N-cadherin, beta-catenin and connexin 43 expression in astrocytic tumours of various grades.

INTRODUCTION: Astrocytic tumors are the most common primary brain tumors, but little is known about their etiology and prognostic factors. N-cadherin and beta-catenin are adhesive proteins, and are often overexpressed in many types of cancers, including breast or colorectal cancer, resulting in better prognosis. Connexin 43 is a gap junction protein involved in cell-cell signaling pathway taking part in the process of carcinogenesis. The aim of the study was to evaluate N-cadherin, beta-catenin and connexin 43 expression in astrocytic tumors of various grades. MATERIALS AND METHODS: We examined 131 cases of astrocytic tumors, including 26 cases of diffuse astrocytoma (group I), 44 anaplasic astrocytomas (group II) and 61 glioblastoma cases (group III)--primary and secondary. To evaluate N-cadherin, beta-catenin and connexin 43 expression, we used immunohistochemical reaction with specific antibodies (Santa Cruz Biotechnology). The obtained results were correlated with clinical and morphological features. RESULTS: Beta-catenin expression was observed in 69.3% of diffuse astrocytomas, 75% of anaplastic astrocytomas, and 82% of glioblastoma cases. N-cadherin expression was observed in 92.3% of diffuse astrocytomas, 90.1% of anaplastic astyrocytomas, and in all glioblastoma cases. Connexin 43 was observed in 76.9% of diffuse astrocytomas, and in all cases of anaplastic astrocytomas and glioblastomas. Beta-catenin expression was significant within the nucleus of neoplastic cells in groups I and II. In group III, staining was observed only in the cellular membranes. N-cadherin and connexin 43 expression was observed only in the cells' membranes. In glioblastomas, both primary and secondary, all protein expression was significant within the cells surrounding the necroses and blood vessels and weak or absent in the tumor's margins. CONCLUSION: Our study shows that beta-catenin nuclear expression in group of diffuse astrocytomas and anaplastic astrocytomas is evidence for transcriptional function of beta-catenin in those groups. Strong N-cadherin and connexin 43 expression in those groups may be evidence for their role in tumor formation and progression. However, in glioblastomas a very important role of all examined proteins is generating intracellular connections to facilitate the escape of tumor cells from the effects of hypoxia or their accumulation around the blood vessels rather than tumor invasion into the brain parenchyma.

New monoallelic combination of KRAS gene mutations in codons 12 and 13 in the lung adenocarcinoma.

PURPOSE: In a retrospective analysis of the prevalence of KRAS mutations in patients with advanced non-small cell lung cancer (NSCLC), we detected a unique and not earlier described case of a double combination of mutations at codons 12 and 13 of the KRAS gene in a patient with lung adenocarcinoma. MATERIAL/METHODS: To determine the molecular characteristics of the infrequent mutation and the mutational status of the KRAS gene in metastatic brain tumors in the same patient, we performed morphological and molecular tests. RESULTS: Molecular analysis of the nature of the double mutation showed that the unique combination of variants is a monoallelic mutation. This type of changes has not yet been registered in the Catalogue of Somatic Mutations in Cancer database. Molecular assessment of the KRAS mutation status in the brain metastatic site in the same patient, showed no mutations in codons 12 and 13. Moreover, we did not find mutation at exon 19 and 21 of EGFR gene, both in primary tumor as well as in secondary metastatic foci in the brain. CONCLUSIONS: The presented case shows an example of KRAS gene molecular mosaicism and heterogeneity of lung adenocarcinoma primary and metastatic tumors. Molecular heterogeneity of lung adenocarcinoma tumors can significantly affect eligibility of patients for targeted therapies.

Increased release of soluble CD163 by the peripheral blood mononuclear cells is associated with worse prognosis in patients with systemic sclerosis.

