Russell L. Dedrick

A CD14-independent LPS receptor cluster

Bacterial lipopolysaccharide (LPS), the major structural component of the outer wall of Gram-negative bacteria, is a potent initiator of an inflammatory response and serves as an indicator of bacterial infection. Although CD14 has been identified as the main LPS receptor, accumulating evidence has suggested the possible existence of other functional receptor(s). In this study, using affinity chromatography, peptide mass fingerprinting and fluorescence resonance energy transfer, we have identified four new proteins that form an activation cluster after LPS ligation and are involved in LPS signal transduction. Here we present evidence that implicates heat shock proteins 70 and 90, chemokine receptor 4 and growth differentiation factor 5 as the main mediators of activation by bacterial lipopolysaccharide.

Effects of administration of a single dose of a humanized monoclonal antibody to CD11a on the immunobiology and clinical activity of psoriasis

BACKGROUND CD11a/CD18 comprise subunits of leukocyte function associated antigen (LFA-1), a T-cell surface molecule important in T-cell activation, T-cell emigration into skin, and cytotoxic T-cell function. OBJECTIVE We explored the immunobiologic and clinical effects of treating moderate to severe psoriasis vulgaris with a single dose of humanized monoclonal antibody against CD11a (hu1124). METHODS This was an open label study with a single dose of hu1124 at doses of 0.03 to 10 mg/kg. Clinical (Psoriasis Area and Severity Index [PASI]) and immunohistologic parameters (epidermal thickness, epidermal and dermal T-cell numbers, and keratinocyte intercellular adhesion molecule 1 [ICAM-1] expression) were followed. RESULTS Treatment with hu1124, at doses higher than 1.0 mg/kg (group III), completely blocks CD11a staining for at least 14 days in both blood and psoriatic plaques. At 0.3 to 1.0 mg/kg, T-cell CD11a staining was completely blocked; however, blockade lasted less than 2 weeks (group II). Only partial saturation of either blood or plaque cellular CD11a was observed at doses of hu1124 between 0.01 and 0.1 mg/kg (group I). This pharmacodynamic response was accompanied by decreased numbers of epidermal and dermal CD3(+) T cells, decreased keratinocyte and blood vessel expression of ICAM-1, and epidermal thinning. Statistically significant drops in PASI compared with baseline were observed in group II patients at weeks 3 and 4 and in group III patients at weeks 2 through 10. No significant drop in PASI score was observed in group 1. Adverse events were mild at doses of 0.3 mg/kg or less and included mild chills, abdominal discomfort, headache, and fever. At a single dose of 0.6 mg/kg or higher, headache was the most common dose-limiting toxicity observed. CONCLUSION Targeting CD11a may improve psoriasis by inhibiting T-cell activation, T-cell emigration into the skin, and cytotoxic T-cell function.

Population pharmacokinetics and pharmacodynamics of the anti-CD11a antibody hu1124 in human subjects with psoriasis

The pharmacokinetics of hu1124, a human anti-CD11a antibody, were investigated in human subjects with psoriasis. CD11a is a subunit of LFA-1, a cell surface molecule involved in T cell mediated immune responses. Subjects received a single dose of 0.03, 0.1, 0.3, 0.6, 1, 2, 3, or 10 mg/kg of hu1124 intravenously over 1–3 hr. Blood samples were collected at selected times from 60 min to 72 days after administration. Plasma samples were assayed for hu1124 by ELISA, and pharmacokinetic analyses were performed on the drug plasma concentrations. As the dose of hu1124 was increased, the clearance decreased from 322 ml/day per kg at 0.1 mg/kg to 6.6 ml/day per kg at 10 mg/kg of hu1124. The plasma hu1124 concentration–time profile suggested that the clearance of hu1124 was saturable above 10 μg/ml. In addition, treatment with hu1124 caused a rapid reduction in the level of CD11a expression on CD3-positive lymphocytes (T cells) to about 25% of pretreatment levels. Regardless of the hu1124 dose administered, cell surface CD11a remained at this reduced level as long as hu1124 was detectable (>0.025 μg/ml) in the plasma. When hu1124 levels fell below 3 μg/ml, the drug was rapidly cleared from the circulation and expression of CD11a returned to normal within 7–10 days thereafter. In vitro, half-maximal binding of hu1124 to lymphocytes was achieved at about 0.1 μg/ml and saturation required more than 10 μg/ml. One of the receptor-mediated pharmacokinetic/pharmacodynamic models which was developed describes the dynamic interaction of hu1124 binding to CD11a, resulting in the removal of hu1124 from the circulation and reduction of cell surface CD11a. The model accounts for the continually changing number of CD11a molecules available for removing hu1124 from the circulation based on prior exposure of cells expressing CD11a to hu1124. In addition, the model also accounts for saturation of CD11a molecules by hu1124 at drug concentrations of approximately 10 μg/ml, thereby reducing the clearance rate of hu1124 with increasing dose.

