The OmpL37 Surface-Exposed Protein Is Expressed by Pathogenic Leptospira during Infection and Binds Skin and Vascular Elastin

Pathogenic Leptospira spp. shed in the urine of reservoir hosts into freshwater can be transmitted to a susceptible host through skin abrasions or mucous membranes causing leptospirosis. The infection process involves the ability of leptospires to adhere to cell surface and extracellular matrix components, a crucial step for dissemination and colonization of host tissues. Therefore, the elucidation of novel mediators of host-pathogen interaction is important in the discovery of virulence factors involved in the pathogenesis of leptospirosis. In this study, we assess the functional roles of transmembrane outer membrane proteins OmpL36 (LIC13166), OmpL37 (LIC12263), and OmpL47 (LIC13050), which we recently identified on the leptospiral surface. We determine the capacity of these proteins to bind to host tissue components by enzyme-linked immunosorbent assay. OmpL37 binds elastin preferentially, exhibiting dose-dependent, saturating binding to human skin (Kd, 104±19 nM) and aortic elastin (Kd, 152±27 nM). It also binds fibrinogen (Kd, 244±15 nM), fibrinogen fragment D (Kd, 132±30 nM), plasma fibronectin (Kd, 359±68 nM), and murine laminin (Kd, 410±81 nM). The binding to human skin elastin by both recombinant OmpL37 and live Leptospira interrogans is specifically enhanced by rabbit antiserum for OmpL37, suggesting the involvement of OmpL37 in leptospiral binding to elastin and also the possibility that host-generated antibodies may promote rather than inhibit the adherence of leptospires to elastin-rich tissues. Further, we demonstrate that OmpL37 is recognized by acute and convalescent leptospirosis patient sera and also by Leptospira-infected hamster sera. Finally, OmpL37 protein is detected in pathogenic Leptospira serovars and not in saprophytic Leptospira. Thus, OmpL37 is a novel elastin-binding protein of pathogenic Leptospira that may be promoting attachment of Leptospira to host tissues

Heterologous expression of pathogen-specific genes ligA and ligB in the saprophyte Leptospira biflexa confers enhanced adhesion to cultured cells and fibronectin

BACKGROUND: In comparison to other bacterial pathogens, our knowledge of the molecular basis of the pathogenesis of leptospirosis is extremely limited. An improved understanding of leptospiral pathogenetic mechanisms requires reliable tools for functional genetic analysis. Leptospiral immunoglobulin-like (Lig) proteins are surface proteins found in pathogenic Leptospira, but not in saprophytes. Here, we describe a system for heterologous expression of the Leptospira interrogans genes ligA and ligB in the saprophyte Leptospira biflexa serovar Patoc. RESULTS: The genes encoding LigA and LigB under the control of a constitutive spirochaetal promoter were inserted into the L. biflexa replicative plasmid. We were able to demonstrate expression and surface localization of LigA and LigB in L. biflexa. We found that the expression of the lig genes significantly enhanced the ability of transformed L. biflexa to adhere in vitro to extracellular matrix components and cultured cells, suggesting the involvement of Lig proteins in cell adhesion. CONCLUSIONS: This work reports a complete description of the system we have developed for heterologous expression of pathogen-specific proteins in the saprophytic L. biflexa. We show that expression of LigA and LigB proteins from the pathogen confers a virulence-associated phenotype on L. biflexa, namely adhesion to eukaryotic cells and fibronectin in vitro. This study indicates that L. biflexa can serve as a surrogate host to characterize the role of key virulence factors of the causative agent of leptospirosis

Leptospira interrogans Endostatin-Like Outer Membrane Proteins Bind Host Fibronectin, Laminin and Regulators of Complement

