Guselkumab demonstrates long-term efficacy and maintenance of treatment response postwithdrawal in systemic treatment-naïve patients and nonresponders to fumaric acid esters: results from parts II and III of a randomized active-comparator-controlled phase IIIb trial (POLARIS).

BACKGROUND: The anti-interleukin-23 antibody guselkumab (GUS) demonstrated favourable week 24 efficacy and safety over fumaric acid esters (FAE) in systemic treatment-naïve patients with moderate-to-severe plaque psoriasis (study part I). OBJECTIVES: To compare, in study part II, the sustainability of treatment responses (weeks 24-32) in GUS- and FAE-treated patients and treatment responses (weeks 32-56) in patients treated with GUS and FAE and in FAE nonresponders switching to GUS; and, in part III, to investigate the maintenance of response through week 100 in patients withdrawn from GUS at week 56. METHODS: At week 0, systemic treatment-naïve patients were randomized 1 : 1 to GUS or FAE as per label. At week 32, patients with a Psoriasis Area and Severity Index (PASI) 75 (≥ 75% improvement in PASI score) response (r) continued assigned treatment (GUSr-GUS; FAEr-FAE), whereas nonresponders (nr) received GUS (FAEnr-GUS; GUSnr-GUS). GUS-treated patients with a week 56 PASI 90 response (≥ 90% improvement in PASI score) were withdrawn (w) and followed until loss of response or week 100. RESULTS: At week 32, 98% (n = 54/55) of GUS- and 41% (n = 14/34) of FAE-treated patients were PASI 75 responders. At week 56, 91%, 50% and 80% of GUSr-GUS, FAEr-FAE and FAEnr-GUS patients, respectively, achieved a PASI 90 response; 72%, 29% and 45%, respectively, achieved a Dermatology Life Quality Index score of 0/1. At week 100, 44 weeks postwithdrawal, 47% (n = 17/36) and 25% (n = 3/12) of GUS-GUSw and FAE-GUSw patients, respectively, maintained a PASI score ≤ 5. Overall, the adverse event and discontinuation rates were lower for GUS than FAE. CONCLUSIONS: In these exploratory analyses, GUS, as a first-line systemic treatment or second-line systemic treatment in FAE nonresponders, was associated with long-term clinical efficacy up to week 100, including a withdrawal period.

The socio-economic burden of H1-antihistamine-refractory chronic spontaneous urticaria in Germany.

BACKGROUND AND STUDY AIM: Data from the AWARE study (A Worldwide Antihistamine-Refractory chronic urticaria patient Evaluation) illustrate a substantial disease burden in German patients with H1-antihistamine (-H1-AH)-refractory chronic spontaneous urticaria (CSU). Detrimental effects on patients' quality of life, poor disease control and impairment in the ability to work and perform other daily activities are reported. Based on these findings, this study aims to quantify the epidemiological and socio-economic burden of H1-AH-refractory CSU in Germany. METHODS: To determine the epidemiological burden of H1-AH-refractory CSU, the age- and gender-specific prevalence of CSU and the proportion of H1-AH-refractory patients in Germany anonymized data from the InGef research database have been used. In a second step, the socio-economic burden in terms of lost numbers of hours in paid and unpaid work was calculated by extrapolating the age- and gender-specific work productivity and activity impairment (WPAI) observed in AWARE to the H1-AH-refractory CSU population in Germany. Finally, productivity losses in paid and unpaid work were monetized using the human capital and the friction cost approach respectively. Moreover, socio-economic burden was calculated depending on symptom control of the patients (measured by urticaria control test [UCT]). RESULTS: In Germany, over 203,000 patients (20 years or older) had H1-AH-refractory CSU in 2018. The avoided lost paid and unpaid work hours attributable to H1-AH-refractory CSU summed up to over 100 million. Overall, the socio-economic burden of H1-AH-refractory CSU in monetary terms was evaluated at € 2.2 billion and the majority of this was due to unpaid work loss. Patients with poor disease control, as indicated by UCT score < 12, were more likely to suffer from high impairment than patients with controlled disease, resulting in a higher socio-economic burden. CONCLUSION: The results of our analyses picture the substantial socio-economic burden of H1-AH-refractory CSU and therefore the tremendous impact it has on daily lives of individuals and society overall.

