Interaction of the photobactericides methylene blue and toluidine blue with a fluorophore in Pseudomonas aeruginosa cells.

BACKGROUND AND OBJECTIVES: The difference in photobactericidal efficacy between methylene blue (MB) and toluidine blue (TB) may be explained by their involvement with proteins, lipopolysaccharides (LPS), and siderophores and siderophore-receptor protein complexes on the bacterial outer membrane. This study aims to determine if this is the case by using the fluorescence given off by a pseudomonal siderophore named pyoverdin. STUDY DESIGN/MATERIALS AND METHODS: Confocal laser scanning microscopy was used to observe the fluorescence of Pseudomonas aeruginosa cells excited at 488 nm in the presence of increasing dye concentrations. RESULTS: Cellular fluorescence at 522 nm progressively decreased with increasing dye concentrations. The Stern-Volmer constants for cellular fluorescence quenching with the dyes were compared to the association constants for dyes complexed with LPS. The quenching of cellular fluorescence was associated with the formation of a ground-state complex between the dyes and pyoverdin-FpvA protein system. MB readily complexed with this system, whereas TB complexed more strongly with LPS. CONCLUSION: The different affinities of the dyes for both pyoverdin-protein and LPS will affect the contributions of the dyes' interactions with these biopolymers to the overall bacterial photodamage.

Treatment of oral candidiasis with methylene blue-mediated photodynamic therapy in an immunodeficient murine model.

OBJECTIVES: The purpose of this study was to evaluate the efficacy of using methylene blue (MB)-mediated photodynamic therapy to treat oral candidiasis in an immunosuppressed murine model, mimicking what is found in human patients. STUDY DESIGN: Seventy-five experimental mice with severe combined immunodeficiency disease were inoculated orally with Candida albicans by swab 3 times a week for a 4-week period. On treatment day, mice were cultured for baseline fungal growth and received a topical oral cavity administration of 0.05 mL MB solution at one of the following concentrations: 250, 275, 300, 350, 400, 450, or 500 microgram/mL. After 10 minutes the mice were recultured and underwent light activation with 664 nm of diode laser light with a cylindrical diffuser. After photodynamic therapy the mice were cultured again for colony-forming units per milliliter and then killed, their tissue harvested for histopathology. RESULTS AND CONCLUSIONS: The results indicate an MB dose-dependent effect. Concentrations from 250 to 400 microgram/mL reduced fungal growth but did not eliminate Candida albicans. MB concentrations of 450 and 500 microgram/mL totally eradicated Candida albicans from the oral cavity, resulting in reductions from 2.5 log(10) and 2.74 log(10) to 0, respectively. These results suggest that MB-mediated photodynamic therapy can potentially be used to treat oral candidiasis in immunodeficient patients.

Comparison of the methylene blue and toluidine blue photobactericidal efficacy against gram-positive and gram-negative microorganisms.

BACKGROUND AND OBJECTIVE: Studies on the photobactericidal efficacy of methylene blue (MB) and toluidine blue (TB) have shown inconsistent results in the literature. This study evaluated the bactericidal efficacy of MB and TB against different bacteria under light and dark conditions to determine the most effective bactericidal dye. STUDY DESIGN/MATERIALS AND METHODS: Suspensions of Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecalis, Hemophilus influenzae, Escherichia coli, and Pseudomonas aeruginosa in saline were treated in dark and red laser light conditions in the presence of each dye using an argon pumped-dye and a diode laser emitting light at 630 and 664 nm, respectively. The effect of dye concentration, dark incubation time, the fluence and intensity of laser light on the destruction of different bacteria were compared. RESULTS: Both dyes eradicated all examined bacteria under laser light. The complete photodestruction of microorganisms was reached at TB concentrations of 1.5-7-fold less than that of MB. CONCLUSION: TB exhibits a greater bactericidal activity than MB against most bacteria in dark and light conditions. Mostly, these results are consistent with their respective dye partition coefficients.

Topical ofloxacin treatment of otorrhea in children with tympanostomy tubes.

