Enzyme-Linked Immunospot Assay as a Complementary Method to Assess and Monitor Cytomegalovirus Infection in Kidney Transplant Recipients on Pre-emptive Antiviral Therapy: A Single-Center Experience.

BACKGROUND: Cytomegalovirus (CMV) disease represents a major cause of post-transplantation morbidity and mortality. To estimate the risk of infection and monitor response to antiviral therapy, current guidelines suggest combination of viral load monitoring with direct assessment of CMV-specific immune response. We used enzyme-linked immunospot (ELISpot) for the evaluation of CMV-specific T-cell response in kidney transplant recipients with CMV viremia and investigated how information gained could help manage CMV infection. METHODS: Seventeen patients on pre-emptive antiviral therapy and CMV quantitative polymerase chain reaction (qPCR) ≥500 copies/mL (first episode after transplantation) were assessed using ELISpot and divided into Weak (9 patients with baseline ELISpot <25 spot-forming colonies [SFCs]/200,000 peripheral blood mononuclear cells [PBMCs]) and Strong Responders (8 patients with baseline ELISpot ≥25 SFCs/200,000 PBMCs). CMV-specific T-cell response, infection severity, viral load, and antiviral therapy were prospectively recorded and compared between groups at 1, 2, and 24 months of follow-up. RESULTS: Demographic and transplant characteristics of Weak and Strong Responders were similar. No episodes of CMV disease were observed. Weak Responders were more likely to experience CMV syndrome (56% vs 36.5%) and late virus reactivation (56% vs 25%) than Strong Responders. Weak Responders showed higher baseline median viral loads (19,700 vs 9265 copies/mL) and needed antiviral therapy for longer (179 vs 59.5 days). T-cell response showed 2 main patterns: early and delayed. CONCLUSIONS: ELISpot provides prognostic information about infection severity, risk of late reactivation, and response to therapy. Randomized trials, evaluating the need for antiviral therapy in kidney transplant recipients with asymptomatic infection and effective virus-specific T-cell immune response, are warranted.

Further evidence about the crucial role of CSF biomarkers in diagnosis of posterior cortical atrophy.

Cerebrospinal fluid (CSF) biomarkers (protein tau, phosphorylated tau and amyloid Beta 1-42) are recognized as a supportive feature in diagnosis of Alzheimer's disease (AD) and their role in identifying atypical variants of AD is currently under investigation. We dosed these proteins in nine patients clinically and instrumentally affected by posterior cortical atrophy (PCA), a rare disorder characterized by a progressive neurodegenerative process that involves primarily the posterior brain regions. We compared the obtained values with a large group of AD patients (N = 117), recruited in our neurological department. Our data revealed no differences in the CSF profile between PCA and AD, showing abnormal values of protein tau, phosphorylated tau and amyloid Beta 1-42 in both groups of patients. This study underlines the diagnostic importance of CSF biomarkers in PCA patients, supporting the hypothesis that PCA is an atypical variant of AD with an onset before the age of 65.

HPV infection in squamous cell carcinomas arising from different mucosal sites of the head and neck region. Is p16 immunohistochemistry a reliable surrogate marker?

BACKGROUND: Human papillomavirus 16 infection has been proven to be associated with oropharyngeal squamous cell carcinomas (SCCs) and is probably the main reason of the reported increase in the incidence. The role of high-risk (HR) HPV for carcinogenesis of other sites in the head and neck awaits confirmation. With the aim to evaluate the prevalence of HPV infection and the reliability of different diagnostic tools in SCCs of different sites, 109 consecutive untreated head and neck SCCs were enrolled, and fresh tumour samples collected. METHODS: Human papillomavirus DNA was detected by Digene Hybrid Capture 2 (HC2). Human papillomavirus E6 and E7 mRNA were detected by NucliSENS EasyQ HPVv1. P16 expression was evaluated by immunohistochemistry. RESULTS: In all, 12.84% of cases were infected by HR genotypes and 1.84% by low-risk genotypes. Human papillomavirus 16 accounted for 87% of HR infections. The overall agreement between DNA and RNA detection is 99.1%. Although p16 expression clearly correlates with HPV infection (P=0.0051), the inter-rater agreement is poor (k=0.27). The oropharynx showed the highest HR HPV infection rate (47.6%) and was also the only site in which p16 immunohistochemistry revealed to be a fair, but not excellent, diagnostic assay (κ=0.61). CONCLUSION: The prognostic role of HR HPV infection in oropharyngeal oncology, with its potential clinical applications, underscores the need for a consensus on the most appropriate detection methods. The present results suggest that viral mRNA detection could be the standard for fresh samples, whereas DNA detection could be routinely used in formalin-fixed, paraffin-embedded samples.

