A versatile method for gene dosage quantification: multiplex PCR and single base extension for copy number and gene-conversion identification of SMN genes.

A comparison of the individual genomes within a species demonstrates that structural variation, including copy number variation (CNV), is a major contributor to phenotypic diversity and evolutionary adaptation. CNVs lead to the under/over-expression of a gene, according to the changes in the gene dosage, which account for the development of a number of genomic disorders. Thus, the development of efficient, rapid and accurate CNV screening is of fundamental importance. We report a method that enables the simultaneous determination of the copy numbers of several different targets as well as the discrimination among highly similar/almost identical targets that differ by only one single nucleotide variant, which establishes their copy numbers. The PCR co-amplification and single-base extension technologies are used to identify the copy number of a target sequence relative to a reference sequence of known genomic copy number in a given sample. This efficient and accurate quantification platform was successfully used to quantify the copy numbers of the primary spinal muscular atrophy-determining gene, SMN1, and the disease modifier gene, SMN2. The reliability, low-cost and potential for high-throughput make our method suitable for screening large populations as well as for use as a tool in clinical settings for genetic diagnosis/prognosis.

The -308 TNFα and the -174 IL-6 promoter polymorphisms associate with effective anti-TNFα treatment in seronegative spondyloarthritis.

The genetic predisposition to a long-term efficacy of anti-tumor necrosis factor (TNF)α treatment in seronegative spondyloarthritis (SpA) was investigated by analysing the possible correlation between several single nucleotide gene polymorphisms and the retention rate of anti-TNFα therapies. We compared patients needing to switch the first anti-TNFα (Sw, No. 64) within at least 12 months of follow-up with patients not needing to switch (NSw, No. 123), observing at least 6 months of treatment to establish anti-TNFα failure, leading to treatment change. Response to treatment was evaluated by standardised criteria (BASDAI for axial involvement, DAS28-EULAR for peripheral involvement). The TNFα -308 A allele and the interleukin (IL)-6 -174GG homozygosis resulted as independent biomarkers predicting survival of the first anti-TNFα therapy in SpA patients (P=0.007, odds ratio (OR): 4.4, 95% confidence interval (CI)=1.5-13.1 and P=0.035, OR: 2.1, 95% CI=1.1-4.4). Also, the male gender (P=0.001, OR: 3.4, 95% CI=1.6-7.1) associated with the NSw phenotype, whereas no association was found either with the specific diagnosis or the predominant joint involvement.

Second trimester amniotic fluid retinol in patients developing preeclampsia.

BACKGROUND: Retinol (ROH) is an essential micronutrient required for normal fetal development and an essential molecule for antioxidant processes. OBJECTIVE: To investigate the putative role of ROH as a marker of preeclampsia in early second trimester amniotic fluid (AF). MATERIALS AND METHODS: Case-control study comparing the concentration of ROH and other antioxidants such as uric acid, vitamin E and malondialdehyde (MDA) in second trimester AF in patients that later developed preeclampsia with normal pregnancies. RESULTS: The concentration of ROH in amniotic fluids of women that later developed preeclampsia was significantly higher than those of uncomplicated pregnancies (66.72 µg/l (49.00-70.56) vs. 44.4 µg/l (31.9-51.17), p < 0.05). No statistical significant difference was found in uric acid, vitamin E and MDA concentration. In the multivariate logistic regression, concentrations of ROH in amniotic fluids directly correlate with the risk of developing preeclampsia (OR 1.13, IC 0.01-1.26, p < 0.05). CONCLUSIONS: Second trimester AF ROH concentration was significantly higher in pregnancies that developed preeclampsia compared to normal pregnancies.

The thyroid lobes: the different twins.

