Comparative cardiovascular safety of strontium ranelate and bisphosphonates: a multi-database study in 5 EU countries by the EU-ADR Alliance.

Strontium ranelate use, compared with oral bisphosphonates, is not associated with increased risk of AMI in patients with no contraindications for SR use. However, current strontium ranelate (compared with current bisphosphonate) appears associated with 25-30% excess risk of VTE and 35% excess risk of CVDeath. INTRODUCTION: Evaluate the risk of cardiac and thromboembolic events among new users of SR and oral BPs without contraindications for SR. METHODS: We conducted three multi-national, multi-database (Aarhus-Denmark, HSD-Italy, IPCI-Netherlands, SIDIAP-Spain, THIN-UK) case-control studies nested within a cohort of new users of SR/BP. We matched cases of acute myocardial infarction (AMI), venous thromboembolism (VTE), and cardiovascular death (CVDeath), up to 10 controls on gender, year of birth, index date, and country. Conditional logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CIs) according to current SR vs current BP use and current vs past SR use, adjusting for potential confounders. Data were pooled using random effects meta-analysis. RESULTS: No excess risk of AMI (5477 cases/54,674 controls) was found with current SR vs current BP (OR 0.89 (95%CI 0.70, 1.12)) nor with current vs past SR use (0.71(0.56, 0.91)). For VTE (5614 cases/6036 controls), an excess risk was found with current SR compared with current BP use, 1.24 (0.96, 1.61), and current vs past SR use, 1.30 (1.04, 1.62). For CVDeath (3019 cases/29,871 controls), an increased risk was seen with current SR vs current BP use, 1.35 (1.02, 1.80), but not with current vs past SR use (0.68 (0.48, 0.96)). CONCLUSION: In patients without contraindications for SR, we found no evidence of an increased risk of AMI but a 25-30% excess risk of VTE and a 35% excess risk of CVDeath with current SR vs current BP users. This is despite a reduction in risk in CVDeath with current vs past SR users. The latter disparity could still be partially explained by cessation of preventative therapies in end-of-life or residual confounding by indication.

Correction to: Impact of risk minimisation measures on the use of strontium ranelate in Europe: a multi-national cohort study in 5 EU countries by the EU-ADR Alliance.

The original version of this article, published on 26 November 2019 contained a mistake.

Impact of risk minimisation measures on the use of strontium ranelate in Europe: a multi-national cohort study in 5 EU countries by the EU-ADR Alliance.

INTRODUCTION: In May 2013 and March 2014, the European Medicines Agency (EMA) issued two decisions restricting the use of strontium ranelate (SR). These risk minimisation measures (RMM) introduced new contraindications and limited the indications of SR therapy. The EMA required an assessment of the impact of RMMs on the use of SR in Europe. Methods design: multi-national, multi-database cohort Setting: electronic medical record databases based on hospital (Denmark) and primary care provenance (Italy, Spain, the Netherlands, UK). PARTICIPANTS: the database source populations were included for population-based analyses, and SR users for patient-level analyses. INTERVENTION: New RMMs included contraindications (ischaemic heart disease, peripheral arterial disease, cerebrovascular disease, uncontrolled hypertension) and restricted SR indication to severe osteoporosis with initiation by experienced physician and not as first line anti-osteoporosis therapy. METHODS: Prevalence and incidence rates of SR use in the population; prevalence of contraindications and restricted indications in SR users, plus 1-year therapy persistence. Drug use measures were calculated in three periods for comparison: reference (2004 to May 2013), transition (June 2013 to March 2014) and assessment (from April 2014 to end 2016). RESULTS: The study population included 143 million person-years(PY) of follow-up and 76,141 incident episodes of SR treatment. Average monthly prevalence rates of SR use dropped by 86.4% from 62.6/10,000 PY (95 CI 62.4-62.9) in the reference to 8.5 (8.5-8.6) in the assessment period. Similarly, the incidence rate of SR use fell by 97.3% from 7.4/10,000 PY (7.4-7.4) to 0.2 (0.2-0.2) between the reference and assessment period. The prevalence of any contraindication decreased, whilst the prevalence of restricted indications increased in these periods. One-year persistence decreased in the assessment compared with reference period. CONCLUSIONS: Our study demonstrates a substantial impact of the regulatory action to restrict use of SR in Europe: SR utilisation overall decreased strongly. The proportion of patients fulfilling the restricted indications, without contraindications, increased after the proposed RMMs.

