Bladder cancer-derived interleukin-1 converts the vascular endothelium into a pro-inflammatory and pro-coagulatory surface.

BACKGROUND: Bladder cancer cells orchestrate tumour progression by pro-inflammatory cytokines. Cytokines modulate the local tumour microenvironment and increase the susceptibility of tumour distant tissues for metastasis. Here, we investigated the impact of human bladder cancer cell derived factors on the ability to modulate and activate human vascular endothelial cells. METHODS: The pro-inflammatory and pro-coagulatory potential of four different bladder cancer cell lines was accessed by qRT-PCR arrays and ELISA. Modulation and activation of endothelial cells was studied in microfluidic devices. Clinical relevance of our findings was confirmed by immune histology in tissue samples of bladder cancer patients and public transcriptome data. RESULTS: The unbalanced ratio between interleukin (IL)-1 and IL-1 receptor antagonist (IL-1ra) in the secretome of bladder cancer cells converted the quiescent vascular endothelium into a pro-adhesive, pro-inflammatory, and pro-coagulatory surface. Microfluidic experiments showed that tumour cell induced endothelial cell activation promoted leukocyte recruitment and platelet adhesion. Human bladder cancer tissue analysis confirmed that loss of IL-1ra and elevated IL-1 expression was associated with enhanced cancer progression. CONCLUSIONS: Our data indicate that IL-1 and IL-1ra were dysregulated in bladder cancer and could facilitate tumour dissemination through endothelial cell activation. Targeting the IL-1/IL-1ra axis might attenuate tumour-mediated inflammation and metastasis formation.

May the morphological findings in the first-trimester abortion materials be indicative of inherited thrombophilia?

PURPOSE: Inherited thrombophilia is associated with severe pregnancy complications including recurrent spontaneous abortion. In the light of this strong association, the impact of thrombophilic mutations on the placenta and their morphological reflections has aroused attention of both clinicians and pathologists. In the present study, we aimed to show the association between placental abnormalities with thrombophilia by examining the morphological findings in a wide range of first-trimester chorionic villi. METHODS: We performed a histological examination on the abortion specimens obtained from 129 patients with recurrent pregnancy losses that were evaluated with respect to inherited thrombophilia based on the presence of Factor V Leiden (G1691A), Prothrombin G20210A and methylenetetrahydrofolate reductase (MTHFR) C677T gene mutations detected by genetic analysis. Abortion materials either with and without thrombophilia were evaluated in terms of the morphological parameters such as hydropic change, vascularity, fibrosis, fibrinoid degeneration, Hofbauer macrophage, syncytiotrophoblast knotting, villitis, calcification, villous contour and villous size, hemorrhage, thrombus, proliferation of trophoblasts, villous stromal or villous vascular karyorrhexis. RESULTS: No statistically significant difference was found between the patient groups with and without thrombophilia in terms of morphological findings except vascularity of chorionic villi. The avascular chorionic villi (<3 vessels per villus) were found in 62.9% and 16.9% obtained from the women with and without thrombophilic mutation, respectively. This difference was statistically significant (P < 0.001). CONCLUSION: As a conclusion, it could be stated that the analysis of morphological findings in the abortion specimen is not a time-wasting process. Particularly, data related with vascularity of chorionic villi would be precious and beneficial. We suggest that highlighting the presence of avascular villi in the pathology report as a parameter would be guiding for clinicians regarding the impact of the thrombophilic gene mutations.

Higher Levels of Serum TLR2 and TLR4 in Patients with Hashimoto's Thyroiditis.

