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Veillonella parvula, prototypical member of the oral and gut microbiota, is at times commensal yet also potentially pathogenic. The definition of the molecular basis tailoring this contrasting behavior is key for broadening our understanding of the microbiota-driven pathogenic and/or tolerogenic mechanisms that take place within our body. In this study, we focused on the chemistry of the main constituent of the outer membrane of V. parvula, the lipopolysaccharide (LPS). LPS molecules indeed elicit pro-inflammatory and immunomodulatory responses depending on their chemical structures. Herein we report the structural elucidation of the LPS from two strains of V. parvula and show important and unprecedented differences in both the lipid and carbohydrate moieties, including the identification of a novel galactofuranose and mannitol-containing O-antigen repeating unit for one of the two strains. Furthermore, by harnessing computational studies, in vitro human cell models, as well as lectin binding solid-phase assays, we discovered that the two chemically diverse LPS immunologically behave differently and have attempted to identify the molecular determinant(s) governing this phenomenon. Whereas pro-inflammatory potential has been evidenced for the lipid A moiety, by contrast a plausible "immune modulating" action has been proposed for the peculiar O-antigen portion.

Veillonella parvula, prototypical member of the oral and gut microbiota, is at times commensal yet also potentially pathogenic. The definition of the molecular basis tailoring this contrasting behavior is key for broadening our understanding of the microbiota driven pathogenic and/or tolerogenic mechanisms that take place within our body. In this study, we focused on the chemistry of the main constituent of the outer membrane of V. parvula, the lipopolysaccharide (LPS). LPS molecules indeed elicit pro-inflammatory and immunomodulatory responses depending on their chemical structures. Here we report about the structural elucidation of the LPS from two strains of V. parvula and show important and unprecedented differences in both the lipid and carbohydrate moieties, including the identification of a novel galactofuranose and mannitol-containing O-antigen repeating unit for one of the two strains. Furthermore, by harnessing computational studies, in vitro human cell models, as well as lectin binding solid phase assays, we discovered that the two chemically diverse LPS immunologically behave differently and have attempted to identify the molecular determinant(s) governing this phenomenon. Whether a pro-inflammatory potential has been evidenced for the lipid A moiety, by contrast a plausible “immune modulating” action has been proposed for the peculiar O-antigen portion.

347 p., Multiple sclerosis (MS) is a chronic inflammatory neurological disease whose underlying cause remains uncertain and can be influenced by different environmental and genetic factors. We have studied two MS-susceptibility genes, IL22RA2 and IL12A, to better understand their regulation and relationship to MS. We demonstrated that the IL22RA2 gene gives rise to three isoforms in monocyte-derived dendritic cells (moDCs).. We also found that CD14++/CD16+ intermediate monocytes produced higher levels of IL22RA2 mRNA than classical and non-classical monocytes. Using transfected human cell lines, we demonstrated that the protein product of IL22RA2 variant 1 (IL22RA2v1), the IL-22BP isoform 1 (IL-22BPi1), was poorly secreted and largely retained in the endoplasmic reticulum (ER). We performed interactome analysis to uncover the mechanism underlying the ER retention of IL-22BPi1 and identified ER chaperones GRP78, GRP94, GRP170 and calnexin as main interactors. Structure-function analysis revealed that, like IL-22BPi2, IL-22BPi1 binds to the substrate-binding domain of GRP78 as well as to the middle domain of GRP94, and thus, we have demonstrated the domain of interaction of IL-22BPi1 and IL-22BPi2 with GRP78 and GRP94. Upon silencing of IL22RA2 expression in moDC, GRP78 levels were significantly reduced, suggesting that native IL22RA2 expression naturally contributes to upregulated GRP78 levels in these cells. Finally, we demonstrated that another variant apart from the canonical IL12A was expressed in moDCs and in different immune and epithelial cells lines. Altogether, our work highlights the relevance of the study of splicing variants of MS susceptibility genes for a better understanding of the effect of the genetic factors in the disease.

Over the course of History, neuroanatomical knowledge has evolved in many ways from philosophical conceptions, over artistic illustrations to medical applications. From human dissections initiated by Andreas Vesalius to modern non-invasive 3-D neuroimaging techniques, our knowledge of the inner structure and function of the nervous system has relied on the continuous development of new Methods. In this Research Topic, we have gathered some of the latest innovations that continue to push the frontiers of the neuroanatomy. For this theme issue “Methods and applications in frontiers in neuroanatomy”, we have selected papers that range from (1) novel approaches to collect, fix, handle and analyze human brains; (2) histological protocols and their applications and (3) approaches to study specific tracks in the nervous system.

262 p., The human IL22RA2 gene co-produces three protein isoforms in dendritic cells (IL-22 binding protein isoform-1 [IL-22BPi1], -2 [IL-22BPi2], and -3 [IL-22BPi3]). Two of these, namely, IL-22BPi2 and IL-22BPi3, are capable of neutralizing the biological activity of IL-22. The function of IL-22BPi1, which differs from IL-22BPi2 through an in-frame, 32-amino-acid insertion provided by an alternatively spliced exon, remains unknown.This thesis focuses on the biochemical characterization of the three isoforms (in silico and in vitro), as well as the potential pharmacological targeting thereof. Additionally, it addresses the functionality of the non-synonymous single nucleotide polymorphism (SNP), rs28385692, which is located in IL22RA2 and has been found to be associated with multiple sclerosis (MS).El gen humano IL22RA2 coproduce tres isoformas en las células dendríticas (la isoforma-1 de la proteína de unión a la IL-22 [IL-22BPi1], la -2 [IL-22BPi2] y la -3 [IL-22BPi3]). Dos de ellas, la IL-22BPi2 y la IL-22BPi3, son capaces de neutralizar la actividad biológica de la IL-22. La función de la IL-22BPi1, que difiere de la IL-22BPi2 por una inserción de 32 aminoácidos mediante splicing alternativo, sigue siendo desconocida. El objetivo general de esta tesis es comprender la función y el destino de la IL-22BP mediante la caracterización bioquímica de sus isoformas. Los obteivos específicos son: estudiar la expresión de los transcritos de IL22RA2 y relacionarlos con la secreción de sus isoformas mediante su caracterización bioquímica, así como identificar los factores clave implicados en su plegamiento y secreción e investigar su secreción y función dentro de la célula; evaluar si las chaperonas identificadas pudieran ser utilizadas como posibles dianas terapéuticas que tuvieran efectos diferenciales en la secreción de las isoformas de IL-22BP; y determinar si la variante de riesgo de la EM, rs28385692, tiene efectos funcionales sobre las isoformas de la IL-22BP y en qué medid

Capítulo 3.1 confidencial .-- Tesis completa 165 p.-- Tesis censurada 156 p., La alergia al polen (polinosis) es una de las alergias más comunes que afecta hasta a un 20 % de la población europea, siendo los pólenes de gramíneas los principales causantes de polinosis. Para el tratamiento de la alergia, la inmunoterapia específica con alérgenos es la única terapia modificadora de la enfermedad con efectos clínicos (mejoría de los síntomas) de larga duración. Sin embargo, los efectos secundarios de la administración de extractos alergénicos limitan la aplicación extensiva de este tipo de inmunoterapia.El objetivo de este trabajo ha sido la generación de una novedosa vacuna para tratar la alergia al polen de gramíneas, a través del desarrollo de una inmunoterapia de segunda generación basada en proteínas recombinantes mediante la estrategia de híbridos hipoalergénicos. En el trabajo se describen las etapas para llevar una idea de laboratorio de investigación a un producto potencialmente industrializable aplicando el concepto de calidad mediante diseño. La inmunoterapia basada en principios activos puros y debidamente caracterizados, permitirá una práctica clínica más segura y eficaz.

Measuring the sources and rates of predation on planktonic protists is of interest from a number of perspectives. In studies of protist population dynamics, mortality rates as well as division rates need to be assessed. From a predator's perspective, the abundance (and specific composition of assemblages) of planktonic protists can markedly influence a predator's growth rate. From the point of view of ecosystem studies, the intensity and selectivity of predation on planktonic protists can alter the pathways of material and energy transfer in pelagic food webs. The present method was developed to measure predation on ciliate protitsts by planktonic suspension-feeding copepods. This prey-predator interaction is addressed because of the suspected importance of ciliates as a prey resource for planktonic metazoans and the likelihood that ciliates are significant internmediaries in the transfer of microbial loop production to larger suspension feeders. The need for new methods to study predation on planktonic ciliates arises because many ciliates are quite fragile and lack recognizable hard parts. Conventional methods for collecting, concentrating, incubating, and fixing planktonic organisms frequently result in lysis of these cells. This is particularly treu fro ciliates in the order Oligotrichida and those members of the Choreotrichida that lack a lorica. Despite the particular focus of the method outlined here, the principles of this immunochemical method should also be applicable to other protist-predator studies.

