Effects of Human LAV-BPIFB4 Gene Therapy on the Epigenetic Clock and Health of Aged Mice.

The homozygous genotype of the Longevity-Associated Variant (LAV) in Bactericidal/Permeability-Increasing Fold-Containing Family B member 4 (BPIFB4) is enriched in long-living individuals of three independent populations and its genetic transfer in C57BL/6J mice showed a delay in frailty progression and improvement of several biomarkers of aging and multiple aspects of health. The C57BL/6J strain is a suitable model for studying therapies aimed at extending healthy aging and longevity due to its relatively short lifespan and the availability of aging biomarkers. Epigenetic clocks based on DNA methylation profiles are reliable molecular biomarkers of aging, while frailty measurement tools are used to evaluate overall health during aging. In this study, we show that the systemic gene transfer of LAV-BPIFB4 in aged C57BL/6J mice was associated with a significant reduction in the epigenetic clock-based biological age, as measured by a three CpG clock method. Furthermore, LAV-BPIFB4 gene transfer resulted in an improvement of the Vitality Score with a reduction in the Frailty Index. These findings further support the use of LAV-BPIFB4 gene therapy to induce beneficial effects on epigenetic mechanisms associated with aging and frailty in aged mice, with potential implications for future therapies to prevent frailty in humans.

Antimetastatic and Antitumor Activities of Orally Administered NAX014 Compound in a Murine Model of HER2-Positive Breast Cancer.

The natural isoquinoline alkaloid Berberine (BBR) has been shown to possess several therapeutic effects, including anticancer activity. Different BBR derivatives have been designed and synthesized in order to obtain new compounds with enhanced anticancer efficacy. We previously showed that intraperitoneal (IP) administration of the BBR-derived NAX014 compound was able to counteract HER-2 overexpressing mammary tumors onset and progression in transgenic mice. However, the IP administration was found to induce organ toxicity at doses higher than 2.5 mg/Kg. In this study, we evaluated the effect of intragastric (IG) administration of 20 mg/kg of NAX014 on both safety and anticancer efficacy in HER-2/neu transgenic mice. Furthermore, cancer cell dissemination and migration, tumor cell senescence and immunological changes were examined. Our results demonstrated that IG NAX014 administration delayed the onset of mammary tumors with no negative effects on health and survival. NAX014 reduced HER-2 overexpressing BC cells migration in vitro and the frequency of lung metastasis in HER-2/neu transgenic mice. A statistically significant increase of senescence-associated p16 expression was observed in tumors from NAX014-treated mice, and the induction of cell senescence was observed in HER-2 overexpressing BC cells after in vitro treatment with NAX014. Although NAX014 did not modulate the presence of tumor-infiltrating lymphocytes, the level of circulating TNF-α and VEGF was found to be reduced in NAX014-treated mice. The overall results address the NAX014 compound as potential tool for therapeutic strategies against HER-2 overexpressing breast cancer.

New Evidence and Insights on Dalbavancin and Wound Healing in a Mouse Model of Skin Infection.

Dalbavancin is an effective antibiotic that is widely used to treat skin infection. Our aim was to determine the effect of dalbavancin administration on wound healing compared to that of vancomycin and to elucidate if epidermal growth factor receptor (EGFR), matrix metalloproteinase 1 (MMP-1), MMP-9, and vascular endothelial growth factor (VEGF) could be involved in its therapeutic mechanism. A mouse model of methicillin-resistant Staphylococcus aureus (MRSA) skin infection was established. Mice were treated daily with vancomycin (10 mg/kg) and weekly with dalbavancin at day 1 (20 mg/kg) and day 8 (10 mg/kg). After 14 days, wounds were excised, and bacterial counts were performed. Wound healing was assessed by histological and immunohistochemical staining, followed by protein extraction and immunoblotting. Our microbiological results confirmed that both dalbavancin and vancomycin are effective in reducing the bacterial load in wounds. The dalbavancin group showed a strong effect compared with infected untreated animals and the vancomycin-treated group. The wounds treated with dalbavancin showed robust epidermal coverage with reconstitution of the regular and keratinized epidermal lining and well-organized granulation tissue with numerous blood vessels, although slightly less than that in the uninfected group. While in the vancomycin-treated group the epithelium appeared, in general, still hypertrophic, the granulation tissue appeared even less organized. We observed elevated EGFR and VEGF expression in both treated groups, although it was higher in dalbavancin-treated mice. MMP-1 and MMP-9 were decreased in uninfected tissue and in both treated tissues compared with untreated infected wounds. This study showed faster healing with dalbavancin treatment that might be associated with higher EGFR and VEGF levels.

