Keratin 8 is a potential self-antigen in the coronary artery disease immunopeptidome: A translational approach.

BACKGROUND: Inflammation is an important risk factor in atherosclerosis, the underlying cause of coronary artery disease (CAD). Unresolved inflammation may result in maladaptive immune responses and lead to immune reactivity to self-antigens. We hypothesized that inflammation in CAD patients would manifest in immune reactivity to self-antigens detectable in soluble HLA-I/peptide complexes in the plasma. METHODS: Soluble HLA-I/peptide complexes were immuno-precipitated from plasma of male acute coronary syndrome (ACS) patients or age-matched controls and eluted peptides were subjected to mass spectrometry to generate the immunopeptidome. Self-peptides were ranked according to frequency and signal intensity, then mouse homologs of selected peptides were used to test immunologic recall in spleens of male apoE-/- mice fed either normal chow or high fat diet. The peptide detected with highest frequency in patient plasma samples and provoked T cell responses in mouse studies was selected for use as a self-antigen to stimulate CAD patient peripheral blood mononuclear cells (PBMCs). RESULTS: The immunopeptidome profile identified self-peptides unique to the CAD patients. The mouse homologs tested showed immune responses in apoE-/- mice. Keratin 8 was selected for further study in patient PBMCs which elicited T Effector cell responses in CAD patients compared to controls, associated with reduced PD-1 mRNA expression. CONCLUSION: An immunopeptidomic strategy to search for self-antigens potentially involved in CAD identified Keratin 8. Self-reactive immune response to Keratin 8 may be an important factor in the inflammatory response in CAD.

Therapeutic effects of lentinan on inflammatory bowel disease and colitis-associated cancer.

In this study, we investigated the therapeutic potential of lentinan in mouse models of inflammatory bowel disease (IBD) and colitis-associated cancer (CAC). Lentinan decreased the disease activity index and macroscopic and microscopic colon tissue damage in dextran sulphate sodium (DSS)-induced or TNBS-induced models of colitis. High-dose lentinan was more effective than salicylazosulfapyridine in the mouse models of colitis. Lentinan decreased the number of tumours, inflammatory cell infiltration, atypical hyperplasia and nuclear atypia in azoxymethane/DSS-induced CAC model. It also decreased the expression of pro-inflammatory cytokines, such as IL-13 and CD30L, in IBD and CAC model mice possibly by inhibiting Toll-like receptor 4 (TLR4)/NF-κB signalling and the expression of colon cancer markers, such as carcinoembryonic antigen, cytokeratin 8, CK18 and p53, in CAC model mice. In addition, lentinan restored the intestinal bacterial microbiotal community structure in IBD model mice. Thus, it shows therapeutic potential in IBD and CAC model mice possibly by inhibiting TLR4/NF-κB signalling-mediated inflammatory responses and disruption of the intestinal microbiotal structure.

A Comparison of Three Methods for the Detection of Circulating Tumor Cells in Patients with Early and Metastatic Breast Cancer.

BACKGROUND: We directly compared CTC detection rates and prognostic significance, using three different methods in patients with breast cancer (BC). METHODS: Early (n=200) and metastatic (n=164) patients were evaluated before initiating adjuvant or first-line chemotherapy, using the CellSearchTM System, an RT-qPCR for CK-19 mRNA detection and by double immunofluorescence (IF) microscopy using A45-B/B3 and CD45 antibodies. RESULTS: Using the CellSearchTM System, 37% and 16.5% of early BC patients were CTC-positive (at ≥1 and ≥2 CTCs/23 ml of blood), 18.0% by RT-qPCR and 16.9% by IF; no agreement was observed between methods. By the CellSearchTM 34.8% and 53.7% (at≥ 5 and ≥ 2 CTCs/7.5 ml) of metastatic patients were CTC-positive, 37.8% by RT-qPCR and 28.5% by IF. A significant agreement existed only between the CellSearchTM and RT-qPCR. In 60.8% of cases, differential EpCAM and CK-19 expression on CTCs by IF could explain the discrepancies between the CellSearchTM and RT-qPCR. CTC-positivity by either method was associated with decreased overall survival in metastatic patients. CONCLUSION: A significant concordance was observed between the CellSearchTM and RT-qPCR in metastatic but not in early BC. Discordant results could be explained in part by CTC heterogeneity. CTC detection by all methods evaluated had prognostic relevance in metastatic patients.

