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Biomedical subjects

R D Rossen

Publications and source records attributed to R D Rossen.

At least 19 recordsLinked to original sources
PubMed2001

Matrix-dependent mechanism of neutrophil-mediated release and activation of matrix metalloproteinase 9 in myocardial ischemia/reperfusion.

BACKGROUND: A key component of reperfusion of myocardial infarction is an immediate inflammatory response, which enhances tissue repair. Matrix turnover is crucial to tissue repair, and matrix metalloproteinases (MMPs) are key enzymes involved in matrix degradation. The hypothesis tested is that one inflammation-based effector of tissue repair is the secretion and activation of MMP-9 by infiltrating neutrophils. METHODS AND RESULTS: Cardiac lymph and tissue were assayed for atent and active MMP-2 and MMP-9 by zymography and immunochemistry. Dual-labeling immunofluorescence determined the cellular source of MMP-9 protein. Isolated canine neutrophils were incubated with preischemic and postischemic cardiac lymph in the presence and absence of collagen-fibronectin pads, and the supernatants were assayed for latent and active MMP-9. MMP-9 increased during the first hours of reperfusion in both lymph supernatants and myocardial extracts, and this increase was of neutrophil origin. MMP-9 in the cardiac lymph remained latent but was activatable. In contrast, MMP-9 in the myocardium was in both latent and active forms. In situ zymography demonstrated that activated MMP-9 surrounded the infiltrated neutrophils. When postischemic cardiac lymph was incubated with neutrophils in vitro, MMP-9 secretion and activation occurred only in the presence of a collagen-fibronectin substrate; preischemic cardiac lymph did not induce significant secretion or activation. CONCLUSIONS: Infiltrating neutrophils are an early source of MMP-9 after reperfusion, and a portion of MMP-9 in the myocardium is active. Infiltrating neutrophils may localize MMP-9 activation by secreting MMP-9 and as a source of activating proteases.

Animals↗
PubMed1999

Fibronectin fragments modulate monocyte VLA-5 expression and monocyte migration.

To identify the mechanisms that cause monocyte localization in infarcted myocardium, we studied the impact of ischemia-reperfusion injury on the surface expression and function of the monocyte fibronectin (FN) receptor VLA-5 (alpha(5)beta(1) integrin, CD49e/CD29). Myocardial infarction was associated with the release of FN fragments into cardiac extracellular fluids. Incubating monocytes with postreperfusion cardiac lymph that contained these FN fragments selectively reduced expression of VLA-5, an effect suppressed by specific immunoadsorption of the fragments. Treating monocytes with purified, 120-kDa cell-binding FN fragments (FN120) likewise decreased VLA-5 expression, and did so by inducing a serine proteinase-dependent proteolysis of this beta(1) integrin. We postulated that changes in VLA-5 expression, which were induced by interactions with cell-binding FN fragments, may alter monocyte migration into tissue FN, a prominent component of the cardiac extracellular matrix. Support for this hypothesis came from experiments showing that FN120 treatment significantly reduced both spontaneous and MCP-1-induced monocyte migration on an FN-impregnated collagen matrix. In vivo, it is likely that contact with cell-binding FN fragments also modulates VLA-5/FN adhesive interactions, and this causes monocytes to accumulate at sites where the fragment concentration is sufficient to ensure proteolytic degradation of VLA-5.

Amino Acid Sequence↗
PubMed1998

Cytokines and the microcirculation in ischemia and reperfusion.

The intense inflammatory reaction following reperfusion of the infarcted myocardium has been implicated as a factor in extension of injury. However, this inflammatory reaction is also critical to tissue repair. The cellular responses that mediate these functions are orchestrated by sequential induction and/or release of cytokines resulting in a closely regulated cytokine cascade. This paper reviews research on these cytokine cascades, their cellular origin, and factors which control the cellular response to their presence. Factors examined include leukotaxis, phenotypic transition of leukocytes, adhesion molecule induction and the role of cytokines in tissue repair and scar formation.

Animals↗
PubMed1997

Phagocytes in ischemia injury.

We are now developing the means to evaluate components of this inflammatory response that may facilitate healing. A key event in the change in the inflammatory response is the development of a cytokine cascade that promotes phenotypic changes in the infiltrating leukocytes, which endow them with the ability to promote fibroblast proliferation and collagen deposition, the hallmarks of healing.

Animals↗
PubMed1997

Transendothelial migration of lymphocytes from HIV-1-infected donors: a mechanism for extravascular dissemination of HIV-1.

