Michael C. Graves

Efficacy of minocycline in patients with amyotrophic lateral sclerosis: a phase III randomised trial

BACKGROUND Minocycline has anti-apoptotic and anti-inflammatory effects in vitro, and extends survival in mouse models of some neurological conditions. Several trials are planned or are in progress to assess whether minocycline slows human neurodegeneration. We aimed to test the efficacy of minocycline as a treatment for amyotrophic lateral sclerosis (ALS). METHODS We did a multicentre, randomised placebo-controlled phase III trial. After a 4-month lead-in phase, 412 patients were randomly assigned to receive placebo or minocycline in escalating doses of up to 400 mg/day for 9 months. The primary outcome measure was the difference in rate of change in the revised ALS functional rating scale (ALSFRS-R). Secondary outcome measures were forced vital capacity (FVC), manual muscle testing (MMT), quality of life, survival, and safety. Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00047723. FINDINGS ALSFRS-R score deterioration was faster in the minocycline group than in the placebo group (-1.30 vs -1.04 units/month, 95% CI for difference -0.44 to -0.08; p=0.005). Patients on minocycline also had non-significant tendencies towards faster decline in FVC (-3.48 vs -3.01, -1.03 to 0.11; p=0.11) and MMT score (-0.30 vs -0.26, -0.08 to 0.01; p=0.11), and greater mortality during the 9-month treatment phase (hazard ratio=1.32, 95% CI 0.83 to 2.10; p=0.23) than did patients on placebo. Quality-of-life scores did not differ between the treatment groups. Non-serious gastrointestinal and neurological adverse events were more common in the minocycline group than in the placebo group, but these events were not significantly related to the decline in ALSFRS-R score. INTERPRETATION Our finding that minocycline has a harmful effect on patients with ALS has implications for trials of minocycline in patients with other neurological disorders, and for how potential neuroprotective agents are screened for use in patients with ALS.

The structure and evolution of the major capsid protein of a large, lipid-containing DNA virus

Paramecium bursaria Chlorella virus type 1 (PBCV-1) is a very large, icosahedral virus containing an internal membrane enclosed within a glycoprotein coat consisting of pseudohexagonal arrays of trimeric capsomers. Each capsomer is composed of three molecules of the major capsid protein, Vp54, the 2.0-Å resolution structure of which is reported here. Four N-linked and two O-linked glycosylation sites were identified. The N-linked sites are associated with nonstandard amino acid motifs as a result of glycosylation by virus-encoded enzymes. Each monomer of the trimeric structure consists of two eight-stranded, antiparallel β-barrel, “jelly-roll” domains related by a pseudo-sixfold rotation. The fold of the monomer and the pseudo-sixfold symmetry of the capsomer resembles that of the major coat proteins in the double-stranded DNA bacteriophage PRD1 and the double-stranded DNA human adenoviruses, as well as the viral proteins VP2-VP3 of picornaviruses. The structural similarities among these diverse groups of viruses, whose hosts include bacteria, unicellular eukaryotes, plants, and mammals, make it probable that their capsid proteins have evolved from a common ancestor that had already acquired a pseudo-sixfold organization. The trimeric capsid protein structure was used to produce a quasi-atomic model of the 1,900-Å diameter PBCV-1 outer shell, based on fitting of the Vp54 crystal structure into a three-dimensional cryoelectron microscopy image reconstruction of the virus.

Ineffective phagocytosis of amyloid-β by macrophages of Alzheimer's disease patients

The defective clearance of amyloid-beta (Abeta) in the brain of Alzheimers disease (AD) patients is unexplained. The immunohistochemical studies of the frontal lobe and hippocampus show perivascular and intraplaque infiltration by blood-borne macrophages containing intracellular Abeta but only inefficient clearance of beta deposits. Neurons and neuronal nuclei, respectively, express interleukin-1beta and the chemokine RANTES, which could induce the inflammatory cell infiltration. To clarify the pathophysiology ofbeta clearance, we examined Abeta phagocytosis by monocytes and macrophages isolated from the blood of age-matched patients and controls. Control monocytes display excellent differentiation into macrophages and intracellular phagocytosis of Abeta followed by beta degradation or export. AD monocytes show poor differentiation and only surface uptake of Abeta and suffer apoptosis. HLA DR and cyclooxygenase-2 are abnormally expressed on neutrophils and monocytes of AD patients. AD patients have higher levels of intracellular cytokines compared to controls. Thus Abeta clearance is not restricted to brain microglia and involves systemic innate immune responses. In AD, however, macrophage phagocytosis is defective, which may elicit compensatory response by the adaptive immune system.

