Cultivation of microbial cultures at high cell density in microbioreactor fermentations to increase protein yield.

Microbioreactors have been proven to be a useful tool in high-throughput applications, such as clone screening, synthetic library testing, and media optimization. Most were designed for low cell density applications, where the optical density of the cultures typically does not exceed an OD600 of 10. In microbial applications, where protein is to be expressed, such a scale is not sufficient to produce material for extensive target molecule testing. Here, we present a method for growing high-cell density Escherichia coli cultures in milliliter-scale bioreactors, to produce milligram quantities of target protein. We used a micro-Matrix system with a starting volume of 3 ml per culture. A combination of defined medium, a fed-batch feeding strategy at low temperature, and an advanced self-adapting control algorithm achieved up to 0.7 g of wet cell weight (WCW) in a 5.7 ml final culture volume, which corresponds to 123 g/L WCW. This translates to an estimated protein yield of 1150 mg of target protein per liter final volume.

Rheumatoid factor and anti-citrullinated peptide antibodies in the general population: hepatitis B and C virus association and 15-year-risk of rheumatoid arthritis.

OBJECTIVES: The study aimed to determine the prevalence of rheumatoid factor (RF) and anti-citrullinated peptides (ACPA), to estimate the association with hepatitis B (HBV) or C (HCV) virus infections and the 15-year risk of developing RA in a large cohort from a Northern Italian region. METHODS: In 1998, 15,907 subjects between the ages of 18 and 75 were randomly selected 1:4 for HBV and HCV testing; more recently, we tested a subgroup of sera for RF (n=2196) and ACPA (n=2525). Administrative databases were searched after 15 years for incident RA diagnoses occurring between 1998 and 2013. RESULTS: RF was positive in 8.1% of cases with 10% of RF-positive subjects having HBsAg (p=0.004) and 9% anti-HCV. ACPA were detected in 4.8% of subjects with 5% of the ACPA-positive subjects having HBsAg and 5.9% anti-HCV. Older subjects had higher positivity rates for both RF and ACPA. HBsAg and anti-HCV were detected in 5.5% and 4.3% of sera, respectively. Over 15 years, 10 RA cases were recorded (9 women, median age at diagnosis 52 years) with RF previously positive in 2/10 and ACPA in 5/10 cases. RF and ACPA were associated with relative risks for developing RA of 5.7 (adjusted for HBsAg status; 95% CI 1.2-26.3) and 13.2 (95% CI 3.8-46.3), respectively. CONCLUSIONS: Our data in a large cohort from an unselected general population confirm a higher risk of RA development associated with ACPA compared to RF. HBV exposure correlates with RF but not with ACPA positivity.

IgA antibodies against CD74 are associated with structural damage in the axial skeleton in patients with axial spondyloarthritis.

OBJECTIVES: To study the association between the presence of antibodies against CD74 and structural damage in the sacroiliac joints and spine in patients with axial spondyloarthritis (axSpA). METHODS: Antibodies against CD74 were measured in the sera of patients with axSpA from 2 cohorts: 1. An observational cohort from Damp in Northern Germany and 2. from a clinical trial (ENRADAS), in which the modified Stoke Ankylosing Spondylitis Spine Score (mSASSS) had been evaluated by two readers blinded to the time point at baseline and two years later. The presence of antibodies against CD74 was correlated with the presence and grade of radiographic sacroiliitis in the observational cohort, and with baseline mSASSS in the ENRADAS cohort. RESULTS: The sensitivity of IgA anti-CD74 antibodies for axSpA was 50% in the Damp cohort and 42% in ENRADAS. The presence of IgA antibodies against CD74 was associated with a higher grade of sacroiliitis (observational cohort) and a higher baseline mSASSS in the ENRADAS cohort. CONCLUSIONS: IgA antibodies against CD74 are not only markers of AS, but are associated with structural damage development in the sacroiliac joints and in the spine.

CD74 is a T cell antigen in spondyloarthritis.

