5-Aminolevulinic acid/sodium ferrous citrate enhanced the antitumor effects of programmed cell death-ligand 1 blockade by regulation of exhausted T cell metabolism in a melanoma model.

Mitochondria are key cytoplasmic organelles. Their activation is critical for the generation of T cell proliferation and cytotoxicity. Exhausted tumor-infiltrating T cells show a decreased mitochondrial function and mass. 5-Aminolevulinic acid (5-ALA), a natural amino acid that is only produced in the mitochondria, has been shown to influence metabolic functions. We hypothesized that 5-ALA with sodium ferrous citrate (SFC) might provide metabolic support for tumor-infiltrating T cells. In a mouse melanoma model, we found that 5-ALA/SFC with a programmed cell death-ligand 1 (PD-L1) blocking Ab synergized tumor regression. After treatment with 5-ALA/SFC and anti-PD-L1 Ab, tumor infiltrating lymphocytes (TILs) were not only competent for the production of cytolytic particles and cytokines (granzyme B, interleukin-2, and γ-interferon) but also showed enhanced Ki-67 activity (a proliferation marker). The number of activated T cells (PD-1+ Tim-3- ) was also significantly increased. Furthermore, we found that 5-ALA/SFC activated the mitochondrial functions, including the oxygen consumption rate, ATP level, and complex V expression. The mRNA levels of Nrf-2, HO-1, Sirt-1, and PGC-1α and the protein levels of Sirt-1 were upregulated by treatment with 5-ALA/SFC. Taken together, our findings revealed that 5-ALA/SFC could be a key metabolic regulator in exhausted T cell metabolism and suggested that 5-ALA/SFC might synergize with anti-PD-1/PD-L1 therapy to boost the intratumoral efficacy of tumor-specific T cells. Our study not only revealed a new aspect of immune metabolism, but also paved the way to develop a strategy for combined anti-PD-1/PD-L1 cancer immunotherapy.

Heterozygous disruption of ALAS1 in mice causes an accelerated age-dependent reduction in free heme, but not total heme, in skeletal muscle and liver.

5-Aminolevulinic acid (ALA) is the rate-limiting intermediate in heme biosynthesis in vertebrate species; a reaction catalyzed by the mitochondrial ALA synthase 1 (ALAS1) enzyme. Previously we reported that knockdown of the ubiquitously expressed ALAS1 gene in mice disrupts normal glucose metabolism, attenuates mitochondrial function and results in a prediabetic like phenotype when animals pass 20-weeks of age (Saitoh et al., 2018). Contrary to our expectations, the cytosolic and mitochondrial heme content of ALAS1 heterozygous (A1+/-) mice were similar to WT animals. Therefore, we speculated that regulatory "free heme" may be reduced in an age dependent manner in A1+/- mice, but not total heme. Here, we examine free and total heme from the skeletal muscle and liver of WT and A1+/- mice using a modified acetone extraction method and examine the effects of aging on free heme by comparing the amounts at 8-12 weeks and 30-36 weeks of age, in addition to the mRNA abundance of ALAS1. We found an age-dependent reduction in free heme in the skeletal muscle and liver of A1+/- mice, while WT mice showed only a slight decrease in the liver. Total heme levels showed no significant difference between young and aged WT and A1+/- mice. ALAS1 mRNA levels showed an age-dependent reduction similar to that of free heme levels, indicating that ALAS1 mRNA expression levels are a major determinant for free heme levels. The free heme pools in skeletal muscle tissue were almost 2-fold larger than that of liver tissue, suggesting that the heme pool varies across different tissue types. The expression of heme oxygenase 1 (HO-1) mRNA, which is expressed proportionally to the amount of free heme, were similar to those of free heme levels. Taken together, this study demonstrates that the free heme pool differs across tissues, and that an age-dependent reduction in free heme levels is accelerated in mice heterozygous for ALAS1, which could account for the prediabetic phenotype and mitochondrial abnormality observed in these animals.

