Molluscum contagiosum: immunomorphological aspects of keratinocytes markers of differentiation and adhesion

Background Molluscum contagiosum (MC) is a Molluscipox virus infection of keratinocytes with hyperplasia and intracytoplasmic inclusions-the molluscum bodies (MBs). Few papers address cytokeratins (K) profile in MC, mainly focusing terminal keratinization process. Methods Forty-one MC lesions were subjected to immunohistochemical technique to verify K1, K10, K14, K16, involucrin, filaggrin, E-cadherin and p63 expression. MC immunolabeling pattern was compared to adjacent normal appearing epidermis (ANAE). Results In MC and ANAE, K1/K10 were expressed in suprabasal layers, K14 was expressed in basal and suprabasal layers and K16 was expressed through all spinous layer. Involucrin and filaggrin were observed in granular, spinous and in basal layer of ANAE and MC. E-cadherin was present up to the first layers of MC while ANAE exhibited E-cadherin labeling at basal and spinous layers. Basal and spinous layers keratinocytes nuclei, in both MC and ANAE, express p63. Conclusion Infection by Molluscipox virus alters keratinocyte differentiation status. The presence of K14 and p63 in spinous layer, as well as early expression of involucrin and filaggrin, associated to a hyperproliferative state disclosed by K16 expression, may be a result of disruption in keratinocytes maturation process. The changes observed at ANAE may represent early events in keratinization disturbance. Callegaro CF, Sotto MN. Molluscum contagiosum: immunomorphological aspects of keratinocytes markers of differentiation and adhesion.

Autopathogenic T helper cell type 1 (Th1) and protective Th2 clones differ in their recognition of the autoantigenic peptide of myelin proteolipid protein

We previously generated a panel of T helper cell 1 (Th1) clones specific for an encephalitogenic peptide of myelin proteolipid protein (PLP) peptide 139-151 (HSLGKWLGHPDKF) that induces experimental autoimmune encephalomyelitis (EAE) upon adoptive transfer. In spite of the differences in their T cell receptor (TCR) gene usage, all these Th1 clones required W144 as the primary and most critical TCR contact residue for the activation. In this study, we determined the TCR contact residues of a panel of Th2/Th0 clones specific for the PLP peptide 139-151 generated either by immunization with the PLP 139-151 peptide with anti-B7-1 antibody or by immunization with an altered peptide Q144. Using alanine-substituted peptide analogues of the native PLP peptide, we show that the Th2 clones have shifted their primary contact residue to the NH2-terminal end of the peptide. These Th2 cells do not show any dependence on the W144, but show a critical requirement for L141/G142 as their major TCR contact residue. Thus, in contrast with the Th1 clones that did not proliferate to A144-substituted peptide, the Th2 clones tolerated a substitution at position 144 and proliferated to A144 peptide. This alternative A144 reactive repertoire appears to have a critical role in the regulation of autoimmune response to PLP 139-151 because preimmunization with A144 to expand the L141/G142-reactive repertoire protects mice from developing EAE induced with the native PLP 139-151 peptide. These data suggest that a balance between two different T cell repertoires specific for same autoantigenic epitope can determine disease phenotype, i.e., resistance or susceptibility to an autoimmune disease.

Conformational analysis of LYS(11-36), a peptide derived from the beta-sheet region of T4 lysozyme, in TFE and SDS

The solution conformation of a peptide LYS(11-36), which corresponds to the beta-sheet region in T4 lysozyme, has been examined in aqueous solution, TFE, and SDS micelles by CD and H-1 NMR spectroscopy. Secondary structure predictions suggest some beta-sheet and turn character in aqueous solution but predict a helical conformation in a more hydrophobic environment. The predictions were supported by the CD and NMR studies which showed the peptide to be relatively unstructured in aqueous solution, although there was some evidence of a beta-turn conformer which was maintained in 200 mM SDS and, to a lesser extent, in 50% TFE. The peptide was significantly helical in the presence of either 50% TFE or 200 mM SDS. TFE and SDS titrations showed that the peptide could form helical, sheet, or extended structure depending on the TFE or SDS concentration. The studies indicate that peptide environment is the determining factor in secondary structure adopted by LYS(11-36).

