Effects of temporal IFNγ exposure on macrophage phenotype and secretory profile: exploring GMP-Compliant production of a novel subtype of regulatory macrophages (MregIFNγ0) for potential cell therapeutic applications.

BACKGROUND: Macrophages are involved in tissue homeostasis, angiogenesis and immunomodulation. Proangiogenic and anti-inflammatory macrophages (regulatory macrophages, Mreg) can be differentiated in-vitro from CD14+ monocytes by using a defined cell culture medium and a stimulus of IFNγ. AIM OF THE STUDY: To scrutinize the potential impact of temporal IFNγ exposure on macrophage differentiation as such exposure may lead to the emergence of a distinct and novel macrophage subtype. METHODS: Differentiation of human CD14+ monocytes to Mreg was performed using a GMP compliant protocol and administration of IFNγ on day 6. Monocytes from the same donor were in parallel differentiated to MregIFNγ0 using the identical protocol but with administration of IFNγ on day 0. Cell characterization was performed using brightfield microscopy, automated and metabolic cell analysis, transmission electron microscopy, flow cytometry, qPCR and secretome profiling. RESULTS: Mreg and MregIFNγ0 showed no differences in cell size and volume. However, phenotypically MregIFNγ0 exhibited fewer intracellular vesicles/vacuoles but larger pseudopodia-like extensions. MregIFNγ0 revealed reduced expression of IDO and PD-L1 (P < 0.01 for both). They were positive for CD80, CD14, CD16 and CD38 (P < 0.0001vs. Mreg for all), while the majority of MregIFNγ0 did not express CD206, CD56, and CD103 on their cell surface (P < 0.01 vs. Mreg for all). In terms of their secretomes, MregIFNγ0 differed significantly from Mreg. MregIFNγ0 media exhibited reduced levels of ENA-78, Osteopontin and Serpin E1, while the amounts of MIG (CXCL9) and IP10 were increased. CONCLUSION: Exposing CD14+ monocytes to an alternatively timed IFNγ stimulation results in a novel macrophage subtype which possess additional M1-like features (MregIFNγ0). MregIFNγ0 may therefore have the potential to serve as cellular therapeutics for clinical applications beyond those covered by M2-like Mreg, including immunomodulation and tumor treatment.

New insights into neuropathology and pathogenesis of autoimmune glial fibrillary acidic protein meningoencephalomyelitis.

Anti-glial fibrillary acidic protein (GFAP) meningoencephalomyelitis (autoimmune GFAP astrocytopathy) is a new autoimmune central nervous system (CNS) disease diagnosable by the presence of anti-GFAP autoantibodies in the cerebrospinal fluid and presents as meningoencephalomyelitis in the majority of patients. Only few neuropathological reports are available and little is known about the pathogenic mechanisms. We performed a histopathological study of two autopsies and nine CNS biopsies of patients with anti-GFAP autoantibodies and found predominantly a lymphocytic and in one autopsy case a granulomatous inflammatory phenotype. Inflammatory infiltrates were composed of B and T cells, including tissue-resident memory T cells. Although obvious astrocytic damage was absent in the GFAP-staining, we found cytotoxic T cell-mediated reactions reflected by the presence of CD8+/perforin+/granzyme A/B+ cells, polarized towards astrocytes. MHC-class-I was upregulated in reactive astrocytes of all biopsies and two autopsies but not in healthy controls. Importantly, we observed a prominent immunoreactivity of astrocytes with the complement factor C4d. Finally, we provided insight into an early phase of GFAP autoimmunity in an autopsy of a pug dog encephalitis that was characterized by marked meningoencephalitis with selective astrocytic damage with loss of GFAP and AQP4 in the lesions.Our histopathological findings indicate that a cytotoxic T cell-mediated immune reaction is present in GFAP autoimmunity. Complement C4d deposition on astrocytes could either represent the cause or consequence of astrocytic reactivity. Selective astrocytic damage is prominent in the early phase of GFAP autoimmunity in a canine autopsy case, but mild or absent in subacute and chronic stages in human disease, probably due to the high regeneration potential of astrocytes. The lymphocytic and granulomatous phenotypes might reflect different stages of lesion development or patient-specific modifications of the immune response. Future studies will be necessary to investigate possible implications of pathological subtypes for clinical disease course and therapeutic strategies.

Characterization of large extracellular vesicles (L-EV) derived from human regulatory macrophages (Mreg): novel mediators in wound healing and angiogenesis?

