Spectrofluorometric method for measuring tobacco smoke particulate matter on cigarette filters and Cambridge pads.

Almost all cigarettes sold have a filter (United States, >98%; worldwide, >95%). In the last 25 years cigarette manufacturers have introduced diverse filters designed to reduce components in tobacco smoke. Today, there exists a need to establish assays to assess the efficacy of cigarette filters to retain total particulate matter (TPM), particularly unique filters of cigarettes that are being marketed as potential reduced exposure products (PREPs). We report the results of studies that were undertaken to test the hypothesis that a technique could be established for dissolving cigarette filters, and that the TPM in the fluid could be quantified by spectrofluorometry. Described here are procedures for assaying TPM on both Cambridge filter pads (glass fibres) of smoking machines and on cigarette filters (cellulose acetate fibres). The principle of the assays is based upon the observation that there exists a direct correlation between the amount of tobacco product emission TPM and fluorescence. In the absence of a tobacco tar or TPM standard, the fluorescent dye acridine orange was confirmed as a useful surrogate. Filters assayed included those of Kentucky reference cigarettes 2R4F and popular US brand cigarettes. The proposed assays are inexpensive, expedient, reproducible and amendable for large-scale studies.

Tobacco flakes on cigarette filters grow bacteria: a potential health risk to the smoker?

Bacterial growth from a single flake of tobacco was documented for cigarettes that had been purchased recently from local vendors and from cigarettes that had been stored for more than six years in a warehouse. In a novel tobacco flake assay, a pack of cigarettes was opened within the sterile environment of a laminar flow hood. A single flake of tobacco was collected randomly and aseptically from the middle of the cigarette column and placed onto the surface of a blood agar plate. The test cigarettes included eight different popular US brands, and these were from three different tobacco companies. After 24 hours of incubation at 37 degrees C, the plates showed bacterial growth for tobacco from all brands of cigarettes. Further, more than 90% of the individual tobacco flakes of a given brand grew bacteria. Likewise, bacteria grew from microparticulate tobacco that had been sieved from cigarettes. Tobacco flakes were observed lying loosely on the cut surface of the filter of cigarettes in newly opened packs, and bacteria grew from cigarette filters that had been touched to the surface of a blood agar plate. In conclusion, the results of these studies predict that diverse microbes and microbial toxins are carried by tobacco microparticulates that are released from the cigarette during smoking, and carried into mainstream smoke that is sucked deep into the lung.

Disparity of mixed lymphocyte reactivity to cultured cells of human T and B lymphoid lines.

Repeated attempts with difference assays and experimental conditions failed to detect significant peripheral blood lymphocyte reactivity in one-way mixed lymphocyte reactions to irradiated or mitomycin C-treated cells of the "leukemic" T lymphoid line RPMI 8402. In contrast, consistently high levels of peripheral blood lymphocyte reactivity were obtained with cells of six B lymphoid lines established from the same blood sample used to initiate this T lymphoid line. Although attempts to define the reason why these cultured T cells did not initiate a mixed lymphocyte reaction were not successful, evidence indicates that this inability may be an intrinsic characteristic common to three other T lymphoid lines (MOLT-4, CCRF-CEM, and CCRF-HSB-2), also established from patients with relapsed acute lymphoblastic leukemia.

Elaboration of pyrimidine-specific nucleosidases by human lymphoblastoid cells of established cultures.

Pyrimidine-specific nucleosidases were released rapidly by human lymphoblastoid cells of established cultures when incubated under certain culture conditions having no adverse affect on their viability or morphology. Nucleosidase production was not restricted to any particular type of lymphoblastoid line; enzymes with a high level of activity were elaborated by cells of cultures initiated from healthy subjects and patients with uncontrolled lymphocytic or myelocytic leukemia, as well as by cells of cultures exhibiting mostly B- or T-cell properties. Tritiated thymine and uracil, which were not incorporated to any appreciable extent by DNA- and RNA-synthesizing cells, were identified by paper chromatography as the primary products arising from nucleosidase degradation of radiolabeled thymidine, uridine, and cytidine. Neither adenosine nor guanosine was catabolized. These heat-labile and ultraviolet-sensitive enzymes with a molecular weight of 5 to 10 X 10(4) did not affect the viability, morphology, or proliferation of lymphocytes in mitogenactivated cultures, lymphoblastoid cells in long-term cultures, or fibroblasts in monolayer cultures.

