Faculty Bibliography

To evaluate the safety and efficacy of passive immunotherapy for advanced human immunodeficiency virus (HIV) infection, a randomized, double-blind, controlled trial of human anti-HIV hyperimmune plasma was conducted. Sixty-three subjects with stage IV HIV disease (AIDS) were randomized to received 250 mL of either HIV-immune plasma or HIV antibody-negative plasma every 4 weeks. Although nonsignificant trends toward improved survival and delayed occurrence of a new opportunistic infection were noted, no significant effects on absolute CD4 lymphocyte counts or quantitative HIV viremia were seen. The only notable toxicity was the allergenicity to be expected from infusing plasma products, usually manifesting as urticaria. Thus, results do not rule out the potential usefulness of passive immunization with different preparations, but did fail to demonstrate clinical benefit of the product studied.

It is has been shown that the major afferent input to the dentate gyrus, the perforant path, excites dentate hilar neurons. However, little is known about the other inputs to hilar cells. Therefore, we examined the responses of hilar neurons to stimulation of the fimbria. We positioned our stimulating electrodes so that granule cells were not excited antidromically by fimbria stimulation, although action potentials were easily triggered in area CA3b and CA3c pyramidal cells by such stimulation. In these experiments, fimbria stimulation evoked responses from every hilar cell tested, including examples of both of the major cell types, the spiny hilar 'mossy' cells (n = 15) and the relatively aspiny, 'fast-spiking' cells (putative interneurons, n = 5). Hilar cell responses consisted primarily of EPSPs that could trigger action potentials, but small IPSPs were also evoked in some cases, particularly in the fast-spiking cells. Excitation was blocked by an antagonist of the AMPA/kainate receptor subtype of excitatory amino acid receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 5 microM, n = 5), whereas the cholinergic antagonist atropine (10 microM) had no effect (n = 4). When sequential intracellular recordings were made from hilar cells and area CA3 pyramidal cells in the same slice, hilar cell EPSPs began after action potentials of CA3b pyramidal cells, and stimulus strengths required to evoke hilar cell EPSPs were above threshold for area CA3b pyramidal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The lysosomal system has often been considered a prominent morphologic marker of distressed or dying neurons. Lysosomes or their constituent hydrolases have been viewed in different neuropathologic states as either initiators and direct agents of cell death, agents of cellular repair and recompensation, effectors of end-stage cellular dissolution, or autolytic scavengers of cellular debris. Limited data and limitations of methodology often do not allow these potential roles to be discriminated. In all forms of neurodegeneration, it may be presumed that lysosomes ultimately rupture and release various hydrolases that promote cell autolysis during the final stages of cellular disintegration. Beyond this perhaps universal contribution to cell death, the degree to which the lysosomal system may be involved in neurodegenerative states varies considerably. In many conditions, morphologic evidence for activation of the lysosomal system is minimal or undetectable. In other cases, lysosomal activation is evident only when other morphologic signs of cell injury are also present. This level of participation may be viewed as either an attempt by the neuron to compensate for or repair the injury or a late-stage event leading to cell dissolution. The early involvement of the lysosomal system in neurodegeneration occurs most commonly in the form of intraneuronal accumulations of abnormal storage profiles or residual bodies (tertiary lysosomes). Very often the lysosomal involvement can be traced to a primary defect or dysfunction of lysosomal components or to accelerated or abnormal membrane breakdown that leads to the buildup of modified digestion-resistant substrates within lysosomes. Because they are often striking, changes in the lysosomal system are a sensitive morphologic indicator of certain types of metabolic distress; however, whether they reflect a salutary response of a compromised neuron or a mechanism to promote cell death and removal of debris from the brain remains to be established for most conditions. Factors that may influence the lysosomal response during lethal neuronal injury include species differences, stage of neuronal development, duration of injury and pace of cell death. The lysosomal system may be more closely coupled to certain forms of neuronal cell death in lower vertebrate or invertebrate systems than in mammalian systems

