RESUMO
Although mortality caused by Sarcoptes scabiei has been reported in European wild rabbit (Oryctolagus cuniculus) and Iberian hare (Lepus granatensis), there is a lack of detailed information regarding the exposure of wild lagomorph species to this parasite. Here, we aimed to determine the seroprevalence and potential risk factors associated with S. scabiei exposure in European wild rabbits and Iberian hares in Mediterranean ecosystems of southern Spain. Between 2018/2019 and 2021/2022 hunting seasons, serum samples from 464 wild rabbits and 132 Iberian hares were collected from 100 hunting grounds in Andalusia (southern Spain). Sera were tested using an in-house indirect ELISA to detect specific anti-S. scabiei antibodies based on the immunodominant protein Ssλ20ΔB3. The overall apparent individual seroprevalence was 15.9% (95/596; 95%CI: 13.0-18.9). Antibodies against S. scabiei were detected in 11.6% (54/464; 95%CI: 8.7-14.5) of the European wild rabbits and 31.1% (41/132; 95%CI: 23.2-39.0) of the Iberian hares. Species (Iberian hare), age (adults) and geographical area (western Andalusia) were identified as risk factors potentially associated with S. scabiei exposure using generalized estimating equation analysis. By applying spatial analysis, two significant cluster of high seropositivity were detected in western and central Andalusia, respectively. The seroprevalence values obtained provide evidence of endemic, widespread and heterogeneous exposure to S. scabiei among wild lagomorph populations in Spanish Mediterranean ecosystems. Our findings underscore the importance of implementing integrated surveillance programs for sarcoptic mange in wild lagomorphs as well as in other sympatric species.
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Animais Selvagens , Sarcoptes scabiei , Escabiose , Animais , Espanha/epidemiologia , Escabiose/veterinária , Escabiose/epidemiologia , Sarcoptes scabiei/imunologia , Fatores de Risco , Coelhos/parasitologia , Estudos Soroepidemiológicos , Animais Selvagens/parasitologia , Lebres/parasitologia , Feminino , Ecossistema , Masculino , Lagomorpha/parasitologiaRESUMO
Tuberculosis BCG vaccination induced non-specific protective effects in humans led to postulate the concept of trained immunity (TRAIM) as an innate type of immune mechanism that triggered by a pathogen, protects against others. Killed vaccines have been considered not to be effective. However, field efficacy of a commercial vaccine against paratuberculosis, as well as of a recently developed M. bovis heat-inactivated vaccine (HIMB) prompted to test whether it could also induce TRAIM. To this, we used a sarcoptic mange rabbit model. Twenty-four weaned rabbits were treated orally or subcutaneously with a suspension of either HIMB (107 UFC) or placebo. Eighty-four days later the animals were challenged with approximately 5000 S. scabiei mites on the left hind limb. Skin lesion extension was measured every 2 weeks until 92 days post-infection (dpi). Two animals were killed at 77 dpi because of extensive skin damage. The rest were euthanized and necropsied and the lesion area and the mite burden per squared cm were estimated. Specific humoral immune responses to S. scabiei and to M. bovis were investigated with the corresponding specific ELISA tests. Subcutaneously and orally HIMB vaccinated animals compared with placebo showed reduced lesion scores (up to 74% and 62%, respectively) and mite counts (-170% and 39%, respectively). This, together with a significant positive correlation (r = 0.6276, p = 0.0031) between tuberculosis-specific antibodies and mite count at 92 dpi supported the hypothesis of non-specific effects of killed mycobacterial vaccination. Further research is needed to better understand this mechanism to maximize cross protection.
