The MCP-4/MCP-1 ratio in plasma is a candidate circadian biomarker for chronic post-traumatic stress disorder.

Post-traumatic stress disorder (PTSD) is psychiatric disease, which can occur following exposure to traumatic events. PTSD may be acute or chronic, and can have a waxing and waning course of symptoms. It has been hypothesized that proinflammatory cytokines and chemokines in the cerebrospinal fluid (CSF) or plasma might be mediators of the psychophysiological mechanisms relating a history of trauma exposure to changes in behavior and mental health disorders, and medical morbidity. Here we test the cytokine/chemokine hypothesis for PTSD by examining levels of 17 classical cytokines and chemokines in CSF, sampled at 0900 hours, and in plasma sampled hourly for 24 h. The PTSD and healthy control patients are from the NIMH Chronic PTSD and healthy control cohort, initially described by Bonne et al. (2011), in which the PTSD patients have relatively low comorbidity for major depressive disorder (MDD), drug or alcohol use. We find that in plasma, but not CSF, the bivariate MCP4 (CCL13)/ MCP1(CCL2) ratio is ca. twofold elevated in PTSD patients compared with healthy controls. The MCP-4/MCP-1 ratio is invariant over circadian time, and is independent of gender, body mass index or the age at which the trauma was suffered. By contrast, MIP-1ß is a candidate biomarker for PTSD only in females, whereas TARC is a candidate biomarker for PTSD only in males. It remains to be discovered whether these disease-specific differences in circadian expression for these specific immune signaling molecules are biomarkers, surrogates, or drivers for PTSD, or whether any of these analytes could contribute to therapy.

Differentiation of adipose-derived stem cells into Schwann cell phenotype induces expression of P2X receptors that control cell death.

Schwann cells (SCs) are fundamental for development, myelination and regeneration in the peripheral nervous system. Slow growth rate and difficulties in harvesting limit SC applications in regenerative medicine. Several molecules, including receptors for neurosteroids and neurotransmitters, have been suggested to be implicated in regulating physiology and regenerative potential of SCs. Adipose-derived stem cells (ASCs) can be differentiated into SC-like phenotype (dASC) sharing morphological and functional properties with SC, thus representing a valid SC alternative. We have previously shown that dASC express γ-aminobutyric-acid receptors, which modulate their proliferation and neurotrophic potential, although little is known about the role of other neurotransmitters in ASC. In this study, we investigated the expression of purinergic receptors in dASC. Using reverse transriptase (RT)-PCR, western blot analyses and immunocytochemistry, we have demonstrated that ASCs express P2X3, P2X4 and P2X7 purinoceptors. Differentiation of ASCs towards glial phenotype was accompanied by upregulation of P2X4 and P2X7 receptors. Using Ca(2+)-imaging techniques, we have shown that stimulation of purinoceptors with adenosine 5'-triphosphate (ATP) triggers intracellular Ca(2+) signals, indicating functional activity of these receptors. Whole-cell voltage clamp recordings showed that ATP and BzATP induced ion currents that can be fully inhibited with specific P2X7 antagonists. Finally, using cytotoxicity assays we have shown that the increase of intracellular Ca(2+) leads to dASC death, an effect that can be prevented using a specific P2X7 antagonist. Altogether, these results show, for the first time, the presence of functional P2X7 receptors in dASC and their link with critical physiological processes such as cell death and survival. The presence of these novel pharmacological targets in dASC might open new opportunities for the management of cell survival and neurotrophic potential in tissue engineering approaches using dASC for nerve repair.

White particulate matter found in blood collection bags consist of platelets and leukocytes.

