Functional alterations of human blood monocytes after exposure to various nickel compounds in vitro: an effect on the production of hydrogen peroxide.

It is generally known that nickel, a metal with distinct carcinogenic properties, can significantly alter the functioning of host defense mechanisms and impair various components of the immune system. In the present study the influence of 3 nickel salts on the production of hydrogen peroxide (H2O2) by human monocytes was examined in in vitro culture. Highly purified, resting and PMA-stimulated normal human monocytes were cultured with subtoxic concentrations of nickel subsulfide nickel sulfate, nickel acetate and manganese chloride. A portion of the cells was cultured with nickel-manganese salt mixture. Following culture cells were tested in an in vitro functional assay for H2O2 production. It has been shown that all nickel salts, used in micromole concentrations, suppressed H2O2 formation both in resting and PMA-stimulated monocytes, while it was not the case when manganese chloride was used for cell cultures. The strongest suppressive effect was manifested by nickel sulfate. The cells subjected to nickel-manganese mixture displayed H2O2 production similar to that of control ones. These results show that nickel salts in micromole concentrations exert a suppressive effect on oxygen-dependent antimicrobial system of human monocytes and manganese prevents this effect.

The effect of nickel compounds on immunophenotype and natural killer cell function of normal human lymphocytes.

In order to elucidate effects of nickel on human lymphocytes in vitro, peripheral blood mononuclear cells from normal donors were initially tested for viability in the presence of increasing concentrations of two selected nickel salts, sparingly-soluble nickel subsulfide (Ni3S2), and promptly-soluble nickel sulfate (NiSO4). After establishing the toxicity profile, the cells were cultured for 24 h with each compound at three nontoxic concentrations, 0.01 mM, 0.02 mM, and 0.04 mM, to determine its effect on lymphocyte immunophenotype and function. Cells were also cultured in the presence of 0.01-0.04 mM magnesium acetate, Mg(CH3COO)2, while still other cell samples were subjected to a mixture of Mg(CH3COO)2 plus either Ni3S2 or NiSO4 at equimolar concentration. Following the culture, the immunophenotype of the cells was determined by indirect immunofluorescence, using monoclonal antibodies to major differentiation antigens of peripheral blood mononuclear cells, and their natural killer activity toward K562 target cells was measured. Both nickel salts were found to exert distinct effects on lymphocyte phenotype. Exposure of cells to Ni3S2 resulted in the decline of CD4 and natural killer cell populations. NiSO4 diminished the abundance of natural killer cells and, to a limited extent, also of CD4 cells. The nickel salts tested suppressed natural cytotoxicity of peripheral blood mononuclear cells, with Ni3S2 acting more strongly than NiSO4. The addition of Mg(CH3COO)2 to a nickel salt during in vitro culture abolished the above inhibitory effects. Nickel and magnesium salts did not affect CD3, CD8, CD20, and CD11a cell populations. The results indicate that nickel salts have deleterious effects on human peripheral blood mononuclear cells in short-term in vitro culture, but the magnitude of these effects varies, depending on the cell subsets.

Tumor infiltrating lymphocytes in HLA+ and HLA- laryngeal cancer--quantitative approach.

In search of factors governing the accumulation of tumor infiltrating lymphocytes (TIL), frozen sections from fresh surgical specimens of laryngeal carcinoma (n = 36) were tested by alkaline phosphatase-anti-alkaline phosphatase (APAAP) immunohistochemistry for monomorphic determinants of HLA class I and class II expression on tumor cells and for the distribution of lymphoid cells bearing CD differentiation antigens. Cell subsets were quantitated in two tumor compartments, tumor mass and tumor stroma, by computer-assisted image analysis. In a portion of examined samples lymphoid cell suspension was isolated from cancerous tissues and assessed by flow cytometry. It has been found that T cells, localized mostly in tumor stroma, were predominant cell population in the tumor microenvironment. Their ability to penetrate tumor mass but not tumor stroma, by CD8+ T cells in particular, but also by natural killer (NK) cells, was associated with HLA class I antigen expression on tumor cells. In flow cytometric analysis activated T lymphocytes (CD3+DR+) were abundant in HLA+ tumors as compared to HLA- ones. In 4 year follow up of 20 patients the mortality was higher in HLA- group but the data were not statistically significant. These results show that HLA class I expression on tumor cells favor penetration of cytotoxic lymphoid cells into tumor mass, at least in the laryngeal cancer.

Evaluation of immunophenotype of lymphoid cells isolated from malignant pleural effusions.

