Staphylococcus aureus is an important cause of infection, particularly in persons colonized with this organism. This study compared the annual prevalence and microbiological characteristics of methicillin-resistant S. aureus (MRSA) nasal colonization in Taiwanese children from 2004 through 2009. Risk factors for MRSA were determined for the overall study period.Children from birth to ≤14 years of age presenting for health maintenance visits or attending 1 of 57 kindergartens were recruited. Nasal swabs were obtained, and a questionnaire was administered. The prevalence and microbiological characteristics of MRSA colonization were also calculated for two 3-year periods: 2004–2006 and 2007–2009.S. aureus in 824 (25.8%) of the 3,200 children, and MRSA colonization was found in 371 (11.6%) children. The prevalence of S. aureus colonization decreased from 28.1% in 2004–2006 to 23.3% in 2007–2009 (p<0.01), whereas the prevalence of MRSA colonization increased from 8.1% to 15.1% during this period (p<0.0001). Multivariate analysis revealed that the independent risk factors for MRSA carriage were different for male and female children, and also among age groups. Most MRSA isolates belonged to sequence type 59 (ST59) (86.3%); however, a multiresistant MRSA clone with ST338 background emerged in 2007–2009. Ten (62.5%) of the 16 MRSA isolates expressed the genotypic profile ST338/staphylococcal cassette chromosome mec VT/Panton-Valentine leukocidin-positive/staphylococcal enterotoxin B-positive, and differed only in their antimicrobial susceptibility patterns.Cultures of the anterior nares were positive for S. aureus. A multiresistant MRSA clone characterized as ST338 was identified from these children.The prevalence of nasal colonization by MRSA increased among healthy Taiwanese children from 2004–2006 to 2007–2009, despite an overall decrease in the prevalence of nasal colonization by  Staphylococcus aureus is a common human pathogen of skin infections and invasive diseases in all age groups, such as pneumonia, osteomyelitis, and endocarditis, in healthcare and community settings S. aureus (MRSA) isolates have been recognized as a source of healthcare-associated infections since the 1960s mec (SCCmec) type IV T SCCmec elements, were found to be the major strains of CA-MRSA S. aureus colonizes the anterior nares and other body sites, but the anterior nares are the most consistent site of colonization S. aureus, including MRSA, is common in children, and genetic evidence supports a relationship between nasal carriage of S. aureus and MRSA and subsequent invasive staphylococcal infection S. aureus and MRSA to both community and hospital environments S. aureus was conducted in 2005 and 2006, and it showed a measurable prevalence of colonization with MRSA in the community S. aureus, to identify the potential risk factors for MRSA nasal colonization, and to describe the evolving epidemiology of nasal colonization by S. aureus and by MRSA specifically.In Taiwan, the first island-wide prevalence survey of nasal colonization with S. aureus colonization to be 30%. Therefore, we calculated that a sample size of 1100 children would be necessary to estimate risk factors for MRSA colonization, based on estimated prevalence ranging from 1% to 3%, with a 95% confidence interval . Study participants were recruited sequentially until the estimated recruitment number was met.This prospective observational study was conducted from 2004 to 2009 at Tri-Service General Hospital, a 1400-bed tertiary medical center in northern Taiwan. The study proposal was reviewed and approved by the National Defense Medical Center Institutional Review Board. Eligible children were 14 years of age or younger with no acute medical problem, who either presented for a health maintenance visit or attended one of 57 kindergartens in Taipei. Written informed consent was obtained from each child's parents or legal representative before nasal specimen collection or interviews. During the 6-year study period, all children who presented for health maintenance visits to our hospital were invited to participate in this study. In addition, the selection of the kindergartens from all kindergartens in Taipei was based on support for the surveillance study by the kindergartens' principals. The number of children sampled per kindergarten was proportional to the number of children attending each kindergarten. Based on a previous study S. aureus colonization status were obtained by interviews with the guardians immediately after consent was obtained and before swabs were collected Individual variables, demographic characteristics of the family, and medical history over the preceding 12 months, including previous hospitalization, medication history prior to receiving the screening test, and any underlying diseases correlated with 2. Cultures on BAPs were screened using Slidex Staph Plus . MRSA identification and antimicrobial susceptibility testing were performed according to Clinical Laboratory Standards Institute  guidelines S. aureus isolates were defined as isolates resistant to three or more antimicrobial classes. All S. aureus isolates were frozen at –70°C for additional testing of organism characteristics.Nasal samples were obtained with a sterile cotton swab, placed in transport medium , and then transported to and processed in the microbiology laboratory within 4 hours. Cotton swabs were plated on mannitol salt agar . Each distinctive morphotype of mannitol-fermenting colony was selected from an MSA plate, subcultured to a trypticase soy agar plus 5% sheep blood agar plate , and incubated at 37°C in a humidified incubator with 5% COlukS-PV and lukF-PV genes encoding PVL components was determined by a polymerase chain reaction (PCR)-based method with the primer pair and thermocycler conditions reported by Lina et al ermA, ermB, ermC and msrA) was determined by PCR according to a previously described method sea to see, seg to sei, sek, seq, eta, etb, and tst, encoding staphylococcal enterotoxins , exfoliative toxins (ETA and ETB), and toxic shock syndrome toxin-1, respectively, were detected using the methods described by Jarraud et al. and Diep et al mec typing was performed using a multiplex PCR strategy with sets of region-specific primers as previously described mec VT was performed with the primer and thermocycler conditions reported by Boyle-Vavra et al. and Huang et al Chromosomal DNA from three to five isolated colonies was prepared using the Puregene DNA purification kit  as recommended by the manufacturer, with lysostaphin at 2 mg/ml and RNase at 4 mg/ml for the lysis step. The presence of the Pulsed-field gel electrophoresis (PFGE) was performed using the CHEF Mapper XA system  according to a published protocol S. aureus and children with MRSA to those with methicillin-susceptible S. aureus (MSSA). All parameters were initially determined by means of univariate analysis; those with p values of <0.05 and those being biologically meaningful were entered into the multivariate analysis. However, parameters with colinearity, tested by correlation matrices, were not simultaneously considered in the final model. In the multivariate analysis, stepwise model comparison was used to determine the best model. Risk factors were assessed using SAS 9.1.3 . All tests were two-tailed, and a p value of <0.05 was defined as statistically significant.Data were entered into Microsoft Access XP software and exported into SPSS statistical software, version 10.0 (SPSS), which was used for data analyses. The categorical variables were compared using the chi-square test, Mantel-Haenszel test or Fisher's exact test. The trend in annual prevalence rates of colonization was examined using Poisson regression analysis. The prevalence and microbiological characteristics of colonization were also determined for two time periods (2004–2006 and 2007–2009) separately, to compare categorical variables between the two survey cycles. In addition, data from 2004 through 2009 were combined to provide more reliable estimates of the microbiological characteristics of and risk factors for MRSA colonization and to increase the study's power to detect statistically significant differences. To analyze the risk factors for carrying MRSA among male and female children in various age groups , we used polytomous logistic regression to compare children with MRSA to those without A total of 3200 children with ages ranging from 1 month to 14 years participated in this study: 1019 were recruited during health maintenance visits and 2181 were recruited from the 57 selected kindergartens. The median age of the participating children was 4.3 years, and 1602 (50.0%) were male.S. aureus. The percentage of MRSA isolates among all S. aureus isolates was 45.0%. From 2004–2006, a total of 1615 children were interviewed and examined; of these, 454 (28.1%) were culture-positive for S. aureus. Similarly, from 2007–2009, a total of 1585 children were interviewed and examined, and 370 (23.3%) of them were culture positive for S. aureus. The annual prevalence rates of MRSA and S. aureus nasal colonization are shown in the p<0.0001, test for trend), whereas the prevalence of S. aureus colonization decreased . Moreover, the prevalence of S. aureus nasal colonization decreased to 23.3% during 2007–2009  from 28.1% during 2004–2006  (p<0.0001).Of the 3200 nares specimens, 371 (11.6%) were positive for MRSA, 453 (14.2%) were positive for MSSA, and 2376 (74.2%) were culture negative for %–30.3%) . HoweverS. aureus and MRSA among children tested from 2004–2006 and 2007–2009. When prevalence was compared across surveys, a significant decrease in S. aureus nasal colonization occurred in children from kindergartens (p<0.01), in female children (p<0.01), and among children aged <1 year and >5 years . In addition, the prevalence of MRSA colonization increased in 2007–2009 among both male and female children , children attending both health maintenance visits and kindergartens , and children aged 1–5 years and >5 years .S. aureus. The risk factors for MRSA colonization identified in the multivariate analysis are shown in p<0.05, aged <1 year; p<0.01, aged 1–5 years; p<0.0001, aged >5 years), atopic dermatitis , and history of skin and soft tissue infection . For male children aged <1 year only, chronic disease was associated with MRSA colonization (p<0.05). For male children aged 1–5 years, MRSA colonization was also associated with hospitalization in past 12 months (p<0.0001). For male children aged >5 years, household contact with hospital staff was