Bernd Alt-Epping, Volker Arndt, Stefan Delorme, Norbert Frickhofen, Susanne Friedrich, Stefan Fuxius, Uwe Haberkorn, Uwe Haverkamp, Robert M. Hermann, Silke Hermann, Christoph Kahl, Matthias Kleiß, Herbert Koch, Florian Lordick, Bernd Oliver Maier, Ute Mons, Sabine Mousset, Birgitt van Oorschot, Franz-Josef Prott, Konrad K. Richter, Ulrich Wedding, Eva C. Winkler, Hendrik A. Wolff, and Stefan Zettl
Martin Hauer-Jensen, Konrad K. Richter, Magne K. Fagerhol, and Junru Wang
We examined whether early radiation-induced granulocyte transmigration (assessed by the fecal transferrin excretion ELISA assay) predicts subsequent development of (consequential) chronic radiation enteropathy. After accounting for the effect of radiation dose, transferrin excretion remained an independent predictor of overall tissue injury, intestinal fibrosis, and mucosal ulcers, but not TGF-β immunoreactivity.
Junru Wang, Konrad K. Richter, Martin Hauer-Jensen, and Ching-Ching Sung
Transforming growth factor beta1 (TGF-beta1) appears to play an important role in the pathogenesis of chronic radiation-induced fibrosis in the intestine and several other organs. TGF-beta1 is secreted as a non-biologically active complex and its function depends on activation. In vitro data suggest that the mannose 6-phosphate/insulin-like growth factor-beta (M6P/IGF-II) receptor is involved in the mechanism of TGF-beta1 activation. Thus, we used a rat model of radiation enteropathy to examine the potential role of the M6P/IGF-II receptor in the in vivo regulation of TGF-beta1 activity and localization.A scrotal hernia containing a loop of small intestine was created in male rats. The intestine in the scrotum was exposed to 0, 12, or 21 Gy single dose X-radiation. Groups of rats were euthanized 1 day and 2, 6 and 26 weeks after irradiation. Histopathologic injury was assessed with a radiation injury score (RIS). Computerized image analysis was used to identify M6P/IGF-II receptor-positive cells and to quantify extracellular matrix-associated TGF-beta1 immunoreactivity. Changes in urokinase plasminogen activator (uPA), tissue-like plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) immunoreactivity were also assessed.In normal (sham-irradiated) intestine, M6P/IGF-II immunoreactivity was confined to relatively weak, but specific epithelial staining. Irradiated intestine exhibited a highly significant time- and dose-dependent increase in the number of M6P/IGF-II receptor-positive cells (P0.001). There was a striking spatial shift of M6P/IGF-II receptor immunoreactivity from epithelium during the early post-radiation phase to stromal cells, most notably fibroblasts during the later stages of injury. Irradiated intestine exhibited distinct co-localization of M6P/ IGF-II receptor-positive cells and extracellular matrix-associated TGF-beta1 in areas of histopathologic injury. There were highly significant associations between the number of M6P/IGF-II receptor-positive stromal cells and TGF-beta1 immunoreactivity (P0.001), radiation-induced fibrosis (P0.001) and RIS (P0.001). Endothelial tPA immunoreactivity decreased significantly after irradiation (P0.001), whereas uPA and PAI-1 immunoreactivity levels appeared to be unchanged.M6P/IGF-II receptor upregulation may be a key factor in the in vivo control of TGF-beta1 activity and responsible for the tissue specificity of TGF-beta1 action after irradiation.
