Loss of glucose-induced insulin secretion and GLUT2 expression in transplanted beta-cells.

Either 200 or 400 syngeneic islets were transplanted under the kidney capsule of normal or streptozocin-induced diabetic B6/AF1 mice. The diabetic mice with 400 islets became normoglycemic, but those with 200 islets, an insufficient number, were still diabetic after the transplantation (Tx). Two weeks after Tx, GLUT2 expression in the islet grafts was evaluated by immunofluorescence and Western blots, and graft function was examined by perfusion of the graft-bearing kidney. Immunofluorescence for GLUT2 was dramatically reduced in the beta-cells of grafts with 200 islets exposed to hyperglycemia. However, it was plentiful in grafts with 400 islets in a normoglycemic environment. Densitometric analysis of Western blots on graft homogenates demonstrated that GLUT2 protein levels in the islets, when exposed to chronic hyperglycemia for 2 weeks, were decreased to 16% of those of normal recipients. Moreover, these grafts had defective glucose-induced insulin secretion, while the effects of arginine were preserved. We conclude that GLUT2 expression in normal beta-cells is promptly down-regulated during exposure to hyperglycemia and may contribute to the loss of glucose-induced secretion of diabetes.

Expression hierarchy of T cell epitopes from melanoma differentiation antigens: unexpected high level presentation of tyrosinase-HLA-A2 Complexes revealed by peptide-specific, MHC-restricted, TCR-like antibodies.

Peptide Ags presented by class I MHC molecules on human melanomas and that are recognized by CD8(+) T cells are the subjects of many studies of antitumor immunity and represent attractive candidates for therapeutic approaches. However, no direct quantitative measurements exist to reveal their expression hierarchy on the cell surface. Using novel recombinant Abs which bind these Ags with a peptide-specific, MHC-restricted manner, we demonstrate a defined pattern of expression hierarchy of peptide-HLA-A2 complexes derived from three major differentiation Ags: gp100, Melan-A/Mart-1, and tyrosinase. Studying melanoma cell lines derived from multiple patients, we reveal a surprisingly high level of presentation of tyrosinase-derived complexes and moderate to very low expression of complexes derived from other Ags. No correlation between Ag presentation and mRNA expression was found; however, protein stability may play a major role. These results provide new insights into the characteristics of Ag presentation and are particularly important when such targets are being considered for immunotherapy. These results may shed new light on relationships between Ag presentation and immune response to cancer Ags.

The activity of S.pombe DSC-1-like factor is cell cycle regulated and dependent on the activity of p34cdc2.

In the eukaryotic cell cycle, there are major control points in late G2 to determine the timing of the initiation of mitosis, and in late G1, regulating entry into S phase. In yeasts, this latter control is called start. Traverse of the start control and progression to S phase is accompanied by an increase in the expression of some of the genes whose products are required for DNA synthesis. In Saccharomyces cerevisiae, the coordinate expression of these genes in late G1 is dependent on a cis-acting sequence element called the MluI cell cycle box (MCB). A transcription factor called DSC-1 binds these elements and mediates cell cycle regulated transcription, though it is unclear whether this is by cell cycle-dependent changes in its activity. A DSC-1-like factor has also been identified in the fission yeast S.pombe. This is composed of at least the products of the cdc10 and sct1/res1 genes, and binds to the promoters of genes whose expression increases prior to S phase. We demonstrate that p85cdc10 is a nuclear protein and that the activity of the S.pombe DSC-1 factor varies through the cell cycle; it is high in cells that have passed start, decreases at the time of anaphase, remains low during the pre-start phase of G1 and increases at the time of the next S phase. We also show that the reactivation in late G1 is dependent on the G1 form of p34cdc2.

Mechanism of reduction of virus release and cell-cell fusion in persistent canine distemper virus infection.

