Rudi Beschorner

Long-term expression of heme oxygenase-1 (HO-1, HSP-32) following focal cerebral infarctions and traumatic brain injury in humans.

Abstract Extracellular heme derived from hemoglobin following hemorrhage or released from dying cells induces the expression of heme oxygenase-1 (HO-1, HSP-32) which metabolizes heme to the gaseous mediator carbon monoxide (CO), iron (Fe) and biliverdin. Biliverdin and its product bilirubin are powerful antioxidants. Thus, expression of HO-1 is considered to be a protective mechanism against oxidative stress and has been described in microglia, astrocytes and neurons following distinct experimental models of pathological alterations to the brain such as subarachnoidal hemorrhage, ischemia and traumatic brain injury (TBI) and in human neurodegenerative diseases. We have now analyzed the expression of HO-1 in human brains following TBI (n = 28; survival times: few minutes up to 6 months) and focal cerebral infarctions (FCI; n = 17; survival time: < 1 day up to months) by ¶immunohistochemistry. Follwing TBI, accumulation of ¶HO-1+ microglia/macrophages at the hemorrhagic lesion was detected as early as 6 h post trauma and was still pronounced after 6 months. In contrast, after FCI HO-1+ microglia/macrophages accumulated within focal hemorrhages only and were absent in non-hemorrhagic regions. Further, HO-1 was weakly expressed in astrocytes in the perifocal penumbra. In contrast to experimental data derived from rat focal ischemia, these results indicate a prolonged HO-1 expression in humans after brain injury.

The neurovascular unit as a selective barrier to polymorphonuclear granulocyte (PMN) infiltration into the brain after ischemic injury

The migration of polymorphonuclear granulocytes (PMN) into the brain parenchyma and release of their abundant proteases are considered the main causes of neuronal cell death and reperfusion injury following ischemia. Yet, therapies targeting PMN egress have been largely ineffective. To address this discrepancy we investigated the temporo-spatial localization of PMNs early after transient ischemia in a murine transient middle cerebral artery occlusion (tMCAO) model and human stroke specimens. Using specific markers that distinguish PMN (Ly6G) from monocytes/macrophages (Ly6C) and that define the cellular and basement membrane boundaries of the neurovascular unit (NVU), histology and confocal microscopy revealed that virtually no PMNs entered the infarcted CNS parenchyma. Regardless of tMCAO duration, PMNs were mainly restricted to luminal surfaces or perivascular spaces of cerebral vessels. Vascular PMN accumulation showed no spatial correlation with increased vessel permeability, enhanced expression of endothelial cell adhesion molecules, platelet aggregation or release of neutrophil extracellular traps. Live cell imaging studies confirmed that oxygen and glucose deprivation followed by reoxygenation fail to induce PMN migration across a brain endothelial monolayer under flow conditions in vitro. The absence of PMN infiltration in infarcted brain tissues was corroborated in 25 human stroke specimens collected at early time points after infarction. Our observations identify the NVU rather than the brain parenchyma as the site of PMN action after CNS ischemia and suggest reappraisal of targets for therapies to reduce reperfusion injury after stroke.

Prognostic Implications of Atypical Histologic Features in Choroid Plexus Papilloma

The prognostic significance of atypical histologic features in choroid plexus tumors remains uncertain. Therefore, a series of 164 choroid plexus tumors was evaluated for the presence of atypical histologic features, including mitotic activity, increased cellularity, nuclear pleomorphism, blurring of papillary growth pattern, and necrosis. The impact of histopathologic and clinical features on the probability of recurrence and survival was investigated. Twenty-four tumors displaying frank signs of malignancy were diagnosed as choroid plexus carcinoma according to World Health Organization criteria. Of 124 choroid plexus papillomas that had not received adjuvant treatment, 46 tumors (37%) displayed at least one atypical feature, including increased cellularity (n = 25 [20%]), mitotic activity (≥2 mitoses per 10 high-power fields; n = 19 [15%]), nuclear pleomorphism (n = 16 [13%]), solid growth (n = 15 [12%]), and necrosis (n = 5 [4%]). Only one tumor-related death, but 10 recurrences, were observed on a mean observation time of 58 months. On univariate analysis, incomplete surgical resection (p = 0.03) and mitotic activity (p < 0.001) were the only clinicopathologic factors associated with recurrence. Using a multivariate model, an independent effect of mitotic activity on the probability of recurrence could be confirmed (p = 0.001). Because mitotic activity is the sole atypical histologic feature independently associated with recurrence, we propose to define atypical choroid plexus papilloma by mitotic activity (≥2 mitoses per 10 high-power fields) corresponding to World Health Organization grade II, thus adjoining other intermediate tumor entities associated with increased mitotic activity such as atypical meningioma. Close follow up of patients harboring atypical choroid plexus papillomas may be warranted.

