Neutrophil-to-lymphocyte ratio is independently associated with carotid atherosclerosis burden in individuals with type 1 diabetes.

BACKGROUND AND AIMS: Recent studies have identified a relationship between innate versus. Adaptative immunity and cardiovascular disease (CVD) in the general population, but information on type 1 diabetes (T1D) is lacking. We aimed to study the relationship between inflammatory biomarkers and preclinical atherosclerosis in this population. METHODS AND RESULTS: Cross-sectional study in T1D individuals without CVD and with ≥1 of the following: ≥40 years, diabetic kidney disease, or ≥10 years of diabetes duration with classical CVD risk factors. Carotid plaques were evaluated by ultrasonography. C-reactive protein, total leukocyte count, neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio and systemic immune-inflammation index were assessed as inflammatory markers. Multivariate-adjusted models including age, sex, and other CVD risk factors were constructed to test their independent associations with atherosclerosis burden. We included 602 subjects (52.8% men, 48.7 ± 10.2 years old and 27.0 ± 10.5 years of diabetes duration). Carotid plaques were found in 41.2% of the individuals (12.8%, ≥3 plaques). The number of carotid plaques (none, 1-2, ≥3 plaques), was directly associated with the leukocyte count (6570 [5445-8050], 6640 [5450-8470] and 7310 [5715-8935] per mm3, respectively; p for trend = 0.021) and the NLR (1.63 [1.28-2.13], 1.78 [1.38-2.25] and 2.14 [1.58-2.92], respectively; p for trend <0.001), but only the NLR remained directly associated in fully-adjusted models (presence of plaques; OR 1.285 [1.040-1.587]; ≥3 plaques, OR 1.377 [1.036-1.829]). CONCLUSIONS: The NLR was independently and directly associated with carotid plaque burden in T1D individuals. Our data support the role of innate versus. Adaptative immunity in atherosclerosis also among the T1D population.

Identification of differences in gene expression implicated in the Adherent-Invasive Escherichia coli phenotype during in vitro infection of intestinal epithelial cells.

Introduction: Adherent-invasive Escherichia coli (AIEC) is strongly associated with the pathogenesis of Crohn's disease (CD). However, no molecular markers currently exist for AIEC identification. This study aimed to identify differentially expressed genes (DEGs) between AIEC and non-AIEC strains that may contribute to AIEC pathogenicity and to evaluate their utility as molecular markers. Methods: Comparative transcriptomics was performed on two closely related AIEC/non-AIEC strain pairs during Intestine-407 cell infection. DEGs were quantified by RT-qPCR in the same RNA extracts, as well as in 14 AIEC and 23 non-AIEC strains to validate the results across a diverse strain collection. Binary logistical regression was performed to identify DEGs whose quantification could be used as AIEC biomarkers. Results: Comparative transcriptomics revealed 67 differences in expression between the two phenotypes in the strain pairs, 50 of which (81.97%) were corroborated by RT-qPCR. When explored in the whole strain collection, 29 DEGs were differentially expressed between AIEC and non-AIEC phenotypes (p-value < 0.042), and 42 genes between the supernatant fraction of infected cell cultures and the cellular fraction containing adhered and intracellular bacteria (p-value < 0.049). Notably, six DEGs detected in the strain collection were implicated in arginine biosynthesis and five in colanic acid synthesis. Furthermore, two biomarkers based on wzb and cueR gene expression were proposed with an accuracy of ≥ 85% in our strain collection. Discussion: This is the first transcriptomic study conducted using AIEC-infected cell cultures. We have identified several genes that may be involved in AIEC pathogenicity, two of which are putative biomarkers for identification.

Advanced lipoprotein profile identifies atherosclerosis better than conventional lipids in type 1 diabetes at high cardiovascular risk.

