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Biomedical subjects

D A Lawrence

Publications and source records attributed to D A Lawrence.

At least 19 recordsLinked to original sources
PubMed1998

Plasminogen activator inhibitor-1 contains a cryptic high affinity binding site for the low density lipoprotein receptor-related protein.

Much of the controversy surrounding the binding of plasminogen activator inhibitor-1 (PAI-1) to the low density lipoprotein receptor-related protein (LRP) may be due to the labile structure of PAI-1 and the distinct conformations that it can adopt. To examine this possibility and to test the hypothesis that PAI-1 contains a specific high affinity binding site for LRP, a sensitive and quantitative assay for PAI-1 binding to LRP was developed. This assay utilizes a unique PAI-1 mutant that was constructed with a hexapeptide tag at the NH2 terminus, which is recognized by the protein kinase, heart muscle kinase and can be specifically labeled with 32P. Our results show that only 32P-PAI-1 in complex with a proteinase binds LRP with high affinity and is efficiently endocytosed by cells, indicating that a high affinity site for LRP is generated on PAI-1 only when in complex with a proteinase. In addition, PAI-1 in complex with different proteinases is shown to cross-compete for LRP binding, demonstrating that the binding site is independent of the proteinase and therefore must reside on the PAI-1 portion of the complex. Finally, mutagenesis of PAI-1 results in loss of LRP binding, confirming that the high affinity binding site is located on PAI-1 and suggesting that the LRP binding site lays within a region of PAI-1 previously shown to contain the heparin binding domain.

Animals↗
PubMed1998

Differential production of interleukin-6 in the brain and spleen of mice treated with lipopolysaccharide in the presence and absence of lead.

The heavy metal lead (Pb) markedly augments the lethality of endotoxin in laboratory animals. Much of the tissue injury produced by endotoxin is thought to be mediated by cytokines. Thus, the effects of Pb on the regulation of interleukin-6 (IL-6), a proinflammatory cytokine that shows high correlation with symptoms of endotoxic shock, and the levels of corticosterone, a hormone produced to prepare the body to cope with stress, upon lipopolysaccharide (LPS; endotoxin) administration were investigated. After intravenous administration of LPS, the kinetics of IL-6 gene expression by Northern blot analysis revealed a rapid increase of IL-6 mRNA, which peaked by 2 h in the spleens and 3 h in the brains of B6C3F1 female mice, with or without Pb exposure. Peak production of IL-6 protein after LPS challenge was observed at 2 h in the spleens and 3 h in the sera regardless of Pb-treatment. However, Pb-exposed mice showed an altered kinetic profile of IL-6 appearance in the brain, in that the levels of IL-6 in the brains peaked at 4 h rather than 3 h, the peak for the control mice. Moreover, at two time points, the amounts of IL-6 were found to be higher in the brains of Pb-treated mice. Increases in IL-6 were detected in multiple areas of the brain, but Pb did not significantly enhance this level in any area. The observation of both IL-6 transcripts and protein in the brains of mice upon peripheral LPS administration is indicative of local de novo synthesis of IL-6 in the brain. IL-6 production in the brain may contribute to the centrally mediated effects of IL-6, since IL-6 in the brain is known to activate the hypothalamus-pituitary-adrenal (HPA) axis. Upon LPS challenge, corticosterone levels peaked at the 2-h time point and stayed elevated for 6 h regardless of Pb exposure. The increases in brain IL-6 and its extended expression by Pb do not appear to have significantly altered the HPA axis on the basis of the corticosterone level, but brain IL-6 is known to affect multiple brain functions such as long-term potentiation.

Animals↗
PubMed1998

Differential effects of lead and cAMP on development and activities of Th1- and Th2-lymphocytes.