Fibers released from cigarette filters: an additional health risk to the smoker?

Tests of 12 popular brands of cigarettes manufactured by 6 companies from the United States have shown that fibers were released from the filters and that there exists probable cause to suggest that fibers are inhaled and/or ingested. Filter fibers, made of cellulose acetate, were implanted in mice for 6 months. The fibers withstood degradation and retained the tobacco-brown color and bright fluorescence of the tobacco tar that had been adsorbed from cigarette smoke. With a confocal laser scanning microscope, we have observed cigarette filter fibers in lung tissue from patients with lung cancer and who were known to be habitual smokers. These findings raise the question as to whether fibers released from cigarettes further jeopardize the health of smokers and document the need to test components of cigarette filters for toxicity and tumorigenicity.

Isolation of mouse T-cell lymphoma lines from different long-term interleukin 2-dependent cultures.

A number of different biological properties have been ascribed to the hormone-like protein interleukin 2 (IL-2). However, the most salient feature of this lymphokine is its ability to sustain the long-term proliferation of T-cells from humans and mice. Reported herein are the results of studies demonstrating the isolation of growth factor-independent cell lines from the long-term IL-2-dependent murine T-cell line CTLL-2 that is used frequently as the source of target cells in IL-2 bioassays. Sustained log-phase growth of these T-cells in vitro has been achieved using Petri dishes of polymethylpentene; growth could not be sustained in similar dishes of glass, untreated polystyrene, polystyrene that had been treated for cell culture, or polycarbonate. The IL-2-independent line grew as a T-cell lymphoma when injected i.p. into pristane-treated, but not untreated, syngeneic C57BL/6 mice. In contrast, cells from the IL-2 parental line CTLL-2 did not grow in vivo. Characterization of the IL-2-independent lines propagated in vitro (denoted as line CEC) or in vivo (denoted as line CEP) demonstrated that they retained their dependency for 2-mercaptoethanol and expressed phenotypic profiles of their parental line CTLL-2 (Thy 1.2+, Lyt-1-; Lyt-2-). Isolation of an IL-2-independent T-cell lymphoma from a CTLL-2 line obtained from another investigator using a protocol that has proven reproducible under carefully controlled laboratory conditions and defined phenotypic traits of the syngeneic T-cell isolates provided evidence that the tumors were not a cross-culture contaminant arising as a result of a laboratory accident. Moreover, karyotypic analysis using a quinacrine:Hoechst banding technique revealed similar marker chromosomes in the IL-2-dependent and -independent lines. IL-2-independent lines have also been established from the IL-2-dependent murine T-cell line CT-6. Accordingly, the results of these studies suggest that, during prolonged cultivation that has included exposure to crude IL-2 preparations known to contain phorbol ester, possibly viruses, and other contaminants, the IL-2-dependent lines have developed subpopulations that are thought to have undergone malignant transformation of unknown etiology to generate IL-2-independent murine T-cell lymphomas that can be passaged repetitively either in vitro or in vivo.

Human recombinant interleukin-2 is mitogenic to human lymphocytes.

Reported herein are the results of studies demonstrating that purified recombinant human interleukin-2 (hrlL-2) is a potent mitogen for lymphocytes of healthy human donors. The specificity of the hrlL-2-induced response was defined in experiments in which mitogenicity of this T cell growth-promoting lymphokine was completely abrogated by blocking the T cell membrane receptor for IL-2 with the anti-Tac monoclonal antibody. Depletion of adherent mononuclear leukocytes markedly reduced lymphocyte reactivity to hrlL-2, but the response could be fully recovered by the addition of interleukin-1 (IL-1). Increased proliferative responses were observed using a combination of hrlL-2 and a monoclonal antibody OKT3 that defines a T cell membrane antigen. These studies demonstrate that hrlL-2, as with antigens and phytomitogens, may serve as the first signal of T cell proliferation.

Expression and capping of a proliferation-associated surface membrane p34 kDa antigen on different human hematopoietic cell lines.

