OBJECTIVE

To assess changes in biomechanical properties of human cornea after treatment of keratoconus with UV-A-riboflavin corneal collagen cross-linking (CXL).

METHODS

Single-center, prospective, interventional study.

METHODS

Ten eyes of 10 patients aged 26.5 +/- 5.7 (mean +/- SD) years with progressive keratoconus were treated with UV-A-riboflavin CXL and assessed with the Ocular Response Analyzer (ORA) that measured corneal hysteresis (CH), corneal resistance factor (CRF), Goldmann-correlated intraocular pressure (IOPg), and corneal compensated intraocular pressure (IOPcc). Intraocular pressure was also measured by Goldmann applanation tonometry (GAT-IOP). Patients were assessed with ORA preoperatively, at week 1, months 1, 3, and 6 after treatment. Postoperative measurements at each visit were compared with preoperative values.

RESULTS

CH and CRF were transiently elevated after cross-linking treatment, with the difference not statistically significant (P>> 0.3). IOPcc and IOPg were statistically significantly higher at 1 week and 1 month but not subsequently (P < 0.04). GAT-IOP was statistically significantly higher at 1 week and at 1 and 3 months (P < 0.01).

CONCLUSIONS

There were no significant differences in corneal biomechanical properties, as measured with the ORA parameters CH and CRF, after CXL in keratoconus. IOPcc, IOPg, and GAT-IOP values were transiently elevated after CXL treatment in our study. Whether this reflects a measurement artifact resulting from corneal changes or true elevation of intraocular pressure is unclear.

BACKGROUND

Food and diet are central issues that concern patients with irritable bowel syndrome (IBS). Few studies have thoroughly analyzed dietary intake in IBS. Our aims were to determine the nutrient intake in IBS patients in comparison to the general population, assess nutritional differences between IBS subgroups based on the predominant bowel habit or symptom severity, as well as to evaluate if their nutrient intake meet nutrition recommendations.

METHODS

We included 187 IBS patients (mean 40.2 years; 139 women). They completed a 4-days food registration record, which was compared with an age-, and gender-matched control group (n = 374; 278 women) from a nation-wide dietary survey and with Nordic Nutrient Recommendations.

RESULTS

Daily nutrient intake in IBS patients was similar to the general population and met national nutrients recommendations. Irritable bowel syndrome patients had similar energy distribution from macronutrients compared to the control group, but the protein percentage tended to be higher. Irritable bowel syndrome patients also had significantly higher daily intake of vitamin E, folate, iron, vitamin C, and dietary fibers, as well as lower intake of vitamin A, riboflavin, calcium, and potassium. There was no association between nutrient intake and IBS subtypes or symptom severity.

CONCLUSIONS

Although many IBS patients state that they avoid food items, this does not seem to influence their intake of nutrients to any large extent. The observed minor differences in nutrient intake indicate a tendency toward higher intake of fruit and vegetables and a lower intake of meat and dairy products in IBS patients.

BACKGROUND

The purpose of this study was to evaluate the safety and efficacy of ultraviolet A irradiation cross-linking on completion for cases of high myopic laser-assisted in situ keratomileusis (LASIK).

METHODS

Forty-three consecutive LASIK cases treated with femtosecond laser flap and the WaveLight excimer platform were evaluated perioperatively for uncorrected visual acuity, best corrected spectacle visual acuity, refraction, keratometry, topography, total and flap pachymetry, corneal optical coherence tomography, and endothelial cell count. All eyes at the completion of LASIK had cross-linking through the repositioned flap, with higher fluence (10 mW/cm(2)) ultraviolet light of an average 370 μm wavelength and 10 mW/cm(2) fluence applied for 3 minutes following an earlier single instillation of 0.1% riboflavin within the flap interface. Mean follow-up duration was 3.5 (range 1.0-4.5) years.

RESULTS

Mean uncorrected visual acuity changed from 0.2 to 1.2, best corrected spectacle visual acuity from 1.1 to 1.2, spherical equivalent from -7.5 diopters (D) to -0.2 D, keratometry from 44.5 D to 38 D, flap pachymetry from 105 μm to, total pachymetry from 525 to 405, and endothelial cell count from 2750 to 2800. None of the cases developed signs of ectasia or significant regression during follow-up.

