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Hemoglobin (Hb), a life-sustaining and highly abundant erythrocyte protein, is not readily fluorescent. A few studies have already reported Two-Photon Excited Fluorescence (TPEF) of Hb, however, the mechanisms through which Hb becomes fluorescent upon interaction with ultrashort laser pulses are not completely understood. Here, we characterized photophysically this interaction on Hb thin film and erythrocytes using fluorescence spectroscopy upon single-photon/two-photon absorption, and UV-VIS single-photon absorption spectroscopy. A gradual increase of the fluorescence intensity, ending up with saturation, is observed upon prolonged exposure of Hb thin layer and erythrocytes to ultrashort laser pulses at 730 nm. When compared to protoporphyrin IX (PpIX) and oxidized Hb by H2O2, TPEF spectra from a thin Hb film and erythrocytes showed good mutual agreement, broad peaking at 550 nm, supporting hemoglobin undergoes degradation and that same fluorescent specie(s) originating from the heme moiety are generated. The uniform square shaped patterns of the fluorescent photoproduct exhibited the same level of the fluorescence intensity even after 12 weeks from the formation, indicating high photoproduct stability. We finally demonstrated the full potential of the formed Hb photoproduct with TPEF scanning microscopy towards spatiotemporally controlled micropatterning in HTF and single human erythrocyte labelling and tracking in the whole blood.

Hemoglobin (Hb), a life-sustaining and highly abundant erythrocyte protein, is not readily fluorescent. A few studies have already reported Two-Photon Excited Fluorescence (TPEF) of Hb, however, the mechanisms through which Hb becomes fluorescent upon interaction with ultrashort laser pulses are not completely understood. Here, we characterized photophysically this interaction on Hb thin film and erythrocytes using fluorescence spectroscopy upon single-photon/two-photon absorption, and UV-VIS single-photon absorption spectroscopy. A gradual increase of the fluorescence intensity, ending up with saturation, is observed upon prolonged exposure of Hb thin layer and erythrocytes to ultrashort laser pulses at 730 nm. When compared to protoporphyrin IX (PpIX) and oxidized Hb by H2O2, TPEF spectra from a thin Hb film and erythrocytes showed good mutual agreement, broad peaking at 550 nm, supporting hemoglobin undergoes degradation and that same fluorescent specie(s) originating from the heme moiety are generated. The uniform square shaped patterns of the fluorescent photoproduct exhibited the same level of the fluorescence intensity even after 12 weeks from the formation, indicating high photoproduct stability. We finally demonstrated the full potential of the formed Hb photoproduct with TPEF scanning microscopy towards spatiotemporally controlled micropatterning in HTF and single human erythrocyte labelling and tracking in the whole blood.

Background: Patients with erythropoietic protoporphyria (EPP) are hypersensitive to long wave ultraviolet (UVA) radiation and visible light and they experience severe skin pain by light exposure. The patients have very limited treatment options. Sunless skin tanning with dihydroxyacetone (DHA) is now being investigated as a possible treatment modality of skin photosensitivity in EPP. Methods: We simulated the theoretical light protection factor provided by DHA application. In addition, we present 19 cases with EPP who were treated at our department with DHA weekly during spring and summer from 2018 to 2021 inclusive. Results: The protection factor against UVA and visible light was estimated to approximately two. Out of the 19 patients with EPP who were treated with DHA in 2018, 11 patients experienced a sustained good effect and continued to use the treatment on a weekly basis in the spring and summer of 2019, 2020, and 2021. Conclusion and perspectives: Both the theoretical estimates and the uncontrolled study suggest that sunless tanning with DHA reduces photosensitivity in patients with EPP. Our hypothesis is that skin treated with DHA can tolerate twice the daylight dose compared to untreated skin before onset of skin symptoms. To validate this conclusion, we plan a randomized clinical trial to determine the effect of DHA application to reduce photosensitivity in patients with EPP under controlled clinical conditions. The study protocol for this trial is presented in the paper.

Erythropoietic protoporphyria (EPP) and X-linked protoporphyria (XLP) are characterized by skin photosensitivity caused by accumulation of protoporphyrin IX. We aimed to review the clinical evidence of efficacy and safety of skin photosensitivity treatments in individuals with EPP or XLP. We systematically searched MEDLINE, Embase, the Cochrane Library, and ClinicalTrials.gov. A total of 40 studies with data on 18 treatment modalities were included. Comprehensive treatment safety data were obtained from the European Medicines Agency and the United States Food and Drug Administration. The studies used different outcome measures to evaluate the sensitivity without a generally accepted method to assess treatment effect on skin photosensitivity. Of the included studies, 13 were controlled trials. Gathered, the trials showed moderate positive effect of inorganic sunscreen application and subcutaneous implant of afamelanotide and no effect of organic sunscreen application, or oral treatment with beta-carotene, cysteine, N-acetylcysteine, vitamin C, or warfarin. Studies without control groups suggested treatment effect of foundation cream, dihydroxyacetone/lawsone cream, narrow-band ultraviolet B phototherapy, erythrocyte transfusion, extracorporeal erythrocyte photodynamic therapy, or oral treatment with zinc sulphate, terfenadine, cimetidine, or canthaxanthin, but the real effect is uncertain. Assessment of treatment effect on photosensitivity in patients with EPP or XLP carries a high risk of bias since experienced photosensitivity varies with both weather conditions, exposure pattern, and pigmentation. Controlled trials of promising treatment options are important although challenging in this small patient population.

Background: Patients with erythropoietic protoporphyria (EPP) are hypersensitive to long wave ultraviolet (UVA) radiation and visible light and they experience severe skin pain by light exposure. The patients have very limited treatment options. Sunless skin tanning with dihydroxyacetone (DHA) is now being investigated as a possible treatment modality of skin photosensitivity in EPP. Methods: We simulated the theoretical light protection factor provided by DHA application. In addition, we present 19 cases with EPP who were treated at our department with DHA weekly during spring and summer from 2018 to 2021 inclusive. Results: The protection factor against UVA and visible light was estimated to approximately two. Out of the 19 patients with EPP who were treated with DHA in 2018, 11 patients experienced a sustained good effect and continued to use the treatment on a weekly basis in the spring and summer of 2019, 2020, and 2021. Conclusion and perspectives: Both the theoretical estimates and the uncontrolled study suggest that sunless tanning with DHA reduces photosensitivity in patients with EPP. Our hypothesis is that skin treated with DHA can tolerate twice the daylight dose compared to untreated skin before onset of skin symptoms. To validate this conclusion, we plan a randomized clinical trial to determine the effect of DHA application to reduce photosensitivity in patients with EPP under controlled clinical conditions. The study protocol for this trial is presented in the paper.

Erythropoietic protoporphyria (EPP) and X-linked protoporphyria (XLP) are characterized by skin photosensitivity caused by accumulation of protoporphyrin IX. We aimed to review the clinical evidence of efficacy and safety of skin photosensitivity treatments in individuals with EPP or XLP. We systematically searched MEDLINE, Embase, the Cochrane Library, and ClinicalTrials.gov. A total of 40 studies with data on 18 treatment modalities were included. Comprehensive treatment safety data were obtained from the European Medicines Agency and the United States Food and Drug Administration. The studies used different outcome measures to evaluate the sensitivity without a generally accepted method to assess treatment effect on skin photosensitivity. Of the included studies, 13 were controlled trials. Gathered, the trials showed moderate positive effect of inorganic sunscreen application and subcutaneous implant of afamelanotide and no effect of organic sunscreen application, or oral treatment with beta-carotene, cysteine, N-acetylcysteine, vitamin C, or warfarin. Studies without control groups suggested treatment effect of foundation cream, dihydroxyacetone/lawsone cream, narrow-band ultraviolet B phototherapy, erythrocyte transfusion, extracorporeal erythrocyte photodynamic therapy, or oral treatment with zinc sulphate, terfenadine, cimetidine, or canthaxanthin, but the real effect is uncertain. Assessment of treatment effect on photosensitivity in patients with EPP or XLP carries a high risk of bias since experienced photosensitivity varies with both weather conditions, exposure pattern, and pigmentation. Controlled trials of promising treatment options are important although challenging in this small patient population.

