Blood Serum Zonulin in Colorectal Cancer, Autoimmune Bowel Diseases, and Irritable Bowel Syndrome.

Zonulin content in blood serum of patients with colorectal cancer (CRC; n=152; 30-84 years) and patients with large bowel adenomas (n=32; 39-82 years) was measured by standardized kit IDK Zonulin ELISA (Immundiagnostik AG). The healthy control group (n=50) comprised volunteers (27 women, 23 men; 25-68 years); pathological control group (n=84) - patients (55 women, 29 men;18-84 years) with irritable bowel syndrome (n=29), Crohn's disease (n=5), and ulcero-necrotic colitis (n=50). In comparison to healthy control group, the level of zonulin was significantly increased in CRC patients (p<0.0000001) and in patients with benign large bowel tumors (p<0.004), as well as in patients with inflammatory intestine diseases and with irritable bowel syndrome (p<0.0002). Zonulin level in blood serum of CRC patients was slightly, but significantly higher (p<0.05) than in the group of pathological control. ROC curve construction revealed that at optimal zonulin cut-off level (52.2 ng/ml), the diagnostic sensitivity of CRC detection was 66.7% and specificity relative to healthy control was 81.8%. The specificity relative to the combined control group (healthy control+non-tumor bowel diseases) was only 68.9%. Thus, no acceptable cut-off levels for differentiation between malignant and benign tumors, as well as between tumor and non-tumor large bowel pathologies were found. Analysis of the associations between serum zonulin level and the main clinical and pathological characteristics of CRC demonstrated that the level of this marker increased with disease progression (p<0.01; Kruskal-Wallis test), but was not associated with individual criteria of the TNM system, tumor localization, histological structure, and malignancy grade.

[The study of mRNA expression profiles of main cell function regulator genes in unchanged colonic mucosa from healthy donors]. / Issledovanie profilei ékspressii mRNK genov, uchastvuiushchikh v reguliatsii osnovnykh kletochnykh funktsii v neizmenennom épitelii tolstoi kishki u zdorovykh donorov.

A comparative analysis of molecular genetic phenotypes of mucous membrane cells in five anatomical regions of the colon in a group of healthy donors was conducted by comparing mRNA expression profiles of 62 genes involved in the regulation of vital cellular function. We used 181 biopsy samples of morphologically unchanged colonic mucosa, obtained from the colon (ascending, transverse-colon, descending, sigmoid) and rectum sections during prophylactic colonoscopy of 58 donors with no colon pathology. The mRNA levels for 62 genes involved in the regulation of apoptosis, proliferation, transcription, differentiation, cell-cell adhesion, and immune response were assessed by RT-PCR. Statistically significant differences were found for the molecular phenotypes of five sections of the colon. The results of the study can serve as a basis for creating a reference database (values of expression profiles), developing methods of differential diagnostics and screening of various pathologies of the colon.

Comparison of Biological Effects of γ-Radiation of Low and Ultra-High Dose Rate on Lymphocytes and Cultured Human Malignant Lymphoma Cells.

We studied the effect of low and high-dose rate photon radiation on activation of cell death by apoptosis and necrosis in malignant cell lines of lymphocytic origin Raji and Jurkat (human B and T-cell lymphomas) and normal human lymphocytes from healthy volunteers. It was shown that photon radiation with ultra-high dose rate induced significantly higher levels of "early" apoptosis and lower levels of necrosis compared to γ-radiation with dose rate used for radiation therapy.

[Comparison of matrix proteinase mRNA expression in morphologically normal, neoplastic, and metastatic colon tissue and colon biopsies from healthy donors]. / Sravnenie ékspressii mRNK matriksnykh metalloproteinaz v morfologicheski normal'noi, neoplasticheskoi i metastaticheskoi tkaniakh tolstogo kishechnika i v bioptatakh zdorovykh donorov.

Matrix metalloproteinases (MMPs) responsible for the extracellular matrix remodeling, the activation of various growth factors, and angiogenesis play an important role in the colorectal cancer (CRC) development. In the present work the comparative analysis of MMP-7, -8, -9, and -11 mRNA as well mRNA of the Ki-67 proliferation marker in tissue samples obtained from CRC patients and healthy individuals. Employing the real time PCR method the expression levels of several MMPs (MMP-7, -8, -9, and -11) and cell proliferation marker, Ki-67, were simultaneously measured in 256 tissue samples obtained from 112 patients with CRC: 112 samples of the primary tumor (CRC), 112 samples of the most distant border of morphologically normal colonic mucosa (MNT), 16 samples of liver metastases) and from 16 healthy volunteers who underwent colonoscopy and biopsy. The expression of both MMPs studied and Ki-67 was found to be elevated in CRC primary tumors and liver metastases compared with the normal mucosa. CRC tumor and metastatic cells exhibited similar proliferative activity. The metastases are characterized by the highest cross-correlation of MMPs among tissue types tested. For the first time it was shown that normal mucosa from healthy individuals and CRC patients varied in the MMP-8 expression level. They also had dissimilar MMP correlation patterns thus suggesting that epithelial cells adjusted to CRC tumor differ from mucosal epithelial cells of healthy individuals.

