Clot composition characterization using diffuse reflectance spectroscopy in acute ischemic stroke.

Acute ischemic stroke caused by large vessel occlusion is treated with endovascular thrombectomy, but treatment failure may occur when clot composition and thrombectomy technique mismatch. In this proof-of-concept study, diffuse reflectance spectroscopy (DRS) is evaluated for identification of clot composition ex vivo. DRS spectra and histology were acquired from 45 clot units retrieved from 29 stroke patients. DRS spectra correlated to clot RBC content, R= 81, p < .001, and could discriminate between RBC-rich and fibrin-rich clots, p < 0.001. Sensitivity and specificity for detection of RBC-rich clots were 0.722 and 0.846 respectively. Applied in an intravascular device, DRS could potentially provide intraprocedural information on clot composition that could increase endovascular thrombectomy efficiency.

Identifying clot composition using intravascular diffuse reflectance spectroscopy in a porcine model of endovascular thrombectomy.

BACKGROUND: Endovascular thrombectomy has revolutionized the management of acute ischemic stroke and proven superior to stand-alone intravenous thrombolysis for large vessel occlusions. However, failed or delayed revascularization may occur as a result of a mismatch between removal technique and clot composition. Determination of clot composition before thrombectomy provides the possibility to adapt the technique to improve clot removal efficacy. We evaluated the application of diffuse reflectance spectroscopy (DRS) for intravascular determination of clot composition in vivo. METHODS: Three clot types, enriched in red blood cells or fibrin or with a mixed content, were prepared from porcine blood and injected into the external carotids of a domestic pig. A guidewire-like DRS probe was used to investigate the optical spectra of clots, blood and vessel wall. Measurement positions were confirmed with angiography. Spectra were analyzed by fitting an optical model to derive physiological parameters. To evaluate the method's accuracy, photon scattering and blood and methemoglobin contents were included in a decision tree model and a random forest classification. RESULTS: DRS could differentiate between the three different clot types, blood and vessel wall in vivo (p<0.0001). The sensitivity and specificity for detection was 73.8% and 98.8% for red blood cell clots, 80.6% and 97.8% for fibrin clots, and 100% and 100% for mixed clots, respectively. CONCLUSION: Intravascular DRS applied via a custom guidewire can be used for reliable determination of clot composition in vivo. This novel approach has the potential to increase efficacy of thrombectomy procedures in ischemic stroke.

Influence of Energy Balance and Glycemic Index on Metabolic Endotoxemia in Healthy Men.

OBJECTIVE: Overfeeding with a high-fat and/or high-carbohydrate (CHO) diet is known to increase plasma concentrations of endotoxin (lipopolysaccharide [LPS]) that may lead to metabolic disturbances like insulin resistance. The impact of CHO quality (i.e., the glycemic index [GI]) independent of fat intake on metabolic endotoxemia remains unclear. In the present study, the effects of changes in energy balance and GI on plasma endotoxin were studied. METHODS: Fifteen healthy young men overconsumed diets containing 65% CHO and 20% fat for 1 week (OF; +50% of energy requirement) followed by 3 weeks of caloric restriction (CR; -50% of energy requirement) and were then randomized to 2 weeks hypercaloric refeeding (RF, +50% of energy requirement) with either a low- or high-GI (40 vs 74) diet. RESULTS: During OF, subjects gained 1.9 ± 0.7 kg body weight (+0.6 ± 0.8% fat mass) followed by a weight loss of 6.1 ± 0.8 kg (-2.0 ± 0.6% fat mass) and weight regain of 4.0 ± 0.6 kg (0.9 ± 0.8% fat mass). Fasting insulin and homeostasis model assessment-insulin resistance (HOMAIR) increased with OF and RF and decreased with CR, MatsudaISI decreased by 37% after RF (all p < 0.05). Endotoxin significantly increased by 30.8% with OF and by 24.7% with RF (both p < 0.05), whereas CR normalized endotoxin levels. No difference in endotoxin levels was observed between refeeding a hypercaloric high- or low-GI diet. Changes in endotoxin levels with RF were not related to changes in insulin sensitivity. CONCLUSION: A hypercaloric diet (OF and RF) increased plasma endotoxin irrespective of GI, whereas a negative energy balance did not reduce endotoxemia. Impaired insulin sensitivity with hypercaloric refeeding on a high-GI diet was not explained by metabolic endotoxemia.

