Reflections on tissue engineering and trauma therapy.

Although all of Trauma Care has remarkably improved during the latter half of the last century the treatment of burn injury has substantially out paced other areas and serves as an excellent example illustrating the improvements taking place in the treatment of injured patients. Although Trauma treatment lagged behind the rest of medicine in mid-century today it rests solidly on the cutting edge of advancing therapeutic knowledge and practice in areas of metabolism, immunology, infection control, critical care, tissue engineering and the delivery of clinical care. It is important to understand what has happened to allow the treatment of injury to be so effective. The bench mark therapeutic moves that have had major effects allowing the present highly effective treatment are: immediate tailored fluid resuscitation, preventive (prophylactic) and topical antibiotics, metabolically designed nutritional therapy and most important early definitive repair of the injury. All must be delivered early after trauma if they are to be optimally effective in preventing the complications of injury that are devastating if encountered. Unfortunately, all problems are not solved by todays treatment of injury, improved as it is. These problems are largely related to an inexact understanding: of the physiologic changes of aging, of the exact pathophysiologic events in inhalation injury and multisystem organ failure and of the technology required to replace those body parts damaged by the injury itself that lead to death or to healing with loss of function. There will be major improvements in the understanding and ability to effectively deal with the problems of aging and inhalation injury through basic and clinical research but perhaps the major improvement in injury treatment will come through the ability to replace worn out, defective or damaged body parts through technologies that resemble regeneration. Here the concepts of Tissue Engineering have much to contribute and it is worth exploring the donation of Tissue Engineering to dermal replacement following burn injury to serve as an example of what types of additions to treatment Tissue Engineering can make.

Artificial skin.

The skin is a complex organ that is difficult to replace when it is irreversibly damaged by burns, trauma, or disease. Although autologous skin transplantation remains the most common form of treatment in patients with significant skin loss, there are now a number of commercially available products that can be used to replace the skin temporarily or permanently. Here we describe several such products under the rubric "artificial skin," focusing on two types of technology that have been applied to the problem of permanent skin replacement.

A novel human astrocyte cell line (A735) with astrocyte-specific neurotransmitter function.

Studies of brain cell function and physiology are hampered by the limited availability of immortal human brain-derived cell lines, as a result of the technical difficulties encountered in establishing immortal human cells in culture. In this study, we demonstrate the application of recombinant DNA vectors expressing SV40 T antigen for the development of immortal human cell cultures, with morphological, growth, and functional properties of astrocytes. Primary human astrocytes were transfected with the SV40 T antigen expression vectors, pSV3neo or p735.6, and cultures were established with an extended lifespan. One of these cultures gave rise to an immortal cell line, designated A735. All the human SV40-derived lines retained morphological features and growth properties of type 1 astrocytes. Immunohistochemical studies and Western blot analysis of the intermediate filament proteins and glutamine synthetase demonstrated a differentiated but immature astrocyte phenotype. Transport of gamma-amino butyric acid and glutamate were examined and found to be by a glial-specific mechanism, consistent with the cell lines' retaining aspects of normal glial function. We conclude that methods based on the use of SV40 T antigen can successfully immortalize human astrocytes, retaining key astrocyte functions, but T antigen-induced proliferation appeared to interfere with expression of glial fibrillary acidic protein. We believe A735 is the first documented nontumor-derived human glial cell line which is immortal.

Application of a rapid method (targeted display) for the identification of differentially expressed mRNAs following NGF-induced neuronal differentiation in PC12 cells.

Nerve growth factor (NGF)-induced differentiation of the rat pheochromocytoma, PC12, cell line presents a model system for the study of early gene expression changes involved in neuronal differentiation. Rapid alterations in mRNA expression patterns were investigated in PC12 cells following exposure to NGF using a set of statistically designed primers that exhibit coding-strand bias, and the products were analyzed on agarose gels. This simple and rapid method (targeted display) generated reproducible expression profiles, indicating a complex pattern of gene regulation, and resulted in the identification of a number of NGF-regulated transcripts. Thirty-two of these were selected at random and sequenced, revealing 19 known and 13 novel genes (or ESTs). Northern blot analysis and RT-PCR confirmed the differential regulation of 22 genes (16 known, 6 novel) and demonstrated 1 false positive result. Antisense application of one isolated gene product, the serine/threonine kinase MARK1, prevented neuronal differentiation in transiently transfected PC12 cells.

Current thinking on chronic renal allograft rejection: issues, concerns, and recommendations from a 1997 roundtable discussion.

