Effects of methylene blue photodynamic therapy on oral carcinoma and leukoplakia cells.

Objective: Methylene blue (MB) is a readily available and affordable substrate that can be used as a photosensitizer for photodynamic therapy (PDT). The objective of this study was to determine if PDT with MB can downregulate matrix metalloproteinases (MMPs) related to oral carcinoma. Methods: Cell cultures of oral squamous cell carcinoma (CA-9-22), oral leukoplakia (MSK-Leuk1), and immortalized keratinocytes (Rhek-1A) were photosensitized with MB and treated with PDT. MMP-9 gene expression was interrogated via qRT-PCR. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to confirm the efficacy of MB PDT. Results: MMP-9 gene expression was found to be significantly decreased in oral carcinoma, leukoplakia, and immortalized keratinocytes with use of MB PDT. Conclusion: This work demonstrates that MB-mediated PDT can downregulate MMPs which are critical to the invasion and metastasis of oral cancer. These results suggest that MB PDT could be a clinically significant and cost-effective treatment for oral leukoplakia and carcinoma. Level of Evidence: NA.

A phase I, single-center, open-label study of RM-1929 photoimmunotherapy in Japanese patients with recurrent head and neck squamous cell carcinoma.

BACKGROUND: To determine the safety, preliminary efficacy, pharmacokinetics, and immunogenicity of a single cycle of RM-1929 photoimmunotherapy, an anti-EGFR antibody cetuximab conjugated with a light-activatable dye (IRDye®700DX), in Japanese patients with recurrent head and neck squamous cell carcinoma (rHNSCC). METHODS: Patients received a single fixed dose (640 mg/m2) of RM-1929 and a fixed light treatment dose (50 J/cm2 for superficial illumination; 100 J/cm fiber diffuser length for interstitial illumination). Safety, tumor response (modified RECIST v1.1 by central radiology review), pharmacokinetics, and immunogenicity were evaluated. RESULTS: Three Japanese patients were enrolled who had failed ≥ 3 prior lines of therapy including radiation, chemotherapy, cetuximab, and immunotherapy. Target lesions were: submental lesion; right superficial cervical node lesion and oropharynx lesion; and external auditory canal lesion. All patients experienced ≥ 1 treatment-emergent adverse event (TEAE), but none were considered dose-limiting. TEAEs were mild to moderate in severity except for one grade 3 application-site pain, which was transient, resolved without sequelae within 24 h, and did not affect study treatment administration. Thirteen of 17 TEAEs reported were possibly or probably related to study treatment. Three patient reports of application-site pain and localized edema were deemed probably related to study treatment. Objective response was observed in two patients (both partial responses). The third patient had disease progression. RM-1929 concentrations and pharmacokinetic parameters were similar in all patients. No patients tested positive for anti-drug antibodies. CONCLUSIONS: RM-1929 photoimmunotherapy showed a manageable safety profile in rHNSCC. Tumor response in these heavily pre-treated patients was clinically meaningful and warrants further investigation. CLINICAL TRIAL REGISTRATION: The trial was registered with the Japanese registry of clinical trials as jRCT2031200133.

Successful Translation of Fluorescence Navigation During Oncologic Surgery: A Consensus Report.

Navigation with fluorescence guidance has emerged in the last decade as a promising strategy to improve the efficacy of oncologic surgery. To achieve routine clinical use, the onus is on the surgical community to objectively assess the value of this technique. This assessment may facilitate both Food and Drug Administration approval of new optical imaging agents and reimbursement for the imaging procedures. It is critical to characterize fluorescence-guided procedural benefits over existing practices and to elucidate both the costs and the safety risks. This report is the result of a meeting of the International Society of Image Guided Surgery (www.isigs.org) on February 6, 2015, in Miami, Florida, and reflects a consensus of the participants' opinions. Our objective was to critically evaluate the imaging platform technology and optical imaging agents and to make recommendations for successful clinical trial development of this highly promising approach in oncologic surgery.

Id1 and NF-κB promote the generation of CD133+ and BMI-1+ keratinocytes and the growth of xenograft tumors in mice.

