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J B Hay

Publications and source records attributed to J B Hay.

At least 55 records · Page 3Linked to original sources
PubMed1991

Long-term tracking of lymphocytes in vivo: the migration of PKH-labeled lymphocytes.

Studies of in vivo cell migration using cell markers such as 51Cr, 111In, FITC, or XRITC have been limited to short time periods due to the elution, toxicity, or rapid loss of label detectability. We have labeled sheep lymphocytes in vitro with PKH-2, a new fluorescent cell membrane label, and, after their intravenous injection back into donor sheep, have been able to detect them in efferent lymph, using flow cytometry, for longer than 38 days. The PKH-2-labeled lymphocytes migrated with similar kinetics, efficiency, and tissue specificity as lymphocytes labeled with cell markers used previously. PKH-2-labeled cells mediated graft versus host reactions indistinguishable from those mediated by unlabeled cells, and cell surface antigens were equally detectable on the surface of labeled and unlabeled lymphocytes. According to the slow, consistent loss of fluorescence intensity of the labeled cells in vivo, we predict that labeled lymphocytes could remain detectable by flow cytometry for greater than 7 weeks with the labeling protocol used in these experiments.

Animals↗
PubMed1991

Lymphatic removal of dialysate from the peritoneal cavity of anesthetized sheep.

Several investigators have suggested that the lymphatic circulation reduces ultrafiltration in continuous ambulatory peritoneal dialysis (CAPD). The purpose of this study was to assess lymphatic drainage of the peritoneal cavity directly in anesthetized sheep under dialysis conditions. Lymph was collected from the caudal mediastinal lymph node and the thoracic duct, both of which are involved in the lymphatic drainage of the ovine peritoneal cavity, and from the prescapular lymph node, which is not involved in peritoneal lymphatic drainage. Fifty ml/kg volumes of a mildly hypertonic dialysis solution (Dianeal 1.5%) containing 25 microCi 125I-human serum albumin were instilled into the peritoneal cavity, and lymph flows and the appearance of labeled protein in the lymphatic and vascular compartments were monitored for six hours. Following the instillation of dialysis fluid there was a tendency for lymph flow rates from the thoracic duct to increase but these changes were not significant. However, flow rates from the caudal lymphatic demonstrated significant increases, especially in the final three hours of the monitoring period. Only about 8% of the radiolabeled albumin was removed from the peritoneal cavity over six hours (that is, 92% was left in the peritoneal space). Of the albumin removed, approximately 17% of this was drained by abdominal visceral lymphatics into the thoracic duct. About 25% passed through the diaphragm into the caudal mediastinal lymph node and into efferent lymph. Since the efferent lymphatic duct of the caudal mediastinal node empties directly into the thoracic duct, about 42% of all protein removed from the peritoneal cavity of the sheep was ultimately transported to the thoracic duct.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Lymphatic drainage of the peritoneal cavity in sheep.

Lymphatic drainage of the peritoneal cavity has been investigated in anesthetized sheep. Studies involving intraperitoneal administration of a complex of Evans blue dye and bovine serum albumin demonstrated the existence of three anatomically distinct pathways. In the first pathway, dye is removed from the peritoneal cavity by diaphragmatic lymphatics that pass into caudal sternal lymph nodes. Efferent lymphatics from these nodes transport the material to cranial sternal lymph nodes. Efferent cranial sternal lymphatics then convey the material either directly or indirectly, via tracheal lymphatic trunks, to the right lymph duct. In the second pathway, the complex is transported from the peritoneal cavity by diaphragmatic lymphatics that pass into the caudal mediastinal lymph node. Efferent lymphatic ducts from this node transport the material to the thoracic duct. The third pathway appears to involve transport of the dye across the mesothelial lining of the abdominal viscera and removal from the interstitium by afferent visceral lymphatics. Material taken up in this manner is ultimately transported to the thoracic duct by efferent visceral lymphatics. Experiments involving measurements of lymphatic absorption of 125I-labeled human serum albumin from the peritoneal cavity indicated that, over the 6-h period studied, 4.55 +/- 1.20 and 1.43 +/- 0.56% of the injected tracer could be recovered in thoracic duct lymph and caudal mediastinal efferent lymph, respectively, and the sum of these values represented 26% of the recovered radioactivity. On the other hand, 16.95 +/- 6.93% of the injected radioactivity could be found in the blood over the same period.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Lymphocyte subset-specific and tissue-specific lymphocyte-endothelial cell recognition mechanisms independently direct the recirculation of lymphocytes from blood to lymph in sheep.

