Hepatotoxicity is a serious complication in patients taking HAART. Coinfection with hepatitis viruses increases the risk of liver toxicity while taking antiretroviral therapy. Baseline transaminases should be checked before beginning antiretorviral therapy and all patients should be screened for pre-existing liver disease, most notably hepatitis B and C infections. Regular monitoring of transaminases is mandatory when commencing antiretroviral therapy. In patients with normal liver function, transaminases may be checked monthly after commencing HAART for the first 3 months. If stable this can be broadened to 3 month intervals. In patients with pre-existing liver disease monitoring should be performed more frequently (every 2 weeks) when initiating therapy. Once stable liver enzymes should be checked monthly. The less hepatotoxic drugs such as lamivudine and abacavir should be preferred in patients at high risk for hepatotoxicity. Risks include co-infection with hepatitis B and C viruses, a previous record of hepatotoxicity, cirrhosis, obesity and female gender. Minor enzyme elevations (< 5-fold upper normal limit) are generally safe to tolerate and usually resolve. Patients must be closely observed with regular liver function tests and a hypersensitivity type drug reaction should be excluded. The onset of clinical symptoms, elevated serum lactate or evidence of severe hepatic dysfunction (coagulopathy or elevation of ammonia levels) are suggestive of severe toxicity and HAART should be withheld. Treatment of suspected HAART related hepatotoxicity should first involve withdrawal of therapy. Hypersensitivity reactions may be treated with corticosteroids. Nucleoside-induced mitochondrial damage may improve with riboflavin or thiamine therapy.

The study examined intrahousehold food behavior in six villages in a rural hill area of mid-Western Nepal. Qualitative and quantitative methodologies taken from both anthropology and nutritional sciences were used to collect data on food belief systems, household allocation of food resources, and the effect of these features on diet and anthropometric status in a sample of 767 individuals in 115 households. Background data were also collected on socioeconomic status and demographic variables such as education levels, occupation, and migration patterns. The core methodological approach used direct structured observations of meals to examine how food is distributed within households. The results document a variety of mechanisms by which some individuals are favored over others through household food distribution, including serving order, serving method, refusing to serve foods, channeling foods and substituting low status foods for high status foods. No differences were observed in mechanisms of food distribution or nutrient intake between male and female children, contrary to evidence in the literature suggesting that male children will be favored. On the other hand, adult women were less likely to meet their nutrient requirements for energy, beta-carotene, riboflavin, and vitamin C than men of the same age. Women's late position in household serving order, channeling of special foods to males and children, and lower total intake of food accounts for these findings.

Energy coupling factor (ECF) transporters are a subgroup of ATP-binding cassette (ABC) transporters involved in the uptake of vitamins and micronutrients in prokaryotes. In contrast to classical ABC importers, ECF transporters do not make use of water-soluble substrate binding proteins or domains but instead employ integral membrane proteins for substrate binding (named S-components). S-components form active translocation complexes with the ECF module, an assembly of two nucleotide-binding domains (NBDs, or EcfA) and a second transmembrane protein. In some cases, the ECF module is dedicated to a single S-component, but in many cases, the ECF module can interact with several different S-components that are unrelated in sequence and bind diverse substrates. The modular organization with exchangeable S-components on a single ECF module allows the transport of chemically different substrates via a common route. The recent determination of the crystal structures of the S-components that recognize thiamin and riboflavin has provided a first clue about the mechanism of S-component exchange. This review describes recent advances and the current views of the mechanism of transport by ECF transporters.

A systematic literature search identified studies validating the methodology used for measuring the usual dietary intake in infants, children and adolescents. The quality of each validation study selected was assessed using a European micronutrient Recommendations Aligned-developed scoring system. The validation studies were categorised according to whether the study used a reference method that reflected short-term intake ( < 7 d), long-term intake (>> or = 7 d) or used biomarkers. A correlation coefficient for each nutrient was calculated from the mean of the correlation coefficients from each study weighted by the quality of the study. Thirty-two articles were included in the present review: validation studies from infants (1-23 months); child preschool (2-5 years); children (6-12 years); adolescents (13-18 years). Validation of FFQ studies in infants and preschool children using a reference method that reflected short-term intake showed good correlations for niacin, thiamin, vitamins B6, D, C, E, riboflavin, Ca, K, Mg, Fe and Zn (with correlations ranging from 0.55 for vitamin E to 0.69 for niacin).Regarding the reference method reflecting short-term intake in children and adolescents, good correlations were seen only for vitamin C (r 0.61) and Ca (r 0.51). Using serum levels of micronutrient demonstrated that the 3 d weighed dietary records was superior to the FFQ as a tool to validate micronutrient intakes. Including supplement users generally improved the correlations between micronutrient intakes estimated by any of the dietary intake methods and respective biochemical indices.

