The Equilibration of PCO2 in Pigs Is Independent of Lung Injury and Hemodynamics.

OBJECTIVES: In mechanical ventilation, normoventilation in terms of PCO2 can be achieved by titration of the respiratory rate and/or tidal volume. Although a linear relationship has been found between changes in respiratory rate and resulting changes in end-tidal cO2 (△PetCO2) as well as between changes in respiratory rate and equilibration time (teq) for mechanically ventilated patients without lung injury, it is unclear whether a similar relationship holds for acute lung injury or altered hemodynamics. DESIGN: We performed a prospective randomized controlled animal study of the change in PetCO2 with changes in respiratory rate in a lung-healthy, lung-injury, lung-healthy + altered hemodynamics, and lung-injury + altered hemodynamics pig model. SETTING: University research laboratory. SUBJECTS: Twenty mechanically ventilated pigs. INTERVENTIONS: Moderate lung injury was induced by injection of oleic acid in 10 randomly assigned pigs, and after the first round of measurements, cardiac output was increased by approximately 30% by constant administration of noradrenalin in both groups. MEASUREMENTS AND MAIN RESULTS: We systematically increased and decreased changes in respiratory rate according to a set protocol: +2, -4, +6, -8, +10, -12, +14 breaths/min and awaited equilibration of Petco2. We found a linear relationship between changes in respiratory rate and △PetCO2 as well as between changes in respiratory rate and teq. A two-sample t test resulted in no significant differences between the lung injury and healthy control group before or after hemodynamic intervention. Furthermore, exponential extrapolation allowed prediction of the new PetCO2 equilibrium and teq after 5.7 ± 5.6 min. CONCLUSIONS: The transition between PetCO2 equilibria after changes in respiratory rate might not be dependent on moderate lung injury or cardiac output but on the metabolic production or capacity of cO2 stores. Linear relationships previously found for lung-healthy patients and early prediction of PetCO2 equilibration could therefore also be used for the titration of respiratory rate on the PetCO2 for a wider range of pathologies by the physician or an automated ventilation system.

A new type of metal recognition by human T cells: contact residues for peptide-independent bridging of T cell receptor and major histocompatibility complex by nickel.

In spite of high frequencies of metal allergies, the structural basis for major histocompatibility complex (MHC)-restricted metal recognition is among the unanswered questions in the field of T cell activation. For the human T cell clone SE9, we have identified potential Ni contact sites in the T cell receptor (TCR) and the restricting human histocompatibility leukocyte antigen (HLA)-DR structure. The specificity of this HLA-DR-promiscuous VA22/VB17+ TCR is primarily harbored in its alpha chain. Ni reactivity is neither dependent on protein processing in antigen-presenting cells nor affected by the nature of HLA-DR-associated peptides. However, SE9 activation by Ni crucially depends on Tyr29 in CDR1alpha, an N-nucleotide-encoded Tyr94 in CDR3alpha, and a conserved His81 in the HLA-DR beta chain. These data indicate that labile, nonactivating complexes between the SE9 TCR and most HLA-DR/peptide conjugates might supply sterically optimized coordination sites for Ni ions, three of which were identified in this study. In such complexes Ni may effectively bridge the TCR alpha chain to His81 of most DR molecules. Thus, in analogy to superantigens, Ni may directly link TCR and MHC in a peptide-independent manner. However, unlike superantigens, Ni requires idiotypic, i.e., CDR3alpha-determined TCR amino acids. This new type of TCR-MHC linkage might explain the high frequency of Ni-reactive T cells in the human population.

T cell receptor (TCR) interaction with haptens: metal ions as non-classical haptens.

