Hepatitis B virus impairs TLR9 expression and function in plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) play a key role in detecting pathogens by producing large amounts of type I interferon (IFN) by sensing the presence of viral infections through the Toll-Like Receptor (TLR) pathway. TLR9 is a sensor of viral and bacterial DNA motifs and activates the IRF7 transcription factor which leads to type I IFN secretion by pDCs. However, during chronic hepatitis B virus (HBV) infection, pDCs display an impaired ability to secrete IFN-α following ex vivo stimulation with TLR9 ligands. Here we highlight several strategies used by HBV to block IFN-α production through a specific impairment of the TLR9 signaling. Our results show that HBV particle internalisation could inhibit TLR9- but not TLR7-mediated secretion of IFN-α by pDCs. We observed that HBV down-regulated TLR9 transcriptional activity in pDCs and B cells in which TLR9 mRNA and protein levels were reduced. HBV can interfere with TLR9 activity by blocking the MyD88-IRAK4 axis and Sendai virus targeting IRF7 to block IFN-α production. Neutralising CpG motif sequences were identified within HBV DNA genome of genotypes A to H which displayed a suppressive effect on TLR9-immune activation. Moreover, TLR9 mRNA and protein were downregulated in PBMCs from patients with HBV-associated chronic hepatitis and hepatocellular carcinoma. Thus HBV has developed several escape mechanisms to avoid TLR9 activation in both pDCs and B lymphocytes, which may in turn contribute to the establishment and/or persistence of chronic infection.

The e-Bug project in France.

The high rates of antibiotic prescriptions and antimicrobial resistance in France motivated its participation in the European e-Bug school project concerning microbes, and infection transmission, prevention and treatment. The prospect of raising awareness among children, helping them to adopt suitable attitudes and behaviour towards infection transmission and treatment starting from childhood, generated enthusiastic support from relevant national educational and health institutions throughout the Project. France was actively involved in every stage: background research showed that the subject matter was best suited to the national science curricula of the fourth and fifth forms in junior schools, and the sixth and ninth forms in senior schools; a focus group study with junior and senior teachers elicited teachers' needs concerning teaching resources; and a qualitative and quantitative evaluation, after translation and pack review, enabled further adaptation of the packs. This evaluation showed an overall enthusiastic reception by teachers and their students in France, and reassured teachers on the ease of use of the Project's resources and students' progress. The e-Bug Project was launched through a national institutional implementation plan in September 2009 and orders for e-Bug tools increased rapidly. By the end of October, 57% of all senior science teachers and 16% of all junior school teachers had ordered the pack. France is one of the most frequent users of the e-Bug web site. The collaboration with both educational and health partners was particularly helpful to implementing the Project, and this was confirmed by the favourable reception and participation of teachers and students in the field.

Hepatitis B virus replication in primary macaque hepatocytes: crossing the species barrier toward a new small primate model.

UNLABELLED: The development of new anti-hepatitis B virus (HBV) therapies, especially immunotherapeutic approaches, has been limited by the lack of a primate model more accessible than chimpanzees. We have previously demonstrated that sylvanus and cynomolgus macaques are susceptible to in vivo HBV infection after intrahepatic HBV DNA inoculation. In this study, we evaluated the susceptibility of primary macaque hepatocytes (PMHs) to HBV infection with a highly efficient HBV genome-mediated transfer system via a recombinant baculovirus (Bac-HBV). Freshly prepared PMHs, isolated from macaque liver tissue by collagenase perfusion, were transduced with Bac-HBV, and intermediates of replication were followed for 9 days post-transduction. Evidence of HBV replication (hepatitis B surface antigen secretion, viral DNA, RNA, and covalently closed circular DNA) was detected from day 1 to day 9 post-transduction. HBV markers were dose-dependent and still detectable at a multiplicity of infection of 10. Importantly, transduced PMHs secreted all typical forms of HBV particles, as evidenced by a cesium chloride gradient as well as transmission electron microscopy. Furthermore, the Toll-like receptor 9 (TLR9) ligand was used to stimulate freshly prepared macaque peripheral blood mononuclear cells to generate TLR9-induced cytokines. We then demonstrated the antiviral effects of both TLR9-induced cytokines and nucleoside analogue (lamivudine) on HBV replication in transduced PMHs. CONCLUSION: Baculovirus-mediated genome transfer initiated a full HBV replication cycle in PMHs; thus highlighted both the baculovirus efficiency in crossing the species barrier and macaque susceptibility to HBV infection. Moreover, our results demonstrate the relevance of thus system for antiviral compound evaluations with either nucleoside analogues or inhibitory cytokines. Cynomolgus macaques are readily available, are immunologically closely related to humans, and may therefore represent a promising model for the development of new immunotherapeutic strategies.

