Clemens L. Bockmeyer

The price of tumor control: an analysis of rare side effects of anti-CTLA-4 therapy in metastatic melanoma from the ipilimumab network

Background Ipilimumab, a cytotoxic T-lymphocyte antigen-4 (CTLA-4) blocking antibody, has been approved for the treatment of metastatic melanoma and induces adverse events (AE) in up to 64% of patients. Treatment algorithms for the management of common ipilimumab-induced AEs have lead to a reduction of morbidity, e.g. due to bowel perforations. However, the spectrum of less common AEs is expanding as ipilimumab is increasingly applied. Stringent recognition and management of AEs will reduce drug-induced morbidity and costs, and thus, positively impact the cost-benefit ratio of the drug. To facilitate timely identification and adequate management data on rare AEs were analyzed at 19 skin cancer centers. Methods and Findings Patient files (n = 752) were screened for rare ipilimumab-associated AEs. A total of 120 AEs, some of which were life-threatening or even fatal, were reported and summarized by organ system describing the most instructive cases in detail. Previously unreported AEs like drug rash with eosinophilia and systemic symptoms (DRESS), granulomatous inflammation of the central nervous system, and aseptic meningitis, were documented. Obstacles included patientś delay in reporting symptoms and the differentiation of steroid-induced from ipilimumab-induced AEs under steroid treatment. Importantly, response rate was high in this patient population with tumor regression in 30.9% and a tumor control rate of 61.8% in stage IV melanoma patients despite the fact that some patients received only two of four recommended ipilimumab infusions. This suggests that ipilimumab-induced antitumor responses can have an early onset and that severe autoimmune reactions may reflect overtreatment. Conclusion The wide spectrum of ipilimumab-induced AEs demands doctor and patient awareness to reduce morbidity and treatment costs and true ipilimumab success is dictated by both objective tumor responses and controlling severe side effects.

Role of increased sphingomyelinase activity in apoptosis and organ failure of patients with severe sepsis

Numerous studies support the notion that an activation of sphingomyelinases and a subsequent increase of the concentration of the bioactive lipid mediator ceramide are critical in the concert of inflammatory stimuli and to the induction of apoptosis during inflammation. Here we show that patients with severe sepsis exhibit an enhanced sphingolytic activity in comparison with controls [262 pmol/(mlxh) vs. 123.6 pmol/(mlxh), P<0.005]. During the clinical course, a further increase was paralleled by the severity of illness and by fatal outcome. Moreover, we show that oxidative stress may partially account for the increased activity through posttranslational modification of the enzyme. In a murine endotoxic shock model, administration of a low molecular weight inhibitor diminished the rise in enzymatic activity and improved the survival rate. In liver specimen, inhibition of activity correlated with a reduced rate of hepato‐cellular apoptosis. Our data support the concept that activation of the plasmatic isoform of sphingomyelinase may play a critical role in the development of apoptosis and organ failure in sepsis. An inhibition of the secreted isoform of sphingomyelinase should be explored further as a potential target in the complicated puzzle of sepsis.

Plexiform Lesions in Pulmonary Arterial Hypertension: Composition, Architecture, and Microenvironment

Pulmonary arterial hypertension (PAH) is a debilitating disease with a high mortality rate. A hallmark of PAH is plexiform lesions (PLs), complex vascular formations originating from remodeled pulmonary arteries. The development and significance of these lesions have been debated and are not yet fully understood. Some features of PLs resemble neoplastic disorders, and there is a striking resemblance to glomeruloid-like lesions (GLLs) in glioblastomas. To further elucidate PLs, we used in situ methods, such as (fluorescent) IHC staining, three-dimensional reconstruction, and laser microdissection, followed by mRNA expression analysis. We generated compartment-specific expression patterns in the lungs of 25 patients (11 with PAH associated with systemic shunts, 6 with idiopathic PAH, and 8 controls) and GLLs from 5 glioblastomas. PLs consisted of vascular channels lined by a continuously proliferating endothelium and backed by a uniform myogenic interstitium. They also showed up-regulation of remodeling-associated genes, such as HIF1a, TGF-β1, VEGF-α, VEGFR-1/-2, Ang-1, Tie-2, and THBS1, but also of cKIT and sprouting-associated markers, such as NOTCH and matrix metalloproteinases. The cellular composition and signaling seen in GLLs in neural neoplasms differed significantly from those in PLs. In conclusion, PLs show a distinct cellular composition and microenvironment, which contribute to the plexiform phenotype and set them apart from other processes of vascular remodeling in patients with PAH. Neoplastic models of angiogenesis seem to be of limited use in further study of plexiform vasculopathy.

