Biomedical subjects

G O Till

Publications and source records attributed to G O Till.

At least 37 records · Page 2Linked to original sources

PubMed1996

Requirement and role of C5a in acute lung inflammatory injury in rats.

The complement activation product, C5a, may play a key role in the acute inflammatory response. Polyclonal antibody to rat C5a was used to define the requirements for C5a in neutrophil-dependent inflammatory lung injury after systemic activation of complement by cobra venom factor (CVF) or after intrapulmonary deposition of IgG immune complexes. In the CVF model, intravenous infusion (but not intratracheal instillation) of anti-C5a produced a dose-dependent reduction in lung permeability and in lung content of myeloperoxidase. In C6-deficient rats, CVF infusion caused the same level of lung injury (measured by leak of 125I-albumin) as found in C6-sufficient rats. In the IgG immune complex model of lung injury, anti-C5a administered intratracheally (but not intravenously) reduced in a dose-dependent manner both the increase in lung vascular permeability as well as the buildup of lung myeloperoxidase. Treatment with anti-C5a greatly suppressed upregulation of lung vascular intercellular adhesion molecule-1 (ICAM-1). This was correlated with a substantial drop in levels of TNFalpha in bronchoalveolar fluids. These data demonstrate the requirement for C5a in the two models of injury. In the IgG immune complex model, C5a is required for the full production of TNFalpha and the corresponding upregulation of lung vascular ICAM-1.

Animals↗

PubMed1995

The role of complement in experimental bullous pemphigoid.

Bullous pemphigoid (BP) is a blistering skin disease associated with an IgG autoimmune response directed against the ectodomain of the hemidesmosomal protein, BP180. An animal model of BP has recently been developed by our laboratory based on the passive transfer of rabbit antimurine BP180 antibodies into neonatal BALB/c mice. The experimental animals develop a blistering disease that reproduces all of the key immunopathological features of BP. In the present study we have investigated the role of complement in the pathogenesis of subepidermal blistering in the mouse model of BP. We demonstrate the following. (a) Rabbit anti-murine-BP180 IgG was effective in inducing cutaneous blisters in a C5-sufficient mouse strain, but failed to induce disease in the syngeneic C5-deficient strain; (b) neonatal BALB/c mice, pretreated with cobra venom factor to deplete complement, became resistant to the pathogenic effects of the anti-BP180 IgG; (c) F(ab')2 fragments generated from the anti-BP180 IgG exhibited no pathogenic activity in the mouse model; and (d) histologic evaluation of the skin of mice described in points b and c above showed minimal or no neutrophilic cell infiltration in the upper dermis. Thus, anti-BP180 antibodies trigger subepidermal blistering in this BP model via complement activation. This experimental model of BP should greatly facilitate future studies on the pathophysiology of autoantibody-mediated diseases of the dermal-epidermal junction.

Animals↗

PubMed1994

Phorbol-stimulated influx of extracellular calcium in rat pulmonary artery endothelial cells.

Stimulation of rat pulmonary artery endothelial cells (RPAEC) with phorbol 12-myristate 13-acetate (PMA) resulted in an increase in intracellular calcium ([Ca2+]i). Unlike the response to bradykinin, C5a and tumor necrosis factor-alpha (TNF-alpha) previously reported (15), the PMA-induced increase in [Ca2+]i was predominantly dependent on extracellular calcium. The PMA response paralleled the BAY K 8644-induced, extracellular calcium-dependent increase in [Ca2+]i. Pretreatment of endothelial cells with the protein kinase C inhibitor staurosporine resulted in a concentration-dependent inhibition of the increase in [Ca2+]i in response to PMA. The ability of PMA analogues to induce significant increase in [Ca2+]i paralleled their ability to induce O2- generation in neutrophils. The PMA-induced influx of extracellular Ca2+ was inhibited by the L-channel selective antagonists diltiazem, nifedipine, nicardipine, and verapamil in a dose-dependent manner. Depolarizing conditions induced by high [K+]o enhanced the calcium response to PMA. The data presented are consistent with the hypothesis that PMA-induced increases in [Ca2+]i in endothelial cells are the result of Ca2+ influx through voltage-dependent L-type Ca2+ channels.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1994

Role of selectins in local and remote tissue injury following ischemia and reperfusion.

