Fluorescence labeled PEI-based gene delivery systems for near infrared imaging in nude mice

Gene therapy is a research area where nucleic acids are transferred into cells to treat neoplastic, metabolic and hereditary diseases. Delivery of genetic material into living organisms can be achieved with viral or non-viral vectors. Viral gene carriers are very efficient but present some major disadvantages due to their pathogenicity and immunogenicity. Nonviral carriers are based on synthetic molecules binding and condensing nucleic acids into small, virus-like particles. The aim of this thesis was to study the biodistribution and tumor targeting properties of non-viral gene vectors based on polyethylenimine (PEI) after systemic injection into mice. The gene vectors were labeled with fluorescent dyes emitting in the near infrared (NIR), which allowed studying their bio-distribution in living animal over time. Owing to its amine groups PEI has a high positive charge density that enables electrostatic interactions with negatively charged nucleic acids and their efficient compaction into nucleic acid-PEI complexes, called polyplexes. The net positive surface charge of these polyplexes permits interactions with negatively charged cell surface molecules, thus leading to their internalization into the cell. To avoid unspecific interactions with blood components and nontarget tissues after intravenous application, polyplexes were shielded with the hydrophilic molecule polyethylene glycol (PEG). PEI-based gene carrier systems were tested on two subcutaneously implanted tumor types: Human Hepatocellular Carcinoma (HUH7) and Murine Neuroblastoma (N2a). HUH7 cells express epidermal growth factor (EGF) receptors, while N2a cells express transferrin (Tf) receptors on their surfaces. To enable targeting of the polyplexes to the tumor cells, polyplexes were generated containing the ligands EGF and Tf for targeting of HUH7 cells and N2a cells respectively. The targeted polyplexes were then intravenously injected into immunodeficient, athymic nu/nu mice in which HUH7 or N2a tumor cells had been previously set under their skin. To monitor the biodistribution of polyplexes throughout the mouse organism and to evaluate their gene delivery capability into the neoplastic cells, polyplexes were labeled with fluorescent dyes (Alexa 750, NIR 797) or near infrared emitting quantum dots (QD), whose fluorescent expression signal was detected and analyzed with a device for imaging in vivo. All fluorescent molecules and quantum dots were biocompatible and non-toxic. They emitted light in the near infrared area of the spectrum, thus avoiding overlapping phenomena with autofluorescent biomolecules or absorption of light by hemoglobin. With all dyes used for polyplex labeling a fluorescent signal could be observed in organs like liver and lung being clearly distinguishable from background fluorescence. Among the fluorescent molecules tested, quantum dots were identified being the most suitable method for in vivo studies, showing the highest signal/noise ratios. PEG-shielding led to best tumor targeting efficiency when administering EGF or Tf-targeted polypelxes in mice bearing HUH7 and N2a tumors respectively. A clear fluorescent signal specific for tumor tissue was detected; the imaging software used allowed quantitative analysis of this signal. For this reason this system is now available for further experimental applications.Gentherapie befasst sich mit der Insertion von Nukleinsäuren in Zellen, um neoplastische, metabolische sowie hereditäre Krankheiten zu behandeln. Der Transfer von genetischem Material in einen lebenden