Assuntos
Mycobacterium bovis , Escabiose , Tuberculose , Humanos , Coelhos , Animais , Escabiose/prevenção & controle , Escabiose/veterinária , Tuberculose/veterinária , Ensaio de Imunoadsorção Enzimática/veterinária , Imunidade Humoral , Vacinas de Produtos Inativados , Vacina BCGRESUMO
The lack of sensitive diagnostic methods to detect Mycobacterium avium subsp. paratuberculosis (Map) subclinical infections has hindered the control of paratuberculosis (PTB). The serum proteomic profiles of naturally infected cows presenting focal and diffuse pathological forms of PTB and negative controls (n = 4 per group) were analyzed using TMT-6plex quantitative proteomics. Focal and diffuse are the most frequent pathological forms in subclinical and clinical stages of PTB, respectively. One (focal versus (vs.) control), eight (diffuse vs. control), and four (focal vs. diffuse) differentially abundant (DA) proteins (q-value < 0.05) were identified. Ingenuity pathway analysis of the DA proteins revealed changes in the acute-phase response and lipid metabolism. Six candidate biomarkers were selected for further validation by specific ELISA using serum from animals with focal, multifocal, and diffuse PTB-associated lesions (n = 108) and controls (n = 56). Overall, the trends of the serum expression levels of the selected proteins were consistent with the proteomic results. Alpha-1-acid glycoprotein (ORM1)-based ELISA, insulin-like growth factor-binding protein 2 (IGFBP2)-based ELISA, and the anti-Map ELISA had the best diagnostic performance for detection of animals with focal, multifocal, and diffuse lesions, respectively. Our findings identify potential biomarkers that improve diagnostic sensitivity of PTB and help to elucidate the mechanisms involved in PTB pathogenesis.
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Bovine paratuberculosis (PTB) is a chronic granulomatous enteritis, caused by Mycobacterium avium subsp. paratuberculosis (Map). The progression of PTB from subclinical to the clinical stage of the disease is determined locally at the level of the granuloma, a host defence hallmark against mycobacterial infection. Therefore, in-depth characterization of distinct cell populations controlling granuloma formation is critical to understanding PTB progression. Confocal laser scanning microscopy (CLSM) has been extensively used to visualize two or more proteins of interest concomitantly within a variety of cellular structures. As such, it is an invaluable tool for the correct identification and characterization of different cell populations. In this study, a novel approach, CLSM of whole-mount small intestinal mucosa samples, is used to characterize three-dimensional (3-D) paratuberculosis granulomas and epithelioid macrophages. Detailed optimized procedures to perform CLSM in whole mount small intestinal mucosa samples and also in formalin fixed paraffin embedded (FFPE) intestinal tissue sections of Holstein Friesian cows presenting different types of PTB-associated histological lesions are described.
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Doenças dos Bovinos , Doenças Inflamatórias Intestinais , Mycobacterium avium subsp. paratuberculosis , Paratuberculose , Feminino , Bovinos , Animais , Paratuberculose/microbiologia , Doenças dos Bovinos/microbiologia , Granuloma/veterinária , Mucosa Intestinal/patologia , Doenças Inflamatórias Intestinais/veterinária , Coloração e Rotulagem/veterinária , Imunofluorescência/veterináriaRESUMO
Bovine paratuberculosis (PTB) is an infectious disease that affects ruminants worldwide and is a burden on the dairy industry. PTB control measures include culling of Mycobacterium avium subsp. paratuberculosis (MAP)-infected animals from the herd and the enhancement of farm-biosecurity measures. Diagnostics tools for the direct detection of MAP are fecal real-time qPCR and bacteriological culture, the last one being considered the gold standard. However, both show limitations for detecting subclinical MAP-infected cattle with low bacterial load in feces and gut tissues. Droplet digital polymerase chain reaction (ddPCR) is a third-generation PCR method that shows high reproducibility for the quantification of low DNA copy numbers. The objective of this study was to design a ddPCR assay to detect and quantify a fragment of the F57 MAP-specific sequence in samples of naturally MAP-infected Holstein cattle. DNA was isolated from whole-blood and fecal samples from control cows with a negative ELISA and qPCR result (N = 75) and from cows with PTB-associated focal (N = 32), multifocal (N = 21), and diffuse lesions (N = 17) in gut tissues. After ddPCR, the DNA extracted from fecal samples of cows with diffuse lesions showed higher mean copies per microliter (13,791.2 copies/µl) than samples from cows with multifocal lesions (78.8 copies/µl), focal lesions (177.1 copies/µl) or control cows (4.8 copies/µl) (P ≤ 0.05). Significant differences in mean DNA copies/µl were also observed in the blood samples from cows with focal lesions (47.7 copies/µl) when compared with cows with multifocal and diffuse lesions; 18.1 and 12.4 copies/µl, respectively. Using a principal component analysis, the results of the fecal ddPCR clustered together with the results of a commercial ELISA for the specific detection of MAP antibodies, fecal and tissue qPCR, and bacteriological culture results. In contrast, blood ddPCR results clustered together with the results of an ELISA for the detection of a biomarker of subclinical PTB, the ABCA13 transporter. Blood ddPCR was the most sensitive tool (sensitivity 71%, specificity 100%) of all the quantitative methods used in the study for the detection of subclinical cows with focal lesions.