Prescribing practices and clinical predictors of glucose-lowering therapy within the first year in people with newly diagnosed Type 2 diabetes.

AIM: To examine prescribing practices and predictors of glucose-lowering therapy within the first year following diagnosis of Type 2 diabetes mellitus in a clinical care setting. METHODS: We followed people enrolled in the Danish Centre for Strategic Research in Type 2 Diabetes (DD2) cohort from outpatient hospital clinics and general practices throughout Denmark in 2010-2013. We used Poisson regression to compute age- and gender-adjusted risk ratios (RRs). RESULTS: Among 1158 new Type 2 diabetes mellitus patients, 302 (26%) did not receive glucose-lowering therapy within the first year, 723 (62%) received monotherapy [685 (95%) with metformin], and 133 (12%) received more than one drug. Predictors of receiving any vs. no therapy and combination vs. monotherapy were: age < 40 years [RR: 1.29 (95% CI: 1.16-1.44) and 3.60 (95% CI: 2.36-5.50)]; high Charlson Comorbidity Index [RRs: 1.20 (95% CI: 1.05-1.38) and 2.08 (95% CI: 1.16-3.72)]; central obesity [RRs: 1.23 (95% CI: 1.04-1.44) and 1.93 (95% CI: 0.76-4.94)]; fasting blood glucose of ≥ 7.5 mmol/l [RRs: 1.25 (95% CI: 1.10-1.42) and 1.94 (95% CI: 1.02-3.71)]; and HbA1c ≥ 59 mmol/mol (≥ 7.5%) [RR: 1.26 (95% CI: 1.20-1.32) and 2.86 (95% CI: 1.97-4.14)]. Weight gain ≥ 30 kg since age 20, lack of physical exercise and C-peptide of < 300 pmol/l also predicted therapy. CONCLUSIONS: Comorbidity, young age, central obesity and poor baseline glycaemic control are important predictors of therapy one year after Type 2 diabetes mellitus debut.

Inactivation of E2a in recombinant adenoviruses improves the prospect for gene therapy in cystic fibrosis.

Although first generation recombinant adenoviruses, deleted of sequences spanning E1a and E1b, have been useful for in vivo applications of gene therapy, expression of the recombinant gene has been transient and often associated with the development of inflammation. We show that with first generation adenovirus-mediated gene transfer to the mouse lung, viral proteins are expressed leading to destructive cellular immune responses and repopulation of the lung with nontransgene containing cells. Second generation E1 deleted viruses further crippled by a temperature sensitive mutation in the E2a gene were associated with substantially longer recombinant gene expression and less inflammation. Stable expression of human CF transmembrane conductance regulator has been achieved in lungs of CF mice instilled with a second generation virus.

Independent and joint effects of antibodies to human heat-shock protein 60 and Chlamydia pneumoniae infection in the development of coronary atherosclerosis.

BACKGROUND: Studies have suggested that the prevalence of antibodies against heat-shock proteins (HSPs), Chlamydia pneumoniae (CPN), and cytomegalovirus (CMV) is associated with coronary artery disease (CAD), but the independent or joint effects of human (h) HSP60 antibodies and these pathogens in patients have not been fully elucidated. METHODS AND RESULTS: A total of 405 subjects (276 patients with CAD and 129 control individuals) were tested for serum antibodies to hHSP60, CPN, and CMV immediate-early-1 (IE1) antigens. Patients were also assessed for serum cholesterol, triglyceride levels, and smoking habit. Significantly elevated levels of antibodies to hHSP60 and CPN but not to CMV-IE1 antigens were documented in CAD patients. Multiple logistic regression analysis and subanalyses of selected subjects showed that these associations were independent of age, sex, smoking, and serum lipid levels. Antibodies to hHSP60 and CPN did not correlate quantitatively; however, the relative risk of disease development was substantially increased in subjects with high antibody levels to both hHSP60 and CPN:, reaching an odds ratio of 82.0 (95% CI 10.6 to 625.0). CONCLUSIONS: High levels of antibodies to hHSP60 and CPN: are independent risk factors for coronary atherosclerosis, but their simultaneous presence substantially increases the risk for disease development.

