RESUMO
INTRODUCTION: Over half of women with surgically managed breast cancer in the UK undergo breast-conserving treatment (BCT). While photographs are shown prior to reconstructive surgery or complex oncoplastic procedures, standard practice prior to breast conservation is to simply describe the likely aesthetic changes. Patients have expressed the desire for more personalized information about likely appearance after surgery. The hypothesis was that viewing a three-dimensional (3D) simulation improves patients' confidence in knowing their likely aesthetic outcome after surgery. METHODS: A randomized, controlled trial of 117 women planning unilateral BCT was undertaken. The randomization was three-way: standard of care (verbal description alone, control group), viewing two-dimensional (2D) photographs, or viewing a 3D simulation before surgery. The primary endpoint was the comparison between groups' median answer on a visual analogue scale (VAS) for the question administered before surgery: 'How confident are you that you know how your breasts are likely to look after treatment?' RESULTS: The median VAS in the control group was 5.2 (i.q.r. 2.6-7.8); 8.0 (i.q.r. 5.7-8.7) for 2D photography, and 8.9 (i.q.r. 8.2-9.5) for 3D simulation. There was a significant difference between groups (P < 0.010) with post-hoc pairwise comparisons demonstrating a statistically significant difference between 3D simulation and both standard care and viewing 2D photographs (P < 0.010 and P = 0.012, respectively). CONCLUSION: This RCT has demonstrated that women who viewed an individualized 3D simulation of likely aesthetic outcome for BCT were more confident going into surgery than those who received standard care or who were shown 2D photographs of other women. The impact on longer-term satisfaction with outcome remains to be determined.Registration number: NCT03250260 (http://www.clinicaltrials.gov).
Most women with breast cancer are able to have an operation to remove the cancer while preserving the breast ('lumpectomy'). Whilst cancer control is the most important goal, appearance after surgery has been shown to affect long-term quality of life and is considered when planning treatment. Currently, surgeons simply describe the likely changes in appearance and, for more complex procedures, photographs of other women are shown. Patients themselves have indicated they would like more information regarding the likely changes to their breast after treatment. The authors have developed a way to simulate appearance following lumpectomy and radiotherapy using three-dimensional (3D) photographs. The study invited women undergoing lumpectomy to be assigned at random to one of three groups receiving standard care (discussion), a two-dimensional photograph, or the 3D simulation before their operation. The authors have demonstrated that showing a woman her simulation prior to surgery improves confidence going into treatment.
Assuntos
Simulação por Computador , Estética , Imageamento Tridimensional , Mamoplastia/psicologia , Mastectomia Segmentar/psicologia , Educação de Pacientes como Assunto/métodos , Idoso , Neoplasias da Mama/psicologia , Neoplasias da Mama/cirurgia , Feminino , Humanos , Pessoa de Meia-Idade , FotografaçãoRESUMO
Extraintestinal pathogenic Escherichia coli (ExPEC) cause a wide range of clinical diseases such as bacteremia and urinary tract infections. The increase of multidrug resistant ExPEC strains is becoming a major concern for the treatment of these infections and E. coli has been identified as a critical priority pathogen by the WHO. Therefore, the development of vaccines has become increasingly important, with the surface lipopolysaccharide constituting a promising vaccine target. This study presents genetic and structural analysis of clinical urine isolates from Switzerland belonging to the serotype O25. Approximately 75% of these isolates were shown to correspond to the substructure O25B only recently described in an emerging clone of E. coli sequence type 131. To address the high occurrence of O25B in clinical isolates, an O25B glycoconjugate vaccine was prepared using an E. coli glycosylation system. The O antigen cluster was integrated into the genome of E. coli W3110, thereby generating an E. coli strain able to synthesize the O25B polysaccharide on a carrier lipid. The polysaccharide was enzymatically conjugated to specific asparagine side chains of the carrier protein exotoxin A (EPA) of Pseudomonas aeruginosa by the PglB oligosaccharyltransferase from Campylobacter jejuni. Detailed characterization of the O25B-EPA conjugate by use of physicochemical methods including NMR and GC-MS confirmed the O25B polysaccharide structure in the conjugate, opening up the possibility to develop a multivalent E. coli conjugate vaccine containing O25B-EPA.