Lymphoid cells, isolated from malignant pleural effusions and collected from patients bearing primary lung carcinoma, were examined by means of indirect immunofluorescence and a panel of monoclonal antibodies vs several CD antigens. The percentages of CD4+ T lymphocytes were found to be significantly depressed in malignant effusions as compared to inflammatory ones. In relation to histological type of cancer it was especially evident in squamous cell and anaplastic carcinomas (small and large cell), in comparison to adenocarcinomas. Expression of T cell antigen receptor tau/delta (TCR-1) on T lymphocytes, demonstrated by BB3 MoAb (vs V delta 2), was significantly higher in malignant effusions as compared with non-malignant ones. This was not the case when A13 MoAb (equivalent of TCS 1) was used (vs V delta 1). Percentage values of NK cells, monocyte/granulocyte series activated cells and B lymphocytes did not differ significantly in malignant and non-malignant effusions. It is concluded that these are T lymphocyte subpopulations which are apparently distinct in both effusion groups examined.

Immunoregulatory effect of T cell subsets on PWM-induced IgG synthesis by blood lymphocytes in lung cancer.

The mechanism of pokeweed mitogen (PWM) dependent decreased IgG production by blood lymphocytes from lung cancer patients was studied in comparison to control patients and blood donors. It has been shown that the depletion of monocytes has some influence on IgG synthesis but is not a decisive factor. Also, quantitative alterations in the CD4 and CD8 lymphocyte subsets do not significantly influence the PWM stimulation index for IgG synthesis. The assessment of T lymphocyte suppressor activity in lung cancer patients was performed by means of a co-culture with blood mononuclear cells, while helper activity was evaluated through co-culture with donor B lymphocytes. It has been found that lung cancer patient T lymphocytes have no increased suppressor activity, however, especially in the CD4 subset, display the decrease of helper function for B lymphocytes in PWM-induced IgG synthesis. The weakened helper function of CD4 lymphocytes may explain the suppression of specific antibody synthesis do novo which is evident in patients with lung cancer.

Assessment of immunophenotype of potentially cytotoxic tumor infiltrating cells in laryngeal carcinoma.

Among host lymphoid cells engaged in anti-tumor defence, tumor infiltrating cells (TIC) are apparently the most suitable for this purpose, due to feasibility of direct contact with tumor cells. The aim of the present study was to evaluate distribution of TIC which express phenotype of cytotoxic cells, in tissues of laryngeal carcinoma. Cryostat sections of surgical tumor samples were subjected to sensitive APAAP immunohistochemistry, following reaction with one of the panel of monoclonal antibodies (MoAbs) vs. several CD antigens and assessed semiquantitatively. It has been found that the content and distribution of potentially cytotoxic cells are quite heterogenous and vary from case to case in examined cancer. CD8+ cells and those bearing NK cell phenotype were the most frequently encountered, mainly within tumor mass. The cells belonging to NK cell subsets, detected by GL183 and EB6 MoAbs could be demonstrated in tumor proximity. TCR-1+ (tau/delta) T lymphocytes were quite a few in part cases. On the other hand, only a scarce number of cells among TIC expressed interleukin-2 receptor. It is concluded that in the vicinity of laryngeal cancer there are fairly large numbers of potentially cytotoxic cells, but at low or nil state of activation.

Protein and mRNA expression of CD56/N-CAM on follicular epithelial cells of the human thyroid.

In order to confirm CD56/N-CAM antigen prevalence in the human thyroid and to compare its expression on thyrocytes and NK cells, an expression of CD56/N-CAM antigen was searched for on isolated thyroid follicular cells and NK cells by flow cytometry. In addition, mRNA for CD56 was searched for in RNA isolated from human thyroid samples and few other organs using dot blot hybridization assay to prove the existence of mechanisms for active synthesis of the protein in question. The isolated cells from the follicular epithelium of 22 various pathological thyroid tissue specimens were examined for the expression of CD56/N-CAM in terms of the percentage of positive cells and the mean fluorescence intensity (MFI). Blood lymphocytes were tested in parallel. The total RNA isolated from thyroid and control tissue specimens was subjected to dot-blot hybridization assay using CD56/N-CAM cDNA probe. All thyroid specimens expressed CD56/N-CAM, but the obtained values differed depending on the tissue examined and the CD56 antibody used. There were no significant differences between the non-malignant thyroid cells of various histology, while cells in the carcinoma group had a much lower MFI, especially the median value. CD56 expression on NK cells from the donors' blood had a homogeneous distribution but the mean and median values of FI were almost three times lower than those on the thyroid cells. Dot blot hybridization came out positive with the RNA isolated from the thyroid specimens and also from the RNA isolated from the tonsil and lymph nodes, but came out negative with the RNA isolated from human and rat kidneys. These results strongly suggest that thyroid follicular epithelial cells express both protein and mRNA of CD56/N-CAM, thus being able to synthesis the relevant antigen. The protein expression seems to be affected by the malignant transformation of the thyroid cells. NK cells have apparently lower CD56/NCAM expression than thyroid cells.

