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A quantitative method for evaluating the degradation of biologic scaffold materials.

Scaffolds derived from naturally occurring extracellular matrix (ECM) have found extensive use in the fields of tissue engineering and regenerative medicine. Many of these scaffolds are designed to degrade rapidly as they are replaced by new host tissue. Other scaffolds are chemically crosslinked to slow the rate of degradation or add strength to the scaffold. Commercially available ECM scaffolds have considerable variability with regards to tissue origin and methods of processing, and little is known about their rate of degradation and the fate of their degradation products. A novel method is described herein to integrally label ECM with a radioactive isotope ((14)C). It was found that a number of tissues are efficiently labeled, including heart, liver, trachea, pancreas, small intestine, and urinary bladder tissue. Of the tissues analyzed, only spleen was not found to contain detectable levels of (14)C. The technique is extremely sensitive, accurate, and safe, but requires access to accelerator mass spectrometry, and is expensive and time consuming. This model represents the first described quantitative method to determine the rate of degradation for an ECM scaffold and to track the fate of the degradation products.

Animals↗

Detection of known thalassemia point mutations by snapback single-strand conformation polymorphism: the feasibility analysis.

OBJECTIVES: To explore if snapback single-strand conformation polymorphism (snapback SSCP) can be applied to the detection of known thalassemia point mutations. DESIGN AND METHODS: We examined 13 types of known thalassemia point mutations by using snapback SSCP. RESULTS: These 13 different thalassemia point mutations could be identified by snapback SSCP. CONCLUSIONS: Snapback SSCP can be applied to the detection of known thalassemia point mutations. The advantages of snapback SSCP are (1) it is simple as compared to PCR-ASO; (2) snapback SSCP is specific and stable once the conditions of snapback SSCP are optimized; (3) samples can be checked at any time without the limit of half-life of radioactive isotope; and (4) since PCR products used in snapback SSCP can be checked by using 2% agarose gel before snapback SSCP, misdiagnoses caused by false positive or false negative PCR can be reduced significantly in snapback SSCP.

Base Sequence↗

Treatment of mantle cell lymphoma: current approach and future directions.

Although mantle cell lymphoma has been described as "moderately aggressive" it has become clear that it carries a worse long-term prognosis than other subtypes of non-Hodgkin's lymphoma. In recent years, this has prompted numerous clinical trials of novel and more aggressive therapies in hopes of impacting these poor outcomes. These include more intensive combination chemotherapy regimens, monoclonal antibody therapy in conjunction with other treatments or conjugated to radioactive isotopes, high-dose chemotherapy followed by autologous or allogeneic stem cell transplantation, and newer targeted therapies based on increasing understanding of the molecular pathways of this malignancy.

Antineoplastic Combined Chemotherapy Protocols↗

Clinical applications of newer radionuclide therapies.

When radio-iodine was first used in the treatment of metastasized thyroid carcinoma in 1943, its success in terms of tumour response, quality of life improvement and survival was considered a 'miracle', as in those days metastatic cancer was generally fatal. Inspired by this, many efforts have been made to apply radioisotope therapy to other tumours. Radionuclide therapy uses radioactive isotopes labelled with specific targeting agents that aim to deliver the irradiation of the isotope to the tumour, while sparing normal tissues. Its unique modality allows to systemically target radiosensitive tumours throughout the body. Another important principle is its so-called 'cross-fire' action, whereby, owing to the larger reach of the radiation in relation to the cell diameter, a tumour cell receives lethal hits also from isotopes in the neighbourhood that are not directly associated with this cell. The treatment is therefore less hampered by inhomogeneous distribution and metabolism than for example chemo- or immunotherapy. The European Association of Nuclear Medicine has issued guidelines on so-called 'established' therapies (www.eanm.org), i.e. hyperthyroidism, thyroid carcinoma, refractory synovitis, bone metastases, mIBG therapy, 32P therapy and Lipiodol therapy. Newer therapies include radio-peptide therapy, radio-immunotherapy of lymphoma and microsphere therapy for liver cancer. The aim of a recently held workshop at the ECCO13 conference 2005 and this review is to inform the oncology community about these new developing therapies.

Forecasting↗

125I used for labelling of proteins in an absorption model changes the absorption rate of insulin aspart.

