Prachi Stafford

HPA-1a antibody potency and bioactivity do not predict severity of fetomaternal alloimmune thrombocytopenia

BACKGROUND: The antenatal management of fetomaternal alloimmune thrombocytopenia (FMAIT) due to HPA‐1a antibodies remains controversial, and a test identifying pregnancies that do not require therapy would be of clinical value.

Immunologic and structural analysis of eight novel domain-deletion beta3 integrin peptides designed for detection of HPA-1 antibodies.

Summary.  Background: The single‐nucleotide polymorphism (SNP) rs5918 in the ITGB3 gene defines the human platelet antigen‐1 (HPA‐1) system encoding a Leu (HPA‐1a) or Pro (HPA‐1b) at position 33. HPA‐1 antibodies are clinically the most relevant in the Caucasoid population, but detection currently requires αIIbβ3 integrin from the platelets of HPA‐genotyped donors.Objectives: We set out to define the β3 integrin domains required for HPA‐1a antibody binding and produce recombinant soluble β3 peptides for HPA‐1 antibody detection.Methods: We designed two sets (1a and 1b) of four soluble β3 domain‐deletion peptides (ΔSDL, ΔβA, PSIHybrid, PSI), informed by crystallography studies and computer modeling. The footprints of three human HPA‐1a‐specific phage antibodies were defined by analyzing binding patterns to the β3 peptides and canine platelets, and models of antibody–antigen interfaces were derived. Specificity and sensitivity for HPA‐1a detection were assessed using sera from 140 cases of fetomaternal alloimmune thrombocytopenia (FMAIT). Results: Fusion of recombinant proteins to calmodulin resulted in high‐level expression in Drosophila S2 cells of all eight β3 peptides. Testing of FMAIT samples indicated that ΔβA‐Leu33 is the superior peptide for HPA‐1a antibody detection, with 96% sensitivity and 95% specificity. The existence of type I and II categories of HPA‐1a antibodies was confirmed by the study of HPA‐1a phage antibody footprints and the reactivity pattern of clinical samples with the four β3‐Leu33 peptides, but there was no correlation between antibody category and clinical severity of FMAIT. Conclusions: Soluble recombinant β3 peptides can be used for detection of clinical HPA‐1a antibodies.

A single-nucleotide polymorphism in the human ITGB3 gene is associated with the platelet-specific alloantigen Vaa (HPA-17bw) involved in fetal maternal alloimmune thrombocytopenia

BACKGROUND: The previously reported platelet (PLT)‐specific antigen, Vaa, was defined by an alloantibody detected in the serum sample of a mother who delivered an infant displaying symptoms of severe fetal maternal alloimmune thrombocytopenia (FMAIT). This PLT antigen was localized to the integrin αIIbβ3 (GPIIbIIIa) but its genetic basis was not defined.

Gingipain-dependent degradation of mammalian target of rapamycin pathway proteins by the periodontal pathogen Porphyromonas gingivalis during invasion.

Porphyromonas gingivalis and Tannerella forsythia are gram-negative pathogens strongly associated with periodontitis. Their abilities to interact, invade and persist within host cells are considered crucial to their pathogenicity, but the mechanisms by which they subvert host defences are not well understood. In this study, we set out to investigate whether P. gingivalis and T. forsythia directly target key signalling molecules that may modulate the host cell phenotype to favour invasion and persistence. Our data identify, for the first time, that P. gingivalis, but not T. forsythia, reduces levels of intracellular mammalian target of rapamycin (mTOR) in oral epithelial cells following invasion over a 4-h time course, via the action of gingipains. The ability of cytochalasin D to abrogate P. gingivalis-mediated mTOR degradation suggests that this effect is dependent upon cellular invasion. We also show that levels of several other proteins in the mTOR signalling pathway are modulated by gingipains, either directly or as a consequence of mTOR degradation including p-4E-BP1. Taken together, our data suggest that P. gingivalis manipulates the mTOR pathway, providing evidence for a potentially novel mechanism by which P. gingivalis mediates its effects on host cell responses to infection.

The roles of HOXD10 in the development and progression of head and neck squamous cell carcinoma (HNSCC)

Background:HOX gene expression is altered in many cancers; previous microarray revealed changes in HOX gene expression in head and neck squamous cell carcinoma (HNSCC), particularly HOXD10.Methods:HOXD10 expression was assessed by qPCR and immunoblotting in vitro and by immunohistochemistry (IHC) in tissues. Low-expressing cells were stably transfected with HOXD10 and the phenotype assessed with MTS, migration and adhesion assays and compared with the effects of siRNA knockdown in high-HOXD10-expressing cells. Novel HOXD10 targets were identified using expression microarrays, confirmed by reporter assay, and validated in tissues using IHC.Results:HOXD10 expression was low in NOKs, high in most primary tumour cells, and low in lymph node metastasis cells, a pattern confirmed using IHC in tissues. Overexpression of HOXD10 decreased cell invasion but increased proliferation, adhesion and migration, with knockdown causing reciprocal effects. There was no consistent effect on apoptosis. Microarray analysis identified several putative HOXD10-responsive genes, including angiomotin (AMOT-p80) and miR-146a. These were confirmed as HOXD10 targets by reporter assay. Manipulation of AMOT-p80 expression resulted in phenotypic changes similar to those on manipulation of HOXD10 expression.Conclusions:HOXD10 expression varies by stage of disease and produces differential effects: high expression giving cancer cells a proliferative and migratory advantage, and low expression may support invasion/metastasis, in part, by modulating AMOT-p80 levels.

