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The hematopoietic growth factor, granulocyte colony-stimulating factor (G-CSF), has become one of the few growth factors approved for clinical use. It has therapeutic potential for numerous neurodegenerative diseases; however, at present the cellular effects of G-CSF on the central nervous system remain unclear and in need of investigation. In the present study, we used spinal cord ischemia, a neurodegenerative model, to examine the effects of intrathecal (i.t.) G-CSF on glial cell (microglia and astrocyte) activation and neuroprotective factor expression, including glial cell line-derived neurotrophic factor (GDNF) and vascular endothelial growth factor A (VEGF-A) protein expression. Our results indicate that i.t. G-CSF could enhance ischemia-induced microglial activation and inhibit ischemia-induced astrocyte activation. Both GDNF and VEGF-A are upregulated after injury, and i.t. G-CSF could enhance GDNF and VEGF-A expressions after injury. Interestingly, our results indicate that performing i.t. G-CSF alone on normal animals could have the effect of microglial and astrocyte activation and enhanced GDNF and VEGF-A expressions. Furthermore, through laser scanning confocal microscopy, we found that astrocytes may contribute to the majority of GDNF and VEGF-A expressions of G-CSF after spinal cord ischemia. Overall, this G-CSF-induced upregulation suggests that activation of endogenous neuroprotective mechanisms could resist neurodegenerative insults. These observations demonstrate the cellular mechanism of i.t. G-CSF after spinal cord ischemia and confirm the neuroprotective effect of G-CSF after spinal cord ischemia injury.

The present study was designed to investigate the pharmacokinetics following oral and intravenous administration of Riccardin D, an anticancer drug candidate isolated from Chinese liverworts Dumortiera hirsute , in Wistar rats. An HPLC-MS/MS analytical method was developed and validated. The results demonstrated that Riccardin D’s bioavailability was 13.4%, 11.4%, and 9.8% after oral administration at 20 mg/kg, 40 mg/kg, and 80 mg/kg, respectively. There was no significant difference in the elimination half-time of Riccardin D at these doses, suggesting that Riccardin D may have linear pharmacokinetic characteristics in rats. The metabolite of Riccardin D in rat was identified as the glucuronide of Riccardin D. Riccardin D showed a wide distribution in various tissues followed by a rapid elimination from most of the tissues tested. Riccardin D was found to distribute widely in the tissues 0.5 h after oral and intravenous administration. The tissue concentrations were markedly decreased 8 h and 6 h after oral and intravenous dosing, respectively. Both Riccardin D and its conjugated metabolite were detected in urine and bile samples while only Riccardin D was detected in feces. Taken together, the study provided valuable pharmacokinetic data for further drug development of Riccardin D.

Summary Objective To study the effects of oral glucosamine sulfate on the development of osteoarthritis (OA) and to examine concomitant changes in the nociceptive behavior of rats. Methods OA was induced in Wistar rats by anterior cruciate ligament transection (ACLT) of the right knee; the left knee was untreated. The OA+glucosamine group received oral glucosamine sulfate (250mg/kg/day) in a 2-g wafer once a day for 10 consecutive weeks starting at week 5 after ACLT. The OA group was treated as above with 2-g wafers (placebo). The control group of naive rats received 2-g wafers only. The glucosamine alone group comprised naive rats receiving glucosamine sulfate only. Nociceptive behavior (mechanical allodynia and weight-bearing distribution of hind paws) during OA development was analyzed pre- and 3, 6, 9, 12, 15, and 18 weeks post-ACLT. Macroscopic and histologic studies were then performed on the cartilage and synovia. Immunohistochemical analysis was performed to examine the effect of glucosamine on expression of mitogen-activated protein kinases (MAPKs) in the articular cartilage chondrocytes. Results OA rats receiving glucosamine showed a significantly lower degree of cartilage degeneration than the rats receiving placebo. Glucosamine treatment also suppressed synovitis. Mechanical allodynia and weight-bearing distribution studies showed significant improvement in the OA+glucosamine group as compared to the OA group. Moreover, glucosamine attenuated p38 and c-Jun N-terminal kinase (JNK) but increased extracellular signal-regulated kinase 1/2 (ERK) expression in OA-affected cartilage. Conclusion Our results indicate that treatment with oral glucosamine sulfate in a rat OA model (1) attenuates the development of OA, (2) concomitantly reduces nociception, and (3) modulates chondrocyte metabolism, possibly through inhibition of cell p38 and JNK and increase of ERK expression.

Granulocyte colony-stimulating factor (G-CSF) is a potent hematopoietic factor. Recently, this factor has been shown to exhibit neuroprotective effects on many CNS injuries. Spinal cord ischemic injury that frequently results in paraplegia is a major cause of morbidity after thoracic aorta operations. In the present study, we examined the neuroprotective role of G-CSF on spinal cord ischemia-induced neurological dysfunctions and changes in the mitogen-activated protein kinase (MAPK) and Akt signaling pathways in the spinal cord. Spinal cord ischemia was induced in male Wistar rats by occluding the descending aorta with a 2F Fogarty catheter for 12 min 30 s. Immediately after ischemia surgery, the rats were administered G-CSF (10 mug) or saline by intrathecal (i.t.) injection. The rats were divided into four groups: control, ischemia plus saline, ischemia plus G-CSF and G-CSF alone. The neurological dysfunctions were assessed by calculating the motor deficit index after ischemia surgery. The expressions of MAPK and Akt were studied using Western blotting and double immunohistochemistry. First, we observed that ischemia plus i.t. G-CSF can significantly reduce the motor function defects and downregulate phospho-p38 and phospho-c-Jun N-terminal kinase protein expressions-this can be compared with the ischemia plus saline group. In addition, G-CSF inhibited the ischemia-induced activation of p38 in the astrocytes. Furthermore, we concluded that i.t. G-CSF produced a significant increase in phospho-Akt and phospho-ERK in the motor neurons and exhibited beneficial effects on the spinal cord ischemia-induced neurological defects.

