Novel role of human CD4 molecule identified in neurodegeneration.

The human CD4 molecule (hCD4) is expressed on T lymphocytes and macrophages and acts as a key component of the cellular receptor for HIV. At baseline, hCD4 transgenic mice expressed hCD4 on microglia, the resident mononuclear phagocytes of the brain, and showed no neuronal damage. Activation of brain microglia by peripheral immune challenges elicited neurodegeneration in hCD4 mice but not in nontransgenic controls. In post-mortem brain tissues from AIDS patients with opportunistic infections, but without typical HIV encephalitis, hCD4 expression correlated with neurodegeneration. We conclude that hCD4 may function as an important mediator of indirect neuronal damage in infectious and immune-mediated diseases of the central nervous system.

Amyloid precursor proteins protect neurons of transgenic mice against acute and chronic excitotoxic injuries in vivo.

The beta-amyloid protein precursor (APP) is well conserved across different species and may fulfill important physiological functions within the CNS. While high-level neuronal expression of amyloidogenic forms of human APP results in beta-amyloid production and neurodegeneration, lower levels of neuronal human APP expression in neurons of transgenic mice may primarily accentuate physiological functions of this molecule. To assess the neuroprotective potential of human APP in vivo, mice from seven distinct transgenic lines expressing different human APP isoforms from the neuron-specific enolase promoter were challenged with systemic kainate injections (n=30) or transgene-mediated glial expression of gp120 (n=32), an HIV-1 protein capable of inducing excitotoxic neuronal damage. To quantitate human APP-mediated neuroprotection. the area of neuropil occupied by presynaptic terminals and neuronal dendrites in the neocortex and hippocampus of each mouse was determined using laser scanning confocal microscopy of double-immunolabelled brain sections and computer-aided image analysis. Compared with gp120 singly transgenic controls, mice from three of three human APP751gp120 bigenic lines expressing the 751 amino acid form of human APP at low levels showed significant protection against degeneration of presynaptic terminals; two of these lines also showed significantly less damage to neuronal dendrites. Two of three human APP695/gp120 bigenic lines expressing human APP695 at low levels were protected against presynaptic and dendritic damage, whereas one low expressor line and a human APP695/gp120 bigenic line expressing human APP695 at higher levels showed no significant protection. In the corresponding human APP singly transgenic lines, overexpressing only specific human APP isoforms, significant protection against kainate-induced degeneration of presynaptic terminals and neuronal dendrites was found in two of three human APP751 lines and not in any of the four human APP695 lines tested. These results indicate that human APP can protect neurons against chronic and acute excitotoxic insults in vivo and that human APP isoforms differ in their neuroprotective potential, at least with respect to specific forms of neural injury. It is therefore possible that impairments of neuroprotective human APP functions or aberrant shifts in human APP isoform ratios could contribute to neurodegeneration.

Spatial learning deficit in mice expressing human 751-amino acid beta-amyloid precursor protein.

Cognitive and other behavioural characteristics of 3-month-old heterozygous male transgenic mice expressing the 751-amino acid isoform of human amyloid precursor protein (hAPP751) under the control of a neurone-specific enolase promoter, were compared with those of age-matched non-transgenic control males. No difference was found between hAPP751 transgenics and non-transgenic controls in passive avoidance learning, or in motor coordination. Significantly decreased measures were found in the open field test and in cage activity indicative of general hypoactivity in hAPP751 transgenics. In water maze training, hAPP751 males required significantly longer to locate the hidden platform. This was not due to decreased swimming velocity in hAPP751 mice, but rather to increased path lengths. This suggests a purely spatial learning deficit in hAPP751 males even though their performance during a final spatial test, the probe trail that followed water maze training, was indistinguishable from that of controls. Decreased activity and impaired spatial learning were also reported in an independent study of hAPP751-expressing transgenics showing beta-amyloid immunoreactive deposits and altered tau protein. Since such histopathological alterations were not found in the transgenic model analysed in this study, our results indicate that beta-amyloid deposition is not required for the development of behavioural and/or cognitive deficits in hAPP751 transgenic mice.

Activity of adefovir dipivoxil against all patterns of lamivudine-resistant hepatitis B viruses in patients.

