John P. Rossiter

Short-Course Radiation plus Temozolomide in Elderly Patients with Glioblastoma.

Background Glioblastoma is associated with a poor prognosis in the elderly. Survival has been shown to increase among patients 70 years of age or younger when temozolomide chemotherapy is added to standard radiotherapy (60 Gy over a period of 6 weeks). In elderly patients, more convenient shorter courses of radiotherapy are commonly used, but the benefit of adding temozolomide to a shorter course of radiotherapy is unknown. Methods We conducted a trial involving patients 65 years of age or older with newly diagnosed glioblastoma. Patients were randomly assigned to receive either radiotherapy alone (40 Gy in 15 fractions) or radiotherapy with concomitant and adjuvant temozolomide. Results A total of 562 patients underwent randomization, 281 to each group. The median age was 73 years (range, 65 to 90). The median overall survival was longer with radiotherapy plus temozolomide than with radiotherapy alone (9.3 months vs. 7.6 months; hazard ratio for death, 0.67; 95% confidence interval [CI], 0.56 to 0.80; P<0.001), as was the median progression‐free survival (5.3 months vs. 3.9 months; hazard ratio for disease progression or death, 0.50; 95% CI, 0.41 to 0.60; P<0.001). Among 165 patients with methylated O6‐methylguanine–DNA methyltransferase (MGMT) status, the median overall survival was 13.5 months with radiotherapy plus temozolomide and 7.7 months with radiotherapy alone (hazard ratio for death, 0.53; 95% CI, 0.38 to 0.73; P<0.001). Among 189 patients with unmethylated MGMT status, the median overall survival was 10.0 months with radiotherapy plus temozolomide and 7.9 months with radiotherapy alone (hazard ratio for death, 0.75; 95% CI, 0.56 to 1.01; P=0.055; P=0.08 for interaction). Quality of life was similar in the two trial groups. Conclusions In elderly patients with glioblastoma, the addition of temozolomide to short‐course radiotherapy resulted in longer survival than short‐course radiotherapy alone. (Funded by the Canadian Cancer Society Research Institute and others; ClinicalTrials.gov number, NCT00482677.)

Expression of Toll-like receptor 3 in the human cerebellar cortex in rabies, herpes simplex encephalitis, and other neurological diseases

There is recent in vitro evidence that human neurons express the innate immune response receptor, Toll-like receptor-3 (TLR-3), and that expression is enhanced in viral infections. The authors examined the immunohistochemical expression of TLR-3 in the cerebellar cortex of postmortem human brains. Purkinje cells were found to express TLR-3 in all cases of rabies (4 of 4) and herpes simplex encephalitis (2 of 2) as well as in cases of amyotrophic lateral sclerosis (1 of 2), stroke (1 of 2), and Alzheimer’s disease (3 of 3). In cases of viral infection, direct viral infection was not necessary for enhanced neuronal TLR-3 expression, suggesting that soluble factors likely play an important role in inducing TLR-3 expression. In addition to neurons, occasional Bergmann glia expressed TLR-3 in some cases. This study has provided evidence that human brain neurons can express TLR-3 in vivo and suggests that neurons may play an important role in initiating an inflammatory reaction in a variety of neurological diseases.

Temozolomide chemotherapy versus radiotherapy in high-risk low-grade glioma (EORTC 22033-26033) : a randomised, open-label, phase 3 intergroup study

Background Outcome of low-grade glioma (LGG, WHO grade II) is highly variable reflecting molecular heterogeneity of the disease. We compared two different single modality treatment strategies: standard radiotherapy (RT) versus primary temozolomide (TMZ) chemotherapy with the aim of tailoring treatment and identifying predictive molecular factors. Methods 477 patients (2005 – 2012, median FU 48 months) with a low-grade glioma (astrocytoma, oligoastrocytoma, oligodendroglioma, WHO grade II) with at least one high-risk feature (age > 40 years, progressive disease, tumor > 5 cm or crossing the midline, neurological symptoms (e.g. focal or mental deficits, increased intracranial pressure or intractable seizures)) were, after stratification by chromosome 1p-status, randomized to either conformal RT (50.4 Gy/28 fractions) or dose-dense TMZ (75 mg/m2 daily × 21 days, q28 days, max. 12 cycles). Random treatment allocation was performed online using a minimization technique. A planned analysis was performed after 246 progression events. All analyses are intent to treat. Primary clinical endpoint was progression-free survival (PFS), correlative analyses included molecular markers (1p/19q co-deletion, MGMT methylation status, IDH1+2 mutations). The trial has been registered at the European Trials Registry (EudraCT 2004-002714-11) and at ClinicalTrials.gov (NCT00182819). Findings Four hundred seventy-seven patients were randomized. Severe hematological toxicity occurred in 14% of TMZ-treated patients, infections in 3% of TMZ-treated patients, and 1% of RT-treated patients. Moderate to severe fatigue was recorded in 3% of patients in the RT group and 7% in the TMZ group. At a median follow-up of 48 months (IQR:31–56), median PFS was 39 months (IQR:16–46) in the TMZ arm and 46 months (IQR:19–48) in the RT group (hazard ratio 1.16, 95% CI, 0.9–1.5; p=0.22). Median OS has not been reached. Exploratory analyses identified treatment-dependent variation in outcome of molecular LGG subgroups (n=318). Interpretation There was no significant difference in outcome of the overall patient population treated with either radiotherapy alone or TMZ chemotherapy alone. Further data maturation is needed for overall survival analyses and evaluation of the full predictive impact of the molecular subtypes for individualized treatment choices. Funding Merck & Co, Swiss-Bridge Award 2011, Swiss Cancer League.