To identify factors that cause HIV-1 to establish perivascular foci of infected cells, we studied the transendothelial migration of blood mononuclear leukocytes (MNL) from 76 HIV+ patients and 41 controls. The fraction of patients' lymphocytes that migrated across endothelial cell monolayers in vitro was significantly increased (p < or = 0.03) compared with that of control donors. Migration of patients' CD4+ T cells was particularly enhanced, whereas the migration of monocytes did not differ between patients and controls. Lymphocyte migration correlated with expression of CD11a/CD18 and CD49d/CD29 and with the quantity of TNF-alpha produced as MNLs migrated through the endothelium. Measurement of HIV-1 proviral DNA copies in the patients' MNLs (n = 26) suggested that in half the cases virus-infected cells accumulated preferentially amidst the migratory leukocytes. We observed the same behavior with normal donor MNLs infected, in vitro, with each of 4 strains of HIV-1. The number of HIV-1 proviral DNA copies per million MNLs was 40 to 178 times higher in the migratory population than in the original population added to the endothelium. To test whether only certain strains of HIV-1 stimulate transendothelial migration of infected cells, we used single strand conformation polymorphism analysis to identify quasispecies of HIV-1 in the MNLs. If all strains of HIV-1 were equal in their ability to stimulate transendothelial migration, we expected to find no differences in the quasispecies present in the original and migratory cell populations. In fact the quasispecies differed in 14 of 19 paired samples, suggesting that only certain HIV-1 quasispecies promote transendothelial migration of infected cells.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Complement C5a, TGF-beta 1, and MCP-1, in sequence, induce migration of monocytes into ischemic canine myocardium within the first one to five hours after reperfusion.

BACKGROUND: Recent studies suggest that reperfusion promotes healing of formerly ischemic heart tissue even when myocardial salvage is no longer possible. Since monocyte-macrophage infiltration is the hallmark of the healing infarct, we have attempted to identify mechanisms that attract monocytes into the heart after reperfusion of ischemic canine myocardium. METHODS AND RESULTS: Isolated autologous 99mTc-labeled mononuclear leukocytes injected into the left atrium localized preferentially in previously ischemic myocardium within the first hour after reperfusion. Histological studies revealed CD64+ monocytes in small venules and the perivascular connective tissue within the first hour after reperfusion. Flow cytometric analysis of cells in cardiac lymph showed systematically increasing numbers of neutrophils and monocytes between 1 and 4 hours after reperfusion; monocyte enrichment was eventually greater than neutrophil enrichment. Monocyte chemotactic activity in cardiac lymph collected in the first hour after reperfusion was wholly attributable to C5a. Transforming growth factor (TGF)-beta 1 contributed significantly to this chemotactic activity after 60 to 180 minutes, and after 180 minutes, monocyte chemotactic activity in lymph was largely dependent on monocyte chemoattractant protein (MCP)-1 acting in concert with TGF-beta 1. CONCLUSIONS: Beginning in the first 60 minutes after reperfusion, C5a, TGF-beta 1, and MCP-1, acting sequentially, promote infiltration of monocytes into formerly ischemic myocardium. These events may promote the healing of myocardial injury facilitated by reperfusion.

Animals↗
PubMed1997

Induction of monocyte chemoattractant protein-1 in the small veins of the ischemic and reperfused canine myocardium.

BACKGROUND: Healing after myocardial infarction is characterized by the presence of macrophages in the infarcted area. Since augmented monocyte influx has been implicated as a potential mechanism for improved healing after reperfusion, we wished to study the induction of monocyte chemoattractant protein-1 (MCP-1) during reperfusion. METHODS AND RESULTS: The cDNA for MCP-1 was cloned from a canine jugular vein endothelial cell (CJVEC) library and exhibited 78% identity with the deduced amino acid sequence of human MCP-1. Samples of myocardium were taken from control and ischemic segments after 1 hour of ischemia and various times of reperfusion; total RNA was isolated from myocardial samples and probed with a cDNA probe for canine MCP-1. Induction of MCP-1 mRNA occurred only in previously ischemic segments within the first hour of reperfusion, peaked at 3 hours, and persisted throughout the first 2 days of reperfusion. In the absence of reperfusion, no significant MCP-1 induction was seen. Both ischemic (but not preischemic) cardiac lymph and human recombinant TNF-alpha induced MCP-1 in CJVECs. MCP-1 was identified by immunostaining on infiltrating cells and venular (but not arterial) endothelium by 3 hours. In contrast, in situ hybridization showed MCP-1 mRNA to be confined to the endothelium of small veins (venules) 10 to 70 microns in diameter. CONCLUSIONS: MCP-1 mRNA is induced in the endothelium of a specific class of small veins immediately after reperfusion. MCP-1 induction is confined to the previously ischemic area that has been reperfused. We suggest a significant role for MCP-1 in monocyte trafficking in the reperfused myocardium.

