Dimitra Beroukas

Identified axo-axonic cells are immunoreactive for GABA in the hippocampus visual cortex of the cat

Chandelier or axo-axonic cells (AACs) are specialized interneurons terminating on the axon initial segments of pyramidal neurons. Two AACs have been localized by Golgi impregnation, one in the CA1 region of the hippocampus and one in the visual cortex of cat, for structural analysis and for the identification of their transmitter. They had 323 and 268 terminal bouton rows, respectively, probably making synapses with an equal number of initial segments. The distribution of the dendrites of the hippocampal cell was strikingly similar to that of pyramidal cells suggesting a similar input. Using an antiserum to GABA and postembedding GABA-immunocytochemistry, developed for Golgi-impregnated neurons, both cells were found to be GABA-immunoreactive. The strategic location of their synapses and the presence of GABA in AACs suggest that in normal cortical tissue they play a major role in GABA-mediated inhibition.

Up-regulation of M3-Muscarinic Receptors in Labial Salivary Gland Acini in Primary Sjögren's Syndrome

M3-muscarinic receptors (M3R) mediate parasympathetic cholinergic neurotransmission to salivary and lacrimal glands, and autoantibodies to these receptors have been implicated in sicca symptoms and autonomic dysfunction in Sjögrens syndrome. We have investigated the expression of M3R in paraffin-embedded labial salivary glands (LSG) from seven patients with primary Sjögrens syndrome (pSS) and five healthy controls using high-resolution confocal microscopy and an affinity-purified goat polyclonal antibody raised against the COOH-terminal sequence of the human M3R. Immunolocalization of M3R was similar in control and pSS glands, with punctate staining of M3R in the basal membrane of acinar cells and in the luminal and abluminal membrane of myoepithelial cells. Bright, granular M3R staining was also detected in the cytoplasm and membranes of all intercalated and striated ducts, and infiltrating lymphocytes in pSS. All immunoreactivity was specifically blocked by the immunizing peptide. An increase in M3R expression specifically in acini in pSS was demonstrated by a 30% increase in receptor number per cluster and a 68% increase in the number of clusters in the membrane. This up-regulation is consistent with inhibition of parasympathetic neurotransmission, possibly by antagonistic autoantibodies to M3R. The up-regulation, rather than down-regulation, of M3R in acini of pSS LSG can explain the effectiveness of muscarinic agonists in treating sicca symptoms in pSS.

Subcellular distribution of aquaporin 5 in salivary glands in primary Sjögren's syndrome.

Secretions from salivary and lacrimal glands are reduced in patients with primary Sjögrens syndrome (PSS). Since aquaporin 5 is involved in transport of water and is present in salivary and lacrimal glands, this protein was thought to have a major role in the pathogenesis of PSS. We used indirect immunofluorescence and an immunoperoxidase technique to assess expression and subcellular localisation of aquaporin 5 in patients and controls. Our results suggest that the distribution and density of aquaporin 5 in salivary glands does not differ between patients with PSS and those without. Thus, the role of aquaporin 5 in the pathogenesis of PSS needs to be reassessed and alternative pathogenetic mechanisms investigated.

Subcellular redistribution of la/SSB autoantigen during physiologic apoptosis in the fetal mouse heart and conduction system: a clue to the pathogenesis of congenital heart block.

OBJECTIVE In isolated congenital heart block, the mechanism by which maternal autoantibodies target the intracellular components of the Ro/La RNP complex is unclear. Previous studies have demonstrated that cultured fetal cardiac myocytes rendered apoptotic bind antibodies to 48-kd La/SSB. This study further investigated the subcellular distribution of the La antigen during apoptosis in the fetal mouse heart and conduction system. METHODS The atrioventricular (AV) node, AV bundle, and sinoatrial (SA) node were identified in serial sections prepared from paraffin blocks of normal mouse fetuses on days 15, 17, and 19 of gestation. Apoptosis was detected by TUNEL assay. Under confocal microscopy, fluorescent labeling of fragmented DNA in apoptotic cells was assessed by TUNEL, and La protein localization was visualized simultaneously using a murine monoclonal antibody or affinity-purified human polyclonal anti-La antibodies. RESULTS Apoptotic cells were detected in and at the periphery of the AV and SA nodes as well as in the fetal heart valve insertions and working myocardium. In contrast, no apoptosis was detected in the adult heart AV node or surrounding myocardium. As expected, the La antigen was predominantly immunolocalized to the nucleus in nonapoptotic cells. However, apoptotic cells showed a marked reduction of nuclear La and redistribution of La to the cytoplasm. High-resolution confocal microscopy revealed that in cells that had undergone apoptosis, La antigen asymmetrically clustered near the surface of TUNEL-positive nuclei and apoptotic bodies. CONCLUSION These data provide the first in vivo demonstration of the subcellular translocation of La autoantigen during apoptosis in the fetal heart and the conduction system under physiologic conditions. This observation supports the hypothesis that subcellular redistribution of La in the normally developing heart facilitates the binding of cognate maternal antibodies and subsequent tissue damage.