PURPOSE: CD163 is a scavenger receptor which is exclusively expressed on monocytes/macrophages and participates in modulation of inflammatory response. We aimed to evaluate ex vivo production of soluble CD163 (sCD163) by peripheral blood mononuclear cells (PBMC) from patients with systemic sclerosis (scleroderma, SSc). MATERIAL/METHODS: Concentration of sCD163 was measured by commercially available ELISA kit in the PBMC suparnates from 23 SSc patients and 16 age- and sex-matched healthy controls (HC). Eighteen SSc patients were subsequently followed for at least three years or until death whichever happened earlier. Disease progression was defined as death due to SSc-related organ complication, development of a new or progression of pre-existing SSc-related organ involvement. RESULTS: PBMC from SSc patients released significantly greater amounts of sCD163 as compared with HC (p<0.05). No significant associations between release of sCD163 by PBMC and baseline clinical or laboratory parameters of the disease could be found. However, concentration of sCD163 in cell culture supernates was significantly higher in 6 SSc patients who experienced subsequent progression of the disease as compared with 12 SSc patients with stable disease course over a 3-year follow-up period (p<0.05). CONCLUSIONS: We show, for the first time, that PBMC from SSc release significantly greater amounts of sCD163 than do PBMC from healthy subjects. Evaluation of sCD163 production by PBMC ex vivo may serve as a new biomarker of disease progression. Further studies are required to evaluate the role of sCD163 in the development of SSc.

Ethanolic extract of propolis, chrysin, CAPE inhibit human astroglia cells.

PURPOSE: The aim of the present study was to examine the effect of freeze dried ethanolic extract of propolis (EEP), chrysin and caffeic acid phenethyl ester (CAPE) dependently on their concentrations on the viability and morphology of human astroglia cells line (SVGp12). MATERIAL AND METHODS: Using gas chromatography - mass spectroscopy (GC-MS) we have established the composition of lyophilisate of EEP collected in Podlasie region (Poland). After 24 h, 48 h and 72 h of exposition to EEP or its ingredients we evaluated the survivability of human astroglia cells (SVGp12) using MTT test. Morphological analysis of human astroglia cells was defined by transmission electron microscope. RESULTS: About 70 ingredients of EEP were evaluated by GC-MS. We obtained the strong decline of viability of astroglia cells SVGp12 approximately to 16% after EEP; 33% after chrysin and 25% after CAPE application. Condensed form of mitochondria observed in transmission electron microscope may indicate activation of intrinsic pathway of apoptosis induced by EEP, chrysin and CAPE in SVGp12 cell line. CONCLUSION: This study showed that EEP, chrysin and CAPE reduced viability of human astroglia cells probably due to apoptosis process.

Peripheral blood mononuclear cells from patients with systemic sclerosis spontaneously secrete increased amounts of vascular endothelial growth factor (VEGF) already in the early stage of the disease.

PURPOSE: To investigate the capacity of the peripheral blood mononuclear cells (PBMC) from patients with systemic sclerosis (SSc) to produce vascular endothelial growth factor (VEGF), and to identify clinical associations of altered production of VEGF by PBMC in SSc. In addition, correlation with another pro-angiogenic cytokine, TNF-related weak inducer of apoptosis (TWEAK), was evaluated. METHODS: PBMC were isolated from 25 patients with SSc and 17 healthy controls (HC). VEGF and TWEAK were measured in the supernatants of cultured PBMC using commercially available ELISA kits. RESULTS: PBMC from SSc patients spontaneously released significantly greater amounts of VEGF as compared with HC. Production of VEGF was comparable between patients with early SSc and those with longer disease duration, and in both SSc groups higher than in HC. Patients without active digital ulcers produced significantly greater amounts of VEGF as compared with HC, while there was no significant difference in the production of VEGF between SSc patients with active digital ulcers and HC. VEGF/TWEAK ratio was significantly higher in PBMC from SSc patients than in HC indicating that high production of VEGF is not paralleled by increased release of TWEAK in SSc. CONCLUSIONS: PBMC form SSc patients produce increased amounts of VEGF already in the early stage of disease. There is an imbalance in the profile of pro-angiogenic mediators produced by PBMC in SSc which might contribute to the pathogenesis of SSc. Further studies should address clinical significance of our findings.

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.

