Sensitivity of osteoblasts, fibroblasts, bone marrow cells, and dendritic cells to 5-aminolevulinic acid based photodynamic therapy

INTRODUCTION: Photodynamic therapy with 5-aminolevulinic acid (5-ALA-PDT) exerts cell type specific effects on target cells. Since chondrocytes were found to be more resistant than osteoblasts to 5-ALA-PDT, the pre-treatment of osteochondral grafts with 5-ALA-PDT may represent a means to devitalize the osseous portion while maintaining functional cartilage. The present study was designed to determine the effects of 5-ALA-PDT in vitro on cell populations residing in skeletal tissues. METHODS: Osteoblasts, fibroblasts, bone marrow cells, and dendritic cells were incubated with 0.5 mM 5-ALA for 4 h. Protoporphyrin IX (PpIX) accumulation and after exposure to light cellular functions were assessed for up to 6 days. RESULTS: Accumulation of PpIX reached a plateau at 0.5 mM in osteoblasts, fibroblasts, and dendritic cells, and at 2.0 mM in bone marrow cells. At 0.5 mM 5-ALA, similar responses to illumination were observed in all cells with a survival rate of less than 12% at a light dose of 20 J/cm(2). The function of osteoblasts (proliferation, levels of mRNA encoding collagen type I, alkaline phosphatase activity) and fibroblasts (proliferation, levels of mRNAs encoding collagens type I and III) was not affected, when the cells were treated with 5-ALA and light doses of < or =10 J/cm(2). Paralleling the reduction of viable cells after 5-ALA-PDT, the capacity of dendritic cells to stimulate T cells in a mixed leukocyte reaction decreased to 4+/-2% at 20 J/cm(2). CONCLUSION: The investigated cell types were sensitive to 5-ALA-PDT and the residual cell debris did not elicit an allogenic response. These findings, together with the resistance of chondrocytes to 5-ALA-PDT, encourage the further investigation of this protocol in the pretreatment of osteochondral allografts.

Regulation of human liver delta-aminolevulinic acid synthase by bile acids

Aminolevulinic acid synthase 1 (ALAS1) is the rate-limiting enzyme of heme synthesis in the liver and is highly regulated to adapt to the metabolic demand of the hepatocyte. In the present study, we describe human hepatic ALAS1 as a new direct target of the bile acid-activated nuclear receptor farnesoid X receptor (FXR). Experiments in primary human hepatocytes and in human liver slices showed that ALAS1 messenger RNA (mRNA) and activity is increased upon exposure to chenodeoxycholic acid (CDCA), the most potent natural FXR ligand, or the synthetic FXR-specific agonist GW4064. Moreover, overexpression of a constitutively active form of FXR further increased ALAS1 mRNA expression. In agreement with these observations, an FXR response element was identified in the 5' flanking region of human ALAS1 and characterized in reporter gene assays. A highly conserved FXR binding site (IR1) within a 175-bp fragment at -13 kilobases upstream of the transcriptional start site was able to trigger an FXR-specific increase in luciferase activity upon CDCA treatment. Site-directed mutagenesis of IR1 abolished this effect. Binding of FXR/retinoid acid X receptor heterodimers was demonstrated by mobility gel shift experiments. Conclusion: These data strongly support a role of bile acid-activated FXR in the regulation of human ALAS1 and, consequently, hepatic porphyrin and heme synthesis. These data also suggest that elevated endogenous bile acids may precipitate neuropsychiatric attacks in patients with acute hepatic porphyrias.

Genotypic and phenotypic characterization of Trypanosoma brucei gambiense isolates from Ibba, South Sudan, an area of high melarsoprol treatment failure rate

Resistance of trypanosomes to melarsoprol is ascribed to reduced uptake of the drug via the P2 nucleoside transporter. The aim of this study was to look for evidence of drug resistance in Trypanosoma brucei gambiense isolates from sleeping sickness patients in Ibba, South Sudan, an area of high melarsoprol failure rate. Eighteen T. b. gambiense stocks were phenotypically and only 10 strains genotypically characterized. In vitro, all isolates were sensitive to melarsoprol, melarsen oxide, and diminazene. Infected mice were cured with a 4 day treatment of 2.5mg/kg bwt melarsoprol, confirming that the isolates were sensitive. The gene that codes for the P2 transporter, TbATI, was amplified by PCR and sequenced. The sequences were almost identical to the TbAT1(sensitive) reference, except for one point mutation, C1384T resulting in the amino acid change proline-462 to serine. None of the described TbAT1(resistant)-type mutations were detected. In a T. b. gambiense sleeping sickness focus where melarsoprol had to be abandoned due to the high incidence of treatment failures, no evidence for drug resistant trypanosomes or for TbAT1(resistant)-type alleles of the P2 transporter could be found. These findings indicate that factors other than drug resistance contribute to melarsoprol treatment failures.