Assuntos
Vacinas contra Escherichia coli/imunologia , Escherichia coli/classificação , Glicoconjugados , Vacinas ConjugadasRESUMO
Shigellosis remains a major cause of diarrheal disease in developing countries and causes substantial morbidity and mortality in children. Vaccination represents a promising preventive measure to fight the burden of the disease, but despite enormous efforts, an efficacious vaccine is not available to date. The use of an innovative biosynthetic Escherichia coli glycosylation system substantially simplifies the production of a multivalent conjugate vaccine to prevent shigellosis. This bioconjugation approach has been used to produce the Shigella dysenteriae type O1 conjugate that has been successfully tested in a phase I clinical study in humans. In this report, we describe a similar approach for the production of an additional serotype required for a broadly protective shigellosis vaccine candidate. The Shigella flexneri 2a O-polysaccharide is conjugated to introduced asparagine residues of the carrier protein exotoxin A (EPA) from Pseudomonas aeruginosa by co-expression with the PglB oligosaccharyltransferase. The bioconjugate was purified, characterized using physicochemical methods and subjected to preclinical evaluation in rats. The bioconjugate elicited functional antibodies as shown by a bactericidal assay for S. flexneri 2a. This study confirms the applicability of bioconjugation for the S. flexneri 2a O-antigen, which provides an intrinsic advantage over chemical conjugates due to the simplicity of a single production step and ease of characterization of the homogenous monomeric conjugate formed. In addition, it shows that bioconjugates are able to raise functional antibodies against the polysaccharide antigen.
Assuntos
Imunogenicidade da Vacina/imunologia , Antígenos O/imunologia , Shigella flexneri/imunologia , Vacinas Conjugadas/imunologia , Animais , Feminino , Antígenos O/química , Ratos , Ratos Sprague-Dawley , Shigella flexneri/química , Shigella flexneri/crescimento & desenvolvimento , Vacinas Conjugadas/químicaRESUMO
Shigellosis remains a major cause of diarrheal disease in developing countries and causes substantial morbidity and mortality in children. Glycoconjugate vaccines consisting of bacterial surface polysaccharides conjugated to carrier proteins are the most effective vaccines for controlling invasive bacterial infections. Nevertheless, the development of a multivalent conjugate vaccine to prevent Shigellosis has been hampered by the complex manufacturing process as the surface polysaccharide for each strain requires extraction, hydrolysis, chemical activation and conjugation to a carrier protein. The use of an innovative biosynthetic Escherichia coli glycosylation system substantially simplifies the production of glycoconjugates. Herein, the Shigella dysenteriae type 1 (Sd1) O-polysaccharide is expressed and its functional assembly on an E. coli glycosyl carrier lipid is demonstrated by HPLC analysis and mass spectrometry. The polysaccharide is enzymatically conjugated to specific asparagine residues of the carrier protein by co-expression of the PglB oligosaccharyltransferase and the carrier protein exotoxin A (EPA) from Pseudomonas aeruginosa. The extraction and purification of the Shigella glycoconjugate (Sd1-EPA) and its detailed characterization by the use of physicochemical methods including NMR and mass spectrometry is described. The report shows for the first time that bioconjugation provides a newly developed and improved approach to produce an Sd1 glycoconjugate that can be characterized using state-of-the-art techniques. In addition, this generic process together with the analytical methods is ideally suited for the production of additional Shigella serotypes, allowing the development of a multivalent Shigella vaccine.
Assuntos
Processamento de Proteína Pós-Traducional , Vacinas Protozoárias/imunologia , Shigella dysenteriae/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Endotoxinas/genética , Endotoxinas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicosilação , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Pseudomonas aeruginosa/enzimologia , Vacinas Conjugadas/imunologiaRESUMO
Recent developments in proteomic techniques have led to the development of mass spectrometry (MS)-based methods to characterize site-specific glycosylation of proteins. However, appropriate analytical tools to characterize acidic and high-molecular-weight (hMW) glycopeptides are still lacking. In this study, we demonstrate that the addition of supercharging reagent, m-nitrobenzyl alcohol (m-NBA), into mobile phases greatly facilitates the analysis of acidic and hMW glycopeptides. Using commercial glycoproteins, we demonstrated that in the presence of m-NBA the charge state of sialylated glycopeptides increased and the chromatographic separation of neutral and acidic glycopeptides revealed a remarkable improvement. Next, we applied this system to the characterization of a glycoconjugate vaccine candidate consisting of a genetically detoxified exotoxin A of Pseudomonas aeruginosa covalently linked to Shigella flexneri type 2a O-antigen (Sf2E) produced by engineered Escherichia coli. The addition of m-NBA, allowed us to identify peptides with glycan chains of unprecedented size, up to 20 repeat units (98 monosaccharides). Our results indicated that incorporation of m-NBA into reversed-phase liquid chromatography (LC) solvents improves sensitivity, charging, and chromatographic resolution for acidic and hMW glycopeptides.