The Na(+)-pumping NADH-ubiquinone oxidoreductase has six polypeptide subunits (NqrA-F) and a number of redox cofactors, including a noncovalently bound FAD and a 2Fe-2S center in subunit F, covalently bound FMNs in subunits B and C, and a noncovalently bound riboflavin in an undisclosed location. The FMN cofactors in subunits B and C are bound to threonine residues by phosphoester linkages. A neutral flavin-semiquinone radical is observed in the oxidized enzyme, whereas an anionic flavin-semiquinone has been reported in the reduced enzyme. For this work, we have altered the binding ligands of the FMNs in subunits B and C by replacing the threonine ligands with other amino acids, and we studied the resulting mutants by EPR and electron nuclear double resonance spectroscopy. We conclude that the sodium-translocating NADH:quinone oxidoreductase forms three spectroscopically distinct flavin radicals as follows: 1) a neutral radical in the oxidized enzyme, which is observed in all of the mutants and most likely arises from the riboflavin; 2) an anionic radical observed in the fully reduced enzyme, which is present in wild type, and the NqrC-T225Y mutant but not the NqrB-T236Y mutant; 3) a second anionic radical, seen primarily under weakly reducing conditions, which is present in wild type, and the NqrB-T236Y mutant but not the NqrC-T225Y mutant. Thus, we can tentatively assign the first anionic radical to the FMN in subunit B and the second to the FMN in subunit C. The second anionic radical has not been reported previously. In electron nuclear double resonance spectra, it exhibits a larger line width and larger 8alpha-methyl proton splittings, compared with the first anionic radical.

OBJECTIVE

To investigate relationships between use of vitamin supplements and the three principal cataract types in a population-based sample.

METHODS

We studied 2873 of the 3654 participants (79%) aged 49 to 97 years attending the cross-sectional Blue Mountains Eye Study who completed a detailed food frequency questionnaire, which included type, dose, and duration of vitamin supplement use. Masked grading of nuclear, cortical, and posterior subcapsular opacities from lens photographs was performed, using the Wisconsin method.

RESULTS

Use of multivitamin supplements was associated with reduced prevalence of nuclear cataract, odds ratio 0.6, 95% confidence interval 0.4 to 1.0, P =.05. For both nuclear and cortical cataract, longer duration of multivitamin use was associated with reduced cataract prevalence (nuclear cataract, trend P =.02; cortical cataract, trend P =.03). Use of thiamin supplements was associated with reduced prevalence of nuclear (odds ratio 0.6, confidence interval 0.4 to 1.0, P =.03, dose trend P =.03) and cortical cataract (odds ratio 0.7, confidence interval 0.5 to 0.9, P =.01, dose trend P =.02). Riboflavin (odds ratio 0.8, confidence interval 0.6 to 1.0, P =.05) and niacin (odds ratio 0.7, confidence interval 0.6 to 1.0, P =.04) supplements exerted a weaker protective influence on cortical cataract. Vitamin A supplements were protective against nuclear cataract (odds ratio 0.4, confidence interval 0.2 to 0.8, P =.01, dose trend P =.01). Folate (odds ratio 0.4, confidence interval 0.2 to 0.9, P =.03) appeared protective for nuclear cataract, whereas both folate (odds ratio 0.6, confidence interval 0.3 to 0.9, P =.01, dose trend P =.04) and vitamin B12 supplements (odds ratio 0.7, confidence interval 0.5 to 1.0, P =.03, dose trend P =.02) were strongly protective against cortical cataract.

