A clinical impact study of dermatologists' use of diagnostic gene expression profile testing to guide patient management.

Aim: Cutaneous melanocytic neoplasms with diagnostic and/or clinical ambiguity pose patient management challenges. Methods: Six randomized case scenarios with diagnostic/clinical uncertainty were described with/without a benign or malignant diagnostic gene expression profile (GEP) result. Results: Clinical impact was assessed by reporting the mean increase/decrease of management changes normalized to baseline (n = 32 dermatologists). Benign GEP results prompted clinicians to decrease surgical margins (84.2%). Malignant GEP results escalated surgical excision recommendations (100%). A majority (72.2%) reduced and nearly all (98.9%) increased follow-up frequency for benign or malignant GEP results, respectively. There was an overall increase in management plan confidence with GEP results. Conclusion: Diagnostic GEP tests help guide clinical decision-making in a variety of diagnostically ambiguous or clinicopathologically discordant scenarios.

Dermatologists' use of diagnostic gene expression profiles for personalized patient care. When your doctor takes a piece of a mole, that mole is looked at under the microscope by a pathologist. The pathologist is responsible for figuring out if the mole is dangerous or not. Dangerous moles are removed with surgery to make sure all the dangerous tissue is gone. Moles without a health threat are left alone. Sometimes figuring out how dangerous a mole is is difficult. The pathologist may not provide the doctor with enough information for them to know how to treat your mole. There is a test that can provide information on whether your mole is unsafe. This test is called diagnostic gene expression profiling or GEP. In this study, GEP is used to help doctors figure out how to treat a mole and how often the patient should be seen in the office for skin checks. With GEP, important changes in patient treatment were identified. These include the need for an additional surgery, how much healthy tissue should be removed during surgery and how often the patient should be seen in the office. For suspicious moles where the pathology report is unclear, GEP can provide information that leads to more appropriate and personalized patient care.

Ancillary diagnostic gene expression profile testing for ambiguous cutaneous melanocytic lesions helps optimize dermatologist recommendations for excision margin and follow-up.

An engineered alpha1 integrin-binding collagenous sequence.

Collagen is an extracellular matrix structural component that can regulate cellular processes through its interaction with the integrins, α1ß1, α2ß1, α10ß1, and α11ß1. Collagen-like proteins have been identified in a number of bacterial species. Here, we used Scl2 from Streptococcus pyogenes serotype M28 strain MGAS6274 as a backbone for the introduction of discrete integrin-binding sequences. The introduced sequences GLPGER, GFPGER, or GFPGEN did not affect triple helix stability of the Scl (Streptococcal collagen-like) protein. Using ELISA and surface plasmon resonance, we determined that Scl2(GLPGER) and Scl2(GFPGER) bound to recombinant human α1 and α2 I-domains in a metal ion-dependent manner and without a requirement for hydroxyproline. We predicted a novel and selective integrin-binding sequence, GFPGEN, through the use of computer modeling and demonstrated that Scl2(GFPGEN) shows specificity toward the α1 I-domain and does not bind the α2 I-domain. Using C2C12 cells, we determined that intact integrins interact with the modified Scl2 proteins with the same selectivity as recombinant I-domains. These modified Scl2 proteins also acted as cell attachment substrates for fibroblast, endothelial, and smooth muscle cells. However, the modified Scl2 proteins were unable to aggregate platelets. These results indicate that Scl2 is a suitable backbone for the introduction of mammalian integrin-binding sequences, and these sequences may be manipulated to individually target α1ß1 and α2ß1.

Potential dissemination of Bacillus anthracis utilizing human lung epithelial cells.

