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Embryonic diapause is a maternally controlled phenomenon. The molecule controlling the onset of the phenomenon is unknown. We demonstrated that overexpression of microRNA let-7a or incubation with let-7g-enriched extracellular vesicles from endometrial epithelial cells prolonged the in vitro survival of mouse blastocysts, which developed into live pups after having been transferred to foster mothers. Similar to in vivo dormant blastocysts, let-7-induced dormant blastocysts exhibited low level of proliferation, apoptosis, and nutrient metabolism. Let-7 suppressed c-myc/mTORC1 and mTORC2 signaling to induce embryonic diapause. It also inhibited ODC1 expression reducing biosynthesis of polyamines, which are known to reactivate dormant embryos. Furthermore, the overexpression of let-7 blocked trophoblast differentiation and implantation potential of human embryo surrogates, and prolonged survival of human blastocysts in vitro, supporting the idea that embryonic diapause was an evolutionary conserved phenomenon. In conclusion, let-7 is the main factor inducing embryonic diapause.

Our aim was to explore the effects of functional groups at carbon-2 (C2) of a sugar on the conformational properties of the peptide backbone. Three monosaccharides, mannose, galactose, and N-acetylgalactosamine (GalNAc), were added separately to the serine side-chain of a hamster prion peptide because it is a sensitive model for comparing the effect of protein modification on the conformational properties of the polypeptide chain. In buffer, this prion peptide goes through a gradual coil-to-β structural conversion and forms amyloid fibrils slowly during incubation. Our results showed that a sugar with an N-acetyl amino group in the equatorial configuration (GalNAc) or with a hydroxyl group in the axial configuration (mannose) on C2 had a greater inhibitory effect on the amyloidogenesis of the prion peptide than a sugar with the hydroxyl group in the equatorial configuration (galactose). We suggest that galactosylation has less effect than mannosylation or GalNAc glycosylation on promoting turn formation at the glycosylation site and on inhibition of amyloidogenesis. The anti-amyloidogenic property of mannose implies that protein mannosylation has an anti-aggregation function.

BACKGROUND Allogeneic bone marrow transplantation is under investigation for a range of nonmalignant indications, including tolerance induction through mixed chimerism. This strategy has so far been tested experimentally only in young recipients. Due to immunosenescence, older patients have an increase in memory T cells (TMEM) as well as other alterations to their immune system, which may influence the potential to induce tolerance. We therefore investigated the impact of immunosenescence on chimerism-based tolerance induction. METHODS Groups of young (2 months) and old (12 months) C57BL/6 recipients received BALB/c bone marrow under nonmyeloablative (3 Gy) and minimal (1 Gy) total body irradiation and treatment with costimulation blockade, T-cell depletion, or rapamycin. Multilineage chimerism, clonal deletion, and lymphocyte subsets were analyzed by flow cytometry. Tolerance was assessed by skin and heart grafts and enzyme-linked immunospot, intracellular cytokine, and mixed lymphocyte reaction assays. RESULTS Unexpectedly, chimerism and tolerance were established in old recipients with comparable-and in some cases increased-efficacy as in young recipients employing costimulation blockade-based or T-cell depletion-based conditioning with 1 or 3 Gy total body irradiation. TMEM reactivity in (naive) old mice was augmented in response to polyclonal but not to allogeneic stimulation, providing a mechanistic underpinning for the susceptibility to chimerism induction despite increased TMEM frequencies. Tolerance in old recipients was associated with peripheral and central clonal deletion and a higher frequency of regulatory T cells. CONCLUSION Advanced age does not impair bone marrow engraftment, thereby widening the clinical potential of experimental protocols inducing transplantation tolerance through mixed chimerism.

Brain death impairs donor organ quality and accelerates immune responses after transplantation. Detailed aspects of immune activation following brain death remain unclear. We have established a mouse model and investigated the immediate consequences of brain death and anesthesia on immune responses.C57JBl/6 mice (n=6/group) were anesthetized with isoflurane (ISF) or ketamine/xylazine (KX); subsequently, animals underwent brain death induction and were followed for 3h under continuous ventilation. Blood pressure was monitored continuously and animals were resuscitated with normal saline to achieve normotension. Immune activation in brain dead animals was analyzed by IFNγ-ELispot, MLR, and flow-cytometry. Sham-operated and naïve animals served as controls.Blood pressure remained stable in both BD/KX and BD/ISF animals during the 3h observation time. Brain death was linked to systemic immune activation: IFNγ-expression of splenocytes and lymphocyte proliferation rates was significantly elevated subsequent to brain death (p0.02,0.01); T-cell activation markers CD28 and CD69 had increased in brain dead animals (p0.03,0.02). Isoflurane treatment in sham controls throughout the observation period (3.5h) revealed anesthesia associated IFNγ-expression and lymphocyte activation which were not observed when animals were treated with ketamine/xylazine (p0.04,0.009).This study reports on a reproducible and hemodynamically stable brain death mouse model. Hemodynamic stability was not impacted through either isoflurane or ketamine/xylazine induction. Of clinical relevance, prolonged anesthesia with isoflurane had been linked to pro-inflammatory cytokine activation. Brain death caused systemic immune activation in organ donors.

The receptor for macrophage colony-stimulating factor (M-CSFR; c-fms) is expressed at increased levels by microglia in Alzheimer's disease (AD) and in mouse models for AD. Increased expression of M-CSFR on cultured microglia results in a strong proinflammatory response, but the relevance of this cell culture finding to intact brain is unknown. To determine the effects of increased microglial expression of M-CSFR in a complex organotypic environment, we developed a system for biolistic transfection of microglia in hippocampal slice cultures. The promoter for the Mac-1 integrin alpha subunit CD11b is active in cells of myeloid origin. In the brain, CD11b expression is restricted to microglia. Constructs consisting of the promoter for CD11b and a c-fms cDNA or an enhanced green fluorescent protein (EGFP) cDNA were introduced into monotypic cultures of microglia, neurons, and astrocytes. Strong CD11b promoter activity was observed in microglia, whereas little activity was observed in other cell types. Biolistic transfection of organotypic hippocampal cultures with the CD11b/c-fms construct resulted in expression of the c-fms mRNA and protein that was localized to microglia. Furthermore, biolistic overexpression of M-CSFR on microglia resulted in significantly increased production by the hippocampal cultures of the proinflammatory cytokines interleukin (IL)-1alpha macrophage inflammatory protein (MIP-1alpha), and trends toward increased production of IL-6 and M-CSF. These findings demonstrate that microglial overexpression of M-CSFR in an organotypic environment induces an inflammatory response, and suggest that increased microglial expression of M-CSFR could contribute to the inflammatory response observed in AD brain.

