Leukocyte Procoagulant Activity Research Articles

Interference of Metal Oxide Nanoparticles with Coagulation Cascade and Interaction with Blood Components

AbstractMetal oxide nanoparticles (NPs) are increasingly used for different purposes, showing a potential risk on human health. The analysis of the interaction of these metal oxide NPs with blood components is a crucial step in the characterization of their biocompatibility, but information available of comparative studies with several doses and different metal oxide NPs is really scarce. In this study, six different metal oxide NPs (TiO2, CeO2, Al2O3, Y2O3, and two different types of ZnO NPs) at different concentrations are used, and their potential adverse effects on blood are determined. Both, prothrombin time (PT) and activated partial thromboplastin time (aPTT), are assessed to understand particle influence on the plasma coagulation cascade. Additional hematocompatibility tests include assessment of thrombin coagulation time, platelet aggregation, leukocyte procoagulant activity (PCA), hemolysis, and complement activation. The results demonstrate that only the ZnO and TiO2 NPs affect the coagulation cascade by increasing the aPTT in a dose‐dependent manner. Moreover, ZnO NPs increase PT, while TiO2 NPs induce a decrease in the PT. In addition to affecting coagulation time, ZnO NPs also induce platelet aggregation and leukocyte PCA. All tested metal oxide NPs do not affect hemolysis and complement activation.

In Vitro Assessment of Nanoparticle Effects on Blood Coagulation.

Blood clotting is a complex process which involves both cellular and biochemical components. The key cellular players in the blood clotting process are thrombocytes or platelets. Other cells, including leukocytes and endothelial cells, contribute to clotting by expressing the so-called pro-coagulant activity (PCA) complex on their surface. The biochemical component of blood clotting is represented by the plasma coagulation cascade, which includes plasma proteins also known as coagulation factors. The coordinated interaction between platelets, leukocytes, endothelial cells, and plasma coagulation factors is necessary for maintaining hemostasis and for preventing excessive bleeding. Undesirable activation of all or some of these components may lead to pathological blood coagulation and life-threatening conditions such as consumptive coagulopathy or disseminated intravascular coagulation (DIC). In contrast, unintended inhibition of the coagulation pathways may lead to hemorrhage. Thrombogenicity is the property of a test material to induce blood coagulation by affecting one or more elements of the clotting process. Anticoagulant activity refers to the property of a test material to inhibit coagulation. The tendency to cause platelet aggregation, perturb plasma coagulation, and induce leukocyte PCA can serve as an in vitro measure of a nanomaterial's likelihood to be pro- or anticoagulant in vivo. This chapter describes three procedures for in vitro analyses of platelet aggregation, plasma coagulation time, and activation of leukocyte PCA. Platelet aggregation and plasma coagulation procedures have been described earlier. The revision here includes updated details about nanoparticle sample preparation, selection of nanoparticle concentration for the in vitro study, and updated details about assay controls. The chapter is expanded to describe a method for the leukocyte PCA analysis and case studies demonstrating the performance of these in vitro assays.

Non-immunogenic dextran-coated superparamagnetic iron oxide nanoparticles: a biocompatible, size-tunable contrast agent for magnetic resonance imaging.

Iron oxide-based contrast agents have been in clinical use for magnetic resonance imaging (MRI) of lymph nodes, liver, intestines, and the cardiovascular system. Superparamagnetic iron oxide nanoparticles (SPIONs) have high potential as a contrast agent for MRI, but no intravenous iron oxide-containing agents are currently approved for clinical imaging. The aim of our work was to analyze the hemocompatibility and immuno-safety of a new type of dextran-coated SPIONs (SPIONdex) and to characterize these nanoparticles with ultra-high-field MRI. Key parameters related to nanoparticle hemocompatibility and immuno-safety were investigated in vitro and ex vivo. To address concerns associated with hypersensitivity reactions to injectable nanoparticulate agents, we analyzed complement activation-related pseudoallergy (CARPA) upon intravenous administration of SPIONdex in a pig model. Furthermore, the size-tunability of SPIONdex and the effects of size reduction on their biocompatibility were investigated. In vitro, SPIONdex did not induce hemolysis, complement or platelet activation, plasma coagulation, or leukocyte procoagulant activity, and had no relevant effect on endothelial cell viability or endothelial–monocytic cell interactions. Furthermore, SPIONdex did not induce CARPA even upon intravenous administration of 5 mg Fe/kg in pigs. Upon SPIONdex administration in mice, decreased liver signal intensity was observed after 15 minutes and was still detectable 24 h later. In addition, by changing synthesis parameters, a reduction in particle size <30 nm was achieved, without affecting their hemo- and biocompatibility. Our findings suggest that due to their excellent biocompatibility, safety upon intravenous administration and size-tunability, SPIONdex particles may represent a suitable candidate for a new-generation MRI contrast agent.