A novel surface membrane nonglycosylated acidic polypeptide (34 kDa), encoded by a structural gene on chromosome 11, has been identified using murine monoclonal antibody (MoAb) 53.6 (IgG2a). MoAb 53.6, raised against uninduced cells of a human erythroleukemia line (HEL), recognizes a surface membrane antigen that is displayed on proliferating (cell cycle phase G1, S, and M + G2 phase) human leukocytes. The expression and redistribution (i.e., patching and capping) of the p34 kDa antigen on 27 different long-term human hematopoietic cell (HHC) lines was defined by fluorescence microscopy. These lines had been established from patients with leukemia or healthy donors and included phenotypically defined populations of T cells, B cells, and myelomonocytic cells. Almost all (greater than 95%) of the leukocytes of the 27 lines reacted strongly with MoAb 53.6. The majority of the leukocytes displayed p34 kDa antigen patching (26/27 lines; patched cells, 96-100%); moreover, 20 of 27 lines exhibited p34 kDa antigen capping (capped cells, 8-96%). Presentation of the p34 kDa antigen on surface membrane ultrastructures, imaged with immunogold using an indirect antibody labeling procedure, was illustrated by scanning electron microscopy, and endocytosis of the gold-tagged antigen-antibody complex was studied by transmission electron microscopy. The HHC lines are thought to represent immortalized populations of different human leukocyte subsets that are in different stages of maturation and/or differentiation; thus these lines should prove useful as models for further characterizing this unique p34 kDa proliferation-associated antigen and for defining the mechanisms and significance of surface membrane antigen redistribution and modulation that has been associated with leukocyte activation and propagation.

Effect of human recombinant tumor necrosis factor on the growth of different human and mouse long-term hematopoietic cell lines.

Previous studies have demonstrated that one of the most salient features of tumor necrosis factor (TNF) is its ability to induce tumor necrosis in vivo, and the specificity of its cytotoxic/cytostatic activity for tumor cells has been demonstrated in in vitro studies in which this lymphokine has been shown to kill cultured cells of malignant lines and to have no effect on cells of normal diploid lines. Studies described herein defined the effect of highly purified human recombinant TNF on cells of 34 different human and murine hematopoietic cell lines, particularly human leukemic T and B cells of long-term lymphoblastoid cultures. Results of these studies demonstrated that TNF at concentrations of 3,600 U/ml had no significant effect on the growth of these cells as defined by cytotoxicity, measured with the use of the trypan blue dye-exclusion assay and as defined by cytostasis, assayed by the enumeration of cells and the uptake of [3H]-thymidine and -uridine. In contrast, positive control cultures of TNF-sensitive cells from a murine tumor (L-M/clone L-929, connective tissue) displayed at 50% (LD50) reduction in growth by TNF at approximately 5 U/ml. Likewise, human tumors (MCF-7, breast, and HT-29, colon) were also highly sensitive (LD50 less than 100 U/ml). These studies demonstrate that T and B cells of lymphoblastoid lines as well as cells of other hematopoietic lines display little or no sensitivity to TNF.

Lysis of autologous human macrophages by lymphokine-activated killer cells: interaction of effector cell and target cell conjugates analyzed by scanning electron microscopy.

Lymphokine (i.e., interleukin 2; IL-2)-activated killer (LAK) cells derived from normal human blood are known to destroy human tumor target cells. Accordingly, immunotherapy modalities using IL-2, either alone or in combination with LAK cells, have been evaluated for eradicating metastatic cancer. In studies conducted to characterize receptors on LAK cell membrane ultrastructures, we observed that LAK cells kill autologous human monocyte-derived macrophages (M phi). In these experiments, peripheral blood mononuclear cells of a healthy adult donor were cultured to generate LAK cells and autologous non-adherent M phi. Thereafter, conjugates were prepared by incubating for 3 h autologous populations of LAK cells and M phi. Examination of the conjugates by scanning electron microscopy (SEM) identified LAK cell-mediated killing of M phi. Moreover, SEM analysis of the LAK cell membrane architecture identified microvilli-like ultrastructures that provided a physical bridge that joined together the LAK cell and M phi. The immunological mechanism(s) underling LAK cell killing of autologous M phi is not known; nevertheless, these conjugates will provide a useful model to study membrane receptors on ultrastructures that mediate the initial stages of cytolysis that include target cell recognition and cell-to-cell adhesion. The results of our observations and the findings of other investigators who have also demonstrated LAK cell killing of autologous normal human leukocytes are discussed in the context of the association of IL-2 and IL-2-activated killer cells with side effects observed in ongoing clinical trials and with autoimmune disorders.

Long-term cultivation of functional human macrophages in Teflon dishes with serum-free media.

