Highlights d Flow cytometry, RNA-seq, and protein and image analyses reveal brain TME complexity d Glioma IDH mutation status and brain metastasis primary tumors shape the brain TME d Microglia and monocyte-derived macrophages exhibit multifaceted activation d TME immune cells show disease-and cell-type-specific expression patterns

Comprehensive knowledge of the human leukocyte antigen (HLA) class-I and class-II peptides presented to T-cells is crucial for designing innovative therapeutics against cancer and other diseases. However methodologies for their purification for mass-spectrometry analysis have been a major limitation. We designed a novel high-throughput, reproducible and sensitive method for sequential immuno-affinity purification of HLA-I and -II peptides from up to 96 samples in a plate format, suitable for both cell lines and tissues. Our methodology drastically reduces sample-handling and can be completed within five hours. We challenged our methodology by extracting HLA peptides from multiple replicates of tissues (n = 7) and cell lines (n = 21, 108 cells per replicate), which resulted in unprecedented depth, sensitivity and high reproducibility (Pearson correlations up to 0.98 and 0.97 for HLA-I and HLA-II). Because of the method's achieved sensitivity, even single measurements of peptides purified from 107 B-cells resulted in the identification of more than 1700 HLA-I and 2200 HLA-II peptides. We demonstrate the feasibility of performing drug-screening by using ovarian cancer cells treated with interferon gamma (IFNγ). Our analysis revealed an augmented presentation of chymotryptic-like and longer ligands associated with IFNγ induced changes of the antigen processing and presentation machinery. This straightforward method is applicable for basic and clinical applications.

HLA-I molecules bind short peptides and present them for recognition by CD8 + T cells. The length of HLA-I ligands typically ranges from 8 to 12 aa, but variability is observed across different HLA-I alleles. In this study we collected recent in-depth HLA peptidomics data, including 12 newly generated HLA peptidomes (31,896 unique peptides) from human meningioma samples, to analyze the peptide length distribution and multiple specificity across 84 different HLA-I alleles. We observed a clear clustering of HLA-I alleles with distinct peptide length distributions, which enabled us to study the structural basis of peptide length distributions and predict peptide length distributions from HLA-I sequences. We further identified multiple specificity in several HLA-I molecules and validated these observations with binding assays. Explicitly modeling peptide length distribution and multiple specificity improved predictions of naturally presented HLA-I ligands, as demonstrated in an independent benchmarking based on the new human meningioma samples.

BackgroundHOX genes are a family of developmental genes that are expressed neither in the developing forebrain nor in the normal brain. Aberrant expression of a HOX-gene dominated stem-cell signature in glioblastoma has been linked with increased resistance to chemo-radiotherapy and sustained proliferation of glioma initiating cells. Here we describe the epigenetic and genetic alterations and their interactions associated with the expression of this signature in glioblastoma.ResultsWe observe prominent hypermethylation of the HOXA locus 7p15.2 in glioblastoma in contrast to non-tumoral brain. Hypermethylation is associated with a gain of chromosome 7, a hallmark of glioblastoma, and may compensate for tumor-driven enhanced gene dosage as a rescue mechanism by preventing undue gene expression. We identify the CpG island of the HOXA10 alternative promoter that appears to escape hypermethylation in the HOX-high glioblastoma. An additive effect of gene copy gain at 7p15.2 and DNA methylation at key regulatory CpGs in HOXA10 is significantly associated with HOX-signature expression. Additionally, we show concordance between methylation status and presence of active or inactive chromatin marks in glioblastoma-derived spheres that are HOX-high or HOX-low, respectively.ConclusionsBased on these findings, we propose co-evolution and interaction between gene copy gain, associated with a gain of chromosome 7, and additional epigenetic alterations as key mechanisms triggering a coordinated, but inappropriate, HOX transcriptional program in glioblastoma.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0583-7) contains supplementary material, which is available to authorized users.

Summary:Objective: Patients with intractable epilepsy due to extensive lesions involving the posterior quadrant (temporal, parietal, and occipital lobes) form a small subset of epilepsy surgery. This study was done with a view to analyze our experience with this group of patients and to define the changes in the surgical technique over the last 15 years. We also describe the microsurgical technique of the different surgical variants used, along with their functional neuroanatomy.Methods: In this series there were 13 patients with a median age of 17 years. All patients had extensive presurgical evaluation that provided concordant evidence localizing the lesion and seizure focus to the posterior quadrant. The objective of the surgery was to eliminate the effect of the epileptogenic tissue and preserve motor and sensory functions.Results: During the course of this study period of 15 years, the surgical procedure performed evolved toward incorporating more techniques of disconnection and minimizing resection. Three technical variants were thus utilized in this series, namely, (i) anatomical posterior quadrantectomy (APQ), (ii) functional posterior quadrantectomy (FPQ), and (iii) periinsular posterior quadrantectomy (PIPQ). After a median follow-up period of 6 years, 12/13 patients had Engel's Class I seizure outcome.Conclusion: The results of surgery for posterior quadrantic epilepsy have yielded excellent seizure outcomes in 92% of the patients in the series with no mortality or major morbidity. The incorporation of disconnective techniques in multilobar surgery has maintained the excellent results obtained earlier with resective surgery.

There are clear indications for hemispherectomy in children. In some instances of incomplete deficit, timing of surgery remains a major concern. The less invasive approach to eliminate the influence of the diseased hemisphere, in our opinion, is with disconnective techniques of hemispherectomy, and among the latter, peri-insular hemispherotomy provides, in our opinion, the best complications-benefits ratio.

