1,3-Diacylglycero-2-phosphocholines--synthesis, aggregation behaviour and properties as inhibitors of phospholipase D.

A series of 1,3-diacylglycero-2-phosphocholines (1,3-PCs) with acyl chain lengths of C8-C18 were synthesised by chemical introduction of the phosphocholine moiety into the regioisomerically pure 1,3-diacylglycerols, which were obtained from glycerol and the vinyl esters of fatty acid by means of lipase from Rhizomucor mihei. The 1,3-PCs being regioisomers of the natural glycerophospholipids were studied with respect to their aggregation behaviour in the absence and in the presence of sodium dodecylsulfate (SDS) as well as their properties as substrates and inhibitors of phospholipase D (PLD) from cabbage. While the main structures of the pure 1,3-PCs were micelles (C8), liposomes (C10, C12) or planar bilayers (C14, C16, C18), the addition of SDS resulted in the formation of mixed micelles (C8, C10) and mixed liposomes (C12, C14, C16, C18). None of the 1,3-PCs was found to be hydrolysed by PLD, whereas all of them showed inhibitory properties in the standard assay for PLD. The inhibitory power was strongest with 1,3-didecanoylglycero-2-phosphocholine (IC50 = 43 microM).

Drug-induced immunological unresponsiveness: selective inhibition of T-cell 'helper function' by cyclophosphamide in mice pretreated with phytohaemagglutinin.

Studies involving the combined use of phytohaemagglutinin (PHA) and cyclophosphamide (CY) indicate that both agents can act together to produce immunological unresponsiveness: following injection of PHA into mice, splenic DNA synthetic responses [14C]thymidine incorporation) and haemolysin plaque formation against sheep red blood cells were determined in daily intervals. Both immunosuppression and DNA synthetic activity were maximally developed 5 days after treatment with PHA. Administration of CY at this time resulted in immunological unresponsiveness lasting for about 18 days. Antibody production could be completely restored with antigen-activated T cells (but not with B cells), thus indicating a selective inhibition of T-cell 'helper function' in mice treated with PHA and CY. This observation is consistent with the general assumption that cells involved in the response to PHA are predominantly T cells. Apparently, these cells are highly sensitive to an inactivation by CY after stimulation with PHA.

[Immunosuppression by means of cytostatic chemotherapy. Inhibition of antibody formation and induction of tolerance in animal experiments]. / Immunsuppression mit zytostatisch wirksamen Chemotherapeutika. Inhibition der Antikörper-Produktion und Toleranz-Induktion im Tierversuch.

Two different experimental models are presented for the induction of immunosuppression and immunological tolerance in mice with antineoplastic drugs: a) an unspecific T cell activation with a phytomitogen (phytohemagglutinin), or b) a partial synchronization of antigen-induced proliferation by mitotic blockade with vincristinsulfate prepare lymphoid tissue for an increased sensitivity to cyclophosphamide. With the first model a complete (antigen-nonspecific) inhibition of antibody production--mediated by an inactivation of T cells which act synergistically with antibody-forming cell precursors during the induction of the humoral immune response ("helper cells") -- has been demonstrated. The second model was shown to be suitable for the induction of immunotolerance, indicating a rather selective suppression of a clone of antigen-specific cells.

[Animal experimental studies on the increase in the humoral immune response caused by whole body irradiation]. / Tierexperimentelle Untersuchungen über die Steigerung der humoralen Immunantwort durch Ganzkörperbestrahlung.

The production of antibodies against heterologous erythrocytes (sheep erythrocytes) can be significantly increased in mice and rats by total body irradiation. This effect depends upon the radiation dose applied and can be demonstrated only in animals immunized first and then irradiated at intervals of six hours. In vitro assays show indirectly that the radiogenic immune stimulation is caused by the varying radiosensitivity of the individual cell populations involved in the humoral immune reaction, leading to a relatively selective inactivation of T-suppressor cells.

[Comparative research on cellular immunostimulation by whole-body irradiation]. / Vergleichende Untersuchungen über die zelluläre Immunstimulation durch Ganzkörperbestrahlung.

The effect of the cellular immune response by total body irradiation was investigated. The transplant survival (skin grafts) was determined as immune parameter. Donors were colony-bred Wistar rats and recipients were colony-bred Sprague-Dawley rats. The investigations were carried out with irradiated rats and with rats irradiated after thymectomy and/or adrenalectomy as well as with animals without irradiation. A single total-body irradiation (1 and 2 Gy) was administered. The skin graft survival in irradiated rats was significant shorter (radiogenic immunostimulation) than in unirradiated rats; there were no significant differences between the operated (thymectomy and/or adrenalectomy) and not operated animals. Including precedent examinations this radiogenic immunostimulation is caused by a relatively selective inactivation of T-suppressor cells.

[Pre-operative irradiation and tumor-specific immune response in hypernephroid renal carcinoma (author's transl)]. / Präoperative Bestrahlung und Tumorspezifische Immunantwort beim hypernephroiden Nierenkarzinom.

