Genetic associations between cathepsin D exon 2 C-->T polymorphism and Alzheimer's disease, and pathological correlations with genotype.

Genetic variations represent major risk factors for Alzheimer's disease (AD). While familial early onset AD is associated with mutations in the amyloid precursor protein and presenilin genes, only the e4 allele of the apolipoprotein E (APOE) gene has so far been established as a genetic risk factor for late onset familial and sporadic AD. It has been suggested that the C-->T (224Ala-->Val) transition within exon 2 of the cathepsin D gene (CTSD) might represent a risk factor for late onset AD. The objective of this study was to investigate whether possession of the CTSD exon 2 T allele increases the risk of developing AD, and to determine whether this modulates the amyloid pathology of the disease in conjunction with, or independent of, the APOE e4 allele. Blood samples were obtained from 412 patients with possible or probable AD and brain tissues from a further 148 patients with AD confirmed by postmortem examination. CTSD and APOE genotyping were performed by PCR on DNA extracted from blood, or from frontal cortex or cerebellum in the postmortem cases. Pathological measures of amyloid beta protein (Abeta), as plaque Abeta40 and Abeta42(3) load and degree of cerebral amyloid angiopathy were made by image analysis or semiquantitative rating, respectively. CTSD genotype frequencies in AD were not significantly different from those in control subjects, nor did these differ between cases of early or late onset AD or between younger and older controls. There was no gene interaction between the CTSD T and APOE e4 alleles. The amount of plaque Abeta40 was greater in patients carrying the CTSD T allele than in non-carriers, and in patients bearing APOE e4 allele compared with non-carriers. Possession of both these alleles acted synergistically to increase levels of plaque Abeta40, especially in those individuals who were homozygous for the APOE e4 allele. Possession of the CTSD T allele had no effect on plaque Abeta42(3) load or degree of CAA. Possession of the CTSD T allele does not increase the risk of developing AD per se, but has a modulating effect on the pathogenesis of the disorder by increasing, in concert with the APOE e4 allele, the amount of Abeta deposited as senile plaques in the brain in the form of Abeta40.

The apolipoprotein E epsilon4 allele selectively increases the risk of frontotemporal lobar degeneration in males.

OBJECTIVE: To determine whether polymorphic variations in the apolipoprotein E gene (APOE) are associated with increased risk of frontotemporal lobar degeneration (FTLD) when mutation in tau gene is absent. METHODS: The APOE gene was genotyped by polymerase chain reaction from DNA routinely extracted from blood or brain tissues. The APOE epsilon4 allele frequency in 198 patients with FTLD not associated with mutations in tau gene was compared with that of a control group of 756 normal individuals drawn from the same geographical region. Analyses were done according to clinical subtype or sex. RESULTS: The APOE epsilon4 allele frequency (19.4%) was increased (p = 0.01) in FTLD v the whole control group (14.1%), while the APOE epsilon2 allele frequency in FTLD (6.5%) was slightly lower than in controls (8.0%) (NS). The APOE epsilon4 allele frequency in men with FTLD (22.3%) was greater (p = 0.002) than in male controls (12.3%); the frequency in women (16.3%) was similar to that in female controls (14.8%) (NS). The APOE epsilon2 allele frequency in men with FTLD was 4.9% while in male controls it was 9.5% (p = 0.06), but there was no difference in women (7.5% v 7.9%, NS). Neither the APOE epsilon2 nor APOE epsilon4 allele frequency varied significantly between any of the clinical subtypes. CONCLUSIONS: In FTLD not associated with mutations in tau gene, possession of APOE epsilon4 allele in men roughly doubles the chances of developing disease, whereas this has no impact upon disease risk in women.

Qualitative neuropsychological performance characteristics in frontotemporal dementia and Alzheimer's disease.