Background and Purpose-Lactate is central for the regulation of brain metabolism and is an alternative substrate to glucose after injury. Brain lactate metabolism in patients with subarachnoid hemorrhage has not been fully elucidated. Methods-Thirty-one subarachnoid hemorrhage patients monitored with cerebral microdialysis (CMD) and brain oxygen (PbtO 2 ) were studied. Samples with elevated CMD lactate (Ͼ4 mmol/L) were matched to PbtO 2 and CMD pyruvate and categorized as hypoxic (PbtO 2 Ͻ20 mm Hg) versus nonhypoxic and hyperglycolytic (CMD pyruvate Ͼ119 mol/L) versus nonhyperglycolytic. Results-Median per patient samples with elevated CMD lactate was 54% (interquartile range, 11%-80%). Lactate elevations were more often attributable to cerebral hyperglycolysis (78%; interquartile range, 5%-98%) than brain hypoxia (11%; interquartile range, 4%-75% Generally considered a product of anaerobic metabolism, endogenous lactate is pivotal for neuronal survival, 3,4 particularly in conditions of acute injury. 5,6 Cerebral microdialysis (CMD) enables quantification of brain metabolites in cerebral extracellular fluid and provides information about energy metabolism and the extent of aerobic versus anaerobic glycolysis. 7 Further insights can be obtained by combining CMD with brain oxygen (PbtO 2 ) monitoring to quantify the extent of brain hypoxia. 8 Brain lactate metabolism after subarachnoid hemorrhage (SAH) has not been fully elucidated. We hypothesized that elevations of brain lactate occur in poor-grade SAH patients either as the consequence of cerebral hyperglycolysis or as the consequence of brain hypoxia, and that differences in the patterns of elevated brain lactate may be associated with outcome. Patients and MethodsWe studied comatose patients with aneurysmal SAH admitted to the Division of Neurocritical Care, Hospital of the University of Pennsylvania, Philadelphia, and in the Department of Critical Care, Lausanne University Hospital, Switzerland, over a 4-year period and who underwent combined CMD-PbtO 2 monitoring. Approval was obtained by local Institutional Review Board. Patients had at least 24 hours of valid intracranial monitoring and were alive for Ͼ5 days. Intracranial monitoring was performed as part of standard care, as previously described. 9 Patients were managed according to a standard protocol in both centers; 10 therapeutic targets were set to avoid cerebral perfusion pressure Ͻ60 mm Hg and PbtO 2 Ͻ20 mm Hg. CMD catheters (CMA 70; CMA Microdialysis AB; flow rate, 0.3 L/min) and PbtO 2 probes (Licox; Integra Neurosciences) were inserted via a triple-lumen bolt and placed into visually normal white matter. CMD samples were collected every 60 minutes and analyzed for concentrations of lactate, pyruvate, and glucose. Outcome at 6 months was assessed with the modified Rankin Scale score by 1 neurologist and 1 neurointensive care nurse who were blinded to physiological data.First hour of monitored data, artifacts, and data points outside physiological ranges were excluded. Brain lactate elevations ...

Background: Elevated intracranial pressure (ICP) is frequent after traumatic brain injury (TBI) and may cause abnormal pupillary reactivity, which in turn is associated with a worse prognosis. Using automated infrared pupillometry, we examined the relationship between the Neurological Pupil index (NPi) and invasive ICP in patients with severe TBI. Methods: This was an observational cohort of consecutive subjects with severe TBI (Glasgow Coma Scale [GCS] < 9 with abnormal lesions on head CT) who underwent parenchymal ICP monitoring and repeated NPi assessment with the NPi-200® pupillometer. We examined NPi trends over time (four consecutive measurements over intervals of 6 h) prior to sustained elevated ICP > 20 mmHg. We further analyzed the relationship of cumulative abnormal NPi burden (%NPi values < 3 during total ICP monitoring time) with intracranial hypertension (ICHT)-categorized as refractory (ICHT-r; requiring surgical decompression) vs. non-refractory (ICHT-nr; responsive to medical therapy)-and with the 6-month Glasgow Outcome Score (GOS). Results: A total of 54 patients were studied (mean age 54 ± 21 years, 74% with focal injuries on CT), of whom 32 (59%) had ICHT. Among subjects with ICHT, episodes of sustained elevated ICP (n = 43, 172 matched ICP-NPi samples; baseline ICP [T − 6 h ] 14 ± 5 mmHg vs. ICPmax [T 0 h ] 30 ± 9 mmHg) were associated with a concomitant decrease of the NPi (baseline 4.2 ± 0.5 vs. 2.8 ± 1.6, p < 0.0001 ANOVA for repeated measures). Abnormal NPi values were more frequent in patients with ICHT-r (n = 17; 38 [3-96]% of monitored time vs. 1 [0-9]% in patients with ICHT-nr [n = 15] and 0.5 [0-10]% in those without ICHT [n = 22]; p = 0.007) and were associated with an unfavorable 6-month outcome (15 [1-80]% in GOS 1-3 vs. 0 [0-7]% in GOS 4-5 patients; p = 0.002). Conclusions: In a selected cohort of severe TBI patients with abnormal head CT lesions and predominantly focal cerebral injury, elevated ICP episodes correlated with a concomitant decrease of NPi. Sustained abnormal NPi was in turn associated with a more complicated ICP course and worse outcome.