Biomarker selection for detection of occult tumour cells in lymph nodes of colorectal cancer patients using real-time quantitative RT–PCR

Accurate identification of lymph node involvement is critical for successful treatment of patients with colorectal carcinoma (CRC). Real-time quantitative RT–PCR with a specific probe and RNA copy standard for biomarker mRNA has proven very powerful for detection of disseminated tumour cells. Which properties of biomarker mRNAs are important for identification of disseminated CRC cells? Seven biomarker candidates, CEA, CEACAM1-S/L, CEACAM6, CEACAM7-1/2, MUC2, MMP7 and CK20, were compared in a test-set of lymph nodes from 51 CRC patients (Dukes' A–D) and 10 controls. Normal colon epithelial cells, primary tumours, and different immune cells were also analysed. The biomarkers were ranked according to: (1) detection of haematoxylin/eosin positive nodes, (2) detection of Dukes' A and B patients, who developed metastases during a 54 months follow-up period and (3) identification of patients with Dukes' C and D tumours using the highest value of control nodes as cutoff. The following properties appear to be of importance; (a) no expression in immune cells, (b) relatively high and constant expression in tumour tissue irrespective of Dukes' stage and (c) no or weak downregulation in tumours compared to normal tissue. CEA fulfilled these criteria best, followed by CK20 and MUC2

Human AdV-20-42-42, a promising novel adenoviral vector for gene therapy and vaccine product development

Preexisting immune responses toward adenoviral vectors limit the use of a vector based on particular serotypes and its clinical applicability for gene therapy and/or vaccination. Therefore, there is a significant interest in vectorizing novel adenoviral types that have low seroprevalence in the human population. Here, we describe the discovery and vectorization of a chimeric human adenovirus, which we call HAdV-20-42-42. Full-genome sequencing revealed that this virus is closely related to human serotype 42, except for the penton base, which is derived from serotype 20. The HAdV-20-42-42 vector could be propagated stably to high titers on existing E1-complementing packaging cell lines. Receptor-binding studies revealed that the vector utilized both CAR and CD46 as receptors for cell entry. Furthermore, the HAdV-20-42-42 vector was potent in transducing human and murine cardiovascular cells and tissues, irrespective of the presence of blood coagulation factor X. In vivo characterizations demonstrate that when delivered intravenously (i.v.) in mice, HAdV-20-42-42 mainly targeted the lungs, liver, and spleen and triggered robust inflammatory immune responses. Finally, we demonstrate that potent T-cell responses against vector-delivered antigens could be induced upon intramuscular vaccination in mice. In summary, from the data obtained we conclude that HAdV-20-42-42 provides a valuable addition to the portfolio of adenoviral vectors available to develop efficacious products in the fields of gene therapy and vaccination

Pentavalent Sialic Acid Conjugates Block Coxsackievirus A24 Variant and Human Adenovirus Type 37–Viruses That Cause Highly Contagious Eye Infections

Coxsackievirus A24 variant (CVA24v) and human adenovirus 37 (HAdV-37) are leading causative agents of the severe and highly contagious ocular infections acute hemorrhagic conjunctivitis and epidemic keratoconjunctivitis, respectively. Currently, neither vaccines nor antiviral agents are available for treating these diseases, which affect millions of individuals worldwide. CVA24v and HAdV-37 utilize sialic acid as attachment receptors facilitating entry into host cells. Previously, we and others have shown that derivatives based on sialic acid are effective in preventing HAdV-37 binding and infection of cells. Here, we designed and synthesized novel pentavalent sialic acid conjugates and studied their inhibitory effect against CVA24v and HAdV-37 binding and infection of human corneal epithelial cells. The pentavalent conjugates are the first reported inhibitors of CVA24v infection and proved efficient in blocking HAdV-37 binding. Taken together, the pentavalent conjugates presented here form a basis for the development of general inhibitors of these highly contagious ocular pathogens

Pentavalent Sialic Acid Conjugates Block Coxsackievirus A24 Variant and Human Adenovirus Type 37 – Viruses That Cause Highly Contagious Eye Infections

Coxsackievirus A24 variant (CVA24v) and human adenovirus 37 (HAdV-37) are leading causative agents of the severe and highly contagious ocular infections acute hemorrhagic conjunctivitis and epidemic keratoconjunctivitis, respectively. Currently, neither vaccines nor antiviral agents are available for treating these diseases, which affect millions of individuals worldwide. CVA24v and HAdV-37 utilize sialic acid as attachment receptors facilitating entry into host cells. Previously, we and others have shown that derivatives based on sialic acid are effective in preventing HAdV-37 binding and infection of cells. Here, we designed and synthesized novel pentavalent sialic acid conjugates and studied their inhibitory effect against CVA24v and HAdV-37 binding and infection of human corneal epithelial cells. The pentavalent conjugates are the first reported inhibitors of CVA24v infection, and proved efficient in blocking HAdV-37 binding. Taken together, the pentavalent conjugates presented here form a basis for the development of general inhibitors of these highly contagious ocular pathogens

The GD1a glycan is a cellular receptor for adenoviruses causing epidemic keratoconjunctivitis.

Adenovirus type 37 (Ad37) is a leading cause of epidemic keratoconjunctivitis (EKC), a severe and highly contagious ocular disease. Whereas most other adenoviruses infect cells by engaging CD46 or the coxsackie and adenovirus receptor (CAR), Ad37 binds previously unknown sialic acid-containing cell surface molecules. By glycan array screening, we show here that the receptor-recognizing knob domain of the Ad37 fiber protein specifically binds a branched hexasaccharide that is present in the GD1a ganglioside and that features two terminal sialic acids. Soluble GD1a glycan and GD1a-binding antibodies efficiently prevented Ad37 virions from binding and infecting corneal cells. Unexpectedly, the receptor is constituted by one or more glycoproteins containing the GD1a glycan motif rather than the ganglioside itself, as shown by binding, infection and flow cytometry experiments. Molecular modeling, nuclear magnetic resonance and X-ray crystallography reveal that the two terminal sialic acids dock into two of three previously established sialic acid-binding sites in the trimeric Ad37 knob. Surface plasmon resonance analysis shows that the knob-GD1a glycan interaction has high affinity. Our findings therefore form a basis for the design and development of sialic acid-containing antiviral drugs for topical treatment of EKC