The mesenchymal stromal cell secretome promotes tissue regeneration and increases macrophage infiltration in acute and methicillin-resistant Staphylococcus aureus-infected skin wounds in vivo.

BACKGROUND AIMS: The prevalence of chronic wounds continues to be a burden in human medicine. Methicillin-resistant Staphylococcus aureus (MRSA) is commonly isolated from infected wounds. MRSA infections primarily delay healing by impairing local immune cell functions. This study aimed to investigate the potential of mesenchymal stromal cell (MSC)-secreted bioactive factors, defined as the secretome, to improve innate immune responses in vivo. MSCs were isolated from the bone marrow of horses, which serve as valuable translational models for wound healing. The MSC secretome, collected as conditioned medium (CM), was evaluated in vivo using mouse models of acute and MRSA-infected skin wounds. METHODS: Punch biopsies were used to create two full-thickness skin wounds on the back of each mouse. Acute wounds were treated daily with control medium or bone marrow-derived MSC (BM-MSC) CM. The antibiotic mupirocin was administered as a positive control for the MRSA-infected wound experiments. Wounds were photographed daily, and wound images were measured to determine the rate of closure. Trichrome staining was carried out to examine wound tissue histologically, and immunofluorescence antibody binding was used to assess immune cell infiltration. Wounds in the MRSA-infected model were swabbed for quantification of bacterial load. RESULTS: Acute wounds treated with BM-MSC CM showed accelerated wound closure compared with controls, as illustrated by enhanced granulation tissue formation and resolution, increased vasculature and regeneration of hair follicles. This treatment also led to increased neutrophil and macrophage infiltration. Chronic MRSA-infected wounds treated with BM-MSC CM showed reduced bacterial load accompanied by better resolution of granulation tissue formation and increased infiltration of pro-healing M2 macrophages compared with control-treated infected wounds. CONCLUSIONS: Collectively, our findings indicate that BM-MSC CM exerts pro-healing, immunomodulatory and anti-bacterial effects on wound healing in vivo, validating further exploration of the MSC secretome as a novel treatment option to improve healing of both acute and chronic wounds, especially those infected with antibiotic-resistant bacteria.

Rational Design and Antimicrobial Potency Assessment of Abaecin Analogues.

The widespread and escalating emergence of multidrug resistance is now recognized as one of the most severe global threats to human health. To address the urgent issue of drug-resistant bacteria and the limitation of effective clinical treatments, antimicrobial peptides (AMPs) have been developed as promising substituents of conventional antibiotics. In this study, rational design strategies were employed to acquire seven cationic and α-helical engineered peptides based on the original template of Abaecin. After investigation, we found that AC7 (LLRRWKKLFKKIIRWPRPLPNPGH) demonstrated potent and broad-spectrum antimicrobial activity. Additionally, it demonstrated low cytotoxicity and hemolysis while maintaining good stability. Notably, AC7 displays the antibacterial mechanism with superior abilities in cell membrane disruption and potential DNA binding in vitro, as well as effectively disrupting biofilms. Moreover, the murine skin wound model infected with drug-resistant Pseudomonas aeruginosa was employed to evaluate the anti-infective efficacy and therapeutic potential of AC7. It was observed that AC7 displays a remarkable capacity to inhibit wound colonization, reduce levels of inflammatory cytokines (TNF-α) and inflammatory cells (white blood cells (WBC), monocytes (MONO), lymphocytes (LYMPH), neutrophils (GRAN)), promote the levels of IL-10 and VEGF, and enhance wound healing. Overall, these findings demonstrate the potential of AC7 as a viable alternative to traditional antibiotics.

Topical application of gemcitabine generates microvesicle particles in human and murine skin.

Chemotherapy has remained the mainstay for the treatment of multiple types of cancers. In particular, topical use of chemotherapy has been used for skin cancers. Though effective, topical chemotherapy has been limited due to adverse effects such as local and even systemic toxicities. Our recent studies demonstrated that exposure to pro-oxidative stressors, including therapeutic agents induces the generation of extracellular vesicles known as microvesicle particles (MVP) which are dependent on activation of the Platelet-activating factor-receptor (PAFR), a G-protein coupled receptor present on various cell types, and acid sphingomyelinase (aSMase), an enzyme required for MVP biogenesis. Based upon this premise, we tested the hypothesis that topical application of gemcitabine will induce MVP generation in human and murine skin. Our ex vivo studies using human skin explants demonstrate that gemcitabine treatment results in MVP generation in a dose-dependent manner in a process blocked by PAFR antagonist and aSMase inhibitor. Importantly, gemcitabine-induced MVPs carry PAFR agonists. To confirm the mechanisms, we employed PAFR-expressing and deficient (Ptafr-/- ) mouse models as well as mice deficient in aSMase enzyme (Spmd1-/- ). Similar to the findings using pharmacologic tools, genetic-based approaches demonstrate that gemcitabine-induced MVP release in WT mice was blunted in Ptafr-/- and Spmd1-/- mice. These findings demonstrate a novel mechanism by which local chemotherapy can generate bioactive components as a bystander effect in a process that is dependent upon the PAFR-aSMase pathway.

