Multi-omics analysis identifies potential microbial and metabolite diagnostic biomarkers of bacterial vaginosis.

BACKGROUND: Bacterial vaginosis (BV) is a common clinical manifestation of a perturbed vaginal ecology associated with adverse sexual and reproductive health outcomes if left untreated. The existing diagnostic modalities are either cumbersome or require skilled expertise, warranting alternate tests. Application of machine-learning tools to heterogeneous and high-dimensional multi-omics datasets finds promising potential in data integration and may aid biomarker discovery. OBJECTIVES: The present study aimed to evaluate the potential of the microbiome and metabolome-derived biomarkers in BV diagnosis. Interpretable machine-learning algorithms were used to evaluate the utility of an integrated-omics-derived classification model. METHODS: Vaginal samples obtained from reproductive-age group women with (n = 40) and without BV (n = 40) were subjected to 16S rRNA amplicon sequencing and LC-MS-based metabolomics. The vaginal microbiome and metabolome were characterized, and machine-learning analysis was performed to build a classification model using biomarkers with the highest diagnostic accuracy. RESULTS: Microbiome-based diagnostic model exhibited a ROC-AUC (10-fold CV) of 0.84 ± 0.21 and accuracy of 0.79 ± 0.18, and important features were Aerococcus spp., Mycoplasma hominis, Sneathia spp., Lactobacillus spp., Prevotella spp., Gardnerella spp. and Fannyhessea vaginae. The metabolome-derived model displayed superior performance with a ROC-AUC of 0.97 ± 0.07 and an accuracy of 0.92 ± 0.08. Beta-leucine, methylimidazole acetaldehyde, dimethylethanolamine, L-arginine and beta cortol were among key predictive metabolites for BV. A predictive model combining both microbial and metabolite features exhibited a high ROC-AUC of 0.97 ± 0.07 and accuracy of 0.94 ± 0.08 with diagnostic performance only slightly superior to the metabolite-based model. CONCLUSION: Application of machine-learning tools to multi-omics datasets aid biomarker discovery with high predictive performance. Metabolome-derived classification models were observed to have superior diagnostic performance in predicting BV than microbiome-based biomarkers.

No effect of switching to high-dose rosuvastatin, add-on nicotinic acid, or add-on fenofibrate on serum vitamin D levels in patients with mixed dyslipidemia.

BACKGROUND: Low 25-hydroxy-vitamin D [25(OH)VitD] levels may represent a novel cardiovascular disease risk factor. Several statins may increase 25(OH)VitD concentration. The effect of other lipid-lowering drugs is unknown. AIM: To investigate whether switching to high-dose rosuvastatin, add-on-statin nicotinic acid or add-on-statin fenofibrate would alter 25(OH)VitD levels in patients with mixed dyslipidemia who are already on a conventional statin dose. METHODS: This is a prespecified analysis of a previously published study. Forty-four patients with mixed dyslipidemia not at treatment goal despite treatment with simvastatin 10-40 mg or atorvastatin 10-20 mg or rosuvastatin 5-10 mg were randomly allocated to switch to rosuvastatin 40 mg (n=17), add-on-statin extended release nicotinic acid (ER-NA)/laropiprant (LRPT) (1000/20 mg first four weeks and 2000/40 mg thereafter) (n=14), or add-on-statin micronized fenofibrate (200 mg) for three months. The endpoint for this analysis was between-group difference in changes in 25(OH)VitD levels. RESULTS: Serum 25(OH)VitD levels did not significantly change in any group. In the switch to the highest dose of rosuvastatin group and the add-on-statin ER-NA/LRPT group there was an insignificant decrease in 25(OH)VitD levels {-4.7% [from 16.8 (3.2-37) to 16.0 (7.9-51.6)] and -14.8% [from 12.8 (2.0-54.8) to 10.9 (2.4-34)], respectively]}, while in the add-on-statin fenofibrate group there was an insignificant increase [+13% (from 14.5 (1.0-42) to 16.4 (4.4-30.4) ng/mL)]. No significant difference between groups was found. CONCLUSION: In patients already on a conventional statin dose, neither switching to high-dose rosuvastatin (40 mg) nor add-on-statin ER-NA/LRPT or fenofibrate were associated with significant changes in 25(OH)VitD serum levels. Hippokratia 2015; 19 (2):136-140.

