Comparison of a multiplexed bead-based assay with an immunofluorescence and an enzyme-immuno assay for the assessment of Epstein-Barr virus serologic status

Dans cette étude, nous avons testé la performance diagnostique d'une nouvelle technique d'analyse multiplexée qui permet la détection d'anticorps de différentes spécificités dans la même réaction. En l'absence de gold standard, nous avons choisi de comparer la performance diagnostique de l'analyse avec deux méthodes de référance que sont l'IF et EIA, et avec un consensus déterminé selon une règle de majorité entre les trois méthodes.¦393 sérums analysés par IF, conservés par congélation, ont été décongelés pour être analysés par EIA et BBA. Pour chaque sérum, les anticorps recherchés ont été les anti-VCA (Viral Capsid Antigen) IgM, anti-VCA IgG et anti-EBNA (Epstein-Barr Nuclear Associated) IgG. Les échantillons ont été classés en cinq groupes selon les résultats de l'IF : séronégatifs, infections aiguës, infections anciennes et deux types d'indéterminés.¦Pour chaque méthode, le résultat numérique (index ou titre) des analyses est converti en termes de positif, négatif ou douteux. Pour le résultat de chaque type d'anticorps, un consensus est établi selon une règle de majorité entre les trois méthodes, permettant une interprétation du stade de l'infection. Puis l'interprétation de chacune des méthodes a été comparée au consensus. Nous avons également comparé les trois méthodes les unes aux autres concernant la détection des anticorps.¦Globalement, nous observons une bonne corrélation qualitative entre les trois approches pour détecter les anti-VCA IgG et IgM. Pour pour les anti-EBNA IgG, il y a une divergence notable entre l'IF et les deux autres méthodes, l'IF apparaissant moins sensible que les autres méthodes, résultant en un nombre accru d'interprétations indéterminées du stade de l'infection.¦L'origine de cette divergence ne peut être due à une perte d'anticorps liée au stockage de longue durée des échantillons. En effet, EIA et BBA restent plus sensibles que IF, dont l'analyse a été faite sur des sérums frais.¦Cette divergence ne semble pas non plus être due aux différents antigènes utilisés par les trois méthodes. EIA et BBA utilisent le même antigène recombinant EBNA-1, alors que l'IF utilise des "cellules lymphoïdes choisies pour leur production sélective d'antigènes EBNA". Ces cellules sont probablement des cellules infectées par EBV qui devraient exprimer plus d'antigènes de latence que seul EBNA-1. Cette différence devrait donc plutôt en principe résulter en une meilleure sensibilité de l'IF par rapport aux deux autres méthodes.¦Les anti-EBNA IgG peuvent disparaître chez les patients immunocompromis chez qui se produit une réactivation d'EBV. Nous avons donc recherché le status immunitaire des patients du groupe dont les sérums étaient négatifs pour anti-EBNA IgG en IF et positifs par les autres méthodes: seulement 28 des 70 patients étaient immunocompromis.¦Par conséquent, il est probable que dans la majorité de ces résultats discordants, les anticorps anti-EBNA IgG détectés par BBA et EIA sont de vrais positifs non décelés par l'IF.¦En conclusion, BBA est meilleur que la méthode de référance qu'est l'IF, et est égal à EIA en ce qui concerne la performance diagnostique. En outre, ces deux nouvelles méthodes offrent une économie de temps en raison de manipulations moindres, et ne requièrent aucune formation en microscopie à fluorescence. Elles sont également plus économes en échantillons que IF. BBA a l'avantage de n'avoir besoin que de deux analyses pour donner un diagnostique, alors que IF et EIA ont en besoin d'une par anticorps. Enfin, BBA dispose de contrôles internes permettant de reconnaître les liaisons non antigène-spécifiques des anticorps. Par contre, BBA nécessite l'achat d'un lecteur par cytométrie de flux assez coûteux.

Assessing multiple sclerosis activity: is the in vitro production of tumor necrosis factor-alpha, interleukins 2, 6, 4, and 10, and immunoglobulin G of value?

