Limitations of cigarette machine smoking regimens.

There is an ongoing debate about the 'usefulness' of the standard machine smoking regimen for cigarettes defined by the US Federal Trade Commission (FTC) and adopted in principal by the International Organization for Standardization (ISO). More intense smoking regimens result in much higher smoke yields, and these higher yields have been suggested to be much closer to human smoke uptake. However, it appears that more intense smoking regimens are less efficient in detecting possible differences in the yield of toxicants. Intense smoking regimens reproducibly decrease the concentration of toxicants in the smoke per unit mass of total particulate matter, tar, or nicotine, most likely as a result of a more complete combustion. The toxicant concentration reaches the same plateau for different cigarette types under the intense smoking regimens. As such, differences in toxicant concentrations due to product changes, which are observable under ISO or FTC conditions, may disappear under intensive smoking regimens. Intense machine smoking regimens might be used for regulatory compliance and consumer information. However, when evaluating the toxicological impact of cigarette product changes, especially to avoid increases in toxicity, they should not be used as a standard (or at least not as the only standard). The type of smoking regimen has to be carefully considered and aligned to the underlying question. Depending on the question a combination of the reviewed regimens or even other approaches might be appropriate.

Cytotoxicity, mutagenicity, and tumorigenicity of mainstream smoke from three reference cigarettes machine-smoked to the same yields of total particulate matter per cigarette.

The particle phase of mainstream smoke from three types of cigarettes was investigated in vitro in the Neutral Red cytotoxicity assay and the Salmonella typhimurium Reverse Mutation Assay (Ames Assay) and in vivo in the two-stage dermal tumorigenicity assay (Skin Painting Assay) in SENCAR mice. The cigarettes used were the Reference Cigarettes 1R5F, 2R4F, and 2R1F from the University of Kentucky, USA, which, when smoked according to the smoking regimen defined by the International Standards Organization (ISO), produce a yield of approximately 2, 12, and 26 mg total particulate matter (TPM)/cigarette, respectively. All cigarettes were machine smoked according to ISO and then again in such a way that the TPM yields per cigarette equaled the ISO TPM yields of the other two cigarette types. The TPM from cigarettes with inherently different smoke yields showed similar in vitro toxicity and in vivo toxicity when, with different smoking regimens, these cigarettes were smoked to the same TPM yield. More intensive smoking conditions were associated with lower in vitro and in vivo activity per gram of TPM. The strongest decrease, and the tightest correlation, in this regard was observed for dermal tumorigenicity (tumor incidence).

A unifying concept for assessing toxicological interactions: changes in slope.

Robust statistical methods are important to the evaluation of toxicological interactions (i.e., departures from additivity) among chemicals in a mixture. However, different concepts of joint toxic action as applied to the statistical analysis of chemical mixture toxicology data or as used in environmental risk assessment often appear to conflict with one another. A unifying approach for application of statistical methodology in chemical mixture toxicology research is based on consideration of change(s) in slope. If the slope of the dose-response curve of one chemical does not change in the presence of other chemicals, then there is no interaction between the first chemical and the others. Conversely, if the rate of change in the response with respect to dose of the first chemical changes in the presence of the other chemicals, then an interaction is said to exist. This concept of zero interaction is equivalent to the usual approach taken in additivity models in the statistical literature. In these additivity models, the rate of change in the response as a function of the i(th) chemical does not change in the presence of other chemicals in a mixture. It is important to note that Berenbaum's (1985, J. Theor. Biol. 114, 413-431) general and fundamental definition of additivity does not require the chemicals in the mixture to have a common toxic mode of action nor to have similarly shaped dose response curves. We show an algebraic equivalence between these statistical additivity models and the definition of additivity given by Berenbaum.

Chemical composition, cytotoxicity and mutagenicity of smoke from US commercial and reference cigarettes smoked under two sets of machine smoking conditions.

