Continuum of care in hard-to-heal wounds by copper dressings: a case series.

OBJECTIVE: The quest for an ideal wound dressing has been a longstanding challenge due to the complex nature of wound healing, including stages of haemostasis, inflammation, maturation and remodelling, with overlapping timelines. This makes it difficult to find a single dressing that optimally supports all phases of wound healing. In addition, the ideal wound dressing should possess antibacterial properties and be capable of effectively debriding and lysing necrotic tissue. Copper is an essential trace element that participates in many of the key physiological wound healing processes. METHOD: Copper stimulates secretion of various cytokines and growth factors, thus promoting angiogenesis, granulation tissue formation, extracellular matrix proteins secretion and re-epithelialisation. Harnessing this knowledge, we have used copper oxide-impregnated wound dressings in numerous cases and observed their benefits throughout the entire wound healing process. RESULTS: This led us to postulate the 'continuum of care' hypothesis of copper dressings. In this study we describe four cases of hard-to-heal wounds of various aetiologies, in which we applied copper dressings consistently across all stages of wound healing, with rapid uneventful healing. CONCLUSION: We believe we have successfully implemented the continuum of care principle.

Copper Ions Ameliorated Thermal Burn-Induced Damage in ex vivo Human Skin Organ Culture.

INTRODUCTION: The zone of stasis is formed around the coagulation zone following skin burning and is characterized by its unique potential for salvation. The cells in this zone may die or survive depending on the severity of the burn and therefore are target for the local treatments of burns. Their low survival rate is consistent with decreased tissue perfusion, hypotension, infection, and/or edema, resulting in a significant increase in the wound size following burning. Copper is an essential trace mineral needed for the normal function of almost all body tissues, including the skin. OBJECTIVE: The aim of the work was to study the effect copper ions have on skin burn pathophysiology. METHODS: Skin obtained from healthy patients undergoing abdominoplasty surgery was cut into 8 × 8 mm squares, and round 0.8-mm diameter burn wounds were inflicted on the skin explants. The burned and control intact skin samples were cultured up to 27 days after wounding. Immediately following injury and then again every 48 h, saline only or containing 0.02 or 1 µM copper ions was added onto the skin explant burn wounds. RESULTS: We found that exposing the wounded sites immediately after burn infliction to 0.02 or 1 µM copper ions reduced the deterioration of the zone of stasis and the increase in wound size. The presence of the copper ions prevented the dramatic increase of pro-inflammatory cytokines (interleukin (IL)-6 and IL-8) and transforming growth factor beta-1 that followed skin burning. We also detected re-epithelialization of the skin tissue and a greater amount of collagen fibers upon copper treatment. CONCLUSION: The deterioration of the zone of stasis and the increase in wound size following burning may be prevented or reduced by using copper ion-based therapeutic interventions.

Stimulation of Healing of Non-Infected Stagnated Diabetic Wounds by Copper Oxide-Impregnated Wound Dressings.

Background and Objective: Copper, a wide spectrum biocide, also plays a key role in angiogenesis and wound healing. Antibacterial wound dressings impregnated with copper oxide microparticles (COD) have been recently cleared by the U.S. FDA and other regulatory bodies for the treatment of acute and chronic wounds, including diabetic wounds. Our objective was to evaluate the capacity of COD in stimulating the healing of non-infected stagnated wounds in diabetic patients initially treated with standard of care (SOC) dressings. Materials and Methods: The trial was divided into the three following phases: 1-2 weeks of screening, during which the patients were treated with SOC dressings; 4 weeks of treatment, during which the COD was applied twice weekly; and 2 weeks of follow-up, during which the patients were again treated with SOC dressings. The wound conditions and sizes were assessed by clinical evaluation and a wound imaging artificial intelligence system. Results: Following 1 month of COD treatment, there was a clear reduction in the mean wound area (53.2%; p = 0.003), an increase in granulation tissue (43.37; p < 0.001), and a reduction in fibrins (47.8%; p = 0.002). In patients with non-weight-bearing wounds, the reduction in wound size was even more dramatic (66.9%; p < 0.001). Conclusions: The results of this study, showing a statistically significant influence of COD on wound healing of hard-to-heal wounds in diabetic patients, strongly supports the notion that copper oxide-impregnated dressings enhance wound healing directly. Further larger controlled studies should be conducted to substantiate our findings.