Tissue-specific and lymphocyte subset-specific lymphocyte recirculation patterns have been analysed simultaneously. Lymphocytes obtained from one lymph compartment were directly labelled with fluorochrome in vitro and returned to the blood of the same animal. Over the next 48-72 h, the recirculation of these cells into both the same lymph compartment and at least one different lymph compartment was monitored. The cells in all of these lymph collections, as well as an aliquot of the cells used for direct fluorescent labelling, were then phenotyped with monoclonal antibodies (mAb) which define the mutually exclusive small CD4+ and CD8+ T-lymphocyte subsets in sheep. All cell samples were analysed by flow cytometry and CD4/CD8 ratios were determined for the recirculated, fluorochrome-labelled population in each lymph collection. The mean CD4/CD8 ratio calculated for each lymph compartment was then compared with the CD4/CD8 ratio calculated for each lymph compartment was then compared with the CD4/CD8 ratio of the transfused, starting population. In one experiment employing efferent prescapular lymph cells, three experiments employing efferent intestinal lymph cells, and two experiments employing afferent intestinal lymph cells, tissue-specific recirculation was observed. In all of these experiments, the pattern of recirculation of small CD4+ and CD8+ T lymphocytes was non-random. Moreover, in each experiment, this non-randomness was completely unrelated to tissue-specific phenomena, since the mean CD4/CD8 ratio of the recirculated population was higher than the CD4/CD8 ratio of the transfused, starting population regardless of the lymph compartment examined. These data are therefore consistent with the hypothesis that tissue-specific and lymphocyte subset-specific lymphocyte-endothelial cell recognition mechanisms independently direct the recirculation of small lymphocytes from blood to lymph.

Animals↗
PubMed1990

Production of plasminogen activator and plasminogen activator inhibitor by bovine lymphatic endothelial cells: modulation by TNF-alpha.

We have investigated whether lymphatic endothelial cells in culture produce plasminogen activators (PAs) and their inhibitors (PAIs) and if these activities can be modulated by the inflammatory cytokine Tumor Necrosis Factor alpha (TNF-alpha). Examination by reverse fibrin autography of the conditioned medium from these cells revealed a PAI of Mr 50 kDa. Also evident by fibrin autography were two species of PAs, of Mr 110 kDa and Mr 60 kDa. The 110 kDa protein co-migrated with the PA-PAI complexes and the 60 kDa protein co-migrated with tissue Plasminogen Activator (tPA). Functional and immunological assays indicated the human TNF-alpha increased the type 1 plasminogen activator inhibitor (PAI-1) in a time dependent manner. Treatment of the cells with recombinant human TNF-alpha for 24 hours resulted in a 3 to 7 fold increase in the amount of PAI released into the conditioned media. Immunoblot analysis identified the PAI in the TNF-alpha treated cell conditioned media, as PAI-1. Deposition of PAI-1 in the extracellular matrix then became apparent. TNF-alpha increased 4 fold the amount of tPA-PAI-1 complexes (Mr 110 kDa) detected in the conditioned media. Free tPA (Mr 60 kDa) decreased to 1/5 of control. Net fibrinolytic activity, as determined by a chromogenic substrate assay, decreased after TNF-alpha treatment. No urokinase type Plasminogen Activator (uPA) activity was detected in control or treated cells. This fibrinolytic activity may be important in maintaining free fluid movement in the interstitium and lymphatic vessels and in inflammatory states this potential may be decreased by the increase in PAI-1.

