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M L Zhao

Publications and source records attributed to M L Zhao.

At least 19 recordsLinked to original sources
PubMed1999

Neuronal nitric oxide synthase expression in developing and adult human CNS.

Neuronal nitric oxide synthase (nNOS) is constitutively expressed by subpopulations of neurons in the CNS and is involved in neurotransmission, learning and memory, and neuronal injury. While the distribution of nNOS neurons has been characterized in the rodent CNS, the expression in human brain has not been well documented. We determined the expression of nNOS in second trimester human fetal and adult brain. In second trimester fetal brain, the nNOS neurons are concentrated in the developing cerebral cortex at the subplate zone and in layer VI, the striatum, and in certain brainstem nuclei. The nNOS neurons are sparsely distributed in the hippocampus, and virtually absent in the cerebellar cortex. The nNOS neurons in the subplate zone extend their processes radially, suggesting a developmental role, perhaps in guidance. The number and distribution of NADPH diaphorase-positive neurons corresponds to that of the nNOS neurons. While the distribution of nNOS neurons in the adult brain is similar to that found in fetal brain, the overall density is lower in the adult. The highest density of nNOS neurons is found in the striatum followed by the neocortex. A region-specific role for nNOS neurons in human brain and a potential developmental role for nNOS in the cerebral cortex are suggested by these data.

Adult↗
PubMed1998

Inducible nitric oxide synthase expression is selectively induced in astrocytes isolated from adult human brain.

Inducible nitric oxide synthase (iNOS) expression has been shown to be differentially regulated among different cell types and species. In cultures of primary human fetal glial cells, we have shown that astrocytes rather than microglia express iNOS. In the present study, we extended these findings to primary cultures of astrocytes and microglia derived from adult human brains. Mixed cultures of adult brain tissue were stimulated with IL-1beta and IFNgamma, a combination known to induce iNOS maximally in human fetal cells, and the expression of iNOS was determined by immunocytochemistry. Cell types were determined by morphology as well as immunocytochemistry for GFAP (astrocytes) and CD68 (microglia). The results showed that in cultures of adult human glia, iNOS was expressed following stimulation with cytokines, and the expression was restricted to astrocytes. Astrocyte iNOS immunoreactivity was detected both in the cytosol and in a discrete paranuclear region, a pattern noted in human fetal astrocytes. These results demonstrate that the ability to express iNOS is common to both fetal and adult human astrocytes.

Adult↗
PubMed1998

Persistence of an encephalitogenic T cell clone in the spinal cord during chronic, relapsing experimental autoimmune encephalomyelitis.

The CDR3 region of the TCR beta-chain of a CD4+, Th1, Vbeta2+ encephalitogenic T cell clone was used as an idiotypic marker to track the location of the clone in vivo. cDNA prepared from the spinal cord, thymus, lymph nodes, spleen, and liver of the recipients at various stages of EAE was amplified using Vbeta2 and Cbeta-region primers, and the products immobilized. The membrane was probed with a 32P-labeled oligonucleotide complementary to the CDR3 region of the T cell clone. The probe reacted strongly with products from the spinal cord, spleen and liver and less strongly with products from lymph nodes and thymus of mice with acute EAE. The signal was greatly diminished in the spinal cord and other tissues during recovery from acute disease and reappeared in the spinal cord at each relapse.

Animals↗
PubMed1998

Acute and relapsing experimental autoimmune encephalomyelitis in IL-4- and alpha/beta T cell-deficient C57BL/6 mice.

Experimental autoimmune encephalomyelitis follows a chronic relapsing course in several inbred strains of mice. To address the role of T cells in recovery and relapse, the clinical course of EAE was compared in C57BL/6 (B6) normal and immunodeficient mice following active immunization with MOG p35-55 or adoptive transfer of encephalitogenic peptide-specific T cell lines. The course of actively-induced EAE in B6 wild-type and IL-4 -/- mice was similar. B6 IL-4 -/- mice recovered normally from acute passive EAE, but did not relapse in contrast to wild-type B6 mice. EAE was progressive in B6 RAG -/- and alpha/beta TCR -/- mice, but the disease course could be arrested by infusion of normal spleen cells. When non-activated MOG peptide-specific T cells were transferred to wild-type or alpha/beta TCR -/- mice, spontaneous disease ensued in the mutants only.

Acute Disease↗
PubMed1998

Pathogenesis of acute passive murine encephalomyelitis I. Importance of host-derived cells as determined by kinetic analysis.

