Contact sensitivity (CS) is a form of delayed‐type hypersensitivity to haptens applied epicutaneously and is thought to be mediated, like classical delayed‐type hypersensitivity responses, by CD4+ T helper‐1 cells. The aim of this study was to identify the effector T cells involved in CS. We studied CS to the strongly sensitizing hapten dinitrofluorobenzene (DNFB) in mice rendered deficient by homologous recombination in either major histocompatibility complex (MHC) class I, MHC class II, or both, and which exhibited deficiencies in, respectively, CD8+, CD4+, or both, T cells. MHC class I single‐deficient and MHC class I/class II double‐deficient mice, both of which have a drastic reduction in the number of CD8+ T cells, were unable to mount a CS response to DNFB. In contrast, both MHC class II‐deficient mice and normal mice treated with an anti‐CD4 monoclonal antibody (mAb) developed exaggerated and persistent responses relative to heterozygous control littermates. Furthermore, anti‐CD8 mAb depletion of class II‐deficient mice totally abolished their ability to mount an inflammatory response to DNFB. Removal of residual CD4+ T cells in class II‐deficient mice by anti‐CD4 mAb treatment did not diminish the intensity of CS. These data clearly demonstrate that class I‐restricted CD8+ T cells are sufficient for the induction of CS to DNFB, and further support the idea that MHC class II‐restricted CD4+ T cells down‐regulate this inflammatory response.

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The purpose of this article is to review, and make recommendations for, the use of relevant skin sensitization test methods, for the purposes of determination of relative potency and the threshold dose necessary for the induction of skin sensitization, and for risk assessment. In addressing the first area, the utility of three guinea pig tests (the guinea pig maximization test, the occluded patch test, and the open epicutaneous test) of the local lymph node assay (LLNA) and of human volunteer testing for the assessment of relative potency and identification of thresholds for sensitization were considered. The following conclusions were drawn. (1) Although attempts have been made to modify the guinea pig maximization test for the purposes of deriving dose-response relationships, this method is usually unsuitable for determination of relative sensitizing potency. (2) Guinea pig methods that do not require the use of adjuvant and which employ a relevant route of exposure (the occluded patch test and the open epicutaneous test) are more appropriate for the assessment of relative skin-sensitizing potency. (3) The LLNA is suitable for the determination of relative skin sensitizing potency, and the adaptation of this method for derivation of comparative criteria such as EC3 values (the estimated concentration of test chemical required to induce a stimulation index of 3 in the LLNA) provides an effective and quantitative basis for such measurements. (4) For all the methods identified above, potency is assessed relative to other chemical allergens of known skin sensitizing potential. The estimation of likely threshold concentrations is dependent upon the availability of suitable benchmark chemicals of known potency for human sensitization. (5) Human testing (and specifically, the Human Repeat Insult Patch Test) can provide information of value in confirming the absence of skin sensitizing activity of formulations and products under specific conditions of use and exposure. Based on the above, the following recommendations are made. (1) If results are already available from suitable guinea pig tests, then judicious interpretation of the data may provide information of value in assessing relative skin sensitizing potency. This option should be explored before other analyses are conducted. (2) The LLNA is the recommended method for new assessments of relative potency, and/or for the investigation of the influence of vehicle or formulation on skin sensitizing potency. (3) Whenever available, human skin sensitization data should be incorporated into an assessment of relative potency. With respect to risk assessment, the conclusion drawn is that all the available data on skin-sensitizing activity in animals and man should be integrated into the risk-assessment process. Appropriate interpretation of existing data from suitable guinea pig studies can provide valuable information with respect to potency, as the first step in the development of a risk assessment. However, for de novo investigations, the LLNA is the method favored fo...

Contact sensitivity to para-phenylenediamine (PPD) is a frequent delayed-type hypersensitivity resulting in contact dermatitis. The aim of the present study, conducted in 16 patients allergic to PPD (as assessed by a positive patch test), was to get better insight into the mechanism of T-cell activation in PPD contact sensitivity. PPD was unable to induce significant proliferation of T cells from a first set of 9 patients. In 7 further patients, lymphocyte proliferation was assessed using PPD and 2 PPD metabolites, namely Bandrowski’s base (BB) and benzoquinone (BQ). BB specifically stimulated T-cell proliferation in a dose-dependent fashion in all 7 patients whereas BQ, like PPD, was ineffective. The peripheral blood mononuclear cells (PBMC) of 8 PPD nonallergic individuals did not respond to either PPD, BB or BQ. We concluded from this study that: (1) the immunogenic hapten in PPD hypersensitivity is not PPD itself, and (2) BB might be the oxidative derivative of PPD endowed with T-cell-activating properties. Further support to this statement was provided by the observation that a T cell line derived from PBMC of a PPD-allergic patient in the presence of PPD responded to BB but not to PPD. Our in vitro results suggest that PPD is a prohapten which when applied on the skin is metabolized and converted into products (such as BB) which are the immunogenic haptens able to activate specific T cells.

