RESUMO
OBJECTIVES: Ground-glass pulmonary opacities (GGOs) are increasingly encountered in routine clinical practice and an accurate differentiation between benign and malignant lesions is crucial. The aim of this study is to evaluate the relationship between radiological features and the actual biological behavior of these nodules. The secondary endpoint is to identify any radiological predictors able to choose the type of surgical resection and the extent of lymphadenectomy. MATERIALS AND METHODS: This single-center retrospective study included all patients, who underwent high resolution computed tomography (HRCT) and surgical resection for GGOs between 2010 and 2020. Histopathological sampling focused on lesion size, histology, growth pattern, amount of lepidic component, percentage of ground-glass (GG), grade of tumor and proliferation index (Ki67). RESULTS: In 56 patients enrolled, 65 lesions (15 pure GG and 50 part-solid) were resected (44 lobectomies, 9 anatomical segmentectomies, 12 wedge resections). A direct significant correlation was found between: the GG at HRCT and the amount of lepidic component (p < 0.0001; R = 0.305), the tumor grading and the lepidic component at HRCT (p = 0.003), the percentage of GG and the expression of Ki67 (p = 0.016), the lepidic percentage and the expression of Ki67 (p = 0.004; R = 0.223). A total of 609 lymph-nodes were removed (stations N1 and N2) and histopathological analysis was negative for nodal involvement in all cases. CONCLUSION: Pure and part-solid GGOs could benefit from less invasive and lung sparing surgery with just nodal sampling. These would reduce surgical complications and guarantee a better quality of life for the patient. The major limitations are the number of patients and the lack of a longer follow-up.
Assuntos
Neoplasias Pulmonares , Humanos , Antígeno Ki-67 , Pulmão/diagnóstico por imagem , Pulmão/patologia , Pulmão/cirurgia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/cirurgia , Pneumonectomia/métodos , Qualidade de Vida , Estudos RetrospectivosRESUMO
PURPOSE: To identify the predictors of malignancy on CT for the evaluation of gastrointestinal stromal tumors (GIST) by correlating CT findings with the mitotic index in order to propose a "CT-based predictive model of Miettinen index." METHODS: One radiologist and one resident in radiology with 14- and 4-year experience in oncological field reviewed the CT findings of 42 patients by consensus, with respect to lesion site, size, contour, tumor growth pattern, enhancing pattern, degree of enhancement of tumor, percentage of tumor necrosis, mesenteric fat infiltration, ulceration, calcification, regional lymphadenopathy, direct invasion to adjacent organs, and distant metastasis. All parameters were correlated with the mitotic index evaluated at histopathological analysis following surgery. Normality of variables was evaluated using Shapiro-Wilk test. Pearson's correlation test was used to assess the interaction between variables. The diagnostic accuracy percentage of tumor necrosis was measured by receiver operating characteristic (ROC) analysis for detecting whether the number of mitosis per 50 high-power fields was > 5. RESULTS: A significant statistical correlation was found between percentage of tumor necrosis and the mitotic index (p < 0.005), dimension, and location of the tumor. CONCLUSION: CT could be an accurate technique in the prediction of malignancy of GIST in a CT risk assessment system, based on the location of the tumor, its size, and the percentage of tumor necrosis.
Assuntos
Tumores do Estroma Gastrointestinal , Medição de Risco , Tomografia Computadorizada por Raios X , Tumores do Estroma Gastrointestinal/diagnóstico por imagem , Humanos , Estudos Retrospectivos , Fatores de RiscoRESUMO
OBJECTIVE: Aim of this study is to evaluate the possibility of limb magnetic resonance lymphography (MRL) to differentiate lymphatic vessels from pathological veins, collect a specimen of the identified lymphatic vessel during operations of super microsurgical lymphatic-venular anastomosis (s-LVA) and perform immunohistochemical stainings to confirm the nature of the collected vessels. PATIENTS AND METHODS: Twenty patients presenting lymphedema were enrolled in this study. Five patients reported lower limb lymphedema and 15 patients reported upper limb lymphedema. All patients had the indication for s-LVA and underwent preoperative MRL imaging of the affected limb. A total of 57 lymphatic vessels were identified by MRL and used to guide s-LVA: all these vessels have also been used to perform an intraoperative biopsy for immunohistochemical evaluation. RESULTS: A total of 53/57 vascular structures resulted compatible with lymphatic vessels at the immunohistochemical study performed with D2-40 antibody; 3/57 specimen showed the absence of the D2-40 antibody. A significant association was found between preoperative MRL and immunohistochemical marker D2-40 on collected specimen. CONCLUSIONS: Most of the articles in the international literature report the concomitant presence of both lymphatic and venous vessels at MRL. However, no one in literature describes the possibility to differentiate venous vessels from lymphatic vessels, and this is a crucial issue for the correct evaluation of the lymphatic system in patients with limb lymphedema undergoing a future surgical correction. In the present study, MRL allowed to identify active lymphatic vessels. MRL was predictive to determine preoperatory lymphatic vessels and to perform successful s-LVA in lymphedema patients. This is the first study to prove the nature of the vessels identified at the preoperative MRL with immunohistochemical stainings.