5-aminolaevulinic acid (ALA), enhances heme oxygenase (HO)-1 expression and attenuates tubulointerstitial fibrosis and renal apoptosis in chronic cyclosporine nephropathy.

BACKGROUND: Cyclosporine-A (CsA) is an immunosuppressant indicated for various immunological diseases; however, it can induce chronic kidney injury. Oxidative stress and apoptosis play a crucial role in CsA-induced nephrotoxicity. The present study evaluated the protective effect of combining 5-aminolaevulinic acid with iron (5-ALA/SFC), a precursor of heme synthesis, to enhance HO-1 activity against CsA-induced chronic nephrotoxicity. METHODS: Mice were divided into three groups: the control group (using olive oil as a vehicle), CsA-only group, and CsA+5-ALA/SFC group. After 28 days, the mice were sacrificed, and blood and kidney samples were collected. In addition to histological and biochemical examination, the mRNA expression of proinflammatory and profibrotic cytokines was assessed. RESULTS: Renal function in the 5-ALA/SFC treatment group as assessed by the serum creatinine and serum urea nitrogen levels was superior to that of the CsA-only treatment group, demonstrating that 5-ALA/SFC significantly attenuated CsA-induced kidney tissue inflammation, fibrosis, apoptosis, and tubular atrophy, as well as reducing the mRNA level of TNF-α, IL-6, TGF-ß1, and iNOS while increasing HO-1. CONCLUSION: The activity of 5-ALA/SFC has important implications for clarifying the mechanism of HO-1 activity in CsA-induced nephrotoxicity and may provide a favorable basis for clinical therapy.

5-ALA/SFC Attenuated Binge Alcohol-Induced Gut Leakiness and Inflammatory Liver Disease in HIV Transgenic Rats.

BACKGROUND: This study aimed to investigate the protective effect of 5-aminolevulinic acid (5-ALA) and sodium ferrous citrate (SFC) against binge alcohol-induced gut leakiness and inflammatory liver disease in HIV transgenic (TG) rats. METHODS: TG rats were treated with 3 consecutive doses of binge ethanol (EtOH) with or without 5-ALA/SFC. Blood and liver tissue samples were collected at 6 hours following the last dose of EtOH. RESULTS: Compared with the wild-type (WT) rats, the TG rats showed increased sensitivity to alcohol-mediated inflammation, as evidenced by the significantly elevated levels of serum endotoxin, AST, ALT, ED1, and ED2 staining in liver. In contrast, 5-ALA/SFC improved the above biochemical and histochemical profiles. 5-ALA/SFC also attenuated the up-regulated mRNA expression of leptin and CCL2. Furthermore, down-regulated intestinal ZO-1 protein expression was also inhibited by 5-ALA/SFC. Moreover, the expressions of HO-1, HO-2, Sirt1, and related signal transduction molecules in liver were increased by 5-ALA/SFC. These results demonstrated that 5-ALA/SFC treatment ameliorated binge alcohol exposure liver injury in a rat model of HIV-infected patients by reducing macrophage activation and expression of inflammatory cytokines/chemokines, and by inducing HO-1, HO-2, and Sirt1 expression. CONCLUSIONS: Taken together, these findings suggested that treatment with 5-ALA/SFC has a potential therapeutic effect for binge alcohol exposure liver injury in HIV-infected patients.

Flow Cytometry-Based Photodynamic Diagnosis with 5-Aminolevulinic Acid for the Detection of Minimal Residual Disease in Multiple Myeloma.

