It is well established that auto/paracrine acetylcholine (ACh) is essential for wound epithelialization, and that the mechanisms include regulation of keratinocyte motility and adhesion via nicotinic ...ACh receptors (nAChRs). Keratinocyte nAChRs can be also activated by non‐canonical ligands, such as secreted mammalian Ly‐6/urokinase‐type plasminogen activator receptor‐related protein (SLURP)‐1 and ‐2. In this study, we determined effects of recombinant (r)SLURP‐1 and‐2 on migration of human epidermal and oral keratinocytes under agarose and epithelialization of cutaneous and oral mucosal excisional wounds in mice, and also identified nAChRs mediating SLURP signals. Both in vitro and in vivo, rSLURP‐1 decreased and SLURP‐2 increased epithelialization rate. The mixture of both peptides accelerated epithelialization even further, indicating that their simultaneous signaling renders an additive physiologic response. The specificity of rSLURP actions was illustrated by similar effects on cutaneous and oral wounds, which feature distinct responses to injury, and also by abrogation of rSLURP effects with neutralizing antibodies. rSLURP‐1 acted predominantly via the α7 nAChR‐coupled up‐regulation of the sedentary integrins α2 and α3, whereas SLURP‐2—through α3, and α9 nAChRs up‐regulating migratory integrins α5 and αV. The biologic effects of rSLURPs required the presence of endogenous ACh, indicating that auto/paracrine SLURPs provide for a fine tuning of the physiologic regulation of crawling locomotion via the keratinocyte ACh axis. Since nAChRs have been shown to regulate SLURP production, cholinergic regulation of keratinocyte migration appears to be mediated by a reciprocally arranged network. The cholinergic peptides, therefore, may become prototype drugs for the treatment of wounds that fail to heal.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK, VSZLJ
Pemphigus vulgaris (PV) is a mucocutaneous blistering disease characterized by IgG autoantibodies against the stratified squamous epithelium. Current understanding of PV pathophysiology does not ...explain the mechanism of acantholysis in patients lacking desmoglein antibodies, which justifies a search for novel targets of pemphigus autoimmunity. We tested 264 pemphigus and 138 normal control sera on the multiplexed protein array platform containing 701 human genes encompassing many known keratinocyte cell-surface molecules and members of protein families targeted by organ-non-specific PV antibodies. The top 10 antigens recognized by the majority of test patients' sera were proteins encoded by the DSC1, DSC3, ATP2C1, PKP3, CHRM3, COL21A1, ANXA8L1, CD88 and CHRNE genes. The most common combinations of target antigens included at least one of the adhesion molecules DSC1, DSC3 or PKP3 and/or the acetylcholine receptor CHRM3 or CHRNE with or without the MHC class II antigen DRA. To identify the PV antibodies most specific to the disease process, we sorted the data based on the ratio of patient to control frequencies of antigen recognition. The frequency of antigen recognition by patients that exceeded that of control by 10 and more times were the molecules encoded by the CD33, GP1BA, CHRND, SLC36A4, CD1B, CD32, CDH8, CDH9, PMP22 and HLA-E genes as well as mitochondrial proteins encoded by the NDUFS1, CYB5B, SOD2, PDHA1 and FH genes. The highest specificity to PV showed combinations of autoantibodies to the calcium pump encoded by ATP2C1 with C5a receptor plus DSC1 or DSC3 or HLA-DRA. The results identified new targets of pemphigus autoimmunity. Novel autoantibody signatures may help explain individual variations in disease severity and treatment response, and serve as sensitive and specific biomarkers for new diagnostic assays in PV patients.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Despite the increasing availability of genome sequences from many human pathogens, the production of complete proteomes remains at a bottleneck. To address this need, a high-throughput PCR ...recombination cloning and expression platform has been developed that allows hundreds of genes to be batch-processed by using ordinary laboratory procedures without robotics. The method relies on high-throughput amplification of each predicted ORF by using gene specific primers, followed by in vivo homologous recombination into a T7 expression vector. The proteins are expressed in an Escherichia coli-based cell-free in vitro transcription/translation system, and the crude reactions containing expressed proteins are printed directly onto nitrocellulose microarrays without purification. The protein microarrays are useful for determining the complete antigen-specific humoral immune-response profile from vaccinated or infected humans and animals. The system was verified by cloning, expressing, and printing a vaccinia virus proteome consisting of 185 individual viral proteins. The chips were used to determine Ab profiles in serum from vaccinia virus-immunized humans, primates, and mice. Human serum has high titers of anti-E. coli Abs that require blocking to unmask vaccinia-specific responses. Naïve humans exhibit reactivity against a subset of 13 antigens that were not associated with vaccinia immunization. Naïve mice and primates lacked this background reactivity. The specific profiles between the three species differed, although a common subset of antigens was reactive after vaccinia immunization. These results verify this platform as a rapid way to comprehensively scan humoral immunity from vaccinated or infected humans and animals.