Nerve detection with optical spectroscopy for regional anesthesia procedures.

BACKGROUND: Regional anesthesia has several advantages over general anesthesia but requires accurate needle placement to be effective. To achieve accurate placement, a needle equipped with optical fibers that allows tissue discrimination at the needle tip based on optical spectroscopy is proposed. This study investigates the sensitivity and specificity with which this optical needle can discriminate nerves from the surrounding tissues making use of different classification methods. METHODS: Diffuse reflectance spectra were acquired from 1563 different locations from 19 human cadavers in the wavelength range of 400-1710 nm; measured tissue types included fascicular tissue of the nerve, muscle, sliding fat and subcutaneous fat. Physiological parameters of the tissues were derived from the measured spectra and part of the data was directly compared to histology. Various classification methods were then applied to the derived parameter dataset to determine the accuracy with which fascicular tissue of the nerve can be discriminated from the surrounding tissues. RESULTS: From the parameters determined from the measured spectra of the various tissues surrounding the nerve, fat content, blood content, beta-carotene content and scattering were most distinctive when comparing fascicular and non-fascicular tissue. Support Vector Machine classification with a combination of feature selections performed best in discriminating fascicular nerve tissue from the surrounding tissues with a sensitivity and specificity around 90 %. CONCLUSIONS: This study showed that spectral tissue sensing, based on diffuse reflectance spectroscopy at the needle tip, is a promising technique to discriminate fascicular tissue of the nerve from the surrounding tissues. The technique may therefore improve accurate needle placement near the nerve which is necessary for effective nerve blocks in regional anesthesia.

Relationships between body roundness with body fat and visceral adipose tissue emerging from a new geometrical model.

OBJECTIVE: To develop a new geometrical index that combines height, waist circumference (WC), and hip circumference (HC) and relate this index to total and visceral body fat. DESIGN AND METHODS: Subject data were pooled from three databases that contained demographic, anthropometric, dual energy X-ray absorptiometry (DXA) measured fat mass, and magnetic resonance imaging measured visceral adipose tissue (VAT) volume. Two elliptical models of the human body were developed. Body roundness was calculated from the model using a well-established constant arising from the theory. Regression models based on eccentricity and other variables were used to predict %body fat and %VAT. RESULTS: A body roundness index (BRI) was derived to quantify the individual body shape in a height-independent manner. Body roundness slightly improved predictions of %body fat and %VAT compared to the traditional metrics of body mass index (BMI), WC, or HC. On this basis, healthy body roundness ranges were established. An automated graphical program simulating study results was placed at http://www.pbrc.edu/bodyroundness. CONCLUSION: BRI, a new shape measure, is a predictor of %body fat and %VAT and can be applied as a visual tool for health status evaluations.

Iron intake and iron status in breastfed infants during the first year of life.

BACKGROUND & AIMS: Breastfed infants may be at particular risk for iron deficiency because breast milk is low in iron. In a secondary analysis of data from a complementary feeding trial, indicators of iron status were examined, with particular focus on the development of iron status in those infants who were fully breastfed during the first 4 months of life. METHODS: In this retrospective analysis of data from a randomized controlled trial infants were stratified according to their predominant milk diet during the first 4 months of life, a subgroup of breastfed infants (group BM, n=53) were compared with a subgroup of infants fed (iron-fortified) formula (group F, n=23). Dietary iron intake and indicators of iron status were analysed at 4 months of age (during the full milk feeding period), and during the complementary feeding period at 7 and 10 months of age. RESULTS: Iron intake was low in the BM group, ranging below the Dietary Reference Intakes throughout the complementary feeding period, with the (estimated) bioavailable iron intake only just achieving the reference requirements. At 4 months, iron deficiency (ID, Ferritin <12.0 ng/mL) was observed in 3 infants in the BM group and in 1 infant in the F group; no infant developed iron deficiency anaemia (IDA, ID and Hb <10.5 g/dl). At 7 and at 10 months of age, iron status was adequate in all infants of the F group. In the BM group, at 7 (10) months of age, ID was diagnosed in 10 (11) infants and IDA was found in 2 (1) infants. CONCLUSIONS: Healthy infants, fully breastfed at 4 months of age, demonstrated ID in about 21% and IDA in up to 6% during the second half of infancy while fed according to the paediatric dietary guidelines. This finding supports the recommendation that supplementation with bioavailable iron via complementary foods should be started early (4-6 months of age) in order to prevent iron deficiency during infancy.

