Allospecific rejection of MHC class I-deficient bone marrow by CD8 T cells.

Avoidance of long-term immunosuppression is a desired goal in organ transplantation. Mixed chimerism offers a promising approach to tolerance induction, and we have aimed to develop low-toxicity, nonimmunodepleting approaches to achieve this outcome. In a mouse model achieving fully MHC-mismatched allogeneic bone marrow engraftment with minimal conditioning (3 Gy total body irradiation followed by anti-CD154 and T cell-depleted allogeneic bone marrow cells), CD4 T cells in the recipient are required to promote tolerance of preexisting alloreactive recipient CD8 T cells and thereby permit chimerism induction. We now demonstrate that mice devoid of CD4 T cells and NK cells reject MHC Class I-deficient and Class I/Class II-deficient marrow in a CD8 T cell-dependent manner. This rejection is specific for donor alloantigens, since recipient hematopoiesis is not affected by donor marrow rejection and MHC Class I-deficient bone marrow that is syngeneic to the recipient is not rejected. Recipient CD8 T cells are activated and develop cytotoxicity against MHC Class I-deficient donor cells in association with rejection. These data implicate a novel CD8 T cell-dependent bone marrow rejection pathway, wherein recipient CD8 T cells indirectly activated by donor alloantigens promote direct killing, in a T cell receptor-independent manner, of Class I-deficient donor cells.

Recipient dendritic cells, but not B cells, are required antigen-presenting cells for peripheral alloreactive CD8+ T-cell tolerance.

Induction of mixed allogeneic chimerism is a promising approach for achieving donor-specific tolerance, thereby obviating the need for life-long immunosuppression for solid organ allograft acceptance. In mice receiving a low dose (3Gy) of total body irradiation, allogeneic bone marrow transplantation combined with anti-CD154 tolerizes peripheral CD4 and CD8 T cells, allowing achievement of mixed chimerism with specific tolerance to donor. With this approach, peripheral CD8 T-cell tolerance requires recipient MHC class II, CD4 T cells, B cells and DCs. Recipient-type B cells from chimeras that were tolerant to donor still promoted CD8 T-cell tolerance, but their role could not be replaced by donor-type B cells. Using recipients whose B cells or DCs specifically lack MHC class I and/or class II or lack CD80 and CD86, we demonstrate that dendritic cells (DCs) must express CD80/86 and either MHC class I or class II to promote CD8 tolerance. In contrast, B cells, though required, did not need to express MHC class I or class II or CD80/86 to promote CD8 tolerance. Moreover, recipient IDO and IL-10 were not required. Thus, antigen presentation by recipient DCs and not by B cells is critical for peripheral alloreactive CD8 T cell tolerance.

HVEM/LIGHT/BTLA/CD160 cosignaling pathways as targets for immune regulation.

Immunosuppression is currently the treatment of choice to attenuate the chronic deterioration of tissue function as a result of the effector mechanisms of the immunological response in transplant rejection and autoimmune diseases. However, global immunosuppression greatly increases the risk of acquiring life-threatening infections and is associated with organ toxicity when used long-term. Thus, alternative approaches that inhibit only the unwanted immune responses and preserve general immunity are highly desirable. The receptor/ligand pairs involved in the cross-talk between DC and T cells have been the focus of intense and exciting research during the last decade. The HVEM/LIGHT/BTLA/CD160 costimulatory/coinhibitory pathway has emerged as a potential target for the development of immune therapeutic interventions. Herein, we will summarize and discuss how blockade of the costimulatory HVEM/LIGHT interaction or agonist signaling through the inhibitory BTLA and CD160 receptors could contribute to the control of deleterious immune responses.

A LabVIEW model incorporating an open-loop arterial impedance and a closed-loop circulatory system.