CONCLUSIONS

Long-term use of multivitamins, B group and vitamin A supplements was associated with reduced prevalence of either nuclear or cortical cataract. A strong protective influence on cortical cataract, from use of folate or vitamin B12 supplements, is a new finding.

OBJECTIVE

Hyperhomocysteinaemia is considered to be a risk factor for cardiovascular disease (particularly stroke) and has been implicated in recurrent miscarriage and osteoporotic fracture, recognized manifestations of coeliac disease (CD). The objective of this study was to compare plasma homocysteine levels and biomarker status of metabolically related B vitamins (folate, vitamin B(12), B(6) and riboflavin) in treated and untreated CD patients and healthy controls.

METHODS

CD patients attending a clinic for either initial or follow-up biopsy (at least 12 months after commencing a gluten-free diet) were categorized into three groups: 1) newly diagnosed (untreated; n=35); 2) persistent villous atrophy (VA) at follow-up (n=24) or 3) recovered VA at follow-up (n=41). Blood samples were analysed for plasma homocysteine, serum and red cell folate and serum vitamin B(12) levels, and for plasma pyridoxal 5-phosphate (PLP, vitamin B(6)) and riboflavin (vitamin B(2)) status.

RESULTS

Homocysteine concentrations were significantly higher (p=0.05) and red cell and serum folate significantly lower (p<0.001) in untreated patients compared with controls, while all three reached normal levels in recovered VA patients. Although untreated and persistent VA patients tended to have lower B(12) levels, these did not reach significance. There was no evidence of compromised B(6) or riboflavin status, even in untreated CD patients. Homocysteine concentrations were inversely associated with both serum (r=-0.421; p<0.001) and red cell (r=-0.459; p<0.001) folate and with serum vitamin B(12) (r=-0.353; p=0.001).

CONCLUSIONS

Gluten exclusion in CD improves folate status and normalizes homocysteine concentrations. Reducing the risk of homocysteine-related disease may be another reason for aggressive diagnosis and treatment of CD.

Here we present new evidence that riboflavin is present as one of four flavins in Na+-NQR. In particular, we present conclusive evidence that the source of the neutral radical is not one of the FMNs and that riboflavin is the center that gives rise to the neutral flavosemiquinone. The riboflavin is a bona fide redox cofactor and is likely to be the last redox carrier of the enzyme, from which electrons are donated to quinone. We have constructed a double mutant that lacks both covalently bound FMN cofactors (NqrB-T236Y/NqrC-T225Y) and have studied this mutant together with the two single mutants (NqrB-T236Y and NqrC-T225Y) and a mutant that lacks the noncovalently bound FAD in NqrF (NqrF-S246A). The double mutant contains riboflavin and FAD in a 0.6:1 ratio, as the only flavins in the enzyme; noncovalently bound flavins were detected. In the oxidized form, the double mutant exhibits an EPR signal consistent with a neutral flavosemiquinone radical, which is abolished on reduction of the enzyme. The same radical can be observed in the FAD deletion mutant. Furthermore, when the oxidized enzyme reacts with ubiquinol (the reduced form of the usual electron acceptor) in a process that reverses the physiological direction of the electron flow, a single kinetic phase is observed. The kinetic difference spectrum of this process is consistent with one-electron reduction of a neutral flavosemiquinone. The presence of riboflavin in the role of a redox cofactor is thus far unique to Na+-NQR.

BACKGROUND

Chronic, low-grade inflammation provides a link between normal ageing and the pathogenesis of age-related diseases. A series of in vitro tests confirmed the strong anti-inflammatory activities of known inhibitors of NF-κB activation (δ-tocotrienol, quercetin, riboflavin, (-) Corey lactone, amiloride, and dexamethasone). δ-Tocotrienol also suppresses β-hydroxy-β-methylglutaryl coenzyme A (HMG-CoA) reductase activity (the rate-limiting step in de novo cholesterol synthesis), and concomitantly lowers serum total and LDL cholesterol levels. We evaluated these compounds in an avian model anticipating that a dietary additive combining δ-tocotrienol with quercetin, riboflavin, (-) Corey lactone, amiloride, or dexamethasone would yield greater reductions in serum levels of total cholesterol, LDL-cholesterol and inflammatory markers (tumor necrosis factor-α [TNF-α], and nitric oxide [NO]), than that attained with the individual compounds.