Kinetics of entry into the CNS of donor- and host-derived T-cells during the onset of acute murine EAE induced by the passive transfer of an encephalitogenic PLP(139-151)-specific T-cell clone was investigated. RT-PCR and spectratypic analysis of total RNA recovered from recipient mice demonstrated the presence in the CNS of donor- and host-derived T-cells 24 h post adoptive transfer. Donor-derived T-cells detected in the CNS decreased days 2-6 post transfer while host-derived T-cells persisted during this time. Beginning 3 days before clinical onset, an increase in the CNS of both T-cell populations was observed which persisted through disease onset. Similar analysis performed on recipients of an nonencephalitogenic PLP(139-151)-specific T-clone demonstrated a transient infiltration of donor- and host-derived T-cells beginning 4 days post transfer (dpt) and returning to background levels by day 7 post transfer. Results presented here suggest the importance of host-derived T-cells in the onset of acute passive murine EAE.

Acute Disease↗
PubMed1997

Alterations in frequency coding and activity dependence of excitability in cultured neurons of Drosophila memory mutants.

Mutants of the Drosophila dunce (dnc) and rutabaga (rut) genes, which encode a cAMP-specific phosphodiesterase and a calcium/calmodulin-responsive adenylyl cyclase, respectively, are deficient in short-term memory. Altered synaptic plasticity has been demonstrated at neuromuscular junctions in these mutants, but little is known about how their central neurons are affected. We examined this problem by using the "giant" neuron culture, which offers a unique opportunity to analyze mutational effects on neuronal activity and the underlying ionic currents in Drosophila. On the basis of instantaneous frequency and first latency of spikes evoked by current steps, four categories of firing patterns (tonic, adaptive, delayed, and interrupted) were identified in wild-type neurons, revealing interesting parallels to those commonly observed in vertebrate CNS neurons. The distinct firing patterns were correlated with expression of different ratios of 4-aminopyridine- and tetraethylammonium-sensitive K+ currents. Subsets of dnc and rut neurons displayed abnormal spontaneous spikes and altered firing patterns. Altered frequency coding in mutant neurons was demonstrated further by using stimulation protocols involving conditioning with previous activity. Abnormal spike activity and reduced K+ current remained in double-mutant neurons, suggesting that the opposite effects on cAMP metabolism by dnc and rut do not counterbalance the mutual functional defects. The aberrant spontaneous activity and altered frequency coding in different stimulus paradigms may present problems in the stability and reliability of neural circuits for information processing during certain behavioral tasks, raising the possibility of modulation in neuronal excitability as a cellular mechanism underlying learning and memory.

Action Potentials↗
PubMed1996

Thymic expression of myelin basic protein (MBP). Activation of MBP-specific T cells by thymic cells in the absence of exogenous MBP.

Previous studies have shown golli-myelin basic protein (MBP) mRNA to be expressed in the thymus of normal SJL mice, but translation of the mRNA was not assessed. To test for the presence of immunoreactive protein, single cell suspensions were prepared from adult SJL thymus and cultured with syngeneic MBP-specific T cells. After 48 h [3H]thymidine was added to the microcultures to assess T cell proliferation. MBP-specific T cell lines proliferated strongly (stimulation index range, 13-31). T cell lines specific for MBP exon 2, MBP peptide 89-101, proteolipid protein peptide 139-151, and OVA gave stimulation indices of 10-13, 5-6, 2-3, and 2-3, respectively. Stimulatory activity could be abrogated by irradiation of either the thymic cells or the MBP-specific T cells. Stimulatory activity was a property of a minor population of plastic-adherent thymic cells. Monoclonal anti-I-As Ab added to the microcultures inhibited the reaction by 77%. MBP-specific T cells cultured with syngeneic nonirradiated thymus cells in the absence of added MBP transferred experimental autoimmune encephalomyelitis adoptively to syngeneic recipients. These findings indicate that golli-MBP mRNA is translated in normal SJL thymus, and that peptides reactive with MBP-specific T cells in the context of class II MHC molecules are expressed.

Amino Acid Sequence↗
PubMed1996

Expression of type II nitric oxide synthase in primary human astrocytes and microglia: role of IL-1beta and IL-1 receptor antagonist.

