Proteomics identifies apoptotic markers as predictors of histological transformation in patients with follicular lymphoma.

Follicular lymphoma (FL) is an indolent lymphoma with a generally favorable prognosis. However, histological transformation (HT) to a more aggressive disease leads to markedly inferior outcomes. This study aims to identify biological differences predictive of HT at the time of initial FL diagnosis. We show differential protein expression between diagnostic lymphoma samples from patients with subsequent HT (subsequently-transforming FL [st-FL]; n = 20) and patients without HT (nontransforming FL [nt-FL]; n = 34) by label-free quantification nano liquid chromatography-tandem mass spectrometry analysis. Protein profiles identified patients with high risk of HT. This was accompanied by disturbances in cellular pathways influencing apoptosis, the cytoskeleton, cell cycle, and immune processes. Comparisons between diagnostic st-FL samples and paired transformed FL (n = 20) samples demonstrated differential protein profiles and disrupted cellular pathways, indicating striking biological differences from the time of diagnosis up to HT. Immunohistochemical analysis of apoptotic proteins, CASP3, MCL1, BAX, BCL-xL, and BCL-rambo, confirmed higher expression levels in st-FL than in nt-FL samples (P < .001, P = .015, P = .003, P = .025, and P = .057, respectively). Moreover, all 5 markers were associated with shorter transformation-free survival (TFS; P < .001, P = .002, P < .001, P = .069, and P = .010, respectively). Notably, combining the expression of these proteins in a risk score revealed increasingly inferior TFS with an increasing number of positive markers. In conclusion, proteomics identified altered protein expression profiles (particularly apoptotic proteins) at the time of FL diagnosis, which predicted later transformation.

IDO1 Protein Is Expressed in Diagnostic Biopsies from Both Follicular and Transformed Follicular Patients.

Follicular lymphoma (FL) is a lymphoid neoplasia characterized by an indolent clinical nature. Despite generally favorable prognoses, early progression and histological transformation (HT) to a more aggressive lymphoma histology remain the leading causes of death among FL patients. To provide a basis for possible novel treatment options, we set out to evaluate the expression levels of indoleamine 2,3-dioxygenase 1 (IDO1), an immunoinhibitory checkpoint molecule, in follicular and transformed follicular biopsies. The expression levels of IDO1 were assessed using immunohistochemical staining and digital image analysis in lymphoma biopsies from 33 FL patients without subsequent HT (non-transforming FL, nt-FL) and 20 patients with subsequent HT (subsequently transforming FL, st-FL) as well as in paired high-grade biopsies from the time of HT (transformed FL, tFL). Despite no statistical difference in IDO1 expression levels seen between the groups, all diagnostic and transformed lymphomas exhibited positive expression, indicating its possible role in novel treatment regimens. In addition, IDO1 expression revealed a positive correlation with another immune checkpoint inhibitor, namely programmed death 1 (PD-1). In summary, we report IDO1 expression in all cases of FL and tFL, which provides the grounds for future investigations of anti-IDO1 therapy as a possible treatment for FL patients.

The role of skin inflammation, barrier dysfunction, and oral tolerance in skin sensitization to gluten-derived hydrolysates in a rat model.

BACKGROUND: Adverse reactions to wheat-containing skin care products have been linked to food allergy development. OBJECTIVES: To determine the role of skin barrier dysfunction and inflammation in sensitization to gluten-derived hydrolysates via the skin in Brown Norway rats with and without oral tolerance to wheat. METHODS: Skin barrier defect was induced by mechanical disruption, and skin inflammation was induced by topical application of SLS or MC903. Unmodified, enzyme hydrolyzed, or acid hydrolyzed gluten products were applied to the skin three times per week for 5 weeks. Subsequently, rats were orally gavaged with unmodified gluten. RESULTS: Wheat-naïve rats were readily sensitized to gluten hydrolysates via the skin. Skin barrier defect and skin inflammation had little effect on the skin sensitization and hydrolysate-specific IgE levels. Oral administration of unmodified gluten promoted the production of unmodified gluten-specific IgE in rats sensitized via the skin. Sensitization through intact skin, disrupted skin barrier, or inflamed skin was unable to break tolerance to unmodified gluten in rats on a wheat-containing diet. CONCLUSIONS: Mechanical skin barrier disruption and skin inflammation play a limited role in experimental skin sensitization to gluten-derived hydrolysates.