Structural Abnormalities in Neurons Are Sufficient To Explain the Clinical Disease and Fatal Outcome of Experimental Rabies in Yellow Fluorescent Protein-Expressing Transgenic Mice

ABSTRACT Under natural conditions and in some experimental models, rabies virus infection of the central nervous system causes relatively mild histopathological changes, without prominent evidence of neuronal death despite its lethality. In this study, the effects of rabies virus infection on the structure of neurons were investigated with experimentally infected transgenic mice expressing yellow fluorescent protein (YFP) in neuronal subpopulations. Six-week-old mice were inoculated in the hind-limb footpad with the CVS strain of fixed virus or were mock infected with vehicle (phosphate-buffered saline). Brain regions were subsequently examined by light, epifluorescent, and electron microscopy. In moribund CVS-infected mice, histopathological changes were minimal in paraffin-embedded tissue sections, although mild inflammatory changes were present. Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling and caspase-3 immunostaining showed only a few apoptotic cells in the cerebral cortex and hippocampus. Silver staining demonstrated the preservation of cytoskeletal integrity in the cerebral cortex. However, fluorescence microscopy revealed marked beading and fragmentation of the dendrites and axons of layer V pyramidal neurons in the cerebral cortex, cerebellar mossy fibers, and axons in brainstem tracts. At an earlier time point, when mice displayed hind-limb paralysis, beading was observed in a few axons in the cerebellar commissure. Toluidine blue-stained resin-embedded sections from moribund YFP-expressing animals revealed vacuoles within the perikarya and proximal dendrites of pyramidal neurons in the cerebral cortex and hippocampus. These vacuoles corresponded with swollen mitochondria under electron microscopy. Vacuolation was also observed ultrastructurally in axons and in presynaptic nerve endings. We conclude that the observed structural changes are sufficient to explain the severe clinical disease with a fatal outcome in this experimental model of rabies.

Apoptotic cell death is an important cause of neuronal injury in experimental Venezuelan equine encephalitis virus infection of mice

Abstract Mice develop a fatal encephalomyelitis after infection with the Trinidad donkey strain of Venezuelan equine encephalitis (VEE) virus. Adult mice were inoculated intraperitoneally with VEE virus and the brains were examined at different time points. Morphological changes were assessed by histological staining. VEE virus antigen was detected with immunoperoxidase staining, and DNA fragmentation was evaluated in situ using the terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) method. VEE antigen was found in many areas of the brain and it was prominent in neurons. There were mild associated inflammatory changes. DNA fragmentation was demonstrated in many of these areas using TUNEL. In areas with TUNEL staining, morphological neuronal changes ranged from nuclear chromatin condensations to nuclear and cellular fragmentation, which are characteristic of apoptosis. There is strong morphological and biochemical evidence of apoptotic cell death in this experimental model of VEE virus infection.

Neuronal apoptosis does not play an important role in human rabies encephalitis

It is generally accepted that there are not prominent features of neuronal cell death in rabies encephalitis. However, Hemachudha and coworkers recently reported widespread apoptosis in the central nervous system of several human rabies cases (BMC Infect Dis 5: 104, 2005). In this study we have evaluated morphological features and markers of neuronal apoptosis in postmortem brain tissue from 12 cases of human rabies who died in four different countries. Histopathological analysis, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling) staining, and immunostaining for cleaved (activated) caspase-3 were performed on paraffin-embedded tissues from the cerebral cortex, hippocampus, and brainstem, and additional regional areas from one of the cases. We did not find morphological evidence of neuronal apoptosis or TUNEL staining in any of the cases of rabies encephalitis. Similarly, immunostained cleaved caspase-3 was not seen in neurons, but prominent staining was observed in microglial processes. We conclude that neuronal apoptosis does not play an important pathogenetic role in human rabies encephalitis.