Oral cholic acid for hereditary defects of primary bile acid synthesis: a safe and effective long-term therapy.

BACKGROUND & AIMS: Oral bile acid replacement has been shown to be an effective therapy in primary bile acid synthesis defects, but to date there have been no reports of the long-term effects of this therapy. The aim of the study was to evaluate the long-term effectiveness and safety of cholic acid (CA) therapy. METHODS: Fifteen patients with either 3beta-hydroxy-Delta(5)-C(27)-steroid oxidoreductase (3beta-HSD) (n = 13) or Delta(4)-3-oxosteroid 5beta-reductase (Delta(4)-3-oxo-R) (n = 2) deficiency confirmed by mass spectrometry and gene sequencing received oral CA and were followed up prospectively. RESULTS: CA therapy was started at a median age of 3.9 years (range, 0.3-13.1 years). The median follow-up with treatment was 12.4 years (range, 5.6-15 years). The mean daily dose of CA was initially 13 mg/kg and was 6 mg/kg at last evaluation. During CA therapy, physical examination findings, laboratory test results, and findings on sonography normalized. Mass spectrometry analysis of urine showed that excretion of the atypical metabolites was reduced by 500-fold and 30-fold in 3beta-HSD and Delta(4)-3-oxo-R deficiency, respectively, and total urinary bile acid excretion decreased dramatically. Liver biopsies performed in 14 patients after at least 5 years of CA therapy showed marked improvement, especially in patients with the 3beta-HSD deficiency. CA was well tolerated with all children developing normally, including 2 women having 4 normal pregnancies during treatment. CONCLUSIONS: Oral CA therapy is a safe and effective long-term treatment of the most common primary bile acid synthesis defects.

Inhibitory effect of the combination of CpG-induced cytokines with lamivudine against hepatitis B virus replication in vitro.

BACKGROUND: Currently approved antiviral monotherapies against chronic hepatitis B fail to eradicate hepatitis B virus (HBV), to overcome the defects in HBV-specific immune responses and to prevent HBV relapse after cessation of therapy. CpG oligodesoxynucleotides (CpG ODN) are synthetic agonists of Toll-like receptor 9 and potent inducers of innate and acquired immunity. Our aim was to establish the proof of concept of the antiviral benefit of combining a nucleoside analogue with CpG-induced cytokines on HBV replication in vitro. METHODS: Peripheral blood mononuclear cells from HBV-negative individuals were stimulated with CpG ODN to generate CpG-induced cytokine supernatants. Proliferating HepaRG and HepG2 cells were transduced with recombinant HBV baculovirus and differentiated HepaRG cells were inoculated with HBV virions. Antiviral effects of CpG-induced cytokine with or without lamivudine were evaluated by analysing HBV DNA, HBV RNA and antigen secretion (hepatitis B surface antigen [HBsAg] and hepatitis B e antigen [HBeAg]). RESULTS: Following transduction or HBV inoculation, CpG-induced cytokines strongly inhibited HBV viral intermediates of replication, as well as HBsAg and HBeAg secretion from infected cells. Strikingly, in transduced HepaRG cells, the combination of CpG-induced cytokines with lamivudine reduced the 50% effective concentration of lamivudine by 100-fold. Importantly, the treatment of CpG-induced cytokines prior to HBV inoculation conferred a partial protection against infection to hepatocytes. CONCLUSIONS: CpG-induced cytokines associated with polymerase inhibitors represent a promising combination to suppress HBV replication. Such an immunotherapeutic strategy should be evaluated in vivo to assess restoration and duration of anti-HBV-specific immune responses.

Immunohistochemical detection of HCV proteins in liver tissue.

