Experimental autoimmune myocarditis in rats and therapeutic histamine H1 - H4 receptor inhibition.

Myocarditis, a life threatening disease, is still not adequately treated. Histamine plays an important role in physiology and pathophysiology of cardiovascular system. All four histamine receptors (H1R - H4R), are present in the heart. Experimental autoimmune myocarditis (EAM) was used to investigate which histamine receptor had a greater impact on the disease's progression. EAM was evoked in Lewis rats by porcine myosin immunization. Mepyramine, ranitidine and ciproxifan were used to inhibit H1R, H2R and H3R receptors, respectively, and 2,4-diaminopyrimidines: ST994, ST1012, ST1006 were ligands of H4R. Quinapril, an ACE inhibitor, served as a reference drug. Drugs were administered daily, either from 0 - 2 weeks or from 2 to 4 weeks post EAM induction. Cardiac dysfunction developed with significant decreases in left ventricular ejection fraction and fractional shortening due to dilatation and wall thickening. EAM rats treated with mepyramine and ST994 in weeks 0 - 2 had the lowest decreases. These treated with ST994, ST1012 or quinapril performed much better the following 2 weeks without therapy than did the other groups. On autopsy their hearts were smaller, less fibrotic, histopathological changes in them of a lower grade. When the treatment started with 2 weeks' delay, the ST994-treated EAM rats showed the highest median survival. H4 receptor antagonism inhibits heart remodelling, preserves heart contractility, improves survival and may be of potent therapeutic relevance in human clinics. The blockade of H1 receptor inhibits heart dilatation but does not prolong the life.

Autophagy-related gene expression in colorectal cancer patients.

There is evidence that autophagy can play a dual role in tumor cells – as a tumor suppressor, and a process involved in tumor cell survival. The aim of this work was to assess the expression of the genes engaged in the autophagy process in biopsies taken from the colon, confirmed as adenocarcinoma, and normal tissue and to relate them to the clinical stage of the tumor. A total of 20 pairs of surgically removed tumors and healthy (marginal) tissue samples from colorectal cancer patients at clinical stages (CS) I-IV were analyzed. Gene expression profile analysis was performed using HG-U133A microarrays. Differentially expressed genes were identified, using the PL-Grid Infrastructure. Only for CSI, there were two specific genes: FOXO1 and BNIP1; further in CSII – LAMP2, MET and BCL2L, in CSIII – HIF1A and 2 ID mRNAs for HGF and 18 genes were specific for CSIV in comparison to controls. PINK1 is the only gene that differentiates all transcriptome groups from controls. Furthermore, examination of the expression of genes associated with the autophagy process may allow for better knowledge and understanding of the processes occurring during the development of colon cancer. The presented genes may be used as prognostic markers of clinical stages of colorectal cancer, contributing to the development of new lines of therapy focused on reducing metastasis of the primary tumor.

The prevalence of Chlamydia pneumoniae in the aortic wall and in peripheral blood of patients scheduled for coronary artery bypass grafting.

Some reports confirm a potential role of Chlamydia pneumoniae (ChP) in atherogenesis. In order to explore possible association between ChP and atherosclerosis, investigations were carried out in which the frequency of ChP in the arterial wall and peripheral blood was assessed in a group of patients with chronic coronary artery disease (CAD). Fifty-seven patients were enrolled in the study, 13 women and 44 men aged 61.8±6.5 (47-74), with previously diagnosed CAD, scheduled for planned coronary artery bypass grafting due to clinical indications. Vessel specimens retrieved from the ascending aorta (as a part of routine proximal venous graft development procedure) and peripheral blood mononuclear cells (PBMCs) from venous blood were evaluated for the presence of ChP DNA. Genomic DNA was extracted from PBMCs and vessel specimens. Quantitative real-time polymerase chain reaction (qPCR) was performed to detect ChP DNA. A statistically more frequent occurrence of ChP was observed in aortic tissues compared to blood samples (70.2% vs 56.1%, respectively). Similarly, the number of ChP DNA genomic copies [n/1µg genomic DNA] was significantly higher in tissue specimens compared to blood samples (89±91 vs 41±77, respectively; p=0.0046). In patients without ChP in blood specimens, we observed significantly higher amounts of ChP in tissue specimens compared to patients with ChP in blood specimens (156±71 vs 107±88, respectively; p=0.0453). No correlation was found between the number of ChP DNA copies [n/1µg genomic DNA] in blood and in aortic specimens. The infection of ChP in the aortic wall was connected with hypercholesterolemia (p=0.029) and diabetes (p=0.03). We conclude that Chlamydia pneumoniae is a pathogen frequently occurring in the aortic wall of patients with CAD. The occurrence of ChP DNA in the aortic tissue is related to classic CAD risk factors such as diabetes and dyslipidemia.