Although differences in size of the right and left thyroid lobes are well defined, differences in morphology, follicles structure, cAMP production, thyrotropin receptor, and protein involved in cell signalling have not previously been reported. This study provides morpho-functional data of right and left thyroid lobes by biochemical, immunohistochemistry, immunoblotting and immunofluorescence analysis. We demonstrate that, in comparison with the left lobe, the right lobe has a higher activation index, is more sensitive to thyrotropin treatment, is rich in thyrotropin receptor and caveolin 1 involved in thyroid hormone synthesis as well as in epithelial thyroid cell homeostasis, is characterised by a high content of molecules involved in cell signalling such as stat3, raf1, sphingomyelinase and sphingomyelin-synthase whose activity ratio is necessary for epithelial cell activity and finally has more areas calcitonin-dependent. The relation between structure/function of right lobe and its susceptibility to the higher risk of pathological modifications with respect the left lobe is discussed.

Buprenorphine/naloxone versus methadone in opioid dependence: a longitudinal survey.

BACKGROUND AND OBJECTIVES: Buprenorphine and methadone are widely used for the treatment of opioid dependence, but their diversion and/or misuse are frequent. In principle, buprenorphine/naloxone combination therapy should be associated with a lower frequency of drug abuse/misuse than methadone. This study assessed the efficacy of the substitution of buprenorphine treatment with the buprenorphine/naloxone combination in opioid-dependent patients. MATERIAL AND METHODS: 3812 drug-addicted outpatients selected from 10 Italian Public Services for Addiction (Ser.T.) centres in Naples (Italy) were enrolled: 3105 (81.5%) were treated with methadone and 707 (18.5%) with buprenorphine. The buprenorphine treatment was switched to buprenorphine/naloxone (4:1), and the patients were followed for about 1 year. The number of subjects still on treatment after 1 year, their status according to social, educational and toxicologic (assessed by a urine toxicology test) parameters were assessed. RESULTS: 1 year after the therapy switch, the number of patients still on treatment was similarly reduced with methadone (2883; -7.5%) and buprenorphine/naloxone (632; -10.6%; p=0.369). However, in patients treated with buprenorphine/naloxone, a significant improvement was reported in social life status (63% versus 39% of the buprenorphine/naloxone and methadone treated subjects, respectively, were married/cohabiting p<0.001), in the educational level (43% of buprenorphine/naloxone treated versus 32% of the methadone treated subjects obtained at least a high school certificate, p<0.001) and in the toxicological conditions (53% of buprenorphine/naloxone treated subject versus 30% of methadone treated individuals had opioid- and cocaine- negative urine tests, p<0.001). DISCUSSION: These preliminary data suggest that buprenorphine/naloxone treatment of opioid dependence reduces the percentage of treated subjects similarly to methadone, and is associated with an improvement in social life, educational and toxicological conditions, compared with methadone treatment. However, we cannot exclude a selection bias, i.e. patients who were more likely to stabilize their opiate dependence switched to buprenorphine/naloxone.

UVC radiation-induced effect on human primary thyroid cell proliferation and HLA-DR expression.

The aim of this study was to examine how UVC irradiation will affect normal human thyroid cell proliferation and HLA-DR expression. Primary human thyroid cells were exposed to UVC (254 nm wavelength) irradiation. In some experiments 0.5 mM buthionine sulfoximine (BSO) was added. Apoptosis was detected measuring annexin V, proteins involved in apoptotic process (p53, Bax, Bcl-2, caspase 3, and 9) by immunoblot analysis and HLA-DR expression by FACS. UVC induced a cell cycle arrest in G0/G1 phase in the first 24 h, accumulation of cells in the S phase 72 h after treatment, and an increase of apoptotic cells. BSO pretreatment showed an earlier appearance and a higher percentage of apoptosis. p53, caspase 3 and 9 were increased, while Bax and Bcl-2 were decreased. We also observed a transient significant increase in HLA-DR expression. UVC inhibited cell proliferation and induced apoptosis in normal human primary thyroid cells. An inhibitor of glutathione synthesis induced an earlier appearance and higher percentage of apoptosis suggesting that oxidative stress may play a role. Apoptotis involved components of the intrinsic mitochondrial pathway. A transient increase in HLA-DR expression after UVC irradiation could play a role in inducing AITD.

Study on the possible role of the -174G>C IL-6 promoter polymorphism in predicting response to rituximab in rheumatoid arthritis.

