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D English

Publications and source records attributed to D English.

At least 55 records · Page 3Linked to original sources
PubMed1999

Nitric oxide reduces the sequestration of polymorphonuclear leukocytes in lung by changing deformability and CD18 expression.

Nitric oxide (NO) influences polymorphonuclear leukocytes (PMN)-endothelial cell interactions. The aim of this study was to evaluate this effect in the lung and investigate this mechanism. PMN sequestration in the lung was evaluated in vivo after the infusion of complement fragments. Rabbits (n = 9) that inhaled 40 ppm of NO were compared with control rabbits (n = 9) over a 2-h period following infusion of complement fragments. Circulating PMN counts immediately decreased after infusion of complement fragments in both groups followed by a recovery to baseline. This recovery was maintained in the NO-treated group compared with the control rabbits (p < 0.05). NO reduced PMN sequestration in the lung measured by both arteriovenous PMN difference across the lung (p < 0.01) and the myeloperoxidase (MPO) content of the lung tissue (p < 0.01). NO had no effect on the complement fragments-induced PMN release from the bone marrow. In vitro studies showed that NO partially inhibited F-actin assembly (p < 0.01) reduced the change in deformability (p < 0.05) and inhibited CD18 upregulation (p < 0.05) but had no effect on the L-selectin shedding of PMN stimulated by complement fragments. We conclude that NO reduces the sequestration of activated PMN by reducing deformability change via inhibition of F-actin assembly and inhibiting the upregulation of CD18.

Actins↗
PubMed1999

Cigarette smoking causes sequestration of polymorphonuclear leukocytes released from the bone marrow in lung microvessels.

Studies from our laboratory have shown that chronic cigarette smoke exposure causes a neutrophilia associated with a shortening of the mean transit time of polymorphonuclear leukocytes (PMN) though the postmitotic pool of the marrow. The present study was designed to test the hypothesis that PMN newly released from bone marrow by smoke exposure preferentially sequestered in pulmonary microvessels. The thymidine analogue 5'-bromo-2'-deoxyuridine (BrdU) was used to label dividing PMN in the marrow of rabbits; their appearance in the circulation was measured using immunocytochemistry, and their sequestration in lung tissue was determined using standard morphometric techniques. Animals exposed to 11 d of cigarette smoke (n = 6) compared with sham-exposed control animals (n = 4) showed no increase in circulating PMN counts but showed an increase in both the percentage of band cells (smoking, 9.8 +/- 1.1% versus control, 5.5 +/- 0.9%; P < 0.05) and BrdU-labeled PMN (PMNBrdU) in the circulation (smoking, 10.8 +/- 0.6% versus control, 7.5 +/- 0.3%; P < 0.05). There were more PMN sequestered in the lungs of smoke-exposed animals (51.7 +/- 3.4 x 10(7)/ml tissue) than in those of control animals (25.1 +/- 1.8 x 10(7)/ ml tissue) (P < 0.05) and a higher percentage of these cells were PMNBrdU (smoking, 16.9 +/- 2. 3% versus control, 9.6 +/- 0.4%; P < 0.05). The percentage of PMNBrdU in the gravity-independent regions (11.7 +/- 1.9%) of the lung was higher than gravity-dependent regions (7.8 +/- 1.8%) in the smoke-exposure group (P < 0.05). Transmission electron microscopy showed pulmonary capillary endothelial damage with adherent PMN in the smoke-exposure group. We conclude that younger PMN released from the bone marrow by cigarette smoking preferentially sequestered in pulmonary microvessels and speculate that these PMN may contribute to the alveolar wall damage associated with smoke-induced lung emphysema.

Animals↗
PubMed1999

Bax and apoptosis in acute and chronic rejection of rat cardiac allografts.