Weed resistance to glyphosate has been a driving force behind the increased use of alternative herbicides in agriculture. Recently, dicamba-tolerant recombinant plants were introduced to the market, which may result in residues of this agrochemical contaminating environmental waters. Given that restrictions on the use of dicamba have consequently been established by regulatory agencies, it is therefore also desirable to conduct extensive controls on dicamba residues. Immunoassays are currently the most powerful bioanalytical technology for the rapid monitoring of chemical residues and contaminants. In the present study, a novel hapten was designed maintaining unaltered all the antigenic moieties of the target molecule, and this was used to generate high-affinity monoclonal antibodies against dicamba for the first time. Additionally, a collection of haptens with different linker composition or linker tethering site was synthesized and conjugated to proteins. Using these novel immunoreagents, a direct competitive enzyme-linked immunosorbent assay with a limit of detection for dicamba of 0.24 ng/mL was developed and validated. Analysis of water samples from different origins afforded recovery values between 90 % and 120 %, and coefficients of variation below 20 % were obtained. These results indicate that the developed immunochemical assay is suitable for the rapid determination of dicamba residues in environmental water samples.

Measuring the sources and rates of predation on planktonic protists is of interest from a number of perspectives. In studies of protist population dynamics, mortality rates as well as division rates need to be assessed. From a predator's perspective, the abundance (and specific composition of assemblages) of planktonic protists can markedly influence a predator's growth rate. From the point of view of ecosystem studies, the intensity and selectivity of predation on planktonic protists can alter the pathways of material and energy transfer in pelagic food webs. The present method was developed to measure predation on ciliate protitsts by planktonic suspension-feeding copepods. This prey-predator interaction is addressed because of the suspected importance of ciliates as a prey resource for planktonic metazoans and the likelihood that ciliates are significant internmediaries in the transfer of microbial loop production to larger suspension feeders. The need for new methods to study predation on planktonic ciliates arises because many ciliates are quite fragile and lack recognizable hard parts. Conventional methods for collecting, concentrating, incubating, and fixing planktonic organisms frequently result in lysis of these cells. This is particularly treu fro ciliates in the order Oligotrichida and those members of the Choreotrichida that lack a lorica. Despite the particular focus of the method outlined here, the principles of this immunochemical method should also be applicable to other protist-predator studies.

262 p., The human IL22RA2 gene co-produces three protein isoforms in dendritic cells (IL-22 binding protein isoform-1 [IL-22BPi1], -2 [IL-22BPi2], and -3 [IL-22BPi3]). Two of these, namely, IL-22BPi2 and IL-22BPi3, are capable of neutralizing the biological activity of IL-22. The function of IL-22BPi1, which differs from IL-22BPi2 through an in-frame, 32-amino-acid insertion provided by an alternatively spliced exon, remains unknown.This thesis focuses on the biochemical characterization of the three isoforms (in silico and in vitro), as well as the potential pharmacological targeting thereof. Additionally, it addresses the functionality of the non-synonymous single nucleotide polymorphism (SNP), rs28385692, which is located in IL22RA2 and has been found to be associated with multiple sclerosis (MS).El gen humano IL22RA2 coproduce tres isoformas en las células dendríticas (la isoforma-1 de la proteína de unión a la IL-22 [IL-22BPi1], la -2 [IL-22BPi2] y la -3 [IL-22BPi3]). Dos de ellas, la IL-22BPi2 y la IL-22BPi3, son capaces de neutralizar la actividad biológica de la IL-22. La función de la IL-22BPi1, que difiere de la IL-22BPi2 por una inserción de 32 aminoácidos mediante splicing alternativo, sigue siendo desconocida. El objetivo general de esta tesis es comprender la función y el destino de la IL-22BP mediante la caracterización bioquímica de sus isoformas. Los obteivos específicos son: estudiar la expresión de los transcritos de IL22RA2 y relacionarlos con la secreción de sus isoformas mediante su caracterización bioquímica, así como identificar los factores clave implicados en su plegamiento y secreción e investigar su secreción y función dentro de la célula; evaluar si las chaperonas identificadas pudieran ser utilizadas como posibles dianas terapéuticas que tuvieran efectos diferenciales en la secreción de las isoformas de IL-22BP; y determinar si la variante de riesgo de la EM, rs28385692, tiene efectos funcionales sobre las isoformas de la IL-22BP y en qué medid

Bakgrund: Glykerat hemoglobin i blod (B-HbA1c) speglar den genomsnittliga blodsocker-nivån de senaste 8 till 12 veckorna. B-HbA1c används för att följa behandlingen vid diabetes mellitus. B-HbA1c kan mätas på olika sätt och mätresultaten kan ibland skilja sig åt. I detta arbete har en ny immunkemisk metod, HbA1c Advanced (Beckman Coulter, USA) (AU-metoden) utvärderats. Metod: Med patientprover och kontroller, dubbelprover, beräknades imprecision och bias samt korrelation med laboratoriets ordinarie jonbyteskromatografiska metod, Tosoh G11 (G11-metoden). Resultat: Imprecision inomserie blev <3 %, total imprecision 5 %, och bias var -2,8 till -5,3 %. AU-metoden gav i medeltal 2,5 mmol/mol (3,9 %) lägre värde än G11-metoden med en korrelationskoefficient på 0,9967. AU-metoden mätte korrekt de vanligaste hemoglobin-varianterna men inte en patologisk ökning av fraktionen HbF. Slutsats: AU-metodens precision och bias uppfyller nationella kvalitetsmål och korrelationen med laboratoriets ordinarie metod är god för såväl prover utan som med de vanligare hemo-globinvarianterna. Som förväntat för immunkemiska metoder ger AU-metoden för lågt värde vid uttalad ökning av HbF. AU-metoden fungerar så väl som en immunkemisk metod kan göra och bedöms vara lämplig för kliniskt bruk för att mäta B-HbA1c., Background: Glycated hemoglobinin blood (B-HbA1c) reflects the average blood sugar level over the last 8 to 12 weeks. B-HbA1c is used to monitor the treatment of diabetes mellitus. B-HbA1c can be measured in different ways with eventual results that sometimes differ from each other. During this project, a new immunochemical method, HbA1c Advanced (Beckman Coulter, USA) (AU method) was evaluated. Methods: With patient samples, precision and bias were calculated as well as correlation with the laboratory's ordinary ion exchange chromatographic method, Tosoh G11 (G11 method). Results: Imprecision in-series was <3 %, total imprecision was 5 %, and bias was -2.8 to -5.3 %. On average, the AU method gave values that were lower than the G11 method (2.5 mmol/mol (3.9 %) with a correlation coefficient of 0.9967. The AU method correctly measured the most common hemoglobin variants but not a pathological increase in the HbF fraction. Conclusion: The precision and bias of the AU method fulfill the national quality objectives and the correlation with the laboratory's ordinary method, the G11 method is good for samples both with and without the most common hemoglobin variants. As expected for immunochemical methods, the AU method gives too low a value for samples with a pronounced increase in HbF. In conclusion, the AU method fulfills the quality goals as is expected for an immunochemical method for measuring B-hbA1c and is concidered appropriate to use in clincical work.