Reported herein are the results of studies demonstrating the utility of a chemically defined, serum-free medium designated as AIM-V (GIBCO) for the long-term (greater than 2 weeks) cultivation of functionally-defined human macrophages. The AIM-V medium is a mixture of HEPES-buffered Dulbecco's Modified Eagle Medium and Ham's Nutrient Mixture F12 that had been supplemented with purified human albumin, transferrin, insulin, and a proprietary mixture of purified factors. Nonadherent macrophages for serial studies were generated in petri dishes with a Teflon liner that had been seeded with a heterogeneous population of peripheral blood mononuclear cells of healthy human adults. For comparison, cultures were initiated with serum-free medium AIM-V and medium supplemented with AB/Rh+ serum or freshly collected autologous serum. Viability was defined by trypan blue dye, glass adherence, and phagocytosis of yeast. Macrophages were characterized by light microscopy, cytochemistry, and phenotypic analysis. Ultrastructural morphology was defined by scanning electron microscopy. These studies demonstrate that functionally defined human macrophages can be sustained in long-term culture with the use of serum-free medium that has not been augmented with mitogenic stimulants, growth-promoting lymphokines/monokines, or differentiation-inducing agents. Serum-free medium AIM-V, which has been approved for generating lymphokine, (i.e., interleukin-2; IL-2)-activated killer cells (LAK) for IL-2/LAK adoptive immunotherapy modalities, may also prove useful for studies defining and isolating regulatory proteins produced by activated monocytes and macrophages and for generating cytolytic macrophages for different antitumor regimens.

Consumer perception of risk associated with filters contaminated with glass fibers.

The filters in Eclipse, a new cigarette-like smoking article marketed by R. J. Reynolds Tobacco Company, are contaminated with glass fibers, fragments, and particles. Reported herein are the results of a study in which consumers were questioned about their opinions as to whether exposure to glass fibers in such a filter poses an added health risk beyond that from smoking and whether the manufacturer has an obligation to inform consumers about the glass contamination problem. The study queried 137 adults who were interviewed while waiting at a Division of Motor Vehicles office in Erie County, New York in 1997. All but one person expressed the view that the presence of glass fibers on the filters poses an added health risk beyond that associated with exposure to tobacco smoke alone. Nearly all expressed the position that the cigarette manufacturer has a duty to inform the public about the potential for glass exposure.

Glass fiber contamination of cigarette filters: an additional health risk to the smoker?

We report here the results of studies documenting the contamination of a cigarette-appearing smoking article labeled Eclipse with glass fibers, fragments, and particles. Eclipse, a product of the R. J. Reynolds Tobacco Company (RJR), was commercialized in June of 1996. Eclipse is unlike conventional cigarettes in that, like its predecessor Premier, it is designed to heat and not burn tobacco. The purpose of Eclipse was to simplify the chemical composition and reduce the biological activity of the mainstream and sidestream smoke and to achieve a significant reduction of environmental tobacco smoke. In Eclipse, tobacco pyrolysis is reduced by a carbon fuel rod that serves as a heat source for generating an aerosol having nicotine and tobacco flavor. The carbon rod, at the tip of the cigarette, is insulated and bound with two wrapping mats of glass fibers. Recently, Eclipse has been modified to address consumer complaints of burdensome draw and off-taste. The redesigned Eclipse, which we have termed the NEW Eclipse, has an unconventional filter-appearing mouthpiece that consists of a cellulose acetate cylindrical bundle with a central hollow tunnel. In our analysis of Eclipse, glass fibers (length:width aspect ratio, > or = 3:1) were: (a) observed protruding from the tip; (b) identified on the white cigarette wrapping paper; (c) viewed on the surface of the cork-appearing tipping paper; (d) found in the pack residue; (e) discovered lying freely on the cut surface of the filter by both light and electron microscopy; (f) harvested from the filter with adhesive tape; and (g) displaced when Eclipse was smoked mechanically. In a study of Eclipse that had not been removed from carefully opened packs, we observed that > or = 95% of the filters were contaminated with glass fibers (Eclipse: Regular, n = 114/120, 95%; Milds, n = 118/120, 98%; Menthol, n = 120/120, 100%). Likewise, 99% of NEW Eclipse had glass fibers on the redesigned filter (Regular, n = 119/120). In contrast, glass fibers were never observed on the filters of conventional United States filter cigarettes that had been used as controls (n = 0/120, 0%). In a study of Eclipse (n = 60), the number of glass fibers contaminating the filter surface ranged from 5 to 55. Glass fibers as well as fiber fracture items [aspect ratio, < 3:1 (e.g., particles, fragments, bits, chips, flakes, specks, and dust)] were discovered in the pack residue. The average number of glass fibers in the residue of a pack of Eclipse was 7,548 (SE +/- 3443; range, 1,164 to 26,725 glass fibers/pack; n = 7 packs). The thin and fragile glass fibers of the insulation mats had most likely been broken and fragmented in the high-speed multiple-step Eclipse manufacturing operation. Invariably, puffing on Eclipse discharged glass fibers and glass particles from the filter into the smoker's mouth. Subsequently, the bioresistant glass fibers and microscopic glass dust are inhaled and/or ingested. Contamination of Eclipse filters with glass fibers and glass dust poses a potential and unnecessary health hazard to uninformed consumers. Eclipse is a paradigm of the health danger that may be imposed by technically complex tobacco articles and nicotine delivery devices promoted by an unregulated industry to smokers worldwide, many of whom are addicted to nicotine and who seek a less hazardous cigarette.

