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Biomedical subjects

E Rector

Publications and source records attributed to E Rector.

At least 19 recordsLinked to original sources
PubMed2002

Regulation of B16F1 melanoma cell metastasis by inducible functions of the hepatic microvasculature.

We have previously shown that circulating intravascular cells generally arrest by mechanical restriction in the hepatic sinusoids, causing rapid release of nitric oxide (NO) which is cytotoxic to these cells and inhibits their growth into metastatic tumours. Here, we present evidence that these NO-dependent cytotoxic mechanisms are susceptible to upregulation by lipopolysaccharide (LPS). Five x 10(5) fluorescently labelled melanoma cells were injected into the mesenteric vein of C57BL/6 mice to effect their localisation in the hepatic microvasculature. Test mice were then given 1 mg/kg LPS intraperitoneally (i.p.) to activate the microvascular cells. By electron paramagnetic resonance (EPR) spectroscopy, the expression of NO in the liver was significantly increased by 8 h in the LPS-treated mice. The non-selective NO synthase inhibitor L-NAME inhibited the induction of NO by LPS, while its inactive enantiomer D-NAME had no significant effect. Using immunohistochemistry (IHC), iNOS-positive microvascular cells were detected in the terminal portal venule (TPV) region of the liver 8 h after LPS stimulation. LPS treatment also increased the retention of melanoma cells in the liver between 8 and 24 h, especially in the TPV region. Eight hours after cell injection, local expression of VCAM-1 and ICAM-1 was detected by double-label immunohistochemistry at the sites of tumour cell arrest. Expression of these adhesion molecules was enhanced in mice treated with LPS. Using flow cytometry, 98% of the B16F1 melanoma cells expressed VLA-4, the counter receptor of VCAM-1, and approximately 1.5% expressed LFA-1, the counter receptor of ICAM-1. LPS did not significantly alter the expression of either counter receptor on melanoma cells in vitro or in vivo. By DNA end-labelling, the rates of melanoma cell apoptosis were significantly increased from 8 to 24 h in the TPV region (but not in the sinusoids) of LPS-treated mice. Fourteen days after tumour cell injection, the LPS-treated mice had a significantly smaller hepatic metastatic tumour burden than the control mice. These data suggest that LPS can inhibit the metastasis of melanoma cells in the liver by inducing the expression of NO and adhesion molecules by the hepatic endothelium. The induction of iNOS and the inducible cytotoxic effect of LPS appear to be primarily located within the TPV region of the liver acinus.

Animals↗
PubMed2000

Functional status of the immune system after chronic administration of 2'-deoxycoformycin in the BB rat.

Insulin-dependent diabetes mellitus (IDDM) is caused by autoimmune destruction of pancreatic beta cells with the primary mechanism being cell mediated. The BB rat develops insulitis and IDDM with many features analogous to the disease in man. In previous studies we reported that weekly administration of 2'-deoxycoformycin (dCF) for four months reduces significantly the incidence of IDDM in the BB rat by 70%, and that the animals remain free of diabetes for a minimum of two months after drug withdrawal. Since the diabetes-prone BB rat is lymphopenic, with a reduction of both CD4 and CD8 cells, the continuous failure of dCF treated animals to develop diabetes may have been due to generalized immunosuppression. To test this possibility, the ability of dCF treated diabetes-free BB rats to mount an immune response after challenge with Ovalbumin was examined five months after drug withdrawal. The results showed that the post-immunization levels of total IgG and specific IgG in these animals did not differ from those observed in non-dCF treated controls nor those of control diabetes-resistant non-lymphopenic BB rats. Moreover, FACS analysis indicated no change in the percentages of total numbers of CD4+ or CD8+ cells between the two groups of animals. Histological assessment of the pancreata of the post-dCF treated animals showed varying degrees of mononuclear cell infiltrates in the islets. These data demonstrate that treatment by dCF is not permanent, and may require intermittent or continuous administration to prevent development of diabetes. Further studies are needed to determine the mechanism of action of dCF in this model of IDDM.

Animals↗
PubMed1998

Airway smooth muscle cell proliferation: characterization of subpopulations by sensitivity to heparin inhibition.