Dissemination of Bacillus anthracis spores from the lung is a critical early event in the establishment of inhalational anthrax. We recently reported that B. anthracis could adhere to and be internalized by cultured intestinal epithelial and fibroblast cells. Here, using gentamicin protection assays and/or electron microscopy, we found that Sterne strain 7702 spores were able to adhere to and subsequently be internalized by polarized A549 cells and primary human small airway epithelial cells. We showed for the first time that internalized spores were able to survive and that spores could translocate across an A549 cell barrier from the apical side to the basolateral side without disrupting the barrier integrity, suggesting a transcellular route. In addition, dormant spores of fully virulent Ames and UT500 strains were able to adhere to A549 cells at a frequency similar to that of 7702, whereas the capsule in germinated Ames and UT500 spores prevented adherence. Fluorescence microscopy also revealed that dormant Ames spores were internalized at a frequency similar to that of 7702. These findings highlight the possibility of a novel route of dissemination in which B. anthracis utilizes epithelial cells of the lung. The implications of these results to B. anthracis pathogenesis are discussed.

In vivo demonstration and quantification of intracellular Bacillus anthracis in lung epithelial cells.

Inhalational anthrax is initiated by the entry of Bacillus anthracis spores into the lung. A critical early event in the establishment of an infection is the dissemination of spores from the lung. Using in vitro cell culture assays, we previously demonstrated that B. anthracis spores are capable of entering into epithelial cells of the lung and crossing a barrier of lung epithelial cells without apparent disruption of the barrier integrity, suggesting a novel portal for spores to disseminate from the lung. However, in vivo evidence for spore uptake by epithelial cells has been lacking. Here, using a mouse model, we present evidence that B. anthracis spores are taken up by lung epithelial cells in vivo soon after spores are delivered into the lung. Immunofluorescence staining of thin sections of lungs from spore-challenged BALB/c mice revealed that spores were associated with the epithelial surfaces in the airway and the alveoli at 2 and 4 h postinoculation. Confocal analysis further indicated that some of the associated spores were surrounded by F-actin, demonstrating intracellular localization. These observations were further confirmed and substantiated by a quantitative method that first isolated lung cells from spore-challenged mice and then stained these cells with antibodies specific for epithelial cells and spores. The results showed that substantial amounts of spores were taken up by lung epithelial cells in vivo. These data, combined with those in our previous reports, provided powerful evidence that the lung epithelia were directly targeted by B. anthracis spores at early stages of infection.

A multifunctional streptococcal collagen-mimetic protein coating prevents bacterial adhesion and promotes osteoid formation on titanium.

The major barriers to the clinical success of orthopedic and dental implants are poor integration of fixtures with bone tissue and biomaterial-associated infections. Although multifunctional device coatings have long been considered a promising strategy, their development is hindered by difficulties in integrating biocompatibility, anti-infective activity and antithrombotic properties within a single grafting agent. In this study, we used cell adhesion assays and confocal microscopy of primary murine osteoblasts and human osteoblast cell lines MG-63 and Saos-2 to demonstrate that a streptococcal collagen-like protein engineered to display the α1 and α2 integrin recognition sequences enhances osteoblast adhesion and spreading on titanium fixtures. By measuring calcium deposition and alkaline phosphatase activity, we also showed that selective activation of α2ß1 integrin induces osteoblast differentiation, osteoid formation and mineralization. Moreover, cell adhesion assays and scanning electron microscopy of clinical isolates Staphylococcus aureus Philips and Staphylococcus epidermidis 9491 indicated that streptococcal collagen-mimetic proteins inhibit bacterial colonization and biofilm formation irrespective of their interaction with integrins. Given that streptococcal collagenous substrates neither interact with platelets nor trigger a strong immune response, this novel bioactive coating appears to have desirable multifaceted properties with promising translational applications.

Augmenting the articular cartilage-implant interface: Functionalizing with a collagen adhesion protein.