CONCLUSIONS

Prophylactic collagen cross-linking for high-risk LASIK cases appears to be a safe and effective adjunctive treatment for refractive regression and potential ectasia. This application may be viewed as prophylactic customization of the biomechanical behavior of corneal collagen.

OBJECTIVE

The loss of corneal endothelial cells associated with aging and possibly other causes has been speculated to be related to exposure to reactive oxygen species (ROS). The current study was conducted to investigate, by use of photosensitizers, the underlying mechanisms involved in the death of bovine corneal endothelial cells (BCENs) caused by ROS.

METHODS

BCEN cells in primary culture were treated with a photosensitizer (riboflavin or rose bengal) with light exposure. The patterns of cell damage and death were assessed using an acridine orange-ethidium bromide differential staining method, TdT-mediated dUTP nick-end labeling (TUNEL) assay, and transmission electron microscopy. The cytotoxicity was assayed by mitochondrial function using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) testing. Antioxidants, including catalase, L-histidine, salicylic acid, and superoxide dismutase, were used to determine the types of ROS involved. Activation of nuclear factor (NF)-kappaB was examined by fluorescent immunocytochemistry with anti-p65 antibody.

RESULTS

Light-irradiated riboflavin or rose bengal resulted in a significant decrease in viability of BCEN cells. Chromosomal condensation and fragmentation were observed in apoptotic cells, and membrane lysis and damage of cell ultrastructures were observed in necrotic cells. Riboflavin induced apoptosis at 30 minutes and thereafter and induced necrosis after 2 hours. Rose bengal was shown to cause similar effects within half the time required for the effects of riboflavin. Catalase and salicylic acid were found to provide protection for BCENs from cytotoxic effects of riboflavin, and L-histidine was found to protect BCENs from cytotoxicity induced by rose bengal. Kinetic studies using immunocytochemistry showed that NF-kappaB was translocated into the nucleus within 15 minutes and 30 minutes after treatment with rose bengal and riboflavin, respectively.

CONCLUSIONS

The cytotoxic effects of photo-irradiated riboflavin and rose bengal are shown to be mediated by two distinct but parallel pathways, one leading to apoptosis and the other to necrosis. Possible involvement of NF-kappaB in cell death is suggested. These findings provide potential leads for future investigation into the molecular mechanisms of loss of corneal endothelial cells related to aging, oxidative stress, and possibly other similar causes.

OBJECTIVE

To determine differences in riboflavin concentration in the anterior, intermediate, and posterior stroma after 3 corneal cross-linking imbibition techniques (standard epithelial [epi]-off, epi-on, and iontophoresis-assisted administration) of 0.1% riboflavin.

METHODS

Experimental laboratory investigation of human cadaver corneas not suitable for transplantation.

METHODS

Ten corneas underwent imbibition with epi-on (n = 3), epi-off (n = 3), iontophoresis (n = 3), and saline exposure (control; n = 1). Femtosecond laser was used to produce 3 8-mm discs of the superficial (0-150 μm), intermediate (150-300 μm), and deep stroma (>300 μm). Riboflavin concentration was measured with high-performance liquid chromatography. The main outcome measure was riboflavin concentration at the 3 evaluated depths.

RESULTS

The overall stromal concentration of riboflavin was 34.1 ± 7.1 μg/g in epi-off, 7.2 ± 3.7 μg/g in epi-on, and 15.0 ± 5.1 μg/g in iontophoresis. The mean riboflavin content in the superficial slice in the epi-off group was about 2-fold greater than that of the iontophoresis group (50.5 ± 5.3 μg/g and 23.6 ± 2.5 μg/g, respectively) and 4-fold greater than that of the epi-on group (11.7 ± 3.3 μg/g). Similar differences among the 3 groups were observed for the intermediate and posterior stromal slices, presenting an evident reduction of riboflavin concentration with increasing depth in all groups. Slice depth-dependent decrease in riboflavin concentration was statistically significant (general linear model (GLM); F1,6 = 62.265, P < .001), as was the group-dependent variation (GLM; F2,6 = 20.268, P = .002) and the slice depth group interaction (GLM; F2,6 = 18.004, P = .002).

CONCLUSIONS

Corneal cross-linking transepithelial iontophoresis imbibition yielded greater and deeper riboflavin saturation with respect to conventional epi-on, while maintaining the advantages of avoiding epithelial removal and shorter procedure time, but did not reach concentrations obtained with standard epi-off.