Canine distemper virus (CDV), a mobillivirus related to measles virus causes a chronic progressive demyelinating disease, associated with persistence of the virus in the central nervous system (CNS). CNS persistence of morbilliviruses has been associated with cell-to-cell spread, thereby limiting immune detection. The mechanism of cell-to-cell spread remains uncertain. In the present study we studied viral spread comparing a cytolytic (non-persistent) and a persistent CDV strain in cell cultures. Cytolytic CDV spread in a compact concentric manner with extensive cell fusion and destruction of the monolayer. Persistent CDV exhibited a heterogeneous cell-to-cell pattern of spread without cell fusion and 100-fold reduction of infectious viral titers in supernatants as compared to the cytolytic strain. Ultrastructurally, low infectious titers correlated with limited budding of persistent CDV as compared to the cytolytic strain, which shed large numbers of viral particles. The pattern of heterogeneous cell-to-cell viral spread can be explained by low production of infectious viral particles in only few areas of the cell membrane. In this way persistent CDV only spreads to a small proportion of the cells surrounding an infected one. Our studies suggest that both cell-to-cell spread and limited production of infectious virus are related to reduced expression of fusogenic complexes in the cell membrane. Such complexes consist of a synergistic configuration of the attachment (H) and fusion (F) proteins on the cell surface. F und H proteins exhibited a marked degree of colocalization in cytolytic CDV infection but not in persistent CDV as seen by confocal laser microscopy. In addition, analysis of CDV F protein expression using vaccinia constructs of both strains revealed an additional large fraction of uncleaved fusion protein in the persistent strain. This suggests that the paucity of active fusion complexes is due to restricted intracellular processing of the viral fusion protein.

Myosin, tubulin and laminin immunoreactivity in the ectoderm of the growing area opaca of the chick embryo

Distribution of myosin, tubulin and laminin immunoreactive cells in the area opaca of the young chick embryo (Stages 4-8 HH) was studied using immunofluorescence technique. For the three markers, the number of stained cells increased with the age of the blastoderm. Cells stained for tubulin and laminin, were distributed throughout the area opaca, showing no supracellular organization. On the contrary, the cells stained for myosin became organized in a ring surrounding the area pellucida. This pattern appeared at the stage 6. Such an heterogenous distribution of the markers suggests a functional diversification of the ectodermal cell monolayer forming at these early developmental stages the area opaca. This idea is also supported by the results of autoradiography for tritiated thymidin which showed that the edge cells did not synthetize DNA and consequently did not divide.

Immunolocalization of GLUTX1 in the testis and to specific brain areas and vasopressin-containing neurons.

GLUTX1 or GLUT8 is a newly characterized glucose transporter isoform that is expressed at high levels in the testis and brain and at lower levels in several other tissues. Its expression was mapped in the testis and brain by using specific antibodies. In the testis, immunoreactivity was expressed in differentiating spermatocytes of type 1 stage but undetectable in mature spermatozoa. In the brain, GLUTX1 distribution was selective and localized to a variety of structures, mainly archi- and paleocortex. It was found in hippocampal and dentate gyrus neurons as well as amygdala and primary olfactory cortex. In these neurons, its location was close to the plasma membrane of cell bodies and sometimes in proximal dendrites. High GLUTX1 levels were detected in the hypothalamus, supraoptic nucleus, median eminence, and the posterior pituitary. Neurons of these areas synthesize and secrete vasopressin and oxytocin. As shown by double immunofluorescence microscopy and immunogold labeling, GLUTX1 was expressed only in vasopressin neurons. By immunogold labeling of ultrathin cryosections microscopy, GLUTX1 was identified in dense core vesicles of synaptic nerve endings of the supraoptic nucleus and secretory granules of the vasopressin positive neurons. This localization suggests an involvement of GLUTX1 both in specific neuron function and endocrine mechanisms.

Behavior of nuclear matrix proteins during camptothecin-induced apoptosis in HL-60 human leukemia cells.

