RESUMO
Free-living amoebae belonging to the genus Acanthamoeba are the causative agents of infections such as amoebic keratitis (AK), granulomatous amoebic encephalitis (GAE) and cutaneous lesions. The mechanisms involved in the establishment of infection are unknown. However, it is accepted that the initial phase of pathogenesis involves adherence to the host tissue. In this work, we analysed surface molecules with an affinity for epithelial and neuronal cells from the trophozoites of Acanthamoeba castellanii. We also investigated the cellular mechanisms that govern the process of trophozoite adhesion to the host cells. We first used confocal and epifluorescence microscopy to examine the distribution of the A. castellanii actin cytoskeleton during interaction with the host cells. The use of drugs, as cytochalasin B (CB) and latrunculin B (LB), revealed the participation of cytoskeletal filaments in the adhesion process. In addition, to identify the proteins and glycoproteins on the surface of A. castellanii, the trophozoites were labelled with biotin and biotinylated lectins. The results revealed bands of surface proteins, some of which were glycoproteins with mannose and N-acetylglucosamine residues. Interaction assays of biotinylated amoebae proteins with epithelial and neuronal cells showed that some surface proteins had affinity for both cell types. The results of this study provide insight into the biochemical and cellular mechanisms of the Acanthamoeba infection process.
Assuntos
Acanthamoeba castellanii/fisiologia , Amebíase/parasitologia , Citoesqueleto/metabolismo , Interações Hospedeiro-Parasita , Acanthamoeba castellanii/efeitos dos fármacos , Acanthamoeba castellanii/patogenicidade , Acetilglucosamina/metabolismo , Citoesqueleto de Actina/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Adesão Celular , Linhagem Celular , Extensões da Superfície Celular/metabolismo , Citocalasina B/farmacologia , Células Epiteliais/parasitologia , Glicoproteínas/metabolismo , Humanos , Lectinas/metabolismo , Manose/metabolismo , Modelos Biológicos , Proteínas de Protozoários/metabolismo , Tiazolidinas/farmacologia , TrofozoítosRESUMO
BACKGROUND: There are no established biomarkers to identify tumour recurrence in stage II colon cancer. As shown previously, the enzymatic activity of the cyclin-dependent kinases 1 and 2 (CDK1 and CDK2) predicts outcome in breast cancer. Therefore, we investigated whether CDK activity identifies tumour recurrence in colon cancer. METHODS: In all, 254 patients with completely resected (R0) UICC stage II colon cancer were analysed retrospectively from two independent cohorts from Munich (Germany) and Leiden (Netherlands). None of the patients received adjuvant treatment. Development of distant metastasis was observed in 27 patients (median follow-up: 86 months). Protein expression and activity of CDKs were measured on fresh-frozen tumour samples. RESULTS: Specific activity (SA) of CDK1 (CDK1SA), but not CDK2, significantly predicted distant metastasis (concordance index=0.69, 95% confidence interval (CI): 0.55-0.79, P=0.036). Cutoff derivation by maximum log-rank statistics yielded a threshold of CDK1SA at 11 (SA units, P=0.029). Accordingly, 59% of patients were classified as high-risk (CDK1SA ≥11). Cox proportional hazard analysis revealed CDK1SA as independent prognostic variable (hazard ratio=6.2, 95% CI: 1.44-26.9, P=0.012). Moreover, CKD1SA was significantly elevated in microsatellite-stable tumours. CONCLUSION: Specific activity of CDK1 is a promising biomarker for metastasis risk in stage II colon cancer.
