Scribd Logo
Carica

Benvenuto in Scribd!

  • Mechanism of Propionibacterium Acne Necrosis by Initiation of Reactive Oxygen Species (ROS) by Porphyrin Absorption

    Caricato da

    Caerwyn Ash
    101 visualizzazioni23 pagine
    Background and Objectives: This study provides scientific evidence to support the clinical effect of photodynamic therapy for acne clearance using 414 nm LEDs and filtered Intense Pulsed Light (IPL). The Propionibacterium acnes (P. acnes) was viewed using a scanning electron microscope (SEM) offering the potential to visualise the destructive mechanism of free radical generation by porphyrin absorption. The benefits of using light to treat acne vulgaris safely and effectively are well known. This study had two objectives, firstly to show the dose response for various wavelengths and energy, secondly to morphologically show microscopic changes in the bacteria due to the effect of wavelengths of light. Study Design/Materials and Methods: Cultured P. acnes NCTC 737 (HPA, Salisbury, UK) were diluted to 0.5 McFarland using 2 ml of Butterfield’s phosphate buffer solution. Following further dilution, this suspension was pipetted into a 15 mm well plate for a dosed illumination using four IPL and LED light sources, each with different cut-off filters. After illumination of the sample, the suspension was dispensed onto blood agar plates and incubated at 37ºC for 72 hours under anaerobic conditions. Quantitative assessment was performed by counting the total number of colony forming units (CFUs) on each agar plate. Three controls samples were produced per experiment. Results: At 2 J/cm2 the short wavelength 400 nm filtered IPL has a 15.6% reduction effect on CFUs compared to 5.7% reduction with a 414 nm LED and a 4.6% reduction with a 530 nm IPL. The unfiltered IPL had a 79% reduction at 2 J/cm2, although this output spectrum of the unfiltered IPL contained a significant amount of DNA-damaging wavelengths below 400 nm. Conclusions: This study showed the mechanism of photodynamic therapy of Propionibacterium at a cellular level. Therefore, illumination of the endogenous coproporphyrin with blue light (400-420 nm) plays a major role in P. acnes photo-inactivation.

    Copyright

    © Attribution Non-Commercial (BY-NC)

    Formati disponibili

    PDF, TXT o leggi online da Scribd

    Hai trovato utile questo documento?

    Questo contenuto è inappropriato?

    Segnala questo documento
    Background and Objectives: This study provides scientific evidence to support the clinical effect of photodynamic therapy for acne clearance using 414 nm LEDs and filtered Intense Pulsed Light (IPL). The Propionibacterium acnes (P. acnes) was viewed using a scanning electron microscope (SEM) offering the potential to visualise the destructive mechanism of free radical generation by porphyrin absorption. The benefits of using light to treat acne vulgaris safely and effectively are well known. This study had two objectives, firstly to show the dose response for various wavelengths and energy, secondly to morphologically show microscopic changes in the bacteria due to the effect of wavelengths of light. Study Design/Materials and Methods: Cultured P. acnes NCTC 737 (HPA, Salisbury, UK) were diluted to 0.5 McFarland using 2 ml of Butterfield’s phosphate buffer solution. Following further dilution, this suspension was pipetted into a 15 mm well plate for a dosed illumination using four IPL and LED light sources, each with different cut-off filters. After illumination of the sample, the suspension was dispensed onto blood agar plates and incubated at 37ºC for 72 hours under anaerobic conditions. Quantitative assessment was performed by counting the total number of colony forming units (CFUs) on each agar plate. Three controls samples were produced per experiment. Results: At 2 J/cm2 the short wavelength 400 nm filtered IPL has a 15.6% reduction effect on CFUs compared to 5.7% reduction with a 414 nm LED and a 4.6% reduction with a 530 nm IPL. The unfiltered IPL had a 79% reduction at 2 J/cm2, although this output spectrum of the unfiltered IPL contained a significant amount of DNA-damaging wavelengths below 400 nm. Conclusions: This study showed the mechanism of photodynamic therapy of Propionibacterium at a cellular level. Therefore, illumination of the endogenous coproporphyrin with blue light (400-420 nm) plays a major role in P. acnes photo-inactivation.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Scarica in formato PDF, TXT o leggi online su Scribd
    Segnala contenuti inappropriati
    101 visualizzazioni23 pagine

