Serum Calretinin and Genetic Variability as a Prognostic and Predictive Factor in Malignant Mesothelioma.

Calretinin is a promising diagnostic biomarker for malignant mesothelioma (MM), but less is known about its prognostic role. Our aim was to evaluate the association between serum calretinin concentration or genetic factors and the survival or outcome of cisplatin-based chemotherapy in MM. Our study included 265 MM patients. Serum calretinin concentration was determined using ELISA. Patients were genotyped for seven polymorphisms in CALB2, E2F2, MIR335, NRF1, and SEPTIN7 using competitive allele-specific PCR. Nonparametric tests, logistic regression, and survival analysis were used for statistical analysis. Higher serum calretinin concentration was associated with shorter progression-free (PFS) (HR = 1.18 (1.02-1.37), p = 0.023) and overall survival (OS) (HR = 1.20 (1.03-1.41), p = 0.023), but the association was not significant after adjusting for clinical factors (HR = 1.05 (0.85-1.31), p = 0.653 and HR = 1.06 (0.84-1.34), p = 0.613, respectively). SEPTIN7 rs3801339 and MIR335 rs3807348 were associated with survival even after adjustment (HR = 1.76 (1.17-2.64), p = 0.007 and HR = 0.65 (0.45-0.95), p = 0.028, respectively). Calretinin concentration was higher in patients who progressed after treatment with cisplatin-based chemotherapy (1.68 vs. 0.45 ng/mL, p = 0.001). Calretinin concentration above 0.89 ng/mL was associated with shorter PFS and OS from the start of chemotherapy (HR = 1.88 (1.28-2.77), p = 0.001 and HR = 1.91 (1.22-2.97), p = 0.004, respectively), even after adjusting for clinical factors (p < 0.05). MIR335 rs3807348 was associated with a better response to chemotherapy (OR = 2.69 (1.17-6.18), p = 0.020). We showed that serum calretinin is associated with survival and chemotherapy treatment outcomes in MM and could serve as a predictive biomarker.

Assessment of miR-103a-3p in leukocytes-No diagnostic benefit in combination with the blood-based biomarkers mesothelin and calretinin for malignant pleural mesothelioma diagnosis.

Malignant pleural mesothelioma (MPM) is a cancer associated with asbestos exposure and its diagnosis is challenging due to the moderate sensitivities of the available methods. In this regard, miR-103a-3p was considered to increase the sensitivity of established biomarkers to detect MPM. Its behavior and diagnostic value in the Mexican population has not been previously evaluated. In 108 confirmed MPM cases and 218 controls, almost all formerly exposed to asbestos, we quantified miR-103-3a-3p levels in leukocytes using quantitative Real-Time PCR, together with mesothelin and calretinin measured in plasma by ELISA. Sensitivity and specificity of miR-103-3a-3p alone and in combination with mesothelin and calretinin were determined. Bivariate analysis was performed using Mann-Whitney U test and Spearman correlation. Non-conditional logistic regression models were used to calculate the area under curve (AUC), sensitivity, and specificity for the combination of biomarkers. Mesothelin and calretinin levels were higher among cases, remaining as well among males and participants ≤60 years old (only mesothelin). Significant differences for miR-103a-3p were observed between male cases and controls, whereas significant differences between cases and controls for mesothelin and calretinin were observed in men and women. At 95.5% specificity the individual sensitivity of miR-103a-3p was 4.4% in men, whereas the sensitivity of mesothelin and calretinin was 72.2% and 80.9%, respectively. Positive correlations for miR-103a-3p were observed with age, environmental asbestos exposure, years with diabetes mellitus, and glucose levels, while negative correlations were observed with years of occupational asbestos exposure, creatinine, erythrocytes, direct bilirubin, and leukocytes. The addition of miR-103a-3p to mesothelin and calretinin did not increase the diagnostic performance for MPM diagnosis. However, miR-103a-3p levels were correlated with several characteristics in the Mexican population.

