Monitoring of effector and target cell stimulation during conjugation by fluorescence polarization

The aim of the present study was to trace early intracellular changes induced in effector and target cells during their conjugation. This was performed by monitoring the intracellular fluorescein fluorescence polarization (IFFP), using the Cellscan apparatus. This apparatus permits the repetitive spectroscopic measurement of individual selected live cells within a population of many cells, while the location of each cell is known and preserved during the various cell manipulations and/or their suspending medium. Both natural killer (NK) and lymphocyte activated killer (LAK) cells were used as effector cells, while NK-sensitive K562 and NK-resistant Daudi cell lines were used as targets. In this study kinetic IFFP measurements were carried out for a period of approximately 4 h following cell attachment. Within minutes following effector-target conjugation, transient reduction of IFFP was observed consecutively, first in the effector and then in the target cells. A continuous reduction of IFFP occurring only in target cells was also found 50 min following conjugation. No reduction in IFFP was observed using NK- and LAK-resistant target cells. Good correlation was found between early stages of conjugation, as assessed by IFFP, and cytolytic efficiency as assessed by 51chromium release assay. When NK-resistant and LAK-resistant target cells were used, no reduction of IFFP was observed.     

Cell activation influences cell staining kinetics

Stimulation of cells has so far been observed, among other methods, by the decrease of the intracellular fluorescein fluorescence polarization (IFFP). It is shown that the rate constant of leakage of the fluorescent marker out of the cells increases with stimulation much more significantly than the polarization decreases; thus it might provide a more sensitive method to observe cells stimulation. It is also shown that due to negligible leakage of the marker out of the cells shortly after initiation of the staining of the cell suspension, the fluorescein fluorescence polarization (FFP) of the cell suspension, is very close to IFFP.     

Cell population changes during atrophy and regeneration of rat parotid gland

Melan-A/MART-1 is a melanoma differentiation antigen that is recognized by a high proportion of cytolytic T lymphocyte (CTL) clones derived from human leukocyte antigen (HLA)-A2+ melanoma patients. Whereas peptide Melan-A/ MART-1(27-35) was originally defined as the immunodominant CTL epitope, we have previously reported that peptide Melan-A/MART-1(26-35) was recognized more efficiently by the majority of tumor-reactive CTL clones. As demonstrated here, CTL populations generated from blood lymphocytes of either melanoma patients or healthy individuals after in vitro stimulation with peptide Melan-A/MART-1(26-35) killed specifically HLA-A2+ Melan-A+ allogeneic melanoma cells, thus suggesting their potential use in adoptive immunotherapy. We characterized the surface phenotype of the circulating CTL precursors (CTLp), which respond to in vitro stimulation with peptide Melan-A/MART-1(26-35). In melanoma patients, these CTLp predominantly expressed the CD45RO memory marker. In contrast, they were mainly, although not exclusively, found in the CD45RA subpopulation of CD8 T cells in healthy individuals. The demonstration that Melan-A/MART-1-specific CTLp in peripheral blood lymphocytes from HLA-A2+ patients with metastatic melanoma express a memory phenotype provides direct evidence that in vivo priming of this antigen may occur during tumor progression.     

Interstitial lung disease in systemic sclerosis: comparison of BALF lymphocyte phenotype and DLCO impairment

Patients with scleroderma (systemic sclerosis-SSc) frequently develop an interstitial lung disease. The role of lymphocytes in fibrosing alveolitis preceding lung fibrosis has been established. The purpose of this work was to evaluate cell profiles and lymphocyte phenotypes in the bronchoalveolar lavage (BAL) fluid and to correlate them with depression in lung function tests detected by depletion of diffusing capacity (DLCO). BAL was carried out in 25 untreated, non-smoking patients suffering from diffuse scleroderma and in 12 healthy non-smoking volunteers. For the analysis of lymphocyte sub-sets flow cytometry and monoclonal antibodies were used. The following cell sub-types were counted: T lymphocytes, B lymphocytes, helper lymphocytes, suppressor/cytotoxic lymphocytes, natural killer cells, cytotoxic T lymphocytes and activated T lymphocytes. The total cell count was higher in the group of patients with mild and moderate impairment in DLCO. The percentage of lymphocytes was greater in patients with DLCO lower than 65% of the predicted value since neutrophilia was found in patients with severe DLCO depletion, i.e. significant when compared with healthy subjects. The proportions of suppressor/cytotoxic lymphocytes and of activated T lymphocytes were higher in patients than in controls. The statistical analysis revealed significant differences between patients with moderate and mild changes in DLCO and the healthy volunteers. A decreased helper/suppressor ratio was noticed in these patients. We concluded that the BALF lymphocyte phenotype analysis may reflect the features of alveolitis in patients with SSc.     