The effects of Bifidobacterium breve on immune mediators and proteome of HT29 cells monolayers.

The use of beneficial microorganisms, the so-called probiotics, to improve human health is gaining popularity. However, not all of the probiotic strains trigger the same responses and they differ in their interaction with the host. In spite of the limited knowledge on mechanisms of action some of the probiotic effects seem to be exerted through maintenance of the gastrointestinal barrier function and modulation of the immune system. In the present work, we have addressed in vitro the response of the intestinal epithelial cell line HT29 to the strain Bifidobacterium breve IPLA20004. In the array of 84 genes involved in inflammation tested, the expression of 12 was modified by the bifidobacteria. The genes of chemokine CXCL6, the chemokine receptor CCR7, and, specially, the complement component C3 were upregulated. Indeed, HT29 cells cocultivated with B. breve produced significantly higher levels of protein C3a. The proteome of HT29 cells showed increased levels of cytokeratin-8 in the presence of B. breve. Altogether, it seems that B. breve IPLA20004 could favor the recruitment of innate immune cells to the mucosa reinforcing, as well as the physical barrier of the intestinal epithelium.

Three distinct subsets of thymic epithelial cells in rats and mice defined by novel antibodies.

AIM: Thymic epithelial cells (TECs) are thought to play an essential role in T cell development and have been detected mainly in mice using lectin binding and antibodies to keratins. Our aim in the present study was to create a precise map of rat TECs using antibodies to putative markers and novel monoclonal antibodies (i.e., ED 18/19/21 and anti-CD205 antibodies) and compare it with a map from mouse counterparts and that of rat thymic dendritic cells. RESULTS: Rat TECs were subdivided on the basis of phenotype into three subsets; ED18+ED19+/-keratin 5 (K5)+K8+CD205+ class II MHC (MHCII)+ cortical TECs (cTECs), ED18+ED21-K5-K8+Ulex europaeus lectin 1 (UEA-1)+CD205- medullary TECs (mTEC1s), and ED18+ED21+K5+K8dullUEA-1-CD205- medullary TECs (mTEC2s). Thymic nurse cells were defined in cytosmears as an ED18+ED19+/-K5+K8+ subset of cTECs. mTEC1s preferentially expressed MHCII, claudin-3, claudin-4, and autoimmune regulator (AIRE). Use of ED18 and ED21 antibodies revealed three subsets of TECs in mice as well. We also detected two distinct TEC-free areas in the subcapsular cortex and in the medulla. Rat dendritic cells in the cortex were MHCII+CD103+ but negative for TEC markers, including CD205. Those in the medulla were MHCII+CD103+ and CD205+ cells were found only in the TEC-free area. CONCLUSION: Both rats and mice have three TEC subsets with similar phenotypes that can be identified using known markers and new monoclonal antibodies. These findings will facilitate further analysis of TEC subsets and DCs and help to define their roles in thymic selection and in pathological states such as autoimmune disorders.

High salt buffer improves integrity of RNA after fluorescence-activated cell sorting of intracellular labeled cells.

Over the past years, massive progress has been made in the ability to collect large-scale gene expression data from a limited sample size. Combined with improvements in multiplex flow cytometry-based techniques, this has made it possible to isolate and characterize specific cellular subtypes within heterogeneous populations, with a great impact on our understanding of different biological processes. However, sorting based on intracellular markers requires fixation and permeabilization of samples, and very often the integrity of RNA molecules is compromised during this process. Many attempts have been made to improve the quality of nucleic acids from such samples, but RNA degradation still remains a limiting factor for downstream analyses. Here we present a method to isolate high quality RNA from cells that have been fixed, permeabilized, intracellularly labeled and sorted. By performing all incubation steps in the presence of a high salt buffer, RNA degradation was avoided and samples with remarkable integrity were obtained. This procedure offers a straightforward and very affordable technique to retrieve high quality RNA from isolated cell populations, which increases the possibilities to characterize gene expression profiles of subpopulations from mixed samples, a technique with implications in a broad range of research fields.

Cytokeratin 8-MHC class I interactions: a potential novel immune escape phenotype by a lymph node metastatic carcinoma cell line.