Vitamin D levels and insulin resistance in children born with severe growth restriction.

This study was designed to examine differences in serum 25(OH)D levels between small-for-gestational-age (SGA) and appropriate-for-gestational-age (AGA) prepubertal children in correlation with birth weight and indices of insulin resistance and ß-cell function. Sixty-five nonobese children were examined at age 5-7.5 years; 27 born SGA and 38 matched AGA. Body weight, height, BMI, and waist circumference were recorded and fasting serum levels of glucose, insulin, 25(OH)D, and parathyroid hormone (PTH) were measured. The homeostasis model assessment for insulin resistance (HOMA-IR) and the ß-cell function index (HOMA-ß%) were estimated. The mean level of 25(OH)D was higher in the SGA group (26.2±10 vs. 17.2±7 ng/ml, p<0.01) but that of PTH was no different. The insulin resistance and ß-cell function indices were higher in the SGA group: HOMA-IR 1.34±0.67 vs. 0.99±0.53, and HOMA-ß% 135±56 vs. 97±60 in the SGA and AGA groups, respectively. In the SGA group, 25(OH)D was correlated with HOMA-ß% but not with HOMA-IR or insulin. In multiple regression, in the total cohort 25(OH)D and HOMA-IR were independently negatively correlated with birth weight (ß= - 0.31, ß= - 0.36, p<0.05) respectively. In conclusion, at prepuberty severely in utero growth restricted children have increased birth weight dependent levels of 25(OH)D, which might exert a regulatory role on ß-cell function.

Serum insulin-like growth factor I (IGF-I), IGF-binding proteins-1 and -3, and postnatal growth of late preterm infants.

Late preterm infants may have impaired early growth. The role of circulating insulin-like growth factors (IGFs) in the regulation of postnatal growth of these infants is unclear. The aim of the study was to investigate prospectively the serum levels of IGFs during the first year of life in late preterm infants and their association with birth weight and early postnatal growth. The study was conducted on 112 infants, born appropriate for gestational age (GA) at GA 32-36 weeks. Serum levels of IGF-I and IGF-binding proteins (IGFBP) -1 and -3, and anthropometric measurements were recorded at the chronological age of 2 and 6 weeks, and 3, 6, 9, and 12 months. The mean levels of both IGF-I and IGFBP-3 were found to be lower at 2 and 6 weeks, 82±44, 100±31 ng/ml, and 1.7±0.8, 2.1±1 µg/ml, respectively, but then rose and remained stable between 3 and 12 months. The levels of IGFBP-1 were lower at the 3 first study points and increased gradually thereafter. Birth weight correlated positively with the level IGF-I at 2 and 6 weeks (R=0.35, 0.37; p<0.01), but negatively at 12 months (R= - 0.34; p<0.01), independent of other factors. At all study points up to 6 months, the level of IGF-I was higher in infants who showed more rapid growth in either body weight or crown heel length. In late preterm infants, the serum IGF-I level is closely related to early accelerated growth. Its diverse associations with birth weight may imply a regulatory effect on regression of growth towards the mean.

Visfatin levels in prepubertal children born small or large for gestational age.

Children born small (SGA) or large (LGA) for gestational age are prone to develop insulin resistance (IR) during childhood. Visfatin, a hormone with insulin-mimetic actions, has been associated with IR. This study was designed to examine whether serum level of visfatin is correlated with metabolic indices of IR, in prepuberty in association with the intrauterine growth pattern. The following parameters were evaluated at a mean age of 6.5±1.2 years in 155 prepubertal children born appropriate for the gestational age (AGA) (n=63), or SGA (n=42), or LGA (n=50): serum levels of visfatin, adiponectin, leptin, fasting glucose (G(F)) and insulin (I(F)), the homeostasis model assessment IR index (HOMA-IR), plasma lipids, anthropometric indices at birth and the time of evaluation, and obesity indices [waist circumference (WC), body mass index (BMI) and skinfold thickness]. The mean serum level of visfatin was lower in the SGA than in the AGA and the LGA children (9±5.2 vs. 11.8±5.1 and 12.7±5.6 ng/ml, respectively, p<0.01). Girls had lower visfatin levels than boys (10.4±4.3 ng/ml vs. 12.5±6.7 ng/ml, p<0.05). Visfatin was not correlated with IR indices. In multiple regression analysis visfatin level was positively correlated with birth weight z-score (t=2.56, beta=0.24, p<0.01) and crown to heel z-score (t=2.46, beta=0.22, p=0.014), independent of age, gender, maternal weight before pregnancy, maternal weight gain during pregnancy, BMI z-score, WC z-score, serum leptin and adiponectin, and HOMA-IR. In conclusion serum visfatin level was lower in prepubertal SGA children but not correlated with IR indices. Low birth weight was an independent predictor of visfatin level.

