Carine J. Peutz-Kootstra

Aggravation of Anti-Myeloperoxidase Antibody-Induced Glomerulonephritis by Bacterial Lipopolysaccharide: Role of Tumor Necrosis Factor-α

Wegeners granulomatosis, microscopic polyangiitis, Churg-Strauss syndrome, and idiopathic pauci-immune necrotizing crescentic glomerulonephritis are associated with myeloperoxidase (MPO)-specific anti-neutrophil cytoplasmic autoantibodies (ANCAs). Clinical and experimental evidence indicates that ANCA and proinflammatory stimuli of infectious origin act synergistically to cause vasculitis. We tested this hypothesis in a recently developed mouse model of anti-MPO IgG-induced glomerulonephritis by using bacterial lipopolysaccharide (LPS) as the proinflammatory stimulus. Systemic administration of LPS dose dependently increased renal injury induced by anti-MPO IgG as demonstrated by increased glomerular crescent formation and glomerular necrosis. In the early phase, LPS enhanced anti-MPO IgG-induced glomerular neutrophil accumulation. Furthermore, a transient induction of circulating tumor necrosis factor (TNF)-alpha levels, followed by a marked increase in circulating MPO levels, was observed on administration of LPS. In vitro, anti-MPO IgG induced a respiratory burst in murine neutrophils only after priming with TNF-alpha. Finally, anti-TNF-alpha treatment attenuated, but did not prevent, the LPS-mediated aggravation of anti-MPO IgG-induced glomerulonephritis. In conclusion, our study demonstrates that ANCA and proinflammatory stimuli act synergistically to induce vasculitic disease and suggests potential benefits of inhibiting TNF-alpha bioactivity in treating human ANCA-associated necrotizing crescentic glomerulonephritis.

The Mannose-Binding Lectin-Pathway Is Involved in Complement Activation in the Course of Renal Ischemia-Reperfusion Injury

Ischemia-reperfusion (I/R) is an important cause of acute renal failure (ARF). The complement system appears to be essentially involved in I/R injury. However, via which pathway the complement system is activated and in particular whether the mannose-binding lectin (MBL)-pathway is activated is unclear. This tempted us to study the activation and regulation of the MBL-pathway in the course of experimental renal I/R injury and in clinical post-transplant ARF. Mice subjected to renal I/R displayed evident renal MBL-depositions, depending on the duration of warm ischemia, in the early reperfusion phase. Renal deposition of C3, C6 and C9 was observed in the later reperfusion phase. The deposition of MBL-A and -C completely co-localized with the late complement factor C6, showing that MBL is involved in complement activation in the course of renal I/R injury. Moreover, the degree of early MBL-deposition correlated with complement activation, neutrophil-influx, and organ-failure observed in the later reperfusion phase. In serum of mice subjected to renal I/R MBL-A, levels increased in contrast to MBL-C levels, which dropped evidently. In line, liver mRNA levels for MBL-A increased, whereas MBL-C levels decreased. Renal MBL mRNA levels rapidly dropped in the course of renal I/R. Finally, in human biopsies, MBL-depositions were observed early after transplantation of ischemically injured kidneys. In line with our experimental data, in ischemically injured grafts displaying post-transplant organ-failure extensive MBL depositions were observed in peritubular capillaries and tubular epithelial cells. In conclusion, in experimental renal I/R injury and clinical post-transplant ARF the MBL-pathway is activated, followed by activation of the complement system. These data indicate that the MBL-pathway is involved in ischemia-induced complement activation.