The lack of integration between implants and articular cartilage is an unsolved problem that negatively impacts the development of treatments for focal cartilage defects. Many approaches attempt to increase the number of matrix-producing cells that can migrate to the interface, which may help to reinforce the boundary over time but does not address the problems associated with an initially unstable interface. The objective of this study was to develop a bioadhesive implant to create an immediate bond with the extracellular matrix components of articular cartilage. We hypothesized that implant-bound collagen adhesion protein (CNA) would increase the interfacial strength between a poly(vinly alcohol) implant and an articular cartilage immediately after implantation, without preventing cell migration into the implant. By way of a series of in vitro immunohistochemical and mechanical experiments, we demonstrated that (i) free CNA can bind to articular cartilage, (ii) implant-bound CNA can bind to collagen type II and (iii) implants functionalized with CNA result in a fourfold increase in interfacial strength with cartilage relative to untreated implants at day zero. Of note, the interfacial strength significantly decreased after 21 days in culture, which may be an indication that the protein itself has lost its effectiveness. Our data suggest that functionalizing scaffolds with CNA may be a viable approach toward creating an initially stable interface between scaffolds and articular cartilage. Further efforts are required to ensure long-term interface stability.

Bacillus anthracis spore entry into epithelial cells is an actin-dependent process requiring c-Src and PI3K.

Dissemination of Bacillus anthracis from the respiratory mucosa is a critical step in the establishment of inhalational anthrax. Recent in vitro and in vivo studies indicated that this organism was able to penetrate the lung epithelium by directly entering into epithelial cells of the lung; however the molecular details of B. anthracis breaching the epithelium were lacking. Here, using a combination of pharmacological inhibitors, dominant negative mutants, and colocalization experiments, we demonstrated that internalization of spores by epithelial cells was actin-dependent and was mediated by the Rho-family GTPase Cdc42 but not RhoA or Rac1. Phosphatidylinositol 3-kinase (PI3K) activity was also required as indicated by the inhibitory effects of PI3K inhibitors, wortmannin and LY294002, and a PI3K dominant negative (DN) mutant Deltap85alpha. In addition, spore entry into epithelial cells (but not into macrophages) required the activity of Src as indicated by the inhibitory effect of Src family kinase (SFK) inhibitors, PP2 and SU6656, and specific siRNA knockdown of Src. Enrichment of PI3K and F-actin around spore attachment sites was observed and was significantly reduced by treatment with SFK and PI3K inhibitors, respectively. Moreover, B. anthracis translocation through cultured lung epithelial cells was significantly impaired by SFK inhibitors, suggesting that this signaling pathway is important for bacterial dissemination. The effect of the inhibitor on dissemination in vivo was then evaluated. SU6656 treatment of mice significantly reduced B. anthracis dissemination from the lung to distal organs and prolonged the median survival time of mice compared to the untreated control group. Together these results described a signaling pathway specifically required for spore entry into epithelial cells and provided evidence suggesting that this pathway is important for dissemination and virulence in vivo.

Bacillus anthracis internalization by human fibroblasts and epithelial cells.

The current model for Bacillus anthracis dissemination in vivo focuses on macrophages as carriers. However, recent evidence suggested that other host cells may also play a role in the process. Here, we tested the possibility of B. anthracis being internalized by a human fibroblast cell line, HT1080 and an epithelial cell line, Caco-2. A combination of gentamicin protection assays, scanning and transmission electron microscopy (EM) and fluorescence microscopy was used. The results demonstrated for the first time that both spores and vegetative cells of B. anthracis Sterne strain 7702 were able to adhere to and be internalized by cultured HT1080 and Caco-2 cells. Spore adherence to and internalization by HT1080 cells were not affected by a germination inhibitor. This suggested that certain features on dormant spores were sufficient for these processes. Vegetative cell adherence to and internalization by both cell lines were growth phase-dependent. EM images suggested that vegetative cells may have the ability to escape phagocytic vacuoles. Finally, we showed that internalization of both spores and vegetative cells required active functions of the host cell cytoskeleton. These results raised the possibility that B. anthracis may disseminate in vivo by directly infecting non-phagocytic cells.