Risk of consuming products with or without precautionary wheat or gluten labelling for persons with coeliac disease.

Persons with Coeliac disease (CD) need to keep a lifelong diet without gluten and therefore need to know which foods are safe to eat. The objective of this study was to analyse ordinary foods products without gluten containing ingredients (PWG) with or without the precautionary allergen statement (PAL) "May contain wheat/gluten" or similar wording and use these data to perform a probabilistic risk assessment. Foods with and without PAL were analysed for gluten. Inputs for the risk assessment were analytical results, daily food consumption and data from a 90-days challenge study in coeliac patients (Catassi et al., 2007).20/77 products with PAL and 10/51 without PAL had gluten ≥5 mg/kg 3/77 with PAL and 3/51 without PAL had gluten ≥20 mg/kg. A coconut cookie brand was outlier with 421 mg gluten/kg. The likelihood of developing mucosal damage in the CD population when eating the same PWG for 90 days in addition to certified gluten-free products was low (0.2-3.2%) with little difference between with or without PAL. Including the coconut cookie results increased the risk to 3.8-9.2%. Caution should be taken in connection to foods where there is an obvious chance of contamination.

Tumor-Tissue Expression of the Hyaluronic Acid Receptor RHAMM Predicts Histological Transformation in Follicular Lymphoma Patients.

Histological transformation (HT) remains the leading cause of mortality in follicular lymphoma (FL), underlining the need to identify reliable transformation predictors. The hyaluronic acid receptors CD44 and the receptor for hyaluronan mediated motility (RHAMM, also known as HMMR and CD168), have been shown to be involved in the pathogeneses of both solid tumors and hematological malignancies. In an attempt to improve risk stratification, expression of RHAMM and CD44 were evaluated by immunohistochemistry and digital image analysis in pre-therapeutic tumor-tissue biopsies from FL patients, either without (nt-FL, n = 34), or with (st-FL, n = 31) subsequent transformation, and in paired biopsies from the transformed lymphomas (tFL, n = 31). At the time of initial diagnosis, samples from st-FL patients had a higher expression of RHAMM compared with samples from nt-FL patients (p < 0.001). RHAMM expression further increased in tFL samples following transformation (p < 0.001). Evaluation of CD44 expression showed no differences in expression comparing nt-FL, st-FL, and tFL samples. Shorter transformation-free survival was associated with high tumoral and intrafollicular RHAMM expression, as well as with low intrafollicular CD44 expression (p = 0.002, p < 0.001, and p = 0.034, respectively). Our data suggest that high tumor-tissue RHAMM expression predicts the risk of shorter transformation-free survival in FL patients already at initial diagnosis.

Acid Hydrolysis of Gluten Enhances the Skin Sensitizing Potential and Drives Diversification of IgE Reactivity to Unmodified Gluten Proteins.

SCOPE: Personal care products containing hydrolyzed gluten have been linked to spontaneous sensitization through the skin, however the impact of the hydrolysate characteristics on the sensitizing capacity is generally unknown. METHODS AND RESULTS: The physicochemical properties of five different wheat-derived gluten products (one unmodified, one enzyme hydrolyzed, and three acid hydrolyzed) are investigated, and the skin sensitizing capacity is determined in allergy-prone Brown Norway rats. Acid hydrolyzed gluten products exhibited the strongest intrinsic sensitizing capacity via the skin. All hydrolyzed gluten products induced cross-reactivity to unmodified gluten in the absence of oral tolerance to wheat, but were unable to break tolerance in animals on a wheat-containing diet. Still, the degree of deamidation in acid hydrolyzed products is associated with product-specific sensitization in wheat tolerant rats. Sensitization to acid hydrolyzed gluten products is associated with a more diverse IgE reactivity profile to unmodified gluten proteins compared to sensitization induced by unmodified gluten or enzyme hydrolyzed gluten. CONCLUSION: Acid hydrolysis enhances the skin sensitizing capacity of gluten and drives IgE reactivity to more gluten proteins. This property of acid hydrolyzed gluten may be related to the degree of product deamidation, and could be a strong trigger of wheat allergy in susceptible individuals.