Poor prediction of resting energy expenditure in obese women by established equations.

The objective of the study was to evaluate the accuracy of established prediction equations that calculate resting energy expenditure (REE) in obese women. This was a cross-sectional study. In 273 mildly to severely obese women (age, 41.7 +/- 13.2 years; body mass index, 42.8 +/- 7.0 kg/m(2)), REE was measured by indirect calorimetry (mREE), along with fat mass (FM) and fat-free mass (FFM) by bioelectrical impedance analysis. Eleven established equations were used to predict REE (pREE), with 9 equations basing on the anthropometric parameters body weight and height and 2 equations including body composition parameters (FM, FFM). All equations provided pREE values that significantly correlated with mREE (r > 0.66, P < .001), although 8 equations systematically underestimated mREE (P < .05). Of note, even the best equation was not able to accurately predict mREE with a deviation of less than +/-10% in more than 70% of the tested women. Furthermore, equations using body composition data were not superior in predicting REE as compared with equations exclusively including anthropometric variables. Multiple linear regression analyses revealed 2 new equations--one including body weight and age and another including FM, FFM, and age--that explained 56.9% and 57.2%, respectively, of variance in mREE. However, when these 2 new equations were applied to an independent sample of 33 obese women, they also provided an accurate prediction (+/-10%) of mREE in only 56.7% and 60.6%, respectively, of the women. Data show that an accurate prediction of REE is not feasible using established equations in obese women. Equations that include body composition parameters as assessed by bioelectrical impedance analysis do not increase the accuracy of prediction. Based on our results, we conclude that calculating REE by standard prediction equations does not represent a reliable alternative to indirect calorimetry for the assessment of REE in obese women.

Complementary food with low (8%) or high (12%) meat content as source of dietary iron: a double-blinded randomized controlled trial.

BACKGROUND: To investigate whether a low meat content of complementary food as accepted by EU law increases the risk of well-nourished infants to develop iron deficiency during the complementary feeding period. METHODS: Term born, healthy infants were randomized into a 'High Meat' Group (HM, n = 48) receiving commercial baby jars with a meat content of 12% by weight (according to pediatric guidelines), and a 'Low Meat' Group (LM, n = 49) receiving meals as marketed (meat 8% by weight, the lowest level of EU law). Intervention was from 4 to 10 months of age. Dietary intake was recorded continuously, repeated blood samples were collected. RESULTS: Estimated intake of bioavailable iron conformed to reference requirements. In the primary analysis of the total sample, iron status was adequate before (4 months), during (7 months), and after (10 months) the intervention. A secondary analysis in the subgroup of infants fully breast-fed for 4-6 months demonstrated an increased risk of low Hb values with 10 months of age in the LM group. INTERPRETATION: Present day low meat content of complementary food does not significantly impair iron status in well-nourished infants but may increase the risk of developing marginal iron status in older infants after fully breast-feeding for 4-6 months, i.e., in the subgroup of infants with the lowest habitual iron intake.

Effect of quercetin on paraoxonase 2 levels in RAW264.7 macrophages and in human monocytes--role of quercetin metabolism.

There is increasing evidence that the intracellular antioxidant enzyme paraoxonase 2 (PON2) may have a protective function in the prevention of atherogenesis. An enhancement of PON2 activity by dietary factors including flavonoids is therefore of interest. In the present study we determined the effect of quercetin on paraoxonase 2 levels in cultured murine macrophages in vitro and in overweight subjects with a high cardiovascular risk phenotype supplemented with 150 mg quercetin/day for 42 days in vivo. Supplementation of murine RAW264.7 macrophages in culture with increasing concentrations of quercetin (1, 10, 20 micromol/L) resulted in a significant increase in PON2 mRNA and protein levels, as compared to untreated controls. Unlike quercetin, its glucuronidated metabolite quercetin-3-glucuronide did not affect PON2 gene expression in cultured macrophages. However the methylated quercetin derivative isorhamnetin enhanced PON2 gene expression in RAW264.7 cells to similar extent like quercetin. Although supplementing human volunteers with quercetin was accompanied by a significant increase in plasma quercetin concentration, dietary quercetin supplementation did not change PON2 mRNA levels in human monocytes in vivo. Current data indicate that quercetin supplementation increases PON2 levels in cultured monocytes in vitro but not in human volunteers in vivo.

