Leucocyte depletion of neocyte-rich red blood cell concentrates by use of immunomagnetic beads.

In a series of pilot experiments we depleted neocyte-rich red blood cell concentrates of contaminating leucocytes by use of immunomagnetic beads. The beads were coated with monoclonal antibodies directed against the leucocyte common antigen CD 45. The number of cells isolated depended on the dose of beads which also corresponded to the decrease in leucocytes in the neocyte concentrates. The required ratio of beads to target cells was approximately 25:1, the depletion rate of leucocytes was about 96.0-99.8%.

A comparison of a solid phase IRC assay and the PSIFT for detection of antibodies to platelets.

A rapid solid phase indicator red cells assay (IRCA) for detection of platelet antibodies was developed and its sensitivity compared with PSIFT. Platelets were attached to the surface of polystyrene microtitre plate wells by means of a sodium carbonate buffer and centrifugation. Uncovered areas were blocked by a gelatin blocking buffer. After serum incubation bound platelet-specific antibodies were made visible by anti-IgG-coated indicator red cells and a brief centrifugation. A positive result, meaning the presence of an anti-platelet antibody was indicated by red cell adherence over the reaction surface. In the absence of serum antibodies to platelets the indicator red cells formed a pellet. The IRCA showed a high sensitivity; the anti-platelet antibody Thrombocyte was detectable until a dilution of 1:1,600 whereas the same antibody in the PSIFT could only be detected until a dilution of 1:400.

SLP assay: a rapid assay for detection of red blood cell antibodies in the serology of pregnancies.

A rapid manual test for the detection of red cell antibodies called SLP assay has been developed and compared with the sensitivity of the antiglobulin assay. Acid-soluble proteins (SLP) from human leukocytes cause aggregation of human red blood cells. SLP represents a group of proteins consisting of 5 fractions of different positively charged macromolecules, which are able to reduce the negative charge of the red cells. Reduction of the negative charge results in a nonspecific hemagglutination of different strengths, depending on the SLP fractions used. This hemagglutination can be reversed by neutralizing the SLP with heparin. In the case of blood group-antibody-mediated aggregation the hemagglutination is nonreversible despite neutralization with heparin and remains stable for several hours. Because of the high sensitivity of the SLP assay all blood group antibodies from the IgM type as well as from the IgG type are detectable even in low concentrations. The sensitivity of the SLP assay is comparable to the antiglobulin assay.

Serum soluble interleukin-2 receptor levels of normal blood donors.

Following T cell activation a part of the IL-2-receptor (sIL-2-R) was released from the T cell membrane. Elevated serum levels of sIL-2-R were observed in some pathological conditions. The serum of healthy blood donors (n = 228) was investigated by ELISA to see whether a correlation existed in normal donors between the age or the sex of the donor and the sIL-2-R. The serum levels found were in the range between 5 and 398 pM. No correlation was found between the different ages or the sex of the donors.

[A new procedure for lymphocyte depletion of thrombocyte concentrates with immunomagnetic beads]. / Neues Verfahren zur Lymphozytendepletion von Thrombozytenkonzentraten durch immunmagnetische Beads.

In a series of pilot experiments we depleted platelet concentrates from contaminating leukocytes by use of immunmagnetic beads. The beads were coated with monoclonal antibodies directed against the leukocyte/lymphocyte antigens CD 2, CD 19 or CD 45. The amount of isolated cells depended on the dose of the used beads and was analogous to the observed decrease of leukocytes in platelet concentrates. The total amount of necessary beads for a complete leukocyte depletion depends on the grade of leukocyte contamination, which shows a high variation among different platelet preparations.

Immunomodulatory action of eicosanoids and other small molecular weight products of macrophages.

There is an increasing body of evidence that T cell-mediated immune response is regulated by a variety of small molecular weight products of macrophages. One of the best known immunoregulatory products in this context is prostaglandin E2 (PGE2) which is known to regulate both T cell and macrophage functions. Recently, ornithine, cysteine, and lactate have also been recognized as immunoregulatory mediators. In analogy to the hormone-like cytokines and lymphokines, all these substances are produced by immunologically relevant cells (macrophages) at a variable and regulated rate; and they have been shown to regulate the functional activities of other cells (T cells and macrophages). This brief review describes the key observations that underscore the important regulatory role of these metabolites in physiological and pathological conditions.

A simple and safe method for single HLA-antigen-typing by a solid phase assay.

A rapid solid phase assay for detection of single HLA-antigens on platelets was developed. The platelets were attached to the surface of polystyrene microtitre plate wells by means of a sodium carbonate buffer and centrifugation. Uncovered areas were blocked by a gelatin blocking buffer. After incubation with commercially available anti-HLA-sera the bound anti-HLA-specific antibodies directed against HLA-antigens present on the platelets were made visible by anti-IgG-coated indicator red cells and a brief centrifugation. A positive result, meaning the presence of an HLA-antigen, was indicated by a slight red cell adherence over the reaction surface. In the absence of the HLA-antigen no binding occurred and the indicator red cells formed a small red disc-like pellet.

Use of stroma for the detection of blood-group antibodies by ELISA.

A micro enzyme-linked immunosorbent assay (ELISA), using lyophilized stroma as carrier of red blood cell antigens, which stay stable longer than usual, using intact erythrocytes, was developed for determination of blood-group antibodies in the AB0, Kell and Lewis-systems. Stroma being fixed on microtiter plates was incubated with antisera and peroxidase-conjugated anti-human globulin. The transformation of the substrate added was determined photometrically. A binding of antibodies to the stroma could be demonstrated up to an antibody dilution of 1:1024 for the ABO-system, of 1:512 for the Kell-system and of 1:64 for the Lewis-system. By standardization of this method the quantitative determination of antibodies becomes possible without being restricted by the limited stability of intact erythrocytes.