Assuntos
Neoplasias do Colo/enzimologia , Quinases Ciclina-Dependentes/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Neoplasias do Colo/patologia , Primers do DNA , Feminino , Imunofluorescência , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Estudos Retrospectivos , Adulto JovemRESUMO
BACKGROUND: We established the cell cycle profiling (C2P) assay for specific activity (SA; activity/expression) of cyclin-dependent kinases (CDKs). C2P risk score (C2P-RS) based on CDK1 and CDK2 SAs was significantly associated with relapse in breast cancer (BC). This study was conducted to investigate the predictive value of C2P-RS for neoadjuvant chemotherapy (NAC). PATIENTS AND METHODS: Among 124 eligible patients, 122 were treated with weekly paclitaxel followed by 5-fluorouracil, epirubicin and cyclophosphamide (P-FEC) and 2 were treated with paclitaxel monotherapy. C2P-RS was determined via C2P using frozen biopsy samples before NAC. RESULTS: Negative estrogen receptor (ER), negative progesterone receptor (PR), positive human epidermal growth factor receptor 2 (HER2), high Ki-67 expression and intermediate + high C2P-RS were significantly associated with high pathological complete response (pCR) rates compared with positive ER (30% versus 9%), positive PR (25% versus 6%), negative HER2 (34% versus 11%), low Ki-67 expression (24% versus 7%) or low C2P-RS (24% versus 9%), respectively. The combination of C2P-RS and Ki-67 had a stronger impact on pCR than each parameter alone, and a multivariate analysis showed that the combination was an independent predictor of pCR (odds ratio 3.3, 95% confidence interval 1.1-9.5). CONCLUSIONS: C2P-RS was significantly associated with pCR after P-FEC and may be a useful predictor for chemotherapy in BCs.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/enzimologia , Proteína Quinase CDC2/metabolismo , Carcinoma Ductal de Mama/enzimologia , Quinase 2 Dependente de Ciclina/metabolismo , Terapia Neoadjuvante , Recidiva Local de Neoplasia/enzimologia , Adulto , Idoso , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/cirurgia , Carcinoma Ductal de Mama/tratamento farmacológico , Carcinoma Ductal de Mama/cirurgia , Ciclofosfamida/administração & dosagem , Epirubicina/administração & dosagem , Feminino , Fluoruracila/administração & dosagem , Humanos , Antígeno Ki-67/metabolismo , Pessoa de Meia-Idade , Análise Multivariada , Recidiva Local de Neoplasia/prevenção & controle , Paclitaxel/administração & dosagem , Receptores de Esteroides/metabolismo , Fatores de Risco , Resultado do TratamentoRESUMO
Amoebic liver abscess (ALA) is the most important extraintestinal complication of Entamoeba histolytica infection. Amoebic liver abscess development causes severe destruction of the liver tissue concomitant with a strong inflammatory reaction. We analyse the in situ expression of TNF-α, IFN-γ, IL-1ß, 1L-8 and IL-10 at different stages of ALA development in a susceptible animal model. Results showed that after inoculation, neutrophils (PMN) and some macrophages infiltrated the liver and were positive for TNF-α and IFN-γ at the acute phase of amoeba infection. The presence of these cytokines was transient and decreased as tissue damage progressed. In contrast, IL-1ß and IL-8 were detected mainly in neutrophils and macrophages from the periods of acute infection to subacute and chronic infection and decreased when granulomas were formed. The IL-10 was expressed in PMN and mononuclear cells and only during a short period at the onset of acute infection. The qRT-PCR of mRNA revealed a relationship with the expression of the cytokines in cells found in the ALA. Furthermore, our data suggest that IL-10 does not regulate local production of these cytokines. Our results indicate that an exacerbated inflammatory milieu is established and contributes to liver tissue damage and probably supports the survival of the parasites.
Assuntos
Citocinas , Entamoeba histolytica/imunologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/imunologia , Inflamação/imunologia , Abscesso Hepático Amebiano/imunologia , Abscesso Hepático Amebiano/metabolismo , Fígado/imunologia , Fígado/metabolismo , Macrófagos/imunologia , Neutrófilos/imunologia , RNA Mensageiro/análise , Animais , Cricetinae , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Entamoeba histolytica/metabolismo , Imuno-Histoquímica , Inflamação/metabolismo , Fígado/parasitologia , Fígado/ultraestrutura , Abscesso Hepático Amebiano/parasitologia , Macrófagos/metabolismo , Masculino , Neutrófilos/metabolismo , Reação em Cadeia da PolimeraseRESUMO
To define the role of CD38 in the migration of neutrophils to the liver and consequently in the induction of an innate immune response during murine hepatic amoebiasis by Entamoeba histolytica, we examined amoebic liver abscess development (ALA), presence of amoebae and neutrophils, and expression levels of cytokines and other inflammation mediators mRNA, in infected wild-type and CD38 Knockout (CD38KO) C57BL/6J mice. Results showed that CD38KO mice undergo a delay in ALA development in comparison with the wild-type strain. The presence of amoebae lasted longer in CD38(-/-), and although neutrophils arrived to the liver in both strains, there was a clear difference in the time between the two strains; whereas in the wild-type strain, neutrophils arrived at early times (6-12 h), in the CD38KO strain, neutrophils arrived later (48-72 h). Cytokines profile during the innate immune response development (TNF-α, IL-1ß, IL-6) was, for WT mice concomitant with, and preceded, for CD38KO mice, the time in which neutrophils were present in the liver lesion. In conclusion, CD38 is important for neutrophils migration during hepatic amoebiasis, and in turn, these cells play an important role in the innate immune response.