Daily quercetin supplementation dose-dependently increases plasma quercetin concentrations in healthy humans.

Our aim was to investigate the effects of an oral supplementation of quercetin at 3 different doses on plasma concentrations of quercetin, parameters of oxidant/antioxidant status, inflammation, and metabolism. To this end, 35 healthy volunteers were randomly assigned to take 50, 100, or 150 mg/d (group Q50-Q150) quercetin for 2 wk. Fasting blood samples were collected at the beginning and end of the supplementation period. Compared with baseline, quercetin supplementation significantly increased plasma concentrations of quercetin by 178% (Q50), 359% (Q100), and 570% (Q150; P < 0.01 for all). High interindividual variation was found for plasma quercetin concentrations (36-57%). Quercetin did not affect concentrations of serum uric acid or plasma alpha- and gamma-tocopherols, oxidized LDL, and tumor necrosis factor-alpha, or plasma antioxidative capacity as assessed by the ferric-reducing antioxidant potential and oxygen radical absorbance capacity assays. In addition, serum lipids and lipoproteins, body composition, and resting energy expenditure did not significantly change during quercetin supplementation. Pharmacokinetics of quercetin were investigated in a subgroup of 15 volunteers. The areas under the plasma concentration-time curves ranged from 76.1 mumol.min.L(-1) to 305.8 mumol.min.L(-1) (50- and 150-mg dosages, respectively). Median maximum plasma concentrations of quercetin (431 nmol/L) were observed 360 min after intake of 150 mg quercetin. In conclusion, daily supplementation of healthy humans with graded concentrations of quercetin for 2 wk dose-dependently increased plasma quercetin concentrations but did not affect antioxidant status, oxidized LDL, inflammation, or metabolism.

Effects of Bifidobacterium lactis Bb12 supplementation on body weight, fecal pH, acetate, lactate, calprotectin, and IgA in preterm infants.

Preterm infants are prone to abnormal bacterial colonization of the intestine with ensuing adverse health effects. To examine whether the oral application of Bifidobacterium lactis Bb12 (probiotic) may improve selected indicators of health status in preterm infants, a double blind, placebo controlled randomized clinical study was performed on 69 preterm infants (<37 gestation wk). Weight gain was defined as the primary outcome measure. In antibiotic-treated infants, probiotic supplementation resulted in a higher body weight compared with placebo (p < 0.001). In the probiotic group, the fecal pH was significantly lower than in the placebo group. The fecal concentrations of acetate and lactate were 42 and 38% higher, respectively, in the probiotic group than in the placebo group. Fecal calprotectin was lower in the probiotic group (p = 0.041), while fecal IgA was higher in this group compared with the placebo group (p = 0.021).

Frequency of 23 human papillomavirus types using DNA microarray in women with and without cytological anomalies.

OBJECTIVE: To evaluate the frequency of single and multiple human papillomavirus (HPV) infections in women with and without cervical dysplasia. MATERIALS AND METHODS: An oligonucleotide microarray system was used to detect 19 types of high-risk HPV (HPV-16/-18/-311-33/-35/-39/-45/-51/ -52/-53/-56/-58/-59/-66/-68/-73/-821-16 variant E-E6-G350/-16 variant E-E6-T350) and 4 types of low-risk HPV (HPV-6/-11/ -42/-44) in 122 consecutive women visiting our colposcopy outpatient clinic classified into controls (normal epithelium, nonspecific cervicitis, metaplasia; n = 56) and cervical intraepithelial neoplasia (CIN) (n = 66). RESULTS: In 78/122 (64%) cervical samples, HPV DNA was detected. Compared to controls, HPV infection was significantly more prevalent among women with CIN (8/56 [14%] versus 49/66 [74%]; p = 0.001). HPV-18 and HPV-16 were the most common HPV types in all specimens (25% [31/122] and 25% [31/122], respectively). Of note, HPV-16 was significantly more frequent in women with CIN compared to controls (35% [23/66] vs. 14% [8/56], respectively; p = 0.02). Double HPV infections were detected in 16/122 (13%) and multiple infections in 43/122 (35%) women. Multiple HPV infections were found significantly more often among women with CIN compared to controls (30/66 [45%] vs. 13/56 [23%], respectively; p = 0.002). Using a univariate and multivariate logistic regression model to estimate the relative risk of CIN vs. HPV type, HPV-16-positive cases were found to have the highest risk of CIN (odds ratio [OR] 3.2; 95% confidence interval [CI] 1.3-7.9; p = 0.002). CONCLUSION: Multiple HPV infections are common in women with and without CIN, but significantly more prevalent among women with CIN compared to controls.

