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Biomedical subjects

M Magnani

Publications and source records attributed to M Magnani.

At least 19 recordsLinked to original sources
PubMed2006

Added value of OCT in evaluating the presence of leakage in patients with age-related macular degeneration treated with PDT.

BACKGROUND: Evaluating the presence of leakage on fluorescein angiography (FA) in patients with age-related macular degeneration (AMD) retreated with photodynamic therapy (PDT) can be difficult. New diagnostic tools such as optical coherence tomography (OCT) might help to optimize PDT management. METHODS: Thirty AMD patients scheduled for regular follow-up FA in conjunction with PDT treatment were also scanned with OCT. Follow-up data at 9 months were retrieved from the patients' medical records. Inter-observer agreement [kappa coefficient] for the presence of leakage on FA, for OCT parameters for leakage, and agreement between FA and OCT evaluations were calculated. The indication for retreatment was evaluated using the leakage analysis based on FA alone, OCT alone, and both examinations combined, and compared to the actual follow-up of the patients at 9 months. RESULTS: Agreement between the two observers for the presence of leakage on FA was moderate (kappa=0.51). OCT agreement between the two observers for the presence of leakage was good (kappa=0.85). Agreement between FA and OCT for the presence of leakage was poor (kappa=0.16). Follow-up data at 9 months on all patients were analyzed. Seven out of 30 patients were not retreated at the time of examination, and four of these patients (57%) remained stable without further treatment. Twenty-three patients did receive a PDT treatment at the time of examination; and eight of these patients did not show leakage on OCT, and five of these patients (62%) remained stable without additional treatment. In contrast, only three out of 15 patients (20%) with leakage on both FA and OCT remained stable during this 9 month follow-up period. CONCLUSIONS: Inter-observer agreement for the presence of leakage was moderate for FA and good for OCT. There was considerable disagreement between leakage as judged by OCT and by FA. OCT could be of help in the decision regarding PDT retreatment. Assuming that 57% of the patients without leakage either on FA or OCT would remain stable without retreatment, the rate of probable ineffective retreatment could be reduced from 35% to 20%.

Aged↗
PubMed2006

A combination of diagnostic tools for rapid screening of ovine listeriosis.

A combined serological and PCR method for the detection of Listeria monocytogenes infection in symptomatic and asymptomatic ovine flocks was evaluated. Seventy-eight milk samples and 157 serum samples were analysed using a L. monocytogenes PCR detection kit and an anti-listeriolysin O IgG immunoassay kit. The combined use of these commercial kits allowed a rapid and effective detection of L. monocytogenes infection in both the early stage, before seroconversion, and in a later phase, even after antibiotic therapy.

Animals↗
PubMed2006

Development of a magnetic capture hybridization-PCR assay for Listeria monocytogenes direct detection in milk samples.

AIMS: A rapid and sensitive method for Listeria monocytogenes direct detection from milk was developed. It is based on a magnetic capture hybridization procedure for selective DNA purification, followed by PCR identification. A comparison with two similar commercial systems from Dynal (Dynabeads) was carried out. METHODS AND RESULTS: The technique used previously developed nanoparticles modified with a 21-mer oligonucleotide. This sequence, sharing homology with all the L. monocytogenes strains, was selected on hlyA gene and located outside the desired specific PCR site to avoid cross-contaminations. Capture probe properties, in term of spacer length and purification, were determined to obtain the highest hybridization efficiency. Its specificity was tested in hybridization experiments with nontarget bacterial species. Any inhibitory effect of the nanoparticles on PCR was also examined. The amplification performed with the purified DNA could reliably identify a 10 CFU ml(-1) contamination rate. CONCLUSIONS: The optimized purification method showed a high specificity and sensitivity, with a detection level one log more sensitive than PCR carried out with nucleic acids obtained using commercial nanoparticles. SIGNIFICANCE AND IMPACT OF THE STUDY: The method, avoiding pre-enrichment, provides a rapid alternative to conventional microbiological detection methods. Furthermore, it is suitable for automation and can be proposed for the screening of a large number of samples.

Adsorption↗
PubMed2006

Capture probe conjugated to paramagnetic nanoparticles for purification of Alexandrium species (Dinophyceae) DNA from environmental samples.

