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Abstract Background By modulating the expression levels of specific signal transduction molecules, the 26S proteasome plays a central role in determining cell cycle progression or arrest and cell survival or death in response to stress stimuli, including ionizing radiation. Inhibition of proteasome function by specific drugs results in cell cycle arrest, apoptosis and radiosensitization of many cancer cell lines. This study investigates whether there is also a concomitant increase in cellular radiosensitivity if proteasome inhibition occurs only transiently before radiation. Further, since proteasome inhibition has been shown to activate caspase-3, which is involved in apoptosis, and caspase-3 can cleave DNA-PKcs, which is involved in DNA-double strand repair, the hypothesis was tested that caspase-3 activation was essential for both apoptosis and radiosensitization following proteasome inhibition. Methods Prostate carcinoma PC-3 cells were treated with the reversible proteasome inhibitor MG-132. Cell cycle distribution, apoptosis, caspase-3 activity, DNA-PKcs protein levels and DNA-PK activity were monitored. Radiosensitivity was assessed using a clonogenic assay. Results Inhibition of proteasome function caused cell cycle arrest and apoptosis but this did not involve early activation of caspase-3. Short-time inhibition of proteasome function also caused radiosensitization but this did not involve a decrease in DNA-PKcs protein levels or DNA-PK activity. Conclusion We conclude that caspase-dependent cleavage of DNA-PKcs during apoptosis does not contribute to the radiosensitizing effects of MG-132.

Traumatic brain injury (TBI) is often associated with sustained attention and memory deficits. As persisting neuroinflammation and neurodegeneration may contribute to posttraumatic psychomotor dysfunction, we studied the relationship of brain cellular reactions three months after a weight-drop closed head injury in male mice with posttraumatic learning and memory using automated home-cage monitoring of socially housed mice in IntelliCages as well as tests for locomotor activity, anxiety and forepaw fine motor skills. One month after TBI, deficits in place learning and cognitive flexibility in reverse learning were clearly detectable in IntelliCages and these memory deficits correlated with the initial trauma severity on the functional neuroscore. While sucrose preference or its extinction were not influenced by TBI, traumatized mice performed significantly worse in a complex episodic memory learning task. In consecutive locomotor and forepaw skilled use tests, posttraumatic hyperactivity and impairment of contralateral paw use were evident. Analysis of cellular reactions to TBI three months after injury in selected defined regions of interest in the immediate lesion, ipsi- and contralateral frontoparietal cortex and hippocampus revealed a persistent microgliosis and astrogliosis which were accompanied by iron-containing macrophages and myelin degradation in the lesion area as well as with axonal damage in the neighboring cortical regions. Microglial and astroglial reactions in cortex showed a positive correlation with the initial trauma severity and a negative correlation with the spatial and episodic memory indicating a role of brain inflammatory reactions in posttraumatic memory deficits., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Anaplastic thyroid carcinoma (ATC) and metastatic poorly differentiated thyroid carcinomas (PDTCs) are rare aggressive malignancies with poor overall survival (OS) despite extensive multimodal therapy. These tumors are highly proliferative, with frequently increased tumor mutational burden (TMB) compared with differentiated thyroid carcinomas, and elevated programmed death ligand 1 (PD-L1) levels. These tumor properties implicate responsiveness to antiangiogenic and antiproliferative multikinase inhibitors such as lenvatinib, and immune checkpoint inhibitors such as pembrolizumab. Patients and Methods: In a retrospective study, we analyzed six patients with metastatic ATC and two patients with PDTC, who received a combination therapy of lenvatinib and pembrolizumab. Lenvatinib was started at 14-24 mg daily and combined with pembrolizumab at a fixed dose of 200 mg every three weeks. Maximum treatment duration with this combination was 40 months, and 3 of 6 ATC patients are still on therapy. Patient tumors were characterized by whole-exome sequencing and PD-L1 expression levels (tumor proportion score [TPS] 1-90%). Results: Best overall response (BOR) within ATCs was 66% complete remissions (4/6 CR), 16% stable disease (1/6 SD), and 16% progressive disease (1/6 PD). BOR within PDTCs was partial remission (PR 2/2). The median progression-free survival was 17.75 months for all patients, and 16.5 months for ATCs, with treatment durations ranging from 1 to 40 months (1, 4, 11, 15, 19, 25, 27, and 40 months). Grade III/IV toxicities developed in 4 of 8 patients, requiring dose reduction/discontinuation of lenvatinib. The median OS was 18.5 months, with three ATC patients being still alive without relapse (40, 27, and 19 months) despite metastatic disease at the time of treatment initiation (UICC and stage IVC). All patients with long-term (>2 years) or complete responses (CRs) had either increased TMB or a PD-L1 TPS >50%. Conclusions: Our results implicate that the combination of lenvatinib and pembrolizumab might be safe and effective in patients with ATC/PDTC and can result in complete and long-term remissions. The combination treatment is now being systematically examined in a phase II clinical trial (Anaplastic Thyroid Carcinoma Lenvatinib Pembrolizumab [ATLEP]) in ATC/PDTC patients.

Based on recent findings that show that depletion of factor XII (FXII) leads to better posttraumatic neurological recovery, we studied the effect of FXII-deficiency on post-traumatic cognitive and behavioral outcomes in female and male mice. In agreement with our previous findings, neurological deficits on day 7 after weight-drop traumatic brain injury (TBI) were significantly reduced in FXII -/- mice compared to wild type (WT) mice. Also, glycoprotein Ib (GPIb)-positive platelet aggregates were more frequent in brain microvasculature of WT than FXII -/- mice 3 months after TBI. Six weeks after TBI, memory for novel object was significantly reduced in both female and male WT but not in FXII -/- mice compared to sham-operated mice. In the setting of automated home-cage monitoring of socially housed mice in IntelliCages, female WT mice but not FXII -/- mice showed decreased exploration and reacted negatively to reward extinction one month after TBI. Since neuroendocrine stress after TBI might contribute to trauma-induced cognitive dysfunction and negative emotional contrast reactions, we measured peripheral corticosterone levels and the ration of heart, lung, and spleen weight to bodyweight. Three months after TBI, plasma corticosterone levels were significantly suppressed in both female and male WT but not in FXII -/- mice, while the relative heart weight increased in males but not in females of both phenotypes when compared to sham-operated mice. Our results indicate that FXII deficiency is associated with efficient post-traumatic behavioral and neuroendocrine recovery.

