Design and chemical evaluation of reduced machine-yield cigarettes.

Experimental cigarettes (ECs) were made by combining technological applications that individually reduce the machine measured yields of specific toxicants or groups of toxicants in mainstream smoke (MS). Two tobacco blends, featuring a tobacco substitute sheet or a tobacco blend treatment, were combined with filters containing an amine functionalised resin (CR20L) and/or a polymer-derived, high activity carbon adsorbent to generate three ECs with the potential for generating lower smoke toxicant yields than conventional cigarettes. MS yields of smoke constituents were determined under 4 different smoking machine conditions. Health Canada Intense (HCI) machine smoking conditions gave the highest MS yields for nicotine-free dry particulate matter and for most smoke constituents measured. Toxicant yields from the ECs were compared with those from two commercial comparator cigarettes, three scientific control cigarettes measured contemporaneously and with published data on 120 commercial cigarettes. The ECs were found to generate some of the lowest machine yields of toxicants from cigarettes for which published HCI smoke chemistry data are available; these comparisons therefore confirm that ECs with reduced MS machine toxicant yields compared to commercial cigarettes can be produced. The results encourage further work examining human exposure to toxicants from these cigarettes, including human biomarker studies.

The use of a novel tobacco-substitute sheet and smoke dilution to reduce toxicant yields in cigarette smoke.

The Institute of Medicine encouraged the pursuit and development of potential reduced-exposure products, tobacco products that substantially reduce exposure to one or more tobacco toxicants and can reasonably be expected to reduce the risk of one or more specific diseases or other adverse health effects. One approach to reducing smoke toxicant yields is to dilute the smoke with glycerol. We report chemical, biological and human exposure data related to experimental cigarettes containing up to 60% of a novel glycerol containing "tobacco-substitute" sheet. Analysis of mainstream smoke from experimental cigarettes showed reductions in yields of most measured constituents, other than some volatile species. In vitro toxicological tests showed reductions in the activity of smoke particulates in proportion to their glycerol content. Human exposure to nicotine was reduced by a mean of 18% as determined by filter studies and by 14% using 24h urinary biomarker analysis. Smoke particulate exposures were reduced by a mean of 29% in filter studies and NNK exposure by similar amounts based on urinary 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol concentrations. These results show that reducing exposure to some smoke toxicants is possible using a tobacco-substitute sheet, although some smoke toxicants, and the sensory attributes of the smoke, remain as technical challenges.

An approach to the validation of biomarkers of harm for use in a tobacco context.

There is both a call and a need for biomarkers of harm that are validated for use in a tobacco context. Currently, there are no validated biomarkers and there is no consensus about which ones may be suitable for this purpose. To advance the science in this area a working definition of biomarkers of harm and a shortlist of candidate biomarkers are proposed. A framework for the validation of biomarkers of harm using of a series of epidemiological studies culminating in a targeted prospective study is outlined. The candidate biomarkers have advanced to preliminary testing although this does not imply that any on the shortlist will become validated. This framework could also be used for the evaluation of proteomic, genomic, transcriptosomic or metabonomic profiles, which may turn out to be the preferred biomarkers for use in harm prediction. Biomarker studies would complement data that are generated from specific in vitro tests and from animal studies to evaluate tobacco products.

Recombinant interleukin-6 increases the intracellular and extracellular growth of Mycobacterium avium.

Human monocytes were isolated from the peripheral blood of normal donors and allowed to differentiate in vitro into macrophages. The susceptibility of these cells to infection with a virulent Mycobacterium avium and its modulation by some soluble factors was monitored. The virulent strain of Mycobacterium avium grew progressively in untreated macrophage monolayers. Interleukin-6 (IL-6) was tested for its ability to modulate the macrophage-mycobacteria interaction. Surprisingly, IL-6 was shown to increase M. avium growth in macrophage monolayers by twofold as compared with untreated cells, when added before or after infection. Moreover, addition of rIL-6 to replicating mycobacteria in vitro enhanced their growth two- to three-fold as compared with cultures treated with rIL-6 and a rabbit antiserum to rIL-6. Treatment with IL-6 and interferon-gamma (IFN-gamma) or IL-4 did not modify the growth promoting effect of IL-6 in human macrophages. Overall, our results suggest that IL-6 may contribute significantly to the pathogenesis of infections with M. avium by promoting mycobacterial growth.

