Human germline heterozygous gain-of-function STAT6 variants cause severe allergic disease.

STAT6 (signal transducer and activator of transcription 6) is a transcription factor that plays a central role in the pathophysiology of allergic inflammation. We have identified 16 patients from 10 families spanning three continents with a profound phenotype of early-life onset allergic immune dysregulation, widespread treatment-resistant atopic dermatitis, hypereosinophilia with esosinophilic gastrointestinal disease, asthma, elevated serum IgE, IgE-mediated food allergies, and anaphylaxis. The cases were either sporadic (seven kindreds) or followed an autosomal dominant inheritance pattern (three kindreds). All patients carried monoallelic rare variants in STAT6 and functional studies established their gain-of-function (GOF) phenotype with sustained STAT6 phosphorylation, increased STAT6 target gene expression, and TH2 skewing. Precision treatment with the anti-IL-4Rα antibody, dupilumab, was highly effective improving both clinical manifestations and immunological biomarkers. This study identifies heterozygous GOF variants in STAT6 as a novel autosomal dominant allergic disorder. We anticipate that our discovery of multiple kindreds with germline STAT6 GOF variants will facilitate the recognition of more affected individuals and the full definition of this new primary atopic disorder.

Non-targeted detection of food adulteration using an ensemble machine-learning model.

Recurrent incidents of economically motivated adulteration have long-lasting and devastating effects on public health, economy, and society. With the current food authentication methods being target-oriented, the lack of an effective methodology to detect unencountered adulterants can lead to the next melamine-like outbreak. In this study, an ensemble machine-learning model that can help detect unprecedented adulteration without looking for specific substances, that is, in a non-targeted approach, is proposed. Using raw milk as an example, the proposed model achieved an accuracy and F1 score of 0.9924 and 0. 0.9913, respectively, when the same type of adulterants was presented in the training data. Cross-validation with spiked contaminants not routinely tested in the food industry and blinded from the training data provided an F1 score of 0.8657. This is the first study that demonstrates the feasibility of non-targeted detection with no a priori knowledge of the presence of certain adulterants using data from standard industrial testing as input. By uncovering discriminative profiling patterns, the ensemble machine-learning model can monitor and flag suspicious samples; this technique can potentially be extended to other food commodities and thus become an important contributor to public food safety.

Human mast cells induce osteoclastogenesis through cell surface RANKL.

OBJECTIVES: We employed the co-culture of CD34+ stem cell-derived human mast cells (HMC) and human monocyte-derived osteoclast precursors to evaluate if mast cells contribute to the pathogenesis of osteoporosis through regulation of osteoclast proliferation and activation. METHODS: Mature HMC and osteoclast precursors were cultured from monocytes isolated from human buffy coat. The osteoclast precursors were incubated with HMC or receptor activator of nuclear factor kappa-B ligand (RANKL) for a week prior to determination of osteoclast maturation through characterization by their morphology and tartrate resistant acid phosphatase (TRAP) expression. The bone absorption activity was determined by pit formation on osteo-assay plate. RESULTS: Mature osteoclasts were identified following co-culture of osteoclast precursors with HMC for one week in the absence of RANKL and they were capable of bone resorption. These actions of HMC on osteoclasts were not affected by mast cell activators such anti-IgE or substance P but could be reversed by osteoprotegerin (OPG) in the co-culture system suggesting the involvement of RANKL. The expression of RANKL on the cell surface of HMC was confirmed by flow cytometry and the density was not affected by activation of HMC. CONCLUSION: Our study provided direct evidence confirming the initiation of osteoclast proliferation and activation by mast cells through cell surface RANKL suggesting that mast cells may contribute to bone destruction in pathological conditions such as osteoporosis.

Degradation of Monocyte Chemoattractant Protein-1 by Tryptase Co-Released in Immunoglobulin E-Dependent Activation of Primary Human Cultured Mast Cells.