Chronic rejection accounts for most renal allograft losses after the first year posttransplantation. On March 24 and 25, 1997, a roundtable of five transplant surgeons, two nephrologists, and one pathologist assembled in Dallas, Texas, to review critical issues surrounding chronic renal allograft rejection. This article summarizes the presentations and relevant discussions of this meeting regarding the cause of chronic rejection, clinical diagnoses, risk factors, future prospects for intervention strategies, and general recommendations for the transplant community. Growing evidence indicates that chronic rejection is the aggregate sum of irreversible immunologic and nonimmunologic injuries to the renal graft over time. A history of acute rejection episodes and inadequate immunosuppression, likely attributable to inconsistent cyclosporine exposure or poor patient compliance, are among the most recognizable immunologic risk factors for chronic rejection. Donor organ quality, delayed graft function, and other donor and recipient variables leading to reduced nephron mass are nonimmunologic factors that contribute to the progressive deterioration of renal graft function. Clinical management of renal transplant recipients should incorporate both immunologic- and nonimmunologic-based intervention strategies aimed at minimizing risk factors to thwart the progression of chronic rejection and improve long-term allograft and patient survival.

Molecular cloning and characterization of an invertebrate homologue of a neuropeptide Y receptor.

Neuropeptide Y is an abundant and physiologically important peptide in vertebrates having effects on food intake, sexual behaviour, blood pressure and circadian rhythms. Neuropeptide Y homologues have been found in invertebrates, where they are very likely to play similar, important roles. Although five neuropeptide Y-receptor subtypes have been identified in mammals, none has been reported from invertebrates. Here we describe the cloning of a neuropeptide Y-receptor from the brain of the snail Lymnaea stagnalis. The identity of the receptor was deduced by expressing the neuropeptide Y-receptor-encoding cDNA in Chinese Hamster Ovary cells, which were subsequently challenged with size-fractionated Lymnaea brain extracts. An active peptide, selected on the basis of its ability to induce changes in cAMP levels, was purified to homogeneity, analysed by mass spectrometry and amino acid sequence determination, and turned out to be a Lymnaea homologue of neuropeptide Y.

Both C3a and C3a(desArg) regulate interleukin-6 synthesis in human peripheral blood mononuclear cells.

Synthesis of complement components is part of the acute-phase response. Interleukin-6 (IL-6) is a critical mediator of the acute-phase response during infections and injuries. Plasma levels of C3a and IL-6 have been proposed as prognostic indicators in sepsis and trauma. The effects of C3a and C3a(des)Arg on IL-6 gene expression and protein production in human peripheral blood mononuclear cells (PBMC) were investigated. Neither C3a nor C3a(des)Arg alone induced detectable IL-6 protein or mRNA levels. However, C3a and C3a(des)Arg affected endotoxin-induced IL-6 synthesis in a dose-dependent manner. In nonadherent PBMC, C3a or C3a(des)Arg suppressed, while in adherent PBMC, C3a or C3a(des)Arg enhanced IL-6 protein and mRNA levels. These results suggest that C3a and C3a(des)Arg may provide a control mechanism of acute-phase responses by enhancing IL-6 synthesis in adherent monocytes at local inflammatory sites and by inhibiting IL-6 synthesis in circulating monocytes.

A 6-month study of low-dose recombinant human erythropoietin alone and in combination with androgens for the treatment of anemia in chronic hemodialysis patients.

Two previous short-term studies (12 weeks and up to 16 weeks) that used androgens to supplement recombinant human erythropoietin (rHuEPO) for the treatment of the anemia associated with end-stage renal disease showed divergent results. Both studies were limited by their brief duration, since the hematopoietic effect of androgens does not peak until 5 months. Therefore, we conducted a 6-month, prospective, randomized trial comparing low-dose rHuEPO alone and in combination with androgens for the treatment of the anemia of end-stage renal failure. Nineteen anemic chronic hemodialysis patients were randomized into two groups. Group A (n = 10) received 1,500 U rHuEPO intravenously three times a week for 26 weeks. Group B (n = 9) received the same dose of rHuEPO plus nandrolone decanoate 100 mg intramuscularly weekly. Baseline transferrin saturation, serum ferritin, intact serum parathyroid hormone, plasma aluminum, and hematocrit levels were not significantly different between the groups. At study completion, both groups showed a significant increase in mean hematocrit compared with baseline (group A: 24.8% +/- 1.4% to 28.3% +/- 2.8%, P = 0.003; group B: 25.1% +/- 1.5% to 33.2% +/- 4.5%, P = 0.001). The increase in hematocrit in the rHuEPO plus androgen-treated group was statistically greater than in the rHuEPO-alone group (8.2% +/- 4.4% v 3.5% +/- 2.8%; P = 0.012). With the exception of mild discomfort at the injection site, there were no significant side effects from nandrolone. We conclude that the combination of low-dose rHuEPO and nandrolone decanoate is effective treatment for the anemia of end-stage renal failure.