Bakgrund: Glykerat hemoglobin i blod (B-HbA1c) speglar den genomsnittliga blodsocker-nivån de senaste 8 till 12 veckorna. B-HbA1c används för att följa behandlingen vid diabetes mellitus. B-HbA1c kan mätas på olika sätt och mätresultaten kan ibland skilja sig åt. I detta arbete har en ny immunkemisk metod, HbA1c Advanced (Beckman Coulter, USA) (AU-metoden) utvärderats. Metod: Med patientprover och kontroller, dubbelprover, beräknades imprecision och bias samt korrelation med laboratoriets ordinarie jonbyteskromatografiska metod, Tosoh G11 (G11-metoden). Resultat: Imprecision inomserie blev <3 %, total imprecision 5 %, och bias var -2,8 till -5,3 %. AU-metoden gav i medeltal 2,5 mmol/mol (3,9 %) lägre värde än G11-metoden med en korrelationskoefficient på 0,9967. AU-metoden mätte korrekt de vanligaste hemoglobin-varianterna men inte en patologisk ökning av fraktionen HbF. Slutsats: AU-metodens precision och bias uppfyller nationella kvalitetsmål och korrelationen med laboratoriets ordinarie metod är god för såväl prover utan som med de vanligare hemo-globinvarianterna. Som förväntat för immunkemiska metoder ger AU-metoden för lågt värde vid uttalad ökning av HbF. AU-metoden fungerar så väl som en immunkemisk metod kan göra och bedöms vara lämplig för kliniskt bruk för att mäta B-HbA1c., Background: Glycated hemoglobinin blood (B-HbA1c) reflects the average blood sugar level over the last 8 to 12 weeks. B-HbA1c is used to monitor the treatment of diabetes mellitus. B-HbA1c can be measured in different ways with eventual results that sometimes differ from each other. During this project, a new immunochemical method, HbA1c Advanced (Beckman Coulter, USA) (AU method) was evaluated. Methods: With patient samples, precision and bias were calculated as well as correlation with the laboratory's ordinary ion exchange chromatographic method, Tosoh G11 (G11 method). Results: Imprecision in-series was <3 %, total imprecision was 5 %, and bias was -2.8 to -5.3 %. On average, the AU method gave values that were lower than the G11 method (2.5 mmol/mol (3.9 %) with a correlation coefficient of 0.9967. The AU method correctly measured the most common hemoglobin variants but not a pathological increase in the HbF fraction. Conclusion: The precision and bias of the AU method fulfill the national quality objectives and the correlation with the laboratory's ordinary method, the G11 method is good for samples both with and without the most common hemoglobin variants. As expected for immunochemical methods, the AU method gives too low a value for samples with a pronounced increase in HbF. In conclusion, the AU method fulfills the quality goals as is expected for an immunochemical method for measuring B-hbA1c and is concidered appropriate to use in clincical work.

Background and aimsThe optimal time interval for diagnostic colonoscopy completion after an abnormal stool-based colorectal cancer (CRC) screening test is uncertain. We examined the association between time to colonoscopy and CRC outcomes among individuals who underwent diagnostic colonoscopy after abnormal stool-based screening.MethodsWe performed a retrospective cohort study of veterans age 50 to 75 years with an abnormal fecal occult blood test (FOBT) or fecal immunochemical test (FIT) between 1999 and 2010. We used multivariable Cox proportional hazards to generate CRC-specific incidence and mortality hazard ratios (HRs) and 95% confidence intervals (CI) for 3-month colonoscopy intervals, with 1 to 3 months as the reference group. Association of time to colonoscopy with late-stage CRC diagnosis was also examined.ResultsOur cohort included 204,733 patients. Mean age was 61 years (SD 6.9). Compared with patients who received a colonoscopy at 1 to 3 months, there was an increased CRC risk for patients who received a colonoscopy at 13 to 15 months (HR 1.13; 95% CI 1.00-1.27), 16 to 18 months (HR 1.25; 95% CI 1.10-1.43), 19 to 21 months (HR 1.28; 95% CI: 1.11-1.48), and 22 to 24 months (HR 1.26; 95% CI 1.07-1.47). Compared with patients who received a colonoscopy at 1 to 3 months, mortality risk was higher in groups who received a colonoscopy at 19 to 21 months (HR 1.52; 95% CI 1.51-1.99) and 22 to 24 months (HR 1.39; 95% CI 1.03-1.88). Odds for late-stage CRC increased at 16 months.ConclusionsIncreased time to colonoscopy is associated with higher risk of CRC incidence, death, and late-stage CRC after abnormal FIT/FOBT. Interventions to improve CRC outcomes should emphasize diagnostic follow-up within 1 year of an abnormal FIT/FOBT result.

Introduction: Colorectal cancer (CRC) is the third cancer with the highest incidence in men and women in the Dominican Republic. A high incidence has been found between the presence of occult blood in stool and the expression of CRC, as well as for colorectal polyps. The purpose of this study was to identify tangible data that will demonstrate the use of this test as a cost-effective tool, capable of detecting premalignant CRC lesions. Methodology: An observational-descriptive, series of cases study was carried out within the Hospital General Plaza de la Salud, where patients were analyzed with a fecal immunochemical test (FIT), followed by a colonoscopy within the period of January 2016 to February 2020. Results: 378 patients were found with a positive FIT and a subsequent colonoscopy, of these, 90 patients didn’t present pathological findings, 128 had polyps, and 160 had other types of pathological findings in colonoscopies. Conclusion: The FITs represents a cost-effective tool for screening and prevention of malignant colorectal lesions, and we recommend its use within early colorectal cancer screening., Introducción: el cáncer colorrectal (CCR) es el tercer cáncer con mayor incidencia en hombres y en mujeres en la República Dominicana. Se ha encontrado una elevada incidencia entre la presencia sangre oculta en heces (SOH) y la expresión de CCR, al igual que para la de pólipos colorrectales. El propósito de este estudio fue identificar datos tangibles que demostraran el uso de esta prueba como herramienta costo-efectiva capaz de detectar lesiones premalignas de CCR. Metodología: se realizó un estudio observacional-descriptivo, serie de casos, dentro del Hospital General Plaza de la Salud, donde se analizaron pacientes con una prueba de sangre oculta en heces por inmunohistoquímica positiva (SOHi), que luego se realizaron una colonoscopia dentro del periodo de enero 2016 a febrero 2020. Resultados: se encontraron 378 con pruebas de SOHi positivas, que luego se realizaron colonoscopia, de estos, 90 no presentaron hallazgos patológicos, 128 presentaron pólipos y 160 pacientes presentaron otros tipos de hallazgos patológicos en las colonoscopias. Conclusión: la prueba de SOHi representa una herramienta costo-efectiva para la detección temprana y prevención de lesiones colorrectales malignas, y recomendamos su uso dentro del tamizaje temprano del cáncer colorrectal.

262 p., The human IL22RA2 gene co-produces three protein isoforms in dendritic cells (IL-22 binding protein isoform-1 [IL-22BPi1], -2 [IL-22BPi2], and -3 [IL-22BPi3]). Two of these, namely, IL-22BPi2 and IL-22BPi3, are capable of neutralizing the biological activity of IL-22. The function of IL-22BPi1, which differs from IL-22BPi2 through an in-frame, 32-amino-acid insertion provided by an alternatively spliced exon, remains unknown.This thesis focuses on the biochemical characterization of the three isoforms (in silico and in vitro), as well as the potential pharmacological targeting thereof. Additionally, it addresses the functionality of the non-synonymous single nucleotide polymorphism (SNP), rs28385692, which is located in IL22RA2 and has been found to be associated with multiple sclerosis (MS).El gen humano IL22RA2 coproduce tres isoformas en las células dendríticas (la isoforma-1 de la proteína de unión a la IL-22 [IL-22BPi1], la -2 [IL-22BPi2] y la -3 [IL-22BPi3]). Dos de ellas, la IL-22BPi2 y la IL-22BPi3, son capaces de neutralizar la actividad biológica de la IL-22. La función de la IL-22BPi1, que difiere de la IL-22BPi2 por una inserción de 32 aminoácidos mediante splicing alternativo, sigue siendo desconocida. El objetivo general de esta tesis es comprender la función y el destino de la IL-22BP mediante la caracterización bioquímica de sus isoformas. Los obteivos específicos son: estudiar la expresión de los transcritos de IL22RA2 y relacionarlos con la secreción de sus isoformas mediante su caracterización bioquímica, así como identificar los factores clave implicados en su plegamiento y secreción e investigar su secreción y función dentro de la célula; evaluar si las chaperonas identificadas pudieran ser utilizadas como posibles dianas terapéuticas que tuvieran efectos diferenciales en la secreción de las isoformas de IL-22BP; y determinar si la variante de riesgo de la EM, rs28385692, tiene efectos funcionales sobre las isoformas de la IL-22BP y en qué medid

Mycotoxins represent a major concern for human and animal health because of their harmful effects and high occurrence in food and feed. Rapid immunoanalytical methods greatly contribute to strengthening the safety of our food supply by efficiently monitoring chemical contaminants, so high-affinity and specific antibodies have been generated for almost all internationally regulated mycotoxins. The only exception is patulin, a mycotoxin mainly produced by Penicillium expansum for which such a target has not yet been achieved. Accordingly, no point-of-need tests commonly used in food immunodiagnostics are commercially available for patulin. In the present study, three functionalized derivatives conforming to generally accepted rules in hapten design were firstly tested to generate suitable antibodies for the sensitive immunodetection of patulin. However, these conventional bioconjugates were unable to elicit the desired immune response, so an alternative strategy that takes advantage of the high electrophilic reactivity of patulin was explored. Patulin was reacted with 4-bromothiophenol, and the obtained adduct was used to produce antibodies with nanomolar affinity values. These results demonstrated for the first time that targeting the adduct resulting from the reaction of patulin with a thiol-containing compound is a promising approach for developing user-friendly immunoanalytical techniques for this elusive mycotoxin.

