This study describes the 24-h changes in plasma prolactin levels, and dopamine (DA), serotonin (5HT), gamma-aminobutyric acid (GABA) and taurine concentration in median eminence and adenohypophysis of newborn male rabbits.Animals were kept under controlled light-dark cycles , housed in individual metal cages, and fed ad libitum with free access to tap water. On day 1 after parturition, litter size was standardized to 8–9 to assure similar lactation conditions during the experiment. Groups of 6–7 suckling male rabbits were killed by decapitation on day 11 of life at six different time points during a 24-h period.Plasma prolactin levels changed significantly throughout the day, showing a peak at the beginning of the active phase (at 01:00 h) and a second maximum during the first part of the resting phase (at 13:00 h). Median eminence DA concentration also changed significantly during the day, peaking at the same time intervals as plasma prolactin. A single maximum (at 13:00 h) was found for adenohypophysial DA concentration. Individual adenohypophysial DA concentrations correlated significantly with their respective plasma prolactin levels. A maximum in median eminence 5HT concentration occurred at 21:00 h whereas adenohypophysial 5HT peaked at 13:00 h. Median eminence 5HT concentration and circulating prolactin correlated inversely. In the median eminence, GABA concentration attained maximal values at 21:00 h, whereas it reached a maximum at 13:00 h in the pituitary gland. Median eminence GABA concentration correlated inversely with circulating prolactin. In the median eminence, taurine values varied in a bimodal way showing two maxima, at the second half of the rest span and of the activity phase, respectively. In the adenohypophysis, minimal taurine levels coincided with the major plasma prolactin peak (at 01:00 h). Circulating prolactin and adenohypophysial taurine levels correlated inversely.The correlations among the changes in the neurotransmitters analyzed and circulating prolactin levels explain the circadian secretory pattern of the hormone in newborn male rabbits. The mechanisms that regulate prolactin secretion are complex . Two majIt is well known that basal secretion of prolactin varies throughout the day, describing a characteristic pattern with maximal values close to the light-dark transition ,8. Such The rat is very immature at birth, so that newborn and suckling rats are very sensitive to manipulations that can affect adulthood -18. CircThe rabbit is probably the best-studied laboratory animal in the wild, due to its abundance, size and importance as an agricultural pest ,24. WildIn contrast to the large amount of information available on circadian rhythms in adult mammals, studies on circadian phenomena in neonates are few ,27. For This study was performed using 24 multiparous, lactating Californian × New Zealand White crossbreed doe rabbits. Animals were housed in research facilities of the Animal Production Department. They were maintained under controlled light-dark cycles , housed in individual metal cages, fed at libitum using a commercial pellet diet  with free access to tap water. On day 1 after parturition, litter size was standardized to 8–9 by adding or removing kits to assure similar lactation conditions during the experiment. This study was performed according to the CEE Council Directive  for the care of experimental animals. Groups of 6–7 suckling male rabbits were killed by decapitation on day 11 of life at six different time points throughout a 24-hour cycle. The brains were quickly removed, and the median eminence and the anterior pituitary were taken out. Anterior pituitaries were weighed and homogenized in chilled (0–1°C) 2 M acetic acid. After centrifugation , the samples were either analyzed for DA and 5HT or boiled for 10 min and further centrifuged at 14000 rpm for 20 min to measure GABA and taurine.L-RP-1. Hormone was labeled with 125I by the chloroamine-T method [Staphylococcus aureus  was used to precipitate the bound fraction [Plasma prolactin levels were measured by a specific homologous RIA method  using AFT method . The volfraction . All sam2/H+ reference electrode were: conditioning electrode: -0.4 V; preoxidation electrode: +0.10; working electrode: +0.35 V. Indoleamine and catecholamine concentration was calculated from the chromatographic peak heights by using external standards and was expressed as pg/μg protein. The linearity of the detector response for DA and 5HT was tested within the concentration ranges found in median eminence and adenohypophysial supernatants.DA and 5HT concentration was measured by high pressure liquid chromatography (HPLC) using electrochemical detection , as described elsewhere . A C-18 Amino acids were isolated and analyzed by HPLC with fluorescence detection after precolumn derivatization with O-phthalaldehyde (OPA) as described elsewhere . An aliqStatistical analysis of results was performed by a one-way analysis of variance (ANOVA) followed by post-hoc Tukey-Kramer's multiple comparisons tests. Curve estimation in regression analysis was made by using SPSS software, version 10.1 . P values lower than 0.05 were considered evidence for statistical significance.Figure Figures 2 = 0.16, b0 = -123.7 and b1= 18.1 .Median eminence DA concentration changed in a bimodal way as a function of time of day, showing two maxima, coinciding with those of plasma prolactin at the active and resting phase of the diurnal cycle .As shown in Figure 2= 0.21, b0 = 25.7 and b1 = -0.22, F = 6.6, p < 0.01).Figure 2= 0.42, b0 = 11.6 and b1 = -0.11, F = 17.4, p < 0.0001).Figure The present study, performed in neonatal male rabbit pups sacrificed at 6 different time intervals during a 24-h cycle, describes for the first time significant changes in plasma prolactin levels throughout the day. In concomitant measurements of median eminence and adenohypophysial concentration of DA, 5HT, GABA and taurine, a clear daily pattern was found in almost every case. Contrasting with neonatal rats that did not display any circadian pattern of plasma prolactin , a dailyIn adult rabbits, daily patterns of prolactin secretion depend on light/dark phases . The preThe activity of several nuclei of rabbit hypothalamus increases with age and with experience of anticipatory arousal . HoweverAmong other possible neuromodulators of prolactin secretion, the arcuate nucleus receives a dense serotonergic innervation consisting of a population of brainstem neurons arising mainly from the midbrain raphe nuclei  and fromTaurine has also been implicated in the regulation of prolactin release ,13,35,36A relatively dense innervation of GABA terminals exists in the external layer of the median eminence , and theGABA acting on specific receptors in the anterior pituitary has been reported to suppress prolactin secretion ,40, althIn suckling male rabbits plasma prolactin and median eminence and anterior pituitary concentration of several neuromodulators change on a daily basis. The existence of significant correlations among several of the neurotransmitters analyzed and plasma prolactin levels may explain the circadian secretory pattern of prolactin at this age in suckling rabbits. Collectively, the present results differ from the reported absence of circadian rhythmicity of prolactin and median eminence and adenohypophysial neuromodulators in rats at a comparable age.The author(s) declare that they have no competing interests.MPA and PC carried out the experiment and the immunoassays and the analysis of catecholamines, indoleamines and amino acids. DPC and AIE designed the experiments. Also, DPC performed the statistical analysis. PR took care of the experimental animals. AIE supervised its technical implementation and drafted the manuscript. All authors read and approved the final manuscript.


There is accumulated evidence that plasma concentration of the sulfur-containing amino-acid homocysteine (Hcy) is a prognostic marker for cardiovascular morbidity and mortality. Both fasting levels of Hcy and post methionine loading levels are used as prognostic markers. The aim of the present study was to investigate the existence of a daily rhythm in plasma Hcy under strictly controlled nutritional and sleep-wake conditions. We also investigated if the time during which methionine loading is performed, i.e., morning or evening, had a different effect on the resultant plasma Hcy concentration.® formula).Six healthy men aged 23–26 years participated in 4 experiments. In the first and second experiments, the daily rhythm in Hcy as well as in other amino acids was investigated under a normal or an inverse sleep-wake cycle. In the third and fourth, Hcy concentrations were investigated after a morning and evening methionine loading. To standardize food consumption in the first two experiments, subjects received every 3 hours 150 ml of specially designed low-protein liquid food (EnsureIn both the first and second experiments there was a significant daily rhythm in Hcy concentrations with a mid-day nadir and a nocturnal peak. Strikingly different 24-h patterns were observed in methionine, leucine, isoleucine and tyrosine. In all, the 24-h curves revealed a strong influence of both the sleep-wake cycle and the feeding schedule. Methionine loading resulted in increased plasma Hcy levels during both morning and evening experiments, which were not significantly different from each other.There is a daily rhythm in plasma concentration of the amino acid Hcy, and this rhythm is independent of sleep-wake and food consumption. In view of the fact that increased Hcy concentrations may be associated with increased cardiovascular risks, these findings may have clinical implications for the health of rotating shift workers. Experimental results accumulated in recent years have revealed that plasma concentration of the sulfur-containing amino-acid homocysteine (Hcy) is a prognostic marker for cardiovascular morbidity and mortality -5. PlasmStudies conducted during the 1960s have demonstrated that plasma levels of several amino acids vary in a daily manner. Feigin, Klainer and Beisel  were theMore recently, plasma Hcy levels were also shown to vary in a daily manner in humans with an evening peak and a morning nadir . SignifiIn the present study, we further investigated the possible existence of a daily rhythm in plasma Hcy under strictly controlled nutritional and sleep-wake conditions. We also investigated if the time during which methionine loading is performed, i.e., morning or evening, had a different effect on the resultant plasma Hcy concentration.2). They were instructed to avoid alcohol and coffee beverages during the 24 hours that preceded each of the experimental periods. The study was approved by the local Human Ethics Committee, and subjects gave written informed conset before being enrolled in the first experiment. Subjects were paid for their participation.Six healthy men aged 23–26 years participated in 4 experiments. All were students who maintained a normal and regular sleep-wake cycle for at least three months prior to the studies. They were screened to ensure an adequate state of health by physical examination, detailed medical history and blood testing. All had a normal body weight (mean body mass index (BMI) = 23.5 ± 1.6 Kg/mIn the first and second experiments, daily rhythms in Hcy as well as in other amino acids were investigated under a normal or an inverse sleep-wake cycle. In the third and fourth experiments, Hcy concentrations were investigated after morning and evening methionine loading.