Antidepressant prescribing in five European countries: application of common definitions to assess the prevalence, clinical observations, and methodological implications.

PURPOSE: Drug utilization studies have applied different methods to various data types to describe medication use, which hampers comparisons across populations. The aim of this study was to describe the time trends in antidepressant prescribing in the last decade and the variation in the prevalence, calculated in a uniform manner, in seven European electronic healthcare databases. METHODS: Annual prevalence per 10,000 person-years (PYs) was calculated for 2001-2009 in databases from Spain, Germany, Denmark, the United Kingdom (UK), and the Netherlands. Prevalence data were stratified according to age, sex, antidepressant type (selective serotonin re-uptake inhibitors [SSRIs] or tricyclic antidepressants [TCAs]) and major indications. RESULTS: The age- and sex-standardized prevalence was lowest in the two Dutch (391 and 429 users per 10,000 PYs) and highest in the two UK (913 and 936 users per 10,000 PYs) populations in 2008. The prevalence in the Danish, German, and Spanish populations was 637, 618, and 644 users per 10,000 PY respectively. Antidepressants were prescribed most often in 20- to 60-year-olds in the two UK populations compared with the others. SSRIs were prescribed more often than TCAs in all except the German population. In the majority of countries we observed an increasing trend of antidepressant prescribing over time. Two different methods identifying recorded indications yielded different ranges of proportions of patients recorded with the specific indication (15-57% and 39-69% for depression respectively). CONCLUSION: Despite applying uniform methods, variations in the prevalence of antidepressant prescribing were obvious in the different populations. Database characteristics and clinical factors may both explain these variations.

Glutathione S-transferase P1 c.313A > G polymorphism could be useful in the prediction of doxorubicin response in breast cancer patients.

BACKGROUND: Identification of predicting factors for anthracyclines-based chemotherapy remains a clinical challenge. Glutathione S-transferase (GSTs) enzymes detoxify chemotherapy drugs and their metabolites. Several polymorphisms in GST genes result in reduced or no activity of the enzymes. Specifically, GSTM1 and GSTT1 genes are polymorphically deleted, the polymorphism GSTP1 c.313A>G (rs1695) determines the amino acid substitution Ile105Val, where the Val-containing enzyme has reduced activity. Also, GSTA1*B allele has reduced levels of GSTA1 enzyme. Several polymorphisms in GSTs have been associated with differences in survival for cancer patients treated with chemotherapy. PATIENTS AND METHODS: We genotyped a total of five polymorphisms in GSTM1, GSTT1, GSTP1 and GSTA1 genes in 159 patients with locally advanced breast cancer, treated with single-agent doxorubicin or docetaxel (Taxotere). Gene expression microarrays were performed in 67 breast tumor samples. We correlate this data with treatment outcome. RESULTS: In multivariate analysis, patients homozygous GG for GSTP1 c.313A>G SNP had a lower risk of chemoresistance when treated with doxorubicin (odds ratio 0.106; confidence interval 0.012-0.898; P=0.040). No association was found in the docetaxel arm. Also, we found that GSTP1 expression varied significantly among breast cancer molecular subtypes. CONCLUSIONS: GSTP1 may constitute another tool contributing to individualized anthracycline-based therapy.

Genomic predictors of response to doxorubicin versus docetaxel in primary breast cancer.

UNLABELLED: Taxanes and anthracyclines improve the outcome of early breast cancer, although the benefit is limited to a small proportion of patients and are toxic. We prospectively looked for predictors of response to these drugs. EXPERIMENTAL DESIGN: Four cycles of doxorubicin (75 mg/m²) or docetaxel (100 mg/m²) were compared as presurgical chemotherapy for breast cancer. Biomarkers were determined by immunohistochemistry and fluorescent in situ hybridization using prechemotherapy core biopsies. Tumors were also classified into one of the molecular intrinsic subtypes using an immunohistochemical panel of five biomarkers and genomic profiles. Single genes and intrinsic subtypes were correlated with response to doxorubicin versus docetaxel. Among the 204 evaluable patients, significant predictors of sensitivity in multivariate analysis were low topo2a expression and ER-negative status for doxorubicin and small tumor size and ER-negative status for docetaxel. Predictors of resistance in multivariate analysis were triple-negative status (ER/PgR/HER2 negative by IHC/FISH) for doxorubicin, and high TNM stage for docetaxel. Triple-negative tumors were associated with topo2a overexpression more than the other subtypes. In 94 patients with gene expression profiles, docetaxel was superior to doxorubicin in the basal-like subtype (good pathological response rate - PCR + class I of 56 vs. 0%; P = 0.034); no significant differences were observed in the other subtypes when comparing these two drugs. Low topo2a expression and ER-negative status were predictors of response to doxorubicin, while small tumor size and ER-negative status predicted response to docetaxel. Docetaxel was superior to doxorubicin in triple-negative/basal-like tumors, while no significant differences were seen in the remaining intrinsic subtypes.

