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1.
Anal Biochem ; 393(1): 73-9, 2009 Oct 01.
Article in English | MEDLINE | ID: mdl-19523916

ABSTRACT

For the bioanalysis of therapeutic monoclonal antibodies in biological matrices, immunoassays--especially enzyme-linked immunosorbent assays (ELISAs)--are the most widely used techniques. Although ELISAs are very sensitive, the obtained sensitivity is not always sufficient. In this study, we have investigated the possibilities of performing a precipitate-enhanced immunoassay (PEIA) with ellipsometric detection for the bioanalysis of the therapeutic monoclonal antibody trastuzumab. Hydrophobic silicon slides were coated with anti-idiotype trastuzumab antibodies. Trastuzumab in serum samples could bind to this primary catcher, and biotinylated anti-idiotype antibodies were used for detection. After binding of streptavidin-poly-horseradish peroxidase (HRP), the precipitating substrate 3,3'-diaminobenzidine tetrahydrochloride (DAB) was added. Precipitation speed was analyzed using a novel prototype eight-cell ellipsometer, and calibration curves were obtained by plotting this speed versus the trastuzumab concentration. Results demonstrate that the PEIA is at least four times more sensitive than the same ELISA using the chromogenic substrate 3,5,3',5'-tetramethylbenzidine (TMB) instead of precipitating DAB. The calibration range of the assay is 11 to 700 pg/ml. Serum samples are diluted 10 times prior to incubation corresponding to 110 to 7000 pg/ml in undiluted serum. Validation results demonstrate that these low concentrations can be analyzed accurately and precisely. In addition, samples of a patient treated with trastuzumab were analyzed with both the PEIA and the ELISA. Results demonstrate excellent correlation (r=0.984) between the methods. Thus, when more sensitivity is required than in a conventional immunoassay, a PEIA with ellipsometric detection may be a useful alternative. The prototype ellipsometer is still in development, and from the data obtained in this study, improvements will be implemented.


Subject(s)
Antibodies, Monoclonal/blood , Immunoassay/methods , Spectrum Analysis/methods , Antibodies, Monoclonal, Humanized , Humans , Trastuzumab
2.
Haematologica ; 93(9): 1351-7, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18641016

ABSTRACT

BACKGROUND: Thrombin generation has been shown to reflect coagulation potential and factor VIII (FVIII) levels in patients with hemophilia A. We hypothesize that thrombin generation in the presence of thrombomodulin reflects plasma FVIII levels better. DESIGN AND METHODS: Plasma FVIII levels were determined chromogenically and thrombin generation was measured with and without thrombomodulin in 12 patients with severe hemophilia A. Blood was sampled at baseline and 15 min, 1, 3, 6, 24 and 48 hours after recombinant FVIII administration. RESULTS: FVIII administration restored the decreased baseline thrombin generation (reflected by endogenous thrombin potential, peak height, slope and time to peak). Lag time did not change. All thrombin generation parameters except time to peak returned to baseline within 48 hours, while plasma FVIII concentration was increased and time to peak shortened. Endogenous thrombin potential and peak height showed wide inter-individual variation, with strong intra-individual correlations. Addition of thrombomodulin to the assay shortened time to peak and decreased endogenous thrombin potential and peak height. The decrease in peak height was almost completely offset by FVIII administration. Multiple linear regression analysis revealed thrombomodulin-modified thrombin generation to be a moderately better predictor of plasma FVIII levels than thrombin generation in the absence of thrombomodulin (adjusted R(2) 0.79 vs. 0.71). CONCLUSIONS: Addition of thrombomodulin has pronounced effects on all parameters of thrombin generation. This thrombomodulin-modified thrombin generation assay better reflects plasma FVIII levels than thrombin generation in the absence of thrombomodulin.


