Performance of peripheral (serum and molecular) blood markers for diagnosis of endometriosis.

PURPOSE: To quantify the mRNA levels of MMP-3, MMP-9, VEGF and Survivin in peripheral blood and the serum levels of CA-125 and Ca19-9 in women with and without endometriosis and to investigate the performance of these markers to differentiate between deep and ovarian endometriosis. METHODS: A case control study enrolled a series of 60 patients. Twenty controls have been matched with 20 cases of ovarian and 20 cases of deep endometriosis. Univariable and multivariable performance of serum CA125 and CA19-9, mRNA for Survivin, MMP9, MMP3 and VEGF genes have been evaluated by means of ROC curves and logistic regression, respectively. RESULTS: No difference in markers' concentration was detected between ovarian and deep endometriosis. In comparison with controls, serum CA125 and CA19 yielded the better sensitivity followed by mRNA for Survivin gene (81.5, 51.9 and 7.5% at 10% false positive rate, respectively). Multivariable estimated odds of endometriosis yielded a sensitivity of 87% at the same false positive rate. CONCLUSIONS: A combination of serum and molecular markers could allow a better diagnosis of endometriosis.

No evidence of HAND2 involvement in nonsyndromic cleft lip with or without cleft palate.

Craniofacial morphogenesis is determined by multistep processes involving signalling molecules and transcription factors, which are organised into highly coordinated pathways. Derailment from this intricate network can lead to congenital malformations. Cells migrate from neural crests to populate different structures, such as branchial arches, involved in embryonal orofacial development. The EDN1 pathway is involved in branchial arch development. Gene knockout and knockdown experiments on EDN1 or its downstream effector dHAND resulted in mice that were characterised by craniofacial defects and cleft palate. Our aim was to evaluate whether the transcription factor HAND2 could be implicated in non-syndromic cleft lip with or without cleft palate (CL/P) aetiology. A sample study composed of 39 multiplex Italian pedigrees was enrolled to test linkage between two microsatellite flanking HAND2 locus and CL/P. No evidence of linkage between HAND2 and CL/P was obtained. Indeed, formal levels of exclusion were obtained with different inheritance models. Investigation results did not support a role of HAND2 in CL/P aetiology. Nevertheless a minor contribute of the gene in clefting could not be ruled out.

No evidence for a role of CRISPLD2 in non-syndromic cleft lip with or without cleft palate in an Italian population.

Non-syndromic cleft lip with or without cleft palate (NSCLP) is a malformation with variable phenotypes, resulting from a mixture of genetic and environmental factors. Some studies have supported a role for the 16q24 region and its candidate gene, CRISPLD2, in clefting. A replication study is necessary to confirm these findings. The aim of the present study was to test, by genetic linkage and association analyses, whether the candidate gene, CRISPLD2, represents a risk factor for NSCLP. The analysis of 39 multigenerational families provided formal exclusion of a linkage between NSCLP and the CRISPLD2 locus under different genetic models and non-parametric analyses. The family-based study of 239 unrelated probands and their parents revealed no association between any particular allele or haplotype and NSCLP. Therefore, the present investigation did not support the hypothesis of the involvement of CRISPLD2 in NSCLP malformation, at least with regard to the Italian population.

Matrix metalloproteinase-3 mRNA: a promising peripheral blood marker for diagnosis of endometriosis.

BACKGROUND/AIMS: Endometriosis is an invasive disease. Its diagnosis depends on laparoscopy, which is traumatic and associated with potential complications. The aim of this study was to develop a rapid, reliable, and less invasive diagnostic test for endometriosis. We hypothesized that genes related to cell invasion would be transcriptionally upregulated in endometriosis, and tested whether blood levels of their transcripts might be used as biomarkers of endometriosis. METHODS: We used quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) to quantify the mRNA levels of vascular endothelial growth factor A (VEGFA), matrix metalloproteinase-3 (MMP-3), and MMP-9 in peripheral blood from 20 patients with mild/intermediate endometriosis, 20 patients with severe endometriosis and 20 endometriosis-free subjects. RESULTS: Our results indicate that circulating mRNA for MMP-3 is significantly higher in patients with endometriosis than in control patients, regardless of the degree of severity. Conversely, the level of circulating mRNA for VEGFA and MMP-9 did not distinguish patients from controls. CONCLUSION: MMP-3 mRNA is a promising peripheral blood marker that discriminates between patients with endometriosis and healthy subjects. Our results support the possibility of finding genes suitable for diagnostic qRT-PCR for endometriosis in peripheral blood and should be explored further.