OBJECTIVE: Hashimoto's thyroiditis (HT) is an autoimmune disorder caused by the interaction between genes and environmental triggers. HT is the most common endocrine disorder, as well as the most common cause of hypothyroidism. Autoimmunity plays a crucial role in the pathogenesis of HT and recent studies suggest that Toll-like receptor (TLR) signals lead to increased inflammatory response. The aim of our study is to investigate whether TLR-2 and TLR-4 levels and gene polymorphisms contribute to the damaged immune response leading to HT. METHODS: Using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, single-nucleotide polymorphisms (SNPs) of TLR2 gene Arg677Trp, Arg753Gln, 196-174 del and TLR4 gene Asp299Gly, Thr399Ile were studied in 100 patients with HT and 100 healthy controls. Also, we investigated serum levels of TLR-2 and TLR-4 in the immunopathogenesis of HT. TLR-2 and TLR-4 serum levels were found to be significantly higher in HT patients than the control group. However, no statistical significance was found between patient and control groups in terms of genotype frequencies and allele frequency distribution of TLR2 gene Arg677Trp, Arg753Gln, 196-174 del and TLR4 gene Asp299Gly, Thr399Ile polymorphisms. RESULT: TLR2 gene Arg677Trp, Arg753Gln, 196-174 del and TLR4 gene Asp299Gly, Thr399Ile polymorphism do not appear to have a role in the development of HT disease. However, in our study, serum levels of TLR-2 and TLR-4 were found to be higher in HT patients than control groups. CONCLUSION: These findings suggest that TLR-2 and TLR-4 play an important role in the immunopathologic mechanism of disease by causing an increase in proinflammatory response.

Predictors of prenatal distress and fear of childbirth among nulliparous and parous women.

BACKGROUND: Prenatal distress and fear of childbirth negatively affect the health of the mother and the fetus. Sociodemographic and pregnancy related characteristics may influence prenatal distress and fear of childbirth. AIM: This study aimed to explore the relationship between fear of childbirth and prenatal distress levels with accompanying factors. SUBJECTS AND METHODS: The study was designed as a cross-sectional survey study and conducted in the outpatient clinic of Obstetrics and Gynecology Department of Pamukkale University Hospital, Denizli, Turkey, between April 2017 and January 2018. Survey data were collected from 103 third-trimester pregnant women who had admitted to the hospital for routine prenatal examination. Sociodemographic Information Form, the Revised Prenatal Distress Questionnaire (NUPDQ), and the Wijma Delivery Expectancy/Experience Questionnaire (W-DEQ) were used to collect data. Sociodemographics, obstetrics, and other variables were summarized by descriptive statistics. Mann-Whitney U-test, Chi-squared test, and Fisher's exact test were used for comparison of data between groups. RESULTS: The mean score of NUPDQ was 7.58 (SD 4.09) in the nulliparous group and 8.17 (SD 5.16) in the multiparous group (P = 0.68). The mean W-DEQ score was 40.46 (SD 21.80) in nulliparous women and 45.55 (SD 26.72) in multiparous women (P = 0.38). The W-DEQ and NUPDQ scores were moderately correlated with a Spearman correlation co-efficient of 0.58 (P < 0.001). CONCLUSIONS: The results of this study revealed that fear of childbirth and prenatal distress were moderately and positively correlated. NUPDQ and W-DEQ can be used during pregnancy to understand if pregnant women have fear or distress. This could help to give a better support to pregnant women.

[The development of real-time multispectral imaging for the diagnostics of bladder cancer]. / Entwicklung der Echtzeitmultispektralbildgebung für die Diagnostik des Harnblasenkarzinoms.

The performance of white light (WL) cystoscopy in the diagnostics of bladder cancer can be optimized by the use of modern imaging modalities, such as photodynamic diagnostics (PDD) and narrow band imaging (NBI). Real-time multispectral imaging (rMSI) enables simultaneous imaging of reflectance and fluorescence modalities in multiple spectral bands. We created a multiparametric cystoscopy image by digital overlapping of several modalities, e.g. WL, enhanced vascular contrast (EVC), raw fluorescence mode, protoporphyrin IX and autofluorescence (AF). The technical development and the subsequent clinical implementation of rMSI required a structured preclinical evaluation process, including both ex vivo and in vivo trials before the technology can be applied in patients. This review article presents the phases of testing, validation and the first clinical application of rMSI in urological endoscopy.

Anaesthetic Management of Patients Undergoing Bariatric Surgery.