Subject(s)
Factor VIII/therapeutic use , Hemophilia A/drug therapy , Hemophilia A/metabolism , Thrombin/metabolism , Thrombomodulin/metabolism , Adolescent , Adult , Aged , Humans , Male , Middle Aged , Models, Biological , Recombinant Proteins/therapeutic use
3.
Hum Gene Ther ; 17(5): 487-99, 2006 May.
Article in English | MEDLINE | ID: mdl-16716106

ABSTRACT

Human lipoprotein lipase (hLPL) deficiency, for which there currently exists no adequate treatment, leads to excessive plasma triglycerides (TGs), recurrent abdominal pain, and life-threatening pancreatitis. We have shown that a single intramuscular administration of adeno-associated virus (AAV) serotype 1 vector, encoding the human LPL(S447X) variant, results in complete, long-term normalization of dyslipidemia in LPL(/) mice. As a prelude to gene therapy for human LPL deficiency, we tested the efficacy of AAV1-LPL(S447X) in LPL(/) cats, which demonstrate hypertriglyceridemia (plasma TGs, >10,000 mg/dl) and clinical symptoms similar to LPL deficiency in humans, including pancreatitis. Male LPL(/) cats were injected intramuscularly with saline or AAV1-LPL(S447X) (1 x 10(11)-1.7 x 10(12) genome copies [GC]/kg), combined with oral doses of cyclophosphamide (0-200 mg/m(2) per week) to inhibit an immune response against hLPL. Within 3-7 days after administration of >or=5 x 10(11) GC of AAV1-LPL(S447X) per kilogram, the visible plasma lipemia was completely resolved and plasma TG levels were reduced by >99% to normal levels (10-20 mg/dl); intermediate efficacy (95% reduction) was achieved with 1 x 10(11) GC/kg. Injection in two sites, greatly limiting the amount of transduced muscle, was sufficient to completely correct the dyslipidemia. By varying the dose per site, linear LPL expression was demonstrated over a wide range of local doses (4 x 10(10)-1 x 10(12) GC/site). However, efficacy was transient, because of an anti-hLPL immune response blunting LPL expression. The level and duration of efficacy were significantly improved with cyclophosphamide immunosuppression. We conclude that AAV1-mediated delivery of LPL(S447X) in muscle is an effective means to correct the hypertriglyceridemia associated with feline LPL deficiency.


Subject(s)
Dependovirus/genetics , Genetic Therapy/methods , Hypertriglyceridemia/therapy , Lipoprotein Lipase/deficiency , Animals , Antibody Formation , Cats , Cyclophosphamide/therapeutic use , Feasibility Studies , Gene Transfer Techniques , Hypertriglyceridemia/genetics , Immunosuppressive Agents/therapeutic use , Lipids/blood , Lipoprotein Lipase/blood , Lipoprotein Lipase/genetics , Lipoprotein Lipase/immunology , Male , Muscle, Skeletal/metabolism , Point Mutation , Transgenes/immunology , Triglycerides/blood
4.
Hum Gene Ther ; 16(11): 1276-86, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16259561

ABSTRACT

Lipoprotein lipase (LPL) deficiency causes hypertriglyceridemia and recurrent, potentially life-threatening pancreatitis. There currently is no adequate treatment for this disease. Previously, we showed that intramuscular administration of an adeno-associated virus serotype 1 (AAV1) vector encoding the human LPL(S447X) variant cDNA (AAV1-LPL(S447X)) normalized the dyslipidemia of LPL-/- mice for more than 1 year. In preparation for a clinical trial, we evaluated the safety and biodistribution of AAV1-LPL(S447X) in wild-type mice and fully characterized six LPL-deficient patients. Toxicological analysis in mice showed that intramuscular administration was well tolerated. Acute inflammatory response markers were transiently increased, and anti- AAV1 antibodies were generated. Histological analyses indicated a dose-dependent reversible spleen hyperplasia, and myositis at the injection sites. Biodistribution data showed short-term vector leakage from injection sites into the circulation, followed by liver-mediated clearance. Persistence of vector DNA was limited to the injected muscle and draining lymph nodes, and spread to reproductive organs was limited. Characterization of LPL-deficient patients showed that all patients presented with hypertriglyceridemia and recurrent pancreatitis. LPL catalytic activity was absent, but LPL protein levels were 20-100% of normal. Myoblasts derived from skeletal muscle biopsies of these patients were efficiently transduced by AAV1-LPL(S447X) and secreted active LPL. These data support the initiation of a clinical trial in LPL-deficient patients, for which regulatory approval has been granted.