AIMS: To develop a rapid, cost-effective and selective Alexandrium DNA extraction procedure from environmental samples in order to provide good-quality template for the downstream PCR-based detection assay. METHODS AND RESULTS: In this study, we tested a DNA extraction method based on silica-coated, superparamagnetic nanoparticles conjugated to a DNA-capture sequence (probe) complementary to a specific region of 5.8S rDNA of the genus Alexandrium. Cultured Alexandrium catenella cells were used as the harmful algal bloom species for the DNA extraction. Then, a PCR assay was performed with primers specific for the genus Alexandrium to assess the specificity and sensitivity of the nucleic acid extraction method. This method was applied to both cultured and field samples, reaching in both cases a detection limit of one A. catenella cell. CONCLUSIONS: The results suggest that the use of probe-conjugated paramagnetic nanoparticles could be effective for the specific purification of microalgal DNA in cultured or environmental samples, ensuring sensitivity and specificity of the subsequent PCR assays. SIGNIFICANCE AND IMPACT OF THE STUDY: The DNA extraction method optimized in this study represents a progress towards the rapid and efficient direct detection of Alexandrium cells in seawater monitoring. In fact, this method requires no other equipment than a magnet and a hybridization oven and, in principle, can be adapted to different toxic microalgal species and can be automated, allowing the processing of a high number of samples.

Animals↗
PubMed2006

Administration of fludarabine-loaded autologous red blood cells in simian immunodeficiency virus-infected sooty mangabeys depletes pSTAT-1-expressing macrophages and delays the rebound of viremia after suspension of antiretroviral therapy.

A major limitation of highly active antiretroviral therapy is that it fails to eradicate human immunodeficiency virus (HIV) infection due to its limited effects on viral reservoirs carrying replication-competent HIV, including monocytes/macrophages (M/M). Therefore, therapeutic approaches aimed at targeting HIV-infected M/M may prove useful in the clinical management of HIV-infected patients. In previous studies, we have shown that administration of fludarabine-loaded red blood cells (RBC) in vitro selectively induces cell death in HIV-infected M/M via a pSTAT1-dependent pathway. To determine the in vivo efficacy of this novel therapeutic strategy, we treated six naturally simian immunodeficiency virus (SIV)-infected sooty mangabeys (SMs) with either 9-[2-(R)-(phosphonomethoxy)propyl]adenine (PMPA) only, fludarabine-loaded RBC only, or PMPA in association with fludarabine-loaded RBC. The rationale of this treatment was to target infected M/M with fludarabine-loaded RBC at a time when PMPA is suppressing viral replication taking place in activated CD4+ T cells. In vivo administration of fludarabine-loaded RBC was well tolerated and did not induce any discernible side effect. Importantly, addition of fludarabine-loaded RBC to PMPA delayed the rebound of viral replication after suspension of therapy, thus suggesting a reduction in the size of SIV reservoirs. While administrations of fludarabine-loaded RBC did not induce any change in the CD4+ or CD8+ T-cell compartments, we observed, in chronically SIV-infected SMs, a selective depletion of M/M expressing pSTAT1. This study suggests that therapeutic strategies based on the administration of fludarabine-loaded RBC may be further explored as interventions aimed at reducing the size of the M/M reservoirs during chronic HIV infection.

Adenine↗
PubMed2006

Perturbations of cell cycle control in T cells contribute to the different outcomes of simian immunodeficiency virus infection in rhesus macaques and sooty mangabeys.

