Biochemical and immunomodulatory insights of extracellular matrix from decellularized human whole cervix: recellularization andin vivoECM remodeling interplay.

Extracellular matrix (ECM) rich whole organ bio-scaffolds, preserving structural integrity and essential growth factors, has potential towards regeneration and reconstruction. Women with cervical anomalies or trauma can benefit from clinical cervicovaginal repair using constructs rich in site specific ECM. In this study, complete human cervix decellularization was achieved using a modified perfusion-based stir bench top decellularization method. This was followed by physico-chemical processes including perfusion of ionic agents, enzymatic treatment and washing using detergent solutions for a duration of 10-12 d. Histopathological analysis, as well as DNA quantification confirmed the efficacy of the decellularization process. Tissue ultrastructure integrity was preserved and the same was validated via scanning electron microscopy and transmission electron microscopy studies. Biochemical analysis and structural characterizations like Fourier transform infrared, Raman spectroscopy of decellularized tissues demonstrated preservation of important proteins, crucial growth factors, collagen, and glycosaminoglycans.In vitrostudies, using THP-1 and human umbilical vein endothelial cell (HUVEC) cells, demonstrated macrophage polarization from M1 to M2 and vascular functional genes enhancement, respectively, when treated with decellularized human cervical matrix (DHCp). Crosslinked DHC scaffolds were recellularized with site specific human cervical epithelial cells and HUVEC, showing non-cytotoxic cell viability and enhanced proliferation. Furthermore, DHC scaffolds showed immunomodulatory effectsin vivoon small rodent model via upregulation of M2 macrophage genes as compared to decellularized rat cervix matrix scaffolds (DRC). DHC scaffolds underwent neo-vascularization followed by ECM remodeling with enhanced tissue integration.

17-ß Estradiol (E2) distinctly regulates the expression of IL-4 and IL-13 in Th2 cells via modulating the interplay between GATA3 and PU.1.

17-ß Estradiol (E2) has long standing known functions in regulating human physiology as well as immune system. E2 is known to elicit a protective role against experimental autoimmune encephalomyelitis (EAE) and has been used as a drug for treatment against multiple sclerosis. Moreover, E2 regulates the adaptive immune system by directly affecting the T helper cell subsets differentiation and antibody secretion mediated by B cells. Reports have shown that E2 promotes Th1 and Treg cell differentiation; whereas it attenuated the Th17 and Tfh cell differentiation. Albeit multiple and contrasting studies, the mechanisms of behind E2 action on Th2 cells remained understudied. Hence, we sought to dissect the impact of E2 in Th2 cell differentiation. In this study, we elucidated the molecular mechanisms behind E2-mediated regulation of the differentiation of Th2 cells. We observed that E2 significantly attenuated the IL-4-secreting Th2 population in an ERα-dependent manner. We validated these findings using ICI 182, 780, an antagonist to ERα, not ERß and ectopically overexpressing ERα in Th2 cells. We further determined that ERα alters the recruitment of GATA3 and PU.1 to Il4 gene by directly interacting with them. This altered recruitment was observed to be stronger at Il4 than Il13 locus. Interestingly, we detected a distinct recruitment of GATA3 and PU.1 at Il13 gene; however, there was no E2-mediated broad alteration in the recruitment of histone-modifiers at Il13 locus. These findings suggest that E2 regulates Il4 in a distinctly separate mechanism as opposed to Il13 locus in Th2 cells.

Biodegradable Multi-layered Silk Fibroin-PCL Stent for the Management of Cervical Atresia: In Vitro Cytocompatibility and Extracellular Matrix Remodeling In Vivo.

Cervical atresia is a rare congenital Müllerian duct anomaly that manifests as the absence or deformed nonfunctional presence of the cervix. Herein, a multi-layered biodegradable stent is fabricated using a homogeneous blend of silk fibroin with polycaprolactone using hexafluoroisopropanol as a common solution. Briefly, a concentric cylinder of 3D honeycomb layer is sandwiched within electrospun sheets for fixing at the cervico-uterine junction to pave the way of cervical reconstruction. An average length of 40 mm with 3 mm diameter is fabricated for the hybrid stent design. SEM evidences an evenly distributed pore architecture of the electrospun layer, and mechanical characterization of stent reveals a tensile strength of 1.7 ± 0.2 MPa, with a Young's modulus of 5.9 ± 0.1 MPa. Physico-chemical characterization confirms the presence of silk fibroin and poly caprolactone within the engineered stent. Following 14 days of pepsin enzymatic degradation, 18% degradation and a contact angle measurement of 97° are observed. In vitro cytocompatibility studies are performed using site-specific primary human cervical squamous, columnar epithelial cells, and human endometrial stromal cells. The study demonstrates non-cytotoxic cells' viability (no significant toxicity), improved cell anchoring, adherence among the stent layers, and proliferation in the 3D microenvironment. Furthermore, in vivo subcutaneous studies in the rodent model indicate that the implanted stent undergoes constructive remodeling, neo-tissue creation, neo-vasculature formation, and re-epithelialization while maintaining patency for 2 months.

