Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 11 de 11
Filter
Add more filters










Publication year range
1.
Ann Med Surg (Lond) ; 77: 103645, 2022 May.
Article in English | MEDLINE | ID: mdl-35637985

ABSTRACT

Background: Curative-intent therapies for hepatocellular carcinoma (HCC) include radiofrequency ablation (RFA), liver resection (LR), and liver transplantation (LT). Controversy exists in treatment selection for early-stage tumours. We sought to evaluate the oncologic outcomes of patients who received either RFA, LR, or LT as first-line treatment for solitary HCC ≤ 3 cm in an intention-to-treat analysis. Materials and methods: All patients with solitary HCC ≤ 3 cm who underwent RFA, LR, or were listed for LT between Feb-2000 and Nov-2018 were analyzed. Cox regression analysis was then performed to compare intention-to-treat (ITT) survival by initial treatment allocation and disease-free survival (DFS) by treatment received in patients eligible for all three treatments. Results: A total of 119 patients were identified (RFA n = 83; LR n = 25; LT n = 11). The overall intention-to-treat survival was similar between the three groups. The overall DFS was highest for the LT group. This was significantly higher than RFA (p = 0.02), but not statistically significantly different from LR (p = 0.14). After multivariable adjustment, ITT survival was similar in the LR and LT groups relative to RFA (LR HR:1.13, 95%CI 0.33-3.82; p = 0.80; LT HR:1.39, 95%CI 0.35-5.44; p = 0.60). On multivariable DFS analysis, only LT was better relative to RFA (LR HR:0.52, 95%CI 0.26-1.02; p = 0.06; LT HR:0.15, 95%CI 0.03-0.67; p = 0.01). Compared to LR, LT was associated with a numerically lower hazard on multivariable DFS analysis, though this did not reach statistical significance (HR 0.30, 95%CI 0.06-1.43; p = 0.13). Conclusion: For treatment-naïve patients with solitary HCC ≤ 3 cm who are eligible for RFA, LR, and LT, adjusted ITT survival is equivalent amongst the treatment modalities, however, DFS is better with LR and LT, compared with RFA. Differences in recurrence between treatment modalities and equipoise in ITT survival provides support for a future prospective trial in this setting.

2.
Clin Vaccine Immunol ; 22(8): 965-73, 2015 Aug.
Article in English | MEDLINE | ID: mdl-26108285

ABSTRACT

Leptospirosis is a global zoonotic disease caused by different Leptospira species, such as Leptospira interrogans, that colonize the renal tubules of wild and domestic animals. Thus far, attempts to develop effective leptospirosis vaccines, both for humans and animals, have failed to induce immune responses capable of conferring protection and simultaneously preventing renal colonization. In this study, we evaluated the protective immunity induced by subunit vaccines containing seven different recombinant Leptospira interrogans outer membrane proteins, including the carboxy-terminal portion of the immunoglobulinlike protein A (LigA(C)) and six novel antigens, combined with aluminum hydroxide (alum) or Salmonella flagellin (FliC) as adjuvants. Hamsters vaccinated with the different formulations elicited high antigen-specific antibody titers. Immunization with LigA(C), either with alum or flagellin, conferred protective immunity but did not prevent renal colonization. Similarly, animals immunized with LigA(C) or LigA(C) coadministered with six leptospiral proteins with alum adjuvant conferred protection but did not reduce renal colonization. In contrast, immunizing animals with the pool of seven antigens in combination with flagellin conferred protection and significantly reduced renal colonization by the pathogen. The present study emphasizes the relevance of antigen composition and added adjuvant in the efficacy of antileptospirosis subunit vaccines and shows the complex relationship between immune responses and renal colonization by the pathogen.


