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1.
BMC Microbiol ; 15: 207, 2015 Oct 09.
Article in English | MEDLINE | ID: mdl-26453184

ABSTRACT

BACKGROUND: The use of indwelling medical devices is associated with a significant risk of infections by Staphylococcus aureus (S. aureus) which possesses a variety of virulence factors including many toxins and the ability to invade eukaryotic cells or to form biofilm on biotic and abiotic surfaces. The virulence factors above described are often related to proteins exposed on the bacterial surface. Blocking S. aureus colonization may reduce the incidence of invasive infectious diseases. Previously reports evaluated the anti-infective properties of serratiopeptidase (Spep), an extracellular metalloprotease produced by Serratia marcescens ATCC 21074 (E-15), in impairing virulence-related staphylococcal properties, such as attachment to inert surfaces and adhesion/invasion on eukaryotic cells. However, to date its mechanism of action is unknown. METHODS: Spep gene was PCR amplified and cloned into expression vector pET28b(+). The mutant EspepA was constructed from plasmid pET28b-Spep applying the one-step overlap extension PCR strategy. There sulting plasmids were costransformed in EcBL21(DE3) cells with the plasmid pRuW4inh1 harboring the Erwinia chrysanthemi secretion system. Bacterial pellets and supernatants were collected and analyzed by SDS-PAGE and zymography. The unambiguous identification and a detailed structure characterization of both the wild type and the mutant Spep were obtained by mass spectrometric analyses. The resultant supernatants sterilized by filtration were separately used to condition biofilm formation of S. aureus. Quantification was based on crystal violet method. RESULTS: In this work we constructed Spep mutant by substituting the glutamic acid in the catalytic site with a residue of alanine. In this manner we were able to evaluate the anti-biofilm activity of Spep mutant in absence of proteolytic activity. As expected, this mutant did not display protease activity but it retained its anti-biofilm properties, suggesting that this action is independent by enzymatic activity. CONCLUSIONS: New knowledge obtained from data reported in this paper calls attention to a novel mechanism of action of Spep. This protein could be developed as a potential "antipathogenic agent" capable to impair the ability of S. aureus to form biofilm on prostheses, catheters and medical devices, exploiting a mechanism different from the proteolytic activity.


Subject(s)
Anti-Infective Agents/metabolism , Biofilms/drug effects , Metalloproteases/metabolism , Peptide Hydrolases/metabolism , Staphylococcus aureus/physiology , Amino Acid Substitution , Cloning, Molecular , Escherichia coli/genetics , Gene Expression , Mass Spectrometry , Metalloproteases/chemistry , Metalloproteases/genetics , Mutagenesis, Site-Directed , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/isolation & purification , Mutant Proteins/metabolism , Peptide Hydrolases/chemistry , Peptide Hydrolases/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Staphylococcus aureus/drug effects
2.
Int J Immunopathol Pharmacol ; 28(1): 104-13, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25816412

ABSTRACT

Staphylococcus epidermidis is recognized as cause of biofilm-associated infections and interest in the development of new approaches for S. epidermidis biofilm treatment has increased. In a previous paper we reported that the supernatant of Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 presents an anti-biofilm activity against S. epidermidis and preliminary physico-chemical characterization of the supernatant suggested that this activity is due to a polysaccharide. In this work we further investigated the chemical nature of the anti-biofilm P. haloplanktis TAC125 molecule. The production of the molecule was evaluated in different conditions, and reported data demonstrated that it is produced in all P. haloplanktis TAC125 biofilm growth stages, also in minimal medium and at different temperatures. By using a surface coating assay, the surfactant nature of the anti-biofilm compound was excluded. Moreover, a purification procedure was set up and the analysis of an enriched fraction demonstrated that the anti-biofilm activity is not due to a polysaccharide molecule but that it is due to small hydrophobic molecules that likely work as signal. The enriched fraction was also used to evaluate the effect on S. epidermidis biofilm formation in dynamic condition by BioFlux system.


Subject(s)
Biofilms/growth & development , Pseudoalteromonas/physiology , Staphylococcus epidermidis/physiology , Antarctic Regions , Polysaccharides/metabolism , Pseudoalteromonas/metabolism , Staphylococcus epidermidis/metabolism , Surface-Active Agents/metabolism
3.
Minerva Med ; 105(2): 167-74, 2014 Apr.
Article in Italian | MEDLINE | ID: mdl-24727881

