Claudia Maletzki

Intracellular persisting Staphylococcus aureus is the major pathogen in recurrent tonsillitis.

Background The two major indications for tonsillectomy are recurrent tonsillitis (RT) and peritonsillar abscess (PTA). Unlike PTAs, which are primarily treated surgically, RT is often cured by tonsillectomy only after a series of failed drug therapy attempts. Although the bacteriological background of RT has been studied, the reason for the lack of success of conservative therapeutic approaches is not well understood. Methods In a prospective study, tonsil specimens from 130 RT patients and 124 PTA patients were examined for the presence of extra- and intracellular bacteria using antibiotic protection assays. Staphylococcus aureus isolates from RT patients were characterized by pulsed-field gel electrophoresis (PFGE), spa-typing and MSCRAMM-gene-PCR. Their ability for biofilm formation was tested and their cell invasiveness was confirmed by a flow cytometric invasion assay (FACS), fluorescent in situ hybridization (FISH) and immunohistochemistry. Findings S. aureus was the predominant species (57.7%) in RT patients, whereas Streptococcus pyogenes was most prevalent (20.2%) in PTA patients. Three different assays (FACS, FISH, antibiotic protection assay) showed that nearly all RT-associated S. aureus strains were located inside tonsillar cells. Correspondingly, the results of the MSCRAMM-gene-PCRs confirmed that 87% of these S. aureus isolates were invasive strains and not mere colonizers. Based upon PFGE analyses of genomic DNA and on spa-gene typing the vast majority of the S. aureus isolates belonged to different clonal lineages. Conclusions Our results demonstrate that intracellular residing S. aureus is the most common cause of RT and indicate that S. aureus uses this location to survive the effects of antibiotics and the host immune response. A German translation of the Abstract is provided as supplementary material (Abstract S1).

Pancreatic cancer regression by intratumoural injection of live Streptococcus pyogenes in a syngeneic mouse model

Background: This study addressed the potential of bacteriolytic therapy using Streptococcus pyogenes in a syngeneic pancreatic carcinoma mouse model. Methods: Panc02 tumours were either infected with S pyogenes or were treated with the equivalent volume of vehicle. In addition to assessment of tumour histology and immunohistochemistry, isolated splenocytes were analysed by flow cytometry. Interferon (IFN) γ secretion as a reaction of splenocytes against tumour cells was shown through the ELISpot technique. A cytotoxic effect of lymphocytes against tumour targets was detected by lactate dehydrogenase (LDH) release. Cytokine levels in serum were measured. Results: A single application of live bacteria into established Panc02 tumours resulted in complete tumour regression. This antitumoural effect was accompanied by massive leucocyte infiltration into the tumours as well as a significant and sustained elevation of systemic levels of the proinflammatory cytokines IFNγ, tumour necrosis factor α and interleukin 6. Lymphocytes obtained from treated mice specifically recognised syngeneic tumour cells in IFNγ-ELISpot, and most importantly in cellular cytotoxicity assays, indicating a tumour-specific immune response. Conclusions: We provide data that both the direct lytic activity of S pyogenes towards tumour cells and the infection-driven infiltration of tumours by cells of the innate immune system lead to damage of tumour cells followed by a dissemination of tumour components. This last outcome allows for the activation of tumour-specific effector cells, most probably in draining lymph nodes, promoted by the proinflammatory context. Taken together, these data indicate that the application of live S pyogenes may be a promising new treatment strategy for advanced pancreatic cancer patients that warrants further investigation.

Ex-vivo Clonally Expanded B Lymphocytes Infiltrating Colorectal Carcinoma Are of Mature Immunophenotype and Produce Functional IgG

Background Tumor infiltrating B cells (TiBc) have not yet been investigated in detail. This may at least in part be due to technical difficulties. Here we describe a straightforward and reproducible method to isolate and culture TiBc from primary colorectal carcinomas (CRC). Methods/Results TiBc cultures were generated by Epstein-Barr virus (EBV) immortalization. With this method, monoclonal TiBc cultures were obtained for 14/19 CRCs. As assessed by flow cytometry and ELISA, TiBc showed an activated immunophenotype (CD23+, CD80+) and produced immunoglobulin (Ig; IgG secretion in 55% of the cultures). In functional in vitro analysis, most of the IgGs specifically bound to allogeneic CRC target cells. These data suggest that TiBc are antigen-experienced and thus may exhibit functionality in situ. Additionally, mini-cultures generated from 12 further CRCs revealed TiBc outgrowth exclusively in the presence of EBV. Conclusion In summary, this simple method provides a cellular tool and our data set the stage for analysing the bivalent role of TiBc; being antigen-presenting cells on the one hand and tumor-specific antibody producers on the other. Additionally, the generation of long-term TiBc cultures and their monoclonal Ig may serve to identify novel tumor-specific antigens.