While numerous computer models exist for the circulatory system, many are limited in scope, contain unwanted features or incorporate complex components specific to unique experimental situations. Our purpose was to develop a basic, yet multifaceted, computer model of the left heart and systemic circulation in LabVIEW having universal appeal without sacrificing crucial physiologic features. The program we developed employs Windkessel-type impedance models in several open-loop configurations and a closed-loop model coupling a lumped impedance and ventricular pressure source. The open-loop impedance models demonstrate afterload effects on arbitrary aortic pressure/flow inputs. The closed-loop model catalogs the major circulatory waveforms with changes in afterload, preload, and left heart properties. Our model provides an avenue for expanding the use of the ventricular equations through closed-loop coupling that includes a basic coronary circuit. Tested values used for the afterload components and the effects of afterload parameter changes on various waveforms are consistent with published data. We conclude that this model offers the ability to alter several circulatory factors and digitally catalog the most salient features of the pressure/flow waveforms employing a user-friendly platform. These features make the model a useful instructional tool for students as well as a simple experimental tool for cardiovascular research.

Energetics and hemodynamic changes of normal and various right heart bypass (Fontan) circulations in lambs under varying respiration parameters.

The Fontan circulation or one of its many modifications is a common surgical procedure implemented in children with congenital heart defects to improve pulmonary circulation. Two modifications are the atriopulmonary connection (AP) and the modified total cavopulmonary connection (TCPC). Successful AP and TCPC operations performed in lambs show respiration has a major influence on systemic and pulmonary hemodynamics. Instantaneous pressure and flow waveforms are obtained before and after the procedure and energies are calculated. Hemodynamic changes in relevant vessels are also acquired. Time delays due to respiration will be calculated and transfer functions computed. The purpose of this study is to examine these waveforms and energy gains/losses under varying respiration parameters in order to aid in determining an optimal surgical protocol as well as assist in the development an accurate computer simulation model.

The effect of incorporating vessel compliance in a computational model of blood flow in a total cavopulmonary connection (TCPC) with caval centerline offset.

BACKGROUND: The total cavopulmonary connection (TCPC), a palliative correction for congenital defects of the right heart, is based on the corrective technique developed by Fontan and Baudet. Research into the TCPC has primarily focused on reducing power loss through the connection as a means to improve patient longevity and quality of life. The goal of our study is to investigate the efficacy of including a caval offset on the hemodynamics and, ultimately, power loss of a connection. As well, we will quantify the effect of vessel wall compliance on these factors and, in addition, the distribution of hepatic blood to the lungs. METHODS: We employed a computational fluid dynamic model of blood flow in the TCPC that includes both the non-Newtonian shear thinning characteristics of blood and the nonlinear compliance of vessel tissue. RESULTS: Power loss in the rigid-walled simulations decayed exponentially as caval offset increased. The compliant-walled results, however, showed that after an initial substantial decrease in power loss for offsets up to half the caval diameter, power loss increased slightly again. We also found only minimal mixing in both simulations of all offset models. CONCLUSIONS: The increase in power loss beyond an offset of half the caval diameter was due to an increase in the kinetic contribution. Reduced caval flow mixing, on the other hand, was due to the formation of a pressure head in the offset region which acts as a barrier to flow.

Diastolic shape of the right ventricle of the heart.

BACKGROUND: Knowledge of right ventricular (RV) shape is important to the understanding of RV mechanical function and for the improvement of clinically important RV volume estimation techniques. Refinements to the simplest conceptions of RV shape are presented statistically here, based on a quantitative analysis of three-dimensional magnetic resonance (MR) images of excised lamb hearts. METHODS: The passive shape of the heart in six freshly excised lamb hearts was studied with MR imaging with independent passive pressurization of both ventricles. Global features of shape were assessed, including measurement of short-axis, cross-sectional shape parameters associated with the pinched-arc model. RESULTS: The slice-area x apex-base length was found to be highly correlated with the volume of the RV, with little sensitivity to the degree of filling of the ventricle or to the exact slice chosen (r = 0.987; n = 22 from five hearts). The RV was shown to follow a clockwise helical path around the left ventricle of 47 +/- 17 degrees, below the outflow tract, as seen from the apical view, progressing from the apex to the base. Based on the pinched-arc model, the anterior arc is shallower than the posterior arc, with a larger radius of curvature and a smaller angle between the arc and the septal axis. As the RV is passively filled, opposite changes in shape occur between the anterior and posterior regions tending to equalize their shapes. CONCLUSIONS: A high degree of regularity of shape does exist in the RV and, thus, can be characterized effectively in terms of a representative cross-sectional shape and in terms of the changes in that shape proceeding from the base to the apex.

