PubMed · 9231464
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P B Dissanayake. 1997. Interference by mannitol in total protein estimation.. https://pubmed.ncbi.nlm.nih.gov/9231464/
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Since human hepatocytes are available only in limited number, the development of a serum-free culture system for long-term cultivation of differentiated and functional hepatocytes is of great importance. Here we describe the culture of human hepatocytes in a chemically defined serum-free medium for up to 5 weeks. Cell morphology was assayed by light and electron microscopy and revealed a well-preserved cellular morphology. Marker proteins for epithelial and bile duct cells, cytokeratin (CK) 18 and 19, and liver-specific proteins, like phosphoenolpyruvate carboxykinase-2 (PCK2) and serum proteins, were expressed. Liver-enriched transcription factors CCAAT/enhancer binding protein alpha (C/EBPalpha) and hepatocyte nuclear factor-4 (HNF-4), cytokine and mitogen activated factors (nuclear factor kappa B) NFkappaB, and activator protein-1 (AP-1) were maintained and active for several weeks in our cultures. In summary, our serum-free culture system allows the culture of differentiated human hepatocytes for several weeks. It may serve as a model system for metabolic, pharmacologic-toxicologic studies, and studies on human pathogens under defined chemical conditions.
Partially quaternized poly[thio-1-(N,N-diethyl-aminomethyl) ethylene]s, Q-P(TDAE)(x) with x indicating the percentage of quaternized subunits, have been proposed as potential carriers for drugs insoluble in water. However these cationic polyelectrolytes form emboli upon intravenous administration. In order to study the mechanism, Q-P(TDAE)(11) was incubated in vitro with red blood cells (RBCs) suspended in various aqueous media such as autologous plasma, autologous serum, albumin dissolved in phosphate buffer, plasma-serum mixtures and Tris buffer. The deformability of the RBC membrane studied by viscometry was not affected by the polycation. Q-P(TDAE)(11)-induced hemagglutination was studied by optical microscopy. It depended on the polymer concentration and on the presence of plasma proteins. As ghosts were formed in some cases, hemolysis was investigated by measuring potassium and hemoglobin released from RBCs. Fibrinogen and serum proteins, except albumin, protected RBCs from hemolysis. Moreover the order of addition of the suspension components modulated dramatically the Q-P(TDAE)(11)-induced hemolysis. Addition of Q-P(TDAE)(11) to whole blood caused hemolysis whereas addition of the polymer to plasma prior to contact with RBCs did not affect the cell integrity. In contrast, addition of the polymer to RBCs suspended in albumin solution caused greater hemolysis than the addition to whole blood, and the contact between Q-P(TDAE)(11) and albumin prior to RBC addition still enhanced cell lysis. Two conclusions can be drawn from these observations: (i) Q-P(TDAE)(11) induces both hemagglutination, probably through electrostatic interaction, and hemolysis, because Q-P(TDAE)(11) disrupted the RBC lipid bilayer; (ii) proteins can decrease or increase the deleterious effects of Q-P(TDAE)(11) on RBCs.
BACKGROUND: Eosinophil cationic protein (ECP) and eosinophil protein X (EPX) or eosinophil-derived neurotoxin (EDN) are released by eosinophil granulocytes in allergic diseases. Serum ECP (s-ECP) levels have been correlated with disease activity in atopic dermatitis (AD) in adults and young patients, and high urinary EPX (u-EPX) levels in asthmatic patients seem to reflect active disease. A relationship between AD severity and u-EPX concentration in young children has not been previously studied. OBJECTIVE: This study was performed to evaluate whether the severity of AD in infants and young children was correlated with s-ECP and u-EPX levels. METHODS: Fifty-four infants and children (mean age, 17.7 months; range, 4-48 months) with AD and without other allergic conditions were evaluated. The severity of AD was measured by using the SCORAD index. S-ECP, serum total IgE, serum-specific IgE for common allergens, and peripheral blood eosinophil counts (PBECs) were determined. In forty-two children u-EPX was also measured. Seven age-matched control patients underwent the same determinations. RESULTS: S-ECP and u-EPX were significantly higher in children with AD than in control children (mean, 23.9 vs 3.5 microg/dL [P <.001] and 57.7 vs 6.0 microg/mmol creatinine [P <.001]). A significant correlation was found between SCORAD and s-ECP (P =.002), u-EPX (P =.01), and PBECs (P =.01) and between symptom index and uEPX (P =.0004). PBECs were strongly correlated to s-ECP and u-EPX (P <.0001). However, 5 patients with moderate and severe AD (11.9%) showed low levels of s-ECP, u-EPX, and PBECs. CONCLUSION: S-ECP and u-EPX were useful markers of AD activity in infants and young children. When taken together, the two determinations could give more information about the clinical course of the illness. Some patients seemed to have clinical exacerbations without an involvement of eosinophils and their products.