A Combination of Essential Fatty Acids, Panax Ginseng Extract, and Green Tea Catechins Modifies Brain fMRI Signals in Healthy Older Adults.

OBJECTIVES: To assess the effects of a combination of omega 3 essential fatty acids, green tea catechins, and ginsenosides on cognition and brain functioning in healthy older adults. DESIGN: Double-blind, placebo-controlled, crossover design randomized controlled trial with 26-day intervention phases and a 30-day washout period. SETTING: The Institute for Dementia Research and Prevention at the Pennington Biomedical Research Center. PARTICIPANTS: Ten independently-living, cognitively-healthy older adults (mean age: 67.3 + 2.01 years). INTERVENTION: Daily consumption of an investigational product (trade name "Cerbella TM") consisting of an emulsified liquid combination of standardized fish oil, panax ginseng extract, and green tea catechins in a flavored base of lecithin phospholipids optimized to maximize bioavailability of the active ingredients. MEASUREMENTS: Before and after supplementation with the investigational product or placebo, participants completed cognitive tests including the Mini Mental State Exam (MMSE), Stroop test, Digit Symbol Substitution Test (DSST), and Immediate and Delayed Recall tests, as well as functional magnetic resonance imaging (fMRI) during a standard cognitive task switching paradigm. RESULTS: Performance on the MMSE, Stroop test, and DSST increased significantly over one month of supplementation with the investigational product (one-sample t tests, p<.05) although differences between these changes and corresponding changes during supplementation with placebo were not significant (two-sample t tests, p>.05). During supplementation with the investigational product, brain activation during task performance increased significantly more than during supplementation with placebo in brain regions known to be activated by this task (anterior and posterior cingulate cortex). Functional connectivity during task execution between task regions (middle frontal gyrus and anterior cingulate cortex) increased significantly during supplementation with the investigational product, relative to placebo. Functional connectivity during rest between task regions (precentral gyrus and middle frontal gyrus) and default mode network regions (medial frontal gyrus and precuneus) decreased during supplementation with the investigational product relative to placebo, suggesting greater segregation of task and rest related brain activity. CONCLUSION: One-month supplementation with a combination of omega 3 essential fatty acids, green tea catechins, and ginsenosides was associated with suggestive changes in cognitive functioning as well as modification of brain activation and brain functional connectivity in cognitively healthy older adults.

The pathogenicity of human immunodeficiency virus (HIV) type 1 Nef in CD4C/HIV transgenic mice is abolished by mutation of its SH3-binding domain, and disease development is delayed in the absence of Hck.

The human immunodeficiency virus type 1 (HIV-1) Nef protein is an important determinant of AIDS pathogenesis. We have previously reported that HIV-1 Nef is responsible for the induction of a severe AIDS-like disease in CD4C/HIV transgenic (Tg) mice. To understand the molecular mechanisms of this Nef-induced disease, we generated Tg mice expressing a mutated Nef protein in which the SH3 ligand-binding domain (P(72)XXP(75)XXP(78)) was mutated to A(72)XXA(75)XXQ(78). This mutation completely abolished the pathogenic potential of Nef, although a partial downregulation of the CD4 cell surface expression was still observed in these Tg mice. We also studied whether Hck, one of the effectors previously found to bind to this PXXP motif of Nef, was involved in disease development. Breeding of Tg mice expressing wild-type Nef on an hck(-/-) (knockout) background did not abolish any of the pathological phenotypes. However, the latency of disease development was prolonged. These data indicate that an intact PXXP domain is essential for inducing an AIDS-like disease in CD4C/HIV Tg mice and suggest that interaction of a cellular effector(s) with this domain is required for the induction of this multiorgan disease. Our findings indicate that Hck is an important, but not an essential, effector of Nef and suggest that another factor(s), yet to be identified, may be more critical for disease development.

The AIDS disease of CD4C/HIV transgenic mice shows impaired germinal centers and autoantibodies and develops in the absence of IFN-gamma and IL-6.