Subject(s)
Genetic Therapy , Hyperlipoproteinemia Type I/therapy , Lipoprotein Lipase/genetics , Animals , Dependovirus/genetics , Feasibility Studies , Female , Genetic Therapy/adverse effects , Genetic Vectors , Injections, Intramuscular , Lipoprotein Lipase/administration & dosage , Lipoprotein Lipase/pharmacokinetics , Male , Mice , Mice, Inbred C57BL , Tissue Distribution
5.
Circulation ; 109(1): 23-5, 2004 Jan 06.
Article in English | MEDLINE | ID: mdl-14691043

ABSTRACT

BACKGROUND: Patients with severe renal dysfunction often have unexplained elevated serum concentrations of cardiac troponin T (cTnT). We investigated whether in vivo fragmentation of cTnT could explain these increases. METHODS AND RESULTS: cTnT, creatine kinase isoenzyme MB, and myoglobin serum concentrations were measured in all 63 dialysis patients of our in-hospital dialysis department. A highly sensitive immunoprecipitation assay, followed by electrophoresis and Western blotting, was used to extract and concentrate cTnT and its possible fragments from serum of these 63 hemodialysis patients. Although creatine kinase isoenzyme MB values excluded recent ischemic myocardial events in 55 of the 63 cases, cTnT fragments ranging in size from 8 to 25 kDa were present in the serum samples of all dialysis patients. CONCLUSIONS: cTnT is fragmented into molecules small enough to be cleared by the kidneys of healthy subjects. Impaired renal function causes accumulation of these cTnT fragments and is very likely the cause of the unexplained elevations of serum cTnT found in patients with severe renal failure.


Subject(s)
Kidney Failure, Chronic/blood , Kidney/metabolism , Troponin T/blood , Aged , Biomarkers/blood , Blotting, Western , Cardiomyopathies/blood , Creatine Kinase/blood , Creatine Kinase, MB Form , Electrophoresis, Polyacrylamide Gel , Female , Humans , Immunoprecipitation , Isoenzymes/blood , Kidney Failure, Chronic/metabolism , Male , Middle Aged , Myoglobin/blood , Peptide Fragments/blood , Peptide Fragments/metabolism , Renal Dialysis , Troponin T/metabolism
6.
FASEB J ; 16(1): 54-60, 2002 Jan.
Article in English | MEDLINE | ID: mdl-11772936

ABSTRACT

Animal models for human neurological and psychiatric diseases only partially mimic the underlying pathogenic processes. Therefore, we investigated the potential use of cultured postmortem brain tissue from adult neurological patients and controls. The present study shows that human brain tissue slices obtained by autopsy within 8 h after death can be maintained in vitro for extended periods (up to 78 days) and can be manipulated experimentally. We report for the first time that 1) neurons and glia in such cultures could be induced to express the reporter gene LacZ after transduction with adeno-associated viral vectors and 2) cytochrome oxidase activity could be enhanced by the addition of pyruvate to the medium. These slice cultures offer new opportunities to study the cellular and molecular mechanisms of neurological and psychiatric diseases and new therapeutic strategies.


Subject(s)
Brain/cytology , Culture Techniques/methods , Neurodegenerative Diseases/pathology , Aged , Cell Count , Cell Survival , Cells, Cultured , Dependovirus/genetics , Electron Transport Complex IV/metabolism , Genetic Vectors , Humans , Kinetics , Middle Aged , Motor Cortex/cytology , Neuroglia/metabolism , Neurons/drug effects , Neurons/metabolism , Postmortem Changes , Pyruvic Acid/pharmacology , Transduction, Genetic , beta-Galactosidase/genetics
7.
Clin Chim Acta ; 352(1-2): 15-35, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15653098

ABSTRACT

BACKGROUND: One of the novel and promising plasma markers for detection of tissue injury is the family of 15 kDa cytoplasmic fatty acid-binding proteins of which various tissue-specific types occur. AIMS AND OBJECTIVES: The present status of heart-type fatty acid-binding protein (H-FABP) as a diagnostic and prognostic marker for acute and chronic cardiac injury, as well as the preliminary diagnostic use of other types of FABP for detecting injury in other organs, is reviewed. METHODS: This review is based on an overview of the literature on clinical diagnostics of various forms of organ injury, and uses additional literature on physiological aspects relevant for the interpretation of plasma marker concentrations. RESULTS: H-FABP not only proves to be an excellent early marker for cardiac injury in acute coronary syndromes, but also allows detection of minor myocardial injury in heart failure and unstable angina. Preliminary results indicate that sensitivity, rule-out power and prognostic value of H-FABP in cardiac injury surpass the performance of the standard early marker myoglobin. The liver only contains liver-type FABP (L-FABP), but co-expression of H-FABP and L-FABP occurs in the kidney. Similarly, intestinal-type FABP (I-FABP) and L-FABP are found in intestines, and brain-type FABP (B-FABP) and H-FABP occur in the brain. Preliminary but promising applications of these proteins have been demonstrated for liver rejection, viability selection of kidneys from non-heart-beating donors (NHBD), inflammatory and ischemic bowel disease, traumatic brain injury and in the prevention of muscle injury in trained athletes. CONCLUSIONS: Further study of the diagnostic and prognostic use of various FABP types is warranted, but their clinical application will require further commercialization of automated and rapid assays.


