Giovanni Piccaro

Activities of Drug Combinations against Mycobacterium tuberculosis Grown in Aerobic and Hypoxic Acidic Conditions

ABSTRACT Mycobacterium tuberculosis is exposed to hypoxia and acidity within granulomatous lesions. In this study, an acidic culture model of M. tuberculosis was used to test drug activity against aerobic 5-day-old (A5) and hypoxic 5-, 12-, and 19-day-old (H5, H12, and H19, respectively) bacilli after 7, 14, and 21 days of exposure. In A cultures, CFU and pH rapidly increased, while in H cultures growth stopped and pH increased slightly. Ten drugs were tested: rifampin (R), isoniazid (I), pyrazinamide (Z), ethambutol (E), moxifloxacin (MX), amikacin (AK), metronidazole (MZ), nitazoxanide (NZ), niclosamide (NC), and PA-824 (PA). Rifampin was the most active against A5, H5, H12, and H19 bacilli. Moxifloxacin and AK efficiently killed A5 and H5 cells, I was active mostly against A5 cells, Z was most active against H12 and H19 cells, and E showed low activity. Among nitrocompounds, NZ, NC, and PA were effective against A5, H5, H12, and H19 cells, while MZ was active against H12 and H19 cells. To kill all A and H cells, A5- and H5-active agents R, MX, and AK were used in combination with MZ, NZ, NC, or PA, in comparison with R-I-Z-E, currently used for human therapy. Mycobacterial viability was determined by CFU and a sensitive test in broth (day to positivity, MGIT 960 system). As shown by lack of regrowth in MGIT, the most potent combination was R-MX-AK-PA, which killed all A5, H5, H12, and H19 cells in 14 days. These observations demonstrate the sterilizing effect of drug combinations against cells of different M. tuberculosis stages grown in aerobic and hypoxic acidic conditions.

Activity of drug combinations against dormant Mycobacterium tuberculosis.

ABSTRACT Aerobic (5-day-old cultures) and nonreplicating (dormant) Mycobacterium tuberculosis (5-, 12-, and 19-day-old cultures) bacteria were treated with rifampin (R), moxifloxacin (MX), metronidazole (MZ), amikacin (AK), or capreomycin (CP) for 7, 14, and 21 days. R-MX-MZ-AK and R-MX-MZ-CP killed both aerobic and dormant bacilli in 21 days, as shown by lack of regrowth in solid and liquid media. R-MX-MZ-AK and R-MX-MZ-CP also caused a strong decrease of nonreplicating bacilli in 7 days in a cell-based dormancy model.

Rifampin Induces Hydroxyl Radical Formation in Mycobacterium tuberculosis

ABSTRACT The antituberculosis (anti-TB) drug rifampin (RIF) binds to the beta subunit of the RNA polymerase (RpoB) of Mycobacterium tuberculosis, but the bactericidal responses triggered after target interaction are not known. To evaluate whether RIF induced an oxidative burst, lysates of RIF-treated M. tuberculosis were tested for determination of reactive oxygen species (ROS) by the electron paramagnetic resonance (EPR) technique using 1-hydroxy-3-carboxy-pyrrolidine (CPH) and 5,5-dimethyl-1-pyrrolidine-N-oxide (DMPO) as spin traps. M. tuberculosis killing by RIF stimulated an increase in the rate of formation of the CPH radical (CP·). Lysate pretreatment with the O2·− and ·OH scavengers superoxide dismutase (SOD) and thiourea (THIO), respectively, or with the metal chelator diethylene triamine pentaacetic acid (DTPA) inhibited CP· formation, arguing in favor of a metal-catalyzed ROS response. Formation of CP· did not increase following treatment of RIF-resistant strains with RIF, indicating that the ROS were induced after RpoB binding. To identify the ROS formed, lysates of RIF-treated bacilli were incubated with DMPO, a spin trap specific for ·OH and O2·−, with or without pretreatment with SOD, catalase, THIO, or DTPA. Superoxide dismutase, catalase, and THIO decreased formation of the DMPO-OH adduct, and SOD plus DTPA completely suppressed it, suggesting that RIF activated metal-dependent O2·−-mediated mechanisms producing ·OH inside tubercle bacilli. The finding that the metal chelator DTPA reduced the bactericidal activity of RIF supported the possibility that ·OH was generated through these mechanisms and that it participated at least in part in M. tuberculosis killing by the drug.