Tumor necrosis factor (TNF) alpha, interleukins (IL) 2, 4, 6, and 10, and IgG oligoclonal bands (IgG OB) in vitro production was assessed, after whole-blood stimulation with lipopolysaccharide or concanavalin A, in 61 patients presenting with relapsing-remitting, relapsing-progressive, or chronic progressive multiple sclerosis. Multiple sclerosis patients were receiving no treatment or azathioprine (AZA), cyclosporin, cyclophosphamide, subcutaneous interferon (IFN) beta 1 a, or corticosteroids (CST). Statistical correlations significantly showed that: (a) AZA lowers TNF-alpha (P = 0.002) and increases IL-4 production (P = 0.0024), and IFN-beta 1 a increases TNF-alpha and decreases IL-4 levels; (b) CST has a negative effect on TNF-alpha, IL-6, and IL-4 synthesis; and (c) AZA, IFN-beta 1 a, and CST diminish IgG OB synthesis (P = 0.001). Although our study of the dynamics of TNF-alpha, IL-2, IL-4, IL-6, and IL-10 in vitro production generally found no statistically significant correlations (partly explained by the limited number of values in the various groups), IL-6 was shown to drop during the periods surrounding relapse (P = 0.05) in the absence of treatment, while TNF-alpha (P = 0.04) and IL-6 (P < 0.05) dropped before exacerbation in the presence of AZA. In vitro production of TNF-alpha was closely and positively correlated with that of IL-6, independently of clinical features. The enhanced production of IL-10 detected before or at relapse with AZA and IFN-beta 1 a (trends) may interfere with initiation of the immune reaction and with the development of new CNS lesions. Some discrepancies with previously published results stress the difficulties in studying the state of stimulation of different populations of leukocytes by using a variety of in vitro stimuli and in establishing a correlation between mRNA studies and the amount of final or active protein produced.

Differential distribution of thyroid hormone receptor isoform in rat dorsal root ganglia and sciatic nerve in vivo and in vitro.

Using autoradiographic techniques carried out under precise conditions we previously demonstrated that both sensory neurons and peripheral glial cells in dorsal root ganglia (DRG) or sciatic nerve, possess specific [125I]-labeled T3 binding sites. Thyroid hormone receptors (TR) include several isoforms (TR alpha(1), TR alpha(2), TR beta(1), TR beta(2...)) The present study demonstrates that while sensory neurons and peripheral glial cells both possess functional TR, they express a differential expression of TR isoforms. Using a panel of antisera to specific for the TR alpha-common (alpha(1) and alpha(2)), TR alpha-1 or TR beta-1 isoforms, we detected TRs isoform localization at the cellular level during DRG and sciatic nerve development and regeneration. Immunohistochemical analysis revealed that during embryonic life, sensory neurons express TR alpha-common and TR beta-1 rather than TR alpha-1. The number of TR alpha-common and TR beta-1 positive neurons as well as the intensity of labeling increased during the first two postnatal weeks and remained more or less stable in adult life. TR alpha-1 immunoreactivity, which was undetectable in embryonic sensory neurons, became discreetly visible in neurons after birth. In developing DRG and sciatic nerves, Schwann cells exhibited TR alpha-common and TR alpha-1 rather than TR beta-1 immunolabeling. The appearance of TR alpha-common and alpha-1 isoform immunoreactivity in the sciatic nerve was restricted to a short period ranging from E17 up to two postnatal weeks. By comparing TR alpha-common and TR alpha-1 immunostaining we can deduce that Schwann cells primarily express TR alpha-1. Afterwards, in adult rat sciatic nerve TR alpha isoforms was no more detected. However transection of sciatic nerve caused a reexpression of TR alpha isoforms in degenerating nerve. The prevalence of TR alpha in Schwann cells in vivo was correlated with in vitro results. The differential expression of TR alpha and beta by sensory neurons and Schwann cells indicates that the feedback regulation of circulating thyroid hormone could occur by binding to either the alpha or beta TR isoforms. Moreover, the presence of multiple receptor isoforms in developing sensory neurons suggests that thyroid hormone uses multiple signaling pathways to regulate DRG and sciatic nerve development.

