Sequenzierung von alpha- und beta2-Toxin-Genen aus C. perfringens-Proben von Kaninchen

Rabbit enterocolitis is a serious gastrointestinal syndrome appearing in France since the end of 1996. The disease is characterized by small quantities of watery diarrhoea followed by a decrease in food intake and finally resulting in intestinal paralysis. It has high mortality rates (30-80%) and spreads very rapidly. There is clear experimental evidence that this disease is produced by Clostridium perfringens. Aim of this work is to characterize the genotype of the Clostridium strains circulating in Germany and sequence the major toxin genes. C. perfringens strains were collected from different rabbit livestocks throughout Germany (including 3 from Italy), and isolated from the intestine of sick rabbits. All samples were typed via PCR using consensus primers of the major toxin genes (alpha, beta, epsilon, iota, cpe and beta2). From a total of 71 strains, all contain the alpha-gene and 25 additionally the beta2-gene. Therefore all are type A Clostridium perfringens. The alpha-toxin genes were isolated and sequenced, including their immediate upstream and downstream surroundings. The upstream region contains all elements necessary for expression, i.e. typical bacterial promoter elements for transcription initiation and a Shine-Dalgarno-sequence necessary for translation. No transcriptional termination signals were found at the end of the coding region, suggesting the alpha-toxin gene to be member of an operon. The deduced amino acid differences within the 71 alpha-toxins are low and restricted to certain areas of the protein. All the functionally important amino acid residues, i.e. the ones involved in metal ion-, membrane-binding and in the conformational activation, are conserved in all the 71 alpha–toxins. Therefore, it can be concluded that all alpha- toxins are equally well active. Studies carried on by Gilbert et al., 1997, showed that beta2 toxin-producing strains are associated with animal diseases such as necrotic enteritis in piglets and enterocolitis in horses. Therefore, the beta2-toxin gene, cpb2, was analyzed in the C. perfringens isolated from sick rabbits as well. The strategy applied to obtain the beta2-gene sequences was analogous to that for the alpha-toxin genes, i.e. PCR amplification of the DNA region of interest including upstream and downstream parts of the gene. After finding the correct PCR primers for the amplification of the beta2-toxin gene types it could be shown that from the 25 beta2-toxin genes 16 belong to the so-called “porcine”- and 9 to the “non-porcine”-type. All the “porcine”- and 5 of the “non-porcine”-type genes were sequenced. They contain all regulatory elements necessary for transcription, i.e. promoter elements and “hairpin”-termination signals indicationg that monocistronic mRNAs are produced. 15 of the 16 “porcine”-type beta2-toxin genes display an A-deletion within the putative signal peptide region, expecting an immediate downstream translational stop to occur. Only n°6 (Wallenhorst) has an undisturbed open reading frame and should therefore be expressed as a complete protein. This is for the first time, that a “porcine-type” beta2-toxin gene isolated from Clostridia grown in non-porcine animals has this feature. Due to non-solvable sequencing problems, only 5 of the 9 non-porcine beta2-toxin genes could be sequenced completely. All display an undisturbed open reading frame. Similar as within the alpha-toxin samples, the deduced amino acid differences within each beta2-toxin-type are very low, although being marked between the two groups. From only the nucleotide sequence data no prediction can be made for the expression of the two beta2-toxin genes. Therefore, they were cloned into the bacterial expression vector pASK-IBA6. The cloned genes were expressed as fusion proteins, offering the possibility to purify the recombinant beta2-toxins. After immunizing animals, anti-beta2-toxin-specific antibodies should become available to clarify the problem of the expression of the beta2-toxin genes. Summarizing all the data on all toxin-specific genes, due to their low sequence variability, the development of a toxoid vaccine against rabbit enterocolitis should become feasible.Die sogenannte “Enterocolitis” des Kaninchens wurde zuerst Ende 1996 in Frankreich beschrieben. Die Erkrankung ist charakterisiert anfangs durch