Barry van der Strate

Reduced inflammatory response in cigarette smoke exposed Mrp1/Mdr1a/1b deficient mice

BackgroundTobacco smoke is the principal risk factor for chronic obstructive pulmonary disease (COPD), though the mechanisms of its toxicity are still unclear. The ABC transporters multidrug resistance-associated protein 1 (MRP1) and P-glycoprotein (P-gp/MDR1) extrude a wide variety of toxic substances across cellular membranes and are highly expressed in bronchial epithelium. Their impaired function may contribute to COPD development by diminished detoxification of noxious compounds in cigarette smoke.MethodsWe examined whether triple knock-out (TKO) mice lacking the genes for Mrp1 and Mdr1a/1b are more susceptible to develop COPD features than their wild-type (WT) littermates. TKO and WT mice (six per group) were exposed to 2 cigarettes twice daily by nose-only exposure or room air for 6 months. Inflammatory infiltrates were analyzed in lung sections, cytokines and chemokines in whole lung homogenates, emphysema by mean linear intercept. Multiple linear regression analysis with an interaction term was used to establish the statistical significances of differences.ResultsTKO mice had lower levels of interleukin (IL)-7, KC (mouse IL-8), IL-12p70, IL-17, TNF-alpha, G-CSF, GM-CSF and MIP-1-alpha than WT mice independent of smoke exposure (P < 0.05). IL-1-alpha, IL-6, IL-8, IL-13, IL-17, TNF-alpha, G-CSF, GM-CSF and MCP-1 increased after smoke exposure in both groups, but the increase in IL-8 was lower in TKO than WT mice (P < 0.05) with a same trend for G-CSF (P < 0.10). Smoke-induced increase in pulmonary inflammatory cells in WT mice was almost absent in TKO mice. The mean linear intercept was not different between groups.ConclusionMrp1/Mdr1a/1b knock-out mice have a reduced inflammatory response to cigarette smoke. In addition, the expression levels of several cytokines and chemokines were also lower in lungs of Mrp1/Mdr1a/1b knock-out mice independent of smoke exposure. Further studies are required to determine whether dysfunction of MRP1 and/or P-gp contribute to the pathogenesis of COPD.

Charge modification of plasma and milk proteins results in antiviral active compounds

Previous studies have shown that acylated plasma and milk proteins with increased negative charge, derived from various animal and human sources, are potent anti‐HIV compounds. The antiviral effects seemed to correlate positively with the number of negative charges introduced into the various polypeptides: proteins with a high content of basic amino acids in which all of the available εNH2 groups were anionized yielded the most potent anti‐HIV compounds. It remained unclear however whether the total net negative charge of the various derivatized proteins, or rather the charge density on the protein backbone, is essential for the observed anti‐HIV activity. Earlier studies have shown that acylated albumins preferentially block the process of HIV/cell fusion through binding to the HIV envelope proteins gp120 and gp41 as well as to the cell surface of the HIV target cells. Some of these polyanionic proteins have been shown to interfere also with the gp120–CD4 mediated virus/cell binding. The relative contribution of these effects to the anti‐HIV activity may depend both on the total negative charge introduced as well as the hydrophobicity of the acylating reagent added to the particular proteins. In this study we show that the higher the charge density of the derivatized proteins, the more potent their HIV replication inhibiting effects are. In contrast, the addition of positive charge to the studied plasma and milk proteins through amination resulted in a reduced anti‐HIV activity but a clearly increased anti‐HCMV activity, with IC50 values in the low micromolar concentration range. Interestingly, native lactoferrin (Lf) was antivirally active against both HIV and HCMV. Acylation or amination of Lf increased the anti‐HIV and anti‐HCMV activity, respectively. The N‐terminal portion of Lf appeared essential for its anti‐HCMV effect: N‐terminal deletion variants of human Lf were less active against HCMV. Circular dichroism of the modified proteins showed that the secondary structure of the tested proteins was only moderately influenced by acylation and/or covalent attachment of drugs, making these (derivatized) proteins useful candidates as antiviral agents and/or intrinsically active drug carriers. The relatively simple chemical derivatization as well as the abundant sources of blood plasma and milk proteins provides attractive opportunities for the preparation of potent and relatively cheap antiviral agents for systemic or local applications. Copyright

Heme oxygenase-1 prevents smoke induced B-cell infiltrates: a role for regulatory T cells?

BackgroundSmoking is the most important cause for the development of COPD. Since not all smokers develop COPD, it is obvious that other factors must be involved in disease development. We hypothesize that heme oxygenase-1 (HO-1), a protective enzyme against oxidative stress and inflammation, is insufficiently upregulated in COPD.The effects of HO-1 modulation on cigarette smoke induced inflammation and emphysema were tested in a smoking mouse model.MethodsMice were either exposed or sham exposed to cigarette smoke exposure for 20 weeks. Cobalt protoporphyrin or tin protoporphyrin was injected during this period to induce or inhibit HO-1 activity, respectively. Afterwards, emphysema development, levels of inflammatory cells and cytokines, and the presence of B-cell infiltrates in lung tissue were analyzed.ResultsSmoke exposure induced emphysema and increased the numbers of inflammatory cells and numbers of B-cell infiltrates, as well as the levels of inflammatory cytokines in lung tissue. HO-1 modulation had no effects on smoke induced emphysema development, or the increases in neutrophils and macrophages and inflammatory cytokines. Interestingly, HO-1 induction prevented the development of smoke induced B-cell infiltrates and increased the levels of CD4+CD25+ T cells and Foxp3 positive cells in the lungs. Additionally, the CD4+CD25+ T cells correlated positively with the number of Foxp3 positive cells in lung tissue, indicating that these cells were regulatory T cells.ConclusionThese results support the concept that HO-1 expression influences regulatory T cells and indicates that this mechanism is involved in the suppression of smoke induced B-cell infiltrates. The translation of this interaction to human COPD should now be pursued.