PD-1 Expression in Pre-Treatment Follicular Lymphoma Predicts the Risk of Subsequent High-Grade Transformation.

PURPOSE: Follicular lymphoma (FL) is an indolent, yet generally incurable neoplasia with a median survival exceeding 10 years. However, a subset of FL patients experiences histological transformation (HT) to a more aggressive lymphoma, in the majority of cases to diffuse large B-cell lymphoma (DLBCL). This affects both the clinical course and the prognostic outcome, resulting in a markedly reduced survival after transformation. Thus, early risk stratification and prediction of patients at risk of HT would be highly valuable in the clinical setting. Here, we investigated the potential of the immune inhibitory programmed death 1 (PD-1) receptor as a biomarker predictive of HT. PATIENTS AND METHODS: Immunohistochemical staining and quantification by digital image analysis of PD-1 was performed on diagnostic tumor-tissue samples from FL patients with and without subsequent transformation (n=34 and n=46, respectively), and on paired samples from the transformed lymphoma (n=34). RESULTS: At the time of initial FL diagnosis, samples from patients with subsequent HT had significantly higher tumor-tissue expression of PD-1 compared with diagnostic FL samples from patients without subsequent HT (p=0.010). At the time of transformation, PD-1 expression was significantly reduced (p<0.001). No difference was observed in intra-follicular PD-1 expression at FL diagnosis between samples from patients with or without HT; however, high intra-follicular levels of PD-1 were associated with significantly shorter transformation-free survival times (p<0.043). CONCLUSION: Our data suggest that pre-treatment tumor-tissue PD-1 expression already predicts the risk of subsequent transformation to DLBCL, as early as the time of FL diagnosis.

An Aldehyde Responsive, Cleavable Linker for Glucose Responsive Insulins.

A glucose responsive insulin (GRI) that responds to changes in blood glucose concentrations has remained an elusive goal. Here we describe the development of glucose cleavable linkers based on hydrazone and thiazolidine structures. We developed linkers with low levels of spontaneous hydrolysis but increased level of hydrolysis with rising concentrations of glucose, which demonstrated their glucose responsiveness in vitro. Lipidated hydrazones and thiazolidines were conjugated to the LysB29 side-chain of HI by pH-controlled acylations providing GRIs with glucose responsiveness confirmed in vitro for thiazolidines. Clamp studies showed increased glucose infusion at hyperglycemic conditions for one GRI indicative of a true glucose response. The glucose responsive cleavable linker in these GRIs allow changes in glucose levels to drive the release of active insulin from a circulating depot. We have demonstrated an unprecedented, chemically responsive linker concept for biopharmaceuticals.

The Validity of Intracerebral Hemorrhage Diagnoses in the Danish Patient Registry and the Danish Stroke Registry.

PURPOSE: To establish the validity of intracerebral hemorrhage (ICH) diagnoses in the Danish Stroke Registry (DSR) and the Danish National Patient Registry (DNPR). PATIENTS AND METHODS: Based on discharge summaries and brain imaging reports, we estimated the positive predictive value (PPV) of a first-ever diagnosis code for ICH (ICD-10, code I61) for all patients in the Region of Southern Denmark (1.2 million) during 2009-2017 according to either DNPR or DSR. We estimated PPVs for any non-traumatic ICH (a-ICH) and spontaneous ICH (s-ICH) alone (ie, without underlying structural cause). We also calculated the sensitivity of these diagnoses in each of the registers. Finally, we classified the location of verified s-ICH. RESULTS: A total of 3,956 patients with ICH diagnosis codes were studied (DSR only: 87; DNPR only: 1,513; both registries: 2,356). In the DSR, the PPVs were 86.5% (95% CI=85.1-87.8) for a-ICH and 81.8% (95% CI=80.2-83.3) for s-ICH. The PPVs in DNPR (discharge code, primary diagnostic position) were 76.2% (95% CI=74.7-77.6) for a-ICH and 70.2% (95% CI=68.6-71.8) for s-ICH. Sensitivity for a-ICH and s-ICH was 76.4% (95% CI=74.8-78.0) and 78.7% (95% CI=77.1-80.2) in DSR, and 87.3% (95% CI=86.0-88.5) and 87.7% (95% CI=86.3-88.9) in DNPR. The location of verified s-ICH was lobar (39%), deep (33.6%), infratentorial (13.2%), large unclassifiable (11%), isolated intraventricular (1.9%), or unclassifiable due to insufficient information (1.3%). CONCLUSION: The validity of a-ICH diagnoses is high in both registries. For s-ICH, PPV was higher in DSR, while sensitivity was higher in DNPR. The location of s-ICH was similar to distributions seen in other populations.

