Poststroke venous thromboembolism and neutrophil activation: an illustrated review.

Patients with acute ischemic stroke are at a high risk of venous thromboembolism (VTE), such as deep vein thrombosis (DVT), estimated to affect approximately 80,000 patients with stroke each year in the United States. The prevalence of symptomatic DVT after acute stroke is approximately 10%. VTE is associated with increased rates of in-hospital death and disability, with higher prevalence of in-hospital complications and increased 1-year mortality in patients with stroke. Current guidelines recommend the use of pharmacologic VTE prophylaxis in patients with acute ischemic stroke. However, thromboprophylaxis prevents only half of expected VTE events and is associated with high risk of bleeding, suggesting the need for targeted alternative treatments to reduce VTE risk in these patients. Neutrophils are among the first cells in blood to respond after ischemic stroke. Importantly, coordinated interactions among neutrophils, platelets, and endothelial cells contribute to the development of DVT. In case of stroke and other related immune disorders, such as antiphospholipid syndrome, neutrophils potentiate thrombus propagation through the formation of neutrophil-platelet aggregates, secreting inflammatory mediators, complement activation, releasing tissue factor, and producing neutrophil extracellular traps. In this illustrated review article, we present epidemiology and management of poststroke VTE, preclinical and clinical evidence of neutrophil hyperactivation in stroke, and mechanisms for neutrophil-mediated VTE in the context of stroke. Given the hyperactivation of circulating neutrophils in patients with stroke, we propose that a better understanding of molecular mechanisms leading to neutrophil activation may result in the development of novel therapeutics to reduce the risk of VTE in this patient population.

Regulating Neutrophil PAD4/NOX-Dependent Cerebrovasular Thromboinflammation.

Background: Neutrophil extracellular trap (NET) production has been implicated in the pathogenesis of thromboinflammatory conditions such as Sickle Cell Disease (SCD), contributing to heightened risk for ischemic stroke. NETs are catalyzed by the enzyme Peptidyl Arginine Deiminase 4 (PAD4) and neutrophil derived reactive oxygen species (ROS), especially NADPH oxidase (NOX) which interacts with PAD4 and is therefore critical for neutrophil function. However, the role that NOX-dependent ROS and NETs play in the accelerated cerebral microvascular thrombosis associated with thromboinflammatory conditions, such as SCD, has not been fully elucidated and is the aim of this study. Methods: The in-vitro effects of targeting PAD4 and NOX were examined using physiologically relevant NET assays with neutrophils isolated from healthy volunteers (control) and SCD patients. In addition, in-vivo intravascular effects of targeting PAD4 and NOX in the cerebral microcirculation of C57BL/6 and sickle transgenic mice (STM) were assessed using a photoactivation thrombosis model (light/dye) coupled with real-time fluorescence intravital microscopy. Results: We found that targeting PAD4 and NOX in human neutrophils significantly inhibited ionomycin dependent H3cit+ neutrophils. Targeting PAD4 and NOX in-vivo resulted in prolonged blood flow cessation in cerebrovascular arterioles as well as venules. Moreover, we were able to replicate the effects of PAD4 and NOX targeting in a clinical model of accelerated thromboinflammation by increasing blood flow cessation times in cerebral microvessels in STM. These findings concurred with the clinical setting i.e. neutrophils isolated from SCD patients, which possessed an attenuation of H3cit+ neutrophil production on targeting PAD4 and NOX. Conclusions: Taken together, our compelling data suggests that PAD4 and NOX play a significant role in neutrophil driven thromboinflammation. Targeting PAD4 and NOX limits pathological H3cit+ neutrophils, which may further explain attenuation of cerebral thrombosis. Overall, this study presents a viable pre-clinical model of prevention and management of thromboinflammatory complications such as ischemic stroke.

Bryostatin-1 Attenuates Ischemia-Elicited Neutrophil Transmigration and Ameliorates Graft Injury after Kidney Transplantation.

