Deregulation of Selected MicroRNAs in Sinonasal Squamous Cell Carcinoma: Searching for Potential Prognostic Biomarkers.

Sinonasal carcinomas are head and neck tumours arising from the nasal cavity and paranasal sinuses characterized by unfavourable outcome, difficult treatment, diagnosis and prognosis. MicroRNAs are key molecules in the regulation of development and progression of cancer and their expression profiles could be used as prognostic biomarkers, to predict the patients' survival and response to treatment. In this study, we used quantitative real­time PCR with TaqMan® Advanced miRNA Assays to investigate the relative expression values of selected micro- RNAs in a unique set of formalin-fixed paraffin-embedded tissue samples obtained from 46 patients with sinonasal squamous cell carcinoma. Our results showed statistically significant up-regulation of three mature microRNAs: miR-9-5p (fold change: 6.80), miR-9-3p (fold change: 3.07) and let-7d (fold change: 3.93) in sinonasal carcinoma patients. Kaplan-Meier survival analysis and logrank test identified association between higher expression of miR-9-5p and longer survival of the patients (P = 0.0264). Lower expression of let-7d was detected in the patients with impaired survival, and higher expression of miR-137 was linked to shorter survival of the patients. We also identified several correlations between expression of the studied microRNAs and recorded clinicopathological data. Higher expression of miR-137 and lower expression of let-7d correlated with local recurrence (P = 0.045 and P = 0.025); lower expression of miR-9-5p and higher expression of miR-155-5p correlated with regional recurrence (P = 0.045 and P = 0.036). Higher expression of miR-9-3p correlated with occupational risk (P = 0.031), presence of vascular invasion (P = 0.013) and perineural invasion (P = 0.031). Higher expression of miR-155-5p was present in the samples originating from maxillary sinus (P = 0.011), cN1-3 classified tumours (P = 0.009) and G2-3 classified tumours (P = 0.017). In conclusion, our study supports the hypothesis of future prospect to use expression of miRNAs as prognostic biomarkers of squamous cell sinonasal carcinoma. In particular, miR-9-5p and miR-9-3p seem to be important members of the sinonasal cancer pathogenesis.

Analysis of DNA methylation and microRNA expression in NUT (nuclear protein in testis) midline carcinoma of the sinonasal tract: a clinicopathological, immunohistochemical and molecular genetic study.

The aim of this study was a detailed clinicopathological investigation of sinonasal NUT midline carcinoma (NMC), including analysis of DNA methylation and microRNA (miRNA) expression. Three (5%) cases of NMC were detected among 56 sinonasal carcinomas using immunohistochemical screening and confirmed by fluorescence in situ hybridization. The series comprised 2 males and 1 female, aged 46, 60, and 65 years. Two tumors arose in the nasal cavity and one in the maxillary sinus. The neoplasms were staged pT1, pT3, and pT4a (all cN0M0). All patients were treated by radical resection with adjuvant radiotherapy. Two patients died 3 and 8 months after operation, but one patient (pT1 stage; R0 resection) experienced no evidence of disease at 108 months. Microscopically, all tumors consisted of infiltrating nests of polygonal cells with vesicular nuclei, prominent nucleoli and basophilic cytoplasm. Abrupt keratinization was present in only one case. Immunohistochemically, there was a diffuse expression of cytokeratin (CK) cocktail, CK7, p40, p63, and SMARCB1/INI1. All NMCs tested negative for EBV and HPV infection. Two NMCs showed methylation of RASSF1 gene. All other genes (APC, ATM, BRCA1, BRCA2, CADM1, CASP8, CD44, CDH13, CDKN1B, CDKN2A, CDKN2B, CHFR, DAPK1, ESR1, FHIT, GSTP1, HIC1, KLLN, MLH1a, MLH1b, RARB, TIMP3, and VHL) were unmethylated. All NMCs showed upregulation of miR-9 and downregulation of miR-99a and miR-145 and two cases featured also upregulation of miR-21, miR-143, and miR-484. In summary, we described three cases of sinonasal NMCs with novel findings on DNA methylation and miRNA expression, which might be important for new therapeutic strategies in the future.

Importance of Tumour Suppressor Gene Methylation in Sinonasal Carcinomas.