Detection and localization of HCV in liver tissue are vital for diagnostic purposes and clinical management of HCV-infected patients, as well as for the elucidation of viropathological mechanisms. The fragility of HCV RNA and the low levels of viral expression in infected tissues are a constant limitation in molecular assays for HCV characterization. HCV antigen detection, by immunochemistry, in liver biopsies is an attractive option for precise localization and quantification of viral proteins with direct access to histological patterns. We describe here a study using a novel immunohistochemical method effective on fixed, archived specimens, including liver biopsies and surgical resection samples. The initial protocol uses a biotin-detection system but can also be used in a polymer-detection system. This protocol offers easy, precise, and strong staining resolution with distinct patterns consistent with the liver pathology, irrespective of the viral HCV genotype examined. This approach provides applications for diagnosis as well as for exploratory pathological studies.

[How to practice health education in a general medicine or a pharmacy setting? Results from an experiment among pregnant women, adolescents and elderly people]. / Comment pratiquer l'education pour la santé en médecine generate ou en pharmacie? Résultats d'une expérimentation auprès de femmes enceintes, d'adolescents et de personnes agées.

This experiment evaluated the development and implementation of 5 pilot health education activities using a comprehensive and population-based approach in general medicine practice and in pharmacies. These interventions involved 185 patients, and they were developed and carried out by 35 general practitioners and pharmacists in 5 sites in France between 2001 and 2004. Health professionals often demonstrated and argued for a comprehensive approach to health: positive vision of health, integration of different aspects of health, and personalized, tailored efforts. Pregnant women, adolescents, and elderly people have certain specificities that must be taken into account in a population-based approach. The activities were based on simple techniques using interview guidelines and informational documents to aid and support both expressiveness and the professional-patient relationship.

Essential role of antigen-presenting cell-derived BAFF for antibody responses.

Antigen-presenting cells (APC) are directly involved in survival, growth and differentiation of naive B cells and in immunoglobulin class switch recombination. Less is known about the contribution of APC to memory B cell responses. We employed an in vitro model to investigate the secondary humoral response against foot-and-mouth disease virus, with cells from a natural host of the virus - the pig. This response is T cell-dependent. Under conditions of limited T cell help, defined as a low T-to-B cell ratio or by the replacement of T cells with interleukin-2 only, the antibody response was dependent on APC. These included monocytes and monocyte-derived DC, but not plasmacytoid DC. APC mediated their help through soluble factors, particularly soluble B cell-activating factor belonging to the TNF family (BAFF). Our results suggest that the 'ménage à trois' concept, saying that both APC and T cells have a direct effect in B cell activation, is also valid for secondary B cell responses, and imply an important role for BAFF under conditions that might be physiologically relevant in secondary lymphoid organs.

Effect of highly active antiretroviral therapy on tacrolimus pharmacokinetics in hepatitis C virus and HIV co-infected liver transplant recipients in the ANRS HC-08 study.

OBJECTIVE: To characterise the interactions between tacrolimus and antiretroviral drug combinations in hepatitis C virus-HIV co-infected patients who had received a liver transplant. DESIGN: An observational, open-label, multiple-dose, two-period, one-sequence design clinical trial in which patients received tacrolimus as an immunosuppressive therapy during the postoperative period and then had an antiretroviral drug regimen added. Tacrolimus pharmacokinetics were evaluated at steady state during these two periods. METHODS: Fourteen patients participated in the study and seven participated in the intensified pharmacokinetic protocol. Patients were included if they had undergone liver transplantation for end-stage chronic hepatitis C, absence of opportunistic infection, a CD4 cell count of >150 cells/microL and an undetectable HIV plasma viral load (<50 copies/mL) under highly active antiretroviral therapy. During the posttransplantation period, the tacrolimus dose was adjusted according to blood concentrations. When liver function and the tacrolimus dose were stable, antiretroviral therapy was reintroduced. RESULTS: When lopinavir/ritonavir were added to the tacrolimus regimen (seven patients), the tacrolimus dose was reduced by 99% to maintain the tacrolimus concentration within the therapeutic range. Only two patients were treated with nelfinavir, which led to a wide variation in inhibition of tacrolimus metabolism. When efavirenz (four patients) or a nucleoside analogue combination (one patient) was added, very little change in tacrolimus dosing was required. CONCLUSION: The lopinavir/ritonavir combination markedly inhibited tacrolimus metabolism, whereas the effect of efavirenz was small. Tacrolimus dosing must be optimised according to therapeutic drug monitoring and the antiretroviral drug combination.