Autism spectrum disorder (ASD) is still diagnosed through behavioral observation, due to a lack of laboratory biomarkers, which could greatly aid clinicians in providing earlier and more reliable diagnoses. Metabolomics on human biofluids provides a sensitive tool to identify metabolite profiles potentially usable as biomarkers for ASD. Initial metabolomic studies, analyzing urines and plasma of ASD and control individuals, suggested that autistic patients may share some metabolic abnormalities, despite several inconsistencies stemming from differences in technology, ethnicity, age range, and definition of "control" status.

ASD-specific urinary metabolomic patterns were explored at an early age in 30 ASD children and 30 matched controls (age range 2-7, M:F = 22:8) using hydrophilic interaction chromatography (HILIC)-UHPLC and mass spectrometry, a highly sensitive, accurate, and unbiased approach. Metabolites were then subjected to multivariate statistical analysis and grouped by metabolic pathway.

Urinary metabolites displaying the largest differences between young ASD and control children belonged to the tryptophan and purine metabolic pathways. Also, vitamin B6, riboflavin, phenylalanine-tyrosine-tryptophan biosynthesis, pantothenate and CoA, and pyrimidine metabolism differed significantly. ASD children preferentially transform tryptophan into xanthurenic acid and quinolinic acid (two catabolites of the kynurenine pathway), at the expense of kynurenic acid and especially of melatonin. Also, the gut microbiome contributes to altered tryptophan metabolism, yielding increased levels of indolyl 3-acetic acid and indolyl lactate.

The metabolic pathways most distinctive of young Italian autistic children largely overlap with those found in rodent models of ASD following maternal immune activation or genetic manipulations. These results are consistent with the proposal of a purine-driven cell danger response, accompanied by overproduction of epileptogenic and excitotoxic quinolinic acid, large reductions in melatonin synthesis, and gut dysbiosis. These metabolic abnormalities could underlie several comorbidities frequently associated to ASD, such as seizures, sleep disorders, and gastrointestinal symptoms, and could contribute to autism severity. Their diagnostic sensitivity, disease-specificity, and interethnic variability will merit further investigation.

Vitamin B2 (riboflavin) is essential for metabolic functions and is synthesized by many bacteria, yeast, and plants, but not by mammals and other animals, which must acquire it from the diet. In mammals, modified pyrimidine intermediates from the microbial biosynthesis of riboflavin are recognized as signature biomarkers of microbial infection. This recognition occurs by specialized lymphocytes known as mucosal associated invariant T (MAIT) cells. The major histocompatibility class I-like antigen-presenting molecule, MR1, captures these pyrimidine intermediates, but only after their condensation with small molecules derived from glycolysis and other metabolic pathways to form short-lived antigens. The resulting MR1-Ag complexes are recognized by MAIT cell antigen receptors (αβ T cell receptors (TCRs)), and the subsequent MAIT cell immune responses are thought to protect the host from pathogens at mucosal surfaces. Here, we review our understanding of how these novel antigens are generated and discuss their interactions with MR1 and MAIT TCRs.

OBJECTIVE

To study the current diet and nutritional status of rural adolescents in India.

METHODS

Cross-sectional study with household as the unit of randomization.

METHODS

National Nutrition Monitoring Bureau collected information in the rural areas of the nine States.

METHODS

In each State, 120 villages were selected from eight districts. From each of the selected villages, 20 households (HHs) were selected from five clusters. The information on socio-demographic profile was collected in all the 20 HHs, while anthropometric data such as weight, height and clinical signs of nutritional deficiency was collected on all the available adolescents in the selected households. In every fourth sampled household, ie five HHs, dietary information on all the members was collected using 24 h dietary recall. The outcome measures for nutritional status were proportion of underweight (<median -2 s.d. of NCHS standards of weight for age), stunted (<median -2 s.d. of NCHS standards of height for age) and body mass index. The nutrient intakes were compared with recommended dietary allowances (RDA).