Coexistence of atheroemboli and minimal-change disease.

We report the first case of coexistent cholesterol emboli and minimal-change disease in an elderly individual with acute renal failure and the nephrotic syndrome. Renal insufficiency heavy proteinuria improved rapidly with oral steroids.

Interaction with autologous platelets multiplies interleukin-1 and tumor necrosis factor production in mononuclear cells.

The effect of activated platelets on cytokine production by human peripheral blood mononuclear cells (PBMC) was investigated. When PBMC were coincubated with activated autologous platelets amid lipopolysaccharide (LPS, 50-100 pg/mL) for 8 h, the production of interleukin (IL)-1alpha increased 11- to 18-fold and tumor necrosis factor (TNF)-alpha 3- to 5-fold compared with PBMC without platelets. Activated platelets in a dual-chamber well that prevented platelet-PBMC contact but permitted passage of soluble factors enhanced IL-1alpha production (P < .01). Platelet-PBMC contact in the chamber resulted in a further enhancement of IL-1alpha production. These data suggest that platelet-PBMC interaction, both directly and with platelet-derived factors, enhances production of shock-producing IL-1alpha and TNF-alpha, albeit differently. The interaction of platelets with monocytes may play an important role in the pathophysiology of sepsis and disseminated intravascular coagulation.

A new biologic role for C3a and C3a desArg: regulation of TNF-alpha and IL-1 beta synthesis.

The complement activation products C3a and C3a desArg are generated in the course of trauma, infection, tissue injury, and ischemia. We have investigated the effects of C3a and C3a desArg on gene expression and protein synthesis of TNF-alpha and IL-1 beta in PBMC. Neither C3a nor C3a desArg alone induced detectable protein or mRNA levels for TNF-alpha and IL-1 beta. C3a modulated LPS-induced TNF-alpha and IL-1 beta synthesis. In nonadherent PBMC, C3a suppressed LPS-induced synthesis of TNF-alpha (20-71% decrease by 0.2-10 microgram/ml of C3a, p less than 0.01) and IL-1 beta (19-57% decrease by 0.5-10 microgram/ml of C3a, p less than 0.01), independently of endogenous production of PGE2. C3a also suppressed LPS-induced mRNA levels for TNF-alpha and IL-1 beta. In contrast, in adherent PBMC, C3a at 5 to 20 microgram/ml enhanced LPS-induced TNF-alpha (75-188% increase, p less than 0.001) and IL-1 beta (119-274% increase, p less than 0.001) synthesis. C3a enhanced TNF-alpha and IL-1 beta mRNA levels in LPS-stimulated adherent cells. Furthermore, C3a desArg shared with C3a the ability to modulate LPS-induced mRNA and protein synthesis for TNF-alpha and IL-1 beta. These results suggest that C3a, thought to be proinflammatory, and C3a desArg, thought to be biologically inactive, are modulators of inflammation. Both C3a and C3a desArg may enhance cytokine synthesis by adherent monocytes at local inflammatory sites, while inhibiting the systemic synthesis of proinflammatory cytokines by circulating cells.

Measurement of muscle protein synthesis by positron emission tomography with L-[methyl-11C]methionine.

Positron emission tomography (PET) with L-[methyl-11C]methionine was explored as an in vivo, noninvasive, quantitative method for measuring the protein synthesis rate (PSR) in paraspinal and hind limb muscles of anesthetized dogs. Approximately 25 mCi (1 Ci = 37 GBq) of L-[methyl-11C]methionine was injected intravenously, and serial images and arterial blood samples were acquired over 90 min. Data analysis was performed by fitting tissue- and metabolite-corrected arterial blood time-activity curves to a three-compartment model and assuming insignificant transamination and transmethylation in this tissue. PSR was calculated from fitted parameter values and plasma methionine concentrations. PSRs measured by PET were compared with arterio-venous (A-V) difference measurements across the hind limb during primed constant infusion (5-6 h) of L-[1-13C, methyl-2H3]methionine. Results of PET measurements demonstrated similar PSRs for paraspinal and hind limb muscles: 0.172 +/- 0.062 vs. 0.208 +/- 0.048 nmol-1.min-1.(g of muscle)-1 (P = not significant). PSR determined by the stable isotope technique was 0.27 +/- 0.050 nmol-1.min-1.(g of leg tissue)-1 (P < 0.07 from PET) and indicated that the contribution of transmethylation to total hind limb methionine utilization was approximately 10%. High levels of L-[methyl-11C]methionine utilization by bone marrow were observed. We conclude that muscle PSR can be measured in vivo by PET and that this approach offers promise for application in human metabolic studies.

