Scientific conduct: contrasts on a gray scale.
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Ethics, Professional↗
Biomedical subjects
F E Bloom
Publications and source records attributed to F E Bloom.
The Koshland years--a decade of progress.
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Administrative Personnel↗
Cytokine-mediated survival from lethal herpes simplex virus infection: role of programmed neuronal death.
The mechanisms responsible for cytokine-mediated antiviral effects are not fully understood. We approached this problem by studying the outcome of intraocular herpes simplex (HSV) infection in transgenic mice that express interferon gamma in the photoreceptor cells of the retina. These transgenic mice showed selective survival from lethal HSV-2 infection manifested in both eyes, the optic nerve, and the brain. Although transgenic mice developed greater inflammatory responses to the virus in the eyes, inflammation and viral titers in their brains were equivalent to nontransgenic mice. However, survival of transgenic mice correlated with markedly lower numbers of central neurons undergoing apoptosis. The protooncogene Bcl2 was found to be induced in the HSV-2-infected brains of transgenic mice, allowing us to speculate on its role in fostering neuronal survival in this model. These observations imply a complex interaction between cytokine, virus, and host cellular factors. Our results suggest a cytokine-regulated salvage pathway that allows for survival of infected neurons.
Animals↗
Rapid induction of tumor necrosis factor alpha in the cerebrospinal fluid after intracerebroventricular injection of lipopolysaccharide revealed by a sensitive capture immuno-PCR assay.
Tumor necrosis factor alpha (TNF-alpha) is an important mediator in many pathophysiologic processes, both in the central nervous system (CNS) and in the periphery. For this study, we have designed a very sensitive immuno-PCR detection system to investigate the time course of TNF-alpha induction in the rat cerebrospinal fluid after intracerebroventricular administration of bacterial lipopolysaccharide (LPS). Immuno-PCR combines antibody specificity with PCR signal amplification and provides a sensitivity in the picomolar range. The enhanced sensitivity of this assay allowed the detection of TNF-alpha in the cerebrospinal fluid as early as 15 min after intracerebroventricular administration of LPS. The present results suggest that the ventricular compartment of the CNS, although confined within the blood-brain barrier, is highly responsive to proinflammatory stimuli such as LPS administration. Insight into the molecular mechanisms underlying this compartmentalization could be key to the pathology and treatment of many CNS diseases, especially the meningitides.
Animals↗
Feline immunodeficiency virus as a model for study of lentivirus infection of the central nervous system.
Feline immunodeficiency virus infects the CNS and results in predictable pathophysiology strikingly similar to that seen with HIV-1 infection of humans. The observed pathophysiology is mimicked in several physiologically assessed modalities, further supporting the validity of the feline model. Peripheral and control evoked potential findings and the occurrence of the sleep architecture changes in both cat and human disease provide an intriguing focus for further investigation. Although structurally diverse in an absolute sense, FIV and HIV-1 share basic structural features and commonalities of their life cycle. It is likely that by understanding the common mechanisms by which these lentiviruses influence CNS function, a more complete understanding of the neurological deficits seen in HIV-1 infected patients will be obtained. The cat model is particularly valuable for study of CNS disease, since it allows detailed analyses of events during the acute phase of infection, under circumstances in which the nature and timing of the infection are carefully controlled. The availability of molecular clones for mutational analysis will facilitate mapping of genomic regions critical to the perturbation of CNS function. It is suggested that development of intervention strategies in the cat model will yield treatment modalities directly applicable to HIV-1 infection of humans.
AIDS Dementia Complex↗
Neuroscience-knowledge management: slow change so far.
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Database Management Systems↗
Rapid assay of phage-derived recombinant human fabs as bispecific antibodies.
