Challenges with Wound Infection Models in Drug Development

: Wound research is an evolving science trying to unfold the complex untold mechanisms behind the wound healing cascade. In particular, interest is growing regarding the role of microorganisms in both acute and chronic wound healing. Microbial burden plays an important role in the persistence of chronic wounds, ultimately resulting in delayed wound healing. It is therefore important for clinicians to understand the evolution of infection science and its various etiologies. Therefore, to understand the role of bacterial biofilm in chronic wound pathogenesis, various in vitro and in vivo models are required to investigate biofilms in wound-like settings. Infection models should be refined comprising an important signet of biofilms. These models are eminent for translational research to obtain data for designing an improved wound care formulation. However, all the existing models possess limitations and do not fit properly in the model frame for developing wound care agents. Among various impediments, one of the major drawbacks of such models is that the wound they possess does not mimic the wound a human develops. Therefore, a novel wound infection model is required which can imitate the human wounds. : This review article mainly discusses various in vitro and in vivo models showing microbial colonization, their advantages and challenges. Apart from these models, there are also present ex vivo wound infection models, but this review mainly focused on various in vitro and in vivo models available for studying wound infection in controlled conditions. This information might be useful in designing an ideal wound infection model for developing an effective wound healing formulation.

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Type VI secretion system and its effectors PdpC, PdpD and OpiA contribute to Francisella virulence in Galleria mellonella larvae

Infection and Immunity ◽

10.1128/iai.00579-20 ◽

2021 ◽

Author(s):

Maj Brodmann ◽

Sophie Schnider ◽

Marek Basler

Keyword(s):

Galleria Mellonella ◽

Response Regulator ◽

Infection Model ◽

Type Vi Secretion System ◽

Infectious Dose ◽

Infection Models

Francisella tularensis causes the deadly zoonotic disease tularemia in humans and is able to infect a broad range of organisms including arthropods, which are thought to play a major role in Francisella transmission. However, while mammalian in vitro and in vivo infection models are widely used to investigate Francisella pathogenicity, a detailed characterization of the major Francisella virulence factor, a non-canonical T6SS, in an arthropod in vivo infection model is missing. Here we use Galleria mellonella larvae to analyze the role of the Francisella T6SS and its corresponding effectors in F. novicida virulence. We report that G. mellonella larvae killing depends on the functional T6SS and infectious dose. In contrast to other mammalian in vivo infection models, even one of PdpC, PdpD or OpiA T6SS effectors is sufficient to kill G. mellonella larvae while sheath recycling by ClpB is dispensable. We further demonstrate that treatment by polyethylene glycol (PEG) activates Francisella T6SS in liquid culture and that this is independent of the response regulator PmrA. PEG-activated IglC secretion is dependent on T6SS structural component PdpB but independent of putative effectors PdpC, PdpD, AnmK and OpiB1-3. The results of larvae infection and secretion assay suggest that AnmK, a putative T6SS component with unknown function, interferes with OpiA-mediated toxicity but not with general T6SS activity. We establish that the easy-to-use G. mellonella larvae infection model provides new insights into function of T6SS and pathogenesis of Francisella.

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Role of Honey in Advanced Wound Care

Molecules ◽

10.3390/molecules26164784 ◽

2021 ◽

Vol 26 (16) ◽

pp. 4784

Author(s):

Hana Scepankova ◽

Patricia Combarros-Fuertes ◽

José María Fresno ◽

María Eugenia Tornadijo ◽

Miguel Sousa Dias ◽

...

Keyword(s):

Wound Healing ◽

Biomedical Applications ◽

Wound Care ◽

Direct Application ◽

Main Role ◽

Pathogenic Microorganisms ◽

Fibrous Membranes

Honey is a natural product rich in several phenolic compounds, enzymes, and sugars with antioxidant, anticarcinogenic, anti-inflammatory, and antimicrobial potential. Indeed, the development of honey-based adhesives for wound care and other biomedical applications are topics being widely investigated over the years. Some of the advantages of the use of honey for wound-healing solutions are the acceleration of dermal repair and epithelialization, angiogenesis promotion, immune response promotion and the reduction in healing-related infections with pathogenic microorganisms. This paper reviews the main role of honey on the development of wound-healing-based applications, the main compounds responsible for the healing capacity, how the honey origin can influence the healing properties, also highlighting promising results in in vitro and in vivo trials. The challenges in the use of honey for wound healing are also covered and discussed. The delivery methodology (direct application, incorporated in fibrous membranes and hydrogels) is also presented and discussed.

