Irmgard Behlau

PhoP/PhoQ transcriptional repression of Salmonella typhimurium invasion genes: evidence for a role in protein secretion.

Previously, the PhoP‐repressed locus prgH was identified as important for signalling epithelial cells to endocytose Salmonella typhimurium. Characterization of prgH revealed that it is an operon of four genes encoding polypeptides of 392 (prgH), 80 (prgl), 101 (prgJ) and 252 amino acid residues (prgK). Synthesis of the 2.6 kb prgHIJK transcript was repressed in bacteria that activate PhoP/PhoQ, indicating that PhoP/PhoQ regulates prgHIJK by transcriptional repression. The prgl, prgJ and prgK predicted gene products were similar to Shigella flexneri and Yersinia enterocolitica proteins required for secretion of lpa and Yop virulence factors. Analysis of the culture supernatants from wild‐type S. typhimurium demonstrated that at least 25 polypeptides larger than 14 kDa could be detected. In contrast, prgH1::TnphoA, phoP‐constitutive and hil‐deletion mutants had significant defects in their supernatant protein profiles. The invasion and supernatant protein profile defects of the prgH1::TnphoA mutant were both complemented by a 5.1 kb plasmid that included prgHIJK. These results suggest that PhoP/PhoQ regulates extracellular transport of proteins by transcriptional repression of secretion determinants and that secreted proteins may be involved in signalling epithelial cells to endocytose bacteria.

A drug-eluting contact lens.

PURPOSE To formulate and characterize a drug-eluting contact lens designed to provide extended, controlled release of a drug. METHODS Prototype contact lenses were created by coating PLGA (poly[lactic-co-glycolic acid]) films containing test compounds with pHEMA (poly[hydroxyethyl methacrylate]) by ultraviolet light polymerization. The films, containing encapsulated fluorescein or ciprofloxacin, were characterized by scanning electron microscopy. Release studies were conducted in phosphate-buffered saline at 37 degrees C with continuous shaking. Ciprofloxacin eluted from the contact lens was studied in an antimicrobial assay to verify antimicrobial effectiveness. RESULTS After a brief and minimal initial burst, the prototype contact lenses demonstrated controlled release of the molecules studied, with zero-order release kinetics under infinite sink conditions for over 4 weeks. The rate of drug release was controlled by changing either the ratio of drug to PLGA or the molecular mass of the PLGA used. Both the PLGA and the pHEMA affected release kinetics. Ciprofloxacin released from the contact lenses inhibited ciprofloxacin-sensitive Staphylococcus aureus at all time-points tested. CONCLUSIONS A prototype contact lens for sustained drug release consisting of a thin drug-PLGA film coated with pHEMA could be used as a platform for ocular drug delivery with widespread therapeutic applications.

Microbial biofilms in ophthalmology and infectious disease.

Most bacterial infections involve biofilms. Biofilms are collections of microorganisms encased in a matrix that is often composed of both bacterial and host materials. They form on natural surfaces such as heart valves or abiotic surfaces such as contact lenses or intraocular lenses. The biofilm matrix promotes adherence of the microbe to smooth surfaces as well as to other cells. Biofilms thereby form large 3-dimensional microbial communities of complex architecture through cell-to-cell communication and coordinated multicellular behavior. The biofilm architecture promotes the exchange of nutrients and waste products. The ability of microorganisms to attach to abiotic surfaces and grow in highly stable communities greatly confounds the medical use of implantable devices. Much effort is now being invested to understand the molecular nature of biofilms, with a view toward designing biofilm-resistant implantable devices and more effective antimicrobials.

Fungal keratitis: changing pathogens and risk factors.