One quantitative trait locus for intra- and interspecific variation in a sex pheromone.

Even though premating isolation is hypothesized to be a major driving force in speciation, its genetic basis is poorly known. In the noctuid moth Heliothis subflexa, one group of sex pheromone components, the acetates, emitted by the female, plays a crucial isolating role in preventing interspecific matings to males of the closely related Heliothis virescens, in which females do not produce acetates and males are repelled by them. We previously found intraspecific variation in acetates in H. subflexa: females in eastern North America contain significantly more acetates than females in Western Mexico. Here we describe the persistence of this intraspecific variation in laboratory-reared strains and the identification of one major quantitative trait locus (QTL), explaining 40% of the variance in acetate amounts. We homologized this intraspecific QTL to our previously identified interspecific QTL using restriction-associated DNA (RAD) tags. We found that a major intraspecific QTL overlaps with one of the two major interspecific QTL. To identify candidate genes underlying the acetate variation, we investigated a number of gene families with known or suspected acetyl- or acyltransferase activity. The most likely candidate genes did not map to our QTL, so that we currently hypothesize that a transcription factor underlies this QTL. Finding a single, large QTL that impacts variation in pheromone blends between and within species is, to our knowledge, the first such example for traits that have been demonstrated to affect premating isolation.

The heme oxygenase/biliverdin reductase system: a potential drug target in Alzheimer’s disease.

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the progressive loss of cognitive function, the inability to perform the activities of daily living and psychiatric symptoms. The formation of toxic aggregates of amyloid-beta-peptide (Abeta), through the activities of beta - and gamma- secretases, is considered as the earlier event in the pathogenesis of the disease. The deposition of both Abeta and the following hyperphosphorylation of tau protein, trigger an exaggerate immune-inflammatory response culminating with the production of excess reactive oxygen and nitrogen species responsible for damage on cellular nucleic acids, proteins and lipids. One of the mechanisms used by neural cells to counteract oxidative/nitrosative damage in AD is the enhancement of the cell stress response. Among the main components of the cell stress response is the heme oxygenase/biliverdin reductase (HO/BVR) axis, which catalyzes the degradation of heme which is toxic if produced in excess or under redox unbalanced conditions. However, the HO/BVR system and its by-products, carbon monoxide and bilirubin, have also been shown to be neuroprotective by activating pro-survival pathways and scavenging free radicals. Nevertheless, recent research demonstrated as both the inducible isoform of HO, known as HO-1, and BVR undergo oxidative/nitrosative/phosphorylative post-translational modifications in AD brain which alter the ability of HO-1 and BVR to activate the cell stress response. In this light, naturally occurring substances or drugs (e.g. statins) that prevent the post-translational modifications leading to a controlled up-regulation of the HO/BVR system have been proposed as potential new tools for the treatment of AD.

Dimensional and microbiological in vitro analysis of a dental implant locking taper connection.

The present study was carried out to compare the differences in contact, height and contact area between the implant-abutment interface and the implant-healing cap interface of an implant system featuring a locking tapered connection by using X-ray micro-tomography. It was also conducted to test in vitro whether the implant-healing cap tapered interface is capable of preventing bacterial leakage from the implant well to the external environment. The images of the samples, acquired by the X-ray micro-tomography, after being processed with a dedicated software, showed a greater contact height (CH) in the implant-abutment sample (3.57 mm) compared to the implant-healing cap sample (2.52 mm). This was also true for the contact area that was equal to 40.63 mm2 in the implant-abutment sample and 25.14 mm2 in the implant-healing cap sample. No bacteria were detected both in the nutrient of the test group and of the negative control after 24 h. An increased contact height and contact area in a tapered connection, between the implant and the abutment, have demonstrated to offer mechanical and biological advantages, in a implant-healing cap tapered connection. The major concern regards the microbiological aspects of this connection. The implant-healing cap tapered connection provides an hermetic barrier to microbial passage in vitro, even though such connection features lower contact height and contact area compared to the implant-abutment connection of the same implant system.

Relationship between antiretroviral plasma concentration and emergence of HIV-1 resistance mutations at treatment failure.

