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Study objectives: To determine the different effects of interleukin (IL)-2, IL-4, IL-7, IL-10, IL-12, and/or T-cell receptor (TCR)-CD3 engagement in recovering the functions of cytotoxic T lymphocytes (CTL) from malignant [...]

This article examines the effects of voluntary wheel running on the natural immune function of spontaneously hypertensive rats. Results indicate that the overall running distance and duration of training is associated with significant improvements in natural immune function but rest periods during prolonged training help to maintain the beneficial effects.

Fetal blood levels of neopterin increase as the baby grows, indicating a functioning immune system. Neopterin is produced by immune cells called macrophages and monocytes.

Background: The transmembrane receptor Fas, together with its protein-binding partner (Fas ligand), is a key regulator of programmed cell death (i.e., apoptosis). Fas and Fas ligand also influence the ability of cytotoxic T lymphocytes and natural killer cells to eliminate tumor cells. However, by inducing apoptosis in activated T cells, the Fas/Fas ligand system may protect some tumor cells from clearance by the immune system. Anticancer drugs enhance Fas ligand expression on the surface of Fas receptor-expressing leukemia cells, thus suggesting that apoptosis caused by these drugs may be mediated via the Fas/fas ligand system. Purpose: This study was conducted to further investigate the relationship between the modulation of Fas receptor gene and protein expression by treatment of cells with cytotoxic drugs and the immune clearance of tumor cells. Methods: Fas expression on human HT29 colon carcinoma cells treated with a variety of anticancer drugs (cisplatin, doxorubicin, mitomycin C, fluorouracil, and camptothecin) was analyzed by use of quantitative flow cytometry. Human HCT8R and HCT116 colon carcinoma cells and human U937 leukemia cells were treated with cisplatin only and analyzed in the same way. Fas ligand messenger RNA and protein levels were studied by use of a reverse transcription-polymerase chain reaction assay and by flow cytometry. Fas gene expression and messenger RNA levels in cisplatin-treated HT29 cells were characterized by use of in vitro nuclear run-on and northern blot hybridization assays. The cytotoxic activities of agonistic anti-fas antibodies, Fas ligand, and allogeneic peripheral blood leukocytes, in the absence or presence of Fas-blocking monoclonal antibodies, against tumor cells were assessed by methylene blue staining and chromium-51 release assays. Results: Clinically relevant concentrations of cisplatin, doxorubicin, mitomycin C, fluorouracil, or camptothecin enhanced Fas receptor expression on the plasma membrane of HT29 cells. Cisplatin-mediated increases in Fas expression were confirmed in HCT8R, HCT116, and U937 cells. The enhancement of Fas protein expression was associated with an increased sensitivity of cisplatin-treated tumor cells to agonistic anti-fas antibodies, to soluble Fas ligand, and to allogeneic peripheral blood leukocyte-mediated cytotoxicity. Each of these effects was blocked by co-treatment of the cells with antagonistic anti-Fas antibody. Conclusion and Implications: In addition to their direct cytotoxic effects, chemotherapeutic drugs sensitize tumor cells to Fas-mediated cytotoxicity and Fas-dependent immune clearance. On the basis of these findings, new strategies might be developed to improve the efficacy of these drugs.

The NOD/Lt mouse, a widely used model of human autoimmune IDDM, was used to establish the mode of Β-cell death responsible for the development of IDDM. Apoptotic cells were present [...]

