Stephen B. Jones

Regulation of procollagen metabolism in the pressure-overloaded rat heart.

To determine the molecular events responsible for the disproportionate accumulation of myocardial fibrillar collagens during sustained hypertension, we examined the in vivo rate of procollagen synthesis, collagen accumulation, and intracellular procollagen degradation 1-16 wk after abdominal aortic banding in young rats. These measurements were correlated with tissue mRNA levels for type I and type III procollagen polypeptides. Banded animals developed moderate, sustained hypertension and mild left ventricular hypertrophy. Increased type III procollagen mRNA levels were detected early after banding and persisted for the entire observation period. Disproportionate collagen accumulation without histological evidence of fibrosis was noted within 1 wk after hypertension induction. Fibrillar collagen accumulation at this time point resulted not from a major increase in procollagen synthesis, but rather a marked decrease in the rate of intracellular procollagen degradation. Interstitial fibrosis, however, was observed 16 wk after banding. Type I procollagen mRNA levels were increased six-fold, but only after 16 wk of hypertension. These results correlated well with the results of in vivo procollagen synthesis experiments at 16 wk, which demonstrated a threefold increase in left ventricular procollagen biosynthesis. We conclude that pretranslational as well as posttranslational mechanisms regulate fibrillar collagen deposition in the myocardial extracellular matrix during sustained hypertension.

Immunologic Responses to Critical Injury and Sepsis

Almost 2 million patients are admitted to hospitals in the United States each year for treatment of traumatic injuries, and these patients are at increased risk of late infections and complications of systemic inflammation as a result of injury. Host response to injury involves a general activation of multiple systems in defending the organism from hemorrhagic or infectious death. Clinicians have the capability to support the critically injured through their traumatic insult with surgery and improved critical care, but the inflammatory response generated by such injuries creates new challenges in the management of these patients. It has long been known that local tissue injury induces systemic changes in the traumatized patient that are often maladaptive. This article reviews the effects of injury on the function of immune system cells and highlights some of the clinical sequelae of this deranged inflammatory-immune interaction.

Local epicardial chemical ablation of vagal input to sino-atrial and atrioventricular regions of the canine heart

In open-chest, pentobarbitalized dogs, right and left cervical vagi were electrically stimulated (20 Hz, 5.0 ms, 4-6 volts) before and after carefully painting phenol (90%) over each of 6-8 narrow strips (2-3 mm width) and over restricted portions of the superior and inferior right atrium. Successive phenol strips were applied until the sino-atrial nodal (SAN) region had been completely surrounded, and also applied over a triangular fat pad at the junction of the inferior vena cava (IVC) and inferior left atrium (ILA). Electrical excitation of the autonomic trunks following each sequential epicardial phenol blockade resulted in successive deletion of cardio-inhibitory responses, a majority of sympathetic excitatory responses remaining intact. We conclude that most vagal fibers reach the SAN region via the superior-posterior right atrium (SVC and superior pulmonary veins) and these can be ablated leaving most vagal fibers to the atrioventricular nodal AVN region unimpaired. Phenol painting at the junction of IVC and ILA abolished vagal inhibition of conduction across the A-V junction. These studies illustrate distinct vagal distributions to the SAN and AVN regions of the canine heart. After all responses to vagal stimulation have been abolished, sympathetic alterations in heart rate and A-V conduction remain, thus revealing important differentiation in sympathetic fiber distribution to these key regions of automaticity and conduction.

Norepinephrine modulates myelopoiesis after experimental thermal injury with sepsis.