TLR9 expression and function is abolished by the cervical cancer-associated human papillomavirus type 16.

Cervical cancer development is linked to the persistent infection by high-risk mucosal human papillomaviruses (HPVs) types. The E6 and E7 major oncoproteins from this dsDNA virus play a key role in the deregulation of the cell cycle, apoptosis, and adaptive immune surveillance. In this study, we show for the first time that HPV type 16 (HPV16), the most carcinogenic type among the high-risk subgroup, interferes with innate immunity by affecting the expression of TLRs. Infection of human primary keratinocytes with HPV16 E6 and E7 recombinant retroviruses inhibits TLR9 transcription and hence functional loss of TLR9-regulated pathways. Similar findings were achieved in HPV16-positive cancer-derived cell lines and primary cervical cancers, demonstrating that this event occurs also in an in vivo context. Interestingly, E6 and E7 from the low-risk HPV type 6 are unable to down-regulate the TLR9 promoter. In addition, E6 and E7 from the high-risk HPV type 18, which are known to persist less competently in the host than HPV16, have reduced efficiency compared with HPV16 in inhibiting TLR9 transcription. Furthermore, a CpG motif derived from the HPV16 E6 DNA sequence activated TLR9, indicating this virus is able to initiate innate responses via the receptor it later down-regulates. This study reveals a novel mechanism used by HPV16 to suppress the host immune response by deregulating the TLR9 transcript, providing evidence that abolishing innate responses may be a crucial step involved in the carcinogenic events mediated by HPVs.

Silencing of natural interferon producing cell activation by porcine circovirus type 2 DNA.

Porcine circovirus type 2 (PCV2) infection of natural interferon producing cells (NIPCs) impairs the induction of interferon (IFN)-alpha and tumour necrosis factor (TNF)-alpha by cytosine-phosphorothioate-guanine (CpG) oligodeoxynucleotides (ODNs), thereby preventing both their autocrine maturation and the paracrine maturation of myeloid dendritic cells (DCs). The present study shows that the PCV2-mediated inhibition of NIPCs was mediated by viral DNA, although it was independent of virus replication. The inhibitory effect of PCV2 DNA was more diversified than if it had simply targeted CpG-ODN-induced cytokines (IFN-alpha, TNF-alpha, interleukin-6, IL-12). A broad spectrum inhibition was noted, affecting responses induced by toll-like receptor (TLR)-7 and TLR9 agonists, as well as viruses including pseudorabies virus, transmissible gastroenteritis virus and classical swine fever virus. From these results, it would appear that PCV2 DNA can induce a dominant negative signal influencing independent pattern recognition receptor-induced activation cascades. Despite a concomitant internalization of PCV2 DNA and CpG-ODNs, no colocalization was observed, indicating that PCV2 DNA and CPG-ODNs may not target the same receptor. This study describes a novel modulation of the innate immune response, which would render the host more susceptible to secondary or concomitant microbial infections.

Cytokine and C-reactive protein profiles induced by porcine circovirus type 2 experimental infection in 3-week-old piglets.

The purpose of this study was to determine serum profiles of cytokines at a protein level and Creactive protein (CRP) during the development of postweaning multisystemic wasting syndrome (PMWS) in experimentally inoculated pigs. Levels of serum IFN-alpha, IL-6, IL-10, and CRP were examined for a 35-day period in 10 piglets experimentally infected with PCV2 at 3 weeks of age. Four of the infected piglets developed severe PMWS at 14 to 21 days post-infection (d.p.i.) and died prior to termination of the experiment. The remaining six PCV2-infected piglets experienced transient fever, but did not display overt clinical signs of PMWS and were considered as subclinically infected. A bioassay was used to detect IL-6 and ELISAs were used to detect IFN-alpha, IL-10, and CRP. There were no significant differences in cytokine or CRP expression from 0 to 7 d.p.i. between the PMWS-affected and the subclinically infected piglets. Levels of IL-10 and CRP were elevated from 10 and 14 d.p.i. respectively in the PMWS-affected piglets compared to the subclinically infected piglets. There were no significant differences in IFN-alpha and IL-6 expression between the PMWS-affected piglets and the subclinically infected piglets. The present study shows that elevated levels of serum CRP and IL-10 were associated with PCV2-infected piglets that subsequently developed severe PMWS. This may help to provide further insight into the immunoaetiogenesis of this syndrome.

