Bone Marrow-Resident Vδ1 T Cells Co-express TIGIT With PD-1, TIM-3 or CD39 in AML and Myeloma.

Background: γδ T cells represent a unique T cell subpopulation due to their ability to recognize cancer cells in a T cell receptor- (TCR) dependent manner, but also in a non-major histocompatibility complex- (MHC) restricted way via natural killer receptors (NKRs). Endowed with these features, they represent attractive effectors for immuno-therapeutic strategies with a better safety profile and a more favorable anti-tumor efficacy in comparison to conventional αß T cells. Also, remarkable progress has been achieved re-activating exhausted T lymphocytes with inhibitors of co-regulatory receptors e.g., programmed cell death protein 1 (PD-1), T cell immunoreceptor with Ig and ITIM domains (TIGIT) and of the adenosine pathway (CD39, CD73). Regarding γδ T cells, little evidence is available. This study aimed to immunophenotypically characterize γδ T cells from patients with diagnosed acute myeloid leukemia (AML) in comparison to patients with multiple myeloma (MM) and healthy donors (HD). Methods: The frequency, differentiation, activation, and exhaustion status of bone marrow- (BM) derived γδ T cells from patients with AML (n = 10) and MM (n = 11) were assessed in comparison to corresponding CD4+ and CD8+ T cells and peripheral blood- (PB) derived γδ T cells from HDs (n = 16) using multiparameter flow cytometry. Results: BM-infiltrating Vδ1 T cells showed an increased terminally differentiated cell population (TEMRAs) in AML and MM in comparison to HDs with an aberrant subpopulation of CD27-CD45RA++ cells. TIGIT, PD-1, TIM-3, and CD39 were more frequently expressed by γδ T cells in comparison to the corresponding CD4+ T cell population, with expression levels that were similar to that on CD8+ effector cells in both hematologic malignancies. In comparison to Vδ2 T cells, the increased frequency of PD-1+-, TIGIT+-, TIM-3+, and CD39+ cells was specifically observed on Vδ1 T cells and related to the TEMRA Vδ1 population with a significant co-expression of PD-1 and TIM-3 together with TIGIT. Conclusion: Our results revealed that BM-resident γδ T cells in AML and MM express TIGIT, PD-1, TIM-3 and CD39. As effector population for autologous and allogeneic strategies, inhibition of co-inhibitory receptors on especially Vδ1 γδ T cells may lead to re-invigoration that could further increase their cytotoxic potential.

Increased cathepsin D protein expression is a biomarker for osteosarcomas, pulmonary metastases and other bone malignancies.

Cancer proteomics provide a powerful approach to identify biomarkers for personalized medicine. Particularly, biomarkers for early detection, prognosis and therapeutic intervention of bone cancers, especially osteosarcomas, are missing. Initially, we compared two-dimensional gel electrophoresis (2-DE)-based protein expression pattern between cell lines of fetal osteoblasts, osteosarcoma and pulmonary metastasis derived from osteosarcoma. Two independent statistical analyses by means of PDQuest® and SameSpot® software revealed a common set of 34 differentially expressed protein spots (p < 0.05). 17 Proteins were identified by mass spectrometry and subjected to Ingenuity Pathway Analysis resulting in one high-ranked network associated with Gene Expression, Cell Death and Cell-To-Cell Signaling and Interaction. Ran/TC4-binding protein (RANBP1) and Cathepsin D (CTSD) were further validated by Western Blot in cell lines while the latter one showed higher expression differences also in cytospins and in clinical samples using tissue microarrays comprising osteosarcomas, metastases, other bone malignancies, and control tissues. The results show that protein expression patterns distinguish fetal osteoblasts from osteosarcomas, pulmonary metastases, and other bone diseases with relevant sensitivities between 55.56% and 100% at ≥87.50% specificity. Particularly, CTSD was validated in clinical material and could thus serve as a new biomarker for bone malignancies and potentially guide individualized treatment regimes.

Aneuploidy-associated gene expression signatures characterize malignant transformation in ulcerative colitis.

BACKGROUND: Malignant transformation in ulcerative colitis (UC) is associated with pronounced chromosomal instability, reflected by aneuploidy. Although aneuploidy can precede primary cancer diagnosis in UC for more than a decade, little is known of its cellular consequences. METHODS: Whole-genome gene expression analysis was applied to noninflamed colon mucosa, mucosal biopsies of patients with UC, and UC-associated carcinomas (UCCs). DNA image cytometry was used to stratify samples into ploidy types. Differentially expressed genes (DEGs) were analyzed by Ingenuity Pathway Analysis and validated by real-time quantitative PCR. RESULTS: Gene expression changes were more pronounced between normal mucosa and UC (2587 DEGs) than between UC and UCC (827 DEGs). Cytometry identified colitis patients with euploid or aneuploid mucosa biopsies, whereas all UCCs were aneuploid. However, 1749 DEGs distinguished euploid UC and UCCs, whereas only 15 DEGs differentiated aneuploid UC and UCCs. A total of 16 genes were differentially expressed throughout the whole sequence from normal controls to UCCs. Particularly, genes pivotal for chromosome segregation (e.g., SMC3 and NUF2) were differentially regulated along aneuploidy development. CONCLUSIONS: The high number of DEGs between normal mucosa and colitis is dominated by inflammatory-associated genes. Subsequent acquisition of aneuploidy leads to subtle but distinct transcriptional alterations, revealing novel target genes that drive genomic instability and thus carcinogenesis. The gene expression signature of malignant phenotypes in aneuploid UC suggests that these lesions might need to be considered as severe as high-grade dysplasia.

