Phosphate-buffered Saline Control Research Articles

Effect of Ceftaroline, Ceftazidime/Avibactam, Ceftolozane/Tazobactam, and Meropenem/Vaborbactam on Establishment of Colonization by Vancomycin-Resistant Enterococci and Klebsiella pneumoniae in Mice.

The potential for promotion of intestinal colonization with healthcare-associated pathogens by new antibiotics used to treat infections due to multidrug-resistant Gram-negative bacilli is unclear. Mice treated for 3 days with daily subcutaneous phosphate-buffered saline (control), ceftazidime/avibactam, ceftolozane/tazobactam, ceftaroline, and meropenem/vaborbactam were challenged with 10,000 colony-forming units (CFU) of vancomycin-resistant Enterococcus (VRE) resistant to each of the antibioics or carbapenemase-producing Klebsiella pneumoniae 1 day after the final treatment dose. The concentrations of VRE or K. pneumoniae in stool were measured on days 1, 3, 6, and 15 after challenge. Control mice had transient low levels of VRE or K. pneumoniae (<3 log10 CFU/g) detected in stool with negative cultures on days 6 and 15 after challenge. In comparison to control mice, each of the antibiotics promoted establishment of high-density colonization with VRE (mean concentration, >8 log10 CFU/g of stool on day 1 after challenge) that persisted at >4 log10 CFU/g of stool through day 15 (P<0.01). In comparison to control mice, meropenem/vaborbactam and ceftaroline promoted high-density colonization with K. pneumoniae (peak concentration, >8 log10 CFU/g of stool) (P<0.01), ceftolozane/tazobactam promoted colonization to a lesser degree (peak concentration, >5 log10 CFU/g of stool), and ceftazidime/avibactam did not promote colonization (P>0.05). Our results suggest that several beta-lactam antibiotics recently developed for treatment of infections with resistant Gram-negative bacilli have the potential to promote colonization by healthcare-associated pathogens. Additional studies are needed to examine the impact of these agents in patients.

C-di-GMP@ZIF-8 nanocomposite injectable hydrogel based on modified chitosan and hyaluronic acid for infected wound healing by activating STING signaling

The treatment of infected wounds relies on antibiotics; however, increasing drug resistance has made therapeutic processes more difficult. Activating self-innate immune abilities may provide a promising alternative to treat wounds with bacterial infections. In this work, we constructed an immunogenic injectable hydrogel crosslinked by the Schiff base reaction of carboxymethyl chitosan (NOCC) and aldehyde hyaluronic acid (AHA) and encapsulated with stimulator of interferon genes (STING) agonist c-di-GMP loaded ZIF-8 nanoparticles (c-di-GMP@ZIF-8). Nanocubic ZIF-8 was screened as the most efficient intracellular drug delivery vector from five differently-shaped morphologies. The NOCC/AHA hydrogel released c-di-GMP@ZIF-8 more quickly (43 %) in acidic environment (pH = 5.5) of infected wounds compared with 34 % in non-infected wound environment (pH = 7.4) at 96 h due to pH-responsive degradation performance. The released c-di-GMP@ZIF-8 was found to activate the STING signaling of macrophages and enhance the secretion of IFN-β, CCL2, and CXCL12 5.8–7.6 times compared with phosphate buffer saline control, which effectively inhibited S. aureus growth and promoted fibroblast migration. In rat models with infected wounds, the c-di-GMP@ZIF-8 nanocomposite hydrogels improved infected wound healing by promoting granulation tissue regeneration, alleviating S. aureus-induced inflammation, and improving angiogenesis. Altogether, this study demonstrated a feasible strategy using STING-targeted and pH-responsive hydrogels for infected wound management.

Antiviral Effects of Ozonated Water for Handwashing Against SARS-CoV-2 Omicron Variant Using the E1052-20 Test Method of the American Society of Testing Materials International

ABSTRACT Alcohol-based hand rubs are generally recommended for infection control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Hand hygiene agents, including ozonated water (OW), have been used as an alternative because alcohol-based products may cause skin damage. A few reports are available on the virucidal activity of OW against SARS-CoV-2; therefore, this study examined the inhibitory effect of 0.4, 1.0, and 4.0 ppm OW on the Omicron variant infection to VeroE6 cells expressing transmembrane protease serine 2 by calculating the median tissue culture infectious dose (TCID50) using the E1052–20 test method of the American Society of Testing Materials International. Additionally, viral RNA copies were measured in culture supernatants of control or OW. Each OW concentration demonstrated ≥5.6 log10 TCID50 reduction, corresponding to ≥99.999% reduction of infectious virus, while the control phosphate-buffered saline showed no inactivation effect. More than 5 log10 copies/mL reduction of viral RNA was observed in the culture supernatant of each OW compared with the control. These findings suggest that exposure of SARS-CoV-2 to at least 0.4 ppm OW for 5 s is sufficient for viral inactivation and that OW can be used as an alternative hand hygiene agent to prevent SARS-CoV-2 infection.