CD44 Expression Predicts Local Recurrence after Radiotherapy in Larynx Cancer

Purpose: To find molecular markers from expression profiling data to predict recurrence of laryngeal cancer after radiotherapy. Experimental Design: We generated gene expression data on pre-treatment biopsies from 52 larynx cancer patients. Patients developing a local recurrence were matched for T-stage, subsite, treatment, gender and age with non-recurrence patients. Candidate genes were then tested by immunohistochemistry on tumor material from a second series of 76 patients. Both series comprised early stage cancer treated with radiotherapy alone. Finally, gene expression data of eight larynx cancer cell lines with known radiosensitivity were analyzed. Results: Nineteen patients with a local recurrence were matched with 33 controls. Gene sets for hypoxia, proliferation and intrinsic radiosensitivity did not correlate with recurrence, whereas expression of the putative stem cell marker CD44 did. In a supervised analysis, probes for all three splice variants of CD44 on the array appeared in the top 10 most significantly correlated with local recurrence. Immunohistochemical analysis of CD44 expression on the independent validation series confirmed CD44s predictive potential. In 8 larynx cancer cell lines, CD44 gene expression did not correlate with intrinsic radiosensitivity although it did correlate significantly with plating efficiency, consistent with a relationship with stem cell content. Conclusions: CD44 was the only biological factor tested which significantly correlated with response to radiotherapy in early stage larynx cancer patients, both at the mRNA and protein levels. Further studies are needed to confirm this and to assess how general these findings are for other head and neck tumor stages and sites. Clin Cancer Res; 16(21); 5329–38. ©2010 AACR.

Activation of the complement system in human nonalcoholic fatty liver disease

Activation of the innate immune system plays a major role in nonalcoholic fatty liver disease (NAFLD). The complement system is an important component of innate immunity that recognizes danger signals such as tissue injury. We aimed to determine whether activation of the complement system occurs in NAFLD, to identify initiating pathways, and to assess the relation between complement activation, NAFLD severity, apoptosis, and inflammatory parameters. Liver biopsies of 43 obese subjects with various degrees of NAFLD and of 10 healthy controls were analyzed for deposition of complement factors C1q, mannose‐binding lectin (MBL), C4d, activated C3, and membrane attack complex (MAC)‐associated C9. Furthermore, hepatic neutrophil infiltration, apoptosis, and pro‐inflammatory cytokine expression were quantified. Whereas complement activation was undetectable in the liver of healthy subjects, 74% of the NAFLD patients showed hepatic deposition of activated C3 and C4d. C1q as well as MBL accumulation was found in most activated C3‐positive patients. Strikingly, 50% of activated C3‐positive patients also displayed MAC‐associated C9 deposition. Deposition of complement factors was predominantly seen around hepatocytes with macrovesicular steatosis. Subjects showing accumulation of activated C3 displayed increased numbers of apoptotic cells. Importantly, hepatic neutrophil infiltration as well as interleukin (IL)‐8 and IL‐6 expression was significantly higher in patients showing activated C3 deposition, whereas patients with C9 deposition additionally had increased IL‐1β expression. Moreover, nonalcoholic steatohepatitis (NASH) was more prevalent in patients showing hepatic C9 or activated C3 deposition. Conclusion: There is widespread activation of the complement system in NAFLD, which is associated with disease severity. This may have important implications for the pathogenesis and progression of NAFLD given the function of complement factors in clearance of apoptotic cells, hepatic fibrosis, and liver regeneration. (HEPATOLOGY 2009.)

Role of arginine in superficial wound healing in man

Arginine supplementation has been identified as advantageous in experimental wound healing. However, the mechanisms underlying this beneficial effect in tissue repair remain unresolved. Animal studies suggest that the beneficial role of arginine supplementation is mediated, at least in part through NO. The latter component mediates processes involved in tissue repair, including angiogenesis, epithelialization and collagen formation. This prospective study is performed to investigate arginine metabolism in acute surgical wounds in man. Expression of enzymes, known to be involved in arginine metabolism, was studied in donor sites of skin grafts of 10 hospitalized patients undergoing skin transplantation. Plasma and wound fluid levels of arginine metabolites (ornithine, citrulline, nitrate and nitrite = NOx) were measured using High Performance Liquid Chromatography. Expression of iNOS, eNOS, arginase-1 and arginase-2 was studied by immunohistochemistry in paraffin sections of skin tissue. Arginase-1 concentration was measured in plasma and wound fluid using ELISA. Arginase-2 was determined using Western blot analysis. We observed increased levels of citrulline, ornithine, NOx and arginase-1 in wound fluid when compared with plasma. Arginase-2 was expressed in both plasma and wound fluid and seemed higher in plasma. iNOS was expressed by neutrophils, macrophages, fibroblasts, keratinocytes and endothelial cells upon wounding, whereas eNOS reactivity was observed in endothelial cells and fibroblasts. Arginase-1 was expressed in neutrophils post-wounding, while arginase-2 staining was observed in endothelial cells, keratinocytes, fibroblasts, macrophages and neutrophils. For the first time, human data support previous animal studies suggesting arginine metabolism for an NO- as well as arginase-mediated reparation of injured skin.

