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1.
Eur J Neurol ; 23(5): 958-63, 2016 May.
Article in English | MEDLINE | ID: mdl-26924156

ABSTRACT

BACKGROUND AND PURPOSE: Chronic inflammatory demyelinating polyneuropathy (CIDP) is an acquired immunomediated condition affecting the peripheral nervous system where probably macrophages are the primary effector cells for demyelination. Reactive oxygen species (ROS), catalyzed by the NOX family of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzymes, can induce peroxidation and are potentially injurious to myelin. Our aim was to assess the activity of NOX2, an isoform of NOX, in a series of CIDP patients and to analyze the effect of intravenous immunoglobulin (IVIg) on NOX2. METHODS: Thirty CIDP patients treated with IVIg and 30 control subjects were enrolled. To evaluate NOX2 activity, neutrophil and monocyte oxidative burst was measured directly in fresh whole blood using the Phagoburst™ assay, a fluorescence-activated cell sorting method. The mean fluorescence intensity, emitted in response to different stimuli, leads to the production of ROS and corresponds to the percentage of oxidizing cells and their enzymatic activity. RESULTS: Mean fluorescence intensity values for granulocyte and monocyte burst in patients (mean 633.3, SD 191; mean 111.8, SD 28.5) were different from those measured in healthy controls (granulocytes, mean 436.6, SD 137.0, P = 0.0003; monocytes, mean 78.2, SD 17.3, P = 0.000001). Moreover, IVIg administration increased both granulocyte (P = 0.005) and monocyte (P = 0.0009) burst. CONCLUSION: Our findings demonstrate that oxidative burst is significantly increased in CIDP patients and that treatment with IVIg enhances oxidative values, thus representing a possible IVIg therapeutic effect linked to a regulatory effect of ROS. Based on this, the development of treatments targeting the specific activation of NOX may be beneficial in autoimmune disorders.


Subject(s)
NADPH Oxidases/metabolism , Polyradiculoneuropathy, Chronic Inflammatory Demyelinating/metabolism , Reactive Oxygen Species/metabolism , Adult , Aged , Enzyme Activation/drug effects , Female , Humans , Immunoglobulins, Intravenous/pharmacology , Immunoglobulins, Intravenous/therapeutic use , Male , Middle Aged , Monocytes/metabolism , Polyradiculoneuropathy, Chronic Inflammatory Demyelinating/drug therapy
2.
Int J Immunopathol Pharmacol ; 21(4): 867-77, 2008.
Article in English | MEDLINE | ID: mdl-19144272

ABSTRACT

T cells from HLA-A2+ healthy donors were co-cultured with autologous dendritic cells (DC) loaded with apoptotic tumor cells expressing rat neu, and were induced to mature by tumor necrosis factor (TNF)alpha and interleukin (IL)-1beta (mDC(neu)) or by the CCL16 chemokine (CCL16/mDC(neu)). Priming by CCL16/mDC(neu) induces a larger population of T cells that express cytoplasmatic interferon (IFN)gamma, TNFalpha, perforin and granzyme B compared to those primed by mDC(neu). T cells primed by CCL16/mDC(neu) release IFNgamma in response to human HER-2+ cells and kill human HER-2+ target cells more efficiently than those primed by mDC(neu). Our results show that both the loading of DC with xenogeneic rat neu and their maturation by CCL16 are two issues of critical importance for the elicitation of an effective response to human HER-2 in T cells from normal donors.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Chemokines, CC/physiology , Receptor, ErbB-2/immunology , Animals , Cell Line , Coculture Techniques , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Humans , Rats
3.
Int J Immunopathol Pharmacol ; 18(2): 339-49, 2005.
Article in English | MEDLINE | ID: mdl-15888256

