Development of a bleeding scale and hemostasis algorithm in cranial neurosurgery.

Effective hemostasis is crucial in neurosurgery as anatomical and functional considerations reduce tolerance for any bleeding. The classification of bleeding severities is a necessary step to enable neurosurgeons to counteract bleeding during surgery. Even though bleeding scales are used for a variety of surgical specialties, they cannot be transferred to cranial neurosurgery without adaption, and no consensus on the nature of such a classification exists to date. Moreover, there is plethora of topical hemostasis products with diverse mechanisms of action and application available. Clinical studies investigating those products used in neurosurgery did not define standardized procedures. This article demonstrates the systematic establishment of both a bleeding scale and a hemostasis algorithm to close this gap in the assessment of intracranial bleeding. The expert panel consisting of 7 members from different neurosurgical centers developed a qualitative bleeding scale with the peculiarities of neurosurgical procedures, based on the experience of each member in daily practice. The hemostasis algorithm is a recommendation for neurosurgeons to aid in the decision-making process to control any sort of bleeding, taking into account the rational use of available hemostatics, depending on type and location of bleeding, as well as the mechanism of action of such agents. Effectiveness of hemostasis, surgery times and economic costs can be optimized by applying the algorithm in daily practice.

Staphylococcus aureus surgical site infection rates in 5 European countries.

OBJECTIVE: To determine the overall and procedure-specific incidence of surgical site infections (SSI) caused by Staphylococcus aureus (S. aureus) as well as risk factors for such across all surgical disciplines in Europe. METHODS: This is a retrospective cohort of patients with surgical procedures performed at 14 European centres in 2016, with a nested case-control analysis. S. aureus SSI were identified by a semi-automated crossmatching bacteriological and electronic health record data. Within each surgical procedure, cases and controls were matched using optimal propensity score matching. RESULTS: A total of 764 of 178 902 patients had S. aureus SSI (0.4%), with 86.0% of these caused by methicillin susceptible and 14% by resistant pathogens. Mean S. aureus SSI incidence was similar for all surgical specialties, while varying by procedure. CONCLUSIONS: This large procedure-independent study of S. aureus SSI proves a low overall infection rate of 0.4% in this cohort. It provides proof of principle for a semi-automated approach to utilize big data in epidemiological studies of healthcare-associated infections. Trials registration The study was registered at clinicaltrials.gov under NCT03353532 (11/2017).

Harmonized procedure coding system for surgical procedures and analysis of surgical site infections (SSI) of five European countries.

BACKGROUND: The use of routine data will be essential in future healthcare research. Therefore, harmonizing procedure codes is a first step to facilitate this approach as international research endeavour. An example for the use of routine data on a large scope is the investigation of surgical site infections (SSI). Ongoing surveillance programs evaluate the incidence of SSI on a national or regional basis in a limited number of procedures. For example, analyses by the European Centre for Disease Prevention (ECDC) nine procedures and provides a mapping table for two coding systems (ICD9, National Healthcare Safety Network [NHSN]). However, indicator procedures do not reliably depict overall SSI epidemiology. Thus, a broader analysis of all surgical procedures is desirable. The need for manual translation of country specific procedures codes, however, impedes the use of routine data for such an analysis on an international level. This project aimed to create an international surgical procedure coding systems allowing for automatic translation and categorization of procedures documented in country-specific codes. METHODS: We included the existing surgical procedure coding systems of five European countries (France, Germany, Italy, Spain, and the United Kingdom [UK]). In an iterative process, country specific codes were grouped in ever more categories until each group represented a coherent unit based on method of surgery, interventions performed, extent and site of the surgical procedure. Next two ID specialist (arbitrated by a third in case of disagreement) independently assigned country-specific codes to the resulting categories. Finally, specialist from each surgical discipline reviewed these assignments for their respective field. RESULTS: A total number of 153 SALT (Staphylococcus aureus Surgical Site Infection Multinational Epidemiology in Europe) codes from 10 specialties were assigned to 15,432 surgical procedures. Almost 4000 (26%) procedure codes from the SALT coding system were classified as orthopaedic and trauma surgeries, thus this medical field represents the most diverse group within the SALT coding system, followed by abdominal surgical procedures with 2390 (15%) procedure codes. CONCLUSION: Mapping country-specific codes procedure codes onto to a limited number of coherent, internally and externally validated codes proofed feasible. The resultant SALT procedure code gives the opportunity to harmonize big data sets containing surgical procedures from international centres, and may simplify comparability of future international trial findings. TRIAL REGISTRATION: The study was registered at clinicaltrials.gov under NCT03353532 on November 27th, 2017.

