Treatment of experimental autoimmune encephalomyelitis with genetically modified memory T cells.

The migratory properties of memory T cells provide a model vector system for site-specific delivery of therapeutic transgene factors to autoimmune inflammatory lesions. Lymph node cells from (SWRxSJL)F1 mice immunized with the p139-151 determinant of myelin proteolipid protein (PLP) were transfected with a DNA construct that placed the anti-inflammatory cytokine interleukin-10 (IL-10) cDNA under control of an antigen-inducible IL-2 promoter region. Isolated T cell clones demonstrated antigen-inducible expression of transgene IL-10 and expressed cell surface markers consistent with the phenotype of normal memory T cells. Upon adoptive transfer, transfected T cell clones were able to inhibit onset of experimental autoimmune encephalomyelitis (EAE) and to treat EAE animals therapeutically after onset of neurologic signs. Semiquantitative immunocytochemistry showed a significant correlation between decreased demyelination and treatment with the transfected T cells. Taken together, these data indicate the autoreactive T cells can be genetically designed to produce therapeutic factors in an antigen-inducible manner resulting in a decreased severity of clinical and histological autoimmune demyelinating disease.

A-kinase anchoring protein 100 (AKAP100) is localized in multiple subcellular compartments in the adult rat heart.

Stimulation of beta-adrenergic receptors activates type I and II cyclic AMP-dependent protein kinase A, resulting in phosphorylation of various proteins in the heart. It has been proposed that PKA II compartmentalization by A-kinase-anchoring proteins (AKAPs) regulates cyclic AMP-dependent signaling in the cell. We investigated the expression and localization of AKAP100 in adult hearts. By immunoblotting, we identified AKAP100 in adult rat and human hearts, and showed that type I and II regulatory (RI and II) subunits of PKA are present in the rat heart. By immunofluorescence and confocal microscopy of rat cardiac myocytes and cryostat sections of rat left ventricle papillary muscles, we localized AKAP100 to the nucleus, sarcolemma, intercalated disc, and at the level of the Z-line. After double immunostaining of transverse cross-sections of the papillary muscles with AKAP100 plus alpha-actinin-specific antibodies or AKAP100 plus ryanodine receptor-specific antibodies, confocal images showed AKAP100 localization at the region of the transverse tubule/junctional sarcoplasmic reticulum. RI is distributed differently from RII in the myocytes. RII, but not RI, was colocalized with AKAP100 in the rat heart. Our studies suggest that AKAP100 tethers PKA II to multiple subcellular compartments for phosphorylation of different pools of substrate proteins in the heart.

The cytoplasmic domain mediates localization of furin to the trans-Golgi network en route to the endosomal/lysosomal system.

To investigate the mechanisms of membrane protein localization to the Golgi complex, we have examined the intracellular trafficking of epitope-tagged forms of the mammalian endopeptidase, furin, in stably transformed rat basophilic leukemia cells. Our studies show that furin is predominantly localized to the trans-Golgi network (TGN) at steady state, with smaller amounts present in intracellular vesicles. Biochemical and morphological analyses reveal that furin is progressively delivered to a lysosomal compartment, where it is degraded. Analyses of furin deletion mutants and chimeric proteins show that the cytoplasmic domain is both necessary and sufficient for localization to the TGN in various cell types. Interestingly, deletion of most of the cytoplasmic domain of furin results in a molecule that is predominantly localized to intracellular vesicles, some of which display characteristics of lysosomes. To a lesser extent, the cytoplasmically deleted molecule is also localized to the plasma membrane. These observations suggest the existence of an additional determinant for targeting to the endosomal/lysosomal system within the lumenal and/or transmembrane domains of furin. Thus, the overall pattern of trafficking and steady state localization of furin are determined by targeting information contained within more than one region of the molecule.

Anti-sense oligonucleotide of p21(waf1/cip1) prevents interleukin 4-mediated elevation of p27(kip1) in low grade astrocytoma cells.

