Lawrence F. Eng

Heterogeneity of Genotypic and phenotypic characteristics of fifteen permanent cell lines derived from human gliomas.

Six new permanent cell lines were established from human gliomas and compared to nine other cell lines from human gliomas. All fifteen lines had individually distinct HLA phenotypes and all but two, which were from a black patient, had type B glucose-6-phosphate-dehydrogenase isoenzymes. Morphologically, the lines could be classified into four patterns descriptively designated as fibroblastic, fascicular, epithelial, or glial. Four of the lines grew progressively and could be serially transplanted when injected into athymic mice; two others grew initially and then regressed. From none to 100% of cells developed elongated tapering processes and showed reduction in nuclear-cytoplasmic ratio in the presence of 1 miu cyclic AMP and theophylline. Levels of 2-3 cyclic nucleotide 3-phosphohydrolase activity ranged from nondetectable to 12.78 ± 1.49 μmoles 2 AMP formed per hr per mgm total protein. None of the lines had detectable S-100 protein, but two had readily demonstrable glial fibrillary acidic protein in indirect immunofluorescence. Fibronectin levels in spent culture supernatanls ranged from undetectable levels to 21.4 μg/ml/105 cells. All but one line shared surface antigens with normal human adult or fetal brain, as detected in absorption analyses with nonhuman primate antiserum raised against glioblastoma multiforme tissue or cell line U-251 MG. Although there were many common properties of the lines, each line had a unique profile of the parameters evaluated. This heterogeneity most likely reflects the individuality of the tumors of origin and individual genotypes and capacity for a range of phenotypic expression of the cells

Reaction of Lewy bodies with antibodies to phosphorylated and non-phosphorylated neurofilaments

The reaction of Lewy bodies in the substantia nigra and the sympathetic ganglia was studied in 10 cases of Parkinsons disease and other Lewy body-related conditions, using monoclonal antibodies to non-phosphorylated (02-40) and phosphorylated (03-44 and 07-5) neurofilaments. Although many Lewy bodies were unstained, a peripheral ring of positive immunoreactivity was frequently observed. This reaction was more common and more intense with antibodies to phosphorylated neurofilaments, suggesting a posttranslational phosphorylation of elements in the Lewy body. Although Lewy bodies appear to have some antigenic sites in common with neurofilaments, certain differences between neurofilaments and Lewy body filaments are emphasized.

Immunohistochemistry of Nervous System-Specific Antigens

In recent years, immunohistochemical detection and localization of specific antigens in tissue sections have had important clinical and experimental applications. Morphological information derived from these studies combined with biochemical data is proving to be an extremely valuable means for studying structural and functional components of tissue. Biochemical data describing brain antigens have prompted many immunohistochemical studies attempting to localize these factors. Whereas most studies have employed the immunofluorescence technique, the increasing requirement for high-resolution localization offered by electron microscopy (EM) necessitates the use of immunoperoxidase methods Immunocytochemical localization of brain antigens is lending a great deal to our understanding of brain morphology, organization, and function. Many but not all of the nervous system antigens being studied by immunohistochemical techniques have been cited in this chapter. The authors have attempted to concentrate on areas of most recent advances and of broad general interest.

Cytokine chemokine expression in contused rat spinal cord

Spinal cord injury within the first few hours, is complicated by inflammatory mechanisms, including the influx of monocyte/macrophages as well as the activation of resident spinal microglia and astrocytes. Numerous studies have suggested that the initial infiltration of the hematogenous cells may be due to the secretion of cytokines and chemokines in the injured CNS. In order to elucidate which chemotactic factors may be expressed following traumatic spinal cord contusion, the presence of mRNA for a number of cytokines, chemokines and growth factors was examined in contused rat spinal cord by reverse transcriptase-polymerase chain reaction and immunohistochemistry. Spinal injury was accompanied by an increase in glial fibrillary acidic protein mRNA suggesting astrocyte activation and astrogliosis. TNFalpha message levels were upregulated as early as 1 h post injury and returned to baseline levels by 3 days post injury (DPI). By immunocytochemistry, staining for TNFalpha increased at 1 and 3 dpi and was predominantly diffuse in the necrotic tissue. The chemokines IP-10, MCP-1, and MIP-1alpha were also detected in the injured spinal cord. mRNA levels of IP-10 peaked around 6 h post injury and were upregulated up to 7 dpi. MCP-1 mRNA was detected at 1 h post injury and its levels returned to baseline by 14 dpi. An increase in MCP-1 staining was observed from 1 to 7 dpi. The staining was also diffuse in the necrotic tissue and also localized to cells near the site of injury. The presence of aFGF and bFGF was also detected in the injured spinal cord. mRNA for aFGF was detected at 0 time, increased at 6 h post injury, peaked at 3 days, and remained elevated up to 21 days. bFGF mRNA was initially detected at 1 h post injury, increased between 6 h and 3 days, declined thereafter and returned to baseline levels by 21 days.

