Paulina Sannomiya

Experimental Models Of Sepsis And Their Clinical Relevance

Sepsis remains a major cause of morbidity and mortality mainly because of sepsis-induced multiple organ dysfunction. In contrast to preclinical studies, most clinical trials of promising new treatment strategies for sepsis have failed to demonstrate efficacy. Although many reasons could account for this discrepancy, the misinterpretation of preclinical data obtained from experimental studies and especially the use of animal models that do not adequately mimic human sepsis may have been contributing factors. In this review, the potentials and limitations of various animal models of sepsis are discussed to clarify to which extent these findings are relevant to human sepsis. Such models include intravascular infusion of endotoxin or live bacteria, bacterial peritonitis, cecal ligation and perforation, soft tissue infection, pneumonia or meningitis models using different animal species including rats, mice, rabbits, dogs, pigs, sheep, and nonhuman primates. Despite several limitations, animal models remain essential in the development of all new therapies for sepsis and septic shock because they provide fundamental information about the pharmacokinetics, toxicity, and mechanism of drug action that cannot be replaced by other methods. New therapeutic agents should be studied in infection models, even after the initiation of the septic process. Furthermore, debility conditions need to be reproduced to avoid the exclusive use of healthy animals, which often do not represent the human septic patient.

Effect of fatty acids on leukocyte function

Fatty acids have various effects on immune and inflammatory responses, acting as intracellular and intercellular mediators. Polyunsaturated fatty acids (PUFAs) of the omega-3 family have overall suppressive effects, inhibiting lymphocyte proliferation, antibody and cytokine production, adhesion molecule expression, natural killer cell activity and triggering cell death. The omega-6 PUFAs have both inhibitory and stimulatory effects. The most studied of these is arachidonic acid that can be oxidized to eicosanoids, such as prostaglandins, leukotrienes and thromboxanes, all of which are potent mediators of inflammation. Nevertheless, it has been found that many of the effects of PUFA on immune and inflammatory responses are not dependent on eicosanoid generation. Fatty acids have also been found to modulate phagocytosis, reactive oxygen species production, cytokine production and leukocyte migration, also interfering with antigen presentation by macrophages. The importance of fatty acids in immune function has been corroborated by many clinical trials in which patients show improvement when submitted to fatty acid supplementation. Several mechanisms have been proposed to explain fatty acid modulation of immune response, such as changes in membrane fluidity and signal transduction pathways, regulation of gene transcription, protein acylation, and calcium release. In this review, evidence is presented to support the proposition that changes in cell metabolism also play an important role in the effect of fatty acids on leukocyte functioning, as fatty acids regulate glucose and glutamine metabolism and mitochondrial depolarization.

Secreted glucocorticoids regulate leukocyte-endothelial interactions in inflammation. A direct vital microscopic study

Leukocytes come into intimate contact with the venular endothelium as they extravasate from blood to the interstitium during inflammation. In exteriorized tissues, the number of leukocytes rolling along the vessel wall was markedly increased in adrenalectomized and metyrapone‐treated animals, relative to sham‐operated and normal animals. During the development of an acute, local inflammatory response, rollers were numerically decreased and a stronger adhesion of the cells to the endothelium, with a concomitant migration into tissues, was observed. Adhesion and migration were much more marked in adrenalectomized and metyrapone‐treated animals than in controls, suggesting that secreted glucocorticoids exert a suppressive effect on leukocyte‐endothelial interactions. The increased number of rolling leukocytes in adrenalectomized and metyrapone‐treated animals apparently resulted in more cells available to migrate into inflamed tissues. The effect appears to involve receptor occupancy and induction of gene expression because normal animals receiving the steroid antagonist RU 38 486, actinomycin D, or cydoheximide behaved as adrenalectomized or metyrapone‐treated animals. Administration to adrenalectomized animals of a monoclonal antibody to the membrane glycoprotein complex GD18 did not affect the number of rolling cells, but dramatically reduced the number of adherent or migrated leukocytes. It is suggested that secreted glucocorticoids, in addition to an effect on rolling behavior of circulating leukocytes, might also modulate the activity of the glycoprotein complex CD18 on white blood cells. The ultimate consequence is a restrictive effect on cell emigration in inflammation. J. Leukoc. Biol. 57: 379–386; 1995.