Assuntos
Glicopeptídeos/análise , Nanotecnologia , Álcoois Benzílicos/química , Cromatografia Líquida de Alta Pressão , Peso Molecular , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The majority of congenital disorders of glycosylation (CDG) are caused by defects of dolichol (Dol)-linked oligosaccharide assembly, which lead to under-occupancy of N-glycosylation sites. Most mutations encountered in CDG are hypomorphic, thus leaving residual activity to the affected biosynthetic enzymes. We hypothesized that increased cellular levels of Dol-linked substrates might compensate for the low biosynthetic activity and thereby improve the output of protein N-glycosylation in CDG. To this end, we investigated the potential of the squalene synthase inhibitor zaragozic acid A to redirect the flow of the polyisoprene pathway toward Dol by lowering cholesterol biosynthesis. The addition of zaragozic acid A to CDG fibroblasts with a Dol-P-Man synthase defect led to the formation of longer Dol-P species and to increased Dol-P-Man levels. This treatment was shown to decrease the pathologic accumulation of incomplete Dol pyrophosphate-GlcNAc(2)Man(5) in Dol-P-Man synthase-deficient fibroblasts. Zaragozic acid A treatment also decreased the amount of truncated protein N-linked oligosaccharides in these CDG fibroblasts. The increased cellular levels of Dol-P-Man and possibly the decreased cholesterol levels in zaragozic acid A-treated cells also led to increased availability of the glycosylphosphatidylinositol anchor as shown by the elevated cell-surface expression of the CD59 protein. This study shows that manipulation of the cellular Dol pool, as achieved by zaragozic acid A addition, may represent a valuable approach to improve N-linked glycosylation in CDG cells.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Defeitos Congênitos da Glicosilação/metabolismo , Dolicóis/metabolismo , Inibidores Enzimáticos/farmacologia , Farnesil-Difosfato Farnesiltransferase/antagonistas & inibidores , Oligossacarídeos/biossíntese , Ácidos Tricarboxílicos/farmacologia , Antígenos CD59/biossíntese , Antígenos CD59/genética , Células Cultivadas , Colesterol/biossíntese , Colesterol/genética , Defeitos Congênitos da Glicosilação/genética , Dolicóis/genética , Farnesil-Difosfato Farnesiltransferase/genética , Farnesil-Difosfato Farnesiltransferase/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glicosilação/efeitos dos fármacos , Humanos , Manosiltransferases/genética , Manosiltransferases/metabolismo , Oligossacarídeos/genéticaRESUMO
N-linked glycosylation is an essential posttranslational modification of proteins in eukaryotes. The substrate of N-linked glycosylation, dolichol pyrophosphate (DolPP)-GlcNAc(2)Man(9)Glc(3), is assembled through a complex series of ordered reactions requiring the translocation of the intermediate DolPP-GlcNAc(2)Man(5) structure across the endoplasmic-reticulum membrane. A young patient diagnosed with a congenital disorder of glycosylation characterized by an intracellular accumulation of DolPP-GlcNAc(2)Man(5) was found to carry a homozygous point mutation in the RFT1 gene. The c.199C-->T mutation introduced the amino acid substitution p.R67C. The human RFT1 protein shares 22% identity with its yeast ortholog, which is involved in the translocation of DolPP-GlcNAc(2)Man(5) from the cytosolic into the lumenal side of the endoplasmic reticulum. Despite the low sequence similarity between the yeast and the human RFT1 proteins, we demonstrated both their functional orthology and the pathologic effect of the human p.R67C mutation by complementation assay in Deltarft1 yeast cells. The causality of the RFT1 p.R67C mutation was further established by restoration of normal glycosylation profiles in patient-derived fibroblasts after lentiviral expression of a normal RFT1 cDNA. The definition of the RFT1 defect establishes the functional conservation of the DolPP-GlcNAc(2)Man(5) translocation process in eukaryotes. RFT1 deficiency in both yeast and human cells leads to the accumulation of incomplete DolPP-GlcNAc(2)Man(5) and to a profound glycosylation disorder in humans.