Severe skin pain when exposed to long wave ultraviolet radiation or visible light is the main symptom of erythropoietic protoporphyria (EPP). Treatment options for EPP are inadequate and new treatments are needed but hampered by the lack of valid efficacy outcomes. Phototesting with well-defined illumination of the skin can be performed reliably. We aimed to provide an overview of phototest procedures used to evaluate EPP treatments. Systematic searches of Embase, MEDLINE and the Cochrane Library were performed. Searches identified 11 studies using photosensitivity as efficacy outcome. The studies used eight different phototest protocols. Illuminations were performed with a filtered high-pressure mercury arc, or a xenon arc lamp equipped with monochromator or filters. Some used broadband, others narrowband illumination. In all protocols phototests were performed on the hands or the back. Endpoints were minimal dose required to induce either first symptom of discomfort, erythema, urticaria or intolerable pain. Other endpoints were change in erythema intensity or diameter of any type of flare after exposure compared to before. In conclusion, protocols displayed extensive variability in illumination set-up and evaluation of phototest reactions. Implementation of a standardized phototest method will allow more consistent and reliable outcome evaluation in future therapeutic research of protoporphyric photosensitivity.

Severe skin pain when exposed to long wave ultraviolet radiation or visible light is the main symptom of erythropoietic protoporphyria (EPP). Treatment options for EPP are inadequate and new treatments are needed but hampered by the lack of valid efficacy outcomes. Phototesting with well-defined illumination of the skin can be performed reliably. We aimed to provide an overview of phototest procedures used to evaluate EPP treatments. Systematic searches of Embase, MEDLINE and the Cochrane Library were performed. Searches identified 11 studies using photosensitivity as efficacy outcome. The studies used eight different phototest protocols. Illuminations were performed with a filtered high-pressure mercury arc, or a xenon arc lamp equipped with monochromator or filters. Some used broadband, others narrowband illumination. In all protocols phototests were performed on the hands or the back. Endpoints were minimal dose required to induce either first symptom of discomfort, erythema, urticaria or intolerable pain. Other endpoints were change in erythema intensity or diameter of any type of flare after exposure compared to before. In conclusion, protocols displayed extensive variability in illumination set-up and evaluation of phototest reactions. Implementation of a standardized phototest method will allow more consistent and reliable outcome evaluation in future therapeutic research of protoporphyric photosensitivity.

Systemic medications used during the treatment of onychomycosis caused by Trichophyton rubrum may have relapse, making them costly and insignificant for the patient. Photodynamic therapy (PDT) is an advantageous therapeutic option for disease control, mainly due to the absence of risk of microbial resistance. The action of PDT is combined by three elements: photosensitizer (PS), visible light (VL) and molecular oxygen, leading to the formation of reactive oxygen species (ROS) or singlet oxygen formation (1O2), both ROS (type I mechanism) as 1O2 (type II mechanism) induce damage and death to microbial cells. This research was proposed as a study through a non-systematic review, to investigate the action of PS "Protoporphyrin IX" (Pp IX) associated with visible light on T. rubrum, contemplating clinical and relevant data regarding the treatment of onychomycosis by PDT. PubMed survey was conducted from June 2021 to April 2022. The research strategy included clinical trials, randomized trials, systematic reviews, meta-analyses and reviews in English. Pp IX has affinity for T. rubrum, which is justified by the incorporation of Pp IX into the cell membrane, which led to inhibition by LV irradiation. Thus, the photodynamic process of Pp IX may lead to cell death by type I and II mechanisms. The data found are promising, however, new studies in vitro and in vivo are suggested, since few studies have been found related to the theme.

Systemic medications used during the treatment of onychomycosis caused by Trichophyton rubrum may have relapse, making them costly and insignificant for the patient. Photodynamic therapy (PDT) is an advantageous therapeutic option for disease control, mainly due to the absence of risk of microbial resistance. The action of PDT is combined by three elements: photosensitizer (PS), visible light (VL) and molecular oxygen, leading to the formation of reactive oxygen species (ROS) or singlet oxygen formation (1O2), both ROS (type I mechanism) as 1O2 (type II mechanism) induce damage and death to microbial cells. This research was proposed as a study through a non-systematic review, to investigate the action of PS "Protoporphyrin IX" (Pp IX) associated with visible light on T. rubrum, contemplating clinical and relevant data regarding the treatment of onychomycosis by PDT. PubMed survey was conducted from June 2021 to April 2022. The research strategy included clinical trials, randomized trials, systematic reviews, meta-analyses and reviews in English. Pp IX has affinity for T. rubrum, which is justified by the incorporation of Pp IX into the cell membrane, which led to inhibition by LV irradiation. Thus, the photodynamic process of Pp IX may lead to cell death by type I and II mechanisms. The data found are promising, however, new studies in vitro and in vivo are suggested, since few studies have been found related to the theme.

Systemic medications used during the treatment of onychomycosis caused by Trichophyton rubrum may have relapse, making them costly and insignificant for the patient. Photodynamic therapy (PDT) is an advantageous therapeutic option for disease control, mainly due to the absence of risk of microbial resistance. The action of PDT is combined by three elements: photosensitizer (PS), visible light (VL) and molecular oxygen, leading to the formation of reactive oxygen species (ROS) or singlet oxygen formation (1O2), both ROS (type I mechanism) as 1O2 (type II mechanism) induce damage and death to microbial cells. This research was proposed as a study through a non-systematic review, to investigate the action of PS "Protoporphyrin IX" (Pp IX) associated with visible light on T. rubrum, contemplating clinical and relevant data regarding the treatment of onychomycosis by PDT. PubMed survey was conducted from June 2021 to April 2022. The research strategy included clinical trials, randomized trials, systematic reviews, meta-analyses and reviews in English. Pp IX has affinity for T. rubrum, which is justified by the incorporation of Pp IX into the cell membrane, which led to inhibition by LV irradiation. Thus, the photodynamic process of Pp IX may lead to cell death by type I and II mechanisms. The data found are promising, however, new studies in vitro and in vivo are suggested, since few studies have been found related to the theme.

Systemic medications used during the treatment of onychomycosis caused by Trichophyton rubrum may have relapse, making them costly and insignificant for the patient. Photodynamic therapy (PDT) is an advantageous therapeutic option for disease control, mainly due to the absence of risk of microbial resistance. The action of PDT is combined by three elements: photosensitizer (PS), visible light (VL) and molecular oxygen, leading to the formation of reactive oxygen species (ROS) or singlet oxygen formation (1O2), both ROS (type I mechanism) as 1O2 (type II mechanism) induce damage and death to microbial cells. This research was proposed as a study through a non-systematic review, to investigate the action of PS "Protoporphyrin IX" (Pp IX) associated with visible light on T. rubrum, contemplating clinical and relevant data regarding the treatment of onychomycosis by PDT. PubMed survey was conducted from June 2021 to April 2022. The research strategy included clinical trials, randomized trials, systematic reviews, meta-analyses and reviews in English. Pp IX has affinity for T. rubrum, which is justified by the incorporation of Pp IX into the cell membrane, which led to inhibition by LV irradiation. Thus, the photodynamic process of Pp IX may lead to cell death by type I and II mechanisms. The data found are promising, however, new studies in vitro and in vivo are suggested, since few studies have been found related to the theme.

Systemic medications used during the treatment of onychomycosis caused by Trichophyton rubrum may have relapse, making them costly and insignificant for the patient. Photodynamic therapy (PDT) is an advantageous therapeutic option for disease control, mainly due to the absence of risk of microbial resistance. The action of PDT is combined by three elements: photosensitizer (PS), visible light (VL) and molecular oxygen, leading to the formation of reactive oxygen species (ROS) or singlet oxygen formation (1O2), both ROS (type I mechanism) as 1O2 (type II mechanism) induce damage and death to microbial cells. This research was proposed as a study through a non-systematic review, to investigate the action of PS "Protoporphyrin IX" (Pp IX) associated with visible light on T. rubrum, contemplating clinical and relevant data regarding the treatment of onychomycosis by PDT. PubMed survey was conducted from June 2021 to April 2022. The research strategy included clinical trials, randomized trials, systematic reviews, meta-analyses and reviews in English. Pp IX has affinity for T. rubrum, which is justified by the incorporation of Pp IX into the cell membrane, which led to inhibition by LV irradiation. Thus, the photodynamic process of Pp IX may lead to cell death by type I and II mechanisms. The data found are promising, however, new studies in vitro and in vivo are suggested, since few studies have been found related to the theme.

