Increased expression of in situ IL-31RA and circulating CXCL8 and CCL2 in pemphigus herpetiformis suggests participation of the IL-31 family in the pathogenesis of the disease.

BACKGROUND: Pemphigus herpetiformis (PH) is a rare clinical subtype of pemphigus with the presence of urticarial plaques, severe pruritus, rare acantholysis and eosinophilic spongiosis. OBJECTIVES: The aim of this study was to investigate the influence of IL-31 and pro-inflammatory cytokines/chemokines in the pathogenesis of PH. METHODS: Twenty-five patients with PH and three groups: pemphigus foliaceus (PF = 14), pemphigus vulgaris (PV = 15) and healthy controls (HC = 20) were selected for this study. The groups were analysed by immunohistochemistry utilizing IL-31, IL-31RA, IL-4, IL-17 and TNF-α antibodies. Serum levels of IL-4, IL-13, TNF, CXCL8, CCL5 and CCL2 were evaluated by cytometric bead array. RESULTS: Analysis of IL-31 family of PH patients revealed the following findings: (i) Enhanced in situ expression of IL-31 in PH samples, compared to PF and to PV (epidermis); (ii) Cutaneous IL-31RA expression in PH samples was higher than in PF, PV and HC groups (epidermis and dermis); (iii) PF patients that evolved to PH showed significant increased IL-31RA epidermal expression during the PH phase. Profile of pro-inflammatory cytokines (IL-4, IL-17 and TNF-α) in PH patients' skin exhibited: (i) Enhanced IL-4 expression, when compared to patients with PF (epidermis and dermis) and with PV (epidermis); (ii) Augmented IL-17 expression than PF and PV patients (epidermis); (iii) Augmented expression of TNF-α when compared to PF at the epidermal level. Evaluation of circulating cytokines and chemokines showed higher levels of CXCL8 and CCL2 in PH sera compared to HC group. CONCLUSIONS: IL-31 and IL-31RA, cytokines related to pruritus, and pro-inflammatory chemokines (CXCL8 and CCL2) seem to exert a role in the pathogenesis of PH. These findings support future studies to clarify the role of IL-31 pathway as a potential therapeutic target for patients with PH.

Exploring the in situ expression of vascular endothelial growth factor and endoglin in pemphigus foliaceus variants and pemphigus vulgaris.

BACKGROUND: Erythroderma is a severe manifestation of pemphigus foliaceus (PF), a blistering disease mediated by IgG autoantibodies against desmoglein 1. Increasing evidence supports the contribution of angiogenic mediators in the pathogenesis of erythroderma. OBJECTIVE: To evaluate the in situ expression of vascular endothelial growth factor (VEGF) and endoglin in patients with PF with erythroderma. METHODS: Formalin-fixed paraffin-embedded skin samples obtained from patients with erythrodermic PF (n = 19; 12 patients with endemic PF), non-erythrodermic PF (n = 17), pemphigus vulgaris (PV; n = 10), psoriasis (n = 10) and healthy individuals (HI; n = 10) were processed in an automated immunohistochemistry platform utilizing anti-VEGF and anti-endoglin as primary antibodies. Reactivity was evaluated both manually (0 = negative; 1+ = mild; 2+ = intense) and through an automated microvessel analysis algorithm. RESULTS: Vascular endothelial growth factor expression in erythrodermic PF was higher than in non-erythrodermic PF (P = 0.034) and in HI (P = 0.004), and similar to psoriasis (P = 0.667) and PV (P = 0.667). In non-erythrodermic PF, VEGF positivity was similar to HI (P = 0.247), and lower than psoriasis (P = 0.049) and PV (P = 0.049). Both erythrodermic and non-erythrodermic PF presented similar endoglin expression (P = 0.700). In addition, endoglin positivity during erythrodermic PF was similar to psoriasis (P = 0.133) and lower than PV (P = 0.0009). Increased expression of in situVEGF suggests that healing processes are triggered in response to tissue damage led by autoantibodies in PF, especially during erythroderma. Reduced endoglin positivity suggests that an unbalanced angiogenesis may occur during erythrodermic PF. Further studies may help to confirm if the regulation of VEGF and endoglin expression in patients with PF can contribute to control the healing process and enable disease remission. CONCLUSION: Overexpression of VEGF in erythrodermic PF as well as in PV and psoriasis points out a dysregulated repair process in severe forms of these diseases and suggests VEGF and endoglin could act as prognostic markers and future therapeutic targets to enable proper healing in PF.

Increased serum levels of vascular endothelial growth factor in pemphigus foliaceus patients with erythroderma.

