Non-Small Cell Lung Cancer Testing on Reference Specimens: An Italian Multicenter Experience.

INTRODUCTION: Biomarker testing is mandatory for the clinical management of patients with advanced non-small cell lung cancer (NSCLC). Myriads of technical platforms are now available for biomarker analysis with differences in terms of multiplexing capability, analytical sensitivity, and turnaround time (TAT). We evaluated the technical performance of the diagnostic workflows of 24 representative Italian institutions performing molecular tests on a series of artificial reference specimens built to mimic routine diagnostic samples. METHODS: Sample sets of eight slides from cell blocks of artificial reference specimens harboring exon 19 EGFR (epidermal growth factor receptor) p.E746_AT50del, exon 2 KRAS (Kirsten rat sarcoma viral oncogene homologue) p.G12C, ROS1 (c-ros oncogene 1)-unknown gene fusion, and MET (MET proto-oncogene, receptor tyrosine kinase) Δ exon 14 skipping were distributed to each participating institution. Two independent cell block specimens were validated by the University of Naples Federico II before shipment. Methodological and molecular data from reference specimens were annotated. RESULTS: Overall, a median DNA concentration of 3.3 ng/µL (range 0.1-10.0 ng/µL) and 13.4 ng/µL (range 2.0-45.8 ng/µL) were obtained with automated and manual technical procedures, respectively. RNA concentrations of 5.7 ng/µL (range 0.2-11.9 ng/µL) and 9.3 ng/µL (range 0.5-18.0 ng/µL) were also detected. KRAS exon 2 p.G12C, EGFR exon 19 p.E736_A750del hotspot mutations, and ROS1 aberrant transcripts were identified in all tested cases, whereas 15 out of 16 (93.7%) centers detected MET exon 14 skipping mutation. CONCLUSIONS: Optimized technical workflows are crucial in the decision-making strategy of patients with NSCLC. Artificial reference specimens enable optimization of diagnostic workflows for predictive molecular analysis in routine clinical practice.

Blood circulating bacterial DNA in hospitalized old COVID-19 patients.

BACKGROUND: Coronavirus disease COVID-19 is a heterogeneous condition caused by SARS-CoV-2 infection. Generally, it is characterized by interstitial pneumonia that can lead to impaired gas-exchange, acute respiratory failure, and death, although a complex disorder of multi-organ dysfunction has also been described. The pathogenesis is complex, and a variable combination of factors has been described in critically ill patients. COVID-19 is a particular risk for older persons, particularly those with frailty and comorbidities. Blood bacterial DNA has been reported in both physiological and pathological conditions and has been associated with some haematological and laboratory parameters but, to date, no study has characterized it in hospitalized old COVID-19 patients The present study aimed to establish an association between blood bacterial DNA (BB-DNA) and clinical severity in old COVID-19 patients. RESULTS: BB-DNA levels were determined, by quantitative real-time PCRs targeting the 16S rRNA gene, in 149 hospitalized older patients (age range 65-99 years) with COVID-19. Clinical data, including symptoms and signs of infection, frailty status, and comorbidities, were assessed. BB-DNA was increased in deceased patients compared to discharged ones, and Cox regression analysis confirmed an association between BB-DNA and in-hospital mortality. Furthermore, BB-DNA was positively associated with the neutrophil count and negatively associated with plasma IFN-alpha. Additionally, BB-DNA was associated with diabetes. CONCLUSIONS: The association of BB-DNA with mortality, immune-inflammatory parameters and diabetes in hospitalized COVID-19 patients suggests its potential role as a biomarker of unfavourable outcomes of the disease, thus it could be proposed as a novel prognostic marker in the assessment of acute COVID-19 disease.

Effect of Cytomegalovirus Reactivation on Inflammatory Status and Mortality of Older COVID-19 Patients.

