Metaproteomics of fecal samples of Crohn's disease and Ulcerative Colitis.

Crohn's Disease (CD) and Ulcerative Colitis (UC) are chronic inflammatory bowel diseases (IBD) of the gastrointestinal tract. This study used non-invasive LC-MS/MS to find disease specific microbial and human proteins which might be used later for an easier diagnosis. Therefore, 17 healthy controls, 11 CD patients and 14 UC patients but also 13 Irritable Bowel Disease (IBS) patients, 8 Colon Adenoma (CA) patients, and 8 Gastric Carcinoma (GCA) patients were investigated. The proteins were extracted from the fecal samples with liquid phenol in a ball mill. Subsequently, the proteins were digested tryptically to peptides and analyzed by an Orbitrap LC-MS/MS. For protein identification and interpretation of taxonomic and functional results, the MetaProteomeAnalyzer software was used. Cluster analysis and non-parametric test (analysis of similarities) separated healthy controls from patients with CD and UC as well as from patients with GCA. Among others, CD and UC correlated with an increase of neutrophil extracellular traps and immune globulins G (IgG). In addition, a decrease of human IgA and the transcriptional regulatory protein RprY from Bacillus fragilis was found for CD and UC. A specific marker in feces for CD was an increased amount of the human enzyme sucrose-isomaltase. SIGNIFICANCE: Crohn's Disease and Ulcerative Colitis are chronic inflammatory diseases of the gastrointestinal tract, whose diagnosis required comprehensive medical examinations including colonoscopy. The impact of the microbial communities in the gut on the pathogenesis of these diseases is poorly understood. Therefore, this study investigated the impact of gut microbiome on these diseases by a metaproteome approach, revealing several disease specific marker proteins. Overall, this indicated that fecal metaproteomics has the potential to be useful as non-invasive tool for a better and easier diagnosis of both diseases.

Viral dominance patterns in chronic hepatitis delta determine early response to interferon alpha therapy.

Chronic hepatitis D is caused by coinfection of hepatitis B and hepatitis D virus. While HDV is the dominant virus over HBV in the majority of cases, mechanisms and consequences of viral dominance are largely unknown. We aimed to investigate associations between viral dominance patterns and patients' characteristics and inflammatory features; 109 HDV-infected patients treated with PEG-IFNa-2α within the international multicentre, prospective HIDIT-2 trial were studied. Patients were classified as D- or B-dominant if the viral load of one virus exceeded that of the other virus by more than 1log10 . Otherwise, no viral dominance (ND) was described. We used Luminex-based multiplex technology to study 50 soluble immune mediators (SIM) in pretreatment samples of 105 HDV RNA-positive patients. Dominance of HDV was evident in the majority (75%) of cases. While only 7% displayed B-dominance, 17% showed nondominance. D-dominance was associated with downregulation of 4 interleukins (IL-2ra, IL-13, IL-16 and IL-18) and 5 chemokines/cytokines (CTACK (CCL27), MCP-1 (CCL2), M-CSF, TRAIL and ICAM-1) while no analyte was increased. In addition, D-dominance could be linked to a delayed HDV RNA response to pegylated interferon as patients with B-dominance or nondominance showed higher early HDV RNA responses (61% at week 12) than D-dominant patients (11%; P < .001). In conclusion, this study revealed unexpected effects of viral dominance on clinical and immunological features in chronic hepatitis delta patients. Individualizing PEG-IFNa-2α treatment duration should consider viral dominance. Overall, our findings suggest an activated but exhausted IFN system in D-dominant patients.

[Intestinal microbiota in individualized therapies]. / Die intestinale Mikrobiota als Ansatz für individuelle Therapien.