BACKGROUND: Large extracellular vesicles (L-EV) with a diameter between 1 and 10 µm are released by various cell types. L-EV contain and transport active molecules which are crucially involved in cell to cell communication. We have shown that secretory products of human regulatory macrophages (Mreg) bear pro-angiogenic potential in-vitro and our recent findings show that Mreg cultures also contain numerous large vesicular structures similar to L-EV with so far unknown characteristics and function. AIM OF THIS STUDY: To characterize the nature of Mreg-derived L-EV (L-EVMreg) and to gain insights into their role in wound healing and angiogenesis. METHODS: Mreg were differentiated using blood monocytes from healthy donors (N = 9) and L-EVMreg were isolated from culture supernatants by differential centrifugation. Characterization of L-EVMreg was performed by cell/vesicle analysis, brightfield/transmission electron microscopy (TEM), flow cytometry and proteome profiling arrays. The impact of L-EVMreg on wound healing and angiogenesis was evaluated by means of scratch and in-vitro tube formation assays. RESULTS: Mreg and L-EVMreg show an average diameter of 13.73 ± 1.33 µm (volume: 1.45 ± 0.44 pl) and 7.47 ± 0.75 µm (volume: 0.22 ± 0.06 pl) respectively. Flow cytometry analyses revealed similarities between Mreg and L-EVMreg regarding their surface marker composition. However, compared to Mreg fewer L-EVMreg were positive for CD31 (P < 0.01), CD206 (P < 0.05), CD103 (P < 0.01) and CD45 (P < 0.05). Proteome profiling suggested that L-EVMreg contain abundant amounts of pro-angiogenic proteins (i.e. interleukin-8, platelet factor 4 and serpin E1). From a functional point of view L-EVMreg positively influenced in-vitro wound healing (P < 0.05) and several pro-angiogenic parameters in tube formation assays (all segment associated parameters, P < 0.05; number of meshes, P < 0.05). CONCLUSION: L-EVMreg with regenerative and pro-angiogenic potential can be reproducibly isolated from in-vitro cultured human regulatory macrophages. We propose that L-EVMreg could represent a putative therapeutic option for the treatment of chronic wounds and ischemia-associated diseases.

Prognosis and histology of sporadic synchronous and metachronous meningiomas and comparative analyses with singular lesions.

Synchronous or metachronous growth of multiple tumors (≥ 2) is found in up to 20% of meningioma patients. However, biological as well as histological features and prognosis are largely unexplored. Clinical and histological characteristics were retrospectively investigated in 95 patients harboring 226 multiple meningiomas (MMs) and compared with 135 cases of singular meningiomas (SM) using uni- and multivariate analyses. In MM, tumors occurred synchronously and metachronously in 62% and 38%, respectively. WHO grade was intra-individually constant in all but two MMs, and histological subtype varied in 13% of grade 1 tumors. MM occurred more commonly in convexity/parasagittal locations, while SM were more frequent at the skull base (p < .001). In univariate analyses, gross total resection (p = .014) and high-grade histology in MM were associated with a prolonged time to progression (p < .001). Most clinical characteristics and rates of high-grade histology were similar in both groups (p ≥ .05, each). Multivariate analyses showed synchronous/metachronous meningioma growth (HR 4.50, 95% CI 2.26-8.96; p < .001) as an independent predictor for progression. Compared to SM, risk of progression was similar in cases with two (HR 1.56, 95% CI .76-3.19; p = .224), but exponentially raised in patients with 3-4 (HR 3.25, 1.22-1.62; p = .018) and ≥ 5 tumors (HR 13.80, 4.06-46.96; p < .001). Clinical and histological characteristics and risk factors for progression do not relevantly differ between SM and MM. Although largely constant, histology and WHO grade occasionally intra-individually vary in MM. A distinctly higher risk of disease progression in MM as compared to SM might reflect different underlying molecular alterations.

Expression of decitabine-targeted oncogenes in meningiomas in vivo.

Treatment of meningiomas refractory to surgery and irradiation is challenging and effective chemotherapies are still lacking. Recently, in vitro analyses revealed decitabine (DCT, 5-aza-2'-deoxycytidine) to be effective in high-grade meningiomas and, moreover, to induce hypomethylation of distinct oncogenes only sparsely described in meningiomas in vivo yet.Expression of the corresponding onco- and tumor suppressor genes TRIM58, FAM84B, ELOVL2, MAL2, LMO3, and DIO3 were analyzed and scored by immunohistochemical staining and RT-PCR in samples of 111 meningioma patients. Correlations with clinical and histological variables and prognosis were analyzed in uni- and multivariate analyses.All analyzed oncogenes were highly expressed in meningiomas. Expression scores of TRIM58 tended to be higher in benign than in high-grade tumors 20 vs 16 (p = .002) and all 9 samples lacking TRIM58 expression displayed WHO grade II/III histology. In contrast, median expression scores for both FAM84B (6 vs 4, p ≤ .001) and ELOVL2 (9 vs 6, p < .001) were increased in high-grade as compared to benign meningiomas. DIO3 expression was distinctly higher in all analyzed samples as compared to the reference decitabine-resistant Ben-Men 1 cell line. Increased ELOVL2 expression (score ≥ 8) correlated with tumor relapse in both uni- (HR: 2.42, 95%CI 1.18-4.94; p = .015) and multivariate (HR: 2.09, 95%CI 1.01-4.44; p = .046) analyses.All oncogenes involved in DCT efficacy in vitro are also widely expressed in vivo, and expression is partially associated with histology and prognosis. These results strongly encourage further analyses of DCT efficiency in meningiomas in vitro and in situ.

