Humoral Autoimmune Responses to the Squamous Cell Carcinoma Antigen Protein Family in Psoriasis

Substantial evidence indicates that psoriasis is a T-lymphocyte-mediated autoimmune disease. However, longstanding data also indicate IgG and complement deposition in upper epidermis of psoriasis plaques. This led us to propose that autoantigen–autoantibody interactions in the skin may also be of pathogenic importance. Here, we have confirmed the presence of IgG in upper lesional epidermis and used high-resolution two-dimensional immunoblotting of extracts from this tissue, and laser desorption mass spectrometry of tryptic peptides, to define a series of epidermal proteins that bind IgG from psoriatic serum. The most prominent of these autoantigens are homologues of the serpin, squamous cell carcinoma antigen (SCCA), the other autoantigens identified including arginase 1, enolase 1, and keratin 10. Blood levels of IgG autoantibodies that bind to SCCA proteins were significantly higher in psoriasis than healthy controls (P=0.005), but were not detectable in sera from patients with active atopic dermatitis. To our knowledge, SCCA proteins have not previously been described as autoantigenic in animals or humans and form complexes with IgG that are associated with complement deposition. These findings expose potentially pathogenic humoral immunologic events and thus possible therapeutic targets in psoriasis

Pneumolysin Activates the NLRP3 Inflammasome and Promotes Proinflammatory Cytokines Independently of TLR4

Pneumolysin (PLY) is a key Streptococcus pneumoniae virulence factor and potential candidate for inclusion in pneumococcal subunit vaccines. Dendritic cells (DC) play a key role in the initiation and instruction of adaptive immunity, but the effects of PLY on DC have not been widely investigated. Endotoxin-free PLY enhanced costimulatory molecule expression on DC but did not induce cytokine secretion. These effects have functional significance as adoptive transfer of DC exposed to PLY and antigen resulted in stronger antigen-specific T cell proliferation than transfer of DC exposed to antigen alone. PLY synergized with TLR agonists to enhance secretion of the proinflammatory cytokines IL-12, IL-23, IL-6, IL-1β, IL-1α and TNF-α by DC and enhanced cytokines including IL-17A and IFN-γ by splenocytes. PLY-induced DC maturation and cytokine secretion by DC and splenocytes was TLR4-independent. Both IL-17A and IFN-γ are required for protective immunity to pneumococcal infection and intranasal infection of mice with PLY-deficient pneumococci induced significantly less IFN-γ and IL-17A in the lungs compared to infection with wild-type bacteria. IL-1β plays a key role in promoting IL-17A and was previously shown to mediate protection against pneumococcal infection. The enhancement of IL-1β secretion by whole live S. pneumoniae and by PLY in DC required NLRP3, identifying PLY as a novel NLRP3 inflammasome activator. Furthermore, NLRP3 was required for protective immunity against respiratory infection with S. pneumoniae. These results add significantly to our understanding of the interactions between PLY and the immune system

Relationship of structure to function in the pore-forming toxin Pneumolysin from Streptococcus pneumoniae

Pneumolysin is an important virulence factor produced by the human pathogen Streptococcus pneumoniae. It belongs to the family of cholesterol dependent cytolysins (CDCs) that damage the target cell membrane, by forming large oligomeric pores of 30 to 80 toxin monomers, where each monomer is thought to contribute at least two p-hairpins. A panel of mutations was done in the two putative transmembrane region of pneumolysin TMH1 and TMH2 located in domain 3 of the toxin monomer, and believed to be lining the pore lumen. The generated mutants exhibited different levels of haemolytic activity, particularly the single mutation W278F, W278D, and the triple mutation (D257N-E258Q-E260Q), largely impaired the haemolytic activity of the wild-type toxin. These mutant toxins along with a previously made lytic deficient mutant W433F were subjected to further studies. Circular dichroism analysis done with those mutants showed that the secondary structure of the native toxin was conserved. The kinetics of release of calcein from liposomes along with the kinetics of lysis of erythrocytes exposed to these mutants was substantially slower than that of the wild-type toxin. Pneumolysin and other CDCs induced pores were studied on model systems like lipid bilayer and liposomes. In this thesis, I demonstrated the formation of pores by pneumolysin on the membrane of a 'real' cell by using the patch-clamp technique. Pneumolysin induced heterogeneous pore on either side of the membrane, of different conductance states, classified as small, medium and large. A stepwise increase in current was observed with early appearance of small conductance channels followed by larger ones. The mutant toxins generated in this work and W433F were also tested with patch clamping. They formed pores of various conductance states with a decrease in the occurrence of large channels, in comparison to the wild-type

Pneumolysin generates multiple conductance pores in the membrane of nucleated cells

Pneumolysin generates multiple conductance pores in the membrane of nucleated cell

Increased blood levels of IgG reactive with secreted Streptococcus pyogenes proteins in chronic plaque psoriasis.

A pathogenic role for Streptococcus pyogenes infections in chronic plaque psoriasis is suspected but poorly defined. We separated cellular and supernatant proteins from S pyogenes cultures by high-resolution two-dimensional gel electrophoresis, and used immunoblotting to demonstrate the diversity of serum or plasma IgGs that react with elements of the proteome of this bacterium. We have shown that a substantial proportion of IgG-reactive proteins from cultured S pyogenes are secreted. The total secreted protein fraction, including diverse IgG-binding elements, was subsequently used in an ELISA to measure blood titers of reactive IgG. This ELISA showed that blood samples from patients with chronic plaque psoriasis contained significantly higher titers of reactive IgG than samples from age- and sex-matched healthy controls (p = 0.0009). In contrast, neither a standard assay measuring antistreptolysin O titers nor ELISAs measuring titers of IgG reactive with protein fractions from Staphylococcus aureus and Staphylococcus epidermidis, were able to distinguish between blood samples from the two groups. These findings justify the hypothesis that S pyogenes infections are more important in the pathogenesis of chronic plaque psoriasis than has previously been recognised, and indicate the need for further controlled therapeutic trials of antibacterial measures in this common skin disease