STAT4 Phosphorylation of T-helper Cells predicts surgical outcomes in Refractory Chronic Rhinosinusitis.

Objective: Chronic rhinosinusitis (CRS) impacts an estimated 5% to 15% of people worldwide, incurring significant economic healthcare burden. There is a urgent need for the discovery of predictive biomarkers to improve treatment strategies and outcomes for CRS patients. Study design: Cohort study of CRS patients and healthy controls using blood samples. Setting: Out-patient clinics. Methods: Whole blood samples were collected for flow cytometric analysis. Mechanistic studies involved the transfection of human primary T cells and Jurkat cells. Results: Our analysis began with a 63-69 year-old female patient diagnosed with refractory CRS,. Despite undergoing multiple surgeries, she continually faced sinus infections. Whole exome sequencing pinpointed a heterozygous IL-12Rb1 mutation situated in the linker region adjacent to the cytokine binding domain. When subjected to IL-12 stimulation, the patient's CD4 T-cells exhibited diminished STAT4 phosphorylation. However, computer modeling or T-cell lines harboring the same IL-12 receptor mutation did not corroborate the hypothesis that IL-12Rb could be responsible for the reduced phosphorylation of STAT4 by IL-12 stimulation. Upon expanding our investigation to a broader CRS patient group using the pSTAT4 assay, we discerned a subset of refractory CRS patients with abnormally low STAT4 phosphorylation. The deficiency showed improvement both in-vitro and in-vivo after exposure to Latilactobacillus sakei (aka Lactobacillus sakei), an effect at least partially dependent on IL-12. Conclusion: In refractory CRS patients, an identified STAT4 defect correlates with poor clinical outcomes after sinus surgery, which can be therapeutically targeted by Latilactobacillus sakei treatment. Prospective double-blind placebo-controlled trials are needed to validate our findings.

Mammalian Target of Rapamycin Pathway Assessment in Antiphospholipid Antibody-Positive Patients with Livedo.

OBJECTIVE: In antiphospholipid antibody (aPL) nephropathy, activation of the mammalian target of rapamycin (mTOR) contributes to endothelial cell proliferation, a key finding of aPL microvascular disease. Here, we examined mTOR activation in the skin of aPL-positive patients with livedo. METHODS: Three patient groups with livedo were studied: (1) persistently aPL-positive with systemic lupus erythematosus (SLE); (2) persistently aPL-positive without SLE; and (3) aPL-negative SLE (control). After collecting aPL-related medical history, two 5-mm skin biopsies of livedo were performed on each patient: (1) peripheral (erythematous-violaceous lesion); and (2) central (nonviolaceous area). We stained specimens for phosphorylated protein kinase B (p-AKT) and phosphorylated S6 ribosomal protein (p-S6RP) as mTOR activity markers, CD31 to identify endothelial cells, and Ki-67 to show cellular proliferation. We counted cells in the epidermis and compared mTOR-positive cell counts between peripheral and central samples, and between patient groups, using Freidman test and Wilcoxon signed-rank test. RESULTS: Ten patients with livedo reticularis were enrolled: 4 aPL-positive without SLE (antiphospholipid syndrome [APS] classification met, n = 3), 4 aPL-positive SLE (APS classification met, n = 3), and 2 aPL-negative SLE (control). In all aPL-positive patients, epidermal p-AKT and p-S6RP staining were significantly increased in both peripheral and central skin samples when compared to aPL-negative SLE controls; both were more pronounced in the lower basal layers of epidermis. CONCLUSION: Our study demonstrates increased mTOR activity in livedoid lesions of aPL-positive patients with or without SLE compared to aPL-negative patients with SLE, with more prominent activity in the lower basal layers of the epidermis. These findings may serve as a basis for further investigating the mTOR pathway in aPL-positive patients.

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.

Basophil activation test: Mechanisms and considerations for use in clinical trials and clinical practice.