Spinal cord ischemic injury usually results in paraplegia, which is a major cause of morbidity after thoracic aorta operations. Ample evidence indicates that massive release of excitatory amino acids (EAAs; glutamate) plays an important role in the development of neuronal ischemic injuries. However, there is a lack of direct evidence to indicate the involvement of EAAs in the glutamate metabolizing system (including the glutamate transporter isoforms, i.e. the Glu-Asp transporter (GLAST), Glu transporter-1 (GLT-1), and excitatory amino acid carrier one (EAAC1); glutamine synthetase (GS); and glutamate dehydrogenase (GDH)) in spinal cord ischemia. In the present results, we found that methylprednisolone (MP; intrathecal (i.t.) injection, 200 mug twice daily administered for 3 days before ischemia), a synthetic glucocorticoid, is the therapeutic agent for the treatment of spinal injuries in humans, can significantly reduce the ischemia-induced motor function defect and down-regulate the glutamate metabolizing system (including GLAST, GLT-1, GS, and GDH) in male Wistar rats. The spinal cord ischemia-induced down-regulation of EAAC1 protein expression in the ventral portion of the lumbar spinal cord was partly inhibited by pretreatment with i.t. MP. However, MP did not affect the down-regulation of EAAC1 in the dorsal portion of the lumbar spinal cord after spinal cord ischemia. The i.t. injection of MP alone did not change the neurological functions and the expression of proteins of the glutamate metabolizing system in the spinal cord. Our results indicate that spinal cord ischemia-induced neurological deficits accompany the decrease in the expression of proteins of the glutamate metabolizing system in the lumbar portion of the spinal cord. The i.t. MP pretreatment significantly prevented these symptoms. These results support the observation that MP delivery through an i.t. injection, is beneficial for the treatment of spinal cord ischemic injuries.

To investigate the role of the synthetic steroid tibolone in the progression of osteoarthritis (OA) and in nociceptive behaviour in an experimental rat model of OA and ovariectomy (OVX)-induced osteoporosis.OA was induced in Wistar rats by anterior cruciate ligament transection (ACLT) of the right knee. Osteoporosis was induced by bilateral OVX. Groups of animals were subjected to ACLT, OVX, sham or OVX + ACLT. In addition, two groups were subjected to OVX + ACLT surgeries and were orally administered 0.1 or 0.5 mg tibolone every other day for 14 consecutive weeks, starting 6 weeks after surgery. Nociceptive behaviours (secondary mechanical allodynia and weight-bearing distribution of the hind paws) were analysed prior to and every 3 weeks after surgery up to 24 weeks. At 24 weeks, histopathological studies were performed on the cartilage and synovial membranes of the knee joints, and bone metabolism was assessed by measuring serum concentrations of calcium, phosphorus and alkaline phosphatase.Rats undergoing ACLT or OVX + ACLT surgeries showed obvious OA changes in the joints. Animals subjected to ACLT + OVX and treated with tibolone had significantly less cartilage degeneration and synovitis and showed improved nociceptive tests compared with animals undergoing ACLT + OVX surgeries alone. OVX increased the severity of the ACLT-induced OA changes. There was a significant increase in serum alkaline phosphatase in the tibolone-treated ACLT + OVX groups.Treatment with tibolone attenuated the development of OA, concomitantly reduced nociception and increased serum alkaline phosphatase in ACLT + OVX rats.

Recently, the hematopoietic factor, granulocyte colony-stimulating factor (G-CSF), has been shown to exhibit neuroprotective effects in CNS injuries. Our previous study demonstrated that intrathecal (i.t.) G-CSF significantly improved neurological defects in spinal cord ischemic rats. Considerable evidence indicates that the release of excessive amounts of excitatory amino acids (EAAs) plays a critical role in neuron injury induced by ischemic insult. In the present study, we used a spinal cord ischemia-microdialysis model to examine whether i.t. G-CSF exerted antiexcitotoxicity effects in a rat model of spinal cord ischemia. I.t. catheters and a microdialysis probe were implanted in male Wistar rats. The results revealed that spinal cord ischemia-induced neurological defects were accompanied by a significant increase in the concentration of EAAs (aspartate and glutamate) in the spinal dialysates from 30 min to 2 days after reperfusion. I.t administration of G-CSF immediately after the performance of surgery designed to induce ischemia led to a significant reduction in ischemia-induced increases in the levels of spinal EAAs. Moreover, i.t. G-CSF also brought about a significant reduction in the elevation of spinal EAA concentrations induced by exogenous i.t. administration of glutamate (10 microl of 500 mM). I.t. G-CSF attenuated spinal cord ischemia-induced downregulation of expression of three glutamate transporters (GTs), glial transporter Glu-Asp transporter (GLAST), Glu transporter-1 (GLT-1), and excitatory amino acid carrier 1 (EAAC1) protein 48 h after spinal cord ischemic surgery. Immunohistofluorescent staining showed that i.t. G-CSF significantly upregulated expression of the three GTs in the gray matter of the lumbar spinal cord from 3 to 24 h after injection. We propose that i.t. G-CSF possesses an ability to reduce the extent of spinal cord ischemia-induced excitotoxicity by inducing the expression of glutamate transporters.