Immunogenicity of the ALLAVGATK (gp100[17-25]) peptide in HLA-A3.1 melanoma patients

A T cell line recognizing autologous and allogeneic HLA-A3.1 melanomas was obtained from a disease-free melanoma patient (patient 15392). By transfection of a tumor cDNA library and in vitro sensitization experiments, the ALLAVGATK gp100/Mel17-derived peptide was found to be the epitope recognized by this melanoma-specific T cell line. The role of the ALLAVGATK peptide in the systemic immune response to melanoma of this patient was evaluated. When pulsed on the autologous peripheral blood mononuclear cells, the ALLAVGATK peptide generated tumor-specific HLA-A3-restricted T lymphocytes and a single restimulation in vitro was sufficient to raise gp100-specific T lymphocytes, indicating a high frequency of epitope-specific T cells. gp100-specific T cells were also induced from T lymphocytes purified from tumor-invaded lymph nodes (tumor-associated lymphocytes, TAL). TAL-derived effectors displayed lower peptide affinity and lower tumor recognition than effectors elicited from peripheral blood lymphocytes (PBL). To further evaluate its immunogenicity, ALLAVGATK was used to stimulate PBL derived from six additional HLA-A3.1 melanoma patients and seven healthy donors. After 7 weeks of peptide stimulation in vitro the generation of anti-gp100 and tumor-specific T cell lines was achieved in one out of the six patients analyzed. Taken together these data indicate that an in vivo priming leading to a systemic immunity against gp100 in HLA-A3 melanoma patients may occasionally occur and that the immunogenicity of ALLAVGATK peptide in melanoma patients is comparable to that of other HLA-A2-restricted epitopes derived from gp100/Mel 17 protein.     

Whole blood production of thromboxane, prostacyclin and leukotriene B4 after dietary fish oil supplementation in man: effect of vitamin E

Antigenic peptides derived from several differentiation antigens of the melanocyte lineage were recently identified in human melanomas as targets for HLA-A2.1-restricted cytotoxic T lymphocytes (CTLs). To examine their potential role in tumour-directed immune responses in vivo, we determined CTL reactivity against seven antigenic peptides derived from the Melan A/MART-1, tyrosinase and gp100/Pmel17 antigens in the peripheral blood of 10 HLA-A2+ healthy controls and 26 HLA-A2+ melanoma patients. The influenza matrix peptide (GILGFVFTL) presented by HLA-A2.1 was used as a control peptide. CTL reactivity was assessed in a mixed lymphocyte 'peptide' culture assay. Reactivity against Melan A/MART-1-derived peptide antigens was readily detectable in both melanoma patients and controls. Reactivity directed against tyrosinase-derived peptide antigens was also detected in both melanoma patients and healthy individuals, but less frequently. A measurable response against gp100/Pmel17-derived antigens was found in 1/10 controls and in 1/26 of the melanoma patients. Reactivity against the influenza matrix peptide was common in both melanoma patients and controls. Our findings show that precursor CTLs against melanocyte differentiation antigens can be detected in peripheral blood of melanoma patients and healthy individuals. The pattern of CTL reactivity directed against melanoma-associated antigens does not seem to be altered in melanoma patients. Despite antigen-specific CTL reactivity, tumour growth was not prevented in melanoma patients and autoimmune phenomena were not detected in healthy individuals. It remains to be determined whether precursor CTLs recognizing melanocyte differentiation antigens can be activated by immunization and lead to effective tumour rejection in vivo.     