The applicability of established clinical and histopathological risk factors for tumor recurrence during long-term postoperative care in meningioma patients.

Risk factors to predict late-onset tumor recurrence in meningioma patients are urgently needed to schedule control intervals during long-term follow-up. We therefore analyzed the value of established risk factors for postoperative meningioma recurrence for the prediction of long-term prognosis. Correlations of clinical and histopathological variables with tumor relapse after 3, 5, and 10 years following microsurgery were analyzed in uni- and multivariate analyses, and compared to findings in the entire cohort. In the entire cohort (N = 1218), skull base location (HR: 1.51, 95%CI 1.05-2.16; p = .026), Simpson ≥ IV resections (HR: 2.41, 95%CI 1.52-3.84; p < .001), high-grade histology (HR: 3.70, 95%CI 2.50-5.47; p < .001), and male gender (HR: 1.46, 95%CI 1.01-2.11; p = .042) were independent risk factors for recurrence. Skull base location (HR: 1.92, 95%CI 1.17-3.17; p = .010 and HR: 2.02, 95%CI 1.04-3.95; p = .038) and high-grade histology (HR: 1.87, 95%CI 1.04-3.38; p = .038 and HR: 2.29, 95%CI 1.07-4.01; p = .034) but not subtotal resection (HR: 1.53, 95%CI .68-3.45; p = .303 and HR: 1.75, 95%CI .52-5.96; p = .369) remained correlated with recurrence after a recurrence-free follow-up of ≥ 3 and ≥ 5 years, respectively. Postoperative tumor volume was related with recurrence in general (p < .001) but not beyond a follow-up of ≥ 3 years (p > .05). In 147 patients with a follow-up of ≥ 10 years, ten recurrences occurred and were not correlated with any of the analyzed variables. Skull base tumor location and high-grade histology but not the extent of resection should be considered when scheduling the long-term follow-up after meningioma surgery. Recurrences ≥ 10 years after surgery are rare, and predictors are lacking.

Disseminated cerebral fusariosis in a liver-transplant patient: A case report and review of the literature.

Fusarium spp. can cause invasive infection with fatal outcomes in immunocompromised patients. Therefore, invasive fusariosis is rare after solid organ transplantation. For this reason, experience and management are limited to single published case reports.We report a 65-year-old female patient with disseminated brain abscesses caused by Fusarium after liver transplantation (LT). The patient underwent LT for secondary sclerosing cholangitis after acute respiratory distress syndrome (ARDS). After a complicated course with aneurysm and thrombosis of the hepatic artery, re-transplantation was performed after one month. Due to inadequate awakening response, cerebral imaging was performed, which showed multiple abscesses. The patient died shortly thereafter, and an autopsy showed fusariosis.

Effects of remote ischemic preconditioning (RIPC) and chronic remote ischemic preconditioning (cRIPC) on levels of plasma cytokines, cell surface characteristics of monocytes and in-vitro angiogenesis: a pilot study.

