Klaus J. Bross

Predictive Value of Bronchoalveolar T Cell Subsets for the Course of Pulmonary Sarcoidosis

To study the value of knowing the proportions of bronchoalveolar T cell subsets when predicting the course of pulmonary sarcoidosis, we subjected 31 patients to clinical, physiologic, and radiographic evaluations, with controls, for at least 12 months. Initially, when all patients were untreated, BALs were performed, and BAL lymphocyte subsets were marked by the following monoclonal antibodies: OKT3 (expressed by all T cells), OKT4 (to mark helper-inducer T cells), OKT8 (to mark suppressor-cytotoxic T cells), and OKIa (to mark Ia antigen-positive, activated T cells). A normal T4/T8 ratio was highly predictive of a favorable course: the conditions of 13 out of 15 patients with normal ratios remained stable or improved, and only 2 of these 15 patients had to be treated because of persistent symptoms. On the other hand, the conditions of 10 out of 16 patients with elevated T4/T8 ratios deteriorated during the follow-up period. The specificity of T cell subsets for predicting deterioration was improved by considering both the T4/T8 ratio and the number of Ia antigen-positive, activated T cells present. Deterioration occurred in 9 out of 11 patients with elevated T4/T8 ratios and elevated levels of activated T cells. These results suggest that the subtyping of BAL lymphocytes may be useful in determining prognosis in pulmonary sarcoidosis.

Demonstration of estrogen and progesterone receptors as well as Ki-67 and p-145 antigens in single tumor cells from blood and pleural effusions using a slide assay.

SummaryWe describe a slide assay that allows the demonstration of antigens localized in the nucleus from isolated white blood cells as well as from single tumor cells derived from malignant effusions. With the antibodies Ki-67 and anti-p-145 an increased rate of nuclear and nucleolar staining resulted in cells from highly malignant lymphomas. An almost identical reaction was obtained when tumor cells from malignant effusions were tested. Cells isolated from the blood of patients with leukemic spread of lymphomas of low malignancy yielded a weak staining comparable to that of normal mesothelial cells from non-tumorous cavity fluids. The detection of estrogen and progesterone receptors (ER and PR) localized in the cell nucleus can be achieved by the same assay. The reaction is enhanced by incubation of the tumor cells for 30 min at 37‡ C prior to fixation. Pleural effusions from 20 patients with breast cancer were tested. ER was positive in 13 and PR was positive in 12 of the 20 samples. In 5 cases there was a divergent reaction with ER and PR antibody. The hormone receptors of the primary tumor were known in 15 (ER) and 14 (PR) patients, respectively. In each cohort there was only one case with a negative reaction of the primary tumor and a positive reaction with the isolated tumor cells from the pleural effusions. These results indicate that the demonstration of hormone receptor proteins in cells from malignant effusions is possible and that there is a correlation with the status of the primary site of cancer.

Expression of the proliferation-associated Ki-67 antigen of transferrin receptors and of DNA polymeraseα in human tumour lines: implications for in vitro chemoresistance

SummaryTo compare the time course of in vitro expression of various proliferation-associated markers including Ki-67 antigen, transferrin receptors (TfR), and DNA polymerase α, six human tumour cell lines of different histological origin were studied under defined conditions. Proliferation markers were demonstrated by peroxidase/anti-peroxidase staining using specific monoclonal antibodies, and their expression was compared to results obtained from [3H]-thymidine incorporation assays and cell counting. Expression of all proliferation markers began to increase during the lag phase, and occurred earlier than elevations of [3H]dT incorporation and cell numbers were recorded. Maximum expression was observed before cell growth reached plateau phase. The time courses of expression of DNA polymerase and Ki-67 were almost identical. The closest correlation of [3H]dT incorporation with time course of expression of proliferation-associated markers was observed, when intranuclear staining of DNA polymerase was analysed. TfR were expressed earlier than the polymerase and Ki-67. Since TfR were also found at remarkable levels in resting cells, they seem less proliferation-specific than Ki-67 and DNA polymerase. While in rapidly growing cell lines more than 95% of the cells expressed Ki-67, TfR, and more than 75% DNA polymerase in cell nuclei, a malignant melanoma and a pleural mesothelioma line displayed fewer than 35% of cells stained for DNA polymerase in cell nuclei during log phase. Determination of growth fractions by monoclonal antibodies may thus contribute to the prediction of chemoresistance by identifying quiescent cells that are not sensitive to S-phasespecific drugs.

Identification of proliferating lymphocyte subpopulations in microcultures by surface marker and autoradiography.

A micromethod is described which allows subpopulation classification of proliferating (3H thymidine incorporating) cells, previously stimulated in microcultures. The technique is based on transferring 1,000 to 5,000 cells from microcultures to poly-L-lysine coated multispot slides. The cells are then stained for surface markers using the immuno-peroxidase method, combined with subsequent autoradiography.

Stimulation of growth of human malignant lymphoma and lymphoid leukemia cells in vitro

SummaryWe have analyzed cultures of malignant lymphoma cells and cells from patients with acute lymphoid leukemia in methylcellulose for their ability to from colonies. Clonogenic growth was examined in the presence or absence of fetal calf serum (FCS), platelet-poor plasma (PPP), medium conditioned by phytohemagglutininstimulated leukocytes (PHA-LCM), or irradiated allogeneic bone marrow stroma cells. Cells from 25 lymphoma patients — 17 with non-Hodgkins lymphoma (NHL), eight with Hodgkins disease (HD) — and from 19 patients with acute lymphocytic leukemia (ALL) were investigated. We show that colony growth can be obtained in a minority of cases (in 3 NHL, 5 HD, and 2 ALL) and that the use of FCS and allogeneic irradiated stroma cells may be required for optimal colony formation.

The effectiveness of drugs and the significance of monoclonal antibodies in eight human small cell lung carcinoma (SCLC) xenografts

The low cure rate of SCLC has not improved in recent years. Experimental models are necessary to select new antitumor drugs or combinations and to understand the tumor biology. Therefore, we developed tumor models by transplant ing 14 human SCLC into nude mice. By histopathological examinations a l l xenografts resembled the or ig inal tumors closely and were typical of SCLC. Tumor take occurred in 8/14 biopsies. The median doubling times in ser ia l passage ranged from 8 to 22 days, the number of ser ial passages from 2 to 29. Original SCLC tumor ce l ls and xenograft tumors were tested by the immunoperoxidase method with MoAbs defining transfe r r in receptor (OKTg), neuronspecific enolase antigen (NSE), the human leucocyte antigen (HLA class I ) , the common leucocyte antigen (HLe-I), tumor associated ant i gens (BASIO, SAMI2), and CEA. There seems to be a pattern of MoAbs associated with SCLC. Al l of the SCLC ce l ls were negative for HLe-I antigen, and most of the ce l ls los t the HLA class I antigens. On the other hand, there is a strong reaction with the fo l lowing MoAbs: OKTg, SAM12, BASIO and NSE. CEA was only expressed on some SCLC tumors. There is no dif ference between antibody patterns of o r i ginal ce l ls and passages of nude mice. The response to drugs was studied in 6 subcutaneously growing tumors in nude mice. 2/6 responded to ADR, 2/3 to CV, 3/3 to VIND, 2/6 to PLAT, 2/3 to VP16, 1/2 to MITO, 2/2 to HECNU. In conclusion 8 models for SCLC were developed and the i r chemotherapeutic response was determined. Using a series of MoAbs, there seems to be a character is t ic pattern for SCLC.