BACKGROUND: Mast cells are key immune effector cells which release chemokines, proteases, and other inflammatory mediators upon activation by immunological stimuli. The aim of this study was to investigate the effects of co-releasing proteases on the kinetics of release of the chemokine monocyte chemoattractant protein-1 (MCP-1) in immunoglobulin E (IgE)-mediated activation of human mast cells. METHODS: Homogenous populations of mature and functional primary human mast cells were generated from CD34+ progenitors originated from buffy coats of healthy adult donors. The releases of MCP-1 from human mast cells in basal conditions and in response to FcεRI cross-linking were assessed at different time points. The effects of different types of protease inhibitors on MCP-1 release from these mast cells under stimulated or unstimulated conditions were also investigated. RESULTS: Cultured human mast cells released MCP-1 in basal conditions and its levels increased in a time-dependent manner. When stimulated by FcεRI cross-linking, the levels of MCP-1 detected in the medium gradually decreased over time after the initial peak induction. Such a decline in MCP-1 levels after IgE-dependent activation was completely prevented by pretreatment with a cocktail of protease inhibitors or the specific tryptase inhibitor APC366. CONCLUSIONS: Direct regulation of MCP-1 expression by co-release of tryptase in cultured human mast cells upon IgE-dependent activation demonstrates a role of the serglycin:serine protease axis in modulation of inflammatory reactions through proteolytic degradation of mediators such as chemokines.

Immobilized Osteopontin Enhances Adhesion but Suppresses Cytokine Release of Anti-IgE Activated Human Mast Cells.

Osteopontin (OPN) is an Arg-Gly-Asp (RGD)-containing extracellular matrix protein which is upregulated in inflamed tissues and has been reported to modulate mast cell activities in mice. Due to the known heterogeneity among mast cells of different species and the important roles of mast cells in allergic reactions, we investigated the effects of human OPN (hOPN) on human mast cell activities. Mature primary human cultured mast cells (HCMC) were derived from peripheral blood CD34+ progenitors and the modulation of their activation by soluble and plate-bound immobilized hOPN were examined by studying their release of inflammatory mediators (histamine, IL-5, IL-8, TNF-α, and VEGF) and matrix adhesion following stimulation by anti-IgE. Immobilized hOPN enhanced the adhesion, but suppressed the release of IL-5, IL-8, and TNF-α of anti-IgE-activated HCMC while soluble hOPN failed to demonstrate any significant effects. By employing cyclic RGD peptide and neutralizing antibodies against different classes of integrin and CD44, we demonstrated that the interaction of immobilized hOPN and HCMC was mediated by the RGD domain of hOPN and integrin but not CD44 on HCMC. Our results suggest that immobilized hOPN anchored to extracellular matrix can regulate adaptive immunity in humans by retaining mast cells at the site of inflammation and suppressing anti-IgE-induced cytokine release from HCMC.

Author Correction: Novel six-week protocol for generating functional human connective tissue-type (MCTC) mast cells from buffy coats.

The online version of the original article can.

Novel six-week protocol for generating functional human connective tissue-type (MCTC) mast cells from buffy coats.

OBJECTIVE: The aim of this study was to develop a novel protocol for generating large populations of fully mature and functional human mast cells (HMC) from CD34+ hematopoietic stem cells which require less culturing time than previously reported methods. METHODS: CD34+ cells isolated from fresh human buffy coats were sequentially cultured with different combinations of SCF, IL-6, IL-3, IL-9 and IL-4 under selected culturing conditions and time periods. Cells were then harvested for immunohistochemical characterization of morphological phenotypes and were functionally characterized by assessing their responses to IgE-dependent and -independent stimuli by measuring the release of inflammatory mediators and cytokines. Moreover, the pharmacological profiles of several classes of anti-inflammatory drugs in inhibiting the activation of these HMC were also characterized. RESULTS: We have developed a novel protocol that can generate large homogenous populations of mature and functional HMC in 6 weeks. These cells expressed both tryptase and chymase and were activated by anti-IgE, cationic peptides and calcium ionophores. Moreover, IgE-dependent activation of these cells was significantly inhibited by anti-inflammatory drugs. The morphological and functional characteristics of these mast cells resembled those of MCTC type or connective tissue-type HMC. DISCUSSION: Our protocol represents a novel time-saving and economical approach for generating large numbers of primary HMC for functional studies of mast cell biology and for profiling novel anti-inflammatory therapeutic agents with mast cell-inhibitory properties in humans.

Differential effects of the Toll-like receptor 2 agonists, PGN and Pam3CSK4 on anti-IgE induced human mast cell activation.