Ischemia (4-hour) followed by reperfusion (4-hour) of rat hind limbs results in local injury as well as remote (lung) injury. It has recently been shown that injury in this model is neutrophil- and cytokine-dependent and requires the beta 2 integrin adhesion molecules CD11a/CD18 and CD11b/CD18. The role of selectins in events leading to injury (as determined by leakage of albumin and by hemorrhage) was assessed either through the use of blocking antibodies to L-, E- or P-selectins or by the use of oligosaccharides that are reactive with selectins. Lung injury was found to be L- and E-selectin-dependent. When the ischemia and reperfusion times were reduced, lung injury was also found to be P-selectin dependent. In the case of hind limb injury involving the crural muscle mass, injury was L-selectin-dependent but independent of requirements for P- and E-selectin. Injury in both organs was blocked by the infusion of sialylated Lewis pentasaccharide, whereas sialyl-N-acetyllactosamine pentasaccharide failed to protect against injury. In general, when selectin-blocking approaches were protective, there were parallel reductions in tissue content of myeloperoxidase. These data underscore the role of selectins in ischemia-reperfusion injury and suggest that selectin requirements may vary with the vascular bed under study.

Animals↗

PubMed1994

Role of leukocyte adhesion molecules in lung and dermal vascular injury after thermal trauma of skin.

Acute second degree thermal injury of rat skin involving 25 to 30% total body surface of anesthetized rats results at 4 hours in evidence of vascular injury both locally (in skin) and remotely (involving lung). The neutrophil dependency for both types of injury has now been established. Monoclonal antibodies to various adhesion molecules have been used to define the requirements for these molecules in the development of vascular injury. In dermal vascular injury, a requirement for Mac-1 (CD11b/CD18) but not for leukocyte function-associated antigen-1 (LFA-1, CD11a/CD18) has been established. In this model requirements have also been demonstrated for intercellular adhesion molecule-1 (ICAM-1) and E- and L-selectin but not for very late arising antigen-4 (VLA-4) or P-selectin. With respect to lung vascular injury, dual requirements for both leukocyte function-associated antigen-1 and Mac-1 were found as well as for ICAM-1 and E- and L-selectin but not for VLA-4 and P-selectin. In the lung, there was a close correlation between neutrophil content of the tissue (as assessed by myeloperoxidase) and the effects of protective interventions (directed against blocking of adhesion molecules). These data emphasize the roles of beta 2 integrins, selectins (L and E), and ICAM-1 in events that lead to neutrophil-mediated vascular injury of dermis and lung after thermal trauma to skin.

Animals↗

PubMed1994

Acute skin injury releases neutrophil chemoattractants.

BACKGROUND: Progressive or ongoing skin necrosis after traumatic injury is well known. Experimental evidence has associated these events with neutrophil activation and secondary oxidant injury. To determine the mechanism by which neutrophils migrate to a site of injury, cytokine release from injured skin was measured. METHODS: Twenty-five skin biopsy specimens of acute partial thickness skin injuries were compared with uninjured skin of the same patient. Conditioned medium from explanted skin was assayed for tumor necrosis factor (TNF), interleukin-6 (IL-6), and IL-8. RESULTS: Acute skin injury resulted in a significant release of IL-8 but not IL-6 or TNF. In eight patients gradient cytokine release was found; IL-8 levels for partial thickness burn were 26.4 ng/ml, for unburned skin adjacent to the burn were 2.1 ng/ml, and for distal normal skin were 0.2 ng/ml. CONCLUSIONS: IL-8 is released from acutely injured skin; IL-6 and TNF are not. This selective release suggests a mechanism whereby neutrophils are recruited into injured tissue. These neutrophils might then induce further injury, increasing the extent of posttraumatic tissue loss.