Aging-Related Expression of Twinfilin-1 Regulates Cholangiocyte Biological Response to Injury.

Disorders of the biliary tree develop and progress differently according to patient age. It is currently not known whether the aging process affects the response to injury of cholangiocytes. The aim of this study was to identify molecular pathways associated with cholangiocyte aging and to determine their effects in the biological response to injury of biliary cells. A panel of microRNAs (miRs) involved in aging processes was evaluated in cholangiocytes of young and old mice (2 months and 22 months of age, respectively) and subjected to a model of sclerosing cholangitis. Intracellular pathways that are common to elevated miRs were identified by in silico analysis. Cell proliferation and senescence were evaluated in Twinfilin-1 (Twf1) knocked-down cells. In vivo, senescence-accelerated prone mice (Samp8, a model for accelerated aging), Twf1-/- , or their respective controls were subjected to DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine). Cholangiocytes from DDC-treated mice showed up-regulation of a panel of aging-related miRs. Twf1 was identified by in silico analysis as a common target of the up-regulated miRs. Twf1 expression was increased both in aged and diseased cholangiocytes, and in human cholangiopathies. Knock-down of Twf1 in cholangiocytes reduced cell proliferation. Senescence and senescence-associated secretory phenotype marker expression increased in Twf1 knocked-down cholangiocytes following pro-proliferative and pro-senescent (10-day lipopolysaccharide) stimulation. In vivo, Samp8 mice showed increased biliary proliferation, fibrosis, and Twf1 protein expression level, whereas Twf1-/- had a tendency toward lower biliary proliferation and fibrosis following DDC administration compared with control animals. Conclusion: We identified Twf1 as an important mediator of both cholangiocyte adaptation to aging processes and response to injury. Our data suggest that disease and aging might share common intracellular pathways.

Role of Daptomycin on Burn Wound Healing in an Animal Methicillin-Resistant Staphylococcus aureus Infection Model.

Prolonged hospitalization and antibiotic therapy are risk factors for the development of methicillin-resistant Staphylococcus aureus (MRSA) infections in thermal burn patients. We used a rat model to study the in vivo efficacy of daptomycin in the treatment of burn wound infections by S. aureus, and we evaluated the wound healing process through morphological and immunohistochemical analysis. A copper bar heated in boiling water was applied on a paraspinal site of each rat, resulting in two full-thickness burns. A small gauze was placed over each burn and inoculated with 5 × 107 CFU of S. aureus ATCC 43300. The study included two uninfected control groups with and without daptomycin treatment, an infected control group that did not receive any treatment, and two infected groups treated, respectively, with intraperitoneal daptomycin and teicoplanin. The main outcome measures were quantitative culture, histological evaluation of tissue repair, and immunohistochemical expression of wound healing markers: epidermal growth factor receptor (EGFR) and fibroblast growth factor 2 (FGF-2). The highest inhibition of infection was achieved in the group that received daptomycin, which reduced the bacterial load from 107 CFU/ml to about 103 CFU/g (P < 0.01). The groups treated with daptomycin showed better overall healing with epithelialization and significantly higher collagen scores than the other groups, and these findings were also confirmed by immunohistochemical data. In conclusion, our results support the hypothesis that daptomycin is an important modulator of wound repair by possibly reducing hypertrophic burn scar formation.