Modelling of patient journey in chronic spontaneous urticaria: Increasing awareness and education by shorten patients' disease journey in Germany.

BACKGROUND: Chronic spontaneous urticaria (CSU) is both physically and emotionally stressful, and guideline recommendations are often not optimally implemented in clinical practice. The objective of this study was to provide an overview on the patient journey in CSU and to develop a mathematical model based on solid data. METHODS: The journey of CSU patients in Germany was traced through literature review and expert meetings that included medical experts, pharmacists and representatives of patient organizations. The current situation's main challenges in the patient journey (education, collaboration and disease management) were discussed in depth. Then, a probabilistic model was developed in a co-creation approach to simulate the impact of three potential improvement strategies: (1) patient education campaign, (2) medical professional education programme and (3) implementation of a disease management programme (DMP). RESULTS: Chronic spontaneous urticaria patients are severely burdened by delays in diagnosis and optimal medical care. Our simulation indicates that in Germany, it takes on average of 3.8 years for patients to achieve disease control in Germany. Modelling all three optimization strategies resulted in a reduction to 2.5 years until CSU symptom control. On a population level, the proportion of CSU patients with disease control increased from 44.2% to 58.1%. CONCLUSION: In principle, effective CSU medications and a disease-specific guideline are available. However, implementation of recommendations is lagging in practice. The approach of quantitative modelling of the patient journey validates obstacles and shows a clear effect of multiple interventions on the patient journey. The data generated by our simulation can be used to identify strategies for improving patient care. Our approach might helping in understanding and improving the management of patients beyond CSU.

Chronic spontaneous urticaria - a management pathway for patients with chronic spontaneous urticaria.

Chronic spontaneous urticaria (CSU) is a common and challenging disease, especially with respect to healthcare provision in the context of the German statutory health insurance system. If treatment with second-generation antihistamines is unsuccessful, current guidelines recommend further therapeutic options. However, most of these are off-label. This discrepancy between treatment according to guidelines and the ability to prescribe drugs at the expense of the statutory health insurance (reimbursability) often leads to uncertainties in everyday clinical practice. In addition, physicians prescribing certain drugs are faced with the difficulty of measuring and documenting therapeutic success/outcome. Respective outcome measurement methods have not yet been established in daily practice. Using a consensus process, a working group composed of dermatologists in private practice and specialized urticaria centers has defined a practical pathway for the implementation of current treatment recommendations based on the 2013 S3 guidelines for urticaria. Here, we present a diagnostic and therapeutic management pathway for CSU. Further, we discuss prescription issues in daily practice, including updosing of antihistamines, with regard to cost-effectiveness and drug approval on the basis of published studies and current legislation. Constituting the highest treatment level, the use of cyclosporine A, montelukast, and omalizumab, which has recently become available as therapeutic option, is reviewed. The urticaria control test (UCT) is presented as a valid outcome measure in routine practice. Our objective was to provide physicians in private practice with a practical guideline-based therapeutic decision tool, taking into account the requirements imposed by the statutory health insurance system. It is not meant to replace individualized history taking or treatment of this heterogeneous disease. Rather, we would like to suggest reference points for clinical diagnosis and treatment of CSU.

Long-term use of fumaric acid esters for the treatment of psoriasis in daily practice.

BACKGROUND: Fumaric acid ester (FAE) is the most commonly prescribed first-line systemic therapy for the treatment of psoriasis in Germany. Although developed in the 1990s, only limited long-term data are available. METHODS: Data of 200 adult psoriatic patients from 10 study centers were collected in a noninterventional, multicenter, retrospective analysis. The inclusion criteria was treatment with FAE in 2015. RESULTS: Eighty-two percent of the patients were naive to systemic treatment. Ten percent of all patients had FAE-treatment for 10 years or longer with an average drug survival of 4.32 years. The maintenance dose was ranging from 1-4 120 mg tablets for 87.5% of the patients. In our population, 14% of the patients stopped therapy during the first six month mainly due to gastro-intestinal side effects. No serious side effects were reported. Seventy-eight percent of the patients responded to FAE therapy with improvement of their psoriasis to mild (61%) or clear (17%). The PASI 75 response was achieved in 44% of the patient during long-term treatment without remarkable differences between moderate or severe plaque psoriasis. CONCLUSION: Our study confirms FAE therapy as a long-term, first-line treatment for moderate-to-severe plaque psoriasis.