OBJECTIVE: Identification of genetic biomarkers of response to biologics in rheumatoid arthritis (RA) is a relevant issue. The -174G>C interleukin-6 (IL-6) promoter polymorphism was investigated in RA patients treated with rituximab (RTX), being IL-6 a key cytokine for B cell survival and proliferation, thus possibly implicated in rituximab efficacy. METHODS: The study was conducted in a real-life retrospective cohort of 142 unselected RA patients (120F/22M) treated with RTX and referred to 7 rheumatologic centres in the north of Italy. One hundred and thirteen (79.6%) patients were rheumatoid factor (RF)-positive and 112 (78.9%) were anti-CCP antibodies positive. The response to therapy was evaluated at the end of the sixth month after the first RTX infusion, by using both the EULAR criteria (DAS28) and the ACR criteria. The IL-6 -174G>C promoter polymorphism was analyzed by RFLP following previously reported methods. RESULTS: Lack of response to RTX at month +6 by EULAR criteria was more prevalent in RA patients with the IL-6 -174 CC genotypes (9/21, 42.8%), than in the GC/GG patients (23/121, 19.0%) (OR 3.196, 95% CI=1.204-8.485; p=0.0234). Similar results were found when evaluating the response by ACR criteria. No differences were found in RA duration, baseline DAS28, baseline HAQ, RF status, anti-CCP status according to the different IL-6 -174 genotypes. CONCLUSION: IL-6 promoter genotyping may be useful to better plan treatment with RTX in RA. Larger replication studies are in course to confirm these preliminary results.

[Multicenter study on the monitoring of in vitro susceptibility to tigeeyeline in Santiago, Chile]. / Estudio multicéntrico de la vigilancia de la susceptibilidad in vitro a tigeciclina en Santiago de Chile.

The objective of this multicenter study was to determine tigecycline susceptibility rates, measured by agar diffusion, in nine hospitals in Santiago and to compare these rates with other antimicrobials. Each center studied 20 strains per month. All intermediate and fully resistant strains as well as 10% of susceptibile strains were also studied by the broth microdilution method. Overall, 2301 strains were studied displaying the following susceptibility rates for tigecycline: 100% for Streptococcus sp, Enterococcus sp, and E. coli respectively, 99.8% for Staphylococcus sp, 93% for Klebsiella and 80% for Acinetobacter baumarmii. For Proteus, Providencia and Morganella the susceptibility rates were 4%. For cefotaxime-resistant Klebsiella and imipenem-resistant A. baumarmii susceptibility rates were 95% and 80% respectively. The agar diffusion and broth dilution method were 100% concordant for tigecycline susceptible strains but only 27% for resistant or intermediate strains represented mostly by Acinetobacter baumannii. The majority of these strains (57/59) proved to be susceptible after retesting. The great majority (96,6%) of strains tested from nine Chilean hospitals proved to be susceptible to tigecycline with exception for Proteus, Providencia and Morganella (66% resistance). Using the agar diffusion method for measuring tigecycline susceptibility to A. baumannii may be misleading.

Anti-ß2-glycoprotein I and anti-phosphatidylserine/prothrombin antibodies exert similar pro-thrombotic effects in peripheral blood monocytes and endothelial cells.

PURPOSE: The introduction of the anti-phosphatidylserine/prothrombin (aPS/PT) antibodies among the routinely investigated anti-phospholipid (aPL) antibodies led to an improvement in anti-phospholipid syndrome (APS) laboratory diagnostic performance; however, their pathogenic mechanism is still substantially undefined. To support clinical data and future inclusion as possible new criteria antibodies, we designed a head-to-head study to directly compare the procoagulant effects sustained in vitro by aPS/PT to those sustained by anti-ß2-glycoprotein I (aß2GpI) domain 1-specific antibodies. METHODS: Blood donors-derived monocytes and endothelial cells (HUVEC) were stimulated with lipopolysaccharides (LPS) alone or in combination with the IgG fractions isolated from the serum of six APS patients, positive only for aß2GpI or for aPS/PT antibodies. As control, cells were incubated with LPS plus the IgG isolated from blood donors. Tissue factor (TF) mRNA expression was measured after four hours incubation by real-time PCR. Nitric oxide (NO) levels were measured in cells supernatant after 16 h incubation by colorimetric assay. RESULTS: aPS/PT and aß2GpI IgG antibodies fractions showed comparable ability to enhance LPS-induced TF mRNA expression, either in monocytes and in HUVEC. Compared to LPS alone, we found that NO levels are strongly overproduced in HUVEC treated with LPS plus aß2GpI and aPS/PT IgG fractions. CONCLUSIONS: Our data support the significant and independent role of aPS/PT in the pathogenesis of the thrombotic events in APS patients, possibly adding new light to the therapeutic management of cases characterized by the sole presence of aPS/PT IgG antibodies.