Objective Serous endometrial cancer (USC) is a challenging malignancy associated with metastasis, recurrence and poor outcome. To identify clinically relevant prognostic biomarkers, we focused on a panel of proteins selected after a comprehensive literature review, for tumour profiling of a homogeneous cohort of USC patients. Methods Protein levels and localization were assessed by immunohistochemistry analysis in 36 hysterectomy samples. Tissue sections were stained with the following antibodies: Aurora A, phospho (T288) Aurora A, BRCA1, CHK1, CIP2A, Cyclin B1, Cyclin E, E2F-1, phospho (S364) E2F-1, FBXW7, FOXM1, phospho (S9) GSK3Beta, PLK1, phospho (T210) PLK1, PPP2R1B, p73, RAD51. Each marker was evaluated as a continuously-scaled variable for association with disease progression and death, using Cox proportional hazards models. The sample consisted of 36 patients with USC, half with stage III or IV disease. Results Results showed that higher CHK1 (Checkpoint kinase 1) expression was associated with a decreased risk of progression and death, after adjusting for stage. Interestingly, analysis of a TCGA data set of 109 USC patients corroborates our results showing a favourable prognostic role of CHEK1 after adjusting for stage. Higher FBXW7 (F-box and WD repeat domain containing 7) expression and higher cytoplasmic expression of PPP2R1B (Protein Phosphatase 2 A, Scaffold Subunit Abeta) were each associated with a decreased risk of progression, after adjusting for stage. Conclusions In conclusion, results from the present study identify new clinically relevant biomarkers and potential drug targets for uterine serous endometrial cancer.

BackgroundIn middle-income countries, the burden of colorectal cancer (CRC) is increasing in parallel with resources for diagnosis and treatment. There is a potential benefit of CRC screening programs in Mexico.ObjectiveSince there are no organized screening programs in the country, we explored the willingness of individuals to complete a faecal immunochemical testing (FIT) based CRC screening program and its potential benefit in Mexico.MethodsWe conducted a CRC screening program pilot in Veracruz, Mexico, during 2015-16 using FIT. Individuals with FIT results >100 ng of haemoglobin/ml buffer were referred for diagnostic colonoscopy.ResultsOf 473 FIT kits distributed to adults aged 50-75, 85.8% (406) were completed by participants and analysed in the laboratory. Of these, 5.9% (24/406) of test results showed >100 ng haemoglobin/ml. Twenty-one participants completed colonoscopy. The positive predictive value of FIT >100 ng haemoglobin/ml for premalignant lesions was 33%.ConclusionThese results provide preliminary evidence of the willingness of individuals to complete FIT-based CRC screening program in Mexico. However, further evaluation of health systems resources will be needed prior to large-scale implementation of CRC screening programs.

IntroductionColorectal cancer (CRC) is a major cause of cancer-related morbidity and mortality in the United States. Although various interventions have improved screening rates, they often require abundant resources and can be difficult to implement. Social psychology and behavioral economics principles offer an opportunity for low-cost and easy-to-implement strategies but are less common in clinical settings.MethodsWe randomized 2,000 patients aged 50-75 years eligible for CRC screening to one of the 2 mailed interventions: a previously used text-based letter describing and offering fecal immunochemical testing (FIT) and colonoscopy (usual care arm); or a letter leveraging social psychology and behavioral economics principles (e.g., implied scarcity and choice architecture), minimal text, and multiple images to offer FIT and colonoscopy (intervention arm). We compared total screening uptake, FIT uptake, and colonoscopy uptake at 1-month intervals in each group.ResultsThere were 1,882 patients included in the final analysis. The mean age was 69.3 years, and baseline characteristics in the 2 groups were similar. Screening completion at 26 weeks was 19.5% in the usual care arm (16.3% FIT vs 3.2% colonoscopy, P < 0.01) and 24.1% in the intervention arm (22.1% FIT vs 2.0% colonoscopy, P < 0.01) (P = 0.02).DiscussionAmong primary care patients aged 50-75 years in an academic setting, mailed CRC outreach employing social psychology and behavioral economics principles led to a higher participation in CRC screening than usual care mailed outreach.Translational impactMailed interventions to increase CRC screening should incorporate social psychology and behavioral economics principles to improve participation.

BackgroundThe fecal immunochemical test (FIT) is the second most commonly used colorectal cancer (CRC) screening modality in the United States; yet, follow-up of abnormal FIT results with diagnostic colonoscopy is underutilized. Our objective was to determine patient-reported barriers to diagnostic colonoscopy following abnormal FIT in an academic healthcare setting.MethodsWe included patients age 50-75 with an abnormal FIT result between 1/1/2015 and 10/31/2017 and no documented follow-up diagnostic colonoscopy. We abstracted demographic data from the electronic health record (EHR). Study personnel conducted telephone surveys with patients to confirm colonoscopy completion and elicit data on notification of FIT results and barriers to colonoscopy. We also provided brief verbal education about diagnostic colonoscopy. We calculated frequencies of demographic data and survey responses and compared survey responses by interest in colonoscopy after education.ResultsWe surveyed 67 patients. Fifty-one were aware of the abnormal FIT result, and a majority learned of the abnormal FIT result by direct communication with providers (19, 37.3%) or EHR messaging (11, 21.6%). Overall, fifty-three patients (79.1%) confirmed lack of colonoscopy, citing provider-related (19, 35.8%), patient-related (16, 30.2%), system-related (1, 1.9%), or multifactorial (17, 32.1%) reasons. Lack of knowledge of FIT result (14, 26.4%) was most common. After brief education, 20 (37.7%) patients requested colonoscopy.ConclusionPatients with an abnormal FIT reported various multi-level barriers to diagnostic colonoscopy after abnormal FIT, including knowledge of FIT results. When provided with brief education, participants expressed interest in diagnostic colonoscopy. Future efforts will evaluate interventions to improve colonoscopy follow-up.

Capítulo 3.1 confidencial .-- Tesis completa 165 p.-- Tesis censurada 156 p., La alergia al polen (polinosis) es una de las alergias más comunes que afecta hasta a un 20 % de la población europea, siendo los pólenes de gramíneas los principales causantes de polinosis. Para el tratamiento de la alergia, la inmunoterapia específica con alérgenos es la única terapia modificadora de la enfermedad con efectos clínicos (mejoría de los síntomas) de larga duración. Sin embargo, los efectos secundarios de la administración de extractos alergénicos limitan la aplicación extensiva de este tipo de inmunoterapia.El objetivo de este trabajo ha sido la generación de una novedosa vacuna para tratar la alergia al polen de gramíneas, a través del desarrollo de una inmunoterapia de segunda generación basada en proteínas recombinantes mediante la estrategia de híbridos hipoalergénicos. En el trabajo se describen las etapas para llevar una idea de laboratorio de investigación a un producto potencialmente industrializable aplicando el concepto de calidad mediante diseño. La inmunoterapia basada en principios activos puros y debidamente caracterizados, permitirá una práctica clínica más segura y eficaz.