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Arginase Activity in Eisenia andrei Coelomocytes: Function in the Earthworm Innate Response

International Journal of Molecular Sciences ◽

10.3390/ijms22073687 ◽

2021 ◽

Vol 22 (7) ◽

pp. 3687

Author(s):

Joanna Homa ◽

Alina Klosowska ◽

Magdalena Chadzinska

Keyword(s):

Immune Response ◽

Wound Healing ◽

Arginase Activity ◽

Eisenia Andrei ◽

Heavy Metals Ions ◽

First Time ◽

Important Marker

Arginase is the manganese metalloenzyme catalyzing the conversion of l-arginine to l-ornithine and urea. In vertebrates, arginase is involved in the immune response, tissue regeneration, and wound healing and is an important marker of alternative anti-inflammatory polarization of macrophages. In invertebrates, data concerning the role of arginase in these processes are very limited. Therefore, in the present study, we focused on the changes in arginase activity in the coelomocytes of Eisenia andrei. We studied the effects of lipopolysaccharide (LPS), hydrogen peroxide (H2O2), heavy metals ions (e.g., Mn2+), parasite infection, wound healing, and short-term fasting (5 days) on arginase activity. For the first time in earthworms, we described arginase activity in the coelomocytes and found that it can be up-regulated upon in vitro stimulation with LPS and H2O2 and in the presence of Mn2+ ions. Moreover, arginase activity was also up-regulated in animals in vivo infected with nematodes or experiencing segment amputation, but not in fasting earthworms. Furthermore, we confirmed that the activity of coelomocyte arginase can be suppressed by l-norvaline. Our studies strongly suggest that similarly to the vertebrates, also in the earthworms, coelomocyte arginase is an important element of the immune response and wound healing processes.

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Development of Cephradine-Loaded Gelatin/Polyvinyl Alcohol Electrospun Nanofibers for Effective Diabetic Wound Healing: In-Vitro and In-Vivo Assessments

Pharmaceutics ◽

10.3390/pharmaceutics13030349 ◽

2021 ◽

Vol 13 (3) ◽

pp. 349

Author(s):

Anam Razzaq ◽

Zaheer Ullah Khan ◽

Aasim Saeed ◽

Kiramat Ali Shah ◽

Naveed Ullah Khan ◽

...

Keyword(s):

Wound Healing ◽

Polyvinyl Alcohol ◽

Mtt Assay ◽

Chronic Wound ◽

Drug Loading ◽

Group Identification ◽

Wound Infections ◽

Diabetic Wound

Diabetic wound infections caused by conventional antibiotic-resistant Staphylococcus aureus strains are fast emerging, leading to life-threatening situations (e.g., high costs, morbidity, and mortality) associated with delayed healing and chronic inflammation. Electrospinning is one of the most widely used techniques for the fabrication of nanofibers (NFs), induced by a high voltage applied to a drug-loaded polymer solution. Particular attention is given to electrospun NFs for pharmaceutical applications (e.g., original drug delivery systems) and tissue regeneration (e.g., as tissue scaffolds). However, there is a paucity of reports related to their application in diabetic wound infections. Therefore, we prepared eco-friendly, biodegradable, low-immunogenic, and biocompatible gelatin (GEL)/polyvinyl alcohol (PVA) electrospun NFs (BNFs), in which we loaded the broad-spectrum antibiotic cephradine (Ceph). The resulting drug-loaded NFs (LNFs) were characterized physically using ultraviolet-visible (UV-Vis) spectrophotometry (for drug loading capacity (LC), drug encapsulation efficiency (EE), and drug release kinetics determination), thermogravimetric analysis (TGA) (for thermostability evaluation), scanning electron microscopy (SEM) (for surface morphology analysis), and Fourier-transform infrared spectroscopy (FTIR) (for functional group identification). LNFs were further characterized biologically by in-vitro assessment of their potency against S. aureus clinical strains (N = 16) using the Kirby–Bauer test and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, by ex-vivo assessment to evaluate their cytotoxicity against primary human epidermal keratinocytes using MTT assay, and by in-vivo assessment to estimate their diabetic chronic wound-healing efficiency using NcZ10 diabetic/obese mice (N = 18). Thin and uniform NFs with a smooth surface and standard size (<400 nm) were observed by SEM at the optimized 5:5 (GEL:PVA) volumetric ratio. FTIR analyses confirmed the drug loading into BNFs. Compared to free Ceph, LNFs were significantly more thermostable and exhibited sustained/controlled Ceph release. LNFs also exerted a significantly stronger antibacterial activity both in-vitro and in-vivo. LNFs were significantly safer and more efficient for bacterial clearance-induced faster chronic wound healing. LNF-based therapy could be employed as a valuable dressing material to heal S. aureus-induced chronic wounds in diabetic subjects.