In vitro resistance of articular chondrocytes to 5-Aminolevulinic acid based photodynamic therapy

OBJECTIVE: 5-Aminolevulinic acid based photodynamic therapy (5-ALA-PDT) has revealed promising results in the treatment of inflammatory joint diseases due to the sensitivity of inflamed synovial tissue. For 5-ALA-PDT to be safe and beneficial for intra-articular applications, resistance of chondrocytes is essential to prevent cartilage damage. As no data yet exist, the aim of the present study was to assess in vitro the response of the chondrocytes to 5-ALA-PDT and to compare with osteoblasts and synovial tissue derived cells. METHODS: Bovine articular chondrocytes, osteoblasts, and synovial cells were subjected to 5-ALA-PDT in cell culture. The PpIX accumulation and the function of the cells were assessed for up to 12 days. RESULTS: Bovine chondrocytes showed lower PpIX fluorescence upon incubation with 5-ALA (0.0-2.0 mM) for 4 hours as compared to osteoblasts and synovial cells suggesting a low PpIX accumulation. After incubation with 0.5 mM 5-ALA and application of light at a dose of 20 J/cm2, chondrocytes were functionally not affected (collagen type II and aggrecan mRNA, glycosaminoglycan synthesis) whereas a decrease in the proportion of viable cells was observed in osteoblasts and synovial cells (2+/-2% and 14+/-8%, respectively; chondrocytes 91+/-13%). Chondrocytes showed a 58% reduction of 5-ALA uptake using [3H]5-ALA as compared to osteoblasts and a lower mitochondrial content as assessed by the activity of the mitochondrial marker enzyme citrate synthase (9.2+/- 3.6 mU/mg protein) than osteoblasts (32.6+/-10.5 mU/mg) and synovial cells (60.0+/-10.8 mU/mg). The reduced uptake of 5-ALA and/or the low mitochondrial content, an adaptation to their in vivo environment and the site of PpIX synthesis, presumably explains the lower PpIX content in chondrocytes and their resistance against 5-ALA-PDT. CONCLUSION: 5-ALA-PDT might represent a treatment strategy in inflammatory joint diseases without endangering the cartilage function. However, further in vitro and in vivo experiments are required to confirm this data in the authentic environment of chondrocytes, the articular cartilage.

Serum lipoproteins during treatment with the antihypertensive agent indapamide

Considering the documented, potentially undesirable influence of various thiazide-type or loop diuretics on serum lipoproteins, we prospectively investigated in 69 men (mean age +/- SEM, 32 +/- 1 years) the metabolic effects of the new diuretic-antihypertensive compound indapamide. Compared to placebo, indapamide (2.5 mg/day) given for 6 to 8 weeks lowered (p less than 0.02 to less than 0.001) blood pressure (supine values from 148/98 +/- 3/2 to 137/93 +/- 3/2) in 29 men with mild to moderate essential hypertension, but not in 40 healthy men. In both groups, significant (p less than 0.05 to less than 0.001) decreases in body weight (-0.8 kg) and plasma potassium (-0.6 mmol/L), and increases in plasma uric acid (+20%), renin activity (+200%), and aldosterone documented good compliance. There were no significant changes in total cholesterol (in all subjects, from 208 +/- 6 to 213 +/- 6 mg/dl), low- or very low-density lipoprotein (VLDL) cholesterol (127 +/- 6 to 129 +/- 6 and 21 +/- 1 to 21 +/- 2 respectively), high-density lipoprotein cholesterol (50 +/- 1 to 51 +/- 1 mg/dl), total triglycerides (Tg) (108 +/- 5 to 112 +/- 6 mg/dl), VLDL-Tg, apoproteins A1 and A2, plasma glucose, epinephrine, norepinephrine, sodium, calcium, magnesium, and creatinine; apoprotein B (84 +/- 2 to 88 +/- 3 mg/dl) and plasma insulin after glucose loading dose tended to be increased minimally. The absence of distinct lipoprotein alterations after short-term indapamide treatment may be of clinical and epidemiological interest.