The weight-drop model is used widely to replicate closed-head injuries in mice; however, the histopathological and functional outcomes may vary significantly between laboratories. Because skull fractures are reported to occur in this model, we aimed to evaluate whether these breaks may influence the variability of the weight-drop (WD) model. Male Swiss Webster mice underwent WD injury with either a 2 or 5 mm cone tip, and behavior was assessed at 2 h and 24 h thereafter using the neurological severity score. The expression of interleukin (IL)-6, IL-1β, tumor necrosis factor-α, matrix metalloproteinase-9, and tissue inhibitor of metalloproteinase-1 genes was measured at 12 h and 1, 3, and 14 days after injury. Before the injury, micro-computed tomography (micro-CT) was performed to quantify skull thickness at the impact site. With a conventional tip diameter of 2 mm, 33% of mice showed fractures of the parietal bone; the 5 mm tip produced only 10% fractures. Compared with mice without fractures, mice with fractures had a severity-dependent worse functional outcome and a more pronounced upregulation of inflammatory genes in the brain. Older mice were associated with thicker parietal bones and were less prone to skull fractures. In addition, mice that underwent traumatic brain injury (TBI) with skull fracture had macroscopic brain damage because of skull depression. Skull fractures explain a considerable proportion of the variability observed in the WD model in mice-i.e., mice with skull fractures have a much stronger inflammatory response than do mice without fractures. Using older mice with thicker skull bones and an impact cone with a larger diameter reduces the rate of skull fractures and the variability in this very useful closed-head TBI model.

Purpose: REQUITE aimed to establish a resource for multi-national validation of models and biomarkers that predict risk of late toxicity following radiotherapy. The purpose of this article is to provide summary descriptive data., Methods: An international, prospective cohort study recruited cancer patients in 26 hospitals in eight countries between April 2014 and March 2017. Target recruitment was 5300 patients. Eligible patients had breast, prostate or lung cancer and planned potentially curable radiotherapy. Radiotherapy was prescribed according to local regimens, but centres used standardised data collection forms. Pre-treatment blood samples were collected. Patients were followed for a minimum of 12 (lung) or 24 (breast/prostate) months and summary descriptive statistics were generated., Results: The study recruited 2069 breast (99% of target), 1808 prostate (86%) and 561 lung (51%) cancer patients. The centralised, accessible database includes: physician- (47,025 forms) and patient- (54,901) reported outcomes; 11,563 breast photos; 17,107 DICOMs and 12,684 DVHs. Imputed genotype data are available for 4223 patients with European ancestry (1948 breast, 1728 prostate, 547 lung). Radiation-induced lymphocyte apoptosis (RILA) assay data are available for 1319 patients. DNA (n = 4409) and PAXgene tubes (n = 3039) are stored in the centralised biobank. Example prevalences of 2-year (1-year for lung) grade ≥2 CTCAE toxicities are 13% atrophy (breast), 3% rectal bleeding (prostate) and 27% dyspnoea (lung)., Conclusion: The comprehensive centralised database and linked biobank is a valuable resource for the radiotherapy community for validating predictive models and biomarkers., Patient Summary: Up to half of cancer patients undergo radiation therapy and irradiation of surrounding healthy tissue is unavoidable. Damage to healthy tissue can affect short- and long-term quality-of-life. Not all patients are equally sensitive to radiation "damage" but it is not possible at the moment to identify those who are. REQUITE was established with the aim of trying to understand more about how we could predict radiation sensitivity. The purpose of this paper is to provide an overview and summary of the data and material available. In the REQUITE study 4400 breast, prostate and lung cancer patients filled out questionnaires and donated blood. A large amount of data was collected in the same way. With all these data and samples a database and biobank were created that showed it is possible to collect this kind of information in a standardised way across countries. In the future, our database and linked biobank will be a resource for research and validation of clinical predictors and models of radiation sensitivity. REQUITE will also enable a better understanding of how many people suffer with radiotherapy toxicity., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Traumatic brain injury (TBI) is characterized by mechanical disruption of brain tissue due to an external force and by subsequent secondary injury. Secondary brain injury events include inflammatory responses and the activation of coagulation resulting in microthrombi formation in the brain vasculature. Recent research suggests that these mechanisms do not work independently. There is strong evidence that FXII and platelet activation connects both, inflammation and the formation of microthrombi. This review summarizes the current knowledge on posttraumatic microthrombus formation and its link to inflammation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Traumatic brain injury (TBI) is a devastating neurological condition and a frequent cause of permanent disability. Posttraumatic inflammation and brain edema formation, two pathological key events contributing to secondary brain injury, are mediated by the contact-kinin system. Activation of this pathway in the plasma is triggered by activated factor XII. Hence, we set out to study in detail the influence of activated factor XII on the abovementioned pathophysiological features of TBI., Methods: Using a cortical cryogenic lesion model in mice, we investigated the impact of genetic deficiency of factor XII and inhibition of activated factor XII with a single bolus injection of recombinant human albumin-fused Infestin-4 on the release of bradykinin, the brain lesion size, and contact-kinin system-dependent pathological events. We determined protein levels of bradykinin, intracellular adhesion molecule-1, CC-chemokine ligand 2, and interleukin-1β by enzyme-linked immunosorbent assays and mRNA levels of genes related to inflammation by quantitative real-time PCR. Brain lesion size was determined by tetrazolium chloride staining. Furthermore, protein levels of the tight junction protein occludin, integrity of the blood-brain barrier, and brain water content were assessed by Western blot analysis, extravasated Evans Blue dye, and the wet weight-dry weight method, respectively. Infiltration of neutrophils and microglia/activated macrophages into the injured brain lesions was quantified by immunohistological stainings., Results: We show that both genetic deficiency of factor XII and inhibition of activated factor XII in mice diminish brain injury-induced bradykinin release by the contact-kinin system and minimize brain lesion size, blood-brain barrier leakage, brain edema formation, and inflammation in our brain injury model., Conclusions: Stimulation of bradykinin release by activated factor XII probably plays a prominent role in expanding secondary brain damage by promoting brain edema formation and inflammation. Pharmacological blocking of activated factor XII could be a useful therapeutic principle in the treatment of TBI-associated pathologic processes by alleviating posttraumatic inflammation and brain edema formation.