Animals↗
PubMed1990

Non-random recirculation of small, CD4+ and CD8+ T lymphocytes in sheep: evidence for lymphocyte subset-specific lymphocyte- endothelial cell recognition.

The migratory properties of small, CD4+ and CD8+ T lymphocyte subsets have been examined in sheep under physiological conditions. Lymphocytes obtained free-floating in lymph were directly labeled with fluorochrome in vitro and returned to the blood of the same animal. Over the next 48 h the lymph collection bottles were replaced at various times. The cells in these collections, as well as an aliquot of the cells used for direct fluorescent labeling, were then phenotyped with monoclonal antibodies (mAbs) which define the mutually exclusive CD4+ and CD8+ T lymphocyte subsets in sheep. Binding of mAbs was detected by using secondary reagents labeled with a fluorochrome of a different colour. All cell samples were analyzed by flow cytometry and CD4/CD8 ratios were determined for the recirculated, fluorochrome-labeled population in each lymph collection. The mean CD4/CD8 ratio was then calculated and compared with the CD4/CD8 ratio of the intravenously infused starting population. In three experiments employing efferent prescapular lymph cells, three experiments employing efferent intestinal lymph cells and two experiments employing afferent intestinal lymph cells, the mean CD4/CD8 ratio of the recirculated, fluorochrome-labeled population was different from the CD4/CD8 ratio of the starting population, thereby indicating that non-random migration of these subsets had occurred. The finding that the CD4/CD8 ratio of the recirculated population was higher than the CD4/CD8 ratio of the transfused starting population in every case provides strong experimental support for the hypothesis that small, CD4+ T cells are extracted from the blood by specialized vascular endothelium with greater efficiency than small, CD8+ T cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

The relevance of lymphoid cell migration to immunodeficiency syndromes.

It has been known for some time that antigen stimulation can alter lymphocyte traffic patterns and that viruses are particularly potent in this respect; such alterations may be a consequence of host-derived factors. The retention of lymphocytes in lymph nodes can be sustained for several hours with locally administered interferon (IFN)alpha. The extravasation of lymphocytes from blood into non-lymphoid tissues can be induced in the skin with IFN gamma and particularly tumor necrosis factor (TNF)alpha. Recent evidence supports the concept that the migratory capacity of CD4+ cells differs from the capacity of CD8+ cells. Agents (cytokines?) which differentially affect the traffic of these two sub-sets have not yet been described but such a possibility has not been adequately tested. Several new molecules have been defined which alter the interactions between lymphocytes and blood vascular endothelial cells, and these may be important in the critical adhesive event in lymphocyte traffic. In both rat and sheep, it has been possible to cultivate post-capillary endothelial cells from lymphoid tissue, and this may be a helpful approach to studying the mechanisms and molecules involved in adhesion. New cell tracking dyes recently available (Zynaxis Cell Science) permit more significant, long-term studies on the life span of lymphocyte sub-sets and their migratory status. In our experiments, labeled lymphocytes can be followed in vivo for over 30 days. Traffic alterations may explain some of the abnormalities in immunodeficiency states.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Isolation, culture, and characterization of endothelial cells derived from the post-capillary venules of sheep mesenteric lymph nodes, Peyer's patches, and associated small bowel.

Lymphocyte recirculation is mediated principally by specialized endothelial cells which line the post-capillary venules of lymph nodes and other secondary lymphoid tissues. The ontogeny and physiology of this process have been characterized in sheep in considerable detail. To further enhance the analytical potential of this experimental system we have isolated endothelial cells from the post-capillary venules of ovine mesenteric lymph nodes by perfusion with small (37-74 microns diameter), sulfonated microcarrier beads. Cells isolated in this manner have been maintained in vitro for greater than 12 months through greater than 30 passages. The endothelial nature of these cells has been conclusively established on the basis of morphologic, metabolic, and immunologic criteria. Virtually all (greater than 99%) cells in primary and passaged cultures metabolized Dil-AC-LDL, a known marker for endothelial cells. Furthermore, nearly all (greater than 95%) cells expressed cell-surface von Willebrand factor and antithrombin III, which are known endothelial antigens. All cells expressed major histocompatibility class I antigens but no cells expressed class II antigens. In vitro lymphocyte-binding studies revealed that these cells bound lymphocytes in a dose-dependent fashion. The microcarrier perfusion technique was also used to isolate endothelial cells from the post-capillary venules of ileal Peyer's patches and associated small bowel in sheep. The majority (70%) of cells isolated in this manner resembled the cells isolated from mesenteric lymph nodes both morphologically and metabolically.