Phase III randomized trial of gabapentin in patients with amyotrophic lateral sclerosis

Background: Preclinical and clinical studies of gabapentin in patients with ALS led the authors to undertake a phase III randomized clinical trial. Methods: Patients were randomly assigned, in a double-blinded fashion, to receive oral gabapentin 3,600 mg or placebo daily for 9 months. The primary outcome measure was the average rate of decline in isometric arm muscle strength for those with two or more evaluations. Results: Two hundred four patients enrolled, 196 had two or more evaluations, and 128 patients completed the study. The mean rate of decline of the arm muscle strength was not significantly different between the groups. Moreover, there was no beneficial effect upon the rate of decline of other secondary measures (vital capacity, survival, ALS functional rating scale, timed walking) nor was there any symptomatic benefit. In fact, analysis of the combined data from the phase II and III trials revealed a significantly more rapid decline of forced vital capacity in patients treated with gabapentin. Conclusion: These data provide no evidence of a beneficial effect of gabapentin on disease progression or symptoms in patients with ALS.

Inflammation in amyotrophic lateral sclerosis spinal cord and brain is mediated by activated macrophages, mast cells and T cells

Recent studies have shown inflammatory markers in affected neural tissues of amyotrophic lateral sclerosis (ALS) patients. We examined immunocytochemically spinal cord tissues of six patients with ALS, two with corticospinal tract degeneration secondary to cerebral infarcts and three control subjects without neuropathologic abnormalities. ALS spinal cords had dense macrophage infiltration (one log greater than control spinal cords) involving the white and gray matter, with heaviest infiltration of lateral and ventral columns and, in one patient, prefrontal gyrus and the occipital lobes of the brain. Macrophages in ALS spinal cord showed strong expression of cyclooxygenase-2 (COX-2) (one log greater than control tissues) and inducible nitric oxide synthase. In the gray matter, macrophages surrounded and appeared to phagocytize neurons (NeuN-positive) that appeared to be dying. Vessels showed damage to the tight junction protein ZO-1 in relation to perivascular CD40 receptor-positive macrophages and CD40 ligand-positive T lymphocytes. ALS spinal cords, but not control cords, were sparsely infiltrated with mast cells. In control cases with corticospinal tract degeneration following hemispheric cerebral infarction, macrophage infiltration of the white matter was COX-2-negative and restricted to lateral and anterior corticospinal tracts. Our data suggest that inflammation in ALS spinal cord and cortex is based on innate immune responses by macrophages and mast cells and adaptive immune responses by T cells.

Phycodnaviridae– large DNA algal viruses

Summary. Members and prospective members of the family Phycodnaviridae are large icosahedral, dsDNA (180 to 560 kb) viruses that infect eukaryotic algae. The genomes of two phycodnaviruses have been sequenced: the 331 kb genome of Paramecium bursaria chlorella virus (PBCV-1) and more recently, the 336 kb genome of the Ectocarpus siliculosus virus (EsV-1). EsV-1 has ∼ 231 protein-encoding genes whereas, the slightly smaller PBCV-1 genome has 11 tRNA genes and ∼ 375 protein-encoding genes. Surprisingly, the two viruses only have 33 genes in common, of which 17 have no counterparts in the databases. The low number of homologous genes between the two viruses can probably be attributed to their different life styles. PBCV-1 is a lytic virus that infects a unicellular, endosymbiotic freshwater green alga whereas, EsV-1 is a lysogenic virus that infects a free-living filamentous marine brown alga. Furthermore, accumulating evidence indicates that the phycodnaviruses and their genes are ancient, thus allowing significant differences to have evolved. This review briefly describes some of the biological properties of the phycodnaviruses, focusing on PBCV-1 and EsV-1, and then compares their genomes.

CXC and CC chemokine receptors on coronary and brain endothelia.

BackgroundChemokine receptors on leukocytes play a key role in inflammation and HIV-1 infection. Chemokine receptors on endothelia may serve an important role in HIV-1 tissue invasion and angiogenesis.Materials and MethodsThe expression of chemokine receptors in human brain microvascular endothelial cells (BMVEC) and coronary artery endothelial cells (CAEC) in vitro and cryostat sections of the heart tissue was determined by light and confocal microscopy and flow cytometry with monoclonal antibodies. Chemotaxis of endothelia by CC chemokines was evaluated in a transmigration assay.ResultsIn BMVEC, the chemokine receptors CCR3 and CXCR4 showed the strongest expression. CXCR4 was localized by confocal microscopy to both the cytoplasm and the plasma membrane of BMVEC. In CAEC, CXCR4 demonstrated a strong expression with predominantly periplasmic localization. CCR5 expression was detected both in BMVEC and CAEC but at a lower level. Human umbilical cord endothelial cells (HUVEC) expressed strongly CXCR4 but only weakly CCR3 and CCR5. Two additional CC chemokines, CCR2A and CCR4, were detected in BMVEC and CAEC by immunostaining. Immunocytochemistry of the heart tissues with monoclonal antibodies revealed a high expression of CXCR4 and CCR2A and a low expression of CCR3 and CCR5 on coronary vessel endothelia. Coronary endothelia showed in vitro a strong chemotactic response to the CC chemokines RANTES, MlP-1α, and MIP-1β.ConclusionsThe endothelia isolated from the brain display strongly both the CCR3 and CXCR4 HIV-1 coreceptors, whereas the coronary endothelia express strongly only the CXCR4 coreceptor. CCR5 is expressed at a lower level in both endothelia. The differential display of CCR3 on the brain and coronary endothelia could be significant with respect to the differential susceptibility of the heart and the brain to HIV-1 invasion. In addition, CCR2A is strongly expressed in the heart endothelium. All of the above chemokine receptors could play a role in endothelial migration and repair.