ABSTRACT

AIM: Vascular dementia (VaD) is defined as a loss of cognitive function resulting from ischemic, hypoperfusive, or hemorrhagic brain lesions due to cerebrovascular disease or cardiovascular pathology. The main types of VaD are: Small Vessel Disease Dementia (sVAD), Large vessel disease dementia, hypoperfusive-ischemic dementia and hemorragic dementia. The sVAD is divided into two main categories: subcortical ischemic vascular dementia (SIVD) and cortical dementia. Currently, no drugs are approved for the treatment of VaD. This study aimed to determine whether rivastigmine, a second generation cholinesterase inhibitor with selectivity for the CNS, with capacity to inhibit both acetylcholinesterase (AChE) and butyryl-cholinesterase (BuChE), slows the rate of cognitive decline associated with VaD. METHODS: Study subjects were 27 male and 43 female outpatients aged 80.03±6.53 years, with Mini-Mental State Examination (MMSE) score ranging batween 22 and 12, affected by VaD. They were included in the study if they were undergoing pharmacological treatment with acetylsalicylic acid 100 mg for at least six months. Patients were divided into two groups: one group was treated with ASA 100 mg and rivastigmine patch 9.5 mg (Rivastigmine group), the other just with ASA 100 mg (ASA group). All patients were followed for 6 months, with a first evaluation (T0) and a second examination after six mounths of treatment (T1). RESULTS: Statistically data proved as the Rivastigmine group showed constant values at MMSE, compared with patients of the ASA group who experienced decline of their cognitive performances. The same result was found in CDR, ADL, GDS and NPI scales. It is remarkable to underline as Rivastigmine-treated patients had a mean improvement in GDS scales, in comparison with patients of the ASA group who showed a worsening of mood. CONCLUSION: Rivastigmine-therapy improves cognitive performance in elderly with SIVD.


Subject(s)
Acetylcholinesterase , Butyrylcholinesterase , Cholinesterase Inhibitors/therapeutic use , Cognition/drug effects , Dementia, Vascular/drug therapy , Neuroprotective Agents/therapeutic use , Phenylcarbamates/therapeutic use , Aged , Aged, 80 and over , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Aspirin/therapeutic use , Dementia, Vascular/classification , Disease Progression , Female , Humans , Male , Mental Status Schedule , Rivastigmine
4.
Int J Immunopathol Pharmacol ; 27(4): 675-82, 2014.
Article in English | MEDLINE | ID: mdl-25572750

ABSTRACT

Betamethasone is an anti-inflammatory steroid drug used in cases of anaphylactic and allergic reactions, of Alzheimer and Addison diseases and in soft tissue injuries. It modulates gene expression for anti-inflammatory activity suppressing the immune system response. This latter effect might decrease the effectiveness of immune system response against microbial infections. Corticosteroids, in fact, mask some symptoms of infection and during their use superimposed infections may occur. Thus, the use of glucocorticoids in patients with sepsis remains extremely controversial. In this study we analyzed the in vitro effect of a commercial formulation of betamethasone (Bentelan) on several Gram positive and Gram negative bacteria of clinical relevance. It was found to be an inhibitor of the growth of most of the strains examined. Also the effect of betamethasone in combination with some classes of antibiotics was evaluated. Antibiotic-steroid combination therapy is, in such cases, superior to antibiotic-alone treatment to impair bacterial growths. Such effect was essentially not at all observable on Staphylococcus aureus or Coagulase Negative Staphylococci (CoNS).


Subject(s)
Anti-Bacterial Agents/pharmacology , Betamethasone/pharmacology , Gram-Negative Bacteria/drug effects , Gram-Positive Bacteria/drug effects , Anti-Bacterial Agents/administration & dosage , Betamethasone/administration & dosage , Drug Therapy, Combination , Gram-Negative Bacteria/growth & development , Gram-Positive Bacteria/growth & development , Microbial Sensitivity Tests
5.
J Appl Microbiol ; 114(1): 266-77, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23057709

ABSTRACT

AIMS: The purpose of this study was to evaluate the antimicrobial efficacy of five different proteases belonging to two different families on Staphylococcus aureus and Staphylococcus epidermidis strains. METHODS AND RESULTS: We used three serine proteases and two metalloproteases in single species biofilm formation assays and in human cell invasion processes. Following each protease incubation with bacterial cells, surface protein patterns were analysed by SDS-PAGE and zymography. Some differently expressed proteins were identified by mass spectrometry. CONCLUSIONS: The effect of tested proteases on biofilm formation was not related to the protease category but was strain-dependent and was related to the biofilm formation capacity of each staphylococcal strain. Some proteases showed a nonspecific and indiscriminate effect on surface proteins, while others induced a discrete and reproducible action on protein profiles. SIGNIFICANCE AND IMPACT OF THE STUDY: The inhibition of the surface-related virulence factors is a promising avenue to overcome persistent infections caused by bacterial biofilms. To this end, we show here that proteases, in particular the metalloprotease serratiopeptidase, can interfere with adhesion and invasion of eukaryotic cells and biofilm formation in staphylococci and their use could represent a viable treatment for the development of novel combination therapies.