PURPOSE: The relationship between antiretroviral pharmacokinetic exposure and acquisition of human immunodeficency virus-1 (HIV-1) drug resistance mutations (DRM) is not fully understood. The aim of this study was to investigate whether antiretroviral plasma concentration could predict the emergence of DRM at treatment failure. METHODS: The study cohort comprised retrospectively selected patients with failing antiretroviral regimens for whom a protease inhibitor (PI) or non-nucleoside reverse transcriptase inhibitor (NNRTI) trough concentration measurement (TDM) had been obtained before failure, a genotypic resistance test (GRT1) had been performed before the TDM, and a genotypic resistance test (GRT2) had been performed at therapeutic failure. Drug levels were classified as undetectable/detectable or subtherapeutic/therapeutic according to limits of quantification of a high-performance liquid chromatography-ultraviolet assay or pre-defined efficacy thresholds, respectively. The number of DRM acquired at treatment failure was evaluated by comparing the results of the GRT2 and GRT1. RESULTS: A total of ten and 57 failure episodes occurred among our patients on NNRTI-based and PI-based regimens, respectively, and included in the evaluation. PI concentration was subtherapeutic in 28.1% of patients, among which the levels were undetectable in 21.1%. Twenty-five (43.9%) patients acquired at least one new PI-DRM according to the GRT2. Patients with undetectable PI levels showed a lower emergence of PI-DRM (minor + major) than those with detectable levels (8.3 vs. 53.3%, p = 0.007). Multivariate analysis confirmed that undetectable PI levels were independent negative predictors of DRM selection. NNRTI measurements were subtherapeutic in 2/10 (20%) patients. NNRTI-DRM were acquired by all patients regardless of NNRTI levels. CONCLUSIONS: A PI measurement showing undetectable drug levels prior to treatment failure predicted the lack of emergence of PI-DRM at failure. These results suggest that PI levels can help clinicians interpret the reasons for treatment failure and guide the type of interventions needed.

Oxidative profile in patients with colon cancer: effects of Ruta chalepensis L.

AIM: To verify the involvement of free radicals in tumor progression and to investigate the effects of an ethanolic extract of Ruta Chalepensis L. and of rutin in blood of patients with colon cancer. MATERIALS AND METHODS: Leaves of Ruta Chalepensis L. were collected in the area around Catania (Italy). For the preparation of the ethanol extract of leaves, an exhaustive extraction of 100 g of the drug was carried out in Soxhlet with 800 ml of 95% ethanol. Fifty-six patients with colorectal cancer were randomly selected for this study; among these, 34 were affected by an early stage (T1 N0 M0 according to scale), while 22 were affected by an advanced stage (T4, N1-2, M0) of cancer. Data obtained from these patients were compared with those of a control group consisting of 20 healthy subjects. Plasma of each sample was used for determining non-proteic antioxidant capacity, thiol groups, lipid hydroperoxides and nitrite/nitrate levels, evaluated by spectrophotometric tests. In addition, percentage of haemolysis was evaluated incubating (for 2 hours at 37 degrees C) erythrocyte suspension with a free radical donor (50 mM 2,2'-azobis-amidino propane chloridrate), in the presence or absence of ethanolic extract of Ruta Chalepensis L. (250 microg/ml) or rutin (1 mM). RESULTS: Non-proteic antioxidant capacity was significantly lower in cancerous patients than in healthy subjects (p < 0.001). This decrease was stage-related. In fact, non-proteic antioxidant capacity resulted lower in advanced than in early colorectal cancer (p < 0.001). The same significant stage-related decrease was observed in plasma thiol groups (p < 0.001). Coherently with the decrease in non-proteic antioxidant capacity and thiol groups, higher levels of lipid hydroperoxides and nitrite/nitrate were observed in patients with colorectal cancer with respect to healthy subjects (p < 0.001) and the increase in these markers of oxidative stress was related to the cancer stadiation. Neoplastic patients also showed an increased percentage of oxidative hemolysis respect to controls and the haemolytic damage was correlated with the stage of colon cancer. Both the extract of Ruta Chalepensis L. and rutin were able to protect erythrocytes from oxidative stress induced by the free radical donor, but the extract of Ruta Chalepensis L. was more effective than rutin. This protective effect was significant only in erythrocytes from patients with early colorectal group, whereas no significant modification was induced by Ruta Chalepensis L. or rutin in red blood cells from advanced colorectal cancer patients exposed to the same experimental conditions. CONCLUSION: Oxidative stress correlates with colon cancer stadiation and both the extract of Ruta chalepensis and rutin are able to protect red blood cells from radical-induced damage. However, their effects are significant in early stages of cancer. So these natural antioxidants might be usefull to prevent carcinogenesis and/or tumor progression.