Multiple myeloma is the cancer of plasma cells. Along with the development of new and effective therapies, improved outcomes in patients with multiple myeloma have increased the interest in minimal residual disease (MRD) monitoring. However, the considerable heterogeneity of immunophenotypic and molecular markers of myeloma cells has limited its clinical application. 5-Aminolevulinic acid (ALA) is a natural compound in the heme biosynthesis pathway. Following ALA treatment, tumor cells preferentially accumulate porphyrins because of the differential activities of aerobic glycolysis, known as Warburg effect. Among various porphyrins, protoporphyrine IX is a strong photosensitizer; thus, ALA-based photodynamic diagnosis has been widely used in various solid cancers. Here, the feasibility of flow cytometry-based photodynamic detection of MRD was tested in multiple myeloma. Among various human cell lines of hematological malignancies, including K562 erythroleukemia, Jurkat T-cell leukemia, Nalm6 pre-B cell leukemia, KG1a myeloid leukemia, and U937 monocytic leukemia, human myeloma cell line, KMS18, and OPM2 abundantly expressed ALA transporters, such as SLC36A1 and SLC15A2, and 1 mM ALA treatment for 24 h resulted in nearly 100% porphyrin fluorescence expression, which could be competitively inhibited by ALA transport with gamma-aminobutyric acid. Titration studies revealed that the lowest ALA concentration required to achieve nearly 100% porphyrin fluorescence in KMS18 cells was 0.25 mM, with an incubation period of 2 h. Under these conditions, incubation of primary peripheral blood mononuclear cells resulted in only 1.8 % of the cells exhibiting porphyrin fluorescence. Therefore, flow cytometry-based photodynamic diagnosis is a promising approach for detecting MRD in multiple myeloma.

Antimalarial Drugs Enhance the Cytotoxicity of 5-Aminolevulinic Acid-Based Photodynamic Therapy against the Mammary Tumor Cells of Mice In Vitro.

Artemisinin and its derivatives, including artesunate (ART) and artemether (ARM), exert anticancer effects in the micromolar range in drug and radiation-resistant cell lines. Artemisinin has been reported to sensitize cervical cancer cells to radiotherapy. In the present study, we determined whether ART and ARM could enhance the cytotoxicity of 5-aminolevulinic acid (5-ALA)-based photodynamic therapy (PDT) against the mammary tumor cells of mice. The corrected PpIX fluorescence intensities in the control, 5-ALA, 5-ALA + ART, and 5-ALA + ARM groups were 3.385 ± 3.730, 165.7 ± 33.45, 139.0 ± 52.77, and 165.4 ± 51.10 a.u., respectively. At light doses of 3 and 5 J/cm2, the viability of 5-ALA-PDT-treated cells significantly decreased with ART (p < 0.01 and p < 0.01) and ARM treatment (p < 0.01 and p < 0.01). Besides, the number of annexin V-FITC and ethidium homodimer III-positive cells was greater in the 5-ALA-PDT with ARM group than that in the other groups. N-acetylcysteine could not significantly inhibit the percentages of apoptotic cells or inviable cells induced by 5-ALA-PDT with ARM. These reactive oxygen species-independent mechanisms might enhance cytotoxicity in 5-ALA-PDT with ARM-treated tumor cells, suggesting that the use of 5-ALA-PDT with ARM could be a new strategy to enhance PDT cytotoxicity against tumor cells. However, as these results are only based on in vitro studies, further in vivo investigations are required.

5-Aminolevulinic acid with ferrous iron improves early renal damage and hepatic steatosis in high fat diet-induced obese mice.

5-Aminolevulinic acid, a natural amino acid, activates mitochondrial respiration and induces heme oxygenase-1 expression. Obesity and type 2 diabetes mellitus are associated with age-related mitochondrial respiration defect, oxidative stress and inflammation. The aim of this study is to investigate the effects of 5-aminolevulinic acid with sodium ferrous citrate on early renal damage and hepatic steatosis. 7-Month-old C57BL/6 mice were fed with a standard diet or high fat diet for 9 weeks, which were orally administered 300 mg/kg 5-aminolevulinic acid combined with 47 mg/kg sodium ferrous citrate (5-aminolevulinic acid/sodium ferrous citrate) or vehicle for the last 5 weeks. We observed that 5-aminolevulinic acid/sodium ferrous citrate significantly decreased body weight, fat weight, hepatic lipid deposits and improved levels of blood glucose and oral glucose tolerance test. In addition, 5-aminolevulinic acid/sodium ferrous citrate suppressed increased glomerular tuft area in high fat diet-fed mice, which was associated with increased heme oxygenase-1 protein expression. Our findings demonstrate additional evidence that 5-aminolevulinic acid/sodium ferrous citrate could improve glucose and lipid metabolism in diabetic mice. 5-Aminolevulinic acid/sodium ferrous citrate has potential application in obesity or type 2 diabetes mellitus-associated disease such as diabetic nephropathy and nonalcoholic fatty liver disease.

