Effect of Hydrocortisone on Mortality and Organ Support in Patients With Severe COVID-19: The REMAP-CAP COVID-19 Corticosteroid Domain Randomized Clinical Trial.

Importance: Evidence regarding corticosteroid use for severe coronavirus disease 2019 (COVID-19) is limited. Objective: To determine whether hydrocortisone improves outcome for patients with severe COVID-19. Design, Setting, and Participants: An ongoing adaptive platform trial testing multiple interventions within multiple therapeutic domains, for example, antiviral agents, corticosteroids, or immunoglobulin. Between March 9 and June 17, 2020, 614 adult patients with suspected or confirmed COVID-19 were enrolled and randomized within at least 1 domain following admission to an intensive care unit (ICU) for respiratory or cardiovascular organ support at 121 sites in 8 countries. Of these, 403 were randomized to open-label interventions within the corticosteroid domain. The domain was halted after results from another trial were released. Follow-up ended August 12, 2020. Interventions: The corticosteroid domain randomized participants to a fixed 7-day course of intravenous hydrocortisone (50 mg or 100 mg every 6 hours) (n = 143), a shock-dependent course (50 mg every 6 hours when shock was clinically evident) (n = 152), or no hydrocortisone (n = 108). Main Outcomes and Measures: The primary end point was organ support-free days (days alive and free of ICU-based respiratory or cardiovascular support) within 21 days, where patients who died were assigned -1 day. The primary analysis was a bayesian cumulative logistic model that included all patients enrolled with severe COVID-19, adjusting for age, sex, site, region, time, assignment to interventions within other domains, and domain and intervention eligibility. Superiority was defined as the posterior probability of an odds ratio greater than 1 (threshold for trial conclusion of superiority >99%). Results: After excluding 19 participants who withdrew consent, there were 384 patients (mean age, 60 years; 29% female) randomized to the fixed-dose (n = 137), shock-dependent (n = 146), and no (n = 101) hydrocortisone groups; 379 (99%) completed the study and were included in the analysis. The mean age for the 3 groups ranged between 59.5 and 60.4 years; most patients were male (range, 70.6%-71.5%); mean body mass index ranged between 29.7 and 30.9; and patients receiving mechanical ventilation ranged between 50.0% and 63.5%. For the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively, the median organ support-free days were 0 (IQR, -1 to 15), 0 (IQR, -1 to 13), and 0 (-1 to 11) days (composed of 30%, 26%, and 33% mortality rates and 11.5, 9.5, and 6 median organ support-free days among survivors). The median adjusted odds ratio and bayesian probability of superiority were 1.43 (95% credible interval, 0.91-2.27) and 93% for fixed-dose hydrocortisone, respectively, and were 1.22 (95% credible interval, 0.76-1.94) and 80% for shock-dependent hydrocortisone compared with no hydrocortisone. Serious adverse events were reported in 4 (3%), 5 (3%), and 1 (1%) patients in the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively. Conclusions and Relevance: Among patients with severe COVID-19, treatment with a 7-day fixed-dose course of hydrocortisone or shock-dependent dosing of hydrocortisone, compared with no hydrocortisone, resulted in 93% and 80% probabilities of superiority with regard to the odds of improvement in organ support-free days within 21 days. However, the trial was stopped early and no treatment strategy met prespecified criteria for statistical superiority, precluding definitive conclusions. Trial Registration: ClinicalTrials.gov Identifier: NCT02735707

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

TLXI, a thaumatin-like xylanase inhibitor : isolation, characterisation and comparison with other wheat (Triticum aestivum L.) xylanase inhibiting proteins

