Application of gene therapy to acute inflammatory diseases.

The application of gene therapy to acute inflammation has not received as much research attention as has the treatment of genetically-based diseases, cancer, and viral infections. However, gene therapy as a drug delivery system offers several theoretical and practical advantages over current protein delivery systems. These include the ability to target therapies to individual tissues or cell types, to locally produce proteins that can act intracellularly or in an autocrine, juxtacrine, or paracrine fashion, and to sustain new protein synthesis for periods up to several weeks after a single administration. Although retrovirus, herpes simplex, and adeno-associated virus have been proposed for gene therapy in cancer and in genetic diseases, nonviral and adenovirus approaches appear most applicable as drug delivery systems due to their rapid onset and short duration of transgene expression. The relative modest transduction efficiencies obtained at present with nonviral approaches, and the inherent inflammatory properties of first-generation adenovirus constructs, however, have limited their usefulness to date. The present review discusses the theoretical and practical benefits of specific gene therapy approaches for the treatment of acute inflammatory diseases, as well as our experiences with liposome:plasmid DNA and adenovirus-based approaches. Although a number of technical and theoretical hurdles remain before it can be evaluated in humans with acute inflammation, gene therapy offers a novel approach for the treatment of acute inflammation, and will likely enter the armamentarium of critical care physicians in the near future.

Angiopeptin, the octapeptide analogue of somatostatin, decreases rat heart endothelial cell adhesiveness for mononuclear cells.

The effect of angiopeptin, a stable analogue of somatostatin, was studied on basal and interleukin-1-beta-induced endothelial cell adhesiveness for mononuclear cells, and compared to the effect of somatostatin. Angiopeptin and somatostatin decreased basal and interleukin-1-beta-induced endothelial cell adhesiveness for mononuclear cells. The decreased mononuclear cells adhesion to endothelial cells exposed to angiopeptin and somatostatin is not due to modulation of the expression of intrecellular adhesion molecule-1 because neither angiopeptin nor somatostatin decreased basal and interleukin-1-beta-induced expression of this adhesion molecule. The effect of angiopeptin in inhibiting endothelial cell adhesiveness for mononuclear cells was abolished by addition of dibutyryl-cyclic AMP. Angiopeptin induced a transient decrease in basal and interleukin-1-beta-induced cyclic AMP levels in endothelial cells. Exposure of unstimulated and interleukin-1-beta-activated endothelial cells to KT5720, a specific inhibitor of cyclic AMP-dependent protein kinase, decreased endothelial cell adhesiveness for mononuclear cells. Thus, angiopeptin most likely diminishes endothelial adhesiveness for mononuclear cells by affecting the cyclic AMP-dependent protein kinase signal transduction pathway. The findings suggest that angiopeptin and somatostatin may modify the development of the immune response by attenuating endothelial cell adhesiveness for mononuclear cells. Angiopeptin may have a potential clinical application as a modulator of some aspects of the immune response due to its long half-life and prolonged inhibitory effect on interleukin-1-beta induced endothelial adhesiveness for mononuclear cells.

Angiopeptin, a somatostatin-14 analogue, decreases adhesiveness of rat leukocytes to unstimulated and IL-1 beta-activated rat heart endothelial cells.

We have previously demonstrated that somatostatin-14 and its octapeptide analogue, angiopeptin, decrease the ability of rat heart endothelial cells to bind leukocytes [Leszczynski, et al., Reg. Pept. 43 (1993) 131-140]. Here, we examined whether exposure of leukocytes to angiopeptin modifies their adhesiveness to the unstimulated and to IL-1 beta-activated endothelium. Monolayers of unstimulated endothelial cells bind 274 +/- 12 leukocytes/mm2. Exposure of leukocytes for 1, 4 and 24 hours to angiopeptin (1 microM) reduced significantly (p < 0.05) adhesion of leukocytes from 274 +/- 12 to 188 +/- 10, 185 +/- 8 and 172 +/- 3 cells/mm2, respectively. Stimulation of endothelial cells with Il-1 beta (100 U/ml) for 24 hours increased endothelial adhesiveness from 274 +/- 12 to 381 +/- 17 adhering leukocytes/mm2. Exposure of leukocytes for 1, 4 and 24 hours to angiopeptin (1 microM) reduced significantly (p < 0.05) binding of leukocytes to IL-1 beta-activated endothelium from 381 +/- 17 to 237 +/- 8, 254 +/- 11 and 248 +/- 13 cells/mm2, respectively. Angiopeptin had no effect on the expression of lymphocyte function-associated molecule-1 (LFA-1; CD11a/CD18) by leukocytes, as assessed by flow cytometry. This suggests that angiopeptin modulates adhesive properties of leukocytes by (1) altering the expression of other than LFA-1 adhesion molecule(s) and/or (2) modulating the affinity of adhesion molecule(s) expressed by leukocytes. In conclusion, our results demonstrate that angiopeptin reduces leukocyte adhesiveness to unstimulated and to IL-1 beta-activated endothelium. It suggests that angiopeptin may suppress immune response via modulation of the leukocyte-endothelial interaction.

