The new HLA-DQB1*05:304 allele identified in an Italian patient.

DQB1*05:304 allele was identical to DQB1*05:02:01 except for a single nucleotide substitution.

Effects of short-term exposure to genistein and overfeeding diet on the neural and retinal progenitor competence of adult zebrafish (Danio rerio).

Neurogenesis is a process that occurs throughout the life of a vertebrate. Among the different factors that may affect the natural occurrence of neurogenesis, obesity seems to decrease the proliferation capacity of progenitor neuronal cells. Conversely, the phytoestrogen genistein is known to attenuate some obesity effects beyond its neuroprotective action. Aiming to improve the understanding of how obesity and genistein trigger an impact on the neural and retinal progenitor competence of adult zebrafish, fish were exposed to genistein (GEN - 2 µg L-1) alone or combined with two dietary groups (control and overfeed - OFD) for up to 9 weeks. Zebrafish were fed once per day with Artemia sp. in the control and GEN (2% of BW, control diet), and three times per day in the OFD and OFD + GEN groups (12% BW, overfeeding diet). To assess obesity induction, BMI, biometric parameters, and PPAR-γ protein were quantified. Afterwards, qRT-PCR and immunohistochemistry were performed to determine the cell proliferation and the presence of stem cells through PCNA and Sox-2. Our findings proved that overfeeding adult zebrafish increased the general growth and induced the development of fatty liver. However, for OFD + GEN, this effect was assuaged through the anti-adipogenic effect of GEN. This finding suggests that phytoestrogens could be beneficial to reduce the negative effects of obesity. Moreover, OF induced negative effects on retinal and brain homeostasis, decreasing the proliferation capacity of progenitor neuronal cells. With regard to retinal progenitor competence, genistein seems to mitigate the negative impacts of obesity, whereas the effects of obesity on the brain were exacerbated by this phytoestrogen which negatively influenced the homeostasis of zebrafish neural progenitor competence. This study highlighted the fact that the effects of phytoestrogens in adult neural progenitor competence are complex and could exhibit dissimilar effects depending on the tissue.

Copper impair autophagy on zebrafish (Danio rerio) gill epithelium.

Copper (Cu) is an essential element for organism's metabolism, being controversially listed as a priority pollutant. Importantly, the toxicity of Cu has been linked to several cell death pathways. Thus, this study aimed to assess if macroautophagic pathways are triggered by Cu in zebrafish gill, the main target of waterborne pollutants. The electron microscopy findings indicated that Cu induced profound impacts on zebrafish gill structure and functions, being this tissue a biomarker sensitive enough to indicate early toxic effects. The findings also support a clear impairment of autophagy, througth the absence of phagossomes and the significant down-regulation mRNA transcript levels of microtubule-associated protein light chain 3 (LC3). The reduction of LC3 levels was often associated to an increase of apoptotic activation, indicating that the inhibition of macroautophagy triggers apoptosis in zebrafish gills. This study highlighted that the autophagic down-regulation might be affected through the activation of other cell death signaling pathway.

Kaolin modulates ABA and IAA dynamics and physiology of grapevine under Mediterranean summer stress.

The foliar exogenous application of kaolin, a radiation-reflecting inert mineral, has proven to be an effective short-term climate change mitigation strategy for Mediterranean vineyards. In this work, we address the hypothesis that kaolin could improve both the hormonal dynamics and physiological responses of grapevines growing in Douro Region, northern Portugal. For this purpose, the leaf water potential, gas exchange and chlorophyll a fluorescence parameters were monitored, as well as the abscisic acid (ABA) and indole-3-acetic acid (IAA) quantification and immunolocalization were assessed. The study revealed a slight decrease in ABA and an increase in IAA in the kaolin treatment, which in turn were associated with the improvement of physiological performance. A month after spraying, kaolin improves the water potential respectively, 30% and 17% in the predawn and midday periods. Besides, plants treated with kaolin showed higher values of stomatal conductance, net CO2 assimilation rate and intrinsic water use efficiency. Kaolin also ameliorates the effective PSII efficiency (67%), as well as the maximum quantum efficiency of photosystem II and the photosynthetic electron transport rate (>73%). These results were consistent with the higher photochemical quenching and the lower non-photochemical quenching observed in treated leaves and with the better performance obtained by the JIP test parameters. Physiological and hormonal analysis confirmed that kaolin effectively enhance grapevine summer stress tolerance.

A nucleotide insertion in Exon 2 is responsible for a new HLA-DRB1 null allele, HLA-DRB1*14:166N.

DRB1*14 is identical to DRB1*14:54:01 except for a single nucleotide insertion of A in position 175 in Exon 2.

Sequence-based typing characterization of the novel HLA-DRB3*01:16 allele, identified in an Italian family.

The novel DRB1*01:16 allele differs for G to T substitution at position 595 from DRB3*01:01P.