Vitamin D and parathormone levels of late-preterm formula fed infants during the first year of life.

BACKGROUND/OBJECTIVES: Preterm infants are at risk for low vitamin D but documentation on late-preterm infants is sparse. This prospective study monitored longitudinally vitamin D and parathormone (PTH) levels in late-preterm formula fed infants during the first year of life, taking into consideration in utero and postnatal growth, and season and diet. SUBJECTS/METHODS: The study population comprised 128 infants of gestational age (GA) 32-36 weeks, of which 102 were appropriate (AGA) and the remaining 26 were small for GA (SGA). Serum levels of vitamin D (25(OH)D), PTH calcium, phosphate (P) and alkaline phosphate were estimated at 2 and 6 weeks, and at 3, 6, 9 and 12 months of age. RESULTS: The 25(OH)D levels were relatively low at 2 and 6 weeks in both AGA and SGA infants (21±11, 20±7 ng/ml and 25±16, 23±8 ng/ml, respectively), but increased at 6 months (45±14, 47±10 ng/ml) and remained stable thereafter. SGA infants had lower 25(OH)D levels at 9 and 12 months (AGA 45±14, 47±18 ng/ml vs SGA 38±13, 37±13 ng/ml, P<0.05). Deficiency of 25(OH)D (<20 ng/ml) was found in 18.5% of measurements in 92 (72%) infants, and its insufficiency (20-32 ng/ml) was found in 29.2% of measurements in 99 (77.3%) infants. Most measurements with vitamin D <32 ng/ml were observed at the first three study points, where PTH showed an inverse association with 25(OH)D, reaching a plateau thereafter. CONCLUSIONS: Late-preterm, formula fed infants may have suboptimal vitamin D levels and elevated PTH, especially, during the first 3 months. Those born SGA may have lower vitamin D levels up to the end of the first year of life.

ANCA-stimulated neutrophils release BLyS and promote B cell survival: a clinically relevant cellular process.

OBJECTIVES: To determine a role for antineutrophil cytoplasmic antibody (ANCA)-activated neutrophils in promoting B cell survival through the release of B lymphocyte stimulator (BLyS). METHODS: Neutrophil BLyS expression was measured by flow cytometry. Concentrations of BLyS in cell supernatants and donor serum samples were measured by ELISA. Cell survival assays were carried out using an L3055 cell line and viability measured by flow cytometry. RESULTS: Tumour necrosis factor α and formyl-Met-Leu-Phe (fMLP) treatment of non-primed neutrophils and treatment of primed neutrophils with anti-PR3 ANCA IgG resulted in a significant increase in surface expression of BLyS within 30 min which returned to basal levels by 2 h. Supernatants from ANCA-stimulated neutrophils were shown to contain increased levels of BLyS and to promote the survival of the centroblast cell line L3055. Serum BLyS concentrations are increased in patients with active ANCA-associated systemic vasculitis and these levels are increased further following 1-3 months of treatment with rituximab. CONCLUSIONS: ANCA specifically causes the release of BLyS from activated neutrophils which can support B cell survival in vitro. The presence of serum BLyS in active disease and its increase following B cell depletion suggest it is an important factor in disease pathogenesis and may facilitate disease relapse.

Growth factors, adiponectin, leptin and body mass index in pre-pubertal children born large for gestational age.

