Vlastimil Kulda

Relevance of miR-21 and miR-143 expression in tissue samples of colorectal carcinoma and its liver metastases

MicroRNAs, which are endogenously expressed regulatory noncoding RNAs, have an altered expression in colorectal cancer. The aim of our study was to assess the relationship of miR-21 and miR-143 expression to the prognostic/clinicopathological features of colorectal carcinoma (CRC) and colorectal liver metastases (CLM). The estimation was performed in 46 paired (tumor and control) tissue samples of CRC. Further, we studied 30 tissue samples of CLM. MiR-21 and miR-143 expressions were quantified by using the quantitative reverse transcription polymerase chain reaction method. Relation of miR-21 and miR-143 expression to disease-free interval (DFI) (Wilcoxon; P = 0.0026 and P = 0.0191, respectively) was recorded. There was shorter DFI in patients with a higher expression of miR-21 and, surprisingly, also in patients with a higher expression of miR-143, which is a putative tumor suppressor. There was a higher expression of miR-21 and lower expression of miR-143 in CRC tissue in comparison with adjacent normal colon tissue (P < 0.0001; P < 0.0001, respectively). Similarly, we observed a higher expression of miR-21 and a lower expression of miR-143 in CLM in comparison with normal colon tissue (P < 0.0001; P < 0.0001, respectively). Our results support the hypothesis about oncogenic function of miR-21 and show its relation to DFI. The role of miR-143 in carcinogenesis seems to be more complex.

May CTC technologies promote better cancer management

In the case of cancer, death is usually not due to the primary tumor itself but due to dissemination. Analysis of the circulating tumor cells (CTCs), i.e., cells responsible for a formation of metastases, should provide information useful for the management of cancer patients, fulfilling the objectives of predictive, preventive, and personalized medicine (PPPM). Despite promising results, the decisions on stage of disease and how to guide the adjuvant treatment still do not include results of CTC assessment. We want to describe two major reasons why the recent diagnostic value of CTC analysis is not sufficient for clinical use. The first reason arises from the biological nature of the tumor itself and the second reason is associated with an interdisciplinary status of CTC diagnostics in the sense that it is neither a theme purely for pathologists nor for haemato-oncologists nor clinical biochemists. We anticipate that there are at least three areas where CTCs can be useful for clinical practice. The first is monitoring of treatment efficacy of cancer patients. The second is a molecular characterization of captured CTCs for targeted treatment, and the third is a cultivation of captured CTCs for drug sensitivity testing. All of these approaches allow researchers recognize and respond to changes of phenotype of cancer cells during disease progression and introduce PPPM into clinical practice.

Intracerebellar application of P19-derived neuroprogenitor and naive stem cells to Lurcher mutant and wild type B6CBA mice

Summary Background Neurotransplantation has great potential for future treatments of various neurodegenerative disorders. Preclinically, the Lurcher mutant mouse represents an appropriate model of genetically-determined olivocerebellar degeneration. The aim of the present study was to assess survival of naïve and neurally differentiated P19 carcinoma stem cells following transplantation into the cerebellum of Lurcher mice and wild type littermates. Material/Methods Adult normal wild type (n=51) and Lurcher mutant mice (n=87) of the B6CBA strain were used. The mean age of the animals at the time of transplantation was 261.5 days. Suspension of naive and neurally differentiated P19 carcinoma stem cells was injected into the cerebellum of the mice. In the Lurcher mutants, 2 depths of graft injection were used. Three weeks after implantation the brains of experimental animals were examined histologically. Results Survival of neuroprogenitor grafts at a depth of 1.6 mm was significantly higher in wild type vs. Lurcher mutant mice. In wild type mice, the typical graft localization was in the middle of the cerebellum, whereas in Lurcher mice the graft was never found inside the degenerated cerebellum and was primarily localized in the mesencephalon. Conclusions We conclude that the appearance and low survival rate of cerebellar P19 carcinoma stem cell grafts in the Lurcher mutant mice weigh against the therapeutic value of this cell line in preclinical studies of neurodegeneration.