Nitric oxide modulates eosinophil infiltration in antigen-induced airway inflammation in rats.

The influence of nitric oxide (NO) on eosinophil infiltration into the airways was investigated in rats actively sensitized with ovalbumin. The animals were treated chronically with the NO synthase inhibitor, N omega-Nitro-L-arginine methyl ester (L-NAME; 75 mumol rat-1 day-1), for 4 weeks. Bronchoalveolar lavage was performed at 6, 24, 48 and 72 h after intratracheal injection of ovalbumin. Intratracheal challenge of the sensitized rats with ovalbumin caused a significant increase in total leucocyte infiltration in bronchoalveolar lavage fluid both 24 and 48 h post-ovalbumin injection. Neutrophils and eosinophils peaked, respectively, at 24 h (29%) and 48 h (30%) in bronchoalveolar lavage fluid whereas the mononuclear cell did not differ significantly from the counts in non-sensitized rats at any time. At both 6 and 24 h post-ovalbumin injection, the chronic treatment of the animals with L-NAME affected neither the total nor the differential leucocyte content. However, at 48 h post-ovalbumin challenge, the total cell count was reduced by approximately 48% in the L-NAME-treated animals and this was associated with a marked inhibition (81%) of the eosinophil influx. Histological examination of the lungs from these animals (48 h post-ovalbumin challenge) also showed a prominent reduction (69.5%; P < 0.05) of the eosinophil infiltration in the respiratory segments. Our results demonstrate that NO plays a pivotal role in the eosinophil infiltration in airways of actively sensitized rats.

Resuscitation Affects Microcirculatory Polymorphonuclear Leukocyte Behavior After Hemorrhagic Shock: Role of Hypertonic Saline and Pentoxifylline

We have previously shown that lung injury following fluid resuscitation either with hypertonic saline (HS) or lactated Ringers (LR) plus pentoxifylline (PTX) attenuated acute lung injury when compared with LR resuscitation. The objective of the present study is to determine whether our previous observations are accompanied by changes in polymorphonuclear leukocyte (PMN) behavior. To study this, PMN-endothelial cell interactions, microcirculatory blood flow, lung histology, lung PMN infiltration (WIPO, Myeloperoxidase), and lung intracellular adhesion molecule-1 (ICAM-1) expression were assessed in a controlled hemorrhagic shock model followed by LR, HS, and LR+PTX resuscitation in rodents. Rats (240-300 g) were bled to a mean arterial pressure (MAP) of 35 mm Hg for 1 hr and then randomized into three groups: HS (7.5% NaCl, 4 ml/kg); LR (3× shed blood); and LR+PTX (25 mg/kg). Additionally, total shed blood was reinfused. A sham group underwent no shock and no treatment. The internal spermatic fascia was exteriorized and the microcirculation was observed by closed-circuit TV coupled to a microscope, 2 and 6 hrs after treatment. The number of leukocytes sticking to the venular endothelium was determined 2 hrs after fluid resuscitation. Microcirculatory blood flow was measured by an optical Doppler velocimeter. Lung histology and lung MPO immunostaining were assessed at 6 hrs, and lung ICAM-1 expression was determined by immunostaining at 2 hrs following fluid resuscitation. Two hours after treatment, HS (1.4 ± 0.4), LR+PTX (1.7 ± 0.3), and sham (0.4 ± 0.2) groups presented significant reductions in leukocyte adherence (cells/100 urn venule length), compared with the LR group (4.0 ± 0.9, P < 0.05). No differences were observed 6 hrs after treatment on leukocyte adherence and microcirculatory blood flow. ICAM-1 expression was significantly higher in LR- treated animals compared with the HS, LR+PTX, and sham groups (P < 0.01). PMN infiltration and overall lung injury were significantly attenuated by HS and LR+PTX. These results support earlier studies that indicated the potential application of HS and PTX in shock therapy and the increase in PMN- endothelial cell interaction and lung injury after LR resuscitation.