5-Aminolevulinic acid regulates the immune response in LPS-stimulated RAW 264.7 macrophages.

BACKGROUND: Macrophages are crucial players in a variety of inflammatory responses to environmental cues. However, it has been widely reported that macrophages cause chronic inflammation and are involved in a variety of diseases, such as obesity, diabetes, metabolic syndrome, and cancer. In this study, we report the suppressive effect of 5-aminolevulinic acid (ALA), via the HO-1-related system, on the immune response of the LPS-stimulated mouse macrophage cell line RAW264.7. RESULTS: RAW264.7 cells were treated with LPS with or without ALA, and proinflammatory mediator expression levels and phagocytic ability were assessed. ALA treatment resulted in the attenuation of iNOS and NO expression and the downregulation of proinflammatory cytokines (TNF-α, cyclooxygenase2, IL-1ß, IL-6). In addition, ALA treatment did not affect the phagocytic ability of macrophages. To our knowledge, this study is the first to investigate the effect of ALA on macrophage function. Our findings suggest that ALA may have high potential as a novel anti-inflammatory agent. CONCLUSIONS: In the present study, we showed that exogenous addition of ALA induces HO-1 and leads to the downregulation of NO and some proinflammatory cytokines. These findings support ALA as a promising anti-inflammatory agent.

Combination of 5-aminolevulinic acid and iron prevents skin fibrosis in murine sclerodermatous graft-versus-host disease.

Scleroderma or systemic sclerosis (SSc) is a clinically heterogeneous rheumatological autoimmune disease affecting the skin, internal organs and blood vessels. There is at present no effective treatment for this condition. Our study investigated the effects of 5-aminolevulinic acid (5-ALA), which is a precursor of haem synthesis, on graft-vs-host disease (GvHD)-induced SSc murine model. Lymphocytes were intravenously injected from donor mice (B10.D2) into recipient BALB/c mice (recombination-activating gene 2 (Rag-2)-null mice) deficient in mature T and B cells to induce sclerodermatous GvHD (scl-GvHD). To investigate the effect of 5-ALA on scl-GvHD, combination of 5-ALA and sodium ferrous citrate (SFC) was orally administered to the recipient mice for 9 weeks. 5-ALA/SFC treatment significantly reduced progressive inflammation and fibrosis in the skin and ears. Furthermore, 5-ALA/SFC suppressed mRNA expression of transforming growth factor-ß, type I collagen and inflammatory cytokines. These results indicate that the 5-ALA/SFC combination treatment has a protective effect against tissue fibrosis and inflammation in a murine scl-GvHD-induced skin and ear inflammation and fibrosis. Furthermore, the efficacy of 5-ALA/SFC suggests important implications of HO-1 protective activity in autoimmune diseases, and therefore, 5-ALA/SFC may have promising clinical applications. These findings suggested that the 5-ALA/SFC treatment may be the potential strategies for SSc.

Artesunate Enhances the Cytotoxicity of 5-Aminolevulinic Acid-Based Sonodynamic Therapy against Mouse Mammary Tumor Cells In Vitro.