Background: Traumatic brain injury (TBI) is a major cause of death and disability. Neuroinflammation contributes to acute damage after TBI and modulates long-term evolution of degenerative and regenerative responses to injury. The aim of the present study was to evaluate the relationship of microglia activation to trauma severity, brain energy metabolism, and cellular reactions to injury in a mouse closed head injury model using combined in vivo PET imaging, ex vivo autoradiography, and immunohistochemistry., Methods: A weight-drop closed head injury model was used to produce a mixed diffuse and focal TBI or a purely diffuse mild TBI (mTBI) in C57BL6 mice. Lesion severity was determined by evaluating histological damage and functional outcome using a standardized neuroscore (NSS), gliosis, and axonal injury by immunohistochemistry. Repeated intra-individual in vivo μPET imaging with the specific 18-kDa translocator protein (TSPO) radioligand [(18)F]DPA-714 was performed on day 1, 7, and 16 and [(18)F]FDG-μPET imaging for energy metabolism on days 2-5 after trauma using freshly synthesized radiotracers. Immediately after [(18)F]DPA-714-μPET imaging on days 7 and 16, cellular identity of the [(18)F]DPA-714 uptake was confirmed by exposing freshly cut cryosections to film autoradiography and successive immunostaining with antibodies against the microglia/macrophage marker IBA-1., Results: Functional outcome correlated with focal brain lesions, gliosis, and axonal injury. [(18)F]DPA-714-μPET showed increased radiotracer uptake in focal brain lesions on days 7 and 16 after TBI and correlated with reduced cerebral [(18)F]FDG uptake on days 2-5, with functional outcome and number of IBA-1 positive cells on day 7. In autoradiography, [(18)F]DPA-714 uptake co-localized with areas of IBA1-positive staining and correlated strongly with both NSS and the number of IBA1-positive cells, gliosis, and axonal injury. After mTBI, numbers of IBA-1 positive cells with microglial morphology increased in both brain hemispheres; however, uptake of [(18)F]DPA-714 was not increased in autoradiography or in μPET imaging., Conclusions: [(18)F]DPA-714 uptake in μPET/autoradiography correlates with trauma severity, brain metabolic deficits, and microglia activation after closed head TBI.

Objective: Traumatic brain injury is a major global public health problem for which specific therapeutic interventions are lacking. There is, therefore, a pressing need to identify innovative pathomechanism-based effective therapies for this condition. Thrombus formation in the cerebral microcirculation has been proposed to contribute to secondary brain damage by causing pericontusional ischemia, but previous studies have failed to harness this finding for therapeutic use. The aim of this study was to obtain preclinical evidence supporting the hypothesis that targeting factor XII prevents thrombus formation and has a beneficial effect on outcome after traumatic brain injury., Methods: We investigated the impact of genetic deficiency of factor XII and acute inhibition of activated factor XII with a single bolus injection of recombinant human albumin-fused infestin-4 (rHA-Infestin-4) on trauma-induced microvascular thrombus formation and the subsequent outcome in 2 mouse models of traumatic brain injury., Results: Our study showed that both genetic deficiency of factor XII and an inhibition of activated factor XII in mice minimize trauma-induced microvascular thrombus formation and improve outcome, as reflected by better motor function, reduced brain lesion volume, and diminished neurodegeneration. Administration of human factor XII in factor XII-deficient mice fully restored injury-induced microvascular thrombus formation and brain damage., Interpretation: The robust protective effect of rHA-Infestin-4 points to a novel treatment option that can decrease ischemic injury after traumatic brain injury without increasing bleeding tendencies. Ann Neurol 2016;79:970-982., (© 2016 The Authors. Annals of Neurology published by Wiley Periodicals, Inc. on behalf of American Neurological Association.)

Traumatic brain injury (TBI) is a major cause of mortality and morbidity worldwide. Despite improvements in acute intensive care, there are currently no specific therapies to ameliorate the effects of TBI. Successful therapeutic strategies for TBI should target multiple pathophysiologic mechanisms that occur at different stages of brain injury. The kallikrein-kinin system is a promising therapeutic target for TBI as it mediates key pathologic events of traumatic brain damage, such as edema formation, inflammation, and thrombosis. Selective and specific kinin receptor antagonists and inhibitors of plasma kallikrein and coagulation factor XII have been developed, and have already shown therapeutic efficacy in animal models of stroke and TBI. However, conflicting preclinical evaluation, as well as limited and inconclusive data from clinical trials in TBI, suggests that caution should be taken before transferring observations made in animals to humans. This review summarizes current evidence on the pathologic significance of the kallikrein-kinin system during TBI in animal models and, where available, the experimental findings are compared with human data.

Inflammation is a pathological hallmark of traumatic brain injury (TBI). Recent evidence suggests that immune cells such as lymphocytes are of particular relevance for lesion development after TBI. FTY720, a sphingosine-1-phosphate (S1P) receptor modulator, sequesters T lymphocytes in lymphoid organs and has been shown to improve outcome in a variety of neurological disease models. We investigated the mode of FTY720 action in models of TBI. Focal cortical cryolesion was induced in C57BL/6 mice treated with FTY720 (1mg/kg) or vehicle immediately before injury. Lesion size was assessed 24h later. Immune cells in the blood and brain were counted by flow cytometry and immunocytochemistry. The integrity of the blood-brain barrier was analyzed using Evans Blue dye. To validate the findings in a diffuse brain trauma model, FTY720-treated mice and controls were subjected to weight drop contusion injury and neurological deficits were assessed until day 7. As expected FTY720 significantly lowered the numbers of circulating lymphocytes and attenuated the invasion of immune cells into the damaged brain parenchyma. However, FTY720 was unable to improve lesion size or functional outcome in both trauma models at either stage, i.e. acute vs chronic. Accordingly, the extent of blood-brain barrier disruption and neuronal apoptosis was similar between FTY720-treated mice and controls. We conclude that pharmacological S1P receptor modulation is an unfavorable strategy to combat TBI. Moreover, our findings put into perspective the pathophysiological relevance of inflammatory cells in traumatic neurodegeneration., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