Background: Epidermal growth factor receptor (EGF-R) perturbation by receptor ligand(s), e.g., epidermal growth factor (EGF) and transforming growth factor-[Alpha] (TGF-[Alpha]), or receptor-specific antibodies accentuates cisplatin-induced toxicity in tumor cells. This sensitization occurs only in tumor cells with high expression of EGF-R but not in those with low expression of EGF-R. Purpose: Therefore, we have studied the role of EGF-R expression on cisplatin-mediated cytotoxicity. Methods: MIDA-468 human breast cancer cells were stably transfected with a p-chloramphenicol acetyl transferase (pact[p]-CAT) vector containing a 4.1-kilobase full-length antisense EGF-R complementary DNA. EGF-R content was assessed by [sup.125.I-EGF] binding and EGF-R immunoblot assays. Cisplatin sensitivity was evaluated by (a) colony-forming assay in vitro, (b) xenograft growth in nude mice, (c) cell cycle distribution of propidium iodide-labeled DNA, (d) DNA fragmentation in agarose gels, and (e) terminal deoxynucleotidyl transferase (Tdt) fluorescence in situ. Cisplatin uptake was measured by atomic absorption spectroscopy, and the levels of drug-DNA intrastrand adducts were determined by a dissociation-enhanced fluoroimmunoassay that utilizes an antibody against cisplatin-modified DNA. Results: Selected clones (MDA-468/AS-EGFR) exhibited more than 90% loss of both [sup.125.I-EGF] binding and receptor content determined by western blot analysis, whereas clones transfected with the vector alone (MDA-468/p-CAT) had EGF-R levels similar to those of the parent cells. By use of a colony-forming assay, the 1-hour [IC.sub.50] (i.e., the concentration of drug required for 1 hour to achieve 50% cell kill) for cisplatin was 2 [Mu]M or less for parental and vector-transfected clones (n = 4), whereas it was 25 [Mu]M or more for all MDA-469/AS-EGFR clones (n = 3). MDA-468/p-CAT clones exhibited internucleosomal DNA fragmentation, enhanced Tdt-end labeling in situ, and [G.sub.2] arrest 48 hours after a 1-hour incubation with 3-30 [Mu]M cisplatin. Under these conditions, apoptosis and [G.sub.2] arrest were undetectable in all MDA-468/AS-EGFR clones. An MDA-468 subline selected after long-term treatment with a TGF-[Alpha]-Pseudomonas exotoxin A fusion protein 40 lacked EGF binding and also exhibited cisplatin resistance (1-hour [IC.sub.50]: [is greater than] 30 [Mu]M) compared with parental cells. This EGF-R-dependent difference in cisplatin response was confirmed in a nude mouse xenograft model by use of high- and low-EGF-R-expressing cell clones. Total intracellular drug accumulation after a 1-hour cisplatin exposure, as measured by atomic absorption spectroscopy, was identical in both groups of cells. Intrastrand drug-DNA adducts, however, were statistically higher in high EGF-R expressors than in low-EGF-R-expressing clones. Conclusions: These data indicate that a critical level of EGF-R signaling, which is amplified in some common human cancers, is necessary for cisplatin-mediated apoptosis in tumor cells and suggest an inhibitory effect of this pathway on the repair of cisplatin-damaged DNA. [J Natl Cancer Inst 1997;89:365-73]

