Biomedical subjects

J Trial

Publications and source records attributed to J Trial.

At least 19 recordsLinked to original sources

PubMed2004

Erythropoietin withdrawal leads to the destruction of young red cells at the endothelial-macrophage interface.

Erythropoietin is a growth factor for endothelial cells as well as for erythroid cells. In contrast to their proliferative response to physiological levels of erythropoietin, endothelial cells may respond to decreased levels by triggering a process called neocytolysis. Neocytolysis is the selective destruction of the youngest circulating red cells, which may be prompted by endothelial cells communicating with macrophages to stimulate phagocytosis of this unusual cell subset. We speculate that this is due to decreased production by endothelial cells of the macrophage-deactivating transforming growth factor-beta. The resulting proinflammatory phenotype may include macrophage production of thrombospondin, which forms bridges between adhesion molecules selectively expressed on young red cells (CD36) and the CD36/alphavbeta3 complex on macrophages that triggers phagocytosis. Alternatively, inflammatory mediators secreted by endothelial cells and macrophages during erythropoietin withdrawal may signal young red cells to expose phosphatidylserine, which would mark them for elimination via the normal pathway for aged red cell destruction. Neocytolysis has been demonstrated in returning astronauts and in polycythemic individuals at high altitude on descent to sea level. It contributes to the anemia of renal disease, is triggered by the rapidly falling levels of erythropoietin seen after intravenous administration, and may be the normal mechanism for reduction of red cell mass in newborns. It may play a role in chronic diseases including malaria and sickle cell anemia. New erythropoietin products and methods of administration avoid the intermittent rapid decreases associated with the stimulus for neocytolysis, but study of this phenomenon may yield further improvements in drug design.

Animals↗

PubMed2001

Erythropoietin withdrawal alters interactions between young red blood cells, splenic endothelial cells, and macrophages: an in vitro model of neocytolysis.

BACKGROUND: We have described the rapid destruction of young red blood cells (neocytolysis) in astronauts adapting to microgravity, in polycythemic high altitude dwellers who descend to sea level, and in patients with kidney disorders. This destruction results from a decrease in erythropoietin (EPO) production. We hypothesized that such EPO withdrawal could trigger physiological changes in cells other than red cell precursors and possibly lead to the uptake and destruction of young red cells by altering endothelial cell-macrophage interactions, most likely occurring in the spleen. METHODS: We identified EPO receptors on human splenic endothelial cells (HSEC) and investigated the responses of these cells to EPO withdrawal. RESULTS: A monolayer of HSEC, unlike human endothelial cells from aorta, glomerulus, or umbilical vein, demonstrated an increase in permeability upon EPO withdrawal that was accompanied by unique morphological changes. When HSEC were cultured with monocyte-derived macrophages (but not when either cell type was cultured alone), EPO withdrawal induced an increased ingestion of young red cells by macrophages when compared with the constant presence or absence of EPO. CONCLUSIONS: HSEC may represent a unique cell type that is able to respond to EPO withdrawal by increasing permeability and interacting with phagocytic macrophages, which leads to neocytolysis.

Adaptation, Physiological↗

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

TNF-alpha released by comigrating monocytes promotes transendothelial migration of activated lymphocytes.

We investigated mechanisms that increase motility and transendothelial trafficking of activated lymphocytes. Freshly isolated lymphocytes stimulated with immobilized anti-CD3 for 2 h migrate into polymerized collagen in 1.99+/-0.25-fold greater numbers and across confluent endothelial monolayers in 4.8+/-0.5-fold greater numbers compared with leukocytes incubated with non-specific IgG. Activated lymphocytes form clusters with monocytes, and their increased motility was dependent on the presence of comigrating monocytes. Five lines of evidence support the idea that monocytes modulate lymphocyte motility through the release of TNF-alpha: 1) flow-cytometric analyses, using highly specific and avid mAbs to probe permeabilized whole blood leukocytes, showed that >80% of circulating monocytes contain intracellular TNF-alpha, whereas <5% contain IL-1 and none contain IL-6; 2) stimulation with immobilized anti-CD3 that was intended to activate lymphocytes also induced monocytes to release increased quantities of TNF-alpha; 3) rTNF-alpha, added in doses of 1 to 20 pg/ml to purified anti-CD3-stimulated lymphocytes, reproduced, in a dose-dependent manner, the motility-enhancing effect of adding monocytes; 4) the transient increase in the expression of TNF R-I on CD3-activated T lymphocytes parallels their transiently increased motility; and 5) addition of anti-TNF-alpha, anti-TNF R-I, anti-TNF R-II, or soluble TNF R-I decreased the motility of stimulated lymphocytes. These results suggest that T lymphocyte stimulation via the CD3-TCR complex signals nearby monocytes to release TNF-alpha, which feeds back on the lymphocytes to increase their locomotor activity.

Antigens, CD↗

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↗

PubMed1996

Repetitive endocytosis and recycling of the beta 2-adrenergic receptor during agonist-induced steady state redistribution.

The human beta 2-adrenergic receptor (beta 2AR) rapidly internalizes after binding agonist, resulting in a dramatic redistribution of receptors from the plasma membrane and into endocytic vesicles. We sought to determine whether intracellular receptors constitute a static pool or represent a fraction of dynamically internalizing and recycling receptors. Using cells expressing a beta 2AR with an epitope tag at its amino-terminal ectodomain, changes in surface receptor levels were measured by flow cytometry and radioligand binding assays. The addition of a saturating level of a strong agonist (isoproterenol) caused the endocytosis of receptors with first-order kinetics (ke for naive cells, 0.222 min-1). After 10 min, the level of surface receptors remained stable at approximately 20% that of untreated cells, even though endocytosis continued with similar kinetics (ke for pretreated cells, 0.258 min-1), suggesting that internalized receptors were cycling in steady state with surface receptors. This prediction was confirmed directly by showing that internalized beta 2ARs recycled to the cell surface in the continued presence of agonist. The calculated transit times (1/k) in the presence of isoproterenol were 3.9 min for endocytosis and 11.2 min for recycling. The endocytic rate constant and the steady state redistribution to the internal pool were much lower after treatment with the partial agonist albuterol, suggesting a correlation between the efficiency of endocytosis and that of receptor coupling to the downstream signal transduction pathway. These findings indicate that in the presence of agonist, beta 2ARs are in a dynamic steady state between the plasma membrane and endosomes that is regulated principally by agonist efficacy.