In vitro demonstration of a tumor-specific immune response was tried using the so-called leucoycte migration test in altogether 32 patients with a hypernephroid renal carcinoma (nontreated before operation or pre-irradiated). The principle of this technique consists in the release of a lymphocyte factor by bringing in contact sensitized lymphocytes with the concerned tumourous tissue, the lymphocyte factor inhibiting the migration capacity of granulocytes. With hypernephroid cancerous tissue from nontreated patients no reaction was obtained in 12 of 13 cases (no inhibition of migration), whereas a positive reaction (inhibition of leuococyte migration) was observed in 17 out of 19 patients with hypernephromas having been irradiated before surgical treatment. This radiation-induced effect on the immune response from hypernephroma and lymphocytes in patients with this neoplasm is specific, as no reaction of irradiated hypernephroid cancerous tissue with lymphocytes of sound donors or of patients with different malignant tumors could be shown in control tests, a significant inhibition of leucocyte migration failing to appear.

[Tumor-specific immune response of hypernephroid renal carcinoma after in vitro irradiation (author's transl)]. / Tumorspezifische Immunantwort nach "In-vitro-Bestrahlung" von hypernephroiden Nierenkarzinomen.

It is possible to reproduce in vitro the induction in the leucocyte migration test of an immune response specific to hypernephroid carcinoma by preoperative radiation treatment of this neoplasm. The leucocyte migration is inhibited by soluble extracts of the tumor, prepared from hypernephroma-tissues (of 11 patients nontreated preoperatively) after in vitro irradiation either with electrons or with 60Co. This effect depends on the dose. The radiation dose producing a maximal inhibition following in vitro irradiation is nearly equal to that delivered by us before surgical treatment.

[Comparative studies on the immunostimulation by whole-body irradiation or vincristine sulphate]. / Vergleichende Untersuchungen über die Immunstimulation durch Ganzkörperbestrahlung oder Vincristinsulfat.

Comparative experimentations were performed on Sprague-Dawley rats in order to study the immunostimulation by whole-body irradiation with 60Co gamma radiation (doses between 0.5 and 2 Gy) or application of vincristine sulphate (doses between 1.5 and 6 micrograms per 100 g body weight). The primary production of antibodies against erythrocytes of sheep was determined as immunity parameter. An immunostimulation was observed in all experiments. The irradiated groups showed the highest stimulation after 1 Gy, and the most stimulating dose of vincristine was 6 micrograms/100 g body weight. The radiogenic stimulation was more significant. As a reason for these stimulations, the authors suppose the different sensitivity of individual lymphocyte populations participating in the induction of the humoral immune response which leads to a relatively greater inactivation of T suppressor lymphocytes.

Immunoglobulin and T cell receptor gene rearrangements in lymphoproliferative disorders.

Thirty-one samples representing Hodgkin's and non-Hodgkin's lymphomas, angioimmunoblastic lymphadenopathy (AILD), and benign follicular hyperplasia in HIV infections were examined for rearrangements of the immunoglobulin (Ig) and T cell receptor (TcR) beta-chain gene loci. In 11 of 12 non-Hodgkin's lymphomas (classified as Burkitt lymphoma (2), centrocytic lymphoma (1), centrocytic-centroblastic lymphoma (5), centroblastic lymphoma (3], only rearranged Ig genes could be detected. The exceptional case was an unclassified high-grade lymphoma, which represented a rearrangement of the TcR beta-chain. We also examined DNA from lymphoid neoplasms in which the lineage of the malignant cell was still controversial. Rearrangement of the TcR could exclusively be demonstrated in all 3 cases of AILD. One Ig gene rearrangement and 4 TcR beta-chain rearrangements were found in 13 samples of Hodgkin's lymphomas (11 lymph nodes, 1 pleura effusion and 1 bone biopsy with proven infiltration). Examination of 3 cases of benign follicular hyperplasia in HIV infection represented one Ig rearrangement.

Phytohaemagglutinin-induced lymphocyte transformation and histologic findings in patients with chronic aggressive hepatitis before and during immunosuppressive therapy.

In vitro lymphocyte transformation (14C-thymidine incorporation rate following stimulation by PHA) was studied in 19 patients with histologically proven chronic aggressive hepatitis. In the majority of the cases measurements were performed prior to and during a combined (azathioprine/corticosteroid) immunosuppressive therapy. In comparison to normal controls, the majority of the patients showed a significantly reduced lymphocyte transformation before the treatment started. During the immunosuppressive therapy there was a normalization of PHA-responsiveness in most of the patients, or an improvement, at least. Bioptic control seems to be the most reliable parameter to prove a therapeutic effect, since the histologic criteria for a decrease of the inflammatory activity were the last to occur. After transient cessation of the therapy, in 4 out of 6 cases a decrease of lymphocytic PHA-responsiveness was developed again, paralleled by histologic activation of the process where biopsy could be performed. This observation favours a long-term therapy for at least several years.