Anti-tumor antibodies in lung cancer patients. Immunofluorescence study using various indicator cells.

By means of indirect immunofluorescence a number of primary lung cancer patient sera and control sera were tested for anti-tumor antibody activity on living tumor cells as a substrate. Antibodies against surface antigens were the most frequently detected in autologous system (in 65%) on cells derived from fresh surgical material of lung cancer. They were also found in 50% of cases using tumor cells from primary short-term culture. When established cell line of lung cancer was used (E-14) in allogeneic system, the antibodies were detected in only 22% of examined lung cancer sera. Absorption of positive sera with homogenates of normal tissues did not abolish their specific activity. Positive reactions were confined to squamous cell type of bronchogenic carcinoma.

Identification of a tumor-related protein antigen in immune complexes derived from pleural effusions of patients with bronchial carcinoma.

Pleural effusions from 15 patients with advanced primary bronchial carcinoma, from 2 patients with metastatic lung cancer and from 6 patients with nonmalignant disease were studied. Immune complexes were found in examined fluids in amounts corresponding to 2.5-210 mg/100 ml of aggregated IgG by means of ELISA solid phase anti C3 and 125ICIq binding radioimmunoassay. Following determination of protein content and salting out by ammonium sulfate of examined fluids, the sediments were subjected to subsequent chromatographic procedure including molecular sieving (Sephadex G-200, Sepharose 4B) and affinity chromatography on Protein A-Sepharose CL-4B. The yield--apparently pure immune complexes--was then split by means of chaotropic agent 2.5 M KSCN. It permitted to obtain 2 fractions: one contained IgG while the other was a non-Ig protein of m. w. = 150 000. The latter isolated from malignant effusions possessed antigenic activity in the leukocyte migration inhibition (LMI) assay. It resulted in inhibition of migration of allogenic peripheral blood leukocytes from lung cancer patients in 87% of cases. It had no activity against leukocytes from nonmalignant disease patients. LMI activity of the final second fraction derived from malignant effusion was significantly different from that of other fractions obtained both from malignant and nonmalignant fluids.

Evaluation of phenotype of mononuclear host cells isolated from primary tumour and peripheral blood of patients with laryngeal carcinoma.

Mononuclear host cells isolated from primary laryngeal carcinoma were assessed by means of indirect immunofluorescence with a panel of monoclonal antibodies against various lymphocyte subsets and macrophages. Tumours of various staging groups were examined in parallel with cells isolated from patient and donor peripheral blood (PBL). It was found that percentage values of cells bearing T3 and T4 phenotype were reduced both in tumour infiltrating cells (TIC) and in PBL population. The fall in T4+ cells in PBL from cancer patients in T3 and T4 staging groups was statistically significant (p less than 0.01) as compared with donor cells. Corresponding values for T8+ cells from TIC were increased in T1 and T2 staging groups of cancer, but showed a gradual fall in advanced stages. The T4+/T8+ cell ratio was decreased in both TIC and PBL cells. The HNK-1+ (NK) cell pattern in TIC was analogous to that for T8+ cells, i.e. the cell percentage decreased with advance in tumour growth. Corresponding values for OKM-1+ were increased in TIC and in patient blood, though in TIC they grew in proportion to tumour growth. Ia+ (HLA-DR+) cells in peripheral blood were significantly increased in patients versus those of donors (p less than 0.01), but only in T3 and T4 staging groups of examined cancer. These results show that subsets of tumour infiltrating cells in laryngeal carcinoma are a complex phenomenon, associated with growth and progression of tumour.

[Immunophenotype of blood lymphocytes in elderly patients with laryngeal cancer]. / Immunofenotyp limfocytów krwi obwodowej u osób w podeszlym wieku chorych na raka krtani.

