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T H Howard

Publications and source records attributed to T H Howard.

At least 55 records · Page 3Linked to original sources
PubMed1987

Actin polymerization and its relationship to locomotion and chemokinetic response in maturing human promyelocytic leukemia cells.

We studied actin polymerization in the HL-60 human promyelocytic leukemia cell line during induced myeloid maturation and its relationship to the rate of locomotion (ROL). The percent G-actin (of total actin) was measured by DNAase I inhibition, F-actin was determined by fluorescence-activated cell sorter (FACS) analysis of nitrobenzoxadiazol (NBD)-phallacidin-stained cells, and ROL was measured by computer-assisted analysis of the tracks of individual cells. Uninduced HL-60 cells moved slowly (2.3 +/- 1.0 microns/min) and showed no change in ROL or in the state of actin polymerization when stimulated by formyl-methionyl-leucyl-phenylalanine (fMLP). Nonstimulated cells induced to differentiate with dimethylformamide had no change in the degree of actin polymerization but exhibited a mean (m) ROL similar to normal human polymorphonuclear leukocytes (PMN) (8.6 +/- 1.4 micron/min [HL-60 cells] v 7.8 +/- 1.8 microns/min [PMN]. When induced HL-60 cells were stimulated with fMLP, actin polymerization occurred. The F-actin content increased, as determined by FACS analysis of NBD-phallacidin-stained cells, and the percentage of G-actin decreased, as determined by a 24.5% decrease in DNAase I inhibitory activity. However, induced HL-60 cells stimulated with fMLP did not increase their mROL. These studies show that, unlike normal human PMN, chemotactic peptides can cause an intracellular biochemical change that is not associated with a chemokinetic response in induced HL-60 cells. The HL-60 cell line may be a useful model to study the development of chemotactic peptide-mediated actin polymerization during myeloid cell maturation.

Actins↗
PubMed1987

F-actin content of neonate and adult neutrophils.

We utilized flow cytometric analysis of NBDphallacidin-stained cells to measure F-actin content, expressed as fluorescent channel numbers, and we compared the microfilamentous cytoskeletal organization in neutrophils from healthy neonates (36 to 38 weeks gestational age) and adults. Basal F-actin content in neonate cord blood neutrophils is higher than that of adults. The elevation is intrinsic to the cell and not related to parturition because basal F-actin content of neonatal cells obtained by venipuncture (days 1 to 8 of age) is also elevated (38 +/- 10, N = 15) when compared to adults (21 +/- 7.0, N = 27). The rate of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced actin polymerization is similar in adult and neonatal neutrophils and is maximal by 30 to 45 seconds at 25 degrees C. Adult, neonatal cord, and neonatal venipuncture neutrophils increase F-actin content to a similar extent following 0.5 mumol/L fMLP activation (52 +/- 18, N = 27; 58.7 +/- 18, N = 18; 51.5 +/- 7.0, N = 15, respectively). However, the relative increase in F-actin content following fMLP activation is much greater in adult (2.37-fold) than neonatal neutrophils (1.28-fold). This difference is due to the elevated basal F-actin content of neonatal cells. Comparison of distribution of F-actin content among basal, neonatal neutrophils reveals two subpopulations of neutrophils with respect to F-actin content--approximately 25% with F-actin content similar to that of adult neutrophils and 75% with F-actin content greater than that of adult cells. Following fMLP activation, the subpopulations disappear. The results suggest that abnormalities in microfilamentous cytoskeletal organization of neonatal cells may, in part, be responsible for decreased chemotactic response of neonatal neutrophils.

Actins↗
PubMed1986

Thrombocytopenia as the presenting manifestation of human T-lymphotropic virus type III infection in infants.