Infants who are passively exposed to morphine or heroin through their addicted mothers usually develop neurobiological changes. The postsynaptic density 95 (PSD-95) protein, a submembranous cytoskeletal specialization, is dynamically linked with N-methyl-d-aspartate receptors (NMDARs) to form a synaptic complex in postsynaptic neurons. This complex serves important neurobiological functions, including mammalian learning and memory. However, the effects of prenatal morphine exposure on this synaptic complex are not well understood. In this study, we determined whether prenatal morphine exposure altered the synaptic complex association between PSD-95 and three major NMDAR subunits (NR1, NR2A, and NR2B), at the mRNA and protein levels, within the hippocampal CA1 subregion (an important integration area for mammalian learning and memory) of rat offspring along with the performance of long-term cognitive functions. Sprague-Dawley rat offspring from morphine-addicted mothers were studied at a younger age (postnatal day 14; P14) and at an older age (P45). Subsequently, an eight-arm radial maze task was applied to analyze the working and cued reference memory in such offspring (P45). The real-time polymerase chain reaction results showed that prenatal morphine exposure caused significant decreases in mRNA levels of the PSD-95 and three NMDAR subunits (NR1, NR2A, and NR2B) in offspring (P14 and P45). Similarly, at the protein level, immunoblotting showed that decreased whole levels of PSD-95 and NMDAR subunits were seen in offspring subjected with prenatal morphine. Furthermore, the protein interaction of the synaptic complex between the PSD-95 and NMDAR subunit, as indicated by coimmunoprecipitation, was less in prenatal morphine samples than in vehicle controls (P14 and P45). The prenatal morphine group also showed poorer performance for an eight-arm radial maze task than the vehicle-control group. These results are particularly important for a better understanding of certain opioid-mediated neurobehavioral cognitive changes in offspring associated with altered protein interaction between PSD-95 and NMDAR subunits within the developing brain.

Similar to many diurnal animals, the daily distribution of activity in Drosophila exhibits a bimodal pattern with clock-controlled morning and evening peaks separated by a midday "siesta." In prior work, we showed that the thermosensitive splicing of a 3'-terminal intron in the RNA product from the Drosophila period (per) gene (dper) is critical for temperature-induced adjustments in the timing of evening activity. Cold temperatures enhance the splicing efficiency of this intron (termed dmpi8, Drosophila melanogaster per intron 8), an event that stimulates the daily accumulation of dper RNA and protein, leading to earlier evening activity. Conversely, warm temperatures attenuate dmpi8 splicing efficiency contributing to delayed evening activity, likely ensuring that flies avoid activity during the hot midday sun when they are at increased risk of desiccation. Here, we discuss the underlying molecular mechanisms governing the thermosensitive splicing of dmpi8 and how it contributes to seasonal changes in the daily activity patterns of Drosophila. On a broader perspective, RNA-RNA interactions likely have fundamental roles in the thermal adaptation of life forms to the daily and seasonal changes in temperature.

It is well known that long-term morphine administration results in tolerance, which limits the clinical use of this drug in pain management.Male Wistar rats were randomly assigned to receive one of four different infusions: morphine [15 microg/h, intrathecal (i.t.)], saline, MK-801 (5 microg/h, i.t.) plus morphine (15 microg/h, i.t.), or MK-801 (5 microg/h, i.t.) alone.Morphine infusion induced a maximal antinociceptive effect on day 1 and tolerance on day 3, and the maximal anti-receptive tolerance was observed on day 5. Co-infusing MK-801 with morphine attenuated morphine's anti-receptive tolerance. Two-dimensional gel electrophoretic analysis of spinal proteins revealed that eight protein spots were up-regulated in morphine-tolerant rats, and that they were significantly inhibited by MK-801 co-infusion. Among the up-regulated proteins, glial fibrillary acid protein (GFAP), a glial-specific maker, was identified by mass spectrometry. This finding was also confirmed by Western blot analysis.Using proteomic analysis, we identified eight GFAP protein spots that were up-regulated in the dorsal horn of morphine-tolerant rat spinal cords. This up-regulation was partly inhibited by N-methyl-D-aspartate receptor antagonist MK-801 co-infusion, which suggests that GFAP protein can be considered to be a pathogenesis marker of morphine tolerance.

Mouse platelet basic protein (CXCL7/mPBP) was cloned from thymic stromal cells and further identification indicated that it was expressed in thymic monocytes/macrophages (Mo/Mphis). Recombinant mPBP was chemoattractive for target cells of polymorphonuclear leucocytes, peritoneal Mo/Mphis and splenic lymphocytes with distinct potencies. CXCR2 was identified to be a cognate receptor for mPBP. Mouse thymocyte subsets of CD4-CD8- double-negative (DN), CD4+CD8+ double-positive (DP), CD4+CD8- single-positive (CD4SP) and CD4-CD8+ single-positive (CD8SP) expressed cell surface CXCR2 with different positive percentages and expression levels. mPBP was chemoattractive for thymocyte subsets with the potency order DN>DP> CD8SP>CD4SP, consistent with the levels of CXCR2 expressed on the respective cells. Thus, mPBP in thymus is functionally redundant with chemokine CXCL12/ SDF-1. Moreover, our finding that thymic Mo/Mphis can produce mPBP implies that they may have other functions apart from acting as scavengers in thymus.

The expression pattern of McAb PF18-3 recognized molecule in activation induced thymocyte apoptosis was analysed. Results indicated that following activation by ConA, thymocytes underwent activation induced apoptosis identified by delayed production of DNA ladder and TUNEL positive staining of thymocytes in late stage of activation. PF18-3 molecule was found to express specifically in subset of apoptotic thymocytes with diploid. Kinetic comparison of expression between PF18-3 molecule and Fas or translocated membrane phospholipid suggested that PF18-3 molecule is different from them and likely to be a novel molecule related to thymocyte apoptosis.

Phenotypic analysis of the medullary-type CD4+CD8- (CD4SP) thymocytes have revealed phenotypic heterogeneity within these cells. The phenotype of mature peripheral T cells is Qa-2+ HSA- CD69-, whereas in the medullary-type CD4SP thymocytes, the expression pattern of many markers were quite different, suggesting that the medullary-type CD4SP thymocytes may undergo phenotypic maturation. According to the results of two-color cytometry, seven discrete phenotypes were defined by the relative expression of Qa-2, HSA, CD69, 3G11 and 6C10: 3G11-6C10+CD69+HSAhi--3G11+6C10+CD69+ HSAhi--3G11+6C10-CD69+HSAint--3G11+6C10- CD69-HSAint Qa-2(-)--3G11+HSAlo/-Qa-2lo, at the same time, 3G11+6C10-CD69-HSAint Qa-2(-)--3G11-HSAlo Qa-2(-)--3G11-HSAlo/- Qa-2hi, the last two Qa-2 positive subsets could exit the thymus and home into periphery.