Lead (Pb) is known to have detrimental effects on the central nervous, hematopoietic, renal, and immune systems. Herein, it is demonstrated that Pb can skew T cell reactivities by preferentially enhancing the development of Th2 cells and inhibiting the development of Th1 cells. When naive splenic CD4+ T cells from DO11.10 ovalbumin-specific transgenic (OVA-tg) mice or OVA-tg/RAG2-/- mice were developed in vitro in the presence of Pb, preferential skewing toward Th2 cells was evident. The Pb-driven skewing toward Th2 was blocked significantly in the presence of exogenous IL-12 or anti-IL-4 mAbs. Although Pb and dibutyryl cAMP (dbcAMP) appear to have similar effects on the development and reactivity of Th1 cells, unlike Pb, dbcAMP did not enhance Th2 development/activity. Further evidence of Pb's differential T cell effects was observed, in that regardless of the activation stimuli (Ag/APC; anti-CD3; PMA + ionomycin), the addition of PbCl2 consistently resulted in significant inhibition of IFN gamma production by a Th1 clone and in increased IL-4 production by a Th2 clone. In vitro addition of IL-12 overcame Pb's inhibition of Th1 cells. Th1 cells treated with a phosphodiesterase inhibitor had significantly elevated [cAMP]i levels following anti-CD3 activation in the presence of Pb, suggesting that Pb may inhibit Th1 development by enhancing adenylate cyclase activity and elevating the [cAMP]i level. Similar to Pb, a low concentration (10 microM) of dbcAMP inhibited IFN gamma production by Th1, which was prevented by IL-12; however, inhibition of protein kinase A activity by KT5720 did not reverse these effects. These results indicate that the environmental toxicant Pb can modify immune reactivities by significantly altering the differentiation of precursor or naive Th cells as well as by directly inhibiting Th1 cells and stimulating Th2 cells.

Animals↗
PubMed1998

Lead potentiates cytokine- and glutamate-mediated increases in permeability of the blood-brain barrier.

We have measured the transendothelial electrical resistance across the blood-brain barrier (BBB) with a microelectrode technique and determined the effects of subcutaneous injections (five injections over ten days) of lipopolysaccharide (LPS, 100 ng/g), recombinant mouse interleukin-6 (IL-6, 5 ng/g), and/or inorganic lead (lead, 2.5 5 micrograms/g) on the ion permeability of arterioles in the temporoparietal cortex of anaesthetized mice between 10 and 40 days of age. In controls the electrical resistance increased with age. It was decreased in animals treated with IL-6, but unaffected by lead at the different ages studied. In IL-6 treated mice, repeated neonatal exposure to lead (five injections between 2 and 10 days after birth) caused a delay in the increase in arteriole resistance with age. LPS injections caused a 36% increase in ion permeability of the BBB in twenty-day-old mice, and lead potentiated this effect of LPS. Intra-arterial injections of glutamate did not alter vascular resistance, but topical applications of glutamate on the cerebrum caused a reversible decrease in the resistance in mice not treated with lead, and an irreversible decrease in mice treated with lead. Injections of glutamate in the lumen of arterial vessels in the parietal and temporoparietal brain areas of mice pretreated with lead and LPS, plus a topical application of glutamate, caused depolarization of neurons in the temporoparietal cortex. These results suggest that disruption of the BBB can allow serum glutamate to penetrate the brain, causing further disruption of the BBB, and that lead irreversibly potentiates this cascade of harmful events.

Animals↗
PubMed1998

Lipopolysaccharide and interleukin-6 enhance lead entry into cerebellar neurons: application of a new and sensitive flow cytometric technique to measure intracellular lead and calcium concentrations.