BACKGROUND: In late January 2003, some blood centers and hospitals throughout the US voluntarily sus-pended the use of some RBC and plasma units for trans-fusion due to the presence of unknown white particulate matter (WPM) in these units. To better understand the WPM phenomena, a number of technologies were used to establish the nature of the particulates observed in Terumo Collection sets. STUDY DESIGN AND METHODS: All AS-5 nonleuko-reduced RBCs and plasma units were visually inspected for WPM by placing the bags on a flat counter, undisturbed, for approximately 10 minutes and then perform-ing a visual examination for particles. Particles were isolated and placed on microscope slides or in plastic tubes for further analysis. Electron microscopy, bright field microscopy, differential interference contrast microscopy, infrared spectroscopy, and flow cytometry procedures were performed to establish the nature of the particulate matter. In addition, leukoreduction filters and blood transfusion sets were used on RBCs units with WPM. RESULTS: The particles were mostly composed of PLTs and WBCs, and fragments of these cells. All macroscopic WPM was removed from RBCs with leukoeduction and transfusion filters. CONCLUSIONS: WPM originated from PLTs and WBCs. Foreign matter (e.g., plastic) was not observed in any of the units. Leukoreduction and transfusion filters can be used to remove macroscopic WPM.

Mechanisms of hemorrhage in dengue without circulatory collapse.

To characterize the molecular basis for the hemostatic defects of dengue infections, a study was conducted in Bangkok, Thailand. Febrile children (n = 68) hospitalized with suspected dengue were enrolled before their clinical syndromes were classified as either dengue fever (DF) or dengue hemorrhagic fever (DHF). Hospital course and outcome were recorded; blood was obtained during the febrile illness (S1), after defervescence (S2), and 1 month after onset of disease (S4). Patients were classified as DF (n = 21) and DHF grades 1, 2, and 3; (DHF1, n = 8; DHF2, n = 30; and DHF3, n = 9). All had marked thrombocytopenia. Bleeding scores were assigned on the basis of bleeding site. Although there was no correlation between bleeding scores and pleural effusion index (a measure of vascular leakage) or bleeding scores and platelet counts, there was a correlation between pleural effusion index and platelet counts. Bleeding scores did not correlate with hemostatic data. Activated partial thromboplastin time was prolonged, with trends toward decreased fibrinogen and increased levels of prothrombin fragment F1.2 in the acute-phase samples. However, no factor level was dramatically decreased. We conclude that most patients with DF or DHF, even without overt hemorrhage, have consumptive coagulopathy. Nevertheless, hemorrhage in dengue without circulatory collapse is most likely due to activation of platelets rather than coagulopathy, which is well compensated. Our data suggest that vascular alteration may be the principal factor involved in the association of thrombocytopenia and hemorrhage with disease severity.

Proteasome inhibitors block the entry of liposome-encapsulated antigens into the classical MHC class I pathway.

Liposome-encapsulated conalbumin (L(conalbumin)) is an antigen that is efficiently phagocytosed by bone marrow-derived macrophages and presented to effector cells as part of the major histocompatibility complex (MHC) class I complex. In this report, we show that the conalbumin component of L(conalbumin) is degraded to small peptide fragments and translocated to the area of the Golgi. Golgi localization is confirmed by co-localization of L(Texas red-conalbumin) (L(TR-conalbumin))with both NBD-ceramide, a lipid Golgi marker, and green fluorescent protein (GFP)-galactosyl transferase, a Golgi resident enzyme. Incubation of the cells with brefeldin A disrupts the Golgi and disperses the TR-conalbumin. Furthermore, when macrophages were incubated with another liposome-encapsulated antigen, L(ovalbumin), ovalbumin peptides were observed in the Golgi area and MHC class I-peptide complexes could be detected on the cell surface by both immunofluorescence microscopy and flow cytometry. The Golgi localization observed in vitro in cultured macrophages is mirrored by the in vivo uptake and Golgi localization of fluorescent L(conalbumin) in macrophages isolated from the spleen of a mouse injected with L(TR-conalbumin). The accumulation of peptide fragments in the Golgi is inhibited by the addition of the proteasome inhibitors, lactacystin and MG-132, demonstrating the role of the proteasome in this activity. In addition, when macrophages or a macrophage-derived cell line, are incubated with liposome-enccapsulated antigens and used as target cells in a cytotoxic T-cell (CTL) assay, the CTLs recognize the processed peptide-MHC complexes and kill the cells. In contrast, specific lysis of target cells by CTLs is inhibited when the target cells are first incubated with lactacystin. These results suggest that uptake and processing of L(antigen) follows the classical MHC class I pathway.