RESULTS

The present results showed that supplementation of control diets with all compounds tested except riboflavin, (-) Corey lactone, and dexamethasone produced small but significant reductions in body weight gains as compared to control. (-) Corey lactone and riboflavin did not significantly impact body weight gains. Dexamethasone significantly and markedly reduced weight gain (>75%) compared to control. The serum levels of TNF-α and NO were decreased 61% - 84% (P < 0.001), and 14% - 67%, respectively, in chickens fed diets supplemented with δ-tocotrienol, quercetin, riboflavin, (-) Corey lactone, amiloride, or dexamethasone as compared to controls. Significant decreases in the levels of serum total and LDL-cholesterol were attained with δ-tocotrienol, quercetin, riboflavin and (-) Corey lactone (13% - 57%; P < 0.05), whereas, these levels were 2-fold higher in dexamethasone treated chickens as compared to controls. Parallel responses on hepatic lipid infiltration were confirmed by histological analyses. Treatments combining δ-tocotrienol with the other compounds yielded values that were lower than individual values attained with either δ-tocotrienol or the second compound. Exceptions were the significantly lower total and LDL cholesterol and triglyceride values attained with the δ-tocotrienol/(-) Corey lactone treatment and the significantly lower triglyceride value attained with the δ-tocotrienol/riboflavin treatment. δ-Tocotrienol attenuated the lipid-elevating impact of dexamethasone and potentiated the triglyceride lowering impact of riboflavin. Microarray analyses of liver samples identified 62 genes whose expressions were either up-regulated or down-regulated by all compounds suggesting common impact on serum TNF-α and NO levels. The microarray analyses further identified 41 genes whose expression was differentially impacted by the compounds shown to lower serum lipid levels and dexamethasone, associated with markedly elevated serum lipids.

CONCLUSIONS

This is the first report describing the anti-inflammatory effects of δ-tocotrienol, quercetin, riboflavin, (-) Corey lactone, amiloride, and dexamethasone on serum TNF-δ and NO levels. Serum TNF-δ levels were decreased by >60% by each of the experimental compounds. Additionally, all the treatments except with dexamethasone, resulted in lower serum total cholesterol, LDL-cholesterol and triglyceride levels. The impact of above mentioned compounds on the factors evaluated herein was increased when combined with δ-tocotrienol.

Many marine and pathogenic bacteria have a unique sodium-translocating NADH:ubiquinone oxidoreductase (Na(+)-NQR), which generates an electrochemical Na(+) gradient during aerobic respiration. Na(+)-NQR consists of six subunits (NqrA-F) and contains five known redox cofactors: two covalently bound FMNs, one noncovalently bound FAD, one riboflavin, and one 2Fe-2S center. A stable neutral flavin-semiquinone radical is observed in the air-oxidized enzyme, while the NADH- or dithionite-reduced enzyme exhibits a stable anionic flavin-semiquinone radical. The NqrF subunit has been implicated in binding of both the 2Fe-2S cluster and the FAD. Four conserved cysteines (C70, C76, C79, and C111) in NqrF match the canonical 2Fe-2S motif, and three conserved residues (R210, Y212, S246) have been predicted to be part of a flavin binding domain. In this work, these two motifs have been altered by site-directed mutagenesis of individual residues and are confirmed to be essential for binding, respectively, the 2Fe-2S cluster and FAD. EPR spectra of the FAD-deficient mutants in the oxidized and reduced forms exhibit neutral and anionic flavo-semiquinone radical signals, respectively, demonstrating that the FAD in NqrF is not the source of either radical signal. In both the FAD and 2Fe-2S center mutants the line widths of the neutral and anionic flavo-semiquinone EPR signals are unchanged from the wild-type enzyme, indicating that neither of these centers is nearby or coupled to the radicals. Measurements of steady-state turnover using NADH, Q-1, and the artificial electron acceptor ferricyanide strongly support an electron transport pathway model in which the noncovalently bound FAD in the NqrF subunit is the initial electron acceptor and electrons then flow to the 2Fe-2S center.