Differential expression of the components of the plasminogen activating system in human thyroid tumour derived cell lines and papillary carcinomas.

We characterised the expression of the plasminogen activators (uPA and tPA), the uPA receptor (uPAR) and the PAs inhibitors (PAI-1 and PAI-2) in human thyroid cell lines derived from normal thyroid, follicular adenoma, follicular, papillary and anaplastic carcinomas. Urokinase PA activity was detected in the supernatant of normal thyrocytes and augmented in those of all tumour cells. Quantitative RT-PCR analysis showed that uPA, uPAR and PAI-1 mRNAs increased in all carcinoma cells. Similar results were found in 13 papillary thyroid carcinoma (PTC) tissues which were mirrored in Western blot experiments. A correlation was found between tumour size and uPA mRNA increase, and higher levels of uPA and uPAR mRNAs were found in metastatic PTC. In conclusion, thyroid carcinoma cell lines and PTC overexpress uPA, uPAR and PAI-1 and the correlation of uPA and its cognate receptor with tumour size and metastasis may suggest their potential prognostic relevance in thyroid cancer.

Expression of the neoplastic phenotype by human thyroid carcinoma cell lines requires NFkappaB p65 protein expression.

We have investigated the role of the NFkappaB complex in the process of thyroid carcinogenesis by analysing thyroid carcinoma cell lines. A significant increase in p65 NFkappaB mRNA and protein expression, compared to normal thyroid cultures or tissue, was found in all of the cancer cell lines. Conversely, only a modest increase in the p50 NFkappaB mRNA and protein was found in most, but not all carcinoma cell lines. The block of p65 protein synthesis with specific antisense oligonucleotides greatly reduced the ability of two undifferentiated carcinoma cell lines to form colonies in agar and reduced their growth rate. On the other hand, no effect was observed in the same cell lines when treated with p50 specific antisense oligonucleotides. These inhibitory effects seem to be mediated by the suppression of c-myc gene expression, since treatment with antisense oligonucleotides for p65 gene interfered negatively with c-myc gene expression. Our results indicate that activation of the NFkappaB complex by overexpression of p65 plays a critical role in the process of thyroid cell transformation.

Subclones of a rat parathyroid cell line (PT-r): regulation of growth and production of parathyroid hormone-related peptide (PTHRP).

Four subclones from a rat parathyroid cell line (PT-r cell) have been isolated, and morphological and functional characteristics have been examined. Subclones 1 and 2 display a polygonal shape, show growth and secretory responses to calcium (half-maximal suppressions at 1.2 and 1.7 mM, respectively), and respond to secretin with cAMP production (14.5-fold and 16.9-fold over basal) and hormone secretion (41 and 58% over basal). Subclone 4 is elongated in form and does not respond to calcium or secretin. Subclone 3 shows mixed morphology, elongated and polygonal shapes, with moderate response to calcium (half-maximal suppression at 1.7 mM) and secretin (cAMP, 3.2-fold increase and hormone secretion, 50% increase over basal). The clones were tested for content of messenger RNA (mRNA) representing parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHRP). Only PTHRP mRNA was found. The peptide released is virtually all PTHRP. PTH mRNA was not detected even with a sensitive RNA probe. The amount of mRNA for PTHRP closely paralleled the amount of PTH-like bioactivity released into the medium from each clone (144.7 +/- 12.1, 110.0 +/- 12.9, 68.0 +/- 5.6, and 39.9 +/- 2.4 pgEq of rat PTH-(-34) per 10(7) cells per 12 h in a medium with 0.7 mM ionized calcium, from subclones 1, 2, 3, and 4, respectively). Culture conditions, low-density passage (less than 1:50 split ratio) or high-density passage (greater than 1:10 split ratio), affected morphology and function of the clones 1 and 2. They became elongated and functionally dedifferentiated like subclone 4 and 3 months of high-density culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyroid-specific gene expression is differentially influenced by intracellular glutathione level in FRTL-5 cells.