Subject(s)
Adjuvants, Immunologic/administration & dosage , Bacterial Outer Membrane Proteins/immunology , Bacterial Vaccines/immunology , Flagellin/administration & dosage , Leptospira interrogans/immunology , Leptospirosis/prevention & control , Aluminum Hydroxide/administration & dosage , Animals , Antibodies, Bacterial/blood , Bacterial Outer Membrane Proteins/genetics , Bacterial Vaccines/administration & dosage , Bacterial Vaccines/genetics , Immunoglobulin G/blood , Kidney/microbiology , Leptospira interrogans/genetics , Leptospirosis/immunology , Male , Mesocricetus , Survival Analysis , Vaccines, Subunit/administration & dosage , Vaccines, Subunit/genetics , Vaccines, Subunit/immunology , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology
3.
Microb Pathog ; 61-62: 29-36, 2013.
Article in English | MEDLINE | ID: mdl-23665291

ABSTRACT

Leptospirosis is a re-emergent zoonosis, caused by pathogenic spirochetes from the genus Lepstospira. To date, there is no protein described to be involved in leptospiral hemorrhagic manifestations, although several proteases have been reported for other bacterial infections. In this study we identified 12 putative metalloproteases from the genome of Leptospira interrogans, and characterized for the first time a putative metalloprotease, here named Leptallo I, as a potential Zn(2+) dependent glycylglycine protease belonging to the M23 metalloendopeptidase family. The native protein was detected in extracts from several pathogenic Leptospira species and further shown to be secreted to the culture medium. We expressed the recombinant protein and its C-terminal fragment containing the metalloprotease domain, and both presented regular secondary structures. The sera of humans with leptospirosis were able to recognize rLeptallo I, indicating that the native protein is expressed and presented to the immune system during infection. The recombinant proteins displayed a significant, though relatively low, elastinolytic activity, and the challenge of hamsters immunized with rLeptallo I conferred 33% protection, suggesting a significant importance of this protein in the pathogenesis. The elastinolytic activity may be important for leptospires-host interaction, because elastin constitutes a significant proportion of total lung and blood vessel proteins.


Subject(s)
Bacterial Proteins/immunology , Bacterial Proteins/metabolism , Elastin/metabolism , Leptospira interrogans/pathogenicity , Leptospirosis/prevention & control , Metalloproteases/metabolism , Pancreatic Elastase/metabolism , Amino Acid Sequence , Animals , Antibodies, Bacterial/blood , Antibodies, Bacterial/immunology , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Cricetinae , Elastin/genetics , Humans , Leptospira interrogans/genetics , Leptospira interrogans/metabolism , Leptospirosis/immunology , Leptospirosis/microbiology , Male , Mesocricetus , Metalloproteases/chemistry , Metalloproteases/genetics , Metalloproteases/immunology , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Pancreatic Elastase/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , Sequence Analysis, DNA
4.
J Atheroscler Thromb ; 17(7): 730-9, 2010 Jul 30.
Article in English | MEDLINE | ID: mdl-20523012

ABSTRACT

AIM: Platelets plays a central role in hemostatic processes and consequently are similarly involved in pathological processes, such as arterial thrombosis and atherosclerosis. Herein we described the synthesis, antiplatelet profile and structure-activity relationship (SAR) of a new series of N'-substitutedphenylmethylene-1H-pyrazolo[3,4-b]pyridine-carbohydrazide derivatives (3a-3k). METHODS: These compounds were synthesized in good yield and tested in platelet aggregation assays using collagen, ADP and arachidonic acid as agonists. We also performed a SAR studies using SPARTAN' 08 program, in silico ADMET screening and the Lipinski " rule of five " using Osiris Property Explorer and molinspiration on-line programs. RESULTS: Interestingly, the new compounds were active against collagen and arachidonic acid (AA) with the two most actives compounds (3a and 3c - IC(50)=61 microM and 68 microM respectively) almost 5-fold more potent than aspirin (IC(50)=300 microM). These derivatives showed low theoretical toxicity risks in in silico ADMET screening and fulfilled the Lipinski rule of five, suggesting good oral biodisponibility. CONCLUSION: This work showed carbohydrazide group as potential for designing new antiplatelets. On that purpose, 3a and 3c may act as prototypes to generate more efficient and safe molecules for treating thrombotic diseases.