Interleukin-2 and granulocyte-macrophage colony-stimulating factor stimulate growth of a virulent strain of Escherichia coli.

The effect of human recombinant interleukin-2 (IL-2) and human recombinant granulocyte-macrophage colony-stimulating factor on the growth of a virulent strain of Escherichia coli in tissue culture medium and in untreated, normal mouse serum was investigated. Both of these cytokines enhanced the growth of the microorganism two- to threefold in tissue culture medium with or without additional fetal calf serum and in untreated mouse serum. IL-4 did not have any effect on the growth of this microbe under the conditions tested. That the enhancement of growth seen with recombinant IL-2 was due to the active cytokine was shown by the following data: (i) addition of an antibody to IL-2 abrogated the growth-promoting effect; (ii) the excipient buffer, which contained everything except the active cytokine, was inactive in modifying bacterial growth; and (iii) heat-inactivated recombinant IL-2 did not promote enhanced microbial growth. The enhancement of growth with IL-2 was significant with concentrations as low as 1 U/ml. Growth of an avirulent strain of E. coli was not stimulated by IL-2. Moreover, addition of IL-2 to growth virulent E. coli in tissue culture medium led to rapid removal of the cytokine from the medium. Collectively, these data suggest that cytokines may act as growth factors for some virulent bacteria.

Identification of cytokines which enhance (CSF-1, IL-3) or restrict (IFN-gamma) growth of intramacrophage Listeria monocytogenes.

Murine peritoneal macrophages were isolated by adherence and their listericidal activity assessed in the presence or absence of selected cytokines. Untreated macrophages were not highly listericidal, showing moderate killing in the first 2 h after infection, and allowed progressive microbial growth thereafter (up to 9 h). Pre-treatment of cells with 10 to 100 U/ml of IFN-gamma allowed macrophages to develop sustained listericidal activity for the 9-h observation period, with a 2-log reduction of Listeria CFU per monolayer. Pulsing of cells with TNF-alpha alone did not result in enhanced microbicidal activity but TNF-alpha potentiated IFN-gamma-induced listericidal activity, resulting in high levels of killing when both cytokines were present. Conversely, macrophages pre-treated with interleukin-3 (IL-3) or colony-stimulating factor-1 (CSF-1) were found to be much more permissive for Listeria growth. Neither IL-3 nor CSF-1 abrogated IFN-gamma-induced listericidal activity. Moreover, neither IL-3 nor CSF-1 had any effect on the ability of macrophages to develop a respiratory burst following Listeria infection, as judged by H2O2 release following in vitro infection. Overall, these results suggest that different cytokines may have opposing effects on intracellular microbial growth, and that the balance of cytokine production in vivo may determine the resistance or susceptibility of the infected host.

Modulation of Mycobacterium avium growth in murine macrophages: reversal of unresponsiveness to interferon-gamma by indomethacin or interleukin-4.

The ability of soluble factors to modulate the growth of a virulent strain of Mycobacterium avium in murine peritoneal macrophages was studied. The virulent strain, TMC 702, grew progressively in the organs of susceptible BALB/C mice. In addition, this strain of M. avium grew progressively in untreated peritoneal macrophages. Treatment of macrophage monolayers with interferon-gamma (IFN-gamma) did not change significantly the intracellular growth of M. avium. Addition of indomethacin to IFN-gamma-treated macrophage monolayers rendered them significantly more bacteriostatic than macrophages treated with interferon alone, suggesting a role for prostaglandins in inducing unresponsiveness to IFN-gamma in infected cells. Additionally, treatment with tumour necrosis factor-alpha led to a modest increase in bacteriostasis, as compared to untreated monolayers. Further experiments with recombinant interleukins showed that interleukin-4 (IL-4), on its own, could increase bacteriostatic activity against M. avium in a reproducible fashion. Experiments with interleukin combinations showed that IFN-gamma and IL-4 treatment of macrophages rendered these cells almost fully bacteriostatic against M. avium, inclusion of scavengers of reactive oxygen species did not modify the beneficial effect of IFN-gamma and IL-4. Overall, our results suggest an important role for interleukins in modulating the interaction between virulent mycobacteria and murine macrophages.

The effect of macrophage activation state on antigen presenting capability as defined by helper T-cell function.

