Search

Gram-negative sepsis ranks as the leading cause of death in intensive care units. Despite the development of new antibiotics, mortality from gram-negative sepsis remains high. The present study aims to investigate the in vivo effects of berberine (BBR) administration on septic death induced by intraperitoneal Escherichia coli injection. The results showed that (i) single 5 mg/kg dose of BBR increases the survival of septic mice, (ii) BBR administration improves the antimicrobial efficacy of antibiotic drug, (iii) BBR pre-treatment prevents improvements of BBR therapy without affecting the pro-survival effects of antibiotic drug. The effects of BBR administration were associated with immunological alterations represented by changes in CD4+ and CD8+ lymphocytes population and IL-6 and TNF-α production. This study highlighted the benefits of berberine administration as antibiotic adjuvant in E. coli sepsis. Furthermore, information about berberine-induced immunological perturbations and their influence on host response to infection and therapy has been shown.

The advent of trastuzumab has significantly improved the prognosis of HER2-positive (HER2+) breast cancer patients; nevertheless, drug resistance limits its clinical benefit. Anti-HER2 active immunotherapy represents an attractive alternative strategy, but effective immunization needs to overcome the patient’s immune tolerance against the self-HER2. Phage display technology, taking advantage of phage intrinsic immunogenicity, permits one to generate effective cancer vaccines able to break immune tolerance to self-antigens. In this study, we demonstrate that both preventive and therapeutic vaccination with M13 bacteriophages, displaying the extracellular (EC) and transmembrane (TM) domains of human HER2 or its Δ16HER2 splice variant on their surface (ECTM and Δ16ECTM phages), delayed mammary tumor onset and reduced tumor growth rate and multiplicity in ∆16HER2 transgenic mice, which are tolerant to human ∆16HER2. This antitumor protection correlated with anti-HER2 antibody production. The molecular mechanisms underlying the anticancer effect of vaccine-elicited anti-HER2 antibodies were analyzed in vitro against BT-474 human breast cancer cells, sensitive or resistant to trastuzumab. Immunoglobulins (IgG) purified from immune sera reduced cell viability mainly by impairing ERK phosphorylation and reactivating retinoblastoma protein function in both trastuzumab-sensitive and -resistant BT-474 cells. In conclusion, we demonstrated that phage-based HER2 vaccines impair mammary cancer onset and progression, opening new perspectives for HER2+ breast cancer treatment.

Background: LL-37 is the only human antimicrobial peptide that belongs to the cathelicidins. The aim of the study was to evaluate the efficacy of LL-37 in the management of MRSA-infected surgical wounds in mice. Methods: A wound on the back of adult male BALB/c mice was made and inoculated with Staphylococcus aureus. Two control groups were formed (uninfected and not treated, C0, infected and not treated, C1) and six contaminated groups were treated, respectively, with: teicoplanin, LL-37, given topically and /or systemically. Histological examination of VEGF expression and micro-vessel density, and bacterial cultures of wound tissues, were performed. Results: Histological examination of wounds in the group treated with topical and intraperitoneal LL-37 showed increased re-epithelialization, formation of the granulation tissue, collagen organization, and angiogenesis. Conclusions: Based on the mode of action, LL-37 has a potential future role in the management of infected wounds.

BACKGROUND:There is an increasing concern about age-related frailty because of the growing number of elderly people in the general population. The Longevity-Associated Variant (LAV) of the human BPIFB4 gene was found to correct endothelial dysfunction, one of the mechanisms underlying frailty, in aging mice whereas the RV-BPIFB4 variant induced opposite effects. Thus, we newly hypothesize that, besides being associated with life expectancy, BPIFB4 polymorphisms can predict frailty.Aim and Results: Here we investigated if the BPIFB4 haplotypes, LAV, wild-type (WT) and RV, differentially associate with frailty in a cohort of 237 elderly subjects from Calabria region in Southern Italy. Moreover, we studied the effect of systemic adeno-associated viral vector-mediated LAV-BPIFB4 gene transfer on the progression of frailty in aging mice. We found an inverse correlation of the homozygous LAV-BPIFB4 haplotype with frailty in elderly subjects. Conversely, carriers of the RV-BPIFB4 haplotype showed an increase in the frailty status and risk of death. Moreover, in old mice, LAV-BPIFB4 gene transfer delayed frailty progression.CONCLUSIONS:These data indicate that specific BPIFB4 haplotypes could represent useful genetic markers of frailty. In addition, horizontal transfer of a healthy gene variant can attenuate frailty in aging organisms.

