Georg A. Roth

Alpha-Gal on bioprostheses: xenograft immune response in cardiac surgery

Background  The α‐Gal (Galα1,3‐Galβ1–4GlcNAc‐R) epitope is the major xenoantigen causing hyperacute rejection of pig organs transplanted into primates. Porcine bioprostheses are utilized in cardiac surgery. However, premature degeneration of bioprostheses has limited utilization in younger patients and the immune response remains elusive. We sought to investigate whether a specific α‐Gal immune response may play a role in this clinical scenario.

Thrombophilia associated with anti-CD154 monoclonal antibody treatment and its prophylaxis in nonhuman primates.

Background. The authors previously reported thromboembolic complications associated with anti-CD154 monoclonal antibody (mAb) treatment in nonhuman primates. The underlying mechanisms of this complication and its management have not been established. Methods. Eighty cynomolgus monkey renal allograft recipients treated with anti-CD154 mAb were studied for the incidence of thrombosis and its prophylaxis. Results. Without anticoagulation prophylaxis, thromboembolic complications were seen in 5 of 11 recipients. With addition of perioperative heparin, the incidence was decreased to 2 of 10. No further improvement was observed by adding intraoperative prostaglandin (PG) E1. However, addition of ketorolac tromethamine to PGE1 and heparin decreased the incidence of thrombosis (one of eight). Most recently, the authors have found that ketorolac administration alone resulted in no thrombosis in 25 consecutive recipients. Conclusions. Ketorolac is remarkably effective in preventing thromboembolism associated with anti-CD154 mAb treatment, suggesting the mechanism underlying this complication may be related to platelet activation leading to enhanced aggregation.

Susceptibility to programmed cell death in T-lymphocytes from septic patients: a mechanism for lymphopenia and Th2 predominance

Abstract Sepsis causes lymphopenia which is inversely correlated with patient survival. The role of apoptosis-specific immune-activation and activation-induced cell-death in sepsis is incompletely understood. Fifteen septic patients and 20 healthy controls were included. T-cell proliferation was measured by [3H]thymidine uptake. Apoptosis and cell phenotype were determined by FACS. sTNFR1, sCD95, interleukin-1β converting enzyme (sICE), and interleukin (IL)-10 were measured by ELISA. PHA and CD3-driven T-cell proliferation were significantly decreased in septic patients. The percentages of CD3+ and CD4+ T cells and CD19+ B cells were significantly reduced. Percent memory T-cells (CD45RO+) and cells undergoing apoptosis (CD95+/annexin-V+) were significantly increased in sepsis. Moreover, sCD95, sTNFRI, and ICE were significantly increased. Anti-CD3 antibody triggering induced a 56% increase of CD4 T-cell death in septic patients vs. 7.5% in controls relative to IgG. Serum level of IL-10, a Th2 cytokine, was enhanced. These findings strongly suggest that in septic patients Th1 T-cells are selectively susceptible to undergo apoptosis. This observation provides an additional pathophysiological concept in the genesis of Th2 dominance.

Elevated serum levels of epithelial cell apoptosis-specific cytokeratin 18 neoepitope m30 in critically ill patients.

Apoptosis of the epithelium is deemed to play a pivotal role in the pathogenesis of sepsis. A neoepitope in cytokeratin 18 (CK18), termed M30 neoantigen, becomes available at an early caspase cleavage event during apoptosis of epithelium-derived cells and is not detectable in vital or necrotic epithelial cells. A monoclonal antibody, M30, specifically recognizes a fragment of CK18 cleaved at Asp396 (M30 neoantigen). We used an enzyme-linked immunosorbent assay (ELISA) to measure M30 antigen levels in the sera of 15 septic patients. Healthy humans and critical ill patients suffering from severe trauma served as controls. Mann-Whitney U test was used to calculate significance, and a P value of <0.01 was considered to be statistically significant. Serum levels of the CK18 neoepitope M30 were significantly increased in septic patients (236.88 ± 47.4 U/L) versus trauma (97.2 ± 17.1 U/L) and healthy controls (66.9 ± 9.2 U/L) (P < 0.01 and P < 0.008, respectively). The increased serum level of the CK18 neoepitope in septic patients indicates a heightened apoptotic turnover in epithelial cells as compared with trauma patients and healthy controls. Interestingly, nonsurviving trauma patients exhibited a significant increase in the M30 neoantigen as compared with survivors and healthy controls (P < 0.003 and P < 0.002, respectively). The detection of CK18 neoepitope M30 in the serum might be a useful marker in tracing apoptotic epithelium in septic patients.

