Compromised intestinal integrity in older adults during daily activities: a pilot study.

BACKGROUND: Malnutrition is a common and significant problem in older adults. Insight into factors underlying malnutrition is needed to develop strategies that can improve the nutritional status. Compromised intestinal integrity caused by gut wall hypoperfusion due to atherosclerosis of the mesenteric arteries in the aging gastrointestinal tract may adversely affect nutrient uptake. The presence of compromised intestinal integrity in older adults is not known. The aim of this study is to provide a proof-of-concept that intestinal integrity is compromised in older adults during daily activities. METHODS: Adults aged ≥75 years living independently without previous gastrointestinal disease or abdominal surgery were asked to complete a standardized walking test and to consume a standardized meal directly afterwards to challenge the mesenteric blood flow. Intestinal fatty acid-binding protein (I-FABP) was measured as a plasma marker of intestinal integrity, in blood samples collected before (baseline) and after the walking test, directly after the meal, and every 15 min thereafter to 75 min postprandially. RESULTS: Thirty-four participants (median age 81 years; 56% female) were included. Of the participants, 18% were malnourished (PG-SGA score ≥ 4), and 32% were at risk of malnutrition (PG-SGA score, 2 or 3). An I-FABP increase of ≥50% from baseline was considered a meaningful loss of intestinal integrity and was observed in 12 participants (35%; 8 females; median age 80 years). No significant differences were observed in either baseline characteristics, walking test scores, or calorie/macronutrient intake between the groups with and without a ≥ 50% I-FABP peak. CONCLUSION: This study is first to indicate that intestinal integrity is compromised during daily activities in a considerable part of older adults living independently.

Females Are More Resistant to Ischemia-Reperfusion-induced Intestinal Injury Than Males: A Human Study.

BACKGROUND AND OBJECTIVE: Sex differences in responses to intestinal ischemia-reperfusion (IR) have been recognized in animal studies. We aimed to investigate sexual dimorphism in human small intestinal mucosal responses to IR. METHODS: In 16 patients (8 men and 8 women) undergoing pancreaticoduodenectomy, an isolated part of jejunum was subjected to IR. In each patient, intestinal tissue and blood was collected directly after 45 minutes of ischemia without reperfusion (45I-0R), after 30 minutes of reperfusion (45I-30R), and after 120 minutes of reperfusion (45I-120R), as well as a control sample not exposed to IR, to assess epithelial damage, unfolded protein response (UPR) activation, and inflammation. RESULTS: More extensive intestinal epithelial damage was observed in males compared to females. Intestinal fatty acid binding protein (I-FABP) arteriovenous (V-A) concentrations differences were significantly higher in males compared to females at 45I-0R (159.0 [41.0-570.5] ng/mL vs 46.9 [0.3-149.9] ng/mL). Male intestine showed significantly higher levels of UPR activation than female intestine, as well as higher number of apoptotic Paneth cells per crypt at 45I-30R (16.4% [7.1-32.1] vs 10.6% [0.0-25.4]). The inflammatory response in male intestine was significantly higher compared to females, with a higher influx of neutrophils per villus at 45I-30R (4.9 [3.1-12.0] vs 3.3 [0.2-4.5]) and a higher gene expression of TNF-α and IL-10 at 45I-120R. CONCLUSION: The human female small intestine seems less susceptible to IR-induced tissue injury than the male small intestine. Recognition of such differences could lead to the development of novel therapeutic strategies to reduce IR-associated morbidity and mortality.

SM22 a Plasma Biomarker for Human Transmural Intestinal Ischemia.

OBJECTIVE: To evaluate the diagnostic potential of smooth muscle protein of 22 kDa (SM22) as plasma biomarker for the detection of transmural intestinal ischemia. BACKGROUND: Acute mesenteric ischemia is an abdominal emergency requiring rapid diagnosis and treatment. Especially, detection of transmural damage is imperative because it mandates emergency surgery. Since early clinical and radiological signs are nonspecific, there is an urgent need for accurate biomarkers. SM22 is a potential marker for transmural damage because of its abundant expression in intestinal smooth muscles. METHODS: SM22 concentrations were measured using a newly built enzyme-linked immunosorbent assay. SM22 release was assessed in plasma and intestinal tissue of rats subjected to intestinal ischemia. Blood and tissue were sampled at baseline and followed up to 24 hours of ischemia. Next, organ-specific SM22 arteriovenous concentration differences were studied in both rats and patients. Finally, plasma from patients with intestinal ischemia, other acute abdominal complaints, and healthy volunteers were tested for SM22. RESULTS: SM22 concentrations were significantly elevated in rats from 4 hours of ischemia onwards. Furthermore, SM22 plasma concentrations closely paralleled the histological increasing degree of intestinal smooth muscle damage. Arteriovenous calculations showed that SM22 was specifically released by the intestines and renally cleared. First data of SM22 release in man demonstrated that patients with transmural intestinal ischemia had significantly higher plasma SM22 levels than patients with only ischemic mucosal injury, other acute abdominal diseases, or healthy controls. CONCLUSIONS: This study shows that SM22 is released into the circulation upon severe ischemia of the intestinal muscle layers. Plasma levels of SM22 are potentially useful for the detection of transmural intestinal damage.

The effect of Mindfulness-Based Stress Reduction on wound healing: a preliminary study.

Psychological factors have been shown to influence the process of wound healing. This study examined the effect of Mindfulness-Based Stress Reduction (MBSR) on the speed of wound healing. The local production of pro-inflammatory cytokines and growth factors was studied as potential underlying mechanism. Forty-nine adults were randomly allocated to a waiting-list control group (n = 26) or an 8-week MBSR group (n = 23). Pre- and post-intervention/waiting period assessment for both groups consisted of questionnaires. Standardized skin wounds were induced on the forearm using a suction blister method. Primary outcomes were skin permeability and reduction in wound size monitored once a day at day 3, 4, 5, 6, 7, and 10 after injury. Secondary outcomes were cytokines and growth factors and were measured in wound exudates obtained at 3, 6, and 22 h after wounding. Although there was no overall condition effect on skin permeability or wound size, post hoc analyses indicated that larger increases in mindfulness were related to greater reductions in skin permeability 3 and 4 days after wound induction. In addition, MBSR was associated with lower levels of interleukin (IL)-8 and placental growth factor in the wound fluid 22 h after wound induction. These outcomes suggest that increasing mindfulness by MBSR might have beneficial effects on early stages of wound healing. Trial Registration NTR3652, http://www.trialregister.nl.

Serum I-FABP Detects Gluten Responsiveness in Adult Celiac Disease Patients on a Short-Term Gluten Challenge.

OBJECTIVES: Response to gluten challenge (GC) is a key feature in diagnostic algorithms and research trials in celiac disease (CD). Currently, autoantibody titers, late responders to GC, and invasive duodenal biopsies are used to evaluate gluten responsiveness. This study investigated the accuracy of serum intestinal-fatty acid binding protein (I-FABP), a marker for intestinal epithelial damage, to predict intestinal damage during GC in patients with CD. METHODS: Twenty adult CD patients in remission underwent a two-week GC with 3 or 7.5 g of gluten daily. Study visits occurred at day -14, 0, 3, 7, 14, and 28. Serum I-FABP, antibodies to tissue transglutaminase (tTG-IgA), deamidated gliadin peptides (IgA-DGP), and anti-actin (AAA-IgA) were assessed at each visit. Villous-height to crypt-depth ratio (Vh:Cd) and intraepithelial lymphocyte (IEL) count were evaluated at day -14, 3, and 14. Forty-three CD-serology negative individuals were included to compare serum I-FABP levels in CD patients on a gluten-free diet (GFD) with those in healthy subjects. RESULTS: Serum I-FABP levels increased significantly during a two-week GC. In contrast, the most pronounced autoantibody increase was found at day 28, when patients had already returned to a GFD for two weeks. IgA-AAA titers were only significantly elevated at day 28. I-FABP levels and IEL count correlated at baseline (r=0.458, P=0.042) and at day 14 (r=0.654, P=0.002) of GC. Neither gluten dose nor time on a GFD influenced I-FABP change during GC. CONCLUSIONS: Serum I-FABP levels increased significantly during a two-week GC in adult CD patients and correlated with IEL count. The data suggest that serum I-FABP is an early marker of gluten-induced enteropathy in celiac patients and may be of use in both clinical and research settings.

Plasma cholesteryl ester transfer protein is predominantly derived from Kupffer cells.

UNLABELLED: The role of Kupffer cells (KCs) in the pathophysiology of the liver has been firmly established. Nevertheless, KCs have been underexplored as a target for diagnosis and treatment of liver diseases owing to the lack of noninvasive diagnostic tests. We addressed the hypothesis that cholesteryl ester transfer protein (CETP) is mainly derived from KCs and may predict KC content. Microarray analysis of liver and adipose tissue biopsies, obtained from 93 obese subjects who underwent elective bariatric surgery, showed that expression of CETP is markedly higher in liver than adipose tissue. Hepatic expression of CETP correlated strongly with that of KC markers, and CETP messenger RNA and protein colocalized specifically with KCs in human liver sections. Hepatic KC content as well as hepatic CETP expression correlated strongly with plasma CETP concentration. Mechanistic and intervention studies on the role of KCs in determining the plasma CETP concentration were performed in a transgenic (Tg) mouse model expressing human CETP. Selective elimination of KCs from the liver in CETP Tg mice virtually abolished hepatic CETP expression and largely reduced plasma CETP concentration, consequently improving the lipoprotein profile. Conversely, augmentation of KCs after Bacille-Calemette-Guérin vaccination largely increased hepatic CETP expression and plasma CETP. Also, lipid-lowering drugs fenofibrate and niacin reduced liver KC content, accompanied by reduced plasma CETP concentration. CONCLUSIONS: Plasma CETP is predominantly derived from KCs, and plasma CETP level predicts hepatic KC content in humans.

Complement activation by ceramide transporter proteins.

C1q is the initiator of the classical complement pathway and, as such, is essential for efficient opsonization and clearance of pathogens, altered self-structures, and apoptotic cells. The ceramide transporter protein (CERT) and its longer splicing isoform CERTL are known to interact with extracellular matrix components, such as type IV collagen, and with the innate immune protein serum amyloid P. In this article, we report a novel function of CERT in the innate immune response. Both CERT isoforms, when immobilized, were found to bind the globular head region of C1q and to initiate the classical complement pathway, leading to activation of C4 and C3, as well as generation of the membrane attack complex C5b-9. In addition, C1q was shown to bind to endogenous CERTL on the surface of apoptotic cells. These results demonstrate the role of CERTs in innate immunity, especially in the clearance of apoptotic cells.

Proteasome inhibition with bortezomib depletes plasma cells and specific autoantibody production in primary thymic cell cultures from early-onset myasthenia gravis patients.

Bortezomib is a potent inhibitor of proteasomes currently used to eliminate malignant plasma cells in multiple myeloma patients. It is also effective in depleting both alloreactive plasma cells in acute Ab-mediated transplant rejection and their autoreactive counterparts in animal models of lupus and myasthenia gravis (MG). In this study, we demonstrate that bortezomib at 10 nM or higher concentrations killed long-lived plasma cells in cultured thymus cells from nine early-onset MG patients and consistently halted their spontaneous production not only of autoantibodies against the acetylcholine receptor but also of total IgG. Surprisingly, lenalidomide and dexamethasone had little effect on plasma cells. After bortezomib treatment, they showed ultrastructural changes characteristic of endoplasmic reticulum stress after 8 h and were no longer detectable at 24 h. Bortezomib therefore appears promising for treating MG and possibly other Ab-mediated autoimmune or allergic disorders, especially when given in short courses at modest doses before the standard immunosuppressive drugs have taken effect.

Colonic metaproteomic signatures of active bacteria and the host in obesity.

