Intestinal Fatty Acid Binding Protein as a Predictor of Early Mesenteric Injury Preceding Clinical Presentation: A Case Report.

Introduction: Diagnosing non-occlusive mesenteric ischaemia (NOMI) in patients is complicated, due to poor signs and symptoms and non-specific laboratory tests, leading to a high mortality rate. This case study presents the rare case of a patient who developed mesenteric ischaemia after an emergency thoracic endovascular aneurysm repair (TEVAR) for a type B aortic dissection (TBAD) and peri-operative cardiogenic shock. Study outcomes revealed that intestinal fatty acid binding protein (I-FABP) identified early mucosal damage two days before the clinical presentation. Report: A 43 year old male patient was admitted to the emergency department with an acute TBAD and a dissection of the superior mesenteric artery (SMA), for which TEVAR was performed with additional stent placement in the SMA. Peri-operatively, the patient went into cardiogenic shock with a sustained period of hypotension. Post-operatively, the plasma I-FABP levels were measured prospectively, revealing an initial increase on post-operative day five (551.1 pg/mL), which continued beyond day six (610.3 pg/mL). On post-operative day seven, the patient developed a fever and demonstrated signs of peritonitis and bowel perforation. He underwent an emergency laparotomy, followed by an ileocaecal resection (<100 cm) with a transverse ileostomy. Pathological analysis confirmed the diagnosis of mesenteric ischaemia. Discussion: The diagnosis of NOMI in critically ill patients is often complicated, and the currently available diagnostic markers lack the specificity and sensitivity to detect early intestinal injury. This case report highlights that elevated I-FABP in plasma levels may indicate the presence of early mesenteric injury. Further research needs to be conducted before I-FABP can be applied in daily practice.

Lipocalin-2 and neutrophil activation in pancreatic cancer cachexia.

Background: Cancer cachexia is a multifactorial syndrome characterized by body weight loss and systemic inflammation. The characterization of the inflammatory response in patients with cachexia is still limited. Lipocalin-2, a protein abundant in neutrophils, has recently been implicated in appetite suppression in preclinical models of pancreatic cancer cachexia. We hypothesized that lipocalin-2 levels could be associated with neutrophil activation and nutritional status of pancreatic ductal adenocarcinoma (PDAC) patients. Methods: Plasma levels of neutrophil activation markers calprotectin, myeloperoxidase, elastase, and bactericidal/permeability-increasing protein (BPI) were compared between non-cachectic PDAC patients (n=13) and cachectic PDAC patients with high (≥26.9 ng/mL, n=34) or low (<26.9 ng/mL, n=34) circulating lipocalin-2 levels. Patients' nutritional status was assessed by the patient-generated subjective global assessment (PG-SGA) and through body composition analysis using CT-scan slices at the L3 level. Results: Circulating lipocalin-2 levels did not differ between cachectic and non-cachectic PDAC patients (median 26.7 (IQR 19.7-34.8) vs. 24.8 (16.6-29.4) ng/mL, p=0.141). Cachectic patients with high systemic lipocalin-2 levels had higher concentrations of calprotectin, myeloperoxidase, and elastase than non-cachectic patients or cachectic patients with low lipocalin-2 levels (calprotectin: 542.3 (355.8-724.9) vs. 457.5 (213.3-606.9), p=0.448 vs. 366.5 (294.5-478.5) ng/mL, p=0.009; myeloperoxidase: 30.3 (22.1-37.9) vs. 16.3 (12.0-27.5), p=0.021 vs. 20.2 (15.0-29.2) ng/mL, p=0.011; elastase: 137.1 (90.8-253.2) vs. 97.2 (28.8-215.7), p=0.410 vs. 95.0 (72.2-113.6) ng/mL, p=0.006; respectively). The CRP/albumin ratio was also higher in cachectic patients with high lipocalin-2 levels (2.3 (1.3-6.0) as compared to non-cachectic patients (1.0 (0.7-4.2), p=0.041). Lipocalin-2 concentrations correlated with those of calprotectin (rs =0.36, p<0.001), myeloperoxidase (rs =0.48, p<0.001), elastase (rs =0.50, p<0.001), and BPI (rs =0.22, p=0.048). Whereas no significant correlations with weight loss, BMI, or L3 skeletal muscle index were observed, lipocalin-2 concentrations were associated with subcutaneous adipose tissue index (rs =-0.25, p=0.034). Moreover, lipocalin-2 tended to be elevated in severely malnourished patients compared with well-nourished patients (27.2 (20.3-37.2) vs. 19.9 (13.4-26.4) ng/mL, p=0.058). Conclusions: These data suggest that lipocalin-2 levels are associated with neutrophil activation in patients with pancreatic cancer cachexia and that it may contribute to their poor nutritional status.