IntroductionColorectal cancer (CRC) is a major cause of cancer-related morbidity and mortality in the United States. Although various interventions have improved screening rates, they often require abundant resources and can be difficult to implement. Social psychology and behavioral economics principles offer an opportunity for low-cost and easy-to-implement strategies but are less common in clinical settings.MethodsWe randomized 2,000 patients aged 50-75 years eligible for CRC screening to one of the 2 mailed interventions: a previously used text-based letter describing and offering fecal immunochemical testing (FIT) and colonoscopy (usual care arm); or a letter leveraging social psychology and behavioral economics principles (e.g., implied scarcity and choice architecture), minimal text, and multiple images to offer FIT and colonoscopy (intervention arm). We compared total screening uptake, FIT uptake, and colonoscopy uptake at 1-month intervals in each group.ResultsThere were 1,882 patients included in the final analysis. The mean age was 69.3 years, and baseline characteristics in the 2 groups were similar. Screening completion at 26 weeks was 19.5% in the usual care arm (16.3% FIT vs 3.2% colonoscopy, P < 0.01) and 24.1% in the intervention arm (22.1% FIT vs 2.0% colonoscopy, P < 0.01) (P = 0.02).DiscussionAmong primary care patients aged 50-75 years in an academic setting, mailed CRC outreach employing social psychology and behavioral economics principles led to a higher participation in CRC screening than usual care mailed outreach.Translational impactMailed interventions to increase CRC screening should incorporate social psychology and behavioral economics principles to improve participation.

BackgroundIn middle-income countries, the burden of colorectal cancer (CRC) is increasing in parallel with resources for diagnosis and treatment. There is a potential benefit of CRC screening programs in Mexico.ObjectiveSince there are no organized screening programs in the country, we explored the willingness of individuals to complete a faecal immunochemical testing (FIT) based CRC screening program and its potential benefit in Mexico.MethodsWe conducted a CRC screening program pilot in Veracruz, Mexico, during 2015-16 using FIT. Individuals with FIT results >100 ng of haemoglobin/ml buffer were referred for diagnostic colonoscopy.ResultsOf 473 FIT kits distributed to adults aged 50-75, 85.8% (406) were completed by participants and analysed in the laboratory. Of these, 5.9% (24/406) of test results showed >100 ng haemoglobin/ml. Twenty-one participants completed colonoscopy. The positive predictive value of FIT >100 ng haemoglobin/ml for premalignant lesions was 33%.ConclusionThese results provide preliminary evidence of the willingness of individuals to complete FIT-based CRC screening program in Mexico. However, further evaluation of health systems resources will be needed prior to large-scale implementation of CRC screening programs.

BackgroundThe fecal immunochemical test (FIT) is the second most commonly used colorectal cancer (CRC) screening modality in the United States; yet, follow-up of abnormal FIT results with diagnostic colonoscopy is underutilized. Our objective was to determine patient-reported barriers to diagnostic colonoscopy following abnormal FIT in an academic healthcare setting.MethodsWe included patients age 50-75 with an abnormal FIT result between 1/1/2015 and 10/31/2017 and no documented follow-up diagnostic colonoscopy. We abstracted demographic data from the electronic health record (EHR). Study personnel conducted telephone surveys with patients to confirm colonoscopy completion and elicit data on notification of FIT results and barriers to colonoscopy. We also provided brief verbal education about diagnostic colonoscopy. We calculated frequencies of demographic data and survey responses and compared survey responses by interest in colonoscopy after education.ResultsWe surveyed 67 patients. Fifty-one were aware of the abnormal FIT result, and a majority learned of the abnormal FIT result by direct communication with providers (19, 37.3%) or EHR messaging (11, 21.6%). Overall, fifty-three patients (79.1%) confirmed lack of colonoscopy, citing provider-related (19, 35.8%), patient-related (16, 30.2%), system-related (1, 1.9%), or multifactorial (17, 32.1%) reasons. Lack of knowledge of FIT result (14, 26.4%) was most common. After brief education, 20 (37.7%) patients requested colonoscopy.ConclusionPatients with an abnormal FIT reported various multi-level barriers to diagnostic colonoscopy after abnormal FIT, including knowledge of FIT results. When provided with brief education, participants expressed interest in diagnostic colonoscopy. Future efforts will evaluate interventions to improve colonoscopy follow-up.

Capítulo 3.1 confidencial .-- Tesis completa 165 p.-- Tesis censurada 156 p., La alergia al polen (polinosis) es una de las alergias más comunes que afecta hasta a un 20 % de la población europea, siendo los pólenes de gramíneas los principales causantes de polinosis. Para el tratamiento de la alergia, la inmunoterapia específica con alérgenos es la única terapia modificadora de la enfermedad con efectos clínicos (mejoría de los síntomas) de larga duración. Sin embargo, los efectos secundarios de la administración de extractos alergénicos limitan la aplicación extensiva de este tipo de inmunoterapia.El objetivo de este trabajo ha sido la generación de una novedosa vacuna para tratar la alergia al polen de gramíneas, a través del desarrollo de una inmunoterapia de segunda generación basada en proteínas recombinantes mediante la estrategia de híbridos hipoalergénicos. En el trabajo se describen las etapas para llevar una idea de laboratorio de investigación a un producto potencialmente industrializable aplicando el concepto de calidad mediante diseño. La inmunoterapia basada en principios activos puros y debidamente caracterizados, permitirá una práctica clínica más segura y eficaz.

This doctoral thesis deals with the phenomenon of unchanged or even decreased trans-epidermal water loss (TEWL) in the aged skin despite a decreased skin barrier lipids content. To address this task, analysis of structural components of the skin barrier was performed along with analysis of the skin barrier properties by using a wide range of methods. Optimal procedures for the skin sampling and subsequent sample handling and analysis were established as the first step. The second step included the analysis of the epidermal tight junction (TJ) proteins expression, lipid content and the skin barrier properties in the skin affected by ageing to select a potential target responsible for this phenomenon. The last part of this thesis challenged the target molecule occludin with regard to TEWL in the skin with diminished lipid barrier by using an ex vivo porcine skin model., Tato dizertační práce se zabývá fenoménem stabilní nebo dokonce snížené transepidermální ztráty vody (TEWL) ve stárnoucí kůži navzdory nižšímu obsahu bariérových lipidů. K řešení této problematiky bylo nutné analyzovat strukturální komponenty kožní bariéry a biofyzikální parametry kůže, k čemuž byla použitá celá řada analytických metod. Jako první krok byla provedena optimalizace postupu pro odběr vzorku a jeho následného zpracování a analýzy. Další krok zahrnoval analýzu exprese proteinů epidermálních těsných spojů, analýzu obsahu lipidů a analýzu bariérových vlastností v kůži ovlivněné stárnutím, což bylo použito k následné selekci možných cílových molekul zodpovědných za výše uvedený fenomén. Poslední část této práce zkoumá potenciální molekulu okludin ve vztahu k TEWL v kůži se sníženým obsahem lipidů za použití ex vivo modelu prasečí kůže., Fakulta chemicko-technologická, Dokončená práce s úspěšnou obhajobou

The gut microbiome likely plays a role in the etiology of multiple health conditions, especially those affecting the gastrointestinal tract. Little consensus exists as to the best, standard methods to collect fecal samples for future microbiome analysis. We evaluated three distinct populations (N = 132 participants) using 16S rRNA gene amplicon sequencing data to investigate the reproducibility, stability, and accuracy of microbial profiles in fecal samples collected and stored via fecal occult blood test (FOBT) or Flinders Technology Associates (FTA) cards, fecal immunochemical tests (FIT) tubes, 70% and 95% ethanol, RNAlater, or with no solution. For each collection method, based on relative abundance of select phyla and genera, two alpha diversity metrics, and four beta diversity metrics, we calculated intraclass correlation coefficients (ICCs) to estimate reproducibility and stability, and Spearman correlation coefficients (SCCs) to estimate accuracy of the fecal microbial profile. Comparing duplicate samples, reproducibility ICCs for all collection methods were excellent (ICCs ≥75%). After 4-7 days at ambient temperature, ICCs for microbial profile stability were excellent (≥75%) for most collection methods, except those collected via no-solution and 70% ethanol. SCCs comparing each collection method to immediately-frozen no-solution samples ranged from fair to excellent for most methods; however, accuracy of genus-level relative abundances differed by collection method. Our findings, taken together with previous studies and feasibility considerations, indicated that FOBT/FTA cards, FIT tubes, 95% ethanol, and RNAlater are excellent choices for fecal sample collection methods in future microbiome studies. Furthermore, establishing standard collection methods across studies is highly desirable.