One hundred and thirty-one post-liver transplantation patients with chronic hepatitis B and failing lamivudine therapy with detectable serum hepatitis B virus (HBV) deoxyribonucleic acid by hybridization assays or > or =1 x 10(6) copies/mL by polymerase chain reaction, and elevated alanine transaminase levels despite continuous lamivudine, were enrolled in an open-label study of adefovir dipivoxil. The B and C domains of HBV polymerase were sequenced for baseline samples to determine the presence of lamivudine resistance mutations. The results showed that 98% of the samples had tyrosine-methionine-aspartate-aspartate (YMDD) mutations, indicating a strong correlation between the above clinical definition of lamivudine treatment failure and the presence of YMDD mutations. In addition to the rtM204V/I and the rtL180M mutations, the mutation rtV173L was identified in 19% of patients. Four major patterns of lamivudine-resistant HBV were identified: rtL180M + rtM204V (60%), rtV173L + rtL180M + rtM204V (19%), rtM204I (9%) and rtL180M + rtM204I (9%). Treatment with adefovir dipivoxil showed similar antiviral efficacy in patients with lamivudine-resistant virus from all four patterns.

In vitro evaluation of hepatitis B virus polymerase mutations associated with famciclovir resistance.

Several mutations (V521L, P525L, L528M, T532S, and V555I) in the gene for hepatitis B virus (HBV) polymerase have been identified in HBV isolated from patients that displayed break-through viremia during famciclovir treatment. To determine whether these mutations cause phenotypic resistance to famciclovir, we compared the inhibition constants (K(i)) of penciclovir triphosphate (PCVTP, the active metabolite of famciclovir) for recombinant wild-type and mutant HBV polymerases containing these mutations. In in vitro enzymatic assays, the V555I mutation displayed the most resistance (with K(i) increased by 6.2-fold) to PCVTP. The V521L and L528M mutations showed moderately decreased sensitivity to PCVTP (K(i) increased by >3-fold). We also analyzed the cross-resistance profiles of these variants for adefovir and lamivudine, two other antiviral agents that also inhibit DNA replication by HBV polymerase. All 5 famciclovir-associated mutations were sensitive to adefovir diphosphate (ADVDP) in in vitro enzymatic assays (<2.3-fold decreased sensitivity). The V521L, L528M, and T532S mutations were also sensitive to lamivudine triphosphate (LAMTP); however, the P525L and V555I mutations displayed moderately decreased sensitivity to LAMTP in enzymatic assays (3.6-fold decreased sensitivity). The lamivudine-resistant mutations M552I, M552V, and L528M+M552V, which were previously shown to display 8- to 25-fold resistance to LAMTP, were less resistant (< or = 3.1-fold) to PCVTP.

Molecular modeling and biochemical characterization reveal the mechanism of hepatitis B virus polymerase resistance to lamivudine (3TC) and emtricitabine (FTC).

Success in treating hepatitis B virus (HBV) infection with nucleoside analog drugs like lamivudine is limited by the emergence of drug-resistant viral strains upon prolonged therapy. The predominant lamivudine resistance mutations in HBV-infected patients are Met552IIe and Met552Val (Met552Ile/Val), frequently in association with a second mutation, Leu528Met. The effects of Leu528Met, Met552Ile, and Met552Val mutations on the binding of HBV polymerase inhibitors and the natural substrate dCTP were evaluated using an in vitro HBV polymerase assay. Susceptibility to lamivudine triphosphate (3TCTP), emtricitabine triphosphate (FTCTP), adefovir diphosphate, penciclovir triphosphate, and lobucavir triphosphate was assessed by determination of inhibition constants (K(i)). Recognition of the natural substrate, dCTP, was assessed by determination of Km values. The results from the in vitro studies were as follows: (i) dCTP substrate binding was largely unaffected by the mutations, with Km changing moderately, only in a range of 0.6 to 2.6-fold; (ii) K(i)s for 3TCTP and FTCTP against Met552Ile/Val mutant HBV polymerases were increased 8- to 30-fold; and (iii) the Leu528Met mutation had a modest effect on direct binding of these beta-L-oxathiolane ring-containing nucleotide analogs. A three-dimensional homology model of the catalytic core of HBV polymerase was constructed via extrapolation from retroviral reverse transcriptase structures. Molecular modeling studies using the HBV polymerase homology model suggested that steric hindrance between the mutant amino acid side chain and lamivudine or emtricitabine could account for the resistance phenotype. Specifically, steric conflict between the Cgamma2-methyl group of Ile or Val at position 552 in HBV polymerase and the sulfur atom in the oxathiolane ring (common to both beta-L-nucleoside analogs lamivudine and emtricitabine) is proposed to account for the resistance observed upon Met552Ile/Val mutation. The effects of the Leu528Met mutation, which also occurs near the HBV polymerase active site, appeared to be less direct, potentially involving rearrangement of the deoxynucleoside triphosphate-binding pocket residues. These modeling results suggest that nucleotide analogs that are beta-D-enantiomers, that have the sulfur replaced by a smaller atom, or that have modified or acyclic ring systems may retain activity against lamivudine-resistant mutants, consistent with the observed susceptibility of these mutants to adefovir, lobucavir, and penciclovir in vitro and adefovir in vivo.