1. The decay of inward currents was studied using the giant patch-clamp technique and a cloned inward rectifier K(+) channel, Kir2.1, expressed in Xenopus oocytes. 2. In inside-out patches, inward currents carried by NH4(+) or Tl(+) decayed over time. When the voltage was more negative, the degree and rate of decay were greater. The rate of NH4(+)-induced decay saturated at a symmetrical [NH4(+)] of approximately 100 mM. The decay rate was slow (2.6 x 10(3) M(-1) s(-1)) at -140 mV with 10 mM [NH4(+)]. 3. Upon a 10 degrees C increase in temperature, the single-channel NH4(+) current amplitude increased by a factor of 1.57, whereas the NH4(+)-induced decay rate increased by a factor of 2.76. In the R148Y Kir2.1 mutant (tyrosine 148 is at the external pore mouth), NH4(+)-induced inactivation was no longer observed. 4. NH4(+) single-channel currents revealed one open and one closed state. The entry rate into the closed state was voltage dependent whereas the exit rate from the closed state was not. An increase of internal [NH4(+)] not only decreased the entry rate into but also elevated the exit rate from the closed state, consistent with the occupancy model modified from the foot-in-the-door model of gating. 5. These results suggest that the decay of NH4(+) current is unlikely to be due to a simple bimolecular reaction leading to channel block. We propose that NH4(+) binding to Kir2.1 channels induces a conformational change followed by channel closure. 6. The decay induced by permeant ions other than K(+) may serve as a secondary selectivity filter, such that K(+) is the preferred permeant ion for Kir2.1 channels.

Traumatic injury to the central nervous system (CNS) results in the breakdown of the blood-brain barrier and recruitment of hematogenous cells at the site of injury. The role of chemokines in this process has been well recognized and they have been regarded as promising targets for development of anti-inflammatory therapies. The expression of monocyte chemoattractant protein (MCP-1), in particular, has been closely linked to macrophage infiltration following trauma in rat brain. In this study we determined whether inhibition of MCP-1 following stab wound injury would reduce macrophage infiltration. Stab wound injured Sprague-Dawley rats were infused with MCP-1 sense or antisense oligonucleotides using an Alzet miniosmotic pump (1 microl/h for 3 days). Three days following injury, widespread gliosis was observed in both groups of rats as judged by glial fibrillary acidic protein (GFAP) immunoreactivity. Immunohistochemistry showed significantly less staining for MCP-1 in antisense treated animals. In addition, the number of macrophages were reduced by 30% in the antisense compared to the sense treated animals (P0.05). These results demonstrate that modulation of MCP-1 expression in stab wound injury directly affects monocytic infiltration and provide a basis for MCP-1 inhibition as a therapeutic strategy for controlling inflammatory events of traumatic brain injury.

Previous studies have shown that the mechanical wounding of 3-week-old cultured rat astrocytes results in cell proliferation and hypertrophy resembling astrocyte responses to a brain injury in vivo. We now report the effects of basic fibroblast growth factor (bFGF) and an anti-bFGF antibody on astrocyte morphology, proliferation, and migration following in vitro wounding of confluent secondary cultures. Addition of bFGF (20 ng/ml) to wounded cultures induced morphological changes characteristic of differentiation in wounded and nonwounded areas of the culture. Combined treatment with bFGF and an anti-bFGF antibody (100 micrograms/ml) prevented this effect. Astrocyte proliferation along the edges of a scratch wound was at maximum 24 hr after wounding in cells growing in Eagle's minimum essential medium (EMEM) containing 10% serum. Low serum concentration and treatment with dibutyryl cyclic adenosine monophosphate (dbc-AMP) reduced injury-associated astrocyte proliferation. Addition of bFGF to cultures in EMEM with serum increased astrocyte proliferation at 18 and 24 hr after wounding. This effect was reduced considerably by treatment of cultures with bFGF in combination with an anti-bFGF antibody. The combined treatment and the antibody alone reduced cell division to a level lower than in control cultures. Twenty-four hr following wounding, astrocytes along the edges of the wound exhibited extension of thick, flat processes into the wound area. At 3 and 5 days after wounding, a bodily migration of astrocytes into the wounded area was observed. Addition of bFGF significantly increased astrocyte migration 1 day after wounding, with maximum effect on day 3 and no subsequent increase on day 5. A combination of bFGF and anti-bFGF antibody as well as the antibody alone reduced astrocyte migration to a level lower than in controls. Immunohistochemical localization and isoform pattern of bFGF in astrocytes did not change with dbc-AMP treatment or wounding. We conclude that mechanically wounded confluent astrocytes respond to bFGF added to the culture medium by enhancing cell division, differentiation, and migration. In addition, the results of the antibody treatment also suggest a role for endogenous bFGF in astrocyte proliferation and migration elicited by wounding in vitro. These results support the notion that in vivo, both bFGF released by injury and endogenous bFGF synthesized by astrocytes, contribute to the cellular responses that lead to astrogliosis.

The survival of colony-forming cells in pig epidermis after irradiation was measured using different electron field sizes. The sensitivity of the colony-forming cells was characterized by D(o) = 2.5-3.0 Gy. There was an effect of field size, described approximately by: Dose (to give five colonies/cm2) (Gy) = (31 +/- 2) x Area-(0.048 +/- 0.015). The effect of field size was less than found previously using tolerance to skin reactions (area exponent = 0.16). This work indicates for the first time that the effects of different large field sizes in skin can be detected at the level of colony-forming cell survival.

In conscious rats, we have previously shown that immunoneutralization of circulating insulin with a rabbit anti-insulin serum abolished the pancreatic exocrine secretion stimulated by a meal or a combination of exogenous secretin and cholecystokinin octapeptide (CCK-8). To investigate the mechanism of endogenous insulin action on the exocrine pancreas, isolated rat pancreata were perfused with intra-arterial infusion of Krebs-Henseleit solution (37 degrees C) at 1.2 ml/min, whereas both pancreatic juice and portal venous effluent were collected separately in 15-min samples. Simultaneous intra-arterial infusion of secretin and CCK-8 in doses of 0.75 and 4.2 pmol/h; respectively, significantly increased volume, bicarbonate, and protein output in 7 rat pancreata (P < 0.01). When a rabbit anti-insulin serum was administered intra-arterially (0.1-ml bolus followed by 0.1 ml for 10 min), pancreatic secretion of volume, bicarbonate, and protein output was profoundly suppressed (n = 7, P < 0.01), whereas a normal rabbit serum failed to influence pancreatic secretion. The decrease in pancreatic secretion by the antiserum coincided with a significant increase in somatostatin in portal venous effluent from 1.4 +/- 0.2 to 4.1 +/- 0.8 pM (n = 6, P < 0.05). The combined administration of a rabbit antisomatostatin serum (0.4 ml) and the anti-insulin serum partially reversed the effect of the anti-insulin serum alone. Thus the pancreatic secretion was significantly greater than that achieved by the anti-insulin serum alone (P < 0.05). These observations strongly suggest that the action of insulin on exocrine pancreas is mediated by its local or paracrine action.(ABSTRACT TRUNCATED AT 250 WORDS)