RESULTS

Anthropometric and socio-economic information on 12 124 adolescent boys and girls and dietary information on 2579 individuals in 1996-1997 was available for the analysis. The major occupation of the heads of the households surveyed was agriculture. More than a third (37.3%) of the families with adolescents did not possess any land. The per capita income per month was about Rs 250/- at 1996-1997 prices. About 23% of the adolescent girls were married before the age of 18 y. About a quarter of the married adolescent girls had short stature and 18.6% were underweight. They considered as 'at risk'. About 39% of the adolescents were stunted (<Median -2 s.d. of NCHS height for age) irrespective of sex. The prevalence of undernutrition (<median -2 s.d. of NCHS weight for age) is higher (53.1%) in boys than in girls (39.5%). The extent of stunting was higher (42.7%) among adolescents belonging to the scheduled caste community. In the case of girls, the extent of underweight was considerably less in each age group than their male counterparts. About 70% of adolescents consumed more than 70% of RDA for energy. The intakes of micronutrients such as vitamin A and <em>riboflavin</em> were woefully inadequate.

CONCLUSIONS

The extent of undernutrition was high among adolescents and was higher among boys than girls. Adolescent girls in the rural areas could be at greater risk of nutritional stress because of early marriage and early conception before completion of their physical growth.

OBJECTIVE

To evaluate possible changes in USDA nutrient content data for 43 garden crops between 1950 and 1999 and consider their potential causes.

METHODS

We compare USDA nutrient content data published in 1950 and 1999 for 13 nutrients and water in 43 garden crops, mostly vegetables. After adjusting for differences in moisture content, we calculate ratios of nutrient contents, R (1999/1950), for each food and nutrient. To evaluate the foods as a group, we calculate median and geometric mean R-values for the 13 nutrients and water. To evaluate R-values for individual foods and nutrients, with hypothetical confidence intervals, we use USDA's standard errors (SEs) of the 1999 values, from which we generate 2 estimates for the SEs of the 1950 values.

RESULTS

As a group, the 43 foods show apparent, statistically reliable declines (R < 1) for 6 nutrients (protein, Ca, P, Fe, riboflavin and ascorbic acid), but no statistically reliable changes for 7 other nutrients. Declines in the medians range from 6% for protein to 38% for riboflavin. When evaluated for individual foods and nutrients, R-values are usually not distinguishable from 1 with current data. Depending on whether we use low or high estimates of the 1950 SEs, respectively 33% or 20% of the apparent R-values differ reliably from 1. Significantly, about 28% of these R-values exceed 1.

CONCLUSIONS

We suggest that any real declines are generally most easily explained by changes in cultivated varieties between 1950 and 1999, in which there may be trade-offs between yield and nutrient content.

Keratoconus is an ectatic corneal disorder for which exciting therapeutic and diagnostic technologies are emerging. However, its pathogenesis is still heterogeneous and elusive. We researched overlooked Asian keratoconus data by literature review of databases (PubMed, MEDLINE, Ovid, Google Scholar, Cornea, and Cochrane) using key words "keratoconus, Asia, epidemiology, treatment, risk factors, genes" and names of Asian countries. Articles and their references were analyzed. Studies showed that keratoconus may be more prevalent, have earlier onset, and have greater disease progression in certain Asian and non-Asian ethnicities, particularly Indians, Pakistanis, Middle Easterners, and Polynesians, compared with white populations. Epidemiological risk factors include ethnicity, age (younger than 30 years), gender (male), positive family history, and eye rubbing. Genetic and disease risk factors include atopy, vernal keratoconjunctivitis, Down syndrome, pellucid marginal corneal degeneration, VSX1 (visual system homeobox 1) gene, and Leber congenital amaurosis. Differentiation of heterogeneous keratoconus subsets with detailed genotype-phenotype characterization may advance understanding. Comprehensive multiethnic population studies with valid large-scale data are needed. New effective treatments (deep anterior lamellar keratoplasty, intrastromal corneal ring segments, and corneal collagen cross-linking with riboflavin) are succeeding previous treatments.

The Escherichia coli gene ssyB was cloned and sequenced. The ssyB63 (Cs) mutation is an insertion mutation in nusB, while the nusB5 (Cs) mutation suppresses secY24, indicating that inactivation of nusB causes cold-sensitive cell growth as well as phenotypic suppression of secY24. The correct map position of nusB is 9.5 min rather than 11 min as previously assigned. It is located at the distal end of an operon that contains a gene showing significant homology with a Bacillus subtilis gene involved in riboflavin biosynthesis.