The pathogenic spirochete Leptospira interrogans disseminates throughout its hosts via the bloodstream, then invades and colonizes a variety of host tissues. Infectious leptospires are resistant to killing by their hosts' alternative pathway of complement-mediated killing, and interact with various host extracellular matrix (ECM) components. The LenA outer surface protein (formerly called LfhA and Lsa24) was previously shown to bind the host ECM component laminin and the complement regulators factor H and factor H-related protein-1. We now demonstrate that infectious L. interrogans contain five additional paralogs of lenA, which we designated lenB, lenC, lenD, lenE and lenF. All six genes encode domains predicted to bear structural and functional similarities with mammalian endostatins. Sequence analyses of genes from seven infectious L. interrogans serovars indicated development of sequence diversity through recombination and intragenic duplication. LenB was found to bind human factor H, and all of the newly-described Len proteins bound laminin. In addition, LenB, LenC, LenD, LenE and LenF all exhibited affinities for fibronectin, a distinct host extracellular matrix protein. These characteristics suggest that Len proteins together facilitate invasion and colonization of host tissues, and protect against host immune responses during mammalian infection

A Randomized Double-Blind Crossover Study of Phase-Shift Sound Therapy for Tinnitus

Objective. The purpose of this study was to compare the efficacy of the treatment of tinnitus with a phase-shifting pure tone to that of the same tone treatment without phase shifting. Study Design. A double-blind crossover randomized controlled trial. Setting. This study was conducted at the University Medical Center Groningen. Subjects and Methods. Twenty-two patients with predominantly tonal tinnitus underwent both intervention and control treatments. Each treatment consisted of three 30-minute sessions in 1 week. The control treatment was identical to the intervention treatment, except that the stimulus was a pure tone without phase shifting. Questionnaires, tinnitus loudness match, and annoyance and loudness ratings were used to measure treatment effects. Results. Pure-tone treatment and phase-shift treatment had no significant effect on tinnitus according to questionnaires (Tinnitus Handicap Index, Tinnitus Reaction Questionnaire, Hospital Anxiety and Depression Scale, and Maastricht Questionnaire), audiological matching procedures, and loudness and annoyance ratings of tinnitus. Furthermore, phase-shift treatment showed no additional significant improvement in comparison with pure-tone treatment. Changes in questionnaire scores due to pure-tone and the phase-shift treatment were correlated. Conclusion. On average across the group, both treatments failed to demonstrate a significant effect. Both treatments were beneficial for some patients. However, a positive effect was not demonstrated that could be attributed to the periodic shifting of the phase of the stimulus tone

A LigA Three-Domain Region Protects Hamsters from Lethal Infection by Leptospira interrogans

The leptospiral LigA protein consists of 13 bacterial immunoglobulin-like (Big) domains and is the only purified recombinant subunit vaccine that has been demonstrated to protect against lethal challenge by a clinical isolate of Leptospira interrogans in the hamster model of leptospirosis. We determined the minimum number and location of LigA domains required for immunoprotection. Immunization with domains 11 and 12 was found to be required but insufficient for protection. Inclusion of a third domain, either 10 or 13, was required for 100% survival after intraperitoneal challenge with Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130. As in previous studies, survivors had renal colonization; here, we quantitated the leptospiral burden by qPCR to be 1.2×103 to 8×105 copies of leptospiral DNA per microgram of kidney DNA. Although renal histopathology in survivors revealed tubulointerstitial changes indicating an inflammatory response to the infection, blood chemistry analysis indicated that renal function was normal. These studies define the Big domains of LigA that account for its vaccine efficacy and highlight the need for additional strategies to achieve sterilizing immunity to protect the mammalian host from leptospiral infection and its consequences

Inflammatory cytokines and aromatase inhibitor-associated musculoskeletal syndrome: a case–control study

The aromatase inhibitor (AI)-associated musculoskeletal syndrome (AIMSS) occurs in approximately 50% of AI-treated patients. Inflammatory mediators are associated with oestrogen signalling and may change with oestrogen depletion. We hypothesised that AIMSS may be associated with changes in circulating inflammatory markers

ZNF93 Increases Resistance to ET-743 (Trabectedin; Yondelis®) and PM00104 (Zalypsis®) in Human Cancer Cell Lines