Obesity is associated with the intestinal microbiota in humans but the underlying mechanisms are yet to be fully understood. Our previous phylogenetic study showed that the faecal microbiota profiles of nonobese versus obese and morbidly obese individuals differed. Here, we have extended this analysis with a characterization of the faecal metaproteome, in order to detect differences at a functional level. Proteins were extracted from crude faecal samples of 29 subjects, separated by 1D gel electrophoresis and characterized using RP LC-MS/MS. The peptide data were analyzed in database searches with two complementary algorithms, OMSSA and X!Tandem, to increase the number of identifications. Evolutionary genealogy of genes: nonsupervised orthologous groups (EggNOG) database searches resulted in the functional annotation of over 90% of the identified microbial and human proteins. Based on both bacterial and human proteins, a clear clustering of obese and nonobese samples was obtained that exceeded the phylogenetic separation in dimension. Moreover, integration of the metaproteomics and phylogenetic datasets revealed notably that the phylum Bacteroidetes was metabolically more active in the obese than nonobese subjects. Finally, significant correlations between clinical measurements and bacterial gene functions were identified. This study emphasizes the importance of integrating data of the host and microbiota to understand their interactions.

The Human Colon Is More Resistant to Ischemia-reperfusion-induced Tissue Damage Than the Small Intestine: An Observational Study.

OBJECTIVE: Aim of this study was to draw comparisons between human colonic and jejunal ischemia-reperfusion sequelae in a human in vivo experimental model. BACKGROUND: In patients, colonic ischemia-reperfusion generally has a milder course than small intestinal ischemia-reperfusion. It is unclear which pathophysiologic processes are responsible for this difference. METHODS: In 10 patients undergoing colonic surgery and 10 patients undergoing pancreaticoduodenectomy, 6 cm colon or jejunum was isolated and exposed to 60 minutes ischemia followed by various reperfusion periods. Morphology (hematoxylin and eosin), apoptosis (M30), tight junctions (zonula occludens 1), and neutrophil influx (myeloperoxidase) were assessed using immunohistochemistry. Quantitative polymerase chain reaction and enzyme-linked immunosorbent assay were performed for interleukin-6 and tumor necrosis factor-α. RESULTS: Hematoxylin and eosin staining revealed intact colonic epithelial lining, but extensive damage in jejunal villus tips after 60 minutes ischemia. After reperfusion, the colonic epithelial lining was not affected, whereas the jejunal epithelium was seriously damaged. Colonic apoptosis was limited to scattered cells in surface epithelium, whereas apoptosis was clearly observed in jejunal villi and crypts, (42 times more M30 positivity compared with colon, P < 0.01). Neutrophil influx and increased tumor necrosis factor-α mRNA expression were observed in jejunum after 30 and 120 minutes of reperfusion (P < 0.05). Interleukin-6 mRNA expression was increased in jejunum after 120 minutes of reperfusion (3.6-fold increase, P < 0.05), whereas interleukin-6 protein expression was increased in both colon (1.5-fold increase, P < 0.05) and small intestine (1.5-fold increase, P < 0.05) after 30 and 120 minutes of reperfusion. CONCLUSIONS: Human colon is less susceptible to IR-induced tissue injury than small intestine.

Plasma cathepsin D levels: a novel tool to predict pediatric hepatic inflammation.

OBJECTIVES: Nonalcoholic steatohepatitis (NASH) is the most severe form of a hepatic condition known as nonalcoholic fatty liver disease (NAFLD). NASH is histologically characterized by hepatic fat accumulation, inflammation, and ballooning, and eventually coupled with fibrosis that, in turn, may progress to end-stage liver disease even in young individuals. Hence, there is a critical need for specific noninvasive markers to predict hepatic inflammation at an early age. We investigated whether plasma levels of cathepsin D (CatD), a lysosomal protease, correlated with the severity of liver inflammation in pediatric NAFLD. METHODS: Liver biopsies from children (n=96) with NAFLD were histologically evaluated according to the criteria of Kleiner (NAFLD activity score) and the Brunt's criteria. At the time of liver biopsy, blood was taken and levels of CatD, alanine aminotransferase (ALT), and cytokeratin-18 (CK-18) were measured in plasma. RESULTS: Plasma CatD levels were significantly lower in subjects with liver inflammation compared with steatotic subjects. Furthermore, we found that CatD levels were gradually reduced and corresponded with increasing severity of liver inflammation, steatosis, hepatocellular ballooning, and NAFLD activity score. CatD levels correlated with pediatric NAFLD disease progression better than ALT and CK-18. In particular, CatD showed a high diagnostic accuracy (area under receiver operating characteristic curve (ROC-AUC): 0.94) for the differentiation between steatosis and hepatic inflammation, and reached almost the maximum accuracy (ROC-AUC: 0.998) upon the addition of CK-18. CONCLUSIONS: Plasma CatD holds a high diagnostic value to distinguish pediatric patients with hepatic inflammation from children with steatosis.