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BACKGROUND: Clinical pathways (CPs) are usually expressed by means of workflow formalisms, providing health care personnel with an easy-to-understand, high-level conceptual model of medical steps in specific patient conditions, thereby improving overall health care process quality in clinical practice. From a standardized perspective, the business process model and notation (BPMN), a widely spread general-purpose process formalism, has been used for conceptual modeling in clinical domains, mainly because of its easy-to-use graphical notation, facilitating the common understanding and communication of the parties involved in health care. However, BPMN is not particularly oriented toward the peculiarities of complex clinical processes such as infection diagnosis and treatment, in which time plays a critical role, which is why much of the BPMN clinical-oriented research has revolved around how to extend the standard to address these special needs. The shift from an agnostic, general-purpose BPMN notation to a natively clinical-oriented notation such as openEHR Task Planning (TP) could constitute a major step toward clinical process improvement, enhancing the representation of CPs for infection treatment and other complex scenarios. OBJECTIVE: Our work aimed to analyze the suitability of a clinical-oriented formalism (TP) to successfully represent typical process patterns in infection treatment, identifying domain-specific improvements to the standard that could help enhance its modeling capabilities, thereby promoting the widespread adoption of CPs to improve medical practice and overall health care quality. METHODS: Our methodology consisted of 4 major steps: identification of key features of infection CPs through literature review, clinical guideline analysis, and BPMN extensions; analysis of the presence of key features in TP; modeling of relevant process patterns of catheter-related bloodstream infection as a case study; and analysis and proposal of extensions in view of the results. RESULTS: We were able to easily represent the same logic applied in the extended BPMN-based process models in our case study using out-of-the-box standard TP primitives. However, we identified possible improvements to the current version of TP to allow for simpler conceptual models of infection CPs and possibly of other complex clinical scenarios. CONCLUSIONS: Our study showed that the clinical-oriented TP specification is able to successfully represent the most complex catheter-related bloodstream infection process patterns depicted in our case study and identified possible extensions that can help increase its adequacy for modeling infection CPs and possibly other complex clinical conditions.
Assuntos
Procedimentos Clínicos , Sepse , Humanos , Modelos Teóricos , Fluxo de TrabalhoRESUMO
Clinical Pathways (CP) provide healthcare personnel with an easy-to-understand high level model of medical steps in specific patient conditions, thereby improving overall process quality in clinical practice. The emergence of new clinical-oriented standards such as openEHR Task Planning (TP) could pose a major step towards clinical process improvement, particularly in complex domains such as infection diagnosis and treatment, where time plays a critical role. In this work, we analyze the suitability of TP to successfully represent time constraints of common process patterns in infections, modelling some of the Catheter-Related Blood Stream Infection (CR-BSI) process patterns as a case study. Our research shows that TP is useful to represent time constraints of infection CPs, although minor improvements could increase its suitability not only for infection processes but for other time-related complex clinical scenarios.