Organismus kann durch die Verwendung viraler oder nicht viraler Vektoren erfolgen. Viren sind zwar sehr effektiv, aber ihre Anwendung birgt auch Probleme, die meist durch ihre Pathogenität und Immunogenität bedingt sind. Nicht virale Vektoren sind synthetische Moleküle, die Nukleinsäuren in kleine, virusähnliche Partikeln einbinden und kondensieren. Das Ziel dieser Doktorarbeit war es die Bioverteilung und die gezielt gegen den Tumor gerichteten Eigenschaften von Genvektoren, die auf Polyethylenimin (PEI) basieren, nach intravenöser Injektion in Mäusen zu untersuchen. Die Genvektoren wurden mit fluoreszierenden Farbstoffen markiert, die Licht innerhalb des Nahinfrarot-Bereiches (NIR) emittieren und im Zeitverlauf die Beobachtung der Bioverteilung in lebenden Tieren erlauben. Dank seiner Aminogruppen hat PEI eine stark positive Ladung, die gute elektrostatische Bindungen zu negativ geladenen Nukleinsäuren erlaubt, mit denen es zum Aufbau eines Nukleinsäure-PEI-Komplexes führt, der Polyplex genannt wird. Die positiv geladene Oberfläche von PEI ermöglicht auch Interaktionen mit negativ geladenen Zellenoberflächen mit der Folge einer Internalisierung des Komplexes. Um unspezifische Interaktionen mit Blutbestandteilen oder anderen als dem Zielgewebe zu verhindern, wurden die Polyplexe mit dem hydrophilen Molekül Polyethylenglycol (PEG) umhüllt. Diese auf PEI-Basis synthetisierten Gencarrier-Systeme wurden dann an der humanen Leberzell Karzinom Linie HUH7 und der Maus Neuroblastom Linie N2a getestet. Die HUH7-Zellinie exprimiert Rezeptoren für den epidermalen Wachstumsfaktor (EGF) und die N2a-Zelllinie Rezeptoren für Transferrin (Tf) auf ihrer Oberfläche. Um ein gezieltes Tumor-Targeting von HUH7- oder N2a-Zellen zu ermöglichen, wurden Polyplexe erstellt, die EGF bzw. Transferrin an ihrer Oberfläche enthalten. Diese oberflächlich modifizierten Komplexe wurden dann intravenös in athymische nu/nu Mäuse verabreicht, die vorher HUH7- oder N2a-Zellen subkutan implantiert bekommen hatten. Um die Verteilung der Polyplexe als experimentelle genetische Medikamente innerhalb des murinen Organismus zu beobachten und die Akkumulation im Tumorgewebe auszumessen, waren sie mit fluoreszierenden Farbstoffen(Alexa 750, NIR 797) oder im Nahinfraroten emittierenden Quantum Dots (QD) markiert, deren Fluoreszenzsignalexpression mittels eines Messungsgerät für in vivo Analysen analysiert wurde. Alle fluoreszierenden Moleküle und Quantum Dots zeigten sich biokompatibel und nicht toxisch und emittierten Licht innerhalb des nahinfraroten Bereichs des Spektrums. Damit konnte die Lichtabsorption durch Hämoglobin ebenso vermieden werden, wie sämtliche Überschneidungsphänomene mit der Fluoreszenz anderer Biomoleküle. Bei allen für die Markierung von Polyplexen verwendeten Farbstoffen und den Quantum Dots wurde ein fluoreszierendes Signal in Organen wie Leber und Lunge beobachtet, das eindeutig von der Hintergrundfluoreszenz unterschieden werden konnte. Unter den getesteten fluoreszierenden Substanzen konnten Quantum Dots als beste Methode für die Fluoreszenzmarkierung festgelegt werden. Hier war das Signal am deutlichsten von störender Hintergrundstrahlung zu unterscheiden. Das PEG-Shielding erlaubte die höchste Effizienz im Tumor-Targeting vor allem bei Applikation von EGF- bzw. Tfmarkierten Polyplexen in Mäusen, die HUH7- bzw. den N2a-Tumor trugen. Ein ausgeprägtes und für Tumorgewebe spezifisches Signal konnte hier beobachtet werden. Die verwendete Imaging-Software für in vivo Applikationen ermöglichte eine quantitative Signalanalyse. Damit steht dieses System für weitere experimentelle Anwendungen zur Verfügung

