Phenotype and function of natural killer cells in patients with bronchogenic carcinoma.

Decreased peripheral blood natural killer (NK) cell lytic activity may be associated with tumor presence. We evaluated peripheral blood NK lytic activity in 38 patients with bronchogenic carcinoma and compared this to ten normal volunteers of comparable age. The patients with carcinoma had significantly (P less than 0.001) less NK activity (20 +/- 17 lytic units at 25% specific lysis)/10(6) peripheral blood lymphocytes) against the K562 tumor target compared to normal (69 +/- 9, SD). NK subpopulations can be defined phenotypically using CD56, CD16, and CD3 monoclonal antibodies and express differing degrees of lytic activity. NK cells from patients with carcinoma had the same absolute number of CD56+ cells and percentage of CD56+CD16+ NK cells (the most lytic subpopulation). However, patients with carcinoma had significantly (P less than 0.001) more CD56+CD16-CD3- cells in their overall NK population. This is of note, since this subpopulation is the least lytic. Patient NK cells bound to tumor cells as effectively as those from normal volunteers; however, the maximum rate of kill of patient NK cells was significantly (P less than 0.001) less. Thus, decreased NK lytic activity in patients with carcinoma was not due to decreased numbers or proportion of NK cells in peripheral blood or to defective tumor cell binding, but rather to the large CD56+CD16-CD3-NK subpopulation which is characterized by minimal lytic activity. The relation of this NK cell population with the presence of carcinoma is currently under investigation.

Regulation of lymphokine-activated killer activity in T-replete and T-cell-depleted human bone marrow by interleukin 4.

Donor-derived lymphokine-activated killer (LAK) cells appear to play a role in mediating an antileukemia effect in recipients of both T-replete and T-cell-depleted (TCD) bone marrow transplants. LAK activity, however, is subject to regulation by cytokines other than interleukin 2 (IL-2). The purpose of this study was to examine the effect of interleukin 4 (IL-4) on the induction of LAK activity in both T-replete and TCD bone marrow. IL-4 inhibited the induction of LAK activity in a time- and dose-dependent manner in both T-replete and TCD bone marrow cultures, although there appeared to be a differential effect, suggesting that T and non-T LAK precursors have different thresholds of sensitivity to IL-4. Single-cell cytotoxicity assays indicated that IL-4 did not inhibit binding of LAK effectors to targets but did reduce the frequency of lytic conjugates. Kinetic analysis techniques demonstrated that IL-4 decreased the maximal rate of target cell lysis by IL-2-activated LAK precursors and inhibited the rate of lytic programming. These data indicate that IL-4 is able to regulate the induction of LAK activity in both T-replete and TCD bone marrow and may play a role in modulating the generation of effector cells with potential antileukemia reactivity in vivo.

Synthesis and biological evaluation of novel potent antagonists of the bombesin/gastrin releasing peptide receptor.

This paper reports the synthesis and antagonist activity of 20 C-terminal analogues of gastrin releasing peptide (GRP). The ability of each analogue to inhibit bombesin (BN) stimulated amylase release from rat pancreatic acini was determined, and those showing antagonist activity were further evaluated for their ability to inhibit BN-stimulated [3H]thymidine uptake in serum-starved 3T3 cells. The assays also included two known peptide antagonists, C (Leu14,psi 13,14]BN) and H (N-pivaloyl-GRP20-25-(R)-2-methyl-4-nonylamide) as positive controls. On the basis of these assays we suggest that a des-Met27,Leu26-psi[CH2NHCOCH3]GRP C-terminal octapeptide imparts antagonist activity. The two most active compounds are peptides 14 ([D-Phe19,Leu26-psi(CH2NHCOCH3)]GRP19-26) and 18 ([D-Phe19,Gln20,Leu26-psi(CH2NHCOCH3)]GRP19++ +-26). In their ability to inhibit BN-stimulated [3H]thymidine uptake, the IC50 of peptides C, H, 14, and 18 were 43.2, 31.2, 2.7, and 32.5 nM, respectively. In conclusion, the novel C-terminal psi[CH2NHCOCH3] bond promises to be a useful peptide backbone modification for imparting antagonism in GRP/BN analogues.