Growth and maturation state of airway smooth muscle cells (SMCs) are determinants of asthma pathophysiology. Heparin reduces airway SMC proliferation and arterial SMC replication and phenotypic modulation. Distinct arterial SMC subtypes, differing in heparin sensitivity, have been characterized. We assessed the cellular mechanisms underlying the growth and phenotype of heparin-treated canine tracheal myocytes in primary culture. Heparin reduced replication by 40%. Immunoblot assay of myosin, actin, and myosin light chain kinase revealed heparin had no effect on rapid spontaneous phenotypic modulation after the cells were plated. Heparin increased cellular protein and vimentin contents in confluent cultures, suggesting that it may induce hypertrophic growth. Cell cycle analysis revealed that heparin decreased serum-stimulated replicating myocyte number by 40%. Also, G2-M transit was 20% slower for the set of SMCs that proceeded past G1 in the presence of heparin. These data indicate that heparin does not inhibit airway SMC replication by blocking modulation from the contractile state. Moreover, airway smooth muscle is composed of distinct SMC populations differing in mitogen and antiproliferative mediator responsiveness. Identification of functionally divergent subgroups suggests that distinct sets of SMCs may contribute differentially to airway physiology and pathophysiology.

Animals↗
PubMed1997

Peripheral blood lymphocyte beta 2 integrin and ICAM expression in inflammatory bowel disease.

The beta 2 integrin intercellular adhesion molecule (ICAM) adhesion pathway is likely pivotal in the immunopathogenesis of inflammatory bowel disease (IBD). We have undertaken a comprehensive study of peripheral blood lymphocyte (PBL) expression of all beta 2 integrins and ICAMs in patients with IBD using flow cytometry and assessed our data on the basis of IBD diagnosis, disease state of activity, and use of corticosteroids. Blood was collected from patients with Crohn's disease (N = 49), ulcerative colitis (N = 43), and normal control volunteers (N = 15). Mononuclear cells were separated using a Ficoll-Hypaque gradient and prepared for flow cytometry. The data were analyzed for percentage expression, mean fluorescent intensity (MFI) as well as for histogram patterns. The analysis was stratified for disease diagnosis, disease activity level, and for use of prednisone among patients with active disease. There was decreased percentage expression of CD11a, CD18, and ICAM-3 in Crohn's disease and ulcerative colitis compared with normal, but an increased MFI for these molecules among patients with Crohn's disease. Active Crohn's disease showed a greater change in this pattern compared with both inactive disease and active ulcerative colitis. CD11a and CD18 histograms typically had two peaks of expression. The predominance of one peak over the other varied with disease diagnosis and activity. CD11b and alpha d expression patterns were not different in IBD compared with normal. CD11c was not expressed by PBLs and, ICAM-2, typically an endothelial ligand, was expressed on PBLs. There were changes in the expression of beta 2 integrins in IBD, which were more evident in Crohn's disease than ulcerative colitis. We hypothesize that the decreased percentage expression and increased MFI of CD11a, CD18, and ICAM-3 may suggest that cells up-regulate these ligands following activation and are egressing into tissue.

Adult↗
PubMed1997

Characterization of molecular determinants of smooth muscle cell heterogeneity.

Broad diversity in contractile and pharmacological properties of different smooth muscles is well recognized. Differences in proliferative capacity, electrophysiology, phenotypic marker protein content, matrix synthesis, and expression of cell-specific transcription factors between individual smooth muscle cells (SMCs) have also been reported. Precise developmental and molecular mechanisms underlying heterogeneity are not known; however, their elucidation is the thrust of much current research involving vascular smooth muscle. In contrast, limited studies of heterogeneity of subtypes of airway SMCs are available. In this report, we review molecular aspects of differentiation that may determine phenotypic heterogeneity of SMCs and also present data from our own studies characterizing heterogeneity in the proliferative capacity and marker protein content of airway SMCs. Using flow cytometry, cell cycle transit was monitored for cultured canine tracheal SMCs. Only 70% of arrested cells responded and traversed the cell cycle when stimulated with 10% fetal bovine serum. Furthermore, heparin inhibited 40% of serum-responsive cells from entering the cell cycle, suggesting that both serum- and heparin-sensitive and -insensitive airway SMCs exist. Flow cytometric analysis of contractile protein and DNA content in freshly dissociated canine tracheal SMCs revealed that diploid (approximately 87%) and tetraploid (approximately 13%) populations exist. Clusters of SMCs having "high" or "low" smooth muscle myosin or alpha-actin content were also discerned, indicating that distinct subtypes of SMCs exist in mature airways. Diversity of SMCs may be a critical factor determining specific responses of smooth muscles to a number of physiological or pathophysiological stimuli that may include, for example, inflammatory mediators in asthmatic airways.