Apoptosis is associated with acute rejection, transplant vascular disease, and the "Quilty effect" in cardiac allografts. However, the causality and mechanisms of apoptosis in the pathogenesis of vascular injury are poorly understood. In the current study, the Lewis-to-F344 rat cardiac allograft model was utilized as a means to immunohistochemically evaluate the expression of Bax, Bcl-2, and factor VIII-related antigen in transplant vascular disease. Apoptosis was detected by in situ labeling of fragmented DNA using in situ terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) in native hearts and grafted hearts of allogeneic and syngeneic recipients. Bax immunostaining was detected in 50% of endothelial cells, in 60% of infiltrating leukocytes associated with acute rejection in the myocardium, and in certain other parenchymal cells, considering all cardiac allografts. More than 75% of infiltrating leukocytes in the intima of vessel walls immunostained positive for Bax. Bcl-2 immunopositivity was not detected in native hearts, allo allo-, or syngrafts. On Days 2, 4, 7, and 14 after transplantation, TUNEL positivity was detected in only about 1% of leukocytes in the interstitial infiltrates, despite the fact that rather severe rejection was observed in Day 14 allografts. The number of apoptotic leukocytes increased significantly by Days 28 and 56 after transplantation, although the severity of histopathological rejection did not increase as compared with Day 14. The apoptotic leukocytes remained isolated or in small clusters, mainly perivascular. TUNEL positivity colocalized with Bax expression in these cells. TUNEL staining was also observed in certain parenchymal cells in the interstitium and in randomly distributed inflammatory cells in vessel walls. TUNEL positivity was detected in rare luminal endothelial cells in transverse sections of vessel walls (about 10% of cells in <1/10 of the vessels studied). Nuclear TUNEL positivity was observed in Bax-negative cardiomyocytes in ischemically damaged areas of myocardium in both allografts and syngrafts. In summary, increased expression of Bax was observed in rat cardiac allografts. The colocalization of TUNEL and Bax suggests that endothelial cell injury and infiltrating leukocyte apoptosis may be regulated in part by the apoptosis-promoting protein, Bax. In the current model, myocyte death due to ischemia and surgical injury in syngrafts and allografts does not seem to involve Bax.

Animals↗
PubMed1998

Release of polymorphonuclear leukocytes from the bone marrow by interleukin-8.

Several studies have shown that interleukin-8 (IL-8) causes a rapid granulocytosis with the release of polymorphonuclear leukocytes (PMN) from the bone marrow (BM) partially responsible for the granulocytosis. This study was designed to quantitate the release of PMN from the BM by IL-8 and measure the transit time of PMN through the marrow after IL-8 administration. The thymidine analogue, 5'-bromo-2'-deoxyuridine (BrdU), was used to label dividing PMN in the marrow and follow their release into the circulation after intravenous IL-8. This allowed us to calculate the transit time of PMN through the mitotic and postmitotic pools of BM. BrdU was infused intravenously into rabbits 24 hours before IL-8 (2.5 microg/kg). IL-8 caused a rapid, transient granulocytopenia (5.9 +/- 0.4 at baseline v 0.2 +/- 0.06 x 10/9L at 5 minutes, P < .05) followed by granulocytosis (8.4 +/- 0.1 at 30 minutes, P < .05) associated with an increased number (0.3 +/- 0.1 at baseline v 1.2 +/- 0.6 x 10(9)/L at 30 minutes, P < .05) and percentage of band cells (P < .05), as well as a rapid increase in the number of BrdU-labeled PMN (PMNBrdU) in the circulation (0.09 +/- 0.05 at baseline to 1.5 +/- 0.6 x 10(9)/L at 60 minutes, P < .05). The transit time of PMN through both the mitotic and postmitotic pools of BM was not affected by IL-8. To determine the marrow compartment from which the PMN were mobilized by IL-8, we quantitated PMN movement from the hematopoietic and sinusoidal compartments into the circulation. The fraction of PMNBrdU in both compartments was higher than in the circulating blood (P < .05) and the fraction and number of PMNBrdU in the sinusoids decreased with IL-8 treatment (P < .05). We conclude that the pool of PMN residing in the BM venous sinusoids are rapidly released into the circulation after administration of IL-8.

Animals↗
PubMed1998

Fundamental cryobiology of human hematopoietic progenitor cells. I: Osmotic characteristics and volume distribution.