Healing of Chronic Wounds by Copper Oxide-Impregnated Wound Dressings-Case Series.

Novel antimicrobial wound dressings impregnated with copper oxide micro-particles have been cleared for treatment of acute and chronic wounds. Our objective is to provide preliminary data regarding the potential benefit of using these novel wound dressings including in non-infected wounds. Methods involved the treatment of wounds that responded partially or poorly to conventional wound healing treatments with copper oxide impregnated wound dressings in patients with a range of etiologies. Ten cases of patients with etiologies such as diabetes mellitus, sickle cell disease, renal failure, and necrotizing fasciitis, in which the application of copper oxide impregnated wound dressings in infected and non-infected wounds, which resulted in significant enhanced wound healing, are presented. This was exemplified by clearing of the wound infections, reduction of the fibrous and/or necrotic tissue and by intense granulation, epithelialization, and wound closure. The described 10 case reports support our hypothesis that the copper oxide-containing wound dressing not only confers protection to the wound and the dressing from microbial contamination, and in some cases may help clear the wound infections, but in addition and more importantly, stimulate skin regeneration and wound healing. Our findings are in line with previous animal and in vitro studies showing that copper plays a key role in angiogenesis and skin regeneration. These case reports support the notion that the use of copper oxide impregnated wound dressings may be an important intervention in the arsenal of wound treatment modalities, especially in hard to heal wounds.

Potent bactericidal efficacy of copper oxide impregnated non-porous solid surfaces.

BACKGROUND: The role of fomites and the environment in nosocomial infections is becoming widely recognized. In this paper we discuss the use of Cupron copper oxide impregnated non-porous solid surface in the hospital setting and present in vitro testing data via USA Environmental Protection Agency (EPA) approved testing protocols that demonstrate the efficacy of these products to assist in reduction in environmental contamination and potentially nosocomial infections. RESULTS: The two countertops tested passed all the acceptance criteria by the EPA (>99.9% kill within 2 hours of exposure) killing a range of bacterial pathogens on the surface of the countertops even after repeated exposure of the countertops to the pathogen, and multiple wet and dry abrasion cycles. CONCLUSIONS: Cupron enhanced EOS countertops thus may be an important adjunct to be used in hospital settings to reduce environmental bioburden and potentially nosocomial infections.

Beneficial regulation of fibrillar collagens, heat shock protein-47, elastin fiber components, transforming growth factor-ß1, vascular endothelial growth factor and oxidative stress effects by copper in dermal fibroblasts.

Skin aging is associated with the loss of the structural collagens and the elastin fiber components that form the extracellular matrix (ECM). It is associated with reduced transforming growth factor-ß (TGF-ß), angiogenesis and increased oxidative stress. Copper has been incorporated into cosmetics for anti-skin aging. This research investigated the mechanism for the anti-skin aging effect copper ions, from cuprous oxide powders. Dermal fibroblasts were exposed to copper and examined for expression (protein and/or promoter levels) of types I, III, V collagen, heat shock protein-47 (HSP-47), elastin, fibrillin-1, and fibrillin-2, TGF-ß1, vascular endothelial growth factor (VEGF), and in addition for membrane damage and lipid peroxidation. The direct antioxidant activity of copper was also determined. The research indicates that copper's anti-skin aging and skin regeneration potential is through its stimulation of ECM proteins, TGF-ß1, VEGF, and inhibition of oxidative stress effects at physiological concentrations; and supports its use in cosmetics.

Antiviral propierties of 5,5'-dithiobis-2-nitrobenzoic acid and bacitracin against T-tropic human immunodeficiency virus type 1.