Characterization of a novel HLA-DQB1*06 allele, HLA-DQB1*06:04:04.

The novel allele human leukocyte antigen(HLA)-DQB1*06:04:04 differs from HLA-DQB1*06:04:01 by a silent nucleotide substitution at codon 75 (TTG → CTG).

The novel HLA-A*03:143 allele, identified by sequence-based typing in an Italian family.

HLA-A*03:143 has one nucleotide change from A*03:01: 01:01 at nt 406 from G to C, resulting in an amino acid change at codon 112 of exon 3 from Gly to Arg.

The novel HLA-C*06:58 allele, identified by sequence-based typing in an Italian family.

The novel HLA-C allele C*06:58 shows one nucleotide change from C*06:02:01:01 at nt 366 from G to A, resulting in an amino acid change at codon 98 of exon 3 from Met to Ile.

Does the term 'trophic' actually mean anti-amyloidogenic? The case of NGF.

The term trophic is widely used to indicate a general pro-survival action exerted on target cells by different classes of extracellular messengers, including neurotrophins (NTs), a family of low-molecular-weight proteins whose archetypal member is the nerve growth factor (NGF). The pro-survival action exerted by NTs results from a coordinated activation of multiple metabolic pathways, some of which have only recently come to light. NGF has been shown to exert a number of different, experimentally distinguishable effects on neurons, such as survival, differentiation of target neurons, growth of nerve fibers and their guidance (tropism) toward the source of its production. We have proposed a more complete definition of the NGF trophic action that should also include its newly discovered property of inhibiting the amyloidogenic processing of amyloid precursor protein (APP), which is among the first hypothesized primary trigger of Alzheimer's disease (AD) pathogenesis. This inhibitory action appears to be mediated by a complex series of molecular events and by interactions among NGF receptors (TrkA and p75), APP processing and tau metabolic fate and function.

Which target for DBS in Parkinson's disease? Subthalamic nucleus versus globus pallidus internus.

We selected 14 patients with advanced idiopathic Parkinson's disease (PD) and examined the clinical effects of STN DBS versus GPi DBS. Nine patients underwent bilateral STN DBS and five underwent bilateral GPi patients. All patients were followed for at least 12 months. The evaluation was performed on and off drug before surgery; on-drug/on-DBS and off-drug/on-DBS at 1, 3, 6 and 12 months after stereotactic surgery. At 1 and 3 months after surgery in off-drug/on-DBS condition, both groups showed an improvement in motor score (UPDRS III). Nevertheless, the results changed after long-term stimulation in the two groups. Chronic STN DBS is superior to GPi DBS in the amelioration of the clinical features and in the decrease of time spent in the off state. The efficacy in reduction of LID was comparable at 1 and 3 months after surgery, but the results were better in STN DBS after chronic stimulation. The L-dopa dose was reduced only in the STN group.

[Knee arthroscopy in children]. / Arthroscopie du genou de l'enfant.

PURPOSE OF THE STUDY: Between 1990 and 1998, 110 knee arthroscopies were performed in children. We analyzed the epidemiology and diagnostic data and studied the correlation between clinical and radiographic findings and the final diagnosis after arthroscopy in order to establish a consensus on use of knee arthroscopy in children. MATERIAL AND METHODS: We made a retrospective analysis of 110 knee arthroscopies performed in children, classing the patients in three age groups: 0-5 years, 5-10 years, 10-17 years. Clinical and radiological findings were compared with the arthroscopy findings. RESULTS: One or more arthroscopies were performed in 56 boys and 48 girls. Mean age at the time of the procedure was 12 years 4 months. There were 11 children aged 0-5 years, 14 aged 5-10 years and 85 aged 10-17 years. The main pathology was arthritis in the 0-5 year and 5-10 year age groups. Trauma was more frequent in the older children. Knee arthroscopy was found to be normal in 19 cases. DISCUSSION: For most surgery teams, arthroscopy is indicated for arthritis of the knee. Arthroscopy may also be needed for hemarthrosis. In these contexts, arthroscopy is both a diagnostic and therapeutic procedure. Our analysis demonstrates that emergency arthroscopy is only warranted for free floating osteochondral fractures and fractures of the tibial articular surfaces, with the exception of the tibial spines. Arthroscopy may be performed later in other cases after careful physical examination and radiographic series. We had 19 normal arthroscopies and 10 that showed femoropatellar chondropathies and plicas that could explain knee pain. We recommend arthrography before arthroscopy to avoid unnecessary procedures. CONCLUSION: Arthritis of the knee is an excellent indication for arthroscopy. Painful and acute hemarthrosis requires attentive physical exams and x-rays before making the decision for surgery.

Granule neuron DNA damage following deafferentation in adult rats cerebellar cortex: a lesion model.

