Human ß-defensin-2 suppresses key features of asthma in murine models of allergic airways disease.

BACKGROUND: Asthma is an airway inflammatory disease and a major health problem worldwide. Anti-inflammatory steroids and bronchodilators are the gold-standard therapy for asthma. However, they do not prevent the development of the disease, and critically, a subset of asthmatics are resistant to steroid therapy. OBJECTIVE: To elucidate the therapeutic potential of human ß-defensins (hBD), such as hBD2 mild to moderate and severe asthma. METHODS: We investigated the role of hBD2 in a steroid-sensitive, house dust mite-induced allergic airways disease (AAD) model and a steroid-insensitive model combining ovalbumin-induced AAD with C muridarum (Cmu) respiratory infection. RESULTS: In both models, we demonstrated that therapeutic intranasal application of hBD2 significantly reduced the influx of inflammatory cells into the bronchoalveolar lavage fluid. Furthermore, key type 2 asthma-related cytokines IL-9 and IL-13, as well as additional immunomodulating cytokines, were significantly decreased after administration of hBD2 in the steroid-sensitive model. The suppression of inflammation was associated with improvements in airway physiology and treatment also suppressed airway hyper-responsiveness (AHR) in terms of airway resistance and compliance to methacholine challenge. CONCLUSIONS AND CLINICAL RELEVANCE: These data indicate that hBD2 reduces the hallmark features and has potential as a new therapeutic agent in allergic and especially steroid-resistant asthma.

PSM Peptides of Staphylococcus aureus Activate the p38-CREB Pathway in Dendritic Cells, Thereby Modulating Cytokine Production and T Cell Priming.

The challenging human pathogen Staphylococcus aureus has highly efficient immune evasion strategies for causing a wide range of diseases, from skin and soft tissue to life-threatening infections. Phenol-soluble modulin (PSM) peptides are major pathogenicity factors of community-associated methicillin-resistant S. aureus strains. In previous work, we demonstrated that PSMs in combination with TLR2 ligand from S. aureus induce tolerogenic dendritic cells (DCs) characterized by the production of high amounts of IL-10, but no proinflammatory cytokines. This in turn promotes the activation of regulatory T cells while impairing Th1 response; however, the signaling pathways modulated by PSMs remain elusive. In this study, we analyzed the effects of PSMs on signaling pathway modulation downstream of TLR2. TLR2 stimulation in combination with PSMα3 led to increased and prolonged phosphorylation of NF-κB, ERK, p38, and CREB in mouse bone marrow-derived DCs compared with single TLR2 activation. Furthermore, inhibition of p38 and downstream MSK1 prevented IL-10 production, which in turn reduced the capacity of DCs to activate regulatory T cells. Interestingly, the modulation of the signaling pathways by PSMs was independent of the known receptor for PSMs, as shown by experiments with DCs lacking the formyl peptide receptor 2. Instead, PSMs penetrate the cell membrane most likely by transient pore formation. Moreover, colocalization of PSMs and p38 was observed near the plasma membrane in the cytosol, indicating a direct interaction. Thus, PSMs from S. aureus directly modulate the signaling pathway p38-CREB in DCs, thereby impairing cytokine production and in consequence T cell priming to increase the tolerance toward the pathogen.

Staphylococcus aureus PSM peptides induce tolerogenic dendritic cells upon treatment with ligands of extracellular and intracellular TLRs.

Dendritic cells (DCs) are key players of the immune system and thus a target for immune evasion by pathogens. We recently showed that the virulence factor phenol-soluble modulin (PSM) produced by community-associated methicillin-resistant Staphylococcus aureus strains induces tolerogenic DCs upon Toll-like receptor (TLR) 2 activation via the p38-CREB-IL-10 pathway. Here, we addressed the question whether this tolerogenic phenotype of DCs induced by PSMs is specific for TLR2 activation. Therefore, bone marrow-derived DCs were treated with various ligands for extracellular and intracellular TLRs simultaneously with PSMα3. We show that PSMα3 modulates antigen uptake, maturation and cytokine production of DCs activated by TLR1/2, TLR2/6, TLR4, TLR7, and TLR9. Pre-incubation of DCs with a p38 MAP kinase inhibitor prevented the PSMα3-induced IL-10 secretion, as well as MHC class II up-regulation upon TLR activation. In consequence, the tolerogenic DCs induced by PSMα3 in response to several TLR ligands promoted priming of regulatory T cells. Thus, PSMs could be useful as inducers of tolerogenic DCs upon TLR ligand stimulation for therapeutic applications.