BACKGROUND AND AIMS: People with type 1 diabetes (T1D) present lipoprotein disturbances that could contribute to their increased cardiovascular disease (CVD) risk. We evaluated the relationship between lipoprotein alterations and atherosclerosis in patients with T1D. METHODS AND RESULTS: Cross-sectional study in subjects with T1D, without previous CVD, but high-risk (≥40 years, nephropathy, or ≥10 years of evolution of diabetes with another risk factor). The presence of plaque (intima-media thickness ≥1.5 mm) in the different carotid segments was determined by ultrasound. The advanced lipoprotein profile was analysed by magnetic resonance imaging (1H NMR). We included 189 patients (42% women, 47.8 ± 10.7 years, duration of diabetes 27.3 ± 10.1 years, HbA1c 7.5% [7-8]). Those with carotid plaques (35%) were older, with longer diabetes duration, had a higher prevalence of hypertension, and showed lower and smaller LDL particles (LDL-P) and HDL particles (HDL-P), but higher VLDL particles (VLDL-P). Some LDL, HDL and VLDL-related parameters were associated with atherosclerosis in sex, age and statin use adjusted models (p < 0.05), but after adjusting for multiple confounders, including conventional lipid parameters, only HDL-P (OR 0.440 [0.204-0.951]; p = 0.037), medium HDL-P (OR 0.754 [0.590-0.963]; p = 0.024), HDL-P cholesterol content (OR 0.692 [0.495-0.968]; p = 0.032), 1H NMR LDL-P number/conventional LDL-cholesterol (OR 1.144 [1.026-1.275]; p = 0.015), and 1H NMR non-HDL particle number/conventional non-HDL-cholesterol ratios (OR 1.178 [1.019-1.361], p = 0.026) remained associated with atherosclerosis. CONCLUSIONS: In adults with T1D at high-risk, variables related to HDL, LDL and total atherogenic particle number are independently associated with preclinical atherosclerosis. Advanced lipoprotein profiling could be used to identify those at the highest risk of CVD.

Nuclear Magnetic Resonance-Based Lipidomics in the Assessment of Cardiometabolic Risk in Type 1 Diabetes: An Exploratory Analysis.

INTRODUCTION: Cardiovascular disease (CVD) is the leading cause of mortality in type 1 diabetes (T1D). However, there is a need for daily practice tools for identifying those more prone to suffer from these events. We aimed to assess the relationships between nuclear magnetic resonance (1H NMR)-based lipidomic analysis and several CVD risk variables (including preclinical carotid atherosclerosis) in individuals with T1D at high risk. METHODS: We included patients with T1D without CVD, with at least one of the following: age ≥ 40 years, diabetic kidney disease, or ≥ 10 years of evolution with another risk factor. The presence of plaque (intima-media thickness > 1.5 mm) was determined by standardized ultrasonography protocol. Lipidomic analysis was performed by 1H NMR. Bivariate and multivariate-adjusted differences in 1H NMR lipidomics were evaluated. RESULTS: We included n = 131 participants (49.6% female, age 46.4 ± 10.3 years, diabetes duration 27.0 ± 9.5 years, 47.3% on statins). Carotid plaques were present in 28.2% of the individuals (n = 12, with ≥ 3 plaques). Glucose (HbA1c), anthropometric (body mass index and waist circumference), and insulin resistance-related (fatty liver index and estimated glucose disposal rate) variables were those most associated with 1H NMR-derived lipidomic analysis (p < 0.01 for all). Regarding preclinical atherosclerosis, sphingomyelin was independently associated with carotid plaque presence (for 0.1 mmol/L increase, OR 0.50 [0.28-0.86]; p = 0.013), even after adjusting for age, sex, hypertension, statin use, mean 5-year HbA1c and diabetes duration. Furthermore, linoleic acid and ω-6 fatty acids remained independently associated with higher plaque burden (≥ 3 plaques) in multivariate models (0.17 [0.03-0.93] and 0.27 [0.07-0.97], respectively; p < 0.05 for both). CONCLUSION: In our preliminary study of individuals with T1D at high risk, several 1H NMR-derived lipidomic parameters were independently associated with preclinical atherosclerosis. Specifically, ω-6 fatty acids and linoleic acid seem promising for identifying those with higher plaque burden.

Prevalence, Abundance, and Virulence of Adherent-Invasive Escherichia coli in Ulcerative Colitis, Colorectal Cancer, and Coeliac Disease.