Immunocytochemical studies of substance P and met-enkephalin in the basal ganglia and substantia nigra in huntington’s, parkinson’s and alzheimer’s diseases

Immunocytochemical studies of the distribution and intensity of Substance P and Met-enkephalin staining in the basal ganglia and substantia nigra were carried out in five cases each of brains from patients with Huntingtons disease, Parkinsons disease, Alzheimers disease, and normal controls. The usefulness of the peroxidase-antiperoxidase method for human autopsy material was confirmed. Substance P and Met-enkephalin fibers were distributed in essentially the same pattern as described in experimental animals and in human brains. In Huntingtons disease brains decreased Substance P staining was found in the internal globus pallidus and the substantia nigra, in agreement with radioimmunoassay studies by others. Met-enkephalin staining in the external globus pallidus was of normal intensity, although present within a shrunken area. In Parkinsons and Alzheimers diseases there was intense immunoreactivity for Substance P in the globus pallidus and substantia nigra, and for Met-enkephalin in the globus pallidus, at variance with reported decreases in Parkinsons disease by radioimmunoassay, but in essential agreement with other immunocytochemical studies. Immunocytochemical methods complement radioimmunoassays of human brain and may help in mapping neuropeptidergic pathways and in pinpointing abnormalities in these pathways in basal ganglia disorders.

Chemokine antagonist infusion attenuates cellular infiltration following spinal cord contusion injury in rat.

Spinal cord injury is accompanied by an initial inflammatory reaction followed by secondary injury that is caused, in part, by apoptosis. Recruitment of leukocytes from the blood compartment to the site of inflammation in the injured spinal cord has been attributed to locally generated chemotactic agents (cytokines and chemokines). In addition to upregulation in the message levels of a number of chemokines, we have found up‐regulation in the message levels of several chemokine receptors following spinal cord contusion injury. To reduce the inflammatory response after spinal cord injury, we have blocked the interaction of chemokine receptors with their ligands using the vMIPII chemokine antagonist. Using a rat model of spinal cord contusion injury, we show that continuous infusion of the antagonist for up to 7 days results in a decrease in infiltrating hematogenous cells at the site of injury. Histological evaluation ofthe tissue showed fewer activated macrophages at the site of injury. Concomitantly, reduced neuronal loss and gliosis were observed in the antagonist infused spinal cord. In addition, increased expression of Bcl‐2 gene, an endogenous inhibitor of apoptosis, was seen in the antagonist infused spinal cord at 7 days post injury. Morphologically, staining with the bisbenzamide dye Hoechst 33342 showed significantly more apoptotic bodies in the vehicle compared to antagonist infused spinal cord. Our data suggest that chemokine antagonist infusion post‐injury results in limiting the inflammatory response following spinal cord contusion injury, thereby attenuating neuronal loss, possibly due to decreased apoptosis. These findings support the contention that disrupting chemokine interactions with their receptors may be an effective approach in reducing the secondary damage after spinal cord injury. J. Neurosci. Res. 59:63–73, 2000

Effects of primary antiserum dilution on staining of "antigenrich" tissues with the peroxidase antiperoxidase technique.

The effect of primary antiserum dilution on staining results with the peroxidase antiperoxidase method were investigated using frozen sections of perfused rat cerebellum and optic nerve. Results comparable to formalin fixed and paraffin embedded tissue were attainable only when low antiserum concentrations were used. Optimal staining of antigen rich tissue, such as frozen sections, with the peroxidase antiperoxidase method required low antiserum concentrations apparently to minimize the binding of both antigen-binding fragments of the bridging antibody to the tissue bound antiserum. It appears that low antiserum concentration insures that sufficient bridge antibody molecules will be only singly bound and thus free to attach the peroxidase antiperoxidase complex.

