With the global prevalence of type 2 diabetes mellitus steeply rising, instances of chronic, hard-healing, or non-healing diabetic wounds and ulcers are predicted to increase. The growing understanding of healing and regenerative mechanisms has elucidated critical regulators of this process, including key cellular and humoral components. Despite this, the management and successful treatment of diabetic wounds represents a significant therapeutic challenge. To this end, the development of novel therapies and biological dressings has gained increased interest. Here we review key differences between normal and chronic non-healing diabetic wounds, and elaborate on recent advances in wound healing treatments with a particular focus on biological dressings and their effect on key wound healing pathways.

Drugs targeting immune checkpoint molecules have been found effective in melanoma, lung cancer, and other malignancies treatment. Recent studies on breast cancer demonstrated the significance of inhibitory anti-CTLA-4 and anti-PD-1 in the regulation of disease progression. However, seemingly the same types of breast cancer do not always respond unambiguously to immunotherapy. Thus, here we set out to analyze the in vitro effects of inhibiting CTLA-4 and PD-1 on interactions between co-cultured lymphocytes and two selected breast adenocarcinoma cell lines. Breast cancer cells were co-cultured with lymphocytes to evaluate the effects of CTLA-4 and PD-1 inhibition. Proliferation, cell cycle, and viability assessment were measured in cancer cells. IFN-gamma, IL-10, perforin, granzyme B production, and CTLA-4 and PD-1 expression were analyzed in lymphocytes. We found that administration of anti-CTLA-4 improved the anti-cancer activity of T cells with reduced proliferation and viability of MDA-MB-231. Lack of response was observed in the context of MCF-7. In addition, differential expression of checkpoint proteins was found between studied cancer cells lines. Inhibition of molecules was followed by IL-10 and IFN-gamma decrease in lymphocytes co-cultured with MDA-MB-231, not demonstrated in reference to MCF-7. Furthermore, CTLA-4 blockage was associated with reduction of CTLA-4+ and PD-1+ lymphocytes in MDA-MB-231, with a significant increase in MCF-7, reduced by anti-PD-1. Altogether, our study revealed that anti-CTLA-4 and anti-PD-1 treatment can improve lymphocytes effects on breast cancer cells. Favorable effects seemed to be related to breast cancer cells features as differential responses were reported. Novel blocking antibodies strategies should be tested for more effective cancer inhibition.

Chronic ulcerative and hard-healing wounds are a growing global concern. Skin substitutes, including acellular dermal matrices (ADMs), have shown beneficial effects in healing processes. Presently, the vast majority of currently available ADMs are processed from xenobiotic or cadaveric skin. Here we propose a novel strategy for ADM preparation from human abdominoplasty-derived skin. Skin was processed using three different methods of decellularization involving the use of ionic detergent (sodium dodecyl sulfate; SDS, in hADM 1), non-ionic detergent (Triton X-100 in hADM 2), and a combination of recombinant trypsin and Triton X-100 (in hADM 3). We next evaluated the immunogenicity and immunomodulatory properties of this novel hADM by using an in vitro model of peripheral blood mononuclear cell culture, flow cytometry, and cytokine assays. We found that similarly sourced but differentially processed hADMs possess distinct immunogenicity. hADM 1 showed no immunogenic effects as evidenced by low T cell proliferation and no significant change in cytokine profile. In contrast, hADMs 2 and 3 showed relatively higher immunogenicity. Moreover, our novel hADMs exerted no effect on T cell composition after three-day of coincubation. However, we observed significant changes in the composition of monocytes, indicating their maturation toward a phenotype possessing anti-inflammatory and pro-angiogenic properties. Taken together, we showed here that abdominoplasty skin is suitable for hADM manufacturing. More importantly, the use of SDS-based protocols for the purposes of dermal matrix decellularization allows for the preparation of non-immunogenic scaffolds with high therapeutic potential. Despite these encouraging results, further studies are needed to evaluate the beneficial effects of our hADM 1 on deep and hard-healing wounds.