Background: Etoposide, an inhibitor of the normal religation activity of the nuclear enzyme topoisomerase II, can induce a secondary acute myeloid leukemia characterized by site-specific DNA rearrangements. The schedule of drug administration appears to be a clinical risk factor for this devastating treatment complication. Purpose: We tested the hypothesis that prolonged exposure of leukemia cells in vitro to low concentrations of etoposide, compared with short exposures to high concentrations, could produce equivalent or greater desired cytotoxic effects, with decreased occurrence of undesired site-specific double-stranded DNA recombinational events (i.e., recombinogenesisi. Methods: We used the frequency of V(D)J (variable-diversity-joining) recombinase-mediated deletions of exons 2 and 3 of the hypoxanthine phosphoribosyltransferase (HPRT) gene as a biomarker of etoposide-induced, nonhomologous, site-specific DNA rearrangement. A polymerase chain reaction-based technique was used to measure exon 2 + 3 deletions in human lymphoid leukemia CCRF-CEM cells 6 days after either 4-hour or 24-hour treatment with etoposide at clinically relevant concentrations. Cytotoxic effects of etoposide (determined by the number of viable cells present in the treated compared with the control [i.e., untreated] cells) were measured 6 days after treatment of the cells. The frequency of the exon 2 + 3 deletion following the two treatment-duration conditions was compared by use of the Mantel-Haenszel statistic. All P values resulted from two-sided tests. Results: Cytotoxicity increased with increasing etoposide concentration and exposure duration, as expected. By day 6, the frequency of exon 2 + 3 deletions was signiricantly higher (global P value = .0003) after the 4-hour treatment than after the 24-hour treatment, regardless of whether the frequency was assessed at etoposide concentrations achieving equivalent (e.g., 95%) cytotoxicity (14.2 x [10.sup.-7] versus 4.1 x [10.sup.-7]) or at equivalent etoposide concentrations (e.g., 1 [mu]MM) (10.8 x [10.suup.-7] versus 1.3 x [10.sup.-7]. Thus, the ratio of desired cytotoxic to undesired recombinogenic effects was higher with the 24-hour schedule. After the treated cells were subeloned at limiting dilutions, the frequency of the exon 2 + 3 deletion increased from 16.3 x [10.sup.-7] to 4.33 x [10.sup.-3] indicating that the recombinational event is not necessarily lethal. Conclusion: For all drug concentrations and levels of cytotoxicity studied in CCRF-CEM cells, there was a greater ratio of cytotoxicity to genetic recombination following prolonged exposure to etoposide than following brief exposure. Implication: These data suggest that recombinogenesis is not inextricably linked to cytotoxicity. If confirmed in the clinical setting, the use of prolonged dosage schedules may provide a means to decrease the risk of etoposide-induced acute myeloid leukemia without compromising treatment efficacy.

BACKGROUND: Laparotomy under general anesthesia is associated with depressed natural killer cell cytotoxicity (NKCC) and compromised clearance of tumor cells. We tested the hypothesis that awake epidural anesthesia (AEA) improves NKCC compared to conventional general endotracheal anesthesia (GEA). PATIENTS AND METHODS: Preoperative, perioperative, and postoperative (day 3) NKCC, plasma epinephrine, norepinephrine, cortisol levels, and 24-hour urinary cortisol levels were measured in 20 patients undergoing open colectomy under either AEA or GEA. RESULTS: Preoperative and postoperative measurements were not significantly different in the two groups. Patients receiving GEA had a significant reduction in NKCC from 36% [+ or -] 4% preoperatively to 22% [+ or -] 4% perioperatively (P = 0.02). Patients receiving AEA had no significant change in NKCC. Perioperative plasma epinephrine and cortisol levels were higher with GEA than AEA. The perioperative 24-hour urinary cortisol excretion values were significantly higher in the group receiving GEA, suggesting a greater stress hormone response in this group compared to AEA patients. CONCLUSIONS: Compared to GEA,AEA appears to preserve perioperative NKCC. This effect may be related to an attenuated stress hormone response associated with AEA. Cancer patients may have improved killing of embolized tumor cells during surgery performed under AEA. Am J Surg. 1996;171:68-73.