The most abundant polysaccharide on earth, next to cellulose, is (arabino)xylan, an important constituent of cereal seed cell walls. It is largely degraded by xylanases. In cereals, these enzymes are mainly responsible for cell wall remodelling and degradation upon germination. The best-studied xylanases, however, originate from bacteria and fungi where they are involved in plant infection and in the decay of dead organic material. They are one of the enzymes produced by microorganisms to degrade the plant cell wall and to, in this manner, invade the plant tissues. Next to their role in plant infection, xylanases have also been well studied because of their application in cereal-based industrial processes. They are used as process, yield or product improvers in a range of different processes. Xylanases are nowadays inherently present in most bread improvers, as they beneficially affect bread volume and processibility of doughs. They can also be used in beer production, feeds and gluten-starch separation. In 1997, the occurrence of xylanase inhibiting proteins in wheat was discovered. Since then, two different types of xylanase inhibitors have been described, namely Triticum aestivum xylanase inhibitor (TAXI) and xylanase inhibitor protein (XIP) type inhibitors. More recently, during the purification of these two xylanase inhibitors from wheat whole meal, a third type of xylanase inhibitors was discovered. The research described in this doctoral thesis aimed at purifying and fully characterising this new type of xylanase inhibitor from wheat. Subsequently, it aimed at contributing to an increased understanding on the physiological role of plant xylanase inhibitors. Two different methods to purify this novel protein, starting from wheat whole meal, were developed. The first method is based on the difference in affinity for xylanases between the three types of xylanase inhibitors, while in the second method TAXI and XIP (and other proteins) are selectively precipitated using a methanolic ammonium acetate solution. The inhibitor is then isolated from the supernatant with affinity chromatography with immobilised xylanases. The first method is very convenient when all three types of inhibitors are needed, but it is rather time consuming when the only objective is to purify the last discovered one. The yield for the latter inhibitor with both methods is in the range of 2 to 4 mg per kg whole meal. Subsequently, the novel inhibitor was characterised. It is a basic protein (pI > 9.3) and has a molecular mass (MM) of approximately 18 kDa, which makes it the smallest xylanase inhibitor from wheat. It occurs in different forms with varying degrees of glycosylation. Its glycan moiety/moieties are mostly O-bound. Little, if any, N-bound sugars are present. The amino acid sequence shows homology to thaumatin and contains the consensus sequence of the thaumatin protein family. Therefore, the new inhibitor is called thaumatin-like xylanase inhibitor (TLXI). Much as for other thaumatin-like proteins (TLPs), TLXI is a very stable protein both under extreme thermal and pH conditions. It is also quite resistant towards proteolytic attack by pepsin. Much as for other TLPs, the stability of TLXI can probably be ascribed to the presence of several intramolecular disulfide bridges. The three dimensional structure model of TLXI, generated with the crystal structure of the thaumatin protein as template, indeed shows that all (ten) cysteine residues are involved in five disulfide bridges. Furthermore, it maintains that TLXI contains 12 β-strands, organised in 3 β-sheets, and no α-helices. Experimental evidence to confirm these structural features was provided. The experimental work allows concluding that TLXI is generally active towards xylanases of glycoside hydrolase family (GH) 11 with low pH optimum (pH ≤ 6.0). No inhibition activity was seen towards GH 10 xylanases, proteases or α-amylases. The inhibition activity of TLXI is generally maximal at pH conditions around pH 5.0 and at temperatures around 40 °C. In contrast to what could be observed for the other cereal xylanase inhibitors, the interaction between TLXI and xylanases does not occur in or near the active site. TLXI inhibits in a non-competitive manner, while TAXI and XIP are competitive inhibitors. In addition, TLXI is slow-tight-binding. The interaction between TLXI and a GH 11 xylanase of T. longibrachiatum (XTL1) occurs in a single slow step and is characterised by a Ki of approximately 60 nM. The Ki value is thus situated in the nanomolar range, much as for the other cereal xylanase inhibitors. The TLXI levels in whole meals of different wheat cultivars are, in general, approximately 10 and 30 times lower than the TAXI and XIP levels, respectively. In other cereals seeds, the levels of TLXI homologues, if present, seem also lower than those of TAXI and XIP homologues, if present. In rye and barley, protein bands that cross-react with antibodies raised against wheat TLXI were detected in the fraction recovered after affinity chromatography with immobilised xylanases. In barley, this protein had a MM of approximately 27 kDa, while that in rye had a MM of 18 kDa. The N-terminal amino acid sequence of the latter was identical to that of TLXI from wheat and that of the protein expressed by the putative rye tlxi gene, confirming the actual expression of this gene in rye. We could not identify a TLXI homologue in barley. Because the accumulation of different TLPs during fruit ripening has been described, different fruits were also studied for the occurrence of TLXI homologues. A 26 kDa kiwi protein, of which the N-terminal amino acid sequence was identical to that of a thaumatin-like kiwi allergen with antifungal activity, binds to immobilised xylanases but does not inhibit xylanases in solution. In contrast, a similar protein fraction from apple displayed xylanase inhibition activity. The cross-reactivity with anti-TLXI antibodies and the MM of the protein present in this fraction suggest that the protein is similar to that in kiwi, but no N-terminal amino acid sequence could be determined. Finally in banana, a 21 kDa protein was found, which shows inhibition activity towards GH 11 xylanases of Bacillus subtilis (XBS) and Aspergillus niger (XAN11), but no cross-reactivity with the anti-TLXI antibodies. This suggests the presence of an unknown type of xylanase inhibitors in banana. During the study of the kinetic parameters, it came to light that TLXI not only interacts with xylanases, but also with arabinoxylan. As polysaccharide binding had already been reported for XIP, a profound study of the interaction of all three xylanase inhibitors with polysaccharides was performed. It revealed that all types of inhibitors can bind to (arabino)xylans and that their affinity increases with decreasing arabinose to xylose ratio. The highest level of binding was observed for TLXI. In addition, interaction with certain β-glucans was observed for all inhibitors, though this binding was less pronounced than that to (arabino)xylans. For TLPs, a correlation exists between the binding to polysaccharides and their antifungal activity. In addition, a role in plant defence has been suggested for xylanase inhibitors, without insight in the exact mechanism. The present PhD research studied their direct effect on fungal growth. The hyphal growth of Fusarium graminearum and Rhizoctonia solani was inhibited by XIP and TAXI, respectively, when a medium with glucose as only carbon source was used. Since xylan was absent in the medium, xylanase inhibition activity is probably not the cause of this antifungal activity. When medium with xylan as only carbon source was used, the halo expansion, caused by xylanase activity of Penicillium expansum and Aspergillus flavus, was retarded by all three inhibitors. No effect on the hyphal growth on the latter medium was observed. In short, this dissertation describes the purification and full characterisation of a recently discovered type of xylanase inhibitors, which belongs to the thaumatin family. Furthermore, it contributes to the unravelling of the function of xylanase inhibitors by demonstrating their binding affinity for polysaccharides and their inhibitory effect on fungal growth.status: publishe