Oligosaccharides of Hyaluronan activate dendritic cells via toll-like receptor 4.

Low molecular weight fragmentation products of the polysaccharide of Hyaluronic acid (sHA) produced during inflammation have been shown to be potent activators of immunocompetent cells such as dendritic cells (DCs) and macrophages. Here we report that sHA induces maturation of DCs via the Toll-like receptor (TLR)-4, a receptor complex associated with innate immunity and host defense against bacterial infection. Bone marrow-derived DCs from C3H/HeJ and C57BL/10ScCr mice carrying mutant TLR-4 alleles were nonresponsive to sHA-induced phenotypic and functional maturation. Conversely, DCs from TLR-2-deficient mice were still susceptible to sHA. In accordance, addition of an anti-TLR-4 mAb to human monocyte-derived DCs blocked sHA-induced tumor necrosis factor alpha production. Western blot analysis revealed that sHA treatment resulted in distinct phosphorylation of p38/p42/44 MAP-kinases and nuclear translocation of nuclear factor (NF)-kappa B, all components of the TLR-4 signaling pathway. Blockade of this pathway by specific inhibitors completely abrogated the sHA-induced DC maturation. Finally, intravenous injection of sHA-induced DC emigration from the skin and their phenotypic and functional maturation in the spleen, again depending on the expression of TLR-4. In conclusion, this is the first report that polysaccharide degradation products of the extracellular matrix produced during inflammation might serve as an endogenous ligand for the TLR-4 complex on DCs.

Inhibition of tumour cell growth by hyperforin, a novel anticancer drug from St. John's wort that acts by induction of apoptosis.

Hyperforin is a plant derived antibiotic from St. John's wort. Here we describe a novel activity of hyperforin, namely its ability to inhibit the growth of tumour cells by induction of apoptosis. Hyperforin inhibited the growth of various human and rat tumour cell lines in vivo, with IC(50) values between 3-15 microM. Treatment of tumour cells with hyperforin resulted in a dose-dependent generation of apoptotic oligonucleosomes, typical DNA-laddering and apoptosis-specific morphological changes. In MT-450 mammary carcinoma cells hyperforin increased the activity of caspase-9 and caspase-3, and hyperforin-mediated apoptosis was blocked by the broad-range caspase inhibitor zVAD.fmk. When added to MT-450 cells, hyperforin, but not paclitaxel, induced a rapid loss of the mitochondrial transmembrane potential Deltapsi(m), and subsequent morphological changes such as homogenization and vacuolization of mitochondria. Monitoring of Deltapsi(m) revealed that the hyperforin-mediated mitochondrial permeability transition can not be prevented by zVAD.fmk. This indicates that mitochondrial permeabilization is a cause rather than a consequence of caspase activation. Moreover, hyperforin was capable of releasing cytochrome c from isolated mitochondria. These findings suggest that hyperforin activates a mitochondria-mediated apoptosis pathway. In vivo, hyperforin inhibited the growth of autologous MT-450 breast carcinoma in immunocompetent Wistar rats to a similar extent as the cytotoxic drug paclitaxel, without any signs of acute toxicity. Owing to the combination of significant antitumour activity, low toxicity in vivo and natural abundance of the compound, hyperforin holds the promise of being an interesting novel antineoplastic agent that deserves further laboratory and in vivo exploration.

Distinct effects of sphingosine-1-phosphate, lysophosphatidic acid and histamine in human and mouse dendritic cells.

Dendritic cells (DC) are specialized antigen presenting cells characterized by their ability to migrate into target sites and secondary lymphoid organs in order to process antigens and activate naive T cells. Previously, we have shown that several secretion products from platelets and mast cells such as histamine, sphingosine-1-phosphate (S1P), and lysophosphatidic acid (LPA) have chemotactic activity towards immature human DC. Furthermore, they limit the capacity of mature human DC to initiate and amplify T helper cell type 1 (Th1) immune responses by inhibition of interleukin (IL)-12 and upregulation of IL-10 secretion. In this study we focused on the effect of these agents on murine DC. In murine DC no influence on IL-10 and IL-12 release by these agents was observed. Moreover, histamine and LPA failed to stimulate chemotaxis and actin reorganization in mouse DC. Instead, S1P had chemotactic activity and induced actin polymerization in immature as well as mature mouse DC. Therefore, our in vitro data implicate that in contrast to humans the function and immunological capacity of murine DC are not so tightly controlled by mast cell and platelet-derived secretion products such as histamine, S1P and LPA. These findings suggest that mouse models might underestimate the complex regulative network between mast cells, platelets and DC.

