A patient-designed tissue-engineered model of the infiltrative glioblastoma microenvironment.

Glioblastoma is an aggressive brain cancer characterized by diffuse infiltration. Infiltrated glioma cells persist in the brain post-resection where they interact with glial cells and experience interstitial fluid flow. We use patient-derived glioma stem cells and human glial cells (i.e., astrocytes and microglia) to create a four-component 3D model of this environment informed by resected patient tumors. We examine metrics for invasion, proliferation, and putative stemness in the context of glial cells, fluid forces, and chemotherapies. While the responses are heterogeneous across seven patient-derived lines, interstitial flow significantly increases glioma cell proliferation and stemness while glial cells affect invasion and stemness, potentially related to CCL2 expression and differential activation. In a screen of six drugs, we find in vitro expression of putative stemness marker CD71, but not viability at drug IC50, to predict murine xenograft survival. We posit this patient-informed, infiltrative tumor model as a novel advance toward precision medicine in glioblastoma treatment.

Intraneural perineurioma of the sciatic nerve: an under-recognized nerve neoplasm with characteristic MRI findings.

Intraneural perineurioma is a benign peripheral nerve neoplasm that typically affects teenagers and young adults and tends to result in a motor-predominant neuropathy. The lesion is rare, but has likely been underdiagnosed due to a lack of familiarity among both clinicians and radiologists. There have been few reports in the radiology literature despite the lesion having a fairly characteristic imaging appearance. We report a case of a 26-year-old woman with an intraneural perineurioma of the left sciatic nerve confirmed with excisional biopsy and pathologic analysis.

Autosomal dominant subcortical gliosis presenting as frontotemporal dementia.

OBJECTIVE: To describe a multigenerational kindred with a frontotemporal dementia clinical syndrome (FTDS), extensive subcortical gliosis pathology, and autosomal dominant genetics. METHODS: Clinical, imaging, and pathologic evaluations of multiple family members. RESULTS: Symptom onset commonly occurred in the fifth or sixth decade, although some kindred members did not develop obvious symptoms until their eighth decade. White matter changes were prominent on both MRI and CT imaging. Results from six brain autopsy evaluations showed consistent but varying degrees of pathology that, while unique, share some histologic similarities with leukodystrophies. These brains were notably devoid of both tau- and ubiquitin-containing inclusions. CONCLUSIONS: Subcortical gliosis in this kindred arises from mutation of a novel gene or else represents a unique frontotemporal dementia clinical syndrome variant caused by mutation of an already known gene. Clinical relevance and research implications are discussed.

Narrowing in on the causative defect of an intriguing X-linked myopathy with excessive autophagy.

BACKGROUND: X-Linked myopathy with excessive autophagy (XMEA) is a childhood-onset slowly progressive disease of skeletal muscle with no cardiac, nervous system, or other organ involvement. Pathology is distinctive: membrane-bound autophagic vacuoles, multifold reduplication of the basement membrane, and intense deposition of membrane attack complex and calcium at the myofiber surface. XMEA has been linked to the most telomeric 10.5 cM of Xq28. The authors now report identification of new families, refinement of the locus, mapping of genes to the region, and screening of candidate genes for mutations. METHODS AND RESULTS: Seven new families were ascertained, including an American family with XMEA. Using 11 new microsatellite genetic markers, the authors fine-mapped a recombination in this family and a common ancestral haplotype in two French families, which localized the gene in a 4.37-Mb region. Sequence data were assembled from public and private databases and a near-continuous sequence derived for the entire region. With this sequence, a gene map of 82 genes and 28 expressed sequence tag clusters was constructed; to date, 12 candidate genes have been screened for mutations. CONCLUSIONS: This study doubles the number of reported families with XMEA and more firmly establishes its distinctive clinicopathologic features. It also advances the search for the XMEA causative defect by reducing the disease locus to approximately half its previous size, assembling an almost complete sequence of the refined region, identifying all known genes in this sequence, and excluding the presence of mutations in 10% of these genes.

Mouse paracentric inversion In(3)55Rk mutates the urate oxidase gene.