Subject(s)
Blood Platelets/drug effects , Platelet Aggregation Inhibitors/pharmacology , Platelet Aggregation/drug effects , Pyrazoles/pharmacology , Pyridines/pharmacology , Thrombosis/drug therapy , Dose-Response Relationship, Drug , Humans , Molecular Structure , Platelet Aggregation Inhibitors/chemical synthesis , Platelet Aggregation Inhibitors/chemistry , Pyrazoles/chemical synthesis , Pyrazoles/chemistry , Pyridines/chemical synthesis , Pyridines/chemistry , Structure-Activity Relationship , Thrombosis/pathology
5.
FEBS Letters ; 583(8): 1381-1385, Apr 17,2009.
Article in English | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP, SESSP-IBACERVO | ID: biblio-1062804

ABSTRACT

The role of TlyA, TlyB and TlyC proteins in the biology of Leptospira is still uncertain. Although these proteins have been considered as putative hemolysins, we demonstrate that leptospiral recombinant TlyB and TlyC do not possess hemolytic activity. However, further experiments showed that TlyC is a surface-exposed protein that seems to bind to laminin, collagen IV and fibronectin. The expression of both proteins was detected both in vitro and in vivo. Our findings suggest that TlyB and TlyC are not directly involved in hemolysis, and that TlyC may contribute to Leptospira binding to extracellular matrix (ECM) during host infection.


Subject(s)
Leptospira interrogans , Leptospirosis/microbiology , Hemolysis , Bacterial Proteins
6.
J. venom. anim. toxins incl. trop. dis ; 15(2): 289-304, 2009. ilus, graf
Article in English | LILACS, Sec. Est. Saúde SP | ID: lil-517287

ABSTRACT

Schistosomes use proteinases to accomplish some tasks such as tissue penetration, tissue digestion for nutrition and evasion of host immune responses. The Cathepsin L is a cysteine proteinase of the papain superfamily detected in the gut lumen indicating that this enzyme contributes to the proteolysis of ingested hemoglobin. Due to these roles they play in the schistosome biology, proteolytic enzymes are considered potential targets to develop and direct anti-schistosomal therapies. In this work, the cDNA coding Cathepsin L1 of Schistosoma mansoni was cloned into the pAE vector that provides high-level expression of heterologous proteins in Escherichia coli. The recombinant protein was expressed as inclusion bodies, purified under denaturing conditions through nickel charged chromatography and used for experimental animal vaccination. ELISA was performed with the pooled sera. Although this protein showed to be immunogenic, mice immunized with three doses of recombinant protein plus aluminum hydroxide as adjuvant did not protect against S. mansoni infection.


Subject(s)
Animals , Female , Mice , Schistosomiasis mansoni/prevention & control , Escherichia coli Proteins/therapeutic use , Vaccines
7.
Braz. j. med. biol. res ; 37(8): 1103-1109, Aug. 2004. ilus, tab
Article in English | LILACS, Sec. Est. Saúde SP | ID: lil-362559

ABSTRACT

We report here the construction of a vector derived from pET3-His and pRSET plasmids for the expression and purification of recombinant proteins in Escherichia coli based on T7 phage RNA polymerase. The resulting pAE plasmid combined the advantages of both vectors: small size (pRSET), expression of a short 6XHis tag at N-terminus (pET3-His) and a high copy number of plasmid (pRSET). The small size of the vector (2.8 kb) and the high copy number/cell (200-250 copies) facilitate the subcloning and sequencing procedures when compared to the pET system (pET3-His, 4.6 kb and 40-50 copies) and also result in high level expression of recombinant proteins (20 mg purified protein/liter of culture). In addition, the vector pAE enables the expression of a fusion protein with a minimal amino-terminal hexa-histidine affinity tag (a tag of 9 amino acids using XhoI restriction enzyme for the 5'cloning site) as in the case of pET3-His plasmid and in contrast to proteins expressed by pRSET plasmids (a tag of 36 amino acids using BamHI restriction enzyme for the 5'cloning site). Thus, although proteins expressed by pRSET plasmids also have a hexa-histidine tag, the fusion peptide is much longer and may represent a problem for some recombinant proteins.