Hepatic Uptake of Rectally Administered Butyrate Prevents an Increase in Systemic Butyrate Concentrations in Humans.

BACKGROUND: Short-chain fatty acids (SCFAs), fermentation products of undigested fibers, are considered beneficial for colonic health. High plasma concentrations are potentially harmful; therefore, information about systemic SCFA clearance is needed before therapeutic use of prebiotics or colonic SCFA administration. OBJECTIVE: The aim of this study was to investigate the effect of rectal butyrate administration on SCFA interorgan exchange. METHODS: Twelve patients (7 men; age: 66.4 ± 2.0 y; BMI 24.5 ± 1.4 kg/m(2)) undergoing upper abdominal surgery participated in this randomized placebo-controlled trial. During surgery, 1 group received a butyrate enema (100 mmol sodium butyrate/L; 60 mL; n = 7), and the other group a placebo (140 mmol 0.9% NaCl/L; 60 mL; n = 5). Before and 5, 15, and 30 min after administration, blood samples were taken from the radial artery, hepatic vein, and portal vein. Plasma SCFA concentrations were analyzed, and fluxes from portal-drained viscera, liver, and splanchnic area were calculated and used for the calculation of the incremental area under the curve (iAUC) over a 30-min period. RESULTS: Rectal butyrate administration led to higher portal butyrate concentrations at 5 min compared with placebo (92.2 ± 27.0 µmol/L vs. 14.3 ± 3.4 µmol/L, respectively; P < 0.01). In the butyrate-treated group, iAUCs of gut release (282.8 ± 133.8 µmol/kg BW · 0.5 h) and liver uptake (-293.7 ± 136.0 µmol/kg BW · 0.5 h) of butyrate were greater than in the placebo group [-16.6 ± 13.4 µmol/kg BW · 0.5 h (gut release) and 16.0 ± 13.8 µmol/kg BW · 0.5 h (liver uptake); P = 0.01 and P < 0.05, respectively]. As a result, splanchnic butyrate release did not differ between groups. CONCLUSION: After colonic butyrate administration, splanchnic butyrate release was prevented in patients undergoing upper abdominal surgery. These observations imply that therapeutic colonic SCFA administration at this dose is safe. The trial was registered at clinicaltrials.gov as NCT02271802.

Nε-(carboxymethyl)lysine-receptor for advanced glycation end product axis is a key modulator of obesity-induced dysregulation of adipokine expression and insulin resistance.

OBJECTIVE: Dysregulation of inflammatory adipokines by the adipose tissue plays an important role in obesity-associated insulin resistance. Pathways leading to this dysregulation remain largely unknown. We hypothesized that the receptor for advanced glycation end products (RAGE) and the ligand N(ε)-(carboxymethyl)lysine (CML) are increased in adipose tissue and, moreover, that activation of the CML-RAGE axis plays an important role in obesity-associated inflammation and insulin resistance. APPROACH AND RESULTS: In this study, we observed a strong CML accumulation and increased expression of RAGE in adipose tissue in obesity. We confirmed in cultured human preadipocytes that adipogenesis is associated with increased levels of CML and RAGE. Moreover, CML induced a dysregulation of inflammatory adipokines in adipocytes via a RAGE-dependent pathway. To test the role of RAGE in obesity-associated inflammation further, we constructed an obese mouse model that is deficient for RAGE (ie, RAGE(-/-)/Leptr(Db-/-) mice). RAGE(-/-)/Leptr(Db-/-) mice displayed an improved inflammatory profile and glucose homeostasis when compared with RAGE(+/+)/Leptr(Db-/-) mice. In addition, CML was trapped in adipose tissue in RAGE(+/+)/Leptr(Db-/-) mice but not in RAGE(-/-)/Leptr(Db-/-). RAGE-mediated trapping in adipose tissue provides a mechanism underlying CML accumulation in adipose tissue and explaining decreased CML plasma levels in obese subjects. Decreased CML plasma levels in obese individuals were strongly associated with insulin resistance. CONCLUSIONS: RAGE-mediated CML accumulation in adipose tissue and the activation of the CML-RAGE axis are important mechanisms involved in the dysregulation of adipokines in obesity, thereby contributing to the development of obesity-associated insulin resistance.