Correlation of immunogenicity and production of ornithine by peritoneal macrophages.

The release of ornithine by macrophages and its correlation with their immunogenicity after treatment with various macrophage-stimulating substances were analyzed. Pristane-elicited peritoneal macrophages (PM) were found to express strong arginase activity and to release L-ornithine into the extracellular space. This activity is strongly reduced within 3 hr after treatment with tetradecanoylphorbol acetate (TPA) but not with lipopolysaccharide (LPS). Resident PM usually express little arginase activity, but this activity is markedly augmented within 24 or 48 hr after treatment with LPS. The release of ornithine by peritoneal cells (PC) (60 to 90% macrophages) was found to be correlated with their immunogenicity as determined by the in vivo immunization for a subsequent in vitro secondary cytotoxic response against minor H antigens. The immunogenicity of pristane-elicited PC is markedly stronger than that of resident PC or TPA-treated, pristane-elicited PC. Moreover, the immunogenicity of the resident PC and TPA-treated elicited PC is substantially augmented by the simultaneous injection of ornithine, whereas the immunogenicity of the untreated elicited PC is not further augmented by exogenous ornithine, indicating that the endogenous production of ornithine by the stimulating cells had a strong influence on the resulting immune response. Injection of glutathione into pristane-treated mice also reduces the ornithine production and immunogenicity of the resulting peritoneal exudate cells. The immunogenicity in this case is at least partly reconstituted by application of exogenous ornithine. Our experiments revealed no correlation between the production of ornithine and prostaglandin E2. Prostaglandin E2 production of resident and pristane-elicited PC is not markedly different and is in either case strongly augmented by TPA. Elicited or resident PM which have been incubated for several days in culture release practically no ornithine; but ornithine production can be induced again by incubation for 24 hr with LPS and to some extent also with interferon-gamma.

Putrescine and its biosynthetic precursor L-ornithine augment the in vivo immunization against minor histocompatibility antigens and syngeneic tumor cells.

Putrescine was found to augment strongly the in vivo priming for secondary in vitro cytotoxic responses by small numbers of syngeneic ESb-D tumor cells and MHC-compatible allogeneic cells (minor H-antigens). The cytotoxic response against minor H-antigens in putrescine-treated mice showed the typical MHC-restriction that has previously been observed after immunization with higher cell doses without putrescine. The injection of putrescine had practically no effect on the subsequent in vitro primary cytotoxic response against an unrelated set of allogeneic stimulator cells. A similar augmentation of the in vivo immunization for secondary in vitro responses was achieved with L-ornithine, the biosynthetic precursor of putrescine. A substantial secondary in vivo cytotoxic activity against minor H-antigens was also obtained by application of L-ornithine shortly after the primary immunization.

Antagonistic regulation of cytotoxic T lymphocyte activation by prostaglandin E2 and L-ornithine.

Two macrophage-mediated immunoregulatory substances, prostaglandin E2 and L-ornithine, were found to mediate antagonistic effects. Both substances were previously shown to inhibit the activation of cytotoxic T lymphocytes under certain conditions. Our experiments now show that high doses of L-ornithine counteract the inhibitory effect of PGE2, and that PGE2 counteracts the inhibitory effect of high doses of L-ornithine; i.e. both substances can also augment cytotoxic responses in the presence of high concentrations of the other component. The direction of the immune regulatory effects of these substances is, therefore, expected to depend on the endogenous levels of PGE2 and L-ornithine in the individual animal at the site of immunization.

Suppression of cytotoxic T lymphocyte activation by L-ornithine.

The effect of L-ornithine on several types of immune reactions was analyzed. L-ornithine was found to suppress the activation of cytotoxic T lymphocytes (CTL) in vivo and in vitro. This suppressive effect was not observed with the structural analogues D-ornithine, L-lysine, or putrescine or with the amino acids L-histidine or L-alanine. The concentration of 9 X 10(-3) M L-ornithine was found to mediate a practically complete suppression of the cytotoxic response in vitro if applied on day 0 or day 1 of the culture, but a comparably weak suppression if applied on day 3. The same concentration of L-ornithine had no effect on the production of the lymphokines interleukin 2 (IL 2) and gamma-interferon (IFN-gamma). This concentration of ornithine had also no substantial effect on several types of proliferative responses, including the allogeneic mixed lymphocyte reaction, the concanavalin A-activated IL 2-dependent proliferation of thymocytes, and IL 2-dependent proliferation of the T cell clone W-2. These observations suggest that L-ornithine inhibits selectively the differentiation of CTL effector cells. By the criteria tested, the immunosuppressive effect of L-ornithine is more selective than that of cyclosporine A, which was previously found to suppress not only the activation of cytotoxic activity but also proliferative responses and the production of the lymphokines IL 2 and IFN-gamma.

Augmentation of cytotoxic responses by prostaglandin E2.

The cytotoxic-T-lymphocyte activity in the spleen cells after in vivo immunization of C3H mice with allogeneic spleen cells ip was consistently very weak. Substantial cytotoxic responses were obtained, however, when prostaglandins (PGE2, PGE1, or PGI2) were injected ip together with or prior to the immunization. An augmentation of cytotoxic responses against allogeneic stimulator cells was also observed in mixed lymphocyte cultures which were provided with an interleukin 2-containing helper factor. This augmentation was observed when PGE2 was added at the start of the culture but not if added 1 day later. Indomethacin was found to be suppressive in these cultures.