To analyze the capability of IL-1 and IL-6 in the induction of chemokine (CF) production by mouse thymus epithelial cell (MTEC1) clones, MTEC1 cells were cloned through one cell culture and individual cell clones were established in long term culture referred to as MTEC1-DW clones. The constitutive production of IL-1, IL-6 and CF by MTEC1-DW clones was evaluated and the patterns of the cytokine production determined. Addition of exogenous IL-1 or IL-6 or both to the cultures of those MTEC1-DW clones that are unable to produce CF, and incubated for 2 days, then, to assess the chemotactic activity in the cell culture supernatants (SNs). In the opposite, addition of anti-IL-1 mAb(s) to the cultures of those MTEC1-DW clones that can produce IL-1 and CF to neutralize secreted IL-1 then, to test chemotactic activity in the SNs after 2-day incubation. The results showed that in the MTEC1-DW clones which were unable to constitutively produce IL-1 or CF, addition of IL-1 could induce these cloned cells to produce CF with high chemotactic activity. By constrast, addition of anti-IL-1 mAb(s) to those MTEC1-DW clones that constitutively produce IL-1 and CF could significantly inhibit them to produce CF. IL-6 only exhibited weak activity in the induction of CF production by those cloned cells. Therefore, in the cytokine network regulation, CF production is mainly induced by endogenously produced IL-1 in MTEC1 cells.

A medullary type mouse thymic epithelial cell line MTEC1 can produce chemokines. Crude culture supernant of MTEC1 cells was concentrated by controlled-pore glass beads. One of these chemotactic proteins was isolated from MTEC1 supernatant and purified to homogeneity by heparin-Sepharose affinity chromatography, cation-exchange FPLC and Reverse Phase-HPLC. The chemotactic factor for both lymphocytes and monocytes was identified as a 30 kD protein by SDS-PAGE analysis under reducing conditions. After cleavage of the NH2-terminally blocked protein with formic acid, the amino acid sequence of the internal fragment was analyzed and found to be identical to the amino acid sequence of mouse MCP-1/JE. The protein was hence identified as a glycosylated MCP-1/JE-like chemokine secreted by thymic epithelial cells. The characterization of chemokines produced by thymic stromal cells will benefit the analysis of the mechanism of thymus homing and the finding of new chemokines.

Using fast cyclic voltammetry (FCV), atomic absorption/flame emission spectrophotometry and high performance liquid chromatography for electrochemical detection, we studied the change in iron content in the substantia nigra (SN) of 6-hydroxydopamine (6-OHDA) lesioned Parkinsonian (PD) rats and the toxic effect of intranigral injection of iron on DA neurons. The neuroprotective effect of desferrioxamine mesylate was also observed. The results are as follows. (1) The iron content in SN on the lesioned side of 6-OHDA-lesioned PD rats was about three times as high as that in nonstandard PD rats. (2) The iron content in caudate putamen (CPu) on the lesioned side of PD rats was not different from that on the unlesioned side. (3) DA release as well as the content of DA and its metabolites were significantly decreased on the lesioned side of PD rats. (4) In the rats pretreated with intracerebroventricular desferrioxamine mesylate before 6-OHDA injection, the release and content of DA on the lesioned side were not significantly different from those on the unlesioned side. (5) Intranigral injection of 40 micrograms FeCl3 resulted in a dramatic reduction of both DA release and content in CPu. The above results strongly suggest that 6-OHDA reduces the DA release from CPu, in which iron plays an important role. Elevation of iron content in SN is one of the mechanisms responsible for the reduction of DA content. Desferrioxamine mesylate may exert a protective action on dopaminergic neurons.

With the use of beta-EP microinjection into the central nervous system, spectrophotometrical analysis and immunochemical method we studied the effect of beta-Endorphin (beta-EP) on plasma sialic acid (SA) level. The results indicate: (1) The plasma SA level was significantly decreased after microinjection of beta-EP into the lateral ventricle (LV); (2) When beta-EP was microinjection into nucleus arcuatus (ARC), the plasma SA level was significantly decreased (P0.05). This effect can be blocked by i.v. atropine or vagotomy; (3) Microinjection of beta-EP into ARC can cause the increase of CD3, CD4 and CD4/CD8, while CD8 was decreased. The above results suggest that the possible mechanism underlying the decrease of plasma SA level is mediated by cholinergic muscarinic receptor, and the cellular immune function is enhanced.

A murine fetal liver stromal cell line, called MFLC, has been established and maintained in DME supplemented with 10%NCS for 2 years. Without exogenous stimulation, the MFLC cells spontaneously secreted several types of cytokines, including IL-6, GM-CSF and chemotactic factor. Of which, IL-6 and chemotactic factor were abundant, GM-CSF at low level. Biological activities of IL-7 and IL-3 were not detected in the MFLC cell's supernatants. MFLC-SN induced colony formation of murine bone marrow cells in a dose dependent fashion in which mixed granulocyte/macrophage/megakaryocyte colonies(CFU-GMM) and granulocyte/macrophage colonies(CFU-GM) were dominant. MFLC-SN also sustained colony formation of bone marrow cells taken from 5-Fu injected mice, suggesting that there was a biological activity similar to SCF in MFLC-SN. The cytokines secreted by MFLC might play an important role in T cell early development in fetal liver. These results will be useful for us to analyse the mechanism of T cell development in fetal liver and to study the cytokine network regulation.