Assuntos
Vasos Linfáticos/diagnóstico por imagem , Linfedema/diagnóstico por imagem , Linfografia , Imageamento por Ressonância Magnética , Microcirurgia , Anastomose Cirúrgica , Feminino , Humanos , Pessoa de Meia-IdadeRESUMO
T-cell Acute Lymphoblastic Leukemia (T-cell ALL) is a rare haematological neoplasia, that affects children and less commonly adults. Female genital tract and particularly uterus involvement in acute ALL is rare. This report presents the CT features of a 64-year-old woman with uterine relapse of T-cell ALL, occurring 11 months after the diagnosis, as a second, unique relapse of disease. The patient was asymptomatic when a CT examination showed a homogenous thickness of the uterine wall in comparison with the previous CT examination. Histology from biopsy specimens, obtained through hysteroscopy, confirmed T-cell ALL localisation (TdT+, CD10+, CD3c+ and CD2+). The uterus could be a site of relapse in patients suffering from ALL. Even though an MRI examination could better demonstrate the disease in cases of suspected female genital tract involvement by ALL, the comparison of differences between a present and a previous CT examination is sufficient to suspect the diagnosis.
Assuntos
Infiltração Leucêmica/diagnóstico por imagem , Leucemia-Linfoma Linfoblástico de Células T Precursoras/diagnóstico por imagem , Tomografia Computadorizada por Raios X , Útero/diagnóstico por imagem , Antígenos de Diferenciação de Linfócitos T/análise , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biópsia , Neoplasias da Mama/tratamento farmacológico , DNA Nucleotidilexotransferase/análise , Feminino , Humanos , Histeroscopia , Imunofenotipagem , Pessoa de Meia-Idade , Segunda Neoplasia Primária , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Linfócitos T/química , Linfócitos T/patologiaRESUMO
AIM: The aim of this paper was to evaluate the influence of a challenging neck on mid-term results using the Endurant I stent-graft system in high risk patients. METHODS: A retrospective study was conducted on a prospectively compiled database of 72 elective patients with challenging neck treated with the Endurant I system (Endurant Stent Graft, Medtronic AVE, Santa Rosa, CA, USA). These patients were compared to a control group (65 patients) without significant neck problems. The endpoints were mid-term 2-years technical, clinical success and the event free survival of all treated patients. RESULTS: Mean age was 76.12 years; 76.6% of patients were males. Risk factors and preoperative variables did not differ significantly between the two groups. Only 4 (5.5%) patients of the study group vs. 2 (3.1%) in the control group developed type I endoleak during the follow-up. Three (4.1%) study group patients developed type III endoleak vs. 2 (3.1%) in the control group. All these patients required an adjunct procedure of relining with a new endograft. No type II endoleaks requiring adjunctive endovascular procedures were detected in our series. The 2-year event free survival rate did not differ statistically between the two groups (P=0.425). CONCLUSION: Treatment with the Endurant stent-graft is technically feasible and safe, yielding satisfactory results even in challenging anatomies. Mid-term results are promising and challenge current opinion concerning the negative influence of challenging neck anatomy on EVAR especially after a longer follow-up.
