Hypertrophic and keloid scars fail to progress from the CD34- /α-smooth muscle actin (α-SMA)+ immature scar phenotype and show gradient differences in α-SMA and p16 expression.

BACKGROUND: Our understanding of the pathogenesis underlying keloid scar formation is still very limited, and the morphological distinction between hypertrophic and keloid scars remains difficult. OBJECTIVES: To test whether hypertrophic and keloid scars may reflect an inability to progress from immaturity to the desired mature normotrophic scar phenotype. METHODS: Using whole-biopsy imaging and an objectively quantifiable way to analyse immunoreactivity, we have compared the immunohistopathological profiles of young immature scars with mature normotrophic scars, hypertrophic scars, and keloids with their surrounding-normal-skin. RESULTS: Abnormal scars (hypertrophic scars and keloids) maintain the immature scar phenotype, characterized by a CD34- (tumour biomarker) and α-smooth muscle actin (α-SMA)+ (myofibroblast) dermal region. This is in contrast to normal skin, surrounding-normal-skin and mature normotrophic scars that were CD34+ / α-SMA- . Immature, hypertrophic and keloid scars showed abnormal epidermal differentiation (involucrin), but only hypertrophic scars and keloids showed increased epidermal thickness. Immature scars did show increased epidermal and dermal proliferation (Ki67), which was absent from abnormal scars, where mesenchymal hypercellularity (vimentin) and senescence (p16) were predominant. Keloidal collagen and α-SMA were previously considered to distinguish between hypertrophic scars and keloids. However, α-SMA staining was present in both abnormal scar types, while keloidal collagen was present mostly in keloids. There were no obvious signs of heterogeneity within keloid scars, and the surrounding-normal-skin resembled normal skin. CONCLUSIONS: Both abnormal scar types showed a unique CD34- /α-SMA+ /p16+ scar phenotype, but the differences between hypertrophic scars and keloids observed in this study were of a gradient rather than absolute nature. This suggests that scar progression to the mature normal scar phenotype is, for as yet unknown reasons, hindered in hypertrophic and keloid scars. What's already known about this topic? Hypertrophic and keloid scars both have sustained epidermal barrier dysfunction, suggesting the persistence of an immature scar phenotype. Morphological distinction between hypertrophic and keloid scars remains a topic of debate, although α-smooth muscle actin (α-SMA) and keloidal collagen have been considered distinguishing features of hypertrophic and keloid scars, respectively. It has been suggested that keloids are not simply homogeneous growths, as heterogeneity within keloid scars and possible involvement of the surrounding-normal-skin have been reported. What does this study add? An extensive whole-biopsy imaging and quantifiable immunohistochemical assessment of immature, mature normal, hypertrophic and keloid scars, including normal skin surrounding keloids. Hypertrophic and keloid scars maintain dermal characteristics of immature scars, rather than transitioning into the normal mature phenotype. Differences between hypertrophic and keloid scars were of a gradient rather than absolute nature, with keloids showing the more extreme phenotype. There was no obvious heterogeneity within keloids, and the normal skin surrounding keloids resembled normal skin. What is the translational message? Keloids remain primarily a clinical diagnosis. A raised scar with the CD34- /α-SMA+ /p16+ phenotype with strong immunoreactivity for p16 and significant amounts of keloidal collagen, together with a thickened and strongly abnormal involucrin-stained epidermis, would sway the diagnosis towards keloid scars. A hypertrophic scar seems more likely when the CD34- /α-SMA+ /p16+ phenotype shows very strong presence of α-SMA+ in large dermal nodules, with lesser p16 staining and absent or negligible keloidal collagen.

Increased epidermal thickness and abnormal epidermal differentiation in keloid scars.