Haptens are classified as low molecular chemicals with an intrinsic potential to covalently modify proteins, and many of them are strong inducers of contact hypersensitivity (CHS). CHS is T cell mediated, and hapten-specific T cells have been shown to interact with hapten-modified, MHC-associated peptides. However, the most common contact sensitizer in the industrialized world is nickel. In contrast to classical haptens, nickel ions do not form covalent bonds to proteins, but rather become caught in reversible coordination complexes. We here review work demonstrating that some T cells, indeed, may react to such Ni complexes on the MHC/peptide-surface absolutely comparable to other haptens. In other cases, Ni ions unlike classical haptens, may activate T cells by crosslinking their receptors to MHC molecules, independent of the nature of the associated peptide. Moreover, Ni-interacting proteins appear to make use of the reversibility of Ni-binding, and to mediate the transfer of Ni-ions to the receptor-MHC interphase. We have demonstrated such properties for human serum albumin (HSA) as well as for transferrin and identified numerous new Ni-binding proteins in human B-cell lines or dendritic cells by affinity purification and mass spectroscopy. These proteins include a notable number of known heat shock proteins and chaperones, implying that Ni may functionally interfere with these stress proteins.

Mechanical load and mechanical integrity of lung cells - experimental mechanostimulation of epithelial cell- and fibroblast-monolayers.

Experimental mechanostimulation of soft biologic tissue is widely used to investigate cellular responses to mechanical stress or strain. Reactions on mechanostimulation are investigated in terms of morphological changes, inflammatory responses and apoptosis/necrosis induction on a cellular level. In this context, the analysis of the mechanical characteristics of cell-layers might allow to indicate patho-physiological changes in the cell-cell contacts. Recently, we described a device for experimental mechanostimulation that allows simultaneous measurement of the mechanical characteristics of cell-monolayers. Here, we investigated how cultivated lung epithelial cell- and fibroblast-monolayers behave mechanically under different amplitudes of biaxial distension. The cell monolayers were sinusoidally deflected to 5%, 10% or 20% surface gain and their mechanical properties during mechanostimulation were analyzed. With increasing stimulation amplitudes more pronounced reductions of cell junctions were observed. These findings were accompanied by a substantial loss of monolayer rigidity. Pulmonary fibroblast monolayers were initially stiffer but were stronger effected by the mechanostimulation compared to epithelial cell-monolayers. We conclude that, according to their biomechanical function within the pulmonary tissue, epithelial cells and fibroblasts differ with respect to their mechanical characteristics and tolerance of mechanical load.

A new device for dynamic ventilation-analogue mechanostimulation of pliant tissue layers.

The experimental mechanostimulation of biological cell and tissue test samples has become a standard method in biomechanics research. In order to apply a static or a dynamic mechanical load on biological tissue a variety of different devices for the mechanostimulation have been developed. While cyclic load applications are typically restricted to sinusoidal or rectangular stimulation patterns, a device for more complex dynamic stimulation patterns which would simulate, for instance, the dynamics during mechanical ventilation does not exist. The dynamic alveolar recruitment/derecruitment has been identified as one of the main causes of ventilator-induced lung injury. Therefore, there is a demand for an experimental ventilation-analogue mechanostimulation of the pulmonary cells and tissue. Here, we present our mechanostimulator combined with a new driving system which is able to produce the ventilation-analogue patterns of a dynamic mechanostimulation. In an experimental setting where the test samples were simulated by silicone-membranes in single-, double- and fourfold membrane configuration, we varied the stimulation amplitude from 5% to 60% surface increase and stimulation frequencies ranging from 15/min to 2000/min. Furthermore, the frequency components of mechanical load applied to the sample at sinusoidal, rectangular and ventilation- analogue mechanostimulations were analyzed by means of a Fast Fourier Transform (FFT). The system allows for a homogeneous mechanostimulation with various temporal profiles which may include frequency components of up to 20 Hz. The relative amount of mechanical load applied to the sample at the main stimulation frequency was 76% during sinusoidal stimulation, 35% during the rectangular stimulation, and 29% to 42% during ventilation analogue stimulation.

Mechanostimulation, electrostimulation and force measurement in an in vitro model of the isolated rat diaphragm.