ObjectiveTo determine whether thermal injury and sepsis cause an increase in bone marrow norepinephrine release and whether such a release influences bone marrow monocytopoiesis. Summary Background DataThe authors previously demonstrated enhanced bone marrow monocytopoiesis after burn with sepsis. They also showed that physiologic stress and bacterial challenge without injury could lead to a dynamic release of norepinephrine from the bone marrow compartment. In this study, they sought to determine the potential cause-and-effect relationship of bone marrow norepinephrine release on increased monocytopoiesis after burn sepsis. MethodsNorepinephrine release from bone marrow was determined by traditional pulse-chase methods. Tissue and bone marrow norepinephrine content was ablated by chemical sympathectomy with 6-hydroxydopamine treatment. Clonogenic potential in response to colony-stimulating factors was determined in total nucleated bone marrow cells. Dual color flow cytometry was used to document the distribution pattern of monocyte progenitors. ResultsBurn sepsis induced increased norepinephrine release in bone marrow, spleen, and heart. Colony-forming assays demonstrated an increase in responsive colonies, which was significantly attenuated when norepinephrine content was reduced in animals before burn sepsis. Flow cytometric analysis of early and late monocyte progenitors showed a significantly altered distribution profile of monocyte progenitors in norepinephrine-depleted mice compared with norepinephrine-intact mice. Abrogation of bone marrow norepinephrine content resulted in a 62% survival rate in burn septic mice compared with no survivors in norepinephrine-intact mice. ConclusionsThese data suggest that enhanced bone marrow norepinephrine release after burn sepsis may play a role in bone marrow monocytopoiesis, thus contributing to the sustenance of inflammation.

Burn injury and pulmonary sepsis: development of a clinically relevant model.

BACKGROUND Despite improvements in the early resuscitation of the critically injured, mortality from multiple organ failure has remained stable, with the lung often the first organ to fail. Early intubation and mechanical ventilation predispose patients to the development of pneumonia and respiratory failure. Our objective was to establish a murine model of combined injury, consisting of burn/trauma and pulmonary sepsis with reproducible end-organ responses and mortality. METHODS Male B6D2F1 mice were divided into four groups: burn/infection (BI), burn (B), infection (I), and sham (S). Burned animals had a full-thickness 15% dorsal scald burn. BI and I groups were inoculated intratracheally with Pseudomonas aeruginosa (3-5 x 103 colony-forming units). S and B animals received saline intratracheally. All animals were resuscitated with 2 mL of intraperitoneal saline. Mortality was recorded at 24, 48, and 72 hours. Bacterial sepsis was confirmed by tissue Grams stain of the lungs and positive organ and blood cultures for Pseudomonas aeruginosa. Femoral bone marrow cells were collected at 72 hours from surviving animals. Clonogenic potential was assessed by response to macrophage (M) colony-stimulating factor (CSF) and granulocyte-macrophage (GM) CSF in a soft agar assay and the data were represented as colonies per femur. Isolated alveolar macrophages and whole lung tissue were assayed for levels of the inflammatory cytokines tumor necrosis factor-alpha and interleukin-6. RESULTS Mortality at 72 hours was 30% in BI, 12% in I, and <10% in B and S groups. Pneumonia was documented in all infected animals at 24 hours by Grams stain and positive tissue cultures for Pseudomonas aeruginosa. Systemic sepsis as confirmed by blood, and remote organ cultures was seen in BI animals only. Significantly increased responsiveness to M-CSF stimulations was noted in all groups (BI, 8,291 +/- 1,402 colonies/femur; B, 6,357 +/- 806 colonies/femur; and I, 8,054 +/- 1,112 colonies/femur; p < 0.05) relative to sham (3,369 +/- 883 colonies/femur, p < 0.05). Maximal responsiveness to GM-CSF stimulation was noted in the BI group (11,932 +/- 982 colonies/femur, p < 0.05), and similar GM responsiveness was noted in all other groups (B, 7,135 +/- 548 colonies/femur; I, 7,023 +/- 810 colonies/femur; and S, 6,829 +/- 1,439 colonies/femur). Alveolar macrophage release of the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-6 increased in all animals, but the magnitude of increase was not proportional to the strength of the inciting stimulus. CONCLUSION Although minimal perturbations were seen after burn or pulmonary infection alone, the combined insult of burn and pulmonary sepsis resulted in statistically significant hematopoietic changes with increased monocytopoiesis. Only the combined injury resulted in systemic sepsis and significantly increased mortality. We have developed a clinically relevant model of trauma and pulmonary sepsis that will allow further clarification of the inflammatory response after injury and infection.