Ameliorating effects of selenium nanoparticle coated by gallic acid on histological and biochemical parameters of testis in azoospermic rat model

This study was designed to examine the effects of selenium nanoparticles (SeNPs) coated with gallic acid (GA) on testis in azoospermic rats. Thirty-six adult Wistar rats were assigned to six groups: control (1 ml intraperitoneal (i.p.) phosphate-buffered saline (PBS) for 7 consecutive days), SHAM (single i.p. injection of 1 ml of 8 % dimethyl sulfoxide (DMSO)), BUS (single i.p. injection of busulfan (BUS) 30 mg/kg body weight), GA (single i.p. injection of BUS 30 mg/kg on day 1, 100 mg/kg body weight GA from days 2–7), SeNPs (single i.p. injection of BUS 30 mg/kg on day 1, 0.5 mg/kg body weight SeNPs from days 2–7), and SeNPs-GA (single i.p. injection of BUS 30 mg/kg on day 1, 0.5 mg/kg body weight SeNPs-GA from days 2–7). Subsequently, serum levels of testosterone and insulin-like growth factor-1 (IGF-1), antioxidant markers, sperm parameters, and histological parameters were evaluated. The results showed that BUS injection induced azoospermia in rats by causing oxidative stress and testicular tissue damage. In contrast, co-administration of SeNPs and GA showed significant improvements in testosterone and IGF-1 levels, antioxidant status, testicular tissue characteristics, and sperm parameters. Overall, the findings suggest that GA-coated SeNPs offer therapeutic potential in BUS-induced azoospermic models.

Abstract 1795: Tumor-targeting Salmonella A1-R selectively delivers recombinant methioninase and inhibits syngeneic-cancer mouse models

Abstract Background: Methionine addiction, termed the Hoffman effect, is a general hallmark of cancer. Methionine restriction (MR) using an MR diet or recombinant methioninase (rMETase) is effective for all types of cancer. However, methionine restriction also inhibits the activity of CD8-positive T-lymphocytes. Therefore, we hypothesized that methionine depletion in the tumor only may be more effective for cancer therapy. We previously developed Salmonella A1-R, which selectively targets and kills tumors. In the present study, we established rMETase-producing Salmonella A1-R, by transfer of the Pseudomonas putida methioninase gene, to target and inhibit syngeneic cancer mouse models. Methods: A plasmid containing the Pseudomonas putida methioninase gene was extracted from rMETase-producing E. coli and inserted into Salmonella A1-R using electroporation. The rMETase-producing Salmonella A1-R (A1-R-rMETase) infected several cancer cell lines in vitro, including HT29, PC-3, MDA-MB-435, and Lewis Lung Carcinoma (LLC). LLC was chosen for in vivo studies as it supported extensive growth of A1-R-rMETase. We determined 108 A1-R-rMETase was a safe dosage in C57BL/6 mice. LLC cells (106) were injected in male C57BL/6 mice aged 4-6 weeks subcutaneously. After tumor growth, 18 mice were divided into three groups of 6. One group was injected with phosphate-buffered saline (PBS) via the tail vein, twice per week as a control. Another group was injected with 108 Salmonella A1-R via the tail vein, twice per week. Another group was injected with 108 A1-R-rMETase via the tail vein, twice per week for two weeks. Tumor size was measured with calipers three times per week for 3 weeks. On day 22, tumor methionine level was measured using HPLC in the PBS control and the mice injected with A1-R-rMETase. Results: The mean LLC tumor size of each group on day 22 was as follows: The PBS control: 741.5 mm3; mice injected with A1-R: 566.3 mm3; and mice injected with A1-R-rMETase: 198.8 mm3. Turkey’s multiple comparisons test showed a significant difference between the PBS control and mice injected with A1-R-rMETase (p&amp;lt;0.0001) and between mice injected with A1-R and mice injected with A1-R-rMETase (p=0.0036). However, the PBS control and the mice injected with A1-R did not show a significant difference (p=0.1794). The mice injected with A1-R-rMETase showed a significantly lower mean methionine level than mice injected with PBS (5.9 nM/mg protein vs. 11.1 nM/mg protein, p=0.0095, Mann Whitney test). Conclusion: Tumor-targeting Salmonella A1-R modified to express the Pseudomonas putida methioninase gene (A1-R-rMETase), inhibited LLC tumor growth in a syngeneic mouse model and reduced the methionine level in the tumor. A1-R-rMETase combines the tumor targeting and killing capability of A1-R itself and restriction methionine selectively in tumors. Citation Format: Yutaro Kubota, Yusuke Aoki, Noriyuki Masaki, Koya Obara, Sei Morinaga, Kohei Mizuta, Motokazu Sato, Ming Zhao, Qinghong Han, Bouvet Michael, Takuya Tsunoda, Robert M. Hoffman. Tumor-targeting Salmonella A1-R selectively delivers recombinant methioninase and inhibits syngeneic-cancer mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1795.