Id1 and NF-κB are highly expressed in oral squamous cell carcinoma (OSCC). Whether they have a synergistic role in the carcinogenesis of OSCC is unclear. The current study was designed to demonstrate the synergy of both Id1 and NF-κB in the underlying disease mechanisms of OSCC using in vitro and in vivo animal models. Id1 and NF-κB strengthened the expression of both CD133 and BMI-1 in OSCC cell cultures. CD133(+) and BMI-1(+) keratinocytes from OSCC tissues and cell cultures initiated the growth of xenograft tumors in SCID/Beige mice. Id1 and NF-κB regulate the expression of CD133 and BMI-1 in an additive or synergistic manner in OSCC, which is associated with the generation of naïve and self-renewable keratinocytes and initiate the growth of xenograft tumors in vivo.

Photodynamic therapy of antibiotic-resistant biofilms in a maxillary sinus model.

BACKGROUND: Chronic rhinosinusitis (CRS) is one of the most common chronic conditions in the United States. There is a significant subpopulation of CRS patients who remain resistant to cure despite rigorous treatment regimens including surgery, allergy therapy, and prolonged antibiotic therapy. Antimicrobial photodynamic therapy (aPDT) is a noninvasive nonantibiotic broad spectrum antimicrobial treatment. Our previous in vitro studies demonstrated that aPDT reduced CRS polymicrobial planktonic bacteria and fungi by >99.9% after a single treatment. However, prior to human treatment, the effectiveness of aPDT to eradicate polymicrobial biofilms in a maxillary sinus cavity must be demonstrated. The objective of this study was to demonstrate the effectiveness of a noninvasive aPDT treatment of antibiotic resistant biofilms known to cause CRS in a novel anatomically correct maxillary sinus in vitro model using an enhanced photosensitizer solution. METHODS: Antibiotic resistant polymicrobial biofilms of Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA) were grown in an anatomically correct novel maxillary sinus model and treated with a methylene blue/ethylenediamine tetraacetic acid (EDTA) photosensitizer and 670-nm nonthermal activating light. Cultures of the biofilms were obtained before and after light treatment to determine efficacy of biofilm reduction. RESULTS: The in vitro maxillary sinus CRS biofilm study demonstrated that aPDT reduced the CRS polymicrobial biofilm by >99.99% after a single treatment. CONCLUSION: aPDT can effectively treat CRS polymicrobial antibiotic resistant Pseudomonas aeruginosa and MRSA biofilms in a maxillary sinus cavity model.

The effect of antimicrobial photodynamic therapy on human ciliated respiratory mucosa.

BACKGROUND: Chronic recurrent sinusitis (CRS) is one of the most common chronic conditions in the United States. There is a significant subpopulation of CRS patients who remain resistant to cure despite rigorous treatment regimens including surgery, allergy therapy, and prolonged antibiotic therapy. Antimicrobial photodynamic therapy (aPDT) is a noninvasive nonantibiotic broad spectrum antimicrobial treatment. Our previous in vitro studies demonstrated that aPDT reduced CRS polymicrobial biofilm and planktonic bacteria and fungi by > 99.9% after a single treatment. Prior to human treatment however, aPDT treatment must be demonstrated to not result in histologic damage to the sinus ciliated respiratory epithelium. The objective of this study was to demonstrate the safety of aPDT treatment on a living human ciliated respiratory mucosal model (EpiAirway). METHODS: A study of aPDT treatment of EpiAirway was performed. Treatment groups included a nontreatment control, laser light alone, photosensitizer alone, and therapeutic photosensitizer and light combination (aPDT). At completion of treatment, the EpiAirway tissue was fixed in 10% formalin, paraffin-embedded, sectioned, H&E stained and mounted. All samples were blinded and microscopically examined by a human pathologist to assess any effect of aPDT on the tissue, cilia, or mucosal glands. The results were correlated with the treatment parameters. RESULTS: The EpiAirway histologic study demonstrated no histologic alteration of the respiratory cilia or mucosal epithelium in any of the treatment groups. CONCLUSIONS: aPDT is a safe treatment for CRS resulting in no histologic alteration of human ciliated respiratory mucosa as is found in the human sinuses.

Reduction of Endotracheal Tube Biofilms Using Antimicrobial Photodynamic Therapy.