Linkage disequilibrium analysis of two genes mapping on OFC3: PVR and PVRL2.

Clefts of the lip with or without cleft palate (CL/P) are one of the most common birth defects, occurring in 1/700-1/1,000 infants born alive. The nature of the genetic contribution is still to be clarified; however, some chromosome regions and candidate genes have been proposed for this malformation. Recently, a couple of genes, PVR and PVRL2, mapping in the candidate region OFC3 on chromosome 19q13.31, have been investigated because of their homology to PVRL1, a gene previously shown to cause the Margarita Island CL/P-ectodermal dysplasia syndrome. In the present work, we investigated PVR and PVRL2 genes by family-based linkage disequilibrium analysis using a sample collected from the Italian population. In contrast to previous analyses on other populations, we could not find any statistically significant association between the markers alleles and non-syndromic clefting.

Lactobacilli, bifidobacteria and E. coli nissle induce pro- and anti-inflammatory cytokines in peripheral blood mononuclear cells.

AIM: To investigate whether the stimulation of peripheral blood mononuclear cells (PBMNC) with the cell debris and cell extraction of different probiotic strains is similar or species specific. METHODS: Three strains of bifidobacteria, 4 strains of lactobacilli, and E. coli nissle were sonicated and centrifuged in order to divide them into cell extract and cell debris. PBMNC were separated by density gradient and incubated for 36 h with either the cell debris or the cell extract of single strains of probiotic bacteria in doses from 10(2) to 10(8) CFU/mL. Cell supernatants were taken and interleukin (IL)-10, IL-1beta, and tumor necrosis factor (TNF)-alpha were determined by ELISA. RESULTS: Depending on the species super-family, the strains had different stimulation patterns. Except for both L. casei strains, the cell extract of bifidobacteria and lactobacilli had less stimulating capacity than cell debris, whereas the cell extract of E. coli nissle had similar stimulating properties to that of the cell debris of the strain and significantly more stimulating capacity than that of bifidobacteria and lactobacilli. The cell debris of bifidobacteria stimulated more cytokine release than the cell debris of lactobacilli. The cell debris of lactobacilli did not have a stimulating capacity when lower concentrations were used. Neither cell extraction nor cell debris had an inhibitory effect on the production of the tested cytokines by stimulated PBMNC. CONCLUSION: The incubation of probiotic strains, which have been used in clinical trials for inflammatory diseases, with immunocompetent cells leads to different species specific reactions. High IL-10 response to cell debris of bifidobacteria and E. coli nissle can be found. This corresponds to positive effects of bifidobacteria and E. coli nissle in clinical trials for inflammatory bowel disease compared to negative outcomes obtained with lactobacilli.

A module of inflammatory cytokines defines resistance of colorectal cancer to EGFR inhibitors.