Subject(s)
Humans , Recombinant Fusion Proteins , Escherichia coli , Genetic Vectors , RNA, Bacterial , Polymerase Chain Reaction , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel
8.
Braz J Med Biol Res ; 37(8): 1103-9, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15273812

ABSTRACT

We report here the construction of a vector derived from pET3-His and pRSET plasmids for the expression and purification of recombinant proteins in Escherichia coli based on T7 phage RNA polymerase. The resulting pAE plasmid combined the advantages of both vectors: small size (pRSET), expression of a short 6XHis tag at N-terminus (pET3-His) and a high copy number of plasmid (pRSET). The small size of the vector (2.8 kb) and the high copy number/cell (200-250 copies) facilitate the subcloning and sequencing procedures when compared to the pET system (pET3-His, 4.6 kb and 40-50 copies) and also result in high level expression of recombinant proteins (20 mg purified protein/liter of culture). In addition, the vector pAE enables the expression of a fusion protein with a minimal amino-terminal hexa-histidine affinity tag (a tag of 9 amino acids using XhoI restriction enzyme for the 5'cloning site) as in the case of pET3-His plasmid and in contrast to proteins expressed by pRSET plasmids (a tag of 36 amino acids using BamHI restriction enzyme for the 5'cloning site). Thus, although proteins expressed by pRSET plasmids also have a hexa-histidine tag, the fusion peptide is much longer and may represent a problem for some recombinant proteins.


Subject(s)
Escherichia coli/metabolism , Genetic Vectors/genetics , Recombinant Fusion Proteins/biosynthesis , Base Sequence , Cloning, Molecular , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Genetic Vectors/metabolism , Humans , Molecular Sequence Data , Plasmids/genetics , Plasmids/metabolism , Polymerase Chain Reaction , Protein Biosynthesis/genetics , Recombinant Fusion Proteins/genetics , Viral Proteins
9.
Biochem Biophys Res Commun ; 262(1): 98-102, 1999 Aug 19.
Article in English | MEDLINE | ID: mdl-10448075

ABSTRACT

PC12 cell line is a cellular model to study neurite outgrowth and neurotransmitter release mechanisms. Molecular motors may be involved in these responses and myosin V could be a candidate to mediate these effects. Overlay experiments using [(125)I]-calmodulin showed that PC12 cells possess several calmodulin-binding proteins, some of them around 190-210 kDa. Western blots using affinity purified polyclonal antibodies raised against chicken brain myosin V revealed a component of 190 kDa, a molecular mass typical of myosin V. Furthermore, Northern blots using a myosin V probe also detected a transcript of around 12 kbp. Immunofluorescence cytochemistry demonstrated the localization of myosin V throughout the cytoplasm, in the neurites, growth cone tips, and with an intense asymmetrical perinuclear labeling. Western blot analyses of PC12 cellular extracts after FGF-2 and/or dibutyryl cAMP treatment revealed variations between myosin V and myosin II expression during neuronal differentiation. These results demonstrated the presence of myosin V in PC12 cells and also suggest a role for this motor molecule in the neuronal differentiation response in PC12 cells.


Subject(s)
Calmodulin-Binding Proteins/metabolism , Myosin Type V , Nerve Tissue Proteins/metabolism , Neurons/cytology , Animals , Blotting, Western , Bucladesine/pharmacology , Calmodulin/metabolism , Calmodulin-Binding Proteins/genetics , Cell Differentiation/drug effects , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Cytoplasm/drug effects , Cytoplasm/metabolism , Fibroblast Growth Factor 2/pharmacology , Fluorescent Antibody Technique , Gene Expression Regulation/drug effects , Growth Cones/drug effects , Growth Cones/metabolism , Molecular Weight , Myosins/metabolism , Nerve Tissue Proteins/genetics , Neurites/drug effects , Neurites/metabolism , Neurons/drug effects , Neurons/metabolism , PC12 Cells , RNA, Messenger/analysis , RNA, Messenger/genetics , Rats
10.
J Cell Biochem ; 57(2): 311-20, 1995 Feb.
Article in English | MEDLINE | ID: mdl-7759568