BACKGROUND: Ventilator-associated pneumonia (VAP) is reported to occur in 12 to 25% of patients who require mechanical ventilation with a mortality rate of 24 to 71%. The endotracheal (ET) tube has long been recognized as a major factor in the development of VAP since biofilm harbored within the ET tube become dislodged during mechanical ventilation and have direct access to the lungs. The objective of this study was to demonstrate the safety and effectiveness of a non-invasive antimicrobial photodynamic therapy (aPDT) treatment method of eradicating antibiotic resistant biofilms from ET tubes in an in vitro model. METHODS: Antibiotic resistant polymicrobial biofilms of Pseudomonas aerugenosa and MRSA were grown in ET tubes and treated, under standard ventilator conditions, with a methylene blue (MB) photosensitizer and 664nm non-thermal activating light. Cultures of the lumen of the ET tube were obtained before and after light treatment to determine efficacy of biofilm reduction. RESULTS: The in vitro ET tube biofilm study demonstrated that aPDT reduced the ET tube polymicrobial biofilm by >99.9% (p<0.05%) after a single treatment. CONCLUSIONS: MB aPDT can effectively treat polymicrobial antibiotic resistant biofilms in an ET tube.

The future of medical diagnostics: review paper.

While histopathology of excised tissue remains the gold standard for diagnosis, several new, non-invasive diagnostic techniques are being developed. They rely on physical and biochemical changes that precede and mirror malignant change within tissue. The basic principle involves simple optical techniques of tissue interrogation. Their accuracy, expressed as sensitivity and specificity, are reported in a number of studies suggests that they have a potential for cost effective, real-time, in situ diagnosis.We review the Third Scientific Meeting of the Head and Neck Optical Diagnostics Society held in Congress Innsbruck, Innsbruck, Austria on the 11th May 2011. For the first time the HNODS Annual Scientific Meeting was held in association with the International Photodynamic Association (IPA) and the European Platform for Photodynamic Medicine (EPPM). The aim was to enhance the interdisciplinary aspects of optical diagnostics and other photodynamic applications. The meeting included 2 sections: oral communication sessions running in parallel to the IPA programme and poster presentation sessions combined with the IPA and EPPM posters sessions.

At the frontiers of surgery: review.

The complete surgical removal of disease is a desirable outcome particularly in oncology. Unfortunately much disease is microscopic and difficult to detect causing a liability to recurrence and worsened overall prognosis with attendant costs in terms of morbidity and mortality. It is hoped that by advances in optical diagnostic technology we could better define our surgical margin and so increase the rate of truly negative margins on the one hand and on the other hand to take out only the necessary amount of tissue and leave more unaffected non-diseased areas so preserving function of vital structures. The task has not been easy but progress is being made as exemplified by the presentations at the 2nd Scientific Meeting of the Head and Neck Optical Diagnostics Society (HNODS) in San Francisco in January 2010. We review the salient advances in the field and propose further directions of investigation.

Antimicrobial photodynamic therapy treatment of chronic recurrent sinusitis biofilms.

BACKGROUND: Chronic recurrent sinusitis (CRS) is an inflammatory disease of the facial sinuses and nasal passages that is defined as lasting longer than 12 weeks or occurring more than 4 times per year with symptoms usually lasting more than 20 days. The National Institute for Health Statistics estimates that CRS is one of the most common chronic conditions in the United States, affecting an estimated 37 million Americans. The potential etiologies of CRS include bacteria, viruses, allergies, fungi, superantigens, and microbial biofilms. In clinical practice there is a significant subpopulation of patients with CRS who remain resistant to cure despite rigorous treatment regimens including surgery, allergy therapy, and prolonged antibiotic therapy. The reason for treatment failure is thought to be related to the destruction of the sinus mucociliary defense by the chronic sinus infection resulting in the development of secondary antibiotic-resistant microbial colonization of the sinuses and biofilm formation. Antimicrobial photodynamic therapy (aPDT) is a nonantibiotic broad-spectrum antimicrobial treatment that has been demonstrated to eradicate antibiotic-resistant bacteria and biofilms. The objective of this study was to demonstrate the effectiveness of a noninvasive aPDT treatment method of eradicating antibiotic resistant biofilms/microorganisms known to cause CRS in an in vitro model. METHODS: Antibiotic-resistant planktonic bacteria and fungi and polymicrobial biofilms of Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA) were grown on silastic sheets and treated with a methylene blue photosensitizer and 670 nm non-thermal-activating light. Cultures of the planktonic microorganisms and biofilms were obtained before and after light treatment to determine efficacy of planktonic bacteria and biofilm reduction. RESULTS: The in vitro CRS planktonic microorganism and biofilm study demonstrated that aPDT reduced the CRS polymicrobial biofilm by >99.9% after a single treatment. CONCLUSION: aPDT can effectively treat CRS polymicrobial antibiotic-resistant bacteria, fungi, and biofilms in vivo. Human clinical studies are currently planned to assess the safety and efficacy of this treatment for CRS.