Subject(s)
Biofilms/drug effects , Metalloproteases/pharmacology , Serine Proteases/pharmacology , Staphylococcus aureus/pathogenicity , Staphylococcus epidermidis/pathogenicity , Bacterial Adhesion , Bacterial Proteins/analysis , Biofilms/growth & development , Genes, Bacterial , HeLa Cells , Humans , Membrane Proteins/analysis , Peptide Hydrolases/pharmacology , Staphylococcus aureus/drug effects , Staphylococcus aureus/genetics , Staphylococcus epidermidis/drug effects , Staphylococcus epidermidis/genetics , Virulence
6.
Biometals ; 25(2): 413-21, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22237941

ABSTRACT

Staphylococcus aureus and Staphylococcus epidermidis are the major cause of infections associated with implanted medical devices. Colonization on abiotic and biotic surfaces is often sustained by biofilm forming strains. Human natural defenses can interfere with this virulence factor. We investigated the effect of human apo-transferrin (apo-Tf, the iron-free form of transferrin, Tf) and holo-transferrin (holo-Tf, the iron-saturated form) on biofilm formation by CA-MRSA S. aureus USA300 type (ST8-IV) and S. epidermidis (a clinical isolate and ATCC 35984 strain). Furthermore S. aureus adhesion and invasion assays were performed in a eukaryotic cell line. A strong reduction in biofilm formation with both Tfs was obtained albeit at very different concentrations. In particular, the reduction in biofilm formation was higher with apo-Tf rather than obtained with holo-Tf. Furthermore, while S. aureus adhesion to eukaryotic cells was not appreciably affected, their invasion was highly inhibited in the presence of holo-Tf, and partially inhibited by the apo form. Our results suggest that Tfs could be used as antibacterial adjuvant therapy in infection sustained by staphylococci to strongly reduce their virulence related to adhesion and cellular invasion.


Subject(s)
Bacterial Adhesion/drug effects , Staphylococcus/drug effects , Transferrins/pharmacology , Biofilms/drug effects , Dose-Response Relationship, Drug , HeLa Cells , Humans , Protein Isoforms/pharmacology , Staphylococcus/physiology
7.
Bioorg Med Chem ; 20(2): 920-6, 2012 Jan 15.
Article in English | MEDLINE | ID: mdl-22182580

ABSTRACT

Use of herbal plant remedies to treat infectious diseases is a common practice in many countries in traditional and alternative medicine. However to date there are only few antimicrobial agents derived from botanics. Based on microbiological screening tests of crude plant extracts we identified four compounds derived from Krameria, Aesculus hippocastanum and Chelidonium majus that showed a potentially interesting antimicrobial activity. In this work we present an in depth characterization of the inhibition activity of these pure compounds on the formation of biofilm of Staphylococcus aureus as well as of Staphylococcus epidermidis strains. We show that two of these compounds possess interesting potential to become active principles of new drugs.


Subject(s)
Anti-Bacterial Agents/chemistry , Biofilms/drug effects , Biological Products/chemistry , Plant Extracts/pharmacology , Plants/chemistry , Staphylococcus aureus/physiology , Staphylococcus epidermidis/physiology , Aesculus/chemistry , Anti-Bacterial Agents/pharmacology , Biological Products/pharmacology , Chelidonium/chemistry , Krameriaceae/chemistry , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/metabolism , Microbial Sensitivity Tests , Plant Extracts/chemistry
8.
Int J Immunopathol Pharmacol ; 24(3): 661-72, 2011.
Article in English | MEDLINE | ID: mdl-21978698

ABSTRACT

Staphylococcus aureus is a flexible microbial pathogen frequently isolated from community-acquired and nosocomial infections. The use of indwelling medical devices is associated with a significant risk of infection by this bacterium which possesses a variety of virulence factors, including many toxins, and the ability to invade eukaryotic cells or to form biofilm on biotic and abiotic surfaces. The present study evaluates the anti-infective properties of serratiopeptidase, a secreted protein of Serratia marcescens, in impairing virulence-related staphylococcal properties, such as attachment to inert surfaces and adhesion/invasion on eukaryotic cells. SPEP seems to exert its action by modulating specific proteins. Proteomic studies performed on surface proteins extracted from SPEP-treated S. aureus cultures revealed that a number of proteins are affected by the treatment. Among these we found the adhesin/autolysin Atl, FnBP-A, SecA1, Sbi, EF-Tu, EF-G, and alpha-enolase. EF-Tu, EF-G and alpha-enolase are known to perform a variety of functions, depending on their cytoplasmic or surface localization. All these factors can facilitate bacterial colonization, persistence and invasion of host tissues. Our results suggest that SPEP could be developed as a potential anti-infective agent capable to hinder the entry of S. aureus into human tissues, and also impair the ability of this pathogen to form biofilm on prostheses, catheters and medical devices.