IL-1 beta-stimulated leucocyte-endothelial adhesion is regulated, in part, by the cyclic-GMP-dependent signal transduction pathway.

It is well known that the exposure of endothelial cells to IL-1 beta induces an increase in endothelial cell adhesiveness for leucocytes. Using rat heart endothelial cells we found that exposure of endothelial cells to IL-1 beta (100 U/ml) induces a 133-fold increase in the intracellular concentration of cyclic-GMP; from 11.5 +/- 0.2 fM to 1530 +/- 117.8 fM (per 10(6) cells). Therefore, we examined whether cyclic-GMP is involved in the regulation of endothelial adhesiveness for leucocytes. Cyclic-GMP analogue, dibutyryl cyclic-GMP Methylene blue, an inhibitor of guanylate cyclaese, and KT5823, a specific inhibitor of cyclic-GMP-dependent protein kinase, inhibited both basal as well as IL-1 beta-induced endothelial cell adhesiveness for leucocytes, and KT5823 abolished the dibutyryl-cyclic-GMP-induced increase in endothelial adhesiveness. The effect of cyclic-GMP, induced by IL-1 beta treatment, on the endothelial adhesiveness may be either direct or indirect because of the time-gap between the rise in cyclic-GMP level and the increase of endothelial adhesiveness. IL-1 beta (100 U/ml) and dibutyryl-cyclic-GMP (0.01 mM) both induced an increase in the expression of intercellular adhesion molecule-1 by endothelial cells. However, the fact that KT5823 failed to prevent this increase, suggests that, although the IL-1 beta-induced increase in adhesiveness is caused by the increase in intracellular levels of cyclic-GMP, it may not be mediated through intercellular adhesion molecule-1. In conclusion, the results obtained indicate that endothelial cell adhesiveness for leucocytes is, in part, regulated by the cyclic-GMP-dependent signal transduction pathway.

Sequence analysis identifies TTRAP, a protein that associates with CD40 and TNF receptor-associated factors, as a member of a superfamily of divalent cation-dependent phosphodiesterases.

CD40 is a member of the tumor necrosis factor (TNF) receptor family. CD40-mediated signal transduction involves the recruitment of several cytoplasmic proteins and induces expression of a large number of genes. TTRAP, a novel protein that interacts with the cytoplasmic domain of CD40 and with TNF-receptor associated factors (TRAFs), has been cloned and shown to inhibit nuclear factor-kappaB activation (NF-kappaB). By using various bioinformatics-based sequence and structure analyses of proteins involved in signaling by the TNF receptor family, we found that TTRAP is a member of a superfamily of Mg(2+)/Mn(2+)-dependent phosphodiesterases. More precisely, our results suggest that TTRAP is related to the human APE1, a Mg(2+)-dependent endonuclease. This potential novel function of TTRAP raises the intriguing possibility for a role of APE1-like DNA-repair endonucleases in TNF receptor family-mediated signaling and functions.

A neutral magnesium-dependent sphingomyelinase isoform associated with intracellular membranes and reversibly inhibited by reactive oxygen species.