Bone Marrow Norepinephrine Mediates Development of Functionally Different Macrophages After Thermal Injury and Sepsis

Objective:We sought to determine the influence of thermal (burn) injury with sepsis and norepinephrine on the clonogenic potential and functional cytokine response to lipopolysaccharide (LPS) stimulation in nonmyeloid committed (CD117+) and myeloid committed (ER-MP12+) bone marrow progenitor cells. Summary and Background Data:We have previously demonstrated that norepinephrine stimulated myelopoiesis after burn injury and sepsis, but the site of this stimulation in monocyte development is unknown. In the present study the influence of norepinephrine on the developmental hierarchy of bone marrow cells after thermal injury and sepsis was determined by assessing the clonogenic potential and LPS-stimulated cytokine responses of mature macrophages derived from CD117+ and ER-MP12+ bone marrow progenitor cells. Methods:Tissue and bone marrow norepinephrine content was ablated by chemical sympathectomy with 6-hydroxydopamine treatment. CD117+ and ER-MP12+ bone marrow cells were isolated using antibody-linked magnetic microbeads. Clonogenic potential in response to colony-stimulating factors was determined. Both progenitor cell types were differentiated to mature macrophages in vitro and tumor necrosis factor (TNF)-α and interleukin (IL)-6 cytokine responses to LPS provocation were determined. Results:The macrophage- and granulocyte-macrophage colony-stimulating factor responsive clonogenic potential was increased with burn sepsis, suggesting an expansion of both progenitor populations. Such increases were greatly reduced with prior depletion of norepinephrine. TNF-α and IL-6 cytokine responses to LPS were markedly influenced by the specific progenitor cells involved as well as the injury conditions and the status of norepinephrine prior to injury. In burn sepsis the depletion of norepinephrine resulted in a dramatic decrease in both IL-6 and TNF-α production by both progenitor-derived macrophages. Conclusions:Depletion of norepinephrine attenuated burn and burn sepsis-induced bone marrow progenitor clonal growth in response to macrophage- and granulocyte-macrophage colony-stimulating factor. Functional phenotypes of bone marrow progenitor-derived macrophages are greatly influenced by norepinephrine and the milieu created by thermal injury and sepsis.

Arterial baroreflexes are not essential in mediating sympathoadrenal activation in conscious endotoxic rats

We examined the contributions of arterial baroreceptor reflexes in mediating sympathoadrenal activation during endotoxicosis. Conscious rats with chronic sinoaortic denervation (SAD) or sham-operation (SHAM) were subject to endotoxin treatment (5 mg/kg, i.v.). Hemodynamic responses, renal sympathetic nerve activity (RSNA) and plasma catecholamines were assessed at different times post endotoxin infusion. In both SAD and sham groups, intravenous endotoxin injection induced a rapid and significant sympathoadrenal activation, as indicated by a parallel elevation of RSNA and plasma catecholamines. Such activation peaked 15-30 min following endotoxin and was sustained throughout the 2-3 h protocol. The early response of the sympathoadrenal system to endotoxin is more profound in SAD rats compared to sham rats. We propose that the afferent neural input from arterial baroreceptors is not essential in mediating sympathoadrenal activation during sepsis. The elimination of feedback buffering mechanisms with SAD may account for the augmented sympathetic response seen in SAD animals.

Functional characteristics and responses to adrenergic stimulation of isolated heart preparations from hypothermic and hibernating subjects

Contemporary studies of isolated hearts from hibernators and nonhibernators are presented. Original experiments with isolated perfused hamster hearts are reported. Such hearts can maintain left ventricular function at temperatures as low as 7 degrees C. Generated left ventricular pressure was 40 +/- 9 mm Hg and heart rate was 7 +/- 1 beats/min. During cooling heart rate dropped dramatically, coronary flow increased, and ventricular pressure decreased initially, plateaued, and then fell as 7 degrees C was approached. Norepinephrine can cause increased heart rate and left ventricular pressure at 22 and 7 degrees C. This positive inotropic and chronotropic response was associated with increased cAMP at 30 sec after stimulation at 22 degrees C but not at 7 degrees C. Furthermore, cAMP was also not changed at peak response at 7 degrees C. Isoproterenol increased cAMP content in 37 degrees C ventricular slices but not at hypothermic temperatures. Possible mechanisms of nonadenylate cyclase mediation of inotropic and chronotropic responses at 7 degrees C are discussed.