Registre d'entrades i eixides per a l'administració pública

Aquesta memòria representa la definició del meu projecte final de carrera amb una aplicació destinada al registre d'entrades i eixides de l'Administració pública. He emprat eines de plataformes lliures i obertes per al desenvolupament del projecte, amb tecnologia J2EE. A més, hi ha l'objectiu de fer servir i provar arquitectures d'última generació com Enterprise JavaBeans Preview_2 (EJB) 3.0 (5/11/04) per a la lògica de negoci, Hibernate 3.0 alpha (actualment hi ha la beta 1.0 publicada el 20/12/04) com a

Abnormal EEG Synchronization Correlates with Demyelination in Alzheimer's Disease

Introduction : The pathological processes caused by Alzheimer's disease (AD) supposedly disrupt communication between and within the distributed cortical networks due to the dysfunction/loss of synapses and myelination breakdown. Indeed, recently (Knyazeva et al. 2008), we have revealed the whole-head topography of EEG synchronization specific to AD. Here we analyze whether and how these abnormalities of synchronization are related to the demyelination of cortico-cortical fibers. Methods : Fifteen newly diagnosed AD patients (CDR 0.5-1) and 15 controls matched for age, participated in the study. Their multichannel (128) EEGs were recorded during 3-5 min at rest. They were submitted to the multivariate phase synchronization (MPS) analysis for mapping regional synchronization. To obtain individual whole-head maps, the MPS was computed for each sensor considering its 2nd nearest topographical neighbors. Separate calculations were performed for the delta, theta, alpha-1/−2, and beta-1/−2 EEG bands. The same subjects were scanned on a 3 Tesla Philips scanner. The protocol included a high-resolution T1-weighted sequence and a Magnetization Transfer Imaging (MTI) acquisition. For each subject, we defined a 3mm thick layer of white matter exactly below the cortical gray matter. The magnetization transfer ratio (MTR) - an estimator of myelination - was calculated for this layer in 39 Brodmann-defined ROIs per hemisphere. To assess the between-group differences, we used a permutation version of Hotelling's T2 test or two-sample T-test (Pcorrected <0.05). For correlation analysis, Spearman Rank Correlation was calculated. Results : In AD patients, we have found an abnormal landscape of synchronization characterized by a decrease in MPS over the fronto-temporal region of the left hemisphere and an increase over the temporo-parieto-occipital regions bilaterally. Also, we have shown a widespread decrease in regional MTR in the AD patients for all the areas excluding motor, premotor, and primary sensory ones. Assuming that AD-related changes in synchronization are associated with demyelination, we hypothesized a correlation between the regional MTR values and MPS values in the hypo- and hyper-synchronized clusters. We found that MPS in the left fronto-temporal hypo-synchronized cluster directly correlates with myelination in BA42-46 of the left hemisphere: the lower the myelination in individual patients, the lower the EEG synchronization. By contrast, in the posterior hyper-synchronized cluster, MPS inversely correlated with myelination, i.e., the lower the myelination, the higher the synchronization. This posterior hyper-synchronization, more characteristic for early-onset AD, probably, results from the initial effect of the disease on cortical inhibition, reducing cortical capacity for decoupling irrelevant connections. Remarkably, it showed different topography of correlations in early- vs. late-onset patients. In the early-onset patients, hyper-synchronization was mainly related to demyelination in posterior BAs, the effect being significant in all the EEG frequency bands. In the late-onset patients, widely distributed correlations were significant for the EEG delta band, suggesting an interaction between the cerebral manifestations of AD and the age of its onset, i.e., topographically selective impairment of cortical inhibition in early-onset AD vs. its wide-spread weakening in old age. Conclusions : Overall, our results document that the degradation of white matter is a significant factor of AD pathogenesis leading to functional dysconnection, the latter being reflected in EEG synchronization abnormalities.

Structural analysis of TCR-ligand interactions studied on H-2Kd-restricted cloned CTL specific for a photoreactive peptide derivative.

To study the interaction of the TCR with its ligand, the complex of a MHC molecule and an antigenic peptide, we modified a TCR contact residue of a H-2Kd-restricted antigenic peptide with photoreactive 4-azidobenzoic acid. The photoreactive group was a critical component of the epitope recognized by CTL clones derived from mice immunized with such a peptide derivative. The majority of these clones expressed V beta 1-encoded beta chains that were paired with J alpha TA28-encoded alpha chains. For one of these TCR, the photoaffinity labeled sites were mapped on the alpha chain as a J alpha TA28-encoded tryptophan and on the beta chain as a residue of the C' strand of V beta 1. Molecular modeling of this TCR suggested the presence of a hydrophobic pocket that harbors this tryptophan as well as a tyrosine on the C' strand of V beta 1 between which the photoreactive side chain inserts. It is concluded that this avid binding principle may account for the preferential selection of V beta 1 and J alpha TA28-encoded TCR.