Pulmonary blood flow profiles with reduced right ventricular function in lambs.

The determinants of right ventricular (RV) performance with damaged RV free wall, such as occurs with RV infarction, are still unclear. Using 20-MHz Doppler ultrasound equipment, we investigated the changes in pulmonary blood flow velocity profiles before and after ligation of the right coronary artery. RV dp/dt, stroke volume, RV stroke work, aortic pressure and cardiac output decreased and central venous pressure rose after the ligation. The RV stroke work-end-diastolic pressure relationship indicated impaired RV function following ligation. We observed shortened acceleration time (65.0 +/- 15.1 vs 54.4 +/- 6.2 ms, P < 0.05) and reduced maximum velocity of forward flow (59.0 +/- 5.9 vs 52.5 +/- 7.6 cm/s, P < 0.05) after the ligation. Acceleration was interrupted earlier after ligation than before ligation. These alterations in flow are thought to be a consequence of the altered movement of the RV free wall and ventricular septum induced by RV infarction.

Hemodynamics of the Fontan connection: an in-vitro study.

The Fontan operation is one in which the right heart is bypassed leaving the left ventricle to drive the blood through both the capillaries and the lungs, making it important to design an operation which is hemodynamically efficient. The object here was to relate the pressure in Fontan connections to its geometry with the aim of increasing the hemodynamically efficiency. From CT or magnetic resonance images, glass models were made of realistic atrio-pulmonary (AP) and cavo-pulmonary (CP) connections in which the right atrium and/or ventricle are bypassed. The glass models were connected to a steady flow loop and flow visualization, pressure and 3 component LDA measurements made. In the AP model the large atrium and curvature of the conduit created swirling patterns, the magnitude of which was similar to the axial velocity. This led to an inefficient flow and a subsequent large pressure loss (780 Pa). In contrast, the CP connection with a small intra-atrial chamber had reduced swirling and a significantly smaller pressure loss (400 Pa at 8 l.min) and was therefore a more efficient connection. There were, however, still pressure losses and it was found that these occurred where there was a large bending of the flow, such as from the superior vena cava to the MPA and from the MPA to the right pulmonary artery.

Effects of chronically elevated pulmonary arterial pressure and flow on right ventricular afterload.

The effects of pulsatile hemodynamics on right ventricle-pulmonary circulation interactions were studied in control lambs and in two lamb models of altered pulmonary hemodynamics induced at infancy: elevated pulmonary arterial pressure (PAP) was created by the infusion of monocrotaline pyrrole (MCTP), and elevated pulmonary arterial blood flow was obtained by the creation of an arteriovenous fistula (Shunt). High-fidelity PAP, midvessel Doppler blood velocity (PAV), and cardiac output (CO) were measured in open-chest, anesthetized lambs. PAV waveforms were normalized to match the measured CO. Measured pressure and flow signals were separated in the time domain into forward and backward components. Pulmonary input impedance and indexes quantifying the timing of the reflected wave pulse (beginning of reflected pulse, duration of reflected pulse in systole, and duration of reflected wave in diastole) were calculated for each group. Results indicate that in control animals the reflected wave returned late in systole and extended through much of diastole, thereby increasing diastolic pressure like a counterpulsation balloon. No significant differences in the timing indexes were found between Shunt and control animals. In the MCTP group, the reflected wave returned significantly earlier than normal with the peak reflected pulse occurring before valve closure. The resulting augmentation of systolic pressure and, therefore, large pulse pressure is consistent with pressure waveforms observed in clinical pulmonary hypertension. We conclude that early wave reflection exerts a detrimental effect in pulmonary hypertension by unfavorably loading the still-ejecting right ventricle.

Induction of right ventricular hypertrophy with obstructing balloon catheter. Nonsurgical ventricular preparation for the arterial switch operation in simple transposition.