Purpose: To describe changes in demographics and pathogens for fungal keratitis cases diagnosed at the Massachusetts Eye and Ear Infirmary. Methods: Patient demographics, clinical and laboratory findings, treatment and outcomes of 46 cases of culture-proven fungal keratitis diagnosed from January 2004 through November 2007 were compared with 23 cases of fungal keratitis previously collected over a similar period from January 1999 through November 2002. Results: During 2004-2007, the rate of fungal keratitis was 1.0 cases per month, an increase from the baseline rate of 0.5 cases per month during 1999-2002. The proportion of cases caused by filamentous fungi increased from 30% (1999-2002) to 65% (2004-2007) (P = 0.01). Soft contact lens wear accounted for 41% of fungal keratitis cases in 2004-2007, as compared with 17% in 1999-2002. The majority of patients (70%) received oral antifungal treatment in addition to topical amphotericin B and natamycin. Seventeen patients (40%) required therapeutic keratoplasty. Patients with a history of corneal transplant had the highest rate of therapeutic keratoplasties (67%) and had the poorest visual outcome (40% counting fingers or less). In the contact lens group, 94% of patients maintained vision of at least 20/40 and only 12% required surgery to control the infection. Conclusions: There has been an increase in fungal keratitis in the Boston area and a change in the causative pathogens and risk factors for infection. Filamentous fungi now account for the majority of fungal keratitis cases, whereas yeasts were the predominant pathogen in the past. Soft contact lens wear is currently the most common risk factor for development of fungal keratitis.

The PhoP virulence regulon and live oral Salmonella vaccines

The PhoP virulence regulon is essential to Salmonella typhimurium mouse typhoid fever pathogenesis and survival within macrophages. This virulence regulon is composed of the PhoP (transcriptional regulator) and PhoQ (environmental sensor) proteins and the genetic loci they positively (pags for PhoP activated genes) and negatively (prgs for PhoP repressed genes) regulate. Three regulated loci pagC, pagD, and prgH, when singly mutated, affect the virulence of S. typhimurium for mice. Strains with phoP locus mutations are effective as live vaccines in mice, and strains with a constitutive regulatory mutation, a point mutation in PhoQ, can protect mice against typhoid fever when only very few organisms are administered. The addition of various PhoP regulon mutations further attenuates aroA mutants of S. typhimurium, suggesting that these mutations would be useful in further attenuating vaccine strains with metabolic pathway mutations. The phoP, phoQ, pagC, and pagD genes are highly conserved between S. typhimurium and S. typhi and may be valuable as mutations in live vaccines for human typhoid fever. A plasmid suicide vector that allows deletion of the pagC gene and stable insertion of heterologous antigen genes within the deleted pagC locus has been constructed and used successfully in S. typhimurium and S. typhi.

Intraocular Pressure Measurement by Radio Wave Telemetry

PURPOSE To determine the biocompatibility of a new wireless intraocular pressure (IOP) transducer (WIT) in rabbit eyes and to correlate its measurements with other pressure-measuring devices. METHODS The WIT is a ring-shaped intraocular device that allows wireless IOP measurements through radiofrequency. It was implanted into six eyes of New Zealand White rabbits after extracapsular lens extraction. A sham rabbit eye with no transducer implanted was used as a control. The animals were observed and examined by microscopy at various intervals up to 25 months after surgery. IOP was measured at various intervals by pneumotonometry, tonometry, WIT, and manometry. The data from the various devices were compared and analyzed for reproducibility. Two eyes were enucleated at 5.5 and 20 months after implantation and analyzed by histology. RESULTS The WIT appears to be well tolerated in the rabbit eye, with no evidence of significant inflammation or scar formation by microscopic in vivo examination. Histology did not reveal intraocular inflammation or membrane formation. Repeated IOP measurements with pneumotonometry, tonometry, and the WIT resulted in SDs of 2.70 mm Hg, 3.35 mm Hg, and 0.81 mm Hg, respectively. The concordance between the WIT and direct manometry measurements was high. A downward drift in IOP measured by the WIT was noted in three rabbits, necessitating recalibration. CONCLUSIONS The WIT is well tolerated by the rabbit eye. Its measurements are reproducible and in close concordance with manometry. A downward IOP drift warrants further investigation.

Biocompatibility and biofilm inhibition of N,N-hexyl,methyl-polyethylenimine bonded to Boston Keratoprosthesis materials

The biocompatibility and antibacterial properties of N,N-hexyl,methyl-polyethylenimine (HMPEI) covalently attached to the Boston Keratoprosthesis (B-KPro) materials was evaluated. By means of confocal and electron microscopies, we observed that HMPEI-derivatized materials exert an inhibitory effect on biofilm formation by Staphylococcus aureus clinical isolates, as compared to the parent poly(methyl methacrylate) (PMMA) and titanium. There was no additional corneal epithelial cell cytotoxicity of HMPEI-coated PMMA compared to that of control PMMA in tissue cultures in vitro. Likewise, no toxicity or adverse reactivity was detected with HMPEI-derivatized PMMA or titanium compared to those of the control materials after intrastromal or anterior chamber implantation in rabbits in vivo.