Epigenetic changes are considered to be a frequent event during tumour development. Hypermethylation of promoter CpG islands represents an alternative mechanism for inactivation of tumour suppressor genes, DNA repair genes, cell cycle regulators and transcription factors. The aim of this study was to investigate promoter methylation of specific genes in samples of sinonasal carcinoma by comparison with normal sinonasal tissue. To search for epigenetic events we used methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) to compare the methylation status of 64 tissue samples of sinonasal carcinomas with 19 control samples. We also compared the human papilloma virus (HPV) status with DNA methylation. Using a 20% cut-off for methylation, we observed significantly higher methylation in RASSF1, CDH13, ESR1 and TP73 genes in the sinonasal cancer group compared with the control group. HPV positivity was found in 15/64 (23.4 %) of all samples in the carcinoma group and in no sample in the control group. No correlation was found between DNA methylation and HPV status. In conclusion, our study showed that there are significant differences in promoter methylation in the RASSF1, ESR 1, TP73 and CDH13 genes between sinonasal carcinoma and normal sinonasal tissue, suggesting the importance of epigenetic changes in these genes in carcinogenesis of the sinonasal area. These findings could be used as prognostic factors and may have implications for future individualised therapies based on epigenetic changes.

The use of a hydrogel matrix for controlled delivery of niacin to the gastrointestinal tract for treatment of hyperlipidemia.

Hyperlipidemia treatment based on niacin requires gastrointestinal administration of relatively high doses. The recommended dietary allowance of niacin as vitamin B3 is 14 to 16 mg daily in adults, while the doses of niacin used in the treatment of hyperlipidemia are generally in the range of 1 to 3 g. Administration of such large doses requires a high concentration of the active compound in the tablet and proper control of the drug release. In this study, a hydrogel matrix based on poly(2-hydroxyethyl methacrylate) and polyvinylpyrrolidone was investigated as delivery vehicle for controlled NA release into the gastrointestinal environment. The prepared hydrogel matrices varied in used monomer and crosslinker types and concentrations. The content of NA in tablets was between 65-80 %. The release profiles of NA from tablets were examined under three different pH values (1, 4.5 and 6.8) over the time period of 30 h. The effects of the monomer ratio, the crosslinking of the polymer network, and the solubility of niacin during drug release under various pH are discussed. The results showed that the release time period can be achieved in a relatively wide range of time and can be adjusted according to the medical requirements.

A comparison of portal vein embolization with poly(2-hydroxyethylmethacrylate) and a histoacryl/lipiodol mixture in patients scheduled for extended right hepatectomy.

To determine whether PHEMA [poly(2-hydroxyethylmethacrylate)] is suitable for portal vein embolization in patients scheduled to right hepatectomy and whether it is as effective as the currently used agent (a histoacryl/lipiodol mixture). Two groups of nine patients each scheduled for extended right hepatectomy for primary or secondary hepatic tumor, had right portal vein embolization in an effort to induce future liver remnant (FLR) hypertrophy. One group had embolization with PHEMA, the other one with the histoacryl/lipiodol mixture. In all patients, embolization was performed using the right retrograde transhepatic access. Embolization was technically successful in all 18 patients, with no complication related to the embolization agent. Eight patients of either group developed FLR hypertrophy allowing extended right hepatectomy. Likewise, one patient in each group had recanalization of a portal vein branch. Histology showed that both embolization agents reach the periphery of portal vein branches, with PHEMA penetrating somewhat deeper into the periphery. PHEMA has been shown to be an agent suitable for embolization in the portal venous system comparable with existing embolization agent (histoacryl/lipiodol mixture).

Intraosseous and intravenous administration of antibiotics yields comparable plasma concentrations during experimental septic shock.