Pharmacokinetic-pharmacodynamic-efficacy analysis of efalizumab in patients with moderate to severe psoriasis

PurposeEfalizumab is a humanized anti-CD11a monoclonal antibody that demonstrated efficacy in the treatment of patients with psoriasis. The objective of this study was to perform a pharmacokinetic (PK)–pharmacodynamic (PD)–efficacy (E) modeling analysis with intersubject variability assessment to increase our understanding of the interaction of efalizumab with CD11a on T cells and consequent reduction in severity of disease in psoriasis patients.MethodsA total of 6,329 samples from 240 patients in five Phase I and II clinical studies were used in the analysis. For the analysis, plasma efalizumab concentration was used as the PK measurement, the percent of predose CD11a was used as the PD measurement, and the psoriasis area and severity index was used as the measure of efficacy. A receptor-mediated PK/PD model was developed that describes the dynamic interaction of efalizumab binding with CD11a. In the efficacy model, the rate of psoriasis skin production is directly proportional to the amount of free surface CD11a on T cells, which is offset by the rate of skin healing. An additional CD11a-independent component to psoriasis skin production accounted for incomplete response to efalizumab therapy. A Monte Carlo parametric expectation maximization method implemented in the ADAPT II program was used to obtain the estimate of population parameters and inter- and intrasubject variability.Results and ConclusionsThe final model described the PK/PD/E data in psoriasis patients reasonably well. In addition, simulations using the final model suggested that efalizumab administered less frequently could possibly be more convenient with similar efficacy.

An Overview of the Pharmacokinetics and Pharmacodynamics of Efalizumab: A Monoclonal Antibody Approved for Use in Psoriasis

Efalizumab is a recombinant humanized monoclonal IgG1 antibody shown to be efficacious for the treatment of moderate to severe chronic plaque psoriasis. Efalizumab, a targeted inhibitor of T cell interactions, binds to the CD11a subunit of lymphocyte function—associated antigen 1 (LFA‐1), thereby preventing LFA‐1 binding to intercellular adhesion molecule 1 (ICAM‐1). The authors review the pharmacokinetic and pharmacodynamic data from the efalizumab clinical development program and discuss how these data led to selection of the optimal weekly subcutaneous (SC) dose of efalizumab (1.0 mg/kg) in adults. Efalizumab SC dosages of 1.0 mg/kg/wk or greater exerted maximal pharmacodynamic effects for CD11a expression and available CD11a binding sites on T lymphocytes. Dosages greater than 1.0 mg/kg/wk SC did not provide additional benefits; moreover, higher doses did not alter the safety profile. During long‐term administration of efalizumab, serum levels were generally stable and pharmacodynamic markers remained maximally affected.

Anti-adhesion antibodies efalizumab, a humanized anti-CD11a monoclonal antibody.

The acquired immune response that leads to graft rejection depends on regulated adhesive interactions between T lymphocytes, endothelial cells, dendritic cells, graft tissue and the extracellular matrix to coordinate cellular trafficking and activation of antigen-reactive T lymphocytes. Inhibiting the function of molecules involved in the adhesion processes offers the potential for interfering with the allograft response. The leukocyte function associated antigen-1 molecule (LFA-1), a heterodimer of CD11a (alphaL) and CD18 (beta2) integrin subunits, is an attractive therapeutic target because it plays an important role in key steps of inflammation and tissue rejection. These include: (1) binding of leukocytes to endothelium; (2) trafficking through activated endothelium; and (3) costimulatory interactions between T lymphocytes and antigen presenting cells. Clinical experience with efalizumab, a humanized anti-CD11a monoclonal antibody (mAb), in patients with chronic plaque psoriasis has shown that anti-CD11a therapy is well tolerated and effective at reducing the severity of the disease without depleting lymphocytes. Initial results in renal transplant patients are also promising.

Adhesion molecules as therapeutic targets for autoimmune diseases and transplant rejection

Inflammatory disorders such as autoimmune diseases and graft rejection are mediated by activated leukocytes, particularly T lymphocytes, which penetrate the inflamed tissue and perpetuate or amplify the immune reaction. In an unstimulated state, leukocytes do not readily adhere to the vascular endothelium. However, inflammatory signals induce the expression of proteins on the endothelial cell surface that promote the adhesion and extravasation of activated immune cells from the circulation into the underlying tissues. Key among these molecules are P- and E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on the endothelial cells, and their respective counter receptors , P-selectin glycoprotein ligand-1 (PSGL-1), leukocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4), on the leukocytes. In vitro blockade of these molecules inhibits the adhesion of leukocytes. In many cases there is attenuation of leukocyte activation as well. Adhesion blockade in animal models prevents or ameliorates graft rejection and disease severity in autoimmune models. Clinical studies with humanised monoclonal antibodies which interfere with LFA-1/ICAM-1 or VLA-4/VCAM-1 interactions have shown significant efficacy and good safety profiles in autoimmune disease, including psoriasis, multiple sclerosis and inflammatory bowel disease. Thus, adhesion blockade is emerging as a useful therapeutic strategy in several inflammatory settings.