The mechanisms responsible for degeneration of germinal centers (GC) and follicular dendritic cell (FDC) networks during progression to AIDS remain elusive. Here, we show that CD4(+) T cells from CD4C/HIV-1 Tg mice, which develop a severe AIDS-like disease, express low levels of CD40 ligand. Accordingly, GC formation, FDC networks, and immunoglobulin isotype switching are impaired in these animals. However, Tg B cells respond to in vitro CD40 stimulation. Total serum IgG levels are reduced in Tg mice, whereas total IgM levels are increased with a significant amount showing DNA specificity. IFN-gamma- and IL-6-deficient CD4C/HIV Tg mice also develop the AIDS-like disease and produce auto-Ab. Thus, CD4C/HIV Tg mice have immune dysfunction accompanied by autoimmune responses.

Nef harbors a major determinant of pathogenicity for an AIDS-like disease induced by HIV-1 in transgenic mice.

Transgenic (Tg) mice expressing the complete coding sequences of HIV-1 in CD4+ T cells and in cells of the macrophage/dendritic lineages develop severe AIDS-like pathologies: failure to thrive/weight loss, diarrhea, wasting, premature death, thymus atrophy, loss of CD4+ T cells, interstitial pneumonitis, and tubulo-interstitial nephritis. The generation of Tg mice expressing selected HIV-1 gene(s) revealed that nef harbors a major disease determinant. The latency and progression (fast/slow) of the disease were strongly correlated with the levels of Tg expression. Nef-expressing Tg thymocytes were activated and alpha-CD3 hyperresponsive with respect to tyrosine phosphorylation of several substrates, including LAT and MAPK. The similarity of this mouse model to human AIDS, particularly pediatric AIDS, suggests that Nef may play a critical role in human AIDS, independently of its role in virus replication.

Transgenic mice expressing human immunodeficiency virus type 1 in immune cells develop a severe AIDS-like disease.

We have constructed transgenic (Tg) mice expressing the entire human immunodeficiency virus type 1 (HIV-1) coding sequences in cells targeted by HIV-1 infection in humans. These Tg mice developed a severe AIDS-like disease leading to early death (< 1 month). They developed muscle wasting, severe atrophy and fibrosis of lymphoid organs, tubulointerstitial nephritis, and lymphoid interstitial pneumonitis. In addition the expression of RANTES was increased in various tissues of these Tg mice relative to that in the normal controls. Disease appearance was correlated with the levels of transgene expression. The numerous pathologies observed in these mice are remarkably similar to those observed in human AIDS and, more specifically, in pediatric AIDS.

The prion protein gene is dispensable for the development of spongiform myeloencephalopathy induced by the neurovirulent Cas-Br-E murine leukemia virus.

The Cas-Br-E murine leukemia virus (MuLV) induces paralysis in susceptible mice that is accompanied by a severe spongiform myeloencephalopathy. These neurodegenerative lesions are very similar to those observed in prion diseases. To determine whether the prion protein gene (Prn-p) product was a downstream effector of this neurovirulent MuLV, we inoculated Prn-p(-/-) knockout homozygote and control heterozygote or wild-type mice with this retrovirus. All groups developed typical paralysis and spongiform encephalopathy, and no differences in clinical or histological phenotypes were observed between these groups. These results indicate that the Cas-Br-E MuLV does not require the prion protein to induce lesions. Thus, MuLV and prion proteins may induce a very similar disease through distinct pathways, or the viral Env protein, which harbors the primary determinant of pathogenicity, may act in a common pathway but downstream of the prion protein.

Identification of the infected target cell type in spongiform myeloencephalopathy induced by the neurotropic Cas-Br-E murine leukemia virus.