Examination of periodontal pathogens in stenotic valve specimens and in whole blood samples in patients affected by aortic valve stenosis and chronic periodontitis.

Periodontitis may be a risk factor for atherosclerosis and coronary heart disease. The influence of periodontal pathogens in cardiovascular diseases needs further investigation. Therefore, the aims of this clinical study are: to test the presence of periodontal bacteria DNA in aortic valves and to assess the concomitant presence of the same periodontal bacteria DNA in whole blood samples in patients affected by aortic valve stenosis and chronic periodontitis. Nineteen consecutive patients (12 males and 7 females, age: 49-85 years) were enrolled in this study after having been subjected to a complete periodontal evaluation to confirm the diagnosis of chronic periodontitis. All patients were scheduled for aortic valve replacement surgery. After clinical and microbial periodontal examination, the aortic valve tissue specimens were obtained by excision during valve replacement surgery and the patients were subjected to the whole blood sampling before the surgery. The polymerase chain reaction technology was used to detect the putative periodontal pathogens Tannerella forshytia, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Fusobacterium nucleatum, Campylobacter rectus, Eikenella corrodens and Treponema denticola. Neither the 19 aortic valve specimens nor the blood samples were positive for the genoma of the selected periodontal pathogens. The selected periodontal pathogens did not colonize the aortic valve of patients affected by stenosis and bacterial genoma was not present in whole blood samples. A high blood pressure at the aortic valve may prevent the adhesion and proliferation of bacterial colonies.

Predictors of successful genotype-guided antiretroviral therapy in treatment-experienced individuals over calendar years: a cohort study.

BACKGROUND: The continuous development of new drugs for use in triple-drug combination antiretroviral therapy (cART) has dramatically decreased morbidity and mortality in HIV-1 infected individuals. However, increasing drug resistance could be associated with a poor outcome. OBJECTIVES: To determine the efficacy of resistance genotype-guided antiretroviral regimens in combination antiretroviral therapy (cART)-failing patients over calendar years and its predictors. STUDY DESIGN: Patients, with an available resistance genotype performed between 1999 and 2008, who failed a highly active antiretroviral therapy (HAART) regimen, changed therapy within 6 months from genotype and maintained the same salvage regimen, were selected from a clinical cohort database. Virologic efficacy was analyzed using time-to virologic suppression (VS, HIV-1 RNA<50 copies/ml). RESULTS: In 270 sequences analyzed from 212 patients, after a median follow-up of 23 weeks, there were 160 patients with VS (59.3%). Mean regimens' genotypic sensitivity score (GSS) increased from 1.86 (SD+/-0.92) in 1999-2001, to 2.29 (SD+/-0.96) in 2005-2008 (p=0.001 for trend). VS was achieved in 39% of those patients genotyped in 1999-2001, and increased to 69% for patients with genotyping performed between 2005 and 2008 (p<0.001). More recent calendar year, younger age and less use of suboptimal therapy were predictors of more effective HAART regimens but only more recent calendar year maintained a trend toward significance in a multivariable model. More recent genotyping calendar year, younger age, lower number of HAART regimens experienced, lower HIV-1 RNA and higher GSS independently conveyed and increased the probability of VS. CONCLUSIONS: Resistance-guided salvage antiretroviral therapy was more effective during more recent calendar years, independent from other measurable confounders, including the GSS of the employed regimen. Convenience and tolerability of newer agents should account for the observed effect.

Recurrent Intraventricular Hemorrhages Caused by an AVM Detected by Angiography with Initially Normal Angiogram.

Primary intraventricular haemorrhage (PIVH) is an uncommon type of non-traumatic cerebral haemorrhage limited to the ventricular system arising in adults and children, with diverse aetiology and variable course. Vascular malformations account for 34% of PIVH, whereas no cause is found in 21-47%. When a primary intraventricular haemorrhage is detected in a young patient an underlying lesion such as an arteriovenous malformation (AVM) must be ruled out. The diagnosis may be suspected when there is sudden onset of headache, nausea and vomiting with or without a reduced level of consciousness. Nevertheless, even if clinical features suggest the diagnosis of PIVH, cerebral computed tomography (CT) scan is required for confirmation, and magnetic resonance imaging (MRI) and catheter angiography are necessary to establish the aetiology. We describe a case of isolated recurrent intraventricular haemorrhages caused by AVM detected by diagnostic intracranial angiography with no abnormality demonstrated on angiography four years earlier.