Assuntos
Aneurisma Aórtico/cirurgia , Implante de Prótese Vascular/instrumentação , Prótese Vascular , Procedimentos Endovasculares/instrumentação , Stents , Idoso , Idoso de 80 Anos ou mais , Aneurisma Aórtico/diagnóstico , Aortografia/métodos , Implante de Prótese Vascular/efeitos adversos , Intervalo Livre de Doença , Endoleak/etiologia , Endoleak/cirurgia , Procedimentos Endovasculares/efeitos adversos , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Desenho de Prótese , Reoperação , Estudos Retrospectivos , Fatores de Tempo , Tomografia Computadorizada por Raios X , Resultado do Tratamento , Ultrassonografia Doppler em CoresRESUMO
Exogenously administered galanin inhibits cholinergic transmission to the longitudinal muscle and reduces peristaltic efficiency in the guinea pig ileum with a mechanism partially mediated by galanin receptor 1 (GAL-R1). We investigated the effect of exogenous galanin 1-16, which has high affinity for GAL-R1, on the ascending excitatory reflex of the circular muscle elicited by radial distension in isolated segments of guinea pig ileum. We used a three-compartment bath that allows dissecting the ascending pathway into the oral (site of excitatory motor neurons), intermediate (site of ascending interneurons) and caudal compartment (site of intrinsic primary afferent neurons). Galanin 1-16 (0.3-3 micromol L(-1)) applied to the oral compartment inhibited in a concentration-dependent manner the ascending excitatory reflex elicited by the wall distension in the caudal compartment. This effect was antagonized by the GAL-R1 antagonist, RWJ-57408 (1 and 10 micromol L(-1)). By contrast, galanin 1-16 was ineffective when added to the intermediate or caudal compartment up to 3 micromol L(-1). GAL-R1 immunoreactive neurons did not contain neuron-specific nuclear protein, a marker for intrinsic primary afferent neurons. These findings indicate that GAL-R1s are present on motor neurons responsible for the ascending excitatory reflex, but not on ascending interneurons and intrinsic primary afferent neurons.
Assuntos
Íleo/inervação , Neurônios Motores/metabolismo , Receptor Tipo 1 de Galanina/metabolismo , Animais , Galanina/farmacologia , Cobaias , Íleo/efeitos dos fármacos , Imuno-Histoquímica , Interneurônios/metabolismo , Masculino , Microscopia Confocal , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Plexo Mientérico/citologia , Plexo Mientérico/metabolismo , Neurônios Aferentes/metabolismo , Técnicas de Cultura de Órgãos , Fragmentos de Peptídeos/farmacologia , Peristaltismo/fisiologia , Reflexo/fisiologiaRESUMO
Galanin actions are mediated by distinct galanin receptors (GAL-R), GAL-R1, -R2 and -R3. We investigated the role of GAL-R1 in gastric motility and the expression of GAL-R1 in the rat stomach. In vivo, in urethane-anaesthetized rats, galanin (equipotent for all GAL-Rs) induced a short inhibition of gastric motility, followed by increase in tonic and phasic gastric motility; the latter was significantly reduced by the GAL-R1 antagonist, RWJ-57408. Galanin 1-16 (high affinity for GAL-R1 and -R2) induced a long-lasting decrease of intragastric pressure, which was not modified by RWJ-57408. In vitro, galanin and galanin 1-16 induced increase of intragastric pressure that was not affected by RWJ-57408. Tetrodotoxin (TTX) did not suppress the galanin excitatory effect, whereas the effect of galanin 1-16 on gastric contraction was increased by TTX- or N-nitro-L-arginine, an inhibitor of nitric oxide synthase. GAL-R1 immunoreactivity was localized to cholinergic and tachykinergic neurons and to neurons immunoreactive for nitric oxide synthase or vasoactive intestinal polypeptide. This study suggests that an extrinsic GAL-R1 pathway mediates the galanin excitatory action, whereas an extrinsic, non GAL-R1 pathway is likely to mediate the galanin inhibitory effect in vivo. GAL-R1 intrinsic neurons do not appear to play a major role in the control of gastric motility.