Introduction: Between-laboratory variation in the quantification of co-migrating paraproteins in the beta-region of serum protein electrophoresis may adversely impact patient care if the patient uses different pathology services. To identify the level of agreement in the reporting of beta-migrating paraproteins, sample exchanges were conducted in five Australian states and New Zealand (NZ). Method(s): A minimum of four samples containing beta-migrating paraproteins were distributed to participating laboratories. Laboratories were invited to report paraprotein concentration using their routine practice and measure involved total immunoglobulin (Ig) using immunochemical methods. Victorian laboratories were requested to also report 'total beta + paraprotein' by densitometry. Result(s): Thirty-one laboratories returned results. Overall, CVs for quantification of the involved total Ig by immunochemical methods performed better than what was routinely reported in the paraprotein field (1.5-13.7% vs 8.2-21.8% for NSW; 4.1-9.0% vs 13-28.6% for NZ; 7.4-29.4% vs 5.7-67.0% for qld; 0.7-7.3% vs 18.2-43.3% for WA; 0.2-7.2% vs 0-23.6% for SA; and 3.2-9.7% vs 13.8-70.5% for VIC). Amongst Victorian laboratories, CVs for 'total beta+paraprotein' quantification were better than what was reported in the paraprotein field (4.0-18.2% vs 13.8-70.5%). Conclusion(s): This study has highlighted the between-laboratory variation in measurement of beta-migrating paraproteins and the need for harmonisation to improve patient care.Copyright © 2018

209 p., Carbohydrate microarrays are well established tools for high-throughput analysis of carbohydrate-protein interactions and for screening the specificity of carbohydrate processing enzymes, In addition, a few groups have also explored the on-chip synthesis of ligands. The use of small amounts of substrates and the absence of long purification step make the microarray a time-saving approach for the parallel synthesis of hundred scaffolds.We developed a versatile platform to quickly synthesize significant number of glycomimetics via chemoenzymatic protocols and test them as potential ligands for C-type lectin receptors (CLRs). The transparent and the conductive hydrophobic-ITO surface allows to perform binding studies with labeled lectins by fluorescent spectroscopy as well as to estimate conversion rates of the on-chip reactions for every printed ligand by MALDI-TOF MS.

Introduction: Between-laboratory variation in the quantification of co-migrating paraproteins in the beta-region of serum protein electrophoresis may adversely impact patient care if the patient uses different pathology services. To identify the level of agreement in the reporting of beta-migrating paraproteins, sample exchanges were conducted in five Australian states and New Zealand (NZ). Method(s): A minimum of four samples containing beta-migrating paraproteins were distributed to participating laboratories. Laboratories were invited to report paraprotein concentration using their routine practice and measure involved total immunoglobulin (Ig) using immunochemical methods. Victorian laboratories were requested to also report 'total beta + paraprotein' by densitometry. Result(s): Thirty-one laboratories returned results. Overall, CVs for quantification of the involved total Ig by immunochemical methods performed better than what was routinely reported in the paraprotein field (1.5-13.7% vs 8.2-21.8% for NSW; 4.1-9.0% vs 13-28.6% for NZ; 7.4-29.4% vs 5.7-67.0% for qld; 0.7-7.3% vs 18.2-43.3% for WA; 0.2-7.2% vs 0-23.6% for SA; and 3.2-9.7% vs 13.8-70.5% for VIC). Amongst Victorian laboratories, CVs for 'total beta+paraprotein' quantification were better than what was reported in the paraprotein field (4.0-18.2% vs 13.8-70.5%). Conclusion(s): This study has highlighted the between-laboratory variation in measurement of beta-migrating paraproteins and the need for harmonisation to improve patient care.Copyright © 2018

209 p., Carbohydrate microarrays are well established tools for high-throughput analysis of carbohydrate-protein interactions and for screening the specificity of carbohydrate processing enzymes, In addition, a few groups have also explored the on-chip synthesis of ligands. The use of small amounts of substrates and the absence of long purification step make the microarray a time-saving approach for the parallel synthesis of hundred scaffolds.We developed a versatile platform to quickly synthesize significant number of glycomimetics via chemoenzymatic protocols and test them as potential ligands for C-type lectin receptors (CLRs). The transparent and the conductive hydrophobic-ITO surface allows to perform binding studies with labeled lectins by fluorescent spectroscopy as well as to estimate conversion rates of the on-chip reactions for every printed ligand by MALDI-TOF MS.

The gut microbiome likely plays a role in the etiology of multiple health conditions, especially those affecting the gastrointestinal tract. Little consensus exists as to the best, standard methods to collect fecal samples for future microbiome analysis. We evaluated three distinct populations (N = 132 participants) using 16S rRNA gene amplicon sequencing data to investigate the reproducibility, stability, and accuracy of microbial profiles in fecal samples collected and stored via fecal occult blood test (FOBT) or Flinders Technology Associates (FTA) cards, fecal immunochemical tests (FIT) tubes, 70% and 95% ethanol, RNAlater, or with no solution. For each collection method, based on relative abundance of select phyla and genera, two alpha diversity metrics, and four beta diversity metrics, we calculated intraclass correlation coefficients (ICCs) to estimate reproducibility and stability, and Spearman correlation coefficients (SCCs) to estimate accuracy of the fecal microbial profile. Comparing duplicate samples, reproducibility ICCs for all collection methods were excellent (ICCs ≥75%). After 4-7 days at ambient temperature, ICCs for microbial profile stability were excellent (≥75%) for most collection methods, except those collected via no-solution and 70% ethanol. SCCs comparing each collection method to immediately-frozen no-solution samples ranged from fair to excellent for most methods; however, accuracy of genus-level relative abundances differed by collection method. Our findings, taken together with previous studies and feasibility considerations, indicated that FOBT/FTA cards, FIT tubes, 95% ethanol, and RNAlater are excellent choices for fecal sample collection methods in future microbiome studies. Furthermore, establishing standard collection methods across studies is highly desirable.

Botulinum neurotoxins (BoNTs) comprise seven agreed-on serotypes, A through G. In 2014, a novel chimeric neurotoxin produced by clostridial strain IBCA10-7060 was reported as BoNT/H, with subsequent names of BoNT/FA or BoNT/HA based on sequence homology of the N-terminus to BoNT/F, the C-terminus to BoNT/A and neutralization studies. The purpose of this study was to define the immunologic identity of the novel BoNT. monoclonal antibodies (mAbs) to the novel BoNT/H N-terminus were generated by antibody repertoire cloning and yeast display after immunization with BoNT/H LC-HN or BoNT/F LC-HN. 21 unique BoNT/H LC-HN mAbs were obtained; 15 from the BoNT/H LC-HN immunized library (KD 0.78 nM to 182 nM) and six from the BoNT/F-immunized libraries (KD 20.5 nM to 1490 nM). A total of 15 of 21 mAbs also bound catalytically inactive BoNT/H holotoxin. The mAbs bound nine non-overlapping epitopes on the BoNT/H LC-HN. None of the mAbs showed binding to BoNT serotypes A-G, nor any of the seven subtypes of BoNT/F, except for one mAb that weakly bound BoNT/F5. The results, combined with the chimeric structure and neutralization by anti-A, but not anti-F antitoxin indicate that immunologically the novel BoNT is BoNT/HA. This determination has significant implications for existing countermeasures and potential vulnerabilities.

Immunoanalytical techniques are key methods of application in clinical diagnostics, genomics, proteomics and other biochemical and molecular biology disciplines. Most often, they are based on the ability of labeled antibodies to bind specific antigens. It is possible to use a large variety of nanomaterials that are designed, synthesized and adapted to allow highly sensitive detection of advanced immunoassays. Detection can be a highly efficient analytical method of laser ablation followed by inductively coupled plasma mass spectrometry (LA-ICP-MS), which allows the detection of elemental tags suitably conjugated to antibodies. The aim of this work was to conjugate model anti-mouse antibody on a surface of 10nm and 60nm gold nanoparticles and choose the better one for conjugation experimentally by using dot-blot immunobinding assay followed by LA-ICP-MS. It has been experimentally proven that 10nm gold nanoparticles are more suitable for conjugation with antibodies because of lower non-specific sorption on a membrane.