Background. Lymphokine-activated killer (LAK) cell function can be generated in peripheral blood mononuclear cells (PBMC) after brief exposure of high dose interleukin-2 (IL-2) over the course of 1 or 2 days, culture in plain culture medium (IL-2-pulsed PBMC). The aim of the present study was to investigate the ability of granulocyte-macrophage-colony stimulating factor (GM-CSF ) to augment LAK induction in low dose IL-2-pulsed PBMC derived from patients with cancer undergoing immunotherapy with IL-2. Methods. Peripheral blood mononuclear cells were collected from patients with cancer receiving a 5-day cycle of local (intraperitoneal or intrapleural) infusions with IL-2. The cells were incubated with IL-2 in the presence or absence of GM-CSF for 1 hour and then tested as effectors against allogeneic tumor cells and LAK-sensitive cell lines. Results. Granulocyte-macrophage-colony stimulating factor at doses between 10 and 100 ng/ml was synergized with low dose IL-2 (100 IU/ml) in the generation of LAK activity in PBMC. Lymphokine-activated killer cell-mediated cytotoxicity derived from PBMC cultures incubated with IL-2 and GM-CSF was significantly higher (up to three-fold) compared with that generated with IL-2 alone. The GM-CSF-induced enhanced LAK activity was maintained when tested at day 5. GM-CSF increased the percentages of IL-2 receptor (R) positive (+) and CD8+ cells in the IL-2-pulsed PBMC. In contrast to CD56+ cells, highly purified CD8+ cells isolated from PBMC pulsed with IL-2 and GM-CSF responded with increased LAK activity, thus representing the cell-type that mediates the augmenting effect of GM-CSF. Major histocompatibility complex (MHC) molecules or the CD3 surface antigens were not involved in the GM-CSF-mediated enhancement of LAK induction because anti-MHC class I and class II monoclonal antibodies (MoAb) or MoAb against the CD3 molecules remained without any effect in this system. The GM-CSF-mediated LAK-enhancement was IL-2-dependent because MoAb against IL-2 receptor completely inhibited the generation of LAK activity. Conclusions. The use of GM-CSF for the enhancement of IL-2-induced LAK activity in 1 hour cultures may improve clinical results in cancer immunotherapy. In addition, implementation of this procedure could eliminate the high cost of cell culture which usually accompanies IL-2/LAK cell therapy as well as eliminate the known toxic side effects associated with this kind of therapy. Cancer 1995;76:1253-60.

Background. Tumor rejection antigens in human melanomas, which are recognized by cytotoxic T lymphocytes (CTLs), have recently been identified. To elucidate the cytotoxic mechanism in tumors other than melanoma, several pairs of CTLs and tumor lines were established. The authors report that HLA-A31 may present a tumor rejection antigen that is recognized by the human autologous gastric signet ring cell carcinoma-specific CTL. They also briefly describe the in vitro enhancing effect of interferon-gamma (INF-[gamma]) on the lysis of tumor cells by autologous CTL. Methods. The MHC Class I-restricted CTL clone, TcHST-2, and autologous gastric signet ring cell carcinoma line, HST-2, were established. Cytotoxicity blocking assays of antibodies reacting against the MHC Class I nonpolymorphic determinant and HLA-A, B, and C haplotype elements, which are expressed on the HST-2 cells, were performed. Results. Lysis of the autologous tumor cells (HST-2) by the CTL clone (TcHST-2) was enhanced when the tumor cells were pretreated with IFN-[gamma]. This lysis was selectively inhibited by the anti-nonpolymorphic MHC Class I determinant monoclonal antibody (MoAb) and anti-HLA-A31 haplotype-specific MoAb. However, TcHST-2 clone was not cytotoxic to HLA-A31+ allogeneic leukemia lines. Conclusion. Pretreatment of target cells with IFN-[gamma] may be a necessary procedure for the efficient lysis of HST-2 cells by autologous TcHST-2 CTL. The data indicate that TcHST-2 was MHC Class I-restricted HST-2 tumor-specific CTL and suggest that the HLA-A31 haplotype element is an antigen-presenting molecule. Also discussed is the nature of the antigenic peptides in gastric signet ring cell carcinoma. Cancer 1995; 75:1484-9.