Epigenetic silencing of endothelin-3 in colorectal cancer.

Endothelins are expressed in a variety of human tissue and are involved in the processes as proliferation, migration and differentiation. The signal transduction pathway is a result of the endothelin-1-3 (ET1-3) binding to their receptors (ETAR, ETBR). ET-3 is a new candidate tumour suppressor gene, which is often downregulated or silenced in human cancer.The aim of the study was to examine DNA methylation of ET-3 genes in colorectal cancer (CRC) tissue samples in relation to the clinical stage (CS) of cancer. The paper is a continuation of our previously published results, which showed a four-fold transcriptional silencing of the ET-3 gene in the samples of colorectal cancer in comparison to normal tissues.A total of 66 paired CRC and normal (surgical margin) tissue samples were used in the study. The tumour tissues were collected from CRC patients in CS I-IV according the 7th edition of UICC TNM Classification of Malignant Tumours (CS I, n = 8; CS II, n = 20; CS III, n = 27; CS IV, n = 11). Assessment of epigenetic silencing of the ET-3 encoding gene was performed in three steps. The silencing of the ET-3 encoding gene was a result from methylation of the promoter sequence using methylation-specific PCR (MS-PCR). Analyses were performed using primers complementary for a CpG island in the first exon of the gene encoding ET-3. An epigenetic silence through methylation of 7.5% (5/66) in comparison to control was observed, including 10% of CS II (2/20), 7% of CS III (2/27) and 9% of CS IV (1/11). The controls and the samples of tumour in CS I showed no epigenetic silencing via methylation. In conclusion, epigenetic silencing of ET-3 in CRC could play a role in the progression than in the induction process. EDN3 would be a future target for epigenetic therapy in colorectal cancer, but further clinical studies are needed.

The profile of melatonin receptors gene expression and genes associated with their activity in colorectal cancer: a preliminary report.

The antiproliferative and immunomodulatory effects of melatonin (MLT) have been demonstrated in a variety of neoplasms including colorectal cancer (CRC). In humans and other mammals, MLT acts on target tissues through membrane and retinoid nuclear receptors. The aim of this study was to evaluate transcription activity of melatonin receptors and genes associated with regulation of their activity in colorectal adenocarcinoma tissues in relation to clinical stage of cancer. A total of 24 pairs of surgically removed tumoral and healthy (marginal) tissue samples from colorectal cancer patients at clinical stages I-II and III-IV were collected. As an additional control, twenty normal samples were tak¬en from people whose large intestine tissues were reported as non-tumoral after colonoscopy. Expression of mRNA genes was studied by microarray HG-U133A analysis. The analysis of gene expression profile was performed using commercially available oligonucleotide microarrays of HG-U133A. High increase of MT1 mRNA expression levels in all cancerous samples vs non-cancerous tissues was observed. The MT2 mRNA expression levels increased slightly in marginal and malignant samples. Among the genes participating in the cascade of signal transfer in cells activated by MLT via melatonin receptors, we found encoding genes (GNA11, OXTR, TPH1) only for differentiating stage III - IV of CRC. Monitoring the expression levels of genes that are related to melatonin receptors may offer a strategy to anticipate tumour development and estimate the molecular changes that occur during carcinogenesis. The mechanism behind this association needs further elucidation.

The effect of maximal physical exercise on relationships between the growth hormone (GH) and insulin-like growth factor 1 (IGF-1) and transcriptional activity of CYP1A2 in young ice hockey players.