Correlation of in vivo and in vitro functions of fresh and stored human platelets.

BACKGROUND: Long-term storage of human platelets has been hindered by the loss of function of the platelets stored under current protocols. Novel preservation methods have encouraged examination of platelet function of cells preserved by cooling and freezing. The function of the platelets was assessed by using both in vitro assays and an in vivo rabbit bleeding model. STUDY DESIGN AND METHODS: Human platelets were stored in the presence or absence of 2 microM: cytochalasin B and 80 microM: EGTA/AM at 4 degrees C for 14 days or by freezing in the presence or absence of 5 percent DMSO. After the storage period, the platelets were resuspended in normal saline and infused into rabbits. Platelet function was assessed in vivo in a kidney bleeding model and in vitro by platelet-induced clot retraction and by platelet aggregation. RESULTS: Platelets stored at either 4 degrees C or -145 degrees C exhibited shorter survival times in the rabbit circulation than did fresh platelets. Platelets cooled to 4 degrees C, in both the presence or absence of cytochalasin B and EGTA/AM treatment, or frozen in the absence of DMSO were not effective in halting bleeding. However, frozen DMSO-treated platelets were as effective as fresh platelets in stopping bleeding. In vitro assays showed that cooled platelets treated with cytochalasin B and egtazic acid/AM and frozen DMSO-treated platelets retained 30 to 40 percent platelet function, while the cooled and frozen control samples exhibited no platelet-induced clot retraction. With thrombin as the agonist, only frozen DMSO-treated platelets exhibited a tendency to aggregate, although at only 22 percent of the aggregation function of fresh platelets. CONCLUSION: It is possible to freeze platelets and retain in vivo efficacy if the cryopreservative DMSO is included in the preparation. In vitro responses were greatly reduced by all of the storage protocols, but it may not be necessary to retain 100 percent in vitro function to have a platelet substitute or storage product that functions satisfactorily in vivo.

Reduction of blood loss by infusion of human platelets in a rabbit kidney injury model.

BACKGROUND: As a first step toward testing the efficacy of stored platelets or platelet substitutes in vivo, a kidney injury model was developed to assess the hemostatic properties of human platelets in normal and thrombocytopenic rabbits. STUDY DESIGN AND METHODS: New Zealand white rabbits were made thrombocytopenic by two consecutive injections of busulfan. Two weeks later, human platelets were transfused to animals whose reticuloendothelial systems were inhibited by the administration of ethyl palmitate. The left kidney was exposed and a slice excised from the anterior pole. The blood was contained in a parafilm boat and absorbed by preweighed gauze to assess blood loss. The percentage of human platelets transfused to the rabbit was determined by flow cytometry on blood collected from the cut site using anti-CD42a (marker for human platelets). The degree of activation of the human platelets was determined using anti-CD62a (marker specific for human p-selectin). RESULTS: Blood loss was similar in normal animals treated with saline alone (35.4 +/- 5.8 g; n = 4); ethyl palmitate and saline (42.5 +/- 5.7 g; n = 6, p = 0.4); or ethyl palmitate and fresh human platelets (45.7 +/- 7.9 g; n = 6, p = 0.3). Bleeding in thrombocytopenic rabbits infused with saline was increased (75.6 +/- 3.9 g; n = 7) as compared with nonthrombocytopenic animals. A significant reduction in blood loss was noted in thrombocytopenic rabbits given fresh human platelets (51.6 +/- 4.5 g; n = 6, p = 0.0023). Transfusion of human platelets to rabbits did not cause activation of the platelets. Furthermore, transfusion of thrombin-activated platelets (60-98% activated) to thrombocytopenic rabbits reduced blood loss (54 +/- 7.3 g; n = 7) to the same extent as fresh platelets. CONCLUSIONS: This is the first report describing a kidney injury model developed to assess the efficacy of fresh and activated human platelets in reducing blood loss in thrombocytopenic rabbits. This model could monitor the efficacy of human platelets prepared by various preservation protocols in suppressing bleeding in rabbits.

