Synthese von tumor-assoziierten MUC1-Mucin-Glycopeptid-Vakzinen und deren immunologische Evaluierung

Eine alternative Methode zur Therapie von Tumorerkrankungen bestünde in einer Immuntherapie ausgelöst durch synthetische Antitumor-Vakzine. Ein vielversprechendes Zielmolekül für eine solche Aktivimmunisierung ist das Glycoprotein MUC1, das auf nahezu allen Epithelgeweben exprimiert und auf Tumorgeweben stark überexprimiert wird. Seine extrazelluläre Domäne enthält eine Vielzahl von Tandem-Repeat-Sequenzen der Art: HGVTSAPDTRPAPGSTAPPA mit fünf potentiellen O-Glycosylierungs-Positionen. Da die Form der Glycosylierung des MUC1 in Tumorzellen stark von der auf normalen Zellen abweicht, liegen auf Tumorzellen eine Reihe tumor-assoziierter Saccharidantigene und Peptidepitope vor.rnIn dieser Arbeit wurden tumor-assoziierte Glycopeptidantigene aus der MUC1-Tandem-Repeat-Region hergestellt. Die synthetisierten MUC1-Glycopeptide tragen in verschiedenen Positionen eine Glycosylierung mit den tumor-assoziierten Tn- und STn-Saccharid-Antigenen. Zur Gewinnung von Vakzinen wurden diese Glycopeptid-Antigene über einen Spacer mit immunstimulierenden Komponenten verknüpft. Als Immunstimulanzien wurden ein T-Zell-Epitop aus dem Ovalbumin (OVA323-339) sowie die Carrier-Proteine Rinderserumalbumin (BSA) und Tetanus-Toxoid (TTox) verwendet. rnDie synthetischen MUC1-Glycopeptide wurden durch Immunisierung von Mäusen einer immunologischen Evaluierung unterzogen. Insbesondere die synthetischen MUC1-Glycopeptid-TTox-Vakzine lösen sehr starke Immunantworten aus. Es konnte gezeigt werden, dass die induzierten Antikörper stark an Tumorzellen und auch an Mammakarzinom-Gewebe binden, was für die Entwicklung von Antitumor-Vakzinen als vielversprechend einzustufen ist.

Synthese und immunologische Evaluierung von Antitumor-Impfstoffen aus dem epithelialen tumorassoziierten Mucin MUC1

Eine Immuntherapie von Tumorerkrankungen, die mit Hilfe von Antitumorimpfstoffen prophylaktisch und therapeutisch erfolgen könnte, wäre eine attraktive Alternative zu den bisher angewendeten Krebsbehandlungen. Aufgrund charakteristisch veränderter Aktivitäten von Glycosyltransferasen in der Glycoprotein-Biosynthese werden auf malignen Zellen stark verkürzte, frühzeitig sialylierte mucinartige Glycoproteine exprimiert. Diese verkürzten Kohlenhydrate repräsentieren tumorassoziierte Antigene. Sie haben zur Folge, dass Peptidepitope der Mucin-Glycoproteine, die auf gesundem Gewebe durch den hohen Glycosylierungsgrad maskiert sind, für das Immunsystem freiliegen. Diese Strukturunterschiede sollten einen selektiven Angriff auf das Tumorgewebe erlauben, ohne dass gesundes Gewebe beeinträchtigt wird, wenn es gelänge, das Immunsystem auf diese veränderten Strukturelemente zu fokussieren. Dabei ist es wichtig, dass die Immunantwort mit synthetisch definierten Glycopeptidepitopen ausgelöst wird, um Autoimmunreaktionen zu vermeiden. Somit ist die Synthese von exakt definierten tumorassoziierten Glycopeptiden von zentraler Bedeutung für die Entwicklung eines Antitumor-Impfstoffes. rnDa die tumorassoziierten Kohlenhydratstrukturen Antigene darstellen, die vom Immunsystem weitgehend toleriert werden, ist es notwendig, ihre Immunogenität mit Hilfe immunstimulierender Epitope so zu erhöhen, dass eine effiziente Immunreaktion erfolgt. Nach diesem Konzept wurden in der vorliegenden Arbeit Methoden und Strategien entwickelt, synthetische Impfstoff-Konjugate zu synthetisieren und immunologisch in Mausexperimenten zu evaluieren. So konnte gezeigt werden, das mit vollsynthetischen Analoga aus tumorassoziierten Glycopeptid-Oberflächenmolekülen in Kombination mit immunstimulierenden Substanzen, wie Trägerproteinen oder Mitogenen, hochselektive humorale Immunantworten ausgelöst werden können.rn