Remote ischemic preconditioning (RIPC) protects the heart against myocardial ischemia/reperfusion (I/R) injury and recent work also suggested chronic remote ischemic conditioning (cRIPC) for cardiovascular protection. Based on current knowledge that systemic immunomodulatory effects of RIPC and the anti-inflammatory capacity of monocytes might be involved in cardiovascular protection, the aim of our study was to evaluate whether RIPC/cRIPC blood plasma is able to induce in-vitro angiogenesis, identify responsible factors and evaluate the effects of RIPC/cRIPC on cell surface characteristics of circulating monocytes. Eleven healthy volunteers were subjected to RIPC/cRIPC using a blood pressure cuff inflated to > 200 mmHg for 3 × 5 min on the upper arm. Plasma and peripheral blood monocytes were isolated before RIPC (Control), after 1 × RIPC (RIPC) and at the end of 1 week of daily RIPC (cRIPC) treatment. Plasma concentrations of potentially pro-angiogenic humoral factors (CXCL5, Growth hormone, IGFBP3, IL-1α, IL-6, Angiopoietin 2, VEGF, PECAM-1, sTie-2, IL-8, MCSF) were measured using custom made multiplex ELISA systems. Tube formation assays for evaluation of in-vitro angiogenesis were performed with donor plasma, monocyte conditioned culture media as well as IL-1α, CXCL5 and Growth hormone. The presence of CD14, CD16, Tie-2 and CCR2 was analyzed on monocytes by flow cytometry. Employing in-vitro tube formation assays, several parameters of angiogenesis were significantly increased by cRIPC plasma (number of nodes, P < 0.05; number of master junctions, P < 0.05; number of segments, P < 0.05) but were not influenced by culture medium from RIPC/cRIPC treated monocytes. While RIPC/cRIPC treatment did not lead to significant changes of the median plasma concentrations of any of the selected potentially pro-angiogenic humoral factors, in-depth analysis of the individual subjects revealed differences in plasma levels of IL-1α, CXCL5 and Growth hormone after RIPC/cRIPC treatment in some of the volunteers. Nevertheless, the positive effects of RIPC/cRIPC plasma on in-vitro angiogenesis could not be mimicked by the addition of the respective humoral factors alone or in combination. While monocyte conditioned culture media did not affect in-vitro tube formation, flow cytometry analyses of circulating monocytes revealed a significant increase in the number of Tie-2 positive and a decrease of CCR2 positive monocytes after RIPC/cRIPC (Tie-2: cRIPC, P < 0.05; CCR2: RIPC P < 0.01). Cardiovascular protection may be mediated by RIPC and cRIPC via a regulation of plasma cytokines as well as changes in cell surface characteristics of monocytes (e.g. Tie-2). Our results suggest that a combination of humoral and cellular factors could be responsible for the RIPC/cRIPC mediated effects and that interindividual variations seem to play a considerable part in the RIPC/cRIPC associated mechanisms.

The Simpson grading: defining the optimal threshold for gross total resection in meningioma surgery.

Classification of the extent of resection into gross and subtotal resection (GTR and STR) after meningioma surgery is derived from the Simpson grading. Although utilized to indicate adjuvant treatment or study inclusion, conflicting definitions of STR in terms of designation of Simpson grade III resections exist. Correlations of Simpson grading and dichotomized scales (Simpson grades I-II vs ≥ III and grade I-III vs ≥ IV) with postoperative recurrence/progression were compared using Cox regression models. Predictive values were further compared by time-dependent receiver operating curve (tdROC) analyses. In 939 patients (28% males, 72% females) harboring WHO grade I (88%) and II/III (12%) meningiomas, Simpson grade I, II, III, IV, and V resections were achieved in 29%, 48%, 11%, 11%, and < .5%, respectively. Recurrence/progression was observed in 112 individuals (12%) and correlated with Simpson grading (p = .003). The risk of recurrence/progression was increased after STR in both dichotomized scales but higher when subsuming Simpson grade ≥ IV than grade ≥ III resections (HR: 2.49, 95%CI 1.50-4.12; p < .001 vs HR: 1.67, 95%CI 1.12-2.50; p = .012). tdROC analyses showed moderate predictive values for the Simpson grading and significantly (p < .05) lower values for both dichotomized scales. AUC values differed less between the Simpson grading and the dichotomization into grade I-III vs ≥ IV than grade I-II vs ≥ III resections. Dichotomization of the extent of resection is associated with a loss of the prognostic value. The value for the prediction of progression/recurrence is higher when dichotomizing into Simpson grade I-III vs ≥ IV than into grade I-II vs ≥ III resections.

Predicting the risk of postoperative recurrence and high-grade histology in patients with intracranial meningiomas using routine preoperative MRI.

Risk factors for prediction of prognosis in meningiomas derivable from routine preoperative magnetic resonance imaging (pMRI) remain elusive. Correlations of tumor and edema volume, disruption of the arachnoid layer, heterogeneity of contrast enhancement, enhancement of the capsule, T2-intensity, tumor shape, and calcifications on pMRI with tumor recurrence and high-grade (WHO grade II/III) histology were analyzed in 565 patients who underwent surgery for WHO grade I (N = 516, 91%) or II/III (high-grade histology, N = 49, 9%) meningioma between 1991 and 2018. Edema volume (OR, 1.00; p = 0.003), heterogeneous contrast enhancement (OR, 3.10; p < 0.001), and an irregular shape (OR, 2.16; p = 0.015) were associated with high-grade histology. Multivariate analyses confirmed edema volume (OR, 1.00; p = 0.037) and heterogeneous contrast enhancement (OR, 2.51; p = 0.014) as risk factors for high-grade histology. Tumor volume (HR, 1.01; p = 0.045), disruption of the arachnoid layer (HR, 2.50; p = 0.003), heterogeneous contrast enhancement (HR, 2.05; p = 0.007), and an irregular tumor shape (HR, 2.57; p = 0.001) were correlated with recurrence. Multivariate analyses confirmed tumor volume (HR, 1.01; p = 0.032) and disruption of the arachnoid layer (HR, 2.44; p = 0.013) as risk factors for recurrence, independent of histology. Subgroup analyses revealed disruption of the arachnoid layer (HR, 9.41; p < 0.001) as a stronger risk factor for recurrence than high-grade histology (HR, 5.15; p = 0.001). Routine pMRI contains relevant information about the risk of recurrence or high-grade histology of meningioma patients. Loss of integrity of the arachnoid layer on MRI had a higher prognostic value than the WHO grading, and underlying histological or molecular alterations remain to be determined.