Eight blended US market cigarettes, two blended reference cigarettes, one Bright tobacco only reference cigarette and an electrically heated prototype cigarette (EHC) were smoked under US Federal Trade Commission (FTC)/International Organisation for Standardisation (ISO) conditions and under Massachusetts Department of Public Health (MDPH) conditions. Smoke was analysed for chemical composition and in vitro toxicity. Yields (quantity/cigarette) of smoke constituents were higher under MDPH conditions compared to FTC/ISO conditions (market and reference average approximately 2.5 times; EHC approximately 1.6 times). Consistent with the higher yields, in vitro toxicity per cigarette was also higher under MDPH conditions. Concentrations (quantity/mg TPM) of nearly all smoke constituents measured decreased with increasing total particulate matter (TPM) yields as regression analyses indicated. Higher TPM yields also tended to be associated with slightly less cytotoxic and mutagenic activity per milligram TPM. Blended reference cigarettes tracked market cigarettes with similar TPM yield. The Bright cigarette displayed high cytotoxicity but low mutagenicity, while in vitro activity of the EHC was remarkably low. The TPM-dependent decreases for the market range of 5-20 mg TPM/cigarette were about 20%, irrespective of whether the increased yields were due to smoking conditions or cigarette construction. At the same TPM yield, the smoke constituent concentrations and in vitro toxicity were similar for low- and high-yield cigarettes.

Analysis of anticonvulsant and neurotoxic responses to combination therapy with carbamazepine, felbamate and phenytoin by response-surface modeling.

Various combinations of carbamazepine (CAS 298-46-4), felbamate (CAS 25451-15-4), and phenytoin (CAS 57-41-0) were evaluated in mice (i.p.) for anticonvulsant activity (maximal electroshock seizure test) and minimal neurotoxicity (rotarod test). The results obtained from these studies were analyzed using response surface methodologies (RSM). The outcomes of these analyses in regard to anticonvulsant activity suggest that, under these experimental study conditions, at 0.5 h post treatment there is a significant carbamazepine/phenytoin synergism even though none of the drugs has a significant dose-response by that time when given alone, and that at 1.0 h post treatment, the combination dose-response is additive. Thus, there appears to be an important dose/time relationship. In regard to the neurotoxic response, the results suggest a significant carbamazepine/phenytoin synergism at 0.25 h post treatment and an additive neurotoxic effect due to the combination of felbamate/carbamazepine/phenytoin at 0.5, 1.0 and 2.0 h post exposure.

Angiofollicular lymph node hyperplasia (Castleman's disease) in an adolescent female. Clinical and immunologic findings.

Angiofollicular lymph node hyperplasia is a heterogeneous disorder of unclear etiology and has a wide spectrum of systemic symptoms. This report describes a case of this disorder in a 15-year-old girl and examines the response of the primary mass, systemic symptoms, and alterations of selected immune parameters at diagnosis, as a result of steroid therapy and radiation therapy (RT). The patient had a 1-year history of growth failure, delayed puberty, and refractory iron deficiency anemia. Computed tomography scan showed a posterior mediastinal mass. Biopsy revealed angiofollicular lymph node hyperplasia of mixed hyaline-vascular and plasma cell type histologic type. Immunoperoxidase studies showed polyclonal B-cells, predominance of T-helper cells (CD4) over cytotoxic/suppressor T-cells (CD8), and the presence of natural killer (NK) cells. Southern blot analysis demonstrated germ line gene configuration for the T-cell antigen receptor and Ig heavy chain. The patient clinically improved with RT after failing to respond to steroids. Immunophenotyping of peripheral blood lymphocytes before therapy revealed a CD4:CD8 ratio of 0.8 with decreased numbers of circulating T-cells; this increased to 1.4 after steroid therapy. The patient's T-lymphocytes had no proliferative response to phytohemagglutinin (PHA) or concanavalin A (Con A) before RT. After RT, a small but significant mitogenic response to these reagents was noticed. The proliferative response to recombinant interleukin-2 (rIL-2) remained similar to that of control lymphocytes. Induction of second messenger signals by activation of protein kinase C (PKC) and elevation of free cytosolic calcium through the use of the phorbol ester, phorbol 12, 13-dibutyrate (PDBu), and ionomycin (Io) resulted in a strong proliferative response at diagnosis and after RT. In vitro cytotoxicity assays revealed diminished NK activity before and after therapy. Lymphokine-activated killer (LAK) activity remained comparable with that of control cells and was not affected by therapy. Before RT patient lymphocytes maintained cytotoxic capabilities after coincubation with rIL-2 and PDBu plus Io, whereas coincubation with these reagents abrogated cytotoxic function of normal cells. This case demonstrates a clinical response to RT as well as improvement in immune parameters. Intact signal transduction mechanisms through PKC activation and elevation of cytosolic calcium were also demonstrated in the circulating lymphocytes.