Acute ischemic injury to the renal microvasculature in human kidney transplantation

Increased understanding of the pathophysiology of ischemic acute kidney injury in renal transplantation may lead to novel therapies that improve early graft function. Therefore, we studied the renal microcirculation in ischemically injured kidneys from donors after cardiac death (DCD) and in living donor kidneys with minimal ischemia. During transplant surgery, peritubular capillaries were visualized by sidestream darkfield imaging. Despite a profound reduction in creatinine clearance, total renovascular resistance of DCD kidneys was similar to that of living donor kidneys. In contrast, renal microvascular perfusion in the early reperfusion period was 42% lower in DCD kidneys compared with living donor kidneys, which was accounted for by smaller blood vessel diameters in DCD kidneys. Furthermore, DCD kidneys were characterized by smaller red blood cell exclusion zones in peritubular capillaries and by greater production of syndecan-1 and heparan sulfate (main constituents of the endothelial glycocalyx) compared with living donor kidneys, providing strong evidence for glycocalyx degradation in these kidneys. We conclude that renal ischemia and reperfusion is associated with reduced capillary blood flow and loss of glycocalyx integrity. These findings form the basis for development of novel interventions to prevent ischemic acute kidney injury.

Histological assessment of preimplantation biopsies may improve selection of kidneys from old donors after cardiac death

Kidneys from old donors after cardiac death (DCD) may increase the donor pool but the prognosis of these kidneys is unsatisfactory. To improve these results, we retrospectively evaluated the diagnostic utility of published selection algorithms for old donor kidneys. We studied all DCD kidney transplantations between January 1, 1994 and July 1, 2005 at our institution (n = 199). Selection algorithms were evaluated in the subset of kidney transplantations from donors aged 60 years or older (n = 52). For histological assessment of kidney biopsies, glomerulosclerosis, tubular atrophy, interstitial fibrosis and vascular narrowing were blindly scored. Functional kidney weight was calculated as renal mass multiplied by the fraction of nonsclerosed glomeruli. Graft function and survival of kidneys from DCD aged 60 years or older were inferior to those from younger DCD. Histological scores were associated with kidney function and graft survival of old DCD kidney transplantations. Functional kidney weight was associated with kidney function but not graft survival, while donor glomerular filtration rate (GFR), donor age and machine perfusion characteristics were associated with neither of the clinical outcomes of interest. We conclude that histological assessment of preimplantation biopsies may improve the selection of kidneys from old DCD and may therefore contribute to expansion of the donor pool.

Transient AT1 receptor-inhibition in prehypertensive spontaneously hypertensive rats results in maintained cardiac protection until advanced age.

Objective In young spontaneously hypertensive rats (SHR), transient angiotensin II type 1 receptor (AT1R) blockade decreases blood pressure for a prolonged period. We tested the hypothesis that transient AT1R blockade in SHR leads to cardiac protection until advanced age. Method Wistar–Kyoto rats, SHR and transiently losartan-treated SHR (SHR-Los) (20 mg/kg per day; weeks 4–8 of age) were followed up until week 72 (n = 9 each group), including repeated echocardiography, radiotelemetric investigations and 24-h urine collection. End-point measurements comprised left ventricular function parameters, left ventricular histomorphology and molecular biology (types I and III collagen, brain natriuretic peptide, AT1R mRNA) as well as renal morphology. Results Prehypertensive treatment with losartan, but not with the general vasodilator hydralazine, reduced blood pressure until age 48 weeks. In untreated SHR, the end-diastolic volume increased from week 36 and the left ventricular ejection fraction fell from week 48. In contrast, age-related changes in end-diastolic volume and ejection fraction were comparable in SHR-Los and Wistar–Kyoto rats up to age 60 weeks. At age 72 weeks, the ejection fraction was reduced in SHR-Los but higher than that in untreated SHR (ejection fraction: Wistar–Kyoto rats, 58 ± 3%; SHR, 39 ± 3%; SHR-Los, 46 ± 3%; P < 0.01 and P < 0.05, respectively). The heart weight/body weight ratio (SHR-Los, 4.7 ± 0.1 g/kg; SHR, 5.2 ± 0.2 g/kg) and cardiac brain natriuretic peptide mRNA levels were improved by treatment. Left ventricular histomorphology and 24-h albuminuria were significantly improved in SHR-Los (41 ± 5 mg/day; SHR, 80 ± 22 mg/day; P < 0.05). Conclusion In young SHR, transient AT1R blockade, not blood pressure lowering, attenuates the development of hypertension and exerts cardioprotective effects up to age 72 weeks.