Synergy of bovine lactoferrin with the anti-cytomegalovirus drug cidofovir in vitro

UNLABELLED Human cytomegalovirus (HCMV) causes severe morbidity and mortality in immunocompromised patients. Treatment of HCMV infections with conventional antiviral drugs like ganciclovir and cidofovir has major drawbacks (i.e. serious side effects). Therefore, combination therapies using drugs with different antiviral mechanisms should be envisaged. Potential synergy between lactoferrin (LF), an antibacterial, antimycotic and antiviral protein, and the antiviral drugs acyclovir, ganciclovir, foscarnet and cidofovir was investigated, using an in vitro test system with the recombinant RC256 HCMV strain. RESULTS Combination of LF with acyclovir and foscarnet resulted in antagonism. When LF and ganciclovir were combined, neither synergy nor antagonism was observed. Strikingly, the combination of LF with cidofovir resulted in marked synergy. The synergistic effect could be explained by inhibition of two subsequent steps in the viral replication cycle: HCMV penetration into the target cells and intracellular synthesis of HCMV DNA. In conclusion, LF might be a potential candidate for combination therapy with cidofovir.

The antiviral protein human lactoferrin is distributed in the body to Cytomegalovirus (CMV) infection-prone cells and tissues

AbstractPurpose. Lactoferrin has anti-Cytomegalovirus (CMV) and -HIV properties in vitro. However, the pharmacokinetic behavior of the 80-kD protein has not been well defined. We, therefore, assessed the plasma decay and body distribution of lactoferrin after intravenous administration to freely moving rats. Furthermore, the systemic availability of lactoferrin after intraperitoneal dosing was determined. Methods and Results. After intravenous injection, human lactoferrin (hLF) was rapidly cleared from the plasma, but higher doses resulted in prolonged plasma levels. Immunohistochemical analysis revealed a pronounced distribution of hLF to endothelial cells in the liver whereas diffuse staining in hepatocytes indicated the presence of considerable amounts in this large cell population. This endothelial association, which also was found in other organ/tissues, including blood vessels, was confirmed by in vitro cell-binding studies. In addition, leukocytes in plasma that were infiltrated in various organs showed binding of hLF. A small fraction of hLF was transported into the lymphatic system. Western blot analysis revealed that hLF, present in the various organs, mainly consisted of an 80-kD protein. After intraperitoneal administration, small amounts of 80-kD hLF distributed to the general circulation. The bioavailability was 0.6% but increased to 3.6% after multiple administrations. Conclusions. The affinity of hLF for endothelial cells and leukocytes, and its penetration into the lymphatic system, indicates that this protein reaches target cells and body compartments that are crucial for CMV and HIV replication. The ability to reach the blood compartment after intraperitoneal dosing offers opportunities for parenteral administration of the protein in future studies on its antiviral effects in vivo.

The 10th GCC Closed Forum: rejected data, GCP in bioanalysis, extract stability, BAV, processed batch acceptance, matrix stability, critical reagents, ELN and data integrity and counteracting fraud

The 10th Global CRO Council (GCC) Closed Forum was held in Orlando, FL, USA on 18 April 2016. In attendance were decision makers from international CRO member companies offering bioanalytical services. The objective of this meeting was for GCC members to meet and discuss scientific and regulatory issues specific to bioanalysis. The issues discussed at this closed forum included reporting data from failed method validation runs, GCP for clinical sample bioanalysis, extracted sample stability, biomarker assay validation, processed batch acceptance criteria, electronic laboratory notebooks and data integrity, Health Canadas Notice regarding replicates in matrix stability evaluations, critical reagents and regulatory approaches to counteract fraud. In order to obtain the pharma perspectives on some of these topics, the first joint CRO-Pharma Scientific Interchange Meeting was held on 12 November 2016, in Denver, Colorado, USA. The five topics discussed at this Interchange meeting were reporting data from failed method validation runs, GCP for clinical sample bioanalysis, extracted sample stability, processed batch acceptance criteria and electronic laboratory notebooks and data integrity. The conclusions from the discussions of these topics at both meetings are included in this report.

Best practices in performing flow cytometry in a regulated environment: feedback from experience within the European Bioanalysis Forum

Flow cytometry is a powerful tool that can be used for the support of (pre)clinical studies. Although various white papers are available that describe the set-up and validation of the instrumentation (the flow cytometer) and validation of flow cytometry methods, to date no guidelines exist that address the requirements for performing flow cytometry in a regulated environment. In this manuscript, the European Bioanalysis Forum presents additional practice guidance on the use of flow cytometry in the support of drug development programs and addresses areas that are not covered in the previous publications. The concepts presented here are based on the consensus of discussions in the European Bioanalysis Forum Topic Team 32, in meetings in Barcelona, Limelette and multiple telephone conferences.

EBF recommendation on practical management of critical reagents for PK ligand-binding assays

Critical reagents play a crucial role in ligand-binding assays; the robustness and reliability of an assay is defined by the quality and long-term availability of these reagents. However, neither regulatory guidelines nor relevant scientific papers provide clear directions for set-up, life cycle management and, more importantly, the acceptance criteria required for the testing of the critical reagents for pharmacokinetic, biomarker and immunogenicity assays. The ambiguity from current guidelines can be a challenge for the bioanalytical community. Members of the European Bioanalysis Forum community undertook a more pragmatic approach on how to assess the impact of critical reagents. In this paper, a review and corresponding gap analysis of the current guidelines and relevant papers will be provided as well as decision trees proposed for lot-to-lot changes of critical reagents for pharmacokinetic assays.