Alteration of human granulocyte functional responses by menadione.

Menadione is a synthetic derivative of the natural vitamins K with antiinflammatory activity among its potentially significant clinical properties. We have found this agent to stimulate the production of superoxide anion (O2-) in human polymorphonuclear leukocytes (PMN) and dimethylsulfoxide-differentiated HL-60 cells in a time-, cell number-, and drug concentration-dependent manner. Conversely, menadione attenuates both O2- production and lysozyme release in cells stimulated by phorbol myristate acetate (PMA), fMet-Leu-Phe, or Ca2+ ionophore. 4-Acetamido-4'-isothiocyano-2-2'-disulfonic acid stilbene and 4,4'-diisothiocyano-2-2'disulfonic acid stilbene, agents which inhibit transmembrane O2-) flux, do not alter menadione's effects on superoxide dismutase (SOD) inhibitable cytochrome c reduction in resting or PMA-stimulated PMN. Likewise, quinone reductase inhibitors, warfarin and dicumarol, known to attenuate vitamin K-dependent responses and enhance quinone-mediated oxidative stress, have no effect upon menadione-stimulated O2- production. Furthermore, menadione-induced suppression of stimulus-mediated lysozyme release is not reversed by cotreatment with oxygen metabolite scavenging enzymes SOD and catalase. Nevertheless, under conditions of restricted oxygen supply, the suppressive effect of menadione on stimulant-induced lysozyme release is greatly diminished. Thus, although pharmacological manipulation suggests otherwise, there appears to exist at least a component of the inhibitory activity of menadione that is oxygen dependent, and may be oxidative stress-related.

Evaluation of the efficacy of two carbamates, physostigmine and pyridostigmine, when used in conjunction for protection against organophosphate exposure.

Recent studies have shown that pretreatment with either pyridostigmine (PYR) or physostigmine (PHY) followed by atropine-oxime therapy is very effective in reducing the lethality of nerve agents. The therapeutic efficacy of a PHY and PYR combination pretreatment was evaluated in guinea pigs challenged with two LD50s of soman. Endpoints measured were percentage of acetylcholinesterase inhibition induced by the pretreatment and survival up to 24 hr postchallenge. Response surface methodology was employed to describe the relationship between each endpoint and the pretreatment combination. Although both carbamates contributed to blood acetylcholinesterase inhibition, PHY alone protected as well as the optimal dose of the combination.

Induction of the expression of differentiation-related antigens on human colon carcinoma cells by stimulating protein kinase C.