BACKGROUND: Recently, a successful result with a rapid two-stage arterial switch operation (ASO) was reported for patients with transposition of the great arteries (TGA) with low left ventricular pressure. In this procedure, the interval between pulmonary arterial banding and ASO was approximately 1 week. This successful result indicates the possibility of a nonsurgical ventricular preparation procedure using an obstructing balloon catheter prior to ASO. METHODS AND RESULTS: A 5F atrioseptostomy catheter was inserted directly into the main pulmonary artery in six lambs aged 20 to 38 days. After the chest was closed, the balloon was inflated twice a day for a period of 2 to 2.5 hours. This procedure was performed for 4 consecutive days. After the final inflation, the ratio of right ventricular weight to total ventricular weight was compared with that in an age-matched control group. After the final inflation, the peak systolic right ventricular pressure and the percentage of peak systolic right ventricular to peak systolic aortic pressure rose to 85.6 +/- 4.7 mm Hg (mean +/- 1 SD) and 79.6 +/- 8.6%, respectively. The percentages of the right ventricular weight to the total ventricular weight were significantly higher after the balloon inflation than those in the control group in terms of wet heart weight (29.5 +/- 1.2% versus 23.0 +/- 1.0%; P < .0001) and dry heart weight (27.0 +/- 2.0% versus 21.0 +/- 1.1%; P < .0001). CONCLUSIONS: The myocardial mass in the right ventricle increased after 4 days of intermittently applied pressure overload. Nonsurgical preparation of the ventricle for ASO in TGA is feasible.

Three-dimensional visualization of pulmonary blood flow velocity profiles in lambs.

For a better understanding of the characteristics of blood flow in the pulmonary artery, we constructed three-dimensional images of velocity profiles of blood flow in the pulmonary artery from pulsed Doppler ultrasound recordings in 14 lambs aged 28-40 days. In 8 lambs, pulmonary hypertension was created by the central venous injection of monocrotaline pyrrole. Six lambs served as unaltered controls. The velocity data were sampled in 2 mm increments along both an anterior-posterior axis and a right-left orthogonal axis in the main pulmonary artery. Using a computer-generated cross-sectional velocity matrix consisting of 0.25 mm square grids, the velocity of blood flow was estimated at each intersection. The cross-sectional velocity matrices were generated at 5 msec intervals during the entire cardiac cycle. In all animals, significant velocity reversal was detected near the posterior wall. In 7 of 14 animals, the peak forward velocity was located near the posterior wall. Three of 8 hypertensive models showed reacceleration during the mid-systolic phase at the center of the velocity waveform, but one reacceleration disappeared at a point only 2 mm away from the center of the vessel toward the posterior wall. Acceleration time correlated well with the mean pulmonary arterial pressure (PAP) (r = -0.85) and the log10 PAP (r = -0.86). Corrected acceleration time (acceleration time divided by the square root of the cardiac cycle length) also correlated with PAP (r = -0.78) and the log10 PAP (r = -0.81).

Intraluminal pulsed Doppler evaluation of the pulmonary artery velocity time curve in a canine model of acute pulmonary hypertension.

The velocity pattern of the blood flow in the pulmonary artery was investigated in an animal model of acute pulmonary hypertension. Nine anesthetized, open-chest dogs were embolized with polystyrene microspheres, and the velocity pattern of the blood flow in the pulmonary artery was studied with use of an invasive pulsed Doppler technique. Phasic intraluminal velocity was recorded with use of a miniature piezoelectric crystal activated by 20-MHz Doppler pulses and mounted on the tip of a needle probe introduced into the pulmonary artery. The recorded Doppler quadrature signals were processed by spectral analysis. Significant increases occurred in mean, systolic, and diastolic pulmonary arterial pressures (p less than 0.0002), in pulmonary vascular resistance (p less than 0.005), and in negative velocity time (duration in milliseconds that the mean velocity was directed toward the pulmonic valve) (p less than 0.002). Significant decreases occurred in right ventricular ejection time (p less than 0.006) and in positive velocity time (duration in milliseconds that the mean velocity was directed away from the pulmonic valve) (p less than 0.005). A significant shortening in the time to peak velocity (acceleration time) was found (p less than 0.005). Second-order regression analyses demonstrated an inverse correlation between the ratio of positive velocity time to negative velocity time and the mean pulmonary artery pressure in all animals (r = 0.71). These findings should be compared with the velocity patterns of the blood flow in the pulmonary artery obtained under pulmonary hypertensive conditions due to various causes to facilitate interpretation and understanding of clinical investigations.