Inhaled cellulosic and plastic fibers found in human lung tissue.

We report the results of studies undertaken to determine whether inhaled plant (i.e., cellulosic; e.g., cotton) and plastic (e.g., polyester) fibers are present in human lungs and, if so, whether inhaled fibers are also present in human lung cancers. Specimens of lung cancer of different histological types and adjacent nonneoplastic lung tissue were obtained from patients undergoing a lung resection for removal of a tumor. With the protection of a laminar flow hood and safeguards to prevent contamination by extraneous fibers, fresh, nonfixed, and nonstained samples of lung tissue were compressed between two glass microscope slides. Specimens in these dual slide chambers were examined with a microscope configured to permit viewing with white light, fluorescent light, polarizing light, and phase-contrast illumination. Near-term fetal bovine lungs and nonlung human tumors were used as controls. In contrast to the observations of these control tissues, morphologically heterogeneous fibers were seen repetitively in freshly excised human lung tissue using polarized light. Inhaled fibers were present in 83% of nonneoplastic lung specimens (n = 67/81) and in 97% of malignant lung specimens (n = 32/33). Thus, of the 114 human lung specimens examined, fibers were observed in 99 (87%). Examination of histopathology slides of lung tissue with polarized light confirmed the presence of inhaled cellulosic and plastic fibers. Of 160 surgical histopathology lung tissue slides, 17 were selected for critical examination; of these, fibers were identified in 13 slides. The inhalation of mineral (e.g., asbestos) fibers has been described by many investigators; we believe, however, that this is the first report of inhaled nonmineral (e.g., plant and plastic) fibers. These bioresistant and biopersistent cellulosic and plastic fibers are candidate agents contributing to the risk of lung cancer.

A simple screening assay for substances affecting lymphocyte reactivity.

A simple method is described for assaying substances modifying lymphocyte reactivity in vitro to mitogens and antigens. This procedure employes whole blood, microtiter plates and an automated cell harvester. The simplicity and expediency afforded by this assay, which requires very little blood, enables the large-scale screening of different test materials which may be of experimental or clinical merit.

Studies of cultured human T lymphocytes. I. Production of the T cell growth-promoting lymphokine interleukin-2.

Described herein is a large-scale procedure that has been successfully employed for producing 62 lots (800-3000 ml) of supernatants containing the T cell growth-promoting factor Interleukin-2 (IL-2). The efficiency of these crude, unconcentrated supernatants was documented in studies in which 70 human long-term (greater than 100 days) IL-2-dependent T cell lines were established from 50 different donors. These included lines initiated from the peripheral blood of healthy subjects (N = 54), blood of children with active acute lymphoblastic leukemia (N = 6) and the thymus of children undergoing surgery to correct congenital heart defects (N = 10). The underlying concept used in constructing this method emphasizes the requirement of the monocyte-derived macrophage and its Interleukin-1 (IL-1) product to mediate IL-2 production by activated T cells. The most salient feature of this technique is the utilization of buffy coat leukocytes that had been pooled from several blood donors and sustained in spinner cultures for several days prior to polyclonal activation with phytohemagglutinin and pooled B cells of established human lymphoblastoid lines.