The hormonal mechanisms mediating protein-stimulated pancreatic exocrine secretion were investigated in four conscious dogs with gastric cannulas and Thomas duodenal cannulas. Pancreatic juice was collected by direct cannulation of the main pancreatic duct in response to intraduodenal infusates prepared with cooked beef liver. When the homogenized liver was administered intraduodenally, cholecystokinin (CCK) in plasma significantly increased. This increase was accompanied by a significant increase in pancreatic exocrine secretion, including volume, bicarbonate, and protein output. The liver homogenate incubated with pancreatic enzymes further increased both plasma CCK and exocrine pancreatic secretion. However, plasma secretin was not affected by the protein digests. Intravenous administration of loxiglumide at the rate of 5.0 and 10.0 mg/kg/h resulted in a significant decrease in the stimulated pancreatic secretion of fluid, bicarbonate, and protein. The study indicates that endogenous CCK released by protein digests exerts not only enzyme secretion but also bicarbonate secretion in dogs.

The risk of acquiring variant Creutzfeldt-Jakob disease is closely related to polymorphism at codon 129 of the human prion gene, because almost all variant Creutzfeldt-Jakob disease patients are Met/Met homozygotes. Although animal transmission experiments corroborated this seeding discrimination, the origin of the differential seeding efficiency of the bovine prion seed for human codon 129 polymorphism remained elusive. Here, we used a short prion protein (PrP) peptide as a model system to test whether seeding discrimination can be found in this simple system. We used a previously developed 'seed-titration method' and time-resolved CD spectroscopy to compare sequence-dependent seeding efficiency regarding codon 129 polymorphism. Our results showed that the Met→Val substitution on the human PrP (huPrP) peptide decreased seeding efficiency by 10 times when fibrils formed from bovine PrP (bPrP) peptide were used as the seed. To explore whether the different seeding barrier is due to the chemical and structural properties of Met and Val or whether another residue is involved in this peptide model, we constructed three bPrP mutants, V112M, L138I and N143S, in each of which one residue was replaced by the corresponding human residue. Our data showed that Leu138 in the bPrP seed might be the key residue causing the different seeding efficiencies related to 129M/V polymorphism and the interference effect of huPrP129V in the huPrP129M/V mixture. We propose a 'surface competition hypothesis' to explain the big seeding barrier caused by 129V in the PrP peptide seeding experiment.

In the present study, we investigated age-related changes in pituitary adenylate cyclase-activating polypeptide (PACAP) immunoreactivity and its protein levels in the gerbil hippocampus at various ages using immunohistochemistry and western blot analysis. In the post-natal month 1 (PM 1) group, PACAP-immunoreactive cells were found in all hippocampal subregions. The number of PACAP-immunoreactive cells was decreased in the PM 3 group and was still more decreased in the PM 6 and 12 groups. Thereafter, in the PM 18 and 24 groups, PACAP-immunoreactive cells were significantly increased again. However, in the mossy fibre zone, PACAP immunostaining was very strong in the adult group, especially in the PM 6 group. In addition, PACAP protein level was highest at PM 6, showing a slight decrease at PM 24. These results indicate that PACAP-immunoreactive cells are lowest in the adult stage and highest in the aged stage. However, PACAP immunoreactivity in the mossy fibre zone and PACAP protein level in the hippocampus are highest in the adult stage.

In the present investigation, we have studied the effect of atropine on the pancreatic secretion stimulated by intraduodenal administration of either sodium oleate or exogenous cholecystokinin (CCK). In four dogs prepared with gastric and Thomas duodenal cannulas, pancreatic juice was collected for measurement of volume, bicarbonate, and protein output, and peripheral venous blood samples were obtained for radioimmunoassay of both secretin and CCK. Volume, bicarbonate, and protein output of the pancreatic juice increased significantly in response to sodium oleate (1-4 mmol/h) in a dose-dependent manner. The increase in pancreatic secretion paralleled the increments in both plasma CCK and secretin. Atropine given intravenously suppressed completely both pancreatic secretion and release of CCK stimulated by sodium oleate, whereas the release of secretin was not affected. Pancreatic secretion was significantly increased in a dose-dependent manner by exogenous CCK octapeptide (CCK-8) at 16, 32, and 64 micrograms (14, 28, and 56 pmol).kg-1.h-1. Atropine inhibited protein output only partially, but it did not influence bicarbonate output. In five additional dogs, the effect of atropine on L-tryptophan-stimulated pancreatic secretion was studied. Interestingly, atropine failed to influence the CCK release and pancreatic secretion of volume and bicarbonate, except for protein secretion, which was significantly inhibited. It was shown previously that atropine inhibited significantly the pancreatic secretion of bicarbonate stimulated by secretin in physiological doses. Thus we conclude that the inhibition by atropine of the pancreatic exocrine secretion stimulated by sodium oleate is mediated by both suppression of CCK release and inhibition of action of secretin on the exocrine pancreas.(ABSTRACT TRUNCATED AT 250 WORDS)

Prion diseases are transmissible neurodegenerative disorders. It is widely accepted that prions are the infectious agents responsible for disease transmission, and the sequence homology between the infectious prion and the host prion protein determines its transmission efficiency across species. However, previous studies have often reported different results regarding seeding efficiency, the efficiency of initiating amyloid propagation by adding pre-existing amyloid fibrils as seed. In the present study, we used synthetic peptides as a simple system to determine the sequence-dependent transmission barrier between hamster and mouse. We found that the heterologous seeding efficiency of hamster and mouse prion peptides was 4 times less than that of homologous seeding. Moreover, residue 139 was not the only residue in determining seeding efficiency. When the seed had Ile at this position, the homology at this position between seed and monomer determined the seeding efficiency. When the seed had Met at this position, homology at residues 109 and 112 determined the seeding efficiency.

The cDNA microarray technique was used to study gene epression in human embryonic pulmonary fibroblasts (HEPF) infected with Hantaan virus (HTNV) under the influence of cordycepin (Cor), an inhibitor of post-transcriptional pre-mRNA polyadenylation. Four apoptotic genes, the insulin-like growth factor binding protein 1, NFkB inhibitor alpha, caspase-3 and NFkB1 were up-regulated in both infected and uninfected Cor-treated cells and two cell cycle-associated genes, CDC-like kinase and beta-induced transforming growth factor were up-regulated in Cor-untreated cells but down-regulated in Cor-treated cells. Cell morphology examination, quantitative RT-PCR, and immunofluorescence (IF) test suggested that following the Cor treatment the HTNV infection took place, but late viral gene expression was slightly reduced. Three parameters, namely caspase-3 activity, annexin V binding, and cell cycle were used to detect apoptosis. The results suggested that the induction of apoptosis in HEPF by HTNV started at 6 hrs post infection (p.i.). Following the Cor treatment, however, the caspase-3 activity began to increase at 24 hrs p.i. Thus it is suggested that inhibition of de novo late viral protein synthesis by Cor changes the apoptosis pathway and cell cycle by delaying caspase-3 gene expression and by up/down-regulating of expression of other apoptotic and cell cycle-associated genes. This implicates that HTNV can induce apoptosis in HEPF even without de novo viral protein synthesis and with a reduced and slowed viral maturation.