Background & Aims: Adherent-invasive E. coli (AIEC) has largely been implicated in the pathogenesis of Crohn's disease (CD). E. coli strains with similar genetic backgrounds and virulence genes profiles have been associated with other intestinal disorders, such as ulcerative colitis (UC), colorectal cancer (CRC), and coeliac disease (CeD), but the role of AIEC in these diseases remains unexplored. We aimed to assess the distribution, abundance, and pathogenic features of AIEC in UC, CRC, and CeD. Methods: The AIEC phenotype was investigated in 4,233 E. coli isolated from the ileum and colon of 14 UC and 15 CRC patients and in 38 fecal E. coli strains obtained from 17 CeD and 10 healthy (H) children. AIEC prevalence and abundance were compared with previous data from CD patients and H controls. Clonality, virulence gene carriage, and phylogenetic origin were determined for the AIEC identified. Results: In UC, AIEC prevalence was intermediate between CD and H subjects (UC: 35.7%, CD: 55.0%, H: 21.4%), and similar to CD patients with colonic disease (C-CD: 40.0%). In CRC, the prevalence was lower (6.7%) than these groups. In patients with AIEC, the estimated abundance was similar across all intestinal conditions. All AIEC strains isolated from UC and CRC belonged to the B1 phylogroup, except for a strain of the A phylogroup, and the majority (75% of clonally distinct AIEC) harbored the Afa/Dr operon and the cdt gene. None of the E. coli isolated from the CeD cohort were AIEC. Nonetheless, E. coli strains isolated from active CeD patients showed higher invasion indices than those isolated from H and inactive CeD pediatric patients. Conclusion: We support the hypothesis that AIEC-like strains can be involved not only in CD but also in UC. Further works are needed to study the virulence particularities of these groups of strains and to determine if there is a causative link between AIEC and UC. In contrast, we rule out the possible association of AIEC with CRC. In addition, to further study the E. coli strains in CeD for their possible pathogenic role would be of interest.

A therapeutic education program with a diabetes specialist nurse for type 2 diabetes patients using insulin in a primary care setting. A diabetes education program with a diabetes specialist nurse in a primary care setting.

INTRODUCTION: Therapeutic education is an essential part in the management of type 2 diabetes mellitus (T2D). Implementing a therapeutic education program with the participation of a diabetes specialist nurse (DSN) addressed to patients with T2D using more than 2 insulin injections and sub-optimal metabolic control in primary care (PC) could improve health care and clinical outcomes. Our purpose was to evaluate the clinical, educational and patient satisfaction outcomes of this program. MATERIAL AND METHODS: A prospective, longitudinal study was performed with an evaluation before and after the intervention. The program had a duration of 6 months and included individual on-site, phone and group visits. RESULTS: 184 subjects were included and 161 were finally evaluated. 89.4% were included due to sub-optimal metabolic control and 10.6% because of repeated hypoglycemia. In the first group, the mean reduction in HbA1c was -1.34%±1.45% without any increase in hypoglycemia episodes. In the second group, a significant reduction in hypoglycemia episodes/week was observed (2.52±1.66 vs. 0.53±1.06; p<0.05) without any increase in HbA1c. Learning skills, lifestyle, adherence to care, and the perception of quality of life had significantly improved at 6 months (p<0.05). The overall program was positively evaluated by patients, the role of DSN being considered essential by 98% of the responders. CONCLUSION: A structured therapeutic education program, including a DSN, addressed to insulin treated T2D patients attending primary care facilities and with sub-optimal metabolic control is associated with beneficial effects in terms of clinical, educational and patient satisfaction endpoints.

A Therapeutic Education Program with a Diabetes Specialist Nurse for Type 2 Diabetes Patients Using Insulin in a Primary Care Setting. A Diabetes Education Program with a Diabetes Specialist Nurse in a Primary Care Setting. / Resultados del programa de educación terapéutica de optimización dirigido a pacientes insulinizados con diabetes tipo 2 desarrollado por enfermería de práctica avanzada en diabetes en el ámbito de atención primaria.