Effects of Bifidobacterium lactis Bb12 supplementation on intestinal microbiota of preterm infants: a double-blind, placebo-controlled, randomized study.

The gastrointestinal microbiota of preterm infants in a neonatal intensive care unit differs from that of term infants. In particular, the colonization of preterm infants by bifidobacteria is delayed. A double-blind, placebo-controlled, randomized clinical study was performed on 69 preterm infants to investigate the role of Bifidobacterium lactis Bb12 supplementation in modifying the gut microbiota. Both culture-dependent and culture-independent approaches were used to study the gut microbiota. Bifidobacterial numbers, determined by fluorescence in situ hybridization, were significantly higher in the probiotic than in the placebo group (log(10) values per g of fecal wet weight: probiotic, 8.18 + 0.54 [standard error of the mean]; placebo, 4.82 + 0.51; P < 0.001). A similar trend for bifidobacterial numbers was also obtained with the culture-dependent method. The infants supplemented with Bb12 also had lower viable counts of Enterobacteriaceae (log(10) values of CFU per g of fecal wet weight: probiotic, 7.80 + 0.34; placebo, 9.03 + 0.35; P = 0.015) and Clostridium spp. (probiotic, 4.89 + 0.30; placebo, 5.99 + 0.32; P = 0.014) than the infants in the placebo group. Supplementation of B. lactis Bb12 did not reduce the colonization by antibiotic-resistant organisms in the study population. However, the probiotic supplementation increased the cell counts of bifidobacteria and reduced the cell counts of enterobacteria and clostridia.

Evidence for an association between mannose-binding lectin 2 (MBL2) gene polymorphisms and pre-term birth.

PURPOSE: Human mannose-binding lectin, encoded by the MBL2 gene, is an important component of innate immunity and an important regulator of inflammatory processes. MBL2 gene polymorphisms are associated with an increased risk of neonatal infections and some data suggest a relation between the maternal MBL2 genotype and the risk of premature delivery. In this study, we evaluated whether there is an association between the fetal MBL2 genotype and prematurity. METHODS: A microarray-based on-chip PCR method was used to simultaneously detect five common MBL2 polymorphisms (codon 52, 54, 57; promoter -550, -221) in 204 DNA samples isolated from archival blood cards. MBL2 genotypes of infants born before the 36th week of pregnancy (N = 102) were compared to a control group of infants born at term after the 37th week (N = 102). RESULTS: The frequency of the codon 52 polymorphism was significantly higher in the pre-term group compared to the term group (10.8% versus 4.9%, P = 0.04), while the frequency of the codon 54 polymorphism was equal in both groups (11.3% versus 11.8%). Interestingly, carriers of genotypes (O/O) likely conferring deficient MBL plasma levels were more common in the group of premature birth (9.8% versus 2.9%, P = 0.05), while the promoter -550 C/C genotype was underrepresented in the pre-term birth group (24.5% versus 39.2%, P = 0.03). CONCLUSION: Our data add to the knowledge about genetic predisposition to prematurity and suggest that the fetal MBL2 genotype might be an additional genetic factor contributing to the risk of premature delivery.

Influence of vitamin C and E supplementation on oxidative stress induced by hyperbaric oxygen in healthy men.