BMP4 and GREM1 are targets of SHH signaling and downstream regulators of collagen in the penis.

Cavernous nerve (CN) injury, caused by prostatectomy and diabetes, initiates a remodeling process (smooth muscle apoptosis and increased collagen) in the corpora cavernosa of the penis of patients and animal models that is an underlying cause of erectile dysfunction (ED), and the Sonic hedgehog (SHH) pathway plays an essential role in the response of the penis to denervation, as collagen increases with SHH inhibition and decreases with SHH treatment. We examined if part of the mechanism of how SHH prevents penile remodeling and increased collagen with CN injury involves bone morphogenetic protein 4 (BMP4) and gremlin1 (GREM1) and examined the relationship between SHH, BMP4, GREM1, and collagen in penis of ED patients and rat models of CN injury, SHH inhibition, and SHH, BMP4, and GREM1 treatment. Corpora cavernosa of Peyronie's disease (control), prostatectomy, and diabetic ED patients were obtained (N= 30). Adult Sprague Dawley rats (n = 90) underwent (1) CN crush (1-7days) or sham surgery; (2) CN injury and BMP4, GREM1, or mouse serum albumin (control) treatment via Affi-Gel beads or peptide amphiphile (PA) for 14days; (3) 5E1 SHH inhibitor, IgG, or phosphate-buffered saline (control) treatment for 2 to 4days; or (4) CN crush with mouse serum albumin or SHH for 9days. Immunohistochemical and Western analysis for BMP4 and GREM1, and collagen analysis by hydroxyproline and trichrome stain were performed. BMP4 and GREM1 proteins were identified in corpora cavernosa smooth muscle of prostatectomy, diabetic, and Peyronie's patients, and in rat smooth muscle, sympathetic nerve fibers, perineurium, blood vessels, and urethra. Collagen decreased 25.4% in rats with CN injury and BMP4 treatment (P = .02) and increased 61.3% with CN injury and GREM1 treatment (P = .005). Trichrome stain showed increased collagen in rats treated with GREM1. Western analysis identified increased BMP4 and GREM1 in corpora cavernosa of prostatectomy and diabetic patients, and after CN injury (1-2days) in our rat model. Localization of BMP4 and GREM1 changed with SHH inhibition. SHH treatment increased the monomer form of BMP4 and GREM1, altering their range of signaling. A better understanding of penile remodeling and how fibrosis occurs with loss of innervation is essential for development of novel ED therapies. The relationship between SHH, BMP4, GREM1, and collagen is complex in the penis. BMP4 and GREM1 are downstream targets of SHH that impact collagen and may be useful in collaboration with SHH to prevent penile remodeling and ED.

Comparative evaluation of rhFGF18 and rhGDF11 treatment in a transient ischemia stroke model.

Pharmacological treatments for ischemic stroke remain limited to thrombolysis, which is associated with increased risk of potentially fatal hemorrhage. Treatments with Recombinant Human Fibroblast Growth Factor 18 (rhFGF18) and Growth and Differentiation Factor 11 (rhGDF11) appear promising based on different preclinical models. The goal of this study was to compare the effects of rhFGF18 and rhGDF11 directly on survival, behavioral deficits, and histological fingerprint of cerebral ischemia in the Wistar rat middle cerebral artery occlusion (MCAO) model of stroke. Ischemia-reperfusion injury was induced using a 2-hour transient MCAO. Animals were administered rhFGF18 (infusion), rhGDF11 (multi-injection), or Phosphate Buffered Saline (PBS) vehicle control and followed for 42 days. Motor-Cognitive deficits were evaluated using the Morris Water Maze at Days 0 (pre-MCAO), 7, 21, and 42. Histopathological assessments were performed on Days 21 and 42. Day 7 post-ischemia water maze performance times increased 38.3%, 2.1%, and 23.1% for PBS, rhFGF18, and rhGDF11-treated groups, respectively. Fraction of neurons with abnormal morphology (chromatolysis, pyknotic nuclei, somal degeneration) decreased in all groups toward Day 42 and was lowest for rhFGF18. AChE-positive fiber density and activity increased over time in the rhFGF18 group, remained unchanged in the rhGDF11 treatment arm, and declined in the PBS control. Metabolic increases were greatest in rhGDF11 treated animals, with both rhFGF18 and rhGDF11 achieving improvements over PBS, as evidenced by increased succinate dehydrogenase and lactate dehydrogenase activity. Finally, rhFGF18 treatment exhibited a trend for reduced mortality relative to PBS (5.6%, 95% CI [27.3%, 0.1% ] vs. 22.2%, 95% CI [47.6%, 6.4% ]). rhFGF18 treatment appears promising in improving survival and promoting motor-cognitive recovery following cerebral ischemia-reperfusion injury.

