Adverse Outcome Pathway for Antimicrobial Quaternary Ammonium Compounds.

Quaternary ammonium compounds (QACs) or quats are a large class of antimicrobial chemicals used in households and institutions as sanitizers and disinfectants. These chemicals are utilized as food processing sanitizers, algicides, in the process of water treatment, and preservatives in cosmetics. The aim of this study was to determine an Adverse Outcome Pathway (AOP) whereby two widely used QACs, alkyl dimethyl benzyl ammonium chloride (ADBAC) and didecyl dimethyl ammonium chloride (DDAC), may result in respiratory tract and gastrointestinal tract effects. When inhaled or ingested, these QACs are incorporated into the epithelial cell membrane at the point of contact. With sufficient dosage, the epithelial membrane is disrupted, reducing its fluidity, and releasing cellular contents. Further, ADBAC and DDAC might disrupt mitochondrial functions leading to decreased ATP production. Both events might lead to cell death, either attributed to direct lysis, necrosis, or apoptosis. Pro-inflammatory mediators are recruited to the tissue, inducing inflammation, edema, and excess mucus production. The primary tissue-level adverse outcome is epithelial degeneration and dysplasia. Most important, no apparent metabolism or distribution is involved in QAC action. Based upon this knowledge, it is suggested to replace default Uncertainty Factors for risk assessments with a set of Data Derived Extrapolation Factors.

Post-market surveillance of consumer products: Framework for adverse event management.

Analysis of spontaneous reports of adverse events is an important source of information that can be used to improve consumer products. Various agencies have adverse event reporting requirements and many companies collect such data directly from consumers. Nonetheless, a universal framework is absent that identifies and evaluates spontaneously reported adverse events, and, most important, assesses the potential association between exposure and adverse events. We are presenting a three-part framework: Phase I - Intake and Documentation of Original Incidents; Phase II - In Depth Review and Follow-up of Phase I Incidents (enhanced, tailored questionnaire); Phase III - Association Assessment. The basis for scoring the strength of association between exposure and adverse events requires assessment of standard factors of association including: temporality; biological, physiological, or pharmacological plausibility; results of de-challenge; results of re-challenge; and consideration of confounding factors. Scores tied to the answers to these questions are totaled for each incident to determine the strength of association between exposure and reported adverse event. We propose that consumer product companies come together to adopt such an association assessment framework to improve adverse event management, obtain maximum value from the data obtained, and use the knowledge derived to improve overall product safety for consumers.

The efficient bovine insulin presentation capacity of bone marrow-derived macrophages activated by granulocyte-macrophage colony-stimulating factor correlates with a high level of intracellular reducing thiols.

Bone marrow-derived macrophages (BMM phi) were shown before to function as antigen-presenting cells. We show here, that the antigen presentation capacity of BMM phi depends on the nature of the antigen and is differently regulated by the lymphokines interferon-gamma (IFN-gamma) and granulocyte/macrophage-colony-stimulating factor (GM-CSF). When bovine insulin (BI) was employed as antigen, only BMM phi treated with GM-CSF (GM-CSF-M phi) were efficient presenters, but when presentation of the antigens ovalbumin and conalbumin was tested, IFN-gamma-pulsed BMM phi (IFN-gamma-M phi) proved superior to GM-CSF-M phi. The lack of efficient BI presentation function of IFN-gamma-M phi was only obvious, when native BI was used as antigen. Preprocessed BI was presented by IFN-gamma-M phi with drastically higher efficiency than by GM-CSF-M phi. Because processing of insulin depends on reduction of disulfide bonds, we analyzed the content of intracellular reducing thiols within IFN-gamma-M phi, GM-CSF-M phi, and untreated BMM phi. Only after stimulation with GM-CSF did the amount of reduced glutathione and cysteine strongly increase, while IFN-gamma did not efficiently augment the intracellular content of both thiols. These findings suggest that the lymphokines IFN-gamma and GM-CSF differently interfere with the processing capacity of BMM phi by differently regulating the intracellular concentration of the thiols reduced glutathione and cysteine. A high level of these thiols induced by GM-CSF correlates with a prominent capacity to present the antigen bovine insulin.

Antigenically activated helper T cells are required as stimulator cells for optimal activation of cytotoxic T lymphocytes under conditions of limiting helper factors.