Increase of n-NOS and i-NOS in Rat Colon After Sacral Neuromodulation.

OBJECTIVE: Sacral neuromodulation (SNM) is proposed to treat different anorectal dysfunctions but its mechanism of action is not yet known. Our previous study demonstrated how SNM can significantly increase neuronal nitric oxide synthase NOS (n-NOS) and inducible NOS (i-NOS) expression in the anus and rectum of rats. There are no reports regarding the relation between SNM and NOS in colonic cells: our aim was to assess NOS expression in colonic rat model after SNM. MATERIALS AND METHODS: Twenty-six female Sprangue-Dawley rats were considered: group I, normal control rats; group II, sham treatment rats, in whom electrodes for electrical stimulation were placed in S1 foramen bilaterally and left in place, without performing neuromodulation; group III, rats in whom SNM was performed. After 14 days, the rats were sacrificed and we evaluated n-NOS and i-NOS in colonic specimens by immunohistochemistry and Western Blot analysis. RESULTS: Western Blot analysis showed that levels of n-NOS and i-NOS were higher in colon of the III group rats respect to the others; in particular, immunohistochemistry revealed that, after neuromodulation, n-NOS expression in the muscle cells and i-NOS expression in glandular epithelium and nervous cells were highly represented (p < 0.05). CONCLUSION: Our study showed that in colon, SNM is able to influence NO synthesis, activating n-NOS expression in muscle cells and i-NOS expression in glandular epithelium and nervous cells. Our study showed a complex colonic response to SNM. This experimental model could be applied to better understand the mechanism of action of SNM in bowel dysfunction.

Antiangiogenic and antitumor activities of berberine derivative NAX014 compound in a transgenic murine model of HER2/neu-positive mammary carcinoma.

Berberine (BBR) is a natural isoquinoline alkaloid with proven antiangiogenic and anticancer activities. We recently demonstrated that BBR and its synthetic derivative 13-(4-chlorophenylethyl)berberine iodide, NAX014, exert antiproliferative activity against HER2-overexpressing breast cancer cells, inducing apoptosis, modulating the expression of cell cycle checkpoint molecules involved in cell senescence, and reducing both HER2 expression and phosphorylation on tumor cells. In this study, we examined the anticancer properties of BBR and NAX014 in a transgenic mouse model which spontaneously develops HER2-positive mammary tumors. Repeated intraperitoneal injections of a safety dose (2.5mg/kg) of NAX014 delayed the development of tumors, reducing both the number and size of tumor masses. In vivo sidestream dark field videomicroscopy revealed a significant lower vessel density in mammary tumors from NAX014-treated mice in comparison with the control group. Immunohistochemical evaluation using CD34 antibody confirmed the reduced vessel density in NAX014 group. Statistically significant increase of senescence associated ß-galactosidase and p16 expression, and reduced expression of heparanase were observed in tumors from NAX014-treated mice than in tumors from control animals. Finally, NAX014 treatment decreased the level of perforine and granzyme mRNA in mammary tumors. Berberine did not show any statistically significant modulation in comparison with control mice. The results of the present study indicate that NAX014 is more effective than BBR in exerting anticancer activity delaying the development of mammary tumors in mice transgenic for the HER-2/neu oncogene. The antitumor efficacy of NAX014 is mainly related to its effect on tumor vascular network and on induction of tumor cell senescence.

Identification and characterization of cancer stem cells from head and neck squamous cell carcinoma cell lines.