Specific anti-tumor and anti-viral activities can be conferred on lymphocytic and myeloid effector cells by retargeting them with bispecific antibodies. These are antibodies which possess an anti-target binding region and a region capable of binding specific effector cell surface markers. For the rapid evaluation of recombinant human Fabs as bispecific antibodies, we have constructed a vector that allows for the conversion of Fabs into protein A fusion proteins. These can be used to generate bispecific antibodies when complexed to appropriate anti-effector cell immunoglobulins. As a model system, a protein A fusion derivative of a human recombinant anti-herpes simplex virus (HSV) Fab was constructed and complexed to OKT3, a T cell-activating antibody specific for CD3. This complex reduced HSV-2 yields in infected cells by about three logs relative to controls when incubated on HSV-2-infected cell monolayers in the presence of IL-2-activated lymphocytes. The system described allows for the rapid evaluation of recombinant human Fabs as bispecific antibodies for therapeutic applications. In addition, Fab-protein A fusion proteins can be used in ELISA and other immuno-assays with increased sensitivity.
Antibodies, Bispecific↗
Exposure of neonatal rats to alcohol by vapor inhalation demonstrates specificity of microcephaly and Purkinje cell loss but not astrogliosis.
The artificial rearing model (AR) of fetal alcohol syndrome (FAS) has been shown to induce several major pathologies in the early postnatal rat brain development: microcephaly, selective neuronal cell loss, and activation of astroglia in the neocortex. The purpose of this study was to determine whether these pathologies were specific to the action of alcohol or, in contrast, could result from confounds attributed to this model of FAS. For this purpose, the pathological effects of AR were compared with those of a vapor inhalation (VI) model of FAS. Our studies showed that the microcephaly that developed after exposure to periodic blood alcohol levels (BALs) of 300-350 mg% during postnatal days 4-9 could be achieved by both AR and IV models of FAS, and thus is independent of the method of alcohol administration. In contrast, the gliosis measured by glial fibrillary acidic protein (GFAP) mRNA levels in cortex, as well as by immunohistochemical staining for GFAP, was found only in the AR-FAS model, but not in the VI model. However, the lack of gliosis in VI was apparently not due to a less intrusive intervention of alcohol, because VI exposure resulted in a reduction in Purkinje cell number comparable with that found after AR or intragastric intubation of alcohol. Based on these observations, we conclude that the activation of gliosis observed after AR is not a specific effect of alcohol, but rather is caused by an interaction of alcohol with as yet unidentified factors present in AR.
Administration, Inhalation↗
Enhanced sensitivity for light and electron microscopic in situ hybridization with multiple simultaneous non-radioactive oligodeoxynucleotide probes.
Although oligonucleotide probes are useful for in situ hybridization, their low sensitivity compared to riboprobes and cDNA remains a problem. We have systematically examined the protocols to provide a general procedure that increases the sensitivity of oligoprobes for light and electron in situ hybridizations by using mixtures of multiple non-overlapping oligonucleotides (multi-oligoprobes). The protocol achieves these improvements with both radioactive and non-radioactive oligoprobes. With 33P-labeled probes in a semiquantitative assay, we found that mixtures of up to six vasopressin-directed multi-oligoprobes, each employed at saturating concentration, led to an additive signal with no significant increase of the background. Using this approach with non-radioactive oligoprobes, we were able to detect in the hypothalamus several low or moderately abundant mRNAs, such as vasopressin heterogeneous nuclear RNA and the galanin, dynorphin, and tyrosine hydroxylase mRNAs. Moreover, we showed that multi-oligoprobes used in a pre-embedding procedure were suitable for studying the ultrastructural compartmentalization of moderately abundant mRNAs. Finally, with the same basic approach we demonstrated that two sets of multi-oligoprobes can be combined for simultaneous detection of two different mRNAs using fluorescent dyes, making this approach suitable for high-resolution confocal analyses. Overall, our data demonstrate that multi-oligoprobes provide a sensitive tool of choice for various applications in which both well-preserved morphology and high sensitivity are needed. In particular, these probes appear ideal for study of the comparative subcellular localization of mRNAs at both the light and the electron microscopic level.
Animals↗
Tumor necrosis factor-alpha and interleukin-1 alpha enhance glucose utilization by astrocytes: involvement of phospholipase A2.