To study the effect of ATP and extra-ribosomal factors (S-140) on type I and type II RNA N-glycosidases in inactivating ribosome.The activity of ATP and S-140 was determined by characterization of R-fragment in gel. An improved two-step method of cell-free protein synthesis system was used to quantitate the requirements of S-140 in ribosome inactivation.IC50 ratios of camphorin, gamma-momorcharin, luffin S, luffin A, trichosanthin (type I); and ricin, ricin A-chain; cinnamonin, cinnamomin A-chain (type II) between the absence and presence of ATP and S-140 were 3108, 151, 51, 45, 15; and 47, 7, 26, 12, respectively.The ribosome-inactivating activity of type II ribosome-inactivating proteins, including intact protein and its A-chain, was promoted by ATP and S-140. Camphorin showed a significant difference from cinnamomin in need of ATP and S-140 for such promoting.

Early intrathymic T cell development is highly coordinated and controlled by the interaction of a variety of molecules. The development of gene targeting technology provided a valuable tool to study the function of these molecules in vivo. The analysis of TCR and CD3 gene targeted mice indicated that CD44-CD25+ is a control point in early T cell development. At this stage, the expression of pre-TCR complex (composed of pre-TCR alpha, TCR beta and CD3) and/or its combination with unknown ligand provide signals for further progression beyond the CD44-CD25+ stage. The deficiency of any component in pre-TCR complex would lead to the blockage of T cell development at early stages.

Our study showed that hemodilution with modified fluid gelatin resulted in an increase in local cerebral blood flow (LCBF), but no change at all in local cerebral oxygen delivery (LCOD) in rats. Hemosome, a lecithin encapsulated hemoglobin having the oxygen-carrying capacity, was developed to improve LCOD by hemodilution. Therefore, we have hypothesized that LCBFLCOD would be increased by hemodilution with hemosome. To test this hypothesis, adult male Sprague-Dawley rats weighing approximately 350g were used and divided into the hemodilution and the control groups. Hemosome was made from pig red blood cells and lecithin. It's mean diameter was approximately 0.3 um and hemoglobin concentration was approximately 4g/dl. Isovolemic hemodilution, which lowered the systemic hematocrit from approximately 50% to approximately 30%, was achieved by rapidly replacing blood with the same volume of hemosome. Ten min later, LCBF in 14 brain structures were measured using the 14C-iodoantipyrine technique. Our results showed that LCBF of the control group ranged from 115 +/- 11 ml/100g/min in the medulla to 260 +/- 31 ml/100g/min in the occipital cortex. LCBFs were generally higher (p0.05, MANOVA) by 16% in the hemodilution group than in the control group. However LCODs were generally decreased (p0.05, MANOVA) by 18% in the hemodilution group than in the control. In conclusion, hemodilution with hemosome indeed improves LCBF but lowers LCOD in awake rats.

The effect of sodium glutamate (Glu) on plasma sialic acid (SA) level was studied by means of microinjection into the nucleus arcuatus (ARC) and ultraviolet spectrophotometry. The results indicate: (1) The plasma SA level was significantly decreased after microinjection of Glu into the ARC (P0.01). With increasing Glu concentration, the onset of the decrease occurred earlier. (2) When Glu was microinjected into ARC after intraventricular injection of apomorphine, the plasma SA level was significantly decreased (P0.01) and the time required to cause the decrease was shortened. (3) When Glu was microinjected into ARC after intraventricular injection of spiperone, no change was found in the plasma SA level (P0.05). The above results suggest that the mechanism underlying the decrease of plasma SA level is probably related to modulated dopamine (DA) release of hypothalamic dopaminergic neurons by DA-D2 receptor.

A natural outbreak of mouse hepatitis virus infection developed in a breeding colony of nude mice in Taiwan. The outbreak was unique in that morbidity was high in both adult and suckling mice, but only sucklings died. In contrast, all suckling heterozygous (nu/+) mice survived, and no lesions were found in adult female heterozygous (nu/+) mice. Adult male nude mice had chronic, active, necrotizing hepatitis with syncytial giant cells, but no lesions were detected in other tissues. Immunohistochemistry with anti-A59 and anti-JHM serum revealed mouse hepatitis virus antigen in the liver of infected adult and suckling nude mice, although less intensively in the kidney of adult nude mice. Suckling BALB/c mice inoculated with filtrates of the liver of adult nude mice developed hepatitis similar to that in the naturally infected nude mice. Virus was isolated by inoculating cell-free liver filtrate from infected adult nude mice onto 3T3 cells. Electron microscopy of purified virus revealed 100-nm-diameter enveloped particles with characteristic petal-shaped surface projections. We conclude that the outbreak was caused by a weakly virulent, highly hepatotropic murine hepatitis virus.

Previous studies in our laboratory showed that decaffeinated green tea and black tea extracts inhibited 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced tumorigenicity in A/J female mice. In order to understand the mechanism of the inhibitory action, we examined the effects of decaffeinated green tea, black tea, and tea components on the metabolic activation of NNK in vitro and in vivo in this animal model. When added to incubation mixtures containing mouse lung microsomes, decaffeinated green tea and black tea extracts and their fractions, at concentrations up to 0.4 mg/ml, inhibited NNK oxidation and NNK-induced DNA methylation. Among the tea components examined, (-)-epigallocatechin-3-gallate was the most potent inhibitor with 50% inhibitory concentrations of about 0.12 mM for both NNK oxidation and DNA methylation. At these concentrations, (-)-epigallocatechin-3-gallate inhibited the catalytic activities of several P450 enzymes and was more potent against P450 1A and 2B1 than 2E1. When decaffeinated green or black tea extracts were given to female A/J mice as the sole source of drinking fluid before an i.p. injection of NNK (100 mg/kg body weight), a statistically significant inhibition of lung DNA methylation, however, was not observed, although a significant reduction in lung tumor multiplicity was observed. The results suggest that, although inhibition of the metabolic activation of NNK and the subsequent DNA alkylation by tea extracts can be demonstrated in vitro, this mechanism may not be important for the inhibitory action of tea against lung tumorigenesis.

