Therapeutic effect of Cerebrolysin on reducing impaired cerebral endothelial cell permeability

Cerebrolysin has been shown to promote neurovascular protection and repair in preclinical models of stroke and neural injury and is demonstrating promise for stroke and neural injury therapeutic application in the clinic. The effect of Cerebrolysin on the human cerebral endothelial cell function has not been investigated. Using an in-vitro cerebral endothelial cell permeability assay and western blot analyses of tight junction and proinflammatory and procoagulant proteins, the present study showed that tissue plasminogen activator (tPA) and fibrin substantially impaired human cerebral endothelial cell barrier function and increased permeability, which persisted for at least 24 h. western blot analysis revealed that tPA and fibrin significantly increased proinflammatory and procoagulation proteins of intercellular adhesion molecule 1, high mobility group box 1, tumor necrosis factor α and phosphorylated nuclear factor kappa B-p65, and significantly reduced tight junction proteins zonular 1, occludin and claudin. However, Cerebrolysin significantly diminished and reversed tPA- and fibrin-impaired endothelial cell permeability, which was associated with significant reductions of tPA- and fibrin-augmented proinflammatory and procoagulation proteins and significant elevations of tPA- and fibrin-decreased tight junction proteins. The beneficial effect of Cerebrolysin appears specific because cerebroprotein hydrolysate, with a distinct peptide composition, failed to show the reduction of tPA- and fibrin-impaired permeability. These data indicate that cererbrolysin has a therapeutic effect on tPA- and fibrin-impaired cerebral endothelial cell permeability by reducing proinflammatory and procoagulation proteins and by elevating tight junction proteins

T Cell Recognition of the Dominant I-Ak–Restricted Hen Egg Lysozyme Epitope: Critical Role for Asparagine Deamidation

Type-B T cells raised against the immunodominant peptide in hen egg lysozyme (HEL48–62) do not respond to whole lysozyme, and this has been thought to indicate that peptide can bind to l-Ak in different conformations. Here we demonstrate that such T cells recognize a deamidated form of the HEL peptide and not the native peptide. The sequence of the HEL epitope facilitates rapid and spontaneous deamidation when present as a free peptide or within a flexible domain. However, this deamidated epitope is not created within intact lysozyme, most likely because it resides in a highly structured part of the protein. These findings argue against the existence of multiple conformations of the same peptide–MHC complex and have important implications for the design of peptide-based vaccines. Furthermore, as the type-B T cells are known to selectively evade induction of tolerance when HEL is expressed as a transgene, these results suggest that recognition of posttranslationally modified self-antigen may play a role in autoimmunity

The Preferred Substrates for Transglutaminase 2 in a Complex Wheat Gluten Digest Are Peptide Fragments Harboring Celiac Disease T-Cell Epitopes

BACKGROUND: Celiac disease is a T-cell mediated chronic inflammatory disorder of the gut that is induced by dietary exposure to gluten proteins. CD4+ T cells of the intestinal lesion recognize gluten peptides in the context of HLA-DQ2.5 or HLA-DQ8 and the gluten derived peptides become better T-cell antigens after deamidation catalyzed by the enzyme transglutaminase 2 (TG2). In this study we aimed to identify the preferred peptide substrates of TG2 in a heterogeneous proteolytic digest of whole wheat gluten. METHODS: A method was established to enrich for preferred TG2 substrates in a complex gluten peptide mixture by tagging with 5-biotinamido-pentylamine. Tagged peptides were isolated and then identified by nano-liquid chromatography online-coupled to tandem mass spectrometry, database searching and final manual data validation. RESULTS: We identified 31 different peptides as preferred substrates of TG2. Strikingly, the majority of these peptides were harboring known gluten T-cell epitopes. Five TG2 peptide substrates that were predicted to bind to HLA-DQ2.5 did not contain previously characterized sequences of T-cell epitopes. Two of these peptides elicited T-cell responses when tested for recognition by intestinal T-cell lines of celiac disease patients, and thus they contain novel candidate T-cell epitopes. We also found that the intact 9mer core sequences of the respective epitopes were not present in all peptide substrates. Interestingly, those epitopes that were represented by intact forms were frequently recognized by T cells in celiac disease patients, whereas those that were present in truncated versions were infrequently recognized. CONCLUSION: TG2 as well as gastrointestinal proteolysis play important roles in the selection of gluten T-cell epitopes in celiac disease