Herpesviridae reactivation such as cytomegalovirus (CMV) has been described in severe COVID-19 (COronaVIrusDisease-2019). This study aimed to understand if CMV reactivation in older COVID-19 patients is associated with increased inflammation and in-hospital mortality. In an observational single-center cohort study, 156 geriatric COVID-19 patients were screened for CMV reactivation by RT-PCR. Participants underwent a comprehensive clinical investigation that included medical history, functional evaluation, laboratory tests and cytokine assays (TNF-α, IFN-α, IL-6, IL-10) at hospital admission. In 19 (12.2%) of 156 COVID-19 patients, CMV reactivation was detected. Multivariate Cox regression models showed that in-hospital mortality significantly increased among CMV positive patients younger than 87 years (HR: 9.94, 95% CI: 1.66-59.50). Other factors associated with in-hospital mortality were C-reactive protein (HR: 1.17, 95% CI: 1.05-1.30), neutrophil count (HR: 1.20, 95% CI: 1.01-1.42) and clinical frailty scale (HR:1.54, 95% CI: 1.04-2.28). In patients older than 87 years, neutrophil count (HR: 1.13, 95% CI: 1.05-1.21) and age (HR: 1.15, 95% CI: 1.01-1.31) were independently associated with in-hospital mortality. CMV reactivation was also correlated with increased IFN-α and TNF-α serum levels, but not with IL-6 and IL-10 serum changes. In conclusion, CMV reactivation was an independent risk factor for in-hospital mortality in COVID-19 patients younger than 87 years old, but not in nonagenarians.

Sensibility and Specificity of the VitaPCR™ SARS-CoV-2 Assay for the Rapid Diagnosis of COVID-19 in Older Adults in the Emergency Department.

(1) Background: During the COVID-19 pandemic, rapid and reliable diagnostic tools are needed for detecting SARS-CoV-2 infection in urgent cases at admission to the hospital. We aimed to assess the performances of the rapid molecular VitaPCR™ test (Menarini Diagnostics) in a sample of older adults admitted to the Emergency Department of two Italian hospitals (2) Methods: The comparison between the rapid VitaPCR™ and the RT-PCR was performed in 1695 samples. Two naso-pharyngeal swab samplings from each individual were obtained and processed using the VitaPCR™ and the RT-PCR for the detection of SARS-CoV-2 (3) Results: VitaPCR™ exhibited good precision (<3% CV) and an almost perfect overall agreement (Cohen's K = 0.90) with the RT-PCR. The limit of detection of the VitaPCR™ was 4.1 copies/µL. Compared to the RT-PCR, the sensitivity, the specificity, and the positive and negative predictive values of VitaPCR™ were 83.4%, 99.9%, 99.2% and 98.3%, respectively (4) Conclusions: The VitaPCR™ showed similar sensitivity and specificity to other molecular-based rapid tests. This study suggests that the VitaPCR™ can allow the rapid management of patients within the Emergency Department. Nevertheless, it is advisable to obtain a negative result by a RT-PCR assay before admitting a patient to a regular ward.

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.

Potential application of berberine in the treatment of Escherichia coli sepsis.

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.

Association of HERV-K and LINE-1 hypomethylation with reduced disease-free survival in melanoma patients.

Aim: To evaluate CpG methylation of long interspersed nuclear elements 1 (LINE-1) and human endogenous retrovirus K (HERV-K) retroelements as potential prognostic biomarkers in cutaneous melanoma. Materials & methods: Methylation of HERV-K and LINE-1 retroelements was assessed in resected melanoma tissues from 82 patients ranging in age from 14 to 88 years. In addition, nevi from eight patients were included for comparison with nonmalignant melanocytic lesions. Results: Methylation levels were lower in melanomas than in nevi. HERV-K and LINE-1 methylation were decreased in melanoma patients with clinical parameters associated with adverse prognosis, while they were independent of age and gender. Hypomethylation of HERV-K (but not LINE-1) was an independent predictor of reduced disease-free survival. Conclusion: HERV-K hypomethylation can be a potential independent biomarker of melanoma recurrence.

Cognitive Impairment, Chronic Kidney Disease, and 1-Year Mortality in Older Patients Discharged from Acute Care Hospital.