Life-threatening interaction between antiretroviral therapy and vinblastine in HIV-associated multicentric Castleman's disease.

BACKGROUND: HIV-infected patients still present a high risk of developing lymphoproliferative malignancies, despite the fact that their prognosis has been considerably improved by highly active antiretroviral therapy (HAART). Potential interactions exist between antiretroviral drugs, in particular protease inhibitors, and anti-neoplastic ones, but their impact in terms of clinical and haematological adverse events remains unclear. METHODS: We report a case of potentially life-threatening interaction between vinblastine and antiretroviral therapy in a patient presenting with HIV-associated multicentric Castleman's disease (MCD). RESULTS: A 55-year-old HIV-1 infected patient was diagnosed with MCD while being treated with a salvage HAART regimen consisting of zidovudine, lamivudine, abacavir, nevirapine and ritonavir-boosted lopinavir. Vinblastine was prescribed as a monotherapy at the usual dose of 6 mg/m2 (i.e. 10 mg) every 3 weeks. The first vinblastine course was performed without HAART with no adverse event. Antiretroviral therapy was resumed for the two following courses which were associated with unexpected severe digestive and haematological toxicities, and moderate renal failure. Vinblastine was then administered alone at increasing doses without toxicity. HAART was finally resumed and we assessed that a decreased vinblastine dose of 2 mg/m2 was well tolerated, with a complete response of MCD. CONCLUSIONS: HAART regimen comprising protease inhibitors, which are potent inhibitors of cytochrome P450 and P-gp, could interfere with anti-neoplastic drugs, as demonstrated with vinblastine in our case report.

Determination of amprenavir total and unbound concentrations in plasma by high-performance liquid chromatography and ultrafiltration.

Amprenavir is an HIV-1 protease inhibitor with high protein binding (90%) in human plasma. This study was designed to develop an assay to measure the concentration of unbound amprenavir, to study variation with time in patients, and to investigate whether ritonavir and lopinavir, other protease inhibitors that could be combined, interact with amprenavir protein binding in vitro. A reverse-phase high-performance liquid chromatography assay to UV detection was developed and validated to measure total amprenavir in plasma, and this method was adapted to quantitate low concentrations of unbound amprenavir in ultrafiltrate aqueous fluid. Equilibrium dialysis and ultrafiltration were used and compared with separate unbound fraction. The latter method was easier to use and was, therefore, subsequently adopted. In 10 patients who received amprenavir 600 mg bid combined with ritonavir, mean amprenavir free-fraction in plasma was 8.6% (range, 4.4-20%). When added to pooled human plasmas at concentrations close to those found in treated patients, the unbound amprenavir fraction was increased in the presence of lopinavir, but remained unaffected by ritonavir. It remains to be seen whether measurement of unbound concentrations, rather than total concentrations, could improve therapeutic drug monitoring.

High variability of indinavir and nelfinavir pharmacokinetics in HIV-infected patients with a sustained virological response on highly active antiretroviral therapy.

OBJECTIVES: To describe plasma concentrations of indinavir alone or combined with ritonavir, and of nelfinavir and its active metabolite M8, and to measure their variabilities in HIV-infected patients treated with a stable antiretroviral regimen and experiencing a sustained virological response for at least 12 months. PATIENTS AND METHODS: In this prospective trial, blood samples were drawn during a 6-hour time interval between two doses at enrolment to assess protease inhibitor (PI) pharmacokinetic parameters, and 4 months later to assess plasma trough and peak concentrations. Safety and adherence assessments and laboratory data were collected during an 8-month period. PI pharmacokinetic characteristics were analysed using a non-compartmental approach. Inter- and intrapatient variabilities were estimated using a linear mixed-effect model. The impact of different covariates on plasma trough concentrations was investigated. Eighty-eight patients were analysed: 42 treated with indinavir and 46 with nelfinavir. RESULTS: The interquartile range (IQR) of the plasma trough concentration corrected for the sampling time (Ccalc) was 116-374 microg/L for indinavir alone and 163-508 microg/L for indinavir/ritonavir. Ritonavir significantly increased indinavir elimination half-life and plasma exposure. For nelfinavir, the IQR of Ccalc was 896-2059 microg/L for three-times-daily administration and 998-2124 microg/L for twice-daily administration. Variabilities were high for both PIs. Intrapatient variability for indinavir alone (and indinavir + ritonavir) was 76% (107%) and interpatient variability was 58% (10%) in adherent patients. Intrapatient variability for nelfinavir three times daily (and twice daily) was 41% (74%) and interpatient variability was 62% (50%). Intrapatient variability was lowered in patients with a high adherence level. CONCLUSION: Although performed in a homogeneous population, this study documented a high interpatient but also intrapatient variability of indinavir and nelfinavir pharmacokinetics, which should be taken into account when interpreting therapeutic drug monitoring. Once patients have reached a sustained virological response, plasma PI monitoring may have a limited impact.