BACKGROUND: The pathogenesis underlying keloid formation is still poorly understood. Research has focused mostly on dermal abnormalities, while the epidermis has not yet been studied. OBJECTIVES: To identify differences within the epidermis of mature keloid scars compared with normal skin and mature normotrophic and hypertrophic scars. METHODS: Rete ridge formation and epidermal thickness were evaluated in tissue sections. Epidermal proliferation was assessed using immunohistochemistry (Ki67, keratins 6, 16 and 17) and with an in vitro proliferation assay. Epidermal differentiation was evaluated using immunohistochemistry (keratin 10, involucrin, loricrin, filaggrin, SPRR2, SKALP), reverse-transcriptase polymerase chain reaction (involucrin) and transmission electron microscopy (stratum corneum). RESULTS: All scars showed flattening of the epidermis. A trend of increasing epidermal thickness correlating to increasing scar abnormality was observed when comparing normal skin, normotrophic scars, hypertrophic scars and keloids. No difference in epidermal proliferation was observed. Only the early differentiation marker involucrin showed abnormal expression in scars. Involucrin was restricted to the granular layer in healthy skin, but showed panepidermal expression in keloids. Normotrophic scars expressed involucrin in the granular and upper spinous layers, while hypertrophic scars resembled normotrophic scars or keloids. Abnormal differentiation was associated with ultrastructural disorganization of the stratum corneum in keloids compared with normal skin. CONCLUSIONS: Keloids showed increased epidermal thickness compared with normal skin and normotrophic and hypertrophic scars. This was not due to hyperproliferation, but possibly caused by abnormal early terminal differentiation, which affects stratum corneum formation. Our findings indicate that the epidermis is associated with keloid pathogenesis and identify involucrin as a potential diagnostic marker for abnormal scarring.

Methotrexate analogues display enhanced inhibition of TNF-α production in whole blood from RA patients.

OBJECTIVES: Although methotrexate (MTX) is the anchor drug in the treatment of rheumatoid arthritis (RA), patients experience clinical resistance to MTX upon prolonged treatment. We explored whether new-generation antifolates elicit superior anti-inflammatory properties when compared to MTX, based on their capacity to inhibit tumour necrosis factor (TNF)-α production. METHOD: T cells in whole blood from 18 RA patients (including MTX-naïve, MTX- responsive, and MTX non-responsive patients) and seven healthy volunteers were stimulated with αCD3/αCD28 antibodies and incubated ex vivo for 72 h with MTX and eight novel antifolate drugs with potentially favourable biochemical and pharmacological properties. Drug concentrations exerting 50% inhibition (IC-50) of TNF-α production (by enzyme-linked immunosorbent assay, ELISA) were determined as an estimate for their anti-inflammatory capacity. In addition, induction of T-cell apoptosis was evaluated by flow cytometry. RESULTS: The new-generation antifolates PT523, PT644, raltitrexed, and GW1843 proved to be potent inhibitors of TNF-α production in activated T cells from all three groups of RA patients and from healthy volunteers. Based on IC-50 values, these antifolates were up to 10.3 times more potent than MTX. The anti-inflammatory effects were observed at drug concentrations that provoked suppression of T-cell activation and induction of apoptosis in 20-40% of activated T cells. CONCLUSION: In an ex-vivo setting, novel antifolates elicited marked inhibition of TNF-α production in activated T cells from RA patients. Further clinical evaluation is warranted to investigate whether a low dosage of these antifolates can elicit immunosuppressive effects equivalent to MTX, and whether they are superior to MTX in patients who fail to respond to MTX.

Palladium-induced Th2 cytokine responses reflect skin test reactivity.

Recently, a crucial role of Th2 responses in nickel allergic contact dermatitis (ACD) was demonstrated. As palladium allergy is an issue of growing interest, the diagnostic potential of Th2 parameters for palladium sensitization was investigated. Palladium (Na(2) [PdCl(4)])-induced lymphocyte proliferation (LPT), Th1 and Th2 cytokine production were correlated with skin test (ST) reactivity in 16 positive and 21 negative controls. Furthermore, the diagnostic potential of these assays was evaluated using receiver operating characteristics (ROC) analysis. For comparison, same experiments were carried out for nickel (NiSO(4)). Correlation coefficients between palladium ST reactivity and IFN-γ, LPT, IL-5, and IL-13 were 0.34, 0.51, 0.69, and 0.78, and overall test accuracies were 68%, 81%, 89%, and 95%, respectively. Both palladium- and nickel-mediated Th2 responses tightly correlate with ST reactivity, supporting recent findings on the crucial role of Th2 involvement in ACD. Therefore, these assays may have great potential as diagnostic tools for future in vitro sensitization testing.

