Martin H. Goldrosen

Murine colon adenocarcinoma. Immunobiology of metastases

Orthotopic transplantation of MCA‐38 murine colonic tumor cells into the submucosa of the cecum results in the growth of a “primary” tumor that metastasizes to the liver. This model system parallels the sequence of events that can occur with human colon carcinoma and provides a means of evaluating the role of the immune system in hepatic metastases formation. Temporal studies of the specific antitumor response detected by a micro‐leukocyte adherence inhibition (LAI) assay revealed two patterns of sensitization as the “primary” tumor grew and hepatic metastases developed. The systemic antitumor response was monophasic and disappeared prior to the formation of hepatic metastases. In contrast, the local and regional antitumor response was biphasic. The breakdown in the local and regional response may play a permissive role in the formation of hepatic metastasis.

Complementary and alternative medicine: assessing the evidence for immunological benefits

With words such as AIDS, allergy and autoimmunity embedded in the popular lexicon, we often equate health with the precision and the tenor of responses to allergens and microorganisms. This leads many people to seek their own solutions to sustain, restore or even boost their immune competence, hoping to live more comfortably and longer. Here, we consider the social and clinical contexts in which these promises of enhanced immunity are pursued through popular practices known as complementary and alternative medicine and the evidence that supports these.

Isolation of human peripheral blood lymphocytes: Modification of a double discontinuous density gradient of Ficoll-Hypaque

Incubation of whole blood with adenosine diphosphate (ADP) and carbonyl iron prior to layering on a double discontinuous gradient of Ficoll-Hypaque selectively reduced the number of platelets and monocytes found at the upper lymphocyte interface after centrifugation. This modification in combination with a double discontinuous gradient results in a lymphocyte isolation method that is simple, rapid and reproducible.

Leukocyte adherence inhibition. An automated microassay demonstrating specific antigen recognition and blocking activity in two murine tumor systems.

This communication describes an automated micro-adherence inhibition assay. Tumor-specific immunity was demonstrated with B16 melanoma and MCA-38 colon adenocarcinoma, both of which are syngeneic to the same strain of mouse (C57B16/J). Abrogation of the leukocyte adherence inhibition (LAI) response of sensitized leukocytes has been demonstrated in the MCA-38 tumor system by the addition of serum from mice bearing MCA-38 progressively growing tumors, a property not present in normal serum. The sensitivity of the system has also been demonstrated by showing that LAI will change prior to a tumor becoming palpable. This microassay has the advantage of being simple, rapid and reproducible, and involves the use of minimal quantities of antigenic preparations and leukocytes, and in addition is amenable to rigorous statistical analysis.

Interleukin-2 lymphokine-activated killer cell therapy of non-Hodgkin's lymphoma and Hodgkin's disease

We conducted a phase II study utilizing interleukin-2 (IL-2) with lymphokine-activated killer (LAK) cells as therapy for non-Hodgkins lymphoma (NHL) and Hodgkins disease (HD). IL-2 was given at a fixed dose of 3 x 10(6) U/m2/day administered as a 24-h continuous intravenous infusion with LAK cells. Nineteen extensively treated patients were entered and 15 were evaluable. In general, this regimen was reasonably well tolerated with mild toxicities that were rapidly reversible. Patients who completed therapy did so without dose attenuations. However, discontinuation of therapy was necessary in four patients due to atypical toxicities that were not clearly dose related. Two patients (one NHL and one HD) had partial remissions of brief duration, four had disease stabilization, and seven had progressive disease. While there were not sufficient numbers to evaluate critically any NHL or HD subtype, this regimen does not appear to have significant activity for either disease.

Renal cell carcinoma treated with continuous-infusion interleukin-2 with ex vivo-activated killer cells

High-dose recombinant interleukin-2 (rIL-2) results in tumor responses in patients with metastatic renal cell carcinoma ranging from 9 to 31%. Continuous infusion regimens of rIL-2 may be less toxic and may result in greater in vivo lymphokine-activated killer (LAK) cell production. The current trial used a continuous infusion of rIL-2 with ex vivo LAK cells. These cells were pretreated with phenylalanine methyl ester to remove monocytes to allow cell culture at higher concentrations. Twenty-three patients were entered into the trial. Two patients had complete responses (9%) lasting 15+ and 20+ months. Four patients had partial responses (17%) of 9+, 6+,3,and 3 months, respectively. One partial responder at 9+ months had only minimal residual retroperitoneal disease that may represent scar tissue. All responders had prior nephrectomies. All but one of the responding patients completed a full cycle of rIL-2 at the highest (starting) dose, 6 x 106 U/m2. This rIL-2/LAK regimen appears to be an effective therapy for metastatic renal cell carcinoma.