In this work, we studied the expression of type II nitric oxide synthase (NOS) in primary cultures of human astrocytes and microglia. Cytokine-activated human fetal astrocytes expressed a 4.5-kb type II NOS mRNA that was first evident at 8 h, steadily increased through 48 h, and persisted through 72 h. The inducing signals for astrocyte NOS II mRNA expression were in the order IL-1beta + IFN-gamma > IL-1beta + TNF-alpha > IL-1beta. SDS-PAGE analysis of cytokine-stimulated astrocyte cultures revealed an approximately 130-kDa single NOS II band that was expressed strongly at 48 and 72 h (72 h > 48 h). Specific NOS II immunoreactivity was detected in cytokine-treated astrocytes, both in the cytosol and in a discrete paranuclear region, which corresponded to Golgi-like membranes on immunoelectron microscopy. In human microglia, cytokines and LPS failed to induce NOS II expression, while the same stimuli readily induced TNF-alpha expression. In cytokine-treated human astrocytes, neither NOS II mRNA/protein expression nor nitrite production was inhibited by TGF-beta, IL-4, or IL-10. In contrast, IL-1 receptor antagonist exerted near complete inhibition of NOS II mRNA and nitrite induction. Monocyte chemoattractant peptide-1 mRNA was induced in TGF-beta-treated astrocytes, demonstrating the presence of receptors for TGF-beta in astrocytes. These results confirm that in humans, cytokines stimulate astrocytes, but not microglia, to express NOS II belonging to the high output nitric oxide system similar to that found in rodent macrophages. They also show that the regulation of type II NOS expression in human glia differs significantly from that in rodent glia. A crucial role for the IL-1 pathway in the regulation of human astrocyte NOS II is shown, suggesting a potential role for IL-1 as a regulator of astrocyte activation in vivo.

Astrocytes↗
PubMed1996

Active and passive experimental autoimmune encephalomyelitis in strain 129/J (H-2b) mice.

Failure of C57BL/6J and C57BL/10Sn (H-2b) mice to exhibit clinical signs of experimental autoimmune encephalomyelitis following immunization with myelin basic protein (MBP) has been interpreted to indicate that mice of this haplotype are resistant to EAE. Recently, we immunized strain 129/J (H-2b) mice with rat MBP and found that clinical signs of EAE were expressed in the majority of animals within 2 to 3 weeks. Passive EAE was readily induced by adoptive transfer of MBP-specific T cell lines to syngeneic recipients. MBP peptide 89-101 and PLP peptide 178-191 induced EAE upon active immunization although proteolipid protein peptide 139-151 was ineffective in this regard. Strain 129/J mice never recovered fully from acute EAE, and signs of relapsing disease were not observed.

Adoptive Transfer↗
PubMed1995

The immune response to a subdominant epitope in myelin basic protein exon-2 results in immunity to intra- and intermolecular dominant epitopes.

Experimental autoimmune encephalomyelitis was induced in SJL/J mice by adoptive transfer of a MBP exon-2 peptide-specific T cell line. The T cell line, when tested for antigen specificity, reacted strongly with exon-2 peptide, but not with MBP peptides pAc1-11, p43-88, p89-101 or PLP p139-151. The specificity of splenic or lymph node T cells isolated from mice with acute or first relapse EAE induced by adoptive transfer of the exon-2-specific T cell line was identical to the transferred line. Splenocytes or lymphocytes isolated from mice at the second relapse were reactive with MBP p43-88, p89-101 and PLP p139-151 in addition to exon-2 peptide and MBP peptide Ac1-11. T cell lines selected by culture with MBP exon-2 peptide or PLP p139-151 from splenic cells from mice with relapsing EAE were weakly encephalitogenic; however, T cell lines selected from the same mice with MBP pAc1-11 were not encephalitogenic. T cells from the exon-2 and p139-151 T cell lines primed recipients for rapid onset severe EAE, whereas the pAc1-11 T cell line did not. T cells from the exon-2-specific line did not express V beta 17a+ TCR; however, peptide-specific T cell lines derived from the spleens of relapsing animals did express this TCR gene segment providing direct evidence of recruitment and sensitization of recipient T cells.

Amino Acid Sequence↗
PubMed1995

Functional expression of Shaker K+ channels in cultured Drosophila "giant" neurons derived from Sh cDNA transformants: distinct properties, distribution, and turnover.

Expression of transgenic Shaker (Sh) channels has not previously been examined in Drosophila neurons. We studied K+ current by whole-cell recording in cultured "giant" neurons derived from germline transformants. Independent lines were generated by using a P-element vector, in which transcription of the 29-4 cDNA, one of the Sh splicing variants (Iverson and Rudy, 1990), was under the control of a heat shock (HS)-inducible promoter. Transformants in wild-type and two different Sh mutant backgrounds all exhibited an HS-inducible, A-type K+ current that was characterized by a much slower recovery from inactivation and a higher sensitivity to 4-aminopyridine than native K+ currents of Sh 29-4 currents expressed in Xenopus oocytes. Despite similarities in the kinetic and pharmacological properties of the HS-induced current in all backgrounds examined, host-dependent differences in the peak current amplitude have been consistently observed between multiple lines of 29-4 ShM and 29-4 Sh120 that might reflect differential channel subunit assembly in different hosts. Isolation of the novel 29-4 currents allowed determination of the channel turnover rate in cultured neurons. These currents persisted for up to 3 d or more, comparable with the durations previously reported for Na+ and Ca2+ channels. Surprisingly, the percentage of cells expressing inactivating K+ currents remained approximately the same with or without HS induction, suggesting that some mechanisms exist to restrict functional expression of inactivating K+ channels, including transgenic Sh channels and those not encoded by the Sh locus, to certain types of neurons.