Rhodococcus equi is a facultative intracellular opportunistic pathogen of immunocompromised people and a major cause of pneumonia in young horses. An effective live attenuated vaccine would be extremely useful in the prevention of R. equi disease in horses. Toward that end, we have developed an efficient transposon mutagenesis system that makes use of a Himar1 minitransposon delivered by a conditionally replicating plasmid for construction of R. equi mutants. We show that Himar1 transposition in R. equi is random and needs no apparent consensus sequence beyond the required TA dinucleotide. The diversity of the transposon library was demonstrated by the ease with which we were able to screen for auxotrophs and mutants with pigmentation and capsular phenotypes. One of the pigmentation mutants contained an insertion in a gene encoding phytoene desaturase, an enzyme of carotenoid biosynthesis, the pathway necessary for production of the characteristic salmon color of R. equi. We identified an auxotrophic mutant with a transposon insertion in the gene encoding a putative dual-functioning GTP cyclohydrolase II-3,4-dihydroxy-2-butanone-4-phosphate synthase, an enzyme essential for riboflavin biosynthesis. This mutant cannot grow in minimal medium in the absence of riboflavin supplementation. Experimental murine infection studies showed that, in contrast to wild-type R. equi, the riboflavin-requiring mutant is attenuated because it is unable to replicate in vivo. The mutagenesis methodology we have developed will allow the characterization of R. equi virulence mechanisms and the creation of other attenuated strains with vaccine potential.

Epidemiological studies have linked low folate intake with an increased risk of epithelial cancers, including colorectal cancer and cervical cancer. Riboflavin has received much less attention, but there is increasing interest in the well-established role that flavins play in folate metabolism and the possible synergy of a protective effect between these 2 vitamins. Folate plays a key role in DNA synthesis, repair, and methylation, and this forms the basis of mechanistic explanations for a putative role for folate in cancer prevention. The role of folate in these processes may be modulated by genotype for the common C677T thermolabile variant of methylene tetrahydrofolate reductase (MTHFR), homozygosity for which is associated with lower enzyme activity, lower plasma and red blood cell folate, and elevated plasma homocysteine. Riboflavin, as FAD, is a cofactor for MTHFR and there is evidently some interaction among riboflavin status, folate status, and genotype in determining plasma homocysteine, a functional marker of folate status. The MTHFR C677T polymorphism appears to interact with folate and riboflavin in modulating cancer risk in a manner that varies according to cancer site. Most evidence points to a protective effect of this polymorphism for risk of colorectal cancer, but the effect on cervical cancer risk is not clear. The effect of this polymorphism on cancer risk seems to be further modulated by other factors, including alcohol and, in the case of cervical cancer, infection with the human papilloma virus. An additional factor determining the effect of diet and genotype interactions on cancer risk may be the stage of cancer development.

BACKGROUND

Biomarkers and metabolites related to B vitamin function and one-carbon metabolism have been studied as predictors of chronic diseases in studies based on samples stored in biobanks. For most biomarkers, stability data are lacking or fragmentary.

METHODS

Degradation and accumulation kinetics of 32 biomarkers were determined at 23 °C in serum and plasma (EDTA, heparin, and citrate) collected from 16 individuals and stored for up to 8 days. In frozen serum (-25 °C), stability was studied cross-sectionally in 650 archival samples stored for up to 29 years. Concentration vs time curves were fitted to monoexponential, biexponential, linear, and nonlinear models.

RESULTS

For many biomarkers, stability was highest in EDTA plasma. Storage effects were similar at room temperature and at -25 °C; notable exceptions were methionine, which could be recovered as methionine sulfoxide, and cystathionine, which decreased in frozen samples. Cobalamin, betaine, dimethylglycine, sarcosine, total homocysteine, total cysteine, tryptophan, asymetric and symmetric dimethyl argenine, creatinine, and methylmalonic acid were essentially stable under all conditions. Most B vitamins (folate and vitamins B2 and B6) were unstable; choline increased markedly, and some amino acids also increased, particularly in serum. The kynurenines showed variable stability. For many biomarkers, degradation (folate and flavin mononucleotide) or accumulation (pyridoxal, riboflavin, choline, amino acids) kinetics at room temperature were non-first order.

CONCLUSIONS

Data on stability and deterioration kinetics for individual biomarkers are required to optimize procedures for handling serum and plasma, and for addressing preanalytical bias in epidemiological and clinical studies.