Ovarian cancer ascites induces skeletal muscle wasting in vitro and reflects sarcopenia in patients.

BACKGROUND: Cachexia-associated skeletal muscle wasting or 'sarcopenia' is highly prevalent in ovarian cancer and contributes to poor outcome. Drivers of cachexia-associated sarcopenia in ovarian cancer remain elusive, underscoring the need for novel and better models to identify tumour factors inducing sarcopenia. We aimed to assess whether factors present in ascites of sarcopenic vs. non-sarcopenic ovarian cancer patients differentially affect protein metabolism in skeletal muscle cells and to determine if these effects are correlated to cachexia-related patient characteristics. METHODS: Fifteen patients with an ovarian mass and ascites underwent extensive physical screening focusing on cachexia-related parameters. Based on computed tomography-based body composition imaging, six cancer patients were classified as sarcopenic and six were not; three patients with a benign condition served as an additional non-sarcopenic control group. Ascites was collected, and concentrations of cachexia-associated factors were assessed by enzyme-linked immunosorbent assay. Subsequently, ascites was used for in vitro exposure of C2C12 myotubes followed by measurements of protein synthesis and breakdown by radioactive isotope tracing, qPCR-based analysis of atrophy-related gene expression, and NF-κB activity reporter assays. RESULTS: C2C12 protein synthesis was lower after exposure to ascites from sarcopenic patients (sarcopenia 3.1 ± 0.1 nmol/h/mg protein vs. non-sarcopenia 5.5 ± 0.2 nmol/h/mg protein, P < 0.01), and protein breakdown rates tended to be higher (sarcopenia 31.2 ± 5.2% vs. non-sarcopenia 20.9 ± 1.9%, P = 0.08). Ascites did not affect MuRF1, Atrogin-1, or REDD1 expression of C2C12 myotubes, but NF-κB activity was specifically increased in cells exposed to ascites from sarcopenic patients (sarcopenia 2.2 ± 0.4-fold compared with control vs. non-sarcopenia 1.2 ± 0.2-fold compared with control, P = 0.01). Protein synthesis and breakdown correlated with NF-κB activity (rs  = -0.60, P = 0.03 and rs  = 0.67, P = 0.01, respectively). The skeletal muscle index of the ascites donors was also correlated to both in vitro protein synthesis (rs  = 0.70, P = 0.005) and protein breakdown rates (rs  = -0.57, P = 0.04). CONCLUSIONS: Ascites of sarcopenic ovarian cancer patients induces pronounced skeletal muscle protein metabolism changes in C2C12 cells that correlate with clinical muscle measures of the patient and that are characteristic of cachexia. The use of ascites offers a new experimental tool to study the impact of both tumour-derived and systemic factors in various cachexia model systems, enabling identification of novel drivers of tissue wasting in ovarian cancer.

Activation of the Complement System in Patients with Cancer Cachexia.

Systemic inflammation is thought to underlie many of the metabolic manifestations of cachexia in cancer patients. The complement system is an important component of innate immunity that has been shown to contribute to metabolic inflammation. We hypothesized that systemic inflammation in patients with cancer cachexia was associated with complement activation. Systemic C3a levels were higher in cachectic patients with inflammation (n = 23, C-reactive protein (CRP) ≥ 10 mg/L) as compared to patients without inflammation (n = 26, CRP < 10 mg/L) or without cachexia (n = 13) (medians 102.4 (IQR 89.4-158.0) vs. 81.4 (IQR 47.9-124.0) vs. 61.6 (IQR 46.8-86.8) ng/mL, respectively, p = 0.0186). Accordingly, terminal complement complex (TCC) concentrations gradually increased in these patient groups (medians 2298 (IQR 2022-3058) vs. 1939 (IQR 1725-2311) vs. 1805 (IQR 1552-2569) mAU/mL, respectively, p = 0.0511). C3a and TCC concentrations were strongly correlated (rs = 0.468, p = 0.0005). Although concentrations of C1q and mannose-binding lectin did not differ between groups, C1q levels were correlated with both C3a and TCC concentrations (rs = 0.394, p = 0.0042 and rs = 0.300, p = 0.0188, respectively). In conclusion, systemic inflammation in patients with cancer cachexia is associated with the activation of key effector complement factors. The correlations between C1q and C3a/TCC suggest that the classical complement pathway could play a role in complement activation in patients with pancreatic cancer.

Lysophosphatidic acid prevents renal ischemia-reperfusion injury by inhibition of apoptosis and complement activation.