Unraveling the regulatory mechanisms underlying tissue-dependent genetic variation of gene expression.

It is known that genetic variants can affect gene expression, but it is not yet completely clear through what mechanisms genetic variation mediate this expression. We therefore compared the cis-effect of single nucleotide polymorphisms (SNPs) on gene expression between blood samples from 1,240 human subjects and four primary non-blood tissues (liver, subcutaneous, and visceral adipose tissue and skeletal muscle) from 85 subjects. We characterized four different mechanisms for 2,072 probes that show tissue-dependent genetic regulation between blood and non-blood tissues: on average 33.2% only showed cis-regulation in non-blood tissues; 14.5% of the eQTL probes were regulated by different, independent SNPs depending on the tissue of investigation. 47.9% showed a different effect size although they were regulated by the same SNPs. Surprisingly, we observed that 4.4% were regulated by the same SNP but with opposite allelic direction. We show here that SNPs that are located in transcriptional regulatory elements are enriched for tissue-dependent regulation, including SNPs at 3' and 5' untranslated regions (P = 1.84×10(-5) and 4.7×10(-4), respectively) and SNPs that are synonymous-coding (P = 9.9×10(-4)). SNPs that are associated with complex traits more often exert a tissue-dependent effect on gene expression (P = 2.6×10(-10)). Our study yields new insights into the genetic basis of tissue-dependent expression and suggests that complex trait associated genetic variants have even more complex regulatory effects than previously anticipated.

Genetic and epigenetic regulation of gene expression in fetal and adult human livers.

BACKGROUND: The liver plays a central role in the maintenance of homeostasis and health in general. However, there is substantial inter-individual variation in hepatic gene expression, and although numerous genetic factors have been identified, less is known about the epigenetic factors. RESULTS: By analyzing the methylomes and transcriptomes of 14 fetal and 181 adult livers, we identified 657 differentially methylated genes with adult-specific expression, these genes were enriched for transcription factor binding sites of HNF1A and HNF4A. We also identified 1,000 genes specific to fetal liver, which were enriched for GATA1, STAT5A, STAT5B and YY1 binding sites. We saw strong liver-specific effects of single nucleotide polymorphisms on both methylation levels (28,447 unique CpG sites (meQTL)) and gene expression levels (526 unique genes (eQTL)), at a false discovery rate (FDR) < 0.05. Of the 526 unique eQTL associated genes, 293 correlated significantly not only with genetic variation but also with methylation levels. The tissue-specificities of these associations were analyzed in muscle, subcutaneous adipose tissue and visceral adipose tissue. We observed that meQTL were more stable between tissues than eQTL and a very strong tissue-specificity for the identified associations between CpG methylation and gene expression. CONCLUSIONS: Our analyses generated a comprehensive resource of factors involved in the regulation of hepatic gene expression, and allowed us to estimate the proportion of variation in gene expression that could be attributed to genetic and epigenetic variation, both crucial to understanding differences in drug response and the etiology of liver diseases.

Murine bladder imaging by 2-photon microscopy: an experimental study of morphology.