UNLABELLED: The aim of this study is to validate the ability of the disappearance model to predict absorption rates of insulin aspart in pigs. The disappearance model is used as a screening tool to estimate absorption rates after subcutaneous injections in humans or pigs especially of insulin and insulin analogues. The disappearance model measures remaining radioactivity at the injection site and therefore radioactive labelling of the insulin analogue is necessary. The labelling is done with 125I. One of the assumptions for the disappearance model to be reliable is that absorption rates of the labelled and non-labelled molecules are comparable. In this study, we compared disappearance data with absorption calculated from plasma samples of insulin aspart. The calculated absorption is based on non-labelled insulin aspart. The absorption rate from the disappearance data was statistical significant (p = 0.0028) different from the absorption rate based on plasma samples. A control study was carried out where 125I labelled insulin aspart was compared to 127I (the natural non-radioactive isotope) insulin aspart. In this study, absorption rate from the disappearance data and absorption rate based on plasma samples were similar (p = 0.63). CONCLUSION: Iodination of insulin aspart changes the subcutaneous absorption rate.

Absorption↗

A non-radioactive method for measuring Cu uptake in HepG2 cells.

At present, all data on Cu uptake and metabolism have been derived from radioactive uptake experiments. These experiments are limited by the availability of the radioactive isotopes 64Cu or 67Cu, and their short half-life (12.5 and 62 h, respectively). In this paper, we investigate an alternative method to study the uptake of Cu with natural isotopes in HepG2 cells, a liver cell line used extensively to study Cu metabolism. In nature, Cu occurs as two stable isotopes, 63Cu and 65Cu (63Cu/65Cu = 2.23). This ratio can be measured accurately using inductively coupled plasma mass spectrometry (ICP-MS). In initial experiments, we attempted to measure the time course of Cu uptake using 65Cu. The change in the 63Cu/65Cu ratio, however, was too small to allow measurement of Cu uptake by the cells. To overcome this difficulty, the natural 63Cu/65Cu ratio in HepG2 cells was altered using long-term incubation with 63Cu. This had a significant effect on Cu concentration in HepG2 cells, changing it from 81.9 +/- 9.46 pmol microg DNA(-1) (week 1) to 155 +/- 8.63 pmol microg DNA(-1) (week 2) and stabilising at 171 +/- 4.82 pmol microg DNA(-1) (week 3). After three weeks of culture with 2 microM 63Cu the 63Cu/65Cu changed from 2.18 +/- 0.05 to 15.3 +/- 1.01. Cu uptake was then investigated as before using 65Cu. Uptake was linear over 60 min, temperature dependent and consistent with previous kinetics data. These observations suggest that stable isotope ICP-MS provides an alternative technique for the study of Cu uptake by HepG2 cells.

Animals↗

Assessment of myocardial perfusion with real-time myocardial contrast echocardiography: methodology and clinical applications.

Real-time myocardial contrast perfusion imaging (RTMCI) with echocardiography is a promising technique for evaluation of patients with known or suspected coronary artery disease. The technique is based on the utilization of small (<10 mum) microbubbles, which are capable of crossing the pulmonary circulation after intravenous injection. Unlike radioactive isotopes, which are taken actively or diffuse passively in the myocytes, myocardial contrast agents remain extracellularly in the capillaries and present a measure of the myocardial capillary blood volume and microvascular integrity. RTMCI has been shown to be a safe and feasible method for the assessment of myocardial perfusion at rest and with pharmacologic stress. Recent studies have shown the value of RTMCI with dobutamine stress in improving overall and regional detection of coronary artery disease and detecting of abnormalities at submaximal stress, therefore improving sensitivity in patients who are unable to achieve the target heart rate. The advantages of the technique include the ability to assess perfusion at bedside in one setting, simultaneous assessment of myocardial function, shorter imaging time, no need for ionizing irradiation, immediate availability of the results, and the ability to determine the ischemic threshold. Recent studies have shown that RTMCI improves the prognostic utility of standard dobutamine stress in addition to wall motion analysis. Patients with normal perfusion had a better outcome than those with normal wall motion. The combination of abnormal wall motion and perfusion identified patients at greatest risk of death and nonfatal myocardial infarction. Perfusion abnormalities were also shown to predict short-term cardiac events in patients presenting to the emergency department with chest pain and no ST-segment elevation. Refinement of imaging techniques is expected to improve the specificity of RTMCI, particularly in differentiating true perfusion defects from artifacts. This review will discuss the physiologic basis, methodology, clinical utility, and limitations of RTMCI in the assessment of patients with known or suspected coronary artery disease.

