Single nucleotide polymorphism detection by optical DNA-based sensing coupled with whole genomic amplification.

The work presented here deals with the optimization of a strategy for detection of single nucleotide polymorphisms based on surface plasmon resonance imaging. First, a sandwich-like assay was designed, and oligonucleotide sequences were computationally selected in order to study optimized conditions for the detection of the rs1045642 single nucleotide polymorphism in the gene ABCB1. Then the strategy was optimized on a surface plasmon resonance imaging biosensor using synthetic DNA sequences in order to evaluate the best conditions for the detection of a single mismatching base. Finally, the assay was tested on DNA extracted from human blood which was subsequently amplified using a whole genome amplification kit. The direct detection of the polymorphism was successfully achieved. The biochip was highly regenerable and reusable for up to 20 measurements. Furthermore, coupling these promising results with the multiarray assay, we can foresee applying this biosensor in clinical research extended to concurrent analysis of different polymorphisms.

Human parvovirus B19 infection: its relationship with systemic lupus erythematosus.

OBJECTIVES: The clinical presentation and outcome of four cases of human parvovirus-B19 (HPV-B19) infection, initially diagnosed as systemic lupus erythematosus (SLE), were reviewed and compared with similar cases previously reported in the literature. The relationship between HPV-B19 infection and SLE is discussed. METHODS: The medical records of four patients with documented HPV-B19 infection, initially diagnosed as SLE, were reviewed and studied in detail. A Medline search from 1985 to 1997 was performed to identify other cases reported in the literature in which a relationship between HPV-B19 and SLE had been identified in both adults and children. RESULTS: In all of our cases, the clinical findings (fever, rash, arthritis and malaise) and hematologic data (leukopenia, thrombocytopenia, anemia, presence of autoantibodies, hypocomplementemia, etc.) had initially suggested a diagnosis of juvenile SLE. Subsequently, evidence of HPV-B19 infection at the time of clinical presentation was ascertained. In three of these cases, the disease course was self-limiting with complete clinical remission and normalization of hematologic abnormalities within 18 months; one case, however, had persistent disease activity and repeated exacerbations. CONCLUSIONS: The occurrence of HPV-B19 infection has been documented in patients with SLE, in particular in relation to disease onset. Similarities in clinical and immunological features of viral infections and SLE at presentation may hinder the differential diagnosis between these two conditions. The family history, a self-limiting disease course and certain disease specific clinical aspects may help the pediatrician formulate an accurate diagnosis. In our patients, HPV-B19 infection may have mimicked the onset of SLE in three cases, but triggered the disease in one.

Reactive arthritis triggered by Yersinia enterocolitica: a review of 18 pediatric cases.

Over a period of four years 18 children seen in our clinic have been diagnosed as suffering from Yersinia enterocolitica reactive arthritis: this group constitutes one third of the total reactive arthritides seen by us. The diagnosis was based on elevated specific antibody titres and the outcome has always been good with anti-inflammatory therapy, symptoms disappearing within a few weeks without any complications or recurrences. This positive outcome could be due to the absence of the HLA B27 haplotype. Yersinia has been described as the most frequent agent causing reactive arthritis, a common complication in an otherwise benign infection in children. It seems that Yersinia may trigger in predisposed hosts an immunological reaction leading to arthritis.

Brain study using magnetic resonance imaging and proton MR spectroscopy in pediatric onset systemic lupus erythematosus.

OBJECTIVE: The aim of the present study was to assess and monitor brain damage in patients with pediatric onset systemic lupus erythematosus (SLE) using non-invasive techniques such as magnetic resonance imaging (MRI) and proton magnetic resonance spectroscopy (H-MRS). METHODS: Twenty-four SLE patients, both symptomatic or asymptomatic for central nervous system (CNS) involvement, and 20 controls were examined. Each individual underwent a diagnostic MRI using a 1.5 T Philips ACS-NT scanner including transverse T2-weighted (T2W) spin echo, transverse FLuid Attenuated Inversion Recovery (FLAIR), and sagittal T2W turbo spin echo 5 mm slices. In addition, single voxel proton MR spectroscopy localized on the supraventricular region was performed in all patients and controls. Patients were re-examined after one year. RESULTS: 75% of SLE patients had clinical CNS involvement; 46% showed abnormal MRI (3 of them, in the absence of neurologic signs); 4 SLE patients showed N-acetylaspartate/Creatine (NAA/Cr) ratios significantly lower than the controls. Among 5 SLE patients examined at the onset of the disease, 1 had MRI alterations and another showed a decrease of NAA/Cr values. Three patients with relapses showed a correlation between the course of the disease and the NAA/Cr ratios. CONCLUSION: MRI and H-MRS are non-invasive techniques that might be useful, in some cases, in detecting CNS involvement in SLE patients and monitoring the disease course and efficacy of pharmacological treatment.