Cicatricial pemphigoid in the upper aerodigestive tract: diagnosis and management in severe laryngeal stenosis.

Pemphigoid is a group of rare, acquired, autoimmune subepithelial blistering diseases. The condition has been subclassified into bullous pemphigoid and cicatricial pemphigoid (CP). Diagnosis is based on clinical presentation, evidence of subepithelialvesicles or bullae on routine histologic analysis, and direct and indirect immunofluorescence studies. Cicatricial pemphigoid is characterized by linear deposition of immunoreactants, principally IgG and complement factor 3, along epithelial basement membranes. Cicatricial pemphigoid usually leads to mucosal scarring. We present a case of severe CP that led to laryngeal and subglottic stenosis and involvement of both eyes and the oral, nasal, and nasopharyngeal mucosae. Treatment with dapsone, corticosteroids, azathioprine sodium, cyclosporine A, cyclophosphamide, methotrexate sodium, and mycophenolate mofetil between 1997 and 2001 only resulted in temporary disease control. The patient has been treated with leflunomide for the past 8 months, and there have been no relapses. Treatment of CP with leflunomide has not been described in the literature until now.

Toll-like receptor and IL-12 signaling control susceptibility to contact hypersensitivity.

Allergic contact hypersensitivity (CHS) is a T cell-mediated inflammatory skin disease. Interleukin (IL)-12 is considered to be important in the generation of the allergen-specific T cell response. Loss of IL-12 function in IL-12Rbeta2-deficient mice, however, did not ameliorate the allergic immune response, suggesting alternate IL-12-independent pathways in the induction of CHS. Because exposure to contact allergens always takes place in the presence of microbial skin flora, we investigated the potential role of Toll-like receptors (TLRs) in the induction of CHS. Using mice deficient in TLR4, the receptor for bacterial lipopolysaccharide (LPS), IL-12 receptor (R) beta2, or both, we show that the concomitant absence of TLR4 and IL-12Rbeta2, but not the absence of TLR4 or IL-12Rbeta2 alone, prevented DC-mediated sensitization, generation of effector T cells, and the subsequent CHS response to 2,4,6-trinitro-1-chlorobenzene (TNCB), oxazolone, and fluorescein isothiocyanate. Introduction of the TLR4 transgene into the TLR4/IL-12Rbeta2 mutant restored the CHS inducibility, showing a requirement for TLR4 in IL-12-independent CHS induction. Furthermore, the concomitant absence of TLR2 and TLR4 prevented the induction of CHS to TNCB in IL-12-competent mice. Finally, CHS was inducible in germ-free wild-type and IL-12Rbeta2-deficient mice, but not in germ-free TLR4/IL-12Rbeta2 double deficient mice, suggesting that the necessary TLR activation may proceed via endogenous ligands.

Switch in syndecan-1 and syndecan-4 expression controls maturation associated dendritic cell motility.

Dendritic cells (DCs) need to mobilize within the extracellular matrix (ECM) during their maturation and concomitant migration from peripheral sites to lymphoid organs. Syndecans are cell surface proteoglycans that mediate the interaction of DCs with the ECM. Here we investigated the influence of syndecans on dendritic cell motility and morphology. Langerhans cells of the epidermis and monocyte-derived DCs were found to undergo a switch in syndecan expression during maturation. Syndecan-1 was downregulated and syndecan-4 was strongly upregulated within the first hours of lipopolysaccharide-induced dendritic cell maturation and during Langerhans cell emigration from human skin, as shown by flow cytometry and qRT-PCR. Syndecan-1 downregulation was inhibited by syndecan-4 siRNA knock-down, indicating a functional interconnection between enhanced syndecan-4 expression and syndecan-1 downregulation. Syndecan-4 upregulation is functionally involved in dendritic cell motility, as inhibition of syndecan-4 function by means of blocking antibodies or through siRNA knock-down decreased dendritic cell motility. In other experiments, the cytoskeletal component a-actinin was observed to be upregulated in DCs as a consequence of the induction of maturation, and was found to colocalize with syndecan-4. Furthermore, lammellopodial spreading by DCs on fibronectin (FN)-coated surfaces was dependent on syndecan-4. This binding of syndecan-4 to FN and its association with the cytoskeleton may be relevant for syndecan-4-dependent dendritic cell motility. We conclude that the switch in syndecan expression during dendritic cell maturation controls the motility of DCs in a way that appears to be crucial for their mobilization from peripheral sites and subsequent migration to lymphoid tissues.