Systemic medications used during the treatment of onychomycosis caused by Trichophyton rubrum may have relapse, making them costly and insignificant for the patient. Photodynamic therapy (PDT) is an advantageous therapeutic option for disease control, mainly due to the absence of risk of microbial resistance. The action of PDT is combined by three elements: photosensitizer (PS), visible light (VL) and molecular oxygen, leading to the formation of reactive oxygen species (ROS) or singlet oxygen formation (1O2), both ROS (type I mechanism) as 1O2 (type II mechanism) induce damage and death to microbial cells. This research was proposed as a study through a non-systematic review, to investigate the action of PS "Protoporphyrin IX" (Pp IX) associated with visible light on T. rubrum, contemplating clinical and relevant data regarding the treatment of onychomycosis by PDT. PubMed survey was conducted from June 2021 to April 2022. The research strategy included clinical trials, randomized trials, systematic reviews, meta-analyses and reviews in English. Pp IX has affinity for T. rubrum, which is justified by the incorporation of Pp IX into the cell membrane, which led to inhibition by LV irradiation. Thus, the photodynamic process of Pp IX may lead to cell death by type I and II mechanisms. The data found are promising, however, new studies in vitro and in vivo are suggested, since few studies have been found related to the theme.

The inhibitory effects of 5-aminolevulinic acid phosphate (5-ALA), an important amino acid for energy production in the host, against viral infections were previously reported. Here, the antiviral effects of 5-ALA against classical swine fever virus (CSFV) belonging to the genus Pestivirus in the Flaviviridae family and its possible mechanisms were investigated. CSFV replication was suppressed in swine cells supplemented with 5-ALA or its metabolite, protoporphyrin IX (PPIX). The infectivity titer of CSFV was decreased after mixing with PPIX extracellularly. In addition, the activities of the replication cycle were decreased in the presence of PPIX based on the CSFV replicon assay. These results showed that PPIX exerted antiviral effects by inactivating virus particles and inhibiting the replication cycle. To evaluate the in vivo efficacy of 5-ALA, pigs were supplemented daily with 5-ALA for 1 week before virus inoculation and then inoculated with a virulent CSFV strain at the 10(7.0) 50% tissue culture infectious dose. The clinical scores of the supplemented group were significantly lower than those of the nonsupplemented group, whereas the virus growth was not. Taken together, 5-ALA showed antiviral effects against CSFV in vitro, and PPIX played a key role by inactivating virus particles extracellularly and inhibiting the replication cycle intracellularly.

The inhibitory effects of 5-aminolevulinic acid phosphate (5-ALA), an important amino acid for energy production in the host, against viral infections were previously reported. Here, the antiviral effects of 5-ALA against classical swine fever virus (CSFV) belonging to the genus Pestivirus in the Flaviviridae family and its possible mechanisms were investigated. CSFV replication was suppressed in swine cells supplemented with 5-ALA or its metabolite, protoporphyrin IX (PPIX). The infectivity titer of CSFV was decreased after mixing with PPIX extracellularly. In addition, the activities of the replication cycle were decreased in the presence of PPIX based on the CSFV replicon assay. These results showed that PPIX exerted antiviral effects by inactivating virus particles and inhibiting the replication cycle. To evaluate the in vivo efficacy of 5-ALA, pigs were supplemented daily with 5-ALA for 1 week before virus inoculation and then inoculated with a virulent CSFV strain at the 10(7.0) 50% tissue culture infectious dose. The clinical scores of the supplemented group were significantly lower than those of the nonsupplemented group, whereas the virus growth was not. Taken together, 5-ALA showed antiviral effects against CSFV in vitro, and PPIX played a key role by inactivating virus particles extracellularly and inhibiting the replication cycle intracellularly.

The inhibitory effects of 5-aminolevulinic acid phosphate (5-ALA), an important amino acid for energy production in the host, against viral infections were previously reported. Here, the antiviral effects of 5-ALA against classical swine fever virus (CSFV) belonging to the genus Pestivirus in the Flaviviridae family and its possible mechanisms were investigated. CSFV replication was suppressed in swine cells supplemented with 5-ALA or its metabolite, protoporphyrin IX (PPIX). The infectivity titer of CSFV was decreased after mixing with PPIX extracellularly. In addition, the activities of the replication cycle were decreased in the presence of PPIX based on the CSFV replicon assay. These results showed that PPIX exerted antiviral effects by inactivating virus particles and inhibiting the replication cycle. To evaluate the in vivo efficacy of 5-ALA, pigs were supplemented daily with 5-ALA for 1 week before virus inoculation and then inoculated with a virulent CSFV strain at the 10(7.0) 50% tissue culture infectious dose. The clinical scores of the supplemented group were significantly lower than those of the nonsupplemented group, whereas the virus growth was not. Taken together, 5-ALA showed antiviral effects against CSFV in vitro, and PPIX played a key role by inactivating virus particles extracellularly and inhibiting the replication cycle intracellularly.

Systemic medications used during the treatment of onychomycosis caused by Trichophyton rubrum may have relapse, making them costly and insignificant for the patient. Photodynamic therapy (PDT) is an advantageous therapeutic option for disease control, mainly due to the absence of risk of microbial resistance. The action of PDT is combined by three elements: photosensitizer (PS), visible light (VL) and molecular oxygen, leading to the formation of reactive oxygen species (ROS) or singlet oxygen formation (1O2), both ROS (type I mechanism) as 1O2 (type II mechanism) induce damage and death to microbial cells. This research was proposed as a study through a non-systematic review, to investigate the action of PS "Protoporphyrin IX" (Pp IX) associated with visible light on T. rubrum, contemplating clinical and relevant data regarding the treatment of onychomycosis by PDT. PubMed survey was conducted from June 2021 to April 2022. The research strategy included clinical trials, randomized trials, systematic reviews, meta-analyses and reviews in English. Pp IX has affinity for T. rubrum, which is justified by the incorporation of Pp IX into the cell membrane, which led to inhibition by LV irradiation. Thus, the photodynamic process of Pp IX may lead to cell death by type I and II mechanisms. The data found are promising, however, new studies in vitro and in vivo are suggested, since few studies have been found related to the theme.

Systemic medications used during the treatment of onychomycosis caused by Trichophyton rubrum may have relapse, making them costly and insignificant for the patient. Photodynamic therapy (PDT) is an advantageous therapeutic option for disease control, mainly due to the absence of risk of microbial resistance. The action of PDT is combined by three elements: photosensitizer (PS), visible light (VL) and molecular oxygen, leading to the formation of reactive oxygen species (ROS) or singlet oxygen formation (1O2), both ROS (type I mechanism) as 1O2 (type II mechanism) induce damage and death to microbial cells. This research was proposed as a study through a non-systematic review, to investigate the action of PS "Protoporphyrin IX" (Pp IX) associated with visible light on T. rubrum, contemplating clinical and relevant data regarding the treatment of onychomycosis by PDT. PubMed survey was conducted from June 2021 to April 2022. The research strategy included clinical trials, randomized trials, systematic reviews, meta-analyses and reviews in English. Pp IX has affinity for T. rubrum, which is justified by the incorporation of Pp IX into the cell membrane, which led to inhibition by LV irradiation. Thus, the photodynamic process of Pp IX may lead to cell death by type I and II mechanisms. The data found are promising, however, new studies in vitro and in vivo are suggested, since few studies have been found related to the theme.

Background: Erythropoietic protoporphyria (EPP) is a rare genetic photodermatosis caused by loss-of-function mutations in the gene for ferrochelatase leading to accumulation of the fluorescent protoporphyrin IX (PpIX) in erythrocytes. The mutations are most often inherited mutations present in all cells causing inherited EPP. In very rare cases EPP are acquired in association with myelodysplastic syndromes or myeloproliferative neoplasms, conditions with genetic instability. Case report: We report a case of acquired EPP in association with hematological disease. We followed erythrocyte PpIX concentration over a year and measured PpIX fluorescence in individual erythrocytes in a blood sample from the case using flow cytometry. The major proportion of erythrocytes did not fluoresce (84%), whereas 13% contained low PpIX fluorescence, 1% contained medium fluorescence, and 2% contained high fluorescence. Discussion: Our observation of the very skewed PpIX distribution in erythrocytes supports the description that acquired EPP is caused by a somatic mutation effecting a clone of hematopoietic cells.