Clinical Implications and Longitudinal Alteration of Peripheral Blood Transcriptional Signals Indicative of Future Cardiac Allograft Rejection

BACKGROUND We have previously demonstrated that a peripheral blood transcriptional profile using 11 distinct genes predicts onset of cardiac allograft rejection weeks to months prior to the actual event. METHODS In this analysis, we ascertained the performance of this transcriptional algorithm in a Bayesian representative population: 28 cardiac transplant recipients who progressed to moderate to severe rejection; 53 who progressed to mild rejection; and 46 who remained rejection-free. Furthermore, we characterized longitudinal alterations in the transcriptional gene expression profile before, during and after recovery from rejection. RESULTS In this patient cohort, we found that a gene expression score (range 0 to 40) of or =3A) rejection; 16 of 53 (30%) from the intermediate group (those who progressed to ISHLT Grade 1B or 2) and 13 of 46 (28%) controls (who remained Grade 0 or 1A) had scores < or =20. A gene score of > or =30 was associated with progression to moderate to severe rejection in 58% of cases. These two extreme scores (< or =20 or > or =30) represented 44% of the cardiac transplant population within 6 months post-transplant. In addition, longitudinal gene expression analysis demonstrated that baseline scores were significantly higher for those who went on to reject, remained high during an episode of rejection, and dropped post-treatment for rejection (p < 0.01). CONCLUSIONS The use of gene expression profiling early after transplantation allows for separation into low-, intermediate- or high-risk categories for future rejection, permitting development of discrete surveillance strategies.

Interactions of bacterial lipopolysaccharide and peptidoglycan with a 70 kDa and an 80 kDa protein on the cell surface of CD14+ and CD14− cells

Bacterial cell wall components, lipopolysaccharide (LPS), lipoteichoic acid (LTA), and peptidoglycan (PGN) are known to stimulate cells of the immune, inflammatory and vascular systems contributing to septic shock. CD14 has been identified as the main LPS receptor, a process that is accelerated by the serum protein LPS-binding protein (LBP). CD14 has also been found to bind LTA and PGN from the cell wall of gram positive bacteria. Recently, toll-like receptor proteins TLR-2 and TLR-4 have been shown to be required for LPS and LTA-induced intracellular signalling. Although CD14 functions as either a glycosylphosphatidylinositol (GPI)-anchored molecule that does not transverse the cell membrane or as a soluble serum protein, the mechanisms by which the CD14-LPS/LTA complex interacts with the TLRs remains to be elucidated. We have looked directly for cell surface protein(s) that bind LPS or LTA in a CD14-dependent manner. Using biochemical approaches we have identified two proteins of molecular weight 70 kDa (LAP-1) and 80 kDa (LAP-2) that can be precipitated from both CD14(+) and CD14(-) cells with LPS- or LTA-specific antibodies. Binding of LPS and LTA to LAP-1 and -2 required serum. While soluble CD14 (sCD14) was sufficient to allow precipitation of these two proteins from CD14(-) cells, serum could not be replaced by purified sCD14 and/or LBP when mCD14-expressing cells were used.

Endotoxin: physical requirements for cell activation

Lipopolysaccharide (LPS) is the eminent lipid component of the outer leaflet of the outer membrane of Gram-negative bacteria and the major initiator of innate immune response to bacterial infection. Below the critical micellar concentration (CMC), LPS is exclusively present as a monomer. Above this concentration, aggregates are formed. Increasing the concentration beyond the CMC leads to an increase in aggregate concentration, whereas the concentration of monomers remains constant or even decreases. The question how LPS activates immune cells and whether the aggregate or the monomer is the biologically active unit has been and still is controversial. To prepare clearly defined monomeric solutions, we utilized a dialysis set-up consisting of a donor and an acceptor chamber, separated by a dialysis diaphragm with a cut-off of 5 kDa, thus allowing only monomers to pass. Human mononuclear cells (MNCs) were then stimulated with equal concentrations of aggregates and monomers, respectively, of deep rough mutant LPS from Escherichia coli strain F515 (Re LPS) and TNF-α release was determined. In contrast to earlier and very recent work of others, we started with a preparation of aggregate-suspensions and pure monomer-solutions and show that monomers are significantly less active than aggregates in the absence and presence of serum proteins at identical concentrations. In our model, we propose that LPS aggregates are detected by membrane-associated LBP and intercalated into the cell membrane to bring LPS into close proximity to signaling proteins in the membrane, thus finally leading to cell activation. To support this model, we present data showing that LBP is indeed present in or at the cell membrane of human macrophages.