Sindbis virus receptor protein of BHK cells.

Exposure of cultured BHK 21 cells to very low concentrations of non-ionic detergent Nonidet P40 resulted in the elution of cellular proteins located on the outer surface of the plasma membrane. One of these proteins, partially purified by affinity ultrafiltration and cosedimentation with Sindbis virions, seems to be the "receptor molecule" of Sindbis virus.

Impaired interleukin 2 production by spleen cells from mice infected with human adenovirus.

Spleen cells from mice infected with human adenovirus type 6 (Ad6) showed defective interleukin 2 (IL2) production 3-5 days after the infection. The response of spleen cells to exogenous IL 2 was also deficient. The impaired capacity of concanavalin A--(Con A)-activated spleen cells from Ad6--infected mice to utilize IL 2 seemed to be related to the depressed capacity of the infected splenocytes to express IL 2 receptors. The immunologic dysfunction following infection with Ad6 may be a consequence of deficiencies in both the elaboration of and response to IL 2.

Involvement of prostaglandins in immunosuppression caused by adenovirus in mice.

The effect of intraperitoneally (i.p.) inoculated human adenovirus type 6 (Ad6) was tested for humoral immune response against sheep red blood cells (SRBC) in normal and indomethacin-treated mice, with the aim to elucidate the mode of the virus action in immunosuppression. The results indicate that inhibition of prostaglandin synthesis slightly influences the immunosuppressive effect of the virus. It is very likely that also other mechanisms are involved in the immunosuppression observed.

Physico-chemical characteristics of interferons induced by human adenovirus in chick fibroblasts and leucocytes.

The physico-chemical characteristics of interferons with different anti-viral activities produced in chick fibroblast cells and leucocytes in response to the human adenovirus type 12 were investigated. The molecular weights of interferons produced by chick fibroblasts and leucocytes were similar: the main anti-viral activity was found at 27000 and 32000 and a low-titered shoulder at 28000 and 23000 respectively. Under our experimental conditions no oligomeric structure of interferons could be detected. The isoelectric pattern of leucocyte interferon was slightly different from that of the fibroblast interferon.

The role of macrophages in adenovirus-induced immunosuppression in mice.

A decreased humoral immune response to sheep red blood cells (SRBC) was demonstrated in CFLP mice inoculated with human adenovirus type 6 (Ad6 virus). The suppressive effect depended on the inoculation sites of the virus and SRBC. Inoculation by the same route, i.e. intraperitoneally (i.p.), resulted in a significant decrease of the humoral immune response to SRBC. When administering the virus and SRBC at different sites, no inhibitory effect could be detected. Immunosuppression was prevented by pretreatment of mice with silica depressing the macrophage function. The results indicate that peritoneal macrophages play an important role in the immunosuppression observed.

Effect of human adenovirus type 6 on the primary immune response in mice.

CFLP and BALB/c mice inoculated intraperitoneally with large doses of adenovirus type 6 (Ad6) showed a decreased humoral immune response to sheep red blood cells (SRBC) and circulating interferon was detected in their serum. The timing of infection was critical. Infection of mice 3--11 days before SRBC administration led to depression of the 19 S haemolytic plaque forming cell (HPFC) response in the spleen. When mice were given Ad6 and SRBC simultaneously on Ad6 14 days before or 1 day after SRBC, there was no decrease in the number of HPFC. The suppressive effect was dependent on the dose of virus and antigen. Heat and UV treatment completely abolished the immunosuppressive effect of the virus, suggesting that a great amount of infectious adenovirus is needed to induce immunosuppression in mice.

Immunoenhancement and suppression induced by adenovirus in chicken.

