Single-chain VαVß T-cell receptors function without mispairing with endogenous TCR chains.

Transduction of exogenous T-cell receptor (TCR) genes into patients' activated peripheral blood T cells is a potent strategy to generate large numbers of specific T cells for adoptive therapy of cancer and viral diseases. However, the remarkable clinical promise of this powerful approach is still being overshadowed by a serious potential consequence: mispairing of the exogenous TCR chains with endogenous TCR chains. These 'mixed' heterodimers can generate new specificities that result in graft-versus-host reactions. Engineering TCR constant regions of the exogenous chains with a cysteine promotes proper pairing and reduces the mispairing, but, as we show here, does not eliminate the formation of mixed heterodimers. By contrast, deletion of the constant regions, through use of a stabilized Vα/Vß single-chain TCR (scTv), avoided mispairing completely. By linking a high-affinity scTv to intracellular signaling domains, such as Lck and CD28, the scTv was capable of activating functional T-cell responses in the absence of either the CD3 subunits or the co-receptors, and circumvented mispairing with endogenous TCRs. Such transduced T cells can respond to the targeted antigen independent of CD3 subunits via the introduced scTv, without the transduced T cells acquiring any new undefined and potentially dangerous specificities.

Infrequent recovery of HIV from but robust exogenous infection of activated CD4(+) T cells in HIV elite controllers.

BACKGROUND. Human immunodeficiency virus (HIV) elite controllers are able to control infection with HIV-1 spontaneously to undetectable levels in the absence of antiretroviral therapy, but the mechanisms leading to this phenotype are poorly understood. Although low frequencies of HIV-infected peripheral CD4(+) T cells have been reported in this group, it remains unclear to what extent these are due to viral attenuation, active immune containment, or intracellular host factors that restrict virus replication. METHODS. We assessed proviral DNA levels, autologous viral growth from and infectability of in vitro activated, CD8(+) T cell-depleted CD4(+) T cells from HIV elite controllers (mean viral load, <50 copies/mL), viremic controllers (mean viral load, <2000 copies/mL), chronic progressors, and individuals receiving highly active antiretroviral therapy. RESULTS. Although we successfully detected autologous virus production in ex vivo activated CD4(+) T cells from all chronic progressors and from most of the viremic controllers, we were able to measure robust autologous viral replication in only 2 of 14 elite controllers subjected to the same protocol. In vitro activated autologous CD4(+) T cells from elite controllers, however, supported infection with both X4 and R5 tropic HIV strains at comparable levels to those in CD4(+) T cells from HIV-uninfected subjects. Proviral DNA levels were the lowest in elite controllers, suggesting that extremely low frequencies of infected cells contribute to difficulty in isolation of virus. CONCLUSIONS. These data indicate that elite control is not due to inability of activated CD4(+) T cells to support HIV infection, but the relative contributions of host and viral factors that account for maintenance of low-level infection remain to be determined.

Tissue engineering with meniscus cells derived from surgical debris.

OBJECTIVE: Injuries to the avascular regions of the meniscus fail to heal and so are treated by resection of the damaged tissue. This alleviates symptoms but fails to restore normal load transmission in the knee. Tissue engineering functional meniscus constructs for re-implantation may improve tissue repair. While numerous studies have developed scaffolds for meniscus repair, the most appropriate autologous cell source remains to be determined. In this study, we hypothesized that the debris generated from common meniscectomy procedures would possess cells with potential for forming replacement tissue. We also hypothesized that donor age and the disease status would influence the ability of derived cells to generate functional, fibrocartilaginous matrix. METHODS: Meniscus derived cells (MDCs) were isolated from waste tissue of 10 human donors (seven partial meniscectomies and three total knee arthroplasties) ranging in age from 18 to 84 years. MDCs were expanded in monolayer culture through passage 2 and seeded onto fiber-aligned biodegradable nanofibrous scaffolds and cultured in a chemically defined media. Mechanical properties, biochemical content, and histological features were evaluated over 10 weeks of culture. RESULTS: Results demonstrated that cells from every donor contributed to increasing biochemical content and mechanical properties of engineered constructs. Significant variability was observed in outcome parameters (cell infiltration, proteoglycan and collagen content, and mechanical properties) amongst donors, but these variations did not correlate with patient age or disease condition. Strong correlations were observed between the amount of collagen deposition within the construct and the tensile properties achieved. In scaffolds seeded with particularly robust cells, construct tensile moduli approached maxima of approximately 40 MPa over the 10-week culture period. CONCLUSIONS: This study demonstrates that cells derived from surgical debris are a potent cell source for engineered meniscus constructs. Results further show that robust growth is possible in MDCs from middle-aged and elderly patients, highlighting the potential for therapeutic intervention using autologous cells.