BACKGROUND: The maximal physical exercise influences on changes in GH, IGF-1 concentrations and activity of CYP1A2. The aim of this study was to answer the question whether the change of endogenous GH and IGF-1 influenced the expression of CYP1A2 and whether these changes correlate with others parameters of blood and test. METHODS: The twenty ice young hockey players were subjected to maximal exercise cycle test. The test duration was mean 21.0 ± 2.4 min, and work done was mean 3261.3 ± 558.3 J/kg. In blood samples collected before and after the exercise were evaluated: concentrations of GH and IGF-1 which were determined in the serum by IRMA method and the transcriptional activity of CYP1A2 in RNA isolated from whole blood by the QRT-PCR reaction. RESULTS: Before the exercise the median of GH was mean 0.31 ± 0.23 µU/mL and after the exercise increased to mean 21.6 ± 16.6 µU/mL (P<0.001). Mean of serum IGF-1 level before the exercise and immediately after test did not change. Statistically significant increase in value of Ct for CYP1A2 (P<0.001) was found. CONCLUSION: The results of the study showed that: an intense exercise of young ice hockey players led to a significant increase in GH concentration, which caused the Ct values of CYP1A2 increase, whereas IGF-1 did not change. GH and IGF-1 levels after the exercise depended on the work done and the relative levels of CYP1A2 expression correlated with the time and the amount of work done by the athletes.

Differential expression of tripartite motif-containing family in normal human dermal fibroblasts in response to porcine endogenous retrovirus infection.

Antiretroviral restriction factors may play an essential role in the safety of xenotransplantation. Therefore, the present study focused on investigation of the changes in the tripartite motif-containing family (TRIM) gene expression in normal human dermal fibroblasts with and without lipopolysaccharide stimulation in response to porcine endogenous retrovirus infection. Analysis of the expression profile of TRIMs was performed using oligonucleotide microarrays and QRT-PCR. Nine (TRIM1, TRIM2, TRIM5, TRIM14, TRIM16, TRIM18, TRIM22, TRIM27 and TRIM31) statistically significantly differentially expressed genes were found (P < 0.05, one-way ANOVA). In conclusion, comprehensive analysis of retroviral restriction factor gene expression in human dermal fibroblasts before and after porcine endogenous retrovirus infection with and without LPS stimulation may suggest association of the selected TRIMs with antiretroviral activity.

Expression of ADAM28 and IGFBP-3 genes in patients with colorectal cancer - a preliminary report.

Adamalisynes (ADAMs) play an important role in inter-membrane interactions, cell adhesion and fusion processes and protein shedding from the cell surface. Many reports indicate that members of the ADAMs family are overexpressed in human cancer. The aim of the present study was to evaluate ADAM28 and Insulin Like Growth Factor Binding Protein-3 (IGFBP-3)) gene expression in colorectal carcinoma tissues with regard to the overweight or obese status of the patients using an oligonucleotide microarray technique. Fresh tissue specimens were obtained from colorectal cancer patients during surgical treatment. Eighteen specimens from tumour and 18 normal tissue specimens from colorectal cancer patients at clinical stages III and IV were analysed. The examined patients were divided into two groups; those with BMI greater than or equal to 25 and those with normal BMI. The control group consisted of 18 specimens of non-neoplastic colon tissues, which were divided between overweight/obese and normal body weight patients. The gene transcriptional activity from the specimens was analysed using an oligonucleotide microarray technique. Microarrays and rinsing and marking solutions were prepared according to the procedure in the Gene Expression Analysis Technical Manual. The following conclusions were made: i) change of ADAM28 and IGFBP-3 genes expression are present in the normal tissue in overweight/obese patients with colorectal cancer only; ii) the observed molecular variability of ADAM28 and IGFBP-3 expression may be an initial process of cancer proliferation; iii) the histopathologically normal surgical margin in this group of patients was not equal to the molecular margin.

CATECHOLAMINES AND ß2-ADRENOCEPTOR GENE EXPRESSION BEFORE AND AFTER MAXIMAL INCREMENTAL CYCLE TEST IN YOUNG ICE HOCKEY PLAYERS: RELATION TO WORK PERFORMED.