In-Ovo Glutamine Administration Enhances Intestinal Development and Functions in Broiler Chickens: Insights from Enteroid Models

BackgroundEarly life events play significant roles in tissue development and animal health in their later life. Early nutrition, through in-ovo delivery, has shown beneficial effects on improving intestinal health in broiler chickens. However, the underlying mechanism is not fully investigated. A recently developed enteroid culture technique allows investigations on intestinal epithelial functions that are close to physiologic conditions. ObjectivesIn this study, we evaluated the short- and long-term effects of in-ovo administration of glutamine (Gln) on intestinal epithelial development and functions by using intestinal enteroid culture and tissue electrophysiologic analysis. MethodsA hundred eggs of commercial Cobb500 broilers were in-ovo injected with 0.2 mL of either phosphate-buffered saline (PBS) or 3% Gln at embryonic day 18 (E18). Chicks were killed on the day of hatch, and at 3- and 14-d posthatch. Enteroids were generated from the small intestine. After 4 d of culture, enteroids were harvested for 5-ethynyl-2′-deoxyuridine proliferation, fluorescein isothiocyanate-4 kDa dextran permeability, and glucose absorption assays. At day 3 (d3) and day 14 (d14), intestinal barrier and nutrient transport functions were measured by the Ussing chamber. The gene expression of epithelial cell markers, nutrient transporters, and tight-junction proteins were analyzed in both intestinal tissues and enteroids. ResultsIn comparison with the PBS control group, in-ovo Gln increased intestinal villus morphology, epithelial cell proliferation, and differentiation, and altered epithelial cell population toward increased number of enteroendocrine and goblet cells while decreasing Paneth cells. Enteroids gene expression of nutrient transporters (B0AT1, SGLT1, and EAAT3), tight junction (ZO2), glucose absorption, and barrier functions were enhanced on the day of hatch. Long-term increases of intestinal di-peptide and alanine transport were observed at day 14 posthatch. ConclusionsTogether our results suggested that the in-ovo injection of Gln stimulated intestinal epithelium proliferation and programmed the epithelial cell differentiation toward absorptive cells.

Electrolyzed Saline Targets Biofilm Periodontal Pathogens In Vitro

Preventing the development and recurrence of periodontal diseases often includes antimicrobial mouthrinses to control the growth of the periodontal pathogens. Most antimicrobials are nonselective, targeting the symbiotic oral species as well as the dysbiosis-inducing ones. This affects the overall microbial composition and metabolic activity and consequently the host–microbe interactions, which can be detrimental (associated with inflammation) or beneficial (health-associated). Consequently, guiding the antimicrobial effect for modulating the microbial composition to a health-associated one should be considered. For such an approach, this study investigated electrolyzed saline as a novel rinse. Electrolyzed saline was prepared from sterile saline using a portable electrolysis device. Multispecies oral homeostatic and dysbiotic biofilms were grown on hydroxyapatite discs and rinsed daily with electrolyzed saline (EOS). Corresponding positive (NaOCl) and negative (phosphate-buffered saline) controls were included. After 3 rinses, biofilms were analyzed with viability quantitative polymerase chain reaction and scanning electron microscopy. Supernatants of rinsed biofilms were used for metabolic activity analysis (high-performance liquid chromatography) through measuring organic acid content. In addition, human oral keratinocytes (HOKs) were exposed to EOS to test biocompatibility (cytotoxicity and inflammation induction) and also to rinsed biofilms to assess their immunogenicity after rinsing. Rinsing the dysbiotic biofilms with EOS could reduce the counts of the pathobionts (>3 log10 Geq/mm2 reduction) and avert biofilm dysbiosis (≤1% pathobiont abundance), leading to the dominance of commensal species (≥99%), which altered both biofilm metabolism and interleukin 8 (IL-8) induction in HOKs. EOS had no harmful effects on homeostatic biofilms. The scanning electron micrographs confirmed the same. In addition, tested concentrations of EOS did not have any cytotoxic effects and did not induce IL-8 production in HOKs. EOS showed promising results for diverting dysbiosis in in vitro rinsed biofilms and controlling key periopathogens, with no toxic effects on commensal species or human cells. This novel rinsing should be considered for clinical applications.

