Developmental regulation of αβ T cell antigen receptor assembly in immature CD4+CD8+ thymocytes

Most lymphocytes of the T cell lineage develop along the CD4/CD8 pathway and express antigen receptors on their surfaces consisting of clonotypic αβ chains associated with invariant CD3-γδε components and ζ chains, collectively referred to as the T cell antigen receptor complex (TCR). Expression of the TCR complex is dynamically regulated during T cell development, with immature CD4⁺CD8⁺ thymocytes expressing only 10% of the number of αβ TCR complexes on their surfaces expressed by mature CD4⁺ and CD8⁺ T cells. Recent evidence demonstrates that low surface TCR density on CD4⁺CD8⁺ thymocytes results from the limited survival of a single TCR component within the ER, the TCRα chain, which has a half life of only 15 minutes in immature thymocytes, compared to >75 minutes in mature T cells. Instability of TCRα proteins in immature CD4⁺CD8⁺ thymocytes represents a novel mechanism by which expression of the multisubunit TCR complex is quantitatively regulated during T cell development. In the current review we discuss our recent findings concerning the assembly, intracellular transport, and expression of αβ TCR complexes in CD4⁺CD8⁺ thymocytes and comment on the functional significance of TCRα instability during T cell development.     

Activation of Unusual Mac-1+CD4− CD8− T Cells Bearing αβ Antigen Receptor in Murine Lungs during Infection with Mycobacterium bovis BCG: Regulation by Interferon-γ

We have recently (Kawakami et al, Immunol. Lett. 1995;46: 143) demonstrated that unusual Mac-1⁺CD4^?CD8^? T cells bearing αβ antigen receptor (Mac-1⁺ αβ T cells) reside in a considerable proportion in murine lungs. The present study was performed to examine the dynamics of accumulation of these cells in the lungs following intravenous administration of Mycobacterium bovis BCG (BCG). Mac-1⁺ αβ T cells accumulated rapidly 24 hr after infection, followed by a gradual increase over the observation period of 15 days. Furthermore, the expression of Ia, ICAM-1 and FcγR II/III on their surface intensified dramatically after BCG infection. The kinetics of enhancement of Ia expression was slower than that of ICAM-1, with the maximum level attained in one day in the latter molecule but in two weeks in the former. Neutralization of endogenous IFN-γ by specific mAb completely blocked the augmented expression of Ia on Mac-1⁺ αβ T cells after BCG infection, but did not have any significant effect on that of ICAM-1. In contrast, in vivo administration of IFN-γ enhanced the expression of ICAM-1 as well as that of Ia. Our results indicate that accumulation of Mac-1 αβ T cells within the lung is associated with a differential change in the expression of surface antigens, and suggest that these cells may play a role in the host defense against mycobacterial infection.     

Blood Platelets Contain and Secrete Laminin-8 (α4β1γ1) and Adhere to Laminin-8 via α6β1 Integrin

Laminins, a family of heterotrimeric proteins with cell adhesive/signaling properties, are characteristic components of basement membranes of vasculature and tissues. In the present study, permeabilized platelets were found to react with a monoclonal antibody to laminin γ1 chain by immunofluorescence. In Western blot analysis of platelet lysates, several monoclonal antibodies to γ1 and β1 laminin chains recognized 220- to 230-kDa polypeptides, under reducing conditions, and a structure with much slower electrophoretic mobility under nonreducing conditions. Immunoaffinity purification on a laminin β1 antibody–Sepharose column yielded polypeptides of 230, 220, 200, and 180 kDa from platelet lysates. In the purified material, mAbs to β1 and γ1 reacted with the two larger polypeptides, while affinity-purified rabbit antibodies to laminin α4 chain recognized the smallest polypeptide. Identity of the polypeptides was confirmed by microsequencing. One million platelets contained on average 1 ng of laminin (approximately 700 molecules per cell), of which 20–35% was secreted within minutes after stimulation with either thrombin or phorbol ester. Platelets adhered to plastic surfaces coated with the purified platelet laminin, and this process was largely inhibited by antibodies to β1 and α6 integrin chains. We conclude that platelets contain and, following activation, secrete laminin-8 (α4β1γ1) and that the cells adhere to the protein by using α6β1 integrin.     