Subject(s)
Carrier Proteins/blood , Wounds and Injuries/blood , Wounds and Injuries/pathology , Animals , Biomarkers/blood , Fatty Acid Binding Protein 3 , Fatty Acid-Binding Proteins , Humans , Organ Specificity/physiology
8.
Hum Gene Ther ; 15(9): 906-19, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15353045

ABSTRACT

Human lipoprotein lipase (LPL) deficiency causes profound hypertriglyceridemia and life-threatening pancreatitis. We recently developed an adult murine model for LPL deficiency: LPL -/- mice display grossly elevated plasma triglyceride (TG) levels (>200-fold) and very low high-density lipoprotein cholesterol (HDL-C < 10% of normal). We used this animal model to test the efficacy of adeno-associated virus-mediated expression of hLPL(S447X) (AAV1-LPL(S447X)) in muscle for the treatment of LPL deficiency. Intramuscular administration of AAV1-LPL(S447X) resulted in dose-dependent expression of hLPL protein and LPL activity (up to 33% of normal murine levels) in postheparin plasma. Remarkably, visible hyperlipidemia was resolved within 1 week; plasma TG was reduced to near-normal levels (from 99.0 to 1.8 mmol/L), and plasma HDL-C was increased 6-fold (from 0.2 to 1.1 mmol/L). At 8 months after administration of AAV1-LPL(S447X), an intravenous lipid challenge showed efficient, near-normal clearance of plasma TG. Histologic analyses of injected muscle further indicated that abnormal muscle morphology observed in LPL -/- mice was reversed after treatment. Expression of therapeutic levels of LPL(S447X), and the subsequent beneficial effect on plasma lipid levels, has lasted for more than 1 year. We therefore conclude that AAV1-mediated transfer of LPL(S447X) into murine skeletal muscle results in long-term near-correction of dyslipidemia associated with LPL deficiency.


Subject(s)
Dependovirus/genetics , Genetic Therapy , Hyperlipoproteinemia Type I/therapy , Lipoprotein Lipase/genetics , Mutation , Animals , Dependovirus/metabolism , Female , Gene Transfer Techniques , Genetic Vectors , Hyperlipidemias/metabolism , Hyperlipidemias/therapy , Hyperlipoproteinemia Type I/metabolism , Injections, Intramuscular , Lipid Metabolism , Lipids/blood , Male , Mice , Muscles/cytology
9.
Thromb Haemost ; 87(6): 978-84, 2002 Jun.
Article in English | MEDLINE | ID: mdl-12083505

ABSTRACT

Plasma concentrations of secretory (non-pancreatic) phospholipase A2 (sPLA2) may rise 1000-fold during inflammation, and this acute phase response has been related to anticoagulant effects. In the present study this hypothesis was further investigated. Prothrombinase activity was measured for model membranes mimicking the phospholipid composition of the outer membrane of resting and activated blood platelets. Using ellipsometry, membrane degradation by sPLA2 could be measured simultaneously with inhibition of thrombin production. The same technique was used to study clotting, by the sudden appearance of fibrin strands on the membrane. Results were compared with the effects of sPLA2 on the activation of washed platelets and platelets in plasma. In buffer solution, model membranes were degraded by (patho)physiological concentrations of sPLA2. Even when only partially degraded, membranes rapidly lost their prothrombinase activity, indicating preferential degradation of phosphatidylserine. Addition of diluted plasma interfered with membrane degradation, and also with inhibition of prothrombinase activity. In agreement with these observations, sPLA2 inhibited thrombin production and annexin V-binding of activated washed platelets, but had no effects on platelet activation or clotting in plasma. These findings indicate that the elevated plasma sPLA2 concentrations observed in inflammatory disease will not reduce hypercoagulability in such patients.