Bacitracin and the membrane-impermeant thiol reagent 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) are agents known to inhibit protein disulfide isomerase (PDI), a cell-surface protein critical in HIV-1 entry therefore they are fusion inhibitors (FI). Here we investigated the possibility that Bacitracin and or DTNB might have other antiviral activities besides FI. By means of residual activity assays, we found that both compounds showed antiviral activity only to viruses T-tropic HIV-1 strain. Cell-based fusion assays showed inhibition on HeLa-CD4-LTR-ß-gal (CD4) and HL2/3 cells treated with Bacitracin, and DTNB with the latest compound we observed fusion inhibition on both cells but strikingly in HL2/3 cells (expressing Env) indicating a possible activity on both, the cell membrane and the viral envelope. A time-of-addition experiment showed that both compounds act on HIV entry inhibition but DTNB also acts at late stages of the viral cycle. Lastly, we also found evidence of long-lasting host cell protection in vitro by DTNB, an important pharmacodynamic parameter for a topical microbicide against virus infection, hours after the extracellular drug was removed; this protection was not rendered by Bacitracin. These drugs proved to be leading compounds for further studies against HIV showing antiviral characteristics of interest.

Drastic Reduction of Bacterial, Fungal and Viral Pathogen Titers by Cuprous Oxide Impregnated Medical Textiles.

Hospital patients and personnel are at risk of nosocomial viral infections, as clearly manifested during the COVID-19 pandemic. Transmission of respiratory viral pathogens can occur through contaminated surfaces, including from medical textiles. Copper has potent biocidal properties, and cuprous oxide impregnated medical textiles (CMT) reduce hospital-acquired bacterial infections. In the current study we confirm the antimicrobial properties of CMT and determine their capacity to reduce infectious titres of human coronavirus (HCoV-229E) in an independent laboratory. The antibacterial and antiviral activities of the CMT were determined according to AATCC TM100-2019 and ISO 18184:2019 standards, respectively. The CMT reduced by 4 logs the viable titers of MRSA, Klebsiella pneumoniae, Enterococcus faecalis, and Candida auris after 2 h of incubation. Viable titers of Clostridium difficile were reduced by 2.3, 3, and 4 logs after 2, 6, and 18 h, respectively. Infectious titers of HCoV-229E exposed to CMT for 2 h were reduced by 2.8 and 4 logs (99.85% and 99.99% reductions) as compared to Time-0 control and initial inoculum, respectively. The CMT retain their antibacterial efficacy even after 100 industrial washings. Use of cuprous oxide impregnated textiles in clinical settings may reduce not only hospital acquired infections caused by bacterial and fungal pathogens, but also, and equally important, those caused by coronavirus and other viruses.

Molecular mechanisms of enhanced wound healing by copper oxide-impregnated dressings.

ABSTRACT Copper plays a key role in angiogenesis and in the synthesis and stabilization of extracellular matrix skin proteins, which are critical processes of skin formation. We hypothesized that introducing copper into wound dressings would enhance wound repair. Application of wound dressings containing copper oxide to wounds inflicted in genetically engineered diabetic mice (C57BL/KsOlaHsd-Lepr(db)) resulted in increased gene and in situ up-regulation of proangiogenic factors (e.g., placental growth factor, hypoxia-inducible factor-1 alpha, and vascular endothelial growth factor), increased blood vessel formation (p<0.05), and enhanced wound closure (p<0.01) as compared with control dressings (without copper) or commercial wound dressings containing silver. This study proves the capacity of copper oxide-containing wound dressings to enhance wound healing and sheds light onto the molecular mechanisms by which copper oxide-impregnated dressings stimulate wound healing.

Copper oxide impregnated wound dressing: biocidal and safety studies.

 Copper plays a key role in angiogenesis and in the expression and stabilization of extracellular skin proteins. Copper also exhibits broad biocidal properties. The authors hypothesized that introducing copper into a wound dressing would not only reduce the risk of wound and dressing contamination, but would also stimulate wound repair. To test this hypothesis, non-stick dressings composed of a highly absorbent internal mesh fabric and an external non-woven fabric were fabricated, and each was impregnated with ~2.65% (weight/weight) copper oxide particles. The application to wounds inflicted in genetically engineered diabetic mice resulted in increased gene and in-situ upregulation of proangiogenic factors, increased blood vessel formation, and enhanced wound closure. The present study reports both the potent broad spectrum antimicrobial and antifungal properties of these wound dressings and the lack of adverse reactions as determined in rabbits and a porcine wound model. The prolonged efficacy of the wound dressing is demonstrated by its capacity to reduce the microbial challenge by more than 99.9% even when spiked 5 consecutive times with a high bacterial titer. The dressing's antimicrobial efficacy is exerted within minutes. The dressing did not cause any skin irritation or sensitization to closed skin. Furthermore, no histological differences were found between open wounds exposed to copper oxide containing wound dressings or control dressings. Therefore, copper containing wound dressings hold significant promise in wound healing and their clinical use should be explored .