A preclinical model to assess the antigenicity of an HLA-A2 melanoma cell vaccine

BACKGROUND: The authors have demonstrated that immunization with melanoma whole-cell vaccine (MCV) augments T-cell responses to melanoma and that cytotoxic T-cells (CTL) recognize allogeneic melanoma-bearing shared HLA-A antigens. A preclinical model was developed to assess CTL activation in vitro using melanoma lines as stimulators. HLA-A2 expression is predominant in melanoma patients and plays a role in HLA class I restricted CTL killing of melanomas. The authors hypothesized that a MCV consisting of allogeneic HLA-A2 melanomas may be as good as autologous melanoma MCV for HLA-A2 patients. METHODS: CTL were generated from peripheral blood lymphocytes of patients with HLA-A2 melanoma by stimulation with autologous melanoma, allogeneic melanoma (HLA-A2 or non-HLA-A2), or allogeneic MCV (mixed HLA-A2 and non-HLA-A2 melanomas). RESULTS: HLA-A2 MCV and autologous melanoma were similar and significantly better stimulators than the others. Specificity also was supported by CTL killing and mixed lymphocyte tumor reaction assays. CONCLUSIONS: These studies provide important information for the studying immunization of patients with HLA-A2 melanoma with an allogeneic HLA-A2 MCV in a Phase I clinical trial.     

T-cell receptor beta variable region diversity in melanoma metastases after interleukin 2-based immunotherapy

Limited T-cell receptor (TCR) repertoire of tumor-infiltrating lymphocytes has been found in melanoma metastases and spontaneously regressing melanoma. Immunotherapy with INF-alpha/interleukin 2 can induce tumor regression in a proportion of patients with metastatic melanoma. We analyzed the gene expression of the TCR-beta variable (Vbeta) region of tumor-infiltrating lymphocytes from 16 melanoma metastases by subgroup-specific semiquantitative RNA PCR to investigate the influence of immunotherapy on the TCR pattern. In five progressing metastases before or after immunotherapy, no overexpression of Vbeta gene families was detectable, whereas in seven of seven metastases responding to IFN-alpha/interleukin 2 one to four Vbeta gene families were overexpressed. Preferential usage of certain Vbeta gene subgroups in patients sharing the same HLA class I molecules suggests a T-cell response to dominant public epitopes. Analysis of multiple specimens from the same patients gives evidence that this strong oligoclonal T-cell selection is induced or at least augmented by immunotherapy, supporting the functional relevance of this finding.     

Halothane macrophage migration inhibtiion factor test in halothane-associated hepatitis

As an index of delayed hypersensitivity in vitro halothane macrophage migration inhibition factor tests (halothane-MIF tests) were performed on peripheral blood lymphocytes from five patients with halothane hepatitis. Twenty-two subjects exposed to halothane, but with no evidence of jaundice, five 'healthy' hospital anaesthetists, nine jaundiced subjects without halothane exposure, and 10 healthy subjects with no history of exposure to halothane were also tested. The halothane-MIF test was positive in four of the five patients with halothane-induced hepatitis; the negative result was in a patient on steroid treatment. The test was negative in all other subjects. Our findings suggest that the halothane-MIF test may be of value in the diagnosis of halothane-induced hepatitis and as a screeening procedure for the identification of susceptible subjects.     

Relaxation of canine pulmonary arteries caused by stimulation of atypical beta-adrenergic receptors

BACKGROUND: Although in some cases delayed hypersensitivity may be observed, beta-lactam antibiotics frequently induce immediate allergic IgE-mediated reactions with the specificity localized in the acyl-side chain structure. Generally, delayed immunologic reactions are related to sensitized T lymphocytes and major histocompatibility complex restricted. OBJECTIVE: To investigate the prevalence of HLA class I and II antigens in patients with delayed hypersensitivity to aminopenicillins in order to evaluate a relationship between major histocompatibility complex immune response genes and aminopenicillins hypersensitivity. METHODS: We assessed 24 patients with history of delayed hypersensitivity to aminopenicillins using (1) skin test with penicilloyl polylysine, minor determinant mixture, benzylpenicillin, amoxicillin, and ampicillin; (2) patch tests with benzylpenicillin, amoxicillin, and ampicillin; (3) RAST for penicilloyls G and V; and (4) oral challenges with amoxicillin, ampicillin, and penicillin V in 18/24 patients. All patients were typed by microlymphotoxicity standard test for HLA class I and II antigens. Statistical analysis by chi2 test 2 x 2 contingency tables, according to Svejgaard, were used for comparison between patients and random Italian population (522 subjects). RESULTS: In the patients group we found higher prevalence of HLA A2 (12/24 = 50%, RR = 6.76 P < .001, EF = 0.425), DRw52 (20/24 = 83.3%, RR = 9.28, P < .001, EF = 0.74), and lower frequency of DR4 (3/24 = 12% ns). CONCLUSIONS: These data suggest that the immune mechanisms involved in adverse reactions to aminopenicillins in vivo are related to genetic markers of immune response and confirms that the presentation of penicillin-hapten determinants to lymphocyte is major histocompatibility complex restricted.     

