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.

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.

Cigarettes with defective filters marketed for 40 years: what Philip Morris never told smokers.

BACKGROUND: More than 90% of the cigarettes sold worldwide have a filter. Nearly all filters consist of a rod of numerous ( > 12 000) plastic-like cellulose acetate fibres. During high speed cigarette manufacturing procedures, fragments of cellulose acetate that form the mouthpiece of a filter rod become separated from the filter at the end face. The cut surface of the filter of nearly all cigarettes has these fragments. In smoking a cigarette in the usual manner, some of these fragments are released during puffing. In addition to the cellulose acetate fragments, carbon particles are released also from some cigarette brands that have a charcoal filter. Cigarettes with filters that release cellulose acetate or carbon particles during normal smoking conditions are defective. OBJECTIVE: Specific goals were to review systematically the writings of tobacco companies to: (a) identify papers that would document the existence of defective filters; (b) characterise the extent of the defect; (c) establish when the defect became known; (d) determine whether the defect exists on cigarettes marketed currently; (e) assess the prevalence of the defect on cigarettes manufactured by different companies; (f) define whether the knowledge of the defect had been withheld by the tobacco company as confidential and not disclosed publicly; and (g) ascertain the feasibility of correcting or preventing the defect. METHODS: Document searches utilised databases of the scientific literature, medical journals, chemical abstracts, US Patents, Tobacco Abstracts, papers presented at tobacco meetings and court documents. RESULTS: Sixty one documents of Philip Morris, Inc were selected for study because they disclosed specifically the "fall-out" of cellulose acetate filter fibres and, for cigarettes with charcoal filters, carbon particles from cigarette filters. The term "fall-out" was defined in 1985 laboratory protocols of Philip Morris, Inc. as "loose fibers (or particles) that are drawn out of the filter during puffing of the cigarette". As early as 1957, the health concern of inhaling cellulose acetate fibres released from cigarette filters was addressed by Philip Morris, Inc. A 1962 document reported the results of laboratory tests conducted by Phillip Morris, Inc that compared the "fall-out" of cellulose acetate fibres from the filters of their cigarettes (Marlboro) and cigarettes of their competitor (Liggett & Meyers). A 1997 overview by Phillip Morris of documents addressing the "fallout of carbon particles and cellulose acetate fibers from filters" stated that they were "essentially routine reports" of cigarette filter assays, and referenced a "Filter Fallout" memo written in 1961-more than 40 years ago. Most likely these tests are being conducted presently as illustrated by a 1999 report that details the revisions of the "fall-out" protocol of Phillip Morris, Inc and reports the results of tests that measured the discharge of cellulose acetate fibres and silica gel from beta cigarettes with a new type of filter. Our analysis of the "fall-out" tests results presented in the 61 "fall-out" documents showed that filter fibres and carbon particles were discharged from the filters of all types of cigarettes tested. These cigarette types (n = 130) included both coded cigarettes and popular brand name cigarettes. No publications were found in the scientific literature of the "fall-out" studies. Thus, the results of the "fall-out" studies are thought to have been withheld as confidential to Philip Morris, Inc. We have identified also other companies that have tested recently cigarettes for defective filters. In addition, our searches have shown that simple, expedient, and inexpensive technologies for decontaminating cigarette filters of loose cellulose acetate fibres and particles from the cut surface of the filter have been developed and described in 1997 and 1998 US patents. What is more important is that these patents also define methods for preventing or reducing the broken plastic-like fibres that arise during cigarette making. Many US patents (n = 607; 1957 to 2001) have been awarded for cigarette filters. Some of these inventions describe novel materials and unique filtration schemes that would eliminate or minimise the discharge of filter materials into mainstream smoke. CONCLUSIONS: We have shown that: (a) the filter of today's cigarette is defective; (b) Philip Morris, Inc has known of this filter defect for more than 40 years; (c) the existence of this filter defect has been confirmed by others in independent studies; (d) many methods exist to prevent and correct the filter defect, but have not been implemented; and (e) results of investigations substantiating defective filters have been concealed from the smoker and the health community. The tobacco industry has been negligent in not performing toxicological examinations and other studies to assess the human health risks associated with regularly ingesting and inhaling non-degradable, toxin coated cellulose acetate fragments and carbon microparticles and possibly other components that are released from conventional cigarette filters during normal smoking. The rationale for harm assessment is supported by the results of consumer surveys that have shown that the ingestion or inhalation of cigarette filter fibres are a health concern to nearly all smokers.