Inflammation-associated ADAMTS13 deficiency promotes formation of ultra-large von Willebrand factor

The findings of this study suggest that systemic inflammation results in ADAMTS13 deficiency, and thereby activates hemostasis. In a prospective, longitudinal study, we investigated the association between decreased ADAMTS13 activity and impaired hemostasis, as well as organ dysfunctions in patients with systemic inflammation due to extracorporeal cardiopulmonary circuit or with severe sepsis. Similar to negative acute phase proteins, ADAMTS13 activity declined stepwise according to the extent of inflammatory responses. A marked imbalance between ADAMTS13 activity and VWF antigen level was associated with the appearance of ultra-large VWF multimers in plasma, with organ dysfunction and lethality. Our data support the view that systemic inflammation results in an ADAMTS13 deficiency which activates hemostasis.

The synthetic Tie2 agonist peptide vasculotide protects against vascular leakage and reduces mortality in murine abdominal sepsis

IntroductionAngiopoietin-1 (Angpt1), the natural agonist ligand for the endothelial Tie2 receptor, is a non-redundant endothelial survival and vascular stabilization factor that reduces endothelial permeability and inhibits leukocyte-endothelium interactions. Here we evaluate the efficacy of a novel polyethylene glycol (PEG)-clustered Tie2 agonist peptide, vasculotide (VT), to protect against vascular leakage and mortality in a murine model of polymicrobial abdominal sepsis.MethodsPolymicrobial abdominal sepsis in C57BL6 mice was induced by cecal-ligation-and-puncture (CLP). Mice were treated with different dosages of VT or equal volume of phosphate-buffered saline (PBS). Sham-operated animals served as time-matched controls.ResultsSystemic administration of VT induced long-lasting Tie2 activation in vivo. VT protected against sepsis-induced endothelial barrier dysfunction, as evidenced by attenuation of vascular leakage and leukocyte transmigration into the peritoneal cavity. Histological analysis revealed that VT treatment ameliorated leukocyte infiltration in kidneys of septic mice, probably due to reduced endothelial adhesion molecule expression. VT-driven effects were associated with significantly improved organ function and reduced circulating cytokine levels. The endothelial-specific action of VT was supported by additional in vitro studies showing no effect of VT on either cytokine release from isolated peritoneal macrophages, or migratory capacity of isolated neutrophils. Finally, administration of VT pre-CLP (hazard ratio 0.39 [95% confidence interval 0.19-0.81] P < 0.001) and post-CLP reduced mortality in septic mice (HR 0.22 [95% CI 0.06-0.83] P < 0.05).ConclusionsWe provide proof of principle in support of the efficacious use of PEGylated VT, a drug-like Tie2 receptor agonist, to counteract microvascular endothelial barrier dysfunction and reduce mortality in a clinically relevant murine sepsis model. Further studies are needed to pave the road for clinical application of this therapeutic concept.