Preferential apoptosis of HIV-1-specific CD4+ T cells

In contrast to other viral infections such as CMV, circulating frequencies of HIV-1-specific CD4+ T cells in peripheral blood are quantitatively diminished in the majority of HIV-1-infected individuals. One mechanism for this quantitative defect is preferential infection of HIV-1-specific CD4+ T cells, although <10% of HIV-1-specific CD4+ T cells are infected. Apoptosis has been proposed as an important contributor to the pathogenesis of CD4+ T cell depletion in HIV/AIDS. We show here that, within HIV-1-infected individuals, a greater proportion of ex vivo HIV-1-specific CD4+ T cells undergo apoptosis compared with CMV-specific CD4+ T cells (45 vs 7.4%, respectively, p < 0.05, in chronic progressors). The degree of apoptosis within HIV-1-specific CD4+ T cells correlates with viral load and disease progression, and highly active antiretroviral therapy abrogates these differences. The data support a mechanism for apoptosis in these cells similar to that found in activation-induced apoptosis through the TCR, resulting in oxygen-free radical production, mitochondrial damage, and caspase-9 activation. That HIV-1 proteins can also directly enhance activation-induced apoptosis supports a mechanism for a preferential induction of apoptosis of HIV-1-specific CD4+ T cells, which contributes to a loss of immunological control of HIV-1 replication.     

Effect of HIF1α on Foxp3 expression in CD4+CD25− T lymphocytes

The aim of the present study was to investigate the effect of HIF1α on Foxp3 expression in CD4⁺CD25^? T lymphocytes. CD4⁺CD25^? T lymphocytes were sorted from PBMC using a CD4⁺CD25⁺ regulatory T cell isolation kit. Lentivirus containing lentiviral vector that overexpressed HIF1α (HIF‐lenti) and those containing empty expression vector (control‐lenti) were produced. Meanwhile, lentivirus that contained lentiviral vector that suppressed HIF1α expression (siHIF‐lenti) and those containing control vector (sicontrol‐lenti) were also generated. The sorted CD4⁺CD25^? T lymphocytes were infected with HIF‐lenti, control‐lenti, siHIF‐lenti, and sicontrol‐lenti, respectively. Approximately 72 hr after transduction, real‐time PCR and Western blot were carried out to analyze the RNA and protein expression level of HIF1α and Foxp3. CD4⁺CD25^? T lymphocytes cultured under 21% O₂, 5% CO₂ (normoxia) and 1% O₂, 5% CO₂ (hypoxia) were used as control. Our results showed that overexpression of HIF1α increased both mRNA and protein expression of Foxp3 and, meanwhile, suppression of HIF1α expression by RNAi could reverse high Foxp3 expression in CD4⁺CD25^? T lymphocytes caused by hypoxic culture. These results suggested that hypoxia could stimulate Foxp3 expression by increasing HIF1α expression in CD4⁺ T lymphocytes which may promote CD4⁺ T lymphocytes to convert to Treg.

     

PARP-2 deficiency affects the survival of CD4+CD8+ double-positive thymocytes

Poly-(ADP-ribose) polymerase-2 (PARP-2) belongs to a large family of enzymes that synthesize and transfer ADP-ribose polymers to acceptor proteins, modifying their functional properties. PARP-2-deficient (Parp-2-/-) cells, similar to Parp-1-/- cells, are sensitive to both ionizing radiation and alkylating agents. Here we show that inactivation of mouse Parp-2, but not Parp-1, produced a two-fold reduction in CD4+CD8+ double-positive (DP) thymocytes associated with decreased DP cell survival. Microarray analyses revealed increased expression of the proapoptotic Bcl-2 family member Noxa in Parp-2-/- DP thymocytes compared to littermate controls. In addition, DP thymocytes from Parp-2-/- have a reduced expression of T-cell receptor (TCR)alpha and a skewed repertoire of TCRalpha toward the 5' Jalpha segments. Our results show that in the absence of PARP-2, the survival of DP thymocytes undergoing TCRalpha recombination is compromised despite normal amounts of Bcl-xL. These data suggest a novel role for PARP-2 as an important mediator of T-cell survival during thymopoiesis by preventing the activation of DNA damage-dependent apoptotic response during the multiple rounds of TCRalpha rearrangements preceding a positively selected TCR.     