ABSTRACT

CCL16 is a CC chemokine originally identified as a liver-expressed chemokine. Its expression has been detected in activated monocytes where it is up-regulated by stimulation with IL-10. This is in contrast with IL-10's inhibition of the expression of most chemokines. CCL16 is chemotactic for monocytes, lymphocyte and dendritic cells. We investigated whether CCL16 displays biological activities other than chemotaxis and whether IL-10 affects monocyte response to CCL16. We show that CCL16 induces the expression of CCL2 at the mRNA and protein level, but does not affect that of CCL5, CCL18 and proinflammatory cytokines. This effect was prevented by treatment with pertussis toxin and may thus be mediated by G-protein-coupled receptors. IL-10 markedly increased CCL2 production induced by CCL16, but suppressed that of CXCL8. It also enhanced the chemotactic response to CCL16. Addition of antibodies blocking CCR1, but not CCR8, prevented this enhanced chemotactic response and suggested that CCR1 is primarily involved. We propose that IL-10 modulates the effects of CCL16 on monocytes by increasing their CCR1-dependent response. The coordinated secretion of CCL16 and IL-10 may thus enhance monocyte infiltration.


Subject(s)
Chemokine CCL2/biosynthesis , Chemokines, CC/pharmacology , Chemotaxis, Leukocyte/drug effects , Interleukin-10/pharmacology , Leukocytes, Mononuclear/drug effects , Cell Line , Chemokine CCL2/immunology , Chemotaxis, Leukocyte/immunology , Enzyme-Linked Immunosorbent Assay , Humans , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Lipopolysaccharides/pharmacology , Recombinant Proteins/pharmacology , Reverse Transcriptase Polymerase Chain Reaction
4.
Kidney Int ; 52(5): 1206-15, 1997 Nov.
Article in English | MEDLINE | ID: mdl-9350643

ABSTRACT

By using immunocytochemical techniques we have been able to localize the V1 vasopressin receptor in the rat kidney. Immunoblotting using an antiserum raised against an affinity-purified vasopressin receptor showed a 55,000 daltons protein band that has a molecular mass similar to that of the liver V1 vasopressin receptor, as demonstrated by cross-linking studies. Immunoblotting of the antibody showed a band of 55,000 daltons in A-10 cells, which contains the V1 subtype, whereas it did not stain LLC-PK1 cells, which possess the V2 subtype, showing that the antibody recognizes the V1 vasopressin receptor. The immunostaining of kidney sections with this antiserum showed a strong reaction of the connecting tubules and cortical and medullary collecting ducts. The immunostaining pattern of connecting tubule and collecting duct cells was different, that is, the former showed a staining of both the apical and basal plasma membrane but also in the cytoplasm, whereas the latter showed a strong reaction mainly in the basolateral membrane. Immunostaining of consecutive serial sections with an antiserum raised against tissue kallikrein, an enzyme present exclusively in connecting tubules, and with the anti-receptor serum allowed us to show, for the first time, the presence of the vasopressin receptor in the connecting tubule cells and their absence in intercalated cells, the other cell type present in connecting tubules. These findings support experiments carried in the eighties on the release of renal tissue kallikrein by AVP.


Subject(s)
Kidney/chemistry , Receptors, Vasopressin/analysis , Animals , Antibody Specificity , Autoradiography , Immune Sera/immunology , Immunohistochemistry , Rabbits , Rats , Receptors, Vasopressin/immunology
7.
Biochem Int ; 26(4): 759-66, 1992 Mar.
Article in English | MEDLINE | ID: mdl-1535196

ABSTRACT

The binding of [3H]vasopressin (AVP) and the 125I-labelled vasopressin antagonist (VP-AT) d(CH2)5[Tyr2(Me),Tyr9(NH2)]AVP to rat liver membranes was examined with or without the addition of milimolar concentrations of divalent cations. The binding of vasopressin was enhanced by Mg2+ and Co2+ and markedly decreased by EGTA. The addition of EGTA and Mg2+ together restored the binding to a value similar to that of Mg2+ alone. On the contrary, the addition of Mg2+, Co2+, EGTA, and the combination of EGTA and Mg2+ decreased the binding of VP-AT to rat liver membranes. Kinetic analyses showed that Mg2+ increased the Kd twofold for VP-AT; that is from 0.13 nM to 0.28 nM. Moreover, it showed that the receptor with or without the addition of Mg2+ consists of a single population of binding sites, indicating that the receptor is switched from a high affinity to a low affinity state for VP-AT in the presence of 10 mM Mg2+. GTP gamma S was unable to block the effect of Mg2+ on the binding of VP-AT. These results suggest that this divalent cation interacts with receptor itself producing a conformational changes which thus modulates the affinity of the receptor.