associated with MRSA colonization (p<0.001).We used polytomous logistic regression to identify risk factors for MRSA colonization by comparing children with MRSA to those with MSSA and children with MRSA to those without carriage of p<0.01). For female children aged 1–5 years, MRSA colonization was significantly associated with atopic dermatitis (p<0.0001), hospitalization in past 12 months (p<0.0001), history of skin and soft tissue infection (p<0.0001), and household contact with hospital staff (p<0.01). Among female children aged >5 years, antibiotic use in the past 12 months (p<0.0001), atopic dermatitis (p<0.01), and history of skin and soft tissue infection (p<0.001) were independently associated with MRSA colonization.The distribution of independent risk factors for MRSA carriage among female children in three different age groups was divergent. Among female children aged <1 year, household contact with hospital staff was associated with MRSA colonization . Among the 371 children colonized with MRSA, the largest proportion, 86.3% , carried ST59 clonal type. ST338 clonal type, a single-locus variant of ST59 (1 nucleotide difference in the gmk locus), was the next most prevalent MLST type and was recovered from 4.3%  of children with MRSA colonization. Children aged >5 years were more likely to be colonized with ST59 clonal type than those aged <1 year and 1–5 years (p<0.05). In addition, colonization with ST338 clonal type was significantly more prevalent in 2007–2009 compared with 2004–2006 (p<0.05), but this difference was not evident in ST59 clonal type.The prevalence and distribution of isolates associated with community transmission among MRSA colonized children are shown in p<0.05), the resistance pattern, distribution of erm genotype and exotoxin profile of MRSA colonizing isolates did not vary significantly by source or survey period (p<0.0001). Moreover, the combined results of molecular analyses showed that the majority (62.5%) of the 16 MRSA ST338 isolates expressed the genotypic profile ST338/SCCmec VT/PVL-positive/SEB-positive.Except for an increase in the multidrug resistance rate in 2007–2009  during 2007–2009 Between 2004–2006 and 2007–2009, the overall prevalence of Few studies have reported the determinants of MRSA colonization in community settings S. aureus was unexpected. These findings might be explained, in part, by the previously reported excessive use of antibiotics in Taiwan It is well recognized that day care centers are efficient settings for the acquisition and transmission of pathogens and several determinants have been suggested, such as crowded conditions, frequent close physical contact, breakdown in appropriate hygiene and intensive exposure to antimicrobials mec IV/VT was not significantly different between 2007–2009  and 2004–2006 . Conversely, ST338 emerged in 2005 and accounted for 6.3% of MRSA colonizing isolates from 2007 to 2009 (p<0.05). More recently, PVL-positive CA-MRSA ST338 has been shown to be correlated with an increasing proportion of S. aureus infections among otherwise healthy children in the southern region of China and the majority of these clones belong to SCCmec V mec VT, which contains a ccrC recombinase gene variant (ccrC2) and mec complex C2 mec V isolates, which may indicate that the background of ST338 clones in Taiwan is different from mainland China. In antibiotic susceptibility testing, MRSA colonizing isolates expressed high incidences of resistance to non-β-lactam antibiotics and multiple drugs. In a previous longitudinal study of 257 MRSA bloodstream infections, Chen et al. reported that multiresistance may have provided an advantage for the CA-MRSA strains to easily enter and rapidly proliferate in healthcare facilities, with resulting blurring of the distinction between CA-MRSA and healthcare-associated MRSA Previous studies throughout Taiwan found that ST59 MRSA isolates were the most common MLST type of MRSA causing CA-MRSA infections and nasal colonization Several study limitations merit consideration. First, the survey design was cross-sectional and children who were only intermittently colonized may not have been detected. Thus, the frequency of colonization with organisms might have been underestimated. Second, recall bias may have occurred when parents or guardians attempted to remember past events and exposures assessed by the questionnaire. In addition, the relatively small number of children less than 1 year of age with MRSA colonization might have limited the ability to detect all risk factors. Third, the persistence of MRSA colonization in the study subjects could not be determined and the incidence of subsequent MRSA infection could not be measured. Finally, this study was conducted at a single site and may not reflect colonization rates throughout the country.S. aureus colonization during the same period. Our investigation provides evidence of the constantly changing epidemiology of staphylococcal colonization in our community. Moreover, the detection of 16 isolates belonging to ST338, a multiresistant clone frequently associated with PVL genes, is of special concern in a young population as it has the potential to sharply limit therapeutic options should it become widespread.In conclusion, this sequential, prospective observational study found the prevalence of MRSA nasal colonization increased in Taiwanese children from 2004–2006 to 2007–2009, despite a decrease in the overall prevalence of


In conclusion, NGF signaling deregulation might play an important role in the development of DPN. EA represents a supportive tool to control DPN development by modulating NGF signaling in diabetes-targeted neurons. Diabetic polyneuropathy (DPN), characterized by early hyperalgesia and increased nerve growth factor (NGF), evolves in late irreversible neuropathic symptoms with reduced NGF support to sensory neurons. Electroacupuncture (EA) modulates NGF in the peripheral nervous system, being effective for the treatment of DPN symptoms. We hypothesize that NGF plays an important pathogenic role in DPN development, while EA could be useful in the therapy of DPN by modulating NGF expression/activity. Diabetes was induced in rats by streptozotocin (STZ) injection. One week after STZ, EA was started and continued for three weeks. NGF system and hyperalgesia-related mediators were analyzed in the dorsal root ganglia (DRG) and in their spinal cord and skin innervation territories. Our results show that four weeks long diabetes increased NGF and NGF receptors and deregulated intracellular signaling mediators of DRG neurons hypersensitization; EA in diabetic rats decreased NGF and NGF receptors, normalized c-Jun N-terminal and p38 kinases activation, decreased transient receptor potential vanilloid-1 ion channel, and possibly activated the nuclear factor kappa-light-chain-enhancer of activated B cells (Nf- Sensory polyneuropathy is a major complication of type 1 diabetes, still lacking adequate treatment –5 associNTR) were found deregulated in experimental diabetes [Nerve growth factor (NGF) is essential for the development and functional maintenance of dorsal root ganglion (DRG) sensory neurons, which are primarily targeted by diabetes , and hasdiabetes , 9. NGF diabetes , 11. Clidiabetes , evidencdiabetes . Thus, tdiabetes , 13, witTraditional Chinese acupuncture and its western derivate electroacupuncture (EA) have been proven effective in the therapy of neuropathic pain , 15, andHere, we investigated the possible regulation of the NGF system in primary sensory neurons and in their innervation targets, in the early phase of experimental diabetes after EA treatment. We also studied the variation of NGF intracellular mediators since their reported involvement in the development of neuropathic symptoms in the STZ diabetes model , 9, 18 aAdult female Sprague-Dawley rats  weighing 200–220 g were housed under constant environmental conditions. Animals care and experiments were conducted in conformity with Italian and international laws and policies . The experimental protocol (no. 10/2011) was approved by the Italian Ministry of Health. All surgeries were performed under chloral hydrate anesthesia, and all efforts were made to reduce the number of animals used and to minimize their sufferance. mg/kg streptozotocin  dissolved in citrate buffer pH 4.5 to induce type 1 diabetes [n = 12) were injected once i.p. with 20 mM citrate, pH 4.5; STZ-injected  STZ-injected, and electroacupuncture-treated rats  were treated twice a week with low-frequency EA for 3 consecutive weeks starting 1 week after diabetes induction. Control of the EA treatment trough minimal or superficial sensory stimulation  in diabetic rats was omitted, since it has been demonstrated that it does not represent an inert intervention, often arising significant responses based on the same anatomic and physiological substrates activated by acupuncture itself [Rats were treated with a single i.p. injection of 65diabetes . One weee itself . The tree itself .Four weeks after STZ and one day after the last EA session, 10 rats in each group were killed by decapitation, serum, hindpaw skin, lumbar (L3–L5) DRG, and lumbar spinal cord, collected and quickly frozen and stored at −80°C until processed for assays. Two rats for each group were anesthetized with 400 mg/kg of chloral hydrate and transcardially perfused with 4% paraformaldehyde in PBS. Samples of the glabrous hindpaw skin  and of the lumbar spinal cord tissues were removed and postfixed for 24 hours. Then, tissue samples were transferred in 30% sucrose/PBS solution and sectioned at a sliding freezing microtome . Forty-micrometer-thick sections were collected in 0.05% sodium azide/PBS and stored at 4°C until used.