Three infants between 8 and 9 months of age developed thrombocytopenia resulting from immune-mediated platelet destruction, as evidenced by the presence of serum antibody to platelets and elevated platelet-associated immunoglobulin G in two patients, and abundant bone marrow megakaryocytes in all patients. The patients had a satisfactory response to corticosteroid therapy, and platelet counts have remained normal during observation after therapy. All patients had serum antibody to human T-lymphotropic virus type III, and HTLV-III was isolated from the peripheral blood lymphocytes in two patients. The HTLV-III infections were presumably acquired via blood transfusions in the neonatal period; none of the patients' mothers belonged to a risk group for HTLV-III infection, and all were HTLV-III seronegative. Although thrombocytopenia was the major clinical manifestation, the patients had a number of immunologic abnormalities characteristic of HTLV-III infection; these included hyperimmunoglobulinemia, a decreased proportion of peripheral blood T cells, and a marked reduction in the proportion of peripheral blood T helper-inducer lymphocytes. We conclude that the patients had immune-mediated thrombocytopenia caused by HTLV-III infection.

Acquired Immunodeficiency Syndrome↗
PubMed1986

The chemokinetic response of human neutrophils.

A computer-assisted single cell assay that allows quantification of the locomotive behavior of individual cells and a flow-through system that allows study of response of individual cells to stimulation were utilized to study the chemokinetic response of neutrophils. The range of basal mean rate of locomotion (mROL) and chemokinetic response to 10(-9) mol/L formylmethionyl leucyl phenylalanine (FMLP) was determined for neutrophils of eight normal adults. The basal mROL was 8.2 +/- 1.5 um/min and 6.2 +/- 1.0 um/min; the rate after 10(-9) mol/L fMLP was 12.1 +/- 2.1 and 9.5 +/- 1.8 um/min in 2.0 g% and 0.05 g% HSA, respectively. The mean increase in ROL for neutrophils was 50%. Assay with the flow-through system shows that the chemokinetic response--increase in mROL of a population of neutrophils in response to 10(-9) mol/L--is due to an increase in ROL when cells are actively moving and not due to a decrease in the amount of time the cell spends inactive. Studies of individual cells within the populations show that chemokinetic response to 10(-9) mol/L fMLP is highly variable. The majority of cells (77%) respond with an increase in ROL; the minority (23%) are nonresponders that characteristically move at ROL greater than or equal to 14 um/min prior to stimulation and do not change ROL or exhibit a net decline in ROL in response to 10(-9) mol/L fMLP. The dose response of a population of neutrophils and of individual neutrophils to serial addition of 10(-10) to 10(-6) mol/L fMLP shows that the fMLP dose dependence for maximal chemokinetic response is highly variable among individual cells. Seventeen percent of cells do not respond to any fMLP concentration; 25% of neutrophils exhibit maximal response to 10(-10) mol/L fMLP, while 50% and 25% of cells showed peak chemokinetic response to 10(-9) mol/L and greater than or equal to 10(-8) mol/L fMLP, respectively. These studies document the variability in the locomotive responses of peripheral blood neutrophils. Understanding the causes of variability in the chemokinetic responsiveness of individual neutrophils may improve our understanding of how the cellular inflammatory response in man can be modulated.

Adult↗
PubMed1985

A method for quantifying F-actin in chemotactic peptide activated neutrophils: study of the effect of tBOC peptide.

The studies presented here characterize a simple, quantitative NBDphallacidin extraction assay for determining the F-actin content of fMLP-activated neutrophils. The NBDphallacidin extraction assay is based upon the specificity of NBDphallacidin binding to F-actin and the solubility of NBDphallacidin in methanol. Cells are fixed, permeabilized, and stained with NBDphallacidin; the cells are then pelleted, the bound NBDphallacidin is extracted into methanol, and the RFI (excite 465; emit 535) of the solution is determined. Binding of NBDphallacidin to neutrophils is saturable and 90% of bound NBDphallacidin is displaced by nonfluorescent phalloidin. The extraction of bound NBDphallacidin into methanol is complete and the excitation/emission characteristics of NBDphallacidin are not altered by extraction. The assay is relatively inexpensive, applicable to the study of cells in suspension or on substratum, allows kinetic studies with 5-10s time resolution, and is not affected by the shape of the cell or the distribution of the probe. We used the NBDphallacidin extraction assay to study the kinetics of fMLP-induced change in the F-actin content of neutrophils and the effect of tBOC peptide, an inhibitor of fMLP binding, on these changes. The extraction assay reveals a rapid, sequential fMLP-induced increase followed by a decrease in F-actin content. The tBOC peptide inhibits fMLP-induced actin polymerization. Addition of tBOC during fMLP-induced polymerization or at times when F-actin content is maximal enhances F-actin depolymerization. The rate of F-actin depolymerization is greater than or equal to fourfold faster in the presence than in the absence of tBOC. The results show that The NBDphallacidin extraction assay is useful for studying the kinetics of change in F-actin content of nonmuscle cells; fMLP receptor occupancy is required for fMLP-dependent polymerization but not depolymerization; and both the actin polymerizing and depolymerizing processes are active in the cell within 5 s after fMLP stimulation. Implications of these observations for understanding the observed increase and, then, decrease in F-actin content of fMLP-activated cells are discussed.