Acute Disease↗

PubMed1993

Evidence that NADPH-dependent methemoglobin reductase and administered riboflavin protect tissues from oxidative injury.

NADPH-dependent methemoglobin reductase, first detected in erythrocytes sixty years ago, has subsequently been purified and characterized as a methylene blue reductase and a flavin reductase. The reductase plays no role in methemoglobin reduction under normal conditions, but its activity serves as the basis for the treatment of methemoglobinemia with methylene blue or flavin. On-going studies demonstrate that this cytosolic protein is also present in liver and that its primary structure distinguishes it from other known proteins. The bovine erythrocyte reductase tightly binds hemes, porphyrins, and fatty acids with resulting loss of activity. Pyrroloquinoline quinone serves as a high-affinity substrate of the reductase, suggesting that this naturally-occurring compound may be a physiological substrate. The ability of the reductase to catalyze the intracellular reduction of administered riboflavin to dihydroriboflavin suggested that this system might be exploited to protect tissues from oxidative damage. This hypothesis was supported by our finding that dihydroriboflavin reacts rapidly with Fe(IV)O and Fe(V)O oxidation states of hemeproteins, states that have been implicated in tissue damage associated with ischemia and reperfusion. Preliminary studies demonstrate that, as predicted, administration of low concentrations of riboflavin protects isolated rabbit heart from reoxygenation injury, rat lung from injury resulting from systemic activation of complement, and rat brain from damage caused by four hours of ischemia. Data from these animal studies suggest that flavin therapy holds promise in protecting tissue from the oxidative injuries of myocardial infarction, acute lung injury, stroke, and a number of other clinical conditions.

Animals↗

PubMed1993

Correlation of the local and systemic cytokine response with clinical outcome following thermal injury.

Eighty-eight patients with acute thermal injury were evaluated. Forty-eight hours after injury, TNF, IL-6, and IL-8 were significantly present in the systemic circulation, lung, normal skin, and thermally injured skin. The presence of TNF, IL-6, and IL-8 proteins in the lung, normal skin, and thermally injured skin were associated with TNF, IL-6, and IL-8 mRNA upregulation. Logistic regression analysis controlling for the Abbreviated Burn Severity Index demonstrated that the presence of IL-8 in the lung was associated with early pulmonary physiologic dysfunction (p = 0.006) and nosocomial pulmonary infection (p = 0.040). We conclude that acute thermal injury initiates an early systemic, lung, and skin response involving TNF, IL-6, and IL-8. The TNF, IL-6, and IL-8 protein present in the lung and skin in response to acute thermal injury are generated locally and do not originate from the systemic cytokine pool. The lung cytokine response to acute thermal injury may initiate local organ failure.

Adult↗

PubMed1993

Requirements for tumor necrosis factor-alpha and interleukin-1 in limb ischemia/reperfusion injury and associated lung injury.

Ischemia in rat hind limbs followed by reperfusion results in local as well as remote organ (lung) injury characterized by increased vascular permeability (125I-labeled bovine serum albumin leakage) and hemorrhage (51Cr-labeled rat erythrocytes extravasation) in skeletal muscle and lung, together with an associated increased tissue content of myeloperoxidase, reflecting neutrophil accumulation. Within 60 minutes of reperfusion following ischemia, tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), and IL-6 plasma levels increased significantly, reaching maximum levels after 2 hours of reperfusion. Polyclonal antibodies to TNF-alpha and IL-1 provided significant protection against vascular injury in both muscle and lung. These results were confirmed by the use of soluble TNF-alpha receptor and IL-1 receptor antagonist. In rat lungs following ischemia and reperfusion, there was immunohistochemical evidence of E-selectin expression in the lung vasculature; this expression was blocked by treatment of animals with anti-TNF-alpha. These data indicate that both local (hind limb) and remote (lung) organ injury after ischemia/reperfusion requires participation of TNF-alpha and IL-1. The cytokines may, in part, be involved in the up-regulation of endothelial adhesion molecules.