BACKGROUND/AIMS: Head and neck squamous cell carcinoma (HNSCC) ranks sixth worldwide for tumor-related mortality. A subpopulation of tumor cells, termed cancer stem cells (CSCs), has the ability to support cancer growth. Therefore, profiling CSC-enriched populations could be a reliable tool to study cancer biology. METHODS: We performed phenotypic characterization of 7 HNSCC cell lines and evaluated the presence of CSCs. CSCs from Hep-2 cell line and HNSCC primary cultures were enriched through sphere formation and sphere-forming cells have been characterized both in vitro and in vivo. In addition, we investigated the expression levels of Nicotinamide N-methyltransferase (NNMT), an enzyme overexpressed in several malignancies. RESULTS: CSC markers were markedly expressed in Hep-2 cell line, which was found to be highly tumorigenic. CSC-enriched populations displayed increased expression of CSC markers and a strong capability to form tumors in vivo. We also found an overexpression of CSC markers in tumor formed by CSC-enriched populations. Interestingly, NNMT levels were significantly higher in CSC-enriched populations compared with parental cells. CONCLUSION: Our study provides an useful procedure for CSC identification and enrichment in HNSCC. Moreover, results obtained seem to suggest that CSCs may represent a promising target for an anticancer therapy.

Sidestream dark field videomicroscopy for in vivo evaluation of vascularization and perfusion of mammary tumours in HER2/neu transgenic mice.

Angiogenesis plays a key role in tumour growth and the formation of metastases. Angiogenesis inhibitors and antivascular agents may prove useful in the treatment of breast cancer. A comprehensive characterization of the vasculature and perfusion of mammary tumours is a prerequisite for developing new specific drugs. We used sidestream dark field videomicroscopy to evaluate in vivo the vascular network of spontaneous mammary tumours in HER2/neu transgenic mice. The tumour masses showed higher vessel density compared with the healthy mammary gland (median (interquartile range) total vessel density 16.8 (13.4-20.5) vs 9.1 (8.1-10.9) mm/mm(2), respectively; P < 0.001). Tumor vessel density was reduced in mice treated with the anti-angiogenesis agent berberine, 12.1 (10.6-13.1) mm/mm(2). Sidestream dark field imaging is a versatile technique that may be useful for understanding the role of angiogenesis in the progression of breast cancer and its relationship with outcome. It may represent a valuable tool for dynamic monitoring of the effects of new anti-angiogenesis therapies.

ER-mitochondria association negatively affects wound healing by regulating NLRP3 activation.

Methicillin-resistant Staphylococcus aureus (MRSA) is the most common causative agent of acute bacterial skin and skin-structure infections (ABSSSI), one of the major challenges to the health system worldwide. Although the use of antibiotics as the first line of intervention for MRSA-infected wounds is recommended, important side effects could occur, including cytotoxicity or immune dysregulation, thus affecting the repair process. Here, we show that the oxazolidinone antibiotic linezolid (LZD) impairs wound healing by aberrantly increasing interleukin 1 ß (IL-1ß) production in keratinocytes. Mechanistically, LZD triggers a reactive oxygen species (ROS)-independent mitochondrial damage that culminates in increased tethering between the endoplasmic reticulum (ER) and mitochondria, which in turn activates the NLR family pyrin domain-containing 3 (NLRP3) inflammasome complex by promoting its assembly to the mitochondrial surface. Downregulation of ER-mitochondria contact formation is sufficient to inhibit the LZD-driven NLRP3 inflammasome activation and IL-1ß production, restoring wound closure. These results identify the ER-mitochondria association as a key factor for NLRP3 activation and reveal a new mechanism in the regulation of the wound healing process that might be clinically relevant.

Effect of annatto-tocotrienols supplementation on the development of mammary tumors in HER-2/neu transgenic mice.