Traumatic brain injury (TBI) induces a strong inflammatory response which includes blood-brain barrier damage, edema formation and infiltration of different immune cell subsets. More recently, microvascular thrombosis has been identified as another pathophysiological feature of TBI. The contact-kinin system represents an interface between inflammatory and thrombotic circuits and is activated in different neurological diseases. C1-Inhibitor counteracts activation of the contact-kinin system at multiple levels. We investigated the therapeutic potential of C1-Inhibitor in a model of TBI. Male and female C57BL/6 mice were subjected to cortical cryolesion and treated with C1-Inhibitor after 1 h. Lesion volumes were assessed between day 1 and day 5 and blood-brain barrier damage, thrombus formation as well as the local inflammatory response were determined post TBI. Treatment of male mice with 15.0 IU C1-Inhibitor, but not 7.5 IU, 1 h after cryolesion reduced lesion volumes by ~75% on day 1. This protective effect was preserved in female mice and at later stages of trauma. Mechanistically, C1-Inhibitor stabilized the blood-brain barrier and decreased the invasion of immune cells into the brain parenchyma. Moreover, C1-Inhibitor had strong antithrombotic effects. C1-Inhibitor represents a multifaceted anti-inflammatory and antithrombotic compound that prevents traumatic neurodegeneration in clinically meaningful settings.

Acute ischemic stroke and traumatic brain injury are a major cause of mortality and morbidity. Due to the paucity of therapies, there is a pressing clinical demand for new treatment options. Successful therapeutic strategies for these conditions must target multiple pathophysiological mechanisms occurring at different stages of brain injury. In this respect, the kallikrein-kinin system is an ideal target linking key pathological hallmarks of ischemic and traumatic brain damage such as edema formation, inflammation, and thrombosis. In particular, the kinin receptors, plasma kallikrein, and coagulation factor XIIa are highly attractive candidates for pharmacological development, as kinin receptor antagonists or inhibitors of plasma kallikrein and coagulation factor XIIa are neuroprotective in animal models of stroke and traumatic brain injury. Nevertheless, conflicting preclinical evaluation as well as limited and inconclusive data from clinical trials suggest caution when transferring observations made in animals into the human situation. This review summarizes current evidence on the pathological significance of the kallikrein-kinin system during ischemic and traumatic brain damage, with a particular focus on experimental data derived from animal models. Experimental findings are also compared with human data if available, and potential therapeutic implications are discussed., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Delayed cerebral vasospasm following subarachnoid hemorrhage (SAH) is a serious medical complication, characterized by constriction of cerebral arteries leading to varying degrees of cerebral ischemia. Numerous clinical and experimental studies have been performed in the last decades; however, the pathophysiologic mechanism of cerebral vasospasm after SAH still remains unclear. Among a variety of experimental SAH models, the double hemorrhage rat model involving direct injection of autologous arterial blood into the cisterna magna has been used most frequently for the study of delayed cerebral vasospasm following SAH in last years. Despite the simplicity of the technique, the second blood injection into the cisterna magna may result in brainstem injury leading to high mortality. Therefore, a modified double hemorrhage model of cisterna magna has been developed in rat recently. We describe here step by step the surgical technique to induce double SAH and compare the degree of vasospasm with other cisterna magna rat models using histological assessment of the diameter and cross-sectional area of the basilar artery.

The two bradykinin receptors B1R and B2R are central components of the kallikrein-kinin system with different expression kinetics and binding characteristics. Activation of these receptors by kinins triggers inflammatory responses in the target organ and in most situations enhances tissue damage. We could recently show that blocking of B1R, but not B2R, protects from cortical cryolesion by reducing inflammation and edema formation. In the present study, we investigated the role of B1R and B2R in a closed head model of focal traumatic brain injury (TBI; weight drop). Increased expression of B1R in the injured hemispheres of wild-type mice was restricted to the later stages after brain trauma, i.e. day 7 (P<0.05), whereas no significant induction could be observed for the B2R (P>0.05). Mice lacking the B1R, but not the B2R, showed less functional deficits on day 3 (P<0.001) and day 7 (P<0.001) compared with controls. Pharmacological blocking of B1R in wild-type mice had similar effects. Reduced axonal injury and astroglia activation could be identified as underlying mechanisms, while inhibition of B1R had only little influence on the local inflammatory response in this model. Inhibition of B1R may become a novel strategy to counteract trauma-induced neurodegeneration.

The method to induce unilateral cryogenic lesions was first described in 1958 by Klatzo. We describe here an adaptation of this model that allows reliable measurement of lesion volume and vasogenic edema by 2, 3, 5-triphenyltetrazolium chloride-staining and Evans blue extravasation in mice. A copper or aluminium cylinder with a tip diameter of 2.5 mm is cooled with liquid nitrogen and placed on the exposed skull bone over the parietal cortex (coordinates from bregma: 1.5 mm posterior, 1.5 mm lateral). The tip diameter and the contact time between the tip and the parietal skull determine the extent of cryolesion. Due to an early damage of the blood brain barrier, the cryogenic cortical injury is characterized by vasogenic edema, marked brain swelling, and inflammation. The lesion grows during the first 24 hours, a process involving complex interactions between endothelial cells, immune cells, cerebral blood flow, and the intracranial pressure. These contribute substantially to the damage from the initial injury. The major advantage of the cryogenic lesion model is the circumscribed and highly reproducible lesion size and location.

Traumatic brain injury (TBI) is a result of an outside force causing immediate mechanical disruption of brain tissue and delayed pathogenic events. In order to examine injury processes associated with TBI, a number of rodent models to induce brain trauma have been described. However, none of these models covers the entire spectrum of events that might occur in TBI. Here we provide a thorough methodological description of a straightforward closed head weight drop mouse model to assess brain injuries close to the clinical conditions of human TBI.