Manipulation of graft-versus-host disease for a graft-versus-leukemia effect after allogeneic bone marrow transplantation in AKR mice with spontaneous leukemia/lymphoma.

Graft-versus-host (GVH) disease can result in a beneficial graft-versus leukemia (GVL) effect after bone marrow transplantation in patients with malignant disease. In this report, we used bacteria-free AKR (H-2k) mice bearing advanced spontaneous T cell leukemia/lymphoma as a moel to evaluate the GVH and GVL effects of bone marrow transplantation using fully incompatible SJL (H-2s) donors. A therapeutic GVL effect, accompanied by increased leukemia-free survival, was obtained only when 0.5 X 10(6) allogeneic lymphocytes (lymph node cells) were added to the marrow inoculum. Transplantation of allogeneic bone marrow without added lymph node cells (or use of syngeneic cells) resulted in a significant increase in leukemia relapse; increasing the dose of allogeneic lymph node cells to 2.0 X 10(6) resulted in significantly higher GVH-associated mortality. Survival and therapeutic benefits were obtained only when the intensity of the GVH reaction was carefully controlled by manipulation of alloreactive lymphocytes present in the marrow. These results suggest, indirectly, that T cell depletion may abolish any GVL effect of marrow transplantation, even if the donor is mismatched with the host at the major histocompatibility complex. The frequency in the spleen of cytotoxic T lymphocytes (CTL) reactive against host alloantigens was estimated using limiting-dilution microcytotoxicity assays at various times after transplantation of allogeneic bone marrow with and without added lymph node cells. The average frequency of CTL was highest in mice that were given marrow plus lymph node cells and tested within the first four weeks after transplantation. The level of CTL activity measured in vitro was dependent on the dose of lymphocytes injected and correlated with both the GVL and GVH effects in vivo. Down-regulation of CTL activity against host, but not third-party, alloantigens in vitro was observed under limiting dilution assay conditions, leading to the suggestion that host-specific regulatory cells may be present in these allogeneic bone marrow chimeras.

Reactivity of in-vitro-expanded alloimmune cytotoxic T lymphocytes and Qa-1-specific cytotoxic T lymphocytes against AKR leukemia in vivo.

The ability of alloimmune spleen cells expanded in mixed leukocyte culture (MLC) and cloned cytotoxic T lymphocytes (CTL) to kill H-2-compatible leukemia in vivo was evaluated. In comparison with fresh alloimmune spleen cells, MLC-expanded cells had a significantly higher frequency of CTL reactive against leukemia targets in vitro. However, the reactivity of MLC-expanded cells against first-passage spontaneous AKR (H-2k) leukemia in vivo was significantly less than when an equivalent number of fresh alloimmune spleen cells was injected. Comparable antileukemia reactivity was observed in vivo only when the inoculum of MLC-expanded cells was 2-3-fold higher than that of fresh spleen cells. This relative ineffectiveness was attributed to the altered migration pattern of cultured cells in vivo. IL-2-dependent cloned CTL, specific for a normal lymphocyte antigen (Qa-1b) also present on leukemia cells, were derived from MLC-expanded cultures and tested in vivo. For cloned CTL, as with MLC-expanded cells, eradication of AKR leukemia in vivo was associated with the tissue distribution pattern of the injected effector cells. That is, an effective antileukemia reaction was achieved only in tissues in which effector and target proximity was maintained. Qa-1b-specific cloned CTL did not interfere with engraftment of autologous or allogeneic bone marrow in lethally irradiated host mice, nor did they cause any clinically evident graft-versus-host disease. These findings suggest that cloned CTL specific for a normal cell surface antigen with limited host tissue distribution, but present on tumor cells, could be used for adoptive immunotherapy, provided CTL and tumor cell proximity can be attained.

Migration patterns and functional activity of cloned cytotoxic T lymphocytes in syngeneic and allogeneic mice.