Subject(s)
Blood Coagulation/drug effects , Lipid Bilayers/metabolism , Phospholipases A/pharmacology , Plasma/metabolism , Blood Platelets/ultrastructure , Fibrin/metabolism , Humans , Kinetics , Models, Biological , Phosphatidylserines/metabolism , Phospholipases A/antagonists & inhibitors , Phospholipases A/metabolism , Phospholipases A2 , Phospholipids/metabolism , Platelet Activation/drug effects , Thrombin/antagonists & inhibitors , Thrombin/biosynthesis
10.
BMC Neurosci ; 5: 4, 2004 Jan 30.
Article in English | MEDLINE | ID: mdl-15005815

ABSTRACT

BACKGROUND: Inactivating genes in vivo is an important technique for establishing their function in the adult nervous system. Unfortunately, conventional knockout mice may suffer from several limitations including embryonic or perinatal lethality and the compensatory regulation of other genes. One approach to producing conditional activation or inactivation of genes involves the use of Cre recombinase to remove loxP-flanked segments of DNA. We have studied the effects of delivering Cre to the hippocampus and neocortex of adult mice by injecting replication-deficient adeno-associated virus (AAV) and lentiviral (LV) vectors into discrete regions of the forebrain. RESULTS: Recombinant AAV-Cre, AAV-GFP (green fluorescent protein) and LV-Cre-EGFP (enhanced GFP) were made with the transgene controlled by the cytomegalovirus promoter. Infecting 293T cells in vitro with AAV-Cre and LV-Cre-EGFP resulted in transduction of most cells as shown by GFP fluorescence and Cre immunoreactivity. Injections of submicrolitre quantities of LV-Cre-EGFP and mixtures of AAV-Cre with AAV-GFP into the neocortex and hippocampus of adult Rosa26 reporter mice resulted in strong Cre and GFP expression in the dentate gyrus and moderate to strong labelling in specific regions of the hippocampus and in the neocortex, mainly in neurons. The pattern of expression of Cre and GFP obtained with AAV and LV vectors was very similar. X-gal staining showed that Cre-mediated recombination had occurred in neurons in the same regions of the brain, starting at 3 days post-injection. No obvious toxic effects of Cre expression were detected even after four weeks post-injection. CONCLUSION: AAV and LV vectors are capable of delivering Cre to neurons in discrete regions of the adult mouse brain and producing recombination.


Subject(s)
Dependovirus/genetics , Gene Transfer Techniques , Integrases/administration & dosage , Integrases/genetics , Lentivirus/genetics , Neurons/metabolism , Viral Proteins/administration & dosage , Viral Proteins/genetics , Animals , Cell Line , Gene Expression , Genes, Reporter , Genetic Vectors/administration & dosage , Genetic Vectors/genetics , Green Fluorescent Proteins , Hippocampus/metabolism , Hippocampus/virology , Integrases/metabolism , Luminescent Proteins/biosynthesis , Luminescent Proteins/genetics , Mice , Mice, Transgenic , Neocortex/metabolism , Neocortex/virology , Neurons/virology , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombination, Genetic/drug effects , Recombination, Genetic/genetics , Transgenes , Viral Proteins/metabolism
11.
Clin Biochem ; 36(7): 529-35, 2003 Oct.
Article in English | MEDLINE | ID: mdl-14563446

ABSTRACT

OBJECTIVES: Intestinal-type fatty acid-binding protein (I-FABP) has been proposed as plasma marker for the detection of acute intestinal injury. However, intestinal mucosa also expresses liver-type FABP (L-FABP). We have investigated the tissue distribution of I-FABP and L-FABP in segments of the human intestine along the duodenal to colonal axis and the potential of both proteins to serve as plasma marker for the diagnosis of intestinal injury. DESIGN AND METHODS: I-FABP and L-FABP were measured with specific immunoassays in autopsy samples of the intestine (duodenum, jejunum, ileum and colon) of 23 subjects and in plasma samples from patients (n = 51) with intestinal and/or hepatic disease. Plasma reference values were established in normal healthy individuals (n = 92). RESULTS: The I-FABP tissue contents in duodenum, jejunum, ileum, proximal colon and distal colon amounted to 2.22, 4.79, 1.04, 0.27 and 0.25 mug/g ww, respectively. L-FABP tissue contents were markedly higher, amounting to 124 and 198 mug/g ww in duodenum and jejunum, and to 58, 26 and 44 mug/g ww in ileum, proximal colon and distal colon, respectively. Elevated plasma levels of both I-FABP and L-FABP were found in patients suffering from intestinal diseases, while only L-FABP was increased in cases of purely hepatocellular injury. CONCLUSIONS: I-FABP and L-FABP show a similar pattern of tissue distribution along the duodenal to colonal axis with highest tissue contents found in the jejunum but in each intestinal segment a >40-fold higher content of L-FABP than of I-FABP. Accordingly, besides I-FABP, also L-FABP is a useful plasma marker for the detection of intestinal injury, especially in patients undergoing intestinal surgery.