Traumatic brain injury, a leading cause of death and disability, is a result of an outside force causing mechanical disruption of brain tissue and delayed pathogenic events which collectively exacerbate the injury. These pathogenic injury processes are poorly understood and accordingly no effective neuroprotective treatment is available so far. Experimental models are essential for further clarification of the highly complex pathology of traumatic brain injury towards the development of novel treatments. Among the rodent models of traumatic brain injury the most commonly used are the weight-drop, the fluid percussion, and the cortical contusion injury models. As the entire spectrum of events that might occur in traumatic brain injury cannot be covered by one single rodent model, the design and choice of a specific model represents a major challenge for neuroscientists. This review summarizes and evaluates the strengths and weaknesses of the currently available rodent models for traumatic brain injury.

The bacterial pathogen Legionella pneumophila responds to environmental changes by differentiation. At least two forms are well described: replicative bacteria are avirulent; in contrast, transmissive bacteria express virulence traits and flagella. Phenotypic analysis, Western blotting, and electron microscopy of mutants of the regulatory genes encoding RpoN, FleQ, FleR, and FliA demonstrated that flagellin expression is strongly repressed and that the mutants are nonflagellated in the transmissive phase. Transcriptome analyses elucidated that RpoN, together with FleQ, enhances transcription of 14 out of 31 flagellar class II genes, which code for the basal body, hook, and regulatory proteins. Unexpectedly, FleQ independent of RpoN enhances the transcription of fliA encoding sigma 28. Expression analysis of a fliA mutant showed that FliA activates three out of the five remaining flagellar class III genes and the flagellar class IV genes. Surprisingly, FleR does not induce but inhibits expression of at least 14 flagellar class III genes on the transcriptional level. Thus, we propose that flagellar class II genes are controlled by FleQ and RpoN, whereas the transcription of the class III gene fliA is controlled in a FleQ-dependent but RpoN-independent manner. However, RpoN and FleR might influence flagellin synthesis on a posttranscriptional level. In contrast to the commonly accepted view that enhancer-binding proteins such as FleQ always interact with RpoN to fullfill their regulatory functions, our results strongly indicate that FleQ regulates gene expression that is RpoN dependent and RpoN independent. Finally, FliA induces expression of flagellar class III and IV genes leading to the complete synthesis of the flagellum.

To transit from intra- to extracellular environments, Legionella pneumophila differentiates from a replicative/non-virulent to a transmissive/virulent form using the two-component system LetA/LetS and the global repressor protein CsrA. While investigating how both regulators act co-ordinately we characterized two ncRNAs, RsmY and RsmZ, that link the LetA/LetS and CsrA regulatory networks. We demonstrate that LetA directly regulates their expression and show that RsmY and RsmZ are functional in Escherichia coli and are able to bind CsrA in vitro. Single mutants have no (ΔrsmY) or a little (ΔrsmZ) impact on virulence, but the ΔrsmYZ strain shows a drastic defect in intracellular growth in Acanthamoeba castellanii and THP-1 monocyte-derived macrophages. Analysis of the transcriptional programmes of the ΔletA, ΔletS and ΔrsmYZ strains revealed that the switch to the transmissive phase is partially blocked. One major difference between the ΔletA, ΔletS and ΔrsmYZ strains was that the latter synthesizes flagella. Taken together, LetA activates transcription of RsmY and RsmZ, which sequester CsrA and abolish its post-transcriptional repressive activity. However, the RsmYZ-CsrA pathway appears not to be the main or only regulatory circuit governing flagella synthesis. We suggest that rather RpoS and LetA, by influencing LetE and probably cyclic-di-GMP levels, regulate motility in L. pneumophila.

Previously, we identified ladC in a cohort of genes that were present in Legionella pneumophila but absent in other Legionella species. Here we constructed a ladC mutant of L. pneumophila and assessed its ability to replicate in mammalian cell lines and Acanthamoeba castellanii. The ladC mutant was recovered in significantly lower numbers than wild-type L. pneumophila at early time points, which was reversed upon transcomplementation with ladC but not ladC(N430A/R434A), encoding a putative catalytically inactive derivative of the protein. In fact, complementation of ladC::Km with ladC(N430A/R434A) resulted in a severe replication defect within human and amoeba cell models of infection, which did not follow a typical dominant negative phenotype. Using differential immunofluorescence staining to distinguish adherent from intracellular bacteria, we found that the ladC mutant exhibited a 10-fold reduction in adherence to THP-1 macrophages but no difference in uptake by THP-1 cells. When tested in vivo in A/J mice, the competitive index of the ladC mutant dropped fivefold over 72 h, indicating a significant attenuation compared to wild-type L. pneumophila. Although localization of LadC to the bacterial inner membrane suggested that the protein may be involved in signaling pathways that regulate virulence gene expression, microarray analysis indicated that ladC does not influence the transcriptional profile of L. pneumophila in vitro or during A. castellanii infection. Although the mechanism by which LadC modulates the initial interaction between the bacterium and host cell remains unclear, we have established that LadC plays an important role in L. pneumophila infection.

Horizontal gene transfer probably contributes to evolution of Legionella pneumophila and its adaptation to different environments. Although horizontal gene transfer was observed in Legionella, the mechanism is still not specified. In this study we identified and analysed a new type of conjugation/type IVA secretion system (trb/tra) of L. pneumophila Corby, a virulent human isolate. Two similar versions of this conjugation system were identified, localized on two different genomic islands (Trb-1, 42,710 bp and Trb-2, 34,434 bp). Trb-1 and Trb-2 are integrated within the tRNA(Pro) gene (lpc2778) and the tmRNA gene (lpc0164), respectively. Both islands exhibit an oriT region and both can be excised from the chromosome forming episomal circles. Trb-1 was analysed in more detail. It is active and can be horizontally transferred to other Legionella strains by conjugation and then integrated into the genome in a site-specific manner within the tRNA(Pro) gene. We characterized the sequence of the excision and integration sites of Trb-1 in three different L. pneumophila strains. Here we demonstrate that L. pneumophila exhibits a functional oriT region and that genomic islands in Legionella can be mobilized and conjugated to other species of Legionella. Thus, we describe for the first time a mechanism that may explain the observed horizontal transfer of chromosomal DNA in Legionella.

