Applications of immortalized cells in basic and clinical neurology

Immortalized cell lines can serve as model systems for studies of neuronal development and restoration of function in models of neurological disease. Cell lines which result from spontaneous or experimentally-induced tumors have been used for these purposes. More recently, the techniques of genetic engineering have resulted in the production of cell lines with specific desired characteristics. This has been accomplished by insertion of a desired gene into a pre-existing immortal cell or by immortalizing primary cells. The production of immortal cell lines using temperature-sensitive immortalizing genes offers an additional method of controlling gene expression, and thereby controlling cell proliferation and differentiation. In the nervous system, these techniques have produced immortal cell lines with neuronal and glial properties.     

Long-term expression of gene introduction into normal human T-lymphocytes by retroviral-mediated gene transfer

Human T-lymphocytes are long lived, easily accessible, mature, and capable of proliferation. They are theoretically a suitable target for retroviral mediated gene transfer. To test this hypothesis, normal human T-cells obtained from bone marrow and peripheral blood were stimulated with phytohemagglutinin (PHA) and infected 24 h later with the retroviral vector N2 which carries the bacterial neo gene. T-lymphocytes were propagated in culture for up to 14 weeks with interleukin-2 (IL-2). Analysis by whole cell RNA dot/blot using a single stranded RNA probe demonstrated persistent expression of the neo gene. Preliminary functional studies revealed that both helper and suppressor functions were preserved in the infected cells in culture. These results demonstrate that normal T-cells are capable of long-term expression of genes introduced by retroviral mediated gene transfer and are potential target cells for somatic gene therapy.     

Evidence for an immunological and functional relationship between superoxide dismutase and a high molecular weight osteoclast plasma membrane glycoprotein.

Large multinucleated osteoclasts are the major cells responsible for bone breakdown and have been reported to produce high levels of superoxides which may contribute to the process of bone resorption (Key et al.: J Bone and Mineral Res 4 [suppl. 1]:S206, 1989). Osteoclasts also possess high levels of superoxide dismutase, a protective enzyme capable of converting toxic superoxides to less dtoxic H2O2 (Fridovich: J Biol Chem 264:7761-7764, 1989). The amino acid sequence of manganese and/or iron superoxide dismutase has a conserved region which exhibits substantial homology with a fragment obtained from a high molecular weight osteoclast surface marker glycoprotein which is reactive with monoclonal antibody 121F. In this report, evidence is presented substantiating immunological, biochemical, and functional similarities between the osteoclast membrane antigen recognized by the 121F monoclonal antibody and superoxide dismutase. Western blot and immunoprecipitation studies show that a monospecific polyclonal antibody generated against immunoaffinity purified antigen is cross-reactive with superoxide dismutase. Both the antigen and a high molecular weight superoxide dismutase activity have been detected in osteoclast plasma membrane preparations. The levels of superoxide dismutase activity and the membrane antigen have been found to correlate in antigen depletion studies and in western blots probing osteoclasts and closely related marrow-derived giant cells. Moreover, regions of osteoclast superoxide dismutase activity identified by electrophoretic zymogram analysis have been shown by gel electrophoresis and western blots to contain the high molecular weight antigen, or complexes of the antigen with the 121F monoclonal antibody when these were premixed prior to nondenaturing electrophoresis. It is proposed that the osteoclast plasma membrane possesses a high molecular weight superoxide dismutase activity. Furthermore, it appears that this activity is associated with the osteoclast antigen recognized by the 121F monoclonal antibody.     

Adaptations of Goldner's Masson Trichrome Stain for the Study of Undecalcified Plastic Embedded Bone

Specialized adaptations for application of Goldner's Masson trichrome stain to plastic embedded undecalcified bone specimens are presented. This stain can be used successfully on methyl-glycol methacrylate, glycol methacrylate and Spurr embedded bones. The stain affords the advantage of good cellular staining due to the hematoxylin component with concomitant sharp discrimination of mature bone matrix which stains green, immature new bone matrix which stains red, and calcified cartilage which stains very pale green. Use of red filters during photomicrography aids in bone-osteoid discrimination in black and white photographs.     

Cortical surface patterns in human and nonhuman primates

An analysis of skulls from several primate species shows that a “worm-track” surface pattern, first identified in the brow region in fossil adult hominids and subsequently in olive baboons, chimpanzees, and macaques, is also present in numerous other species. Fine cancellous bone and its attendant vermiculate surface pattern have been observed in subadult and adult gelada baboons, gibbons, gorillas, and orangutans as well as in modern Homo sapiens and several Plio-Pleistocene fossil hominids. In contemporary primates, fine cancellous bone has been identified not only in the brow region, but also along the zygomatic arch, on the pterygoid plates, on the maxilla, along the temporal line, on the mastoid process, and in the region of inion. Given the widespread distribution of this trait, caution is advised when using it as a diagnostic indicator of the evolutionary or functional significance of craniofacial morphology.     