This study was undertaken to determine whether the phorbol diester, phorbol 12-myristate 13-acetate (PMA), causes differentiation of the human colon carcinoma cell line, SW 48. Under routine growth conditions, the cells are round, have a high nuclear-to-cytoplasmic ratio, and lack cytoplasmic vacuoles. After treatment for 1 hour with 100 nmol/L of PMA at 37 degrees C, the cells assumed a spread-out, flasklike shape, displayed a low nuclear-to-cytoplasmic ratio, and exhibited cytoplasmic vacuoles. An inert but lipophilic phorbol diester, 4 phorbol 12,13-didecanoate, failed to induce these morphological changes. Cell kinetic studies showed that whereas SW 48 cells have a doubling time of 35 hours, those incubated with 100 nmol/L of PMA have a doubling time of 90 hours. Although the flow cytometry histograms were similar until 8 hours into the cell cycle, the PMA-treated cells ultimately spent proportionately less time in S and more in G2/M. Finally, under routine growth conditions, SW 48 cells express neither carcinoembryonic antigen nor G7 antigen. These antigens, which are present on the surface of well-differentiated cells, were expressed after treatment of SW 48 with PMA. The data suggest that PMA causes profound changes in structure, cell growth kinetics, and antigen expression, consistent with induction of differentiation of the cell line SW 48.

Evaluation of a two-drug combination pretreatment against organophosphorus exposure.

A pretreatment combination of physostigmine and azaprophen (6-methyl-6-azabicyclo[3.2.1]octan-3-ol-2,2-diphenylpropionate), a novel cholinolytic, was evaluated for its ability to minimize soman-induced incapacitation and lethality in guinea pigs. This was accomplished by using response surface methodology to model and analyze the combination, varying physostigmine from 0 to 194 micrograms/kg, azaprophen from 0 to 5 mg/kg, and soman from 30 to 150 micrograms/kg. One hundred percent survival was achieved against 5 LD50 of soman using as little as 100 micrograms/kg of physostigmine in the presence of 5 mg/kg azaprophen. Both survival and soman-induced incapacitation were similarly affected by this pretreatment combination. For both endpoints, greater efficacy was achieved with the combination than could be achieved with either component alone (therapeutic synergism). This suggests that such a pretreatment combination may prove very efficacious against soman-induced lethality and incapacitation in higher species.

Assessing the efficacy of azaprophen and physostigmine as a pretreatment for soman-induced incapacitation in guinea pigs by response-surface modeling.

Physostigmine (PHY) has the advantage over pyridostigmine of minimizing OP-induced incapacitation because it penetrates into the CNS. However, physostigmine is behaviorally toxic at relatively low concentrations. It is anticipated that this could be offset by a cholinolytic to prevent behavioral deficit due to the carbamate pretreatment alone. The therapeutic efficacy of physostigmine/azaprophen pretreatment therapy was evaluated in soman-challenged guinea pigs. Response surface methodology was employed to describe the relationship of the pretreatment combination with duration of incapacitation. The significance of the combination relative to PHY alone was evaluated in addition to dose combinations that yield optimal time to recovery. Analysis of the fitted response surface indicated that combination pretreatment with these compounds significantly reduces the time to recovery after soman challenge versus pretreatment with PHY alone.

Comparing the response surfaces of two cholinolytics when used in combination with physostigmine as a pretreatment against organophosphate challenge.

Physostigmine (PHY) is currently being evaluated as a potential pretreatment against nerve agent challenge. Although it might provide increased protection against nerve agent-induced incapacitation, it is nonetheless behaviorally toxic itself. Addition of a cholinolytic adjunct is expected to significantly reduce this behavioral toxicity. The efficacy of pretreatment regimens (PRG) (composed of PHY in combination with either scopolamine (SCP) or trihexyphenidyl (artane; ART] was evaluated in guinea pigs challenged with soman (98 ug/kg, sc; 3.5 LD50) to determine the dose of PHY and adjunct required for optimal efficacy as well as the better of the two adjuncts. Three different endpoints were measured on each animal: (1) whether or not the animal survived up to 24 hours post challenge, (2) severity of incapacitation, and (3) time to recovery. The survival data revealed no significant differences between the two adjuncts and PHY against soman, but the data suggests that a pretreatment combination of PHY and ART is likely to give a greater therapeutic index than one containing SCP. Furthermore, since ART is less toxic behaviorally, it would most likely be better tolerated as a pretreatment with PHY in non-agent exposed subjects. The findings in this report are not to be construed as an official Department of the Army position unless so designated by other authorized documents. In conducting the work described in this report, the investigators adhered to the "Guide for the Care and Use of Laboratory Animals" as promulgated by the Committee on Revision of the Guide for Laboratory Animal Facilities and Care of the Institute of Laboratory Animal Resources, National Research Council.