Subject(s)
Carrier Proteins/analysis , Intestines/chemistry , Liver/chemistry , Tumor Suppressor Proteins , Adult , Aged , Carrier Proteins/blood , Enzyme-Linked Immunosorbent Assay , Fatty Acid-Binding Protein 7 , Fatty Acid-Binding Proteins , Female , Health , Humans , Intestinal Diseases/diagnosis , Intestinal Diseases/metabolism , Intestinal Diseases/surgery , Intestines/injuries , Intestines/pathology , Intestines/surgery , Male , Middle Aged , Reference Values , Sex Characteristics , Time Factors
14.
Br J Haematol ; 131(1): 91-9, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16173968

ABSTRACT

To study the state and diagnostic value of plasma tissue factor (TF) in patients with acute coronary syndromes (ACS), we quantitatively compared plasma TF antigen and TF activity in 90 early-hospitalised patients with chest pain. Using high-affinity antibodies, a sensitive assay for TF antigen was developed with a detection limit of 40 fmol/l. One of the antibodies was used to capture TF from plasma and, after elution and dialysis-free reconstitution in phospholipid-glucoside micelles, absolute amounts of TF activity could be measured with a detection limit of 80 fmol/l. All TF in plasma was found to be exposed, and a value of 2.5(1.1-14.8) pmol/l (median with range) was found for TF antigen. Most of this TF antigen (70-80%) circulated in a (potentially) functional state. Left in its in vivo state, however, TF captured from plasma was totally inactive, probably because of the lack of a procoagulant matrix. Compared with controls with non-cardiac chest pain, TF activity was unchanged and TF antigen about 25% elevated in ACS patients. Combined with the markers prothrombin fragment F1+2 and fatty acid-binding protein, TF did not improve the early diagnosis of ACS.


Subject(s)
Antigens/blood , Chest Pain/blood , Myocardial Infarction/blood , Thromboplastin/analysis , Acute Disease , Adult , Aged , Aged, 80 and over , Biomarkers/blood , Case-Control Studies , Electrocardiography , Enzyme-Linked Immunosorbent Assay/methods , Female , Hospitalization , Humans , Male , Middle Aged , Predictive Value of Tests , ROC Curve , Thromboplastin/immunology , Troponin T/blood
15.
Biol Chem ; 383(2): 337-41, 2002 Feb.
Article in English | MEDLINE | ID: mdl-11934273

ABSTRACT

We demonstrate for the first time that ellipsometry and confocal fluorescence correlation spectroscopy (FCS) are complementary methods for the characterisation of supported planar phospholipid bilayers (SPBs) formed on mica, a mineral used in atomic force microscopy investigations of SPBs. Addition of small unilamellar vesicles containing 20% dioleoyl-phosphatidylserine (DOPS) and 80% dioleoyl-phosphatidylcholine (DOPC) to an oxidised borosilicate surface, on the other hand, results in a planar lipid system characterised by lateral diffusion coefficients which are three time smaller than those obtained for SPBs. Moreover, seven labelled phospholipids were tested for their suitability in the FCS characterisation of vesicles as well as of SPBs.


Subject(s)
Aluminum Silicates/chemistry , Lipid Bilayers/chemistry , Phospholipids/chemistry , Boron Compounds/chemistry , Membrane Fusion , Microscopy, Confocal , Microscopy, Fluorescence , Phosphatidylcholines/chemistry , Phosphatidylserines/chemistry , Silicates/chemistry , Surface Properties
16.
Anal Biochem ; 326(2): 257-61, 2004 Mar 15.
Article in English | MEDLINE | ID: mdl-15003565