Fc gamma RII-dependent sensitisation of natural interferon-producing cells for viral infection and interferon-alpha responses.

Natural interferon-producing cells (NIPC), also called plasmacytoid dendritic cells, are the most potent producers of IFN-alpha in response to viral and bacterial components, serving an important function in innate immune defences. The present work demonstrates that NIPC responsiveness can be primed by immunisation, increasing their capacity to produce IFN-alpha after viral infection. NIPC isolated from pigs immunised against classical swine fever virus (CSFV), a member of the Flaviviridae, were more receptive to viral infection and produced higher levels of IFN-alpha than NIPC from immunologically naive animals. This sensitisation of NIPC was maintained for at least 8 months after immunisation. IFN-alpha production was dependent on live virus and required replication, and the "immune" NIPC responded to lower infectious doses of virus. Co-localisation of the virus with Fc(gamma)RII (CD32) in polarised structures was observed with "immune" NIPC only. This Fc(gamma)RII-dependent virus capture and sensitisation of NIPC, evidently mediated through cytophilic CSFV-specific antibodies, was inhibited by non-specifically aggregated immunoglobulin as well as by pre-formed virus-antibody complexes. In conclusion, these results demonstrate that NIPC not only represent a major player in innate immunity but are also functionally linked to the immunological memory of the adaptive immune system.

Subset-dependent modulation of dendritic cell activity by circovirus type 2.

Viral interactions with dendritic cells (DCs) have important consequences for immune defence function. Certain single-stranded DNA viruses that associate with a number of species, including humans and pigs, exhibit interesting characteristics in this context. Porcine circovirus type 2 (PCV2) can persist within myeloid DCs in the absence of virus replication. Internalization was observed with both conventional blood DCs and plasmacytoid DCs [natural interferon-producing cells (NIPCs)], as well as DC precursors. This PCV2-DC interaction neither induced nor inhibited DC differentiation. The maturation of myeloid DCs induced by a cocktail of interferon-alpha/tumour necrosis factor-alpha (IFN-alpha/TNF-alpha), and the ability to process and present antigen to T lymphocytes, remained intact in the presence of PCV2. The virus was clearly internalized by the DCs, a process noted with both mature and immature cells. This suggested a non-macropinocytic uptake, confirmed by an insensitivity to wortmannin but sensitivity to cytochalasin D, chlorpromazine and bafilomycin. Nevertheless, PCV2 was immunomodulatory, being effected through the reaction of NIPC to danger signals. When NIPCs responded to the CpG-oligonucleotide (CpG-ODN), their costimulatory function which induces myeloid DC maturation was clearly impaired by the presence of PCV2. This was caused by a PCV2-induced inhibition of the IFN-alpha and TNF-alpha normally produced following interaction with CpG-ODN. Thus, the immunomodulatory activity of PCV2 is mediated through the disruption of NIPC function. This would impair the maturation of associated myeloid DC and have major implications for the efficient recognition of viral and bacterial danger signals, favouring the establishment of infections additional to that of PCV2.

Interactions between atazanavir-ritonavir and tenofovir in heavily pretreated human immunodeficiency virus-infected patients.

