Rachel G. Khadaroo

Soluble Fibrinogen-Like Protein 2/Fibroleukin Exhibits Immunosuppressive Properties: Suppressing T Cell Proliferation and Inhibiting Maturation of Bone Marrow-Derived Dendritic Cells

Fibrinogen-like protein 2 (fgl2)/fibroleukin is a member of the fibrinogen-related protein superfamily. In addition to its established role in triggering thrombosis, it is known to be secreted by T cells. The soluble fgl2 (sfgl2) protein generated in a baculovirus expression system bound to both T cells and bone marrow-derived dendritic cells (DC) in a specific manner. sfgl2 exhibited immunomodulatory properties capable of inhibiting T cell proliferation stimulated by alloantigens, anti-CD3/anti-CD28 mAbs, and Con A in a dose-dependent manner; however, it had no inhibitory effects on CTL activity. The time- and dose-dependent inhibitory effect of sfgl2 on alloreactive T cell proliferation could be neutralized by a mAb against mouse fgl2. Polarization toward a Th2 cytokine profile with decreased production of IL-2 and IFN-γ and increased production of IL-4 and IL-10 was observed in sfgl2-treated allogeneic cultures. Exposure of immature DC to sfgl2 abrogated the expression of CD80high and MHC class IIhigh molecules and markedly inhibited NF-κB nuclear translocation, thus inhibiting their maturation. sFgl2-treated DC had an impaired ability to stimulate allogeneic T cell proliferation. Maximal inhibition of proliferation was observed when allogeneic T cells were cultured with sfgl2-treated DC and sfgl2 protein was added in the culture. These data provide the first evidence to demonstrate that sfgl2 exerts immunosuppressive effects on T cell proliferation and DC maturation.

Twenty-five percent albumin prevents lung injury following shock/resuscitation.

ObjectiveTo evaluate novel indications for the use of human albumin solutions in the prevention and treatment of acute lung injury following shock/resuscitation and to test the hypothesis that 25% human albumin is an effective resuscitation fluid as well as an immunomodulatory agent protective against lung injury in our model. DesignA previously developed rodent model of acute lung injury in which resuscitated shock primes for increased lung injury in response to a small dose of intratracheal lipopolysaccharide. SettingUniversity-affiliated hospital. SubjectsSprague Dawley rats weighing 300–350 g. InterventionsAnimals were bled to a mean arterial pressure of 40 mm Hg and maintained in a shock phase for 1 hr. Animals then were resuscitated by transfusion of the shed blood plus an equal volume of Ringer’s lactate or their shed blood plus 3 mL/kg volume of 25% albumin or their shed blood plus 15 mL/kg of 5% human albumin over a period of 2 hrs. To test for the possible role of 25% albumin as an antioxidant, we also performed resuscitation with Ringer’s lactate supplemented with N-acetylcysteine or 25% albumin depleted of its antioxidant properties by N-ethylmaleimide. Mean arterial pressure was monitored continuously. One hour after resuscitation, 100 &mgr;g of lipopolysaccharide in 200 &mgr;L of saline was administered intratracheally. Measurements and Main ResultsResuscitation with 25% albumin significantly reduced transpulmonary protein flux, bronchoalveolar lavage fluid neutrophil counts, and the degree of histopathological injury compared with resuscitation with Ringer’s lactate or 5% albumin. To delineate the underlying mechanism of this beneficial effect, the production of cytokine-induced neutrophil chemoattractant as well as nuclear translocation of its critical transcription factor nuclear factor-&kgr;B was measured. Both cytokine-induced neutrophil chemoattractant messenger RNA concentrations and nuclear factor-&kgr;B translocation were diminished following 25% albumin resuscitation. Furthermore, 25% albumin significantly decreased lipid peroxidation in plasma as measured by 8-isoprostane concentrations. N-ethylmaleimide modified 25% albumin, possessing lesser antioxidant activity, exhibited an attenuated protection from lung injury. ConclusionsResuscitation with 25% albumin attenuates lung injury in this rat model. The beneficial effect was due to reduced neutrophil sequestration. The antioxidant properties of the 25% albumin preparation appeared to be partially responsible for the effects observed. These studies suggest a novel role for 25% albumin as an anti-inflammatory agent in neutrophil-mediated diseases, such as acute respiratory distress syndrome.

Oxidative Stress Reprograms Lipopolysaccharide Signaling via Src Kinase-dependent Pathway in RAW 264.7 Macrophage Cell Line