Sonodynamic therapy (SDT) kills tumor cells through the synergistic effects of ultrasound (US) and a sonosensitizer agent. 5-Aminolevulinic acid (5-ALA) has been used as a sonodynamic sensitizer for cancer treatment. However, studies have shown that 5-ALA-based SDT has limited efficacy against malignant tumors. In this study, we examined whether artesunate (ART) could enhance the cytotoxicity of 5-ALA-based SDT against mouse mammary tumor (EMT-6) cells in vitro. In the ART, ART + US, ART + 5-ALA, and ART + 5-ALA + US groups, the cell survival rate correlated with ART concentration, and decreased with increasing concentrations of ART. Morphologically, many apoptotic and necrotic cells were observed in the ART + 5-ALA + US group. The percentage of reactive oxygen species-positive cells in the ART + 5-ALA + US group was also significantly higher than that in the 5-ALA group (p = 0.0228), and the cell death induced by ART + 5-ALA + US could be inhibited by the antioxidant N-acetylcysteine. These results show that ART offers great potential in enhancing the efficacy of 5-ALA-based SDT for the treatment of cancer. However, these results are only based on in vitro studies, and further in vivo studies are required.

5-Aminolevulinic acid with sodium ferrous citrate induces autophagy and protects cardiomyocytes from hypoxia-induced cellular injury through MAPK-Nrf-2-HO-1 signaling cascade.

BACKGROUND: Hypoxia causes cardiac disease via oxidative stress and mitochondrial dysfunction. 5-Aminolevulinic acid in combination with sodium ferrous citrate (ALA/SFC) has been shown to up-regulate heme oxygenase-1 (HO-1) and decrease macrophage infiltration and renal cell apoptosis in renal ischemia injury mice. However, its underlying mechanism remains largely unknown. The aim of this study was to investigate whether ALA/SFC could protect cardiomyocytes from hypoxia-induced apoptosis by autophagy via HO-1 signaling. MATERIALS & METHODS: Murine atrial cardiomyocyte HL-1 cells were pretreated with ALA/SFC and then exposed to hypoxia. RESULTS: ALA/SFC pretreatment significantly attenuated hypoxia-induced cardiomyocyte apoptosis, reactive oxygen species production, and mitochondrial injury, while it increased cell viability and autophagy levels. HO-1 expression by ALA/SFC was associated with up-regulation and nuclear translocation of Nrf-2, whereas Nrf-2 siRNA dramatically reduced HO-1 expression. ERK1/2, p38, and SAPK/JNK pathways were activated by ALA/SFC and their specific inhibitors significantly reduced ALA/SFC-mediated HO-1 upregulation. Silencing of either Nrf-2 or HO-1and LY294002, inhibitor of autophagy, abolished the protective ability of ALA/AFC against hypoxia-induced injury and reduced ALA/SFC-induced autophagy. CONCLUSION: Taken together, our data suggest that ALA/SFC induces autophagy via activation of MAPK/Nrf-2/HO-1 signaling pathway to protect cardiomyocytes from hypoxia-induced apoptosis.

5-Aminolevulinic acid combined with ferrous iron ameliorate ischemia-reperfusion injury in the mouse fatty liver model.

BACKGROUND: The fatty liver could increase the risk of serious acute ischemia reperfusion (I/R) injury, and hepatic steatosis is indeed a major risk factor for hepatic failure after grafting a fatty liver. MATERIALS & METHODS: Fatty liver models of methionine- and choline-deficient high-fat mice were subjected to I/R injury with or without 5-aminolevulinic acid (5-ALA)/sodium ferrous citrate (SFC) treatment. Levels of hepatic enzymes, lipid peroxidation and apoptosis, inflammatory cytokines and heme oxygenase (HO)-1, and the carbon monoxide (CO) in the liver, and reactive oxygen species (ROS), inflammatory cytokines and members of the signaling pathway in isolated Kupffer were assessed. RESULTS: Alanine aminotransferase and aspartate aminotransferase levels, the number of necrotic areas, thiobarbituric acid reactive substance content, TUNEL-positive cells, infiltrated macrophages, and the inflammatory cytokine expression after I/R injury were dramatically decreased, whereas the endogenous CO concentrations and the HO-1 expression were significantly increased by 5-ALA/SFC treatment. The expression of toll-like receptors 2 and 4, NF-κB and inflammatory cytokines and ROS production in Kupffer cells were significantly decreased with 5-ALA/SFC treatment. CONCLUSION: 5-ALA/SFC significantly attenuates the injury level in the fatty liver after I/R injury.

