Biomedical subjects
Publications and source records attributed to V Ghetie.
By the use of rabbit polymerized albumin labelled with fluorescein isothiocyanate, or coated on sheep red blood cells, specific receptors on rabbit liver cells were demonstrated. The possible biological role of these receptors is discussed.
Three methods for detection and quantitation of anti-albumin autoantibodies (AAA) in human sera have been developed based on their reaction with a new antigenic site of glutaraldehyde-polymerized human serum albumin (HSA). The first is a double diffusion technique (ID-HSA) were the sera to be tested are reacted with a copolymer of glutaraldehyde activated HSA (copoly-HSA). The reaction is estimated by the lowest copoly HSA concentration giving precipitation with the undiluted sera. The second method is based on agglutination (HA) by AAA positive sera of human red blood cells coated with glutaraldehyde-polymerized HSA (EPA). The reaction is quantified as the highest dilution of sera giving agglutination. The third method uses glutaraldehyde insolubilized HSA as an immunoadorbent able to bind AAA in positive sera. The quantity of protein adsorbed, expessed in microng antibody/ml serum, measures the level of AAA in sera. Significant correlation between the values of AAA as determined by all three methods presented above were recorded.
Mouse spleen lymphocytes treated with rabbit IgG anti-sheep erythrocytes (SRBC) complexed with protein A of Staphylococcus aureus (SpA) form rosettes with SRBC. The attachment of SRBC to lymphocytes was due to the binding of the SpA-IgG antibody complex to the surface of the lymphocytes and was thus considered "arming" of the cells. Normal mouse spleen cells "armed" with SpA-rabbit IgG anti-chicken erythrocytes (CRBC) kill specifically 51Cr-labeled CRBC "in vitro" in the absence of free antibodies. The killing by these "armed" cells is an effect of the cell-bound SpA-IgG antibody complex. Both the SRBC rosette formation and the cell-mediated CRBC killing was dependent on the concentration of the SpA-IgG antibody complexes used for "arming" the cells. A 100-fold increase in rosette formation or in killing of target cells was recorded for lymphocytes treated with SpA-IgG antibody complexes in comparison with cells treated with noncomplexed IgG antibodies. The specific binding of SpA-IgG antibody complexes to the Fc receptors of mouse spleen cells was demonstrated by inhibition studies. More than 60% inhibition of the rosette formation and in the killing of target cells was shown for cells treated with normal rabbit IgG or its Fc fragment before addition of the SpA-IgG antibody complex.
A new rosette technique for identification of Fc-receptor-bearing cells is based on the ability of sheep erythrocytes coated with protein A of Staphylococcus aureus (ES) to form rosettes with cells treated with monomeric IgG or aggregated IgG. The IgG is attached to lymphocytes through its Fc region and binds to a trypsin-resistant but pronase-sensitive receptor (considered an Fc receptor). The ES rosette technique facilitates studies of the interaction of IgG with Fc receptor sites; the binding of any IgG preparation reacting with protein A of Staphylococcus aureus (SpA) can be studied by the technique. Inhibition of rosette formation by SpA was used for quantitation of IgG fixed to the cell surface (that is, the number of Fc receptors/cell). The sensitivity of the method permits quantitation of less than 10(5) IgG molecules bound to the Fc receptors. The relative affinity constant between Fc receptors and IgG ligands was estimated by plotting the percentage of ES rosettes as a function of IgG concentration and calculating the reciprocal of the IgG concentration giving half the maximal number of ES rosettes.
A human lymphoid cell ine (S95) with surface-located IgG is described. The line was derived from a patient with Hodgkin's disease and is aneuploid. Its characteristics are suggestive of a malignant B lymphocyte derivation, but no ultimate proof of malignancy can be forwarded. The line produces immunoglobulin (IgG) which appears to be devoid of light chain. The amount of IgG detectable at the cell surface by inhibition of the rosette formation by protein A-coated sheep red blood cells with protein A was 5-10 ng/10(6) cells corresponding to 70 000 - 140 000 molecules/cell. The immunoglobulin is membrane bound. Partial characterization of the IgG-like protein shows that it has a molecular weight of 50 000 Daltons and that the molecule lacks some of the normal gamma-chain determinants.