Alteration of the redox potential has been proposed as a mechanism influencing gene expression. Reduced glutathione (GSH) is one of the cellular scavengers involved in the regulation of the redox potential. To test the role that GSH may play in thyroid cells, we cultured a differentiated rat thyroid cell strain (FRTL-5) in the presence of L-buthionine-(S,R)-sulfoximine (BSO). BSO affects GSH synthesis by irreversibly inhibiting gamma-glutamylcysteine synthetase (EC 6.3.2.2), a specific enzyme involved in GSH synthesis. BSO-treated FRTL-5 cells show a great decrease in the GSH level, whereas malondialdehyde increases in the cell culture medium as a sign of lipid peroxidation. In these conditions the activity of two thyroid-specific promoters, thyroglobulin (Tg) and thyroperoxidase (TPO), is strongly reduced in transient transfection experiments. As both Tg and TPO promoters depend upon the thyroid-specific transcription factors, thyroid-specific transcription factor-1 (TTF-1) and Pax-8 for full transcriptional activity, we tested whether reduction of GSH concentration impairs the activity of these transcription factors. After BSO treatment of FRTL-5 cells, both transcription factors fail to trans-activate the respective chimerical targets, C5 and B-cell specific activating protein promoters, containing, respectively, multimerized TTF-1- or Pax-8-binding sites only as well as the Tg and TPO natural promoters. Northern analysis revealed that endogenous Tg messenger RNA (mRNA) expression is also reduced by BSO treatment, whereas endogenous TPO expression is not modified. Furthermore, the Pax-8 mRNA steady state concentration does not change in BSO-treated cells, whereas TTF-1 mRNA slightly decreases. Immunoblotting analysis of FRTL-5 nuclear extracts does not show significant modification of the Pax-8 concentration in BSO-treated cells, whereas a decrease of 25% in TTF-1 protein is revealed. Furthermore, BSO treatment decreases the DNA-binding activity to the respective consensus sequence of both transcription factors. Finally, different mechanisms seem to act on TTF-1 and Pax-8 functional impairment in BSO-treated cells. Indeed, with a lowered GSH concentration, the overexpressed Pax-8 still activates transcription efficiently, whereas, on the contrary, the overexpressed TTF-1 does not recover its transactivation capability when the respective chimerical target sequences are used (C5 and BSAP). When the natural Tg and TPO promoter sequences are used, overexpression of Pax-8 parallels the effect on both promoters observed using the chimeric target sequences, whereas overexpression of TTF-1 increases TPO promoter transcriptional activity only.

A new model of human aortic endothelial cells in vitro.

Vascular endothelial cells play an important role in coagulation regulation of vascular tone and in a variety of synthetic and metabolic functions. Endothelial cells also have a pivotal role in immunological diseases atherogenesis and tumor angiogenesis. Endothelial cells are often used as system to study the pathophysiology of late complications in diabetes mellitus atherosclerotic damages and leukocyte adhesion in inflammatory diseases. Most of the studies have been performed on primary arterial and venous endothelial cell cultures with problems such as availability of autoptic material and reproducibility of cell cultures. We have isolated and characterized a novel system of proliferating long-term cultures of human aortic endothelial cells that maintain their differentiated characteristics for many generations in vitro. They produce antithrombotic and thrombotic factors such as t-PA and PAI-1 and respond to TNFalpha, an important factor correlated with the inflammatory process by modifying growth characteristics by producing cytokines such as GM-CSF by expressing ICAM-1 on the surface and by producing large amounts of nitric oxide and endothelin. This new system may be very useful to understand and study the molecular mechanisms involved in many vascular alteration pathologies and in the aging process.

