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

Immunogenicity assays are required to measure antidrug antibodies that are generated against biotherapeutic modalities. As for any ligand-binding assays, critical reagents (CR) play a crucial role in immunogenicity assays, as the robustness and reliability of an assay are defined by the quality and long-term availability of these reagents. The current regulatory guidelines do not provide clear directions on how to implement and verify lot-to-lot changes of CR during an assay life cycle, or the acceptance criteria that should be used when implementing new lots of CR. These aspects were extensively discussed within the European Bioanalysis Forum community. In this paper, CR for immunogenicity assays are identified and the minimum requirements for introducing new lots of CR in immunogenicity assays are described

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

<p>Abstract</p> <p>Background</p> <p>Tobacco 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.</p> <p>Methods</p> <p>We examined whether triple knock-out (TKO) mice lacking the genes for <it>Mrp1 </it>and <it>Mdr1a/1b </it>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.</p> <p>Results</p> <p>TKO 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 (<it>P </it>< 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 (<it>P </it>< 0.05) with a same trend for G-CSF (<it>P </it>< 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.</p> <p>Conclusion</p> <p><it>Mrp1/Mdr1a/1b </it>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 <it>Mrp1/Mdr1a/1b </it>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.</p

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

Purpose. 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

Dependence of neovascularization mechanisms on the molecular microenvironment

In vivo vascularization of implanted (bio)artificial constructs is essential for their proper function. Vascularization may rely on sprouting angiogenesis, vascular incorporation of bone marrow-derived endothelial cells (BMDECs), or both. Here we investigated the relative contribution of these 2 mechanisms to neovascularization in a mouse model of a foreign body reaction (FBR) to subcutaneously implanted Dacron and in hind limb ischemia (HLI) in relation to the molecular microenvironment at these neovascularization sites. Neovascularization was studied in C57B1/6 mice reconstituted with enhanced green fluorescent protein (EGFP) transgenic bone marrow. Sprouting angiogenesis, detected using nuclear incorporation of bromodeoxyuridine in endothelial cells was present in both models, whereas vascular incorporation of EGFP(+) BMDECs was restricted to HLI. In HLI, the presence of a pro-angiogenic molecular microenvironment comprising vascular endothelial growth factor, fibroblast growth factor 2, and granulocyte colony-stimulating factor corroborated the importance of these factors for vascular BMDEC incorporation, whereas this microenvironment was absent in FBR. Enhanced mobilization of BMDECs by granulocyte-macrophage colony-stimulating factor administration or by combining HLI and FBR with Dacron did not induce incorporation of BMDECs in FBR neovessels. We conclude that the efficacy of BMDEC-based therapy is not generally warranted, but it depends on the molecular microenvironment in the targeted tissue

Circulating CD34(+) progenitor cells modulate host angiogenesis and inflammation in vivo

Within the phenotypically and functionally heterogeneous group of circulating progenitor cells (CPC), a subclass of cells with vascular repair potential have been identified. These CPC are detected and isolated based on single or combined expression of CD34, CD 133 and VEGFR-2, and referred to as endothelial progenitor cells. Here we asked whether CPC subsets defined by single expression of these markers exhibit functional heterogeneity. As functional parameters, we chose the capacity of CPC to differentiate into endothelial cells. Moreover, we studied their role in remodeling by recruitment of inflammatory cells, an aspect that has been little explored. We established an in vivo model in which the intrinsic functional capacity of these human CPC subsets was studied. Human CD34(+) CPC, but not CD133(+) or VEGFR-2(+) CPC, seeded in Matrigel pellets and transplanted subcutaneously in a nude mouse host, contributed little to donor-derived neovascularization. However, host angiogenesis in the Matrigel implant, as demonstrated by the presence of capillaries containing erythrocytes and expressing mouse CD31, was strong in response to implantation of human CD34(+) CPC and significantly lower in response to the other two CPC subsets. Moreover, the CD34(+) CPC subset was significantly superior to CD133(+) CPC and VEGFR-2(+) CPC in the recruitment of host monocytes/macrophages. These three CPC populations were further dissected into seven discrete subsets, based on three-parameter flow cytometry analysis of combined expression patterns of CD34, CD133 and VEGFR-2. In conclusion, in our system, CD34(+) CPC contribute marginally to neovascularization by differentiation but are potent regulators of the host angiogenic and pro-inflammatory response, suggesting a possible role for these cells in the remodeling of vascular lesions. (c) 2006 Elsevier Inc. All rights reserved