Background and objectives: Mitochondria require oxygen to generate energy for essential cellular processes. An in-vivo non-invasive technique to monitor mitochondrial oxygen tension (mitoPO2) is the protoporphyrin IX-triplet state lifetime technique. Protoporphyrin IX (PpIX), one of the key components of heme, is naturally present in the mitochondria. Topical administration of 5-aminolevulinic acid hydrochloride (ALA) increases PpIX concentration in the skin. After excitation with a laser pulse, PpIX emits red light with an oxygen-dependent delayed fluorescence lifetime. This signal is detected by a photomultiplier and the mitoPO2 can be derived from this. A clinical device has been developed using this technique. Although this device has been proven feasible, improvements in reliability and practicality are needed. This work aims to investigate possible improvements to the components of this device for better application in a clinical setting. The main areas of improvement are the excitation wavelength, the detection of the signal, and the light source. Excitation wavelength: First, wavelengths from 415 nm to 695 nm are explored as possible excitation wavelengths. Second, four excitation wavelengths that exhibit low noise are investigated in more detail. For a better comparison, the pulse energy per wavelength is equated. The excitation wavelengths 415 nm and 515 nm showed low noise, no oversaturation, and accurate fits. These two excitation wavelengths are then validated on three test subjects with skin types II, IV, and V on the Fitzpatrick scale. Oxygen-dependent lifetimes are obtained in all test subjects with an excitation wavelength of 415 nm. Detection: A new detector is explored to replace the large and expensive photomultiplier. The detector is tested in a fluorescent solution and on human skin. The first experiments resulted in oversaturation of the detector. To prevent this, several combinations of band-pass, long-pass, and absorption filters are, Technical Medicine | Sensing and Stimulation

Background: Erythropoietic protoporphyria (EPP) is a rare genetic photodermatosis caused by loss-of-function mutations in the gene for ferrochelatase leading to accumulation of the fluorescent protoporphyrin IX (PpIX) in erythrocytes. The mutations are most often inherited mutations present in all cells causing inherited EPP. In very rare cases EPP are acquired in association with myelodysplastic syndromes or myeloproliferative neoplasms, conditions with genetic instability. Case report: We report a case of acquired EPP in association with hematological disease. We followed erythrocyte PpIX concentration over a year and measured PpIX fluorescence in individual erythrocytes in a blood sample from the case using flow cytometry. The major proportion of erythrocytes did not fluoresce (84%), whereas 13% contained low PpIX fluorescence, 1% contained medium fluorescence, and 2% contained high fluorescence. Discussion: Our observation of the very skewed PpIX distribution in erythrocytes supports the description that acquired EPP is caused by a somatic mutation effecting a clone of hematopoietic cells.

Several trials for the COVID-19 pandemic are found. Prof. Shunji Nakamura, Tokushima University was awarded the 2014 Nobel prize in Physics for light-emitting diodes (LEDs). Deep ultraviolet LED (DUV-LED) causes SARS-CoV-2 inactivation as 99.9% by 265 nm. Gc protein-derived macrophage-activating factor (GcMAF)-based immunotherapy has a wider application for serious infection, chronic fatigue syndrome, and cancers. Covid center in Naples submitted a phase II report concerning GcMAF and COVID-19 in Feb 2021. 5-aminolevulinic acid (5-ALA) and its metabolite protoporphyrin IX (PpIX) show anti-inflammatory and antiviral effects for Zika, Dengue, influenza A viruses, and SARS-CoV-2. These results hopefully contribute to the research development for COVID-19.

5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX) fluorescence is widely used for the intraoperative detection of malignant tumors. However, the fluorescence emission profiles of the accompanying necrotic regions of these tumors have yet to be determined. To address this, we performed fluorescence and high-performance liquid chromatography (HPLC) analyses of necrotic tissues of squamous cancer after 5-ALA administration. In resected human lymph nodes of metastatic squamous cell carcinoma, we found a fluorescence peak at approximately 620 nm in necrotic lesions, which was distinct from the PpIX fluorescence peak at 635 nm for viable cancer lesions. Necrotic lesions obtained from a subcutaneous xenograft model of human B88 oral squamous cancer also emitted the characteristic fluorescence peak at 620 nm after light irradiation: the fluorescence intensity ratio (620 nm/635 nm) increased with the energy of the irradiation light. HPLC analysis revealed a high content ratio of uroporphyrin I (UPI)/total porphyrins in the necrotic cores of murine tumors, indicating that UPI is responsible for the 620 nm peak. UPI accumulation in necrotic tissues after 5-ALA administration was possibly due to the failure of the heme biosynthetic pathway. Taken together, fluorescence imaging of UPI after 5-ALA administration may be applicable for the evaluation of tumor necrosis.

Background and Objectives: Thermo-mechanical fractional injury (TMFI) impacts the skin barrier and may increase cutaneous drug uptake. This study investigated the potential of TMFI in combination with 5-aminolevulinic acid (ALA) cream and gel formulations to enhance Protoporphyrin IX (PpIX) fluorescence at the skin surface and in the skin. Study Design/Materials and Methods: In healthy volunteers (n = 12) a total of 144 test areas were demarcated on the upper back. Test areas were randomized to (i) TMFI (6 milliseconds, 400 µm at a single pass) or no pretreatment and (ii) 20% ALA in cream or gel formulations. Skin surface PpIX fluorescence was quantified by PpIX fluorescence photography and photometry in 30-minute intervals until 3 hours. PpIX fluorescence microscopy quantified separate PpIX fluorescence in the epidermis, and in superficial-, mid-, and deep- dermis from punch biopsies sampled after 3 hours of ALA incubation. Local skin reactions (LSR) and pain intensities (numerical rating scale 0–10) were evaluated immediately, at 3 hours and 14 days after the intervention. Results: TMFI exposure before photosensitizer application significantly increased skin surface PpIX fluorescence, both for ALA cream (TMFI-ALA-cream 7848 arbitrary units [AU] vs. ALA-cream 5441 AU, 3 hours, P < 0.001) and ALA gel (TMFI + ALA-gel 4591 AU vs. ALA-gel 3723 AU, 3 hours, P < 0.001). The TMFI-mediated increase in PpIX fluorescence was similar for ALA-cream and -gel formulations (P = 0.470) at the skin surface. In the epidermis, PpIX fluorescence intensities increased from combination treatment with TMFI and ALA-cream (TMFI + ALA-cream 421 AU vs. ALA-cream 293 AU, P = 0.034) but not from combination with TMFI and ALA-gel (TMI + ALA-gel 264 AU vs. ALA-gel 261 AU, P = 0.791). Dermal fluorescence intensities (superficial-, mid-, or deep dermis) were unaffected by TMFI pretreatment in both ALA-cream and ALA-gel exposed skin (P = 0.339). ALA-cream generally induced higher PpIX fluo

Background: In glioblastoma (GB), tissue is required for accurate diagnosis and subtyping. Tissue can be obtained through resection or (stereotactic) biopsy, but these invasive procedures provide risks for patients. Extracellular vesicles (EVs) are small, cell-derived vesicles that contain miRNAs, proteins, and lipids, and possible candidates for liquid biopsies. GB-derived EVs can be found in the blood of patients, but it is difficult to distinguish them from circulating non-tumor EVs. 5-aminolevulinic acid (5-ALA) is orally administered to GB patients to facilitate tumor visualization and maximal resection, as it is metabolized to fluorescent protoporphyrin IX (PpIX) that accumulates in glioma cells. In this study, we assessed whether PpIX accumulates in GB-derived EVs and whether these EVs could be isolated and characterized to enable a liquid biopsy in GB. Methods: EVs were isolated from the conditioned media of U87 cells treated with 5-ALA by differential ultracentrifugation. Blood samples were collected and processed from healthy controls and patients undergoing 5-ALA guided surgery for GB. High-resolution flow cytometry (hFC) enabled detection and sorting of PpIX-positive EVs, which were subsequently analyzed by digital droplet PCR (ddPCR). Results: PpIX-positive EVs could be detected in conditioned cell culture media as well as in patient samples after administration of 5-ALA. By using hFC, we could sort the PpIX-positive EVs for further analysis with ddPCR, which indicated the presence of EVs and GB-associated miRNAs. Conclusion: GB-derived EVs can be isolated from the plasma of GB patients by using 5-ALA induced fluorescence. Although many challenges remain, our findings show new possibilities for the development of blood-based liquid biopsies in GB patients.