Heightened levels of circulating 20S proteasome in critically ill patients

Background  Recently, circulating proteasome core particles (20S proteasome) have been suggested as a marker of cell damage and immunological activity in autoimmune diseases. Aberrant leucocyte activation and increased lymphocyte apoptosis with consecutive T‐cell unresponsiveness is deemed to play a pivotal role in the sepsis syndrome. Moreover sepsis‐induced muscle proteolysis mainly reflects ubiqutin proteasome‐dependent protein degradation. We therefore sought to investigate serum levels of 20S proteasome in critical ill patients.

Aberrant T cell activation and heightened apoptotic turnover in end-stage renal failure patients: a comparative evaluation between non-dialysis, haemodialysis, and peritoneal dialysis

Patients in end-stage renal disease (ESRD) have a high incidence of bacterial and viral infections. Fifteen non-dialysed (ND), 15 haemodialysed (HD), 15 patients with peritoneal dialysis (PD), and 15 healthy controls were included. T cell proliferation was measured by [3H]thymidine uptake. Apoptosis and cell phenotype were determined by FACS. sTNF-R1, sCD95, interleukin-1beta-converting enzyme (sICE), and interleukin (IL)-10 were measured by ELISA. PHA and CD3-driven T cell proliferation were significantly decreased in ESRD patients. CD3(+), CD19(+) B cells, and percentage of CD4(+) T cells were significantly reduced. Percent memory T cells (CD45RO(+)) and cells undergoing apoptosis (CD95(+)/Annexin V+) were significantly increased in ESRF. Moreover, sCD95, sTNFRI, and ICE were significantly increased. Serum level of IL-10, a Th2 cytokine, was enhanced. These findings strongly suggest that in ESRD patients Th1 T cells are selectively susceptible to undergo apoptosis. This observation provides an additional pathophysiological concept in the genesis of Th2 dominance.

Intravenous and intramyocardial injection of apoptotic white blood cell suspensions prevents ventricular remodelling by increasing elastin expression in cardiac scar tissue after myocardial infarction

Congestive heart failure developing after acute myocardial infarction (AMI) is a major cause of morbidity and mortality. Clinical trials of cell-based therapy after AMI evidenced only a moderate benefit. We could show previously that suspensions of apoptotic peripheral blood mononuclear cells (PBMC) are able to reduce myocardial damage in a rat model of AMI. Here we experimentally examined the biochemical mechanisms involved in preventing ventricular remodelling and preserving cardiac function after AMI. Cell suspensions of apoptotic cells were injected intravenously or intramyocardially after experimental AMI induced by coronary artery ligation in rats. Administration of cell culture medium or viable PBMC served as controls. Immunohistological analysis was performed to analyse the cellular infiltrate in the ischaemic myocardium. Cardiac function was quantified by echocardiography. Planimetry of the infarcted hearts showed a significant reduction of infarction size and an improvement of post AMI remodelling in rats treated with suspensions of apoptotic PBMC (injected either intravenously or intramoycardially). Moreover, these hearts evidenced enhanced homing of macrophages and cells staining positive for c-kit, FLK-1, IGF-I and FGF-2 as compared to controls. A major finding in this study further was that the ratio of elastic and collagenous fibres within the scar tissue was altered in a favourable fashion in rats injected with apoptotic cells. Intravenous or intramyocardial injection of apoptotic cell suspensions results in attenuation of myocardial remodelling after experimental AMI, preserves left ventricular function, increases homing of regenerative cells and alters the composition of cardiac scar tissue. The higher expression of elastic fibres provides passive energy to the cardiac scar tissue and results in prevention of ventricular remodelling.