Activation of neutral sphingomyelinase(s) and subsequent generation of ceramide has been implicated in a wide variety of cellular responses. Although this enzyme(s) has not been purified and cloned from higher organisms, one mammalian cDNA has been previously isolated based on its similarity to the bacterial enzyme. To further elucidate the function of this neutral sphingomyelinase, we studied its relationship with enzymes present in mammalian cells and tissues, its subcellular localization, and properties that could be important for the regulation of its activity. Using specific antibodies, it is suggested that the enzyme could represent one of several forms of neutral sphingomyelinases present in the extract from brain particulate fraction. In PC12 cells, the enzyme is localized in the endoplasmic reticulum and is not present in the plasma membrane. The same result has been obtained in several cell lines transfected or microinjected with plasmids encoding this enzyme. The molecular and enzymatic properties of the cloned neutral magnesium-dependent sphingomyelinase, produced using baculovirus or bacterial expression systems, have been analyzed, demonstrating the expected ion dependence and substrate specificity. The enzyme activity also has a strong requirement for reducing agents and is reversibly inhibited by reactive oxygen species and oxidized glutathione. The studies demonstrate that the cellular localization and some properties of this enzyme are distinct from properties previously associated with neutral magnesium-dependent sphingomyelinases in crude or partially purified preparations.

Products of cyclooxygenase-2 catalysis regulate postoperative bowel motility.

Laparotomy involving manipulation of the small intestine causes injury, initiating an inflammatory cascade in the small bowel wall, which generates eicosanoids and proinflammatory cytokines. We have shown that ketorolac and salsalate, nonselective cyclooxygenase (COX) inhibitors, ameliorate postoperative small bowel ileus in a rodent model. Others have shown that interleukin-1 receptor antagonism improves postoperative gastric emptying. We examined whether inhibition of the proinflammatory cytokines, tumor necrosis factor alpha (TNFalpha) and interleukin-1 (IL-1), or selective blockade of cyclooxygenase-2 (COX-2), the COX isoform induced during inflammation, would accelerate postoperative small bowel transit in our model. Duodenostomy tubes were inserted into male Sprague-Dawley rats. One week later, animals were randomized to receive TNF-binding protein (TNF-bp), IL-1 receptor antagonist (IL-1ra), or saline (NS) prior to standardized laparotomy. Additional rats were gavaged preoperatively with a selective COX-2 inhibitor (NS-398) or NS. Small intestinal transit was measured as the geometric center (GC) of distribution of (51)CrO(4) at 30 min, 3 h, or 6 h (n = 5-9 rats/group) following laparotomy. Selective inhibition of COX-2 significantly increased postoperative small bowel transit compared to controls (GC 2.9 +/- 0.3 vs 2.2 +/- 0.1 at 30 min, GC 2.9 +/- 0.3 vs 2.5 +/- 0.2 at 3 h, and GC 3.3 +/- 0.3 vs 2.8 +/- 0.2 at 6 h, P < 0.05). In contrast, neither TNF-bp nor IL-1ra altered postoperative small intestinal transit in this model. Use of selective COX-2 inhibitors may accelerate recovery of postoperative bowel dysmotility without the undesirable effects (e.g., gastrointestinal irritation and anti-platelet effect) of nonselective COX inhibitors.

Morphologic and biomechanical comparison of tendons used as free grafts.

The palmaris longus (n = 10), plantaris (n = 11), and extensor digitorum longus (n = 10) tendons were harvested from cadaver limbs for morphologic and biomechanical comparisons. Eleven flexor digitorum profundus tendons from the index finger were also harvested for comparison of biomechanical properties of the free graft tendons with those of a typical digital flexor. Maximal tendon length, cross-sectional area, volume, stiffness, and modulus of elasticity were determined by measurement, laser micrometry, and tensile testing. The plantaris and extensor digitorum longus yielded the longest grafts, 334 and 325 mm, respectively, compared with the palmaris longus, which yielded only 161 mm of length. Cross-sectional areas were as follows: palmaris longus, 3.1 mm2; plantaris, 1.4 mm2; extensor digitorum longus, 3.3 mm2; and flexor digitorum profundus, 10.6 mm2. Mean volumes were as follows: palmaris longus, 529 mm3; plantaris, 557 mm3; and extensor digitorum longus, 1006 mm3. The palmaris longus and the extensor digitorum longus showed greater stiffness, 42.0 and 47.8 N/mm, respectively, than the plantaris, which had a stiffness of 25.5 N/mm. However, the flexor digitorum profundus showed significantly greater stiffness than all of the graft tendons. The modulus of elasticity ranged from 1161.6 to 1673.0 MPa, with no significant difference between tendons tested. The findings in this study provide data that may be useful in the selection of a specific donor for free tendon grafting.