4-1BB-mediated expansion affords superior detection of in vivo primed effector memory CD8+ T cells from melanoma sentinel lymph nodes.

We have been studying the re-activation of tumor-associated antigen (TAA)-specific CD8(+) T cells in sentinel lymph nodes (SLN) of melanoma patients upon intradermal administration of the CpG-B oligodeoxynucleotide PF-3512676. To facilitate functional testing of T cells from small SLN samples, high-efficiency polyclonal T cell expansion is required. In this study, SLN cells were expanded via classic methodologies with plate- or bead-bound anti-CD3/CD28 antibodies and with the K562/CD32/4-1BBL artificial APC system (K32/4-1BBL aAPC) and analyzed for responsiveness to common recall or TAA-derived peptides. K32/4-1BBL-expanded T cell populations contained significantly more effector/memory CD8(+) T cells. Moreover, recall and melanoma antigen-specific CD8(+) T cells were more frequently detected in K32/4-1BBL-expanded samples as compared with anti-CD3/CD28-expanded samples. We conclude that K32/4-1BBL aAPC are superior to anti-CD3/CD28 antibodies for the expansion of in vivo-primed specific CD8(+) T cells and that their use facilitates the sensitive monitoring of functional anti-tumor T cell immunity in SLN.

The proteasome inhibitor bortezomib inhibits the release of NFkappaB-inducible cytokines and induces apoptosis of activated T cells from rheumatoid arthritis patients.

OBJECTIVE: The proteasome is a multicatalytic proteinase complex regulating the intracellular breakdown of many proteins, including those mediating the activation of pro-inflammatory signaling pathways (e.g. NFkappaB), cell proliferation and survival. Conceptually, proteasome inhibitors may therefore elicit potential anti-inflammatory properties by inhibiting these processes and thereby impair the cellular release of pro-inflammatory cytokines such as Tumor Necrosis Factor-alpha (TNF-alpha) in RA patients. METHODS: Whole-blood from 19 RA patients (including methotrexate-responsive and non-responsive patients) and 7 healthy volunteers was incubated ex-vivo with the proteasome inhibitor bortezomib after T-cell stimulation with alphaCD3/CD28. Inhibition of cytokine production by bortezomib was measured after 24 and 72 hours by ELISA. Effects of bortezomib on apoptosis and T-cell activation (CD25 expression) were measured by FACS-analysis. RESULTS: Bortezomib proved to be a rapid (<24 hour) and potent inhibitor of the release of several NFkappaB-inducible cytokines (including TNF-alpha, IL-1Beta, IL-6 and IL-10) by activated T-cells from healthy volunteers and RA patients, regardless of their clinical responsiveness to methotrexate. Median concentrations of bortezomib required to inhibit TNF-alpha production by 50% (mIC-50) were 12 nM (range: 8-50 nM) for healthy volunteers and 46 nM (range: 18-60 nM) for RA patients. A reduction of T cell activation and a marked induction of T-cell apoptosis were revealed as late effects after bortezomib incubations beyond 24 hours. CONCLUSION: Proteasome inhibitors represented by bortezomib may elicit potential anti-inflammatory properties that deserve further exploration in experimental therapies for RA.

Progress on the development of human in vitro dendritic cell based assays for assessment of the sensitizing potential of a compound.

Allergic contact dermatitis is the result of an adaptive immune response of the skin to direct exposure to an allergen. Since many chemicals are also allergens, European regulations require strict screening of all ingredients in consumer products. Until recently, identifying a potential allergen has completely relied on animal testing (e.g.: Local Lymph Node Assay). In addition to the ethical problems, both the 7th Amendment to the Cosmetics Directive and REACH have stimulated the development of alternative tests for the assessment of potential sensitizers. This review is aimed at summarising the progress on cell based assays, in particular dendritic cell based assays, being developed as animal alternatives. Primary cells (CD34(+) derived dendritic cells, monocyte derived dendritic cells) as well as dendritic cell-like cell lines (THP-1, U-937, MUTZ-3, KG-1, HL-60, and K562) are extensively described along with biomarkers such as cell surface markers, cytokines, chemokines and kinases. From this review, it can be concluded that no single cell based assay nor single marker is yet able to distinguish all sensitizers from non-sensitizers in a test panel of chemicals, nor is it possible to rank the sensitizing potential of the test chemicals. This suggests that sensitivity and specificity may be increased by a tiered assay approach. Only a limited number of genomic and proteomic studies have been completed until now. Such studies have the potential to identify novel biomarkers for inclusion in future assay development. Although progress is promising, this review suggests that it may be difficult to meet the up and coming European regulatory deadlines.