Functional maturation of mouse CD4~+CD8~- thymocytes induced by medullary-type thymus epithelial cells

Murine CD4+CD8- (CD4SP) thymocyte subset is a heterogeneous population, in which the Qa-2- cells are less functional, whereas the Qa-2+ cells are fully functional. Evidence is provided here that the transition from Qa-2- to Qa-2+ CD4SP thymocytes is an intrathymic process of differentiation induced by thymic medullary-type epithelial cells. The separated Qa-2-CD4SP could be induced to express Qa-2 molecules up to 84%- 89% of the total viable celb after cocultured for 3d with MTEC1 cells, a murine thymic medullary type epithelial cell line established in our laboratory. Kinetic study showed that both the percentage of Qa-2+ cells and the density of the expressed Qa-2 molecules on CD4SP thymocytes induced by MTEC1 were progressively increasing in 72-h cultures. The MTECl-induced Qa-2+CD4SP thymocytes were fully functional, which exhibited capabilities of proliferation and cytokine secretion in response to Con A stimulation as high as those of freshly isolated Qa-2+CD4SP thymocytes. The profile of cytokine     

Activation of CD 4-, CD 8- thymocytes with IL 4 vs IL 1 + IL 2

Thymocytes from C57BL/6 mice were highly purified to obtain the CD 4-, CD 8- subpopulation which constitutes only 5% of all thymocytes. Substantial proliferation was induced in vitro with either IL-1 + IL-2 or with IL-4 in the presence of PMA. IL-1 and IL-2 synergized in inducing proliferation of these purified CD 4-, CD 8- thymocytes whereas neither synergized with IL-4. In order to determine whether stimulation with IL-1 + IL-2 acted via IL-4 or vice versa, cultures were treated reciprocally with affinity-purified anti-IL-2 or anti-IL-4 antibodies. Cultures with IL-4 were inhibited by anti-IL-4 but were unaffected by anti-IL-2. The CD 4-, CD 8- thymocytes cultured with IL-1 + IL-2 + anti-IL-2 were inhibited to baseline IL-1 stimulation. At low concentrations of IL-1 (1 U/ml) and IL-2 (100 U/ml), anti-IL-4 had no effect, whereas at higher levels of IL-1 (2 U/ml IL-1), and 100 or 200 U/ml IL-2, anti-IL-4 significantly reduced DNA synthesis. This result suggests that at higher concentrations the combination of IL-1 + IL-2 can induce cells to produce IL-4 which then contributes to overall proliferation. When CD 4-, CD 8- thymocytes were cultured with the low doses of IL-1 + IL-2 for 72 h, 62% expressed cell surface T3 complex (vs 11% at initiation) and 27% were F23.1+ (vs 5% at initiation). In contrast, culture with IL-4 led to no increase in numbers of T3+ cells and none were F23.1+; however, there was coexpression of Thy1 and 6B2 on 20% of cells at the end of culture (vs 4% at initiation). Thus, IL-1 + IL-2 causes expansion of a CD 4-, CD 8- thymocyte population expressing the alpha, beta-T cell receptor, whereas IL-4 induces cells to express a phenotype present in small numbers in the periphery of normal mice and in larger numbers in mice bearing the lpr gene.     

Lymphocyte transendothelial migration toward smooth muscle cells in interleukin-1 β-stimulated co-cultures is related to fibronectin interactions with α4β1 and α5β1 integrins

We previously reported infiltration of immune-inflammatory cells in coronary arteries from cardiac allografts, associated with increased endothelial and smooth muscle cell fibronectin synthesis regulated by interleukin (IL)-1b?. We now investigate, using a porcine endothelial-smooth muscle cell co-culture system, whether IL-1b?-stimulated fibronectin production is functionally important in lymphocyte transendothelial migration. Lymphocytes were harvested from porcine peripheral blood and, in the unactivated state or following activation with phorbol myristic acetate (PMA) and IL-2, were characterized by fluorescence-activated cell sorter (FACS) analysis and added to a confluent endothelial monolayer on the upper chamber of a transwell system. Endothelial cells, as well as smooth muscle cells (in the bottom of the chamber), were stimulated with IL-1b?. Then transendothelial lymphocyte migration was determined in the presence of CS1 and RGD (fibronectin) peptides, blocking α₄b?₁ and α₅b?₁ integrin receptors on lymphocyte surfaces, respectively. A 55-70% inhibition of lymphocyte migration was observed when compared to control peptides. The combination of CS1 and RGD peptides did not significantly enhance the inhibitory effect of either peptide alone. A similar decrease in lymphocyte transendothelial migration toward smooth muscle cells was documented using a monoclonal antibody to cellular fibronectin. Furthermore, using smooth muscle cell conditioned medium; we reproduced the enhanced transendothelial lymphocyte migration as well as the inhibition with blocking peptides or fibronectin antibodies. Our data suggest that cytokine-mediated fibronectin synthesis in vascular cells recruits inflammatory cells through interactions of specific peptides with cell surface α₄b?₁ α₅b?₁ integrins. © 1995 Wiley-Liss, Inc.     