Two Decades of Brain Tumour Imaging with O-(2-[18F]fluoroethyl)-L-tyrosine PET: The Forschungszentrum Jülich Experience.

O-(2-[18F]fluoroethyl)-L-tyrosine (FET) is a widely used amino acid tracer for positron emission tomography (PET) imaging of brain tumours. This retrospective study and survey aimed to analyse our extensive database regarding the development of FET PET investigations, indications, and the referring physicians' rating concerning the role of FET PET in the clinical decision-making process. Between 2006 and 2019, we performed 6534 FET PET scans on 3928 different patients against a backdrop of growing demand for FET PET. In 2019, indications for the use of FET PET were as follows: suspected recurrent glioma (46%), unclear brain lesions (20%), treatment monitoring (19%), and suspected recurrent brain metastasis (13%). The referring physicians were neurosurgeons (60%), neurologists (19%), radiation oncologists (11%), general oncologists (3%), and other physicians (7%). Most patients travelled 50 to 75 km, but 9% travelled more than 200 km. The role of FET PET in decision-making in clinical practice was evaluated by a questionnaire consisting of 30 questions, which was filled out by 23 referring physicians with long experience in FET PET. Fifty to seventy per cent rated FET PET as being important for different aspects of the assessment of newly diagnosed gliomas, including differential diagnosis, delineation of tumour extent for biopsy guidance, and treatment planning such as surgery or radiotherapy, 95% for the diagnosis of recurrent glioma, and 68% for the diagnosis of recurrent brain metastases. Approximately 50% of the referring physicians rated FET PET as necessary for treatment monitoring in patients with glioma or brain metastases. All referring physicians stated that the availability of FET PET is essential and that it should be approved for routine use. Although the present analysis is limited by the fact that only physicians who frequently referred patients for FET PET participated in the survey, the results confirm the high relevance of FET PET in the clinical diagnosis of brain tumours and support the need for its approval for routine use.

A multicenter cohort study of early complications after cranioplasty: results of the German Cranial Reconstruction Registry.

OBJECTIVE: Cranioplasty (CP) is a crucial procedure after decompressive craniectomy and has a significant impact on neurological improvement. Although CP is considered a standard neurosurgical procedure, inconsistent data on surgery-related complications after CP are available. To address this topic, the authors analyzed 502 patients in a prospective multicenter database (German Cranial Reconstruction Registry) with regard to early surgery-related complications. METHODS: Early complications within 30 days, medical history, mortality rates, and neurological outcome at discharge according to the modified Rankin Scale (mRS) were evaluated. The primary endpoint was death or surgical revision within the first 30 days after CP. Independent factors for the occurrence of complications with or without surgical revision were identified using a logistic regression model. RESULTS: Traumatic brain injury (TBI) and ischemic stroke were the most common underlying diagnoses that required CP. In 230 patients (45.8%), an autologous bone flap was utilized for CP; the most common engineered materials were titanium (80 patients [15.9%]), polyetheretherketone (57 [11.4%]), and polymethylmethacrylate (57 [11.4%]). Surgical revision was necessary in 45 patients (9.0%), and the overall mortality rate was 0.8% (4 patients). The cause of death was related to ischemia in 2 patients, diffuse intraparenchymal hemorrhage in 1 patient, and cardiac complications in 1 patient. The most frequent causes of surgical revision were epidural hematoma (40.0% of all revisions), new hydrocephalus (22.0%), and subdural hematoma (13.3%). Preoperatively increased mRS score (OR 1.46, 95% CI 1.08-1.97, p = 0.014) and American Society of Anesthesiologists Physical Status Classification System score (OR 2.89, 95% CI 1.42-5.89, p = 0.003) were independent predictors of surgical revision. Ischemic stroke, as the underlying diagnosis, was associated with a minor rate of revisions compared with TBI (OR 0.18, 95% CI 0.06-0.57, p = 0.004). CONCLUSIONS: The authors have presented class II evidence-based data on surgery-related complications after CP and have identified specific preexisting risk factors. These results may provide additional guidance for optimized treatment of these patients.

