Comparison of pleural fluid pH values obtained using blood gas machine, pH meter, and pH indicator strip.

STUDY PURPOSE: The purpose of this study was to compare the pleural fluid pH values obtained with a blood gas machine (pHbg), with a pH meter (pHmet), and with a pH indicator strip (pHstrip), to determine if the pleural fluid pH measured by a pH meter or a pH indicator strip was sufficiently accurate for clinical decisions. METHODS: The pleural fluid pH was determined, within 20 min after being collected anaerobically, by a blood gas machine (CIBA-Corning model 288), pH meter (Corning pH meter 610A), and pH indicator strip (Baxter Diagnostic) following routine laboratory procedures in 50 pleural fluids. Pleural fluid pH was determined in seven additional samples with the blood gas machine and a pH meter at 25 and 37 degrees C respectively, initially, and after 30 min. RESULTS: The mean pHbg (7.42+/-0.01) was significantly less than the mean pHmet (7.58+/-0.02) or the mean pHstrip (8.23+/-0.06). There were significant differences between the pHbg and the pHmet (p < 0.001), and between the pHbg and the pHstrip (p < 0.001). Analysis of the additional seven samples demonstrated that when the blood gas machine was set at 25 degrees C, the pHbg (pHbg = 7.54+/-0.02) and the pHmet (7.53+/-0.01) were almost identical. CONCLUSION: When the pleural fluid pH is going to be used for decision making, only the pH values provided by the blood gas machine are sufficiently accurate.

Patients with features similar to Huntington's disease, without CAG expansion in huntingtin.

OBJECTIVE: To describe characteristics of gene-negative patients with clinical features of Huntington's disease (HD), exploring likely etiologies. BACKGROUND: When a direct gene test became definitive for diagnosis of HD, we discovered a number of patients in our clinics in Baltimore, MD, and Cambridge, UK, believed or suspected to have HD who did not have the triplet repeat expansion. METHODS: Patients were examined using standardized instruments, and given full neurologic and psychiatric evaluations. Those negative for HD were tested for dentatorubro-pallidoluysian atrophy, SCA-1, SCA-3, SCA-2, SCA-6, and other conditions as indicated. RESULTS: Of 15 patients, 7 received specific diagnoses or appear to be sporadic cases, 4 have a possible but uncertain relation to HD, and 4 have unknown familial progressive movement disorders. CONCLUSIONS: This last group of patients might be properly described as phenocopies of HD, some of which may be caused by unidentified triplet repeat expansions.

Frontal lobe volume in patients with Huntington's disease.

Neuropathologic and neuroimaging studies have suggested that frontal lobes are affected in Huntington's disease (HD), and that atrophy in this region may be associated with some of the cognitive impairment and clinical decline observed in patients with HD. We measured gray and white matter volumes within the frontal lobes on MRI for 20 patients with HD (10 mildly affected and 10 moderately affected) and 20 age- and sex-matched control subjects. We also correlated frontal lobe measurements with measures of symptom severity and cognitive function. Patients who were mildly affected had frontal lobe volumes (both gray and white matter) essentially identical to those of control subjects, despite clearly abnormal basal ganglia. Patients who were moderately affected demonstrated significant reductions in total frontal lobe volume (17%) and frontal white matter volume (28%). Frontal lobe white matter volume reductions, but not total frontal lobe volume reductions, were disproportionately greater than overall brain volume reductions (17%). Frontal lobe volume correlated with symptom severity and general cognitive function, but these correlations did not remain significant after taking into account total brain volume. We conclude that cognitive impairment and symptom severity are associated with frontal lobe atrophy, but this association is not specific to the frontal lobes. Frontal lobe atrophy (like total brain atrophy) occurs in later stages of increasing HD symptom severity and this atrophy primarily involves white matter.

Dentatorubral and pallidoluysian atrophy (DRPLA). Clinical and neuropathological findings in genetically confirmed North American and European pedigrees.