AIM: To investigate the effect of a 4-week vitamin C and E supplementation on oxidative stress induced by hyperbaric oxygen (HBO). METHODS: 19 healthy men were exposed to 3 sequential protocols, i.e. HBO (100% O2, 2.4 bar, 131 min) before (T1) and after 4 weeks of daily supplementation with 500 mg slow-release vitamin C and 272 IU vitamin E (T2). A normoatmospheric protocol (21% O2, 1.0 bar, 131 min) served as control treatment (nonexposed). Blood samples were taken before (B) and immediately after (A) treatment. Plasma levels of vitamin A, C, E, beta-carotene, reduced glutathione and malondialdehyde were measured by HPLC. Antioxidative capacity and lipid peroxides in plasma were analyzed by ELISA. RESULTS: HBO decreased vitamin C and antioxidative capacity (T1). At T1, Delta A - B of vitamin C and lipid peroxides was different from nonexposed. Vitamin supplementation increased plasma levels of vitamin C and E by 28 and 37%, respectively. Vitamin supplementation led to decreased concentrations of lipid peroxides and reduced glutathione. After supplementation, HBO decreased vitamin C and reduced glutathione. At T2, Delta A - B of vitamin C and lipid peroxides was significantly different from nonexposed. CONCLUSION: In humans, oxidative stress decreased plasma levels of vitamin C and antioxidative capacity and increased plasma lipid peroxides. Supplementation with vitamin C and E did not prevent these effects.

Microarray-based detection of mannose-binding lectin 2 (MBL2) polymorphisms in a routine clinical setting.

The assessment of allelic variants in the human mannose-binding lectin 2 (MBL2) gene is of great clinical importance in newborns or immune-suppressed patients at high risk for a variety of infections. Here, we present a study on the genotyping accuracy of a DNA microarray-based on-chip PCR method suited for the detection of five different polymorphisms in the MBL2 gene. We tested 153 genomic DNA samples, prepared from archival blood spots on Guthrie cards, for the presence of allelic variants in the human MBL2 gene by the on-chip PCR method and compared the obtained results of three variants to standard DNA capillary sequencing. The genotyping power of the described assay was readily comparable to DNA sequencing (453/459 correct genotype calls in 153 DNA samples; 98.7% accuracy), mainly due to intrinsic technical benefits of microarrays such as high number of test replicates and automated data analysis. This study demonstrates, for the first time, the accuracy and reliability of a microarray-based on-chip PCR genotyping assay for measuring allelic variants in a routine clinical setting.

Dissecting progressive stages of 5-fluorouracil resistance in vitro using RNA expression profiling.

Resistance to anticancer drugs such as the widely used antimetabolite 5-fluorouracil (FU) is one of the most important obstacles to cancer chemotherapy. Using GeneChip arrays, we compared the expression profile of different stages of FU resistance in colon cancer cells after in vitro selection of low-, intermediate- and high-resistance phenotypes. Drug resistance was associated with significant changes in expression of 330 genes, mainly during early or intermediate stage. Functional annotation revealed a majority of genes involved in signal transduction, cell adhesion and cytoskeleton with subsequent alterations in apoptotic response, cell cycle control, drug transport, fluoropyrimidine metabolism and DNA repair. A set of 33 genes distinguished all resistant subclones from sensitive progenitor cells. In the early stage, downregulation of collagens and keratins, together with upregulation of profilin 2 and ICAM-2, suggested cytoskeletal changes and cell adhesion remodeling. Interestingly, 6 members of the S100 calcium-binding protein family were suppressed. Acquisition of the intermediate-resistance phenotype included upregulation of the well-known drug resistance gene ABCC6 (ATP-binding cassette subfamily C member 6). The very small number of genes affected during transition to high resistance included the primary FU target thymidylate synthase. Although limited to an in vitro model, our data suggest that resistance to FU cannot be explained by known mechanisms alone and substantially involves a wide molecular repertoire. This study emphasizes the understanding of resistance as a time-depending process: the cell is particularly challenged at the beginning of this process, while acquisition of the high-resistance phenotype seems to be less demanding.

Estrogen-metabolizing gene polymorphisms in the assessment of breast carcinoma risk and fibroadenoma risk in Caucasian women.

