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The Use of Tracers in Transport Studies
Published in Joan Gil, Models of Lung Disease, 2020
Maya Simionescu, Nicolae Ghinea
The main sources of cholesterol for surfactant biosynthesis are the plasma lipoproteins (Hass and Longmore, 1980), such as low-density lipoproteins (LDL). To find out how circulating LDL is transported through endothelium, the lipoprotein was adsorbed to 5 nm colloidal gold (LDL-Au). The conjugate retains the same immunoreactivity as native LDL as shown by immunoelectrophoresis, Ouchterlony double immunodiffusion, and Laurrell immunoelectrophoresis against antihuman LDL (Fig. 11). By negative staining, one or two colloidal gold particles appeared attached to an LDL particle (Fig. 2a). The LDL-Au conjugate was recirculated in situ through the pulmonary vasculature, and its presence on and within the endothelium was recorded at 7, 30 and 60 min. (Nistor and Simionescu, 1986). At 7 min, LDL-Au labeled a large number of coated pits and plasmalemmal vesicles open to the luminal front or apparently internalized (Fig. 12a,b). After 30 and 60 min of recirculation, in addition to the features mentioned, the conjugate appeared associated with coated vesicles, plasmalemmal vesicles opened to the abluminal front, and in structures resembling endosomes and lysosomes (Fig. 12c,d). LDL-Au could occasionally be found in the pericapillary space. Similar results were obtained in experiments in which native untagged LDL was perfused and visualized by tissue treatment with 1% tannic acid previous to osmication (Nistor and Simionescu, 1986). The results showed that the features involved in uptake and transport of LDL in alveolar endothelium are those known to be effective in receptor-mediated endocytosis (i.e., coated pits and vesicles, endosomes, and lysosomes) and fluid phase transcytosis (i.e., plasmalemmal vesicles). These data, together with those from experiments in which radiolabeled LDL was used, showed that LDL is partially taken up from the circulation by a receptor-mediated process that is saturable and time and concentration dependent. When heparin was added to the washing buffer to remove receptor-bound LDL (Goldstein et al., 1976), it was found that 50% of LDL uptake is receptor independent. The fractional contribution of each of these processes for uptake and transport of LDL by the lung in vivo remains to be established.
Current understanding and recent advances in myositis-specific and -associated autoantibodies detected in patients with dermatomyositis
Published in Expert Review of Clinical Immunology, 2020
Takahisa Gono, Masataka Kuwana
Identification of individual MSAs/MAAs should be considered based on clinical presentation and results of ANA testing. Table 1 lists staining patterns on indirect immunofluorescence test and detection methods for MSAs/MAAs found in DM patients . A variety of assays are used for the identification of individual antibodies and include RNA and protein immunoprecipitation (IP) assays, enzyme immunoassay (EIA), line-blot immunoassay (LIA), and Ouchterlony double immunodiffusion (DID). All MSAs/MAAs were first identified by DID or IP assay, which are regarded as gold standards. The procedures of IP assays are complicated and time-consuming, and require the use of a radioisotope. DID is highly specific but is less sensitive, and sensitivity varies depending on the antigen preparation lots used. Therefore, those assays are mainly conducted in specialized research laboratories. EIA is a convenient technique widely used for the measurement of many autoantibodies, including NSAs/MAAs, because of its ability to handle a large number of samples simultaneously in a short time. EIA, which uses enzyme-conjugated secondary anti-human immunoglobulin antibodies, has been the mainstream, but commercial kits utilizing more sensitive chemiluminescent immunoassays and fluorescence enzyme immunoassays have become available in recent years. Another advantage of EIA over IP assays is to provide quantitative results, which are shown to correlate with disease activity and clinical outcomes [87–89]. LIA is considered the first generation multianalyte immunoassay and is commercially available for detection of a series of MSAs/MAAs (EUROLINE, Euroimmune) [90]. This system adopts the principle of immunoblots, in which membrane strips are coated with thin parallel lines of a panel of purified antigens. It can be performed manually, but automated systems have become available. LIA is principally a nonquantitative assay, but the intensity of the bands is shown to correlate with the antibody titer. The majority of commercial EIA kits represent both sensitivity and specificity >95%, in comparison with results measured by the gold standard IP assay [52,91,92]. However, the concordance rates of the results between the LIA and IP assays are shown to be lower than the rates between the EIA and IP assay results [93]. A recent study has reported that anti-OJ antibody is virtually undetectable by LIA because of the lack of antigenicity of the OJ antigen, since autoantibodies in the patients’ sera recognize a conformational epitope formed on a multiprotein complex [94]. In addition, measurement of anti-PM-Scl antibody by LIA results in a high false-positive rate [95]. Recently, particle-based multianalyte technology (PMAT) has been established as the next generation multianalyte immunoassays for the detection of the autoantibodies, and high concordance between the results of PMAT and LIA or IP assays was reported [96]. In general, we can use either EIA or LIA depending on availability, but we need to recognize that comprehensive studies comparing the sensitivity and specificity of individual commercial kits are lacking. If autoantibody results were not consistent with ANA titer/pattern and/or patients’ clinical presentation, additional measurement using other methods is highly recommended.