Trafficking of liposomal antigen to the trans-Golgi of murine macrophages requires both liposomal lipid and liposomal protein.

Major histocompatibility complex (MHC) class I molecules found on antigen-presenting cells present peptides derived from cytoplasmic proteins to T cells. In contrast, peptides from exogenous proteins are mostly presented by class II molecules. It has been well established that liposomes can serve as an efficient delivery system for entry of exogenous protein antigens into the MHC class I pathway. Our previous studies utilizing fluorophore-labeled proteins encapsulated in liposomes demonstrated that after phagocytosis of the liposomes by bone marrow-derived macrophages (BMs), the processed peptides were subsequently visualized in the trans-Golgi, while free conalbumin was excluded from the trans-Golgi area. In the present study, we investigated whether liposomal lipids follow the same intracellular route as the liposomal proteins after phagocytosis by BMs. Multilamellar liposomes with different lipid compositions that also contained fluorescent phospholipids (empty liposomes) were incubated with murine BMs. Our results indicate that although empty liposomes were avidly phagocytosed by macrophages, the fluorescent liposomal lipids did not localize to any particular area of the cell but were distributed throughout the cell. In contrast, when a protein was encapsulated in the liposomes, the liposomal lipids were no longer dispersed throughout the cell, but were concentrated and localized in the trans-Golgi area. Furthermore, when the liposomes contained a fluorescent-labeled protein, the fluorescent peptides also localized to the trans-Golgi. These results demonstrate that the combination of both liposomal lipids and liposomal protein is required for Golgi-specific targeting of liposomal antigens. Transport of both liposomal lipids and liposomal proteins to the Golgi complex, a major subcellular organelle in the passage of MHC class I molecules, might explain why antigens encapsulated in liposomes readily induce cytotoxic T lymphocytes.

Post-chemotherapy and cytokine pretreated marrow stromal cell layers suppress hematopoiesis from normal donor CD34+ cells.

Marrow stromal layers were used to investigate the potential role of negative regulators produced by the marrow microenvironment as one potential cause of hematopoietic suppression after chemotherapy and cytokines. Stromal layers were established from marrow of normal or prechemotherapy donors and breast cancer patients after hematological recovery from one cycle of 5-fluorouracil, leucovorin, doxorubicin, and cyclophosphamide and GM-CSF or PIXY321 (GM-CSF/IL-3 fusion protein). Normal donor CD34+ cells were placed in contact with stromal layers, and the number of colony-forming units for granulocytes and macrophages (CFU-GM) was determined. There were 25-79% fewer CFU-GM in post-chemotherapy stromal layer cocultures than in no chemotherapy cocultures. With neutralizing antibody to TNF-alpha the number of CFU-GM in no chemotherapy and post-chemotherapy stromal cocultures was, respectively, 96 +/- 7% (n = 5) and 142 +/- 8% (n = 5) of the number with no antibody treatment. PIXY321 and GM-CSF pretreated stromal layers also suppressed production of CFU-GM. Anti-TNF-alpha promoted an increase in CFU-GM numbers from GM-CSF, but not PIXY321, pretreated stromal cocultures. The results demonstrate that post-chemotherapy marrow stromal layers were deficient in supporting in vitro hematopoiesis and suggest that negative regulators induced by chemotherapy and cytokines may be one cause for this defect.

Survival of fresh human platelets in a rabbit model as traced by flow cytometry.