Induction of tumoricidal activity and alterations of growth by interleukin-2 and manipulation of protein kinase C and cytosolic calcium in childhood acute lymphocytic leukemia cells.

Signals from many receptor-ligand interactions are mediated by enhancement of phospholipid hydrolysis which generates metabolic intermediates stimulating protein kinase C (PKC) and elevating cellular calcium. Pharmacologic agents such as phorbol 12, 13-dibutyrate (PDBu) and ionomycin selectively stimulate PKC and elevate intracellular calcium to directly stimulate downstream mechanisms critical to cell growth and function. This study examines the effects of PDBu, ionomycin, and rIL-2 on childhood ALL blasts of early B lineage with respect to various aspects of cell activation, including DNA synthesis, induction of non-MHC restricted tumoricidal activity, and changes in morphology and phenotype. Five childhood ALL samples were tested. A marked heterogeneity was seen among the ALL samples with respect to in vitro growth following manipulation with PDBu, ionomycin, and/or rIL-2, whereas normal peripheral blood lymphocytes (PBL) were consistently stimulated to grow with the combination of PDBu and ionomycin. Growth responsiveness did not appear to correlate with morphologic or phenotypic classification of the leukemia samples. Four of the five leukemia samples developed substantial non-MHC restricted cytotoxicity to K562 (natural killer cell (NK) sensitive) and Daudi (NK resistant) targets in response to rIL-2. This functional cytotoxic response correlated with morphologic changes in the cells and the appearance of granules. Phenotypic analyses of the ALL samples at the time of their peak cytotoxic function were consistent with the fresh ALL phenotype and showed no major change in cell populations. Three of the five ALL samples also retained rIL-2 induced cytotoxic capabilities when exposed simultaneously to the combination of PDBu and ionomycin, whereas rIL-2 induced tumoricidal activity in normal PBL and bone marrow cultures was inhibited by these reagents. These data show that morphologically and phenotypically similar ALL blasts have heterogeneous proliferative responses to the PKC and calcium modulators PDBu and ionomycin, as well as to rIL-2. Cytotoxic responses are also different from those of normal PBL and bone marrow cells with respect to kinetics and responsiveness to inducing agents. Thus current morphologic and phenotypic classifications of ALL may not adequately reflect the heterogeneity of this disorder as described here.

Thermal regulation of FMLP receptors on human neutrophils.

Polymorphonuclear leukocytes (PMNs) from subjects diagnosed as having juvenile periodontitis (JP) have been categorized on the basis of their chemotactic (CTX) response to f-met-leu-phe (FMLP) when assayed concurrently with PMNs from periodontally healthy subjects (HP). When PMNs from JP groups demonstrating depressed CTX were assayed for lysosomal enzyme secretion (LES) in response to FMLP, there were no significant differences with respect to rate or amount. Significant differences were observed between HP and chemotactically depressed JP cells when assessed for FMLP receptor ligand binding at 23 degrees C, but not at 4 degrees C. Receptor differences observed at 23 degrees C in HP cells included an increase in amount of total binding, number of receptors, and available displaceable binding sites, compared with the chemotactically depressed JP PMNs, whereas the receptor affinities were similar. These data suggest that differences in FMLP receptor density in JP PMN that are chemotactically depressed may be related to processes that modulate receptor mobility and/or expression.

Interactive effects of pertussis toxin and the phorbol ester tumour promotor, phorbol dibutyrate, on T-lymphocyte mitogenesis and the expression of phenotypic determinants.