Heat shock protein 90 is a new potential target of anti-rejection therapy in allotransplantation.

The critical roles of heat shock protein 90 (HSP90) in immune reactions associated with viral infection and autoimmune disease are well known. To date, however, its roles in the alloimmune response and the immunosuppressive effect of HSP90 inhibitors in allotransplantation have remained unknown. The purpose of this study was to examine the therapeutic efficacy of the HSP90 inhibitor 17-DMAG in allotransplantation models. C57BL/6 (H-2b) and BALB/c (H-2d) mice were used as donors for and recipients of skin and heart transplantation, respectively. Treatment with 17-DMAG (daily i.p.) or a vehicle was initiated 3 days before transplantation. Immunological outcomes were assessed by histopathological examinations, flow cytometric analysis, quantitative RT-PCR, ELISA, ELISPOT assay, and MLR. 17-DMAG treatment significantly prolonged the survival of both skin and heart allografts. In 17-DMAG-treated mice, donor-reactive splenocytes producing IFN-γ were significantly reduced along with the intragraft mRNA expression level and serum concentration of IFN-γ. Intragraft mRNA expression of cytokines and chemokines associated with both innate and adaptive immunity was suppressed in 17-DMAG-treated group. MLR showed suppression of the donor-specific proliferation of CD4 + T and CD19 + B cells in the spleens of 17-DMAG-treated mice. 17-DMAG treatment also reduced the number of activated NK cells. Furthermore, the treatment lowered the titers of donor-specific antibodies in the serum and prolonged a second skin allograft in mice sensitized by previous skin transplantation. HSP90 inhibition by 17-DMAG can affect various immune responses, including innate immunity, adaptive immunity, and humoral immunity, suggesting its therapeutic potential against acute rejection in allotransplantation.

In Vitro and In Vivo Activity of 14-O-[(4,6-Diamino-pyrimidine-2-yl) thioacetyl] Mutilin against Methicillin-Resistant Staphylococcus aureus.

Staphylococcus aureus (S. aureus) is a major human pathogen that requires new antibiotics with unique mechanism. A new pleuromutilin derivative, 14-O-[(4,6-Diamino-pyrimidine-2-yl) thioacetyl] mutilin (DPTM), has been synthesized and proved as a potent antibacterial agent using in vitro and in vivo assays. In the present study, DPTM was further in vitro evaluated against methicillin-resistant Staphylococcus aureus (MRSA) isolated from dairy farms and outperformed tiamulin fumarate, a pleuromutilin drug used for veterinary. Moreover, a murine skin wound model caused by MRSA infection was established, and the healing effect of DPTM was investigated. The results showed that DPTM could promote the healing of MRSA skin infection, reduce the bacterial burden of infected skin MRSA and decrease the secretion of IL-6 and TNF-α inflammatory cytokines in plasma. These results provided the basis for further in-depth drug targeted studies of DPTM as a novel antibacterial agent.

A Murine Skin Infection Model Capable of Differentiating the Dermatopathology of Community-Associated MRSA Strain USA300 from Other MRSA Strains.

USA300 is a predominant and highly virulent community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strain that is a leading cause of skin and soft tissue infections. We established a murine intradermal infection model capable of demonstrating dermatopathological differences between USA300 and other MRSA strains. In this model, USA300 induced dermonecrosis, uniformly presenting as extensive open lesions with a histologically documented profound inflammatory cell infiltrate extending below the subcutis. In contrast, USA400 and a colonizing control strain M92 caused only localized non-ulcerated skin infections associated with a mild focal inflammatory infiltrate. It was also determined that the dermonecrosis induced by USA300 was associated with significantly increased neutrophil recruitment, inhibition of an antibacterial response, and increased production of cytokines/chemokines associated with disease severity. These results suggest that induction of severe skin lesions by USA300 is related to over-activation of neutrophils, inhibition of host antibacterial responses, and selective alteration of host cytokine/chemokine profiles.

The inhibitory effects and mechanisms of polymannuroguluronate sulfate against human papillomavirus infection in vitro and in vivo.