® formula) with the following composition: proteins , fat , carbohydrates , vitamins and minerals, in 77 ml water. No other food except for water was allowed.Subjcts were admitted to the laboratory at 1800 for a period of 24 hours, after having a normal day. A catheter was inserted into an antecubital vein and was kept patent by a drip of saline. Electrodes were attached for polysomnographic monitoring to determine sleep stages. These included EEG, EMG, EOG, respiration by respiratpry belt and nasal thermistor, and oximetry. Starting at 1900, 5-ml blood samples were drawn every hour until 1900 on the next day. Thoughout this period subjects were either in a supine or a sitting position in individual rooms where they could read, use their personal computers or watch television. From 2300 to 0800 the room lights were turned off during the sleep period. Blood samples were collected into EDTA treated tubes, immediately centrifuged at 4°C, and plasma was stored at -70°C until assay. Hourly blood sampling during sleep continued with minimal disturbance to subjects' sleep. To standardize food consumption and to provide adequate energy intake, subjects received every 3 hours 150 ml of specially designed liquid food . At the start of each methionine loading test, subjects were administered 100 mg/kg body weight methionine, mixed in fruit juice. Blood samples (5 ml) were taken into EDTA treated tubes before methionine loading, designated as time 0, and then at +2, +4, +6 and +8 hours after methionine administration. Light carbohydrate rich meals were provided at +1 and +6 hours after the methionine loading in each of the test periods.12 were also measured in all samples of all subjects using commercially available kits from Abbott. The assays were performed on an Abbott IMX analyzer that utilizes ion capture technology for folate determination and microparticle enzyme immunoassay (MEIA) technology for B12. The assays were performed according to the manufacturers' instructions and used quality control sera supplied by Abbott.Plasma amino acids levels  were measured in duplicates using a Biochrom 20 Amino-Acid analyzer  as described before . The meaRepeated measurements ANOVA was used to compare the means of the amino acids between the first two experiments. To obtain the average 24-h Hcy curves, each individual data point was replaced by a z-transformation based on the individual 24-h mean and standard deviation, before averaging across subjects. Then, each of the individual time series was subjected to Cosinor analysis to determine its amplitude and acrophase. Since Experiment 1 was perfomed during the summer (August) and Experiment 2 was performed during early winter (late November), approximately 2 months after the change from Summer daylight-saving time to Winter time, during which the clock in Israel was advanced by one hour, the 24-h curves of the first experiment were advanced by 1 hour before the analysis. Then repeated measurements ANOVA was used to determine differences in acrophase between the experiments. In the third and fourth experimens, the concentrations of Hcy at times 0, 2, 4, 6, and 8 hours after methionine loading were analysed by repeated measures ANOVA to determine if there were any significant morning-evening differences in Hcy levels.All subjects successfully completed the four experiments. In experiment 1 when they slept from 2300 to 0700, average sleep latency was 22.2 ± 7.3 min, total sleep time was 407.3 ± 51.8 min, and sleep efficiency was 77.7 ± 9.2%. In experiment 2 when they slept from 0720 to 1500, average sleep latency was 4 ± 3.1 min, total sleep time was 371.5 ± 59.4 min, and sleep efficiency was 83.6 ± 12.2%. In spite of the reversal of the sleep-wake cycle, the 24 h means and coefficients of variation of Hcy in the two experiments were very similar to each other, 8.82 μmol/L and 29.7% and 8.51 μmol/L and 27.7%, in experiments 1 and 2, respectively. None of the subjects had abnormal Hcy levels (>15 μmol/L) at any point across the 24 hours.Figure Strikingly different 24-h patterns were observed for the other amino acids: methionine, leucine, isoleucine and tyrosine. In all, the average z-transformed 24-h curves revealed a strong influence of both the sleep-wake cycle and the feeding schedule. Their level was notably lower during the sleep period, regardless of its timing, and increased every two hours in synchrony with the times of feeding. This pattern is exemplified in Figure 12 and folic acid. While folic acid showed a linear increase throughout the study period, the 24-h pattern of B12 was rather constant with slight elevation during the night time (data not shown).We did not find any evidence for rhythmicity in the concentrations of BAs expected, methionine loading resulted in increased plasma Hcy levels during both morning and evening experiments Figure . AnalysiThe present study demonstrated that under strictly controlled dietary conditions plasma levels of Hcy shows significant daily rhythmicity, which is independent of the 24-h cycle of sleep and wake, with a peak at around 2200 to 2400. Previously, similar rhythmicity in Hcy with an evening peak was reported in obese diabetic patients by Bremner et al  and with12 dependent while the reaction with betaine is not. In the transsulforation pathway, Hcy condenses with serine to form cystationine in an irreversible reaction catalyzed by the pyridoxal-5'-phosphate (PLP)-containing enzyme, cystationine beta synthase. Although we do not have any information as yet on the underlying mechanism responsible for the daily rhythm in plasma Hcy, it is most probably related to the balance between its rates of production and disposal. A high Hcy concentration could be due to an elevated production rate, a decreased rate of transsulforation, a decreased rate of remethylation to methionine, or any combination of these processes.Homocysteine is a non-protein sulfur containing amino acid, and an intermediate in the metabolism of the essential amino acid methionine. The metabolism of Hcy is accomplished by two major pathways, remethylation into methionine and transsulfuration to cystationine . In reme12 deficiencies, or by methylenetetrahydrofolate reductase thermolability. The variations in plasma vitamin concentrations, however, could not provide an explanation for the daily rhythms in Hcy. The 24-pattern of folate levels showed a linear increase from the beginning to the end of the study. Although the plasma concentrations of vitamin B12 varied across the 24 hours – in contrast however to what was expected if B12 were involved in the daily rhythm in Hcy, ie, increasing levels of B12 associated with decreasing levels of Hcy – the 24-h pattern in B12 was parallel to that of Hcy with a daytime nadir and a night time peak. Thus, it is unlikely that a daily rhythm in plasma vitamin concentrations can  explain the daily rhythm in Hcy.The fact that the range of the daily variations in the plasma levels of Hcy is on the same order of magnitude as those seen in mild hyperhomocysteinemia, may suggest that the two phenomena share a common underlying mechanism. Mild hyperhomocystenemia seen under fasting conditions is due to mild impairement in the methylation pathway. This may be caused by folate or BThe methionine loading test has been used to test the individual's ability to dispose of methionine through the transsulforation pathway . The facA different possibility that cannot be ruled out at this point is the involvement of the Hcy cellular export mechanism. The small amount of plasma Hcy is the result of a cellular export mechanism that is essential for keeping intracellular concentrations low to avoid potentially Hcy cytotoxic effects. Thus the daily rhythm in plasma Hcy may reflect variations in the activity of the cellular export mechanism, which result in varying levels of Hcy disposed to the plasma at different phases of the 24 hours rather than in its rate of metabolism. Further studies are needed to test this possibility.Finally, what may be the clinical implications of the present findings? We would like to suggest that the existence of a daily rhythm in Hcy concentration may have possible health-related consequences to shift workers, who were shown to be at an increased cardiovascular risk . FirstlySecondly, we do not know how the desynchronization between the circadian system and the enviornment which occurs in rotating shift workers may affect the rhythm in Hcy concentrations and its overall plasma concentration. Recently, Martins et al  reportedIn view of the fact that Hcy is a risk factor for cardiovascular morbidity, more research is needed on the possible role of hyperhomocysteinemia as a cardiovascular risk factor in shift workers.12 and folic acid. Methionine loading at 9 AM and 9 PM produced a comparable time-dependent increase in Hcy concentrations with a tendency toward a higher increase in the evening during the first 2 hours after loading. In view of the possible involvement of Hcy in cardiovascular morbidity, and of the increased cardiovascular morbidity in shift wokers, these findings may have implications to shift workers health.Our results demonstrated a daily rhythm in plasma concentrations of Hcy with a nocturnal peak that was independent of sleep-wake cycle and food consumption. There were no comparable rhythms in the concentrations of methionine, leucine, isoleucine and tyrosine, nor in the concentrations of BHcy – homocysteineEEG – ElectroencephalographyEMG – electromyographyEOG – electrooculographyEDTA – ethylanediaminetetraacetic acidCV – coefficient of variationANOVA – analysis of varianceNone declared.PL and LL co-designed the study, supervised the data collection and data analysis and wrote the paper.