Acute bacterial skin and skin structure infections in internal medicine wards: old and new drugs

Skin and soft tissue infections (SSTIs) are a common cause of hospital admission among elderly patients, and traditionally have been divided into complicated and uncomplicated SSTIs. In 2010, the FDA provided a new classification of these infections, and a new category of disease, named acute bacterial skin and skin structure infections (ABSSSIs), has been proposed as an independent clinical entity. ABSSSIs include three entities: cellulitis and erysipelas, wound infections, and major cutaneous abscesses This paper revises the epidemiology of SSTIs and ABSSSIs with regard to etiologies, diagnostic techniques, and clinical presentation in the hospital settings. Particular attention is owed to frail patients with multiple comorbidities and underlying significant disease states, hospitalized on internal medicine wards or residing in nursing homes, who appear to be at increased risk of infection due to multi-drug resistant pathogens and treatment failures. Management of ABSSSIs and SSTIs, including evaluation of the hemodynamic state, surgical intervention and treatment with appropriate antibiotic therapy are extensively discussed

Fattori di rischio nell\u2019ambito dell\u2019infezione materno fetale ( IMF ).

The results of microbiologic cultures from 186 vaginal samples obtained from 93 labouring women have been compared with the results of the microbiologic cultures from meconium of their newborns, to test possible risk-factors for IMF. The high frequency of positive cultures from vaginal samples (83.3%), constantly with strong bacterial charge, and the proved direct mother to foetus transmission for many potential pathogens (23.6%), cause various interpretative problems and require further studies

The most appropriate therapeutic strategy for acute lower respiratory tract infections: a Delphi-based approach

Lower respiratory tract infections (LRTIs) cause high morbidity and mortality worldwide. Empiric therapy often base the choice of antibiotic treatment on antibacterial spectrum of the agent rather than on its pharmacological properties or the pathogen resistance profile. Inappropriate prescribing leads to therapeutic failure and antibiotic resistance, with increasing direct and indirect health costs. A consensus on appropriate prescribing in LRTI therapy was appraised by this Delphi exercise, based on a panel of 70 pulmonologists, coordinated by a Scientific Committee of nine experts in respiratory medical care. Full or very high consensus was reached on several issues, including the role of oral cephalosporins in first-line treatments of LRTIs and the appropriateness of cefditoren, with balanced spectrum and high intrinsic activity, in LRTI treatment. Evidence-based medicine approach and a comprehensive process of disease management, from diagnosis to therapy and follow-up, should guide antibiotic prescribing

Viral growth assay to evaluate the replicative capacity of HIV-1 isolates.

The replicative capacity of HIV is studied by carrying out replication-competition experiments with the insertion of the gene of interest. These assays cannot capture the complicated patterns of mutations of different genes.A cross sectional study was carried out on 10 HIV-infected nai;ve patients and on 15 patients failing HAART. The CD8-depleted PBMCs, with known proviral DNA and cellular HIV-RNA copy numbers, were cultured. A reference curve was determined using the data obtained from 10 nai;ve patients. The replicative capacity was calculated as the ratio multiplied by 100 of the p24 antigen level of isolates over the p24 antigen level determined on the reference curve.A linear correlation between p24 antigen level and the infectious doses of HIV-DNA alone or plus cellular RNA copy number of PBMCs was found in naive patients (r=0.63, P<0.001 and r=0.67, P<0.001, respectively). Although all patients failing therapy had strains with impaired replicative capacity, a wide range of values (0.1-74.5%) was detected. All strains with a replicative capacity above 10% had non-nucleoside reverse transcriptase inhibitors related mutations.A viral assay to evaluate the HIV replicative capacity is described. The high variability of replicative capacity confirms the need to undertake replicative capacity assay using the whole virus

Replication capacity, biological phenotype, and drug resistance of HIV strains isolated from patients failing antiretroviral therapy

The fitness of human immunodeficiency virus (HIV) in vivo depends on the interaction of a multitude of viral and host factors. The aim of this study was to analyze the biological phenotype and the intrinsic capacity of the HIV isolates with drug-resistance mutations to replicate efficiently in the absence of drugs. An open label multi-center cross-sectional study was undertaken on 28 HIV-infected patients failing antiretroviral treatment. The subjects were studied for CD4(+) cell count, HIV viral load, syncytium-inducing phenotype, genotypic drug-resistance assay, and replication capacity of HIV isolates assessed by co-culture assay. All HIV isolates showed a decreased replication capacity compared with wildtype strains. The lowest replication capacity was detected in HIV strains with more than five drug-resistance mutations. The highest replication capacity was observed in strains carrying the K103N and Y181C primary mutations that emerged after treatment with non-nucleoside analogue inhibitors. Isolates with R5 biological phenotype had a higher number of resistant mutations than X4 isolates (P=0.004). Particularly, the R5 phenotype was detected in all 6 isolates with more than 14 drug-resistance mutations. Patients with R5 strains had plasma viral load similar to patients with X4 strains, but marginally higher CD4(+) cell counts, and their HIV isolates had significantly lower replication capacity of HIV isolates (P=0.008). No patient carrying HIV with a maintained replication capacity had a viral load less than 30,000 copies/ml. In patients failing HAART, the detection of HIV isolates with the R5 biological phenotype correlates with CD4(+) cell count, an impaired replication capacity, and a high number of drug-resistance mutations. (C) 2003 Wiley-Liss, Inc