ABSTRACT

Standard enzyme immunoassays (EIAs) require washing steps to remove excess enzyme-antibody complexes. Such washing is laborious, lengthens assay time, and increases assay scatter. Recently, so-called precipitate-enhanced immunoassays (PEIAs) were introduced. Instead of color formation due to enzymatic conversion of a chromogenic substrate, this technique measures the rate of precipitate formation due to conversion of a substrate with a precipitating product. Such precipitation can be measured in the presence of active enzyme-antibody complexes in the buffer and no washing is required. In the present study this technique was used in a one-step PEIA, without washing steps, for the measurement of plasma concentrations of fatty-acid-binding protein. Horseradish peroxidase was used as tagging enzyme and diaminobenzidine as precipitating substrate. Precipitate formation was measured by ellipsometry. Assay time of the one-step PEIA was much shorter than that for an existing standard EIA. Test results can be obtained within minutes, depending on the sensitivity required. Assay precision of the one-step PEIA was better than that of the standard EIA. In the one-step assay, loss of surface-bound conjugate due to washing is prevented, which could explain part of the improved sensitivity compared to that of the two-step PEIA. More importantly, the presence of substrate-converting enzyme-antibody complexes in the buffer caused a large enhancement of precipitation.


Subject(s)
Enzyme-Linked Immunosorbent Assay/methods , Immunoenzyme Techniques , Proteins/analysis , Biomarkers/blood , Chemical Precipitation , Dose-Response Relationship, Immunologic , Enzyme-Linked Immunosorbent Assay/instrumentation , Humans , Silicon/chemistry , Surface Properties
17.
Neurobiol Dis ; 15(2): 394-406, 2004 Mar.
Article in English | MEDLINE | ID: mdl-15006710

ABSTRACT

Rubrospinal neurons (RSNs) undergo marked atrophy after cervical axotomy. This progressive atrophy may impair the regenerative capacity of RSNs in response to repair strategies that are targeted to promote rubrospinal tract regeneration. Here, we investigated whether we could achieve long-term rescue of RSNs from lesion-induced atrophy by adeno-associated viral (AAV) vector-mediated gene transfer of brain-derived neurotrophic factor (BDNF). We show for the first time that AAV vectors can be used for the persistent transduction of highly atrophic neurons in the red nucleus (RN) for up to 18 months after injury. Furthermore, BDNF gene transfer into the RN following spinal axotomy resulted in counteraction of atrophy in both the acute and chronic stage after injury. These novel findings demonstrate that a gene therapeutic approach can be used to reverse atrophy of lesioned CNS neurons for an extended period of time.


Subject(s)
Atrophy/therapy , Brain-Derived Neurotrophic Factor/genetics , Gene Transfer Techniques , Genetic Vectors/genetics , Nerve Regeneration/genetics , Spinal Cord Injuries/therapy , Acute Disease , Animals , Atrophy/metabolism , Atrophy/physiopathology , Axotomy , Brain-Derived Neurotrophic Factor/metabolism , Brain-Derived Neurotrophic Factor/therapeutic use , Chronic Disease , Dependovirus/genetics , Disease Models, Animal , Efferent Pathways/growth & development , Efferent Pathways/pathology , Efferent Pathways/physiopathology , Genetic Vectors/therapeutic use , Male , Nerve Regeneration/drug effects , Neurons/drug effects , Neurons/metabolism , Rats , Reaction Time/genetics , Receptor, trkB/metabolism , Red Nucleus/growth & development , Red Nucleus/pathology , Red Nucleus/physiopathology , Retrograde Degeneration/metabolism , Retrograde Degeneration/physiopathology , Retrograde Degeneration/therapy , Spinal Cord/growth & development , Spinal Cord/pathology , Spinal Cord/physiopathology , Spinal Cord Injuries/metabolism , Spinal Cord Injuries/physiopathology
18.
Exp Neurol ; 189(2): 303-16, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15380481

ABSTRACT

Following avulsion of a spinal ventral root, motoneurons that project through the avulsed root are axotomized. Avulsion between, for example, L2 and L6 leads to denervation of hind limb muscles. Reimplantation of an avulsed root directed to the motoneuron pool resulted in re-ingrowth of some motor axons. However, most motoneurons display retrograde atrophy and subsequently die. Two neurotrophic factors, glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF), promote the survival of motoneurons after injury. The long-term delivery of these neurotrophic factors to the motoneurons in the ventral horn of the spinal cord is problematic. One strategy to improve the outcome of the neurosurgical reinsertion of the ventral root following avulsion would involve gene transfer with adeno-associated viral (AAV) vectors encoding these neurotrophic factors near the denervated motoneuron pool. Here, we show that AAV-mediated overexpression of GDNF and BDNF in the spinal cord persisted for at least 16 weeks. At both 1 and 4 months post-lesion AAV-BDNF- and -GDNF-treated animals showed an increased survival of motoneurons, the effect being more prominent at 1 month. AAV vector-mediated overexpression of neurotrophins also promoted the formation of a network of motoneuron fibers in the ventral horn at the avulsed side, but motoneurons failed to extent axons into the reinserted L4 root towards the sciatic nerve nor to improve functional recovery of the hind limbs. This suggests that high levels of neurotrophic factors in the ventral horn promote sprouting, but prevent directional growth of axons of a higher number of surviving motoneurons into the implanted root.