Oxidative stress generated during ischemia/reperfusion injury has been shown to augment cellular responsiveness. Whereas oxidants are themselves known to induce several intracellular signaling cascades, their effect on signaling pathways initiated by other inflammatory stimuli remains poorly elucidated. Previous work has suggested that oxidants are able to prime alveolar macrophages for increased NF-κB translocation in response to treatment with lipopolysaccharide (LPS). Because oxidants are known to stimulate the Src family of tyrosine kinases, we hypothesized that the oxidants might contribute to augmented NF-κB translocation by LPS via the involvement of Src family kinases. To model macrophage priming in vitro, the murine macrophage cell line, RAW 264.7, was first incubated with various oxidants and then exposed to low dose LPS. These studies show that oxidant stress is able to augment macrophage responsiveness to LPS as evidenced by earlier and increased NF-κB translocation. Inhibition of the Src family kinases by either pharmacological inhibition using PP2 or through a molecular approach by cell transfection with Csk was found to prevent the augmented LPS-induced NF-κB translocation caused by oxidants. Interestingly, while Src kinase inhibition was able to prevent the LPS-induced NF-κB translocation in oxidant-treated macrophages, this strategy had no effect on NF-κB translocation caused by LPS in the absence of oxidants. These findings suggested that oxidative stress might divert LPS signaling along an alternative signaling pathway. Further studies demonstrated that the Src-dependent pathway induced by oxidant pretreatment involved the activation of phosphatidylinositol 3-kinase. Involvement of this pathway appeared to be independent of traditional LPS signaling. Together, these studies provide a novel potential mechanism whereby oxidants might prime alveolar macrophages for altered responsiveness to subsequent inflammatory stimuli and suggest different cellular targets for immunomodulation following ischemia/reperfusion.

Sarcopenia is a predictor of outcomes in very elderly patients undergoing emergency surgery

BACKGROUND With the increasing aging population, the number of very elderly patients (age ≥80 years) undergoing emergency operations is increasing. Evaluating patient-specific risk factors for postoperative morbidity and mortality in the acute care surgery setting is crucial to improving outcomes. We hypothesize that sarcopenia, a severe depletion of skeletal muscles, is a predictor of morbidity and mortality in very elderly patients undergoing emergency surgery. METHODS A total of 170 patients older than the age of 80 underwent emergency surgery between 2008 and 2010 at a tertiary care facility; 100 of these patients had abdominal computed tomography images within 30 days of the operation that were adequate for the assessment of sarcopenia. The impact of sarcopenia on the operative outcomes was evaluated using both univariate and multivariate analysis. RESULTS The mean patient age was 84 years, with an in-hospital mortality of 18%. Sarcopenia was present in 73% of patients. More sarcopenic patients had postoperative complications (45% sarcopenic versus 15% nonsarcopenic, P = .005) and more died in hospital (23 vs 4%, P = .037). There were no differences in duration of stay or requirement for intensive care unit postoperatively. After we controlled for confounding factors, increasing skeletal muscle index (per incremental cm(2)/m(2)) was associated with decreased in-hospital mortality (odds ratio ∼0.834, 95% confidence interval 0.731-0.952, P = .007) in multivariate analysis. CONCLUSION Sarcopenia was independently predictive of greater complication rates, discharge disposition, and in-hospital mortality in the very elderly emergency surgery population. Using sarcopenia as an objective tool to identify high-risk patients would be beneficial in developing tailored preventative strategies and potentially resource allocation in the future.

Hypertonic preconditioning prevents hepatocellular injury following ischemia/reperfusion in mice: A role for interleukin 10

Ischemia/reperfusion (I/R) of the liver occurs in many clinical scenarios including trauma, elective surgery, and transplantation. Events initiated by this process can lead to inflammation in the liver, culminating in local injury as well as distant organ dysfunction. Recent studies have suggested that hypertonic saline exerts anti‐inflammatory effects, which may be beneficial in preventing organ injury. In the present study, we examine the effect of hypertonic saline on the development of liver inflammation following I/R in both rat and mouse models. Hypertonic pretreatment was shown to prevent liver enzyme release concomitant with a reduction in liver neutrophil sequestration. Hypertonic saline appeared to exert this effect by inhibiting liver tumor necrosis factor α (TNF‐α) generation, an effect that culminated in reduced liver adhesion molecule expression. Hypertonic saline pretreatment was shown to augment liver interleukin 10 (IL‐10) expression following I/R, as a potential mechanism underlying its anti‐inflammatory effect. To examine the role of IL‐10 in the protective effect of hypertonic saline on liver I/R injury, we used a murine model of I/R. In wild type mice, hypertonic pretreatment similarly prevented liver injury induced by I/R. However, in IL‐10 knockout animals, hypertonic pretreatment was unable to prevent the liver enzyme release, TNF‐α generation, or neutrophil sequestration induced by I/R. In conclusion, these findings define a novel mechanism responsible for the anti‐inflammatory effects of hypertonic saline and also suggest a potential clinical role for hyperosmolar solutions in the prevention of liver injury associated with I/R. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2004;40:211–220.)

ARDS and the multiple organ dysfunction syndrome: Common mechanisms of a common systemic process

The multiple organ dysfunction syndrome is a common but poorly understood complication of critical illness. Its evolution reflects the interactions of an acute, life-threatening insult, the response of the host to that insult, and the therapeutic measures instituted to restore normal homeostasis. Although the cellular mechanisms remain elusive, processes such as inflammation, microvascular thrombosis, apoptosis, and fibrosis and tissue repair contribute to its clinical expression. In the lung, these forces create the characteristic changes of ARDS; that common disorder, however, is better seen as one manifestation of a systemic process than as an isolated problem of the lung. Therapy, in the absence of a more sophisticated understanding of pathologic mechanism, is supportive. The growing recognition that iatrogenic factors contribute to the expression of MODS has highlighted the need for the clinician to be aware of the potential for harm inherent with every intervention.