The B oligomer of pertussis toxin serves as a weak mitogen in the T lymphocyte, an effect which is associated with an early rise in cytosolic free calcium concentrations, as monitored by Fura-2 fluorescence. Upon co-administration of phorbol dibutyrate, a phorbol ester tumour promotor which activates protein kinase C, pertussis toxin-induced proliferation was synergistically enhanced, as measured by the increased uptake of [3H]thymidine, into cellular DNA. Although phorbol ester co-administration has often been associated with an inhibition of Ca2+-mobilizing pathways, phorbol dibutyrate pretreatment had no inhibitory effect on the pertussis toxin-induced calcium flux and may actually have enhanced this response slightly. Flow cytometric analysis of cell populations expanded by the combined regimen did not provide evidence for the preferential expansion of cells bearing either CD4 or CD8, the T-cell determinants representative of the helper-inducer and cytotoxic-suppressor subsets, respectively. Pertussis toxin and phorbol dibutyrate appear, therefore, to elicit polyclonal stimulation, rather than the selective activation of a given lymphocyte subset. Expression of the transferrin receptor, a marker for nutrient uptake, and CD25, the Tac component of the interleukin-2 (IL-2) receptor, was, however, synergistically enhanced in cells activated by the co-treatment procedure.

Coordination and reversibility of signals for proliferative activation and interleukin-2 mRNA production in resting human T lymphocytes by phorbol ester and calcium ionophore.

Sequential stimulation and washout procedures were employed to examine the kinetics and reversibility of pharmacologically manipulated second messenger signals mediating phenotypic changes and proliferative activation of resting human T lymphocytes. Phorbol dibutyrate (PDBu) was used to stimulate protein kinase C (Ca2+/phospholipid-dependent enzyme) while ionomycin was used to manipulate intracellular Ca2+ levels. Stimulation by PDBu alone induced phosphorylation of several endogenous substrates and altered expression of phenotypic markers, downregulating expression of CD4 and CD3 while increasing expression of CD2 and the interleukin 2 (IL-2) receptor. Stimulation with ionomycin alone caused an increase in intracellular Ca2+ levels but did not induce proliferation or cause major changes in the expression of phenotypic markers (CD2, CD3, CD4, CD8, IL-2, and transferrin receptors). Analysis of endogenous PDBu stimulated phosphosubstrates indicated that some substrates (pp92, pp82, pp55) underwent dephosphorylation, returning to base-line levels following PDBu removal while others (pp61, pp65) showed only partial dephosphorylation, while one (pp28) remained phosphorylated. Washing ionomycin-stimulated cells resulted in an approximately 75% reduction of intracellular Ca2+. Ionomycin exposure did not alter the affinity (KD = 22.3 +/- 7.4 nM) or number of receptors (53,497 +/- 8,291 receptors/cell) for [3H]PDBu. These data suggest that signals induced by PDBu or ionomycin are reversible following removal of the stimulating agents with respect to proliferative activation of T lymphocytes. Furthermore, a transcriptional mechanism regulating the production of IL-2 mRNA requires simultaneous activation of protein kinase C and elevation of intracellular Ca2+.

Macrophage spreading disparity: alveolar vs peritoneal.

Macrophage spreading over glass surfaces is a recognized in vitro manifestation of activation. We examined macrophage spreading using a simplified assay. Hartley guinea pigs (n = 15) were anesthetized with pentobarbital (0.4 mg/gm) IM. Macrophages were obtained by lavaging the peritoneal (PM) and alveolar (AM) spaces with sterile 0.9% NaCl (NS). AM or PM (150,000 cells) were placed into chambers of Lab-Tek microtiter slides +/- phorbol myristate acetate (PMA). Slides were incubated /37 degrees C in 5% CO2 for 20 minutes. Macrophages with a diameter greater than 2 x control cells were considered spread. Compared to PM, resident AM show increased spontaneous spreading (16 +/- 2% vs 79 +/- 2%) respectively. AM demonstrated no significant concentration-dependent response to PMA stimulation. The PM has served as the basis for much of the morphological and functional observations attributed to macrophages in general. The above spreading data support the concept of disparity of macrophage function dependent upon various factors, including site of origin. Our observations suggest that extrapolation of macrophage characteristics to cells from discordant sources may not be possible.