In contrast to human immunodeficiency virus (HIV) infection of humans and experimental simian immunodeficiency virus (SIV) infection of rhesus macaques (RMs), SIV infection of sooty mangabeys (SMs), a natural host African monkey species, is typically nonpathogenic and associated with preservation of CD4+ T-cell counts despite chronic high levels of viral replication. In previous studies, we have shown that the lack of SIV disease progression in SMs is related to lower levels of immune activation and bystander T-cell apoptosis compared to those of pathogenic HIV/SIV infection (G. Silvestri, D. Sodora, R. Koup, M. Paiardini, S. O'Neil, H. M. McClure, S. I. Staprans, and M. B. Feinberg, Immunity 18:441-452, 2003; G. Silvestri, A. Fedanov, S. Germon, N. Kozyr, W. J. Kaiser, D. A. Garber, H. M. McClure, M. B. Feinberg, and S. I. Staprans, J. Virol. 79:4043-4054, 2005). In HIV-infected patients, increased T-cell susceptibility to apoptosis is associated with a complex cell cycle dysregulation (CCD) that involves increased activation of the cyclin B/p34-cdc2 complex and abnormal nucleolar structure with dysregulation of nucleolin turnover. Here we report that CCD is also present during pathogenic SIV infection of RMs, and its extent correlates with the level of immune activation and T-cell apoptosis. In marked contrast, naturally SIV-infected SMs show normal regulation of cell cycle control (i.e., normal intracellular levels of cyclin B and preserved nucleolin turnover) and a low propensity to apoptosis in both peripheral blood- and lymph node-derived T cells. The absence of significant CCD in the AIDS-free, non-immune-activated SMs despite high levels of viral replication indicates that CCD is a marker of disease progression during lentiviral infection and supports the hypothesis that the preservation of cell cycle control may help to confer the disease-resistant phenotype of SIV-infected SMs.

Animals↗
PubMed2006

Antiviral and immunomodulatory properties of new pro-glutathione (GSH) molecules.

Reduced glutathione (GSH) is present in millimolar concentrations in mammalian cells. It is involved in many cellular functions such as detoxification, amino acid transport, production of coenzymes, and the recycling of vitamins E and C. GSH acts as a redox buffer to preserve the reduced intracellular environment. Decreased glutathione levels have been found in numerous diseases such as cancer, viral infections, and immune dysfunctions. Many antioxidant molecules, such as GSH and N-acetylcysteine (NAC), have been demonstrated to inhibit in vitro and in vivo viral replication through different mechanisms of action. Accumulating evidence suggests that intracellular GSH levels in antigen-presenting cells such as macrophages, influence the Th1/Th2 cytokine response pattern, and more precisely, GSH depletion inhibits Th1-associated cytokine production and/or favours Th2 associated responses. It is known that GSH is not transported to most cells and tissues in a free form. Therefore, a number of different approaches have been developed in the last years to circumvent this problem. This review discusses the capacity of some new molecules with potent pro-GSH effects either to exert significant antiviral activity or to augment GSH intracellular content in macrophages to generate and maintain the appropriate Th1/Th2 balance. The observations reported herein show that pro-GSH molecules represent new therapeutic agents to treat antiviral infections and Th2-mediated diseases such as allergic disorders and AIDS.

Adjuvants, Immunologic↗
PubMed2005

Interleukin 1B gene (IL-1B) and interleukin 1 receptor antagonist gene (IL-1RN) polymorphisms in Helicobacter pylori-negative gastric cancer of intestinal and diffuse histotype.

BACKGROUND: Polymorphisms in the interleukin 1beta gene (IL-1B-31T/C and IL-1B-511C/T single nucleotide changes) and in the interleukin 1 receptor anatagonist gene (IL-1RN2 variable number of tandem repeats) have been studied with respect to gastric cancer susceptibility. Available data support an aetiologic role of these genetic variants in the presence of concomitant Helicobacter pylori infection. Their contribution without H. pylori infection is still an open field of investigation. MATERIALS AND METHODS: IL-1B and IL-1RN polymorphisms were investigated in 138 H. pylori-negative Italian patients with sporadic gastric cancer and 100 H. pylori-negative controls. Unconditional regression with odd ratios (OR) and 95% confidence intervals (CI), haplotype and linkage disequilibrium analyses were used to investigate the association of the polymorphisms with disease. RESULTS: In all gastric cancer cases, carriers of the homozygous IL-1B-511T/T genotype showed a significant risk for the development of the disease (OR 3.2 with 95% CI 1.27-8.05). In cases with intestinal-type gastric cancer, however, both IL-1B-511T and IL-1RN2 alleles were associated with disease. In this subgroup, the odds ratio for carriers of both IL-1B-511T and IL-1RN2 was 6.49 (95% CI 2.07-20.4). Haplotype analysis supported the aetiologic contribution of these alleles in gastric cancer of the intestinal histotype. CONCLUSIONS: In conclusion, IL-1B-511T and IL-1RN2 may contribute to intestinal gastric cancer risk in the absence of concomitant H. pylori infection. In this setting, future epidemiologic studies should consider dietary habits and exposure to carcinogens interacting with pro-inflammatory host genotypes.