[Background] The 5-aminolevulinic acid (ALA)-based photodynamic diagnosis is based on the accumulation of photosensitizing protoporphyrin IX in the tumor after ALA administration. However, the mechanisms connecting exogenous ALA and tumor fluorescence in pancreatic cancer remain unclear. We aimed to elucidate the mechanism underlying the ALA-induced fluorescent. [Methods] Human pancreatic duct epithelial cells (hPDECs) and pancreatic cancer cell lines were used. The expressions of ALA-associated enzymes and membrane transporters in these cell lines were investigated. ALA-induced fluorescence was also investigated. [Results] The expression of oligopeptide transporter-1 (PEPT-1), through which ALA is absorbed, was significantly higher in AsPC-1 cells and lower in MIA PaCa-2 cells than in hPDECs. AsPC-1 cells showed rapid and intense fluorescence after ALA administration, and that was attenuated by PEPT-1 inhibition. ALA-induced fluorescence was not sufficiently strong in MIA PaCa-2 cells to distinguish the cells from hPDECs. [Conclusion] We revealed the association of PEPT-1 with ALA-induced fluorescence. Cancers expressing PEPT-1 could be easily distinguished by this technique from normal cells. These findings help develop novel diagnostic modalities for pancreatic cancer.

Background: In glioblastoma (GB), tissue is required for accurate diagnosis and subtyping. Tissue can be obtained through resection or (stereotactic) biopsy, but these invasive procedures provide risks for patients. Extracellular vesicles (EVs) are small, cell-derived vesicles that contain miRNAs, proteins, and lipids, and possible candidates for liquid biopsies. GB-derived EVs can be found in the blood of patients, but it is difficult to distinguish them from circulating non-tumor EVs. 5-aminolevulinic acid (5-ALA) is orally administered to GB patients to facilitate tumor visualization and maximal resection, as it is metabolized to fluorescent protoporphyrin IX (PpIX) that accumulates in glioma cells

Purpose5-aminolevulinic acid (5-ALA) fluorescence-guided surgery (FGS) appears to be a promising treatment for glioma. However, 5-ALA-mediated fluorescence cannot always be detected in grade II/III gliomas. We hypothesized that gene expression patterns in the Protoporphyrin IX (PpIX) synthesis pathway may be associated with intraoperative fluorescence status of grade II/III gliomas, and then attempted to identify the key molecule of 5-ALA-mediated fluorescence.MethodsUsing 50 surgically obtained specimens, which were diagnosed as grade II and III gliomas, we analyzed gene expression within the PpIX synthesis pathway to identify candidate molecules according to intraoperative 5-ALA-mediated fluorescence status. The most likely candidate gene was selected and confirmed by protein expression analysis. To evaluate the biological function of the molecule in PpIX synthesis, functional analysis was performed using specific, small interference (si)RNA in the SW-1783human grade III glioma cell line.ResultsAmong the genes involved in the porphyrin synthesis pathway, the mRNA expression of Peptide transporter 2 (PEPT2) in FGS fluorescence-positive gliomas was significantly higher than that in fluorescence-negative gliomas. Protein expression of PEPT2 was also significantly higher in the fluorescence-positive gliomas, which was confirmed by western blot analysis and immunofluorescence analysis. The siRNA-mediated downregulation of the mRNA and protein expression of PEPT2 led to decreased PpIX fluorescence intensity, as confirmed by fluorescence spectrum analysis.ConclusionsThe results suggest PEPT2 is an important candidate molecule in 5-ALA-mediated FGS in grade II/III gliomas. As the overexpression of PEPT2 was associated with higher PpIX fluorescence intensity, PEPT2 may improve fluorescence-guided resection in grade II/III gliomas.

Purpose5-aminolevulinic acid (5-ALA) fluorescence-guided surgery (FGS) appears to be a promising treatment for glioma. However, 5-ALA-mediated fluorescence cannot always be detected in grade II/III gliomas. We hypothesized that gene expression patterns in the Protoporphyrin IX (PpIX) synthesis pathway may be associated with intraoperative fluorescence status of grade II/III gliomas, and then attempted to identify the key molecule of 5-ALA-mediated fluorescence.MethodsUsing 50 surgically obtained specimens, which were diagnosed as grade II and III gliomas, we analyzed gene expression within the PpIX synthesis pathway to identify candidate molecules according to intraoperative 5-ALA-mediated fluorescence status. The most likely candidate gene was selected and confirmed by protein expression analysis. To evaluate the biological function of the molecule in PpIX synthesis, functional analysis was performed using specific, small interference (si)RNA in the SW-1783human grade III glioma cell line.ResultsAmong the genes involved in the porphyrin synthesis pathway, the mRNA expression of Peptide transporter 2 (PEPT2) in FGS fluorescence-positive gliomas was significantly higher than that in fluorescence-negative gliomas. Protein expression of PEPT2 was also significantly higher in the fluorescence-positive gliomas, which was confirmed by western blot analysis and immunofluorescence analysis. The siRNA-mediated downregulation of the mRNA and protein expression of PEPT2 led to decreased PpIX fluorescence intensity, as confirmed by fluorescence spectrum analysis.ConclusionsThe results suggest PEPT2 is an important candidate molecule in 5-ALA-mediated FGS in grade II/III gliomas. As the overexpression of PEPT2 was associated with higher PpIX fluorescence intensity, PEPT2 may improve fluorescence-guided resection in grade II/III gliomas.

Purpose5-aminolevulinic acid (5-ALA) fluorescence-guided surgery (FGS) appears to be a promising treatment for glioma. However, 5-ALA-mediated fluorescence cannot always be detected in grade II/III gliomas. We hypothesized that gene expression patterns in the Protoporphyrin IX (PpIX) synthesis pathway may be associated with intraoperative fluorescence status of grade II/III gliomas, and then attempted to identify the key molecule of 5-ALA-mediated fluorescence.MethodsUsing 50 surgically obtained specimens, which were diagnosed as grade II and III gliomas, we analyzed gene expression within the PpIX synthesis pathway to identify candidate molecules according to intraoperative 5-ALA-mediated fluorescence status. The most likely candidate gene was selected and confirmed by protein expression analysis. To evaluate the biological function of the molecule in PpIX synthesis, functional analysis was performed using specific, small interference (si)RNA in the SW-1783human grade III glioma cell line.ResultsAmong the genes involved in the porphyrin synthesis pathway, the mRNA expression of Peptide transporter 2 (PEPT2) in FGS fluorescence-positive gliomas was significantly higher than that in fluorescence-negative gliomas. Protein expression of PEPT2 was also significantly higher in the fluorescence-positive gliomas, which was confirmed by western blot analysis and immunofluorescence analysis. The siRNA-mediated downregulation of the mRNA and protein expression of PEPT2 led to decreased PpIX fluorescence intensity, as confirmed by fluorescence spectrum analysis.ConclusionsThe results suggest PEPT2 is an important candidate molecule in 5-ALA-mediated FGS in grade II/III gliomas. As the overexpression of PEPT2 was associated with higher PpIX fluorescence intensity, PEPT2 may improve fluorescence-guided resection in grade II/III gliomas.

Purpose5-aminolevulinic acid (5-ALA) fluorescence-guided surgery (FGS) appears to be a promising treatment for glioma. However, 5-ALA-mediated fluorescence cannot always be detected in grade II/III gliomas. We hypothesized that gene expression patterns in the Protoporphyrin IX (PpIX) synthesis pathway may be associated with intraoperative fluorescence status of grade II/III gliomas, and then attempted to identify the key molecule of 5-ALA-mediated fluorescence.MethodsUsing 50 surgically obtained specimens, which were diagnosed as grade II and III gliomas, we analyzed gene expression within the PpIX synthesis pathway to identify candidate molecules according to intraoperative 5-ALA-mediated fluorescence status. The most likely candidate gene was selected and confirmed by protein expression analysis. To evaluate the biological function of the molecule in PpIX synthesis, functional analysis was performed using specific, small interference (si)RNA in the SW-1783human grade III glioma cell line.ResultsAmong the genes involved in the porphyrin synthesis pathway, the mRNA expression of Peptide transporter 2 (PEPT2) in FGS fluorescence-positive gliomas was significantly higher than that in fluorescence-negative gliomas. Protein expression of PEPT2 was also significantly higher in the fluorescence-positive gliomas, which was confirmed by western blot analysis and immunofluorescence analysis. The siRNA-mediated downregulation of the mRNA and protein expression of PEPT2 led to decreased PpIX fluorescence intensity, as confirmed by fluorescence spectrum analysis.ConclusionsThe results suggest PEPT2 is an important candidate molecule in 5-ALA-mediated FGS in grade II/III gliomas. As the overexpression of PEPT2 was associated with higher PpIX fluorescence intensity, PEPT2 may improve fluorescence-guided resection in grade II/III gliomas.