Assuntos
ADP-Ribosil Ciclase 1/deficiência , Entamoeba histolytica/imunologia , Imunidade Inata , Abscesso Hepático Amebiano/imunologia , Fígado/imunologia , Glicoproteínas de Membrana/deficiência , Neutrófilos/imunologia , ADP-Ribosil Ciclase 1/imunologia , Animais , Citocinas/biossíntese , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Mediadores da Inflamação/imunologia , Masculino , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de TempoRESUMO
In a Japanese study, cyclin-dependent kinase (CDK) based risk determined by CDK 1 and 2 activities was associated with risk of distance recurrence in early breast cancer patients. The aim of our study was to validate this risk categorization in European early breast cancer patients. We retrospectively analyzed frozen breast cancer specimens of 352 Dutch patients with histologically confirmed primary invasive early breast cancer. CDK-based risk was determined in tumour tissues by calculating a risk score (RS) according to kinases activity and protein mass concentration assay without the knowledge of outcome. Determination of CDK-based risk was feasible in 184 out of 352 (52%) tumours. Median follow-up of these patients was 15 years. In patients not receiving systemic treatment, the proportions of risk categories were 44% low, 16% intermediate, and 40% high CDK-based risk. These groups remained significant after univariate and multivariate Cox-regression analysis. Factors associated with a shorter distant recurrence-free period were positive lymph nodes, mastectomy with radiotherapy, and high CDK-based risk. There was no significant correlation with overall survival (OS). CDK-based risk is a prognostic marker of distance recurrence of patients with early breast cancer. More validation would be warranted to use of CDK-based risk into clinical practice.
Assuntos
Neoplasias da Mama/enzimologia , Quinases Ciclina-Dependentes/metabolismo , População Branca , Adulto , Neoplasias da Mama/patologia , Feminino , Humanos , Pessoa de Meia-Idade , Prognóstico , Fatores de Risco , Análise de SobrevidaRESUMO
OBJECTIVE: To evaluate the effectiveness and usefulness of transnasal endoscopic surgery for the treatment of odontogenic maxillary cysts. METHODS: Between February 2003 and February 2008, transnasal endoscopic surgery was performed under general anesthesia in 13 patients (male 6 and female 7, 19 to 75 years old) with odontogenic maxillary cysts that extended to the maxillary sinus. Ten patients had a radicular cyst and three patients had a dentigerous cyst. After the resection of anterior edge of the inferior turbinate, the lateral wall of the inferior nasal meatus was opened. Then, the cyst wall of the maxillary sinus was partially or completely removed under the endoscope. RESULTS: The cyst walls were completely removed in five often patients with a radicular cyst and in all three patients with a dentigerous cyst. Five patients with a radicular cyst received partial resection of the cyst wall. The affected teeth could be preserved in seven of ten patients with a radicular cyst and in one of three patients with a dentigerous cyst. There were no complications, and postoperative courses were uneventful. Follow-up period ranged from 11 to 72 months (mean 42 months), and no recurrence has been noted in any of the cases. CONCLUSIONS: Endoscopic transnasal surgery for the odontogenic maxillary cyst is less invasive than conventional dental approach, and most of the affected teeth can be preserved. This technique appears to be a simple and highly effective surgical treatment for the treatment of patients with odontogenic cysts that extend to the maxillary sinus.