Animals↗

PubMed1993

Role of beta 2 integrins and ICAM-1 in lung injury following ischemia-reperfusion of rat hind limbs.

Ischemia/reperfusion involving the hind limbs of rats results in both local injury to skeletal muscle as well as injury to lungs, as measured by increased vascular permeability (125I-labeled bovine serum albumin leakage) and hemorrhage (extravasation of 51Cr-labeled rat erythrocytes). In the current study, we have focused on events in lungs occurring during reperfusion of hind limbs. Analysis of blood neutrophils obtained 4 hours after reperfusion has indicated up-regulation of CD11b and CD18 but not CD11a. Plasma from the same animals demonstrate the ability to induce similar effects in normal blood neutrophils, indicative of the presence of a neutrophil-activating agent in plasma. During reperfusion, lung injury, which develops progressively over a 4-hour period, has been shown to be neutrophil-dependent and requires CD11a/CD18 and CD11b/CD18 as well as intercellular adhesion molecule-1. These data suggest that ischemia and reperfusion injury of rat lower extremities causes systemic changes that result in neutrophil-dependent lung injury that is beta 2 integrin- (leukocyte function antigen-1, Mac-1) and intercellular adhesion molecule-1-dependent.

Animals↗

PubMed1993

Identification of xanthine oxidase activity following reperfusion in human tissue.

In this series of experiments, we surveyed xanthine oxidase activity after microvascular transfer in the venous effluent after reperfusion of human rectus abdominis muscle (n = 8) and jejunum (n = 4). Enzyme activity was correlated with duration of ischemia and biochemical markers of cellular injury. Xanthine oxidase (XO) activity was measured spectrofluorometrically using a pterin assay, whereas cellular injury was measured with commercial creatinine phosphokinase activity assay and lipid peroxidation products using a spectrophotometer. The data demonstrated that XO activity was statistically significantly increased in muscle flaps kept at room temperature during ischemia compared with muscle flaps that were cooled (p < 0.05). Creatinine phosphokinase activity was also increased after 15 minutes of reperfusion in muscle flaps that were not cooled (p < 0.05). Two of the jejunal free flaps had ischemia times of > 1 hour and had elevated XO activity after reperfusion despite cooling (p < 0.05). Two other jejunal flaps had ischemia times of < 1 hour, but in one case, the XO activity was increased before harvest. The other case had no increase in XO activity.

Abdominal Muscles↗

PubMed1993

Requirements for neutrophil products and L-arginine in ischemia-reperfusion injury.

Ischemia followed by reperfusion in rat limb results in evidence of vascular injury in the limb as well as in the lung as measured by leakage of [125I]albumin and extravasation of [51Cr] red blood cells. Vascular injury in lung and limb was proportional to the time of limb reperfusion and was associated with accumulation of myeloperoxidase, as well as evidence of complement consumption. In this model, the rank order of protective interventions was: neutrophil depletion > catalase + superoxide dismutase = allopurinol > dimethylthiourea = dimethylsulfoxide > deferoxamine = complement depletion. These data suggest that toxic oxygen products of neutrophils are related to the development of vascular injury. There was a reasonable correlation between protective effects of interventions and reduced tissue content of myeloperoxidase. Systemic treatment with the L-arginine antagonists, NG-monomethyl-L-arginine or nitro-L-arginine methyl ester, was also protective against vascular injury, suggesting that metabolic products of L-arginine participate in events leading to injury.

Animals↗

PubMed1992

Roles of beta 2 integrins of rat neutrophils in complement- and oxygen radical-mediated acute inflammatory injury.