BACKGROUND: Genes encoding enzymes involved in estrogen metabolism are held to be candidate genes for associations with breast disease. In these candidate genes, no critical combination of single-nucleotide polymorphisms (SNPs) for assessing breast carcinoma risk has been reported to date. METHODS: In a large case-control study, the authors investigated 10 estrogen-metabolizing SNPs in 396 patients with breast carcinoma, 154 patients with fibroadenoma, and 1936 healthy control patients without breast carcinoma in their personal history. The following 10 SNPs were analyzed using sequencing-on-chip technology via a solid-phase polymerase chain reaction assay performed on oligonucleotide microarrays: catechol-O-methyltransferase Val158Met G-->A, 17-beta-hydroxysteroid dehydrogenase type 1 vIV A-->C, cytochrome P-450 (CYP) family 17 A2 allele T-->C, CYP1A1-1 MspI restriction fragment length polymorphism (RFLP) T-->C, CYP1A1-2 Ile462Val A-->G, CYP19-1 Trp39Arg T-->C, CYP19-2 Arg264Cys C-->T, CYP19-3 Cys1558Thr C-->T, steroid-5-alpha reductase type 2 Val89Leu G-->C, and vitamin D receptor BsmI RFLP. A total of 21,350 genotypes were evaluated. Associations and two-way interaction models were calculated using stepwise logistic regression. RESULTS: In a multiple model, CYP1A1-1 (P = 0.004) and CYP1A1-2 (P = 0.03) were found to be associated with significantly decreased and increased risks of breast carcinoma, respectively. When two-way interactions involving investigated SNPs were ascertained, no significant interactions among polymorphisms were noted. Comparison of patients with fibroadenoma with control patients revealed significantly increased and decreased risks of fibroadenoma when the mutant alleles of CYP17 (P = 0.02) and CYP1A1-1 (P = 0.04), respectively, were present. CONCLUSIONS: The authors obtained the first SNP data indicating that CYP17 and CYP1A1-1 play a role in the pathogenesis of fibroadenoma. Although the authors were not able to develop interaction models involving SNPs, they did provide evidence that CYP1A1 is a low-penetrance susceptibility gene with respect to breast carcinoma in a large series of Caucasian women.

Microarray-based identification of bacteria in clinical samples by solid-phase PCR amplification of 23S ribosomal DNA sequences.

The rapid identification of the bacteria in clinical samples is important for patient management and antimicrobial therapy. We describe a DNA microarray-based PCR approach for the quick detection and identification of bacteria from cervical swab specimens from mares. This on-chip PCR method combines the amplification of a variable region of bacterial 23S ribosomal DNA and the simultaneous sequence-specific detection on a solid phase. The solid phase contains bacterial species-specific primers covalently bound to a glass support. During the solid-phase amplification reaction the polymerase elongates perfectly matched primers and incorporates biotin-labeled nucleotides. The reaction products are visualized by streptavidin-cyanine 5 staining, followed by fluorescence scanning. This procedure successfully identified from pure cultures 22 bacteria that are common causes of abortion and sterility in mares. Using the on-chip PCR method, we also tested 21 cervical swab specimens from mares for the presence of pathogenic bacteria and compared the results with those of conventional bacteriological culture methods. Our method correctly identified the bacteria in 12 cervical swab samples, 8 of which contained more than one bacterial species. Due to the higher sensitivity of the on-chip PCR, this method identified bacteria in five cervical swab samples which were not detected by the conventional identification procedure. Our results show that this method will have great potential to be incorporated into the routine microbiology laboratory.

Microarrayed allergens for IgE profiling.

Diagnosis of type I allergy is based on anamnesis, provocation testing, and serological determination of total and specific IgE. Currently, in vivo and in vitro diagnostic tests employ allergen extracts prepared from various allergen sources (e.g., pollen, mites, animal dander, moulds, foods, venoms, etc.). The application of recombinant DNA technology to the field of allergen characterization has allowed to reveal the molecular nature of the most common allergens. To date a continuously increasing number of allergen sequences has become available and panels of recombinant allergens-assembling the epitope complexity of natural allergens sources-can be produced. The use of recombinant allergens instead of crude, natural extracts for allergy diagnosis allows us to determine the individual IgE reactivity profile of each patient. To enable a comprehensive analysis of the patient's IgE binding pattern to a large number of individual allergens, a new type of serological test is required. In this paper, we applied microarray technology to create a multi-allergen test system, based on microarrayed recombinant allergens.