Circulating free DNA and p53 antibodies in plasma of patients with ovarian epithelial cancers.

BACKGROUND: This study was conducted in order to evaluate the significance of circulating free DNA (CFDNA), blood plasma p53 antibodies (p53-Ab) and mutations of KRAS gene in the prognosis of ovarian epithelial cancers. PATIENTS AND METHODS: A total of 126 patients were included in this study. KRAS mutations and CFDNA were detected by means of the PCR-restriction fragment length polymorphism (PCR-RFLP) and enriched by the PCR-RFLP method. Enzyme-linked immunosorbent assay was used to analyze plasma p53-Ab. RESULTS: KRAS mutations were detected in 27 (21.4%) of examined tumors. The frequency of KRAS mutations was especially high in mucinous cancers (P < 0.001). CFDNA and p53-Ab were frequently detected in patients with serous cancers in high grade (P < 0.001). The overall survival rate was significantly lower for patients with serous tumors and CFDNA and p53-Ab-positive than negative tumors (P = 0.022 and P < 0.001, respectively). In mucinous ovarian cancer, a worse overall survival was correlated with the KRAS mutations (P = 0.03). CONCLUSIONS: The results of the present study suggested that a presence of KRAS mutations in mucinous ovarian cancer and CFDNA and p53-Ab in serous tumors was correlated with the highest risk of cancer progression.

Disturbances in some gene expression in T regulatory cells separated from children with metabolic syndrome.

The metabolic syndrome (MS) is defined as a cluster of risk factors, including abdominal obesity, dyslipidaemia, glucose intolerance and hypertension, which increase the risk for coronary heart disease. The immunological aspects of obesity and MS, including the role of T regulatory cells, have been intensively investigated. The aim of this study was to determine whether there is any disturbance in T regulatory cells number and/or function in children with MS. The percentages of T regulatory cells in the peripheral blood of children fulfilling the International Diabetes Federation criteria of the disease (n = 47) were assessed. Treg cells were also separated for further analysis of multiple genes important in their function with the use of real-time RT-PCR. We did not observe any difference in Treg percentages between study and control group but there was lower expression of some molecules including transforming growth factor-beta and interleukin-12 family members in Treg cells separated from children with MS compared to the healthy subjects. Our study is the first to report significant disturbances in some gene expression in T regulatory cells separated from children with MS. The results should be useful for further research in this field, including immunotherapeutic interventions.

Acute lymphoblastic leukemia-derived dendritic cells express tumor associated antigens: PNPT1, PMPCB, RHAMM, BSG and ERCC1.

In all types of leukemia both in children and adults there is a need for novel therapies that could reduce the risk of relapse after standard treatment. Acute lymphoblastic leukemia (ALL) cells are ineffective antigen presenting cells, but as shown by many authors including results from our laboratory, stimulation with CD40L restores their antigen expressing capacity. The development of T-cell therapies for leukemic patients can be based on discovery of leukemia-associated antigens (LAA) which could be recognized by the host immune system. The aim of our present study was to test the hypothesis that leukemia-derived dendritic cells maintain the expression of tumor associated antigens. Twenty five children with B-cell precursor ALL were prospectively enrolled into the study. The mononuclear cells from peripheral blood or bone marrow were cultured and stimulated (or not) with CD40L and IL-4. The assessment of costimulatory/adhesion molecules with the use of flow cytometry and real-time RT PCR were used to confirm the possibility of turning ALL cells into dendritic-like cells. Additionally 22 tumor associated antigens mRNA levels were determined by real-time PCR technique with the TaqMan chemistry using ready-to-use Low Density Arrays for Gene Expression. The results of the study showed maintained expression and even up-regulation of some (PNPT1, PMPCB, HMMR/RHAMM, BSG and ERCC1) tumor associated antigens in CD40-activated leukemic cells. CD40L stimulation leading to the differentiation of leukemic cells into DCs which combine both antigen presenting function and expression of tumor associated antigens represents an interesting approach in cancer immunotherapy.

Expression of selected tumor suppressor and oncogenes in endometrium of women with endometriosis.

BACKGROUND: It is becoming increasingly evident that the eutopic endometrium of women with endometriosis shows certain genetic alterations which are not found in the endometrium of disease-free women. The aim of the study was to compare the expression level of mammalian target of rapamycin (mTOR) tumor suppressor and oncogene-related genes in the endometrium of women with and without endometriosis as well as in ovarian endometriosis. METHODS: A total of 81 regularly menstruating patients were recruited in the study. We applied the micro fluidic gene array to examine the expression of 15 human tumor suppressor and oncogenes in eutopic endometrium of 40 women with endometriosis and 41 controls without endometriosis. In 14 patients with endometriosis, gene expression was also studied in matched ovarian lesions. We studied the following genes: NF1, RHEB, mTOR, PTEN, TSC1, TSC2, KRAS, S6K1, TP53, EIF4E, LKB1, PIK3CA, BECN1, 4EBP1 and AKT1. Immunohistochemical studies were subsequently performed for selected proteins. RESULTS: Of the 15 studied genes, we found significantly higher levels of oncogene AKT1 (P = 0.006) and tumor suppressor gene 4EBP1 (P = 0.01) mRNAs in the eutopic endometrium of women with endometriosis compared with control patients. Immunohistochemistry showed that 4EBP1 and AKT1 proteins were expressed in eutopic endometrium. CONCLUSIONS: Our results suggest that up-regulation of AKT1 and 4EBP1 in eutopic endometrium may be associated with the pathogenesis of endometriosis, but their precise role remains to be established.