Tocotrienols (T3), the lesser known isomers of vitamin E, have been reported to possess anticancer activity both in in vitro and in vivo experimental models of rodents transplanted with parental tumors or treated with carcinogens. We investigated the effects of dietary supplementation with annatto-T3 (90% δ-T3 and 10% γ-T3) on the spontaneous development of mammary tumors in HER-2/neu transgenic mice. Underlying mechanisms of the antitumor effect were evaluated by studying apoptosis, senescent-like growth arrest, immune modulation, oxidative effect and the expression of HER-2/neu in tumoral mammary glands of transgenic mice and in vitro in human and mice tumor cell lines. Annatto-T3 supplementation delayed the development of mammary tumors, reducing the number and size of mammary tumor masses and those of lung metastases. In annatto-T3-supplemented mice, both apoptosis and senescent-like growth arrest of tumor cells were increased in mammary glands while no immune modulation was observed. In vitro, a dose-dependent inhibition of cell growth, increased apoptosis and senescent-like growth arrest and a time-dependent accumulation of reactive oxygen species were observed in tumor cells treated with annatto-T3 or purified δ-T3. Annatto-T3 reduced both HER-2/neu mRNA and p185(HER-2/neu) protein in tumors and in tumor cell lines. The results show that the antitumor effect of annatto-T3 supplementation in HER-2/neu transgenic mice is mainly related to the direct induction of oxidative stress, senescent-like growth arrest and apoptosis of tumor cells rather than to an immune modulation.

The efficacy of the quorum sensing inhibitor FS8 and tigecycline in preventing prosthesis biofilm in an animal model of staphylococcal infection.

We investigated the efficacy of tigecycline and FS8, alone or combined, in preventing prosthesis biofilm in a rat model of staphylococcal vascular graft infection. Graft infections were established in the back subcutaneous tissue of adult male Wistar rats by implantation of Dacron prostheses followed by topical inoculation with 2 x 107 colony-forming units of Staphylococcus aureus, strain Smith diffuse. The study included a control group, a contaminated group that did not receive any antibiotic prophylaxis, and three contaminated groups that received: (i) intraperitoneal tigecycline, (ii) FS8-soaked graft, and (iii) tigecycline plus FS8-soaked graft, respectively. Each group included 15 animals. The infection burden was evaluated by using sonication and quantitative agar culture. Moreover, an in vitro binding-study was performed to quantify the how much FS8 was coated to the surface of the prosthesis. Tigecycline, combined with FS8, against the adherent bacteria showed MICs (2.00 mg/L) and MBCs (4.00 mg/L) four-fold lower with respect to tigecycline alone in in vitro studies. The rat groups treated with tigecycline showed the lowest bacterial numbers (4.4 x 104 ± 1.2 x 104 CFU/mL). The FS8-treated group showed a good activity and significant differences compared to control group with bacterial numbers of 6.8 x 104 ± 2.0 x 104 CFU/mL. A stronger inhibition of bacterial growth was observed in rats treated with a combined FS8 and tigecycline therapy than in those that were singly treated with bacterial numbers of 101 CFU/mL graft. In conclusion, the ability to affect biofilm formation as well, its property to be an antibiotic enhancer suggests FS8 as alternative or additional agent to use in conjunction with conventional antimicrobial for prevention of staphylococcal biofilm related infection.

Comprehensive longitudinal non-invasive quantification of healthspan and frailty in a large cohort (n = 546) of geriatric C57BL/6 J mice.

Frailty is an age-related condition characterized by a multisystem functional decline, increased vulnerability to stressors, and adverse health outcomes. Quantifying the degree of frailty in humans and animals is a health measure useful for translational geroscience research. Two frailty measurements, namely the frailty phenotype (FP) and the clinical frailty index (CFI), have been validated in mice and are frequently applied in preclinical research. However, these two tools are based on different concepts and do not necessarily identify the same mice as frail. In particular, the FP is based on a dichotomous classification that suffers from high sample size requirements and misclassification problems. Based on the monthly longitudinal non-invasive assessment of frailty in a large cohort of mice, here we develop an alternative scoring method, which we called physical function score (PFS), proposed as a continuous variable that resumes into a unique function, the five criteria included in the FP. This score would not only reduce misclassification of frailty but it also makes the two tools, PFS and CFI, integrable to provide an overall measurement of health, named vitality score (VS) in aging mice. VS displays a higher association with mortality than PFS or CFI and correlates with biomarkers related to the accumulation of senescent cells and the epigenetic clock. This longitudinal non-invasive assessment strategy and the VS may help to overcome the different sensitivity in frailty identification, reduce the sample size in longitudinal experiments, and establish the effectiveness of therapeutic/preventive interventions for frailty or other age-related diseases in geriatric animals.