DESIGN AND SYNTHESIS OF NOVEL SYNTHETIC ANTIOXIDANTS FOR THE TREATMENT OF OXIDATIVE STRESS RELATED DISEASES

Free radicals play an important role in many physiological processes that occur in the human body such as cellular defense responses to infectious agents and a variety of cellular signaling pathways. While at low concentrations free radicals are involved in many significant metabolic reactions, high levels of free radicals can have deleterious effects on biomolecules like proteins, lipids, and DNA. Many physiological disorders such as diabetes, ageing, neurodegenerative diseases, and ischemia-reperfusion (I/R) injury are associated with oxidative stress.1 In particular, the deleterious effects caused by I/R injury developed during organ transplantation, cardiac infarct, and stroke have become the main cause of death in the United States and Europe.1,2 In this context, we synthesized and characterized a series of novel indole-amino acid conjugates as potential antioxidants for I/R injury. The synthesis of indole-phenol conjugate compounds is also discussed. Phenolic derivatives such as caffeic acid, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), resveratrol, and its analogues are known for their significant antioxidative properties. A series of resveratrol analogues have been designed and synthesized as potential antioxidants. The radical scavenging mechanisms for potential antioxidants and assays for the in vitro evaluation of antioxidant activities are also discussed.

Treatment of hypercholesterolemia in patients with primary biliary cirrhosis might be more beneficial than indicated

Cholesterol circulating levels are elevated in most of the patients with primary biliary cirrhosis. This review questions whether hypercholesterolaemia represents a cardiovascular risk in primary biliary cirrhosis and whether it should be treated. The published evidence indicates that hypercholesterolaemia in patients with primary biliary cirrhosis should be considered a cardiovascular risk factor only when other factors are present. Ursodeoxycholic acid the standard treatment of primary biliary cirrhosis improves the cholestasis and hereby lowers circulating levels of cholesterol. Primary biliary cirrhosis is not a contraindication to prescribe statins or fibrates to these patients. Interestingly, these two classes of drugs have been shown to improve not only the lipid profile but also the liver tests. In particular fibrates have been found to normalize liver tests in patients responding incompletely to ursodeoxycholic acid. Statins as well as fibrates possess specific anti-inflammatory properties which may be beneficial in primary biliary cirrhosis. In conclusion, hypercholesterolaemia in the absence of other cardiovascular risk factors does not require specific therapeutic intervention in patients with primary biliary cirrhosis. However, statins as well as fibrates seem to have beneficial effects on the primary biliary cirrhosis itself and deserve formal testing within clinical trials.

Toxicity of valproic acid in mice with decreased plasma and tissue carnitine stores

The aim of this study was to investigate whether a decrease in carnitine body stores is a risk factor for valproic acid (VPA)-associated hepatotoxicity and to explore the effects of VPA on carnitine homeostasis in mice with decreased carnitine body stores. Therefore, heterozygous juvenile visceral steatosis (jvs)(+/-) mice, an animal model with decreased carnitine stores caused by impaired renal reabsorption of carnitine, and the corresponding wild-type mice were treated with subtoxic oral doses of VPA (0.1 g/g b.wt./day) for 2 weeks. In jvs(+/-) mice, but not in wild-type mice, treatment with VPA was associated with the increased plasma activity of aspartate aminotransferase and alkaline phosphatase. Furthermore, jvs(+/-) mice revealed reduced palmitate metabolism assessed in vivo and microvesicular steatosis of the liver. The creatine kinase activity was not affected by treatment with VPA. In liver mitochondria isolated from mice that were treated with VPA, oxidative metabolism of l-glutamate, succinate, and palmitate, as well as beta-oxidation of palmitate, were decreased compared to vehicle-treated wild-type mice or jvs(+/-) mice. In comparison to vehicle-treated wild-type mice, vehicle-treated jvs(+/-) mice had decreased carnitine plasma and tissue levels. Treatment with VPA was associated with an additional decrease in carnitine plasma (wild-type mice and jvs(+/-) mice) and tissue levels (jvs(+/-) mice) and a shift of the carnitine pools toward short-chain acylcarnitines. We conclude that jvs(+/-) mice reveal a more accentuated hepatic toxicity by VPA than the corresponding wild-type mice. Therefore, decreased carnitine body stores can be regarded as a risk factor for hepatotoxicity associated with VPA.