    Mechanism of Propionibacterium Acne Necrosis by Initiation of Reactive Oxygen Species (ROS) by Porphyrin Absorption

    Caricato da

    Caerwyn Ash
    Background and Objectives: This study provides scientific evidence to support the clinical effect of photodynamic therapy for acne clearance using 414 nm LEDs and filtered Intense Pulsed Light (IPL). The Propionibacterium acnes (P. acnes) was viewed using a scanning electron microscope (SEM) offering the potential to visualise the destructive mechanism of free radical generation by porphyrin absorption. The benefits of using light to treat acne vulgaris safely and effectively are well known. This study had two objectives, firstly to show the dose response for various wavelengths and energy, secondly to morphologically show microscopic changes in the bacteria due to the effect of wavelengths of light. Study Design/Materials and Methods: Cultured P. acnes NCTC 737 (HPA, Salisbury, UK) were diluted to 0.5 McFarland using 2 ml of Butterfield’s phosphate buffer solution. Following further dilution, this suspension was pipetted into a 15 mm well plate for a dosed illumination using four IPL and LED light sources, each with different cut-off filters. After illumination of the sample, the suspension was dispensed onto blood agar plates and incubated at 37ºC for 72 hours under anaerobic conditions. Quantitative assessment was performed by counting the total number of colony forming units (CFUs) on each agar plate. Three controls samples were produced per experiment. Results: At 2 J/cm2 the short wavelength 400 nm filtered IPL has a 15.6% reduction effect on CFUs compared to 5.7% reduction with a 414 nm LED and a 4.6% reduction with a 530 nm IPL. The unfiltered IPL had a 79% reduction at 2 J/cm2, although this output spectrum of the unfiltered IPL contained a significant amount of DNA-damaging wavelengths below 400 nm. Conclusions: This study showed the mechanism of photodynamic therapy of Propionibacterium at a cellular level. Therefore, illumination of the endogenous coproporphyrin with blue light (400-420 nm) plays a major role in P. acnes photo-inactivation.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Scarica in formato PDF, TXT o leggi online su Scribd
    Segnala contenuti inappropriati
    di 23

    Mechanism of Propionibacterium Acne Necrosis by initiation of Reactive Oxygen Species (ROS) by Porphyrin Absorption

    Caerwyn Ash PhD1, Llinos Harris PhD2, Thierry Maffeis PhD3, Marc Clement PhD1, Gareth Stockman PhD1, Mike Kiernan PhD1, Godfrey Town4
    1. CyDen Institute of Light Therapy 2. Medical Microbiology & Infectious Diseases, Swansea University 3. School of Nanotechnology, Swansea University 4. University of Wales, Faculty of Applied Design & Engineering, Swansea Metropolitan, Swansea, SA1 6ED, UK

    2011 Conference, Dallas Texas

    Statement of Disclosure
    The following potential conflict of interest relationships are germane to my presentation: Salary, equipment, travel expenses paid by CyDen Ltd, Swansea, UK

    2011 Conference, Dallas Texas

    Background

    Collaboration:

    2011 Conference, Dallas Texas

    Background

    Collaboration: knowledge transfer

    2011 Conference, Dallas Texas

    Background
    Investigative Study
    In-Vitro Dose response
    Various Fluence Various Wavelengths

    Scanning Electron Microscope


    Back scattered electrons Transmission electron microscopy

    2011 Conference, Dallas Texas

    Absorption Coefficients

    2011 Conference, Dallas Texas

    Absorption Coefficients
    Inter-Cellular Porphyrin

    2011 Conference, Dallas Texas

    In-Vitro Methodology
    Stock syrup kept at -80, agar plated and incubated for 72 hours at 37C Grade 0.5 McFarland solution using 2ml of Butterfields buffer 10,000 fold dilution with butterfield solution illuminate 300l in a 15mm well plate (random sequence) Plate 50l onto blood agar petri dish using a WASP Incubate for 72hrs at 37C under dark anaerobic conditions Count number of Colony forming units (CFU) Various wavelengths and fluence values

