Impact of human immunodeficiency virus type 1 gp41 amino acid substitutions selected during enfuvirtide treatment on gp41 binding and antiviral potency of enfuvirtide in vitro.

Enfuvirtide (ENF), a novel human immunodeficiency virus type 1 (HIV-1) fusion inhibitor, has potent antiviral activity against HIV-1 both in vitro and in vivo. Resistance to ENF observed after in vitro passaging was associated with changes in a three-amino-acid (aa) motif, GIV, at positions 36 to 38 of gp41. Patients with ongoing viral replication while receiving ENF during clinical trials acquired substitutions within gp41 aa 36 to 45 in the first heptad repeat (HR-1) of gp41 in both population-based plasma virus sequences and proviral DNA sequences from isolates showing reduced susceptibilities to ENF. To investigate their impact on ENF susceptibility, substitutions were introduced into a modified pNL4-3 strain by site-directed mutagenesis, and the susceptibilities of mutant viruses and patient-derived isolates to ENF were tested. In general, susceptibility decreases for single substitutions were lower than those for double substitutions, and the levels of ENF resistance seen for clinical isolates were higher than those observed for the site-directed mutant viruses. The mechanism of ENF resistance was explored for a subset of the substitutions by expressing them in the context of a maltose binding protein chimera containing a portion of the gp41 ectodomain and measuring their binding affinity to fluorescein-labeled ENF. Changes in binding affinity for the mutant gp41 fusion proteins correlated with the ENF susceptibilities of viruses containing the same substitutions. The combined results support the key role of gp41 aa 36 to 45 in the development of resistance to ENF and illustrate that additional envelope regions contribute to the ENF susceptibility of fusion inhibitor-naïve viruses and resistance to ENF.

Long-term consequences of early postnatal seizures on hippocampal learning and plasticity.

Neural activity influences the patterning of synaptic connections and functional organization of developing sensory and motor systems, but the long-term consequences of intense neural activity such as seizures in the developing hippocampus are not adequately understood. To evaluate the possibility that abnormal neural activity during early development may have long-term functional effects in hippocampal circuitry that plays a role in learning, memory and epilepsy, functional properties of hippocampal circuitry were assessed in adult rats that had experienced seizures induced by kainic acid on specific days during early postnatal development. Although previous studies have suggested that the immature hippocampus is relatively resistant to seizure-induced alterations compared with adults, independent behavioural and physiological experiments demonstrated that seizures evoked by kainic acid during early postnatal development induced a long-term loss of hippocampal plasticity manifesting as reduced capacity for long-term potentiation, reduced susceptibility to kindling, and impaired spatial learning, which was associated with enhanced paired-pulse inhibition in the dentate gyrus. The enhancement of inhibition and loss of plasticity were maximal when the seizures occurred on the first day of life, but were also observed when seizures were induced as late as postnatal day 14, which delimited a period of postnatal susceptibility in the developing rat hippocampus when disruption of normal neural activity by seizures produced consistent effects on a hippocampal-dependent behaviour and several forms of hippocampal plasticity implicated in learning, memory and the development of epilepsy in adulthood.

Increased dentate granule cell neurogenesis following amygdala kindling in the adult rat.

Structural neuronal network plasticity is associated with epileptogenesis during limbic kindling, but the full extent of network changes is not well understood. We investigated whether dentate granule cell (DGC) neurogenesis, which continues into adulthood in the rodent, is altered in the amygdala kindling model of epileptogenesis. Adult rats were stimulated to either 4-6, 9-10 or 19-20 class 4/5 (generalized) kindled seizures. 5-Bromo-2'-deoxyuridine labeling showed that cell proliferation increased in the dentate gyrus only in animals that experienced nine or more class 4/5 kindled seizures. Immunocytochemistry for neuronal markers revealed that many of the newly generated cells differentiated into DGCs in the inner aspect of the DGC layer. The lack of increased DGC neurogenesis after fewer kindled seizures or at early timepoints following kindling suggests that this process is not involved in kindling development. Instead, newly generated DGCs may be important for maintenance of the kindled state or the increased susceptibility to spontaneous recurrent seizures.

A point mutation (D79N) of the alpha2A adrenergic receptor abolishes the antiepileptogenic action of endogenous norepinephrine.

Norepinephrine serves as a neurotransmitter for a population of neurons the cell bodies of which reside in a brainstem nucleus and the axons of which project widely to discrete subsets of forebrain neurons. Norepinephrine powerfully inhibits epileptogenesis in the kindling model. Pharmacological methods have demonstrated that the antiepileptogenic actions of norepinephrine are exerted via alpha2 adrenergic receptors residing on targets of noradrenergic neurons. The existence of three alpha2 adrenergic receptor subtypes together with the lack of subtype-specific ligands has precluded understanding the role of individual alpha2 adrenergic receptor subtypes in the antiepileptogenic actions of norepinephrine. Gene targeting was used to introduce a point mutation into the alpha2A adrenergic subtype in the mouse genome. The mutation produced a marked enhancement of epileptogenesis and abolished the proepileptogenic actions of the alpha2 adrenergic receptor antagonist idazoxan. These studies reveal the crucial contribution of the alpha2A receptor subtype in suppression of epileptogenesis. Development of agents that promote selective activation of the alpha2A receptor subtype may provide novel therapeutic strategies for the prophylaxis of epilepsy.

Glutamate receptor GluR3 antibodies and death of cortical cells.

Rasmussen's encephalitis (RE), a childhood disease characterized by epileptic seizures associated with progressive destruction of a single cerebral hemisphere, is an autoimmune disease in which one of the autoantigens is a glutamate receptor, GluR3. The improvement of some affected children following plasma exchange that removed circulating GluR3 antibodies (anti-GluR3) suggested that anti-GluR3 gained access to the central nervous system where it exerted deleterious effects. Here, we demonstrate that a subset of rabbits immunized with a GluR3 fusion protein develops a neurological disorder mimicking RE. Anti-GluR3 IgG isolated from serum of both ill and healthy GluR3-immunized animals promoted death of cultured cortical cells by a complement-dependent mechanism. IgG immunoreactivity decorated neurons and their processes in neocortex and hippocampus in ill but not in healthy rabbits. Moreover, both IgG and complement membrane attack complex (MAC) immunoreactivity was evident on neurons and their processes in the cortex of a subset of patients with RE. We suggest that access of IgG to epitopes in the central nervous system triggers complement-mediated neuronal damage and contributes to the pathogenesis of both this animal model and RE.