During recent years, the analysis of the human microbiota has been receiving more and more scientific focus. Deep sequencing analysis enables characterization of microbial communities in different environments without the need of culture-based methods. Hereby, information about microbial communities is increasing enormously. Numerous studies in humans and animal models revealed the important role of the microbiome in emergence and natural course of diseases such as autoimmune diseases and metabolic disorders, e. g., the metabolic syndrome. The identification of causalities between the intestinal microbiota composition and function, and diseases in humans and animal models can help to develop individualized therapies targeting the microbiome and its modification. Nowadays, it is established that several factors influence the composition of the microbiota. Diet it is one of the major factors shaping the microbiota and the use of pro- and prebiotica may induce changes in the microbial community. Fecal microbiome transfer is the first approach targeting the intestinal microbiota which is implemented in the clinical routine for patients with therapy-refractory infections with Clostridium difficile. Herewith, the recipient's microbiota can be changed permanently and the patient can be cured from the infection.

Development and evaluation of a baseline-event-anticipation score for hepatitis delta.

Hepatitis delta is considered the most severe form of viral hepatitis, but variables associated with disease progression are poorly defined. This study aimed to identify risk factors associated with worse clinical outcome in patients with hepatitis delta and to develop a clinical score to determine their risk of experiencing liver-related morbidity or mortality. We followed 75 HBsAg-anti-HDV-positive patients with hepatitis delta for up to 16 years (median 5 years). The baseline-event-anticipation score (BEA score) was developed based on variables associated with the development of liver-related clinical complications. Age, region of origin, presence of cirrhosis, albumin, INR, hyperbilirubinemia and thrombocytopenia were all associated with the development of an event in the training cohort. The BEA score included age, sex, region of origin, bilirubin, platelets and INR. Points were allocated according to hazard ratios, and three risk groups were defined: BEA-A mild risk, BEA-B moderate risk and BEA-C high risk. Hazard ratios of BEA-B and BEA-C patients for liver-related clinical endpoints were 9.01 and 25.27 vs BEA-A with an area under curve of the receiving operating characteristic curve of 0.88. The accuracy of the BEA score was confirmed in two independent validation cohorts followed in Barcelona (n = 77) and Düsseldorf (n = 62). Delta hepatitis is associated with a very severe long-term outcome. The BEA score is easy to apply and predicts with a very high accuracy the development of liver-related complications in patients with hepatitis delta.

Hepatitis C virus core antigen testing in liver and kidney transplant recipients.

HCV RNA levels correlate with the long-term outcome of hepatitis C in liver transplant recipients. Nucleic acid testing (NAT) is usually used to confirm HCV reinfection and to examine viral loads after liver transplantation. HCV core antigen (HCVcoreAg) testing could be an alternative to NAT with some potential advantages including very low intra- and interassay variabilities and lower costs. The performance of HCVcoreAg testing in organ transplant recipients is unknown. We prospectively studied 1011 sera for HCV RNA and HCVcoreAg in a routine real-world setting including 222 samples obtained from patients after liver or kidney transplantation. HCV RNA and HCVcoreAg test results showed a consistency of 98% with a very good correlation in transplanted patients (r > 0.85). The correlation between HCV RNA and HCVcoreAg was higher in sera with high viral loads and in samples from patients with low biochemical disease. Patients treated with tacrolimus showed a better correlation between both parameters than individuals receiving cyclosporine A. HCV RNA/HCVcoreAg ratios did not differ between transplanted and nontransplanted patients, and HCV RNA and HCVcoreAg kinetics were almost identical during the first days after liver transplantation. HCVcoreAg testing can be used to monitor HCV viral loads in patients after organ transplantation. However, the assay is not recommended to monitor antiviral therapies.

Virological and clinical characteristics of delta hepatitis in Central Europe.