Epidermal Growth Factor Receptor (EGFR) activates a robust signalling network to which colon cancer tumours often become addicted. Cetuximab, one of the monoclonal antibodies targeting this pathway, is employed to treat patients with colorectal cancer. However, many patients are intrinsically refractory to this treatment, and those who respond develop secondary resistance along time. Mechanisms of cancer cell resistance include either acquisition of new mutations or non genomic activation of alternative signalling routes. In this study, we employed a colon cancer model to assess potential mechanisms driving resistance to cetuximab. Resistant cells displayed increased ability to grow in suspension as colonspheres and this phenotype was associated with poorly organized structures. Factors secreted from resistant cells were causally involved in sustaining resistance, indeed administration to parental cells of conditioned medium collected from resistant cells was sufficient to reduce cetuximab efficacy. Among secreted factors, we report herein that a signature of inflammatory cytokines, including IL1A, IL1B and IL8, which are produced following EGFR pathway activation, was associated with the acquisition of an unresponsive phenotype to cetuximab in vitro. This signature correlated with lack of response to EGFR targeting also in patient-derived tumour xenografts. Collectively, these results highlight the contribution of inflammatory cytokines to reduced sensitivity to EGFR blockade and suggest that inhibition of this panel of cytokines in combination with cetuximab might yield an effective treatment strategy for CRC patients refractory to anti-EGFR targeting.

Probiotic therapy in the prevention of pouchitis onset: decreased interleukin-1beta, interleukin-8, and interferon-gamma gene expression.

BACKGROUND: Probiotic therapy has been shown to prevent the onset of pouchitis and to improve the quality of life in ulcerative colitis patients who required ileal pouch anal anastomosis. Pouchitis has been associated with elevated levels of proinflammatory cytokines and chemokines. METHODS: In this retrospective analysis of archived endoscopic samples from responding patients enrolled in the above-mentioned trial, we were interested in studying mucosal gene expression of the pleiotropic proinflammatory cytokines (interleukin-1beta, interleukin-6), TH1 cytokines (interferon-gamma, tumor necrosis factor-alpha, interleukin-12), regulatory cytokines (interleukin-10, transforming growth factor-beta), and the chemokine interleukin-8. In addition to assessment of cytokine gene expression, the presence of polymorphonuclear cells in the mucosal tissue was evaluated. RESULTS: Data show that patients who were treated with probiotics had significant lower mucosal mRNA expression levels of interleukin-1beta, interleukin-8, and interferon-gamma compared with placebo-treated patients. In addition, a lower number of polymorphonuclear cells was present in the tissue of patients within the probiotic group compared with the number of polymorphonuclear cells in the tissue of patients receiving placebo and patients having an episode of pouchitis. CONCLUSIONS: These data suggest that probiotic treatment regulates the mucosal immune response by reducing mucosal levels of neutrophil-chemoattractant IL-8 and tissue influx of polymorphonuclear cells, and may further act by inhibition of T-cell activation, by reinforcement of barrier function and by a tight control of the potent pro-inflammatory cytokine IL-1beta.

Immunomodulatory effects of probiotic bacteria DNA: IL-1 and IL-10 response in human peripheral blood mononuclear cells.

A new therapeutic approach for inflammatory bowel diseases is based on the administration of probiotic bacteria. Prokaryotic DNA contains unmethylated CpG motifs which can activate immune responses, but it is unknown whether bacterial DNA is involved in the beneficial effects obtained by probiotic treatment. Peripheral blood mononuclear cells (PBMC) from healthy donors were incubated with pure DNA of eight probiotic strains and with total bacterial DNA from human feces collected before and after probiotic ingestion. Cytokine production was analyzed in culture supernatants. Modification of human microflora after probiotic administration was proven by polymerase chain reaction analysis. Here we show that Bifidobacterium genomic DNA induced secretion of the antiinflammatory interleukin-10 by PBMC. Total bacterial DNA from feces collected after probiotic administration modulated the immune response by a decrease of interleukin-1 beta and an increase of interleukin-10.

P-15 cell-binding domain derived from collagen: analysis of MG63 osteoblastic-cell response by means of a microarray technology.