μsec, a length of 0.1 sec, and internal burst frequency of 80 Hz. The intensity (1.0–1.5 mA) was monitored by checking for local muscle contractions to reflect the activation of muscle-nerve afferents. Control and STZ rats were manipulated and sedated in a manner similar to the STZ + EA rats.Rats from the EA group were exposed to 30 minutes of EA stimulation twice a week for 3 weeks . During Glucose content in plasma derived from blood collected when animals were sacrificed was checked by a commercially available blood glucose analyzer . Insulin levels were assessed by Insulin Rat ELISA test (S-1238) from Bachem AG , following manufacturer's instructions.n = 10 for each experimental group) was measured by commercial ELISA , following manufacturer's instructions.DRGs were homogenized as previously described , and proNTR, ERK1-2, Akt, JNK, p38, phospho-IκB-α, phospho-NF-κB-p65, TRPV-1, and GAPDH protein tissue contents  were diluted with loading buffer [http://rsb.info.nih.gov/ij/) was used for gel densitometry and protein quantification following the method described in [n = 9 for each experimental group).DRGs extracts were also used for Western blots of NGF receptors. TrkA and p75ribed in . The GAPμg total RNA using the GoScript Reverse Transcription System  following manufacturer's instructions.Total RNA was extracted from DRG, paw skin, and spinal cord samples by SV Total RNA Isolation Kit , following manufacturer's instructions. DNase I (included in the kit) digestion was performed to eliminate DNA contamination. RNA concentration was determined by NanoDrop ND-1000 . First-strand cDNA was synthesized from 0.5 NTR in the DRGs were analyzed by real-time PCR using inventoried TaqMan assays from Applied Biosystems . The assays codes were Rn01533872_m1 (NGF), Rn00561634_m1 (p75NTR), and RN00572130_m1 (TrkA). GAPDH  was used as an endogenous control to allow for relative gene expression quantification. Thermal cycling and fluorescence detection were performed with an ABI Prism 7900HT Sequence Detection System with SDS Software 2.1 (Applied Biosystems). Thermal cycling conditions were 2 min at 50°C and 10 min at 94.5°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. Relative gene expression was calculated using the 2−ΔΔCt method [Gene expression of NGF in the spinal cord and hindpaw skin and of TrkA and p75t method .NTR distribution in the hind paw skin and spinal cord were analyzed by immunofluorescence. Forty-micrometer-thick tissue sections were preincubated with 10% of donkey serum in PBS containing 0.1% Triton X-100 (PBST) for 2 hours and then incubated overnight at 4°C with primary antibodies against TrkA (1 : 100) and p75NTR (1 : 200) listed in TrkA and p75NTR and TrkA double immunofluorescence was performed by Imaris 7.4 (Bitplane A.G.) software on five different images derived from each experimental group. For the skin, a mask was manually drawn on the basal cell layer and for the spinal cord on the laminae I to III. All the parameters were selectively evaluated only on these histological structure. Morphological parameters under analysis were tissue intensity, vesicles/terminals intensity, vesicles/terminals number, diameter, and colocalization which were evaluated by using surface, spots, and Coloc Imaris modules. To determine fluorescent signal colocalization between different channels, a median filter was applied to reduce the background noise, and the degree of overlap was characterized by Manders' and Pearson's coefficient.Image analysis of p75n = 9 for each experimental group), ELISA and real-time PCR , and image analysis  data were then evaluated by one-way ANOVA and data expressed as mean ± SD. Post hoc comparisons within different experimental groups were performed using Tukey's HSD test. A P value less than 0.05 was considered significant. Data were analyzed by the GraphPad 5 software . Western blot (P < 0.05) that was concomitant with a significant decrease of plasma insulin . EA induced a slight, nonsignificant improvement in plasma glucose  and did not affect plasma insulin .STZ treatment induced a significant increase of plasma glucose,  and spinal cord . EA did not correct such reductions, suggesting a possible control of the EA on the NGF protein production or uptake/transport rather than on the NGF gene expression.STZ caused a significant +33% increase of NGF protein content in the DRGs , that waP < 0.05). Western blots densitometry , while EA did not exert significant effects on TrkA protein content . The pTyr496-TrkA, the activated form of TrkA, was significantly increased by diabetes , indicating an augmented NGF signaling. EA induced a significant reduction of pTyr496-TrkA in diabetic animals , indicating an inhibitory effect of EA upon TrkA activation.TrkA mRNA in DRGs  showed aitometry  revealedNTR receptor, known to be activated by NGF in neuropathic conditions [NTR  above controls by STZ, while EA decreased p75NTR content in the DRGs well below control level , indicating a parallel effect of STZ and EA on the p75NTR and its ligand NGF  revealed a selective increase of p75NTR immunoreactivity in the epidermal basal cell layer after STZ which was partially reverted by EA , as vesicles intensity (+401%), and as the number of immunopositive vesicles (+206%) in the cellular cytoplasm  only the terminals fluorescence intensity  compared to the controls , suggesting a diabetes-induced repression in the activity of the Nf-κB. EA in diabetic animals induced an increased phosphorylation of the complex Nf-κB-p65  and a concomitant increase in the phosphorylation of the IκB-α , suggesting an EA-induced inactivation of the IκB-α with a parallel increase in the activity of the Nf-κB.In addition, we analyzed the activation of the transcription factor Nf-hic pain , 28. Thehe Nf-κB . STZ indP < 0.05) in DRGs (P < 0.05) and a more substantial increase of phosphorylated p38 , the p38 , and the phospho-p38 , suggesting a normalizing action of the therapy on the p38 activation and in the expression of the pain-mediator TRPV1.NGF-induced hyperalgesia could be concomitant to the activation of the MAPK p38 that in turn provokes the overexpression of the ion channel TRPV1 in DRG neurons . We inve in DRGs  that wasNTR, and by increased TrkA phosphorylation. These diabetes-induced NGF modifications are associated with the activation of JNK and p38, with the repression of the transcription factor Nf-κB and with the upregulation of the ion channel TRPV1. Of particular interest, our data demonstrate that three weeks treatment with low-frequency EA counteracts deregulations of both NGF and NGF receptors, normalizing STZ-induced JNK and p38 activation, decreasing TRPV1, and possibly activating the Nf-κB transcription factor (Our data demonstrate that in the primary sensory neuron circuitry: (1) early experimental diabetes is characterized by molecular modifications of the NGF signaling system; (2) EA treatment is able to revert the majority of such modifications . The earn factor .NTR presence, increased MAPKs activity, the possible repression of Nf-κB, and upregulation of the TRPV1. As a limit of our study, it should be stated that these data may represent a mere correlative picture of the NGF system activation after STZ. Indeed, further studies are necessary to clarify: (i) if the effects of STZ and EA on MAPKs and TRPV1 are only mediated by NGF signaling modulation or if other signaling systems are involved; (ii) if neurobehavioural features other than thermal hyperalgesia known to be altered in late diabetic neuropathy and affected by NGF—that is, decreased motor and sensory nerve conduction velocity [κB have been linked to p75NTR-mediated hyperalgesia [2+ overload mediated by TRPV1 [NTR activation, the possible consequent JNK activation, and Nf-κB repression could play a major role since its well-known role in neurodegenerative diseases [As suggested by our previous  and presvelocity  or increvelocity —would haralgesia , and theby TRPV1 , 30. Actby TRPV1 . The emediseases . Indeed,NTR upregulation following STZ indicates that NGF signaling was shifted from a TrkA/p75NTR physiological state to a p75NTR/TRkA “prevalent” state. p75NTR is widely thought to mediate cell death and degenerative phenomena when its activation is concomitant to a high p75NTR/TrkA ratio [Data from confocal microscopy give clues about the dynamics of NGF signaling in primary sensory circuitry during the development of experimental DPN. In the skin, the selective p75kA ratio . Thus, okA ratio , 39 as akA ratio , 41. In kA ratio .κB activity [EA could revert neuropathic symptoms in the early diabetes , and itsactivity . Our resactivity , 46–49, activity  and a poactivity , 30.NTR upregulation in the skin, suggesting a selective contrast to NGF deregulation. Possible mechanisms that remain to be specifically addressed in this latter EA action encompass EA-regulated antidromic modulation of sensory activity [The anatomical location for EA-induced NGF modulation may be the spinal cord. We previously demonstrated that early STZ-induced thermal hyperalgesia could be reverted by EA and that this effect was parallel with a decrease in NGF and TRPV1 content and TrkA activation, all found in the spinal cord rather than in the paw skin . Our preactivity  and/or sactivity  to the sactivity . Of noteThe previous literature, both on animal models and in humans, shows that late DPN is characterized by a defective NGF support to sensory neurons, due to reduced NGF production and/or transport from target tissues . SupportIn conclusion, NGF-triggered DRG neuron hyperactivity could be of primary importance in the establishment of DPN and in the early diabetes-associated neuronal distress. Physical therapies based on sensory stimulation, such as EA, could be valid supportive tools in controlling DPN development and progression and hopefully for the neuro-protection of diabetes-targeted DRG neurons.NTR double immunofluorescence in the epidermis/dermis in STZ, STZ+EA and Control groups.Supplemental Figure 1: Low magnification confocal images of TrkA and p75NTR was distributed to the both epidermal and dermal regions. However, many of the sensory, glandular and muscular structures present in the dermis expressed a high intensity p75NTR immunofluorescence and a medium intensity TrkA immunofluorescence. TrkA in the epidermis was densely distributed in the granular-squamous and basal epidermal layers as a medium tissue background decorated by small vesicles endowed of medium to high intensity fluorescence. On this tissue background, and selectively confined to the proliferating basal cell layer, small vesicles of high intensity fluorescence densely filled the cellular cytoplasm giving more evidence to the basal cell layer. However, very often, in this latter layer, rows of cells which did not show the intense TrkA immunofluorescence were alternated to rows of cells showing the intense TrkA immunofluorescence. p75NTR immunoreactivity was present as low-medium intensity tissue background in the epidermal region with the exception of few positive vesicles located in the basal cell layer. Of interest, in the dermal papillae and in juxtaposition to the epidermal basal cell layer, several sensorial and glandular structures often showed an intense p75NTR immunoreactivity. In all three groups, TrkA immunoreactivity appeared mainly confined to the epidermis, while p75Click here for additional data file.


