The transfer of antibiotic resistance from food to humans: facts, implications and future directions.

The food chain, from production to the consumer's kitchen, can be an important contributor to the development, persistence and dissemination of antibiotic-resistant (ART) microbes, including both ART foodborne pathogens and commensal bacteria. Many factors in the food chain, such as the antimicrobial compounds used and how they were used, microbial co-selection, fitness and persistence mechanisms, host lifestyle, and food treatment conditions, influence the antibiotic resistance (AR) cycle. Targeted mitigation strategies, such as those used in the dairy processing industry, can be effective in reducing the AR gene pool.

Industry challenge to best practice risk communication.

UNLABELLED: Effective food safety and food defense risk communication helps to inform consumers without causing panic and alarm. The Risk Communication Team of the Natl. Center for Food Protection and Defense has developed a list of 11 best practices recommended for effective risk communication. These practices, designed for a food defense crisis, are currently applied to food safety issues, since fortunately a food defense crisis has yet to occur. IFT examined the utility of these best practices and the limitations on their use during food safety and food defense crises by academics, trade associations, and the government. It was hypothesized that legal and business considerations as well as the nature of the event would determine the implementation of the best practices. Through the use of focus group meetings, it was discovered that there was a low level of awareness of the best practices. However, stakeholders practiced some aspects of the recommended practices. Participants felt some of the practices were related and could be consolidated. They also agreed that a food defense event will increase the urgency of the communication and include players not typically involved in food safety issues. The challenges reported by the stakeholders varied, but legal liability, as well as the impact their communications could have on an industry, were often cited. From the government perspective, their need to act within their authorities drove some of their actions with respect to communication. Determining the differences in communication limitations during food safety against food defense events can provide key information to further developing and refining risk communications and specific messages targeted for a food defense incident. PRACTICAL APPLICATION: Effective food safety and food defense risk communication helps to inform consumers without causing panic and alarm. Determining the differences in communication limitations during food safety against food defense events can provide key information to further developing and refining risk communications and specific messages targeted for a food defense incident.

Development of a risk-ranking framework to evaluate potential high-threat microorganisms, toxins, and chemicals in food.

Through a cooperative agreement with the U.S. Food and Drug Administration, the Institute of Food Technologists developed a risk-ranking framework prototype to enable comparison of microbiological and chemical hazards in foods and to assist policy makers, risk managers, risk analysts, and others in determining the relative public health impact of specific hazard-food combinations. The prototype is a bottom-up system based on assumptions that incorporate expert opinion/insight with a number of exposure and hazard-related risk criteria variables, which are propagated forward with food intake data to produce risk-ranking determinations. The prototype produces a semi-quantitative comparative assessment of food safety hazards and the impacts of hazard control measures. For a specific hazard-food combination the prototype can produce a single metric: a final risk value expressed as annual pseudo-disability adjusted life years (pDALY). The pDALY is a harmonization of the very different dose-response relationships observed for chemicals and microbes. The prototype was developed on 2 platforms, a web-based user interface and an Analytica(R) model (Lumina Decision Systems, Los Gatos, Calif., U.S.A.). Comprising visual basic language, the web-based platform facilitates data input and allows use concurrently from multiple locations. The Analytica model facilitates visualization of the logic flow, interrelationship of input and output variables, and calculations/algorithms comprising the prototype. A variety of sortable risk-ranking reports and summary information can be generated for hazard-food pairs, showing hazard and dose-response assumptions and data, per capita consumption by population group, and annual p-DALY.

Ballistic helmets and aspects of their design.

The head represents approximately 9% of the body area exposed in combat yet receives approximately 20% of all "hits." The desirability of protecting this vital structure would appear self-evident. Helmet design is a complex issue. Factors that designers of United States Army helmets thoughtfully consider include weight, ballistic qualities of the construction material, balance, helmet-to-person interface (comfort), maintenance of vision and hearing, equipment and weapon compatibility, ease of modification, available materials and manufacturing techniques, durability, ease of decontamination, disposability, and cost. The envisioned future role of the infantryman will make the interplay among these factors even more daunting.

Olfactory receptor neurons in partially purified epithelial cell cultures: comparison of techniques for partial purification and identification of insulin as an important survival factor.