Cerebrospinal fluid biomarkers of central nervous system dysfunction in HIV-infected patients

Background :¦In addition to opportunistic infections of the central nervous system (CNS), which are due to immunosuppression related to HIV, the latter virus, itself, can cause neuropathological abnormalities which are located mainly in the basal ganglia and are characterized by microglial giant cells, reactive astrocytosis and perivascular monocytes. This HIV encephalopathy is characterized, clinically, by psycho-motor slowing, memory loss, difficulties in complex tasks requiring executive functions, as well as motor disorders .These cognitive deficits are grouped under the acronym of HIV-associated neurocognitive disorders (HAND). In fact, HANDs are subdivided in three groups in accordance with the severity of the cognitive impairment: Asymptomatic Neurocognitive Impairment (ANI), Mild/moderate Neurocognitive Disorders (MND) and HIV Associated Dementia (HAD).¦While the incidence of HAD has significantly decreased in the era of combined antiretrobiral therapy (cART), the prevalence of milder forms of HIV-associated neurocognitive disorders HAND seem to have increased. There are many potential reasons to explain this state of facts.¦An important question is to understand how soon the brain may be affected by HIV. Since performing a biopsy in these patients is not an issue, the study of the CSF represents the best available way to look at putative biomarkers of inflammation/neurodegeneration in the CNS. Here, we wanted to examined the putative usefulness of different biomarkers as early indicators of anti-retroviral failure at the level of the CNS. We chose to study the CSF levels of:¦Amyloid-β 1-42 (Aβ42), Tau total (tTau), phosphorylated Tau (pTau), Neopterin and S100-β.¦Indeed, these molecules are representative biomarkers of the major cells of the CNS, i.e. neurons,¦macrophages/microglia and astrocytes.¦To examine how sensitive were these CSF biomarkers to indicate CNS insults caused by HIV, we proposed to take advantage of the MOST (Monotherapy Switzerland/Thailand study) study, recently published in AIDS. Thus, we collaborated with Prof. Pietro Vernazza in St-Gall. In MOST study, monotherapy (MT) consisting in ritonavir-boosted lopinavir (LPV/r) was compared to continuous conventional antiretroviral therapy including several molecules, hereafter referred as CT¦Methods :We tested 61 cerebrospinal fluid (CSF) samples from 52 patients enrolled in MOST, including 34 CSF samples of CT and 27 of MT (mean duration on MT: 47+20 weeks) in patients who maintained full VL suppression in blood (<50cps/ml). Using enzyme-linked immunosorbent assay (ELISA), we determined the CSF concentration of S100-beta (astrocytosis), neopterin (microglia, inflammation), total Tau (tTau), phosphorylated Tau (pTau), and amyloid-beta 1-42 (Abeta), the latter three markers indicating neuronal damages. The CSF samples of 37 HIV-negative patients with Alzheimer dementia (AD) served as controls. Results are expressed in pg/ml and reported as median ± interquartile range. Mann Whitney-U test was used to compare the results of a given biomarker between two groups and the Fisher test to compare frequencies.¦Results: We found a higher concentration of S100-beta (570±1132) and neopterin (2.5±2.9) in the CSF of MT versus CT (0±532, p=0.002 and 1.2±2.5, p=0.058, respectively). A cutoff of 940 pg/ml for S100-beta allowed to discriminate MT (11 above versus 16 below) from CT (1 vs 33, p=0.0003). At a lesser extent, a cutoff of 11 pg/ml for neopterin separated MT (4 above versus 23) from CT (0 vs 34, p=0.034) (Figure).¦In AD, tTau was higher (270±414) and Abeta lower (234±328) than in CT (150±153, p=0.0078, and 466±489, p=0.007, respectively). Such as for CT, Abeta was lower in AD than in MT (390±412, p=0.01). However, contrasting with CT, the levels of tTau were not different between AD and MT (199±177, p=0.11). S100b (173±214; p=0.0006) and neopterin (1.1±0.9; p=0.0014) were lower in AD than MT.¦Conclusions: Despite full VL-suppression in blood, HIV monotherapy is sufficient to trigger inflammation and, especially, astrocytosis. CSF markers of patients on CT have the same profile as reported for healthy subjects, suggesting that CT permits a good control of HIV in the brain. Finally, the levels of tTau, which are relatively similar between AD and MT patients, suggest that neurons are damaged during monotherapy.

Interplay between keratinocytes and immune cells--recent insights into psoriasis pathogenesis.