Subject(s)
Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Bacterial Adhesion/drug effects , Membrane Proteins/metabolism , Staphylococcus aureus/drug effects , Bacterial Proteins/metabolism , Biofilms/drug effects , Blotting, Western , Electrophoresis, Polyacrylamide Gel , HeLa Cells , Humans , Microscopy, Electron, Scanning , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Staphylococcus aureus/pathogenicity , Virulence Factors/genetics
9.
Int J Immunopathol Pharmacol ; 23(1): 383-6, 2010.
Article in English | MEDLINE | ID: mdl-20394708

ABSTRACT

The main problem associated with artificial vascular devices is the risk of bacterial infections, mostly sustained by coagulase negative Staphylococci or Staphylococcus aureus. Many efforts have been made to identify materials refractory to bacterial adhesion. The aim of our study is to verify the antimicrobial properties of two kinds of vascular prosthesis to prevent early onset infections and the efficacy of the concomitant action of a systemic antibiotic treatment. Adult male Wistar rats were used. We subcutaneously implanted in four groups a silver-coated prosthesis fragment, and a rifampicin-soaked prosthesis fragment in the remaining four groups. We inoculated in the site of implant a high bacterial burden of S. aureus in four groups and a low burden in the remaining groups. Systemic levofloxacin was administered for seven days in four groups representing the two kinds of prosthesis; after 21 days the rats were sacrificed, prosthesis fragments were sonicated and the corresponding supernatants were plated for bacterial counts. The rifampicin-soaked prostheses explanted from rats treated with levofloxacin were sterile, regardless of the bacterial inoculum. In other groups some prostheses were colonized. In the experimental rat model used, the action of local and systemic antibiotic treatment was able to reduce colonisation of artificial prostheses.


Subject(s)
Antibiotic Prophylaxis , Blood Vessel Prosthesis/adverse effects , Levofloxacin , Ofloxacin/therapeutic use , Rifampin/pharmacology , Silver/pharmacology , Animals , Male , Prosthesis-Related Infections/prevention & control , Rats , Rats, Wistar , Staphylococcal Infections/drug therapy
10.
Int J Immunopathol Pharmacol ; 22(1): 145-51, 2009.
Article in English | MEDLINE | ID: mdl-19309561

ABSTRACT

Herpes simplex virus infections are prevalent viral infections in humans. HSVs are also the most common cause of sporadic viral encephalitis (HSE). Magnetic resonance is the imaging method of choice for HSE because it provides the most sensitive method for detecting early lesions. The objective of this study is to set-up and in vitro test an experimental contrast agent specific for antigens present on HSV-infected cells, bound with a paramagnetic agent detectable by MR imaging. A selected anti-HSV HrFab was labelled with Alexa Fluor 488, 125I and Gd3+Cl6. In order to assess anti-HSV affinity and specificity, ELISA assays were performed. Vero cells infected with HSV strains were visualized by MRI using anti-HSV HrFab/Gd3+Cl6 complex. Results of the ELISA tests demonstrated that the anti-HSV HrFab labelled with Gd3+Cl6 showed similar affinity for the antigens while the 125I immunoconjugate showed reduced affinity. MRI confirmed high affinity and specificity of antibody for the detection of HSV infections.


Subject(s)
Gadolinium , Herpesvirus 1, Human/isolation & purification , Herpesvirus 2, Human/isolation & purification , Magnetic Resonance Imaging/methods , Animals , Antibodies, Viral/immunology , Chlorocebus aethiops , Enzyme-Linked Immunosorbent Assay , Image Enhancement , Immunoglobulin Fab Fragments/immunology , Recombinant Proteins/immunology , Vero Cells
11.
Infection ; 36(5): 467-71, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18791660

ABSTRACT

BACKGROUND: In recent years, several reports have suggested, but never definitely demonstrated that dental units (DU) could be potential sources of viral cross-infections sustained by viral agents including HBV, HCV and HIV. This work aims at assessing the risk of HCV cross-infection by dental unit water lines (DUWLs). MATERIALS AND METHODS: Ten anti-HCV positive viremic patients were submitted to dental treatment on three different DU (one unit fully equipped to minimize viral contamination risk). A PCR method using primers for UTR and E2 regions was used to evaluate HCV RNA presence in DUWLs sprays. A modified RNA extraction protocol was developed to eliminate the risk of low sensibility due to the presence of inhibitors in saliva. Sequences obtained from E2 PCR products amplified from blood and oral fluids were analyzed and compared. RESULTS: Fluids collected from three different DU before treatment were always negative for the presence of HCV RNA; after treatment viral contamination was detected in six out of ten cases in conventional DU, in three out of ten cases on the reduced-retraction DU while was never detected in sprays taken from fully equipped DU. Comparison of E2 region sequences obtained from blood and DUWLs sprays showed identity in each patient. CONCLUSION: Here we demonstrate that fixed DUWLs and handpieces can be contaminated by viral agents and become a vehicle of cross-infection and that a specific online active decontamination system developed for both handpieces and fixed waterlines can eliminate this risk.