BACKGROUND: Erythroderma is a clinical skin syndrome shared by patients with cutaneous disorders of distinct aetiologies as a result of the combined actions of chemokines, adhesion molecules, and cytokines, such as vascular endothelial growth factor (VEGF). OBJECTIVE: To evaluate the profile of serum levels of VEGF and soluble vascular endothelial growth factor receptor 1 (sVEGFR-1) in pemphigus foliaceus (PF) patients with erythroderma. METHODS: We conducted a retrospective study, which included (i) a chart review of all PF patients from the Autoimmune Blistering Clinic, University of Sao Paulo, Brazil, from January 1991 to December 2014, together with an evaluation of demographic variables, hospitalization duration and complications and (ii) analysis of the circulating VEGF and sVEGFR-1 levels in PF patients with erythroderma by ELISA. The controls included patients with pemphigus vulgaris or psoriasis. RESULTS: We observed higher serum VEGF levels in PF patients during erythroderma than during the non-erythrodermic phase. PF patients showed increased serum levels of sVEGFR-1 during the erythrodermic phase in comparison to controls. Interestingly, the sVEGFR-1 and antidesmoglein-1 levels were positively correlated during the non-erythrodermic period. CONCLUSION: Erythroderma, which represents one clinical form of PF, implies more severe outcomes. The circulating levels of VEGF, a potent endothelial activator, are increased in PF patients with erythroderma; this result suggests the contribution of the blood vessel endothelium to the pathogenesis of this clinical syndrome. Interestingly, our findings showed a positive correlation between the sVEGFR-1 and antidesmoglein-1 antibody levels, indicating a suppressive response to VEGF augmentation during the erythrodermic phase of PF.

Toxicity and outcomes after chemoradiation for esophageal cancer in patients age 75 or older.

Randomized trials of chemoradiation for esophageal cancer have included very few patients age > or = 75. In this retrospective study, we describe the outcomes and toxicity of full-dose chemoradiation in elderly patients with esophageal cancer. Patients, age > or = 75, treated with full-dose chemoradiation for esophageal carcinoma from 2002 to 2008 were retrospectively reviewed. Thirty-four patients were identified with a median age of 79.5 (range 75-89). The median Eastern Cooperative Oncology Group performance status was 1 (range 0-3) and the median Adult Comorbidity Evaluation-27 score was 1 (range 0-3). Twenty-eight patients received definitive and six received neoadjuvant chemoradiation. The median radiation dose delivered was 50.4 Gray (range 3.6-68.4 Gray). Platinum-based chemotherapy was used in 79.4% of patients. Fifty percent of the patients completed all planned radiation therapy (RT) and chemotherapy; 85.3% completed RT. Acute toxicity > or = grade 4 occurred in 38.2% of patients, and 70.6% of the patients required hospitalization, emergency department visit, and/or RT break. Median follow-up was 14.5 months among 7 survivors, and median survival was 12.0 months (95% confidence interval [CI]: 9.7 to 24.1 months). The actuarial overall survival at 2 years was 29.7% (95% CI: 16.6 to 52.6%). There were four treatment-related deaths. The median time to any recurrence was 10.4 months. Nineteen patients had a local and/or distant recurrence. In conclusion, elderly patients experienced substantial morbidity from chemoradiation, and long-term survival was low. Future efforts to improve treatment tolerability in the elderly are needed.

Clinicopathological evaluation of in vivo epidermal nuclear fluorescence.

BACKGROUND: Nuclear fluorescence in keratinocytes is an occasional phenomenon, often present in autoimmune diseases, especially in connective-tissue disease (CTD); however, its clinical significance remains unclear. AIM: To investigate the profile of patients with positive nuclear staining on direct immunofluorescence (DIF) of skin samples. METHODS: A retrospective analysis of 28 patient records from our immunodermatology laboratory was performed between May 2003 and June 2006. Inclusion criteria were the presence of autoantibodies (IgG, IgA or IgM) or complement (C3) binding keratinocyte nuclei on DIF. RESULTS: The most prevalent diseases related to the nuclear keratinocyte DIF staining were systemic lupus erythematosus (n = 9), mixed CTD (n = 3), overlap syndrome (n = 3), Sjögren's syndrome (n = 1), and CREST (calcinosis, Raynaud's phenomenon, oesophageal dysmotility, sclerodactyly and telangiectasia) syndrome (n = 1). Serum antinuclear antibody (ANA) was positive in 20 of 28 patients, with titres varying from 1 : 160 to 1 : 1280. Of the 20 patients with positive anti-nuclear antibodies (ANA), 17 were positive for anti-extractable nuclear antigen antibodies, 12 had anti-SSA/Ro, 11 had anti-SSB/La and 8 had anti-ribonucleoprotein. Eight patients were negative for ANA. Positive predictive value of in vivo ANA for systemic CTDs was 75%. CONCLUSION: The present data suggest that in vivo ANA evaluation is an additional and feasible auxiliary tool for diagnosing CTDs.