Time-dependent vascular regression and permeability changes in established human tumor xenografts induced by an anti-vascular endothelial growth factor/vascular permeability factor antibody

The hyperpermeability of tumor vessels to macromolecules, compared with normal vessels, is presumably due to vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) released by neoplastic and/or host cells. In addition, VEGF/VPF is a potent angiogenic factor. Removal of this growth factor may reduce the permeability and inhibit tumor angiogenesis. To test these hypotheses, we transplanted a human glioblastoma (U87), a human colon adenocarcinoma (LS174T), and a human melanoma (P-MEL) into two locations in immunodeficient mice: the cranial window and the dorsal skinfold chamber. The mice bearing vascularized tumors were treated with a bolus (0.2 ml) of either a neutralizing antibody (A4.6.1) (492 micrograms/ml) against VEGF/VPF or PBS (control). We found that tumor vascular permeability to albumin in antibody-treated groups was lower than in the matched controls and that the effect of the antibody was time-dependent and influenced by the mode of injection. Tumor vascular permeability did not respond to i.p. injection of the antibody until 4 days posttreatment. However, the permeability was reduced within 6 h after i.v. injection of the same amount of antibody. In addition to the reduction in vascular permeability, the tumor vessels became smaller in diameter and less tortuous after antibody injections and eventually disappeared from the surface after four consecutive treatments in U87 tumors. These results demonstrate that tumor vascular permeability can be reduced by neutralization of endogenous VEGF/ VPF and suggest that angiogenesis and the maintenance of integrity of tumor vessels require the presence of VEGF/VPF in the tissue microenvironment. The latter finding reveals a new mechanism of tumor vessel regression-i.e., blocking the interactions between VEFG/VPF and endothelial cells or inhibiting VEGF/VPF synthesis in solid tumors causes dramatic reduction in vessel diameter, which may block the passage of blood elements and thus lead to vascular regression.     

Tumor angiogenesis predicts recurrence in invasive colorectal cancer when controlled for Dukes staging

Tumor angiogenesis is associated with metastasis in several types of solid tumors, including melanoma, breast, prostate, lung, bladder, and oral-cavity tumors. The purpose of this study was to determine whether tumor angiogenesis could predict recurrence following curative surgery for colorectal cancer. Thirty-five patients were studied, including 13 patients with recurrent tumor and 22 without. Representative formalin-fixed, paraffin-embedded sections of invasive colorectal cancers from these patients were sectioned. The endothelial cells of microvessels within the tumors were highlighted by immunohistochemical staining for CD31. The most active areas were identified and the microvessels counted in a x 400 field (0.152 mm2) by two observers in a blinded fashion. Tumor microvessel count (p = 0.0062). Dukes' staging (p = 0.0004), vascular invasion (p = 0.0280), and tumor grade (p = 0.0559) were all significantly associated with tumor recurrence. Tumor microvessel counts > or = 65 per x 400 field were associated with tumor recurrence (p = 0.0035, relative risk [RR] = 11.3). Controlling for Dukes' stage, a multivariate logistic regression model revealed that a tumor microvessel count > or = 65 is an important predictor of tumor recurrence (p = 0.0783, RR = 6.0). A backwards elimination proportional hazards model revealed that a microvessel count > or = 65 shows a trend toward independent prediction of time to tumor recurrence (p = 0.1203, RR = 2.967) when controlled for Dukes' staging (p = 0.0029, RR = 9.089). Despite the small number of patients studied, these results suggest that the number of microvessels in sections of invasive colorectal adenocarcinoma immunohistochemically stained with CD31 may be an important independent predictor of tumor recurrence and time to recurrence.     