Subject(s)
Cross Infection/prevention & control , Dental Equipment/virology , Equipment Contamination/prevention & control , Hepacivirus/isolation & purification , Infection Control, Dental , Cross Infection/virology , Fresh Water/virology , Hepacivirus/genetics , Hepatitis C/prevention & control , Hepatitis C/transmission , Hepatitis C/virology , Humans , Pilot Projects
12.
Clin Microbiol Infect ; 10(12): 1081-8, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15606635

ABSTRACT

Staphylococcus epidermidis is an important cause of catheter-associated infections, which are attributed to its ability to form a multilayered biofilm on polymeric surfaces. This ability depends, in part, on the activity of the icaADBC locus and the icaR gene, which are involved in the production of the polysaccharide intercellular adhesin (PIA) that is functionally necessary for cell-to-cell adhesion and biofilm accumulation. The present study determined: (1) the prevalence of the icaADBC operon in S. epidermidis isolates from catheter-related and other nosocomial infections; (2) the correlation between the presence of this operon, biofilm production and resistance to antibiotics; (3) the expression of ica genes and biofilm production; and (4) the genetic relatedness of the isolates. The results showed that icaRADBC was present in 45% of the isolates included in the study, and that such isolates were significantly more resistant to the main antibiotics tested than were ica-negative isolates. The presence of the entire cluster did not always correlate with biofilm production, determined under different culture conditions, but there was evidence to suggest a correlation when at least two genes (icaAD) were co-transcribed. Eight of 18 ica-positive isolates had the entire operon in the same restriction fragment after pulsed-field gel electrophoresis, but the isolates were not clonal. Estimation of genetic relatedness indicated that ica-positive S. epidermidis isolates belonged to different lineages, distributed in only one of two major clusters, with a genetic distance of c. 0.12.


Subject(s)
Operon/genetics , Staphylococcus epidermidis/genetics , Bacterial Adhesion/genetics , Bacterial Adhesion/physiology , Biofilms/growth & development , Catheters, Indwelling/microbiology , Drug Resistance, Bacterial/genetics , Humans , Operon/physiology , Staphylococcal Infections/microbiology , Staphylococcus epidermidis/physiology
13.
Dig Liver Dis ; 34(8): 570-6, 2002 Aug.
Article in English | MEDLINE | ID: mdl-12502213

ABSTRACT

BACKGROUND: Infection with transfusion transmitted virus, a new member of the Parvoviridae family, has been found in patients both with chronic and fulminant post-transfusion cryptogenic hepatitis. AIM: To evaluate the prevalence and clinical impact of transfusion transmitted virus infection in Italy. PATIENTS AND METHODS: Studies were carried out on 256 patients and control subjects from three centres from Northern, Central and Southern Italy (92 nonA-nonC chronic hepatitis, 10 acute non fulminant cryptogenic hepatitis, 41 hepatitis C virus-related chronic hepatitis and 113 blood donors). Serum transfusion transmitted virus was detected by nested polymerase chain reaction using two overlapping sets of primers. RESULTS: A total of 52 of the 92 patients (54.3%) with chronic cryptogenic liver disease and 17 of the 41 hepatitis C virus chronic hepatitis patients (41.4%) were transfusion transmitted virus-DNA positive. Transfusion transmitted virus co-infection in hepatitis C virus patients was not associated with either a higher severity of liver histology or higher alanine transaminase levels or signs of cholestasis, transfusion transmitted virus was found in 48 out of 113 (42.4%) blood donors. In the majority of samples, transfusion transmitted virus DNA was detected with only one of the two sets of primers used. Genotyping and phylogenetic analysis performed on 21 randomly selected viral isolates showed the presence of both type 1 and type 2 transfusion transmitted virus and allowed identification of two isolates with high homology to genotype 6, described, so far, mostly in Japan. CONCLUSIONS: Transfusion transmitted virus type 1 and 2 infection is common among blood donors and patients with liver disease in Italy. The pathogenic potential of transfusion transmitted virus type 1 and 2 in nonA-nonC hepatitis patients is unlikely but further studies are needed to evaluate the epidemiological and clinical impact of other transfusion transmitted virus subtypes.


Subject(s)
Blood Donors , DNA Virus Infections/epidemiology , Hepatitis, Chronic/virology , Torque teno virus/genetics , Adolescent , Adult , Aged , DNA, Viral/analysis , Female , Hepatitis C, Chronic/virology , Humans , Italy/epidemiology , Male , Middle Aged , Prevalence , Torque teno virus/isolation & purification
14.
Psychiatry Res ; 105(1-2): 45-55, 2001 Dec 15.
Article in English | MEDLINE | ID: mdl-11740974

