Maria Antonietta Tufano

Dry powders based on PLGA nanoparticles for pulmonary delivery of antibiotics: Modulation of encapsulation efficiency, release rate and lung deposition pattern by hydrophilic polymers

Although few experimental studies have been handled so far to exploit the potential of poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) in the production of dry powders for antibiotic inhalation, there has been no comprehensive study on the role played by NP composition. In this work, we try to shed light on this aspect by designing and developing a pulmonary delivery system for antibiotics, such as tobramycin (Tb), based on PLGA NPs embedded in an inert microcarrier made of lactose, referred to as nano-embedded micro-particles (NEM). At nanosize level, helper hydrophilic polymers were used to impart the desired surface, bulk and release properties to PLGA NPs prepared by a modified emulsion-solvent diffusion technique. Results showed that poly(vinyl alcohol) (PVA) and chitosan (CS) are essential to optimise the size and modulate the surface properties of Tb-loaded PLGA NPs, whereas the use of alginate (Alg) allows efficient Tb entrapment within NPs and its release up to one month. Optimized formulations display good in vitro antimicrobial activity against P. aeruginosa planktonic cells. Furthermore, spray-drying of the NPs with lactose yielded NEM with peculiar but promising flow and aerosolization properties, while preserving the peculiar NP features. Nonetheless, in vivo biodistribution studies showed that PVA-modified Alg/PLGA NPs reached the deep lung, while CS-modified NPs were found in great amounts in the upper airways, lining lung epithelial surfaces. In conclusion, PLGA NP composition appears to play a crucial role in determining not only the technological features of NPs but, once processed in the form of NEM, also their in vitro/in vivo deposition pattern.

Toll-like receptor 2 (TLR2) mediates intracellular signalling in human keratinocytes in response to Malassezia furfur.

Toll-like receptors (TLRs) are crucial players in the innate immune response to microbial invaders. The lipophilic yeast Malassezia furfur has been implicated in the triggering of scalp lesions in psoriasis. The aim of the present study was to assess the role of TLRs in the defence against M. furfur infection. The expression of the myeloid differentiation factor 88 (MyD88) gene, which is involved in the signalling pathway of many TLRs, was also analysed. In addition, a possible correlation of antimicrobial peptides of the β-defensin family to TLRs was tested. Human keratinocytes infected with M. furfur and a variety of M. furfur-positive psoriatic skin biopsies were analysed by RT-PCR, for TLRs, MyD88, human β-defensin 2 (HBD-2), HBD-3 and interleukin-8 (IL-8) mRNA expression. When keratinocytes were infected with M. furfur, an up-regulation for TLR2, MyD88, HBD-2, HBD-3 and IL-8 mRNA was demonstrated, compared to the untreated cells. The same results were obtained when psoriatic skin biopsies were analysed. The M. furfur-induced increase in HBD-2 and IL-8 gene expression is inhibited by anti-TLR2 neutralising antibodies, suggesting that TLR2 is involved in the M. furfur-induced expression of these molecules. These findings suggest the importance of TLRs in skin protection against fungi and the importance of keratinocytes as a component of innate immunity.

Antimicrobial human beta-defensin-2 stimulates migration, proliferation and tube formation of human umbilical vein endothelial cells

Human beta-defensin-2 (hBD-2) is an antimicrobial peptide which is released upon microbial invasion and contributes to mucosal and epithelial defense modulating both innate and adaptive immunity. We found that hBD-2 stimulates chemotaxis of human endothelial cells with an extent similar to that exerted by the vascular endothelial growth factor (VEGF). The hBD-2-dependent chemotaxis is dose-dependent, maximal effect being reached at 500 ng/ml concentration. In the absence of any growth factor, hBD-2 favors wound healing of endothelial cells, causing an about 2-fold increase in the speed of wound closure with respect to the control. Furthermore, hBD-2 promotes endothelial cell proliferation, although at a minor extent as compared to VEGF. When plated on matrigel enriched with angiogenic factors, endothelial cells form a three-dimensional network of tubes that gives rise to capillary-like structures. Similarly to VEGF, hBD-2 promotes capillary-like tube formation of human endothelial cells. Pro-angiogenic effect promoted by hBD-2 is dose-dependent, peaks at a 500 ng/ml hBD-2 concentration and is prevented by blocking anti-alphavbeta3 monoclonal antibody. However, hBD-2-induced pro-angiogenic activity is not due to endogenously produced VEGF because it is not prevented by neutralizing anti-VEGF antibodies. Overall, our findings suggest that hBD-2 could link inflammation and the host defense through its pro-angiogenic activity.

