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Biomedical subjects

E J Brown

Publications and source records attributed to E J Brown.

At least 37 records · Page 2Linked to original sources
PubMed2000

Membrane raft association of CD47 is necessary for actin polymerization and protein kinase C theta translocation in its synergistic activation of T cells.

CD47 is a ubiquitously expressed membrane protein with an extracellular Ig domain and a multiple membrane-spanning domain that can synergize with antigen to induce interleukin (IL)-2 secretion by T lymphocytes. Ligation of CD47 induced actin polymerization and increased protein kinase Ctheta (PKCtheta) association with the cytoskeleton independent of antigen receptor ligation, but ligation of mutant forms of the molecule missing either the Ig domain or the multiple membrane-spanning domain did not. Simultaneous ligation of CD47 and CD3 led to additive effects on F-actin and synergistic effects on PKCtheta cytoskeletal association. Disruption of membrane rafts by removal of cholesterol with cyclodextrin blocked CD47-induced actin polymerization, and mutant forms of CD47 that localized poorly to rafts failed to effect cytoskeletal rearrangement. However, raft association alone was not sufficient, because a raft-localized CD47 Ig domain bound to the membrane by a glycan phosphoinositol anchor was unable to induce actin polymerization. A mutant form of CD47 without its Ig domain that did not induce actin polymerization or localize to rafts still enhanced T cell receptor (TCR)-dependent tyrosine phosphorylation of PLCgamma and associated Ca(2+) signaling but did not augment IL-2 secretion. Thus, CD47 synergy with TCR to increase [Ca(2+)](i) is independent of actin and rafts but is insufficient to explain CD47 cooperation with TCR in IL-2 synthesis. Full synergy with TCR requires CD47 localization to membrane rafts where ligation leads to TCR-independent signals causing actin polymerization and PKCtheta translocation.

Actins↗
PubMed2000

CD47, a ligand for the macrophage fusion receptor, participates in macrophage multinucleation.

The macrophage fusion receptor (MFR), also called P84/BIT/SIRPalpha/SHPS-1, is a transmembrane glycoprotein that belongs to the superfamily of immunoglobulins. Previously, we showed that MFR expression is highly induced at the onset of fusion in macrophages, and that MFR appears to play a role in macrophage-macrophage adhesion/fusion leading to multinucleation. The recent finding that IAP/CD47 acts as a ligand for MFR led us to hypothesize that it interacts with CD47 at the onset of cell-cell fusion. CD47 is a transmembrane glycoprotein, which, like MFR, belongs to the superfamily of immunoglobulins. We show that macrophages express the hemopoietic form of CD47, the expression of which is induced at the onset of fusion, but to a lower level than MFR. A glutathione S-transferase CD47 fusion protein engineered to contain the extracellular domain of CD47, binds macrophages, associates with MFR, and prevents multinucleation. CD47 and MFR associate via their amino-terminal immunoglobulin variable domain. Of the nine monoclonal antibodies raised against the extracellular domain of CD47, three block fusion, as well as MFR-CD47 interaction, whereas the others have no effect. Together, these data suggest that CD47 is involved in macrophage multinucleation by virtue of interacting with MFR during adhesion/fusion.

Animals↗
PubMed2000

Role of a bacillus Calmette-Guérin fibronectin attachment protein in BCG-induced antitumor activity.

