Baseline computed tomography screening and blood microRNA predict lung cancer risk and define adequate intervals in the BioMILD trial.

BACKGROUND: Large randomized trials have demonstrated that lung cancer (LC) screening with low-dose computed tomography (LDCT) reduces LC mortality in heavy smokers. We previously showed in the MILD screening trial that the combination of a prespecified circulating microRNA (miRNA) signature classifier (MSC) and LDCT improves the accuracy of LDCT alone. The primary aim of the prospective BioMILD study was to assess the additional value of the blood MSC assay at the time of baseline LDCT with the goal of personalizing LC screening intervals. PATIENTS AND METHODS: The study enrolled 4119 volunteers from January 2013 to March 2016, with a median follow-up of 5.3 years. Baseline LDCT and miRNAs stratified participants into four groups: CT-/MSC- (n = 2664; 64.7%); CT-/MSC+ (n = 800; 19.4%); CT+/MSC- (n = 446; 10.8%); and CT+/MSC+ (n = 209; 5.1%). As per the protocol, those in the CT-/MSC- and CT-/MSC+ groups were allocated to LDCT repeat at 3-year and 1-year intervals; CT+ participants were allocated for 1-year or earlier intervals on the basis of LDCT features independent of MSC results. RESULTS: CT+ participants had a 15.8-fold higher 4-year LC incidence than CT- participants (95% confidence interval 10.34-24.05), and MSC+ participants had a 2.0-fold higher 4-year LC incidence than MSC- participants (95% confidence interval 1.40-2.90); there was no evidence that the MSC effect differed between CT+ and CT- participants. LC incidence at 4 years was 0.8% in CT-/MSC-, 1.1% in CT-/MSC+, 10.8% in CT+/MSC-, and 20.1% in CT+/MSC+ participants. LC mortality rates at 5 years in the four risk groups were 0.5 in CT-/MSC-, 1.5 in CT-/MSC+, 4.2 in CT+/MSC-, and 10.1 in CT+/MSC+. CONCLUSION: The combined use of LDCT and blood miRNAs at baseline predicts individual LC incidence and mortality, with a major effect of MSC for LDCT-positive individuals. These findings may have important implications in personalizing screening intervals.

Whole genome sequencing of Shigella sonnei through PulseNet Latin America and Caribbean: advancing global surveillance of foodborne illnesses.

OBJECTIVES: Shigella sonnei is a globally important diarrhoeal pathogen tracked through the surveillance network PulseNet Latin America and Caribbean (PNLA&C), which participates in PulseNet International. PNLA&C laboratories use common molecular techniques to track pathogens causing foodborne illness. We aimed to demonstrate the possibility and advantages of transitioning to whole genome sequencing (WGS) for surveillance within existing networks across a continent where S. sonnei is endemic. METHODS: We applied WGS to representative archive isolates of S. sonnei (n = 323) from laboratories in nine PNLA&C countries to generate a regional phylogenomic reference for S. sonnei and put this in the global context. We used this reference to contextualise 16 S. sonnei from three Argentinian outbreaks, using locally generated sequence data. Assembled genome sequences were used to predict antimicrobial resistance (AMR) phenotypes and identify AMR determinants. RESULTS: S. sonnei isolates clustered in five Latin American sublineages in the global phylogeny, with many (46%, 149 of 323) belonging to previously undescribed sublineages. Predicted multidrug resistance was common (77%, 249 of 323), and clinically relevant differences in AMR were found among sublineages. The regional overview showed that Argentinian outbreak isolates belonged to distinct sublineages and had different epidemiologic origins. CONCLUSIONS: Latin America contains novel genetic diversity of S. sonnei that is relevant on a global scale and commonly exhibits multidrug resistance. Retrospective passive surveillance with WGS has utility for informing treatment, identifying regionally epidemic sublineages and providing a framework for interpretation of prospective, locally sequenced outbreaks.

Mir-660 is downregulated in lung cancer patients and its replacement inhibits lung tumorigenesis by targeting MDM2-p53 interaction.