Subject(s)
Magnesium/pharmacology , Receptors, Angiotensin/drug effects , Animals , Arginine Vasopressin/analogs & derivatives , Arginine Vasopressin/metabolism , Cations, Divalent , Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology , In Vitro Techniques , Kinetics , Liver/metabolism , Magnesium/metabolism , Rats , Receptors, Angiotensin/metabolism , Receptors, Vasopressin , Vasopressins/antagonists & inhibitors
8.
Biochemistry ; 30(35): 8611-6, 1991 Sep 03.
Article in English | MEDLINE | ID: mdl-1832297

ABSTRACT

Chemical and photoaffinity cross-linking experiments as well as ligand affinity blotting techniques were used to label the V1 vasopressin receptor. In order to determine the optimal reaction conditions, pig liver membranes were incubated with 5 nM [8-lysine]vasopressin (LVP) labeled with 125I and then cross-linked with the use of DMS (dimethyl suberimidate), EGS [ethylene glycol bis(succinimidyl succinate)] or HSAB (hydroxysuccinimidyl p-azidobenzoate) at different final concentrations. Consistently, EGS was found to label with high yield one band of Mr 60,000 in rat and pig liver membranes when used at a final concentration between 0.05 and 0.25 mM. The protein of Mr 60,000 is labeled in a concentration-dependent manner when pig liver membranes are incubated with increasing concentrations of 125I-LVP and then cross-linked with EGS. The label was displaced by increasing concentrations of unlabeled LVP or d(CH2)5 [Tyr2(Me),-Tyr9(NH2)]AVP (V1/V2 antagonist). A protein band of similar molecular mass was cross-linked with 125I-LVP in rat liver membranes. The reaction was specific since the incorporation of label into the protein of Mr 60,000 was inhibited by LVP, [8-arginine]vasopressin (AVP), the V1/V2-antagonist, and the specific V1-antagonist d(CH2)5 [Tyr2(Me)]AVP, only partially by [des-Gly9]AVP (V2-agonist) and by oxytocin, and not at all by angiotensin II. Incubation of nitrocellulose containing membrane proteins from pig liver with 125I-LVP showed the labeling of a band of Mr 58,000 that is inhibited by an excess of unlabeled LVP. This band of Mr 58,000 seems to correspond with the protein of Mr 60,000 revealed by the cross-linking experiment.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Affinity Labels , Cross-Linking Reagents , Lypressin/metabolism , Receptors, Angiotensin/analysis , Animals , Binding, Competitive , Collodion , Electrophoresis, Polyacrylamide Gel , Liver/chemistry , Membrane Proteins/isolation & purification , Molecular Weight , Rats , Receptors, Vasopressin , Swine
9.
Neurosci Lett ; 98(3): 241-6, 1989 Apr 10.
Article in English | MEDLINE | ID: mdl-2725945

ABSTRACT

Membrane proteins from bovine neurosecretory granules isolated by density gradient centrifugation were separated by polyacrylamide gel electrophoresis. A doublet of 120 kDa and and 67 kDa bands were identified as specific proteins of the neurosecretory granule membrane. Antibodies against the 120 kDa doublet were raised in rabbits and characterized by western blotting and immunocytochemistry. Analysis of the antiserum by western blotting showed that this recognizes mainly the 120 kDa doublet and some other minor components which seem to be degradation products. The antiserum against the 120 kDa proteins stained, by immunocytochemistry, specifically the supraoptic and paraventricular neurons of the rat hypothalamo-neurohypophysial system. In the neural lobe the immunoreaction was found around blood vessels on structures which appear to be nerve endings and on Herring bodies. Immunoelectron microscopy using protein A-gold showed that the 120 kDa antigens are located on the membrane of neurosecretory granules in sections of rat neural lobes. The presence of the 120 kDa antigens exclusively in the hypothalamo-neurohypophysial system suggests that these proteins are probably not involved in a general secretory mechanism and that they might be a result of the tissue-specific expression of proteins.