OBJECTIVES: Spondyloarthritis (SpA) is a chronic inflammatory disease of unknown aetiology. Previously, we identified autoantibodies against CD74 in sera of SpA patients. The aim of this study was to evaluate CD74 as a T cell antigen in SpA. METHODS: Recombinant CD74 protein and a panel of selected peptides representing its amino acid residues were examined for their capability to stimulate peripheral blood mononuclear cells from patients with SpA. In particular, cytokine production by CD4+ T cells was evaluated with flow cytometric detection of intracellular TNF-α, IFNγ, TGFß and IL-17A. Patients' sera were tested for antibodies against CD74 using ELISA. Samples from patients with rheumatoid arthritis and healthy blood donors were similarly tested as controls. RESULTS: Significantly more CD4+ T cells from SpA patients produced TNF-α, IFNγ and IL-17A in response to recombinant CD74 than patients with rheumatoid arthritis or healthy blood donors. Among evaluated epitopes, the most promiscuous one lies within the peptide of the amino acid residues 142-185, which appeared more immunogenic. Further, the proportion of cytokine producing CD4+ T cells was significantly higher among SpA patients with autoantibodies against CD74. CONCLUSIONS: CD74 is a T cell antigen in SpA, eliciting Th1 and Th17 responses, which may be relevant in disease pathogenesis. Recognition of the highly immunogenic amino acid residues of CD74 may contribute to our understanding of autoimmune responses of T helper cells in SpA.

Processed Food Additive Microbial Transglutaminase and Its Cross-Linked Gliadin Complexes Are Potential Public Health Concerns in Celiac Disease.

Microbial transglutaminase (mTG) is a survival factor for microbes, but yeasts, fungi, and plants also produce transglutaminase. mTG is a cross-linker that is heavily consumed as a protein glue in multiple processed food industries. According to the manufacturers' claims, microbial transglutaminase and its cross-linked products are safe, i.e., nonallergenic, nonimmunogenic, and nonpathogenic. The regulatory authorities declare it as "generally recognized as safe" for public users. However, scientific observations are accumulating concerning its undesirable effects on human health. Functionally, mTG imitates its family member, tissue transglutaminase, which is the autoantigen of celiac disease. Both these transglutaminases mediate cross-linked complexes, which are immunogenic in celiac patients. The enzyme enhances intestinal permeability, suppresses mechanical (mucus) and immunological (anti phagocytic) enteric protective barriers, stimulates luminal bacterial growth, and augments the uptake of gliadin peptide. mTG and gliadin molecules are cotranscytosed through the enterocytes and deposited subepithelially. Moreover, mucosal dendritic cell surface transglutaminase induces gliadin endocytosis, and the enzyme-treated wheat products are immunoreactive in CD patients. The present review summarizes and updates the potentially detrimental effects of mTG, aiming to stimulate scientific and regulatory debates on its safety, to protect the public from the enzyme's unwanted effects.

[THE GUT FEELING OF THE EYES: GUT-EYE AXIS].

INTRODUCTION: In intestinal diseases there are ophthalmological abnormalities. In celiac disease, for example, the eyes' pathologies are expressed by motoric, neurological, inflammatory, autoimmune, vision sharpness, dryness, redness, conjunctivitis and cataract tendency etc. It appears that intestinal luminal components and processes can irradiate to peripheral organs, including to the eyes, inducing functional abnormalities in this target organ. The present review describes the luminal and mucosal constituents and processes, the sensing mechanisms of those generated signals and the routes to deliver those messages to remote organs, eyes included. The gut-eye axis is very challenging and its exploration might bring future therapeutic strategies to treat ophthalmological disease.

Challenges in gluten-free diet in coeliac disease: Prague consensus.

BACKGROUND: New treatments in coeliac disease are being vigorously pursued to either replace or facilitate the difficult-tofollow gluten-free diet. DESIGN: The present review intends to summarise the challenges in gluten-free diet adherence during the transitional period, as reflected in the last Prague consensus, published in 2016. RESULTS: The honourable panel members recommended that dietary adherence and the consequences of nonadherence represent key components for discussion in the transitional period setting. CONCLUSIONS: There are numerous difficulties in adhering to gluten withdrawal, but the transition period from adolescence to young adulthood is considered a fragile and high-risk period for intentional and unintentional gluten intake.

Anti-ribosomal-phosphoprotein autoantibodies penetrate to neuronal cells via neuronal growth associated protein, affecting neuronal cells in vitro.

