Matthias Schulte

Innate Defense Regulator Peptide 1018 in Wound Healing and Wound Infection

Innate defense regulators (IDRs) are synthetic immunomodulatory versions of natural host defense peptides (HDP). IDRs mediate protection against bacterial challenge in the absence of direct antimicrobial activity, representing a novel approach to anti-infective and anti-inflammatory therapy. Previously, we reported that IDR-1018 selectively induced chemokine responses and suppressed pro-inflammatory responses. As there has been an increasing appreciation for the ability of HDPs to modulate complex immune processes, including wound healing, we characterized the wound healing activities of IDR-1018 in vitro. Further, we investigated the efficacy of IDR-1018 in diabetic and non-diabetic wound healing models. In all experiments, IDR-1018 was compared to the human HDP LL-37 and HDP-derived wound healing peptide HB-107. IDR-1018 was significantly less cytotoxic in vitro as compared to either LL-37 or HB-107. Furthermore, administration of IDR-1018 resulted in a dose-dependent increase in fibroblast cellular respiration. In vivo, IDR-1018 demonstrated significantly accelerated wound healing in S. aureus infected porcine and non-diabetic but not in diabetic murine wounds. However, no significant differences in bacterial colonization were observed. Our investigation demonstrates that in addition to previously reported immunomodulatory activities IDR-1018 promotes wound healing independent of direct antibacterial activity. Interestingly, these effects were not observed in diabetic wounds. It is anticipated that the wound healing activities of IDR-1018 can be attributed to modulation of host immune pathways that are suppressed in diabetic wounds and provide further evidence of the multiple immunomodulatory activities of IDR-1018.

Expression Profile of Human Beta-Defensin 3 in Oral Squamous Cell Carcinoma

Although it is known that innate immunity is important for protecting the body against foreign agents such as bacteria, little is known about elements of the innate immune system that have antitumor activity. This prospective study was designed to investigate the function of human beta-defensin 3 (hBD-3), an important component of the innate immune response, in oral squamous cell carcinoma (OSCC). Paired cancerous and noncancerous specimens of 45 patients who underwent surgical treatment for OSCC were examined for hBD-3 expression on protein and mRNA. Clinical and pathological features such as age, gender, tumor and lymph node status, UICC stage, and histological grading were correlated. hBD-3 was significantly overexpressed in tumors in comparison to healthy tissue examined with real-time quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) analysis (p = .004). Immunohistochemical stain for hBD-3 was much more pronounced in tumors than in corresponding healthy mucosa. The results illustrate that hBD-3 is frequently overexpressed in oral squamous cell carcinomas and seems to be related to oncogenesis. Increased expression of hBD-3 in oral squamous cell carcinomas suggests its potential role in the pathogenesis of oral cancer. This might be a starting point for novel pharmacological/molecular treatment modalities.

Polybrene improves transfection efficacy of recombinant replication‐deficient adenovirus in cutaneous cells and burned skin

The hostile environment found in acute and chronic wounds decreases the physiological half‐life of purified synthetic or recombinant peptides dramatically. Gene therapy, on the other hand, may be a viable option since it relies on the cellular machinery of the host to locally manufacture the proteins of interest. The aim of this study was to evaluate and optimize the local administration of transient cutaneous adenoviral gene delivery in wounds.

A bioartificial surgical patch from multilayered human amniotic membrane—In vivo investigations in a rat model

The study was performed to evaluate the suitability of glycerol-cryopreserved human amniotic membrane (HAM) as a surgical patch, far from its common use in ophthalmic surgery. In vivo experiments in rat models were performed to study the degradation patterns, biocompatibility, postoperative tissue formation and its suitability for abdominal wall closure. Degradation and thickness of the membranes were assessed over a period of 60 days after subdermal implantation of monolayer and multilayer HAM in 96 immunocompetent and immunosuppressed rats. The tissue response was mild, and histological analysis evaluated that multilayer application and immunosuppression prolonged graft survival significantly. In a second rat model, another 18 animals were monitored over a period of 28 days after abdominal wall reconstruction with multilayered HAM. Polypropylene mesh (Prolene) and polyglactin910/polydioxanon patches (Ethisorb) served as controls. Gross examination and histological analysis proved that multilayer HAM was a sufficient material for abdominal wall closure in comparison with the polypropylene mesh and was superior to the polyglactin910/polydioxanon patch. Additionally, significantly reduced postoperative intraabdominal adhesions were observed when compared to the polyglactin910/polydioxanon patch. This study demonstrates that HAM is a biocompatible, resorbable surgical patch in a rat xenotransplantation model and serves as a mechanically sufficient material for abdominal wall closure in a small animal model. These findings are encouraging and justify further research for the use of cryopreserved human amniotic membrane in soft tissue repair.