Hepatitis D virus (HDV) or delta hepatitis has mainly been studied in Asian and Mediterranean cohorts, but data on virological and clinical characteristics of HDV-infected Central and Northern European patients are limited. We investigated virological patterns, as well as biochemical and clinical features of liver disease in 258 HDV infected patients recruited over a period of 15 years at Hannover Medical School. Virological parameters were compared to 2083 anti-HDV negative hepatitis B surface antigen (HBsAg) positive individuals. In this cohort, (i) HDV infection was associated with both suppressed hepatitis B virus (HBV) and hepatitis C virus (HCV) replication, (ii) the suppression of HBV-DNA and HCV-RNA was not related to HDV-RNA replication, (iii) mean HBsAg levels did not significantly differ between HBV-monoinfected patients and individuals with delta hepatitis, (iv) HCV coinfection was rather frequent as about one third of our delta hepatitis patients tested anti-HCV positive, however, without being associated with more advanced liver disease, (v) delta hepatitis patients presented in a high frequency with an advanced stage of liver disease, and (vi) the course of delta hepatitis did not differ between Turkish-born, Eastern European (EE)-born and German-born patients. In summary, in this cohort of patients which is the largest so far Central European single centre group of delta hepatitis patients, we confirm the presence of frequently severe disease and describe novel virological profiles which require consideration in the management of this difficult to treat group of patients.

Gene expression profiling of liver metastases and tumour invasion in pancreatic cancer using an orthotopic SCID mouse model.

The prognosis of pancreatic adenocarcinoma is affected by early metastases and local tumour invasion beyond surgical margins. Gene expression profiling in pancreatic cancer tissue is complicated due to the high amount of RNAses being present in human tissue and that of suitable models. In order to demonstrate early metastases, the models should take into account the anatomical environment of the tumour. Using the orthotopic transplantation of pancreatic tumour cells in SCID (severe combined immunodeficiency) mice, these interactions are taken into consideration. In order to identify genes associated with local tumour invasion and metastases in ductal pancreatic cancer, we investigated a human pancreatic tumour cell line derived from an orthopic pancreatic tumour model in SCID mice. Differential gene expression was performed on the basis of microarray technique. The human MiaPaca-2 cell line was implanted orthotopically in SCID mice. Transcriptional profiling was performed on fresh frozen tissue derived from the primary tumour, the tumour invasion front and the liver metastases. Differentially expressed genes were identified using statistical analyses, and were validated with external databases and with immunohistochemistry. A total of 1066 of 14 500 genes were significantly differentially expressed. Comparing the primary tumour with the tumour invasion front, there were 614 statistically significant up- and 348 downregulated genes. Twenty-five statistically significant up- and 181 downregulated genes were identified comparing the liver metastases with the primary tumour. Eight genes (PAI-1, BNIP3l, VEGF, NSE, RGS4, HSP27, GADD45A, PTPN14) were chosen and validated in a semi-quantitative immunohistochemical analysis, which revealed a positive correlation to the array data. Overrepresentation analyses revealed a total of 66 significantly regulated pathways associated with cell proliferation, cell stress, cell communication metabolic and cytokine function. In conclusion, model marker genes for local invasion and liver metastases can be identified using transcriptional profiling in the SCID mouse. Overrepresentation analysis secures a good and fast overview about the significantly regulated genes and can assign genes to certain pathways. These marker genes can be related to the apoptotic cascade, angiogenesis and cell interaction.

An orthotopic model of ductal adenocarcinoma of the pancreas in severe combined immunodeficient mice representing all steps of the metastatic cascade.

INTRODUCTION: Clinically relevant animal models are needed to evaluate new therapeutic strategies against pancreatic adenocarcinoma, which is almost incurable by established treatment. AIMS: To establish and characterize a metastatic orthotopic transplant model for pancreatic ductal adenocarcinoma in severe combined immunodeficient (SCID) mice. METHODOLOGY: Human pancreatic ductal carcinoma cells, PancTu 1, were implanted either subcutaneously or orthotopically into the pancreas. RESULTS: After 4 weeks, orthotopic transplantation resulted in an extensive local tumor growth of an undifferentiated ductal adenocarcinoma with slight to moderate desmoplastic reaction. The tumor growth and spread resembled the situation in humans, including invasion into adjacent organs causing biliary and stomach obstruction. In addition, tumor metastases to regional lymph nodes of the pancreas, lung, liver, mesentery, and diaphragm, and attached to the kidneys, spleen, and reproductive organs were observed. In contrast, no invasion or metastases could be demonstrated by subcutaneous implanted PancTu I cells. Using immunohistochemical analysis, even single human tumor cells could be detected in blood vessels and metastatic organs, providing evidence that the orthotopic transplant model appropriately reflects the entire process of the metastatic cascade. CONCLUSION: This cancer model in SCID mice appears to be a powerful tool to investigate the identity of metastasis-associated genes and to evaluate preclinically the potency of novel antimetastatic agents in ductal adenocarcinoma of the pancreas.