In vivo curative and protective potential of orally administered 5-aminolevulinic acid plus ferrous ion against malaria.

5-Aminolevulinic acid (ALA) is a naturally occurring amino acid present in diverse organisms and a precursor of heme biosynthesis. ALA is commercially available as a component of cosmetics, dietary supplements, and pharmaceuticals for cancer diagnosis and therapy. Recent reports demonstrated that the combination of ALA and ferrous ion (Fe(2+)) inhibits the in vitro growth of the human malaria parasite Plasmodium falciparum. To further explore the potential application of ALA and ferrous ion as a combined antimalarial drug for treatment of human malaria, we conducted an in vivo efficacy evaluation. Female C57BL/6J mice were infected with the lethal strain of rodent malaria parasite Plasmodium yoelii 17XL and orally administered ALA plus sodium ferrous citrate (ALA/SFC) as a once-daily treatment. Parasitemia was monitored in the infected mice, and elimination of the parasites was confirmed using diagnostic PCR. Treatment of P. yoelii 17XL-infected mice with ALA/SFC provided curative efficacy in 60% of the mice treated with ALA/SFC at 600/300 mg/kg of body weight; no mice survived when treated with vehicle alone. Interestingly, the cured mice were protected from homologous rechallenge, even when reinfection was attempted more than 230 days after the initial recovery, indicating long-lasting resistance to reinfection with the same parasite. Moreover, parasite-specific antibodies against reported vaccine candidate antigens were found and persisted in the sera of the cured mice. These findings provide clear evidence that ALA/SFC is effective in an experimental animal model of malaria and may facilitate the development of a new class of antimalarial drug.

Proposed Robust Entanglement-Based Magnetic Field Sensor Beyond the Standard Quantum Limit.

Recently, there have been significant developments in entanglement-based quantum metrology. However, entanglement is fragile against experimental imperfections, and quantum sensing to beat the standard quantum limit in scaling has not yet been achieved in realistic systems. Here, we show that it is possible to overcome such restrictions so that one can sense a magnetic field with an accuracy beyond the standard quantum limit even under the effect of decoherence, by using a realistic entangled state that can be easily created even with current technology. Our scheme could pave the way for the realizations of practical entanglement-based magnetic field sensors.

5-Aminolevulinic acid enhances cell death under thermal stress in certain cancer cell lines.

5-aminolevulinic acid (5-ALA) is contained in all organisms and a starting substrate for heme biosynthesis. Since administration of 5-ALA specifically leads cancer cells to accumulate protoporphyrin IX (PpIX), a potent photosensitizer, we tested if 5-ALA also serves as a thermosensitizer. 5-ALA enhanced heat-induced cell death of cancer cell lines such as HepG2, Caco-2, and Kato III, but not other cancer cell lines including U2-OS and normal cell lines including WI-38. Those 5-ALA-sensitive cancer cells, but neither U2-OS nor WI-38, accumulated intracellular PpIX and exhibited an increased reactive oxygen species (ROS) generation under thermal stress with 5-ALA treatment. In addition, blocking the PpIX-exporting transporter ABCG2 in U2-OS and WI-38 cells enhanced their cell death under thermal stress with 5-ALA. Finally, a ROS scavenger compromised the cell death enhancement by 5-ALA. These suggest that 5-ALA can sensitize certain cancer cells, but not normal cells, to thermal stress via accumulation of PpIX and increase of ROS generation.

