In vitro–generated soft tissue could provide alternate therapies for soft tissue defects. The aim of this study was to evaluate methacrylated gelatin/hyaluronan as scaffolds for soft tissue engineering and their interaction with human adipose–derived stem cells (hASCs). ASCs were incorporated into methacrylated gelatin/hyaluronan hydrogels. The gels were photocrosslinked with a lithium phenyl-2,4,6-trimethylbenzoylphosphinate photoinitiator and analyzed for cell viability and adipogenic differentiation of ASCs over a period of 30 days. Additionally, an angiogenesis assay was performed to assess their angiogenic potential. After 24 h, ASCs showed increased viability on composite hydrogels. These results were consistent over 21 days of culture. By induction of adipogenic differentiation, the mature adipocytes were observed after 7 days of culture, their number significantly increased until day 28 as well as expression of fatty acid binding protein 4 and adiponectin. Our scaffolds are promising as building blocks for adipose tissue engineering and allowed long viability, proliferation, and differentiation of ASCs.

Methods for human skin gene therapy requires efficient and stable introduction of genes into skin cells. Transient cutaneous gene therapy is an attractive approach in the treatment of skin diseases. The ‘Achilles heel’ of adenoviral gene therapy is its immunogenicity and many aspects of adenovirus induced cutaneous immune reaction still remain unanswered, particularly the role of keratinocytes. Therefore, human keratinocytes were transfected with adenoviral DNA and cytokine expression was analyzed. Moreover, adenoviral transduction of full-skin was performed ex vivo and in vivo. We observed cytokine induction after cytoplasmatic internalization of adenoviral DNA into epidermal cells. Inhibition of AIM2, NALP3, DAI or mda5 downregulated the cytokine response. Transduction of immunocompetent mice led to a detectable transgene expression for 12 days. Re-application of the vector led to a decrease in intensity and duration of transgene expression limited to 4 days and an increased cytokine expression. In contrast, immunodeficient mice showed a reduced expression of cytokines after DNA internalization. AIM2, NALP3, DAI and mda5 are essential in the induction of an innate immune response towards adenoviral DNA. This immune reaction leads to a decrease in transduction efficiency of the vector after re-application and modulation of these receptor systems stabilizes transgene expression.

High pre-existing antibodies against viral vectors reduce their functionality and may lead to adverse complications. To circumvent this problem in future gene therapy approaches, we tested the seroprevalence of a large range of human adenovirus types in patients with neuromuscular disorders (NMDs) to find appropriate viral vector candidates for gene replacement therapy for NMDs. Binding and neutralizing antibodies against 39 human adenovirus types were tested in the sera of 133 patients with NMDs and 76 healthy controls aged 17–92 years. The influence of age, sex, and NMDs on antibody levels was analyzed. The seroprevalence of different adenoviruses in the cohort varied widely. The highest levels of binding antibodies were detected against HAdV-D27, -C1, -D24, -D70, -B14, -C6, -D13, -B34, and -E4, whereas the lowest reactivity was detected against HAdV-F41, -A31, -B11, -D75, -D8, -D65, -D26, -D80, and -D17. The highest neutralizing reactivity was observed against HAdV-B3, -C2, -E4, -C1, -G52, -C5, and -F41, whereas the lowest neutralizing reactivity was observed against HAdV-D74, -B34, -D73, -B37, -D48, -D13, -D75, -D8, -B35, and -B16. We detected no influence of sex and only minor differences between different age groups. Importantly, there were no significant differences between healthy controls and patients with NMDs. Our data show that patients with NMDs have very similar levels of binding and neutralizing antibodies against HAdV compared to healthy individuals, and we identified HAdV-A31, -B16, -B34, -B35, -D8, -D37, -D48, -D73, -D74, -D75, and -D80 as promising candidates for future vector development due to their low binding and neutralizing antibody prevalence.

The process of wound healing consists of several well distinguishable and finely tuned phases. For most of these phases specific proteins have been characterized, although the underlying mechanisms of regulation are not yet fully understood. It is an open question as to whether deficits in wound healing can be traced back to chronic illnesses such as diabetes mellitus. Previous research efforts in this field focus largely on a restricted set of marker proteins due to the limitations detection by antibodies imposes. For mechanistic purposes the elucidation of differences in acute and chronic wounds can be addressed by a less restricted proteome study. Mass spectrometric (MS) methods, e.g. multi dimensional protein identification technology (MudPIT), are well suitable for this complex theme of interest. The human wound fluid proteome is extremely complex, as is human plasma. Therefore, high-abundant proteins often mask the mass spectrometric detection of lower-abundant ones, which makes a depletion step of such predominant proteins inevitable.

Myofibrillar myopathies (MFM) are a group of chronic muscle diseases pathophysiologically characterized by accumulation of protein aggregates and structural failure of muscle fibers. A subtype of MFM is caused by heterozygous mutations in the filamin C (FLNC) gene, exhibiting progressive muscle weakness, muscle structural alterations and intracellular protein accumulations. Here, we characterize in depth the pathogenicity of two novel truncating FLNc variants (p.Q1662X and p.Y2704X) and assess their distinct effect on FLNc stability and distribution as well as their impact on protein quality system (PQS) pathways. Both variants cause a slowly progressive myopathy with disease onset in adulthood, chronic myopathic alterations in muscle biopsy including the presence of intracellular protein aggregates. Our analyses revealed that p.Q1662X results in FLNc haploinsufficiency and p.Y2704X in a dominant-negative FLNc accumulation. Moreover, both protein-truncating variants cause different PQS alterations: p.Q1662X leads to an increase in expression of several genes involved in the ubiquitin-proteasome system (UPS) and the chaperone-assisted selective autophagy (CASA) system, whereas p.Y2704X results in increased abundance of proteins involved in UPS activation and autophagic buildup. We conclude that truncating FLNC variants might have different pathogenetic consequences and impair PQS function by diverse mechanisms and to varying extents. Further studies on a larger number of patients are necessary to confirm our observations.

Background: Adenoviral vectors have provided effective methods for in vivo gene delivery in therapeutic applications. However, these vectors can induce immune responses that may severely affect the ability of vector re-application. There is limited information about the mechanisms and signal transduction pathways involved in adenoviral recognition. For optimization of cutaneous gene therapy it is necessary to investigate molecular mechanisms of virus recognition in epidermal cells. The aim of this study was to investigate the signal transduction of the innate immunity after adenoviral DNA internalization in keratinocytes. Methods: In vitro, keratinocytes were transfected with DNA, in the presence and absence of inhibitors for signalling molecules. In vivo, immunocompetent and athymic mice (n = 3 per group) were twice transduced with an Ad-vector. Results: The results show an acute induction of type-I-interferon after in vitro transfection. Inhibition of PI3K, p38 MAPK, JNK and NFkappaB resulted in a decreased expression of type-I-interferon. In contrast to immunocompetent mice, athymic mice demonstrated a constant transgene expression and reduced inflammatory response in vivo. Conclusion: The results suggest an induction of the innate immunity triggered by cytoplasm localised DNA which is mediated by PI3K-, p38 MAPK-, JNK-, NFkappaB-, JAK/STAT- and ERK1/2-dependent pathways. A stable transgene expression and a reduced inflammatory response in immunodeficient mice have been observed. These results provide potential for an effective adenoviral gene delivery into immunosupressed skin.