Dentatorubral and pallidoluysian atrophy (DRPLA) is an autosomal dominant disorder that clinically overlaps with Huntington's disease (HD) and manifests combinations of chorea, myoclonus, seizures, ataxia, and dementia. DRPLA is caused by a CAG triplet repeat (CTG-B37) expansion coding for polyglutamine on chromosome 12 and exhibits the genetic phenomenon of anticipation. This neurodegenerative disease has only rarely been reported in non-Japanese pedigrees, and there are only a few neuropathological studies in genetically confirmed patients. We report 10 cases of DRPLA from two North American and two British pedigrees in which CTG-B37 expansions have been demonstrated within each kindred (54-83 repeats), individually in 8 of the 10 cases, and describe the neuropathological findings in 4 cases. Members of DRPLA kindreds have a wide range of clinical phenotypes and markedly variable ages at onset. The neuropathological spectrum is centered around the cerebellifugal and pallidofugal systems, but neurodegenerative changes can be found in many nuclei, tracts, and systems. Evidence of CTG-B37 triplet repeat expansion should be sought in HD-like cases that are negative for expanded triplet repeats within the HD IT15 gene or in autopsy cases with degeneration of the dentatorubral or pallidoluysian systems.

Expansion of polyglutamine repeat in huntingtin leads to abnormal protein interactions involving calmodulin.

Huntington's disease (HD) is an inherited neurodegenerative disorder associated with expansion of a CAG repeat in the IT15 gene. The IT15 gene is translated to a protein product termed huntingtin that contains a polyglutamine (polyGln) tract. Recent investigations indicate that the cause of HD is expansion of the polyGln tract. However, the function of huntingtin and how the expanded polyGln tract causes HD is not known. We investigate potential protein-protein interactions of huntingtin using affinity resins. Huntingtin from brain extracts is retained on calmodulin(CAM)-Sepharose in a calcium-dependent fashion. We purify rat huntingtin to apparent homogeneity using a combination of DEAE-cellulose column chromatography, ammonium sulfate precipitation, and preparative SDS/PAGE. Purified rat huntingtin does not interact with CAM directly as revealed by 125I-CAM overlay. Huntingtin forms a large CAM-containing complex of over 1,000 kDa in the presence of calcium, which partially disassociates in the absence of calcium. Furthermore, an increased amount of mutant huntingtin from HD patient brains is retained on CAM-Sepharose compared to normal huntingtin from control patient brains, and the mutant allele is preferentially retained on CAM-Sepharose in the absence of calcium. These results suggest that huntingtin interacts with other proteins including CAM and that the expansion of polyGln alters this interaction.

DRPLA gene (atrophin-1) sequence and mRNA expression in human brain.

Dentatorubral pallidoluysian atrophy (DRPLA, Smith's disease) is one of five disorders currently known to result from expansion of a CAG trinucleotide repeat encoding glutamine. The reported full length cDNA sequence encodes a serine repeat and a region of alternating acidic and basic amino acids, as well as the glutamine repeat. We now report the nucleic acid and deduced amino acid sequences of the open reading frame of this gene, obtained from a series of independently isolated and sequenced cDNA clones. Eight nucleotide differences from the originally published sequence result in a change of 34 amino acids, most prominently in the region of alternating acidic and basic residues. Northern analysis and in situ hybridization indicate that atrophin-1 mRNA is expressed in multiple brain regions. The level of mRNA expression as determined by in situ hybridization in a DRPLA-diseased brain is indistinguishable from the level observed in a matched control brain. These results indicate that the correlation between atrophin-1 expression and regions of pathology in DRPLA is at best partial, and that the expanded allele does not cause a major loss of mRNA expression. The pathology of the disorder may therefore arise from the altered structure and function of the abnormal protein.

Expression of the Huntington's disease (IT15) protein product in HD patients.

