Biomedical subjects

D F Gruber

Publications and source records attributed to D F Gruber.

At least 19 recordsLinked to original sources

PubMed1991

In vitro modulation of canine polymorphonuclear leukocyte function by granulocyte-macrophage colony stimulating factor.

Granulocyte-macrophage colony stimulating factor (GMCSF) promotes the growth of granulocytes and macrophages from undifferentiated bone marrow cells and modulates the oxidative responses of polymorphonuclear leukocytes (PMN) to endogenous chemoattractants. We found that, in vitro, naturally occurring glycolsylated human GMCSF does not disturb the resting canine PMN membrane potential, may attentuate PMN oxidative responses to PMA, and is, to a small degree, chemotaxigenic. GMCSF, however, inhibits PMN chemotaxis to zymosan-activated plasma (ZAP). Compared to temperature controls, GMCSF (1-100 U/ml) produced up to 1.5-fold increases in H2O2 production after 15 minutes, while phorbol myristate acetate (PMA) treated cells increased H2O2 production 8-12-fold after 15 minutes. Preincubation of cells with GMCSF (1-100 U/ml) prior to PMA stimulation significantly reduced the H2O2 levels induced by PMA. H2O2 production was inhibited up to 15% after 15 minutes of GMCSF preincubation and up to 40% after 60 minutes of preincubation. As a chemotaxigenic agent, GMCSF (10-1000 U/ml) was able to elicit 49%-102% increases in quantitative cellular migration, compared to random migration. Total cellular chemotaxis to GMCSF was less than 30% of the response to ZAP. Preincubation of PMNs with GMCSF for 15 minutes significantly inhibited ZAP-induced cellular migration. Human GMCSF does not appear to activate canine PMN in vitro and may actually down-regulate PMN inflammatory responses.

Animals↗

PubMed1990

Biochemical and physiological alterations in canine neutrophils separated by lysis or Percoll gradient isolation technologies.

Neutrophils were isolated from the peripheral blood of clinically normal canines by hypotonic lysis or density gradient (Percoll) centrifugation techniques. As a function of preparative technique, separated neutrophils were examined in vitro for alterations in membrane lipid integrity, and both granular and cytosolic specific enzymes. Membrane lipid disorder was assessed by merocyanine 540 (MC540), a fluorescent bioprobe, which intercalates into disrupted cellular lipid membranes. Evidence of membrane lipid disorder was based upon comparisons of mean cellular fluorescence exhibited by unstimulated cells. Based upon comparisons of mean cellular fluorescence the isolation methodologies did not appear significantly different. Significant levels of membrane disruption, due to preparative technique, were evident upon neutrophil stimulation with phorbol myristate acetate (PMA). Neutrophils which were Percoll-separated and PMA-stimulated demonstrated significant levels of membrane disruption not apparent in lysis-separated stimulated aliquots of cells. Cellular isolation methods did not significantly alter cellular myeloperoxidase (MPO) levels based on comparisons of cellular totals. Extracellular lactate dehydrogenase (LDH) levels of lysis-separated cells were four-times those of Percoll-separated neutrophils.

Animals↗

PubMed1990

Effects of collection methods and storage on the in vitro stability of canine plasma catecholamines.

Norepinephrine (NE) and epinephrine (EPI) collected from dogs were sequentially and temporally measured in blood and plasma at 24 C. Heparin and EDTA anticoagulants, in combination with reduced glutathione and EDTA as a preservative, were also compared. Norepinephrine and EPI concentrations were measured by high-pressure liquid chromatography with electrochemical detection. In heparinized plasma, NE and EPI concentrations were relatively stable in the absence or presence of preservative after 24 hours at 24 C. In EDTA plasma, NE and EPI values were less stable when compared with those in heparinized samples. Norepinephrine concentrations in EDTA plasma without preservative decreased by 163.2 +/- 8.88 pg over 24 hours, compared with an 86.6 +/- 7.92 pg loss of NE in heparinized plasma. The degradation of EPI in EDTA plasma without preservative was also twofold greater, compared with that in heparinized plasma. Addition of preservative had no stabilizing effect on NE or EPI in heparinized or EDTA plasma. During long-term storage at -70 C, plasma NE and EPI values decreased less than 0.6 and less than 0.1 pg/d, respectively. Norepinephrine and EPI values were stable in heparinized blood for 6 hours but decreased to less than 25% and less than 6% of initial base line values, respectively, when plasma separation was delayed 24 hours.