Escherichia coli 0157:H7 is a food-borne pathogen that can cause severe complications in vulnerable populations. Mouse infection models of E. coli 0157:H7 are usually developed under severe animal suffering classification by depleting the normal flora, in which age plays a role. Objective To develop a refined method for longitudinal monitoring of E. coli 0157:H7 in young and old mice with intact flora. Methods We applied discriminant analysis and computed composite standardized scores from 19 variables obtained from physiological parameters, analysis of locomotor activity, grip strength measurement and fecal shedding in 16 aged and 16 young C57BL/6 mice after two mild oral challenges of E. coli 0157:H7. The resulting scores were validated in another experiment performed in 24 aged and 24 young mice including a group (8 aged and 8 young mice) treated with oxytetracycline. Results We show that our scores are significantly affected in the post-infection period and that can be used to measure and compare the recovery time after a treatment. The scores are most sensitive when separately developed in young and aged mice. Conclusions We developed a method that minimizes the level of animal suffering and that can be applied in preclinical testing of new therapies.

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.

Fibrosis is a condition that affects the connective tissue in an organ or tissue in the restorative or responsive phase as a result of injury. The consequences of excessive fibrotic tissue growth may lead to various physiological complications of deformity and impairment due to hypertrophic scars, keloids, and tendon adhesion without understating the psychological impact on the patient. However, no method accurately quantifies the rate and pattern of subcutaneous induced hypertrophic fibrosis. We, therefore, devised a rodent excisional model to evaluate the extent of fibrosis with talc. Tissue specimens were set on formalin, and paraffin sections for histological, immunohistochemical, and molecular analysis talc was used to induce the fibroproliferative mechanism typical of hypertrophic scars. This pathway is relevant to the activation of inflammatory and fibrotic agents to stimulate human hypertrophic scarring. This model reproduces morpho-functional features of human hypertrophic scars to investigate scar formation and assess potential anti-scarring therapies.

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.

This corrects the article "Anastomotic healing in a rat model of peritonitis after non-steroidal anti-inflammatory drug administration " in volume 64(1):3085 In the published article “Anastomotic healing in a rat model of peritonitis after non-steroidal anti-inflammatory drug administration” Eur J Histochem 2020;64(1):3085, https://doi.org/10.4081/ejh.2020.3085,” one affiliation was published incorrectly. The authors apologize for any inconvenience that it may have caused. Roberto Ghiselli,1 Guendalina Lucarini,2 Monica Ortenzi,1 Eleonora Salvolini,3 Stefania Saccomanno,2 Fiorenza Orlando,4 Mauro Provinciali,4 Fabio Casciani,1 Mario Guerrieri1 1Clinic of Surgery, Marche Polytechnic University, Ancona2Department of Clinical and Molecular Sciences, Marche Polytechnic University, Ancona3Department of Odontostomatologic and Specialized Clinical Sciences, Marche Polytechnic University, Ancona4Experimental Animal Models for Aging Units, Research Department, Italian National Institute on Aging (INRCA) IRCCS, Ancona, Italy The affiliation should be corrected as follows: 4Experimental Animal Models for Aging Units, Scientific Technological Area, IRCCS INRCA, Ancona, Italy