The choice of anaesthesia for glioblastoma surgery does not impact the time to recurrence.

Anaesthetics used during cancer surgery may influence tumour cells and immunological response. The aim of this study was to evaluate a potential influence of the anaesthetic method (inhaled anaesthetics versus total-intravenous anaesthesia using propofol) on recurrence-free and overall survival in glioblastoma patients. We retrospectively identified patients undergoing resection of contrast enhancing glioblastoma under general anaesthesia followed by standard adjuvant treatment between January 2010 and February 2017 at two University Hospitals. Matched pairs of patients receiving either balanced with volatile anaesthetics or total intravenous anaesthesia were generated according to the known prognostic factors (extent of resection, methyl-guanine-methyl-transferase (MGMT) promoter methylation, age, Karnofsky performance score). Groups were compared using chi-square and Whitney-Man-U test. Time to recurrence was calculated using Kaplan Meier estimates. Log Rank test was used to assess the influence of the anaesthetic method. One hundred and fifty-eight (79:79) patients were included. Groups showed no significant difference in recurrence-free (volatiles: 8.0 (95% CI 6.5-9.8) vs. propofol: 8.4 (95% CI 7.9-10.1) months; p = 0.54) or overall survival (propofol: 17.4 (95% CI 14.0-20.7) vs. volatiles: 16.9 (95% CI 13.9-20.1) months; p = 0.85). In contrast to potential beneficial effects in some other solid tumours, the choice of anaesthetic method had no impact on survival in patients with glioblastoma in a well-defined cohort.

A case report of recurrent membranoproliferative glomerulonephritis after kidney transplantation due to ventriculoatrial shunt infection.

BACKGROUND: Transplant failure requires the consideration of numerous potential causes including rejection, acute tubular necrosis, infection, and recurrence of the original kidney disease. Kidney biopsy is generally required to approach these differential diagnoses. However, the histopathological findings on their own do not always lead to a definite diagnosis. Consequently, it is crucial to integrate them with clinical findings and patient history when discussing histopathological patterns of injury. The histopathologic finding of a membranoproliferative glomerulonephritis (MPGN) is one of the most challenging constellations since it does not refer to a specific disease entity but rather reflects a pattern of injury that is the result of many different causes. Whilst MPGN is occasionally classified as immune complex mediated, careful evaluation usually reveals an underlying disorder such as chronic infection, plasma cell dyscrasia, complement disorders, and autoimmune disease. CASE PRESENTATION: We describe the case of a 43-year-old woman who was referred to us because of a slowly rising serum creatinine 4 years after kidney transplantation. As in the native kidney, the biopsy revealed an MPGN pattern of injury. The cause of this finding had not been established prior to transplantation leading to a classification as idiopathic MPGN in the past. Further workup at the time of presentation and allograft failure revealed chronic infection of a ventriculoatrial shunt as the most probable cause. CONCLUSION: This case underlines the fact that MPGN is not a disease but a histopathological description. Consequently, the causative disorder needs to be identified to avoid kidney failure and recurrence after transplantation.

Current treatment concepts for iatrogenic ventriculitis: a nationwide survey in Germany.