Exogenous glycosaminoglycans (GAG) differentially modulate GAG synthesis by anchorage-independent cultures of the outer cells from neonatal rat calvaria in the absence and presence of TGF-β

In anchorage-dependent (AD) cultures of the outer cell population (OCP) from neonatal rat calvaria, transforming growth factor-₁ (TGF-) specifically upregulated the synthesis of chondroitin sulfate (CS) proteoglycan (PG) and uncoupled the inhibitory effect of increasing cell density on CS PG synthesis (reference #30). Utilizing the same cell population, we have further examined the possibility that glycosaminoglycans (GAG) known to be synthesized and secreted by bone cells might exert feedback effects on GAG synthesis and/or its stimulation by TGF-. Although addition of TGF- alone stimulated net synthesis of HA and CS in both AD and anchorage-independent (AI) cultures, significant alterations of basal and TGF--stimulated GAG synthesis by exogenous GAGs were observed only in AI cultures. In AI cultures exogenously added hyaluronic acid (HA) markedly enhanced the basal synthesis of HA and CS while heparin (H) suppressed the basal synthesis of HA, CS as well as dermatan sulfate (DS). Also, the addition of HA markedly potentiated the stimulation by TGF- of HA and CS synthesis as did heparan sulfate (HS) for CS and DS synthesis. H suppressed the stimulation of the synthesis of HA, CS and DS by TGF-. Overall, our results indicate specific effects of individual GAGs on basal and TGF--stimulated GAG synthesis in OCP cultures. We suggest that some of the GAGs in the OCP microenvironment (which with the exception of HA are covalently linked to protein cores of secreted PGs), acting in concert with TGF-, may serve as an amplification system for upregulating GAG synthesis in the rapidly growing neonatal calvarium.     

A comparative study of the temporal bone in three populations of man

A comparative study was made to determine race and sex differences in the temporal bone, to investigate growth relationships, and to establish a basis for phylogenetic studies of the temporal bone and the temporal lobe of the brain. Data on Eskimo, Indian, and white crania were collected from radiographs and directly from the skulls. Of the 25 variables studied, only the minimum diameter of porus failed to demonstrate some difference among the races. Variation between sexes was found for all measurements except the cranial base angle (of deflection) and three angles related to the petrous pyramids. Correlation coefficients indicated that none of the angles are related in any consistent manner to the other variables studied. This is interpreted as further evidence of cranial base stability. The Indians have the lowest, longest squamae, differing most from the whites. The position of squama is more anterior in the Eskimos. Females of each race possess more anteriorly positioned squamae than males. When the squama is more anteriorly located, the porus is in a more posterior position within the squama itself. Strong race variation exists in the shape of porus. In order to establish a basis for phylogenetic studies of the temporal lobe of the brain better reference points for reflecting its size and shape must be found.     

Maladaptive innate immune training of myelopoiesis links inflammatory comorbidities

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An optimal dietary non-phytate phosphorus level of broilers fed a conventional corn–soybean meal diet from 4 to 6 weeks of age

It is imperative to evaluate precise nutrient requirements of animals in order to optimize productivity and minimize feed cost and nutrient excretions. The current non-phytate phosphorus (NPP) recommendation for broilers is based on the papers published 30 years ago. However, today’s commercial birds are quite different from those before 30 years. Therefore, the present experiment was conducted with growing male broiler chickens to evaluate an optimal dietary NPP level of broiler chickens fed a conventional corn–soybean meal diet from 4 to 6 weeks of age. The 1-day-old chicks were fed corn–soybean meal diet containing 0.39% NPP from 1 to 3 weeks of age. At 22 days of age, 360 birds were selected and randomly allotted by BW to one of 10 dietary treatments with six replicate cages of six birds per cage for each treatment. Birds were fed the P-unsupplemented corn–soybean meal basal diet and the basal diet supplemented with inorganic P as CaHPO₄·H₂O ranging from 0.00% to 0.45% with 0.05% increment from 4 to 6 weeks of age. The dietary NPP levels were 0.09%, 0.14%, 0.20%, 0.24%, 0.30%, 0.34%, 0.38%, 0.45%, 0.49% and 0.54%, respectively, and the dietary Ca level was fixed at 0.90% for all treatments. The results showed that average daily gain, serum inorganic P concentration, tibia bone strength, tibia ash percentage and P percentage, tibia bone mineral content (BMC) and density (BMD), middle toe ash percentage and P percentage, middle toe BMC, total body BMC and BMD were affected (P<0.0001) by dietary NPP level, and increased linearly (P<0.0001) and quadratically (P<0.003) as dietary NPP levels increased. Optimal dietary NPP levels estimated based on fitted broken-line models (P<0.0001) of the above indices are 0.21%, 0.29%, 0.29%, 0.29%, 0.29%, 0.31%, 0.29%, 0.30%, 0.27%, 0.29% and 0.28%, respectively. It is suggested that the total body BMC and BMD, and middle toe ash P and BMC might be new, sensitive and non-invasive criteria to evaluate the dietary NPP requirements of broilers. The optimal dietary NPP level would be 0.31% for broiler chickens fed a conventional corn–soybean meal diet from 4 to 6 weeks of age.     

Lithium attenuates lead induced toxicity on mouse non-adherent bone marrow cells

Lead is a poisonous heavy metal that occurs in all parts of environment and causes serious health problems in humans. The aim of the present study was to investigate the possible protective effect of lithium against lead nitrate induced toxicity in non-adherent bone marrow stem cells. Trypan blue and MTT assays represented that exposure of the cells to different concentrations of lead nitrate decreased viability in a dose dependent manner, whereas, pretreatment of the cells with lithium protected the cells against lead toxicity. Lead reduced the number and differentiation status of bone marrow-derived precursors when cultured in the presence of colony stimulating factor (CSF), while the effect was attenuated by lithium. The cells treated with lead nitrate exhibited cell shrinkage, DNA fragmentation, anion superoxide production, but lithium prevented lead action. Moreover, apoptotic indexes such as PARP cleavage and release of HMGB1 induced by lead, were protected by lithium, suggesting anti-apoptotic effect of lithium. Immunoblot analysis of histone H3K9 acetylation indicated that lithium overcame lead effect on acetylation. In conclusion, lithium efficiently reduces lead toxicity suggesting new insight into lithium action which may contribute to increased cell survival. It also provides a potentially new therapeutic strategy for lithium and a cost-effective approach to minimize destructive effects of lead on bone marrow stem cells.