Computer Systems↗

Evaluation of the effect of chronic exposure to 137Cesium on sleep-wake cycle in rats.

Since the Chernobyl accident, the most significant problem for the population living in the contaminated areas is chronic exposure by ingestion of radionuclides, notably (137)Cs, a radioactive isotope of cesium. It can be found in the whole body, including the central nervous system. The present study aimed to assess the effect of (137)Cs on the central nervous system and notably on open-field activity and the electroencephalographic pattern. Rats were exposed up to 90 days to drinking water contaminated with (137)Cs at a dosage of 400 Bq kg(-1), which is similar to that ingested by the population living in contaminated territories. At this level of exposure, no significant effect was observed on open-field activity. On the other hand, at 30 days exposure, (137)Cs decreased the number of episodes of wakefulness and slow wave sleep and increased the mean duration of these stages. At 90 days exposure, the power of 0.5-4 Hz band of (137)Cs-exposed rats was increased in comparison with controls. These electrophysiological changes may be due to a regional (137)Cs accumulation in the brain stem. In conclusion, the neurocognitive effects of (137)Cs need further evaluation and central disorders of population living in contaminated territories must be considered.

Animals↗

Sentinel lymph node biopsy using lymphoscintigraphy.

Cancer surgery has existed as an elective treatment modality for more than 150 years, yet it is relatively unsophisticated in light of the advances seen in other medical disciplines. Using the nuclear medicine technique of lymphoscintigraphy (i.e., mapping lymph nodes using a radioactive isotope), the surgeon can perform a sentinel lymph node biopsy that targets only the lymph nodes that first drain a primary tumor site. This procedure can provide accurate staging information without risking the potential morbidity associated with lymph node dissection. This type of precise biopsy is made possible by using a gamma-detecting instrument coupled with an ultrasensitive probe designed for intraoperative use.

Biopsy↗

Radiolabeled monoclonal antibodies.

Monoclonal antibodies (MoAbs) are biologically engineered proteins designed to bind to antigens emanating from tumor cells. Selected radioactive isotopes are fused with MoAbs to allow radioimmunodetection of external imaging of metastatic deposits in patients with colon cancer. For the past 10 years, radiolabeled MoAbs have improved tumor localization techniques and influenced the clinical management of surgical patients. Intraoperatively, surgeons use appropriately shielded, handheld, gamma detection probes to locate radiolabeled MoAbs and corresponding colon cancers (ie, residual, recurrent tumors). Using gamma detection probes intraoperatively, surgeons can localize nonpalpable occult tumors and disease not suspected from external antibody scans or other traditional diagnostic methods. This success confirms the need for complete tumor resections, thorough scanning of entire tumor beds, and ex vivo scanning of surgical specimens to assess for potential nodal metastases.

Animals↗

Prenatal analysis of rhesus CcDEe blood groups by heteroduplex generator.

OBJECTIVE: Our purpose was to determine the efficacy of the heteroduplex generator in the prenatal analysis of rhesus CcDEe blood groups. STUDY DESIGN: A cross-sectional study was performed evaluating fetal samples from 85 women undergoing prenatal diagnosis and comparing the results with standard immunologic serotyping on cord blood delivery. RESULTS: Of the 85 samples, 64 were tested, for all CcDEe alleles: one case was discrepant. Twenty-one cases were tested solely for the D antigen. Two novel genotypes were detected in the population by heteroduplex generator and confirmed by deoxyribonucleic acid sequencing. Five cases were indeterminate because of an indistinct banding pattern. CONCLUSIONS: Heteroduplex analysis can identify rhesus blood group alleles and is inexpensive, rapid, and does not use radioactive isotopes.

Alleles↗

A novel sandwich immunosensing method for measuring cardiac troponin I in sera.