Dura mater repair represents a final and crucial step in cranial surgery: an inadequate dural reconstruction determines dreadful consequences that significantly increase morbidity and mortality rates. Different dural substitutes have been used with poor results. To overcome this issue, in previous studies we proposed a laser-based approach to the bonding of porcine dura mater, evidencing the feasibility of the laser assisted procedure. In this work, we present the optimization of the laser bonding approach ex vivo in porcine dura mater and in vivo in rats. An 810 nm cw laser was used to weld the ICG stained chitosan patch to the dura. The ex vivo tests enabled to optimize the laser parameters, using histology and leak pressure evaluation to study the bonding effect. The in vivo tests were performed on 32 adult Wistar rats: laser bonding was carried out in 16 rats, while a collagen matrix was used for duroplasty in the control group. After the treatment, the animals were left to recover and were observed in a 15 and 90 days follow up study. At sacrifice, the rats were anesthetized for fluid leakage pressure test; treated tissue was harvested and underwent standard histology. The results of this study pointed out that the laser bonding procedure can be used to close the dura mater, both ex vivo and in vivo. The thermal effect is limited and spatially confined. The technique can thus be proposed as a valid alternative to standard method for the closuring of dura mater in cranial surgery.

Gram-negative sepsis ranks as the leading cause of death in intensive care units. Despite the development of new antibiotics, mortality from gram-negative sepsis remains high. The present study aims to investigate the

The tissue inflammatory response can influence the outcome of anastomotic healing. Anastomotic leakage represents a dreadful complication after gastrointestinal surgery, in particular sepsis and intra-abdominal infections impair the restorative process of colic anastomoses. It has been debated whether the administration of non-steroidal anti-inflammatory drugs (NSAIDs) is a risk factor for dehiscence, since many patients receive NSAIDs in the early postoperative period. Our aim was, for the first time, to analyze the morpho-functional effects of postoperative administration of two commonly used NSAIDs, Diclofenac and Ketorolac, on the healing process of colo-colic anastomoses constructed under condition of fecal peritonitis in a rat model. Sixty adult male rats underwent two surgical procedures: peritonitis induction and colo-colic anastomosis, and were divided into three groups: 20 rats received saline; 20 rats 4 mg/kg Diclofenac and 20 rats 5 mg/kg Ketorolac. We assessed anastomosis strength, morphological features of tissue wound healing, immunohistochemical metalloproteinase 9 (MMP9) expression and collagen deposition and content by Sirius red staining and hydroxyproline level. We found no significant difference in bursting pressure, collagen content and organization and morphological features between the groups, except a significantly reduced presence of inflammatory cells and MMP9 expression in the groups treated with NSAIDs. Our findings showed that Diclofenac and Ketorolac administration did not affect post-surgical healing and did not increase the leakage risk of colo-colic anastomoses during peritonitis.

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

We investigated the efficacy of colistin combined with teicoplanin or daptomycin in an experimental mouse model of multiresistant Acinetobacter baumannii infection. Animal received intraperitoneally 1ml saline containing 2×1010CFU of A. baumannii. Colistin, daptomycin, teicoplanin, and colistin plus daptomycin or teicoplanin were given by intraperitoneal administration 2h after bacterial challenge. A control group received sodium chloride solution. In the in vitro study A. baumannii showed to be susceptible only to colistin with MIC of 2mg/l. In the in vivo study, colistin alone showed a good antimicrobial efficacy. When combined with teicoplanin or daptomycin, colistin produced the lowest bacterial and the best survival rates. In immunological studies, when colistin was associated to daptomycin or teicoplanin, both the number and the cytotoxic activity of NK cells increased. In conclusion, colistin combined with teicoplanin or daptomycin may improve the therapy of multiresistant A. baumannii infection.