Antibacterial effects of dentifrices against Streptococcus mutans in children: a comparative in vitro study.

Fluoridated dentifrices have antibacterial effects on children's teeth. On the other hand, the side effects encountered with the use of them have led researchers to look for safe alternatives. This study aimed to determine the antibacterial effect of different commercially available fluoride-free dentifrices on Streptococcus mutans (S. mutans) in comparison with different concentrations of fluoridated dentifrices. Study groups comprised of fluoride-free dentifrices, which contain Probiotic (Activated Charcoal Probiotic Dentifrice-Group P), Aloe Vera-Group AV and Salivary Proteins-Group SP. Fluoridated dentifrices containing 1450 ppm fluoride-Control Group 1 and 500 ppm fluoride-Control Group 2 served as control groups. Antibacterial activity was assessed by Minimum Inhibitory Concentrations and agar well diffusion assays on S. mutans. Biofilm inhibition assay was performed with dentifrices, which had antibacterial activities, and a negative control phosphate-buffered saline (Group PBS) on sterile hydroxyapatite discs against S. mutans. Statistical evaluation was performed. Only group AV showed an antibacterial effect on S. mutans, while control groups showed a similar antibacterial effect. The mean number of viable bacteria present in S. mutans biofilm in Control Group 1 and 2 and Group AV were statistically significantly lower than that in Group PBS, but there were no statistically significant differences between Control Groups and Group AV. Antibacterial activity of commercial dentifrices against S. mutans may be exerted by antibacterial components other than fluoride. Aloe vera-containing toothpaste showed an antibacterial effect on S. mutans, although not as much as the fluoride-containing toothpastes in the control groups. However, further in vivo and long-term studies are required.

Prophylactic vitamin C supplementation regulates DNA demethylation to protect against cisplatin-induced acute kidney injury in mice

Cisplatin-induced acute kidney injury (AKI) restricts the use of cisplatin as a first-line chemotherapeutic agent. Our previous study showed that prophylactic vitamin C supplementation may act as an epigenetic modulator in alleviating cisplatin-induced AKI in mice. However, the targets of vitamin C and the mechanisms underlying the epigenetics changes remain largely unknown. Herein, whole-genome bisulfite sequencing and bulk RNA sequencing were performed on the kidney tissues of mice treated with cisplatin with prophylactic vitamin C supplementation (treatment mice) or phosphate-buffered saline (control mice) at 24 h after cisplatin treatment. Ascorbyl phosphate magnesium (APM), an oxidation-resistant vitamin C derivative, was found that led to global hypomethylation in the kidney tissue and regulated different functional genes in the promoter region and gene body region. Integrated evidence suggested that APM enhanced renal ion transport and metabolism, and reduced apoptosis and inflammation in the kidney tissues. Strikingly, Mapk15, Slc22a6, Cxcl5, and Cd44 were the potential targets of APM that conferred protection against cisplatin-induced AKI. Moreover, APM was found to be difficult to rescue cell proliferation and apoptosis caused by cisplatin in the Slc22a6 knockdown cell line. These results elucidate the mechanism by which vitamin C as an epigenetic regulator to protects against cisplatin-induced AKI and provides a new perspective and evidence support for controlling the disease process through regulating DNA methylation.

Folic Acid-Modified Long-Circulating Liposomes Loaded with Sulfasalazine For Targeted Induction of Ferroptosis in Melanoma.

Melanoma is a malignant tumor that originates from melanocytes. The incidence of melanoma is increasing worldwide, partially because of its insensitivity to radiotherapy or chemotherapy. Therefore, effective treatments for melanoma are urgently required. In this study, we employed folic acid-modified sulfasalazine long-circulating liposomes (FA-SSZ-Lips) to precisely target drug delivery to melanoma cells, eliciting ferroptosis effectively. The synthesized FA-SSZ-Lips were characterized as small spheres of a double-layer membrane, a particle size of 110.1 nm, and a ζ-potential of -22.8 ± 0.66 mV. FA-SSZ-Lips are effective drug carriers with SSZ-loading ratio and SSZ release rate of 6.2 ± 0.10%, and 72.63 ± 1.40%, respectively. The liposomes enhanced SSZ solubility, and the folic acid modifications increased the liposome targeting to melanoma cells. Compared with SSZ alone, FA-SSZ-Lips more strongly inhibited B16F10 cell growth, significantly disrupted the intracellular redox balance, and induced ferroptosis. After treatment, considerable differences were observed in the tumor volumes between FA-SSZ-Lips and phosphate-buffered saline control groups. The tumor growth-inhibition value of the FA-SSZ-Lips group reached 70.09%. Thus, FA-SSZ-Lips exhibited favorable antitumor effects in vitro and in vivo and are a promising strategy for melanoma treatment.