Intravesical Mycobacterium bovis bacillus Calmette-Gu*erin (BCG) is the treatment of choice for superficial bladder cancer. Previous studies showed that attachment of BCG to fibronectin within the bladder was necessary for mediation of the antitumor response. Further studies identified a bacterial receptor, fibronectin attachment protein (FAP), as an important mediator of BCG attachment to fibronectin. In vitro studies showed that a stable BCG/fibronectin interaction was dependent on FAP binding to fibronectin; however, no role for FAP in the attachment of BCG in vivo has been characterized. We now report the cloning of the M. bovis BCG FAP (FAP-B) and demonstrate an important role for FAP in the in vivo attachment of BCG to the bladder wall and in the induction of BCG-mediated antitumor activity. The predicted amino acid sequence for FAP-B shows 61% and 71% homology, respectively, with Mycobacterium avium FAP (FAP-A) and Mycobacterium leprae FAP (FAP-L). Rabbit polyclonal antibodies against Mycobacterium vaccae FAP (FAP-V) reacted with all 3 recombinant FAP proteins on Western blots. Functional studies show FAP-B to bind fibronectin via the highly conserved attachment regions previously identified for FAP-A and FAP-L and also to competitively inhibit attachment of BCG to matrix fibronectin. In vivo studies show FAP to be a necessary protein for the stable attachment of BCG to the bladder wall. Moreover, stable binding of BCG via FAP was shown to be necessary for the expression of BCG-induced antitumor activity. Our results demonstrate a biological role for FAP in the mediation of BCG-induced antitumor activity.

Adhesins, Bacterial↗
PubMed2000

ATR disruption leads to chromosomal fragmentation and early embryonic lethality.

Although a small decrease in survival and increase in tumor incidence was observed in ATR(+/-) mice, ATR(-/-) embryos die early in development, subsequent to the blastocyst stage and prior to 7.5 days p.c. In culture, ATR(-/-) blastocysts cells continue to cycle into mitosis for 2 days but subsequently fail to expand and die of caspase-dependent apoptosis. Importantly, caspase-independent chromosome breaks are observed in ATR(-/-) cells prior to widespread apoptosis, implying that apoptosis is caused by a loss of genomic integrity. These data show that ATR is essential for early embryonic development and must function in processes other than regulation of p53.

Animals↗
PubMed2000

The role of p53 in megakaryocyte differentiation and the megakaryocytic leukemias of Down syndrome.

Sequence analysis of the p53 tumor suppressor gene was performed on diagnostic blood and bone marrow samples from nine children with Down syndrome and acute megakaryoblastic leukemia. p53 sequence alterations were not observed in any of seven cases of transient leukemia; however, samples from two of three patients with acute megakaryoblastic leukemia harbored sequence alterations. The acquisition of both mutations (Gly245Val and Arg72Pro) in the transformation from transient leukemia to overt acute megakaryoblastic leukemia suggests a functional role of mutant p53 in the evolution of this disease.

Amino Acid Sequence↗
PubMed2000

African American grandmothers' perceptions of caregiver concerns associated with rearing adolescent grandchildren.

In 1996, grandparents were the sole providers of care for 269,000 grandchildren. Research findings indicate that grandmothers are usually the primary caretakers of grandchildren. Previous issues associated with raising grandchildren vary, but usually relate to the grandparents' health, financial concerns, or lack of social support. This qualitative descriptive study was designed to describe African American grandmothers' perceptions about the burdens of raising adolescent grandchildren. Using this approach, the researchers were able to study the richness and diversity of the grandmothers' perceptions. Data were collected from 35 grandmothers through a focus group (n = 7) and during a 4-day intervention research study (n = 28) entitled "Grandmother-Grandchild Health Promotion Program." Numerous concerns were voiced by both groups, but those stated most frequently were associated with the well-being of grandchildren (grandchildren's sexual behavior, potential drug use, and potential risk of encountering violence) and relational conflicts with adult children. Unexpected findings were the absence of stated concerns associated with the grandmothers' financial situation or health. Recommendations for intervention, clinical practice, and research are discussed.

Adolescent↗
PubMed2000

The role of Mycobacterium avium complex fibronectin attachment protein in adherence to the human respiratory mucosa.