Current and forward looking experimental approaches in gluten-free bread making research

Research efforts on gluten-free bread making have rapidly increased during the last decade. A lot of different approaches are being used to improve the quality of these products. The techniques used in gluten-free bread making research vary widely. This review focuses on the methodological aspects of gluten-free bread making research and extracts relevant data from all Web of Science peer reviewed research articles on gluten-free bread published from 2010 to date. Recipes and methodologies are grouped by (main) starch source and list other ingredients, additives and treatments used. The focus lies on the experimental setups typically used to analyze batter/dough and end product. Small deformation rheological measurements are typically performed on gluten-free batter/dough, along with several other batter/dough properties, but there is no clear link between these characteristics and the bread quality which typically is determined by volume and texture analysis or sensory evaluation. Some more recent techniques that have already been used on wheat bread or other bakery products are discussed as well. Their application in gluten-free bread making research may help extend the current knowledge.status: publishe

The impact of baking time and bread storage temperature on bread crumb properties

Two baking times (9 and 24min) and storage temperatures (4 and 25°C) were used to explore the impact of heat exposure during bread baking and subsequent storage on amylopectin retrogradation, water mobility, and bread crumb firming. Shorter baking resulted in less retrogradation, a less extended starch network and smaller changes in crumb firmness and elasticity. A lower storage temperature resulted in faster retrogradation, a more rigid starch network with more water inclusion and larger changes in crumb firmness and elasticity. Crumb to crust moisture migration was lower for breads baked shorter and stored at lower temperature, resulting in better plasticized biopolymer networks in crumb. Network stiffening, therefore, contributed less to crumb firmness. A negative relation was found between proton mobilities of water and biopolymers in the crumb gel network and crumb firmness. The slope of this linear function was indicative for the strength of the starch network.publisher: Elsevier articletitle: The impact of baking time and bread storage temperature on bread crumb properties journaltitle: Food Chemistry articlelink: http://dx.doi.org/10.1016/j.foodchem.2013.06.031 content_type: article copyright: Copyright © 2013 Elsevier Ltd. All rights reserved.status: publishe

Impact of egg white and soy protein on crumb firming in gluten-free breads based on rice flour or a mixture of cassava and potato starch

Oral Presentation.status: publishe

Wheat (Triticum aestivum L.) puroindoline functionality in bread making and its impact on bread quality

Wheat puroindolines (PINs) spontaneously adsorb at air/water interfaces and show excellent foaming properties. They can positively impact bread quality, in which the formation of stable foam is important for product quality. The impact of endogenous PINs on bread quality was studied by preparing gluten–starch blends from isolated gluten and starch fractions with different PIN levels, which allowed largely retaining the interaction between PINs and flour components. Our results indicate that blends with high PIN levels yielded more homogeneous crumb structures with fine gas cells than bread made with blends containing medium or low PIN levels. However, the mechanism by which PINs exert this crumb improving effect is not clear. Varying PIN levels impacted neither dough extensibility nor did it result in different PIN levels in dough liquor. Lipid removal yielded bread with a less homogeneous crumb gas cell distribution, indicating that lipids also are required to obtain good crumb structure.status: publishe

Fermentation affects the composition and foaming properties of the aqueous phase of dough from soft wheat flour

status: publishe

Xylanase inhibitors bind to nonstarch polysaccharides

This study is an in-depth investigation of the interaction between polysaccharides and the proteinaceous xylanase inhibitors, Triticum aestivum xylanase inhibitor (TAXI), xylanase inhibitor protein (XIP), and thaumatin-like xylanase inhibitor (TLXI). The binding affinities of all three known types of xylanase inhibitors from wheat are studied by measuring the residual xylanase inhibition activity after incubation of the inhibitors in the presence of different polysaccharides, such as beta-glucans and (arabino)xylans. The binding affinities of all three xylanase inhibitors for (arabino)xylans increased with a decreasing arabinose/xylose ratio (A/X ratio). This phenomenon was observed both with water-extractable and water-unextractable (arabino)xylans. The inhibitors also interacted with different soluble and insoluble beta-glucans. None of the inhibitors tested had the ability to hydrolyze the polysaccharides investigated. The present findings contribute to the unraveling of the function of xylanase inhibitors in nature and to the prediction of the effect of added xylanases in cereal-based biotechnological processes, such as bread making and gluten-starch separation.status: publishe