Gut microbiota in mucosa and feces of newly diagnosed, treatment-naïve adult inflammatory bowel disease and irritable bowel syndrome patients.

The knowledge on how gut microbes contribute to the inflammatory bowel disease (IBD) at the onset of disease is still scarce. We compared gut microbiota in newly diagnosed, treatment-naïve adult IBD (Crohn's disease (CD) and ulcerative colitis (UC)) to irritable bowel syndrome (IBS) patients and healthy group. Mucosal and fecal microbiota of 49 patients (13 UC, 10 CD, and 26 IBS) before treatment initiation, and fecal microbiota of 12 healthy subjects was characterized by 16S rRNA gene sequencing. Mucosa was sampled at six positions, from terminal ileum to rectum. We demonstrate that mucosal microbiota is spatially homogeneous, cannot be differentiated based on the local inflammation status and yet provides bacterial footprints superior to fecal in discriminating disease phenotypes. IBD groups showed decreased bacterial diversity in mucosa at all taxonomic levels compared to IBS. In CD and UC, Dialister was significantly increased, and expansion of Haemophilus and Propionibacterium characterized UC. Compared to healthy individuals, fecal microbiota of IBD and IBS patients had increased abundance of Proteobacteria, Enterobacteriaceae, in particular. Shift toward reduction of Adlercreutzia and butyrate-producing taxa was found in feces of IBD patients. Microbiota alterations detected in newly diagnosed treatment-naïve adult patients indicate that the microbiota changes are set and detectable at the disease onset and likely have a discerning role in IBD pathophysiology. Our results justify further investigation of the taxa discriminating between disease groups, such as H. parainfluenzae, R. gnavus, Turicibacteriaceae, Dialister, and Adlercreutzia as potential biomarkers of the disease.

A chromatographic approach to development of 5-aminosalicylate/folic acid fixed-dose combinations for treatment of Crohn's disease and ulcerative colitis.

Medication adherence is an important factor in inflammatory bowel disease therapy, which includes regular supplementation of malabsorbed vitamins. Absorption of folic acid is limited due to the damaging of the gastrointestinal tract, which can increase the chances to develop megaloblastic anaemia and colorectal cancer. In this work, 5-aminosalicylates (mesalazine, balsalazide, sulfasalazine and olsalazine) and folic acid were characterized regarding their pharmacokinetic related properties (hydrophobicity, phospholipid and plasma protein binding) using the biomimetic chromatographic approach. Despite the high binding percentage of 5-aminosalicylates for human serum albumin (> 61.44%), results have shown that folic acid binding to human serum albumin protein is far greater (69.40%) compared to α1-acid-glycoprotein (3.45%). Frontal analysis and zonal elution studies were conducted to provide an insight into the binding of folic acid to human serum albumin and potential competition with 5-aminosalicylates. The analytical method for the simultaneous determination of assay in proposed fixed-dose combinations was developed and validated according to ICH Q2 (R1) and FDA method validation guidelines. Separation of all compounds was achieved within 16 min with satisfactory resolution (Rs > 3.67) using the XBridge Phenyl column (150 × 4.6 mm, 3.5 µm). High linearity (r > 0.9997) and precision (RSD < 2.29%) was obtained, whilst all recoveries were within the regulatory defined range by British (100.0 ± 5.0%) and United States Pharmacopeia (100.0 ± 10.0%).

Occurrence of Anaemia in the First Year of Inflammatory Bowel Disease in a European Population-based Inception Cohort-An ECCO-EpiCom Study.