Amino Acid Sequence↗
PubMed1995

In vitro evaluation of the role of humoral immunity against Bartonella henselae.

The contribution of humoral immunity against Bartonella henselae was evaluated by examining the in vitro bactericidal activity of sera and the ability of these microorganisms to activate complement and stimulate phagocytosis and an oxidative burst in polymorphonuclear leukocytes. The organism was killed by complement-mediated cytolysis. Complement activation preferentially proceeded by the alternative pathway. The presence of specific antibodies did not increase the serum bactericidal activity or complement activation. However, phagocytosis and the subsequent production of oxygen radicals, evaluated by flow cytometry, were significantly enhanced in the presence of bacteria previously opsonized with immune sera.

Adult↗
PubMed1995

Phenotypic and functional changes in peripheral blood monocytes during progression of human immunodeficiency virus infection. Effects of soluble immune complexes, cytokines, subcellular particulates from apoptotic cells, and HIV-1-encoded proteins on monocytes phagocytic function, oxidative burst, transendothelial migration, and cell surface phenotype.

We postulated that changes in the cell surface display of molecules that facilitate cell-cell and cell-matrix adhesions may reflect the changing immunosurveillance capacity of blood monocytes during progression of human immunodeficiency virus (HIV) infections. In Centers for Disease Control (CDC) stage A patients, whose monocytes' ability to phagocytose bacteria and generate reactive oxygen intermediates is often increased, the frequency of monocytes expressing CD49d, HLA-DP, HLA-DQ, and an activation epitope of CD11a/CD18 was increased and monocyte transendothelial migration was unimpaired. In CDC stage B/C patients, whose monocytes' ability to phagocytose bacteria and migrate across confluent endothelial monolayers was diminished, surface expression of CD49e and CD62L and the percentage of monocytes expressing CD18, CD11a, CD29, CD49e, CD54, CD58, CD31, and HLA-I were significantly decreased. Incubating normal donor monocytes with immune complexes in vitro reproduced the phenotypic and functional abnormalities seen in stage B/C patients. By contrast, in vitro stimulation with subcellular particulates released by apoptotic lymphocytes reproduced changes seen in stage A patients' monocytes. Although circulating monocytes appear to be activated at all stages, these data suggest that the high levels of circulating immune complexes, found predominantly in the later stages of HIV infection, may be particularly instrumental in reducing the monocyte's capacity to maintain surveillance against infection.

Antigen-Antibody Complex↗
PubMed1994

Phenotypic and functional activation of monocytes in HIV-1 infection: interactions with neural cells.

To investigate mechanisms that facilitate transendothelial migration of HIV-infected leukocytes and their interactions with neural tissues early in the disease, we studied peripheral blood from Centers for Disease Control class A patients. Patients' monocytes displayed increased quantities of the adhesion molecules CD11a, CD11b, and very late antigen 4 (VLA-4). Expression of these correlated directly with the numbers of monocytes that migrated through confluent endothelium. These ligands also mediated leukocyte interactions with cultured human neural cell lines. Although patients' cells bound in greater numbers, there was no evidence of target cell injury. To evaluate the direct effect of HIV-1 on monocyte neuroadhesion, we compared infected with uninfected monocytoid (U-937,THP-1) and T lymphoblastoid (MT-4) cell lines. HIV infection increased the neuroadhesiveness of monocytoid lines only. By using lines with more than 95% HIV-infected cells, we demonstrated that HIV-1 gp120 participates with lymphocyte function-associated antigen 1 and VLA-4 to mediate monocyte-neural cell interactions.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Cardiolipin-protein complexes and initiation of complement activation after coronary artery occlusion.