Neuronal programmed cell death is regulated by a neurotrophic supply from targets and afferent inputs. The relative contribution of each component varies according to neuronal type and age. We have previously reported that primary cultures of cerebellar granule cells undergo apoptosis when deprived of depolarising KCl concentrations, suggesting a significant role of afferent inputs in the control of cerebellar granule cells survival. This issue was investigated by setting up various in vivo lesional paradigms in order to obtain partial or total deafferentation of the cerebellar granule layer in adult rats. At different times after surgery, cerebellar sections were subjected to TUNEL staining in order to detect possible DNA damage. One week after unilateral pedunculotomy, few scattered groups of apoptotic granule neurons were observed in the homolateral hemisphere. On the contrary, total deafferentation obtained by a new experimental paradigm based on an "L-cut" lesion induced massive and widespread apoptotic death in the granule layer of the deafferentated area. The time window of DNA fragmentation in granule layer was one to seven days after the "L-cut". Selective Purkinje cell deafferentation obtained by 3-acetylpyridine injection did not result in TUNEL staining in the cerebellar cortex. The current finding that mossy fiber axotomy induces granule cell apoptotic death points out for the first time the crucial role of afferent inputs in mature granule cell survival. Moreover, the in vivo lesional model described here may prove to be an useful tool for investigating cellular and molecular mechanisms of neuronal death triggered by deafferentation.

Neuroantibodies: ectopic expression of a recombinant anti-substance P antibody in the central nervous system of transgenic mice.

Recombinant antibodies are efficiently secreted by cells of the nervous system. Thus, their local expression in the CNS of transgenic mice could be used to perturb the function of the corresponding antigen. As a first application of this approach, we have generated transgenic mice that express antibodies against the neuropeptide substance P, under the transcriptional control of the promoter of the neuronal gene vgf. The transgenic antibodies are expressed in a tissue-specific and developmentally regulated manner and are effective in competing with the endogenous substance P, as demonstrated by a marked inhibition of neurogenic inflammation and by motor deficits. This phenotypic knockout approach may provide a complementary alternative to gene knockout by homologous recombination.

A recombinant cell line expressing a form of the Y13-259 anti-p21ras antibody which binds protein A and may be produced as ascites.

We have recently reported the molecular cloning of the immunoglobulin genes encoding the variable regions of the rat anti-p21ras antibody, Y13-259. These genes were reassembled into expression vectors supplying DNA sequences encoding human gamma 1 and kappa constant domains, as well as the leader sequence for antibody secretion, thus yielding Hu-Y13-259, a secretory anti-p21ras antibody containing human constant regions. We now report the creation of a recombinant cell line, NS0/Hu-Y13-259/B6, secreting high levels of the Hu-Y13-259 Ig. The antigen specificity of this recombinant antibody was demonstrated to be identical to that of the parental Y13-259, i.e. the amino acid sequence 60-76 of the p21ras protein. Unlike the parental cell line, the recombinant cells could be grown as ascites in mice, allowing the production of large quantities of the protein A-binding Hu-Y13-259 antibody.

Neuroantibodies: molecular cloning of a monoclonal antibody against substance P for expression in the central nervous system.

We present a strategy to study functional and/or developmental processes occurring in the nervous system, as well as in other systems, of mice. This strategy is based on the local expression of specific monoclonal antibodies (mAbs) by cells of the nervous system. As an application of this strategy, we report the cloning of the anti-substance P rat mAb NC1/34HL. Functional substance P-binding antibodies were reconstituted from the cloned variable domains by using vectors for expression in myeloma cells. With these and other vectors a general system for the cloning and expression of mAbs under a series of promoters (of the rat VGF8a gene, the neurofilament light-chain gene, and the methallothionein gene) has been created. The activity of these plasmids was confirmed by expressing the recombinant NC1/34HL mAb in GH3 pituitary cells, PC12 pheochromocytoma cells, and COS cells. DNA from the described constructs can be used to target the expression of the NC1/34HL mAb to the central nervous system of transgenic mice. This procedure will allow us to perturb substance P activity in a controlled way in order to dissect its multiple roles.

IFN-gamma facilitates NGF-induced neuronal differentiation in PC12 cells.

Natural or recombinant murine interferon-gamma causes a reversible arrest of proliferation of PC12 cells. Treatment with other antimitotics (AraC, colchicine, mitomycin C, hydroxyurea) or removal of serum, on the contrary, leads to mitotic arrest followed by cell death. IFN-gamma-treated PC12 cells respond more rapidly to NGF in terms of speed of neuronal outgrowth. On the other hand, NGF potentiates the action of IFN-gamma in stimulating the enzyme 2',5'-A synthetase which shifts from an average of 4.4-fold stimulation at 48 h with IFN-gamma alone to increments varying between 5- and 18-fold when PC12 cells are treated for 48 h with IFN-gamma and NGF. NGF alone, on the contrary, does not exert any detectable effect on this enzyme. From the findings we propose the use of a combined treatment of PC12 cells with NGF and IFN-gamma for a more rapid induction of neuronal differentiation.