BACKGROUND: Iatrogenic ventriculitis is a common complication of the external ventricular drainage. While the procedure and indications for external ventricular drains (EVD) are highly standardized, the treatment of ventriculitis is not clearly defined. OBJECTIVE: To depict the treatment of iatrogenic ventriculitis currently performed in German hospitals. METHODS: A standardized questionnaire consisting of 18 multiple choice questions, each with the ability to provide additional individual answers, covering the diagnosis and treatment of iatrogenic ventriculitis as well as general handling of EVDs, was sent to 121 neurosurgical hospitals registered in the German Society for Neurosurgery (DGNC). RESULTS: Thirty-three out of 121 hospitals returned the questionnaire. While diagnostics are performed similarly in most hospitals, the treatment varies remarkably. Ten of the 33 (30%) units never applied antibiotics intrathecally and 12 (36%) only in selected (1-20%) cases, while 7 (21%) do this routinely, and the remaining 4 centers vary their treatment. While the targeted systemic therapy after pathogen identification and resistance testing is similar, the choice of empiric antibiotics varies as does the type of drug used for intrathecal therapy. Among the applied systemic antibiotics, vancomycin [n = 23 (70%)] and meropenem [n = 22 (67%)] were the most common, but many others, including ceftriaxone, metronidazol, linezolid, piperacillin/tazobactam, fosfomycin and ceftazidim, are used. There is no standard practice regarding EVD handling. Twelve (36%) hospitals do not replace the EVD after a new diagnosis of ventriculitis, 13 (39%) do so once after the diagnosis, and 8 (24%) regularly switch EVDs after a defined time span (7-20 days), even without signs of infection. CONCLUSION: Treatment concepts for iatrogenic ventriculitis are very heterogeneous. Thus, there is an urgent need for generating outcome data and defining a standard treatment algorithm with the recently published practice guideline being an important first step.

Glioblastoma multiforme from diagnosis to death: a prospective, hospital-based, cohort, pilot feasibility study of patient reported symptoms and needs.

PURPOSE: Glioblastoma (GBM) patients have many palliative care (PC) issues. To date, there are no studies examining the prospective usage of validated PC assessment tools as patient reported outcome measures for GBM patients. METHODS: GBM patients' PC issues were assessed from diagnosis to death or for at least 12 months every 7 weeks (±8 days) using semi-structured interviews and the Hospice and Palliative Care Evaluation (HOPE, including Eastern Cooperative Oncology Group (ECOG) performance status, 17 items) and the Palliative Outcome Scale (POS, 11 items). Data from patients who died within 12 months of the last patient's enrollment were evaluated using summarizing content analysis, visual graphical analysis (VGA), and linear mixed models for repeated measures. RESULTS: Nineteen of 33 patients screened were enrolled; two dropped out and four were still alive at the end of the study. The remaining 13 were assessed at 59 points until death (time range 4-68 weeks; 1-10 contacts per patient; assessment: self, 33; joint, 8; external, 18). VGA of the HOPE and POS data, including all 1,652 assessed item data, showed consistent trajectory profiles for 14 of 28 items: 10 were increasing (meaning symptom worsening) and comprised predominantly psychosocial issues and care dependency. Type of assessment partly interacted with time, however, not qualitatively so. Analysis of semi-structured interviews revealed delayed interactions with PC/hospice services and numerous neuropsychiatric problems not detected by HOPE and POS. CONCLUSIONS: Prospective self-assessment of GBM patients' PC issues is feasible. However, disease progression may necessitate further, external assessment. Modification of existing PC assessment tools is needed to detect GBM-specific issues.

Intracerebral administration of heat-inactivated Staphylococcus epidermidis enhances oncolysis and prolongs survival in a 9L orthotopic gliosarcoma model.

BACKGROUND/AIMS: The association between postoperative infection and prolonged survival in high-grade glioma is still a matter of debate. Previously we demonstrated that the intracerebral (i.c.) injection of heat-inactivated staphylococcal epitopes (HISE) resulted in a well-defined infux of immunocompetent cells across the blood-brain barrier. The present study investigated the potential antitumoral effect of HISE-immunostimulation in an experimental glioma model. METHODS: Wistar rats were intracerebrally implanted with 9L gliosarcoma cells (n=6), 9L cells mixed with HISE (n=12), or phosphate buffered saline (n=4). Tumor growth was measured by serial magnetic resonance imaging (MRI). After death due to the tumor burden, the brains were histopathologically assessed for inflammation and oncolysis. A toxicity assay was performed to quantify potential impairment of HISE on tumor cell growth in vitro. RESULTS: Animals treated by HISE showed a significant increase in average survival and even complete regression of an already established mass in one case. Naïve 9L gliosarcomas failed to recruit significant numbers of systemic immune cells. In contrast, concomitant intracerebral HISE inoculation lead to a oncolysis and a distinct peri- and intratumoral infiltration of macrophages, CD8 and CD4 co-expressing T-lymphocytes in two thirds of the tumor-bearing animals. The toxicity screening showed HISE-mediated oncolysis to be ineffective ex vivo. CONCLUSION: This study describes a novel approach for combatting malignant glioma using inactivated staphylococci as potent immunomodulators. Our results provide an outline for investigating the strategic potential of bacteria as emerging future therapeutics.