BACKGROUND AND AIMS: Anaemia is an important complication of inflammatory bowel disease [IBD]. The aim of this study was to determine the prevalence of anaemia and the practice of anaemia screening during the first year following diagnosis, in a European prospective population-based inception cohort. METHODS: Newly diagnosed IBD patients were included and followed prospectively for 1 year in 29 European and one Australian centre. Clinical data including demographics, medical therapy, surgery and blood samples were collected. Anaemia was defined according to the World Health Organization criteria. RESULTS: A total of 1871 patients (Crohn's disease [CD]: 686, 88%; ulcerative colitis [UC]: 1,021, 87%; IBD unclassified [IBDU] 164. 81%) were included in the study. The prevalence of anaemia was higher in CD than in UC patients and, overall, 49% of CD and 39% of UC patients experienced at least one instance of anaemia during the first 12 months after diagnosis. UC patients with more extensive disease and those from Eastern European countries, and CD patients with penetrating disease or colonic disease location, had higher risks of anaemia. CD and UC patients in need of none or only mild anti-inflammatory treatment had a lower risk of anaemia. In a significant proportion of patients, anaemia was not assessed until several months after diagnosis, and in almost half of all cases of anaemia a thorough work-up was not performed. CONCLUSIONS: Overall, 42% of patients had at least one instance of anaemia during the first year following diagnosis. Most patients were assessed for anaemia regularly; however, a full anaemia work-up was frequently neglected in this community setting.

Prevalent hypovitaminosis D in Crohn's disease correlates highly with mediators of osteoimmunology.

PURPOSE: In the context of osteoimmunology in Crohn's disease, an association was hypothesized among vitamin D and members of the TNF-α family, known as the RANK (receptor-activator of nuclear factor- κB)-RANK ligand-osteoprotegerin pathway. METHODS: This was a cross-sectional study of 95 patients with Crohn's disease (80 with long-standing disease and 15 newly diagnosed, never treated) and two control groups (healthy volunteers, n=30; and ulcerative colitis patients, n=30). Spine and hip bone mineral density was measured by dual-energy x-ray absorptiometry. Serum 25-hydroxyvitamin-D3, TNF-α, IL-6, sRANKL, osteoprotegerin levels and biochemical markers of bone turnover were analyzed. RESULTS: The precursor metabolite, 25(OH)D3, was measured in 95 young adult CD patients (47 men, 48 women; median age 30 years). A suboptimal 25(OH)D3 level was observed in 90% of CD patients, of whom 40% had a serious deficiency. There was no significant difference in 25(OH)D3 levels between CD patients and those with ulcerative colitis. Analysis revealed an association between 25(OH)D3 deficiency and the increased biogenesis of osteoclastically-active sRANKL (p=0.014) and the proinflammatory cytokines TNF-α (p=0.015) and IL-6 (p=0.029) . CD patients with low bone mineral density had a mean 25(OH)D3 (35±18 nmol/l) in the range of serious deficiency to insufficiency, whereas mean 25(OH)D3 was higher (49±28 nmol/l) in patients with healthy bone status, although levels were still inadequate (p=0.004). The logistic model reported low levels of 25(OH)D3 to be a significant predictor of bone disease [odds ratio=2.66(6.8), p < 0.009]. In the multivariable analysis, adjusted for several confounding factors, 25(OH)D3, sRANKL, IL-6 and TNF-α were independently associated with a likelihood of bone disease [odds ratio (range): 1.02(2.75); 1.09(3.71); 1.27(6.95) respectively, p=0.001]. CONCLUSION: The presented findings suggest that a 25(OH)D3 deficiency accompanying an inflammatory state in CD is a high risk condition for metabolic bone disease.

Initial disease course and treatment in an inflammatory bowel disease inception cohort in Europe: the ECCO-EpiCom cohort.

BACKGROUND: The EpiCom cohort is a prospective, population-based, inception cohort of inflammatory bowel disease (IBD) patients from 31 European centers covering a background population of 10.1 million. The aim of this study was to assess the 1-year outcome in the EpiCom cohort. METHODS: Patients were followed-up every third month during the first 12 (±3) months, and clinical data, demographics, disease activity, medical therapy, surgery, cancers, and deaths were collected and entered in a Web-based database (www.epicom-ecco.eu). RESULTS: In total, 1367 patients were included in the 1-year follow-up. In western Europe, 65 Crohn's disease (CD) (16%), 20 ulcerative colitis (UC) (4%), and 4 IBD unclassified (4%) patients underwent surgery, and in eastern Europe, 12 CD (12%) and 2 UC (1%) patients underwent surgery. Eighty-one CD (20%), 80 UC (14%), and 13 (9%) IBD unclassified patients were hospitalized in western Europe compared with 17 CD (16%) and 12 UC (8%) patients in eastern Europe. The cumulative probability of receiving immunomodulators was 57% for CD in western (median time to treatment 2 months) and 44% (1 month) in eastern Europe, and 21% (5 months) and 5% (6 months) for biological therapy, respectively. For UC patients, the cumulative probability was 22% (4 months) and 15% (3 months) for immunomodulators and 6% (3 months) and 1% (12 months) for biological therapy, respectively in the western and eastern Europe. DISCUSSION: In this cohort, immunological therapy was initiated within the first months of disease. Surgery and hospitalization rates did not differ between patients from eastern and western Europe, although more western European patients received biological agents and were comparable to previous population-based inception cohorts.