OBJECTIVE: To describe perioperative anaesthetic management with laparoscopic sleeve gastrectomy (LSG). STUDY DESIGN: An observational study. PLACE AND DURATION OF STUDY: Department of Anesthesiology, Ondokuz Mayis University, Turkey, between January 2012 and December 2017. METHODOLOGY: Patients who underwent LSG at the study centre were considered. Hospital records were retrospectively reviewed. Information was collected on demographic characteristics, comorbidities, haemodynamic parameters, airway and anaesthetic management and complications. RESULTS: The study included 95 patients (mean age, 37.4±12.1 years; mean body mass index, 46 Kg/m2). Despite high airway assessment scores in some patients, 93 patients (98%) were conventionally intubated using our modified ramp position. Anaesthesia induction involved propofol, and anaesthesia maintenance involved inhalation anaesthetics (remifentanil supplementation). Additionally, rocuronium and sugammadex were used. Postoperative pain was managed with multimodal analgesia. Dose calculations were mostly based on lean/ideal body weight. Significant differences were found in the mean arterial pressure, heart rate and arterial oxygen saturation before induction and 5 min after induction. Intraoperatively, 3 patients (3.2%) developed bronchospasm and 1 (1.1%) developed bradycardia. There were no postoperative complications. CONCLUSION: Inhalational anaesthesia with remifentanil and rocuronium-sugammadex is a safe option in bariatric surgery. Although conventional techniques are sufficient to establish the airway in most cases, preparations for difficult intubation should be made. Furthermore, careful patient selection, preoperative anaesthetic management planning and appropriate postoperative monitoring are necessary.

THE RELATIONSHIP BETWEEN CONGENITAL HEART DEFECTS AND e-NOS GENE IN DOWN SYNDROME.

The aim of the study was to compare the effects of three eNOS gene polymorphisms associated with congenital heart defects, between Down syndrome patients with and without cardiac anomalies. Transthoracic echocardiographic examinations and eNOS single-nucleotide polymorphisms were investigated on seventy-five patients, prospectively. The frequencies of mutant alleles in the eNOS promoter (the -786T/C polymorphism) and exon 7 mutant alleles (the 894G--->T polymorphism) were significantly higher in Down syndrome patients with and without cardiac anomalies. The frequency of the intron GIOT polymorphism did not significantly differ between patients with and without cardiac anomalies. We found a significant relationship between eNOS gene polymorphisms and the congenital heart defects in patients with Down syndrome. Screening for the presence or absence of eNOS polymorphisms may be useful to obtain preliminary data on the risk of congenital heart defects in patients with Down syndrome.

Does higher NORs expression affect the developmental stages of Down syndrome infants?

BACKGROUND: The nucleolar organizer regions (NORs) are localized at the secondary constriction of the five pairs of acrocentric chromosomes (13, 14, 15, 21 and 22) in human. MATERIALS AND METHODS: To evaluate whether increasing AgNOR protein synthesis effects or not the development of babies/children, 25 Down syndrome patients were included in this study. Firstly, the Ankara Development Screening Inventory (AGTE) test was performed. Then the buccal epithelial cells of patients were taken via a sterile toothpick on clean glass slides and spreaded and AgNOR staining technique was applied to the slides of each individual. Mean NOR area/Total nucleus area (NORa/TNa) were evaluated for each nucleus using a special computer program. RESULTS: The mean NORa/TNa was found to be 3.8+/-1.16. According to these data, a significant correlation was not evident between the NORa/TNa and developmental stages (p>0.05). CONCLUSIONS: There is no correlation between extra energy spending for NOR protein synthesis and developmental deficiency.

Differential regulation of human and mouse telomerase reverse transcriptase (TERT) promoter activity during testis development.

In adult testis, telomerase activity is exclusively detectable during the early steps of spermatogenesis and is downregulated during differentiation to spermatozoa. Knowledge about telomerase activity during testis development from birth to adulthood is still scarce. Telomerase activity is regulated primarily via the transcriptional initiation of TERT expression which encodes its catalytic subunit. We used the hTERTp-lacZ transgenic mouse model that expresses the bacterial lacZ reporter gene under the control of an 8.0-kbp human TERT promoter fragment to analyze simultaneously endogenous mouse Tert gene expression as well as human TERT promoter activity during mouse testis development. We show that human TERT promoter activity increased during puberty and was highest in adult mouse testis whereas mouse Tert expression and telomerase activity were found to be high in testis from the earliest time point tested (6 days postpartum). Histological analysis revealed that beta-galactosidase expression, encoded by the lacZ reporter gene, is present in all seminiferous tubules in adult testis, but in a subset of tubules before puberty. We further analyzed the expression of SCF/ c-KIT, which was described to regulate spermatogonia proliferation and mouse Tert expression, in prepubertal and adult testis by immunohistochemistry. Whereas SCF and c-KIT were detectable in all seminiferous tubules, a spatial and preclusive expression pattern of human TERT promoter activity and activated c-KIT (p-c-KIT Tyr 567/569) was observed in prepubertal testes.