Increased pro-collagen 1, elastin, and TGF-ß1 expression by copper ions in an ex-vivo human skin model.

BACKGROUND: Clinical studies demonstrated that continued exposure to copper oxide-embedded textiles, such as pillowcases, significantly reduces depth of facial wrinkles and skin sagging and enhances skin elasticity. OBJECTIVE: Study the mechanisms by which the exposure to copper ions improve the well-being of the skin. METHODS: Human skin explants, cultured ex-vivo, were exposed topically to saline alone or saline containing 0.02 or 1 µmol/L copper ions. The skin explants viability, histology and secretion of elastin, pro-collagen 1, and TGF-ß1 to the culture medium were determined at various time intervals. RESULTS: Exposure to saline containing 0.02 or 1 µmol/L copper ions did not affect the viability or morphological profile of the explants as compared to control explants treated with saline only. Notably, exposure of the skin grafts to 0.02 or to 1 µmol/L of copper ions resulted in ~100% and ~20% increases in elastin and pro-collagen 1 concentrations, respectively, in the culture supernatants already after 1 day of incubation, which remained statistically significantly elevated also after 6 days on incubation, as compared to the control explants. In addition, ~2- and ~4-fold increases in TGF-ß1 levels in the culture supernatants of explants exposed to the copper ions were detected after 4 and 6 days of culture, as compared to the explants exposed to saline alone. CONCLUSION: This study substantiated the anti-aging effect that copper ions have on the skin and gave insights into the mechanisms by which exposure of the skin to copper ions improves the skin well-being.

NeoR6 inhibits HIV-1-CXCR4 interaction without affecting CXCL12 chemotaxis activity.

Aminoglycoside-arginine conjugates (AACs) are multi-target HIV-1 inhibitors. The most potent AAC is neomycin hexa-arginine conjugate, NeoR6. We here demonstrate that NeoR6 interacts with CXCR4 without affecting CXCL12-CXCR4 ordinary chemotaxis activity or loss of CXCR4 cell surface expression. Importantly, NeoR6 alone does not affect cell migration, indicating that NeoR6 interacts with CXCR4 at a distinct site that is important for HIV-1 entry and mAb 12G5 binding, but not to CXCL12 binding or signaling sites. This is further supported by our modeling studies, showing that NeoR6 and CXCL12 bind to two distinct sites on CXCR4, in contrast with other CXCR4 inhibitors, e.g. T140 and AMD3100. This complementary utilization of chemical, biology, and computation analysis provides a powerful approach for designing anti-HIV-1 drugs without interfering with the natural function of CXCL12/CXCR4 binding.

Chronic parasite infections cause immune changes that could affect successful vaccination.

Several important issues must be considered when performing any vaccination in areas with high prevalence of geohelminths. Immunization of populations infected with geohelminths could be sub-optimal if the immune background is not taken into consideration. Immune modulation and deworming might be essential for effective protective vaccination. In addition, further animal models and clinical studies addressing these issues are required. Underscoring the importance of these issues, a recent report has highlighted several vaccination studies in which nematode-infected mice or pigs failed to mount efficient protective immune responses.

Biocidal textiles can help fight nosocomial infections.