Beta‐hexosaminidase activity of the oral pathogen Tannerella forsythia influences biofilm formation on glycoprotein substrates

Tannerella forsythia is an important pathogen in periodontal disease. Previously, we showed that its sialidase activity is key to utilization of sialic acid from a range of human glycoproteins for biofilm growth and initial adhesion. Removal of terminal sialic acid residues often exposes β-linked glucosamine or galactosamine, which may also be important adhesive molecules. In turn, these residues are often removed by a group of enzymes known as β-hexosaminidases. We show here that T. forsythia has the ability to cleave glucosamine and galactosamine from model substrates and that this activity can be inhibited by the hexosaminidase inhibitor PugNAc (O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino N-phenyl carbamate). We now demonstrate for the first time that β-hexosaminidase activity plays a role in biofilm growth on glycoprotein-coated surfaces because biofilm growth and initial cell adhesion are inhibited by PugNAc. In contrast, adhesion to siallo-glycoprotein-coated surfaces is unaltered by PugNAc in the absence of sialidase activity (using a sialidase-deficient mutant) or surprisingly on the clinically relevant substrates saliva or serum. These data indicate that β-hexosaminidase activity has a significant role in biofilm formation in combination with sialidase activity in the biofilm lifestyle of T. forsythia.

Role of OmpA2 surface regions of Porphyromonas gingivalis in host–pathogen interactions with oral epithelial cells

Outer membrane protein A (OmpA) is a key outer membrane protein found in Gram‐negative bacteria that contributes to several crucial processes in bacterial virulence. In Porphyromonas gingivalis, OmpA is predicted as a heterotrimer of OmpA1 and OmpA2 subunits encoded by adjacent genes. Here we describe the role of OmpA and its individual subunits in the interaction of P. gingivalis with oral cells. Using knockout mutagenesis, we show that OmpA2 plays a significant role in biofilm formation and interaction with human epithelial cells. We used protein structure prediction software to identify extracellular loops of OmpA2, and determined these are involved in interactions with epithelial cells as evidenced by inhibition of adherence and invasion of P. gingivalis by synthetic extracellular loop peptides and the ability of the peptides to mediate interaction of latex beads with human cells. In particular, we observe that OmpA2‐loop 4 plays an important role in the interaction with host cells. These data demonstrate for the first time the important role of P. gingivalis OmpA2 extracellular loops in interaction with epithelial cells, which may help design novel peptide‐based antimicrobial therapies for periodontal disease.

Molecular characterization of the variable domains of an αIIbβ3‐specific immunoglobulin M κ platelet cold agglutinin in a follicular lymphoma patient with treatment refractory autoimmune thrombocytopenia: idiotypic overlap between αIIbβ3 integrin antibodies

BACKGROUND: Cold hemagglutinins are generally immunoglobulin M (IgM) κ antibodies reactive at temperatures below 37°C and if of high titer may cause hemolysis. Platelet (PLT) cold agglutinins (CAs) are rare and poorly characterized. A detailed molecular characterization of the variable domains of a pathologic, PLT‐reactive, CA is presented.

Platelet immunology, present and future

The detection of antibodies against human platelet antigens (HPAs) is of clinical significance in neonatal alloimmune thrombocytopenia (NAIT), post-transfusion purpura (PTP) and platelet refractoriness (PR). Their rapid and sensitive detection and identification is imperative for good clinical care. This review is about HPA antibody detection and HPA genotyping, the possible value of HPA-1a antibody quantification to predict the severity of NAIT and the relation between clinical management and outcome. Finally new developments of recombinant HPA antigens for antibody detection and possible novel therapeutic strategies for severe NAIT are discussed. Molecular basis of HPAs

Platelet integrin α 2 I-domain specific antibodies produced via domain specific DNA vaccination combined with variable gene phage display

Antibodies are a powerful tool for structure/function studies of platelet proteins. However, classic immunisation frequently elicits antibody responses against domains of minor functional interest. Robust strategies to generate antibodies against defined domains would be of significant interest in post-genome research. In this study, we report a new strategy using a combination of DNA vaccination and V gene phage display that allows the rapid generation of domain specific single-chain Fv antibodies (scFvs). This system was validated using the I-domain of alpha2 integrin as a model. The alpha2beta1 integrin, which is expressed on many cell types, is the dominant collagen attachment receptor on platelets, functioning in close interplay with the collagen signalling receptor glycoprotein VI. A novel set of I-domain specific antibodies was obtained by a DNA vaccination/V gene repertoire cloning approach. Mice were first immunised with a DNA vaccine in which the alpha2 I-domain is expressed as a fusion protein with fragment C of tetanus toxoid (FrC-TT). Then the heavy and kappa light chain variable gene repertoires were rescued from immune splenocytes using antibody phage display. A total of four alpha2 I-domain specific scFvs were isolated by selection on recombinant I-domain or native platelet alpha2beta1 integrin. Characterisation of the scFvs indicated that they recognised distinct epitopes that had profound differences in accessibility between native and recombinant I-domain. Our data suggest DNA immunisation and phage display represent versatile alternatives to protein immunisation and hybridoma-fusion techniques for the isolation of recombinant antibody reagents. This approach will be particularly useful for the generation of domain or splice-variant specific antibodies that recognise native protein.