Elevation of the cyclin-dependent kinase (cdk) inhibitor, p27(kip1) is necessary for Interleukin (IL)-4-mediated growth arrest of human low grade astrocytoma (RTLGA) cells and occurs at 24 h of treatment. Pathways involved in IL4 alteration of p27(kip1) are unknown, however. Here we investigated whether other cdk inhibitors contributed to the actions of IL-4 on RTLGA cells. By 12 h of IL-4 treatment, both cdk4 and cdk2 kinase activities against the retinoblastoma protein (pRb) were reduced and nuclear entry of pRb was prohibited. Twelve-hour cdk complexes contained elevated p21(waf1/cip1) but not p27(kip1), p15(ink4B) or p16(ink4A). IL-4 increased p21(waf1/cip1) but not p27(kip1) mRNA levels, and stimulated luciferase activity of a p21(waf1/cip1) promoter-luciferase reporter. In p53-mutant WITG3 cells, IL-4 did not alter p21(waf1/cip1) mRNA and promoter-luciferase activity or p27(kipl) protein, suggesting a need for functional p53. STAT6 phosphorylation by IL-4, however, occurred in both p53-mutant WITG3 and p53-functional RTLGA cells. Pre-treatment of RTLGA with anti-sense but not missense p21(waf1/cip1) oligonucleotide prior to IL-4: (a) restored cdk activities; (b) reduced cdk4-associated p21(waf1/cip1) levels; (c) prevented p27(kipl) elevation; and (d) reversed growth arrest. These results are the first to suggest that p21(waf1/cip1) is essential for IL-4-mediated elevation of p27(kip) and growth arrest of astrocytoma cells.

Human defensin 5 expression in intestinal metaplasia of the upper gastrointestinal tract.

BACKGROUND: Upper gastrointestinal tract intestinal metaplasia (IM) is termed Barrett's oesophagus (BO) or gastric intestinal metaplasia (GIM), depending on its location. BO and GIM are associated with chemical exposure resulting from gastro-oesophageal reflux and chronic Helicobacter pylori infection, respectively. Paneth cells (PCs), characterised by cytoplasmic eosinophilic granules, are found in a subset of IM at these sites, but histology may not accurately detect them. AIM: To determine human defensin 5 (HD5; an antimicrobial peptide produced by PCs) expression in BO and GIM, and to investigate its association with H pylori infection. METHODS: Endoscopic biopsies from 33 patients with BO and 51 with GIM, and control tissues, were examined by routine histology and for H pylori infection and HD5 mRNA and protein expression. RESULTS: In normal tissues, HD5 expression was specific for PCs in the small intestine. Five patients with BE and 42 with GIM expressed HD5, but few HD5 expressing cells in IM had the characteristic histological features of PCs. Most HD5 positive specimens were H pylori infected and most HD5 negative specimens were not infected. CONCLUSIONS: HD5 immunohistochemistry was often positive in IM when PCs were absent by conventional histology. Thus, HD5 immunohistochemistry may be superior to histology for identifying metaplastic PCs and distinguishing GIM from BO. The higher frequency of HD5 expression in GIM than in BO is associated with a higher frequency of H pylori infection, suggesting that in IM PCs may form part of the mucosal antibacterial response.

Adenosine A1 receptors are located predominantly on axons in the rat hippocampal formation.