Assuntos
Motilidade Gastrointestinal/fisiologia , Receptor Tipo 1 de Galanina/fisiologia , Animais , Relação Dose-Resposta a Droga , Galanina/farmacologia , Motilidade Gastrointestinal/efeitos dos fármacos , Técnicas In Vitro , Masculino , Rede Nervosa/efeitos dos fármacos , Rede Nervosa/fisiologia , Fragmentos de Peptídeos/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Galanina/agonistasRESUMO
The functioning of enteric neuronal circuitries has been elucidated in the recent past. Evidence is now gathering to explain how dysfunction of the enteric nervous system (ENS) may lead to human gastrointestinal motor disorders. These conditions include achalasia, congenital hypertrophic pyloric stenosis, chronic intestinal pseudo-obstruction, Hirschsprung's disease, chronic idiopathic constipation, and probably irritable bowel syndrome. Degenerative, inflammatory and genetic mechanisms exert a critical role in ENS dysfunction underlying gut dysmotility. The study of the ENS abnormalities in gut dysmotility provides a framework to better understand the mechanisms involved in degeneration and neuronal loss and fosters the development of targeted therapeutic options.
Assuntos
Doenças do Sistema Nervoso Autônomo/patologia , Doenças do Sistema Nervoso Autônomo/fisiopatologia , Sistema Nervoso Entérico/patologia , Gastroenteropatias/etiologia , Motilidade Gastrointestinal/fisiologia , Gastroenteropatias/patologia , Gastroenteropatias/fisiopatologia , HumanosRESUMO
OBJECTIVES: To test the hypothesis that genital prolapse may be related to peripheral nerve abnormalities, we examined the changes occurring to peptide-containing nerve processes supplying the periurethral muscles in women with stress urinary incontinence associated with prolapse. METHODS: Thirty patients with genital prolapse and 10 age-matched control subjects entered the study. All patients were evaluated by urodynamic investigations. Ten of 30 patients had pure stress urinary incontinence; none of the control subjects was incontinent. During surgery, four biopsy samples were obtained from each woman from the periurethral and perirectal muscles. The muscle sections were processed for immunohistochemistry using specific antibodies to glial (S-100 protein) and general neuronal markers (neuron-specific enolase) and neuropeptides, including neuropeptide Y, vasoactive intestinal polypeptide, and substance P. The evaluation of immunolabeled nerves was based on a semiquantitative analysis that allowed for a four-point ordinate scale score. RESULTS: S-100 and neuron-specific enolase immunoreactive nerve fibers, running either singly or in small bundles, along with a dense network of neural processes containing neuropeptide Y, vasoactive intestinal polypeptide, and substance P, were found throughout the connective tissue and striated muscle of the control specimens. In contrast, in the muscle specimens from those with genitourinary prolapse, both the density and the intensity of neuropeptide Y, vasoactive intestinal polypeptide, and substance P immunoreactive nerves were markedly reduced compared with the control specimens. CONCLUSIONS: The evidence of a reduced peptide-containing nerve supply to the perineal muscles provides a morphologic basis suggesting that neural abnormalities contribute to the pathogenesis of genital prolapse and urinary incontinence.
Assuntos
Denervação , Proteínas do Tecido Nervoso/análise , Neurônios/química , Neuropeptídeos/análise , Diafragma da Pelve/inervação , Doenças do Sistema Nervoso Periférico/diagnóstico , Incontinência Urinária por Estresse/etiologia , Prolapso Uterino/etiologia , Idoso , Biomarcadores , Biópsia , Peso ao Nascer , Tecido Conjuntivo/inervação , Feminino , Humanos , Pessoa de Meia-Idade , Modelos Neurológicos , Músculo Esquelético/inervação , Músculo Esquelético/patologia , Neuropeptídeo Y/análise , Obesidade/complicações , Paridade , Doenças do Sistema Nervoso Periférico/complicações , Doenças do Sistema Nervoso Periférico/metabolismo , Fosfopiruvato Hidratase/análise , Pós-Menopausa , Reto/inervação , Proteínas S100/análise , Substância P/análise , Uretra/inervação , Incontinência Urinária por Estresse/fisiopatologia , Incontinência Urinária por Estresse/cirurgia , Prolapso Uterino/fisiopatologia , Prolapso Uterino/cirurgia , Peptídeo Intestinal Vasoativo/análiseRESUMO