High prevalence of genotype 4 among hepatitis C virus-infected intravenous drug users in north-eastern Poland.

The aim of this study was to describe the distribution of hepatitis C genotypes among intravenous drug users in north-eastern Poland. The study group included intravenous drug users recruited at a drug treatment center and a clinic for HIV-infected patients. HCV infection was confirmed by qualitative nested RT-PCR to test for the presence of HCV RNA. Genotypes were determined by 5'UTR sequencing and comparing the results with known genotype sequences. Among 111 HCV-infected and HCV-RNA-positive intravenous drug users, the most prevalent genotypes were 1 (38.7%), 3 (37.8%), and 4 (23.4%). Most infections with genotype 4 (88.5%) were found among HCV-HIV-coinfected drug users. The study demonstrated a high prevalence of genotype 4 (23.4%) among HCV-infected Polish drug users.

B-cell chronic lymphocytic leukemia-derived dendritic cells stimulate allogeneic T-cell response and express chemokines involved in T-cell migration.

UNLABELLED: Despite discovery of new therapeutic agents, including nucleoside analogs and monoclonal antibodies, the B-cell chronic lymphocytic leukemia (B-CLL) remains incurable. In recent years, some effort has been made in developing T-cell specific immunity against neoplasmatic cells. Reconstitution of effective costimulation and immunological response of host T-cells against CLL cells could be a potential approach in immunotherapeutic trials. CD40/CD40L system is involved in the survival and proliferation of normal and neoplasmatic B-cells. Some preclinical studies have shown that CD40 stimulation can differentiate leukemic cells into dendritic cells (DCs) and result in host response. In this study, we sought to determine whether B-CLL cells could be turned into efficient and functional antigen presenting cells, as well as to assess the type of allogeneic T-cell response against B-CLL - derived DCs. MATERIAL AND METHODS: B-CLL cells from 25 patients were cultured with or without the presence of CD40L and IL-4 for 96 hours and then cultured in mixed lymphocyte reaction with allogeneic T-cells. RESULTS: 1) after CD40 stimulation B-CLL cells achieved phenotypical and functional characterization of DCs (i.e. upregulated co-stimulatory and adhesion molecules at mRNA and protein level) 2) leukemia-derived DCs expressed higher amount of mRNA for chemokines involved in T-cell migration (MDC, TARC and CCR7) 3) the proliferating response of Tcells against leukemia-derived DCs consisted of CD4 and CD8 cells (upregulation of HLA-DR and OX40). CONCLUSIONS: our experiment confirm that B-CLL cells can be turned into dendritic-like cells, additionally, these cells express chemokines involved in T-cell migration and stimulate allogeneic response.

HBV-DNA and sFas, sFasL concentrations in serum of healthy HBsAg carriers.

PURPOSE: Increased HBV-DNA concentration is a prognostic factor of disease progression in chronic hepatitis B patients. Moreover, active hepatic inflammation during HBV replication influences apoptosis intensification. The aim of this study was to estimate occurrence of HBV replication among carriers of HBsAg. Furthermore, we analysed the correlation between HBV replication and HBeAg or anti-HBe presence as well as known apoptosis indicators--sFas and sFasL concentration. MATERIAL AND METHODS: The study included 34 HBV infected patients, aged 20-43 yrs defined as HBsAg healthy carriers. HBV-DNA was extracted from patients' serum using two different DNA isolation kits: the QIAamp DNA Mini Kit (QIAGEN Ltd, USA) and the Gene Elute Mammalian Genomic DNA Miniprep Kit (Sigma, USA). HBV-DNA concentration in serum was measured by RT-PCR based on TaqMan Universal Master Mix (Applied Biosystems). The detection limit of this system was as few as 10 HBV-DNA copies/mL of serum. HBV-DNA concentration was calculated from a linear standard curve obtained between 10 and 10(8) DNA copies/reaction. HBeAg and anti-HBe in serum were detected by MEIA method (ABBOTT, Germany). The concentration of sFas and sFasL in serum was-estimated by ELISA method (Bender MedSystems, Austria). RESULTS: HBV active replication was detected in 79% HBsAg carriers. The HBV-DNA levels exceeding 10(5) copies/mL were observed in 64% patients. Among HBsAg carriers presenting HBeAg, HBV replication occurred more often and was more intensify than in HBsAg carriers presenting anti-HBe antibodies. The sFasL occurrence in serum of 56% HBsAg carriers shows an active apoptosis, independent from ALT and AST activity within normal ranges.