Resident peritoneal cells were obtained from BALB/c mice and enriched for cells of the macrophage lineage by adherence onto 96 well tissue culture plates. Adherent cells were then exposed to various recombinant cytokines or supernatants from cell cultures, for 24 h. The ability of such adherent antigen presenting cells (APC) to support proliferation and development of helper function in T-lymphocyte populations, primed with sheep erythrocytes (SRBC), was examined. The addition of cytokines to the APC population did not enhance either proliferation of the T-cells nor helper function, assessed by assay of polyclonal IgG secretion in second cultures, beyond that obtained with control APC. The potent macrophage activators interferon-gamma and lipopolysaccharide caused a significant decrease in both parameters of T-cell activity. This effect was caused by a prostaglandin-mediated pathway inasmuch as indomethacin (1-5 microM) prevented it. Further analysis showed that this negative signal predominated until macrophages were diluted below 5% of the total cell population. At 0.5% macrophages, interferon-gamma stimulated APC function of these cells compared with untreated macrophages. Despite the relative difficulty in manipulating the T-cell response by attempted modulation of the APC with cytokines, the simple manoeuvre of incubation of otherwise responsive, primed T-cells with a high dose (10%) of SRBC during in vitro restimulation, caused the proliferation and helper function of these T-cells to be markedly decreased. This phenomenon was seen regardless of the cytokine used to stimulate the APC population. These studies further clarify the dual role the macrophage in regulation of T-cell responses.

Studies on cytokine activation of listericidal activity in murine macrophages.

The ability of a variety of soluble factors, alone or in combination, to endow murine resident peritoneal macrophages with listericidal activity was assessed. Inhibition of growth and (or) killing of Listeria in infected macrophages was determined by the uptake of [3H]uracil following lysis of the infected macrophage monolayers. Interferon-gamma was shown to induce modest listericidal activity in murine resident macrophages as compared with untreated monolayers. Treatment with tumour necrosis factor alpha also induced significant listericidal activity in this system. Among other cytokines tested, IL-4 induced an ability to inhibit growth of Listeria in resident macrophages. The ability of cytokines tested, IL-4 induced an ability to inhibit growth of Listeria in resident macrophages. The ability of cytokines to act in an additive or synergistic fashion with IFN-gamma was also investigated. Combinations of IFN-gamma and IL-4 and IFN-gamma and IL-2 induced listericidal activity not greater than that seen with IFN-gamma alone. IFN-gamma and TNF-alpha were shown to increase bactericidal activity in an additive fashion. However, elicited macrophages were shown to spontaneously exert a significant listericidal activity that was not enhanced by cytokine treatment. Collectively, these findings show that cytokine treatment induced rather modest enhancement in listericidal activity in murine resident peritoneal macrophages and no enhancement whatsoever in elicited macrophages. Thus, in in vivo situations where Listeria organisms are completely cleared from the infected organs, mechanisms other than lymphokine-induced listericidal activity of resident macrophages would seem to be operating.

Recombinant tumour necrosis factor-alpha decreases whereas recombinant interleukin-6 increases growth of a virulent strain of Mycobacterium avium in human macrophages.

The ability of a virulent strain of Mycobacterium avium to infect and replicate within human monocyte-derived macrophages of normal donors was assessed. Moreover, the ability of selected cytokines to modulate the intracellular growth of M. avium was investigated. Our virulent strain of M. avium grew progressively in human macrophages. Treatment of macrophage monolayers with interferon-gamma (IFN-gamma) did not lead to any significant change in the infection pattern. Conversely, treatment with tumour necrosis factor-alpha (TNF-alpha) led to a significant reduction in the growth of M. avium in the macrophages. In contrast, treatment of macrophages with interleukin-6 (IL-6) enhanced their susceptibility to M. avium significantly. This finding was substantiated by other results which showed that IL-6 increased the growth of M. avium in tissue culture medium. These results suggest that cytokines may influence the M. avium-macrophage interaction, in a positive or negative manner.

Immunomodulatory properties of platelet factor 4: prevention of concanavalin A suppressor-induction in vitro and augmentation of an antigen-specific delayed-type hypersensitivity response in vivo.