The paracentric inversion In(3)55Rk on mouse Chromosome 3 (Chr 3) was induced by cesium irradiation. Genetic crosses indicate the proximal breakpoint cosegregates with D3Mit324 and D3Mit92; the distal breakpoint cosegregates with D3Mit127, D3Mit160, and D3Mit200. Giemsa-banded chromosomes show the inversion spans approximately 80% of Chr 3. The proximal breakpoint occurs within band 3A2, not 3B as reported previously; the distal breakpoint occurs within band 3H3. Mice homozygous for the inversion exhibit nephropathy indicative of uricase deficiency. Southern blot analyses of urate oxidase, Uox, show two RFLPs of genomic mutant DNA: an EcoRI site between exons 4-8 and a BamHI site 3' to exon 6. Mutant cDNA fails to amplify downstream of base 844 at the 3' end of exon 7. FISH analysis of chromosomes from inversion heterozygotes, using a cosmid clone containing genomic wild-type DNA for Uox exons 2-4, shows that a 5' segment of the mutated Uox allele on the inverted chromosome has been transposed from the distal breakpoint region to the proximal breakpoint region. Clinical, histopathological, and Northern analyses indicate that our radiation-induced mutation, uox(In), is a putative null.

A green fluorescent protein kinase substrate allowing detection and localization of intracellular ERK/MAP kinase activity.

We describe a versatile intracellular reporter of ERK/MAP kinase activity: a cDNA construct, pGFP.MBP, encoding amino acids 85-144 of the human myelin basic protein fused to the C-terminus of an enhanced green fluorescent protein (GFP). The fused fragment of myelin basic protein contains a single consensus ERK/MAP kinase phosphorylation motif (PRTP, where the threonine is phosphorylated). Phosphorylation of the specific motif can be detected via immunoblotting or immunofluorescence with a commercially available phospho-specific monoclonal antibody. When expressed in mammalian cells by either transient or stable transfection, the fusion protein acts as a bona fide kinase substrate, as demonstrated by rapid serum-induced phosphorylation that is blocked by a specific MEK inhibitor. Moreover, the localization of the total substrate pool is easily visualized by GFP autofluorescence and the extent of its phosphorylation simultaneously detected within intact fixed cells by immunofluorescence using the commercially available phospho-specific antibody. The approach described should be generally applicable to the intracellular analysis of many specific protein kinase substrates for which phospho-specific antibodies have been produced.

Mechanical trauma induces rapid astroglial activation of ERK/MAP kinase: Evidence for a paracrine signal.

Astrogliosis is a prominent and ubiquitous reaction of astrocytes to many forms of CNS injury, often implicated in the poor regenerative capacity of the adult mammalian CNS. Transmembrane signals that rapidly trigger and maintain astroglial responses to injury are largely undefined. Several candidate inducers of astrogliosis, including growth factors and neuropeptides, act via the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway. We previously observed chronically activated ERK/MAPK in human reactive astrocytes. To investigate mechanisms of pathway activation in a defined in vitro model, primary cultured astroglial monolayers were subjected to focal mechanical injury. Within 2-10 min, ERK/MAPK was activated, but only in cells near the wound edge. By 30 min, the entire monolayer showed activation, which persisted for 4 to 8 h. ERK/MAPK activation was specifically blocked by application of the MEK inhibitors, PD98059 and U0126. Cell-cell contact was not necessary for intercellular spread of ERK/MAPK activation, and ERK/MAPK-stimulating activity was found in the injury-conditioned medium. The activating factor was shown to have a native size of 50-100 kD and did not signal through the classical EGF receptor. Injury-induced signaling to ERK/MAPK required Ras, as demonstrated by specific blockade after transient transfection with a dominant negative Ha-RasN17 construct. Finally, we demonstrated that focal lesioning of adult rat cortex induces a rapid activation and spreading of astroglial ERK/MAPK, suggesting that similar mechanisms may operate in astroglial activation following acute brain injury.

Phosphorylation causes subtle changes in solvent accessibility at the interdomain interface of methylesterase CheB.

The crystal structure of the unphosphorylated state of methylesterase CheB shows that the regulatory domain blocks access of substrate to the active site of the catalytic domain. Phosphorylation of CheB at Asp56 results in a catalytically active transiently phosphorylated enzyme with a lifetime of approximately two seconds. Solvent accessibility changes in this transiently phosphorylated state were probed by MALDI-TOF-detected amide hydrogen/deuterium exchange. No changes in solvent accessibility were seen in the regulatory domain upon phosphorylation of Asp56, but two regions in the catalytic domain (199-203 and 310-317) became more solvent accessible. These two regions flank the active site and contain domain-domain contact residues. Comparison with results from the isolated catalytic domain-containing C-terminal fragment of CheB (residues 147-349) showed that the increased solvent accessibility was less than would have occurred upon detachment of the regulatory domain. Thus, phosphorylation causes subtle changes in solvent accessibility at the interdomain interface of CheB.