Galanin effects are mediated by distinct receptors, galanin receptor 1 (GAL-R1), GAL-R2 and GAL-R3. Here, we analyzed 1) the role of GAL-R1 in cholinergic transmission and peristalsis in the guinea-pig ileum using longitudinal muscle-myenteric plexus preparations and intact segments of the ileum in organ bath, and 2) the distribution of GAL-R1 immunoreactivity in the myenteric plexus with immunohistochemistry and confocal microscopy. Galanin inhibited electrically stimulated contractions of longitudinal muscle-myenteric plexus preparations with a biphasic curve. Desensitization with 1 microM galanin suppressed the high potency phase of the curve, whereas the GAL-R1 antagonist, RWJ-57408 (1 microM), inhibited the low potency phase. Galanin (3 microM) reduced the longitudinal muscle contraction and the peak pressure, and decreased the compliance of the circular muscle. All these effects were antagonized by RWJ-57408 (1 or 10 microM). RWJ-57408 (10 microM) per se did not affect peristalsis parameters in normal conditions, nor when peristalsis efficiency was reduced by partial nicotinic transmission blockade with hexamethonium. In the myenteric plexus, GAL-R1 immunoreactivity was localized to neurons and to fibers projecting within the plexus and to the muscle. GAL-R1 was expressed mostly by cholinergic neurons and by some neurons containing vasoactive intestinal polypeptide or nitric oxide synthase. This study indicates that galanin inhibits cholinergic transmission to the longitudinal muscle via two separate receptors; GAL-R1 mediates the low potency phase. The reduced peristalsis efficiency could be explained by inhibition of the cholinergic drive, whereas the decreased compliance is probably due to inhibition of descending neurons and/or to the activation of an excitatory muscular receptor. Endogenous galanin does not appear to affect neuronal pathways subserving peristalsis in physiologic conditions via GAL-R1.
Assuntos
Galanina/farmacologia , Íleo/fisiologia , Plexo Mientérico/efeitos dos fármacos , Peristaltismo/fisiologia , Receptor Tipo 1 de Galanina/metabolismo , Acetilcolina/metabolismo , Animais , Relação Dose-Resposta a Droga , Estimulação Elétrica , Cobaias , Imuno-Histoquímica , Microscopia Confocal , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Plexo Mientérico/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Técnicas de Cultura de Órgãos , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologiaRESUMO
AIMS: To develop a real-time polymerase chain reaction (PCR) method for rapid detection and quantification of Oenococcus oeni in wine samples for monitoring malolactic fermentation. METHODS AND RESULTS: Specific primers and fluorogenic probe targeted to the gene encoding the malolactic enzyme of O. oeni were developed and used in real-time PCR assays in order to quantify genomic DNA either from bacterial pure cultures or wine samples. Conventional CFU countings were also performed. The PCR assay confirmed to be specific for O. oeni species and significantly correlated to the conventional plating method both in pure cultures and wine samples (r = 0.902 and 0.96, respectively). CONCLUSIONS: The DNA extraction from wine and the real-time PCR quantification assay, being performed in ca 6 h and allowing several samples to be concurrently processed, provide useful tools for the rapid and direct detection of O. oeni in wine without the necessity for sample plating. SIGNIFICANCE AND IMPACT OF THE STUDY: Rapid quantification of O. oeni by a real-time PCR assay can improve the control of malolactic fermentation in wines allowing prompt corrective measures to regulate the bacterial growth.
Assuntos
Cocos Gram-Positivos/isolamento & purificação , Vinho/microbiologia , Contagem de Colônia Microbiana/métodos , DNA Bacteriano/análise , DNA Bacteriano/isolamento & purificação , Microbiologia de Alimentos , Genes Bacterianos , Cocos Gram-Positivos/crescimento & desenvolvimento , Leuconostoc/crescimento & desenvolvimento , Leuconostoc/isolamento & purificação , Malato Desidrogenase/genética , Reação em Cadeia da Polimerase , Sensibilidade e EspecificidadeRESUMO
A phase IV trial with 5% amikacin gel was carried out on 100 adult in patients of both sexes suffering from venous infected leg ulcers. After 2 weeks' therapy the microbiological culture tests were negative for more than 80% of the patients. The mean ulcer surface area was reduced by 34% and the accompanying symptoms of erythema, inflammation and pain were improved. Only very mild unwanted local effects were reported by four out of the 100 patients. Five percent amikacin gel was judged a safe and effective topical treatment for curing infected venous leg ulcers.