OBJECTIVE: To determine the safety and efficacy of ofloxacin otic solution in the treatment of acute otorrhea in children with tympanostomy tubes. DESIGN: Multicenter study with an open-label, prospective ofloxacin arm and retrospective historical and current practice arms. SETTING: Ear, nose, and throat pediatric and general practice clinics and office-based practices. SUBJECTS: Children younger than 12 years with acute purulent otorrhea of presumed bacterial origin and tympanostomy tubes. INTERVENTION: Instillation of 0.3% ofloxacin, 0.25 mL, twice daily for 10 days in the prospective arm; review of medical records in the retrospective arms. MAIN OUTCOME MEASURES: The primary index of clinical efficacy was absence (cure) or presence (failure) of otorrhea at 10 to 14 days after therapy. The primary index of microbiologic efficacy (in the ofloxacin arm only) was eradication of pathogens isolated at baseline. Safety was evaluated in the ofloxacin arm only. RESULTS: Significantly more clinically evaluable ofloxacin-treated subjects were cured (84.4%; 119/141) than were historical practice subjects (64.2%; 140/218) (P< or =.001) or current practice subjects (70%; 33/47) (P< or =.03). All baseline pathogens were eradicated in 103 (96.3%) of 107 microbiologically evaluable ofloxacin subjects. Adverse events considered "possibly" or "probably" treatment related occurred in 29 (12.8%) of 226 ofloxacin-treated subjects. CONCLUSION: Ofloxacin is safe and significantly more effective than treatments used in historical or current practice for acute purulent otorrhea in children with tympanostomy tubes.

Evaluation of the microbiology of chronic maxillary sinusitis.

Chronic paranasal sinusitis is a disease that afflicts a significant percentage of the population and causes considerable long-term morbidity. With the common use of multiple courses of broad-spectrum oral antibiotics to treat this condition, there is a possibility of an alteration in the pathogens that promote a persistence of chronic sinusitis. One hundred seventy-four consecutive patients with a diagnosis of chronic maxillary sinusitis requiring an endoscopic surgical procedure were prospectively evaluated. At the time of surgery, cultures of mucopus in the maxillary sinus were aseptically obtained endoscopically and cultured for aerobes, anaerobes, and fungus. Two hundred seventeen isolates from 174 patients were obtained. Coagulase-negative staphylococci were the most common isolates (36%), followed by Staphylococcus aureus (25%), Streptococcus viridans (8.3%), Corynebacterium (4.6%), and anaerobes (6.4%). Although coagulase-negative Staphylococcus is not considered a pathogen, of the 24 isolates that had sensitivity testing performed, 13 demonstrated resistance to multiple antibiotics, including cephalothin, erythromycin, oxacillin, sulfonamides, and clindamycin. This study demonstrates that aerobic rather than anaerobic bacteria are the more common pathogens in chronic sinusitis. In addition, coagulase-negative Staphylococcus may be a pathogen in the disease process, and sensitivities should be obtained of this isolate for evaluation and possible treatment.

Photodynamic therapy and the treatment of head and neck neoplasia.

OBJECTIVE: To present the theory, technique, and results of photodynamic therapy for the treatment of oral, laryngeal, and head and neck cancers. STUDY DESIGN: Retrospective review of the literature of more than 500 patients with head and neck cancer treated with photodynamic therapy, as well as a retrospective review of the author's 107 patients treated with photodynamic therapy for head and neck neoplasia between 1990 and 1997. METHODS: The literature was retrospectively reviewed, as were patient records, and tabulated for age, sex, site, and staging of lesions, with special focus on post-photodynamic therapy treatment outcome, long-term disease-free survival, and complications. RESULTS: Twenty-five patients with carcinoma in situ and T1 squamous cell carcinoma of the true vocal cord who underwent photodynamic therapy treatment for cure obtained a complete response after a single photodynamic therapy treatment. Only one patient has had recurrence to date, with a cure rate to 79-month follow-up of 95%. Twenty-nine patients with carcinoma in situ and T1 recurrent squamous cell carcinomas of the oral cavity and tongue were treated. All obtained a complete response after a single photodynamic therapy treatment; however, five patients developed local recurrence with follow-up to 70 months, for an 80% cure rate. A review of 217 patients with early squamous cell carcinomas of the head and neck treated with photodynamic therapy in the literature demonstrated an 89.5% complete response rate. The most common complication in these patients was limited prolonged skin photosensitivity without any permanent sequelae. CONCLUSIONS: Photodynamic therapy is effective for treating carcinoma in situ and T1 squamous cell carcinoma of the larynx and oral cavity and may be of benefit as an adjuvant intraoperative treatment of stages III and IV tumors of the head and neck in conjunction with surgery and radiation therapy to improve cure rates. Further controlled studies need to be performed to further demonstrate the effectiveness of photodynamic therapy and the treatment of head and neck cancers.

Methylation of the HIC-1 candidate tumor suppressor gene in human breast cancer.