Quality assurance of hematopoietic stem cells by macrophages determines stem cell clonality.

Tissue-specific stem cells persist for a lifetime and can differentiate to maintain homeostasis or transform to initiate cancer. Despite their importance, there are no described quality assurance mechanisms for newly formed stem cells. We observed intimate and specific interactions between macrophages and nascent blood stem cells in zebrafish embryos. Macrophage interactions frequently led to either removal of cytoplasmic material and stem cell division or complete engulfment and stem cell death. Stressed stem cells were marked by surface Calreticulin, which stimulated macrophage interactions. Using cellular barcoding, we found that Calreticulin knock-down or embryonic macrophage depletion reduced the number of stem cell clones that established adult hematopoiesis. Our work supports a model in which embryonic macrophages determine hematopoietic clonality by monitoring stem cell quality.

All-Ion Monitoring-Directed Low-Abundance Protein Quantification Reveals CALB2 as a Key Promoter in Hepatocellular Carcinoma Metastasis.

Because of the wide abundance range of the proteome, achieving high-coverage quantification of low-abundance proteins is always a major challenge. In this study, a complete pipeline focused on all-ion monitoring (AIM) is first constructed with the concept of untargeted parallel-reaction monitoring, including the seamless connection of protein sample preparation, liquid chromatography mass spectrometry (LC-MS) acquisition, and algorithm development to enable the in-depth quantitative analysis of low-abundance proteins. This pipeline significantly improves the reproducibility and sensitivity of sample preparation and LC-MS acquisition for low-abundance proteins, enabling all the precursors ions fragmented and collected. Contributed by the advantages of the AIM method with all the target precursor acquisition by the data-dependent acquisition (DDA) approach, together with the ability of data-independent acquisition to fragment all precursor ions, the quantitative accuracy and precision of low-abundance proteins are greatly enhanced. As a proof of concept, this pipeline is employed to discover the key differential proteins in the mechanism of hepatocellular carcinoma (HCC) metastasis. On the basis of the superiority of AIM, an extremely low-abundance protein, CALB2, is proposed to promote HCC metastasis in vitro and in vivo. We also reveal that CALB2 activates the TRPV2-Ca2+-ERK1/2 signaling pathway to induce HCC cell metastasis. In summary, we provide a universal AIM pipeline for the high-coverage quantification of low-abundance functional proteins to seek novel insights into the mechanisms of cancer metastasis.

CLDN15 is a novel diagnostic marker for malignant pleural mesothelioma.

Malignant mesothelioma is a cancer with a poor survival rate. It is difficult to diagnose mesotheliomas because they show a variety of histological patterns similar to those of various other cancers. However, since currently used positive markers for mesotheliomas may show false positives or false negatives, a novel mesothelial positive marker is required. In the present study, we screened 25 claudins and found that claudin-15 is expressed in the mesothelial cells. We made new rat anti-human claudin-15 (CLDN15) monoclonal antibodies that selectively recognize CLDN15, and investigated whether CLDN15 is a good positive marker for malignant pleural mesotheliomas (MPMs) using MPM tissue samples by immunohistochemistry and semi-quantification of the expression level using an immunoreactive score (IRS) method. Of 42 MPM samples, 83% were positive for CLDN15. The positive ratio was equal to or greater than other positive markers for MPMs including calretinin (81%), WT-1 (50%), and D2-40 (81%). In 50 lung adenocarcinoma sections, four cases were positive for CLDN15 and the specificity (92%) was comparable with other markers (90-100%). Notably, CLDN15 was rarely detected in 24 non-mesothelial tumors in the tissue microarray (12/327 cases). In conclusion, CLDN15 can be used in the clinical setting as a positive marker for MPM diagnosis.

Whole-genome sequencing of African Americans implicates differential genetic architecture in inflammatory bowel disease.