Inhibition of eosinophil chemotaxis by chronic blockade of nitric oxide biosynthesis

The effect of chronic N omega-nitro-L-arginine methyl ester (L-NAME) treatment on the in vivo eosinophil migration induced by bradykinin, platelet-activating factor (PAF), lipopolysaccharide and carrageenin has been investigated in the rat using the pleurisy model. The in vitro (microchemotaxis chamber) eosinophil migration induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP), PAF and zymosan-activated serum was also evaluated in the rat. The eosinophils were obtained from the peritoneal cavity of male Wistar rats and isolated on a discontinuous metrizamide gradient. Chronic inhibition of nitric oxide biosynthesis was achieved by adding L-NAME to the drinking water to give an intake of approximately 75 mumol/rat/day for 4 weeks. Rats treated chronically with L-NAME developed a significant level of hypertension (163 +/- 4.8 mmHg; P < 0.01) compared with animals which received either the same dose of the inactive enantiomer D-NAME (124 +/- 3.2 mmHg) or tap water alone (119 +/- 1.6 mmHg). The intrapleural injection of bradykinin (50 micrograms), PAF (1 microgram), lipopolysaccharide (0.25 microgram) and carrageenin (125 micrograms) into untreated rats in vivo induced a significant level of eosinophil migration by 24 h post-injection. This migration was markedly reduced in L-NAME-treated rats. Eosinophils obtained from untreated rats showed a significant level of migration in vitro in response to fMLP (5 X 10(-8) M), PAF (10(-8) M) and zymosan-activated serum (27 microliters). In contrast, the migration induced by these chemotactic agents was markedly reduced in cells isolated from animals treated chronically with L-NAME. L-Arginine (5.5 mM), but not D-arginine (5.5 mM), restored the ability of eosinophils from L-NAME-treated animals to migrate in response to fMLP. Our results indicate that nitric oxide plays a major role in the in vivo and ex vivo migration of eosinophils.

Inhibition of leukocyte chemotaxis by serum factor in diabetes mellitus: selective depression of cell responses mediated by complement-derived chemoattractants.

Rat neutrophil chemotactic responses to N-formyl-methionyl-leucyl-phenylalanine (FMLP), leukotriene (LT) B4, and lipopolysaccharide-activated serum (LPS-AS) were quantitatively assessed using the micropore filter system. Cells were suspended in either normal or diabetic rat serum for testing. Diabetic donor serum did not affect migration of neutrophils in a concentration gradient of the synthetic chemotactic agents. In contrast, the migratory responses to LPS-AS were significantly less than normal in this circumstance. Summation of effects was observed when FMLP and LPS-AS, or LTB4 and LPS-AS were simultaneously added to the test chamber, with cells suspended in normal serum. Suspended in diabetic rat serum neutrophils responded normally to the synthetic chemoattractants but the response to the activated serum was blocked. Cells previously incubated in the presence of diabetic donor serum then transferred to a culture medium for testing, presented reduced migratory responses to LPS-AS. Supramaximal, inhibitory concentrations of FMLP and LTB4, did not influence the response of neutrophils to LPS-AS.In vivo, suppression of cellular emigration to an inflamed area was observed from the early stages of the diabetic state. The inhibitory activity of chemotaxis in diabetes mellitus was previously reported to be associated with a protein factor in plasma of the animals. It is suggested that the inhibitory factor of chemotaxis in diabetes mellitus interacts with neutrophil receptors for complement-derived chemoattractants to induce blockade of cell-oriented locomotion eitherin vitro orin vivo.