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models continue to be an essential tool for developing new antifibrotic strategies. Here we provide an effective method to investigate in vivo antifibrotic activity of human mesenchymal stromal cells obtained from whole umbilical cord (hUC-MSC) in attenuating bleomycin-induced lung injury. C57BL/6 mice receive a single endotracheal injection of bleomycin (1.5 U/kg body weight) followed by a double infusion of hUC-MSC (2.5 x 105) into the tail vein, 24 h and 7 days after the bleomycin administration. Upon sacrifice at days 8, 14, or 21, inflammatory and fibrotic changes, collagen content, and hUC-MSC presence in explanted lung tissue are analyzed. The injection of bleomycin into the mouse trachea allows the direct targeting of the lungs, leading to extensive pulmonary inflammation and fibrosis. The systemic administration of a double dose of hUC-MSC results in the early blunting of the bleomycin-induced lung injury. Intravenously infused hUC-MSC are transiently engrafted into the mouse lungs, where they exert their anti-inflammatory and antifibrotic activity. In conclusion, this protocol has been successfully applied for the preclinical testing of hUC-MSC in an experimental mouse model of human pulmonary fibrosis. However, this technique can be easily extended both to study the effect of different endotracheally administered substances on the pathophysiology of the lungs and to validate new anti-inflammatory and antifibrotic systemic therapies.

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.

Dysfunction of endothelial progenitor cells (EPCs) has been reported either in aging or diabetes, though the influence of an "old" environment on numerical and functional changes of diabetes associated EPCs is not known. We evaluated the effect of both aging and early stage of streptozotocin-induced diabetes on the number of bone marrow-derived CD117+ progenitor cells, and on their differentiation in vitro toward EPCs. The phenotype of progenitor cells and the uptake of acetylated-low density lipoprotein (Ac-LDL) were evaluated after cell culture in VEGF, FGF-1, and IGF-1 supplemented medium. Hyperglycemia similarly reduced the number of CD117+ cells both in young and old mice. CD117+ cells from young mice differentiated better than those from old animals "in vitro", with a greater reduction of CD117+ cells and an higher increase of CD184+VEGFR-2+ cells. In diabetic mice, in vitro CD117+ cells differentiation was significantly reduced in young animals. Diabetes did not impact on the scarce differentiation of CD117+ cells from old mice. Hyperglycemia reduced the uptake of acLDL by EPCs greatly in young than in old mice. These findings indicate that part of the EPCs functional alterations induced by hyperglicemia in young mice are observed in normal aged mice.

We investigated the efficacy of colistin combined with pexiganan in experimental mouse models of Acinetobacter baumannii infection.Adult male BALB/c mice received intraperitoneally 1 mL saline containing 2 × 10 CFU of susceptible and multiresistant A. baumannii. Two hours after bacterial challenge, animals received 1 mg/kg of colistin, 1 mg/kg of pexiganan, or 1 mg/kg of colistin plus 1 mg/kg of pexiganan.Blood culture positivity, the quantities of bacteria in the intra-abdominal fluid, the rate of lethality and immunological studies, such as immunophenotyping and NK cytotoxicity, were evaluated.In the in vitro study, A. baumannii showed susceptibility to colistin and pexiganan and a strong synergy was observed by testing colistin combined with pexiganan with fractionary inhibitory concentration index of 0.312 for both strains.In the in vivo study colistin or pexiganan alone showed a good antimicrobial efficacy. When colistin was combined with pexiganan, the positive interaction produced low bacterial counts that were statistically significant versus singly treated groups. For both strains the highest rate of survival was observed in combined-treated groups (90%).Pexiganan increased NK cytotoxic activity over the levels of infected and colistin-treated animals.In conclusion, pexiganan combined with colistin was found to be efficacious against A. baumannii infection.