Curcuminoids enhance amyloid-β uptake by macrophages of Alzheimer's disease patients

Treatment of Alzheimers disease (AD) is difficult due to ignorance of its pathogenesis. AD patients have defects in phagocytosis of amyloid-beta (1-42) (Abeta) in vitro by the innate immune cells, monocyte/macrophages and in clearance of Abeta plaques [5]. The natural product curcuminoids enhanced brain clearance of Abeta in animal models. We, therefore, treated macrophages of six AD patients and 3 controls by curcuminoids in vitro and measured Abeta uptake using fluorescence and confocal microscopy. At baseline, the intensity of Abeta uptake by AD macrophages was significantly lower in comparison to control macrophages and involved surface binding but no intracellular uptake. After treatment of macrophages with curcuminoids, Abeta uptake by macrophages of three of the six AD patients was significantly (P<0.001 to 0.081) increased. Confocal microscopy of AD macrophages responsive to curcuminoids showed surface binding in untreated macrophages but co-localization with phalloidin in an intracellular compartment after treatment. Immunomodulation of the innate immune system by curcuminoids might be a safe approach to immune clearance of amyloidosis in AD brain.

Cocaine opens the blood-brain barrier to HIV-1 invasion.

Cocaine abuse has been associated with vasculitis and stroke, and is suspected to influence the progression of AIDS dementia. Cocaine may enhance HIV-1 neuroinvasion by actions directed at the blood-brain barrier. HIV-1 appears to penetrate the human brain microvascular endothelial cell barrier by a paracellular route breached by tumor necrosis factor-alpha (TNF-alpha). Cocaines effects on the blood-brain barrier were investigated using human brain microvascular endothelial cells and peripheral blood monocytes. Cocaine (10(-5) M and 10(-6) M) increased molecular permeability of the barrier and viral invasion by the macrophage-tropic HIV-1(JR-FL) into the brain chamber. Cocaine also augmented apoptosis of brain endothelial cells and monocytes, increased secretion of four chemokines (interleukin-8, interferon-inducible protein-10, macrophage inflammatory protein-1alpha, and monocyte chemoattractant protein-1) and the cytokine, TNF-alpha, by human monocytes. TNF-alpha enhanced invasion of the brain compartment by macrophage-tropic, lymphotropic, and bitropic HIV-1 strains. These data indicate that HIV-1 neuroinvasion can be increased by (a) cocaines direct effects on brain microvascular endothelial cells and (b) paracrine effects of cocaine-induced pro-inflammatory cytokines and chemokines on the blood-brain barrier.

Phase II/III randomized trial of TCH346 in patients with ALS

Background: TCH346 exerts antiapoptotic effects by binding to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and blocking the apoptotic pathway in which GAPDH is involved. Apoptosis is considered to be a key pathogenic mechanism in neurodegenerative diseases including ALS. Methods: Patients were randomly assigned in a double-blind fashion to receive either placebo or one of four doses of TCH346 (1.0, 2.5, 7.5, or 15 mg/day) administered orally once daily for at least 24 weeks. The primary outcome measure was the rate of change in the revised ALS functional rating scale (ALSFRS-R). The trial design included a 16-week lead-in phase to determine each patients rate of disease progression. The between treatment comparison was adjusted for the individual pretreatment rates of progression. The study was powered to detect a 25% reduction in the rate of decline of the ALSFRS-R as compared with placebo. Secondary outcome measures included survival, pulmonary function, and manual muscle testing (MMT). Results: Five hundred ninety-one patients were enrolled at 42 sites in Europe and North America. There were no differences in baseline variables. There were no significant differences between placebo and active treatment groups in the mean rate of decline of the ALSFRS-R or in the secondary outcome measures (survival, pulmonary function, and MMT). Conclusion: The trial revealed no evidence of a beneficial effect of TCH346 on disease progression in patients with ALS.