Lung cancer represents the leading cause of cancer-related death in developed countries. Despite the advances in diagnostic and therapeutic techniques, the 5-year survival rate remains low. The research for novel therapies directed to biological targets has modified the therapeutic approach, but the frequent engagement of resistance mechanisms and the substantial costs, limit the ability to reduce lung cancer mortality. MicroRNAs (miRNAs) are small noncoding RNAs with known regulatory functions in cancer initiation and progression. In this study we found that mir-660 expression is downregulated in lung tumors compared with adjacent normal tissues and in plasma samples of lung cancer patients with poor prognosis, suggesting a potential functional role of this miRNA in lung tumorigenesis. Transient and stable overexpression of mir-660 using miRNA mimics reduced migration, invasion, and proliferation properties and increased apoptosis in p53 wild-type lung cancer cells (NCI-H460, LT73, and A549). Furthermore, stable overexpression using lentiviral vectors in NCI-H460 and A549 cells inhibited tumor xenograft growth in immunodeficient mice (95 and 50% reduction compared with control, respectively), whereas the effects of mir-660 overexpression were absent in H1299, a lung cancer cell line lacking p53 locus, both in in vitro and in vivo assays. We identified and validated mouse double minute 2 (MDM2) gene, a key regulator of the expression and function of p53, as a new direct target of mir-660. In addition, mir-660 expression reduced both mRNA and protein expression of MDM2 in all cell lines and stabilized p53 protein levels resulting in an upregulation of p21(WAF1/CIP1) in p53 wild-type cells. Our finding supports that mir-660 acts as a tumor suppressor miRNA and we suggest the replacement of mir-660 as a new therapeutic approach for p53 wild-type lung cancer treatment.

Pathology of striped dolphins (Stenella coeruleoalba) infected with Brucella ceti.

Seventeen striped dolphins (Stenella coeruleoalba) displaying swimming disorders compatible with neurological syndromes were investigated for Brucella infection. Sixteen dolphins had meningoencephalomyelitis. Serum antibody against Brucella antigen was detected in all 14 animals tested and Brucella ceti was isolated from eight out of nine animals. Brucella antigen was detected in the brain by immunofluorescence, but not by immunohistochemical labelling. By contrast, Brucella antigen was demonstrated by immunohistochemistry in the trophoblast of animals with severe placentitis and in the mitral valve of animals with myocarditis. The microscopical lesions observed in the tissues of the infected dolphins were similar to those of chronic brucellosis in man. The severity of brucellosis in S. coeruleoalba indicates that this dolphin species is highly susceptible to infection by B. ceti.

[Intravesical Electromotive Drug Administration®(EMDA)with Mitomycin-C for non-muscle invasive bladder cancer].

Electromotive Drug Administration® (EMDA) offers a means of controlling and enhancing the tissue transport of certain drugs, when applied to a surface epithelium, where they have a local therapeutic effect, in order to increase their efficacy. One application option is the treatment of non-muscle invasive bladder cancer with intravesical mitomycin-C (MMC). Laboratory studies demonstrated that EMDA/MMC can reduce the variability and enhance the drug administration rate into all layers of the bladder wall, and that the applied electric current causes no histological damage to tissue and no chemical modification of MMC. A prospective randomized study, performed in patients with in situ carcinoma, validated the prediction that electromotive enhancement of MMC delivery would provide results superior to those achieved using passive MMC transport. A further randomized study in patients with pT1 bladder cancer demonstrated that a regimen combining intravesical BCG and EMDA/MMC increased the disease-free interval and reduced the recurrence rate, as well as the disease progression and mortality rate if compared with BCG alone. The possibility that BCG may enhance the efficacy of MMC against high-grade pT1 transitional cell carcinoma and in situ carcinoma represents an important new therapeutic perspective in the high-risk non-muscle invasive bladder cancer.

Regulation of Brucella virulence by the two-component system BvrR/BvrS.