Mast cells in peritoneal fluid in rats with experimentally induced peritoneal adhesions.

Mast cells (MC) produce, store and release many biologically active substances, especially inflammatory factors, chemotactic substances for neutrophiles, cytokines and prostaglandins. They play very important role in fibrinosis and they are an important factor in peritoneal adhesions formation and lysis. In this study we tried to evaluate role of mast cells in peritoneal adhesions formation. We estimated number of mast cells in peritoneal fluid in rats with experimentally developed peritoneal adhesions. The number of mast cells per ml was counted in flow cytometry in specimens of peritoneal fluid taken from operated rats. The fluid was taken in standardized conditions the same for each group at the first operation and during reoperation. Peritoneal cavity was washed with 0.9% Saline solution. MC were visualized using indirect immunohistochemical method LSAB with mouse antibody. The animals were divided into 4 groups. 1 st group was control group (n=20) on which the abdomen was opened and closed without any manipulations, and the reoperation was done after 72 hours. The other groups (2, 3, 4; n=20 for each group) were operated and scarification of the partial peritoneum and serosa was performed. The rats were brought back to conscious and then were reoperated respectively after 24, 72 and 168 hours after first surgery. After the laparotomy and damage of the peritoneum we observed formation of the peritoneal adhesions between intestine loops and between intestines and damaged parietal peritoneum. Also the higher number of mast cells was observed in the groups of animals with damaged peritoneum. The highest number of peritoneal adhesions was observed in the group of animals reoperated after 72 hours. After 72 and 168 hours the higher number of MC and neutrophils was observed. The difference was statistically significant. The percentage of mast cells was increasing during the experiment duration. It was different from other cells populations which decreased after 168 hours. The MC and neutrophils were cell population which changed significantly after manipulations in peritoneal cavity. It is very probable that they play an important role in peritoneal adhesions formation.

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.

Comparison of serology assays and polymerase chain reaction for the monitoring of active cytomegalovirus infection in renal transplant recipients.

BACKGROUND: Cytomegalovirus (CMV) infection is a common complication of renal transplantation. It can be diagnosed serologically, mainly based on seroconversion or by the detection of viral antigen via CMV-DNA amplification (polymerase chain reaction [PCR]). AIM: We sought diagnosis of an active CMV infection in renal transplant patients comparing serologic assays of CMV-IgM antibodies with CMV-DNA amplification. METHODS: We retrospectively studied renal transplant recipients 26 (including 15 women) hospitalized with clinical suspicion of CMV disease. The diagnosis of CMV infection was suspected on the basis of nonspecific symptoms, including fever, leukopenia, hyperbilirubinemia, and alanine aminotransferase elevation, alone or in combination. At the time of admission, all patients were screened for CMV-IgM antibody (immunoassays AxSYM/IMx) and CMV-DNA (qualitative PCR). RESULTS: The confirmation of CMV infection by the two methods (immunoassay and PCR) was obtained in only three patients (11.5%), its unambiguous exclusion--in four cases (15.4%). Nineteen patients (73.1%) were positive for CMV-IgM and negative for CMV-DNA. CONCLUSION: Detection of CMV-IgM antibodies by various immunoassays is not sensitive enough for diagnosis and cannot be used for monitoring during the active period in renal transplant recipients. This observation supported the prolonged presence of IgM antibodies after recent CMV infection in this patient group.

Acute lymphoblastic leukaemia cells express CCR7 but not higher amounts of IL-10 after CD40 ligation.