Establishment, Characterization and Chemosensitivity of Three Mismatch Repair Deficient Cell Lines from Sporadic and Inherited Colorectal Carcinomas

Background Colorectal cancer (CRC) represents a morphologic and molecular heterogenic disease. This heterogeneity substantially impairs drug effectiveness and prognosis. The subtype of mismatch repair deficient (MMR-D) CRCs, accounting for about 15% of all cases, shows particular differential responses up to resistance towards currently approved cytostatic drugs. Pre-clinical in vitro models representing molecular features of MMR-D tumors are thus mandatory for identifying biomarkers that finally help to predict responses towards new cytostatic drugs. Here, we describe the successful establishment and characterization of three patient-derived MMR-D cell lines (HROC24, HROC87, and HROC113) along with their corresponding xenografts. Methodology MMR-D cell lines (HROC24, HROC87, and HROC113) were established from a total of ten clinicopathological well-defined MMR-D cases (120 CRC cases in total). Cells were comprehensively characterized by phenotype, morphology, growth kinetics, invasiveness, and molecular profile. Additionally, response to clinically relevant chemotherapeutics was examined in vitro and in vivo. Principal Findings Two MMR-D lines showing CIMP-H derived from sporadic CRC (HROC24: K-raswt, B-rafmut, HROC87: K-raswt, B-rafmut), whereas the HROC113 cell line (K-rasmut, B-rafwt) was HNPCC-associated. A diploid DNA-status could be verified by flow cytometry and SNP Array analysis. All cell lines were characterized as epithelial (EpCAM+) tumor cells, showing surface tumor marker expression (CEACAM+). MHC-class II was inducible by Interferon-γ stimulation. Growth kinetics as well as invasive potential was quite heterogeneous between individual lines. Besides, MMR-D cell lines exhibited distinct responsiveness towards chemotherapeutics, even when comparing in vitro and in vivo sensitivity. Conclusions These newly established and well-characterized, low-passage MMR-D cell lines provide a useful tool for future investigations on the biological characteristics of MMR-D CRCs, both of sporadic and hereditary origin. Additionally, matched patient-derived immune cells allow for comparative genetic studies.

Tumor-infiltrating B cells: The ignored players in tumor immunology.

B cells infiltrating into solid tumors are poorly investigated despite their described positive prognostic value. Whether this antitumor potential comes from either the antigen presentation or the antibody production capacity of B cells, or both, is unknown. Our recently published method on tumor-infiltrating B lymphocyte cloning may prove helpful in unraveling the actual relevance of these cells for tumor development and response to therapy.

S100 Proteins as Diagnostic and Prognostic Markers in Colorectal and Hepatocellular Carcinoma

Context Clinical and experimental studies have suggested a link between S100 gene expression and neoplastic disorders, however, the molecular mechanisms of this association are not well understood. The aim of this review was to conduct a comprehensive literature search in order to understand the possible underlying molecular mechanisms of this association. We also discuss their application as diagnostic and prognostic markers in colorectal and hepatocellular carcinoma. Evidence Acquisitions We searched Pubmed (NLM) and Web of Science (ISI Web of Knowledge). Results S100 genes display a complex expression pattern in colorectal and hepatocel lular carcinoma. They are expressed in tumor and/or tumor stroma cells, and they exert both pro- and antitumorigenic actions. In view of this complexity, it becomes clear that S100 proteins might act as both friend and foe. The biological role of the S100 genes is predicted to depend on the relative contributions of the different cell types at specific stages of tumor progression. Conclusions Further research is required in order to uncover the functional role of S100 genes in tumorigenesis. Answers to this issue are needed before we can more fully understand the clinical relevance of S100 protein expression within epithelial tumors, with regard to their potential applicability as biomarkers for diagnosis and therapy decisions.

An MSI Tumor Specific Frameshift Mutation in a Coding Microsatellite of MSH3 Encodes for HLA-A0201-Restricted CD8+ Cytotoxic T Cell Epitopes

Background Microsatellite instability (MSI) resulting from inactivation of the DNA mismatch repair system (MMR) characterizes a highly immunological subtype of colorectal carcinomas. Those tumors express multiple frameshift-mutated proteins which present a unique pool of tumor-specific antigens. The DNA MMR protein MSH3 is frequently mutated in MSI+ colorectal tumors, thus making it an attractive candidate for T cell-based immunotherapies. Methodology/Principal Findings FSP-specific CD8+ T cells were generated from a healthy donor using reverse immunology. Those T cells specifically recognized T2 cells sensitized with the respective peptides. Specific recognition and killing of MSI+ colorectal carcinoma cells harbouring the mutated reading frame was observed. The results obtained with T cell bulk cultures could be reproduced with T cell clones obtained from the same cultures. Blocking experiments (using antibodies and cold target inhibition) confirmed peptide as well as HLA-A0201-specificity. Conclusions We identified two novel HLA-A0201-restricted cytotoxic T cell epitopes derived from a (-1) frameshift mutation of a coding A(8) tract within the MSH3 gene. These were 386-FLLALWECSL (FSP18) and 387-LLALWECSL (FSP19) as well as 403-IVSRTLLLV (FSP23) and 402-LIVSRTLLLV (FSP31), respectively. These results suggest that MSH3(-1) represents another promising MSI+-induced target antigen. By identifying two distinct epitopes within MSH3(-1), the sustained immunogenicity of the frameshift mutated sequence was confirmed. Our data therefore encourage further exploitation of MSH3 as a piece for peptide-based vaccines either for therapeutic or –even more important– preventive purposes.