IL-10/IL-6 ratio from nasal & oral swab samples, acts as an inflammatory indicator for COVID-19 patients infected with the delta variant.

Background: Hyper-inflammatory immune response of SARS-CoV-2 is often characterized by the release of multiple pro-inflammatory cytokines with an impact on the expression of numerous other interleukins (ILs). However, from oral and nasal swab samples the specific quantitative association of the different IL-markers with the disease progression and its relationship with the status of vaccination remains unclear. Materials and methods: Patients' combined oral and nasal swab samples were collected from both non-vaccinated and double-vaccinated individuals with high (Ct value < 25) and low (Ct value > 30) viral loads, along with uninfected donors. None of the patients were critically ill, or needed ICU support. The expression of different cytokines (IL6, IL10, IL1B, IFNG) and mucin (MUC5AC, MUC1) markers were assessed between different groups by qRT-PCR. The important cytokine markers differentiating between vaccinated and non-vaccinated patients were identified by PCA. Conclusion: IL6 expression was higher in non-vaccinated COVID-19 patients infected with delta-variant irrespective of their viral-load compared to uninfected individuals. However, in double-vaccinated patients, only in high viral-load patients (Ct value < 25), IL6 expression increased. In high viral-load patients, irrespective to their vaccination status, IL10 expression was lower compared to the uninfected control group. Surprisingly, IL10 expression was lower in double-vaccinated patients with Ct value > 30. IL1B, and IFNG expression remained unaltered in uninfected and infected individuals. However, MUC5AC expression was lower in non-vaccinated patients with Ct value < 25 compared to control group. Our study unveiled that IL10/IL6 ratio can be used as a biomarker for COVID-19 patients upon proper establishment of it in a clinical setting.

Synthesis and characterization of two self-assembled [Cu6Gd3] and [Cu5Dy2] complexes exhibiting the magnetocaloric effect, slow relaxation of magnetization, and anticancer activity.

Two new paths for coordination driven self-assembly reactions under the binding support of 2-((1-hydroxy-2-methylpropan-2-ylimino)methyl)-6-methoxyphenol (H2L) have been discovered from the reactions of Cu(ClO4)2·6H2O, NEt3 and GdCl3/DyCl3·6H2O in MeOH/CHCl3 (2 : 1) medium. A similar synthetic protocol is useful to provide two different types of self-aggregated molecular clusters [Cu6Gd3(L)3(HL)3(µ3-Cl)3(µ3-OH)6(OH)2]ClO4·4H2O (1) and [Cu5Dy2(L)2(HL)2(µ-Cl)2(µ3-OH)4(ClO4)2(H2O)6](ClO4)2·2NHEt3Cl·21H2O (2). The adopted reaction procedure established the importance of the HO- and Cl- ions in the mineral-like growth of the complexes, derived from solvents and metal ion salts. In the case of complex 1, one GdIII center has been trapped at the central position of the core upheld by six µ3-OH and three µ3-Cl groups, whereas for complex 2 one CuII center was trapped using four µ3-hydroxo and two µ-chlorido groups. The magnetothermal behavior of 1 has been examined for a magnetocaloric effect of -ΔSm = 11.3 J kg-1 K-1 at 2 K for ΔH = 7 T, whereas the magnetic susceptibility measurements of 2 showed slow magnetic relaxation with Ueff = 15.8 K and τ0 = 9.8 × 10-7 s in zero external dc field. Cancer cell growth inhibition studies proved the potential of both the complexes with interestingly high activity for the Cu6Gd3 complex against human lung cancer cells. Both complexes 1 and 2 also exhibited DNA and human serum albumin (HSA) binding abilities in relation to the involved binding sites and thermodynamics.

Performance and cell toxicity studies for the use of graphene oxide-bimetallic oxide hybrids in the absorptive removal of Pb(II) from wastewater: fixed-bed column study with regeneration.