Assuntos
Amicacina/uso terapêutico , Antibacterianos/uso terapêutico , Úlcera Varicosa/tratamento farmacológico , Adulto , Feminino , Géis , Humanos , Masculino , Resultado do TratamentoRESUMO
In this study, we tested the effectiveness of Rifaximin, a surface antibiotic which is not absorbed when given orally, in the eradication of Helicobacter pylori (HP). The drug was combined in triple therapy either with Amoxicillin and Omeprazole or with Erythromycin-ethylsuccinate and Omeprazole. Twenty-three patients complaining of dyspeptic symptoms and gastric infection due to HP were evaluated. The patients were randomly given one of the following therapeutic protocols: Rifaximin susp. 600 mg/day x 3/day (at least two hours after meals: 10:00 am, 2:00 pm, 9:00 pm), Amoxicillin tab. 1 g x 2/day (at least two hours after meals: 10:00 am, 9:00 pm), Omeprazole tab. 40 mg/day (in the morning before breakfast) (protocol A) and Rifaximin susp. 600 mg/day x 3/day and Erythromycin-ethylsuccinate tab. 600 mg x 3/day (at least two hours after meals: 10:00 am, 2:00 pm, 9:00 pm), Omeprazole tab. 40 mg/day (in the morning before breakfast) (protocol B). Both therapeutic protocols were prescribed for two weeks. At least one month after the end of the treatment the patients were controlled to ascertain eradication of the infection. The follow-up carried out after treatment showed that HP infection was eradicated in 6 of 10 patients in the first group (protocol A) and in 1 of 10 in the second group (protocol B). These patients were HP-negative in all the tests performed: histological, CP-TEST, culture test. The data collected showed a reasonable level of effectiveness of the protocol using the combination Rifaximin-Amoxicillin and Omeprazole. However, they do not differ from the reported data in the literature which show a similar effectiveness of the combination Omeprazole-Amoxicillin at the same doses. Different formulations that makes it possible for the drug to reach these "protected areas" would probably be more effective.
Assuntos
Quimioterapia Combinada/uso terapêutico , Infecções por Helicobacter/tratamento farmacológico , Helicobacter pylori , Rifamicinas/uso terapêutico , Adolescente , Adulto , Criança , Quimioterapia Combinada/efeitos adversos , Feminino , Infecções por Helicobacter/microbiologia , Humanos , Masculino , Rifamicinas/efeitos adversos , RifaximinaRESUMO
We have previously documented a dramatic elevation in the activity of alpha 2,6-sialytransferase towards Gal beta 1,4GlcNAc (EC 2.4.99.1) (alpha 2,6ST) in CaCo-2 cells maintained in culture for several days after confluence to elicit a high degree of enterocytic differentiation phenotype. Northern analysis performed with a probe complementary to a region of human alpha 2,6ST mRNA common to all known transcripts demonstrated that the expression of alpha 2,6ST mRNA in CaCo-2 cells increased with the degree with the degree of differentiation. When probes complementary to 5'-untranslated exons (Y + Z or X) previously identified in transcripts isolated from human placenta and from several human lymphoblastoid cell lines were used, no hybridization signal with mRNA of CaCo-2 cells was found, as reported for the mRNA of hepatoma cell line HepG2 (Wang XC, Vertino A, Eddy RL, Byers MG, Jani-Sait SN, Shows TB, Lau JTY (1993) J Biol Chem 268: 4355-61). These results support the notion that the major alpha 2,6ST transcript of CaCo-2 cells was the hepatoma isoform or a new one, so far unreported. Consistent with the differentiation-dependent increase in alpha 2,6ST-mRNA expression, an elevation of the reactivity with Sambucus nigra agglutinin of differentiated CaCo-2 cell-surface was observed, indicating an enhanced alpha 2,6-sialylation of membrane glycoconjugates.