Regulated ADAM17-dependent EGF family ligand release by substrate-selecting signaling pathways.

Ectodomain cleavage of cell-surface proteins by A disintegrin and metalloproteinases (ADAMs) is highly regulated, and its dysregulation has been linked to many diseases. ADAM10 and ADAM17 cleave most disease-relevant substrates. Broad-spectrum metalloprotease inhibitors have failed clinically, and targeting the cleavage of a specific substrate has remained impossible. It is therefore necessary to identify signaling intermediates that determine substrate specificity of cleavage. We show here that phorbol ester or angiotensin II-induced proteolytic release of EGF family members may not require a significant increase in ADAM17 protease activity. Rather, inducers activate a signaling pathway using PKC-α and the PKC-regulated protein phosphatase 1 inhibitor 14D that is required for ADAM17 cleavage of TGF-α, heparin-binding EGF, and amphiregulin. A second pathway involving PKC-δ is required for neuregulin (NRG) cleavage, and, indeed, PKC-δ phosphorylation of serine 286 in the NRG cytosolic domain is essential for induced NRG cleavage. Thus, signaling-mediated substrate selection is clearly distinct from regulation of enzyme activity, an important mechanism that offers itself for application in disease.

Pivotal role of phospholipase D1 in tumor necrosis factor-α-mediated inflammation and scar formation after myocardial ischemia and reperfusion in mice.

Myocardial inflammation is critical for ventricular remodeling after ischemia. Phospholipid mediators play an important role in inflammatory processes. In the plasma membrane they are degraded by phospholipase D1 (PLD1). PLD1 was shown to be critically involved in ischemic cardiovascular events. Moreover, PLD1 is coupled to tumor necrosis factor-α signaling and inflammatory processes. However, the impact of PLD1 in inflammatory cardiovascular disease remains elusive. Here, we analyzed the impact of PLD1 in tumor necrosis factor-α-mediated activation of monocytes after myocardial ischemia and reperfusion using a mouse model of myocardial infarction. PLD1 expression was highly up-regulated in the myocardium after ischemia/reperfusion. Genetic ablation of PLD1 led to defective cell adhesion and migration of inflammatory cells into the infarct border zone 24 hours after ischemia/reperfusion injury, likely owing to reduced tumor necrosis factor-α expression and release, followed by impaired nuclear factor-κB activation and interleukin-1 release. Moreover, PLD1 was found to be important for transforming growth factor-ß secretion and smooth muscle α-actin expression of cardiac fibroblasts because myofibroblast differentiation and interstitial collagen deposition were altered in Pld1(-/-) mice. Consequently, infarct size was increased and left ventricular function was impaired 28 days after myocardial infarction in Pld1(-/-) mice. Our results indicate that PLD1 is crucial for tumor necrosis factor-α-mediated inflammation and transforming growth factor-ß-mediated collagen scar formation, thereby augmenting cardiac left ventricular function after ischemia/reperfusion.

Ex vivo gene delivery to synovium using foamy viral vectors.

BACKGROUND: Gene transfer technologies have the potential to fundamentally improve current therapies for arthritic conditions, although this is essentially dependent on safe and efficient vector systems. The foamy virus (FV)-based vectors have many safety features that favour their use in the treatment of arthritis. In the present study, we investigated the use of safe prototype foamy viral vectors (FVV) for indirect gene delivery to articular tissues. METHODS: We generated recombinant FVV encoding enhanced green fluorescent protein (EGFP) or human interleukin 1 receptor antagonist protein (IL1RA) cDNA under the control of the spleen focus forming virus U3 promoter and explored their transgene expression profile following ex vivo gene delivery to knee joints of Wistar and athymic nude rats. RESULTS: FVV efficiently transduced primary rat synovial fibroblasts using the EGFP and the IL1RA transgene in vitro. FVV-mediated IL1RA expression was functional in blocking IL1 effects in vitro. After the transplantation of FVV transduced synovial fibroblasts, the intra-articular transgene expression in Wistar rats was initially high and declined after approximately 3 weeks for both transgenes. By contrast, FVV-mediated expression of EGFP and IL1RA persisted for at least 12 weeks at high levels in immunocompromised nude rats. FVV-meditated gene delivery was well tolerated by all animals without extra-articular transgene expression, arguing for the safety of this approach. CONCLUSIONS: Our results indicate that FVV are capable of efficient ex vivo gene transfer to synovium and merit further investigation as a means to provide long-term intra-articular transgene expression for arthritis treatment.