Differential regulation of hyaluronan metabolism in the epidermal and dermal compartments of human skin by UVB irradiation.

Hyaluronan (HA), a major component of the cutaneous extracellular-matrix, is involved in tissue repair. Human skin is exposed to and damaged by UVB-irradiation. Here, we investigate the regulation of HA metabolism in human skin during acute UVB-induced inflammation. Expression of HA synthesizing (HAS) and degrading enzymes hyaluronidase (HYAL) as evaluated by quantitative reverse transcribed PCR in response to UVB differed when fibroblasts and HaCaT-keratinocytes, representative cell types in dermis and epidermis, respectively, were compared. Both demonstrated temporally different expression patterns of these genes 3- and 24-hours post-irradiation. This resulted 24-hours post-irradiation in an increase in HAS gene expression in both fibroblasts and HaCaT-keratinocytes, and an increase in HYAL expression only in fibroblasts. HA-production as analyzed by the HA content of conditioned medium was reduced in HaCaT and fibroblast cultures 3-hours post-irradiation, whereas HA increased in HaCaT-cultures 24-hours post-irradiation but remained suppressed in fibroblasts-cultures. Consistently, immunohistochemical staining for HA in human skin 24-hours post-irradiation demonstrated an increased epidermal HA, but a decrease in the dermal compartment. Moreover, analysis of the HA content of dermal microdialysis-fluid revealed increased accumulation of HA degradation products 24-hours post-irradiation. These data demonstrate that there is a complex temporal and spatial regulation of HA-metabolism in skin in response to UVB irradiation.

Mature monocyte-derived dendritic cells respond more strongly to CCL19 than to CXCL12: consequences for directional migration.

The chemokine receptor CCR7 is crucial for migration of mature dendritic cells (DC) directed toward secondary lymphoid organs; however, there is little knowledge about the function of the homeostatic chemokine receptor CXCR4 in DC and its contribution to directional migration of DC during inflammation. By comparing the impact of chemokine receptor engagement on mature DC we found that the CCR7 ligand CCL19 holds a stronger chemotactic potency than the CXCR4 ligand CXCL12. Moreover, CCL19 elicited rapid, steep and long-lasting mobilization of intracellular calcium in individual cells and induced intense phosphorylation of extracellular signal-regulated kinase 1/2 and protein kinase B, while the intracellular signals elicited by CXCL12 were in part distinct and significantly weaker. Analysis of chemokine receptor expression revealed that although CCR7 and CXCR4 were expressed by a similar percentage of DC, the mean fluorescence intensity of CCR7 was up to six times higher, suggesting a higher receptor density. Based on these correlations we propose that the type of chemokine signal in conjunction with the expression and functional activity of the respective chemokine receptor is also determining the migration rate and potency of a chemotactic response in mature DC. In conclusion, our data support the fundamental role of CCR7 for rapidly guiding DC toward secondary lymphoid organs at an extra- and intracellular molecular level and on the contrary render CXCR4 a weaker contributor to directional migration of DC during inflammation.

In situ profiling and quantification of cytokines released during ultraviolet B-induced inflammation by combining dermal microdialysis and protein microarrays.

In skin, an evolving inflammatory or immune response is triggered by early release of a cytokine cascade into the extracellular space. Investigation of extracellular cytokine secretion in situ has been limited by low cut-off filtering membranes and sample volume size and the inability to monitor changes in cytokine protein levels in real-time in situ. Here, we combine for the first time the methods of intradermal microdialysis and antibody protein arraying to profile the early cascade of multiple cytokines in a complex inflammatory response exemplified by ultraviolet B (UVB)-induced inflammation. We observed significant differences of the cytokine and growth factor responses after tissue injury by catheter placement and UVB-induced inflammation. UVB irradiation initiates a rapid proinflammatory response followed by a mixed TH1/TH2 response in which ultimately TH2 cytokines IL-4 and IL10 predominated after 24 h. This most likely indicates the termination and self limitation of the inflammatory response. We conclude that the combination of dermal microdialysis and protein microarray offers a powerful tool to analyze in real-time the complex and rapidly changing interstitial protein milieu during cutaneous inflammatory responses.