Quantitative comparison of the expression of antimicrobial peptides in the oral mucosa and extraoral skin

Antimicrobial peptides (AMP) defend epithelial surfaces against pathological micro-organisms. We know of no comparison of their expression between the oral mucosa and extraoral epithelium, but knowledge of differences in their quantities is of interest, possibly as a starting point for new treatments. Expression of AMP human beta-defensin (hBD)-1/-2/-3 and psoriasin in the oral mucosa and extraoral epithelium of the head and neck were measured by real-time polymerase chain reaction (RT-PCR) (n=14), immunohistochemistry (n=6), and western blot (n=8). RT-PCR showed that all the genes investigated were expressed significantly more in the oral mucosa than in the skin (hBD-1: p=0.002; hBD-2: p=0.006; hBD-3: p=0.035; psoriasin: p=0.02). Immunohistochemistry and western blot showed differential concentrations of proteins: hBD-2 (p=0.021) and hBD-3 (p=0.043) were pronounced in the oral mucosa, whereas psoriasin was raised in the extraoral skin (p=0.021). There was no difference in protein concentrations for hBD-1 (p=0.08). The observed differences in the expression of AMP may be important for new treatments such as topical application of AMP derivatives.

Expression of host defence peptides in the lip vermilion mucosa during early infancy

Emerging resistance to antibiotics has become a major problem. Host defence peptides (HDPs), which are effector molecules of the innate immune system, show broad antimicrobial activity. Synthetic derivates are currently being investigated as new anti-infectious agents. In infants, the use of conventional antibiotics is limited to a few substances because of adverse reactions. The new HDP substances might become alternatives to conventional antibiotics, but knowledge of the physiological quantities of the HDPs in infants is essential because of a narrow therapeutic index of currently available derivates. This study compares the mRNA levels of five major HDPs between infants and adults to test the hypothesis that HDP gene expression differs between these groups. Expression profiles of human beta-defensin (hBD)-1, hBD-2 and hBD-3, psoriasin and RNase 7 were assessed in the lip vermilion mucosa of infants (n = 15) and adult controls (n = 15) using real-time polymerase chain reaction. A significantly lower expression of hBD-2 (P = 0.043), hBD-3 (P = 0.014) and psoriasin (P = 0.018) was found in infants. No difference between the groups was noted with respect to transcript levels of hBD-1 and RNase 7. In conclusion, several HDPs are expressed at lower levels in infants, but not all. The results emphasize the need to adjust the dose of agents based on the specific HDP level for the treatment of infantile infections.

Evaluation of signal transduction pathways after transient cutaneous adenoviral gene delivery

BackgroundAdenoviral 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.MethodsIn 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.ResultsThe 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.ConclusionThe 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.

Low-energy extracorporeal shockwave therapy (ESWT) improves metaphyseal fracture healing in an osteoporotic rat model

Purpose As result of the current demographic changes, osteoporosis and osteoporotic fractures are becoming an increasing social and economic burden. In this experimental study, extracorporeal shock wave therapy (ESWT), was evaluated as a treatment option for the improvement of osteoporotic fracture healing. Methods A well-established fracture model in the metaphyseal tibia in the osteoporotic rat was used. 132 animals were divided into 11 groups, with 12 animals each, consisting of one sham-operated group and 10 ovariectomized (osteoporotic) groups, of which 9 received ESWT treatment. Different energy flux intensities (0.15 mJ/mm2, 0.35 mJ/mm2, or 0.55 mJ/mm2) as well as different numbers of ESWT applications (once, three times, or five times throughout the 35-day healing period) were applied to the osteoporotic fractures. Fracture healing was investigated quantitatively and qualitatively using micro-CT imaging, quantitative real-time polymerase chain reaction (qRT-PCR) analysis, histomorphometric analysis and biomechanical analysis. Results The results of this study show a qualitative and quantitative improvement in the osteoporotic fracture healing under low-energy (energy flux intensity: 0,15 mJ/mm2) ESWT and with fewer treatment applications per healing period. Conclusion In conclusion, low-energy ESWT seems to exhibit a beneficial effect on the healing of osteoporotic fractures, leading to improved biomechanical properties, enhanced callus-quantity and -quality, and an increase in the expression of bone specific transcription factors. The results suggest that low-energy ESWT, as main treatment or as adjunctive treatment in addition to a surgical intervention, may prove to be an effective, simple to use, and cost-efficient option for the qualitative and quantitative improvement of osteoporotic fracture healing.