OBJECTIVE: To examine whether the IGF axis in pre-pubertal children born large for gestational age (LGA) differs from that of those born appropriate for gestational age (AGA). RESEARCH DESIGN AND METHODS: The study population consisted of 98 non-obese children aged 5.5-8 yr, of whom 37 were LGA, with birth weight (BW) > 90th percentile, and 61 AGA. The LGA children were subdivided into two subgroups, with BW 90th-97th percentile (no.=24) and BW > 97th percentile (no.=13), respectively. Total and free IGF-I, their binding proteins 1 and 3 (IGFBP-1 and IGFBP-3), leptin, adiponectin, fasting glucose (GF) and insulin (IF) were measured, and the homeostasis model assessment for insulin resistance (HOMA-IR index) was determined. RESULTS: IGF-I, free IGF-I and IGFBP-1 were similar in both groups. Both LGA subgroups had lower IGFBP-3 levels than the AGA group (2.34 ± 0.61 and 2.70 ± 0.90, respectively, vs 3.92 ± 1.1 µg/ml, p < 0.01). Adiponectin was higher in the 90th-97th percentile LGA subgroup than the AGA group (p<0.01). GF and IF were higher in the LGA group (86.5 ± 5.6 mg/dl, p < 0.01, and 5.84 ± 2.13 µU/ml, respectively, p < 0.05) than in the AGA group (82.6 ± 7.7 mg/dl and 4.62 ± 1.9 µU/ml, respectively), as was the HOMA-IR index (1.27 ± 0.60 vs 0.94 ± 0.43, p < 0.01). These three parameters were also found higher in the >97th percentile LGA subgroup. CONCLUSION: The IGF axis was not different in pre-pubertal children born LGA or AGA, with the exception of IGFBP-3, which was lower in the LGA children. In LGA pre-pubertal children the severity of intrauterine overgrowth was associated with the insulin resistance indices.

Changes in red cell phosphate metabolism of preterm and fullterm infants with perinatal problems during their first month of life.

The effects of perinatal problems on red cell phosphate metabolism were studied in two groups of infants (preterms B and fullterms D) during the first month of life. All infants started milk feeding from day three after birth. The results were compared to those of healthy preterms (A) and fullterms (C), respectively. Comparisons were also made between the preterm and fullterm groups B and D. The preterms with perinatal problems (B) showed a significant delay in catching up with the plasma and red cell inorganic phosphate (Pi) levels of controls (A) throughout the first month of life (p < 0.05). In parallel, the erythrocyte 2,3 diphosphoglycerate (2,3-DPG) concentrations of the sick preterms lagged significantly behind those of controls (p < 0.001); but the ATP levels were comparable between the two groups. The fullterms behaved slightly differently. No significant differences in plasma Pi (Pl Pi) and red cell 2,3-DPG were seen between the sick and healthy neonates during the month of study, while red cell Pi (RBC Pi) and ATP were found to be lower in the sick ones (p < 0.05). The fullterms with perinatal problems (D) had significantly higher Pl Pi (p < 0.05) and RBC Pi (p < 0.01) than preterms with problems (B) from the first week of life and continued in a similar pattern until the end of the month. Red cell 2,3-DPG concentrations were found to be significantly correlated with Pl Pi and RBC Pi in both preterm groups (p < 0.01) and in the sick fullterms (p < 0.001) during the time of the study. In the healthy fullterms 2,3-DPG was found to correlate only with red cell Pi (p < 0.05). Perinatal problems seem to affect Pi metabolism to a different degree in preterm and fullterm neonates in the first month of life.

Correlation of estrogen receptor beta gene polymorphisms with spinal bone mineral density in peri- and post-menopausal Greek women.