4-Aminopyridine↗
PubMed1995

Cryptococcus neoformans survive and replicate in human microglia.

BACKGROUND: Cryptococcus neoformans (CN) is an opportunistic pathogen that can cause fatal meningoencephalitis in patients with immune deficiency. Among the central nervous system (CNS) cells that are infected by CN are perivascular microglia and macrophages. Little is known about the interaction of CN and CNS phagocytes at the cellular level. EXPERIMENTAL DESIGN: To better understand the fate of CN in microglia, we followed Ab-opsonized CN in human fetal microglial culture by phase-contrast microscopy, combined lighted microscopy and transmission electron microscopy of plastic-embedded monolayers, and immunocytochemistry for localization of capsular Ag. RESULTS: Phase-contrast microscopy revealed that microglia initially internalized and contained Ab-opsonized yeast cells within phagolysosomes. However, CN escaped from microglia and resumed extracellular growth 16 to 24 hours after being phagocytosed. Transmission electron microscopy/1-mu epoxy sections revealed that intracellular CN were localized in two types of phagosomes in microglia: spacious phagosomes (SP) and close-fitting phagosomes (CP). Three lines of evidence indicate that SP are the primary sites for intracellular CN survival and replication: (a) SP contained multiple, budding yeast cells, whereas CP contained only single yeast cells within a tightly bound phagosomal membrane; (b) the number of SP and the number of CN within SP increased considerably at 24 hours compared with at 2 hours; and (c) microglial cultures challenged with heat- or amphotericin B-treated CN had significantly fewer SP than those challenged with live CN. Both SP and CP phagosomes fused with lysosomes, suggesting that CN survival in SP was not due to failure of phagolysosomal fusion. In SP, there was attenuation and diffusion of capsular polysaccharide within the phagosome, whereas in CP the fungal capsules remained compact and homogeneous. Immunocytochemistry with an mAb directed to capsular glucoronoxylomannan supported continued synthesis of polysaccharide within SP. CONCLUSION: The results suggest that, in human microglia, CN survive and replicate within SP. Modification of CN capsular polysaccharide within SP may be a contributing factor to this aberrant microglial-CN interaction.

Animals↗
PubMed1994

Immunoreactivity profile of hippocampal CA2/3 neurites in diffuse Lewy body disease.

Ubiquitin-immunoreactive dystrophic neurites in the CA2/3 region of the hippocampus are characteristic of diffuse Lewy body disease (DLBD). The origin of dystrophic CA2/3 neurites is unknown, but their extent correlates with the number of cortical Lewy bodies (LBs). To examine the molecular composition of these lesions, hippocampal sections were obtained at postmortem from cases of DLBD, Parkinson's disease and Alzheimer's disease. The tissue samples were fixed in a variety of fixatives and immunostained with antibodies to ubiquitin, ubiquitin C-terminal hydrolase (PGP9.5), neurofilament protein subunits, tau protein, paired helical filaments and tyrosine hydroxylase (TH). In addition to being ubiquitin positive, both cortical LBs and CA2/3 dystrophic neurites were positive with a neurofilament monoclonal antibody (RM032) and PGP9.5; however, fewer lesions were detected with these antibodies compared to ubiquitin immunocytochemistry. The dystrophic CA2/3 neurites were not stained with antibodies to tau proteins, paired helical filaments or TH. Absence of TH immunoreactivity suggests that CA2/3 neuritic processes are not derived from brain stem dopaminergic afferents to the hippocampus. Since CA2/3 neurites are immunologically similar to cortical LB, the pathogenesis of these lesions may be similar. Characterization of dystrophic CA2/3 neurites and cortical LBs may clarify how these lesions contribute to the emergence of dementia in DLBD.

Alzheimer Disease↗
PubMed1994

Encephalitogenicity of myelin basic protein exon-2 peptide in mice.