BACKGROUND: We aimed to investigate whether comparable antibiotic concentrations could be reached with intraosseous and intravenous administration during septic shock. METHODS: In this randomized, prospective experimental study conducted at an animal research laboratory at the University Hospital of Uppsala, eight anesthetized pigs, weighing 21.2 to 29.1 kg (mean: 25.2 ± 2.3 kg), received endotoxin infusion at 4 µg/kg/h for 6 h. At the onset of clinical shock, alternatively after 3 h of endotoxemia, they received 75 mg/kg of cefotaxime and 7 mg/kg of gentamicin either in a proximal tibial intraosseous catheter or in a peripheral intravenous catheter. Mixed venous samples were taken after 5, 15, 30, 60, 120 and 180 min and analyzed for antibiotic concentrations. RESULTS: For both antibiotics, plasma concentrations after intraosseous and intravenous administration followed similar curves throughout the observation period, and peak concentrations were comparable. Mean concentration area under the curve (AUC mg × h/l) for cefotaxime was 108.1 ± 19.5 after intraosseous and 116.5 ± 11.1 after intravenous administration; ratio 0.93, (95% CI 0.71-1.19). Mean AUC for gentamicin was 28.1 ± 6.8 for intraosseous and 32.2 ± 3.5 for intravenous administration; ratio 0.87 (95% CI 0.62-1.19). CONCLUSIONS: In this porcine septic shock model, intraosseous and intravenous administration of gentamicin and cefotaxime yielded comparable concentrations. In an emergency, intraosseous administration of these antibiotics may be considered in severe infections when venous access is difficult.

3D microscopic imaging and evaluation of tubular tissue architecture.

3D microscopy and image analysis provide reliable measurements of length, branching, density, tortuosity and orientation of tubular structures in biological samples. We present a survey of methods for analysis of large samples by measurement of local differences in geometrical characteristics. The methods are demonstrated on the structure of the capillary bed in a rat brain.

Type 2 diabetes is associated with vertebral fractures in a sample of clinic- and hospital-based Latinos.

Latinos are the fastest growing ethnic population in the United States and type 2 diabetes is a major health burden in this population, but little effort has been made to study the prevalence of diabetic vertebral fragility in Latinos. We performed a cross-sectional study to determine vertebral fracture prevalence in a hospital-based population of South Texas residents (N = 296). We defined fractures in X-rays as a >20% reduction in vertebral body height. Numerous variables were recorded, including age, body mass index, indicators of diabetes management and others. 71% of the sample (N = 296) was Latino. The prevalence of vertebral fracture was increased in diabetic subjects relative to non-diabetic subjects (diabetic 27.9%, non-diabetic 13.8%) and, regardless of sex and diabetics status, decreased in Latinos relative to non-Latinos (Latino 16.7%, non-Latino 26.4%). These data suggest that vertebral fractures may be a growing concern for diabetic Latinos as well as diabetics of any racial/ethnic background.

Concordance of psychiatric symptom ratings between a subject and informant, relevancy to post-mortem research.

Investigators are interested in determining whether lifetime behavioral traits and specific mood states experienced close to death affect brain gene and protein expression as assessed in post-mortem human brains. Major obstacles to conducting this type of research are the uncertain reliability of the post-mortem psychiatric diagnoses and clinical information because of the retrospective nature of the information. In this study, we addressed the concordance of clinical information obtained through an informant compared with information obtained through a clinician interview of the subject. To test this, we measured both lifetime and within the week psychiatric symptoms of subjects (n=20) and an informant, their next-of-kin (n=20) who were asked identical questions. We found Diagnostic and Statistical Manual (DSM)-IV axis 1 diagnoses by Mini-International Neuropsychiatric Interview proportion of positive agreement for major depression was 0.97, bipolar disorder was 0.81, whereas proportion of negative agreement was 0.97 for schizophrenia. Symptom scale intra-class correlation coefficients and 95% confidence interval were: Bipolar Inventory of Signs and Symptoms=0.59 (0.23, 0.81), Brief Psychiatric Rating Scale=0.58 (0.19, 0.81), Hamilton Depression Rating Scale=0.44 (0.03, 0.72), Montgomery Asberg Depression Rating Scale=0.44 (0.03, 0.72), Young Mania Rating Scale=0.61 (0.30, 0.82), Barratt Impulsiveness Score=0.36 (-0.11, 0.70) and Childhood Trauma Questionnaire=0.48 (-0.15, 0.83). We show that DSM-IV diagnoses; lifetime impulsivity severity, childhood trauma score and symptom scores were significantly consistent between the subjects and their informants. These data suggest, with some limitations, that both retrospective and informant obtained information can provide useful clinical information in post-mortem research.

Protein kinase C iota as a therapeutic target in alveolar rhabdomyosarcoma.