Animals↗

PubMed1990

Hypermetabolic priming of canine neutrophils by 7-S nerve growth factor.

Canine circulating neutrophils, isolated by a blood lysing technique, were incubated with 7-S nerve growth factor (NGF), at final concentrations between 12.5 and 800 ng/ml, for 30 minutes at 37 C. Neutrophil cytosolic H2O2 production, measured by flow cytometry, after 7-S NGF incubation was not significantly different from that produced at 37 C (baseline temperature controls) alone. Phorbol myristate acetate (PMA; 100 ng/ml) stimulation of neutrophils produced cytosolic H2O2 concentrations almost 13 times that of baseline temperature control neutrophils. Preincubation of neutrophils with 7-S NGF (100 to 800 ng/ml, 30 minutes, 37 C) and subsequent stimulation by PMA resulted in augmented H2O2 production in excess of twice that of neutrophils treated with PMA alone, and almost 30 times that of baseline temperature controls.

Animals↗

PubMed1989

In vitro effects of leukotriene B4 (LTB4) on canine PMN effector function(s).

The canine has become an accepted research model for the examination of a number of human clinical conditions. Despite it's status as a research model, little is known regarding the peripheral effects of inflammatory mediator substances. Products of arachidonic acid metabolism (leukotrienes) are reported capable of altering leukocyte functions. Because of the emerging importance of the canine research model and leukotrienes we examined the effects of leukotriene B4 (LTB4) on several in vitro functions of isolated canine peripheral polymorphonuclear leukocytes (PMN). Changes in forward angle light scatter properties of the cells were used as one measure of PMN activation. Other functional changes examined following LTB4 pretreatment included chemotactic capability, the electrophysiological state of the cell plasma membrane, and the metabolic oxidative response (i.e. H2O2 production). Random cellular movement of PMNs increased by 120% and 72% following preincubation with 10(-7) and 10(-9) M LTB4, respectively. LTB4 between 10(-7) and 10(-13) M did not significantly alter cellular resting membrane potential. Between 10(-7) and 10(-9) M LTB4 elicited significant levels of cellular H2O2 production. Although significant, H2O2 production was less than 40% that induced by phorbol myristate acetate (PMA). In numerous respects, canine in vitro PMN responses parallel previous reports of human cell function(s) in the presence of inflammatory mediators and may represent an attractive alternative for investigation of PMN dysfunctions.

Animals↗

PubMed1989

Aminophylline induced oxidative metabolism in isolated canine polymorphonuclear leukocytes.

Adenosine reportedly mediates myocardial and skeletal blood flow, bronchoconstriction, and cellular production of toxic oxygen radicals. Cellular effects of adenosine can be antagonized by the methylxanthines, which are widely used in the clinical treatment of obstructive airway diseases. Methylxanthine compounds such as aminophylline and theophylline inhibit the cyclic nucleotide phosphodiesterase of smooth muscle, reversing pathogenic states of bronchoconstriction. Recent techniques in flow cytometry allow examination of individual cells for the electrophysiological and metabolic cellular side effects of methylxanthine therapy. We report that the flow cytometric examination of isolated canine peripheral neutrophils, in the presence of therapeutic concentrations of aminophylline resulted in small but significant membrane depolarization and almost fivefold increases in baseline cytosolic H202 levels. If aminophylline is capable of direct in vitro activation of isolated canine neutrophils it may have the capacity to potentiate neutrophil activation in vivo: indirectly by competing with circulating modifiers, such as adenosine, for cell surface receptor sites and directly by the induction of toxic oxygen radicals as demonstrated here. H202 induction by aminophylline and other xanthine derivatives may become clinically important in instances of vascular occlusion, stasis, or instances of reperfusion where neutrophils may become activated. In an activated state, neutrophils could contribute to pathogenicity and tissue damage by indiscriminantly releasing oxygen-reactive species.

Aminophylline↗

PubMed1989

Tropism of canine neutrophils to xanthine oxidase.

