Polyclonal human antibodies reduce bacterial attachment to soft contact lens and corneal cell surfaces.

Bacterial keratitis due to Pseudomonas aeruginosa is a potentially serious complication of extended-wear contact lens use. Adhesion of P. aeruginosa to soft contact lens materials or corneal endothelial cells in the presence of pooled human immunoglobulins and/or neutrophils in artificial tear fluid was studied in vitro as a potential method to treat contact lens-associated infection. Soft hydrophilic contact lens materials equilibrated in sterile saline were soaked in artificial tear fluid for 18 h prior to use. P. aeruginosa IFO 3455 was added to groups of lenses or confluent cultured bovine corneal endothelial cells with varying amounts of human polyclonal immunoglobulin (IgG) and human blood neutrophils or serum albumin as a control. After 2 or 4 h incubation, adherent viable bacteria on lenses were quantified. Fluorescence microscopy was used to assess bacterial adherence to bovine corneal endothelial cells in the presence and absence of IgG and neutrophils. Various concentrations of albumin had no effect on adhesion. Human immunoglobulin solutions (25 mg/ml) reduced P. aeruginosa adhesion by nearly 1 log and 2 logs after 2 and 4 h incubations, respectively. Neutrophils in combination with 25 mg/ml IgG reduced bacterial adhesion approximately 1 log over reduction in adhesion by neutrophils alone. Diluted human IgG (10 mg/ml) did not significantly decrease bacterial adhesion after 2 or 4 h, but did reduce adhesion in combination with human neutrophils at both time points. Similar reductions in amounts of fluorescently labeled bacteria adhered to cultured monolayers of corneal endothelial cells under these conditions were qualitatively observed.

Correlating fibronectin adsorption with endothelial cell adhesion and signaling on polymer substrates.

Fibronectin (Fn) adsorption was studied on different commercial polymer surface chemistries, including tissue culture polystyrene (TCPS), bacteriologic polystyrene (BPS), fluoropolymer Teflon AF, and poly-L-lactide (PLLA). Antibody probes detected the availability of Fn's cell binding domain on adsorbed Fn in the competitive presence and absence of bovine serum albumin (BSA). Domain availability was highest for Fn adsorbed on TCPS, especially in the presence of either serum albumin or dilute serum. Attachment and growth efficiencies for human umbilical venous endothelial cells (HUVECs) cultured on surfaces preadsorbed with Fn in serum and serum-free media correlated with antibody cell-binding domain availability: TCPS > BPS, Teflon AF > PLLA. Intracellular signaling from the GTPase, RhoA, was highest (RhoA:RhoGDI inhibitor ratio) in cells cultured on the Teflon AF surfaces, indicating that despite lower attached cell numbers on Teflon AF compared to TCPS, cell signaling remained activated after 24 h of growth. Up-regulated cellular Fn mRNA messages, assessed using RT-PCR techniques, supported HUVECs' producing the endogenous extracellular matrix (ECM) protein Fn in order to attach and survive on the suboptimal Teflon AF culture surfaces.

Mouse protein arrays from a TH1 cell cDNA library for antibody screening and serum profiling.

The mouse is the premier genetic model organism for the study of disease and development. We describe the establishment of a mouse T helper cell type 1 (T(H)1) protein expression library that provides direct access to thousands of recombinant mouse proteins, in particular those associated with immune responses. The advantage of a system based on the combination of large cDNA expression libraries with microarray technology is the direct connection of the DNA sequence information from a particular clone to its recombinant, expressed protein. We have generated a mouse T(H)1 expression cDNA library and used protein arrays of this library to characterize the specificity and cross-reactivity of antibodies. Additionally, we have profiled the autoantibody repertoire in serum of a mouse model for systemic lupus erythematosus on these protein arrays and validated the putative autoantigens on highly sensitive protein microarrays.

High throughput identification of potential Arabidopsis mitogen-activated protein kinases substrates.

Mitogen-activated protein kinase (MAPK) cascades are universal and highly conserved signal transduction modules in eucaryotes, including plants. These protein phosphorylation cascades link extracellular stimuli to a wide range of cellular responses. However, the underlying mechanisms are so far unknown as information about phosphorylation substrates of plant MAPKs is lacking. In this study we addressed the challenging task of identifying potential substrates for Arabidopsis thaliana mitogen-activated protein kinases MPK3 and MPK6, which are activated by many environmental stress factors. For this purpose, we developed a novel protein microarray-based proteomic method allowing high throughput study of protein phosphorylation. We generated protein microarrays including 1,690 Arabidopsis proteins, which were obtained from the expression of an almost nonredundant uniclone set derived from an inflorescence meristem cDNA expression library. Microarrays were incubated with MAPKs in the presence of radioactive ATP. Using a threshold-based quantification method to evaluate the microarray results, we were able to identify 48 potential substrates of MPK3 and 39 of MPK6. 26 of them are common for both kinases. One of the identified MPK6 substrates, 1-aminocyclopropane-1-carboxylic acid synthase-6, was just recently shown as the first plant MAPK substrate in vivo, demonstrating the potential of our method to identify substrates with physiological relevance. Furthermore we revealed transcription factors, transcription regulators, splicing factors, receptors, histones, and others as candidate substrates indicating that regulation in response to MAPK signaling is very complex and not restricted to the transcriptional level. Nearly all of the 48 potential MPK3 substrates were confirmed by other in vitro methods. As a whole, our approach makes it possible to shortlist candidate substrates of mitogen-activated protein kinases as well as those of other protein kinases for further analysis. Follow-up in vivo experiments are essential to evaluate their physiological relevance.