Circulating angiopoietins in idiopathic pulmonary arterial hypertension

AIMS To determine the diagnostic utility of circulating angiopoietin-1 (Ang-1) and its antagonist angiopoietin-2 (Ang-2) as potential biomarkers of disease severity or response to treatment in idiopathic pulmonary arterial hypertension (IPAH). Imbalances in angiogenic factors including vascular endothelial cell growth factor (VEGF) and the angiopoetin-Tie2 receptor system have been implicated in the pathogenesis of IPAH. METHODS AND RESULTS Plasma Ang-1, Ang-2, soluble Tie2 (sTie2), and VEGF were determined by in-house immunoassays in two cohorts of IPAH patients: a retrospective cohort (n = 81) and a prospective cohort (n = 25). Ten patients with normal pulmonary artery pressures and 14 apparently healthy subjects served as controls. Plasma levels of all angiogenic factors were elevated in IPAH patients compared with controls (all P < 0.005). Angiopoietin-2, but not Ang-1, sTie2, and VEGF correlated with cardiac index (r = -0.53, P < 0.001), pulmonary vascular resistance (PVR) (r= 0.60, P < 0.001), and mixed venous oxygen saturation (SvO(2)) (r= -0.63, P < 0.001). In multivariate analysis, elevated Ang-2 was an independent risk factor of mortality (P = 0.004). The patients in the prospective cohort were studied longitudinally at baseline and 3 months after initiation of therapy. Changes in Ang-2 after initiation of therapy correlated with changes in mean right atrial pressure (r = 0.6, P = 0.008), PVR (r = 0.51, P = 0.04), and inversely related to changes in SvO(2) (r = -0.75, P < 0.001). Histological studies showed that the expression of Ang-2 mRNA and protein was up-regulated in plexiform lesions from IPAH lung tissue samples. CONCLUSION Ang-2 may be involved in the pathogenesis of IPAH, and plasma Ang-2 might serve as a promising new biomarker of disease severity and response to treatment in patients with IPAH.

Plexiform vasculopathy of severe pulmonary arterial hypertension and microRNA expression

BACKGROUND Recent studies have revealed that microRNAs (miRNAs) play a key role in the control of angiogenesis and vascular remodeling. Specific miRNAs in plexiform vasculopathy of severe pulmonary arterial hypertension (PAH) in humans have not yet been investigated. METHODS We analyzed expression of miR-143/145 (vascular smooth muscle-specific), miR-126 (endothelial-specific) and related mRNAs in plexiform (PLs) and concentric lesions (CLs), which had been laser-microdissected from specimens of formalin-fixed, paraffin-embedded, explanted lungs of PAH patients (n = 12) and unaffected controls (n = 8). Samples were analyzed by real-time polymerase chain reaction, and protein expression was determined by immunohistochemistry. RESULTS Expression levels of miR-143/145 and its target proteins (e.g., myocardin, smooth muscle myosin heavy chain) were found to be significantly higher in CLs than in PLs, whereas miR-126 and VEGF-A were significantly up-regulated in PLs when compared with CLs, indicating a more prominent angiogenic phenotype of PL. This correlates with a down-regulation of miR-204 as well as an up-regulation of miR-21 in PLs, which in turn corresponds to enhanced cell proliferation. CONCLUSIONS Our findings show that morphologic changes of plexiform vasculopathy in the end-stage PAH lung are reflected by alterations at the miRNA level.

Characteristics of Clinical Sepsis Reflected in a Reliable and Reproducible Rodent Sepsis Model