Lipophilic but not hydrophilic statins selectively induce cell death in gynaecological cancers expressing high levels of HMGCoA reductase.

Recent reports have suggested that statins induce cell death in certain epithelial cancers and that patients taking statins to reduce cholesterol levels possess lower cancer incidence. However, little is known about the mechanisms of action of different statins or the effects of these statins in gynaecological malignancies. The apoptotic potential of two lipophilic statins (lovastatin and simvastatin) and one hydrophilic statin (pravastatin) was assessed in cancer cell lines (ovarian, endometrial and cervical) and primary cultured cancerous and normal tissues. Cell viability was studied by MTS assays and apoptosis was confirmed by Western blotting of PARP and flow cytometry. The expressions of key apoptotic cascade proteins were analysed. Our results demonstrate that both lovastatin and simvastatin, but not pravastatin, selectively induced cell death in dose- and time-dependent manner in ovarian, endometrial and cervical cancers. Little or no toxicity was observed with any statin on normal cells. Lipophilic statins induced activation of caspase-8 and -9; BID cleavage, cytochrome C release and PARP cleavage. Statin-sensitive cancers expressed high levels of HMG-CoA reductase compared with resistant cultures. The effect of lipophilic statins was dependent on inhibition of enzymatic activity of HMG-CoA reductase since mevalonate pre-incubation almost completely abrogated the apoptotic effect. Moreover, the apoptotic effect involved the inhibition of synthesis of geranylgeranyl pyrophosphate rather than farnesyl pyrophosphate. In conclusion, lipophilic but not hydrophilic statins induce cell death through activation of extrinsic and intrinsic apoptotic cascades in cancerous cells from the human female genital tract, which express high levels of HMG-CoA reductase. These results promote further investigation in the use of lipophilic statins as anticancer agents in gynaecological malignancies.

Longitudinal Cytokine Profiling Identifies GRO-α and EGF as Potential Biomarkers of Disease Progression in Essential Thrombocythemia.

Myeloproliferative neoplasms (MPNs) are characterized by deregulation of mature blood cell production and increased risk of myelofibrosis (MF) and leukemic transformation. Numerous driver mutations have been identified but substantial disease heterogeneity remains unexplained, implying the involvement of additional as yet unidentified factors. The inflammatory microenvironment has recently attracted attention as a crucial factor in MPN biology, in particular whether inflammatory cytokines and chemokines contribute to disease establishment or progression. Here we present a large-scale study of serum cytokine profiles in more than 400 MPN patients and identify an essential thrombocythemia (ET)-specific inflammatory cytokine signature consisting of Eotaxin, GRO-α, and EGF. Levels of 2 of these markers (GRO-α and EGF) in ET patients were associated with disease transformation in initial sample collection (GRO-α) or longitudinal sampling (EGF). In ET patients with extensive genomic profiling data (n = 183) cytokine levels added significant prognostic value for predicting transformation from ET to MF. Furthermore, CD56+CD14+ pro-inflammatory monocytes were identified as a novel source of increased GRO-α levels. These data implicate the immune cell microenvironment as a significant player in ET disease evolution and illustrate the utility of cytokines as potential biomarkers for reaching beyond genomic classification for disease stratification and monitoring.

Δ9-tetrahydrocannabinol modulates the proteasome system in the brain.