Adult↗
PubMed2004

Effect of listeriolysin O-loaded erythrocytes on Mycobacterium avium replication within macrophages.

OBJECTIVE: To evaluate the efficacy of erythrocytes loaded with the haemolytic toxin listeriolysin O against Mycobacterium avium replication within human macrophages. METHODS: Recombinant listeriolysin O was loaded in human erythrocytes by a procedure of hypotonic dialysis and isotonic resealing. Loaded erythrocytes were modified to allow them to be recognized and taken up by human macrophages infected with M. avium. The antimycobacterial activity of the erythrocytes loaded with listeriolysin O was evaluated by supernatant and intracellular cfu counts on days 4 and 7 post-erythrocyte administration. RESULTS: Recombinant listeriolysin O was encapsulated in human erythrocytes to reach final concentrations ranging from 1 to 4 ng/mL of erythrocytes. Erythrocytes loaded with increasing quantities of recombinant protein were able to reduce (at most by 50%) M. avium replication in a dose-dependent fashion when administered to infected macrophages. CONCLUSIONS: Erythrocytes loaded with listeriolysin O are effective against M. avium replication within macrophages. We are confident that the strategy presented could be useful against mycobacteria other than M. avium (such as Mycobacterium tuberculosis and Mycobacterium leprae) by itself or as part of an antimycobacterial treatment.

Bacterial Toxins↗
PubMed2004

Combined analysis of E-cadherin gene (CDH1) promoter hypermethylation and E-cadherin protein expression in patients with gastric cancer: implications for treatment with demethylating drugs.

BACKGROUND: Hypermethylation is studied as a new, relevant mechanism for silencing tumor suppressor genes. It is a potentially reversible epigenetic change and it is the target of novel anticancer compounds with demethylating activity. In this perspective, we investigated E-cadherin gene (CDH1) promoter hypermethylation in gastric carcinomas and its correlation with E-cadherin protein expression. METHODS: Consecutive cases of gastric carcinoma with assessable paraffin-embedded tumor blocks and paired normal mucosa were considered eligible for study entry. CDH1 promoter hypermethylation and E-cadherin protein expression were determined by methylation-specific polymerase chain reaction and immunohistochemistry, respectively. RESULTS: CDH1 promoter hypermethylation was found in 20 out of 70 gastric carcinomas and the epigenetic change occurred in the early, as well as in the locally advanced disease. In five cases, hypermethylation was also detected in the normal mucosa. Eighteen out of 20 hypermethylated tumors were of the diffuse histotype (P=0.0001). Of 24 tumors with reduced or negative E-cadherin expression, 19 were hypermethylated and 5 were unmethylated (P=0.0001). CONCLUSIONS: CDH1 promoter hypermethylation frequently occurs in gastric carcinomas of the diffuse histotype and it is significantly associated with downregulated E-cadherin expression. The knowledge on the hypermethylation status of tumor suppressor genes may be relevant to the development of demethylating drugs and novel chemopreventive strategies in solid tumors.

Adult↗
PubMed2004

Locked nucleic acids (LNA): versatile tools for designing oligonucleotide decoys with high stability and affinity.

Double-stranded oligodeoxynucleotides (ODN) containing the consensus binding sequence of a transcription factor are valuable tools for the manipulation of gene expression at the transcriptional level by means of the decoy strategy. The approach involves flooding the cells with enough ODN decoy to compete for binding of the transcription factor with its consensus sequence in target genes. The technique has been proven effective in vitro and in vivo, suggesting its use in therapy. Therefore, great attention has been recently focused on chemical modifications enhancing biostability of the oligonucleotides so as to improve their pharmacological properties. Unfortunately, benefits in terms of nuclease resistance have been often negated by a decrease in the affinity and/or specificity of transcription factor binding. To circumvent these problems, circular dumbbell and chimeric decoy oligonucleotides, the latter comprising a central stretch of transcription factor recruiting deoxynucleotides flanked by modified nucleotides, have been introduced. Nevertheless, these approaches have the limitation of leaving the ODNs still sensitive to endonuclease cleavage. To address these concerns, we have recently investigated the use of a new class of nucleotide analogs termed locked nucleic acids (LNA) in the designing of decoy molecules for the transcription factor kappaB (NF-kappaB). In this article, we review the advantages of LNA in the design of ODN decoys pointing to the feasibility of introducing modifications in the transcription factor binding sequence in order to obtain molecules with increased stability compared to end-capped ODNs, while remaining efficiently recognized by NF-kappaB.