BACKGROUND: Frontotemporal dementia (FTD) and Alzheimer's disease are clinically distinct disorders, yet neuropsychological studies have had variable success in distinguishing them. A possible reason is that studies typically rely on overall accuracy scores, which may obscure differences in reasons for failure. OBJECTIVES: To explore the hypothesis that analysis of qualitative performance characteristics and error types, in addition to overall numerical scores, would enhance the neuropsychological distinction between FTD and Alzheimer's disease. METHODS: 38 patients with FTD and 73 with Alzheimer's disease underwent assessment of language, visuospatial abilities, memory, and executive function, using a neuropsychological screening instrument and standard neuropsychological tests. In each of these cognitive domains, performance characteristics and error types were documented, in addition to numerical scores on tests. RESULTS: Whereas comparison of neuropsychological test scores revealed some group differences, these did not occur consistently across tests within cognitive domains. However, analysis of performance characteristics and error types revealed qualitative differences between the two groups. In particular, FTD patients displayed features associated with frontal lobe dysfunction, such as concrete thought, perseveration, confabulation, and poor organisation, which disrupted performance across the range of neuropsychological tests. CONCLUSIONS: Numerical scores on neuropsychological tests alone are of limited value in differentiating FTD and Alzheimer's disease, but performance characteristics and error types enhance the distinction between the two disorders. FTD is associated with a profound behavioural syndrome that affects performance on cognitive assessment, obscuring group differences. Qualitative information should be included in neuropsychological research and clinical assessments.

Potential for respirable quartz exposure from North Carolina farm soils.

Sandy-loam soils from six active farms in the coastal plains of North Carolina (USA) were analyzed for aerodynamic equivalent diameter and quartz content and compared with results to similar analyses of clay soils of the Piedmont and sandy soils from the sand hills of North Carolina to see whether respirable quartz content varies with soil type. The respirable fraction of sandy loam-soils averaged 0.04 (SD 0.02) versus 0.13 (SD 0.03) for clay soils and 0.04 (SD 0.03) for sandy soils. Quartz content in the 4.25 mu m fraction of sandy-loam soils averaged 15.2 (SD 4.1) % versus 2.2 (SD 0.8) % in clay soils and 29.0 (SD 11.1) % in sandy soils. The mass of respirable quartz in sandy-loam soils averaged 0.7 (SD 0.4)% versus 0.3 (SD 0.1)% in clay soils and 1.0 (SD 0.4) % in sandy soils. These results suggest that, during dusty farm activities, there is a potential for greater respirable quartz exposures associated with work with sandy or sandy-loam soils than from work with clay soils.

3-Deazaadenosine 5'-triphosphate: a novel metabolite of 3-deazaadenosine in mouse leukocytes.

Evidence has been obtained for the metabolic formation of small amounts (1-2% of the ATP pool) of 3-deazaadenosine 5'-triphosphate (c3ATP) from 3-deazaadenosine (c3Ado) in mouse cytolytic lymphocytes and mouse resident peritoneal macrophages. With intact leukocytes, pharmacological evidence was obtained that adenosine kinase was not the enzyme chiefly responsible for the phosphorylation of c3Ado. Moreover, in the presence of MgCl2, NaCl and IMP, purified rat liver 5'-nucleotidase catalyzed the phosphorylation of c3Ado to 3-deazaadenosine 5'-monophosphate (c3AMP). Two lines of evidence suggest that the metabolic formation of c3ATP is not involved in the inhibition of leukocyte function caused by c3Ado. First, the inhibitory action of c3Ado on antibody-dependent phagocytosis and lymphocyte-mediated cytolysis was reversed markedly upon removal of the drug from the medium. However, the intracellular content of c3ATP remained constant in lymphocytes and macrophages after removal of c3Ado. Second, in macrophages and in lymphocytes, similar intracellular amounts of c3ATP were formed from both c3Ado and 3-deazaadenine under conditions in which the former was biologically active and the latter was essentially inactive. Thus, it appears unlikely that the novel c3ATP metabolite is of relevance for the mechanism of action of c3Ado in mouse leukocytes.

Locomotion elicited by lateral hypothalamic stimulation in the anesthetized rat does not require the dorsal midbrain.

Locomotor stepping elicited by lateral hypothalamic stimulation in the anesthetized rat is blocked by lesions in the anterior ventromedial midbrain. This study determined in acute experiments whether the dorsal midbrain regions implicated in locomotion were also part of the necessary pathway. Rats were anesthetized with Nembutal and held in a stereotaxic apparatus so that stepping responses rotated a wheel. Stepping was elicited by stimulation of the lateral hypothalamus (up to 100 microA, 0.5 ms cathodal pulses, 50 Hz, 10-s train length). Nine rats received unilateral lesions ipsilateral to the locomotor electrode and 3 rats received bilateral lesions. None of the dorsal midbrain lesions reduced locomotion elicited by ipsilateral lateral hypothalamic stimulation. Therefore the following regions are unnecessary for this type of locomotion: the dorsal and lateral central gray, the tegmentum lateral to the central gray, and in particular the area cuneiformis and the dorsal aspect of the pedunculopontine region. The neural systems required for lateral hypothalamic locomotion are located ventral to the superior cerebellar peduncle.