The current study involves the removal of Pb(II) ions from an aqueous solution using GO/Mn-Fe hybrids in a fixed bed column study. The capability of the hybrid in the Pb removal was examined using a continuous flow fixed bed column which revealed that the hybrid had the maximum adsorption capacity of 172.768 mg/g at a flow rate of 2 mL/min, bed height of 1 cm, and influent concentration of 200 mg/L. The breakthrough curves obtained from the experiments were examined using three different models, i.e., Bohart-Adams model, Thomas Model, and Yoon-Nelson model, wherein all the models showed high correlation coefficient values. Three consecutive adsorption-desorption cycles in the column yielded regeneration efficiencies of 91.71%, 88.31%, and 85.41%. The column life factor indicated that the fixed bed would have enough capacity to avoid a zero breakthrough time for up to 9 cycles, implying that GO/Mn-Fe could be used as a cheap and efficient adsorbent in the removal of Pb(II) from contaminated water. The adsorption mechanism was postulated based on the characterization of the spent adsorbent by FTIR and SEM. The phenomenon of the adsorption process can be described in accordance with the surface complex formation theory, which suggests that an increase in pH decreases the competition between metal ions and protons, favoring metal ion adsorption. The toxicity of the synthesized hybrid was evaluated on HeLa cells and compared to the toxicity of GO. Increasing the concentration of GO/Mn-Fe hybrid from 50 to 250 g/mL resulted in a decrease in cell viability from 91.90 to 56.52%, whereas increasing the concentration of GO resulted in a decrease in cell viability from 61.59 to 37.19%. The study clearly demonstrates the use of GO/Mn-Fe hybrid as an adsorbent for efficient sequestration of Pb(II) ions with lower environmental toxicity.

COVID-19 pandemic: the delta variant, T-cell responses, and the efficacy of developing vaccines.

BACKGROUND: The mayhem COVID-19 that was ushered by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) was declared pandemic by the World Health Organization in March 2020. Since its initial outbreak in late 2019, the virus has affected hundreds of million adults in the world and killing millions in the process. After the approval of newly developed vaccines, severe challenges remain to manufacture and administer them to the adult population globally in quick time. However, we have witnessed several mutations of the virus leading to 'waves' of viral spread and mortality. WHO has categorized these mutations as variants of concern (VOCs) and variants of interest (VOIs). The mortality due to COVID-19 has also been associated with various comorbidities and improper immune response. This has created further complications in understanding the nature of the SARS-CoV2-host interaction that has fuelled doubts in the efficacy of the approved vaccines. Whether there is requirement of booster dose and whether the impending wave could affect the children are some of the hotly debated topics. MATERIALS AND METHODS: A systematic literature review of PubMed, Medline, Scopus, Google Scholar was utilized to understand the nature of Delta variant and how it alters our T-cell responses and cytokine production and neutralizes vaccine-generated antibodies. CONCLUSION: In this review, we discuss the variants of SARS-CoV2 with specific focus on the Delta variant. We also specifically review the T-cell response against the virus and bring a narrative of various factors that may hold the key to fight against this marauding virus.

Piecewise Isothermal Nucleic Acid Testing (PINAT) for Infectious Disease Detection with Sample-to-Result Integration at the Point-of-Care.

We developed a piecewise isothermal nucleic acid test (PINAT) as a platform technology for diagnosing pathogen-associated infections, empowered by an illustrative novel methodology that embeds an exclusive DNA-mediated specific probing reaction with the backbone of an isothermal reverse transcription cum amplification protocol for detecting viral RNA. In a point-of-care format, this test is executable in a unified single-step, single-chamber procedure, leading to seamless sample-to-result integration in an inexpensive, scalable, pre-programmable, and customizable portable device, with mobile-app-integrated interpretation and analytics involving minimal manually operative procedures. The test exhibited a high sensitivity and specificity of detection when assessed using 200 double-blind patient samples for detecting SARS-CoV-2 infection by the Indian Council of Medical Research (ICMR), and subsequently using 170 double-blind patient samples in a point-of-care format outside controlled laboratory settings as performed by unskilled technicians in an organized clinical trial. We also established its efficacy in detecting Influenza A infection by performing the diagnosis at the point of collection with uncompromised detection rigor. The envisaged trade-off between advanced laboratory-based molecular diagnostic procedures and the elegance of common rapid tests renders the method ideal for deployment in resource-limited settings towards catering the needs of the underserved.