Assuntos
Glicoproteínas de Membrana/deficiência , Doenças Metabólicas/genética , Açúcares de Poli-Isoprenil Fosfato/metabolismo , Processamento de Proteína Pós-Traducional/genética , Adolescente , Sequência de Aminoácidos , Análise Mutacional de DNA , Feminino , Teste de Complementação Genética , Glicosilação , Humanos , Glicoproteínas de Membrana/genética , Dados de Sequência Molecular , Linhagem , Mutação Puntual , Saccharomyces cerevisiae/genéticaRESUMO
Colonic diverticular disease is extremely common in developed countries. Although the majority of patients with diverticulosis remain asymptomatic, about one-third of the patients manifest the disease with either hemorrhage or inflammation. Diverticulitis may be uncomplicated or complicated by abscess formation, perforation with peritonitis, fistula, intestinal obstruction, or stricture. Controversy exists regarding the aggressiveness of diverticulitis during recurrent attacks of the disease as well as in special groups of patients including immunocompromised patients, young patients, and patients with right-sided disease. Clinical characteristics of symptomatic uncomplicated disease can be similar to irritable bowel syndrome, while acute diverticulitis is sometimes difficult to distinguish from segmental colitis associated with diverticulosis. The considerable clinical overlap between those entities with diverticular disease demonstrates that there are still areas of uncertainty in their physiopathology.
Assuntos
Doença Diverticular do Colo/complicações , Fístula/etiologia , Hemorragia Gastrointestinal/etiologia , Obstrução Intestinal/etiologia , Fatores Etários , Colite/etiologia , Doença Diverticular do Colo/patologia , Doença Diverticular do Colo/cirurgia , Humanos , RecidivaRESUMO
Diverticular disease is a common problem in the western population and sometimes leads to serious complications such as hemorrhage, bowel stenosis, obstruction, abscesses, fistulae, bowel perforation, and peritonitis. The severity of these complications can differ, and it is not always clear which procedure is indicated in each case and what measures should be followed before bringing the patient into the operating room. Certain operations have high rates of morbidity and mortality, especially in compromised patients. Along with advancements in imaging and minimally invasive techniques, the indications for surgery have currently being adapted to "damage limitation" or "down-staging" protocols, which seem to offer improved results. There are still some questions to be solved in the following years by prospective studies, such as the usefulness of laparoscopic lavage in purulent peritonitis or of Hartmann's procedure in fecal peritonitis. These indications, based on current literature, are systematically discussed in the present review.
Assuntos
Doença Diverticular do Colo/complicações , Fístula/cirurgia , Hemorragia Gastrointestinal/terapia , Obstrução Intestinal/cirurgia , Perfuração Intestinal/cirurgia , Fatores Etários , Constrição Patológica/etiologia , Constrição Patológica/cirurgia , Fístula/etiologia , Hemorragia Gastrointestinal/diagnóstico , Hemorragia Gastrointestinal/etiologia , Humanos , Obstrução Intestinal/etiologia , Perfuração Intestinal/etiologia , RecidivaRESUMO
BACKGROUND: Participation in research can be beneficial for patients and healthcare providers, but may prove demanding at patient, clinician and organizational levels. Patient representatives are supportive of online research to overcome these challenges. The aim of this pilot study was to develop an online recruitment platform and test its feasibility and acceptability while evaluating the accuracy of participant-reported data. METHODS: The online research platform was developed in a 1-day 'hackathon' with a digital design company. Women who underwent implant-based breast reconstruction in 2011-2016 were invited by letter containing the web address (URL) of the study site and their unique study number. Once online, participants learned about the study, consented, entered data on demographics, treatment received and patient-reported outcome measures (BREAST-Q™), and booked an appointment for a single hospital visit for three-dimensional surface imaging (3D-SI). Real-time process evaluation was performed. The primary endpoint was recruitment rate. RESULTS: The recruitment rate was 40 per cent. Of the 100 women, 50 logged on to the platform and 40 completed the process through to 3D-SI. The majority of discontinuations after logging on occurred between consenting and entering demographics (3 women, 6 per cent), and between completing the BREAST-Q and booking an appointment for 3D-SI using the online calendar (3 women, 6 per cent). All women completed the online BREAST-Q™ once started. Participants took a median of 23 minutes to complete the online process. Patient-reported clinical data were accurate in 12 of 13 domains compared with electronic records (95 per cent concordance). Process evaluation demonstrated acceptability. CONCLUSION: The results of this pilot demonstrate the online platform to be acceptable, feasible, and accurate for this population from a single institution. The low-burden design may enable participation from centres with less research support and participants from hard-to-reach groups or dispersed geographical locations, but with online access.
