Aggregation of human osteoblasts unlocks self-reliant differentiation and constitutes a microenvironment for 3D-co-cultivation with other bone marrow cells.

Skeletal bone function relies on both cells and cellular niches, which, when combined, provide guiding cues for the control of differentiation and remodeling processes. Here, we propose an in vitro 3D model based on human fetal osteoblasts, which eases the study of osteocyte commitment in vitro and thus provides a means to examine the influences of biomaterials, substances or cells on the regulation of these processes. Aggregates were formed from human fetal osteoblasts (hFOB1.19) and cultivated under proliferative, adipo- and osteoinductive conditions. When cultivated under osteoinductive conditions, the vitality of the aggregates was compromised, the expression levels of the mineralization-related gene DMP1 and the amount of calcification and matrix deposition were lower, and the growth of the spheroids stalled. However, within spheres under growth conditions without specific supplements, self-organization processes occur, which promote extracellular calcium deposition, and osteocyte-like cells develop. Long-term cultivated hFOB aggregates were free of necrotic areas. Moreover, hFOB aggregates cultivated under standard proliferative conditions supported the co-cultivation of human monocytes, microvascular endothelial cells and stromal cells. Overall, the model presented here comprises a self-organizing and easily accessible 3D osteoblast model for studying bone marrow formation and in vitro remodeling and thus provides a means to test druggable molecular pathways with the potential to promote life-long bone formation and remodeling.

Body impedance analysis to estimate malnutrition in inflammatory bowel disease patients - A cross-sectional study.

OBJECTIVE: Malnutrition is a common clinical problem in patients with inflammatory bowel diseases (IBD). However, a gold standard for the detection of malnutrition in IBD patients is lacking. METHODS: A cross-sectional study to assess malnutrition in patients with IBD and healthy controls (HCs). Clinical characteristics (Montreal classification, disease activity, previous surgery) and mutations in the NOD2 gene in patients with Crohn's disease (CD) were obtained. We performed a nutritional assessment with screening for nutritional risk and diagnosis for malnutrition (Malnutrition Universal Screening Tool [MUST]) score, NRS-2002, European Society for Clinical Nutrition and Metabolism (ESPEN), and Global Leadership Initiative on Malnutrition (GLIM) criteria and performed body impedance analysis (BIA). RESULTS: 101 IBD patients (57 CD and 44 ulcerative colitis (UC) and 50 HC were included in a single northern German tertiary center. GLIM criteria detected malnutrition significantly more often compared to the ESPEN criteria. Active disease, a long-standing disease course, and previous surgery were associated with reduced muscle mass. IBD patients had a higher fat mass index compared to HC. Mutations in the NOD2 gene had no effect on nutritional status. CONCLUSIONS: The GLIM criteria detect malnutrition at a higher rate compared to ESPEN. Specific disease factors might put IBD patients at a higher risk for the development of malnutrition, so these patients might benefit from a frequently performed screening, which might result in a favorable disease course.

Genetic association analysis of CLEC5A and CLEC7A gene single-nucleotide polymorphisms and Crohn's disease.

BACKGROUND: Crohn's disease (CD) is characterized by a multifactorial etiology and a significant impact of genetic traits. While NOD2 mutations represent well established risk factors of CD, the role of other genes is incompletely understood. AIM: To challenge the hypothesis that single nucleotide polymorphisms (SNPs) in the genes CLEC5A and CLEC7A, two members of the C-type lectin domain family of pattern recognition receptors, may be associated with CD. METHODS: SNPs in CLEC5A, CLEC7A and the known CD risk gene NOD2 were studied using real time PCR-based SNP assays. Therefore, DNA samples from 175 patients and 157 healthy donors were employed. Genotyping data were correlated with clinical characteristics of the patients and the results of gene expression data analyses. RESULTS: In accordance with previous studies, rs2066844 and rs2066847 in NOD2 were found to be significantly associated with CD (allelic P values = 0.0368 and 0.0474, respectively). Intriguingly, for genotype AA of rs1285933 in CLEC5A, a potential association with CD (recessive P = 0.0523; odds ratio = 1.90) was observed. There were no associations between CD and SNPs rs2078178 and rs16910631 in CLEC7A. Variants of rs1285933 had no impact on CLEC5A gene expression. In contrast, genotype-dependent differences of CXCL5 expression in peripheral blood mononuclear cells were observed. There is no statistical interaction between the tested SNPs of NOD2 and CLEC5A, suggesting of a novel pathway contributing to the disease. CONCLUSION: Our data encourage enlarged follow-up studies to further address an association of SNP rs1285933 in CLEC5A with CD. The C-type lectin domain family member also deserves attention regarding a potential role in the pathophysiology of CD.

Mutations in the NOD2 gene are associated with a specific phenotype and lower anti-tumor necrosis factor trough levels in Crohn's disease.

OBJECTIVE: Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) gene mutations are known to be an important risk factor in the pathogenesis of Crohn's disease (CD). Specific disease phenotypes are associated with the presence of NOD2 gene mutation. One treatment option is to use an anti-tumor necrosis factor (TNF)-α agent. Therapeutic drug monitoring (TDM) is usually performed in cases of a loss of response. Our aim was to explore whether NOD2 gene mutations have an effect on the disease phenotype, vitamin D levels, and on TDM in CD patients. METHODS: This was a retrospective genotype-phenotype association study on NOD2 gene mutations in 161 patients with CD. RESULTS: Altogether 55 (34.2%) patients carried at least one mutant allele of NOD2. NOD2 gene mutations were associated with ileocecal disease, ileocecal resection, stricturing and perianal disease, and patients with NOD2 gene mutation had significantly less frequent colonic disease and received an ostomy less frequently. TDM in patients with NOD2 gene mutation showed more frequent anti-TNF trough levels in the subtherapeutic range and lower anti-TNF trough levels than in NOD2 wild-type (WT) patients. CONCLUSIONS: CD patients with NOD2 gene mutation have a specific clinical phenotype and they may require higher doses of anti-TNF agents to achieve sufficient anti-TNF trough levels. They may therefore benefit from a proactive TDM than a reactive approach. This could be another step in the direction of personalized medicine.