Actins↗
PubMed1985

The kinetics of chemotactic peptide-induced change in F-actin content, F-actin distribution, and the shape of neutrophils.

Formyl-met-leu-phe (fMLP) induces actin assembly in neutrophils; the resultant increase in F-actin content correlates with an increase in the rate of cellular locomotion at fMLP concentrations less than or equal to 10(-8) M (Howard, T.H., and W.H. Meyer, 1984, J. Cell Biol., 98:1265-1271). We studied the time course of change in F-actin content, F-actin distribution, and cell shape after fMLP stimulation. F-actin content was quantified by fluorescence activated cell sorter analysis of nitrobenzoxadiazole-phallacidin-stained cells (Howard, T.H., 1982, J. Cell Biol., 95(2, Pt. 2:327a). F-actin distribution and cell shape were determined by analysis of fluorescence photomicrographs of nitrobenzoxadiazole-phallacidin-stained cells. After fMLP stimulation at 25 degrees C, there is a rapid actin polymerization that is maximal (up to 2.0 times the control level) at 45 s; subsequently, the F-actin depolymerizes to an intermediate F-actin content 5-10 min after stimulation. The depolymerization of F-actin reflects a true decrease in F-actin content since the quantity of probe extractable from cells also decreases between 45 s and 10 min. The rate of actin polymerization (3.8 +/- 0.3-4.4 +/- 0.6% increase in F-actin/s) is the same for 10(-10) - 10(-6) M fMLP and the polymerization is inhibited by cytochalasin D. The initial rate of F-actin depolymerization (6.0 +/- 1.0-30 +/- 5% decrease in F-actin/min) is inversely proportional to fMLP dose. The F-actin content of stimulated cells at 45 s and 10 min is greater than control levels and varies directly with fMLP dose. F-actin distribution and cell shape also vary as a function of time after stimulation. 45 s after stimulation the cells are rounded and F-actin is diffusely distributed; 10 min after stimulation the cell is polarized and F-actin is focally distributed. These results indicate that actin polymerization and depolymerization follow fMLP stimulation in sequence, the rate of depolymerization and the maximum and steady state F-actin content but not the rate of polymerization are fMLP dose dependent, and concurrent with F-actin depolymerization, F-actin is redistributed and the cell changes shape.

Actins↗
PubMed1985

Isolation of bluetongue virus from bull semen.

The efficacy of inoculation of Vero cell cultures or intravenous inoculation of chicken embryos in the isolation and titration of seminal bluetongue virus (BTV) was studied, as was the toxicity of bull semen for these 2 isolation systems. Frozen and thawed BTV-contaminated ejaculates collected during periods of viremia from 2 bulls experimentally infected with cell culture-adapted BTV serotype 17 were used in isolation, titration and fractionation studies. Blood collected from the 2 bulls concurrently with the semen was titrated in chicken embryos. Bull semen was toxic for both isolation systems. Toxicity was associated with both the spermatozoa and seminal plasma. Dilution of the semen at least 1:25, addition of peptone or tryptose broth to the diluent, limitation of adsorption time and postinoculation washing of cell culture monolayers all reduced the destructive effects of semen. Isolation of BTV was successful from 11 ejaculates and was titratable in 9 of these. Blind passage of surviving embryos or cell cultures at the endpoints of the titrations produced BTV isolations in 4 instances. The virus was never isolated from semen in the absence of concurrent viremia. Peak seminal BTV titers of 10(5.5) CEIVLD50/ml and 10(5.7) TCID50/ml were observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Bluetongue virus and embryo transfer in cattle.