Diagnostic and Prognostic Value of microRNA-21 in Colorectal Cancer: An Original Study and Individual Participant Data Meta-analysis

Background: We aimed to systematically summarize the diagnostic and prognostic value of circulating/tissue miR21 in patients with colorectal cancer. Methods: An original study was conducted to explore the potential value of circulating miR21 in colorectal cancer diagnosis and tissue miR21 in colorectal cancer prognosis. PUBMED and EMBASE were searched (to August, 2013) to identify eligible studies. To explore the diagnostic performance of circulating miR21, meta-analysis methods were used to pool sensitivity, specificity, positive and negative likelihood ratio, diagnostic OR and to construct a summary ROC curve. For prognostic meta-analysis, study-specific HRs of tissue miR21 for survival were summarized. Subgroup and sensitivity analyses were applied to explore heterogeneity. Results: Finally, 14 studies (including our study) were included in the meta-analyses. The pooled sensitivity, specificity, and AUC of circulating miR21 were 0.76 [95% confidence interval (CI), 0.59–0.88], 0.81 (95% CI, 0.76–0.85), and 0.81 (95% CI, 0.78–0.85) in diagnosing colorectal cancer. Patients with higher expression of tissue miR21 had significant inferior overall survival (OS; pooled HR, 1.56; 95% CI, 1.16–2.11) and disease-free survival (DFS; pooled HR, 1.35; 95% CI, 1.08–1.69). The individual participant data (IPD) meta-analysis demonstrated that tissue miR21 level was independently associated with worse colorectal cancer OS (HR, 1.69; 95% CI, 1.07–2.67; P = 0.023), whereas this association seems to be confined to males (P = 0.007) but not for females (P = 0.845). Conclusions: Circulating miR21 level has potential value for colorectal cancer early detection, whereas high tissue miR21 level is associated with adverse colorectal cancer prognosis. Impact: miR21 is a promising biomarker for early detection and prognosis of colorectal cancer. Cancer Epidemiol Biomarkers Prev; 23(12); 2783–92. ©2014 AACR.

Morphological analysis of embryonic cerebellar grafts in SCA2 mice.

SCA2 transgenic mice are thought to be a useful model of human spinocerebellar ataxia type 2. There is no effective therapy for cerebellar degenerative disorders, therefore neurotransplantation could offer hope. The aim of this work was to assess the survival and morphology of embryonic cerebellar grafts transplanted into the cerebellum of adult SCA2 mice. Four month-old homozygous SCA2 and negative control mice were treated with bilateral intracerebellar injections of an enhanced green fluorescent protein-positive embryonic cerebellar cell suspension. Graft survival and morphology were examined three months later. Graft-derived Purkinje cells and the presence of astrocytes in the graft were detected immunohistochemically. Nissl and hematoxylin-eosin techniques were used to visualize the histological structure of the graft and surrounding host tissue. Grafts survived in all experimental mice; no differences in graft structure, between SCA2 homozygous and negative mice, were found. The grafts contained numerous Purkinje cells but long distance graft-to-host axonal connections to the deep cerebellar nuclei were rarely seen. Relatively few astrocytes were found in the center of the graft. No signs of inflammation or tissue destruction were seen in the area around the grafts. Despite good graft survival and the presence of graft-derived Purkinje cells, the structure of the graft did not seem to promise any significant specific functional effects. We have shown that the graft is available for long-term experiments. Nevertheless, it would be beneficial to search for ways of enhancement of connections between the graft and host.

Comparison of fibroblast and osteoblast response to cultivation on titanium implants with different grain sizes

The in vitro response of human fibroblast cell line HFL1 and human osteoblast cell line hFOB 1.19 on nanostructured titanium with different grain sizes has been compared in the present study. Used samples of titanium produced by equal channel angular (ECA) pressing have grain sizes of 160 nm, 280 nm, and 2400 nm with cross- and longitudinal sections. Similar cellular behaviour was observed on all studied biomaterials. There were significant differences related to the initial phase of attachment, but not in proliferation. Furthermore, the results indicate that osteoblasts grow best on material with grain size of 160nm with a longitudinal section in comparison with other examined materials. Therefore, this material could be recommended for further evaluation with respect to osseointegration in vivo.

Differentiated expression of the lactate dehydrogenase subunit M in pleural fluids of neoplastic aetiology.

Objective. An anaerobic type of glycolysis exemplified by hyperproduction of the lactate dehydrogenase (LDH) subunit M has been detected in lung tumours, while a similar pattern has been found in concomitant pleural effusions (PE). The aim of this study was to verify the presence of the LDH subunit M in PEs of different aetiology and to compare its expression with markers of inflammation. Material and methods. LDH isoenzymes were estimated and the LDH5/LDH1 coefficient was calculated in paraneoplastic PEs (n = 99), including subgroups with a different tumour ultrastructure, origin and pleural involvement. The expression pattern was compared with parainflammatory PEs (n = 21), transudates (n = 16) and with the expression of 13 inflammatory markers in PEs. Results. The LDH5/LDH1 coefficient was higher in PEs associated with non‐small‐cell lung cancer (NSCLC) and with pleura‐invading tumours, and lower in PEs of small‐cell lung cancer and tumours without a confirmed pleural involvement. The LDH5/LDH1 coefficient positively correlated with uPA, IL‐8, IL‐10, sICAM, sVCAM, MPO and MMP‐9. Conclusions. In accordance with inflammatory markers, it appears that the expression of LDH and its isoenzymes in PEs reflects the host reaction in pleural space and, in NSCLC, may also feature the anaerobic phenotype of cancer cells.