Lesion stage-dependent causes for impaired remyelination in MS.

Multiple sclerosis (MS) is the most frequent demyelinating disease and a leading cause for disability in young adults. Despite significant advances in immunotherapies in recent years, disease progression still cannot be prevented. Remyelination, meaning the formation of new myelin sheaths after a demyelinating event, can fail in MS lesions. Impaired differentiation of progenitor cells into myelinating oligodendrocytes may contribute to remyelination failure and, therefore, the development of pharmacological approaches which promote oligodendroglial differentiation and by that remyelination, represents a promising new treatment approach. However, this generally accepted concept has been challenged recently. To further understand mechanisms contributing to remyelination failure in MS, we combined detailed histological analyses assessing oligodendroglial cell numbers, presence of remyelination as well as the inflammatory environment in different MS lesion types in white matter with in vitro experiments using induced-pluripotent stem cell (iPSC)-derived oligodendrocytes (hiOL) and supernatants from polarized human microglia. Our findings suggest that there are multiple reasons for remyelination failure in MS which are dependent on lesion stage. These include lack of myelin sheath formation despite the presence of mature oligodendrocytes in a subset of active lesions as well as oligodendroglial loss and a hostile tissue environment in mixed active/inactive lesions. Therefore, we conclude that better in vivo and in vitro models which mimic the pathological hallmarks of the different MS lesion types are required for the successful development of remyelination promoting drugs.

Extrinsic immune cell-derived, but not intrinsic oligodendroglial factors contribute to oligodendroglial differentiation block in multiple sclerosis.

Multiple sclerosis (MS) is the most frequent demyelinating disease in young adults and despite significant advances in immunotherapy, disease progression still cannot be prevented. Promotion of remyelination, an endogenous repair mechanism resulting in the formation of new myelin sheaths around demyelinated axons, represents a promising new treatment approach. However, remyelination frequently fails in MS lesions, which can in part be attributed to impaired differentiation of oligodendroglial progenitor cells into mature, myelinating oligodendrocytes. The reasons for impaired oligodendroglial differentiation and defective remyelination in MS are currently unknown. To determine whether intrinsic oligodendroglial factors contribute to impaired remyelination in relapsing-remitting MS (RRMS), we compared induced pluripotent stem cell-derived oligodendrocytes (hiOL) from RRMS patients and controls, among them two monozygous twin pairs discordant for MS. We found that hiOL from RRMS patients and controls were virtually indistinguishable with respect to remyelination-associated functions and proteomic composition. However, while analyzing the effect of extrinsic factors we discovered that supernatants of activated peripheral blood mononuclear cells (PBMCs) significantly inhibit oligodendroglial differentiation. In particular, we identified CD4+ T cells as mediators of impaired oligodendroglial differentiation; at least partly due to interferon-gamma secretion. Additionally, we observed that blocked oligodendroglial differentiation induced by PBMC supernatants could not be restored by application of oligodendroglial differentiation promoting drugs, whereas treatment of PBMCs with the immunomodulatory drug teriflunomide prior to supernatant collection partly rescued oligodendroglial differentiation. In summary, these data indicate that the oligodendroglial differentiation block is not due to intrinsic oligodendroglial factors but rather caused by the inflammatory environment in RRMS lesions which underlines the need for drug screening approaches taking the inflammatory environment into account. Combined, these findings may contribute to the development of new remyelination promoting strategies.

Sirt1 protects from K-Ras-driven lung carcinogenesis.