The quantitative distribution of cytolytic T lymphocytes (CTL) generated in mixed leukocyte culture (MLC) and an interleukin-2 (IL-2)-dependent, CTL clone (WRL-A3) was investigated in various tissues of irradiated syngeneic and allogeneic mice. In addition, the ability of the WRL-A3 CTL clone to remain viable and retain antigen specificity following in vivo passage was evaluated. Injection i.v. of 51Cr-labeled cultured CTL resulted in: (1) extensive deposition of cells in the lungs with significantly more lymphocytes being recovered in allogeneic as compared with syngeneic lung tissue; (2) minimal accumulation in spleen with more in syngeneic than in allogeneic tissue; and (3) no localization in blood, femurs, thymus, or lymph nodes. The migration rate of cultured CTL exiting the lung during the first 4 hr was markedly faster in syngeneic than in allogeneic recipients and was directly associated with the distribution of these cells in other tissues at 24 hr. The WRL-A3 CTL clone recovered from irradiated syngeneic and allogeneic lung tissue at 1, 3, 6, 8, and 13 days after i.v. injection remained viable, even though no exogenous IL-2 was administered to the recipient mice. The recovered cells proliferated when recultured with IL-2, and retained their antigen specificity for Qed-1b target cells after in vivo passage. These findings indicate that restricted and undesirable tissue distribution, rather than impaired viability or loss of antigenic specificity, is the major obstacle to successful use of cultured CTL for adoptive immunotherapy of disseminated cancer.

Lymphokine-activated killer cell activity in lung cancer.

This study evaluates local pulmonary immune effector cell lytic activity. Purified lymphocyte populations were isolated from BALF obtained from 18 patients with bronchogenic carcinoma, six patients with lung disorders other than cancer, and ten normal control volunteers matched for age and smoking history. These cells were evaluated for NK and LAK cell lytic activity against NK-resistant LAK-sensitive tumor targets (A549 pulmonary tumor and Daudi tumor cells) and an NK-sensitive tumor (K562); LAK activity was detected in BALF from 6 of the 18 patients with cancer. The remaining patients with cancer, the subjects with pulmonary disease other than cancer, and the normal volunteers had no detectable lytic activity. Peripheral blood lymphocytes from all subjects had only NK lytic activity and did not kill the pulmonary tumor target; AMs were not tumoricidal. Interleukin-2, which is required for LAK cell activation, was detected only in BALF recovered from the six patients with pulmonary LAK lytic activity. These results demonstrate that activated LAK cells, capable of killing pulmonary tumor cells, are present in BALF of some patients with bronchogenic carcinoma. This lytic LAK cell population represents a local pulmonary response against the lung cancer in the absence of systemic tumoricidal activity. The functional status of pulmonary immune effector cells, as well as the type and quantities of cytokines in the lung determine local responsiveness to bronchogenic carcinoma and may well control the course of this disease.

Elevated prostaglandin E2 levels in bronchoalveolar lavage fluid of patients with bronchogenic carcinoma.

Understanding local pulmonary immunoregulatory mechanism(s) in patients with carcinoma of the lung is an important step towards the development of innovative methods of treatment. Prostaglandin E2 plays an integral role in immunoregulation. Therefore, we evaluated PGE2 concentrations in BALF from 18 patients with bronchogenic carcinoma, compared to that from six patients with pulmonary diseases other than carcinoma and ten normal smokers of similar age. The level of PGE2 in patients with lung carcinoma (158.1 +/- 88.7 pg/ml) was significantly (p less than 0.001) higher than the other two groups (16.2 +/- 6.9 and 4.4 +/- 3.4 pg/ml). Levels of PGE2 also varied among patients with carcinoma of different cell types. Patients with SQCA had significantly (p less than 0.001) higher levels of PGE2 (242.7 +/- 29.4 pg/ml) than patients with ADCA or SCCA (82.3 +/- 27.9 and 66.3 +/- 15.2 pg/ml, respectively). Furthermore, there was a marked difference in PGE2 concentration between carcinomatous lung and clinically noninvolved lung in patients with SQCA and ADCA. Further study is warranted to determine the interactions between PGE2 and other cytokines (interleukin-1, IL-2, and tumor necrosis factor), as well as the activity of cytolytic lymphocytes (LAK cells) in the lungs of patients with bronchogenic carcinoma.