Foamy virus-adenovirus hybrid vectors for gene therapy of the arthritides.

BACKGROUND: Genetic treatments of chronic arthritic conditions are essentially dependent on safe and efficient vector systems. To combine features of the efficient transduction of adenovirus vectors with the advantage of stable integration into the host cell genome of apathogenic prototype foamy virus vectors, hybrid vectors (FAD) have been established. In the present study, we have generated and investigated the use of safe FAD vectors for direct gene delivery to joints. METHODS: We generated recombinant FAD encoding enhanced green fluorescent protein (EGFP) or human interleukin 1 receptor antagonist protein (IL1RA) cDNA, and explored their transgene expression profile, as well as the bioactivity of the IL1RA transgene in vitro. The feasibility of IL1RA gene delivery to articular tissues was investigated in a pilot study employing direct FAD injections to the knee joints of Wistar rats. RESULTS: FAD vectors efficiently transduced human or rat fibroblasts with EGFP or IL1RA transgene in vitro. Levels of IL1RA transgene expression were high, stable and functional in vitro. Transduced synovial fibroblasts and high levels of IL1RA protein (10-35 ng/ml) could be detected in vivo in the synovium of Wistar rats 3-5 days after injection of FAD vectors to the knee joints. CONCLUSIONS: Our results indicate that FAD vectors are capable of efficient in vivo gene transfer to synovium and merit further investigation as a means of providing efficient and long-term intra-articular transgene expression for treatment of the arthritides.

Heparan sulfate is an attachment factor for foamy virus entry.

The cellular receptor of foamy viruses (FVs) is unknown. The broad spectrum of permissive cells suggests that the cellular receptor is a molecular structure with almost ubiquitous prevalence. Here, we investigated the ability of heparan sulfate (HS), a glycosaminoglycan (GAG) present on the extracellular matrix of many cells, to bind FV particles and to permit prototype FV (PFV) and feline FV (FFV) entry. Permissivity of different cell lines for FV entry correlated with the amount of heparan sulfate present on the cell surface. The resulting 50% cell culture infectious doses (CCID(50)s) were distributed over a range of 4 logs, which means that the most susceptible cell line tested (HT1080) was more than 10,000 times more susceptible for PFV infection than the least susceptible cell line (CRL-2242). HS surface expression varied over a range of 2 logs. HS expression and FV susceptibility were positively correlated (P < 0.001). Enzymatic digestion of heparan sulfate on HT1080 cells diminished permissivity for PFV entry by a factor of at least 500. Using fast protein liquid chromatography (FPLC), we demonstrated binding of FV vector particles to a gel filtration column packed with heparin, a molecule structurally related to heparan sulfate, allowing for the purification of infectious particles. Both PFV and FFV infection were inhibited by soluble heparin. Our results show that FVs bind to HS and that this interaction is a pivotal step for viral entry, suggesting that HS is a cellular attachment factor for FVs.

Human ß-Defensin 2 Mediated Immune Modulation as Treatment for Experimental Colitis.

Defensins represents an integral part of the innate immune system serving to ward off potential pathogens and to protect the intestinal barrier from microbial encroachment. In addition to their antimicrobial activities, defensins in general, and human ß-defensin 2 (hBD2) in particular, also exhibit immunomodulatory capabilities. In this report, we assessed the therapeutic efficacy of systemically administered recombinant hBD2 to ameliorate intestinal inflammation in three distinct animal models of inflammatory bowel disease; i.e., chemically induced mucosal injury (DSS), loss of mucosal tolerance (TNBS), and T-cell transfer into immunodeficient recipient mice. Treatment efficacy was confirmed in all tested models, where systemically administered hBD2 mitigated inflammation, improved disease activity index, and hindered colitis-induced body weight loss on par with anti-TNF-α and steroids. Treatment of lipopolysaccharide (LPS)-activated human peripheral blood mononuclear cells with rhBD2 confirmed the immunomodulatory capacity in the circulatory compartment. Subsequent analyzes revealed dendritic cells (DCs) as the main target population. Suppression of LPS-induced inflammation was dependent on chemokine receptor 2 (CCR2) expression. Mechanistically, hBD2 engaged with CCR2 on its DC target cell to decrease NF-κB, and increase CREB phosphorylation, hence curbing inflammation. To our knowledge, this is the first study showing in vivo efficacy of a systemically administered defensin in experimental disease.

