It is widely accepted that humans exposed to known concentrations of ozone under controlled conditions exhibit reversible changes that affect the large and small airways as well as the alveolar region of the lung. Among the reversible changes, the induction of inflammatory responses in the lung are of major concern. Many of the cell types found in the lining of the nasopharyngeal region are similar to cells of the tracheal and bronchial lining. Therefore, it has been suggested that the cellular responses in the nose to toxicants are likely to be similar to the lower airway at the same dose of the agent. If these pollutants are respiratory irritants, capable of causing cellular damage, effects may therefore be detected in the nasal passage. Experimental studies have shown that the inflammatory response in the nose may be predictive for the situation in the lung. In this paper we described the results of a feasibility study on the use of nasal lavage for epidemiological studies. Nasal lavages were performed in 12 volunteers, 5-7 times per volunteer during 2 months. Polymorph nuclear leukocytes (PMN's), immune mediators and markers for exudation were monitored in the nasal lavage (NAL). It was found that the procedure of the nasal lavage technique was minimally invasive, very well tolerated and no adverse side effects were observed. The leukocytes, the proteins myeloperoxidase (MPO), eosinophil cationic protein (ECP) and interleukin-8 (IL-8) were detectable in NAL of most volunteers, while tryptase IgE and IL-6 were not detectable. Exudation markers albumin, urea and uric acid were also detectable. The coefficient of variance (CV) values of the various cells and mediators varied between 13% and 137%. It was calculated that, except for the number of leukocytes and the concentration of ECP, it should be possible to detect ozone effects with a study- protocol of 6 repeated measurements among 35 children and an assumed 26% increase in cells or mediators per 100 μg O3 per m3. To measure increase in leukocytes number or in ECP concentration more children are needed. In conclusion, this pilot study has shown that it is possible to measure relevant biomarkers in NAL, and that these assays can be easily incorporated in epidemiological studies

Intralesional injection of BCG into an established line-10 hepatocellular carcinoma in the strain-2 guinea pig causes regression of the tumor and induction of line-10 immunity. We found that the animals were already protected for a second challenge with line-10 tumor cells 7 days after BCG treatment. We studied whether this early induction of immunity was correlated with the expression of MHC class II antigens on line-10 tumor cells and was correlated with an increased expression of MHC class II antigens on leukocytes in the primary tumor and in the regional lymph node (Ln. axillaris accessorius). The MHC class II antigens and the leukocyte subpopulations were measured with monoclonal antibodies and flow cytofluorometry. In the draining lymph node the number of nucleated cells increased about 10-fold during the first 5 days after intralesional injection of BCG. At this time the MHC class II antigen expression of these cells was increased from 21%-32% in the naive controls to 39%-53% in animals with BCG-treated tumors. This implies that the number of MHC-class-II-positive cells increased about 20-fold in the draining lymph node. Surprisingly, the increase in percentage of MHC-class-II-antigen-positive cells was mainly due to an increase of IgM-positive B cells from 8%-11% to 22%-41% and an increase of IgG-positive B cells from 7%-27% to 25%-44%. In the tumor, BCG treatment induced a small increase of MHC-class-II-antigen-positive cells from 11%-12% to 15%-20%. Probably this increase came not from tumor cells but mainly from a BCG-induced infiltration of mononuclear cells, as an increase of T cells from 14% to 20%, an increase of macrophages from 8% to 18%, and an increase of B cells from 0 to 6% was observed. We conclude that the potentiation of anti-(line-10 tumor cell) immunity correlated with a 20-fold increase of MHC-class-II-antigen-positive cells in the lymph nodes and a small increase in the number of MHC-class-II-antigen-positive tumor-infiltrating cells.

No abstract

Tumor immunity induced by bacillus Calmette-Guérin was studied in the line 10 hepatocellular carcinoma (line 10) in the strain-2 guinea pig. Line 10 immunity was investigated in vitro with a lymphocyte proliferation assay using line 10 tumor protein extracted with 3 M KCl and in vivo by adoptive transfer of line-10-immune spleen cells. Monoclonal antibodies against guinea pig leucocyte markers were used to block functional properties of the immune cells in order to determine which cell types or cell markers are involved in the immune response to the line 10 tumor. In vitro cells from the spleen, peripheral blood and regional lymph node of immune animals reacted with a proliferative response to line 10 protein. This antigen-specific response was caused by T cells and was regulated by major histocompatibility complex (MHC) class II molecules. In blocking experiments it was found that CT5 (anti-PanT), or MSgp4 [anti-(MHC class I antigen)] monoclonal antibodies did not block but sometimes stimulated the proliferative response. The effect of H159 (anti-PanT) was irregular, while H155 [anti-(T helper)], and 5C3 [anti-(IL-2 receptor)] monoclonal antibodies blocked the response almost completely. We studied the relevance of the results in vitro obtained and found that mAb 5C3 [anti-(IL-2 receptor)] inhibited the adoptive transfer of line 10 immunity, suggesting that the rejection of line 10 cells is caused by a mechanism that is interleukin-2 (IL-2)-dependent. Moreover, complement lysis of MHC-class-II-antigen-positive immune spleen cells inhibited completely the rejection of the line 10 tumor cell challenge in the adoptive-transfer experiments. In conclusion, our data show that MHC class II molecules or cells possessing these molecules are involved in immunity against line 10 tumor cells, as (a) monoclonal antibodies against MHC class II antigens inhibited the in vitro proliferative response of T cells to tumor antigens and (b) removal of MHC-class-II-positive immune spleen cells abrogated the antitumor effect in the adoptive-transfer experiments. Interleukin-2-dependent proliferation of immune T cells is required for the rejection of line 10 tumor cells.

We investigated the cellular composition and the major histocompatibility complex (MHC) class II antigen expression in the draining lymph node and the tumour during potentiation of the immune response by intralesional bacillus Calmette-Guérin (BCG) administration in the line 10 hepatocellular carcinoma in the strain 2 guinea-pig. Five days after its injection BCG induced a ninefold increase in the number of draining lymph node cells and an increased MHC class II expression. This increased MHC class II expression was mostly due to the selective increase of B cells in the lymph nodes, and to a lesser extent to the increase of T cells expressing MHC class II antigens. Taking into account this nine-fold increase, intralesional treatment of BCG increased considerably the number of T helper/inducer (anti-CT7) and T suppressor/cytotoxic (anti-CT6) lymph node cells expressing MHC class II antigen. The percentage of tumour-infiltrating T cells expressing MHC class II antigen in the tumour was higher than the percentage of T cells in the regional draining lymph node of non-treated guinea-pigs, indicating the presence of activated T cells in the tumour. After treatment with BCG no further increase in MHC class II expression was measured in the tumour, nor was any phenotypical change of the tumour-infiltrating T cells found. In conclusion, with the use of two-colour flow cytofluorometry we have shown that the potentiation of the already existing immune response to line 10 is accompanied by a considerable increase in T helper/inducer, T suppressor/cytotoxic cells and MHC class II antigen in the regional lymph node. Whether this is essential for the potentiation of the immune response causing tumour regression and long-lasting immunity is a subject for further study.

Offprint requests to: P. A. Steerenberg approach of in vitro expanded TIL cells should take into consideration the requirement of two T cell subsets.

No abstract

No abstract