This review focuses on the studies to investigate the feasibility of 5-aminolevulinic acid （5-ALA）-mediated photodynamic diagnosis（PDD） in particular, for detecting gastric cancer and upper GI tumors in the earlier stage. Using a prototype endoscope equipped with a blue laser light excitation for protoporphyrin IX, a photosensitizer, 5-ALA-mediated PDD was conducted in 30 cases of gastric cancer and upper GI tumors. 5-ALA solution was given orally 3 hours before PDD. Red fluorescence signal and intensity was assessed as for clinicopathological features of the cases. The laser-based endoscopy could detect upper GI tumors as red fluorescent navigation （PDD-positive） in 77%（23/30）．There were nominal adverse events. Thus, 5-ALA-PDD using the blue laser-equipped endoscope offers a promising diagnostic tool for the other digestive tumors such as colonic and pancreatic tumors. Further studies on the basic mechanisms of the difference in fluorescence intensity are warrant to be executive.

5-aminolevulinic-acid-(5-ALA)-induced protoporphyrin IX (PpIX) fluorescence may be used to improve stereotactic brain tumor biopsies. In this study, the sensitivity of PpIX-based tumor detection has been investigated for two potential excitation wavelengths (405 nm, 633 nm). Using a 200 μm fiber in contact with semi-infinite optical phantoms containing ink and Lipovenös, PpIX detection limits of 4.0 nM and 200 nM (relating to 1 mW excitation power) were determined for 405 nm and 633 nm excitation, respectively. Hence, typical PpIX concentrations in glioblastomas of a few μM should be well detectable with both wavelengths. Additionally, blood layers of selected thicknesses were placed between fiber and phantom. Red excitation was shown to be considerably less affected by blood interference: A 50 μm blood layer, for instance, blocked the 405- nm-excited fluorescence completely, but reduced the 633-nm-excited signal by less than 50%. Ray tracing simulations demonstrated that - without blood layer - the sensitivity advantage of 405 nm rises for decreasing fluorescent volume from 50-fold to a maximum of 100-fold. However, at a tumor volume of 1 mm3, which is a typical biopsy sample size, the 633-nm-excited fluorescence signal is only reduced by about 10%. Further simulations revealed that with increasing fiber-tumor distance, the signal drops faster for 405 nm. This reduces the risk of detecting tumor tissue outside the needle's coverage, but diminishes the overlap between optically and mechanically sampled volumes. While 405 nm generally offers a higher sensitivity, 633 nm is more sensitive to distant tumors and considerably superior in case of blood-covered tumor tissue., Funding agencies|German Ministry of Education and Research (BMBF)

Fluorescence diagnosis may be used to improve the safety and reliability of stereotactic brain tumor biopsies using biopsy needles with integrated fiber optics. Based on 5-aminolevulinic-acid-induced protoporphyrin IX (PpIX) fluorescence, vital tumor tissue can be localized in vivo during the excision procedure to reduce the number of necessary samples for a reliable diagnosis. In this study, the practical suitability of two different PpIX excitation wavelengths (405 nm, 633 nm) was investigated on optical phantoms. Violet excitation at 405 nm provides a 50-fold higher sensitivity for the bulk tumor; this factor increases up to 100 with decreasing fluorescent volume as shown by ray tracing simulations. Red excitation at 633 nm, however, is noticeably superior with regard to blood layers obscuring the fluorescence. Experimental results on the signal attenuation through blood layers of well-defined thicknesses could be confirmed by ray tracing simulations. Typical interstitial fiber probe measurements were mimicked on agarose-gel phantoms. Even in direct contact, blood layers of 20-40 µm between probe and tissue must be expected, obscuring 405-nm-excited PpIX fluorescence almost completely, but reducing the 633-nm-excited signal only by 25.5%. Thus, 633 nm seems to be the wavelength of choice for PpIX-assisted detection of high-grade gliomas in stereotactic biopsy. PpIX signal attenuation through clinically relevant blood layers for 405 nm (violet) and 633 nm (red) excitation., Funding agencies: German Ministry of Education and Research (BMBF) [01DJ14012B]

Fluorescence diagnosis may be used to improve the safety and reliability of stereotactic brain tumor biopsies using biopsy needles with integrated fiber optics. Based on 5-aminolevulinic-acid-induced protoporphyrin IX (PpIX) fluorescence, vital tumor tissue can be localized in vivo during the excision procedure to reduce the number of necessary samples for a reliable diagnosis. In this study, the practical suitability of two different PpIX excitation wavelengths (405 nm, 633 nm) was investigated on optical phantoms. Violet excitation at 405 nm provides a 50-fold higher sensitivity for the bulk tumor; this factor increases up to 100 with decreasing fluorescent volume as shown by ray tracing simulations. Red excitation at 633 nm, however, is noticeably superior with regard to blood layers obscuring the fluorescence. Experimental results on the signal attenuation through blood layers of well-defined thicknesses could be confirmed by ray tracing simulations. Typical interstitial fiber probe measurements were mimicked on agarose-gel phantoms. Even in direct contact, blood layers of 20-40 µm between probe and tissue must be expected, obscuring 405-nm-excited PpIX fluorescence almost completely, but reducing the 633-nm-excited signal only by 25.5%. Thus, 633 nm seems to be the wavelength of choice for PpIX-assisted detection of high-grade gliomas in stereotactic biopsy. PpIX signal attenuation through clinically relevant blood layers for 405 nm (violet) and 633 nm (red) excitation., Funding agencies: German Ministry of Education and Research (BMBF) [01DJ14012B]

The oligomeric Mg chelatase (MgCh), consisting of the subunits CHLH, CHLI, and CHLD, is located at the central site of chlorophyll synthesis, but is also thought to have an additional function in regulatory feedback control of the tetrapyrrole biosynthesis pathway and in chloroplast retrograde signaling. In Arabidopsis thaliana and Chlamydomonas reinhardtii, two genes have been proposed to encode the CHLI subunit of MgCh. While the role of CHLI1 in A. thaliana MgCh has been substantially elucidated, different reports provide inconsistent results with regard to the function of CHLI2 in Mg chelation and retrograde signaling. In the present report, the possible functions of both isoforms were analyzed in C. reinhardtii Knockout of the CHLI1 gene resulted in complete loss of MgCh activity, absence of chlorophyll, acute light sensitivity, and, as a consequence, down-regulation of tetrapyrrole biosynthesis and photosynthesis-associated nuclear genes. These observations indicate a phenotypical resemblance of chli1 to the chlh and chld C. reinhardtii mutants previously reported. The key role of CHLI1 for MgCh reaction in comparison with the second isoform was confirmed by the rescue of chli1 with genomic CHLI1 Because CHLI2 in C. reinhardtii shows lower expression than CHLI1, strains overexpressing CHLI2 were produced in the chli1 background. However, no complementation of the chli1 phenotype was observed. Silencing of CHLI2 in the wild-type background did not result in any changes in the accumulation of tetrapyrrole intermediates or of chlorophyll. The results suggest that, unlike in A. thaliana, changes in CHLI2 content observed in the present studies do not affect formation and activity of MgCh in C. reinhardtii.

The oligomeric Mg chelatase (MgCh), consisting of the subunits CHLH, CHLI, and CHLD, is located at the central site of chlorophyll synthesis, but is also thought to have an additional function in regulatory feedback control of the tetrapyrrole biosynthesis pathway and in chloroplast retrograde signaling. In Arabidopsis thaliana and Chlamydomonas reinhardtii, two genes have been proposed to encode the CHLI subunit of MgCh. While the role of CHLI1 in A. thaliana MgCh has been substantially elucidated, different reports provide inconsistent results with regard to the function of CHLI2 in Mg chelation and retrograde signaling. In the present report, the possible functions of both isoforms were analyzed in C. reinhardtii Knockout of the CHLI1 gene resulted in complete loss of MgCh activity, absence of chlorophyll, acute light sensitivity, and, as a consequence, down-regulation of tetrapyrrole biosynthesis and photosynthesis-associated nuclear genes. These observations indicate a phenotypical resemblance of chli1 to the chlh and chld C. reinhardtii mutants previously reported. The key role of CHLI1 for MgCh reaction in comparison with the second isoform was confirmed by the rescue of chli1 with genomic CHLI1 Because CHLI2 in C. reinhardtii shows lower expression than CHLI1, strains overexpressing CHLI2 were produced in the chli1 background. However, no complementation of the chli1 phenotype was observed. Silencing of CHLI2 in the wild-type background did not result in any changes in the accumulation of tetrapyrrole intermediates or of chlorophyll. The results suggest that, unlike in A. thaliana, changes in CHLI2 content observed in the present studies do not affect formation and activity of MgCh in C. reinhardtii.