Whether or not populations diverge with respect to the genetic contribution to risk of specific complex diseases is relevant to understanding the evolution of susceptibility and origins of health disparities. Here, we describe a large-scale whole-genome sequencing study of inflammatory bowel disease encompassing 1,774 affected individuals and 1,644 healthy control Americans with African ancestry (African Americans). Although no new loci for inflammatory bowel disease are discovered at genome-wide significance levels, we identify numerous instances of differential effect sizes in combination with divergent allele frequencies. For example, the major effect at PTGER4 fine maps to a single credible interval of 22 SNPs corresponding to one of four independent associations at the locus in European ancestry individuals but with an elevated odds ratio for Crohn disease in African Americans. A rare variant aggregate analysis implicates Ca2+-binding neuro-immunomodulator CALB2 in ulcerative colitis. Highly significant overall overlap of common variant risk for inflammatory bowel disease susceptibility between individuals with African and European ancestries was observed, with 41 of 241 previously known lead variants replicated and overall correlations in effect sizes of 0.68 for combined inflammatory bowel disease. Nevertheless, subtle differences influence the performance of polygenic risk scores, and we show that ancestry-appropriate weights significantly improve polygenic prediction in the highest percentiles of risk. The median amount of variance explained per locus remains the same in African and European cohorts, providing evidence for compensation of effect sizes as allele frequencies diverge, as expected under a highly polygenic model of disease.

Mesothelioma Biomarkers: Discovery in Search of Validation.

Malignant pleural mesothelioma (MPM) is an asbestos-related neoplasm that can only be treated successfully when correctly diagnosed and treated early. The asbestos-exposed population is a high-risk group that could benefit from sensitive and specific blood- or tissue-based biomarkers. We review recent work with biomarker development in MPM and literature of the last 20 years on the most promising blood- and tissue-based biomarkers. Proteomic, genomic, and epigenomic platforms are covered. SMRP is the only validated blood-based biomarker with diagnostic, monitoring and prognostic value. To strengthen development and testing of MPM biomarkers, cohorts for validation must be established by enlisting worldwide collaborations.

[Mutant calreticulin and the molecular mechanisms in development of myeloproliferative neoplasms].

This review aimed to evaluate the molecular mechanism underlying the development of myeloproliferative neoplasms (MPN) caused by mutant calreticulin (CALR). This mutation is found in a subset of patients with Philadelphia chromosome-negative MPNs, and it encodes a molecular chaperone. However, it is essentially impossible to elucidate the oncogenic property of mutant CALR from the wild-type CALR function. Studies have reported that mutant CALR forms a homomultimeric complex via intermolecular interaction between novel domains acquired due to a frameshift mutation, gains a high binding affinity for myeloproliferative leukemia protein (MPL), the thrombopoietin receptor, through a presumptive structural change, and acts as an agonist for MPL. In this review, I would like to describe the course of the discovery of this unique molecular mechanism and discuss future scope of research on mutant CALR.

Modulation of Calretinin Expression in Human Mesothelioma Cells Reveals the Implication of the FAK and Wnt Signaling Pathways in Conferring Chemoresistance towards Cisplatin.

Malignant mesothelioma (MM) is an aggressive asbestos-linked neoplasm, characterized by dysregulation of signaling pathways. Due to intrinsic or acquired chemoresistance, MM treatment options remain limited. Calretinin is a Ca2+-binding protein expressed during MM tumorigenesis that activates the FAK signaling pathway, promoting invasion and epithelial-to-mesenchymal transition. Constitutive calretinin downregulation decreases MM cells' growth and survival, and impairs tumor formation in vivo. In order to evaluate early molecular events occurring during calretinin downregulation, we generated a tightly controlled IPTG-inducible expression system to modulate calretinin levels in vitro. Calretinin downregulation significantly reduced viability and proliferation of MM cells, attenuated FAK signaling and reduced the invasive phenotype of surviving cells. Importantly, surviving cells showed a higher resistance to cisplatin due to increased Wnt signaling. This resistance was abrogated by the Wnt signaling pathway inhibitor 3289-8625. In various MM cell lines and regardless of calretinin expression levels, blocking of FAK signaling activated the Wnt signaling pathway and vice versa. Thus, blocking both pathways had the strongest impact on MM cell proliferation and survival. Chemoresistance mechanisms in MM cells have resulted in a failure of single-agent therapies. Targeting of multiple components of key signaling pathways, including Wnt signaling, might be the future method-of-choice to treat MM.