Effect of once-yearly zoledronic acid 5 mg on fracture risk and change in femoral neck bone mineral density

Context: In the Health Outcomes and Reduced Incidence with Zoledronic Acid Once Yearly - Pivotal Fracture Trial (HORIZON-PFT), zoledronic acid (ZOL) 5 mg significantly reduced fracture risk. Objective: To identify factors associated with greater efficacy during ZOL 5 mg treatment. Design, Setting and Patients: Subgroup analysis (preplanned and post hoc) of a multicenter, double-blind, placebo-controlled, 36-month trial in 7765 women with postmenopausal osteoporosis. Intervention: Single infusion of ZOL 5 mg or placebo at baseline, 12 and 24 months. Main Outcome Measures: Primary endpoints: new vertebral fracture and hip fracture. Secondary endpoints: non-vertebral fracture, change in femoral neck bone mineral density (BMD). Baseline risk factor subgroups: age, BMD T-score and vertebral fracture status, total hip BMD, race, weight, geographical region, smoking, height loss, history of falls, physical activity, prior bisphosphonates, creatinine clearance, body mass index (BMI), concomitant osteoporosis medications. Results: Greater ZOL induced effects on vertebral fracture risk with younger age (treatment-by-subgroup interaction P=0.05), normal creatinine clearance (P=0.04), and BMI >/=25 kg/m(2) (P=0.02). There were no significant treatment-factor interactions for hip or non-vertebral fracture or for change in BMD. Conclusions: ZOL appeared more effective in preventing vertebral fracture in younger women, overweight/obese women and women with normal renal function. ZOL had similar effects irrespective of fracture risk factors or femoral neck BMD.

Effects of ursodeoxycholic acid in combination with vitamin E on adipokines and apoptosis in patients with nonalcoholic steatohepatitis

BACKGROUND/AIMS: Adipokines and hepatocellular apoptosis participate in the pathogenesis of nonalcoholic steatohepatitis (NASH). In a randomized trial ursodeoxycholic acid (UDCA) with vitamin E (VitE) improved serum aminotransferases and hepatic histology. The present work evaluates the effect of this combination on adipokines and hepatocellular apoptosis. METHODS: Circulating levels of adiponectin, resistin, leptin, interleukin (IL)-6, IL-8, retinol binding protein-4, monocyte chemoattractant protein-1 and tumour necrosis factor-alpha were measured by enzyme-linked immunoassays at the beginning and after 2 years of treatment with either UDCA+VitE, UDCA+placebo (P) or P+P. Apoptosis was assessed by immunohistochemistry for activated caspase-3 and circulating levels of apoptosis-associated cytokeratin 18 fragments (M30). RESULTS: Levels of adiponectin increased in patients treated with UDCA+VitE, whereas they decreased in the two other groups (P<0.04) and correlated with the improvement of liver steatosis (P<0.04). M30 levels worsened in the P/P group and improved in the other two groups. They correlated with hepatocellular apoptosis (P<0.02) and steatosis (P<0.02) as well as negatively with adiponectin levels (P<0.04). CONCLUSIONS: UDCA+VitE improves not only aminotransferase levels and liver histology of patients with NASH, but also decreases hepatocellular apoptosis and restores circulating levels of adiponectin. These results suggest that the UDCA+VitE combination has metabolic effects in addition to its beneficial cytoprotective properties.