The nucleoside adenosine exerts potent biological effects via specific receptors, including the inhibitory A1 adenosine receptor (A1AR). In the hippocampus A1ARs play an important role in regulating neuronal activity. However, the cellular sites of hippocampal A1ARs are undefined. Using in situ hybridization, receptor autoradiography, and single- and double-label immunocytochemistry techniques, we have characterized the cellular sites of A1AR expression in the rat hippocampus. In situ hybridization and receptor autoradiography studies revealed strikingly different patterns of labeling. In situ hybridization studies revealed heaviest labeling of cell bodies in the granular layer of the dentate gyrus and the pyramidal layers of Ammon's horn. In contrast, using [3H]DPCPX, we observed heavy specific labeling over the neuropil in the dentate hilus stratum moleculare, stratum lacunosum-moleculare, stratum radiatum, and stratum oriens, and little labeling over cell bodies. Using single-label immunocytochemistry, A1AR immunoreactivity was found to be heaviest over fibers in regions corresponding with heavy [3H]DPCPX labeling. Double-label florescent confocal microscopy was then used to determine the identity of labeled fibers. A1AR immunoreactivity was found to co-localize with SMI-31 that labels axons, but not with MAP2a,b that labels cell bodies and dendrites, or with synaptophysin that labels synapses. These data identify axons as the predominant site of A1AR expression in hippocampus. Activation of A1ARs may be a powerful mechanism by which adenosine alters axonal transmission to inhibit neurotransmitter release.

Increased alveolar macrophage nuclear factor-kappa B activation and macrophage inhibitory protein-1alpha levels in lung transplant patients.

BACKGROUND: Lung transplantation is increasingly used as the treatment for many end-stage pulmonary diseases. A major cause of morbidity and mortality in patients who undergo lung transplantation is rejection of the allograft. Proinflammatory macrophage-derived cytokines may sustain and/or enhance the immunological response to lung allograft antigens. Nuclear factor-kappa B (NF-kappaB) is a transcription factor that regulates the production of many of these cytokines and growth factors in alveolar macrophages (AMs). The aim of our study was to evaluate the activation of NF-kappaB in AMs and the levels of one of the proinflammatory cytokines whose production it controls, macrophage inhibitory protein-1alpha (MIP-1alpha), in AMs from transplanted lungs compared to those from healthy controls. METHODS: Twenty-eight (28) transplant recipients were included in the study. NFkappaB activation was evaluated by electrophoretic mobility shift assay of whole cell extracts and by immunohistochemical analysis on cytospin preparations. Concentrated bronchoalveolar lavage fluid was analyzed by enzyme-linked immunosorbent assay for MIP-1alpha levels. RESULTS: NF-kappaB was activated in alveolar macrophages from transplant patients as compared to healthy controls. MIP-1alpha levels in epithelial-lining fluid were elevated in transplant patients as compared to healthy controls. Increased MIP-1alpha levels correlated with viral infections in the transplant patients. Neither finding was found to correlate with acute rejection by transbronchial biopsy. CONCLUSIONS: These results demonstrate that NF-kappaB activation and MIP-1alpha levels are increased in transplanted lungs and may play a role in the inflammatory cytokine cascade that leads to the long-term tissue damage and allograft rejection in these patients.

Evidence for physiologically active axonal adenosine receptors in the rat corpus callosum.

Several neurotransmitter receptors have been identified on axons, and emerging evidence suggests that central axonal conduction may be modulated by neurotransmitters. We have recently demonstrated the presence of extra-synaptic adenosine Al receptors along rat hippocampal axons. We now present immunocytochemical evidence for Al receptors on rat corpus callosum axons and show that these receptors actively modulate axon physiology. Using rat brain coronal slices, we stimulated the corpus callosum and recorded the evoked extracellular compound action potential. The lipid-soluble, Al-specific adenosine receptor agonist cyclopentyladenosine, dose-dependently decreased the compound action potential amplitude, an effect reversed by the specific Al antagonist 8-cyclopentyl-1, 3-dipropylxanthine. These data provide the first direct evidence that axonal Al adenosine receptors modulate axon physiology in the adult mammalian brain. Influencing axonal transmission is a potentially powerful mechanism of altering information processing in the nervous system.

Regional variation in brain capillary density and vascular response to ischemia.