A 2-year retrospective study on pleural effusions: A cancer centre experience.

BACKGROUND: Cytopathological examination of pleural effusions is a fast and minimally invasive method for verification of the presence of neoplastic cells. We report our 2-year experience using a categorised diagnostic system and reporting risks of malignancy (ROMs) for each defined category. METHODS: Cytological reports of patients between November 2016 and October 2018 were collected, with results primarily classified into a five-tiered classification scheme. Immunohistochemistry markers used in cytology and their results were also recorded. Final agreement to histology and overall test performance was calculated for cases with available concomitant (up to 3 months) pleural biopsies. RESULTS: A total of 519 samples from 385 patients were collected, being 29 (5.6%) classified as non-diagnostic, 291 (56%) as negative, 28 (5.4%) as atypical, 30 (5.8%) as suspicious and 141 (27.2%) as positive. Most requested markers were calretinin, TTF1, Ber-EP4 and Gata-3, being conclusive in 45 (76.3%) cases. Total cyto-histological agreement was achieved in 49 (80.3%) specimens, with an overall sensitivity and specificity of 69.4% and 93.3%, respectively. Positive predictive value was 96.2% and negative predictive value was of 56%. ROM for each diagnostic category was 50% for non-diagnostic, 44% for negative, 50% for atypical, 83.3% for suspicious and 96.2% for positive. CONCLUSIONS: Our 2-year retrospective study has shown a high specificity and positive predictive value for pleural cytology. The use of a five-tiered system has also shown to be highly effective, with a concordantly progressive higher ROM for the assigned diagnostic categories.

Transplantation of Human Embryonic Stem Cell-Derived Retinal Tissue in the Subretinal Space of the Cat Eye.

To develop biological approaches to restore vision, we developed a method of transplanting stem cell-derived retinal tissue into the subretinal space of a large-eye animal model (cat). Human embryonic stem cells (hESC) were differentiated to retinal organoids in a dish. hESC-derived retinal tissue was introduced into the subretinal space of wild-type cats following a pars plana vitrectomy. The cats were systemically immunosuppressed with either prednisolone or prednisolone plus cyclosporine A. The eyes were examined by fundoscopy and spectral-domain optical coherence tomography imaging for adverse effects due to the presence of the subretinal grafts. Immunohistochemistry was done with antibodies to retinal and human markers to delineate graft survival, differentiation, and integration into cat retina. We successfully delivered hESC-derived retinal tissue into the subretinal space of the cat eye. We observed strong infiltration of immune cells in the graft and surrounding tissue in the cats treated with prednisolone. In contrast, we showed better survival and low immune response to the graft in cats treated with prednisolone plus cyclosporine A. Immunohistochemistry with antibodies (STEM121, CALB2, DCX, and SMI-312) revealed large number of graft-derived fibers connecting the graft and the host. We also show presence of human-specific synaptophysin puncta in the cat retina. This work demonstrates feasibility of engrafting hESC-derived retinal tissue into the subretinal space of large-eye animal models. Transplanting retinal tissue in degenerating cat retina will enable rapid development of preclinical in vivo work focused on vision restoration.

A triplex probe-based TaqMan qPCR assay for Calreticulin type I and II mutation detection.