Defective human leukocyte antigen (HLA) class I expression in malignant cells facilitates their escape from destruction by CD8(+) cytotoxic T lymphocytes. In this study, a post-translational mechanism of HLA class I abnormality that does not involve defects in the HLA subunits and antigen processing machinery components was identified and characterized. The marked HLA class I downregulation phenotype of a metastatic carcinoma cell line can be readily reversed by trypsin, suggesting a masking effect by serine protease-sensitive HLA class I-interacting factors. Co-immunoprecipitation, combined with LC-tandem mass spectrometry and immunoblotting identified these factors as cytokeratin (CK) 8 and its heterodimeric partners CK18 and CK19. Ectopic CK8/18 or CK8/19 expression in HEK293 cells resulted in surface CK8 expression with an HLA class I downregulation phenotype, while redirecting CK8/18 and CK8/19 to the endoplasmic reticulum (ER) had no such effect. This observation and the failure to constrain CK8/18 and CK8/19 membrane trafficking by an ER-Golgi transport inhibitor suggested an ER-independent route for CK8 access to HLA class I molecules. Monoclonal antibody mapping revealed a potential CK8 blockade of HLA class I-CD8 and -TCR contacts. These findings, along with the emerging role of cell surface CK8 in cancer metastasis, may imply a dual strategy for tumor cell survival in the host.

Identification of a mimotope for circulating anti-cytokeratin 8/18 antibody and its usage for the diagnosis of breast cancer.

A novel circulating tumor-associated autoantibody, K94, obtained from a hepatocellular carcinoma (HCC) mouse model was characterized. The target antigen of K94 autoantibody was expressed in various tumor cell lines including liver cancer, and its secretion was detectable using MCF-7 breast carcinoma cells. Proteomic analysis revealed that the protein bands reactive to K94 included cytokeratin (CK) 8 and 18, which are known to be related to tumorigenesis and form a heterotypic complex with each other. However, K94 showed no activity toward CK8 or CK18 separately. The epitope of the K94 antibody was only presented by a complex between CK8 and CK18, which was confirmed by analysis using recombinant CK8 and CK18 proteins. To formulate an assay for anti-CK8/18 complex autoantibody, a mimotope peptide reactive to K94 was selected from loop-constrained heptapeptide (-CX7C-) display phage library, of which sequence was CISPDAHSC (K94p1). A mimotope enzyme-linked immunosorbent assay (ELISA) using phage-displayed K94p1 peptide as a coating antigen was able to discriminate breast cancer (n=30) patients from normal subjects (n=30) with a sensitivity of 50% and a specificity of 82.61%. CA15.3 was detected at very low levels in the same breast cancer subjects and did not discriminate breast cancer patients from normal subjects, although it is a conventional biomarker of breast cancer. These results suggest that a mimotope ELISA composed of K94p1 peptide may be useful for the diagnosis of breast cancer.

A simple multicolor flow cytometry protocol for detection and molecular characterization of circulating tumor cells in epithelial cancers.

Circulating tumor cells (CTCs) might not only serve as prognostic marker but could also be useful for monitoring treatment efficacy. A multicolor flow cytometry protocol for their detection and molecular characterization in peripheral blood was developed which consisted of erythrocyte lysis followed by staining of cells with fluorochrome-labeled antibodies against CD45 and the epithelial markers EpCam and cytokeratin 7/8. For reducing the number of events acquired by flow cytometry, an electronic threshold for the fluorescent signals from the epithelial markers was applied. After establishment of the protocol by using spiking experiments, its suitability to determine the absolute number of CTCs as well as their expression of epidermal growth factor receptor (EGFR) and its phosphorylated form (phospho-EGFR) in blood samples from patients with squamous cell carcinoma of the head and neck (SCCHN) was validated. Spiking experiments demonstrated an excellent recovery (mean 85%) and a linear performance (R(2) = 0.98) of the protocol. Sensitivity and specificity were comparable to our former protocol using immunomagnetic CTC pre-enrichment. The analysis of 33 SCCHN patient samples revealed the presence of CTCs in 33.3% of cases with a mean ± SD of 1.5 ± 0.5 CTCs per 3.75 ml blood. EGFR was expressed in 100% and phospho-EGFR in 36.4% of the CTC+ cases. We have established a simple and sensitive multicolor flow cytometry protocol for detection of CTCs in patients with epithelial cancers including SCCHN which will allow their detailed molecular characterization.