Cannabis is the most consumed illicit drug worldwide. Its principal psychoactive component, Δ9-tetrahydrocannabinol (THC), affects multiple brain functions, including cognitive performance, by modulating cannabinoid type-1 (CB1) receptors. These receptors are strongly enriched in presynaptic terminals, where they modulate neurotransmitter release. We analyzed, through a proteomic screening of hippocampal synaptosomal fractions, those proteins and pathways modulated 3 h after a single administration of an amnesic dose of THC (10 mg/kg, i.p.). Using an isobaric labeling approach, we identified 2040 proteins, 1911 of them previously reported in synaptic proteomes, confirming the synaptic content enrichment of the samples. Initial analysis revealed a significant alteration of 122 proteins, where 42 increased and 80 decreased their expression. Gene set enrichment analysis indicated an over-representation of mitochondrial associated functions and cellular metabolic processes. A second analysis focusing on extreme changes revealed 28 proteins with altered expression after THC treatment, 15 of them up-regulated and 13 down-regulated. Using a network topology-based scoring algorithm we identified those proteins in the mouse proteome with the greatest association to the 28 modulated proteins. This analysis pinpointed a significant alteration of the proteasome function, since top scoring proteins were related to the proteasome system (PS), a protein complex involved in ATP-dependent protein degradation. In this regard, we observed that THC decreases 20S proteasome chymotrypsin-like protease activity in the hippocampus. Our data describe for the first time the modulation of the PS in the hippocampus following THC administration under amnesic conditions that may contribute to an aberrant plasticity at synapses.

Genotype and phenotype relationship in MLSb resistance in Streptococcus pyogenes and Streptococcus agalactiae isolated in central Italy.

The aim of this work is to study a correlation between phenotype and genotype in clinical isolates of erythromycin-resistant Streptococcus spp. Among the 25 erythromycin-resistant S. pyogenes, we detected six strains with iMLSB, nine with cMLSB and two with M phenotypes. Among 14 erythromycin-resistant S. agalactiae, we detected five strains with iMLSB, seven with cMLSB and none with an M phenotype. Moreover, 8 S. pyogenes and 2 S. agalactiae showed a phenotype not matching the known ones described in literature, defining an unknown phenotype. Upon examination, the genetic profiles, erm(A), erm(B) and mef(A), of the clinical isolates did not easily correlate with a specific phenotype. Our findings highlighted that the whole matter of phenotypic diversity in macrolide-resistant S. pyogenes and S. agalactiae strains and the correlation with their genetic profiles should be submitted to a more careful analysis of phenotypic and genotypic characterization.

Melaleuca alternifolia essential oil possesses potent anti-staphylococcal activity extended to strains resistant to antibiotics.

Melaleuca alternifolia Cheel essential oil (TTO) and its major component terpinen-4-ol were examined against a large number of clinical isolates of Staphylococcus aureus to establish their anti-staphylococcal activities. Classic and established procedures were used to study M.I.C., time-kill curves, synergism and mutational frequency. The anti-staphylococcal activity of terpinen-4-ol and TTO were superior to those of antibiotics belonging to the major families (all the tested drugs are for topical use or included in ointments, eye drops or used during surgery); terpinen 4-ol and TTO were active against strains resistant to mupirocin, fusidic acid, vancomycin, methicillin and linezolid. TTO and terpinen-4-ol were bactericidal as revealed by time-kill curves; the frequency of mutational frequency to TTO was < 2.9 x 10 9. The study demonstrates good anti-staphylococcal activity of TTO and terpinen-4-ol against a large number of S.aureus isolates and suggests the possible application of these agents for topical treatment of staphylococcal infections. This is the first extensive study on the anti-staphylococcal activity of TTO. The results suggest that this compound may have application as a topical agent for the control of superficial staphylococcal infections, including activity against organisms resistant to antibiotics which can be used, or are specific, for topical use.

An automated classification of the structure of protein loops.

