Different Patterns of Induction of Fibroblast Growth Factor-2 and Brain-Derived Neurotrophic Factor Messenger RNAs During Kindling Epileptogenesis, and Development of a Herpes Simplex Vector for Fibroblast Growth Factor-2 Gene Transfer in Vivo

Summary: Purpose : To investigate the gene expression patterns of brain-derived neurotrophic factor (BDNF) and fibroblast growth factor-2 (FGF-2) in the kindling model, and to construct a replication-defective herpes simplex virus vector to induce expression of FGF-2 in vivo.
Methods : RNase protection assay and herpes simplex virus vector (TH FGF-2) deleted in the immediate-early genes ICP4, ICP22, and ICP27, with FGF-2 inserted in tk under the control of the human cytomegalovirus immediate-early promoter.
Results : A single kindling stimulation did not modify BDNF gene expression, whereas it increased FGF-2 messenger RNA (mRNA) levels in the hippocampus, the cortex, and the hypothalamus. BDNF and FGF-2 gene expression were not altered in kindled animals left unstimulated for 1 week. In contrast, kindled seizures produced a great increase in hippocampal and cortical BDNF mRNA levels, but FGF-2 mRNA was increased only in the ipsilateral cortex. Infection of Vero cells with TH FGF-2 resulted in a long-lasting increase in FGF-2 levels. Protein extracts of infected cells induced neuronal differentiation of PC12 cells, indicating that the newly synthesized FGF-2 was biologically active. Robust transient transgene expression was observed in the rat hippocampus after inoculation with TH FGF-2 in the absence of significant toxicity.
Conclusions : BDNF and FGF-2 are recruited at different stages of kindling and, accordingly, may play different roles in the adaptive changes taking place during epileptogenesis. TH FGF-2 is suitable for studies of FGF-2 involvement in kindling epileptogenesis.     

Maintenance of T cell specification and differentiation requires recurrent notch receptor-ligand interactions

Notch signaling has been shown to play a pivotal role in inducing T lineage commitment. However, T cell progenitors are known to retain other lineage potential long after the first point at which Notch signaling is required. Thus, additional requirements for Notch signals and the timing of these events relative to intrathymic differentiation remain unknown. Here, we address this issue by culturing subsets of CD4 CD8 double negative (DN) thymocytes on control stromal cells or stromal cells expressing Delta-like 1 (Dll1). All DN subsets were found to require Notch signals to differentiate into CD4+ CD8+ T cells. Using clonal analyses, we show that CD44+ CD25+ (DN2) cells, which appeared committed to the T cell lineage when cultured on Dll1-expressing stromal cells, nonetheless gave rise to natural killer cells with a progenitor frequency similar to that of CD44+ CD25- (DN1) thymocytes when Notch signaling was absent. These data, together with the observation that Dll1 is expressed on stromal cells throughout the thymic cortex, indicates that Notch receptor-ligand interactions are necessary for induction and maintenance of T cell lineage specification at both the DN1 and DN2 stages of T cell development, suggesting that the Notch-induced repression of the B cell fate is temporally separate from Notch-induced commitment to the T lineage.     

Humoral immunity and correlation between ELISA, hemagglutination inhibition, and neutralization tests after vaccination against tick-borne encephalitis virus in children

Vaccination against tick-borne encephalitis (TBE) virus is the measure of choice for disease control in endemic areas, as no treatment is available. In Italy, the province of Belluno is one of the most active TBE virus infection foci. In this study sera were examined from vaccinated children living in areas around Belluno in order to monitor the immune response after anti-TBE vaccination. For the assessment of neutralizing antibodies, a plaque reduction neutralization test (PRNT) was optimized and the correlation between enzyme-linked immunosorbent assay (ELISA), hemaglutination inhibition (HI), and neutralizing antibodies titers was evaluated. All children had high serum levels of TBE IgG in ELISA test after the vaccination, in agreement with previous studies. HI and PRNT titers ranged between very low and high levels. A good correlation between HI and PRNT titers, and with IgG ELISA titers, was observed. PRNT is an useful assay for monitoring protective immunity after the completion of anti-TBE vaccination. This type-specific assay is an important tool for differential diagnosis in cases where the presence of cross-reactive antibodies due to other flavivirus infections or vaccinations cannot be excluded.     