Embryos recovered nonsurgically from donor cattle infected with bluetongue virus (BTV) were transferred to seronegative, uninfected recipients. High titers of BTV were found in donor blood at the time of embryo recovery and BTV could be isolated from the recovery medium obtained from many of the donors. Bluetongue virus was not isolated from the blood of any of the 39 recipients that received embryos from infected donors, and none of these recipients seroconverted within 60 days of transfer. There was no evidence that BTV was transmitted to the early embryo in viremic donors. These results indicate that, under standard conditions of embryo transfer, transmission of BTV from infected donors to recipients is unlikely to occur.

Animals↗
PubMed1985

Seminal shedding of bluetongue virus in experimentally infected bulls.

Twenty bulls were inoculated with bluetongue virus (BTV) to study the frequency, duration and pathogenesis of seminal shedding of the virus. BTV was isolated from 19 of 181 ejaculates collected from these bulls during the period of viremia, but from none of 79 ejaculates collected after the viremia. Three categories of bulls were recognized on the basis of frequency and duration of seminal shedding of BTV. Virus was never isolated from semen in a majority (13 of 20) of bulls. In another group (5 of 20), virus was isolated from semen in low titer and from only 1 to 3 ejaculates. However, 2 bulls did shed relatively large quantities of virus in semen over a period of 2 to 3 weeks. Seminal shedding of BTV in infected bulls closely followed peak virus titers in blood and in no case was virus isolated from semen without its concurrent isolation from blood. Semen was processed and frozen from 1 bull that shed BTV. Six of 9 heifers inseminated at estrus with this semen became pregnant, and 3 of 9 became viremic and seroconverted. An additional heifer developed antibody to the virus, but did not become detectably viremic. No evidence of fetal infection was found in the heifer that became pregnant and infected, or in 5 heifers that became pregnant, but did not became infected.

Animals↗
PubMed1984

Chemotactic peptide modulation of actin assembly and locomotion in neutrophils.

To determine the relationship between the state of actin polymerization in neutrophils and the formyl-methionyl-leucyl-phenylalanine (fMLP)-induced changes in the locomotive behavior of neutrophils, the mean rate of locomotion (mROL), the percent G-actin, and the relative F-actin content of neutrophils were determined. The mROL was quantified by analysis of the locomotion of individual cells; the percentage of total actin as G-actin was measured by DNase I inhibition; and the F-actin was determined by fluorescence-activated cell sorter (FACS) analysis of nitrobenzoxadiazol (NBD)-phallacidin-stained neutrophils. Neutrophils stimulated with fMLP exhibit a change in their mROL that is biphasic and dose dependent. The mROL of neutrophils exposed to 10(-8) M fMLP, the KD, is 11.9 +/- 2.0 micron/min (baseline control 6.2 +/- 1.0 micron/min). At 10(-6) M fMLP, the mROL returns to baseline levels. Stimulation of neutrophils with fMLP also induces action polymerization. Evidence for actin polymerization includes a 26.5% reduction in G-actin and a twofold increase in the amount of NBD-phallacidin staining of cells as determined by FACS analysis. The NBD-phallacidin staining is not due to phagocytosis, is inhibited by phalloidin, requires cell permeabilization, and is saturable at NBD-phallacidin concentrations greater than 10(-7)M. The fMLP-induced increase in NBD-phallacidin staining occurs rapidly (less than 2 min), is temperature dependent, and is not due to cell aggregation. Since NBD-phallacidin binds specifically to F-actin, the increase in fluorescent staining of cells likely reflects an increase in the F-actin content of fMLP-stimulated cells. FACS analysis of NBD-phallacidin-stained cells shows that the relative F-actin content of neutrophils stimulated with 10(-11)-10(-8)M fMLP increases twofold and remains increased at concentrations greater than 10(-8)M fMLP. Therefore, the fMLP-induced increase in F-actin content of neutrophils as determined by FACS analysis of NBD-phallacidin-stained cells coincides with a decrease in G-actin and correlates with increased mROL of neutrophils under some (10(-11)-10(-8)M fMLP) but not all (greater than 10(-8)M fMLP) conditions of stimulation. Quantification of the F-actin content of nonmuscle cells by FACS analysis of NBD-phallacidin-stained cells may allow rapid assessment of the state of actin polymerization and correlation of that state with the motile behavior of nonmuscle cells.