Differential Cellular Accumulation of Connective Tissue Growth Factor Defines a Subset of Reactive Astrocytes, Invading Fibroblasts, and Endothelial Cells Following Central Nervous System Injury in Rats and Humans

In brain injury, the primary trauma is followed by a cascade of cellular and molecular mechanisms resulting in secondary injury and scar formation. Astrogliosis and expression of transforming growth factor beta (TGF-beta) are key components of scar formation. A cytokine mediating the effects of TGF-beta is connective tissue growth factor (CTGF), a fibrogenic peptide encoded by an immediate early gene with suggested roles in tissue regeneration and aberrant deposition of extracellular matrix. In order to investigate CTGF in traumatic lesions, we evaluated 20 human brains with traumatic brain injury (TBI) and 18 rat brains with stab wound injury. Compared to remote areas and unaltered control brains, CTGF+ cells accumulated in border zones of the traumatic lesion site (p < 0.0001). In the direct peri-lesional rim, CTGF expression was confined to invading vimentin+, GFAP- fibroblastoid cells, endothelial and smooth muscle cells of laminin+ vessels, and GFAP+ reactive astrocytes. In the direct peri-lesional rim, CTGF+ astrocytes (>80%) co-expressed the activation associated intermediate filaments nestin and vimentin. In injured rat brains, numbers of CTGF+ cells peaked at day 3 and 7 and decreased to almost base level 3 weeks postinjury, whereas in humans, CTGF+ cells remained persistently elevated up to 6 months (p < 0.0001). The restricted accumulation of CTGF+-reactive astrocytes and CTGF+ fibroblastoid cells lining the adjacent laminin+ basal lamina suggests participation of these cells in scar formation. Furthermore, peri-lesional upregulation of endothelial CTGF expression points to a role in blood-brain barrier function and angiogenesis. In addition, CTGF appears to be a sensitive marker of early astrocyte activation.

The Gene of the Ubiquitin-Specific Protease 8 Is Frequently Mutated in Adenomas Causing Cushing's Disease

CONTEXT We have recently reported somatic mutations in the ubiquitin-specific protease USP8 gene in a small series of adenomas of patients with Cushings disease. OBJECTIVE To determine the prevalence of USP8 mutations and the genotype-phenotype correlation in a large series of patients diagnosed with Cushings disease. DESIGN We performed a retrospective, multicentric, genetic analysis of 134 functioning and 11 silent corticotroph adenomas using Sanger sequencing. Biochemical and clinical features were collected and examined within the context of the mutational status of USP8, and new mutations were characterized by functional studies. PATIENTS A total of 145 patients who underwent surgery for an ACTH-producing pituitary adenoma. MAIN OUTCOMES MEASURES Mutational status of USP8. Biochemical and clinical features included sex, age at diagnosis, tumor size, preoperative and postoperative hormonal levels, and comorbidities. RESULTS We found somatic mutations in USP8 in 48 (36%) pituitary adenomas from patients with Cushings disease but in none of 11 silent corticotropinomas. The prevalence was higher in adults than in pediatric cases (41 vs 17%) and in females than in males (43 vs 17%). Adults having USP8-mutated adenomas were diagnosed at an earlier age than those with wild-type lesions (36 vs 44 y). Mutations were primarily found in adenomas of 10 ± 7 mm and were inversely associated with the development of postoperative adrenal insufficiency. All the mutations affected the residues Ser718 or Pro720, including five new identified alterations. Mutations reduced the interaction between USP8 and 14-3-3 and enhanced USP8 activity. USP8 mutants diminished epidermal growth factor receptor ubiquitination and induced Pomc promoter activity in immortalized AtT-20 corticotropinoma cells. CONCLUSIONS USP8 is frequently mutated in adenomas causing Cushings disease, especially in those from female adult patients diagnosed at a younger age.

Infiltrating CD14+ monocytes and expression of CD14 by activated parenchymal microglia/macrophages contribute to the pool of CD14+ cells in ischemic brain lesions

CD14, a key pattern recognition receptor of the innate immune system, is a surface molecule on monocytic cells involved in cellular activation. We investigated 18 autopsy cases of focal cerebral infarctions (FCI) by immunohistochemistry to examine CD14 expression following ischemia. Controls confirmed constitutive CD14 expression by few perivascular cells. In contrast to quiescent CD14- parenchymal microglial cells, following ischemia activated microglia/macrophages expressed abundant CD14. In FCI, CD14+ cells increased both in perivascular spaces and in brain parenchyma within 1-2.5 days and remained elevated until late stages. Early CD14 expression suggests an essential part of CD14 in the acute inflammatory response following stroke.

Allograft-inflammatory-factor-1 is upregulated in microglial cells in human cerebral infarctions.