Common methods for monitoring human cardiac troponin I (cTn I) are based on using antibodies against cTn I labeled with horseradish peroxidase, radioactive isotopes, or other labels. In this study, a novel label-free sandwich immunosensing method for measuring cTn I was developed. Three monoclonal antibodies (mAbs 9F5, 2F11, and 8C12) against human cTn I were generated by the commonly used hybridoma technique and characterized by a surface plasmon resonance (SPR) biosensor. An optimal pair of mAbs for measuring human cTn I was selected, as both mAbs have high affinities for cTn I and do not compete against each other for cTn I binding. An optical immunosensor for measuring cTn I in sera based on SPR was developed by using avidin as an intermediate layer and biotinylated-2F11 as the capturing antibody. Two detection methods for cTn I with the immunosensor were performed: (1) the direct detection of cTn I with a detection range of 2.5 to 40 microg/L and (2) the sandwich immunosensing method. In the sandwich assay mode, the second antibody 9F5 biologically amplified the sensor response. As a result, the sandwich assay showed a sensitivity of 0.25 microg/L and a detection range of 0.5 to 20 microg/L with within-run variation of 4.9 to 6.7% and between-run variation of 5.2 to 8.4%. This method has greatly enhanced the sensitivity for detection compared to that previously reported in the literatures.

Antibodies, Monoclonal↗

[Inductively coupled plasma mass spectrometry. Perspectives in analysis and in biology].

ICP-MS is an instrumental method of multi-elementary qualitative and quantitative analysis. It associates with a mass spectrometer (MS) an ion source composed of a plasma torch fed with inductive coupling with a high frequency electromagnetic generator (ICP), similar to that used as a light source in highly-successful Atomics Emission Spectrometry (AES). ICP-MS can be applied to simultaneous analysis of numerous metallic and metalloid elements (80 or so). Its sensitiveness is all in all far better than that available with previous spectrometric techniques, which nevertheless remain more advantageous for processing certain low atomic mass elements. Thanks to its broad dynamic range, ICP-MS allows quantification of an array of elementary concentrations within a single sample. ICP-MS offers particularly interesting perspectives in geochemistry and metal processing, as well as in biochemistry and food or toxicology and environmental analysis. Implementation is rapid and the technique is suitable for series or continuous analyses, or for analysis of any evolving medium such as effluents from gas or liquid chromatography or from capillary electrophoresis, making it a valuable tool for speciation analyses. Finally it enables non-radioactive isotopic labeling, essential for nutritional studies of trace elements, and sufficiently accurate isotopic dilutions, even with more accessible machines.

Animals↗

Nephrotoxic and pneumotoxic antibodies and their acute action on lungs. III. Organ localization of antibodies after experimental administration of lethal doses.

Experimental intravenous administration of large doses of heterologous anti-glomerular and anti-lung antibodies induces acute exsudative haemorrhagic changes in the lungs resulting in death of the experimental animals. The specific localization of these antibodies labelled with radioactive isotopes of iodine and administered using the paired-isotope technique with radioiodinated normal IgG, displayed high values in the lungs, liver and kidneys. Pulmonary changes developing after administration of the same dose of anti-glomerular antibodies into the left heart ventricle are of lesser extent and the animals survive. On this mode of administration, the specific localization of antibodies presents lower values in the lungs, whereas the values in the kidneys and liver are approximately the same as seen after intravenous administration. Antibody localization in the kidneys and liver is facilitated by the morphologic structure of the capillaries, whereas in the lungs a direct transendothelial transfer must be considered, since in this organ capillary endothelium forms a coherent layer. Attention is being drawn to the high specific localization of antibodies in the liver and to the potential effect of these antibodies on the liver.

Animals↗

A novel flow cytometric assay for quantitation and multiparametric characterization of cell-mediated cytotoxicity.