Ching-Ching Sung, Bradley M. Hughes, Konrad K. Richter, Louis M. Fink, and Martin Hauer-Jensen
Background and purpose: Radiation enteropathy is characterized by locally elevated levels of inflammatory and fibrogenic cytokines. Microvascular injury may sustain these alterations through persistent local hypercoagulopathy, platelet aggregation, leukocyte adhesion and release of biologically active mediators. This study assessed the relationship of endothelial thrombomodulin (TM), a key regulator of the protein C anticoagulant pathway and marker of endothelial function, with transforming growth factor β (TGF-β) immunoreactivity and morphologic alterations in radiation enteropathy. Materials and methods: Small bowel resection specimens from 9 patients with radiation enteropathy were analyzed by computerized quantitative immunohistochemistry using antibodies against TM, von Willebrand factor (vWF) and TGF-β. Identical measurements were performed on intestinal resection specimens from otherwise healthy penetrating trauma victims and on archived small intestines. A previously validated image analysis technique was used to assess submucosal vessels for TM and vWF immunoreactivity, and the intestinal wall for total extracellular matrix-associated TGF-β immunoreactivity. Results: Specimens from irradiated patients showed prominent submucosal and subserosal thickening and fibrosis, and obliterative vasculopathy. Control specimens were histopathologically normal. Vascular density and vWF immunoreactivity were similar in radiation enteropathy patients and controls. The image-analysis techniques were highly reproducible, with correlation coefficients for repeated measurements ranging from 0.86 to 0.93. Radiation enteropathy specimens exhibited a highly significant reduction in the number and proportion of TM-positive submucosal vessels per unit area (P < 0.0001) and increased intestinal wall TGF-β immunoreactivity (P = 0.002). Conclusions: These data support the theory that sustained endothelial dysfunction is involved in the molecular pathogenesis of radiation enteropathy, and point to TM as important in the chronic nature of radiation enteropathy and a potential target for prophylactic and therapeutic interventions.
Konrad K. Richter, J.C. Carr, Ching-Ching Sung, Magne K. Fagerhol, J.M. Winkler, and Martin Hauer-Jensen
Inflammatory cells are involved in the pathogenesis of tissue injury through release of cytokines and biologically active compounds. This study used a novel, noninvasive method to assess the association between granulocyte transmigration and structural and molecular changes in radiation enteropathy. A 4 cm loop of rat small intestine was exposed to 0, 2.8, 12, or 23 Gy localized irradiation. Feces was collected in metabolic cages before and 3, 7, 14, 28, and 42 days after irradiation. Granulocyte marker protein (GMP) was measured in buffer extracts of feces by enzyme-linked immunosorbent assay (ELISA). Irradiated and shielded intestine were procured at 2 and 26 weeks and assessed for histopathologic injury [radiation injury score (RIS)], ED-2 positive macrophages, and interleukin-1 alpha (IL-1 alpha) positive cells. Irradiated intestine exhibited characteristic histopathologic alterations and increased numbers of macrophages and IL-1 alpha positive cells. There was a highly significant dose-dependent increase in post-radiation GMP (P < 0.0001). Maximal GMP excretion occurred 3-7 days after irradiation. Six weeks after irradiation, GMP excretion had returned to normal in the 2.8 and 12 Gy groups, but was still 3.5 times higher in the 23 Gy group than in controls. The associations between early GMP excretion and RIS and fibrosis at 26 weeks were highly significant (P < 0.001 and P < 0.0001, respectively). Post-radiation granulocyte transmigration is dose-dependent and correlates with structural and molecular changes, as well as with subsequent chronic injury. The GMP assay is a sensitive, non-invasive indicator of acute intestinal radiation injury and a promising biological predictor of chronic toxicity. Our data underscore the importance of consequential mechanisms in radiation enteropathy.
Konrad K, Richter, Alexander, Dempster, Angelo P D, Tos, Rakesh, Premkumar, and Christopher, Jackson
The differential diagnosis of submucosal stomach lesions includes gastrointestinal stromal tumour (GIST), leiomyoma, synovial sarcomas, perineurioma, myxoid chondrosarcoma, myoepithelial tumour and other rare mesenchymal tumours. GISTs are well-defined lesions with distinctive morphologic and histogenetic characteristics that show 95% positive staining for CD117. Differential diagnosis of wild-type GISTs can be challenging. Here, we present two stomach tumours that were operated on in our surgical department. Both presented with positive immunoreactivity for CD117. In one tumour, c-Kit mutation analysis demonstrated positivity of exon 11_c.1674_1676delGGT, thus confirming the diagnosis of a GIST. Mutational analysis of the second stomach lesion demonstrated negativity for all known c-KIT and PDGFRA exons. In situ hybridisation ruled out a synovial sarcoma. An additional immunohistochemical staining for epithelial membrane antigen eventually confirmed the diagnosis of an extremely rare reticular perineurioma in the stomach, so far reported for the second time worldwide. Both patients have not shown any signs of recurrence 2 years after surgery. The presented cases emphasise the benefits of performing a mutational analysis in difficult GISTs, including wt-GISTs, and demonstrates the importance and challenges in differentiating GISTs from other mesenchymal tumours.