Mycobacterium avium complex (MAC) are opportunistic respiratory pathogens that infect non-immunocompromised patients with established lung disease, although they can also cause primary infections. The ability to bind fibronectin is conserved among many mycobacterial species. We have investigated the adherence of a sputum isolate of MAC to the mucosa of organ cultures constructed with human tissue and the contribution of M. avium fibronectin attachment protein (FAP) to the process. MAC adhered to fibrous, but not globular mucus, and to extracellular matrix (ECM) in areas of epithelial damage, but not to intact extruded cells and collagen fibres. Bacteria occasionally adhered to healthy unciliated epithelium and to cells that had degenerated exposing their contents, but never to ciliated cells. The results obtained with different respiratory tissues were similar. Two ATCC strains of MAC gave similar results. There was a significant reduction (P < 0.05) in the number of bacteria adhering to ECM after preincubation of bacteria with fibronectin and after preincubation of the tissue with M. avium FAP in a concentration-dependant manner. The number of bacteria adhering to fibrous mucus was unchanged. Immunogold labelling demonstrated fibronectin in ECM as well as in other areas of epithelial damage, but only ECM bound FAP. A Mycobacterium smegmatis strain had the same pattern of adherence to the mucosa as MAC. When the FAP gene was deleted, the strain demonstrated reduced adherence to ECM, and adherence was restored when the strain was transfected with an M. avium FAP expression construct. We conclude that MAC adheres to ECM in areas of epithelial damage via FAP and to mucus with a fibrous appearance via another adhesin. Epithelial damage exposing ECM and poor mucus clearance will predispose to MAC airway infection.

Adenoids↗
PubMed2000

Early angiotensin converting enzyme inhibitor therapy enhances the benefits of late coronary artery reperfusion on infarct expansion.

BACKGROUND: Individually, both late reperfusion and early angiotensin converting enzyme (ACE) inhibitor treatment prevent infarct expansion after acute myocardial infarction. OBJECTIVE: To examine the effect and mechanism of early post-myocardial infarction ACE inhibitor treatment, when used in combination with late coronary artery reperfusion, on infarct expansion. METHODS: Sprague-Dawley rats underwent 8 h of coronary occlusion followed by permanent reperfusion. The treatment group received enalapril, started 1 h after coronary occlusion and continued for 13 days. A control group received placebo. Two weeks after acute myocardial infarction, hemodynamic, morphometric and histologic analyses were performed. RESULTS: Hemodynamic parameters were similar in both groups (P = NS). Infarct size was similar in the ACE inhibitor and placebo treatment groups (44 +/- 4% compared with 39 +/- 4%, P = NS). Septal thickness was also similar in the two groups (2.8 +/- 0.3 mm compared with 2.7 +/- 0.3 mm, P = NS). The ACE inhibitor-treated group had thicker infarcts than those in the placebo-treated group (0.93 +/- 0.07 mm compared with 0.76 +/- 0.04 mm, P < 0.05) and these infarcts were less expanded (expansion index 1.17 +/- 0.12 compared with 1.57 +/- 0.12, P < 0.05). ACE inhibitor treatment was associated with hypertrophy of viable myocytes within the scar compared with placebo treatment (cell diameter 11.1 +/- 0.5 microns compared with 8.9 +/- 0.4 microns, P < 0.01). CONCLUSIONS: Early post-myocardial infarction ACE inhibitor treatment enhances the benefits of late coronary reperfusion on infarct expansion. The benefits may be related to hypertrophy of still-viable myocytes within the infarcted zone.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed2000

Target outcomes for long-term oral health care in dementia: a Delphi approach.

This study developed a list of target outcomes for long-term oral health care in persons with dementia. A three-round Delphi study was used to develop a list of target outcomes. Participants included 99 staff and 171 family members associated with the Dementia Special Care Unit in Bedford, MA. In Round 1 participants were asked to list five outcomes for long-term oral health care. Items were grouped, redundancies removed, and fed back in Round 2, when participants scored the items from 1 (least important) to 10 (most important). Round 2 responses were tabulated and the top 20 were fed back for scoring in Round 3. The top 10 target outcomes in decreasing order of importance were: patient will be free from oral pain, patient will not be at risk for aspiration, emergency dental treatment will be available when needed, prevent mouth infections, daily mouth care is as much a part of daily care as shaving or brushing hair, prevent discomfort from loose teeth or sore gums, teeth will be brushed thoroughly once a day, staff will be able to provide oral hygiene care as needed, provide dental care to prevent problems eating, and recognize oral problems early. Family and professional caregivers were remarkably consistent in their identification of the top 10 outcomes. Further work is needed to ensure broad international and interdisciplinary acceptance (including families and the long-term care residents themselves) of target outcomes for long-term oral health care in persons with dementia.