Hyperhomocysteinemia is an important and independent risk factor for vascular disease. About 35% of patients with stroke and 47% of patients with peripheral arterial disease have elevated plasma homocysteine (HCY) concentrations. The relationship between plasma HCY and the methylentetrahydrofolate reductase (MTHFR) C677T polymorphism is still unclear, especially in regard to screening/diagnostic power.This case-control study was performed on 96 patients, who underwent surgery due to asymptomatic or symptomatic high grade stenosis of the internal carotid artery (ICA), and 96 healthy age and sex-matched, controls. Plasma HCY concentration was determined using a commercial kit for fully automated analysis . The C677T polymorphism of the MTHFR-gene was assessed by PCR.The mean plasma HCY concentration was significantly higher in the group with stenosis of ICA compared to the controls, 12.43 ± 6.96 μM and 10.16 ± 3.16 μM, respectively, (p < 0.05). An HCY plasma concentration of 1.5 SD above the mean value of the control group, was defined as cut-off for a pathological versus physiological plasma concentration. The sensitivity and specificity of HCY was 0.27 and 0.94, respectively. The positive predictive value was 0.82. There was no significant difference in the frequency of the MTHFR 677 CT and TT genotype between patients and controls . Carriers of the T-allele (CT and TT genotypes) have significantly higher plasma HCY concentrations than CC patients, 14.1 ± 7.6 μM and 10.29 ± 5.2 μM, respectively, p < 0.05. Sensitivity and specificity of the MTHFR C677T polymorphism  were 0.56 and 0.40, respectively. The positive predictive value was 0.48. There was no significant difference in plasma HCY or genotype frequency of the MTHFR C677T polymorphism between asymptomatic and symptomatic patients.Our study shows that in a population with a given pretest disease probability of 50%, the determination of plasma HCY concentration, with a positive predictive value of 0.82, is more suitable for screening of patients at risk than analysis of the MTHFR C677T polymorphism. Stroke is a frequent cause of death in the developed world with a prevalence of 1–5% in the general population. In Germany, there are about 160,000 new cases of stroke recorded per year, with an annual cost over 11.7 billion dollars . About 1In 1969, McCully reported an autopsy case of a 7-week-old-new-born with advanced atherosclerotic lesions and very high plasma homocysteine concentration . Plasma With respect to genetic factors, homocysteinemia may be the result of enzyme deficiency of the homocysteine intermediary metabolism. Apart from rare inherited metabolic disorders , the most common disorder investigated has been a deficiency of methylentetrahydrofolate reductase . Frosst et al.  identifiWe performed a case-control study in the patients who were admitted for surgical treatment of high grade ICA stenosis to assess the screening and/or diagnostic power of the MTHFR C677T polymorphism against plasma homocysteine concentration. This study was conducted in order to improve our knowledge on the role of the MTHFR C677T polymorphism and plasma HCY in patients with ICA stenosis, to rationalize the diagnostic work-up and to subsequently reduce unnecessary costs in routine diagnostics.The patient group consisted of 96 persons (age range 40–87 years) who were admitted to our hospital for surgery due to a high-grade carotid artery stenosis (at least 80%). Thirty-four patients were asymptomatic and 62 symptomatic and had already experienced TIA or stroke. The diagnosis of ICA stenosis was confirmed by doppler and duplex sonography in all patients and, additionally, by digital subtraction angiography (DAS) or magnetic resonance angiography (MRA) in some cases. All patients had a cranial computed tomography (CT) or magnetic resonance imaging (MRI) performed to evaluate preoperative brain damage. Ninety-six healthy volunteers (age range 45–83 years) without evidence of atherosclerosis or deep vein thrombosis represented the control group. The sex distribution was uniform in both groups, 23% (n = 22) women and 77% (n = 74) men, respectively.A careful anamnestic evaluation and physical examination were performed in all participants including evaluation of nutrition. Patients with vitamin and mineral supplementation and subjects with known disorders of B6, B12 and folate metabolism, as well as patients suffering from hematological and oncological disorders were excluded. Furthermore, subjects under continuous or recent medication, which are known to influence HCY, folic acid and vitamins B metabolism, women with hormone contraception or any other form of hormone substitution were also excluded. This study was performed according to the Helsinki declaration and was approved by the local ethical committee. A written consent has been obtained from each participant.Venous blood was obtained by puncture of the cubital vein, collected in EDTA tubes and immediately centrifuged at 2500 rpm for 10 min at 4°C. Cells were further assessed for genetic polymorphism. The plasma fraction was aliquoted for biochemical analysis. One aliquot was stored at -40°C to await analysis of homocysteine, folic acid or B12.Homocysteine plasma concentration was measured using a standard commercial diagnostic kit (AxSYM Homocystein) for the fully automated Abbott AxSYM system . Briefly, the assay included 3 analytical steps: 1) reduction of protein-bound homocysteine and other homocysteine fractions to free homocysteine using dithiothreitol, 2) enzymatic conversion using synthetic activity of S-adensylhomocysteine (SAH)-hydrolase in excess of adenosine, 3) a fluorescence polarisation immunoassay (mouse anti-SAH antibody) for determination of S-adenosylhomocysteine. Linearity of homocysteine determinations in the water and human whole plasma were proven using serial dilutions of HCY standards (range 0.6–600 μM). Intra- and inter-assay variations and possible effects of freezing and thawing were also assessed. According to the results published by others and following the manufacturer's recommendations, the cut-off value for an elevated HCY concentration was set at 1.5 SD above the mean value of the control group.Folic acid and vitamine B12 plasma concentrations were assessed in one aliquot of stored plasma using standard commercial Abbott kits, the AxSYM Folate Assay and the AxSYM B12 Assay. AxSYM Folate assay is a Ion-Capture-Assay for quantitative determination of folic acid in human sera or plasma and AxSYM B12 is a microparticle enzyme intrinsic factor assay (MEIA) for quantitative determination of vitamin B12 in human serum or plasma. Both assays were conducted according to the manufacturer's instruction using AxSYM analyzer.® Roche Diagnostics GmbH, Mannheim, Germany) and hybridisation probes for genotyping of the C677T point mutation in exon 4 of the MTHFR gene in human DNA. A 233 bp fragment of the MTHFR gene is amplified from genomic DNA using specific primers and the PCR product is detected by fluorescence (Lightcycler -Red 640) using a specific pair of hybridisation probes.The MTHFR C677T polymorphism was assessed using the Lightcycler-MTHFR C677T mutation detection protocol, previously described in detail . BrieflyThe data are presented as means ± SD. Statistical significance of the results was analysed using the Student's t-test. In cases where the data distribution was not compatible with the application of Student's t-test, Wilcoxon's test of the sum of ranks was applied. Regression analysis was based on individual measurements using the Spearman's rank correlation coefficient. In addition, non-linear regression analysis was compared with the rank correlation analysis. Statistical analyses were performed using SPSS for Windows, version 6.0.1. A P-value below 0.05 (two-tailed) was assumed to indicate a significant difference.2-CVmeth2)1/2. For evaluation of the HCY plasma concentration and MTHFR C677T polymorphism, as a marker of enhanced risk of ICA stenosis, Bayes theorem was used [Within both, the patient and the control group, interindividual differences of the plasma HCY concentration were expressed as the coefficient of variation (CVobs). To obtain the real biological variability of homocysteine plasma concentration, the observed variability was corrected for methodological variability, which was assessed separately using plasma sample standards with known concentrations of homocysteine and expressed as the coefficient of variation of the method (CVmeth). A real biological coefficient of variation was calculated as CVbiol = , which may affect homocysteine distribution among protein-bound, free and mixed disulphides had no influence on the accuracy of the assay. Intra- and inter-assay variations as well as the coefficients of variation with and without freezing, were identical, yielding a coefficient of variation of the method (CVmeth) of 0.05. Descriptive statistical data for total plasma HCY and MTHFR C677T polymorphism are summarized in the Table In the control group, male subjects showed higher total plasma HCY concentrations than female subjects (10.8 ± 3.35 μM vs. 9.30 ± 2.03 μM p < 0.05). This gender-related significant difference in the total plasma HCY concentration was not observed in the group with stenosis of ICA. Male patients had no significantly different plasma HCY as compared to females . In the control group, HCY correlated significantly with age , while in the patient group no such a relationship with age was observed . However, in the control and the patient groups no significant correlation was found between the HCY concentration and age, when related to gender . An interesting observation was found in regard to plasma HCY and folate concentration. In both groups, a significant inverse relation between folate and plasma HCY was observed . Between patients with low and high folate concentrations (defined as < of 40 % and % > 140% of the mean plasma folate concentration), a statistical difference in the plasma HCY concentration was observed, 14.04 ± 6.41 vs. 10.15 ± 4.21 μM, p < 0.01, respectively. HCY in both examined groups did not show significant correlation with plasma B12 concentration, r = 0.01.The frequency distributions of the MTHFR C677T polymorphism are summarized in Table The relation between MTHFR C677T polymorphism and plasma HCY as well as plasma B12 and folate concentration in the patient group is presented in the Table For further evaluation, the patient population was stratified according to the clinical severity of ICA stenosis (TIA or stroke). Compared to asymptomatic patients, symptomatic patients had higher HCY plasma concentrations, but the difference was not statistically significant . The frequency of MTHFR 677 CT or TT genotypes did not differ between asymptomatic and symptomatic patients .To evaluate the total plasma HCY concentration and the MTHR polymorphism as a marker of ICA stenosis, Bayes theorem was used . The cutAccording to the discrepancy between recruited subjects in regard to gender distribution  a separate statistical analysis was performed.The analysis revealed that homocysteine plasma concentrations between male patients and male controls showed a significant statistical difference, 12.72 ± 7.34 μM and 10.8 ± 3.35 μM, p < 0.05, respectively. The sensitivity and specificity in the male subgroup compared to the whole collective were 0.26 and 0.27, 0.96 and 0.94, respectively. A positive predictive value, calculated in the male subgroup, was 0.86 compared to the 0.82 in the all subjects. Similarly, a separate analysis of the MTHFR 677T polymorphism in the male subgroup did not influence the results. Sensitivity and specificity were 0.54 and 0.38 and did not differ significantly compared to the whole group . The positive predictive value was 0.46 (0.48 in the whole group).In this study we explored the association between the MTHFR C677T polymorphism and plasma HCY concentration in patients with ICA stenosis. The main aim was to evaluate the relevance of this genetic marker in comparison to plasma HCY and to assess the screening and/or diagnostic power of the MTHFR C677T polymorphism against plasma homocysteine concentration.The study provided the following major results:1. Homocysteine plasma concentration was significantly higher in the patient group compared to the control.2. The genotype frequency of the MTHFR C677T polymorphism revealed no difference between the patient group and the group of healthy individuals.3. Patients, carrying the T-allele had significantly higher plasma HCY concentrations than CC patients.4. There was no statistically significant difference between symptomatic and asymptomatic patients in regard to plasma HCY concentration and genotype frequency of the MTHFR C677T polymorphism.5. The determination of plasma HCY concentration with a positive predictive value of 0.82 was more suitable for screening and monitoring of patients at risk than analysis of the MTHFR C677T polymorphism .In order to evaluate total plasma HCY and MTHFR C677T polymorphism as marker of ICA stenosis, this study (index test) was performed according to "gold standard" procedures . This reAs it is evident from Table However, higher plasma HCY concentration and CV of plasma HCY in the group of patients could not be explained by the MTHFR C677T polymorphism. A careful data evaluation showed that there was no consistent relationship between genotype frequency, vitamin status and plasma HCY. An inconsistent relation between examined determinants might be explained by the presence of some other unidentified genetic linkage or unknown risk factors, which could have an impact on the development of hyperhomocysteinemia.One possibility is that other mutations affect the MTHFR gene and have influence on the plasma HCY concentration. Van der Put et al.  for firsNevertheless, the application of Bayes theorem in our study design indicated that determination of MTHFR 677T polymorphism as screening or diagnostic tool is of minor importance.Our study showed that in a population with a given pretest disease probability of 50%, the determination of plasma HCY concentration, with a positive predictive value of 0.82, is more suitable for screening and monitoring of patients at risk than an analysis of the MTHFR C677T polymorphism.From our results we concluded that the measurement of the total plasma HCY concentration does not serve as a screening test for ICA stenosis in the general population but rather as a screening test and/or monitoring marker in certain risk groups .The author(s) declare that they have no competing interests.RL: initiated the study, designed, coordinated and drafted the manuscript.BTM: participated in the design of the study and recruited patients.RBZ: performed the statistical analysis and helped to draft the manuscript.CS: recruited patients and performed the measurement.WS: participated in the design of the study.RES: participated in the design and coordination of the study.