New insights into disease pathogenesis in crusted (Norwegian) scabies: the skin immune response in crusted scabies

Background  Crusted scabies is a rare and severely debilitating disease characterized by infestation of the skin with up to millions of Sarcoptes scabiei mites, high total IgG levels, extremely high total IgE levels, and the development of hyperkeratotic skin crusts that may be loose, scaly and flaky or thick and adherent.

Selective Down-Regulation of Aquaporin-1 in Salivary Glands in Primary Sjögren's Syndrome

Salivary and lacrimal gland secretions are reduced in primary Sjögren’s syndrome (pSS). Aquaporins (AQPs) are involved in transmembrane water transport, and different isoforms show specific cellular and subcellular distributions in salivary and lacrimal glands. Changes in expression of AQP molecules have therefore been suggested to contribute to the glandular dysfunction in pSS. AQP-5 is present in the apical membrane of acinar cells, where it mediates fluid outflow; however, we have recently shown that its expression is not altered in pSS. We therefore studied whether expression of other isoforms of AQP would be altered in pSS. Using high-resolution confocal microscopy, we determined the distribution of AQP-1 and AQP-3 in labial salivary gland biopsies from 11 patients with pSS and 9 healthy controls. AQP-1 is present in myoepithelial cells surrounding acini, and its expression in these cells was decreased by 38% in pSS glands. By contrast, expression of AQP-1 in endothelial cells of nonfenestrated capillaries was not altered in pSS. AQP-3 was expressed in the basolateral membrane of acinar epithelial cells, and its expression was not altered in disease. We therefore conclude that AQP-1 expression in myoepithelial cells is selectively down-regulated in pSS and that myoepithelial cell dysfunction may play a crucial role in the pathology of this disease.

Characterisation of a dendritic cell subset in synovial tissue which strongly expresses Jak/STAT transcription factors from patients with rheumatoid arthritis

Objectives: To characterise the phenotype of the putative dendritic cells strongly expressing Jak3 and STAT4, which have been previously identified in the synovial tissue of patients with active rheumatoid arthritis (RA). Methods: Synovial biopsy specimens were obtained at arthroscopy from 30 patients with active RA (42 synovial biopsies). Immunohistological analysis was performed using monoclonal antibodies to detect dendritic cell subsets, including activation markers and cytokines relevant to dendritic cell function. Co-localisation of cell surface markers and cytokines was assessed primarily using sequential sections, with results confirmed by dual immunohistochemistry and immunofluorescence with confocal microscopy. Results: The dendritic cells identified in RA synovial tissue that strongly express Jak3 also strongly express STAT4 and STAT 6 and are correlated with the presence of serum rheumatoid factor. These cells are not confined to a single dendritic cell subset, with cells having phenotypes consistent with both myeloid- and plasmacytoid-type dendritic cells. The activation status of these dendritic cells suggests that they are maturing or mature dendritic cells. These dendritic cells produce IL12 as well as interferon α and γ. Conclusions: The close correlation of these dendritic cells with the presence of serum rheumatoid factor, a prognostic factor for worse disease outcome, and the strong expression by these cells of components of the Jak/STAT transcription factor pathway suggest a potential therapeutic target for the treatment of RA.

Changes in synovial tissue Jak-STAT expression in rheumatoid arthritis in response to successful DMARD treatment.