BACKGROUND: P-15 is an analog of the cell-binding domain of collagen. P-15 has been shown to facilitate physiological processes in a way similar to collagen; to serve as an anchorage for cells; and to promote the binding, migration, and differentiation of cells. METHODS: Expression profiling by DNA microarray is a molecular technology that allows the analysis of gene expression in a cell system. By using DNA microarrays containing 19,200 genes, we identified in osteoblast-like cell line (MG-63) cultured with P-15 several genes whose expression was significantly up- or downregulated. RESULTS: The differentially expressed genes cover a broad range of functional activities: 1) signaling transduction, 2) differentiation, 3) apoptosis, and 4) cell-cycle regulation. It was also possible to detect some genes whose function is unknown. CONCLUSIONS: The data reported are, to our knowledge, the first genetic portrait of P-15 effects. They can help us to better understand the molecular mechanism of osteogenesis and can serve as a model for comparing different cell cultures and/or other materials with similar effect.

Cross-talk between interleukin-6 and transforming growth factor-beta3 regulates extracellular matrix production by human fibroblasts from subjects with non-syndromic cleft lip and palate.

BACKGROUND: Transforming growth factor-beta (TGF-beta) interference with interleukin 6 (IL-6) activity and the role of the latter in early human embryonic development prompted us to examine the effects IL-6 on matrix synthesis and the effects of TGF-beta3 on IL-6 expression human cleft lip and palate (CLP) fibroblasts. METHODS: Collagen and glycosaminoglycan (GAG) synthesis were determined by radiolabeled precursors and biglycan expression by Northern blotting before and after adding IL-6. The effects of TGF-beta3 on IL-6 production were assayed by evaluating IL-6 transcript by Northern blotting and IL-6 protein secretion by enzyme-linked immunosorbent assay. RESULTS: The results showed that IL-6 elicited an inhibitory effect on collagen and GAG levels in CLP fibroblasts by lowering hyaluronan and dermatan sulfate secretion. IL-6 up-regulated biglycan expression, but less strongly than TGF-beta3. TGF-beta3 significantly down-regulated IL-6 transcript and secretion in CLP fibroblasts. CONCLUSIONS: These data suggest the increase in matrix components that characterize the CLP fibroblast phenotype might be due to a concerted TGF-beta3-IL-6 action. We hypothesize changes in cross-talk between TGF-beta3 and IL-6 signal transduction pathways are involved in the induction of cleft palate.

PLAC4 and beta-HCG mRNA levels are not altered in the maternal circulation of pregnancies with trisomy 21.

OBJECTIVE: Beta-human chorionic gonadotropin (HCG) and pregnancy-associated plasma protein (PAPP-A) are placentally produced proteins whose levels are altered in pregnancies with trisomy 21. PLAC4 is located on chromosome 21 and its expression is restricted to the placenta. Here we investigated whether the levels of beta-HCG-, PAPP-A- and PLAC4 mRNA could be able to discriminate pregnancies whose fetus is affected by trisomy 21. METHOD: Hundred and forty-three blood samples from normal pregnancies and eight samples from trisomic pregnancies were collected. Total RNA was extracted from whole maternal blood, reverse-transcribed and the three mRNAs were quantified by real-time quantitative PCR. Hundred and nine controls were also tested for the serum levels of PAPP-A and HCG proteins. RESULTS: Beta-HCG and PLAC4 mRNAs were detected in all samples, in higher amounts than in plasma, whereas the detection rate for PAPP-A mRNA was below 10%. The levels of beta-HCG mRNA significantly correlated with the circulatory concentrations of the HCG protein. However, neither beta-HCG- nor PLAC4 mRNAs show a significant difference between cases and controls. CONCLUSION: Maternal blood levels of beta-HCG-, PLAC4- and PAPP-A mRNAs are not useful markers for the screening of pregnancies with trisomy 21 as their concentrations are either not significantly altered (beta-HCG and PLAC4) or too low to be detected (PAPP-A).

Genetic effect of anatase on osteoblast-like cells.