Structural characterisation of galactoglucomannan secreted by suspension-cultured cells of Nicotiana plumbaginifolia

Galactoglucomannan (GGM) from cultures of Nicotiana plumbaginifolia has Man:Glc:Gal:Ara:Xyl in 1.0:1.1:1.0:0.1:0.04 ratio. Linkage analysis contained 4- and 4,6-Manp, 4-Glcp, terminal Galp and 2-Galp, small amounts and terminal Arap and terminal Xylp, and similar to 0.03 mol acetyl per mol of glucosyl residue. Treatment with alpha- and beta-D-galactosidases showed that the majority of the side-chains were either single Galp-alpha-(1 --> residues or the disaccharide Galp-beta-(1 --> 2)-Galp-alpha-(1 --> linked to O-6 of the 4-Manp residues of the glucomannan backbone. Analysis of the oligosaccharides generated by endo-(1 --> 4)-beta-mannanase digestion confirmed that the GGM comprises a backbone of predominantly alternating --> 4)-D-Manp-beta-(1 --> and --> Lt)-D-Glcp-beta-(1 --> branched at O-6 of 65% of the 4-Manp residues. The major oligosaccharide identified was D-Glcp-beta-(1 --> 4)-[D-Galp-beta-(1 --> 2)-D-Galp-alpha-(1 --> 6)]-D-Manp-beta-(1 --> 4)-D-Glcp-beta-(I --> 4)-[D-Galp-alpha-(1 --> 6)]-D-Manp-beta-(1 --> (27%), and most of the other oligosaccharides produced in significant quantities were based on this structure. (C) 1997 Elsevier Science Ltd.

Intestinal mucosa-associated microflora in ulcerative colitis patients before and after restorative proctocolectomy with an ileoanal pouch

PURPOSE: This study was designed to identify the mucosa-associated microflora in patients with severe ulcerative colitis before and after restorative proctocolectomy with ileoanal pouch construction in comparison with historic controls. METHODS: Ten patients with a diagnosis of ulcerative colitis were evaluated. Mucus was collected during colonoscopy from all segments of the colon and terminal ileum before surgery, and from the ileal pouch two and eight months after ileostomy closure. The prevalence and mean concentration of the mucosa-associated microflora were compared over time and with historic controls. RESULTS: Veillonella sp was the most prevalent bacterium in patients and controls. Klebsiella sp was significantly more prevalent in the ileum of controls, was not found in patients with ulcerative colitis, and after proctocolectomy returned to values found in controls. Some bacteria such as Enterobacter sp, Staphylococcus sp (coag-), Bacteroides sp (npg), Lactobacillus sp, and Veillonella sp had higher mean concentrations in the ileal pouch of patients after surgery than in controls. CONCLUSION: No bacterium was identified that could be exclusively responsible for the maintenance of the inflammatory process. The mucosa-associated microflora of patients with ulcerative colitis underwent significant changes after proctocolectomy with ileal pouch construction and returned to almost normal values for some bacteria.

Hepatitis D and B virus genotypes in chronically infected patients from the Eastern Amazon Basin