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Mitochondrion-Dependent Cell Death in TDP-43 Proteinopathies

Biomedicines ◽

10.3390/biomedicines9040376 ◽

2021 ◽

Vol 9 (4) ◽

pp. 376

Author(s):

Chantal B. Lucini ◽

Ralf J. Braun

Keyword(s):

Cell Death ◽

Oxygen Species ◽

In Vivo Models ◽

Dependent Cell ◽

Cell Death Mechanisms ◽

Sting Pathway ◽

Mitochondrial Membrane Permeabilization

In the last decade, pieces of evidence for TDP-43-mediated mitochondrial dysfunction in neurodegenerative diseases have accumulated. In patient samples, in vitro and in vivo models have shown mitochondrial accumulation of TDP-43, concomitantly with hallmarks of mitochondrial destabilization, such as increased production of reactive oxygen species (ROS), reduced level of oxidative phosphorylation (OXPHOS), and mitochondrial membrane permeabilization. Incidences of TDP-43-dependent cell death, which depends on mitochondrial DNA (mtDNA) content, is increased upon ageing. However, the molecular pathways behind mitochondrion-dependent cell death in TDP-43 proteinopathies remained unclear. In this review, we discuss the role of TDP-43 in mitochondria, as well as in mitochondrion-dependent cell death. This review includes the recent discovery of the TDP-43-dependent activation of the innate immunity cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) pathway. Unravelling cell death mechanisms upon TDP-43 accumulation in mitochondria may open up new opportunities in TDP-43 proteinopathy research.

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Photo-Crosslinked Polymeric Matrix with Antimicrobial Functions for Excisional Wound Healing in Mice

Nanomaterials ◽

10.3390/nano8100791 ◽

2018 ◽

Vol 8 (10) ◽

pp. 791 ◽

Cited By ~ 3

Author(s):

Ming-Hsiang Chang ◽

Yu-Ping Hsiao ◽

Chia-Yen Hsu ◽

Ping-Shan Lai

Keyword(s):

Wound Healing ◽

Wound Infection ◽

Polymer Solution ◽

Wound Dressing ◽

Glycol Diacrylate ◽

Excisional Wound ◽

Poly Ethylene ◽

Systemic Infections

Wound infection extends the duration of wound healing and also causes systemic infections such as sepsis, and, in severe cases, may lead to death. Early prevention of wound infection and its appropriate treatment are important. A photoreactive modified gelatin (GE-BTHE) was synthesized by gelatin and a conjugate formed from the 3,3′,4,4′-benzophenone tetracarboxylic dianhydride (BTDA) and the 2-hydroxyethyl methacrylate (HEMA). Herein, we investigated the photocurable polymer solution (GE-BTHE mixture) containing GE-BTHE, poly(ethylene glycol) diacrylate (PEGDA), chitosan, and methylene blue (MB), with antimicrobial functions and photodynamic antimicrobial chemotherapy for wound dressing. This photocurable polymer solution was found to have fast film-forming property attributed to the photochemical reaction between GE-BTHE and PEGDA, as well as the antibacterial activity in vitro attributed to the ingredients of chitosan and MB. Our in vivo results also demonstrated that untreated wounds after 3 days had the same scab level as the GE-BTHE mixture-treated wounds after 20 s of irradiation, which indicates that the irradiated GE-BTHE mixture can be quickly transferred into artificial scabs to protect wounds from an infection that can serve as a convenient excisional wound dressing with antibacterial efficacy. Therefore, it has the potential to treat nonhealing wounds, deep burns, diabetic ulcers and a variety of mucosal wounds.

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Activity of the Pore-Forming Virulence Factor Listeriolysin O Is Reversibly Inhibited by Naturally Occurring S-Glutathionylation

Infection and Immunity ◽

10.1128/iai.00959-16 ◽

2017 ◽

Vol 85 (4) ◽

Cited By ~ 15

Author(s):

Jonathan L. Portman ◽

Qiongying Huang ◽

Michelle L. Reniere ◽

Anthony T. Iavarone ◽

Daniel A. Portnoy

Keyword(s):