BACKGROUND: Calreticulin (CALR) exon 9 frameshift mutations have recently been identified in 30-40% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF) without JAK2 or MPL mutations. We aimed to develop a qPCR assay to screen type I and II mutations of CALR. METHODS: Three different fluorescent-labeled hydrolysis probes and one pair of primers in a closed-tube system were developed to detect CALR type I and II mutations and distinguish them from wild-type. The sensitivity and specificity were validated using TA-cloning plasmids containing CALR wild-type and type I and II mutants, respectively. Fifty-nine ET and PMF specimens were screened by TaqMan qPCR and sequenced by Sanger sequencing. For intra-assay validation, 20 replicates of the assay were performed with each sample. For inter-assay validation, four replications of each sample were carried out and repeated continuously for 5 days. RESULTS: We found that triplex probe-based TaqMan qPCR was reliable in detecting CALR type I and II mutants within DNA that was diluted to 1% of total DNA with the wild-type DNA as background. In 59 patient specimens, six of the observed mutations of CALR were type I and five were type II. Genotyping results obtained from TaqMan qPCR were 100% concordant with Sanger sequencing. The intra- and inter-assay CVs of TaqMan qPCR were less than 3%, respectively. CONCLUSIONS: Triplex probe-based TaqMan qPCR is an accurate and sensitive method for screening ET or PMF patients with type I and II mutations in CALR.

The Effect of the MicroRNA-183 Family on Hair Cell-Specific Markers of Human Bone Marrow-Derived Mesenchymal Stem Cells.

Hearing loss is considered the most common sensory disorder across the world. Nowadays, a cochlear implant can be an effective treatment for patients. Moreover, it is often believed that sensorineural hearing loss in humans is caused by loss or disruption of the function of hair cells in the cochlea. In this respect, mesenchymal cells can be a good candidate for cell-based therapeutic approaches. To this end, the potential of human bone marrow-derived mesenchymal stem cells to differentiate into hair cells with the help of transfection of microRNA in vitro was investigated. MicroRNA mimics (miRNA-96, 182, and 183) were transfected to human bone marrow-derived mesenchymal stem cells using Lipofec-tamine as a common transfection reagent following the manufacturer's instructions at 50 nM for microRNA mimics and 50 nM for the scramble. The changes in cell morphology were also observed under an inverted microscope. Then, the relative expression levels of SOX2, POU4F3, MYO7A, and calretinin were assayed using real-time polymerase chain reaction according to the ΔΔCt method. The ATOH1 level was similarly measured via real-time polymerase chain reaction and Western blotting. The results showed that increased expression of miRNA-182, but neither miRNA-96 nor miRNA-183, could lead to higher expression levels in some hair cell markers. The morphology of the cells also did not change in this respect, but the evaluation of gene expression at the levels of mRNA could promote the expression of the ATOH1, SOX2, and POU4F3 markers. Furthermore, miRNA-182 could enhance the expression of ATOH1 at the protein level. According to the results of this study, it was concluded that miRNA-182 could serve as a crucial function in hair cell differentiation by the upregulation of SOX2, POU4F3, and ATOH1 to promote a hair cell's fate.

Cell differentiation in cardiac myxomas: confocal microscopy and gene expression analysis after laser capture microdissection.