Advances in RNA Vaccines for Preventive Indications: A Case Study of A Vaccine Against Rabies.

: There is a global need for effective and affordable rabies vaccines, which is unmet by current vaccines due to limitations in their production capacities, required administration schedules, storage requirements, and cost. Many different experimental approaches previously used for bacterial and viral vaccines have been applied to rabies, but with variable success. One of the most promising new concepts is the use of messenger RNA (mRNA) in encoding the main rabies virus antigen, the envelope glycoprotein (RABV-G). CureVac has applied their proprietary technology platform for the production of mRNA to this problem, resulting in the rabies vaccine candidate CV7201. Following preclinical studies in mice and pigs showing that CV7201 could induce neutralizing immune responses that protected against rabies virus, different dosages and routes of administration of CV7201 were tested in a phase 1 human study. This clinical study proved that mRNA vaccination was safe and had an acceptable reactogenicity profile, but immune responses depended on the mode of administration, and they did not unequivocally support CV7201 for further development as a prophylactic vaccine with this particular formulation. Further, preclinical studies using RABV-G mRNA encapsulated in lipid nanoparticles (LNPs) showed an improved response in both mice and nonhuman primates, and these encouraging results are currently being followed up in clinical studies in humans. This review summarizes the recent advances in mRNA vaccines against rabies.

PSM Peptides From Community-Associated Methicillin-Resistant Staphylococcus aureus Impair the Adaptive Immune Response via Modulation of Dendritic Cell Subsets in vivo.

Dendritic cells (DCs) are key players of the immune system and thus a target for immune evasion by pathogens. We recently showed that the virulence factors phenol-soluble-modulins (PSMs) produced by community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strains induce tolerogenic DCs upon Toll-like receptor activation via the p38-CREB-IL-10 pathway in vitro. Here, we addressed the hypothesis that S. aureus PSMs disturb the adaptive immune response via modulation of DC subsets in vivo. Using a systemic mouse infection model we found that S. aureus reduced the numbers of splenic DC subsets, mainly CD4+ and CD8+ DCs independently of PSM secretion. S. aureus infection induced upregulation of the C-C motif chemokine receptor 7 (CCR7) on the surface of all DC subsets, on CD4+ DCs in a PSM-dependent manner, together with increased expression of MHCII, CD86, CD80, CD40, and the co-inhibitory molecule PD-L2, with only minor effects of PSMs. Moreover, PSMs increased IL-10 production in the spleen and impaired TNF production by CD4+ DCs. Besides, S. aureus PSMs reduced the number of CD4+ T cells in the spleen, whereas CD4+CD25+Foxp3+ regulatory T cells (Tregs) were increased. In contrast, Th1 and Th17 priming and IFN-γ production by CD8+ T cells were impaired by S. aureus PSMs. Thus, PSMs from highly virulent S. aureus strains modulate the adaptive immune response in the direction of tolerance by affecting DC functions.

Staphylococcus aureus PSM Peptides Modulate Human Monocyte-Derived Dendritic Cells to Prime Regulatory T Cells.

Staphylococcus aureus (Sa), as one of the major human pathogens, has very effective strategies to subvert the human immune system. Virulence of the emerging community-associated methicillin-resistant Sa (CA-MRSA) depends on the secretion of phenol-soluble modulin (PSM) peptide toxins e.g., by binding to and modulation of innate immune cells. Previously, by using mouse bone marrow-derived dendritic cells we demonstrated that PSMs in combination with various Toll-like receptor (TLR) ligands induce a tolerogenic DC phenotype (tDC) characterized by the production of IL-10 and impaired secretion of pro-inflammatory cytokines. Consequently, PSM-induced tDCs favored priming of CD4+CD25+FoxP3+ Tregs with suppressor function while impairing the Th1 response. However, the relevance of these findings for the human system remained elusive. Here, we analyzed the impact of PSMα3 on the maturation, cytokine production, antigen uptake, and T cell stimulatory capacity of human monocyte-derived DCs (moDCs) treated simultaneously with either LPS (TLR4 ligand) or Sa cell lysate (TLR2 ligand). Herein, we demonstrate that PSMs indeed modulate human moDCs upon treatment with TLR2/4 ligands via multiple mechanisms, such as transient pore formation, impaired DC maturation, inhibited pro- and anti-inflammatory cytokine secretion, as well as reduced antigen uptake. As a result, the adaptive immune response was altered shown by an increased differentiation of naïve and even CD4+ T cells from patients with Th1/Th17-induced diseases (spondyloarthritis and rheumatoid arthritis) into CD4+CD127-CD25hiCD45RA-FoxP3hi regulatory T cells (Tregs) with suppressor function. This Treg induction was mediated most predominantly by direct DC-T-cell interaction. Thus, PSMs from highly virulent Sa strains affect DC functions not only in the mouse, but also in the human system, thereby modulating the adaptive immune response and probably increasing the tolerance toward the bacteria. Moreover, PSMα3 might be a novel peptide for tolerogenic DC induction that may be used for DC vaccination strategies.