Alveolar rhabdomyosarcoma is an aggressive pediatric cancer exhibiting skeletal-muscle differentiation. New therapeutic targets are required to improve the dismal prognosis for invasive or metastatic alveolar rhabdomyosarcoma. Protein kinase C iota (PKCι) has been shown to have an important role in tumorigenesis of many cancers, but little is known about its role in rhabdomyosarcoma. Our gene-expression studies in human tumor samples revealed overexpression of PRKCI. We confirmed overexpression of PKCι at the mRNA and protein levels using our conditional mouse model that authentically recapitulates the progression of rhabdomyosarcoma in humans. Inhibition of Prkci by RNA interference resulted in a dramatic decrease in anchorage-independent colony formation. Interestingly, treatment of primary cell cultures using aurothiomalate (ATM), which is a gold-containing classical anti-rheumatic agent and a PKCι-specific inhibitor, resulted in decreased interaction between PKCι and Par6, decreased Rac1 activity and reduced cell viability at clinically relevant concentrations. Moreover, co-treatment with ATM and vincristine (VCR), a microtubule inhibitor currently used in rhabdomyosarcoma treatment regimens, resulted in a combination index of 0.470-0.793 through cooperative accumulation of non-proliferative multinuclear cells in the G2/M phase, indicating that these two drugs synergize. For in vivo tumor growth inhibition studies, ATM demonstrated a trend toward enhanced VCR sensitivity. Overall, these results suggest that PKCι is functionally important in alveolar rhabdomyosarcoma anchorage-independent growth and tumor-cell proliferation and that combination therapy with ATM and microtubule inhibitors holds promise for the treatment of alveolar rhabdomyosarcoma.

[Comparison of central corneal thickness and keratometric measurements using the Scheimpflug HR imaging system, laser interferometry, automatic keratometry and ultrasound pachymetry]. / Srovnání merení tloustky a zakrivení rohovky pomocí Scheimpflugovy kamery, laserové interferometrie, automatické keratometrie a ultrazvukové pachymetrie.

INTRODUCTION: The aim of our study was to compare keratometry and central corneal thickness measurements obtained with three different ophthalmic devices and to decide if they can be used interchangeably in clinical practice. METHODS: 43 healthy persons were included in the study (29 women and 14 men, average age 25 ± 3.5 years). Central corneal thickness (CCT) was measured with the Scheimpflug HR imaging system (Pentacam), Allegro BioGraph and with ultrasound pachymetry (RXP OcuScan). Keratometry in two main meridians of the cornea (K1, K2) was measured with Pentacam, Allegro BioGraph and automated keratometry. RESULTS: The mean difference in K1-readings was 0.01 ± 0.31 D for BioGraph vs. automated keratometry, 0.06 ± 0.23 D for BioGraph vs. Pentacam and 0.05 ± 0.34 D for automated keratometry and Pentacam. The mean difference in K2-readings was 0.29 ± 0.45 D for BioGraph vs. automated keratometry, 0.11 ± 0.28 D for BioGraph vs. Pentacam and 0.19 ± 0.44 D for automated keratometry and Pentacam. The interdevice differences were in all cases statistically significant (p < 0.05). The mean difference in CCT was 4.57 ± 7.84 µm for BioGraph vs. ultrasound, 4.33 ± 7.55 µm for BioGraph vs. Pentacam and 8.90 ± 7.49 µm for ultrasound vs. Pentacam. The interdevice differences in CCT were also statistically significant (p < 0.05). CONCLUSION: Our results suggest that the measurements of keratometry and CCT may differ significantly between the tested machines and therefore should not be used interchangeably in clinical practice.

[New knowledge of the pathogenesis of Crohn's disease]. / Nové poznatky v patogenezi Crohnovy choroby.

Crohns disease is a complex chronic inflammatory disease of the gastrointestinal tract with multifactorial pathogenesis. Over the recent years, there has been rather a sharp increase in the incidence of Crohn's disease and, even though this disease had been known for some time, the cause remains unknown. Studies exploring genetic basis of Crohn's disease have provided new knowledge of the pathogenesis of this disease, suggesting that this may be associated with a failure of mechanisms behind symbiosis of gut microflora and intestinal mucosal immune system. Crohn's disease seems to be caused by inadequate immune response to intestinal flora in genetically predisposed individuals. Crohn's disease has been linked to a number of genes. Many of them are related to the modulation of non-specific immune response, defects of which are considered to be key in Crohn's disease pathogenesis. The aim of this review paper is to summarize the new knowledge on the pathogenesis of Crohn's disease at the level of polymorphisms of the NOD2, ATG16L1 genes and the IL23-Th17-lymfocytes signalling pathway genes and to consider further research directions in this disease.