This paper deals with the question of how antigenically activated helper cells interact with cytotoxic T-lymphocyte (CTL) precursor cells in an environment where helper factor is limiting. Experiments in culture systems with limiting concentrations of helper factor indicated that the (optimal) activation of CTL required antigenically activated helper T cells as stimulator cells. These experiments were performed partly in macrocultures which contained prostaglandin E2 (PGE2) and partly in microcultures with small numbers of responder cells and without additional helper factors. The results showed that a strong activation of CTL against TNP-haptenated syngeneic or semiallogeneic cells occurred only if the cultures contained TNP-haptenated stimulator cells from euthymic but not athymic donors and if the haptenated stimulator cells were exposed to allogeneic determinants. Moreover, combinations of F1-hybrid stimulator cells and parental responder cells generated no substantial cytotoxic responses against determinants of the other parent, unless the cultures were supplemented with a source of I-region determinants which were foreign to the semiallogeneic stimulator cells. Strong responses against haptenated syngeneic or semiallogeneic stimulator cells were obtained, however, when helper factors were added to the cultures. It was concluded that our cultures with limiting concentrations of helper factors required a close proximity between helper T cells and CTL precursor cells; and this proximity was obviously provided by the receptors of the CTL precursor cells with no detectable contribution from the helper-T-cell receptors. Allogeneically activated helper T cells in the responder cell population or in a second irradiated spleen cell population which did not bear the target antigens delivered no substantial helper effect to the CTL precursor cells under test.

Inhibition of a primary DNA-synthesis response by trinitrobenzosulfonate (TNBS)-activated suppressor cells; characterization of the inhibitory cells.

Repeated intravenous injections of high doses of trinitrobenzosulfonate (TNBS) or dinitrobenzosulfonate (DNBS) activate suppressor cells which inhibit the in vivo activation of a primary DNA synthesis response against trinitrochlorobenzene (TNCB) and dinitrochlorobenzene (DNCB), respectively, almost completely and the delayed type hypersensitivity (DH) response only partially. When tested on the DNA-synthesis response, the suppressor cells show excellent specificity with little cross reactivity of TNBS (or DNBS) induced suppressor cells for DNCB- (or TNCB-) specific responses. TNBS- and DNBS-specific suppressor activity is found in spleen cells, mesenteric lymph nodes and peripheral lymph nodes. The activation of suppressor cells is resistant to the early effects of adult thymectomy (ATx), but sensitive to pretreatment with cyclophosphamide (CyP). The suppressor cells are not simply haptenated cells. They need several days for their activation and are inactivated by incubation for 30 minutes at 56 degrees C or by 2,000 R irradiation. Attempts to obtain soluble suppressor factors by in vitro incubation or extraction of these suppressor cells failed.

Surface carbohydrate composition of murine thymocytes and of lymph node cells from normal and athymic (nude) mice.

Surface glycoproteins (papain digests) have been isolated from lymph node cells of normal mice which contain mainly T cells, and from lymph node cells of nude (athymic) mice, which essentially represent B cells. Gaschromatographic analysis revealed that the glycoproteins from the lymph node cells of the euthymic mice contain less galactose than the glycoproteins from lymph node cells of the athymic mice, but contain still more galactose than glycoproteins from thymocytes. Lymph node cells from both sources contain about equal amounts of neuraminic acid, while thymocytes contain slightly less sialic acid. The observed differences provide a molecular explanation for the different reactivity of murine B cells and T cells towards soybean agglutinine and other galactose-binding plant lectins.

"Early T cells" and "late T cells"; suggestive evidence for two T cell lineages with separate developmental pathways.

Peripheral lymphocytes from mice have been analyzed by a combination of cell electrophoresis and size distribution analysis and the data have been processed with a computer into two-dimensional distribution patterns (fingerprints). The fingerprints of lymph node cells revealed the existence of at least three major classes of small lymphocytes. Cells with similar physical properties were found in the spleen and thoracic duct lymph. The data also allowed calculation of the quantitative ratios of the three cell types. In the normal mouse, the two electrophoretically faster cell classes represent essentially two subclasses of T cells. The quantitative ratios are here always in agreement with the known percentages of B and T cells. Lethally irradiated mice that have been reconstituted with syngeneic bone marrow or fetal liver cells often lack one of the T cell subclasses. This has two important implications. 1) It indicates that the widely used syngeneic or allogeneic bone marrow chimeras may be incomplete in respect to their T cell repertoire and that experiments with these models should be interpreted with caution. 2) The deficiency was found to correlate strictly with a deficiency in a physically defined subset of small cortical thymocytes that has been described previously. This correlation suggests strongly that the fast peripheral T cells and the "early small cortical thymocytes" represent two developmental stages of a distinct cell lineage ("early T cell lineage"), while the thymic pool of "late small cortical thymocytes" gives rise to electrophoretically slow peripheral T cells, which may be called "late T cells). The possibility that both T cell lineages are derived from different classes of prethymic stem cells in the reconstitlting stem cell preparation is discussed. The proportion of "early T cells" in the lymph nodes decreases with age. The ratio of "late" and "early T cells" is similar in the spleen and in the lymph nodes, and both cell classes contain a significant proportion of cells that are not affected by the early effects of adult thymectomy. Thus, they do not correspond to T1 and T2 cells.

The antigen-inexperienced thymic suppressor cells: a class of lymphocytes in the young chicken thymus that inhibits antibody production and cell-mediated immune responses.