The aim of this study was to assess the plasma adrenaline and noradrenaline concentrations as well as whole blood ß2-adrenoceptor gene (ADRB2) expression in young ice hockey players before and immediately after exercise in relation to performed work. Nineteen Youth National Team ice hockey players were subjected to the maximal incremental cycloergometer exercise. The test was done in the pre-competitive phase of training. Among many parameters the plasma adrenaline and noradrenaline concentrations and ADRB2 gene expression in peripheral blood mononuclear cells (PBMC) were determined before and after exercise. The average performed work was 3261.3 ± 558.3 J · kg(-1) and maximal oxygen consumption (VO2max) for all players was 53.85 ± 3.91 mL · kg(-1) min(-1). The geometric mean of the ADRB2 gene expression was statistically significantly different before and after exercise (P ≤ 0.05), while adrenaline and noradrenaline levels in plasma significantly increased after exercise. In the analysed group of athletes we found that initial level of plasma noradrenaline correlated with the performed work (r = - 0.55, P < 0.014) and normalized ADRB2 expression before the exercise correlated with the work done by them (r = 0.48, P<0.039). However, no statistically significant correlations were found between the plasma adrenaline or noradrenaline concentrations and ADRB2 gene expression in peripheral blood of the players. The performed work in the maximal incremental exercise test of regularly training young ice hockey players depends on the initial levels of noradrenaline in plasma and ADRB2 mRNA in PBMC.

Expression of genes associated with H factor in fibroblasts infected with Borrelia spirochaetes.

Infection with Borrelia spirochaetes leads to the launch of specific and non-specific immunological response in humans. Activation of the complement system is one of the first defence mechanisms against penetrating pathogens. The aim of this study was to select genes related to the alternative pathway of the complement system [including complement factor H (CFH)], differentiating the type of infection in the system model, that is, a culture of normal human diploid fibroblasts infected with three different spirochaete genospecies: Borrelia afzelii, Borrelia garinii and Borrelia burgdorferii sensu stricto, by comparing the infected fibroblast culture with the control fibroblast one. With the use of oligonucleotide microarrays HGU-133A, the differences in the expression of genes selected on the basis of a scientific database Affymetrix were studied by comparing transcriptomes from the four cultures of fibroblasts. In the result of infection of fibroblast cultivation with a specific Borrelia genospecies, a variable expression of certain CFH and complement system-associated genes, specific for one genospecies only, B. afzelii- C1QBP, CD59, C2, CD46 and FHL1; B. garinii - C1S and CLU; Borrelia burgeforii- CFB, A2M and VSIG4, was observed. CFH differentiates infections induced by B. afzelii and B. garinii from infections induced by B. burgdorferii sensu stricto.

Posttraumatic temporal TGF-ß mRNA expression in lens epithelial cells of paediatric patients.

The aim of the study was to determine temporal TGFB1, TGFB2 and TGFB3 gene expression profiles in the anterior lens capsule of paediatric patients with posttraumatic cataract. The patient group comprised 22 children selected with a fragment of anterior lens capsule obtained during elective cataract surgery and sampled for molecular analysis. The levels of TGF-ß isoforms in the anterior lens capsule were determined based on the number of mRNA copies per 1 µg total RNA by real-time qRTPCR. Three time-related result clusters were identified based on hierarchical cluster analysis: 2.2, 4.4 and 15.0 months (time span from injury to anterior capsule sampling during surgery) and compared with regard to temporal gene expression profile and quantitative relations of TGF-ß1, 2 and 3 mRNAs. TGF-ß1, TGF-ß2, and TGF-ß3 mRNAs were detected in all anterior lens capsule samples. A comparative analysis revealed: TGF-ß1>TGF-ß2>TGF-ß3 during the entire observation period. The TGF-ß mRNA levels continued to increase up to four months after injury, then returning close to the base levels after around 15 months. The expression patterns of TGF-ß isoforms showed a similar tendency. Differences in the expression levels of TGF-ß1 and TGF-ß2 between the particular clusters were statistically significant. Posttraumatic transcriptional activities of TGF-ß1 and TGF-ß2 in the anterior lens capsule of paediatric patients depend on the time elapsing from injury. Our findings indicate that the transcriptional activities of TGFB family genes show a transient period of over-expression during the months after injury. TGF-ß1 is a dominant isoform expressed in lens epithelial cells following injury.