Actins↗
PubMed1983

Embryo transfer from cattle infected with bluetongue virus.

Embryos recovered nonsurgically from donor cattle during the peak of bluetongue viremia were surgically transferred to seronegative recipients 7 to 8 or 10 to 11 days after the onset of donor estrus. Virus was isolated from the uterine flushing medium recovered from 11 of the 20 donors. Bluetongue virus was not isolated from the blood of any of 39 recipients, nor did any recipient seroconvert to the virus following transfer. The number of recipients that became pregnant after transfer of embryos from infected donors (21 of 39) was not significantly different from contemporary controls. Virus antigen was not detected by immunofluorescence in any of 63 embryos and oocytes recovered from viremic donors. These results indicate that under standard embryo transfer conditions, transmission of bluetongue virus from infected donors to uninfected recipients is unlikely to occur.

Animals↗
PubMed1983

Seminal shedding of bluetongue virus in experimentally infected mature bulls.

Sixteen bulls ranging in age from 2 to 11 years were experimentally inoculated with bluetongue virus to investigate the frequency and duration of seminal shedding of the virus. All bulls developed typical viremia, lasting 21 to 58 days, and they seroconverted 2 to 3 weeks after inoculation. Virus isolation was attempted from a total of 232 ejaculates, 163 (70%) of which were collected during the period of viremia. Bluetongue virus was not isolated from any of the ejaculates collected from 11 of the 16 bulls. Virus was isolated from 9 of 52 ejaculates collected from the other 5 bulls during the period of viremia. In no instance was virus isolated from semen without concurrent isolation from blood.

Animals↗
PubMed1983

Changes in actin content during induced myeloid maturation of human promyelocytes.

Actin is an important cytoskeletal protein; new actin synthesis occurs during differentiation of many motile cells. To better understand the process of myeloid maturation, the change in actin content during induced maturation of HL-60 human promyelocytic leukemia cells was studied. HL-60 cells induced toward myeloid maturation by a 5-day exposure to dimethylformamide showed an 86% increase in a 43,000 mol wt protein comigrating with rabbit muscle actin on dodecyl sulfate polyacrylamide gels. To further demonstrate that this was an increase in actin content, the total actin content of lysed HL-60 cells was measured by the ability of actin to inhibit DNAase I. Using this assay, actin content of HL-60 cells increased 96% during induced differentiation. The amount of incorporation of 3H-leucine into actin doubled after a 5-day exposure to dimethylformamide, suggesting the increase in actin was due primarily to new synthesis. Total new protein synthesis increased 2-7-fold during differentiation. Additional analysis of polyacrylamide gels showed increased quantities and new synthesis of a high molecular weight protein comigrating with rabbit muscle myosin. This study shows that actin content increases during myeloid maturation. It also demonstrates that the HL-60 cell line is a useful model to study both functional and biochemical events during human myeloid differentiation.

Actins↗
PubMed1982

Antibiotic control of Campylobacter fetus by three extenders of bovine semen.