Down-regulation of mast cell activation and airway reactivity in diabetic rats: role of insulin

Hormones play a modulating role in allergic inflammation. An inverse relationship between atopy and diabetes mellitus was reported. The mechanisms regulating this interaction are not completely understood. This study examined whether insulin influences mast cell activation following antigen challenge in rats. The experimental design included alloxan-induced diabetic rats and matching controls. Experiments were performed 30 days after alloxan injection. The animals were sensitised by s.c. injection of ovalbumin (OA) and aluminium hydroxide. OA-induced airway contraction, morphometric analysis of airway mast cells and tissue histamine quantification were evaluated in the isolated main bronchus and intrapulmonary bronchus upon exposure to antigen in vitro. Relative to controls, a reduced contraction to OA was observed in bronchial segments isolated from diabetic rats. This was accompanied by a 50% reduction in the number of degranulated mast cells and in histamine release. A complete recovery of the impaired responses was observed under the influence of insulin. In conclusion, the data suggested that insulin might modulate the controlling of mast cell degranulation; therefore, the early-phase response to antigen provocation, which represents a new insight into a better understanding of the mechanisms, accounted for the decreased risk of asthma among type-1 diabetic patients.

ROLE OF CHEMOTACTIC FACTORS IN NEUTROPHIL ACTIVATION AFTER THERMAL INJURY IN RATS

Acute thermal trauma is well known to produce evidence of a “systemic inflammatory response” in vivo, as manifested by evidence of complement activation, appearance in plasma of a variety of inflammatory factors, and development of multi-organ injury. The current studies were focused on acute thermal injury of rat skin and factors responsible for accompanying activation of blood neutrophils. Acute thermal injury of rat skin resulted in a time-dependent loss of L-selectin and up-regulation of Mac-1 (CD11b/CD18) on blood neutrophils, with no changes in LFA-1 (CD11a/CD18). The loss of L-selectin was prevented by blockade of C5a but not by blockade of the α-chemokine, macrophage inflammatory protein-2 (MIP-2). C5a, the α chemokines, MIP-2 and keratinocyte-derived cytokine (KC), and platelet activating factor (PAF) contributed to up-regulation of blood neutrophil Mac-1. Blocking interventions against these mediators also blunted the degree of neutropenia developing after thermal trauma. These data suggest that activation of blood neutrophils after thermal trauma is related to the role of several chemotactic mediators. These studies may provide clues regarding factors responsible for development of the “systemic inflammatory response syndrome” after thermal injury in the experimental model employed.

Aminoguanidine and the prevention of leukocyte dysfunction in diabetes mellitus: a direct vital microscopic study

1 Defective leukocyte‐endothelial interactions are observed in experimental diabetes mellitus. The present study investigated the effect of aminoguanidine, an inhibitor of advanced glycation end products formation, on leukocyte‐endothelial interactions in alloxan‐induced diabetic rats. 2 In rats anaesthetized with sodium pentobarbitone, the internal spermatic fascia was exteriorized and the microcirculation was observed by a closed‐circuit TV coupled to a microscope. The number of leukocytes rolling along the venular endothelium and sticking to the vascular wall was determined after topical application of zymosan‐activated plasma (1 mg ml−1), as well as the number of adherent and migrated cells after an irritative stimulus (carrageenan 100 μg). 3 The diabetic state decreased the number of rolling, sticking and migrated leukocytes. Pretreatment of diabetic animals with aminoguanidine (250 mg kg−1 day−1, for 18 days) normalized these values. To be effective, aminoguanidine had to be administered chronically, starting treatment before induction of the diabetic state. 4 The preventive effect was unrelated to the number of circulating leukocytes, or to the hyperglycaemia or to the hyperosmolality secondary to hyperglycaemia. 5 A non‐dialyzed (>12,000‐Mr) material in plasma from diabetic, but not normal animals, decreased the number of rolling, sticking and migrated leukocytes in recipient rats. This effect was completely abolished by chronic treatment of diabetic plasma donors with aminoguanidine. 6 The results suggest that a protein modified by glycosylation (>12 kDa) is associated with leukocyte dysfunction in diabetes mellitus and that the ability of aminoguanidine to prevent such dysfunction is related to an inhibitory effect on advanced glycation end products formation.