Real world data on histological transformation in patients with follicular lymphoma: incidence, clinico-pathological risk factors and outcome in a nationwide Danish cohort.

We investigated incidence, risk factors and outcome for follicular lymphoma (FL) patients with histologic transformation (HT) found at primary diagnosis (discordant/composite, dc-tFL) or sequentially (s-tFL). Between 2000 and 2015, 2773 patients were identified. The majority of patients (2252, 81%) did not experience HT (nt-FL), while 224 (8%) had dc-tFL and 297 (11%) s-tFL. The risk of HT was 2.2% per year and 9.6% at 5 years. Age ≥60, a high FLIPI risk score and LDH-elevation were associated with increased risk of HT. Calculated from primary diagnosis and compared with nt-FL, 5-year overall survival (OS) was inferior in both s-tFL and dc-tFL (nt-FL: 82%, s-tFL: 68%, dc-tFL: 68%; p = .001), whereas 5-year progression-free survival (PFS) was worse only in s-tFL (s-tFL: 18%, dc-tFL: 58%, nt-FL: 60%). Calculated from time of HT, s-tFL had inferior outcome compared to dc-tFL for both OS (s-tFL: 47%, dc-tFL: 68%, p = .001) and PFS (s-tFL: 35%, dc-tFL: 58%, p = .001).

Can we define a level of protection for allergic consumers that everyone can accept?

Substantial progress has been made in characterising the risk associated with exposure to allergens in food. However, absence of agreement on what risk is tolerable has made it difficult to set quantitative limits to manage that risk and protect allergic consumers effectively. This paper reviews scientific progress in the area and the diverse status of allergen management approaches and lack of common standards across different jurisdictions, including within the EU. This lack of regulation largely explains why allergic consumers find Precautionary Allergen Labelling confusing and cannot rely on it. We reviewed approaches to setting quantitative limits for a broad range of food safety hazards to identify the reasoning leading to their adoption. This revealed a diversity of approaches from pragmatic to risk-based, but we could not find clear evidence of the process leading to the decision on risk acceptability. We propose a framework built around the criteria suggested by Murphy and Gardoni (2008) for approaches to defining tolerable risks. Applying these criteria to food allergy, we concluded that sufficient knowledge exists to implement the framework, including sufficient expertise across the whole range of stakeholders to allow opinions to be heard and respected, and a consensus to be achieved.

Acute Neurofilament Light Chain Plasma Levels Correlate With Stroke Severity and Clinical Outcome in Ischemic Stroke Patients.

Background: Ischemic stroke causes increased blood-brain barrier permeability and release of markers of axonal damage and inflammation. To investigate diagnostic and prognostic roles of neurofilament light chain (NF-L), we assessed levels of NF-L, S100B, interleukin-6 (IL-6), E-selectin, vascular endothelial growth factor-A (VEGF-A), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) in patients with acute ischemic stroke or transient ischemic attack (TIA) and healthy controls. Methods: We studied neurofilament (NF) expression in 2 cases of human postmortem ischemic stroke, representing infarcts aged 3- to >7-days. In a prospective study, we measured plasma NF-L and inflammatory markers <8 h of symptom onset and at 72 h in acute ischemic stroke (n = 31), TIA (n = 9), and healthy controls (n = 29). We assessed whether NF-L, S100B, and IL-6 were associated with clinical severity on admission (Scandinavian Stroke Scale, SSS), diagnosis of ischemic stroke vs. TIA, and functional outcome at 3 months (modified Rankin Scale, mRS). Results: NF expression increased in ischemic neurons and in the infarcted brain parenchyma after stroke. Plasma NF-L levels were higher in stroke patients than in TIA patients and healthy controls, but IL-6 levels were similar. Higher acute NF-L levels were associated with lower SSS scores at admission and higher mRS scores at 3 months. No correlation was observed between NF-L and S100B, NF-L and IL-6, nor between S100B or IL-6 and SSS or mRS. Compared to controls, stroke patients had significantly higher VEGF-A and VCAM-1 at <8 h that remained elevated at 72 h, with significantly higher VEGF-A at <8 h; ICAM-1 was significantly increased at <8 h, while S100B and E-selectin were unchanged. Conclusions: Plasma NF-L levels, but not IL-6 and S100B, were significant predictors of clinical severity on admission and functional outcome at 3 months. Plasma NF-L is a promising biomarker of functional outcome after ischemic stroke.

