Effects of Streptolysin O On Extracellular Matrix Gene Expression in Normal Human Epidermal Keratinocytes

ML-05 is a non-hemolytic form of streptolysin O, the membrane-damaging extracellular toxin produced by certain streptococci. ML-05 stimulates keratinocyte migration and proliferation in wound-healing scratch assays and promotes wound healing in a human skin organ culture wound model. Pathway-focused DNA microarrays were used to elucidate ML-05’s mechanism of action in wound healing processes. Normal human epidermal keratinocytes (NHEK) were treated with varying concentrations of ML-05 for 24 hours, followed by RNA extraction and cRNA production. Gene expression profiling utilized microarrays containing nucleic acid probes for 113 extracellular matrix (ECM) genes. Microarrays yielded 6 upregulated and 4 downregulated genes with ≥2-fold changes and p<0.05 in t-tests. Quantitative real-time polymerase chain reactions (qPCR) were used to verify gene regulation. Upregulated genes of interest were VCAN (formerly CSPG2, encoding versican), CD44 (encoding hyaluronan receptor), ICAM1 (encoding intercellular adhesion molecule-1) and CTGF (encoding connective tissue growth factor). All four upregulated genes encode proteins involved in promoting keratinocyte migration and proliferation. Downregulated genes of interest were MMP9 (encoding matrix metalloproteinase 9) and SPP1 (encoding osteopontin). ML-05 may enhance wound healing through the expression of specific genes encoding proteins capable of promoting keratinocyte migration, proliferation, and other activities related to maintaining ECM structure and function

Streptolysin Effects on Keratinocyte ECM Gene Expression

ML-05 is a non-hemolytic form of streptolysin O, the membrane-damaging extracellular toxin produced by certain streptococci. ML-05 stimulates keratinocyte migration and proliferation in wound-healing scratch assays and promotes wound healing in a human skin organ culture wound model. Pathway-focused DNA microarrays were used to elucidate ML-05’s mechanism of action in wound healing processes. Normal human epidermal keratinocytes (NHEK) were treated with varying concentrations of ML-05 for 24 hours, followed by RNA extraction and cRNA production. Gene expression profiling utilized microarrays containing nucleic acid probes for 113 extracellular matrix (ECM) genes. Microarrays yielded 6 upregulated and 4 downregulated genes with ≥2-fold changes and p<0.05 in t-tests. Quantitative real-time polymerase chain reactions (qPCR) were used to verify gene regulation. Upregulated genes of interest were VCAN (formerly CSPG2, encoding versican), CD44 (encoding hyaluronan receptor), ICAM1 (encoding intercellular adhesion molecule-1) and CTGF (encoding connective tissue growth factor). All four upregulated genes encode proteins involved in promoting keratinocyte migration and proliferation. Downregulated genes of interest were MMP9 (encoding matrix metalloproteinase 9) and SPP1 (encoding osteopontin). ML-05 may enhance wound healing through the expression of specific genes encoding proteins capable of promoting keratinocyte migration, proliferation, and other activities related to maintaining ECM structure and function

Streptolysin O enhances keratinocyte migration and proliferation and promotes skin organ culture wound healing in vitro

ML-05, a modified form of the hemolytic and cytotoxic bacterial toxin, streptolysin O, is currently being investigated as a treatment for collagen-related disorders such as scleroderma and fibrosis. Furthermore, ML-05 may be effective in promoting wound healing and alleviating the formation of hypertrophic scars and keloids. To investigate the effects of ML-05 on wound-healing processes, in vitro wound-healing scratch assays (using human primary epidermal keratinocytes and dermal fibroblasts) and a human skin organ culture wound model were utilized. ML-05 markedly enhanced keratinocyte migration and proliferation in wound scratch assays. ML-05 did not affect either proliferation or migration of dermal fibroblasts, indicating that ML-05's effects on cell migration/proliferation may be keratinocyte-specific. ML-05 was tested in a dose-dependent manner in a skin organ culture wound model using two different application methods: Through the culture media (dermal exposure) or direct topical treatment of the wound surface. ML-05 was found to accelerate wound healing as measured by reepithelialization, particularly after topical application. Therefore, ML-05 may have potential as a wound-healing agent that promotes reepithelialization through stimulation of keratinocyte migration and proliferation

