Intra-accumbens infusions of antisense oligodeoxynucleotides to one isoform of glutamic acid decarboxylase mRNA, GAD65, but not to GAD67 mRNA, impairs sustained attention performance in the rat

The effects of bilateral infusions of antisense oligodeoxynucleotides (ODNs) for the two isoforms of glutamic acid decarboxylase (GAD65; GAD67) into the nucleus accumbens on the performance of intact rats in a task designed to assess sustained attention were tested. The task required the animals to discriminate between signal and non-signal events. Signals and non-signals were presented randomly and unpredictably. The task generated all four response types of a sustained attention task, i.e., hits, misses, correct rejections, false alarms. Infusions of the scrambled sequence ODNs did not affect performance. Likewise, infusions of the GAD67 ODNs failed to produce any effect. However, infusions of the GAD65 ODNs into the nucleus accumbens resulted in a robust and reliable decrease in the relative number of hits. Similarly, the combined infusion of GAD65+67 ODNs impaired the hit rate but did not affect the animals' ability to reject non-signals. Following each treatment series, performance rapidly returned to baseline, further indicating the specificity and reversibility of the effects of the infusions of the ODNs. While these data suggest that translation arrest of specifically the GAD65 isoform of the enzyme in the nucleus accumbens impairs attentional performance, the neuronal mechanisms mediating these effects remain unsettled.     

Redirecting effector T cells through their IL-2 receptors

Fusion proteins constructed of a tumor-specific Ab joined to IL-2 (Ab-IL-2) have been used in the past to deliver cytokine directly to the site of tumor cells in vivo. These molecules mimic the activity of IL-2 and assist in activating and expanding antitumor effector cells. To enhance the cytolytic activity of CTL specific for peptide epitopes of the Her-2/neu tumor Ag presented by HLA-A*0201 molecules, a fusion protein was constructed consisting of a single chain Ab specific for Her-2/neu, linked to IL-2 (neu-Ab-IL-2). When added to a mixture of tumor cells and Her-2/neu-specific CTL, the protein was found to augment lysis of tumor cells. In addition, the hybrid molecule also promoted lysis of Her-2/neu expressing tumors by non-tumor-specific cloned T cell lines, including Th1 CD4 cells. Analysis of the mechanism of cytotoxicity revealed that the fusion protein mediates the formation of stable conjugates between T cells expressing IL-2R and tumor cells expressing Her-2/neu, resulting in lysis through the Fas-Fas ligand pathway. Lysis induction was independent of specific engagement by the TCR. When tested for its ability to enhance tumor cell eradication by Her-2/neu-specific CD8+ T cells in an adoptive transfer model in SCID mice, neu-Ab-IL-2 facilitated the elimination of tumor cells in vivo. Surprisingly, the combination of non-tumor-specific CD8+ T cells and fusion protein also induced a significant delay of tumor growth. This represents a novel approach for redirecting non-tumor-specific T cells to eliminate tumors.     

Effects of maternal antibodies on protection and development of antibody responses to human rotavirus in gnotobiotic pigs

Although maternal antibodies can protect against infectious disease in infancy, they can also suppress active immune responses. The effects of circulating maternal antibodies, with and without colostrum and milk antibodies, on passive protection and active immunity to human rotavirus (HRV) were examined in gnotobiotic pigs. Pigs received intraperitoneal injections of high-titer serum (immune pigs [groups 1 and 2]) from immunized sows, low-titer serum from naturally infected sows (control pigs [groups 3 and 4]), or no serum (group 5). Immune or control colostrum and milk were added to the diet of groups 2 and 4, respectively. After inoculation (3 to 5 days of age) and challenge (postinoculation day [PID] 21) with virulent HRV, the effects of maternal antibodies on protection (from diarrhea and virus shedding), and on active antibody responses (measured by quantitation of antibody-secreting cells [ASC] in intestinal and systemic lymphoid tissues by ELISPOT) were evaluated. Groups 1 and 2 had significantly less diarrhea and virus shedding after inoculation but higher rates of diarrhea and virus shedding after challenge than did groups 3 and 5. Group 1 and 2 pigs had significantly fewer immunoglobulin A (IgA) ASC in intestinal tissues at PID 21 and at postchallenge day (PCD) 7 compared to group 5. Significantly fewer IgG ASC were present in the intestines of group 2 pigs at PID 21 and PCD 7 compared to group 5. There was a trend towards fewer ASC in intestinal tissues of group 2 than group 1, from PID 21 on, with significantly fewer IgA ASC at PCD 7. IgG ASC in the duodenum and mesenteric lymph nodes of group 3 and 4 pigs were significantly fewer than in group 5 at PCD 7. These decreases in ASC emphasize the role of passive antibodies in impairing induction of ASC rather than in merely suppressing the function of differentiated B cells. To be successful, vaccines intended for populations with high titers of maternal antibodies (infants in developing countries) may require higher titers of virus, multiple doses, or improved delivery systems, such as the use of microencapsulation or immune stimulating complexes, to overcome the suppressive effects of maternal antibodies.     