The distribution of intracellular ionized lead (Pb) and calcium in dissociated cerebellar cells of ten-day-old mice was measured by flow cytometry. There are no fluorescent probes specific for lead, whereas commonly used fluorescent calcium indicators bind heavy metals with greater affinity than they do calcium, which impedes discrimination of lead- and calcium-induced fluorescence changes. Therefore, we developed a method to determine [Pb2+]i and [Ca2+]i by employing a combination of the calcium indicator fluo-3 and the heavy-metal chelator TPEN. Using these methods, we studied the effects of multiple in vivo exposure (five subcutaneous injections over 10 days) to lipopolysaccharide (LPS, 100 ng/g), recombinant mouse interleukin-6 (IL-6, 5 ng/g) and/or inorganic lead (lead, 2.5 micrograms/g) on lead and calcium concentrations. Control cells had [Cai] of 112 nM. Lead exposure alone had little effect on [Ca2+]i and resulted in a mean [Pb2+]i of about 7 pM, and did not alter cell volume. A significant fraction of cells (about 44% of living cells) from animals treated with lead plus LPS were swollen, as determined by analysis of the light scattering pattern, and there was a small increase in the number of dead cells, identified with the nucleic acid stain, 7-aminoactinomycin. While [Ca2+]i was not significantly increased in animals treated with either only LPS or IL-6, lead and calcium concentrations were increased in animals exposed to lead and LPS or IL-6 in both the non-swollen and swollen cells, with a mean value of (Pb2+)i of 32 pM and (Ca2+)i of 155 nM in cells not swollen. Electrophysiological analysis showed that LPS injections caused decreases in the membrane potential of endothelial cells of the blood-brain barrier (BBB) and lead potentiated the effect of LPS. IL-6 mimicked the effects of LPS, but was less potent. Thus these experiments indicate a synergistic interaction between lead and cytokines on biophysical properties of both neurons and endothelial cells of the BBB.

Animals↗
PubMed1997

Neuroserpin, a brain-associated inhibitor of tissue plasminogen activator is localized primarily in neurons. Implications for the regulation of motor learning and neuronal survival.

A cDNA clone for the serine proteinase inhibitor (serpin), neuroserpin, was isolated from a human whole brain cDNA library, and recombinant protein was expressed in insect cells. The purified protein is an efficient inhibitor of tissue type plasminogen activator (tPA), having an apparent second-order rate constant of 6. 2 x 10(5) M-1 s-1 for the two-chain form. However, unlike other known plasminogen activator inhibitors, neuroserpin is a more effective inactivator of tPA than of urokinase-type plasminogen activator. Neuroserpin also effectively inhibited trypsin and nerve growth factor-gamma but reacted only slowly with plasmin and thrombin. Northern blot analysis showed a 1.8 kilobase messenger RNA expressed predominantly in adult human brain and spinal cord, and immunohistochemical studies of normal mouse tissue detected strong staining primarily in neuronal cells with occasionally positive microglial cells. Staining was most prominent in the ependymal cells of the choroid plexus, Purkinje cells of the cerebellum, select neurons of the hypothalamus and hippocampus, and in the myelinated axons of the commissura. Expression of tPA within these regions is reported to be high and has previously been correlated with both motor learning and neuronal survival. Taken together, these data suggest that neuroserpin is likely to be a critical regulator of tPA activity in the central nervous system, and as such may play an important role in neuronal plasticity and/or maintenance.

Animals↗
PubMed1997

In vivo the environmental pollutants lead and mercury induce oligoclonal T cell responses skewed toward type-2 reactivities.