Biodistribution of 131I-labeled anti-CK8 monoclonal antibody in HNSCC in xenotransplanted SCID mice.

BACKGROUND: A new promising approach to improve the outcome of head and neck squamous cell carcinoma (HNSCC) is the application of radio-labeled antibodies directed against tumor-associated antigens. Cytokeratin 8 (CK8), an intermediate filament forming protein, is shown to be de novo expressed in dysplastic lesions as well as in HNSCC. Therefore like the epithelial cell adhesion molecule CK8 seems to be a suitable anchor molecule for targeted radioimmunotherapy (RIT). The aim of this study was to investigate the biodistribution of a radio-labeled Cytokeratin 8-specific monoclonal antibody (mAb) in a SCID (severe combined immunodeficiency disease) mouse model. MATERIALS AND METHODS: The mAb against CK8 was labeled with (131)I and biodistribution was tested in established HNSCC xenografts in SCID mice. The biodistribution of the mAb in the tumor and different organs was determined with a gamma counter and was calculated as % injected dose/gram tissue. RESULTS: Initially, after systemic administration of (131)I-anti CK8 monoclonal antibody high activity was seen in all the organs. Over time the general activity decreased, whereas activity accumulated in the tumor. This activity decayed compared to the other tissues with a two- to threefold prolonged radioactive half-life. CONCLUSION: Specific antibody-antigen-binding is probably responsible for the prolonged radioactive half-life in the tumor and the resulting cumulative activity due to enrichment of the (131)I-anti CK8 mAb, so that Cytokeratin 8 seems to be a suitable anchor molecule for radioimmunotherapy in HNSCC.

The role of liver biopsy in the diagnosis and prognosis of patients with acute deterioration of alcoholic cirrhosis.

BACKGROUND & AIMS: The aim of this study was to systematically assess the diagnostic and prognostic value of early liver biopsy in patients who require hospital admission with acute deterioration of alcoholic cirrhosis. METHODS: Sixty-eight patients with acute deterioration of alcoholic cirrhosis underwent a liver biopsy within 7 days and the biopsies were processed using routine stains and K8/18 immunohistochemistry to characterize balloon degeneration. The biopsies were scored by two independent histopathologists using pre-defined criteria. The patients were managed according to institutional protocols and followed until the time of hospital discharge or death. RESULTS: With use of K8/18 immunohistochemistry, very high concordance rate for the diagnosis of balloon degeneration was reached (r = 0.7; p = 0.0001). The presence of a systemic inflammatory response (SIRS) suggestive of acute alcoholic steatohepatitis (ASH), predicts severe ASH histologically in only 50% patients. Moreover, in 41% of SIRS negative patients who were thought not to have ASH, a diagnosis of ASH was subsequently confirmed on histological grading. Patients that have SIRS criteria but no evidence of histological ASH are more likely to develop infection which may be indicated by the severity of canalicular cholestasis. Nineteen patients died during follow up. Patients manifesting ASH on biopsy who were also SIRS positive, had a significantly greater risk of mortality compared to those that were SIRS positive but ASH negative (p < 0.01) and those that were SIRS negative (p < 0.0001). CONCLUSIONS: The use of K8/18 immunostaining allows grading of the severity of alcoholic steatohepatitis. Early liver biopsy in these patients presenting with acute deterioration of cirrhosis is safe and provides important diagnostic and prognostic information.

Construction of divalent anti-keratin 8 single-chain antibodies (sc(Fv)(2)), expression in Pichia pastoris and their reactivity with multicellular tumor spheroids.