geringfügigen wässrigen Durchfall, gefolgt von reduzierter Nahrungsaufnahme mit am Ende völligen Zusammenbruchs intestinaler Aktivität (Darmlähmung). Mortalitätsraten (30-80%) und die Ausbreitungsgeschwindigkeit sind sehr hoch. Klare experimentelle Evidenz weist auf Clostridium perfringens als den Erreger der Erkrankung hin. Ziel dieser Arbeit ist die Charakterisierung der Toxin Genotypen möglichst vieler in deutschen Kaninchenbeständen kursierender C. perfringens Stämme und die Sequenzierung ihrer Haupttoxin-Gene. 71 C.perfringens Isolate aus infizierten Kaninchenbeständen (68 aus Deutschland, 3 aus Italien) wurden gesammelt und mittels PCR unter Verwendung von „consensus“ Oligonukleotiden, charakteristisch für die Haupttoxin Gene (alpha, ß, ε, i, cpe and beta2), typisiert. Alle 71 Proben enthalten das alpha-Toxin-, 25 zusätzlich das ß2-Toxin-Gen. Daher gehören alle zum Clostridium perfringens Typ-A. Die alpha-Toxin Gene wurden amplifiziert, zum Teil kloniert und anschließend sequenziert, einschließlich der strom-aufwärts und –abwärts Regionen. Die Stromaufwärtsregionen enthalten alle zur Transkription erforderlichen Elemente, wie bakterielle Promotoren und das für die Translation notwendige Shine-Dalgarno-Äquivalent. Am Ende des kodierenden Berichs waren keine Transkriptionsterminationselemente vorhanden, was darauf hinweist, dass das alpha-Toxin Gen Bestandteil eines Operons ist. Was den kodierenden Bereich betrifft, ist zu konstatieren, dass die aus der Nukleotidsequenz abgleiteten Aminosäure-Unterschiede in den 71 alpha-Toxinen gering sind und zudem auf bestimmte Bereiche des Proteins beschränkt bleiben. Alle funktionell wichtigen Aminosäure-Reste, also diejenigen, die involviert sind in Metallionen-, Membranbindung und Aktivierung, sind konserveirt. Daraus ist zu schließen, dass alle 71 alpha-Toxine ähnlich hoch aktiv sind. Studien von Gilbert et al (1997) zeigten, dass beta2-Toxin-Gen enthaltende C. perfingens Stämme mit Enteritiden bei Ferkeln und Pferden assoziiert sind. Deshalb wurden die beta2-Toxin Gene von C.perfringens-Isolaten aus erkrankten Kaninchen ebenfalls analysiert. Die dabei angewandte Strategie entsprach der bei der Charakterisierung der alpha-Toxin Gene verwendeten, d.h., PCR- Amplifizierung der entsprechenden DNA-Region unter Einbeziehung strom-aufwärts und-abwärts liegender Genregionen. Nach zum Teil experimenteller Ermittlung der geeigneten PCR-Starter konnte gezeigt werden, dass von den 25 beta2-Toxin Genen 16 zum sog. „Schwein“- und 9 zum „Nicht-Schwein“-Typ gehören. Alle „Schwein“-Typ- und 5 der 9 „Nicht-Schwein“-Typ beta2-Gene wurden sequenziert. Sie enthalten alle die für die Transkription notwendigen Promoter- und Terminations-elemente, was darauf hinweist, dass eine monocistronische mRNA synthetisiert wird. 15 der 16 „Schwein“-Typ beta2-Toxin Gene zeigen eine A-Deletion innerhalb der Signalpeptid-kodierenden Region, was zu einem sehr schnellen Translationsstop führen sollte. Nur die Probe 6 (Wallenhorst) besitzt einen nicht unterbrochenen offenen Leserahmen, sodass erwartet werden kann, dass hier das komplette beta2-Toxin synthetisiert wird. Dies ist das erste Mal, dass ein „Schwein“-Typ beta2-Toxin Gen von Clostridien, die aus Nicht-Schwein-Tieren isoloiert wurden, eine solche Charakterisitik besitzt. Auf Grund nicht lösbarer Sequenzierprobleme konnten nur 5 der 9 „Nicht- Schwein“-Typ beta2-Toxin Gene komplett sequenziert werden. Alle weisen einen kompletten offenen Leserahmen auf. Ähnlich wie bei den alpha-Toxin Genen sind die Aminosäure-Unterschiede innerhalb der Mitglieder jedes beta2-Toxin-Typs sehr gering, allerdings deutlich zwischen Mitgliedern verschiedener beta2 -Toxin-Typen. Aus den Nukleinsäuresequenzdaten allein kann keine Voraussage über die Synthese der beiden beta2-Toxine getroffen werden. Deshalb wurden die Gene in den bakteriellen Expressionsvektor pASK-IBA6 kloniert. Alle drei Toxin-Gene ließen sich als Fusionsproteine exprimieren. Dadurch bietet sich die Möglichkeit die rekombinanten Toxine aufzureinigen und durch anschließende Immunisierung anti-beta2-Toxin-spezifische Antikörper zu erhalten. Mit ihrer Hilfe sollte sich das Problem der beta2-Toxin-Expression zweifelsfrei klären lassen. Fasst man alle Sequenzdaten über die Toxin-spezifischen Gene zusammen, so sollte auf Grund der geringen Sequenzvariabilität die Entwicklung einer Toxoid-Vakzine gegen die Enterocolitis des Kaninchens machbar sein

