H.-D. Flad

Discontinuous density gradient separation of human minonuclear leucocytes using percoll® as gradient medium

The use of a new commerically available medium (Percoll) for fractionation of human mononuclear leucocytes is described. Cells can be fractionated on the basis of their densities with high reproducibility. The separated cells were characterized by morphological and functional criteria. Monocytes can be obtained in the low density fractions with a purity of 70%--90%. Lymphocytes were found in high density fractions with a purity up to 99%. No separation between E-rosette forming (E-RFC) and surface immunoglobulin-bearing lymphocytes was obtained. However, a reduced number of high avidity E-rosette forming lymphocytes (HAE-RFC) was found within low density lymphocytes. Best spontaneous DNA synthesis and reaction in mixed leucocyte culture (MLC) were obtained with cells isolated from the 1.066/1.068 density interface, whereas the stimulation with phytohemagglutinin (PHA) or pokeweed mitogen (PWM) had a peak response with cells from the 1.064/1.066 density interface. Colony forming myelopoietic stem cells and colony forming T-lymphocytes were detected in fractions of low density.

BACILLUS-CALMETTE-GUERIN (BCG) AND 3D TUMORS: AN IN VITRO MODEL FOR THE STUDY OF ADHESION AND INVASION

PURPOSEnTo study adhesion, penetration and internalization of BCG and effector-cells to and into three-dimensional in vitro cell aggregates from benign and malignant urothelial origin mimicking small in vitro tumors.nnnMATERIALS AND METHODSnMulticellular spheroids (MCS) were generated by liquid-overlay technique. Adhesion and penetration of viable FITC-labelled BCG into MCS from urothelial cancer cell lines and normal urothelial cells was studied by electron microscopy (TEM) and fluorescence microscopy. Spheroid growth during BCG-co-incubation was determined by light microscopy. Peripheral blood mononuclear cells (PBMC) were stimulated with BCG to generate BCG-activated-killer (BAK) cells. The infiltration of these effectors and of lymphokine-activated killer (LAK) cells into MCS was examined at different intervals by means of immunohistochemistry. The resulting cytotoxicity was judged in a 3H-l-methionine release assay.nnnRESULTSnBCG adhered to MCS from tumor cells but not to benign cell MCS. Intracellular internalization of the bacteria was detectable in superficial tumor cell-layers (1-5) whereas BCG was not found in deeper layers. Proliferation of malignant MCS was reduced in the presence of BCG. Benign MCS showed contact inhibition growth arrest, which was not altered by BCG. BAK and LAK effector cells both infiltrated tumor cell MCS as opposed to unstimulated PBMC. In contrast to LAK cells, BAK cells did not infiltrate into benign cell MCS and were not cytotoxic towards them.nnnCONCLUSIONnWith regard to the clinical situation the selective adhesion and internalization of BCG to malignant cells might explain why BCG has been rarely found in follow-up biopsies in tumor free patients. More interestingly, the selective adhesion of BCG to and infiltration of BAK effector cells into malignant cell spheroids suggests a selective mode of action of BCG.

Bone marrow transplantation for aplastic anaemia from a HL-A and MLC-identical unrelated donor

SummaryBone-marrow transplantation (BMT) from an unrelated, HL-A-phenotype-identical, MLC-negative donor was performed in a 31 year old woman with severe longlasting aplastic anemia. In vitro assays failed to demonstrate humoral or cellular sensitization of the recipient against donor-type antigens. Following conditioning with cyclophosphamide, prompt but only transient engraftment of the transplant occurred accompanied by signs of mild graft-versus-host-disease (GVHD) of the liver. The results of a second bone marrow transplantation from the same donor cannot be evaluated due to early death of the recipient.It is concluded that bone marrow from unrelated, HL-A- and MLC-identical donors may engraft without severe GVHD. Rejection of the graft in our patient may have been related to greater antigenic differences that can be expected to exist between HL-A-and MLC-identical unrelated individuals than between HL-A-and MLC-identical siblings. However, insufficient preparative immunosuppression with cyclophosphamide due to severe hepatic hemosiderosis appears equally likely as the cause of graft rejection. The possibly increased risk of graft rejection or severe GVHD should not preclude the use of unrelated HL-A-and MLC-identical marrow donors, when histocompatible sibling donors are not available; but more potent immunosuppressive regimens than the cyclophosphamide protocol may be necessary to ensure permanent engraftment.ZusammenfassungBei einer 31 jährigen Patientin wurde wegen schwerer progredienter Panmyelopathie eine Knochenmarktransplantation von einer nichtverwandten, HL-A phänotypisch identischen, MLC-negativen Spenderin durchgeführt. Humorale oder zelluläre Sensibilisierung der Empfängerin gegen zellgebundene Antigene der Spenderin war in vitro nicht nachweisbar. Nach Konditionierung mit Cyclophosphamid waren zeitgerechtes, jedoch nur vorübergehendes Angehen des Transplantates und Symptome einer leichten Transplantat-gegen-Wirt-Reaktion (GVHD) zu beobachten. Der Ausgang einer Retransplantation von Knochenmark derselben Spenderin ist wegen des frühen Todes der Patientin nicht zu beurteilen.Nach Konditionierung mit Cyclophosphamid kann das Knochenmark eines nicht verwandten, HL-A-und MLC-identischen Spenders angehen, ohne daß eine schwere GVHD-Reaktion auftritt. Ob die Abstoßung des Transplantates bei unserer Patientin darauf zurückzuführen ist, daß zwischen Empfänger und HL-A-und MLC-identischem, nichtverwandtem Spender größere, in vitro nicht erkennbare Antigendifferenzen bestehen, als zwischen HL-A-und MLC-identischen Geschwistern, kann nicht entschieden werden. Ungenügende Immunsuppression durch Cyclophosphamid als Folge einer schweren Hämosiderose der Leber mag ebenso zur Transplantatabstoßung geführt haben. Das möglicherweise höhere Risiko einer Transplantatabstoßung oder einer schweren GVHD-Reaktion sollte nicht von der Transplantation des Knochenmarks eines nichtverwandten, HL-A-und MLC-identischen Spenders abhalten, wenn histokompatible Geschwister nicht zur Verfügung stehen; jedoch mag dann stärkere Immunsuppression erforderlich sein, als sie mit Cyclophosphamid zu erzielen ist, um ein permanentes Angehen des transplantierten Knochenmarks zu gewährleisten.

