Mario Zucca

Interaction of Bartonella henselae with the Murine Macrophage Cell Line J774: Infection and Proinflammatory Response

ABSTRACT Bartonella henselae is the causative agent of cat scratch disease (CSD), a self-limiting condition characterized by a subacute regional lymphadenopathy that may develop into disseminated bartonellosis in immunocompromised subjects. Mice experimentally infected with B. henselaedisplay typical liver and spleen granulomas rich in T cells and macrophages. So far there are no data on the interaction between bartonellae and macrophages. In order to clarify this topic, we investigated the interaction of B. henselae with J774, a mouse macrophage cell line. Analysis of bacterial uptake by functional assays and transmission electron microscopy indicates that bartonellae can enter and survive inside J774. Entry occurred within 30 min postinfection and reached a plateau at 160 min. Infection of J774 was followed by a dose-dependent release of the proinflammatory cytokines tumor necrosis factor alpha, interleukin 1β (IL-1β), and IL-6. Bartonellae persisted intracellularly without loss of viability for at least 8 h, and their number slightly decreased 24 h postinfection. Gamma interferon (IFN-γ) treatment of J774 significantly decreased the number of recoverable bacteria at 8 and 24 h. This enhancement of macrophage bactericidal activity was associated with nitric oxide (NO) release and was prevented by the addition of the competitive inhibitor of NO synthesis NG-monomethyll-arginine. These findings suggest that IFN-γ-mediated activation of macrophages may be important for the clearing ofB. henselae infection and that anti-B. henselae microbicidal activity of IFN-γ-activated macrophages is mediated to a large extent by NO production.

Novel approaches for the design and discovery of quorum-sensing inhibitors

Introduction: The spread of antibiotic resistance, together with the lack of antibiotics based on novel molecular scaffolds, marks the so-called ‘post-antibiotic era’. Interference with bacterial virulence has emerged as an attractive approach among the current potential strategies for developing new anti-infective drugs. Furthermore, the discovery that virulence gene expression is mostly regulated by quorum sensing (QS) has raised a lot of interest and prompted a lot of research aimed at finding inhibitors of this mechanism. Areas covered: This paper deals with the most recent strategies aimed at discovering new inhibitors able to disrupt the different steps of the QS system, targeting signal production, signal molecules and signal receptors. The authors provide an overview of the literature, including research papers, mostly dealing with inhibitors of the Staphylococcus aureus and Pseudomonas aeruginosa QS systems, and reviews dealing with the application of the newest technologies in the field. They also highlight the fields latest prospects and emerging concerns regarding their possible clinical applications. Expert opinion: QS inhibition is a promising strategy against infections. However, despite the discovery of a huge number of QS inhibitors, with about 40 patents, the potential of QS inhibition is still to be fully assessed. The current validation methods of QS inhibitors must be optimized, and the discovery that QS disruption may favor or select more virulent strains must be investigated in depth. Given the current market-dependent situation, it should be possible to develop hits into licensed drugs through joint ventures between private companies, academia and public institutions.

Imbalance between activin A and follistatin drives postburn hypertrophic scar formation in human skin

Abstract:  Hypertrophic scarring is a skin disorder characterized by persistent inflammation and fibrosis that may occur after wounding or thermal injury. Altered production of cytokines and growth factors, such as TGF‐β, play an important role in this process. Activin A, a member of the TGF‐β family, shares the same intra‐cellular Smad signalling pathway with TGF‐β, but binds to its own specific transmembrane receptors and to follistatin, a secreted protein that inhibits activin by sequestration. Recent studies provide evidences of a novel role of activin A in inflammatory and repair processes. The aim of this study was to evaluate the importance of activin A and follistatin expression in the different phases of scar evolution. Immunostaining of sections obtained from active phase hypertrophic scars (AHS) revealed the presence of a high number of α‐SMA+ myofibroblasts and DC‐SIGN+ dendritic cells coexpressing activin A. Ex‐vivo AHS fibroblasts produced more activin and less follistatin than normal skin or remission phase hypertrophic scar (HS) fibroblasts, both in basal conditions and upon TGF‐βs stimulation. We demonstrate that fibroblasts do express activin receptors, and that this expression is not affected by TGF‐βs. Treatment of HS fibroblasts with activin A induced Akt phosphorylation, promoted cell proliferation, and enhanced α‐SMA and type I collagen expression. Follistatin reduced proliferation and suppressed activin‐induced collagen expression. These results indicate that the activin/follistatin interplay has a role in HS formation and evolution. The impact of these observations on the understanding of wound healing and on the identification of new therapeutic targets is discussed.