The Cas-Br-E murine leukemia virus (MuLV) induces a progressive hindlimb paralysis accompanied by a spongiform myeloencephalopathy in susceptible mice. In order to better understand the pathological process leading to these neurodegenerative lesions, we have investigated the nature of the cell type(s) infected by the virus during the course of the disease in CFW/D and SWR/J mice. For this purpose, we used in situ hybridization with virus-specific probes in combination with cell-type-specific histochemical (lectin) and immunological markers as well as morphological assessment. In the early stage of infection, endothelial cells represented the main cell type expressing viral RNA in the central nervous system (CNS). With disease progression and the appearance of lesions, microglial cells became the major cell type infected, accounting for up to 65% of the total infected cell population in diseased areas. Morphologically, these cells appeared activated and were frequently found in clusters. Infection and activation of microglial cells were almost exclusively restricted to diseased regions of the CNS. Neurons in diseased regions were not discernibly infected with virus at either early or late times of disease progression. Similarly, the proportion of infected astrocytes was typically < 1%. Although some endothelial cells and oligodendrocytes were infected by the virus, their infection was not limited to diseased CNS regions. These results are consistent with a model of indirect motor neuron degeneration, subsequent to the infection of nonneuronal CNS cells and especially of microglial cells. Infected microglial cells may play a role in the disease process by releasing not only virions or viral env-gene-encoded gp70 proteins but also other factors which may be directly or indirectly toxic to neurons. Parallels between microglial cell infection by MuLV and by lentiviruses, and specifically by human immunodeficiency virus, are discussed.

Neurological disease induced in transgenic mice expressing the env gene of the Cas-Br-E murine retrovirus.

The Cas-Br-E murine leukemia virus induces a spongiform myeloencephalopathy in susceptible mice. We constructed transgenic mice harboring either the viral genome (in a replication-defective form) or only its env gene. Low levels of expression of either transgene resulted in mild neuropathology and/or signs of neurological disease in more than half of these mice. These results indicate that the disease can occur in the absence of virus replication and strongly suggest that the env gp70/p15E complex is sufficient to induce disease.

The majority of cells infected with the defective murine AIDS virus belong to the B-cell lineage.

Murine AIDS (MAIDS) is caused by a defective retrovirus which encodes a gag fusion protein (Pr60gag). We previously reported that this virus induced an oligoclonal proliferation of infected cells and suggested that this cell expansion was an important event in the pathogenesis of MAIDS. To identify these target cells, we constructed novel defective viruses whose genomes could be detected with specific probes. Helper-free stocks of these viruses induced MAIDS. Using in situ hybridization and immunocytochemistry and Southern analysis, we found that most infected cells belong to the B-cell lineage. Transformation of these B cells appears to be the primary event responsible for the development of immunodeficiency. This animal model may be relevant to our understanding of AIDS, of the immunodeficiencies associated with B-cell lymphoproliferative disorders, and of the role of B-cell proliferation and transformation in the effects of superantigens, since Pr60gag appears to be a superantigen.

Retrovirus-induced spongiform myeloencephalopathy in mice: regional distribution of infected target cells and neuronal loss occurring in the absence of viral expression in neurons.

The Cas-Br-E murine leukemia virus (MuLV) induces a spongiform myeloencephalopathy resulting in a progressive hindlimb paralysis. We have used in situ hybridization with a Cas-Br-E MuLV-specific probe to study viral expression in the central nervous system. Infected cells were concentrated in regions where spongiform lesions and gliosis are detected (lumbosacral spinal cord, brainstem, deep cerebellar regions), suggesting a causative link between the level of virus expression and the degree of pathological changes in this disease. However, viral expression was not in itself sufficient to cause disease, since significant viral expression was observed in regions that did not exhibit pathological changes (cerebellar cortex, hippocampus, corpus callosum, peripheral nervous system). In both diseased and nondiseased regions, endothelial and glial cells were identified as the main target cells. Neurons in diseased regions did not show viral expression. The regional distribution of the spongiform changes appears to be laid down very early following infection, since expression could be detected at 10 days postinfection in regions that become diseased. These results indicate that nonneuronal cells have distinct properties in various regions of the central nervous system and suggest an indirect mechanism of neuronal loss consequent to viral expression in nonneuronal cells.

Substitution of the U3 long terminal repeat region of the neurotropic Cas-Br-E retrovirus affects its disease-inducing potential.