Identification of seven Treponema species in health- and disease-associated dental plaque by nested PCR

Species-specific nested PCR was used to detect Treponema amylovorum, Treponema denticola, Treponema maltophilum, Treponema medium, Treponema pectinovorum, Treponema socranskii, and Treponema vincentii in dental plaque. Subjects with periodontitis harbored all species, but T. pectinovorum and T. vincentii were not found in plaque from disease-free subjects.     

Fulminant hepatic failure associated with bicalutamide

Bicalutamide is a new, nonsteroidal antiandrogen with a favorable toxicity profile. We report the first case of fulminant hepatic failure associated with its use.     

Alloantigen-stimulated anti-HIV activity

A number of studies have suggested that an immune response to human leukocyte antigen (HLA) alloantigens may contribute to protection against HIV infection. In the present study, we examined the effect of alloantigen-stimulated cell lines obtained from peripheral blood mononuclear cells (PBMC) of HIV-uninfected (HIV-) individuals and the soluble factors produced by these cell lines on HIV-1 replication. Multiple in vitro restimulation with irradiated allogeneic PBMC from HIV- donors resulted in the expansion of CD8(+) T-cell lines that inhibited HIV-1 replication when cocultured with either autologous or heterologous in vitro-infected phytohemagglutinin (PHA) blasts. Supernatants from the alloantigen-stimulated cell lines also inhibited HIV replication in both PHA blasts and a chronically infected cell line. The alloantigen-stimulated cell lines and the factors they produced inhibited both T-cell-tropic (T) and macrophage-tropic (M) isolates of HIV-1. Blocking experiments using anti-chemokine antibodies suggested that this inhibition of HIV replication was not due to the beta-chemokines present in cocultures of cell lines with HIV-infected blasts. These results indicate that alloantigen-stimulation of PBMC from HIV- individuals activates CD8(+) T cells that produce soluble factor(s) that inhibit HIV replication of a wide spectrum of HIV-1 isolates through a chemokine-independent mechanism. This is a US government work. There are no restrictions on its use.     

ATP-dependent proteolysis in mitochondria. m-AAA protease and PIM1 protease exert overlapping substrate specificities and cooperate with the mtHsp70 system

To analyze protein degradation in mitochondria and the role of molecular chaperone proteins in this process, bovine apocytochrome P450scc was employed as a model protein. When imported into isolated yeast mitochondria, P450scc was mislocalized to the matrix and rapidly degraded. This proteolytic breakdown was mediated by the ATP-dependent PIM1 protease, a Lon-like protease in the mitochondrial matrix, in cooperation with the mtHsp70 system. In addition, a derivative of P450scc was studied to which a heterologous transmembrane region was fused at the amino terminus. This protein became anchored to the inner membrane upon import and was degraded by the membrane-embedded, ATP-dependent m-AAA protease. Again, degradation depended on the mtHsp70 system; it was inhibited at non-permissive temperature in mitochondria carrying temperature-sensitive mutant forms of Ssc1p, Mdj1p, or Mge1p. These results demonstrate overlapping substrate specificities of PIM1 and the m-AAA protease, and they assign a central role to the mtHsp70 system during the degradation of misfolded polypeptides by both proteases.     