An increased concentration of plasma homocysteine (Hcy) has toxic effects on vascular endothelium. This seems to be a risk factor of cardiovascular disease, premature stroke and venous thrombosis. The risk is higher in coincidence with other factors like chronic diseases and familiar hypercholesterolemia. The aim of our study was to evaluate plasma Hcy concentration in patients with juvenile idiopathic arthritis (JIA) and its correlation with methotrexate (MTX) therapy, serum folate and B12 vitamin, and hyperlipidemia.th percentile observed in the control group.Fifty-one patients  with JIA and 52 healthy controls  were included in the study. Thirty-two patients were using weekly MTX (mean doses: 0.1–1 mg/kg). For statistical analysis both JIA and control groups were distributed in three subgroups according to age . The laboratory investigation included measurement of erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), plasma Hcy, serum folate, vitamin B12, triglycerides, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL). For data analysis, we considered raised Hcy values ≥ 12.56 μmol/L, which corresponds to the 90The mean plasma Hcy concentration was 9.3 ± 3.16 μmol/L in JIA patients and 8.9 ± 2.42 μmol/L in healthy controls (p = 0.615). Higher concentration of Hcy was observed in the subgroup of 13 – 18 years . We did not find correlation between MTX use and plasma Hcy concentration. With regard to vitamin B12 concentration, we detected normal values in both patients and controls while serum folate concentration was higher in patients (p < 0.001). With regard to the lipidogram, lower concentration of HDL was found in patients (p = 0.007) and higher levels of VLDL (p = 0.014) and triglycerides (p = 0.001) were observed in controls. We did not observe correlation among plasma Hcy concentration, clinical findings, ESR and CRP.JIA patients do not present significant increased concentration of Hcy despite the use of MTX, probably due to the folate supplementation. The mild abnormalities in the lipidogram may reflect a current concern with diet and health. Homocysteine (Hcy) is a sulphydryl amino acid derived from the essential amino acid methionine during its conversion to cysteine . Its maiHcy has many functions: threshold of the auto-oxidation of low-density lipoprotein (LDL) , promoteBoth in children and in adults increased concentration of plasma Hcy has direct and indirect toxic effects on the vascular endothelium and seems to be one more risk factor of cardiovascular disease, premature stroke and venous thrombosis , especiaJuvenile idiopathic arthritis (JIA) is a chronic inflammatory disease in which clinical manifestations may persist for decades in some patients despite adequate treatment . There aThe paucity of studies about this subject and the conflicting results published to the present date stimulated us to evaluate this metabolite in children and adolescents with JIA.A cross-sectional study involving 51 children and adolescents with JIA  selected consecutively from our outpatient Pediatric Rheumatology Unit was performed. All of them were diagnosed according to the International League of Associations for Rheumatology (ILAR) . Fifty-tAll patients were clinically evaluated by a board certified pediatric rheumatologist. We defined active JIA when the patient presented one or more joints with arthritis at the time of the study. We also measured the ESR and CRP of all patients.Patients and controls samples were identified by an unknown code by the laboratory staff. Thirty milliliters of blood were collected from fasting participants and centrifuged at 3000 rpm for 5 minutes. The plasma was separated in aliquots and immediately frozen and stored at -80°C. The time between the beginning and the end of this process did not exceed one hour.One portion of each blood sample was used to determine the total Hcy concentration, the concentration of vitamin B12 and serum folate. The leftover blood sample was used to determine the total cholesterol level (TC) and its fractions, and triglycerides (TG).th percentile of the control group).Total plasma Hcy concentration was measured according the method proposed by Pfeiffer et al, which uses the High Performance Liquid Chromatography (HPLC) with fluorimetric detection and isocratic elution . Increas® of Abbot Laboratories). Serum folate concentrations were measured by ionic capture (Imx Folate ® of Abbot Laboratories).Vitamin B12 concentration was performed by an enzyme immunoassay method with micro particles , LDL, very low-density lipoprotein (VLDL), serum creatinine as well as TG were measured by the enzymatic colorimetric method.For statistical analysis both JIA and control groups were distributed into 3 subgroups according to age .th percentile of the control group.To establish a reference value (cutting point) in order to make comparisons between the groups of patients and controls, a receiver operating characteristics (ROC) curve analysis was conducted and after that, the categorization by the 9010) due to the variability and asymmetry of values, in order to obtain a normal probability distribution.Some variables like Hcy concentration, TC and its fractions, TG, vitamin B12, serum folate concentrations and serum creatinine were transformed into logarithm , the Fisher's exact test was used. The Student's t-test was used for the comparison of continuous variables. The relation between Hcy concentration and other continuous variables was analyzed by Spearman's correlation coefficient.To evaluate the relation between high and normal Hcy concentration groups of normal and high values and the other variables in each group, the logistic regression analysis with logic model was used to estimate odds ratio. Univariate and multivariate analysis were conducted using the stepwise variables selection criteria.The level of significance adopted for the statistical tests was 5%, that is, p < 0.05.The study protocol was approved by the Ethics Committee of the Universidade Federal de São Paulo. A consent form was obtained for each patient and control either from a parent, guardian or responsible adult before inclusion in the study.The demographic and clinical characteristics of the patients are presented in table Average serum Hcy concentration was 9.3 ± 3.16 μmol/L in JIA patients and 8.9 ± 2.42 μmol/L in healthy controls (p = 0.615)   Table . A lowerJIA is a chronic inflammatory disease that affects young children and adolescents. In this disease the mortality is low, and many patients have a good quality of life in their adult years. Studies with normal adults and rheumatoid arthritis patients have shown a correlation between plasma Hcy concentration and the presence of cardiovascular and thromboembolic events ,12. ThisIn this study we observed an elevated concentration of plasma Hcy in some children with JIA and also in some controls. We found a significant difference between two groups of patients: 3 to 7 year-old and 13 to 18 year-old (p < 0.001). Some studies relate age to an increase in Hcy levels, especially in children who are over 10 . In the In the present study we observed a progressive increase in the average concentration of Hcy in patients with JIA and controls related to age.There are few articles about the subject. Huemer et al  studied Hcy serum concentration can be a result of several factors including: genetic aspects ,16, age One of the main determinant factors of the Hcy concentration is folate , due to It is known that MTX takes part in Hcy metabolism increasing its plasmatic concentration . Van EdeCorticosteroids, diuretics and immunosuppressive drugs like azathioprine and cyclosporine may interfere with Hcy concentration . In our Chiang et al  reportedDyslipidemia may be found in some rheumatic diseases especially related to disease activity and corticosteroid use . Its relJIA patients do not present significant increased concentration of Hcy despite the use of MTX, probably due to the folate supplementation. The mild abnormalities in the lipidogram may reflect a current concern with diet and health.Our data is not conclusive. Multicentric studies involving a higher number of patients will help to indicate with more accuracy the significance of Hcy in patients with JIA.Hcy = homocysteineLDL = low-density lipoproteinJIA = juvenile idiopathic arthritisMTX = methotrexate (MTX)ILAR = International League of Associations for RheumatologyTC = total cholesterol levelTG = triglyceridesHPLC = High Performance Liquid ChromatographyHDL = high-density lipoproteinVLDL = very low-density lipoproteinROC = receiver operating characteristicsSLE = systemic lupus erythematosusNSAIDs = nonsteroidal anti-inflammatory drugsSD = standard deviationThe author(s) declare that they have no competing interests.MG, VDA, MOEH were involved in the development of the protocol. MG was responsible to see the patients at outpatient clinic. VDA, EMGS and LCG performed the laboratory tests. MG, CAL and MOEH carried out the literature search. MG, CAL and MOEH prepared and revised the manuscript. CAL coordinated the submission of the article. All authors participated in critical editing of the manuscript and read and approved the final version.


The aim of the study was to assess the plasma levels of homocysteine in patients with multiple sclerosis (MS) and to investigate whether an association with depression exists.Plasma homocysteine (Hcy), vitamin B12 and plasma folate were measured in 65 moderately disabled patients with relapsing/remitting MS (RR-MS) and 60 healthy controls. All subjects were assessed with the Beck Depression Inventory (BDI).Hcy levels were significantly increased in MS patients compared to controls . A significant correlation was found between Hcy levels and BDI scores . Plasma Hcy was not related to Extended Disability Status Scale (EDSS) score, age, disease duration or vitamin B12 and folate.Moderately disabled MS patients with elevated Hcy levels are particularly prone to develop depressive symptomatology. Further study is warranted in order to elucidate the prognostic and therapeutic implications of this novel finding. Homocysteine is a non-essential sulfur-containing amino acid derived from methionine by demethylation. Vitamins B12 and B6 as well as folate play an important role in the metabolic pathway of homocysteine . As a reAmongst neurological conditions, multiple sclerosis (MS) has been extensively investigated with regard to homocysteine metabolism, with conflicting results. Some studies demonstrated elevated plasma homocysteine (Hcy) levels in MS patients with ,7 or witWith regard to psychiatric disorders, depression has been linked in particular to increased Hcy plasma levels in the context of altered methylation reactions -13. No sAccordingly, the present study was designed in order to assess Hcy plasma levels in MS patients and investigate whether an association with depression and clinical disability or disease duration exists.et al. [A total of 65 relapsing remitting MS (RR-MS) patients with a mean age 39.2 ± 8.3 years, entered the study after providing informed consent for the procedures. All patients met the criteria for clinically definite MS according to Poser et al. , were inA total of 60 healthy volunteers with a mean age of 38.2 ± 7.7 years served as a control group. The demographic characteristics of MS patients and healthy controls are shown in Table After overnight fasting, blood samples were drawn from a peripheral vein and were kept on ice prior to separation (maximum 30 min after drawing). Total homocysteine (free plus protein-bound) was quantified using a sandwich enzyme immunoassay method based on a monoclonal/polyclonal antibody pair . Values higher than 15 μmol/l were considered increased according to relevant literature and manufacturer's instructions.Serum folate and B12 levels were quantified with a paramagnetic particle chemiluminescence's immunoassay .Assessment of depressive symptomatology in all subjects was performed by the Beck Depression Inventory (BDI) , a self-Statistical testing was performed by Fisher's exact test for categorical variables whereas an unpaired t test was used for comparing continuous variables. Correlations between Hcy levels and other investigated parameters were derived using Pearson correlation analysis. The level of statistical significance was set at p = 0.05. All statistical analyses were performed with SPSS version 10.0 for Windows .Plasma Hcy levels were significantly increased in MS patients compared to controls  involving monoamines and various neurotransmitters, amongst other cellular constituents. Thus, increased Hcy levels in depression are thought to reflect functional folate and/or B12 deficiency, which may ultimately cause an imbalance at the monoamine or neurotransmitter level.At the biochemical level, the rise in homocysteine levels observed in patients with depression is ascribed to failure of methylation of Hcy to methionine due to a shortage of supply of methyl groups from methyl folate or lack of the vitamin B12 cofactor for this methylation reaction . MethionThe mechanism of increased Hcy levels in MS patients, particularly those with depression, seems to be different since folate and/or B12 deficiency was not documented in the context of the present study. Irrespective of the precise pathogenetic mechanisms, however, increased Hcy may induce neuronal damage through its neurotoxic action . It is aThe clinical implications of our finding are not entirely clear. Since Hcy is associated with an atherogenic propensity, MS patients with elevated Hcy and depression might represent a subgroup of MS patients with a predisposition for atherothrombotic cerebrovascular disease. In addition, since our patients were not entered into a treatment study, it is currently unknown whether vitamin treatment combined with antidepressants is the optimal way to treat this subgroup of MS patients.In conclusion, our results suggest that moderately disabled MS patients with elevated Hcy levels are particularly prone to develop depressive symptomatology, reflected by higher BDI scores. Further studies and follow up of this subgroup of MS patients are warranted in order to elucidate the prognostic and therapeutic implications of this finding.The authors declare that they have no competing interests.NT conceived of the study, and participated in its design and coordination, carried out the statistical analysis and drafted the manuscript. MEE and VKK participated in the design of the study and drafted the manuscript. EK and MS participated in data acquisition. FB and CN carried out the immunoassays. KNF participated in the design of the study and drafted the manuscript. CS, NV and DV participated in the interpretation of data. All authors read and approved the final manuscript