Mucoepidermoid Carcinoma of the Parotid Gland: A Rare Presentation in a Young Child

Although mucoepidermoid carcinoma (MEC) is the most common malignant salivary gland neoplasm in childhood and adolescence, it is rarely found in children under the age of 10. A 6-year-old girl had an asymptomatic neck mass for 5 months. Clinical examination findings showed a 1.5-cm smooth and firm but mobile nontender mass located in the upper left anterior cervical triangle, clinically separate from the parotid gland. Ultrasound examination findings showed a vascular mass, with a cystic component, possibly within the tail of the parotid gland. An excisional biopsy was performed and frozen section showed a low-grade MEC. A left superficial parotidectomy was then performed. Final histopathologic examination showed one positive resection margin. Subsequently, reexcision of the surgical site and an upper modified neck dissection was undertaken. This unusual presentation of MEC as a neck mass in one of the youngest reported patients illustrates that the anatomic region for parotid tumors is large. Possibly some of these tumors may arise from heterotopic or accessory parotid tissue.

Impaired induction of IL-10 expression in the lung following hemorrhagic shock.

The balance between pro- and anti-inflammatory cytokines is considered to be an important determinant of the magnitude of inflammation in a number of disease states. We previously showed that resuscitated hemorrhagic shock augmented LPS-induced release of proinflammatory molecules by alveolar macrophages (AM). In the present studies, we evaluated the expression and regulation of the counter inflammatory cytokine IL-10 in the lung using this model. We hypothesized that impaired up-regulation of IL-10 in shock/resuscitated animals might serve as a mechanism contributing to accentuated lung inflammation. In a rodent model, animals exposed to LPS alone exhibited enhanced IL-10 mRNA levels in lung tissue as well as in AM, but antecedent shock/resuscitation delayed and attenuated the LPS-induced IL-10 mRNA levels. The ability of shock to attenuate LPS-stimulated IL-10 was also seen in the protein levels. This effect correlated with an augmented expression of cytokine-induced neutrophil chemoattractant (CINC) mRNA. Shock/resuscitated animals given exogenous IL-10 had reduced proinflammatory response, as shown by decreased expression of CINC mRNA and decreased neutrophil sequestration in the lung. Shock/resuscitation plus LPS markedly reduced the transcription rate of IL-10 mRNA compared to LPS alone but did not affect IL-10 mRNA stability. Reduced IL-10 transcription was not caused solely by impaired nuclear translocation of STAT3 and Sp1/Sp3 transcription factors because LPS-induced nuclear translocation of these factors was augmented by antecedent shock. Considered together, these findings show that shock/resuscitation suppresses LPS-induced IL-10 expression by AM in the lung by inhibiting IL-10 gene transcription. Failed up-regulation of counter inflammatory cytokines may contribute to augmented organ dysfunction in trauma patients.

Surgical Site Infections

Surgical site infections are a major contributor to morbidity and mortality in postsurgical care. Risk for surgical site infection is multifactorial and includes a host of microbial, patient-related, and procedure-related factors. Prevention of surgical infection relies on optimization of patient factors and use of a variety of evidence-based pharmacologic and nonpharmacologic measures. At the forefront of these measures is antimicrobial prophylaxis, which has been shown to be effective at reducing risk of surgical site infection. As guidelines for prevention of surgical site infection become increasingly complex and nuanced, surgical infection outcomes are increasing tied to quality outcome and performances measures.

Gastrointestinal dysfunction in the critically ill: can we measure it?

Critical care physicians are increasingly facing patients receiving oral anticoagulation for either cessation of major haemorrhage or to reverse the effects of vitamin K antagonists ahead of emergency surgery. Rapid reversal of anticoagulation is particularly essential in cases of life-threatening bleeding. In these situations, guidelines recommend the concomitant administration of prothrombin complex concentrates (PCCs) and oral or intravenous vitamin K for the fastest normalisation of the international normalised ratio (INR). Despite their universal recommendation, PCCs remain underused by many physicians who prefer to opt for fresh frozen plasma despite its limitations in anticoagulant reversal, including time to reverse INR and high risk of transfusion-related acute lung injury. In contrast, the lower volume required to normalise INR with PCCs and the room temperature storage facilitate faster preparation and administration time, thus increasing the speed at which haemorrhages can be treated. PCCs therefore allow faster, more reliable and complete reversal of vitamin K anticoagulation, especially when administered immediately following confirmation of haemorrhage. In the emergency setting, probabilistic dosing may be considered.Gastrointestinal dysfunction is an intuitively important, yet descriptively elusive component of the multiple organ dysfunction syndrome. Reintam and colleagues have attempted to quantify this dimension using a combination of intolerance of enteral feeding, and the development of intra-abdominal hypertension. While they show that both parameters are associated with an increased risk of death (and therefore that, in combination, the risk of death is even greater), they fall short in developing a novel descriptor of gastrointestinal dysfunction. Nonetheless, and even with its shortcomings, their effort is a welcome contribution to the surprisingly complex process of describing the morbidity of critical illness.