Comparative effects of micafungin, caspofungin, and anidulafungin against a difficult-to-treat fungal opportunistic pathogen, Candida glabrata.

The aim of this study was to compare the in vitro and in vivo activities of micafungin, caspofungin, and anidulafungin against Candida glabrata. The MICs against 28 clinical isolates showed that the overall susceptibilities to caspofungin and to micafungin were not statistically different in the absence of human serum, whereas the isolates were less susceptible to micafungin than to caspofungin in its presence. Minimum fungicidal concentrations, as well as time-kill experiments, showed that caspofungin was more active than anidulafungin, while micafungin was superior to either caspofungin or anidulafungin without serum; its addition rendered caspofungin and micafungin equally effective. A murine model of systemic candidiasis against a C. glabrata-susceptible isolate was performed to study the effects of all three echinocandins, and kidney burden counts showed that caspofungin, micafungin, and anidulafungin were active starting from 0.25, 1, and 5 mg/kg of body weight/day, respectively. Two echinocandin-resistant strains of C. glabrata were selected: C. glabrata 30, a laboratory strain harboring the mutation Fks2p-P667T, and C. glabrata 51, a clinical isolate harboring the mutation Fks2p-D666G. Micafungin activity was shown to be as effective as or more effective than that of caspofungin or anidulafungin in terms of MICs. In vivo studies against these resistant strains showed that micafungin was active starting from 1 mg/kg/day, while caspofungin was effective only when administrated at higher doses of 5 or 10 mg/kg/day. Although a trend toward colony reduction was observed with the highest doses of anidulafungin, a significant statistical difference was never reached.

In vivo electroporation restores the low effectiveness of DNA vaccination against HER-2/neu in aging.

Experimental evidence has been provided that cancer vaccines are less effective at older age than in young adults. In this study, we evaluated the possibility to recover the low effectiveness of DNA immunization against HER-2/neu increasing plasmid uptake by cells from old mice through electroporation with the aim to enhance the activation of specific immune responses. Young and old Balb/c mice received two immunizations with a pCMV-ECDTM DNA plasmid using plasmid intramuscular injection followed by electroporation (IM + E) or plasmid intramuscular injection alone (IM), and successively, they were challenged with syngeneic HER-2/neu overexpressing TUBO cells. Young mice were completely protected whereas less than 60% protection was observed in old mice after IM immunization. IM + E immunization completely protected old mice against a TUBO cell challenge. The protection was associated with increased transgene expression in the site of immunization and with the induction of both humoral and cell-mediated immunity in old mice. We conclude that the effectiveness of anticancer DNA vaccination in old ages may be improved increasing plasmid uptake and transgene expression through electroporation, suggesting the relevant role of the first steps of the immunization process in the success of cancer vaccines at older age.

In vitro and in vivo effects of echinocandins against Candida parapsilosis sensu stricto, Candida orthopsilosis and Candida metapsilosis.