Huntington's disease (HD) is an inherited, neurodegenerative disorder caused by expansion of a CAG repeat in the IT15 gene, leading to an expanded glutamine repeat in the HD protein. The mechanism by which the expanded repeat causes expression of the disease is not known, though there do not appear to be changes in the mRNA levels. We have conducted quantitative Western blot analyses of HD patients and controls. Expression of the IT15 protein is essentially equal in control and HD frontal cortex. In caudate from HD patients, IT15 protein is decreased in parallel with the decrease in a neuronal marker, suggesting that loss of IT15 protein is secondary to neuronal loss. In order to determine expression of the two alleles of the IT15 protein we used Western blots of 4% polyacrylamide gels. Both alleles of the IT15 protein were expressed at similar levels in HD lymphoblastoid cell lines and HD post-mortem hippocampus and cerebellum (regions relatively spared in HD), indicating that even very long CAG repeats can be translated into polyglutamine. In contrast, in cerebral cortex and caudate (regions severely affected in HD), in the longer expanded repeat cases the expanded allele of the IT15 protein was present at a significantly lower level (compared with the normal length allele), often with a smear of more slowly migrating reactivity above it. These data suggest the possibility of altered structure, abnormal processing or abnormality of protein-protein interactions involving the IT15 protein with the expanded glutamine repeat.

Widespread expression of Huntington's disease gene (IT15) protein product.

Huntington's Disease (HD) is caused by expansion of a CAG repeat within a putative open reading frame of a recently identified gene, IT15. We have examined the expression of the gene's protein product using antibodies developed against the N-terminus and an internal epitope. Both antisera recognize a 350 kDa protein, the predicted size, indicating that the CAG repeat is translated into polyglutamine. The HD protein product is widely expressed, most highly in neurons in the brain. There is no enrichment in the striatum, the site of greatest pathology in HD. Within neurons, the protein is diminished in nuclei and mitochondria and is present in the soluble cytoplasmic compartment, as well as loosely associated with membranes or cytoskeleton, in cell bodies, dendrites, and axons. It is concentrated in nerve terminals, including terminals within the caudate and putamen. Thus, the normal HD gene product may be involved in common intracellular functions, and possibly in regulation of nerve terminal function. The product of the expanded allele is expressed, consistent with a gain of function mechanism for HD at the protein level.

Expression of the mutant allele of IT-15 (the HD gene) in striatum and cortex of Huntington's disease patients.

Huntington's disease (HD) is an inherited neurodegenerative disorder expressed when a trinucleotide repeat in the gene IT-15 is expanded. The mechanism by which the expanded repeat causes the expression of the disease is unknown. Possible mechanisms include alterations in the amount of the mRNA, potentially resulting from changes in gene transcription or abnormal mRNA stability. In order to determine whether the expanded IT-15 allele is present in mRNA, we isolated total RNA from the cortex and striatum of patients and controls. To distinguish the two alleles of the IT-15 transcript in HD patients, we amplified across a region containing a dimorphic single triplet deletion observed on some chromosomes and found that the relative intensity of the two PCR bands amplified from genomic DNA and those amplified from first strand cDNA from brain tissue were essentially equal. In order to determine whether the exon containing the expanded CAG repeat is present in IT-15 mRNA from HD patients, we amplified across this region and demonstrated the presence of the expanded repeat in cDNA from both striatum and cortex. Based on this evidence, we suggest that the mechanism of disease expression does not occur during transcription or in the stability of the RNA, but rather occurs during translation or postranslationally.

Selective loss of [3H]kainic acid and [3H]AMPA binding in layer VI of frontal cortex in Huntington's disease.

Excitatory amino acid neurotoxicity has been proposed to cause the neostriatal neuronal degeneration of Huntington's disease (HD); N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), and kainate receptors have been hypothesized to play important roles in this process. We have recently reported a loss of neurons in layer VI of the cerebral cortex in HD. Using quantitative autoradiographic methods, we have now measured NMDA, AMPA, and kainate receptor binding in the frontal cerebral cortex of the brains of controls and individuals with HD. We find no change in NMDA receptor binding but a selective decrease in kainate and AMPA receptor binding in layer VI. These data suggest that cerebral cortical neurons possessing kainate or AMPA receptors may be selectively vulnerable in individuals with HD.

Huntington's disease gene (IT15) is widely expressed in human and rat tissues.