Calcitriol and Retinoic acid antagonize each other to suppress the production of IL-9 by Th9 cells.

Distinct T helper cells, including Th9 cells help maintain homeostasis in the immune system. Vitamins play pivotal role in the immune system through many mechanisms, including regulating the differentiation of T helper cells. Calcitriol (1,25-dihydroxyvitamin D3) and retinoic acid possess hormone-like properties and are the bioactive metabolites of vitamin D and A, respectively, that signal through heterodimers containing the common retinoid X receptor. In contrast to individual treatment with the vitamins that significantly attenuates IL-9 production from Th9 cells, Th9 cells treated with both vitamins demonstrated IL-9 production similar to untreated Th9 cells. This is associated with reciprocal expression of PU.1 and Foxp3. While the recruitment of PU.1 was significantly impaired to the Il9 gene in the presence of calcitriol or retinoic acid in Th9 cells, addition of both vitamins together increased the recruitment of PU.1 to the Il9 gene. Calcitriol and retinoic acid together impaired the recruitment of HDAC1 to the Il9 gene without impacting Gcn5 recruitment. Importantly, retinoic acid negated the effect of calcitriol and impaired the binding of VDR on the Il9 gene by dampened VDR-RXR formation. Collectively, our data show that calcitriol and retinoic acid antagonize each other to regulate the differentiation of Th9 cells.

Calcitriol attenuates TLR2/IL-33 signaling pathway to repress Th9 cell differentiation and potentially limits the pathophysiology of rheumatoid arthritis.

There is limited information regarding the TLR2 signaling pathway involved in Th9 cell differentiation. The role of calcitriol in regulating TLR2-mediated Th9 cell development is unknown. Thus, we aimed to unravel the TLR2 signaling pathway in Th9 cells and its regulation by calcitriol. We have used n = 5-6 animals for each murine experiment. Human studies involved five healthy volunteers. Moreover, ten healthy individuals and ten RA patients were included in the study. Murine and human Th9 cells were treated with Calcitriol (100 nM) and Pam3CSK4 (2 µg/mL). The number of IL-9+ve cells was determined by flow cytometry. Real-time PCR was used to assess the gene expression. Serum 25(OH)D3 levels were determined by HPLC. We observed that TLR2 signals via IL-33/ST2 in Th9 cells. Increased TLR2 expression associated with increased IL9 expression and augmented disease severity in RA patients. Calcitriol attenuated TLR2 signaling in murine and human Th9 cells. Low serum vitamin D3 level negatively associated with increased IL-9 and TLR2 expression and disease severity in RA patients. Our data suggest a potential role of calcitriol to ameliorate the disease severity of RA patients.

In vitro and in silico studies on the structural and biochemical insight of anti-biofilm activity of andrograpanin from Andrographis paniculata against Pseudomonas aeruginosa.

Microbial infections have become a global threat to drug-tolerant phenomena due to their biofilm formatting capacity. In many cases, conventional antimicrobial drugs fail to combat the infection, thus necessitating the discovery of some alternative medicine. Over several decades, plant metabolites have played a critical role in treating a broad spectrum of microbial infections due to its low cytotoxicity. Andrograpanin, a secondary metabolite, is a diterpenoid present in the leaf of Andrographis paniculata. In this study, andrograpanin (0.15 mM) exhibited significant inhibition on biofilm production by Pseudomonas aeruginosa in the presence of gentamicin (0.0084 mM). The impaired production of extracellular polymeric substances and several virulence factors of Pseudomonas aeruginosa were investigated to understand the mechanism of action mediated by andrograpanin. The structural alteration of biofilm was evaluated by using fluorescence microscopy, atomic force microscopy and field emission scanning electron microscopy. The in silico molecular simulation studies predicted interaction of andrograpanin with quorum sensing proteins such as RhlI, LasI, LasR, and swarming motility protein BswR of Pseudomonas aeruginosa. Overall the studies indicate that andrograpanin could be used as a therapeutic molecule against biofilm development by Pseudomonas aeruginosa.

Calcitriol Regulates the Differentiation of IL-9-Secreting Th9 Cells by Modulating the Transcription Factor PU.1.