The Use of Streptolysin O for the Treatment of Scars, Adhesions and Fibrosis: Initial Investigations Using Murine Models of Scleroderma

Diseases and conditions involving the deposition of excessive amounts of collagen include scleroderma, fibrosis, and scar and surgical adhesion formation. Diseases such as scleroderma may result from acute and chronic inflammation, disturbances in the normal parenchymal area, and activation of fibroblasts. ML-05, a modified form of the hemolytic and cytotoxic bacterial toxin, streptolysin O, is being developed for the treatment of such collagen-related disorders. At sublytic concentrations in vitro, ML-05 was shown to activate CD44 expression. This may modulate production of collagen, hyaluronate, and their associated enzymes to allow a restoration of normal extracellular matrices within tissues. More importantly, ML-05 appeared to decrease skin collagen levels in two in vivo models of collagen disorders, the tight skin mouse (Tsk) model of scleroderma, and the bleomycin-induced mouse skin fibrosis model. In the Tsk model, levels of hydroxyproline (a measure of total collagen) decreased by 25% in the Tsk+ML-05 treatment group relative to the Tsk+saline control group over a 3-month period. In the bleomycin-induced skin fibrosis study, hydroxyproline levels decreased from 15–22% over a 6-week period in a bleomycin-induced ML-05 treatment group (relative to levels in a bleomycin-induced, untreated control group). Hydroxyproline levels in samples from this treatment group were only slightly greater than levels in an uninduced control group at 8 weeks. Thus, ML-05 treatment appeared to reduce collagen levels in two separate mouse skin fibrosis models, one genetically based and the other chemically induced

A Comparative Study on Antimicrobial Resistance in Escherichia coli Isolated from Channel Catfish and Related Freshwater Fish Species

Antimicrobial resistance (AMR) trends in 114 generic Escherichia coli isolated from channel catfish and related fish species were investigated in this study. Of these, 45 isolates were from commercial-sized channel catfish harvested from fishponds in Alabama, while 69 isolates were from Siluriformes products, accessed from the U.S. Department of Agriculture Food Safety and Inspection Service’ (FSIS) National Antimicrobial Resistance Monitoring System (NARMS) program. Antibiotic susceptibility testing and whole genome sequencing were performed using the GenomeTrakr protocol. Upon analysis, the fishpond isolates showed resistance to ampicillin (44%), meropenem (7%) and azithromycin (4%). The FSIS NARMS isolates showed resistance to tetracycline (31.9%), chloramphenicol (20.3%), sulfisoxazole (17.4%), ampicillin (5.8%) and trimethoprim-sulfamethoxazole, nalidixic acid, amoxicillin-clavulanic acid, azithromycin and cefoxitin below 5% each. There was no correlation between genotypic and phenotypic resistance in the fishpond isolates, however, there was in NARMS isolates for folate pathway antagonists: Sulfisoxazole vs. sul1 and sul2 (p = 0.0042 and p < 0.0001, respectively) and trimethoprim-sulfamethoxazole vs. dfrA16 and sul1 (p = 0.0290 and p = 0.013, respectively). Furthermore, correlations were found for tetracyclines: Tetracycline vs. tet(A) and tet(B) (p < 0.0001 each), macrolides: Azithromycin vs. mph(E) and msr(E) (p = 0.0145 each), phenicols: Chloramphenicol vs. mdtM (p < 0.0001), quinolones: Nalidixic acid vs. gyrA_S83L=POINT (p = 0.0004), and β-lactams: Ampicillin vs. blaTEM-1 (p < 0.0001). Overall, we recorded differences in antimicrobial susceptibility testing profiles, phenotypic-genotypic concordance, and resistance to critically important antimicrobials, which may be a public health concern