Background: Modulation of Jak-STAT signalling may provide an effective therapeutic strategy in inflammatory arthritis (IA). Objective: To examine the effect of successful disease-modifying antirheumatic drug (DMARD) treatment on the expression of Jak-STAT in a cohort of patients with active rheumatoid arthritis. Methods: Synovial tissue biopsy specimens from 16 patients with active rheumatoid arthritis, taken before and after initiation of DMARD treatment, were examined for the presence of janus kinase (Jak)3, signal transducer and activator of transcription (STAT)1, STAT4 and STAT6 expression using immunohistochemistry. Results: Successful treatment with DMARDs results in reduction in STAT1 expression in the lining, and STAT1 and STAT6 in the sublining of rheumatoid arthritis synovial tissue. Although the overall expression of STAT4 and Jak3 was not significantly altered by DMARD treatment, there was a significant reduction in the expression of the STAT4 and Jak3 bright cells, thought to be an activated dendritic cell subpopulation. Conclusion: Results show that Jak3, STAT1, STAT4 expression and STAT6 sublining expression decrease in response to successful treatment of rheumatoid arthritis with standard DMARDs. Therefore, altering the expression of these pathways may represent an alternative treatment option, either through promoting up-regulation of inhibitory pathways, or suppressing inflammatory paths.

Cognate T Cell Help Is Sufficient to Trigger Anti-Nuclear Autoantibodies in Naive Mice

The mechanisms involved in the initiation of anti-nuclear autoantibodies are unknown. In this study, we show that one factor allowing anti-nuclear autoantibodies to develop is the incomplete nature of immune tolerance to many of these proteins. Immune responses in mice toward the ubiquitous nuclear autoantigen La/SS-B are much weaker than responses to the xenoantigen, human La (hLa; 74% identical). However, in transgenic (Tg) mice expressing hLa, the Ab response to this neo-autoantigen was reduced to a level resembling the weak autoimmune response to mouse La. Partial tolerance to endogenous La autoantigen was restricted to the T compartment because transfer of CD4+ T cells specific for one or more hLa determinants into mice bearing the hLa transgene was sufficient to elicit production of anti-hLa autoantibodies. Notably, only hLa- specific T cells from non-Tg mice, and not T cells from hLa Tg mice, induced autoantibody production in hLa Tg mice. These findings confirm partial Th tolerance to endogenous La and indicate the existence in normal animals of autoreactive B cells continuously presenting La nuclear Ag. Therefore, the B cell compartment is constitutively set to respond to particular nuclear autoantigens, implicating limiting Th responses as a critical checkpoint in the development of anti-nuclear autoantibodies in normal individuals.

Sphingosine-1-phosphate-induced nociceptor excitation and ongoing pain behavior in mice and humans is largely mediated by S1P3 receptor.

The biolipid sphingosine-1-phosphate (S1P) is an essential modulator of innate immunity, cell migration, and wound healing. It is released locally upon acute tissue injury from endothelial cells and activated thrombocytes and, therefore, may give rise to acute post-traumatic pain sensation via a yet elusive molecular mechanism. We have used an interdisciplinary approach to address this question, and we find that intradermal injection of S1P induced significant licking and flinching behavior in wild-type mice and a dose-dependent flare reaction in human skin as a sign of acute activation of nociceptive nerve terminals. Notably, S1P evoked a small excitatory ionic current that resulted in nociceptor depolarization and action potential firing. This ionic current was preserved in “cation-free” solution and blocked by the nonspecific Cl− channel inhibitor niflumic acid and by preincubation with the G-protein inhibitor GDP-β-S. Notably, S1P3 receptor was detected in virtually all neurons in human and mouse DRG. In line with this finding, S1P-induced neuronal responses and spontaneous pain behavior in vivo were substantially reduced in S1P3−/− mice, whereas in control S1P1 floxed (S1P1fl/fl) mice and mice with a nociceptor-specific deletion of S1P1−/− receptor (SNS-S1P1−/−), neither the S1P-induced responses in vitro nor the S1P-evoked pain-like behavior was altered. Therefore, these findings indicate that S1P evokes significant nociception via G-protein-dependent activation of an excitatory Cl− conductance that is largely mediated by S1P3 receptors present in nociceptors, and point to these receptors as valuable therapeutic targets for post-traumatic pain.