Activities of Daily Living↗
PubMed2000

Comprehensive STD/HIV prevention education targeting US adolescents: review of an ethical dilemma and proposed ethical framework.

Adolescents are increasingly at risk for sexually transmitted diseases (STDs), including human immunodeficiency virus (HIV) infection. The prolonged latency period, sometimes in excess of five years, and the incubation period of up to 10 years before the manifestation of symptoms, may foster adolescents' false sense of invincibility and denial as they often do not see the devastating effects of the disease in their peers until they are older. In turn, their practice of safer sex may be hindered and thereby contribute to the escalation of this public health crisis among sexually active adolescents. Prevention-focused recommendations were made in the USA as a result of this crisis. Recommendations were made to: (1) include STD/HIV education in the curricula of grades kindergarten to 12; (2) increase to at least 75% the proportion of primary care and mental health professionals who provide age-appropriate STD/HIV prevention counselling to adolescents; and (3) expand HIV prevention services to include age-appropriate HIV education curricula for students in grades 4-12 in 95% of schools. Yet, in the USA, the provision of school-based comprehensive STD/HIV education has been difficult to achieve owing to certain limitations and, in some instances, legal action. These limitations include: limited student access; restricted content; and the implementation of sporadic and/or brief educational programmes. Given these recommendations and the fact that adolescents are acquiring STDs and HIV infections at increasing rates, and despite the limitations and legal actions, do health care professionals not have an ethical obligation to provide adolescents with comprehensive STD/HIV prevention education? This ethical dilemma will be discussed using the ethical decision-making principles of 'autonomy' and 'beneficence', and a decision-making model proposed by Thompson and Thompson, and by Chally and Loric.

Adolescent↗
PubMed2000

HIV among people with mental illness: contributing factors, prevention needs, barriers, and strategies.

1. Findings suggest that PWMI, including those with serious mental illness, are sexually active although some individuals, including mental health professionals, perceive them to be asexual or not sexually active. 2. PWMI may be particularly susceptible to HIV risk-related behaviors due to poor judgment, limited impulse control, deficits in problem-solving skills, and suicidal intent and self-destructive behavior. 3. Nurses, due to their close interaction with PWMI, are in key positions to document HIV risk-related behaviors and to advocate for integrating HIV risk reduction strategies into the therapeutic milieu.

Acquired Immunodeficiency Syndrome↗
PubMed2000

Late coronary artery reperfusion: benefits and mechanisms.

In patients with acute myocardial infarction (AMI), coronary artery reperfusion beyond 4 to 6 h after the onset of symptoms is believed to have little or no effect on infarct size. However, benefits have been demonstrated even when reperfusion is performed late and without significant myocardial salvage. Late reperfusion prevents infarct expansion and left ventricular remodelling. The mechanism is multifactorial and partially related to salvage of small islets of still viable myocytes within the scar. Other benefits of late reperfusion are electrical stability and prevention of arrhythmias. The survival benefits of reperfusion are strongly dependent on the time after the onset of symptoms until treatment (better outcomes are obtained with earlier reperfusion). Based on the demonstration of a significant reduction in mortality, the current practice is to perform reperfusion up to 12 h after the onset of symptoms. The survival benefits of reperfusion performed 13 to 24 h after the onset of AMI are less certain; however, large AMIs, particularly anterior infarcts, have the maximum risk of remodelling. Thus, benefits of reperfusion on left ventricular remodelling, arrhythmogenicity and prevention of congestive heart failure justify reperfusion in this group of patients up to 24 h after the onset of symptoms.

Animals↗
PubMed1999

Subversion of monocyte functions by coxiella burnetii: impairment of the cross-talk between alphavbeta3 integrin and CR3.