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.

Immunotoxicology of cigarette smoke condensates: suppression of macrophage responsiveness to interferon gamma.

We have investigated systematically the effects of short-term exposure to main stream cigarette smoke condensates (CSC-MS) on basal and inducible functional capacities of murine peritoneal exudate macrophages. Macrophages treated with CSC-MS form granules that fluoresce orange under blue excitation, consistent with the speculation that they are polycyclic aromatic hydrocarbons (PAH). CSC-MS selectively suppressed interferon gamma (IFN gamma) induction of four macrophage functional capacities: enhanced phagocytosis of immunoglobulin-opsonized sheep red blood cells, TPA-induced H2O2 production, class II major histocompatibility complex expression, and nitric oxide synthesis. In contrast, two macrophage functions that are not induced by IFN gamma, basal electron transport and LPS-induced TNF alpha production, were enhanced by treatment with CSC-MS. These results suggest that the suppressive effects of CSC-MS on macrophage responsiveness were selective and were not due to nonspecific inhibition of general functions such as RNA or protein synthesis. Since macrophage responsiveness to IFN gamma can result in induction of functional capacities that are fundamental to immunity, the data suggest that CSC-MS maybe deleterious to the general health of the smoker.

Release of carbon granules from cigarettes with charcoal filters.

OBJECTIVE: To inspect cigarettes with a triple granular filter for charcoal granules on the cut filter surface and, if present, to determine whether the charcoal granules on the filter are released during smoking. DESIGN: 400 Lark cigarettes in 20 packs were examined individually by each of three investigators for the presence of charcoal granules on the cut surface of the cellulose acetate filter. Without removing the cigarettes from the pack, the filters were examined with a stereo zoom microscope for charcoal granules. The percentage of cigarettes that had charcoal granules was defined, and charcoal granules on each filter were counted. Randomly selected cigarettes were then smoked by consenting adult smokers to assess whether the charcoal granules were released during smoking. Lark cigarettes were smoked with a conventional cigarette holder that had been configured to contain an in-line membrane. After smoking, the membrane was analysed microscopically for charcoal granules and other components of the filter that had been released during smoking. RESULTS: Charcoal granules were observed in 79.8% (319/400) of the cigarettes examined. The number of granules per cigarette was 3.3 (SD 3.7). Gaps between the tipping papers--the wrapping papers that surround the filter--were often seen (70%; 242 (71); n = 400 cigarettes). Further, the charcoal cavity was about 60% empty. For all smokers (n = 8/8), charcoal granules were released during smoking. The number of charcoal granules captured on the membranes was 22.5 (16.2) per cigarette. CONCLUSIONS: Charcoal granules are incorporated into cigarette filters to aid in removing toxins in cigarette smoke. In studies of Lark, a popular American cigarette with a charcoal filter, charcoal granules were observed on the filter surface, and were released from the filter when the cigarettes were smoked. During smoking, the toxin-containing charcoal granules are inhaled or ingested. The specific adverse health effects of inhaling or ingesting carbon granules have not been addressed; nevertheless, the smoker, as an educated consumer, should be informed of the possible health risks.

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.

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)

Tumour-cytolytic human monocyte-derived macrophages: a simple and efficient method for the generation and long-term cultivation as non-adherent cells in a serum-free medium.