Subject(s)
Antibodies/isolation & purification , Cytoplasmic Granules/analysis , Hypothalamo-Hypophyseal System/analysis , Membrane Proteins/isolation & purification , Animals , Cattle , Cell Fractionation , Cytoplasmic Granules/ultrastructure , Immunohistochemistry , Membrane Proteins/immunology , Pituitary Gland, Posterior/analysis , Pituitary Gland, Posterior/ultrastructure , Rabbits , Rats , Supraoptic Nucleus/analysis , Supraoptic Nucleus/ultrastructure
10.
Gen Comp Endocrinol ; 67(3): 348-55, 1987 Sep.
Article in English | MEDLINE | ID: mdl-2822524

ABSTRACT

By using antisera against human pituitary hormones in immunocytochemistry in combination with classical cytochemical techniques, we have been able to identify the different cell types in the adenohypophysis of the Austromenidia laticlavia and to determine their location. Antisera against prolactin and growth hormones did not stain cells in the pituitary of Austromenidia, whereas antisera against beta-endorphin, LH, and beta-TSH selectively cross-reacted with cells which have a specific location within the adenohypophysis. The beta-endorphin antiserum stained the periodic acid-Schiff (PAS)-negative cells in the pars intermedia and also, though faintly, the PAS-negative cells in the internal border of the rostral pars distalis (RPD). Human beta-TSH antiserum showed a discrete population of small PAS-positive cells in the proximal pars distalis (PPD). Antiserum against human LH stained PAS-positive cells located in the most ventral zone of the PPD and around the pars intermedia (PI). The distribution of the different cell types is similar to that of other teleosts. The phylogenetic implications of the degree of cross-reactivity of the antisera against human pituitary hormones with specific cells of the teleost fish pituitary is discussed.


Subject(s)
Fishes/physiology , Pituitary Gland/cytology , Pituitary Hormones/analysis , Adrenocorticotropic Hormone/analysis , Animals , Female , Immunoenzyme Techniques , Luteinizing Hormone/analysis , Male , Thyrotropin/analysis
11.
Anal Biochem ; 157(2): 316-22, 1986 Sep.
Article in English | MEDLINE | ID: mdl-3777435

ABSTRACT

A procedure for the subfractionation of neurosecretory granules into membrane and content components is described. The procedure involves the hypotonic lysis of the secretory granule fraction and further purification of the membranes by centrifugation through a discontinuous sucrose gradient. The neurosecretory granule membranes represented 5.2% of the total proteins of the neurosecretory granule fraction and were highly enriched in cytochrome b561. Electron microscopic analysis of the purified membranes showed vesicles devoid of electrodense content.


Subject(s)
Cytoplasmic Granules/ultrastructure , Neurosecretory Systems/ultrastructure , Animals , Cattle , Cell Fractionation/methods , Centrifugation, Density Gradient , Cytochrome b Group/metabolism , Cytoplasmic Granules/metabolism , Intracellular Membranes/metabolism , Intracellular Membranes/ultrastructure , Neurosecretory Systems/metabolism , Pituitary Gland, Posterior/metabolism , Pituitary Gland, Posterior/ultrastructure
12.
Rev. argent. cir ; 47(5): 220-9, 1984.
Article in Spanish | BINACIS | ID: bin-33386

ABSTRACT

Las dilataciones quisticas de la via biliar son alteraciones congenitas que si bien no constituyen una patologia frecuente, conviene que el cirujano las conozca para que al enfermo se le resuelva su problema de la mejor manera y en una sola operacion


Subject(s)
Adult , Middle Aged , Humans , Male , Female , Cysts , Dilatation , Common Bile Duct Diseases , Surgical Procedures, Operative
13.
Rev. argent. cir ; 47(5): 220-9, 1984.
Article in Spanish | LILACS | ID: lil-25131

ABSTRACT

Las dilataciones quisticas de la via biliar son alteraciones congenitas que si bien no constituyen una patologia frecuente, conviene que el cirujano las conozca para que al enfermo se le resuelva su problema de la mejor manera y en una sola operacion


Subject(s)
Adult , Middle Aged , Humans , Male , Female , Cysts , Dilatation , Common Bile Duct Diseases , Surgical Procedures, Operative
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