Cardiac myxomas are rare tumors with a heterogeneous cell population including properly neoplastic (lepidic), endothelial and smooth muscle cells. The assessment of neoplastic (lepidic) cell differentiation pattern is rather difficult using conventional light microscopy immunohistochemistry and/or whole tissue extracts for mRNA analyses. In a preliminary study, we investigated 20 formalin-fixed and paraffin-embedded cardiac myxomas by means of conventional immunohistochemistry; in 10/20 cases, cell differentiation was also analyzed by real-time RT-PCR after laser capture microdissection of the neoplastic cells, whereas calretinin and endothelial antigen CD31 immunoreactivity was localized in 4/10 cases by double immunofluorescence confocal microscopy. Gene expression analyses of α-smooth muscle actin, endothelial CD31 antigen, alpha-cardiac actin, matrix metalloprotease-2 (MMP2) and tissue inhibitor of matrix metalloprotease-1 (TIMP1) was performed on cDNA obtained from either microdissected neoplastic cells or whole tumor sections. We found very little or absent CD31 and α-Smooth Muscle Actin expression in the microdissected cells as compared to the whole tumors, whereas TIMP1 and MMP2 genes were highly expressed in both ones, greater levels being found in patients with embolic phenomena. α-Cardiac Actin was not detected. Confocal microscopy disclosed two different signals corresponding to calretinin-positive myxoma cells and to endothelial CD31-positive cells, respectively. In conclusion, the neoplastic (lepidic) cells showed a distinct gene expression pattern and no consistent overlapping with endothelial and smooth muscle cells or cardiac myocytes; the expression of TIMP1 and MMP2 might be related to clinical presentation; larger series studies using also systematic transcriptome analysis might be useful to confirm the present results.

Regulation of calretinin in malignant mesothelioma is mediated by septin 7 binding to the CALB2 promoter.

BACKGROUND: The calcium-binding protein calretinin (gene name: CALB2) is currently considered as the most sensitive and specific marker for the diagnosis of malignant mesothelioma (MM). MM is a very aggressive tumor strongly linked to asbestos exposure and with no existing cure so far. The mechanisms of calretinin regulation, as well as its distinct function in MM are still poorly understood. METHODS: We searched for transcription factors binding to the CALB2 promoter and modulating calretinin expression. For this, DNA-binding assays followed by peptide shotgun-mass spectroscopy analyses were used. CALB2 promoter activity was assessed by dual-luciferase reporter assays. Furthermore, we analyzed the effects of CALB2 promoter-binding proteins by lentiviral-mediated overexpression or down-regulation of identified proteins in MM cells. The modulation of expression of such proteins by butyrate was determined by subsequent Western blot analysis. Immunohistochemical analysis of embryonic mouse lung tissue served to verify the simultaneous co-expression of calretinin and proteins interacting with the CALB2 promoter during early development. Finally, direct interactions of calretinin with target proteins were evidenced by co-immunoprecipitation experiments. RESULTS: Septin 7 was identified as a butyrate-dependent transcription factor binding to a CALB2 promoter region containing butyrate-responsive elements (BRE) resulting in decreased calretinin expression. Accordingly, septin 7 overexpression decreased calretinin expression levels in MM cells. The regulation was found to operate bi-directionally, i.e. calretinin overexpression also decreased septin 7 levels. During murine embryonic development calretinin and septin 7 were found to be co-expressed in embryonic mesenchyme and undifferentiated mesothelial cells. In MM cells, calretinin and septin 7 colocalized during cytokinesis in distinct regions of the cleavage furrow and in the midbody region of mitotic cells. Co-immunoprecipitation experiments revealed this co-localization to be the result of a direct interaction between calretinin and septin 7. CONCLUSIONS: Our results demonstrate septin 7 not only serving as a "cytoskeletal" protein, but also as a transcription factor repressing calretinin expression. The negative regulation of calretinin by septin 7 and vice versa sheds new light on mechanisms possibly implicated in MM formation and identifies these proteins as transcriptional regulators and putative targets for MM therapy.

Utility of cell block to detect malignancy in fluid cytology: Adjunct or necessity?