Calcium-activated neutral proteinases (CANPs or calpains) are believed to be key enzymes in intracellular signaling cascades and potential mediators of calcium-induced neuronal degeneration. To investigate their involvement in Alzheimer disease, we identified three isoforms of muCANP (calpain I) in human postmortem brain corresponding to an 80-kDa precursor and two autolytically activated isoforms (78 and 76 kDa). As an index of changes in the in vivo activity of muCANP in Alzheimer disease, the ratio of the 76-kDa activated isoform of muCANP to its 80-kDa precursor was measured by immunoassay in selected brain regions from 22 individuals with Alzheimer disease and 18 normal controls. This muCANP activation ratio was elevated 3-fold in the prefrontal cortex from patients with Alzheimer disease but not from patients with Huntington disease. The activation ratio was also significantly elevated, but to a lesser degree, in brain regions where Alzheimer pathology is milder and has not led to overt neuronal degeneration. These findings indicate that muCANP activation is not simply a consequence of cellular degeneration but may be associated with dysfunction in many neurons before gross structural changes occur. The known influences of CANPs on cytoskeleton and membrane dynamics imply that persistent CANP activation may contribute to neurofibrillary pathology and abnormal amyloid precursor protein processing prior to causing synapse loss or cell death in the most vulnerable neuronal populations. Pharmacological modulation of the CANP system may merit consideration as a potential therapeutic strategy in Alzheimer disease

A major goal in assessing biological determinants of behavior lies in studying the effect(s) of rearing on the development of the central nervous system. Specifically, a series of neuroanatomic analyses have been undertaken to identify potential neuropathological changes seen in monkeys exposed to early social deprivation, which leads to profound psychopathology and inappropriate responses to stress. The animals used in this study were either raised with their mother and peers (socially reared) or raised without maternal/peer contact (socially deprived). Within this context, the distribution of tuberoinfundibular dopaminergic neurons in the hypothalamic paraventricular nucleus and arcuate nucleus of rhesus monkeys was determined by immunohistochemistry using an antibody against the enzyme tyrosine hydroxylase, a marker for dopamine-containing systems. Additionally, the distribution of corticotropin-releasing factor-containing neurons in the paraventricular nucleus was assessed immunohistochemically. The majority (97.5%) of dopaminergic neurons in the paraventricular nucleus were parvicellular, with a small (2.5%), but consistently observed population of magnocellular neurons immunoreactive for tyrosine hydroxylase. Within the arcuate nucleus, tyrosine hydroxylase-immunoreactive neurons were similar in morphology to the parvicellular neurons of the paraventricular nucleus. Qualitative assessment of corticotropin-releasing factor-immunoreactive neurons in the paraventricular nucleus revealed a parvicellular population of neurons located in medial aspects of the nucleus, similar to what has been observed in the rat. Quantitative analysis revealed no differences in the number of tyrosine hydroxylase- and corticotropin-releasing factor-immunoreactive neurons between rearing conditions, suggesting that these neurons were not affected, in terms of overall cell counts, by the early environmental insult of social deprivation.

Eleven patients with senile dementia of the Alzheimer type and 11 age-matched control subjects were given the thyrotropin-releasing hormone (TRH) test. The two groups did not differ with respect to peak thyrotropin (TSH) response or TSH levels at baseline, 20, 30, and 45 min after TRH injection. There were significant differences between the groups on Hamilton Depression Rating Scale scores (p < 0.03), although neither group met clinical criteria for depression. Items that were significantly different pertained to depressed mood, loss of interest, loss of insight, suicidal ideation, and obsessional symptoms

We investigated the relative inhibition of purified human mu CANP and mCANP by five cysteine proteinase inhibitors including N-acetyl-Leu-Leu-nor-leucinal (C-I) and N-acetyl-Leu-Leu-methioninal (C-II), calpeptin, E64, and leupeptin. Based on IC50 measurements, calpeptin and C-I were stronger inhibitors by one to two orders of magnitude than C-II, leupeptin or E64. None of the five inhibitors, however, exhibited greater specificity for human mu CANP or mCANP. These results indicate that, although the inhibition of a given cellular event by these compounds may suggest CANP involvement, effects on mu CANP cannot be discriminated from those on mCANP