INTRODUCTION: Therapeutic education is an essential part in the management of type 2 diabetes mellitus (T2D). Implementing a therapeutic education program with the participation of a diabetes specialist nurse (DSN) addressed to patients with T2D using more than 2insulin injections and sub-optimal metabolic control in primary care (PC) could improve health care and clinical outcomes. Our purpose was to evaluate the clinical, educational and patient satisfaction outcomes of this program. MATERIAL AND METHODS: A prospective, longitudinal study was performed with an evaluation before and after the intervention. The program had a duration of 6 months and included individual on-site, phone and group visits. RESULTS: 184 subjects were included and 161 were finally evaluated. 89.4% were included due to sub-optimal metabolic control and 10.6% because of repeated hypoglycemia. In the first group, the mean reduction in HbA1c was -1.34±1.45% without any increase in hypoglycemia episodes. In the second group, a significant reduction in hypoglycemia episodes/week was observed (2.52±1.66 vs. 0.53±1.06; P<.05) without any increase in HbA1c. Learning skills, lifestyle, adherence to care, and the perception of quality of life had significantly improved at 6 months (P<.05). The overall program was positively evaluated by patients, the role of DSN being considered essential by 98% of the responders. CONCLUSION: A structured therapeutic education program, including a DSN, addressed to insulin treated T2D patients attending primary care facilities and with sub-optimal metabolic control is associated with beneficial effects in terms of clinical, educational and patient satisfaction endpoints.

Why the discovery of adherent-invasive Escherichia coli molecular markers is so challenging?

Adherent-invasive Escherichia coli (AIEC) strains have been extensively related to Crohn's disease (CD) etiopathogenesis. Higher AIEC prevalence in CD patients versus controls has been reported, and its mechanisms of pathogenicity have been linked to CD physiopathology. In CD, the therapeutic armamentarium remains limited and non-curative; hence, the necessity to better understand AIEC as a putative instigator or propagator of the disease is certain. Nonetheless, AIEC identification is currently challenging because it relies on phenotypic assays based on infected cell cultures which are highly time-consuming, laborious and non-standardizable. To address this issue, AIEC molecular mechanisms and virulence genes have been studied; however, a specific and widely distributed genetic AIEC marker is still missing. The finding of molecular tools to easily identify AIEC could be useful in the identification of AIEC carriers who could profit from personalized treatment. Also, it would significantly promote AIEC epidemiological studies. Here, we reviewed the existing data regarding AIEC genetics and presented those molecular markers that could assist with AIEC identification. Finally, we highlighted the problems behind the discovery of exclusive AIEC biomarkers and proposed strategies to facilitate the search of AIEC signature sequences.

Study of a classification algorithm for AIEC identification in geographically distinct E. coli strains.

Adherent-invasive Escherichia coli (AIEC) have been extensively implicated in Crohn's disease pathogenesis. Currently, AIEC is identified phenotypically, since no molecular marker specific for AIEC exists. An algorithm based on single nucleotide polymorphisms was previously presented as a potential molecular tool to classify AIEC/non-AIEC, with 84% accuracy on a collection of 50 strains isolated in Girona (Spain). Herein, our aim was to determine the accuracy of the tool using AIEC/non-AIEC isolates from different geographical origins and extraintestinal pathogenic E. coli (ExPEC) strains. The accuracy of the tool was significantly reduced (61%) when external AIEC/non-AIEC strains from France, Chile, Mallorca (Spain) and Australia (82 AIEC, 57 non-AIEC and 45 ExPEC strains in total) were included. However, the inclusion of only the ExPEC strains showed that the tool was fairly accurate at differentiating these two close pathotypes (84.6% sensitivity; 79% accuracy). Moreover, the accuracy was still high (81%) for those AIEC/non-AIEC strains isolated from Girona and Mallorca (N = 63); two collections obtained from independent studies but geographically close. Our findings indicate that the presented tool is not universal since it would be only applicable for strains from similar geographic origin and demonstrates the need to include strains from different origins to validate such tools.

Amino Acid Substitutions and Differential Gene Expression of Outer Membrane Proteins in Adherent-Invasive Escherichia coli.