AIM OF THE STUDY: Cell block (CB) technique when supplemented with conventional smear, provides increased cellularity, preservation of architectural pattern with excellent morphology, and a clear background. We compare the utility of CB technique compared to conventional smear in detection of malignancy in serous effusions. MATERIALS AND METHODS: An institution-based observational and analytical study was carried out over 1 year on 50 patients with effusions. The residual amount of centrifuged deposit after preparation of conventional smear was mixed with 10% alcohol-formalin solution, and CBs were prepared. Calretinin and cytokeratin 5 were used for reactive mesothelial cells and Wilms tumor 1, thyroid transcription factor 1, CDX2, and estrogen receptor were used to confirm the adenocarcinoma cells. RESULTS: Maximum patients belonged to the age group of 61-70 years. Male:female ratio 1:1.17. Most common cause of malignant peritoneal effusion was due to ovarian malignancies in females and adenocarcinoma of stomach in males while, in case of pleural effusion, it was breast carcinoma in females and lung carcinoma in males. Thirteen suspicious cases were subjected to immunohistochemistry (IHC). In 70% cases, CB findings were consistent with the findings of conventional smears. In 20% cases, the conventional smears were suspicious for malignancy, and malignancy was confirmed by CB technique, whereas in 10% cases, both smears and CB were suspicious for malignancy and the original nature of the lesion was confirmed by the IHC. Sensitivity and specificity of CB compared to conventional smear were 88.88% and 86.98%, respectively. CONCLUSION: CB produced significantly better results (P = 0.0271) while detecting malignant lesions and reducing suspicious results (P = 0.0226).

The Identification of Interferon-γ as a Key Supportive Factor for Retinal Differentiation of Murine Mesenchymal Stem Cells.

Retinal disorders represent the main cause of decreased quality of vision and even blindness worldwide. The loss of retinal cells causes irreversible damage of the retina, and there are currently no effective treatment protocols for most retinal degenerative diseases. A promising approach for the treatment of retinal disorders is represented by stem cell-based therapy. The perspective candidates are mesenchymal stem cells (MSCs), which can differentiate into multiple cell types and produce a number of trophic and growth factors. In this study, we show the potential of murine bone marrow-derived MSCs to differentiate into cells expressing retinal markers and we identify the key supportive role of interferon-γ (IFN-γ) in the differentiation process. MSCs were cultured for 7 days with retinal extract and supernatant from T-cell mitogen concanavalin A-stimulated splenocytes, simulating the inflammatory site of retinal damage. MSCs cultured in such conditions differentiated to the cells expressing retinal cell markers such as rhodopsin, S antigen, retinaldehyde-binding protein, calbindin 2, recoverin, and retinal pigment epithelium 65. To identify a supportive molecule in the supernatants from activated spleen cells, MSCs were cultured with retinal extract in the presence of various T-cell cytokines. The expression of retinal markers was enhanced only in the presence of IFN-γ, and the supportive role of spleen cell supernatants was abrogated with the neutralization antibody anti-IFN-γ. In addition, differentiated MSCs were able to express a number of neurotrophic factors, which are important for retinal regeneration. Taken together, the results show that MSCs can differentiate into cells expressing retinal markers and that this differentiation process is supported by IFN-γ.

Regulation of retinal pigment epithelial cell phenotype by Annexin A8.

The retinoic acid derivative fenretinide (FR) is capable of transdifferentiating cultured retinal pigment epithelial (RPE) cells towards a neuronal-like phenotype, but the underlying mechanisms are not understood. To identify genes involved in this process we performed a microarray analysis of RPE cells pre- and post-FR treatment, and observed a marked down-regulation of AnnexinA8 (AnxA8) in transdifferentiated cells. To determine whether AnxA8 plays a role in maintaining RPE cell phenotype we directly manipulated AnxA8 expression in cultured and primary RPE cells using siRNA-mediated gene suppression, and over-expression of AnxA8-GFP in conjunction with exposure to FR. Treatment of RPE cells with AnxA8 siRNA recapitulated exposure to FR, with cell cycle arrest, neuronal transdifferentiation, and concomitant up-regulation of the neuronal markers calretinin and calbindin, as assessed by real-time PCR and immunofluorescence. In contrast, AnxA8 transient over-expression in ARPE-19 cells prevented FR-induced differentiation. Ectopic expression of AnxA8 in AnxA8-depleted cells led to decreased neuronal marker staining, and normal cell growth as judged by phosphohistone H3 staining, cell counting and cleaved caspase-3 levels. These data show that down-regulation of AnxA8 is both necessary and sufficient for neuronal transdifferentiation of RPE cells and reveal an essential role for AnxA8 as a key regulator of RPE phenotype.