Transcription profile analysis of the endometrium revealed molecular markers of the personalized 'window of implantation' during in vitro fertilization.

To determine the most informative markers for assessing the functional state of endometrium during the 'window of implantation' and creating a model for assessment of the readiness of endometrium for embryo implantation. Forty-seven women with tubal infertility and a successful IVF pregnancy participated in the study. Pipelle endometrial sample was performed during the supposed 'window of implantation' in natural cycle with subsequent histological study, and transcriptional profile of genes GPX3, PAEP, DPP4, TAGLN, HABP2, IMPA2, AQP3, HLA-DOB, MSX1, POSTN determined by real-time quantitative polymerase chain reaction (qRT-PCR). Differences in the level of mRNA expression of all the studied genes in the receptive endometrium were found in comparison to the prereceptive one, which allowed us to classify two functional states of the endometrium. The results of histological examination responded to the stage of maturation of the endometrium in 78.7% of cases. Receptive endometrial status can be determined based on the integral evaluation of mRNA expression level of 4 PAEP, DPP4, MSX1, and HLA-DOB genes. The model for determining a personalized `window implantation' is offered for practical application in ART.

[Mammaglobin in peripheral blood and tumor in breast cancer patients]. / Mammaglobin v perifericheskoi krovi i opukholi pri rake molochnoi zhelezy.

Currently, no molecular biological markers do exist for early diagnosis of breast cancer. One of the possible candidates for the marker of early breast cancer is mammaglobin (MGB1) or SCGB2A2 (secretoglobin, family 2A, member 2), characterized by the maximal expression level in early breast cancer. Using the RT-PCR method MGB1 mRNA expression was examined in 57 tumor tissue samples and 57 samples of morphologically non-malignant tissue (MNT) of breast cancer (BC) patients. Specificity and sensitivity of the MGB1 mRNA assay in peripheral blood of BC patients was evaluated by nested PCR. 169 blood samples (from 95 BC patients, 22 from patients with benign breast tumors, 28 from patients with tumors of other localizations, and 24 samples from healthy donors) have been analyzed. MGB1 expression was significantly higher in BC tissue samples compared to MNT (p=0.0019). The maximal expression level was in the samples T1 (p=0.013), stage I BC (p=0.037), GI (p=0.0019). The MGB1 expression positively correlated with expression of estrogen (p = 0,034) and progesterone (p=0.0004) receptors. Sensitivity and specificity of the MGB1 mRNA assay in peripheral blood were 60.6% and 92.3%, respectively. Expression of MGB1 was higher in BC than MNT and it decreased during BC progression. The sensitivity and specificity of the MGB1 mRNA assay may be used as an additional diagnostic method.

Characteristics of new monomolecular chimeric T-cell receptors to carcinoembryonic antigen.

We described two original genetic constructs encoding chimeric monomolecular T-cell receptors, where the effector T-cell receptor fragment was linked with the antigen-recognizing part consisting of two variable fragments of two different antibodies to carcinoembryonic antigen. Following transfection, these receptors were expressed on the cell surface and bound carcinoembryonic antigen. Human peripheral blood lymphocytes transfected with the above constructs demonstrated high cytotoxic activity against HCT116 cells expressing carcinoembryonic antigen.

[Risk factors for hematologic toxicity and multifaceted analysis of blood in Hodgkin's disease: a model for hematologic toxicity].

Basic hematological features of CEA/ABVD medication for Hodgkin's disease were studied. An effective model was worked out on the principle of data discrimination for predicting different leukocytic toxicities induced by cytostatics-1 administration, once in two weeks. It might predict individual limits (dosage and intervals) of a chemotherapy course unless a colony-stimulation technique is used.

[Features and risk factors of post-mastectomy, locally advanced breast cancer relapse].

Multivariate analysis of risk of locally-advanced breast cancer IA-IIIB progression was carried out in 444 patients 10 years after mastectomy in the framework of two randomized studies. Combination therapy (median 156.3 months) included mastectomy, different modalities of neoadjuvant and adjuvant chemohormonal and radiotherapy.

[Effect of metabolizable peptide p16INK4a on short-lived human tumor cultures].

The study was concerned with antitumor action of internalized peptide incorporating a fragment of p161INK4a using a model of short-lived human tumor cultures sampled from resected material. Renal cancer sample showed the greatest therapeutic interval.

[Multivariate analysis of risk of cardiac complications in Hodgkin's lymphoma].