[Tacrolimus--a new therapeutic option for Hailey-Hailey-disease?]. / Tacrolimus--Neue Therapieoption beim Morbus Hailey-Hailey?

Hailey-Hailey disease is a chronic inflammatory skin disorder. Only symptomatic measures are available for long-term treatment. Topical tacrolimus has been shown to be effective in a number of inflammatory skin diseases, such as atopic eczema and can be used over long periods of time because of its safety profile. We tested tacrolimus ointment in a patient who had problems with Hailey-Hailey disease for many years. After only five days of treatment with topical tacrolimus, improvement was apparent. After four weeks of treatment, the pain and erosions were significantly ameliorated. The efficacy of topical tacrolimus in Hailey-Hailey disease may provide new insights into the pathogenesis of the disorder.

Hyperforin acts as an angiogenesis inhibitor.

Hyperforin is a plant compound from Hypericum perforatum that inhibits tumor cell proliferation in vitro by induction of apoptosis. Here, we report that hyperforin also acts as an angiogenesis inhibitor in vitro and in vivo. In vitro, hyperforin blocked microvessel formation of human dermal microvascular endothelial cells (HDMEC) on a complex extracellular matrix. Furthermore, hyperforin reduced proliferation of HDMEC in a dose-dependent manner, without displaying toxic effects or inducing apoptosis of the cells. To evaluate the antiangiogenic activity of hyperforin in vivo, Wistar rats were subcutaneously injected with MT-450 mammary carcinoma cells and were treated with peritumoral injections of hyperforin or solvent. Hyperforin significantly inhibited tumor growth, induced apoptosis of tumor cells and reduced tumor vascularization, as shown by in situ staining of CD31-positive microvessels in the tumor stroma. These data suggest that, in addition to the induction of tumor cell apoptosis, hyperforin can also suppress angiogenesis by a direct, non-toxic effect on endothelial cells.

Induction of CD4+ T cell apoptosis as a consequence of impaired cytoskeletal rearrangement in UVB-irradiated dendritic cells.

Low dose UVB irradiation of dendritic cells (DC) dose-dependently decreases their allostimulatory capacity and inhibits alloreactive T cell proliferation. The reduction of the stimulatory capacity is not associated with a perturbation of CD28 costimulation. To examine the underlying mechanism, cell cycle analysis of T cells from cocultures with UVB-irradiated DC (UVB-DC) was performed, revealing no cell cycle arrest, but an increased number of apoptotic T cells in sub-G(0) phase. We confirmed T cells to undergo apoptosis after coincubation with UVB-DC by TUNEL staining and DNA laddering. To analyze whether T cell apoptosis requires the Fas/Fas ligand (FasL) pathway, MLRs were performed with Fas-, FasL-deficient, and wild-type DC and T cells. No differences were found on comparison of wild-type DC with Fas-/FasL-deficient DC or T cells. Likewise, addition of a neutralizing anti-TNF-alpha mAb to cocultures could not overcome inhibition of T cell proliferation by UVB-DC, excluding involvement of the TNF-alpha/TNF-alphaR pathway. FACS analysis of CD69 and CD25 revealed no up-regulation on T cells cocultured with UVB-DC, suggesting a perturbation of early T cell activation. Analysis of UVB-DC by confocal microscopy demonstrated impaired filamentous actin bundling, a process critical for T cell stimulation. To investigate the functional relevance of these observations, time lapse video microscopy was performed. Indeed, calcium signaling in CD4(+) T cells was significantly diminished after interaction with UVB-DC. In conclusion, UVBR of DC impairs their cytoskeletal rearrangement and induces apoptosis in CD4(+) T cells by disruption of early DC-T cell interaction, resulting in a reduced Ca(2+) influx in T cells.

Hyaluronan fragments stimulate endothelial recognition of injury through TLR4.

