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.

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.

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.

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.

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.

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.

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.

Vocal quality, articulation and audiological characteristics of children and young adults with diagnosed allergies.

This study details vocal quality, articulation errors, and hearing disorders in 80 children and young adults with diagnosed allergies. Results indicated that almost 50% had abnormalities in vocal quality and/or articulation and 13% had reduced auditory acuity. Vocal quality disorders showed a significant relationship to bronchial asthma in association with other allergic reactions. All subjects with diminished hearing had allergic rhinitis either singly or in combination with another disorder. Findings suggest that bronchial asthma and allergic rhinitis are related to the development of vocal quality disorders and that allergic rhinitis is associated with misarticulations and diminished hearing. The presence of speech sound omissions in allergic rhinitis patients above age 8 may predict the presence or previous history of fluctuating hearing loss.

Advances in burn care management: role of the speech-language pathologist.

Because the inclusion of the speech-language pathologist in a burn management team is not widely practiced, we discuss our successes as members of a burn team. We also review speech-language evaluation and treatment strategies and present two patients with head and neck burns who gained from our intervention.