Effect of 5-aminolevulinic acid on erythropoiesis: a preclinical in vitro characterization for the treatment of congenital sideroblastic anemia.

Congenital sideroblastic anemia (CSA) is a hereditary disorder characterized by microcytic anemia and bone marrow sideroblasts. The most common form of CSA is attributed to mutations in the X-linked gene 5-aminolevulinic acid synthase 2 (ALAS2). ALAS2 is a mitochondrial enzyme, which utilizes glycine and succinyl-CoA to form 5-aminolevulinic acid (ALA), a crucial precursor in heme synthesis. Therefore, ALA supplementation could be an effective therapeutic strategy to restore heme synthesis in CSA caused by ALAS2 defects. In a preclinical study, we examined the effects of ALA in human erythroid cells, including K562 cells and human induced pluripotent stem cell-derived erythroid progenitor (HiDEP) cells. ALA treatment resulted in significant dose-dependent accumulation of heme in the K562 cell line. Concomitantly, the treatment substantially induced erythroid differentiation as assessed using benzidine staining. Quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis confirmed significant upregulation of heme-regulated genes, such as the globin genes [hemoglobin alpha (HBA) and hemoglobin gamma (HBG)] and the heme oxygenase 1 (HMOX1) gene, in K562 cells. Next, to investigate the mechanism by which ALA is transported into erythroid cells, quantitative RT-PCR analysis was performed on previously identified ALA transporters, including solute carrier family 15 (oligopeptide transporter), member (SLC15A) 1, SLC15A2, solute carrier family 36 (proton/amino acid symporter), member (SLC36A1), and solute carrier family 6 (neurotransmitter transporter), member 13 (SLC6A13). Our analysis revealed that SLC36A1 was abundantly expressed in erythroid cells. Thus, gamma-aminobutyric acid (GABA) was added to K562 cells to competitively inhibit SLC36A1-mediated transport. GABA treatment significantly impeded the ALA-mediated increase in the number of hemoglobinized cells as well as the induction of HBG, HBA, and HMOX1. Finally, small-interfering RNA-mediated knockdown of ALAS2 in HiDEP cells considerably decreased the expression of HBA, HBG, and HMOX1, and these expression levels were rescued with ALA treatment. In summary, ALA appears to be transported into erythroid cells mainly by SLC36A1 and is utilized to generate heme. ALA may represent a novel therapeutic option for CSA treatment, particularly for cases harboring ALAS2 mutations.

5-Aminolevulinic acid combined with ferrous iron induces carbon monoxide generation in mouse kidneys and protects from renal ischemia-reperfusion injury.

Renal ischemia reperfusion injury (IRI) is a major factor responsible for acute renal failure. An intermediate in heme synthesis, 5-aminolevulinic acid (5-ALA) is fundamental in aerobic energy metabolism. Heme oxygenase (HO)-1 cleaves heme to form biliverdin, carbon monoxide (CO), and iron (Fe(2+)), which is used with 5-ALA. In the present study, we investigated the role of 5-ALA in the attenuation of acute renal IRI using a mouse model. Male Balb/c mice received 30 mg/kg 5-ALA with Fe(2+) 48, 24, and 2 h before IRI and were subsequently subjected to bilateral renal pedicle occlusion for 45 min. The endogenous CO concentration of the kidneys from the mice administered 5-ALA/Fe(2+) increased significantly, and the peak concentrations of serum creatinine and blood urea nitrogen decreased. 5-ALA/Fe(2+) treatments significantly decreased the tubular damage and number of apoptotic cells. IRI-induced renal thiobarbituric acid-reactive substance levels were also significantly decreased in the 5-ALA/Fe(2+) group. Furthermore, mRNA expression of HO-1, TNF-α, and interferon-γ was significantly increased after IRI. Levels of HO-1 were increased and levels of TNF-α and interferon-γ were decreased in the 5-ALA/Fe(2+)-pretreated renal parenchyma after IRI. F4/80 staining showed reduced macrophage infiltration, and TUNEL staining revealed that there were fewer interstitial apoptotic cells. These findings suggest that 5-ALA/Fe(2+) can protect the kidneys against IRI by reducing macrophage infiltration and decreasing renal cell apoptosis via the generation of CO.