The Cas-Br-E and ts-Mo BA-1 murine leukemia viruses (MuLV) induce a spongiform neurodegenerative disease with different clinical manifestations, namely, either hind limb paralysis (Cas-Br-E) or tremors, spasticity, and hind limb weakness (ts-Mo Ba-1). We constructed the chimeric NEBA-1 MuLV by replacing the long terminal repeat of Cas-Br-E MuLV with that of ts-Mo BA-1 MuLV. In SWR/J or CFW/D mice, NEBA-1 MuLV induced an ataxic neurological disease characterized by clinical signs different from those induced by both parents. Although NEBA-1 MuLV did not induce lesions in novel brain areas, the spongiform lesions were more severe in deep cerebellar nuclei and in the spinal cord than those found in paralyzed mice inoculated with Cas-Br-E MuLV. By in situ hybridization, we found that the distribution of the spongiform lesions closely correlated with the distribution of the infected central nervous system cells. In the spinal cord, a close correlation was found between the number of infected cells and the severity of the spongiform degeneration. Sequencing of the substituted ts-BA-1 MuLV fragment and comparison with homologous sequences of Cas-Br-E and Moloney MuLV showed differences mainly in the U3 tandem direct repeats. Our results show that a few modifications within the U3 long terminal repeat allow the virus to cause more severe lesions in some central nervous system regions and that the severity of the spongiform degeneration correlates with the level of viral replication.

Selective degradation of insulin within rat liver endosomes.

To characterize the role of the endosome in the degradation of insulin in liver, we employed a cell-free system in which the degradation of internalized 125I-insulin within isolated intact endosomes was evaluated. Incubation of endosomes containing internalized 125I-insulin in the cell-free system resulted in a rapid generation of TCA soluble radiolabeled products (t1/2, 6 min). Sephadex G-50 chromatography of radioactivity extracted from endosomes during the incubation showed a time dependent increase in material eluting as radioiodotyrosine. The apparent Vmax of the insulin degrading activity was 4 ng insulin degraded.min-1.mg cell fraction protein-1 and the apparent Km was 60 ng insulin.mg cell fraction protein-1. The endosomal protease(s) was insulin-specific since neither internalized 125I-epidermal growth factor (EGF) nor 125I-prolactin was degraded within isolated endosomes as assessed by TCA precipitation and Sephadex G-50 chromatography. Significant inhibition of degradation was observed after inclusion of p-chloromercuribenzoic acid (PCMB), 1,10-phenanthroline, bacitracin, or 0.1% Triton X-100 into the system. Maximal insulin degradation required the addition of ATP to the cell-free system that resulted in acidification as measured by acridine orange accumulation. Endosomal insulin degradation was inhibited markedly in the presence of pH dissipating agents such as nigericin, monensin, and chloroquine or the proton translocase inhibitors N-ethylmaleimide (NEM) and dicyclohexylcarbodiimide (DCCD). Polyethylene glycol (PEG) precipitation of insulin-receptor complexes revealed that endosomal degradation augmented the dissociation of insulin from its receptor and that dissociated insulin was serving as substrate to the endosomal protease(s). The results suggest that as insulin is internalized it rapidly but incompletely dissociates from its receptor. Dissociated insulin is then degraded by an insulin specific protease(s) leading to further dissociation and degradation.

Ligand-mediated internalization, recycling, and downregulation of the epidermal growth factor receptor in vivo.