Single-chain variable fragments (scFvs) are small monovalent recombinant antibody fragments that retain the specificity of their parent immunoglobulins. ScFvs are excellent building blocks for new and improved immunodiagnostic and therapeutic proteins. However, the monovalency and the rapid renal elimination of scFvs result in poor tumor accumulation and retention. Engineering divalent antibody fragments is an excellent way to address these shortcomings. In this study, covalent divalent single-chain variable fragments (sc(Fv)(2)s), were constructed from the monovalent anti-keratin 8 scFvs, TS1-218 and its mutant, HE1-Q. The scFvs and sc(Fv)(2)s were expressed in the methylotrophic yeast Pichia pastoris, utilizing the alpha-factor secretion signal (α-factor) for extracellular secretion. The immunoreactivity and specificity of the antibody fragments were analyzed with enzyme-linked immunosorbent assay (ELISA) and the uptake and retention of the (125)I labeled antibody fragments were evaluated using HeLa HEp-2 multicellular tumor spheroids (MCTSs). Analysis of the antibody fragments demonstrated that parts of the α-factor remained at the N-terminal of the antibody fragments. Despite incomplete processing of the α-factor, the antibody fragments were functional where the sc(Fv)(2)s gave a three-fold stronger signal in ELISA compared to their scFv counterparts and the mutant antibodies demonstrated a stronger signal than their initial wild types. In addition, the sc(Fv)(2)s DiTS1-218 and DiHE1-Q displayed an approximately two-fold higher uptake and were retained to a larger extent in the MCTS, demonstrating a 3.9 and 9.4-fold increase in half-life respectively compared to their corresponding scFvs. In conclusion, expression in P. pastoris improved the yield 20-fold and facilitated the purification of the antibody fragments. Furthermore, the sc(Fv)(2)s presented a higher functional affinity to K 8 both in ELISA and MCTS compared to the scFvs with DiHE1-Q being the best candidate for further studies.

Localization of complexed anticytokeratin 8 scFv TS1-218 to HeLa HEp-2 multicellular tumor spheroids and experimental tumors.

Recombinant single-chain fragment variable (scFv) antibodies with specificity to tumor antigens can be used to target tumors in vivo. The approach to use administration of complexes of idiotypic-anti-idiotypic scFvs when targeting tumors has not been tested earlier, and from a theoretical point it could contribute to longer in vivo circulation and improved targeting efficiency by dissociation, when in contact with the target antigen. In this study two models to evaluate the targeting efficiency of such complexes were used. HeLa HEp-2 tumor cells were grown as multicellular tumor spheroids (MCTS) and exposed to the antibody constructs in vitro. The behavior in vivo was tested in an in vivo tumor xenograft model. To increase the size of the anticytokeratin 8 scFv, TS1-218, complexes were formed between TS1-218 and its anti-idiotype, alphaTS1 scFv. The functionality of (125)I-labeled TS1-218 alone and in complex was studied in both models. The uptake patterns were similar in both models. The idiotypic TS1-218 was able to localize to the MCTS and xenografted tumors, both alone and in complex with alphaTS1 scFv. TS1-218 in complex, however, demonstrated a significantly higher uptake than the monomeric TS1-218 in both models (p < 0.0005 and p < 0.0089, respectively). When complexes were administered in vivo, a slower clearance and an increased tumor half-life could be observed. The present investigation indicates that administration of targeting antibodies, with initially blocked antigen-binding sites by complex formation with their anti-idiotypes, may improve targeting efficiency.

Prognostic impact of regional lymph node micrometastasis in patients with node-negative biliary cancer.

OBJECTIVE: To immunohistochemically identify regional lymph node micrometastases in patients with regional node-negative biliary cancer who underwent curative resection, and to evaluate their clinical significance. SUMMARY BACKGROUND DATA: The clinical significance of immunohistochemically detected lymph node micrometastasis has recently been evaluated in various tumors. However, few reports have focused on this issue with regard to biliary cancer. METHODS: A total of 1421 regional lymph nodes from 151 patients with biliary cancer with negative regional nodes (as determined by conventional methods) were immunostained with antibody against cytokeratins 7 and 8 (CAM5.2). Prognostic impact was evaluated among patients with no metastasis, micrometastasis, and obvious metastasis detected by hematoxylin and eosin staining. Immunostained tumor foci were classified as small micrometastasis or large micrometastasis according to size (above or below 0.2 mm). RESULTS: CAM5.2-positive occult carcinoma cells in regional lymph nodes were detected in 33 (22%) of 151 patients and 49 (3%) of 1421 regional lymph nodes. Small micrometastases were detected in 23 patients, whereas large micrometastases were found in 10 patients. Survival for patients with micrometastasis was significantly worse than that for patients without (P = 0.0051), but was significantly better than that for patients with overt metastasis (P = 0.0092). No significant difference in postoperative survival was seen between patients with small and large micrometastases (P = 0.4221). CONCLUSIONS: Occult cancer cells were present in regional lymph nodes of 22% patients with regional node-negative biliary cancer, and were associated with significantly worse survival. Patients with micrometastases should be treated as carefully as node-positive patients.