Cytokines can be produced within the nervous system by various cell types, including astrocytes, which secrete them in response to pathological processes such as viral infections. Astrocytes are known to play an important role in the homeostasis of the nervous system, in particular, by contributing to the regulation of local energy metabolism. We report that tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 alpha (IL-1 alpha) markedly stimulate glucose uptake and phosphorylation in primary cultures of neonatal murine astrocytes, as determined with [3H]-2-deoxyglucose ([3H]2DG). This effect is both concentration dependent, with observed EC50 values of 8 ng/ml for TNF-alpha and 30 pg/ml for IL-1 alpha, and time dependent, with a maximal response observed 24 hr after cytokine application. The effects of TNF-alpha and IL-1 alpha on glucose uptake and phosphorylation appear to be mediated by the phospholipase A2 signal transduction pathway. Evidence in support of this includes (i) inhibition by mepacrine, a phospholipase A2 inhibitor, of [3H]2DG uptake evoked by TNF-alpha and IL-1 alpha, and (ii) stimulation of [3H]arachidonic acid release by TNF-alpha and IL-1 alpha. Protein kinase C activation does not appear to be involved as the specific protein kinase C inhibitor Ro 31-7549 does not abolish TNF-alpha- or IL-1 alpha-induced increase in [3H]2DG uptake and phosphorylation. The additional glucose imported by astrocytes on exposure to TNF-alpha and IL-1 alpha is neither stored as glycogen nor released as glycolytically derived lactate, suggesting that it is processed through the tricarboxylic acid cycle or pentose phosphate pathway. These results demonstrate that TNF-alpha and IL-1 alpha can fundamentally perturb the energy metabolism of astrocytes, possibly impairing their ability to provide adequate energy substrates for neurons.
Animals↗
Alcohol selectively attenuates stress-induced c-fos expression in rat hippocampus.
The ability of ethyl alcohol to modify responses to stress has been well documented (cf. Pohorecky, 1990). However, the structural substrate mediating these effects of alcohol remains undefined. Using immediate early gene (IEG) expression in the brain as a marker of altered neuronal response, we investigated the effect of acute alcohol exposure on the activity of brain regions of rats exposed to 15 min of restraint stress. Immunocytochemical localization c-Fos protein demonstrated that restraint stress led to an induction of c-Fos expression in several brain structures including cingulate and piriform cortex, cortico-amygdaloid and hippocampo-amygdaloid transition zones, hippocampus, hypothalamus, supramammillary nucleus, and centromedial nucleus of thalamus. An intraperitoneal injection of 2 g/kg alcohol prior to stress decreased c-Fos expression in several but not all of these structures. In particular, alcohol strongly attenuated the stress-induced expression of c-Fos in hippocampus and cingulate cortex. Using slot-blot hybridization, significant induction of c-fos mRNA after restraint stress was demonstrated both in hippocampus and cortex, but prior alcohol exposure specifically attenuated c-fos induction only in the hippocampus. The response of c-fos mRNA expression to stress and alcohol differed from the effects on jun-B, c-jun and jun-D mRNA levels. Perhaps surprisingly, acute exposure to alcohol in otherwise unstressed rats did not induce significant changes in expression of IEGs in comparison to control (saline-injected) animals even with doses sufficient to elevate plasma corticosterone. In summary, these studies demonstrate a selective sensitivity of stress-induced activity of neurons of hippocampus and cingulate cortex to acute alcohol exposure.
Animals↗
Interleukin-2 (IL-2) induces corticotropin-releasing factor (CRF) release from the amygdala and involves a nitric oxide-mediated signaling; comparison with the hypothalamic response.