Differences in brain neuroarchitecture have been extensively studied and recent results demonstrated that regional differences in the physiological properties of glial cells are equally common. Relatively little is known on the topographic differences in vascular supply, distribution and density of brain capillaries in different CNS regions. We developed a simple method consisting of intravascular injection of fluorescent dyes coupled to immunocytochemical techniques that allows for simultaneous observation of glia-neuronal-vascular interactions in immersion-fixed brain specimens from small rodents. This technique permits quantitative evaluation of regional differences in glial/neuronal distribution and the study of their relationship to vascular densities. Variations of this technique also allow the detection of abnormal microvasculature (i.e. 'leaky' vessels), a useful feature for studies of blood-brain barrier function in health and disease. By use of quantitative confocal microscopy, the three-dimensional geometry of cortical and hippocampal structures revealed remarkable differences in vascularization between cortical gray/white matter junction, and hippocampal formation (CA1 and CA3 regions). Significant differences were also observed within the same investigative region: CA1 was characterized by low capillary density compared to neighboring CA3. Following an ischemic insult, CA1 vessels had more extensive blood-brain barrier leakage than CA3 vessels. We conclude that in addition to neuronal and glial heterogeneity, cortical structures are also endowed with region-specific vascular patterns characterized by distinct pathophysiological responses.

Growth cone interactions with purified cell and substrate adhesion molecules visualized by interference reflection microscopy.

The migration of growth cones on substrates consisting of naturally occurring cell adhesion molecules has been extensively studied in cell culture. However, relatively little is known about how growth cones contact the substrate or how the patterns of contact change as growth cones move forward. We have examined the interactions of chick retinal ganglion cell growth cones with laminin, merosin, N-cadherin, L1 and poly-L-lysine by time-lapse interference reflection microscopy (IRM) using a laser scanning confocal microscope. In images obtained by IRM, areas of a cell that are closely apposed to the substrate appear dark whereas areas that are farther away appear light. Growth cones on Jaminin and merosin were almost uniformly light, indicating that very little of the membrane was in close contact with the substrate. Growth cones on N-cadherin had a mottled appearance with some relatively large dark gray areas. The proximal portions of filopodia often were dark, in contrast to those on laminin and merosin which were light. In addition, growth cones on N-cadherin had numerous dark gray punctate regions of close association with the substrate. Growth cones on L1 had darker regions than growth cones on other substrates and these comprised a larger fraction of their area. There also were differences in the temporal dynamics of growth cone interactions with different substrates and these differences correlated with differences in rates of growth. None of the contacts observed in growth cones were as dark or stable as focal contacts of fibroblasts.

Gene deletion of inositol hexakisphosphate kinase 2 predisposes to aerodigestive tract carcinoma.

Inositol hexakisphosphate kinase 2 (IP6K2), a member of the inositol hexakisphosphate kinase family, functions as a growth suppressive and apoptosis-enhancing kinase during cell stress. We created mice with a targeted deletion of IP6K2; these mice display normal embryogenesis, development, growth and fertility. Chronic exposure to the carcinogen 4-nitroquinoline 1-oxide (4-NQO, a UV-mimetic compound) in drinking water resulted in fourfold increased incidence of invasive squamous cell carcinoma (SCC) formation in the oral cavity and esophagus of the knockout (KO) mice compared to the wild-type (WT) littermates. Paradoxically, KO mice displayed relative resistance to ionizing radiation and exhibit enhanced survival following 8-10 Gy total body irradiation. Primary KO fibroblasts displayed resistance to antiproliferative effects of interferon-beta and increased colony forming units following ionizing radiation. Radioresistance of KO fibroblasts was associated with accelerated DNA repair measured by comet assay. Direct microinjection of 5-PP-Ins(1,2,3,4,6)P(5) (the enzymatic product of IP6K2), but not InsP(6) (the substrate of IP6K2) induced cell death in SCC22A squamous carcinoma cells.

The role of cell adhesion molecules in neurite outgrowth on Müller cells.