Chickens injected intravenously (i.v.) with human adenovirus type 6 (Ad6) reveal a 2-17-fold increase in the number of plaque-forming cells producing antibody (Ab) against sheep red blood cells (SRBC) 2-6 days after virus infection. Further, polyclonal B-cell activation has been demonstrated by the quantitation of immunoglobulin-producing cells (IgPC) and cells producing immunoglobulin (Ig) of IgM isotype (IgPC mu) in the spleen of chicken inoculated with Ad6. Ad6 infection in chicken results in immunosuppression against SRBC when this unrelated antigen is given after virus infection. It seems that coincidence occurs between the B-cell mitogenic activation and the immunosuppression caused by Ad6, as the most pronounced change in both activities appears on the fourth day following virus infection. These findings suggest that the B-cell mitogenicity of the virus contributes to the impairment of the humoral immune response to SRBC.

An adenovirus-herpes simplex virus glycoprotein B recombinant (Ad-HSV.gB) protects mice against a vaccinia HSV.gB and HSV challenge.

We investigated the mechanism by which mice immunized with an adenovirus-herpes simplex glycoprotein B recombinant (Ad-HSV.gB) are protected from challenge with a vaccinia (Vac)-HSV.gB and the cognate virus, HSV. C57/BL mice immunized intraperitoneally with Ad-HSV.gB were protected against an intracerebrally inoculated lethal dose of a Vac-HSV.gB or HSV-1. CD8+ cytotoxic T lymphocytes but not interferon-gamma-dependent mechanisms play a major role in the clearance of both viruses from the central nervous system. These results demonstrate that the administration of two recombinant viruses carrying the same foreign insert for either immunization or challenge in a mouse protection model provides useful information about the protective nature of the inserted gene product.

Comparative study of antiproliferative effects of chlorpromazine, 7,8-dioxochlorpromazine, amantadine-N-mustard, rutin-N-mustard and alpha, beta and gamma interferon on K-562 cells in vitro.

The effects of rutin-N-mustard, amantadine-N-mustard, chlorpromazine and human interferon types alpha, beta and gamma (IFN-alpha, -beta and -gamma) were studied on the DNA, RNA and protein synthesis of K-562 cells. Monocyte-mediated cytotoxicity and immune spleen cell activity were examined in the presence of the same compounds (except for IFN-beta). The natural killer (NK) cell activity was tested in the presence of the two chlorpromazine compounds and the two N-mustard derivatives. Only 7,8-dioxochlorpromazine exerted an inhibitory effect on DNA synthesis. The protein synthesis of the cells was inhibited in the presence of IFN-alpha, -beta and -gamma. 7,8-Dioxochlorpromazine exerted some inhibition on both NK and immune spleen cell activity, while monocyte-mediated cytolysis was not altered. IFN-alpha, -beta and -gamma activated the cytolytic activity of monocytes and the NK activity in control experiments. Chlorpromazine, rutin-N-mustard and amantadine-N-mustard were ineffective in both tests in vitro. Rutin-N-mustard, 7,8-dioxochlorpromazine and the interferons may be assumed to have quite different antiproliferative mechanisms of actions.

Murine cytotoxic T cell response specific for human cytomegalovirus glycoprotein B (gB) induced by adenovirus and vaccinia virus recombinants expressing gB.

A murine model of the cytotoxic T lymphocyte (CTL) response to glycoprotein B (gB) of human cytomegalovirus (HCMV) was developed based on the use of adenovirus (Ad) and vaccinia virus (Vac) recombinants expressing gB. Mice of different major histocompatibility haplotypes [CBA (H-2k), BALB/k (H-2k) and BALB/c (H-2d)] infected with the Ad-gB recombinant developed an Ad-specific CTL response. However, only the H-2k mice developed a significant HCMV gB-specific CTL response, as indicated by the major histocompatibility complex class I-restricted lysis of Vac strain Copenhagen (VacC)-gB recombinant-infected target cells by H-2k mouse immune spleen cells. The VacC-gB recombinant elicited only a weak gB-specific CTL response in these mice, indicating that the observed gB-specific CTL response in mice is dependent on the expression vector used for immunization. The gB-specific cytotoxicity observed in H-2k mice was mediated by the CD8 lymphocyte subset.

Roles of interleukin-12 and gamma interferon in murine Chlamydia pneumoniae infection.