Insulin-dependent diabetes mellitus (IDDM) results from a T-cell-mediated destruction of the insulin-producing β-cells. In this study, we designed a sensitive assay to detect and identify islet cell-reactive T-cells in patients with newly diagnosed IDDM. The relation between T-cell recognition of β-cell antigens with IDDM and the pathogenesis of the disease (the β-cell destruction process) was tested in a large group of IDDM patients and compared with T-cell responses in nondiabetic children with other chronic inflammations and in immunologically normal, age-matched control subjects. The results demonstrate that peripheral blood T-cells reacting with a β-cell membrane preparation enriched for insulin-secretory granule antigen were detectable in the majority of newly diagnosed IDDM patients (27 of 40 [67%]; mean stimulation index [SI] 37.0). Such reactivity was reduced post-onset in IDDM patients proportionally to the duration of the disease (11 of 30 l37%]; mean SI 8.7). Nondiabetic age-matched control subjects showed no responses or moderate responses to the granule preparation (4 of 48 [8%]; mean SI 3.4). The magnitude of the T-cell response was significantly greater in newly diagnosed IDDM patients than in IDDM patients tested at least 2 years postonset (P < 0.001). Two children in remission for insulin dependency (so-called honeymoon period) displayed exceptionally high proliferative responses to insulin-secretory granules (mean SI 86.7). These results imply that T-cell recognition of insulin-secretory granule antigens is associated with IDDM and in particular with the immune-mediated process of β-cell destruction. Diabetes 44:278-283, 1995, Insulin-dependent diabetes mellitus (IDDM) is the result of a genetically controlled autoimmune-mediatec process in which the insulin-producing pancreati β-cells are thought to be destroyed by autoreactive T-cells (1-3). The disease [...]

Background. The nonsteroidal antiestrogenic drug, tamoxifen, inhibits the growth of estrogen receptor-positive tumors by interfering with the growth-stimulatory effect of estradiol. However, there is compelling evidence that tamoxifen treatment also is beneficial for patients with estrogen receptor-negative tumors. The hypothesis that tamoxifen is capable of enhancing the immunologic defense of tumor-bearing hosts was been investigated as a possible method for targeting receptor-negative neoplasms. Methods. Natural killer (NK) cells in the spleen of female Fisher and Wistar--Furth rats were used against the YAC-1 murine lymphoma target in [.sup.51Cr-release] assays. The effect of various concentrations of estradiol and tamoxifen (1 nM--1 [micro]M) and of the metabolic inhibitors actinomycin D and cyclohexamide on target-cell killing was investigated. Results. Tamoxifen enhanced and estradiol inhibited killing if applied for the entire 5 hours of the cytotoxic reaction. When applied jointly in this experimental setup, estradiol interfered with the enhancing effect of tamoxifen. Pretreatment of target cells with tamoxifen led to highly significant enhancement of cytotoxicity; estradiol also enhanced target cell killing, but to a lesser extent. After joint treatment, the level of cytotoxicity was comparable with that obtained with tamoxifen alone. Both pharmacologic (100 nM and 1 [micro]M) and physiologic (1 or 10 nM) concentrations of estradiol and equimolar tamoxifen enhanced target cell lysis. However, pharmacological levels of estradiol inhibited effector cells when applied alone or in combination with tamoxifen. Highly significant enhancement of target-cell destruction occurred if both target and effector cells were pretreated with tamoxifen, whereas estradiol treatment of both cell types led to slight enhancement or no effect on cytotoxicity. Treatment of the target cells with actinomycin D or cycloheximide inhibited the lysis of untreated and tamoxifen- or estradiol-exposed cells. Treatment of YAC-1 target cells with tamoxifen or estradiol also enhanced the NK cell-mediated release of the nuclear label, [.sup.3H-thymidine,] indicating DNA degradation. Similarly treated P815 cells resisted lysis by NK cells, but showed sensitization when the NK cells were stimulated by interleukin-2 for 48 hours before the lytic reaction. Estradiol and tamoxifen changed the kinetics of [.sup.3H-thymidine] incorporation by YAC-1 cells, but the cells were capable of growing with the highest drug concentrations (1 [micro]M) used in the cytotoxicity experiments. YAC-1 cells have no cytosolic estradiol receptors and are weakly positive for cytosolic progesterone receptors. Conclusions. These experiments indicate that NK cell-mediated target-cell destruction can be enhanced by tamoxifen primarily through sensitizing the target for lysis. Estradiol also sensitizes the target but inhibits the effector cells simultaneously so that little or no change results in cytolysis. Target-cell sensitization is not mediated by classical estrogen receptors and requires the active metabolic participation of the cells treated. A likely mechanism of this phenomenon is priming the target cell for apoptosis.