    2011 Conference, Dallas Texas

    In-Vitro Methodology
    Counting plates was time consuming Custom software to count total number of Colony Forming units (CFU) Also provided average, minimum and maximum of colony sizes. Information key for studying effect of metabolic changes

    In-Vitro Results

    2011 Conference, Dallas Texas

    In-Vitro Results

    IPL covers Q-bands 414nm diode LED (soret) IPL (soret & Q-band) IPL (soret & Q-band also UV)

    2011 Conference, Dallas Texas

    In-Vitro Results - Summary


    Results of 400-1100nm IPL, 530-1100nm IPL, and 414nm diode LED are linear wrt to increased fluence. Thus suggesting a photochemical reaction proportional to delivered photons. 330-1100nm IPL (unfiltered flashlamp) had large clearance of bacteria in-vitro, but relationship clearly different from other wavelengths. Colony size was smaller on average, suggesting a metabolic change, DNA Damage of short wavelengths is a hypotenuse.

    2011 Conference, Dallas Texas

    Scanning Electron Microscope (SEM)


    Scanning Electron Microscope
    Back scattered electrons Transmission electron microscopy

    2011 Conference, Dallas Texas

    Bacteria Fixing for SEM


    Stock syrup kept at -80C, , agar plated and incubated for 72 hours at 37C Grade 2 McFarland solution using 2ml of Butterfield buffer SEM Attach to Aluminium Stubs Add to well plate with Aluminium slides

    Illumination Control 414nm LED 330 1100nm IPL 400 1100nm IPL
    Centrifuge at 4000rpm for 5min Aspirate solution Wash with Butterfield buffer

    2.5% glutaraldehyde paraformaldehyde


    Add 500l of Butterfield buffer Centrifuge at 4000rpm for 5min

    2011 Conference, Dallas Texas

    SEM Results 330-1100nm

    Asymmetrical separation

    2011 Conference, Dallas Texas

    SEM Results 330-1100nm

    Asymmetrical separation

    2011 Conference, Dallas Texas

    SEM Results 330-1100nm

    Asymmetrical separation

    2011 Conference, Dallas Texas

    SEM Results 400-1100nm

    Elongated Cells due to fluid loss

    2011 Conference, Dallas Texas

    SEM Results - Transmission


    Samples was fixed with 1% osmium tetraoxide and uranyl acetate. The cells were dehydrated with ethanol and embedded in Epon resin. Thin sectioned slices of samples were prepared using an ultratome LKB.

    Unable to supply images! Images show asymmetrical separation of treated cells and cellular leakage

    2011 Conference, Dallas Texas

    SEM Results - Summary This study has observed structural damages to membranes in illuminated bacteria's

    Morphological changes Asymmetrical separation of treated cells (budding phase) Elongation of cells and cellular leakage

    2011 Conference, Dallas Texas

    Discussion
    This collaborative effort proved productive and beneficial to all groups Although effective in-vitro penetration depth in-vivo is an issue for short wavelengths The Soret band key in effective results in-vitro Consecutive treatments yield better results (data not presented) Illumination did not induce heat into bacteria or media.

    2011 Conference, Dallas Texas

    Conclusions
    This study bears relevance on current clinical research and better understanding on Acne pathology. Illumination of porphyrin with photons in the Soret region play a major role in P.acne cell necrosis. Benefits of using light therapy for acne treatment is well known wrt current antibiotics. Light of 407-420nm is not phototoxic to human cells For a one fold decrease in viable bacteria 73J/cm2 for 530-1100nm IPL 18.2J/cm2 for 414nm diode LED 10J/cm2 for 400-1100nm IPL
    2011 Conference, Dallas Texas

    (Q-bands) (Soret) (Soret & Q-bands)

    Thank you
    E: caerwynash@yahoo.co.uk

    2011 Conference, Dallas Texas

    Potrebbero piacerti anche