RESUMO
Volumetric laser endomicroscopy (VLE) uses optical coherence tomography (OCT) for real-time, microscopic cross-sectional imaging. A US-based multi-center registry was constructed to prospectively collect data on patients undergoing upper endoscopy during which a VLE scan was performed. The objective of this registry was to determine usage patterns of VLE in clinical practice and to estimate quantitative and qualitative performance metrics as they are applied to Barrett's esophagus (BE) management. All procedures utilized the NvisionVLE Imaging System (NinePoint Medical, Bedford, MA) which was used by investigators to identify the tissue types present, along with focal areas of concern. Following the VLE procedure, investigators were asked to answer six key questions regarding how VLE impacted each case. Statistical analyses including neoplasia diagnostic yield improvement using VLE was performed. One thousand patients were enrolled across 18 US trial sites from August 2014 through April 2016. In patients with previously diagnosed or suspected BE (894/1000), investigators used VLE and identified areas of concern not seen on white light endoscopy (WLE) in 59% of the procedures. VLE imaging also guided tissue acquisition and treatment in 71% and 54% of procedures, respectively. VLE as an adjunct modality improved the neoplasia diagnostic yield by 55% beyond the standard of care practice. In patients with no prior history of therapy, and without visual findings from other technologies, VLE-guided tissue acquisition increased neoplasia detection over random biopsies by 700%. Registry investigators reported that VLE improved the BE management process when used as an adjunct tissue acquisition and treatment guidance tool. The ability of VLE to image large segments of the esophagus with microscopic cross-sectional detail may provide additional benefits including higher yield biopsies and more efficient tissue acquisition. Clinicaltrials.gov NCT02215291.
Assuntos
Esôfago de Barrett/diagnóstico por imagem , Padrões de Prática Médica/estatística & dados numéricos , Tomografia de Coerência Óptica/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Esôfago de Barrett/patologia , Esôfago de Barrett/terapia , Biópsia , Tomada de Decisão Clínica , Sistemas Computacionais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Sistema de Registros , Tomografia de Coerência Óptica/estatística & dados numéricos , Estados UnidosAssuntos
Oclusão com Balão , Colangiopancreatografia Retrógrada Endoscópica , Hemobilia/terapia , Stents , Idoso , Humanos , MasculinoRESUMO
A novel monoclonal antibody has been developed that reacts strongly with human prostatic cancer, especially tumors of high grade. This antibody (7E11C-5) is currently in Phase 3 trials as an imaging agent for metastatic disease. We have cloned the gene that encodes the antigen that is recognized by the 7E11C-5 monoclonal antibody and have designated this unique protein prostate-specific membrane (PSM) antigen. PSM antigen is a putative class II transmembranous glycoprotein exhibiting a molecular size of Mr 94,000. Functionally, class II membrane proteins serve as transport or binding proteins or have hydrolytic activity. Preliminary studies have demonstrated binding of pteroylmonoglutamate (folate) to membrane fractions that also cross-reacted with the PSM monoclonal antibody. We observed substantial carboxypeptidase activity as folate hydrolase associated with PSM antigen. The purpose of our study was to demonstrate that human prostatic carcinoma cells expressing PSM antigen exhibit folate hydrolase activity using methotrexate triglutamate (MTXGlu3) and pteroylpentaglutamate (PteGlu5) as substrates. Isolated membrane fractions from four human prostate cancer cell lines (LNCaP, PC-3, TSU-Prl, and Duke-145) were examined for folate hydrolase activity using capillary electrophoresis. After timed incubations at various pH ranges and in the presence and absence of thiol reagents, separation of pteroyl(glutamate)n derivatives was achieved with an electrolyte of sodium borate and SDS, while absorbance was monitored at 300 nm. The results demonstrate clearly that LNCaP