While methods for the cryopreservation of hematopoietic stem cells are well established, new sources of progenitor cells, such as umbilical cord blood, fetal tissue, and ex vivo expanded progenitor cells, may require refined protocols to achieve optimal recovery after freezing. To predict optimal protocols for cryopreservation of human hematopoietic progenitors, knowledge of fundamental cryobiological characteristics including cell osmotic characteristics, water and cryoprotectant permeability coefficients of cell membrane, and activation energies of these coefficients is required. In this study, we used CD34+CD33- cells isolated from human bone marrow as hematopoietic progenitor cell models/representatives to study the osmotic characteristics of the progenitor cells. Volume distribution and osmotic behavior of the CD34+CD33- cells were determined using two different methods: (a) a shape-independent electronic sizing technique and (b) a shape-dependent optical image analysis. The cell diameter was measured to be 8.2 +/- 1.1 microns (mean +/- SD, n = 1,091,475, the number of donors = 8) using the electronic sizing technique or 8.7 +/- 1.2 microns (mean +/- SD, n = 1508, the number of donors = 6) by image analysis at initial (isotonic) osmolality, 325 mosm/kg. The cell volume change was measured after the cells were exposed and equilibrated to different anisosmotic conditions. The cell volume was found to be a linear function of the reciprocal of the extracellular osmolality (Boyle van't Hoff plot) ranging from 163 to 1505 mosm/kg. The volume fraction of intracellular water which is osmotically active was determined to be 79.5% of the cell volume. It was concluded that human CD34+CD33- cells osmotically behave as ideal osmometers. This information coupled with cell water and cryoprotectant permeability coefficients as well as their activation energies (to be determined in the ongoing research projects) will be used to design optimum conditions for cryopreservation of human hematopoietic progenitor cells.

Antigens, CD↗
PubMed1998

Telemedicine evaluation of cutaneous diseases: a blinded comparative study.

BACKGROUND: Numerous telemedicine programs have been created in the United States, but studies documenting the fidelity and effectiveness of telemedicine for evaluation of skin diseases are lacking. OBJECTIVE: We attempted to determine the percentage of encounters in which two different dermatologists, one using telemedicine and one on-site, could independently arrive at the same primary diagnosis. METHODS: Two clinical telemedicine sites linked through the Georgia Statewide Telemedicine Program were used in this study of 60 patients with skin problems. One dermatologist evaluated the patients on telemedicine (interactive television) and a second then took the patients into a separate examination room and evaluated them on-site. Each investigator recorded their diagnoses with no discussion with each other. As a control group, the investigators independently and in a blinded fashion (to each other's diagnoses) recorded diagnoses for a group of patients from a third dermatologist's clinic. Raw data were evaluated and classified by this third dermatologist who assigned diagnoses to categories of complete agreement, partial agreement, or disagreement. RESULTS: There were no significant differences with regard to disagreement. However, there was a higher probability of complete agreement between the two dermatologists when each examined the patient on-site and in person than when one evaluated the patient on telemedicine and one examined the patient on-site and in person. CONCLUSION: Our results suggest that telemedicine is an effective means of diagnosing cutaneous diseases. However, because partial interobserver agreement on diagnoses was greater for the telemedicine group than for the control group (p < 0.05), it is likely that optimum use of medical assistants at the remote site will be necessary to increase the likelihood of complete agreement on diagnoses among dermatologists using interactive television.

Dermatology↗
PubMed1998

Bacteremic pneumococcal pneumonia: bone marrow release and pulmonary sequestration of neutrophils.