Ulmus pumila (U. pumila) was extracted with ethanol, and then the antimicrobial effects were tested on clinically isolated 12 MRSA strains and 1 standard MRSA strain. U. pumila showed antibacterial activities against all MRSA strains. Minimum inhibitory concentration (MIC) of U. pumila root bark against all MRSA strains revealed a range from 125 to 250 μg/mL. These results may provide the scientific basis on which U. pumila root bark has traditionally been used against infectious diseases in Korea. In real-time PCR analysis, the sub-MIC (64–125 μg/mL) concentrations of U. pumila root bark extract showed the inhibition of the genetic expressions of virulence factors such as mecA, sea, agrA, and sarA in standard MRSA. Phytochemical analyses of U. pumila root bark showed relatively strong presence of phenolics, steroids, and terpenoids. These results suggest that the ethanol extract of U. pumila root bark may have antibacterial activity against MRSA, which may be related to the phytochemicals such as phenolics, steroids, and terpenoids. Further studies are needed to determine the active constituents of U. pumila root bark responsible for such biomolecular activities.In this study, root bark of  Staphylococcus aureus (S. aureus) is one of the most common bacteria in humans. S. aureus is normally present in the skin, nasal cavity, or laryngopharynx of healthy men and opportunistically causes a local or systemic infection [S. aureus is a causative bacteria of nosocomial infection and occupies more than 80% of pyogenic infection such as abscess and septicemia [nfection , 2. Howepticemia .Staphylococcus aureus (MRSA) [β-lactams, or aminoglycosides. Treatment of patients infected with these bacterial strains is known to be very difficult [β-lactamase, which inactivate the β-lactam antibiotics and (2) the possession of mecA gene that produces penicillin-binding proteins, such as PBP, PBP2′, or PBP2a, which have low affinity to β-lactam antibiotics [Penicillin was developed in 1941 and has been used as a therapeutic agent indicated for bacterial infections. Since then, numerous antibiotic agents have been developed and are effective against bacterial infections, but the appearance of antibiotic-resistant bacterial strains caused a big problem in the treatment of patients , 7. The s (MRSA) . These bifficult , so MRSAibiotics . We haveibiotics –14.Ulmus pumila (U. pumila) is a natural herb that has traditionally been used for the treatment of infections in Korea. U. pumila, belonging to the botanical classification of Ulmaceae, is distributed in Korea, Japan, northern China, Sakhalin, and East Siberia. This tree grows to 15 meters and its bark is dark brown in color. Owing to its antibacterial and anti-inflammatory reaction, U. pumila has been traditionally used for abscess, infection, edema, rhinitis, empyema, and otitis media. It has also been used for gastric and duodenal ulcers as well as gastric cancer [c cancer . U. pumila was extracted with ethanol, and then the antimicrobial effects of U. pumila ethanol extract were tested on clinically isolated 12 MRSA strains and 1 standard MRSA strain, and phytochemical analysis was performed.In this study, U. pumila was obtained from the oriental drug store, Dae Hak Yak Kuk . The identity was confirmed by Dr. Bong-Seop Kil at the Department of Natural Science, Wonkwang University.The bark of U. pumila (100 g) was chopped into small pieces and was extracted 2 times with 1000 mL of ethanol for 72 h at room temperature. The filtration of the extracted solution and evaporation under reduced pressure yielded ethanol extracts (9.3 g). After the extract was thoroughly dried for complete removal of solvent, the dry extract was then stored in a deep freezer (−70°C).Voucher specimen (number 09-03-26) has been deposited at the Herbarium of Department of Oral Biochemistry in Wonkwang University. Dried bark of mecA gene and their resistance to oxacillin [β-Lactamase activity was also determined using the DrySlide Beta Lactamase test  according to manufacturer's specification. After culturing on Mueller-Hinton agar (Difco Laboratories), the bacteria were suspended in Mueller-Hinton broth (Difco Laboratories) and used for inoculation. All MRSA strains used in this study are identified as MRSA [Staphylococcal strains listed in xacillin . β-Lacta as MRSA .μL of different amounts  of the extracts dissolved in dimethyl sulfoxide (DMSO) and were left to dry for 12 h at 37°C in a sterile room. Bacterial suspensions were diluted to match the 0.5 MacFarland standard scale (approximately 1.5 × 108 CFU/mL) and they were further diluted to obtain a final inoculum. After Mueller-Hinton agar was poured into Petri dishes to give a solid plate and inoculated with 100 μL of suspension containing 1 × 108 CFU/mL of bacteria, the discs treated with extracts were applied to Petri dishes. Ampicillin and oxacillin were used as positive controls and paper discs treated with DMSO were used as negative controls. The plates were then incubated at 35°C for 24 h in a incubator . Inhibition zone diameters around each disc were measured and recorded at the end of the incubation time.As the first screening, the paper disc diffusion method was used to determine antibacterial activity, which is based on the method described previously , 19. Ste4 CFU/mL was spotted with a multipoint inoculator  onto agar plates. The plates were then incubated at 35°C for 24 h in the incubator . The MIC was defined as the lowest concentration of extracts at which no visible growth was observed. The minimum concentration of extracts that inhibited 50% and 90% of the isolates tested was defined as MIC50 and MIC90, respectively.MICs were determined by the agar dilution method, which is based on the method described previously , 20. MICU. pumila extract on gene expression, a real-time PCR assay was performed. The sub-MIC (32–125 μg/mL) of U. pumila extract was used to treat and culture MRSA ATCC 33591 for 24 h. Total RNA was isolated from MRSA by using Trizol reagent (Gibco-BRL) according to the manufacturer's instructions and was treated with DNase to digest contaminated DNA. Then, cDNA was synthesized using a reverse transcriptase reaction . The DNA amplifications were carried out using an ABI-Prism 7000 Sequence Detection System with Absolute QPCR SYBR Green Mixes . The primer pairs that were used in this study were described by previous reports [To determine the effect of  reports –23 and aPhytochemical tests of extracts were performed as previously described , 25. Mayt-test.All experiments were carried out in triplicate. Data were analyzed using the statistical package for social sciences (SPSS). Differences between means of the experimental and control groups were evaluated by the Student's U. pumila on clinically isolated MRSA strain 12 and standard MRSA strain 1 (ATCC 33591) was examined. As a result of measuring antibacterial activity of U. pumila using the disc diffusion method, U. pumila showed antibacterial activities against all strains  concentrations of U. pumila extract on the genetic expressions of virulence factors in standard MRSA (ATCC 33591). The expressions of mecA, sea, agrA, and sarA were significantly decreased in MRSA when it was treated with the sub-MIC (63–125 μg/mL) concentrations of U. pumila extract  concentrations of U. pumila extract on the genetic expression of mecA was determined by real-time PCR analysis. The expression of mecA was significantly decreased in standard MRSA (ATCC 33591) when it was treated with concentration higher than 64 μg/mL. Additional investigation is necessary to determine whether U. pumila may have multidrug resistance inhibitors. Several mechanisms by which microorganisms can overcome antimicrobial agents are known. These mechanisms include production of drug insensitive enzymes, modification of targets for drug, and multidrug resistance pump which discharge the antimicrobial agents entered in bacterial cells. ce genes . Recent ce genes . In the sea, encodes Staphylococcal enterotoxin A which is one of major virulence factors in MRSA [U. pumila extract significantly inhibited sea expression. sea gene expression in MRSA is regulated by global regulators such as agr and sarA genes [μg/mL) concentrations of U. pumila extract showed the inhibition of agrA and sarA expressions in MRSA. agrA encodes accessory gene regulator A which positively regulates exotoxin-encoding genes. sarA also upregulates expression of virulence factor genes. Previous research has shown that inhibition of agrA or sarA expression by some chemicals such as thymol or clindamycin reduces transcription of exotoxin-encoding genes [U. pumila extract on sea gene expression may, in part, be related with the inhibitory effect of U. pumila extract on agrA and sarA expressions [A virulence factor gene,  in MRSA . StaphylrA genes . In our ng genes . In the ressions .U. pumila has antibacterial effects against MRSA, which may be related to the phytochemicals such as phenolics, steroids, or terpenoids which are highly present in U. pumila.In conclusion,


Background. Serum levels of the mannose-binding lectin (MBL), which is an activator of the complement system, have been considered as a pathogenic factor in a broad range of diseases, and means of modulating MBL are therefore being evaluated. In this study we examine the effects of weight loss on MBL levels, and in continuation of this if MBL is synthesized in human adipose tissue. Methods. 36 nondiabetic obese subjects received a very low-calorie diet (VLCD) of 800 kcal/day for 8 weeks. Blood samples were collected at baseline and after VLCD. Furthermore, we measured MBL mRNA levels by the real-time RT-PCR on human adipose tissue compared to liver tissue. Results. The mean body weight was reduced from 106.3 ± 2.6 kg to 92.8 ± 2.4 kg, P < 0.0001. Median MBL at baseline was 746 μg/L (IQR 316–1190) versus 892 μg/L (IQR 336–1511) after 8 weeks, P = 0.23. No correlations were found between weight loss and changes in MBL . MBL real-time RT-PCR showed no expression of mRNA in adipose tissue, but as expected a good expression in liver tissue was seen. Conclusions. MBL levels are not affected by weight loss and MBL is not synthesized in human adipose tissue. Mannose-binding lectin (MBL) is a serum protein known to be synthesized by hepatocytes. MBL exerts an important role in the innate immune system, where it upon binding to carbohydrate patterns of microorganisms activates the lectin pathway of the complement system through MBL-associated serine protease 1, 2, and 3 [MBL2, the interindividually expressed serum level of MBL ranges from a few μg/L to several thousand μg/L. In healthy subjects approximately 56% have a high MBL encoding genotype (homozygote wild type), 40% have an intermediate MBL encoding genotype , and 4% have a low MBL encoding genotype , the latter being functionally deficient in MBL [Serum MBL level is dependent on genotype and, because of commonly occurring polymorphisms in the MBL encoding gene Several clinical studies have shown low MBL levels to be associated with increased susceptibility or to poor outcome of infections. This lack of defense against invading microorganisms may be important especially in certain situations, for example, in infants with an immature immune system –7 and inUnder disease circumstances other than an infection, activation of the complement system can be adverse and low MBL level may here confer an advantage. Keller et al. found that healthy men with low MBL levels are protected from cardiovascular disease .Normally, MBL does not bind to the body's own cells, but may react with altered self-structures, for example, cells exposed to ischaemia/reperfusion injury , 12 or tIn a prospective study with 15 years of followup, our group documented an increased mortality among type 2 diabetic patients with high MBL levels compared with those with low MBL levels . A plausBecause of MBL's possible pathogenic involvement in a broad range of diseases it might comprise a potential target of treatment. Studies of