cells, which highly express PSM, hydrolyze gamma-glutamyl linkages of MTXGlu3. The membrane-bound enzyme is an exopeptidase, because it progressively liberates glutamates from MTXGlu3 and PteGlu5 with accumulation of MTX and PteGlu1, respectively. The semipurified enzyme has a broad activity from pH 2.5 to 9.5 and exhibits activity maxima at pH 5 and 8. Enzymatic activity is maintained in the presence of reduced glutathione, homocysteine, and p-hydroxymercuribenzoate (0.05-0.5 mm) but was inhibited weakly by DTT (>/=0.2 mm). By contrast to LNCaP cell membranes, membranes isolated from other human prostate adenocarcinoma cells (PC-3, Duke-145, and TSU-Pr1) did not exhibit comparable hydrolase activity, nor did they react with 7E11-C5 monoclonal antibody. After transfection of PC-3 cells with a full-length 2.65-kb PSM cDNA subcloned into a pREP7 eukaryotic expression vector, non-PSM antigen-expressing PC-3 cells developed immunoreactivity to 7E11-C5 monoclonal antibody and demonstrated folate hydrolase activities and optimum pH activity profiles identical to those of LNCaP cells. The membrane-bound enzymes from both LNCaP- and PC-3-transfected cells also have a capacity to hydrolyze an alpha-linked glutamyl moiety from N-acetyl-alpha-aspartylglutamate. We have identified that PSM antigen is a pteroyl poly-gamma-glutamyl carboxypeptidase (folate hydrolase) and is expressed strongly in human prostate cancer. Cancer cells that express this enzyme are resistant to methotrexate therapy. Those developing future therapeutic strategies in the treatment of prostate cancer that utilize folate antagonists need to consider this mechanism of resistance.
Assuntos
Antígenos de Superfície , Carboxipeptidases/metabolismo , Carboxipeptidases/química , Fracionamento Celular , Membrana Celular/química , Membrana Celular/enzimologia , Ácido Fólico/metabolismo , Glutamato Carboxipeptidase II , Humanos , Concentração de Íons de Hidrogênio , Hidrolases/metabolismo , Imuno-Histoquímica , Masculino , Metotrexato/análogos & derivados , Metotrexato/química , Metotrexato/metabolismo , Ácido Poliglutâmico/análogos & derivados , Ácido Poliglutâmico/química , Ácido Poliglutâmico/metabolismo , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Ácidos Pteroilpoliglutâmicos/química , Ácidos Pteroilpoliglutâmicos/metabolismo , Especificidade por Substrato , Células Tumorais Cultivadas/química , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/enzimologia , gama-Glutamil Hidrolase/metabolismoRESUMO
Heparan sulfate proteoglycans (HSPGs) are ubiquitously present within the perivascular basement membrane, and have been shown to be altered in patients with Alzheimer's Disease (AD). Although the HSPG agrin clearly orchestrates the differentiation of the neuromuscular junction, its role in the brain remains unclear. Growing evidence suggests that agrin may be an important vascular basement membrane (VBM)-associated HSPG. In previous studies, we demonstrated that agrin is present throughout the brain microvasculature, as well as in neuronal cell bodies. AD brains exhibited fragmentation of VBM-associated agrin. Agrin immunoreactivity was also seen within senile plaques and neurofibrillary tangles. These changes were accompanied by the appearance of an additional pool of insoluble agrin. In the present study, we provide further evidence for microvascular damage in AD, by examining the distribution of agrin and laminin within the VBM, and by measuring the agrin concentration within hippocampus and prefrontal cortex. Furthermore, we assessed blood-brain-barrier (BBB) leakage by examining the perivascular distribution of prothrombin immunoreactivity. Soluble agrin levels were increased approximately 30% in Braak stage III-VI AD patients relative to age-matched controls. Furthermore, agrin and laminin exhibited identical patterns of VBM fragmentation in AD and colocalized with beta-amyloid in senile plaques. Microvascular changes were associated with the appearance of perivascular prothrombin immunoreactivity. Our data suggest that agrin is an important VBM-associated HSPG in the brain and that agrin levels are altered in association with microvascular damage in AD.