A novel experimental in vivo model of cerebral immunomodulation induced by inactivated Staphylococcus epidermidis.

The genesis and appropriate treatment of neuroinflammation in various infectious and non-infectious disorders of the central nervous system is still a matter of debate. We introduce an alternative and simple experimental model for the investigation of the cellular inflammatory response to bacterial antigens by stereotactic intracerebral injection of heat-inactivated Staphylococcus epidermidis (HISE). HISE-injection resulted in well-circumscribed intraparenchymal deposits encompassed by an early micro- and astroglial response and a selective but sustained opening of the blood-brain barrier (BBB). After 24h, the HISE collections were densely infiltrated by granulocytes and few circumjacent macrophages that became the predominating immunocompetent cell type from day 4 on. CD8a+ lymphocytes peaked at day 4, whereas CD4+ and CD20+ lymphocytes increased gradually in number, developing a scattered infiltrate until day 17, indicating the initiation of an adaptive immune response. MHC class II presenting cells were abundantly recruited from day 1 and eventually shaped an increasingly dense accumulation within the lesion. Intracerebral HISE administration provides a controlled, highly reproducible and well defined influx of immunocompetent cells across the BBB leading to a distinct and condensed inflammatory reaction. The technique is straightforward, easily feasible and may significantly enable further investigations of the initiation, maintenance and therapeutic modulation of acute neuroinflammation.

Hypertrophy of the lumbar ligamentum flavum is associated with inflammation-related TGF-ß expression.

BACKGROUND: Despite the significance of hypertrophy of the ligamentum flavum (HLF) in the disease progress of neurogenic claudication, the cellular mechanisms underlying the gradual fibrotic thickening of the ligamentum flavum remain poorly understood. The aim of our study was to get insight into the contribution of inflammatory mechanisms to the development of hypertrophy. METHODS: Specimens of hypertrophied ligamenta flava were obtained at surgery from 20 patients with acquired lumbar osteoligamentous spinal canal stenosis from the central part of the ligament. Paraffin sections were stained with hematoxylin and eosin and Elastica van Gieson to evaluate extracellular matrix architecture, and immunohistochemistry was performed to characterize the inflammatory reaction and the sources of transforming growth factor beta (TGF-ß) expression. Sections of normal ligamenta flava obtained from corresponding anatomical sites and stained in parallel served as a control. RESULTS: HLF was characterized by a considerable distortion of the elastic matrix and fibrotic transformation by extracellular collagen deposition. All specimens showed highly inflammatory cellular infiltrates confined to regions exhibiting marked degeneration of the elastic matrix composed mainly of macrophages, scattered T lymphocytes, and neovascularization, thus representing a chronic inflammation. Surprisingly, macrophages as well as vascular endothelial cells but not fibroblasts showed a strong expression of TGF-ß, a strong inducer of extracellular collagen deposition. CONCLUSIONS: Macrophages were identified as a major cellular source of TGF-ß in advanced HLF and may perpetuate further hypertrophy. This finding suggests that modulating the immune response locally or systemically could prove to be effective for impeding the disease progress.

Extract derived from rat brains in the acute phase following traumatic brain injury impairs survival of undifferentiated stem cells and induces rapid differentiation of surviving cells.