HIC-1 (hypermethylated in cancer) is a candidate tumor suppressor gene which is located at 17p13.3, a region which frequently undergoes allelic loss in breast and other human cancers. HIC-1 is proposed to be commonly inactivated in human cancers by hypermethylation of a normally unmethylated dense CpG island which encompasses the entire gene. To study whether HIC-1 inactivation may be important to the development of breast cancer, we first measured methylation of the HIC-1 gene in normal breast ductal tissues from microdissected frozen breast tissues and from epithelial cells purified from mammoplasty specimens. Surprisingly, in all normal breast ductal tissues we found approximately equal amounts of densely methylated HIC-1 and completely unmethylated HIC-1. This is in contrast to most normal tissues, in which all copies of HIC-1 are completely unmethylated. We then evaluated 39 primary breast cancer tissues and found virtually complete methylation of the HIC-1 gene in 26 (67%) of the cases. We also found loss of heterozygosity at the telomeric portion of chromosomal arm 17p in 22 of the 26 cases with strongly methylated HIC-1, suggesting that loss of an unmethylated HIC-1 allele may contribute to the inactivation of HIC-1 in cells with a pre-existing methylated allele. Finally, by RNase protection analysis, HIC-1 was found to be expressed in microdissected normal breast ductal tissues and unmethylated tumors but not in tumors with hypermethylation of the HIC-1 gene. These results indicate that hypermethylation of HIC-1 and associated loss of HIC-1 expression is common in primary breast cancer. Furthermore, the HIC-1 gene is densely methylated in approximately one-half of the alleles in normal breast epithelium, which may predispose this tissue to inactivation of this gene by loss of heterozygosity.

Tissue-specific expression of human achaete-scute homologue-1 in neuroendocrine tumors: transcriptional regulation by dual inhibitory regions.

Malignancies with neuroendocrine (NE) features such as medullary thyroid cancer (MTC) and small cell lung cancer (SCLC) are prototypic neoplasms arising from peripheral endocrine cells. The mechanisms that regulate the NE phenotype in these tumors and their cellular precursors are not well understood. However, a basic helix-loop-helix transcription factor that is homologous to Drosophila neural fate determination proteins may have a central role. Human achaete-scute homologue-1 (hASH1), a human homologue of the Drosophila achaete-scute complex, is highly expressed in MTC, SCLC, and pheochromocytomas. To determine what mechanisms allow constitutive expression of hASH1 in NE tumors, we cloned human genomic DNA fragments containing the hASH1 gene and characterized its promoter region. We show that hASH1 expression is restricted to NE cell lines by a transcriptionally regulated mechanism. Dual promoters initiate hASH1 transcription, with the predominant site being an evolutionarily conserved initiator (INR) element. Transient transfection studies provide evidence for a generalized enhancer region that has high activity in all cell lines tested. Restriction of hASH1 expression to NE tumor cells depends on two tissue-specific repressor regions, present in the proximal and distal (> 13.5 kb) 5'-flanking region. Understanding the mechanisms of tissue-specific control of hASH1 gene expression provides a useful model to explore regulatory cascades influencing both normal nervous system development and the NE phenotype of tumors such as MTC and SCLC.

HIC1 hypermethylation is a late event in hematopoietic neoplasms.

HIC1, a candidate tumor suppressor gene on 17p13.3, is hypermethylated and silenced in a large number of solid tumors. To determine its potential role in leukemias, we studied its methylation status in normal and neoplastic hematopoietic cells. We found HIC1 to be unmethylated in peripheral blood cells, bone marrow cells, and CD34+ cells. HIC1 was rarely methylated in newly diagnosed acute myelogenous leukemias (10%) but was relatively frequently methylated in newly diagnosed non-Hodgkin's lymphoma (25%), acute lymphocytic leukemia (ALL; 53%), and chronic-phase chronic myelogenous leukemia (50%). By contrast, HIC1 was hypermethylated in 100% of recurrent ALL and 100% of blast crisis chronic myelogenous leukemia. In two patients with ALL for whom paired diagnosis/relapse samples were available, HIC1 was unmethylated at diagnosis but was highly methylated at relapse after a chemotherapy-induced complete remission. HIC1 methylation, therefore, seems to be a progression event in hematopoietic neoplasms.

Photodynamic therapy as an adjuvant intraoperative treatment of recurrent head and neck carcinomas.

OBJECTIVE: To demonstrate the feasibility of adjuvant intraoperative photodynamic therapy in the treatment of large, infiltrating, recurrent carcinomas of the head and neck. DESIGN: A nonrandomized trial involving 5 patients treated with intraoperative adjuvant photodynamic therapy with 29-month follow-up. SETTING: A tertiary care referral center in a private practice setting. PATIENTS: Five patients with massive, recurrent, infiltrating squamous cell carcinomas of the head and neck and skull base. INTERVENTION: Photodynamic therapy was used as an adjuvant intraoperative treatment after resection of tumors. MAIN OUTCOME MEASURE: Time of survival after treatment without recurrence of disease. RESULTS: Four of the 5 patients have remained free of recurrent disease without complications up to 24 months after treatment. CONCLUSION: Adjuvant intraoperative photodynamic therapy may improve cure rates in patients with recurrent head and neck malignancies by providing larger tumor-free margins of resection, while preserving normal structures.