Animals↗
PubMed1997

[Evaluation of the antinociceptive effect of xylazine after epidural administration in dogs under general anesthesia with isoflurane].

Seven dogs anaesthetized with isoflurane in oxygen (1.9 Vol.-% ET) were examined before and after lumbosacral injection of xylazine (0.25 mg/kg) into the epidural space. Over a 240 minute period the dogs were first stimulated in a visceral manner (extension of the colon descendens by a balloon-catheter) every 15 minutes. In a second part, the dogs were stimulated somatically by pressure to the interdigital skin of a hindlimb. Heart rate and mean arterial pressure were used to determine the analgesic properties of epidurally injected xylazine. Furthermore, plasma xylazine concentration was controlled in both series. Before epidural anaesthesia, heart rate and mean arterial pressure increased during stimulation significantly in both groups. After epidural injection of xylazine, hemodynamic reactions were distinctly diminished, in the somatically stimulated group more than in the viscerally stimulated group. Up to 30 minutes after epidural injection, plasma xylazine concentrations of about 100 ng/ml were found in both groups. Up to 60 minutes after epidural injection, plasma xylazine concentrations of more than 60 ng/ml still were found. From the 120th minute on, xylazine concentration decreased below 30 ng/ml. From these results, it can be concluded that there is a well-suited analgesic effect of epidurally applicated xylazine for approximately 240 minutes.

Analgesia↗
PubMed1996

[Pharmacokinetics and effects of xylazine (Rompun) in dogs].

Six beagle dogs were treated with xylazine hydrochloride (1 mg/kg i.m.). The plasma xylazine concentration was measured by HPLC. Additionally, clinical effects were registered (cardiac rate, respiratory activity, electrocardiogram, body temperature, motoric activity, attention, analgesia). Maximum plasma concentrations were measured after 15 minutes (476 ng/ml). The plasma half-life was 24 minutes. Sedation was registered over one hour (xylazine concentration of more than 150 ng/ml). Within the first 30 minutes after treatment (xylazine concentration of more than 300 ng/ml), a low-grade analgesia was observed. In contrast, cardiac and respiratoric depression and also significantly diminished body temperature were registered over 2 to 3 hours.

Analgesia↗
PubMed1993

Major histocompatibility complex class II-restricted cytotoxicity by self-myelin basic protein-reactive T-cell hybridomas: evidence for a tumour necrosis factor-independent nucleolytic mechanism.

Direct cytotoxicity by class II-restricted T cells has been proposed as a potential mechanism in autoimmune tissue damage, as well as in immunoregulation. We used I-A(s)-restricted non-granular cytotoxic T-cell hybridomas (BP24.29 and BP47.7), specific for self-determinants on myelin basic protein (MBP), and different monoclonal targets, in order to characterize the mechanism of killing used by these cells. An early lesion at the level of the target cell nucleus was indicated by the fact that target DNA lysis ([3H]thymidine release) proceeded 2-2.5-fold as rapidly as cytoplasmic lysis (51Cr release) over the first 14 hr after stimulation. Cytotoxicity was relatively resistant to inhibition by anti-calcium agents (TMB-8 and verapamil), even under conditions which blocked interleukin-2 (IL-2) release. Although tumour necrosis factor (TNF) has been proposed as one mediator of class II-restricted cytotoxicity, these cells (i) released no detectable TNF after stimulation with antigen, concanavalin A (Con A), or anti-CD3, (ii) readily lysed TNF-resistant targets (A20 and LS-102.9), and (iii) had no cytotoxic effect on TNF-sensitive cells (L929). Substantial 'bystander' killing of I-A-mismatched targets was observed, which was 13-37% of the cognate (I-A(s)-restricted) cytotoxicity measured in parallel. This finding may indicate an effector mechanism in autoimmune demyelination, since the myelin-forming oligodendrocytes of the central nervous system are not inducible for major histocompatibility complex (MHC) class II expression.