Estrogens play a significant role in bone physiology. Their action is mainly exerted through their receptors. Estrogen receptor alpha (ERalpha) plays a major role in bone homeostasis and there is evidence suggesting that estrogen receptor beta (ERbeta) has also an effect on BMD. We investigated the possible effect of two ERbeta gene polymorphisms on spinal bone mineral density (BMD) and metabolic bone markers in Greek women. Spine BMD as well as biochemical bone markers were measured in 147 healthy peri- and post-menopausal women [mean age (S.D.) 54 (7.9) years]. Genotyping was performed for two restriction fragment length polymorphisms (RFLPs) of ERbeta gene, RsaI in exon 5 and AluI in exon 8. For each polymorphism studied the cohort was divided into two groups: the "wild-type" group (RR and AA, respectively) and the "carrier" group including subjects with at least one allele with the restriction site (Rr&rr and Aa&aa, respectively). The distribution of RsaI genotypes was RR: 91.2% (n = 134), Rr: 8.2% (n = 12), and rr: 0.6% (n = 1) and of AluI genotypes AA: 36.7% (n = 54), Aa: 57.2% (n = 84), and aa: 6.1% (n = 9). No linkage disequilibrium was found between the two polymorphic sites studied. Spine BMD did not differ significantly in the two groups of either polymorphism, after adjusting for age, weight, height, and years since menopause [mean BMD (S.D.) for RR 0.841 (0.17) g/cm(2) versus Rr&rr 0.798 (0.13) g/cm(2), p = 0.25, and mean BMD (S.D.) for AA 0.828 (0.16)g/cm(2) versus Aa&aa 0.848 (0.17) g/cm(2), p = 0.32]. No significant differences were noted in metabolic bone markers except for a marginal difference of RR versus Rr/rr in urinary hydroxyproline/creatinine ratio [median (IQR) 3.88 (6.04) micromol/mmol in RR versus 8.2 (4.32) micromol/mmol in Rr/rr, p = 0.05]. Furthermore, no interaction between the two polymorphisms on BMD was found. In conclusion, in a Greek female post-menopausal population, the two ERbeta gene polymorphisms were not associated with BMD, or metabolic bone markers.

Vitamin D: a necessity for children and adolescents in Greece.

Children and adolescents with the high bone turnover comprise a high risk population for vitamin D insufficiency. A sample of 178 clinically healthy children aged 3 to 18 years who came from public schools and lived in North West of Greece participated in the study. They were grouped into three age groups (I: 3-10, II: 11-14 and III: 15-18 years of age). Blood samples were taken during winter and summer months for determining calciotropic hormones, calcium, phosphate and biochemical markers of bone synthesis.A high percentage (47%) of the subjects aged 15-18 years was found to have 25OHD <10 ng/ml in winter but much less (13-14%) of the younger ages (13-14 years), while in the summer they were all >10 ng/ml. The prevalence was even higher in the girls of the older group accompanied by lower Pi concentrations again in winter (win:1.19+/-0.03, sum:1.93+/-0.03 mmol/l, p < 0.001). The 24,25(OH)(2)D levels were changing in parallel to 25OHD, but again in the older subjects, during winter, they were by 2/3 lower than the summer ones (0.73+/-0.10 vs. 2.41+/-0.20 ng/ml, p < 0.001). No significant differences were found between seasons and groups in the 1,25(OH)(2)D levels. The biochemical markers of bone synthesis, osteocalcin (OC) and total alkaline phosphatase (ALP), were found significantly lower in the girls of the older group both in winter and summer respectively. Even in a sunny country like Greece the adolescents living in an urban area are in high risk for vitamin D deficiency during winter. Supplementation with vitamin D of milk, of popular beverages and perhaps some foods would be of help.

Apolipoprotein E polymorphism is not associated with spinal bone mineral density in peri- and postmenopausal Greek women.