Expression and characterization of molluscan insulin-related peptide VII from the mollusc Lymnaea stagnalis.

A complementary DNA clone encoding molluscan insulin-related peptide VII was identified from a complementary DNA library of the cerebral ganglia of the CNS of the freshwater snail, Lymnaea stagnalis. The novel molluscan insulin-related peptide VII complementary DNA encodes a preprohormone resembling the organization of preproinsulin, with a putative signal sequence, and an A and B chain, and is connected by an unusual long C peptide. The A and B chains, as well as the C peptide of molluscan insulin-related peptide VII, differ remarkably in primary structure with the previously identified molluscan insulin-related peptides. The C peptide of molluscan insulin-related peptide VII shares no significant sequence identity with counterparts in other molluscan insulin-related peptides. Both molluscan insulin-related peptide VII and the other molluscan insulin-related peptides exhibit structural features which make them a unique class of the insulin superfamily. Molluscan insulin-related peptide VII complementary DNA was shown to hybridize in situ with messenger RNA present in the cerebral light green cells, neuroendocrine cells that control growth and that have previously been shown to produce molluscan insulin-related peptides I-III and V. Uniquely, the molluscan insulin-related peptide VII gene is also expressed in neurons that may form part of the feeding circuitry in Lymnaea, indicating that it may function as a neurotransmitter/neuromodulator.

Whole body arginine metabolism and nitric oxide synthesis in newborns with persistent pulmonary hypertension.

Despite the potential relevance of the L-arginine-nitric oxide (NO) pathway in the pathophysiology of pulmonary hypertension, no in vivo studies of the kinetics of arginine and NO have been conducted previously in this population. The terminal guanidino N-atom of L-arginine is the precursor for NO, which is oxidized to the stable inorganic nitrogen oxides, nitrite (NO2-) and nitrate (NO3-). Thus, synthesized NO is detected in serum or urine as NO2- and NO3-. The purpose of this investigation was to compare studies of whole body arginine metabolism twice in nine patients with persistent pulmonary hypertension of the newborn (PPHN), using a primed constant i.v. infusion of L-[guanidino-15N2,5,5(2)H2]arginine and L-[5,5,5(2)H3]leucine, first during acute pulmonary vasoconstriction and again during convalescence, and thereby to characterize quantitative aspects of whole body arginine kinetics and NO production, as estimated from the rate of transfer of the 15N-guanidino-label of arginine to urinary nitrate (15NO3-). Arginine flux rates were 84.1 +/- 8.6 mumol.kg-1 h-1 (mean +/- SEM) during acute pulmonary hypertension and increased to 125 +/- 13.2 (p < 0.05) during convalescence, whereas leucine fluxes were unchanged (168.5 +/- 15 versus 178.8 +/- 10.2 mumol.kg-1 h-1), and comparable to those reported in healthy newborns. During convalescence total urinary nitrate excreted increased by 66% (p < 0.05), urinary 15NO3- increased from 0.29 +/- 0.07 to 0.74 +/- 0.15 mumol.d-1 (p < 0.05), and the rate of plasma arginine conversion to NO increased from 10.3 +/- 2.2 to 45.6 +/- 13 mumol.d-1 (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

The acutely burned hand: management and outcome based on a ten-year experience with 1047 acute hand burns.

Optimal hand function has a very positive impact on the quality of survival after burn injury. Over a 10-year period, 659 patients with 1047 acutely burned hands were managed at the Sumner Redstone Burn Center of the Massachusetts General Hospital. Our approach to acutely burned hands emphasizes ranging and splinting throughout hospitalization, prompt sheet autograft wound closure as soon as practical, and the selective use of axial pin fixation and flaps. This approach is associated with normal function in 97% of those with superficial injuries and 81% of those with deep dermal and full-thickness injuries requiring surgery. Although only 9% of those with injuries involving the extensor mechanism, joint capsule, or bone had normal functional outcomes, 90% were able to independently perform activities of daily living.

Cell cycle progression, morphology and contact inhibition are regulated by the amount of SV40 T antigen in immortal human cells.