Assuntos
Procedimentos Clínicos , Registros Eletrônicos de Saúde , Humanos , RegistrosRESUMO
Bovine paratuberculosis (PTB) is a chronic enteritis caused by Mycobacterium avium subspecies paratuberculosis (Map) that causes a heavy economic impact worldwide. Map infected animals can remain asymptomatic for years while transmitting the mycobacteria to other members of the herd. Therefore, accurate detection of subclinically infected animals is crucial for disease control. In a previous RNA-Seq study, we identified several mRNAs that were overexpressed in whole blood of cows with different PTB-associated histological lesions compared with control animals without detected lesions. The proteins encoded by two of these mRNAs, ATP binding cassette subfamily A member 13 (ABCA13) and Matrix Metallopeptidase 8 (MMP8) were significantly overexpressed in whole blood of animals with focal histological lesions, the most frequent pathological form in the subclinical stages of the disease. In the current study, the potential of sensitive early diagnostic tools of commercial ELISAs, based on the detection of these two biomarkers, was evaluated in serum samples of 704 Holstein Friesian cows (566 infected animals and 138 control animals from PTB-free farms). For this evaluation, infected animals were classified into three groups, according to the type of histological lesions present in their gut tissues: focal (n = 447), multifocal (n = 59), and diffuse (n = 60). The ELISA based on the detection of ABCA13 was successfully validated showing good discriminatory power between animals with focal lesions and control animals (sensitivity 82.99% and specificity 80.43%). Conversely, the MMP8-based ELISA showed a poor discriminatory power between the different histological groups and non-infected controls. The ABCA13-based ELISA showed a higher diagnostic value (0.822) than the IDEXX ELISA (0.517), the fecal bacterial isolation (0.523) and the real-time PCR (0.531) for the detection of animals with focal lesions. Overall, our results indicate that this ABCA13 ELISA greatly improves the identification of subclinically infected animals with focal lesions that are undetectable using current diagnostic methods.
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Short bowel syndrome (SBS) is a rare condition characterised by extensive loss of intestinal mass secondary to congenital or acquired disease. The outcomes are determined by dependency on parenteral nutrition (PN), its possible complications and factors that influence intestinal adaptation. In order to achieve the best results, patients should be managed by a specialised multidisciplinary team with the aims of promoting growth and development, stimulating intestinal adaptation and preventing possible complications. This involves timely surgical management aimed at rescuing maximum bowel length and eventually re-establishing intestinal continuity where appropriate. A combination of enteral and parenteral nutrition needs to be targeted towards maintaining a balance between fulfilling the nutritional and metabolic needs of the child while preventing or at least minimising potential complications. Enteral nutrition and establishment of oral feeding play a fundamental role in stimulating bowel adaptation and promoting enteral autonomy. Other measures to promote enteral autonomy include the chyme recycling in patients where bowel is not in continuity, autologous gastrointestinal reconstruction and pharmacological treatments, including promising new therapies like teduglutide. Strategies such as lipid reduction, changing the type of lipid emulsion and cycling PN are associated with a reduction in the rates of intestinal failure-associated liver disease. Even though vast improvements have been made in the surgical and medical management of SBS, there is still lack of consensus in many aspects and collaboration is essential.
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Paratuberculosis (PTB), a chronic granulomatous enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP), is responsible for important economic losses in the dairy industry. Our previous RNA-sequencing (RNA-Seq) analysis showed that bovine intelectin 2 (ITLN2) precursor gene was overexpressed in ileocecal valve (ICV) samples of animals with focal (log2 fold-change = 10.6) and diffuse (log2 fold-change = 6.8) PTB-associated lesions compared to animals without lesions. This study analyzes the potential use of ITLN2, a protein that has been described as fundamental in the innate immune response to infections, as a biomarker of MAP infection. The presence of ITLN2 was investigated by quantitative immunohistochemical analysis of ICV samples of 20 Holstein Friesian cows showing focal (n = 5), multifocal (n = 5), diffuse (n = 5) and no histological lesions (n = 5). Significant differences were observed in the mean number of ITLN2 immunostained goblet and Paneth cells between the three histopathological types and the control. The number of immunolabelled cells was higher in the focal histopathological type (116.9 ± 113.9) followed by the multifocal (108.7 ± 140.5), diffuse (76.5 ± 97.8) and control types (41.0 ± 81.3). These results validate ITLN2 as a post-mortem biomarker of disease progression.