Achromobacter xylosoxidans respiratory tract infections in cystic fibrosis patients

Achromobacter xylosoxidans is a ubiquitous Gram‐negative non‐fermenting rod, recently characterized as an emerging pathogen in cystic fibrosis (CF) patients. Its pathogenic potential and prevalent transmission routes are still unclear. This study investigated the PFGE genetic pattern and antimicrobial resistance profile of 42 A. xylosoxidans isolates obtained over 4 years from the respiratory tract of 22 CF patients. By genotypic analysis, 31 isolates were attributed to 8 distinct PFGE patterns (A–H), whereas 11 isolates were not typable because their DNA was not restricted by XbaI and DraI restriction enzymes. The majority of the isolates showed multidrug resistance; imipenem and piperacillin were the most active drugs. During the course of A. xylosoxidans chronic infection forced expiratory volume and body mass index values were not significantly lowered. The demonstration of widespread antibiotic resistance underscores the importance of antibiogram‐directed therapy. Our data suggest that in some cases the infection may have been acquired from other patients or from a common contaminated source. Further epidemiological studies may be important for the design and implementation of prophylactic measures in CF centers.

Current Developments in the Therapy of Protozoan Infections

Protozoan parasites cause serious human and zoonotic infections, including life-threatening diseases such as malaria, African and American trypanosomiasis, and leishmaniasis. These diseases are no more common in the developed world, but together they still threaten about 40% of the world population (WHO estimates). Mortality and morbidity are high in developing countries, and the lack of vaccines makes chemotherapy the only suitable option. However, available antiparasitic drugs are hampered by more or less marked toxic side effects and by the emergence of drug resistance. As the main prevalence of parasitic diseases occurs in the poorest areas of the world, the interest of the pharmaceutical companies in the development of new drugs has been traditionally scarce. The establishment of public-private partnerships focused on tropical diseases is changing this situation, allowing the exploitation of the technological advances that took place during the past decade related to genomics, proteomics, and in silico drug discovery approaches. These techniques allowed the identification of new molecular targets that in some cases are shared by different parasites. In this review we outline the recent developments in the fields of protease and topoisomerase inhibitors, antimicrobial and cell-penetrating peptides, and RNA interference. We also report on the rapidly developing field of new vectors (micro and nano particles, mesoporous materials) that in some cases can cross host or parasite natural barriers and, by selectively delivering new or already in use drugs to the target site, minimize dosage and side effects.

Clinical and Environmental Burkholderia Strains: Biofilm Production and Intracellular Survival

Bacteria belonging to the Burkholderia species are important pulmonary pathogens in cystic fibrosis (CF) patients. Their ability to establish chronic and sometimes fatal infections seems linked to the quorum sensing-regulated expression of virulence factors. We examined 23 Burkholderia isolates, 19 obtained from CF patients and 4 from the environment, to evaluate their ability to form biofilm and to penetrate and replicate inside J774 macrophagic cells. Our results indicate that biofilm formation and intracellular survival are behavioral traits frequently expressed by Burkholderia strains isolated from CF patients. Successive isolates obtained from each of four chronically infected patients yielded bacteria consistently belonging to the same strain but showing increasing ability to replicate intracellularly and to produce biofilm, possibly due to in vivo bacterial microevolution driven by the selective lung environmental conditions. Protection against antimicrobials granted to burkholderiae by the expression of these two virulence factors might account for the frequent failures of antibiotic treatment in CF patients.