Drug-resistant tuberculosis among foreign-born persons in Italy

To the Editors: Over the last few years, drug-resistant tuberculosis (TB) has emerged as an important threat to public health in industrialised countries. In Italy, the most recent data on resistance to the first-line drugs (FLDs) streptomycin (S), isoniazid (H), rifampicin (R) and ethambutol (E) were reported for the period 1998–2001 [1]. These studies determined the prevalence of resistance among new cases and previously treated cases, but no information was available on the contribution of immigration, which plays an important role on TB epidemiology in low-incidence countries [2]. In the last decade, while the notified incidence of TB in Italy was stable at approximately seven cases per 100,000 people annually, the proportion of foreign-born persons (FBPs) with TB increased from 22% in 1999 to 46% in 2008 [3]. In the same period, the proportion of African-born persons with TB decreased from 51% to 30%, whereas the proportion of European cases increased from 16% to 33%, most of them being born in Eastern Europe, including Former Soviet Union (FSU) countries. Eastern European countries are among those with the highest TB rates caused by multidrug-resistant (MDR) Mycobacterium tuberculosis strains ( i.e. resistant to at least H and R) and extensively drug-resistant (XDR) strains ( i.e. MDR strains resistant to any fluoroquinolone and to at least one injectable second-line drug (SLD): kanamycin (KM), capreomycin (CM), amikacin (AK)) [4]. Reliable drug susceptibility testing (DST) is essential to diagnose TB caused by drug-resistant strains. In Italy, a network of laboratories coordinated by the World Health Organization (WHO) Supranational Reference Laboratory (SRL) in Rome performs drug susceptibility proficiency testing for S, H, R, E (five rounds from 1997 to 2010) and SLD (KM, AK, CM and ofloxacin (OFL)) (one round in 2010) [5]. In order to understand …

Gender-dependent resiliency to stressful and metabolic challenges following prenatal exposure to high-fat diet in the p66Shc−/− mouse

Metabolic stressful challenges during susceptible time windows, such as fetal life, can have important implications for health throughout life. Deletion of the p66Shc gene in mice leads to reduced oxidative stress (OS), resulting in a healthy and lean phenotype characterized by increased metabolic rate, resistance to high-fat diet (HFD)-induced obesity and reduced emotionality at adulthood. Here we hypothesize that p66Shc−/− (KO) adult offspring might be protected from the detrimental effects induced by maternal HFD administered before and during pregnancy. To test such hypothesis, we fed p66Shc+/+ (WT) and KO females with HFD for 13 weeks starting on 5 weeks of age until delivery and tested adult male and female offspring for their metabolic, neuroendocrine, and emotional profile. Prenatal diet affected stress responses and metabolic features in a gender-dependent fashion. In particular, prenatal HFD increased plasma leptin levels and decreased anxiety-like behavior in females, while increasing body weight, particularly in KO subjects. KO mice were overall characterized by metabolic resiliency, showing a blunted change in glycemia levels in response to glucose or insulin challenges. However, in p66Shc−/− mice, prenatal HFD affected glucose tolerance response in an opposite manner in the two genders, overriding the resilience in males and exacerbating it in females. Finally, KO females were protected from the disrupting effect of prenatal HFD on neuroendocrine response. These findings indicate that prenatal HFD alters the emotional profile and metabolic functionality of the adult individual in a gender-dependent fashion and suggest that exposure to high-caloric food during fetal life is a stressful condition interfering with the developmental programming of the adult phenotype. Deletion of the p66Shc gene attenuates such effects, acting as a protective factor.

Mycobacterium tuberculosis PstS1 amplifies IFN-γ and induces IL-17/IL-22 responses by unrelated memory CD4+ T cells via dendritic cell activation.

The immunological mechanisms that modulate protection during Mycobacterium tuberculosis (Mtb) infection or vaccination are not fully understood. Secretion of IFN‐γ and, to a lesser extent, of IL‐17 by CD4+ T cells plays a major role both in protection and immunopathology. Few Mtb Ags interacting with DCs affect priming, activation, and regulation of Ag‐unrelated CD4+ T‐cell responses. Here we demonstrate that PstS1, a 38 kDa‐lipoprotein of Mtb, promotes Ag‐independent activation of memory T lymphocytes specific for Ag85B or Ag85A, two immunodominant protective Ags of Mtb. PstS1 expands CD4+ and CD8+ memory T cells, amplifies secretion of IFN‐γ and IL‐22 and induces IL‐17 production by effector memory cells in an Ag‐unrelated manner in vitro and in vivo. These effects were mediated through the stimulation of DCs, particularly of the CD8α− subtype, which respond to PstS1 by undergoing phenotypic maturation and by secreting IL‐6, IL‐1β and, to a lower extent, IL‐23. IL‐6 secretion by PstS1‐stimulated DCs was required for IFN‐γ, and to a lesser extent for IL‐22 responses by Ag85B‐specific memory T cells. These results may open new perspectives for immunotherapeutic strategies to control Th1/Th17 immune responses in Mtb infections and in vaccinations against tuberculosis.