Anti-Prokineticin1 Suppresses Liver Metastatic Tumors in a Mouse Model of Colorectal Cancer with Liver Metastasis.

Multidisciplinary treatment for colorectal cancer (CRC) has undergone significant advances, and molecularly targeted drugs have substantially improved patient prognosis. However, one problem with current molecularly targeted therapeutics is that they must be used in combination with anticancer agents. New molecular targeted therapies that can be used alone are needed. We have previously identified prokineticin1 (PROK1) factor as a therapeutic potential target for CRC. PROK1 factor is involved in the angiogenesis of tissues surrounding CRC tumors. Additionally, PROK1 receptors 1 and 2 are expressed in CRC cell lines, playing roles in cell proliferation via an autocrine mechanism and in the signaling system. In this study, a liver metastasis mouse model was developed using human colorectal cancer cell lines, and mice were divided into anti-PROK1 antibody administration and control groups. Mice were treated intraperitoneally with antibodies or phosphate-buffered saline (control) every three days. The number, size, and cell proliferation ability of metastatic lesions were analyzed. Our results suggested that the number, size, and cancer cell proliferation ability of metastatic lesions decreased, and the survival time significantly increased in the antibody-treated group compared to those in the control group. Thus, the anti-PROK1 antibody therapy suppressed the cell proliferation ability of liver metastatic lesions in a CRC mouse model, suggesting its potential as a novel treatment strategy.

Bacillus-based probiotic cleansers reduce the formation of dry biofilms on common hospital surfaces.

In the absence of liquid suspension, dry biofilms can form upon hard surfaces within a hospital environment, representing a healthcare-associated infection risk. Probiotic cleansers usinggenerally recognized as safe organisms, such as those of the Bacillus genus, represent a potential strategy for the reduction of dry biofilm bioburden. The mechanisms of action and efficacy of these cleaners are, however, poorly understood. To address this, a preventative dry biofilm assay was developed using steel, melamine, and ceramic surfaces to assess the ability of a commercially available Bacillus spp. based probiotic cleanser to reduce the surface bioburden of Escherichia coli and Staphylococcus aureus. Via this assay, phosphate-buffered saline controls were able to generate dry biofilms within 7 days of incubation, with the application of the probiotic cleanser able to prevent >97.7% of dry biofilm formation across both pathogen analogs and surface types. Further to this, surfaces treated with the probiotic mixture alone also showed a reduction in dry biofilm across both pathogen and surface types. Confocal laser scanning microscopy imaging indicated that the probiotic bacteria were able to germinate and colonize surfaces, likely forming a protective layer upon these hard surfaces.

Recombinant-methioninase-producing Escherichia coli Instilled in the Microbiome Inhibits Triple-negative Breast Cancer in an Orthotopic Cell-line Mouse Model.

Methionine restriction by diet and recombinant methioninase (rMETase) are effective for cancer therapy by themselves or combined with chemotherapy drugs. We previously showed that oral administration of rMETase-producing Escherichia coli JM109 (E. coli JM109-rMETase) can be installed in the mouse microbiome and inhibit colon-cancer growth in a syngeneic mouse model. In the present report, we investigated the efficacy of oral administration of E. coli JM109-rMETase in an orthotopic triple-negative breast cancer (TNBC) cell-line mouse model. First, we established orthotopic 4T1 mouse triple-negative breast cancer on an abdominal mammary gland in female athymic nu/nu nude mice aged 4-6 weeks. After tumor growth, 15 mice were divided into three groups of 5. Group 1 was administered phosphate-buffered saline (PBS) orally by gavage twice daily as a control; Group 2 was administered non-recombinant E. coli JM109 competent cells orally by gavage twice daily as a control; Group 3 was administered E. coli JM109-rMETase cells by gavage twice daily for two weeks. Tumor size was measured with calipers twice per week. On day 15, blood methionine level was examined using an HPLC method. Oral administration of E. coli JM109-rMETase inhibited 4T1 TNBC growth significantly compared to the PBS and E. coli JM109 control groups. On day 15, the blood methionine level was significantly lower in the mice administered E. coli JM109-rMETase than in the PBS control. E. coli JM109-rMETase lowered blood methionine levels and inhibited TNBC growth in an orthotopic cell-line mouse model, suggesting future clinical potential against a highly recalcitrant cancer.

Probiotic Potential of Bacillus sp. 62A Isolated from a Marine Extreme Environment.