BACKGROUND: In the event of hemorrhage and blood loss, platelets play a vital role in the coagulation process. However, there are currently no acceptable protocols for long-term storage of platelets. As a first step toward testing the efficacy of stored platelets or platelet substitutes in vivo, a flow cytometric technique was developed to detect human platelets in rabbit blood. STUDY DESIGN AND METHODS: Human platelets were transfused to rabbits whose reticuloendothelial system was inhibited by the administration of ethyl palmitate. Because human and rabbit platelets display surface molecules with different epitopes, human platelets were selectively labeled with antibodies specific for glycoprotein IX (CD42a). As this antibody does not label rabbit platelets, it allows discrimination of human from rabbit platelets in samples of rabbit blood containing both types of platelets. RESULTS: Survival of human platelets in rabbits was monitored by flow cytometry and fluorescence microscopy in blood drawn at various times after the platelet transfusion. Fresh human platelets transfused to untreated control rabbits (n = 3) were removed from circulation within 10 minutes of the completion of the transfusion. Fresh platelets (1 day old) transfused to rabbits treated with ethyl palmitate (n = 5) survived for 24 hours with an average half-life of 8.6 hours. In contrast, 8-day-old platelets were cleared from the circulation sooner with an average half-life of 2.9 hours (n = 4). CONCLUSION: This report describes a rapid and efficient method of assessing the survival of human platelets in a rabbit model using flow cytometry. This technique will enable the monitoring in rabbits of human platelets prepared by various preservation protocols.

Liposomes containing lipid A serve as an adjuvant for induction of antibody and cytotoxic T-cell responses against RTS,S malaria antigen.

Encapsulation of soluble protein antigens in liposomes was previously shown to result in processing of antigen via the major histocompatibility complex class I pathway, as evidenced by costaining of the trans-Golgi region of murine bone marrow-derived macrophages (BMs) by fluorophore-labeled liposomal antigen and by a trans-Golgi-specific fluorescent lipid. Evidence is presented here that free or liposome-encapsulated RTS,S, a particulate malaria antigen consisting of hepatitis B particles coexpressed with epitopes from the Plasmodium falciparum circumsporozoite protein, also was localized in the trans-Golgi after incubation with BMs, suggesting processing by the class I pathway. An in vivo cytotoxic T-lymphocyte (CTL) response was detected, however, only after immunization with RTS,S encapsulated in liposomes containing lipid A and not after immunization with free RTS,S or with RTS,S encapsulated in liposomes lacking lipid A. Therefore, intracellular delivery of antigen containing CTL epitopes to the Golgi of BMs does not necessarily result in a CTL response in vivo unless an additional adjuvant, such as liposomes containing lipid A, is utilized. Encapsulation of RTS,S in liposomes containing monophosphoryl lipid A (MPL) resulted in a dose-dependent enhancement of the NANP-specific immunoglobulin G (IgG) antibody response compared to that of free RTS,S. The IgG1 and IgG2a subclasses predominated after immunization with RTS,S encapsulated in liposomes containing MPL. These results demonstrate that encapsulation of a lipid-containing particulate antigen, such as RTS, S, in liposomes containing lipid A can enhance both humoral and cellular immune responses.

Activation of human platelets causes post-translational modifications to cytoplasmic dynein.

In our studies of human platelets we have detected the presence of the molecular motors kinesin and dynein. Dynein is present at a concentration (0.8 microg/g tissue) that is approximately 1/3 the concentration reported for neuronal tissue. Immunofluorescence microscopy of resting platelets shows that, while platelet microtubules are arranged in coiled hoops forming the marginal band in the cortical region of the platelet, dynein is distributed in a pattern of punctate staining throughout the cytoplasm of the platelets. Fractionation of unactivated platelets shows that dynein partitions to the soluble fraction. Stimulation of platelets with thrombin, ADP or epinephrine causes a partial translocation of dynein from the soluble fraction to the particulate fraction with thrombin being the most efficient agent at promoting this shift. Dynein intermediate chain recovered in the soluble fraction of disrupted platelets following activation displays a transient, time-dependent phosphorylation. In contrast, dynein intermediate chain recovered in the particulate fraction shows decreased phosphorylation. These results indicate that human platelets contain a complex microtubule-based system of motor proteins that is an integral part of the physiological changes occurring during platelet activation.