Impaired T cell proliferation, increased soluble death-inducing receptors and activation-induced T cell death in patients undergoing haemodialysis

Haemodialysis is a widespread option for end‐stage renal disease (ESRD). Long‐term success of dialysis is, however, limited by a high rate of serious bacterial and viral infections. We compared T cell functions in ESRD patients undergoing haemodialysis (n = 20), or were not dialysed and received conventional medical treatment (n = 20). Healthy volunteers (n = 15) served as controls. The T cell phenotype was examined by immunofluorescence using fluorochrome‐labelled monoclonal antibodies and FACS analysis. The concentration of soluble CD95/Fas and of tumour necrosis factor‐α receptor type 1 (sTNFR1) in the sera was quantified by ELISA. Activation‐induced programmed T cell death was triggered by anti‐CD3/CD28 antibodies and measured by 7‐AAD staining. All immunological tests were performed at least 1 month after dialysis initiation. T cell proliferation in response to phytohaemagglutinin or anti‐CD3 monoclonal antibodies was moderately diminished in non‐dialysed patients and markedly reduced in haemodialysis patients compared to healthy controls (P < 0·01 and P < 0·001, respectively). In a mixed lymphocyte culture the proliferative response of T cells from dialysed patients was significantly diminished (P < 0·001). T cells of both non‐dialysed and dialysed patients have augmented CD95/Fas and CD45RO expression, increased sCD95/Fas and sTNFR1 release and spontaneously undergo apoptosis. Culture of T cells from haemodialysis patients with anti‐CD3/CD28 antibodies increased the proportion of CD4+ T cells committing activation‐induced cell death by a mean 7·5‐fold compared to T‐helper cells from non‐dialysed patients (P < 0·001). Renal failure and initiation of haemodialysis results in a reduced proliferative T cell response, an aberrant state of T cell activation and heightened susceptibility of CD4+ T cells to activation‐induced cell death.

HSP27 and HSP70 serum and urine levels in patients suffering from chronic kidney disease.

BACKGROUND Chronic kidney disease (CKD) is a condition associated with inflammation and high levels of uremic toxins and reactive oxygen species. As a counterregulation to systemic stress heat shock proteins (HSP) are increased expressed to minimize cell death and preserve cell integrity by inhibiting apoptotic pathways. The aim of this study was to determine HSP27 and HSP70 concentrations in sera and urine of patients suffering from CKD. METHODS Concentrations of HSP27 and HSP70 in urine and serum were determined in 119 patients with CKD stages 1 to 5 and 23 healthy volunteers by using ELISA technique. RESULTS HSP27 serum levels were significantly elevated in patients suffering from CKD stages 3 to 5 as well as fractional HSP27 excretion in stages 2-5 versus healthy controls. Absolute HSP70 urinary values were significantly elevated in stages 4 and 5 and fractional HSP70 excretion was increased in stage 5 compared to controls. Moreover, ROC curve analysis showed the potential of urine and especially serum HSP levels to identify various stages of CKD. CONCLUSION We provide evidence for elevated HSP27 concentrations in serum and urine and increased HSP70 excretion levels in patients suffering from CKD. Moreover, our results show that HSP levels might offer potential to examine the stages of CKD as well as the disease course which could further promote individually adjusted treatment planning.

Increased plasma zonulin in patients with sepsis

Introduction: Zonulin is a eukaryotic protein structurally similar to Vibrio cholerae’s zonula occludens toxin. It plays an important role in the opening of small intestine tight junctions. The loss of gut wall integrity during sepsis might be pivotal and has been described in various experimental as well as human studies. Increased levels of zonulin could be demonstrated in diseases associated with increased intestinal inflammation, such as celiac disease and type 1 diabetes. We therefore investigated the role of plasma levels of zonulin in patients with sepsis as a non-invasive marker of gut wall integrity. Materials and methods: Plasma level of zonulin was measured in 25 patients with sepsis, severe sepsis or septic shock according to ACCP/SCCM criteria at the first day of diagnosed sepsis. 18 non-septic post-surgical ICU-patients and 20 healthy volunteers served as control. Plasma levels were determined by using commercially available ELISA kit. Data are given as median and interquartile range (IQR). Results: Significantly higher plasma concentration of zonulin were found in the sepsis group: 6.61 ng/mL (IQR 3.51–9.46), as compared to the to the post-surgical control group: 3.40 ng/mL (IQR 2.14–5.70) (P = 0.025), as well as to the healthy group: 3.55 ng/mL (IQR 3.14–4.14) (P = 0.008). Conclusion: We were able demonstrate elevated levels of plasma zonulin, a potential marker of intestinal permeability in septic patients. Increased zonulin may serve as an additional mechanism for the observed increased intestinal permeability during sepsis and SIRS.