Human urinary metabolomic profile of PPARalpha induced fatty acid beta-oxidation

Activation of the peroxisome proliferator-activated receptor alpha (PPARalpha) is associated with increased fatty acid catabolism and is commonly targeted for the treatment of hyperlipidemia. To identify latent, endogenous biomarkers of PPARalpha activation and hence increased fatty acid beta-oxidation, healthy human volunteers were given fenofibrate orally for 2 weeks and their urine was profiled by UPLC-QTOFMS. Biomarkers identified by the machine learning algorithm random forests included significant depletion by day 14 of both pantothenic acid (>5-fold) and acetylcarnitine (>20-fold), observations that are consistent with known targets of PPARalpha including pantothenate kinase and genes encoding proteins involved in the transport and synthesis of acylcarnitines. It was also concluded that serum cholesterol (-12.7%), triglycerides (-25.6%), uric acid (-34.7%), together with urinary propylcarnitine (>10-fold), isobutyrylcarnitine (>2.5-fold), (S)-(+)-2-methylbutyrylcarnitine (5-fold), and isovalerylcarnitine (>5-fold) were all reduced by day 14. Specificity of these biomarkers as indicators of PPARalpha activation was demonstrated using the Ppara-null mouse. Urinary pantothenic acid and acylcarnitines may prove useful indicators of PPARalpha-induced fatty acid beta-oxidation in humans. This study illustrates the utility of a pharmacometabolomic approach to understand drug effects on lipid metabolism in both human populations and in inbred mouse models.

Serum metabolomics reveals irreversible inhibition of fatty acid beta-oxidation through the suppression of PPARalpha activation as a contributing mechanism of acetaminophen-induced hepatotoxicity

Metabolic bioactivation, glutathione depletion, and covalent binding are the early hallmark events after acetaminophen (APAP) overdose. However, the subsequent metabolic consequences contributing to APAP-induced hepatic necrosis and apoptosis have not been fully elucidated. In this study, serum metabolomes of control and APAP-treated wild-type and Cyp2e1-null mice were examined by liquid chromatography-mass spectrometry (LC-MS) and multivariate data analysis. A dose-response study showed that the accumulation of long-chain acylcarnitines in serum contributes to the separation of wild-type mice undergoing APAP-induced hepatotoxicity from other mouse groups in a multivariate model. This observation, in conjunction with the increase of triglycerides and free fatty acids in the serum of APAP-treated wild-type mice, suggested that APAP treatment can disrupt fatty acid beta-oxidation. A time-course study further indicated that both wild-type and Cyp2e1-null mice had their serum acylcarnitine levels markedly elevated within the early hours of APAP treatment. While remaining high in wild-type mice, serum acylcarnitine levels gradually returned to normal in Cyp2e1-null mice at the end of the 24 h treatment. Distinct from serum aminotransferase activity and hepatic glutathione levels, the pattern of serum acylcarnitine accumulation suggested that acylcarnitines can function as complementary biomarkers for monitoring the APAP-induced hepatotoxicity. An essential role for peroxisome proliferator-activated receptor alpha (PPARalpha) in the regulation of serum acylcarnitine levels was established by comparing the metabolomic responses of wild-type and Ppara-null mice to a fasting challenge. The upregulation of PPARalpha activity following APAP treatment was transient in wild-type mice but was much more prolonged in Cyp2e1-null mice. Overall, serum metabolomics of APAP-induced hepatotoxicity revealed that the CYP2E1-mediated metabolic activation and oxidative stress following APAP treatment can cause irreversible inhibition of fatty acid oxidation, potentially through suppression of PPARalpha-regulated pathways.

Effects of decontamination and implant surface characteristics on re-osseointegration following treatment of peri-implantitis

BACKGROUND: Although considerable bone fill may occur following treatment of peri-implantitis, re-osseointegration appears to be limited and unpredictable. Objectives: To evaluate the effects of various decontamination techniques and implant surface configurations on re-osseointegration of contaminated dental implants. MATERIAL AND METHODS: Three months after tooth extraction, implants consisting of a basal part and an exchangeable intraosseous implant cylinder (EIIC) were placed in the mandibles of dogs. The EIIC was machined (M), sandblasted and acid-etched (SLA), or titanium plasma sprayed (TPS). Ligature-induced peri-implantitis was initiated 8 weeks post-implantation and lasted until bone loss reached the junction of the two implant parts. Three treatment modalities were applied: (T1) the EIIC was exchanged for a pristine EIIC; (T2) the EIIC was sprayed in situ with saline; and (T3) the EIIC was removed, cleansed outside the mouth by spraying with saline, steam-sterilized, and remounted. A collagen barrier was placed over each fixture, and 3 months later, samples were processed for histology and histomorphometry. RESULTS: T2 revealed the highest bone-to-implant contact (BIC) level (significantly better than T1 and T3). T2 also yielded the highest bone crest level (significantly better than T1), followed by T3 (significantly better than T1). SLA showed the highest BIC level (significantly better than M), followed by TPS. There were no statistically significant differences in bone crest height between implant types. CONCLUSIONS: Both SLA implants and in situ cleansing resulted in the best re-osseointegration and bone fill of previously contaminated implants.