Campylobacter fetus were added to raw semen in two factorial experiments that evaluated seminal extender, initial antibiotic treatment of raw semen, and inclusion of antibiotics in the extender. Extenders were conventional egg yolk citrate, complete egg yolk Tes-Tris, and complete egg yolk Tris. Raw semen was treated with 2000 micrograms streptomycin sulfate and 1000 units of polymyxin B sulfate per milliliter. Antibiotic-treated extender contained 500 units of polymyxin B sulfate, 1000 micrograms streptomycin sulfate, and 500 units of potassium penicillin G per milliliter. Aliquots of extended semen were cultured immediately prior to freezing in .5-ml French straws and after thawing. Treatment of raw semen or extender alone with antibiotics was not consistently effective in controlling Campylobacter fetus. The dual treatment of raw semen and incorporation of antibiotics into the extender were effective with egg yolk citrate and egg yolk Tes-Tris. However, Campylobacter fetus survived in semen that had been treated with antibiotics and then extended with egg yolk Tris containing Antibiotics. Thus, the procedure required by Certified Semen Services for antibiotic treatment of bovine semen was ineffective in controlling Campylobacter fetus in semen extended in this complete egg yolk Tris. Efficacy should be demonstrated, rather than assumed, for each procedure for extending and processing bovine semen.

Animals↗
PubMed1982

Interaction of bluetongue virus with preimplantation embryos from mice and cattle.

Preimplantation embryos from mice and cattle were exposed to bluetongue virus in vitro to determine whether the virus would replicate in these early embryos and, if so, what pathologic consequences would ensue. A high proportion of zona pellucida-free, 2-cell embryos and morulae from mice, and morulae from cattle became infected. The infection was rapidly cytopathic in embryos from both species. Indirect immunofluorescence was used to demonstrate accumulation of virus antigen in the blastomeres of these embryos. The zona pellucida of both murine and bovine embryos provided effective protection from virus present in culture fluid.

Animals↗
PubMed1982

Quantification of the locomotive behavior of polymorphonuclear leukocytes in clot preparations.

Time-lapse videotape recordings of polymorphonuclear leukocytes (PMNs) from clot preparations were used to quantify the locomotive behavior of individual PMNs from normal subjects. Tracings derived from the videotapes allow one to quantify multiple parameters of the locomotive behavior of PMNs--direction, distance, rate, and angle of turn. The results obtained are reproducible from subject-to-subject and from preparation-to-preparation. The method allows the investigator to record the locomotive behavior of 100 cells simultaneously within a 5-min period and analyze the recording as time permits. We utilized this technique to compare the locomotive behavior of slow and fast PMNs (arbitrarily defined as cells that move less than or equal to 7.0 micrometer/min and greater than 7.0 micrometer/min mean rate of locomotion, respectively). The studies show that slow and fast PMNs, thus defined, differ not only in mean rate of locomotion but also in their rate of locomotion during periods of active locomotion, in the number of periods of inactivity/PMN/5 min (slow = 1.65 +/- 0.31; fast = 0.36 +/- 0.12), and in their turning behavior as measured by angle of turn (slow = 92 degrees +/- 39 degrees; fast = 39 degrees +/- 35 degrees). These results show that human PMNs from clot preparations are remarkably heterogeneous in their locomotive behavior, and the results suggest this heterogeneity is due to endogenous differences within cells.

Adult↗
PubMed1981

Correlation of the biologic effects and binding of cytochalasins to human polymorphonuclear leukocytes.

Treatment of human PMNs with cytochalasins (CE, CD, CB, and H2CB) results in alteration of cell morphology and inhibition of cell motility. Morphological changes are similar to those reported for nonamoeboid fibroblasts--rounding, zeiosis, and arborization. Mean cell velocity of PMNs, as measured by quantitative analysis of time-lapse videotape recordings, was reduced to 0.1 micron/min (control, 7.3 +/- 4.2 micron/min). Phagocytosis by PMNs, as measured by phagocytosis of latex beads, was inhibited by 75%. The relative potency of the cytochalasins for inducing morphological change or for inhibiting locomotion and phagocytosis is similar to their relative potencies for affecting non-amoeboid cells: CE greater than CD greater than CB greater than or equal to H2CB. Quantitative binding of 3H-CB to purified PMNs under equilibrium conditions reveal two types of specific CB binding sites: high-affinity sites (KD approximately 3 x 10(-7) M, 3 x 10(6) sites/cell) and low affinity sites (KD approximately 2 x 10(-6) M). The relative affinities of the cytochalasins for the high-affinity and low-affinity CB binding sites parallel their relative potencies for inducing biologic effects (i.e. CE greater than CD greater than CB greater than or equal to H2CB).

Cell Movement↗