Our previous results showed that embryotrophic factor-3 (ETF-3) from human oviductal cells increased the size and hatching rate of mouse blastocysts in vitro. The present study investigated the production of ETF-3 by an immortalized human oviductal cell line (OE-E6/E7) and the effects of ETF-3 on the mRNA expression of mouse embryos. The ETF-3 was purified from primary oviductal cell conditioned media using sequential liquid chromatographic systems, and antiserum against ETF-3 was raised. The ETF-3-supplemented Chatot-Ziomek-Bavister medium was used to culture Day 1 MF1 x BALB/c mouse embryos for 4 days. The ETF-3 treatment significantly enhanced the mouse embryo blastulation and hatching rate. The antiserum, at concentrations of 0.03-3%, abolished the embryotrophic effect of ETF-3. Positive ETF-3 immunoreactivity was detected in the primary oviductal cells, OE-E6/E7, and blastocysts derived from ETF-3 treatment. Vero cells (African Green Monkey kidney cell line), fibroblasts, and embryos cultured in control medium did not possess ETF-3 immunoreactivity. The mRNA expression patterns of the treated embryos were studied at the blastocyst stage by mRNA differential display reverse transcription-polymerase chain reaction (DDRT-PCR). The DDRT-PCR showed that some of the mRNAs were differentially expressed after ETF-3 treatment. Twelve of the differentially expressed mRNAs that had high homology with cDNA sequences in the GenBank were selected for further characterization. The differential expression of seven of these mRNAs (ezrin, heat shock 70-kDa protein, cytochrome c oxidase subunit VIIa-L precursor, proteinase-activated receptor 2, eukaryotic translation initiation factor 2beta, cullin 1, and proliferating cell nuclear antigen) was confirmed by semiquantitative RT-PCR. In conclusion, immortalized oviductal cells produce ETF-3, which influences mRNA expression of mouse blastocyst.

Microglia are important in the inflammatory response in Alzheimer's disease (AD). We showed previously that macrophage colony-stimulating factor receptor (M-CSFR), encoded by the c-fms protooncogene, is overexpressed on microglia surrounding amyloid beta (Abeta) deposits in the APP(V717F) mouse model for AD. The M-CSFR is also increased on microglia after experimental brain injury and in AD. To determine the relevance of these findings, we transiently expressed M-CSFR on murine BV-2 and human SV-A3 microglial cell lines using an SV40-promoted c-fms construct. M-CSFR overexpression resulted in microglial proliferation and increased expression of inducible nitric-oxide synthase, the proinflammatory cytokines interleukin-1alpha, macrophage inflammatory protein 1-alpha, and interleukin-6 and of macrophage colony-stimulating factor (M-CSF) itself. Antibody neutralization of M-CSF showed that the M-CSFR-induced proinflammatory response was dependent on M-CSF in the culture media. By using a co-culture of c-fms-transfected murine microglia and rat organotypic hippocampal slices and a species-specific real time reverse transcriptase-polymerase chain reaction assay and enzyme-linked immunosorbent assay, we showed that M-CSFR overexpression on exogenous microglia induced expression of interleukin-1alpha by the organotypic culture. These results show that increased M-CSFR expression induces microglial proliferation, cytokine expression, and a paracrine inflammatory response, suggesting that in APP(V717F) mice increased M-CSFR on microglia could be an important factor in Abeta-induced inflammatory response.

Recombinant adeno-associated viral (rAAV) vectors are capable of long-term expression of secreted and intracellular proteins following delivery to muscle, liver, and the central nervous system. In this study, we have evaluated subcutaneous injection of rAAV encoding a variety of transgenes as an alternative route of administration for the systemic delivery of therapeutic proteins.rAAV vectors encoding the human factor IX, human interferon-alpha 2a, murine erythropoietin (epo), and Escherichia coli lacZ genes were used for subcutaneous delivery into mature immunocompetent mice. Expression of factor IX and interferon in mouse serum was measured by ELISA. Expression of Epo was monitored by an increase in hemotocrit and by RIA. The tissue tropism of AAV transduction was determined by histochemistry following administration of the lacZ vector.Long-term protein expression (at least one year) is demonstrated in the serum of immunocompetent mice following subcutaneous delivery of AAV vectors encoding the human factor IX and interferon genes. The murine epo gene delivered via this route resulted in levels of Epo that correlate with increased hematocrits of up to 90% for a duration of nine months. rAAV encoding the lacZ gene revealed that the panniculus carnosus, a skeletal muscle layer of the skin, was transduced upon subcutaneous administration.This study shows that long-term expression of secreted proteins can be achieved using rAAV vectors injected subcutaneously as a single administration. The observation that the panniculus carnosus is the primary tissue transduced by rAAV illustrates the high tropism of rAAV for skeletal muscle.

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.

The aim of the present study was to examine the in vivo effect of interleukin (IL)-12 on a murine model of asthma induced by Dermatophagoides pteronyssinus-derived Der p 1 allergen. C57BL/6 mice immunized with Der p 1 allergen adsorbed to alum/pertussis toxin developed a T-helper type 2 (Th2)-dominant immune response characterized by the presence of IgE antibody, airway eosinophil infiltration and increased production of Th2 cytokine. Intraperitoneal injection of IL-12 (1 or 0.1 microg per day) for 5 days (day -1 to +3) simultaneously with each immunization, inhibited the production of IgE and IgG1 antigen-specific antibodies, whereas production of IgG2a was strongly enhanced. In addition, mice receiving both doses of IL-12 showed a strong inhibition of IL-5 but up-regulation of IFN-gamma production by spleen cells stimulated with antigen. Administration of IL-12 also prevented antigen-induced eosinophil infiltration into the bronchoalveolar area in a dose-dependent manner and the primary inflammatory mediator serotonin in bronchoalveolar lavage (BAL) fluids was also reduced significantly. Taken together, the data indicate that IL-12 has a potent immunomodulatory effect on house-dust-mite-induced allergic disorders and may be used as an efficient agent for immunotherapy.