Elimination kinetics of volatile organics in humans using breath measurements

During the past decade significant strides have been made toward understanding the sources and factors which lead to volatile organic chemical (VOC) exposure in the general population. Less is known, however, about the impact of low-level environmental exposure on human health. Investigations are underway in a number of laboratories in an effort to determine the uptake, distribution, metabolism, and elimination kinetics for VOCs in humans. We examined the elimination kinetics for the third phase for ten VOCs--1,1,-trichloroethane, trichloroethylene, tetrachloroethylene, benzene, toluene, m,p-xylenes, o-xylene, ethylbenzene, p-dichlorobenzene, and limonene--in human subjects. Subjects were exposed to a variety of common consumer products and breath samples were collected post-exposure while the subjects spent up to 10 hr in a clean air environment. VOCs from breath samples were collected into canisters or onto Tenax GC cartridges and analyzed by gas chromatography-mass spectrometry. Exponential modeling of the decay data was performed to obtain kinetic parameters. The half-lives for trichloroethylene and 1,1,1-trichloroethane were approximately 5 to 8 hr for the four subjects. In general, the magnitude and range of variability was larger for toluene, limonene, and p-dichlorobenzene than for the other VOCs; the elimination rate spanning a few hours to a day or two. Thus, VOCs exhibit relatively short residence times in the body relative to other halo-carbons, such as polychlorinated biphenyls and dioxins.     

Effects of age, sex, and ethnicity on the association between apolipoprotein E genotype and Alzheimer disease. A meta-analysis. APOE and Alzheimer Disease Meta Analysis Consortium

OBJECTIVE: To examine more closely the association between apolipoprotein E (APOE) genotype and Alzheimer disease (AD) by age and sex in populations of various ethnic and racial denominations. DATA SOURCES: Forty research teams contributed data on APOE genotype, sex, age at disease onset, and ethnic background for 5930 patients who met criteria for probable or definite AD and 8607 controls without dementia who were recruited from clinical, community, and brain bank sources. MAIN OUTCOME MEASURES: Odds ratios (ORs) and 95% confidence intervals (CIs) for AD, adjusted for age and study and stratified by major ethnic group (Caucasian, African American, Hispanic, and Japanese) and source, were computed for APOE genotypes epsilon2/epsilon2, epsilon2/epsilon3, epsilon2/epsilon4, epsilon3/epsilon4, and epsilon4/epsilon4 relative to the epsilon3/epsilon3 group. The influence of age and sex on the OR for each genotype was assessed using logistic regression procedures. RESULTS: Among Caucasian subjects from clinic- or autopsy-based studies, the risk of AD was significantly increased for people with genotypes epsilon2/epsilon4 (OR=2.6, 95% CI=1.6-4.0), epsilon3/epsilon4 (OR=3.2, 95% CI=2.8-3.8), and epsilon4/epsilon4 (OR=14.9, 95% CI= 10.8-20.6); whereas, the ORs were decreased for people with genotypes epsilon2/epsilon2 (OR=0.6, 95% CI=0.2-2.0) and epsilon2/epsilon3 (OR=0.6, 95% CI=0.5-0.8). The APOE epsilon4-AD association was weaker among African Americans and Hispanics, but there was significant heterogeneity in ORs among studies of African Americans (P<.03). The APOE epsilon4-AD association in Japanese subjects was stronger than in Caucasian subjects (epsilon3/epsilon4: OR=5.6, 95% CI=3.9-8.0; epsilon4/epsilon4: OR=33.1, 95% CI=13.6-80.5). The epsilon2/epsilon3 genotype appears equally protective across ethnic groups. We also found that among Caucasians, APOE genotype distributions are similar in groups of patients with AD whose diagnoses were determined clinically or by autopsy. In addition, we found that the APOE epsilon4 effect is evident at all ages between 40 and 90 years but diminishes after age 70 years and that the risk of AD associated with a given genotype varies with sex. CONCLUSIONS: The APOE epsilon4 allele represents a major risk factor for AD in all ethnic groups studied, across all ages between 40 and 90 years, and in both men and women. The association between APOE epsilon4 and AD in African Americans requires clarification, and the attenuated effect of APOE epsilon4 in Hispanics should be investigated further.