Pulmonary blood velocity profile variability in open-chest dogs: influence of acutely altered hemodynamic states on profiles, and influence of profiles on the accuracy of techniques for cardiac output determination.

Clinical investigations focused on finding characteristics of noninvasively obtained measurements of pulmonary blood velocity that can be used to quantitate pulmonary blood flow and/or pulmonary pressure have often yielded results whose imprecision has been attributed to flow pattern variability. To determine flow pattern variability in an in vivo animal model in varying hemodynamic states, main pulmonary artery blood velocity waveforms were recorded in 17 dogs at 2-mm intervals along an anterior to posterior wall-oriented axis using a 20-MHz pulsed Doppler needle probe. Control data were obtained before the animals were subjected to altered flow (atrial level shunts) and pressure (10% O2 inhalation) states. Instantaneous velocity profiles were computed throughout the cardiac cycle. Estimates of pulmonary blood flow were obtained assuming an elliptical model of the pulmonary artery which allowed computation of velocity at all points in the cross section, based on the measured values along the axis. Model-based estimates were compared to measured values and estimates obtained in the traditional fashion, i.e., the product of centerline velocity and cross-sectional area. Results clearly showed marked interanimal variability, even in control states. Reverse flow in the posterior half of the vessel, which tended to become more pronounced with increased pulmonary artery pressure, was observed during late systole and early diastole. Elevated pulmonary blood flow tended to increase the maximum velocities along the anterior wall relative to midline velocities. Neither estimate of cardiac output yielded consistently accurate results (r = 0.77 for model-based method, r = 0.80 for area times central velocity method). Findings of this study, which highlight the dependency of waveform characteristics on sampling site, the large degree of intersubject variability, and the need for large or multiple sample volumes for pulmonary blood flow determination, help clarify inconsistencies observed by clinicians and suggest that future work with animal models will facilitate a greater understanding of the determinants of human pulmonary velocity waveforms.

Analysis of T cell receptor-gene rearrangement in T cells from the cerebrospinal fluid of patients with multiple sclerosis.

Characterization of T cells present in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) may contribute to an understanding of the immunopathologic role of these cells. To analyze the T cells in the CSF of MS patients, 30 cloned T cell lines from each of two MS patients were surveyed for their patterns of T cell receptor (TcR) beta-chain gene rearrangement. DNA from the (CSF-derived) T cell clones was digested with a number of restriction endonucleases and the gene rearrangement patterns were analyzed with a T cell receptor beta-chain probe. Southern blot analysis of the DNA of these T cell clones indicated that all had rearrangements of the TcR beta-chain genes, but none of the rearrangements were identical. These results suggest that, if a few clones of specific T cells are involved, they must form a tiny minority in comparison to the total number of T cells in the CSF of MS patients.

The acute effects of pneumonectomy on pulmonary vascular impedance in the dog.

Pulmonary vascular impedance is a measure of the pulsatile characteristic of pressure and flow that occurs in the proximal pulmonary arteries. Pulmonary vascular resistance (PVR) is most influenced by the distal circulation of the lung. This study was performed to evaluate the changes that occurred in pulmonary vascular impedance, as well as in other hemodynamic variables, following pneumonectomy by a closed-chest method in 10 anesthetized dogs. The following observations were made (numbers compare mean values for the 10 dogs before and after pneumonectomy): (1) PVR increased from 447 to 761 dyne sec cm-5 (p = .02); (2) the oscillatory work of the right ventricle increased from 1.23 to 1.76 J/min (p = .006); (3) the mean pulmonary artery pressure increased from 14 to 18.8 mm Hg (p = .0001); and (4) cardiac output and heart rate remained unchanged. Surprisingly, the estimated characteristic impedance (the impedance to oscillatory flow in the proximal bed) did not change significantly (279 to 296 dyne sec cm-5). This observation cannot be explained by the usual lumped compartmental models classically used to characterize the pulmonary vascular bed.