Psoriasis is one of the most common human inflammatory skin diseases characterised by hyperproliferation and aberrant differentiation of keratinocytes. The trigger of the typical epidermal changes seen in psoriasis was considered to be a dysregulated immune response with Th-1/Tc1 cells playing a central role. Recent studies have provided new insights into psoriasis pathogenesis in defining intraepidermal alpha(1)beta(1)+ T cells as key effectors driving keratinocyte changes. Critical roles for IFN-alpha secreted by plasmacytoid dendritic cells and the IL-23/Th-17 axis were postulated. Initially, these subsequent stages are at least partially driven by the endogenous antimicrobial peptide LL37 that converts inert self-DNA into a potent trigger of interferon production by binding and delivering the DNA into plasmacytoid dendritic cells to trigger toll-like receptor 9. As LL37 is expressed by keratinocytes upon various stimuli, keratinocytes might regain momentum as instigators of an aberrant immune response which then precedes the characteristic changes in the epidermis. Data from these new studies indicate a complex interplay between keratinocytes overexpressing antimicrobial peptides and immune cells driving epidermal hyperproliferation and aberrant keratinocyte differentiation in the pathogenesis of psoriasis.

Differential T cell receptor photoaffinity labeling among H-2Kd restricted cytotoxic T lymphocyte clones specific for a photoreactive peptide derivative. Labeling of the alpha-chain correlates with J alpha segment usage.

Using a direct binding assay based on photoaffinity labeling, we studied the interaction of T cell receptor (TCR) with a Kd-bound photoreactive peptide derivative on living cells. The Kd-restricted Plasmodium berghei circumsporozoite (PbCS) peptide 253-260 (YIPSAEKI) was reacted NH2-terminally with biotin and at the TCR contact residue Lys259 with photoreactive iodo, 4-azido salicylic acid (IASA) to make biotin-YIPSAEK(IASA)I. Cytotoxic T lymphocyte (CTL) clones derived from mice immunized with this derivative recognized this conjugate, but not a related one lacking the IASA group nor the parental PbCS peptide. The clones were Kd restricted. Recognition experiments with variant conjugates, lacking substituents from IASA, revealed a diverse fine specificity pattern and indicated that this group interacted directly with the TCR. The TCR of four clones could be photoaffinity labeled by biotin-YIPSAEK(125IASA)I. This labeling was dependent on the conjugates binding to the Kd molecule and was selective for the TCR alpha (2 clones) or beta chain (1 clone), or was common for both chains (1 clone). TCR sequence analysis showed a preferential usage of J alpha TA28 containing alpha chains that were paired with V beta 1 expressing beta chains. The TCR that were photoaffinity labeled at the alpha chain expressed these J alpha and V beta segments. The tryptophan encoded by the J alpha TA28 segment is rarely found in other J alpha segments. Moreover, we show that the IASA group interacts preferentially with tryptophan in aqueous solution. We thus propose that for these CTL clones, labeling of the alpha chain occurs via the J alpha-encoded tryptophan residue.

Induction of transendothelial migration in normal and malignant human T lymphocytes.

Activated CD 3+ enriched human peripheral blood T cells exhibited potent capacity for transendothelial migration through HUVEC layers in the absence of T cell ***. In contrast, malignant human T cell lines *** no or negligible ability of transendothelial migration in the absence of chemoattractants. Time lapse studies of transendothelial migration of activated CD 3+ enriched peripheral blood T cells through a HUVEC layer showed that the first T cells were detected in the lower compartment of a tissue culture insert after 1 hour and that migration increased to reach a maximum of 25 x 10(4) T cells/hr after 24 hours. Adhesion assays of human T cell lines demonstrated that all T cell lines were capable of adhesion to HUVEC and that adhesion of T cells to HUVECs was primarily mediated by CD11a/CD18 and ICAM-1 interactions. Furthermore, transendothelial migration of CD 3+ enriched human peripheral blood T cells was inhibited by pretreating the T cells with anti-CD 18 monoclonal antibodies. The inability of malignant T cells to migrate through HUVEC layers in the absence of chemoattractants was not due to poor motility per se, since both normal and malignant T cells migrated well on extracellular matrix components as determined by using Boyden chambers. Crosslinking of alpha 1 beta 2 and alpha 4 beta 1 with immobilized monoclonal antibodies induced motile behaviour in activated CD 3 enriched human peripheral blood T cells but not in malignant T cell lines. In conclusion, the differences in the ability of transendothelial migration between normal and malignant human T cells in the absence of chemoattractants is primarily due to the differences in the capacity of alpha 1 beta 2 and alpha 4 beta 1 to trigger motile behaviour in the separate cell types.