Several intracellular pathogens exploit macrophages as a niche for survival and replication. The success of this strategy requires the subversion or the avoidance of microbicidal functions of macrophages. Coxiella burnetii, the agent of Q fever, is a strictly intracellular bacterium that multiplies in myeloid cells. The survival of C. burnetii may depend on the selective use of macrophage receptors. Virulent C. burnetii organisms were poorly internalized but survived successfully in human monocytes, whereas avirulent variants were efficiently phagocytosed but were also rapidly eliminated. The uptake of avirulent organisms was mediated by leukocyte response integrin (alphavbeta3 integrin) and CR3 (alphaMbeta2 integrin), as demonstrated by using specific Abs and RGD sequence-containing peptides. The phagocytic efficiency of CR3 depends on its activation via alphavbeta3 integrin and integrin-associated protein. Indeed, CR3-mediated phagocytosis of avirulent C. burnetii was abrogated in macrophages from integrin-associated protein-/- mice. In contrast, the internalization of virulent C. burnetii organisms involved the engagement of alphavbeta3 integrin but not that of CR3. The pretreatment of monocytes with virulent C. burnetii organisms prevented the CR3-mediated phagocytosis of zymosan particles and CR3 activation assessed by the expression of the 24 neo-epitope. We conclude that the virulence of C. burnetii is associated with the engagement of alphavbeta3 integrin and the impairment of CR3 activity, which probably results from uncoupling alphavbeta3 integrin from integrin-associated protein. This study describes a strategy not previously reported of phagocytosis modulation by intracellular pathogens.

Antigens, CD↗
PubMed1999

Human signal-regulatory protein is expressed on normal, but not on subsets of leukemic myeloid cells and mediates cellular adhesion involving its counterreceptor CD47.

Signal-regulatory proteins (SIRPs) comprise a novel transmembrane glycoprotein family involved in the negative regulation of receptor tyrosine kinase-coupled signaling pathways. To analyze the expression and function of SIRPs, we prepared soluble recombinant fusion proteins of the extracellular regions of SIRPalpha1 and SIRPalpha2, as well as a variety of monoclonal antibodies (MoAbs) against these domains. The antibodies reacted predominantly with monocytes, granulocytes, dendritic cells, and their precursors, as well as with bone marrow CD34(+), AC133(+), CD90(+) hematopoietic stem/progenitor cells. In contrast, SIRP expression was absent or significantly reduced on the majority of myeloid blasts from patients with acute myeloid leukemia (AML) or chronic myeloid leukemia (CML). Functional studies showed that the extracellular domains of SIRPalpha1 and SIRPalpha2 support adhesion of a number of primary hematopoietic cells and cell lines. This interaction could be blocked by 4 of 7 SIRPalpha1-reactive MoAbs. In addition, SIRPalpha1 and SIRPalpha2 competed for the same cell binding site, suggesting a common widely expressed SIRP ligand. In an approach to identify this molecule, MoAbs were generated against the SIRP-binding cell line CCRF-CEM, and MoAb CC2C6 was selected because of its capacity to inhibit cell binding to SIRPalpha1. Further analysis showed that this antibody recognized CD47, a ubiquitously expressed plasma membrane protein previously implicated in integrin function, host defense action, and neutrophil migration. In this study, we identify CD47 as the extracellular ligand for human SIRP and show that these two counterreceptors are involved in cellular adhesion.

Antigens, CD↗
PubMed1999

Immune complex-induced integrin activation and L-plastin phosphorylation require protein kinase A.