direct inhibition of human MBL in vitro  the rat MBL level is, as mentioned above, mostly dependent on genotype and though it is known to be affected by hormonal stimuli  and is aSo far the effects of weight loss and changes in insulin resistance on MBL levels have only been poorly investigated and with conflicting results. Regarding weight loss induced by bariatric surgery in obese subjects, both increased levels  and unchWe investigated the effect of weight loss and change in insulin resistance on MBL levels in an intervention study including 36 healthy and nondiabetic obese subjects (18 women and 18 men). The subjects received a very low-calorie diet (VLCD) of ~800 kcal/day for 8 weeks. Anthropometric data was obtained at baseline and after the 8 weeks of VLCD. Body composition (fat mass) and the percentage of body fat (fat mass/weight × 100%) were estimated by bioelectrical impedance using a multifrequency bioimpedance spectroscopy analyzer  as described by Heitmann .We obtained fasting blood samples from the antecubital vein at baseline and after the 8 weeks of VLCD. Serum was separated and frozen at −80°C until the time of the analysis.HOMA-IR was calculated as the product of the fasting plasma insulin level (microU/mL) and the fasting plasma glucose level (mmol/L), divided by 22.5.Furthermore, to investigate if MBL is synthesized in adipose tissue real-time RT-PCR for MBL mRNA expression was performed on human adipose tissue obtained from fat biopsies from a subset of the participants and compared to commercial available human liver RNA.The study was conducted according to the Declaration of Helsinki and was approved by the local Ethical Committee. Study participants gave a written informed consent before entering the study.Serum glucose was measured by standard methods at the Clinical Biochemical Department, Aarhus University Hospital.Serum insulin was measured by AutoDELFIA Insulin kit B080-101 .μg/L. In brief, microtiter wells were coated with monoclonal anti-MBL antibody followed by incubation with samples diluted 200-fold. After washing, monoclonal anti-MBL antibody labeled with europium was added, and after incubation and washing, the amount of bound, labeled antibody was assessed by time-resolved fluorometry. A number of control serum samples covering different MBL levels were included in all assays.Serum MBL levels were measured by an immunoassay, as described previously , using aFor determination of MBL mRNA expression, RNA was isolated from adipose tissue obtained from a subset of the participants using TRIzol reagent as previously described . IsolateReverse transcription was performed using random hexamer primers as described by the manufacturer . Then, PCR-mastermix containing the specific primers and Taq DNA polymerase  was added. β-actin: ACGGGGTCACCCACACTGTGC and CTAGAAGCATTTGCGGTGGACGATG, product length 658 bp. Real-time quantitation of a target gene to β-actin mRNA was performed with a SYBR-Green real-time PCR assay using an ICycler from Bio-Rad. The target gene and β-actin mRNA were amplified in separate tubes. The increase in fluorescence was measured in real time during the extension step. The threshold cycle (Ct) was calculated and the relative gene expression was calculated essentially as described in the User Bulletin number 2, 1997, from Perkin Elmer . Briefly, the target gene (X0)-to-β-actin (R0) ratio in each sample before amplification was calculated as X0/R0 = kx1/((2**deltaCt)); delta Ct is the difference between Ct-target and Ct-reference, and k is a constant, set to 1. All samples were amplified in duplicate. A similar setup was used for negative controls except that the reverse transcriptase was omitted and no PCR products were detected under these conditions.The following primers were designed using the primer analysis software Oligo version 6.64: MBL2: GCAAACAGAAATGGCACGTAT and AGAGGCCTGGAACTTGACA, product length 139 bp and Values are presented as means ± SE, except for MBL, which was nonnormally distributed, and values are given as medians with interquartile ranges (IQR). t-test was used for normally distributed variables as appropriate. For nonnormally distributed variables, comparisons between groups were performed using the Wilcoxon signed ranks test or the Mann-Whitney U-test as appropriate. Pearson's correlation was used for normally distributed variables, whereas Spearman's correlation with two-tailed probability values was used to estimate the strength of association between nonnormally distributed variables.Paired or unpaired P < 0.05. IBM SPSS version 20 was used for all calculations.Statistical significance was assumed for 2  (range from 29.5 to 40.8 kg/m2).The participants' mean age was 43 years (range from 24 to 62 years). Their characteristics at baseline and after 8 weeks of VLCD are presented in P < 0.001), 2 versus 29.9 ± 0.6 kg/m2, P < 0.001) and the insulin resistance (HOMA-IR) was reduced by 50.2% . After 8 weeks of VLCD the mean body weight was reduced by 12.7%  were also significantly reduced after 8 weeks. μg/L versus 892 (336–1511) μg/L, P = 0.23), Median serum MBL levels did not change significantly during the 8 weeks of VLCD (746 (316–1190) Median serum MBL levels did not differ significantly between men and women at baseline nor after 8 weeks and the change in MBL levels remained insignificant when looking at men and women separately (data not shown).MBL levels did not correlate with age (data not shown).r = 0.595, P < 0.001).As expected, change in HOMA-IR correlated to change in weight . Furthermore, the study concluded that the change in MBL levels were positively associated with the increase in insulin sensitivity . In contWe found no evidence that MBL is synthesized in the human adipose tissue. This further supports our finding of MBL not being related to weight loss.The contradictory impacts of MBL under various conditions support a hypothesis of duality of MBL in a disease, and interventions, changing the MBL level, are currently being explored. Up to now, most of the focus has been made on antibodies to MBL or downstream complement factors, whereas the possible effect of lifestyle interventions remains sparsely explored.We suggest that MBL levels may not be significantly modifiable by lifestyle interventions, such as weight loss, and that obesity and insulin resistance are not associated with MBL levels. MBL is not synthesized in human adipose tissue. Together, this indicates that decreased MBL levels are neither the result nor a contributing cause of obesity and insulin resistance. Rather interindividual differences in MBL depend primarily on the MBL genotype and constitute a preexisting condition, which might be an advantage or a disadvantage under different circumstances of health and disease.


Objective. The aim of this study was to compare SUDOSCAN, a new device to evaluate sweat function , with conventional measures of peripheral and cardiac neuropathy in patients with type 2 diabetes. Methods. 265 diabetic patients were tested for symptoms and clinical signs of neuropathy using Michigan Neuropathy Screening Instrument (MNSI), vibration perception threshold (VPT) using biothesiometer, and cardiac autonomic neuropathy (CAN) using Ewing's protocol. Sudomotor function was investigated with SUDOSCAN through measurement of electrochemical skin conductance (ESC) of hands and feet. Lower ESC is suggestive of sudomotor dysfunction. Results. Lower ESC at feet was significantly associated both with increasing symptoms (MNSI A) and increasing score on physical abnormalities (MNSI B). Lower ESC at feet was also significantly associated with increasing VPT by biothesiometry (P < 0.01), and with higher number of abnormal CAN results (P < 0.05). ESC was associated with postural fall in blood pressure  (P < 0.05), but not with heart rate variability (HRV) tests . Conclusions. Sudomotor dysfunction testing may be a simple test to alert physicians to peripheral nerve and cardiac sympathetic dysfunction. Ease of performance could make it useful in a busy diabetic clinic. Further studies with hard clinical outcomes are indicated. Neuropathy is a common complication in diabetes mellitus (DM), with 60%–70% of patients affected over lifetime. Symptoms of neuropathy are very common, and subclinical neuropathy is more common than clinical neuropathy . NeuropaSweat glands are innervated by thin and unmyelinated sympathetic C fibers that could be affected early in the course of diabetes, as demonstrated in patients with prediabetes . The conWe compared SUDOSCAN findings with conventional measures of peripheral and cardiac neuropathy in patients with type 2 diabetes, in a proof of principle study to investigate the usefulness of this measure in clinical practice.The study was conducted in Diabetes Unit, King Edward Memorial (KEM) Hospital Research Centre, Pune, India from September 2009 to February 2010. KEM Hospital Research Centre ethics committee approved the study, and informed consent was obtained from each subject.Type 2 diabetes patients between 21 and 75 yrs of age, with or without symptoms of neuropathy, were enrolled in the study. Exclusion criteria included treatment with drugs that would have an effect on the sympathetic system such as beta blockers (to avoid interference in CAN testing), amputation of arms or legs, implantation of electrical implantable devices (pacemaker/defibrillator), sensitivity to nickel or any other standard electrodes, history of seizures or epilepsy, presence of proliferative retinopathy, myocardial infarction (MI) and/or stroke in the past 6 months, arrhythmia's, treatment with anti-arrhythmic drugs, and any advanced systemic condition.A medical officer examined the patients. Height, weight, waist, and hip circumference were measured using standardised methods and used to calculate body mass index (BMI) and waist hip ratio (WHR). Blood pressure was recorded in supine position after 5 minutes rest using oscillometry. Medication history  was noted.We used the MNSI questionnaire to record neuropathic symptoms (MNSI A) . ClinicaVPT was measured on both sides (left and right) using a biothesiometer on the plantar side of the great toe on a continuous scale. We used mean of the two sides for analysis.R-R intervals during inhalation and exhalation are calculated. The longest R-R interval is determined during expiration (R-R max) and the shortest interval during inspiration (R-R min). The ratio of the longest to shortest R-R is called as E/I ratio. Normal values ≥ 1.21.HRV during deep breathing (E/I Ratio): R-R interval is measured after standing which is around the 15th beat. This is followed by bradycardia which is indicated by the longest R-R interval around 30th beat. The ratio of longest to shortest R-R is calculated which is called as 30/15 ratio. Normal values ≥ 1.03.HRV to standing (30/15 Ratio): shortest R-R interval after maneuver to shortest R-R interval during the maneuver is calculated. Normal values ≥ 1.20.HRV during Valsalva maneuver: in this test the ratio of longest Blood pressure response to standing (orthostatic blood pressure response): the postural fall in the blood pressure is taken as the difference between the systolic blood pressure lying and standing. Normal fall is ≤20 mmHg.International Diabetes Federation (IDF) (2005) recommends the use of resting heart rate and heart rate response to provocation tests , and lying and standing blood pressure difference for diagnosis of CAN . ADA hasμS) for the hands, and the feet (right and left sides). We used mean of left and right readings for the analysis.SUDOSCAN device  consists1c was measured using HPLC method  calibrated against  NGSP. Urine albumin was measured using an immunoprecipitation assay, and albumin-creatinine ratio (UACR) was calculated.Nonfasting blood sample was collected in EDTA vacutainer and processed to obtain plasma. Plasma aliquots were stored (−70°C) until further analysis. Hemoglobin was measured on whole blood on a Beckman Coulter Analyzer . Plasma glucose, uric acid, and creatinine, were measured on