Binding Sites↗
PubMed2003

Red blood cell-mediated delivery of recombinant HIV-1 Tat protein in mice induces anti-Tat neutralizing antibodies and CTL.

The immunotherapeutic potential of biologically active HIV-1 Tat protein coupled to autologous red blood cells (RBCs) was evaluated in a mouse model. HIV-1 Tat expressed in Escherichia coli and purified to homogeneity was found to be active in viral trans activation and efficiently internalised by monocyte-derived dendritic cells (MDDCs). The product of HIV-Tat biotinylation and coupling to RBCs by means of a biotin-avidin-biotin bridge, (RBC-Tat), showed no trans activation activity and was still efficiently internalized by MDDCs as compared to uncoupled Tat.Balb/c mice were then immunized with 10 microg of soluble Tat in complete Freund's adjuvant or with 40 ng of Tat coupled on RBCs surface and boosted at week 3, 6 and 25 with 5 microg soluble Tat in incomplete Freund's adjuvant or with 20 ng of RBC-coupled Tat, respectively. Anti-Tat antibody response was similar in both groups; however, 2/6 animals immunized with soluble Tat and 6/6 animals immunized with RBC-Tat developed anti-Tat neutralizing antibodies. In addition, at week 28 cytolytic anti-Tat CTLs were detected in all animals although they were slightly higher in mice immunized with RBC-Tat. These results indicate that RBC-mediated delivery of HIV-1 Tat, in amounts 250 times lower than soluble Tat, is safe and induces specific CTL responses and neutralizing antibodies.

AIDS Vaccines↗
PubMed2002

Macrophage protection by addition of glutathione (GSH)-loaded erythrocytes to AZT and DDI in a murine AIDS model.

Monocyte-macrophages play a central role in HIV-1 infection because they are among the first cells to be infected and because later they are important reservoirs for the virus. Thus, newly designed therapies should take into account the protection of this cell compartment. Herein, we report the results obtained in a murine AIDS model, by the addition to AZT+DDI of a system (GSH-loaded erythrocytes) able to protect macrophages against HIV-1 infection. Five groups of LP-BM5-infected mice were treated as follows: one group was treated by AZT, one group was treated by DDI, one group was treated by the combination of both, another by GSH-loaded erythrocytes, and finally, one by the combination of all three. After 10 weeks of infection the parameters of the disease were studied and the proviral DNA content in different organs and in macrophages of bone marrow and of the peritoneal cavity was quantified. The results obtained show that mice treated with AZT+DDI+GSH-loaded erythrocytes showed proviral DNA content in the brain and in macrophages of bone marrow that was significantly lower than in mice treated with AZT+DDI. This study may help developing strategies aimed at blocking HIV-1 replication in its reservoirs in the body.

Animals↗
PubMed2002

Erythrocyte-mediated delivery of drugs, peptides and modified oligonucleotides.

An important determinant for the success of every new therapy is the ability to deliver the molecules of interest to the target cells or organ. This selective delivery is even more complex when the therapeutic agents are peptides, modified oligonucleotides or genes. In this paper we summarize the possibility of using autologous erythrocytes for the delivery and targeting of new and conventional therapeutics. In fact, a number of macromolecules can be encapsulated by different procedures into human erythrocytes. These modified cells can then be re-infused into the same or a compatible recipient where they can circulate for several weeks. However, drug-loaded erythrocytes can also be modified to be selectively recognized by tissue macrophages. These phagocyte cells recognize the modified drug-loaded erythrocytes which are able to release their content into the macrophage. The feasibility and safety of the use of erythrocytes as drug delivery systems was evaluated in 10 cystic fibrosis patients, where a sustained release of corticosteroids from dexamethasone 21-phosphate-loaded erythrocytes was obtained. In vitro human erythrocytes were found to be able to deliver ubiquitin analogues and modified oligonucleotides to macrophages. Thus, drug-loaded erythrocytes are safe and useful carriers of new and conventional therapeutics and can be advantageous delivery systems for new clinical applications where proteins and oligonucleotides are therapeutic agents.