[The role of anti-TNF therapy in ulcerative colitis]. / Mjesto anti-TNF terapije u lijecenju ulceroznog kolitisa.

Anti-TNF-alfa molecules are currently being used to treat ulcerative colitis regarding to the fact that TNF-alpha has an important role in the pathogenesis of IBD. Although these drugs improved the therapy of patients, immunogenicity limits their potential for clinical use. Infliximab and adalimumab are effective for induction and maintenance of remission in outpatients with moderate to severe steroid-refractory ulcerative colitis. Biologics can be a drug of choice for patients with refractory proctitis and refractory pouchitis. In hospitalized patients with steroid-resistant severe ulcerative colitis who are candidates for colectomy, infliximab may be second-line option. Adequate long-term maintenance therapy with anti-TNF is required after rescue therapy for a sustained benefit. Regarding to the known risk for side-effects of anti-TNF drugs especially in patients concomitantly treated with thiopurines it is urgent future research.

Simultaneous analysis of mitotane and its main metabolites in human blood and urine samples by SPE-HPLC technique.

Adrenocortical carcinoma (ACC) is a rare malignancy with an incompletely understood pathogenesis and a poor prognosis. The adrenalytic activity of mitotane has made it the most important single drug in the treatment of ACC. Unfortunately, the exact mechanism of mitotane action is still unknown. It is believed that mitotane belongs to the class of drugs that require metabolic transformation by cytochrome P450 for therapeutic action; therefore determination of plasma levels of not only mitotane but also its metabolites would help in carrying out the treatment. The objective of this work was to develop and validate an SPE-HPLC method for simultaneous determination of mitotane and its metabolites in different biological fluids. The sample preparation consisted of a solid-phase extraction on a Discovery DSC(18) cartridge, while analysis of extracts was performed on a Symmetry C(18) column. The usefulness of the proposed method was confirmed by analysis of plasma, red cell and urine samples from patient chronically treated with 1.5 g of mitotane. The patient involved in this study had a high plasma concentration of mitotane and none of the investigated metabolites were found. In order to investigate whether the polymorphism of CYP2C9 and CYP2C19 enzymes could be related to the metabolism of mitotane, RT-PCR analysis was performed.

Proinflammatory cytokines and receptor activator of nuclear factor kappaB-ligand/osteoprotegerin associated with bone deterioration in patients with Crohn's disease.

OBJECTIVES: The high incidence of bone disease and the increasing evidence of Crohn's disease (CD) bone decline in corticosteroid users and nonusers suggest that bone metabolism is affected by inflammatory process. The aim of the study was to compare serum levels of proinflammatory cytokines, markers of bone turnover and regulatory molecules of osteoclast biogenesis, receptor activator of nuclear factor kappaB-ligand (RANKL) and osteoprotegerin (OPG), between naïve and long-standing CD patients. METHODS: The study included 95 CD patients, 15 of them with newly diagnosed and previously untreated CD. The spine and hip bone mineral density was measured by dual-energy X-ray absorptiometry. Biochemical markers were determined by immunoassay. RESULTS: Osteopenia was recorded at diagnosis in 53% of naïve patients and osteoporosis was found in 26% of long-standing CD patients. The newly diagnosed patients showed correlation between TNF-alpha and soluble RANKL (sRANKL) (r=0.5; P=0.04), and this positive relationship characterized the study population as a whole (r=0.3; P=0.003). Analysis of the OPG and sRANKL relationship showed absence of correlation in patients with healthy skeleton, whereas an inverse correlation was found in those with osteopenia (r=-0.31; P=0.033) and osteoporosis (r=-0.48; P=0.028). In naïve patients with reduced T score, the correlation between sRANKL and OPG was highly inverse (r=-0.8; P=0.02) and these patients were characterized by lower BMI, significantly higher level of proinflammatory cytokines, elevated C-reactive protein, and increased activity of free sRANKL and OPG. CONCLUSION: Bone disease that accompanies CD at diagnosis suggests that bone metabolism is affected by the underlying inflammatory process per se, as probably confirmed by our finding of the central proinflammatory cytokine TNF-alpha being strongly associated with the osteoclastogenic mediator RANKL, and inversely with bone density.

