Melvin Madsen

A methodological study of E-rosette formation using AET-treated sheep red blood cells

The influence of some of the well knwon technical variables on the E-rosette technique was examined using sheep red blood cells (SRBC) treated with 2-aminoethylisothiouronium bromide (AET). With AET treatment, E-rosette formation becomes less dependent on time and temperature and on the presence of serum. The mechanical stability of the rosettes is enhanced, and the number of SRBC attached to each rosette-forming lymphocyte (RFC) is markedly increased, leading to a sharper distinction between RFC and non-RFC. Ultimately, significantly more E-receptor carrying lymphocytes become detectable. Evidence is provided that the specificity of the E-rosette technique is unchanged after AET treatment of SRBC, in spite of the enhanced binding. A simple and reliable method for E-RFC identification is documented.

Isolation of human T and B lymphocytes by E-rosette gradient centrifugation. Characterization of the isolated subpopulations

Some methodological aspects of E-rosette gradient centrifugation for separation of human B and T lymphocytes are described. Treatment of sheep red blood cells (SRBC) with 2-aminoethylisothiouronium bromide (AET) accelerates and enhances E-rosette formation providing an effective and time saving method which takes less than 2 h to perform. Depletion of E-rosette forming cells (E-RFC) is almost complete, leaving less than 1% of the initial E-RFC at the interphase layer. By use of the lymphocyte donors autologous serum containing naturally occurring anti-SRBC antibody and complement the SRBC in the rosetted T lymphocyte fraction are easily and completely lysed without damage to the lymphocytes. This procedure is shown to have little effect on the properties of the isolated T cells. The isolated lymphocyte subpopulations have been characterized by tests for lymphocyte and monocyte markers.

Lymphocyte subpopulations in man: suppression of PWM-induced B-cell proliferation by infectious mononucleosis T cells.

The in vitro polyclonal pokewced milogen (PWM)‐induced activation of human B lymphocytes is enhanced by addition of autologous or allogeneic: irrudiated T cells. This model for B/T‐cell cooperation may he used tu define and describe the balance between T helper and T suppressor phenomena. The present study investigates the helper and suppressor capacities of mononuclear cells isolated from peripherial blood of infectious mononucleosis patients during the acute disease and the reconvalescence period. During the acute disease, we found a functional lack of T helper capacity; furthermore, the T cells were able to suppress the PWM and T‐cell‐dependent B‐cell proliferation of healthy donor cells. The suppression was non‐cytotoxic: i.e. not due to destruction of the responder cells. This phenomenon of non‐cytotoxic suppression was found for all seven patients studied and disappeared during the reconvalescence period, indicating that the T lympliocyltosis seen in infectious mononucleosis includes an expansion of T suppressor cells.

Lymphocyte Subpopulations in Man: B-Cell Stimulation Induced by Pokeweed Mitogen and Irradiated T Cells

The enhanced stimulasion of human B lymphocytes by pokeweed mitogen in the presence of irradiated T helper lymphocytes has been studied, revealing that the proliferative responses measured hy incorporation of thymidine in cultures of B lymphocytes and irradiated T lymphocytes in a 1:2 or 1:4 ratio is mosily a function of the B cells. Only a minimal number, if any, of the T cells contaminating the B cell suspensions is stimulated to proliferation. This is in contrast to stimulation ofihe B‐cell suspensions without addition of irradiated T cells, where both B cells and T cells proliferate. The irradiated T helper cells have no FcR for antigen‐bound IgG, and as well allogeneic as aucologous T ceils exhibit helper capacity of equal strength. The test system described makes it possible selectively to lest one B‐cell function and the corresponding T helper capacity.

Activation of human alloreactive cytotoxic precursor T lymphocytes

The requirements for allogeneic T-cell activation have been studied in experiments with T and/or B cells as stimulator. Although target determinants (TDs, defined by CTL effectors in CML) are present on B and T cells used as target cells, this study indicates that TDs are functionally different when expressed on B and T cells used as stimulator cells, as only B cells can activate CTL precursors. Further, the study confirms that inducing TDs and strong lymphocyte-activating determinants (LADs, defined by proliferation in MLC) can be distinct structures found on two different stimulator B cells. The study suggests that binding of cytotoxic precursor T cells to TDs per se does not allow any detectable activation or start of proliferation and differentiation but requires another function of the stimulator cells in the non-T-cell compartment. The nature of this function is unknown, but it is the background for the first signal received by the TD-specific clones of CTL precursors, resulting in the expression of growth receptors for T-cell growth factor or interleukin 2 which is the second signal necessary for clonal expansion and differentiation.

Modeling surface imperfections in thin films and nanostructured surfaces

Accurate scatterometry and ellipsometry characterization of non-perfect thin films and nanostructured surfaces are challenging. Imperfections like surface roughness make the associated modelling and inverse problem solution difficult due to the lack of knowledge about the imperfection on the surface. Combining measurement data from several instruments increases the knowledge of non-perfect surfaces. In this paper we investigate how to incorporate this knowledge of surface imperfection into inverse methods used in scatterometry and ellipsometry using the Rigorous Coupled Wave Analysis. Three classes of imperfections are examined. The imperfections are introduced as periodic structures with a super cell periods ten times larger than the simple grating period. Two classes of imperfections concern the grating and one class concern the substrate. It is shown that imperfections of a few nanometers can severely change the reflective response on silicon gratings. Inverse scatterometry analyses of gratings with imperfection using simulated data with white noise have been performed. The results show that scatterometry is a robust technology that is able to characterize grating imperfections provided that the imperfection class is known.