In an in vitro model of the entire rat diaphragm, diaphragmatic contraction forces at defined preload levels were investigated. A total of 24 excised rat diaphragms were electrically stimulated inside a two-chamber strain-applicator. The resulting contraction forces were determined on eight adjusted preload levels via measuring the elicited pressure in the chamber below the diaphragm. Subsequently, diaphragms were exposed for 6 h to one of four treatments: (1) control, (2) cyclic mechanical stretch, (3) intermittent electrical stimulation or (4) combination of cyclic mechanical stretch and electrical stimulation. Diaphragmatic contraction force increased from 116 ± 21 mN at the lowest preload level to 775 ± 85 mN at the maximal preload level. After 6 h maximal muscle contraction forces were smallest after non-electrostimulated treatment (control: 81 ± 15 mN, mechanical deflection: 94 ± 12 mN) and largest after electrostimulation treatment (mere electrostimulation: 165 ± 20 mN, combined mechano- and electro-stimulation: 164 ± 14 mN). We conclude that our model allows force measurements on isolated rat diaphragms. Furthermore, we conclude that by intermediate electrical stimulation diaphragmatic force generation was better preserved than by mechanical stimulation.

Characteristics of highly flexible PDMS membranes for long-term mechanostimulation of biological tissue.

Measurement of mechanical properties of soft biological tissue remains a challenging task in mechanobiology. Recently, we presented a bioreactor for simultaneous mechanostimulation and analysis of the mechanical properties of soft biological tissue samples. In this bioreactor, the sample is stretched via deflection of a flexible membrane. It was found that the use of highly compliant membranes increases accuracy of measurements. Here, we describe the production process and characteristics of thin and flexible membranes of polydimethylsiloxane (PDMS) designed to improve the signal-to-noise ratio of our bioreactor. By a spin-coating process, PDMS membranes were built by polymerization of a two component elastomer. The influence of resin components proportion, rotation duration, and speed of the spinning were related to the membrane mechanics. Membranes of 22 mm inner diameter and 33 to 36 microm thickness at homogeneous profiles were produced. Isolated rat diaphragms served as biological tissue samples. Mechanical properties of the membranes remained constant during 24 h of mechanostimulation. In contrast, time- and strain-dependent mechanical properties of the diaphragms were found.

Drug interaction with T-cell receptors: T-cell receptor density determines degree of cross-reactivity.

BACKGROUND: Immune-mediated adverse reactions to drugs are often due to T-cell reactivity, and cross-reactivity is an important problem in pharmacotherapy. OBJECTIVE: We investigated whether chemical inert drugs can stimulate T cells through their T-cell receptor (TCR) and analyzed the cross-reactivities to related compounds. METHODS: We transfected human TCRs isolated from two drug-reactive T-cell clones (TCCs) by PCR into a TCR-negative mouse T-cell hybridoma. The TCCs were isolated from a patient with drug hypersensitivity to the antibacterial sulfonamide sulfamethoxazole (SMX). RESULTS: The transfectants reacted to SMX only in the presence of antigen-presenting cells (APCs). Glutaraldehyde-fixed APCs, however, were sufficient to elicit T-cell stimulation, indicating a processing-independent direct interaction of the drug with the TCR and MHC molecule. The transfected hybridomas secreted IL-2 in a drug dose-dependent manner, whereas the degree of reactivity was dependent on the level of TCR expression. One transfectant reacted not only to SMX but also to related sulfonamide compounds. Interestingly, high TCR expression increased cross-reactivity to other structurally related compounds. In addition, SMX-specific TCR cross-reacted only with sulfonamides bearing a sulfanilamide core structure but not with sulfonamides such as celecoxib, furosemide, or glibenclamide. CONCLUSIONS: These results demonstrate that the T-cell reactivity to drugs is solely determined by the TCR. Moreover, these results show that cross-reactivity of structurally similar compounds correlates with the density of the TCR. Stably transfected T-cell hybridomas may represent a powerful screening tool for cross-reactivity of newly generated sulfonamide-containing compounds such as celecoxib.