Pro-metastatic signaling by c-Met through RAC-1 and reactive oxygen species (ROS).

Overexpression of the c-Met/hepatocyte growth factor receptor(HGF-R) proto-oncogene and abnormal generation of intracellular oxygen species (reactive oxygen species (ROS)) have been linked, by independent lines of evidence, to cell transformation and to malignant growth. By comparing two subpopulations of the B16 mouse melanoma (B16-F0 and B16-F10) endowed with different lung metastasis capacities (low and high, respectively) we found that both the expression/phosphorylation of c-Met and the steady-state levels of ROS positively correlated with metastatic growth. shRNA-mediated downregulation of c-Met in F10 cells led to a parallel decrease in the generation of oxygen species and in metastatic capacity, suggesting that oxidants may mediate the pro-metastatic activity of the HGF receptor. c-Met activation by a ligand elicits the formation of oxidant species through the oxidase-coupled small GTPase Rac-1, a relevant downstream target of the HGF-R. Moreover, cell treatment with the catalytic ROS scavengers EUK-134 and EUK-189 attenuates Met signaling to ERKs and inhibits the anchorage-independent growth of F10 cells, consistent with a critical role for oxygen species in HGF signaling and in aggressive cell behavior. Finally, genetic manipulation of the Rac-ROS cascade at different levels demonstrated its crucial role in the pro-metastatic activity of c-Met in vivo. Thus, we have outlined a novel cascade triggered by c-Met and mediated by ROS, linked to metastasis and potentially targetable by new antimetastatic, redox-based therapies.

Detection of antibodies to phospholipase B in patients infected with Cryptococcus neoformans by enzyme-linked immunosorbent assay (ELISA).

Secreted phospholipases are virulence factors of several fungi, including Cryptococcus neoformans. We describe for the first time the detection by ELISA of antibodies to cryptococcal phospholipase B in the serum of patients infected with C. neoformans or C. gattii. Sixty-nine sera from 25 patients with cryptococcosis, 23 patients with Candida infections and 26 with bacterial or viral infections were tested. The sensitivity of the ELISA in patients with cryptococcosis was 100% in immunocompetent hosts and 64.3% in immunosuppressed patients with cryptococcal meningitis. Absorbance readings were significantly higher in immunocompetent patients (P<0.001). Titres remained positive for up to 2 years. Positive results were noted in 10 episodes of invasive candidiasis, three patients colonised with Candida, and three cases of bacterial infection. Mean absorbance readings were significantly lower in patients with bacterial infection (P <0.001). We conclude that phospholipase B is produced in vivo during cryptococcal infection and that serum phospholipase B antibodies are a sensitive marker of present or past infection. Cross-reactivity of the ELISA with sera from patients with candidiasis indicates that common epitopes are present on cryptococcal and candidal phospholipase B, hence fungal phospholipase B may constitute a new therapeutic target.

Bacterial and viral DNA in periodontal disease: a study using multiplex PCR.

Recent studies have suggested an association between periodontal disease and the presence of Herpesviruses, in particular: Epstein-Barr virus (EBV) and Human Cytomegalovirus (CMV) (Contreras et al., 1999--Contreras et al., 2000--Slots et al., 2000--Ting et al., 2000). In the work reported in this paper, we use a multiplex Polymerase Chain Reaction (PCR) to compare the presence of Herpesviruses and putative bacterial pathogens in patients with periodontal disease and in healthy individuals. Direct detection of microorganisms with PCR is shown to offer significant advantages in terms of time, effort and cost. The study detected no statistically significant differences between the prevalence of EBV and CMV in patients and controls. The failure to replicate previous findings may be due to differences in the age composition and the geographical and social origins of the study groups. The study detected a significant excess of HSV-1 in periodontal patients. This suggests that the role of Herpesviruses in the pathogenesis of periodontal disease deserves further investigation. The bacterial assay confirmed the results of previous studies showing a strong association between periodontitis and the presence of A. actinomycetemcomitans, P. gingivalis and P. intermedia.