Assuntos
Células CACO-2/enzimologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sialiltransferases/genética , Sequência de Bases , Sequência de Carboidratos , Diferenciação Celular , Primers do DNA/genética , Expressão Gênica , Glicoconjugados/química , Glicoconjugados/metabolismo , Humanos , Cinética , Dados de Sequência Molecular , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Sialiltransferases/metabolismo , Especificidade por Substrato , beta-D-Galactosídeo alfa 2-6-SialiltransferaseRESUMO
We have previously shown that human colon carcinoma CaCo-2 cells express the Sda-beta 1,4-N-acetylgalactosaminyltransferase (Sda-beta GalNAc-transferase) and that the enzyme activity correlates with the degree of enterocytic differentiation. Here we report that a large amount of this glycosyltransferase is released in soluble form, particularly when CaCo-2 cells are maintained in culture for more than 3 weeks in order to ensure an higher degree of enterocyte differentiation. The soluble enzyme was concentrated and partially purified by Blue-Sepharose and fetuin-Sepharose chromatography. The substrate specificity of the partially purified enzyme was similar to that of Sda-enzyme from epithelial cells of colon mucosa, and for its activity strictly required the presence in acceptors of NeuAc in alpha 2,3-linkage to subterminal galactose. Among the low molecular glycans tested, NeuAc alpha 2,3Gal beta 1,4GlcNAc appeared to be the best acceptor, whereas sialyl-Lewisx and sialyl-Lewisa did not serve as acceptors, indicating that the fucosylation of sub-terminal GlcNAc hindered the transferase activity. Contrary to this, the activity towards a disialylated acceptor such as di-sialyl-lacto-N-tetraose was reduced but not abolished. When CaCo-2 cells were cultured on porous membranes and the transferase activity assayed in medium collected from chambers corresponding to either the apical or basolateral face of highly differentiated CaCo-2 cells, a preferential release from the basolateral surface was found. Considering that Sda-beta GalNAc-transferase is mainly located in the large intestine, current results support the notion that colonic cells largely contribute to the presence of the enzyme in human plasma.
Assuntos
N-Acetilgalactosaminiltransferases/metabolismo , Células CACO-2 , Sequência de Carboidratos , Diferenciação Celular/fisiologia , Humanos , Dados de Sequência Molecular , Solubilidade , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
The high occurrence in large intestine epithelial cells from pig of a beta-N-acetylgalactosaminyltransferase with a substrate specificity very similar to that of the Sda beta 1,4-N-acetylgalactosaminyltransferase from other tissues is reported. The enzyme strictly recognized the NeuAc alpha 2,3Gal beta terminal sequence of N- and O-linked oligosaccharides bound to glycoproteins. The transferase activity required Mn2+ and an optimum pH of 7.4. In contrast to the kidney Sda-enzyme from humans and other mammals, the microsomal fraction of pig colonic cells expressed a very high activity even in the absence of Triton X-100. A rapid procedure is presented for the large scale preparation of GalNAc beta 1,4(NeuAc alpha 2,3)Gal beta 1,4Glc from NeuAc alpha 2,3Gal beta 1,4Glc. The biosynthesized tetrasaccharide was completely resistant to the action of neuraminidase from Vibrio cholerae, whereas about 60% of N-acetylneuramic acid was cleaved by neuraminidase from Newcastle disease virus. HPLC separation of different compounds is reported.
Assuntos
Antígenos de Grupos Sanguíneos/metabolismo , Ceco/enzimologia , Colo/enzimologia , Mucosa Intestinal/enzimologia , N-Acetilgalactosaminiltransferases/metabolismo , Oligossacarídeos/biossíntese , Animais , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão , Epitélio/enzimologia , Humanos , Técnicas In Vitro , Rim/enzimologia , Dados de Sequência Molecular , Oligossacarídeos/química , Oligossacarídeos/isolamento & purificação , Especificidade de Órgãos , Ratos , Especificidade da Espécie , Especificidade por Substrato , SuínosRESUMO
In previous works we established that the alpha 2,6-sialyltransferase acting on N-acetyllactosaminic sequences [alpha 2,6(N)ST, E.C. 2.4.99.1] behaves, in colonic cells, as an oncodevelopmentally regulated enzyme. Subpopulations of the human colon cancer cell line HT-29 adapted to grow in 10(-5) M methotrexate (MTX), permanently retain the ability to differentiate as mucus-secreting cells when kept confluent for extended periods of time [Lesuffleur et al. (1991) J. Cell Biol. 115, 1409-1418]. In this study we have compared the activities of five sialyltransferases acting on N- or O-linked chains of glycoproteins in parental HT-29 and in the 10(-5) M MTX-resistant variant. Both cell lines were studied during the exponential phase of growth as well as after a long period of postconfluent culture (28-30 days). Regardless the culture conditions, resistance to 10(-5) M MTX is associated with a virtual disappearance of alpha 2,6(N)ST activity. This change results in a dramatic reduction of the reactivity of cell membranes with the fluorescent lectin from Sambucus nigra, specific for alpha 2,6-sialylated structures. The activity of the alpha 2,3-sialyltransferase which acts on N-acetyllactosaminic sequences increases about two times in postconfluent cultures of 10(-5) M MTX-resistant cells, suggesting a close relationship with the differentiation degree. No significative changes were observed in the activity of other sialyltransferases.