3-Deazaadenosine-induced disorganization of macrophage microfilaments.

3-Deazaadenosine (c3Ado) has been reported to inhibit a number of cellular functions. These biological effects of c3Ado have generally been attributed to its ability to act as inhibitor and substrate of S-adenosylhomocysteine hydrolase. In this report, it is revealed by fluorescence microscopy that c3Ado caused disorganization of the microfilament system of mouse macrophages at concentrations (greater than or equal to 5 microM) similar to those that inhibited antibody-dependent phagocytosis and zymosan-stimulated H2O2 production by these cells. Inhibition of phagocytosis and perturbation of microfilaments by c3Ado were completely abrogated by washing the macrophages free of this agent and allowing the cells a 30-min recovery period. Furthermore, these effects of c3Ado on phagocytosis and microfilaments appeared to be independent of the increase in S-adenosylhomocysteine and S-3-deazaadenosylhomocysteine that occurred in these macrophages. First, periodate-oxidized adenosine and 3-deaza(+/-)aristeromycin, two other inhibitors of S-adenosylhomocysteine hydrolase that caused greater increases in macrophage S-adenosylhomocysteine than did c3Ado, had no effect on either phagocytosis or microfilaments. Second, pretreatment of macrophages with periodate-oxidized adenosine (to inhibit S-adenosylhomocysteine hydrolase) prevented the subsequent metabolism of c3Ado to S-3-deazaadenosylhomocysteine but did not diminish the effects of c3Ado on phagocytosis or microfilaments. These results demonstrate that c3Ado can perturb the microfilament system of cells and provide an alternative mechanism for the biological effects of c3Ado.

Locomotor stepping elicited by electrical stimulation of the lateral hypothalamus requires an ipsilateral descending pathway.

The effects of unilateral midbrain lesions on stepping produced by ipsilateral medial forebrain bundle (mfb) stimulation was determined in acute experiments. Rats were anesthetized with nembutal, mounted in a stereotaxic apparatus and suspended over a wheel which rotated when the rat stepped. Stimulation consisted of 10-sec trains of 50 Hz cathodal pulses (0.5 msec) at currents up to 200 microA delivered through monopolar electrodes. The stimulation sites were in the mfb at the level of the subthalamic nucleus. One side of the midbrain was randomly selected to receive a radio-frequency lesion. Certain midbrain lesions (N = 11) abolished or severely reduced stepping elicited from ipsilateral mfb stimulation but produced no effect on contralaterally elicited stepping. Areas of common damage in these lesions included the medial half of the medial lemniscus and the ventral tegmental area (VTA). Lesions that were without effect (N = 18) generally spared the dorsal VTA but damaged extensively either the ventrolateral VTA, medial lemniscus, substantia nigra, red nucleus, or central gray. A supplemental experiment further implicated a critical descending system since neither unilateral nor bilateral mfb lesions blocked stepping elicited by VTA stimulation. Together these results suggest that a descending path through the ipsilateral dorsal VTA mediates stepping elicited by mfb stimulation in the anesthetized rat.

Antagonism by taxol of effects of microtubule-disrupting agents on lymphocyte cAMP metabolism and cell function.

Several microtubule-disrupting agents (colchicine, demecolcine, vinblastine, vincristine, podophyllotoxin, and nocodazole) have been shown to inhibit lymphocyte-mediated cytolysis. These agents also enhanced the prostaglandin E1-induced rise in cAMP levels in these cytolytic lymphocytes. Taxol, a natural product alkaloid that has been shown to enhance microtubule polymerization and to stabilize microtubules, antagonized both of these effects of the microtubule-disrupting agents in the cytolytic lymphocytes. Taxol also antagonized the enhancement of cAMP increases by colchicine in lymphocytes stimulated by 2-chloroadenosine, isoproterenol, and cholera toxin. The enhancement of the prostaglandin E1-induced cAMP response caused by treatment of the lymphocytes with either cytochalasin B or 3-deazaadenosine in the presence or absence of L-homocysteine was not antagonized by taxol. Taxol, colchicine, or the combination of these two agents did not affect ATP levels in cytolytic lymphocytes. These results support a modulatory role for microtubules in both the cytolytic process and the production of cAMP in these lymphocytes.