Photodynamic therapy and the treatment of head and neck cancers.

Photodynamic therapy is an effective curative treatment for early carcinomas of the head and neck and may be of benefit as an adjuvant intraoperative therapy to increase cure rates of large head and neck tumors. Eighty-seven patients with neoplastic diseases of the larynx, oral cavity, pharynx, and skin have been treated with photodynamic therapy (PDT) with follow-up to 66 months. Patients with carcinoma-in-situ and T1 carcinomas obtained a complete response after one PDT treatment. All but two have remained free of disease. Ten patients with massive neck recurrences of squamous cell carcinomas were treated with intraoperative adjuvant PDT following tumor resection. Only three developed recurrence with 40-month follow-up, but only one recurrence was in the field of surgery and PDT.

Photodynamic therapy of head and neck cancers.

Sixty-five patients with neoplastic diseases of the larynx, oral cavity, pharynx, and skin have been treated with photodynamic therapy (PDT) with follow-up to 56 months. Patients with carcinoma in situ (CIS) and T1 carcinomas obtained a complete response after one PDT treatment. All but two have remained free of disease. Eight patients with T2 and T3 carcinomas treated with PDT obtained a complete response, but they all recurred locally. This is due to the inability to adequately deliver laser light to the depths of the tumor bed. Five patients with massive neck recurrences of squamous cell carcinomas were treated with intraoperative adjuvant PDT following tumor resection. Only one developed recurrence with 24-month follow-up. PDT is highly effective for the curative treatment of early carcinomas (CIS, T1) of the head and neck. Also, intraoperative adjuvant PDT may increase cure rates of large infiltrating carcinomas of the head and neck.

p53 activates expression of HIC-1, a new candidate tumour suppressor gene on 17p13.3.

For several human tumour types, allelic loss data suggest that one or more tumour suppressor genes reside telomeric to the p53 gene at chromosome 17p13.1. In the present study we have used a new strategy, involving molecular analysis of a DNA site hypermethylated in tumour DNA, to identify a candidate gene in this region (17p13.3). Our approach has led to identification of HIC-1 (hypermethylated in cancer), a new zinc-finger transcription factor gene which is ubiquitously expressed in normal tissues, but underexpressed in different tumour cells where it is hypermethylated. Multiple characteristics of this gene, including the presence of a p53 binding site in the 5' flanking region, activation of the gene by expression of a wild-type p53 gene and suppression of G418 selectability of cultured brain, breast and colon cancer cells following insertion of the gene, make HIC-1 gene a strong candidate for a tumour suppressor gene in region 17p13.3.

Photodynamic therapy and the treatment of neoplastic diseases of the larynx.

Photodynamic therapy (PDT) is an innovative treatment involving the use of light-sensitive drugs to selectively identify and destroy diseased cells. Therefore, photodynamic therapy has the potential to treat and cure precancerous and early cancerous lesions (carcinoma in situ [CIS], T1 and T2) of the larynx while preserving normal tissue. Eleven patients with recurrent leukoplakia and carcinomas of the larynx were treated with PDT with follow-up to 27 months. One patient with a T1 verrucous carcinoma, 5 patients with T1 squamous cell carcinomas of the vocal cord failing radiotherapy, 1 patient with a T2 squamous cell carcinoma of the vocal cord failing radiotherapy, and 3 patients with CIS and severe atypia were treated with PDT and obtained a complete response and are disease free. One patient with a T3 carcinoma of the larynx was treated with PDT but died 4 weeks post-treatment of unrelated causes and could not be assessed. Photodynamic therapy is a promising therapy for treatment of precancerous and cancerous lesions of the larynx. This therapy may be particularly beneficial for the treatment of recurrent carcinomas of the larynx that have failed conventional radiotherapy, thereby preserving voice and eliminating the need for destructive laryngeal surgery.

Effect of radiation therapy and Photofrin on tissue response in a rat model.