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Toxoplasma gondii is an obligate intracellular parasite that can infect almost all warm-blooded animals, avian species and humans. Toxoplasmosis is asymptomatic in healthy individuals, whereas it may ...lead to death in immune suppressed or deficient patients. A vaccine against T. gondii is required to prevent consequences of the infection. The aim of this study is to generate a multivalent recombinant protein vaccine against T. gondii. 49 previously discovered antigenic proteins of T gondii were evaluated by their expression level in E. coli and by comprehensive bioinformatics analyses to determine antigenic epitopes. Based on these analyses, six vaccine candidate proteins were selected to generate a hexavalent recombinant protein vaccine adjuvanted with Montanide ISA 50 V. Humoral and cellular immune responses were determined by flow cytometry and ELISA. Vaccinated mice were challenged with T. gondii Ankara strain tachyzoites. In mice vaccinated with hexavalent vaccine, strong total IgG (P 0.0001) and IgG2a (P 0.001) responses were induced compared to controls, the ratio of CD4.sup.+ and CD8.sup.+ T lymphocytes secreting IFN-gamma increased, and significantly higher extracellular IFN-gamma secretion was achieved compared to the controls (P 0.001). The survival time of the vaccinated mice increased to 8.38 + or - 2.13 days which was significantly higher than controls (P 0.01). Altogether, these results show that the hexavalent vaccine which is developed for the first time against T. gondii induced strong and balanced Th1 and Th2 immune responses as well as conferred significant protection against challenge with lethal toxoplasmosis in murine model.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Is Grover's disease an autoimmune dermatosis? Phillips, Courtney; Kalantari-Dehaghi, Mina; Marchenko, Steve ...
Experimental dermatology,
12/2013, Volume:
22, Issue:
12
Journal Article
Peer reviewed
Open access
Grover's disease (GD) is a transient or persistent, monomorphous, papulovesicular, asymptomatic or pruritic eruption classified as non‐familial acantholytic disorder. Contribution of autoimmune ...mechanisms to GD pathogenesis remains controversial. The purpose of this study was to investigate antibody‐mediated autoimmunity in 11 patients with GD, 4 of which were positive for IgA and/or IgG antikeratinocyte antibodies by indirect immunofluorescence. We used the most sensitive proteomic technique for an unbiased analysis of IgA‐ and IgG‐autoantibody reactivities. Multiplex analysis of autoantibody responses revealed autoreactivity of all 11 GD patients with cellular proteins involved in the signal transduction events regulating cell development, activation, growth, death, adhesion and motility. Semiquantitative fluorescence analysis of cultured keratinocytes pretreated with sera from each patient demonstrated decreased intensity of staining for desmoglein 1 and/or 3 and PCNA, whereas 4 of 10 GD sera induced BAD expression, indicating that binding of autoantibodies to keratinocytes alters expression/function of their adhesion molecules and activates apoptosis. We also tested the ability of GD sera to induce visible alterations of keratinocyte shape and motility in vitro but found no specific changes. Thus, our results demonstrated that humoral autoimmunity in GD can be mediated by both IgA and IgG autoantibodies. At this point, however, it is impossible to conclude whether these autoantibodies cause or are caused by the disease. Antidesmoglein antibodies may be triggered by exposure to immune system of sequestered antigens due to disintegration of desmosomes during primary acantholysis. Clarifying aetiology of GD will help improve treatment, which currently is symptomatic and of marginal effectiveness.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
To elucidate how the nicotinic acetylcholine receptors expressed on bronchial and oral epithelial cells targeted by the tobacco nitrosamine (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone) (NNK) ...facilitate carcinogenic transformation.
Since NNK-dependent transformation can be abolished by the nicotinergic secreted mammalian Ly-6/urokinase plasminogen activator receptor related protein-1 (SLURP-1), we compared effects of NNK and recombinant (r)SLURP-1 on the expression of genes related to tumorigenesis in human immortalized bronchial and oral epithelial cell lines BEP2D and Het-1A, respectively.
NNK stimulated expression of oncogenic genes, including MYB and PIK3CA in BEP2D, ETS1, NRAS and SRC in Het-1A, and AKT1, KIT and RB1 in both cell types, which could be abolished in the presence of rSLURP-1. Other cancer-related genes whose upregulation by NNK was abolishable by rSLURP-1 were the growth factors EGF in BEP2D cells and HGF in Het-1A cells, and the transcription factors CDKN2A and STAT3 (Het-1A only). NNK also upregulated the anti-apoptotic BCL2 (Het-1A) and downregulated the pro-apoptotic TNF (Het-1A), BAX and CASP8 (BEP2D), all of which could be abolished, in part, by rSLURP-1. NNK decreased expression of the CTNNB1 gene encoding the intercellular adhesion molecule β-catenin (BEP2D), as well as tumor suppressors CDKN3 and FOXD3 in BEP2D cells and SERPINB5 in Het-1A cells. These pro-oncogenic effects of NNK were abolished by rSLURP-1 that also upregulated RUNX3.