Assuntos
Resistência a Medicamentos/fisiologia , Metotrexato/toxicidade , Lectinas de Plantas , Sialiltransferases/metabolismo , Configuração de Carboidratos , Sequência de Carboidratos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Neoplasias do Colo , Citometria de Fluxo , Humanos , Cinética , Lectinas , Dados de Sequência Molecular , Oligossacarídeos/biossíntese , Proteínas Inativadoras de Ribossomos , Especificidade por Substrato , Células Tumorais Cultivadas , beta-D-Galactosídeo alfa 2-6-Sialiltransferase , beta-Galactosídeo alfa-2,3-SialiltransferaseRESUMO
The distribution of glycoprotein B (gB) among different human herpesvirus 6 (HHV-6) strains was analysed with a panel of three monoclonal antibodies (MAbs) derived from mice immunized with U1102-infected lymphocytes. MAb 2D9 reacted specifically by immunofluorescence and immunoprecipitation with the U1102 and GS isolates, and failed to react with Z29 and the variant B strains Hashimoto and SF. In addition, Z29, Hashimoto and SF gB had a lower M(r) than U1102 and GS gB. MAb 2D9 also failed to react with the exanthem subitum isolate CV, included in this study as an as yet poorly characterized isolate. Consistent with this result, CV failed to react with the variant A-specific MAb to gp82-105 and behaved as a variant B virus even with respect to the diagnostic HindIII endonuclease restriction cleavage site located in a fragment hybridizing to the pZVH14 probe. By contrast with MAb 2D9, MAbs 2B9 and 2D10 reacted with all of the isolates tested, strengthening the argument tha they have common epitopes. Based on the antigenic and M(r) specificities of gB, the HHV-6 isolates tested were arranged into two non-overlapping clusters, which closely parallel the variant A and B strain groups, defined previously by several criteria, including restriction endonuclease polymorphism, antigenic variations, growth in in vitro cultures and sequence analyses.
Assuntos
Antígenos Virais/análise , Herpesvirus Humano 6/imunologia , Proteínas do Envelope Viral/análise , Anticorpos Monoclonais , Variação Antigênica , Herpesvirus Humano 6/classificação , Humanos , Proteínas do Envelope Viral/imunologiaRESUMO
Monoclonal antibody 2D10 (MAb 2D10) raised toward human herpesvirus-6(U1102) [HHV6(U1102)] immunoprecipitated three glycosylated peptides, M(r) 112,000, 64,000, and 58,000, designated as gp112 from U1102-infected lymphocytes. Pulse-chase experiments suggest that the M(r) 64,000 and 58,000 polypeptides are very likely generated by post-translational cleavage of the M(r) 112,000 polypeptide. MAb 2D10 neutralized virion infectivity in the presence of complement, suggesting that gp112 is located in the virion envelope. MAb 2D10 did not prevent the appearance of HHV6-specific cytopathic effect. MAb 2D10 was reactive with denatured gp112 in immunoblots. HHV6 isolates form two clusters (Schimer, Wyatt, Yamanishi, Rodriguez, and Frenkel, Proc. Natl. Acad. Sci. USA 88, 5922; Ablashi, Balachandran, Josephs, Hung, Krtueger, Kramarsky, Salahuddin, and Gallo, Virology 184, 545). MAb 2D10 reacted by immunofluorescence and immunoprecipitation with the prototypes of each cluster, GS and Z29. Whereas the proteins immunoprecipitated by MAb 2D10 from GS-infected lymphocytes had an electrophoretic pattern very similar to that of U1102 gp112, the homologous glycoprotein immunoprecipitated from Z29-infected lymphocytes consisted of three polypeptides with M(r) 102,000, 59,000, and 50,000. The data suggest a variation among HHV6 isolates as far as this glycoprotein is concerned.