by persistent blood and/or tissue eosinophilia associated with organ dysfunction. Approximately one-third of HES cases are caused by neoplastic diseases, with the remaining cases classified as reactive or idiopathic. Lung involvement is seen in up to 67% of cases and may be the presenting manifestation of the disorder. CONCLUSION: The differential diagnosis of pulmonary eosinophilia is broad and requires a multidisciplinary approach with clinicopathologic-radiologic correlation.

Perinatal Case of Fatal Simpson-Golabi-Behmel Syndrome with Hyperplasia of Seminiferous Tubules.

BACKGROUND Simpson-Golabi-Behmel syndrome (SGBS) is a rare X-linked recessive syndrome characterized by fetal overgrowth. CASE REPORT We present a case of a male infant with SGBS. Abnormal prenatal ultrasound (including congenital diaphragmatic hernia) prompted microarray testing of amniotic fluid cells, which showed deletion on chromosome Xq26.2 affecting the glypican-3 gene consistent with SGBS type I. The infant died six hours after birth and at autopsy showed features of SGBS, including macrosomia, organomegaly, diaphragmatic hernia with consequent pulmonary hypoplasia, cleft palate, large tongue with a midline groove, a supernumerary nipple, Meckel's diverticulum, and abnormal phalanges. Additionally, we observed features that have previously not been described in SGBS, including testes with hyperplastic seminiferous tubules and Mullerian remnants, and placenta with incipient fetal thrombotic vasculopathy. CONCLUSIONS While most patients with SGBS type I survive into childhood or even adulthood, the severe course in our patient was ascribed to pulmonary hypoplasia secondary to the bilateral diaphragmatic hernia.

Regulation of Siglec-8-induced intracellular reactive oxygen species production and eosinophil cell death by Src family kinases.

RATIONALE: Siglec-8 is a surface receptor predominantly expressed on human eosinophils where its ligation induces reactive oxygen species (ROS) formation and cell death. Since Siglec-8 has intracellular tyrosine-based motifs, we hypothesized that Src family kinases (SFKs) are involved in ROS formation and cell death induced by Siglec-8 engagement. METHODS: Human peripheral blood eosinophils were purified and incubated with anti-Siglec-8 monoclonal antibodies (mAb, agonist), IL-5, and SFK pharmacological inhibitors. We focused on Siglec-8-induced cell death in short-term IL-5-activated cells leading to a regulated necrosis-type cell death. ROS production was determined by dihydrorhodamine (DHR) 123 labeling and flow cytometry, or by chemiluminescence using Amplex red. Activation of SFK was determined using phospholuminex and Western blotting. RESULTS: In order to determine cellular localization of ROS production, we measured intra and extracellular ROS. While an ETosis stimulus (calcium ionophore A23187) led to extracellular ROS (ecROS) production, Siglec-8-engagement in short-term IL-5 activated cells led to intracellular ROS (icROS) accumulation. Consistently, inhibition of extracellular ROS by catalase inhibited ETosis, but not IL-5-primed Siglec-8-induced cell death. In order to determine signaling events for Siglec-8, we performed Western blotting and found SFK phosphorylation in lysates from eosinophils stimulated with anti-Siglec-8 mAb±IL-5. In order to identify which SFKs were involved, we used the phospholuminex assay and found increased levels of phosphorylated Fgr in the cytoplasmic fraction of cells co-stimulated with anti-Siglec-8 and IL-5 for 3 hours compared with cells stimulated with IL-5 alone. To test the involvement of SFKs in ROS production and cell death, we used SFK inhibitors PP2 and dasatinib, both of which completely inhibited eosinophil ROS production and cell death induced by anti-Siglec-8 and IL-5 co-stimulation. CONCLUSION: Siglec-8 engagement in short-term IL-5-activated eosinophils causes icROS production and SKF phosphorylation, and both are essential in mediating Siglec-8-induced cell death.

Cyclophilin D regulates necrosis, but not apoptosis, of murine eosinophils.

Eosinophil degranulation and clusters of free extracellular granules are frequently observed in diverse diseases, including atopic dermatitis, nasal polyposis, and eosinophilic esophagitis. Whether these intact granules are released by necrosis or a biochemically mediated cytolysis remains unknown. Recently, a peptidyl-prolyl isomerase located within the mitochondrial matrix, cyclophilin D (PPIF), was shown to regulate necrotic, but not apoptotic, cell death in vitro in fibroblasts, hepatocytes, and cardiomyocytes. Whether cyclophilin D regulates necrosis in hematopoietic cells such as eosinophils remains unknown. We used PPIF-deficient (Ppif(-/-)) mice to test whether cyclophilin D is required for regulating eosinophil necrosis. PPIF deficiency did not affect eosinophil development or maturation at baseline. After in vitro ionomycin or H2O2 treatment, Ppif(-/-) eosinophils were significantly protected from Ca(2+) overload- or oxidative stress-induced necrosis. Additionally, Ppif(-/-) eosinophils demonstrated significantly decreased necrosis, but not apoptosis, in response to Siglec-F cross-linking, a stimulus associated with eosinophil-mediated processes in vitro and in vivo. When treated with apoptosis inducers, Ppif(+/+) and Ppif(-/-) eosinophils exhibited no significant difference in apoptosis or secondary necrosis. Finally, in a dextran sodium sulfate-induced colitis model, although levels of colitogenic cytokines and eosinophil-selective chemokines were comparable between Ppif(+/+) and Ppif(-/-) mice, the latter exhibited decreased clinical outcomes. This correlated with significantly reduced eosinophil cytolysis in the colon. Collectively, our present studies demonstrate that murine eosinophil necrosis is regulated in vitro and in vivo by cyclophilin D, at least in part, thus providing new insight into the mechanism of eosinophil necrosis and release of free extracellular granules in eosinophil-associated diseases.

Utility of frozen section analysis for fungal organisms in soft tissue wound debridement margin determination.

BACKGROUND: Zygomycetes cause different patterns of infection in immunosuppressed individuals, including sino-orbito-cerebral, pulmonary, skin/soft tissue infection and disseminated disease. Infections with Zygomycetes have a high mortality rate, even with prompt treatment, which includes anti-fungal agents and surgical debridement. In some centers, clear margins are monitored by serial frozen sections, but there are no specific guidelines for the use of frozen sections during surgical debridement. Studies in fungal rhinosinusitis found 62.5-85 % sensitivity of frozen section analysis in margin assessment. However, the utility of frozen section analysis for margin evaluation in debridement of skin/soft tissue infection has not been published. METHODS: We present a case of zygomycosis of decubitus ulcers in which we assessed statistical measures of performance of frozen section analysis for presence of fungal organisms on the margin, compared with formalin-fixed paraffin embedded (FFPE) sections as gold standard. A total of 33 specimens (94 blocks) were sectioned, stained with H&E and evaluated by both frozen and FFPE analysis. Negative interpretations were confirmed by Gomori methenamine silver stain on FFPE sections. RESULTS: H&E staining of frozen sections had 68.4 % sensitivity and 100 % specificity for assessing margins clear of fungal organisms. The negative and positive predictive values were 70.0 % and 100 %, respectively. Using presence of acute inflammation and necrosis as markers of fungal infection improved sensitivity (100 %) at the expense of specificity (42.9 %). CONCLUSION: Use of intraoperative assessment of skin and soft tissue margins for fungal infection is a valuable tool in the management of skin and soft tissue fungal infection treatment.