The roles of beta 2 integrin molecules in neutrophil accumulation and tissue injury have been examined by the use of antibodies that are reactive with human CD11b and CD18 and cross-react with the homologous epitopes on rat neutrophils. Adherence to rat pulmonary artery endothelial cells by human neutrophils and endothelial cell killing by phorbol ester-activated human neutrophils required CD11b, CD11c, and CD18. Companion adherence studies between rat neutrophils and endothelial cells revealed a requirement for both CD11b and CD18. Neither anti-CD11b nor anti-CD18 depressed in vitro responses (O2- generation and chemotactic migration) of rat neutrophils. The accumulation of neutrophils in glycogen-induced peritoneal exudates was diminished substantially in rats treated with either anti-CD18 or anti-CD11b. In oxidant-mediated acute lung injury induced by rapid intravascular infusion of cobra venom factor, treatment of rats with either anti-CD18 or anti-CD11b significantly attenuated injury as assessed by increases in vascular permeability and hemorrhage. These protective effects correlated morphologically with diminished adhesion of neutrophils to interstitial intrapulmonary capillary endothelial cells. In studies of immune complex (BSA-anti-BSA)-induced alveolitis and dermal vasculitis, anti-CD18 had protective effects at all doses of anti-BSA employed. The protective effects of anti-CD18 correlated with diminished neutrophil accumulation in tissues at lower doses of anti-BSA. Although anti-CD11b was not effective under the same experimental conditions, intratracheal administration of this antibody conveyed protection against immune complex-induced lung injury, suggesting that both CD11b and CD18 are required for the full expression of injury. The current studies also demonstrated that when surface-bound IgG immune complexes were treated with fresh rat serum, the increment in O2- and TNF alpha generated by alveolar macrophages was suppressed by anti-CD18, but not by anti-CD11b, suggesting a heretofore unrecognized role for CD18 in the O2- and TNF-alpha responses of alveolar macrophages. Thus, neutrophil beta 2 integrins play a requisite role for the full expression of complement-dependent and oxygen radical-mediated injury of the lung and dermal vasculature.

Animals↗

PubMed1992

Superoxide responses of endothelial cells to C5a and TNF-alpha: divergent signal transduction pathways.

There is increasing evidence that endothelial cells respond to a variety of mediators. In the current studies rat pulmonary artery endothelial cells (RPAEC) responded to human recombinant C5a and tumor necrosis factor-alpha (TNF-alpha) with the generation of superoxide (O2-). RPAEC responsiveness was dependent on whether cells had been obtained from confluent or subconfluent cell monolayers. RPAEC responded to C5a and TNF-alpha in a dose-dependent manner, with increases in intracellular Ca2+ (Cai2+), formation of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], and generation of O2-. Optimal O2- responses occurred in cells that had been pretreated with the inhibitor of superoxide dismutase (SOD), diethyldithiocarbamate, and O2- responses were allopurinol insensitive. Pertussis toxin pretreatment abolished the ability of C5a to cause increases in Ins(1,4,5)P3 and Cai2+ and formation of O2- but did not inhibit the changes in Cai2+ and formation of O2- after addition of TNF-alpha. The O2- response to C5a but not to TNF-alpha was abolished by pretreatment with the inhibitor of protein kinase C, staurosporine. These data indicate that signal transduction events in response to C5a and TNF-alpha were fundamentally different.

Alkaloids↗

PubMed1992

Adhesion molecules in experimental phacoanaphylactic endophthalmitis.

Intraocular accumulation of inflammatory neutrophils is an important feature of experimental phacoanaphylactic endophthalmitis (EPE). Increasing evidence suggests that localization of neutrophils to the site of inflammation requires the participation of neutrophil and endothelial adhesion molecules. These studies were undertaken to determine if blocking of adhesion molecules on neutrophils (CD18) or endothelium (ELAM-1) could attenuate EPE in Lewis rats. Treatment of experimental animals with anti-CD18 or anti-ELAM-1 significantly suppressed intraocular neutrophil accumulation, retinal hemorrhage, and vasculitis, and attenuated retinal edema formation by 48% and 70%, respectively. These observations demonstrate that antibodies directed against adhesion molecules on the neutrophil (CD18) or the vascular endothelial cell (ELAM-1) exhibit potent anti-inflammatory effects, resulting in a striking amelioration of injury in EPE in rats.