Background: Pseudomonas aeruginosa is a gram-negative pathogen, associated with a severe mortality rate. It is also difficult to treat due to numerous resistance mechanisms to a wide range of antibiotics. Objective: Evaluate the activity of pexiganan, an antimicrobial peptide, in combination with two clinical antibiotics (azithromycin and tigecycline) that are not active against P. aeruginosa. Methods: Ten clinical P. aeruginosa were isolated from urinary tract infections, blood culture, skin infections and respiratory tract infections. Minimum inhibitory concentrations (MICs) and synergies were evaluated by broth microdilution, checkerboard assays and time-kill studies. In vitro synergy was confirmed with an in vivo experiment using a murine model of sepsis. Results: Pexiganan MICs were included between 2 and 16 mg/L. Tigecycline and azithromycin MICs were high as expected (4-64 mg/L and 32-256 mg/L, respectively). Pexiganan and azithromycin combination resulted to be additive or indifferent while tigecycline and pexiganan combination was synergic against seven out of ten P. aeruginosa and additive against the other strains. In vivo experiment confirmed the in vitro synergy, denoting a significative reduction of bacteria in mice treated with pexiganan and tigecycline combination. Conclusion: Antimicrobial peptides are molecules that could be useful in the fight against infections and pexiganan seems to be one of the most promising. Our results demonstrated that, in association with tigecycline, pexiganan administration could overcome antibiotic resistance and increase the effectiveness of treatment against P. aeruginosa sepsis.

Complicated skin and soft tissue infections constitute a heterogeneous group of severe disorders, with surgical site infections being the most common hospital-acquired ones. The aim of our study was to investigate the synergistic and bactericidal activities of antimicrobial combinations of fosfomycin with rifampicin and tigecycline against Enterococcus faecalis, Enterococcus faecium and methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates, and also to evaluate their in vivo effects in a mouse wound infection model. In in vitro studies, the combinations of fosfomycin with rifampicin and tigecycline were both synergistic. These synergies were confirmed in in vivo studies: the drug combinations showed the highest antimicrobial effects compared to monotherapy. In conclusion, the efficacy of fosfomycin combinations, also confirmed in our in vivo model, may suggest new directions in the treatment of infected skin and a possible alternative way to control bacterial skin infection.

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 × 10 7 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 10 7 CFU/ml to about 10 3 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.

Cancer vaccines are less effective at old than at young age because of immunosenescence. Besides, in preliminary observations we showed that the immunization with HER-2/neu DNA plasmid in transgenic young mice (standard immunization, SI) delays but not abrogate spontaneous mammary tumours progressively appearing during aging. In this study we evaluated whether booster immunizations (BI) of HER-2/neu transgenic mice with HER-2/neu DNA plasmids every 6 (ECD6), 3 (ECD3), or 1.5 (ECD1.5) months after SI induce a protective immunity that could be maintained over life span. The long term BI significantly improved the effect of SI increasing the number of tumour free mice at 110 weeks of age from 13% (SI) to 58% (BI). Both the number and the volume of tumour masses were reduced in BI than in SI groups. The protective effect of BI was associated with increased antibody production with isotype switching to IgG2a, augmented CD4 T cells, and increased in vivo cytotoxicity of HER-2/neu specific cytotoxic T lymphocytes, mainly in ECD1.5 and ECD3 groups. The transfer of sera from ECD1.5 mice to untreated HER-2/neu mice highly protected against tumour development than sera from SI mice. We conclude that BI induce a protective immunity effective over life span.

Background Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of skin and soft-tissue infection worldwide. An adequate immune response acts as a first line of defence against infections and therefore plays an essential role in the maintenance of health. Tocotrienols (T3s), the lesser known isomers of vitamin E, possess many biological properties and have been recognized as immunomodulators. Purpose The aim of this study was to investigate whether the in vivo supplementation with a mixture of 87.1% δ- and 12.9% γ-T3s extract from seeds of Bixa orellana, (T3s) could be effective in increasing the effect of daptomycin (DAP) in a mouse model of wound infection due to MRSA. Study design/methods Bacteria were inoculated onto full-thickness wound on the dorsal side of BALB/c mice at 5 × 106 CFU per mouse. Mice were randomized into five groups: an uninfected group, an infected-untreated group, a T3s-pretreated group with no antibiotics given after challenge, a T3s-pretreated group plus DAP given after challenge, a group only given DAP after challenge. Main outcome measures were: bacterial load on the wounds, analysis of Natural Killer (NK) cytotoxicity, immunological phenotype and markers of tissue repair. Results Our results showed that bacterial load in wounds from mice receiving T3s or DAP alone was 1- or 3-log10 lower, respectively, compared with the infected-untreated group. T3s plus daptomycin showed the highest efficacy, achieving a 4-log10 decrease in bacterial load. This higher antimicrobial effect was associated with increased levels of NK cytotoxicity and markers of wound repair. Conclusion These data suggest that treatment with T3s may be useful for the management of infected wounds as immune adjuvants in combination with DAP.