Potential method to determine irritant potency in vitro - Comparison of two reconstructed epidermal culture models with different barrier competency.

Testing chemicals for their ability to cause skin irritation is required for all ingredients of products that come into contact with the skin. Here, we describe a potential method for determining the irritant potency of a chemical in vitro and apply the method to two different reconstructed epidermis models which exhibit different barrier properties. Two surfactants: sodium dodecyl sulphate, Triton X100 and two non-surfactants: 2-4-di-nitro-chloro-benzene, cinnamaldehyde were applied topically in a dose response for 24h. Biomarkers IL-1alpha, IL-1RA, IL-8 and MTT were assessed and EC(50) values determined. Variation in barrier properties between the epidermal models led to variation in the extent of penetration of surfactants, but not of non-surfactants which in turn influenced the EC(50) value obtained from surfactants. Furthermore, EC(50) values showed that no single biomarker could be classed as the most sensitive biomarker since biomarker sensitivity differed between the different chemicals studied. However, the ranking of the chemicals in order of strong to weak irritant was the same irrespective of the model used and also independent of the biomarker used (Triton X100>DNCB>SDS>CA). This study describes a method which not only distinguishes an irritant from a non-irritant but which may possibly also be used to determine irritant potency.

Differential suppression of dendritic cell cytokine production by anti-inflammatory drugs.

BACKGROUND: Various anti-inflammatory drugs are available for the treatment of skin disorders. In these diseases, untoward immune responses to endogenous and/or environmental antigens are initiated by maturation and polarization of dendritic cells (DC). OBJECTIVE: To explore the suppressive effects of anti-inflammatory drugs on DC maturation and, in particular, polarization. METHODS: Exposure of DC to nickel in vitro results in DC maturation and secretion of both type 1 and type 2 cytokines, thereby providing a model to study the effects of anti-inflammatory drugs on DC responses. The inhibitory effects of anti-inflammatory drugs (ciclosporin, dexamethasone, diclofenac, dimethylfumarate, hydrocortisone, lactoferrin, 1-alpha,25-dihydroxyvitamin D3) on DC maturation (CD83, CD86, HLA-DR, CXCL8) and polarization (type 1: IL-12p70, TNF-alpha; type 2: IL-10, CCL17) were studied. RESULTS: All anti-inflammatory drugs, except for lactoferrin, had inhibitory effects on DC maturation. Hydrocortisone and dexamethasone exclusively suppressed the release of type 1 cytokines. A less pronounced, but similar profile was observed for dimethylfumarate and 1-alpha,25-dihydroxyvitamin D3. Ciclosporin suppressed both type 1 and 2 cytokines. In contrast, diclofenac suppressed only type 2 DC cytokine secretion. CONCLUSION: The present results give more insight into the pharmacological effects of immunosuppressive drugs on the immune system, and can thereby contribute to a more rational selection of anti-inflammatory drugs for the treatment of inflammatory skin disorders.

A cytotoxic analysis of antiseptic medication on skin substitutes and autograft.