Human papillomaviruses (HPVs) are non-enveloped DNA viruses that infect epithelia and can cause a wide variety of benign and pre-malignant epithelial tumours. The sulfated polysaccharides such as carrageenans were reported to be able to interfere with the binding process of HPV to the cell surface. In this study, brown seaweed derived polysaccharides polymannuroguluronate sulfate (PMGS) were prepared, and their anti-HPV effects were explored in vitro and in vivo. The results indicated that PMGS effectively inhibited high-risk HPV16 and HPV45 infection with very low toxicity. PMGS may inactivate HPV particles or block the binding and entry process of HPV through direct interaction with viral capsid proteins. PMGS can enter into HeLa cells and down-regulate the expression levels of viral oncogene proteins E6 and E7. In addition, PMGS also dramatically inhibited HPV infection on the skin of BALB/c Nude Mice. Thus, marine derived polysaccharide PMGS possessed anti-HPV activities in vitro and in vivo, and may block HPV infection via targeting viral capsid L1 protein, suggesting that it has great potential to be developed into a novel anti-HPV agent in the future.

Quantification and characterization of radical production in human, animal and 3D skin models during sun irradiation measured by EPR spectroscopy.

Sun radiation is indispensable to our health, however, a long term and high exposure could lead to erythema, premature skin aging and promotion of skin tumors. An underlying pathomechanism is the formation of free radicals. First, reactive oxygen species (*OH, *O2-) and then, secondary lipid oxygen species (C centered radicals, CCR) are formed. A high amount of free radicals results in oxidative stress with subsequent cell damage. In dermatological research different skin models are used, however, comparative data about the cutaneous radical formation are missing. In this study, the radical formation in porcine-, (SKH-1) murine-, human- ex vivo skin and reconstructed human skin (RHS) were investigated during simulated sun irradiation (305-2200 nm), with X-band EPR spectroscopy. The amount of radical formation was investigated with the spin probe PCA exposed to a moderate sun dose below one minimal erythema dose (MED, ~25 mJ/cm2 UVB) in all skin models. Furthermore, the *OH and *CCR radical concentrations were measured with the spin trap DMPO within 0-4 MED (porcine-, human skin and RHS). The highest amount of radicals was found in RHS followed by murine and porcine, and the lowest amount in human ex vivo skin. In all skin models, more *OH than CCR radicals were found at 0-4 MED. Additionally, this work addresses the limitations in the characterization with the spin trap DMPO. The measurements have shown that the most comparable skin model to in vivo human skin could differ depending on the focus of the investigation. If the amount of radial production is regarded, RHS seems to be in a similar range like in vivo human skin. If the investigation is focused on the radical type, porcine skin is most comparable to ex vivo human skin, at an irradiation dose not exceeding 1 MED. Here, no comparison to in vivo human skin is possible.

Development of tensile strength methodology for murine skin wound healing.

In this study, a methodology was evaluated and improved to quickly measure the tensile strength of murine skin in a biomechanical assay for an incisional wound healing model. The aim was to streamline and enhance the wound model, skin specimen preparation, and tensile test so that large numbers of fresh tissue could be tested reliably and rapidly. Linear incisions of 25-mm length were made in the dorsal skin of mice along the spine and metallic staples were used to close the wound. After 20 days, the mice were sacrificed, and a square-shaped section of skin containing the linear incision was excised. Two metallic punches were fabricated and used to punch 15-mm long strips of skin of 2 mm width whose length was orthogonal to the direction of incision. The tensiometer configuration was modified to expedite tensile measurements on fresh skin, and load-to-failure was measured for each strip of skin from the cephalad to the caudal region. We evaluated sources of error in the animal model and the testing protocol and developed procedures to maximize speed and reproducibility in tensile strength measurements. This report provides guidance for efficient and reproducible tensile strength measurement of large numbers of skin specimens from freshly sacrificed animals. •Tattoo placement to identify the two ends of the healing incisional wound assisted in decreasing error in the position and orientation of tensile strips.•Custom-made punches to prepare skin strips for tensile testing helped conduct tensile tests of fresh tissue rapidly.•Alteration of the manual grips of the tensile tester enabled specimens to be gripped rapidly to significantly accelerate testing for each skin strip.

Wedelolactone mitigates UVB induced oxidative stress, inflammation and early tumor promotion events in murine skin: plausible role of NFkB pathway.