The roles of neural cell adhesion molecule (NCAM), L1, N-cadherin, and integrin in neurite outgrowth on various substrates were studied. Antibodies against these cell surface molecules were added to explants of chick retina and the neurites from retinal ganglion cells were examined for effects of the antibodies on neurite length and fasciculation. On laminin, an anti-integrin antibody completely inhibited neurite outgrowth. The same antibody did not inhibit neurite outgrowth on polylysine or Müller cells. Antibodies to NCAM, L1, and N-cadherin did not significantly inhibit neurite outgrowth on laminin but produced significant inhibition on Müller cells. The inhibition of neurite outgrowth on glia by anti-L1 antibodies supports the hypothesis that L1 is capable of acting in a heterophilic binding mechanism. On laminin, both anti-N-cadherin and anti-L1 caused defasciculation of neurites from retinal ganglion cells, while anti-NCAM did not. None of these antibodies produced defasciculation on Müller cells. The results indicate that these three cell adhesion molecules may be very important in interactions with glia as axons grow from the retina to the tectum and may be less important in axon-axon interactions along this pathway. No evidence was found supporting the role of integrins in axon growth on Müller cells.

Studies of the developing chick retina using monoclonal antibody 8A2 that recognizes a novel set of gangliosides.

A monoclonal antibody, Mab 8A2, that recognizes a novel set of gangliosides was produced by immunizing a mouse with Embryonic Day 14 chick optic nerve. Immunohistochemical studies of the developing chick retina revealed a complex pattern of Mab 8A2 immunoreactivity. Initially, staining is concentrated in the optic fiber layer in the central retina. Later in development, the most intense staining is seen at the periphery of the retina and 8A2 immunoreactivity appears in other retina layers. In the adult retina, 8A2 immunoreactivity is lost from the optic fiber layer but persists in the inner plexiform layer, inner nuclear layer, and outer plexiform layer. Cell culture experiments showed intense staining of neurites from retinal ganglion cells but no staining of Muller cells. Biochemical characterization of the epitope recognized by Mab 8A2 suggests that it includes a 9-O-acetyl group that is present on five different gangliosides. The 8A2 immunoreactive gangliosides are distinct from and have slower mobilities on thin-layer chromatographs than those recognized by Mab D1.1 which recognizes 9-O-acetyl GD3.

Specific interaction of heterogeneous nuclear ribonucleoprotein particle U with the leader RNA sequence of vesicular stomatitis virus.

The 3' ends of the genome and antigenome RNA of vesicular stomatitis virus (VSV) serve as the promoter sites for the RNA-dependent RNA polymerase in the initiation of transcription and replication, respectively. The leader RNA, the first transcript synthesized during the RNA synthetic step, contains sequences to initiate encapsidation with the nucleocapsid protein, which is a prerequisite for replication. It also plays a role in the inhibition of cellular RNA synthesis. To search for a specific cellular factor(s) which may interact with the leader RNA sequences and regulate these processes, we used a gel mobility shift assay to identify such a protein(s). By using nuclear extract, it was found that in addition to the previously reported La protein, a 120-kDa nuclear protein specifically interacts with the leader RNA. Biochemical and immunological studies identified the 120-kDa protein as heterogeneous nuclear ribonucleoprotein particle U (hnRNP U), which is involved in pre-mRNA processing. We also demonstrate that hnRNP U is associated with the leader RNA in the nuclei of VSV-infected cells and also packaged within the purified virions. By double immunofluorescence labeling and confocal microscopy, hnRNP U appears to colocalize with the virus in the cytoplasm of infected cells. These results strongly suggest that hnRNP U plays an important role in the life cycle of VSV.

Normal and reactive NG2+ glial cells are distinct from resting and activated microglia.

We have previously used antibodies to the NG2 proteoglycan and the alpha receptor for platelet-derived growth factor (PDGF alpha receptor) to identify oligodendroglial progenitor cells in vivo and in vitro. It has recently become evident that the GD3 antigen, which has been widely used as a marker for oligodendrocyte progenitor cells, is also expressed by microglial cells. In this study we have examined the relationship between the NG2+/PDGF alpha receptor+ glial progenitor cells and microglial cells in normal developing and mature rat brain and in inflammatory lesions in mice with experimental autoimmune encephalomyelitis (EAE). Double-labeling of sections from normal rat brain using anti-NG2 antibodies and lectin from Griffonia simplicifolia (GSA I-B4) or monoclonal antibody 4H1 indicated that there is no overlap between NG2+ glial progenitor cells and microglia in the parenchyma of the central nervous system. In EAE lesions, both NG2+ cells and microglia, identified by antibodies to F4/80 and CD45, displayed reactive changes characterized by increased cell number and staining intensity and shortening and thickening of cell processes. Both cell types were found surrounding perivascular infiltrates of lymphocytes. Double-labeling EAE sections for NG2 and F4/80 or CD45 failed to reveal cells that co-expressed both antigens, suggesting that reactive NG2+ cells are distinct from activated microglia. However, a close spatial relationship between NG2+ cells and microglia was observed in the normal brain and to a greater extent in EAE, where processes of an activated microglial cell were sometimes seen to encircle an NG2+ cell. These observations are indicative of a functional interaction between microglia and the NG2+ glial cells.

Specific interaction in vitro and in vivo of glyceraldehyde-3-phosphate dehydrogenase and LA protein with cis-acting RNAs of human parainfluenza virus type 3.

Human parainfluenza virus type 3 (HPIV3) genome RNA is transcribed and replicated by the virus-encoded RNA-dependent RNA polymerase, and specific cellular proteins play a regulatory role in these processes. To search for cellular proteins potentially interacting with HPIV3 cis-acting regulatory RNAs, a gel mobility shift assay was used. Two cellular proteins specifically interacted with the viral cis-acting RNAs containing the genomic 3'-noncoding region and the plus-sense leader sequence region. Surprisingly, by biochemical and immunological analyses, one of the cellular proteins was identified as the key glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The other protein was characterized as the autoantigen, LA protein. Both GAPDH and LA protein also interacted with the same cis-acting RNA sequences in vivo and were found to be associated with the HPIV3 ribonucleoprotein complex in the infected cells. By double immunofluorescent labeling, GAPDH was found to be co-localized with viral ribonucleoprotein in the perinuclear region. These observations strongly suggest that cellular GAPDH and LA Protein participate in the regulation of HPIV3 gene expression.

Selective coexpression of NMDAR2A/B and NMDAR1 subunit proteins in dysplastic neurons of human epileptic cortex.

NR1 and NR2 are the two gene families for the NMDA receptor. In vitro studies show that while NR2 alone is nonfunctional, NR1 alone produces weak currents to glutamate or NMDA. We previously showed by immunocytochemistry (ICC) that in normal appearing, nonepileptic human cortical neurons, only NR1 and not NR2 proteins were expressed, in contrast to the presence of both NR1 and NR2 in normal rat cortical neurons. We also showed, in dysplastic epileptic cortex, that both NR1 and NR2 were highly expressed using ICC on adjacent 30-microm sections. However, the relative coexpressions of NR1 and NR2 proteins in single neurons in single sections of human epileptic cortex were unknown. In this study, we used double-labeled immunofluorescence and confocal microscopy to examine the distribution and coexpression of subunit proteins for NR1 and NR2A/B in both nondysplastic (control comparison) and dysplastic regions of human brain resected for the treatment of intractable epilepsy (11 patients). In nondysplastic regions, cortical neurons did not have immunoreactivity (ir) for NR2A/B, whereas NR1-ir was abundant. By contrast, dysplastic neurons in the regions with epileptic cortical dysplasia showed intense NR2A/B-ir in the somata and their dendritic processes. These same NR2A/B-ir dysplastic neurons were colabeled by NR1. These results demonstrate directly that dysplastic neurons express both NR2A/B and NR1 proteins, whereas nondysplastic cortical neurons express only NR1 proteins. Selective coexpression of NR2A/B and NR1 in dysplastic neurons suggests that NR2A/B may form heteromeric NR1-NR2 coassemblies and hyperexcitability in dysplastic neurons that could contribute to focal seizure onset.