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Bovine paratuberculosis (PTB) is a chronic granulomatous enteritis, caused by Mycobacterium avium subsp. paratuberculosis (MAP), responsible for important economic losses in the dairy industry. Current diagnostic methods have low sensitivities for detection of latent forms of MAP infection, defined by focal granulomatous lesions and scarce humoral response or MAP presence. In contrast, patent infections correspond to multifocal and diffuse types of enteritis where there is increased antibody production, and substantial mycobacterial load. Our previous RNA-Seq analysis allowed the selection of five candidate biomarkers overexpressed in peripheral blood of MAP infected Holstein cows with focal (ABCA13 and MMP8) and diffuse (FAM84A, SPARC and DES) lesions vs. control animals with no detectable PTB-associated lesions in intestine and regional lymph nodes. The aim of the current study was to assess the PTB diagnostic potential of commercial ELISAs designed for the specific detection of these biomarkers. The ability of these ELISAs to identify animals with latent and/or patent forms of MAP infection was investigated using serum from naturally infected cattle (n = 88) and non-infected control animals (n = 67). ROC analysis revealed that the ABCA13-based ELISA showed the highest diagnostic accuracy for the detection of infected animals with focal lesions (AUC 0.837, sensitivity 79.25% and specificity 88.06%) and with any type of histological lesion (AUC 0.793, sensitivity 69.41% and specificity 86.57%) improving on the diagnostic performance of the popular IDEXX ELISA and other conventional diagnostic methods. SPARC and MMP8 showed the highest diagnostic accuracy for the detection of animals with multifocal (AUC 0.852) and diffuse lesions (AUC 0.831), respectively. In conclusion, our results suggest that quantification of ABCA13, SPARC and MMP8 by ELISA has the potential for implementation as a diagnostic tool to reliably identify MAP infection, greatly improving early detection of MAP latent infections when antibody responses and fecal shedding are undetectable using conventional diagnostic methods.
Assuntos
Doenças dos Bovinos/diagnóstico , Bovinos/microbiologia , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Paratuberculose/diagnóstico , Animais , Biomarcadores/análise , Doenças dos Bovinos/patologia , Ensaio de Imunoadsorção Enzimática , Fezes/microbiologia , Feminino , Paratuberculose/patologia , Curva ROCRESUMO
The plastics used in drug packaging systems and medical devices are composed of homologous polymers and generally contain additives such as antioxidants, plasticizers and others, to improve their physicochemical properties. However, these additives have potential drawbacks due to possible migration or leaching towards the drug product. Leaching can cause a change in the chemical composition of the drug which, in turn, could modify its therapeutic action and, in some cases, its organoleptic properties. Leachables may also be considered a health hazard due to their inherent toxicological properties. The analytical characterization (detection, identification, typification/qualification and quantification) of leachable substances is mandatory and this information must be included in the application dossier for the drug before it can receive regulatory approval. The main aim of this paper is to collect and contextualise the reported analytical approaches for characterising and/or controlling organic leachables from plastic materials in contact with drugs. We also describe the state of the art of leachables in conjunction with a valuable, broad-based compilation of directives and guidelines. We end by presenting an updated collection of leachables both gas and liquid chromatography studies as separation techniques over the last eight years. We decided to focus our review exclusively on organic leachables as there is already a wide body of research on inorganic impurities.