The biological significance of the signals triggered by the interaction of cell surface expressed LFA-1 and ICAM-1 has been investigated in Con A stimulated murine thymocytes. Addition of anti-LFA-1 or/and anti-ICAM-1 McAb to the thymocyte culture in the presence of Con A, the cell proliferation was profoundly inhibited. Such inhibitory effect was more significant by the addition of anti-LFA-1 than by the addition of anti-ICAM-1 McAb. FACS analysis revealed that the proliferation of CD8+ T cells was more sensitive to the inhibitory effect of anti-LFA-1 McAb than did the proliferation of CD4+ T cells. The anti-LFA-1 McAb imposed inhibitory effect, however, could be reversed by the supplementation of IL-2 in the initiation of the culture. The proliferation of cultured Con A activated T blast cells could no longer be inhibited by the addition of anti-LFA-1 McAb. Furthermore, neither anti-LFA-1 nor anti-ICAM-1 McAb could block PMA+A23187 induced thymocyte proliferation. The results imply that the ligand-receptor pair of LFA-1-ICAM-1 molecules also takes part in the early events of Con A activated signal transduction pathway which leads to the proliferation of thymocytes.

The cytokine production by MTSC 4 cells, a murine thymic dendritic cell line established in our laboratory, has been investigated. Without exogenous stimulation, the MTSC 4 cells constitutively produce multiple types of cytokines, including IL-1, IL-6, IL-7, CSF, IFN and Chemotactic factor (CF). Of which, IL-6 and IFN were abundant: IL-1 and Chemokine, moderate: IL-7 and CSF at low level. MTSC4 cells could not produce detectable either IL-3 of TNF alpha. The establishment of MTSC 4 may be useful in the analysis of the mechanism of thymus selection, and in the study of the network regulation of autocrined cytokines.

Blumeatin (Blu, 5,3',5'-trihydroxy-7-methoxy-dihydro-flavone) was first isolated from Blumea balsamifera DC by Department of Chemistry, Sunyatsen University of China. Blu ip inhibited the increase of serum alanine aminotransferase (AAT) and liver triglyceride and increased serum triglyceride, beta-lipoprotein, and liver glycogen content in CCl4-intoxicated rats. Histological lesions of liver were less severe than those of hepatic injury control. Blu ip 0.65 and 3.25 mg.kg-1 inhibited the increase of serum AAT and hepatic TG in thioacetamide (TAA)-intoxicated mice. Blu ip shortened the pentobarbital sleeping time in CCl4-intoxicated mice. It suggested that Blu could protect liver against injury induced by CCl4 and TAA.

An untransformed murine thymic epithelial cell line (MTEC1) has been established. Without exogenous stimulation, the MTEC1 cells constitutively produced multiple types of cytokines, including IL-1, IL-6, IL-7, GM-CSF and chemotactic factor(s). Of which, IL-6, GM-CSF and chemotactic factor(s) were abundant; IL-1, moderate; and IL-7 at low level. MTEC1 cells neither produced detectable IL-3 nor TNF alpha. Thus, the MTEC1 cells may be useful not only in the evaluation of the signals required for thymic selection in vitro, but also useful in the analysis of the endogenous regulation of the autocrine cytokine production cascade.

A previous report concluded that a new cytokine, designated IL-10, is a growth cofactor for thymocytes, spleen, and lymph node cells. In this report, we have focused on the effects of IL-10 on CD8+ spleen T cells. We first observed that IL-10 enhances the growth of CD8+ T cells to IL-2. We then investigated the effect of murine rIL-10 on the induction of murine effector CTL from CTL precursors (CTL-p) using both bulk and filler cell-free limiting-dilution cultures. IL-10 alone could not induce Con A-activated FACS-sorted CD8+ T cells either to proliferate or to generate effector CTL. In combination with IL-2, however, IL-10 augmented the cytolytic activity of effector CTL generated from Con A-activated spleen CD8+ T cells in bulk cultures incubated for 5 days. In limiting-dilution cultures (using solid-phase anti-CD3 mAb as stimulus), IL-10, in combination with IL-2, substantially increased the CTL-p frequency and augmented the cytolytic activity per clone expanded from one CD8+ T cell when compared with cells cultured in IL-2 alone. Kinetic studies showed that IL-10 is required at both early and late culture stages for optimal generation of effector CTL. The potentiating effects of IL-10 on CTL function were neutralized by an anti-IL-10 mAb. These results indicate that IL-10 has direct effects on mature T cells, and suggest that IL-10 also functions as a cytotoxic T cell differentiation factor, which promotes a higher number of IL-2-activated CTL-p to proliferate and differentiate into effector CTL. In contrast, IL-10 did not enhance significantly the lymphokine-activated killer cell activity of IL-2-grown CD8+ cytotoxic T cells.

The intact omentum of 13 monkeys was lengthened, placed subcutaneously, and laid on the left cerebral hemisphere prior to occluding the left middle cerebral artery. Two of these 13 monkeys developed left cerebral infarct and a right hemiparesis. Nine other monkeys had their left middle cerebral artery occluded without omental protection. All of these 9 developed a left cerebral infarct and 8 of them a right hemiparesis. Intact omentum may prevent a cerebral infarction when placed on the brain prior to MCA occlusion.