Cnr1, encoding CB1, causes retention of embryos in the oviduct. The role of the endocannabinoids in tubal implantation in humans is not known.Embryo retention in the Fallopian tube (FT) is thought to lead to ectopic pregnancy (EP), a considerable cause of morbidity. In mice, genetic/pharmacological silencing of cannabinoid receptor CNR1 was examined by TaqMan analysis of genomic DNA from the whole blood samples. In normal FT, CB1 mRNA was higher in luteal compared to follicular-phase (p<0.05). CB1 protein was located in smooth muscle of the wall and of endothelial vessels, and luminal epithelium of FT. In FT from women with EP, CB1 mRNA expression was low. CB1 mRNA expression was also significantly lower (p<0.05) in endometrium of women with EP compared to intrauterine pregnancies (IUP). Although of 1359G/A (rs1049353) polymorphisms of CNR1 gene suggests differential distribution of genotypes between the small, available cohorts of women with EP and those with IUP, results were not statistically significant.Timed FT biopsies (n = 18) were collected from women undergoing gynecological procedures for benign conditions. Endometrial biopsies and whole blood were collected from women undergoing surgery for EP (n = 11); management of miscarriage (n = 6), and termination of pregnancy (n = 8). Using RT-PCR and immunohistochemistry, CB1 mRNA and protein expression levels/patterns were examined in FT and endometrial biopsies. The distribution of two polymorphisms of CNR1 gene and EP, suggests a possible genetic predisposition to EP that warrants replication in a larger sample pool.CB1 mRNA shows temporal variation in expression in human FT, likely regulated by progesterone. CB1 mRNA is expressed in low levels in both the FT and endometrium of women with EP. We propose that aberrant endocannabinoid-signaling in human FT leads to EP. Furthermore, our finding of reduced mRNA expression along with a possible association between polymorphism genotypes of the  Tubal ectopic pregnancy remains a common cause of morbidity and occasional mortality Exposure to marijuana and its cannabinoid derivatives is reported to have many adverse effects on reproductive functions, including reduced fertilizing capacity of sperm, retarded development of the embryo, fetal loss and pregnancy failure Nonetheless, in the mouse oviduct, it has been shown that a finely regulated endocannabinoid tone mediated by CB1 regulates normal oviductal transport of embryos CNR1 gene encoding for CB1 . All women were aged 18–45 years (mean 38). Written and informed consent was obtained from all patients before sample collection. Fallopian tube biopsies, endometrial biopsies  and sera (for measurement of circulating estradiol and progesterone concentrations for endocrine staging) were collected from women undergoing gynecological procedures for benign conditions see  and womeTotal RNA was extracted from the Fallopian tube and endometrial biopsies as detailed in the manufacturers' protocol . The concentration and quality of the extracted RNA was assessed using an Agilent bioanalyser. All samples were standardised for quality control and assigned an RNA integrity number (RIN). RNA samples were considered to be of good quality when a mean RIN value of 7.5 was obtained Frozen tissue sections were fixed for 30 minutes in cold acetone at −20°C then allowed to air-dry for two hours. Sections were washed in TBS+0.5% Triton-X (TBSTX) for 10 minutes before blocking of non-specific endogenous peroxidase activity with 3% hydrogen peroxide  in distilled water for 10 minutes at room temperature. Following a further wash in TBSTX, a non-immune blocking solution of normal goat serum was applied for 20 minutes prior to application of cannabinoid receptor 1 antibody  diluted 1∶300 in normal goat serum. Sections were incubated overnight at 4°C then washed in TBSTX. For the negative control sections, the primary antibody (diluted 1∶300) was pre-incubated in antibody diluent for three hours at room temperature with fifty times the amount of cannabinoid receptor 1 peptide  prior to the overnight incubation. A biotinylated goat anti-rabbit secondary antibody  was applied for 30 minutes at room temperature, then washed in TBSTX before a horseradish peroxidase detection system was applied for 30 minutes . Immunoreactivity was detected using the chromagen, 3,3′-diaminobenzidine  followed by counterstaining of sections with Harris's haematoxylin  and mounted in Pertex .Genomic DNA was purified from 2 ml frozen whole blood samples using the QIAamp Blood Midi Kit  as per the manufacturers' protocol. The DNA was quantified using a ND-1000 spectrophotometer .CNR1 gene were genotyped using the Applied Biosystems TaqMan SNP Genotyping Assays (C_ 1652590_10 and C_8943804_10). The recommended protocols were followed using an ABI 7500 Real Time PCR System with reaction volumes of 25 rather than 50 µl and all reagent volumes reduced by 1/2. The genotypes were compared using Likelihood Ratio and Pearson analyses.The 1359G/A (rs1049353) single nucleotide polymorphism (SNP) encoding a synonymous Thr at amino acid 453 residue and the rs806368 C/T SNP in the 3′ UTR of the of the T 15.42±SEM 0.42, n = 5) compared to the follicular phase  (p<0.05) (see T 19.17±SEM 0.41) (p<0.05), mimicking that of the Fallopian tube in the follicular phase  see . In Fallhase see .T 20.32±SEM 0.47, n = 11) compared to the endometrium from intra-uterine pregnancies  (n = 14)  in the decidualized endometrium of women with ectopic pregnancies (mean ΔC 14) see . Thus, lCB1 protein was located in the smooth muscle of the wall, the smooth muscle of the endothelial vessels and luminal epithelium of the Fallopian tube see . ExpressCNR1 SNPs (rs1049353 and rs806368) . The rs806368 SNP was not informative as all ectopic and 13/14 intra-uterine subject DNAs were TT homozygotes.The distribution of two 368) see  was comp368) see , differeCNR1, may possibly contribute to a predisposition to ectopic pregnancy.To date, study of the role of the endocannabinoids in female reproduction has mostly been limited to mice Our other finding of a temporal variation of expression of CB1 in the Fallopian tube reflects that of many genes in the endometrium suggesting a role for progesterone in the human endocannabinoid system In our study, we also show that CB1 protein is expressed primarily in the smooth muscle of the wall and blood vessels of the Fallopian tube, with some expression in the cytoplasm of the luminal epithelium. This is reassuringly consistent with murine data CB1 mRNA was expressed in low levels in both the Fallopian tube and pregnant endometrium of women with ectopic pregnancies. The most obvious difference in the endometrium of the women with ectopic compared to intra-uterine pregnancies is the absence of trophoblast. There are no published data to our knowledge to suggest that CB1 expression in the uterus is regulated by local trophoblast signals. Nevertheless, the coordinated down-regulation of embryonic CB1 and uterine AEA levels are important for both embryo activation and uterine receptivity  so it is not inconceivable that uterine CB1 expression could be under embryonic control CNR1 gene encoding CB1 warrants further investigation that also excludes the possibility that some subjects with ectopic pregnancy and reduced mRNA expression genotyped as 1359G/G homozygotes may actually be G/deletion heterozygotes. To date, the 1359G/A SNP is considered as the unique haplotype tagging SNP of the CNR1 gene but a direct role of this SNP tagging of the CNR1 gene has still to be demonstrated CNR1 gene, and indeed CB1, is important for embryo transport and successful intra-uterine implantation.However, our observation of suggestive differences in distribution of SNP alleles of the The demonstration of a potential role for CB1 in the etiology of human ectopic pregnancy is important. Cigarette smoking is a major risk factor for ectopic pregnancy and there is evidence of altered oviductal transport in rats exposed to nicotine


We experimentally investigated a simple and new technique for the fabrication of micro-ridge long-period gratings (MRLPGs) based on polarization-maintaining fibers (PMFs). The cladding region of the PMFs was etched periodically using a wet etching technique resulting in the periodic formation of micro-ridges on the surface of the PMF. The PMF-based MRLPGs has two resonant peaks because of the birefringence of the PMF. The extinction ratios of two resonant peaks of the PMF-based MRLPGs were effectively improved by increasing the applied strain because of the photoelastic effect. Long-period fiber gratings (LPGs) have attracted much attention in optical communication systems and optical sensors because of their many advantages, such as low cost, ease of fabrication, and electromagnetic immunity -3. SinceT) can be written as [Mode coupling in the MRLPGs is based on the photoelastic effect. After the formation of the periodic micro-ridges in the cladding of the optical fiber, the different cross-sections between the etched and the unetched claddings can essentially induce the periodic index modulation based on the photoelastic effect when strain is applied to the optical fiber . Consequitten as pe is a photoelastic coefficient, re and ru are the radii of the etched and the unetched regions, respectively, ϵ is the applied strain, and l is a grating length. Since the periodic micro-ridges are structurally formed in the cladding region, the averaged cladding mode index should be considered and the structural index change in the core region is negligible [nPMF), the resonant wavelength of the PMF-based MRLPG can be written aswhere gligible . When thΛ is a grating period, and κco are the averaged self-coupling coefficients of the cladding and core modes, respectively. From Equation 2, it is evident that the resonant wavelengths should be determined by the birefringence of the PMF [where  the PMF .Figure 2O3-based stress region was etched higher than that of the silica cladding [Figure cladding ,7. The dThe periodic micro-ridges in the PMF can induce the periodic index modulation based on the photoelastic effect resulting in the mode coupling between the core and the cladding modes when strain is applied. The transmission characteristics of the PMF-based MRLPG were measured using the experimental setup as shown in Figure Figure We proposed and experimentally demonstrated a fabrication method for the PMF-based MRLPG using the double coating and the wet etching processes, which has the great potential for mass production. The transmission characteristics of the PMF-based MRLPG with variations in strain were measured. Two resonant peaks of the PMF-based MRLPG were observed in the transmission spectrum of the PMF-based MRLPG because of the birefringence of the PMF. The extinction ratios of two resonant peaks of the PMF-based MRLPG were enhanced by increasing the applied strain without variation in their resonant wavelengths because of the photoelastic effect. The variation of the extinction ratios of two resonant peaks at wavelengths of 1,395 and 1,471 nm were measured to be −10.16 and −14.13 dB, respectively, when the applied strain was 840 μϵ. We believe that the experimental results are very useful for applications to fiber optic sensors, optical switch filters, etc.The authors declare that they have no competing interests.O-JK and MS participated in the experimental fabrication. Y-GH wrote and corrected the manuscript and conceived and supervised the study. All authors read and approved the final manuscript.