Ferritin-Fe(III) was rapidly and quantitatively reduced and liberated as Fe(II) by FMNH(2), FADH(2) and reduced riboflavin. Dithionite also released Fe(II) from ferritin but at less than 1% of the rate with FMNH(2). Cysteine, glutathione and ascorbate gave a similar slower rate and yielded less than 20% of the total iron from ferritin within a few hours. The reduction of ferritin-Fe(III) by the three riboflavin compounds gave complex second-order kinetics with overlapping fast and slow reactions. The fast reaction appeared to be non-specific and may be due to a reduction of Fe(III) of a lower degree of polymerization, equilibrated with ferritin iron. The amount of this Fe(3+) ion initially reduced was small, less than 0.3% of the total iron. Addition of FMN to the ferritin-dithionite system enhanced the reduction; this is due to the reduction of FMN by dithionite to form FMNH(2) which then reduces ferritin-Fe(III). A comparison of the thermodynamic parameters of FMNH(2)-ferritin and dithionite-ferritin complex formation showed that FMNH(2) required a lower activation energy and a negative entropy change, whereas dithionite required 50% more activation energy and showed a positive entropy change in ferritin reduction. The effectiveness of FMNH(2) in ferritin-Fe(III) reduction may be due to a specific binding of the riboflavin moiety to the protein portion of the ferritin molecule.

An investigation was carried out to determine whether there were significant changes in nutrient intake over 17 years of adult life. The Medical Research Council National Survey of Health and Development is a longitudinal study of a nationally representative cohort of singleton births in the UK in 1946. Of this cohort, 1253 survey members provided information on diet recorded in a 5 d diary at age 36 years in 1982, 43 years in 1989 and 53 years in 1999. The outcome measures were mean intakes of energy, macronutrients, minerals and vitamins. There were significant changes in the intake of most nutrients in 1999 compared with previous years. Intakes of fat, Na, Fe and Cu have fallen, but there was a rising trend in the intakes of Ca, P, carotene, thiamin, pyridoxine, folic acid and vitamins C, D and E in both men and women. Additionally, intakes of K, Mg and vitamin K1 have risen in women. There were significant gender differences, women showing a higher percentage rise in the intakes of carotene, riboflavin, folic acid, vitamin C and vitamin E. These changes were related to changes in the consumption of certain key foods, such as the increased consumption of fruit and vegetables and a shift away from whole milk, butter and red meat. Most of these trends are in line with accepted nutritional guidelines. How far these changes are due to consumer choice and real changes in food composition or are due to artefacts inherent in the methodology is discussed.

OBJECTIVE

To examine the influence of a new crosslinking treatment on corneal swelling properties that correlate with the degree of crosslinking.

METHODS

Department of Ophthalmology, Vivantes-Klinikum Neukölln, Berlin, Germany.

METHODS

Twenty freshly enucleated porcine eyes were crosslinked by applying the photosensitizer riboflavin and ultraviolet-A (UVA) light (370 nm, 3 mW/cm2) for 30 minutes. After the eyes were treated and incubated for 24 hours in a moist chamber, 15 eyes were examined by biomicroscopy and optical coherence tomography (OCT); 5 eyes were examined by light microscopy. Five control eyes were included.

RESULTS

Using light microscopy, a characteristic swelling pattern with 3 zones was identified in the crosslinked porcine cornea: an anterior intensely crosslinked zone of 242 microm, an intermediate partially crosslinked zone of 238 microm (hydration factor 2.2), and a noncrosslinked posterior zone of 1355 microm (hydration factor 2.7). A condensed OCT signal was demonstrated in the treated portion of the anterior stroma to a depth of 520 microm with a pronounced line at 540 microm, correlating with the combined anterior and intermediate layers after hydration in the histological analysis. In the nonhydrated state of the crosslinked cornea, the anterior zone was deduced to be 242 microm; the intermediate zone, 109 microm; and the posterior zone, 501 microm. Therefore, the maximum depth of the crosslinking effect was 351 microm.

CONCLUSIONS

Collagen crosslinking using riboflavin and UVA led to a significant change in the swelling behavior of the anterior stroma, confirming prior findings that the crosslinking effect is strongest in the anterior half of the stroma. Crosslinked cornea did not induce a specific signal on OCT, and OCT is therefore not suited for clinical controls of the crosslinking effect.

BACKGROUND

Compliance with medication in pharmacotherapy trials of alcoholism has been shown to be equal to, or more, important than in other areas of medicine. Research has suggested that naltrexone's effectiveness can be greatly influenced by the compliance of participants in clinical trials. Presently, we compare 2 compliance measurement methods [urine riboflavin and medication event monitoring system (MEMS)] used simultaneously to evaluate naltrexone's efficacy and the impact of compliance on the size of observable treatment effects.