an automated biochemistry analyzer , using standard enzymatic methods. HbAData are presented as mean (sd). Normality of the variables was checked, and appropriate transformations were done before analysis. following variables needed transformation: log transformation for VPT and HRV tests including 30/15 standing ratio and the Valsalva maneuver, log-log transformation for E/I Ratio, and square root transformation for MNSI A score. Agreement between left and right readings (VPT and ESC) was investigated using mean percent difference and coefficient of variation (CV). Simple linear regression analysis and Chi-square proportional trend test were used to study the association of ESC with continuous and categorical measurements of biological determinants  and conventional measures of peripheral and cardiac neuropathy . Agreement between MNSI, biothesiometer readings, and ESC values was studied using simple linear regression analysis. In this we express results as prediction interval by calculating percentage of observation outside the prediction limit. Low percentage indicates good agreement. Association between ESC and biological determinants was also tested using multiple linear regression analysis. Area under the curve (AUC) of the ROC curve was calculated to measure the efficiency of ESC in diagnosing patients with and without neuropathy against VPT . StatistWe studied 265 type 2 diabetic patients  who were found eligible for the study and gave informed consent. Clinical and biochemical characteristics of the subjects are described in 2, of whom 6 (2.2%) had eGFR < 60 mL/min/1.73 m2. Sixty patients (22.5%) had nonproliferative diabetic retinopathy (NPDR). Seventy-four patients had low levels of plasma vitamin B12 (<150 pmol/L) or were on vitamin B12 supplements, and they had similar nerve function  compared to those who had higher levels of vitamin B12 and are included in the analysis.Thirty six patients (13%) showed microalbuminuria, 7 (2.6%) had macroalbuminuria. None had serum creatinine > 1.5 mg%, 53 (20%) had eGFR < 90 mL/min/1.73 mTwo hundred and thirty (86.8%) patients had at least one symptom of neuropathy. One hundred and forty-nine patients (56.2%) had abnormal appearance on foot inspection, 57 (21.5%) had reduced ankle reflex, 41 (15.5%) showed reduced vibration sensation using tuning fork, and 15 (5.7%) showed reduced touch sensation using SWME on at least one foot. One hundred and forty-four patients (54.3%) showed MNSI B score >2 “Clinical neuropathy”. Patients with neuropathy were older, had longer duration of diabetes, and had higher VPT (biothesiometer) and lower ESC (SUDOSCAN)  Table 2Table 2.P < 0.001, both), higher HbA1c concentration (P < 0.05), higher serum creatinine and lower eGFR, and with decreasing ESC .On biothesiometry, 90 (34.0%) patients had a VPT reading of ≤10 V which is considered normal, 93 (35.1%) patients had a reading between >10 and ≤15 V, 60 (22.6%) had a reading between >15 and ≤25 V, and 22 (8.3%) had a reading >25 V. Increasing VPT was associated with higher age and longer duration of diabetes  had abnormal E/I ratio (<1.21), 84 (32%) had abnormal 30/15 ratio (<1.03), and 21 (8%) had orthostatic hypotension (>20 mmHg). Valsalva test was performed satisfactorily by only 171 patients, out of which only 1 was abnormal (<1.20). Of 171 on whom all test results were available, in 102 all tests were normal, 46 had one test abnormal, and 23 had two or more tests abnormal.Conductance measurements were normally distributed in hands and feet. Mean percent difference between measurements on right and left side was 9.5% for hands and 6.0% for feet while it was 14.2% for VPT measurements . The CV For further analysis, only the feet ESC results are used, and similar results were observed for hands ESC.1c, and lower hemoglobin concentrations, but were not related to gender, BMI, WHR, non-fasting plasma glucose, uric acid, creatinine concentrations and eGFR. On multiple linear regression analysis, lower ESC was independently associated with higher age and higher HbA1c , but not with gender, anthropometric and other biochemical parameters.r = −0.12, P < 0.05), increasing score on physical abnormalities, suggestive of peripheral neuropathy (MNSI B) , and increasing score on VPT . Compared to patients with ESC ≥ 40 μS, patients with ESC < 40 μS were more than two times likely to have MNSI B score > 2, (OR = 2.3 (1.28–4.14)), more than three times likely to have VPT > 25 V (OR = 3.97 (1.61–9.99)), and more than 4 times likely to have 2 or more CAN tests abnormal (OR = 4.41 (1.72–11.29)). Of the four CAN tests, only postural fall in blood pressure was associated with lower ESC .Lower ESC was significantly associated with both increasing symptoms (MNSI A)  and VPT on ESC and calculated the predicted VPT and MNSI measurements for any observed value of ESC. Percentage of observations outside prediction interval was 2.6% for MNSI A, 1.5% for MNSI B, and 4.5% for VPT, which indicates good agreement.μS which indicates that 73% of neuropathy patients and 62% of without neuropathy patients were correctly diagnosed by SUDOSCAN.Efficiency of sweat function measurement in diagnosing patients with and without neuropathy was checked using ROC curve. The AUC of ESC, calculated against continuous scale of VPT, was 71% at VPT = 21 V. The sensitivity and specificity, calculated on the continuous scale of ESC, were 73% and 62%, respectively, at ESC = 52 In this proof of principle study in diabetic patients, we found that lower ESC on SUDOSCAN (reflecting sweat gland dysfunction) was significantly associated with symptoms of peripheral neuropathy and conventional clinical tests of vibration perception and monofilament touch. The association of ESC with vibration perception (biothesiometer) was continuous and graded, similar to the findings of a previous study performed in France . Lower E1c) predict low ESC, indirectly supporting that it is a reliable measure of diabetic neuropathy.SUDOSCAN evaluates sudomotor function by measuring ESC resulting from an electrochemical reaction between sweat chloride and nickel electrodes after a low DC stimulation. Importance of sweat chloride in this reaction was well demonstrated in a study performed with cystic fibrosis (CF) patients who had significantly higher conductance compared with healthy subjects . Sweat gTuning fork and monofilament tests are easy to perform but qualitative. Biothesiometry is quantitative. But all these tests are subjective, requiring patient's attention and cooperation , and theAdmittedly, this proof of principle study has some limitations in applicability because of the design and methodology. The need to compare the results with conventional HRV tests (Ewing's protocol) necessitated exclusion of patients who were on medications which could affect HRV. It is also true that Ewing's protocol is somewhat outdated though it remains the “standard” method for autonomic testing. The use of a DC in the testing required exclusion of patients who suffered from seizures or were carrying an electrical medical device. Patients with foot ulcers or amputation had to be excluded because of the technical requirement of the test. Comparison of vibration perception (biothesiometer) and sudomotor function meant that we compared affection of large, myelinated type A fibers with that of small, unmyelinated type C fibres. However, this comparison allowed us to test the performance of a novel method with established clinical methods. It would be informative to compare SUDOSCAN with other methods of sweat gland function like QSART   or NeuroFurther clinical application of sudomotor testing in a diabetic clinic will depend on conducting larger unbiased studies in representative patient populations and relating the findings to varied clinical outcomes including foot ulceration, amputations, cardiovascular events, and sudden death, in all of which autonomic neuropathy is thought to play a significant role.Assessment of sudomotor function using SUDOSCAN, a quick and noninvasive test, could be considered as an additional screening test to alert physicians to peripheral nerve and cardiac sympathetic dysfunction. Ease of performance could make it useful in a busy diabetic clinic to identify patients with small fibre neuropathy who may be at higher risk of foot problems or cardiac neuropathy.


S-phase kinase-associated protein 2 (Skp2) is a member of mammalian F-box proteins. The purpose of this study is to clarify the prognostic significance of expression of Skp2 related to gender, estrogen receptor (ER) and progesterone receptor (PGR) in soft tissue sarcomas (STS). Skp2 has been demonstrated to display an oncogenic function since its overexpression has been observed in many human cancers. Optimized treatment of STS requires better identification of high-risk patients who will benefit from adjuvant therapy. The prognostic significance of Skp2 related to ER and PGR in STS has not been sufficiently investigated.Tissue microarrays from 193 STS patients were constructed from duplicate cores of viable and representative neoplastic tumor areas. Immunohistochemistry was used to evaluate the expression of Skp2, ER and PGR.In univariate analyses, high tumor expression of Skp2 correlated (p = 0.050) with reduced disease-specific survival (DSS). In subgroup analyses expression of PGR in males (p = 0.010) and in patients older than 60 years (p = 0.043) were negative prognostic factors for DSS. Expression of ER in females was a positive prognostic factor for DSS (p = 0.041). In co-expression analyses in the whole cohort, low expression of Skp2 in combination with low expression of ER was positive for DSS (p = 0.049). In females high expression of Skp2 in combination with low expression of ER was a negative prognosticator (p = 0.021). In the multivariate analyses, age (p = 0.012), malignancy grade (p < 0.001), wide resection margins (P = 0.010), ER negative / PGR positive co-expression profile (p = 0.002) and ER positive / PGR negative co-expression profile (p = 0.015) were independent negative prognostic factors for DSS. In females expression of Skp2 (p = 0.006) was associated with shorter DSS.We found diverse prognostic impacts of expression of Skp2, ER, PGR and DSS in male and female patients with STS. In men, but not women, ER positive / PGR negative co-expression profile was an independent negative prognostic factor for DSS. In women, but not men, high expression of Skp2 was associated with reduced DSS. S-phase kinase-associated protein 2 (Skp2), a mammalian F-box protein, displays S-phase-promoting function, through ubiquitin-mediated proteolysis of the CDK inhibitor p27. Skp2 has been shown to regulate cellular proliferation by targeting several cell cycle-regulated proteins for ubiquitination and degradation. Skp2 has also been demonstrated to display an oncogenic function since its overexpression has been observed in many human cancers . High exThe purpose of this study is to clarify the prognostic significance of expression of Skp2 related to age, gender and female steroid hormone receptors (ER and PGR) in non-gastrointestinal stromal tumor (non-GIST) STS. To achieve this, we analyzed the expression of these markers in 193 patients with non-GIST STS in relation to demographic and other clinicopathological variables. Our major hypothesis is that a different prognostic significance of Skp2 in men and women exists and is related to diverse gender expressions of ER and PGR.Primary tumor tissues from patients diagnosed with STS at the University Hospital of North Norway (UNN) from 1973 to 2006 and the Hospitals of Arkhangelsk region, Russia, were used in this retrospective study. In total, 496 potentially suitable patient records were identified from the hospitals’ databases. Of these, 247 patients were excluded due to missing clinical data (n = 86) or inadequate material for histological examination (n = 161). In addition, 33 were excluded because of metastasis at the time of the diagnosis, 13 were excluded because they had no surgery, and 10 