Analysis of expression profile of gene encoding proteins of signal cascades activated by insulin-like growth factors in colorectal cancer.

The aim of the study is to analyse gene typing with the use of the microarray technique (HG-U133A, Affymetrix), differentiating colorectal cancer tissues from tissues assessed histopathologically as healthy ones among a panel of 93 mRNA of gene encoding proteins involved in the activation of cellular signal transduction pathways by insulin-like growth factors. The study was conducted on a group of 8 colorectal cancer patients. Frozen tumor and healthy specimens from the patients were used in molecular tests. Transcript IGF2 differentiated cancer from healthy tissue. Among the genes participating in the cascade of signal transfer in cells activated by IGF, GRB10, PIK3R3, PIK3R1, and IRS1 were qualified as differentiating transcripts. IRS1 indicated over-expression in tumour. Transcript SMAD2 showed a significant changed in tumour samples (increased expression).

Statistical analysis of differential gene expression in colorectal cancer using CLEAR-test.

CLEAR test provides a novel method of analysis by combining inference for differential expression and variability. Frozen tumor specimens from 14 (3 coded Stage I, 5 Stage II, 2 Stage III and 4 Stage IV) colon cancer patients were obtained. Archived primary tumor samples were collected at the time of surgery and normal colon mucosae (controls specimens) were also collected. The studied transcriptomes were clustered using hierarchical agglomeration with Ward's method and Tchebychev distance. The separable groups of transcriptomes were classified as high clinical stage of adenocarcinoma (HCS; stages II-IV), low clinical stage of adenocarcinoma (LCS; stages I and 3 controls), and two normal colon mucosae (controls N1 and N2). The results of the CLEAR-test algorithm in normal colon specimens and adenocarcinoma specimens with low and high clinical stage showed 50 most and 50 least significant genes. The list of differential genes (p<0.01) in normal colon specimens and adenocarcinoma specimens with low and high clinical stage presented 58 genes.

Leptin and its receptors in obese patients with colorectal cancer.

The purpose of this study is to examine serum concentration of leptin and that of the soluble form, the Ob-Re receptor, in patients with colorectal cancer, as well as to examine the level of leptin mRNA and that of its receptors, Ob-Ra and Ob-Rb, in large intestine specimens collected from patients with colorectal cancer, depending on cancer clinical and pathological progression and BMI. A total of 146 patients with colorectal cancer in a I-IV stage scale according to the TNM Classification were enrolled. The patients were divided into two groups according to BMI calculations based on body weight and height: a Study group (BMI greater than or equal to 25 kg/m2) of 75 patients aged 57 plus or minus 4.5 years and a Control group (20 less than BMI less than 25 kg/m2) of 71 patients aged 60 plus or minus 5 years. The experimental part of the work was performed in two stages: Stage I regarding the assay of leptin concentration and that of its soluble receptor, Ob-Re, in the serum of patients with the use of the ELISA method; and Stage II to determine the number of leptin mRNA copies and two isoforms of leptin receptors, Ob-Ra and Ob-Rb, using the QRT-PCR method in tissue specimens collected from 146 patients. In our results the concentration of serum leptin and Ob-Re was not dependent on the stage of clinical and pathological progression of the cancer. There was a statistically significant higher serum leptin level in colon cancer patients who were overweight or obese compared to patients with normal weight. No presence of mRNA of the gene encoding leptin was found in tissues collected from colorectal cancer patients. The number of mRNA copies of Ob-Rb was statistically significantly higher in all the study groups compared to the reference tissues.

Transcriptional activity of telomerase complex in CD34- stem cells of cord blood in dependence of preparation time.