Die Synthese von Mucin-Glycopeptid-Vakzinkonjugaten und Adjuvanzien für eine potentielle Krebsimmuntherapie

Die vorliegende Arbeit beschäftigt sich mit der Synthese von unterschiedlichen tumorassoziierten Antigenen, die in Form von glycosylierten Aminosäurebausteinen durch Festphasensynthese in den tandem repeat des epithelialen Mucin MUC1 eingebaut wurden. Zur Erzeugung von selektiven Immunantworten wurden synthetische MUC1-Glycopeptide über einen nicht immunogenen Spacer mit einer Reihe von Immunstimulanzien umgesetzt. Es wurden hierbei zwei Ziele verfolgt. Zum einen sollten mit Hilfe von synthetischen MUC1-Glycopeptiden und Immunstimulanzien cytotoxische T-Zellen ausgebildet werden, die Tumorzellen mit überexprimiertem MUC1 am Wachstum hindern und sie in den programmierten Zelltod treiben. Zum anderen sollte mit Hilfe von TH2-Epitopen eine adaptive humorale B-Zell-vermittelte Immunantwort in Form von Antikörpern ausgelöst werden. Zur Ausbildung von cytotoxischen T-Zellen kamen Vakzinkonjugate zum Einsatz, die aus dem IL-1β(163 – 169), dem TLR2-Agonisten (Pam3Cys-Ser-Lys4) oder dem TLR4-Agonisten (spacergebundenes MPL A) bestehen und jeweils an MUC1-Glycopeptide konjugiert wurden. Darüber hinaus wurde eine neue moderne Syntheseroute für das MPL-A entwickelt, welches als Adjuvans in den verschiedenen Vakzinen, wie Cervarix®, Fendrix® und Stimuvax®, Verwendung findet und die Immunantwort der Vakzinkonstrukte nochmals verstärken sollte. Zur Induktion von Antikörpern kamen die OVA(323 – 339)-, die Tetanus-Toxoid- und die BSA-Konjugate zur Anwendung, an denen MUC1-Glycopeptide angeknüpft wurden.

Role of amylase, mucin, IgA and albumin on salivary protein buffering capacity: a pilot study

It has been suggested that proteins serve as major salivary buffers below pH5. It remains unclear, however, which salivary proteins are responsible for these buffering properties. The aim of this pilot study was to evaluate the correlation between salivary concentration of total protein, amylase, mucin, immunoglobulin A (IgA), albumin and total salivary protein buffering capacity at a pH range of 4-5. In addition, the buffering capacity and the number of carboxylic acid moieties of single proteins were assessed. Stimulated saliva samples were collected at 9:00, 13:00 and 17:00 from 4 healthy volunteers on 3 successive days. The buffering capacities were measured for total salivary protein or for specific proteins. Also, the concentration of total protein, amylase, mucin, IgA and albumin were analysed. Within the limits of the current study, it was found that salivary protein buffering capacity was highly positively correlated with total protein, amylase and IgA concentrations. A weak correlation was observed for both albumin and mucin individually. Furthermore, the results suggest that amylase contributed to 35 percent of the salivary protein buffering capacity in the pH range of 4-5.

Mucin-producing adenocarcinoma arising in an atrial myxoma

We describe the unique autopsy findings of a patient who died of a metastasizing giant right atrial adenocarcinoma containing few areas of typical myxoma. That no mucin-producing extracardiac tumor was detected pointed to the atrial adenocarcinoma as being the primary. We hypothesize that the adenocarcinoma may have developed from coexistent bland glandular structures within the myxoma.

Regulation of mucin gene expression by CREB via a nonclassical retinoic acid signaling pathway.