The immunomodulatory properties of platelet factor 4 (PF4) have been examined in vitro and in vivo. This agent prevented the induction of concanavalin A (Con A)-induced suppressor cells in vitro in a dose-dependent manner but it did not affect the function of established Con A suppressor cells. This effect was not due to an enhanced production of interleukin-2 (IL-2) by lymphocytes exposed to PF4. The delayed-type hypersensitivity (DTH) reaction to sheep erythrocytes (SRBC) was used as a model for the generation of antigen-specific suppression in vivo. PF4 enhanced the magnitude of the swelling following SRBC challenge 10 days after sensitization by the i.p. route or following sensitization by both the s.c. and i.p. routes. These studies show that PF4 has immunomodulatory activities in well-defined models of cell-mediated immunity and suggest that this agent has a potential use in the dissection of events in antigen-specific suppression.

Cytokine modulation of Mycobacterium tuberculosis growth in human macrophages.

This study was concerned with the handling of ingested tubercle bacilli by normal human macrophages. Intracellular growth was determined after exposure of macrophages to viable bacilli in vitro and the effect of various cytokines, alone or in combination, on bacilli growth/survival was determined. It was found that Mycobacterium tuberculosis (M.tb) grew quite readily in untreated cultured human macrophages. Treatment with soluble factors showed that a crude lymphokine containing supernatant elicited with Concanavalin A (Con A) was ineffective at reducing growth of M.tb in vitro; similarly a crude lymphokine preparation from M.tb lysate-stimulated mononuclear cells failed to induce any mycobacteriostatic activity in human monocyte-derived macrophages. Recombinant cytokines were then evaluated for their ability to modulate growth of the tubercle bacilli in human macrophages. Recombinant interferon-gamma (IFN-gamma), interleukin-2 (IL-2) and recombinant interleukin 4 (IL-4) were all ineffective at modifying M. tuberculosis growth in human macrophages. Recombinant tumour necrosis-alpha (TNF-alpha) curbed the growth of the bacilli in human macrophages in a reproducible fashion. No cytokine combination was more efficient than TNF-alpha alone. These studies thus highlight the resistance of virulent mycobacteria against different mechanisms of cytokine-induced macrophage bactericidal activity.

Whole ricin and recombinant ricin A chain idiotype-specific immunotoxins for therapy of the guinea pig L2C B cell leukemia.

The therapeutic efficacy of whole ricin, or recombinant ricin A chain, coupled to a monoclonal antibody that reacts with the idiotype of the surface IgM expressed on guinea pig L2C lymphoblasts, was assessed. In vitro studies were done to characterize the immunotoxins (IT) and to demonstrate their specificity before use in vivo. The concentration of whole ricin IT (M6-Ricin) that inhibited protein synthesis by 50% (IC50) in L2C cells was 1.4 X 10(-9) M, in a 5-hr assay, in the presence of lactose to block non-antibody-directed toxicity. M6-Ricin did not inhibit protein synthesis in two control guinea pig cell lines that did not express the idiotype, nor did a whole ricin IT prepared with an isotype-matched monoclonal antibody of irrelevant specificity inhibit protein synthesis in L2C cells. Two recombinant ricin A chain IT, which differed from one another by a factor of 2 to 3 in the number of A chains conjugated per antibody molecule, were less effective in vitro than M6-Ricin (IC50 of greater than 5 X 10(-8) M). For in vivo experiments, the IT were given by the i.p. route 24 hr after the i.p. inoculation of 1 X 10(5) L2C cells. The highest doses of M6-Ricin and M6-Ricin A chain IT tested, 30 micrograms/kg and 3000 micrograms/kg, respectively, were within fourfold to fivefold of their maximum tolerated doses; no deaths or ill effects due to ricin toxicity were noted. These doses increased the median survival time of L2C-bearing guinea pigs to 31 to 34 days, compared with 15 days for untreated animals. This magnitude of increase in survival indicates that 99.999% (5 logs) of injected tumor cells were eliminated, thus accounting for the 12% long-term survival rate obtained. Median survival times for guinea pigs treated with 30 micrograms/kg of the A chain IT were 18 and 21 days for the two conjugates tested, and the median survival for guinea pigs treated with 3000 micrograms/kg of unconjugated antibody was 18 days. Our data demonstrate that recombinant A chain IT are active in vivo and that the B chain of ricin can potentiate IT activity in vivo. Although the potency differs by 100-fold, the therapeutic index of the intact ricin IT is similar to that of the ricin A chain IT.

Generation of lymphokine-activated killer cells in strain 2 guinea pigs and their use in the therapy of L2C, an acute B-cell leukemia.