The aim of the study was to determine whether the expression of telomerase subunits encoding genes changes during the process of cord blood preparation. It should establish if the commonly accepted 24 hours time interval in stem cells kriopreservation procedure significantly influences their immortalization and so decreases the "quality" of cord blood stem cells. Investigation includes 69 women. Spontaneous labour was the inclusion condition. The material was collected at birth after clamping of umbilical cord by direct vasopuncture. CD34- cells were extracted from cord blood (MACS, Miltenyi Biotec; Bisley, Surrey, UK). The expression profile of telomerase activators and inhibitors encoding genes was determined using HG_U133A oligonucleotide microarray (Affymetrix). We used a real-time quantitative RT-PCR assay to quantify the telomerase TERT, hTR and TP1 subunits mRNA copy numbers in CD34- cells in 0, 6, 12 and 24 hours after cord blood collection. We observed significant decrease of numbers of copies of TERTA+B mRNA within the successive hours of observation. Significant decrease of numbers of TERTA mRNA copies was confirmed after 24 hours. However, we observed significant increase of numbers of copies of TERTB mRNA after 6 hours of observation. Similar level was maintained during another 6h. The significantly lower number of copies of TERTB mRNA was observed after 24h. We also observed significant increase of number of copies of TERT mRNA after 6 hours. Number of copies of TERT mRNA significantly decreased after another 6h, remaining, however, on a higher then initial one. The significant lower number of copies of TERT mRNA was observed 24h after delivery. The possible explanation of those results is discussed in the paper.

Transcriptional activity of genes-encoding kinin B1 and B2 receptors and kinin-dependent genes in nasal polyps.

PURPOSE: The pro-inflammatory effects of kinins are mediated by two bradykinin receptors: BR1 and BR2. The aim of this study was to evaluate the expression profile of kinin receptor genes by an estimation of mRNA levels in human nasal polyps (NP) and normal mucosa (NM). MATERIAL AND METHODS: BR1 and BR2-dependent genes differentially transcribed in NP were investigated using oligonucleotide microarray technology. The mRNA copy number of BR1, BR2 and TIMP1 genes was assessed by QRT-PCR. Thirty six eosinophilic (ENP), 17 neutrophilic nasal polyps (NNP) and 28 NM samples were included into the study. RESULTS: Among 92 genes encoding proteins involved in signal transduction via B1 and B2 kinin receptors TIMP1 was found to be 2,63-fold higher in the NP than in NM. Increased TIMP1 gene expression was proved by QRT-PCR (p=0,003). Moreover two genes: FOS and PTGS1 presented higher (3,82- and 4,27-fold, respectively) expression in NM compared to NP tissues. In QRT-PCR analysis insignificantly higher expression of gene encoding BR1 in ENP [2564 mRNA copies/microg RNA (22-32863)] compared with NM [1426 copies mRNA (15-27995)] was found. mRNA expression for the BR2 in ENP [9872 copies mRNA (19-244832)] was insignificantly higher than in NM [5753 copies (46-199658)]. BR2 mRNA was the predominant transcript in most NP and NM samples followed by BR1 mRNA (p<0,01). There was a positive correlation between the expression of BR1 and BR2 in the ENP (r=0,91; p<0,01) and NNP (r=0,6; p<0,01). CONCLUSIONS: We did not document any changes in the expression profile of kinin receptors in the analyzed groups, which may suggest that kinin receptors do not make an important contribution in the etiology of NP.

Toll-like receptors and the tendency of normal mucous membrane to transform to polyp or colorectal cancer.

The aim of this study was to identify differences in Toll-like receptor (TLR) expression patterns in normal and diseased tissues of patients with polyps and colorectal cancer. Eight patients were included in the study group (aged 38 to 72 years). Sixteen HG-U133A oligonucleotide microarrays were analysed including four of colonic polyps, four of adenocarcinoma with different degree of histological differentiation (2 poorly and 2 highly differentiated), and eight of macroscopically normal tissue. The levels of selected TLR mRNA transcripts were analysed. An analysis of all per cent variability values with regard to malignancy stage increasing from polyp to stages I to III adenocarcinoma, and normal colon mucosa shows a statistically significant relationship for TLR2 (increasing) and TLR3 (decreasing). In polyps, copy numbers of TLR3, TLR4 and TLR5 mRNA were the highest and TLR7 mRNA the lowest. In normal colon mucosa of polyposis patients the highest mRNA copy numbers were observed for TLR3, and the lowest for TLR7. TLR3 may serve as a marker of colon tissue metaplasia and may indicate the tendency of normal tissue to form polyps transforming to colorectal cancer.