Vitamin A and its metabolite retinoic acid (RA) are essential elements for normal lung development and the differentiation of lung epithelial cells. We previously showed that RA rapidly activated cyclic AMP response element-binding protein (CREB) in a nonclassical manner in normal human tracheobronchial epithelial (NHTBE) cells. In the present study, we further demonstrated that this nonclassical signaling of RA on the activation of CREB plays a critical role in regulating the expression of airway epithelial cell differentiation markers, the MUC2, MUC5AC, and MUC5B genes. We found that RA rapidly activates the protein kinase Calpha isozyme and transmits the activation signal to CREB via the Raf/MEK/extracellular signal-regulated kinase/p90 ribosomal S6 kinase (RSK) pathway. Activated RSK translocated from the cytoplasm to the nucleus, where it phosphorylates CREB. Activated CREB then binds to a cis-acting replication element motif on the promoter (at nucleotides [nt] -878 to -871) of the MUC5AC gene. The depletion of CREB using small interfering RNA abolished not only the RA-induced MUC5AC but also RA-induced MUC2 and MUC5B. Taken together, our findings demonstrate that CREB activation via this nonclassical RA signaling pathway may play an important role in regulating the expression of mucin genes and mediating the early biological effects of RA during normal mucous differentiation in NHTBE cells.

Quantification of soluble fibre in feedstuffs for rabbits and evaluation of the interference between the determinations of soluble fibre and intestinal mucin.

This work compared the quantification of soluble fibre in feeds using different chemical and in vitro approaches, and studied the potential interference between soluble fibre and mucin determinations. Six ingredients: sugar beet pulp (SBP), SBP pectins, insoluble SBP, wheat straw, sunflower hulls and lignocellulose, and seven rabbit diets, differing in soluble fibre content, were evaluated. In experiment 1, ingredients and diets were analyzed for total dietary fibre (TDF), insoluble dietary fibre (IDF), soluble dietary fibre (SDF), aNDFom (corrected for protein, aNDFom-cp) and 2-step pepsin/pancreatin in vitro DM indigestibility (corrected for ash and protein, ivDMi2). Soluble fibre was estimated by difference using three procedures: TDF?IDF (SDFIDF), TDF?ivDMi2 (SDFivDMi2), and TDF?aNDFom-cp (SDFaNDFom-cp). Soluble fibre determined directly (SDF) or by difference as SDFivDMi2 were not different (109 g/kg DM, on average). However, when it was calculated as SDFaNDFom-cp the value was 40% higher (153 g/kg DM, P menor que 0.05), whereas SDFIDF (124 g/kg DM) did not differ from any of the other methods. The correlation between the four methods was high (r ? 0.96; P ? 0.001; n = 13), but it decreased or even disappeared when SBP pectins and SBP were excluded and a lower and more narrow range of variation of soluble fibre was used. In experiment 2, the ivDMi2 using crucibles (reference method) were compared to those made using individual or collective ankom bags in order to simplify the determination of SDFivDMi2. The ivDMi2 was not different when using crucibles or individual or collective ankom bags. In experiment 3, the potential interference between soluble fibre and intestinal mucin determinations was studied using rabbit intestinal raw mucus, digesta and SBP pectins, lignocelluloses and a rabbit diet. An interference was observed between the determinations of soluble fibre and crude mucin, as contents of TDF and apparent crude mucin were high in SBP pectins (994 and 709 g/kg DM) and rabbit intestinal raw mucus (571 and 739 g/kg DM). After a pectinase treatment, the coefficient of apparent mucin recovery of SBP pectins was close to zero, whereas that of rabbit mucus was not modified. An estimation of the crude mucin carbohydrates retained in digesta TDF is proposed to correct TDF and soluble fibre digestibility. In conclusion, the values of soluble fibre depend on the methodology used. The contamination of crude mucin with soluble fibre is avoided using pectinase.

Muc1, a mucin-like protein that is regulated by Mss10, is critical for pseudohyphal differentiation in yeast.

Pseudohyphal differentiation in Saccharomyces cerevisiae was first described as a response of diploid cells to nitrogen limitation. Here we report that haploid and diploid starch-degrading S. cerevisiae strains were able to switch from a yeast form to a filamentous pseudohyphal form in response to carbon limitation in the presence of an ample supply of nitrogen. Two genes, MSS10 and MUC1, were cloned and shown to be involved in pseudohyphal differentiation and invasive growth. The deletion of MSS10 resulted in extremely reduced amounts of pseudohyphal differentiation and invasive growth, whereas the deletion of MUC1 abolished pseudohyphal differentiation and invasive growth completely. Mss10 appears to be a transcriptional activator that responds to nutrient limitation and coregulates the expression of MUC1 and the STA1-3 glucoamylase genes, which are involved in starch degradation. MUC1 encodes a 1367-amino acid protein, containing several serine/threonine-rich repeats. Muc1 is a putative integral membrane-bound protein, similar to mammalian mucin-like membrane proteins that have been implicated to play a role in the ability of cancer cells to invade other tissues.