CYP26A1-specific antagonist influence on embryonic implantation, gene expression and endogenous retinoid concentration in rats.

Retinoids are essential in vertebrate reproduction and embryonic development. All-trans-retinoic acid (ATRA) is tightly regulated during these processes. CYP26A1 is mainly responsible for its degradation. To study the role of CYP26A1 during implantation, we applied R115866, a CYP26A1-specific antagonist, to rats during early gestation days (GD). On GD 6.5 and 12 samples were collected and the number of embryos was evaluated. ATRA concentration increased in uterus and serum, mRNA expression of CYP26A1 and CRABP2 increased in the liver, but not in the uterus. Uterine COX1 and 17ßHSD mRNA expression was decreased. The number of embryos on GD 12 was not altered in this setting. It can be concluded that uterine expression of the analyzed retinoid-response genes during early gestation is not altered by this R115866 treatment and instead indirectly via ATRA. From our experiment we cannot confirm that ATRA obtains a major influencing role in the regulation of embryonic implantation.

High Frequency of Aneuploidy Defines Ulcerative Colitis-Associated Carcinomas: A Prognostic Comparison to Sporadic Colorectal Carcinomas.

OBJECTIVE: Aneuploidy is an independent risk factor for forthcoming carcinogenesis in ulcerative colitis (UC). An inferior prognosis of patients with ulcerative colitis-associated colorectal cancer (UCC) compared with those with sporadic colorectal cancer (SCC) has been reported, but remains controversial. This prompted us to investigate if aneuploidy can be observed in UCCs as frequently as in their sporadic counterpart and if aneuploidy per se might be a driving feature of poor prognosis in UCC. BACKGROUND DATA: We obtained clinical follow-up for 257 SCC patients (average observation time 57 months) and 31 UCC patients (51 months). Touch preparation slides or tissue sections were prepared of all 288 carcinomas for ploidy analysis. METHODS: Ploidy status was assessed for 260 SCCs and 31 UCCs by image cytometry and correlated to clinical features. Survival data were analyzed using Kaplan-Meier estimates. RESULTS: Aneuploidy was detected in 74.6% of SCCs and in all 31 UCCs. Logistic regression analysis yielded age (odds ratio [OR], 1.05; 95% CI, 1.02-1.09; P = 0.003) and aneuploidy (OR, 4.07; 95% CI, 1.46-11.36; P = 0.007) as independent prognostic factors for R0-resected patients devoid of metastases. Diploid SCCs had a more favorable 5-year survival (88.2%) than aneuploid SCCs (69.0%) and UCCs (73.1%) (P = 0.074). CONCLUSIONS: UC-associated carcinomas presented aneuploidy at significantly higher frequency than sporadic colorectal carcinomas (P < 0.0006). UCCs and aneuploid SCCs share a similar prognosis inferior to that of diploid SCCs. Aneuploidy proved to be the strongest independent prognostic marker for R0-resected colorectal cancer patients overall.

Retinoid receptor-activating ligands are produced within the mouse thymus during postnatal development.

Vitamin A deficiency is known to be accompanied with immune deficiency and susceptibility to a wide range of infectious diseases. Experimental evidence suggests that the active metabolites of vitamin A that mediate its effects on the immune system are the retinoic acids (RA), which are ligands for the nuclear RA receptor (RAR) family. RA were previously shown both to promote proliferation and to regulate apoptosis of thymocytes. In this study we detected the age-dependent mRNA expression of retinaldehyde dehydrogenases (RALDH1 and 2), cellular RA binding protein-II and CYP26A, proteins responsible for the synthesis, nuclear transport and degradation of RA in the postnatally developing thymus. RALDH1 was located in thymic epithelial cells. However, the amount of all-trans RA in thymic homogenates was close to the detection limit, suggesting that in this tissue all-trans RA is not the main RAR-regulating product of retinol metabolism. At the same time, by measuring the induction of a RAR-responsive transgene in two independent transgenic mouse strains, we demonstrated the production of an RAR-activating ligand, which was age and RALDH dependent. Our data provide evidence for the existence of endogenous retinoid synthesis in the thymus and suggest that retinoids similar to glucocorticoids might indeed be involved in the regulation of thymic proliferation and selection processes by being present in the thymus in functionally effective amounts.