OBJECTIVES: Neutrophils have been implicated in the pathogenesis of acute lung injury in bacteremic pneumococcal pneumonia. The characteristics of the population of neutrophils that injure the lung are still not known. This study was designed to compare the bone marrow release and lung sequestration of neutrophils during bacteremic pneumococcal pneumonia with nonbacteremic pneumonia and isolated bacteremia. DESIGN: Prospective, controlled, experimental study. SETTING: University research laboratory. SUBJECTS: Female New Zealand white rabbits (n = 17; weight 2.3 to 2.7 kg). INTERVENTIONS: The rabbits were pretreated with intravenous 5'-bromo-2-deoxyuridine (BrdU 100 mg/kg i.v.) to pulse label dividing neutrophils in the bone marrow. Twenty hours after the treatment with BrdU, the rabbits were anesthetized and pneumonia was induced by instilling Streptococcus pneumoniae (1.5 x 10(9) organisms) into the lower lobe of the lung. Four hours after pneumonia, bacteremia was induced by infusing S. pneumoniae (3.0 x 10(9) organisms) into the circulation (pneumonia + bacteremia: n = 6). These animals were compared with those with just pneumonia (n = 5) or bacteremia (n = 6). MEASUREMENTS AND MAIN RESULTS: White blood cell, neutrophil, and differential count. BrdU-labeled neutrophils (neutrophilBrdU) were identified using immunohistochemistry. Cells in tissues were examined microscopically, using sequential level stereologic analysis. The pneumonia + bacteremia group developed a leukopenia (7.3 +/- 0.7 to 2.4 +/- 0.2 x 10(9)/L) following the bacteremia that was associated with an increase in circulating band cells and neutrophilBrdU (2.3 +/- 0.8% to 33.5 +/- 2.8%) which were both higher than those in the other groups (p < .005). Bone marrow smears showed accelerated maturation of neutrophils in the pneumonia + bacteremia group (neutrophilBrdU increased from 11.6 +/- 1.0 to 45.3 +/- 2.1%). Morphometric studies of the lung showed increased neutrophil sequestration in the untreated lung tissue of the pneumonia + bacteremia group (16 +/- 0.8 x 10(8)/mL tissue) compared with the pneumonia (6.6 +/- 0.3 x 10(8)/mL tissue) and bacteremia (12 +/- 0.6 x 10(8)/mL tissue) groups (p < .0001). NeutrophilBrdU preferentially sequester in the lungs of all groups but were slow to migrate into the alveolar air spaces (p < .05). CONCLUSIONS: During bacteremic pneumococcal pneumonia there is an accelerated maturation of neutrophils in the bone marrow with an enhanced release of neutrophils into the circulation. These newly released neutrophils preferentially sequester in lung microvessels but are slow to migrate into the alveolar air space.

Animals↗
PubMed1998

Pulmonary sequestration of polymorphonuclear leukocytes released from bone marrow in bacteremic infection.

We examined the bone marrow response and the sequestration of polymorphonuclear leukocytes (PMNs) in lung using a bacteremic infection model in rabbits. PMNs were labeled with the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) in the bone marrow, and the bone marrow release and the sequestration of BrdU-labeled PMNs were measured using immunohistochemistry. A focal subcutaneous infection (S) was induced, and the bacteremia (B) was produced 4 h later with Streptococcus pneumoniae (S+B). This S+B group was compared with other groups with only subcutaneous infection or only bacteremia. The S+B group developed a profound leukopenia after the bacteremia that was associated with an increase in circulating BrdU-labeled PMNs. Morphometric studies showed more PMN sequestration in the lung of the S+B group compared with the others (P < 0.05). Compared with unlabeled PMNs, BrdU-labeled PMNs, which represent newly released PMNs, preferentially sequestered in lung (P < 0.05) and were slow to migrate into the infected tissues (P < 0.05). We conclude that bacteremic infection is associated with an accelerated release of PMNs from the bone marrow and that these newly released PMNs preferentially sequester in lung and are slow to migrate into infected tissues.

Analysis of Variance↗
PubMed1998

Adherent neutrophils activate endothelial myosin light chain kinase: role in transendothelial migration.

