Limiting dilution assays Experimental design and statistical analysis

• Blood transfusion (BT) is reported to cause immunosuppression. We postulated that BT might therefore adversely affect the prognosis of carcinomas of the colon and rectum. We analyzed the overall and recurrence-free survival of 366 patients whose colorectal carcinomas were resected. The 199 patients who received perioperative BTs appeared to survive less well than the 167 patients who received no blood products. However, Cox analysis, which adjusts for other prognostic variables, shows that the difference was not statistically significant. Although 43% of transfused patients survived, as compared with 56.5% of nontransfused patients, this difference was due to variables other than transfusion. This study did not support the hypothesis that BT has an adverse effect on survival of patients with colorectal cancer. ( Arch Surg 1985;120:734-738)

Perioperative Allogeneic Blood Transfusions: Survival in Patients With Resected Carcinomas of the Colon and Rectum

Expression of Ia molecules by astrocytes during acute experimental allergic encephalomyelitis in the Lewis rat

IL-1α stimulated TNFα production by cultured human proximal tubular epithelial cells

Two computer programs are presented which can be used for the determination of the design and the statistical evaluation of data in the context of limiting and serial dilution analysis. The first program (DESIGN) gives the experimenter the opportunity to set up different designs, to improve a design according to several suggestions, to make a picture of a design and to evaluate the results of artificial data, obtained from a random experiment corresponding with the chosen design, until (s)he is convinced of having a suitable design for a particular experiment. The second program (EVALUATE) evaluates the experimental data. The design method and the statistical methods used in the programs have been chosen on practical and theoretical grounds.

Computer aided design and evaluation of limiting and serial dilution experiments

Analysis of buoyant density of canine peripheral blood leukocytes with PVP-Silica (Percoll) density gradients.

Data are presented on the ability of arterial and venous endothelial cells to stimulate allogeneic leukocytes. Mixed cultures of allogeneic endothelium and lymphocytes result in proliferation of lymphocytes and generation of cell-mediated cytotoxicity, which do not occur in cultures of syngeneic combinations of endothelium and lymphocytes. Studies of kinetics showed a peak in proliferation at days 6-7. The optimal responder-stimulator ratio appeared to be 15:1. Lymphocytes stimulated with venous endothelial cells were cytotoxic both for arterial and for venous endothelial cells and PHA blasts of the stimulator dog, whereas lymphocytes stimulated with arterial endothelial cells lysed only arterial endothelial cells and PHA blasts of the stimulator. Lysis of syngeneic or third-party allogeneic control targets was virtually absent. Optimal conditions for long-term culture of effector cells from mixed leukocyte endothelial cell cultures were analyzed. Addition of Il 2 every 3 days and the original stimulating antigen every 6 days permitted continuous proliferation of these cytotoxic lymphocytes with preservation of the cytotoxicity pattern.

In vitro stimulation of lymphocytes by vascular endothelial cells. A study with canine arterial and venous endothelial cells.

A sensitive limiting dilution assay for the determination of canine cytotoxic T lymphocyte precursors (CTL-P) has been developed. Murine second MLC supernatant as a source of Interleukin 2 (IL 2) was used to expand stimulated CTL-P to numbers that were easily detectable with the classic 51Cr lysis assay. The frequencies of CTL-P that reacted to allogeneic stimulator cells in canine peripheral blood ranged from 1:1000 to 1:2000 lymphocytes. During in vitro stimulation in a mixed leukocyte culture a rapid increase in frequency was noted, and at day 7 a frequency as high as 1:5.4 was found. The presence of irradiated stimulator cells during limiting dilution culture restricted proliferation of the CTL-P; only those which recognized the stimulator cells proliferated. The determination of cytotoxicity in large numbers of individual microcultures with CTL-P seeded at clonal conditions toward 2 allogeneic target cells permitted quantitation of the frequency of CTL-P with specificity for different alloantigens. These techniques greatly facilitate monitoring of cellular immune reactions after renal allografting because both determination of the influence of cytostatic drug treatment on CTL-P frequency, and analysis of the specificity and function of allograft infiltrating cells are now possible.

Limiting dilution analysis of canine alloantigen-reactive T lymphocytes.

In this study significantly prolonged canine renal allograft survival has been demonstrated after transfusion of 100 ml of third-party whole blood given peroperatively. Peroperative transfusions of third-party leukocyte-free blood or pure lymphocyte cell suspensions did not influence graft survival. Furthermore, no improvement in graft survival has been found after a peroperative transfusion of irradiated whole blood (2500 rad). These data suggest that delayed graft rejection after blood transfusions can only be expected after the administration of whole blood. The role of competent lymphocytes in whole blood is questionable, since a transfusion or irradiated whole blood in combination with nonirradiated lymphocytes did not lead to prolonged graft survival. Immunosuppression of the recipient directly after transfusion seems to be essential to induce the beneficial effect of blood transfusions. This has been demonstrated for a transfusion of whole blood 14 days before transplantation. A single transfusion of 100 ml of whole blood 14 days before transplantation could effectively prolong graft survival if immunosuppression with azathioprine and prednisone was started on the day of transfusion. No improvement in graft survival has been found with such a transfusion if preoperative immunosuppression has been omitted.

Effect Of Blood Transfusions On Canine Renal Allograft Survival

Cellular cytotoxicity toward kidney cell targets has been studied in a model using in vitro cultured canine kidney cells obtained after perfusion trypsinization of kidneys from one-haplotype-mismatched beagles To study whether cytotoxic effector cells recognize identical antigens on kidney cells as on phytohemagglutinin (PHA)-stimulated lymphoblasts, adsorption studies with different monolayers have been performed Both leukocyte and kidney cell monolayers reduced cytotoxicity against 51Cr-labeled PHA-stimulated lymphoblasts very effectively The average reduction of cytotoxicity was 86% in six consecutive experiments after one adsorption on either one of these two types of target-specific monolayers Nonspecific monolayers reduced cytotoxicity only for 13% Specific kidney cell monolayers reduced cytotoxicity against kidney cells almost completely, however leukocyte monolayers reduced cytotoxicity toward kidney cells for only 40% There results and cold target inhibition data strongly suggest that kidney cells present antigens to which a selective population of cytotoxic T lymphocytes (CTLs) is directed These CTLs are not cytotoxic for PHA-stimulated lymphoblasts It is discussed whether the relevant antigens on the kidney cells are organ-specific antigens comparable to the endothelial monocyte antigen system as described by Moreas and Stastny or that class II antigens are involved in cytotoxicity toward kidney cells

Vegt Pa

Papers

Analysis of buoyant density of canine peripheral blood leukocytes with PVP-Silica (Percoll) density gradients.

In vitro stimulation of lymphocytes by vascular endothelial cells. A study with canine arterial and venous endothelial cells.

Limiting dilution analysis of canine alloantigen-reactive T lymphocytes.

Effect Of Blood Transfusions On Canine Renal Allograft Survival

Cell-mediated cytotoxicity toward canine kidney epithelial cells.