Olfactory neurons have the rare property of being replaced throughout life. Factors regulating different developmental stages of olfactory receptor neurons (ORNs) are of great interest, because such factors might be used to extend regeneration in the post-developmental brain and spinal cord. Also, these factors may potentially be exploited to treat various smell disorders arising from changes in the olfactory epithelium. Characterization of trophic factors for ORNs requires cell culture systems that are simple and easy to manipulate. We have compared four different cell culture preparations, using two different enzymes and two different media to develop a simple culture system of olfactory epithelial cells. Our preferred preparation, which produces partially purified olfactory epithelial cultures, uses trypsin dissociation and a serum-free keratinocyte growth medium (KGM) supplemented with insulin. These conditions support ORN survival up to 1 week. They also supported other elements of the olfactory epithelium such as Bowman's gland cells and horizontal basal cells. Olfactory epithelial cells predominate, while contaminating mesenchymal cells (glia and fibroblasts) are present in low numbers. Using these cultures, it was determined that insulin was required for ORN survival in vitro. The simplicity of the epithelial cultures will be useful for further studies of insulin and other ORN trophic factors.

Identification of a 9 kDa gamma-crystallin fragment in human lenses.

The degraded polypeptides (M(r) less than 14 kDa) were isolated by a preparative SDS-PAGE method from water soluble (WS) and water insoluble (WI) proteins of human lenses from donors of ages between 5 and 75 years. SDS-PAGE analysis showed the presence of a major 9 kDa polypeptide species that showed an age-related increase in levels in WS-polypeptide preparations. In order to identify the parent crystallin of the 9 kDa polypeptide, the immunoreactivities of the WS- and WI-degraded polypeptides to immuno-affinity-purified anti-human alpha-, beta- and gamma-crystallin antibodies were determined by the Western blot method. The WS- and WI-9 kDa polypeptides showed immunoreactivity to only the anti-gamma-crystallin antibody suggesting it to be a fragment of gamma-crystallin. A 9 kDa species was purified by Sephadex G-50 chromatography from the WS-protein fraction of lenses from 20-30-year-old donors. The purified polypeptide showed a single protein band during SDS-PAGE and also an apparent single spot on two-dimensional gel-electrophoresis (IEF followed by SDS-PAGE). The purified preparation also showed a single major peak during reverse phase HPLC chromatography. The purified 9 kDa polypeptide showed immunoreactivity to only the anti-gamma-crystallin antibody. A polyclonal antibody raised against the purified 9 kDa polypeptide showed immunoreactivity only to a 20 kDa gamma-crystallin species. The partial N-terminal sequence analysis of the 9 kDa polypeptide showed it to be a fragment of gamma D-crystallin. Together these results show that a 9 kDa gamma D-crystallin fragment exists in increasing quantities in human lenses during aging.

Covalent binding to alpha-macroglobulins of a protein with free SH groups produced by activated B cells: blocking by D-penicillamine and gold compounds.

alpha 2-Macroglobulin (alpha 2M) complexed with proteinases or modified by the action of amines has been shown to affect immune responses in vitro, though as yet the mechanisms are poorly understood. Supernates from rabbit lymphoid cells cultured in medium with normal rabbit serum and 35S-methionine (or 14C-leucine) were found to contain intensely radiolabeled alpha-macroglobulins (alpha M) (alpha 1 and alpha 2) on electrophoresis. When human alpha 2 M, instead of rabbit serum, was added to cultures, it also appeared radiolabeled, suggesting that lymphocyte-produced proteins (LyP) formed complexes with serum alpha M. These alpha M-associated LyP were produced in greater quantity when lymphocytes were cultured in the presence of mitogens; they were not produced by cells cultured in the presence of cycloheximide; they were produced primarily by B cells rather than T cells or macrophages. Pretreatment of serum or alpha M with methylamine, enhanced rather than inhibited the formation of LyP-alpha M complexes, a finding which is contrary to that expected if the LyP were a proteinase. Since this methylamine treatment of alpha M also results in the generation of free SH groups from the internal thioester bonds of alpha M, the formation of disulfide bonds between LyP and alpha M was considered. Indeed, (a) the LyP-alpha M complex formation was inhibited by N-ethylmaleimide, aurothiomalate, sodium aurothioglucose or D-penicillamine; (b) blocking the SH groups with NEM, of either culture fluid supernates or serum, had an inhibitory effect on the formation of these complexes; (c) the LyP-alpha M complexes were dissociated by sodium dodecyl sulfate (SDS) only after their reduction with 2-mercaptoethanol (2-ME). Thus, a disulfide bond was formed between alpha M and LyP with free SH groups (SH-LyP). Molecular sieving by high performance liquid chromatography (HPLC) of the serum-free radiolabeled supernates indicated that SH-LyP eluted at a position corresponding to a polypeptide of mol. wt of about 22,000. However, SDS-PAGE of the 22,000 mol. wt HPLC fraction showed that the major protein was approximately mol. wt 11,000 under both reducing and non-reducing conditions. In addition, the SH-LyP reduced by 2-ME from its binding site on alpha 2M had a mol. wt of about 11,000 in SDS-PAGE, suggesting that it was a non-covalent homodimer of mol. wt 11,000 polypeptides. We suggest that alpha 2M as well as SH-LyP may affect the immune system by functioning as SH-reactive agents.