Assuntos
Cistos/cirurgia , Doenças Maxilares/cirurgia , Procedimentos Cirúrgicos Otorrinolaringológicos/métodos , Cisto Radicular/cirurgia , Adulto , Idoso , Cisto Dentígero/cirurgia , Endoscopia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Gels are a soft elastic material consisting of a three-dimensional polymer network with nanometer-sized pores and are used in a variety of applications. However, gel networks typically have a substantial level of defects because the network formation reaction proceeds stochastically. In this study, we present a general scheme to fabricate gels with extremely low levels of defects by applying geometric constraints into pregel solution based on the "bond percolation" concept. In the formed gel, stationary laser speckles, which are an indicator of spatial defects, were not observed at all. In addition, we found that the concentration fluctuations of the polymer chains were ergodic across the whole gel network. In such a homogeneous gel, both the spatial and temporal correlations of polymer chains are the same before and after gelation.
RESUMO
Epithelial cells lining the intestinal mucosa constitute a selective-semipermeable barrier acting as first line of defense in the organism. The number of those cells remains constant during physiological conditions, but disruption of epithelial cell homeostasis has been observed in several pathologies. During colitis, epithelial cell proliferation decreases and cell death augments. The mechanism responsible for these changes remains unknown. Here, we show that the pro-inflammatory cytokine IFNγ contributes to the inhibition of epithelial cell proliferation in intestinal epithelial cells (IECs) by inducing the activation of mTORC1. Activation of mTORC1 in response to IFNγ was detected in IECs present along the crypt axis and in colonic macrophages. mTORC1 inhibition enhances cell proliferation, increases DNA damage in IEC. In macrophages, mTORC1 inhibition strongly reduces the expression of pro-inflammatory markers. As a consequence, mTORC1 inhibition exacerbated disease activity, increased mucosal damage, enhanced ulceration, augmented cell infiltration, decreased survival and stimulated tumor formation in a model of colorectal cancer CRC associated to colitis. Thus, our findings suggest that mTORC1 signaling downstream of IFNγ prevents epithelial DNA damage and cancer development during colitis.
RESUMO
Platelet regeneration represents an important and separate element in the engraftment process for allogeneic stem cell transplantation. Fully automated flow cytometry using blood cell counters now allows reliable quantification of reticulated platelets, expressed as the immature platelet fraction (IPF). We studied the kinetics of IPF in six patients grafted with allogeneic peripheral blood stem cell transplantation (PBSCT), 12 patients with bone marrow transplantation (BMT) and seven patients with cord blood transplantation (CBT). Preconditioning therapy caused an immediate and rapid fall in tri-lineage hematopoiesis. IPF rose transiently above 3% after a mean duration of 11 days post-PBSCT, 18 days post-BMT and 19 days post-CBT. This was 1, 4 and 13 days earlier than platelet engraftment, respectively. A linear correlation model showed a close association between the rise of IPF and tri-lineage engraftment after transplantation. IPF counting may thus provide an accessible measure of thrombopoietic activity, leading to early evaluation of marrow function and allowing monitoring of platelet regeneration.
Assuntos
Plaquetas/fisiologia , Transplante de Medula Óssea/fisiologia , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Contagem de Plaquetas , Transfusão de Plaquetas , Transplante de Células-Tronco , Adolescente , Adulto , Idoso , Contagem de Eritrócitos , Feminino , Humanos , Cinética , Masculino , Pessoa de Meia-Idade , Fatores de Tempo , Transplante HomólogoRESUMO
The intestinal epithelium constitutes a first line of defense of the innate immune system. Epithelial dysfunction is a hallmark of intestinal disorders such as inflammatory bowel diseases (IBDs). The actin cytoskeleton controls epithelial barrier integrity but the function of actin regulators such as cortactin is poorly understood. Given that cortactin controls endothelial permeability, we hypothesized that cortactin is also important for epithelial barrier regulation. We found increased permeability in the colon of cortactin-KO mice that was accompanied by reduced levels of ZO-1, claudin-1, and E-cadherin. By contrast, claudin-2 was upregulated. Cortactin deficiency increased RhoA/ROCK1-dependent actomyosin contractility, and inhibition of ROCK1 rescued the barrier defect. Interestingly, cortactin deficiency caused increased epithelial proliferation without affecting apoptosis. KO mice did not develop spontaneous colitis, but were more susceptible to dextran sulfate sodium colitis and showed severe colon tissue damage and edema formation. KO mice with colitis displayed strong mucus deposition and goblet cell depletion. In healthy human colon tissues, cortactin co-localized with ZO-1 at epithelial cell contacts. In IBDs patients, we observed decreased cortactin levels and loss of co-localization with ZO-1. Thus, cortactin is a master regulator of intestinal epithelial barrier integrity in vivo and could serve as a suitable target for pharmacological intervention in IBDs.