Elite control of HIV infection: implications for vaccine design.

BACKGROUND: 'Elite controllers' are rare HIV-infected individuals who are able to spontaneously control HIV replication without medication, maintaining viral loads that are consistently below the limits of detection by currently available commercial assays. OBJECTIVE: To examine studies of elite controllers that may elucidate mechanisms of HIV immune control useful in designing a vaccine. METHODS: Recent literature on HIV controllers and studies that have evaluated aspects of viral and host immunology that correlate with viral control are examined. RESULTS/CONCLUSIONS: Although many elements of innate and adaptive immunity are associated with control of HIV infection, the specific mechanism(s) by which elite controllers achieve control remain undefined. Ongoing studies of elite controllers, including those examining host genetic polymorphisms, should facilitate the definition of an effective HIV-specific immune response and guide vaccine design.

alpha beta T cell receptor ligand-specific oligomerization revisited.

The mechanism of T cell receptor signaling is unclear. Included among models for TCR signaling is ligand-induced oligomerization in a fashion analogous to other cell surface receptors. Published kinetic, saturation binding, and light scattering experiments have been interpreted to suggest a propensity for soluble alpha beta TCR/peptide/MHC ectodomain complexes to oligomerize. Upon performing these experiments with soluble ectodomains of human class I and class II restricted alpha beta TCRs, we find no evidence for dimerization or oligomerization of complexes. Apparently, oligomerization in solution to a detectable extent is not a general property of soluble alpha beta TCRs or their complexes with ligand. Our results suggest that membrane-anchored, fully assembled TCRs should be studied to determine the role oligomerization plays in T cell signaling.

Identification of a crucial energetic footprint on the alpha1 helix of human histocompatibility leukocyte antigen (HLA)-A2 that provides functional interactions for recognition by tax peptide/HLA-A2-specific T cell receptors.

Structural studies have shown that class I major histocompatibility complex (MHC)-restricted peptide-specific T cell receptor (TCR)-alpha/betas make multiple contacts with the alpha1 and alpha2 helices of the MHC, but it is unclear which or how many of these interactions contribute to functional binding. We have addressed this question by performing single amino acid mutagenesis of the 15 TCR contact sites on the human histocompatibility leukocyte antigen (HLA)-A2 molecule recognized by the A6 TCR specific for the Tax peptide presented by HLA-A2. The results demonstrate that mutagenesis of only three amino acids (R65, K66, and A69) that are clustered on the alpha1 helix affected T cell recognition of the Tax/HLA-A2 complex. At least one of these three mutants affected T cell recognition by every member of a large panel of Tax/HLA-A2-specific T cell lines. Biacore measurements showed that these three HLA-A2 mutations also altered A6 TCR binding kinetics, reducing binding affinity. These results show that for Tax/HLA-A2-specific TCRs, there is a location on the central portion of the alpha1 helix that provides interactions crucial to their function with the MHC molecule.

Conversion of a T cell antagonist into an agonist by repairing a defect in the TCR/peptide/MHC interface: implications for TCR signaling.