Background. Patients with testicular cancer can be cured by cisplatin-based chemotherapy in many cases. Thus, concern about possible late toxicities of treatment is warranted. Methods. In this investigation, the absolute number of natural killer (NK) cells according to their CD56+ phenotype, NK cell activity, and antibody dependent cellular cytotoxicity (ADCC) were investigated in 29 patients with seminomas or nonseminomatous germ cell tumors (NSGCT) a median of 31 months (range, 5-73 months) after termination of chemotherapeutic treatment. Results. No difference in the absolute number of NK cells, NK cell activity, and ADCC was found between patients with testicular cancer after either standard polychemotherapy consisting of bleomycin, etoposide, and cisplatin (BEP) or monotherapy with carboplatin and healthy control subjects. When patients were analyzed further using multivariate analysis, a significant (P < 0.05) detrimental influence of BEP polychemotherapy versus carboplatin monotherapy on NK cell activity was found. Moreover, NK cell activity also depended significantly (P < 0.05) on the time elapsed after chemotherapy was administered, but normalized with time. Because the absolute number of NK cells was not affected, is was assumed that polychemotherapy induced a functional defect. In a subanalysis of patients with NSGCTs, metastases at diagnosis resulted in a significant (P < 0.05) and persistent stimulation of NK cell activity but not of ADCC. Conclusion. Cytostatic chemotherapy in patients with testicular cancer did not lead to compromised lytic effector cell function as assessed by NK cell activity and ADCC. However, a short, time-dependent reduction was found that also depended on the intensity of chemotherapeutic treatment. This finding related to the observation of a long-lasting stimulus of NK cell activity by initial metastases in patients with NSGCTs.

CD4+ lymphocytes appear to be selectively destroyed by cytotoxic T-lymphocytes (CTL) in HIV-infected individuals. CD4+ lymphocytes are one of several kinds of lymphocytes, which make up about one-quarter of the white blood cells in the human body. Lymphocytes act as part of the immune system that fights disease infection. Infection with HIV causes an increase in the number of CD8+ cells and also CTL. Research indicates that the CTL produced as part of the expansion of CD8+ lymphocytes targets HIV-infected CD4+ cells in particular. CTL have little or no effect on CD8+ cells or other lymphocytes. The reduction in CD4+ lymphocytes is an important part of the progression from HIV infection to AIDS.

Background: Camptothecins belong to a group of anticancer agents with a unique mechanism of action: poisoning of eukaryotic DNA topoisomerase I. Two camptothecin derivatives, topotecan (TPT) and CPT-11, are in clinical trials and their chemotherapeutic efficacy appears promising. Purpose: Our aim was to compare simultaneously the molecular and cellular pharmacology of the various camptothecin derivatives that are presently in clinical trials. Methods: Cytotoxicity of drugs toward human colon carcinoma HT-29 cells was determined by colony-forming assays. DNA single-strand breaks (SSB) were measured by alkaline elution. Drug potency to induce topoisomerase 1-mediated DNA cleavage and the sequence selectivity of the breaks were determined by sequencing gel autoradiography. Results: SN-38 38 CPT were more cytotoxic than 9-AC and TPT, and CPT-11 was almost inactive toward HT-29 cells. [IC.sub.50] values were 8.8 nM for SN-38, 10 nM for CPT, 19 nM for 9-AC, 33 nM for TPT, and greater than 100 nM for CPT-11. In drug-induced DNA damage measured by alkaline elution drug concentrations producing 1000-rad-equivalents ([C.sub.1000]), values were 0.037 [mu] M for SN-38, 0.051 [mu] M for CPT, 0.085 [mu] for 9-AC, 0.28 [mu] M for TPT, and greater than 1 [mu] M for CPT-11. SN-38 remained the most potent compound in isolated nuclei, and CPT-11 was still inactive. The potency ranking was the same as in whole cells, and the [C.sub.1000] values were 0.0025 KM for SN-38, 0.012 M for CPT, 0.021 M for 9-AC, 0.44 M for TPT, and greater than 0.1 JAM for CPT-11. Potency difference between SN-38 and the other compounds was greater in isolated nuclei than in whole cells. Conclusions: Kinetics of the reversal of drug-induced SSB in isolated nuclei suggest that dissociation of SN-38 from cleavable complexes is much slower than that of CPT. Cleavage patterns of CPT and 9-AC were similar but differed from those of TPT and SN-38. Although in vitro analyses do not necessarily reflect chemotherapeutic efficacy, this study found that SN-38 is the most potent compound and that 9-AC and TPT are less active than CPT in this system. The effect of CPT-11 is minimal. Therefore, the clinical activity of CPT-11 may strongly depend on its hydrolysis to SN-38. Differences in DNA sequence selectivity and the stability of cleavable complexes induced by the drugs may also contribute to differences among CPT derivatives.