OBJECTIVE: Anti-ribosomal-phosphoprotein antibodies (anti-Ribos.P Abs) are detected in 10-45% of NPSLE patients. Intracerebroventricular administration of anti-ribosomal-P Abs induces depression-like behaviour in mice. We aimed to discern the mechanism by which anti-Ribos.P Abs induce behavioural changes in mice. METHODS: Anti-Ribos.P Abs were exposed to human and rat neuronal cell cultures, as well as to human umbilical vein endothelial cell cultures for a control. The cellular localization of anti-Ribo.P Abs was found by an immunofluorescent technique using a confocal microscope. Identification of the target molecules was undertaken using a cDNA library. Immunohistochemistry and an inhibition assay were carried out to confirm the identity of the target molecules. Neuronal cell proliferation was measured by bromodeoxyuridine, and Akt and Erk expression by immunoblot. RESULTS: Human anti-Ribos.P Abs penetrated into human neuronal cells and rat hippocampal cell cultures in vitro, but not to endothelial cells as examined. Screening a high-content human cDNA-library with anti-Ribos.P Abs identified neuronal growth-associated protein (GAP43) as a target for anti-Ribos.P Abs. Ex vivo anti-Ribos.P Abs bind to mouse brain sections of hippocampus, dentate and amygdala. Anti-Ribos.P Abs brain-binding was prevented by GAP43 protein. Interestingly, GAP43 inhibited in a dose-dependent manner the anti-Ribos.P Abs binding to recombinant-ribosomal-P0, indicating mimicry between the ribosomal-P0 protein and GAP43. Furthermore, anti-Ribos.P Abs reduced neuronal cell proliferation activity in vitro (P < 0.001), whereas GAP43 decreased this inhibitory activity by a factor of 7.6. The last was related to Akt and Erk dephosphorylation. CONCLUSION: Anti-Ribos.P Abs penetrate neuronal cells in vitro by targeting GAP43. Anti -Ribos.P Abs inhibit neuronal-cell proliferation via inhibition of Akt and Erk. Our data contribute to deciphering the mechanism for anti-Ribos.P Abs' pathogenic activity in NPSLE.

Anti-ribosomal-P antibodies accelerate lupus glomerulonephritis and induce lupus nephritis in naïve mice.

BACKGROUND: Lupus nephritis is known to be associated with several antibodies including autoantibodies that target the DNA, C1q and histone, α-actinin, and the nucleosome. In addition, circulating anti-phosphoribosomal protein antibodies (anti-Ribos.P) were found to be associated with lupus nephritis. STUDY OBJECTIVE: We have assessed the direct role of anti-Ribos.P in the development of glomerulonephritis in-vitro and in animal models. STUDY DESIGN: NZBxW/F1 lupus prone mice were immunized with recombinant Ribos.P0 (rRibos.P). Evaluation of renal disease included mice evaluation for proteinuria and histologic analysis of the kidneys. Anti-Ribos.P monoclonal Ab was prepared from the rRibos.P immunized NZBxW/F1 mice by hybridoma technology. MAPKs expression was analyzed by MAPKs protein array and confirmed by real-time PCR and western blot. To elucidate whether anti-Ribos.P induce glomerulonephritis, naïve C3H mice were immunized with recombinant rRibos.P and the glomerulonephritis was followed up as described above. RESULTS: The immunized NZBxW/F1 lupus prone mice developed anti-Ribos.P which was cross reactive with Sm and not dsDNA. The mice developed accelerated glomerulonephritis manifested by early proteinuria and immunoglobulin deposites in the mesangium of the kidneys. Anti-Ribos.P deposited in the glomerular mesangium were eluted from the kidney. The Ribos.P immunized naïve C3H/Hen mice developed glomerulonephritis manifested by circulating autoantibodies directed to Ribos.P, dsDNA and Sm. The anti Ribos.P were cross reactive with Sm but not with dsDNA, and were deposited in the glomeruli. Interestingly these mice developed alopecia. In vitro. Primary mesangial cells exposed to mouse anti-Ribos.P mAb originated from the immunized lupus mice and to human anti-Ribos.P Abs, induced activation of mesangial cells via p38α, JNK, AKT and HSP27 MAPKs expression pathway. CONCLUSIONS: Our data show for the first time that anti-Ribos.P are nephritogenic autoantibodies, as illustrated by in-vitro and in-vivo experiments: a) They accelerate the development of glomerulonephritis in lupus prone mice; b) They induce nephritis in naïve mice. c) Anti-Ribos.P Abs trigger MAPKs expression in primary mesangial cells. These data contribute a direct mechanistic link between anti-Ribos.P and nephritis in lupus mice.

Association between Food-Specific Immunoglobulin G4 Antibodies in Adults with Self-Reported Signs and Symptoms Attributed to Adverse Reactions to Foodstuffs.