The prognostic interaction between chronic kidney disease (CKD) and cognitive impairment is still to be elucidated. We investigated the potential interaction of overall cognitive impairment or defective constructional praxis and CKD in predicting 1-year mortality among 646 older patients discharged from hospital. The estimated glomerular filtration rate (eGFR) was calculated using the Berlin Initiative Study (BIS) equation. Cognitive impairment was assessed by the Mini Mental State Exam (MMSE) and defective constructional praxis was ascertained by the inherent MMSE item. The study outcome was 1-year mortality. Statistical analysis was carried out using Cox regression. After adjusting for potential confounders, the co-occurrence of eGFR <30 and overall cognitive impairment (Hazard Ratio (HR) = 3.12, 95% Confidence Interval (CI) = 1.26-7.77) and defective constructional praxis (HR = 2.50, 95% CI = 1.08-5.77) were associated with the outcome. No significant prognostic interaction of eGFR < 30 with either overall cognitive impairment (HR = 1.99, 95% CI = 0.38-10.3) or constructional apraxia (HR = 1.68, 95% CI = 0.33-8.50) was detectable, while only cognitive deficits were found significantly associated with the outcome in the interaction models (HR = 3.12, 95% CI = 1.45-6.71 for overall cognitive impairment and HR = 2.16, 95% CI = 1.05-4.45 for constructional apraxia). Overall cognitive impairment and defective constructional praxis may be associated with increased risk of 1-year mortality among older hospitalized patients with severe CKD. However, no significant prognostic interaction between CKD and cognitive impairment could be observed.

Kidney function and cognitive impairment among older hospitalized patients: a comparison of four glomerular filtration rate equations.

BACKGROUND: The relationship between the estimated glomerular filtration rate (eGFR) and cognitive impairment may change as a function of the equation used. We aimed at investigating the association between four different eGFR equations and cognitive impairment among older hospitalized patients. METHODS: Our series consisted of 795 older patients consecutively admitted to 7 geriatric and internal medicine acute care wards. The eGFR was calculated by Chronic Kidney Disease Epidemiologic Collaboration (CKD-EPI), Cockcroft-Gault (CG), Berlin Initiative Study (BIS) and Full Age Spectrum (FAS) equations. Study outcomes were total Mini Mental State Examination (MMSE) < 24 and sub-scores related to orientation to time, orientation to space, registration, calculation, three words recall, language and constructional praxis. Statistical analysis was carried out by logistic or Poisson regressions when appropriate. The accuracy of eGFR equations in identifying cognitive outcomes was investigated by calculating the area (AUC) under the receiver operating characteristic (ROC) curve for each equation. RESULTS: After adjusting for potential confounders, eGFR < 30 was significantly associated with MMSE < 24 only with CKD-EPI equation (OR 2.03, 95% CI 1.04-3.96). eGFR < 30 was significantly associated with constructional apraxia with all study equations (CKD-EPI: OR 3.62, 95% CI 1.73-7.56; BIS: OR 2.86, 95% CI 1.31-6.26; FAS: OR 2.83, 95% CI 1.44-5.56; CG: OR 2.08, 95% CI 1.09-3.99). The accuracy of eGFR < 30 in identifying patients with defective constructional praxis was poor with all (BIS: AUC 0.54, 95% CI 0.52-0.55; CKD-EPI: AUC 0.55, 95% CI 0.53-0.57; CG: AUC 0.58, 95% CI 0.55-0.61; FAS: AUC 0.56, 95% CI 0.54-0.58). CONCLUSIONS: Constructional apraxia may characterize the cognitive profile of older patients with severe CKD. The accuracy in identifying patients with constructional apraxia is only fair, and studies including other biomarkers of kidney function are needed.

Nutritional Factors Modulating Alu Methylation in an Italian Sample from The Mark-Age Study Including Offspring of Healthy Nonagenarians.

Alu hypomethylation promotes genomic instability and is associated with aging and age-related diseases. Dietary factors affect global DNA methylation, leading to changes in genomic stability and gene expression with an impact on longevity and the risk of disease. This preliminary study aims to investigate the relationship between nutritional factors, such as circulating trace elements, lipids and antioxidants, and Alu methylation in elderly subjects and offspring of healthy nonagenarians. Alu DNA methylation was analyzed in sixty RASIG (randomly recruited age-stratified individuals from the general population) and thirty-two GO (GeHA offspring) enrolled in Italy in the framework of the MARK-AGE project. Factor analysis revealed a different clustering between Alu CpG1 and the other CpG sites. RASIG over 65 years showed lower Alu CpG1 methylation than those of GO subjects in the same age class. Moreover, Alu CpG1 methylation was associated with fruit and whole-grain bread consumption, LDL2-Cholesterol and plasma copper. The preserved Alu methylation status in GO, suggests Alu epigenetic changes as a potential marker of aging. Our preliminary investigation shows that Alu methylation may be affected by food rich in fibers and antioxidants, or circulating LDL subfractions and plasma copper.