Isolation of interleukin 2 (IL-2) from human and mouse lymphocyte culture supernatants by batch adsorption onto silicic acid.

Described herein is a simple, efficient and inexpensive batch adsorption procedure for the isolation and partial purification of the hydrophobic T cell growth-promoting lymphokine interleukin 2 (IL-2) from crude culture supernatants (SN) of freshly isolated human lymphocytes and leukemic T cells of established lines including human Jurkat J6.2, Gibbon ape MLA-144 and mouse EL-4. In this method, IL-2 was isolated by batch adsorption onto microparticulate silicic acid (SA) by stir-mixing the SA with SN (10 mg/ml; 30 min; 37 degrees C). Thereafter, the SA was pelleted by centrifugation and washed twice with phosphate-buffered saline (PBS). The IL-2 was eluted by adding to the pelleted IL-2-binding SA 5 vols. of ethylene glycol (EG; 50%, v/v) in PBS (pH 7.2) with high salt (1.4 M NaCl). The lymphokine-rich concentrate was then dialyzed (6 kDa MWCO) against PBS to remove the EG and low molecular weight growth inhibitors. The application of the proposed procedure was further defined in experiments in which SA was successfully employed for recovering IL-2 from SN of cultures in which the medium had been supplemented with fetal calf (FCS) or human serum to achieve maximal lymphokine production. Also presented are the results of experiments defining the SA adsorption of proteins from whole sera (e.g., FCS, calf, human and horse) and the relative affinity of different purified proteins for this matrix (e.g., bovine serum albumin, human serum albumin, casein hydrolysate, bovine gamma-globulin and bovine beta-lactoglobulin). The proposed SA procedure may prove useful for isolating other hydrophobic immunoregulatory molecules, and a 2-step purification scheme is anticipated in which the SA adsorption procedure will be used as a preparative method preceding reverse phase high performance liquid chromatography and monoclonal antibody affinity chromatography.

A method for isolating human lung macrophages and observations of fluorescent phagocytes from the lungs of habitual cigarette smokers.

We report the development of a simple, efficient, expedient, and inexpensive procedure for isolating a large and relatively pure population of macrophages (Mphi) from residual (i.e., non-tumor) lung tissue obtained from lung cancer patients undergoing either a lobectomy or pneumonectomy. The proposed technique was founded on observations by fluorescent microscopy of fresh, non-fixed, and non-stained human lung tissue. Examinations of 74 specimens from different patients revealed that most of the Mphi reside as non-adherent cells within the sponge-like lung stroma. Very few Mphi were observed in the lungs of nonsmokers. In contrast, many Mphi were visible in the lungs of habitual smokers. For most specimens from smokers, a few of the Mphi were present as randomly distributed single cells; the majority of the Mphi, however, were in clusters that ranged from a dozen to several hundred cells. The Mphi could be released readily by different mechanical techniques. In the procedure reported herein, pulmonary leukocytes (> 75% Mphi) were dislodged easily from lung tissue with the use of an inexpensive, hand-operated, tissue grinder. The grinder consisted of a glass mortar and Teflon pestle that provided sufficient clearance between the mortar and pestle so as to avert damaging the displaced leukocytes. The leukocytes were then segregated by centrifugation on a density gradient. Further purification was achieved by harvesting Mphi that had been allowed to adhere to serum-coated polystyrene culture dishes (> 90% Mphi). In most experiments, the Mphi yield (approximately 5 x 10(6) Mphi /gr of lung) and Mphi viability (> 85%) were good. A significant advantage of this technique is that it avoids jeopardizing the cells to the hazards associated with enzymes that have been used in techniques employed previously for isolating Mphi from the lung and other organs. Thus, the proposed method provides numerous lung Mphi for detailed studies of their morphology, phenotype, and function. Moreover, lung Mphi were cultured as non-adherent, single cells in a serum- and cytokine-free tissue culture medium for more than 6 weeks. Lung Mphi from habitual smokers displayed a high level of fluorescence that was readily apparent when viewed with a fluorescence microscope that had been configured with either a fluorescein or rhodamine filter. Serial sections of single, living Mphi obtained with the use of a confocal laser scanning microscope revealed that the fluorescence originated from cytoplasmic inclusions. Relative fluorescence intensity was measured by cytometry.(ABSTRACT TRUNCATED AT 400 WORDS)