Martin Hauer-Jensen, Junru Wang, Marjan Boerma, and Konrad K. Richter
Purpose: Intestinal radiation injury (radiation enteropathy) is relevant to cancer treatment, as well as to radiation accidents and radiation terrorism scenarios. This study assessed the protective efficacy of orazipone, a locally-acting small molecule immunomodulator. Methods and Materials: Male rats were orchiectomized, a 4-cm segment of small bowel was sutured to the inside of the scrotum, a proximal anteperistaltic ileostomy was created for intraluminal drug administration, and intestinal continuity was re-established by end-to-side anastomosis. After three weeks postoperative recovery, the intestine in the “scrotal hernia” was exposed locally to single-dose or fractionated X-radiation. Orazipone (30 mg/kg/day) or vehicle was administered daily through the ileostomy, either during and after irradiation, or only after irradiation. Structural, cellular, and molecular aspects of intestinal radiation toxicity were assessed two weeks after irradiation. Results: Orazipone significantly ameliorated histologic injury and transforming growth factor-β immunoreactivity levels, both after single-dose and fractionated irradiation. Intestinal wall thickness was significantly reduced after single-dose and nonsignificantly after fractionated irradiation. Mucosal surface area and numbers of mast cells were partially restored by orazipone after single-dose irradiation. Conclusions: This work (1) demonstrates the utility of the ileostomy rat model for intraluminal administration of response modifiers in single-dose and fractionated radiation studies; (2) shows that mucosal immunomodulation during and/or after irradiation ameliorates intestinal toxicity; and (3) highlights important differences between single-dose and fractionated radiation regimens.
Louis Fink, Konrad K. Richter, Albertson Cm, Martin Hauer-Jensen, and Kudryk Bj
Thrombomodulin (TM) plays an important role in anticoagulation by forming a complex with thrombin, which subsequently activates protein C. TM is inactivated and downregulated by inflammatory cell mediators. This study examined whether bronchopneumonia is associated with changes in TM immunoreactivity, and whether a decrease in TM is accompanied by evidence of hypercoagulability, i.e. local deposition of fibrin. Double antibody staining for TM and fibrin was performed on lung tissue sections from patients who had died of pneumonia and from patients who had died rapidly, secondary to trauma. Inflammatory changes were assessed histologically and immunohistochemically using antibodies against interleukin-1alpha, tumor necrosis factor-alpha, and myeloperoxidase. Areas with bronchopneumonia exhibited markedly decreased endothelial TM staining of alveolar walls and small vessels. These changes were associated with prominent fibrin immunoreactivity. Some areas exhibited mild to moderate inflammation with little fibrin deposition and variable amounts of TM in adjacent vessels. This study is the first to relate changes of TM immunoreactivity levels to fibrin deposition in a human disease process. These data may have implications for pulmonary pathophysiology in patients with bronchopneumonia.