The rates of nosocomial infections, especially by those caused by antibiotic resistant bacteria, are increasing alarmingly over the globe. Although more rigorous infection control measures are being implemented, it is clear that the current modalities to reduce nosocomial infections are not sufficient. Textiles are an excellent substrate for bacterial growth under appropriate moisture and temperature conditions. Patients shed bacteria and contaminate their pyjamas and sheets. The temperature and humidity between the patients and the bed are appropriate conditions allowing for effective bacterial proliferation. Several studies have found that personnel in contact with contaminated textiles were the source of transmission of the micro-organisms to susceptible patients. Furthermore, it has been reported that bed making in hospitals releases large quantities of micro-organisms into the air, which contaminate the immediate and non-immediate surroundings. Contaminated textiles in hospitals can thus be an important source of microbes contributing to endogenous, indirect-contact, and aerosol transmission of nosocomial related pathogens. We hypothesize that the use of antimicrobial textiles, especially in those textiles that are in close contact with the patients, may significantly reduce bioburden in clinical settings and consequently reduce the risk of nosocomial infections. These textiles should possess broad spectrum biocidal properties. They should be safe for use and highly effective against antibiotic resistant micro-organisms, including those that are commonly involved in hospital-acquired infections, and they should not permit the development of resistant micro-organisms to the active compound.

Could chronic wounds not heal due to too low local copper levels?

Copper is an essential trace element involved in numerous human physiological and metabolic processes. It plays a key role in many of the processes that together comprise wound healing, including induction of endothelial growth factor, angiogenesis and expression and stabilization of extracellular skin proteins. We hypothesize that in individuals with diabetic ulcers, decubitus, peripheral vascular, or other wounds which might have compromised circulation to the wound site, that part of the incapacity of the wounds to heal is due to low local copper levels. Contamination of wounds is also an important factor causing impaired wound healing. Importantly, copper has potent broad biocidal properties. In contrast, the risk of adverse skin reactions due to exposure to copper is extremely low. We thus hypothesize that introducing copper into wound dressings would not only reduce the risk of wound and dressing contamination, as silver does but, more importantly, would stimulate faster wound repair directly. This would be done by the release of copper from the wound dressings directly into the wound site inducing angiogenesis and skin regeneration.

Structure-function relationship of novel X4 HIV-1 entry inhibitors - L- and D-arginine peptide-aminoglycoside conjugates.

We present the design, synthesis, anti-HIV-1 and mode of action of neomycin and neamine conjugated at specific sites to arginine 6- and 9-mers D- and L-arginine peptides (APACs). The d-APACs inhibit the infectivity of X4 HIV-1 strains by one or two orders of magnitude more potently than their respective L-APACs. D-arginine conjugates exhibit significantly higher affinity towards CXC chemokine receptor type 4 (CXCR4) than their L-arginine analogs, as determined by their inhibition of monoclonal anti-CXCR4 mAb 12G5 binding to cells and of stromal cell-derived factor 1alpha (SDF-1alpha)/CXCL12 induced cell migration. These results indicate that APACs inhibit X4 HIV-1 cell entry by interacting with CXCR4 residues common to glycoprotein 120 and monoclonal anti-CXCR4 mAb 12G5 binding. D-APACs readily concentrate in the nucleus, whereas the l-APACs do not. 9-mer-D-arginine analogues are more efficient inhibitors than the 6-mer-D-arginine conjugates and the neomycin-D-polymers are better inhibitors than their respective neamine conjugates. This and further structure-function studies of APACs may provide new target(s) and lead compound(s) of more potent HIV-1 cell entry inhibitors.

Reduction of health care-associated infection indicators by copper oxide-impregnated textiles: Crossover, double-blind controlled study in chronic ventilator-dependent patients.

BACKGROUND: Copper oxide has potent wide-spectrum biocidal properties. The purpose of this study is to determine if replacing hospital textiles with copper oxide-impregnated textiles reduces the following health care-associated infection (HAI) indicators: antibiotic treatment initiation events (ATIEs), fever days, and antibiotic usage in hospitalized chronic ventilator-dependent patients. METHODS: A 7-month, crossover, double-blind controlled trial including all patients in 2 ventilator-dependent wards in a long-term care hospital. For 3 months (period 1), one ward received copper oxide-impregnated textiles and the other received untreated textiles. After a 1-month washout period of using regular textiles, for 3 months (period 2) the ward that received the treated textiles received the control textiles and vice versa. The personnel were blinded to which were treated or control textiles. There were no differences in infection control measures during the study. RESULTS: There were reductions of 29.3% (P = .002), 55.5% (P < .0001), 23.0% (P < .0001), and 27.5% (P < .0001) in the ATIEs, fever days (>37.6°C), days of antibiotic treatment, and antibiotic defined daily dose per 1,000 hospitalization days, respectively, when using the copper oxide-impregnated textiles. CONCLUSIONS: Use of copper oxide-impregnated biocidal textiles in a long-term care ward of ventilator-dependent patients was associated with a significant reduction of HAI indicators and antibiotic utilization. Using copper oxide-impregnated biocidal textiles may be an important measure aimed at reducing HAIs in long-term care medical settings.