In the Wnt of Paneth Cells: Immune-Epithelial Crosstalk in Small Intestinal Crohn's Disease.

Paneth cells, specialized secretory epithelial cells of the small intestine, play a pivotal role in host defense and regulation of microbiota by producing antimicrobial peptides especially-but not only-the human α-defensin 5 (HD5) and HD6. In small intestinal Crohn's disease (CD) which is an entity of inflammatory bowel diseases, the expression of HD5 and HD6 is specifically compromised leading to a disturbed barrier and change in the microbial community. Different genetically driven but also non-genetic defects associated with small intestinal CD affect different lines of antimicrobial Paneth cell functions. In this review, we focus on the mechanisms and the crosstalk of Paneth cells and bone marrow-derived cells and highlight recent studies about the role of the Wnt signaling pathway in this connection of ileal CD. In summary, different lines of investigations led by us but also now numerous other groups support and reconfirm the proposed classification of this disease entity as Paneth's disease.

Rescued Chondrogenesis of Mesenchymal Stem Cells under Interleukin 1 Challenge by Foamyviral Interleukin 1 Receptor Antagonist Gene Transfer.

Background: Mesenchymal stem cells (MSCs) and their chondrogenic differentiation have been extensively investigated in vitro as MSCs provide an attractive source besides chondrocytes for cartilage repair therapies. Here we established prototype foamyviral vectors (FVV) that are derived from apathogenic parent viruses and are characterized by a broad host range and a favorable integration pattern into the cellular genome. As the inflammatory cytokine interleukin 1 beta (IL1ß) is frequently present in diseased joints, the protective effects of FVV expressing the human interleukin 1 receptor antagonist protein (IL1RA) were studied in an established in vitro model (aggregate culture system) of chondrogenesis in the presence of IL1ß. Materials and Methods: We generated different recombinant FVVs encoding enhanced green fluorescent protein (EGFP) or IL1RA and examined their transduction efficiencies and transgene expression profiles using different cell lines and human primary MSCs derived from bone marrow-aspirates. Transgene expression was evaluated by fluorescence microscopy (EGFP), flow cytometry (EGFP), and ELISA (IL1RA). For evaluation of the functionality of the IL1RA transgene to block the inhibitory effects of IL1ß on chondrogenesis of primary MSCs and an immortalized MSC cell line (TERT4 cells), the cells were maintained following transduction as aggregate cultures in standard chondrogenic media in the presence or absence of IL1ß. After 3 weeks of culture, pellets were harvested and analyzed by histology and immunohistochemistry for chondrogenic phenotypes. Results: The different FVV efficiently transduced cell lines as well as primary MSCs, thereby reaching high transgene expression levels in 6-well plates with levels of around 100 ng/ml IL1RA. MSC aggregate cultures which were maintained in chondrogenic media without IL1ß supplementation revealed a chondrogenic phenotype by means of strong positive staining for collagen type II and matrix proteoglycan (Alcian blue). Addition of IL1ß was inhibitory to chondrogenesis in untreated control pellets. In contrast, foamyviral mediated IL1RA expression rescued the chondrogenesis in pellets cultured in the presence of IL1ß. Transduced MSC pellets reached thereby very high IL1RA transgene expression levels with a peak of 1087 ng/ml after day 7, followed by a decrease to 194 ng/ml after day 21, while IL1RA concentrations of controls were permanently below 200 pg/ml. Conclusion: Our results indicate that FVV are capable of efficient gene transfer to MSCs, while reaching IL1RA transgene expression levels, that were able to efficiently block the impacts of IL1ß in vitro. FVV merit further investigation as a means to study the potential as a gene transfer tool for MSC based therapies for cartilage repair.