Protein docking using continuum electrostatics and geometric fit.

The computer program DOT quickly finds low-energy docked structures for two proteins by performing a systematic search over six degrees of freedom. A novel feature of DOT is its energy function, which is the sum of both a Poisson-Boltzmann electrostatic energy and a van der Waals energy, each represented as a grid-based correlation function. DOT evaluates the energy of interaction for many orientations of the moving molecule and maintains separate lists scored by either the electrostatic energy, the van der Waals energy or the composite sum of both. The free energy is obtained by summing the Boltzmann factor over all rotations at each grid point. Three important findings are presented. First, for a wide variety of protein-protein interactions, the composite-energy function is shown to produce larger clusters of correct answers than found by scoring with either van der Waals energy (geometric fit) or electrostatic energy alone. Second, free-energy clusters are demonstrated to be indicators of binding sites. Third, the contributions of electrostatic and attractive van der Waals energies to the total energy term appropriately reflect the nature of the various types of protein-protein interactions studied.

Solvent accessibility of the thrombin-thrombomodulin interface.

The kinetics of solvent accessibility at the protein-protein interface between thrombin and a fragment of thrombomodulin, TMEGF45, have been monitored by amide hydrogen/deuterium (H/2H) exchange detected by MALDI-TOF mass spectrometry. The interaction is rapid and reversible, requiring development of theory and experimental methods to distinguish H/2H exchange due to solvent accessibility at the interface from H/2H exchange due to complex dissociation. Association and dissociation rate constants were measured by surface plasmon resonance and amide H/2H exchange rates were measured at different pH values and concentrations of TMEGF45. When essentially 100% of the thrombin was bound to TMEGF45, two segments of thrombin became completely solvent-inaccessible, as evidenced by the pH insensitivity of the amide H/2H exchange rates. These segments form part of anion-binding exosite I and contain the residues for which alanine substitution abolishes TM binding. Several other regions of thrombin showed slowing of amide exchange upon TMEGF45 binding, but the exchange remained pH-dependent, suggesting that these regions of thrombin were rendered only partially solvent-inaccessible by TMEGF45 binding. These partially inaccessible regions of thrombin form both surface and buried contacts into the active site of thrombin and contain residues implicated in allosteric changes in thrombin upon TM binding.

Meningiomas of the orbit: contemporary considerations.

Meningiomas are the most frequently occurring benign intracranial neoplasms. Compared with other intracranial neoplasms they grow slowly, and they are potentially amenable to a complete surgical cure. They cause neurological compromise by direct compression of adjacent neural structures. Orbital meningiomas are interesting because of their location. They can compress the optic nerve, the intraorbital contents, the contents of the superior orbital fissure, the cavernous sinus, and frontal and temporal lobes. Because of its proximity to eloquent neurological structures, this lesion often poses a formidable operative challenge. Recent advances in techniques such as preoperative embolization and new modifications to surgical approaches allow surgeons to achieve their surgery-related goals and ultimately optimum patient outcome. Preoperative embolization may be effective in reducing intraoperative blood loss and in improving intraoperative visualization of the tumor by reducing the amount of blood obscuring the field and allowing unhurried microdissection. Advances in surgical techniques allow the surgeon to gain unfettered exposure of the tumor while minimizing the manipulation of neural structures. Recent advances in technology--namely, frameless computer-assisted image guidance--assist the surgeon in the safe resection of these tumors. Image guidance is particularly useful when resecting the osseous portion of the tumor because the tissue does not shift with respect to the calibration frame. The authors discuss their experience and review the contemporary literature concerning meningiomas of the orbit and the care of patients harboring such lesions.

Maximally efficient two-stage screening.

In DNA library screening, blood testing, and monoclonal antibody generation, significant savings in the number of assays can be realized by employing group sampling. Practical considerations often limit the number of stages of group testing that can be performed. We address situations in which only two stages of testing are used. We define efficiency to be the expected number of positives isolated per assay performed and assume gold-standard tests with unit sensitivity and specificity. Although practical tests never are golden, polymerase chain reaction (PCR) methods provide procedures for screening recombinant libraries that are strongly selective yet retain high sensitivity even when samples are pooled. Also, results for gold-standard tests serve as bounds on the performance of practical testing procedures. First we derive formulas for the efficiency of certain extensions of the popular rows-and-columns technique. Then we derive an upper bound on the efficiency of any two-stage strategy that lies well below the classical upper bound for situations with no constraint on the number of stages. This establishes that a restriction to only two stages necessitates performing many more assays than efficient multistage procedures need. Next, we specialize the bound to cases in which each item belonging only to pools that tested positive in stage 1 must be tested individually in stage 2. The specialized bound for such positive procedures is tight because we show that an appropriate multidimensional extension of the rows-and-columns technique achieves it. We also show that two-stage positive procedures in which the stage-1 groups are selected at random perform suboptimally, thereby establishing that efficient tests must be structured carefully.