Treatment of advanced carcinomas of the head and neck may benefit from adjuvant photodynamic therapy and brachyradiotherapy. To date, however, there has been no controlled study to evaluate whether high-dose irradiation can be safely accomplished without major tissue reaction in the presence of high circulating doses of Photofrin, the photosensitizing agent used in photodynamic therapy. Thirty adult male white rats were involved in the study. Fifteen rats received Photofrin 5 mg/kg intravenously, and 15 rats received the same volume of sterile saline intravenously. At 48 hours following injection, each rat received 1,000 cGy of radiation to a 3 x 5 cm area of dorsal skin using a cobalt linear accelerator unit. Skin changes postradiation were observed for degree of erythema, blistering, necrosis, and sloughing. Five rats from the Photofrin and control radiation groups were sacrificed on days 2, 7, and 21 postradiotherapy. Skin changes in each animal were identical with mild erythema lasting from 10-14 days postradiotherapy. There was no evidence of blistering, necrosis, or sloughing of skin in any of the animals studied. Histologic evaluation of the irradiated skin after sacrifice demonstrated no difference between the Photofrin and saline-irradiated groups. As well, the histologic recovery from acute radiation injury was also identical. This controlled study demonstrates that radiation therapy may be safely administered without increased morbidity when tissue concentrations necessary to perform photodynamic therapy are present.

Postoperative radiation-associated changes in free jejunal autografts.

Free jejunal autografts are a preferred method of pharyngoesophageal reconstruction. Ten adult mongrel dogs underwent free jejunal transplantations to the neck, five being controls and five receiving a 55-Gy equivalent dose of radiation after 3 weeks. Histologic changes 10 months after radiotherapy included simplified and blunted villi with normal architecture loss; fibrous replacement of the lymphatics and microvasculature in the intravillous space; goblet cell increase; significant increase in lamina propria thickness and muscularis mucosa fibrous plates; focal destruction and replacement of muscle layers with fibrosis; gross hypertrophy of the myenteric plexus with increased fibrous tissue about the hypertrophied neural tissue; and significant perivascular fibrosis. Controls demonstrated only minimal changes. These adverse delayed effects of irradiation on revascularized jejunal autografts should be considered in planning the method of pharyngoesophageal reconstruction as well as timing of adjuvant radiotherapy.

Gross morphologic and functional effects of postoperative radiation on free jejunal autografts.

Twenty adult mongrel dogs underwent free jejunal transplants to the neck; 10 either died of or had to be sacrificed as a result of postoperative complications, 5 received a 55-Gy-equivalent dose of radiation after 3 weeks, and 5 were followed as controls. Serial manometric and endoscopic evaluations were performed over a 9-month period. Progressive deterioration of the quality and amplitude of peristalsis of the jejunal autografts was observed only in the radiated group. In addition, all dogs in the radiated group developed severe jejunal circumferential constriction and stricture formation. These delayed effects of irradiation on revascularized jejunal autografts should be considered in planning the method of pharyngoesophageal reconstruction as well as the timing of adjuvant radiotherapy.

The evaluation of pharmacologic agents on composite graft survival.

This study assessed the efficacy of various pharmacologic agents in improving composite graft viability in 60 New Zealand white rabbits given bilateral auricular composite grafts. Treatments included methylprednisolone preoperatively and for 3 days postoperatively or for 7 days postoperatively only; chlorpromazine, dimethylsulfoxide, or superoxide dismutase preoperatively; or indomethacin preoperatively and for either 3 or 7 days postoperatively. Four treatment modalities yielded a statistically significant decrease in percentage of necrosis. Methylprednisolone, when given preoperatively and continued postoperatively, produced the greatest increase (77%) in composite graft tissue survival. Dimethylsulfoxide, chlorpromazine, and indomethacin for 3 days were also effective, but to a much lesser extent. It is speculated that these agents may help stabilize cell membranes during the anoxic phase of plasmic imbibition and minimize cellular edema until revascularization can occur.

Maintaining long-term vessel patency in microvascular surgery using tissue-type plasminogen activator.

The primary cause of free flap failure remains vascular thrombosis at the microanastomosis site. Four-hour local infusion of tissue plasminogen activator (t-PA) has been proved to effectively lyse thromboses in microvascular studies of animals; however, rethrombosis occurs once the infusion of t-PA has been terminated. The present study was designed to examine the efficacy of 48 hours of a continuous local t-PA infusion in maintaining long-term venous patency. Our previously described modified arterial inversion graft reanastomosed into the venous system was used in rabbits to form venous thrombi. Three mg of t-PA was infused over 48 hours in eleven rabbits. Seven of eleven grafts were patent at 48 hours and four remained patent at 1 week. In comparing the patency rates in this study with the overall patency rate using the modified arterial inversion graft model (1/22), there are statistically significant differences at both 48 hours (p less than 0.001) and 7 days (p less than 0.05). We conclude that lengthening the infusion time of t-PA may increase the long-term patency rate in this animal model.