Contrasting effect of transforming growth factor type beta 1 (TGF-β1) on proliferation and interleukin-2 receptor expression in activated and rapidly cycling immature (CD3−CD4−CD8−) thymocytes

Transforming growth factor β (TGF-β) is a cytokine with immunoregulatory properties that acts negatively on T lymphocyte proliferation. However, with the EL 4–6.1 variant of the murine thymoma EL 4 activated with phorbol ester and/or interleukin-1 (IL-1), we recently found that it up-regulates interleukin-2-receptor (IL-2R) expression. Since EL 4–6.1 cells share phenotypic and functional characteristics with the immature thymic subset lacking CD4 and CD8 accessory molecules (DN), we investigated the effect of TGF-β1 on the IL-2R 55kD α chain expression and proliferation of activated DN cells and especially in DN cells that do not express CD3. We observed that TGF-β1 was able to increase both the percentage of CD3^?DN cells expressing IL-2Rα chains and the expression of IL-2Rα chain in these cells. This stimulatory effect of TGF-β1 was distal from early transduction events. In addition, TGF-β1 was found to modulate CD3^?DN cell proliferation. During differentiation in the thymus, CD3^?DN cells transiently express the IL-2Rα chain of the IL-2R and these IL-2R⁺ CD3^?DN cells are preprogrammed to down-regulate the IL-2Rα chain and up-regulate the CD4 and CD8 accessory molecule. We thus also tested the effect of TGF-β1 on IL-2Rα chain expression in these in vitro differentiating CD3^?DN cells. We found that TGF-β1 neither significantly affected IL-2R expression nor changed CD4 or CD8 expression. Hence, in CD3^?DN cells, the effect of TGF-β1 on IL-2R expression seems to be restricted to proliferating cells. © 1993 Wiley-Liss, Inc.     

Studies on the 5α-androstane-3β, 17β-diol-6α-hydroxylase and on the 5α-androstane-3β, 17β-diol-7α-hydroxylase in anterior hypophysis of male rats.

In anterior pituitaries from male rats, it appeared that 5α-androstane-3β, 17β-diol was quickly metabolized into 5α-androstane-3β,6α-17β-triol and 5α-androstane-3β,7α, 17β-triol by action of 6α- and 7α-hydroxylases. Hydroxysteroid hydroxylases were located in endoplasmic reticulum and were dependent on NADPH⁺. Their optimum pH was 8.0, optima temperature, 37°C, and their apparent Km was 2.7 μM. Hydroxylative reactions were not reversible and not modified by gonadectomy. Hydroxylation seemed an efficient control of the pituitary level of 5α-andros-tane-3β, 17β-diol.     

Subpopulations of mature murine thymocytes: properties of CD4-CD8+ and CD4+CD8- thymocytes lacking the heat-stable antigen