Variations in the sequence and/or the expression of outer membrane proteins (OMPs) may modulate bacterial virulence. OmpA and OmpC have been involved in the interaction of adherent-invasive Escherichia coli (AIEC) strain LF82 with intestinal epithelial cells (IECs). Scarce data exist about OMPs sequence variants in a collection of AIEC strains, and no study of OMPs expression during infection exists. We aimed to determine whether particular mutations or differential expression of OMPs are associated with AIEC virulence. The ompA, ompC, and ompF genes in 14 AIEC and 30 non-AIEC strains were sequenced by Sanger method, and the protein expression profile was analyzed by urea-SDS-PAGE. Gene expression was determined during in vitro bacterial infection of intestine-407 cells by RT-qPCR. The distribution of amino acid substitutions in OmpA-A200V, OmpC-S89N, V220I, and W231D associated with pathotype and specific changes (OmpA-A200V, OmpC-V220I, D232A, OmpF-E51V, and M60K) correlated with adhesion and/or invasion indices but no particular variants were found specific of AIEC. OMPs protein levels did not differ according to pathotype when growing in Mueller-Hinton broth. Interestingly, higher OMPs gene expression levels were reported in non-AIEC growing in association with cells compared with those non-AIEC strains growing in the supernatants of infected cultures (p < 0.028), whereas in AIEC strains ompA expression was the only increased when growing in association with cells (p = 0.032), but they did not significantly alter ompC and ompF expression under this condition (p > 0.146). Despite no particular OMPs sequence variants have been found as a common and distinctive trait in AIEC, some mutations could facilitate a better interaction with the host. Moreover, the different behavior between pathotypes regarding OMPs gene expression at different stages of infection could be related with the virulence of the strains.

Genetic and Phenotypic Features to Screen for Putative Adherent-Invasive Escherichia coli.

To date no molecular tools are available to identify the adherent-invasive Escherichia coli (AIEC) pathotype, which has been associated with Crohn's disease and colonizes the intestine of different hosts. Current techniques based on phenotypic screening of isolates are extremely time-consuming. The aim of this work was to search for signature traits to assist in rapid AIEC identification. The occurrence of at least 54 virulence genes (VGs), the resistance to 30 antibiotics and the distribution of FimH and ChiA amino acid substitutions was studied in a collection of 48 AIEC and 56 non-AIEC isolated from the intestine of humans and animals. χ2 test was used to find frequency differences according to origin of isolation, AIEC phenotype and phylogroup. Mann-Whitney test was applied to test association with adhesion and invasion indices. Binary logistic regression was performed to search for variables of predictive value. Animal strains (N = 45) were enriched in 12 VGs while 7 VGs were more predominant in human strains (N = 59). The prevalence of 15 VGs was higher in AIEC (N = 49) than in non-AIEC (N = 56) strains, but only pic gene was still differentially distributed when analyzing human and animal strains separately. Among human strains, three additional VGs presented higher frequency in AIEC strains (papGII/III, iss and vat; N = 22) than in non-AIEC strains (N = 37). No differences between AIEC/non-AIEC were found in FimH variants. In contrast, the ChiA sequence of LF82 was shared with the 35.5% of AIEC studied (N = 31) and only with the 7.4% of non-AIEC strains (N = 27; p = 0.027). Binary logistic regression analysis, using as input variables all the VGs and antibiotic resistances tested, revealed that typifying E. coli isolates using pic gene and ampicillin resistance was useful to correctly classify strains according to the phenotype with a 75.5% of accuracy. Although there is not a molecular signature fully specific and sensitive to identify the AIEC pathotype, we propose two features easy to be tested that could assist in AIEC screening. Future work using additional strain collections would be required to assess the applicability of this method.

Comparative genomics reveals new single-nucleotide polymorphisms that can assist in identification of adherent-invasive Escherichia coli.

Adherent-invasive Escherichia coli (AIEC) have been involved in Crohn's disease (CD). Currently, AIEC are identified by time-consuming techniques based on in vitro infection of cell lines to determine their ability to adhere to and invade intestinal epithelial cells as well as to survive and replicate within macrophages. Our aim was to find signature sequences that can be used to identify the AIEC pathotype. Comparative genomics was performed between three E. coli strain pairs, each pair comprised one AIEC and one non-AIEC with identical pulsotype, sequence type and virulence gene carriage. Genetic differences were further analysed in 22 AIEC and 28 non-AIEC isolated from CD patients and controls. The strain pairs showed similar genome structures, and no gene was specific to AIEC. Three single nucleotide polymorphisms displayed different nucleotide distributions between AIEC and non-AIEC, and four correlated with increased adhesion and/or invasion indices. Here, we present a classification algorithm based on the identification of three allelic variants that can predict the AIEC phenotype with 84% accuracy. Our study corroborates the absence of an AIEC-specific genetic marker distributed across all AIEC strains. Nonetheless, point mutations putatively involved in the AIEC phenotype can be used for the molecular identification of the AIEC pathotype.