The present study evaluates the physiological effects of magnetic stimulation on astrocyte cultures. Cell cultures were exposed to pulsed magnetic stimulation (10 Hz, 10 sec) at the following levels: 0.10 tesla (T; Group A); 0.21 T (Group B); 0.42 T (Group C); and 0.63 T (Group D). Glial fibrillary acidic protein (GFAP) levels from immunoblots, total protein concentrations, and cellular morphology were analyzed at 0, 1, 3, 5, 7, 13, and 20 days poststimulation. Significantly higher GFAP levels were observed in Group D at day 3 (P = 0.0114). The change was transient as the GFAP levels quickly returned to control levels by day 5. No other significant changes in GFAP levels were observed. In comparison to control protein levels at day 0, concentrations from Groups B, C, and D were significantly lower (P0.006), whereas at day 3, Groups C and D were significantly higher (P0.02). Differences in astrocyte morphology due to magnetic stimulation were not observed. This study demonstrated that high intensity magnetic stimulation for only 10 sec induced a transient biological response.

Mice carrying copies of the human glial fibrillary acidic protein (hGFAP) gene driven by its own promoter have been generated that express the human transgene at different levels (Messing et al.: 152:391-398, 1998). Lines that expressed high levels of the gene died shortly after birth. Astrocyte cultures prepared from a low overexpressor (Tg73.2) exhibited abnormal cytoplasmic inclusions identical to those seen in vivo in the high overexpressors. Astrocytes in the Tg73.2 cultures appear odd-shaped and enlarged, express increased levels of GFAP (both human and mouse), and express alphaB crystallin protein, Hsp27 protein, and vimentin protein. At the light microscopic level, the Tg73.2 astrocytes are filled with eosinophilic deposits surrounded by positive GFAP immunostain. Ultrastructurally, the Tg73.2 astrocytes contain osmophilic deposits on a bed of intermediate filaments identical to Rosenthal fibers found in the brain in Alexander's disease. It seems that Tg73.2 mouse astrocytes in culture do not require additional stress from external sources or contact with other neuroectodermal cells to produce Rosenthal fibers. This suggests that the added hGFAP gene is sufficient to induce Rosenthal fibers and that an excess of GFAP in astrocytes may be detrimental to normal function. We hypothesize that the normal mechanism for GFAP turnover may be insufficient to handle the excess GFAP, thus causing an accumulation of stress proteins. The increased amounts of stress proteins and GFAP results in the formation of Rosenthal fibers, similar to those found in Alexander's disease.

In this study, we provide direct evidence that the magnitude and nature of the immune response to a DNA vaccine can be differentially regulated by codelivery of various mouse cytokine genes. Mice immunized with a hepatitis B virus (HBV) DNA vaccine and the IL-12 or IFN-gamma gene exhibited a significant enhancement of Th1 cells and increased production of anti-HBV surface IgG2a Ab, as well as a marked inhibition of Th2 cells and decreased production of IgG1 Ab. In contrast, coinjection of the IL-4 gene significantly enhanced the development of specific Th2 cells and increased production of IgG1 Ab, whereas Th1 differentiation and IgG2a production were suppressed. Coinjection of the IL-2 or the granulocyte-macrophage-CSF gene enhanced the development of Th1 cells, while the development of Th2 cells was not affected, and the production of IgG1 and IgG2a Ab were both increased. The CTL activity induced by HBV DNA vaccination was most significantly enhanced by codelivery of the IL-12 or IFN-gamma gene, followed by the IL-2 or granulocyte-macrophage-CSF gene, whereas codelivery of the IL-4 gene suppressed the activity. When challenged with HBV surface Ag (HBsAg)-expressing syngeneic tumors, significant reduction of tumor growth was observed in mice that were coadministered the IL-12 gene but not the IL-4 gene. Taken together, these results demonstrate that application of a cytokine gene in a DNA vaccine formulation can influence the differentiation of Th cells as well as the nature of an immune response and may thus provide a strategy to improve its prophylactic and therapeutic efficacy.

A traumatic injury to the adult mammalian central nervous system (CNS), such as a stab wound lesion, results in reactive astrogliosis and the migration of hematogenous cells into the damaged neural tissue. The roles of cytokines and growth factors released locally by the damaged endogenous cells are recognized in controlling the cellular changes that occur following CNS injury. However, the role of chemokines, a novel class of chemoattractant cytokines, is only recently being studied in regulating inflammatory cell invasion in the injured/diseased CNS (1). The mRNAs for several chemokines have been shown to be upregulated in experimental allergic encephalomyelitis (EAE), an inflammatory demyelinating disease of the CNS, but chemokine expression in traumatic brain injury has not been studied in detail. Astrocytes have been demonstrated to participate in numerous processes that occur following injury to the CNS. In particular, astrocytic expression of cytokines and growth factors in the injured CNS has been well reviewed (2). Recently a few studies have detected the presence of chemokines in astrocytes following traumatic brain injury (3,4). These studies have suggested that chemokines may represent a promising target for future therapy of inflammatory conditions. This review summarizes the events that occur in traumatic brain injury and discusses the roles of resident and non-resident cells in the expression of growth factors, cytokines and chemokines in the injured CNS.

A stable cationic lipid/DNA complex has been developed for in vivo gene transfer. The formulation capitalizes on a previously described procedure to obtain stable lipid/DNA complexes for in vitro gene transfer (1).Conditions for DNA/lipid complex formation were modified to yield a DNA concentration of 1 mg/ml. Heat stable alkaline phosphatase (AP) under a CMV promoter was used as a reporter gene.The resulting complex was completely insensitive to serum inactivation. Tail vein injection of a 80 micrograms DNA into Balb C mice yielded significant levels of reporter enzyme activity in the lung, heart, spleen, muscle, and liver. Less AP activity was observed in the kidney. No AP activity was observed in blood, bone marrow or brain. A titration of the lipid (DOSPA) to DNA-nucleotide ratio showed the optimal molar ratio for in vivo gene transfer to be 1/1. Using this ratio in a dose response study showed approximately 80 micrograms of DNA/mouse yielded the highest level of gene expression. Using this dose at a 1/1 lipid to DNA nucleotide ratio, the time course for alkaline phosphatase activity was determined. Maximal AP activity was observed 24 hours after injection for all tissues. By day 5, the activity dropped approximately 10 fold for all tissues. By day 7, residual activity was detected in the lung, heart, and muscle. Histology of the lung showed both interstitial and endothelial cells to be transfected. In all other tissues, however, endothelial cells were the only transfected cell type.These results demonstrate that reformulation of an existing cationic lipid can result in the formation of a stable lipid/DNA complex, which is able to reproducibly transfect lung, heart, spleen, and liver upon intravenous administration.