Gamma rays and electrons with kinetic energy up to 10 MeV are routinely used to sterilize biomaterials. To date, the effects of irradiation upon human acellular dermal matrices (hADMs) remain to be fully elucidated. The optimal irradiation dosage remains a critical parameter affecting the final product structure and, by extension, its therapeutic potential. ADM slides were prepared by various digestion methods. The influence of various doses of radiation sterilization using a high-energy electron beam on the structure of collagen, the formation of free radicals and immune responses to non-irradiated (native) and irradiated hADM was investigated. The study of the structure changes was carried out using the following methods: immunohistology, immunoblotting, and electron paramagnetic resonance (EPR) spectroscopy. It was shown that radiation sterilization did not change the architecture and three-dimensional structure of hADM; however, it significantly influenced the degradation of collagen fibers and induced the production of free radicals in a dose-dependent manner. More importantly, the observed effects did not disrupt the therapeutic potential of the new transplants. Therefore, radiation sterilization at a dose of 35kGy can ensure high sterility of the dressing while maintaining its therapeutic potential.

The management of hard-to-heal wounds is a significant clinical challenge. Acellular dermal matrices (ADMs) have been successfully introduced to enhance the healing process. Here, we aimed to develop protocol for the preparation of novel ADMs from abdominoplasty skin. We used three different decellularization protocols for skin processing, namely, 1M NaCl and sodium dodecyl sulfate (SDS, in ADM1); 2M NaCl and sodium dodecyl sulfate (SDS, in ADM1); and a combination of recombinant trypsin and Triton X-100 (in hADM 3). We assessed the effectiveness of decellularization and ADM’s structure by using histochemical and immunochemical staining. In addition, we evaluated the therapeutic potential of novel ADMs in a murine model of wound healing. Furthermore, targeted transcriptomic profiling of genes associated with wound healing was performed. First, we found that all three proposed methods of decellularization effectively removed cellular components from abdominoplasty skin. We showed, however, significant differences in the presence of class I human leukocyte antigen (HLA class I ABC), Talin 1/2, and chondroitin sulfate proteoglycan (NG2). In addition, we found that protocols, when utilized differentially, influenced the preservation of types I, III, IV, and VII collagens. Finally, we showed that abdominoplasty skin-derived ADMs might serve as an effective and safe option for deep wound treatment. More importantly, our novel dressing (ADM1) improves the kinetics of wound closure and scar maturation in the proliferative and remodeling phases of wound healing. In conclusion, we developed a protocol for abdominoplasty skin decellularization suitable for the preparation of biological dressings. We showed that different decellularization methods affect the purity, structure, and therapeutic properties of ADMs.

The months-long treatment for tuberculosis is partially attributed to a minor Mycobacterium tuberculosis (Mtb) subpopulation exhibiting phenotypic drug resistance, although origins of these bacilli in hosts remain unknown. Previously, we observed that mycobacteria respond to growth-restrictive limitations of zinc by accumulating Mrf, and inducing Mpy recruitment to the decoding center of the ribosome, thereby hibernating the ribosome in an inactive state. Mrf accumulation – regulated at both transcriptional and post-translational levels by Zur and Clp, respectively, in a zinc-dependent manner – facilitates the formation of stable Mpy-70S ribosome complex in zinc-starved cells. Here, using a translational fusion of Mrf with a fluorescent-reporter protein we visualized zinc-starved mycobacteria with a physiology conducive for Mpy-dependent ribosome hibernation. About half of zinc-starved mycobacterial population expressed microscopically detectable levels of the reporter and exhibited Mpy-dependent slowdown of growth and isoniazid tolerance in vitro. In chronic, high-burden mouse lung infections, ~3% of Mtb bacilli expressed the reporter, and the frequency increased to ~35% upon isoniazid treatment. Transcriptomics of mouse lungs suggests an important role of S100A8/A9 and ZIP2 in neutrophils in zinc mobilization during Mtb infections. Moreover, depletion of neutrophils, or loss of S100A9 caused significant attenuation of zinc starvation response in Mtb. Altogether, the study offers a new approach for visualizing and manipulating slow-replicating Mtb in host tissues.