Association of increased prostaglandin E2 content in bronchoalveolar lavage fluid and intrathoracic malignancy.

Prostaglandin E2 (PGE2), a metabolite of arachidonic acid, plays an important role in immunoregulation. We have previously reported an increased PGE2 content in bronchoalveolar lavage fluid (BALF) in 18 patients who had primary lung cancer as compared with 6 patients who had benign lung diseases and 10 normal smokers. In this article, we report our further observation, including additional 73 patients with primary lung cancer, 11 patients who had carcinoma other than lung cancer with and without lung metastases, and 24 patients with various benign lung diseases. Prostaglandin E2 content of BALF in patients with primary lung cancer and patients with extrathoracic cancer with lung metastases was markedly increased to 283.7 +/- 423.8 and 278.3 +/- 303.4 pg/ml, respectively. In contrast, patients who had extrathoracic cancer without lung metastases and patients with benign lung diseases had PGE2 content in BALF of 22.6 +/- 6.6 and 27.2 +/- 12.4 pg/ml, respectively. Values between the subgroups of patients with lung cancer (primary and metastatic) and patients with benign diseases or extrathoracic cancer without lung metastasis were significantly different (p < .001). Follow up of eight patients who successfully underwent surgical resection of cancer showed decreased levels of PGE2 in all but two patients. Although the exact cause and mechanism of the observed increase of PGE2 in BALF of patients with lung cancer are not clear at this point, current study together with our previous observations clearly indicates that the presence of lung cancer, both primary and metastatic, is associated with an elevated PGE2 content of BALF. The clinical significance of this observation is not known. However, an increased amount of PGE2 in BALF might adversely affect the course of lung cancer, because PGE2 is a down regulator of immune response against infection and neoplasm. Further investigations into the mechanism(s) and the role of PGE2 in BALF are warranted.

Comparison of four cytokine regimens for mobilization of peripheral blood stem cells: IL-3 alone and combined with GM-CSF or G-CSF.

Improved methods for mobilization may reduce the number of aphereses required to collect adequate numbers of peripheral blood stem cells (PBSC) and hasten engraftment following high-dose therapy. Mobilization with cytokines alone enables engraftment after myeloablative therapy. The optimal cytokine regimen for mobilization has not been established. The study evaluated the effects of four interleukin-3-containing cytokine regimens administered during steady state hematopoiesis on PBSC mobilization in 30 patients with breast cancer or lymphoid malignancies. These regimens included IL-3 alone (Arm 1), sequential IL-3 --> G-GSF (Arm 2), sequential IL-3 --> GM-CSF (Arm 3) and combined IL-3 + G-CSF (Arm 4). Consecutive days of apheresis were performed until a target of 4-6 x 10(8) mononuclear cells/kg were collected. All patients received intravenous GM-CSF after PBSC infusion. Median days to an ANC > or = 500/ microliters in Arm 3(22 days) was significantly slower than for patients in Arm 2 (13 days) but not significantly different from patients in Arm 1 or Arm 4. There was no significant difference in platelet engraftment or days of hospitalization between the study arms. Addition of GM-CSF to IL-3-containing mobilization regimens results in collection of PBSC that lead to delayed engraftment. Further development of Arms 1, 2, and 4 appear warranted.

Immunotherapy with neuraminidase-treated cells and bacillus Calmette-Guerin.

Experiments were designed to determine the effectiveness of active immunotherapy, both specific (neuraminidase-treated cells) and nonspecific [bacillus Calmette-Guerin (BCG) organisms] in the L1210-BDF1 tumor-host system. Tumor burden was minimized with chemotherapy (1,3-bis-(20chloroethyl)-1-nitrosourea) prior to immunotherapy. The effectiveness of immunotherapy was dependent on the amount of drug used to minimize tumor burden. An interval 36 hours between chemotherapy and immunotherapy produced the maximum number of survivors. A single immunization with 10(4) neuraminidase-treated cells was superior to other single or multiple immunizations. BCG was most effective when mice were given 393 X 10(5) organisms. Beneficial effects of immunotherapy were observed only when immunizations were given by an intraperitoneal route. All mice cured of tumor developed tumor-specific immunity. The highest levels of immunity were observed in mice given both neuraminidase-treated cells and BCG organisms after chemotherapy.