The basophil activation test (BAT) is a functional assay that measures the degree of degranulation following stimulation with allergen or controls by flow cytometry. It correlates directly with histamine release. From the dose-response curve resulting from BAT in allergic patients, basophil reactivity (%CD63+ basophils) and basophil sensitivity (EC50 or similar) are the main outcomes of the test. BAT takes into account all characteristics of IgE and allergen and thus can be more specific than sensitization tests in the diagnosis of allergic disease. BAT reduces the need for in vivo procedures, such as intradermal tests and allergen challenges, which can cause allergic reactions of unpredictable severity. As it closely reflects the patients' phenotype in most cases, it may be used to support the diagnosis of food, venom and drug allergies and chronic urticaria, to monitor the natural resolution of food allergies and to predict and monitor clinical the response to immunomodulatory treatments, such as allergen-specific immunotherapy and biologicals. Clinical application of BAT requires analytical validation, clinical validation, standardization of procedures and quality assurance to ensure reproducibility and reliability of results. Currently, efforts are ongoing to establish a platform that could be used by laboratories in Europe and in the USA for quality assurance and certification.

Enzyme replacement therapy reverses B lymphocyte and dendritic cell dysregulations in patients with Gaucher Disease.

Gaucher disease (GD) is caused by mutations in the GBA gene encoding lysosomal enzyme, ß-glucocerebrosidase (GCase). GCase deficiency results in accumulation of its substrates in cells of macrophage lineage, affecting multiple organ systems. Enzyme replacement therapy (ERT) with recombinant human GCase is the standard of care to treat GD. In GD, it is well established that there are immune alterations, clinically presenting as lymphadenopathy, gammopathies, and predisposition to hematological cancers. We examined the effect of ERT on immune dysregulations in treatment-naïve GD patients longitudinally after the initiation of ERT. Immunophenotyping was performed in peripheral blood samples obtained before and after ERT. T and B lymphocyte subsets, NK, NKT and dendritic cells were evaluated. In all treatment naïve patients at baseline, transitional B cells, characterized by CD21low expression were markedly elevated. After establishment of stable-dose therapy, CD21low cells were significantly reduced and subsequent increase in CD21Hi B lymphocytes indicated improved B cell maturation. Class-switching and memory B cell defects which were noted prior to treatment were found to be normalized. An increase in dendritic cells also resulted after the treatment. Our data shows that GD affects across various immune cell types and ERT or its effects directly improve affected immunological parameters.

Persistent immune alterations and comorbidities in splenectomized patients with Gaucher disease.

Gaucher disease (GD) is an autosomal recessive disorder caused by mutations in the gene encoding acid-ß-glucosidase, resulting in functional disruptions in degradation of glycosphingolipids and lysosomal accumulation of the substrates. The most frequent clinical presentations of GD are thrombocytopenia, splenomegaly and bone pain. Prior to advent of enzyme replacement therapy, splenectomy was performed for complications of hypersplenism such as severe thrombocytopenia and transfusion dependency. Though there is evidence about worsening bone disease after splenectomy, there is no systematic study to assess its effects on the immune system in GD patients. In order to investigate the long-term immunological effects of splenectomy, we used flow cytometry to compare the immunophenotypes of GD patients who had undergone splenectomy (SGD) to those with intact spleen. The results show that SGD patients have significantly fewer CD27(+)/IgM(+) B-cells but more CD4(+)/CD45RO(+) and CD8(+)/CD45RO(+) T-cells. The most surprising finding was an almost complete absence of circulating dendritic cells in SGD patients. In addition, splenectomized subjects had comorbidities, the most common being monoclonal gammopathy of undetermined significance (MGUS). Taken together, these results highlight the persistence of multiple immune alterations and comorbidities coexisting in higher frequency in the SGD group and they are not affected by GD specific therapy.

Moderate dose inhaled corticosteroid-induced symptomatic adrenal suppression: case report and review of the literature.

Inhaled corticosteroids (ICS) are drugs of choice for persistent asthma. Less than 500 µg/d of fluticasone are believed to be safe. We found 92 cases of adrenal suppression in PubMed; among these cases there were 13 children who took 500 µg/d or less of fluticasone. Adrenal insufficiency was diagnosed in a 7-year-old boy on 460 µg ICS for 16 months, with a diagnosis of chronic persistent asthma. A random cortisol was nondetectable as was an early morning cortisol. ICS have greatly improved the day-to-day lives of children with chronic persistent asthma. Parents of children younger than 12 years, who use at least 400 µg of inhaled fluticasone (or bioequivalent), must be given oral and written instructions about warning symptoms of hypocortisolism. Major stress such as surgery, gastrointestinal, bronchopulmonary, or other systemic infections, and heat stress may mandate a written plan of action for use by hospital physicians.