Quantitative evaluation of neutrophil chemotaxis was performed on cells obtained by hypotonic-lysis techniques from heparinized blood samples from clinically normal dogs. The techniques resulted in neutrophil recovery rates between 60 and 80%. Chemotaxis comparisons were based on cellular migration in microchambers equipped with polycarbonate membranes with 5-micron pores. Chemo-attractant comparisons were based on neutrophil migration to medium, normal canine plasma, zymosan-activated plasma, and xanthine oxidase. Cellular migration to zymosan-activated plasma in buffer (1:100 dilution) was significantly (P less than 0.001) enhanced over random baseline medium migration. Neutrophil migrations to normal canine plasma and xanthine oxidase were quantitatively less than to zymosan-activated plasma, but were equivalent to each other and significantly greater than for random migration. Migration to xanthine oxidase was maximal at concentrations near 1 U/ml within 30 minutes.

Animals↗

PubMed1989

Xanthine oxidase potentiation of reactive oxygen intermediates in isolated canine peripheral neutrophils.

Oxygen-derived free radicals are believed to contribute to reperfusion injury based, in part, upon results conferred by the pharmacologic administration of allopurinol. Allopurinol inhibits xanthine oxidase (XO) activity in ischemic tissues. The possible role of XO as a pathologic mediator prompted examination of its effects on isolated peripheral canine neutrophils. In contrast to neutrophils alone, or following stimulation with phorbol myristate acetate (PMA), it was determined that XO affected both the membrane potential and the metabolism significantly. Membrane potential assay showed that at 5-10 min, PMA depolarized 89-96% of the canine neutrophils between 32-48%. Incubation with 0.5 U/ml XO involved fewer cells (54-86%), but at substantially increased cellular depolarization levels (76-90%). Metabolic assay showed that XO concentrations as low as 0.124 U induced significant cellular H2O2 production compared with temperature controls. At 0.25-0.5 U XO/10(6) cells, cytosolic H2O2 increases were almost three times those of PMA.

Animals↗

PubMed1988

Flow cytometric characterization of rat thymus cells in a radiation-dominated model of combined injury.

Thymuses of rats that had been: a) gamma-irradiated [500 cGy whole-body radiation (R)], or b) thermally injured [20% BSA dorsal, scald burn (TI)], or c) combined injured [irradiation followed by burn (CI)] were studied for involution and recovery processes after sublethal treatments. The expression of surface antigens on thymic cells before and after injuries was evaluated using the monoclonal antibodies (mcAB) MRC OX4, MRC OX7, MRC OX8, W3/13 HLK, and W3/25 and flow cytometric analysis. Thymic cellularity decreased to less than 1% of normal (N), age-matched rats by 4 days after R or CI. Recovery reached 60% to 70% of N by 28 days post treatments. TI caused a biphasic thymic recovery pattern with nadirs of 40% of N on days 7 and 21. Recovery at day 28 was similar to that after R and CI. Expression of OX7, OX8, W3/13, and W3/25 antigens all reached nadirs of 40% of N by day 4 after R and CI. Recovery of antigen expression, except for W3/25, was near completion by day 7 after R and CI. Changes in antigen expression after TI were less pronounced for all mcAB tested. Decreases in labeling of thymocytes with the helper T-cell marker, W3/25, observed after TI, could not be correlated with elevated expressions of the suppressor/cytotoxic T-lymphocyte antigen, OX8. Variations in relative labeling of nonlymphoid thymic cells with OX4 (Ia-antigen) reflected the disappearance and recovery of radiosensitive lymphoid thymocytes. The similarity of results after R and CI demonstrate that the model of CI is 'radiation-dominated.' The addition of burn injury to radiation trauma had no synergistically damaging effect on the parameters studied.

Animals↗

PubMed1988

Changes in canine neutrophil function(s) following cellular isolation by Percoll gradient centrifugation or isotonic lysis.

Analysis of cellular effector function(s) often requires their isolation from other cellular types. Cell separatory techniques could mask, or select out, clinically important functional lesions. We examined differences in canine peripheral blood neutrophil functions, i.e. migration and H202 production, following two commonly used cell separation techniques: isotonic lysis or density gradient (Percoll) centrifugation. Separation methodology was observed to have a significant impact on both metabolic and mobility functions. In comparison to isotonic lysis, Percoll separation caused near 100% increases in random migration, near 40% decreases in chemotaxis and 70% increases in H202 production.