Huntington's Disease (HD) is notable for selective neuronal vulnerability in the basal ganglia and cerebral cortex. We have investigated in human and rodent tissues the expression of the gene (IT15) whose mutation causes HD. IT15 is widely expressed, with highest levels of expression in brain, but also in lung, testis, ovary, and other tissues. Within the brain, expression is widespread with a neuronal pattern and is not enriched in the basal ganglia. Expression of IT15 is not reduced in the brain of HD patients when corrected for actin (though it is slightly decreased in the striatum when uncorrected, consistent with neuronal loss). Thus, the widespread distribution of IT15 expression does not correspond with the restricted distribution of neuropathologic changes in HD. We suggest that pathophysiology may relate to abnormal cell type-specific protein interactions of the HD protein.

Aged non-human primates: an animal model of age-associated neurodegenerative disease.

Aged non-human primates develop age-associated behavioral and brain abnormalities similar to those that occur in aged humans and, to a greater extent, in individuals with Alzheimer's disease. Declines in performance on cognitive and memory tasks begin at the monkey equivalent of late-middle life. As occurs in elderly humans, significant differences have been demonstrated in levels of performance between animals within older age groups. The brains of old monkeys show degenerative changes in neurons, abnormal axons and neurites (particularly in telencephalic areas), and deposits of amyloid in senile plaques and around blood vessels. Moreover, in some older animals, decrements occur in markers of specific neurotransmitter circuits, including the basal forebrain cholinergic system. It has been suggested that alterations in these cholinergic neurons contribute to the memory deficits that occur in older individuals. Because axotomy-induced retrograde degeneration of these neurons can be prevented by the administration of nerve growth factor, we have begun studies to determine whether administration of nerve growth factor improves performance of aged animals on memory tasks. This review describes the complementary nature of studies of non-human primates and human subjects, illustrating how these investigations can clarify factors that influence behavior and brain biology in age-associated diseases.

Nigral dopamine type-1 receptors are reduced in Huntington's disease: a postmortem autoradiographic study using [3H]SCH 23390 and correlation with [3H]forskolin binding.

Intrastriatal injection of excitatory amino acids, particularly quinolinic acid, has been proposed as an animal model of Huntington's disease. Such neurotoxic lesions of caudate-putamen result in marked dopamine type-1 (D1) receptor losses in the injected nuclei as well as in the ipsilateral substantia nigra pars reticulata. Postmortem human substantia nigra from Huntington's disease brains and from control brains were examined using in vitro autoradiography. A marked reduction in [3H]SCH 23390 binding (labeling D1 receptors) in the substantia nigra of postmortem brains of Huntington's patients was identified, thus paralleling the alterations seen in the animal models. A positive, statistically significant correlation was also encountered between D1 receptor binding (labeled by [3H]SCH 23390) and [3H]forskolin binding (which identifies adenylate cyclase, a second messenger system linked to D1 receptor activation). The results suggest that in the human--as in lower vertebrates--D1 receptors are located on striatonigral terminals and that D1 receptor loss tends to be paralleled by a reduction in adenylate cyclase. Radioactive agents selective for the D1 receptor may prove useful in future studies of Huntington's disease using positron emission tomography scanning.

Laminar organization and age-related loss of cholinergic receptors in temporal neocortex of rhesus monkey.

Using in vitro receptor autoradiography, the distributions of cholinergic muscarinic [3H-N-methyl scopolamine (NMS), 3H-pirenzepine (PZ), and 3H-oxotremorine-M (OXO-M)] and nicotinic [3H-acetylcholine (ACh)] receptors were mapped in the temporal cortices of rhesus monkeys (Macaca mulatta) ranging from 2-22 years of age. Although high-affinity 3H-PZ, low-affinity 3H-NMS binding (M1 sites) and high-affinity 3H-OXO-M, high-affinity 3H-NMS binding (M2 sites) occurred across all layers of the temporal neocortex, the laminar distribution of M1 and M2 receptor binding sites was different. M1 muscarinic receptor binding was concentrated in layers II and III, whereas M2 muscarinic receptor binding was greatest in layers IV and V. The concentration of both muscarinic (M1 and M2) and nicotinic receptor binding sites declined with increasing age, and decrements were uniform across all cortical layers. This investigation provides evidence for a decrease in cholinergic receptor binding with age in temporal cortices of rhesus monkeys. Moreover, these changes appear to precede previously reported age-associated memory deficits and neuropathological changes that occur in this species.