Assuntos
Contaminação de Medicamentos , Embalagem de Medicamentos , Plásticos/química , Técnicas de Química Analítica , Contaminação de Medicamentos/prevenção & controle , Preparações FarmacêuticasRESUMO
BACKGROUND: The over-use of antibiotics in clinical domains is causing an alarming increase in bacterial resistance, thus endangering their effectiveness as regards the treatment of highly recurring severe infectious diseases. Whilst Clinical Guidelines (CGs) focus on the correct prescription of antibiotics in a narrative form, Clinical Decision Support Systems (CDSS) operationalize the knowledge contained in CGs in the form of rules at the point of care. Despite the efforts made to computerize CGs, there is still a gap between CGs and the myriad of rule technologies (based on different logic formalisms) that are available to implement CDSSs in real clinical settings. OBJECTIVE: To helpCDSS designers to determine the most suitable rule-based technology (medical-oriented rules, production rules and semantic web rules) with which to model knowledge from CGs for the prescription of antibiotics. We propose a framework of criteria for this purpose that is extensible to more generic CGs. MATERIALS AND METHODS: Our proposal is based on the identification of core technical requirements extracted from both literature and the analysis of CGs for antibiotics, establishing three dimensions for analysis: language expressivity, interoperability and industrial aspects. We present a case study regarding the John Hopkins Hospital (JHH) Antibiotic Guidelines for Urinary Tract Infection (UTI), a highly recurring hospital acquired infection. We have adopted our framework of criteria in order to analyse and implement these CGs using various rule technologies: HL7 Arden Syntax, general-purpose Production Rules System (Drools), HL7 standard Rule Interchange Format (RIF), Semantic Web Rule Language (SWRL) and SParql Inference Notation (SPIN) rule extensions (implementing our own ontology for UTI). RESULTS: We have identified the main criteria required to attain a maintainable and cost-affordable computable knowledge representation for CGs. We have represented the JHH UTI CGs knowledge in a total of 12 Arden Syntax MLMs, 81 Drools rules and 154 ontology classes, properties and individuals. Our experiments confirm the relevance of the proposed set of criteria and show the level of compliance of the different rule technologies with the JHH UTI CGs knowledge representation. CONCLUSIONS: The proposed framework of criteria may help clinical institutions to select the most suitable rule technology for the representation of CGs in general, and for the antibiotic prescription domain in particular, depicting the main aspects that lead to Computer Interpretable Guidelines (CIGs), such as Logic expressivity (Open/Closed World Assumption, Negation-As-Failure), Temporal Reasoning and Interoperability with existing HIS and clinical workflow. Future work will focus on providing clinicians with suggestions regarding new potential steps for CGs, considering process mining approaches and CGs Process Workflows, the use of HL7 FHIR for HIS interoperability and the representation of Knowledge-as- a-Service (KaaS).
Assuntos
Gestão de Antimicrobianos/organização & administração , Sistemas de Apoio a Decisões Clínicas/organização & administração , Sistemas Inteligentes , Guias de Prática Clínica como Assunto/normas , Antibacterianos/uso terapêutico , Gestão de Antimicrobianos/normas , Infecção Hospitalar/tratamento farmacológico , Sistemas de Apoio a Decisões Clínicas/normas , Humanos , Estudos de Casos Organizacionais , Infecções Urinárias/tratamento farmacológicoRESUMO
BACKGROUND & AIMS: Children on home parenteral nutrition and their parents not only engage with complex nutritional issues but also have to manage difficult social and financial problems with social and clinical support that may not always meet their needs. Baxter's HPN-QOL questionnaire, assesses the QOL of adult patients treated with HPN, and has been developed rigorously using standard guidelines, measuring various dimensions of QOL. Our aim was to use this tool to explore how HPN influences the QOL of paediatric patients. METHODS: The HPN-QOL questionnaire was modified to suit a paediatric HPN population. Data on demographics, aetiology of intestinal failure and duration of HPN were collected from a departmental database. Quality-of-Life grading of functional and symptom scales, HPN specific items and overall QOL Numerical Rating Scales were determined. RESULTS: Fourteen out of 17 families returned the completed questionnaires. QOL was significantly impaired by increased dependency regarding items of daily living such as eating, dressing, washing, and mobility, but was not affected in the domains of school attendance, general fatigue, pain and body image. There were no significant differences in QOL when patients with and without enterostomy were compared. Patients felt well supported by the hospital nutrition team in managing logistics related to HPN. CONCLUSIONS: QOL in HPN patients was not significantly affected by the medical aspects of care. This descriptive study highlights the need for further integration of medical and social care in order to support families of children receiving HPN as QOL was impaired in relation to activities of daily living and social functioning.