The NAD+-dependent deacetylase SIRT1 can be oncogenic or tumor suppressive depending on the tissue. Little is known about the role of SIRT1 in non-small cell lung carcinoma (NSCLC), one of the deadliest cancers, that is frequently associated with mutated K-RAS Therefore, we investigated the effect of SIRT1 on K-RAS-driven lung carcinogenesis. We report that SIRT1 protein levels are downregulated by oncogenic K-RAS in a MEK and PI3K-dependent manner in mouse embryo fibroblasts (MEFs), and in human lung adenocarcinoma cell lines. Furthermore, Sirt1 overexpression in mice delays the appearance of K-RasG12V-driven lung adenocarcinomas, reducing the number and size of carcinomas at the time of death and extending survival. Consistently, lower levels of SIRT1 are associated with worse prognosis in human NSCLCs. Mechanistically, analysis of mouse Sirt1-Tg pneumocytes, isolated shortly after K-RasG12V activation, reveals that Sirt1 overexpression alters pathways involved in tumor development: proliferation, apoptosis, or extracellular matrix organization. Our work demonstrates a tumor suppressive role of SIRT1 in the development of K-RAS-driven lung adenocarcinomas in mice and humans, suggesting that the SIRT1-K-RAS axis could be a therapeutic target for NSCLCs.

Brain invasion in meningiomas: does surgical sampling impact specimen characteristics and histology?

Brain invasion (BI) is a new criterion for atypia in meningiomas and therefore potentially impacts adjuvant treatment. However, it remains unclear whether surgical practice and specimen characteristics influence histopathological analyses and the accuracy of detecting BI. Tumor location, specimen characteristics, and rates of BI were compared in meningioma samples obtained from 2938 surgeries in different neurosurgical departments but diagnosed in a single neuropathological institute. Non-skull base tumor location was associated with CNS tissue on the microscopic slides (OR 1.45; p < .001), increasing specimen weight (OR 1.01; p < .001), and remaining tissue not subjected to neuropathological analyses (OR 2.18; p < .001) but not with BI (OR 1.29; p = .199). Specimen weight, rates of residual tissue not subjected to histopathological analyses, of BI and of brain tissue, on the microscopic slides differed among the neurosurgical centers (p < .001, each). Frequency of BI was increased in one department (OR 2.07; p = .002) and tended to be lower in another (OR .61; p = .088). The same centers displayed the highest and lowest rates of brain tissue in the specimen, respectively (p < .001). Moreover, the correlation of BI with the neurosurgical center was not confirmed when only analyzing specimen with evidence of brain tissue in microscopic analyses (p = .223). Detection of BI was not correlated with the intraoperative use of CUSA in subgroup analyses. Rates of brain invasion in neuropathological analyses are not associated with tumor location but differ among some neurosurgical centers. Evidence raises that surgical nuances impact specimen characteristics and therefore the accuracy of the detection of BI.

Real-time in vivo kinetics of protoporphyrin IX after administration of 5-aminolevulinic acid in meningiomas and comparative analyses with glioblastomas.

BACKGROUND: The usefulness of 5-aminolevulinic acid (5-ALA)-mediated fluorescence-guided surgery (FGS) in meningiomas is intensely discussed. However, data about kinetics of 5-ALA and protoporphyrin (Pp) IX in meningiomas are lacking. METHODS: As the first study so far, we performed longitudinal intraoperative real-time ex situ measurements of fluorescence intensity and PpIX concentrations during FGS of ten benign and two atypical meningiomas. Kinetics were subsequently compared with data from 229 glioblastomas. RESULTS: Spectroscopy revealed fluorescence (median 2945.65 a.u.) and PpIX accumulation (median 18.31 µg/ml) in all 43 analyzed samples. Fluorescence intensity (2961.50 a.u. vs 118.41 a.u.; p < .001) and PpIX concentrations (18.72 µg/ml vs .98 µg/ml; p < .001) were higher in samples with (N = 30) than without (N = 2) visible intraoperative tumor fluorescence. ROC curve analyses revealed a PpIX cut-off concentration of 3.85 µg/ml (AUC = .992, p = .005) and a quantitative fluorescence cut-off intensity of 286.73 a.u. (AUC = .983, p = .006) for intraoperative visible tumor fluorescence. Neither fluorescence intensity (p = .356) nor PpIX (p = .631) differed between atypical and benign meningiomas. Fluorescence and PpIX peaked 7-8 h following administration of 5-ALA. Meningiomas displayed a higher fluorescence intensity (p = .012) and PpIX concentration (p = .005) than glioblastomas 5-6 h after administration of 5-ALA. Although fluorescence was basically maintained, PpIX appeared to be cleared faster in meningiomas than in glioblastomas. CONCLUSIONS: Kinetics of PpIX and fluorescence intensity differ between meningiomas and glioblastomas in the early phase after 5-ALA administration. Modification of the timing of drug administration might impact visibility of intraoperative fluorescence and helpfulness of FGS and should be investigated in future analyses.

Remote ischemic preconditioning attenuates intestinal mucosal damage: insight from a rat model of ischemia-reperfusion injury.