BACKGROUND: There is an increasing demand for the clinical application of human skin substitutes (HSSs) for treating ulcers, burns and surgical wounds. Due to this increasing demand and due to the simultaneous requirement for the administration of topical antiseptic medications, there is a need to determine potential cytotoxic effects of these medications on HSSs compared with autograft skin. OBJECTIVES: To perform such an evaluation. METHODS: Two different HSSs were used (autologous reconstructed epidermis on fibroblast-populated human dermis and allogeneic reconstructed epidermis on a fibroblast-populated rat collagen gel) and were compared with conventional full-thickness autograft. Twelve different antiseptics were applied topically to the stratum corneum in vitro for 24 h. The degree of cytotoxicity was analysed as detrimental changes in histology, metabolic activity (MTT assay) and RNA staining of tissue sections. RESULTS: The antiseptic medications tested showed different degrees of cytotoxicity. Acticoat, Aquacel Ag, Dermacyn, Fucidin, 0.5% silver nitrate solution and chlorhexidine digluconate were not cytotoxic for either HSS or autograft, and can therefore be used as required. Flamazine and zinc oxide cream resulted in moderate cytotoxicity. However, application of Betadine((R)), cerium-silver sulfadiazine cream, silver sulfadiazine cream with 1% acetic acid and Furacine resulted in a substantial decrease in cell viability and a detrimental effect on tissue histology when applied to autograft and especially to HSS. CONCLUSIONS: Due to the potential cytotoxic effect of some antiseptics on HSS, it is advised that clinicians balance the cytotoxicity of the medication, its antiseptic properties and the severity of colonization in choosing which one to apply.

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.

Autologous full-thickness skin substitute for healing chronic wounds.

BACKGROUND: Chronic wounds represent a major problem to our society. Therefore, advanced wound-healing strategies for the treatment of these wounds are expanding into the field of tissue engineering. OBJECTIVES: To develop a novel tissue-engineered, autologous, full-thickness skin substitute of entirely human origin and to determine its ability to heal chronic wounds. METHODS: Skin substitutes (fully differentiated epidermis on fibroblast-populated human dermis) were constructed from 3-mm punch biopsies isolated from patients to be treated. Acellular allodermis was used as a dermal matrix. After a prior 5-day vacuum-assisted closure therapy to prepare the wound bed, skin substitutes were applied in a simple one-step surgical procedure to 19 long-standing recalcitrant leg ulcers (14 patients; ulcer duration 0.5-50 years). RESULTS: The success rate in culturing biopsies was 97%. The skin substitute visibly resembled an autograft. Eleven of the 19 ulcers (size 1-10 cm2) healed within 8 weeks after a single application of the skin substitute. The other eight larger (60-150 cm2) and/or complicated ulcers healed completely (n = 5) or continued to decrease substantially in size (n = 3) after the 8-week follow-up period. Wound healing occurred by direct take of the skin substitute (n = 12) and/or stimulation of granulation tissue/epithelialization (n = 7). Skin substitutes were very well tolerated and pain relief was immediate after application. CONCLUSIONS: Application of this novel skin substitute provides a promising new therapy for healing chronic wounds resistant to conventional therapies.

Expression of drug pathway proteins is independent of tumour type.

Current clinicopathological staging systems have the advantage of standardized criteria for assessing tumour stage, and a relationship between advancing tumour stage and poor prognosis has been established for most cancers. However, these tools have not led to clear criteria for therapy selection in individual patients. Indeed, the concept of therapy based on anatomical location seems quaint. Therefore, a representative drug pathway (irinotecan) was evaluated across common tumour types to test the hypothesis that pharmacological proteins are expressed independent of anatomical location. Many enzymes are involved in controlling the disposition of irinotecan, including the cellular target (TOP1), metabolism enzymes (CES2, UGT1A1, CYP3A4, CYP3A5), and cellular transporters of the anti-cancer agent (ABCB1, ABCC1, ABCC2, ABCC3, ABCC5, ABCG2). These 11 proteins were evaluated in tissue microarrays containing colon, breast, prostate, ovary, and lung cancers; brain tumours; melanoma; lymphoma; and selected normal tissues. A total of 255 tumours and 37 normal tissue samples were evaluable for all proteins. Linear discriminant analysis designed to predict the tissue type from the protein expression levels revealed a 49.6% misclassification rate, indicating that protein expression of this drug pathway is not associated with tissue type. Cluster analysis identified a variety of tumours with the same pharmacological profile. The anatomy independence of drug pathways stimulates efforts to move away from our traditional approaches to the selection of cancer therapy.

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.