Animals↗
PubMed1986

A quantitative lymphocyte localization assay.

Multiple lesions (up to 100 sites) were induced in the skin of sheep using either allogeneic lymphocytes or, in BCG-sensitized animals, tuberculin. Cells recovered from an indwelling lymph catheter draining a prescapular lymph node were labeled with 111-indium, returned to venous blood, and allowed to circulate for 3 hours. Sheep were killed, and the skin lesions and lymph nodes were removed and counted in a gamma spectrometer. High levels of radioactivity (up to 38,000 cpm/lesion) were recovered from lesions, and only a few hundred cpm were recovered from comparable normal skin sites. Dose-response relationships and time kinetics were demonstrated for these lesions, and the radioactivity on blood and lymph cells was measured. The contribution of cell-free radioactivity was negligible. Using replicate injection sites, analytical, internally controlled studies can now be initiated to study the induction, promotion, and suppression of lymphocyte traffic.

Animals↗
PubMed1985

A dual laser analysis of the migration of XRITC-labeled, FITC-labeled, and double-labeled lymphocytes in sheep.

Substituted rhodamine isothiocyanate (XRITC) has been used to study lymphocyte migration in sheep. After being labeled in vitro with XRITC, lymphocytes appeared in the efferent lymph of single lymph nodes with the same kinetics as cells labeled with fluorescein isothiocyanate (FITC). The recovery of intravenously injected XRITC-labeled cells was followed in lymph for several days. The kinetics and recoveries were compared with data obtained using FITC, chromium-51, and indium-111. XRITC was found to be a suitable label and, using dual laser (argon and krypton) flow cytometry, it could be analyzed simultaneously with FITC. In addition, it was possible to relabel FITC-stained cells with XRITC after they were recovered in lymph. The migratory characteristics of such double-labeled cells were not different from single-labeled cells.

Animals↗
PubMed1984

Distribution of radiolabelled lymph cells in lymph nodes and the migratory properties of blood lymphocytes in sheep.

The results found in this study supported the concept of lymphocyte populations with preferential migratory pathways. Preferential localization of lymphocytes in lymph nodes could not explain the non-random lymphocyte migration patterns observed in sheep. The migration of lymphocytes isolated from the blood was similar to that of lymphocytes in the efferent lymph of a subcutaneous lymph node, but was different from the migration of lymphocytes isolated from efferent intestinal lymph. The subcutaneous lymph node did not need to be selective for lymphocytes which entered from blood. However, there must be a selective entry of a population of lymphocytes with preference for migrating through mesenteric lymph nodes into efferent intestinal lymph in order to observe the differential lymphocyte migration patterns exhibited by the lymphocytes in the efferent intestinal lymph of sheep.

Animals↗
PubMed1983

Plasminogen activator and thromboplastin activity from sheep alveolar macrophages.

Alveolar lavage cells from normal sheep were found to be composed of over 95% macrophages. When the cells were cultured, fibrinolytic and thromboplastin-like activities could be detected within 2-4 hours of incubation. As the number of cultured cells was increased the two activities in the conditioned medium increased proportionately. The cells were separated into two distinct subpopulations by means of a sedimentation velocity cell fractionation technique. The macrophage subpopulations were examined for differences in size, morphology, esterase staining and ability to release plasminogen activator and procoagulant activity respectively. These activities were confined to the large cell subpopulation. The fibrinolytic activity was shown to be plasminogen-dependent and could be inhibited by DFP. On the basis of this the fibrinolytic activity has been designated as plasminogen activator. The procoagulant activity was shown to be thromboplastin in nature because it was Factor VII dependent, inactivated by phospholipase C and not inhibited by DFP. The procoagulant activity has been designated as macrophage thromboplastin. The two activities could be distinguished on the basis of DFP inhibition.