Titanium is the gold standard among materials used for prosthetic devices, because of its good mechanical and chemical properties. When exposed to oxygen, titanium becomes an oxide that is biocompatible and able to induce osseointegration. Three allotropic forms of titanium dioxide exist, that is brookite, rutile, and anatase. Anatase can be prepared as a colloidal suspension and then used to coat surfaces. Anatase coating (AC) can potentially have specific biological effects. Here we are testing the effect of AC on osteoblast-like cells (MG63) by using microarray techniques to identify genes that are differently regulated in osteoblasts exposed to AC. By using DNA microarrays containing 20,000 genes, we identified in osteoblast-like cell lines (MG-63) cultured on AC, several genes whose expression was significantly up- or downregulated. They cover a broad range of functional activities: signaling transduction, immunity, cell cycle regulation, lysosomes composition and vesicular transport, cell adhesion, cytoskeleton and extracellular matrix components, proliferation, and apoptosis. The data reported constitute, to our knowledge, the first genetic portrait of AC effects. They can be relevant to a better understanding of the molecular mechanism of bone regeneration and as a model for comparing other materials with similar clinical effects.

Performance of a panel of maternal serum markers in predicting preeclampsia at 11-15 weeks' gestation.

OBJECTIVE: We evaluated whether a discriminant model of prediction based on quantitative distribution of a panel of biomolecules in maternal serum can discriminate normal pregnancies from those who will develop preeclampsia (PE) prior to onset of clinical symptoms at 11-15 weeks' gestation. METHODS: Case control study encompassing 56 women destined to develop PE cases matched 1:3 for gestational age with 168 controls. After multiple of median (MoM) conversion of all available markers, comprising total Activin A (t-activin A), P-selectin, and vascular endothelial growth factor receptor (VEGFR) the combined likelihood ratios generated for each marker were used to calculate, for each patient enrolled in the study, the odds of being affected given a positive results (OAPR) of developing PE. For all the analyses performed, the type II error was < 20% with a type I error fixed at 5%. RESULTS: Data were expressed in MoM of controls. P-selectin was identified as the marker with the best discriminant ability between controls and PE, followed by (t-activin A). No significant differences in VEGFR were observed between cases and controls. By using a 3% prevalence of PE (or, about 1:33) we found that the median OAPR of developing PE for the 56 cases was 1:9 or 10% (1:1-1:417). The median OAPR of PE for controls was 1:40 or 2.5% (range, 1:6-1:4205). Detection rate of the statistical model, with a 5% false-positive rate was 59%. CONCLUSION: This analysis revealed that maternal serum markers assessed at the first and second trimester of pregnancy in asymptomatic patients can improve the early detection of cases at higher risk of developing PE.

Rapid clearance of mRNA for PLAC1 gene in maternal blood after delivery.

OBJECTIVE: To evaluate (1) whether the presence of mRNA for the specific trophoblast gene PLAC1 in maternal whole blood is pregnancy-specific, and (2) whether delivery would result in the clearance of mRNA from maternal blood. METHODS: Sixteen pregnant women at term (41 completed weeks' gestation) were enrolled in the study. Blood samples were obtained before the onset of labor and 24 h after delivery. Eight healthy donors (3 males and 5 non-pregnant women) were used as controls. Total RNA was extracted by means of ABI Prism 6100. A quantitative evaluation was obtained by means of real-time PCR. Wilcoxon test was used to evaluate differences between time intervals. RESULTS: Median concentrations of PLAC1 mRNA relative to the standardization curve (see below) were 44 (2.9-675) ng/ml and 0.48 (0.05-10.7) ng/ml respectively for pre- and post-delivery samples (p value <0.001). Male and non-pregnant female controls did not show any signal of cDNA amplification. CONCLUSION: mRNA transcripts from a placenta-expressed specific gene are detectable in maternal blood and rapidly disappear after delivery. Such an mRNA provides a gender-independent marker for non-invasive prenatal gene expression profiling, and can open new perspectives to monitor those conditions associated to trophoblast damage as well as preeclampsia.

Testing normality of fetal DNA concentration in maternal plasma at 10-12 completed weeks' gestation: a preliminary approach to a new marker for genetic screening.