Dramatic cerebral responses following brain injury (TBI) comprise inflammation, cell death, and modulation of trophic factor release. These cerebral modulations might induce and/or attenuate acute neuronal damage. Here, we investigated the effect of tissue extract derived from healthy (HBE) or injured rat brain (TBE) on the differentiation of cultured embryonic stem cells in vitro. Rats were sacrificed at t = 45 minutes following lateral fluid-percussion injury and extracts of cerebral tissue were prepared from 4-6 healthy or injured rat brain hemispheres. Murine embryonic stem cells (CGR8) cultured in serum-free medium were then conditioned for a week with HBE or TBE. Omission of serum from the culture medium induced neural differentiation of CGR8 stem cells, as indicated by a significant time dependent down-regulation of oct-4 with a concomitant upregulation of nestin after 7 days. In parallel cell loss was observed that seemed to be largely due to apoptotic cell death. In TBE treated cells, on the other hand, a significant amplification of apoptotic cell death, enhancement of nestin and MAP2 expression and marked morphological changes such as axonal-like outgrowth was observed within 3 days of conditioning. Treatment of stem cells with HBE resulted in less pronounced neuronal differentiation processes. Axonal-like outgrowth was not observed. Our data suggest that during the early acute phase of traumatic injury the cerebral environment is disposed to detrimental as well as potent protective signals that seem to rapidly induce neurogenic processes.

Developmental potential of the murine embryonic stem cells transplanted into the healthy rat brain--novel insights into tumorigenesis.

Although engraftment of undifferentiated pluripotent embryonic stem cells (ESCs) into the injured central nervous system (CNS) may lead to targeted cell replacement of lost/damaged cells, sustained proliferative activity combined with uncontrolled differentiation of implanted cells presents a risk of tumor formation. As tumorigenic potential is thought to be associated with pluripotency of embryonic stem cells, pre-differentiation may circumvent this problem. Recently, it has been demonstrated that tumorigenesis occurs despite pre-differentiation if the neural precursor cells are implanted into the brain of a homologous animal (e.g., mouse to mouse). However, xenotransplantation (e.g., mouse to rat) without pre-differentiation, lead to the development of healthy neuronal cells, in absence of tumor formation, suggesting that tumor-suppressive effects of host tissue on engrafted ESCs may play a role in transplant tumorigenesis. We critically investigated tumorigenesis and possible mechanisms of anticipated tumor-suppressive effect under conditions analogous to previously published studies. Xenotransplantation of D-3 murine ESCs into uninjured adult rat brains lacking any preliminary inflammatory potential was found to lead to tumor formation in 5 out of 8 of animals within 2 weeks postimplantation. Tumor-suppressive effects, reflected by Erdo et. al could possibly be ascribed to immunomodulatory activity of macrophages scavenging the tumorigenic fraction of the implanted cells. The importance of number of engrafted cells, implantation site and immunosuppressive effects are discussed as possible variables determining tumorigenic outcome after ESC transplantation.

Degeneration of cholinergic rat basal forebrain neurons after experimental subarachnoid hemorrhage.

OBJECTIVE: The reasons for neuropsychological deficits after subarachnoid hemorrhage (SAH) are fairly unknown. Cholinergic basal forebrain (BFB) neurons are essential for attention, memory, and emotion. We investigated possible changes in the cholinergic BFB and its hippocampal and neocortical terminals after experimental SAH. METHODS: SAH was induced in 19 male Wistar rats by stereotactic injection of 150 microL of autologous blood into the prechiasmatic cistern. Five control animals received 150 microL of saline. Continuous monitoring of brain tissue oxygen tension, intracranial pressure, and cerebral perfusion pressure was performed. After 4 and 14 days, the BFB was analyzed for cholinergic and gamma-aminobutyric acid-ergic cell counts. The number of cholinergic terminals in the hippocampus and neocortex was calculated by optical densitometry. RESULTS: SAH resulted in a 20 to 30% decrease in cholinergic BFB neurons in the medial septum and diagonal band at 4 and 14 days. A similar decline in the density of hippocampal and neocortical cholinergic terminals was demonstrated. Animals treated with saline did not exhibit significant cholinergic cell loss, and gamma-aminobutyric acid-ergic neurons appeared unaffected by the SAH. Courses of intracranial pressure and cerebral perfusion pressure did not differ between animals injected with blood and saline, but brain tissue oxygen tension decreased considerably and continued to stay below baseline in SAH, although it returned to normal values after saline injection. CONCLUSION: The present study provides evidence for a decrease of cholinergic BFB neurons after SAH. The direct effect of blood in the basal cisterns seemed to result in an enduring tissue hypoxia as a significant mechanism for cholinergic degeneration.