In this study we focused our attention on the behavior of four nuclear matrix proteins during the various stages of apoptosis in the HL-60 cell line exposed to the DNA topoisomerase I inhibitor, camptothecin. We have examined the following antigens by immunocytochemical techniques: (i) the 180-kDa nucleolar isoform of DNA topoisomerase II; (ii) a 126-kDa polypeptide of nuclear bodies; (iii) a 125-kDa protein; and (iv) a 160-kDa polypeptide which are known to be components of the matrix inner network. Indirect immunofluorescence experiments were performed to follow these nuclear matrix antigens during apoptosis. Moreover, the ultrastructural localization of both 125- and 160-kDa proteins was investigated by electron microscope immunocytochemistry with gold-conjugated secondary antibodies. While the antibody to the nucleolar isoform of DNA topoisomerase II gave a fluorescent pattern that was well-maintained until the late phases of apoptosis, the other three nuclear antigens showed marked modifications in their distribution. A common feature, particularly evident for 125- and 160-kDa proteins, was their absence from cap-shaped chromatin marginations, whereas they were present in the areas of remaining decondensed chromatin. The 126-kDa polypeptide concentrated progressively in an irregular mass at the opposite side of the crescentic caps and then broke up in fine spots. The 125- and 160-kDa proteins localized in the nucleolus and precisely within certain granules which are known to appear in the nucleolar area after camptothecin administration. These results show that, in addition to the well-known chromatin changes, nuclear organization undergoes other rearrangements during the apoptotic process.

Macular and retinal dysfunction of unknown origin in adults with normal fundi: evidence for an autoimmune pathophysiology.

Here we report the discovery of and phenotypic characterization of a retinal disorder of unknown origin in adults using clinical, electrophysiological and psychophysical techniques, and to seek the presence of circulating retinal autoantibodies in the sera of these patients. Sixteen patients were identified with progressive bilateral visual loss over a period of months. Ten of the patients were male, and the average age was 55.3 years (range from 43 to 76 years). Known causes such as carcinoma-associated retinopathy, acute zonal occult outer retinopathy and hereditary cone dystrophy appeared unlikely. Investigations included electrophysiology, fundus autofluorescence imaging and psychophysical tests. The sera of these patients were analyzed with indirect immunocytochemistry and Western immunoblot analysis on murine (BALB/c) retinal tissue for the presence of retinal autoantibodies. Bilateral visual loss and photophobia progressed over a period of months to years (average 28.7 months, range 3-67) and subsequently stabilized. No abnormality was observed by biomicroscopy, angiography or autofluorescence imaging. Electrophysiology indicated predominant cone-system dysfunction, either macular or generalized, and post-phototransduction involvement in 9 patients (56%). Photopic and scotopic visual fields and dark adaptation kinetics showed both cone and rod system involvement in all cases. Heterogeneous immunohistochemical staining patterns were seen with the sera of these patients as compared with controls. A majority of the affected patients (9/15) stained with an antinuclear pattern. The retinal autoantibodies from the sera of most patients reacted with the retinal proteins of molecular weight between 34 and 40 kDa. The aetiology of this distinctive retinal disorder therefore appears to be mediated through an autoimmune mechanism.

Detection of serum antibodies against Leishmania 94 kDa antigen in visceral and cutaneous leishmaniosis due to Leishmania infantum.

Leishmania promastigotes polypeptides are analyzed by immunoblotting with sera from patients infected with different Leishmania species and presenting visceral or cutaneous infections. These sera recognize Leishmania polypeptides in several molecular masses. The major findings of this study are as follow. 1) The Leishmania 94 kDa antigen, which is specifically recognized by all sera from L. infantum-infected patients with visceral infection, is recognized by some sera from L. infantum-infected patients presenting cutaneous infection. 2) All patients with cutaneous infections due to L. tropica, L. amazonensis, or L. guyanensis do not develop anti-94 kDa antibodies, whatever the Leishmania species used as antigens. 3) Difference in electrophoretic mobilities is seen between the 94 kDa antigen identified by sera from Leishmania infantum-infected patients, and the antigen both recognized by the Concavalin A lectin and a rabbit antiserum raised against deglycosylated Promastigote Surface Protease.