Antimicrobial resistance is an important health concern globally, and probiotics are considered an alternative to minimize it. The present study examined the in vitro probiotic characteristics and in vivo immunomodulatory potential of Bacillus sp. 62A - an extremophile bacterium. Bacillus sp. 62A was evaluated in vitro for its cytotoxicity, hemolytic activity, antibiotic susceptibility, and resistance to gastrointestinal conditions (bile salts, low pH, and intestinal adherence). Additionally, the immunomodulatory effect of Bacillus sp. 62A was studied in mice. The animals were supplemented daily with phosphate-buffered saline (control) and Bacillus sp. 62A at 1 × 108 colony forming units (CFU). Samples were taken on days 5 and 10. Isolated splenocytes were challenged with Escherichia coli for immunological analyses and immune-related gene expression. Serum and feces were collected for IgA and IgG determination. Bacillus sp. 62A did not show cytotoxicity, hemolytic activity, or resistance to antibiotics. Furthermore, the bacterium has autoaggregation and intestinal adhesion capacities and grows in the presence of bile salts and low pH. Bacillus supplementation in mice improved respiratory burst activity, nitric oxide production, and IL-1β and IL-6 gene expressions, mainly at 10days. After E. coli challenge, Bacillus supplementation in mice induced an anti-inflammatory response through a decrease in immunological parameters and an increase in IL-10 gene expression. Moreover, serum IgA and IgG and fecal IgG augmented in supplemented mice. In conclusion, Bacillus sp. 62A has biosafe and immunomodulatory probiotic potential.

Probiotic Enterococcus faecalis surface-delivering key domain of EtMIC3 proteins: immunoprotective efficacies against Eimeria tenella infection in chickens.

Enterococcus faecalis MDXEF-1 strain was used as host bacteria to deliver surface-displayed key domains BC1 and C4D of the Eimeria tenella microneme 3 (EtMIC3) protein, BC1 fusion to dendritic cell (DC) targeting peptides (DCpep) (DC-BC1) and DC-C4D, respectively. The expression of target proteins in MDXEF-1/BC1-CWA, MDXEF-1/DC-BC1-CWA, MDXEF-1/C4D-CWA, and MDXEF-1/DC-C4D-CWA was verified. The systemic immune responses induced by oral immunization with recombinant E. faecalis were recorded. The immunized chickens were challenged with 4.0 × 104 E. tenella-sporulated oocysts. The immune protections were evaluated by calculating average weight gain and oocyst reduction rate, observing gross and histopathological changes in the cecal tissues, and quantifying levels of inflammatory cytokines in the cecal tissues. The results showed that proteins BC1, C4D, DC-BC1, and DC-C4D were expressed on the surface of E. faecalis, respectively. Oral immunizations with recombinant E. faecalis, especially MDXEF-1/DC-BC1-CWA and MDXEF-1/DC-C4D-CWA, stimulated higher levels of antigen-specific humoral and intestinal mucosal immune responses, higher levels of Th1, Th2, and Th17-type cytokines, and higher proliferation of peripheral blood lymphocytes than phosphate-buffered saline (PBS) and vector control groups (P < 0.01). The groups immunized with four recombinant E. faecalis showed better protective effects against homologous infection than the vector control and infection control groups, and the MDXEF-1/DC-BC1-CWA group displayed the best effects. These results demonstrated that probiotic E. faecalis delivering DCpep fused with the key domain of the EtMIC3 protein could be a potential approach for the prevention of Eimeria infection. IMPORTANCE Avian coccidiosis caused by Eimeria brings huge economic losses to the poultry industry. Although live vaccines and anti-coccidial drugs were used for a long time, Eimeria infection in chicken farms all over the world commonly occurred. The exploration of novel, effective vaccines has become a research hotspot. Eimeria parasites have complex life cycles, and effective antigens are particularly critical to developing anti-coccidial vaccines. Microneme proteins (MICs), secreted from microneme organelles located at the parasite apex, are considered immunodominant antigens. Eimeria tenella microneme 3 (EtMIC3) contains four conserved repeats (MARc1, MARc2, MARc3, and MARc4) and three divergent repeats (MARa, MARb, and MARd), which play a vital role during the Eimeria invasion. Enterococcus faecalis is a native probiotic in animal intestines and can regulate intestinal flora. In this study, BC1 and C4D domains of EtMIC3, BC1 or C4D fusing to dendritic cells targeting peptides, were surface-displyed by E. faecalis, respectively. Oral immunizations were performed to investigate immune protective effects against Eimeria infection.

A HER2-targeted Antibody-Drug Conjugate, RC48-ADC, Exerted Promising Antitumor Efficacy and Safety with Intravesical Instillation in Preclinical Models of Bladder Cancer.