Photodynamic diagnosis based on 5-aminolevulinic acid-induced protoporphyrin IX has been clinically applied in many fields based upon its evidenced efficacy and adequate safety. In order to establish a personalized medicine approach for treating gastric cancer patients, rapid intraoperative detection of malignant lesions has become important. Feasibility of photodynamic diagnosis using 5-aminolevulinic acid for gastric cancer patients has been investigated, especially for the detection of peritoneal dissemination and lymph node metastasis. This method enables intraoperative real-time fluorescence detection of peritoneal dissemination, exhibiting higher sensitivity than white light observation without histopathological examination. The method also enables detection of metastatic foci within excised lymph nodes, exhibiting a diagnostic accuracy comparable to that of a current molecular diagnostics technique. Although several complicating issues still need to be resolved, such as the effect of tissue autofluorescence and the insufficient depth penetration of excitation light, this simple and rapid method has the potential to become a useful diagnostic tool for gastric cancer, as well as urinary bladder cancer and glioma.

Fluorescence diagnosis may be used to improve the safety and reliability of stereotactic brain tumor biopsies using biopsy needles with integrated fiber optics. Based on 5-aminolevulinic-acid-induced protoporphyrin IX (PpIX) fluorescence, vital tumor tissue can be localized in vivo during the excision procedure to reduce the number of necessary samples for a reliable diagnosis. In this study, the practical suitability of two different PpIX excitation wavelengths (405 nm, 633 nm) was investigated on optical phantoms. Violet excitation at 405 nm provides a 50-fold higher sensitivity for the bulk tumor; this factor increases up to 100 with decreasing fluorescent volume as shown by ray tracing simulations. Red excitation at 633 nm, however, is noticeably superior with regard to blood layers obscuring the fluorescence. Experimental results on the signal attenuation through blood layers of well-defined thicknesses could be confirmed by ray tracing simulations. Typical interstitial fiber probe measurements were mimicked on agarose-gel phantoms. Even in direct contact, blood layers of 20-40 µm between probe and tissue must be expected, obscuring 405-nm-excited PpIX fluorescence almost completely, but reducing the 633-nm-excited signal only by 25.5%. Thus, 633 nm seems to be the wavelength of choice for PpIX-assisted detection of high-grade gliomas in stereotactic biopsy. PpIX signal attenuation through clinically relevant blood layers for 405 nm (violet) and 633 nm (red) excitation., Funding agencies: German Ministry of Education and Research (BMBF) [01DJ14012B]

Fluorescence diagnosis may be used to improve the safety and reliability of stereotactic brain tumor biopsies using biopsy needles with integrated fiber optics. Based on 5-aminolevulinic-acid-induced protoporphyrin IX (PpIX) fluorescence, vital tumor tissue can be localized in vivo during the excision procedure to reduce the number of necessary samples for a reliable diagnosis. In this study, the practical suitability of two different PpIX excitation wavelengths (405 nm, 633 nm) was investigated on optical phantoms. Violet excitation at 405 nm provides a 50-fold higher sensitivity for the bulk tumor; this factor increases up to 100 with decreasing fluorescent volume as shown by ray tracing simulations. Red excitation at 633 nm, however, is noticeably superior with regard to blood layers obscuring the fluorescence. Experimental results on the signal attenuation through blood layers of well-defined thicknesses could be confirmed by ray tracing simulations. Typical interstitial fiber probe measurements were mimicked on agarose-gel phantoms. Even in direct contact, blood layers of 20-40 µm between probe and tissue must be expected, obscuring 405-nm-excited PpIX fluorescence almost completely, but reducing the 633-nm-excited signal only by 25.5%. Thus, 633 nm seems to be the wavelength of choice for PpIX-assisted detection of high-grade gliomas in stereotactic biopsy. PpIX signal attenuation through clinically relevant blood layers for 405 nm (violet) and 633 nm (red) excitation., Funding agencies: German Ministry of Education and Research (BMBF) [01DJ14012B]

Fluorescence diagnosis may be used to improve the safety and reliability of stereotactic brain tumor biopsies using biopsy needles with integrated fiber optics. Based on 5-aminolevulinic-acid-induced protoporphyrin IX (PpIX) fluorescence, vital tumor tissue can be localized in vivo during the excision procedure to reduce the number of necessary samples for a reliable diagnosis. In this study, the practical suitability of two different PpIX excitation wavelengths (405 nm, 633 nm) was investigated on optical phantoms. Violet excitation at 405 nm provides a 50-fold higher sensitivity for the bulk tumor; this factor increases up to 100 with decreasing fluorescent volume as shown by ray tracing simulations. Red excitation at 633 nm, however, is noticeably superior with regard to blood layers obscuring the fluorescence. Experimental results on the signal attenuation through blood layers of well-defined thicknesses could be confirmed by ray tracing simulations. Typical interstitial fiber probe measurements were mimicked on agarose-gel phantoms. Even in direct contact, blood layers of 20-40 µm between probe and tissue must be expected, obscuring 405-nm-excited PpIX fluorescence almost completely, but reducing the 633-nm-excited signal only by 25.5%. Thus, 633 nm seems to be the wavelength of choice for PpIX-assisted detection of high-grade gliomas in stereotactic biopsy. PpIX signal attenuation through clinically relevant blood layers for 405 nm (violet) and 633 nm (red) excitation., Funding agencies: German Ministry of Education and Research (BMBF) [01DJ14012B]

Background Photodynamic therapy (PDT) delays ultraviolet (UV) radiation-induced squamous cell carcinomas (SCCs) in hairless mice. Efficacy may be enhanced by combining PDT with antineoplastic or pro-differentiating agents. We investigated if pretreatment with 5-fluorouracil (5FU), imiquimod (IMIQ) or calcipotriol (CAL) before PDT further delays tumor onset. Methods Hairless mice (n = 224) were exposed 3 times weekly to 3 standard erythema doses (SED) of UV radiation. Methyl-aminolevulinate (MAL)-PDT sessions were given on days 45 and 90 before SCC development. Three applications of topical 5FU, IMIQ or CAL were given before each PDT session. Fluorescence photography quantified protoporphyrin IX (PpIX) formation. Results PDT delayed UV-induced SCC development by 59 days (212 days UV-MAL-PDT vs. 153 days UV-control, P < 0.001). Pretreatment with 5FU, IMIQ or CAL before PDT did not further delay SCC onset compared to PDT alone (207 days UV-5FU-MAL-PDT, 215 days UV-IMIQ-MAL-PDT, 206 days UV-CAL-MAL-PDT vs. 212 days UV-MAL-PDT, P = ns). PpIX fluorescence intensified by 5FU-pretreatment (median 21,392 au UV-5FU-MAL-PDT, P = 0.011), decreased after IMIQ-pretreatment (12,452 au UV-IMIQ-MAL-PDT, P < 0.001), and was unaffected by CAL-pretreatment (19,567 au UV-CAL-MAL-PDT, P = ns) compared to MAL alone (18,083 au UV-MAL-PDT). Conclusions Short-term three-day pretreatment with 5FU, IMIQ and CAL before PDT does not further delay tumor onset in UV-exposed hairless mice.