A novel small animal model for HIV-1 infection.

Lethally irradiated normal BALB/c mice, reconstituted with murine SCID bone marrow and engrafted with human PBMC (Trimera mice), were used to establish a novel murine model for HIV-1 infection. The Trimera mice were successfully infected with different clades and primary isolates of T- and M-tropic HIV-1, with the infection persisting in the animals for 4-6 wk. Rapid loss of the human CD4+ T cells, decrease in CD4/CD8 ratio, and increased T cell activation accompanied the viral infection. All HIV-1 infected animals were able to generate both primary and secondary immune responses, including HIV specific human humoral and cellular responses. In addition to testing the efficacy of new antiviral compounds, this new murine HIV-1 model may be used for studying host-virus interactions and, most importantly, for screening and developing potential HIV-1 protective vaccines and adjuvants (Ayash-Rashkovsky et al., http://www.fasebj.org/cgi/doi/10.1096/fj.04-3185fje; doi:10.1096/fj.04-3185fje.).

Enhanced HIV-1 specific immune response by CpG ODN and HIV-1 immunogen-pulsed dendritic cells confers protection in the Trimera murine model of HIV-1 infection.

We have recently developed a novel small animal model for HIV-1 infection (Ayash-Rashkovsky et al., http://www.fasebj.org/cgi/doi/10.1096/fj.04-3184fje; doi:10.1096/fj.04-3184fje). The mice were successfully infected with HIV-1 for 4-6 wk with different clades of either T- or M-tropic isolates. HIV-1 infection was accompanied by rapid loss of human CD4+ T cells, decrease in CD4/CD8 ratio, and increased T cell activation. HIV specific human humoral and cellular immune responses were observed in all HIV-1 infected animals. In the present study, HIV specific human immune responses, both humoral and cellular, were generated in noninfected Trimera mice, after their immunization with gp120-depleted HIV-1 antigen, presented by autologous human dendritic cells. Addition of CpG ODN to the antigen-pulsed DCs significantly enhanced (by 2- to 30-fold) the humoral and cellular HIV-1 specific immune responses. Only mice immunized with the HIV-1 immunogen and CpG were completely protected from infection with HIV-1 after challenge with high infection titers of the virus. This novel small animal model for HIV-1 infection may thus serve as an attractive platform for rapid testing of candidate HIV-1 vaccines and of adjuvants and may shorten the time needed for the development and final assessment of protective HIV-1 vaccines in human trials.

TLR9 expression is related to immune activation but is impaired in individuals with chronic immune activation.

Millions of individuals in developing countries are infected with helminths and other chronic infectious diseases, such as HIV-1, which lead to persistent immune activation and unbalanced immune state. We have suggested that the capacity of chronically immune activated individuals to protect themselves, cope with infections, and mount protective immunity following vaccination, is highly impaired. Here we examined the expression of toll-like receptor 9 (TLR9), as an essential component in the recognition of immunostimulating bacterial CpG-DNA motifs, in different subsets of human peripheral blood mononuclear cells (PBMC) obtained from chronically immune activated and non-activated individuals. TLR9 expression was correlated to immune cell activation and was upregulated following phytohemagglutinin or anti-CD3 activation. PBMC obtained from chronically immune activated individuals had a different overall pattern of TLR9 expression, including reduced upregulation of this receptor following additional immune activation, and diminished responsiveness to CpG-DNA stimulation, in comparison to non-activated individuals. These differences may partly account for the reduced capacity of chronically immune activated individuals to mount effective immune responses and strengthen the notion that the host immune background should be considered in the design and trial of potential adjuvants and vaccines.