In vivo killing of Staphylococcus aureus using a light-activated antimicrobial agent.

BACKGROUND: The widespread problem of antibiotic resistance in pathogens such as Staphylococcus aureus has prompted the search for new antimicrobial approaches. In this study we report for the first time the use of a light-activated antimicrobial agent, methylene blue, to kill an epidemic methicillin-resistant Staphylococcus aureus (EMRSA-16) strain in two mouse wound models. RESULTS: Following irradiation of wounds with 360 J/cm(2) of laser light (670 nm) in the presence of 100 microg/ml of methylene blue, a 25-fold reduction in the number of viable EMRSA was seen. This was independent of the increase in temperature of the wounds associated with the treatment. Histological examination of the wounds revealed no difference between the photodynamic therapy (PDT)-treated wounds and the untreated wounds, all of which showed the same degree of inflammatory infiltration at 24 hours. CONCLUSION: The results of this study demonstrate that PDT is effective at reducing the total number of viable EMRSA in a wound. This approach has promise as a means of treating wound infections caused by antibiotic-resistant microbes as well as for the elimination of such organisms from carriage sites.

In vitro photodynamic eradication of Pseudomonas aeruginosa in planktonic and biofilm culture.

Photodynamic disinfection (PDD) is a nonantibiotic approach to treating drug-resistant bacterial infections. Pseudomonas aeruginosa, an opportunistic pathogen, is problematic because of its propensity to develop antibiotic resistance and its ability to secrete a protective biofilm matrix. This study examined the ability of PDD to eradicate planktonic and biofilm cultures of P. aeruginosa in vitro. Planktonic P. aeruginosa cultures were briefly exposed to a methylene blue-based photosensitizer formulation and subjected to energy doses ranging from 1.7 to 20.6 J cm(-2) using a 670 nm nonthermal diode laser. Biofilms were grown for 24 and 48 h and exposed to photosensitizer for 30 s before illumination with 13.2 or 26.4 J of energy. A single exposure of planktonic P. aeruginosa to photosensitizer at >15.5 J cm(-2) resulted in 100% eradication (>7 log(10) reduction from control), an effect that could be decreased significantly in the presence of the singlet oxygen quenchers l-tryptophan and sodium azide. Decreasing the energy dose below this threshold by varying both power density and illumination duration resulted in a dose-dependent decrease in bacterial kill. In addition, 24 h biofilm viability was reduced by 99% with single exposure and 99.9% with double exposure, while 48 h biofilm viability was reduced by >99.999% with both single and double exposures. This study shows that PDD is effective in eradicating planktonic and biofilm cultures of P. aeruginosa, supporting the concept that translation into clinical practice for indications such as otitis externa and wound disinfection is a viable option.

Islet graft assessment in the Edmonton Protocol: implications for predicting long-term clinical outcome.

The success of the Edmonton Protocol for islet transplantation has provided new hope in the treatment of type 1 diabetes. This study reports on the assessment of 83 human islet grafts transplanted using the Edmonton Protocol since 1999. Cellular composition, as assessed by immunohistochemistry, showed a lower islet purity (approximately 40%) than has been reported in previous studies using dithizone staining to quantitate islet equivalents. Furthermore, grafts were found to contain substantial populations of exocrine and ductal tissue. Total cellular insulin transplanted was 8,097.6 +/- 3,164.4 microg/patient, and was significantly lower in bottom gradient layer grafts than top gradient layer or whole/combined grafts (P < 0.0005). A static incubation test for islet function gave a stimulation index of 3-4, although this measure did not correlate with posttransplant metabolic outcome. Furthermore, we confirmed a previously reported trend in which donor age affects islet yield and purity. It is important to note that a significant positive correlation was observed between the number of islet progenitor (ductal-epithelial) cells transplanted and long-term metabolic success as assessed an by intravenous glucose tolerance test at approximately 2 years posttransplant. In summary, careful assessment of islet graft composition is needed in a clinical transplantation program to accurately estimate islet purity and assess the contribution of other cell types present, such as islet progenitor cells.

Stem cell-based approaches to solving the problem of tissue supply for islet transplantation in type 1 diabetes.

Type 1 diabetes is a debilitating condition, affecting millions worldwide, that is characterized by the autoimmune destruction of insulin-producing pancreatic islets of Langerhans. Although exogenous insulin administration has traditionally been the mode of treatment for this disease, recent advancements in the transplantation of donor-derived insulin-producing cells have provided new hope for a cure. However, in order for islet transplantation to become a widely used technique, an alternative source of cells must be identified to supplement the limited supply currently available from cadaveric donor organs. Stem cells represent a promising solution to this problem, and current research is being aimed at the creation of islet-endocrine tissue from these undifferentiated cells. This review presents a summary of the research to date involving stem cells and cell replacement therapy for type 1 diabetes. The potential for the differentiation of embryonic stem (ES) cells to islet phenotype is discussed, as well as the possibility of identifying and exploiting a pancreatic progenitor/stem cell from the adult pancreas. The possibility of creating new islets from adult stem cells derived from other tissues, or directly form other terminally differentiated cell types is also addressed. Finally, a model for the isolation and maturation of islets from the neonatal porcine pancreas is discussed as evidence for the existence of an islet precursor cell in the pancreas.

Energy dose parameters affect antimicrobial photodynamic therapy-mediated eradication of periopathogenic biofilm and planktonic cultures.