Factors influencing the prescription of hormone replacement therapy.

OBJECTIVE: To assess the effects of age, bone mineral density, risk of cardiovascular disease, and of breast cancer on the prevalence of hormone replacement therapy (HRT) prescriptions. METHODS: Seventeen charts of postmenopausal women were summarized. For each chart, we constructed 36 different cases by modifying the age (two levels), the bone mineral density (three levels), the cardiovascular risk (three levels), and the breast cancer risk (two levels). Twelve cases of these 612 files were sent to each Belgian gynecologist (n = 1010). RESULTS: Overall, HRT was prescribed in 67% of the cases. It was prescribed in 54.6% of women who had a normal bone mass, 67.9% of women with a low bone mass, and 79.0% of those with osteoporosis (P < .001). The prescription rate was higher in younger women (mean +/- standard deviation 55 +/- 4 years) than in their peers who were 10 years older (79.3% versus 55.2%; p < .001). No significant variation was observed in relation to the cardiovascular risk profile or to breast cancer risk. CONCLUSION: Osteoporosis is associated with an increased rate and older age with a decreased rate of HRT prescription, whereas no difference is observed in association with cardiovascular or breast cancer risk.

Prescription attitudes among gynecologists towards two particular risk factors of osteoporosis: the patient's age and her bone mineral density.

OBJECTIVE: The present report assesses, among Belgian gynecologists, the effect of age and bone mineral density on osteoporosis prescription strategy in postmenopausal women. METHODS: Charts of postmenopausal women were summarized. We constructed cases by modifying some parameters. Ten years of age were added or subtracted to the real age of the patient. The bone mineral density (BMD) result was also modified (three levels: normal BMD, osteopenia, osteoporosis). A total of 612 cases were constructed. Twelve cases were sent out of these 612 files to every Belgian gynecologist (n = 1010). For each chart the gynecologists were asked whether they would treat the patient with HRT. They were also asked whether they would prescribe other therapies than HRT and if so, which ones. RESULTS: The chance to have an osteoporosis prevention or treatment prescribed increased when BMD decreased (respectively 57.4% for normal BMD, 73.1% for osteopenia cases and 89.4% for osteoporosis cases; P < 0.001). HRT was the most frequently prescribed medication (67% of the cases), but its prescription rate does not reflect only osteoporosis prevention. Nevertheless, for similar cases with osteopenia, the HRT prescription rate increased by a factor 1.25 and for similar cases with osteoporosis, HRT prescription rate increased by a factor 1.39. Calcium was the 2nd most frequent prescribed regimen. It was prescribed in 17% of the cases. A 3.4-fold increase for osteopenia cases and 7.6-fold increase for osteoporosis cases was observed, compared to women with normal BMD. When calcium was prescribed, it was in association with HRT in 64% of the osteopenia cases and in 76% of osteoporosis cases. Other drugs were less often prescribed. For the "younger age group", that is, with a mean age of 55 years, a prescription rate of 82.9% for any osteoporosis regimen was reached, whereas in the age group that was 10 years older a 20% lower prescription rate was reached (62.6%, P < 0.001). This was mostly due to a decrease in HRT prescription. CONCLUSIONS: Prescription of medication known to reduce osteoporosis occurred more often in cases with low BMD. In the older patients with osteoporosis, gynecologists prescribed HRT less frequently. This was not compensated by a higher prescription rate of other medication.

A virtual reality surgical trainer for navigation in laparoscopic surgery.

A virtual reality trainer was designed to familiarize students and surgeons with surgical navigation using an angled laparoscopic lens and camera system. Previous laparoscopic trainers have been devoted to task or procedure training. Our system is exclusively devoted to laparoscope manipulation and navigation. Laparoscopic experts scored better than novices in this system suggesting construct validity. The trainer received favorable subjective ratings. This simulator may provide for improved navigation in the operating room and become a useful tool for residents and practicing surgeons.