The distribution of noradrenergic processes within the hypothalamus of rhesus monkeys (Macaca mulatta) was examined by immunohistochemistry with an antibody against dopamine-beta-hydroxylase. The results revealed that the pattern of dopamine-beta-hydroxylase immunoreactivity varied systematically throughout the rhesus monkey hypothalamus. Extremely high densities of dopamine-beta-hydroxylase-immunoreactive processes were observed in the paraventricular and supraoptic nuclei, while relatively lower levels were found in the arcuate and dorsomedial nuclei and in the medial preoptic, perifornical, and suprachiasmatic areas. Moderate levels of dopamine-beta-hydroxylase immunoreactivity were found throughout the lateral hypothalamic area and in the internal lamina of the median eminence. Very few immunoreactive processes were found in the ventromedial nucleus or in the mammillary complex. Other midline diencephalic structures were found to have high densities of dopamine-beta-hydroxylase immunoreactivity, including the paraventricular nucleus of the thalamus and a discrete subregion of nucleus reuniens, the magnocellular subfascicular nucleus. A moderate density of dopamine-beta-hydroxylase immunoreactive processes were found in the rhomboid nucleus and zona incerta whereas little dopamine-beta-hydroxylase immunoreactivity was found in the fields of Forel, nucleus reuniens, or subthalamic nucleus. The differential distribution of dopamine-beta-hydroxylase-immunoreactive processes may reflect a potential role of norepinephrine as a regulator of a variety of functions associated with the nuclei that are most heavily innervated, e.g., neuroendocrine release from the paraventricular and supraoptic nuclei, and gonadotropin release from the medial preoptic area and mediobasal hypothalamus. Additionally, quantitative analysis of dopamine-beta-hydroxylase-immunoreactive varicosities was performed on a laser scanning microscope in both magnocellular and parvicellular regions of the paraventricular nucleus of the hypothalamus. The methodology employed in this study allowed for the high resolution of immunoreactive profiles through the volume of tissue being analyzed, and was more accurate than conventional light microscopy in terms of varicosity quantification. Quantitatively, a significant difference in the density of dopamine-beta-hydroxylase-immunoreactive varicosities was found between magnocellular and parvicellular regions, suggesting that parvicellular neurons received a denser noradrenergic input. These differential patterns may reflect an important functional role for norepinephrine in the regulation of anterior pituitary secretion through the hypothalamic-pituitary-adrenal stress axis.

We have assessed the properties of three intracellular markers, horseradish peroxidase, biocytin/Neurobiotin, and Lucifer Yellow, and have compared their usefulness as neuronal markers for light and electron microscopic visualization. Neurons in the acute slice preparation of rat hippocampus were filled with one of these markers, and the marker was converted to an optical and electron-dense reaction product. Dimethylsulfoxide (DMSO) greatly facilitated penetration of recognition reagents while preserving membrane integrity. The markers were compared with respect to injection parameters, mobility and recognition, stability and visibility, and ultrastructural clarity. Horseradish peroxidase (HRP)-labeled neurons, recognized histochemically with diaminobenzedine (DAB), were easily visualized by the density of the DAB reaction product; however, the electron density was often so great as to obscure ultrastructural details. Biocytin (BC)-/Neurobiotin (NB)-labeled neurons were recognized by avidin-HRP, followed by histochemical localization of HRP with DAB. The optically dense reaction product gave complete visualization of the soma and processes at the light microscopic level. The electron density was homogeneously distributed throughout the cell, so that ultrastructural features were easily identified. Lucifer Yellow (LY), a fluorescent marker, was converted to an optical and electron-dense reaction product via immunocytochemical staining with a rabbit anti-LY antibody, followed by goat anti-rabbit IgG-HRP and DAB histochemical localization. Similar to BC/NB, the reaction product was evenly dispersed, providing good light microscopic and ultrastructural clarity. Under our experimental conditions, BC/NB and LY were superior markers that could be used routinely to label neurons, and give excellent visualization not only at the light but also at the electron microscopic level

A decrease in protein kinase C activity caused either by treatment with inhibitors, such as staurosporine or H-7, or by prolonged exposure to phorbol diesters has been proposed to be involved in the early events of SH-SY5Y neuroblastoma cell differentiation. Because eight distinct isoforms of protein kinase C with discrete subcellular and tissue distributions have been described, we determined which isoforms are present in SH-SY5Y cells and studied their modifications during differentiation. The alpha, beta 1, delta, and epsilon isoforms were present in SH-SY5Y cells, as well as in rat brain. Protein kinase C-alpha and -beta 1 were the most abundant isoforms in SH-SY5Y cells, and immunoreactive protein kinase C-delta and -epsilon were present in much smaller amounts than in rat brain. Subcellular fractionation and immunocytochemistry demonstrated that all four isoforms are distributed bimodally in the cytoplasm and the membranes. Immunocytochemical analysis showed that the alpha isoform is associated predominantly with the plasma membrane and the processes extended during treatment with 12-tetradecanoyl-13-acetyl-beta-phorbol or staurosporine, and that protein kinase C-epsilon is predominantly membrane-bound. Its localization did not change during differentiation. Western blots of total SH-SY5Y cell extracts and of subcellular fractions probed with isoform-specific polyclonal antibodies showed that when SH-SY5Y cells acquired a morphologically differentiated phenotype, protein kinase C-alpha and -epsilon decreased, and protein kinase C-beta 1 did not change. These data suggest distinct roles for the different protein kinase C isoforms during neuronal differentiation, as well as possible involvement of protein kinase alpha and epsilon in neuritogenesis