Immunological findings in Kawasaki disease: an evaluation in a cohort of Italian children.

OBJECTIVE: Multiple humoral and cellular abnormalities in Kawasaki disease (KD) have already been described. In this study an analysis of immunological findings in a cohort of 34 Italian children affected with KD is reported, and the potential clinical significance of such alterations in predicting the development of coronary aneurysm and the prognosis of the disease is evaluated. METHODS: Levels of circulating immune complexes (CIC), antinuclear antibodies (ANA), anticardiolipin (aCL), antineutrophil cytoplasmic antibodies (ANCA), and anti-endothelial cells (AECA) and the T cell profile were determined in both the acute and the convalescent phases, and were compared to febrile, sex- and age-matched children. RESULTS: CIC were present in 66% of the patients, 18 of whom were in the acute phase and 13 in the convalescent phase. In the control group CIC were detected in 47% of the children. ANA were negative in both the KD and in the febrile group. ANCA were present in 8%, AECA in 26%, and aCL in 30% of KD patients (IgG aCL antibodies were found in 14 patients, IgM aCL in, 1 and 1 had both). Among the controls, aCL antibodies were found in 5 patients (22%); in particular 1 (4.4%) had IgG and 4 (17.4%) had IgM aCL. An altered T cell profile, with an inverted CD4/CD8 ratio, was found in all KD children. All of the immune alterations showed a lower incidence in the convalescent than in the acute phase. No significant relationship between any of these immune findings and cardiac involvement or any other clinical manifestations was found. CONCLUSION: Our data confirms the previously reported immunological anomalies in KD both in the acute and the convalescent phases, with a decreased incidence of such alterations in the convalescent phase. No prognostic significance for the occurrence of aneurysm could be demonstrated.

Preneoplastic lesions and prognostic factors of breast cancer.

The Authors have investigated preneoplastic lesions and the problem of the identification of benign lesions considered with or without preneoplastic potential, comparing for the occasion the presence of transformation markers in these lesions with the positivity of tumor markers in neoplastic tissue. The dosages have been valued on neoplastic tissue (180 cases), dysplastic tissue (50 cases), on cystic fluid (70 cases), and on nipple discharge (80 cases). The data obtained show a marked positivity to the tumor markers and the ionic and hormonal content (K+, Na+ DHEAS, PRL evaluation). The positivity of some lesions, not properly preneoplastic (or atypical) towards some markers of neoplasia could show some differences of great interest between benignity and malignity.

[Hormone replacement therapy (HRT). The effects at 12 months on the risk factors for menopausal osteoporosis]. / Terapia ormonale sostitutiva (HRT). Effetti a dodici mesi sugli indici di rischio per l'osteoporosi menopausale.

Cessation of ovarian activity is accompanied by a more or less marked and more or less accelerated reduction in bone mass; the degree and speed of the process--which occurs in all women--depends on individual (genetic factors influencing peak bone mass, duration of child-bearing period), iatrogenic (treatment with corticosteroids or thyroid hormones) or accidental factors (post-traumatic immobilization). Whatever the factor that triggers off the process, the end result is the destruction of bone tissue. This process may be documented by hematochemical (Nordin's test) and instrumental parameters (MOC and similar techniques). Oestroprogestin (and to a lesser extent calcitonin) hormone replacement therapy has been demonstrated to be highly efficacious in countering this involutive process. The authors report data obtained following the evaluation of 35 women in menopause undergoing. Nordin's test and measurement of the plasma level of estradiol using RIA, before and after twelve months after the start of osteoprotective treatment. Of the 35 patients 9 received only progestin, 22 an oestroprogestin combination (of these 18 patients received estrogen transdermally and 4 orally), and 4 calcitonin administered parenterally. A statistically significant positive correlation with Nordin's test was only found in the group receiving oestrogen therapy. In conclusion, it may be affirmed that in the absence of contraindications oestrogens represent the elective form of treatment for menopausal osteoporosis. Acceptable results have been reported in the literature also using calcitonin, but this treatment could not be evaluated in this study owing to the reduced number of the sample treated.

[The effects of hormone replacement therapy (HRT) on lipid metabolism in the menopause]. / Effetti della terapia ormonale sostitutiva (HRT) sul metabolismo lipidico in menopausa.