BACKGROUND:The effectiveness of colorectal cancer screening with fecal immunochemical tests (FITs) of stool blood depends on high rates of colonoscopy follow-up for abnormal FITs and the use of high-quality tests. This study characterized colonoscopy referral and completion among patients with abnormal FITs and the types of FITs implemented in a sample of Southern California Federally Qualified Health Centers (FQHCs). METHODS:FQHCs in San Diego, Imperial, and Los Angeles Counties were invited to define a cohort of ≥150 consecutive patients with abnormal FITs in 2015-2016 and to provide data on sex, insurance status, diagnostic colonoscopy referrals and completion within 6 months of abnormal FITs, and the types (brands) of FITs implemented. The primary outcomes were the proportions with colonoscopy referrals and completion for all patients at each FQHC and in aggregate. RESULTS:Eight FQHCs provided data for 1229 patients with abnormal FITs; 46% were male, and 20% were uninsured. Among patients with abnormal FITs, 89% (1091 of 1229; 95% confidence interval [CI], 0.87-0.91) had a colonoscopy referral, and 44% (539 of 1229; 95% CI, 0.41-0.47) had colonoscopy completion. Across FQHCs, the range for colonoscopy referral was 73% to 96%, and the range for completion was 18% to 57%. Six of the 8 FQHCs (75%) reported FIT brands with limited data to support their effectiveness. CONCLUSIONS:In a sample of Southern California FQHCs, diagnostic colonoscopy completion after abnormal FITs was substantially below the nationally recommended benchmark to achieve 80% completion, and the use of FIT brands with limited data to support their effectiveness was high. These findings suggest a need for policies and multilevel interventions to promote diagnostic colonoscopy among individuals with abnormal FITs and the use of higher quality FITs.

Background & aimsThe fecal immunochemical test (FIT) is an alternative to colonoscopy and can increase overall screening for colorectal cancer (CRC). However, little is known about the frequency of and reasons for mishandled FIT samples.MethodsWe performed a prospective study, nested within a randomized controlled trial of patients, recruited from December 2015 through August 2017, who were not up to date with colorectal cancer screening (50-75 years old). The patients were randomly assigned to usual care or outreach groups that received a mailed FIT with low literacy level instructions or a reminder call, or both. We examined frequency of and reasons for mishandled FIT samples, including absence of collection date; time from collection to laboratory receipt of more than 14 days; or mishandling of stool, buffer, or cap. The outcomes were the frequency of mishandled FIT samples, effects of outreach on mishandling, and positive results from the FIT among proper and mishandled samples.ResultsFIT samples were returned from 1871 patients assigned to usual care and 3045 who received the low literacy level instructions and a reminder call. In total, 19.8% of samples were mishandled; most of these (93.7%) had not labeled the date of stool collection but were still processed. Of the received samples, 1.2% of were not processed because the time from patient collection to laboratory receipt was more than 14 days. Outreach was associated with a lower proportion of mishandled samples (16.5% vs 25.0% for usual care; P < .0001). The proportion of mishandled samples was lowest among patients who received the low literacy level instruction and a reminder call (12.8%, P < .0001). There was no significant difference in proportions of positive results between properly processed samples (7.5%) and improperly processed samples (6.2%) (P = .14).ConclusionIn a prospective study of patients who were not up to date with colorectal cancer screening, we found that almost 20% of

Laser ablation followed by inductively coupled plasma mass spectrometry is an analytical method suitable for low concentration measurement of elements in a sample. This can be used not only for detection of elemental distribution within a sample but also as a detection means for immunoanalytical techniques using nanoparticle-antibody conjugates as recognition elements. The aim of this work was to prepare a conjugate of 10nm gold nanoparticles with anti-p53 antibody, to verify the antigen binding ability, and to use this construct for identification of p53 in a real sample of cell lysate. It has been experimentally verified that it is possible to use nanoparticle-modified antibodies for immunochemical analysis coupled to LA-ICP-MS detection. However, the nonspecific sorption of nanoparticles on the sample surface needs to be minimized by optimization of the blocking step.

Laser ablation followed by inductively coupled plasma mass spectrometry is an analytical method suitable for low concentration measurement of elements in a sample. This can be used not only for detection of elemental distribution within a sample but also as a detection means for immunoanalytical techniques using nanoparticle-antibody conjugates as recognition elements. The aim of this work was to prepare a conjugate of 10nm gold nanoparticles with anti-p53 antibody, to verify the antigen binding ability, and to use this construct for identification of p53 in a real sample of cell lysate. It has been experimentally verified that it is possible to use nanoparticle-modified antibodies for immunochemical analysis coupled to LA-ICP-MS detection. However, the nonspecific sorption of nanoparticles on the sample surface needs to be minimized by optimization of the blocking step.

Laser ablation followed by inductively coupled plasma mass spectrometry is an analytical method suitable for low concentration measurement of elements in a sample. This can be used not only for detection of elemental distribution within a sample but also as a detection means for immunoanalytical techniques using nanoparticle-antibody conjugates as recognition elements. The aim of this work was to prepare a conjugate of 10nm gold nanoparticles with anti-p53 antibody, to verify the antigen binding ability, and to use this construct for identification of p53 in a real sample of cell lysate. It has been experimentally verified that it is possible to use nanoparticle-modified antibodies for immunochemical analysis coupled to LA-ICP-MS detection. However, the nonspecific sorption of nanoparticles on the sample surface needs to be minimized by optimization of the blocking step.

Immunoanalytical techniques are key methods of application in clinical diagnostics, genomics, proteomics and other biochemical and molecular biology disciplines. Most often, they are based on the ability of labeled antibodies to bind specific antigens. It is possible to use a large variety of nanomaterials that are designed, synthesized and adapted to allow highly sensitive detection of advanced immunoassays. Detection can be a highly efficient analytical method of laser ablation followed by inductively coupled plasma mass spectrometry (LA-ICP-MS), which allows the detection of elemental tags suitably conjugated to antibodies. The aim of this work was to conjugate model anti-mouse antibody on a surface of 10nm and 60nm gold nanoparticles and choose the better one for conjugation experimentally by using dot-blot immunobinding assay followed by LA-ICP-MS. It has been experimentally proven that 10nm gold nanoparticles are more suitable for conjugation with antibodies because of lower non-specific sorption on a membrane.

Immunoanalytical techniques are key methods of application in clinical diagnostics, genomics, proteomics and other biochemical and molecular biology disciplines. Most often, they are based on the ability of labeled antibodies to bind specific antigens. It is possible to use a large variety of nanomaterials that are designed, synthesized and adapted to allow highly sensitive detection of advanced immunoassays. Detection can be a highly efficient analytical method of laser ablation followed by inductively coupled plasma mass spectrometry (LA-ICP-MS), which allows the detection of elemental tags suitably conjugated to antibodies. The aim of this work was to conjugate model anti-mouse antibody on a surface of 10nm and 60nm gold nanoparticles and choose the better one for conjugation experimentally by using dot-blot immunobinding assay followed by LA-ICP-MS. It has been experimentally proven that 10nm gold nanoparticles are more suitable for conjugation with antibodies because of lower non-specific sorption on a membrane.

Immunoanalytical techniques are key methods of application in clinical diagnostics, genomics, proteomics and other biochemical and molecular biology disciplines. Most often, they are based on the ability of labeled antibodies to bind specific antigens. It is possible to use a large variety of nanomaterials that are designed, synthesized and adapted to allow highly sensitive detection of advanced immunoassays. Detection can be a highly efficient analytical method of laser ablation followed by inductively coupled plasma mass spectrometry (LA-ICP-MS), which allows the detection of elemental tags suitably conjugated to antibodies. The aim of this work was to conjugate model anti-mouse antibody on a surface of 10nm and 60nm gold nanoparticles and choose the better one for conjugation experimentally by using dot-blot immunobinding assay followed by LA-ICP-MS. It has been experimentally proven that 10nm gold nanoparticles are more suitable for conjugation with antibodies because of lower non-specific sorption on a membrane.

Laser ablation followed by inductively coupled plasma mass spectrometry is an analytical method suitable for low concentration measurement of elements in a sample. This can be used not only for detection of elemental distribution within a sample but also as a detection means for immunoanalytical techniques using nanoparticle-antibody conjugates as recognition elements. The aim of this work was to prepare a conjugate of 10nm gold nanoparticles with anti-p53 antibody, to verify the antigen binding ability, and to use this construct for identification of p53 in a real sample of cell lysate. It has been experimentally verified that it is possible to use nanoparticle-modified antibodies for immunochemical analysis coupled to LA-ICP-MS detection. However, the nonspecific sorption of nanoparticles on the sample surface needs to be minimized by optimization of the blocking step.