The aim of the present study was to assess the pharmacokinetic behavior of atazanavir-ritonavir when it is coadministered with tenofovir disoproxil fumarate (DF) in human immunodeficiency virus (HIV)-infected patients. Eleven patients enrolled in Agence Nationale de Recherche sur le SIDA (National Agency for AIDS Research, Paris, France) trial 107 were included in this pharmacokinetic study. They received atazanavir at 300 mg and ritonavir at 100 mg once a day (QD) from day 1 to the end of study. For the first 2 weeks, their nucleoside analog reverse transcriptase inhibitor (NRTI) treatments remained unchanged. Tenofovir DF was administered QD from day 15 to the end of the study. Ongoing NRTIs were selected according to the reverse transcriptase genotype of the HIV isolates from each patient. The values of the pharmacokinetic parameters for atazanavir and ritonavir were measured before (day 14 [week 2]) and after (day 42 [week 6]) initiation of tenofovir DF and are reported for the 10 patients who completed the study. There was a significant decrease in the area under the concentration-time curve from 0 to 24 h (AUC(0-24)) for atazanavir with the addition of tenofovir DF (AUC(0-24) ratio, 0.75; 90% confidence interval, 0.58 to 0.97; P = 0.05). There was a trend for a decrease in the minimum concentrations of atazanavir and ritonavir in plasma when they were combined with tenofovir, but none of the differences reached statistical significance. The median decreases in the HIV RNA loads at week 2 and week 6 were 0.1 and 0.2 log copies/ml, respectively. In summary, our data are consistent with the existence of a significant interaction between atazanavir and tenofovir DF.

Interlaboratory testing of porcine sera for antibodies to porcine circovirus type 2.

A panel of 20 porcine sera was distributed to 5 laboratories across Europe and Canada. Each center was requested to test the sera for the presence of porcine circovirus type 2 antibodies using the routine assays, indirect immunofluorescence assay (IFA) and indirect immunoperoxidase monolayer assay (IPMA), and to determine the titer of each serum. Results from all centers were then compiled and correlated. They demonstrate a wide variation in the titers obtained between laboratories. These differences were dependent on the assay used and the choice of fixative. In general, IPMA gave higher titers than did IFA, and paraformaldehyde gave higher titers than did acetone or ethyl alcohol. This report highlights the need for standardized procedures and biologicals for this virus.

Interactions between amprenavir and the lopinavir-ritonavir combination in heavily pretreated patients infected with human immunodeficiency virus.

OBJECTIVE: This pharmacokinetic study was designed to characterize interactions between amprenavir and the lopinavir-ritonavir combination in patients infected with human immunodeficiency virus in whom previous antiretroviral therapy had failed. METHODS: Twenty-seven patients included in a randomized clinical trial (ANRS [National Agency for AIDS Research] Protocol 104) participated in this study. They were randomized to receive ritonavir at a dose of either 100 mg twice daily or 200 mg twice daily. For the first 2 weeks of therapy, they were randomly assigned to receive lopinavir (400 mg twice daily) and ritonavir (100 mg twice daily), amprenavir (600 mg twice daily) plus ritonavir (100 mg twice daily), lopinavir (400 mg twice daily) and ritonavir (100 mg twice daily) plus additional ritonavir (100 mg twice daily), or amprenavir (600 mg twice daily) plus ritonavir (200 mg twice daily). From week 3 onward, all patients received amprenavir plus lopinavir-ritonavir with or without an additional ritonavir dose (100 mg twice daily). The pharmacokinetics of the 3 drugs was studied in weeks 2 and 6 of therapy. RESULTS: Median amprenavir concentrations decreased by 54% (P =.004) when lopinavir was added to the amprenavir-ritonavir regimen. Lopinavir weakly displaced amprenavir from plasma proteins: The average unbound fraction of amprenavir was 0.089 in week 2 and 0.114 in week 6 (P =.03), but this did not fully account for the observed interaction. Increasing the ritonavir dose did not affect the amprenavir concentration. The relationship between lopinavir and ritonavir concentrations fitted a maximum effect (E(max)) model;the average concentration of ritonavir that yielded a lopinavir concentration of 8119 ng/mL (50% of E(max)) was 602 ng/mL (coefficient of variation, 22%). There was a significant relationship between the lopinavir inhibitory quotient and the virologic response in week 2 (P =.005). CONCLUSION: Lopinavir markedly decreases the amprenavir concentration during amprenavir and lopinavir-ritonavir combination therapy. The inhibitory quotients were more predictive of the short-term virologic response than was the level of drug exposure.