OBJECTIVES: The aim of the present study was to compare, in vitro and in vivo, the effects of caspofungin, micafungin and anidulafungin against Candida parapsilosis complex isolates. METHODS: In vitro activities of all three echinocandins were assessed against C. parapsilosis sensu stricto (n = 4), Candida orthopsilosis (n = 4) and Candida metapsilosis (n = 3) using broth microdilution susceptibility testing, minimum fungicidal concentration determination and a killing-curve assay, in the absence and in the presence of 50% human serum. Then, the activities of all drugs were investigated in an immunocompromised murine model of systemic candidiasis. Animals were infected with six isolates (two for each species) and treated with the echinocandins administered at 0.25, 1, 5 and 10 mg/kg/day for six consecutive days. Fungal burdens were assessed in kidney tissues on day 7 post-infection. RESULTS: Geometric mean MICs of caspofungin, micafungin and anidulafungin for C. parapsilosis sensu lato were, respectively, 0.09, 0.14 and 0.20 mg/L without serum, and 0.70, 3.92 and 5.84 mg/L with serum. The fungicidal activity of all three echinocandins was variable; however, the addition of serum reduced the fungicidal effects against these species. In vivo studies showed that caspofungin at 5 and 10 mg/kg/day significantly decreased the kidney burdens with respect to the controls for all isolates, while micafungin was active at 5 and/or 10 mg/kg/day only against C. metapsilosis. CONCLUSIONS: Our susceptibility testing showed that caspofungin was the most active echinocandin against all three species. Also, caspofungin resulted in significant therapeutic effects for treatments of experimental systemic infections due to the three species, while micafungin was effective only against C. metapsilosis.

Tigecycline accelerates staphylococcal-infected burn wound healing through matrix metalloproteinase-9 modulation.

OBJECTIVES: We investigated the in vivo efficacy of tigecycline, a new glycylcycline (a tetracycline derivative), in the management of methicillin-resistant Staphylococcus aureus (MRSA)-infected experimental surgical wounds in rats. The main outcome measures were quantitative bacterial culture, histological examination and immunohistochemical expression of matrix metalloproteinase-9 (MMP-9) and collagen IV. METHODS: An animal model was used to compare the in vivo efficacy of teicoplanin and tigecycline in the treatment of burn wound infections by S. aureus. A copper bar, heated in boiling water, was placed on the paraspinal site of each rat, resulting in full thickness burns. A small gauze was placed over each burn and then inoculated with 5 × 10(7) cfu of S. aureus ATCC 43300. To mimic the clinical situation in burn patients, surgical debridement was performed 48 h after the injury. The wounds were left to heal by secondary intention. The study included an uninfected control group that did not receive any treatment, a contaminated group that did not receive any treatment, and two contaminated groups treated with intraperitoneal tigecycline (2 mg/kg) and teicoplanin (7 mg/kg), respectively. RESULTS: All antibiotic treatments were significantly effective. Tigecycline showed the highest antimicrobial activity, with a better impact on histological results. Infected rats treated with tigecycline showed a significant decrease in MMP-9 expression both in epithelium and in dermis compared with rats treated with teicoplanin. CONCLUSIONS: Tigecycline, besides its antimicrobial activity, exerts an important modulatory effect on MMP-9, accelerating wound healing in staphylococcal-infected burns.

Nitric oxide synthase expression in rat anorectal tissue after sacral neuromodulation.

BACKGROUND: Sacral neuromodulation is becoming established as a valid treatment option for patients with anorectal disorders. Nevertheless, despite its efficacy, little is known regarding its mechanism of action. The purpose of this study was to evaluate whether chronic sacral neuromodulation is able to influence the expression of nitric oxide synthetase (NOS) in the anorectum of rats. MATERIALS AND METHODS: Twenty-six female Sprague-Dawley rats were divided into three groups; normal control rats (n = 6); sham treatment (n =10) and group in whom, electrical sacral neuromodulation was performed (n = 10). Bilateral electrode wires were placed in the S1 and electrical stimulation was performed for 14 d. At the end of the procedures the rats were sacrificed, proctectomy was performed, and anorectal specimens were sent to the laboratory for immunostaining with n-NOS and i-NOS. RESULTS: In the anal and rectal specimens, n-NOS and i-NOS expression was significantly increased in epithelial and muscle cells after neuromodulation of the anus and rectum of the animals. CONCLUSION: Our results showed that this model can be applied in further experimental studies to better understand the mechanism of action of sacral neuromodulation in anorectal disorders.