Visualization of peptides derived from liposome-encapsulated proteins in the trans-Golgi area of macrophages.

Exogenous proteins are generally not presented through the major histocompatibility complex (MHC) class I pathway, yet several recent studies show that particle-associated antigens induce a CD8+ T-cell response. Therefore, a pathway must exist in vivo for the presentation of exogenous antigens on class I molecules. In the present study, we investigated the intracellular fate of liposome-encapsulated Texas Red (TR)-conjugated protein in cultured bone marrow-derived macrophages (BMs). After phagocytosis of liposomes, the fluorescent liposomal protein, initially associated with the liposomal lipids in phagosomes, later entered the cytoplasm, and the processed protein was subsequently visualized in the trans-Golgi as a fluorescent peptide. Experiments performed with BMs from transporter associated with antigen processing (TAP1) knock-out mice demonstrated that the translocation of peptides into the trans-Golgi area was dependent upon TAP1 protein. We conclude that delivery of liposomal proteins or peptides to the cytoplasm of phagocytes and subsequent transport of peptides to the Golgi via the classical MHC class I pathway involving TAP proteins might explain the known propensity of liposomal antigens to induce cytotoxic T-lymphocytes (CTLs).

Dengue-2 virus infection of human bone marrow: characterization of dengue-2 antigen-positive stromal cells.

Dengue is often associated with neutropenia and thrombocytopenia, suggesting that cells of the bone marrow may be targets of dengue viral infections. In this study we infected long-term marrow cultures with dengue type-2 (DEN-2) virus and characterized the viral antigen-positive cells. Using immunofluorescence microscopy and immunohistochemical staining, we demonstrated two types of stromal cells that were positive for DEN-2 virus antigens. The first was a population of relatively small (approximately 25 microns) CD11b/CD18 (MAC-1)-positive cells. When stained with anti-DEN-2 polyclonal antibody, these cells showed viral antigen-positive inclusions and, when stained with anti-tubulin or anti-vimentin antibodies, they showed a diffuse pattern of fluorescence, consistent with mobile dendritic cells with phagocytic functions. The second population of DEN-2 antigen-positive cells comprised a smaller proportion of the total cells. It was made up of larger cells (> 100 microns) that had a well-formed cytoskeletal system as demonstrated by intense staining with anti-actin, anti-tubulin, and anti-vimentin antibodies. When stained with anti-DEN-2 antibody, these cells showed a more diffuse pattern of fluorescence in the perinuclear and Golgi regions, consistent with ongoing virus replication. These large, strongly adherent cells were positive for nerve growth factor receptor, consistent with their identification as adventitial reticular cells. The molecule that mediates the virus interaction with susceptible cells has not previously been identified. Using plasma membrane proteins isolated from K562 cells, virus-binding studies suggest that an approximately 100-kD membrane protein may be involved in the initial virus-cell interaction.

Inhibitory effects of HIV-1-infected stromal cell layers on the production of myeloid progenitor cells in human long-term bone marrow cultures.

This report presents the results of studies using long-term bone marrow cultures (LTBMC) of human bone marrow cells to investigate the effect of HIV-1 on in vitro hematopoiesis. Confluent stromal cell layers established from human bone marrow cells were irradiated to eliminate residual hematopoietic progenitor cells and exposed to HIV-1ADA or to HIV-1IIIB, monocytotropic and lymphocytotropic strains of HIV-1, respectively. A productive infection did not develop in cultures exposed to HIV-1IIIB but did for cultures exposed to HIV-1ADA as there was a progressive increase in HIV-1 p24 antigen. Stromal cell layers infected with HIV-1ADA were also cocultured with autologous CD34+ bone marrow cells. Four days, 1, 2, and 3 weeks later, the number of colony-forming units granulocyte/macrophage (CFU-GM) in non- and HIV-infected LTBMC was determined. The number of CFU-GM increased during the first week in both non- and HIV-infected LTBMC. One week after the coculture of CD34+ cells with stromal cell layers infected with HIV-1ADA, the number of CFU-GM in six out of eight experiments was reduced compared to noninfected control LTBMC. In those six experiments, the number of CFU-GM was 53 +/- 6% standard error of the mean (SEM) of the number in noninfected LTBMC. A reduced number of CFU-GM was observed in the nonadherent fraction of HIV-infected LTBMC for at least 2 weeks. These results demonstrate that some cells in the stromal cell layers of LTBMC were targets for HIV-1 and that HIV-infected stromal cell layers suppressed or delayed the production of CFU-GM.