An oligoclonal utilization of Vbetas has been reported for pathogenesis of several autoimmune diseases, anti-tumorigenic activity, and superantigen-regulation of thymic T cell development. Altered ratios of Th1 and Th2 cells also are observed in immunodysregulations, leading to impaired cell-mediated immunity with an increased incidence of infectious disease or cancer and/or aberrant immunity that could culminate with an autoimmune disease. Lead (Pb) and mercury (Hg) are known pollutants with immunodisrupting activities; Hg is known to cause autoimmune glomerulonephritis. Both metals are known to suppress host resistance to pathogens. To further evaluate the manner by which these metals cause in vivo immunomodulation, their in vivo effects on Vbeta expression were evaluated along with the Th1 and Th2 frequency. Exposure of BALB/c mice to PbCl2 or HgCl2 induced an oligoclonal response with increases of Vbeta 5+, Vbeta 7+, and Vbeta 13+ CD4+ splenic, but not thymic, T cells. A significantly skewed frequency of Pb-induced splenic Th2 cells expressing Vbeta 7 or Vbeta 13 over Th1 cells was determined by limiting dilution analysis, but this Th2 predominance was not observed with CD4+ T cells expressing Vbeta 8. DO11.10 transgenic mouse exposed to Pb and antigen also demonstrated a skewed type-2 response evidenced by significantly increased IgE levels, lowered IFN-gamma levels, and increased IgG1 and lowered IgG2a anti-OVA levels. Even in the absence of specific T cell responses to a Pb-induced antigen, due to the restricted T cell specificity in the transgenic mouse model, Pb still was able to skew the response toward type-2 reactivity. However, this skewing occurred only in the presence of antigen. Therefore, the Pb-induced oligoclonal T cell response in BALB/c mice which must be initiated by self-antigens and was predominately type-2 may be responsible for autoantibody production and the detrimental health effects associated with Pb exposure.

Animals↗
PubMed1997

Transforming growth factor-beta1 enhances the lethal effects of DNA-damaging agents in a human lung-cancer cell line.

In tissue culture conditions, exogeneous active transforming growth factor-beta1 (TGF-beta1) enhances the lethal effect of DNA-damaging agents (UV-C, gamma rays, cisplatin, methotrexate and 5-fluorouracil) toward human A549 cells and mink Mv1Lu cells, as detected by the loss of their capacity to give rise to colonies; both these cell lines harbor a wild-type p53, as determined by immunoprecipitation. Contrastingly, the sole effect of the cytokine used alone is to inhibit reversibly the multiplication of the same cells without further impairing, once withdrawn from their environment, their capacity to divide and give rise to colonies. The lethal synergy between TGF-beta1 and UV-C was studied on mink and human cell lines, and the biomodulation by TGF-beta1 of cell killing by cisplatin, gamma rays, 5-fluorouracil or methotrexate was tested only on human cells. As investigated with UV-C-irradiated human A549 cells, TGF-beta1 appears to enhance apoptosis rather than to disturb the repair of DNA photolesions (mainly pyrimidine dimers) by the nucleotidic excision repair pathway according to results of nucleosomal ladder and comet tests. Our data raise the possibility that, in vivo, TGF-beta1 might affect the curative and/or undesirable secondary side effects of cancer therapy.

Animals↗
PubMed1997

Serpin conformational change in ovalbumin. Enhanced reactive center loop insertion through hinge region mutations.

Ovalbumin is a noninhibitory member of the serpin superfamily that does not spontaneously undergo the loop-to-sheet conformational change upon cleavage of its reactive center that is characteristic of inhibitory serpins. We tested the hypothesis that ovalbumin could be turned into a proteinase inhibitor by increasing the rate of loop insertion through hinge region mutations alone. We found that none of the three variants examined showed any detectable proteinase inhibitory properties. However, replacement of the P14 arginine residue of ovalbumin by serine, either alone or in combination with changes of P12-P10 to alanine, resulted in a large increase in the rate of loop insertion into beta-sheet A following cleavage at the P1-P1' bond by porcine pancreatic elastase (PPE), as shown by the spontaneous formation of a loop-inserted form upon cleavage that has increased the thermal stability. From the magnitude of the increase in stability of the cleaved, loop-inserted forms of the P14 ovalbumin variants, as well as the accessibility of the P1-P1'-cleaved reactive center loop to further proteolysis at P8-P7, we concluded that the reactive center loop can only partially insert into beta-sheet A and therefore that ovalbumin is also defective in the ability of beta-sheet A to expand to fully accommodate the whole of the reactive center loop. This defect, through its effect on the extent and/or rate of loop insertion, is likely to be a principal reason for ovalbumin not being a proteinase inhibitor.