Specific rabbit anti-cardiolipin (anti-CL) antibodies were used to investigate the hypothesis that cardiolipin, associated with mitochondrial membrane proteins, binds C1 and facilitates activation of the complement cascade following reperfusion of ischemic myocardium. By immunoelectron microscopy, anti-CL localized to subsarcolemmal mitochondria, emerging through breaks in membranes of damaged cardiac myocytes. Anti-CL reacted with > 15 mitochondrial constituents, most of which comigrated with the proteins that bind C1q in transblots of subsarcolemmal mitochondria, fractionated by polyacrylamide gel electrophoresis under reducing conditions in the presence of sodium dodecyl sulfate. A subset of the C1q-binding proteins > 24 to 37 kDa served as stable sites for assembly of C3, C5, and C9. Cardiac lymph, collected during the first hour after reperfusion of ischemic myocardium, contained proteins of diverse size that reacted with both anti-CL and C1q. Cardiac lymph, collected before occlusion and 4 to 5 hours after reperfusion, in comparison, had few if any C1q or anti-CL reactive proteins. Treatment with phospholipase suppressed the C1q-binding activity and anti-CL reactivity of the proteins in reperfusion lymph and those with similar properties in mitochondrial extracts. Our data suggest that during ischemia, mitochondria, extruded through breaks in the sarcolemma, unfold and release membrane fragments in which cardiolipin and protein are intimately associated. By binding C1 and supplying sites for the assembly of later-acting complement components, these fragments provide the means to disseminate the complement-mediated inflammatory response to ischemic injury.

Animals↗
PubMed1994

Auditory P300 abnormalities and leukocyte activation in HIV infection.

To evaluate whether P300 testing might serve as a screening modality for the early detection of HIV-related neuropathology, we tested 26 HIV-infected men (23 without neurologic symptoms, 2 with peripheral neuropathy, 1 with AIDS-associated dementia) and 15 controls. Although they had no overt neurologic symptoms, the P300 latency was delayed or undetectable in 30% of patients without clinically evident neurologic disease. P300 latencies did not correlate with peripheral blood CD4 T-cell count, serum quinolinic acid or p24 antigen levels, or the numbers of activated peripheral blood monocytes. Three individuals with abnormal P300 latencies had been HIV-seropositive for < or = 1 year, suggesting that delayed evoked responses detect early neurologic dysfunction. P300 responses do not predict imminent dementia. In only one previously asymptomatic individual with abnormal P300 waveforms have overt neurologic symptoms developed during a 2-year followup. Extended longitudinal studies will be necessary to define the predictive value of P300 latencies in the development of AIDS-related dementia. However, the sensitivity, quantitative nature, and speed of administration of this test suggest that it may be useful for identification of early neurologic involvement in HIV infection.

Acoustic Stimulation↗
PubMed1993

Increased phagocytosis and generation of reactive oxygen products by neutrophils and monocytes of men with stage 1 human immunodeficiency virus infection.

Flow cytometry was used to study phagocytic function and release of reactive oxygen products following phagocytosis by neutrophils (PMNL) and monocytes of heparinized whole blood from stage 1 human immunodeficiency virus type 1 (HIV-1)-infected men. Phagocytic capacity was assessed by measuring uptake of Texas red-labeled bacteria. Reactive oxygen generation after phagocytosis was estimated by the quantity of dichlorofluorescein diacetate converted to dichlorofluorescein intracellularly. Compared with results in samples from age- and sex-matched controls, PMNL and monocytes from HIV-1-infected patients exhibited a significantly increased capacity to phagocytose Staphylococcus aureus and Escherichia coli and generate reactive oxygen products. These results are consistent with the hypothesis that stimuli associated with early HIV-1 infection enhance the nonspecific response of phagocytic cells to potential bacterial pathogens.

Flow Cytometry↗
PubMed1993

Measles virus-induced changes in leukocyte function antigen 1 expression and leukocyte aggregation: possible role in measles virus pathogenesis.

Measles virus (MV) infection of U937 cell or peripheral blood leukocyte cultures was shown to induce changes in the expression of leukocyte function antigen 1 (LFA-1) and cause marked aggregation of these cells. Addition of selected monoclonal antibodies specific for LFA-1 epitopes that did not neutralize MV in standard neutralization assays were found to block both virus-induced leukocyte aggregation and virus dissemination. These data suggest that MV modulation of LFA-1 expression on leukocytes may be an important step in MV pathogenesis.

Antibodies, Monoclonal↗
PubMed1993

Cellular basis for the negative inotropic effects of tumor necrosis factor-alpha in the adult mammalian heart.

To define the mechanism(s) responsible for the negative inotropic effects of tumor necrosis factor-alpha (TNF alpha) in the adult heart, we examined the functional effects of TNF alpha in the intact left ventricle and the isolated adult cardiac myocyte. Studies in both the ventricle and the isolated adult cardiac myocyte showed that TNF alpha exerted a concentration- and time-dependent negative inotropic effect that was fully reversible upon removal of this cytokine. Further, treatment with a neutralizing anti-TNF alpha antibody prevented the negative inotropic effects of TNF alpha in isolated myocytes. A cellular basis for the above findings was provided by studies which showed that treatment with TNF alpha resulted in decreased levels of peak intracellular calcium during the systolic contraction sequence; moreover, these findings did not appear to be secondary to alterations in the electrophysiological properties of the cardiac myocyte. Further studies showed that increased levels of nitric oxide, de novo protein synthesis, and metabolites of the arachidonic acid pathway were unlikely to be responsible for the TNF alpha-induced abnormalities in contractile function. Thus, these studies constitute the initial demonstration that the negative inotropic effects of TNF alpha are the direct result of alterations in intracellular calcium homeostasis in the adult cardiac myocyte.