UVB (Ultra-violet B) radiation is one of the major etiological factors in various dermal pathology viz. dermatitis, actinic folliculitis, solar urticaria, psoriasis and cancer among many others. UVB causes toxic manifestation in tissues by inciting inflammatory and tumor promoting events. We have designed this study to assess the anti-inflammatory and anti-tumor promotion effect of Wedelolactone (WDL) a specific IKK inhibitor. Results indicate significant restoration of anti-oxidative enzymes due to WDL treatments. We also found that WDL was effective in mitigating inflammatory markers consisting of MPO (myeloperoxidase), Mast cells trafficking, Langerhans cells suppression and COX 2 expression up regulation due to UVB exposure. We also deduce that WDL presented a promising intervention in attenuating early tumor promotion events caused by UVB exposure as indicated by the results of ODC (Ornithine Decarboxylase), Thymidine assay, Vimentin and VEGF (Vascular-endothelial growth factor) expression. This study was able to provide substantial cues for the therapeutic ability of Wedelolactone against inflammatory and tumor promoting events in murine skin depicting plausible role of NFkB pathway.

A natural herbal remedy modulates angiogenic activity of bronchoalveolar lavage cells from sarcoidosis patients.

Sarcoidosis is a systemic inflammatory disease with abnormally high angiogenic activity of inflammatory cells. Reumaherb preparation consisting of three herbs: Echinacea purpurea, Harpagophytum procumbens, and Filipendula ulmaria, and it exerts anti-inflammatory, antioxidant, and analgesic activity and stimulates regenerative and immunological processes. The aim of this paper was to estimate the effect of Reumaherb on immunological angiogenesis induced by bronchoalveolar lavage (BAL) cells collected from six patients with sarcoidosis and grafted into Balb/c mice skin. After grafting, the animals were fed for three days with 0.6 or 1.2 mg of Reumaherb (calculated from recommended human daily dose) daily, suspended in 40 µl of water, or 40 µl of water alone (control group). A significant reduction of newly formed blood vessels was obtained in four cases for 1.2 mg and in three cases for 0.6 mg daily dose of this remedy. Thus, we hypothesise that Reumaherb promotes anti-angiogenic activity and may potentially be used in diseases associated with excessive blood vessel formation.

Efficacy of gelatin gel sheets sustaining epidermal growth factor for murine skin defects.

BACKGROUND: Epidermal growth factor (EGF) plays an important role in wound healing. However, EGF must be applied daily due to rapid inactivation in vivo. We investigated the sustained release of EGF from gelatin gel sheets (GGSs) and the efficacy of GGSs impregnated with EGF for promoting wound healing. MATERIALS AND METHODS: GGSs impregnated with EGF were prepared by cross-linking via glutaraldehyde to gelatin solution containing EGF. The sustained release of EGF and the bioactivity of released EGF were evaluated. Then, three kinds of GGSs containing NSS (normal saline solution; NSS group), 2.5 µg of EGF (EGF-L group), or 25 µg of EGF (EGF-H group) were applied to full-thickness skin defects created on the backs of mice. The wounds covered with polyurethane film without GGS were used as a control (PUF group). The wound area, neoepithelium length, regenerated granulation tissue, and newly formed capillaries were evaluated. RESULTS: EGF was sustained and released from GGS as it degraded. The bioactivity of released EGF was confirmed. EGF-L group promoted the neoepithelium length, regenerated granulation tissue, and newly formed capillaries compared with those in the PUF and NSS groups. The area of regenerated granulation tissue in the NSS group (week 1: 2.6 + 0.2 mm(2), week 2: 2.8 + 0.3 mm(2)) was larger than that in the PUF group (week 1: 0.6 + 0.1 mm(2), week 2: 1.0 + 0.1 mm(2)). The area of newly formed capillaries in the EGF-L group (9967 + 1903 µm(2)) was larger than that of the EGF-H group (3485 + 1050 µm(2)). CONCLUSIONS: GGSs impregnated with EGF-L showed promising results regarding wound healing.

The tumor promoting activity of the EP4 receptor for prostaglandin E2 in murine skin.

To determine whether the EP4 receptor for prostaglandin E2 (PGE2) contributes to the tumor promoting activity of PGs in murine skin, EP4 over-expressing mice (BK5.EP4) were generated and subjected carcinogenesis protocols. An initiation/promotion protocol resulted in 25-fold more squamous cell carcinomas (SCCs) in the BK5.EP4 mice than wild type (WT) mice. An increase in SCCs also occurred following treatment with initiator alone or UV irradiation. The initiator dimethylbenz[a]anthracene caused cytotoxicity in BK5.EP4, but not WT mice, characterized by sloughing of the interfollicular epidermis, regeneration and subsequent SCC development. A comparison of transcriptomes between BK5.EP4 and WT mice treated with PGE2 showed a significant upregulation of a number of genes known to be associated with tumor development, supporting a pro-tumorigenic role for the EP4 receptor.