Mathematical and biostatistical methods for designing and analyzing complex chemical interactions.

In this presentation, statistical methods for designing and analyzing experiments evaluating a mixture of drugs/chemicals are discussed. These methods are promising in that they are not limited by the number of interacting agents in the combination. Several examples are given and a discussion of the results follows.

Alteration of human lymphokine-activated killer cell activity by manipulation of protein kinase C and cytosolic Ca2+.

We have examined the effects of protein kinase C (PK-C) stimulation and cytosolic Ca2+ elevation on the in vitro induction of non-histocompatibility-restricted tumoricidal activity from human peripheral blood lymphocytes. The tumor cytolytic activity, as well as the number of cells recovered from interleukin 2 (IL-2)-stimulated cultures, was enhanced by the addition of the PK-C stimulator, phorbol dibutyrate (PDBu), but not non-PK-C-activating phorbol ester analogues while the Ca2+ ionophore, ionomycin, did not significantly alter development of IL-2-induced tumor cytolytic activity nor enhance cell yield. Neither PDBu nor ionomycin, alone or in combination, induced tumoricidal activity. The addition of both PDBu and ionomycin to recombinant interleukin 2 (rIL-2)-exposed cultures produced a strong mitogenic response and high cell yield, although Daudi cell killing measured at Day 5 was completely abolished. This abrogation of lymphokine-activated killer cell activity was seen as early as 24 h following exposure to PDBu and ionomycin, reaching 50% following 2 days of exposure. When lymphocytes mitogenically expanded by primary exposure to PDBu and ionomycin and then washed free of these agents were further cultured with rIL-2 alone, proliferation continued, and substantial cytolytic activity for Daudi cells was induced. The development of this postexpansion cytotoxic activity was not dependent on the addition of exogenous rIL-2 during the primary cultures. Fractionation of cells into large granular lymphocytes and small T-lymphocytes indicated that only the large granular lymphocytes proliferate in response to rIL-2 alone. Both large granular lymphocytes and small T-lymphocytes proliferate in response to the addition of PDBu and ionomycin, and both populations of cells developed tumor cytolytic activity following removal of PDBu and ionomycin and subsequent culture in rIL-2. These data suggest that PK-C and Ca2+ signals play key roles in the regulation and/or proliferation of tumor cytotoxic lymphocytes or their precursors and that manipulation of those signals can be utilized to produce substantially more tumoricidal activity from lymphocyte populations than can be achieved with rIL-2 alone.

Responsiveness of guinea pig alveolar cells.

Guinea pig alveolar cells were obtained in situ via bronchoalveolar lavage. The cells were 86% macrophages (GPAM), (greater than 97% viability) with the remainder of the population comprised of lymphocytes and eosinophils. The following battery of functional assays were studied in GPAM: chemotaxis was stimulated by N-formyl-methionyl-leucine-phenylalanine (FMLP) and by phorbol-12-myristate-13-acetate (PMA) in a concentration-related manner; cytotoxicity as measured by 51Cr release from target cells +/- PMA was induced in P815 mastocytoma cells and less strongly in 3T3 normal mouse fibroblasts; release of N-acetyl-beta-D-glucosaminidase (NAGA) was stimulated by the calcium ionophore A23187, but not by PMA or the combination of PMA + A23187; superoxide anion production as measured by the reduction of ferricytochrome C was stimulated 25-fold by PMA; phagocytosis of opsonized 51Cr sheep red blood cells occurred in a time-related manner and reached its maximum after 120 min; and cell spreading, which exhibited a high rate of spontaneous spreading (76%), was only minimally stimulatable by PMA. The responsiveness of the GPAM, the ease of retrieval, and the large numbers of cells available make the guinea pig an ideal system for future study.