Assuntos
Actomiosina/metabolismo , Colite/imunologia , Cortactina/metabolismo , Doenças Inflamatórias Intestinais/imunologia , Mucosa Intestinal/patologia , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Apoptose , Proliferação de Células , Colite/induzido quimicamente , Cortactina/genética , Citoesqueleto/metabolismo , Sulfato de Dextrana , Modelos Animais de Doenças , Humanos , Imunidade Inata , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína da Zônula de Oclusão-1/metabolismo , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Functional differentiation is found in the spinal cord. A unique set of neurological deficits follows a multi-focal injury. Clinically, sensory and motor disturbance present independently, often resulting in sensory and motor deficit dissociation. This study examined 103 spinal decompression illness (DCI) cases. The neurological deficit dissociation was classified as follows: 1) Cases with sensory impairment only, or motor dysfunction alone, were tagged as having "dissociation" (44 cases); when a case was with both sensory and motor dysfunction, the spinal level of the sensory impairment was determined and was matched with the spinal segments responsible for the motor dysfunction; 2) If the two spinal areas did not match (i.e. with no regional overlap), they were tagged as having "dissociation" for each motor dysfunction (32 cases). In total, dissociation was present in 76 out of 103 cases. We concluded that clinical neurological deficit dissociation is frequently observed in spinal DCI.
Assuntos
Doença da Descompressão/fisiopatologia , Hipestesia/fisiopatologia , Doença dos Neurônios Motores/fisiopatologia , Doenças da Medula Espinal/fisiopatologia , Adolescente , Adulto , Doença da Descompressão/complicações , Feminino , Humanos , Hipestesia/etiologia , Masculino , Pessoa de Meia-Idade , Doença dos Neurônios Motores/etiologia , Estudos Prospectivos , Doenças da Medula Espinal/etiologia , Estatísticas não ParamétricasRESUMO
Here we evaluated the ability of L-theanine in preventing experimental hepatic cirrhosis and investigated the roles of nuclear factor-κB (NF-κB) activation as well as transforming growth factor ß (TGF-ß) and connective tissue growth factor (CTGF) regulation. Experimental hepatic cirrhosis was established by the administration of carbon tetrachloride (CCl4) to rats (0.4 g/kg, intraperitoneally, three times per week, for 8 weeks), and at the same time, adding L-theanine (8.0 mg/kg) to the drinking water. Rats had ad libitum access to water and food throughout the treatment period. CCl4 treatment promoted NF-κB activation and increased the expression of both TGF-ß and CTGF. CCl4 increased the serum activities of alanine aminotransferase and γ-glutamyl transpeptidase and the degree of lipid peroxidation, and it also induced a decrease in the glutathione and glutathione disulfide ratio. L-Theanine prevented increased expression of NF-κB and down-regulated the pro-inflammatory (interleukin (IL)-1ß and IL-6) and profibrotic (TGF-ß and CTGF) cytokines. Furthermore, the levels of messenger RNA encoding these proteins decreased in agreement with the expression levels. L-Theanine promoted the expression of the anti-inflammatory cytokine IL-10 and the fibrolytic enzyme metalloproteinase-13. Liver hydroxyproline contents and histopathological analysis demonstrated the anti-fibrotic effect of l-theanine. In conclusion, L-theanine prevents CCl4-induced experimental hepatic cirrhosis in rats by blocking the main pro-inflammatory and pro-fibrogenic signals.