Subject(s)
Brain-Derived Neurotrophic Factor/genetics , Motor Neurons/metabolism , Nerve Growth Factors/genetics , Nerve Regeneration/genetics , Radiculopathy/therapy , Spinal Cord/metabolism , Animals , Gene Transfer Techniques , Genetic Vectors , Glial Cell Line-Derived Neurotrophic Factor , Growth Cones/metabolism , Growth Cones/ultrastructure , Lumbar Vertebrae , Male , Motor Neurons/cytology , Neuronal Plasticity/genetics , Radiculopathy/metabolism , Radiculopathy/pathology , Rats , Rats, Wistar , Recovery of Function/genetics , Sciatic Nerve/cytology , Sciatic Nerve/physiology , Spinal Cord/pathology , Spinal Nerve Roots/injuries , Spinal Nerve Roots/pathology , Spinal Nerve Roots/surgery
19.
Clin Chem ; 50(9): 1568-75, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15217991

ABSTRACT

BACKGROUND: Detection of brain injury by serum markers is not a standard procedure in clinical practice, although several proteins, such as S100B, neuron-specific enolase (NSE), myelin basic protein, and glial fibrillary acidic protein, show promising results. We investigated the tissue distribution of brain- and heart-type fatty acid-binding proteins (B-FABP and H-FABP) in segments of the human brain and the potential of either protein to serve as plasma marker for diagnosis of brain injury. METHODS: B-FABP and H-FABP were measured immunochemically in autopsy samples of the brain (n = 6) and in serum samples from (a) patients with mild traumatic brain injury (MTBI; n = 130) and (b) depressed patients undergoing bilateral electroconvulsive therapy (ECT; n = 14). The protein markers S100B and NSE were measured for comparison. Reference values of B-FABP and H-FABP were established in healthy individuals (n = 92). RESULTS: The frontal, temporal, and occipital lobes, the striatum, the pons, and the cerebellum had different tissue concentrations of B-FABP and of H-FABP. B-FABP ranged from 0.8 microg/g wet weight in striatum tissue to 3.1 microg/g in frontal lobe. H-FABP was markedly higher, ranging from 16.2 microg/g wet weight in cerebellum tissue to 39.5 microg/g in pons. No B-FABP was detected in serum from healthy donors. H-FABP serum reference value was 6 microg/L. In the MTBI study, serum B-FABP was increased in 68% and H-FABP in 70% of patients compared with S100B (increased in 45%) and NSE (increased in 51% of patients). In ECT, serum B-FABP was increased in 6% of all samples (2 of 14 patients), whereas H-FABP was above its upper reference limit (6 microg/L) in 17% of all samples (8 of 14 patients), and S100B was above its upper reference limit (0.3 microg/L) in 0.4% of all samples. CONCLUSIONS: B-FABP and H-FABP patterns differ among brain tissues, with the highest concentrations in the frontal lobe and pons, respectively. However, in each part of the brain, the H-FABP concentration was at least 10 times higher than that of B-FABP. Patient studies indicate that B-FABP and H-FABP are more sensitive markers for minor brain injury than the currently used markers S100B and NSE.


Subject(s)
Brain Injuries/diagnosis , Brain Injuries/metabolism , Brain/metabolism , Carrier Proteins/metabolism , Adult , Aged , Biomarkers/blood , Blotting, Western , Brain Injuries/blood , Carrier Proteins/blood , Electroconvulsive Therapy , Fatty Acid Binding Protein 3 , Fatty Acid-Binding Proteins , Female , Humans , Immunoassay , Male , Middle Aged , Phosphopyruvate Hydratase/blood , Reference Values , S100 Proteins/blood , Statistics, Nonparametric , Tissue Distribution
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