Signs and symptoms attributed to adverse reactions to foodstuffs (ARFS) need tools for research and evaluation in clinical practice. The objectives of this study were (a) to evaluate the most frequent self-reported signs and symptoms attributed to ARFS in Spanish adults, (b) to determine the prevalence of food-specific IgG4 antibody reactions (AbRs), and (c) to investigate the association between self-reported ARFS symptomatology and food-specific IgG4 AbRs. Food-specific IgG4 AbRs against 57 common food and beverages (AESKUCARE-T2FA® in vitro point-of-care test kit, Aesku.Diagnostics GmbH, Germany) were determined in capillary blood samples of 205 volunteers living in the Region of Madrid (Spain). The most frequent self-reported signs and symptoms were related to skin (43%), digestive (41%), and nervous system (NS, 33%) problems. The prevalence of food-specific IgG4 AbRs was cow's milk (73%), sheep's milk (70%), casein (66%), and goat's milk (56.10%). Positive IgG4 AbRs against tomato had a profile consisting of 3/4 of skin problems, more than half of digestive, and 2/5 of NS self-reported signs and symptoms. In conclusion, at least 1/3 of the studied sample reported skin, digestive, and NS signs and symptoms. The most frequent food-specific IgG4 AbRs were related to dairy. Skin problems were more frequent in positive tomato IgG4 AbRs.

Safety, tolerability and pharmacokinetics of the oligomer modulator anle138b with exposure levels sufficient for therapeutic efficacy in a murine Parkinson model: A randomised, double-blind, placebo-controlled phase 1a trial.

BACKGROUND: Synucleinopathies such as Parkinson ́s disease (PD), Dementia with Lewy bodies (DLB) and Multiple System Atrophy (MSA) are characterized by deposition of misfolded and aggregated α-synuclein. Small aggregates (oligomers) of α-synuclein have been shown to be the most relevant neurotoxic species and are targeted by anle138b, an orally bioavailable small molecule compound which shows strong disease-modifying effects in animal models of synucleinopathies. METHODS: Anle138b was studied in a single-centre, double-blind, randomised, placebo-controlled single ascending dose (SAD) and multiple ascending dose (MAD) study in healthy subjects. Eligible participants were randomly assigned (1:1 for sentinel subjects and 1:5 for main group) to placebo or anle138b (dose range 50 mg to 300 mg per day), respectively. In addition, the effect of food on the pharmakokinetics of anle138b in healthy subjects was examined in doses of 150 mg per day. Participants were randomized to treatment sequence (fed→fasted) or (fasted→fed). Treatment was administered orally in hard gelatine capsules containing either 10 mg or 30 mg of anle138b or excipient only. The primary endpoints were safety and tolerability, the secondary endpoint was pharmakokinetics. Data from all randomized individuals were evaluated. CLINICALTRIALS: gov-identifier: NCT04208152. EudraCT-number: 2019-004218-33. FINDINGS: Between December 17th, 2019 and June 27th, 2020 196 healthy volunteers were screened and 68 participants were enrolled. Of these, all completed the study per protocol. There were no major protocol deviations. Adverse events in this healthy volunteer trial were mostly mild and all fully recovered or resolved prior to discharge. From baseline to completion of the trial no medically significant individual changes were observed in any system organ class. Already at multiple doses of 200 mg, exposure levels above the fully effective exposure in the MI2 mouse Parkinson model were observed. INTERPRETATION: The favourable safety and PK profile of anle138b in doses resulting in exposures above the fully effective plasma level in a mouse Parkinson model warrant further clinical trials in patients with synucleinopathies. FUNDING: This study was funded by MODAG GmbH and by the Michael J. Fox foundation for Parkinson's Research.

The temperature and pH repertoire of the transglutaminase family is expanding.

Transglutaminases (TGs) play important roles in the food industry, pharmacology, and biotechnology, but as protein cross-linkers, their complexes are stable, resistant, immunogenic, and potentially pathogenic. Many TGs have been characterized, but they operate in narrow temperature and pH range limits. In a research article in this issue, Clemens Furnes and colleagues describe a novel cold-adapted TG from Atlantic cod, which expands the operating boundaries to a lower temperature and a wider pH. In this accompanying commentary, we discuss how this TG opens new applications in cold environments and can be deactivated by heating. New sources of TGs should be explored in hot environments like hot springs, in order to increase the temperature and widen the pH ranges for human and industrial benefits.