Bilateral changes in neocortical [3H]pirenzepine and [3H]oxotremorine-M binding following unilateral lesions of the rat nucleus basalis magnocellularis: an autoradiographic study.

Neocortical choline acetyltransferase (ChAT) activity and muscarinic [3H]pirenzepine, [3H]oxotremorine-M, [3H]N-methylscopolamine ([3H]NMS; both high- and low-affinity agonist (carbachol) sites) and nicotinic [3H]acetylcholine binding were assessed both ipsi- and contralaterally 1 week and 13 weeks after unilateral ibotenic acid lesions of the rat nucleus basalis magnocellularis (NBM). Ipsilateral ChAT activity was reduced to 49% of control values 1 week postlesion but by 13 weeks had recovered to 80% of control values. Contralateral ChAT activity did not change significantly at either 1 week or 13 weeks postlesion. At 1 week postlesion, [3H]oxotremorine-M binding was increased by 33% and 54% in ipsilateral and contralateral neocortex, respectively. By week 13, both ipsi- and contralateral [3H]oxotremorine-M binding had returned to normal but [3H]pirenzepine binding was significantly decreased by 31% and 39% in the ipsilateral and contralateral hemispheres, respectively. The binding of [3H]NMS and [3H]acetylcholine did not differ significantly from control values at either 1 week or 13 weeks postlesion. These data suggest that none of the cholinergic binding sites studied is preferentially localized presynaptically and that there may be interhemispheric regulation of neocortical cholinergic binding sites.

The neural basis of memory decline in aged monkeys.

Nonhuman primates experience changes in behavior as they progress into old age. Visual recognition, spatial learning, habit formation, and visuospatial manipulation are impaired in aged rhesus monkeys relative to young controls. We have begun to study the possible neural substrate for these changes, focusing on brain areas that are known, from lesion studies, to be essential for the successful performance of specific tasks. Aged nonhuman primates develop senile plaques, most commonly in amygdala, hippocampus, and neocortex. Our preliminary data suggest that the density of plaques may be related to poor behavioral performance in some aged monkeys. However, behavioral decline begins before the appearance of significant numbers of senile plaques, suggesting that other factors may interfere with cognition. Numerous studies of several genera have shown that receptors for neurotransmitters decline in number between the adolescent years and old age. Our autoradiographic analyses of primate temporal neocortex demonstrate loss of muscarinic, nicotinic, dopaminergic and serotoninergic receptor binding sites between the ages of 2 and 22 years. Preliminary data indicate that markers for adenyl cyclase and phosphatidyl inositol second-messenger systems also are reduced in temporal cortex. Although these declines represent a potential substrate for behavioral changes, no studies have directly related a decrease in receptor number to deficits in learning and memory in aged primates. Other changes in the aging brain include loss of neurons, reduced neurochemical markers, and decreased content of neuronal ribonucleic acid (RNA). All of these decrements may be interrelated to some extent in that decreased RNA could result in changes in neurochemical markers and receptors and, eventually, in dysfunction and death of neurons. These observations underscore the importance of establishing a time course for age-associated neural abnormalities, examining regions of brain in which changes are most likely to occur, and studying their relationship to the progression of behavioral dysfunction. Detailed anatomical analyses of the distribution of in situ uptake/receptor binding sites and messenger RNA (mRNA) in aged nonhuman primates may clarify some of the factors that most likely contribute to behavioral changes in elderly humans.

Reductions in [3H]nicotinic acetylcholine binding in Alzheimer's disease and Parkinson's disease: an autoradiographic study.

In Alzheimer's disease (AD) and Parkinson's disease (PD), dysfunction in the basal forebrain cholinergic system is accompanied by a consistent loss of presynaptic cholinergic markers in cortex, but changes in cholinergic receptor binding sites are poorly understood. In the present study, we used receptor autoradiography to map the distribution of nicotinic [3H]acetylcholine binding sites in cortices of individuals with AD and PD and matched control subjects. In both diseases, a profound loss of nicotinic receptors occurs in all cortical layers, particularly the deepest layers.