Renal ischemia-reperfusion (I/R) injury is an important cause of acute renal failure as observed after renal transplantation, major surgery, trauma, and septic as well as hemorrhagic shock. We previously showed that the inhibition of apoptosis is protective against renal I/R injury, indicating that apoptotic cell-death is an important feature of I/R injury. Lysophosphatidic acid (LPA) is an endogenous phospholipid growth factor with anti-apoptotic properties. This tempted us to investigate the effects of exogenous LPA in a murine model of renal I/R injury. LPA administered at the time of reperfusion dose dependently inhibited renal apoptosis as evaluated by the presence of internucleosomal DNA cleavage. I/R-induced renal apoptosis was only present in tubular epithelial cells with evident disruption of brush border as assessed by immunohistochemistry for active caspase-7 and filamentous actin, respectively. LPA treatment specifically prevented tubular epithelial cell apoptosis but also reduced the I/R-induced loss of brush-border integrity. Besides, LPA showed strong anti-inflammatory effects, inhibiting the renal expression of tumor necrosis factor-alpha and abrogating the influx of neutrophils. Next, LPA dose dependently inhibited activation of the complement system. Moreover, treatment with LPA abrogated the loss of renal function in the course of renal I/R. This study is the first to show that administration of the phospholipid LPA prevents I/R injury, abrogating apoptosis and inflammation. Moreover, exogenous LPA is capable of preventing organ failure because of an ischemic insult and thus may provide new means to treat clinical conditions associated with I/R injury in the kidney and potentially also in other organs.

Complement factor C5a mediates renal ischemia-reperfusion injury independent from neutrophils.

The complement system has been shown to mediate renal ischemia-reperfusion (I/R) injury. However, the contribution of complement factor C5a to I/R injury, in particular in the kidney, remains to be established. In this study, we investigated the impact of blocking the C5aR pathway on the inflammatory response and on the renal function in a murine model of I/R injury. First, we analyzed C5aR expression in kidneys of healthy mice. Intriguingly, we found expression on mesangial, as well as on tubular epithelial, cells. After I/R injury, C5aR expression was up-regulated in tubular epithelial cells. In addition, mRNA levels of CXC chemokines and TNF-alpha increased significantly and kidneys were heavily infiltrated by neutrophils. Blocking the C5aR pathway by a specific C5a receptor antagonist (C5aRA) abrogated up-regulation of CXC chemokines but not of TNF-alpha and reduced neutrophil infiltration by >50%. Moreover, application of the C5aRA significantly reduced loss of renal function. This improvement of function was independent of the presence of neutrophils because neutrophil depletion by mAb NIMP-R14 did not affect the protective effect of C5aRA treatment. Furthermore, blocking of the C5aR pathway had no influence on renal apoptosis. These data provide evidence that C5a is crucially involved in the pathogenesis of renal I/R injury by modulation of neutrophil-dependent as well as neutrophil-independent pathways, which include the regulation of CXC chemokines but not TNF-alpha or apoptotic pathways.

Inhibition of complement factor C5 protects against renal ischemia-reperfusion injury: inhibition of late apoptosis and inflammation.

BACKGROUND: Complement has been implicated in the pathophysiology of renal ischemia-reperfusion (I/R) injury. However, the mechanism underlying complement-mediated renal I/R injury is thus far unknown. To investigate the involvement of complement in I/R injury, we studied the activation and deposition of complement in a murine model of renal I/R injury. Furthermore, we examined the effect of inhibition of complement-factor C5 on renal I/R injury. METHODS: Mice were subjected to 45 min of unilateral ischemia and subsequent contralateral nephrectomy and reperfusion for 2, 12, or 24 hr. Mice were control treated or treated with BB5.1, a monoclonal antibody that prevents cleavage of complement factor C5, thereby preventing C5a generation and formation of the membrane attack complex (MAC). RESULTS: Renal I/R induced extensive deposition of C3 early after reperfusion, whereas C6 and C9 deposition (MAC formation) occurred relatively late. I/R-induced complement deposition was mainly localized to tubular epithelium. Treatment with BB5.1 totally prevented MAC formation but also reduced C3 deposition. Inhibition of C5 strongly inhibited late inflammation, as measured by neutrophil influx and induction of the murine CXC chemokines macrophage inflammatory protein-2, KC, and lipopolysaccharide-induced CXC chemokine. Anti-C5 treatment furthermore abrogated late I/R-induced apoptosis, whereas early apoptosis was not affected. Moreover, BB5.1 treatment significantly protected against I/R-induced renal dysfunction. CONCLUSIONS: Renal I/R is followed by activation of the complement system and intrarenal deposition of C3 and MAC. Complement activation plays a crucial role in the regulation of inflammation and late apoptosis. Complement inhibition, by preventing C5 activation, abrogates late apoptosis and inflammation, being strongly protective against renal function loss.