Animals↗
PubMed1983

Involvement of circulating phospholipase A2 in the pathogenesis of the hemodynamic changes in endotoxin shock.

This study examined the role of plasma phospholipase A2 (PLA2) in the mediation of the hypotension associated with experimental endotoxin shock in rabbits. Endotoxin shock was induced in rabbits, and mean arterial blood pressure and plasma PLA2 levels were monitored. Serial plasma PLA2 determinations over 5 h showed an 11-fold increase in circulating enzyme activity, and the rise in circulating enzyme activity was directly related to the fall in mean arterial blood pressure. Pretreatment of rabbits with glucocorticoids abrogated the hypotensive effect of endotoxin, and also inhibited a rise in plasma PLA2 activity. To determine if the rise in PLA2 activity was simply a mechanistically unrelated epiphenomenon, the effect of infusion of exogenous PLA2 (purified from the blood of rabbits in endotoxin shock) was investigated. Infusion of the exogenous enzyme into normal rabbits caused a fall in mean arterial blood pressure, and the rate of fall of blood pressure paralleled that induced by endotoxin itself. Treatment of the PLA2-active fraction, prior to infusion with the PLA2 inhibitor, p-bromophenacyl bromide, protected against this hypotensive effect. These data are consistent with the postulate that the endotoxin-induced release of massive amounts of PLA2 into the systemic circulation in rabbits contributes significantly to the hypotension associated with septic shock.

Animals↗
PubMed1982

Human skin androgen metabolism and preliminary evidence for its control by two forms of 17 beta-hydroxysteroid oxidoreductase.

Human forehead skin incubated in vitro is known to metabolize testosterone to 17-oxosteroids faster than the reverse reaction, while axillary skin rapidly metabolizes androstenedione to 17 beta-hydroxysteroids, such as testosterone and 5 alpha-dihydrotestosterone. While this has been confirmed using a larger number of patients, some indication has been found that 17 beta-hydroxysteroid oxidoreductase activity declines with age in the axilla. The relative rates of 17 beta-oxidation and reduction (direction of operation of skin 17 beta-hydroxysteroid oxidoreductase activity) were not altered by variety of incubation conditions. Large amounts of a membrane-bound 17 beta-hydroxysteroid oxidoreductase, showing preference for NAD as coenzyme and testosterone (rather than androstenedione) as steroid substrate, were found in forehead skin from one patient. On the other hand, the main axillary skin enzyme in skin from another patient was soluble and showed preference for NADP and androstenedione. It is postulated that 17 beta-oxidation and reduction in skin is controlled by the relative amount, the coenzyme preferences and the kinetic properties of these two enzymes.

17-Hydroxysteroid Dehydrogenases↗
PubMed1982

Mechanism of action of alpha-melanocyte-stimulating hormone in rat preputial glands: the role of androgen metabolism.

The metabolism of testosterone and 5 alpha-dihydrotestosterone has been studied in vitro in preputial glands of posterior hypophysectomized, totally hypophysectomized and control sham-operated rats. The level of C19 steroid 5 alpha-reductase activity/unit of preputial gland DNA did not fall after removal of the neurointermediate lobe and rose after total hypophysectomy. It was concluded from this that the androgen unresponsiveness of the preputial glands of hypophysectomized rats was not due to a near-total lack of 5 alpha-reductase and hence that the combined synergistic action of testosterone and alpha-melanocyte-stimulating hormone (alpha-MSH) on preputial gland activity was unlikely to be due to an alpha-MSH-mediated restoration of 5 alpha-reductase levels in hypophysectomized rats. Levels of 3 alpha and 3 beta-hydroxysteroid dehydrogenase but not of 17 beta-hydroxysteroid dehydrogenase appeared to be altered by hypophysectomy.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