patients had both metastasis and no surgery, leaving a total of 193 patients eligible for this study. This report includes data for 131 Norwegian patients and 62 Russian patients followed until September 2009. The median follow-up was 38 (range 0–392) months. Complete demographic and clinical data were collected retrospectively. Formalin-fixed and paraffin-embedded tumor specimens were obtained from the archives of the Departments of Pathology at UNN and Arkhangelsk. The tumors were graded according to the French Fédération Nationales des Centres de Lutte Contre le Cancer (FNCLCC) system . Wide resection margins were defined as wide local resection with free microscopic margins or amputation of the affected limb or organ. Non-wide resection margins were defined as either marginal or intralesional resection margins.Two pathologists (AV and SWS) reviewed the histology of all soft tissue sarcoma cases. Tissue microarrays (TMAs) were constructed for high-throughput molecular pathology research . The mosStudies suggest that punching multiple 0.6 mm cores from different regions captures the heterogeneity of the tumors more accurately than a single 2 to 4 mm core . We therThe applied antibodies were subjected to in-house validation by the manufacturer of IHC analysis on paraffin-embedded material. All staining was performed in the Ventana Benchmark XT automated slide stainer . Before staining, the sections were incubated over night at 60 degrees Celsius. Tissue sections were incubated with primary mouse monoclonal antibodies recognizing Skp2 , ER  and PGR . The incubation periods were 40 minutes for Skp2, 32 minutes for ER and 24 min for PGR. This was followed by application of liquid diaminobenzidine as substrate-chromogen, yielding a brown reaction product at the site of the target antigen . iVIEW DAB Detection Kit is an indirect biotin streptavidin system for detecting mouse and rabbit primary antibodies. The DAB chromogen produces a dark brown precipitate that is readily visualized by light microscopy. All reagents are provided pre-diluted by the manufacturer for use in Ventana Benchmark XT. Finally, slides were counterstained with hematoxylin to visualize the nuclei. For each antibody, including negative controls, all TMA staining were performed in a single experiment. In the TMA we also used cores from carcinomas and normal tissue as positive and negative controls.The ARIOL imaging system  was used to scan the slides for antibody staining of the TMAs. The specimens were scanned at a low resolution 1.25×) and a high resolution (20×) using an Olympus BX 61 microscope with an automated platform (Prior). The slides were loaded in the automated slide loader (Applied Imaging SL 50). Representative and viable tissue sections were scored manually on a computer screen semi-quantitatively for nuclear and/or cytoplasmic staining. The expression of Skp2, ER and PGR was scored as: 0, negative; 1, weak; 2, intermediate and 3, strong , version 18. The IHC scores from each observer were compared for inter-observer reliability by use of a two-way random effects model with absolute agreement definition. The intra-class correlation coefficient (reliability coefficient) was obtained from these results.Chi-square and Fisher exact tests were used to examine the association between molecular marker expression and various clinicopathological parameters. Univariate analyses were done using the Kaplan-Meier method, and statistical significance between survival curves was assessed by the log rank test. Disease-specific survival (DSS) was determined from the date of histologically confirmed STS diagnosis. Correlation of marker expression was done using the Pearson correlation (2-tailed) at the 0.05 and 0.01 levels.Multivariate analysis was carried out using the Cox proportional hazards model to assess the specific impact of each pre-treatment variable on survival in the presence of other variables. Variables of significant value from the univariate analysis were entered into the Cox regression analysis. Probability for stepwise entry and removal was set at 0.05 and 0.10, respectively. The significance level used was p < 0.05.The National Cancer Data Inspection Board and The Regional Committee for Research Ethics (REK nord) approved the study. The material was collected from our approved biobank for paraffin embedded material and slides. The Regional Committee approved that written consent from the patients for their information to be stored in the hospital database and used for research was not needed because most of the material was more than 10 years old, and most of the patients being dead. The ethics committee specifically waived the need for consent. Data were analyzed anonymously.Demographic, clinical, and histopathological variables are shown in Table There was good scoring agreement between the two investigating pathologists. The IHC scores from each observer were compared using a two-way random effects model with absolute agreement definition. The intra-class correlation coefficients  obtained from these results were 0.94 for Skp2 (p < 0.001), 0.92 for ER (p < 0.001) and 0.96 for PGR (p < 0.001).Nationality, histology, tumor size, malignancy grade and surgical margins were all significant indicators for disease-specific survival (DSS) in univariate analyses Table . Table 2In univariate analyses, increased expression of Skp2 (p = 0.050) correlated significantly with reduced DSS, , men had better 5-year survival (69%) compared to women , while there were no differences (p = 0.376) between men and women in patients with high expression of ER (N = 72). In patients with low expression of PGR (N = 132), men had better 5-year survival (69%) compared to women , while there were no differences (p = 0.271) between men and women in patients with high expression of PGR (N = 57). There were no differences in survival between men and women in univariate analyses of patients with low  or high  expression of Skp2 (data not shown).In co-expression analyses Table  Skp2 negTaking into consideration the possible distortion of results by gender-related sarcomas (i.e. leiomyosarcoma in uterus) we have attempted to exclude these sarcomas and recalculate all analyses. There were no significant differences in the results compared to those obtained without exclusion of gender-related sarcomas (data not shown).Significant demographic, clinicopathological and expression variables from the univariate analyses were entered into the multivariate Cox regression analysis Table . In the In this large-scale study, we evaluated the prognostic significance of expression of Skp2 related to age, gender, ER and PGR in 193 STS patients. Our hypothesis was confirmed. We found diverse prognostic DSS impacts from gender related expression of Skp2, ER, PGR and DSS in STS. In men, but not women, an ER positive/PGR negative co-expression profile was an independent negative prognostic factor for DSS. In women, but not men, high expression of Skp2 was associated with reduced DSS. High expression of ER reduced the negative impact of Skp2 in women. While women with the Skp2 positive / ER positive phenotype had favorable survival, women with the Skp2 positive / ER negative phenotype had poor survival. To the best of our knowledge, this is the first prognostic evaluation of Skp2 related to the female hormone receptors ER and PGR in STS.Expression of ER and PGR is a routinely investigated indicator of endocrine therapy success in breast cancer ,13 and aWe have modified the Allred score for STS and used 1% positivity as cut-off value ,16. The Huang et al. suggested that the therapeutic strategies designed to reduce Skp2 may play an important clinical role in treatment of breast cancer cells, especially ER/HER2 negative breast cancers . Voduc eper se and in co-expression with TGF-β, fascin and Akt isoforms [In our previous work we have shown that ER and PGR expression possess variable prognostic significance depending on gender, both isoforms -9. In thisoforms , while iThe data collection introduced problems in identifying adequate numbers of similar patients with similar tumors and with the same treatment traditions. These are well known problems when conducting STS studies. Our findings are in large hypothesis generating, and to be more conclusive future STS studies must be based on large, multi-institutional and multinational studies with possibilities to establish adequately sized STS patient cohorts of homogenous tumor groups. However, all tumors investigated herein had mesenchymal derivation and belong to the same generic group.In conclusion, there were different prognostic impacts of expression of Skp2, ER, PGR and DSS in male and female patients with STS. In men, but not in women, ER positive / PGR negative co-expression was an independent negative prognostic factor for DSS. In women, but not in men, expression of Skp2 was associated with reduced DSS.The authors declare that they have no competing interests.SWS, TK, AV, TD, RMB and LTB participated in the design of the study. TK, ES and AV collected clinical information. SWS and AV reviewed all the histological diagnosis, histological grading, selected and marked the slides for TMA construction. SWS, TK and AV performed the experiments. SWS, TK, AV, TD, RMB and LTB performed the statistical analysis. SWS, TK, AV, TD, ES, KAS and LTB contributed reagents/materials/analysis tools. SWS, TD, ES, KAS, RMB and LTB drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6890/13/9/prepub


Indigenous Australians experience poorer health than other Australians. Poor diet may contribute to this, and be related to their generally lower socioeconomic status (SES). Even within Indigenous populations, SES may be important. Our aim was to identify factors associated with plasma carotenoids as a marker of fruit and vegetable intake among urban dwelling Indigenous Australians, with a particular focus on SES.Cross sectional study in urban dwelling Indigenous Australians participating in the DRUID (Darwin Region Urban Indigenous Diabetes) Study. An SES score, based on education, employment, household size, home ownership and income was computed and plasma carotenoids measured by high performance liquid chromatography in 897 men and women aged 15 - 81 years . Linear regression analysis was used to determine the relationship between SES and plasma carotenoids, adjusting for demographic, health and lifestyle variables, including frequency of intakes of food groups .SES was positively associated with plasma concentrations of lutein/zeaxanthin (p trend <0.001), lycopene (p trend = 0.001), α- and ß-carotene (p trend = 0.019 and 0.026 respectively), after adjusting for age, sex, glucose tolerance status, smoking, alcohol use, hypercholesterolemia, dyslipidemia, self-reported health, waist to hip ratio and body mass index. These associations remained after adjustment for self-reported frequency of intake of fruit, vegetables, takeaway foods and fruit juice, which all showed some association with plasma carotenoids. Even in the highest SES quintile, concentrations of all carotenoids (except lycopene) were lower than the mean concentrations in a non-Indigenous population.Even within urban Indigenous Australians, higher SES was associated with higher concentrations of plasma carotenoids. Low plasma carotenoids have been linked with poor health outcomes; increasing accessibility of fruit and vegetables, as well as reducing smoking rates could increase concentrations and otherwise improve health, but our results suggest there may be additional factors contributing to lower carotenoid concentrations in Indigenous Australians. Indigenous Australians are at increased risk of cardiovascular disease, type 2 diabetes and renal disease compared with other Australians ,2. WhileLow plasma carotenoid concentrations were found to be associated with urinary albumin excretion in a study of Australian Aborigines from north west Australia, indicating a potential association with vascular renal disease . An inveIn remote Indigenous