Animals↗
PubMed1992

Tumour necrosis factor alpha restores granulomas and induces parasite egg-laying in schistosome-infected SCID mice.

Schistosomiasis (bilharzia) is a parasitic disease caused by several species of schistosome worms (blood flukes). The key pathogenic event in this disease is the formation of granulomas around schistosome eggs trapped in portal venules of the liver. Granulomas are a distinctive form of chronic inflammation characterized by localized aggregation of activated macrophages around an inciting stimulus. Each granuloma evolves to form a fibrous scar; in schistosomiasis, the result is widespread hepatic fibrosis and portal hypertension. To identify the specific immune signal molecules necessary for granuloma formation, we studied schistosome infections in severe combined immunodeficient (SCID) mice, which have normal macrophages but lack functional B or T lymphocytes. Here we report that the immunoregulatory cytokine tumour necrosis factor alpha is necessary and sufficient to reconstitute granuloma formation in schistosome-infected SCID mice. Moreover, we find that the parasitic worms require tumour necrosis factor alpha for egg-laying and for excretion of eggs from the host. The implication of this latter result is that the parasite has adapted so successfully to its host that it uses a host-derived immunoregulatory protein as a signal for replication and transmission.

Animals↗
PubMed1990

Mapping of epitopes on Poa p I and Lol p I allergens with monoclonal antibodies.

Allergen Poa p I isolated from the dialysed aqueous extract of Kentucky blue grass pollen by affinity chromatography with an anti-Lol p I murine monoclonal antibody (MAb) 290A-167 was previously shown to consist of a 35.8-kilodalton (kD) component with a pI of 6.4, designated as Poa p Ia, and a 33-kD component with a pI of 9.1, designated as Poa p Ib. The present study reports on the comparative antigenic analyses of these two components, using MAbs produced separately against Poa p I and Lol p I. Thus, anti-Poa p I MAbs 60 and 61 and anti-Lol p I MAb 290A-167 recognized Poa p Ia and Poa p Ib whereas anti-Poa p I MAbs 62, 63 and 64 and anti-Lol p I MAb 348A-6 recognized only Poa p Ia. The specificities of the MAbs were further resolved by comparing their respective abilities to inhibit the binding of 125I-Poa p I or 125I-Lol p I to the different MAbs prepared in the form of solid phase. These studies revealed that at least 4 distinct epitopes (designated as E1, E2, E3 and E4) were shared by both Poa p I and Lol p I. All 4 epitopes were present on Poa p Ia whereas only E1 and E3 were detected on Poa p Ib. E1 was recognized by MAbs 60 and 61, E2 by MAbs 62, 63 and 64, E3 by MAb 290A-167 and E4 by MAb 348A-6.(ABSTRACT TRUNCATED AT 250 WORDS)

Allergens↗
PubMed1990

[The ototoxicity of cisplatin: a clinical study].

The ototoxic side effects of cisplatin are evaluated in 23 patients by a conventional and high-frequency audiogram. The ototoxicity of cisplatin consists of a high frequency loss which was found to be dose and frequency related. The hearing loss started predominantly in the higher frequencies (12-16 kHz) and the ototoxicity increased with increasing dose of cisplatin. The ototoxicity of cisplatin appears to be inversely related with the patient's age, although it is difficult to draw definite conclusions because age and dose are closely related to each other in our study.

Adult↗
PubMed1986

Isolation and characterization of an alpha-satellite repeated sequence from human chromosome 22.

We constructed a library in lambda L47.1 with DNA isolated from flow-sorted human chromosome 22. Over 50% of the recombinants contained the same highly repetitive sequence. When this sequence was used to probe Southern blots of EcoRI-digested genomic DNA, a ladder of bands with increments of about 170 bp was observed. This sequence comigrates with satellite III in Ag+/Cs2SO4 gradients and may account for at least part of the 170 bp Hae III ladder seen in isolated satellite III DNA. Partial sequence analysis revealed homology to the 171 bp monomeric repeat unit of alpha-R1-DNA and the X specific alpha-satellite consensus sequence. After low stringency in situ hybridization, silver grains were found over the centromeres of a number of chromosomes. Under high stringency conditions, however, the labeling was concentrated over the centromeric region of chromosome 22. This localization was confirmed using DNA from a panel of human/hamster cell lines which showed that the homologous 2.1 and 2.8 kb EcoR1 restriction fragments were chromosome 22 specific. These clones therefore contain chromosome 22 derived alpha-satellite sequences analogous to other chromosome-specific satellite sequences described previously.