The heat-stable antigen (HSA), recognized by the monoclonal antibodies M1/69, B2A2, and J11d, is low or absent on the surface of most murine peripheral T cells but present on all but 3% of thymocytes. The CD4-CD8+ and CD4+CD8- or "single positive" thymic populations may be divided into further subgroups based on surface HSA expression. One group, CD4-CD8+ and expressing very high levels of HSA (HSA++), is an immature, T cell antigen receptor (TcR) negative, outer cortical blast cell. However, a further subdivision of CD4-CD8+ and CD4+CD8- single positives may be made, into those negative to low for HSA (HSA-) and those expressing moderate amounts of HSA (HSA+). The proportion of HSA- single positives is low in the thymus of young mice, whereas the proportion of HSA+ single positives is similar to that of the adult. Both the HSA- and the HSA+ subsets of single positive thymocytes from adult mice are CD3+ and express the normal peripheral T cell incidence of V beta 8 determinants on the TcR. On stimulation with concanavalin A in limit-dilution culture both HSA- and HSA+ subsets of single positive thymocytes give a high frequency of proliferating clones, and the clones from both HSA- and HSA+ subsets of CD4-CD8+ thymocytes are cytotoxic. Thus both HSA- and HSA+ single positive thymocytes are functionally mature. The HSA- subsets of single positive thymocytes differ from the HSA+ subsets in being slightly larger in size, in expressing higher levels of MEL-14, in binding more peanut agglutinin, and in including a proportion of cells expressing high levels of the Pgp-1 glycoprotein. It is suggested that HSA- CD4-CD8+ and HSA- CD4+CD8- thymocytes are more mature than their HSA+ counterparts, and might represent a previously activated or "memory" thymic subpopulation.     

Selection of CD4+CD8+ thymocytes by complex formation with medulla-derived epithelial cells

The role of lymphostromal complexes in T-cell differentiation is far from elucidated, mainly because a clear association of a particular stromal cell type with a distinct thymocyte subset has never been identified. Using an in vitro system, detecting the adherence of thymocytes to a thymic medullary epithelial cell line (E-5), we showed that the phenotype of these thymocytes was that of cortical type: Thy-1hi, LFA-1+, PNAhi, CD4+CD8+, MEL-14-/lo, IL-2R-, CD3-/lo, and TcR V beta 8-/lo. They were enriched in cells in G2/M at the time of complex formation, showed a higher basal proliferation in culture, and did not respond to PHA, IL-2 and only marginally to Con A. These data show that complex formation with mouse thymic medullary epithelium selects for CD4+CD8+ thymocytes, as shown by the marked decrease in CD4+CD8-/CD4-CD8+ thymocytes, and the incapacity of CD4-CD8- thymocytes to adhere.     

Induction of mouse β integrin expression following transfection with human α4 chain

We report here an analysis of the expression and function of the α chain of human VLA-4 in stable mouse L cell transfectants and the requirement for the β chain in these processes. L cells were transfected with human α4 cDNA or α4 and human β1 cDNA. Unexpectedly, human α4 cDNA, when transfected alone, could induce de novo surface expression of host β7 and increased expression of host β1. Induction of mouse β7 and β1 surface expression was not due to de novo gene activation, but instead represented α4/β intracellular subunit association and transport to the cell surface. Transfection with human β1 prevented surface expression of mouse β integrins. Whereas human α4 and human β1 subunits associated very tightly in anti-α4 immunoprecipitates, human α4 and mouse β subunits were only partially associated. Furthermore, binding of human/mouse chimeric receptors to recombinant VCAM, a major ligand for α4β7 and α4β1, was very poor, whereas human α4/human β1 receptors bound strongly to VCAM. One α4 transfectant, which exhibited a tight human α4/mouse β1 association, could be induced, but only after PMA activation, to bind strongly to VCAM. These results indicate that α4 subunits have specific affinity for β7 and β1 integrins and require β subunits for surface expression as well as high affinity ligand binding activity. Our results indicate that a tight association between the α4 and β subunit appears to be critical for ligand binding, consistent with a direct as well as regulatory role for the β subunit in ligand binding. Furthermore, these studies demonstrate that expression of foreign recombinant proteins can alter host cell protein expression resulting in de novo surface protein expression. © 1996 Wiley-Liss, Inc.     

Cyclic strain induces reorganization of integrin α5β1 and α2β1 in human umbilical vein endothelial cells