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Nutrição Parenteral no Domicílio , Qualidade de Vida , Atividades Cotidianas , Adolescente , Criança , Pré-Escolar , Estudos Transversais , Enterostomia , Feminino , Hospitais , Humanos , Lactente , Enteropatias , Intestino Delgado , Masculino , Estado Nutricional , Nutrição Parenteral no Domicílio/psicologia , Projetos Piloto , Síndrome do Intestino Curto , Inquéritos e QuestionáriosRESUMO
In many flowering plants, xyloglucan is a major component of primary cell walls, where it plays an important role in growth regulation. Xyloglucan can be degraded by a suite of exoglycosidases that remove specific sugars. In this work, we show that the xyloglucan backbone, formed by (1â4)-linked ß-d-glucopyranosyl residues, can be attacked by two different Arabidopsis (Arabidopsis thaliana) ß-glucosidases from glycoside hydrolase family 3. While BGLC1 (At5g20950; for ß-glucosidase active against xyloglucan 1) is responsible for all or most of the soluble activity, BGLC3 (At5g04885) is usually a membrane-anchored protein. Mutations in these two genes, whether on their own or combined with mutations in other exoglycosidase genes, resulted in the accumulation of partially digested xyloglucan subunits, such as GXXG, GXLG, or GXFG. While a mutation in BGLC1 had significant effects on its own, lack of BGLC3 had only minor effects. On the other hand, double bglc1 bglc3 mutants revealed a synergistic interaction that supports a role for membrane-bound BGLC3 in xyloglucan metabolism. In addition, bglc1 bglc3 was complemented by overexpression of either BGLC1 or BGLC3 In overexpression lines, BGLC3 activity was concentrated in a microsome-enriched fraction but also was present in soluble form. Finally, both genes were generally expressed in the same cell types, although, in some cases, BGLC3 was expressed at earlier stages than BGLC1 We propose that functional specialization could explain the separate localization of both enzymes, as a membrane-bound ß-glucosidase could specifically digest soluble xyloglucan without affecting the wall-bound polymer.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Membrana Celular/enzimologia , Glucanos/metabolismo , Xilanos/metabolismo , beta-Glucosidase/metabolismo , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Glucuronidase/metabolismo , Mutação/genética , Ligação Proteica , Solubilidade , alfa-L-Fucosidase/metabolismoAssuntos
Antidiarreicos/uso terapêutico , Diarreia/tratamento farmacológico , Síndromes de Malabsorção/tratamento farmacológico , Microvilosidades/patologia , Mucolipidoses/tratamento farmacológico , Tiorfano/análogos & derivados , Pré-Escolar , Diarreia/etiologia , Humanos , Masculino , Tiorfano/uso terapêuticoRESUMO
Host cell proteins (HCPs) are bioprocess-related impurities arising from cell-death or secretion from nonhuman cells used for recombinant protein production. Clearance of HCPs through downstream purification (DSP) is required to produce safe and efficacious therapeutic proteins. While traditionally measured using anti-HCP ELISA, more in-depth approaches for HCP characterization may ensure that risks to patients from HCPs are adequately assessed. Mass spectrometry methods provide rationale for targeted removal strategies through the provision of both qualitative and quantitative HCP information. A high pH, low pH, reversed-phase data independent 2D-LC-MS(E) proteomic platform was applied to determine HCP repertoires in the Protein A purified monoclonal antibody (mAb) samples as a function of culture harvest time, elution buffer used for DSP and also following inclusion of additional DSP steps. Critical quality attributes (CQAs) were examined for mAbs purified with different Protein A elution buffers to ensure that the selected buffers not only minimized the HCP profile but also exhibited no adverse effect on product quality. Results indicated that an arginine based Protein A elution buffer minimized the levels of HCPs identified and quantified in a purified mAb sample and also demonstrated no impact on product CQAs. It was also observed that mAbs harvested at later stages of cell culture contained higher concentrations of HCPs but that these were successfully removed by the addition of DSP steps complementary to Protein A purification. Taken together, our results showed how mass spectrometry based methods for HCP determination in conjunction with careful consideration of processing parameters such as harvest time, Protein A elution buffers, and subsequent DSP steps can reduce the HCP repertoire of therapeutic mAbs.