Telomeres and telomerase. A survey about methods and recent advances in cancer diagnostic and therapy.

Since the discovery that telomerase is repressed in most normal human somatic cells but strongly expressed in most human tumours, telomerase emerged as an attractive target for diagnostic, prognostic and therapeutic purposes to combat human cancer. In this review, a synopsis of methods detecting telomerase is presented evaluating their potential for diagnostic and prognostic use. Also, the most promising telomerase therapeutics are discussed in the light of recent advances in the field.

Characterization of the mouse gene for the heavy metal-responsive transcription factor MTF-1.

MTF-1 is a zinc finger transcription factor that mediates the cellular response to heavy metal stress; its targeted disruption in the mouse leads to liver decay and embryonic lethality at day E14. Recently, we have sequenced the entire MTF-1 gene in the compact genome of the pufferfish Fugu rubripes. Here we have defined the promoter sequences of human and mouse MTF-1 and the genomic structure of the mouse MTF-1 locus. The transcription unit of MTF-1 spans 42 kb (compared to 8.5 kb in Fugu) and is located downstream of the gene for a phosphatase (INPP5P) in mouse, human, and fish. In all of these species, the MTF promoter region has the features of a CpG island. In both mouse and human, the 5' untranslated region harbors conserved short reading frames of unknown function. RNA mapping experiments revealed that in these two species, MTF-1 mRNA is transcribed from a cluster of multiple initiation sites from a TATA-less promoter without metal-responsive elements. Transcription from endogenous and transfected MTF-1 promoters was not affected by heavy metal load or other stressors, in support of the notion that MTF-1 activity is regulated at the posttranscriptional level. Tissue Northern blots normalized for poly A+ RNA indicate that MTF-1 is expressed at similar levels in all tissues, except in the testes, that contain more than 10-fold higher mRNA levels.

Expression of the hTERT gene is regulated at the level of transcriptional initiation and repressed by Mad1.

Telomerase, an enzymatic activity responsible for the replication of chromosome end structures, is strongly upregulated in most human cancers. In contrast, most differentiated tissues are telomerase negative. The rate-limiting step for telomerase activity seems to be the expression of the catalytic subunit of the enzyme, encoded by the human telomerase reverse transcriptase (hTERT) gene. The precise mechanism of how hTERT is regulated has not been elucidated yet. We show here that the down-regulation of hTERT mRNA during 12-O-tetradecanoylphorbol-13-acetate-induced differentiation of human U937 cells is a consequence of a fast decrease in the rate of transcription rather than changes in its half-life. The only transcription factor that has so far been implicated in the regulation of hTERT expression is the c-Myc oncoprotein. Our analysis shows that another member of the myc/marx/mad network, mad1, encoding a transcriptional repressor that is significantly increased by 12-O-tetra-decanoylphorbol-13-acetate treatment, represses hTERT promoter-driven reporter gene activity in transient transfection assays. This effect is dependent on the NH2 terminal domain of Madl, which mediates the association with the transcriptional corepressor mSin3. Our findings suggest the involvement of an additional transcription factor in the regulation of hTERT expression and may provide a model for how hTERT activity is controlled during the differentiation process in human somatic tissues.

Embryonic lethality and liver degeneration in mice lacking the metal-responsive transcriptional activator MTF-1.

We have shown previously that the heavy metal-responsive transcriptional activator MTF-1 regulates the basal and heavy metal-induced expression of metallothioneins. To investigate the physiological function of MTF-1, we generated null mutant mice by targeted gene disruption. Embryos lacking MTF-1 die in utero at approximately day 14 of gestation. They show impaired development of hepatocytes and, at later stages, liver decay and generalized edema. MTF-1(-/-) embryos fail to transcribe metallothionein I and II genes, and also show diminished transcripts of the gene which encodes the heavy-chain subunit of the gamma-glutamylcysteine synthetase, a key enzyme for glutathione biosynthesis. Metallothionein and glutathione are involved in heavy metal homeostasis and detoxification processes, such as scavenging reactive oxygen intermediates. Accordingly, primary mouse embryo fibroblasts lacking MTF-1 show increased susceptibility to the cytotoxic effects of cadmium or hydrogen peroxide. Thus, MTF-1 may help to control metal homeostasis and probably cellular redox state, especially during liver development. We also note that the MTF-1 null mutant phenotype bears some similarity to those of two other regulators of cellular stress response, namely c-Jun and NF-kappaB (p65/RelA).