ET-743 (trabectedin, Yondelis) and PM00104 (Zalypsis) are marine derived compounds that have antitumor activity. ET-743 and PM00104 exposure over sustained periods of treatment will result in the development of drug resistance, but the mechanisms which lead to resistance are not yet understood.Human chondrosarcoma cell lines resistant to ET-743 (CS-1/ER) or PM00104 (CS-1/PR) were established in this study. The CS-1/ER and CS-1/PR exhibited cross resistance to cisplatin and methotrexate but not to doxorubicin. Human Affymetrix Gene Chip arrays were used to examine relative gene expression in these cell lines. We found that a large number of genes have altered expression levels in CS-1/ER and CS-1/PR when compared to the parental cell line. 595 CS-1/ER and 498 CS-1/PR genes were identified as overexpressing; 856 CS-1/ER and 874 CS-1/PR transcripts were identified as underexpressing. Three zinc finger protein (ZNF) genes were on the top 10 overexpressed genes list. These genes have not been previously associated with drug resistance in tumor cells. Differential expressions of ZNF93 and ZNF43 genes were confirmed in both CS-1/ER and CS-1/PR resistant cell lines by real-time RT-PCR. ZNF93 was overexpressed in two ET-743 resistant Ewing sarcoma cell lines as well as in a cisplatin resistant ovarian cancer cell line, but was not overexpressed in paclitaxel resistant cell lines. ZNF93 knockdown by siRNA in CS-1/ER and CS-1/PR caused increased sensitivity for ET-743, PM00104, and cisplatin. Furthermore, ZNF93 transfected CS-1 cells are relatively resistant to ET-743, PM00104 and cisplatin.This study suggests that zinc finger proteins, and ZNF93 in particular, are involved in resistance to ET-743 and PM00104

The Multifunctional LigB Adhesin Binds Homeostatic Proteins with Potential Roles in Cutaneous Infection by Pathogenic Leptospira interrogans

Leptospirosis is a potentially fatal zoonotic disease in humans and animals caused by pathogenic spirochetes, such as Leptospira interrogans. The mode of transmission is commonly limited to the exposure of mucous membrane or damaged skin to water contaminated by leptospires shed in the urine of carriers, such as rats. Infection occurs during seasonal flooding of impoverished tropical urban habitats with large rat populations, but also during recreational activity in open water, suggesting it is very efficient. LigA and LigB are surface localized proteins in pathogenic Leptospira strains with properties that could facilitate the infection of damaged skin. Their expression is rapidly induced by the increase in osmolarity encountered by leptospires upon transition from water to host. In addition, the immunoglobulin-like repeats of the Lig proteins bind proteins that mediate attachment to host tissue, such as fibronectin, fibrinogen, collagens, laminin, and elastin, some of which are important in cutaneous wound healing and repair. Hemostasis is critical in a fresh injury, where fibrinogen from damaged vasculature mediates coagulation. We show that fibrinogen binding by recombinant LigB inhibits fibrin formation, which could aid leptospiral entry into the circulation, dissemination, and further infection by impairing healing. LigB also binds fibroblast fibronectin and type III collagen, two proteins prevalent in wound repair, thus potentially enhancing leptospiral adhesion to skin openings. LigA or LigB expression by transformation of a nonpathogenic saprophyte, L. biflexa, enhances bacterial adhesion to fibrinogen. Our results suggest that by binding homeostatic proteins found in cutaneous wounds, LigB could facilitate leptospirosis transmission. Both fibronectin and fibrinogen binding have been mapped to an overlapping domain in LigB comprising repeats 9–11, with repeat 11 possibly enhancing binding by a conformational effect. Leptospirosis patient antibodies react with the LigB domain, suggesting applications in diagnosis and vaccines that are currently limited by the strain-specific leptospiral lipopolysaccharide coats

Compound Semiconductor Materials and Devices

Contains table of contents for Part I, table of contents for Section 1, an introduction, reports on fourteen research projects and a list of publications.Defense Advanced Research Projects Agency/National Center for Integrated Photonics TechnologyJoint Services Electronics Program Grant DAAH04-95-1-0038MIT Lincoln LaboratoryNational Science Foundation Graduate FellowshipU.S. Navy - Office of Naval ResearchAT&T Bell Laboratories FellowshipU.S. Army - Ft. MeadeNTT CorporationNational Science FoundationLockheed-Martin Corporatio

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy