Biomedical subjects

J Drazba

Publications and source records attributed to J Drazba.

13 recordsLinked to original sources

PubMed2005

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.

Adult↗

PubMed2001

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.

Animals↗

PubMed2001

Epidermal growth factor regulates astrocyte expression of the interleukin-4 receptor via a MAPK-independent pathway.

Human astrocytes express the interleukin (IL)-4 receptor alpha chain (IL-4R alpha) in vitro and in vivo but mechanisms governing astrocyte IL-4R alpha expression have not been established. We hypothesized that epidermal growth factor (EGF) and IL-4, agents that profoundly affect astrocyte proliferation, might also alter IL-4R alpha expression. Exposure to EGF for 24 h enhanced IL-4R alpha mRNA levels; in contrast, IL-4 yielded no increase. Immunoblotting demonstrated that EGF but not IL-4 increased astrocyte IL-4R alpha protein after 2--4 days of exposure. Similarly, EGF but not IL-4 strongly activated phosphorylation of p42/p44 extracellular regulated kinase isoforms, a reaction blocked by the mitogen-activated protein kinase (MAPK) inhibitor, PD98059. PD98059 also blocked EGF-stimulated DNA synthesis but not IL-4R alpha mRNA levels, while antibody to the EGF receptor (erbB1) blocked both EGF effects. Data suggest that astrocyte IL-4R alpha expression is upregulated by EGF but not by IL-4 in an EGF-receptor-dependent manner and that mechanisms are independent of MAPK activation.

Astrocytes↗

PubMed2000

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.

Bronchoalveolar Lavage Fluid↗

PubMed2000

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.

Blotting, Western↗

PubMed2000

Brain plasticity and cellular mechanisms of epileptogenesis in human and experimental cortical dysplasia.

PURPOSE: The cellular mechanisms that may contribute to epilepsy in resected human cortical dysplasia (CD) were compared with the in utero radiated rat CD model. In human and rat focal hippocampal epilepsy, postsynaptic N-methyl-D-aspartate receptors are up-regulated and presynaptic axon collaterals hyperinnervate them. We hypothesized that in both human and rat CD: (a) the N-methyl-D-aspartate receptor subunits NR1 and NR2A/B would be increased and coassembled, and (b) aberrant axons would be in regions of CD. METHODS: Tests for presynaptic and postsynaptic changes in human and rat CD included the following: (a) cytology, (b) immunocytochemistry, (c) coimmunoprecipitation, (d) double-labeled immunofluorescence, and (e) Timm histochemistry of hippocampal mossy fibers. Within-patient comparisons were made between epileptic tissue, identified by subdural electro-encephalographic seizure onsets, and nonepileptic tissue remote from the focus but within the therapeutic resection. Rats were radiated at embryonic day 17, and offspring were studied postnatally. Statistical comparisons were made against normal rats matched for age and tissue processing. RESULTS: In focal CD patients, NR2A/B subunits and their coassemblies with NR1 were increased significantly more than for the remote nonepileptic cortex. Confocal microscopy showed that NR1-NR2A/B colabeled single dysplastic neurons in both human and rat. In CD rats, mossy fibers innervated the anomalously oriented hippocampal neurons. CONCLUSIONS: Human epileptic CD exhibits a spectrum of abnormal cell orientations and laminations that must require plastic axodendritic changes during development. These altered circuits and receptors could account for the seizures and cognitive deficits found in patients with CD. The radiated rat CD model with cortical dyslaminations and NR2A/B subunit increases would allow the development and testing of drugs targeted at only the NR2A/B subunit or at decoupling the NR1-NR2 coassembly, which could provide a specific antiepileptic drug for dysplastic circuits without inducing general depression of all brain neurons.

Abnormalities, Radiation-Induced↗

PubMed1999

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.

Animals↗

PubMed1999

Nitric oxide regulation of asthmatic airway inflammation with segmental allergen challenge.

BACKGROUND: Despite evidence of increased nitric oxide (NO) in asthmatic compared with healthy individuals, the role of NO in airway inflammation is unclear. OBJECTIVE: The purpose of the study was to determine the in vivo effects of localized allergen challenge on airway NO levels and transcription factor activation. METHODS: In this study localized allergen challenge was used as a model of asthmatic exacerbation to determine the relationship of NO to airway inflammation. RESULTS: With allergen challenge, asthmatic patients had a rise in airway NO levels, whereas NO levels in healthy controls did not change. The increased NO in asthma with allergen challenge compared with healthy control subjects was associated with an increase in inflammatory cytokines (GM-CSF and macrophage inflammatory protein-1) in epithelial lining fluid and eosinophilic infiltrate in bronchoalveolar lavage fluid (BAL) and biopsy specimens. To investigate the mechanisms of cytokine gene expression, activation of the transcription factors activator protein-1 and nuclear factor-kappaB (NF-kappaB) in cells from BAL were evaluated. Activator protein-1 was not activated before or after local allergen challenge. In contrast, NF-kappaB activation was less in BAL cells from asthmatic patients with increased NO in comparison with controls. CONCLUSION: Our studies are the first to suggest an inverse correlation between NF-kappaB and airway NO in a localized segmental allergen challenge model in allergic asthmatic patients. The current study demonstrates that activation of the inflammatory response (eg, cytokines, cellular infiltrate) in allergic asthmatic patients is temporally associated with increased airway NO. We propose that NO that is up-regulated by cytokines is part of an autoregulatory feedback loop (ie, allergen challenge stimulates inflammatory cytokine production, which in turn stimulates NO production, and NO down-regulates cytokine production).

Adult↗

PubMed1999

Nitric oxide blocks nuclear factor-kappaB activation in alveolar macrophages.

Nitric oxide (NO) is an important endogenous regulatory molecule implicated in both proinflammatory and antiinflammatory processes in the lung. Previously, we demonstrated that in human alveolar macrophages (AM), NO decreased inflammatory cytokine production, including that of interleukin-1beta, tumor necrosis factor-alpha and macrophage inflammatory protein-1alpha. One mechanism by which NO could regulate such diverse cytokine production is through effects on the transcription factor nuclear factor-kappaB (NF-kappaB), which controls the expression of the genes for these inflammatory cytokines and growth factors. We therefore investigated whether NO affects NF-kappaB activation in AM in vitro and in vivo. In vitro studies with AM showed that NF-kappaB activation by lipopolysaccharide (LPS) is decreased by NO in a dose-dependent manner. NO prevented an LPS-mediated decrease in the NF-kappaB inhibitory protein IkappaB-alpha. In asthma, airway NO levels are increased, whereas in primary pulmonary hypertension (PPH), airway NO levels are lower than in healthy lungs. In vivo investigations were conducted with freshly isolated AM from healthy controls, asthmatic individuals, and PPH patients. Healthy individuals had airway NO levels of 8 +/- 2 ppb (mean +/- SEM), which is associated with low NF-kappaB activation. Asthma patients with airway NO levels > 17 ppb showed minimal NF-kappaB activation, whereas asthmatic individuals with NO levels </= 17 ppb showed greater NF-kappaB activation. PPH patients with low NO (1 +/- 1 ppb) had prominent NF-kappaB activation. These in vivo studies in asthma and PPH support the in vitro observation of an inverse relationship between NO and NF-kappaB activation. One mechanism by which NO blocks cytokine production involves IkappaB.

Asthma↗

PubMed1997

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.

Animals↗

PubMed1994

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.

Amino Acid Sequence↗

PubMed1991

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.

Animals↗

PubMed1990

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.

Animals↗