Animals↗
PubMed1985

Detection and characterization of monoclonal antibodies specific to IgE receptors on human lymphocytes by flow cytometry.

BALB/c mice were immunized with human lymphoblastoid cells (RPMI 8866 cells) expressing surface receptors for IgE (Fc epsilon R). Spleen cells from animals displaying high titres of anti-Fc epsilon R antibodies were fused with HGPRT-deficient NSI myeloma cells. Anti-Fc epsilon R antibodies were identified by a flow cytometric assay based on their ability to block the binding of IgE-coated fluorescent latex particles to Fc epsilon R-positive cells. Fourteen monoclonal hybridoma cell lines secreting antibody of the required specificity were amplified in tissue culture and then grown in the peritoneal cavity of BALB/c mice in order to obtain ascitic fluids with high antibody titres. The specificity of each monoclonal antibody (Mab) to lymphocyte Fc epsilon R was shown by the following observations: (i) the intact monoclonal antibody molecule or, in some cases, its F(ab')2 fragments blocked the binding of IgE to several Fc epsilon R(+) cell lines different from that employed for the initial immunization; (ii) the Mab bound directly to all the Fc epsilon R(+) cell lines tested, but not to several Fc epsilon R(-) cells as determined by indirect immunofluorescence; (iii) the binding of Mab to Fc epsilon R(+) cells was selectively blocked by IgE, but not by the other classes of Ig; and (iv) Mab had no effect on the binding of IgG to Fc gamma R on normal human peripheral blood mononuclear cells (PBMC).

Animals↗
PubMed1984

In vitro synthesis of IgE by human lymphocytes. III. IgE-potentiating activity of culture supernatants from Epstein-Barr virus (EBV) transformed B cells.

Seven Epstein--Barr virus (EBV)-transformed B cell lines were derived from circulating lymphocytes of two atopic and two non-atopic individuals, two preparations of cord blood lymphocytes and one tonsillar lymphocyte preparation. All the cell lines contained a significant proportion of cells expressing Fc epsilon R as detected by rosette formation with IgE-coated bovine erythrocytes (E-IgE) and by flow cytometry using IgE-linked to fluorescent microspheres. None of the cell lines displayed FcR for IgA, IgM or IgG. The cell-free supernatants (CFS) of EBV-transformed cells contained IgE-binding factors (IgE-BFs) detected by their ability to inhibit the binding to RPMI 8866 cells of either E-IgE or IgE-linked to microspheres. Whereas these CFS enhanced the synthesis of IgE and suppressed the synthesis of IgG by purified B lymphocytes isolated from the blood of allergic donors and cultured in the absence of stimulant, their effect on the synthesis of IgA or IgM was not predictable. CFS significantly enhanced the secretion of IgE by the U266 myeloma cell line without interfering with secretion of IgM, IgG or IgA by EBV-transformed cells. These data are in accord with similar properties of RPMI 8866 cells and suggest that B lymphocytes might play a regulating role in the IgE synthesis.

B-Lymphocytes↗
PubMed1984

In vitro synthesis of IgE by human lymphocytes. II. Enhancement of the spontaneous IgE synthesis by IgE-binding factors secreted by RPMI 8866 lymphoblastoid B cells.