ABSTRACT

Endooligopeptidase A is a putative neuropeptide-metabolizing enzyme. It converts small enkephalin-containing peptides into the corresponding enkephalins and inactivates biopeptides such as bradykinin and neurotensin in vitro. We investigated the presence of endooligopeptidase A in PC12 cells. This cell line was derived from a rat pheochromocytoma tumor and resembles fetal chromaffin cell. Depending on the supplements added to the cell culture, this cell line can be differentiated into mature chromaffin cell or sympathetic neuron-like cell. Endooligopeptidase A activity was measured in soluble cellular extracts using a specific fluorogenic substrate QF-ERP7. The PC12 endooligopeptidase A-like activity shared similar but not identical biochemical properties with rabbit brain endooligopeptidase A. Similarly to rabbit brain endooligopeptidase A, the PC12 endooligopeptidase A-like activity was enhanced by DTT, totally inhibited by DTNB and 1-10 Phenanthroline, partially inhibited by cFP-AAF-pAb, and not affected by PMSF. Furthermore, the PC12 endooligopeptidase A-like activity displayed identical elution profile as rabbit brain endooligopeptidase A in gel filtration and anion-exchange chromatography. In addition, an antiserum raised against rabbit brain endooligopeptidase A cross-reacted with a 71 kDa component from PC12 cell extracts in Western blotting and was also able to partially neutralize the PC12 endooligopeptidase A-like activity. Treatment of PC12 cells with basic fibroblast growth factor (bFGF), a neurotrophic factor for this cell line, did not modify the specific activity of this enzyme. However, cAMP analogs decreased the specific activity of the enzyme. These results indicate the presence of an endooligopeptidase A-like activity in PC12 cells which is modulated by cAMP but not by bFGF.


Subject(s)
8-Bromo Cyclic Adenosine Monophosphate/pharmacology , Bucladesine/pharmacology , Cyclic AMP/physiology , Fibroblast Growth Factor 2/pharmacology , Metalloendopeptidases/metabolism , Adrenal Gland Neoplasms , Animals , Blotting, Western , Brain/enzymology , Cell Differentiation , Chromatography, Gel , Dithionitrobenzoic Acid/pharmacology , Dithiothreitol/pharmacology , Ethylenediamines , Immune Sera , Kinetics , Metalloendopeptidases/isolation & purification , Oligopeptides , PC12 Cells , Phenanthrolines/pharmacology , Pheochromocytoma , Rabbits , Rats
11.
Journal of Cellular Biochemistry ; 57(2): 311-320, Feb.1995.
Article in English | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP, SESSP-IBACERVO | ID: biblio-1064216

ABSTRACT

Endooligopeptidase A is a putative neuropeptide-metabolizing enzyme. It converts small enkephalin-containing peptides into the corresponding enkephalins and inactivates biopeptides such as bradykinin and neurotensin in vitro. We investigated the presence of endooligopeptidase A in PC12 cells. This cell line was derived from a rat pheochromocytoma tumor and resembles fetal chromaffin cell. Depending on the supplements added to the cell culture, this cell line can be differentiated into mature chromaffin cell or sympathetic neuron-like cell. Endooligopeptidase A activity was measured in soluble cellular extracts using a specific fluorogenic substrate QF-ERP7. The PC12 endooligopeptidase A-like activity shared similar but not identical biochemical properties with rabbit brain endooligopeptidase A. Similarly to rabbit brain endooligopeptidase A, the PC12 endooligopeptidase A-like activity was enhanced by DTT, totally inhibited by DTNB and 1-10 Phenanthroline, partially inhibited by cFP-AAF-pAb, and not affected by PMSF. Furthermore, the PC12 endooligopeptidase A-like activity displayed identical elution profile as rabbit brain endooligopeptidase A in gel filtration and anion-exchange chromatography. In addition, an antiserum raised against rabbit brain endooligopeptidase A cross-reacted with a 71 kDa component from PC12 cell extracts in Western blotting and was also able to partially neutralize the PC12 endooligopeptidase A-like activity. Treatment of PC12 cells with basic fibroblast growth factor (bFGF), a neurotrophic factor for this cell line, did not modify the specific activity of this enzyme. However, cAMP analogs decreased the specific activity of the enzyme. These results indicate the presence of an endooligopeptidase A-like activity in PC12 cells which is modulated by cAMP but not by bFGF.


Subject(s)
Animals , Rabbits , Rats , /pharmacology , Bucladesine/pharmacology , Cerebrum/enzymology , Dithiothreitol/pharmacology , /pharmacology , Phenanthrolines/pharmacology , Adrenal Gland Neoplasms , Dithionitrobenzoic Acid/pharmacology , Cyclic AMP/physiology , Chromatography, Gel , Cell Differentiation , Fibroblast Growth Factors , Metalloendopeptidases/isolation & purification , Metalloendopeptidases/metabolism , Immune Sera , Blotting, Western
SELECTION OF CITATIONS
SEARCH DETAIL
...