OBJECTIVES: A number of studies have shown a positive relation between ApoE gene and osteoporosis or fracture risk but this finding has not been uniform in all populations studied. The aim of the present study was to determine the possible effect of ApoE gene polymorphism on spinal bone mineral density and metabolic bone markers in Greek women. METHODS: One hundred and forty-seven healthy peri- and postmenopausal women (mean age 54.3 +/- 7.8 years) participated in the study. In all participants, ApoE gene genotype was determined and spinal bone mineral density (BMD) as well as biochemical bone markers were measured. The ApoE genotypes distribution was 0.7% (n = 1) for E2/2, 5.4% (n = 8) for E2/3, 2% (n = 3) for E2/4, 73.5% (n = 108) for E3/3, 16.3% (n = 24) for E3/4 and 2% (n = 3) for E4/4. Participants were divided in two groups according to the presence of the E4 haplotype: E4 carriers (n = 30) and E4 non-carriers (n = 117). RESULTS: Spinal BMD was similar in the two groups, after adjusting for age, weight, height and years since menopause (mean +/- S.D., 0.835 +/- 0.16 g/cm2 in E4 non-carriers versus 0.831 +/- 0.16 g/cm2 in E4 carriers, P = 0.99). Serum osteocalcin levels did not differ significantly in the two groups (median (interquartile range, IQR), 0.55 (0.58) nmol/l in E4 non-carriers versus 0.51 (0.43) nmol/l in E4 carriers), whereas urinary hydroxyproline/creatinine ratio was significantly higher in the E4 non-carriers group (median (IQR), 5.18 (6.04) micromol/mmol in E4 non-carriers versus 1.73 (3.45) micromol/mmol in E4 carriers, P < 0.01). Urinary pyridinoline/creatinine and deoxypyridinoline/creatinine ratios, measured in a subgroup of 51 women, were similar between ApoE carriers and non-carriers, respectively (median (IQR), 25.1 (9.3) nmol/mmol in E4 non-carriers versus 21.8 (7) nmol/mmol in E4 carriers and 6.7 (3.1) nmol/mmol in E4 non-carriers versus 7 (2.2) nmol/mmol in E4 carriers). CONCLUSION: In conclusion, in a Greek female postmenopausal population, ApoE gene does not seem to play an important role in determining BMD and neither does it affect the majority of metabolic bone markers.

Biochemical markers of bone metabolism in infants and children under intravenous corticosteroid therapy.

The short-term effects of corticosteroids (CS) administered intravenously (IV) on biochemical parameters of bone metabolism were followed in infants and children. Forty-nine patients from 2 months to 10 years of age, admitted to Pediatrics Department for bronchiolitis, viral-associated wheezing and croup, were treated with IV hydrocortisone or methylprednisolone (10 or 2 mg/Kg/day, respectively) for 3 days. Blood and fasting urine were collected on admission (day 1), 2 days later (day 3) and 12 days after the end of therapy (day 15). Fifty-one children of similar age and gender without respiratory problems or bone diseases were used as controls. On day 3, suppression of the bone formation markers osteocalcin (OC) (P < 0.001) and total alkaline phosphatase (ALP) (P < 0.05) was observed, but not of the bone resorption markers of hydroxyproline, pyridinoline and calcium excretion (UHyp/UCr, UPYD/UCr and UDPD/UCr, UCa/UCr). Significant decreases were indicated in serum phosphate (Pi) and the maximum renal tubular Pi reabsorption (TmP/GFR) compared to basal (P < 0.001). No significant changes were noticed in the circulating levels of calcium (Ca), parathyroid hormone (iPTH), 25OHD, 24,25(OH)2D, 1,25(OH)2D, the insulin-like growth factor-I (IGF-I) and its binding protein-3 (IGFBP-3). Two weeks after therapy, the increase of OC to higher than basal (P < 0.01) indicated a probable activation of the osteoblasts. Serum Pi and the TmP/GFR index values that had significantly decreased by day 3 returned to pretreatment levels by day 15. When assessing the effects of the CS in relation to age, no changes were detected in the levels of OC and total ALP in the <12-month-old children, but a fall of OC was observed in the >1-year-old group (P < 0.001). In contrast to the OC, the effects on serum and renal tubular reabsorption of phosphate were similar for both groups. In conclusion, short-term IV administered CS led to significant but reversible inhibition of bone formation markers, especially detectable in the >1-year-old children, without affecting the bone resorption ones. The adverse effects on phosphate metabolism were also significant, but temporal and irrespective of age.

Effects of intranasal salmon calcitonin in juvenile idiopathic arthritis: an observational study.