Signal transduction of the innate and adaptive immune system after transient cutaneous adenoviral gene delivery

Introduction: Adenoviral vectors have provided effective methods for in vivo gene delivery for therapeutic purposes. Adenovirus vector can induce significant immune responses and this has severely affected the ability of these vectors to induce long-term gene expression. There is limited information about the mechanisms and signal transduction pathways involved in adenoviral induced immune reaction. In addition, the role of non-specialised immune cells in innate immunity still remains unclear. The aim of this study was the identification of potentially cytoplasmatic DNA-detection involved signal transduction molecules and pathways. Material and methods: For in vitro analysis, human keratinocytes were stimulated with adenoviral, fungal, mammalian and bacterial DNA. Additionally, potentially in DNA detection involved signal transduction molecules were blocked prior to tranfection of the cells. Isolation of total RNA and cDNA synthesis was performed 6 and 15 h post transfection. Expression of type-I-interferon and toll like receptor dependent signal transduction molecules was measured via RT-PCR. For in vivo experiments 1010 IU of adenoviral vectors encoding the green fluorescent protein (GFP) were intradermally injected into hairless mice (n=12). GFP expression was localized and quantified every second day via imager. A reapplication of the same vector dose occurred on day 14. 1–48 h after second injection the transgene expression was quantified and treated skin areals were removed, total RNA was isolated, transcribed into cDNA and transgene expression was measured via RT-PCR. Results: In vitro transfection with adenoviral and fungal DNA resulted in an acute induction of type-I-IFN and MyD88. An induction of TLR-2,-7,-9 and TRIF was not detected. Moreover, an inhibition of PI3K, p38 MAPK, JNK and NFκB resulted in a significantly decreased expression of type-I-IFN. In vivo, the transgene was detected up to 12 days post transduction via imager. A decreased GFP fluorescence was detected 48 h after reapplication, whereas the GFP mRNA was measured on a constant level up to 48 h. Conclusion: Our results suggest an induction of innate immunity via cytoplasmatic localized DNA which is independent of TLR-2,-7 and-9 in epithelial cells. A PI3K and MyD88 induced activation of p38 MAPK, JNK and NFκB leads to an acute type-I-IFN expression. A manipulation of these molecules seems to be an interesting approach for an improvement of adenoviral gene delivery. In regard to the constant GFP mRNA level, there is a possibility to improve adenoviral gene delivery on a translational level.

Host Defense peptides are upregulated in tumors of the upper pharynx

Inflammation and infection have long been associated with the development of cancer. Inflammation contributes to cancer initiation by inducing the release of a variety of cytokines and chemokines that alert the vasculature to release inflammatory cells and factors into the tissue milieu and are in the genesis of neoplastic tumors. Many host defense peptides (HDPs) demonstrate direct antimicrobial activity against bacteria, fungi, eukaryotic parasites and/or viruses. They have a key modulatory role in the innate immune response and present an important link between the innate and adaptive immune responses. The aim of this study was to quantify the local expression of HDP to characterise the role of the innate immune system in development of squamous cell carcinoma (SCC). Material and Methods: During surgical resection (n=46) human tumor tissue and healthy squamous tissue from the contralateral site was harvested from the same donor using a 6mm punch-biopsy. Tissue was stored in RNAlater reagent for mRNA measurements or 4% neutral buffered formaldehyde for histological analysis. After mRNA extraction and cDNA synthesis, LL-37, hBD-1-4, Dermcidin, RNase7 and Psoriasin (S100A7) expression were quantified by qRT-PCR and normalized to 18SrRNA. Additionally fluorescent immunohistochemical staining was performed for hBD-2 and −3, RNase7 and Psoriasin for corresponding localisation of protein expression. Patients who underwent maxillofacial surgery were used as an additional healthy control group (n=23). Results: Expression of hBD-1, −2, −3 and Psoriasin was up to 5-fold increased compared with the corresponding and healthy tissue (p<0.05). LL-37, hBD-4 and Dermcidin could not be detected and for RNase7 no differences were observed. Immunohistochemistry demonstrated specific staining for the investigated peptides confirming the expression level of the qRT-PCR experiments.