ABSTRACT

Immunotherapy with interferon-alpha (IFNalpha) may induce depressive symptoms, anxiety and major depression when administered for at least 1-3 months at a dose of 3-10 MUI daily, twice or three times a week. Previously, it has been shown that immunotherapy with interleukin-2 (IL-2) significantly induces the cytokine network, as measured by increases in serum IL-6, IL-10 and the IL-2 receptor (IL-2R), and that the immunotherapy-induced changes in the cytokine network are significantly correlated with the increases in depression ratings. The main aim of this study was to examine the effects of immunotherapy with IFNalpha on the cytokine network in relation to changes in depression and anxiety ratings. Fourteen patients, affected by chronic active C-hepatitis, were treated with IFNalpha (3-6 MUI s.c. three/six times a week for 6 months) and had measurements of serum IFN-gamma (IFNgamma), IL-2, IL-6, IL-6R, IL-8 and IL-10 before starting therapy and 2, 4, 16 and 24 weeks after immunotherapy with IFNalpha. Severity of depression and anxiety were measured with the Montgomery-Asberg Depression Rating Scale (MADRS) and the Hamilton Anxiety Rating Scale (HAM-A), respectively. Repeated measure (RM) design ANOVAs showed significantly higher MADRS and HAM-A scores 2-4 weeks and 4-6 months after starting IFNalpha-based immunotherapy than at baseline. RM design ANOVAs showed significantly higher serum IL-6 and IL-8 levels 2-4 weeks after starting IFNalpha-based immunotherapy and higher serum IL-10 levels 2-4 weeks and 4-6 months after starting therapy than at baseline. There were significant relationships between the IFNalpha-induced changes in serum IL-6 or IL-8 and the depression and anxiety scores. The findings show that IFNalpha-based immunotherapy induces the cytokine network and that IFNalpha-induced increases in IL-6 predicts the development of depressive symptoms. Depressive symptoms following IFNalpha treatment may be secondary to cytokine induction, including that of IL-6.


Subject(s)
Anxiety/chemically induced , Cytokines/blood , Depression/chemically induced , Hepatitis C, Chronic/drug therapy , Immunotherapy , Interferon-alpha/adverse effects , Adult , Anxiety/immunology , Anxiety/psychology , Depression/immunology , Depression/psychology , Dose-Response Relationship, Drug , Drug Administration Schedule , Female , Hepatitis C, Chronic/immunology , Hepatitis C, Chronic/psychology , Humans , Injections, Subcutaneous , Interferon alpha-2 , Interferon-alpha/administration & dosage , Male , Middle Aged , Recombinant Proteins
15.
Dig Dis Sci ; 46(11): 2389-95, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11713941

ABSTRACT

The aim of this study was to analyze of HCV kinetics during interferon treatment administered daily or three times weekly. Seventy-seven naive patients were randomized to two treatment courses starting with four weeks of high-dose interferon administered daily or three times weekly. Twenty-two patients (28.6%) achieved end-of-treatment response and nine (11.7%, four of whom received daily induction) sustained response. The initial decline of viral load was sharper in patients receiving daily induction, but the rates of early RNA clearance were independent of treatment schedule, being higher in patients with genotype non-1. Detectable HCV RNA during treatment predicted nonresponse more significantly than high pretreatment viral load or genotype 1. HCV RNA at week 2 was the best predictor (100% sensitivity in patients receiving daily induction). In conclusion, daily induction increased the HCV decline slope, but not the rate of virological response. HCV RNA at week 2 reliably identified nonresponders.


Subject(s)
Antiviral Agents/administration & dosage , Hepacivirus/isolation & purification , Hepatitis C, Chronic/drug therapy , Interferon-alpha/administration & dosage , RNA, Viral/blood , Alanine Transaminase/blood , Antiviral Agents/therapeutic use , Drug Administration Schedule , Female , Hepatitis C, Chronic/blood , Humans , Interferon alpha-2 , Interferon-alpha/therapeutic use , Male , Middle Aged , Predictive Value of Tests , Recombinant Proteins , Sensitivity and Specificity , Time Factors
16.
Mol Psychiatry ; 6(4): 475-80, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11443537

ABSTRACT

We have shown that treatment with interleukin-2 (IL-2) or interferon-alpha (IFN alpha) may induce depressive symptoms and activation of the cytokine network and that IL-2 treatment may diminish serum dipeptidyl pepdidase IV (DPP IV) activity. DPP IV (EC 3.4.14.5) is a membrane bound serine protease which catalyzes the cleavage of some cytokines and neuroactive peptides which modulate T cell activity. The aims of the present study were to examine the effects of IFN alpha-based immunotherapy on serum DPP IV activity in relation to induction of the inflammatory response system. In 18 patients with chronic active hepatitis C, we determined the Montgomery and Asberg Rating Scale (MADRS), the Hamilton Anxiety Rating Scale (HAM-A), serum DPP IV activity, the kynurenine/tryptophan (K/T) quotient, which is an indicator of cytokine (in particular IFN)-induced catabolism of tryptophan, and serum interleukin-8 (IL-8) before starting therapy and 2, 4, 16 and 24 weeks after immunotherapy with IFN alpha. IFN alpha-immunotherapy significantly suppressed serum DPP IV 2--4 weeks and 16--24 weeks after starting IFN alpha-based immunotherapy. The reduction in serum DPP IV activity was more pronounced 16--24 weeks after starting immunotherapy than after 2--4 weeks. The IFN alpha-induced suppression of serum DPP IV activity was significantly correlated to IFN alpha-induced increases in the MADRS and HAM-A and increases in the K/T quotient and serum IL-8. In conclusion, long-term immunotherapy with IFN alpha suppresses serum DPP IV activity and the immunotherapy-induced changes in DPP IV are related to increases in severity of depression, anxiety and activation of the inflammatory response system.