A traumatic injury to the adult mammalian central nervous system (CNS) results in reactive astrogliosis and the migration of hematogenous cells into the damaged neural tissue. Chemokines, a novel class of chemoattractant cytokines, are now being recognized as mediators of the inflammatory changes that occur following injury. The expression of MCP-1 (macrophage chemotactic peptide-1), a member of the beta family of chemokines, has recently been demonstrated in trauma in the rat brain (Berman et al.: J Immunol 156:3017-3023, 1996). Using a stab wound model for mechanical injury, we studied the expression of two other beta chemokines: RANTES (Regulated on Activation, Normal T cell Expressed and Secreted) and MIP-1 beta (macrophage inflammatory protein-1 beta) in the rat brain. The stab wound injury was characterized by widespread gliosis and infiltration of hematogenous cells. Immunohistochemical staining revealed the presence of RANTES and MIP-1 beta in the injured brain. RANTES and MIP-1 beta were both diffusely expressed in the necrotic tissue and were detected as early as 1 day post-injury (dpi). Double-labeling studies showed that MIP-1 beta, but not RANTES, was expressed by reactive astrocytes near the lesion site. In addition, MIP-1 beta staining was also detected on macrophages at the site of injury. The initial expression of the chemokines closely correlated with the appearance of inflammatory cells in the injured CNS, suggesting that RANTES and MIP-1 beta may play a role in the inflammatory events of traumatic brain injury. This study also demonstrates for the first time MIP-1 beta expression in reactive astrocytes following trauma to the rat CNS.

Tumor necrosis factor-alpha is a pluripotent cytokine that is reportedly mitogenic to astrocytes. We examined expression of the astrocyte intermediate filament component glial fibrillary acidic protein in astrocyte cultures and the U373 glioblastoma cell line after treatment with tumor necrosis factor-alpha. Treatment with tumor necrosis factor-alpha for 72 h resulted in a decrease in content of glial fibrillary acidic protein and its encoding mRNA. At the same time, tumor necrosis factor-alpha treatment increased the expression of the cytokine interleukin-6 by astrocytes. The decrease in glial fibrillary acidic protein expression was greater when cells were subconfluent than when they were confluent. Thymidine uptake studies demonstrated that U373 cells proliferated in response to tumor necrosis factor-alpha, but primary neonatal astrocytes did not. However, in both U373 cells and primary astrocytes tumor necrosis factor-alpha induced an increase in total cellular protein content. Treatment of astrocytes and U373 cells for 72 h with the mitogenic cytokine basic fibroblast growth factor also induced a decrease in glial fibrillary acidic protein content and an increase in total protein level, demonstrating that this effect is not specific for tumor necrosis factor-alpha. The decrease in content of glial fibrillary acidic protein detected after tumor necrosis factor-alpha treatment is most likely due to dilution by other proteins that are synthesized rapidly in response to cytokine stimulation.

Macrophage inflammatory protein 1 (MIP-1) is a recently characterized inflammatory and chemokinetic cytokine. Proinflammatory stimuli have been shown to induce expression of MIP-1 by macrophages. We hypothesized that microglia and astrocytes express MIP-1 alpha because of their many immunologic similarities to macrophages. MIP-1 alpha mRNA was examined with quantitative reverse transcription and polymerase chain reaction in an immortalized mouse microglial cell line (BV-2) and in mouse cortical astrocyte cultures. We found that in both the BV-2 microglial cell line and in astrocyte cultures, MIP-1 alpha mRNA was strongly induced by lipopolysaccharide and the phorbol ester PMA. MIP-1 alpha mRNA was reduced by dBcAMP, interferon-gamma, and PGE1. Dexamethasone decreased MIP-1 alpha mRNA levels in astrocyte cultures, but not in BV-2 microglial cells. Interleukin-1 beta, tumor necrosis factor alpha, and MIP-1 alpha had no effect on MIP-1 alpha mRNA expression. These findings demonstrate that MIP-1 alpha mRNA is expressed by cultured glial cells and is regulated by proinflammatory and anti-inflammatory stimuli. MIP-1 alpha may be expressed by microglia and astrocytes in vivo, and may help modulate cerebral inflammation.

Leukemia inhibitory factor (LIF) is a multifunctional cytokine synthesized by a variety of cell types. In the nervous system LIF affects neuronal differentiation, and may be important during cerebral infection and inflammation. To clarify the cellular source of LIF in the brain, we examined the expression of LIF mRNA by primary cortical astrocyte cultures and an immortalized microglial cell line. The microglial cell line did not express LIF mRNA in response to pro-inflammatory agents such as lipopolysaccharide (LPS) that induced expression of other cytokine mRNAs. In contrast, primary astrocyte cultures grown in serum-containing medium expressed LIF mRNA constitutively, and this expression was regulated by pro-inflammatory and anti-inflammatory stimuli. Agents which activate the cAMP and protein kinase C second messenger systems also increased LIF mRNA in astrocyte cultures. These results suggest that astrocytes, but not microglia, may be an important source of LIF during cerebral inflammation and infection.

Involvement of the IEGs in brain injury and ischemia is under intensive investigation (Gubits et al., 1993). There are several families of the IEGs. They include the fos, jun, and zinc finger genes that encode transcription factors. Products of the fos family (c-fos, fra-1, fra-2, and fos B) bind to members of the jun family (c-jun, jun B, jun D) via leucine zippers, and this dimer then binds to the AP-1 site (consensus sequence -TGACTCA-) in the promoter of target genes, which in turn regulate the expression of late response genes that produce long-term changes in cells. For example, c-fos may regulate the long-term expression of preproenkephalin, nerve growth factor, dynorphin, vasoactive intestinal polypeptide, tyrosine hydroxylase and other genes with AP-1 sites in their promoters (Curran and Morgan, 1987; Sheng and Greenberg, 1990). It is likely that the c-fos gene up-regulation observed in ischemic astrocytes leads to the changes observed in the expressions of hsp and cytoskeleton protein genes in this experimental model. This is supported by the findings of Sarid (1991) and Pennypacker et al. (1994) who have shown that AP-1 DNA binding activity in hippocampus recognized an AP-1 sequence from the promoter region of the GFAP which is a potential target gene. van de Klundert et al. (1992) also suggested the involvement of AP-1 in transcriptional regulation of vimentin. IEGs can be induced within minutes by extracellular stimuli including transmitters, peptides, and growth factors. In this study, we have shown that c-fos induction by ischemia was rapid and transient.(ABSTRACT TRUNCATED AT 250 WORDS)

Despite the definite etiologic link between apolipoprotein (apo) E mutations and type III hyperlipoproteinemia (HLP), it is not clear what additional factors are involved in the development of florid hyperlipidemia and how to explain the wide variability in the expression of the hyperlipidemic phenotype in carriers of receptor binding-defective apoE variants. The present study was designed to determine whether the overexpression of cholesteryl ester transfer protein (CETP), a plasma protein that transfers cholesteryl esters from the high density lipoproteins (HDL) to the very low density lipoproteins (VLDL) and whose activity is increased in hyperlipidemic states, plays a role in the development of hyperlipidemia and beta-VLDL accumulation in type III HLP. We produced double-transgenic mice that co-expressed high levels of simian CETP and either high or low levels of a human receptor binding-defective apoE variant, apoE(Cys-142). We previously reported that apoE(Cys-142) high-expresser mice showed spontaneous hyperlipidemia and accumulation of beta-VLDL, whereas the low-expresser mice showed only a modest increase in VLDL cholesterol. Co-expression of CETP induced a massive transfer of cholesteryl esters from the HDL to the VLDL in both lines of double-transgenic mice. As a result, HDL cholesterol and apoA-I levels were reduced to about 50% of normal, VLDL cholesterol increased 2.5-fold, and the cholesteryl ester content of VLDL reached values similar to those observed in human beta-VLDL. The ratio of defective to normal apoE in VLDL was unaffected by CETP co-expression and was higher in animals expressing high apoE levels. Finally, in spite of an increased accumulation of beta-VLDL in the high-expresser mice, the VLDL of the low-expresser mice maintained pre-beta mobility upon co-expression of CETP. The results of this study demonstrate that the ratio of defective to normal apoE on the VLDL, rather than the cholesteryl ester content of VLDL, is the major factor determining the development of severe hyperlipidemia and the formation and accumulation of beta-VLDL in type III HLP.