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Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Proteômica/métodos , Animais , Anticorpos Monoclonais/imunologia , Soluções Tampão , Células CHO , Bovinos , Cricetulus , Humanos , Concentração de Íons de Hidrogênio , Fatores de TempoRESUMO
A twoplex method using (12)C6 and (13)C6 stable isotope analogues (Δmass = 6 Da) of 2-aminobenzoic acid (2-AA) is described for quantitative analysis of N-glycans present on monoclonal antibodies and other glycoproteins using ultra performance liquid chromatography with sequential fluorescence and accurate mass tandem quadrupole time of flight (QToF) mass spectrometric detection.
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Anticorpos Monoclonais/química , Cromatografia Líquida/métodos , Glicoproteínas/química , Marcação por Isótopo/métodos , Polissacarídeos/análise , Espectrometria de Massas em Tandem/métodos , ortoaminobenzoatos/química , Radioisótopos de Carbono , Glicosilação , HumanosRESUMO
In growing cells, xyloglucan is thought to connect cellulose microfibrils and regulate their separation during wall extension. In Arabidopsis (Arabidopsis thaliana), a significant proportion of xyloglucan side chains contain ß-galactose linked to α-xylose at O2. In this work, we identified AtBGAL10 (At5g63810) as the gene responsible for the majority of ß-galactosidase activity against xyloglucan. Xyloglucan from bgal10 insertional mutants was found to contain a large proportion of unusual subunits, such as GLG and GLLG. These subunits were not detected in a bgal10 xyl1 double mutant, deficient in both ß-galactosidase and α-xylosidase. Xyloglucan from bgal10 xyl1 plants was enriched instead in XXLG/XLXG and XLLG subunits. In both cases, changes in xyloglucan composition were larger in the endoglucanase-accessible fraction. These results suggest that glycosidases acting on nonreducing ends digest large amounts of xyloglucan in wild-type plants, while plants deficient in any of these activities accumulate partly digested subunits. In both bgal10 and bgal10 xyl1, siliques and sepals were shorter, a phenotype that could be explained by an excess of nonreducing ends leading to a reinforced xyloglucan network. Additionally, AtBGAL10 expression was examined with a promoter-reporter construct. Expression was high in many cell types undergoing wall extension or remodeling, such as young stems, abscission zones, or developing vasculature, showing good correlation with α-xylosidase expression.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/enzimologia , Glucanos/metabolismo , Xilanos/metabolismo , Xilosidases/genética , beta-Galactosidase/metabolismo , Agrobacterium tumefaciens/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Parede Celular/enzimologia , Parede Celular/genética , Ativação Enzimática , Flores/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Genes Reporter , Mutagênese Insercional , Fenótipo , Filogenia , Pichia/genética , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Caules de Planta/enzimologia , Caules de Planta/genética , Caules de Planta/crescimento & desenvolvimento , Regiões Promotoras Genéticas , Xilosidases/metabolismo , beta-Galactosidase/genéticaRESUMO
Ormond's disease (OD) is an uncommon process with an annual incidence nearing 1 per million inhabitants. The etiology in most of the cases is unknown and several pathogenic mechanisms are implicated in secondary OD. Ormond disease is characterized by a fibrotic and inflammatory mass with three different clinical features: i) retroperitoneal fibrosis, ii) perianeurysmatic retroperitoneal fibrosis and iii) inflammatory abdominal aortic aneurysms. Classic management is based on surgical treatment associated or not with steroids. Immunosuppressive agents have been used in the last years with unclear results. We report five cases from the University Hospital of Salamanca occurring during 2000-2008. We highlight the lack of trials designed to establish clinical guidelines for the treatment of the disease and improvement of outcome.