PURPOSE: We developed 2-photon laser scanning microscopy analysis of the native murine bladder. MATERIALS AND METHODS: Bladder tissue from wild-type mice was imaged by 2-photon laser scanning microscopy autofluorescence and second harmonic generation microscopy. Bladder wall layers and structures were analyzed using differences in color, size, shape and morphology. RESULTS: Autofluorescence of the urothelium, nerve structures and muscles was visible in the green spectral channel due to autofluorochromes such as NAD(P)H and elastin. Second harmonic generation of collagen was seen in the blue spectral channel. Imaging from the mucosal side revealed umbrella cells at 0 and 30 µm, of which the high cellular NAD(P)H content allows autofluorescence detection. Below that a network-like connective tissue layer was visualized up to 50 µm that contained vessels with a diameter of 10 to 40 µm and nerves with a diameter of 1 to 6 µm. Imaging from the adventitial side revealed a radiant collagen layer covered with nerves and macrophages at 0 to 20 µm. Below at 20 to 25 µm we visualized a thick muscle layer containing elastic fibers and macrophages. Findings were also represented in 3-dimensional reconstructions, providing information on structure localization, orientation and interconnection. CONCLUSIONS: Two-photon laser scanning microscopy imaging using autofluorescence of the murine bladder is a promising technique to provide new insight into structures and morphology. It opens avenues to identify structural changes in bladder pathology.

Clonal heterogeneity of thymic B cells from early-onset myasthenia gravis patients with antibodies against the acetylcholine receptor.

Myasthenia gravis (MG) with antibodies against the acetylcholine receptor (AChR-MG) is considered as a prototypic autoimmune disease. The thymus is important in the pathophysiology of the disease since thymus hyperplasia is a characteristic of early-onset AChR-MG and patients often improve after thymectomy. We hypothesized that thymic B cell and antibody repertoires of AChR-MG patients differ intrinsically from those of control individuals. Using immortalization with Epstein-Barr Virus and Toll-like receptor 9 activation, we isolated and characterized monoclonal B cell lines from 5 MG patients and 8 controls. Only 2 of 570 immortalized B cell clones from MG patients produced antibodies against the AChR (both clones were from the same patient), suggesting that AChR-specific B cells are not enriched in the thymus. Surprisingly, many B cell lines from both AChR-MG and control thymus samples displayed reactivity against striated muscle proteins. Striational antibodies were produced by 15% of B cell clones from AChR-MG versus 6% in control thymus. The IgVH gene sequence analysis showed remarkable similarities, concerning VH family gene distribution, mutation frequency and CDR3 composition, between B cells of AChR-MG patients and controls. MG patients showed clear evidence of clonal B cell expansion in contrast to controls. In this latter aspect, MG resembles multiple sclerosis and clinically isolated syndrome, but differs from systemic lupus erythematosus. Our results support an antigen driven immune response in the MG thymus, but the paucity of AChR-specific B cells, in combination with the observed polyclonal expansions suggest a more diverse immune response than expected.

Plasma intestinal fatty acid-binding protein levels correlate with morphologic epithelial intestinal damage in a human translational ischemia-reperfusion model.

BACKGROUND AND AIM: Intestinal fatty acid-binding protein (I-FABP) is a useful marker in the detection of intestinal ischemia. However, more insight into the test characteristics of I-FABP release is needed. This study aimed to investigate the relationship between plasma I-FABP levels and the severity of ischemic mucosal injury, and define the clinical usefulness of systemic I-FABP following ischemia. METHODS: In a human experimental model, 6 cm of the jejunum, to be removed for surgical reasons, was selectively exposed to either 15, 30, or 60 minutes of ischemia (I) followed by 30 and 120 minutes of reperfusion (R). Blood and tissue was sampled at all time points. Arteriovenous (V-A) concentration differences of I-FABP were measured. Tissue sections were stained with hematoxylin/eosin, and villus height was measured to score epithelial damage. RESULTS: Histologic analysis showed only minor reversible intestinal damage following 15 I and 30 I; however, severe irreversible epithelial damage was observed in the jejunum exposed to 60 I. I-FABP V-A differences paralleled the degree of tissue damage over time [7.79 (± 1.8) ng/mL, 128.6 (± 44.2) ng/mL, 463.3 (± 139.8) ng/mL for 15 I, 30 I and 60 I, respectively]. A good correlation was found between histologic epithelial damage and V-A I-FABP (r=-0.82, P<0.001). Interestingly, systemic I-FABP levels were significantly increased after 60 I of this short small intestinal segment. CONCLUSIONS: This study demonstrates the relationship between the duration of ischemia and the extent of tissue damage, which is reflected by I-FABP V-A plasma levels. In addition, systemic I-FABP levels appear valuable in detecting irreversible intestinal ischemia-reperfusion damage.