Subject(s)
Antiviral Agents/therapeutic use , Anxiety Disorders/chemically induced , Depression/chemically induced , Dipeptidyl Peptidase 4/blood , Hepatitis C, Chronic/blood , Hepatitis C, Chronic/drug therapy , Hepatitis C, Chronic/psychology , Interferon-alpha/therapeutic use , Adult , Analysis of Variance , Antiviral Agents/adverse effects , Biomarkers/blood , Dose-Response Relationship, Drug , Female , Hepatitis C, Chronic/genetics , Humans , Inflammation , Interferon alpha-2 , Interferon-alpha/adverse effects , Interleukin-8/blood , Male , Psychiatric Status Rating Scales , Recombinant Proteins , Regression Analysis , Time Factors
17.
J Hepatol ; 30(6): 1023-7, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10406179

ABSTRACT

BACKGROUND/AIMS: Contradictory data have been reported about the predictive value of the variability in interferon sensitivity determining region (ISDR) of hepatitis C virus (HCV) genotype-1b on response to interferon-alpha (IFN-alpha) therapy. The aim of this study was to examine this issue in a series of patients with long-term response to IFN treatment. METHODS: We retrospectively analyzed 24 patients with chronic HCV genotype-1b infection treated with IFN-alpha (total dose median 677, range 216-1350 MU) selected in 6 Italian Liver Units. These patients were defined as true long-term responders (LTR) since they showed persisting biochemical and virological responses to IFN treatment (mean follow-up 38 months). HCV genomes from pretreatment serum samples were amplified and directly sequenced. The ISDR amino-acid sequences obtained were aligned and compared with the published sequence of HCV-J. RESULTS: Amino-acid substitutions were found in 23 of the 24 patients, and 22 of them showed an H to R amino-acid change at codon 2218. Fourteen patients showed only one mutation (at codon 2218), two had 2, five had 3, one had 4 and one had 5 mutations. When we compared the ISDR sequences from the 24 LTR with those of non-responders (NR), we found no significant correlation between the number of mutations and the response to therapy. CONCLUSIONS: Our results demonstrate that the persisting efficacy of IFN treatment in patients with chronic HCV is not related to the number of ISDR amino acid substitutions of the infecting viruses. Further studies are needed to verify whether other NS5A sequences outside the ISDR might be involved in the mechanisms of IFN resistance.


Subject(s)
Hepatitis C, Chronic/genetics , Hepatitis C, Chronic/therapy , Interferon-alpha/therapeutic use , RNA-Dependent RNA Polymerase/genetics , Viral Nonstructural Proteins/genetics , Adult , Aged , Amino Acid Sequence , Codon , Female , Follow-Up Studies , Humans , Interferon-alpha/administration & dosage , Male , Middle Aged , Mutation , Time Factors
18.
Hepatology ; 30(2): 510-6, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10421661

ABSTRACT

A sustained response to standard interferon therapy for chronic hepatitis C has been demonstrated in no more than 25% of patients. To improve interferon alfa (IFN-alpha) antiviral effect, a number of combination therapies with IFNs plus other drugs have been proposed for both relapser and nonresponder hepatitis C virus (HCV)-infected patients. Although the causes of IFN resistance in subsets of HCV-infected patients are unknown, both viral and host factors have been involved, including defects in IFN signal transduction and IFN-alpha/beta receptor down-regulation. Here, we report that nonsteroidal anti-inflammatory drugs (NSAIDs), which have been proposed for IFN-alpha combination therapy in nonresponders, potentiate IFN-alpha signaling. We found that, in the hepatoma cell lines, CCL13/Chang and HepG2, indomethacin, a selective cyclo-oxygenase 1 and 2 (COX-1 and COX-2) inhibitor, increases IFN-alpha stimulation of interferon-stimulated response element (ISRE)-dependent transcription in a dose-dependent manner. Interestingly, maximal potentiation was observed with suboptimal IFN-alpha concentrations. Indomethacin exerts its effects by synergizing with IFN-alpha in inducing STAT1 activation by phosphorylation, without affecting concurrent Jak1 phosphorylation. Our data indicate that blockade of arachidonic acid (AA) metabolism by indomethacin activates a signaling pathway that converges on STAT1 activation to potentiate IFN-alpha-dependent gene activation.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , DNA-Binding Proteins/metabolism , Indomethacin/pharmacology , Interferon-alpha/pharmacology , Trans-Activators/metabolism , Arachidonic Acid/metabolism , Cyclooxygenase Inhibitors/pharmacology , Humans , Phospholipases A/physiology , Phosphorylation , Response Elements , STAT1 Transcription Factor , Transcriptional Activation/drug effects , Tumor Cells, Cultured
19.
J Immunol ; 162(11): 6681-9, 1999 Jun 01.
Article in English | MEDLINE | ID: mdl-10352286