RIP140 is a transcriptional coregulator involved in energy homeostasis and ovulation which is controlled at the transcriptional level by several nuclear receptors. We demonstrate here that RIP140 is a novel target gene of the E2F1 transcription factor. Bioinformatics analysis, gel shift assay, and chromatin immunoprecipitation demonstrate that the RIP140 promoter contains bona fide E2F response elements. In transiently transfected MCF-7 breast cancer cells, the RIP140 promoter is transactivated by overexpression of E2F1/DP1. Interestingly, RIP140 mRNA is finely regulated during cell cycle progression (5-fold increase at the G1/S and G2/M transitions). The positive regulation by E2F1 requires sequences located in the proximal region of the promoter (−73/+167), involves Sp1 transcription factors, and undergoes a negative feedback control by RIP140. Finally, we show that E2F1 participates in the induction of RIP140 expression during adipocyte differentiation. Altogether, this work identifies the RIP140 gene as a new transcriptional target of E2F1 which may explain some of the effect of E2F1 in both cancer and metabolic diseases. RIP140  is a nuclear protein of 1158 amino acids, initially identified as a transcription cofactor of estrogen receptors which, despite its recruitment in the presence of agonist ligands, exhibits a strong transcriptional repressive activity of various nuclear receptors  or repressors (E2F4–8) The present study further deciphers the role of RIP140 in the E2F signaling pathway. Our data identify the RIP140 gene as a novel transcriptional target of E2F1 that might be involved in a wide range of pathological processes regulated by this transcription factor such as cancer or metabolic diseases.3-TK-Luc) were obtained from Dr L. Fajas . The pEFc-mycRIP140 expression vectors and the plasmid containing the RIP140 promoter (RIP900) have been described previously The pcDNA3-E2F1 and 4, pCMV-DP1, pCMV-Rb expression vectors were given by Dr C. Sardet  and the pCMV-E2F2, 3, 5 and E2F6 expression vectors by Dr K. Helin . The reporter plasmids (CyclinE-luc and (E2F)www.genomatix.de). Alignment of human and mouse NRIP1 promoter sequences and identification of the evolutionary conserved transcription factor binding elements were performed using Genomatix DiAlign Program (www.genomatix.de).Localization of human 6 cells HeLa cells were grown in 100 mm culture dishes to 60% of confluence in medium containing 0.5% FCS (serum starvation) and 2 mM HU (Boehringer Mannheim) was added during 24 hr to induce cell synchronization. When cells were at 80% of confluence, medium was removed to release the block. At each time point, cells were washed, trypsinized and total RNA was extracted. Cell cycle analysis was performed using a FACs-Vatange flow cytometer . For each sample, 2.104 events were collected and the CellQuest software was used to analyze the list-mode data. ModFit software was used to determine the percentage of each G0/G1, S and G2/M phase in the population.MCF-7 and HeLa human cancer cell lines were purchased from the American Type Culture Collection and routinely maintained in the laboratory as previously described Gel shift assays were performed as previously described For ChIP analysis, MCF-7 cells (70% confluent) were cross-linked with 3,7% formaldehyde during 10 min at 37°C. The ChampionChIP One-Day Kit (SABiosciences) was then used according to the manufacturer’s recommendations. Immunoprecipitations were performed using the KH95 (sc-251 SantaCruz), C-20 (sc-866 Santa-Cruz) or 2656C6a (sc-81370 Santa-Cruz) antibodies against E2F1, E2F4 and RIP140 respectively. It should be noted that although the anti-E2Fs antibody were given by the supplier as being specific, we could not eliminate with certainty the possibility that they may cross-react with other E2Fs. As a negative control, we performed IP with no antibody . Quantitative PCR was then performed using the Power SYBR Green PCR master mix, on an Applied Biosystems 7300 thermal cycler with 2 µl of material per point. Primers flanking the E2F site of the RIP140 and cyclin A2 promoters are given in MCF-7 cells were plated in 96-well plates . 24 h later, plasmid tests containing firefly luciferase reporter gene (25 ng), pRLCMVBis (25 ng per well) used as internal standard, and 25 ng each of different factors  and 200 ng each of either RIP140, pRb, p130 proteins to a total of 0.25 µg total DNA per well, were cotranfected using Jet-PEI. 48 h after transfection, cells were lysed and firefly luciferase values were measured and normalized with the Renilla luciferase activity; all triplicate point values were expressed as mean + SD.Expression plasmids for E2F1, 2, 3, 4 or DP1 were transfected in MCF-7 cells using JetPEI. After cell lysis in 25 mM Tris-HCl, pH7.8, 2 mM EDTA, 2 mM DTT, 10% Glycerol, 1% Triton, supplemented with protease inhibitors, whole cell extracts were diluted in Laemmli sample buffer 2X and resolved by SDS–PAGE. Western blotting detection was performed using primary antibodies against E2Fs . To check for gel loading, we used the anti-actin (A2066 from Sigma) or anti-TBP (MA1-21516 from ThermoScientific) antibodies.Total RNA was extracted from cells as previously described −/− and wild-type mouse embryonic fibroblasts (MEFs) were isolated from 13.5 days-old embryos in compliance with the French guidelines for experimental animal studies (agreement B34-172-27). Three different MEF cultures were obtained from E2F1 wild-type and knock-out embryos and grown in F12-DMEM supplemented with 10% fetal calf serum and 1.5% HEPES. For adipocyte differentiation experiments, MEFs (at passage 3) were seeded in 6-well plates. Two days after confluence, differentiation was induced by treating cells for 2 days with a cocktail containing 0.5 mM IBMX (3-isobutyl-1-methylxanthine), 10 µg/mL insulin, 1 µM dexamethasone and 1 µM BRL49653. Every two days, medium was changed with F12-DMEM, 10% serum, 1.5% HEPES, 10 µg/mL insulin and 1 µM BRL49653. Differentiated cells were visualized with Oil Red O staining (Sigma). Nuclear protein extracts were prepared using the NE-PER kit (Thermo Scientific) and 20 µg of nuclear cell extracts were analyzed by Western blotting using primary antibodies against E2F1 (sc-193 from Santa-Cruz Biotechnology) and RIP140 (H300 from Santa-Cruz Biotechnology). Total RNA was extracted using the Quick-RNA Miniprep (Zymo Research) and 1 µg was analyzed for RIP140 mRNA levels by real-time quantitative Polymerase Chain Reaction on an Applied Biosystems 7300 thermal circler. Results were corrected for RS9 mRNA levels used as a reference gene.E2F1Statistical analysis was performed using the Student t test except for We previously reported the cloning and characterization of the human RIP140 gene promoter with the identification of various response elements bona fide E2F binding sites. Using gel shift experiments, we demonstrated that E2F1 strongly interacts with oligonucleotides encompassing some of the putative binding sites . As shown in We then checked the ability of the five different sites from the human promoter to act as In order to demonstrate that the interaction of E2F1/DP1 also occurred in intact cells, we performed chromatin immunoprecipitation experiments. As shown in To demonstrate a transcriptional regulation of the RIP140 promoter by E2Fs, we transiently transfected MCF-7 breast cancer cells with the RIP900 reporter construct containing the 900 bp of the RIP140 promoter fused to the luciferase coding sequence. This construct (which encompassed the five E2F binding sites that we identified) was cotransfected with expression vectors encoding the different members of the E2F family . When coTo strengthen our results on the transiently transfected RIP140 promoter, we analyzed the regulation of the endogenous RIP140 mRNA upon overexpression of E2F1 and DP1 by transient transfection. As shown in 1 and S and between S and G2/M , we then defined the cis-acting elements required for the positive regulation by E2Fs. In a first step, we generated mutants of the RIP140 promoter with point mutations in the different E2F response elements . When weWe then analyzed reporter transactivation when a single functional E2F response element was left intact . When coDeletion analysis of the promoter confirmed these data . Indeed,E2F transcription factors are believed to act as heterodimers with DP proteins which are ubiquitously expressed i.e. the DHFR and ARF promoters. Interestingly, the negative effect observed with DP1 overexpression on the RIP140 promoter was also observed with the ARF promoter but not with the DHFR promoter  i.e. when heterodimerization was forced) but was significantly less efficient to activate transcription from the RIP140 promoter when overexpressed alone i.e. when Sp1-mediated transactivation took place  increase transcription from the RIP140 promoter through binding to the proximal promoter region. Several evidences (mutagenesis of Sp1 response elements and use of an E2F1 mutant defective for Sp1 binding) strongly supported the involvement of Sp1 transcription factors in the regulation of RIP140 by E2F1. In addition, the use of mithramycin which has been described as an inhibitor of Sp1 binding to DNA Based on in vitro and by coimmunoprecipitation via Sp1 sites et al. have shown that the great majority of E2F1 binding sites are in CpG islands (which are highly enriched in Sp1 sites) and lack the consensus binding site motif The interaction between Sp1 and E2F1 has been previously demonstrated An indirect recruitment of E2F1 is supported by our observation highlighting the repressive role of DP1 overexpression in the regulation of RIP140 promoter. This effect is also observed with the mouse RIP140 promoter  and appein vitro results clearly demonstrate that RIP140 is a direct target of E2F1. This conclusion is reinforced by our data demonstrating that the accumulation of RIP140 mRNA varies during cell cycle progression although it remains to be demonstrated whether E2Fs are involved in the two peaks of RIP140 expression observed in synchronized cells at the G1/S and G2/M transition. This is conceivable since recent data from Nevins’ laboratory clearly demonstrated that E2Fs are key actors in gene regulation during the G2/M transition Altogether, our Homo sapiens, Pan troglodytes, Bos Taurus, Oryctolagus cuniculus, Mus musculus and Rattus norvegicus). As shown in Interestingly, a bioinformatic analysis was conducted on the RIP140 promoter sequence using six different species  MCF-7 cells were transiently transfected with the different human RIP140 promoter reporter plasmids  together or not (Ctrl) with expression vectors for E2F1 and DP1. Relative luciferase activities are expressed as percent of control and are the mean (±SD) of several independent experiments (n = 5). (C) MCF-7 cells were transiently transfected with the human RIP900 reporter plasmids together with expression vectors for E2F1 in the presence or absence of DP1. Relative luciferase activities are expressed as percent of control and are the mean (±SD) of several independent experiments (n = 10). The paired t-test was used for statistical analysis.(TIF)Click here for additional data file.Figure S2Effect of the E2F1 mutant (E132) on the human RIP140 and cyclin E promoters. (A) MCF-7 cells were transiently transfected with the human RIP140 or cyclin E promoter reporter plasmids (25 ng) together with expression vectors for E2F1wt or E132 mutant and DP1 (25 ng each). Relative luciferase activity was normalized with renilla luciferase activity as described in (TIF)Click here for additional data file.Figure S3Effect of activator E2Fs and DP1 on the murine RIP140 promoter. (A) MCF-7 cells were transiently transfected with the murine RIP140 promoter reporter plasmids (25 ng) together with expression vectors for E2F1, E2F2, E2F3 and DP1 (25 ng each). Results are expressed as described in legend of (TIF)Click here for additional data file.Figure S4Binding of E2F4 on the distal site of the RIP140 promoter. (A) Electromobility shift assay was used to analyze E2F1/DP1 or E2F4/DP1 binding on the adenoviral E2F response element (Ad2E2F) or on the E2Fa site of the RIP140 promoter (see oter see . (B) ChI(TIF)Click here for additional data file.Figure S5Conservation of the NRIP1 promoter sequence among mammals. The NRIP1 promoter regions of six mammalian species (from exon 1 b up to about 1 kbp) have been aligned using the Multalin program . Genome assembly used for each species were Homo sapiens (hg19), Pan troglodytes (panTro3), Bos taurus (bosTau6), Oryctolagus cuniculus (oryCun2), Mus musculus (mm9), and Ratus norvegicus (rn4) as indicated at the left of the aligned sequences that were extracted from the UCSC genome browser (http://genome.ucsc.edu/). Localization of putative E2F and Sp1 transcription binding sites was performed using MathInspector pattern search program from Genomatix (http://www.genomatix.de). E2F and Sp1 binding sites are shown in grey and white boxes respectively, with names above the sequence according to (TIF)Click here for additional data file.Table S1Oligonucleotide sequences. The table shows the sequences of all the oligonucleotides used in the different assays . The corresponding species (human or mouse) is indicated as well as the orientation of oligonucleotides i.e. sense/forward (fwd) or reverse (rev). The name of the target  is also presented.(TIF)Click here for additional data file.