In a group of 42 patients with surgically treated laryngeal carcinoma, who were divided into those before their 50 and after 60 years of age, main lymphocyte subsets of peripheral blood lymphocytes were assessed by means of flow cytometry. In the group after 60 years of age significantly increased percentage and raised total number of NK (natural killer) cells were found. In the whole group increased total number of all lymphocyte subsets was found. These data indicate that older patients despite an increased number of total to lymphocytes NK cells show higher percentage and total number as compared with normal population at that age.

[An attempt of immunological monitoring of patients with laryngeal carcinoma by flow cytometry]. / Próba monitorowania immunologicznego chorych na raka krtani przy pomocy cytometrii przeplywowej.

In a group of 60 patients with surgically treated laryngeal carcinoma blood lymphocyte subsets were assessed on the day of surgery (day 0) and six weeks thereafter by means of flow cytometry. Blood cells at day 0 were also compared to tumor infiltrating lymphocytes (TIL) isolated from surgical specimens. Significant alterations were found in postoperative period as compared to day 0 manifested by the fall of B cells, increase of activated T lymphocytes and NK cells. There were also marked changes between blood cells at day 0 and TIL, evidence in rise of B cells, of activated T lymphocytes and decline of NK cells within the latter. These data suggest that the assessment of lymphocyte subsets may be of value in immunological monitoring of laryngeal carcinoma patients.

Gamma-aminobutyric acid concentrations in benign parotid tumours and unstimulated parotid saliva.

OBJECTIVE: Apart from its role as an inhibitory neurotransmitter, γ-aminobutyric acid is also thought to regulate various stages of cell proliferation and differentiation in the brain and periphery. The present study aimed to assess the levels of γ-aminobutyric acid and its biochemical precursor glutamic acid (glutamate) in benign parotid tumours and in unstimulated parotid saliva. METHOD: Unstimulated parotid saliva was collected bilaterally, using the swab method, in 20 patients with unilateral pleomorphic adenoma or Warthin's tumour. Samples of tumour and adjacent salivary tissue were collected during tumour resection. RESULTS: Concentrations of γ-aminobutyric acid and glutamate, but not aspartate, were significantly higher in the tumour tissue than in the non-tumour tissue. There was no significant difference in salivary concentrations of γ-aminobutyric acid, glutamate or aspartate, comparing the involved and non-involved side. CONCLUSION: The present results provide preliminary evidence that γ-aminobutyric acid may be involved in the growth of benign parotid tumours.

Glutamate concentration in whole saliva and taste responses to monosodium glutamate in humans.

It is universally accepted that saliva plays an important role in taste sensations. However, interactions between constituents of whole saliva and the five basic taste modalities are still poorly understood. The aim of the present study was to evaluate possible relationship between endogenous glutamate (Glu) levels in whole saliva and taste responses to a prototypic umami substance, monosodium glutamate (MSG; 0.03-10.0%). Rated intensity and pleasantness of MSG taste was studied in healthy volunteers divided into a high glutamate (HG) in saliva (HG; n = 19) and low glutamate in saliva (LG; n = 18) group based on the median split level of salivary Glu. The HG and LG group did not differ in terms of electrogustometric thresholds, rated intensity of the MSG samples and pleasantness of distilled water and the lower MSG concentrations (0.03-1.0%). Perceived intensity of water taste was significantly (P < 0.05) higher in the LG subjects. The LG group rated the higher MSG concentrations (3.0-10.0%) as more unpleasant (P < 0.01). The difference remained significant after controlling for a between-group difference in age. The present results suggest that individual differences in salivary Glu levels may alter hedonic responses to suprathreshold MSG concentrations.

Surface antigens and cytotoxic natural killer cell (NK) activity of blood lymphocytes in heavy cigarette smokers.

Surface phenotypes of lymphocytes and the assessment of cytotoxic NK activity were determined in peripheral blood leukocytes in a group of heavy smokers and respective non-smoking people control group. Cell phenotypes were evaluated by a panel of monoclonal antibodies by indirect immunofluorescence on cell sediments. Cytotoxic activity was assessed by single cell cytotoxic assay on target K 562 cells. There were no significant differences in T cell (CD 3+) as well as in CD 43+ ones (large sialoglycoprotein) per cent values. The cells possessing receptor for sheep red blood cell (CD 2+) were however more numerous in smokers as compared to non-smokers. Per cent value of B lymphocytes in the former group was significantly decreased vs control one. There was no difference in per cent values of activated and immature cells in both examined groups. Per cent values of NK cell activity were higher in non-smokers in relation to smokers. It was reflected by an increase of cytotoxicity of effector cells, while frequency of incidence of NK cells was comparable in both groups examined.