Glycolytic biomarkers predict transformation in patients with follicular lymphoma.

Follicular lymphoma (FL) is an indolent neoplasia comprising approximately 20% of lymphomas. FL is generally considered incurable, with a median survival exceeding 10 years. A subset of FL patients experiences histological transformation (HT) to a more aggressive lymphoma, resulting in markedly poorer clinical outcome, with a reduced median survival after transformation of 1-2 years. Early, reliable prediction of HT would be valuable in the clinical setting, allowing pre-emptive therapeutic intervention. We previously used proteomics to identify the glycolytic enzymes fructose-bisphosphate aldolase A (aldolase A) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as candidate predictors of FL transformation. Now, we use immunohistochemistry to evaluate expression of these enzymes in paired primary FLs from patients with (n = 41) or without subsequent HT (n = 49), to test their value as predictive biomarkers. At initial FL diagnosis, patients with subsequent HT had significantly higher expression of aldolase A and GAPDH (p<0.001 and p<0.01) compared with patients without HT. Furthermore, high expression of aldolase A and GAPDH was associated with significantly shorter transformation free survival (p = 0.018, p = 0.001). These data suggest that high expression of aldolase A and GAPDH, may indicate increased metabolic turnover, and that these enzymes may be useful biomarkers in primary FL for predicting the risk of subsequent lymphoma transformation.

The use of aluminium hydroxide as adjuvant modulates the specific antibody response-A Brown Norway rat study with native and denatured cow's milk allergens.

There is a need for efficient methods to treat food allergy; however, no immunotherapeutic method has yet been satisfactory due to the high rate of unpredictable severe reactions and the limited efficacy. Therefore, modified versions of food allergens have been suggested as alternatives to the parent proteins for immunotherapy. The aim of the study was to compare the inherent allergenicity of the native and denatured version of the cow's milk proteins ß-lactoglobulin and α-lactalbumin, and to study the impact of the use of Al(OH)3 as an adjuvant. Brown Norway rats were immunized intraperitoneally with either native or denatured ß-lactoglobulin or α-lactalbumin, with or without the use of Al(OH)3 as adjuvant. Antibody responses were analysed in various ways by means of different ELISAs. Both the immunogenicity and the sensitizing capacity of the cow's milk allergens were influenced by their globular folding, with the native version being more allergenic than the denatured counterpart. The native folded proteins mainly raised antibodies against conformational epitope, whereas the denatured versions predominantly raised antibodies against linear epitopes. Most interestingly, the study showed that the use of Al(OH)3 , besides increasing immunogenicity and sensitizing capacity of the cow's milk allergens, caused a modification of the specificity of the antibodies raised against the native version of the proteins. Adsorption of the native forms of the allergens to Al(OH)3 caused a significant greater proportion of antibodies raised against linear epitopes, stressing that the adsorption induced a partly unfolding of the proteins. This may have implications for IT safety and efficacy.

Gluten tolerance prevents oral sensitization with enzymatic or acid hydrolyzed gluten: A study in Brown Norway rats.