A case-control study of esophageal cancer was conducted among the black male residents of Washington, D.C., to find reasons for the exceptionally high risk in this population. The next of kin of 120 esophageal cancer cases who died during 1975-77 and of 250 D.C. black males who died of other causes were interviewed. Five indicators of general nutritional status--fresh or frozen meat and fish consumption, dairy product and egg consumption, fruit and vegetable consumption, relative weight (wt/ht2), and number of meals eaten per day--were each significantly and inversely correlated with the relative risk of esophageal cancer. Associations with other food groups were not apparent. The least nourished third of the study population, defined by any of these five measures, was at twice the risk of the most nourished third. None of these associations was markedly reduced by controlling for ethanol consumption, the other major risk factor in this population; smoking; socioeconomic status; or the other nutrition measures. When the three food group consumption measures were combined into a single overall index of general nutritional status, the relative risk of esophageal cancer between extremes was 14. Estimates of the intake of vitamin A, carotene, vitamin C, thiamin, and riboflavin were inversely associated with relative risk; but each micronutrient index was less strongly associated with risk than were the broad food groups that provide most of the micronutrient. Thus no specific micronutrient deficiency was identified. Instead, generally poor nutrition was the major dietary predictor of risk and may partially explain the susceptibility of urban black men to esophageal cancer.

OBJECTIVE

This was a qualitative investigation of corneal microstructural modifications in keratoconic patients undergoing experimental transepithelial crosslinking (TE CXL).

METHODS

Ten patients with keratoconus intolerant to gas-permeable rigid contact lenses were enrolled. Corneal thickness was in the range 350-390 µm at the thinnest point measured by Visante AC optical coherence tomography system (Zeiss, Jena, Germany). All patients underwent TE CXL with 0.1% riboflavin-15% dextran solution supplemented with TRIS plus sodium EDTA (Ricrolin TE, Sooft Italia) according to Siena protocol. In vivo Heidelberg retinal tomograph II laser scanning confocal analysis (Rostock Cornea Module, Heidelberg, Germany) was performed with the following follow-up: preoperative and postoperative assessments at 1, 3, and 6 months. The following morphologic parameters were evaluated: epithelium, subepithelial, and anterior stroma nerve plexi, keratocytes apoptosis, stromal changes, and the endothelium.

RESULTS

After TE CXL, epithelial cells showed apoptosis, with mosaic alterations gradually disappearing. Keratocytes apoptosis was variable, superficial, and uneven, with a maximum depth of penetration at about 140 µm, measured from the surface of epithelium. Treatment respected subepithelial and stromal nerves that did not disappear. No variation in cell count or endothelial mosaic was observed.

CONCLUSIONS

In vivo confocal analysis of corneal modifications induced by TE CXL showed a limited apoptotic affect of this treatment, about one-third of classic epi-off crosslinking procedure. The TE CXL respected sub-basal and anterior stroma nerve fibers, resulting safe for corneal endothelium. According to limited penetration, its mid- to long-term efficacy needs to be determined in different clinical settings related to patient age and keratoconus progression.

The biosynthesis of the polyketide antibiotic actinorhodin by Streptomyces coelicolor involves the oxidative dimerization and hydroxylation of a precursor, most likely dihydrokalafungin, as the final steps in its formation. Mutations in the actVB gene block these last steps, and the mutants secrete kalafungin as a shunt product. To investigate the role of the actVB gene in these transformation, we have overexpressed the gene in Escherichia coli and purified and characterized the recombinant protein. ActVB was shown to catalyze the reduction of FMN by NADH to give NAD and FMNH2, which, unusually, is released into solution. The protein contains no chromogenic cofactors and exhibits no requirements for added metal ions. The reaction obeys simple kinetics and proceeds through the formation of a ternary complex; Km values for FMN and NADH are 1.5 and 7.3 microM, respectively, and kcat is about 5 s-1. FAD and riboflavin are also substrates for the enzyme, although they have much higher Km values. The subunit structure of the enzyme was investigated by analytical ultracentrifugation, which showed the protein to exist in rapid equilibrium between monomer and dimer forms. The possible role of this oxidoreductase in the oxidative chemistry of actinorhodin biosynthesis is discussed.