A computer database was created to take care of a wide range of protocols for combined treatment of Hodgkin's disease stage I-IV (n=1,573). Early-onset radiation-related injuries (pneumonitis) and exposure of lung tissues to radiation were identified as the main risk factors for cardiopathology development. It is suggested that total focal dosage used after chemotherapy be reviewed since total dosage for the entire lymph collector in excess of 30 Gy might contribute to hazards of cardiopathology. However, a locally administered TTD ranging 36-44 Gy to deal with residual tumor offers best advantage in preventing local relapse. Nor does it increase the risk of future complications. Our approach might promote individualization of prognosis as far as cardiac complications involved in Hodgkin's lymphoma are concerned.

Cytostatic and cytotoxic properties of chimeric peptides containing cyclin-inhibiting fragments.

Properties of chimeric peptides containing cell-penetrating sequences and pl6INK4a and E2F fragments were studied in vitro on immurtal cultures of human cells. Both sequences exhibit cytostatic activity. The peptide containing fragment p16INK4a inhibited proliferation during the G0/G1 stage of the cell cycle, while sequence E2F suppressed proliferation in S phase. Both sequences exhibit cytotoxic properties and can induce apoptosis.

Cytokeratin 19 mRNA concentration in lymph nodes as a diagnostic marker of metastases.

Comparison of the sensitivity of cytological and molecular genetic methods (19 specimens of lymph nodes from 8 patients with breast cancer and suspected metastases obtained by transcutaneous fine-needle aspiration biopsy under ultrasound guidance) showed that molecular genetic and cytological studies produced true results in 95 and 84% specimens, respectively. True-positive and true-negative results were obtained in 8 and 7 patients, respectively. Expression of cytokeratin 19 was detected in 3 specimens with negative cytological results and confirmed metastases in lymph nodes. Our results indicate that molecular genetic diagnostic study for lymph node metastases should be used in small amounts of biopsy specimens, presence of marginal metastases in lymph nodes, and negative results of repeated cytological examination.

[Radiation-induced DNA double-stranded breaks and the dynamics of apoptotic death of human peripheral lymphocytes].

DNA double-stranded breaks and their association with the development of radiation-induced peripheral lymphocyte apoptosis were studied in healthy donors exposed to in vitro gamma-irradiation in a dose of 1 Gy. It was shown that irradiation in 1-Gy dose caused a significant (p < 0.05) increase in the frequency of cells in late apoptosis 4 hours after irradiation and a rise in their frequency in early apoptosis 24 hours following this procedure. A significant correlation (r = 0.52, p < 0.05) was recorded between the primary level of radiation-induced DNA double-stranded breaks and the frequency of cells in late apoptosis following 4 hours, which suggests that DNA double-stranded breaks as a signal to trigger cell apoptotic death are of great importance.

[Cytotoxicity of chimera peptides incorporating sequences of cyclin kinases inhibitors].

The study is concerned with proapoptotic properties of chimera peptides which incorporate sequences of inhibitors of cyclin kinases p161NK4a and p21CIP/WAF1 as well as internalized sequences (Antp and tat). Sequences of the p16 type appeared to be more cytotoxic than the p21 one. Cytotoxic effect proved dependent on orientation with respect to the C or N terminal point of a polypeptide chain rather than on chimera sequence extent. Although p16 endogenous synthesis did not influence chimera peptide levels, apoptosis did not take place in certain cellular lines. Due to the rather unsophisticated nature of such synthesis, it might be used in designing individually-tailored chemotherapeutic drugs.

[Use of flow cytometry in diagnosis of urinary bladder cancer].

Urinary bladder cancer ranks 11th (10-15 cases per 100,000 annually) in the oncological morbidity registry. Morphological diagnosis is generally confirmed by cytological assay of urinary sediment or lavage liquid. Yet, the method is effective only with low-differentiated cell tumor. Flow cytometry was used at the Center's Clinic (2005-2006) to test urinary sediment from 32 patients diagnosed with bladder cancer. In addition, all patients were tested 3 times by the standard cytological procedure using a mix of azure and eosin (Pappenheim). The sensitivity of the latter method proved to be 31.3% as compared with 65.62% for flow cytometry.

[Monoclonal antibody therapy with mabtera of patients with b-cell low grade non-Hodgkin's lymphoma].

The data on monoclonal antibody monotherapy (mabtera, rituximab) in 44 patients with B-cell low grade non-Hodgkin's lymphoma were assessed. Thirty-four of them had received several courses of second- or third line chemotherapy: mabtera was used as first-line therapy in 10. Mabtera was administered in a dose of 375 mg/m2 body surface, once a week, by slow intravenous infusion, 4-14 times depending on effect. Each relapsing patient received, on the average, 4 infusions, while each refractory one--8 weekly infusions. Overall response in the first group was 44%. Median overall relapse-free survival was 9 months. Apparenti effect of treatment was reported in 50% of primary patients (median overall relapse-free survival--15 months). Therapy was well tolerated. Fever and shivering stage I and II were among the most frequent post-infusion effects. High level of CD-20+B-lymphocytes should be used as prognostic indicator for effectiveness of therapy.