Proporción de cáncer de mama en mujeres de 50 a 69 años de Girona según el método de detección / Proportion of breast cancer in women aged 50 to 69 years from Girona, Spain, according to detection method

Fundamento y objetivo: El objetivo de este estudio fue determinar el estadio tumoral, la proporción de casos y la tasa específica por edad de las pacientes con cáncer de mama (CM) según el método de detección. Material y método: Los datos se obtuvieron del Registro de Cáncer de Girona de base poblacional. Se incluyeron las mujeres de 50 a 69 años diagnosticadas de CM en la provincia de Girona durante el período 1999-2006 (n = 1.254). Se clasificaron los CM según el método de detección: cáncer de cribado, cáncer de intervalo y otros. Se calculó la proporción de casos y la tasa específica por edad según el método de detección.Resultados: Durante los años 2002-2006, un 42,2% de los CM diagnosticados en Girona fueron cánceres de cribado, el 52,0% se detectaron fuera del Programa de Detección Precoz del Cáncer de Mama (PDPCM), y el 5,8% fueron cánceres de intervalo. Con la implementación del PDPCM disminuyó la incidencia del CM diagnosticado fuera del programa, aumentó la de los cánceres de cribado y poco después incrementó la de los cánceres de intervalo. Conclusiones: Durante los primeros años del funcionamiento del PDPCM (2002-2006) los casos de cáncer de intervalo representaron un porcentaje bajo (5,8%) respecto el total de CM diagnosticados en mujeres de 50 a 69 años en la provincia de Girona (AU)

Background and objective: The aim of this study was to determine the tumor stage, the proportion of cases and the age specific rate of breast cancer (BC) cases according to detection method. Material and method: Cases of women aged 50 to 69 years diagnosed with BC in the Girona province during 1999-2006 were extracted from the population-based Girona Cancer Registry (n = 1,254). BC was classified by detection method: screen-detected cancer, interval cancer and others. Proportion of cases and age-specific incidence were calculated according to detection method. Results: During the period 2002-2006, the proportion of screen-detected cancers, interval cancers and other cancers were 42.2%, 5.8% and 52.2%, respectively. After implementation of the early detection of breast cancer program (PDPCM), the incidence of screen-detected cases raised; thereafter, interval cancers also increased and the rate of other cancers decreased. Conclusions: In the Girona province during the fully implemented PDPCM period (2002-2006), interval cancers represented a low proportion (5.8%) of women diagnosed with BC at 50 to 69 years old (AU)

[Proportion of breast cancer in women aged 50 to 69 years from Girona, Spain, according to detection method]. / Proporción de cáncer de mama en mujeres de 50 a 69 años de Girona según el método de detección.

BACKGROUND AND OBJECTIVE: The aim of this study was to determine the tumor stage, the proportion of cases and the age specific rate of breast cancer (BC) cases according to detection method. MATERIAL AND METHOD: Cases of women aged 50 to 69 years diagnosed with BC in the Girona province during 1999-2006 were extracted from the population-based Girona Cancer Registry (n=1,254). BC was classified by detection method: screen-detected cancer, interval cancer and others. Proportion of cases and age-specific incidence were calculated according to detection method. RESULTS: During the period 2002-2006, the proportion of screen-detected cancers, interval cancers and other cancers were 42.2%, 5.8% and 52.2%, respectively. After implementation of the early detection of breast cancer program (PDPCM), the incidence of screen-detected cases raised; thereafter, interval cancers also increased and the rate of other cancers decreased. CONCLUSIONS: In the Girona province during the fully implemented PDPCM period (2002-2006), interval cancers represented a low proportion (5.8%) of women diagnosed with BC at 50 to 69 years old.