ABSTRACT

CTL responses against multiple hepatitis C virus (HCV) epitopes were detected in 7 of 29 (24.1%) healthy family members (HFM) persistently exposed to chronically HCV-infected patients (HCV-HFM). These precursor CTL were at very low or undetectable frequencies, as determined by limiting dilution analysis. However, when HCV-specific effector CD8+ T cells, freshly isolated from PBMC of HCV-HFM, were assessed by a sensitive enzyme-linked immunospot assay, their frequencies were severalfold higher than those of precursor CTL. These results indicate that the two assays detect two functionally distinct T cell populations and that the effector cells are not assayed by the 51Cr-release assay. Furthermore, the combination of cell depletion and enzyme-linked immunospot analyses showed that the effector cells were confined into a CD8+ CD45RO+ CD28- population. The persistence of effector CD8+ T cells specific for both the structural and nonstructural viral proteins in uninfected HCV-HFM, suggest that: 1) an immunological memory is established upon a subclinical infection without any evidence of hepatitis, in a large cohort of HCV-exposed individuals; 2) because these cells required neither restimulation nor the addition of particular cytokines in vitro for differentiating in effectors, they should be capable of prompt HCV-specific effector function in vivo, possibly providing antiviral protection; and 3) the maintenance of effector T cell responses may be sustained by persisting low-level stimulation induced by inapparent infections.


Subject(s)
Hepacivirus/immunology , Hepatitis C Antibodies/blood , T-Lymphocytes, Cytotoxic/immunology , Adolescent , Adult , Aged , Antigen Presentation , CD28 Antigens/analysis , Cell Line , Cell Separation , Cytotoxicity Tests, Immunologic , Epitopes, T-Lymphocyte/biosynthesis , Epitopes, T-Lymphocyte/metabolism , Female , HLA-A2 Antigen/immunology , HLA-A2 Antigen/metabolism , Hepatitis C Antigens/biosynthesis , Hepatitis C Antigens/metabolism , Humans , Leukocyte Common Antigens/analysis , Leukocytes, Mononuclear/immunology , Lymphocyte Activation , Lymphocyte Count , Male , Middle Aged , Oligopeptides/immunology , Oligopeptides/metabolism , Stem Cells/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocytes, Cytotoxic/metabolism
20.
Dig Dis Sci ; 44(5): 1013-9, 1999 May.
Article in English | MEDLINE | ID: mdl-10235612

ABSTRACT

In chronic hepatitis C the rate of relapse after an end-of-treatment response to interferon may exceed 50%. The usefulness of retreatment of relapsers with interferon in obtaining a complete sustained response and the role of clinical, virological and immunological features in determining long-term efficacy of retreatment are unclear. We aimed to assess the efficacy of interferon retreatment in obtaining a complete sustained response, to evaluate whether increasing the dose may enhance responsiveness, and to identify possible predictors of sustained response. We enrolled 42 patients with biopsy-proven chronic hepatitis C without cirrhosis who had previously responded to a six-month course of Interferon-alpha2b (total dose: group A, 22 patients, 234 MU; group B, 20 patients, 468 MU) and then relapsed. All, except one, were HCV-RNA negative at the end of first cycle of interferon; most (31/42, 74%) were infected by HCV 1b. Subjects were randomly allocated to receive another cycle of interferon either at the original dose (group A1: 234 MU, 11 patients; group B1 468 MU, 10 patient) or twice the original dose (group A2: 468 MU, 11 patients; group B2: 936 MU, 10 patients). At the end of the second cycle of interferon, 24 subjects (57%) had normal ALT and were HCV-RNA negative, and 16 (39%) had normal ALT, but were HCV-RNA positive. A complete sustained response was obtained in eight patients (19%), at a similar rate in all treatment groups. Complete sustained responders were different from the other patients in terms of age (35.9 +/- 10.4 vs 44.1 +/- 8.8, P = 0.027), rate of infection with non-1b HCV (6/8 vs 5/34, P = 0.0005), serum HCV-RNA (74,016 vs 321,428 median copies/ml, P = 0.037) and serum levels of 90K/MAC-2 BP (5.76 +/- 3.01 vs 10.25 +/- 5.16 units/ml, P = 0.02), an N-glycoprotein implicated in cellular defense functions. Multivariate logistic analysis validated age and HCV genotype as independent predictors of CSR. Among noncirrhotic relapsers who received a total interferon dose > or = 234 MU in the first cycle, retreatment usually induced end-of-treatment response. A complete sustained response was obtained in only one of every five subjects. Increasing the dose of interferon above that of the first cycle did not enhance the rate of sustained response. In conclusion we might assert that young subjects infected by non-1b HCV and with low levels of HCV-RNA and of 90K/MAC-2 BP are the best candidates for retreatment.


Subject(s)
Antiviral Agents/therapeutic use , Hepatitis C, Chronic/therapy , Interferon-alpha/therapeutic use , Adult , Antiviral Agents/administration & dosage , Female , Glycoproteins/blood , Hepacivirus/genetics , Humans , Immunoradiometric Assay , Interferon alpha-2 , Interferon-alpha/administration & dosage , Male , Middle Aged , RNA, Viral/analysis , ROC Curve , Recombinant Proteins , Recurrence , Sensitivity and Specificity , Treatment Outcome , Viral Load
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