Protein Function ◽

Inhibitory Effect ◽

Cysteine Residue ◽

Infection Model ◽

Listeriolysin O ◽

Content Type ◽

Pore Forming Proteins

ABSTRACT Cholesterol-dependent cytolysins (CDCs) represent a family of homologous pore-forming proteins secreted by many Gram-positive bacterial pathogens. CDCs mediate membrane binding partly through a conserved C-terminal undecapeptide, which contains a single cysteine residue. While mutational changes to other residues in the undecapeptide typically have severe effects, mutation of the cysteine residue to alanine has minor effects on overall protein function. Thus, the role of this highly conserved reactive cysteine residue remains largely unknown. We report here that the CDC listeriolysin O (LLO), secreted by the facultative intracellular pathogen Listeria monocytogenes, was posttranslationally modified by S-glutathionylation at this conserved cysteine residue and that either endogenously synthesized or exogenously added glutathione was sufficient to form this modification. When recapitulated with purified protein in vitro, this modification completely ablated the activity of LLO, and this inhibitory effect was fully reversible by treatment with reducing agents. A cysteine-to-alanine mutation in LLO rendered the protein completely resistant to inactivation by S-glutathionylation, and a mutant expressing this mutation retained full hemolytic activity. A mutant strain of L. monocytogenes expressing the cysteine-to-alanine variant of LLO was able to infect and replicate within bone marrow-derived macrophages indistinguishably from the wild type in vitro, yet it was attenuated 4- to 6-fold in a competitive murine infection model in vivo. This study suggests that S-glutathionylation may represent a mechanism by which CDC-family proteins are posttranslationally modified and regulated and help explain an evolutionary pressure to retain the highly conserved undecapeptide cysteine.

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Sanguiin H-6 Fractionated from Cloudberry (Rubus chamaemorus) Seeds Can Prevent the Methicillin-Resistant Staphylococcus aureus Biofilm Development during Wound Infection

Antibiotics ◽

10.3390/antibiotics10121481 ◽

2021 ◽

Vol 10 (12) ◽

pp. 1481

Author(s):

John Jairo Aguilera-Correa ◽

Sara Fernández-López ◽

Iskra Dennisse Cuñas-Figueroa ◽

Sandra Pérez-Rial ◽

Hanna-Leena Alakomi ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Wound Infection ◽

Preventive Measure ◽

Surgical Site Infections ◽

Infection Model ◽

Growth Media ◽

Methicillin Resistant ◽

Biofilm Development

Staphylococcus aureus is the most common cause of surgical site infections and its treatment is challenging due to the emergence of multi-drug resistant strains such as methicillin-resistant S. aureus (MRSA). Natural berry-derived compounds have shown antimicrobial potential, e.g., ellagitannins such as sanguiin H-6 and lambertianin C, the main phenolic compounds in Rubus seeds, have shown antimicrobial activity. The aim of this study was to evaluate the effect of sanguiin H-6 and lambertianin C fractionated from cloudberry seeds, on the MRSA growth, and as treatment of a MRSA biofilm development in different growth media in vitro and in vivo by using a murine wound infection model where sanguiin H-6 and lambertianin C were used to prevent the MRSA infection. Sanguiin H-6 and lambertianin C inhibited the in vitro biofilm development and growth of MRSA. Furthermore, sanguiin H-6 showed significant anti-MRSA effect in the in vivo wound model. Our study shows the possible use of sanguiin H-6 as a preventive measure in surgical sites to avoid postoperative infections, whilst lambertianin C showed no anti-MRSA activity.

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Multifunctional Zinc Oxide/Silver Bimetallic Nanomaterial-Loaded Nanofibers for Enhanced Tissue Regeneration and Wound Healing

Journal of Biomedical Nanotechnology ◽

10.1166/jbn.2021.3152 ◽

2021 ◽

Vol 17 (9) ◽

pp. 1840-1849

Author(s):

Mao Li ◽

Min Hu ◽

Honglian Zeng ◽

Bo Yang ◽

Yi Zhang ◽

...

Keyword(s):

Wound Healing ◽

Zinc Oxide ◽

Tissue Regeneration ◽

Ag Nanoparticles ◽

Wound Care ◽

Fluid Handling ◽

Skin Repair ◽

Wound Healing Effect

Native skin repair requires wound care products that not only protect the wound from bacterial infection, but also accelerate wound closure and minimize scarring. Nanomaterials have been widely applied for wound healing due to their multifunctional properties. In a previous study, we prepared and characterized electrospinning zinc oxide/silver/polyvinylpyrrolidone/polycaprolactone (ZnO/Ag/PVP/PCL) nanofibers using ZnO and Ag nanoparticles, and evaluated their antibacterial effect in vitro. In this work, further characterization studies were performed, which confirmed that the ZnO/Ag nanoparticles were physically embedded and evenly distributed in the ZnO/Ag/PVP/PCL nanofibers, enabling the sustained release of Ag and Zn. In addition, the bimetallic nanofibers showed satisfactory fluid handling and flexibility. In vivo wound healing and histology studies showed that the ZnO/Ag/PVP/PCL nanofibers had a better anti-inflammatory, skin tissue regeneration, and wound healing effect than monometallic nanofibers or a commercially available wound plaster (Yunnan Baiyao). Therefore, ZnO/Ag/PVP/PCL bimetallic nanofibers may be a safe, efficient biomedical dressing for wound healing.

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