Conversion of MCI to dementia: Role of proton magnetic resonance spectroscopy

Mild cognitive impairment (MCI) represents a heterogeneous group of cognitive disturbances at high risk of dementia. The amnestic subtype (aMCI) might be a prodromal state of Alzheimer's disease (AD). The aim of this study is the identification, by proton magnetic resonance spectroscopy (1H MRS), of modifications in brain metabolites able to detect subjects with aMCI at risk of conversion towards AD. Twenty-five subjects with aMCI and 29 normal elderly were enrolled; they underwent a comprehensive clinical and instrumental assessment, a cerebral 1H MRS scan to measure N-acetyl aspartate (NAA), choline (Cho), myo-inositol (mI) and creatine (Cr) in the paratrigonal white matter, bilaterally. After 1 year, 5 MCI subjects became demented (progressive MCI, pMCI). Their baseline levels of metabolites were compared with those evaluated in stable MCI (sMCI) and in controls. We observed a significant difference of the NAA/Cr ratio between pMCI (1.48+/-0.08) and sMCI (1.65+/-0.12) and between pMCI and controls (1.63+/-0.16) in the left hemisphere, suggesting that this metabolic alteration can be detected before the clinical appearance of dementia.     

Comparison of different commercial FFDM units by means of physical characterization and contrast-detail analysis

The purpose of this study was to perform a complete evaluation of three pieces of clinical digital mammography equipment. Image quality was assessed by performing physical characterization and contrast-detail (CD) analysis. We considered three different FFDM systems: a computed radiography unit (Fuji "FCR 5000 MA") and two flat-panel units, the indirect conversion a-Si based GE "Senographe 2000D" and the direct conversion a-Si based IMS "Giotto Image MD." The physical characterization was estimated by measuring the MTF, NNPS, and DQE of the detectors with no antiscatter grid and over the clinical range of exposures. The CD analysis was performed using a CDMAM 3.4 phantom and custom software designed for automatic computation of the contrast-detail curves. The physical characterization of the three digital systems confirms the excellent MTF properties of the direct conversion flat-panel detector (FPD). We performed a relative standard deviation (RSD) analysis, for investigating the different components of the noise presented by the three systems. It turned out that the two FPDs show a significant additive component, whereas for the CR system the statistical noise is dominant. The multiplicative factor is a minor constituent for all the systems. The two FPDs demonstrate better DQE, with respect to the CR system, for exposures higher than 70 microGy. The CD analysis indicated that the three systems are not statistically different for detail objects with a diameter greater than 0.3 mm. However, the IMS system showed a statistically significant different response for details smaller than 0.3 mm. In this case, the poor response of the a-Se detector could be attributed to its high-frequency noise characteristics, since its MTF, NEQ, and DQE are not inferior to those of the other systems. The CD results were independent of exposure level, within the investigated clinical range. We observed slight variations in the CD results, due to the changes in the visualization parameters (window/level and magnification factor). This suggests that radiologists would benefit from viewing images using varied window/level and magnification.     