BACKGROUND: There are several reports describing allergy to hydrolyzed wheat products. After a large outbreak in Japan it was established that sensitization was caused by skin contact with acid hydrolyzed gluten in soap. It is still not clear if other forms of hydrolyzed gluten may sensitize, and if the skin is the only relevant route of sensitization in humans and to what extent oral tolerance to wheat play a role. OBJECTIVES: The aim of the present study was to examine if wheat-tolerant rats may be sensitized via the oral or i.p. route when exposed to gluten, enzymatic or acid hydrolyzed gluten. METHODS: Brown Norway rats, tolerant to wheat, were dosed by three i.p. injections without adjuvant or by oral gavage daily for 35 days with the three gluten products, respectively. Sera were analyzed by ELISA for specific IgG1 and IgE. In addition inhibition and avidity ELISAs were performed. Results were compared to a similar study in rats naïve to wheat. RESULTS: More than half the animals had measurable IgG1 at the start of the dosing period. I.p. immunization resulted in significant specific IgG1 and IgE to the antigen used for immunization but significantly lower than in naïve rats. The results of inhibition and avidity ELISA's indicate that the underlying tolerance to epitopes common to the three products influences the immune response. Oral dosing did not induce significant changes in response to either gluten or the hydrolyzed gluten product used for dosing. CONCLUSIONS: The study shows that i.p. immunization with the three products can break the underlying tolerance to wheat. Exposure by the oral route to enzymatic or acid hydrolyzed gluten is very unlikely to break an already established tolerance to gluten and induce sensitization.

Predictive Markers of Atrial Fibrillation in Patients with Transient Ischemic Attack.

BACKGROUND: Atrial fibrillation (AF) is a major cause of cardio-embolism in patients with stroke and transient ischemic attack (TIA). Insertable cardiac monitors (ICM) make long-term monitoring for AF possible, but limited health care resources make patient selection important. AF is associated with atherosclerosis and markers of this could potentially be used to guide AF monitoring. METHODS AND RESULTS: One-hundred fourteen TIA-patients without AF were thoroughly monitored for AF with ECG, 72-hour Holter monitoring and ICM with a median monitoring time of 2.2 years. Patients with AF (n = 18) were significantly older than patients without AF (age 71.1 versus 64.4 years, P = .008) but were otherwise similar in regards to comorbidities. AF patients had significantly thicker carotid intima-media and also more often presence of carotid plaques than patients without AF, but no difference was found after adjusting for age and sex. No difference in noncontrast cardiac CT calculated coronary artery calcium score was found between the 2 groups. Serum biomarkers did not differ between groups, except for brain natriuretic peptide (BNP), where patients with BNP in the upper tertile were more likely to have AF than patients with BNP in the lowest tertile, odds ratio 5.96 (95% confidence interval 1.04-34.07, P = .045). CONCLUSIONS: Carotid intima-media thickness and coronary artery calcium score were poor predictors of AF in patients with TIA. Apart from BNP, the examined biomarkers (hs-CRP, MR-proADM, c-TnI, copeptin) had no predictive value, but larger scale studies are needed to confirm these findings.

Frequentist and Bayesian approaches for food allergen risk assessment: risk outcome and uncertainty comparisons.

Peer-reviewed probabilistic methods already predict the probability of an allergic reaction resulting from an accidental exposure to food allergens, however, the methods calculate it in different ways. The available methods utilize the same three major input parameters in the risk model: the risk is estimated from the amount of food consumed, the concentration of allergen in the contaminated product and the distribution of thresholds among allergic persons. However, consensus is lacking about the optimal method to estimate the risk of allergic reaction and the associated uncertainty. This study aims to compare estimation of the risk of allergic reaction and associated uncertainty using different methods and suggest improvements. Four cases were developed based on the previous publications and the risk estimations were compared. The risk estimation was found to agree within 0.5% with the different simulation cases. Finally, an uncertainty analysis method is also presented in order to evaluate the uncertainty propagation from the input parameters to the risk.

Can dietary strategies in early life prevent childhood food allergy? A report from two iFAAM workshops.

Food allergy affects a small but significant number of children and adults. Food allergy is responsible for considerable morbidity and is the commonest cause of anaphylaxis in children. One of the aims of the European Union-funded "Integrated Approaches to Food Allergen and Allergy Risk Management" (iFAAM) project was to improve our understanding of the best way to prevent the development of food allergy. Groups within the project worked on integrating the current prevention evidence base as well as generating new data to move our understanding forward. This paper from the iFAAM project is a unique addition to the literature on this topic as it not only outlines the recently published randomized controlled trials (as have previous reviews) but also summarizes two iFAAM-associated project workshops. These workshops focused on how we may be able to use dietary strategies in early life to prevent the development of food allergy and summarized the range of opinions amongst experts in this controversial area.