Immunization with a synthetic peptide with an amino acid sequence corresponding to mouse myelin basic protein exon-2 induced mild experimental allergic encephalitis (EAE) in B10.RIII mice, very mild disease in SJL/J mice and no disease in (SJL x PL)F1 hybrid mice. In contrast, adoptive transfer of an exon-2 peptide-specific T cell line from SJL mice induced severe relapsing EAE in syngeneic recipients. The T cell line was specific for exon-2 peptide and did not cross-react appreciably with an MBP preparation consisting of the 18.5 and 14-kDa isoforms. mRNA for exon-2 containing isoforms could be demonstrated in the spinal cord of SJL/J and B10.RIII mice by amplification using exon-2 and exon-4 oligonucleotide primers. On a relative basis, the level of exon-2 cDNA was lower than that of exon-1 cDNA in the same spinal cord preparations from both strains of mice.

Amino Acid Sequence↗
PubMed1994

Cytoskeletal alterations in human fetal astrocytes induced by interleukin-1 beta.

Previous studies in this and other laboratories have shown that interleukin-1 beta (IL-1 beta) is a selective and potent activator of human astrocytes with respect to induction of cytokines and hematopoietic growth factors. To study the effect of recombinant human IL-1 beta (rhIL-1 beta) on astrocyte morphology, glial fibrillary acidic protein (GFAP) and vimentin expression, and actin organization, we conducted a systematic survey using dissociated human fetal astrocyte cultures. Within hours of stimulation with IL-1 beta, the majority of astrocytes converted from flat, polygonal cells to small, contracted, highly branched cells. This change in morphology was more striking when serum was eliminated from the medium. Complete dissolution of filamentous actin occurred simultaneously with the change in cell shape, as demonstrated by fluorescein-phalloidin binding. These "activated" astrocytes displayed intense GFAP and vimentin immunoreactivity in the small perikarya and processes. In contrast, the large, flat astrocytes in control cultures showed diffuse pale immunoreactivity for GFAP and vimentin. To quantify the changes in GFAP and vimentin content with IL-1 beta stimulation, densitometric analyses of northern and western blots were performed. Northern blot analysis of IL-1 beta-stimulated astrocytes revealed a transient, marked decrease in steady-state levels of mRNA for GFAP, vimentin, and microtubule-associated protein 4. The decrease in mRNA levels was evident by 4-8 h and fell to the lowest level at 16-24 h (80-98% decrease by densitometry) with partial recovery by 72 h. By immunoblotting, a significant decrease in both GFAP and vimentin protein content was observed after IL-1 beta stimulation. Furthermore, metabolic labeling studies revealed an almost total loss of GFAP synthesis following stimulation with IL-1 beta for 16 h. These observations are consistent with the idea that increases in immunoreactivity were related to factors such as redistribution of epitope, rather than increases in total protein content. We hypothesize that in IL-1 beta-stimulated astrocytes, synthesis of other proteins, e.g., inflammatory cytokines, occurs at the expense of structural proteins and that the decrease in content of cytoskeletal proteins may reflect an "activated" state of astrocytes.

Astrocytes↗
PubMed1993

Epitope specificity and TCR V beta gene utilization in the encephalitogenic response of B10.RIII(71NS)/SnJ mice.

A high proportion of peptide 1-11 specific T cells from H-2u (V beta 8+, H-2u) mice express the V beta 8 TCR chain. Peptide 89-101 is immunodominant for B10.RIII (V beta 8+, H-2r) mice; thus, it was of interest to determine whether V beta 8 TCR would be over-represented in a population of peptide 89-101-specific T cells of this strain. Second, it was asked whether MBP peptides other than 89-101 would induce EAE in these mice. Of 70 B10.RIII(71NS)/SnJ mice immunized with mouse myelin basic protein (MBP), 32 of 41 males (78%) and 11 of 29 females (38%) showed clinical signs of experimental allergic encephalomyelitis (EAE). All mice immunized with peptide 89-101 showed clinical signs. One of six mice immunized with peptide 91-103 showed clinical signs, and 9 of 16 mice, all males, responded with EAE when immunized with peptide 38-88. No clinical EAE was observed in mice immunized with peptide 43-67, 68-88, 55-74, 1-37 or 1-20. A peptide 89-101-specific T cell line was established. At the initial stimulation the line was 29% V beta 8+ versus 21% in normal controls, and the line did not transfer EAE adoptively. After five in vitro stimulations, the percentage of V beta 8+ T cells had increased to 54%, and the line was encephalitogenic. Encephalitogenicity was partially blocked by anti-V beta 8 monoclonal antibody. Thus, over-representation of V beta 8+ TCR by encephalitogenic peptide-specific T cells is not limited to peptide 1-11-specific T cells from H-2u mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