Hepatitis D virus (HDV) is a defective hepatotropic virus whose infectivity is dependent on hepatitis B virus (HBV). HDV super- or co-infiection leads to an increased risk of fulminant hepatitis or progression to severe chronic liver disease in HBV infected patients. The Brazilian Amazon Basin has been reported to be endemic for HBV and HDV, especially in the Western Amazon Basin. In this region, HDV infection is frequently associated with acute fulminant hepatitis with characteristic histologic features. HDV is classified into seven major clades (HDV-1 to HDV-7) and HBV is subdivided into eight genotypes (A-H). HDV and HBV genotypes have been shown to have a distinct geographic distribution. The aim of this study was to determine the HBV and HDV genotypes harbored by chronically infected patients from the Eastern Amazon Basin, Brazil. We studied 17 serum samples from HBV and HDV chronically infected patients admitted to a large public hospital (Santa Casa de Misericordia) at Belem, state of Para, Brazil, between 1994 and 2002. HDV-3 and HBV genotype A (subtype adw2) have been identified in all cases, in contrast to previous studies from other regions of the Amazon, where HBV genotype F has been found co-infecting patients that harbored HDV-3. The HDV-3/HBV-A co-infection suggests that there is not a specific interaction between HBV and HDV genotypes, and co-infection might merely reflect the most frequent genotypes found in a particular geographic area. The analysis of the carboxy-terminal region of the large hepatitis D antigen (L-HDAg), which interacts with the hepatitis B surface antigen (HBsAg) and is essential for HDV assembly, showed some diversity between the different isolates from the Eastern Amazon. This diversity is not observed among HDV-3 sequences from other South American regions. (C) 2008 Elsevier B.V. All rights reserved.

The structure of versutoxin (delta-atracotoxin-Hv1) provides insights into the binding of site 3 neurotoxins to the voltage-gated sodium channel

Background: Versutoxin (delta-ACTX-Hv1) is the major component of the venom of the Australian Blue Mountains funnel web spider, Hadronyche versuta. delta-ACTX-Hv1 produces potentially fatal neurotoxic symptoms in primates by slowing the inactivation of voltage-gated sodium channels; delta-ACTX-Hv1 is therefore a useful tool for studying sodium channel function. We have determined the three-dimensional structure of delta ACTX-Hv1 as the first step towards understanding the molecular basis of its interaction with these channels. Results: The solution structure of delta-ACTX-Hv1, determined using NMR spectroscopy, comprises a core beta region containing a triple-stranded antiparallel beta sheet, a thumb-like extension protruding from the beta region and a C-terminal 3(10) helix that is appended to the beta domain by virtue of a disulphide bond. The beta region contains a cystine knot motif similar to that seen in other neurotoxic polypeptides. The structure shows homology with mu-agatoxin-l, a spider toxin that also modifies the inactivation kinetics of vertebrate voltage-gated sodium channels. More surprisingly, delta-ACTX-Hv1 shows both sequence and structural homology with gurmarin, a plant polypeptide. This similarity leads us to suggest that the sweet-taste suppression elicited by gurmarin may result from an interaction with one of the downstream ion channels involved in sweet-taste transduction. Conclusions: delta-ACTX-Hv1 shows no structural homology with either sea anemone or alpha-scorpion toxins, both of which also modify the inactivation kinetics of voltage-gated sodium channels by interacting with channel recognition site 3. However, we have shown that delta-ACTX-Hv1 contains charged residues that are topologically related to those implicated in the binding of sea anemone and alpha-scorpion toxins to mammalian voltage-gated sodium channels, suggesting similarities in their mode of interaction with these channels.

Endomyocardial fibrosis: pathological and molecular findings of surgically resected ventricular endomyocardium

Endomyocardial fibrosis (EMF) is a restrictive cardiomyopathy of unknown etiology prevalent in tropical regions affecting the inflow tract and apex of one or both ventricles, which show fibrous thickening of the endocardium and adjacent myocardium. Surgical treatment is recommended for patients in functional classes III or IV (New York Heart Association). The gross and histological features of the heart have been comprehensively studied in autopsies, but studies in surgical samples are still lacking. Histological and immunohistochemical features of EMF in surgical samples collected from 32 patients were described and correlated with clinical data. Polymerase chain reaction (PCR) and reverse transcription-PCR, performed on formalin fixed endomyocardial samples, were used retrospectively to detect genomes of certain cardiotropic viruses and Toxoplasma gondii. Ventricular endocardium was thickened by superficial acellular hyaline collagen fibers type I and III, with predominance of the former type. Besides fibrosis, a chronic inflammatory process and an anomalous lymphatic rich vascular pattern were observed in the deep endocardium, connected to the terminal coronary circulation of the myocardium, which might be an important pathological finding concerning EMF pathogenesis. Molecular analysis of the endomyocardium revealed high incidence of cardiotropic infective agents (6/12, 50%); however, their role in the disease pathogenesis is still controversial.