Response to thyrotropin of normal thyroid follicular cell strain FRTL5 in hypergravity.

Thyroid hormones control every cell in the organisms and, as indicated by many hormonal changes in astronauts during and shortly after space missions, its complex regulation may be influenced by gravity. To test in vitro the effects of gravity environment on thyroid, we selected a unique cultured cell system: the FRTL5, a normal follicular thyroid cell strain in continuous culture, originally derived from adult rat thyroids. To establish if modifications of the gravitational environment may interfere with post-receptorial signal transduction mechanisms in normal mammalian cultured cells, following our previous microgravity experiments, we exposed thyrotropin-stimulated and unstimulated FRTL5 cells to hypergravity (5 g and 9 g) in a special low-speed centrifuge. At all thyrotropin doses tested, we found significant increases in terms of cyclic AMP production in FRTL5 thyroid cells. The data here reported correlate well with our previous microgravity data, showing that the FRTL5 cells functionally respond to the variable gravity force in a dose-dependent manner in terms of cAMP production following TSH-stimulation.

Sod and GSH inhibit the high glucose-induced oxidative damage and the PDGF increased secretion in cultured human endothelial cells.

Poor control of blood glucose has been established as a key pathogenetic mechanism in the vascular complications of diabetes. It has been reported that glucose may autooxidize generating free radicals which have been suggested to delay proliferation, to modify mobility, to influence platelet-derived growth factor and other secretory protein production in a variety of cell systems. Platelet-derived growth factor, in turn, may induce proliferation and migration of vascular smooth muscle cells and thus play a role in atherogenesis. In the present study the effects of antioxidants on the high glucose-dependent oxidative cell damage and increased platelet-derived growth factor secretion have been investigated using cultured human endothelial cells. Our findings show that rising the glucose concentration in the culture medium from 5 mM to 20 mM, increased the production of free radicals cell damage markers, such as malondialdehyde and conjugated dienes, as well as the production of platelet-derived growth factor. The addition of superoxide dismutase or glutathione prevents both such effects. These results suggest that antioxidants may be a helpful therapeutic adjuvant to reduce the vascular complications of diabetes.

Induction of follicle formation in long-term cultured normal human thyroid cells treated with thyrotropin stimulates iodide uptake but not sodium/iodide symporter messenger RNA and protein expression.

Iodide uptake by the sodium/iodide symporter (NIS) in thyrocytes is essential for thyroid hormone production. Reduced NIS activity has been reported in thyroid diseases, including thyroid cancer and congenital hypothyroidism. The study of iodide uptake in thyrocytes has been limited by the availability of appropriate in vitro models. A new culture technique was recently developed that allows normal human thyroid primary culture cells to grow as monolayer cells and express differentiated functions for more than 3 months. We used this technique to study the effect of follicle formation and TSH on iodide uptake in these cells. Iodide uptake by the cells grown in monolayer was very low. Follicle formation was induced from monolayer cells, and electron micrographs demonstrated cell polarity in the follicles. No significant increase in iodide uptake was observed after TSH treatment of cells in monolayer or when follicle formation was induced without TSH. TSH stimulation of follicles, however, significantly increased iodide uptake ( approximately 4. 4-fold; P<0.001). Compared with iodide uptake in monolayers, the combination of follicle formation and TSH treatment stimulated iodide uptake synergistically to 12.0-fold (P<0.001). NIS messenger RNA (mRNA) and protein levels were almost the same in both monolayer cells and follicles. TSH treatment of monolayers and follicles produced significant (P<0.05) stimulation of mRNA ( approximately 4. 8- and approximately 4.3-fold respectively) and protein ( approximately 6.8- and 4.9-fold respectively). TSH stimulated NIS protein levels in both monolayer and follicles, however, stimulation of functional iodide uptake was only seen with TSH stimulation of follicles. The function of NIS may involve post-transcriptional events, such as intracellular sorting, membrane localization of NIS or another NIS regulatory factor. Polarized functions, such as iodide efflux into follicular lumina, may also contribute to the increased iodide concentration after follicle formation.