Therapy with Minocycline Aggravates Experimental Rabies in Mice

ABSTRACT Minocycline is a tetracycline derivative with antiapoptotic and anti-inflammatory properties, and the drug has been shown to have beneficial effects in a variety of models of neurological disorders. The potentially neuroprotective role of minocycline was assessed in experimental in vitro and in vivo models of rabies virus infection. In this study, 5 nM minocycline did not improve the viability of embryonic mouse cortical and hippocampal neurons infected in vitro with the attenuated SAD-D29 strain of rabies virus, based on assessments using trypan blue exclusion. Two-day-old ICR mice were inoculated in the right hind limb thigh muscle with SAD-D29, and they received daily subcutaneous injections of either 50 mg/kg minocycline or vehicle (phosphate-buffered saline). Infected minocycline-treated mice experienced an earlier onset of neurologic signs and greater mortality (83% versus 50%) than those receiving vehicle (log rank test, P = 0.002 and P = 0.003, respectively). Immunohistochemical analysis of rabies virus antigen distribution was performed at early time points and in moribund mice. There were greater numbers of infected neurons in the regional brain areas of minocycline-treated mice than in vehicle-treated mice, which was significant in the CA1 region of the hippocampus. There was less apoptosis (P = 0.01) and caspase 3 immunostaining (P = 0.0008) in the midbrains of mice treated with minocycline than in mice treated with vehicle, consistent with a neuroprotective role of neuronal apoptosis that may have had a mild effect of inhibiting viral spread. Reduced infiltration of CD3+ T cells was observed in the pons/medulla of moribund mice that received minocycline therapy (P = 0.008), suggesting that the anti-inflammatory actions of minocycline may intensify the neurologic disease. These findings indicate that minocycline has important detrimental effects in the therapy of experimental rabies. Empirical therapy with minocycline should therefore be approached with caution in cases of human rabies and possibly other viral encephalitides until more experimental data become available.

Selective vulnerability of dorsal root ganglia neurons in experimental rabies after peripheral inoculation of CVS-11 in adult mice

The involvement of dorsal root ganglia was studied in an in vivo model of experimental rabies virus infection using the challenge virus standard (CVS-11) strain. Dorsal root ganglia neurons infected with CVS in vitro show prolonged survival and few morphological changes, and are commonly used to study the infection. It has been established that after peripheral inoculation of mice with CVS the brain and spinal cord show relatively few neurodegenerative changes, but detailed studies of pathological changes in dorsal root ganglia have not previously been performed in this in vivo experimental model. In this study, adult ICR mice were inoculated in the right hindlimb footpad with CVS. Spinal cords and dorsal root ganglia were evaluated at serial time points for histopathological and ultrastructural changes and for biochemical markers of cell death. Light microscopy showed multifocal mononuclear inflammatory cell infiltrates in the sensory ganglia and a spectrum of degenerative neuronal changes. Ultrastructural changes in gangliocytes included features characteristic of the axotomy response, the appearance of numerous autophagic compartments, and aggregation of intermediate filaments, while the neurons retained relatively intact mitochondria and plasma membranes. Later in the process, there were more extensive degenerative neuronal changes without typical features of either apoptosis or necrosis. The degree of degenerative neuronal changes in gangliocytes contrasts with observations in CNS neurons in experimental rabies. Hence, gangliocytes exhibit selective vulnerability in this animal model. This contrasts markedly with the fact that they are, unlike CNS neurons, highly permissive to CVS infection in vitro. Further study is needed to determine mechanisms for this selective vulnerability, which will give us a better understanding of the pathogenesis of rabies.

Immunolabelling of the cytoplasm and processes of apoptotic facial motoneurons following axotomy in the neonatal rat.