Efficacy and biodistribution analysis of intracerebroventricular administration of an optimized scAAV9-SMN1 vector in a mouse model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive disease of variable severity caused by mutations in the SMN1 gene. Deficiency of the ubiquitous SMN function results in spinal cord α-motor neuron degeneration and proximal muscle weakness. Gene replacement therapy with recombinant adeno-associated viral (AAV) vectors showed therapeutic efficacy in several animal models of SMA. Here, we report a study aimed at analyzing the efficacy and biodistribution of a serotype-9, self-complementary AAV vector expressing a codon-optimized human SMN1 coding sequence (coSMN1) under the control of the constitutive phosphoglycerate kinase (PGK) promoter in neonatal SMNΔ7 mice, a severe animal model of the disease. We administered the scAAV9-coSMN1 vector in the intracerebroventricular (ICV) space in a dose-escalating mode, and analyzed survival, vector biodistribution and SMN protein expression in the spinal cord and peripheral tissues. All treated mice showed a significant, dose-dependent rescue of lifespan and growth with a median survival of 346 days. Additional administration of vector by an intravenous route (ICV+IV) did not improve survival, and vector biodistribution analysis 90 days postinjection indicated that diffusion from the cerebrospinal fluid to the periphery was sufficient to rescue the SMA phenotype. These results support the preclinical development of SMN1 gene therapy by CSF vector delivery.

Role of BCL9L in transforming growth factor-ß (TGF-ß)-induced epithelial-to-mesenchymal-transition (EMT) and metastasis of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) has a low overall survival rate, which is approximately 20% during the first year and decreases to less than 6% within five years of the disease. This is due to premature dissemination accompanied by a lack of disease-specific symptoms during the initial stages. Additionally, to date there are no biomarkers for an early prognosis available.A growing number of studies indicate that epithelial to mesenchymal transition (EMT), triggered by WNT-, TGF-ß- and other signaling pathways is crucial for the initiation of the metastatic process in PDAC. Here we show, that BCL9L is up-regulated in PDAC cell lines and patient tissue compared to non-cancer controls. RNAi-induced BCL9L knockdown negatively affected proliferation, migration and invasion of pancreatic cancer cells. On a molecular basis, BCL9L depletion provoked an increment of E-cadherin protein levels, with concomitant increase of ß-catenin retention at the plasma membrane. This is linked to the induction of a strong epithelial phenotype in pancreatic cancer cells upon BCL9L knockdown even in the presence of the EMT-inducer TGF-ß. Finally, xenograft mouse models of pancreatic cancer revealed a highly significant reduction in the number of liver metastases upon BCL9L knockdown. Taken together, our findings underline the key importance of BCL9L for EMT and thus progression and metastasis of pancreatic cancer cells. Direct targeting of this protein might be a valuable approach to effectively antagonize invasion and metastasis of PDAC.

Suprabasal spongiosis in acute eczematous dermatitis: cFLIP maintains resistance of basal keratinocytes to T-cell-mediated apoptosis.

In acute eczematous dermatitis, keratinocyte (KC) apoptosis caused by dermis-infiltrating, activated T cells plays a crucial pathogenetic role in the development of spongiosis, the histopathological hallmark of acute eczema. Remarkably, T-cell-mediated apoptosis of single KC, as well as spongiosis, is located predominantly in suprabasal epidermal layers, suggesting that antiapoptotic mechanisms protect basal KC. The cellular Flice-inhibitory protein (cFLIP) is known to block apoptotic CD95-signaling, and may therefore account for such a protection of basal KC. HaCaT KCs retrovirally transduced with the long form of cFLIP were effectively protected against T-cell-mediated apoptosis in KC monolayer/CD4(+) T-cell cocultures. In situ correlation of cFLIP protein expression and KC apoptosis in lesional eczematous skin showed a highly restricted expression of cFLIP in basal KC, whereas cleaved caspase-3 (as a surrogate marker of apoptosis) was detected predominantly in suprabasal epidermal layers. Thus, the modulation of the CD95 signaling pathway by the cell-intrinsic caspase-8 inhibitor cFLIP in basal KC may explain the spatial localization of spongiosis in suprabasal epidermal layers, and provides new insights into the pathogenesis of spongiosis formation in eczematous dermatitis.