Classification of lung cancer patients and controls by chromatography of modified nucleosides in serum.

A wide spectrum of modified nucleosides has been quantified by high-performance liquid chromatography in serum of 49 male lung cancer patients, 35 patients with other cancers, and 48 patients hospitalized for nonneoplastic diseases. Data for 29 modified nucleoside peaks were normalized to an internal standard and analyzed by discriminant analysis and stepwise discriminant analysis. A model based on peaks selected by a stepwise discriminant procedure correctly classified 79% of the cancer and 75% of the noncancer subjects. It also demonstrated 84% sensitivity and 79% specificity when comparing lung cancer to noncancer subjects, and 80% sensitivity and 55% specificity in comparing lung cancer to other cancers. The nucleoside peaks having the greatest influence on the models varied dependent on the subgroups compared, confirming the importance of quantifying a wide array of nucleosides. These data support and expand previous studies which reported the utility of measuring modified nucleoside levels in serum and show that precise measurement of an array of 29 modified nucleosides in serum by high-performance liquid chromatography with UV scanning with subsequent data modeling may provide a clinically useful approach to patient classification in diagnosis and subsequent therapeutic monitoring.

Phase I clinical trial of an MAF-containing lymphoblastoid cell line supernatant.

A preliminary Phase I evaluation of macrophage activating factor (MAF) derived from the partially purified supernatant of human RPMI-1788 lymphoblastoid cell line was performed in 4 parts in 39 patients with advanced cancer. The first two parts used subcutaneous routes of administration and the second two parts used a 4-h intravenous infusion method. Individual doses ranged from 0.1 ml (1.7 mg protein) to 100 ml (1,700 mg protein). Subcutaneous dose was limited by the volume of administered material, and an attempt to use a concentrate of the supernatant resulted in severe local skin reactions. Larger doses given intravenously were well tolerated. Resultant toxicity was mild and consisted of transient fever and chills. One patient with malignant melanoma had a complete response of a 3-cm skin metastasis; one patient with breast cancer had disappearance of a skin nodule while visceral disease progressed; and one patient with histiocytic lymphoma had resolution of a conjunctival lesion. Treatment in many patients was associated with an increase in absolute peripheral lymphocytes. In the high-dose intravenous group, a statistically significant increase in the phagocytic index of peripheral blood leukocytes was noted. Lymphoblastoid MAF appears to be relatively safe to administer and has promise both as an antitumor agent and in the treatment of other altered immune conditions.

Phase I clinical trial of MAF containing preparation of RPMI-1788 B-cell human lymphoblastoid lymphokine in advanced cancer patients.

We report a summary of a Phase I clinical trial of the partially purified MAF-containing supernatant of RPMI-1788 human B-cell lymphoblastoid lymphokine conducted in 39 patients with advanced cancer. The trial was conducted in four parts: two subcutaneous and two intravenous routes and schedules of administration. Dose limiting toxicity in the subcutaneous trials was volume dependent. The intravenous trial was remarkably well tolerated so that the maximum tolerable dose exceeded expectations. The study demonstrated modification of the immune system including an increase in skin test reactivity, an increase in absolute lymphocyte counts, alteration in the monoclonal lymphocyte antibody markers, and an increase in MAF activity. Toxicity in the intravenous trial was limited to transient febrile reactions that were ameliorated by increasing the infusion time. Three objective tumor responses were observed.

Anaphylaxis due to chloramphenicol.

A patient with group B Streptococcal meningitis and a history of penicillin allergy sustained an anaphylactic reaction following intravenous chloramphenicol. The purity of the infusate was confirmed by reverse-phase high-speed liquid chromatography. Anaphylaxis is a rare event following chloramphenicol administration, but physicians should be aware of this complication, especially in patients with prior exposure to the drug.

Lymphokine-stimulated macrophage phagocytosis of fluorescent microspheres: a rapid new assay.

A highly sensitive and rapid in vitro macrophage phagocytosis assay is described for screening of lymphokine preparations with macrophage activating properties. Non-induced mouse peritoneal macrophages were incubated on glass slides for 60 min in the presence of fluorescent 2 micron latex beads and partially purified lymphokine fractions or media control. Lymphokine samples were prepared from culture supernatants of the B lymphoblastoid cell line RPMI 1788 by high performance liquid chromatography of a soluble trichloroacetic acid extract of concentrated culture supernatant. Phagocytosis was measured by direct counts of the percent phagocytic cells and the number of intracellular beads per 100 cells as seen by fluorescence microscopy. Phagocytic uptake in the presence of as little as 0.1 ng of active lymphokine could readily be determined qualitatively by correlation plots and quantitatively by appropriate statistical programs for data processing by computer. This technique provides a rapid, reproducible, screening assay for biological activity of macrophage activating lymphokines and other compounds affecting macrophage phagocytosis.