Vitamin D can modulate the innate and adaptive immune system. Vitamin D deficiency has been associated with various autoimmune diseases. Th9 cells are implicated in the pathogenesis of numerous autoimmune diseases. Thus, we investigated the role of calcitriol (active metabolite of vitamin D) in the regulation of Th9 cell differentiation. In this study, we have unraveled the molecular mechanisms of calcitriol-mediated regulation of Th9 cell differentiation. Calcitriol significantly diminished IL-9 secretion from murine Th9 cells associated with downregulated expression of the Th9-associated transcription factor, PU.1. Ectopic expression of VDR in Th9 cells attenuated the percentage of IL-9-secreting cells. VDR associated with PU.1 in Th9 cells. Using a series of mutations, we were able to dissect the VDR domain involved in the regulation of the Il9 gene. The VDR-PU.1 interaction prevented the accessibility of PU.1 to the Il9 gene promoter, thereby restricting its expression. However, the expression of Foxp3, regulatory T cell-specific transcription factor, was enhanced in the presence of calcitriol in Th9 cells. When Th9 cells are treated with both calcitriol and trichostatin A (histone deacetylase inhibitor), the level of IL-9 reached to the level of wild-type untreated Th9 cells. Calcitriol attenuated specific histone acetylation at the Il9 gene. In contrast, calcitriol enhanced the recruitment of the histone modifier HDAC1 at the Il9 gene promoter. In summary, we have identified that calcitriol blocked the access of PU.1 to the Il9 gene by reducing its expression and associating with it as well as regulated the chromatin of the Il9 gene to regulate expression.

Rheumatoid arthritis: 'melting pot' of T helper subsets.

Rheumatoid arthritis (RA) is an autoimmune disorder that affects joints associated with inflammation leading to poor quality of life. The phenotype of RA is distinct from osteoarthritis (OA), the degenerative joint disorder. The annual incidence of RA is approximately 4 in 10,000 individuals. Studies suggest dysregulated T cell activation in the initiation and progression of RA. Distinct RA-associated allelic variants encode molecules involved in T-cell activation pathways. Additionally, RA is also associated with aberrant regulation and function of T helper cells. The interplay of distinct T helper cell subsets adds complexity to the regulation of RA. In this review we have aimed to understand the currently known biology of different Th subsets in the context of an autoimmune disease like rheumatoid arthritis and find potential therapeutic approaches to tackle the disease through modulation of responsible T cells.

Size-dependent cellular uptake and TLR4 attenuation by gold nanoparticles in lung adenocarcinoma cells.

AIM: To elucidate uptake mechanisms and immunomodulatory potential of differently sized gold nanoparticles (GNPs) in lung adenocarcinoma cells (A549) to enable their use as an adjunct therapy for treating inflammation-linked lung cancer. METHODS: Internalization of the synthesized (5, 15 and 30 nm) GNPs by various endocytosis pathways was determined. Immunomodulatory mechanisms induced by differently sized GNPs in A549 cells in the presence of TLR4 and TLR9 ligands were evaluated. RESULTS: GNPs were size-dependently internalized efficiently by A549 cells. Various sized GNPs downregulated the expression of proinflammatory signaling molecules (5 nm most potent). Mechanistically, 5-nm GNPs attenuated TLR4 signaling by downregulating TLR4 expression in A549 cells. CONCLUSION: Our study suggests the use of immunomodulatory GNPs as an adjunct therapy against inflammation-linked lung cancer.

A Decade of Th9 Cells: Role of Th9 Cells in Inflammatory Bowel Disease.

T helper cell subsets play a critical role in providing protection against offending pathogens by secreting specific cytokines. However, unrestrained T helper cell responses can promote chronic inflammation-mediated inflammatory diseases. Dysregulated T helper cell responses have been suggested to be involved in the pathogenesis of multiple inflammatory diseases, including allergic airway inflammation, rheumatoid arthritis, and inflammatory bowel disease (IBD) among others. Aberrant pro-inflammatory responses induced by Th1, Th2, and Th17 subsets are known to trigger IBD. IBD is a chronic inflammatory disease characterized by weight loss, diarrhea, pain, fever, and rectal bleeding. It poses a major health burden worldwide owing to the increased risk of colorectal cancer development. Despite numerous therapeutic advancements, IBD still remains a major health burden due to the inefficiency of the conventional therapies. Recently, IL-9-secreting Th9 cells are known to be involved in the pathogenesis of IBD. However, the role of Th9 cells and their secretory cytokine IL-9 in IBD is unclear. The functional relevance of Th9 cells is also relatively understudied in IBD. Thus, investigating the actual role of various T helper cell subsets including Th9 cells in IBD is essential to develop novel therapies to treat IBD. Here, we highlight the role of Th9 cells in promoting IBD. We discuss the mechanisms that might be employed by Th9 cells and IL-9 in promoting IBD and thereby propose potential targets for the treatment of Th9 cell-mediated IBD.