Assuntos
Intoxicação por Tetracloreto de Carbono/patologia , Intoxicação por Tetracloreto de Carbono/prevenção & controle , Doença Hepática Induzida por Substâncias e Drogas/patologia , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Fator de Crescimento do Tecido Conjuntivo/biossíntese , Glutamatos/uso terapêutico , Cirrose Hepática/patologia , Cirrose Hepática/prevenção & controle , NF-kappa B/biossíntese , Fator de Crescimento Transformador beta/biossíntese , Alanina Transaminase/sangue , Animais , Antioxidantes/metabolismo , Aspartato Aminotransferases/sangue , Fator de Crescimento do Tecido Conjuntivo/efeitos dos fármacos , Citocinas/biossíntese , Regulação para Baixo/genética , Peroxidação de Lipídeos/efeitos dos fármacos , Cirrose Hepática/induzido quimicamente , Masculino , Metaloproteinase 13 da Matriz/biossíntese , NF-kappa B/efeitos dos fármacos , Ratos , Ratos Wistar , Fator de Crescimento Transformador beta/efeitos dos fármacosRESUMO
We analyzed whether acute treatment with serotonergic agonists would improve motor function in rats with transected spinal cords (spinal rats) and in rats that received transplants of fetal spinal cord into the transection site (transplant rats). Neonates received midthoracic spinal transections within 48 hr of birth; transplant rats received fetal (embryonic day 14) spinal cord grafts at the time of transection. At 3 weeks, rats began 1-2 months of training in treadmill locomotion. Rats in the transplant group developed better weight-supported stepping than spinal rats. Systemic administration of two directly acting agonists for serotonergic 5-HT(2) receptor subtypes, quipazine and (+/-)-1-[2, 5]-dimethoxy-4-iodophenyl-2-aminopropane), further increased weight-supported stepping in transplant rats. The improvement was dose-dependent and greatest in rats with poor to moderate baseline weight support. In contrast, indirectly acting serotonergic agonists, which block reuptake of 5-HT (sertraline) or release 5-HT and block its reuptake (D-fenfluramine), failed to enhance motor function. Neither direct nor indirect agonists significantly improved locomotion in spinal rats as a group, despite equivalent upregulation of 5-HT(2) receptors in the lumbar ventral horn of lesioned rats with and without transplants. The distribution of immunoreactive serotonergic fibers within and caudal to the transplant did not appear to correspond to restoration of motor function. Our results confirm our previous demonstration that transplants improve motor performance in spinal rats. Additional stimulation with agonists at subtypes of 5-HT receptors produces a beneficial interaction with transplants that further improves motor competence.
Assuntos
Transplante de Tecido Fetal/fisiologia , Atividade Motora/fisiologia , Quipazina/farmacologia , Receptores de Serotonina/fisiologia , Agonistas do Receptor de Serotonina/farmacologia , Traumatismos da Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/terapia , Medula Espinal/fisiologia , Medula Espinal/transplante , Anfetaminas/farmacologia , Animais , Animais Recém-Nascidos , Fenfluramina/farmacologia , Atividade Motora/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Serotonina/farmacologia , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Sertralina/farmacologia , Suporte de Carga/fisiologiaRESUMO
Pressure-driven and temperature-driven transitions of two thermoresponsive polymers, poly(N-isopropylacrylamide) (pNIPAM) and poly(N-vinylisobutyramide) (pNVIBA)), in both a soluble linear polymer form and a cross-linked hydro-gel form, were examined by a dynamic light-scattering method and direct microscopic observation, respectively. Their behavior was compared with that of protein systems. Changes in some characteristic parameters in the time-intensity correlation functions of dynamic light-scattering measurement of aqueous solutions of pNIPAM at various pressures and temperatures showed no essential differences during temperature and pressure scanning and, as a whole, the motions of polymers in aqueous solutions were similar in two types of transitions until chain shrinkage occurred. The gels (cross-linked polymer gels) prepared from the thermoresponsive polymers also showed similar volume transitions responding to the pressure and temperature increase. In temperature transitions, however, gels showed drastic volume shrinkage with loss of transparency, while pressure-induced transition showed a slow recovery of transparency while keeping the size, after first transient drastic volume shrinkage with loss of transparency. At a temperature slightly higher than the transition under atmospheric temperature, so-called reentry of the volume change and recovery of the transparency were observed during the pressure-increasing process, which implies much smaller aggregation or non-aggregated collapsed polymer chains in the gel at higher pressures, indicating a certain mechanistic difference of the dehydration processes induced by temperature and pressure.