Melanoma-specific tumor-infiltrating lymphocytes but not circulating melanoma-specific T cells may predict survival in resected advanced-stage melanoma patients.

PURPOSE: To study the effect of autologous tumor cell vaccinations on the presence and numbers of circulating CD8+ T cells specific for tumor-associated antigens (TAA) in metastatic melanoma patients. To investigate the correlation between the presence of tumor-infiltrating lymphocytes (TIL) and circulating TAA-specific CD8+ T cells before and after autologous tumor cell vaccination with overall survival. EXPERIMENTAL DESIGN: Twenty-five stage III and resected stage IV metastatic melanoma patients were adjuvantly treated with a series of intracutaneously injected autologous tumor cell vaccinations, of which the first two contained BCG as an immunostimulatory adjuvant. Tumor samples and blood samples obtained before and after vaccination of these patients were studied for the presence of TAA-specific T cells using HLA-tetramers and results were correlated with survival. RESULTS: In 5 of 17 (29%) melanoma patients, circulating TAA-specific T cells were detectable prior to immunizations. No significant changes in the frequency and specificity were found during the treatment period in all patients. Presence of circulating TAA-specific T cells was not correlated with survival (log rank, P=0.215). Inside melanoma tissue, TAA-specific TIL could be detected in 75% of 16 available tumor samples. In case of detectable TAA-specific TIL, median survival was 22.5 months compared to median survival of 4.5 months in case of absence of TAA-specific T cells (log rank, P=0.0094). In none of the patients, TAA-specific T cells were found both in tumor tissue and blood at the same time. CONCLUSIONS: These data suggest that the presence of TAA-specific TILs forms a prognostic factor, predicting improved survival in advanced-stage melanoma patients. The absence of TAA-specific T cells in the circulation suggests that homing of the tumor-specific T cell population to the tumor site contributes to the effectiveness of antitumor immunity.

CXCL8 secretion by dendritic cells predicts contact allergens from irritants.

Monocyte-derived dendritic cell functions have been explored for identification of contact allergens in vitro. Current methods, including measurement of changes in cell surface marker expression (e.g. CD83, CD86) do not provide a sensitive method for detecting the sensitising potential of a chemical. In this study, we investigated whether chemokine production by monocyte-derived dendritic cells is increased upon maturation and whether chemokine production can provide methodology for the detection of allergens. Monocyte-derived dendritic cells were exposed to allergens (nickel sulphate, cobalt chloride, palladium chloride, copper sulphate, chrome-(III)-chloride, potassium dichromate, p-phenylenediamine and dinitrochlorobenzene) and irritants (sodium dodecyl sulphate, dimethylsulphoxide, benzalkoniumchloride and propane-1-ol). CD83 and CD86 expression was analysed by flow cytometry and chemokine production (CXCL8, CCL5, CCL17, CCL18, CCL19, CCL20, CCL22) was determined by ELISA. Significant up regulation of CD83 and CD86 expression could only be induced by three out of seven and five out of seven allergens, respectively. In contrast, CXCL8 production was significantly increased after stimulation with all allergens tested, whereas irritant exposure led to decreased CXCL8 production. All other chemokines tested, failed in identifying contact allergens. In conclusion, CXCL8 production, next to CD83 and CD86 up regulation, by monocyte-derived dendritic cells provides a promising in vitro tool for discrimination between allergens and irritants.

Regulation of nickel-induced T-cell responsiveness by CD4+CD25+ cells in contact allergic patients and healthy individuals.

In this study, we investigated the capacity of CD4+CD25+ regulatory T cells to suppress nickel-specific effector T cells, both in nickel-allergic patients and healthy controls. CD4+ cells isolated from allergic patients showed an increased proliferative response to nickel, whereas CD4+ cells from negative controls did not respond to allergen. When CD4+CD25+ cells were depleted, nickel-specific responsiveness was strongly increased both in allergic and in non-allergic individuals, with the most pronounced effect in allergic patients. These regulatory T cells were anergic to nickel but inhibited nickel-specific CD4+CD25- effector T cells in coculture experiments. CD4+CD25+ cells from nickel-allergic patients showed only a limited capacity to suppress effector T-cell responsiveness, because an increased nickel reactivity could still be detected in these cocultures. None of the isolated CD4+CD25+ cells, either isolated from healthy controls or allergic patients, produced IL-10 in response to nickel. Overall, these results support the view that CD4+CD25+ cells can control the activation of nickel-specific effector T cells in non-allergic individuals, whereas this regulatory capacity is impaired in allergic patients. To investigate the presence of allergen-specific regulatory T cells in truly naïve, non-sensitized individuals, T-cell reactivity should also be studied with non-environmental contact allergens, such as para-phenylenediamine.