Chemoproteomics-enabled covalent ligand screen reveals a cysteine hotspot in reticulon 4 that impairs ER morphology and cancer pathogenicity.

Chemical genetics has arisen as a powerful approach for identifying novel anti-cancer agents. However, a major bottleneck of this approach is identifying the targets of lead compounds that arise from screens. Here, we coupled the synthesis and screening of fragment-based cysteine-reactive covalent ligands with activity-based protein profiling (ABPP) chemoproteomic approaches to identify compounds that impair colorectal cancer pathogenicity and map the druggable hotspots targeted by these hits. Through this coupled approach, we discovered a cysteine-reactive acrylamide DKM 3-30 that significantly impaired colorectal cancer cell pathogenicity through targeting C1101 on reticulon 4 (RTN4). While little is known about the role of RTN4 in colorectal cancer, this protein has been established as a critical mediator of endoplasmic reticulum tubular network formation. We show here that covalent modification of C1101 on RTN4 by DKM 3-30 or genetic knockdown of RTN4 impairs endoplasmic reticulum and nuclear envelope morphology as well as colorectal cancer pathogenicity. We thus put forth RTN4 as a potential novel colorectal cancer therapeutic target and reveal a unique druggable hotspot within RTN4 that can be targeted by covalent ligands to impair colorectal cancer pathogenicity. Our results underscore the utility of coupling the screening of fragment-based covalent ligands with isoTOP-ABPP platforms for mining the proteome for novel druggable nodes that can be targeted for cancer therapy.

Clinical evaluation of microcystic macular edema in patients with glaucoma.

PurposeTo investigate the prevalence of microcystic macular edema (MME) in patients with glaucoma and the relationship between glaucomatous visual field defects and MME.Patients and methodsWe analyzed 636 eyes of 341 glaucoma patients who underwent spectral domain optical coherence tomography (SD-OCT). MME was defined as vacuoles observed in the inner nuclear layer (INL) on SD-OCT. Quantitative assessment of MME area was performed using en-face imaging obtained swept-source OCT (SS-OCT) and Adobe Photoshop CS6 Extended software. These values were compared with the visual field results with the Humphrey field analyzer.ResultsMME was observed in 1.6% of eyes. The visual field mean deviation (MD), pattern standard deviation (PSD) and visual acuity was significantly worse (P= 0.023, P=0.037, and P=0.018, respectively) in eyes with MME. The average MME area was 2.38±1.43%. There was no significant correlation between visual field deficits and MME area.ConclusionsThe MME detection rate based on general inspection was 1.6%. MME in glaucomatous eyes were associated with worse MD, PSD, and visual acuity. Further research is needed to increase the number of cases to allow for more detailed analysis.

Top-down cortical input during NREM sleep consolidates perceptual memory.

During tactile perception, long-range intracortical top-down axonal projections are essential for processing sensory information. Whether these projections regulate sleep-dependent long-term memory consolidation is unknown. We altered top-down inputs from higher-order cortex to sensory cortex during sleep and examined the consolidation of memories acquired earlier during awake texture perception. Mice learned novel textures and consolidated them during sleep. Within the first hour of non-rapid eye movement (NREM) sleep, optogenetic inhibition of top-down projecting axons from secondary motor cortex (M2) to primary somatosensory cortex (S1) impaired sleep-dependent reactivation of S1 neurons and memory consolidation. In NREM sleep and sleep-deprivation states, closed-loop asynchronous or synchronous M2-S1 coactivation, respectively, reduced or prolonged memory retention. Top-down cortical information flow in NREM sleep is thus required for perceptual memory consolidation.

Analysis of the NADPH oxidase components during differentiation of HL-60 cells to eosinophilic lineage.