Requirement for CARMA1 in antigen receptor-induced NF-kappa B activation and lymphocyte proliferation.

Ligation of antigen receptors (TCR, BCR) on T and B lymphocytes leads to the activation of new transcriptional programs and cell cycle progression. Antigen receptor-mediated activation of NF-kappa B, required for proliferation of B and T cells, is disrupted in T cells lacking PKC theta and in B and T cells lacking Bcl10, a caspase recruitment domain (CARD)-containing adaptor protein. CARMA1 (also called CARD11 and Bimp3), the only lymphocyte-specific member in a family of membrane-associated guanylate kinase (MAGUK) scaffolding proteins that interact with Bcl10 by way of CARD-CARD interactions, is required for TCR-induced NF-kappa B activation in Jurkat T lymphoma cells. Here we show that T cells from mice lacking CARMA1 expression were defective in recruitment of Bcl10 to clustered TCR complexes and lipid rafts, in activation of NF-kappa B, and in induction of IL-2 production. Development of CD5(+) peritoneal B cells was disrupted in these mice, as was B cell proliferation in response to both BCR and CD40 ligation. Serum immunoglobulin levels were also markedly reduced in the mutant mice. Together, these results show that CARMA1 has a central role in antigen receptor signaling that results in activation and proliferation of both B and T lymphocytes.

Crohn's disease-associated polymorphism within the PTPN2 gene affects muramyl-dipeptide-induced cytokine secretion and autophagy.

BACKGROUND: The single nucleotide polymorphism (SNP) rs2542151 within the gene locus region encoding protein tyrosine phosphatase non-receptor type 2 (PTPN2) has been associated with Crohn's disease (CD), ulcerative colitis (UC), type-I diabetes, and rheumatoid arthritis. We have previously shown that PTPN2 regulates mitogen-activated protein kinase (MAPK) signaling and cytokine secretion in human THP-1 monocytes and intestinal epithelial cells (IEC). Here, we studied whether intronic PTPN2 SNP rs1893217 regulates immune responses to the nucleotide-oligomerization domain 2 (NOD2) ligand, muramyl-dipeptide (MDP). MATERIALS AND METHODS: Genomic DNA samples from 343 CD and 663 non-IBD control patients (male and female) from a combined German, Swiss, and Polish cohort were genotyped for the presence of the PTPN2 SNPs, rs2542151, and rs1893217. PTPN2-variant rs1893217 was introduced into T(84) IEC or THP-1 cells using a lentiviral vector. RESULTS: We identified a novel association between the genetic variant, rs1893217, located in intron 7 of the PTPN2 gene and CD. Human THP-1 monocytes carrying this variant revealed increased MAPK activation as well as elevated mRNA expression of T-bet transcription factor and secretion of interferon-γ in response to the bacterial wall component, MDP. In contrast, secretion of interleukin-8 and tumor necrosis factor were reduced. In both, T(84) IEC and THP-1 monocytes, autophagosome formation was impaired. CONCLUSIONS: We identified a novel CD-associated PTPN2 variant that modulates innate immune responses to bacterial antigens. These findings not only provide key insights into the effects of a functional mutation on a clinically relevant gene, but also reveal how such a mutation could contribute to the onset of disease.

Changes in D-serine levels and localization during postnatal development of the rat vestibular nuclei.

The patterns of development of the vestibular nuclei (VN) and their main connections involving glutamate neurotransmission offer a good model for studying the function of the glial-derived neuromodulator D-serine in synaptic plasticity. In this study we show that D-serine is present in the VN and we analyzed its distribution and the levels of expression of serine racemase and D-amino acid oxidase (D-AAO) at different stages of postnatal (P) development. From birth to P21, high levels of D-serine were detected in glial cells and processes in all parts of the VN. This period corresponded to high expression of serine racemase and low expression of D-AAO. On the other hand, in the mature VN D-serine displayed very low levels and was mainly localized in neuronal cell bodies and dendrites. This drop of D-serine in adult stages corresponded to an increasing expression of D-AAO at mature stages. High levels of glial D-serine during the first 3 weeks of postnatal development correspond to an intense period of plasticity and synaptogenesis and maturation of VN afferents, suggesting that D-serine could be involved in these phenomena. These results demonstrate for the first time that changes in D-serine levels and distribution occur during postnatal development in the central nervous system. The strong decrease of D-serine levels and the glial-to-neuronal switch suggests that D-serine may have distinct functional roles depending on the developmental stage of the vestibular network.