OBJECTIVES: To test the distribution of fetal DNA in maternal plasma expressed as gen/eq in a population of normal pregnancies. METHODS: Peripheral blood samples were obtained from 63 women (85% > or =35 years of age at delivery) bearing a euploid male fetus. Each patient underwent chorionic villus sampling (CVS) for karyotype analysis and/or beta thalassemia screening. Ultrasound scanning was used to determine gestational age. At 10-12 weeks' gestation, a peripheral blood sample was collected followed by CVS. To detect the Y chromosome specific sequences (SRY) quantitative polymerase chain reaction (PCR) analysis was used. Normal distribution of the data was tested by means of the Kolmogorov-Smirnov (KS) test. A Symmetry test (reliability p>0.05) was used to evaluate the reliability of the median. RESULTS: Only after natural logarithmic transformation did the data display a normal distribution. The median value of fetal DNA was 23.3 gen/eq (range 2.08-195), interquartile range 18.57-45.4. A Pearson test showed a significant correlation between gestational age and fetal DNA concentration (r=0.25, p=0.045). CONCLUSION: The present finding is a preliminary step towards a possible integration of fetal DNA with other variables (biochemical and/or ultrasound). It may serve to improve the discrimination of the screening for genetic diseases in the first trimester. Because of the relatively high dispersion, adjustments for possible covariates would appear to be necessary in further studies.

Analysis of MG63 osteoblastic-cell response to a new nanoporous implant surface by means of a microarray technology.

Surface implant modifications have been shown to have a relevant importance in modifying cell response. Expression profiling by DNA microarray is a new molecular technology that allows the analysis of gene expression in a cell system. By using DNA microarrays containing 19,200 genes, we identified in osteoblast-like cells line (MG-63) on new implant surface (nanoPORE, Out-Link, Sweden and Martina, Due Carrare, Padova, Italy), several genes whose expressions were significantly down-regulated. The differentially expressed genes cover a broad range of functional activities: (a) immunity, (b) vesicular transport (c) apoptosis and cell cycle regulation. It was also possible to detect some genes whose function is unknown. The data reported are, to our knowledge, the first genetic portrait of an implant surface. They can be relevant to better understand the molecular mechanism of implant osseointegration and as a model for comparing other materials.

Cell-free fetal DNA concentration in plasma of patients with abnormal uterine artery Doppler waveform and intrauterine growth restriction--a pilot study.

OBJECTIVE: To evaluate if an increased amount of fetal DNA concentration can be found in women screened positive for intrauterine growth restriction because of abnormal uterine artery Doppler waveforms. METHODS: We enrolled eight pregnant women (each bearing a male fetus), with the evidence of abnormal uterine artery Doppler waveforms, and 16 control patients for a case-control study matched for gestational age (1 : 2). Uterine artery Doppler was carried out at 20 to 35 weeks' gestation (median 29). The mean uterine artery resistance index (RI) was subsequently calculated, and a value >0.6 was considered positive for the clinical features of pre-eclampsia. The SRY locus was used to determine the amount of male fetal DNA in the maternal plasma at the time of Doppler analysis. RESULTS: Two controls (normal Doppler) were excluded from the final analysis because they had a pre-term delivery. One case (abnormal Doppler) had evidence of intrauterine growth restriction at the time of enrolment. In four out of eight cases (abnormal Doppler), intrauterine growth restriction was subsequently observed. Multiples of median (MoM) conversion of the fetal DNA values showed an increase of 1.81 times in the cases when compared to the controls. An increase of 2.16 times was instead observed for the cases with a growth-restricted fetus (5 cases out of 8) in comparison with the controls (14 cases). CONCLUSIONS: In subjects positive to uterine artery Doppler velocimetry analysis (Doppler analysis for pre-eclampsia screening), the fetal DNA concentration is higher than expected, in the absence of any other clinical feature. Since the increase in fetal DNA seems to be related to the presence or to the future development of intrauterine growth restriction, this paper suggests a possible integration between ultrasound and molecular markers for predicting the disease in some cases.