Increased vascular endothelial cell (EC) permeability and neutrophilic leukocyte (PMN) diapedesis through paracellular gaps are cardinal features of acute inflammation. Activation of the EC contractile apparatus is necessary and sufficient to increase vascular permeability in specific models of EC barrier dysfunction. However, it is unknown whether EC contraction with subsequent paracellular gap formation is required for PMN transendothelial migration in response to chemotactic factors. To test this possibility, we assessed migration of human PMNs across confluent bovine pulmonary arterial EC monolayers. Transendothelial PMN migration in the absence of a chemotactic gradient was minimal, whereas abluminal addition of leukotriene B4 (LTB4; 5 microM) resulted in significantly increased PMN migration. Reductions in EC myosin light chain kinase (MLCK) activity by EC monolayer pretreatment with specific MLCK inhibitors (KT-5926 or ML-7) or by increases in cAMP-protein kinase A activity (cholera toxin) significantly reduced PMN transmigration (30-70% inhibition). In contrast, pretreatment with the myosin-associated phosphatase inhibitor calyculin resulted in the accumulation of phosphorylated myosin light chains, EC contraction, and significantly enhanced PMN migration. Finally, the interaction of PMNs with 32P-labeled EC monolayers was shown to directly increase EC myosin phosphorylation in a time-dependent fashion. Taken together, these results are consistent with the hypothesis that the phosphorylation status of EC myosin regulates PMN migration and further indicate that EC MLCK is activated by chemoattractant-stimulated PMNs. Neutrophil-dependent activation of the EC contractile apparatus with subsequent paracellular gap formation may be a key determinant of transendothelial PMN migration responses to chemotactic agents.

Alkaloids↗
PubMed1998

Children who prosper in unfavorable environments: the relationship to social capital.

OBJECTIVE: Social capital describes the benefits that are derived from personal social relationships (within families and communities) and social affiliations. This investigation examined the extent to which social capital is associated with positive developmental and behavioral outcomes in high-risk preschool children. DESIGN: A cross-sectional case-control analysis of young children "doing well" and "not doing well" at baseline in four coordinated longitudinal studies. PARTICIPANTS: A total of 667 2- to 5-year-old children (mean age, 4.4 years) and their maternal caregivers who are participating in the Longitudinal Studies of Child Abuse and Neglect Consortium. At recruitment, all children were characterized by unfavorable social or economic circumstances that contributed to the identification of the children as high risk. MEASURES: Social capital was defined as benefits that accrue from social relationships within communities and families. A social capital index was created by assigning one point to each of the following indicators: 1) two parents or parent-figures in the home; 2) social support of the maternal caregiver; 3) no more than two children in the family; 4) neighborhood support; and 5) regular church attendance. Outcomes were measured with the Child Behavior Checklist, a widely used measure of behavioral/emotional problems, and with the Battelle Developmental Inventory Screening Test, a standardized test that identifies developmental deficits. Children were classified as doing well if their scores on these instruments indicated neither behavioral nor developmental problems. RESULTS: Only 13% of the children were classified as doing well. The individual indicators that best discriminated between levels of child functioning were the most direct measures of social capital-church affiliation, perception of personal social support, and support within the neighborhood. The social capital index was strongly associated with child well-being, more so than any single indicator. The presence of any social capital indicator increased the odds of doing well by 29%; adding any two increased the odds of doing well by 66%. CONCLUSIONS: Our findings suggest that social capital may have an impact on children's well-being as early as the preschool years. In these years it seems to be the parents' social capital that confers benefits on their offspring, just as children benefit from their parents' financial and human capital. Social capital may be most crucial for families who have fewer financial and educational resources. Our findings suggest that those interested in the healthy development of children, particularly children most at risk for poor developmental outcomes, must search for new and creative ways of supporting interpersonal relationships and strengthening the communities in which families carry out the daily activities of their lives.

Case-Control Studies↗
PubMed1997

Phosphatidic acid elicits calcium mobilization and actin polymerization through a tyrosine kinase-dependent process in human neutrophils: a mechanism for induction of chemotaxis.