The structure of the A6 alphabetaTCR/HTLV-1 Tax-peptide/MHC I complex with proline 6 of Tax substituted with alanine (P6A), an antagonist, is nearly identical to the structure with wild-type Tax agonist. Neither the proline in the agonist nor the alanine in the antagonist is contacted by the alphabetaTCR. Here, we demonstrate that antagonist activity of P6A is associated with low affinity of the A6 alphabetaTCR for Tax-P6A/HLA-A2. We show that stepwise repair of a packing defect in the TCR/MHC interface using N-alkylated amino acids results in stepwise increases in TCR affinity and activity. Kinetic and thermodynamic measurements suggest that for some ligands the range of T cell outcomes does not correlate with either their alphabetaTCR affinity or the half-life of the alphabetaTCR/peptide/MHC complex.

Enzymatic synthesis of methylbutenol from dimethylallyl diphosphate in needles of Pinus sabiniana.

Methylbutenol (2-methyl-3-buten-2-ol) is an abundant volatile organic compound released from Western U.S. pines. To understand the mechanism of methylbutenol formation, we developed a sensitive gas chromatographic assay for its detection and determined that needles of gray pine (Pinus sabiniana) contain an enzyme that catalyzes the synthesis of methylbutenol from dimethylallyl diphosphate (DMAPP). The methylbutenol synthase activity was partially purified; its pH optimum was 7-8, and, like other prenyl diphosphate utilizing enzymes, it was dependent on the presence of a divalent cation, preferably Mn2+. The enzyme also required K+ or NH4+ for activity. The Km values for DMAPP and Mn2+ were about 4.8 and 6 mM, respectively. Geranyl diphosphate was not a substrate for the enzyme, so it is distinct from linalool synthase, a plant enzyme that catalyzes an analogous reaction. The methylbutenol synthase reaction may be responsible for the majority of light-dependent methylbutenol production by many pine species in the Western United States.

The energetics of phosphate binding to a protein complex.

The heat of binding the serine protease, porcine pancreatic elastase, by the inhibitor, turkey ovomucoid third domain, is dependent on the presence of inorganic phosphate. This dependence is saturable and can be accurately modeled as the phosphate binding to a single site on the protease-inhibitor complex; thus, the elastase-ovomucoid system provides a unique opportunity to study phosphate-protein interactions. We have used isothermal titration calorimetry to investigate this binding, thereby providing one of the few complete thermodynamic characterizations of phosphate interacting with proteins. The binding is characterized by a small favorable deltaG degrees, a large unfavorable deltaH degrees, and a positive deltaCp, thermodynamics consistent with the release of water being linked to phosphate binding. These measurements provide insight into the binding of phosphotyrosine containing peptides to SH2 domains by suggesting the energetic consequences of binding phosphate free from other interactions.

The structure and stability of an HLA-A*0201/octameric tax peptide complex with an empty conserved peptide-N-terminal binding site.

The crystal structure of the human class I MHC molecule HLA-A2 complexed with of an octameric peptide, Tax8 (LFGYPVYV), from human T cell lymphotrophic virus-1 (HTLV-1) has been determined. This structure is compared with a newly refined, higher resolution (1.8 A) structure of HLA-A2 complexed with the nonameric Tax9 peptide (LLFGYPVYV) with one more N-terminal residue. Despite the absence of a peptide residue (P1) bound in the conserved N-terminal peptide-binding pocket of the Tax8/HLA-A2 complex, the structures of the two complexes are essentially identical. Water molecules in the Tax8 complex replace the terminal amino group of the Tax9 peptide and mediate a network of hydrogen bonds among the secondary structural elements at that end of the peptide-binding groove. Thermal denaturation measurements indicate that the Tax8 complex is much less stable, DeltaTm = 16 degrees C, than the Tax9 complex, but both can sensitize target cells for lysis by some Tax-specific CTL from HTLV-1 infected individuals. The absence of a P1 peptide residue is thus not enough to prevent formation of a "closed conformation" of the peptide-binding site. TCR affinity measurements and cytotoxic T cell assays indicate that the Tax8/HLA-A2 complex does not functionally cross-react with the A6-TCR-bearing T cell clone specific for Tax9/HLA-A2 complexes.

Four A6-TCR/peptide/HLA-A2 structures that generate very different T cell signals are nearly identical.