Prognostic value of tumor infiltrating lymphocytes in the vertical growth phase of primary cutaneous melanoma

BACKGROUND: Primary cutaneous melanoma is often infiltrated lymphocytes that provide the opportunity to study what may be the local immunologic reaction to the tumor and to correlate the presence of these lymphocytes with overall survival. In an attempt to delineate the histologic diagnostic criteria, to classify different categories of lymphocytic infiltrates, previously described by Elder et al. at brisk, nonbrisk, and absent, and to verify their prognostic significance, we reviewed 285 consecutive cases of primary cutaneous melanomas (American Joint Committee on Cancer Stage I and II). METHODS: In addition to clinical variables (age, sex, and location of tumor) and the presence of tumor infiltrating lymphocytes in the vertical growth phase, the histopathologic attributes reviewed included mitotic rate, thickness, and regression. The results were derived from independent histopathologic review by two pathologists (C.G.C., M.C.M., Jr.) on separate occasions. A multivariate analysis of survival was performed with the Cox's regression model. RESULTS: The 5- and 10-year rates for melanoma with a vertical growth phase and a brisk infiltrate were 77% and 55%, respectively. For tumors with a nonbrisk infiltrate, the 5- and 10-year survival rates were 53% and 45%, respectively, and for tumors with absent tumor infiltrating lymphocytes, the 5- and 10-year survival rates were 37% and 27%, respectively. Mitotic index, thickness, and tumor infiltrating lymphocytes were statistically (univariate analysis) significant prognostic factors (P = 0.003, 0.000001, 0.0003, respectively), whereas the presence or absence of regression is not. In the univariate statistical analysis, the sex of patients and site of melanoma also were statistically significant (P = 0.00001 and 0.002 respectively), whereas age (P = 0.98) was not statistically significant. The multivariate analysis of thickness, mitotic rate, and tumor infiltrating lymphocytes showed that thickness and presence tumor infiltrating lymphocytes were significant and independent histologic prognostic factors. With regard to the clinical factors, sex retained its independent prognostic significance. The histologic characteristics of melanoma with vertical growth phase (brisk, nonbrisk, and absent) are exemplified. CONCLUSIONS: We demonstrated that when categories of tumor infiltrating lymphocytes are strictly defined, they indeed have very strong predictive value for primary cutaneous melanomas with a vertical growth phase. This work confirms the work of Clark et al. and fully illustrates the brisk, nonbrisk, and absent categories of infiltration. Finally, a multivariate analysis comparing thickness, mitotic rate and presence of tumor infiltrating lymphocytes showed that only thickness and presence of tumor infiltrating lymphocytes are significant and independent positive histologic prognostic factors.     

Therapy of primary central nervous system lymphoma with pre-irradiation methotrexate, cyclophosphamide, doxorubicin, vincristine, and dexamethasone (MCHOD)

Recently, Khayat et al. reported that high-dose recombinant interleukin-2 (rIL-2) i.v. may induce tumour regressions in metastatic melanoma patients through an association with cisplatin (CDDP) and alpha-interferon (alpha-IFN). Treatment-related toxicities are, however, important. Previous studies have demonstrated that rIL-2 toxicity may be reduced through a subcutaneous injection. In order to evaluate the effectiveness of low subcutaneous rIL-2 doses in a chemoimmuno-hormonotherapeutic combination, 36 metastatic melanoma patients were treated with CDDP, rIL-2, alpha-IFN and tamoxifen (TAM). The overall response rate was 47.2%: five patients had complete response (14%), 12 partial response (33%) and 13 stable disease (36%). Median response duration was 6.4 months (range: 2-29+). Median overall survival was 10 months (range: 3-36+). The CDDP/rIL-2/alpha-IFN/TAM regimen was effective both on soft tissue and visceral metastases. Toxicity was low and patient management did not require an intensive care unit. A statistically significant increase in both percentage and absolute values of lymphocytes, eosinophils, CD3+/CD4+, CD25+, CD16/56+ and HLA-DR+ cells was found in all patients after two treatment courses. This study shows that lower doses of subcutaneous rIL-2, as well as CDDP and alpha-IFN, associated with TAM, may have similar anticancer efficacy with respect to Khayat's schedule but lower toxicity.     