OBJECTIVE

Keratoconus disease or post-LASIK corneal ectasia are increasingly treated using UV-A/riboflavin-induced corneal collagen cross-linking (CXL). However, this treatment suffers from a lack of techniques to provide an assessment in real-time of the CXL effects. Here, we investigated the potential interest of corneal elasticity as a biomarker of the efficacy of this treatment.

METHODS

For this purpose, supersonic shear wave imaging (SSI) was performed both ex vivo and in vivo on porcine eyes before and after CXL. Based on ultrasonic scanners providing ultrafast frame rates (~30 kHz), the SSI technique generates and tracks the propagation of shear waves in tissues. It provides two- and three-dimensional (2-D and 3-D) quantitative maps of the corneal elasticity.

RESULTS

After CXL, quantitative maps of corneal stiffness clearly depicted the cross-linked area with a typical 200-μm lateral resolution. The CXL resulted in a 56% ± 15% increase of the shear wave speed for corneas treated in vivo (n = 4).

CONCLUSIONS

The in vivo CXL experiments performed on pigs demonstrated that the quantitative estimation of local stiffness and the 2-D elastic maps of the corneal surface provide an efficient way to monitor the local efficacy of corneal cross-linking.

OBJECTIVE

To evaluate the effectiveness of accelerated corneal collagen crosslinking (CXL) with riboflavin for keratoconus by the change in dioptric power and corneal topography.

METHODS

Private practice, Tokyo, Japan.

METHODS

Case series.

METHODS

The accelerated CXL treatments (KXL system) were performed using a 10-minute riboflavin 0.1% (Vibex Rapid) soak and a 3-minute ultraviolet-A (UVA) irradiance at a level of 30 mW/cm(2). This corresponds to a total radiant exposure of 5.4 J/cm(2). Preoperative and 1, 3, and 6 months postoperative examinations were performed.

RESULTS

The study enrolled 39 eyes of 22 patients. The mean uncorrected distance visual acuity showed a statistically significant improvement, from 1.11 ± 0.42 logMAR preoperatively to 0.89 ± 0.53 logMAR 6 months postoperatively (P<.01). The mean maximum keratometry readings also changed significantly, from 49.95 ± 6.11 diopters (D) preoperatively to 49.19 ± 5.82 D at 6 months (P<.01). There were no statistically significant changes in the endothelial cell density between preoperatively and postoperatively.

CONCLUSIONS

The changes after accelerated CXL were similar to those after conventional CXL. Thus, accelerated CXL has the potential to efficiently treat and halt the progression of keratoconus and may be an effective, efficient therapeutic option for treating corneal ectatic disease.

BACKGROUND

No author has a financial or proprietary interest in any material or method mentioned.

A 27-year-old man presented with corneal ectasia in his left eye 4 years after myopic laser in situ keratomileusis (LASIK) and was treated with riboflavin-ultraviolet-A (crosslinking). During the first post-treatment days, diffuse lamellar keratitis (DLK) (stage III) developed. The microbiology culture was negative. After intensive treatment with topical corticosteroids, the DLK resolved during the following 2 weeks. Crosslinking for post-LASIK corneal ectasia may induce DLK. Early diagnosis and appropriate treatment with intensive topical corticosteroids is essential to successfully manage this post-crosslinking complication.

OBJECTIVE

To assess the efficacy of corneal collagen cross-linking (CXL) in the management of infectious keratitis.

METHODS

Comprehensive literature search was performed in MEDLINE/PubMed and Cochrane Central Register of Controlled Trials using combinations of the following search terms: "corneal collagen cross linking" or "photoactivated riboflavin" or "UVA light and riboflavin" and "infectious keratitis" or "corneal ulcer." Last search was on March 19, 2015. Extracted data from individual studies were summarized and summary proportions of eyes healed and complications for different subgroups were estimated.