Molecular basis of infantile reversible cytochrome c oxidase deficiency myopathy

Childhood-onset mitochondrial encephalomyopathies are usually severe, relentlessly progressive conditions that have a fatal outcome. However, a puzzling infantile disorder, long known as `benign cytochrome c oxidase deficiency myopathy` is an exception because it shows spontaneous recovery if infants survive the first months of life. Current investigations cannot distinguish those with a good prognosis from those with terminal disease, making it very difficult to decide when to continue intensive supportive care. Here we define the principal molecular basis of the disorder by identifying a maternally inherited, homoplasmic m.14674T > C mt-tRNA(Glu) mutation in 17 patients from 12 families. Our results provide functional evidence for the pathogenicity of the mutation and show that tissue-specific mechanisms downstream of tRNA(Glu) may explain the spontaneous recovery. This study provides the rationale for a simple genetic test to identify infants with mitochondrial myopathy and good prognosis.

Trypan blue staining for capsulorhexis: Ultrastructural effect on lens epithelial cells and capsules

PURPOSE: To evaluate the ultrastructural effect of trypan blue 0.1% staining for capsulorhexis on lens epithelial cells (LECs) and capsules SETTING: Division of Ophthalmology. University of Sao Paulo, Sao Paulo, Brazil METHODS: Before capsulorhexis, patients were randomly assigned to 1 of 2 groups Trypan blue 0 1% staining was performed in the treatment group No trypan blue was used in the control group Samples of capsules with LECs were fixed and analyzed with routine optical microscopy techniques. immunohistochemistry for beclin-1 expression (a marker of autophagy), terminal deoxynucleotidyl transf erase-mediated dUTP-biotin nick-end labeling to detect apoptosis, and transmission electron microscopy (TEM) Morphometric analyses were performed. and the 2 sets of data were compared. RESULTS: Each group comprised 15 patients Cell death by autophagy and apoptosis was observed in the treatment group but not in the control group The TEM images of subcapsular epithelium cells showed mitochondria` rupture, dilation of the cisterns of the endoplasmic reticulum, increased cytoplasmic and nuclear electron density, and abnormalities in the nuclear profile of trypan blue-stained cells. Morphometric analysis showed statistically significant differences between the 2 groups in the longest nuclear axes and the ratio between the total nuclear perimeter and the cell area (P = .03) The difference in capsule thickness between groups was not significant. CONCLUSION: Trypan blue caused LEC death, which supports the hypothesis that staining with trypan blue 0 1% can help reduce the incidence of posterior capsule pacification after cataract surgery

Early keratocyte apoptosis after epithelial scrape injury in the human cornea

Animal studies in mice, rats, rabbits, pigs and hens demonstrated that anterior keratocytes undergo programmed cell death or apoptosis after corneal epithelial injury. Many other wound healing changes subsequently follow the keratocyte apoptosis response. This study evaluated early keratocyte apoptosis after corneal epithelial scrape injury in human eyes scheduled for enucleation for malignancy. Two eyes had corneal epithelial scrape 1 h prior to the enucleation and another eye served as a control and had no corneal scrape prior to enucleation. One additional eye was enucleated, washed with balanced salt solution, and then had the corneal epithelium scraped 1 h prior to processing for analysis. Apoptosis was identified by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and confirmed by transmission electron microscopy (TEM). Anterior keratocyte apoptosis was detected in the three corneas that had epithelial scrape injury, but not in the control unwounded cornea. This study confirmed that keratocyte apoptosis is also an early response to corneal epithelial injury in humans and showed that tears are not essential for keratocyte apoptosis to occur in response to epithelial injury. (C) 2009 Elsevier Ltd. All rights reserved.