Autoantibodies in lung cancer patients demonstrated on fixed tissue culture cells. An immunofluorescent study.

A panel of sera derived from 138 patients with primary bronchogenic carcinoma, non-neoplastic lung conditions and from blood donors was tested for presence of autoantibodies by indirect immunofluorescence on fixed cells of established lung cancer cell line and lung fibroblasts as a substrate. Autoantibodies were detected in 87% and 64% out of patient sera respectively and in 9% of donor sera. Immunofluorescence patterns permitted to distinguish 3 antibody specificities: anti-nucleolar, anti-cytoplasmic and anti-nuclear ones. The major differences were noted in incidence of anti-nucleolar antibodies, which were present in 77% of lung cancer patients and only in 14% of patients with non-neoplastic lung conditions. The autoantibodies in question belonged to IgG and to lesser degree IgA class of immunoglobulin and were not apparently cancer specific because absorption with normal tissue homogenates removed their activity.

Surface markers and cytotoxic activity of blood natural killer cells studied at the single cell level in Hodgkin's disease.

Purified peripheral blood lymphocytes (PBL) from nine untreated patients with Hodgkin's disease (HD), two HD patients in complete remission and 17 healthy donors were studied for natural killer (NK) cell activity against the K-562 cell line using a single cell cytotoxic assay, which allowed enumeration of effector cells and characterization of their surface membrane phenotypes after staining with monoclonal antibodies. The frequency of NK cells was significantly lower in HD patients than in controls (mean % +/- s.d., 1.9 +/- 0.9 and 2.8 +/- 1.2, respectively), while the fraction of target binding cells was similar in the two groups. The fraction of cytotoxic lymphocytes increased after pre-treatment of PBL with 500 iu leucocyte interferon in all tested control donors (n = 12) and the two patients in remission but only in four of seven untreated patients. No relation between the impaired NK cell frequency and age, tumour histology and clinical stage could be revealed. Subtyping of the target cell binding NK cells by monoclonal antibodies disclosed a marked heterogeneity of effector cells. NK effector cells reactive with M1 and anti-Ia antibodies were enriched while T3+ and T4+ NK lymphocytes tended to be reduced as compared to PBL. There was no difference between patients and healthy donors with regard to the surface antigen patterns of NK cells. Interferon treatment did not alter significantly the phenotypic characteristics of cytotoxic lymphocytes in patients and controls. It is concluded that the impairment of NK cell activity in HD is partly attributed to a lower frequency of cytotoxic effector cells among a normal number of target binding cells. The defect could not be attributed to a selective defect of effector cell subsets.

Assessment of phenotype of blood lymphocyte subsets prior and after PWM stimulation in patients with lung carcinoma.

Surface phenotypes of peripheral blood lymphocytes of lung cancer patients and those of two control groups were assessed by means of indirect immunofluorescence with monoclonal antibodies, prior and after 10 day pokeweed mitogen (PWM) in vitro stimulation. There was no significant alteration in pan T cell per cent values prior and after mitogen stimulation in all groups tested. CD4+ cells in lung cancer group were however significantly decreased as compared to blood donor group (37.3% vs 44.9%, p less than 0.05). This decline was even more pronounced in III/IVo stage of tumour progression according to TNM classification (36.8%, p less than 0.05). These changes, however were not cancer specific, because similar decrease of CD4+ cells was seen in a group of patients with nonneoplastic lung conditions (35.7%, p less than 0.01). Following 10 day PWM culture per cent values of CD4+ cells did not change significantly. The assessment of CD8+ lymphocytes has shown marked differences in two subgroups of lung cancer, namely in II (17.4%) and III/IV (26.2%) of tumour progression (p less than 0.05), which returned to normal values following PWM culture. CD4/CD8 ratio was distinctly depressed in cancer patients in relation to donors. The evaluation of surface markers of B lymphocytes activated cells and monocytes did not show significant alterations in all groups examined. Per cent of HNK1+ cells was heightened in cancer group, especially in III/IV stage of tumour progression in relation to donors (21.7% and 22.8% vs 17.3%, p less than 0.05 respectively). PWM stimulation resulted in marked fall of HNK1+ cells to values corresponding to those in donor group. This study indicates some alterations in per cent values of blood lymphocytes subpopulations belonging mainly to T cell lineage in lung cancer patients linked to tumour staging which only partially return to normal following PWM stimulation.