HL-60 cells were induced to differentiate into eosinophil-like cells with sodium butyrate after passage under mild alkaline condition. The differentiating cells gradually possessed the Luxol-fast-blue (LFB) staining-positive granules and the capacity to produce superoxide. The increase in the amounts of cytochrome b-558 paralleled the superoxide anion generating activity. Immunoblot analysis demonstrated that p47-phox cytosolic oxidase protein appeared 1 day after differentiation, and increased up to 7 days. On the other hand, p67-phox cytosolic oxidase protein appeared in 3 days, and increased gradually up to 7 days. The oxidase activity did not appear until p67-phox protein was expressed in the cytosol during eosinophilic differentiation, indicating that p67-phox protein is likely to be a key protein of cytosolic factors also in eosinophilic differentiating cells. The amounts of p47-phox and p67-phox translocated to the plasma membrane in response to phorbol myristate acetate (PMA) increased with increasing amounts of cytochrome b-558 in the membrane. Our data demonstrate that the appearance of NADPH oxidase activity during eosinophilic differentiation is dependent on the levels of p47-phox and p67-phox cytosolic proteins translocated to the plasma membrane and the amount of cytochrome b-558 in the membrane as observed with neutrophils and monocytes.

Dissecting cellular processes using small molecules: identification of colchicine-like, taxol-like and other small molecules that perturb mitosis.

BACKGROUND: Understanding the molecular mechanisms of complex cellular processes requires unbiased means to identify and to alter conditionally gene products that function in a pathway of interest. Although random mutagenesis and screening (forward genetics) provide a useful means to this end, the complexity of the genome, long generation time and redundancy of gene function have limited their use with mammalian systems. We sought to develop an analogous process using small molecules to modulate conditionally the function of proteins. We hoped to identify simultaneously small molecules that may serve as leads for the development of therapeutically useful agents. RESULTS: We report the results of a high-throughput, phenotype-based screen for identifying cell-permeable small molecules that affect mitosis of mammalian cells. The predominant class of compounds that emerged directly alters the stability of microtubules in the mitotic spindle. Although many of these compounds show the colchicine-like property of destabilizing microtubules, one member shows the taxol-like property of stabilizing microtubules. Another class of compounds alters chromosome segregation by novel mechanisms that do not involve direct interactions with microtubules. CONCLUSIONS: The identification of structurally diverse small molecules that affect the mammalian mitotic machinery from a large library of synthetic compounds illustrates the use of chemical genetics in dissecting an essential cellular pathway. This screen identified five compounds that affect mitosis without directly targeting microtubules. Understanding the mechanism of action of these compounds, along with future screening efforts, promises to help elucidate the molecular mechanisms involved in chromosome segregation during mitosis.

Establishment of a human T-cell hybridoma that produces human macrophage activating factor for superoxide production and translation of messenger RNA of the factor in Xenopus laevis oocyte.

Human monoblast-like histiocytic lymphoma cell line U937 was induced by a macrophage activating factor for O2- production (MAF-O) to produce O2- in response to phorbol myristate acetate stimulation. A MAF-O-producing human T-cell hybridoma, F4-29-4, was established which was also found to produce macrophage activating factor for glucose consumption (MAF-G) and colony stimulating factor (CSF) when assayed against mouse bone marrow cells. MAF-O could be successfully separated from CSF but not from MAF-G by phenyl-Sepharose CL-4B chromatography (Phenyl-EG-fraction). To differentiate MAF-O from MAF-G and to explore a route for large scale production of MAF-O and its structural elucidation, total messenger RNA was extracted from a human T-cell hybridoma, clone F4-29-4. This messenger RNA was fractionated on 5-30% sucrose gradient and each fraction was microinjected into Xenopus laevis oocytes. MAF-O activity was found in the supernatant of oocytes injected with messenger RNA sedimentated at about 13.0S, while MAF-G messenger RNA was found to be about 10.5S. The MAF-O activity, synthesized from the injected messenger RNA, was not neutralized with an excess amount of anti-human IFN-gamma anti-serum, suggesting that MAF-O is antigenically different from human IFN-gamma.

Swine influenza virus strains recognize sialylsugar chains containing the molecular species of sialic acid predominantly present in the swine tracheal epithelium.

We determined the ratio of N-glycolylneuraminic acid (Neu5Gc) to N-acetylneuraminic acid (Neu5Ac) in swine respiratory epithelia by fluorometric high-performance liquid chromatography, and examined the binding specificity of swine influenza virus strains for gangliosides containing different molecular species of sialic acid (Neu5Ac and Neu5Gc), and for bovine erythrocyte sialoglycoprotein 2 (GP-2) containing Neu5Gc as its predominate sialic acid (96% of total sialic acids). The presence of Neu5Gc, which had not been detected in human tracheal epithelia, and Neu5Ac in swine tracheal epithelia was observed in a 1:1 ratio. The swine influenza virus H1 and H3 isolates tested, except for A/swine/Iowa/15/30 (H1N1), displayed a marked binding ability for sialylsugar chains containing Neu5Gc compared with that of the human influenza virus strains. These results suggest that swine influenza viruses recognize sialylsugar chains containing the molecular species of sialic acid present predominantly in the swine tracheal epithelium.