EGF receptor internalization, recycling,a nd downregulation were evaluated in liver parenchyma as a function of increasing doses of injected EGF. The effect of ligand occupancy in vivo on the kinetics and extent of internalization was studied with changes in the receptor content of isolated plasmalemma and endosome fractions evaluated by direct binding, Scatchard analysis, and Western blotting. For all doses of injected EGF, receptor was lost from the plasmalemma and accumulated in endosomes in a time- and dose-dependent fashion. However, at doses of injected EGF equivalent to less than or equal to 50% surface receptor occupancy (i.e., less than or equal to 1 microgram/100 g body weight), receptor levels returned by 120 min to initial values. This return was resistant to cycloheximide and therefore did not represent newly synthesized receptor. Neither was the return due to replenishment by an intracellular pool of low-affinity receptors as such a pool could not be detected by Scatchard analysis or Western blotting. Therefore, receptor return was due to the recycling of previously internalized receptor. At doses of injected EGF greater than 50% receptor occupancy, net receptor loss-i.e., downregulation-was observed by evaluating the receptor content of total particulate fractions of liver homogenates. At the higher saturating doses of injected EGF (5 and 10 micrograms/100 g body weight), the majority of surface receptor content was lost by 15 min and remained low for at least an additional 105 min. As the kinetics of ligand clearance from the circulation and liver parenchyma were similar for all doses of EGF injected, then the ligand-mediated regulation of surface receptor content and downregulation were not a result of a prolonged temporal interaction of ligand with receptor. Rather, the phenomena must be a consequence of the absolute concentrations of EGF interacting with receptor at the cell surface and/or in endosomes.

Epidermal growth factor receptor kinase translocation and activation in vivo.

The rat liver epidermal growth factor (EGF) receptor was assessed for EGF-dependent autophosphorylation as well as phosphorylation of a defined exogenous substrate in purified plasmalemma and Golgiendosome fractions isolated from rat liver homogenates. While EGF-dependent kinase activity was readily detected in plasmalemma the corresponding activity in Golgi-endosome fractions required detergent. Consequent to the systemic injection of EGF in vivo, the majority (approximately 60%) of receptor as evaluated by 125I-EGF binding was rapidly lost (T 1/2 approximately 8 min) from the plasmalemma and correspondingly accumulated in the Golgi-endosome fraction in a dose-dependent manner. Electron microscope radioautography of 125I-EGF uptake into Golgi-endosome fractions identified internalization into lipoprotein-filled vesicles of heterogenous size and shape but not into stacked saccules of the Golgi apparatus. Evaluation of receptor kinase activity in plasmalemma fractions isolated at various times after EGF injection in vivo showed more rapid loss of EGF-dependent autophosphorylation activity (T 1/2 approximately 10 s) than of receptor content (T 1/2 approximately 8 min). In contrast to the EGF receptor kinase of the plasmalemma fraction, kinase activity accumulating in endosomes was activated, i.e. maximally stimulated, in the absence of EGF or Triton X-100 in vitro. Furthermore, following the peak time of accumulation of EGF receptor kinase in endosomes (5-15 min) EGF-dependent autophosphorylation activity and EGF receptor content were lost more slowly (T 1/2 approximately 27 and 87 min for the loss of autophosphorylation activity and receptor content, respectively). The rapidity of translocation of activated EGF receptor into endosomes (30 s) and the dose response to low levels (1 microgram) of EGF injected are consistent with a physiological role for internalized EGF receptor kinase activity.

In vivo uptake of insulin into hepatic Golgi fractions: application of the diaminobenzidine-shift protocol.

The hypothesis that insulin is internalized into the hepatic Golgi apparatus was tested by the diaminobenzidine-shift protocol of Courtoy et al. (1984, J. Cell Biol. 98, 870). Highly purified Golgi fractions were isolated after the coinjection of [125I]insulin and the synthetic ligand, galactose-bovine serum albumin-horseradish peroxidase. Golgi fractions were subsequently reacted in the presence or absence of diaminobenzidine, then subjected to Percoll gradient centrifugation. For incubations carried out in the absence of diaminobenzidine, [125I]insulin-containing components were found at a low density (peak density congruent to 1.042) identical to that of the Golgi marker enzyme galactosyltransferase. However after incubations carried out in the presence of diaminobenzidine, the majority of [125I]insulin-containing components was shifted to a higher density of greater than 1.06 while that of galactosyltransferase remained unchanged (peak congruent to 1.042). These observations indicate that the majority of internalized insulin is not located in galactosyltransferase-containing Golgi components.