Involvement of actin microfilaments in the replication of human parainfluenza virus type 3.

Several studies indicate that paramyxoviruses require a specific cellular factor(s) for transcription of their genomic RNAs. We previously reported that the cellular cytoskeletal protein actin, in its polymeric form, participates in the transcription of human parainfluenza virus type 3 (HPIV3) in vitro. In the present study, we investigated the role of the polymeric form of actin, i.e., the actin microfilaments of the cytoskeletal framework, in the reproduction of HPIV3 in vivo. Pulse-chase labeling analyses indicate that the viral nucleocapsid-associated proteins, NP and P, are present predominantly in the cytoskeletal framework during infection. By in situ hybridization, we found that viral mRNAs and genomic RNA were synthesized from the nucleocapsids that were bound to the cytoskeletal framework. Double immunofluorescent labeling and confocal microscopy of the cytoarchitecture revealed that the viral nucleocapsids are specifically localized on the actin microfilaments. Treatment of cells with the actin-depolymerizing agent, cytochalasin D, resulted in the inhibition of viral RNA synthesis and ribonucleoprotein accumulation. These results strongly suggest that actin microfilaments play an important role in the replication of HPIV3.

A Plasmodium falciparum protein located in Maurer's clefts underneath knobs and protein localization in association with Rhop-3 and SERA in the intracellular network of infected erythrocytes.

We report on the characterization of monoclonal antibodies against Plasmodium falciparum schizonts, which recognize parasite proteins of 130 kDa and 20 kDa. The 130-kDa protein was released by alkaline sodium carbonate treatment, suggesting that the protein is a peripheral membrane protein, while the 20-kDa protein remained associated with the membranes following alkali treatment, suggesting it may be an integral membrane protein. Both proteins were localized to large cytoplasmic vesicles within the cytoplasm of trophozoite and schizont-infected erythrocytes by immunofluorescence assay and confocal microscopy. Both proteins colocalized with Bodipy-ceramide in trophozoite and immature schizont-infected erythrocytes, but not in segmenters. The 130-kDa protein was localized by immunoelectron microscopy (IEM) to Maurer's clefts underneath knobs in a knobby and cytoadherent (K +/ C+) P. falciparum strain. No IEM reactivity was obtained in a knobless and non-cytoadherent (K-/C-)

Interleukin 10 (IL-10)-mediated inhibition of inflammatory cytokine production by human alveolar macrophages.

Alveolar macrophages are an important source of inflammatory cytokines in the lung. IL-10 has been shown to inhibit inflammatory cytokine production by human alveolar macrophages, but mechanisms are unclear. The purpose of the present study was to investigate whether IL-10 modified cytokine production by interference with transcriptional pathways. Alveolar macrophages were obtained from healthy controls by fiberoptic bronchoscopy and incubated with LPS+/-IL-10. Results indicated that steady state mRNA levels of tumour necrosis factor-alpha (TNF) and interleukin 1-beta (IL-1) decreased in the presence of IL-10. Consequently, electrophoretic mobility shift assays were performed using end-labelled nuclear factor-kappa B (NF-kappa B) or activator protein-1 (AP-1) probe. NF-kappa B binding was decreased in extracts from macrophages incubated for 4 h with LPS+IL-10 in comparison to those incubated with LPS alone. IL-10 also inhibited TNF secretion and NF-kappa B activation induced by another stimulus, staphylococcal toxin. Supershift assays revealed the presence of both p50 and p65 subunits of NF-kappa B. AP-1 was not affected by IL-10. Further examination of mechanisms indicated that IL-10 delayed the LPS-mediated degradation of the inhibitor protein I kappa B, thus delaying the nuclear translocation of the p65 subunit. These observations provide the first evidence that IL-10 antagonizes cytokine transcription in human alveolar macrophages by impeding the nuclear translocation of NF-kappa B by delaying the degradation of I kappa B.