The local lymph node assay in practice: a current regulatory perspective.

Following the formal acceptance of the local lymph node assay (LLNA) as an Organization for Economic Cooperation and Development (OECD) guideline in April 2002, the UK Health and Safety Executive (HSE) informed notifiers that this was now the method of choice for the assessment of skin sensitization potential under the EU notification scheme for new industrial chemicals (NONS). This paper summarizes the experience of the HSE for the 2-year period immediately following the issuing of this statement, during which 48 LLNA study reports were assessed for notification purposes. The issues discussed here include adherence to the OECD guideline, interpretation of results, and classification outcomes. Generally, notifying laboratories followed the OECD guideline successfully, with regard to the sex/ strain/numbers of mice used, the precise process used for measurement of cell proliferation, and the use of recommended vehicles and positive controls. Initially, use of the individual animal approach (measuring the cell proliferation in each animal rather than for a pooled dose group) highlighted problems caused by technical inexperience, but these were overcome by practice. Toxicity or irritation were found to be minor factors in dose selection; more important was the choice of vehicle to correctly maximize the test substance concentration, while maintaining appropriate application properties. Contrary to concerns that the LLNA would prove to be less sensitive or more sensitive than the traditionally used Guinea Pig Maximization Test (GPMT), the proportion of new substances classified as skin sensitizers was within the range observed in previous years. Although the sample size is relatively small, the experience of the HSE indicates that the LLNA is satisfactory for routine regulatory use.

Dendritic cells and skin sensitisation hazard assessment.

Allergic contact dermatitis is an important occupational and environmental health disease. There is a need, therefore, to identify skin sensitisation hazard, and to assess accurately likely risks to human health. During the past 15 years very significant advances have been made in our understanding of the cellular and molecular mechanisms that serve to initiate and regulate cutaneous immune responses, including the acquisition of skin sensitisation. This has facilitated parallel advances in the identification and characterisation of skin sensitising chemicals and the development of more robust approaches to risk assessment. It is relevant to consider whether advances in immunobiology provide opportunities also for the design of alternative approaches to the toxicological evaluation of skin sensitisation, including the development of in vitro methods. Here we review the potential use of strategies based on analysis of responses induced in Langerhans cells and dendritic cells; professional antigen processing and presenting cells that are known to play pivotal roles during the induction phase of adaptive immune responses.

Alternative approaches to the identification and characterization of chemical allergens.

Chemical allergy can take a variety of forms, those of greatest importance in an occupational setting being skin sensitization resulting in allergic contact dermatitis and sensitization of the respiratory tract associated with asthma and other symptoms. In both cases there is a need for predictive test methods that allow the accurate identification of sensitizing chemicals. Well characterized methods are available for skin sensitization testing, and although to date no tests for respiratory sensitization have been formally validated, progress has been made in defining suitable animal models. In recent years there have been significant advances in our understanding of the cellular and molecular mechanisms through which allergic sensitization to chemicals is induced and regulated. Such progress provides us now with new opportunities to consider alternative approaches to sensitization testing, including the design of in vitro test methods. The greatest investment has been in exploring novel methods for the identification of contact sensitizers and it is upon this aspect of chemical allergy that this article is focused. Described here are some of the general requirements of in vitro test methods for skin sensitization, and progress that has been made in developing suitable approaches with particular emphasis on the utility of dendritic cell culture systems.

Peripheral cytokine responses to Trichuris muris reflect those occurring locally at the site of infection.

The study of human cellular immune responses to parasite infection under field conditions is very complex. Often, the only practical site from which to sample the cellular responses is the peripheral blood. Sampling peripheral blood lymphocytes (PBL) relies on the assumption that these peripheral responses accurately reflect the immune responses acting locally at the site of infection. This is a particularly important point for the human intestinal helminth Trichuris trichiura, which solely inhabits the cecum and large intestine and so will stimulate a localized immune response. Using the well-defined model of T. trichiura, T. muris in the mouse, we have demonstrated that the dominant cytokine responses of the mesenteric lymph nodes (MLN) can be detected by sampling PBL. Resistant mice which mount a type 2 cytokine response in their MLN had PBL producing interleukin-4 (IL-4), IL-5, and IL-9, with negligible levels of gamma interferon (IFN-gamma). Conversely, susceptible mice which mount a type 1 cytokine response in their MLN had PBL producing IFN-gamma and negligible levels of type 2 cytokines. We have also shown that the PBL are capable of mounting a functional immune response against T. muris. PBL from immune mice were capable of transferring immunity to T. muris-infected severe combined immunodeficient (C.B-17 scid/scid) mice. Sampling PBL responses is therefore a viable option for monitoring human intestinal immune responses during T. trichiura infection in the field.

Stat6 signaling promotes protective immunity against Trichinella spiralis through a mast cell- and T cell-dependent mechanism.