Interleukin-2 plus chemotherapy for patients with metastatic melanoma.

We studied the activity of recombinant interleukin-2 (IL2) in combination with multiagent chemotherapy in the treatment of patients with disseminated malignant melanoma. Patients were randomized to receive the same dose of lymphokine by constant 24 h intravenous infusion (CI) or by subcutaneous bolus (SB) injection. Twenty-two patients, 18 males and four females with a median age of 44 years (range 32-73 years) were randomized to receive IL2 5 million units/m2 once daily by SB injection or by CI, 5 days/week for 2 weeks. All patients received a chemotherapy regimen consisting of lomustine (CCNU) 75 mg/m2 on day 14, bleomycin 10 units/day by CI for 5 days (days 14-19) and cisplatin 75 mg/m2 on day 19. Patients were retreated after a 3 week interval. There were four complete responses and one partial response in the CI arm and two partial responses in the SB arm. The median duration of response was 38 weeks (range 26-107 weeks). The median duration of survival was 6.7 months in non-responders and 11.1 months in responders. The overall response rate was 32%. Since responses were brief and all the responding patients progressed after a median of 38 weeks, the study was terminated before accrual goals were met.

Experimental study of umbilical cord length as a marker of fetal alcohol syndrome.

Umbilical cord length has long been investigated as a potential marker of intrauterine events that may place the neonate at risk for future adverse developmental sequelae. Experimentally, significantly shortened cords have been reported in association with prenatal exposure to common drugs of abuse. This study in rats reports the time course of effects on umbilical cord length of a daily maternal ethanol gavage (3,200 mg/kg) from gestational day 6 through termination of pregnancy at either day 17, 18, 19, or 20. A total of 786 fetuses derived from 60 litters were examined. Control fetuses demonstrated a linear increase in umbilical cord length and body weight gain during late gestation, findings that support previous studies. The body weights of the ethanol-exposed fetuses were reduced significantly on all gestational days examined, indicating intrauterine growth retardation, a characteristic of fetal alcohol syndrome. Similarly, acute fetal akinesia as well as long-term sequelae stemming from impaired neurological development would result from the elevated blood ethanol levels achieved in this study. The umbilical cords of ethanol-exposed fetuses were significantly shorter on gestational days 19 and 20 in comparison to their controls, while cord lengths on days 17 and 18 were not shortened significantly. A stretch hypothesis has been proposed suggesting that the degree of fetal activity is the main determinant of umbilical cord length. In rats, there is a physiologic diminution of the volume of amniotic fluid (oligohydramnios) in late gestation (day 19 to term), which restricts fetal movements but does not appear to alter the linear relationships between gestational age and cord length in controls, thus arguing against the stretch hypothesis. However, cord lengths in the ethanol-exposed fetuses plateaued in late gestation, suggesting possible adherence to a stretch hypothesis. This dichotomy is discussed emphasizing fetal growth and activity as well as intrauterine space.

Sinus histiocytosis (Rosai-Dorfman disease) of the suprasellar region: MR imaging findings--a case report.

Sinus histiocytosis with massive lymphadenopathy (SHML) is an uncommon disorder that typically manifests as systemic symptoms and lymphadenopathy. Extranodal, intracranial disease is uncommon. The authors report on a 15-year-old adolescent girl who had a suprasellar mass at magnetic resonance imaging. Biopsy results demonstrated lymphophagocytosis consistent with a diagnosis of SHML. The clinical, radiologic, and histologic aspects of the disease are discussed.

Histologic evaluation of platinum coil embolization in an aneurysm model in rabbits.