BALB/c and strain 129 mice infected intranasally with Chlamydia pneumoniae displayed a moderate-to-severe inflammation in the lungs and produced interleukin-12 (IL-12), gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and IL-10, with peak levels on days 1 to 3 postinfection (p.i.), returning to basal levels by day 16 p.i. Anti-IL-12 treatment resulted in less-severe pathological changes but higher bacterial titers on days 3 and 7 p.i. By day 16 p.i., the inflammatory responses of control antibody-treated mice subsided. The bacterial titers of both anti-IL-12- and control antibody-treated mice decreased within 3 weeks to marginally detectable levels. Anti-IL-12 treatment significantly reduced lung IFN-gamma production and in vitro spleen cell IFN-gamma production in response to either C. pneumoniae or concanavalin A. In gamma-irradiated infected mice, cytokine production was delayed, and this delay correlated with high bacterial titers in the lungs. Following C. pneumoniae infection, 129 mice lacking the IFN-gamma receptor alpha chain gene (G129 mice) produced similar IL-12 levels and exhibited similarly severe pathological changes but had higher bacterial titers than 129 mice. However, by day 45 p.i., bacterial titers became undetectable in both wild-type 129 and G129 mice. Thus, during C. pneumoniae lung infection, IL-12, more than IFN-gamma, plays a role in pulmonary-cell infiltration. IFN-gamma and IL-12, acting mostly through its induction of IFN-gamma and Th1 responses, play an important role in controlling acute C. pneumoniae infection in the lungs, but eventually all mice control the infection to undetectable levels by IL-12- and IFN-gamma-independent mechanisms.

Early atherosclerotic plaques in the aorta following cytomegalovirus infection of mice.

We show here that BALB/c mice inoculated with murine cytomegalovirus (MCMV) express viral antigens in the endothelial and smooth muscle cells of the aortic wall, and that accumulation of inflammatory cells in the aortic lumen, similar to that seen in early atherosclerotic lesions in humans, colocalizes with the site of virus antigen expression. Immunosuppression of the mice at the time of virus infection increased the expression of viral antigens and the size of early atherosclerotic lesions in the intima. The percentage of the low-density lipoprotein cholesterol (LDL-C), the major lipid contributor to atherosclerotic plaques, was significantly increased in the serum of MCMV-infected mice, whether or not the mice were fed a high cholesterol diet. Human cytomegalovirus (HCMV) significantly increased the esterified cholesterol component of the total cholesterol in a human arterial smooth muscle cell line infected in vitro with HCMV. These results suggest that CMV infection is involved in two of the major mechanisms that lead to development of atherosclerosis, i.e., immune injury and high LDL-C.

High expression of human cytomegalovirus (HCMV)-gB protein in cells infected with a vaccinia-gB recombinant: the importance of the gB protein in HCMV immunity.

The human cytomegalovirus (HCMV), Towne strain, glycoprotein B (gB) gene was cloned into a vaccinia vector (Copenhagen strain) under the control of the H6 early and late vaccinia promoters (Vac-gB recombinant). The gB protein was expressed in a high percentage of the Vac-gB-infected cells throughout the virus replication cycle. Cytosine-arabinoside (ara-C) did not influence the expression of the gB protein early after infection (5 h), but did inhibit it later in viral replication (7-29 h). The Vac-gB recombinant induced HCMV neutralizing antibodies in guinea-pigs. Cells infected with the Vac-gB recombinant absorbed 50-88% of neutralizing activity of human sera obtained from volunteers previously inoculated with the Towne or Toledo strains and from naturally seropositive individuals.

Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy.

An important limitation that has emerged in the use of adenoviruses for gene therapy has been loss of recombinant gene expression that occurs concurrent with the development of pathology in the organ expressing the transgene. We have used liver-directed approaches to gene therapy in mice to study mechanisms that underlie the problems with transient expression and pathology that have characterized in vivo applications of first-generation recombinant adenoviruses (i.e., those deleted of E1a and E1b). Our data are consistent with the following hypothesis. Cells harboring the recombinant viral genome express the transgene as desired; however, low-level expression of viral genes also occurs. A virus-specific cellular immune response is stimulated that leads to destruction of the genetically modified hepatocytes, massive hepatitis, and repopulation of the liver with nontransgene-containing hepatocytes. These findings suggest approaches for improving recombinant adenoviruses that are based on further crippling the virus to limit expression of nondeleted viral genes.