In vitro activation of CMV-specific human CD8(+) T cells by adenylate cyclase toxoids delivering pp65 epitopes.

Human CMV infects between 50-85% of healthy individuals and can cause live-threatening infections in immunocompromised patients. Therefore, peptide vaccination is being developed as a promising immunotherapeutic approach for treatment of patients at risk of CMV disease. The enzymatically inactive toxoid of Bordetella adenylate cyclase (CyaA-AC(-)) was shown to be an efficient tool for delivery of peptide epitopes and stimulation of Ag-specific T-cell immune responses. We investigated here the capacity of two CyaA-AC(-) constructs to deliver epitopes derived from the CMV phosphoprotein pp65 for activation of human T cells in vitro. Expansion of γ-IFN-secreting CMV-specific CD8(+) T cells, as well as increase of total IFN-γ and TNF-α production by PBMCs from CMV-seropositive donors were observed after in vitro stimulation with CyaA-AC(-) constructs carrying CMV epitopes, whereas limited activation of immune response occurred with free peptides. The activation of immune response was confirmed by expansion of CMV-specific T-cell clones and anti-CMV cytotoxic effect of stimulated PBMCs. These data open the way to clinical evaluation of CyaA-AC(-) constructs as tools for detection and expansion of CMV-specific T-cell immune responses for diagnostic and immunotherapeutic applications against CMV-associated diseases.

Assessing the invasiveness of NOTES perforated viscus repair: a comparative study of NOTES and laparoscopy.

BACKGROUND: Natural orifice transluminal endoscopic surgery (NOTES) repair of perforated peptic ulcers may decrease surgical invasiveness and improve patient outcomes. METHODS: Full thickness gastrotomy was created laparoscopically in swine followed by soilage time. Repair proceeded with a laparoscopic (n = 14) or the NOTES (n = 14) approach. For NOTES repair, the omentum was endoscopically pulled into the gastric lumen and clipped. Intraoperative and postoperative parameters were recorded, including arterial blood gas (ABG) analysis and serum samples for white blood cell (WBC), TNF-α, IL-1, and IL-6 analysis. RESULTS: Twenty-four of 28 animals thrived to study completion. NOTES repair could not be accomplished in one animal. At necropsy, all repairs were intact. Blood pressure was equivalent between groups. Pulse examined during the last 30 min of each procedure revealed a slightly higher mean pulse in the animals undergoing NOTES procedures (NOTES, 102 ± 28; laparoscopy, 83 ± 24). ABG obtained at the conclusion of the procedure revealed a pH of 7.47 in NOTES animals and 7.43 in the laparoscopy animals (p = 0.06), a change from baseline in both groups. The final pCO(2) was lower in the NOTES group (NOTES, 40.62; laparoscopy, 47.49, p = 0.03). WBC counts were comparable on postoperative day (POD) 1 (NOTES, 21.1; laparoscopy, 19.0; p = 0.49). Mean TNF-α serum levels were equivalent at all time points between groups; however, TNF-α varied significantly from baseline to POD 7 (p = 0.002). CONCLUSION: NOTES omental repair appears comparable to that of laparoscopy. The lower arterial pCO(2) at the conclusion of the NOTES procedure may be advantageous in critically ill patients.

Increased platelet:RBC ratios are associated with improved survival after massive transfusion.