Conformational clusters and consensus sequences for protein loops have been derived by computational analysis of their structures in a non-redundant set of 233 proteins with less than 25% sequence homology (X-ray resolution better than 2.5 A). Loops have been classified into five types (alpha-alpha, beta-beta links, beta-beta hairpins, alpha-beta and beta-alpha) according to the secondary structures they embrace. Four variables have been used to describe the loop geometry, three angles and one distance between the secondary structure elements embracing the loop. Ramachandran angles (phi, psi) are used to define the loop conformations within each brace geometry. All loops from the non-redundant set have been clustered by means of these geometric features. A total of 56 classes (9 alpha-alpha, 11 beta-beta links, 14 beta-beta hairpins, 13 alpha-beta and 9 beta-alpha) were identified with consensus Ramachandran angles in the loops. These classes were divided into subclasses based on the brace geometry. This clustering procedure captures most of the clusters analysed by predominantly visual inspection methods and finds other clusters that have hitherto not been described. Consensus sequence patterns were identified for the subclasses. An extensive characterisation of loop conformations has therefore been achieved and the computational approach is readily open to the incorporation of information from newly determined structures. These clusters should also enhance model building by comparison studies.

Conformational flexibility in a highly mobile protein loop of foot-and-mouth disease virus: distinct structural requirements for integrin and antibody binding.

The G-H loop of foot-and-mouth disease virus VP1 protein is a highly mobile peptide, that extends from the capsid surface and that in native virions is invisible by X-ray crystallography. In serotype C, this segment contains a hypervariable region with several continuous, overlapping, B-cell epitopes that embrace the conserved Arg-Gly-Asp (RGD) cell attachment motif. The solvent-exposed positioning of this peptide by selective insertion into different structural frameworks of E. coli beta-galactosidase, generates a spectrum of antigenic variants which react distinctively with a panel of anti-VP1 monoclonal antibodies and exhibit different efficiencies as cell ligands. The cell attachment efficiency is much less restricted by the different positioning of the viral segment at the insertion sites. A molecular model of an inserted stretch reveals a highest flexibility of the RGD tripeptide segment compared with the flanking sequences, that could allow a proper accommodation to integrin receptors even in poorly antigenic conformations. The non-converging structural requirements for RGD-mediated integrin binding and antibody recognition, explains the dynamism of the generation of neutralisation-resistant antigenic variants in the viral quasi-species, arising from a conformational space of integrin-binding competent peptides. This might be of special relevance for foot-and-moth disease virus evolution, since unlike in other picornaviruses, the cell binding motif and the major neutralising B-cell epitopes overlap in a solvent-exposed peptide accessible to the host immune system, in a virion lacking canyons and similar hiding structures.

Automated classification of antibody complementarity determining region 3 of the heavy chain (H3) loops into canonical forms and its application to protein structure prediction.

A computer-based algorithm was used to cluster the loops forming the complementarity determining region (CDR) 3 of the heavy chain (H3) into canonical classes. Previous analyses of the three-dimensional structures of CDR loops (also known as the hypervariable regions) within antibody immunoglobulin variable domains have shown that for five of the six CDRs there are only a few main-chain conformations (known as canonical forms) that show clear relationships between sequence and structure. However, the larger variation in length and conformation of loops within H3 has limited the classification of these loops into canonical forms. The clustering procedure presented here is based on aligning the Ramachandran-coded main-chain conformation of the residues using a dynamic algorithm that allows the insertion of gaps to obtain an optimum alignment. A total of 41 H3 loops out of 62 non-identical loops, extracted from the Brookhaven Protein Data Bank, have been automatically grouped into 22 clusters. Inspection of the clusters for consensus sequences or intra-loop interactions or invariant conformation led to the proposal of 13 canonical forms representing 31 loops. These canonical forms include a consideration of the geometry of both the take-off region adjacent to the bracing beta-strands and the remaining loop apex. Subsequently a new set of 15 H3 loops not included in the initial analysis was considered. The clustering procedure was repeated and nine of these 15 loops could be assigned to original clusters, including seven to canonical forms. A sequence profile was generated for each canonical form from the original set of loops and matched against the sequences of the new H3 loops. For five out of the seven new H3 loops that were in a canonical form, the correct form was identified at first rank by this predictive scheme.

Protein similarities beyond disulphide bridge topology.

Structural superimposition is an important procedure to analyse the relationships between proteins. A new approach and program, KNOT-MATCH, has been developed for automated structural superimposition of proteins by means of their disulphide bridge topology. As a result of the superimposition, regular secondary structures, loops and clusters of residues become correctly aligned. This fact allows us to find out important structural overlaps of residues, sometimes with functional significance, not only among proteins belonging to the same family but also between apparently non-related proteins. Different disulphide-rich protein families, such as EGF-like, defensin-like and plant protease inhibitors, have been self or cross analysed with this approach. Some amino acids that have been experimentally determined to be structural and/or functional key residues for these proteins are conserved in the three-dimensional space after superimposition by KNOT-MATCH. The program can be very useful for finding relationships among proteins that would be hidden to the current alignment methods based on sequence and on main-chain topology.