Purpose: We retrospectively analyzed the impact of intraoperative radiation therapy (IORT) on long-term survival in patients with resectable gastric cancer., Methods and Materials: From 1991 to 2001, a total of 84 patients with gastric neoplasms underwent gastectomy or subtotal resection with IORT (23 Gy, 6-15 MeV; IORT-positive [IORT(+)] group). Patients with a history of additional neoadjuvant chemotherapy, histologically confirmed R1 or R2 resection, or reoperation with curative intention after local recurrence were excluded from further analysis. The remaining 61 patients were retrospectively matched with 61 patients without IORT (IORT-negative [IORT(-)] group) for Union Internationale Contre le Cancer (UICC) stage, patient age, histologic grading, extent of surgery, and level of lymph node dissection. Subgroups included postoperative UICC Stages I (n = 31), II (n = 11), III (n = 14), and IV (n = 5)., Results: Mean follow-up was 4.8 years in the IORT(+) group and 5.0 years in the IORT(-) group. The overall 5-year patient survival rate was 58% in the IORT(+) group vs. 59% in the IORT(-) group (p = 0.99). Subgroup analysis showed no impact of IORT on 5-year patient survival for those with UICC Stages I/II (76% vs. 80%; p = 0.87) and III/IV (21% vs. 14%, IORT(+) vs. IORT(-) group; p = 0.30). Perioperative mortality rates were 4.9% and 4.9% in the IORT(+) vs. IORT(-) group. Total surgical complications were more common in the IORT(+) than IORT(-) group (44.3% vs. 19.7%; p < 0.05). The locoregional tumor recurrence rate was 9.8% in the IORT(+) group., Conclusions: Use of IORT was associated with low locoregional tumor recurrence, but had no benefit on long-term survival while significantly increasing surgical morbidity in patients with curable gastric cancer.

Legionella spp. the causative agent of Legionnaires' disease is naturally found in fresh water where the bacteria parasitize intracellularly within protozoa. Upon aerosol formation via man-made water systems, Legionella can enter the human lung and cause a severe form of pneumonia. Here we review results from systematic comparative genome analysis of Legionella species with different pathogenic potentials. The complete genomes reveal that horizontal gene transfer has played an important role during the evolution of Legionella and indicate the importance of secretion machineries for the intracellular lifestyle of this pathogen. Moreover, we highlight recent findings on the in vivo transcriptional program of L. pneumophila and the regulatory networks involved in the biphasic life cycle. In order to understand how Legionella effectively subvert host cell functions for its own benefit the transcriptional host cell response upon infection of the model amoeba Dictyostelium discoideum was studied. The use of this model organism made it possible to develop a roadmap of host cell factors which significantly contribute to the uptake of L. pneumophila and the establishment of an ER-associated replicative vacuole.

Purpose: Recent reports suggest that cancer control may worsen if erythropoietin is administered. We investigated whether erythropoietin receptor expression on cancer cells may correlate with this unexpected finding., Patients and Methods: Cancer tissue from patients with advanced carcinoma of the head and neck (T3, T4, or nodal involvement) and scheduled for radiotherapy was assayed retrospectively for erythropoietin receptor expression by immunohistochemistry. Patients were anemic and randomized to receive epoetin beta (300 U/kg) or placebo under double-blind conditions, given three times weekly starting 10 to 14 days before and continuing throughout radiotherapy. We administered 60 Gy following complete resection or 64 Gy subsequent to microscopically incomplete resection; 70 Gy were given following macroscopically incomplete resection or for definitive radiotherapy alone. We determined if the effect of epoetin beta on locoregional progression-free survival was correlated with the expression of erythropoietin receptors on cancer cells using a Cox proportional hazards regression model., Results: We studied 154 of 157 randomly assigned patients; 104 samples were positive, and 50 were negative for receptor expression. Locoregional progression-free survival was substantially poorer if epoetin beta was administered to patients positive for receptor expression compared with placebo (adjusted relative risk, 2.07; 95% CI, 1.27 to 3.36; P < .01). In contrast, epoetin beta did not impair outcome in receptor-negative patients (adjusted relative risk, 0.94; 95% CI, 0.47 to 1.90; P = .86). The difference in treatment associated relative risks (2.07 v 0.94) was borderline statistically significant (P = .08)., Conclusion: Erythropoietin might adversely affect prognosis of head and neck cancer patients if cancer cells express erythropoietin receptors.

Background: We studied the quality of web sites containing information on gastrointestinal cancer, focusing on the way these web sites dealt with the special concerns of these patients., Materials and Methods: Searching the Internet for German-language gastroenterological cancer web sites, we collected 9,947 web pages from 14 search engines. Evaluation was done with a 36-item questionnaire. Information quality, availability of the web sites, and web site attributes considering patients' concerns and potential embarrassment were analyzed using a scoring system., Results: Belonging to 165 web sites, 1,763 of 9,947 (17.7%) web pages found by search engines provided relevant information. Five hundred forty-seven (5.5%) hits were partly relevant, and 7,637 (76.8%) were irrelevant or not available. Most web sites reported about surgery (92.1%), chemotherapy (88.5%), and radiotherapy (73.9%). Of the web sites, 46.7% (n=77), 34.6% (n=57), and 21.8% (n=36) gave information about the author(s) itself, their qualifications, and references of their information, respectively. Search engines ranked web sites giving no information on evidence-based medicine higher than other web sites, whereas web sites providing this information accurately showed higher link popularities. Patients' concerns and potential embarrassment were best addressed by gastrointestinal web sites initiated by private individuals or web sites directed to both a patient and physician audience., Conclusions: With regard to gastrointestinal cancer web sites, many search engines may be ineffective, and patient emotional needs and concerns are often disregarded. Also, physicians should guide their patients through the Internet to find high-quality information and use link-popularity-based search strategies.