BACKGROUND: Several recent military and civilian trauma studies demonstrate that improved outcomes are associated with early and increased use of plasma-based resuscitation strategies. However, outcomes associated with platelet transfusions are poorly characterized. We hypothesized that increased platelet:red blood cells (RBC) ratios would decrease hemorrhagic death and improve survival after massive transfusion (MT). METHODS: A transfusion database of patients transported from the scene to 22 Level I Trauma Centers over 12 months in 2005 to 2006 was reviewed. MT was defined as receiving ≥ 10 RBC units within 24 hours of admission. To mitigate survival bias, 25 patients who died within 60 minutes of arrival were excluded from analysis. Six random donor platelet units were considered equal to a single apheresis platelet unit. Admission and outcome data associated with the low (>1:20), medium (1:2), and high (1:1) platelet:RBC ratios were examined. These groups were based on the median value of the tertiles for the ratio of platelets:RBC units. RESULTS: Two thousand three hundred twelve patients received at least one unit of blood and 643 received an MT. Admission vital signs, INR, temperature, pH, Glasgow Coma Scale, Injury Severity Score, and age were similar between platelet ratio groups. The average admission platelet counts were lower in the patients who received the high platelet:RBC ratio versus the low ratio (192 vs. 216, p = 0.03). Patients who received MT were severely injured, with a mean (± standard deviation) Injury Severity Score of 33 ± 16 and received 22 ± 15 RBCs and 11 ± 14 platelets within 24 hours of injury. Increased platelet ratios were associated with improved survival at 24 hours and 30 days (p < 0.001 for both). Truncal hemorrhage as a cause of death was decreased (low: 67%, medium: 60%, high: 47%, p = 0.04). Multiple organ failure mortality was increased (low: 7%, medium: 16%, high: 27%, p = 0.003), but overall 30-day survival was improved (low: 52%, medium: 57%, high: 70%) in the high ratio group (medium vs. high: p = 0.008; low vs. high: p = 0.007). CONCLUSION: Similar to recently published military data, transfusion of platelet:RBC ratios of 1:1 was associated with improved early and late survival, decreased hemorrhagic death and a concomitant increase in multiple organ failure-related mortality. Based on this large retrospective study, increased and early use of platelets may be justified, pending the results of prospective randomized transfusion data.

Imbalance in T-cell and cytokine profiles in patients with relapsing-remitting multiple sclerosis.

Multiple sclerosis (MS) is characterized by autoimmune attack leading to demyelination of the white matter in the central nervous system with devastating clinical consequences. Several immune-mediated destruction mechanisms were previously proposed including different T-cell subsets but complex view on immune system function in patients with MS is missing. In the present study, T-lymphocyte populations and pro-inflammatory as well as suppressive cytokine profiles were evaluated in detail in previously untreated patients with relapsing-remitting MS (RRMS). CD4(+) and CD8(+) naïve, central memory (Tcm), effector memory (Tem), terminal effector memory (Ttem), CD4(+) regulatory T-cells (Treg) and CD8(+) T-suppressor cells (Ts) were analysed using flow cytometry, and levels of ten plasma cytokines were determined using fluorescent bead-based immunoassay. We evaluated two groups of RRMS with minor (n=33) and major (n=25) clinical impairment and compared them with healthy controls (n=40) in order to detect any correlation between severity of MS clinical symptoms and immune disturbances. Significant differences were noted in CD4(+)CD45RA(+)CCR7(+) naïve T-cells, CD4(+)CD45RO(+)CCR7(-) and CD8(+)CD45RO(+)CCR7(-) Tem cells, while no differences were recognized in Tcm, Ttem, Treg and Ts cells in RRMS patients. Nine out of ten studied cytokines were disturbed in plasma samples of patients with RRMS. In conclusion, we demonstrate complex immune dysbalances in untreated MS patients.

Optimization of immunomagnetic selection of myeloma cells from bone marrow using magnetic activated cell sorting.

Plasma cells (PCs) enrichment from bone marrow samples of multiple myeloma (MM) patients is frequently performed by immunomagnetic separation (magnetic activated cell sorting, MACS) using anti-CD138 MicroBeads. The aim of our work was to find optimal strategy for immunomagnetic separation of PCs and determine optimal algorithm of separation techniques for samples with various percentage of neoplastic cells. From 2007 to 2008, selection of PCs using separation programs Possels and Posseld(2) was carried out on 234 bone marrow samples obtained from 208 MM patients. In 2008, an optimal algorithm for separation programs was introduced based on the analysis of the previous experiments. The Possels program is applicable for samples with >10% PCs in the mononuclear fraction, while the Posseld(2) program is used for samples with 5-10% PCs in the mononuclear fraction. Median purity of 92.6% for the positive fraction of cells (range 14.5-99.6%) and median recovery of 60.4% (range 25.7-99.5%) were obtained when the Possels program was applied (n = 45). A total of 80% (36/45) of processed samples had purity of >70%. Median purity for the positive fraction of 83.7% (range 14.3-99.7%) and median recovery of 14.3% (range 3.6-50.0%) were achieved using the Posseld(2) program (n = 99). A total of 68% (67/99) of processed samples reached >70% purity. This separation strategy enabled us to obtain sufficient amounts of highly purified PCs required for subsequent research purposes. The MACS method has been unsuccessful if the percentage of PCs in the initial sample was <5%. These samples were processed by fluorescence activated cell sorting (FACS).