Animals↗
PubMed1997

Characterization of the binding of different conformational forms of plasminogen activator inhibitor-1 to vitronectin. Implications for the regulation of pericellular proteolysis.

Plasminogen activator inhibitor type 1 (PAI-1), the primary physiologic inhibitor of plasminogen activation, is associated with the adhesive glycoprotein vitronectin (Vn) in plasma and the extracellular matrix. In this study we examined the binding of different conformational forms of PAI-1 to both native and urea-purified vitronectin using a solid-phase binding assay. These results demonstrate that active PAI-1 binds to urea-purified Vn with approximately 6-fold higher affinity than to native Vn. In contrast, inactive forms of PAI-1 (latent, elastase-cleaved, synthetic reactive center loop peptide-annealed, or complexed to plasminogen activators) display greatly reduced affinities for both forms of adsorbed Vn, with relative affinities reduced by more than 2 orders of magnitude. Structurally, these inactive conformations all differ from active PAI-1 by insertion of an additional strand into beta-sheet A, suggesting that it is the rearrangement of sheet A that results in reduced Vn affinity. This is supported by the observation that PAI-1 associated with beta-anhydrotrypsin, which does not undergo rearrangement of beta-sheet A, shows no such decrease in affinity, whereas PAI-1 complexed to beta-trypsin, which does undergo sheet A rearrangement, displays reduced affinity for Vn similar to PAI-1.plasminogen activator complexes. Together these data demonstrate that the interaction between PAI-1 and Vn depends on the conformational state of both proteins and suggest that the Vn binding site on PAI-1 is sensitive to structural changes associated with loss of inhibitory activity.

Hydrolysis↗
PubMed1997

Assessment of immunotoxicity by multiparameter flow cytometry.

Flow cytometry is a unique technology useful in the examination of effects of immunotoxic agents on target cells of the immune system. The purpose of this workshop was to provide an overview of the use of flow cytometry in new and established models of immunotoxicity, with emphasis on the potential applications, assay validation, and potential pitfalls. This overview begins with a discussion of methods useful in the assessment of Ca2+-dependent mechanisms of lymphoid cell activation in surface marker-defined human B cells, T cells, and monocytes. A discussion of the use of flow cytometry in analysis of apoptosis is also presented in this paper. The second paper presents data on the development and use of flow cytometry as an alternative to a Cr51 release assay for an assessment of cytotoxic T cell activation. The use of surface markers for characterizing and distinguishing the effects of chemical irritants from sensitizers is next presented, followed by an overview of the use of fluorescent probes to assess cell thiol status and overall oxidant-induced injury to lymphoid cells. Finally, an interlaboratory study designed to compare and evaluate the use of flow cytometry procedures in rat splenic cell subtyping is presented. Overall, these studies demonstrate the utility of flow cytometry assays in immunotoxicologic research, but further efforts are needed in the validation of many of these assays for routine use in immunotoxicologic testing.

Allergens↗
PubMed1997

Modulation of T-helper cell populations: potential mechanisms of respiratory hypersensitivity and immune suppression.

Information presented at this symposium indicates that modulation of Th cell responses is one means by which xenobiotics may cause immunotoxicity. A shift from Th1 to Th2 responses can enhance both infectious and allergic disease. Hence, in some cases, a common mechanism may be responsible for effects that are generally considered to be very different. Because cytokines produced in the inflammatory process play a role in modulation of Th cell responses, there is a mechanism by which agents that appear to have only local effects at the portal of entry may, in fact, affect immune responses systemically. An understanding of conditions which trigger certain cytokine responses may be useful not only in understanding inflammation but also in predicting certain kinds of immunosuppressive and allergic responses. Future studies in this area are likely to provide insights into many areas of immunotoxicology.

Air Pollutants↗
PubMed1997

Interleukin-12 promotes enhanced resistance to Listeria monocytogenes infection of lead-exposed mice.