Phospholipids mediate important effects as extracellular messengers in diverse biological systems. We investigated the effects of phosphatidic acid, a biologically active phospholipid potentially involved in the inflammatory process, on calcium mobilization and actin polymerization in human neutrophils and correlated these effects with induction of chemotactic migration. Intermediate-chain length phosphatidic acid (DiC10-PA) induced a biphasic increase in intracellular Ca2+ characterized by a rapid rise commencing immediately upon addition of stimulus followed by a secondary increase which, unlike the initial response, was eliminated by chelation of extracellular Ca2+. Neither of these responses were induced by C10-lysophosphatidic acid or diacylglycerol. The tyrosine kinase inhibitor herbimycin-A (5-10 microg/ml) completely blunted the initial but not the delayed response effected by DiC10-PA. Long-chain phosphatidic acid (DiC18:1) induced only an initial rapid increase in intracellular Ca2+ and this response was similarly markedly attenuated by herbimycin-A. Among several physiologically relevant phospholipids, only phosphatidic acid was able to induce Ca2+ mobilization; phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol -- used individually or in mixed liposomes -- were without effect. Phosphatidic acid conferred calcium-mobilizing activity upon inactive liposome preparations and phosphatidic acid-enriched cellular plasma membranes possessed similar calcium-mobilizing activity. Both DiC10-PA and DiC18:1-PA induced actin polymerization in neutrophils at rates which mirrored the influence of each agent on Ca2+ mobilization. Herbimycin-A blunted the initial increase in actin polymerization effected by phosphatidic acid but had no effect on the delayed, EGTA-sensitive phase. DiC10-PA and DiC18:1-PA also induced neutrophil migration along a concentration gradient. Phospholipids that failed to induce a calcium transient, including phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, and phosphatidylinositol, likewise failed to induce either actin polymerization or chemotactic migration. Unlike chemotaxis induced by zymosan-activated human serum, phosphatidate-induced chemotaxis was strongly inhibited by pretreatment of cells with herbimycin-A. Consistent with these observations, phosphatidic acid induced the tyrosine phosphorylation of several proteins as early as 10 s after stimulation. Phosphorylation of two distinct proteins with approximate molecular sizes of 72 and 82 kDa was inhibited by levels of herbimycin A used to effectively inhibit calcium mobilization, actin polymerization and chemotaxis. Thus, in neutrophilic leukocytes, extracellular phosphatidic acid induces a unique tyrosine kinase-based signalling pathway that results in calcium mobilization and actin polymerization. These processes may promote directed cellular migration as a consequence of the interaction of phosphatidic acid with neutrophil plasma membranes.

Actins↗
PubMed1997

Characterization and purification of neutrophil ecto-phosphatidic acid phosphohydrolase.

Phosphatidic acid and its derivatives play potentially important roles as extracellular messengers in biological systems. An ecto-phosphatidic acid phosphohydrolase (ecto-PAPase) has been identified which effectively regulates neutrophil responses to exogenous phosphatidic acid by converting the substrate to diacylglycerol. The present study was undertaken to characterize this ecto-enzyme on intact cells and to isolate the enzyme from solubilized neutrophil extracts. In the absence of detergent, short chain phosphatidic acids were hydrolysed most effectively by neutrophil plasma membrane ecto-PAPase; both saturated and unsaturated long chain phosphatidic acids were relatively resistant to hydrolysis. Both long (C18:1) and short (C8) chain lyso-phosphatidic acids were hydrolysed at rates comparable with those observed for short chain (diC8) phosphatidic acid. Activity of the ecto-enzyme accounted for essentially all of the N-ethylmaleimide-insensitive, Mg2+-independent PAPase activity recovered from disrupted neutrophils. At 37 degrees C and pH7.2, the apparent Km for dioctanoyl phosphatidic acid (diC8PA) was 1. 4x10(-3) M. Other phosphatidic acids and lysophosphatidic acids inhibited hydrolysis of [32P]diC8PA in a rank order that correlated with competitor solubility, lysophosphatidic acids and unsaturated phosphatidic acids being much more effective inhibitors than long chain saturated phosphatidic acids. Dioleoyl (C18:1) phosphatidic acid was an unexpectedly strong inhibitor of activity, in comparison with its ability to act as a direct substrate in the absence of detergent. Other inhibitors of neutrophil ecto-PAPase included sphingosine, dimethyl- and dihydro-sphingosine, propranolol, NaF and MgCl2. Of several leucocyte populations isolated from human blood by FACS, including T cells, B cells, NK lymphocytes and monocytes, ecto-PAPase was most prevalent on neutrophils; erythrocytes were essentially devoid of activity. A non-hydrolysable, phosphonate analogue of phosphatidic acid, phosphonate 1, efficiently solubilized catalytic activity from intact neutrophils without causing cell disruption or increasing permeability. Enzyme activity in solubilized extracts was purified in the absence of detergent by successive heparin-Sepharose, gel filtration and anion exchange chromatography. By assaying activity in renatured SDS/polyacrylamide gel slices, the molecular mass of neutrophil ecto-PAPase was estimated to be between 45 and 52 kDa, similar to the molecular mass of previously purified plasma membrane PAPases. Since a large portion of neutrophil plasma membrane PAPase is available for hydrolysis of exogenous substrates, ecto-PAPase may play an important role in regulating inflammatory cell responses to extracellular phosphatidic acid in biological systems.