Calretinin as a blood-based biomarker for mesothelioma.

BACKGROUND: Malignant mesothelioma (MM) is a deadly cancer mainly caused by previous exposure to asbestos. With a latency period up to 50 years the incidence of MM is still increasing, even in countries that banned asbestos. Secondary prevention has been established to provide persons at risk regular health examinations. An earlier detection with tumor markers might improve therapeutic options. Previously, we have developed a new blood-based assay for the protein marker calretinin. Aim of this study was the verification of the assay in an independent study population and comparison with the established marker mesothelin. METHODS: For a case-control study in men, a total of 163 cases of pleural MM and 163 controls were available from Australia, another 36 cases and 72 controls were recruited in Germany. All controls had asbestosis and/or plaques. Calretinin and mesothelin were determined by ELISA (enzyme-linked immunosorbent assay) in serum or plasma collected prior to therapy. We estimated the performance of both markers and tested factors potentially influencing marker concentrations like age, sample storage time, and MM subtype. RESULTS: Calretinin was able to detect all major subtypes except for sarcomatoid MM. Calretinin showed a similar performance in Australian and German men. At a pre-defined specificity of 95% the sensitivity of calretinin reached 71% and that of mesothelin 69%, when excluding sarcomatoid MM. At 97% specificity, the combination with calretinin increased the sensitivity of mesothelin from 66% to 75%. Sample storage time did not influence the results. In controls the concentrations of calretinin increased 1.87-fold (95% CI 1.10-3.20) per 10 years of age and slightly more for mesothelin (2.28, 95% CI 1.30-4.00). CONCLUSIONS: Calretinin could be verified as a blood-based marker for MM. The assay is robust and shows a performance that is comparable to that of mesothelin. Retrospective analyses would not be limited by storage time. The high specificity supports a combination of calretinin with other markers. Calretinin is specific for epithelioid and biphasic MM but not the rarer sarcomatoid form. Molecular markers like calretinin and mesothelin are promising tools to improve and supplement the diagnosis of MM and warrant further validation in a prospective study.

Interactions between calcium intake and polymorphisms in genes essential for calcium reabsorption and risk of colorectal neoplasia in a two-phase study.

The SLC8A1 (solute carrier family 8, member 1) gene, encoding Na+ /Ca2+ exchanger, is essential in regulating calcium reabsorption and homeostasis. Calcium homeostasis plays a key role in cell proliferation and apoptosis. We hypothesized that polymorphisms in five calcium-regulating genes (SLC8A1, ATP2B1, CALB1, CALB2, and CABP1) interact with calcium intake in relation to the risk of colorectal neoplasia. A two-phase (discovery and replication) study was conducted within the Tennessee Colorectal Polyp Study, including a total of 1275 cases and 2811 controls. In Phase I, we identified six out of 135 SNPs that significantly interacted with calcium intake in relation to adenoma risk. In Phase II, the calcium intake by rs4952490 (SLC8A1) interaction was replicated (Pinteraction = 0.048). We found an inverse association between calcium intake (1000-2000 mg/day) and colorectal adenomas, particularly for multiple/advanced adenomas, among the G-allele carriers but not among homozygous carriers of the common variant (A) in rs4952490. In the joint analysis of SLC8A1, KCNJ1, and SLC12A1 SNPs, carriers of variant alleles in at least two genes and with calcium intake above the DRI (1000 mg/day) were approximately 30-57% less likely to have adenomas than those whose calcium intake was below the DRI. The association was stronger for multiple/advanced adenomas. No association was found among those who did not carry any variant alleles in these genes when calcium intake was below 2500 mg/day. These findings, if confirmed, may provide a new avenue for the personalized prevention of colorectal adenoma and cancer.