Interleukin-2 (IL-2)-like immunoreactivity and IL-2 receptor immunoreactivity have been reported in different brain regions, under normal and pathophysiological conditions. IL-2 stimulates hypothalamic corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) release and that of pituitary adrenocorticotropin. The amygdala, known to contain high levels of CRF, is involved in stress-related reactions, including regulation of the hypothalamo-pituitary-adrenal axis. IL-2 will release AVP from both the hypothalamus and the amygdala, which further supports a role for cytokine effects in the amygdala in neuroimmune interactions. In the present study, we compared the effects of IL-2, acetylcholine and norepinephrine on the in vitro release of CRF from the amygdala or hypothalamus. In addition, we used these release systems to evaluate the possible involvement of nitric oxide (NO)-mediated signaling in CRF release. IL-2 stimulates CRF release in both regions, in a calcium- and dose-dependent manner. Nitroprusside, an NO generator, also induces CRF release. This IL-2-induced CRF release is antagonized by Ng-methyl-L-arginine and hemoglobin, known NO antagonists. Finally, norepinephrine and acetylcholine induce CRF release. The norepinephrine-induced CRF release is antagonized by phentolamine and propanolol and the acetylcholine-induced release by atropine and mecamylamine, which suggests the involvement of both alpha and beta adrenergic receptors and both muscarinic and nicotinic receptors. The acetylcholine-induced CRF release is antagonized by Ng-methyl-L-arginine, but the norepinephrine-induced response is not. These data support the suggestion that the amygdala may participate in communications between the neuroendocrine and immune systems.
Acetylcholine↗
Sleep patterns are disturbed in cats infected with feline immunodeficiency virus.
Human immunodeficiency virus (HIV)-related sleep disturbances have been reported early in AIDS. Likewise, the feline immunodeficiency virus (FIV), a natural lentivirus pathogen of cats, produces a similar immunodeficiency syndrome with neurological sequelae. To identify the neurophysiological substrate of FIV infection in brain, pathogen-free cats were infected with the Maryland strain of FIV. Eight weeks after inoculation, all FIV-infected cats seroconverted and virus was detected in the cerebrospinal fluid and in the mononuclear cells of peripheral blood. Ten to 12 months after the FIV inoculation, inoculated and control cats were surgically implanted with electrodes to record the sleep/wake cycle. These sleep recordings were obtained under conditions controlling for environmental variables and instrumental adaptation. FIV-infected cats spent 50% more time awake than the sham-inoculated controls and exhibited many more sleep/waking stage shifts--i.e., 40% more than controls. In addition, FIV-infected cats showed approximately 30% of rapid eye movement (REM) sleep reduction compared to controls. The latency to sleep and REM sleep onset was also significantly delayed in FIV-infected cats. In addition, a remarkable increase in cortically recorded spindle activity (8-13 Hz) was observed during slow-wave sleep in some infected subjects, similar to changes described in HIV-infected humans. Moreover, infected cats exhibited no overt signs of systemic morbidity, such as hyperpyrexia or body weight loss. These results indicate that FIV-infected cats exhibit sleep abnormalities similar to the sleep disturbances previously described in AIDS patients and further support the feline preparation as a valuable animal model of HIV infection of the central nervous system.
Animals↗
Four structurally distinct neuron-specific olfactomedin-related glycoproteins produced by differential promoter utilization and alternative mRNA splicing from a single gene.
Four structurally related neuron-specific 1B426b mRNAs, designated AMY, BMY, AMZ, and BMZ, have been isolated from rat brain cDNA libraries. The four mRNAs are related to one another by their shared M region and by two pairs of alternative 5' (A, B) or 3' (Y, Z) regions. All four possible combinations were detected. The four transcripts are derived by differential promoter utilization (to generate A or B 5' ends) and alternative splicing (to generate Y or Z 3' ends) of the primary transcripts of the single D2Sutle gene. All four mRNAs were detected in most brain regions, but were enriched within the cortex and hippocampus. In the pituitary only the two A-type and in the adrenal glands only the two B-type mRNAs were detected. In situ hybridization shows a highly heterogeneous distribution across brain regions, paralleling the Northern blot results and additionally identifying the reactive cells as neurons. The cDNAs encode related glycoproteins of 125, 153, 457, and 485 amino acids, which have been detected immunochemically. The AMZ and BMZ proteins show significant sequence similarity with olfactomedin, an extracellular matrix protein of bullfrog olfactory epithelium, suggesting the possibility of a matrix-related function for these rat glycoproteins in neurons and neurosecretory cells.