Aim: To evaluate the long-term outcome of standard 5-FU based adjuvant or neoadjuvant radiochemotherapy and to identify the predictive factors, especially anemia before and after radiotherapy as well as hemoglobin increase or decrease during radiotherapy., Methods: Two hundred and eighty-six patients with Union International Contre Cancer (UICC) stage II and III rectal adenocarcinomas, who underwent resection by conventional surgical techniques (low anterior or abdominoperineal resection), received either postoperative (n=233) or preoperative (n=53) radiochemotherapy from January 1989 until July 2002. Overall survival (OAS), cancer-specific survival (CSS), disease-free survival (DFS), local-relapse-free (LRS) and distant-relapse-free survival (DRS) were evaluated using Kaplan-Meier, Log-rank test and Cox's proportional hazards as statistical methods. Multivariate analysis was used to identify prognostic factors. Median follow-up time was 8 years., Results: Anemia before radiochemotherapy was an independent prognostic factor for improved DFS (risk ratio 0.76, P=0.04) as well as stage, grading, R status (free radial margins), type of surgery, carcinoembryonic antigen (CEA) levels, and gender. The univariate analysis revealed that anemia was associated with impaired LRS (better local control) but with improved DFS. In contrast, hemoglobin decrease during radiotherapy was an independent risk factor for DFS (risk ratio 1.97, P=0.04). During radiotherapy, only 30.8% of R0-resected patients suffered from hemoglobin decrease compared to 55.6% if R1/2 resection was performed (P=0.04). The 5-year OAS, CSS, DFS, LRS and DRS were 47.0%, 60.0%, 41.4%, 67.2%, and 84.3%, respectively. Significant differences between preoperative and postoperative radiochemotherapy were not found., Conclusion: Anemia before radiochemotherapy and hemoglobin decrease during radiotherapy have no predictive value for the outcome of rectal cancer. Stage, grading, R status (free radial margins), type of surgery, CEA levels, and gender have predictive value for the outcome of rectal cancer.

Increased formation of prostaglandin E2 (PGE2) is a key part of hyperprostaglandin E syndrome/antenatal Bartter syndrome (HPS/aBS), a renal disease characterized by NaCl wasting, water loss, and hyperreninism. Inhibition of PGE2 formation by cyclo-oxygenase inhibitors significantly lowers patient mortality and morbidity. However, the pathogenic role of PGE2 in HPS/aBS awaits clarification. Chronic blockade of the Na-K-2Cl co-transporter NKCC2 by diuretics causes symptoms similar to HPS/aBS and provides a useful animal model. In wild-type (WT) mice and in mice lacking distinct PGE2 receptors (EP1-/-, EP2-/-, EP3-/-, and EP4-/-), the effect of chronic furosemide administration (7 d) on urine output, sodium and potassium excretion, and renin secretion was determined. Furthermore, furosemide-induced diuresis and renin activity were analyzed in mice with defective PGI2 receptors (IP-/-). In all animals studied, furosemide stimulated a rise in diuresis and electrolyte excretion. However, this effect was blunted in EP1-/-, EP3-/-, and EP4-/- mice. Compared with WT mice, no difference was observed in EP2-/- and IP-/- mice. The furosemide-induced increase in plasma renin concentration was significantly decreased in EP4-/- mice and to a lesser degree also in IP-/- mice. Pharmacologic inhibition of EP4 receptors in furosemide-treated WT mice with the specific antagonist ONO-AE3-208 mimicked the changes in renin mRNA expression, plasma renin concentration, diuresis, and sodium excretion seen in EP4-/- mice. The GFR in EP4-/- mice was not changed compared with that in WT mice, which indicated that blunted diuresis and salt loss seen in EP4-/- mice were not a consequence of lower GFR. In summary, these findings demonstrate that the EP4 receptor mediates PGE2-induced renin secretion and that EP1, EP3, and EP4 receptors all contribute to enhanced PGE2-mediated salt and water excretion in the HPS/aBS model.

Background: This analysis was undertaken to evaluate the impact of pre-radiotherapy CEA and CA 19-9 values on clinical outcome of locally advanced rectal cancer., Patients and Methods: Retrospective data were collected from patients (n=203) with UICC stage II and III rectal adenocarcinomas, who underwent low anterior or abdominoperineal resection and received post-operative or pre-operative radiochemotherapy from January 1989 until July 2002. The rates of survival and distant and local recurrences were evaluated using Kaplan-Meier survival analysis, Log-rank test and Cox's proportional hazards (median follow-up 8 years). Multivariate analysis was used to assess the prognostic value of CEA and CA 19-9., Results: The 5-year actuarial rates for patients with normal (n =118) and elevated (n=88) CEA values were as follows: overall survival 62.4% and 32.0% (p<0.001), local control 73.5% and 55.0% (p=0.007), and absence of distant metastasis 83.3% and 88.0% (n.s.), respectively. Similar results were obtained for patients with normal (n=82) and elevated (n = 10) CA 19-9 values: overall survival 60.7% and 14.0% (p=0.007), local control 83.7% and 80.0% (n.s.), and absence of distant metastasis 64.9% and 75.0% (n.s.), respectively. After adjustment for TNM stage, sex, age, LDH, tumor site and grading, the elevation of CEA proved to be an independent prognostic factor for overall survival (relative risk of 1.01 per ng/ml, CI 1.002 - 1.01; p=0.005)., Conclusion: This study confirmed the prognostic value of pre-radiotherapy CEA and CA 19-9 in patients with stage II or III rectal carcinoma.

Aim: To identify the prognostic factors with regard to survival for patients with brain metastasis from primary tumors of the gastrointestinal tract., Methods: Nine hundred and sixteen patients with brain metastases, treated with whole brain radiation therapy (WBRT) between January 1985 and December 2000 at the Department of Radiation Oncology, University Hospital Freiburg, were analyzed retrospectively., Results: Fifty-seven patients presented with a primary tumor of the gastrointestinal tract (esophagus: n = 0, stomach: n = 10, colorectal: n = 47). Twenty-six patients had a solitary brain metastasis, 31 patients presented with multiple brain metastases. Surgical resection was performed in 25 patients. WBRT was applied with daily fractions of 2 Gray (Gy) or 3 Gy to a total dose of 50 Gy or 30 Gy, respectively. The interval between diagnoses of the primary tumors and brain metastases was 22.6 mo vs 8.0 mo for patients with primary tumors of the colon/rectum vs other primary tumors, respectively (P<0.01, log-rank). Median overall survival for all patients with brain metastases (n = 916) was 3.4 mo and 3.2 mo for patients with gastrointestinal neoplasms. Patients with gastrointestinal primary tumors presented significantly more often with a solitary brain metastasis than patients with other primary tumors (P<0.05, log-rank). In patients with gastrointestinal neoplasms (n = 57), the median overall survival was 5.8 mo for patients with solitary brain metastasis vs 2.7 mo for patients with multiple brain metastases (P<0.01, log-rank). The median overall survival for patients with a Karnofsky performance status (KPS) >=70 was 5.5 mo vs 2.1 mo for patients with KPS <70 (P<0.01, log-rank). At multivariate analysis (Cox Model) the performance status and the number of brain metastases were identified as independent prognostic factors for overall survival., Conclusion: Brain metastases occur late in the course of gastrointestinal tumors. Pretherapeutic variables like KPS and the number of brain metastases have a profound influence on treatment outcome.