High Prevalence of M184 Mutation among Patients with Viroimmunologic Discordant Responses to Highly Active Antiretroviral Therapy and Outcomes after Change of Therapy Guided by Genotypic Analysis

Whether highly active antiretroviral therapy (HAART) should be modified in patients with persistent increases in CD4(+) T cells despite detectable viral loads is an unresolved question. Forty-three heavily pretreated human immunodeficiency virus (HIV)-infected patients with virologic failure during HAART were studied before a change of therapy guided by genotypic analysis and during follow-up. Patients with an increase in CD4(+) cell count (>100 cells/ml) over pre-HAART values were considered to be discordant patients (20 individuals), whereas patients with a lower increase or no increase in CD4(+) cell count were considered failing patients (23 individuals). Based on univariate analysis, a high CD4(+) cell count before antiretroviral treatment, homosexual behavior as a risk factor for HIV infection, reduced drug exposure to nonnucleoside reverse transcriptase inhibitors, low replicative capacity of HIV isolates, and more frequent detection of HIV isolates with a non-B subtype, an R5 biological phenotype, and M184V and T215Y/F mutations were factors associated with a discordant response to HAART. Based on multivariate analysis, only the M184V mutation remained significantly associated with a viroimmunologic discordant response (odds ratio, 25.48; 95% confidence interval, 1.43 to 453.93). No difference in lamivudine exposure was found between discordant (95%) and failing (91%) patients. Twelve months after the genotypic analysis-guided change of therapy, 3 discordant (15%) and 6 failing patients (26%) achieved undetectable viral loads (<50 copies/ml), whereas in patients with HIV RNA loads of >500 copies/ml, discordant responses were observed in 5 out of 15 discordant patients and in 4 out of 16 failing patients. A relationship between the M184V mutation and a viroimmunologic discordant response to HAART was found. After the genotypic analysis-driven change of therapy, similar rates of virologic suppression were detected in the two groups

Error in An Italian consensus for invasive candidiasis management (ITALIC)

Introduction: Invasive candidiasis (IC) has primarily been studied in intensive care unit (ICU) patients, although, in reality, a vast majority of these infections occur outside of the ICU. The recent publication of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) guidelines also deal with the non-ICU population, but many uncertainties remain on the management of IC, particularly in non-critically ill patients. Methods: The Italian Society of Antimicrobial Therapy, Società Italiana di Terapia Antimicrobica (SITA), produced practical, hospital-wide recommendations on the management of Candida infection in non-immunocompromised patients in the hospital ward. Results and discussion: Our focus is on patient stratification in terms of risk factors for IC and of clinical severity, emphasising a high index of suspicion to ensure early diagnosis, early treatment and de-escalation when a patient is clinically stable, in order to optimise resource allocation. © 2013 Springer-Verlag

The current role of glycopeptides in the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections in not neutropenic adults: the viewpoint o. group of Italian experts

Staphylococcus aureus is still an important problem in clinical and therapeutic area, worldwide. In Italy, in recent years, methicillin resistance remained stable, yet considerably high, the percentage of strains of MRSA being around 40%. It was deemed interesting and timely to carry ou. consensus conference using the RAND/UCLA method to collect the opinion o. group of experts in infectious diseases on the role of glycopeptides in the management of MRSA infections within several clinical scenarios and namely in pneumonia, bacteremia and endocarditis, joint replacement infections, skin and soft tissue infections, diabetic foot, abdominal infections and central nervous system infections. The scenarios proposed by the Scientific Committee have been validated b. group of experts in infectious diseases and then voted in three meetings of infectious disease specialists. The results obtained on each individual condition were analyzed and therapeutic recommendations on each of these were released