Martin Hauer-Jensen, Huaien Zheng, Konrad K. Richter, Ching-Ching Sung, and Junru Wang
The three mammalian transforming growth factor (TGF)-beta isoforms (TGF-beta1, TGF-beta2, and TGF-beta3) differ in their putative roles in radiation-induced fibrosis in intestine and other organs. Furthermore, tissue specificity of TGF-beta action may result from temporal or spatial changes in production and/or activation. The present study examined shifts in the cell types expressing TGF-beta mRNA relative to TGF-beta immunoreactivity and histopathological injury during radiation enteropathy development. A 4-cm loop of rat small intestine was locally exposed to O, 12, or 21-Gy single doses of x-irradiation. Sham-irradiated and irradiated intestine were procured 2 and 26 weeks after irradiation. Cells expressing the TGF-beta1, TGF-beta2, or TGF-beta3 transcripts were identified by in situ hybridization with digoxigenin-labeled riboprobes. Intestinal wall TGF-beta immunoreactivity was measured using computerized image analysis, and structural radiation injury was assessed by quantitative histopathology. Normal intestinal epithelium expressed transcripts for all three TGF-beta isoforms. Two weeks after irradiation, regenerating crypts, inflammatory cells, smooth muscle cells, and mesothelium exhibited increased TGF-beta1 expression and, to a lesser degree, TGF-beta2 and TGF-beta3 expression. Twenty-six weeks after irradiation, TGF-beta2 and TGF-beta3 expression had returned to normal. In contrast, TGF-beta1 expression remained elevated in smooth muscle, mesothelium, endothelium, and fibroblasts in regions of chronic fibrosis. Extracellular matrix-associated TGF-beta1 immunoreactivity was significantly increased at both observation times, whereas, TGF-beta2 and TGF-beta3 immunoreactivity exhibited minimal postradiation changes. Intestinal radiation injury is associated with overexpression of all three TGF-beta isoforms in regenerating epithelium. Radiation enteropathy was also associated with sustained shifts in the cellular sources of TGF-beta1 from epithelial cells to cells involved in the pathogenesis of chronic fibrosis. TGF-beta2 and TGF-beta3 did not exhibit consistent long-term changes. TGF-beta1 appears to be the predominant isoform in radiation enteropathy and may be more important in the mechanisms of chronicity than TGF-beta2 and TGF-beta3.
Martin Hauer-Jensen, Konrad K. Richter, Ching-Ching Sung, and Carl W. Langberg
Irradiated intestine consistently exhibits increased immunoreactivity of transforming growth factor beta-1 (TGF-beta 1). It is not known whether this increase occurs secondary to mucosal barrier disruption (consequential injury) or to injury in late-responding tissue compartments (primary radiation enteropathy). This study therefore assessed the association between TGF-beta immunoreactivity and specific consequential and primary histopathologic alterations. A small bowel loop was fixed inside the scrotum in male rats and subsequently exposed to either 18 daily fractions of 2.8 Gy or nine daily fractions of 5.6 Gy orthovoltage X-radiation. Radiation-induced induced intestinal complications were recorded and groups of animals were euthanized 2 and 26 weeks post-irradiation. Radiation injury was assessed with a histopathologic radiation injury score (RIS). Total TGF-beta was detected immunohistochemically and measured with interactive computerized image analysis. The image analysis technique yielded highly reproducible quantitation data. The 2.8-Gy group maintained mucosal integrity and had fewer intestinal complications, lower RIS and lower TGF-beta levels than the 5.6-Gy group. There was highly significant correlation between TGF-beta immunoreactivity and radiation injury at both observation times (P0.001 and P0.0001). At 2 weeks, TGF-beta immunoreactivity correlated with mucosal ulceration (P = 0.002), epithelial atypia (P = 0.005), and serosal thickening (P = 0.0004). At 26 weeks, TGF-beta levels correlated significantly with six of seven histopathologic parameters, most strikingly with vascular sclerosis (P = 0.0003). We conclude that mucosal barrier breakdown is closely associated with increased TGF-beta immunoreactivity in consequential radiation enteropathy. The highly significant correlation between TGF-beta expression levels and alterations in late-responding tissue compartments also suggest a role for TGF-beta in primary radiation enteropathy.