Recovery from mild Escherichia coli O157:H7 infection in young and aged C57BL/6 mice with intact flora estimated by fecal shedding, locomotor activity and grip strength.

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.

Efficacy of Pexiganan Combination with Tigecycline in a Mouse Model of Pseudomonas aeruginosa Sepsis.

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.

Anti-inflammatory Activity of Tocotrienols in Age-related Pathologies: A SASPected Involvement of Cellular Senescence.

Tocotrienols (T3) have been shown to represent a very important part of the vitamin E family since they have opened new opportunities to prevent or treat a multitude of age-related chronic diseases. The beneficial effects of T3 include the amelioration of lipid profile, the promotion of Nrf2 mediated cytoprotective activity and the suppression of inflammation. All these effects may be the consequence of the ability of T3 to target multiple pathways. We here propose that these effects may be the result of a single target of T3, namely senescent cells. Indeed, T3 may act by a direct suppression of the senescence-associated secretory phenotype (SASP) produced by senescent cells, mediated by inhibition of NF-kB and mTOR, or may potentially remove the origin of the SASP trough senolysis (selective death of senescent cells). Further studies addressed to investigate the impact of T3 on cellular senescence "in vitro" as well as in experimental models of age-related diseases "in vivo" are clearly encouraged.

Antitumor activity of NAX060: A novel semisynthetic berberine derivative in breast cancer cells.

Breast cancer (BC) is the most common malignancy and the most common cause of cancer death in elderly women. We recently demonstrated that innovative compounds structurally related to and semisynthetically derived from the plant alkaloid berberine represent a promising unexplored resource for novel therapeutic tools in BC therapy. In this study, we analyzed the effectiveness of new 13-dichlorophenylalkyl berberine semisynthetic derivatives (NAX060, NAX103, NAX111, and NAX114) on the viability of BC cell lines. Our results demonstrated that the new compounds effectively inhibited the growth of a variety of human BC cell lines. In particular, the viability of HER-2 overexpressing SK-BR-3 cells was significantly reduced by the treatment with NAX060, the most active compound, in a dose and time-dependent manner. In the same tumor cell line, NAX060 induced a strong increase in sub-G1 population while G0/G1 and G2/M phase cells remarkably decreased. NAX060 withdrawal after 72 h of treatment resulted in an irreversible cell proliferation arrest and increasing cell death. Real-time PCR analyses showed that NAX060 induced the expression of some cell-cycle checkpoint molecules involved in cell senescence such as p21WAF1, p27, p16INK4a, and PAI-1. Furthermore, the HER-2 protein expression and phosphorylation, as well as the level of heparanase expression, were remarkably reduced on SK-BR-3 cells. NAX060 was effective also on HER-2 negative tumor cells, and, in particular, on human triple-negative MDA-MB-231 cells. These data suggest a potential therapeutic effect of NAX060 compound in the management of BC malignancies. Interestingly, NAX060 may represent a new useful tool also in triple-negative BC. © 2018 BioFactors, 44(5):443-452, 2018.

Measuring zinc in biological nanovesicles by multiple analytical approaches.