Assuntos
Mamoplastia , Medidas de Resultados Relatados pelo Paciente , Telemedicina , Adulto , Idoso , Estudos de Viabilidade , Feminino , Humanos , Pessoa de Meia-Idade , Projetos PilotoRESUMO
Dolichols (Dol) are polyprenol lipids that are essential structural components of eukaryotic membranes. In addition, the phosphorylated derivatives of Dol function as lipid anchors of mono- and oligosaccharide precursors involved in protein glycosylation. The biological importance of Dol phosphates (Dol-P) is illustrated by the severe outcome of human disorders linked to Dol biosynthetic defects, such as Dol-kinase deficiency. For characterization of inherited human diseases and evaluation of therapeutic trials, cultured cells often serve as a sole possible source for experimentation. Limited amounts of cell culture material render the quantitative analysis of Dol a challenging task. Here, we present HPLC- and mass spectrometry-based approaches to analyze and quantitate Dol-P from cultured human cells. The composition of naturally occurring Dol-P and the saturation state of the alpha-isoprene units was identified by negative-ion electrospray ionization mass spectrometry. Furthermore, fluorescently labeled Dol-P were separated by HPLC and quantified by comparison to known amounts of the internal standard polyprenol-P. The effect of pravastatin, a 3-hydroxy-3-methyl-glutaryl coenzyme-A reductase inhibitor, on the formation of Dol-P in HeLa cells was investigated. As expected, this treatment led to a decrease of Dol-P down to 35% of normal levels.
Assuntos
Fosfatos de Dolicol/análise , Espectrometria de Massas/métodos , Antracenos/metabolismo , Cromatografia Líquida de Alta Pressão , Fosfatos de Dolicol/química , Flavonoides/análise , Flavonoides/química , Células HeLa , Humanos , Fenóis/análise , Fenóis/química , Polifenóis , Pravastatina/farmacologia , Padrões de ReferênciaRESUMO
PURPOSE: To examine the experiences of outpatients with bipolar disorder recording a 10-minute film to show their "being" in a euthymic mood state. DESIGN AND METHODS: A multicenter qualitative study, in the context of a feasibility study for a newly developed intervention. Data were analyzed using the Stevick-Colaizzi-Keen method. FINDINGS: Participants experienced the recording as positive and valuable. Although camera anxiety was mentioned frequently, the overall conclusion is that recording a film in the context of a newly developed film intervention is valid. PRACTICE IMPLICATIONS: Clear information and support for the patient should be provided during the preparatory conversation and recording.
Assuntos
Transtorno Bipolar/terapia , Comunicação , Psicoterapia , Gravação em Vídeo , Adulto , Feminino , Humanos , Entrevistas como Assunto , Masculino , Pessoa de Meia-Idade , Pacientes Ambulatoriais/psicologia , Pesquisa Qualitativa , Adulto JovemRESUMO
Defects in the biosynthesis of the oligosaccharide precursor for N-glycosylation lead to decreased occupancy of glycosylation sites and thereby to diseases known as congenital disorders of glycosylation (CDG). In the last 20 years, approximately 1,000 CDG patients have been identified presenting with multiple organ dysfunctions. This review sets the state of the art by listing all mutations identified in the 15 genes (PMM2, MPI, DPAGT1, ALG1, ALG2, ALG3, ALG9, ALG12, ALG6, ALG8, DOLK, DPM1, DPM3, MPDU1, and RFT1) that yield a deficiency of dolichol-linked oligosaccharide biosynthesis. The present analysis shows that most mutations lead to substitutions of strongly conserved amino acid residues across eukaryotes. Furthermore, the comparison between the different forms of CDG affecting dolichol-linked oligosaccharide biosynthesis shows that the severity of the disease does not relate to the position of the mutated gene along this biosynthetic pathway.