Antibodies to myeloid precursor cells in autoimmune neutropenia.

Antibodies to mature blood neutrophils and to bone marrow myeloid cells have been described in the sera of some patients with apparent autoimmune neutropenia. To further explore the prevalence and specificities of antibodies to myeloid precursor cells, we evaluated sera from 148 patients with suspected autoimmune neutropenia for the presence of antibodies to neutrophils, to cultured myeloid cell lines, and to highly purified bone marrow myeloid progenitor cells. Using an immunofluorescence flow cytometric assay, we identified IgG antibodies in 42 (28%) of these sera that bound specifically to K562 cells, a multilineage cell line originally derived from a patient with chronic myelogenous leukemia. Twenty-two (15%) of the sera also contained IgG antibodies that bound specifically to the primitive myelomonocytic leukemia cell line KG1a. Twenty-five (17%) of the sera had IgG antibodies to myeloid cell lines in the absence of antibodies to mature neutrophils. There was a trend toward more severe neutropenia in patients with antibodies to K562 cells, without antineutrophil antibodies. In further studies, antibodies from 12 sera bound to mononuclear CD34+ cells that had been purified from normal human bone marrow by an immunomagnetic separation procedure. Moreover, two of these sera suppressed the growth of granulocyte-macrophage colony-forming units (CFU-GM) in methylcellulose cultures. The presence of antibodies to primitive hematopoietic cells in the sera of some patients with suspected immune neutropenia suggests that these antibodies may have a role in the pathogenesis of the neutropenia observed.

Characterization of influenza A virus binding sites on human neutrophils.

Exposure of human neutrophils (PMN) to influenza A virus (IAV) triggers discrete responses in these cells that interfere with their normal host defense functions. Because the restricted host range and tissue specificities of many viruses are determined by cell surface molecules acting as virus receptors on target cells, it seemed plausible that IAV might interact with neutrophils via specific plasma membrane glycoproteins that bind to viral hemagglutinin. When the binding of intact IAV (ATCC strain A/PR/8/34 (H1N1)) to PMNs was examined by flow cytometry, virus binding was found to be saturable and to be diminished after extensive desialation of the cells with neuraminidase. Stimulation of PMNs with FMLP (0.1 microM) caused a transient increase in IAV binding that was maximal (> 200%) at 2 min after stimulation. When neutrophil membrane proteins were separated by gel electrophoresis and transferred to nitrocellulose, IAV bound selectively to two polypeptide bands of approximately 125 and 160 kDa. Relative binding to these two bands was modified and ultimately eliminated by treatment of PMN membrane proteins with neuraminidase before electrophoresis and blotting. Intact virus precipitated a limited number of proteins from solubilized PMN plasma membrane preparations, and Abs specific for sialophorin (CD43) recognized virus-precipitated PMN membrane proteins of the same apparent m.w. as those detected in virus-membrane protein blots. These findings indicate that IAV binds to human PMNs through interactions with a limited number of PMN membrane glycoproteins, which include sialophorin (CD43).

Affinity purification and subcellular localization of kinesin in human neutrophils.