The interactions of three singly substituted peptide variants of the HTLV-1 Tax peptide bound to HLA-A2 with the A6 T cell receptor have been studied using T cell assays, kinetic and thermodynamic measurements, and X-ray crystallography. The three peptide/MHC ligands include weak agonists and antagonists with different affinities for TCR. The three-dimensional structures of the three A6-TCR/peptide/HLA-A2 complexes are remarkably similar to each other and to the wild-type agonist complex, with minor adjustments at the interface to accommodate the peptide substitutions (P6A, V7R, and Y8A). The lack of correlation between structural changes and the type of T cell signals induced provides direct evidence that different signals are not generated by different ligand-induced conformational changes in the alphabeta TCR.

Prediction of binding energetics from structure using empirical parameterization.

We have presented an empirical method that can be used to predict the binding energetics for protein-protein or protein-peptide interactions from three-dimensional structures. The approach differs from other empirical methods in yielding a thermodynamic description of the binding process, including delta Cp, delta H degree, and delta S degree, rather than predicting delta G degree alone. These thermodynamic terms can provide a wealth of detail about the nature of the interaction, and, if sufficient experimental data are available for comparison, a greater assessment of the accuracy of the calculations. A recurring theme throughout this article is the need for more complete thermodynamic and structural characterizations of protein-ligand interactions. This includes not only characterization of the binding delta H degree, delta S degree, and delta Cp, but a thorough investigation into equilibria linked to binding, such as protonation, ion binding, and conformational changes. Sufficient data will allow parameterization on binding data rather than protein unfolding data. Further inclusion of information obtained from unfolding studies is not likely to generate significant improvement in the accuracy of the calculations. As additional binding data become available, the parameterization can be further extended to include relationships derived from analyses of these data. Not only will this increase accuracy and thus confidence, but allow extension of the method of additional types of interactions.

Dissecting the energetics of a protein-protein interaction: the binding of ovomucoid third domain to elastase.

An understanding of the structural basis for protein-protein interactions, and molecular recognition in general, requires complete characterization of binding energetics. Not only does this include quantification of the changes that occur in all of the thermodynamic parameters upon binding, including the enthalpy, entropy and heat capacity, but a description of how these changes are modulated by environmental conditions, most notably pH. Here, we have investigated the binding of turkey ovomucoid third domain (OMTKY3), a potent serine protease inhibitor, to the serine protease porcine pancreatic elastase (PPE) using isothermal titration calorimetry and structure-based thermodynamic calculations. We find that near neutral pH the binding energetics are influenced by a shift in the pKa of an ionizable group, most likely histidine 57 in the protease active site. Consequently, the observed binding energetics are strongly dependent upon solution conditions. Through a global analysis, the intrinsic energetics of binding have been determined, as have those associated with the pKa shift. The protonation energetics suggest that the drop in pKa is largely due to desolvation of the histidine residue. The resulting deprotonation is necessary for the enzymatic function of elastase. Intrinsically, at 25 degrees C the binding of OMTKY3 to PPE is characterized by an almost negligible enthalpy change, a large positive entropy change, and a large negative heat capacity change. These parameters are consistent with a model of the OMTKY3-PPE complex, which shows a large and significantly apolar protein-protein interface. Thermodynamic calculations based upon changes that occur in polar and apolar solvent-accessible surface area are in very good agreement with the measured intrinsic binding energetics.

Evaluation of linked protonation effects in protein binding reactions using isothermal titration calorimetry.

A theoretical development in the evaluation of proton linkage in protein binding reactions by isothermal titration calorimetry (ITC) is presented. For a system in which binding is linked to protonation of an ionizable group on a protein, we show that by performing experiments as a function of pH in buffers with varying ionization enthalpy, one can determine the pK(a)'s of the group responsible for the proton linkage in the free and the liganded states, the protonation enthalpy for this group in these states, as well as the intrinsic energetics for ligand binding (delta H(o), delta S(o), and delta C(p)). Determination of intrinsic energetics in this fashion allows for comparison with energetics calculated empirically from structural information. It is shown that in addition to variation of the ligand binding constant with pH, the observed binding enthalpy and heat capacity change can undergo extreme deviations from their intrinsic values, depending upon pH and buffer conditions.