209 p., Carbohydrate microarrays are well established tools for high-throughput analysis of carbohydrate-protein interactions and for screening the specificity of carbohydrate processing enzymes, In addition, a few groups have also explored the on-chip synthesis of ligands. The use of small amounts of substrates and the absence of long purification step make the microarray a time-saving approach for the parallel synthesis of hundred scaffolds.We developed a versatile platform to quickly synthesize significant number of glycomimetics via chemoenzymatic protocols and test them as potential ligands for C-type lectin receptors (CLRs). The transparent and the conductive hydrophobic-ITO surface allows to perform binding studies with labeled lectins by fluorescent spectroscopy as well as to estimate conversion rates of the on-chip reactions for every printed ligand by MALDI-TOF MS.

Standardization programs for thyroid hormones have revealed bias between immunochemical methods and the reference method ED-ID-LC/MS. Lack of standardization between methods, suboptimal reference intervals and replacement of serum with plasma may compromise the capability of the immunochemical thyroid methods to diagnose thyroid disease. To accommodate the demand for faster turn-around times for laboratory replies, we replaced serum with plasma on some serum CE marked thyroid methods. This forced us to do on-board analytical correction for the plasma total T4 (TT4) method on ADVIA Centaur ® XP. We, next, validated the capability of the ADVIA Centaur ® XP thyroid methods on plasma by (1) first carrying out a prospective method comparison with the ED-ID-LC/MS reference method using collected plasma samples, (2) we verified the clinical reference intervals by analyzing collected plasma samples from healthy individuals, and (3) retrospectively compared laboratory results from two different time periods using serum TT4 and serum total triiodothyronine (TT3) versus plasma free thyroxine (FT4) and plasma TT3, respectively, to diagnose thyroid disease. The plasma FT4 method displayed a negative concentration-dependent bias against the reference method. This bias was apparently counteracted by a fitted reference interval for the plasma FT4 method. Indeed, overt hyperthyroid disease was found in 1.0% and 1.1% of the cases using serum and plasma and overt hypothyroid condition were in 1.3% and 0.6% of the cases using serum and plasma, respectively. In conclusion, the ADVIA Centaur ® XP FT4 method displayed a negative bias at high plasma FT4 concentrations against the reference method, but the diagnostic performance was not compromised due to a fitted reference interval.

IntroductionColorectal cancer (CRC) is a common but largely preventable disease with suboptimal screening rates despite national guidelines to screen individuals age 50-75. Single-component interventions aimed to improve screening uptake only modestly improve rates; data suggest that multi-modal approaches may be more effective.MethodsWe designed, implemented, and evaluated the impact of a multi-modal intervention on CRC screening uptake among unscreened patients in a large managed care population. Patient-level components included a mailed letter with education about screening options and pre-colonoscopy telephone counseling. For providers, we facilitated communication of screening test results and work-flow for abnormal results. System-level modifications included establishment of a patient navigator, expedited work-up for abnormal results, and stream-lined colonoscopy scheduling. We measured the rate of screening uptake overall, screening uptake by modality, change in the proportion of the population screened, and positive fecal immunochemical test (FIT) follow-up rates in the 1-year study period.ResultsThere were 5093 patients in the intervention cohort. Of these, 33.2% participated in FIT or colonoscopy screening within 1 year of the mailing. A total of 1078 (21.2%) participants completed a FIT and 611 (12.0%) completed a screening colonoscopy. The screening rate in the managed care population increased from 65.1 to 76.6%. Fifty-nine patients (5.5%) had a positive FIT, of which 30 (50.8%) completed a diagnostic colonoscopy.ConclusionMulti-modal interventions can result in substantial improvement in CRC screening uptake in large and diverse managed care populations.Translational impactHealth systems should shift their focus from single-level to multi-level interventions when addressing barriers to CRC screening.

184 p., O-glycans from the helminth Schistosoma mansoni represent promising leads in the development of immunomodulatory compounds via the interaction with C-type lectin receptors (CLRs), as reported for DC-SIGN, L-SIGN, Langerin and MGL. We undertook the chemoenzymatic synthesis of an O-glycan library inspired from structures previously isolated from the helminth. We described the synthesis of parasitic O-glycan cores mucin core 2 and the S.mansoni specific core. Enzymatic elongations towards a library of O-glycans were limited to the Galß-1,6GalNAc arm of the cores owing to an unanticipated acceptor specificity of the recombinant glycosyltransferase LgtA_X. Synthesized as aminopentyl glycosides, eight O-glycans were obtained and were printed on-chip alongside N-glycans available from our laboratory. The array was enzymatically modified using a FucT from H.pylori in conjunction with either a fucose or 6-azido-fucose donor and arrayed against a selection of CLRs, revealing interesting features in the CLR specificity and suggesting the existence of exploitable chemical space for CLR targeting.

184 p., O-glycans from the helminth Schistosoma mansoni represent promising leads in the development of immunomodulatory compounds via the interaction with C-type lectin receptors (CLRs), as reported for DC-SIGN, L-SIGN, Langerin and MGL. We undertook the chemoenzymatic synthesis of an O-glycan library inspired from structures previously isolated from the helminth. We described the synthesis of parasitic O-glycan cores mucin core 2 and the S.mansoni specific core. Enzymatic elongations towards a library of O-glycans were limited to the Galß-1,6GalNAc arm of the cores owing to an unanticipated acceptor specificity of the recombinant glycosyltransferase LgtA_X. Synthesized as aminopentyl glycosides, eight O-glycans were obtained and were printed on-chip alongside N-glycans available from our laboratory. The array was enzymatically modified using a FucT from H.pylori in conjunction with either a fucose or 6-azido-fucose donor and arrayed against a selection of CLRs, revealing interesting features in the CLR specificity and suggesting the existence of exploitable chemical space for CLR targeting.

IntroductionColorectal cancer (CRC) is a common but largely preventable disease with suboptimal screening rates despite national guidelines to screen individuals age 50-75. Single-component interventions aimed to improve screening uptake only modestly improve rates; data suggest that multi-modal approaches may be more effective.MethodsWe designed, implemented, and evaluated the impact of a multi-modal intervention on CRC screening uptake among unscreened patients in a large managed care population. Patient-level components included a mailed letter with education about screening options and pre-colonoscopy telephone counseling. For providers, we facilitated communication of screening test results and work-flow for abnormal results. System-level modifications included establishment of a patient navigator, expedited work-up for abnormal results, and stream-lined colonoscopy scheduling. We measured the rate of screening uptake overall, screening uptake by modality, change in the proportion of the population screened, and positive fecal immunochemical test (FIT) follow-up rates in the 1-year study period.ResultsThere were 5093 patients in the intervention cohort. Of these, 33.2% participated in FIT or colonoscopy screening within 1 year of the mailing. A total of 1078 (21.2%) participants completed a FIT and 611 (12.0%) completed a screening colonoscopy. The screening rate in the managed care population increased from 65.1 to 76.6%. Fifty-nine patients (5.5%) had a positive FIT, of which 30 (50.8%) completed a diagnostic colonoscopy.ConclusionMulti-modal interventions can result in substantial improvement in CRC screening uptake in large and diverse managed care populations.Translational impactHealth systems should shift their focus from single-level to multi-level interventions when addressing barriers to CRC screening.

Regular use of colorectal cancer screening can reduce incidence and mortality, but participation rates remain low among low-income, Spanish-speaking Latino adults. We conducted two distinct pilot studies testing the implementation of evidence-based interventions to promote fecal immunochemical test (FIT) screening among Latinos aged 50-75 years who were not up-to-date with CRC screening (n = 200) at a large Federally Qualified Health Center (FQHC) in San Diego, CA. One pilot focused on an opportunistic clinic visit in-reach intervention including a 30-min session with a patient navigator, review of an educational flip-chart, and a take-home FIT kit with instructions. The second pilot was a system-level outreach intervention consisting of mailed materials (i.e., FIT kit, culturally and linguistically tailored instructions, and a pre-paid return envelope). Both received follow-up calls to promote screening completion and referrals for additional screening and treatment if needed. The primary outcome was FIT kit completion and return within 3 months assessed through electronic medical records. The in-reach pilot consisted of mostly insured (85%), women (82%), and Spanish-speaking (88%) patients. The outreach pilot consisted of mostly of Spanish-speaking (73%) women (64%), half of which were insured (50%). At a 3-month follow-up, screening completion was 76% for in-reach and 19% for outreach. These data demonstrate that evidence-based strategies to promote CRC screening can be implemented successfully within FQHCs, but implementation (particularly of mailed outreach) may require setting and population-specific optimization. Patient, provider, and healthcare system related implementation approaches and lessons learned from this study may be implemented in other primary care settings.