Tuberculosis in migrants from 106 countries to Italy, 2008-2014

Tuberculosis (TB) is a major infectious disease worldwide. Over recent years, TB caused by multidrug-resistant (MDR) Mycobacterium tuberculosis strains (resistant to at least isoniazid and rifampicin) and extensively drug-resistant (XDR) strains (MDR strains resistant to any fluoroquinolone and to at least one injectable second-line drug (SLD), i.e. kanamycin, capreomycin or amikacin) has emerged as a public health concern in industrialised countries, due to increasing migration from regions where TB is endemic. In migrants coming to Italy from 106 countries, MDR-TB was high from the former Soviet Union and low from Africa http://ow.ly/WZDbo

Proficiency testing of first- and second-line anti-tuberculosis drugs in Italy

To the Editors: The emergence of drug-resistant tuberculosis (TB) is an increasing threat to public health in industrialised countries; thus, it is important to supervise mycobacteriology laboratories by performing periodic proficiency of anti-TB drug susceptibility testing (DST). In 1994, the World Health Organization (WHO) and the International Union against Tuberculosis and Lung Diseases developed a global project of anti-TB drug resistance surveillance to assist countries via a network of supranational reference laboratories (SRLs). Proficiency test (PT) results of first-line anti-TB drugs have been reported for the SRL network [1] and for some individual countries [2, 3]. The SRL in Rome, Italy, coordinated two PTs of first-line drugs in endemic countries in 2002–2006 [4] and two PTs of first-line drugs in Italy in 1998–2000 [5, 6]. The present study aims to verify whether the quality of DST in Italy changed after that time; to this end, a comprehensive survey of five PTs during a 13-yr period (1998–2010) is reported here, together with a pilot round of second-line drug PTs in 2010. Laboratories covering 18 out of 20 Italian regions participated in the PT exercise: 22 laboratories in 1998, 20 in 2000, 28 in 2003, 29 in 2007 and 30 in 2010. To maintain knowledge and skills, the laboratories were selected by the SRL on the basis of the number of patient samples analysed for DST. For instance, a mean of 88 first-line DSTs per laboratory (range 21–357) were performed in 2009. In 2010, 13 laboratories with a mean of 113 first-line and six second-line DSTs per laboratory in 2009 also performed the second-line drug PT. A mean of nine second-line DSTs per laboratory was performed in 2010. The Mycobacterium tuberculosis panels for first- and second-line drug PTs distributed by the Rome …

Human melanoma cells express FGFR/Src/Rho signaling that entails an adhesion-independent caveolin-1 membrane association

Caveolae have been indicated as a center of cytoskeleton regulation for Src kinase/Rho GTPase signaling. In addition, Src recruitment on intact cortical actin cytoskeleton appears to be required for bFGF/FGFR signal activation. Recently, we established a relationship between caveolin‐1 (Cav‐1) expression and cell migration in human malignant melanoma, constitutively activated by a bFGF autoregulatory loop. This work intends to investigate whether caveolaes asset, through bFGF/FGFR/c‐Src/Rho signaling, could be related to melanoma cell anchorage. Accordingly, we revealed the existence of a FGFR/Src kinase pathway in Cav‐1 enriched detergent‐resistant membranes (DRMs) of Me665/1 metastatic melanoma cells, as confirmed by FGFR silencing. Moreover, we determined the expression and phosphorylation levels of Cav‐1/Src/Erk signal pathway as a function of FGFR activation and cell density. A sucrose density gradient ultracentrifugation was employed to monitor Cav‐1 membrane association and buoyancy in Me665/1 cells treated for actin fragmentation or for altered phosphorylation signals. As a result, melanoma cells show remarkable resistance to Cav‐1 disassembly, together with persisting cell signal activity, being Src and Cav‐1 crucial modulators of Rho GTPases. In conclusion, our study primarily highlights, in a metastatic melanoma cell line expressing caveolin, the circumstances whereby caveola structural and functional endurance enables the FGFR/Src/Rho GTPases pathway to keep on cell progression.

The M. tuberculosis Phosphate-Binding Lipoproteins PstS1 and PstS3 Induce Th1 and Th17 Responses That Are Not Associated with Protection against M. tuberculosis Infection

The M. tuberculosis phosphate-binding transporter lipoproteins PstS1 and PstS3 were good immunogens inducing CD8+ T-cell activation and both Th1 and Th17 immunity in mice. However, this antigen-specific immunity, even when amplified by administration of the protein with the adjuvant LTK63 or by the DNA priming/protein boosting regimen, was not able to contain M. tuberculosis replication in the lungs of infected mice. The lack of protection might be ascribed with the scarce/absent capacity of PstS1/PstS3 antigens to modulate the IFN-γ response elicited by M. tuberculosis infection during which, however, PstS1-specific IL-17 secreting cells were generated in both unvaccinated and BCG-vaccinated mice. In spite of a lack of protection by PstS1/PstS3 immunizations, our results do show that PstS1 is able to induce IL-17 response upon M. tuberculosis infection which is of interest in the study of anti-M. tuberculosis immunity and as potential immunomodulator in combined vaccines.