BACKGROUND: Remote ischemic preconditioning (RIPC) is a phenomenon, whereby repeated, non-lethal episodes of ischemia to an organ or limb exert protection against ischemia-reperfusion (I/R) injury in distant organs. Despite intensive research, there is still an apparent lack of knowledge concerning the RIPC-mediated mechanisms, especially in the intestine. Aim of this study was to evaluate possible protective effects RIPC on intestinal I/R injury. METHODS: Thirty rats were randomly assigned to four groups: I/R; I/R + RIPC; Sham; Sham + RIPC. Animals were anesthetized and the superior mesenteric artery was clamped for 30 min, followed by 60 min of reperfusion. RIPC-treated rats received 3 × 5 min of bilateral hindlimb I/R prior to surgery, sham groups obtained laparotomy without clamping. After I/R injury serum/tissue was analyzed for: Mucosal damage, Caspase-3/7 activity, expression of cell stress proteins, hydrogen peroxide (H2O2) and malondialdehyde (MDA) production, Hypoxia-inducible factor-1α (HIF-1α) protein expression and matrix metalloproteinase (MMP) activity. RESULTS: Intestinal I/R resulted in increased mucosal injury (P < 0.001) and elevated Caspase-3/7 activity (P < 0.001). RIPC significantly reduced the histological signs of intestinal I/R injury (P < 0.01), but did not affect Caspase-3/7 activity. Proteome profiling suggested a RIPC-mediated regulation of several cell stress proteins after I/R injury: Cytochrome C (+ 157%); Cited-2 (- 39%), ADAMTS1 (+ 74%). Serum concentrations of H2O2 and MDA remained unchanged after RIPC, while the reduced intestinal injury was associated with increased HIF-1α levels. Measurements of MMP activities in serum and intestinal tissue revealed an attenuated gelatinase activity at 130 kDa within the serum samples (P < 0.001) after RIPC, while the activity of MMPs within the intestinal tissue was not affected by I/R injury or RIPC. CONCLUSIONS: RIPC ameliorates intestinal I/R injury in rats. The underlying mechanisms may involve HIF-1α protein expression and a decreased serum activity of a 130 kDa factor with gelatinase activity.

Combined intracavitary thermotherapy with iron oxide nanoparticles and radiotherapy as local treatment modality in recurrent glioblastoma patients.

BACKGROUND: There is an increasing interest in local tumor ablative treatment modalities that induce immunogenic cell death and the generation of antitumor immune responses. METHODS: We report six recurrent glioblastoma patients who were treated with intracavitary thermotherapy after coating the resection cavity wall with superparamagnetic iron oxide nanoparticles ("NanoPaste" technique). Patients underwent six 1-h hyperthermia sessions in an alternating magnetic field and, if possible, received concurrent fractionated radiotherapy at a dose of 39.6 Gy. RESULTS: There were no major side effects during active treatment. However, after 2-5 months, patients developed increasing clinical symptoms. CT scans showed tumor flare reactions with prominent edema around nanoparticle deposits. Patients were treated with dexamethasone and, if necessary, underwent re-surgery to remove nanoparticles. Histopathology revealed sustained necrosis directly adjacent to aggregated nanoparticles without evidence for tumor activity. Immunohistochemistry showed upregulation of Caspase-3 and heat shock protein 70, prominent infiltration of macrophages with ingested nanoparticles and CD3+ T-cells. Flow cytometric analysis of freshly prepared tumor cell suspensions revealed increased intracellular ratios of IFN-γ to IL-4 in CD4+ and CD8+ memory T cells, and activation of tumor-associated myeloid cells and microglia with upregulation of HLA-DR and PD-L1. Two patients had long-lasting treatment responses > 23 months without receiving any further therapy. CONCLUSION: Intracavitary thermotherapy combined with radiotherapy can induce a prominent inflammatory reaction around the resection cavity which might trigger potent antitumor immune responses possibly leading to long-term stabilization of recurrent GBM patients. These results warrant further investigations in a prospective phase-I trial.

Clinical, radiological, and histopathological predictors for long-term prognosis after surgery for atypical meningiomas.