Assuntos
Erros Inatos do Metabolismo dos Carboidratos/genética , Dolicóis/metabolismo , Oligossacarídeos/biossíntese , Sequência de Aminoácidos , Erros Inatos do Metabolismo dos Carboidratos/enzimologia , Glicosilação , Humanos , Dados de Sequência MolecularRESUMO
The medical significance of N-glycosylation is underlined by a group of inherited human disorders called Congenital Disorders of Glycosylation (CDG). One key step in the biosynthesis of the Glc(3)Man(9)GlcNAc(2)-PP-dolichol precursor, essential for N-glycosylation, is the translocation of Man(5)GlcNAc(2)-PP-dolichol across the endoplasmic reticulum membrane. This step is facilitated by the RFT1 protein. Recently, the first RFT1-deficient CDG (RFT1-CDG) patient was identified and presented a severe N-glycosylation disorder. In the present study, we describe three novel CDG patients with an RFT1 deficiency. The first patient was homozygous for the earlier reported RFT1 missense mutation (c.199C>T; p.R67C), whereas the two other patients were homozygous for the missense mutation c.454A>G (p.K152E) and c.892G>A (p.E298 K), respectively. The pathogenic character of the novel mutations was illustrated by the accumulation of Man(5)GlcNAc(2)-PP-dolichol and by reduced recombinant DNase 1 secretion. Both the glycosylation pattern and recombinant DNase 1 secretion could be normalized by expression of normal RFT1 cDNA in the patients' fibroblasts. The clinical phenotype of these patients comprised typical CDG symptoms in addition to sensorineural deafness, rarely reported in CDG patients. The identification of additional RFT1-deficient patients allowed to delineate the main clinical picture of RFT1-CDG and confirmed the crucial role of RFT1 in Man(5)GlcNAc(2)-PP-dolichol translocation.
Assuntos
Erros Inatos do Metabolismo dos Carboidratos/genética , Glicoproteínas de Membrana/genética , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Feminino , Teste de Complementação Genética , Humanos , Masculino , FenótipoRESUMO
BACKGROUND: Extra-intestinal pathogenic Escherichia coli (ExPEC) are major human pathogens; however, no protective vaccine is currently available. We assessed in animal models the immunogenicity and safety of a 4-valent E. coli conjugate vaccine (ExPEC-4V, serotypes O1, O2, O6 and O25 conjugated to Exotoxin A from Pseudomonas aeruginosa (EPA)) produced using a novel in vivo bioconjugation method. METHODS: Three doses of ExPEC-4V (with or without aluminum hydroxide) were administered to rabbits (2µg or 20µg per O-antigen, subcutaneously), mice (0.2µg or 2µg per O-antigen, subcutaneously) and rats (0.4µg or 4µg per O-antigen, intramuscularly). Antibody persistence and boostability were evaluated in rats using O6-EPA monovalent conjugate (0.4µg O-antigen/dose, intramuscularly). Toxicity was assessed in rats (16µg total polysaccharide, intramuscularly). Serum IgG and IgM antibodies were measured by ELISA. RESULTS: Robust antigen-specific IgG responses were observed in all animal models, with increased responses in rabbits when administered with adjuvant. O antigen-specific antibody responses persisted up to 168days post-priming. Booster immunization induced a rapid recall response. Toxicity of ExPEC-4V when administered to rats was considered to be at the no observed adverse effect level. CONCLUSIONS: ExPEC-4V conjugate vaccine showed good immunogenicity and tolerability in animal models supporting progression to clinical evaluation.
