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Exosomes, also called extracellular vesicles (EVs), are membranous structures measuring between 40 and 100 nm. Exosomes, secreted by various cells of the human body into body fluids, contain protein, mRNA, miRNA, and signaling molecules. Physiologically, exosomes assist in the intercellular transport of protein and RNA. Immunologically, exosomes exhibit antigen-presenting capability. In recent studies, exosomes were found to be associated with the pathophysiology of cardiovascular, renal, neurological, and ocular diseases. In addition, exosomes may play a major role in cancer metastasis. Due to the extremely small size and scarcity of exosomes in living samples, many early studies utilized sucrose density gradient ultracentrifugation for exosome collection. However, sucrose density gradient ultracentrifugation is rather time consuming and requires large biological sample quantities. Newer exosome studies combined immunoaffinity and microfluidic system approaches for more efficient exosome collection. Our review summarizes existing methods for EV isolation and notes their advantages and disadvantages. These promising approaches are all characterized by isolation efficiency, and savings in cost, labor, and time. Optimization of current methods is a necessary step toward clinically-relevant diagnostic product production, but the fact that EVs are already widely used in disease diagnosis and treatment encourages continued efforts.

An extreme form of hyperalphalipoproteinemia was studied in a patient affected by multiple symmetric lipomatosis (MSL); four relatives and three MSL controls were also evaluated. Plasma lipids and apolipoproteins were measured and overall lipoprotein profile was assessed by density gradient ultracentrifugation. The patient showed a plasma HDL-cholesterol of 138 mg/dl and an apo A-I of 218 mg/dl; moreover significantly high HDL levels were found in two unaffected relatives. The hypobetalipoproteinemia trait was also found both in the patient and in one of his daughters. We suggest that some pre-existing conditions may enhance lipoprotein metabolism alterations in this lipid storage disease.

Dear Editor, We recently reported that C-reactive protein (CRP), when present with the small-molecule compound phosphoethanolamine (PEt) (Sigma-Aldrich), acquired the capability to bind to native low-density lipoprotein (LDL), and that PEt enhanced the binding of CRP to enzymatically-modified LDL (E-LDL) [1-3]. Subsequently, we found that, in the presence of PEt, CRP bound to a variety of other proteins. We randomly selected fibronectin, IgG, leptin, FcγRIIa, activated complement component C3b, complement protein factor H, serum amyloid P component, oxidized LDL, lipoprotein (a), amyloid precursor protein α and amyloid β peptide (Aβ)(Sigma-Aldrich, St. Louis, MO), and determined the binding of CRP to these proteins. CRP did not bind to any of these proteins. However, in the presence of PEt, CRP bound to all of these proteins. As a representative, we present here the data on the binding of CRP to Aβ (Fig. 1A). Fig. 1 CRP is functional at acidic pH. (A) CRP binds to amyloid β protein fragment 1-38 (Aβ) in the presence of phosphoethanolamine (PEt). Microtiter wells were coated with 10 μg/ml of Aβ in 10 mmol/l Tris-HCl, 150 mmol/l NaCl, ... During our attempts to understand the PEt phenomenon, we got hints that the addition of PEt to 10 mmol/l Tris-HCl, 150 mmol/l NaCl, pH 7.2 (TBS) containing 0.1% gelatin, 0.02% Tween-20 and 2 mmol/l CaCl2 (TBS-Ca), the buffer which we used in the experiments, might be lowering the pH, and, indeed, that was the case (Fig. 1B). Accordingly, we determined the binding of CRP to E-LDL as a function of pH and found that the binding of CRP to E-LDL was dramatically enhanced at acidic pH (Fig. 1C). Additional experiments confirmed that the previously reported enhancement in the binding of CRP to E-LDL in the presence of PEt was solely due to acidic pH and not due to the binding of PEt to CRP. At acidic pH, CRP also bound to many other proteins that we examined so far, including Aβ (Fig. 1D). The binding of CRP to an array of unrelated proteins, at acidic pH, was puzzling. We hypothesized that CRP, at acidic pH, recognized a pattern. We noticed that the only component that was common in our experiments was the technique, the ELISA-based solid-phase ligand-binding assay, in which we immobilized the protein ligands on polystyrene microtiter wells (see legend to Fig. 1). Because the immobilization of proteins results in conformational alteration of proteins, in addition to deposition and aggregation of proteins on the walls of the wells, our data suggest that, at acidic pH, CRP binds to deposited, aggregated and conformationally altered proteins, irrespective of the identity of the protein. If this interpretation is correct, then the enhancement seen in the binding of CRP to E-LDL at acidic pH is likely due to the property of CRP to bind to deposited, aggregated and conformationally altered proteins. It is important to note, however, that the acidic pH is not a requirement for the binding of CRP to E-LDL and that CRP prevents formation of E-LDL-loaded macrophage foam cells at normal physiological pH [2,3]. It is also important to note that when sucrose density gradient ultracentrifugation was used to evaluate the binding of CRP to native LDL in serum, in the presence of PEt, and hence at acidic pH, CRP bound to native LDL, but not to HDL [1]. Collectively, our findings suggest that CRP has two native pentameric states: one at normal physiological pH and another at acidic, pathological pH. At acidic pH, CRP changes its conformational state to gain functions distinct from the functions of CRP at normal physiological pH. The significance of the binding of CRP, at acidic pH, to deposited, aggregated and conformationally altered proteins is currently under investigation.

The properties of the membrane associated enzyme complexes were investigated. The enzyme complexes of alkaline phosphatase, arylamidase, and l-gamma-glutamyltranspeptidase were excluded by gel filtration on Sephadex G-200. The electrophoretic mobility of the enzyme complexes was found to be in the alpha-1 portion and these enzyme activities were immunologically fixed by anti-IgA and anti-light chain lambda antibodies. Heterogeneity in density was observed by density gradient ultracentrifugation. The activities of membrane-associated enzymes were demonstrated in both high (d > 1.21) and low density particles (1.063 < d < 1.21), and the enzyme activities of a high density particle were immunologically fixed by anti-IgA and anti-light chain lambda antibodies. The heterogeneity in density was based on the presence of immunoglobulin A molecule in the complexes. The specificity of the immunoglobulin which was found in the enzyme complexes proved to be exclusively of the alpha heavy chain classes and lambda light chain types respectively. This isotype specificity of immunoglobulin A is similar to the specificity observed in enzyme-linked immunoglobulins, and simple non-specific binding of immunoglobulin and membrane moiety was eliminated. This isotype specificity is of interest in connection with the properties of the circulating autoantibodies.

Sedimentation coefficients of the amylases present in the sera of 22 patients with macroamylasemia were measured by means of sucrose density gradient ultracentrifugation using beef liver catalase (11.3 S) and the 7 S serum proteins as markers. The sedimentation coefficients of the macroamylases in 18 of the cases ranged from 7.7 S to 10.7 S; in four cases the activity peaks were too broad to assign a definite sedimentation coefficient. One exceptional case (Case 6) exhibited two peaks of amylase activity: one had a sedimentation coefficient greater than the 19 S serum proteins and the other a coefficient of 10.7 S. The normal-size amylase component seen on dextran gel filtration was no longer discernible after ultracentrifugation in eight of the cases. These observations, in contrast to those on the paper electrophoretic pattern of those sera, suggest that the macroamylase complexes in patients with macroamylasemia are not homogeneous. The observations also indicate that dextran gel filtration may lead to erroneous concepts regarding the component fractions of serum amylase and their molecular sizes because of interaction between amylase and dextran.