R. J. Scheper

Analysis of effector and regulatory immune reactivity to nickel

Background Diagnostic patch testing in allergic contact dermatitis faces the risk of boosting existing hypersensitivities or active sensitization. Risk‐free and reliable in vitro assays using peripheral blood are, therefore, wanted.

Vascular colocalization of P-glycoprotein, multidrug-resistance associated protein 1, breast cancer resistance protein and major vault protein in human epileptogenic pathologies.

Multidrug transporters, such as P‐glycoprotein (P‐gp), multidrug‐resistance associated protein 1 (MRP1) and breast cancer resistance protein (BCRP), are associated with multidrug resistance in cancers; other molecules, such as major vault protein (MVP), have a similar association with drug‐resistant cancer. These proteins are postulated to generate drug resistance in epilepsy. They have been shown individually to be up‐regulated in epileptogenic brain tissue. In any consideration of the function, inhibition or evasion of the activity of such proteins, the colocalization of such proteins needs to be understood. We systematically determined the presence of such colocalization, focusing on microvascular endothelium from epileptogenic human brain tissue. Double labelling immunofluorescence and confocal laser scanning microscopy were used to determine colocalization of P‐gp, MRP1, BCRP and MVP in one case of hippocampal sclerosis and two cases of focal cortical dysplasia type IIb. Endothelial colocalization was examined with double labelling using antibodies to CD34 and Factor VIII. The presence of P‐gp, BCRP and MVP in microvascular endothelium was confirmed. P‐gp, BCRP and MVP colocalized in microvascular endothelium, though not all proteins appeared to be identically distributed within this tissue. MRP1 did not colocalize to endothelium. These findings were not unexpected but required formal confirmation. The demonstrated colocalization of P‐gp, BCRP and MVP in microvascular endothelium in epileptogenic human brain tissue has important implications for functional experiments (including single knock‐out mice studies), work with specific and broad‐spectrum inhibitors of transport function, and any eventual trials of treatment of refractory epilepsy involving modulation of the function of these proteins.

Delayed time course of irritation by sodium lauryl sulfate: Observations on threshold reactions

Irritant reactions to sodium lauryl sulfate were induced on the backs of 20 volunteers by means of patch test occlusion for 24 h. Different concentrations ranging from 0.259% to 2% were used, the lowest concentration being borderline irritant. The skin tests were read at 24, 48 and 72 h. Both the % of responding individuals and the intensity of the skin reactions were maximal at 48 h for all test concentrations. It is concluded that irritants may provoke inflammatory reactions which are not completely developed after 24 h, and are thus very similar to allergic patch test reactions.

IFN-gamma-producing human invariant NKT cells promote tumor-associated antigen-specific cytotoxic T cell responses.

CD1d-restricted invariant NKT (iNKT) cells can enhance immunity to cancer or prevent autoimmunity, depending on the cytokine profile secreted. Antitumor effects of the iNKT cell ligand α-galactosylceramide (αGC) and iNKT cell adoptive transfer have been demonstrated in various tumor models. Together with reduced numbers of iNKT cells in cancer patients, which have been linked to poor clinical outcome, these data suggest that cancer patients may benefit from therapy aiming at iNKT cell proliferation and activation. Herein we present results of investigations on the effects of human iNKT cells on Ag-specific CTL responses. iNKT cells were expanded using αGC-pulsed allogeneic DC derived from the acute myeloid leukemia cell line MUTZ-3, transduced with CD1d to enhance iNKT cell stimulation, and with IL-12 to stimulate type 1 cytokine production. Enhanced activation and increased IFN-γ production was observed in iNKT cells, irrespective of CD4 expression, upon stimulation with IL-12-overexpressing dendritic cells. IL-12-stimulated iNKT cells strongly enhanced the MART-1 (melanoma Ag recognized by T cell 1)-specific CD8+ CTL response, which was dependent on iNKT cell-derived IFN-γ. Furthermore, autologous IL-12-overexpressing dendritic cells, loaded with Ag as well as αGC, was superior in stimulating both iNKT cells and Ag-specific CTL. This study shows that IL-12-overexpressing allogeneic dendritic cells expand IFN-γ-producing iNKT cells, which may be more effective against tumors in vivo. Furthermore, the efficacy of autologous Ag-loaded DC vaccines may well be enhanced by IL-12 overexpression and loading with αGC.

Defective Differentiation of Myeloid and Plasmacytoid Dendritic Cells in Advanced Cancer Patients is not Normalized by Tyrosine Kinase Inhibition of the Vascular Endothelial Growth Factor Receptor

Tumor-derived vascular endothelial growth factor (VEGF) has previously been identified as a causative factor in the disturbed differentiation of myeloid dendritic cells (DC) in advanced cancer patients. Here, we investigated the potential of vascular endothelial growth factor receptor (VEGFR) tyrosine kinase (TK) inhibition to overcome this defective DC differentiation. To this end, peripheral blood DC (PBDC) precursor and subset frequencies were measured in 13 patients with advanced cancer before and after treatment with AZD2171, a TK inhibitor (TKI) of VEGFR, coadministered with gefitinib, and an epidermal growth factor receptor (EGFR) TKI. Of note, not only myeloid DC but also plasmacytoid DC frequencies were significantly reduced in the blood of the cancer patients prior to treatment, as compared to healthy controls. Moreover, besides an accumulated population of immature myeloid cells (ImC), a population of myeloid suppressor cells (MSC) was significantly increased. Upon systemic VEGFR TK inhibition, DC frequencies did not increase, whereas the rate of circulating MSC showed a slight, but not significant, decrease. In conclusion, TK inhibition of VEGFR with AZD2171 does not restore the defective PBDC differentiation observed in advanced cancer patients.