Staphylococcal protein A (SpA) labeled with FITC was used to detect IgG antibodies to tissue and cell surface antigens. Radioiodinated SpA was employed in a radioimmuno technique to assay IgG antibodies which had reacted with cell membrane antigens. Red blood cells coated with SpA were used as an indicator system in the mixed haemadsorption technique to detect IgG antibodies on tissue culture cells. The results showed that SpA was highly specific in the various systems tested, although it was not as sensitive in the immunofluorescence test as FITC anti-IgG. It was confirmed that SpA reacts with IgG antibodies of human, monkey, rabbit, sheep and mice origin. The reactivity with human and monkey IgG was stronger than with IgG of the other species. We consider labeled SpA as an alternative to monospecific anti-IgG for detection and assaying of IgG antibodies to tissue and cell surface antigens.
A method for cell separation according to cell surface markers is described. Heat-aggregated human IgG or IgG antibodies, raised to surface markers of mouse lymphocytes-for example, Ig or theta antigen-are allowed to react with the lymphocytes. When these cells are incubated with sheep erythrocytes coated with protein A of Staphylococcus aureus, rosettes are formed. These rosettes can be separated from nonrosetted lymphocytes by density gradient centrifugation. The erythrocytes of the rosettes are disrupted by complement action or by osmotic shock, and the lymphocytes are recovered. Immunoglobulin-protein A complexes are shed off the cell surfaces by cultivation. In this way cells with, as well as those without, surface markers could be obtained with high purity (up to 80%) and with high viability.
A technique was developed for the isolation of lymphocytes according to their surface antigenic markers. The method was based on the general reaction of protein A (SpA from S. aureus) with the Fc-part of an IgG antibody. Monolayers of S. aureus or Spa-coated sheep red blood cells (SpA-SRBC) fix antibody-charged cells specifically; non-fixed cells are easily removed by washing. Alternatively, the monolayers can be treated with a cell surface specific antibody prior to addition of non-charged cells. Monolayer-adhered cells were detached by the addition of lysostaphin (bacterial monolayer) or ammonium chloride (SpA-SRBC monolayer). As an example, Ig-bearing cells were isolated from mouse spleen lymphocytes using a specific rabbit anti-mouse Ig serum for charging either the cells or the monolayers. The recovery of Ig-bearing cells was approximately 84%. The purity of the cells was approximately 83% and the viability 89%. Antibody-SpA complexes on the surface of isolated cells were removed either by trypsin treatment or by cultivation. Ig on the cell surface is restored after 16 hr of cultivation.
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A cell separation technique was designed based on the interaction between cell-surface-bound IgG and protein A of Staphylococcus aureus. The density of lymphoid cells coated with IgG antibodies against one of the surface markers was increased by adherence of staphylococci. Cells with adhering bacteria were separated from cells without bacteria by density gradient centrifugation in 11.5% sodium metrizoate. Bacteria were removed from the lymphoid cells by lysostaphin digestion. The purity of separated cells was approximately 95% even when the proportion of a specific cell population was below 10% in the initial mixture. The viability and the ability of cells to multiply in vitro were not significantly impaired by the fractionating procedure. The technique can generally be applied for cell separation, provided antibodies of the IgG class against specific surface markers are available.
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We have previously reported that after in vitro treatment with deglycosylated ricin A chain (dgRTA), human umbilical vein endothelial cells (HUVECs) undergo changes in morphology including cell rounding and disruption of monolayers. The present studies were carried out to determine whether these changes were related to the disruptions in endothelial cell (EC) interactions with the extracellular matrix. To this end, we examined the effect of dgRTA on HUVECs in the presence of fibronectin (Fn), an extracellular matrix protein, which plays a role in the maintenance of vascular integrity. The addition of exogenous Fn greatly inhibited dgRTA-mediated morphological changes in HUVEC monolayers, dgRTA-mediated inhibition of [3H]thymidine incorporation in HUVECs and the binding of 125I-dgRTA to HUVECs. Should the same phenomenon occur with RTA-based immunotoxins (ITs) in vivo, this might shed light on the development of dgRTA-mediated vascular leak syndrome (VLS) during IT therapy and provide new insights into how to decrease this toxicity in patients.