Cystic Fibrosis↗
PubMed2001

The potency of acyclovir can be markedly different in different cell types.

Acyclovir is an acyclic guanine analog with a considerable activity against herpes simplex viruses. We studied the antiherpetic activity of acyclovir in macrophages and fibroblast cell lines. Utilising a plaque reduction assay we found that acyclovir potently inhibited the HSV-1 replication in macrophages (EC50) = 0.0025 microM) compared to Vero (EC50 = 8.5 microM) and MRC-5 (EC50 = 3.3 microM) cells. The cytotoxicity of acyclovir was not detected at concentrations < or = 20 microM, thus the selective index in macrophages was >8000. This marked difference in antiherpetic activity between macrophages and fibroblasts was not observed with Foscarnet and PMEA. We suggest that this potent antiviral effect of acyclovir is mainly due to a proficient phosphorylation of the drug and/or a favourable dGTP/acyclovir triphosphate ratio in macrophage cells.

Acrylates↗
PubMed2001

Abnormal intracellular kinetics of cell-cycle-dependent proteins in lymphocytes from patients infected with human immunodeficiency virus: a novel biologic link between immune activation, accelerated T-cell turnover, and high levels of apoptosis.

Human immunodeficiency virus (HIV)-infection is characterized by loss of CD4+ T cells associated with high levels of immune activation, T-cell proliferation, and lymphocyte apoptosis. To investigate the role of intrinsic perturbations of cell-cycle control in the immunopathogenesis of acquired immunodeficiency syndrome (AIDS), we studied the expression of cell-cycle-dependent proteins in lymphocytes from HIV-infected patients. Cyclin B1 expression, Nucleolar Organizer Regions (NORs) number, and NORs area of distribution were all consistently increased in HIV-infected patients, but returned to normal after effective antiretroviral therapy, suggesting that viral replication is directly implicated in the genesis of the observed changes. Analysis of cyclin B1 intracellular turnover showed that the increased cyclin B1 expression is (1) caused by defective degradation in the presence of normal rates of synthesis, and (2) is temporally associated with decreased levels of ubiquitination. After in vitro activation of lymphocytes from healthy individuals, cyclin B1 and cdc25 expression and ubiquitination, p34 cdc2 activity, NORs morphology, and C23/nucleolin localization showed a 72- to 96-hour cyclic pattern that led to a biologic state similar to baseline. On the contrary, complex but consistent changes of the same indices followed activation of T lymphocytes from HIV-infected patients, resulting in a 5-fold increase in apoptosis. Overall, our data indicate that a profound dysregulation of cell-cycle control is present in lymphocytes from HIV-infected patients. This finding may provide a novel biologic link between immune activation, accelerated lymphocyte turnover, and increased apoptosis during HIV infection.

Anti-HIV Agents↗
PubMed2001

Identification of the main ubiquitination site in human erythroid alpha-spectrin.

Erythroid spectrin is the main component of the red cell membrane skeleton, which is very important in determining the shape, resistance to mechanical stresses and deformability of red cells. Previously we demonstrated that human erythroid alpha-spectrin is ubiquitinated in vitro and in vivo, and using recombinant peptides we identified on repeat 17 the main ubiquitination site of alpha-spectrin. In order to identify the lysine(s) involved in the ubiquitination process, in the present study we mutated the lysines by site-directed mutagenesis. We found that ubiquitination was dramatically inhibited in peptides carrying the mutation of lysine 27 on repeat 17 (mutants K25,27R and K27R). We also demonstrated that the correct folding of this protein is fundamental for its recognition by the ubiquitin conjugating system. Furthermore, the region flanking lysine 27 showed a 75% similarity with the leucine zipper pattern present in many regulatory proteins. Thus, a new potential ubiquitin recognition motif was identified in alpha-spectrin and may be present in several other proteins.

Amino Acid Sequence↗