Background and Purpose: Informed consent, especially in oncology, is predominantly seen from a legal point of view. Such a limited perspective runs the risk of reducing informed consent to some tiresome formalism. The present article highlights how the relationship between patient and physician might be enriched by a comprehensive historicocultural understanding of informed consent. The authors show in which future directions the practice of informed consent might develop., Material and Methods: Analysis of historical and forensic literature regarding informed consent., Results: With the terms "information" and "consent" the last 2500 years of medical history can schematically be divided in three epochs: the first epoch started around 500 years BC, lasted until the 19th century AC and was dominated by the principle of "salus aegroti suprema lex". The patient's benefit was exclusively defined by the treating physician. Formal consent was not required in those times. The era of enlightment brought new ideas to Europe, especially the principle of individual autonomy. In 1894, the Supreme Court of the German Reich decided that any medical intervention without the patient's consent was regarded as physical injury and was thus illegal. Systematic requirements regarding patient information on planned medical interventions were not known. The beginning of the third epoch is marked by the introduction of the term "informed consent" in modern medicine in 1957. Since then, a comprehensive information of the patient is seen as a prerequisite for consent. The patient's right of self-determination is attributed a higher legal and moral value than the physician's concept of the proposed treatment. Nowadays, the debate regarding informed consent is dominated by the continuing differentiation of modern medicine, the development of medical practice as part of the service sector, and the changing ways how patients see themselves., Conclusions: Social and legal developments have strongly influenced medical practice in the past. The importance of informed consent will continue to rise in the future, while the emphasis of the physician's task will shift from information to counseling. Informed consent will be increasingly established as independent service.

Background: Radiation treatment may induce acute skin reactions. There are several methods of managing them. Validity of these methods, however, is not sufficiently studied. We therefore investigated, whether moist skin care with 3% urea lotion will reduce acute radiation skin toxicity., Patients and Methods: 88 patients with carcinomas of the head and neck undergoing radiotherapy with curative intent (mean total dose 60 Gy, range: 50-74 Gy) were evaluated weekly for acute skin reactions according to the RTOG-CTC score. In 63 patients, moist skin care with 3% urea lotion was performed. The control group consisted of 25 patients receiving conventional dry skin care. The incidence of grade I, II, and III reactions and the radiation dose at occurrence of a particular reaction were determined and statistically analyzed using the log-rank test. The dose-time relations of individual skin reactions are described., Results: At some point of time during radiotherapy, all patients suffered from acute skin reactions grade I, > 90% from grade II reactions. 50% of patients receiving moist skin care experienced grade I reactions at 26 Gy as compared to 22 Gy in control patients (p = 0.03). Grade II reactions occurred at 51 Gy versus 34 Gy (p = 0.006). Further, 22% of the patients treated with moist skin care suffered from acute skin toxicity grade III as compared to 56% of the controls (p = 0.0007)., Conclusion: Moist skin care with 3% urea lotion delays the occurrence and reduces the grade of acute skin reactions in percutaneously irradiated patients with head and neck tumors.

Background: Vitiligo is one of the most common skin disorders. However, the etiology of vitiligo is still unknown. Current hypotheses discuss autoimmune, autotoxic and neuronal mechanisms. Here we report the case of radiation-induced depigmentation of the skin of a patient with Hodgkin's disease and 25-year history of vitiligo., Patient and Method: We compared possible differences in skin color, skin moisture, microcirculation and skin elasticity between normal skin and skin exhibiting persistent depigmentation in a 37-year-old patient 40 months after completion of external beam radiotherapy., Results: Colormetrically we found a dose-dependent decrease of the red/green and yellow/blue saturation combined with an overall increase in brightness in depigmented skin when compared with normal skin. This was in agreement with a loss of melanocytes in vitiligo. Depigmentation was complete in areas receiving 40 Gy. Areas which received 30 Gy showed depigmentation only if the skin dose was increased by the loss of depth of the build-up dose region in areas with direct contact with the irradiation table. We could not show any change in skin moisture, microcirculation or skin elasticity., Conclusions: Complete radiation-induced depigmentation of skin from patients suffering from vitiligo is a side effect of radiation therapy. Patients should be informed about this side effect by the radiooncologist. Preventing the loss of depth of the build-up dose region might improve the cosmetic results of radiation therapy in patients with history of vitiligo.

Background: The levels of mRNA-expression of multidrug resistance (MDR1) and glutathione-S-transferase pi (GST-pi) were measured and correlated with the immunohistochemical expressions of tumour markers., Materials and Methods: Analysis of total mRNA was performed by Northern and slot blots. The expression of carcinoembryonic antigen (CEA) and other tumour markers was assessed by immunohistochemistry. The tumour panel comprised tumours of different histologies., Results: CEA-positive tumours showed a significantly higher ex-pression of MDR1 and GST-pi than CEA-negative tumours. Wilcoxon-Test: mean rank of the MDR1 expression (14.3 vs. 7.8; p < 0.05) and GST-pi expression (15.3 vs. 5.9; p < 0.001). No other correlation could be found., Conclusions: The relationship of MDR1 and GST-pi with the tumour marker CEA implies that evaluation of CEA can help in discriminating between tumours with high or low expression of drug resistance. Furthermore, correlation between MDR1, GST-pi and CEA indicates that there might be a common mechanism, regulating drug resistance and expression of CEA.