Chemokine antagonist infusion promotes axonal sparing after spinal cord contusion injury in rat

Spinal cord injury produced by mechanical contusion causes the onset of acute and chronic degradative events. These include blood brain barrier disruption, edema, demyelination, axonal damage and neuronal cell death. Posttraumatic inflammation after spinal cord injury has been implicated in the secondary injury that ultimately leads to neurologic dysfunction. Studies after spinal cord contusion have shown expression of several chemokines early after injury and suggested a role for them in the ordered recruitment of inflammatory cells at the lesion site (McTigue et al. [1998] J. Neurosci. Res. 53:368–376; Lee et al., [2000] Neurochem Int). We have demonstrated previously that infusion of the broad‐spectrum chemokine receptor antagonist (vMIPII) in the contused spinal cord initially attenuates leukocyte infiltration, suppresses gliotic reaction and reduces neuronal damage after injury. These changes are accompanied by increased expression of bcl‐2, the endogenous apoptosis inhibitor, and reduced neuronal apoptosis (Ghirnikar et al. [2000] J. Neurosci. Res. 59:63–73). We demonstrate that 2 and 4 weeks of vMIPII infusion in the contusion‐injured spinal cord also results in decreased hematogenous infiltration and is accompanied by reduced axonal degeneration in the gray matter. Luxol fast blue and MBP immunoreactivity indicated reduced myelin breakdown in the dorsal and ventral funiculi. Increased neuronal survival in the ventral horns of vMIPIl infused cords was seen along with increased bcl‐2 staining in them. Immunohistochemical identification of fiber phenotypes showed increased presence of calcitonin gene related peptide, choline acetyl transferase and tyrosine hydroxylase positive fibers as well as increased GAP43 staining in treated cords. These results suggest that sustained reduction in posttraumatic cellular infiltration is beneficial for tissue survival. A preliminary report of this study has been published (Eng et al. [2000] J. Neurochem. 74(Suppl):S67B). In contrast to vMIPII, infusion of MCP‐1 (9‐76), a N‐terminal analog of the MCP‐1 chemokine showed only a modest reduction in cellular infiltration at 14 and 21 dpi without significant tissue survival after spinal cord contusion injury. Comparing data on tissue survival obtained with vMIPII and MCP‐1 (9‐76) further validate the importance of the use of broad‐spectrum antagonists in the treatment of spinal cord injury. Controlling the inflammatory reaction and providing a growth permissive environment would enhance regeneration and ultimately lead to neurological recovery after spinal cord injury. J. Neurosci. Res. 64:582–589, 2001. Published 2001 Wiley‐Liss, Inc.

ALTERATIONS IN MYELIN FATTY ACIDS AND PLASMALOGENS IN MULTIPLE SCLEROSIS

The many attempts to elucidate the pathogenesis of multiple sclerosis (MS) by chemical analysis of brain, blood and spinal fluid have been amply reviewed recently by Scheinberg and Korey. The present studies have been directed toward the alteration of lipids that precede visible demyelination. Their results, in conjunction with findings of other authors, might permit tentative conclusions or, at least, conjecture about the biochemical pathway of demyelination in MS. In addition, the relevance of the early chemical lesions to function and to clinical remission merits attention. Supplementary to the lipid analyses done earlier,2 the 2-hydroxy fatty acids (HFA) were studied in detail to see whether further evidence for selective removal of lipids3 could be obtained. It has not been established that the lipid deficits found in grossly intact white matter of MS bra inP5 are all located in myelin proper. In fact, there is a paucity of data on the lipid composition of this structure. Consequently, a comprehensive analysis of purified myelin fractions was undertaken.

Glial fibrillary acidic protein in regenerating teleost spinal cord.

Immunohistological and ultrastructural studies were carried out on normal and regenerating spinal cord of the gymnotid Sternarchus albifrons, and in the brain and spinal cord of the goldfish Carassius auratus, to examine the distribution of glial fibrillary acidic protein (GFAP) in these tissues. Sections of normal goldfish brain and spinal cord exhibited positive staining for GFAP. In normal Sternarchus spinal cord, electron microscopy has revealed filament-filled astrocytic processes; however, such astrocytic profiles were more numerous in regenerated cord. Likewise, while normal Sternarchus spinal cord showed only a small amount of GFAP staining, regenerated cords were strongly positive for GFAP. Positive staining with anti-GFAP was observed along the entire length of the regenerated cord in Sternarchus, and was especially strong in the transition zone between regenerated and unregenerated cord. Both regeneration of neurites and production of new neuronal cell bodies occur readily in such regenerating Sternarchus spinal cords (Anderson MJ, Waxman SG: J Hirnforsch 24: 371, 1983). These results demonstrate that the presence of GFAP and reactive astrocytes in Sternarchus spinal cord does not prevent neuronal regeneration in this species.