Trans-eQTLs reveal that independent genetic variants associated with a complex phenotype converge on intermediate genes, with a major role for the HLA.

For many complex traits, genetic variants have been found associated. However, it is still mostly unclear through which downstream mechanism these variants cause these phenotypes. Knowledge of these intermediate steps is crucial to understand pathogenesis, while also providing leads for potential pharmacological intervention. Here we relied upon natural human genetic variation to identify effects of these variants on trans-gene expression (expression quantitative trait locus mapping, eQTL) in whole peripheral blood from 1,469 unrelated individuals. We looked at 1,167 published trait- or disease-associated SNPs and observed trans-eQTL effects on 113 different genes, of which we replicated 46 in monocytes of 1,490 different individuals and 18 in a smaller dataset that comprised subcutaneous adipose, visceral adipose, liver tissue, and muscle tissue. HLA single-nucleotide polymorphisms (SNPs) were 10-fold enriched for trans-eQTLs: 48% of the trans-acting SNPs map within the HLA, including ulcerative colitis susceptibility variants that affect plausible candidate genes AOAH and TRBV18 in trans. We identified 18 pairs of unlinked SNPs associated with the same phenotype and affecting expression of the same trans-gene (21 times more than expected, P<10(-16)). This was particularly pronounced for mean platelet volume (MPV): Two independent SNPs significantly affect the well-known blood coagulation genes GP9 and F13A1 but also C19orf33, SAMD14, VCL, and GNG11. Several of these SNPs have a substantially higher effect on the downstream trans-genes than on the eventual phenotypes, supporting the concept that the effects of these SNPs on expression seems to be much less multifactorial. Therefore, these trans-eQTLs could well represent some of the intermediate genes that connect genetic variants with their eventual complex phenotypic outcomes.

Ischaemia-induced mucus barrier loss and bacterial penetration are rapidly counteracted by increased goblet cell secretory activity in human and rat colon.

OBJECTIVE: Colonic ischaemia is frequently observed in clinical practice. This study provides a novel insight into the pathophysiology of colon ischaemia/reperfusion (IR) using a newly developed human and rat experimental model. DESIGN: In 10 patients a small part of colon that had to be removed for surgical reasons was isolated and exposed to 60 min of ischaemia (60I) with/without different periods of reperfusion (30R and 60R). Tissue not exposed to IR served as control. In rats, colon was exposed to 60I, 60I/30R, 60I/120R or 60I/240R (n=7 per group). The tissue was snap-frozen or fixed in glutaraldehyde, formalin or methacarn fixative. Mucins were stained with Periodic Acid Schiff/Alcian Blue (PAS/AB) and MUC2/Dolichos biflorus agglutinin (DBA). Bacteria were studied using electron microscopy (EM) and fluorescent in situ hybridisation (FISH). Neutrophils were studied using myeloperoxidase staining. qPCR was performed for MUC2, interleukin (IL)-6, IL-1ß and tumour necrosis factor α. RESULTS: In rats, PAS/AB and MUC2/DBA staining revealed mucus layer detachment at ischaemia which was accompanied by bacterial penetration (in EM and FISH). Human and rat studies showed that, simultaneously, goblet cell secretory activity increased. This was associated with expulsion of bacteria from the crypts and restoration of the mucus layer at 240 min of reperfusion. Inflammation was limited to minor influx of neutrophils and increased expression of proinflammatory cytokines during reperfusion. CONCLUSIONS: Colonic ischaemia leads to disruption of the mucus layer facilitating bacterial penetration. This is rapidly counteracted by increased secretory activity of goblet cells, leading to expulsion of bacteria from the crypts as well as restoration of the mucus barrier.