Animals↗

PubMed1992

Xanthine oxidase: its role in the no-reflow phenomenon.

This study was designed to probe the hypothesis that oxygen-derived free radicals are involved in initiation of the no-reflow phenomenon. We developed a reproducible model of no reflow in the rat hind limb. Laser Doppler studies confirmed that the hind limbs perfused well after 2 or 4 hours of ischemia, but perfusion ceased in the first 10 minutes after 6 hours of ischemia. Venous blood samples and biopsy specimens of skin and muscle were taken after 2 and 4 hours of ischemia to study tissue injury. Blood samples were evaluated for xanthine oxidase (XO), xanthine dehydrogenase, and creatine phosphokinase (CPK) activities. Conjugated dienes and iodine 125-labeled albumin extravasation were quantified in tissue samples. Groups of animals were treated with inhibitors of XO (allopurinol), antioxidant enzymes (superoxide dismutase plus catalase), and free radical scavengers (dimethyl sulfoxide and dimethyl thiourea) to assess the roles of free radicals in ischemia-reperfusion injury in the hind limbs. After 4 hours of ischemia followed by reperfusion, plasma XO activity rose threefold over preischemia levels (p less than 0.05). Xanthine dehydrogenase activity did not change; conjugated diene levels in muscle rose twofold; CPK levels rose sixfold, and 125I albumin extravasation rose twofold (p less than 0.05). Pretreatment with the XO inhibitor allopurinol reduced XO activity to negligible levels and significantly attenuated conjugated diene levels, CPK levels, and albumin extravasation. Albumin extravasation was also significantly attenuated by pretreating animals with superoxide dismutase together with catalase, dimethyl thiourea, and dimethyl sulfoxide. In all animals pretreated with allopurinol or superoxide dismutase and catalase, reperfusion persisted after 6 hours of ischemia. These data suggest that, in ischemia followed by reperfusion, tissue injury is related to oxygen products derived from XO activity.

Allopurinol↗

PubMed1991

Role of oxygen radicals in tourniquet-related ischemia-reperfusion injury of human patients.

In the current study we evaluated effluent blood from extremities of human patients undergoing reconstructive surgical treatment which is routinely accompanied by upper extremity exsanguination and application of a tourniquet. Following tourniquet release (reperfusion), there were immediate increases in the plasma levels of xanthine oxidase activity, uric acid, and histamine. Xanthine dehydrogenase activity was not detectable. Plasma also contained products consistent with the formation of oxygen-derived free radicals, namely hemoglobin and fluorescent compounds. Our data indicate in humans that ischemia-reperfusion events are associated with the appearance of xanthine oxidase activity and its products in the plasma effluent.

Adult↗

PubMed1991

Modulation of IgM antibody formation by lipid peroxidation products from burn plasma.

Lipid peroxides (conjugated dienes) have been shown to increase in the plasma of thermally injured animals and in humans. The purpose of this study was to determine if conjugated diene-rich fractions extracted from the plasma of burned rats 3 hours after injury would alter humoral immune function in unburned animals. Plasma diene extracts from burned or normal rats were dissolved in ethanol and given intraperitoneally to Long-Evans rats. Fifteen minutes after diene injection, the rats were immunized with sheep erythrocytes. Five days later, serum and spleen cells obtained from these immunized rats were tested for IgM immunity against sheep erythrocytes. Both plaque-forming cells from spleen and hemagglutination titers in serum were significantly enhanced in the group treated with dienes obtained from the thermally injured rats. These data suggest that conjugated diene-bearing lipid fractions from burn plasma may modulate immune function and support the concept that humoral immunity is enhanced after thermal injury.

Animals↗