BACKGROUND Sepsis models are frequently based on induction of peritonitis, with cecal ligation and puncture reflecting the prototypical model. However, there is an ongoing discussion about the limitations of these models due to their variability in progression and outcome. Since standardization is a cornerstone of experimental models, we aimed to develop a reliable and reproducible procedure for induction of peritonitis. MATERIALS AND METHODS A human stool batch was processed for -80° storage. For induction of peritonitis in fluid-resuscitated rats, a defined volume of stool suspension from this batch was injected intraperitoneally. For characterization of the model, physiologic and inflammatory changes were evaluated after sepsis induction. Survival analyses with the same batch were repeated in four independent experiments over a time period of 16 mo. RESULTS The polymicrobial infection resulted in severe peritoneal inflammation with a systemic increase in cytokines. The mortality rate at 15 h was 29% and this was reproducible over a 16 mo time period. If antibiotic treatment was applied, a 50% survival was achieved. Laboratory markers indicated a progressive multi-organ dysfunction, while blood gas analysis showed respiratory compensation of a metabolic acidosis, and maintenance of PaO(2). Intravital microscopy of the liver revealed an impaired microcirculation. A decreased hemostatic potential was demonstrated by rotational thromboelastometry. Despite clinical recovery within 3 d, surviving animals showed laboratory and histologic signs of persisting inflammation even after 2 wk. CONCLUSIONS This model reflects many features of human sepsis. Application of an infectious focus that is both quantitatively and qualitatively defined assures high reproducibility. Moreover, the procedure is simple and can be easily standardized.

Variations in the ratio between von Willebrand factor and its cleaving protease during systemic inflammation and association with severity and prognosis of organ failure.

Von Willebrand factor (VWF) and related parameters as well as the protease activity regulating its biological activity were measured in plasma of healthy controls and patients with different cause and severity of systemic inflammation to examine the efficacy of the measures to detect highly prothrombotic states including thrombotic microangiopathy (TMA), one of the sequelae of sepsis. Plasma levels of VWF increased with increasing severity of systemic inflammation, probably due to activation of the endothelium. In parallel, the proteolytic activity of VWF inactivating protease, ADAMTS13, stepwise declined with the severity of inflammation, emphasizing the role of VWF-triggered platelet aggregation on the endothelium subsequently followed by development of TMA. As a consequence, the ratio of VWF antigen level and ADAMTS13 activity was significantly higher in patients with inflammation and sepsis, suggesting that this ratio might be more useful for the diagnosis of highly prothrombotic states including TMA than VWF multimer analysis alone. These findings suggest that ADAMTS13, VWF and related parameters, even in a combined approach, might be useful for the diagnosis and the therapeutic monitoring of patients with sepsis associated thrombotic microangiopathy.

The Balance between von-Willebrand Factor and its Cleaving Protease ADAMTS13: Biomarker in Systemic Inflammation and Development of Organ Failure?

PURPOSE This review investigates and highlights the activity of Willebrand factor (VWF) and its cleaving protease as biomarkers of the development of multiple organ dysfunction in infectious and noninfectious systemic inflammatory response syndrome. STATE OF THE ART Ultra-large VWF (ULVWF) multimers activate platelets resulting in a prothrombotic situation. Systemic inflammation is associated with increased ULVWF plasma level and a decreased ADAMTS13 activity. The potential role of ADAMTS13 as a diagnostic and prognostic marker of disseminated intravascular coagulopathy is largely underestimated. SUMMARY VWF is an acute phase protein and its plasma level increases in systemic inflammation. When released from endothelial cells and platelets, the native multimeric glycoprotein is mostly present in the ultralarge form (ULVWF), which may have a major clinical significance under proinflammatory conditions. ULVWF-multimers may activate endothelial cells and platelets simultaneously. The multimers undergo limited proteolysis by a specific plasma metalloprotease known as ADAMTS13 (a disintegrin and metalloprotease with thrombospondin motif), thus, in healthy individuals only marginal amounts of circulating ULVWF are detectable. Severe hereditary or acquired ADAMTS13 deficiency causes thrombotic thrombocytopenic purpura (TTP), which contributes to prothrombotic coagulation abnormalities preceding organ dysfunction systemic inflammatory response syndrome (SIRS). In proinflammatory conditions, ADAMTS13 activity decreases due to various mechanisms, (i) down regulation on a transcriptional level, (ii) proteolytic degradation, and (iii) consumption due to the high substrate level. Marked dysbalance as found in patients with severe sepsis or septic shock results in substantial amounts of plasma ULVWF. This level of dysbalance is negatively correlated with platelet count and positively correlated with the severity of inflammation and the degree of organ failure.