Ischemia reperfusion injury (IRI) is a form of sterile inflammation whose severity determines short- and long-term graft fates in kidney transplantation. Neutrophils are now recognized as a key cell type mediating early graft injury, which activates further innate immune responses and intensifies acquired immunity and alloimmunity. Since the macrolide Bryostatin-1 has been shown to block neutrophil transmigration, we aimed to determine whether these findings could be translated to the field of kidney transplantation. To study the effects of Bryostatin-1 on ischemia-elicited neutrophil transmigration, an in vitro model of hypoxia and normoxia was equipped with human endothelial cells and neutrophils. To translate these findings, a porcine renal autotransplantation model with eight hours of reperfusion was used to study neutrophil infiltration in vivo. Graft-specific treatment using Bryostatin-1 (100 nM) was applied during static cold storage. Bryostatin-1 dose-dependently blocked neutrophil activation and transmigration over ischemically challenged endothelial cell monolayers. When applied to porcine renal autografts, Bryostatin-1 reduced neutrophil graft infiltration, attenuated histological and ultrastructural damage, and improved renal function. Our novel findings demonstrate that Bryostatin-1 is a promising pharmacological candidate for graft-specific treatment in kidney transplantation, as it provides protection by blocking neutrophil infiltration and attenuating functional graft injury.

Endovascular Intervention in Acute Ischemic Stroke: History and Evolution.

Stroke is a leading cause of serious long-term disability in the US. Endovascular therapy (EVT), in the form of mechanical thrombectomy, is now a standard of care for patients with acute ischemic stroke with a large vessel occlusion. This article reviews the evolution of EVT in the management of acute ischemic stroke and how it has led to the concept of tissue window over the widely publicized time window.

The Spectrum of Acute Cerebrovascular Disease in Patients with COVID-19.

(1) Background: COVID-19 infection is responsible for the ongoing pandemic and acute cerebrovascular disease (CVD) has been observed in COVID-19 patients. (2) Methods: We conducted a retrospective, observational study of hospitalized adult patients admitted to our hospital with SARS-CoV-2 and acute cerebrovascular disease. All clinical data were reviewed including epidemiology, clinical features, laboratory data, neuroradiological findings, hospital management and course from 32 patients hospitalized for COVID-19 management with acute cerebrovascular disease. (3) Results: Acute CVD with COVID-19 was associated with higher NIH stroke scale on discharge compared to non-COVID-19 CVDs. Seizures complicated the hospital course in 16% of COVID-19 patients with CVD. The majority of the acute CVDs were ischemic (81%) in nature followed by hemorrhagic (22%). Acute CVD with COVID-19 resulted in average hospital stays greater than twice that of the control group (13 days in COVID-19, 5 days in control). Acute CVD with COVID-19 patients had worse clinical outcomes with 31% patient deaths and 6% discharged to hospice. In the control group, 6% of patients died. (4) Conclusions: Acute CVD associated with COVID-19 tends to be more complicated with unique and adverse clinical phenotype, longer hospital admissions, and worse clinical outcomes.

Endovascular Management of Hemorrhagic Stroke.

Significant advances in endovascular neurosurgery tools, devices, and techniques are changing the approach to the management of acute hemorrhagic stroke. The endovascular treatment of intracranial aneurysms emerged in the early 1990s with Guglielmi detachable coils, and since then, it gained rapid popularity that surpassed open surgery. Stent-assisted coiling and balloon remodeling techniques have made the treatment of wide-necked aneurysms more durable. With the introduction of flow diverters and flow disrupters, many aneurysms with complex geometrics can now be reliably managed. Arteriovenous malformations and fistulae can also benefit from endovascular therapy by embolization using n-butyl cyanoacrylate (NBCA), Onyx, polyvinyl alcohol (PVA), and coils. In this article, we describe the role of endovascular treatment for the most common causes of intracerebral and subarachnoid hemorrhages, particularly ruptured aneurysms and vascular malformations.

Neutrophils and Platelets: Immune Soldiers Fighting Together in Stroke Pathophysiology.

Neutrophils and platelets exhibit a diverse repertoire of functions in thromboinflammatory conditions such as stroke. Most cerebral ischemic events result from longstanding chronic inflammation secondary to underlying pathogenic conditions, e.g., hypertension, diabetes mellitus, obstructive sleep apnea, coronary artery disease, atrial fibrillation, morbid obesity, dyslipidemia, and sickle cell disease. Neutrophils can enable, as well as resolve, cerebrovascular inflammation via many effector functions including neutrophil extracellular traps, serine proteases and reactive oxygen species, and pro-resolving endogenous molecules such as Annexin A1. Like neutrophils, platelets also engage in pro- as well as anti-inflammatory roles in regulating cerebrovascular inflammation. These anucleated cells are at the core of stroke pathogenesis and can trigger an ischemic event via adherence to the hypoxic cerebral endothelial cells culminating in aggregation and clot formation. In this article, we review and highlight the evolving role of neutrophils and platelets in ischemic stroke and discuss ongoing preclinical and clinical strategies that may produce viable therapeutics for prevention and management of stroke.