Lymphokine-activated killer (LAK) cells were induced by incubating strain 2 guinea pig splenocytes or lymph node-derived cells in recombinant human interleukin-2 (IL-2) for 3-5 days. These effector cells had the morphology of lymphoblasts and were able to lyse murine P815 tumor cell targets. Fresh, unstimulated, guinea pig effectors were not capable of lysing these targets. The therapy of the L2C leukemia, an acute B-lymphoblastic leukemia of strain 2 guinea pigs, using LAK cells and recombinant IL-2 was examined. Antitumor effects were demonstrated by premixing LAK and tumor cells prior to intradermal injection in Winn type assays and then measuring the growth of local tumor and survival of the animals. In further experiments i.p. administration of LAK cells, 4 h following tumor cell inoculation by the i.p. route, prolonged the survival of treated animals. The best results in this i.p. therapy model were obtained with a 10-fold excess of LAK cells over tumor cells plus additional treatment with 1000 units of IL-2 for 20 days. This resulted in a 10-day increase in median survival of treated animals. Despite these in vivo antitumor effects, lytic activity of LAK effector populations against L2C targets could not be demonstrated in vitro. The potential synergy between LAK cells, IL-2, and a monoclonal antibody directed against the idiotype of the neoplastic cell surface immunoglobulin was also investigated. In these experiments enhanced survival of the combined treatment group, beyond that of either singly treated group, was not found. This study shows that LAK cells are useful agents in the therapy of a widely disseminated, aggressive, B-cell lymphoblastic leukemia. The use of such effectors, even in cases where in vitro lysis of the target tumor cell cannot be demonstrated, is encouraged by these results.

Immunoglobulin negative follicle centre cell lymphoma.

Immunoglobulin (Ig) could not be detected on the surface or in the cytoplasm of neoplastic cells from five cases of follicle centre cell lymphoma with centroblastic/centrocytic follicular histology when examined by immunohistology of frozen or wax embedded sections. Examination by fluorescein labelled antibodies of cells in suspensions prepared from the biopsies revealed a monotypic surface Ig positive population in one case and a surface or cytoplasmic Ig kappa:lambda light chain imbalance in a further two cases consistent with neoplastic B cell involvement: in all cases the proportion of cells failing to express Ig or T cell markers ranged from 24 to 75%. The monoclonal antibodies B1 (Pan B cell), FMC4 (HLA class II) and J5 (cALL antigen) stained the majority of cells in suspension with residual cells staining with UCHT1 or OKT11 (T cell monoclonal antibodies). In frozen sections, neoplastic follicular cells did not stain with UCHT1. However, in the one case tested these cells stained with the antibodies B1 and FMC4. In paraffin sections J chain could be demonstrated in the cytoplasm of three out of five cases. Cells from four cases were cultured in vitro for Ig production: two failed to produce Ig and monotypic light chains were the sole Ig product of the remaining two cases. The failure to express Ig by the majority of the neoplastic cells from the cases described in this report is at variance with the follicular histology of these neoplasms. Mechanisms responsible for this failure are discussed with reference to current models of B cell differentiation.

Secretion of immunoglobulin by neoplastic B lymphocytes from lymph nodes of patients with lymphoma.

An investigation has been made into the ability of neoplastic B lymphocytes obtained from lymphoid tissue of patients with non-Hodgkin's lymphoma (NHL) to secrete immunoglobulin (Ig) in vitro. The majority of the cell populations secreted IgM (17/24 patients), identified as pentameric in three cases examined, and free monotypic light chains (23/24 patients) of the same type as the surface Ig. Secretion of IgD (6/21 patients) and IgG (3/21 patients) was found less frequently. The amounts of Ig secreted were variable and there was no significant difference in the patterns of secretion of cells from NHL patients when compared to previous studies of chronic lymphocytic leukaemia (CLL), nor was there any clear correlation with the histological type. For four of the patients, anti-idiotypic antibody was produced and was used to demonstrate the idiotypic nature of the secreted Ig, and also to show its presence in the serum. The level of idiotypic IgM was measured in one patient during chemotherapy and appeared to correlate well with disease. Such idiotypic Ig must be taken into account when planning treatment of B cell neoplasms with antiidiotypic antibody since it could act as a block to antibody attack. Assessment of the ability of tumour cells to secrete Ig in vitro provides a useful preliminary screen when choosing such patients since a high secretion rate together with extensive disease could lead to unacceptable levels of serum idiotypic Ig.