communities, limited availability and high cost of fresh fruit and vegetables are a major contributor to poor quality dietary intake . In urbaThe primary aim of this study was to evaluate the relationship between SES and plasma carotenoids in an urban Indigenous population, and the extent to which this relationship is mediated by self-reported intake of fruit and vegetables.Data were collected in 2003-2005 as part of the DRUID (Darwin Region Urban Indigenous Diabetes) Study, which has been described in detail elsewhere . BrieflyA total of 1,004 eligible people participated and provided at least one measurement, of these, 107 people did not have carotenoid data, leaving 280 men and 617 women with a mean age of 36.6(standard deviation (SD) 14.6) years, who were included in these analyses.For participants who had fasted for at least 10 hours, fasting blood samples were collected. Up to 13 ml of blood was collected in tubes appropriate for each assay. Blood samples were stored on ice until centrifuged at 3000 RPM for 15 minutes. Where possible this was completed within 1 hour of collection.Socioeconomic status was represented in this analysis by self-reported information on education, employment, household size (as a proxy for overcrowding), home ownership and income . Weekly A 2-hour oral glucose tolerance test was administered to all consenting participants except those using medication for previously diagnosed diabetes, and those who were pregnant. Glucose was measured in plasma and diabetes was defined as: 1) fasting plasma glucose ≥ 7.0 mmol/L; 2) 2-hour post-glucose load plasma glucose ≥ 11.1 mmol/L; or 3) previously diagnosed as having diabetes and currently taking tablets and/or insulin for diabetes. Impaired glucose tolerance (IGT) was defined as fasting glucose <7.0 mmol/L and 2-hour glucose ≥ 7.8 and <11.1 mmol/L; impaired fasting glucose (IFG) as fasting glucose ≥ 6.1 and <7.0 mmol/L, and 2-hour glucose <7.8 mmol/L. Normal glucose tolerance (NGT) included all those with fasting glucose <6.1 mmol/l and 2-hour glucose <7.8 mmol/l.Total cholesterol, triglycerides, and HDL cholesterol were measured in fasting serum as previously described . Blood s3:MeOH:CH3CN (30:35:35). Fifty μl was injected into the high performance liquid chromatograph (HPLC) (Shimadzu HPLC machine equipped with an SPD-M20A PDA Detector and a NovoPak C18 column) with absorbance detection at 292 nm for the tocopherols, 325 nm for retinol and 450 nm for the carotenoids. The carotenoids, retinol and tocopherols were eluted from the column using a mobile phase of 0.0125% ammonium acetate in MeOH (A), 100% CHCl3 (B) and CH3CN with 0.1% triethylamine (C) in three linear gradient steps: from 0 to 5 min, A 50%, C decreased from 50% to 44% and B increased to 6%; from 5 to 16 min, A increased to 55%, C decreased from 44% to 30% and B increased from 6% to 15%. Wash with A:C 50/50 mixture for 3 mins. Retinol and tocopherols in plasma are not good biomarkers of diet [The extraction and analysis of individual carotenoids , retinol and tocopherols  was based on the method of Su et al . Briefly2 for both men and women.Body weight was recorded to the nearest 0.1 kilogram using a digital scale weighing up to 200 kg , and height to the nearest 0.1 cm using a portable stadiometer . Waist and hip circumferences were measured to the nearest 0.1 cm using a 2-metre non-stretch fiberglass tape. The waist was defined as the mid-point between the iliac crest and the costal margin, and the hips as the widest circumference over the buttocks and below the iliac crest. Abdominal obesity was defined as a waist/hip ratio (WHR) of >0.9 for men and >0.8 for women or waist circumference >102 cm for men or >88 cm for women. Participants were classified as underweight if body mass index (BMI) <20, normal weight if BMI ≥ 20 and ≤25, overweight if BMI >25 and ≤ 30, and obese if BMI >30 kg/mSitting blood pressure was measured using a Welch Allyn Spot Vital Signs monitor . Hypertension was defined as systolic blood pressure ≥ 140 mmHg and/or diastolic blood pressure ≥ 90 mm Hg and/or current anti-hypertensive medication .Current tobacco smokers and alcohol consumers were identified by self-report using a simple yes/no response. Self-rated health  was reported. The dietary data was based on questions frequency of consumption of selected food groups. For 'Fruit' and 'Vegetables' consumed per day, response options were 'None' '1 serve or less', '2-3 serves', 4-5 serves', '6 or more serves' or 'Don't know/not sure'. Frequency of consumption of food groups 'Takeaway foods', 'Snacks' and 'Fruit/vegetable juice' was recorded using response options 'Never or rarely', Less than once a week', '1 time per week', '2-3 times per week', '4-6 times per week', 1 time per day' '2 times per day', '3 or more times per day'.The study was approved by the Human Research Ethics Committee of the Northern Territory Department of Health & Community Services and Menzies School of Health Research. It was considered and approved by both the Aboriginal sub-committee, which has absolute right of veto, and by the main committee. The study's governance structure included an Indigenous Steering Group, as well as partnerships with key Indigenous organisations . All parInitially, associations between SES variables and plasma carotenoids were evaluated separately to identify those that showed associations (p < 0.05): education, employment, household size, home ownership and income. Subsequently a combined score was developed by including the five variables identified as having univariate associations in factor analysis using the principal factor method  and thenth and 75th percentiles were used to reflect the distributions. Analysis of variance using natural log transformed carotenoids was used to identify variables associated with plasma carotenoid concentrations. Linear regression models including all variables showing univariate associations with at least one of the carotenoids were then computed. Because younger people could not be properly scored on the SES variable as many were still completing their education, the models were also run excluding people aged less than 20 years, leaving 722 for analysis. The results are presented for non-transformed carotenoid variables so that ß-coefficients are more readily interpreted. Using natural log transformed variables did not materially change the patterns of relationships. Models included: the combined SES score as quintile groupings, age (10 yr age groups), gender, BMI , WHR-obese (Y/N), hypercholesterolaemia (Y/N), dyslipidaemia (Y/N), glucose tolerance , diabetes), alcohol consumption (Y/N) and smoking (Y/N), all of which have been associated with carotenoid concentrations and are CVD risk factors. People with missing values were classified separately. A second model was computed for each carotenoid variable including frequency of intakes of vegetables, fruit, takeaway food  were seen between education, employment, home ownership and income, while household size was weakly inversely associated with equivalised income. This suggests that it is appropriate to combine them. Only one SES factor with an Eigen value greater than 1 (1.43) was identified. Positive loadings were seen for home ownership (0.37), educational level (0.45), equivalised income (0.76) and work status  (0.69). Household size showed a weak inverse loading (-0.17). As shown in Figure In the univariate analysis (data not shown), plasma concentrations of all carotenoids increased with increasing frequency of vegetable or fruit consumption (serves/day); with p < 0.05 for ANOVA in all but lycopene. There were similar trends for consumption of fruit or vegetable juice. Intake of snack food was not associated with plasma carotenoids, but frequency of consumption of takeaway foods (times/week) was inversely associated with all carotenoids except lycopene.In multivariate models not including dietary intake variables Table , plasma With the addition of dietary data to the model, most of these associations were retained: β-coefficient for quintile 5 vs quintile 1 and p trend for lutein/zeaxanthin , β-cryptoxanthin , lycopene , α-carotene  and β-carotene . Takeaway intake was inversely associated with lutein/zeaxanthin and α-carotene concentrations, while fruit and juice intakes were positively associated with concentrations of ß-cryptoxanthin. The multivariate models, including dietary variables explained only 15 to 22% of the variance in plasma carotenoid concentrations.Of the 897 people classified, 46 (5%) were in the top 25% for all plasma carotenoids and classified as the 'hi carotenoid' group. Using the same variables as previously and after excluding 23 people with missing data for hypercholesterolemia or dyslipidemia, being in the hi-carotenoid group was directly associated with being female , hypercholesterolaemic (versus not hypercholesterolaemic) and SES score (top 20% versus bottom 20%), and inversely associated with smoking (versus not smoking) and abnormal glucose tolerance   and 26% (ß-carotene) of variance in carotenoids, similar to our results [A recent review of diet quality and social class found that groups with a lower socioeconomic status consumed less fruit and vegetables and with less variety than those of higher SES . High enThere are other factors beyond cost that may contribute to lower fruit and vegetable intakes in lower SES groups, for example, processed and packaged foods are generally easier to store, last longer, and many require minimal preparation. Exposure to light reduces the levels of carotenoids in fruit and vegetables  and limiWhile the low participation rate in DRUID is a weakness of this study, it is difficult to imagine how this could produce the associations between SES and plasma carotenoids observed. The distribution of participants by age, place of residence, Indigenous group and household income was generally similar to that of the local Indigenous population as measured in the 2001 Census . If partAs intakes were only recorded at food group levels we were not able to determine the carotenoid contents of the fruits and vegetables consumed. Furthermore, we only have data on the frequency of intake, which does not take into account the amount of foods actually consumed. However, the dietary data did show the expected associations with plasma carotenoids. As explained previously by Cunningham et al , there aThe combined SES score has not been validated; however, it combines variables commonly used in other composite indices of SES . The useA strength of this study is that it is one of few examining an urban Indigenous population and includes a wide range of SES, from people with no qualifications, a very low income, a large household and who do not own their home, to those with university degrees, in full-time work, a reasonable income and buying or owning their own house. As noted previously, even those classified as high SES in DRUID would not be considered high SES relative to the general Australian population, and would probably be in the middle of the national SES spectrum .In urban Indigenous Australians, higher SES is associated with higher concentrations of plasma carotenoids. This association was not fully explained by dietary intake data. Our data also suggests that even the most well-off among the DRUID participants have lower plasma carotenoids than a sample from the general population in Queensland. Low plasma carotenoids have been linked with poor health outcomes and may explain some of the heavy burden of diabetes and CVD risk observed in the DRUID population . More reThe authors declare that they have no competing interests.AH analysis plan, data analysis, manuscript preparation. LMB data acquisition, drafting and revising manuscript. JC original study design and data acquisition, drafting and revising manuscript. TD original study design, drafting and revising manuscript. KOD original study design, drafting and revising manuscript. All authors have approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/11/76/prepub