Hypotensive resuscitation using a polymerized bovine hemoglobin-based oxygen-carrying solution (HBOC-201) leads to reversal of anaerobic metabolism.

BACKGROUND: Traditional resuscitation regimens have been recently challenged. This study evaluates hypotensive resuscitation with a hemoglobin-based oxygen-carrying (HBOC) solution after severe hemorrhage in a porcine model. We hypothesized that HBOC-201 restores tissue perfusion at a lower mean arterial pressure than standard resuscitation fluids. METHODS: Yorkshire swine (55-65 kg, n = 30), were rapidly hemorrhaged to a mean arterial pressure (MAP) of 30 mm Hg, maintained hypotensive for 45 minutes, and randomized into groups. Group I was resuscitated with an HBOC solution to a MAP of 60 mm Hg. Groups II and III were resuscitated to a MAP of 80 mm Hg with lactated Ringer's solution (LR) alone or LR (40 mL/kg) followed by shed blood, respectively. Group IV was resuscitated with shed blood alone to a MAP of 60 mm Hg. Group V received an HBOC solution to a MAP of 50 mm Hg. Hemodynamic variables, Swan-Ganz parameters, blood gas samples, and lactate levels were followed for 5 hours. Data were analyzed by analysis of variance/Duncan multiple range test. RESULTS: There were no significant differences in mortality between any groups. Groups I, IV, and V had lower (p < 0.05) cardiac output, pulmonary artery wedge pressure, and MAP than either group II or group III. Svo2 was significantly lower in the HBOC groups. There were no significant differences in arterial pH or lactate between groups I, III, and IV. Lactate levels, base excess, and arterial pH were significantly worse in the LR-alone and HBOC-50 groups. CONCLUSION: Hypotensive resuscitation with HBOC-201 at a MAP of 60 mm Hg after a controlled hemorrhage in swine provides sufficient tissue perfusion and oxygen delivery to reverse anaerobic metabolism on the basis of global physiologic markers despite continued hypotension, hypovolemia, and low cardiac output.

Nitric oxide contributes to adriamycin's antitumor effect.

UNLABELLED: Recently, several antitumor drugs have been shown to stimulate nitric oxide (NO) production. PURPOSE: To determine if adriamycin induces NO production in breast cancer cells in vitro and whether NO contributes to adriamycin's antitumor effect in vivo. METHODS: Murine breast cancer cells (EMT-6) were incubated with adriamycin (ADRIA, 0, 10, 100, 1000 microM) in the presence or absence of the NO synthase inhibitor aminoguanidine (AG, 1 mM). Twenty-four hours later nitrite accumulation (Greiss reagent) and cell viability (MTT assay) were assessed. Supernatants from adriamycin-stimulated cells were also analyzed at 6, 8, and 24 hr for TNF, IL-1, and IFN gamma (ELISA). For in vivo experiments, 10(5) EMT-6 cells were injected into the flank of BALB/c mice (n = 20) and 1 hr later mice received one of four treatments: (1) saline, (2) ADRIA (10 mg/kg ip), (3) AG (100 mg/kg sc BID), or (4) ADRIA (10 mg/kg ip) and AG (100 mg/kg sc BID). Two weeks later tumor size was measured and in situ tumor cell apoptosis was determined by fluorescent microscopy and flow cytometry. RESULTS: Adriamycin was cytotoxic to EMT-6 cells with 100 microM resulting in nearly 100% killing (P < 0.01). Adriamycin also stimulated nitrite accumulation with 100 microM producing 6.5 +/- 0.26 microM nitrite (P < 0.001). AG blocked adriamycin-stimulated nitrite accumulation (P < 0.05), but did not inhibit cytotoxicity in vitro. In vivo, adriamycin inhibited tumor size by nearly 400% (P < 0.001), while AG attenuated adriamycin's effect on tumor growth (P < 0.05). There was no difference in the detection of apoptotic tumor cells between the adriamycin and adriamycin and AG groups as determined by immunohistochemistry and flow cytometry. CONCLUSIONS: These findings suggest that adriamycin stimulated NO production in EMT-6 cells, but adriamycin's cytotoxicity in vitro was NO-independent. In vivo, adriamycin inhibited tumorigenesis partially via an NO-dependent, nonapoptotic mechanism.