Animals↗

PubMed1987

Rat monocytes in a model of combined injury express the OX8 antigen.

We have analyzed peripheral blood mononuclear cell preparations from a rat model of combined injury (CI) [whole-body irradiation (500 cGy 60Co) followed by a thermal injury (20% body surface area, dorsal, scald burn)] for the expression of OX8 antigens. Ficoll-separated mononuclear fractions were labeled with monoclonal antibodies MRC OX8, MRC OX19, W3/13 HLK, or W3/25 for flow cytometric analysis. Combined-injury trauma resulted in decreased mononuclear cells to 6% of normal. This effect was due to the rapid decrease in radiosensitive lymphocytes from 83% to 10%. The relative numbers of monocytes increased from a normal 13% to 70% at day 4 after CI. Labeling of cells with OX8 after CI shifted to a population which was significantly larger in volume than normal lymphocytes. At the same time the mean fluorescence intensity of OX8-positive cells was considerably reduced. With the use of a F(ab) fragment of OX8 as a probe, these results could be partially explained as unspecific binding of the whole molecule of OX8 to Fc receptors expressed by activated monocytes. But double-labeling and cell-sorting experiments also revealed the expression of OX8 antigens by a subset of OX8+/OX19- monocytes after CI.

Animals↗

PubMed1985

Hematopoietic colony-forming cells from mice after wound trauma.

The changes produced in the pluripotential and progenitor cell compartments of the hind leg bone marrow and spleen of skin-wounded mice were examined over a 2-week post-trauma period. Pluripotent cells (colony-forming unit-spleen, CFU-s) were significantly increased in the spleen and slightly reduced in the leg marrow the first week after trauma. Granulocyte macrophage colony-forming cells (GM-CFC) were significantly increased in the spleen throughout the 2-week period and were increased in the leg marrow during the first post-trauma week. Macrophage colony-forming cells (M-CFC) were significantly decreased in the spleen during the 2-week period and were slightly elevated in the leg marrow during that time. The peripheral blood contained significantly increased concentrations of CFU-s and GM-CFC but not M-CFC. Serum of wounded mice supported growth of GM-CFC but not M-CFC. The growth-promoting factor was extractable by CHCl3 treatment. Serum C-reactive protein concentrations were significantly increased for a 5-day period after wound trauma.

Animals↗

PubMed1985

Modulations in mouse hemopoiesis after engraftment with Lewis lung (3LL) carcinoma cells.

Hemopoietic changes in male C57BL/6Cum BR mice engrafted with Lewis lung carcinoma (3LL) were evaluated between day 7, when palpable tumors were present, to day 30 postengraftment. All experimental animals demonstrated decreasing hematocrits (down 40% by day 30) with concurrent leukocytosis which by day 30 postengraftment had reached levels 13.4 times normal. The myelocytic/erythrocytic ratio for normal animals was 1:3 (bone marrow: spleen). The ratio for engrafted animals ranged between 10:1 and 40:1. This apparent shift in production priorities is even more significant in light of the fact that femoral bone marrow cellularity had decreased by 33% on day 17. Splenomegaly, evident by day 7, was seven times control by day 17. Clonogenic analysis of erythroprogenitor cell concentrations revealed an inverse relationship between bone marrow and spleen. 27 days after engraftment, splenic populations demonstrated significant increases in colony forming unit-erythroid (115-fold), burst forming unit-erythroid (7.4-fold), whereas bone marrow concentrations had decreased (6-fold). This report suggests that initiation of 3LL tumor in mice results in a change in the degree of hematopoietic priorities and participation of erythroid organs.

Animals↗

PubMed1985

Radiation abrogation of myelopoietic inhibitors found in thymus-conditioned medium.