Induction of cytokine (interleukin-1alpha and tumor necrosis factor-alpha) and chemokine (CCL20, CCL27, and CXCL8) alarm signals after allergen and irritant exposure.

The immune system is called into action by alarm signals generated from injured tissues. We examined the nature of these alarm signals after exposure of skin residential cells to contact allergens (nickel sulfate and potassium dichromate) and a contact irritant [sodium dodecyl sulfate (SDS)]. Nickel sulfate, potassium dichromate, and SDS were applied topically to the stratum corneum of human skin equivalents. A similar concentration-dependent increase in chemokine (CCL20, CCL27, and CXCL8) secretion was observed for all three chemicals. Exposure to nickel sulfate and SDS was investigated in more detail: similar to chemokine secretion, no difference was observed in the time- and concentration-dependent increase in pro-inflammatory cytokine [interleukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha)] secretion. Maximal increase in IL-1alpha secretion occurred within 2 h after exposure to both nickel sulfate and SDS and prior to increased chemokine secretion. TNF-alpha secretion was detectable 8 h after chemical exposure. After allergen or irritant exposure, increased CCL20 and CXCL8, but not CCL27, secretion was inhibited by neutralizing human antibodies to either IL-1alpha or TNF-alpha. Our data show that alarm signals consist of primary and secondary signals. IL-1alpha and TNF-alpha are released as primary alarm signals, which trigger the release of secondary chemokine (CCL20 and CXCL8) alarm signals. However, some chemokines, for example, CCL27 can be secreted in an IL-1alpha and TNF-alpha independent manner. Our data suggest that skin residential cells respond to both allergen and irritant exposure by releasing mediators that initiate infiltration of immune responsive cells into the skin.

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. OBJECTIVES: Here, we studied new approaches for in vitro monitoring of nickel-specific effector ad regulatory cell functions in allergic patients and potentially tolerized individuals. METHODS: Lymphocyte proliferation assays were carried out with the allergen and additional IL-12/IL-7 or IL-4/IL-7 cytokine supplements. Release of IFN-gamma and IL-5 were assessed as measures for type-1 and type-2 effector T cell function, respectively, and IL-10 and TGF-beta1 to monitor possible regulatory T cell function reflecting immunological tolerance. After optimization of in vitro cut-off values, potency of these parameters was evaluated as compared with conventional nickel patch testing. RESULTS: One hundred and fifty six outpatients were included in this study, 74 of whom presenting with a positive history of nickel allergy. Nickel-sulphate patch test results showed positive reactions in 43 patients, of whom 40 had a positive history (test sensitivity 54%; specificity 96%; overall accuracy 76%). Proliferation tests without cytokine supplementation showed an accuracy of 68%, which was further improved by supplementing IL-4/IL-7 (82%). IFN-gamma and IL-5 cytokine production, as revealed in IL-12/IL-7 and IL-4/IL-7 supplemented cultures, respectively, showed accuracies of 70% and 83%. As to the production of putatively immunoregulatory cytokines, IL-10 was most informative, with highest production rates in nickel-skin test negative individuals with long-lasting mucosal metal contact preceding skin piercing. CONCLUSIONS: These results indicate that measuring both T cell proliferation and cytokine secretion profiles, in particular IL-5 release using IL-4/IL-7 supplemented medium, offers a promising improvement of the in vitro diagnostic options in monitoring nickel contact sensitization. Since oral nickel contact has been shown earlier to induce active tolerization, nickel-induced in vitro IL-10 production may help identify nickel-tolerized individuals.