Structural requirements for catalysis and membrane targeting of mammalian enzymes with neutral sphingomyelinase and lysophospholipid phospholipase C activities. Analysis by chemical modification and site-directed mutagenesis.

The sequence similarity with bacterial neutral sphingomyelinase resulted in the isolation of putative mammalian counterparts and, subsequently, identification of similar molecules in a number of other eukaryotic organisms. Based on sequence similarities and previous characterization of the mammalian enzymes, we have chemically modified specific residues and performed site-directed mutagenesis in order to identify critical catalytic residues and determinants for membrane localization. Modification of histidine residues and the substrate protection experiments demonstrated the presence of reactive histidine residues within the active site. Site directed mutagenesis suggested an essential role in catalysis for two histidine residues (His-136 and His-272), which are conserved in all sequences. Mutations of two additional histidines (His-138 and His-151), conserved only in eukaryotes, resulted in reduced neutral sphingomyelinase activity. In addition to sphingomyelin, the enzyme also hydrolyzed lysophosphatidylcholine. Exposure to an oxidizing environment or modification of cysteine residues using several specific compounds also inactivated the enzyme. Site-directed mutagenesis of eight cysteine residues and gel-shift analysis demonstrated that these residues did not participate in the catalytic reaction and suggested the involvement of cysteines in the formation/breakage of disulfide bonds, which could underlie the reversible inactivation by the oxidizing compounds. Cellular localization studies of a series of deletion mutants, expressed as green fluorescent protein fusion proteins, demonstrated that the transmembrane region contains determinants for the endoplasmic reticulum localization.

Increased leptin expression in mice with bacterial peritonitis is partially regulated by tumor necrosis factor alpha.

Plasma leptin and ob gene mRNA levels were increased in mice following bacterial peritonitis, and blocking an endogenous tumor necrosis factor alpha (TNF-alpha) response blunted the increase. However, plasma leptin concentrations did not correlate with the associated anorexia. We conclude that leptin expression is under partial regulatory control of TNF-alpha in peritonitis, but the anorexia is not dependent on increased leptin production.

Transfection of the type II TGF-beta receptor into colon cancer cells increases receptor expression, inhibits cell growth, and reduces the malignant phenotype.

OBJECTIVE: To determine if transfection of SW48 colon cancer cells with the type II transforming growth factor-beta (TGF-beta) receptor restores growth inhibition and reverses the in vitro and in vivo malignant phenotype. SUMMARY BACKGROUND DATA: The authors have previously shown that SW48 colon cancer cells that are replication error positive in both alleles lack functional cell surface TGF-beta type I (RI) and type II (RII) receptors and are insensitive to TGF-beta1-induced growth inhibition. METHODS: SW48 cells were stably transfected with the cDNA for the normal type II TGF-beta receptor (RII). Once transfected, the cells were evaluated for in vitro phenotypic changes and in vivo changes in tumor growth. RESULTS: Denaturing sequencing gel electrophoresis of the reverse transcriptase-polymerase chain reaction product from SW48 cells revealed that the RII coding sequence contained a single base deletion mutation. When these cells were transfected with normal RII cDNA, Northern and Western blot analyses revealed increased levels of RII mRNA and protein. Affinity labeling techniques revealed that RII-transfected SW48 cells produced functional RI and RII protein. Transfection of SW48 cells also led to changes in cell phenotype, as shown by inhibition of both in vitro growth rate and incorporation of [3H]-thymidine. SW48 cells expressing normal RII also exhibited reduced cloning efficiency in semisolid medium and reduced growth as a xenograft in NOD/LtSz-scid/J mice. CONCLUSIONS: The results confirm that RII is a tumor-suppressor protein that is required for TGF-beta-induced growth inhibition in SW48 colon cancer cells.

Lipopolysaccharide and D-galactosamine-induced hepatic injury is mediated by TNF-alpha and not by Fas ligand.