Substitution of amino acid residue in influenza A virus hemagglutinin affects recognition of sialyl-oligosaccharides containing N-glycolylneuraminic acid.

Sialic acids are essential components of cell surface receptors used by influenza viruses. To determine the molecular mechanisms of viral recognition of two major species of sialic acids, N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), we tested the binding reactivity of nine human H3 influenza A viruses to sialylglycolipids containing type II sugar chain and different molecular species of terminal sialic acids. All human H3 viruses tested except A/Memphis/1/71 bound both Neu5Ac and Neu5Gc. Nucleotide sequence analysis suggests that amino acids at 143, 155, and 158 are linked to the viral recognition of Neu5Gc.

Thujaplicin-copper chelates inhibit replication of human influenza viruses.

The effects of alpha-, beta- and gamma-thujaplicins and six of their metal chelates on human influenza virus-induced apoptosis in Madin-Darby canine kidney (MDCK) cells were examined by DNA fragmentation and flow cytometry. Among the compounds tested, thujaplicin copper chelates inhibited apoptosis induced in the infected MDCK cells with influenza A/PR/8/34(H1N1), A/Shingapol/1/57(H2N2), A/Aichi/2/68(H3N2) and B/Lee/40 viruses, at concentrations of more than 5 microM. These results indicate that the copper chelates inhibit influenza virus-induced apoptosis and that the inhibitory effects may be independent of influenza virus subtype or types. Furthermore, the copper chelates also inhibited the release of the viruses from the infected MDCK cells during apoptosis. The anti-apoptotic effects of the copper chelates may occur 2 4 h postinfection, suggesting that the copper chelates affect MDCK cells directly in the early stage of influenza virus-induced apoptosis. In this study, we demonstrated that thujaplicin-copper chelates inhibit influenza virus-induced apoptosis of MDCK cells and also inhibit virus replication and release from the infected cells.

Cloning and expression of human lymphotoxin mRNA derived from a human T cell hybridoma.

We cloned human lymphotoxin (LT) cDNA from a cDNA library prepared from phorbol myristate acetate (PMA) and Con A-stimulated human T cell hybridoma (AC5-8) cells, The nucleotide sequence of the cDNA insert in the plasmid, pLT13, was determined and compared with those of peripheral blood mononuclear cell derived LT cDNA and the LT gene. Four stretches, containing 14 nucleotides in total, were different among the three sequences. The differences included one base change from C to A in the coding region. Because this change was expected to result in a change in the amino acid at position 26 from Thr to Asn, we constructed an E. coli expression plasmid (pLT13tac6-8.2) and a mammalian cell expression plasmid (pSV2-LT) in order to confirm that pLT13 contains the coding sequence of human LT. Both plasmids were found to synthesize active LT molecules after transfection into JM105 and COS-1 cells, respectively, and their lytic activities were found to be completely neutralized by anti human LT serum. Using an insertion mutant and a deletion mutant, we examined the role of the C terminal domain in the LT activity. The results obtained strongly suggested that the intactness of the C terminal less than 10 residues is required for the LT activity.

A unique phosphatidylinositol bearing a novel branched-chain fatty acid from Rhodococcus equi binds to influenza virus hemagglutinin and inhibits the infection of cells.

From the aquatic bacterium Rhodococcus equi strain S(420), we isolated a substance that strongly binds to influenza viruses. Structural analyses revealed that it is a unique type of phosphatidylinositol (PtdIns) bearing a branched-chain fatty acid (14-methyloctadecanoic acid). In a TLC/virus-binding immunostaining assay, this PtdIns bound to all subtypes of hemagglutinin (HA) of influenza A viruses tested, isolated from humans, ducks and swine, and also to human influenza B viruses. Furthermore, the PtdIns significantly prevented the infection of MDCK cells by influenza viruses, and also inhibited the virus-mediated hemagglutination and low pH-induced hemolysis of human erythrocytes, which represents the fusogenic activities of the viral HA. We also used purified hemagglutinin instead of virions to examine the interaction between viral HA and PtdIns, showing that the PtdIns binds to hemagglutinin. These findings indicate that the inhibitory mechanism of PtdIns on the influenza virus infection may be through its binding to viral HA spikes and host cell endosomal/lysosomal membranes, which are mediated by the function of viral HA.