To determine the impact of enhanced apolipoprotein (apo) E secretion on the mechanism of remnant lipoprotein uptake, rat hepatoma cells (McA-RH7777) were stably transfected with normal human apoE3 or receptor binding-defective apoE-Leiden. After a 2-h incubation, the human apoE secreted from the transfected hepatocytes was 10-12 times greater than the endogenous rat apoE. The apoE3-transfected cells bound and internalized rabbit beta-very low density lipoproteins (beta-VLDL) to a much greater degree than did apoE-Leiden-transfected cells and nontransfected cells. The apoE3-secreting cells displayed a 2-3.5-fold enhancement of cell-associated beta-VLDL compared to either the apoE-Leiden-transfected or nontransfected cells. Fluorescently labeled beta-VLDL were observed to concentrate within intracellular granules of the apoE3-transfected cells, presumably within endosomes and lysosomes. Furthermore, electron microscopy revealed that the apoE3-secreting cells displayed abundant beta-VLDL and chylomicron remnants on their cell surfaces and microvilli, in contrast to non-transfected or apoE-Leiden-secreting cells. Electron microscopy also revealed an abundance of chylomicron remnants within intracellular vesicles and multivesicular bodies of apoE3-transfected hepatocytes. Heparinase treatment (3 units/ml) completely abolished the increased association of beta-VLDL with apoE3-transfected cells but did not affect the limited association of beta-VLDL with apoE-Leiden-transfected or nontransfected cells. We established that the apoE3-enriched beta-VLDL were bound to cell surface heparan sulfate proteoglycans, as was the newly synthesized and secreted apoE3 (approximately 12% of the total secreted apoE3 was released by heparinase and suramin; 4% by heparin). In addition, reisolation of beta-VLDL by fast performance liquid chromatography after their incubation with exogenous apoE3, with medium from apoE3-secreting cells, or with the apoE3-secreting cells themselves revealed that the particles were enriched in apoE3 and displayed enhanced binding. These results suggest a secretion-capture role for apoE and indicate an important role for heparan sulfate proteoglycans on the cell surface for remnant lipoprotein metabolism.

The reverse transcription and polymerase chain reaction technique (RT-PCR) was assessed for the quantification of changes in mRNA levels from primary astrocyte cultures. The effects of dibutyryl cyclic AMP (dBcAMP) on glial fibrillary acidic protein (GFAP) mRNA and the effects of tumor necrosis factor-alpha (TNF-α), interlekin-1 beta (IL-1β), and lipopolysaccharide (LPS) on interleukin-6 (IL-6) mRNA were examined. Two quantitative PCR methods were used: one involved carrying out the reaction in the exponential phase and the other involved the coamplification of a competitive target sequence. Increased GFAP mRNA in response to chronic dBcAMP treatment and increased IL-6 mRNA in response to TNF-α/IL-1β were readily detected. Both RT-PCR techniques were found to be suitable for the detection of large as well as smaller (twofold) changes in mRNA levels. The advantages and limitations of RT-PCR for mRNA quantification are discussed. © 1993 Wiley-Liss, Inc.

In October 1989, an epizootic duckling disease with high mortality occurred in Taiwan. The disease was characterized by droopiness, inappetence, ataxia, ruffled feathers, and watery diarrhea. Affected ducklings were lame, were unable to stand, showed opisthotonos, and often died 3 or 4 days after the onset of the disease. Tolerant maturing ducklings displayed atrophic upper bills with a protruding tongue and became stunted as they reached maturity. No diagnostic histopathologic lesions were found in these ducklings. Fourteen parvovirus isolates, 33 duck viral hepatitis virus (DVHV) isolates, two adenovirus isolates, and two reovirus isolates were obtained and identified from more than 500 sick ducklings in the epizootic. The epizootic was diagnosed as a co-outbreak of duck parvovirus infections and duck viral hepatitis. The high mortality in ducklings and the bill atrophy syndrome were reproduced in ducklings by inoculating the parvovirus isolates alone. The epizootic was controlled by an emergency immunization program of ducklings with sera collected from recovered ducks or a bivalent inactivated vaccine composed of local DVHV and parvovirus isolates.

Brucella abortus may be useful as a component of vaccines. This is because it possesses several unique properties as a carrier that enable it to stimulate human B cells even in the relative absence of T cells. Human immunodeficiency virus type 1 proteins conjugated to B. abortus could induce neutralizing antibodies against human immunodeficiency virus type 1. Recently we showed that the characteristics of lipopolysaccharide (LPS) derived from B. abortus are similar to those of the whole bacterium in that the LPS acts as a T-independent type 1 carrier in mice. In this study we wanted to determine whether LPS derived from B. abortus is associated with the adverse effects seen with other bacterial endotoxins. LPS purified from B. abortus by butanol extraction was shown to have less than 2% (wt/wt) contamination by protein and less than 1% (wt/wt) contamination by nucleic acids and to contain 1% (wt/wt) ketodeoxyoctanic acid. Compared with LPS derived from Escherichia coli, B. abortus LPS was 10,000-fold less potent in eliciting fever in rabbits, 268-fold less potent in killing D-galactosamine-sensitized mice, and 1,400-fold and 400-fold less potent in inducing interleukin-1 beta and tumor necrosis factor alpha production, respectively. These results suggest that B. abortus LPS is much less likely than the LPS from E. coli to evoke endotoxic shock; therefore, it may be feasible to incorporate B. abortus as a component of vaccines.