Assuntos
Infecções por Escherichia coli/prevenção & controle , Vacinas contra Escherichia coli/imunologia , Imunogenicidade da Vacina , Antígenos O/imunologia , ADP Ribose Transferases/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Antibacterianos/sangue , Toxinas Bacterianas/imunologia , Escherichia coli , Exotoxinas/imunologia , Feminino , Imunização Secundária , Camundongos , Camundongos Endogâmicos ICR , Nível de Efeito Adverso não Observado , Coelhos , Ratos , Ratos Sprague-Dawley , Testes de Toxicidade , Vacinas Conjugadas/imunologia , Fatores de Virulência/imunologia , Exotoxina A de Pseudomonas aeruginosaRESUMO
In a previous large scale screen for differentially expressed genes in pancreatic cancer, we identified a gene highly overexpressed in cancer encoding a novel protein with four K-homologous (KH) domains. KH-domains are found in a subset of RNA-binding proteins, including pre-mRNA-binding (hnRNP) K protein and the fragile X mental retardation gene product (FMR1). By fluorescence in situ hybridization (FISH) the identified gene named koc (KH domain containing protein overexpressed in cancer) was assigned to chromosome 7p11.5. Two pseudogenes were localised on chromosome 6 and 11. The cloned koc cDNA has a 250 bp 5'-UTR, a 1740 bp ORF and a 2168 bp 3'-UTR. The AU-rich 3'-untranslated region of koc contains eight AUUUA and four AUUUUUA reiterated motifs. The deduced koc protein with 580 amino-acids has a relative molecular mass (Mr) of approximately 65,000 (65 K). The koc transcript is highly overexpressed in pancreatic cancer cell lines and in pancreatic cancer tissue as compared to both, normal pancreas and chronic pancreatitis tissue. High levels of expression were as well found in tissue samples of other human tumours. As the KH domain has been shown to be involved in the regulation of RNA synthesis and metabolism, we speculate that koc may assume a role in the regulation of tumour cell proliferation by interfering with transcriptional and or posttranscriptional processes. However, the precise role of koc in human tumour cells is unknown and remains to be elucidated.
Assuntos
Neoplasias Pancreáticas/genética , Proteínas de Ligação a RNA/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Complementar , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Proteínas de Neoplasias , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Células Tumorais CultivadasRESUMO
The present review summarizes our strategies aimed at identifying and characterizing genetic alterations occurring at the transcriptional and chromosomal level in pancreatic cancer. To study transcriptional alterations we have used a number of techniques including modified versions of differential hybridizations and cDNA RDA (representational difference analysis). These approaches have led to the identification of more than 500 genes with differential expression in pancreatic cancer. To study chromosomal aberrations occurring in pancreatic cancer tissues we used comparative genomic hybridization (CGH). This allowed the identification of a number of chromosomal regions containing putative tumor suppressor genes or oncogenes. Genes isolated in both approaches represent potential new disease genes for pancreatic cancer and are at present being characterized by individual or serial analysis.
Assuntos
Neoplasias Pancreáticas/genética , Sequência de Aminoácidos , Animais , Regulação Neoplásica da Expressão Gênica , Humanos , Dados de Sequência MolecularRESUMO
High-titre equine immune sera were used to screen a lambda gt 11 expression library of Babesia equi cDNA fragments. Two cDNA clones which did not cross-hybridize to each other were studied. Both clones hybridized specifically to DNA from B. equi but not to DNA from B. caballi, B. divergens or B. ovis. Recombinant proteins were expressed as glutathione S-transferase (GST) fusion proteins with apparent molecular weights of 40 kDa and 75 kDa. Polyclonal antibodies directed against the 40 kDa and 75 kDa recombinant proteins detected native antigens of 55 kDa and 50 kDa respectively in crude lysates of B. equi merozoites indicating that neither cDNA clone was full length (GST = 26 kDa). In Western blotting experiments the 75 kDa protein showed cross-reactivity with sera from horses infected with B. caballi and was not further investigated. The 40 kDa protein was additionally tested in an enzyme-linked immunosorbent assay (ELISA). A test was developed which had a calculated specificity of 99% and a sensitivity of 88% with sera from horses infected with the homologous strain of B. equi. The ELISA did not recognize sera from horses infected with B. equi strains from Brazil and Morocco.