Interleukin-2 with ex vivo activated killer cells: therapy of advanced non-small-cell lung cancer.

A phase II study was conducted to examine the efficacy of interleukin-2 (IL-2) with lymphokine-activated killer (LAK) cells as therapy for advanced non-small-cell lung carcinoma (NSCLC). IL-2 was administered at a fixed dose of 6 x 10(6) U/M2 per day as a 24 h continuous intravenous infusion (CIV) with LAK cells. Eleven patients were entered onto this study and six were evaluable. One patient had a near complete response of 18 months duration. Only two patients were able to complete the regimen without dose reduction. This regimen was poorly tolerated with pulmonary toxicity being the major problem. The partial responder was the only patient to undergo more than one course of therapy. IL-2/LAK therapy may have activity in NSCLC and further studies are warranted in this uniformly fatal disease. However, future studies will have to incorporate less toxic IL-2 regimens.

A radio (51Cr) micro-tube leukocyte adherence inhibition assay: Specific tumor-associated immunity in 3 murine tumor systems

A radio (51Cr) micro-tube leukocyte adherence inhibition assay is described. In this study, murine mononuclear cells were labeled with 51Cr, plated into tissue culture plates with different tumor extracts and counts/min (cpm) of the non-adherent cells were used as a parameter of adherence inhibition. This assay was used to measure anti-tumor immunity, in vitro, in 3 murine tumor systems: MCA-38 colon adenocarcinoma, L1210 lymphoma and P815 mastocytoma. Tumor immunity was detected using 3 doses (0.01-0.001 mg/ml) of tumor extract in the MCA-38 tumor model, and using 2 doses (0.1-0.05 mg/ml) of tumor extract in both the L1210 and P815 tumor models. It was observed that specific tumor-associated adherence inhibition could be measured in the MCA-38 tumor model between days 7 and 22 of tumor growth and in the L1210 and P815 tumor models between days 7 and 17 of tumor growth. The radio-LAI assay described is an easy, specific and reproducible way to measure tumor-associated adherence inhibition, in vitro.

Tumor associated lymphocyte cytotoxicity correlated with stage of disease in patients with colon adenocarcinoma. A preliminary report.

Abstract Whole lymphocyte populations and T and non-T subpopulations from 22 colorectal carcinoma patients ( 8 preoperative; 6 postoperative disease free; 8 postoperative residual disease) and 6 control Stage III melanoma patients were tested in parallel on a colon cell line (P- 4788 ) and a melanoma cell line (M- 7292 ). All three effector populations from the four patient groups were cytotoxic on both cell lines. Within the different donor groups, the unfractionated lymphocytes from preoperative colon patients displayed significantly greater cytotoxicity than the Stage III melanoma patients on the colon cell line. Conversely, the Stage III melanoma patients displayed significantly greater cytotoxicity than the postoperative disease free colon patients on the melanoma cell line. Furthermore, the unfractionated lymphocytes and non-T subfraction from preoperative and postoperative disease free colon carcinoma patients showed significantly stronger cytotoxic effects on the colon cell line than on the melanoma cell line. These two lines of evidence suggest that both tumor associated cytotoxicity and spontaneous or non-selective cytotoxicity are present; and the presence of tumor associated cytotoxicity is dependent on the stage of the disease. Furthermore, thr level of cytotoxicity associated with both postoperative colon carcinoma groups was lower than the preoperative colon carcinoma groups. However, this depression was not evident in subfractions of lymphocytes that demonstrated apparent tumor associated cytotoxicity and thus may reflect a decline in general immune competence.

Semi-automated method for the enumeration of cytotoxicity results☆

This communication provides information on the design and use of a counting machine that enumerates adherent cells in cytotoxicity assays. To date, investigators have relied on inverted microscopes or quantimat automatic image analysis. The former method is tedious and time consuming while the latter method is prohibitively expensive. The counting machine which consists of projection microscope attached to an electronic counter is as accurate as the inverted microscope but is both less tedious and less time consuming.