Mistletoe lectin has a shiga toxin-like structure and should be combined with other Toll-like receptor ligands in cancer therapy

Mistletoe extract (ME) is applied as an adjuvant treatment in cancer therapy in thousands of patients each year in Europe. The main immunostimulating component of mistletoe extract, mistletoe lectin, recently has been shown to be a pattern recognition receptor ligand and hence is binding to an important class of pathogen-sensing receptors. Pattern recognition receptor ligands are potent activators of dendritic cells. This activation is a prerequisite for a full-blown T-cell response against cancer cells. Pattern recognition receptor ligands are increasingly recognized as important players in cancer immunotherapy. We collect evidence from case studies on spontaneous regression, from epidemiology, from experiments in a mouse cancer model, and from protein structure comparisons to argue that a combination of mistletoe therapy with other pattern recognition receptor ligand substances leads to an increased immune stimulatory effect. We show that mistletoe lectin is a plant protein of bacterial origin with a 3D structure very similar to shiga toxin from Shigella dysenteriae, which explains the remarkable immunogenicity of mistletoe lectin. Secondly, we show that a combination of pattern recognition receptor ligands applied metronomically in a cancer mouse model leads to complete remission, while single pattern recognition receptor ligands slowed tumor growth. Taken together, we propose to combine mistletoe drugs with other pattern recognition receptor ligand drugs to increase its efficacy in adjuvant or even primary cancer therapy.

Frameshift-derived neoantigens constitute immunotherapeutic targets for patients with microsatellite-instable haematological malignancies: frameshift peptides for treating MSI+ blood cancers.

PURPOSE Microsatellite instability (MSI) resulting from loss of functional DNA mismatch repair was recently found in various haematological disorders. In coding sequences, MSI leads to frameshift mutations (FSMs) and the production of C-terminally altered proteins which are foreign to the immune system. Here, we wondered whether these frame-shifted peptide (FSP) sequences represent tumour-specific antigens also for MSI(+) leukaemia and lymphomas (L/L). MATERIAL AND METHODS A total of 33 coding region microsatellites were examined in MSI(+) L/L cell lines for the presence of FSMs. Thereafter, recognition of MSI(+) cells by established FSP-specific CD8(+) T cell lines was quantified using interferon (IFN)-γ enzyme-linked immunospot (ELISpot) assays. In each experiment, MSI(+) L/L cell lines and T2 targets exogenously loaded with the cognate peptide (=internal control) were employed. Supplementary, lytic activity towards tumour cells was analysed by standard chromium release assay ((51)Cr). RESULTS Mutational profiling of 33 coding microsatellite loci in nine MSI(+) L/L cell lines revealed instability in at least nine microsatellites. In each cell line, a distinct mutational profile was observed. Only three of the 33 loci were stable. FSP-specific and human leukocyte antigen-A2 (HLA-A2)-restricted T cells specifically recognised MSI(+) L/L cells endogenously expressing TGFβRII(-1), Caspase 5 (-1) and MSH3 (-1) in ELISpot assays. Moreover, specific killing of Caspase 5 (-1) and MSH3 (-1) expressing L/L cell lines was achieved in functional cytotoxicity assays. CONCLUSION Data presented here expand the importance of FSPs as shared and general tumour-specific antigens. Consequently, they open new avenues for specific immunotherapies not only for solid but also for MSI(+) haematological malignancies.

Prophylactic Application of Bovine Colostrum Ameliorates Murine Colitis via Induction of Immunoregulatory Cells

Epidemiologic studies suggest a relationship between early weaning and the incidence of inflammatory bowel disease. Herein, we addressed the question whether bovine colostrum, the first milk produced by mammals, is able to prevent dextran sulfate sodium (DSS)-induced colitis in mice. Prior to induction of colitis with 5% DSS, Naval Medical Research Institute mice were fed bovine colostrum [BV-20 or 200 mg/kg body weight (BW)], BSA (20 mg/kg BW), or water (100 μL) daily by oral gavage for 2 wk. The clinical severity of colitis was determined by scoring changes in BW and colon length reduction. Following 2 wk of observation, the colons were removed for histologic and immunohistochemical evaluation of inflammation. Flow cytometric phenotyping of leukocyte subsets was performed from peripheral blood, mesenteric lymph nodes, and spleens. Administration of bovine colostrum improved the clinical and histologic severity of colorectal inflammation. Compared with BSA-fed and water-fed controls, BV-20 pretreated mice had significantly less severe weight loss and decreased colon shortening. Beneficial effects were accompanied by redistribution of immunoregulatory, peripheral and splenic γδ TCR(+) cells, and CD11b(+)Gr1(+) cells. Higher colostrum doses did not affect disease activity. In summary, prophylactic administration of colostrum improved clinical symptoms of colorectal inflammation in a well-established mouse model of DSS-induced colitis. Further investigations will target the underlying immunomodulatory mechanisms to our approach.