NOD2- and disease-specific gene expression profiles of peripheral blood mononuclear cells from Crohn's disease patients.

AIM: To investigate disease-specific gene expression profiles of peripheral blood mononuclear cells (PBMCs) from Crohn's disease (CD) patients in clinical remission. METHODS: Patients with CD in clinical remission or with very low disease activity according to the Crohn's disease activity index were genotyped regarding nucleotide-binding oligomerization domain 2 (NOD2), and PBMCs from wild-type (WT)-NOD2 patients, patients with homozygous or heterozygous NOD2 mutations and healthy donors were isolated for further analysis. The cells were cultured with vitamin D, peptidoglycan (PGN) and lipopolysaccharide (LPS) for defined periods of time before RNA was isolated and subjected to microarray analysis using Clariom S assays and quantitative real-time PCR. NOD2- and disease-specific gene expression profiles were evaluated with repeated measure ANOVA by a general linear model. RESULTS: Employing microarray assays, a total of 267 genes were identified that were significantly up- or downregulated in PBMCs of WT-NOD2 patients, compared to healthy donors after challenge with vitamin D and/or a combination of LPS and PGN (P < 0.05; threshold: ≥ 2-fold change). For further analysis by real-time PCR, genes with known impact on inflammation and immunity were selected that fulfilled predefined expression criteria. In a larger cohort of patients and controls, a disease-associated expression pattern, with higher transcript levels in vitamin D-treated PBMCs from patients, was observed for three of these genes, CLEC5A (P < 0.030), lysozyme (LYZ; P < 0.047) and TREM1 (P < 0.023). Six genes were found to be expressed in a NOD2-dependent manner (CD101, P < 0.002; CLEC5A, P < 0.020; CXCL5, P < 0.009; IL-24, P < 0.044; ITGB2, P < 0.041; LYZ, P < 0.042). Interestingly, the highest transcript levels were observed in patients with heterozygous NOD2 mutations. CONCLUSION: Our data identify CLEC5A and LYZ as CD- and NOD2-associated genes of PBMCs and encourage further studies on their pathomechanistic roles.

Clinical factors are associated with vitamin D levels in IBD patients: A retrospective analysis.

OBJECTIVE: There is growing evidence that vitamin D deficiency plays a role in the development and the course of inflammatory bowel disease (IBD). However, the correlation between vitamin D deficiency and clinical parameters in IBD is still not completely understood. METHODS: A retrospective study of IBD patients was performed. Vitamin D values were analyzed, regardless of vitamin D substitution administration, and correlated with clinical parameters such as medical therapy, anatomical situation, location of the disease and disease activity. Level of 25-hydroxyvitamin D [25(OH)D] <50 nmoL/L was regarded as vitamin D deficiency and <75 nmoL/L as insufficiency. RESULTS: In total, 208 IBD patients were analyzed, including 123 with Crohn's disease (CD) and 85 with ulcerative colitis (UC). Therapy with azathioprine did not affect the vitamin D values of either disease entity. But CD patients benefited from therapy with tumor necrosis factor-α inhibitor and exhibited significantly higher vitamin D levels than those without. Furthermore, significantly lower vitamin D levels were found if CD was located in the small bowel or if the small bowel had been resected. Moreover, significantly lower levels of vitamin D were detectable for high disease activity (reflected by high simple clinical colitis activity index values) in patients with UC. CONCLUSIONS: Vitamin D deficiency is common in patients with IBD. However, certain clinical situations lead to significantly lower vitamin D levels and may therefore require close monitoring for vitamin D deficiency.

Vedolizumab Treatment for Ulcerative Colitis in an Elderly Multimorbid Patient with Hemophilia A.

The treatment of inflammatory bowel diseases (IBD) can be challenging, especially in elderly multimorbid patients. Since incidence and prevalence rates of IBD are rising steadily, treatment of older patients with relevant and also rare comorbidities will be of increasing relevancy for caregivers. Here we report on a 74-year-old multimorbid patient with severe ulcerative colitis (UC) and hemophilia A. Because of the chronic active disease, therapy with a tumor necrosis factor-α inhibitor was started. He suffered from a severe infectious complication (pneumonia) under therapy with infliximab. The therapy was changed to vedolizumab, with which the patient stayed in long-term clinical and endoscopic remission. Because the patient had a non-ST-segment elevation myocardial infarction in April 2016, he received dual platelet inhibitor therapy with aspirin and clopidogrel. Because of consecutive aspirin intolerance, the therapy was changed to clopidogrel monotherapy. Although the UC was treated appropriately with vedolizumab and the patient was in endoscopic mucosal remission, recurrent bleeding episodes from multiple inflammatory pseudopolyps occurred. The bleeding episodes resolved quickly after immediate treatment with factor VIII (Kogenate®). In conclusion, we describe the first patient in the literature with UC and hemophilia A who stayed in long-term remission under therapy with vedolizumab. From our point of view, vedolizumab can be safely administered in the setting of UC and hemophilia A. Antiplatelet drugs which inhibit primary hemostasis must be used with caution in this setting. Bleeding episodes can be treated safely and effectively with factor VIII (Kogenate®).