Lead suppresses perlecan expression via EGFR-ERK1/2-COX-2-PGI2 pathway in cultured bovine vascular endothelial cells.

Vascular endothelial cell growth is essential for the repair of intimal injury. Perlecan, a large heparan sulfate proteoglycan, intensifies fibroblast growth factor-2 (FGF-2) signaling as a co-receptor for FGF-2 and its receptor, and promotes the proliferation of vascular endothelial cells. Previously, we reported that 2 µM of lead, a toxic heavy metal, downregulated perlecan core protein expression and then suppressed the growth of vascular endothelial cells. However, since the mechanisms involved in the repression of perlecan by lead remains unclear, we analyzed its detailed signaling pathway using cultured bovine aortic endothelial cells. Our findings indicate that 2 µM of lead inhibited protein tyrosine phosphatase (PTP) activity and induced cyclooxygenase-2 (COX-2) via phosphorylation of the epidermal growth factor receptor (EGFR) and its downstream extracellular signal-regulated kinases (ERK1/2). In addition, among the prostanoids regulated by COX-2, prostaglandin I2 (PGI2) specifically contributes to the downregulation of perlecan expression by lead. This study revealed an intracellular pathway-the EGFR-ERK1/2-COX-2-PGI2 pathway activated by inhibition of PTP by lead-as a pathway that downregulates endothelial perlecan synthesis. The pathway is suggested to serve as a mechanism for the repression of perlecan expression, which leads to a delay in cell proliferation by lead.

Comparison of the efficacy of ALA-PDT using an excimer-dye laser (630 nm) and a metal-halide lamp (600 to 740 nm) for treatment of Bowen's disease.

BACKGROUND/PURPOSE: Topical 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT) is an effective treatment for Bowen's disease (BD). In order to compare the efficacy of two different light sources, using either an excimer-dye laser (EDL) (630 nm) or a metal-halide lamp (MHL) (600 to 740 nm) a protocol for topical ALA-PDT for treatment of BD of the extremities was established, and responses during 12 months follow-up were assessed. METHODS: From 25 patients a total of 26 lesions that had been histopathologically diagnosed as BD from 2005 to 2010 in the Department of Dermatology at the Aichi Medical University Hospital were randomly selected. The light source used for the topical ALA-PDT was EDL in 17 lesions and MHL in 9 lesions. The photosensitizing protoporphyrin IX that is produced within BD lesions 4 h after application of 20% ALA cream was mostly consumed after exposure to 100 J/cm(2) irradiation using 630 nm EDL. Each lesion was irradiated once a week for 3 weeks, for a total dosage of 300 J/cm(2) (100 mW/cm(2)). Patients were followed up clinically every 3 months for 12 months, and at 1 month after the final treatment lesions were evaluated histopathologically. RESULTS: Histologically, the complete response (CR) rate at 1-month follow-up was 82% (14/17 lesions) in the EDL treatment group and 100% (9/9 lesions) in the MHL treatment group (P > 0.05). The recurrence rate at 12 months after PDT was 46% (6/13 lesions, one patient lost to follow-up) in the EDL group and 0% in the MHL group (P < 0.05) (χ(2) test with Fisher's exact test). The average period before recurrence after EDL treatment was 6.5 months. CONCLUSION: A novel protocol for topical ALA-PDT in Japanese in Asian patients with BD was developed and implemented. The protocol improved the CR rate compared with previous studies. Moreover, the present results indicate that the efficacy of topical ALA-PDT using MHL was superior to that using EDL for BD patients.

Antiviral Effects of 5-Aminolevulinic Acid Phosphate against Classical Swine Fever Virus: In Vitro and In Vivo Evaluation.

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 107.0 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.