Prostaglandin E2 promotes integrin alpha Vbeta 3-dependent endothelial cell adhesion, rac-activation, and spreading through cAMP/PKA-dependent signaling.

We have recently reported that the inhibition of endothelial cell COX-2 by non-steroidal anti-inflammatory drugs suppresses alpha(V)beta(3)- (but not alpha(5)beta(1)-) dependent Rac activation, endothelial cell spreading, migration, and angiogenesis (Dormond, O., Foletti, A., Paroz, C., and Ruegg, C. (2001) Nat. Med. 7, 1041-1047). Here we investigated the role of the COX-2 metabolites PGE(2) and TXA2 in regulating human umbilical vein endothelial cell (HUVEC) adhesion and spreading. We report that PGE(2) accelerated alpha(V)beta(3)-mediated HUVEC adhesion and promoted Rac activation and cell spreading, whereas the TXA2 agonist retarded adhesion and inhibited spreading. We show that the cAMP level and the cAMP-regulated protein kinase A (PKA) activity are critical mediators of these PGE(2) effects. alpha(V)beta(3)-mediated adhesion induced a transient COX-2-dependent rise in cAMP levels, whereas the cell-permeable cAMP analogue 8-brcAMP accelerated adhesion, promoted Rac activation, and cell spreading in the presence of the COX-2 inhibitor NS-398. Pharmacological inhibition of PKA completely blocked alpha(V)beta(3)-mediated adhesion. A constitutively active Rac mutant (L61Rac) rescued alpha(V)beta(3)-dependent spreading in the presence of NS398 or, but did not accelerate adhesion, whereas a dominant negative Rac mutant (N17Rac) suppressed spreading without affecting adhesion. alpha(5)beta(1)-mediated HUVEC adhesion, Rac activation, and spreading were not affected by PGE(2), 8-brcAMP, or the inhibition of PKA. In conclusion, these results demonstrate that PGE(2) accelerates alpha(V)beta(3)-mediated endothelial cell adhesion through cAMP-dependent PKA activation and induces alpha(V)beta(3)-dependent spreading via cAMP- and PKA-dependent Rac activation and may contribute to the further understanding of the regulation of vascular integrins alpha(V)beta(3) by COX-2/PGE(2) during tumor angiogenesis and inflammation.

Comparative effects of teriparatide and ibandronate on spine bone mineral density (BMD) and microarchitecture (TBS) in postmenopausal women with osteoporosis: a 2-year open-label study.

Treatment effects over 2 years of teriparatide vs. ibandronate in postmenopausal women with osteoporosis were compared using lumbar spine bone mineral density (BMD) and trabecular bone score (TBS). Teriparatide induced larger increases in BMD and TBS compared to ibandronate, suggesting a more pronounced effect on bone microarchitecture of the bone anabolic drug. INTRODUCTION: The trabecular bone score (TBS) is an index of bone microarchitecture, independent of bone mineral density (BMD), calculated from anteroposterior spine dual X-ray absorptiometry (DXA) scans. The potential role of TBS for monitoring treatment response with bone-active substances is not established. The aim of this study was to compare the effects of recombinant human 1-34 parathyroid hormone (teriparatide) and the bisphosphonate ibandronate (IBN), on lumbar spine (LS) BMD and TBS in postmenopausal women with osteoporosis. METHODS: Two patient groups with matched age, body mass index (BMI), and baseline LS BMD, treated with either daily subcutaneous teriparatide (N = 65) or quarterly intravenous IBN (N = 122) during 2 years and with available LS BMD measurements at baseline and 2 years after treatment initiation were compared. RESULTS: Baseline characteristics (overall mean ± SD) were similar between groups in terms of age 67.9 ± 7.4 years, body mass index 23.8 ± 3.8 kg/m(2), BMD L1-L4 0.741 ± 0.100 g/cm(2), and TBS 1.208 ± 0.100. Over 24 months, teriparatide induced a significantly larger increase in LS BMD and TBS than IBN (+7.6 % ± 6.3 vs. +2.9 % ± 3.3 and +4.3 % ± 6.6 vs. +0.3 % ± 4.1, respectively; P < 0.0001 for both). LS BMD and TBS were only weakly correlated at baseline (r (2) = 0.04) with no correlation between the changes in BMD and TBS over 24 months. CONCLUSIONS: In postmenopausal women with osteoporosis, a 2-year treatment with teriparatide led to a significantly larger increase in LS BMD and TBS than IBN, suggesting that teriparatide had more pronounced effects on bone microarchitecture than IBN.