Tissues must quickly recognize injury to respond to the rapid pace of microbial growth. In skin, dermal microvascular endothelial cells must also react to danger signals from the surrounding tissue and immediately participate by initiating the wound repair process. Components of the extracellular matrix such as hyaluronan are rapidly broken down into smaller molecular weight oligosaccharides in a wound, and these can activate a variety of biological processes. This study set out to determine if hyaluronan fragments released following injury can stimulate endothelial cells and what mechanism is responsible for this response. Using genechip microarray analysis, a response to hyaluronan fragments was detected in endothelial cells with the most significant increase observed for the chemokine IL-8. This observation was verified with qualitative reverse transcriptase-PCR and ELISA in human endothelial cell culture, and in a mouse model by observing serum levels of MIP-2 and KC following hyaluronan fragment administration in vivo. Activation was TLR4-dependent, as shown by use of TLR4 blocking antibody and TLR4-deficient mice, but not due to the presence of undetected contaminants as shown by inactivation following digestion with the hyaluronan-degrading enzyme chondroitinase ABC or incubation with the hyaluronan-specific blocking peptide Pep-1. Inactivation of LPS activity failed to diminish the action of hyaluronan fragments. These observations suggest that endogenous components of the extracellular matrix can stimulate endothelia to trigger recognition of injury in the initial stages of the wound defense and repair response.

Targeting dendritic cells with CD44 monoclonal antibodies selectively inhibits the proliferation of naive CD4+ T-helper cells by induction of FAS-independent T-cell apoptosis.

CD44 is a multifunctional adhesion molecule that has been shown to be a costimulatory factor for T-cell activation in vitro and in vivo. The aim of the present study was to expand these findings by characterizing the role of CD44 during dendritic cell (DC) antigen presentation to naive, resting T cells. Certain monoclonal antibodies (mAbs) directed against all CD44 isoforms (pan CD44), or against the epitope encoded by the alternatively spliced exon v4 (CD44v4), dose-dependently inhibited the capacity of murine DC to induce proliferation of naive alloreactive T cells. Preincubation of the T cells or DC with these CD44 mAbs revealed that the effect was dependent upon mAb binding to DC, but not to T cells. DC treated with anti-pan CD44 and anti-CD44v4 mAbs induced CD4+ T-cell apoptosis, as shown by annexin V staining and TdT-mediated biotin-dUTP nick-end labelling (TUNEL) assays. However, CD4+ T-cell apoptosis was not dependent on the Fas/Fas ligand (Fas/FasL) system, as DC from FasL-deficient (Gld) mice and T cells from Fas-deficient (Lpr) mice were still susceptible to apoptosis induced by CD44-treated DC. To investigate whether CD44 treatment of DC affects early T-cell/DC interactions, time-lapse video microscopy was performed using peptide-specific T cells from T-cell receptor (TCR) transgenic mice. Interestingly, calcium signalling in CD4+ T cells was significantly diminished following interaction with CD44 mAb-treated DC, but this was not observed in CD8+ T cells. Taken together, we found that perturbation of distinct epitopes of CD44 on DC interfere with early Ca2+ signalling events during the activation of CD4+ T cells, resulting in T-cell apoptosis.

Early cytoskeletal rearrangement during dendritic cell maturation enhances synapse formation and Ca(2+) signaling in CD8(+) T cells.

The interplay between dendritic cells (DC) and T cells is a dynamic process critically depending on DC maturation. Ca(2+) influx is one of the initial events occurring during DC/T cell contacts. To determine how DC maturation influences DC/T cell contacts, time-lapse video microscopy was established using TCR-transgenic CD8(+) T cells from P14 mice. DC maturation shifted DC/T cell contacts from short-lived interactions with transient Ca(2+) influx in T cells to long-lasting interactions and sustained Ca(2+) influx of 30 min and more. Follow-up of DC/T cell interactions after 2 h using confocal microscopy revealed that long-lasting Ca(2+) responses in T cells were preferentially associated with the formation of an immunological synapse involving CD54 and H2-K(b) at the DC/T cell interface. Such synapse formation preceded MHC or B7 up-regulation, since DC developed into potent Ca(2+) stimulators 7 h after initiation of maturation. Instead, the enhanced capacity of 7 h-matured DC to induce sustained Ca(2+) responses in CD8(+) T cells is critically dependent on the polarization and rearrangement of the cytoskeleton, as shown by Clostridium difficile toxin B inhibitor experiments. These data indicate that already very early after receiving a maturation stimulus, DC display enhanced cytoskeletal activity resulting in the rapid formation of immunological synapses and effective CD8(+) T cell stimulation.