METHODS

One hundred and thirty-seven of 160 randomized alcoholic patients completed 12-weeks (84 days) of naltrexone or placebo and cognitive behavioral therapy (CBT) or motivational enhancement therapy (MET). Urine riboflavin was determined during study weeks 2, 6, and 12. The MEMS provided a detailed computerized record of when a participant opened their medication bottle throughout the trial. Baseline predictors of MEMS (80% openings) and urine riboflavin >>or=1,500 ng/mL by fluorimetry) compliance were examined. The effects of the treatments in the compliant participants defined by one, the other, or both methods were compared and contrasted with a previously reported intent-to-treat analysis where compliance was not taken into account.

RESULTS

Age was predictive of compliance. 105 participants were deemed compliant via urine riboflavin criteria, 87 via MEMS, and 77 when both criteria were met, with no significant differences between treatment groups. The most compliant participants showed a significant medication by therapy interaction. Those treated with naltrexone/CBT showed more abstinence days (p<0.03), less heavy drinking days (p<0.03) and less total drinks (p<0.03) than the other groups. The effect size of this interaction increased from about 0.2 in the intent-to-treat analysis, to about 0.4 to 0.5 in the compliant group analyses, with little difference between compliance measurement methods.

CONCLUSIONS

Compliance measurement does appear to influence the evaluation of the efficacy of naltrexone within the context of CBT. Treatment effect sizes approximately doubled in the most compliant individuals. Measuring compliance by either of 2 distinct methods provides approximately similar results. As compliance with naltrexone within the context of CBT has such a large impact of treatment outcome, methods of enhancing compliance during treatment should be given the utmost attention.

1. 2,4-Dichlorophenol hydroxylase has been purified 13-fold from Acinetobacter grown on 2,4-dichlorophenoxyacetic acid as sole carbon source. The enzyme was estimated to be 80-90% pure by electrophoresis. 2. The enzyme has a relative molecular mass of about 240 000 and consists of four subunits of identical size. 3. The enzyme contains FAD as the prosthetic group. FAD could not be replaced by riboflavin or FMN in reconstituting active enzyme from apoenzyme. 4. The reaction catalysed is an NADPH-dependent hydroxylation of 2,4-dichlorophenol with the formation of 3,5-dichlorocatechol as product. The reaction stoichiometry is typical of a monooxygenase with an external electron donor. NADPH is the preferred reduced pyridine nucleotide substrate but the enzyme can function with NADH. 5. The enzyme possesses broad effector specificity. In addition to 2,4-dichlorophenol, 4-chlorophenol and 4-chloro-2-methylphenol are true substrates for the enzyme. A number of 'non-substrate effectors' has been found. 6. The enzyme is sensitive to thiol-inhibiting reagents.

The pathways of carbohydrate metabolism in Spirochaeta stenostrepta, a free-living, strictly anaerobic spirochete, were studied. The organism fermented glucose to ethyl alcohol, acetate, lactate, CO(2), and H(2). Assays of enzymatic activities in cell extracts, and determinations of radioactivity distribution in products formed from (14)C-labeled glucose indicated that S. stenostrepta degraded glucose via the Embden-Meyerhof pathway. The spirochete utilized a clostridial-type clastic reaction to metabolize pyruvate to acetyl-coenzyme A, CO(2), and H(2), without production of formate. Acetyl-coenzyme A was converted to ethyl alcohol by nicotinamide adenine dinucleotide-dependent acetaldehyde and alcohol dehydrogenase activities. Phosphotransacetylase and acetate kinase catalyzed the formation of acetate from acetyl-coenzyme A. Hydrogenase and lactate dehydrogenase activities were detected in cell extracts. A rubredoxin was isolated from cell extracts of S. stenostrepta. Preparations of this rubredoxin stimulated acetyl phosphate formation from pyruvate by diethylaminoethyl cellulose-treated extracts of S. stenostrepta, an indication that rubredoxin may participate in pyruvate cleavage by this spirochete. Nutritional studies showed that S. stenostrepta fermented a variety of carbohydrates, but did not ferment amino acids or other organic acids. An unidentified growth factor present in yeast extract was required by the organism. Exogenous supplements of biotin, riboflavin, and vitamin B(12) were either stimulatory or required for growth.