Changes in the enterocyte cytoskeleton in newborn rats exposed to ethanol in utero

BACKGROUND: Cytoskeletal changes after longterm exposure to ethanol have been described in a number of cell types in adult rat and humans. These changes can play a key part in the impairment of nutrient assimilation and postnatal growth retardation after prenatal damage of the intestinal epithelium produced by ethanol intake. AIMS: To determine, in the newborn rat, which cytoskeletal proteins are affected by longterm ethanol exposure in utero and to what extent. ANIMALS: The offspring of two experimental groups of female Wistar rats: ethanol treated group receiving up to 25% (w/v) of ethanol in the drinking fluid and control group receiving water as drinking fluid. METHODS: Single and double electron microscopy immunolocalisation and label density estimation of cytoskeletal proteins on sections of proximal small intestine incubated with monoclonal antibodies against actin, alpha-tubulin, cytokeratin (polypeptides 1, 5, 6, 7, 8, 10, 11, and 18), and with a polyclonal antibody anti-beta 1,4-galactosyl transferase as trans golgi (TG) or trans golgi network (TGN) marker, or both. SDS-PAGE technique was also performed on cytoskeletal enriched fractions from small intestine. Western blotting analysis was carried out by incubation with the same antibodies used for immunolocalisation. RESULTS: Intestinal epithelium of newborn rats from the ethanol treated group showed an overexpression of cytoskeletal polypeptides ranging from 39 to 54 kDa, affecting actin and some cytokeratins, but not tubulin. Furthermore, a cytokeratin related polypeptide of 28-29 kDa was identified together with an increase in free ubiquitin in the same group. It was noteworthy that actin and cytokeratin were abnormally located in the TG or the TGN, or both. CONCLUSIONS: Longterm exposure to ethanol in utero causes severe dysfunction in the cytoskeleton of the developing intestinal epithelium. Actin and cytokeratins, which are involved in cytoskeleton anchoring to plasma membrane and cell adhesion, are particularly affected, showing overexpression, impaired proteolysis, and mislocalisation.

Temporal changes in the expression and distribution of adhesion molecules during liver development and regeneration

We have compared by immunocytochemistry and immunoblotting the expression and distribution of adhesion molecules participating in cell-matrix and cell-cell interactions during embryonic development and regeneration of rat liver. Fibronectin and the fibronectin receptor, integrin alpha 5 beta 1, were distributed pericellularly and expressed at a steady level during development from the 16th day of gestation and in neonate and adult liver. AGp110, a nonintegrin fibronectin receptor was first detected on the 17th day of gestation in a similar, nonpolarized distribution on parenchymal cell surfaces. At that stage of development haemopoiesis is at a peak in rat liver and fibronectin and receptors alpha 5 beta 1 and AGp110 were prominent on the surface of blood cell precursors. During the last 2 d of gestation (20th and 21st day) hepatocytes assembled around lumina. AGp110 was initially depolarized on the surface of these acinar cells but then confined to the lumen and to newly-formed bile canaliculi. At birth, a marked increase occurred in the canalicular expression of AGp110 and in the branching of the canalicular network. Simultaneously, there was enhanced expression of ZO-1, a protein component of tight junctions. On the second day postpartum, presence of AGp110 and of protein constituents of desmosomes and intermediate junctions, DGI and E-cadherin, respectively, was notably enhanced in cellular fractions insoluble in nonionic detergents, presumably signifying linkage of AGp110 with the cytoskeleton and assembly of desmosomal and intermediate junctions. During liver regeneration after partial hepatectomy, AGp110 remained confined to apical surfaces, indicating a preservation of basic polarity in parenchymal cells. A decrease in the extent and continuity of the canalicular network occurred in proliferating parenchyma, starting 24 h after resection in areas close to the terminal afferent blood supply of portal veins and spreading to the rest of the liver within the next 24 h. Distinct acinar structures, similar to the ones in prenatal liver, appeared at 72 h after hepatectomy. Restoration of the normal branching of the biliary tree commenced at 72 h. At 7 d postoperatively acinar formation declined and one-cell-thick hepatic plates, as in normal liver, were observed.