Quantification of dental erosions in patients with GERD using optical coherence tomography before and after double-blind, randomized treatment with esomeprazole or placebo

OBJECTIVES: Dental erosion, the chemical dissolution of enamel without bacterial involvement, is a rarely reported manifestation of gastroesophageal reflux disease (GERD), as well as of recurrent vomiting and dietary habits. It leads to loss of tooth substance, hypersensitivity, functional impairment, and even tooth fracture. To date, dental erosions have been assessed using only very basic visual methods, and no evidence-based guidelines or studies exist regarding the prevention or treatment of GERD-related dental erosions. METHODS: In this randomized, double-blind study, we used optical coherence tomography (OCT) to quantify dental tissue demineralization and enamel loss before and after 3 weeks of acid-suppressive treatment with esomeprazole 20 mg b.i.d. or placebo in 30 patients presenting to the Berne University Dental Clinic with advanced dental erosions and abnormal acid exposure by 24-h esophageal pH manometry (defined as >4% of the 24-h period with pH<4). Enamel thickness, reflectivity, and absorbance as measures of demineralization were quantified by OCT before and after therapy at identical localizations on teeth with most severe visible erosions as well as several other predefined changes in teeth. RESULTS: The mean+/-s.e.m. decrease of enamel thickness of all teeth before and after treatment at the site of maximum exposure was 7.2+/-0.16 black trianglem with esomeprazole and 15.25+/-0.17black trianglem with placebo (P=0.013), representing a loss of 0.3% and 0.8% of the total enamel thickness, respectively. The change in optical reflectivity to a depth of 25 black trianglem after treatment was-1.122 +/-0.769 dB with esomeprazole and +2.059+/-0.534 dB with placebo (P 0.012), with increased reflectivity signifying demineralization. CONCLUSIONS: OCT non-invasively detected and quantified significantly diminished progression of dental tissue demineralization and enamel loss after only 3 weeks of treatment with esomeprazole 20 mg b.i.d. vs. placebo. This suggests that esomeprazole may be useful in counteracting progression of GERD-related dental erosions. Further validation of preventative treatment regimens using this sensitive detection method is required, including longer follow-up and correlation with quantitative reflux measures.

Glycyrrhetinic acid food supplementation lowers serum potassium concentration in chronic hemodialysis patients

Hyperkalemia is a common life-threatening problem in hemodialysis patients. Because glycyrrhetinic acid (GA) inhibits the enzyme 11beta-hydroxy-steroid dehydrogenase II and thereby increases cortisol availability to the colonic mineralocorticoid receptor, it has the potential to lower serum potassium concentrations. To test this, 10 patients in a 6 month prospective, double-blind, placebo-controlled crossover study were given cookies or bread rolls supplemented with glycyrrhetinic acid or placebo. Twenty-four-hour blood pressure measurements were performed at baseline and week 6 and 12 of each treatment period. The ratio of plasma cortisol/cortisone was significantly increased in all patients on GA as compared to baseline or placebo, indicating appropriate enzyme inhibition. Nine of the 10 patients had a persistent decrease in predialysis serum potassium concentration. On GA, mean predialysis serum potassium was significantly lower than at baseline or on placebo. On placebo, serum potassium was significantly elevated above the upper limit of normal in 76% compared to 30% of measurements during GA treatment. Furthermore, on this treatment the frequency of severe hyperkalemia significantly decreased from 9% to 0.6%. No differences were found in parameters reflecting sodium retention. Although these studies show that prolonged GA supplementation persistently lowers serum potassium in dialysis patients, a long-term toxicity study will be mandatory before we recommend the routine use of this treatment.