The impact of thrombo-inflammation on the cerebral microcirculation.

The intertwined processes of thrombosis and inflammation (termed "thrombo-inflammation") are significant drivers of cerebrovascular diseases, and as such, they represent prime targets for drug discovery programs focusing on treatment and management of cerebrovascular diseases. Most cerebrovascular events result from chronic systemic microcirculatory dysfunction due to underlying conditions, for example, hypertension, diabetes mellitus, coronary artery disease, dyslipidemia, and sickle cell disease. Immune cells especially neutrophils play a critical role in the onset and maintenance of neuroinflammatory responses in the microcirculation. Neutrophils have the ability to drive both inflammatory and anti-inflammatory/pro-resolution effects depending on the underlying vascular state (physiological vs. pathological). In this article, we highlight the pathophysiological role of neutrophils in stroke and discuss ongoing pharmacotherapeutic strategies that are focused on identifying potential therapeutic targets for enhancing neuroprotection, mitigating inflammatory pathways, and enabling resolution.

Targeting the AnxA1/Fpr2/ALX pathway regulates neutrophil function, promoting thromboinflammation resolution in sickle cell disease.

Neutrophils play a crucial role in the intertwined processes of thrombosis and inflammation. An altered neutrophil phenotype may contribute to inadequate resolution, which is known to be a major pathophysiological contributor of thromboinflammatory conditions such as sickle cell disease (SCD). The endogenous protein annexin A1 (AnxA1) facilitates inflammation resolution via formyl peptide receptors (FPRs). We sought to comprehensively elucidate the functional significance of targeting the neutrophil-dependent AnxA1/FPR2/ALX pathway in SCD. Administration of AnxA1 mimetic peptide AnxA1Ac2-26 ameliorated cerebral thrombotic responses in Sickle transgenic mice via regulation of the FPR2/ALX (a fundamental receptor involved in resolution) pathway. We found direct evidence that neutrophils with SCD phenotype play a key role in contributing to thromboinflammation. In addition, AnxA1Ac2-26 regulated activated SCD neutrophils through protein kinase B (Akt) and extracellular signal-regulated kinases (ERK1/2) to enable resolution. We present compelling conceptual evidence that targeting the AnxA1/FPR2/ALX pathway may provide new therapeutic possibilities against thromboinflammatory conditions such as SCD.

Targeting AnxA1/Formyl Peptide Receptor 2 Pathway Affords Protection against Pathological Thrombo-Inflammation.

Stroke is a leading cause of death and disability globally and is associated with a number of co-morbidities including sepsis and sickle cell disease (SCD). Despite thrombo-inflammation underlying these co-morbidities, its pathogenesis remains complicated and drug discovery programs aimed at reducing and resolving the detrimental effects remain a major therapeutic challenge. The objective of this study was to assess whether the anti-inflammatory pro-resolving protein Annexin A1 (AnxA1) was able to reduce inflammation-induced thrombosis and suppress platelet activation and thrombus formation in the cerebral microvasculature. Using two distinct models of pathological thrombo-inflammation (lipopolysaccharide (LPS) and sickle transgenic mice (STM)), thrombosis was induced in the murine brain using photoactivation (light/dye) coupled with intravital microscopy. The heightened inflammation-induced microvascular thrombosis present in these two distinct thrombo-inflammatory models was inhibited significantly by the administration of AnxA1 mimetic peptide AnxA1Ac2-26 (an effect more pronounced in the SCD model vs. the endotoxin model) and mediated by the key resolution receptor, Fpr2/ALX. Furthermore, AnxA1Ac2-26 treatment was able to hamper platelet aggregation by reducing platelet stimulation and aggregation (by moderating αIIbß3 and P-selectin). These findings suggest that targeting the AnxA1/Fpr2/ALX pathway represents an attractive novel treatment strategy for resolving thrombo-inflammation, counteracting e.g., stroke in high-risk patient cohorts.

Targeting of Formyl Peptide Receptor 2 for in vivo imaging of acute vascular inflammation.