The obtained results identified target genes for both NNK and SLURP-1 and shed light on the molecular mechanism of their reciprocal effects on tumorigenic transformation of bronchial and oral epithelial cells.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
: We performed partial evaluation of pemphigus vulgaris (PV) autoantibody profile using the protein array technology. The sera from seven patients with acute PV and five healthy donors were probed ...for the presence of autoantibodies characteristic of the organ‐non‐specific autoimmune disorders rheumatoid arthritis, lupus erythematosus, scleroderma, diabetes and some other autoimmune disorders, but not to desmosomal proteins. The array targeted 785 human genes amplified using Mammalian Gene Clone Collection with gene‐specific primers containing 20‐bp nucleotide extension complementary to ends of linear pXT7 vector. The array identified PV antibodies significantly (P < 0.05) differentially reactive with 16 antigens, most of which were cell‐surface proteins, such as CD2, CD31, CD33, CD36, CD37, CD40, CD54, CD66c and CD84 molecules, nicotinamide/nicotinic acid mononucleotide adenylyltransferase, immunoglobulin heavy chain constant region gamma 2 and others. Reactivity with Fc‐IgG helps explain an ability of the chimeric desmoglein constructs to absorb out all disease‐causing PV antibodies. Anti‐M1 muscarinic receptor antibody was also identified, consistent with the facts that while blockade of this receptor causes keratinocyte detachment, its activation is therapeutic in PV. Further proteomics analysis of PV antibodies should help elucidate the immunopathogenic mechanisms underlying keratinocyte detachment and blistering.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
Introduction: Humoral immune responses play a pivotal role in naturally acquired immunity to malaria. Understanding which humoral responses are impaired among individuals at higher risk for malaria ...may improve our understanding of malaria immune control and contribute to vaccine development.
Methods: We compared humoral responses with 483 Plasmodium falciparum antigens between adults in, Kisumu (high, year‐long malaria transmission leading to partial immunity), and adults in Kisii (low, seasonal malaria transmission). Then within each site, we compared malaria‐specific humoral responses between those at higher risk for malaria (CD4+≤500) and those at lower risk for malaria (CD4+>500). A protein microarray chip containing 483 P. falciparum antigens and 71 HIV antigens was used. Benjamini–Hochberg adjustments were made to control for multiple comparisons.
Results: Fifty‐seven antigens including CSP, MSP1, LSA1 and AMA1 were identified as significantly more reactive in Kisumu than in Kisii. Ten of these antigens had been identified as protective in an earlier study. CD4+ T‐cell count did not significantly impact humoral responses.
Conclusion: Protein microarrays are a useful method to screen multiple humoral responses simultaneously. This study provides useful clues for potential vaccine candidates. Modest decreases in CD4 counts may not significantly impact malaria‐specific humoral immunity.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
The development of nonhormonal treatment of pemphigus vulgaris (PV) has been hampered by a lack of clear understanding of the mechanisms leading to keratinocyte (KC) detachment and death in ...pemphigus. In this study, we sought to identify changes in the vital mitochondrial functions in KCs treated with the sera from PV patients and healthy donors. PV sera significantly increased proton leakage from KCs, suggesting that PV IgGs increase production of reactive oxygen species. Indeed, measurement of intracellular reactive oxygen species production showed a drastic increase of cell staining in response to treatment by PV sera, which was confirmed by FACS analysis. Exposure of KCs to PV sera also caused dramatic changes in the mitochondrial membrane potential detected with the JC-1 dye. These changes can trigger the mitochondria-mediated intrinsic apoptosis. Although sera from different PV patients elicited unique patterns of mitochondrial damage, the mitochondria-protecting drugs nicotinamide (also called niacinamide), minocycline, and cyclosporine A exhibited a uniform protective effect. Their therapeutic activity was validated in the passive transfer model of PV in neonatal BALB/c mice. The highest efficacy of mitochondrial protection of the combination of these drugs found in mitochondrial assay was consistent with the ability of the same drug combination to abolish acantholysis in mouse skin. These findings provide a theoretical background for clinical reports of the efficacy of mitochondria-protecting drugs in PV patients. Pharmacological protection of mitochondria and/or compensation of an altered mitochondrial function may therefore become a novel approach to development of personalized nonhormonal therapies of patients with this potentially lethal autoimmune blistering disease.
Background: Previous studies suggested that mitochondrial antibodies contribute to pemphigus vulgaris (PV).
Results: PV sera elicited mitochondrial damage, and mitochondria-protecting drugs exhibited protective effect in cell culture and mouse skin.
Conclusion: PV antibodies altered O2 respiration, disrupted electron transfer chain, and increased reactive oxygen species.
Significance: Results provide the mechanism of therapeutic action and justify the use of mitochondria-protecting drugs in PV.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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