Integrins in resting leukocytes are poorly adhesive, and cell activation is required to induce integrin-mediated adhesion. We recently demonstrated a close correlation between phosphorylation of Ser(5) in L-plastin (LPL), a leukocyte-specific 67-kDa actin bundling protein, and activation of alpha(M)beta(2)-mediated adhesion in polymorphonuclear neutrophils (PMN) (Jones, S. L., Wang, J., Turck, C. W., and Brown, E. J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 9331-9336). However, the kinase that phosphorylates LPL Ser(5) has not been identified. We found that cAMP-dependent protein kinase (PKA), but not a variety of other serine kinases, can specifically phosphorylate LPL and LPL-derived peptides on Ser(5) in vitro. The cell-permeable cAMP analog 8-bromo-cAMP and the adenylate cyclase activator forskolin both induce LPL phosphorylation in cells. Two PKA inhibitors, H89 and KT5720, inhibited immune complex (IC)-stimulated LPL phosphorylation as well as IC-induced activation of alpha(M)beta(2)-mediated adhesion in PMN. The dose response of H89 inhibition of PMN adhesion correlated with its inhibition of LPL phosphorylation in response to IC. IC stimulation also transiently increased intracellular cAMP concentration in PMN. Thus, PKA functions in an integrin activation pathway initiated by IC binding to Fcgamma receptors in addition to its better known role as a negative regulator of cell activation by G protein-coupled receptors. In contrast, LPL Ser(5) phosphorylation and PMN adhesion induced by formylmethionyl-leucylphenylalanine or phorbol myristate acetate were not affected by PKA inhibitors, suggesting that a different kinase(s) is responsible for LPL phosphorylation in response to these agonists. Phosphoinositidyl 3-kinase also is required for FcgammaR but not formylmethionyl-leucylphenylalanine- or phorbol myristate acetate-induced LPL phosphorylation and activation of alpha(M)beta(2). Two phosphoinositidyl 3-kinase inhibitors blocked FcgammaR-induced cAMP accumulation, demonstrating that this kinase acts upstream of PKA. These data demonstrate a necessary role for PKA in IC-induced integrin activation and LPL phosphorylation.

Amino Acid Sequence↗
PubMed1999

Role of cholesterol in formation and function of a signaling complex involving alphavbeta3, integrin-associated protein (CD47), and heterotrimeric G proteins.

Integrin-associated protein (CD47) is a multiply membrane spanning member of the immunoglobulin superfamily that regulates some adhesion-dependent cell functions through formation of a complex with alphavbeta3 integrin and trimeric G proteins. Cholesterol is critical for the association of the three protein components of the supramolecular complex and for its signaling. The multiply membrane spanning domain of IAP is required for complex formation because it binds cholesterol. The supramolecular complex forms preferentially in glycosphingolipid-enriched membrane domains. Binding of mAb 10G2 to the IAP Ig domain, previously shown to be required for association with alphavbeta3, is affected by both the multiply membrane spanning domain and cholesterol. These data demonstrate that cholesterol is an essential component of the alphavbeta3/IAP/G protein signaling complex, presumably acting through an effect on IAP conformation.

Antibodies, Monoclonal↗
PubMed1999

Regulation of integrin-mediated T cell adhesion by the transmembrane protein tyrosine phosphatase CD45.

The transmembrane protein tyrosine phosphatase CD45 is required for Ag receptor signal transduction in lymphocytes. Recently, a role for CD45 in the regulation of macrophage adhesion has been demonstrated as well. To investigate further the role of CD45 in the regulation of adhesion, we examined integrin-mediated adhesion to fibronectin of two T cell lines and their CD45-deficient variants. The absence of CD45 correlated with enhanced adhesion to fibronectin via integrin alpha5beta1 (VLA-5), but not alpha4beta1 (VLA-4) in both cell lines. Adhesion returned to normal levels upon transfection of wild-type CD45 into the CD45-deficient lines. Transfection of chimeric or mutant molecules expressing some, but not all, CD45 domains and activities demonstrated that both the transmembrane domain and the tyrosine phosphatase activity of CD45 were required for regulation of integrin-dependent adhesion, but the highly glycosylated extracellular domain was dispensable. In contrast, only a catalytically active CD45 cytoplasmic domain was required for TCR signaling. Transfectants that restored normal levels of adhesion to fibronectin coimmunoprecipitated with the transmembrane protein known as CD45-associated protein. These studies demonstrate a novel role for CD45 in adhesion regulation and suggest a possible function for its association with CD45-associated protein.

Animals↗