Striking the right immunological balance prevents progression of tuberculosis.

INTRODUCTION: Tuberculosis (TB) caused by infection with Mycobacterium tuberculosis (Mtb) is a major burden for human health worldwide. Current standard treatments for TB require prolonged administration of antimycobacterial drugs leading to exaggerated inflammation and tissue damage. This can result in the reactivation of latent TB culminating in TB progression. Thus, there is an unmet need to develop therapies that would shorten the duration of anti-TB treatment and to induce optimal protective immune responses to control the spread of mycobacterial infection with minimal lung pathology. FINDINGS: Granulomata is the hallmark structure formed by the organized accumulation of immune cells including macrophages, natural killer cells, dendritic cells, neutrophils, T cells, and B cells to the site of Mtb infection. It safeguards the host by containing Mtb in latent form. However, granulomata can undergo caseation and contribute to the reactivation of latent TB, if the immune responses developed to fight mycobacterial infection are not properly controlled. Thus, an optimal balance between innate and adaptive immune cells might play a vital role in containing mycobacteria in latent form for prolonged periods and prevent the spread of Mtb infection from one individual to another. CONCLUSION: Optimal and well-regulated immune responses against Mycobacterium tuberculosis may help to prevent the reactivation of latent TB. Moreover, therapies targeting balanced immune responses could help to improve treatment outcomes among latently infected TB patients and thereby limit the dissemination of mycobacterial infection.

Th9 Cells: New Member of T Helper Cell Family.

T Helper cells (CD4+ T cells) constitute one of the key arms of adaptive immune responses. Differentiation of naïve CD4+ T cells into multiple subsets ensure a proper protection against multitude of pathogens in immunosufficient individual. After differentiation, T helper cells secrete specific cytokines that are critical to provide immunity against various pathogens. The recently discovered Th9 cells secrete the pleiotropic cytokine, IL-9. Although IL-9 was cloned more than 25 years ago and characterized as a Th2 cell-specific cytokine, not many studies were carried out to define the function of IL-9. This cytokine has been demonstrated to act on multiple cell types as a growth factor. After the discovery of Th9 cells as an abundant source of IL-9, renewed focus has been generated. In this chapter, I discuss the biology and development of IL-9-secreting Th9 cells. Furthermore, I highlight the role of Th9 cells and IL-9 in health and diseases.

IL-9 Signaling Pathway: An Update.

Since the discovery of IL-9 almost three decades back as a growth factor, we have come a long way to understand its pleiotropic functions in the immune system. Despite its many functions, IL-9 still remains as an understudied cytokine. In the last decade, renewed emphasis has been provided to understand the biology of IL-9. Any growth factor or cytokine signals via its cognate receptor to mediate biological functions. In this chapter, we discuss the IL-9 signal transduction in different cell types, which would then exert its distinct functions.

Systemic innate immune activation in food protein-induced enterocolitis syndrome.

BACKGROUND: Food protein-induced enterocolitis syndrome (FPIES) is a non-IgE-mediated food allergy of infancy whose pathophysiology is poorly understood. OBJECTIVES: We set out to identify and phenotype allergen-responsive cells in peripheral blood of a cohort of subjects undergoing supervised food challenge for FPIES. METHODS: We profiled antigen-responsive cells in PBMCs by flow cytometry, and examined cells in whole blood obtained before and after challenge by CyTOF mass cytometry and RNAseq. RESULTS: Using a CD154-based detection approach, we observed that milk, soy, or rice-responsive T cells, and TNF-α-producing CD154+ T cells, were significantly lower in those with outgrown FPIES compared with those with active FPIES. However, levels were within the normal range and were inconsistent with a role in the pathophysiology of FPIES. Profiling of whole blood by CyTOF demonstrated profound activation of cells of the innate immune system after food challenge, including monocytes, neutrophils, natural killer cells, and eosinophils. Activation was not observed in children with outgrown FPIES. We confirmed this pattern of innate immune activation in a larger cohort by RNAseq. Furthermore, we observed pan-T-cell activation and redistribution from the circulation after a positive food challenge but not in those who had outgrown their FPIES. CONCLUSIONS: Our data demonstrate a compelling role of systemic innate immune activation in adverse reactions elicited by foods in FPIES. Further investigation is needed to identify the mechanism of antigen specificity of adverse reactions to foods in FPIES.