Transfer of thymus cells from young chickens in combination with a light whole body irradiation (360 R) was found to suppress the rejection of skin grafts across strong histocompatibility (B) differences. On the average, the suppressed animals also showed decreased serum hemagglutinin titers against erythrocytes of the skin donor strain and a decreased graft-versus-host (GvH) reactivity against embryos of this strain. The thymic suppressor cells can be obtained from animals that have not experienced the antigen under test. However, after transfer and contact to the antigen (skin graft) they can lead to the formation of specific ("activated") suppressor cells and can mediate in the long run a specific inhibition of the response to this antigen. The suppressive activity is associated with a bursa-dependent cellular subpopulation in the thymus that is different from B lymphocytes, B precursor cells or GvH-reactive T cells. The bursa dependency of the thymic suppressor cell suggests that functionally different lineages of thymic and thymus-derived lymphocytes are derived from different sources of prethymic stem cells. The suppressor cells are predominantly found in the young chicken thymus and already detectable in the 16-day-old embryo, while poor suppressive activity is found in the adult thymus. The suppressive effect can be obtained with thymus cells from either syngeneic or allogeneic donors. Embryonic allogeneic donors provide suppressive cell preparations free of GvH reactivity. The possibility that the thymus suppressor cells mediate self tolerance and "neonatal tolerance" is discussed.

Suppressor cells in the embryonic thymus mediate allograft tolerance.

Thymus cells from non-immunized young chickens suppress the allograft rejection in lightly irradiated syngeneic or allogeneic recipients and mediate longlasting skingraft survival in a significant proportion of recipients across a strong histocompatibility difference. Suppressive activity of this kind is already found in the embryonic thymus and is therefore believed to mediate also self tolerance and neonatal allograft tolerance.

Immunosuppressive effect of syngeneic thymus cells on allograft rejection.

Transfer of thymus cells from young chickens to syngeneic recipients suppresses the allograft rejection between strains differing at the major histocompatibility (B) locus. Thymus cell transfer in combination with a light whole body irradiation (360 R) prolongs significantly the mean rejection time of skin allografts and leads in a proportion of recipients to long-lasting graft survival (greater than 200 days). Three weeks after the cell transfer, the suppression appears to be antigen specific, as judged by the normal reactivity against third-party skin grafts. From the types of thymus cells preparations that are effective in these experiments, it is inferred that the suppressor cell is a bursa-dependent lymphocyte, which is predominantly found in the young chicken thymus and which is different from B-lymphocytes, B-precursor cells, or graft-versus-host-reactive T-cells.

Surface carbohydrate composition of different types of chicken lymphocytes.

A simple and quick procedure was used to analyse the carbohydrate composition of surface glycoproteins from chicken lymphocytes. The procedure included papain digestion of the cells, a two-step purification of the supernatant material and a sugar analysis by gaschromatography. The method made it possible to analyse samples of about 10(9) lymphocytes. The surface glycoproteins from different chicken lymphocyte preparations were found to differ significantly in their sugar composition. Lymphocytes from thymus, bursa, spleen or blood were characterized by typical relative amounts of glucose, galactosamine, fucose and sialic acid. The values for mannose, glucosamine and galactose, however, were approximately 1:1:1 for all four lymphocyte preparations. Bursectomy or thymectomy in combination with whole body irradiation altered the carbohydrate composition significantly. The results suggest the possibility that the carbohydrate composition can be used as a marker for different lymphocyte populations. The results are also discussed in respect to the hypothesis that carbohydrate determinants on the cell surface determine the migration of lymphocytes.

Lymphocyte subpopulations in the thymus.

The thymus of mice and chickens contains at least four discrete populations of lymphoid cells: Two distinct cortical populations of small lymphocytes (early and late population), a hydrocortisone resistant and presumably medullary population of small lymphocytes, and a population of medium sized lymphocytes (prolymphocytes) (see Table I and Figure 3). These four cell types were identified with preparative cell separation techniques (e.g. cell electrophoresis, BSA-density gradient centrifugation, and velocity sedimentation) in combination with size distribution analysis. The combination of these techniques provides two-dimensional distribution patterns ('fingerprints') with high power of resolution. At present the two cortical populations of small lymphocytes cannot be identified as distinct populations by any other method. The physical parameters also provide useful markers for the identification and comparison of cellular subpopulations in animals of different ages, different strains, and to a certain degree even of different species. It is believed that each of these subpopulations is in itself heterogeneous in respect to antigen specificity, and it is proposed to call lymphocytes with different antigen specificity but identical physical characteristics 'isotypic lymphocytes'. The medium and large thymocytes serve as progenitors of the small thymic lymphocytes, as shown by different investigators. Small and larger lymphocytes are thus believed to represent different stages on developmental pathways (vertical heterogeneity). The different populations of small thymocytes, on the other hand, are believed to represent different independent pathways (horizontal heterogeneity). There is clearly the possibility that functionally distinct sublines of peripheral T-cells are generated by separate developmental pathways in the thymus, and the functional properties of single thymic cell types (e.g. of the thymic suppressor cells) may accordingly correspond to the functional properties of their peripheral progeny.