PURPOSE: To characterize the histologic response to platinum coil embolization by using a rabbit aneurysm model. MATERIALS AND METHODS: Saccular aneurysms were created in New Zealand White rabbits by using vessel ligation with intraluminal elastase incubation. Aneurysms were subsequently embolized by using platinum coils. Subjects were sacrificed at various intervals up to 12 weeks following coil embolization. The aneurysm cavities and adjacent vessels were embedded in methylmethacrylate, were sectioned, and were stained for histologic examination. RESULTS: Two weeks following coil implantation, aneurysms were filled predominantly with unorganized thrombus. Six weeks following coil implantation, histologic features included complete filling of the aneurysm lumen with either prominent laminated but unorganized thrombus or areas of unorganized thrombus interspersed among areas of cellular infiltration. At 12 weeks following coil implantation, aneurysms were filled with the loosely packed, disordered cells contained within the extracellular matrix. Fibrosis or smooth muscle cell infiltration was not present in any of the 6- or 12-week samples. CONCLUSION: Platinum coils placed into experimental saccular aneurysms in New Zealand White rabbits failed to elicit a fibrotic response. This model can be used for the testing of biologic modifications of platinum coils aimed at increasing intra-aneurysmal fibrosis.

Endovascular creation of an in vivo bifurcation aneurysm model in rabbits.

PURPOSE: To develop a rabbit model of an intracranial bifurcation aneurysm to test new endovascular therapies. MATERIALS AND METHODS: An experimental aneurysm model was created in rabbits by means of endovascular balloon occlusion of the left common carotid artery, which created an aneurysm at the bifurcation formed by the aortic arch and the brachiocephalic trunk. A total of 18 aneurysms were created. In eight rabbits, the aneurysms were incubated with intraluminal elastase to induce degeneration of the elastic laminae. The animals were followed up with angiography for as long as 3 months. The animals were sacrificed at various times, and histologic evaluation of the aneurysm was performed. RESULTS: Ten aneurysms created without elastase infusion were all very small or completely closed at 1-3 months. Six aneurysms created with elastase infusion had long-term patency (two were patent at 1 month and four, at 3 months). The elastase aneurysms had a mean width of 3 mm (range, 2-3.5 mm) and a mean length of 5 mm (range, 3-7 mm). Histologic evaluation revealed destruction of the normal elastin layers, which allowed the artery to become aneurysmal. CONCLUSION: This aneurysm model re-created the hemodynamic forces and size of human cerebral bifurcation aneurysms and maintained the integrity of the endothelium. The creation of the aneurysms was rapid, reliable, and reproducible.

Recurrent Epstein-Barr virus-associated post-transplant lymphoproliferative disorder: report of a patient with histologically similar but clonally distinct metachronous abdominal and brain lesions.

A liver transplant patient developed a single central nervous system (CNS) intraparenchymal lesion 5 months after the diagnosis of an intraabdominal diffuse large B-cell post-transplant lymphoproliferative disorder (PTLD). Biopsy of the new CNS lesion showed a diffuse large B-cell PTLD morphologically and immunohistochemically indistinguishable from the abdominal lesion. In addition, both lesions were positive for Epstein-Barr virus (EBV) DNA by polymerase chain reaction (PCR) and for EBV-encoded RNA by in situ hybridization. Although these results were consistent with a metastatic origin for the CNS lesion, the finding of an intraparenchymal lesion without leptomeningeal or dural spread was suggestive of a new primary CNS lymphoma. Proof that the brain lesion was a second primary and not a metastasis was obtained by immunoglobulin gene rearrangement studies and assessment of EBV clonality. Multiple primary lymphoid neoplasms arise at higher frequency in the setting of immunosuppression, and molecular investigations of tumor clonality can provide clinically relevant staging and prognostic information.

Upper ureteral reconstructive surgery.

Upper ureteral reconstructive surgery encompasses a wide variety of procedures directed at the correction of abnormal processes and structural defects in the proximal ureter. Although some of these techniques have strict indications for specific causes, technical innovations have led to development of numerous alternatives in upper ureteral reconstructive surgery. These innovations provide the practicing urologist with various options from which to choose for the management of upper ureteral disease.

Activation of mitogen-activated protein kinase associated with prostate cancer progression.

Using an antibody specific for dually phosphorylated extracellular-regulated kinases 1 and 2, we have examined 82 primary and metastatic prostate tumor specimens for the presence of activated mitogen-activated protein (MAP) kinase. Nonneoplastic prostate tissue showed little or no staining with activated MAP kinase antiserum. In prostate tumors, the level of activated MAP kinase increased with increasing Gleason score and tumor stage. In a separate analysis, tumor samples from two patients showed no activation of MAP kinase before androgen ablation therapy; however, following androgen ablation treatment, high levels of activated MAP kinase were detected in the recurrent tumors. Collectively, these data suggest an increase in the activation of the MAP kinase signal transduction pathway as prostate cancer progresses to a more advanced and androgen-independent disease.