Failure of first-line eradication treatment significantly increases prevalence of antimicrobial-resistant Helicobacter pylori clinical isolates

Objectives: Helicobacter pylori infection is a major health problem worldwide, and effective eradication of the infection is mandatory. The efficacy of recommended eradication regimens is approximately 70%. To avoid treatment failure and the consequent development of secondary resistance(s), it is important to choose the most appropriate first-line treatment regimen. This choice should also be made based on the knowledge of the antimicrobial resistance peculiar to a given geographical area. We evaluated the prevalence of antimicrobial-resistant H pylori strains isolated from naive patients and from patients with previous unsuccessful treatments. Methods: This study examined 109 H pylori-infected subjects (Group 1) who had never received an eradication treatment and 104 H pylori-infected subjects (Group 2) who had failed one or more eradication treatments. Resistance to amoxicillin (AMO), tetracycline (TET), clarithromycin (CLA), metronidazole (MET) and levofloxacin (LEV) was determined using the epsilometer test. The significance of differences was evaluated by the χ2 test. Results: The prevalence of antimicrobial resistance was 0% versus 3.1% to AMO, 0% versus 2% to TET, 27% versus 41.3% to MET (p<0.05), 18% versus 45.8% to CLA (p<0.05) and 3% versus 14.6% to LEV (p<0.05) in Group 1 vs Group 2, respectively. In Group 2, there was an increased prevalence of H pylori strains resistant to multiple antimicrobials. Conclusions: This study confirms the high prevalence of H pylori strains resistant to CLA and MET, and indicates that unsuccessful treatments significantly increase resistance. Choosing eradication regimens other than standard triple therapy as a first-line therapy should be advisable in areas with high primary antimicrobial resistance prevalence.

Pretreatment antimicrobial susceptibility testing is cost saving in the eradication of Helicobacter pylori

Abstract Background & Aims: The major obstacle to 100% effective eradication of Helicobacter pylori infection is represented by antimicrobial-resistant H. pylori strains. This randomized study was designed to evaluate whether regimens based on pretreatment susceptibility testing were more effective and cost saving compared with standard nonsusceptibility testing-based therapy in the eradication of H. pylori infection. Methods: We studied 150 consecutive H. pylori -infected dyspeptic subjects. Patients were randomly assigned to omeprazole 20 mg twice daily, clarithromycin 500 mg twice daily, and metronidazole 500 mg twice daily for 7 days or to omeprazole 20 mg twice daily and 2 antimicrobials chosen based on susceptibility testing. H. pylori status was reevaluated 12 weeks after the end of treatment by the 13 C-urea breath test. Results: Susceptibility testing-based regimens led to the following results. (1) Eradication rates were 97.3% (95% confidence interval [CI], 91.2%–99.5%) (71 of 73) and 94.6% (95% CI, 87.6%–98.3%) (71 of 75) in the per-protocol and intention-to-treat analysis, respectively. These were significantly higher ( P Conclusions: Pretreatment antimicrobial susceptibility testing is more effective and cost saving and, in health systems that confirm cost advantage, microbial susceptibility testing should be routinely used for eradication of H. pylori infection.

Malassezia furfur induces the expression of β-defensin-2 in human keratinocytes in a protein kinase C-dependent manner

Antimicrobial peptides of the β-defensin family are expressed in all human epithelial tissues tested to date and have recently been the subject of vigorous investigation. Their localization and characteristics support the hypothesis that these peptides play a role in mucosal and skin defense. The lipophilic yeast Malassezia furfur is a saprophyte found in normal human cutaneous flora. Malassezia furfur is not only a saprophyte, but is also associated with several diseases such as Malassezia folliculitis, seborrheic dermatitis and some forms of atopic dermatitis, psoriasis and confluent and reticulate papillomatosis. Little is known about the mechanism by which M. furfur overcomes the natural barrier of the skin. To further define the role of the β-defensins in the innate human skin immune response, we analyzed the mRNA expression of two human β-defensins HBD-1 and HBD-2 in human keratinocytes treated with M. furfur. In addition, we looked into how M. furfur of TGF-β1 and IL-10, cytokines that interfere with the development of protective cell immunity, regulate their expression. Finally, we examined the signal transduction mechanisms involved during M. furfur uptake. Cultured human keratinocytes were treated with M. furfur. The mRNA and protein expression were analyzed, respectively, by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting. Our data demonstrate that M. furfur does not modify HBD-1 expression, whereas it up-regulates, via protein kinase C (PKC), the expression of HBD-2, TGFβ-1 and IL-10 48 h after treatment. Our results suggest that β-defensins are integral components of innate host defenses. They play an essential part in the resistance of the human skin surfaces against M. furfur uptake and other microbial invasion.