BACKGROUND: Despite considerable rates of recurrence and mortality in atypical meningiomas, reliable predictors for estimating postoperative long-term prognosis remain elusive. METHODS: Clinical, histopathological, and radiological variables from 138 patients, including 64 females and 74 males (46% and 54%, median age 62 years), who underwent surgery for intracranial atypical meningioma were retrospectively analyzed. Associations between variables and recurrence and mortality were investigated using uni- and multivariate analyses. RESULTS: Gross total (GTR) and subtotal resection (STR) was achieved in 81% and 19% of cases, respectively. Within a median follow-up of 62 months, recurrence occurred in 52 (38%) and mortality in 22 (16%) cases. In patients who did not receive adjuvant irradiation, recurrence rates were higher after STR than after GTR (32% vs 63%, p = 0.025). In univariate analyses, only intratumoral calcifications on preoperative MRI (p = 0.012) and the presence of brain invasion in the absence of other histological grading criteria (p = 0.010) were correlated with longer progression-free intervals (PFI). In multivariate analyses, patient age was positively (HR 1.03, 95%CI 1.04-1.05; p = 0.018) and the presence of brain invasion as the only grading criterion (HR 0.37, 95%CI 0.19-0.74; p = 0.005) was negatively related with progression, while rising age at the time of surgery (HR 1.07, 95%CI 1.03-1.12; p = 0.001) was prognostic for mortality. CONCLUSIONS: PFI was longer in brain invasive but otherwise histological benign meningiomas and in tumors displaying calcifications on preoperative MRI. Advancing patient age and lower Karnofsky Performance Score were associated with higher overall mortality.

Anaplastic astrocytoma with piloid features, a novel molecular class of IDH wildtype glioma with recurrent MAPK pathway, CDKN2A/B and ATRX alterations.

Tumors with histological features of pilocytic astrocytoma (PA), but with increased mitotic activity and additional high-grade features (particularly microvascular proliferation and palisading necrosis) have often been designated anaplastic pilocytic astrocytomas. The status of these tumors as a separate entity has not yet been conclusively demonstrated and molecular features have only been partially characterized. We performed DNA methylation profiling of 102 histologically defined anaplastic pilocytic astrocytomas. T-distributed stochastic neighbor-embedding (t-SNE) and hierarchical clustering analysis of these 102 cases against 158 reference cases from 12 glioma reference classes revealed that a subset of 83 of these tumors share a common DNA methylation profile that is distinct from the reference classes. These 83 tumors were thus denominated DNA methylation class anaplastic astrocytoma with piloid features (MC AAP). The 19 remaining tumors were distributed amongst the reference classes, with additional testing confirming the molecular diagnosis in most cases. Median age of patients with MC AAP was 41.5 years. The most frequent localization was the posterior fossa (74%). Deletions of CDKN2A/B (66/83, 80%), MAPK pathway gene alterations (49/65, 75%, most frequently affecting NF1, followed by BRAF and FGFR1) and mutations of ATRX or loss of ATRX expression (33/74, 45%) were the most common molecular alterations. All tumors were IDH1/2 wildtype. The MGMT promoter was methylated in 38/83 tumors (45%). Outcome analysis confirmed an unfavorable clinical course in comparison to PA, but better than IDH wildtype glioblastoma. In conclusion, we show that a subset of histologically defined anaplastic pilocytic astrocytomas forms a separate DNA methylation cluster, harbors recurrent alterations in MAPK pathway genes in combination with alterations of CDKN2A/B and ATRX, affects patients who are on average older than those diagnosed with PA and has an intermediate clinical outcome.

The evolution of cranial meningioma surgery-a single-center 25-year experience.

BACKGROUND: There have been major developments in diagnostic and surgical and non-surgical techniques used in the management of meningiomas over last three decades. We set out to describe these changes in a systematic manner. METHOD: Clinical and radiological data, surgical procedures, complications, and outcome of 817 patients who underwent surgery for primarily diagnosed meningioma between 1991 and 2015 were investigated. RESULTS: Median age at diagnosis increased significantly from 56 to 59 years (p = .042), while tumor location and preoperative Karnofsky performance status did not change during the observation period. Availability of preoperative MRI increased, and rates of angiography and tumor embolization decreased (p < .001, each). Median duration of total, pre-, and postoperative stay was 13, 2, and 9 days, respectively, and decreased between 1991 and 2015 (p < .001, each). Median incision-suture time varied annually (p < .001) but without becoming clearly longer or shorter during the entire observation period. The use of intraoperative neuronavigation and neuromonitoring increased, while the rates of Simpson grade I and III surgeries decreased (p < .001). Rates of postoperative hemorrhage (p = .997), hydrocephalus (p = .632), and wound infection (p = .126) did not change, while the frequency of early postoperative neurological deficits decreased from 21% between 1991 and 1995 to 13% between 2011 and 2015 (p = .003). During the same time, the rate of surgeries for postoperative cerebrospinal fluid leakage slightly increased from 2 to 3% (p = .049). Within a median follow-up of 62 months, progression was observed in 114 individuals (14%). Progression-free interval did not significantly change during observation period (p > .05). Multivariate analyses confirmed the lack of correlation between year of surgery and tumor relapse (HR: 1.1, p > .05). CONCLUSIONS: Preoperative diagnosis and surgery of meningiomas have been substantially evolved. Although early neurological outcome has improved, long-term prognosis remains unchanged.