Humoral SPARC/osteonectin protein in plasma cell dyscrasias.

The matricellular protein SPARC (secreted protein acidic and rich in cysteine)/osteonectin was determined in patients with multiple myeloma and related disease to assess the hypothesized role of SPARC as a possible marker of tumor burden and disease progression. Soluble SPARC was measured by competitive enzyme-linked immunosorbent assay (ELISA) in plasma of 42 patients, including sequential measurements in individual patients, and in 20 healthy controls. SPARC values were heterogeneous in multiple myeloma patients showing a decline from baseline levels recorded in controls (456+/-195 vs 600+/-63 ng/ml, p=0.00023). A SPARC showed a significant positive correlation with platelet count (r=0.72, p=0.000000, n=42), hemoglobin (r=0.52, p=0.00037, n=42), and IgG level (r=0.43, p=0.0085, n=42) and negative correlation with beta(2)-microglobulin (r=-0.46, p=0.0023, n=42), aspartate aminotransferase (AST) (r=-0.42, p=0.0061, n=41), interleukin (IL)-6 (r=-0.41, p=0.008, n=42), lactate dehydrogenase (LDH) (r=-0.36, p=0.02, n=41), and percentage of plasma cells in bone marrow aspirate (r=-0.34, p=0.029, n=42). No correlation was found between SPARC and "M" component or disease stage. Investigations performed during the course of disease, including sequential measurements in individual patients, showed a trend to downregulation by disease progression, with the lowest level recorded in the terminal stage (217+/-107 ng/ml, n=11). Patients with established osteolytic lesions had lower plasma SPARC at diagnosis (309+/-197 vs 581+/-293, p=0.021), which correlated with osteocalcin by disease progression (r=0.31, p=0.026). The results of this pilot study revealed abnormalities in the level of humoral SPARC in multiple myeloma and an overall trend to downregulation in the advanced stage of the disease. The regulation of SPARC seems to be opposite to the markers of tumor burden and of aggressive multiple myeloma phenotype.

[Occurrence of chronic B-cell lymphocytic leukemia in Hodgkin's disease: case report]. / Pojava B-kronicne limfocitne leukemije u Hodgkinovoj bolesti: prikaz bolesnice.

We present a case of a female patient (79 years) with pathohistologic diagnosis of Hodgkin's lymphoma (HL) (stage IIIB, histologic type MC) for which she was treated with chemotherapy according to LVPP protocol (6 cycles) with good therapeutic response. Unexpectedly, 18 months after HL diagnosis leukocytosis occurred (19.4 x 10(9)/L) with 65% of lymphocytes with lymphoplasmocytic differentiation. Immunophenotype of these cells is typical for B-chronic lymphocytic leukemia (B-CLL) (CD5/CD19+, CD23-, CD38 +/-; with weak expression of monoclonal light chains lambda). Molecular analysis confirmed clonal immunoglobulin heavy chain gene (IgH) rearrangement of peripheral blood lymphocytes. The diagnosis of B-CLL imposed the question of the connection between two neoplasms of lymphocytic origin. Molecular analysis of lymph node biopsy taken at the time of lymphoma diagnosis revealed clonal population of B lymphocytes. That test undeniably proved coexistence of both diseases from the beginning. The latest PCR analysis of archive peripheral blood smears confirmed B lymphocyte clonality without diagnostic criteria for lymphoproliferative disease of CLL type. This finding etiologically excludes secondary leukemia. The possibility of untypical presentation of CLL in transformation to Richter's syndrome with morphologic characteristics of HL from the beginning stays unconfirmed. The hypothetical question that remains unanswered is: "Was it one disease in different clinical forms?"