The heavy metal lead (Pb) has been shown to downregulate various parameters of cell-mediated immune (CMI) responses. This inhibition of CMI responses by Pb is exemplified by a higher mortality rate upon infections with sublethal doses of a variety of pathogens. Unlike Pb, which lowers host resistance, interleukin-12 (IL-12) exerts a substantial stimulatory influence on the host response to intracellular bacteria such as Listeria monocytogenes. To explore the influence of IL-12 in mice rendered susceptible to Listerial infection by oral exposure to Pb, we determined bacterial burdens and production of interferon gamma (IFN-gamma). As expected, Pb-exposed mice had increased morbidity due to higher Listerial titers as compared to control mice. However, administration of exogenous IL-12 reversed the Pb-induced inhibition of host defense and boosted the resistance of the non-Pb-treated mice. The enhanced CMI responses observed in both IL-12-treated groups were accompanied with elevations of IFN-gamma in the sera and spleens. Significant reduction in the number of viable Listeria in Pb-exposed mice upon IL-12 administration suggests that the processes downstream of IL-12 production were intact in the Pb-exposed mice and that the inhibition by Pb was due to the lack of functional IL-12. Alternatively, the exogenous IL-12 may have overcome a downstream effect by enhancing an secondary pathway. Support for the former hypothesis is based on the observation that Pb induced elevated levels of p40 splenic messenger RNA since increased p40 expression would result from lack of IL-12 formation. Contrary to the IFN-gamma levels, significantly higher levels of IL-6 and corticosterone were observed in the sera and spleens of Pb-exposed mice upon infection, suggesting heightened stress in the absence of IL-12. Overall, the results suggest that an environmental pollutant such as Pb can enhance the stress response, which naturally occurs during an infection, and can further compromise health by lowering host resistance by altering cytokine levels.

Animals↗
PubMed1997

In utero management of hydrops fetalis caused by critical aortic stenosis.

Hydrops fetalis is rarely associated with congestive heart failure caused by obstructive left-sided heart lesions. There are rare cases of live born neonates with critical congenital valvar aortic stenosis and hydrops reported in the literature, all with fatal outcomes. This report describes, to the best of our knowledge, the first two newborns who were diagnosed prenatally to have hydrops fetalis caused by critical valvar aortic stenosis, who were treated prenatally with digoxin and who postnatally had successful percutaneous balloon aortic valvuloplasty. Both patients had not only left but right ventricular dysfunction. We speculate that right ventricular dysfunction was a contributing factor in the development of hydrops in these patients and in utero medical therapy with digoxin is associated with resolution of the hydrops before delivery.

Angioplasty, Balloon↗
PubMed1997

Flow cytometric measurements of neuronal death triggered by PCBs.

We have examined the effects of several individual polychlorinated biphenyl (PCB) congeners and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on viability and intracellular calcium concentration in acutely dissociated cerebellar granule cell neurons obtained from 7-14 day old rat pups, using a flow cytometer. Cell death was determined using the fluorescent dye, 7-aminoactinomycin-D (7-AAD), while intracellular calcium concentration was determined using Fluo-3-AM. When acutely isolated neurons were exposed to 2,4,4' trichlorobiphenyl (2,4,4', there was a dose- and time-dependent loss of viability beginning within 5 min. At a concentration of 100 microM, 80% of neurons were dead within 45 min. Loss of viability was preceded by an apparent increase in cell granularity. No significant cell death was observed upon exposure to the highly dioxin-like coplanar PCB, 3,4,5,3',4', at a concentration of 100 microM or by TCDD at a concentration of 0.1 microM. A slight loss of viability was seen with the ortho 2,6,2',6' and the coplanar 3,4,3',4', but for both it was small. All of the PCB congeners caused an early, transient increase in intracellular free calcium concentration, although TCDD did not. A small, late increase in intracellular free calcium was seen with 2,4,4', probably reflecting events leading to loss of viability. We conclude that at least one PCB congener, 2,4,4', is very neurotoxic and can cause rapid death of cerebellar granule cells.

Animals↗