Factors influencing tumor response to photodynamic therapy sensitized by intratumor administration of methylene blue.

BACKGROUND AND OBJECTIVES: We examined tumor response to methylene blue (MB)-mediated photodynamic therapy (PDT) in a murine tumor model. The goal was to investigate the effects of drug-light interval (DLI), injection vehicle, and fluence on tumor destruction. Fluorescence and reflectance spectroscopy informed our understanding. MATERIALS AND METHODS: EMT6 tumor cells were implanted intradermally on the backs of female BALB/c mice and grown to ∼4-mm diameter. Mice were given a 35 µl, single site, intratumor injection of 500 µg/ml MB administered in either a water or a 5% ethanol-5% Cremophor-90% saline vehicle. PDT was begun either immediately or after a 1-hour DLI with a fluence rate of 60 mW/cm(2). Each animal received a fluence of 240 or 480 J/cm(2). Fluorescence and reflectance spectra were captured before and during irradiation. RESULTS: A protocol consisting of the Cremophor-based vehicle, 0 DLI, and a fluence of 480 J/cm(2) was the most effective, with a 55% cure rate as measured by no evidence of tumor 90 days after PDT. Use of the water vehicle with this fluence and DLI reduced the cure rate to 20%. Reducing the fluence to 240 J/cm(2) similarly reduced treatment efficacy with 0 and 1-hour DLIs. Univariate Cox proportional hazards analysis identified increased fluence, 0 versus 1-hour DLI, and the Cremophor versus water vehicle as highly significant independent predictors of long term tumor control (P < 0.01 in each case). Multivariate analysis with model selection revealed fluence and injection vehicle as the best predictors of survival hazards. Fluorescence spectroscopy in vivo showed that MB fluorescence decreased monotonically during a 2-hour dark interval but was restored by irradiation. Reflectance spectroscopy revealed that MB at this injected concentration attenuates the treatment beam significantly. CONCLUSION: Sensitizer delivery vehicle, drug-light interval, and fluence contribute significantly to the tumor response to MB-mediated PDT.

Photodynamic therapy of bacterial and fungal biofilm infections.

Biofilms have been found to be involved in a wide variety of microbial infections in the body, by one estimate 80% of all infections. Infectious processes in which biofilms have been implicated include common problems such as urinary tract infections, catheter infections, middle-ear infections, sinusitis, formation of dental plaque, gingivitis, coating contact lenses, endocarditis, infections in cystic fibrosis, and infections of permanent indwelling devices such as joint prostheses and heart valves. Bacteria living in a biofilm usually have significantly different properties from free-floating bacteria of the same species, as the dense and protected environment of the film allows them to cooperate and interact in various ways. One benefit of this environment is increased resistance to detergents and antibiotics, as the dense extracellular matrix and the outer layer of cells protect the interior of the community. In some cases antibiotic resistance can be increased 1000-fold. Also, the biofilm bacteria excrete toxins that reversibly block important processes such as translation and protecting the cell from bactericidal antibiotics that are ineffective against inactive targets. In the head and neck area, biofilms are a major etiologic factor in periodontitis, wound infections, oral candidiasis, and sinus and ear infections. For the past several decades, photodynamic treatment has been reported in the literature to be effective in eradicating various microorganisms using different photosensitizers, different wavelengths of light, and different light sources. PDT has been further studied to demonstrate its effectiveness for the eradication of both Gram-negative and Gram-positive antibiotic-resistant bacteria. This chapter will focus on the use of PDT in the treatment of antibiotic-resistant biofilms, antibiotic-resistant wound infections, and azole-resistant oral candidiasis using methylene blue-based photodynamic therapy.

Photodynamic therapy of head and neck cancers.