RESULTS

Twenty-five studies were included (2 randomized controlled trials, 13 case series, and 10 case reports) with a total of 210 eyes of 209 patients, of which 175 eyes underwent CXL. Causative microorganisms were bacteria, fungi, acanthamoeba, and Herpes simplex virus in 96, 32, 11, and 2 cases, respectively. Coinfections were present in 13 and cause was inconclusive in 21 cases. Sixteen of 175 eyes received no additional antibiotics, whereas 159 underwent CXL as an adjunct to antimicrobial treatment. Proportion of eyes healed with CXL was 87.2% (95% confidence interval (CI), 81.9%, 91.8%). For bacterial keratitis, the proportion of eyes healed was 85.7% (95% CI, 78.5%, 91.7%), whereas 10/11 and 25/32 eyes with acanthamoeba and fungal keratitis, respectively, were healed (available data not sufficient to provide a valid proportion analysis). Treatment resulted in corneal melting and tectonic keratoplasty in both Herpes simplex virus cases.

CONCLUSIONS

CXL seems promising in the management of infectious keratitis, excluding viral infections. However, more randomized controlled trials are required to assess its efficacy.

Changes in the biomechanical properties of the human cornea play an important role in the pathogenesis of corneal ectatic diseases. A variety of conditions in primary acquired (keratoconus and pellucid marginal degeneration) or secondary induced (iatrogenic keratectasia after excimer refractive laser surgery) corneal ectatic disorders lead to reduced biomechanical resistance. Corneal collagen crosslinking (CXL) has emerged as a promising technique to slow or even to stop the progression of these corneal ectatic pathologies. In this procedure, riboflavin (vitamin B2) is administered in conjunction with ultraviolet A light (UVA, 365 nm). This interaction causes the formation of reactive oxygen species, leading to the formation of additional covalent bonds between collagen molecules, with consequent biomechanical stiffening of the cornea. Although this method is not yet accepted as an evidence-based medicine modality for the treatment of corneal primary or secondary ectasias, the results of prospective, randomized studies of CXL used in the treatment of these pathologic entities show significant changes in the properties of corneal tissue. This procedure is currently the only etiopathogenetic approach in ectatic eyes that can delay or stop the process of cornea destabilization, reducing the necessity for keratoplasty. Despite promising results, CXL is associated with issues that include long-term safety and duration of the stabilizing effect. Combination of CXL with vision-improving procedures, such as topography-guided custom ablation and implantation of intracorneal ring segments of phakic intraocular lenses, may expand the indications for this procedure.

Exposure to cyanide causes a spectrum of cardiac, neurological, and metabolic dysfunctions that can be fatal. Improved cyanide antidotes are needed, but the ideal biological pathways to target are not known. To understand better the metabolic effects of cyanide and to discover novel cyanide antidotes, we developed a zebrafish model of cyanide exposure and scaled it for high-throughput chemical screening. In a screen of 3120 small molecules, we discovered 4 novel antidotes that block cyanide toxicity. The most potent antidote was riboflavin. Metabolomic profiling of cyanide-treated zebrafish revealed changes in bile acid and purine metabolism, most notably by an increase in inosine levels. Riboflavin normalizes many of the cyanide-induced neurological and metabolic perturbations in zebrafish. The metabolic effects of cyanide observed in zebrafish were conserved in a rabbit model of cyanide toxicity. Further, humans treated with nitroprusside, a drug that releases nitric oxide and cyanide ions, display increased circulating bile acids and inosine. In summary, riboflavin may be a novel treatment for cyanide toxicity and prophylactic measure during nitroprusside treatment, inosine may serve as a biomarker of cyanide exposure, and metabolites in the bile acid and purine metabolism pathways may shed light on the pathways critical to reversing cyanide toxicity.

The Escherichia coli-based Fur titration assay (FURTA), although a powerful tool for identification of genes regulated by the ferric uptake regulator (Fur), was unsuccessful for the gastric pathogen Helicobacter pylori. The FURTA was modified by construction of an E. coli indicator strain producing H. pylori Fur only. The promoter regions of the ferric citrate receptor homolog fecA2 and the riboflavin synthesis gene ribBA were both positive in the modified FURTA, but negative in the original FURTA. Transcription of fecA2 and ribBA was demonstrated to be iron-repressed in H. pylori. This type of modification should allow FURTA analysis for bacteria with Fur binding sequences poorly recognized by E. coli Fur.

OBJECTIVE

Riboflavin-ultraviolet A (UVA) treatment induces cross-linking and stiffens the corneal stroma. A parallel reduction in stromal swelling and increased resistance to microbial and enzymatic degradation has been suggested. The purpose of this study was to evaluate the potential of riboflavin-UVA treatment in the management of corneal disorders, in particular edema due to endothelial decompensation and non-healing ulcers.