Cell-mediated cytotoxicity is a crucial mechanism involved in several fundamental immunological processes such as protection against intracellular pathogens or termination of an immune response. This phenomenon is classically evaluated by the 51Cr release assay, which requires a radioactive isotope and does not permit the characterization of cells involved in the cytotoxic reaction. We describe a new flow cytometry method, developed in the context of CD95-mediated cell death, which allows the precise quantitation of cell-mediated cytotoxicity and the detection of intracellular events involved in the cytotoxic process. This assay uses a combination of two dyes, i.e. 5- (and 6-) carboxyfluorescein diacetate succinimydyl ester (CFSE) to label effector cells and 7-amino actinomycin D (7-AAD) to stain apoptotic target cells. We show that this assay is more sensitive than the 51Cr release assay and makes it possible to quantitate the percentage of cell lysis and, concomitantly, to immunophenotype target cells. It also facilitates the analysis of some events of the apoptotic pathway such as caspase activation or the expression of mitochondrial molecules. This new assay should contribute to a better understanding of the mechanisms involved in cell-mediated cytotoxicity in normal and pathological situations.

Animals↗

Improved ELISA proliferation assay (EPA) for the detection of in vitro cell proliferation by a new Ki-67-antigen directed monoclonal antibody (Ki-S3).

We describe a simplified and improved proliferation assay based on a conventional ELISA system for the in vitro measurement of cellular proliferation (ELISA proliferation assay = EPA). The assay is based on a new monoclonal antibody (Ki-S3) to the proliferation-specific Ki-67-antigen and is carried out in 96-well microtiter plates using conventional immunoenzymatic methods. Ki-S3 is an immunoprecipitating monoclonal mouse IgG1 antibody, which recognizes a formalin-resistant epitope of the Ki-67 antigen. It can be used to measure proliferating cells in the cell cycle phases G1, S, G2 and M. In phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) the absorbance values obtained with the EPA show a statistically significant correlation to the number of Ki-S3 positive cells in simultaneously immunostained cytospin slides (r = 0.88). A direct comparison with [3H]thymidine labeling reveals the test to be an equally sensitive method for monitoring cellular proliferation (r = 0.91). This assay is an improved ELISA proliferation assay, which is easy to perform, does not require time-consuming pretreatments and avoids the hazards of radioactive isotopes.

Animals↗

An enzyme based assay for the measurement of complement mediated binding of immune complexes to red blood cells.

A new in vitro method is presented for measuring directly the ability of sera to induce binding of immune complexes (ICs) to erythrocytes (ICRB assay). The assay measures the binding of alkaline phosphatase (AP)-anti-alkaline phosphatase (anti-AP) complexes formed in the presence of the test sera to the complement receptor 1 (CR1) on normal human red blood cells (RBCs). By using a standard serum source, the assay can also be used to measure the IC binding ability of RBCs from different donors. As compared to the traditional CH50 method, the ICRB assay generally showed more pronounced abnormality in 10 individuals tested, of whom 5 had primary deficiency of classical pathway components. Seven out of ten individuals had systemic lupus erythematosus (SLE) and 2/10 had other rheumatic diseases without primary complement deficiency. The ICRB measured in samples from 9 other patients with SLE was significantly decreased when compared to values from 80 normal individuals. ICRB in serum samples from a C2 deficient SLE patient collected during plasma infusion treatment reflected closely the rising amount of C2 in the serum. Using RBCs from different donors ICRB activity correlated well with the numbers of CR1 as measured by a flow cytometric assay (FCA). These methods should be valuable for measuring the overall IC clearance capacity of the blood and have the advantage that the use of radioactive isotopes is avoided.

Animals↗

A novel method for measuring CTL and NK cell-mediated cytotoxicity using annexin V and two-color flow cytometry.

An assay based on two-color flow cytometry has been developed to measure CTL and NK cell-mediated cytotoxicity. After effector/target cells are incubated together, CTL or NK populations are stained with an effector cell specific PE-conjugated mAb. Subsequently, annexin V-FITC binds to cells expressing phosphatidylserine (an early marker of apoptosis) on the cell surface. Target cells are gated upon as PE-negative and quantified with respect to their annexin V positivity. The shift from annexin Vneg to annexin Vhi is a discrete event such that all target cells fall within discernible populations with respect to annexin V. There is a strong correlation between cytotoxicity measured with our assay and a standard 51Cr release assay (r2 = 0.989). The PE/annexin V assay shows increased sensitivity at early timepoints after target/effector cell mixing. In addition, this method allows for analysis of target cells at the single cell level. Therefore, we have described a promising new technique to measure in vitro cell-mediated cytotoxicity. It avoids the potential difficulties of working with radioactive isotopes, and offers increased sensitivity and versatility.

Annexin A5↗