Serum amyloid A: a novel biomarker for endometrial cancer

The authors investigated the expression of serum amyloid A (SAA) in endometrial endometrioid carcinoma and evaluated its potential as a serum biomarker. SAA gene and protein expression levels were evaluated in endometrial endometrioid carcinoma and normal endometrial tissues, by real-time polymerase chain reaction (PCR), immunohistochemistry (IHC), and flow cytometry. SAA concentration in 194 serum samples from 50 healthy women, 42 women with benign diseases, and 102 patients including 49 grade 1, 38 grade 2, and 15 grade 3 endometrial endometrioid carcinoma was also studied by a sensitive bead-based immunoassay. SAA gene expression levels were significantly higher in endometrial endometrioid carcinoma when compared with normal endometrial tissues (mean copy number by real-time PCR = 182 vs 1.9; P = .001). IHC revealed diffuse cytoplasmic SAA protein staining in poorly differentiated endometrial endometrioid carcinoma tissues. High intracellular levels of SAA were identified in primary endometrial endometrioid carcinoma cell lines evaluated by flow cytometry, and SAA was found to be actively secreted in vitro. SAA concentrations (microg/mL) had medians of 6.0 in normal healthy women and 6.0 in patients with benign disease (P = .92). In contrast, SAA values in the serum of endometrial endometrioid carcinoma patients had a median of 23.7, significantly higher than those of the healthy group (P = .001) and benign group (P = .001). Patients harboring G3 endometrial endometrioid carcinoma were found to have SAA concentrations significantly higher than those of G1/G2 patients. SAA is not only a liver-secreted protein, but is also an endometrial endometrioid carcinoma cell product. SAA is expressed and actively secreted by G3 endometrial endometrioid carcinoma, and it is present in high concentration in the serum of endometrial endometrioid carcinoma patients. SAA may represent a novel biomarker for endometrial endometrioid carcinoma to monitor disease recurrence and response to therapy

Overexpression of Epithelial Cell Adhesion Molecule in Primary, Metastatic, and Recurrent/Chemotherapy-Resistant Epithelial Ovarian Cancer Implications for Epithelial Cell Adhesion Molecule-Specific Immunotherapy

To evaluate the potential of epithelial cell adhesion molecule (Ep-CAM/TROP-1)-specific immunotherapy against epithelial ovarian carcinomas (EOCs), we have analyzed the expression of Ep-CAM at RNA and protein level in patients harboring primary, metastatic, and chemotherapy-resistant/recurrent EOC. Epithelial cell adhesion molecule expression was evaluated by real-time polymerase chain reaction and immunohistochemistry in 168 fresh-frozen biopsies and paraffin-embedded tissues. In addition, Ep-CAM Surface expression was evaluated by flow cytometry in several freshly established ovarian carcinoma cell lines derived from patients harboring tumors resistant to chemotherapy in vivo as well as in vitro. Epithelial cell adhesion molecule transcript was found significantly overexpressed in primary, metastatic, and recurrent EOC when compared with normal human ovarian surface epithelium cell lines and fresh-frozen normal ovarian tissue (P < 0.001). Similarly, by immunohistochemistry, Ep-CAM protein expression was found significantly higher in primary, metastatic, and recurrent EOC when compared with normal ovarian tissues. Of interest, metastatic/recurrent tumors were found to express significantly higher levels of Ep-CAM protein when compared with primary ovarian carcinomas (P < 0.001). Finally, a high surface expression of Ep-CAM was found in 100% (5/5) of the chemotherapy-resistant ovarian carcinoma cell lines studied by flow cytometry. These results demonstrate high Ep-CAM overexpression in ovarian carcinoma, especially in metastatic and recurrent/chemotherapy-resistant ovarian disease. The lack of Ep-CAM expression on the chelomic epithelium in the peritoneal cavity, combined with the recent development of fully human monoclonal antibodies against this Surface molecule, Suggest Ep-CAM as a promising target for antibody-mediated therapies in ovarian carcinoma patients harboring tumors refractory to standard treatment modalities

Erratum: By promoting cell differentiation, miR-100 sensitizes basal-like breast cancer stem cells to hormonal therapy