B and T lymphocyte colony formation in agar: 2-Mercaptoethanol-activated albumin can substitute for 2-mercaptoethanol

Abstract The effect of 2-mercaptoethanol (2-ME) activated albumin (MaSF) on mouse B lymphocyte colony (BLC) formation and on human phytohemagglutinin (PHA)-induced T lymphocyte colony formation (TLC) formation in semisolid agar medium was studied. MaSF was found to stimulate colony formation comparable to the stimulation obtained with 2-ME. MaSF could not substitute for serum in any of the agar culture systems. BLC formation stimulated by MaSF was obtained with spleen cells from athymic nude mice and nonadherent spleen and lymph node cells of normal mice suggesting a direct effect of MaSF on the colony-forming B cell without interference with T cells or macrophages. The results suggest that the stimulatory effect of 2-ME in the BLC and TLC agar systems is mediated by 2-ME-activated albumin present in the culture medium.

Adjuvant and suppressor activity of the polycation protamine hydrochloride in the primary immune response of mice.

The effect of protamine hydrochloride (PH), a polycation, on the primary immune response of mice to sheep red blood cells (SRBC) was studied. Like other adjuvants, PH enhances or depresses the immune response depending on the time of injection of PH in relation to the antigenic challenge. The suppressive effect of PH correlated with the appearance of activated macrophages in the peritoneal cavity. A second injection of PH together with antigen abolished this suppressive effect. Transfer of activated macrophages resulted in an enhanced immune response when antigen was administered at the time of transfer, and in a depressed response when the antigen was given 1 or 2 days after transfer. It is concluded that the duration of the influence of activated macrophages on T or B cells determines the subsequent immune reactivity. It is discussed that the basic mechanism of action of PH is its ability to release lysosomal enzymes from macrophages. The mechanism by which these enzymes amplify the immune response remains to be clarified.

T-lymphocyte colony formation of murine spleen cells in a one-stage micro agar culture

A one-stage agar culture technique for stimulation of T-lymphocyte colony-forming units (TL-CFU) derived from murine spleen cells is described. The cultures are in glass capillaries in a volume of 30 microliter. Both, phytohemagglutinin (PHA) and concanavalin A (Con A) stimulate growth of TL-CFU. The technique does not require prestimulation of lymphocytes in liquid culture or addition of growth factor(s). The most important requirements for optimal colony growth are a low concentration of agar (less than 0.1%) and the appropriate number of seeded cells (60 X 10(3) per culture). This one-stage micro agar culture is economical and easily handled. Many cultures can be prepared and rapidly evaluated. The plating efficiency is about 10 colonies per 10(4) nucleated spleen cells. The T-cells character of the TL-CFU was demonstrated by treatment of the spleen cells with a monoclonal anti-Thy-1.2 antibody (anti-Thy-1.2) and complement before assaying for colony growth and also by staining the cells in the cultures with anti-Thy-1.2-FITC. Growth characteristics of TL-CFU indicate that cell interactions are operative during activation and proliferation. The nature of the cells cooperating in this system is discussed.

Suppression of in vitro primary immune response by L1210 cells and their culture supernatant: Evidence for cytotoxic effects

Abstract L1210 cells and their culture supernatants were found to inhibit the generation of PFC in the in vitro primary immune response of spleen cells to SRBC. As few as 1% of L1210 cells and 1% of culture fluid were inhibitory. Inhibition of DNA or protein synthesis of L1210 cells did not abolish their immunosuppressive activity, excluding exhaustion of culture medium as a possible mechanism of inhibition of PFC. Heating of the supernatant completely abrogated the suppressive effect and resulted in a marked increase of PFC. Daily evaluation of cell viability in the cultures revealed that, in the presence of L1210 and supernatants, the fraction of surviving cells is markedly reduced. We conclude that a direct cytotoxic effect on splenic lymphocytes and macrophages is the predominant immunosuppressive mechanism of L1210 cells and their culture supernatants.

Promotion of L1210 tumour growth by macrophages.

Low numbers (10(4)) of peritoneal exudate L1210 mouse lymphoma cells were injected into DBA/2 mice subcutaneously and the development of tumours was followed. Tumour takes occurred in 100% of the animals within 9 days after tumour transplantation. The latent period of tumour development was prolonged by 6-10 days when tumour cells of the peritoneal exudate, depleted of adherent/phagocytic cells, were used in the inoculum or when tumour cells derived from continuous cell cultures were used. Addition of adherent cells in high numbers to in-vitro-derived L1210 cells accelerated tumour growth. This effect was found to be not specific for adherent/phagocytic cells, as liver cells had the same influence on tumour growth. It is concluded that, under certain experimental conditions, a cell population with the functional properties of macrophages is able to promote tumour development, most likely due to their non-specific effect on the micro-environment of the growing tumour.