Animals↗
Sensory maps in the human brain.
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Adaptation, Physiological↗
Noninvasive detection of cerebral plasticity in adult human somatosensory cortex.
After upper limb deafferentation, adult macaques exhibit substantial reorganization of cortical somatosensory topography with enlargement of cortical areas responsive to facial stimuli. In the present study non-invasive magnetic source imaging technology has been used to map in detail the bilateral somatosensory homunculi in four neurologically normal controls and two upper arm amputees. Bilateral homuncular maps of normals and of the unaffected hemisphere of both amputees showed a wide hand area. The affected hemisphere of both amputees showed marked intrusion of facial representations into the digit and hand area consistent with the earlier observations in macaques. Our findings provide the first evidence of massive somatosensory plasticity in human adults and suggest a mechanism for post-amputation perceptual changes.
Adolescent↗
Recombinant human Fab to glycoprotein D neutralizes infectivity and prevents cell-to-cell transmission of herpes simplex viruses 1 and 2 in vitro.
Herpes simplex viruses 1 and 2 (HSV-1 and -2) are associated with a number of conditions of varying severity, which are only partially responsive to current therapies. Human antibodies to the viruses offer a potential alternative. We describe here the generation of panels of human monoclonal Fab fragments to HSV-1 and -2 by panning a phage display combinatorial antibody library against whole lysates from the two viruses. Each lysate selected a largely distinct set of Fabs, although all of the Fabs were cross-reactive with both viruses. In a plaque-reduction assay, one Fab neutralized HSV-1 at 0.25 microgram/ml (50% reduction) and HSV-2 at 0.05 microgram/ml. This Fab also inhibited plaque formation when applied to virus-infected monolayers, completely abolishing HSV-2 plaque development at 25 micrograms/ml 72 hr postinfection, indicating the ability of the Fab to prevent cell-to-cell spread of virus. The Fab was shown to recognize viral glycoprotein D and to neutralize virus primarily by a postattachment mechanism. Recombinant Fabs may be useful for topical administration, although whole antibody will probably be required for systemic use.
Amino Acid Sequence↗
Detection and regulation of tyrosine hydroxylase mRNA in catecholaminergic terminal fields: possible axonal compartmentalization.
Reverse transcriptase coupled with nested polymerase chain reaction amplification (RT/nested-PCR) was used to detect mRNA encoding tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, in adult rat cerebellum, striatum, and pituitary neurointermediate lobe (NIL). These regions receive catecholaminergic innervation from the locus coeruleus, substantia nigra, and arcuate and periventricular nuclei of the hypothalamus, respectively, but are devoid of catecholamine-synthesizing cells. The RT/nested-PCR products, which were generated using primers located on different exons of the tyrosine hydroxylase gene, indicate that the tyrosine hydroxylase mRNA detected is devoid of introns and, hence, is processed. These findings raise the possibility that tyrosine hydroxylase mRNA may be axonally transported. Using the same RT/nested-PCR protocol, we were unable to detect mRNA encoding dopamine beta-hydroxylase, a different catecholaminergic biosynthetic enzyme, in either cerebellum, striatum, or NIL pituitary tissue. Thus, the detection of tyrosine hydroxylase mRNA in catecholamine terminal regions is biochemically specific. We were unable to detect tyrosine hydroxylase mRNA in optic nerve, indicating some degree of anatomical specificity as well. Expression of tyrosine hydroxylase mRNA in the cerebellum was markedly increased by subcutaneous administration of the catecholamine-depleting agent, reserpine, suggesting that tyrosine hydroxylase mRNA in catecholamine terminal regions may be functionally important. This finding also indirectly supports the hypothesis that tyrosine hydroxylase mRNA can be axonally transported since the ability of reserpine to induce expression of this transcript in conventional catecholamine cell groups is considered secondary to catecholamine depletion, and cerebellar cells do not synthesize catecholamines. Finally, lesions of the nigrostriatal pathway significantly decreased levels of tyrosine hydroxylase mRNA in the striatum, providing strong additional support for this hypothesis.
Animals↗