An abundantly expressed mucin-like protein from Toxocara canis infective larvae: the precursor of the larval surface coat glycoproteins.

Evasion of host immunity by Toxocara canis infective larvae is mediated by the nematode surface coat, which is shed in response to binding by host antibody molecules or effector cells. The major constituent of the coat is the TES-120 glycoprotein series. We have isolated a 730-bp cDNA from the gene encoding the apoprotein precursor of TES-120. The mRNA is absent from T. canis adults but hyperabundant in larvae, making up approximately 10% of total mRNA, and is trans-spliced with the nematode 5' leader sequence SL1. It encodes a 15.8-kDa protein (after signal peptide removal) containing a typical mucin domain: 86 amino acid residues, 72.1% of which are Ser or Thr, organized into an array of heptameric repeats, interspersed with proline residues. At the C-terminal end of the putative protein are two 36-amino acid repeats containing six Cys residues, in a motif that can also be identified in several genes in Caenorhabditis elegans. Although TES-120 displays size and charge heterogeneity, there is a single copy gene and a homogeneous size of mRNA. The association of overexpression of some membrane-associated mucins with immunosuppression and tumor metastasis suggests a possible model for the role of the surface coat in immune evasion by parasitic nematodes.

Induction of cellular immunity in chimpanzees to human tumor-associated antigen mucin by vaccination with MUC-1 cDNA-transfected Epstein-Barr virus-immortalized autologous B cells.

Aberrant glycosylation of the mucin molecule (encoded by the gene MUC-1) on human epithelial cell tumors leads to the exposure of tumor-associated epitopes recognized by patients' antibodies and cytotoxic T cells. Consequently, these epitopes could be considered targets for immunotherapy. We designed a cellular vaccine, employing, instead of tumor cells, autologous Epstein-Barr virus (EBV)-immortalized B cells as carriers of tumor-associated mucin, to take advantage of their costimulatory molecules for T-cell activation. The vaccine was tested in chimpanzees because of the identity of the human and chimpanzee MUC-1 tandem repeat sequence. EBV-immortalized B cells derived from two chimpanzees were transfected with MUC-1 cDNA, treated with glycosylation inhibitor phenyl-N-acetyl-alpha-D-galactosaminide to expose tumor-associated epitopes, irradiated, and injected subcutaneously four times at 3-week intervals. One vaccine preparation also contained cells transduced with the interleukin 2 (IL-2) cDNA and producing low levels of IL-2. Already after the first injection we found in the peripheral blood measurable frequency of cytotoxic T-cell precursors specific for underglycosylated mucin. The highest frequency observed was after the last boost, in the lymph node draining the vaccination site. Delayed-type hypersensitivity reaction to the injected immunogens was also induced, whereas no appearance of mucin-specific antibodies was seen. Long-term observation of the animals yielded no signs of adverse effects of this immunization. Autologous antigen-presenting cells, like EBV-immortalized B cells, expressing tumor-associated antigens are potentially useful immunogens for induction of cellular anti-tumor responses in vivo.

Expression of the cell surface mucin gene family in adenocarcinomas

Cell surface mucins are complex glycoproteins expressed on the apical membrane surface of mucosal epithelial cells. In malignant epithelial cells they are thought to influence cell adhesion, and are clinical targets for tumor immunotherapy and serum tumor marker assays. We have compared expression of MUC1, MUC3, MUC4, MUC11, MUC12 and MUC13 mRNA in epithelial cancers and/or cell lines with non-malignant tissues. In non-malignant tissues, MUC3, 4, 11, 12 and 13 were expressed at highest levels in gastrointestinal tissues, whereas MUC1 was more widely distributed. Significant down-regulation of the MUC4, MUC12 and MUC13 genes was observed in colonic cancers compared with normal tissue, whereas MUC1 was upregulated. In rectal cancers, levels of all six mucin genes were not significantly different to those in normal rectal tissues. Both MUC1 and MUC4 were down-regulated in gastric cancers, whereas cancer and normal tissue levels were similar for MUC3, 11, 12 and 13. In esophageal cancers there was a general trend toward higher levels than in normal tissue for MUC1, 3, 12 and 13. In ovarian cancers MUC1 levels were very high, whereas only low levels of all other mucins were observed. We also report expression in renal cell carcinomas, bladder carcinomas and breast cancer cell lines. The reported expression profiles of the cell surface mucin gene family will help direct biological and clinical studies of these molecules in mucosal biology, and in malignant and inflammatory diseases of epithelial tissues.