Collagen detection in histological sections and its quantitative estimation by computer-aided image analysis represent important procedures to assess tissue localization and distribution of connective fibers. Different histochemical approaches have been proposed to detect and quantify collagen deposition in paraffin slices with different degrees of satisfaction. The present study was performed to compare the qualitative and quantitative efficiency of three histochemical methods available for collagen staining in paraffin sections of colon. van Gieson, Sirius Red and Sirius Red/Fast Green stainings were carried out for collagen detection and quantitative estimation by morphometric image analysis in colonic specimens from normal rats or animals with 2,4-dinitrobenzenesulfonic acid (DNBS) induced colitis. Haematoxylin/eosin staining was carried out to assess tissue morphology and histopathological lesions. Among the three investigated methods, Sirius Red/Fast Green staining allowed to best highlight well-defined red-stained collagen fibers and to obtain the highest quantitative results by morphometric image analysis in both normal and inflamed colon. Collagen fibers, which stood out against the green-stained non-collagen components, could be clearly appreciated, even in their thinner networks, within all layers of normal or inflamed colonic wall. The present study provides evidence that, as compared with Sirius Red alone or van Gieson staining, the Sirius Red/Fast Green method is the most sensitive, in terms of both qualitative and quantitative evaluation of collagen fibers, in paraffin sections of both normal and inflamed colon. Collagen is one of the major component in the adult gastro-intestinal wall, starting from the early stages of organogenesis  up to noThe purpose of the present investigation was to compare three conventional histochemical methods, suitable for collagen staining, in terms of their efficiency for qualitative and quantitative detection of collagen fibers by computer-aided morphometric image analysis in light microscopy. In particular, staining protocols, carried out by van Gieson and Sirius Red, alone or combined with Fast Green, were compared for their capability of revealing and allowing the quantification of collagen deposition in paraffin sections of colon from normal rats or with colitis induced by 2,4-dinitrobenzenesulfonic acid (DNBS).Albino male Sprague—Dawley rats, 200–250 g body weight, were used throughout the study. The animals were fed standard laboratory chow and tap water ad libitum, and were not employed for at least one week after their delivery to the laboratory. They were housed, three in a cage, in temperature-controlled rooms on a 12-h light cycle at 22–24°C and 50–60% humidity. All experimental protocols were approved by the Animal Care and Use Committee of the University of Pisa, and were in compliance with the national and European guidelines for handling and use of experimental animals. All efforts were made to minimize stress and suffering. Colitis was induced in accordance with the method previously described by Fornai et al. . MicroscColonic samples were immediately fixed in cold 4% neutral formalin diluted in phosphate-buffered saline at 4°C and routinely processed for paraffin embedding and cross-sectioned to obtain 3 μm-thick sections with circular layer and myenteric ganglia cut longitudinally. Before use, sections were deparaffinized, rehydrated and processed for routine haematoxylin/eosin, and histochemical staining.Tissue collagen deposition was detected by applying the following histochemical staining protocols:Colonic sections were treated with haematoxylin for 10 min, washed in tap water and incubated for 2 min in a picrofuchsia acid solution (1% acid fuchsin in acqueous saturated picric acid). Sections, dehydrated and mounted with DPX Mounting, showed collagen fibers pink-stained, nuclei and muscle black- and yellow-coloured, respectively.The slides were incubated with a 0.1% Sirius Red solution dissolved in acqueous saturated picric acid for 1 hour, washed in acidified water (0.5% hydrogen chloride), dehydrated and mounted with DPX Mounting. Collagen and non-collagen components were red- and orange-stained, respectively.Collagen fibers were detected in colonic tissues as previously reported . Brieflytunica muscularis, respectively, were quantitatively estimated for collagen fibers and cellular non-collagen proteins in double Sirius Red/Fast Green staining, within the respective colonic areas. To detect the specific threshold of different colors , a square was applied upon the color of interest and recorded by Image Analysis System ‘L.A.S. software v.4’. Positive tissue areas were automatically estimated on the basis of the total pixel number and intensity. The whole wall and tunica muscularis areas were manually circumscribed and automatically calculated. Data were expressed as percentage of Σ of positive-stained area / Σ of tissue area examined of whole wall or tunica muscularis in three colonic sections (5 fields/each) for each animal.Quantitative estimations of histochemical stainings were carried out independently by two blind investigators (C.S. and C.I.). Each investigator analyzed all tissue specimens under study. The respective values were then averaged and plotted in graphs in accordance with previously described criteria . BrieflyComparisons between groups were performed using the Wilcoxon signed rank test for paired data. All data are given in scatter plot graphs as mean ± SEM  and a P-value ≤ 0.05 was considered statistically significant. All statistical analysis were carried out using the Statistical Package for Social Science .tunica mucosa and submucosa  and serosa. The inflamed colon showed abundant inflammatory infiltrations with high percentage of eosinophils throughout the whole wall  have been found to underestimate collagen content . Therefotunica mucosa and submucosa, while Sirius Red revealed the presence of collagen fibers throughout the wall, highlighting also the thin collagen network within the muscle compartment. The latter observations are in line with the accurate and reliable staining characteristics displayed by Sirius Red in previous studies on the quantification of hepatic collagen [tunica muscularis, against the brilliant, green background of non-collagen components. Of note, collagen fibers stained by Sirius Red alone appeared as red areas surrounded by a lot of reddish nuances, which could not recorded by the image analysis, being the threshold set up on the red area. By contrast, the double Sirius Red/Fast Green coloration was able to stain the collagen fibers more homogeneously, yielding red areas without blending, and allowed to record overall red-stained collagen areas that were larger than those stained by Sirius Red alone. Besides the best qualitative results, the quantitative estimation of Sirius Red/Fast Green-stained sections by image analysis yielded values that outnumbered significantly those obtained by van Gieson and Sirius Red alone. Of note, the choice of a staining method, which allows to specifically stand out the collagen fibers over the tissue background, plays an important role not only for their morphological identification, but also for a better quantitative assessment as compared to Sirius Red alone. In this respect, the Fast Green dye, which selectively stains non-collagen tissue components, gives rise to a useful color contrast and visualization of red-stained collagen fibers, as well as an optimal threshold for counting the positive pixels and, therefore, to obtain the best quantitative estimation of collagen content by image analysis. Consistently with this view, the Sirius Red/Fast Green staining has been widely used for the in situ computer-aided microscopic evaluation of abnormal collagen deposition, such as in kidney [Based on these considerations, and considering the lack of comparative microscopic evaluations of histochemically stained-collagen, we deemed it interesting to perform a comparative image analysis of three known histochemical techniques for collagen detection  in terms of sensitivity to reveal and quantify collagen deposition in paraffin colonic sections. In order to verify whether the staining properties of the dyes were independent from the histologic appearance of the sample, normal colonic tissues were compared to pathologic specimens obtained from the inflamed colon were examined. In normal colonic samples, the van Gieson technique stained collagen fibers only within the collagen  or collacollagen . Under on kidney  and colon kidney –23. Of nn kidney , for coln kidney , lung [2n kidney  and colon kidney ,34.In the present study, a comparative analysis of the collagen-staining capability by the above mentioned techniques was carried out also on the inflamed colon from DNBS-treated rats. The morphological analysis of colitis samples disclosed features compatible with inflammatory lesions with extensive mucosal and submucosal alterations and eosinophil infiltration . In thisWith regard for the detection of collagen in tissue sections, besides the conventional histochemical staining procedures, it must be acknowledged that immunohistochemistry allows a detailed phenotypic analysis of specific connective fibers and their distribution patterns. Nevertheless, multi-antibody immuno-labelling is affected by several disadvantages and pitfalls as compared to histochemistry, such as complex and long-lasting protocols, expensive reagents, and a marked variability in staining patterns of collagen fibers in assays with different commercially available antibodies .In conclusion, the present study provides evidence that histochemical staining carried out by Sirius Red combined with Fast Green represents an excellent method for standing out collagen fibers in paraffin sections of the colon, under both normal and inflammatory conditions, being a technique more sensitive than van Gieson or Sirius Red alone in terms of both morphological and quantitative evaluations.