Comparison of two in vitro dendritic cell maturation models for screening contact sensitizers using a panel of methacrylates

Abstract: Allergen‐induced emigration and maturation of dendritic cells (DC) are pivotal steps in sparking off allergic contact dermatitis. In vitro models, reflecting these steps, may provide tools for assessment of sensitizing capacities of putative contact allergens. Here, we evaluated the applicability of such models for a panel of methacrylate congeners, the sensitizing properties of which were established previously in clinical and experimental animal studies. First, using interleukin‐4 (IL‐4)/granulocyte–macrophage colony‐stimulating factor (GM‐CSF)‐induced, blood monocyte‐derived DC, hapten‐induced up‐regulation of maturation/activation markers, including CD80, CD83, CD86, chemokine receptors CXCR4 and CCR5, as well as the drug resistance related molecules P‐glycoprotein (Pgp) and lung resistance protein (LRP), were monitored by flow cytometry. Of note, whereas CD86 and CXCR4 were most sensitive in discriminating between the contact sensitizers and irritants included in the panel, i.e. sodium dodecyl sulphate (SDS) and croton oil (CO), assessment of CD83 and LRP expression reflected the relatively lower sensitizing capacity of methyl methacrylate. Second, using ex vivo skin explant cultures, allergen‐induced LC migration from epidermal to basal membranous and dermal skin structures was most reliably monitored by CD1a, as compared with Pgp, LRP, HLA‐DR or CD54 staining. The extent of CD1a+ LC migration was found to closely correlate with the sensitizing capacities of the panel of test compounds. These results support the view that both in vitro models can provide valuable data on contact sensitizing properties, and add chemokine receptors and drug resistance related molecules to the list of DC membrane markers revealing allergenic signaling.

Allergic reactions, ‘spillover’ reactions, and T-Cell subsets

SummaryA strong positive, allergic patch-test reaction was elicited in 15 patients with an established allergy for a particular allergen. Patches with a marginally irritating concentration of sodium lauryl sulfate (SLS) were applied at fixed distances. The SLS patch situated adjacent to the allergic reaction was significantly enhanced in 12 of 15 patients (P<0.01) compared to more distant SLS reactions (‘spillover’). Only quantitative differences were observed in the histologic pictures of the different types of reaction. The infiltrate consisted of lymphocytes and histiocytes, mainly located perivascular in the upper dermis.T-cell subsets were assessed with monoclonal antibodies using an immunoperoxidase technique. The distribution of the different T cells was the same for both reaction types.T cells located outside the perivascular infiltrates (e.g., in the epidermal vesicles) were OKT-8-positive (cytotoxic/suppressor T lymphocytes). Immunofluorescence examination did not show different patterns for the allergic or ‘enhanced toxic’ reactions with regard to the presence of immunoglobulins and complement.The ‘spillover’ phenomenon may cause falsepositive patch-test reactions.

Treatment with tumour infiltrating lymphocytes and interleukin-2 in patients with metastatic melanoma: A pilot study

Tumour infiltrating lymphocytes (TIL) were isolated and expanded from biopsy samples of 4 patients with metastatic melanoma. The patients were treated with autologous expanded TIL and continuous or bolus infusion of Interleukin 2 (IL-2) at a dose of 18 × 106 International Units/m2/day for 5 days starting 36–48 hours after administration of cyclophosphamide at a dose of 1 g/m2. The number of TIL infused ranged from 1010 to 5,56 × 1010 cells. Two patients had stable disease (SD) lasting for 2 1/2 and 4 months respectively and they died 24 and 13 months after therapy. One patient died during therapy due to a pseudomonas septicaemia and another patient developed progressive disease (PD). He died 3 months after the start of therapy. The side effects were substantial but most of them were reversible upon cessation of the treatment.The majority of the expanded TIL of all patients were of the CD8+ phenotype. Cutaneous metastases from two patients, removed after treatment with IL-2 and TIL, showed moderate lymphocytic infiltration also mainly of CD8+ T cells.The treatment with IL-2 and TIL is feasible, but further investigations should continue in an attempt to improve the efficacy of the therapy, to reduce toxicity and to diminish the costs and labour of the culture methods.

The angry back syndrome--a retrospective study.

Irrelevant reactions occurred in 68 of 157 patients (43%) with strong positive patch test reactions and concomitant weak positive reactions. A lower figure, 13 of 45 (29%) was found in patients with only weak positive patch test reactions (0.1 > P > 0.05). An allergen eliciting a weak positive reaction should therefore be retested when a conclusion has to be drawn about the epidermal sensitivity of a patient.

Augmentation of antigen‐specific lymphoproliferative responses in vitro by biological response modifiers

The detection of antigen‐specific T cell responsiveness, particularly of resting memory lymphocytes, in cultures of peripheral blood mononuclear cells (PBMC) may be hampered by a less than optimal antigen presentation in vitro. Augmented sensitivity of the test system may be achieved by the addition of reagents with a beneficial effect on lymphocyte and antigen‐presenting cell (APC) functions. In this study the effect of several biological response modifiers on antigen‐specific T cell proliferation was determined, using nickel sulphate and tetanus toxoid as lest antigens. IL‐lα (100 U/ml). interferon‐gamma (IFN‐γ) (10 U/ml), and indomethacin (2 μM) were found to significantly enhance nickel‐induced proliferation in PBMC cultures from nickel‐hypersensitive donors (n = 6). Tetanus‐induced proliferation (n = 5) was similarly enhanced, both by the above supplements and by the addition of polyethylene glycol (PEG) or a neuraminidase treatment of the PBMC before culture. The addition to PBMC cultures of a combination of IL‐lα (30 U/ml), IFN‐γ (10 U/ml), and indomethacin (2 μM) is recommended to specifically enhance antigen‐induced lymphoproliferative signals.