Summary Objective To study the effect of intra-articular injection of meloxicam (Mobic) on the development of osteoarthritis (OA) in rats and examine concomitant changes in nociceptive behavior and the expression of mitogen-activated protein kinases (MAPKs) in articular cartilage chondrocytes. Methods OA was induced in Wistar rats by right anterior cruciate ligament transection (ACLT); the left knee was not treated. The OA + meloxicam (1.0 mg) group was injected intra-articularly in the ACLT knee with 1.0 mg of meloxicam once a week for 5 consecutive weeks starting 5 weeks after ACLT. The OA + meloxicam (0.25 mg) group was treated similarly with 0.25 mg meloxicam. The sham group underwent arthrotomy only and received vehicle of 0.1 mL sterile 0.9% saline injections, whereas the naive rats in meloxicam-only groups were treated similarly with 1.0- and 0.25-mg meloxicam. Nociception was measured as secondary mechanical allodynia and hind paw weight-bearing distribution at before (pre-) and 5, 10, 15, and 20 weeks post-ACLT. Histopathology of the cartilage and synovia was examined 20 weeks after ACLT. Immunohistochemical analysis was performed to examine the effect of meloxicam on MAPKs (p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK)) expression in the articular cartilage chondrocytes. Results OA rats receiving intra-articular meloxicam treatment showed significantly less cartilage degeneration and synovitis than saline-treated controls. Nociception were improved in the OA + meloxicam groups compared with the OA group. Moreover, meloxicam attenuated p38 and JNK but enhanced ERK expression in OA-affected cartilage. Conclusions Intra-articular injection of meloxicam (1) attenuates the development of OA, (2) concomitantly reduces nociception, and (3) modulates chondrocyte metabolism, possibly through inhibition of cellular p38 and JNK, but enhances ERK expression.

SummaryObjectiveThe present study aimed to determine the role of excitatory amino acids (EAAs) and EAA transporters (EAATs) in an osteoarthritis (OA) model of rabbit knees.MethodsOA was induced in New Zealand white male rabbits by anterior cruciate ligament transection (ACLT) in one knee of one hind limb; the other knee left unoperated. Rabbits that received ACLT of knee were assigned to the ACLT group (n=6), while a sham-operated group (n=6) underwent arthrotomy with no ACLT. Six naïve rabbits that received no surgery were used as normal control. The width of the knee joint was measured to determine the severity of joint inflammation. Before operation and at 10, 20, and 30 weeks after operation, knee joint dialysates were collected by microdialysis and assayed for EAAs by high-performance liquid chromatography. Gross morphology and histopathology and EAATs glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) expression in the articular cartilage of the knees were evaluated by immunohistochemistry and western blot analysis.ResultsIn the ACLT knees, a significant increase in the joint width was observed (5.3±0.9mm, P

Fifteen selcted local isolates and five known Newcastle disease virus strains were examined for their cytopathic effects in chick embryo kidney (CEK) cells, egg-infectious units in chick embryos (CE), virulence by mean death time, intracerebral and intravenous pathogenicity indexes for CE and chicks, and ability to cause polykaryocytosis of fusion from within (FFWI) or fusion from without (FFWO) in CEK and BHK-21 monolayer cells. The capacity of the different virus strains to induce cell FFWI at 15 hr post-infection was related to their virulence for CE and chicks, but cell FFWO did not seem to be any relationship with the virulence of the strains.

The frequency of all precursors of T cells capable of proliferation (PTL-p) and of all cytotoxic T-cell clones (CTL-p) was determined for different thymocyte subpopulations using a high cloning-efficiency, Con A and growth factor driven, limit-dilution assay and a lectin-mediated, non-specific cytotoxic readout. As shown previously, more than 99% of precursors were confined to the medullary-type fraction, isolated by fluorescence activated cell sorting as the 14-15% of thymocytes showing low binding of peanut agglutinin (PNA). However, 20-50% of medullary-type cells appeared incapable of responding in a culture system allowing all peripheral T cells to grow, suggesting that the absolute size of the functional pool was 7-12% of all thymocytes. The 3-4% cortisone-resistant fraction of thymus gave a high precursor frequency (PTL-p 1 in 1.3; CTL-p 1 in 6) and a high cloning efficiency per Thy 1 positive cell (80%) which was nevertheless below that of peripheral T cells. However, only 20-25% of the total thymic PTL-p and CTL-p could be recovered in this fraction. Functional precursors were therefore within both the cortisone-sensitive and the cortisone-resistant subgroups of medullary-type thymocytes. Attempts to induce function in PNA+ cortical-type thymocytes by increasing the level of T-cell growth factors in the cultures gave only a marginal increase, the bulk of small cortical cells remained functionally inert. However, the low frequency of precursors found in the PNA+ fraction (around 1% of that in PNA- thymocytes) was not entirely due to PNA- contaminants since a PNA+, high H-2 blast fraction, representing about 4% of all thymocytes, showed a significant, although still low, PTL-p and CTL-p frequency amounting to less than 1% of the total thymus precursor pool. The relevance of this minor subset is discussed.

Monoclonal antibodies to pseudorabies viral capsid protein were prepared by fusing Sp2/0-Ag14 myeloma cells with pseudorabies virus (PrV)-primed Balb/c splenocytes. These monoclonal antibodies were specific for PrV. No crossreaction with Herpes Simplex Virus (HSV, type I) or infectious bovine rhinotrachitis virus (IBR) was found. The reaction between these monoclonal antibodies and PrV was demonstrated by immunofluorescent staining technique and enzyme-linked immunoblotting assay. These antibodies could be a useful diagnostic reagent for PrV as well as a good tool for the study of the capsid protein of PrV.

Cell-associated herpesvirus related to Marek's disease (MD) was isolated from the direct culture of kidney cells of naturally infected chickens at Taoyuan or by inoculation of clinical specimens to chick kidney (CK) and chick embryo fibroblast cells. The virus isolates replicated in CK or chick embryo kidney cell cultures were identified to be MD by the fluorescent-antibody technique.