Assuntos
Agrina/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Idoso , Idoso de 80 Anos ou mais , Peptídeos beta-Amiloides/metabolismo , Química Encefálica/fisiologia , Capilares/patologia , Ensaio de Imunoadsorção Enzimática , Feminino , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Imuno-Histoquímica , Laminina/metabolismo , Masculino , Pessoa de Meia-Idade , Protrombina/metabolismoRESUMO
APOE4 homozygosity has been associated with an increased risk of sporadic Alzheimer's disease through a mechanism, which has yet to be defined. Recent evidence has suggested that microvascular basement membrane injury may be a critical factor in the pathogenesis of AD-related dementia. In previous studies, we have shown that the synaptic organizing protein agrin can be found in neurons, and is a major component of the brain microvascular basement membrane. Here, we compare the basement membrane surface area of cortical microvasculature in AD brains by staining with an anti-agrin antibody. Quantitative morphometric analysis was used to determine the mean basement area (micro(2)) of prefrontal cortical microvessels. An average of 10 capillaries was measured in each of 35 cases of AD genotyped for APOE status. APOE4,4 homozygotes had smaller capillary basement membrane areas (17.4 micro(2))+/-6.2) than APOE3,3 homozygotes (26.9 micro(2)+/-6.5), p<0.001. The capillary basement membrane areas (CBMA) of heterozygotes APOE3,4 did not differ significantly from APOE3,3 or APOE4,4. Braak stage did not contribute significantly to CBMA. However, a preliminary analysis suggests an interaction between APOE4,4 and Braak V-VI producing smaller CBMA, a finding which needs to be confirmed with a larger sample. These data support the hypothesis that APOE4,4 is associated with thinning of the microvascular basement membrane in Alzheimer's disease.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Apolipoproteínas E/genética , Idoso , Idoso de 80 Anos ou mais , Membrana Basal/patologia , Capilares/patologia , Progressão da Doença , Feminino , Genótipo , Humanos , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Córtex Pré-Frontal/patologiaRESUMO
Thinning and discontinuities within the vascular basement membrane (VBM) are associated with leakage of the plasma protein prothrombin across the blood-brain barrier (BBB) in Alzheimer's disease (AD). Prothrombin immunohistochemistry and ELISA assays were performed on prefrontal cortex. In severe AD, prothrombin was localized within the wall and neuropil surrounding microvessels. Factor VIII staining in severe AD patients indicated that prothrombin leakage was associated with shrinkage of endothelial cells. ELISA revealed elevated prothrombin levels in prefrontal cortex AD cases that increased with the Braak stage (Control=1.39, I-II=1.76, III-IV=2.28, and V-VI=3.11 ng prothrombin/mg total protein). Comparing these four groups, there was a significant difference between control and Braak V-VI (p=0.0095) and also between Braak stages I-II and V-VI (p=0.0048). There was no significant difference in mean prothrombin levels when cases with versus without cerebral amyloid angiopathy (CAA) were compared (p-value=0.3627). When comparing AD patients by APOE genotype (ApoE3,3=2.00, ApoE3,4=2.49, and ApoE4,4=2.96 ng prothrombin/mg total protein) an analysis of variance indicated a difference between genotypes at the 10% significance level (p=0.0705). Tukey's test indicated a difference between the 3,3 and 4,4 groups (p=0.0607). These studies provide evidence that in advanced AD (Braak stage V-VI), plasma proteins like prothrombin can be found within the microvessel wall and surrounding neuropil, and that leakage of the blood-brain barrier may be more common in patients with at least one APOE4 allele.