In vivo and in vitro antitumor activity expressed by cells of concomitantly immune mice.

Growth of a primary tumor is often accompanied by the development of resistance to subsequent challenge implants of the same tumor, i.e., concomitant immunity. Using the P815 mastocytoma tumor, the kinetics of concomitant immunity was found to be governed by duration of exposure to the tumor and tumor mass. By implanting small "challenges" prior to the immunizing tumor, resistance to the growth of existing tumor foci was demonstrated. Winn-type assays revealed that antitumor activity was present in cell populations from the peritoneal exudate and lymph node draining the tumor. Peritoneal exudate cells, when infused systemically, were also able to confer protection against P815 mastocytoma challenge, suggesting their role as mediators of concomitant immunity. The 51Cr release technique indicated that cytolytic activity in lymph node cells, peritoneal exudate cells, and the spleen was present over a time course parallel to the kinetics of in vivo challenge. The peritoneal resident cell population was only slightly active; thus, effectors accumulated in the inflammatory exudate. Removal of specific subsets of cells from effector populations with antibody to surface markers and complement produced similar effects on both Winn and cytolytic assays. Anti-Thy 1.2 ablated measurable activity. It was substantially but not completely reduced by anti-Lyt 1.1 and only to a small degree by anti-Lyt 2.1.

Inhibition of lymphocyte function by 9-deazaadenosine.

9-Deazaadenosine (c9Ado), a novel C-nucleoside, has been found to inhibit lymphocyte-mediated cytolysis (LMC) in a time-dependent manner. c9Ado inhibited LMC by 50% at concentrations of 10 and 0.07 microM after drug-pretreatment periods of 3 and 22 hr, respectively, although a 1-hr pretreatment of cytolytic lymphocytes with 100 microM c9Ado had no effect upon this lymphocyte function. c9Ado was metabolized rapidly and extensively to 9-deazaadenosine 5'-triphosphate (c9ATP) both by mouse cytolytic lymphocytes and by human erythrocytes. Adenosine kinase purified from rabbit liver phosphorylated c9Ado with a Km of 200 microM and a Vmax of 8% that for adenosine. The metabolic buildup of c9ATP in lymphocytes was accompanied by a large, time-dependent decrease in cellular ATP and by smaller percentage decreases in CTP, UTP and GTP. Among other biochemical effects examined, c9Ado was found to cause a decrease in lymphocyte cAMP content and appeared to be neither an inhibitor nor a substrate for S-adenosylhomocysteine hydrolase. Consistent with this latter result, L-homocysteine thiolactone had no effect on the inhibition of LMC by c9Ado. Neither the inhibition of LMC by c9Ado nor the metabolic formation of c9ATP in lymphocytes was affected by erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), indicating that c9Ado is not a substrate for adenosine deaminase. 5-Iodotubercidin, a non-competitive inhibitor (Kis = 9 nM, Ku = 20 nM) of adenosine kinase, prevented the above effects of c9Ado on lymphocyte function, c9ATP formation, and ATP levels. Either complete preservation (with coformycin) or partial replenishment (with adenosine plus EHNA) of ATP levels in c9Ado-treated lymphocytes resulted in partial restoration of cytolytic function to cells containing large amounts of c9ATP. These results suggest that c9Ado is inhibitory to LMC both because it causes a decrease in the absolute concentration of ATP within the cytolytic lymphocytes and because it permits the establishment within these cells of an unfavorable c9ATP:ATP ratio which impedes the utilization of ATP in a reaction essential to the execution of this lymphocyte function.

In vitro cytotoxicity expressed by cells active against established tumors in vivo.

Although antitumor activity by host cells has been documented in vivo and in vitro, the cellular relationships between these two classes of studies are not clear. Cells capable of causing the regression of solid tumors are generated in lymph nodes draining sites of immunization with Corynebacterium parvum:irradiated P815 mastocytoma admixtures. These cells are active in a 51Cr release assay at a low effector:target ratio producing a characteristic low level of specific 51Cr release which required 24 hr for optimal development. The activity is immunologically specific for the immunizing tumor and is mediated by nonadherent, rapidly dividing (vinblastine-sensitive) cells. They are absent in thymectomized animals and susceptible to alpha-Thy 1.2 antibody and complement. They are present in peritoneal exudates, consistent with the systemic resistance demonstrable in the animal model. The properties and development kinetics of effector cells measured by 51Cr release correlate closely with those of cells showing in vivo activity, supporting the identity of the two populations.