The aim of this study was to follow the changes in bone mineral density (BMD) and biochemical markers of bone turnover in 10 children (7.5-17.5 years of age) with severe juvenile idiopathic arthritis (JIA), during a 3-year therapy with salmon calcitonin (100 IU/day 2 months on and 2 off for a year and 200 IU/day for 2 years) and calcium (500 mg/day). All patients were functional classes III and IV and were measured at yearly intervals with a dual photon absorptiometer at the lumbar spine. The changes observed were 7.2-9.5% per year for BMD and 2.0-6.0% for volumetric bone mineral density (BMDvol). The bone resorption markers showed significant decreases after a year's treatment (Pyr/Cr from 175+/-15 to 108+/-15 nm/mm, P < 0.001, Pyr-D/Cr from 24.3+/-3.5 to 13.3+/-1.9 nm/mm, P < 0.05, and OHPr/Cr from 57.4+/-11 to 35.1+/-8.4 microg/mg) and smaller changes thereafter. No significant changes were observed in the bone formation markers of osteocalcin and alkaline phosphatase. Serum iPTH, the vitamin D metabolites, and calcium concentrations fluctuated within normal, while calcium excretion increased from 0.3+/-0.1 to 1.9+/-0.4 mg/kg/24 hours, P < 0.001. In conclusion, the present study, despite its limitations of not being placebo controlled, shows possible beneficial effects of intranasal calcitonin on bone resorption and pain relief in JIA patients.

Identification and characterization of roundabout orthologs in zebrafish.

The Roundabout (Robo) family of receptors and their extracellular ligands, the Slit protein family, play important roles in repulsive axon guidance. First identified in Drosophila, Robo receptors form an evolutionarily conserved sub-family of the immunoglobulin (Ig) superfamily that are characterized by the presence of five Ig repeats and three fibronectin-type III repeats in the extracellular domain, a transmembrane domain, and a cytoplasmic domain with several conserved motifs that play important roles in Robo-mediated signaling (Cell 92 (1998) 205; Cell 101 (2000) 703). Robo family members have now been identified in C. elegans, Xenopus, rat, mouse, and human (Cell 92 (1998) 205; Cell 92 (1998) 217; Cell 96 (1999) 807; Dev. Biol. 207 (1999) 62). Furthermore, multiple robo genes have been described in Drosophila, rat, mouse and humans, raising the possibility of potential redundancy and diversity in robo gene function. As a first step in elucidating the role of Robo receptors during vertebrate development, we identified and characterized two Robo family members from zebrafish. We named these zebrafish genes robo1 and robo3, reflecting their amino acid sequence similarity to other vertebrate robo genes. Both genes are dynamically expressed in the developing nervous system in distinct patterns. robo3 is expressed during the first day of development in the hindbrain and spinal cord and is later expressed in the tectum and retina. robo1 nervous system expression appears later in development and is more restricted. Moreover, both genes are expressed in non-neuronal tissues consistent with additional roles for these genes during development.

Reduced serum insulin-like growth factor-1 (IGF-1) and IGF-binding protein-3 levels in adults with inflammatory bowel disease.

In the present study, the changes in circulating IGF-1 and its binding protein IGFBP-3 were determined in adult patients with active inflammatory bowel disease (IBD) in order to assess the effect of this inflammatory condition on the IGF system. IGF-1 and IGFBP-3, as well as interleukin-6 (IL-6) were measured in serum obtained from 22 consecutive newly diagnosed patients (mean age 41.3 years) with active IBD, including 10 patients with Crohn's disease (CD), and 12 with ulcerative colitis (UC). For comparison the same parameters were determined in 30 healthy volunteers matched for age, sex and Body Mass Index (BMI). Serum IGF-1 and IGFBP-3 levels were similar in the two subgroups of patients and the values from all patients were combined for comparison with those from the control group. The mean (+/- SD) serum IGF-1 concentration (178 +/- 91 ng/ml) in the patients with IBD was lower compared with that in the controls (227 +/- 79 ng/ml, P<0.035). Similarly, the mean IGFBP-3 concentration in the patients was lower than in the controls (1.6 +/- 0.6 ng/ml vs 3.2 +/- 0.7 ng/ml respectively, P<0.001), Serum IL-6 levels were higher in the patients compared with the controls (5.5 +/- 4.2 vs 0.65 +/- 0.11 pg/ml, P<0.0001). The reduced IGF-1 and IGFBP-3 levels in patients with active IBD suggest that this systemic inflammatory condition is associated with a degree of acquired GH resistance, possibly induced by inflammatory cytokines.

CD40 ligand, Bcl-2, and Bcl-xL spare group I Burkitt lymphoma cells from CD77-directed killing via Verotoxin-1 B chain but fail to protect against the holotoxin.