Detection of herpesvirus DNA in peripheral blood mononuclear cells and skin lesions of patients with pemphigus by polymerase chain reaction

Pemphigus is an autoimmune disease where both endogenous (genetic) and exogenous (environmental) factors play a part. Viral infections, in particular herpesvirus infections, have been identified as a possible triggering factor for pemphigus. In this study, using the polymerase chain reaction, we studied peripheral blood mononuclear cells (PBMC) and skin biopsies from patients with pemphigus, and in some of these were able to demonstrate the presence of DNA sequences of herpes simplex virus 1/2 (50% in PBMC and 71% in skin biopsies), Epstein–Barr virus (15% in PBMC and 5% in skin biopsies) and human herpesvirus 6 (20% in PBMC only). However, the inability to detect herpesvirus DNA consistently in these cases suggests that viral infection may only be an occasional factor triggering the outbreak or exacerbation of the disease. The possible role of interferons and interleukins in the pathogenesis of virus‐induced pemphigus is discussed.

Prevalence of antimicrobial resistance in eighty clinical isolates of Helicobacter pylori

We studied the in vitro susceptibility of 80 Helicobacter pylori clinical isolates to several antimicrobials commonly used to treat the infection, using the Epsilometer test (E-test). We also compared E-test and disk diffusion test in determining H. pylori susceptibility to the same antibiotics. We found a high prevalence of H. pylori strains resistant to metronidazole (23.7%), whereas the prevalence of H. pylori strains resistant to clarithromycin was 10%. Also, a significant correlation was found between MICs obtained with the disk diffusion test and E-test for metronidazole and clarithromycin. In conclusion, our study confirms a high prevalence of metronidazole- or clarithromycin-resistant H. pylori strains. Also, our data suggest that the E-test is a single, reliable, and cost-effective method to assess in vitro susceptibility of H. pylori to antimicrobial agents commonly used to eradicate the infection.

Possible role of Malassezia furfur in psoriasis: modulation of TGF‐β1, integrin, and HSP70 expression in human keratinocytes and in the skin of psoriasis‐affected patients

Background:  Psoriasis is a disease characterized by an abnormal pattern of keratinocyte growth and differentiation. Malassezia furfur forms part of the normal human skin flora. It may also be involved in the pathogenesis of psoriasis. To define the role of M. furfur in the pathogenesis of psoriasis, we investigated how M. furfur regulates molecules involved in cell migration and proliferation. The experiments were performed using human keratinocytes and skin biopsies from M. furfur‐positive and ‐negative psoriasis‐affected patients. In addition, we examined the signal transduction mechanisms involved.

Artemisinin reduces human melanoma cell migration by down-regulating αVβ3 integrin and reducing metalloproteinase 2 production

SummaryArtemisinin and its derivatives are well known antimalarial drugs, particularly useful after resistance to traditional antimalarial pharmaceuticals has started to occur in Plasmodium falciparum. In recent years, anticancer activity of artemisinin has been reported both in vitro and in vivo. Artemisinin has inhibitory effects on cancer cell growth and anti-angiogenetic activity. In the present investigation, we analyzed the inhibitory effects of artemisinin on migratory ability of melanoma cell lines (A375P and A375M, low and medium metastatic properties, respectively). We demonstrate that artemisinin induces cell growth arrest in A375M, and affects A375P cells viability with cytotoxic and growth inhibitory effects, while it was not effective in contrasting proliferation of other tumor cell lines (MCF7 and MKN). In addition, artemisinin affected the migratory ability of A375M cells by reducing metalloproteinase 2 (MMP-2) production and down-regulating αvβ3 integrin expression. These findings introduce a potential of artemisinin as a chemotherapeutic agent in melanoma treatment.