Structure–function relationships of the competence lipoprotein ComL and SSB in meningococcal transformation

Neisseria meningitidis, the meningococcus, is naturally competent for transformation throughout its growth cycle. The uptake of exogenous DNA into the meningococcus cell during transformation is a multi-step process. Beyond the requirement for type IV pilus expression for efficient transformation, little is known about the neisserial proteins involved in DNA binding, uptake and genome integration. This study aimed to identify and characterize neisserial DNA binding proteins in order to further elucidate the multi-factorial transformation machinery. The meningococcus inner membrane and soluble cell fractions were searched for DNA binding components by employing 1D and 2D gel electrophoresis approaches in combination with a solid-phase overlay assay with DNA substrates. Proteins that bound DNA were identified by MS analysis. In the membrane fraction, multiple components bound DNA, including the neisserial competence lipoprotein ComL. In the soluble fraction, the meningococcus orthologue of the single-stranded DNA binding protein SSB was predominant. The DNA binding activity of the recombinant ComL and SSB proteins purified to homogeneity was verified by electromobility shift assay, and the ComL–DNA interaction was shown to be Mg2+-dependent. In 3D models of the meningococcus ComL and SSB predicted structures, potential DNA binding sites were suggested. ComL was found to co-purify with the outer membrane, directly interacting with the secretin PilQ. The combined use of 1D/2D solid-phase overlay assays with MS analysis was a useful strategy for identifying DNA binding components. The ComL DNA binding properties and outer membrane localization suggest that this lipoprotein plays a direct role in neisserial transformation, while neisserial SSB is a DNA binding protein that contributes to the terminal part of the transformation process

The Molecular Basis for Oat Intolerance in Patients with Celiac Disease

BACKGROUND: Celiac disease is a small intestinal inflammatory disorder characterized by malabsorption, nutrient deficiency, and a range of clinical manifestations. It is caused by an inappropriate immune response to dietary gluten and is treated with a gluten-free diet. Recent feeding studies have indicated oats to be safe for celiac disease patients, and oats are now often included in the celiac disease diet. This study aimed to investigate whether oat intolerance exists in celiac disease and to characterize the cells and processes underlying this intolerance. METHODS AND FINDINGS: We selected for study nine adults with celiac disease who had a history of oats exposure. Four of the patients had clinical symptoms on an oats-containing diet, and three of these four patients had intestinal inflammation typical of celiac disease at the time of oats exposure. We established oats-avenin-specific and -reactive intestinal T-cell lines from these three patients, as well as from two other patients who appeared to tolerate oats. The avenin-reactive T-cell lines recognized avenin peptides in the context of HLA-DQ2. These peptides have sequences rich in proline and glutamine residues closely resembling wheat gluten epitopes. Deamidation (glutamine→glutamic acid conversion) by tissue transglutaminase was involved in the avenin epitope formation. CONCLUSIONS: We conclude that some celiac disease patients have avenin-reactive mucosal T-cells that can cause mucosal inflammation. Oat intolerance may be a reason for villous atrophy and inflammation in patients with celiac disease who are eating oats but otherwise are adhering to a strict gluten-free diet. Clinical follow-up of celiac disease patients eating oats is advisable