Murine models for the assessment of the contact sensitizing properties of chemicals rely on mouse ear swelling tests (Mest), which are not sensitive enough to detect weak sensitizers. The aim of the present study was to develop in mice an adjuvant-free Mest appropriate for in vivo detection of any type of sensitizer (weak to strong), and useful for in vitro assessment of contact sensitivity (CS). 3 haptens were tested: dinitrochlorobenzene (DNCB), para-phenylenediamine (pPD) and isoeugenol. We compared various protocols for induction of the CS reaction, differing by the site of induction, the number of applications and the concentrations of the 3 haptens. Comparison of the induction site for optimal CS reaction showed that, in Balb/c mice, the back was a better site of induction than the abdomen. Detection of the sensitizing properties of weak sensitizers (pPD, isoeugenol) was possible using an adjuvant-free protocol, provided that the induction phase comprised hapten applications on 3 consecutive days on the backs of animals. For DNCB, one application was sufficient to obtain optimal CS reaction. For all 3 haptens, a secondary response in vitro was obtained using semi-purified lymph node T cells from animals sensitized 5 days before with the optimized Mest. These results demonstrate that the Mest could be a useful experimental model for the study of all types of contact sensitizers.

Leucin-rich, glioma inactivated 1 (LGI1) is a secreted protein linked to human seizures of both genetic and autoimmune aetiology. Mutations in the LGI1 gene are responsible for autosomal dominant temporal lobe epilepsy with auditory features, whereas LGI1 autoantibodies are involved in limbic encephalitis, an acquired epileptic disorder associated with cognitive impairment. We and others previously reported that Lgi1-deficient mice have early-onset spontaneous seizures leading to premature death at 2-3 weeks of age. Yet, where and when Lgi1 deficiency causes epilepsy remains unknown. To address these questions, we generated Lgi1 conditional knockout (cKO) mice using a set of universal Cre-driver mouse lines. Selective deletion of Lgi1 was achieved in glutamatergic pyramidal neurons during embryonic (Emx1-Lgi1cKO) or late postnatal (CaMKIIα-Lgi1cKO) developmental stages, or in gamma amino butyric acidergic (GABAergic) parvalbumin interneurons (PV-Lgi1cKO). Emx1-Lgi1cKO mice displayed early-onset and lethal seizures, whereas CaMKIIα-Lgi1cKO mice presented late-onset occasional seizures associated with variable reduced lifespan. In contrast, neither spontaneous seizures nor increased seizure susceptibility to convulsant were observed when Lgi1 was deleted in parvalbumin interneurons. Together, these data showed that LGI1 depletion restricted to pyramidal cells is sufficient to generate seizures, whereas seizure thresholds were unchanged after depletion in gamma amino butyric acidergic parvalbumin interneurons. We suggest that LGI1 secreted from excitatory neurons, but not parvalbumin inhibitory neurons, makes a major contribution to the pathogenesis of LGI1-related epilepsies. Our data further indicate that LGI1 is required from embryogenesis to adulthood to achieve proper circuit functioning.

In synucleinopathies, including Parkinson's disease, partially ubiquitylated α-synuclein species phosphorylated on serine 129 (PS129-α-synuclein) accumulate abnormally. Parkin, an ubiquitin-protein ligase that is dysfunctional in autosomal recessive parkinsonism, protects against α-synuclein-mediated toxicity in various models.We analyzed the effects of Parkin deficiency in a mouse model of synucleinopathy to explore the possibility that Parkin and α-synuclein act in the same biochemical pathway. Whether or not Parkin was present, these mice developed an age-dependent neurodegenerative disorder preceded by a progressive decline in performance in tasks predictive of sensorimotor dysfunction. The symptoms were accompanied by the deposition of PS129-α-synuclein but not PS87-α-synuclein in neuronal cell bodies and neuritic processes throughout the brainstem and the spinal cord; activation of caspase 9 was observed in 5% of the PS129-α-synuclein-positive neurons. As in Lewy bodies, ubiquitin-immunoreactivity, albeit less abundant, was invariably co-localized with PS129-α-synuclein. During late disease stages, the disease-specific neuropathological features revealed by ubiquitin- and PS129-α-synuclein-specific antibodies were similar in mice with or without Parkin. However, the proportion of PS129-α-synuclein-immunoreactive neuronal cell bodies and neurites co-stained for ubiquitin was lower in the absence than in the presence of Parkin, suggesting less advanced synucleinopathy. Moreover, sensorimotor impairment and manifestation of the neurodegenerative phenotype due to overproduction of human α-synuclein were significantly delayed in Parkin-deficient mice.These findings raise the possibility that effective compensatory mechanisms modulate the phenotypic expression of disease in parkin-related parkinsonism.

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