Over 1,500 patients have been treated with PDT using Photofrin, HPD, ALA, or Foscan for head and neck cancers. These patients include a mixture of presentations including primary, recurrent, and metastatic lesions. The predominant histology is squamous cell carcinoma, but other histologies treated include mucosal melanoma, Kaposi's sarcoma, adenocarcinoma, metastatic breast carcinoma, and adenoid cystic carcinoma. Several multi-institutional phase II clinical trials evaluating PDT treatment of head and neck cancers have demonstrated the efficacy of this minimally invasive therapy in the treatment of early oropharyngeal primary and recurrent cancers as well as the palliative treatment of refractory head and neck cancers. Patients with early stage cancers or early recurrences in the oral cavity and larynx (Cis, T1, T2) tend to have an excellent response to PDT. Of 518 patients treated with Cis, T1, or T2 cancers of the oral cavity, larynx, pharynx, and nasopharynx, 462 (89.1%) obtained a complete clinical response after one PDT treatment. Laryngeal cancers, comprising 171 patients in this group, obtained a durable complete response rate of 89% with up to a 16-year follow-up. Photodynamic therapy is as effective as conventional therapies for the treatment of early (Cis, T1, T2) squamous cell cancers of the head and neck. It is also a promising therapy to be used in association with surgery to increase tumor-free margins and therefore increase cure rates.

Photodynamic therapy for head and neck dysplasia and cancer.

OBJECTIVE: To determine the response of dysplasia, carcinoma in situ (CIS), and T1 carcinoma of the oral cavity and larynx to photodynamic therapy with porfimer sodium. DESIGN: Prospective trial. SETTING: A National Cancer Institute-designated cancer institute. PATIENTS: Patients with primary or recurrent moderate to severe oral or laryngeal dysplasia, CIS, or T1N0 carcinoma. INTERVENTION: Porfimer sodium, 2 mg/kg of body weight, was injected intravenously 48 hours before treatment. Light at 630 nm for photosensitizer activation was delivered from an argon laser or diode laser using lens or cylindrical diffuser fibers. The light dose was 50 J/cm(2) for dysplasia and CIS and 75 J/cm(2) for carcinoma. MAIN OUTCOME MEASURES: Response was evaluated at 1 week and at 1 month and then at 3-month intervals thereafter. Response options were complete (CR), partial (PR), and no (NR) response. Posttreatment biopsies were performed in all patients with persistent and recurrent visible lesions. RESULTS: Thirty patients were enrolled, and 26 were evaluable. Mean follow-up was 15 months (range, 7-52 months). Twenty-four patients had a CR, 1 had a PR, and 1 had NR. Three patients with oral dysplasia with an initial CR experienced recurrence in the treatment field. All the patients with NR, a PR, or recurrence after an initial CR underwent salvage treatment. Temporary morbidities included edema, pain, hoarseness, and skin phototoxicity. CONCLUSION: Photodynamic therapy with porfimer sodium is an effective treatment alternative, with no permanent sequelae, for oral and laryngeal dysplasia and early carcinoma. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00530088.

Head & neck optical diagnostics: vision of the future of surgery.

Review paper and Proceedings of the Inaugural Meeting of the Head and Neck Optical Diagnostics Society (HNODS) on March 14th 2009 at University College London. The aim of our research must be to provide breakthrough translational research which can be applied clinically in the immediate rather than the near future. We are fortunate that this is indeed a possibility and may fundamentally change current clinical and surgical practice to improve our patients' lives.

Photodynamic therapy treatment of early oral and laryngeal cancers.

Photodynamic therapy (PDT) is a nonsurgical, minimally invasive treatment that uses a light source to activate light-sensitive drugs or photosensitizers in the treatment of cancer and other diseases. PDT has been successfully employed to treat early carcinomas of the oral cavity and larynx preserving normal tissue and vital functions of speech and swallowing. Two hundred seventy-six patients with early carcinomas of the oral cavity and larynx were treated from 1990 to 2006. Cure rates with a single treatment for early laryngeal and oral cancers were 91% and 94%, respectively. PDT is an effective primary and alternative treatment modality for early oral cavity and laryngeal cancers.

Open-label, long-term safety study of cevimeline in the treatment of postirradiation xerostomia.