From the genome to the proteome--biomarkers in colorectal cancer.

BACKGROUND AND AIMS: Colorectal cancer is the second leading cause of cancer-related death. Current clinical practice in colorectal cancer screening (fecal occult blood test, FOBT; colonoscopy) has contributed to a reduction of mortality. However, despite these screening programs, about 70% of carcinomas are detected at advanced tumor stages (UICC III/IV) presenting poor patient prognosis. Thus, innovative tools and methodologies for early cancer detection can directly result in improving patient survival rates. PATIENTS/METHODS: Biomedical research has advanced rapidly in recent years with the availability of technologies such as global gene and protein expression profiling. Comprehensive tumor profiling has become a field of intensive research aiming at identifying biomarkers relevant for improved diagnostics and therapeutics. RESULTS: In this paper, we report a comprehensive review of genomic, transcriptomic, and proteomic approaches for biomarker identification in tissue and blood with a main emphasis on two-dimensional gel-electrophoresis (2-DE) and mass spectrometry analyses. CONCLUSION: Proteomics-based technologies enable to distinguish the healthy patient from the tumor patient with high sensitivity and specificity and could greatly improve common classification systems and diagnostics. However, this progress has not yet been transferred from bench to bedside but could open the door to a more accurate and target specific personalized medicine with improved patient survival.

Regulation of expression of the retinoic acid metabolizing enzyme CYP26A1 in uteri of ovariectomized mice after treatment with ovarian steroid hormones.

The retinoic acid (RA) synthesizing enzymes, retinaldehyde dehydrogenases (RALDH), are expressed in specific spatial and temporal patterns in uterine tissues during estrous cycle and early pregnancy in mice. Expression of RALDH1 and 2 has been shown to be induced by estrogen treatment within the uterus. In this study, we determined the influence of progesterone and 17-ss-estradiol on the uterine expression of the RA-metabolizing enzyme CYP26A1 after specific time intervals (1, 4, 24, and 48 hr after treatment of ovariectomized mice). In a following experiment, we investigated the influence of gestagen (promegestone 0.3 mg/kg body weight), estrogen (estradiol 3 microg/kg), their combination, as well as the antagonizing anti-progesterone hormone (RU 486 10 mg/kg) on the uterine expression of CYP26A1. Expression of CYP26A1 was localized using in situ hybridization and quantified using RT-PCR. CYP26A1 mRNA expression was strongly--although transiently--induced in uterine endometrial epithelial and glandular cells after administration of gestagen or the combination of gestagen + estrogen, but not by estrogen alone. These observations were confirmed by semi-quantitative RT-PCR experiments on whole uteri. Thus, we show that the expression of CYP26A1 in endometrial epithelial cells is regulated by progesterone and not significantly influenced by co-administration of estrogen. These data indicate an additional level of hormonal control of endogenous RA levels in the mouse uterus, where its synthesis would rely on estrogen-dependent expression of RALDH enzymes, whereas its active metabolism would be triggered by progesterone-induced CYP26A1 expression.

Regulation of expression of the retinoic acid-synthesising enzymes retinaldehyde dehydrogenases in the uteri of ovariectomised mice after treatment with oestrogen, gestagen and their combination.

The active metabolite of vitamin A, retinoic acid (RA), plays an important role in the female reproductive system. The synthesis of RA is tightly regulated by the activity of retinaldehyde dehydrogenases (Raldh). Among these, Raldh1 and Raldh2 exhibit specific temporal and spatial expression patterns in the mouse uterus, both during the oestrous cycle and early pregnancy. In the present study, we have assessed whether oestradiol and progesterone directly influence the uterine expression of Raldh1 and Raldh2 in ovariectomised mice. We investigated the effect of gestagen (promegestone 0.3 mg kg(-1) bodyweight), oestrogen (oestradiol 3 microg kg(-1) bodyweight) and their combination on the uterine expression of Raldh2. Expression was analysed using in situ hybridisation and quantified using real-time detection reverse transcription-polymerase chain reaction. The results show that the expression of Raldh2 is rapidly (within 1-4 h) induced in stromal cells by oestrogen, but not by gestagen, treatment, whereas combined oestrogen + gestagen treatment leads to a more prolonged (48 h) response. In contrast, oestrogen, but not progesterone, treatment downregulates (within 4-24 h) Raldh1 expression in the uterine glandular epithelium. We conclude that the uterine RA concentrations are regulated by oestrogens via an effect on the expression of the Raldh synthesising enzymes. Such a regulation is consistent with the natural fluctuations of Raldh expression during the oestrous cycle, early pregnancy and blastocyst implantation.