Increased cancer mortality following a history of nonmelanoma skin cancer

CONTEXT: Cancer registries have reported an increased incidence of melanoma and certain noncutaneous cancers following nonmelanoma skin cancer (NMSC). Whether these findings were attributable to intensified surveillance, shared risk factors, or increased cancer susceptibility remains unclear. OBJECTIVE: To determine whether a history of NMSC predicts cancer mortality. DESIGN: Prospective cohort with 12-year mortality follow-up adjusted for multiple risk factors. SETTING: Cancer Prevention Study II, United States and Puerto Rico. PARTICIPANTS: Nearly 1.1 million adult volunteers who completed a baseline questionnaire in 1982. MAIN OUTCOME MEASURE: Deaths due to all cancers and common cancers. RESULTS: After adjusting for age, race, education, smoking, obesity, alcohol use, and other conventional risk factors, a baseline history of NMSC was associated with increased total cancer mortality (men's relative risk [RR], 1.30; 95% confidence interval [CI], 1.23-1.36; women's RR, 1.26; 95% CI, 1.17-1.35). Exclusion of deaths due to melanoma reduced these RRs only slightly. Mortality was increased for the following cancers: melanoma (RR, 3.36 in men, 3.52 in women); pharynx (RR, 2.77 in men, 2.81 in women); lung (RR, 1.37 in men, 1.46 in women); non-Hodgkin lymphoma (RR, 1.32 in men, 1.50 in women); in men only, salivary glands (RR, 2.96), prostate (RR, 1.28), testis (RR, 12.7), urinary bladder (RR, 1.41), and leukemia (RR, 1.37); and in women only, breast (RR, 1.34). All-cause mortality was slightly increased (adjusted men's RR, 1.03 [95% CI, 1.00-1.06]; women's RR, 1.04 [95% CI, 1.00-1.09]). CONCLUSIONS: Persons with a history of NMSC are at increased risk of cancer mortality. Although the biological mechanisms are unknown, a history of NMSC should increase the clinician's alertness for certain noncutaneous cancers as well as melanoma.     

Ultrastructural study of anti-tumor effects of tamoxifen in two malignant melanoma patients

In order to clarify the mode of action (tumor cell death) of tamoxifen in treatment for estrogen receptor (ER) negative malignant melanoma, we administered the usual adult dose (20 mg/day) or a low dose, 1/4 of the usual dose (5 mg/day), of tamoxifen for 2 months to 2 male patients and investigated ultrastructural changes in their melanoma cells from metastatic lesions before and after the treatment. After the 2-month administration, metastatic nodules in both patients were reduced in size by approximately 50%. Histologically, their reduced nodules presented coagulation necrosis around the blood vessels. Electron microscopy of the necrosis revealed that melanoma cells were degenerated and disappeared; numerous aggregated melanosomes, free melanosomes, granular endoplasmic reticula, and lysosomes were present in the extracellular matrix and in the space between collagen fibers. The remaining melanoma cells had swollen cytoplasm and mitochondria with vacuolar changes. Cristae of mitochondria had disappeared. There was no infiltration of lymphocytes into the nodules. The organic changes of necrosis lesions were not observed. Because our two patients were ER negative, these effects of tamoxifen could be attributable to an action not mediated by ER.     