Molecular cytogenetic analysis of two primary squamous cell carcinomas of the lung using multicolor fluorescence in situ hybridization.

To fully characterize the numerous chromosomal aberrations in two human squamous cell carcinomas (SCCs) of the lung, molecular cytogenetic characterization was carried out utilizing conventional banding analysis and multicolor fluorescence in situ hybridization (mFISH), providing simultaneous color discrimination of all 24 human chromosomes. Both tumors displayed complex aneuploid karyotypes with a host of numerical and structural chromosome abnormalities. Structural aberrations common to both SCCs included rearrangements of chromosomes 1, 3p, 7q, and 8q, contributing to net loss of chromosomal sequences on 1p, 3p, and 8p, and a net gain of 8q. The recently introduced mFISH technique enabled the disclosure of cryptic translocations and the chromosomal composition of previously unrecognized marker chromosomes. Furthermore, mFISH greatly enhanced the ability to delineate chromosomal breakpoints when integrating banding information from conventional banding analysis. Eventually, the application of mFISH as a powerful approach to refine complex tumor karyotypes is expected to result in a more detailed and complete picture of cytogenetic events associated with the development and progression of solid tumors.

Species-specific detection of Legionella using polymerase chain reaction and reverse dot-blotting.

Legionella pneumophila and some other Legionella species are capable of causing Legionnaire's disease, a potentially fatal pneumonia. The identification of legionellae by standard laboratory techniques such as culture is difficult and time consuming. In the present work, the DNA sequence of the 23S-5S spacer region was determined for 43 Legionella isolates, and the sequence information was used to develop a species-specific detection system using PCR and reverse dot-blotting which employs just one PCR amplicon to perform genus- and species-specific detection. L. pneumophila serogroups 1-16 as well as 21 non-pneumophila isolates could be identified and differentiated at the species level using this system.

Chlamydia pneumoniae as a cause of community-acquired pneumonia in hospitalized patients in Berlin.

The incidence of community-acquired pneumonia due to Chlamydia pneumoniae was evaluated in a 1-year prospective study of 236 hospitalized patients with 237 manifestations of pneumonia. The microbiological diagnosis was based on results of cultures of blood and sputum or bronchoalveolar lavage fluid and results of complement fixation tests and indirect immunofluorescence of acute- and convalescent-phase sera for C. pneumoniae, Mycoplasma pneumoniae, Legionella species, Coxiella burnetii, and respiratory viruses. Diagnosis of acute C. pneumoniae infection was made on the basis of the results of microimmunofluorescence of paired serum samples. A microbiological diagnosis was obtained in 160 cases (67.5%). C. pneumoniae was the causative agent in 27 patients (11.4%). The following organisms were the other etiologic agents of pneumonia: Streptococcus pneumoniae, 30 cases (12.7%) (bacteremia occurred in 53.3%); Mycoplasma pneumoniae, 22 (9.3%); respiratory viruses, 22 (9.3%); and Enterobacteriaceae, 18 (7.6%). The prevalence of C. pneumoniae antibody in our study population was 47.5%. As has been increasingly reported in recent years and confirmed by this study, C. pneumoniae appears to be a common etiologic agent of community-acquired pneumonia and upper respiratory tract infection in Germany.