METHODS

Two clinical series are reported, 11 eyes with endothelial decompensation and 14 eyes with non-healing ulcers. Treatment comprised a) abrasion of epithelium (if present), b) instillation of 0.1% riboflavin in saline, and c) irradiation at 365 nm UVA light over approximately 30 minutes (3 mW/cm(2)). Four eyes in the first group were treated twice. Postoperatively, all eyes were followed for at least 3 months.

RESULTS

Reduction in corneal thickness was observed in 10 of 11 eyes with stromal edema and the majority also experienced improvement in vision. The effect occurred over weeks and lasted for months. Fourteen patients with non-healing ulceration were similarly treated; 6 healed, 8 showed no clear effect.

CONCLUSIONS

In addition to the stiffening of keratoconic and ectatic cornea, riboflavin-UVA treatment is effective in reducing corneal edema and has the potential to heal corneal ulcers.

OBJECTIVE

In recent years, the desire to develop methods to inactivate pathogens in blood components has continued to grow. Several of these proposed approaches have been introduced or are currently in clinical studies. The use of chemical inactivating agents must be considered in terms of the current safety of the blood supply and the potential risks that the introduction of new chemical entities into blood components may carry. The impact which these treatment procedures have on the in vitro and in vivo performance of these products must also be considered relative to the potential benefit of the pathogen inactivation potential they offer. This paper will discuss one possible approach for inactivating pathogens in blood using vitamin B2, Riboflavin, and light.

METHODS

We have used Riboflavin for treating plasma and platelets and evaluated protein quality and platelet function in vitro. Initial toxicology tests to assess the impact of infusion of photoproducts generated in these processes have also been conducted in rodents. Cytotoxicity evaluations have been used to assess the possible impact of photoproduct toxicity in vivo. Virus and bacteria spiking studies using a variety of human and animal model pathogens have been conducted in order to asses the efficacy of this process.

RESULTS

Initial toxicology assessment of the photoproducts of Riboflavin generated under the proposed treatment conditions have been favorable. Virus and bacteria clearance studies have demonstrated efficacy of the procedure against a wide range of human and animal pathogens, including intracellular HIV-1. Studies with platelet and plasma function indicated reductions in vitro comparable to other proposed treatment approaches.

CONCLUSIONS

The use of Riboflavin in a photochemical decontamination process for blood components shows promise.

The most prominent biochemical consequence of riboflavin deficiency in rats is a drastic decrease in various acyl-CoA dehydrogenase activities, especially that of short chain and isovaleryl-CoA dehydrogenase (IVD). As a result, oxidation of fatty acids and leucine is severely inhibited. We studied the effects of FAD at various stages of acyl-CoA dehydrogenase biogenesis. Immunoblot revealed severe losses of various acyl-CoA dehydrogenases and electron transfer flavoprotein in riboflavin-deficient rat liver mitochondria. The decreases in IVD and short chain acyl-CoA dehydrogenase were particularly severe, reaching values of 17 and 34% of controls, respectively. With the exception of IVD, the rate of in vitro transcription of the respective genes and the amounts of mRNAs of these flavoproteins in tissues increased 3-8.5-fold over controls. The amount of IVD mRNA and its transcription rate remained unchanged, suggesting that IVD expression is regulated separately from other acyl-CoA dehydrogenases. When riboflavin was depleted, in vitro translation of acyl-CoA dehydrogenase and electron transfer flavoprotein alpha-subunit mRNAs was moderately inhibited. Translation of non-flavoproteins was also inhibited. The stability of precursor acyl-CoA dehydrogenases and their mitochondrial import/processing were unaffected. However, mature acyl-CoA dehydrogenases degraded markedly faster in deficient mitochondria than in controls. Regardless of whether precursors were translated under riboflavin-depleted or riboflavin replete conditions, mature acyl-CoA dehydrogenases survived well when imported into normal mitochondria but degraded faster when imported into deficient mitochondria. These findings indicate that FAD ligand binds to mature acyl-CoA dehydrogenase inside the mitochondria.