Quantification of the mRNA encoding Tumor Necrosis Factor alpha (TNFalpha) and its receptors in human nasal polyps.

PURPOSE: The object of the study was to assess the expression of the genes encoding TNFalpha and its receptors (TNF-R1 and TNF-R2) in patients with nasal polyps (NP). MATERIAL AND METHODS: The number of the mRNA copies was assessed by QRT-PCR in RNA extracts from 16 eosinophilic (ENP) and 5 neutrophilic nasal polyps (NNP), and 9 normal mucosa (NM) samples. The expression of corresponding proteins was demonstrated using immunohistochemistry. RESULTS: The mean level of mRNA copies for TNFalpha in ENP (82229c/microg) was not significantly higher when compared with controls (74869c/microg). NNP demonstrated significantly lower mean TNFalpha gene expression (7021c/microg) than the controls (p<0.05). A statistically higher mRNA TNFalpha copy number in ENP than in NNP was also revealed (p<0.01). A noticeably lower mRNA expression of TNF-R1 in ENP and NNP was seen as compared to the control group (10198c/microg vs. 30749c/microg, p<0.05 and 3440c/microg vs. 30749c/microg; p<0.05 respectively). In ENP the mean TNF-R2 mRNA copy number was markedly higher than in NNP (185c/microg vs. 7.6c/microg, p<0.05). TNF-R2 mRNA level did not differ significantly between ENP and the control group (185c/microg vs. 469c/microg). TNF-R1 expression was significantly higher than TNF-R2 at the mRNA (p<0.01) and protein (p<0.05) level both in ENP and NNP. No significant correlations in proteins expression were detected between ENP and NNP. CONCLUSIONS: TNF-R1 has been identified to be a prevalent form of the TNFalpha receptor in nasal polyps which may reflect the apparent dominance of this form in TNFalpha signalling. The findings raise the possibility that the eosinophils from NP may influence biological responses through TNFalpha-dependent mechanisms. The differences between ENP and NNP relating to TNFalpha and the expression of its receptors may reflect the distinct character of those diseases.

Quantitative analysis of transforming growth factor beta isoforms mRNA in the human corneal epithelium.

TGF-beta is an important mediator of cell growth, differentiation, and proliferation and plays a significant role in both normal and pathological corneal tissue. However, the quantitative relations between TGF-beta1, -beta2 and -beta3 isoforms in human cornea still remain unclear. Therefore, the aim of this study was to determine the gene expression profile of TGF-betas in order to evaluate quantitative relations between the examined transcripts in human corneal epithelium. Transcriptional activity of TGF-beta1, 2, 3, GAPDH and beta-actin genes was estimated on the basis of mRNA copy number per 1 microg of total RNA using the real-time QRT-PCR technique with the SYBR Green I chemistry. Specificity of RT-PCR reaction was confirmed by determination of the characteristic melting temperature for each amplimer. Additionally, the RT-PCR products were separated on 6% polyacrylamide gels and visualized with silver salts. Expression of all TGF-beta genes for the corneal epithelium was determined. Comparable analysis of mRNA copies/1 mug of total RNA for each TGF-beta isoform showed that: TGF-beta1 > TGF-beta2; TGF-beta3 > TGF-beta2; TGF-beta1 = TGF-beta3 (ANOVA test P < 0.0001; post-hoc Tukey's test: TGF-beta1 and TGF-beta2, P = 0.0306; TGF-beta3 and TGF-beta2, P = 0.0045; TGF-beta1 and TGF-beta3 NS). We found different expression of the TGF-beta1, -2 and -3 isoforms in the human corneal epithelium. Such differential expression of TGF-betas suggests that each of them may play a specific role in corneal tissue.