The aim of our study was to evaluate the in vitro activity of IB-367 and its bactericidal effect for Pseudomonas aeruginosa and Escherichia coli, associated to a synergic study to test the antibiotic combinations between the peptide and colistin or imipenem. Minimum inhibitory concentrations (MICs), the minimum bactericidal concentrations (MBCs), the synergy test and killing study were carried out to evaluate the IB-367 activity. In the in vivo model, a wound was incised through the panniculus carnosus of BALB/c mice, and then inoculated with 5 × 107 colony-forming units of P. aeruginosa and E. coli. For each strain, the study included an infected or not infected group that did not receive any treatment, and five contaminated groups treated with local IB- 367, intraperitoneal imipenem, intraperitoneal colistin, topical IB-367 local plus intraperitoneal imipenem or intraperitoneal colistin. All isolates were inhibited by IB-367 at concentrations of 4-64 mg/l. Killing by IB-367 was shown to be very rapid: its activity on all Gram-negative bacteria was completed within a 40 min exposure period at a concentration of 2 × MIC/l. Synergy was demonstrated when IB-367 was combined with colistin or imipenem. In in vivo studies, the groups treated with topical IB-367 and intraperitoneal colistin showed the best results in terms of bacterial load inhibition either for Pseudomonas or for E. coli. The good in vitro activity and in vivo efficacy, as well as, the synergic interactions with antibiotics suggest that IB-367 is a promising candidate for potential application in the treatment of wound Gram-negative infections.

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.

In staphylococci, quorum sensing regulates both biofilm formation and toxin production, moreover it has been demonstrated to be inhibited by RNAIII inhibiting peptide (RIP). Aim our study was to evaluate the in vitro activity and its in vivo efficacy of the combined administration of FS10, a novel RIP derivative, and tigecycline in an animal model of methicillin-resistant (MR) and methicillin-sensitive (MS) Staphylococcus aureus wound infection. Using a 1.x2 cm template, one full thickness wound was established through the panniculus carnosus on the back subcutaneous tissue of each animal. Infection was determined by inoculation of 5x107 CFU/ml of bacteria, that produced an abscess within 24 h, after this, treatment was initiated. The study included, for each strain, a control group without infection, a control infected group that did not receive any treatment and a control infected group with drug-free foam dressing, and three infected groups treated, respectively, with: FS10-soaked foam dressing (containing 20 μg FS10), daily intraperitoneal tigecycline (7 mg/Kg), FS10-soaked foam dressing (containing 20 μg FS10) and daily intraperitoneal injections of tigecycline (7 mg/Kg). The main outcome measures were quantitative culture and histological examination of tissue repair. The highest inhibition of infection was achieved in the group that received FS10-soaked and parenteral tigecycline reducing the bacterial load from 107 CFU/ml to about 103 CFU/g for MSSA and to about 104 CFU/g for MRSA. The group treated with FS10-soaked foam dressing associated with parenteral tigecycline showed, histologically, better overall healing with epithelialization and collagen scores significantly higher than those of the other groups in both strains. In conclusion, the combined use of topical FS10 with i.p. tigecycline induced positive interaction in vivo, resulting in an enhanced therapeutic benefit versus staphylococcal infections in murine wound models.