The number of CD8+CD28- T-cells in the blood and lungs of HIV-positive patients is higher than in healthy patients. A study of 13 HIV-positive patients and 14 healthy volunteers compared the presence of these lymphocytes using blood samples and bronchial washings. The number of CD8+CD28- cells constituted only 25% of total CD8+ cells in the blood of healthy subjects, compared with 74% of the total in the HIV patients. Higher numbers of these T-cells were also found in the lungs. The higher percentage in HIV-positive individuals may indicate activation by the immune system or expansion of a existing CD28- subset group. The lack of CD28 surface expression on HIV patients' T-cells may have significant and adverse consequences for the body's ability to produce T-cells.

Activation of HIV-1-specific cytotoxic T cells (CTL) may induce replication of HIV type 1 (HIV-1) in chronically infected T cells. CTLs are cells of the immune system that can recognize and lyse target cells with specific types of antigens on their surface. A study examined the effect of HIV-1-specific CTLs on HIV-1 replication in chronically infected T cells in the laboratory. Activation of CTLs with subunits of protein induced replication of HIV-1. Increased replication of HIV-1 was associated with elevated production of tumor necrosis factor-alpha (TNF-alpha) by CTLs. Antibodies against TNF-alpha could inhibit the effect of activated CTLs on HIV-1 replication.

Background. Previous studies have demonstrated that tumor cells have different degrees of sensitivity and resistance to various cytotoxic agents. The acquisition of drug resistance is a major concern in cancer treatment. The current study investigates the cytotoxic effect of cisdiamminedichloroplatinum (II) (CDDP) and tumor necrosis factor-alpha (TNF-[Alpha) used in combination on CDDP-resistant human ovarian tumor cell lines. Methods. Cytotoxicity was determined by the microculture tetrazolium dye assay. Synergy was assessed by isobolographic analysis. TNF-mRNA was examined by Northern blot analysis. Results. Treatment of the CDDP-resistant C30 cells with CDDP and TNF-[Alpha] overcame the resistance of C30 cells to CDDP or TNF-[Alpha]. In addition, the combination of CDDP and TNF-[Alpha] resulted in a synergistic effect on the C30-resistant line, the CDDP-sensitive parental cell line A2780, and two freshly derived ovarian carcinoma cell cultures. Treatment of C30 cells with CDDP followed by TNF-[Alpha] showed a synergistic effect, whereas treatment with TNF-[Alpha] followed by CDDP demonstrated a less cytotoxic effect. A possible mechanism of resistance to TNF-[Alpha] in tumor cells is the induction of TNF-[Alpha] mRNA and protein. C30 cells do not produce mRNA constitutively for TNF-[Alpha]; however, treatment of C30 cells with TNF-[Alpha] induces the expression of TNF-[Alpha] mRNA. When CDDP was used in combination with TNF-[Alpha], the level of TNF-[Alpha] mRNA induced by TNF-[Alpha] was reduced significantly. Conclusions. This study shows that the combination of CDDP and TNF-[Alpha] can overcome the CDDP resistance of tumor cells and that downregulation of TNF-[Alpha] mRNA by CDDP may play a role in the enhanced cytotoxicity seen with the combination of CDDP and TNF-[Alpha]. The synergistic effect obtained with established ovarian tumor cell lines and in short-term cultures of freshly isolated ovarian tumors suggests that combination treatment with TNF-[Alpha] and CDDP may have clinical applications in the treatment of drug-resistant tumors.