Differential activity of P-glycoprotein in normal blood lymphocyte subsets

To better understand the phenomenon of P-glycoprotein (P-170) expression we investigated lymphocyte subpopulations for P-170 function in healthy volunteers. Studies were based on three-colour flow cytometry including the fluorescent probe rhodamine 123 (Rh123), which is transported by P-170. Marked Rh123 efflux was detected in CD8+ T lymphocytes with CD8+/CD45RA+ T cells (naive cells) showing significantly higher P-170 activity as compared with CD8+/CD45RA- cells (P<0.04). Vice versa, CD8+/CD45RO+ T cells (memory cells) demonstrated less P-170 activity than CD8+/CD45RO- cells (P<0.04). P-170 function was less prominent in CD4+ T cells, however, Rh123 efflux was higher in the CD4+/CD45RA+ and CD4+/CD45RO- subpopulations (P<0.025) corresponding to the CD8+ results. Dye efflux differed significantly between activated and non-activated CD8+ and CD4+ as well as CD8+/CD11b+ and CD8+/CD11b- T lymphocytes. Since CD16+ natural killer cells (NK) expressed the highest level of P-170, the NK cytotoxicity against 51Cr-labelled K562 target cells was assayed in the presence or absence of P-170 inhibitors. NK related cytotoxicity was significantly reduced in the presence of R-verapamil and dexnigaldipine-HCP in a dose-dependent manner. The differential expression of P-170 activity in naive and memory T cells together with the reduced NK related cytotoxicity in the presence of MDR-modulators suggest a physiological role of P-170 in immunological functions of these lymphocyte subsets. Consequently, the addition of MDR modulators to conventional chemotherapy as a strategy to overcome drug resistance should consider possible adverse immunosuppressive effects.     

Human tumor cells grown in fetal calf serum and human serum: influences on the tests for lymphocyte cytotoxicity, serum blocking and serum arming effects

Peripheral blood lymphoid cells (PBL) from cancer patients and normal donors were tested against three melanoma cell lines grown in either 10% fetal calf serum (FCS) or 2.5-5% human AB serum in order to determine if the heterologous membrane (HM) antigen or other FCS antigens acquired from the bovine serum supplement could influence lymphoid cell-mediated cytotoxicity in vitro. FCS-grown melanoma cells were more susceptible than the AB serum-grown subline to lymphocyte cytotoxic effects. Arming effects by autologous sera on normal donor lymphocytes and to a lesser extent on lymphocytes of cancer patients were more pronounced on the FCS-grown M12 melanoma cells. This effect was abrogated when the cells were grown in human AB serum for at least 8 weeks. The non-HM tumor-associated antigen remained at the same original low level. Blocking effects were more evident on the AB-grown M14 melanoma line. These data suggest that the FCS antigens on the cell surface may have been responsible for the augmented PBL cytotoxicity. The anti-FCS antibody present in normal and cancer patients' blood induced an antibody-dependent cellular cytotoxicity (ADCC). Elimination of arming activity against HM or other FCS antigens from AB-grown cells may have made the serum blocking factors more apparent. However, cytotoxicity against tumor cells by PBL from normal donors was still apparent even on the human serum-grown cells, suggesting that a different antigen-antibody system was also responsible for this "non-specific" activity.     

Interleukin-2 and lymphocyte-induced eosinophil proliferation and survival in asthmatic patients

BACKGROUND: Eosinophils are increased in the airways and blood of asthmatic patients. However, the mechanism of regulation of eosinophilia is incompletely understood. METHODS: To study the potential effect of asthmatic lymphocytes on eosinophils, lymphocytes from the blood of asthmatic patients in exacerbation, or from healthy subjects, were isolated and cultured in medium alone (LC-CM) or with interleukin-2 (IL-2-CM) (125 U/ml), and the effect of supernatant obtained from these cultures on eosinophil proliferation from progenitors and survival was studied. RESULTS: IL-2-CM from asthmatic patients significantly increased eosinophil colony formation from asthmatic blood but had no effect on colony formation from the blood of healthy subjects. IL-2-CM from asthmatic patients also significantly prolonged the survival of eosinophils. IL-2 alone and IL-2-CM from healthy subjects had no effect on eosinophil proliferation and survival. Asthmatic lymphocytes had more IL-2 receptors (CD25) than normal lymphocytes, and this difference persisted even after culture in IL-2. However, upregulation of the CD25 receptor on normal lymphocytes by incubation with concanavalin A led to the production of IL-2-CM, which did not increase eosinophil proliferation from progenitors. CONCLUSIONS: Lymphocytes from asthmatic patients but not from normal blood can significantly increase eosinophil proliferation and survival. The effects on eosinophil proliferation do not seem to be directly related to the presence of increased CD25 expression on lymphocytes.