Abstract A polyclonal antibody intended to recognize c-Jun (Oncogene Science, c-jun/AP-1, Ab-2) has previously been shown to recognize an apparently novel “apoptosis-specific protein” (ASP) in the cytoplasm of cells undergoing apoptotic cell death in vitro. We have investigated whether this antibody would also serve as a reliable marker for apoptotic motoneurons in vivo. Following transection of the left facial nerve in anesthetized neonatal rat pups, which results in over 90% death of the facial motoneurons, we performed immunohistochemistry on frozen brain stem sections with Oncogene Science Ab-1 and Ab-2 antibodies which are raised against different peptide fragments of c-Jun. While Ab-1/c-Jun labelling was seen in the nuclei of the majority of axotomized motoneurons, Ab-2/ASP immunoreactivity was present only in scattered cells, all of which had characteristic apoptotic morphology. Furthermore, Ab-2/ASP immunoreactivity was cytoplasmic and frequently included the dendrites and axons of dying neurons. Some cerebellar granule cells undergoing postnatal developmental cell death were also Ab-2/ASP positive. The time course of the number of Ab-2/ASP-labelled motoneurons corresponded relatively closely with our previous data on DNA fragmentation in these cells, as assessed by an in situ end labelling (ISEL) technique. When facial nerve axotomy was performed at 7 and 14 days postnatum, resulting in reduced cell death, the number of Ab-2/ASP immunoreactive cells decreased correspondingly. Although the exact identity of the epitope recognized by Ab-2 is unclear, we conclude that, by labelling the cytoplasmic and neuritic components of apoptotic motoneurons, Ab-2/ASP immunohistochemistry is a valuable complementary technique to existing in situ methods based on the detection of fragmented DNA in the cell nucleus.

Familial herpes simplex encephalitis

Herpes simplex encephalitis (HSE) is a sporadic encephalitis with an incidence of about 2 to 4 cases per million population per year. The occurrence of HSE in two members of a family has only rarely been reported. We have identified HSE in a mother and her son within an interval of over 21 years. A 14-year-old boy developed an illness in July 1979 with fever, headache, nausea, confusion, and partial seizures. An initial computed tomography head scan was normal and an electroencephalogram showed intermittent theta and polymorphous delta activity with predominance over the right hemisphere. Cerebrospinal fluid analysis showed 241 white blood cells (95% lymphocytes and 5% polymorphonuclear leukocytes) and 10 red blood cells/ L; cerebrospinal fluid protein was 70mg/dL and glucose 90mg/dL. Therapy was initiated with intravenous adenosine arabinoside. He developed status epilepticus within 72 hours of admission. Electroencephalograms showed epileptiform activity over the left temporal region that extended widely across the left hemisphere and, later, sporadic epileptiform activity was also noted from the right hemisphere. A repeat computed tomography head scan showed hypodense lesions in the left frontal and temporal lobes with mass effect and contrast enhancement. He died after a 2-week hospital course. Postmortem examination showed hemorrhagic necrosis bilaterally in the temporal lobes. Microscopy revealed leptomeningeal and perivascular inflammatory cell infiltrates and scattered neuronal intranuclear inclusions. Immunohistochemistry demonstrated strong labeling for herpes simplex virus 1 antigen in multiple neurons (Fig A) and glial cells; ultrastructural studies showed virions about 100nm in diameter consistent with herpes simplex virus. In November 2000, his mother at age 68 developed an illness with headache, mild aphasia, confusion, and mild fever. She had a generalized seizure after admission to hospital and an electroencephalogram showed irregularly repetitive sharp wave and focal delta activity in the left temporal region. A magnetic resonance imaging scan showed lesions in the left medial temporal lobe and the insular cortex on T2-weighted and fluid-attenuated inversion recovery (FLAIR) sequences (see Fig B). Cerebrospinal fluid showed 173 white blood cells (100% mononuclear cells) and 326 red blood cells/per L; cerebrospinal fluid protein was 97mg/dL and glucose 74mg/dL. Herpes simplex virus 1 DNA was detected in the cerebrospinal fluid with polymerase chain reaction amplification. She was treated with a 21day course of intravenous acyclovir and showed clinical improvement. She had mild residual impairment of memory and mild aphasia, and she was able to continue to live independently. Familial HSE has been the subject of two previous reports. The first described HSE in 2 sisters with an interval of 8 years; the second described HSE in 2 brothers within a 13-year interval. We now report fatal HSE in a 14-year-old boy followed by HSE affecting his mother over 21 years later, and we believe that this is the first report of HSE in both a parent and child. In combination with the two previous reports of familial HSE, this suggests the possibility of an increased genetic susceptibility to the occurrence of HSE in these cases. Alternatively, strains of herpes simplex virus with enhanced neuroinvasive or neurovirulent properties may be transmitted between family members or they may share a common source, and these strains may be more likely to cause HSE upon reactivation years later. These familial cases raise suspicion that there are biologic factors involving the host, the virus, or both, that favor familial occurrence of HSE and that familial cases are not merely random occurrences.