The frequency of all precursors of T cells capable of proliferation (PTL-p), and of all precursors of cytotoxic T-cell clones (CTL-p), was determined for mouse thymic and peripheral T-cell subsets differing in Ly phenotype. A high cloning efficiency, concanavalin A (Con A) and growth factor driven limit dilution culture system was used. A lectin-mediated non-specific lysis readout was used for detecting cytotoxic clones. This approach provided a balance sheet of the overall distribution of functional cells regardless of specificity. Subsets of splenic T lymphocytes were isolated by fluorescence-activated cell sorting (FACS) after two-colour staining with monoclonal anti-Thy 1 and anti-Ly 2 antibodies. Both the Ly 1+2- and Ly 1+2% subsets responded by clonal proliferation, but cytotoxic activity was almost exclusively limited to the Ly 1+2% derived clones. Four subclasses of thymocytes were isolated by FACS after two-colour staining with peanut agglutinin (PNA) and monoclonal anti-Ly 2 antibody. These were PNA+Ly 2+, PNA+ Ly 2-, PNA- Ly 2+ and PNA- Ly 2-, representing 80, 5, 5 and 10% of total thymocytes, respectively. Their respective PTL-p frequencies were 1 in 333, 1 in 200, 1 in 5.3 and 1 in 3.2, values which included a significant activity loss on labeling and isolation. The slight activity in PNA+ cells may have been contaminants. The PNA- Ly 2+ subset formed larger clones than the PNA- Ly 2- subset. CTL-p frequency was 1 in 5 for PNA- Ly 2+ and 1 in 400 for PNA- Ly 2-. The few cytotoxic clones derived from the Ly 2- cells appeared to be genuine and not a result of contamination with Ly 2+ cells. Thus although both Ly subsets of medullary-type thymocytes were able to proliferate, the Ly 2+ subset contributed almost all of the cytotoxic activity of the unfractionated thymocytes. Medullary-type thymocytes display an Ly phenotype development and a level of functional maturation approaching that of peripheral T cells.

The microfilament structures of Newcastle disease virus (NDV)-infected BHK-21 cells were studied in the presence (5 micrograms/ml) or absence of cytochalasin B (CB) by means of phase contrast, indirect immunofluorescence and thin-section immunoelectron microscopy. The results indicated that CB treatment not only impaired virus infections titers and antiactin fluorescence strength but also disrupted cytoplasmic membrane and untagged ferritin-conjugated antibody on the surface of NDV specific antigens.

The frequency and capacity for clonal expansion of several murine thymocyte subpopulations responsive to various IL (fetal day 15, and adult CD4-8-, CD4+8- and CD4-8+) were investigated using a single-cell limiting-dilution cell culture system without filler cells. This assay requires the presence of PMA and ionomycin. The main conclusions of these studies are the following: 1) IL-4 is a better growth factor than IL-2 for immature thymocytes (fetal day 15 or adult CD4-8-). 2) IL-2 is a better growth factor than IL-4 for mature phenotype thymocytes (CD4+8- and CD4-8+). 3) IL-4 is a relatively poor growth factor for adult CD4-CD8- thymocytes and CD4+CD8- thymocytes, while it induced strong responses in fetal day 15 and CD4-8+ thymocytes. 4) IL-6 enhanced the response of CD4+8- thymocytes to either IL-2 or IL-4. 5) Cortisone-resistant thymocytes grown initially with IL-4 and then switched to IL-2 showed a significant decrease in cloning efficiency. No inhibitory effect was observed when cells were cultured first with IL-2 and then switched to IL-4. 6) Finally, supernatant from Con-A stimulated rat spleen cells induced maximal growth of all adult thymocyte populations tested, suggesting that unidentified thymocyte growth factor(s) remain to be characterized. These results indicate that the maturational stage of thymocytes determines their requirements for activation and proliferation.

Using fluorescein-conjugated peanut agglutinin (PNA) and the fluorescence-activated cell sorter, we have shown that the cytotoxic T cells generated by culturing thymocytes (about 85% PNA+ cells) with Con A and medium conditioned by Con A-stimulated spleen cells were mostly PNA+. Cytotoxic T cells generated in parallel cultured of PNA- thymocytes or normal lymph node cells (in which the T cells are PNA-), however, were also mostly PNA+. The development of PNA+ cells from PNA- mature peripheral T cells was confirmed by using cultures of PNA- T cells separated on a fluorescence-activated cell sorter from lymph node cells. Immunization in vivo also resulted in the appearance of PNA+ T cells in the lymph node. These results indicate that receptors for PNA can no longer be regarded as unambiguous markers of T cell immaturity, or of the origin of a T cell from a PNA+ precursor. The possibility that a variable proportion of the PNA+ cells in the thymus may be generated by intrathymic activation of PNA- medullary thymocytes is discussed.

Results of this experiment showed that a high fiber bran diet had the ability to lower significantly the incidence of pathologic changes in the colon of mice receiving 1,2-dimethylhydrazine, a drug known for its specificity for causing carcinoma of the colon. This finding adds support to the hypothesis which clains that the addition of bran to the diet has the ability to lower the indicence of carcinoma of the large intestine.

Cell-mediated immunity to pseudorabies virus (PrV) was studied by means of the leukocyte migration-inhibition (LMI) agarose test. Migration of leukocytes from swine vaccinated with 10 TCID50 inactivate PrV and from normal control swine was not inhibited in the presence of specific PrV antigen. Cellular immune responses were detected in 80% of the vaccinated and 40% of the control group animals at the 1st week after challenge exposure. At the 2nd week, both groups of swine showed a peak level (p less than 0.001) immune response in the LMI test. This level of response remained until the 3rd week in the vaccinated group and the 2nd week in the control group. The averages of migration indices at the 1st, 2nd, 3rd and 4th weeks after challenge exposure were 0.74, 0.22, 0.60 and 0.89 in the vaccinated and 0.82, 0.29, 1.01 and 1.12 in the control group, respectively. Low concentrations of virus-neutralizing (VN) antibodies were present in 30% of the vaccinated swine at the 1st week, and 60% at the 2nd week after vaccination. Anamnestic antibody and primary VN antibody responses occurred after challenge in vaccinated and normal swine, respectively.