Studies of granule-microtubule interactions in human neutrophils have suggested that mechanochemical ATPases such as kinesin or dynein may play a role in granule mobilization during neutrophil activation by inflammatory signals. In this study we show that proteins extracted from the surface of neutrophil granules, found previously to contain microtubule-dependent ATPase activity, caused microtubules polymerized from phosphocellulose-purified rat brain tubulin to move across glass slides. Antibodies were generated against peptides based on two regions of the amino acid sequence of Drosophila kinesin: the ATPase active site (amino acids 86-99) in the head of the kinesin heavy chain and the tail of the heavy chain (residues 913-933). These antibodies were found to recognize kinesin in rat brain extracts as well as kinesin-like polypeptides in extracts of human neutrophils. Furthermore, when used in immunoaffinity chromatography, these antibodies permitted the isolation of a protein from neutrophil granule extracts that was recognized by Drosophila kinesin antibodies. Subcellular localization by immunofluorescence microscopy showed this protein to be associated principally with the cytoplasmic granules of neutrophils.

Rapid and reversible tubulin tyrosination in human neutrophils stimulated by the chemotactic peptide, fMet-Leu-Phe.

Neutrophil activation by specific stimuli, such as the oligopeptide chemotactic factor fMet-Leu-Phe (fMLF), is associated with an increased enzymatic addition of tyrosine to tubulin alpha-subunits, as measured by 14C tyrosine uptake. In studies using immunoblots we have found that this increased tyrosine uptake into tubulin in activated neutrophils reflects an increase in the proportion of cellular tubulin that is tyrosinated rather than simply an increase in the turnover of tyrosinated subunits. However, the increased accumulation of tyrosinated tubulin was also found to follow an initial depletion of tyrosinated tubulin and concomitant increase in detyrosinated tubulin between 0 and 60 sec following stimulation of neutrophils with fMLF. Immunogold electron microscopy studies of intact microtubules recovered from activated neutrophils demonstrated that these rapid changes in the relative content of tubulin isoforms in the cells were not associated with the formation or disappearance of microtubule microdomains composed of only one form of tubulin. Previously, we have shown that under conditions of fMLF-stimulated exocytosis there is an increased binding of neutrophil granules to endogenous microtubules. Since neutrophil activation by fMLF is associated with increased tyrosination of alpha-tubulin subunits, we speculated that rapid changes in the levels of tyrosinated tubulin in the microtubules of activated neutrophils might have a role in the regulation of granule-microtubule interactions. When the binding of purified neutrophil granules to reconstituted rat brain microtubules containing approximately 50% tyrosinated tubulin was measured by electron microscopy and compared with granule binding to microtubules that contained no detectable tyrosinated tubulin, granule-microtubule associations were found to be significantly favored by detyrosinated vs. tyrosinated tubulin. These findings indicate that interactions between cytoplasmic granules and microtubules in activated neutrophils may be modulated by rapid changes in the relative content of detyrosinated and tyrosinated tubulin in the microtubule network of the cells.

Copolymerization of two distinct tubulin isotypes during microtubule assembly in vitro.

Cells contain multiple tubulin isotypes that are the products of different genes and posttranslational modifications. It has been proposed that tubulin isotypes become segregated into different classes of microtubules each adapted to specific activities and functions. To determine if mixtures of tubulin isotypes segregate into different classes of polymers in vitro, we used immunoelectron microscopy to examine the composition of microtubule copolymers that assembled from mixtures of purified tubulin subunits from chicken brain and erythrocytes, each of which has been shown to exhibit distinct assembly properties in vitro. We observed that (a) the two isotypes coassemble rapidly and efficiently despite the fact that each isotype exhibits its own unique biochemical and assembly properties; (b) at low monomer concentrations the ratio of tubulin isotypes changes along the lengths of elongating copolymers resulting in gradients in immuno-gold labeling; (c) two distinct classes of copolymers each containing a distinct ratio of isotypes assemble simultaneously in the same subunit mixture; and (d) subunits and polymers of different isotypes associate nearly equally well with each other, there being only a slight bias favoring interactions among subunits and polymers of the same isotype. The observations agree with previous studies on the homogeneous distribution of multiple isotypes within cells and suggest that if segregation of isotypes does occur in vivo, it is most likely directed by cell-specific microtubule-associated proteins (MAPs) or specialized intracellular conditions.