We report a simple and efficient culture procedure for the generation of tumour-cytolytic human monocyte-derived macrophages (MAC). In this method, normal human peripheral blood mononuclear cells, isolated using a conventional Ficoll-Hypaque density gradient procedure, are cultured as a heterogenous leukocyte population in Teflon or other hydrophobic cultureware, in a commercially available serum-free culture medium (M-SFM) that has been formulated specifically for the cultivation and ex vivo stimulation of human monocytes and MAC, and in the absence of exogenous mitogens, antigens, cytokines or other stimulants. This procedure features a negative-selection technique that takes advantage of the differential survival of blood leukocytes. Using the prescribed in vitro conditions, lymphocytes survived relatively poorly, whereas monocytes differentiated in the absence of exogenous stimulants into mature tumour-cytolytic MAC. The MAC were present as non-adherent, single cells that expressed good viability (greater than 95%) for a prolonged period (greater than 60 days). When compared to conventional procedures for generating MAC, the prescribed technique is thought to offer several important advantages in that it: (a) eliminates the tedious and cumbersome monocyte isolation procedures, thus providing a significant savings not only in time and money but also in eliminating repetitive cell manipulations that have often been associated with damage to monocyte morphology and/or function; (b) reduces the loss of monocyte subsets that are not recovered during specific isolation procedures; (c) facilitates harvesting a single cell, non-adherent suspension of immunocompetent MAC suitable for various examinations including analyses defining MAC morphology, cytochemistry, phenotype and function; and (d) eliminates variability and artifacts associated with different sera that are utilised frequently as medium supplements. The utility of the prescribed method is illustrated by the results of ongoing studies in which scanning electron microscopy and confocal laser scanning microscopy are being used to define MAC function in different immunological reactions, and examples of these observations are presented herein.

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.

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.

Scanning electron microscopic study of immunogold-labeled human leukocytes.

For many years critical point drying (CPD) has been the method of choice for preparing cells for scanning electron microscopy (SEM). Described herein is a simple, efficient, inexpensive, reproducible, and safe procedure using Peldri II, a proprietary fluorocarbon compound that is solid at room temperature and a liquid above 25 degrees C, as a sublimation dehydrant for processing specimens for SEM. The utility of Peldri II was demonstrated in studies using leukocytes from the blood of healthy donors and patients with leukemia as well as from long-term lymphoblastoid cell lines. The application of the proposed Peldri II procedure was further documented in SEM studies in which the expression and distribution of the interleukin-2 receptor (IL-2R) on leukocyte surface membranes was imaged using colloidal gold-labeled antibodies (i.e., immunogold). When compared with current SEM preparation procedures using CPD, Peldri II is a useful alternative that is thought to offer several important advantages.

Tumor-cytolytic human macrophages cultured as nonadherent cells: potential for the adoptive immunotherapy of cancer.

Tumor-cytolytic lymphokine (e.g., interleukin-2; IL-2)-activated killer cells are currently being evaluated in IL-2/LAK cell adoptive immunotherapy regimens for the treatment of cancer. Monocyte-derived macrophages (M phi) are also known to be efficient tumor killer cells; accordingly, M phi that have been activated in vitro may also be of therapeutic merit. However, attempts to cultivate M phi for morphological and functional studies have often been compromised because M phi adhere rapidly and tenaciously to cultureware. Studies that we have conducted to address this problem have proven successful in developing procedures for the long-term cultivation of non-adherent immunocompetent M phi in serum-free medium using petri dishes containing a thin Teflon liner. The utility of this technology is documented by the results of studies presented herein in which light and scanning electron microscopy was used to analyze tumor-cytolytic human M phi. In these experiments, we demonstrated that nonadherent immunocompetent human M phi can be prepared for detailed examinations of their pleomorphic membrane architecture. Moreover, nonadherent human M phi could readily be collected for preparing conjugates of M phi and tumor cells. It is anticipated that this technology should prove useful for future structure-function studies defining the topographical location and spatial distribution of antigens and receptors on M phi membrane ultrastructures, particularly the microvilli-like projections that bridge together an immunocompetent effector M phi and target cell (e.g., tumor cells and microbial pathogens) and which provide the physical interaction required for the initial phases of a cellular immune response that includes antigen recognition and cell-to-cell adhesion.