Inhibition of phospholipase A(2) increases Tau phosphorylation at Ser214 in embryonic rat hippocampal neurons

Background: Arachidonic acid is released from cellular membranes by the action of phospholipase A(2) (PLA(2)) and is implicated in microtubule-associated protein Tau phosphorylation. Tau hyperphosphorylation affects its ability to stabilize microtubules. Objective: To determine the effect of PLA(2) inhibition on the phosphorylation state of Tau phosphoepitopes in primary cultures of hippocampal neurons. Methods: 4 DIC neurons were incubated at different concentrations of methyl-arachidonylfluorophosphonate (MAFP), an irreversible inhibitor of cPLA(2) and iPLA(2). Changes on Tau phosphorylation were determined by Western blotting with a panel of anti-Tau antibodies (C-terminal, Ser199/202, Ser202/205, Ser396 and Ser214). Results: The Ser214 site was hyperphosphorylated upon MAFP treatment. Significant differences were observed with 10 mu M (p = 0.01), 50 mu M (p = 0.01) and 100 mu M (p = 0.05) of MAFP. Less-intense changes were found in other phosphoepitopes. Conclusion: The present findings indicate that the phosphorylation of Ser214 is regulated by c- and/or iPLA(2), whereas other phosphoepitopes primarily regulated by GKS3b were not affected. (C) 2009 Elsevier Ltd. All rights reserved.

Characterization of leptospiral proteins that afford partial protection in hamsters against lethal challenge with Leptospira interrogans

Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira. The whole-genome sequence of Leptospira interrogans serovar Copenhageni together with bioinformatic tools allow us to search for novel antigen candidates suitable for improved vaccines against leptospirosis. This study focused on three genes encoding conserved hypothetical proteins predicted to be exported to the outer membrane. The genes were amplified by PCR from six predominant pathogenic serovars in Brazil. The genes were cloned and expressed in Escherichia coli strain BL21-SI using the expression vector pDEST17. The recombinant proteins tagged with N-terminal 6xHis were purified by metal-charged chromatography. The proteins were recognized by antibodies present in sera from hamsters that were experimentally infected. Immunization of hamsters followed by challenge with a lethal dose of a virulent strain of Leptospira showed that the recombinant protein rLIC12730 afforded statistically significant protection to animals (44 %), followed by rLIC10494 (40 %) and rLIC12922 (30 %). Immunization with these proteins produced an increase in antibody titres during subsequent boosters, suggesting the involvement of a T-helper 2 response. Although more studies are needed, these data suggest that rLIC12730 and rLIC10494 are promising candidates for a multivalent vaccine for the prevention of leptospirosis.

Impact of obesity on airway and lung parenchyma remodeling in experimental chronic allergic asthma

The impact of obesity on the inflammatory process has been described in asthma, however little is known about the influence of diet-induced obesity on lung remodeling. For this purpose, 56 recently weaned A/J mice were randomly divided into 2 groups. In the C group, mice were fed a standard chow diet, while OB animals received isocaloric high-fat diet to reach 1.5 of the mean body weight of C. After 12 weeks, each group was further randomized to be sensitized and challenged with ovalbumin (OVA) or saline. Twenty-four hours after the last challenge, collagen fiber content in airways and lung parenchyma, the volume proportion of smooth muscle-specific actin in alveolar ducts and terminal bronchiole, and the number of eosinophils in bronchoalveolar lavage fluid were higher in OB-OVA than C-OVA. In conclusion, diet-induced obesity enhanced lung remodeling resulting in higher airway responsiveness in the present experimental chronic allergic asthma. (C) 2011 Elsevier B.V. All rights reserved.

Mutations in NOTCH2 cause Hajdu-Cheney syndrome, a disorder of severe and progressive bone loss

We used an exome-sequencing strategy and identified an allelic series of NOTCH2 mutations in Hajdu-Cheney syndrome, an autosomal dominant multisystem disorder characterized by severe and progressive bone loss. The Hajdu-Cheney syndrome mutations are predicted to lead to the premature truncation of NOTCH2 with either disruption or loss of the C-terminal proline-glutamate-serine-threonine-rich proteolytic recognition sequence, the absence of which has previously been shown to increase Notch signaling.