OBJECTIVE: Production of cytokines that support T-cell activation and proliferation and migration to lymph nodes is one of the most important terms of cancer vaccine development. In previous studies we and others used CD40 ligation to obtain higher expression of co-stimulatory and adhesion molecules on leukaemic cells from children with acute lymphoblastic leukaemia (ALL). This time we assess the cytokine and chemokine gene expression profile in CD40-stimulated ALL cells. MATERIAL AND METHODS: Malignant cells from 25 children with BCP-ALL were stimulated (or not) with huCD40LT and rIL-4 for 96 h. Eleven different molecule, cytokine and chemokine mRNAs levels (CCR7, IL-23, TGF-beta-IP, IFN-gamma, IL-10, CD1a, CD40, CD54, CD80, CD83, CD86) were determined using the real-time PCR technique with TaqMan chemistry using ready-to-use low-density arrays for gene expression by Applied Biosystems. RESULTS: 1) Increases in mRNA levels for CD40, CD54 and CD80 after CD40L and IL-4 stimulation were observed, 2) CCR7 mRNA expression was higher after CD40 ligation than before the culture (p = 0.002), 3) IL-10 mRNA expression was higher after the culture with medium than before the culture (p = 0.01). CONCLUSIONS: The results show that leukaemia-derived dendritic cells obtained with CD40 ligation express CCR7 - chemokine is involved in migration to lymph nodes and does not produce higher amounts of IL-10, a potent immunosuppressive cytokine. Our preclinical findings could be used in the design of immunotherapy trials for the treatment of children with ALL.

Low expression of costimulatory molecules and mRNA for cytokines are important mechanisms of immunosuppression in acute lymphoblastic leukemia in children?

UNLABELLED: Mechanisms leading blasts of acute lymphoblastic leukemia to escape from immune surveillance are still unknown. Only few reports showed that ALL cells are inefficient antigen presenting cells. The aim of the study was to assess expression of critical costimulatory/adhesion molecules and mRNA for main pro- and anti-inflammatory cytokines in ALL cells. Children with B-cell precursor ALL (n=20) were prospectively enrolled into the study. Expression of costimulatory/adhesion molecules (CD1a, CD11c, CD40, CD54, CD80, CD83, CD86, CD123, HLA class I and II) was assessed by flow cytometry and mRNA for cytokines (IFN-gamma, IL-10, IL-4, TGF-beta) - with real-time PCR. RESULTS: 1) high expression was observed for HLA I and II class, moderate for CD40, CD83, CD86 and low or no expression for CD80, CD54, CD1a, CD11c and CD123; 2) we found expression of mRNA for IFN-gamma, IL-10, IL-4 and TGF-beta in blasts cells (but not in all specimens). We noted relatively lower expression of all assessed cytokines comparing to T-cells obtained from healthy donors but interestingly expression for IL-10 was higher in normal B-cells than in blast cells, and IFN-gamma and IL-4 were not found in normal B-cells. In summary we suggest that ALL-blasts present low expression of costimulatory/adhesion molecules and mRNA for cytokines and this probably contribute to the absence of host T- cells stimulation to immune response.

The -675 4G/5G plasminogen activator inhibitor-1 promoter polymorphism in house dust mite-sensitive allergic asthma patients.

BACKGROUND: Plasminogen activator inhibitor (PAI)-1 plays an important role in inflammation and tissue remodeling. Recently, the -675 4G/5G PAI-1 polymorphism has been linked with asthma. OBJECTIVE: This study was undertaken to evaluate associations of the -675 4G/5G PAI-1 polymorphism with functional and immunologic parameters of newly diagnosed house dust mite-sensitive allergic asthmatics (HDM-AAs). METHODS: This study was performed in 127 HDM-AAs, who responded with at least 20% fall of forced expiratory volume during the first second (FEV(1)) to a bronchial challenge with Dermatophagoides pteronyssinus allergen and during the follow up observation fulfilled GINA criteria for mild-moderate asthma. About 89 healthy control nonatopic subjects (HCs) were used as controls. RESULTS: The frequency of 4G allele was greater in HDM-AAs (0.69; 95% CI: 0.62-0.76) than in HCs (0.55; 95% CI: 0.48-0.62; P = 0.0034). The PAI-1 polymorphism was associated with an increased risk of HDM-AA; adjusted for sex and age odds ratio was 2.62; (95% CI: 1.16-5.92) for 4G/5G genotype and 3.48 (95% CI: 1.54-7.89) for 4G/4G genotype compared with 5G/5G genotype. Total serum immunoglobulin E (tsIgE) level in 4G/4G homozygotes (557 +/- 343 kU/l) was significantly greater than in 5G/5G homozygotes (241 +/- 288 kU/l; P < 0.001). Both nonspecific and allergen-specific bronchial reactivities were greater in 4G/4G homozygotes than in 5G/5G homozygotes. 4G/4G genotype was associated with significantly higher morning plasma PAI-1 concentration in HDM-AAs and HCs. Morning plasma PAI-1 concentration correlated significantly with log(PC20) (r = -0.39; P = 0.0001) and with log(tsIgE) (r = 0.247; P = 0.0117). CONCLUSION: These results support the hypothesis linking the 4G/4G PAI-1 genotype with an increased risk of allergic asthma, bronchial hyperreactivity, and increased tsIgE levels.