Cell Membrane↗
PubMed1997

Counterflow centrifugal elutriation as a method of T cell depletion may cause loss of immature CD34+ cells.

Counterflow centrifugal elutriation (CCE) is capable of separating cells on the basis of size. CCE has been used successfully to deplete allogeneic bone marrow (BM) grafts of T lymphocytes to decrease the risk of acute graft-versus-host disease. Previous studies have shown that more immature CD34+ cells in human BM tend to be smaller than more mature CD34+ cells. Human BM was subjected to CCE with the 4 ml standard chamber at constant rotor speed (2300 r.p.m.) and increasing flow-rate (14-23 ml/min, rotor-off). The eleven fractions collected were assayed for CD34+ and CD3+ cells, and for CFU-GM, HPP-CFC and long-term culture initiating cells (LTC-IC). The CD3+ T cells were enriched in the early (small-cell) fractions 14-17 ml/min. CD34+ cells were enriched in fractions 17-21 ml/min, and CFU-GM were concentrated in the same fractions. HPP-CFC and LTC-IC showed nearly identical CCE profiles, with enrichment in fractions 16-18 ml/min. When fraction < or = 17 ml/min was chosen as cut-off, the small-cell fraction contained 94.0% of all CD3+ cells, 44.4% of total cells, 33.2% of CD34+ cells and 34.7% of CFU-GM; however, 67.6% of HPP-CFC and 72.4% of LTC-IC were recovered in this small-cell fraction. These data suggest that T cell depletion through CCE as used by us, while losing only minor proportions of CD34+ cells and CFU-GM, carries the risk of losing the majority of more immature progenitor cells. This may lead to an increased risk of graft failure, in particular in HLA-mismatched transplants.

Antigens, CD34↗
PubMed1997

Lineage commitment of HLA-DR/CD38-defined progenitor cell subpopulations in bone marrow and mobilized peripheral blood assessed by four-color immunofluorescence.

We used four-color fluorescence analysis to compare lineage antigen expression in relationship to CD38 and HLA-DR on CD34+ progenitor cells in adult human bone marrow and mobilized peripheral blood. Each of four progenitor cell subpopulations defined by HLA-DR and CD38 intensity (CD38-/HLA-DR-, CD38-/HLA-DR+, CD38+/HLA-DR+, and CD38+/HLA-DR-) were present in both progenitor cell sources in similar ratios. The most prevalent subpopulation consisted of cells that expressed both CD38 and HLA-DR. Virtually all progenitor cells that lacked CD38 also lacked lineage antigens regardless of their HLA-DR expression. In contrast, the majority of the cells within both CD38+ progenitor cell subpopulations possessed either lineage antigens or the proliferation-associated antigen, CD71. Furthermore, CD71 was expressed on three times the number of CD38+/HLA-DR- cells when compared with the CD38-/HLA-DR- subpopulation. Within CD34+ progenitor cell subpopulations defined by the expression of CD38 and HLA-DR, the CD38+/HLA-DR- component appears to be the most mature, based on the expression of CD71 and various lineage-associated antigens, including representative markers characterizing early lymphoid, myeloid, and erythroid precursors. Thus, selection of the most immature CD34+ progenitor cells based solely on the lack of HLA-DR expression results in isolation of two distinct cell populations with markedly different maturation status and resultant growth characteristics.

ADP-ribosyl Cyclase↗
PubMed1997

The nature of leukocyte shape changes in the pulmonary capillaries.