Microsatellite instability in pediatric and adult high-grade gliomas.

About 15% of sporadic gastrointestinal and endometrial tumors show the microsatellite instability (MSI) phenotype because of loss of DNA mismatch repair (MMR) function. The incidence of MSI in tumors of the central nervous system still remains controversial. Previous studies reported a particular high frequency of MSI (approximately 25%) in young patients suffering from high-grade gliomas. Based on these data and the fact that in different tumor entities MMR deficiency defines a subgroup of tumors with distinct pathogenesis and particular clinicopathological features that may have impact on prognosis and therapy, we screened 624 gliomas from 71 young and 553 adult patients for MMR deficiency by MSI analysis using three highly sensitive diagnostic markers. Alterations of MMR protein expression was examined by immunohistochemistry. A malignant glioma from an adult patient displayed MSI and concomitant loss of nuclear MSH2 and MSH6 protein expression (0.16%; 1/619). No evidence for MSI or loss of MMR protein expression was observed in 71 gliomas from young patients (0%; 0/71) including 41 high-grade astrocytic tumors. Overall, we observed a much lower incidence of MSI among high-grade pediatric gliomas than initially reported and suggest that MMR deficiency does not play a major role in the pathogenesis of glial neoplasms.

Potentiated gene delivery to tumors using herpes simplex virus/Epstein-Barr virus/RV tribrid amplicon vectors.

The development and use of gene transfer techniques creates an opportunity to achieve better treatment modalities for numerous disease entities. Promising results for treatment in tumor cells in culture and in small animal models have been reported. Nevertheless, the lack of widespread vector distribution throughout tumor tissue is one of the current limitations for successful clinical application of gene therapy paradigms. The use of migratory tumor cells themselves as vector delivery vehicles may allow wider vector distribution in tumors. In addition, continuous release of retrovirus vectors on-site could generate a high local virion concentration over an extended time period with consequent increases in transduction efficiency. In this paper, we present in culture and in vivo data of a herpes simplex virus-Epstein-Barr virus hybrid amplicon vector containing retrovirus vector components (tribrid vector) that allows conversion of tumor cells into retroviral producer cells. With this method, we were able to achieve a local fourfold amplification of stable transgene expression in tumors. The application of this system, which can integrate a transgene cassette into tumors with therapeutic bystander effects, could increase the local amplification effect to a level of clinical relevance.

Generation of stable retrovirus packaging cell lines after transduction with herpes simplex virus hybrid amplicon vectors.

BACKGROUND: A number of properties have relegated the use of Moloney murine leukemia virus (Mo-MLV)-based retrovirus vectors primarily to ex vivo protocols. Direct implantation of retrovirus producer cells can bypass some of the limitations, and in situ vector production may result in a large number of gene transfer events. However, the fibroblast nature of most retrovirus packaging cells does not provide for an effective distribution of vector producing foci in vivo, especially in the brain. Effective development of new retrovirus producer cells with enhanced biologic properties may require the testing of a large number of different cell types, and a quick and efficient method to generate them is needed. METHODS: Moloney murine leukemia virus (Mo-MLV) gag-pol and env genes and retrovirus vector sequences carrying lacZ were cloned into different minimal HSV/AAV hybrid amplicons. Helper virus-free amplicon vectors were used to co-infect glioma cells in culture. Titers and stability of retrovirus vector production were assessed. RESULTS: Simultaneous infection of two glioma lines, Gli-36 (human) and J3T (dog), with both types of amplicon vectors, generated stable packaging populations that produced retrovirus titers of 0.5-1.2 x 10(5) and 3.1-7.1 x 10(3) tu/ml, respectively. Alternatively, when cells were first infected with retrovirus vectors followed by infection with HyRMOVAmpho amplicon vector, stable retrovirus packaging populations were obtained from Gli-36 and J3T cells producing retrovirus titers comparable to those obtained with a traditional retrovirus packaging cell line, Psi CRIPlacZ. CONCLUSIONS: This amplicon vector system should facilitate generation of new types of retrovirus producer cells. Conversion of cells with migratory or tumor/tissue homing properties could result in expansion of the spatial distribution or targeting capacity, respectively, of gene delivery by retrovirus vectors in vivo.