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.

Use of synaptophysin immunohistochemistry in intestinal motility disorders.

The purpose of this study was to describe synaptophysin (SY) immunoreactivity in colonic specimens from patients with Hirschsprung's disease (HD), chronic constipation (CC), or anal atresia (AA). This membrane protein is specific for the synaptic vesicles in the central and peripheral nervous system and responsible for neurotransmission. Biopsy specimens of the intestinal wall were obtained from 18 patients (age range, 2 days to 7 years). Immunohistochemistry was performed using rabbit anti-human antibodies specific for synaptophysin (DAKO). In the ganglionic colon of HD patients and others the immunoreactivity of SY-positive synapses was abundantly present in the smooth muscle layers. Distinct immunoreactivity showed ganglion cells and nerve fibers inside circular and longitudinal muscle layers. In some non-HD patients' colonic specimens SY-positive synapses were present in the muscularis mucosae. In the aganglionic colonic segment of HD-patients no immunoreactivity of synapses and ganglions was seen. In the transition zone, where ganglion cells appeared sporadically, synapses were very rarely present. In two patients from the CC group the amount of visualized synapses was clearly smaller and the concentration of ganglion cells within ganglions in these cases was much lower than usual (but still within normal ranges). In the AA group in the distal part of the atretic rectum (at the place where the fistula was cut) SY-positive synapses were present in smooth muscle layers and small dysplastic ganglions were seen in the submucosal and muscular region, but not in large numbers. These patients had a normal distribution of ganglion cells and synapses at the place of colostomy. Synaptophysin immunohistochemistry is an indirect labeling method with a high detection rate for intestinal ganglion cells by demonstrating their synapses. Changed intestinal distributions of SY-positive synaptic vesicles usually accompany colonic ganglion cell disorders. The pattern of SY-positive synapses distribution in circular and longitudinal colonic muscles and intermuscular ganglions can reflect functional disturbances of large bowel motility and could be helpful in the description of the innervation status of colonic specimens in HD patients.

Attempts to detect Helicobacter pylori in atherosclerotic plaques.

Cardiovascular and cerebrovascular diseases are regarded to be the main causes of mortality in developed countries, atherosclerosis being at their pathological base. During the recent years, attention was paid to the role of bacterial infections, including Helicobacter pylori, in the process of atherogenesis and coronary heart disease development. The aim of the study was an evaluation of H. pylori presence--by means of PCR technique--in atherosclerotic changes, obtained by endarterectomy, performed during coronary artery bypass grafting (CABG). In the analysed group of patients, the following risk factors were found: hyperlipidaemia, smoking, hypertension, obesity, diabetes mellitus, cardiac infarction. No DNA of the bacteria was traced in any of the patients.

Clinical usefulness of K-RAS mutation detection in colorectal cancer and in surgical margins of the colon.

The incidence of K-RAS gene mutations in tumour and surgical margins was investigated in 63 patients with adenocarcinomas of varied clinical stage and histological grade. Point mutations of codon 12 K-RAS gene were detected, using the PCR-RFPL technique in cancer tissue in 23 patients (36.5%) and in colon margin mucosa in 1 patient (3.7%), out of 27 examined subjects. No significant correlations were found between the mutations and clinical features. Tumours, located in the left colon, and mucinous neoplasms displayed a higher incidence of mutations. No correlation was observed with either Dukes or TMN clinical advancement.