Number of lymphoid infiltrates in paraffin sections of lungs of WT mice and TKO mice that were exposed to cigarette smoke or air for 6 months

<p><b>Copyright information:</b></p><p>Taken from "Reduced inflammatory response in cigarette smoke exposed Mrp1/Mdr1a/1b deficient mice"</p><p>http://respiratory-research.com/content/8/1/49</p><p>Respiratory Research 2007;8(1):49-49.</p><p>Published online 7 Jul 2007</p><p>PMCID:PMC1950505.</p><p></p> The number of smoke-induced lymphoid infiltrates in lungs of WT was higher compared to TKO mice (interaction,= 0.02).

Cytokine and chemokine levels in lung homogenates after 6 months smoke or air exposure

<p><b>Copyright information:</b></p><p>Taken from "Reduced inflammatory response in cigarette smoke exposed Mrp1/Mdr1a/1b deficient mice"</p><p>http://respiratory-research.com/content/8/1/49</p><p>Respiratory Research 2007;8(1):49-49.</p><p>Published online 7 Jul 2007</p><p>PMCID:PMC1950505.</p><p></p> IL-8 levels were elevated by cigarette smoke in total group (WT and TKO) of smokers (= 0.01). IL-8 levels are higher in the total group (Sm and NSm) of WT mice compared to TKO mice (= 0.01). The smoke-induced upregulation of IL-8 in lungs of WT was higher compared to TKO mice (interaction, < 0.05). Granulocyte-colony stimulating factor (G-CSF) levels were elevated by cigarette smoke in total group (WT and TKO) of smokers (= 0.02). G-CSF levels are higher in the total group (Sm and NSm) of WT mice compared to TKO mice (= 0.03). There was a trend to higher smoke-induced upregulation of G-CSF in lungs of WT compared to TKO mice (interaction, < 0.10). TNFα levels were elevated by cigarette smoke in total group (WT and TKO) of smokers (= 0.01) and TNFα levels were higher in the total group (Sm and NSm) of WT mice compared to TKO mice (= 0.00). There was no difference in upregulation of smoke-induced TNFα in lungs of WT versus TKO mice (no interaction). MIP1α levels in total group of smokers compared to non-smokers was not significant, but levels are higher in the total group (Sm and NSm) of WT mice compared to TKO mice (= 0.00). There was no difference in smoke-induced upregulation of MIP1α in lungs of WT versus TKO mice (no interaction).

Emphysema measurement with the Lmi method in mice that were exposed to cigarette smoke or air for 6 months

<p><b>Copyright information:</b></p><p>Taken from "Reduced inflammatory response in cigarette smoke exposed Mrp1/Mdr1a/1b deficient mice"</p><p>http://respiratory-research.com/content/8/1/49</p><p>Respiratory Research 2007;8(1):49-49.</p><p>Published online 7 Jul 2007</p><p>PMCID:PMC1950505.</p><p></p> Paraffin sections were stained with H&E and digital images (19 to 32 images per lung) were morphometrically analyzed. No significant differences were measured between the four groups, although a slight increase in airspace size was detected in both WT and TKO mice that were exposed to smoke.

Histological pictures of paraffin sections of lungs of WT mice and TKO mice that were exposed to cigarette smoke for 6 months

<p><b>Copyright information:</b></p><p>Taken from "Reduced inflammatory response in cigarette smoke exposed Mrp1/Mdr1a/1b deficient mice"</p><p>http://respiratory-research.com/content/8/1/49</p><p>Respiratory Research 2007;8(1):49-49.</p><p>Published online 7 Jul 2007</p><p>PMCID:PMC1950505.</p><p></p> H&E staining of lungs of WT and TKO mice. In WT mice there were markedly more inflammatory infiltrates than in lungs of TKO mice. Two types of infiltrates could be distinguished in paraffin sections, infiltrates mainly consisting of pigmented (smoke particles positive) macrophages (m) and infiltrates mainly consisting of lymphoid (ly) cells. Lymphoid infiltrates mainly consisted of B-cells (B220 antibody) which were far more present in lungs of WT mice compared to TKO mice, see arrows. Pigmented macrophages stained positive with specific antibodies (Mac-3), and were far more present in lungs of WT mice compared to TKO mice, see arrows.