Assuntos
Hidrogel de Polietilenoglicol-Dimetacrilato/química , Pressão , Desnaturação Proteica , Proteínas/química , Temperatura , Resinas Acrílicas/metabolismo , Luz , Dobramento de Proteína , Espalhamento de RadiaçãoRESUMO
In the present investigation, we studied whether neurotrophin-3 (NT-3) contributes to the rescue of axotomized Clarke's nucleus (CN) neurons in adult rats. A significant (24%) loss of CN neurons occurred at L-1 ipsilateral to T-8 hemisection by 14 days, which reached 31% at 2 months and then stabilized. Axotomized CN neurons had also atrophied by 14 days, but mean cell size did not decrease further. Animals that received gelfoam soaked in nerve growth factor, brain derived neurotrophic factor, or ciliary neurotrophic factor at the lesion site also showed a 30% neuron loss at 2 months, and a 40% reduction in average cell area. Rats receiving NT-3 showed a 15% neuron loss, which was not improved by additional neurotrophins in combination with NT-3. None of the treatments prevented neuron atrophy. Bioassay of the gelfoam showed that NT-3 bioactivity remained at 5 days after surgery but not at 14 days. Additional rats with hemisections that received NT-3 continuously via mini-pump for 2 months showed a 15% neuron loss, the same as with NT-3 given via gelfoam. These results indicate that even limited exposure of axotomized CN neurons to NT-3 produces permanent rescue of 50% of the neurons. The virtually complete rescue that we had previously observed with transplants of fetal central nervous system (CNS) tissues may, therefore, be due at least in part to NT-3, but the exogenous administration of a single neurotrophic factor or a combination of neurotrophic factors is less effective than transplants in producing long-term survival of axotomized CNS neurons.
Assuntos
Fatores de Crescimento Neural/farmacologia , Neurônios/citologia , Ratos Sprague-Dawley/fisiologia , Medula Espinal/patologia , Animais , Atrofia , Axotomia , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Contagem de Células , Sobrevivência Celular/efeitos dos fármacos , Fator Neurotrófico Ciliar , Feminino , Proteínas do Tecido Nervoso/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Neurotrofina 3 , Ratos , Fatores de TempoRESUMO
In order to study the initial events during infection of target cells by the enteric pathogen Entamoeba histolytica, we developed a quantitative adhesion assay based on the use of a human colonic cell line (CaCo-2) and biotinylated amoebae tagged with fluorescein. To prevent the strong and rapid lytic activity of Entamoeba histolytica on colonic cells, which would otherwise impede the study of the primary adhesion steps, parasites were mildly fixed, biotinylated and labelled with streptavidin-FITC. After labelled parasites have bound to enterocytes, nonadhered amoebae are removed by washing and attached parasites quantified by means of an automated fluorescence plate reader. The bioassay is simple, nonhazardous and can be completed in 1.5 h. We were able to detect ranges from 200 to 20,000 fluorescent parasites per microwell in a 96-well plate, containing approximately 10(5) colonic cells. Fluorescence intensity (arbitrary units) increased in direct relationship to the number of parasites added per well, and was not limited by the size of the culture plate (96, 24 or six wells). As an example of the value of this assay, two proinflammatory cytokines (interleukin-1, (IL-1 beta) and interferon-gamma (IFN-gamma) known to influence the adhesion properties of endothelial and epithelial cells, were used to assess their effects upon enterocyte-entamoeba binding. The increase in amoebae binding revealed by cytokine treatment to enterocytes suggests that the parasite may take advantage of inflammatory stimuli in order to increase its binding to colonic epithelium. We believe this rapid, sensitive and simple method offers the potential for large scale screening assays to study the immunobiology of this protozoal infection by analysing the mechanisms involved in the primary interactions between Entamoeba histolytica and enterocytes.