Generation of myeloma-specific T cells using dendritic cells loaded with MUC1- and hTERT- drived nonapeptides or myeloma cell apoptotic bodies.

Dendritic cells are able to induce anti-tumor immune responses by presenting tumor-specific antigens to T-lymphocytes. Various tumor-associated antigens have been studied in multiple myeloma in an effort to find a strong antigen capable of generating clinically meaningful responses in vaccinated patients. The aim of our study was to generate myeloma-specific cytotoxic T lymphocytes in vitro using dendritic cells loaded with peptide antigens or apoptotic bodies. Peripheral blood mononuclear cells from HLA-A2+ healthy donors were used for isolation and culture of dendritic cells (DCs) and T lymphocytes. DCs were loaded with hTERT- and MUC1-derived nonapeptides or apoptotic bodies from myeloma cells. Repeated stimulation of T lymphocytes led to their activation characterized by interferon-gamma production. Activated T lymphocytes were separated immunomagnetically and expanded in vitro. Specific cytotoxicity of the expanded T lymphocytes was tested against a myeloma cell line. There was evidence of cytotoxicity for all three types of antigens used for T lymphocyte priming and expansion. No statistically significant differences were observed in T lymphocyte cytotoxicity for any of the antigens. We present a method for the priming and expansion of myeloma-specific T lymphocytes using dendritic cells loaded with different types of tumor antigens. Cytotoxic T lymphocytes and/or activated dendritic cells generated by the described methods can be applied for cellular immunotherapy against multiple myeloma and other malignancies.

MicroRNA-181 family predicts response to concomitant chemoradiotherapy with temozolomide in glioblastoma patients.

MicroRNAs are endogenously expressed regulatory noncoding RNAs. Previous studies showed altered expression levels of several microRNAs in glioblastomas. In this study, we examined the expression levels of selected microRNAs in 22 primary glioblastomas and six specimens of adult brain tissue by real-time PCR method. In addition, we examined methylation status of MGMT promoter by methylation-specific real-time PCR, as this has been shown to be a predictive marker in glioblastomas. MGMT methylation status was not correlated with response to concomitant chemoradiotherapy with temozolomide (RT/TMZ). MiR-221 (p=0.016), miR-222 (p=0.038), miR-181b (p=0.036), miR-181c (p=0.043) and miR-128a (p=0.001) were significantly down-regulated in glioblastomas. The most significant change was observed for up-regulation in miR-21 expression in glioblastomas (p<0.001). MiR-181b and miR-181c were significantly down-regulated in patients who responded to RT/TMZ (p=0.016; p=0.047, respectively) in comparison to patients with progredient disease. Our data indicate for the first time that expression levels of miR-181b and miR-181c could serve as a predictive marker of response to RT/TMZ therapy in glioblastoma patients.

Changes of corneal optical properties after UVB irradiation investigated spectrophotometrically.

Ozone depletion leads to an increase in UV rays of solar radiation reaching the surface of the Earth which is harmful to biological systems. Of the eye, the cornea is directly open to increased amount of UV rays of which mainly UVB rays are capable to induce reactive oxygen species damaging the cells. Previous studies showed that the irradiation of the cornea with UVB rays leads to morphological as well as metabolic disturbances of the cornea. Also, corneal hydration and corneal light absorption are increased after UVB rays. These changes were observed after five days of repeated irradiation of the cornea with UVB rays. The aim of the present paper was to examine how early the changes of corneal hydration and light absorption occur after UVB irradiation. The rabbit corneas were irradiated with UVB rays for one, two, three or four days. Corneal light absorption was examined spectrophotometrically and corneal hydration measured by pachymeter (as corneal thickness). Results show that changes of corneal hydration and light absorption appear early after UVB irradiation and increase along with the number of irradiations. In conclusion, irradiation of the rabbit cornea with UVB rays leads to harmful changes of its optical properties.