Assuntos
Babesia/genética , Babesiose/diagnóstico , Clonagem Molecular/métodos , Genes de Protozoários , Animais , Antígenos de Protozoários/análise , Babesia/isolamento & purificação , Babesia/fisiologia , Babesiose/sangue , Southern Blotting , DNA Complementar , DNA de Protozoário/análise , DNA de Protozoário/genética , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Eritrócitos/parasitologia , Expressão Gênica , Biblioteca Gênica , Glutationa Transferase/biossíntese , Cavalos , Leucócitos/parasitologia , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/isolamento & purificação , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Especificidade da EspécieRESUMO
HISTORY AND CLINICAL FINDINGS: A 71-year-old woman was admitted with the suspected diagnosis of pancreatic carcinoma. As a child she had had repeated attacks of abdominal pain of undetermined cause. When aged 48 years she had developed diabetes mellitus. Her now 42-year-old daughter had from the age of 9 years suffered from repeated attacks of acute pancreatitis that had finally led to chronic pancreatitis. The patient's 15-year-old grandchild was having recurrent bouts of abdominal pain. INVESTIGATIONS: Imaging procedures revealed calcifications in the pancreas and an infiltrating space-occupying lesion, about 3 cm in diameter, in the head of the pancreas with lymph node and liver metastases. Cytological analysis of material aspirated from the space-occupying mass showed typical findings of ductal pancreatic carcinoma. FURTHER TESTS, TREATMENT AND COURSE: At first the patient's course was not typical for a genetically-determined disease, but the family history raised the suspicion of hereditary pancreatitis. A genetic test (Afl-III-RFLP test) demonstrated the mutation Arg 117 His in the cationic trypsinogen gene in all diseased or symptomatic family members. The patient died of the complications of the pancreatic cancer. CONCLUSION: Genetic tests are valuable in the diagnosis of hereditary pancreatitis, because the increased cancer risk can be met by frequent examinations in affected family members.
Assuntos
Pancreatite/genética , Mutação Puntual , Tripsinogênio/genética , Dor Abdominal/etiologia , Doença Aguda , Adolescente , Adulto , Idoso , Calcinose/diagnóstico por imagem , Doença Crônica , Família , Evolução Fatal , Feminino , Humanos , Pâncreas/diagnóstico por imagem , Pancreatopatias/diagnóstico por imagem , Neoplasias Pancreáticas/diagnóstico por imagem , Neoplasias Pancreáticas/etiologia , Pancreatite/complicações , Pancreatite/diagnóstico , Polimorfismo de Fragmento de Restrição , Recidiva , Fatores de Risco , Tomografia Computadorizada por Raios X , UltrassonografiaRESUMO
BACKGROUND/AIM: Genomic imprinting is a chromosomal modification causing differential expression of maternal and paternal alleles. Loss of imprinting (LOI) of IGF-II and H19 has been suggested to be an early oncogenic event in cancerogenesis. Aim of the present study was to describe the status of IGF-II and H19 imprinting in adult human pancreatic tissues. METHODS: Allele-specific gene expression was studied using RNA and DNA from human pancreatic cancer, chronic pancreatitis, and normal pancreas tissues heterozygous for ApaI (IGF-II) or RsaI (H19) restriction fragment length polymorphism. Reverse-transcriptase polymerase chain reaction products were digested with either ApaI or RsaI and analyzed on agarose gels to study the status of allelic expression. The expression level of H19 and IGF-II was studied on Northern blots or by polymerase chain reaction. RESULTS: H19 was imprinted in normal pancreas and in chronic pancreatitis. H19 LOI was observed in 1 of 4 informative cancer tissues and was not associated with increased H19 transcript levels. Biallelic expression of IGF-II was found in 6 of 10 informative cancer tissues and in 6 of 9 informative normal tissues. In chronic pancreatitis, the IGF-II gene was imprinted in all informative samples. IGF-II mRNA was not overexpressed in the tissues showing LOI. CONCLUSION: Low frequencies of H19 LOI and the lack of correlation between biallelic expression and overexpression observed for both H19 and IGF-II suggest that LOI of H19 and IGF-II is not a relevant oncogenic factor during human exocrine pancreatic cancerogenesis.