The Brucella BvrR/BvrS two-component regulatory system is highly similar to the regulatory and sensory proteins of Sinorhizobium and Agrobacterium necessary for endosymbiosis and pathogenicity in plants, and very similar to a putative system present in the animal pathogen Bartonella. Mutations in the bvrR or bvrS genes hamper the penetration of B. abortus in non-phagocytic cells and impairs intracellular trafficking and virulence. In contrast to virulent Brucella, BvrR/BvrS mutants do not recruit small GTPases of the Rho subfamily required for actin polymerization and penetration to cells. Dysfunction of the BvrR/BvrS system alters the outer membrane permeability, the expression of several group 3 outer membrane proteins and the pattern of lipid A acylation. Constructs of virulent B. abortus chimeras containing heterologous LPS from the bvrS(-) mutant demonstrated an altered permeability to cationic peptides similar to that of the BvrR/BvrS mutants. We hypothesize that the Brucella BvrR/BvrS is a system devoted to the homeostasis of the outer membrane and, therefore in the interface for cell invasion and mounting the required structures for intracellular parasitism.

The two-component system BvrR/BvrS essential for Brucella abortus virulence regulates the expression of outer membrane proteins with counterparts in members of the Rhizobiaceae.

The Brucella BvrR/BvrS two-component regulatory system is homologous to the ChvI/ChvG systems of Sinorhizobium meliloti and Agrobacterium tumefaciens necessary for endosymbiosis and pathogenicity in plants. BvrR/BvrS controls cell invasion and intracellular survival. Probing the surface of bvrR and bvrS transposon mutants with monoclonal antibodies showed all described major outer membrane proteins (Omps) but Omp25, a protein known to be involved in Brucella virulence. Absence of Omp25 expression was confirmed by two-dimensional electrophoresis of envelope fractions and by gene reporter studies. The electrophoretic analysis also revealed reduction or absence in the mutants of a second set of protein spots that by matrix-assisted laser desorption ionization MS and peptide mass mapping were identified as a non-previously described Omp (Omp3b). Because bvrR and bvrS mutants are also altered in cell-surface hydrophobicity, permeability, and sensitivity to surface-targeted bactericidal peptides, it is proposed that BvrR/BvrS controls cell envelope changes necessary to transit between extracellular and intracellular environments. A genomic search revealed that Omp25 (Omp3a) and Omp3b belong to a family of Omps of plant and animal cell-associated alpha-Proteobacteria, which includes Rhizobium leguminosarum RopB and A. tumefaciens AopB. Previous work has shown that RopB is not expressed in bacteroids, that AopB is involved in tumorigenesis, and that dysfunction of A. tumefaciens ChvI/ChvG alters surface properties. It is thus proposed that the BvrR/BvrS and Omp3 homologues of the cell-associated alpha-Proteobacteria play a role in bacterial surface control and host cell interactions.

GTPases of the Rho subfamily are required for Brucella abortus internalization in nonprofessional phagocytes: direct activation of Cdc42.

Members of the genus Brucella are intracellular alpha-Proteobacteria responsible for brucellosis, a chronic disease of humans and animals. Little is known about Brucella virulence mechanisms, but the abilities of these bacteria to invade and to survive within cells are decisive factors for causing disease. Transmission electron and fluorescence microscopy of infected nonprofessional phagocytic HeLa cells revealed minor membrane changes accompanied by discrete recruitment of F-actin at the site of Brucella abortus entry. Cell uptake of B. abortus was negatively affected to various degrees by actin, actin-myosin, and microtubule chemical inhibitors. Modulators of MAPKs and protein-tyrosine kinases hampered Brucella cell internalization. Inactivation of Rho small GTPases using clostridial toxins TcdB-10463, TcdB-1470, TcsL-1522, and TcdA significantly reduced the uptake of B. abortus by HeLa cells. In contrast, cytotoxic necrotizing factor from Escherichia coli, known to activate Rho, Rac, and Cdc42 small GTPases, increased the internalization of both virulent and non-virulent B. abortus. Expression of dominant-positive Rho, Rac, and Cdc42 forms in HeLa cells promoted the uptake of B. abortus, whereas expression of dominant-negative forms of these GTPases in HeLa cells hampered Brucella uptake. Cdc42 was activated upon cell contact by virulent B. abortus, but not by a noninvasive isogenic strain, as proven by affinity precipitation of active Rho, Rac, and Cdc42. The polyphasic approach used to discern the molecular events leading to Brucella internalization provides new alternatives for exploring the complexity of the signals required by intracellular pathogens for cell invasion.

