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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Inflammation, aging, and cancer vaccines.

Immunosenescence is characterized by a series of changes of immune pathways, including a chronic state of low-grade inflammation. Mounting evidence from experimental and clinical studies suggests that persistent inflammation increases the risk of cancer and the progression of the disease. Cancer vaccination, which came into view in the last years as the most intriguing means of activating an immune response capable of effectively hampering the progression of the preclinical stages of a tumour, has been shown to be less effective in older age than in young adults. Available evidence on the use of inhibitors of inflammation has indicated their potential enhancement of cancer vaccines, suggesting the possibility to improve the low effectiveness of cancer vaccines in old age employing pharmacological or natural compounds-based anti-inflammatory intervention. This review addresses the effects of age and inflammation on cancer development and progression, and speculates as to whether the modulation of inflammation may influence the response to cancer immunization.

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.

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.

Increased urokinase-type plasminogen activator receptor and epidermal growth factor receptor in serum of patients with prostate cancer.

PURPOSE: Prostate cancer cell motility and invasion have been linked to the up-regulated signaling of epidermal growth factor receptor and urokinase-type plasminogen activator receptor. We analyzed the expression of serum urokinase-type plasminogen activator receptor and epidermal growth factor receptor in the serum of patients with clinical suspicion of prostate cancer to evaluate the possible role as prostate cancer markers. MATERIALS AND METHODS: Serum was collected from 79 consecutive patients referred to our institution for transrectal ultrasound guided prostate biopsy. All blood samples were obtained before prostate biopsy. Total urokinase-type plasminogen activator receptor and epidermal growth factor receptor antigen in serum were measured by specific enzyme-linked immunosorbent assays. Gleason score, the number of positive cores, maximum percent of cancer and inflammation were considered on biopsy. Patients determined to have prostate adenocarcinoma underwent radical retropubic prostatectomy. Gleason score, pathological stage (extraprostatic extension), surgical margins, seminal vesicle involvement, perineural infiltration, lymphovascular invasion and cancer volume were evaluated in radical retropubic prostatectomy specimens. RESULTS: The 30 patients with prostate cancer had significantly higher levels of serum urokinase-type plasminogen activator receptor and epidermal growth factor receptor in comparison to those without prostate cancer but not significantly higher levels of prostate specific antigen. Urokinase-type plasminogen activator receptor and epidermal growth factor receptor levels closely correlated in the serum of patients with prostate cancer. In a multivariate model high serum epidermal growth factor receptor increased the probability of positive biopsies by 1.9 times. ROC analysis revealed that serum epidermal growth factor receptor had 93.3% sensitivity and 98% specificity for detecting prostate cancer at a cutoff of 67.9 ng/ml. Urokinase-type plasminogen activator receptor and epidermal growth factor receptor were significantly higher in patients with extraprostatic extension, seminal vesicle involvement and perineural infiltration in the radical retropubic prostatectomy specimens. Serum urokinase-type plasminogen activator receptor was the only independent predictive serum marker of extraprostatic extension, seminal vesicle involvement and perineural infiltration. CONCLUSIONS: The measurement of urokinase-type plasminogen activator receptor and epidermal growth factor receptor in the serum of patients with clinical suspicion of prostate cancer might provide clinically relevant information on the state of the prostate gland. Measuring serum epidermal growth factor receptor could help predict which patients have prostate cancer, while serum urokinase-type plasminogen activator receptor over expression seems to be related to tumor extraprostatic spread.

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.

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.