Expression of Simian Virus 40 (SV40) T antigen in human dermal fibroblasts over-rides the normal controls on cell division leading to changes in cellular proliferation and life span. These changes are accompanied by other changes in cell morphology, expression of cell specific functions, and altered cell-cell interactions. In this study, we have examined the effects of different amounts of T antigen on cell cycle progression, life span and morphology in human dermal fibroblasts and demonstrated T antigen to be a concentration dependent regulator of the cell cycle. Using a novel, metal inducible episomal expression vector (p735.6) which produces low basal levels of protein but high (greater than 100-fold) levels of induction, we have compared the effects of low and high levels of T antigen expression in a precrisis and immortalised human line (1BRMT1). The presence of inducing agent led to maximal levels of T antigen expression and resulted in cultures with a high rate of proliferation, an extended in vitro life span, a loss of contact inhibition of growth and a morphology characteristic of SV40-transformed cells. In the absence of inducing agent, read-through of the T antigen gene resulted in low but detectable levels of protein. The reduction in T antigen levels was accompanied by a 50% or greater reduction in the proliferative rate and restoration of cell morphology and contact inhibition similar to that found in non-transfected cells. The results presented here demonstrate that low amounts of T antigen are sufficient to maintain cell viability and prevent the re-expression of the senescent phenotype seen in the absence of T antigen. Similarly, the ability of T antigen to extend the in vitro life span is not dependent on high level expression of T antigen. In contrast, the rate of proliferation of human cells as well as the cell morphology and contact inhibition are dependent on the amount of T antigen present. Many of the cellular effects can be minimised or reversed by reducing T antigen expression.

Altered interleukin-1 and tumor necrosis factor production and secretion during pyrogenic tolerance to LPS in rabbits.

Rabbits were injected intravenously with 10 micrograms/kg of endotoxin [lipopolysaccharide (LPS)] on days 0, 1, and 7, and rectal temperatures were monitored. The febrile responses were compared with circulating levels of interleukin-1 beta (IL-1 beta) and tumor necrosis factor (TNF) and in vitro synthesis of these cytokines by peripheral blood mononuclear cells (PBMC) isolated just before the injection of LPS. Fever after the first LPS injection was biphasic on day 0, attenuated and monophasic after the second LPS injection on day 1, and augmented after third injection of LPS on day 7. On day 1, circulating TNF and IL-1 beta levels were significantly (P < 0.05) decreased compared with those on days 0 and 7. Similarly, TNF and IL-1 beta synthesis by LPS-stimulated PBMC were significantly reduced on day 1. On day 7, cellular synthesis and secretion of IL-1 beta were significantly increased compared with that on day 0. A significant positive correlation was observed between fever index and total in vitro IL-1 beta synthesis by LPS-stimulated PBMC (r = 0.866, P = 0.001). These data demonstrate that pyrogenic tolerance in the rabbit after a single LPS injection is associated with decreased circulating IL-1 beta and TNF levels as well as decreased production of these cytokines in vitro. In addition, the pyrogenic hyperresponsiveness to LPS after 7 days is associated with increased synthesis and secretion of IL-1 beta from PBMC in vitro.

Thermal injury induces very early production of interleukin-1 alpha in the rat by mechanisms other than endotoxemia.

BACKGROUND: Cytokines are putative mediators of thermal injury-induced systemic changes. We studied the effects of thermal injury on cytokine activation in vivo with a sensitive radioimmunoassay specific for rat interleukin-1 alpha (IL-1 alpha). METHODS: We characterized the organ distribution and expression kinetics of IL-1 alpha in rats submitted to either 20% total body surface area cutaneous burn, muscle burn, or endotoxic shock. Rats were killed at various time points, and liver, lung, spleen, ileum, thymus, kidney, skin, and plasma were harvested. Tissues were homogenized, and the supernates were assayed for rat IL-1 alpha. The assay detection limit was 1.5 ng/gm wet tissue (WT). RESULTS: Thermal injury induced marked elevations of IL-1 alpha levels in the liver and lung, and maximal levels were reached at 2.5 hours when compared with controls. In the liver mean IL-1 alpha levels in cutaneous burn injury were 16.5 +/- 6.2 ng/gm WT, whereas in sham injury they were 1.7 +/- 0.1 ng/gm WT, p < or = 0.05; in the lung IL-1 alpha levels with cutaneous burn injury were 10.3 +/- 1.3 ng/gm WT, whereas sham injury levels were 1.9 +/- 0.8 ng/gm WT, p < or = 0.002). Levels in all other organs and plasma were below detection limits. Muscle burn injury had similar elevated levels of IL-1 alpha in the liver at 1 hour, indistinguishable from cutaneous burn. In contrast, endotoxin challenge resulted in dramatic elevation of IL-1 alpha levels in all organs tested except for the kidney, whereas the skin maintained its usual large amounts of IL-1 alpha. CONCLUSIONS: These data indicate that thermal or mechanical injury induce very early and organ-specific association of IL-1 alpha in vivo by mechanisms other than endotoxemia.