Follicular fluid levels of anti-Müllerian hormone, insulin-like growth factor 1 and leptin in women with fertility disorders

ABSTRACT Anti-Müllerian hormone (AMH), insulin-like growth factor 1 (IGF1) and leptin are produced in the granulosa cells of follicles and play an important role in the growth and maturation of follicles. The aim of our study was to monitor AMH, IGF1 and leptin levels in a group of healthy women and compare them to a group of women with fertility disorders. The second aim was the evaluation of biomarker levels in relation to the identified cause of infertility. Totally, 146 females were enrolled into our study. Seventy-two healthy controls and seventy-four females with fertility disorders were divided into four subgroups: anovulation, endometriosis, fallopian tube damage, unknown reason. IGF1 was the only biomarker with significantly lower levels throughout the entire group with fertility disorders. We did not identify any statistically significant differences for AMH and leptin. Regarding subgroups, significant differences were only observed in the group of anovulatory women. AMH and leptin showed higher levels while IGF1 showed lower levels. In conclusion, levels of AMH, IGF1 and leptin found in follicular fluid are sensitive markers for anovulatory fertility disorders. AMH, IGF1 and leptin levels in follicular fluid have no relation to the fertility disorders caused by endometriosis, fallopian tube damage or disorders with unknown etiology. Abbreviations: AMH: anti-Müllerian hormone; IGF1: insulin-like growth factor 1; PCOS: polycystic ovary syndrome

Trends in gene expression changes during adipogenesis in human adipose derived mesenchymal stem cells under dichlorodiphenyldichloroethylene exposure

BackgroundsExposure to lipophilic environmental pollutants has been explored as a risk factor of development of diabetes mellitus in obese. Adipose tissue is a reservoir of bioaccumulative lipophilic contaminants including p,pʹ-dichlorodiphenyldichloroethylene (DDE). Our aim was to analyze the effect of DDE (in concentrations 0.1 μM, 1 μM, and 10 μM) on adipocyte differentiation and insulin signalling pathway on in vitro adipogenic model of human adipose derived mesenchymal stem cells (hADMSC).MethodsThe effect of DDE was monitored by analysis of expression (RT qPCR, Western blotting) of genes involved in adipocyte differentiation and insulin signalling pathway including lipid metabolism on days 0, 4, 10, 21, 28 of differentiation.ResultsThe main observation was significant increase of INSR, LIPE, FASN, SREBP1, OCT4 and AKT2 expression under influence of DDE. We did not record any increase of the active form of Akt.ConclusionOur findings suggest that DDE exposure changes the differentiation of adipocytes, enhances the lipid metabolism and so may play a role in the development of obesity and metabolic diseases by affecting the insulin signalling pathway. The influence of DDE seems to be similar to the effect of insulin itself. However, further studies to elucidate the mechanism of action of DDE are necessary.

Proliferation of Osteoblasts on Laser-Modified Nanostructured Titanium Surfaces

Nanostructured titanium has become a useful material for biomedical applications such as dental implants. Certain surface properties (grain size, roughness, wettability) are highly expected to promote cell adhesion and osseointegration. The aim of this study was to compare the biocompatibilities of several titanium materials using human osteoblast cell line hFOB 1.19. Eight different types of specimens were examined: machined commercially pure grade 2 (cpTi2) and 4 (cpTi4) titanium, nanostructured titanium of the same grades (nTi2, nTi4), and corresponding specimens with laser-treated surfaces (cpTi2L, cpTi4L, nTi2L, nTi4L). Their surface topography was evaluated by means of scanning electron microscopy. Surface roughness was measured using a mechanical contact profilometer. Specimens with laser-treated surfaces had significantly higher surface roughness. Wettability was measured by the drop contact angle method. Nanostructured samples had significantly higher wettability. Cell proliferation after 48 hours from plating was assessed by viability and proliferation assay. The highest proliferation of osteoblasts was found in nTi4 specimens. The analysis of cell proliferation revealed a difference between machined and laser-treated specimens. The mean proliferation was lower on the laser-treated titanium materials. Although plain laser treatment increases surface roughness and wettability, it does not seem to lead to improved biocompatibility.