Martin Hauer-Jensen, Etsuko Abe, James W. Hardin, Konrad K. Richter, Junru Wang, and Ching-Ching Sung
Chronic intestinal radiation injury is associated with locally increased TGF-beta1 immunoreactivity that correlates with morphological alterations. However, the underlying mechanisms are not known. This study examined changes in intestinal TGF-beta1 immunoreactivity, steady-state TGF-beta1 mRNA levels, and cellular localization of TGF-beta1 mRNA during development of chronic radiation enteropathy in a rat model. A loop of small bowel was fixed inside the scrotum of orchiectomized male rats. The intestine was subsequently exposed locally to 0, 12 or 21 Gy X radiation. Intestine was procured at 24 h and 2, 6 and 26 weeks and subjected to histopathological analysis, quantitative immunohistochemistry with computerized image analysis, assessment of steady-state TGF-beta1 mRNA levels with quantitative reverse transcriptase polymerase chain reaction, and identification of cell types expressing TGF-beta1 mRNA with in situ hybridization. Intestine from the 21-Gy group exhibited more histopathological injury and increased TGF-beta immunoreactivity 2-26 weeks after irradiation compared to the 12-Gy group and sham-irradiated controls. TGF-beta1 mRNA in irradiated intestine increased up to six times relative to controls at 24 h and 2 weeks, was less at 6 weeks, and did not differ from controls at 26 weeks. In situ hybridization detected TGF-beta1 mRNA in epithelial and Paneth cells in control intestine. Irradiated intestine exhibited additional TGF-beta1 mRNA in inflammatory and fibroblast-like cells. We conclude that there is a radiation-induced shift in the cellular sources of TGF-beta1, and that Tgfb1 gene expression is increased mainly during the early phases of radiation enteropathy, preceding the increase in immunoreactivity and histopathological injury. Translational or post-translational mechanisms are likely involved in sustaining increased TGF-beta1 immunoreactivity levels during the chronic phase of radiation enteropathy.
Konrad K. Richter, Ching-Ching Sung, Martin Hauer-Jensen, and Carl W. Langberg
Driver SGW, Jackson MR, Richter K, Tomlinson P, Brockway B, Halliday BJ, Markie DM, Robertson SP, and Wade EM
Connective tissue disorders are a spectrum of diseases that affect the integrity of tissues including skin, vasculature, and joints. They are often caused by variants that disrupt genes encoding components of extracellular matrix (ECM). The fibulin glycoproteins are ECM proteins important for integrity of tissues including dermis, retina, fascia, and vasculature. The fibulin family consists of seven members (fibulins-1 to -7) and is defined by a fibulin-type domain at the C-terminus. The family is associated with human diseases, for instance a variant in FBLN1, encoding fibulin-1, is associated with synpolydactyly, while one in EFEMP1, encoding fibulin-3, causes Doyne honeycomb degeneration of the retina. Loss-of-function of fibulins-4 and -5 causes cutis laxa, while variants in fibulins-5 and -6 are associated with age-related macular degeneration. Of note, EFEMP1 is not currently associated with any connective tissue disorder. Here we show biallelic loss-of-function variants in EFEMP1 in an individual with multiple and recurrent abdominal and thoracic herniae, myopia, hypermobile joints, scoliosis, and thin translucent skin. Fibroblasts from this individual express significantly lower EFEMP1 transcript than age-matched control cells. A skin biopsy, visualised using light microscopy, showed normal structure and abundance of elastic fibres. The phenotype of this individual is remarkably similar to the Efemp1 knockout mouse model that displays multiple herniae with premature aging and scoliosis. We conclude that loss of EFEMP1 function in this individual is the cause of a connective tissue disorder with a novel combination of phenotypic features, and can perhaps explain similar, previously reported cases in the literature.