Apoptotic Cell Death Is Initiated During Normothermic Ischemia in Human Kidneys

Ischemic damage plays an important role in post‐transplant organ failure. Activation of the apoptotic cascade is crucially involved in post‐ischemic inflammation resulting in tissue damage and organ dysfunction. Here we investigate the initiation of the apoptotic cascade during normothermic ischemia in human kidneys using a model for normothermic ischemia with kidneys nephrectomized because of renal cell carcinoma. Ex vivo, kidneys were stored at 37°C, and consecutive biopsies were taken from disease‐free tissue. Pro‐ and anti‐apoptotic proteins were assessed by Western blotting and immunofluorescence. During normothermic ischemia the pro‐apoptotic proteins Bax and activated caspase‐9 increased with ischemia time, whereas caspase‐8 was not activated. The anti‐apoptotic proteins Bcl‐2 and cFLIP decreased in time. Data on Bcl‐2 and Bax were supported by immunofluorescence for Bcl‐2 and activated Bax. However, activation of the central effector caspase‐3, essential for execution of the apoptotic process, was not detected. In conclusion, during normothermic ischemia the apoptotic cascade in the human kidney is initiated, but not fulfilled. Our data show that the duration of ischemia significantly correlates with activation of the apoptotic cascade. These findings provide insight in the initiation of apoptotic cell‐death during warm ischemia and may be useful in the assessment of ischemic injury.

P16(INK4A) immunostaining is a strong indicator for high-risk-HPV-associated oropharyngeal carcinomas and dysplasias, but is unreliable to predict low-risk-HPV-infection in head and neck papillomas and laryngeal dysplasias

Human papillomavirus (HPV) is a risk factor for the development of benign and malignant mucosal head and neck lesions. P16INK4A is often used as a surrogate marker for HPV‐infection, although there is still controversy with respect its reliability. Our aim was to determine if p16INK4A overexpression can accurately predict both high‐risk and low‐risk‐HPV‐presence in (pre)malignant and benign head and neck lesions. P16INK4A immunohistochemistry was performed on paraffin‐embedded tissue sections of 162 oropharyngeal squamous cell carcinomas (OPSCC), 14 tonsillar and 23 laryngeal dysplasias, and 20 tonsillar and 27 laryngeal papillomas. PCR, enzyme‐immunoassay and FISH analysis were used to assess HPV‐presence and type. Of the 162 OPSCC and 14 tonsillar dysplasias, 51 (31%) and 10 (71%) were HPV16‐positive, respectively. All tonsillar papillomas were HPV‐negative and four laryngeal dysplasias and 26 laryngeal papillomas were positive for HPV6 or −11. P16INK4A immunohistochemistry revealed a strong nuclear and cytoplasmic staining in 50 out of 51 HPV16‐positive and 5 out of 111 HPV‐negative OPSCC (p < 0.0001) and in all HPV16‐positive tonsillar dysplasias, whereas highly variable staining patterns were detected in the papillomas and laryngeal dysplasias, irrespective of the HPV‐status. In addition, the latter lesions generally showed a higher nuclear than cytoplasmic p16INK4A immunostaining intensity. In conclusion, our data show that strong nuclear and cytoplasmic p16INK4A overexpression is a reliable surrogate indicator for HPV16 in OPSCC and (adjacent) dysplasias. For HPV6 or −11‐positive and HPV‐negative benign and premalignant lesions of the tonsil and larynx, however, p16INK4A immunostaining is highly variable and cannot be recommended to predict HPV‐presence.