EGFR interacts with the fusion protein of respiratory syncytial virus strain 2-20 and mediates infection and mucin expression.

Respiratory syncytial virus (RSV) is the major cause of viral lower respiratory tract illness in children. In contrast to the RSV prototypic strain A2, clinical isolate RSV 2-20 induces airway mucin expression in mice, a clinically relevant phenotype dependent on the fusion (F) protein of the RSV strain. Epidermal growth factor receptor (EGFR) plays a role in airway mucin expression in other systems; therefore we hypothesized that the RSV 2-20 F protein stimulates EGFR signaling. Infection of cells with chimeric strains RSV A2-2-20F and A2-2-20GF or over-expression of 2-20 F protein resulted in greater phosphorylation of EGFR than infection with RSV A2 or over-expression of A2 F, respectively. Chemical inhibition of EGFR signaling or knockdown of EGFR resulted in diminished infectivity of RSV A2-2-20F but not RSV A2. Over-expression of EGFR enhanced the fusion activity of 2-20 F protein in trans. EGFR co-immunoprecipitated most efficiently with RSV F proteins derived from “mucogenic” strains. RSV 2-20 F and EGFR co-localized in H292 cells, and A2-2-20GF-induced MUC5AC expression was ablated by EGFR inhibitors in these cells. Treatment of BALB/c mice with the EGFR inhibitor erlotinib significantly reduced the amount of RSV A2-2-20F-induced airway mucin expression. Our results demonstrate that RSV F interacts with EGFR in a strain-specific manner, EGFR is a co-factor for infection, and EGFR plays a role in RSV-induced mucin expression, suggesting EGFR is a potential target for RSV disease.

Refining the diagnosis and EGFR status of non-small cell lung carcinoma in biopsy and cytologic material, using a panel of mucin staining, TTF-1, cytokeratin 5/6, and P63, and EGFR mutation analysis.

INTRODUCTION: The dichotomization of non-small cell carcinoma (NSCLC) subtype into squamous (SQCC) and adenocarcinoma (ADC) has become important in recent years and is increasingly required with regard to management. The aim of this study was to determine the utility of a panel of commercially available antibodies in refining the diagnosis on small biopsies and also to determine whether cytologic material is suitable for somatic EGFR genotyping in a prospectively analyzed series of patients undergoing investigation for suspected lung cancer. METHODS: Thirty-two consecutive cases of NSCLC were first tested using a panel comprising cytokeratin 5/6, P63, thyroid transcription factor-1, 34betaE12, and a D-PAS stain for mucin, to determine their value in refining diagnosis of NSCLC. After this test phase, two further pathologists independently reviewed the cases using a refined panel that excluded 34betaE12 because of its low specificity for SQCC, and refinement of diagnosis and concordance were assessed. Ten cases of ADC, including eight derived from cytologic samples, were sent for EGFR mutation analysis. RESULTS: There was refinement of diagnosis in 65% of cases of NSCLC to either SQCC or ADC in the test phase. This included 10 of 13 cases where cell pellets had been prepared from transbronchial needle aspirates. Validation by two further pathologists with varying expertise in lung pathology confirmed increased refinement and concordance of diagnosis. All samples were adequate for analysis, and they all showed a wild-type EGFR genotype. CONCLUSION: A panel comprising cytokeratin 5/6, P63, thyroid transcription factor-1, and a D-PAS stain for mucin increases diagnostic accuracy and agreement between pathologists when faced with refining a diagnosis of NSCLC to SQCC or ADC. These small samples, even cell pellets derived from transbronchial needle aspirates, seem to be adequate for EGFR mutation analysis.