Mitogenic effect of interleukin-2 on unstimulated human T cells: an editorial review.

The most salient feature of the lymphokine interleukin-2 (IL-2), a hormone-like protein, is its ability to sustain the proliferation of immunocompetent T cells. Results of initial studies characterizing IL-2 in vitro led investigators to conclude that IL-2 had no known effects on lymphocytes that had not been previously activated by exposure to a mitogen or antigen. Several groups postulated that T cell growth required two signals. The first signal, delivered by a mitogen or antigen, induced T cell activation. Resting T cells, which were thought to lack the membrane receptor for interleukin-2 (IL-2R), progressed from the G0-G1 phase to the S phase, during which time they converted from IL-2R- to IL-2R+ cells. Thereafter, the second signal, served by IL-2, induced T cell proliferation of the IL-2R+ cells. Recently, a number of investigators have demonstrated that highly purified preparations of both natural and recombinant IL-2 induced high levels of T cell proliferation in the absence of any known mitogens or antigens. Presented herein is a review of these studies and an overview of the hypotheses of the mechanisms whereby IL-2 alone induces T cell activation and proliferation.

Interleukin-2 induced mitogenesis of human peripheral blood T-lymphocytes: role of accessory cells.

We and others have shown that interleukin-2 (IL-2) is mitogenic to a subset of unstimulated T lymphocytes in human peripheral blood (1-7). We extend our work here in showing that prolonged continuous exposure of human peripheral blood lymphocytes to exogenous IL-2 throughout the 7-8 day culture is not necessary since mitogenesis occurs reproducibly after short term (2-3 hr) pulse exposure. The mitogenic effect of pulse exposure to IL-2 is not significantly reduced by inclusion of anti-Tac monoclonal antibody in the pulsing medium. However, anti-Tac monoclonal antibody markedly inhibits the response if present continuously throughout the 7 day culture. The mitogenic effect of IL-2 is dependent on the presence of accessory cells (monocytes) but the accessory cell requirement can be replaced by the phorbol ester TPA (10(-8 to 10(-11) M). Purified monocytes subjected to short term pulse exposure to IL-2 can cause proliferative response in unprimed autologous lymphocytes in co-cultures. The mitogenic effect of IL-2 pulsed monocytes can not be suppressed by inclusion of anti-Tac antibody in the pulsing medium although the same concentration of the antibody suppresses the effect if present throughout culture. The response of lymphocytes to IL-2 pulsed monocytes is not inhibitable by the continuous presence of a monoclonal antibody to human HLA-DR antigens (OK-Ia1) in culture.

IL-2-induced polyclonal proliferation of human peripheral blood lymphocytes: functional and phenotypic characteristics of proliferating cells.

Unstimulated human peripheral blood lymphocytes (HPBL) were found to proliferate when cultured in vitro with interleukin-2 (IL-2). In bulk long-term cultures of HPBL cultured with IL-2, cell numbers usually doubled after 8-11 days of culture, and a 10-fold increase in cell number occurred between the second and third weeks of culture. These cells retained their ability to respond to a panel of T-cell dependent antigens, phytomitogens and allogeneic cells up to Day 21 of culture. The proliferating cells predominantly expressed the T-cell antigens (CD3, CD4 and CD8), but not antigens of natural killer (NK) cells, B cells or mononuclear phagocytic cells. The proportion of cells expressing CD3 and CD4 antigens progressively increased with length of culture. Purified lymphocytes expressing either CD4 or CD8 antigens were also found to be capable of showing a proliferative response to IL-2, especially when provided with autologous accessory cells. However, purified human peripheral blood B cells expressing the Leu 12 antigen did not respond with or without autologous accessory cells. Unlike the responses to phytomitogen, soluble antigens or allogeneic cells, the proliferative responses of HPBL to IL-2 were not inhibited by a monoclonal antibody (OK-Ia-1) to the non-polymorphic part of human class II histocompatibility antigens.