Genetic variability among Blastoschizomyces capitatus isolates from different clinical sources.

Sixteen clinical isolates and nine ATCC reference strains of Blastoschizomyces capitatus were analysed genetically, examined for the cellobiose, arbutin and salicin assimilation and tested for the aspartyl-proteinase secretion. The restriction endonuclease analysis (REA) with HpaII and HinfI enzymes and the electrophoretic karyotype (EK) were investigated. Both the restriction enzymes revealed two groups (I, II) constituted by the same isolates: 17 isolates (68%) in group I and 8 (32%) in group II. The EK analysis revealed sixteen groups. These data prompts for a genetic variability of the isolates of Blastoschizomyces capitatus and their account in two distinct genetic groups as suggested by REA. This grouping was confirmed by examining the utilisation of cellobiose, arbutin and salicin. The tests for secretory aspartyl proteinase (Sap) were positive only for three isolates, suggesting a marginal role of this specific enzyme in pathogenesis for these isolates.

Alpha 2-interferon in B-cell chronic lymphocytic leukemia: clinical response, serum cytokine levels, and immunophenotype modulation.

Fifteen patients with B-cell chronic lymphocytic leukemia (B-CLL) have been treated with alpha 2b-interferon (alpha IFN) for one year (3 mega units subcutaneously three times a week). The hematological response and the modulation of immunophenotype, serum levels of soluble interleukin-2 receptor (sIL-2R) and tumour necrosis factor (TNF) have been monitored. Hematologically 67% of cases were classified as responders, although no complete responses were observed; three cases progressed during treatment, and two patients showed stable disease. Both peripheral lymphocytes and CD24+ cell absolute number significantly decreased after twelve months of IFN treatment (40.7 +/- 17 x 10(9)/l versus 15.8 +/- 6 x 10(9)/l, mean values +/- sd, p < 0.01, and 30.4 +/- 5.5 x 10(9)/l versus 8.1 +/- 2.8 x 10(9)/l, p < 0.05, respectively), while CD24+ cell percentage did not change (72.1% +/- 4.6 versus 67.5% +/- 8.8, p not significant). In the majority of cases myelomonocytic markers (CD11c, CD14, CD11b) transitorily decreased during the treatment. Serum sIL-2R levels, elevated in all cases before IFN treatment, increased in responders. Serum TNF levels decreased in patients showing high values before the treatment. The explanation of these findings and their possible implication are discussed.

Peripheral blood CD38 expression predicts survival in B-cell chronic lymphocytic leukemia.

CD38 expression was investigated in 161 untreated patients with B-cell chronic lymphocytic leukemia (B-CLL). A score system, devised ad hoc by integrating the percentage and the mean fluorescence intensity (MFI) values of CD38(+) cells, indicated that B-CLL patients with a CD38 score < or =3 are characterized by a significantly longer survival compared to those with a CD38 score >3 (P=0.0026). Thirty-seven percent of patients with a CD38 score < or =3 and 58% of those with a score >3 were dead at 10 years. Multivariate analysis indicates that only the CD38 score successfully predicts survival (P=0.0028), with an estimated 3.8-fold greater risk of death for those cases with CD38 score >3.

Variation of the prion gene in chimpanzees and its implication for prion diseases.

In humans, familial prion diseases are linked to mutations in the PRNP gene. We have sequenced part of this gene in a large sample of common chimpanzee, Pan troglodytes (n=130 chromosomes). No variation in codons 129 and 219 has been observed: all chimpanzees were homozygous for the Met allele, which in humans increases susceptibility to Creutzfeldt-Jakob disease. We found two sequence variants: one is a synonymous polymorphism unique to the chimpanzee at codon 226, TAC to TAT (Y), with a TAC allele frequency of 80.6%; the other is a non-synonymous change at codon 148 (R148H) that falls in the target epitope for some common commercial antibodies used for prion diagnostics, and is highly conserved across species. The pathogenicity of this mutation is still unknown.