We have previously reported conditioned medium from cultures of murine 'nonlymphoid' adherent thymus cells to be a source of myelopoietic inhibitor substance(s). The inhibitor has a molecular weight of less than 1000 daltons. It is dialyzable. and its effects are reversible with washing, but it is not significantly cytotoxic. The inhibitor(s) abrogate both the 10-day granulocyte-macrophage colony-forming cell (GM-CFC) and the 25-day monocyte-macrophage colony-forming cell (M-CFC). Morphologically, cultures appear to be lymphocyte-free after 10-12 days, and consist primarily (80%) of nonspecific esterase positive cells and epitheloid appearing (20%), nonspecific esterase negative cells. Radiation of thymic cultures at 12 days reduces levels of inhibitor(s) found in the medium. Inhibitor levels from pooled samples collected over 4 days demonstrate reductions of 10% at 100 rads 15% at 200 rads, 25% at 300 rads, and 85% at 400 rads. The steepest area of radiation reduction occurs between 300 and 400 rads, with the interpolated 50% radiation reduction level at 342 rads. Analysis of once-harvested radiated culture supernates showed no significant inhibitors present when medium was examined 5-10 days postradiation.

Animals↗

PubMed1984

Thymic inhibition of myelopoietic proliferation.

Homeostasis may be viewed as the dynamic balance reached between cellular production and loss. The effectors of proliferation are thought to be tissue-specific humoral substances. Inhibitory substances appear to negate the proliferate effects of activator substances. We report here that conditioned medium from cultures of murine "nonlymphoid" adherent thymus cells in a source of a potent myelopoietic inhibitor substance(s). Examination of the inhibitor(s) in the soft-agar clonogenic assay show it to abrogate both the 10-day granulocyte-macrophage colony-forming cell (GM-CFC) and the 25-day monocyte-macrophage colony-forming cell (M-CFC). The inhibition is most significant when the thymus-conditioned medium (TCM) is present upon culture initiation. TCM added at a time after soft-agar initiation (day 6) also results in significant clonogenic inhibition. We have characterized the inhibitor as potent on the basis that volume ratios of inhibitor to L-cell colony-stimulating factor (CSF) as low as 1 part to 40 parts will give near total inhibition of both GM-CFC and M-CFC clonogenic growth. The inhibitor is dialyzable, has a molecular weight of less than 1000, is not significantly cytotoxic, and its effects are reversible with washing.

Animals↗

PubMed1983

Submandibular gland-conditioned medium enhancement of bone marrow colony-forming cells in tumor-bearing murine recipients.

Soft-agar clonogenic assay techniques were used to examine 2 sources of colony-stimulating activity (CSA), submandibular gland-conditioned medium (SMG-CM) and pregnant mouse uterine extract (PMUE), for potentiation of granulocyte-macrophage (GM-CFUc) or monocyte-macrophage (M-CFUc) progenitor cell populations. The femoral populations being examined were aspirated from normal mice and from those bearing 1 of 2 types of tumor: Lewis lung carcinoma (3LL) or thymic lymphoma ascites tumor (EL-4). When PMUE was used as a CSA source, normal animals showed a greater clonogenic response per population than either of the tumor-bearing groups. When SMG-CM was used as a CSA source, the pattern of GM-CFUc response was much different: GM-CFUc magnitudes increased by fourfold to sixfold over normal levels in tumor-bearing animals. M-CFUc response patterns were also significant, being similar in response but smaller.

Animals↗

PubMed1977

Erythropoietin, thrombopoietin and colony stimulating factor in fetal mouse liver culture media.

Fetal mouse livers, days 13 to 19 of gestation, were cultured for 21 days and the culture media tested for erythropoietin (Ep), thrombopoietin (TSF) and colony stimulating factor (CSF). High levels of both Ep and CSF were released into the culture media. However, no detectable TSF was found. Maximum Ep culture activity was detected in day 14 and day 15 fetal liver cultures while maximum CSF was found in the day 16 fetal liver culture. These studies indicate that fetal liver cells in culture are capable of producing and/or releasing both Ep and CSF but not TSF.

Animals↗

PubMed1977

Identification of erythropoietin producing cells in fetal mouse liver cultures.

Fetal mouse liver explants were cultured and the culture media shown to possess an erythropoietically active substance neutralizable by antiserum to sheep plasma erythropoietin, suggesting that the media contain erythropoietin. Immunofluorescent and carbon particle ingestion techniques suggest that the erythropoietin was elaborated by macrophages or Kupffer cells of the hepatic reticuloendothelial system.

Animals↗