INTRODUCTION AND AIMS: During menopause the number of cardiovascular attacks increases parallel to the elimination of estradiol production. The administration of the latter reverses this tendency owing to a compound mechanism (improved HDL) cholesterol/total cholesterol ratio, diminished vasal resistance). MATERIALS AND METHODS: The authors present a study performed in 34 patients in menopause receiving oestroprogestinic replacement therapy using an oral or transdermal route. Metabolic status (serum concentrations of total cholesterol (TC) and HDL cholesterol (C.HDL) was evaluated in all patients before treatment and after 12 months. RESULTS: A statistically significant positive correlation (p < 0.0001) was found between the use of oestrogen and serum levels of HDL. This correlation appeared to be more evident in patients using transdermal treatment compared to the oral form. CONCLUSIONS: The authors conclude that in the absence of contraindications, hormone replacement therapy in menopause exercises a beneficial effect on the lipid status, contributing to diminishing the risk of cardiovascular attacks. The possibility of an increased incidence of breast cancer is now being evaluated, whereas effective protection of the endometrium against the risk of hyperplasia and cancer was shown using the doses of progestin used in this study, which coincide with those currently prescribed in the literature.

Direct detection of genomic DNA by surface plasmon resonance imaging: an optimized approach.

The direct detection of specific sequences in genomic DNA samples is very challenging in the biosensor-based approach. In this work we developed an optimized strategy for the direct detection of DNA sequences in human genomic samples by a surface plasmon resonance imaging technology. As model study, the target analyte was identified in a DNA sequence mapping the human ABCB1 gene. The computed-assisted approach was here applied for probe design. After a preliminary evaluation of the probe functioning by the complementary synthetic target, the system was applied to the direct detection of the target sequence in human genomic DNA extracted from lymphocytes. To achieve this result, several steps aimed to improve the analytical performances of the biosensor were studied and optimized. The immobilization chemistry, based on thiolated probes, was adapted here to non-amplified sequence detection. DNA sample pre-treatments, i.e. genomic fragmentation by ultrasounds and dsDNA denaturation by thermal treatment were also investigated. A sandwich-like strategy, by using a secondary probe, was also applied to understand and confirm the selectivity of the developed biosensor in detecting ABCB1 gene in genomic samples. Finally, a reliable calibration curve of ABCB1 was obtained with an experimental detection limit of 140 aM. Furthermore, the biosensor was well regenerable, assuring up to thirty cycles of effective measurements.

SPR detection of human hepcidin-25: a critical approach by immuno- and biomimetic-based biosensing.

The human hepcidin-25 hormone has a key role in iron regulation in blood. The clinical relevance of this hepatic ~2.8 kDa cysteine-rich peptide is rapidly increasing, since altered levels can be associated with inflammatory events and iron dysfunctions, such as hereditary hemochromatosis and iron overload. Moreover, hepcidin has also attracted the anti-doping field for its possible role as indirect marker of erythropoietin blood doping. Methods currently reported are based on immunoassays (ELISA and RIA), or various types of mass spectroscopy (MS)-based protocols, semi-quantitative or quantitative. Despite the great effort in optimizing robust and simple assays measuring hepcidin in real matrices, at present this challenge remains still an open issue. To explore the possibility to face hepcidin detection through the development of affinity-based biosensors, we set up a comparative study by surface plasmon resonance (SPR) technology. An immuno-based, on anti-hepcidin-25 IgG, and a biomimetic-based, on a synthetic peptide corresponding to the hepcidin-binding site on ferroportin (HBD), biosensors were developed. Here we report behaviors and analytical performances of the two systems, discussing limits and potentialities.

A rational approach in probe design for nucleic acid-based biosensing.

Development of nucleic acid-based sensing attracts the interest of many researchers in the field of both basic and applied research in chemistry. Major factors for the fabrication of a successful nucleic acid sensor include the design of probes for target sequence hybridization and their immobilization on the chip surface. Here we demonstrate that a rational choice of bioprobes has important impact on the sensor's analytical performances. Computational evaluations, by a simple and freely available program, successfully led to the design of the best probes for a given target, with direct application to nucleic acid-based sensing. We developed here an optimized and reproducible strategy for in silico probe design supported by optical transduction experiments. In particular Surface Plasmon Resonance imaging (SPRi), at the forefront of optical sensing, was used here as proof of principle. Five probes were selected, immobilized on gold chip surfaces by widely consolidated thiol chemistry and tested to validate the computational model. Using SPRi as the transducting component, real-time and label free analysis was performed, taking the Homo sapiens actin beta (ACTB) gene fragment as model system in nucleic acid detection. The experimental sensor behavior was further studied by evaluating the strength of the secondary structure of probes using melting experiments. Dedicated software was also used to evaluate probes' folding, to support our criteria. The SPRi experimental results fully validate the computational evaluations, revealing this approach highly promising as a useful tool to design biosensor probes with optimized performances.