Infectious endophthalmitis in Boston keratoprosthesis: incidence and prevention

To determine the cumulative worldwide incidence of infectious endophthalmitis and associated vision loss after Boston keratoprosthesis (B‐KPro) Type I/II implantation and to propose both safe and inexpensive prophylactic antibiotic regimens.

In Vitro and In Vivo Assessment of Titanium Surface Modification for Coloring the Backplate of the Boston Keratoprosthesis

PURPOSE Recent use of a titanium (Ti) backplate has improved the design and biocompatibility of the Boston Keratoprosthesis (BKpro). Titaniums shiny metallic appearance, however, makes the cosmetic outcome less favorable. The purpose of this study was to develop and test a coloring surface modification of Ti. METHODS Ti coloring was achieved using electrochemical anodization. Color assessment included scanning electron microscopy, atomic force microscopy (AFM), x-ray diffraction crystallography (XRD), and Fourier transform infrared spectroscopy (FTIR). Biocompatibility assessment of Ti disks included in vitro proliferation and cytotoxicity in coculture with human corneal limbal epithelial (HCLE) cells, primary human corneal fibroblasts, and immortalized human corneal endothelial cells (HCEnCs), and in vivo intralamellar implantation in rabbit corneas. Histologic appearance (hematoxylin-eosin and trichrome staining) and presence of cell inflammation (CD45), apoptosis (TUNEL), and corneal neovascularization (CD31) were evaluated 27 and 53 days post implantation. RESULTS Blue and brown coloration of Ti was achieved. Analysis showed the presence of a nanoporous oxide surface with no chemical change of the modified Ti surface. In vitro assessment showed no significant differences in cell proliferation and cytotoxicity between anodized and nonanodized Ti (P > 0.05; ANOVA for all cell types). Analysis of corneal tissues harboring the Ti disks showed normal cellular appearance, and lack of CD45, TUNEL, and CD31-positive cells. CONCLUSIONS A biocompatible Ti backplate coloring was achieved by electrochemical anodization. In vitro and in vivo results suggest that the anodized Ti is equally biocompatible and as safe as the standard nonanodized Ti. The color modification of the BKpro may improve the cosmesis and acceptance of the BKpro by patients.

Hydroxyapatite for Keratoprosthesis Biointegration

PURPOSE Integration of keratoprosthesis with the surrounding cornea is very important in preventing bacterial invasion, which may cause ocular injury. Here the authors investigated whether hydroxyapatite (HAp) coating can improve keratoprosthesis (KPro) biointegration, using polymethyl methacrylate (PMMA)--the principal component of the Boston KPro--as a model polymer. METHODS HAp coatings were induced on PMMA discs after treatment with concentrated NaOH and coating with poly-dopamine (PDA) or polydopamine and then with 11-mercaptoundecanoic acid (11-MUA). Coatings were characterized chemically (Fourier transform infrared spectroscopy [FTIR], energy dispersive X-ray spectroscopy [EDX]) and morphologically (SEM) and were used as substrates for keratocyte growth in vitro. Cylinders of coated PMMA were implanted in porcine corneas ex vivo for 2 weeks, and the force required to pull them out was measured. The inflammatory reaction to coated discs was assessed in the rabbit cornea in vivo. RESULTS FTIR of the coatings showed absorption bands characteristic of phosphate groups, and EDX showed that the Ca/P ratios were close to those of HAp. By SEM, each method resulted in morphologically distinct HAp films; the 11-MUA group had the most uniform coating. The hydroxyapatite coatings caused comparable enhancement of keratocyte proliferation compared with unmodified PMMA surfaces. HAp coating significantly increased the force and work required to pull PMMA cylinders out of porcine corneas ex vivo. HAp coating of implants reduced the inflammatory response around the PMMA implants in vivo. CONCLUSIONS These results are encouraging for the potential of HAp-coated surfaces for use in keratoprostheses.