RPMI 8866 lymphoblastoid cells, known to express surface Fc epsilon R, were tested for their ability to regulate the in vitro synthesis of human IgE. Cell-free supernatants (CFS) of RPMI 8866 cells enhanced in a dose-dependent fashion the spontaneous IgE synthesis by B cells of allergic individuals. For maximum activity the CFS had to be added during the first 3 days of culture. CFS did not significantly alter the spontaneous synthesis of IgM or IgG, but they suppressed IgA synthesis both in B cell cultures and in pokeweed mitogen-stimulated peripheral blood mononuclear cells cultures. Cyclosporin A did not suppress either the spontaneous Ig production by B cells nor the IgE-potentiating activity of CFS. The enhancing activity of CFS was related to its content in IgE binding factors (IgE-BFs); these factors were detected by their ability to inhibit the rosetting of RPMI 8866 cells with IgE-coated erythrocytes (E-IgE). Both the IgE-BFs and the IgE-potentiating activity of the supernatants of RPMI 8866 cell cultures could be removed by absorption with IgE-Sepharose, from which they could subsequently be eluted with glycine-HCl buffer. IgE-BFs were identified as glycoproteins on the basis of their sensitivity to trypsin and to neuraminidase. By filtration of the RPMI 8866 cell supernatants through a Sephadex G75 column, IgE-binding activity was found to be associated with two fractions with molecular sizes in the range of 10,000-15,000 and 30,000-40,000. The IgA-suppressing activity of the RPMI 8866 culture filtrates could be absorbed with sIgA-Sepharose from which it was subsequently recovered by elution with glycine-HCl buffer. Most unexpectedly, sIgA-Sepharose also removed IgE-BFs and IgE-potentiating activity from the RPMI 8866 supernatants; both could be recovered by subsequent elution from sIgA-Sepharose with gycline-HCl buffer. These data are provisionally interpreted as indicating that the IgE-BFs secreted by RPMI 8866 cells had affinity for both IgE and sIgA and that they exerted a reciprocal effect on the in vitro synthesis of IgE and IgA.

B-Lymphocytes↗
PubMed1984

In vitro synthesis of IgE by human lymphocytes. IV. Suppression of the spontaneous IgE synthesis by IgE-binding factors secreted by tunicamycin-treated RPMI 8866 cells.

It was previously shown that RPMI 8866 cells released IgE-binding factors (IgE-BFs) capable of enhancing the spontaneous in vitro synthesis of IgE by purified B lymphocytes isolated from allergic individuals. In the present study, the influence of tunicamycin, an inhibitor of protein glycosylation, on RPMI 8866 cells was investigated with regard to: (i) the expression of surface receptors for IgE; (ii) the release of IgE-BFs into the culture supernatants, and (iii) the biological activity of IgE-BFs. After preincubation for 60 min with tunicamycin (1 microgram/ml), RPMI 8866 cells were cultured for 48 hr in HB 101 serum-free medium; the culture supernatant was then filtered, concentrated, and its biological activity was compared to that of a parallel culture supernatant from untreated RPMI 8866 cells. The results of these experiments indicate that exposure of RPMI 8866 cells to tunicamycin resulted in: (i) a reduction of surface Fc epsilon R; (ii) no effect on the release of IgE-BFs into the culture supernatant, and (iii) the conversion of IgE-potentiating factors into IgE-suppressing factors. The latter factors suppressed the IgE secretion by U266 myeloma cells and completely inhibited the activity of IgE-potentiating factors on B lymphocytes from allergic individuals. IgE-BFs secreted by tunicamycin-treated cells had no effect on the production of IgG, IgA or IgM by normal or EBV-transformed B cells.

Antibodies, Neoplasm↗
PubMed1984

In vitro synthesis of IgE by human lymphocytes. I. The spontaneous secretion of IgE by B lymphocytes from allergic individuals: a model to investigate the regulation of human IgE synthesis.

In view of the controversial data in the literature regarding the in vitro IgE synthesis by human lymphocytes, the conditions for culture of lymphocytes and the methodology for measurement of the IgE produced are described in detail. In the absence of any added mitogen, enriched B cell preparations derived from 70% of allergic donors actively secreted 100 to 3200 pg/ml of IgE after culture for 7 days, at which time the cell viability was higher than 85%. In comparable B cell cultures derived from non-allergic donors, only trace amounts of de novo synthesized IgE were detected in 20% of the cases. All B cell cultures actively secreted IgG, IgA, IgM and there was no apparent relationship between the secretion of IgE and that of the other classes of Ig. By contrast, the synthesis of IgE by unfractionated peripheral blood mononuclear cells of allergic individuals, which were stimulated with pokeweed mitogen (PWM) under several experimental conditions, was not consistently reproducible, i.e. the spontaneous synthesis of IgE in such cultures was either suppressed or enhanced by PWM. The most important finding was that the secretion of IgE was selectively enhanced by supplementing the B cell cultures with cell-free supernatants (CFS) of cultures of neonatal lymphocytes which had been preincubated with 10 micrograms/ml IgE. It is, therefore, concluded that B cell cultures from allergic individuals constitute an appropriate model for investigations of the mechanisms underlying the regulation of human IgE synthesis.

Adult↗