The majority of infants’ breastfeeding from their HIV-infected mothers do not acquire HIV-1 infection despite exposure to cell-free virus and cell-associated virus in HIV-infected breast milk. Paradoxically, exclusive breastfeeding regardless of the HIV status of the mother has led to a significant decrease in mother-to-child transmission (MTCT) compared with non-exclusive breastfeeding. Although it remains unclear how these HIV-exposed infants remain uninfected despite repeated and prolonged exposure to HIV-1, the low rate of transmission is suggestive of a multitude of protective, short-lived bioactive innate immune factors in breast milk. Indeed, recent studies of soluble factors in breast milk shed new light on mechanisms of neonatal HIV-1 protection. This review highlights the role and significance of innate immune factors in HIV-1 susceptibility and infection. Prevention of MTCT of HIV-1 is likely due to multiple factors, including innate immune factors such as lactoferrin and elafin among many others. In pursuing this field, our lab was the first to show that soluble toll-like receptor 2 (sTLR2) directly inhibits HIV infection, integration, and inflammation. More recently, we demonstrated that sTLR2 directly binds to selective HIV-1 proteins, including p17, gp41, and p24, leading to significantly reduced NFκB activation, interleukin-8 production, CCR5 expression, and HIV infection in a dose-dependent manner. Thus, a clearer understanding of soluble milk-derived innate factors with known antiviral functions may provide new therapeutic insights to reduce vertical HIV-1 transmission and will have important implications for protection against HIV-1 infection at other mucosal sites. Furthermore, innate bioactive factors identified in human milk may serve not only in protecting infants against infections and inflammation but also the elderly; thus, opening the door for novel innate immune therapeutics to protect newborns, infants, adults, and the elderly. In addition to turition  dependinturition –24.The past decade has shown significant progress in the prevention of mother-to-child transmission (PMTCT) of HIV globally through improved access to ARV therapies for women and infants, as well as the universal promotion of exclusive breastfeeding (EBF) when safe and sustainable alternatives are not readily available. As a result of these prevention strategies, for the first time the elimination of MTCT of HIV is considered a realistic public health goal . For exaet al.  and cell-associated virus (CAV), it seems paradoxical that with prolonged and repeated exposure to HIV during EBF can significantly decrease postnatal MTCT of HIV compared with mixed feeding or non-exclusive breastfeeding (nEBF) –24. Thuset al.  showed t , mucin, and soluble toll-like receptor 2 (sTLR2) -SIGN-mediated transmission of HIV from DCs to activated CD4+ T cells  –44  . Specifi (sTLR2) , has beereceptor . Lactofereceptor , 48. Simreceptor , which ial entry . Another T cells .Another recently identified innate factor in breast milk is Tenascin-C (TNC), which has the capability to neutralize HIV-1 virions by binding to the chemokine co-receptor binding site on the HIV-1 envelope . Like lain vitro  indirectly inhibits HIV infection tachment . Howevertachment . Converstachment . Furthertachment , thus inOver the past decade, more than two million HIV-exposed uninfected (HEU) infants were born each year. In sub-Saharan Africa, HEU infants suffer up to four times increased risk of dying in the first 2 years of life and increased risk for infectious morbidity . The etiet al. concluded that EBF of HIV-exposed infants decreased the likelihood of them falling ill to pneumonia  and high complexity in human milk. More than 200 different HMOs have been identified and are unique to human milk –71. TogeCampylobacter jejuni, which binds to α1,2-fucosylatd glycan. Although recently contested  blood group antigens  and mature breast milk (105–106/ml), and mammary epithelial cells (MECs). The majority of leukocytes in breast milk have an activated phenotype  in milk from full-term mothers , and both levels correlate with postpartum MTCT of HIV , 119. Imin vitro , 120–123in vitro , 124. CAin vitro  and is sin vitro , thus inin vitro , 128, whin vitro , 130. Thet al. showed that at 6 months, CFV was more heavily associated with HIV transmission. Up until 6 months, however, the viral load levels were analogous  that recognize highly conserved pathogen-associated molecular patterns (PAMPs). They are part of the first line of defense against pathogen invasion and trigger innate immune responses and subsequent antigen-specific adaptive immunity. The 10 TLRs in humans recognize highly conserved molecules broadly shared and also expressed by pathogens, but not found in mammals, such as dsRNA, ssRNA, flagellin, CpG DNA, and LPS either intracellularly or extracellularly.et al.  from HIV-infected patients and in latently infected cells (et al. showed that a selected group of miRNAs, including miR-28, miR-125b, miR-150, miR223, and miR-382, bound to the 3′ UTR of viral mRNAs and showed that activation of resting CD4+ T cells resulted in downregulation of these miRNAs, which correlated with enhanced HIV-1 susceptibility (MicroRNAs were shown to play a critical role in innate antiviral defense –178. Thied cells . Interesed cells . These stibility . Furthertibility .Other modulators of anti-HIV-1 miRNAs include cytokines and TLR ligands. For example, stimulation of TLR3 was shown to induce an anti-HIV effect in primary macrophages, partially through upregulation of miR-28, miR-125b, miR-150, and miR-382 . More reThe majority of infants’ breastfeeding from their HIV-infected mothers do not acquire HIV. Indeed, EBF has been one of the most successful interventions in protecting infants in resource-limited countries from a broad range of infectious diseases, including HIV. Although the reason for this remains unclear, coordination of a number of innate immune factors in breast milk seem crucial for providing protection when infants are most vulnerable. Identification and characterization of natural immune factors that protect susceptible individuals from acquiring HIV might facilitate the production of novel innate immunotherapeutics in the near future. Given that a number of innate factors have demonstrated anti-HIV activity, and ensuing decreased MTCT, it can be concluded that innate factors are indeed a viable option to pursue as protective therapies. In addition, other factors such as sTLR2 and IL-15 have shown promising results and should be pursued to further understand their mechanisms of binding and blocking HIV-1 MTCT.Human milk is a gold mine of uncharacterized, natural innate bioactive factors that have great promise to be developed and utilized for therapy or treatment of infections and inflammatory conditions in infants as well as adults and the elderly.KR initiated the review article, contributed to the design and conductance of the study, collection of relevant literature, and writing and editing of the manuscript. BH played a key role in design and conductance of the study. She performed experiments noted in the review, analysis of the data as well as contributing to the writing and editing of the review. Similarly, X-DY carried out much of the experimentation and bench research. She also made major contributions to analysis of the data and writing of the manuscript. Both BH and X-DY spent substantial time working on our projects in Kenya and South Africa. LN and AR were students who contributed to collecting, reading, and identifying relevant literature for the review. All authors contributed to editing the review. LN is now a resident physician at McMaster University Health Sciences Centre.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ultraconserved noncoding elements (UCNEs) constitute less than 1 Mb of vertebrate genomes and are impervious to accumulating mutations. About 4000 UCNEs exist in vertebrate genomes, each at least 200 nucleotides in length, sharing greater than 95% sequence identity between human and chicken. Despite extreme sequence conservation over 400 million years of vertebrate evolution, we show both ordered interspecies and within-species interindividual variation in DNA methylation in these regions. Here, we surveyed UCNEs with high CpG density in 56 species finding half to be intermediately methylated and the remaining near 0% or 100%. Intermediately methylated UCNEs displayed a greater range of methylation between mouse tissues. In a human population, most UCNEs showed greater variation than the LINE1 transposon, a frequently used epigenetic biomarker. Global methylation was found to be inversely correlated to hydroxymethylation across 60 vertebrates. Within UCNEs, DNA methylation is flexible, conserved between related species, and relaxed from the underlying sequence selection pressure, while remaining heritable through speciation. Nucleotides(nt)Ultraconserved noncoding elements(UCNEs)Single nucleotide polymorphism(SNP)5’ Cytosine DNA methylation(5mC)5’ Cytosine DNA hydroxymethylation(5hmC)Ultraconserved noncoding elements (UCNEs) are an especially unusual feature of vertebrate genomes. Of the genome, 1.2% codes for proteins and the remaining 98.8% is noncoding sequence. Interspecies comparisons reveal that 8.2% of the human genome is constrained and conserved when compared across mammals . Their dHOXD gene. Over a 7 kb range, 30 UCNEs are dispersed upstream of HOXD, a gene that has been highly conserved over the course of vertebrate evolution .The database of ultraconserved noncoding elements and genomic regulatory blocks (UCNEbase) provided sequence and nomenclature for 4351 UCNEs used in this study [All 4351 UCNEs were downloaded as fasta sequence. GC content was calculated for UCNE group and compared to the 22 autosomes + XY of the human genome. CpG frequency was calculated using a custom Perl script, applied to all UCNE sequences, and also human coding regions (CDS), human introns, and human promoters defined as 1000 nt upstream of coding regions sourced from the UCSC knownGene table of genes using human genome build GRCh/hg38. CpG density is defined as number of CpG sites / length of sequence (or x100 = CpG sites per 100 bp). The single UCNE with highest CpG density, 12.4 per 100 bp (chr1_Aida) was removed from analysis upon discovery that it was misclassified as noncoding. All UCNEs, positions, and CpG density measures can be found in Table S2. T-tests and figures were generated in R version 3.3.3.Fifty-six species were selected for pyrosequencing analysis. DNA was standardized at 200 ng/μl and converted with a Zymo Research 96-well lightning DNA conversion kit according to manufacturer's instructions.All primers were designed using Qiagen Pyromark Assay Design software version 2.0 against the human derived sequence of each UCNE. Primers were optimized for PCR by using bisulfite converted mouse DNA and tested for single-band amplification across all species. The parameters for each reaction included a thermocycler protocol of 95°C for 30 seconds, the optimal temperature for 30 seconds, and 72°C for 30 seconds repeated for 35–40 cycles. Primer sequence, and conditions can be found in Table S1. Conservation values were derived from UCNEbase.PCR products were quantitated for DNA methylation level on a Qiagen Pyromark Q96 ID instrument. Controls consisted of a “no template control” (NTC) and two wells containing bisulfite converted 100% or 0% methylated control human DNA from EpiGentek. Some sites failed assessment in some species due to species-specific mutations preventing either PCR amplification or pyrosequencing. CpG methylation values from pyrosequencing were collated under criteria that the Pyromark software defined as ‘check’ or ‘passing’, with these values retained for analysis, and discarded if ‘failed’. For mouse tissues, UCNEs were run with 6 biological replicates, 3 males and 3 females. Results were averaged over all replicates, and split by sex. Position 3 in VRK1_Siddhartha failed uniformly in mouse and was removed. Pyrosequencing for the human LINE1 was also performed on the human samples using previously developed primers . We analMissing values as seen in http://timetree.org with branch lengths based on evolutionary time scales derived from multiple publications [Phylogenetic signal as measured by Pagel's lambda (λ) was calculated using the average methylation value of all CpG sites per UCNE . The R cications .