Allograft inflammatory factor-1 (AIF-1) is a 17-kDa-peptide identified in rat cardiac allografts undergoing chronic rejection and in activated microglial cells in inflammatory autoimune disease of the CNS. We have investigated the expression of AIF-1 in 18 autopsy cases of human focal cerebral infarction. AIF-1-positive cells show the morphology of microglia and are CD68- but not GFAP-positive. The peptide is expressed at a low level in normal brain. In infarctions, activated microglial cells in the area of glial reaction show strongly enhanced cytoplasmic immunoreactivity. The density of AIF-1-expressing cells increases during the first three days post infarction and remains elevated until chronic cystic stages. The upregulation of AIF-1-immunoreactivity precedes the rise in expression of the S-100-protein MRP-8. We conclude that AIF-1 is a sensitive marker of human microglial activation not only in inflammation but also in non-inflammatory lesions of the CNS.

Water diffusivity: comparison of primary CNS lymphoma and astrocytic tumor infiltrating the corpus callosum.

OBJECTIVE The purpose of this study was to determine whether lymphoma and astrocytic tumor infiltrating the corpus callosum can be reliably differentiated with measurement of water diffusivity. MATERIALS AND METHODS Echo-planar diffusion-weighted MR images of 27 patients with glioblastoma multiforme, five patients with low-grade astrocytoma, five patients with gliomatosis cerebri, and nine patients with primary lymphoma infiltrating the corpus callosum were reviewed retrospectively. Regions of interest were drawn on apparent diffusion coefficient (ADC) maps inside the callosal tumor. ADCs were normalized by calculation of the ratio between the ADC of the tumor and the ADC of an uninvolved region of corpus callosum. RESULTS The mean ADC of glioblastoma multiforme was 1.13 +/- 0.31 (SD) x 10(-3) mm(2)/s, and the mean tumor to corpus callosum ADC ratio was 1.51 +/- 0.46; of low-grade astrocytoma, 1.14 +/- 0.23 x 10(-3) mm(2)/s and 1.54 +/- 0.28; gliomatosis cerebri, 1.01 +/- 0.20 x 10(-3) mm(2)/s and 1.31 +/- 0.36; and lymphoma, 0.71 +/- 0.13 x 10(-3) mm(2)/s and 0.93 +/- 0.19. The difference between the mean tumor to corpus callosum ADC ratio of lymphoma and that of all grades of astrocytoma (1.48 +/- 0.43) was statistically significant (p < 0.001). The optimal ADC threshold for discriminating astrocytic tumor and lymphoma was 0.90 x 10(-3) mm(2)/s (sensitivity, 84%; specificity, 89%). The optimal threshold for tumor to corpus callosum ADC ratio was 1.22 (sensitivity, 73%; specificity, 100%). CONCLUSION The water diffusivity and the ADC ratio of the tumor to normal-appearing corpus callosum of astrocytic tumor differ significantly from those of lymphoma infiltrating the corpus callosum, allowing reliable differentiation of the two types of tumor.

In vivo visualization of prostate-specific membrane antigen in glioblastoma

The expansion of nutrient vessels is an important factor in the growth of glioblastomas (GBM), which is one of the most vascularized tumours [1]. Therefore, new treatment concepts that include tumour vasculature targets are currently under investigation. Prostate-specific membrane antigen (PSMA) is overexpressed in the tumour vasculature of GBM, which therefore might be considered as a potential endothelial target for treatment with PSMA-based agents [2, 3]. However, according to the limited data so far available, PSMA expression in brain tumours varies with glioma grade [4]. PSMAexpressionwas evaluated in a patient suffering fromGBM prior to surgery by simultaneous PET/MR with [Ga]

Dynamics of expression patterns of AQP4, dystroglycan, agrin and matrix metalloproteinases in human glioblastoma

In human glioblastoma, the blood–brain barrier (BBB) is disturbed. According to our concept, the glio-vascular relationships and thus the control of the BBB are essentially dependent on the polarity of astroglial cells. This polarity is characterized by the uneven distribution of the water channel protein aquaporin-4 (AQP4), dystroglycan and other molecules. Recently, we were able to show that the extracellular matrix component agrin is important for the construction and localization of the so-called orthogonal arrays of particles (OAPs), which consist in AQP4. Here, combining freeze-fracture electron microscopy, immunohistochemistry and Western blotting, we describe alterations of expression and distribution of AQP4, dystroglycan, agrin and the matrix metalloproteinases (MMP) 2, 3 and 9 in human primary glioblastomas (eight primary tumours, six recurrent tumours). Increase of MMP3- and MMP2/9 immunoreactivities went along with loss of agrin and dystroglycan respectively. On the protein level, AQP4 expression was increased in glioblastoma compared to control tissue. This was not accompanied by an increase of OAPs, suggesting that AQP4 can also occur without forming OAPs. The results underline our concept of the loss of glioma cell polarity as one of the factors responsible for the disturbance of the neurovascular unit and as an explanation for the formation of edemas in the glioblastoma.