Adrenergic beta-Agonists↗

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

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↗

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↗

PubMed1990

Suppressor cell clones specific for ultraviolet radiation-induced tumors. Function and surface proteins.

Lymphocytes were cloned from animals bearing UV-induced skin tumors. These cells were I-J+, CD4-, CD8-, and had become growth factor independent. Extracts, but not supernatants, of these clones suppressed primary immune reactions in vitro against UV-induced tumors, but not methylcholanthrene-induced tumors. The cells therefore had the functional characteristics of afferent suppressor T cells directed against a common Ag on UV-induced tumors. Surface iodination of the clones revealed an extremely low level expression of molecules that might be TCR or related molecules.

Animals↗

PubMed1990

Two ultraviolet tumor-specific suppressor cell clones. One expresses Ig RNA and the other expresses T cell receptor-alpha and -beta RNA.

Two splenic cell clones were derived from mice bearing UV-induced skin tumors; both clones are I-J+, Thy-1+, and J11D+. Cellular extracts, but not culture supernatants, from the two clones, designated T4 and T5, specifically suppress primary cytotoxic and proliferative responses to UV-induced tumor targets. Therefore, clones T4 and T5 would appear to function as putative I-J+ suppressor cells with specificity for an antigenic epitope common to several UV-induced tumors. To derive more information concerning the status of Ag-receptor genes in the suppressive clones, we analyzed the rearrangement and expression of Ig and TCR genes. The first clone, T4, expressed Thy-1, a T cell-surface marker, yet it had not rearranged its TCR-beta, -gamma, and -delta gene families, nor were TCR-alpha, -beta, -gamma, or -delta transcripts detectable. Instead, clone T4 contained rearranged Ig H chain genes and expressed mu RNA. Because neither lambda nor kappa L chain transcripts were detected, this clone could not utilize a complete Ig molecule as its Ag receptor. On the other hand, clone T5 contained exclusively TCR gene rearrangements; it expressed alpha and beta transcripts of the correct sizes. However, the level of RNA that could encode the delta-chain of the CD3 accessory molecule was below detection, so it is unlikely that this TCR was expressed on the cell surface. The rearrangement of Ig genes in one clone and TCR genes in the other clone does not necessarily indicate that these genes encode the Ag receptors used by these clones. However, it suggests either that suppressor-like cells can derive from different lymphocyte precursor lineages or that rearrangements of the known TCR genes or the Ig genes are irrelevant to the function of this cell lineage.

Animals↗

PubMed1988

Cooperation between early acting delayed-type hypersensitivity T-cells and cultured effector cells in tumor rejection.

Delayed-type hypersensitivity (DTH) to allogeneic tumor cells exhibits two phases, an early reaction peaking at 2 h, and a late phase at 24 h after challenge with viable tumor cells. The purpose of this study was to determine whether DTH to syngeneic tumors also displays two phases and whether either reactivity is involved in tumor rejection in vivo. When normal animals were immunized and challenged with syngeneic regressor tumors induced by UV radiation, a tumor-specific DTH exhibiting both early and late phases was seen. The cells mediating both reactivities were Thy-1+ and L3T4+, but the early DTH cells manifested their reactivity sooner after immunization than the late DTH cells. Adult-thymectomized, X-irradiated mice did not show any greater ability to reject tumors after immune-system reconstitution with either immune population than after reconstitution with normal cells. However, small numbers of the early acting DTH cells acted synergistically with small numbers of cultured T-cells, the latter capable of in vitro cytolysis, to prevent tumor growth. This indicates that the early phase of DTH may be a valuable tool for the potentiation of immunotherapy using cultured cells.

Animals↗

PubMed1988

In vitro cytotoxic activity of interleukin 2-dependent murine Thy-1+ dendritic epidermal cell lines.

Thy-1+ dendritic epidermal cells (Tdec) are bone marrow-derived cells of T-cell lineage. In order to investigate the function of these cells, two interleukin 2-dependent Tdec lines (AU4 and AU16) were established from ear skin of C3H/HeN (MTV-) mice and examined for cytotoxicity against various murine tumor cells. The original phenotype of the Tdec, Thy-1+, AsGM1+, T3+, Lyt-1-, Lyt-2-, Lyt-5+, and L3T4-, was maintained in the cell lines. The Tdec cell lines (AU4 and AU16) lysed a wide spectrum of tumor cell lines in a 4-hr 51Cr-release assay. AU16 was more effective in killing a syngeneic UV-induced skin tumor (UV-2240) and an allogeneic, natural killer cell-resistant tumor (EL-4) than the YAC-1 cell line, which is highly sensitive to natural killer cells. Neither syngeneic nor allogeneic lymphoblasts were killed by AU16. Cytotoxicity was blocked by fixation of AU16 with paraformaldehyde but not by pretreatment of the cells with puromycin or mitomycin C. The finding that two Thy-1+ cell lines derived independently from murine epidermis have unique cytotoxic activity against murine tumor cells suggests that Tdec could play a role in local immune surveillance against skin cancers.

Animals↗