Cyclic strain has been shown to modulate endothelial cell (EC) morphology, proliferation, and function. We have recently reported that the focal adhesion proteins focal adhesion kinase (pp125^FAK) and paxillin, are tyrosine phosphorylated in EC exposed to strain and these events regulate the morphological change and migration induced by cyclic strain. Integrins are also localized on focal adhesion sites and have been reported to induce tyrosine phosphorylation of pp125^FAK under a variety of stimuli. To study the involvement of different integrins in signaling induced by cyclic strain, we first observed the redistribution of α and β integrins in EC subjected to 4 h cyclic strain. Human umbilical vein endothelial cells (HUVEC) seeded on either fibronectin or collagen surfaces were subjected to 10% average strain at a frequency 60 cycles/min. Confocal microscopy revealed that β₁ integrin reorganized in a linear pattern parallel with the long axis of the elongated cells creating a fusion of focal adhesion plaques in EC plated on either fibronectin (a ligand for α₅β₁) or collagen (a ligand for α₂β₁) coated plates after 4 h exposure to cyclic strain. β₃ integrin, which is a vitronectin receptor, did not redistribute in EC exposed to cyclic strain. Cyclic strain also led to a reorganization of α₅ and α₂ integrins in a linear pattern in HUVEC seeded on fibronectin or collagen, respectively. The expression of integrins α₅, α₂, and β₁ did not change even after 24 h exposure to strain when assessed by immunoprecipitation of these integrins. Cyclic strain-induced tyrosine phosphorylation of pp125^FAK occurred concomitant with the reorganization of β₁ integrin. We concluded that α₅β₁ and α₂β₁ integrins play an important role in transducing mechanical stimuli into intracellular signals. J. Cell. Biochem. 64:505–513. © 1997 Wiley-Liss, Inc.     

Engineered cystine knot peptides that bind αvβ3, αvβ5, and α5β1 integrins with low‐nanomolar affinity

There is a critical need for compounds that target cell surface integrin receptors for applications in cancer therapy and diagnosis. We used directed evolution to engineer the Ecballium elaterium trypsin inhibitor (EETI‐II), a knottin peptide from the squash family of protease inhibitors, as a new class of integrin‐binding agents. We generated yeast‐displayed libraries of EETI‐II by substituting its 6‐amino acid trypsin binding loop with 11‐amino acid loops containing the Arg‐Gly‐Asp integrin binding motif and randomized flanking residues. These libraries were screened in a high‐throughput manner by fluorescence‐activated cell sorting to identify mutants that bound to α_vβ₃ integrin. Select peptides were synthesized and were shown to compete for natural ligand binding to integrin receptors expressed on the surface of U87MG glioblastoma cells with half‐maximal inhibitory concentration values of 10–30 nM. Receptor specificity assays demonstrated that engineered knottin peptides bind to both α_vβ₃ and α_vβ₅ integrins with high affinity. Interestingly, we also discovered a peptide that binds with high affinity to α_vβ₃, α_vβ₅, and α₅β₁ integrins. This finding has important clinical implications because all three of these receptors can be coexpressed on tumors. In addition, we showed that engineered knottin peptides inhibit tumor cell adhesion to the extracellular matrix protein vitronectin, and in some cases fibronectin, depending on their integrin binding specificity. Collectively, these data validate EETI‐II as a scaffold for protein engineering, and highlight the development of unique integrin‐binding peptides with potential for translational applications in cancer. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Involvement of integrins α3β1 and α5β1 and glycoprotein IIb in megakaryocyte‐induced osteoblast proliferation

As the prevalence of osteoporosis is expected to increase over the next few decades, the development of novel therapeutic strategies to combat this disorder becomes clinically imperative. These efforts draw extensively from an expanding body of knowledge pertaining to the physiologic mechanisms of skeletal homeostasis. To this body of knowledge, we contribute that cells of hematopoietic lineage may play a crucial role in balancing osteoblastic bone formation against osteoclastic resorption. Specifically, our laboratory has previously demonstrated that megakaryocytes (MKs) can induce osteoblast (OB) proliferation in vitro, but do so only when direct cell‐to‐cell contact is permitted. To further investigate the nature of this interaction, we have effectively neutralized several adhesion molecules known to function in the analogous interaction of MKs with another cell type of mesenchymal origin—the fibroblast (FB). Our findings implicate the involvement of fibronectin/RGD‐binding integrins including α3β1 (VLA‐3) and α5β1 (VLA‐5) as well as glycoprotein (gp) IIb (CD41), all of which are known to be expressed on MK membranes. Furthermore, we demonstrate that interleukin (IL)‐3 can enhance MK‐induced OB activation in vitro, as demonstrated in the MK–FB model system. Taken together, these results suggest that although their physiologic and clinical implications are very different, these two models of hematopoietic–mesenchymal cell activation are mechanistically analogous in several ways. J. Cell. Biochem. 109: 927–932, 2010. © 2010 Wiley‐Liss, Inc.