Assuntos
Colo/parasitologia , Citocinas/farmacologia , Entamoeba histolytica/fisiologia , Animais , Adesão Celular/efeitos dos fármacos , Separação Celular , Células Cultivadas , Colo/citologia , Colo/metabolismo , Entamoeba histolytica/metabolismo , Entamoeba histolytica/patogenicidade , Citometria de Fluxo , Fluoresceína-5-Isotiocianato , Fluorescência , Interações Hospedeiro-Parasita , Humanos , Fenótipo , Sensibilidade e Especificidade , VirulênciaRESUMO
The protozoan parasite Entamoeba histolytica is the etiological agent of human amebiasis. The pathology of the disease starts with the cytolysis of the host target cells by amoebae. It is initiated by the adhesion of trophozoites to the host cells, through surface lectin via specific receptors. These adherence lectins have been demonstrated to be highly conserved, and can be recognised by serum antibodies from patients with invasive amebiasis. Some of these molecules have been used as antigens in serologic studies, which has been very helpful in the diagnosis of invasive intestinal amebiasis. However, false-positive serologic reactivity can occur using E. histolytica extracts and purified antigens. Additional problems are because the extracts display a great enzymatic activity. Several diagnostic methods, using different molecules and techniques, have been described. However, the problem still remains since these tests are not capable of differentiating between amoebic liver abscess (ALA) and intestinal amebiasis.Here, the research has been addressed to the 66-kDa antigen, which is a part of the outer membrane proteins from the E. histolytica strain HM1-IMSS trophozoites. First of all, we characterized the 66-kDa antigen in order to prove the relevance. We found that the 66-kDa antigen is a part of the plasma membranes and is distributed rather homogeneously on the cell surface of trophozoites. Apparently, the 66-kDa antigen is a glycoprotein. Using a monoclonal antibody (MAb), we found 25% of inhibition in the erythrophagocytosis by the trophozoites. Starting form one monoclonal antibody, we prepared an anti-idiotype (anti-Id) antibody reagent, with the purpose of searching for the different expressions of the idiotype between the sera from ALA and the intestinal amebiasis patients. Moreover, we produced the antibody Ab3 that is capable of recognising the 66-kDa antigen; it means that the Ab2 displays the internal image of the antigen. We found that 91.6% of the serum from ALA patients displayed the expression of the Id. In contrast, 15.7% of the E. histolytica asymtomatic cyst carriers displayed the Id expression, 6.6% of the patients with another parasite infection, and 11% of the negative controls (serum from umbilical cords of newborn babies). Our results showed that the expression of the Id could be differentiated among the AHA patients from the other groups with a 91.6% sensibility and 88.3% specificity.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Antígenos de Protozoários/imunologia , Entamoeba histolytica/imunologia , Entamoeba histolytica/isolamento & purificação , Entamebíase/diagnóstico , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Antígenos de Protozoários/análise , Entamebíase/imunologia , Humanos , Imunoensaio , Camundongos , Camundongos Endogâmicos BALB C , Sensibilidade e EspecificidadeRESUMO
Interleukin-1 (IL-1), one of the most important inflammatory cytokines, promotes glia to express intercellular adhesion molecule-1 (ICAM-1) in vitro. IL-1 is known to be produced in situ immediately after brain insults and recently we have found that glia, including astrocytes, express ICAM-1 in vivo following cortical stab wounds. To evaluate the participation of IL-1 beta in posttraumatic ICAM-1 expression on glia in vivo, we performed the following experiments. A cortical stab wound was made in the brain of a mouse. ICAM-1-immunopositive glia began to emerge around the wound from 6 h postlesioning. The number of cells reached a maximum at 48 h and persisted until 7 days postlesioning. Next, a neutralizing monoclonal antibody against IL-1 beta was infused into the wound immediately following the injury. This treatment resulted in a significant reduction of ICAM-1-positive glia at 24 and 48 h postlesioning. We conclude, therefore, that IL-1 beta affects ICAM-1 expression on glia in vivo after experimental brain injury and presumably plays an important role in brain wound repair.
Assuntos
Lesões Encefálicas/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-1/fisiologia , Neuroglia/metabolismo , Animais , Anticorpos/imunologia , Lesões Encefálicas/patologia , Contagem de Células , Feminino , Interleucina-1/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Fatores de TempoRESUMO
To determine whether embryonic spinal cord transplants retained the ability to prevent retrograde death of Clarke's nucleus (CN) neurons if supplied after a delay, we hemisected adult rats at the T8 spinal cord segment and placed transplants of fetal tissue into the hemisection cavity immediately or up to 14 days later. Transplants provided in the first 7 days after injury prevented virtually all of the 30% loss of CN neurons at L1 ipsilateral to hemisection that occurs without a transplant. Transplants supplied at 14 days post-hemisection were ineffective. Because prevention of retrograde neuron death is one mechanism by which transplants may contribute to locomotor recovery after spinal cord injury, this window of effectiveness should be considered in the design of clinical trials.