Characterization of human lymphocyte derived chemotactic factors for mononuclear phagocytes--II. Chemical characterization.

Supernatants of PHA-stimulated human mononuclear cells were fractionated by Sephadex G-100 chromatography and isoelectric focusing. Chemotactic activities for human mononuclear phagocytes were identified at various molecular weight ranges. Major activities were found at mol. wt 45, 30, 25, 18, 12.5, 8 and 6 K. After isoelectric focusing chemotactic activities were recovered predominantly at pH 3.7-5.5 and 7.8-8.5. Fractionation by high performance liquid chromatography (HPLC) on the molecular sieve column I-125 and basic ion exchange column SAX 300 confirmed these results. Furthermore, after absorption of crude supernatants on the reversed-phase column RP 300, chemotactic activity was recovered quantitatively and thus could be separated from the bulk of other materials. In kinetic experiments supernatants were harvested after 5, 27, 42 and 63 hr and fractionated on a molecular sieve column. It was found that only low molecular weight chemotactic activity was released after 5 hr. After 27 hr most activity was found between 10-20,000 after 42 hr activity was found over the whole fractionation range. It is concluded that multiple molecular species of chemotactic factors acting on mononuclear phagocytes are released by activated lymphocyte cultures, whose chemical nature and function remains to be studied.

Lymphokine-mediated immunotherapy studies in mouse tumor systems.

Studies have been performed using two subcutaneously implanted mouse tumor models to investigate the immunotherapeutic potential of lymphokine-containing culture supernatants from long-term human lymphoblast cell cultures. Human lymphoblastoid cell line, RPMI 1788, was used as a cell culture source of lymphokines. Supernatants were removed from cultures at the stationary phase of growth and concentrated on Amicon filters retaining molecules above 10,000 Daltons. This concentrate was applied to a Sephadex G-25 column, equilibrated with ammonium bicarbonate buffer, for removal of salts and dye from the culture medium. The effluent was lyophilized and reconstituted for use in further purification by affinity chromatography and SDS-PAGE gels. Such preparations were used to inject DBA/2 mice bearing subcutaneous L-1210 tumors. In addition, the B-16 melanoma was used as a model of a solid tumor in C57Bl/l mice. Animals were treated intralesionally and intraperitoneally with lymphokines containing preparations and control solutions. Tumors growing subcutaneously were susceptible to lymphokine-induced inflammation-mediated regression without additional therapy. In the study of L-1210 subcutaneous tumors, reduction in tumor size was followed by complete regression, prolonged survival, immunity to additional inoculation, and cures in 20--40% of the treated mice. Tumor regression and prolongation of survival were also noted in mice bearing B-16 melanomas. These studies support the use of mouse tumors as bioassays for antitumor inflammatory activity of human lymphokine preparations and help to quantitate their potential use in human tumor immunotherapy.

The use of leukocyte cell line culture supernatant for skin reaction testing in malignant melanoma.

A study was conducted to determine some of the potential applications of a human leukocyte culture supernatant or "lymphokine" preparation in cancer patients. The application evaluated in this study was the use of this preparation as a skin test reagent for evaluation of the inflammatory response following intradermal injection. The preparation was derived from the supernatant of a long-term cultured lymphoblastoid cell line with migration inhibition factor (MIF) and other lymphokine activities. Dose response, histology and toxicity studies were done in 53 patients with malignant melanoma stage IIIB and IV. A dose response curve was observed for both erythema and induration at 12 and 24 hours, but not at 48 hours. An optimal intradermal dose for eliciting inflammation was determined and found to be five units. Histopathological evaluation of biopsy specimens showed a mixed cell reaction including granulocytes, eosinophils, lymphocytes and monocytes differing in lymphocyte content from the classical delayed type hypersensitivity (DTH) reaction in man. Compared with the response to recall antigens, only a weak correlation with the DTH response to the recall antigens was found. Our results support the conclusion that lymphokines may be used in the future to evaluate the ability to develop nonspecific inflammation in cancer patients, and that this inflammatory response can be obtained in a number of patients no longer capable of responding to recall antigens.

Purification of alpha-2 macroglobulin by immunoadsorbent chromatography.

A method for preparation of alpha-2 macroglobulin (alpha2M) by immunoadsorbent chromatography utilizing rabbit--anti-human alpha2M-conjugated Sepharose 4B is described. This procedure offers a rapid, simple and inexpensive method for purification of alpha2M from as little as 2.0 ml of human serum. By multiple applications to the absorbent column, as much as 10 mg of purified alpha2M may be easily prepared in one day. This technique is especially useful for isolation of alpha2M from individual sera and for studies of cultured cell supernatants containing human alpha2M.