Binding of excess cadmium(II) to Cd7-metallothionein from recombinant mouse Zn7-metallothionein 1. UV-VIS absorption and circular dichroism studies and theoretical location approach by surface accessibility analysis.

A mouse metallotbionein (MT) 1 expression system has been constructed that renders recombinant MT as a high purity Zn-coordinated protein. Spectral changes in absorption and circular dichroism following the addition of up to 7 mol equivalents of Cd2+ to recombinant Zn7-MT showed that it behaves like the native protein. Exposure of Cd7-MT to Cd2+ resulted in further binding of these ions to the protein, although saturation was not achieved on the addition of up to 22 mol equivalents of Cd2+ to Zn7-MT. Spectral data are compatible with a model in which the first four additional Cd2+ ions are bound to Cd7-MT via sulfur atoms, and indicate that no further thiol groups are involved in the binding of the excess Cd(II) over 11. Cd2+ ions bound in excess to Cd7-MT appear to have lower binding constants as exposure of Cdn-MT (n > 7) species to Cbelex-100 retrieved Cd7-MT. Based on the X-ray data, the accessible surface areas of sulfur atoms in Cd5,Zn2-MT 2 were calculated. This led us to propose that the coordination of the first three additional Cd(II) ions to Cd7-MT proceeds by means of S-Met1-O-Met1, S-Cys7-S-Cys13 and S-Cys5-S-Cys26 pairs. Finally, comparison of the behavior of the entire MT with that of the recombinant alpha MT and beta MT subunits indicates that mutual influences may not be negligible.

Multicentric study of seroprevalence of Borrelia burgdorferi and Anaplasma phagocytophila in high-risk groups in regions of central and southern Italy.

The aim of this study was to evaluate the seroprevalence of B. burgdorferi and A. phagocytophila in populations of workers from 4 Italian regions, known to be exposed to tick bites. A total of 712 serum samples collected were divided as follows: 387 samples were obtained from workers at risk for tick bites and 325 from individuals that were not considered to be at risk of ticks bites and served as the control group. Antibodies against B. burgdorferi were found in 29 (7.5%) of the 387 risk workers and in 4 (1.2%) of the 325 control group. Antibodies reactive with the HGE agent were found in 22 (5.7%) of the 387 risk workers and in 3 (0.9%) of the 325 control group. Antibodies to both B. burgdorferi and A. phagocytophila were found in 1.6% of the forestry workers confirming the possibility of coinfection or concurrent infection. The present finding show significant differences between seroprevalence of the risk workers and that of the people with no risk for tick exposure.

Beta-lactamase induction antagonizes beta-lactam susceptibilities in Citrobacter diversus and Enterobacter cloacae clinical isolates.

Inducible beta-lactamases were obtained after exposure to several beta-lactams in clinical isolates of Enterobacter cloacae and Citrobacter diversus. Enzyme production was related to the inducer and medium composition. beta-lactamase is able to inactivate only labile compounds, thus generating minimum inhibitory concentrations higher than in the absence of the inducer; imipenem susceptibilities usually were not changed.

Expression and some properties of beta-lactamase from Mycobacterium fortuitum.

Several species of mycobacteria have been reported to produce beta-lactamases, but only those of M. smegmatis have been purified and partially characterized. This study is a preliminary report of the presence of beta-lactamase activity in M. fortuitum, strain Cow 18. A partial purification of the beta-lactamase has also been achieved. M. fortuitum was grown in either Sauton or glucose-yeast extract medium (GYM) and sonicated cells or culture filtrates were assessed for the presence of beta-lactamase activity using a chromogenic compound (PADAC) as substrate. Cells growing in GYM medium released a detectable amount of enzyme, whereas microorganisms showed only intracellular beta-lactamase activity. The enzyme present in the culture filtrate of M. fortuitum Cow 18 was concentrated by Amicon ultrafiltration and partially purified through Sephadex G-75 and QAE-Sephadex A-50 ion exchanger columns. The spectrum of activity of this enzyme included some cephalosporins (cephaloridine, cephalothin) and some penicillins, the hydrolysis of the former being generally more pronounced. Furthermore, cefoxitin, ceftazidime and cefotaxime were not hydrolysed.