Animals↗
PubMed1992

Inhibition of transcription of HIV-1 in infected human cells by oligodeoxynucleotides designed to form DNA triple helices.

The effect on human immunodeficiency virus 1 (HIV-1) viral transcription and subsequent gene expression mediated by mixed purine-pyrimidine oligodeoxyribonucleotides (oligodeoxynucleotides) designed to form collinear DNA triplexes with purine-rich elements in the viral promoter was evaluated in intact mammalian cell lines (MT4 and U937). Oligonucleotides HIV31 (5'-GTTTTTGGGTGTTGTGGGTGTGTGTGGTTTG-3') and HIV38 (5'-TGGGTGGGGTGGGGTGGGGGGGTGTGGGGTGTGGGGTG-3') were designed to interact with the transcription initiation site (-16 to + 13) and nuclear factor Sp1 binding site (-81 to -44) of HIV-1, respectively. Oligonucleotides, synthesized with a 3' amine blocking group (5'-R-O-PO2-OCH (CHOH)-CH2-NH+3-3') to prevent degradation by cellular nucleases, were readily taken up by MT4 cells from the culture medium, achieving measured intranuclear concentrations higher than the medium in less than 2 h of incubation. The 3' amine modified oligonucleotides were recoverable from the cells after 24 h as greater than 90% intact material. Treatment of acutely infected MT4 cells with either HIV31 or HIV38 significantly inhibited viral-associated cytopathology and P24 antigen production (p less than 0.001). Additionally, inhibition of P24 antigen release, culture supernatant viral titer, and expression of the intact 9.2-kb HIV-1 mRNA was observed when the chronically infected promonocyte cell line, U937, was treated with 10 microM HIV38. Control oligonucleotides with similar base composition did not inhibit virus expression in either cell line. Furthermore, inhibition of viral expression was not due to alpha-interferon induction resulting from oligonucleotide treatment. Both HIV31 and HIV38 associate with their respective DNA target duplexes at micromolar concentrations, and a strong negative ellipticity near 210 nm, characteristic of DNA triplexes, was observed in the circular dichroism spectrum of either target-oligonucleotide complex. These observations suggest that oligonucleotides, designed to form nucleic acid triplexes with specific proviral target sequences, can selectively inhibit transcription of viral mRNA in intact cells and suppress accumulation of viral products.

Base Sequence↗
PubMed1992

Kinetics of C5a release in cardiac lymph of dogs experiencing coronary artery ischemia-reperfusion injury.

Previous studies of myocardial ischemia suggest that complement activation may play a central role in the inflammatory response during reperfusion. Our previous work has demonstrated neutrophil chemotactic activity to be present in reperfusion canine cardiac lymph after myocardial ischemia and infarction. To evaluate the contribution of the complement-dependent anaphylatoxin C5a to this neutrophil chemotactic activity, rabbit antiserum to canine C5a was prepared. At dilutions > 1:500 but < 1:2,000, the antiserum abolished the ability of zymosan-activated dog serum to induce a ruffled, bipolar morphology in isolated neutrophils used as a bioassay of chemotactic stimulation. This antiserum did not affect similar morphological changes in neutrophils exposed to platelet activating factor (10(-7)-10(-6) M) or recombinant human interleukin-8 (10(-9)-10(-8) M); thus, we deemed it functionally specific for canine C5a. In a pattern similar to what we previously reported, cardiac lymph collected before a 1-hour ligation of the left circumflex coronary artery had little ability to alter the morphology of canine neutrophils (shape change index, 11.3 +/- 4.6, mean +/- SEM; n = 7), but by 1 hour of reperfusion, lymph activated neutrophils significantly in five of seven dogs (mean shape change index, 72.6 +/- 17.7; p < 0.01). At 2 hours of reperfusion, neutrophil activation by lymph occurred in six of seven dogs (mean shape change index, 103.1 +/- 22.2). At 3 hours of reperfusion, cardiac lymph of only three of six dogs caused neutrophil activation, and at 4 hours of reperfusion, this activity was evident in lymph from only two of five dogs.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