Efficacy of tigecycline and rifampin alone and in combination against Enterococcus faecalis biofilm infection in a rat model of ureteral stent.

BACKGROUND: We investigated the efficacy of tigecycline and rifampin alone or combined in preventing ureteral stent infection due to Enterococcus faecalis. MATERIALS AND METHODS: The activities of the two antibiotics were previously studied in vitro in absence or in presence of biofilm. For in vivo research, the study included a control group without bacterial challenge to evaluate the sterility of surgical procedure, a challenged control group that did not receive any antibiotic prophylaxis and, for each bacterial strain, three challenged groups that received: (1) 2 mg/kg intraperitoneal tigecycline, immediately after stent implantation; (2) rifampin-coated ureteral stents where 0.2 cm(2) sterile ureteral stents were incubated in 10 mg/L rifampin solution for 30 min immediately before implantation; and (3) intraperitoneal tigecycline plus rifampin-coated ureteral stent at the above concentrations. Ureteral stents were explanted at d 5 following implantation and biofilm bacteria enumerated. RESULTS: The in vitro studies showed that the biofilm was strongly affected by the presence of rifampin and, in its presence, tigecycline had MICs and MBCs lower than those obtained in the absence of rifampin. Intraperitoneal tigecycline exerted stronger effect than rifampin on bacterial numbers. The combination rifampin plus tigecycline showed efficacies higher than that of each single compound. CONCLUSION: These results highlight the potential usefulness of tigecycline in preventing enterococcal ureteral stent infections and the role of rifampin as an interesting antibiotic enhancer.

Transfer of the longevity-associated variant of BPIFB4 gene rejuvenates immune system and vasculature by a reduction of CD38+ macrophages and NAD+ decline.

As we age, our body experiences chronic, systemic inflammation contributing to the morbidity and mortality of the elderly. The senescent immune system has been described to have a causal role in driving systemic aging and therefore may represent a key therapeutic target to prevent pathological consequences associated with aging and extend a healthy lifespan. Previous studies from our group associated a polymorphic haplotype variant in the BPIFB4 gene (LAV-BPIFB4) with exceptional longevity. Transfer of the LAV-BPIFB4 in preclinical models halted the progression of cardiovascular diseases (CVDs) and frailty by counterbalancing chronic inflammation. In the present study, we aimed to delineate the action of systemic adeno-associated viral vector-mediated LAV-BPIFB4 gene transfer (AAV-LAV-BPIFB4) on the deleterious age-related changes of the immune system and thereby the senescence-associated events occurring in C57BL/6J mice aged 26 months. Our in vivo data showed that 26-months-old mice had a higher frequency of CD45+SA-beta Gal+ immune cells in peripheral blood than young (4-months-old) C57BL/6J mice. Notably, AAV-LAV-BPIFB4 gene transfer in aged mice reduced the pool of peripheral immunosenescent cells that were shown to be enriched in the spleen. In addition, the proper tuning of the immune secretory phenotype (IL1ßlow, IL6low, IL10high) associated with a significant reduction in SA-beta Gal-positive area of aorta from AAV-LAV treated mice. At the functional level, the reduction of senescence-associated inflammation ensured sustained NAD+ levels in the plasma of AAV-LAV-BPIFB4 old mice by preventing the NADase CD38 increase in F4/80+ tissue-resident macrophages and Ly6Chigh pro-inflammatory monocytes of the spleen and bone marrow. Finally, to validate the clinical implication of our findings, we showed that Long-living-individuals (LLIs, >95 years), which delay CVDs onset, especially if LAV-carriers, were characterized by high NAD+ levels. In conclusion, the new senotherapeutic action of LAV-BPIFB4 may offer a valuable therapeutic tool to control aging and reduce the burden of its pathophysiological disorders, such as CVDs.