Inflammatory conditions are associated with a variety of diseases and can significantly contribute to their pathophysiology. Neutrophils are recognised as key players in driving vascular inflammation and promoting inflammation resolution. As a result, neutrophils, and specifically their surface formyl peptide receptors (FPRs), are attractive targets for non-invasive visualization of inflammatory disease states and studying mechanistic details of the process. Methods: A small-molecule Formyl Peptide Receptor 2 (FPR2/ALX)-targeted compound was combined with two rhodamine-derived fluorescent tags to form firstly, a targeted probe (Rho-pip-C1) and secondly a targeted, pH-responsive probe (Rho-NH-C1) for in vivo applications. We tested internalization, toxicity and functional interactions with neutrophils in vitro for both compounds, as well as the fluorescence switching response of Rho-NH-C1 to neutrophil activation. Finally, in vivo imaging (fluorescent intravital microscopy [IVM]) and therapeutic efficacy studies were performed in an inflammatory mouse model. Results: In vitro studies showed that the compounds bound to human neutrophils via FPR2/ALX without causing internalization at relevant concentrations. Additionally, the compounds did not cause toxicity or affect neutrophil functional responses (e.g. chemotaxis or transmigration). In vivo studies using IVM showed Rho-pip-C1 bound to activated neutrophils in a model of vascular inflammation. The pH-sensitive ("switchable") version termed Rho-NH-C1 validated these findings, showing fluorescent activity only in inflammatory conditions. Conclusions: These results indicate a viable design of fluorescent probes that have the ability to detect inflammatory events by targeting activated neutrophils.

Development, characterisation and in vitro evaluation of lanthanide-based FPR2/ALX-targeted imaging probes.

We report the design, preparation and characterisation of three small-molecule, Formyl Peptide Receptor (FPR)-targeted lanthanide complexes (Tb·14, Eu·14 and Gd·14). Long-lived, metal-based emission was observed from the terbium complex (τH2O = 1.9 ms), whereas only negligible lanthanide signals were detected in the europium analogue. Ligand-centred emission was investigated using Gd·14 at room temperature and 77 K, leading to the postulation that metal emission may be sensitised via a ligand-based charge transfer state of the targeting Quin C1 unit. Comparatively high longitudinal relaxivity values (r1) for octadentate metal complexes of Gd·14 were determined (6.9 mM-1 s-1 at 400 MHz and 294 K), which could be a result of a relative increase in twisted square antiprism (TSAP) isomer prevalence compared to typical DOTA constructs (as evidenced by NMR spectroscopy). In vitro validation of concentration responses of Tb·14via three key neutrophil functional assays demonstrated that the inflammatory responses of neutrophils (i.e. chemotaxis, transmigration and granular release) remained unchanged in the presence of specific concentrations of the compound. Using a time-resolved microscopy set-up we were able to observe binding of the Tb·14 probe to stimulated human neutrophils around the cell periphery, while in the same experiment with un-activated neutrophils, no metal-based signals were detected. Our results demonstrate the utility of Tb·14 for time-resolved microscopy with lifetimes several orders of magnitude longer than autofluorescent signals and a preferential uptake in stimulated neutrophils.

Novel Role for the AnxA1-Fpr2/ALX Signaling Axis as a Key Regulator of Platelet Function to Promote Resolution of Inflammation.

BACKGROUND: Ischemia reperfusion injury (I/RI) is a common complication of cardiovascular diseases. Resolution of detrimental I/RI-generated prothrombotic and proinflammatory responses is essential to restore homeostasis. Platelets play a crucial part in the integration of thrombosis and inflammation. Their role as participants in the resolution of thromboinflammation is underappreciated; therefore we used pharmacological and genetic approaches, coupled with murine and clinical samples, to uncover key concepts underlying this role. METHODS: Middle cerebral artery occlusion with reperfusion was performed in wild-type or annexin A1 (AnxA1) knockout (AnxA1-/-) mice. Fluorescence intravital microscopy was used to visualize cellular trafficking and to monitor light/dye-induced thrombosis. The mice were treated with vehicle, AnxA1 (3.3 mg/kg), WRW4 (1.8 mg/kg), or all 3, and the effect of AnxA1 was determined in vivo and in vitro. RESULTS: Intravital microscopy revealed heightened platelet adherence and aggregate formation post I/RI, which were further exacerbated in AnxA1-/- mice. AnxA1 administration regulated platelet function directly (eg, via reducing thromboxane B2 and modulating phosphatidylserine expression) to promote cerebral protection post-I/RI and act as an effective preventative strategy for stroke by reducing platelet activation, aggregate formation, and cerebral thrombosis, a prerequisite for ischemic stroke. To translate these findings into a clinical setting, we show that AnxA1 plasma levels are reduced in human and murine stroke and that AnxA1 is able to act on human platelets, suppressing classic thrombin-induced inside-out signaling events (eg, Akt activation, intracellular calcium release, and Ras-associated protein 1 [Rap1] expression) to decrease αIIbß3 activation without altering its surface expression. AnxA1 also selectively modifies cell surface determinants (eg, phosphatidylserine) to promote platelet phagocytosis by neutrophils, thereby driving active resolution. (n=5-13 mice/group or 7-10 humans/group.) Conclusions: AnxA1 affords protection by altering the platelet phenotype in cerebral I/RI from propathogenic to regulatory and reducing the propensity for platelets to aggregate and cause thrombosis by affecting integrin (αIIbß3) activation, a previously unknown phenomenon. Thus, our data reveal a novel multifaceted role for AnxA1 to act both as a therapeutic and a prophylactic drug via its ability to promote endogenous proresolving, antithromboinflammatory circuits in cerebral I/RI. Collectively, these results further advance our knowledge and understanding in the field of platelet and resolution biology.