Human immune cells mediate catecholamine secretion from adrenal chromaffin cells

OBJECTIVES To determine the ability of human mononuclear cells to produce factors that cause catecholamine secretion from adrenomedullary chromaffin cells; to determine conditions that stimulate mononuclear cells to produce such factors; and to compare these results with catecholamine secretion in response to the cytokines interleukin (IL)-1 and IL-2. DESIGN Randomized, controlled, prospective study using in vitro conditions. SETTING University research laboratory. SUBJECTS Human mononuclear cells and porcine chromaffin cells. INTERVENTIONS Circulating human mononuclear cells were isolated and cultured overnight in RPMI media. Cell-free media from these cultures (conditioned media) were then tested for the ability to cause epinephrine secretion from porcine chromaffin cells. Mononuclear cells were stimulated with phytohemagglutinin or by mixing cells from two different individuals while suppression was tested with dexamethasone. Catecholamine secretion in response to IL-1 and IL-2 (50 and 500 units/well, respectively), or nicotinic agonist dimethylphenylpiperazinium (10 microM, which mimics the action of acetylcholine), was tested for comparison. MEASUREMENTS AND MAIN RESULTS Isolated porcine chromaffin cells had stable catecholamine content at the time of secretion measurements, and catecholamine release from cells into the media was measured using electrochemical detection after high-performance liquid chromatography separation. Catecholamine secretion was expressed as a percentage of the total cellular content. Epinephrine secretion due to human conditioned media was 6.9 +/- 1.0% compared with 1.4 +/- 0.6% for control media (p < .05) and 14.6 +/- 3.3% for dimethylphenylpiperazinium (p < .05). Epinephrine secretion with conditioned media from mixed cells (mixed leukocyte reaction) was 16.6 +/- 1.2%, which was higher than the epinephrine secretion caused by media from a single donor (6.9% +/- 1.0, p < .001). Pretreatment with dexamethasone inhibited the formation of bioactive products from mixed mononuclear cell preparations. Cytokines IL-1 and IL-2 did not stimulate chromaffin cell epinephrine secretion above background release with control media incubation. In all cases, norepinephrine secretion was similar to that of epinephrine, and results are included in all figures. CONCLUSIONS Factors released from human immune cells can mediate epinephrine and norepinephrine release from adrenomedullary cells through a nonneural mechanism. Such immune cell factor release can be modulated by immunostimulation and steroid suppression. Release of such factors in vivo may contribute to increased circulating epinephrine in response to infectious challenge and may be an important factor in the critically ill patient.

Overdrive suppression of atrial pacemaker tissues in the alert, awake dog before and chronically after excision of the sinoatrial node☆

Abstract In characterizing pacemakers of the canine right atrium, cycle lengths after rapid atrial pacing were measured in the alert, conscious dog. Using implanted bipolar electrodes on the atrial appendage, pacing was established at a rate 100 percent above the spontaneous heart rate and was continued for 30, 60, 120 or 180 seconds. The average control cycle length subtracted from the first recovery cycle after cessation of pacing was termed the corrected recovery time. To study comparative responses of the Sinoatrial (S-A) node and subsidiary atrial pacemakers, corrected recovery time was measured before and at intervals after excision of the S-A node. Junctlonal regions frequently (but not invariably) assumed control instantaneously after sinus nodal excision, with subsidiary atrial pacemaker dominance occurring within a few minutes to hours; such control was relatively unstable for a few days. Subsidiary atrial pacemaker dominance gradually stabilized with development of consistent P waves and P-R intervals of 80 to 100 ms. Corrected recovery time after 1 minute of rapid atrial pacing averaged 267 ms before excision of the S-A node, 3,500 to 4,500 ms immediately after and for the 1st week after excision, but progressively decreased toward control levels during the 1st to 20th week thereafter. Thus, corrected recovery time first increased greatly, then regularly decreased with time after excision of the S-A node, but remained high for several weeks in subsidiary atrial pacemakers as compared with control dominance by S-A pacemaker tissues. However, the ultimate assumption of comparable corrected recovery time by the subsidiary pacemakers implies important alterations in electrophysiologic characteristics. One minute of fast pacing elicited longer periods of overdrive suppression than did 30 seconds of pacing, but these periods were not systematically further increased by 2 or 3 minutes of pacing. Although atropine greatly attenuated overdrive suppression in the awake dog model, propranolol altered it little or not at all during quiet rest, with modest exaggeration during alert attention.