Direct Thy-1/alphaVbeta3 integrin interaction mediates neuron to astrocyte communication.

Thy-1 is an abundant neuronal glycoprotein of poorly defined function. We recently provided evidence indicating that Thy-1 clusters a beta3-containing integrin in astrocytes to induce tyrosine phosphorylation, RhoA activation and the formation of focal adhesions and stress fibers. To date, the alpha subunit partner of beta3 integrin in DI TNC1 astrocytes is unknown. Similarly, the ability of neuronal, membrane-bound Thy-1 to trigger astrocyte signaling via integrin engagement remains speculation. Here, evidence that alphav forms an alphavbeta3 heterodimer in DI TNC1 astrocytes was obtained. In neuron-astrocyte association assays, the presence of either anti-alphav or anti-beta3 integrin antibodies reduced cell-cell interaction demonstrating the requirement of both integrin subunits for this association. Moreover, anti-Thy-1 antibodies blocked stimulation of astrocytes by neurons but not the binding of these two cell types. Thus, neuron-astrocyte association involved binding between molecular components in addition to the Thy-1-integrin; however, the signaling events leading to focal adhesion formation in astrocytes depended exclusively on the latter interaction. Additionally, wild-type (RLD) but not mutated (RLE) Thy-1 was shown to directly interact with alphavbeta3 integrin by Surface Plasmon Resonance analysis. This interaction was promoted by divalent cations and was species-independent. Together, these results demonstrate that the alphavbeta3 integrin heterodimer interacts directly with Thy-1 present on neuronal cells to stimulate astrocytes.

Intravitreous injection of PLGA microspheres encapsulating GDNF promotes the survival of photoreceptors in the rd1/rd1 mouse.

PURPOSE: To evaluate the potential delay of the retinal degeneration in rd1/rd1 mice using recombinant human glial cell line-derived neurotrophic factor (rhGDNF) encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) microspheres. METHODS: rhGDNF-loaded PLGA microspheres were prepared using a water in oil in water (w/o/w) emulsion solvent extraction-evaporation process. In vitro, the rhGDNF release profile was assessed using radiolabeled factor. In vivo, rhGDNF microspheres, blank microspheres, or microspheres loaded with inactivated rhGDNF were injected into the vitreous of rd1/rd1 mice at postnatal day 11 (PN11). The extent of retinal degeneration was examined at PN28 using rhodopsin immunohistochemistry on whole flat-mount retinas, outer nuclear layer (ONL) cell counting on histology sections, and electroretinogram tracings. Immunohistochemical reactions for glial fibrillary acidic protein (GFAP), F4/80, and rhodopsin were performed on cryosections. RESULTS: Significant delay of rod photoreceptors degeneration was observed in mice receiving the rhGDNF-loaded microspheres compared to either untreated mice or to mice receiving blank or inactivated rhGDNF microspheres. The degeneration delay in the eyes receiving the rhGDNF microspheres was illustrated by the increased rhodopsin positive signals, the preservation of significantly higher number of cell nuclei within the ONL, and significant b-wave increase. A reduction of the subretinal glial proliferation was also observed in these treated eyes. No significant intraocular inflammatory reaction was observed after the intravitreous injection of the various microspheres. CONCLUSIONS: A single intravitreous injection of rhGDNF-loaded microspheres slows the retinal degeneration processes in rd1/rd1 mice. The use of injectable, biodegradable polymeric systems in the vitreous enables the efficient delivery of therapeutic proteins for the treatment of retinal diseases.