Ischemia-Reperfusion Injury in Sickle Cell Disease: From Basics to Therapeutics.

Sickle cell disease (SCD) is one of the most common hereditary hemoglobinopathies worldwide, affecting almost 400,000 newborns globally each year. It is characterized by chronic hemolytic anemia and endothelial dysfunction, resulting in a constant state of disruption of the vascular system and leading to recurrent episodes of ischemia-reperfusion injury (I/RI) to multiple organ systems. I/RI is a fundamental vascular pathobiological paradigm and contributes to morbidity and mortality in a wide range of conditions, including myocardial infarction, stroke, acute kidney injury, and transplantation. I/RI is characterized by an initial restriction of blood supply to an organ, which can lead to ischemia, followed by the subsequent restoration of perfusion and concomitant reoxygenation. Recent advances in the pathophysiology of SCD have led to an understanding that many of the consequences of this disease can be explained by mechanisms associated with I/RI. The following review focuses on the evolving pathobiology of SCD, how various complications of SCD can be attributed to I/RI, and the role of timely therapeutic intervention(s) based on targeting mediators or pathways that influence I/R insult.

Hydrogen Sulfide-Synthesizing Enzymes Are Altered in a Case of Oral Cavity Mucoepidermoid Carcinoma.

Mucoepidermoid carcinoma (MEC) is the most common malignant epithelial neoplasm of the salivary glands. MECs of the mouth floor are rare, with only a few cases reported. Here we report a MEC of the mouth floor in a 55-year-old woman. Since several studies have shown that hydrogen sulfide (H2S)-synthesizing enzymes are often increased in malignant tumors compared to benign counterpart tissues, we used western blotting to compare the protein levels of cystathionine-ß-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST) in a mouth floor MEC to adjacent benign oral mucosae. We also used high-performance liquid chromatography to quantify possible differences in tissue sulfur fraction concentrations between the two biopsy types. Last, we used western blotting to examine nicotinamide phosphoribosyl transferase (Nampt), mitoNEET, and phospho-ser727-Stat3 levels in the biopsies. We found that all the proteins and phospho-ser727-Stat3 are increased in the MEC compared to benign mucosae. Interestingly, free H2S levels, acid-labile, and the sulfane sulfur factions were essentially the same between the MEC and benign tissue. Although limited to a single and unusual tumor type, to our knowledge this is only the third time H2S concentrations were directly quantified inside a human tumor. Last, our results replicate those of two previous studies where the H2S-synthesizing enzymes are increased in a malignant tumor, while free H2S is either not increased or only slightly increased, suggesting that malignant tumors rapidly metabolize H2S as part of tumor maintenance and growth.

Hydrogen Sulfide and Hydrogen Sulfide-Synthesizing Enzymes Are Altered in a Case of Oral Adenoid Cystic Carcinoma.