Dual regulation of osteopontin production by TLR stimulation in dendritic cells

OPN, a cytokine produced, among others, by DCs, is involved in inflammation and defense against pathogens. Here, we report that the activation of the MyD88 pathway by TLR2, TLR5, and TLR7/8 agonists or IL‐1β induces high levels of OPN in human DCs. Conversely, LPS and Poly I:C, two TLR3 and TLR4 agonists that engage the TRIF pathway, were ineffective. TLR2 agonists were the strongest OPN inducers, and OPN production was highly stimulated by TLR2‐triggering bacteria (Staphylococcus aureus) but not by TLR4‐triggering Escherichia coli. Costimulation experiments revealed that TLR3 and TLR4 agonists, beyond being inactive by themselves, sharply limited TLR2‐dependent OPN production by activating a TRIF‐dependent inhibition of the MyD88‐dependent OPN production. MyD88 silencing impaired TLR2‐dependent OPN induction, whereas TRIF pathway blockage by chloroquine, dynasore, or TRIF knockdown prevented the TLR3/4 agonist‐mediated inhibition, which was independent from the endogenous production of type I IFN, IL‐29, IL‐10, or TGF‐β. LPS and Poly I:C inhibitory activity was associated with the release of a >10‐kDa protein factor(s). We also demonstrated that the higher OPN levels produced by S. aureus‐treated DCs compared with E. coli‐treated DCs were responsible for a markedly increased production of IL‐17 by CD4+ T cells. These results highlight the biological relevance of the differential OPN induction by TLR2 and TLR4 agonists and emphasize the importance of TLR cross‐talk in OPN induction. This implies that OPN regulation by TLR signaling is critical in shaping inflammatory responses and may modulate IL‐17 production in response to pathogens.

Novel avenues for Clostridium difficile infection drug discovery

Introduction: Clostridium difficile is the etiologic agent of nosocomial and community-acquired diarrhea associated with exposure to antibiotics that disrupt the normal colonic flora. As antibacterials currently used for primary C. difficile infections favor recurrences, new agents able to neutralize the bacterium without affecting the gut microbiota are badly needed. Areas covered: This article investigates the most promising strategies aimed at developing therapies with minimal or no effect on intestinal flora. These therapies include new narrow-spectrum antibiotics and antimicrobial peptides, bacteriophages and phage lysins, virulence-targeting factors such as riboswitch ligands and quorum sensing-interfering factors. It also reviews bacteriotherapy based on probiotics, fecal transplants, and toxin-targeting molecules. Expert opinion: Beyond the development of new antibiotics, virulence-targeting factors or phage cocktails seem promising strategies, which could replace antibiotics avoiding the emergence of resistant strains and the onset of C. difficile infection (CDI). Until broad-spectrum antimicrobials will be in use, C. difficile-specific lytic phages could help to prevent CDI by eliminating C. difficile in patients and in the hospital staff, and for the prevention and treatment of recurrences. Phage therapy is not currently available in Western countries, but, in our opinion, it should have a new chance. Fecal therapy is emerging as a very effective and readily available treatment for recurrences. The shift is from a standardized, drug-based antibacterial therapy toward the forthcoming less expensive and nonpatentable procedures of a more personalized medicine. This will imply profound changes affecting both patient–physician interactions and the current profit-oriented approach to the pharmacologic therapy of infections.

New antimicrobial frontiers.

New antimicrobials able to counteract bacterial resistance are needed to maintain the control of infectious diseases. The last 40 years have seen the systematic tailoring and refinement of previously identified antibiotics, to produce a multitude of semi-synthetic derivatives that share their mechanism of action with the original molecules. The major limit of this approach is the emergence of multi- and cross-resistant bacterial strains, favoured by the selective pressure inherent to the targeting of specific enzymes. The most promising new strategies aim to the development of molecules that, targeting essential bacterial structures instead of specific enzymatic activities, achieve infection control without enforcing a selective pressure on bacteria. This review, based on the consultation of the up-to-date literature, deals with antimicrobial peptides and some antivirulence factors.

Survey of phenotypic and genetic features of Streptococcus pyogenes strains isolated in northwest Italy

Streptococcus pyogenes (group A Streptococcus [GAS]) is an important pathogen whose virulence is related to the production of exotoxins and the presence of particular surface components. One hundred eighty-two GAS strains were collected in northwestern Italy between 1994 and 2002 and analyzed for phenotypic characteristics (opacity factor, proteolyic activity, and antimicrobial susceptibility) and by polymerase chain reaction for the presence of genes responsible for the production of exotoxins implicated in pathogenesis speA and speF and of prtF1 (encoding fibronectin-binding protein F1). All strains were speF positive and 19.2% were speA positive and prtF1 negative, whereas the prtF1 gene was identified in 39.5% of the other strains. Of these, approximately half revealed the same pulse-field gel electrophoresis (PFGE) pattern but differed in both speA gene and macrolide resistance.