Glial fibrillary acidic protein (GFAP) accumulation is a prominent feature of astrocytic gliosis. The inhibition or delay in GFAP synthesis might delay scar formation resulting from an insult such as spinal cord injury or central nervous system (CNS) demyelination. The delay in the formation of a physical barrier might allow the neurons and oligodendrocytes to reestablish a functional environment. We delivered antisense GFAP RNA complexed with LipofectinTM (LF), a cationic liposome, into cerebral astrocytes in culture and tested the feasibility of inhibiting GFAP synthesis. Our results demonstrate that LF facilitated antisense RNA uptake into astrocytes. Astrocytes took up 3H-antisense GFAP RNA alone and reached an equilibrium of 7–8.8 ηg per mg protein after 2.5 hr. When complexed with LF, astrocytes could increase the uptake to 14 ηg per mg protein and the time for reaching this quantity was shortened to 10 min. This uptake level was further enhanced if experiments were carried out in HEPES buffered saline (HBS). All uptake studies were dose- and time-dependent. Dibutyryl cyclic AMP (dBcAMP) is known to induce an increase of GFAP content in cultured astrocytes. We studied the effect of LF/antisense GFAP RNA on the GFAP content in dBcAMP (0.25 mM)-treated astrocytes. Cultures of astrocytes treated with dBcAMP contained almost twice as much GFAP as untreated cultures after 2 days. Similar cultures treated with LF/antisense RNA in HBS did not show an increase but a 30–40% decrease in GFAP content 2 days after treatment. A similar decrease in GFAP content was obtained in cultures grown in a chemically defined medium, another condition known to induce an increase in GFAP content in cultures. This study has demonstrated that antisense GFAP RNA can inhibit GFAP synthesis in astrocytes and may be useful for regulating astrogliosis immediately following CNS injury.

Whereas organs from donors must be removed almost immediately after death to maximize organ viability in the recipient, there is a slightly longer window for tissue allograft recovery. To determine the maximum safe interval after death within which bone allografts may be harvested for clinical use, an experimental model was devised using adult Sprague-Dawley (SD) rats and duplicating cadaveric storage techniques. Allografts were procured at increasing time intervals after death. The grafts were then transplanted to 80 living SD rats, and the animals killed at 7 weeks to evaluate any increase in the risk of infection and bacterial colonization. None of the allografts procured within 48 h after death were colonized with bacteria, while 12% of grafts procured at 96 h and 50% of allografts procured at 1 week were colonized. The results suggest that it may be possible to extend the safe period within which cadaveric tissue may be procured for transplantation to up to 96 h following death, provided scrupulous measures to prevent and detect microorganism contamination are followed.

Glial fibrillary acidic protein (GFAP) is the major constituent of glial filaments and is restricted within the CNS to astrocytes. As with other classes of intermediate filament proteins, the regulation of GFAP expression is poorly understood. Utilizing highly purified cultures of astrocytes and a chemically defined (CD) medium, we have demonstrated that the expression of GFAP is subject to regulation by hormones and growth factors. The concentration of GFAP/mg protein was induced 2-4-fold in the presence of hydrocortisone, putrescine, prostaglandin F-2 alpha (PGF2 alpha), and pituitary fibroblast growth factor (FGF). Augmentation of the levels of GFAP continued for up to 3 weeks after conversion to CD medium and paralleled the morphological maturation of astrocytes. The accumulation of GFAP resulted from an increase in its specific rate of synthesis. Conversion of astrocytes from serum-supplemented (SS) to CD medium did not alter its rate of degradation. GFAP appeared quite stable under both sets of conditions, exhibiting a half-life of approximately 7.5 days. The data demonstrate that GFAP expression in astrocytes is subject to hormonal regulation, which may have implications for gliosis.

Cells cultured from the caudalmost area of regenerating teleost spinal cord differ both morphologically and in terms of molecular architecture from those of more rostral (more fully differentiated) areas of the cord. The caudalmost regenerating cord consists of an ependymal tube. Cells from this region have flattened or partially flattened cell somas and short spike projections in vitro; they do not exhibit the rounded cell somas nor the long, thin, branching neurites typical of differentiated neurons. A series of cultures taken from different areas along the length of regenerating spinal cord were examined for molecular differentiation by staining with a monoclonal antibody against non-phosphorylated neurofilament protein (SMI 32). None of the cells from the caudalmost culture of the regenerating spinal cord stained with antibody SMI 32. In cultures of more rostral regenerated cord, the cells with neuronal morphology do stain positively with the anti-neurofilament antibody. This result suggests that the cells in the cultures of caudalmost cord represent relatively undifferentiated ependymal cells, or ependyma-derived cells, which later differentiate into neurons and glia in the regenerating spinal cord.

Neonatal rat brain astrocyte secondary cultures were established on nitrocellulose membrane filters (13-mm diameter Millipore disk) and on plastic coverslips in serum-supplemented medium. On these substrata, cultured astrocytes changed their shape from flat and polygonal to stellate in the absence of hormones or growth factor supplements. Cultures became confluent after 4 days, and astrocytes on nitrocellulose filters continued to differentiate morphologically and biochemically, as evidenced by extensive cytoplasmic process formation and glial fibrillary acid protein (GFAP) accumulation. Cultures were immunostained for GFAP and vimentin. mRNAs to GFAP, vimentin, alpha and beta tubulin, and actin also were detected by in situ hybridization with biotinylated cDNA probes. The astrocyte culture method on nitrocellulose provides a simple, versatile means of comparing undifferentiated, morphologically mature, reactive, and neoplastic astrocytes in vitro.

The rate of clearance of porcine myelin basic protein (MBP) from plasma of rabbits was determined following intravenous injection of 20 mg MBP. The MBP level in the plasma was measured by a 2-site immunoradiometric assay with specific antibody to guinea pig MBP produced in rabbits. Plasma MBP-antibody levels were determined by competitive binding radioimmune assay (RIA). Unsensitized and those sensitized with complete Freund's adjuvant (CFA), with porcine MBP in CFA, and with whole porcine spinal cord in CFA were studied. Unsensitized and CFA sensitized rabbits exhibited maximum MBP levels in the plasma within two minutes after injection with rapid decrease to undetectable levels in one hour. Thirty-nine of the unsensitized (control) rabbits exhibited normal, rapid clearance and no subsequent physical signs of EAE while one of the control rabbits exhibited a slightly retarded clearance rate. Histologic examination of autopsy tissues from the control group revealed that five rabbits showed lesions which could be attributed to Encephalitozoan cuniculi or Toxoplasma and one rabbit autopsied 65 days after clearance had minimal EAE lesions. Rabbits sensitized with MBP exhibited a retarded rate of clearance at the acute stage of EAE and following recovery. Rabbits sensitized with whole spinal cord in CFA also exhibited a retarded rate of MBP clearance. Anti (MBP) antibodies were detected in the plasma of all rabbits which exhibited a retarded rate of MBP clearance. Significant rates of retardation were not detected until approximately three weeks after sensitization with CFA-MBP or CFA-spinal cord. While MBP antibody levels in most animals were not detected by the immunodiffusion technique, antibodies were demonstrated by RIA. The 20 mg MBP given intravenously is probably in great antigen excess and conducive to the formation of soluble MBP-anti (MBP) complexes in the blood.