NF1 gene analysis based on DHPLC

The high mutation rate at the NF1 locus results in a wide range of molecular abnormalities. The majority of these mutations are private and rare, generating elevated allelic diversity with a restricted number of recurrent mutations. In this study, we have assessed the efficacy of denaturing high-performance liquid chromatography (DHPLC), for detecting mutation in the NF1 gene. DHPLC is a fast and highly sensitive technique based on the detection of heteroduplexes in PCR products by ion pair reverse-phase HPLC under partially denaturing conditions. We established theoretical conditions for DHPLC analysis of all coding exons and splice junctions of the NF1 gene using the WAVEmaker software version 4.1.40 and screened for mutations a panel of 40 unrelated NF1 patients (25 sporadic and 15 familial), genetically uncharacterized. Disruptive mutations were identified in 29 individuals with an overall mutation detection rate of 72.5%. The mutations included eight deletions (exons 4b, 7, 10a, 14, 26, and 31), one insertion (exon 8), nine nonsense mutation (exons 10a, 13, 23.1, 27a, 29, 31, and 36), six missense mutations (exons 15, 16, 17, 24, and 31), four splice errors (exons 11, 14, 36, and 40) and a complex rearrangement within exon 16. Eighteen (62%) of the identified disruptive mutations are novel. Seven unclassified and three previously reported polymorphisms were also detected. None of the missense mutations identified in this study were found after screening of 150 controls. Our results suggest that DHPLC provides an accurate method for the rapid identification of NF1 mutations.     

Genital Chlamydia trachomatis infection is related to poor sexual quality of life in young sexually active women

IntroductionChlamydia trachomatis (Ct) genital infection has been related to several diseases in young sexually active women. It could be related to their sexual quality of life.AimTo assess whether genital Ct infection can induce sexual function alterations in women.MethodsNine hundred ninety‐eight women (mean age 29.4, range 18–43) attending our Sexually Transmitted Disease Centre were enrolled in this observational case‐control study. All participants were clinically and microbiologically investigated due to their sexual relationships with a subject affected by chronic bacterial prostatitis. All participants underwent microbiological cultures, DNA and antibodies evaluation for common bacteria and Ct on vaginal swab and urine samples. They completed the Female Sexual Function Index [FSFI] questionnaire. On the basis of microbiological investigation results, all patients were split into three groups: Group A—genital Ct infection, Group B—genital common bacteria/yeast infection, and Group C—negative for Ct and bacteria/yeast infection.Main Outcome MeasuresFSFI questionnaire.ResultsTwo hundred ninety‐one women were classified in Group A, 276 in Group B, and 431 in Group C. Group A patients were statistically, significantly different from Group B and Group C patients in terms of pain during sexual intercourse and sexual satisfaction. Group C patients had significantly higher FSFI scores (27.1 ± 1.3) (P < 0.001) for both desire (4.9 ± 1.0) and lubrication domain (3.8 ± 1.1) (P < 0.001, P < 0.003, respectively) when compared with Group A patients. Multivariate analysis demonstrated that negative Ct infection marker in female patients must be considered as an independent prognostic factor in predicting a subsequent optimal FSFI questionnaire score (P = 0.002).ConclusionsPositive values of Ct infection markers are associated with lower FSFI scores for sexual desire, lubrication, and overall sexual function. Genital Ct infection could induce pain during sexual intercourse, reducing sexual satisfaction and sexual quality of life in young sexually active women. Cai T, Mondaini N, Migno S, Meacci F, Boddi V, Gontero P, Malossini G, Geppetti P, Mazzoli S, and Bartoletti R. Genital Chlamydia trachomatis infection is related to poor sexual quality of life in young sexually active women.     

Magnetic alginate microspheres: system for the position controlled delivery of nerve growth factor

The use of polymeric carriers containing dispersed magnetic nanocrystalline particles for targeted delivery of drugs in clinical practice has attracted the interest of the scientific community. In this paper a system comprised of alginate microparticles with a core of magnetite and carrying nerve growth factor (NGF) is described. The magnetic properties of these microspheres, typical of superparamagnetic materials, allow precise and controlled delivery to the intended tissue environment. Experiments carried out on PC12 cells with magnetic alginate microspheres loaded with NGF have confirmed the induction of cell differentiation which is strongly dependent on the distance from the microsphere cluster. In addition, finite element modelling (FEM) of the release profile from the microspheres in culture, indicated the possibility of creating defined and predictable NGF gradients from the loaded microspheres. These observations on the carriage and release of growth factors by the proposed microparticles open new therapeutic options for both neuronal regeneration and of the development of effective neuronal interfaces.