A study was undertaken to attempt to relate the distribution of exogenous factors to the varying incidences of esophageal cancer in the Caspian Littoral of Iran. For the study, 38 villages were chosen by random sampling in 14 regions defined by their esophageal cancer incidence and environmental characteristics. Information was obtained on the dietary, social, and cultural features of each village. In addition, an extensive 5-day study of 6 randomly selected households in each of the 38 villages was conducted. The study consisted of measured dietary intake, a historical food consumption questionnaire, and clinical examinations of adult occupants. Preliminary results show no single factor responsible for the etiology of esophageal cancer. However, there were some major dietary differences between the regions of different esophageal cancer risk. Bread was the chief staple food in high-incidence areas; rice, in low-incidence areas. In high-incidence areas, there was a low intake of vitamins A and C, riboflavin, animal protein, and fresh vegetables and fruit, but a greater consumption of sheeps and goat's milk. Analyses of food samples for aflatoxins, polycylic aromatic hydrocarbons, and nitrosamines showed low levels of these carcinogens in areas of high and low incidences. The use of tobacco and alcohol was not found to be of significance.

The liver is an important immunological organ that remains sterile and tolerogenic in homeostasis, despite continual exposure to non-self food and microbial-derived products from the gut. However, where intestinal mucosal defenses are breached or in the presence of a systemic infection, the liver acts as a second 'firewall', because of its enrichment with innate effector cells able to rapidly respond to infections or tissue dysregulation. One of the largest populations of T cells within the human liver are mucosal-associated invariant T (MAIT) cells, a novel innate-like T-cell population that can recognize a highly conserved antigen derived from the microbial riboflavin synthesis pathway. MAIT cells are emerging as significant players in the human immune system, associated with an increasing number of clinical diseases of bacterial, viral, autoimmune and cancerous origin. As reviewed here, we are only beginning to investigate the potential role of this dominant T-cell subset in the liver, but the reactivity of MAIT cells to both inflammatory cytokines and riboflavin derivatives suggests that MAIT cells may have an important role in first line of defense as part of the liver firewall. As such, MAIT cells are promising targets for modulating the host defense and inflammation in both acute and chronic liver diseases.

Migraine is the most frequent type of headache in children. In the 1980s, scientists first hypothesized a connection between migraine and mitochondrial (mt) disorders. More recent studies have suggested that at least some subtypes of migraine may be related to a mt defect. Different types of evidence support a relationship between mitochondria (mt) and migraine: (1) Biochemical evidence: Abnormal mt function translates into high intracellular penetration of Ca(2+), excessive production of free radicals, and deficient oxidative phosphorylation, which ultimately causes energy failure in neurons and astrocytes, thus triggering migraine mechanisms, including spreading depression. The mt markers of these events are low activity of superoxide dismutase, activation of cytochrome-c oxidase and nitric oxide, high levels of lactate and pyruvate, and low ratios of phosphocreatine-inorganic phosphate and N-acetylaspartate-choline. (2) Morphologic evidence: mt abnormalities have been shown in migraine sufferers, the most characteristic ones being direct observation in muscle biopsy of ragged red and cytochrome-c oxidase-negative fibers, accumulation of subsarcolemmal mt, and demonstration of giant mt with paracrystalline inclusions. (3) Genetic evidence: Recent studies have identified specific mutations responsible for migraine susceptibility. However, the investigation of the mtDNA mutations found in classic mt disorders (mt encephalomyopathy with lactic acidosis and stroke-like episodes, myoclonus epilepsy with ragged red fibers, Kearns-Sayre syndrome, and Leber hereditary optic neuropathy) has not demonstrated any association. Recently, 2 common mtDNA polymorphisms (16519C→T and 3010G→A) have been associated with pediatric cyclic vomiting syndrome and migraine. Also, POLG mutations (eg, p.T851 A, p.N468D, p.Y831C, p.G517V, and p.P163S) can cause disease through impaired replication of mtDNA, including migraine. Further studies to investigate the relationship between mtDNA and migraine will require very large sample sizes to obtain statistically significant results. (4) Therapeutic evidence: Several agents that have a positive effect on mt metabolism have shown to be effective in the treatment of migraines. The agents include riboflavin (B2), coenzyme Q10, magnesium, niacin, carnitine, topiramate, and lipoic acid. Further study is warranted to learn how mt interact with other factors to cause migraines. This will facilitate the development of new and more specific treatments that will reduce the frequency or severity or both of this disease.