This article has been corrected: Due to errors in image assembly, the flow cytometry profiles depicting the isotype matched control (IMC) for CD49f on both scramble (scr) and miR-100 reported in Figure 6 are incorrect. Additionally, we noticed that the IMC reported for CD24 and CD10 in miR-100 are the same. Being both CD24 allophycocianin (APC) and CD10 APC IgG1, they should have the same IMC, as already reported. The corrected Figure 6 is shown below. The authors declare that these corrections do not change the results or conclusions of this paper

Human Papillomavirus Type 16 (HPV-16) Virus-Like Particle L1-Specific CD8+ Cytotoxic T Lymphocytes (CTLs) Are Equally Effective as E7-Specific CD8+ CTLs in Killing Autologous HPV-16-Positive Tumor Cells in Cervical Cancer Patients: Implications for L1 Dendritic Cell-Based Therapeutic Vaccines▿

Papillomavirus-like particles (VLPs) based on L1 capsid protein represent a promising prophylactic vaccine against human papillomavirus (HPV) infections. However, cell-mediated immune responses against this antigen are believed to be of limited therapeutic value in established HPV-infected cervical lesions and, for this reason, have not been intensively investigated in cervical cancer patients. In this study we analyzed and quantified by real-time PCR (RT-PCR) the RNA expression levels of E6, E7, and L1 genes in flash-frozen HPV-16 cervical carcinomas. In addition, the kinetics of expression of E6, E7, and L1 in HPV-16-infected primary cell lines established as long-term cultures in vitro was also evaluated at RNA and protein levels. Finally, in order to evaluate the therapeutic potential of L1-specific CD4+ and CD8+ T lymphocytes responses in cervical cancer patients, L1 VLP-loaded dendritic cells (DCs) were used to stimulate peripheral blood lymphocytes from cervical cancer patients and such responses were compared to those elicited by the E7 oncoprotein. We show that 22 of 22 (100%) flash-frozen cervical biopsy samples collected from HPV-16-positive cervical cancer patients harbor L1, in addition to E6 and E7 RNA, as detected by RT-PCR. E7 RNA copy number (mean, 176.2) was significantly higher in HPV-16-positive cervical cancers compared to the E6 RNA copy number (mean, 47.3) and the L1 copy number (mean, 58.3) (P < 0.0001 and P < 0.001, respectively). However, no significant differences in expression levels between E6 and L1 were found. Kinetic studies of E6, E7, and L1 RNA and protein expression levels in primary tumors showed a sharp reduction in L1 expression after multiple in vitro passages compared to E6 and E7. Autologous DCs pulsed with HPV-16 VLPs or recombinant full-length E7 elicited strong type 1 L1- and E7-specific responses in CD4+ and CD8+ T cells from cervical cancer patients. Importantly, L1 VLP-specific CD8+ T lymphocytes expressed strong cytolytic activity against autologous tumor cells and were as effective as E7-specific cytotoxic T lymphocytes in lysing naturally HPV-16-infected autologous tumor cells. Taken together, these data demonstrate a consistent expression of L1 in primary cervical tumors and the possibility of inducing effective L1/tumor-specific CD4+ and CD8+ T-lymphocyte responses in patients harboring HPV-infected cervical cancer. These results may have important implications for the treatment of patients harboring established HPV-infected lesions with L1 VLPs or combined E7/L1 DC-based vaccinations

Rituximab Treatment Prevents Lymphoma Onset in Gastric Cancer Patient-Derived Xenografts.

Patient-Derived Xenografts (PDXs), entailing implantation of cancer specimens in immunocompromised mice, are emerging as a valuable translational model that could help validate biologically relevant targets and assist the clinical development of novel therapeutic strategies for gastric cancer. More than 30% of PDXs generated from gastric carcinoma samples developed human B-cell lymphomas instead of gastric cancer. These lymphomas were monoclonal, Epstein Barr Virus (EBV) positive, originated tumorigenic cell cultures and displayed a mutational burden and an expression profile distinct from gastric adenocarcinomas. The ability of grafted samples to develop lymphomas did not correlate with patient outcome, nor with the histotype, the lymphocyte infiltration level, or the EBV status of the original gastric tumor, impeding from foreseeing lymphoma onset. Interestingly, lymphoma development was significantly more frequent when primary rather than metastatic samples were grafted. Notably, the development of such lympho-proliferative disease could be prevented by a short rituximab treatment upon mice implant, without negatively affecting gastric carcinoma engraftment. Due to the high frequency of human lymphoma onset, our data show that a careful histologic analysis is mandatory when generating gastric cancer PDXs. Such care would avoid misleading results that could occur if testing of putative gastric cancer therapies is performed in lymphoma PDXs. We propose rituximab treatment of mice to prevent lymphoma development in PDX models, averting the loss of human-derived samples