Mast cells are pivotal in the pathogenesis of allergy and inflammation. In addition to the classical IgE-dependent mechanism involving crosslinking of the high-affinity receptor for IgE (FcεRI), mast cells are also activated by Toll-like receptors (TLRs) which are at the center of innate immunity. In this study, we demonstrated that the response of LAD2 cells (a human mast cell line) to anti-IgE was altered in the presence of the TLR2 agonists peptidoglycan (PGN) and tripalmitoyl-S-glycero-Cys-(Lys)4 (Pam3CSK4). Pretreatment of PGN and Pam3CSK4 inhibited anti-IgE induced calcium mobilization and degranulation without down-regulation of FcεRI expression. Pam3CSK4 but not PGN acted in synergy with anti-IgE for IL-8 release when the TLR2 agonist was added simultaneously with anti-IgE. Studies with inhibitors of key enzymes implicated in mast cell signaling revealed that the synergistic release of IL-8 induced by Pam3CSK4 and anti-IgE involved ERK and calcineurin signaling cascades. The differential modulations of anti-IgE induced mast cell activation by PGN and Pam3CSK4 suggest that dimerization of TLR2 with TLR1 or TLR6 produced different modulating actions on FcεRI mediated human mast cell activation.

Double EGFR mutants containing rare EGFR mutant types show reduced in vitro response to gefitinib compared with common activating missense mutations.

Epidermal growth factor receptor (EGFR) mutations are common in lung adenocarcinomas, especially from nonsmoking women of Asian descent. We have previously shown EGFR mutations occur in >70% of lung adenocarcinoma from nonsmokers in our population with a complex mutational profile, including 13% of EGFR double mutations. In this study, we investigated the in vitro gefitinib response of four EGFR double mutants identified in untreated patients, including Q787R+L858R, E709A+G719C, T790M+L858R, and H870R+L858R. The phosphorylation profiles of EGFR and downstream effectors AKT, STAT3/5, and ERK1/2 were compared by immunoblot analyses among the single and double mutants transfected into H358 cells. Results showed that mutants responded to in vitro gefitinib treatment with different sensitivities. The G719C and L858R single mutants showed the highest gefitinib sensitivity compared with the corresponding coexisting single mutants E709A, Q787R, H870R, and T790M. The double mutants E709A+G719C, Q787R+L858R, and H870R+L858R showed attenuated responses to gefitinib in the EGFR and downstream effector phosphorylation profiles compared with G719C or L858R alone. T790M+L858R showed strong resistance to gefitinib. Clinically, the patient whose tumor contained H870R+L858R showed tumor stabilization by 250 mg oral gefitinib daily but cerebral metastasis developed 6 months later. Correlation with the in vitro phosphorylation profile of H870R+L858R suggested that treatment failure was probably due to inadequate suppression of EGFR signaling by the drug level attainable in the cerebrospinal fluid at the given oral dosage. Overall, the findings suggested that rare types of EGFR substitution mutations could confer relative gefitinib resistance when combined with the common activating mutants.

SRC promotes survival and invasion of lung cancers with epidermal growth factor receptor abnormalities and is a potential candidate for molecular-targeted therapy.

Molecular-targeted therapy using tyrosine kinase inhibitors against epidermal growth factor receptor (EGFR) is an effective therapy for non-small cell lung cancer that harbor EGFR mutations. This study aimed to investigate the role of Src, a close EGFR associator, as a drug target in NSCLC cells with different EGFR genomic statuses. Src inhibition was achieved using 4-(4'-Phenoxyanilino)-6,7-dimethoxyquinazolinee (SKI-1) and the specificity of action was verified by RNA interference. The results showed that SKI-1 induced significant apoptosis in a dose-dependent manner in cancer cells with high basal Src activation. Activation of FAK and p130Cas was involved in Src-mediated invasion in SKI-1-sensitive cells. SKI-1 inhibited phosphorylation of EGFR as well as EGFR downstream effectors, such as signal transducers and activators of transcription 3/5, extracellular signal-regulated kinase 1/2 and AKT in the mutant cells but not the wild-type cells. This inhibition profile of EGFR implicates that induction of apoptosis and sensitivity of mutant cells to SKI treatment is mediated by EGFR and EGFR downstream pathways. Cotreatment with SKI-1 and gefitinib enhanced apoptosis in cancer cells that contained EGFR mutation and/or amplification. SKI-1 treatment alone induced significant apoptosis in H1975 cells known to be resistant to gefitinib. Src phosphorylation was shown by immunohistochemistry in around 30% of primary lung carcinomas. In 152 adenocarcinomas studied, p-Src was associated with EGFR mutations (P = 0.029). Overall, the findings indicated that Src could be a useful target for treatment of non-small cell lung cancer. Besides EGFR genomic mutations, other forms of EGFR and related family member abnormalities such as EGFR amplification might enhance SKI sensitivity.