PURPOSE: To assess the safety of long-term cevimeline treatment of radiation-induced xerostomia in patients with head-and-neck cancer; and to assess the efficacy of cevimeline in these patients. METHODS AND MATERIALS: A total of 255 adults with head-and-neck cancer who had received more than 40 Gy of radiation 4 months or more before entry and had clinically significant salivary gland dysfunction received cevimeline hydrochloride 45 mg t.i.d. orally for 52 weeks. Adverse events (AEs), their severity, and their relationship to the study medication were assessed by each investigator. The efficacy assessment was based on subjects' global evaluation of oral dryness on a scale of 0 (none) to 3 (severe). RESULTS: Overall, 175 subjects (68.6%) experienced expected treatment-related AEs, most mild to moderate. The most frequent was increased sweating (47.5%), followed by dyspepsia (9.4%), nausea (8.2%), and diarrhea (6.3%). Fifteen subjects (5.9%) experienced Grade 3 treatment-related AEs, of which the most frequent was increased sweating. Eighteen subjects (7.1%) reported at least one serious AE, and 45 subjects (17.6%) discontinued study medication because of an AE. The global efficacy evaluation at the last study visit showed that cevimeline improved dry mouth in most subjects (59.2%). Significant improvement was seen at each study visit in the mean change from baseline of the numeric global evaluation score (p < 0.0001). CONCLUSIONS: Cevimeline 45 mg t.i.d. was generally well tolerated over a period of 52 weeks in subjects with xerostomia secondary to radiotherapy for cancer in the head-and-neck region.

Cevimeline for the treatment of postirradiation xerostomia in patients with head and neck cancer.

PURPOSE: To study the efficacy and safety of cevimeline in two double-blind trials (Studies 003 and 004) enrolling patients with head and neck cancer in whom xerostomia developed after radiotherapy. METHODS AND MATERIALS: Subjects were randomly assigned to receive cevimeline, 30 mg three times daily, or placebo for 12 weeks, with the possibility of dose escalation to 45 mg three times daily at 6 weeks. The primary efficacy endpoint was the patient's final global evaluation of oral dryness; change in unstimulated salivary flow was a secondary endpoint. RESULTS: Five hundred seventy subjects (284 in Study 003 and 286 in Study 004) were randomized. Significantly more cevimeline-treated subjects than placebo recipients (47.4% vs. 33.3%, p = 0.0162) in Study 003 reported improvement in dry mouth in the final global evaluation of oral dryness. No significant difference between groups in the final global evaluation was seen in Study 004, in which a high placebo response rate of 47.6% was observed. In both studies, cevimeline-treated subjects had significantly greater increases in the objective measure of unstimulated salivary flow than placebo recipients (p = 0.0093 [Study 003] and p = 0.0215 [Study 004]), whereas no significant differences in stimulated salivary flow were observed. The most frequent adverse event was increased sweating. CONCLUSION: Cevimeline was well tolerated by patients with xerostomia after radiotherapy for head and neck cancer, and oral administration of 30-45 mg of cevimeline three times daily increased unstimulated salivary flow.

Effect of Ca+ on the photobactericidal efficacy of methylene blue and toluidine blue against gram-negative bacteria and the dye affinity for lipopolysaccharides.

BACKGROUND AND OBJECTIVES: Methylene blue (MB) and toluidine blue (TB) form metachromatic complexes with lipopolysaccharides (LPS). The greater photobactericidal efficacy of TB may be explained by its affinity for LPS. This study aims to elucidate the difference in photobactericidal efficacies between the dyes using Ca(2+) as a competitor for dye-binding sites on the bacterial outer membrane. STUDY DESIGN/MATERIALS AND METHODS: Fixed dye concentration solutions with gram-negative bacteria and increasing concentrations of CaCl(2) were exposed to red laser light. Bacterial survival and spectrophotometry were used to describe the effect of Ca(2+) on dye interaction with bacteria and LPS. RESULTS: MB-mediated photokilling was inhibited more significantly than that of TB. CaCl(2) inhibited dye photobleaching and suppressed the metachromatic reaction between the dyes and LPS, in particular TB. CONCLUSIONS: CaCl(2) inhibits bacterial photokilling by binding with LPS, as well as other anionic polymers including outer membrane proteins. LPS is chiefly involved in TB-mediated photokilling, whereas outer membrane proteins probably are more involved in MB-mediated photokilling.