The size discrepancy between leukocytes [white blood cells (WBCs)] and pulmonary capillaries requires WBCs to deform. We investigated the persistence of this deformation on cells leaving the capillary bed and the role played by the cytoskeleton. Isolated rabbit lungs were perfused in situ via the pulmonary artery with effluent fractions collected from the left ventricle. Washout curves from cell counts in each fraction confirmed that WBCs are preferentially retained over erythrocytes. WBC deformation present on exit from the circulation was compared with that present after recovery in paired fractions, fixed either immediately or 60 min later. These cells were compared with cells recovered from the capillary in perfused fixative or fixed in peripheral blood. Our results show that leukocyte deformation persisted after the cells exited the pulmonary circulation. This deformation was associated with minimal submembranous F-actin staining, and microtubule distribution and cell polarization were unchanged. We conclude that cytoskeletal changes that occur during WBC deformation in the pulmonary capillaries are minimal and differ from those known to occur in actively migrating cells during chemotaxis.

Animals↗
PubMed1997

The effect of cigarette smoking on the bone marrow.

Chronic cigarette smoking produces a 20 to 25% increase in the number of peripheral blood leukocytes, and there is increasing evidence that these leukocytes are activated in the lung by the inhalation of cigarette smoke. The present study was designed to measure the effect of cigarette smoke inhalation on the rate of production and release of polymorphonuclear leukocytes (PMN) from the bone marrow into the peripheral blood. The thymidine analogue 5'-bromo-2'-deoxyuridine (BrdU) was used to pulse-label the dividing cells in the marrow of rabbits, measure their appearance in the peripheral blood, and calculate the time that PMN spend in the mitotic and postmitotic pools of the bone marrow. Comparison of animals exposed to 2 wk of cigarette smoke (n = 8) with sham-exposed controls (n = 9) showed that smoking decreased the mean transit time of PMNBrdU through the bone marrow from 97.3 +/- 3.0 h to 89.6 +/- 5.8 h (p < 0.001) by reducing the transit time of PMN in the postmitotic pool from 66.7 +/- 3.9 h to 53.7 +/- 0.7 h (p < 0.001). Both the mitotic (p < 0.05) and postmitotic (p < 0.05) pools of PMN increased in size following cigarette-smoke exposure. We conclude that chronic cigarette smoking stimulates the bone marrow, increases the size of the mitotic and postmitotic pools of PMN, and reduces the time PMN spend in the postmitotic pool in the marrow. These changes may contribute to the leukocytosis seen in cigarette smokers.

Animals↗
PubMed1997

Phagocytosis of small carbon particles (PM10) by alveolar macrophages stimulates the release of polymorphonuclear leukocytes from bone marrow.

Recent studies have shown that an increased concentration of ambient particulate matter (PM10) is related to decreased pulmonary function and respiratory and cardiovascular mortality. The mechanisms responsible for this excess mortality are unknown and the relationship between the level of PM10 and the circulating leukocyte counts has not been previously investigated. We postulated that the deposition of PM10 in the peripheral lung stimulates alveolar macrophages (AM), which results in polymorphonuclear leukocyte (PMN) release from bone marrow (BM). To test this hypothesis, either colloidal carbon (CC) (n = 3) or saline (n = 4) was instilled into the lungs of rabbits and PMN release from BM was evaluated by using 5'-bromo-2'-deoxyuridine (BrdU). CC instillation in the lung shortened the transit time of PMN through the BM to 71.0 +/- 6.9 h compared with the saline controls (85.5 +/- 2.8 h, p < 0.01). The role of AM in this response was further investigated by incubating isolated AM in tissue culture medium either with or without the presence of CC, and measuring the effect of the supernatants on the release of PMN from the BM. The supernatant of AM incubated with CC shortened the PMN transit time through the BM to 74.9 +/- 3.7 h (p < 0.05) compared with the supernatant from the unstimulated AM (98.6 +/- 1.9 h) and medium alone (94.3 +/- 3.7 h). We conclude that the phagocytosis of CC by AM releases mediators (cytokines) that stimulate the BM to release PMN. We speculate that these newly released PMN may play an important role in the decline in lung function and high mortality seen in populations exposed to high concentrations of atmospheric PM10.

Animals↗