OBJECTIVE: This study evaluated the in vitro efficacy of a commercially available aPDT system in eradication of the periopathogens Porphyromonas gingivalis, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans in both planktonic and biofilm cultures. BACKGROUND DATA: Antimicrobial photodynamic therapy (aPDT) is an effective antibacterial approach in vitro; however, few data are available regarding effective light-energy parameters. MATERIALS AND METHODS: Planktonic and biofilm cultures of periopathogens were exposed to a methylene blue-based formulation and irradiated with a 670-nm nonthermal diode laser. Energy doses were varied from 2.3 to 9.4 J/cm(2) through adjustments in illumination time and a constant power density. Controls consisted of no treatment, light only, and photosensitizer only. Temperature changes were recorded in experimental samples before and after illumination. RESULTS: aPDT with an energy dose of 9.4 J/cm(2) was effective in eradicating P. gingivalis, F. nucleatum, and A. actinomycetemcomitans in biofilm and planktonic form. Reductions from control in planktonic cultures at this energy dose were 6.8 +/- 0.7, 5.2 +/- 0.6, and 1.9 +/- 0.6 log(10), respectively, whereas biofilm reductions were 4.5 +/- 1.2, 3.4 +/- 1.1, and 4.9 +/- 1.4 log(10). Decreasing the treatment time produced an energy dose-dependent killing effect in both models. Changes in sample temperature did not exceed 3 degrees C under these exposure parameters. CONCLUSION: This study demonstrated that three important periopathogens are susceptible to aPDT-mediated killing, regardless of whether they are present in planktonic or biofilm form. Furthermore, a clear energy dose-dependence exists with this treatment that should to be taken into account when determining optimal treatment times in clinical application.

Antimicrobial photodynamic therapy may promote periodontal healing through multiple mechanisms.

BACKGROUND: Antimicrobial photodynamic therapy (aPDT) as an adjunctive treatment in addition to scaling and root planing for the treatment of periodontitis has been shown to be clinically useful. Its beneficial effect is reported to be due to its potent bactericidal activity. However, aPDT treatment has the potential to inactivate bacterial and host factors that contribute to disease. In this report, we demonstrate that aPDT treatment can simultaneously kill Porphyromonas gingivalis and inactivate its virulence-associated protease. It also inactivates host destructive cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1 beta. METHODS: We developed a 96-well-based bacterial killing and protease inactivation assay that determined aPDT bactericidal and protease inactivation from the same sample. A cytokine inactivation assay that measured E-selectin expression in response to TNF-alpha and IL-1 beta was developed to measure the ability of aPDT to inactivate cytokine function. RESULTS: A single aPDT treatment in vitro potently inactivated protease activity and resulted in a 4-log(10) reduction in the viability of P. gingivalis. Dose and time-of-exposure experiments revealed that protease inactivation occurred at lower concentrations of photosensitizer and less time of light exposure. Also, aPDT treatment potently and functionally inactivated IL-1 beta and TNF-alpha. CONCLUSIONS: aPDT treatment may augment periodontal treatment by increasing bacterial killing, inactivating bacterial virulence factors, and inactivating host cytokines that impair periodontal restoration. Therefore, aPDT treatment may provide a more favorable healing environment.

Stem cells: a promising source of pancreatic islets for transplantation in type 1 diabetes.

Diabetes is a disease that affects millions and causes a major burden on the health care system. Type 1 diabetes has traditionally been managed with exogenous insulin therapy, however factors such as cost, lifestyle restriction, and life threatening complications necessitate the development of a more efficient treatment alternative. Pancreas transplantation, and more recently transplant of purified pancreatic islets, has offered the potential for independence from insulin injections. Islet transplantation is gaining acceptance as it has been shown to be effective for certain patients with type 1 diabetes. One obstacle, however, is the fact that there is an inadequate supply of cadaveric human islets to implement this procedure on a widespread clinical basis. A promising source of transplantable islets in the future will come through the use of adult or embryonic stem cells. This chapter presents an overview of the advancements made in the development of a stem cell based application to islet transplantation. Advantages and limitations are discussed regarding the use of embryonic stem cells, adult pancreatic stem/progenitor cells, and the use of nonpancreatic tissues based on current experimental models in the literature. It is concluded that stem cells offer the greatest potential for the development of an abundant source of pancreatic islets, although specific obstacles must be overcome before this can become a reality.

Enriched human pancreatic ductal cultures obtained from selective death of acinar cells express pancreatic and duodenal homeobox gene-1 age-dependently.

Adult pancreatic ductal cells are believed to be islet precursors. Our aim was to obtain an enriched human ductal cell population in defined culture conditions, and to characterize these cultures for the presence of pancreatic developmental transcription factors. Non-endocrine adult human pancreatic digest was cultured for 4 days in serum-containing and serum-free media. During this time, analysis was done for phenotypic changes, cell death, and expression of islet and islet precursor markers. Culture in serum-supplemented and serum-free media gave similar recoveries of an enriched ductal population after 4 days. Extensive cell death due to apoptosis and necrosis was also observed over this time period. A donor-age dependent expression of pancreatic and duodenal homeobox gene-1 (PDX-1) in ductal cells was seen at 4 days whereby donors <25 yr expressed significantly more than donors >25 yr. Analysis of gene expression by RT-PCR showed the presence of islet developmental transcription factors neuroD, Nkx6.1, and PDX-1, as well as mature islet hormones. While acinar-ductal transdifferentiation of some cells cannot be ruled out, we provide evidence that the predominant mechanism for the derivation of enriched human ductal cultures in our culture conditions is selective acinar cell death. Furthermore, we have shown that ductal cultures from younger donors exhibit greater plasticity through expression of PDX-1, and may be of greater value in attempts to induce islet neogenesis. The presence, however, of insulin and glucagon mRNA indicates that contaminating endocrine cells remain in these cultures and underscores the need to use caution when assessing differentiation potential.