Owing to its lineage and differentiation stage-restricted expression, CD77 has been mooted as a therapeutic target in Burkitt lymphoma (BL). The recognition that the globotriaosyl moiety of this neutral glycosphingolipid is a receptor for Escherichia coli-derived Verotoxin-1 (Shiga-Like Toxin-1) offers a potential delivery system for the attack. Here we show that CD77-expressing Group I BL cells which are normally susceptible to activation-induced death on binding Verotoxin-1 B chain are protected in the presence of CD40 ligand. Ectopic expression of either bcl-2 or bcl-xL also afforded resistance to the actions of the B chain. In total contrast, neither of the survival genes nor a CD40 signal - even when acting in concert - protected against killing mediated by the holotoxin. These findings indicate that while therapeutic modalities for CD77-expressing B cell tumors (which include follicular lymphoma) based on the use of Verotoxin-1 B chain might be compromised by the activation of endogenous or exogenous survival pathways, those exploiting the holotoxin should be left unscathed.

CD5-positive and CD5-negative human B cells converge to an indistinguishable population on signalling through B-cell receptors and CD40.

Whether CD5 on B cells marks a subset functionally distinct from the conventional CD5 negative (CD5neg) adult population or is more an indicator of activation, remains contentious. Here we have investigated whether CD5 positive (CD5pos) and CD5neg B cells can be distinguished in terms of their response to surrogate signals aimed to model, in vitro, T-cell dependent (TD) and T-independent (TI) encounters with antigen in vivo: the predominantly CD5pos B-cell population found in cord blood, CD5 B cells positively selected from tonsils and their CD5neg counterparts, were compared. Neonatal B cells displayed a near-identical phenotype to that of adult CD5pos B cells, being characterized by uniform immunoglobulin M (IgM), immunoglobulin D (IgD), CD23 and CD44 coexpression. When cultured with anti-IgM maintained at high density on CD32-tranfected mouse L cells to model TI responses or on CD40 ligand (CD40L)-bearing L cells (with or without captured anti-IgM) to model TD encounters, DNA synthesis was stimulated to a similar extent in all three populations. Focusing on CD5 and CD23, we found that - although the signals delivered promoted distinct profiles of expression - under each condition of activation, the phenotypes that emerged for adult CD5pos and CD5neg B cells were remarkably similar. Neonatal B cells displayed a greater diminution in CD5 expression than adult CD5pos B cells following CD40 signals but otherwise the two populations again behaved similarly. The inclusion of interleukin-4 (IL-4) to cultures where cells were costimulated via surface (s)IgM and CD40 resulted in a complete loss of CD5 expression and a corresponding hyperexpression of CD23, irrespective of the population studied. The near-identical response of CD5pos and CD5neg B cells to surrogate TD or TI signals in vitro and their convergence to indistinguishable phenotypes is wholly supportive of CD5 being a fluctuating marker of activation rather than it delineating functionally distinct subsets.

Blueberry juice used per os in upper abdominal MR imaging: composition and initial clinical data.

The aim of this study was to evaluate the use of a commercially available blueberry juice (BJ) both as a positive and negative oral contrast agent and to present the exact contents of paramagnetic ions. The concentration of Mn and Fe were determined in tinned myrtilles in syrup (atomic absorption). Nine healthy volunteers and 12 patients (age range 20-65 years) were examined using a 1-T MR scanner before and after per os administration of 430 ml of BJ. A qualitative analysis of signal alterations in the stomach, duodenum, and proximal small intestine was performed. In addition, a quantitative analysis was assessed in terms of signal-to-noise ratio calculation. The mean concentration (x +/- SD) of the ions found in the content of the three cans were 3.3 +/- 0.4 microg/g for iron and 20.6 +/- 2.6 microg/g for manganese. Based on the qualitative evaluation, signal alteration on T1-weighted images after administration of BJ was statistically significant in the stomach and duodenum, but not in the proximal small bowel. Signal alteration on T2-weighted images was not statistically significant in any part of the gastrointestinal tract. The quantitative analysis of the T1- and T2 shortening showed that BJ is efficient with only T1-weighted sequences, and this applied to the stomach, duodenum, and proximal small bowl. Blueberry juice can be used as an oral contrast agent in upper abdominal MR for T1-weighted imaging.