Kinetic analysis of K+ ion channel function in lymphokine-activated killer (LAK) cells.

K+ ion channels of lymphocytes have been implicated in cellular differentiation, activation and cytolytic functions. We previously demonstrated that K+ channel blockers modulate lytic activity of CTLs and LAK cells. In the present study, we define and quantitate the inhibitory effects of ion channel blockers on the lytic process using kinetic analysis of lysis. The K+ channel blocker, 4-aminopyridine, the neuroendocrine monoamine, serotonin, its agonist, quipazine, and the Ca++ dependent K+ channel blocker, quinidine were found to non-competitively inhibit the lytic process in a dose-dependent manner. These compounds inhibit lytic activity by causing a decrease in the maximum velocity (Vmax) by which LAK cells lyse tumor targets. These ion channel blockers did not alter effector or target cell viability or the binding of LAK cells to tumor cells. The inhibitory effects occurred at the effector cell level, since preincubation of LAK effector cells resulted in a dose-dependent decrease in Vmax which was related to a slower rate of target cell lytic programming (k2) by the LAK effector cells. Modulation of LAK cell lytic function occurs at a post-binding step, perhaps in the generation or release of the lytic signal.

Kinetic analysis of lymphokine-activated killer (LAK) cells: lytic parameters and determination of LAK cell frequency.

Kinetic analysis was used to define lytic events in murine lymphokine-activated killer (LAK) cell-mediated tumour cell lysis. The maximum rate of target cell lysis (Vmax) and Km (target cell number resulting in 1/2 Vmax) were determined. Single cell lytic assays demonstrated that only LAK effector cells bound to target cells (i.e. non-lytic, bystander lymphocytes did not influence the determination of kinetic parameters) in contrast to natural killer (NK) cell lysis. This finding allowed for LAK cell frequency determinations where Km approximates the concentration of lytic LAK effector cells within a given number of lymphocytes. Frequencies determined in this manner were not significantly different from those obtained using the more cumbersome single cell lytic assay. Furthermore, frequencies determined for the same lymphocyte population against four different NK-resistant tumour targets, that varied in their sensitivity to LAK cell lysis, were not significantly different. In addition, LAK cell lytic programming of target cells was found to be the rate limiting lytic event. This study provides a means of determining reliable estimates of LAK cell frequencies within a lymphocyte population, which will be useful in studies evaluating LAK cytolytic mechanisms and the effects of drugs, biological response modifiers, or disease states on LAK cell lytic activity.

Kinetic analysis of cloned cytotoxic T lymphocyte reactivity against normal and leukaemic target cells.

The kinetics of unrestricted killing of normal and leukaemic lymphocyte target cells by a Qa-1b-specific murine cytotoxic T lymphocyte (CTL) clone were evaluated in a manner analogous to enzyme kinetic assays in which the effector and target cells corresponded to the enzyme and substrate, respectively. In order to apply the enzyme-substrate analogy to clonal cytolytic reactions, it was first established that the lytic reactions exhibited initial steady-state velocity of lysis at the effector and target cell concentrations used. The lytic reaction maintained linearity for velocity of lysis during the first 90 min of incubation, then plateaued. Vmax (the maximal rate of target cell lysis achieved by a given effector population) and Km (the target cell number resulting in 1/2Vmax) values were determined over a wide range of target and effector cell concentrations. Both parameters were found to be directly proportional to the number of effector cells. At a given concentration of cloned CTL, the lytic parameters of Vmax and Km were not significantly different for normal or leukaemic target cells that express Qa-1b. Additional kinetic parameters for lysis of normal and leukaemic target cells by a cloned CTL were also compared. The lytic efficiency of the CTL clone (i.e. maximal killing rate with an infinite number of targets) and the intrinsic affinity between effector and target cells were the same with either normal or leukaemic targets. However, the maximal lysis of target cells at an infinite number of effectors was significantly less for normal compared with leukaemic targets. This suggests that the normal target cells were more heterogeneous in their expression of the target (Qa-1b) antigen. Enzyme-like kinetic analysis of cell-mediated lysis reactions can be useful for comparing the relative affinities of effector and target cells obtained from various sources.