Rhodes J, Winder P, Stratheran M, McMillan M, Richter K, and McLeod M
Bayer O, Schummer C, Richter K, Fröber R, and Schummer W
Objective: Central venous catheterization is associated with a significant incidence of complications (5%-20%). The incidence of perforation is approximately 0.25% to 0.4%. To prevent cardiac tamponade associated with a high risk of death, Food and Drug Administration guidelines state that the tip of a central venous catheter (CVC) should not be placed in, or allowed to migrate into, the heart. Therefore, in order to prevent cardiac tamponade, a catheter should be placed above the pericardial reflection. Thus, the intrapericardial length of the superior vena cava (SVC) was studied. Neither the pericardial reflection nor the exact entrance to the right atrium (RA) can be identified by chest x-ray. The goal of this study was to evaluate the variability of the intrapericardial section in relation to the SVC., Design: Observational study., Interventions: The absolute length of the SVC, the upper edge of the pericardial reflection on the SVC, and the lateral and the medial intrapericardial sections of the SVC were recorded and statistically analyzed., Setting: Medical school: dissecting room at the Department of Anatomy., Study Population: Eighteen formalin-preserved adult cadavers., Measurements and Main Results: The median lengths measured were as follows: total SVC, 61 mm; intrapericardial section of the medial SVC, 32.5 mm; and lateral SVC, 20.5 mm. The intrapericardial section was related to the total length of the SVC on both sides (Spearman rank order, p < 0.05). The median difference of the SVC covered with pericardium between the lateral and medial side was 11 mm (range, 5-21). In 15 of 18 cadavers, the pericardial reflection ran within the medial third of the SVC. The lower third of the SVC was regularly covered by the pericardium. The duplication of the pericardium crossed the SVC in the medial third at a diagonal to horizontal angle., Conclusions: Catheters ending below the pericardial reflection, hence positioned in the caudal third of the SVC, are likely to run along the long axis of the vein and the risk for perforation is minimized. Therefore, the authors recommend placing all catheters below the pericardial reflection. According to the present data, CVCs placed approximately 30 mm above the RA border, thus complying with the Food and Drug Administration guidelines, still may have their tips positioned below the pericardial reflection. In this position, pericardial tamponade still may occur. Perforation above the pericardial reflection will result in a hemo- or hydrothorax/mediastinum. A bedside method to determine the position of the CVC with respect to the pericardial reflection (eg, electrocardiographic guidance) should be used.
Glei M, Klenow S, Sauer J, Wegewitz U, Richter K, and Pool-Zobel BL
Epidemiological findings have indicated that red meat increases the likelihood of colorectal cancer. Aim of this study was to investigate whether hemoglobin, or its prosthetic group heme, in red meat, is a genotoxic risk factor for cancer. Human colon tumor cells (HT29 clone 19A) and primary colonocytes were incubated with hemoglobin/hemin and DNA damage was investigated using the comet assay. Cell number, membrane damage, and metabolic activity were measured as parameters of cytotoxicity in both cell types. Effects on cell growth were determined using HT29 clone 19A cells. HT29 clone 19A cells were also used to explore possible pro-oxidative effects of hydrogen peroxide (H2O2) and antigenotoxic effects of the radical scavenger dimethyl sulfoxide (DMSO). Additionally we determined in HT29 clone 19A cells intracellular iron levels after incubation with hemoglobin/hemin. We found that hemoglobin increased DNA damage in primary cells (> or =10 microM) and in HT29 clone 19A cells (> or =250 microM). Hemin was genotoxic in both cell types (500-1000 microM) with concomitant cytotoxicity, detected as membrane damage. In both cell types, hemoglobin and hemin (> or =100 microM) impaired metabolic activity. The growth of HT29 clone 19A cells was reduced by 50 microM hemoglobin and 10 microM hemin, indicating cytotoxicity at genotoxic concentrations. Hemoglobin or hemin did not enhance the genotoxic activity of H2O2 in HT29 clone 19A cells. On the contrary, DMSO reduced the genotoxicity of hemoglobin, which indicated that free radicals were scavenged by DMSO. Intracellular iron increased in hemoglobin/hemin treated HT29 clone 19A cells, reflecting a 40-50% iron uptake for each compound. In conclusion, our studies show that hemoglobin is genotoxic in human colon cells, and that this is associated with free radical mechanisms and with cytotoxicity, especially for hemin. Thus, hemoglobin/hemin, whether available from red meat or from bowel bleeding, may pose genotoxic and cytotoxic risks to human colon cells, both of which contribute to initiation and progression of colorectal carcinogenesis.
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