Exosomes are nanovesicles known to mediate intercellular communication. Although it is established that zinc ions can act as intracellular signaling factors, the measurement of zinc in circulating nanovesicles has not yet been attempted. Providing evidence of the existence of this zinc fraction and methods for its measurement might be important to advance our knowledge of zinc status and its relevance in diseases. Exosomes from 0.5 ml of either fresh or frozen human plasma were isolated by differential centrifugation. A morphological and dimensional evaluation at the nanoscale level was performed by atomic force microscopy (AFM) and Transmission Electron Microscopy (TEM). Energy Dispersive X-Ray Microanalysis (EDX) revealed the elemental composition of exosomes and their respective total Zinc content on a quantitative basis. The zinc mole fraction (in at%) was correlated to the phosphorous mole fraction, which is indicative for exosomal membrane material. Both fresh (Zn/P 0.09 ±â€¯0.01) and frozen exosomes (Zn/P 0.08 ±â€¯0.02) had a significant zinc content, which increased up to 1.09 ±â€¯0.12 for frozen exosomes when treated with increasing amounts of zinc (100-500 µM; each p < 0.05). Interestingly, after zinc addition, the Calcium mole fractions decreased accordingly suggesting a possible exchange by zinc. In order to estimate the intra-exosomal labile zinc content, an Imaging Flow Cytometry approach was developed by using the specific membrane permeable zinc-probe Fluozin-3AM. A labile zinc content of 0.59 ±â€¯0.27 nM was calculated but it is likely that the measurement may be affected by purification and isolation conditions. This study suggests that circulating nano-vesicular-zinc can represent a newly discovered zinc fraction in the blood plasma whose functional and biological properties will have to be further investigated in future studies.

Inducers of Senescence, Toxic Compounds, and Senolytics: The Multiple Faces of Nrf2-Activating Phytochemicals in Cancer Adjuvant Therapy.

The reactivation of senescence in cancer and the subsequent clearance of senescent cells are suggested as therapeutic intervention in the eradication of cancer. Several natural compounds that activate Nrf2 (nuclear factor erythroid-derived 2-related factor 2) pathway, which is involved in complex cytoprotective responses, have been paradoxically shown to induce cell death or senescence in cancer. Promoting the cytoprotective Nrf2 pathway may be desirable for chemoprevention, but it might be detrimental in later stages and advanced cancers. However, senolytic activity shown by some Nrf2-activating compounds could be used to target senescent cancer cells (particularly in aged immune-depressed organisms) that escape immunosurveillance. We herein describe in vitro and in vivo effects of fifteen Nrf2-interacting natural compounds (tocotrienols, curcumin, epigallocatechin gallate, quercetin, genistein, resveratrol, silybin, phenethyl isothiocyanate, sulforaphane, triptolide, allicin, berberine, piperlongumine, fisetin, and phloretin) on cellular senescence and discuss their use in adjuvant cancer therapy. In light of available literature, it can be concluded that the meaning and the potential of adjuvant therapy with natural compounds in humans remain unclear, also taking into account the existence of few clinical trials mostly characterized by uncertain results. Further studies are needed to investigate the therapeutic potential of those compounds that display senolytic activity.

Supplementation with tocotrienols from Bixa orellana improves the in vivo efficacy of daptomycin against methicillin-resistant Staphylococcus aureus in a mouse model of infected wound.

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.

Changes in Zn homeostasis during long term culture of primary endothelial cells and effects of Zn on endothelial cell senescence.

Endothelial cell senescence and Zn nutritional status influence cardiovascular disease. The influence of Zn appears dichotomous, hence it is imperative to understand the relationship with cellular senescence to improve knowledge about the molecular and cellular basis of the disease. Here we aimed to determine: 1) the impact of chronic exposure to a moderately high dose of Zn on senescence of endothelial cells; 2) the changes in Zn homeostasis during the lifespan of primary cultured endothelial cells; and 3) the susceptibility of proliferating and senescent endothelial cells to cell death after short term exposure to increasing doses of Zn and of the Zn chelator TPEN. Chronic exposure to Zn accelerated senescence and untreated cells at later passages, where doubling time had increased, displayed relocation of labile Zn and altered expression of genes involved in the response to Zn toxicity, including SLC30A1, SLC39A6, SLC30A5, SLC30A10 and metallothioneins, indicating that senescent cells have altered zinc homeostasis. Most Zn-dependent genes that were expressed differently between early and late passages were correlated with changes in the expression of anti-apoptotic genes. Short-term treatment with a high dose of Zn leads to cell death, but only in the population of cells at both earlier and later passages that had already entered senescence. In contrast, Zn depletion led to death of cells at earlier but not later passages, which suggests that there are sub-populations of senescent cells that are resistant to Zn depletion. This resistant senescent cell population may accumulate under conditions of Zn deficiency and contribute to vascular pathology.

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.

Booster immunizations with DNA plasmids encoding HER-2/neu prevent spontaneous mammary cancer in HER-2/neu transgenic mice over life span.

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.