Identification of neisserial DNA binding components

Neisseria meningitidis, a causative agent of meningitis and septicaemia, expresses type IV pili, a feature correlating with the uptake of exogenous DNA from the environment by natural transformation. The outer membrane complex PilQ, through which pili are extruded and retracted, has previously been shown to bind DNA in its pore region. In order to further elucidate how DNA is transported across the membranes, we searched for DNA binding proteins within the meningococcal inner membrane. Inner membrane fractions from a panel of neisserial strains were subjected to a solid-phase overlay assay with DNA substrates, and MS was subsequently employed to identify proteins that bind DNA. A number of DNA binding components were detected, including the pilus biogenesis component PilG, the competence protein ComL, and the cell division ATP-binding protein FtsE, as well as two hypothetical proteins. The DNA binding activity of these components was not dependent on the presence of the neisserial DNA uptake sequence. Null mutants, corresponding to each of the proteins identified, were constructed to assess their phenotypes. Only mutants defective in pilus biogenesis were non-competent and non-piliated. The DNA binding activity of the pilus biogenesis components PilQ and PilG and the phenotypes of their respective null mutants suggest that these proteins are directly involved as players in natural transformation, and not only indirectly, through pilus biogenesis

Anti-PAD4 autoantibodies in rheumatoid arthritis: levels in serum over time and impact on PAD4 activity as measured with a small synthetic substrate

Isoform 4 of the human peptidylarginine deiminase (hPAD4) enzyme may be responsible for the citrullination of antigens in rheumatoid arthritis (RA) and has been shown to be itself the target of disease-specific autoantibodies. Here, we have tested whether the level of serum anti-hPAD4 antibodies in RA patients is stable over a period of 10 years and whether the antibodies influence hPAD4-mediated deimination of the small substrate N-α-Benzoyl-l-arginine ethyl ester. RA sera (n = 128) obtained at baseline and after 10 years were assessed for anti-hPAD4 antibodies by a specific immunoassay. For 118 RA patients, serum anti-hPAD4 IgG levels were stable over 10 years. Seven patients who were negative for anti-PAD4 IgG at baseline had become positive after 10 years. Further, total IgG from selected RA patients and controls were purified, and a fraction was depleted for anti-hPAD4 antibodies. Kinetic deimination assays were performed with total IgG and depleted fractions. The kcat and Km values of hPAD4-mediated deimination of N-α-Benzoyl-l-arginine ethyl ester were not affected by the depletion of the anti-hPAD4 antibodies from the total IgG pool. In conclusion, RA patients remain positive for anti-hPAD4 antibodies over time and some patients who are initially anti-hPAD4 negative become positive later in the disease course. The anti-hPAD4 antibodies did not affect the enzymatic activity of hPAD4 when the small substrate N-α-Benzoyl-l-arginine ethyl ester was used. However, this finding may not exclude an effect of these autoantibodies on citrullination of protein substrates in RA

Gluten T cell epitope targeting by TG3 and TG6; implications for dermatitis herpetiformis and gluten ataxia

Transglutaminase 2 (TG2) is well characterized as the main autoantigen of celiac disease. The ability of TG2 to deamidate and crosslink gluten peptides is essential for the gluten-dependent production of TG2 specific autoantibodies. In patients with primarily extraintestinal manifestation of gluten sensitivity the repertoire of autoantibodies may be different. In dermatitis herpetiformis (DH), TG3 appears to be the target autoantigen whereas in gluten ataxia (GA) autoantibodies reactive with TG6 are present. A functional role for TG3 and TG6 in these diseases has yet to be described. It is also not known whether these enzymes can use gluten peptides implicated in the pathology as substrates. We here report that similar to TG2, TG3 and TG6 can specifically deamidate gluten T cell epitopes. However, the fine specificities of the enzymes were found to differ. TG2 can form covalent complexes with gluten by iso-peptide and thioester bonds. We found that both TG3 and TG6 were able to complex with gluten peptides through thioester linkage although less efficiently than TG2, whereas TG6 but not TG3 was able to form iso-peptide linked complexes. Our findings lend credence to the notion that TG3 and TG6 are involved in the gluten-induced autoimmune responses of DH and GA