Mutants in position 69 of the Trp repressor of Escherichia coli K12 with altered DNA-binding specificity.

Structural analysis by X-ray crystallography has indicated that direct contact occurs between Arg69, the second residue of the first helix of the helix-turnhelix (HTH) motif of the Trp repressor, and guanine in position 9 of the alpha-centred consensus trp operator. We therefore replaced residue 69 of the Trp repressor with Gly, Ile, Leu or Gln and tested the resultant repressor mutants for their binding to synthetic symmetrical alpha- or beta-centred trp operator variants, in vivo and in vitro. We present genetic and biochemical evidence that Ile in position 69 of the Trp repressor interacts specifically with thymine in position 9 of the alpha-centred trp operator. There are also interactions with other bases in positions 8 and 9 of the alpha-centred trp operator. In vitro, the Trp repressor of mutant RI69 binds to the consensus alpha-centred trp operator and a similar trp operator variant that carries a T in position 9. In vivo analysis of the interactions of Trp repressor mutant RI69 with symmetrical variants of the beta-centred trp operator shows a change in the specificity of binding to a beta-centred symmetrical trp operator variant with a gua-nine to thymine substitution in position 5, which corresponds to position 9 of the alpha-centred trp operator.

Co-operative binding of two Trp repressor dimers to alpha- or beta-centred trp operators.

The alpha-centred trp operator binds one dimer of the Trp repressor, whereas the beta-centred trp operator binds two dimers of the Trp repressor (Carey et al., 1991; Haran et al., 1992). The Trp repressor with a Tyr-Gly-7 substitution binds almost as well as the wild-type Trp repressor to the alpha-centred trp operator, but it does not bind to the beta-centred trp operator. This confirms that Tyr-7 is involved in the interaction between Trp repressor dimers, as seen in the crystal structure (Lawson and Carey, 1993). Further experiments with alpha-centred trp operator variants showed that positions +/-1 of the alpha-centred trp operators play a crucial role in tetramerisation. The two innermost base pairs of the alpha-centred trp operator are not involved in contacts with the dimer of the Trp repressor binding to it. However, substitutions in these positions (T-A to G-T) effectively transform the alpha-centred trp operator into a beta-centred trp operator, and thus encourage the binding of two Trp repressor dimers to this operator. Finally, we demonstrate, with suitable heterodimers, that one subunit of each dimer suffices to bind to a beta-centred trp operator.

The possible roles of residues 79 and 80 of the Trp repressor from Escherichia coli K-12 in trp operator recognition.

We constructed mutants of the Trp repressor from Escherichia coli K-12 with all possible single amino acid exchanges at positions 79 and 80 (residues 1 and 2 of the recognition helix). We tested these mutants in vivo by measuring the repression of synthesis of beta-galactosidase with symmetric variants of alpha- and beta-centered trp operators, which replace the lac operator in a synthetic lac system. The Trp repressor carrying a substitution of isoleucine 79 by lysine, showed a marked specificity change with respect to base pair 7 of the alpha-centered trp operator. Gel retardation experiments confirmed this result. Trp repressor mutant IR79 specifically recognizes a trp operator variant with substitutions in positions 7 and 8. Another mutant, with glycine in position 79, exhibited loss of contact at base pair 7. We speculate that the side chain of Ile79 interacts with the AT base pairs 7 and 8 of the alpha-centered trp operator, possibly with the methyl groups of thymines. Replacement of thymine in position 7 or 8 by uracil confirms the involvement of the methyl group of thymine 8 in repressor binding. Several Trp repressor mutants in position 80 (i.e. A180, AL80, AM80 and AP80) broaden the specificity of the Trp repressor for alpha-centered trp operator variants with exchanges in positions 3, 4 and 5.