Anti-phospholipid antibody prevalence and association with subclinical atherosclerosis and atherothrombosis in the general population.

BACKGROUND: There is no agreement on the prevalence of anti-phospholipid antibodies (aPLs) and the correlation with atherosclerosis and cardiovascular (CV) events in the general population. METHODS: We performed a cross-sectional study on 1712 randomly enrolled subjects from a Northern Italian city to investigate the presence of aPLs and the association with subclinical atherosclerosis (using the carotid artery intima media thickness measured as inter-adventitia common carotid artery diameters - ICCAD) and retrospectively collected CV factors and events (i.e. acute myocardial infarction, stroke, and peripheral obliterans arterial vasculopathy) using physician-assisted questionnaires. We tested serum IgG, IgM, and IgA anti-cardiolipin, anti-beta2glycoprotein I (aGPI), and anti-phosphatidylserine-prothrombin antibodies. RESULTS: Positive aPLs were found in 15.1% of the subjects, with no differences between sex but with higher rates in older subjects. Carotid subclinical atherosclerosis was more frequent in aPL positive subjects; more specifically, aGPI IgA were associated with higher ICCAD average (adjusted beta 0.51, 95% confidence interval (CI)0.17-0.84; p = 0.003). A positive history of CV events was also more frequent in aPL positive subjects (odds ratio (OR) 1.67, 95%CI 1.08-2.54; p = 0.012), particularly peripheral obliterans arterial vasculopathy (OR 2.02; 95%CI 1.14-3.57; p = 0.015). Among subjects with a Framingham risk score >20, and/or diabetes, and/or body mass index >35 kg/m2, aPL positivity was associated to the highest risk of CV events (OR 2.52, 95%CI 1.24-5.11; p = 0.011). CONCLUSIONS: APL prevalence in the general population is higher than previously reported. CV events and subclinical atherosclerosis are more frequent in the presence of aPL, particularly when a high CV risk coexists.

Microbial transglutaminase should be considered as an environmental inducer of celiac disease.

Due to the recent interest in food additives that can act as triggering factors in autoimmune diseases including celiac disease (CD), the present letter to the editor expands on the microbial transglutaminase (mTG). It is heavily consumed by a plethora of food processing industries as "glue of proteins" thus improving product's stability, texture and shelf life. However, more and more information is accumulated lately, questioning its safety. Its cross-linked gliadin complexes are immunogenic in CD. The enzyme increases gliadin uptake, is transported in a trans-epithelial way and deposited below the enterocyte's line, has anti- phagocytic activity, enhances intestinal permeability and creates luminal resistant isopeptide bonds. No doubt that mTG is beneficial to food industries but a caveat to public health is highly recommended.

Navigating the Gluten-Free Boom: The Dark Side of Gluten Free Diet.

In gluten dependent conditions the gluten free diet is the cornerstone of therapy, decreasing disease activity, improving health and quality of life and treating or preventing the associated complications. Gluten withdrawal implies strict and lifelong elimination not only of wheat, barley, rye, and wheat-contaminated oats, but also of numerous non-nutritional products where components of wheat are often added. Due to multiple reasons the diet is difficult to follow and the long-term adherence is decreased with time. The present review summarizes the dark side of gluten restriction where nutritional deficiencies, toxicity, morbidity, mortality, and mental health problems are reported. The aim being to increase awareness, avoid, detect and treat the side effects and to promote a healthier nutrition, for the patient's benefits.

Probiotics: If It Does Not Help It Does Not Do Any Harm. Really?

Probiotics per definition should have beneficial effects on human health, and their consumption has tremendously increased in the last decades. In parallel, the amount of published material and claims for their beneficial efficacy soared continuously. Recently, multiple systemic reviews, meta-analyses, and expert opinions expressed criticism on their claimed effects and safety. The present review describes the dark side of the probiotics, in terms of problematic research design, incomplete reporting, lack of transparency, and under-reported safety. Highlighted are the potential virulent factors and the mode of action in the intestinal lumen, risking the physiological microbiome equilibrium. Finally, regulatory topics are discussed to lighten the heterogeneous guidelines applied worldwide. The shift in the scientific world towards a better understanding of the human microbiome, before consumption of the probiotic cargo, is highly endorsed. It is hoped that better knowledge will extend the probiotic repertoire, re-confirm efficacy or safety, establish their efficacy and substantiate their beneficial effects.