Generation of an improved foamy virus vector by dissection of cis-acting sequences.

In contrast to other retroviruses, foamy viruses (FVs) generate their Pol protein precursor independently of the Gag protein from a spliced mRNA. The exact mechanism of Pol protein incorporation into the viral capsid is poorly understood. Previously, we showed that Pol encapsidation critically depends on the packaging of (pre-) genomic RNA and identified two distinct signals within the cis-acting sequences (CASI and CASII), Pol encapsidation sequences (PESI and PESII), which are required for Pol capsid incorporation. Here, we investigated whether the presence of PESI and PESII in an FV vector is sufficient for Pol encapsidation and whether the rather extended CASII element can be shortened without loss of functionality. Our results indicate that (i) the presence of PESI and II are not sufficient for Pol encapsidation, (ii) prototype FV vectors with a shortened CASII element retain Pol incorporation and full functionality, in particular upon transducing fibroblasts and primary human mesenchymal stem cells, (iii) the presence of the central poly purine tract significantly increased the transduction rates of FV vectors and (iv) Pol encapsidation and RNA packaging can be clearly separated. In essence, we designed a new FV vector that bears approximately 850 bp less of CAS than previously established vectors and is fully functional when analysed to transduce cell lines and primary human cells.

Cyclosporin A abolishes CD28-mediated resistance to CD95-induced apoptosis via superinduction of caspase-3.

Costimulation of T cells via CD28 promotes both proliferation and resistance to apoptosis. In this study, we show that the immunosuppressive drug cyclosporin A (CsA) fully reverses resistance to CD95-mediated cell death after TCR/CD28 costimulation or superagonistic anti-CD28 mAb stimulation of primary rat lymph node T cells. This effect correlated with a pronounced superinduction of caspase-3 on both mRNA and protein levels, whereas its main antagonist, X chromosome-linked inhibitor of apoptosis, was unaffected by inclusion of CsA. Apoptosis triggered by CD95 cross-linking was characterized by robust caspase-3 activation. Furthermore, CsA sensitization to CD95-mediated apoptosis of CD28-activated T cells did not alter mRNA stability of superinduced caspase-3 mRNA, suggesting a transcriptional regulation of the caspase-3 gene. Addition of Ca(2+) ionophores to TCR/CD28 or superagonistic CD28-stimulated cells reduced caspase-3 levels, further supporting a role for Ca(2+)-dependent signaling pathways in negatively regulating caspase-3. Taken together, these findings suggest that CsA promotes sensitivity to CD95-mediated apoptosis in CD28-stimulated T cells by superinduction of the caspase-3 gene via a mechanism involving suppression of the calcineurin pathway.

Welwitschia mirabilis: CAM or not CAM - what is the answer?

After more than 20 years of extensive study we found clear evidence that Welwitschia mirabilis Hook.f. is able to take up CO2 at night in both of its natural ecosystems, the Namib desert and the Mopane savannah, and hence should be classified a crassulacean acid metabolism (CAM) plant. At six different sites, 85 W. mirabilis plants were marked and the growth rate of their leaves and leaf ribbons were measured over a period of 2.5 years. The slowest and the fastest growing plant of these 85 plants were from the Mopane savannah and from the north-west of the Brandberg massif, respectively. These were selected for the gas-exchange measurements of this study. Within the course of a year nocturnal CO2 uptake was found only in December and January when the nights were shortest and plants were flowering. CO2 uptake during the night was not pronounced and never accounted for more than 4% of the total CO2 uptake over 24 h. Maximum rates of nocturnal CO2 uptake never exceeded 0.1 µmol m-2 s-1 for the slowest and 0.2 µmol m-2 s-1 for the fastest growing plant. Neither water availability in the soil nor night temperature was found to determine nocturnal CO2 uptake in terms known for CAM plants. Regardless of the growing site all leaves of W. mirabilis contained high amounts of malic and citric acid. Small increases of acids over night as calculated from the gas exchange measurements are masked by the extremely uneven distribution of these acids in the leaves, making the feature of an overnight malic or citric acid accumulation an unsuited test for CAM in W. mirabilis. An increase in 13C discrimination with increasing distance from the coast was confirmed. Photorespiration was extremely high and followed air temperature around the leaf. Although the debate whether or not W. mirabilis is a CAM plant can be closed, no answer could be given why W. mirabilis makes so little use of CAM.