Catheter ablation of clinical intraatrial reentrant tachycardias resulting from previous atrial surgery: localizing and transecting the critical isthmus.

OBJECTIVES: We sought to evaluate the efficacy of anatomically based radiofrequency catheter ablation for the treatment of intraatrial reentrant tachycardia in patients with previous atrial surgery. BACKGROUND: Intraatrial reentrant tachycardias, a common late complication of atrial surgery, are often refractory to standard medical management. Data from experimental animals and from humans indicate that anatomic barriers resulting from residual atrial scars provide a substrate for intraatrial reentry. We speculated that these tachycardias require a narrow isthmus of tissue between surgical scars and native nonconductive boundaries and that transection of this isthmus with radiofrequency ablation would therefore constitute an effective treatment. METHODS: Fourteen patients with a history of atrial surgery and clinical intraatrial reentrant tachycardia underwent electrophysiologic testing. From activation mapping, putative surgical scars and patches that served as boundaries of reentrant circuits were identified. Radiofrequency lesions were then placed to transect the narrowest isthmus of conducting tissue between a surgical scar and an anatomic barrier. Catheter ablation was attempted only for tachycardias consistent with the patient's clinical arrhythmias. RESULTS: Radiofrequency catheter ablation was attempted for 17 (55%) of 31 tachycardias identified; it successfully terminated tachycardias in 13 (93%) of 14 patients (95% confidence interval [CI] 79% to 99%). There were clinical recurrences in six patients (46%, 95% CI 19% to 73%), each of whom underwent a repeat ablation that was successful. Twelve (86%) of 14 patients (95% CI 67% to 99%) have remained free of intraatrial reentrant tachycardia for a mean of 7.5 +/- 5.3 months. CONCLUSIONS: Anatomically guided radiofrequency catheter ablation is an effective technique for definitive management of intraatrial reentrant tachycardia in patients with previous atrial surgery.

A case for permitting altruistic surrogacy.

Canada's Royal Commission on New Reproductive Technologies rejects all forms of surrogacy arrangement under the rubric of objecting to commercial surrogacy. Noncommercial surrogacy arrangements, however, can be defended against the commission's objections. They can be viewed as cases of giving a benefit or service to another in a way that expresses benevolence, and establishes a relationship between surrogates and prospective 'social' parents that allows mutual understanding and reciprocal personal interaction between them.

Nonpharmacologic approaches to the treatment of atrial fibrillation and atrial flutter.

The high prevalence of atrial fibrillation, the associated morbidity and mortality, the absence of safe and effective drug therapy, and an increased understanding of the pathophysiologic basis of atrial fibrillation and flutter have collectively led to the development of novel nonpharmacologic treatments for the management of these arrhythmias, including the CORRIDOR and MAZE surgical procedures, catheter-based ablation and modification of AV conduction, catheter-based ablation of atrial flutter and fibrillation, and internal atrial defibrillation. These surgical and catheter-based techniques offer potentially curative therapy while sparing the long-term risk of antiarrhythmic drug therapy. For patients with typical atrial flutter, catheter ablation affords to cure rate in excess of 70%. As technological innovations further facilitate identification and ablation of the critical isthmus in the floor of the right atrium, success rates should improve substantially. For patients with atrial fibrillation, AV junction ablation with implantation of a rate-responsive ventricular pacemaker should be considered palliative therapy, as should modification of AV junction conduction. The MAZE procedure offers very high cure rates, but because it currently involves open heart surgery, patient selection is critical. Catheter-based procedures emulating aspects of the MAZE procedure may one day offer cure rates comparable to those of the surgery itself, but additional research and technological development are necessary to further define and refine the minimal effective procedure, and then to facilitate the placement of contiguous, full-thickness lesions in precise three-dimensional configurations. In the interim, the implantable automatic atrial defibrillator may offer a means for rapidly restoring sinus rhythm without the risks of long-term antiarrhythmic drug therapy.