Adenoid cystic carcinomas (ACC) constitute 1% of all head and neck malignancies and are very rare in the oral cavity. With < 60 oral ACCs described, their pathobiology is incompletely understood. Here, we report a case of oral cavity ACC in a 54-year-old woman. Since recent studies have demonstrated that several human tumors overexpress the hydrogen sulfide (H2S)-synthesizing enzymes cystathionine-ß-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST), and also show dysregulated H2S levels, we examined these biomarkers in the oral ACC and compared the results to those of adjacent benign oral epithelium. Western blotting was used to compare the protein expression of CBS, CSE, 3-MST, nicotinamide phosphoribosyl transferase, and mitoNEET in ACC and adjacent benign oral mucosae. High-performance liquid chromatography was used to quantify the differences in tissue H2S concentrations between the two biopsy types. We found that all the proteins examined here were increased in the ACC compared to adjacent benign oral mucosae. Interestingly, H2S concentrations were decreased approximately 30% in ACC compared to benign mucosae. Thus, in one example of this rare tumor type, the enzymes that synthesize H2S are increased, while tissue H2S levels are lower than those found in adjacent benign oral mucosae. Although limited to a single rare tumor type, to our knowledge this is the second time H2S concentrations have been directly quantified inside a human tumor. Last, our results may indicate that alterations in H2S synthesis and metabolism may be important in the pathobiology of ACC.

Therapeutic Potential of Annexin A1 in Ischemia Reperfusion Injury.

Cardiovascular disease (CVD) continues to be the leading cause of death in the world. Increased inflammation and an enhanced thrombotic milieu represent two major complications of CVD, which can culminate into an ischemic event. Treatment for these life-threatening complications remains reperfusion and restoration of blood flow. However, reperfusion strategies may result in ischemia-reperfusion injury (I/RI) secondary to various cardiovascular pathologies, including myocardial infarction and stroke, by furthering the inflammatory and thrombotic responses and delivering inflammatory mediators to the affected tissue. Annexin A1 (AnxA1) and its mimetic peptides are endogenous anti-inflammatory and pro-resolving mediators, known to have significant effects in resolving inflammation in a variety of disease models. Mounting evidence suggests that AnxA1, which interacts with the formyl peptide receptor (FPR) family, may have a significant role in mitigating I/RI associated complications. In this review article, we focus on how AnxA1 plays a protective role in the I/R based vascular pathologies.

Sickle cell disease: a malady beyond a hemoglobin defect in cerebrovascular disease.

INTRODUCTION: Sickle cell disease (SCD) is a devastating monogenic disorder that presents as a multisystem illness and affects approximately 100,000 individuals in the United States alone. SCD management largely focuses on primary prevention, symptomatic treatment and targeting of hemoglobin polymerization and red blood cell sickling. Areas covered: This review will discuss the progress of SCD over the last few decades, highlighting some of the clinical (mainly cerebrovascular) and psychosocial challenges of SCD in the United States. In addition, focus will also be made on the evolving science and management of this inherited disease. Expert commentary: Until recently hydroxyurea (HU) has been the only FDA approved therapy for SCD. However, advancing understanding of SCD pathophysiology has led to multiple clinical trials targeting SCD related thrombo-inflammation, abnormal endothelial biology, increased oxidant stress and sickle cell mutation. Yet, despite advancing understanding, available therapies are limited. SCD also imposes great psychosocial challenges for the individual and the affected community, which has previously been under-recognized. This has created a pressing need for complementary adjuvant therapies with repurposed and novel drugs, in addition to the establishment of comprehensive clinics focusing on both the medical treatment and the psychosocial issues associated with SCD.

Non-celiac gluten sensitivity: All wheat attack is not celiac.

Currently, 1% of the United States population holds a diagnosis for celiac disease (CD), however, a more recently recognized and possibly related condition, "non-celiac gluten sensitivity" (NCGS) has been suggested to affect up to 6% of the United States public. While reliable clinical tests for CD exist, diagnosing individuals affected by NCGS is still complicated by the lack of reliable biomarkers and reliance upon a broad set of intestinal and extra intestinal symptoms possibly provoked by gluten. NCGS has been proposed to exhibit an innate immune response activated by gluten and several other wheat proteins. At present, an enormous food industry has developed to supply gluten-free products (GFP) with GFP sales in 2014 approaching $1 billion, with estimations projecting sales to reach $2 billion in the year 2020. The enormous demand for GFP also reflects a popular misconception among consumers that gluten avoidance is part of a healthy lifestyle choice. Features of NCGS and other gluten related disorders (e.g., irritable bowel syndrome) call for a review of current distinctive diagnostic criteria that distinguish each, and identification of biomarkers selective or specific for NCGS. The aim of this paper is to review our current understanding of NCGS, highlighting the remaining challenges and questions which may improve its diagnosis and treatment.