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.

Background. Locomotion of peripheral blood lymphocytes (PBL) through peritumoral matrix is obligatory for tumor cell killing. The authors investigated the effect of surgery on lymphocyte locomotion and compared it with the effect on natural killer cell cytotoxicity (NKCC). Methods. PBL from 12 patients with cancer were assessed for locomotion in Type I rat-tail collagen. Preoperative and postoperative locomotion (after 20 hours of incubation) and NKCC were estimated. Results. Locomotion of lymphocytes through collagen increased significantly after operation in 6 of 12 patients, whereas only 1 of 12 had a decrease (P < 0.001). Short-term (20-hour incubation) exposure of the locomotory HSB cell line to patient plasma samples did not affect their migration. NKCC, as estimated against K562 target cells with the use of the [sup.51]Cr-release assay, decreased 5-50% after operation in 9 of 12 patients (P = 0.006). No correlation could be demonstrated between the changes in locomotion and NKCC (regression analyses), nor were identifiable clinical factors associated with these changes. Conclusions. Locomotion of PBL through collagen increases after operation in patients with cancer, whereas possibly independent factors may decrease postoperative NKCC. Cancer 1993; 71:2833-7.

Background. Previously, autologous Burkitt lymphoma-specific cytotoxic T-lymphocytes (CTL) were found to express the gamma and delta T-cell receptor and recognize tumor idiotype in a major histocompatibility complex (MHC) unrestricted fashion. Methods. In this study, the authors established autologous CTL lines and clones specific for a B-cell follicular lymphoma. Results. These CTL are tumor specific and inhibited by antiimmunoglobulin monoclonal antibodies, but unlike the Burkitt lymphoma-specific CTL, they are MHC restricted and express the alpha and beta T-cell receptor. Conclusions. These studies suggest that different B-cell lymphomas can induce CTL of different phenotypes and MHC restriction. Cancer 1992; 70:2181-2186.

In this study the characteristics of the cytotoxic function in a series of patients with metastatic renal cell carcinoma (RCC) were analyzed and the possibility of modulating this capacity in vitro with the use of biologic response modifiers (BRM) such as alpha-interferon([alpha]-IFN) and recombinant interleukin-2 (rIL-2) was verified, with the ultimate goal of providing a rationale for a therapeutic approach to this disease with these molecules. Peripheral blood mononuclear cells (PBMC) of patients with advanced RCC were tested for natural killer (NK) and lymphokine-activated killer (LAK) activity both before and after [alpha]-IFN therapy. In addition, surface markers of unstimulated and stimulated cells were analyzed and in vitro assays were performed to determine the proliferative capacity in response to the stimulus with rIL-2. During an evaluation before treatment, defective NK activity was observed that could be corrected by incubating the cells with rIL-2. In these subjects, LAK cells could be consistently generated after PBMC were activated with this cytokine in vitro. No changes in NK and LAK activity were found after alpha-IFN therapy. In contrast, treatment with [alpha]-IFN affected the proliferative response of PBMC to rIL-2, and a significant decrease in this in vitro capacity was observed during follow-up. The ability to restore NK activity and obtain an adequate LAK cytotoxicity from the PBMC of patients with RCC supports a therapeutic approach with BRM. However, the fact that this type of treatment affects the proliferative response of PBMC to rIL-2 must be considered when clinical trials are designed for patients with RCC. Cancer 1992; 69:2525-2531.