Tumor necrosis factor (TNF)-alpha and Fas ligand (FasL) are trimeric proteins that induce apoptosis through similar caspase-dependent pathways. Hepatocytes are particularly sensitive to inflammation-induced programmed cell death, although the contribution of TNF-alpha and/or FasL to this injury response is still unclear. Here, we report that D-galactosamine and lipopolysaccharide-induced liver injury in C57BL/6 mice is associated with increased hepatic expression of both TNF-alpha and FasL mRNA. Pretreatment of mice with a TNF-binding protein improved survival, reduced plasma aspartate aminotransferase concentrations, and attenuated the apoptotic liver injury, as determined histologically and by in situ 3' OH end labeling of fragmented nuclear DNA. In contrast, pretreatment of mice with a murine-soluble Fas fusion protein (Fasfp) had only minimal effect on survival, and apoptotic liver injury was either unaffected or exacerbated depending on the dose of Fasfp employed. Similarly, mice with a spontaneous mutation in FasL (B6Smn.C3H-Fasl(gld) derived from C57BL/6) were equally sensitive to D-galactosamine/lipopolysaccharide-induced shock. We conclude that the shock and apoptotic liver injury after D-galactosamine/lipopolysaccharide treatment are due primarily to TNF-alpha release, whereas increased FasL expression appears to contribute little to the mortality and hepatic injury.

Modulation of the acute phase response by altered expression of the IL-1 type 1 receptor or IL-1ra.

A complete understanding of the role for endogenously produced interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), and IL-1 receptor antagonist (IL-1ra) in the acute phase response to inflammation remains unknown. In the present studies, knockout mice lacking either a functional IL-1 type I receptor (IL-1RI(-/-)), a TNF type I receptor (TNFR-I(-/-)), or both IL-1 type I and TNF type I receptors (IL-1RI(-/-)/TNFR-I(-/-)) received a turpentine abscess. Additional mice deficient in IL-1ra protein (IL-1ra(-/-)) or overexpressing IL-1ra protein (IL-1ra(tg)) were similarly treated. After a turpentine abscess, IL-1 receptor knockout mice exhibited an attenuated inflammatory response compared with wild-type or animals lacking a functional TNFR-I. Mice overexpressing IL-1ra also had an attenuated hepatic acute phase protein response, whereas IL-1ra knockout mice had a significantly greater hepatic acute phase response. We conclude that the inflammatory response to a turpentine abscess is the result of a balance between IL-1ra expression and IL-1 binding to its type I receptor. Endogenously produced IL-1ra plays a central role in mitigating the magnitude of the IL-1-mediated inflammatory response and, ultimately, the outcome to a turpentine abscess.

Disparate roles for TNF-alpha and Fas ligand in concanavalin A-induced hepatitis.

Apoptosis is a physiologic process that serves to eliminate cells during development or in response to immunologic regulation. In acute inflammation, however, apoptosis triggered by the overproduction of "death factors" such as TNF-alpha or Fas ligand (FasL) may contribute to tissue injury. Both TNF-alpha and FasL are presumed to convey an apoptotic signal by activating a cascade of cysteine-aspartate proteases, which includes IL-1beta-converting enzyme or caspase-1. In the present study, we evaluated the contribution of TNF-alpha and FasL, as well as the role of caspase-1, in Con A-induced hepatitis. We report here that TNF-alpha and FasL mRNA and protein levels are both increased in the livers of Con A-challenged mice. Using a novel inhibitor of TNF-alpha, we can confirm that Con A-induced hepatitis is primarily TNF-alpha dependent. Blockade of FasL with a soluble Fas immunoadhesin does not prevent liver injury in animals treated with Con A alone. However, administration of a matrix metalloproteinase inhibitor exacerbates liver injury, in part through a FasL-dependent process, since pretreatment with the soluble Fas immunoadhesin reduces liver injury in this model. In addition, mice lacking functional caspase-1 are resistant to Con A-induced hepatitis, even after pretreatment with a matrix metalloproteinase inhibitor. We conclude that TNF-alpha plays a predominant role in Con A-induced liver injury, although concomitant activation of FasL can also lead to apoptotic injury. Furthermore, Con A-induced hepatitis is caspase-1 dependent.