Studies in mice infected with the gastrointestinal nematode parasite Nippostrongylus brasiliensis demonstrated that IL-4/IL-13 activation of Stat6 suppresses development of intestinal mastocytosis and does not contribute to IL-4/IL-13 production, but is still essential for parasite expulsion. Because expulsion of another gastrointestinal nematode, Trichinella spiralis, unlike N. brasiliensis expulsion, is mast cell dependent, these observations suggested that T. spiralis expulsion would be Stat6 independent. Instead, we find that Stat6 activation by IL-4/IL-13 is required in T. spiralis-infected mice for the mast cell responses that induce worm expulsion and for the cytokine responses that induce intestinal mastocytosis. Furthermore, although IL-4 induces N. brasiliensis expulsion in the absence of B cells, T cells, and mast cells, mast cells and T cells are required for IL-4 induction of T. spiralis expulsion. Thus, Stat6 signaling is required for host protection against N. brasiliensis and T. spiralis but contributes to expulsion of these two worms by different mechanisms. The induction of multiple effector mechanisms by Stat6 signaling provides a way for a cytokine response induced by most gastrointestinal nematode parasites to protect against most of these parasites, even though different effector mechanisms are required for protection against different nematodes.

Mast cells, eosinophils and antibody-mediated cellular cytotoxicity are not critical in resistance to Trichuris muris.

The murine intestinal nematode Trichuris muris provides an invaluable model of human infection with T. trichiura. Hence, analysis of the immunological responses in the mouse may elucidate the mechanisms of immunity to trichuriasis in man. The work described here investigates the roles of eosinophils, mast cells and antibody-dependent cell-mediated cytotoxicity (ADCC) in the elimination of T. muris from the host gut. Following ablation of IL-5, and hence eosinophilia, mice usually resistant to T. muris infection remained so. Further, blocking the stem cell factor receptor, c-kit, to facilitate complete ablation of mast cells over the period of parasite expulsion in resistant mice had no effect on the development of protective immunity. Therefore it can be deduced that eosinophils and mast cells are not critical in resistance. In addition to these studies, the role of antibody-mediated cellular cytotoxic mechanisms was investigated via the analysis of an infection time course in Fc gamma R-/- mice. These animals, on a resistant background, were fully immune and expelled the parasites before development of the adult stage. Thus this model provides evidence against a major role for ADCC in resistance to infection with T. muris. The studies described here have eliminated some of the major effector mechanisms traditionally associated with helminth infection, and work continues to elucidate the critical immune responses associated with resistance.

Th1 cytokine mRNA expression dominates in the skin-draining lymph nodes of C57BL/6 mice following vaccination with irradiated Schistosoma mansoni cercariae, but is down-regulated upon challenge infection.

Vaccination of C57BL/6 mice with irradiated cercariae of Schistosoma mansoni results in the induction of high levels of immunity to subsequent infection. The events occurring in the lymph nodes draining the exposure site have been analysed ex vivo by reverse transcription-polymerase chain reaction (RT-PCR) and the timing of cytokine gene expression following exposure has been established. After vaccination, spatial separation of the T-helper type 1 (Th1) and Th2 responses was evident, with interferon-gamma (IFN-gamma), interleukin-2 (IL-2) and IL-12 mRNA peaking earlier than mRNA for IL-4, IL-5 and IL-10. In contrast to the profiles observed post-vaccination, following challenge the IL-4 mRNA was predominant in the draining lymph nodes, with IFN-gamma message levels barely detectable above the naive level. These observations are confirmed by the analysis of IL-4 and IFN-gamma mRNA using competitive PCR. From these studies it is clear that irradiated cercariae are more able to promote a protective Th1 response, with normal parasites eliciting higher IL-4 and IL-5 expression upon both primary and secondary stimulation.

Antibody isotype responses to Trichuris infection in humanized SCID mice.

We have developed a humanized SCID mouse model of trichuriasis involving the injection of peripheral blood lymphocytes (PBLs) from normal healthy donors into C. B. 17 severe combined immunodeficiency (SCID) mice followed by vaccination and infection of these mice with the murine species of Trichuris, Trichuris muris. Optimal results with respect to parasite specific antibody production and peripheral engraftment were achieved by injecting intraperitoneally 2 x 10(7) PBLs which had been incubated overnight on anti-human CD3 coated plates. Mice were immunized three weeks post reconstitution with parasite antigen in Freund's Incomplete Adjuvant and infected two weeks later. At autopsy human T cells could be detected in the spleens of engrafted animals and anti-T. muris antibody detected. The dominant IgG isotype responses were shown to be IgG1 and IgG2, providing a similar IgG isotype profile to that seen in humans infected with T. trichiura in the field. In several cases engrafted animals showed the remarkable ability to expel their parasite load. The model will thus be useful for analysing human immune responses to trichuriasis under highly controlled laboratory conditions impossible to achieve in the field.

Pharmacologic management of hyperglycemia in diabetes mellitus: implications for physical therapy.

Diabetes mellitus is a costly, chronic disease that affects millions of Americans each year. The classic triad of diabetes management includes diet, exercise, and pharmacological intervention. A variety of complications may result due to this chronic disease, and manipulation of the triad of treatment factors may be necessary in order to effectively treat the individual patient. Physical therapists are consulted in both the primary care of patients with diabetes and in the case of complications; therefore, an understanding of the various forms of the disease, the complications, and the treatment approaches is necessary for comprehensive patient management. The purposes of this article are to give an overview of the disease and its common complications and to discuss the various treatment approaches with emphasis on the pharmacological interventions and physical therapy concerns.