In vivo proteolytic degradation of the Escherichia coli acyltransferase HlyC.

Escherichia coli hemolysin (HlyA) is the prototype toxin of a major family of exoproteins produced by Gram-negative bacteria known as "repeats in toxins." Only fatty acid-acylated HlyA molecules at residues Lys564 and Lys690 are able to damage the target cell membrane. Fatty acylation of pro-HlyA is dependent on the co-synthesized acyltransferase HlyC and the acylated form of acyl-carrier protein. By using a collection of hlyA and hlyC mutant strains, the processing of HlyC was investigated. HlyC was not detected by Western blot in an E. coli strain encoding hlyC and hlyA, but it was present in a strain encoding only hlyC. The hlyC mRNA pattern, however, was similar in both strains indicating that the turnover of HlyC does not occur at the transcriptional level. HlyC was detected in Western blots of cell lysates from an E. coli strain encoding HlyC and a HlyA derivative where both acylation sites were substituted. Similar results were obtained when HlyC was expressed in a hlyA mutant strain lacking part of a putative HlyC binding domain, indicating that this particular HlyA region affects HlyC stability. We did not detect HlyC in cell lysates from hlyC mutants with different abilities to acylate pro-HlyA, suggesting that the degradation of HlyC is not related to the HlyA acylation process. The protease systems ClpAP, ClpXP, and FtsH were found to be responsible for the HlyA-dependent processing of HlyC.

Incomplete activation of Escherichia coli hemolysin (HlyA) due to mutations in the 3' region of hlyC.

Mutational analysis of the carboxy-terminal region of Escherichia coli HlyC was performed by site-directed mutagenesis. Replacement of residue Val-127 or Lys-129 reduced the activity of HlyC to about 30 or 60%, respectively, of that of the wild type, while replacement of Gly-128 reduced the activity to less than 1% of the wild-type level. Complete inactivation of HlyC was caused by a double mutation, replacement of Gly-128 with valine and of Lys-129 with isoleucine. Analysis of culture supernatants from mutants with reduced hemolytic activity by two-dimensional gel electrophoresis revealed the production and simultaneous secretion of nonacylated, monoacylated, and fully acylated HlyA forms, demonstrating impairment of the acylation reaction, possibly due to a decreased affinity of HlyC for the individual HlyA acylation sites.

Chemoprophylaxis of bacterial infections in granulocytopenic cancer patients using norfloxacin.

Sixty-five cancer patients pretreated with chemo or radiotherapy, with granulocytopenia less than 1000/mm3 and without fever, were entered into this study: 30 of them were submitted to prophylaxis with norfloxacin while the remaining 35 patients were used as a control group. 20% of the treated subjects versus 68.6% of the controls presented a subsequent infection (P less than 0.001), the lung representing the most frequent site of the infectious disease in both groups (3/6 and 14/24 respectively). These data strongly suggest the use of norfloxacin as an effective prophylactic drug in nonfebrile, granulocytopenic cancer patients, especially as far as gram-negative infections are concerned. Because of the high prevalence of lung cancer in the patients of our study, and a related prevalence of lung infections, at the present time, a wider use of this antibiotic in every kind of solid tumor cannot be generalized.

[Neoplastic occlusions of the large intestine (clinical study)]. / Le occlusioni neoplastiche del grosso intestino (contributo clinico).

The authors do a clinico-epidemiologic reviewing of 24 cases of neoplastic obstruction of large intestine, they had the opportunity to observe. Such patients represent 12.9% of all cases of mechanic intestinal obstruction. The obstructive complication, which in 70% of the observations involved sigma and rectosigmoid junction, was the clinical beginning of 9,6% of colonic and rectal carcinomata. Such disease arose preferably in old patients and in those suffering from one or more concomitant diseases. Some reflections about the surgical treatment, which, in this casuistry, determined a mortality of 25%, lead the Authors to think the requirements of the treatment of high risk patients and the rigid observance of the rules of colonic surgery should be put before the requirements of oncological radicalness.