The EML4-ALK fusion gene is involved in various histologic types of lung cancers from nonsmokers with wild-type EGFR and KRAS.

BACKGROUND: The echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) fusion gene resulting from the chromosome inversion inv(2)(p21;p23) recently was identified in nonsmall cell lung cancer (NSCLC). The authors of this study investigated the frequency, genetic and clinicopathologic profiles of EML4-ALK in Chinese patients with NSCLC. METHODS: EML4-ALK was investigated in 266 resected primary NSCLC, including adenocarcinomas (AD), lymphoepithelioma-like carcinomas, squamous cell carcinomas, mucoepidermoid carcinomas, and adenosquamous carcinomas, by reverse transcriptase-polymerase chain reaction and was verified by sequencing. EML4-ALK protein expression was studied by immunohistochemistry. RESULTS: Thirteen tumors (4.9%) had EML4-ALK comprising 4 fusion transcript variants with fusion of the variable segments from 5' EML4 to 3' ALK and with preservation of the ALK kinase domain. The most common variant consisted of 8 tumors with variant 3 that involved EML4 exon 6. The others included 2 tumors with variant 1 (exon 13), 2 tumors with variant 2 (exon 20), and 1 tumor with the novel variant 5 (exon 18). There were 11 ADs and 2 unusual carcinomas with mixed squamous and glandular components. Immunohistochemistry demonstrated diffuse ALK fusion proteins in the tumor cell cytoplasm. EML4-ALK was associated with nonsmokers (P = .009). Tumors with the fusion gene had the wild-type epidermal growth factor receptor (EGFR) (P = .001) and v-Ki-ras2/Kirsten rat sarcoma viral oncogene homolog (KRAS) genes. Patients who had EML4-ALK-positive AD had a younger median age (P = .018) compared with patients who did not have the fusion gene. CONCLUSIONS: The EML4-ALK fusion gene was present in various histologic types of NSCLC. It occurred in mutual exclusion to EGFR and KRAS mutations and was associated with nonsmokers. The authors concluded that EML4-ALK may be useful for predicting the potential response to ALK inhibitors as a therapeutic option for patients with lung cancer.

Distinct epidermal growth factor receptor and KRAS mutation patterns in non-small cell lung cancer patients with different tobacco exposure and clinicopathologic features.

PURPOSE: This study evaluated the mutational profile of epidermal growth factor receptor (EGFR) and KRAS in non-small cell lung cancers in Hong Kong and determined their relation with smoking history and other clinicopathologic features. EXPERIMENTAL DESIGN: Mutational profile of exons 18 to 21 of EGFR and codons 12, 13, and 61 of KRAS were determined in 215 adenocarcinomas, 15 squamous cell (SCC), and 11 EBV-associated lymphoepithelioma-like carcinomas (LELC). RESULTS: EGFR mutations were prevalent in adenocarcinomas (115 of 215), uncommon in LELC (1 of 11), and not found in SCC (P < 0.001). Among adenocarcinomas, mutations were associated with nonsmokers (83 of 111; P < 0.001), female gender (87 of 131; P < 0.001), and well-differentiated (55 of 86) compared with poorly differentiated (11 of 41) tumors (P < 0.001). Decreasing mutation rates with increasing direct tobacco exposure was observed, with 74.8% (83 of 111) in nonsmokers, 61.1% (11 of 18) in passive, 35.7% (10 of 28) in previous, and 19.0% (11 of 58) in current smokers. There were 53% amino acid substitutions, 43% in-frame deletions, and 4% insertions. Complex patterns with 13% double mutations, including five novel substitutions, were observed. For KRAS, mutations occurred in adenocarcinoma only (21 of 215) and were associated with smokers (11 of 58; P = 0.003), men (14 of 84; P = 0.009) and poorly differentiated (7 of 41) compared with well-differentiated (4 of 86) tumors (P = 0.037). EGFR and KRAS mutations occurred in mutually exclusive tumors. Regression analysis showed smoking history was the significant determinant for both mutations, whereas gender was a confounding factor. CONCLUSION: This study shows EGFR mutations are prevalent in lung adenocarcinoma and suggests that it plays an increasing oncogenic role with decreasing direct tobacco damage.