More than half of non-muscle-invasive bladder cancer (NMIBC) patients eventually relapse even if treated with surgery and BCG without optional bladder-preserving therapy. This study aims to investigate the antitumor activity and safety of a HER2-targeted antibody-drug conjugate, RC48-ADC, intravesical instillation for NMIBC treatment. In this preclinical study, itis revealed that human epidermal growth factor receptor 2 (HER2) expression scores of 1+, 2+, and 3+ are recorded for 16.7%, 56.2%, and 14.6% of NMIBC cases. The antitumor effect of RC48-ADC is positively correlated with HER2 expression in bladder cancer (BCa) cell lines and organoid models. Furthermore, RC48-ADC is revealed to exert its antitumor effect by inducing G2/M arrest and caspase-dependent apoptosis. In an orthotopic BCa model, tumor growth is significantly inhibited by intravesical instillation of RC48-ADC versus disitamab, monomethyl auristatin E, epirubicin, or phosphate-buffered saline control. The potential toxicity of intravesical RC48-ADC is also assessed by dose escalation in normal nude mice and revealed that administration of RC48-ADC by intravesical instillation is safe within the range of effective therapeutic doses. Taken together, RC48-ADC demonstrates promising antitumor effects and safety with intravesical administration in multiple preclinical models. These findings provide a rational for clinical trials of intravesical RC48-ADC in NMIBC patients.

Impact of intravenous/intranasal polyinosinic–polycytidylic acid administration on the mediastinal fat-associated lymphoid clusters and lung tissue in healthy mice

BackgroundPolyinosinic–polycytidylic acid (pIC) is a synthetic analog of double-stranded RNA. It is used as a synthetic adjuvant to induce an adaptive immune response. However, the effect of pIC on the development of mediastinal fat-associated lymphoid clusters (MFALCs) that regulate intrathoracic hemostasis has remained unidentified. MethodsWe investigated the impact of intranasal (i.n.) administration (pIC i.n. group) and intravenous (i.v.) administration (pIC i.v. group) of pIC on both MFALCs and lung tissue. ResultsCompared with the control phosphate-buffered saline (PBS) groups, both pIC-administered groups displayed a significant increase in the MFALC size (particularly in the pIC i.n. group), area of MFALC high endothelial venules (HEVs), area of lymphatic vessels (LVs), number of proliferating cells (particularly in the pIC i.v. group), and number of immune cells (B220+ B-lymphocytes, CD3+ T-lymphocytes, Iba1+ macrophages, and Gr-1+ granulocytes) in both MFALCs and lung tissues. In addition, a positive correlation was detected between MFALC size and proliferating cells, immune cell population, LVs, and HEVs within MFALCs in both groups. Except for the proliferating cell and B-lymphocyte populations in the i.n. administered group and granulocyte populations in both i.n. and i.v. administered routes, such correlations were significant. ConclusionIn all, our data indicate that local or systemic administration of pIC induces the development of MFALCs and can be used as an immunostimulant therapeutic strategy.

0.01% Hypochlorous Acid Treats Aspergillus fumigatus Keratitis in Rats by Reducing Fungal Load and Inhibiting the Inflammatory Response.

To investigate the antifungal and anti-inflammatory effects of 0.01% hypochlorous acid (HCLO) on rats with Aspergillus fumigatus keratitis. The time-kill assay and broth microdilution procedures were used in vitro to demonstrate that 0.01% HCLO was fungicidal and fungistatic. The severity of the disease was evaluated in vivo using a clinical score and slit-lamp photographs. Fungal load, polymorphonuclear neutrophil infiltration, and the production of related proteins were determined using colony plate counting, in vivo confocal microscopy, periodic acid-Schiff staining, fungal fluorescence staining, immunofluorescence staining, myeloperoxidase assay, and Western blotting. In vitro, 0.01% HCLO can destroy A. fumigatus spores in 1minute. The optical density of the 0.01% HCLO group was significantly lower than that of the phosphate-buffered saline control group (P < 0.01), and no visible mycelium was observed using a fluorescence microscope. 0.01% HCLO reduced the severity of A. fumigatus keratitis in rats by decreasing the clinical score, fungal loading (periodic acid-Schiff, plate count, and fungal fluorescence staining), and inhibiting neutrophil infiltration and activity (immunofluorescence staining and myeloperoxidase). Furthermore, the Western blot analysis revealed that 0.01% HCO decreased protein expression levels of tumor necrosis factor-α and IL-1β. According to our findings, 0.01% HCLO can kill A. fumigatus spores in vitro. It has antifungal and anti-inflammatory effects on A. fumigatus keratitis in rats. It also inhibited A. fumigatus growth; decreased neutrophil infiltration, tumor necrosis factor-α, and IL-1β expression; and provided a potential treatment for fungal keratitis. This study provides a potential treatment for fungal keratitis in the clinic.