Background Using 5-aminolevulinic acid (ALA) as an intraoperative fluorescence contrast has been proven to improve the resection of glioblastoma and contribute to prolonged patient survival. ALA accumulates as protoporphyrin IX (PpIX) in the tumor cells and is administered in an advised dose of 20 mg/kg body weight (b.w.) for brain tumor resection using fluorescence surgical microscopes. PpIX fluorescence availability and intensities of a four folds lower ALA dose (5 mg/kg b.w.) has been investigated in glioblastomas and skin using a spectroscopy system adapted for surgical guidance. Methods A total of 30 adult patients diagnosed with high grade gliomas were included in the analysis. ALA was orally administered in doses of 5 mg/kg b.w. (n = 15) dissolved in orange juice or 20 mg/kg b.w. (n = 15) dissolved in water. A fluorescence spectroscopy system with a handheld fiber-optical probe was used for performing the quantitative fluorescence measurements. Results The binominal comparison of the diagnostic performance parameters showed no significant statistical difference (p > 0.05). The median fluorescence values in tumor were 2-3 times higher for the high ALA dose group. No PpIX was detected in the skin of the patients in the low dose group (0/4) while PpIX was detected in the skin of the majority of the patients in the high ALA dose group (13/14). Conclusions Application of 5 mg/kg ALA was evaluated as equally reliable as the higher dose regarding the diagnostic performance when guidance was performed using a spectroscopic system. Moreover, no PpIX was detected in the skin of the patients.

Background Using 5-aminolevulinic acid (ALA) as an intraoperative fluorescence contrast has been proven to improve the resection of glioblastoma and contribute to prolonged patient survival. ALA accumulates as protoporphyrin IX (PpIX) in the tumor cells and is administered in an advised dose of 20 mg/kg body weight (b.w.) for brain tumor resection using fluorescence surgical microscopes. PpIX fluorescence availability and intensities of a four folds lower ALA dose (5 mg/kg b.w.) has been investigated in glioblastomas and skin using a spectroscopy system adapted for surgical guidance. Methods A total of 30 adult patients diagnosed with high grade gliomas were included in the analysis. ALA was orally administered in doses of 5 mg/kg b.w. (n = 15) dissolved in orange juice or 20 mg/kg b.w. (n = 15) dissolved in water. A fluorescence spectroscopy system with a handheld fiber-optical probe was used for performing the quantitative fluorescence measurements. Results The binominal comparison of the diagnostic performance parameters showed no significant statistical difference (p > 0.05). The median fluorescence values in tumor were 2-3 times higher for the high ALA dose group. No PpIX was detected in the skin of the patients in the low dose group (0/4) while PpIX was detected in the skin of the majority of the patients in the high ALA dose group (13/14). Conclusions Application of 5 mg/kg ALA was evaluated as equally reliable as the higher dose regarding the diagnostic performance when guidance was performed using a spectroscopic system. Moreover, no PpIX was detected in the skin of the patients.

Because it is highly infiltrative, malignant glioma is a cancer with a poor prognosis despite multidisciplinary treatment strategies, such as aggressive surgery and chemoradiotherapy, necessitating new therapeutic approaches to control migration of tumor cells. In our study, we investigated the efficacy of sonodynamic therapy of glioma cells in vitro using porphyrin derivatives, including 5-aminolevulinic acid, protoporphyrin IX and talaporfin sodium, as sonosensitizers. These substances have been known to accumulate in glioma cells and are expected to have cytotoxic effects on sonication. Our study found that the cytotoxicity of sonication of glioma cells is enhanced by each sonosensitizer and that the efficacy of sonodynamic therapy may depend on the degree of intracellular accumulation of sonosensitizer. Also, the study suggests that induction of apoptosis is a major mechanism underlying cell death. Though further investigations are necessary, our preliminary result indicates a potential for sonodynamic therapy with sonosensitizers in glioma treatment.

Background Using 5-aminolevulinic acid (ALA) as an intraoperative fluorescence contrast has been proven to improve the resection of glioblastoma and contribute to prolonged patient survival. ALA accumulates as protoporphyrin IX (PpIX) in the tumor cells and is administered in an advised dose of 20 mg/kg body weight (b.w.) for brain tumor resection using fluorescence surgical microscopes. PpIX fluorescence availability and intensities of a four folds lower ALA dose (5 mg/kg b.w.) has been investigated in glioblastomas and skin using a spectroscopy system adapted for surgical guidance. Methods A total of 30 adult patients diagnosed with high grade gliomas were included in the analysis. ALA was orally administered in doses of 5 mg/kg b.w. (n = 15) dissolved in orange juice or 20 mg/kg b.w. (n = 15) dissolved in water. A fluorescence spectroscopy system with a handheld fiber-optical probe was used for performing the quantitative fluorescence measurements. Results The binominal comparison of the diagnostic performance parameters showed no significant statistical difference (p > 0.05). The median fluorescence values in tumor were 2-3 times higher for the high ALA dose group. No PpIX was detected in the skin of the patients in the low dose group (0/4) while PpIX was detected in the skin of the majority of the patients in the high ALA dose group (13/14). Conclusions Application of 5 mg/kg ALA was evaluated as equally reliable as the higher dose regarding the diagnostic performance when guidance was performed using a spectroscopic system. Moreover, no PpIX was detected in the skin of the patients.

Because it is highly infiltrative, malignant glioma is a cancer with a poor prognosis despite multidisciplinary treatment strategies, such as aggressive surgery and chemoradiotherapy, necessitating new therapeutic approaches to control migration of tumor cells. In our study, we investigated the efficacy of sonodynamic therapy of glioma cells in vitro using porphyrin derivatives, including 5-aminolevulinic acid, protoporphyrin IX and talaporfin sodium, as sonosensitizers. These substances have been known to accumulate in glioma cells and are expected to have cytotoxic effects on sonication. Our study found that the cytotoxicity of sonication of glioma cells is enhanced by each sonosensitizer and that the efficacy of sonodynamic therapy may depend on the degree of intracellular accumulation of sonosensitizer. Also, the study suggests that induction of apoptosis is a major mechanism underlying cell death. Though further investigations are necessary, our preliminary result indicates a potential for sonodynamic therapy with sonosensitizers in glioma treatment.

Background Using 5-aminolevulinic acid (ALA) as an intraoperative fluorescence contrast has been proven to improve the resection of glioblastoma and contribute to prolonged patient survival. ALA accumulates as protoporphyrin IX (PpIX) in the tumor cells and is administered in an advised dose of 20 mg/kg body weight (b.w.) for brain tumor resection using fluorescence surgical microscopes. PpIX fluorescence availability and intensities of a four folds lower ALA dose (5 mg/kg b.w.) has been investigated in glioblastomas and skin using a spectroscopy system adapted for surgical guidance. Methods A total of 30 adult patients diagnosed with high grade gliomas were included in the analysis. ALA was orally administered in doses of 5 mg/kg b.w. (n = 15) dissolved in orange juice or 20 mg/kg b.w. (n = 15) dissolved in water. A fluorescence spectroscopy system with a handheld fiber-optical probe was used for performing the quantitative fluorescence measurements. Results The binominal comparison of the diagnostic performance parameters showed no significant statistical difference (p > 0.05). The median fluorescence values in tumor were 2-3 times higher for the high ALA dose group. No PpIX was detected in the skin of the patients in the low dose group (0/4) while PpIX was detected in the skin of the majority of the patients in the high ALA dose group (13/14). Conclusions Application of 5 mg/kg ALA was evaluated as equally reliable as the higher dose regarding the diagnostic performance when guidance was performed using a spectroscopic system. Moreover, no PpIX was detected in the skin of the patients.

Background Using 5-aminolevulinic acid (ALA) as an intraoperative fluorescence contrast has been proven to improve the resection of glioblastoma and contribute to prolonged patient survival. ALA accumulates as protoporphyrin IX (PpIX) in the tumor cells and is administered in an advised dose of 20 mg/kg body weight (b.w.) for brain tumor resection using fluorescence surgical microscopes. PpIX fluorescence availability and intensities of a four folds lower ALA dose (5 mg/kg b.w.) has been investigated in glioblastomas and skin using a spectroscopy system adapted for surgical guidance. Methods A total of 30 adult patients diagnosed with high grade gliomas were included in the analysis. ALA was orally administered in doses of 5 mg/kg b.w. (n = 15) dissolved in orange juice or 20 mg/kg b.w. (n = 15) dissolved in water. A fluorescence spectroscopy system with a handheld fiber-optical probe was used for performing the quantitative fluorescence measurements. Results The binominal comparison of the diagnostic performance parameters showed no significant statistical difference (p > 0.05). The median fluorescence values in tumor were 2-3 times higher for the high ALA dose group. No PpIX was detected in the skin of the patients in the low dose group (0/4) while PpIX was detected in the skin of the majority of the patients in the high ALA dose group (13/14). Conclusions Application of 5 mg/kg ALA was evaluated as equally reliable as the higher dose regarding the diagnostic performance when guidance was performed using a spectroscopic system. Moreover, no PpIX was detected in the skin of the patients.