Assuntos
Doença de Alzheimer/complicações , Doença de Alzheimer/patologia , Barreira Hematoencefálica/fisiopatologia , Transtornos Cerebrovasculares/etiologia , Transtornos Cerebrovasculares/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/metabolismo , Análise de Variância , Apolipoproteínas E/genética , Membrana Basal/metabolismo , Membrana Basal/patologia , Córtex Cerebral/metabolismo , Transtornos Cerebrovasculares/metabolismo , Progressão da Doença , Ensaio de Imunoadsorção Enzimática/métodos , Fator VIII/metabolismo , Feminino , Humanos , Imuno-Histoquímica/métodos , Masculino , Pessoa de Meia-Idade , Músculo Liso/metabolismo , Mudanças Depois da Morte , Protrombina/metabolismoRESUMO
Agrin is a heparan sulfate proteoglycan that is widely expressed in neurons and microvascular basal lamina in the rodent and avian central nervous system. Agrin induces the differentiation of nerve-muscle synapses, but its function in either normal or diseased brains is not known. Alzheimer's disease (AD) is characterized by loss of synapses, changes in microvascular architecture, and formation of neurofibrillary tangles and senile plaques. Here we have asked whether AD causes changes in the distribution and biochemical properties of agrin. Immunostaining of normal, aged human central nervous system revealed that agrin is expressed in neurons in multiple brain areas. Robust agrin immunoreactivity was observed uniformly in the microvascular basal lamina. In AD brains, agrin is highly concentrated in both diffuse and neuritic plaques as well as neurofibrillary tangles; neuronal expression of agrin also was observed. Furthermore, patients with AD had microvascular alterations characterized by thinning and fragmentation of the basal lamina. Detergent extraction and Western blotting showed that virtually all the agrin in normal brain is soluble in 1% SDS. In contrast, a large fraction of the agrin in AD brains is insoluble under these conditions, suggesting that it is tightly associated with beta-amyloid. Together, these data indicate that the agrin abnormalities observed in AD are closely linked to beta-amyloid deposition. These observations suggest that altered agrin expression in the microvasculature and the brain parenchyma contribute to the pathogenesis of AD.
Assuntos
Agrina/metabolismo , Doença de Alzheimer/patologia , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Circulação Cerebrovascular , Microcirculação/patologia , Neurônios/metabolismo , Idoso , Agrina/química , Doença de Alzheimer/fisiopatologia , Encéfalo/patologia , Feminino , Humanos , Masculino , Microcirculação/metabolismo , Neurônios/patologia , Valores de Referência , SolubilidadeRESUMO
The choroid plexus plays a key role in supporting neuronal function by secreting cerebrospinal fluid (CSF) and may be involved in the regulation of various soluble factors. Because the choroid plexus is involved in growth factor secretion as well as CSF dynamics, it is important to understand how growth factors in CSF interact with the brain parenchyma as well as with cells in direct contact with the flowing CSF, i.e., choroid plexus and arachnoid villi. While the existence of growth factors in the choroid plexus has been documented in several animal models, the presence and distribution of growth factors in the human choroid plexus has not been extensively examined. This study describes the general distribution and possible functions of a number of key proteins in the human choroid plexus and arachnoid villi, including basic fibroblast growth factor, FGF receptor, and vascular endothelial growth factor. FGF and VEGF could both be readily demonstrated in choroid plexus epithelial cells. The presence of FGF and VEGF within the choroid plexus was also confirmed by ELISA analysis. Since Alzheimer's disease (AD) is known to be associated with a number of growth factor abnormalities, we examined the choroid plexus and arachnoid villi from AD patients. Immunohistochemical studies revealed the presence of FGF and VEGF within the AD choroid plexus and an increased density of FGFr in both the choroid plexus and the arachnoid villi of AD patients. No qualitative changes in the distribution of FGF and VEGF were observed in the AD choroid plexus. The appearance of FGFr in AD arachnoid was associated with robust amyloid and vimentin immunoreactivity. These findings confirm the presence of FGF and VEGF within the normal and AD choroid plexus and suggest that the alteration of growth factors and their receptors may contribute to the pathogenesis of the hydrocephalus ex vacuo that is characteristically seen in AD.