Contributions of phosphatidylserine-positive platelets and leukocytes and microparticles to hypercoagulable state in gastric cancer patients.

Hypercoagulability in gastric cancer is a common complication and a major contributor to poor prognosis. This study aimed to determine procoagulant activity of blood cells and microparticles (MPs) in gastric cancer patients. Phosphatidylserine-positive blood cells and MPs, and their procoagulant properties in particular, were assessed in 48 gastric cancer patients and 35 healthy controls. Phosphatidylserine-positive platelets, leukocytes, and MPs in patients with tumor-node-metastasis stage III/IV gastric cancer were significantly higher than those in stage I/II patients or healthy controls. Moreover, procoagulant activity of platelets, leukocytes, and MPs in stage III/IV patients was significantly increased compared to the controls, as indicated by shorter clotting time, higher intrinsic and extrinsic factor tenase, and prothrombinase complex activity. Interestingly, lactadherin, which competes with factors V and VIII to bind phosphatidylserine, dramatically prolonged clotting time of the cells and MPs by inhibiting factor tenase and prothrombinase complex activity. Although anti-tissue factor antibody significantly attenuated extrinsic tenase complex activity of leukocytes and MPs, it only slightly prolonged clotting times. Meanwhile, treatment with radical resection reduced phosphatidylserine-positive platelets, leukocytes, and MPs, and prolonged the clotting times of the remaining cells and MPs. Our results suggest that phosphatidylserine-positive platelets, leukocytes, and MPs contribute to hypercoagulability and represent a potential therapeutic target to prevent coagulation in patients with stage III/IV gastric cancer.

The role of tissue thromboplastin in the development of DIC accompanying neoplastic diseases.

Procoagulant activity of gastric cancer tissues and leukocytes obtained from various types of leukemia have been studied with special reference to TTP. The following results were obtained. Homogenates of APL leukocytes and gastric cancer tissues contained strong procoagulant activities, most of which have been identified as TTP since the activities were neutralized by a specific antibody against purified human placenta TTP, inactivated by the removal of phospholipid with heptane-butanol mixture, and inactivated by the addition of phospholipase C. The delipidated homogenates regained procoagulant activities by relipidation procedures. These results also confirmed that TTP from APL leukocytes and gastric cancer tissues have the same lipoprotein properties as those of TTP in normal tissues. Though slight proteolytic activity and fibrinolytic activity were demonstrated in the homogenate of gastric cancer tissues, it was noted that the TTP activity was different from these two activities by partial purification of TTP from gastric cancer tissues. The TTP activity of 9 homogenates of gastric cancer tissues was 301 +/- 289 (mean +/- SD) units per mg protein, being higher in homogenates of mucinous adenocarcinoma and signet-ring cell carcinoma than in those of tubular and poorly differentiated adenocarcinoma. The mean TTP activity of leukocyte homogenates from 14 patients with APL and one out of 4 patients with CML in blastic crisis was 81 +/- 76 units/10(7) cells. The TTP activity of the homogenates of leukocytes from 7 out of 18 patients with AML and another patient with CML in blastic crisis ranged from one to six units/10(7) cells with a mean of 3.3 +/- 1.2. The TTP activity of leukocyte homogenates from the other 11 cases of AML, two cases of CML in blastic crisis, 6 cases of CML, and one case each of ALL and CLL were less than one unit/10(7) cells. In leukemic patients, all cases with a value of more than 202 for the product of units of TTP activity per 10(7) cells and differential count (%) of leukemic cells in the bone marrow smear (MU value) were accompanied by DIC. The MU value of leukemic patients correlated well to the plasma fibrinogen and serum FDP levels. All patients with a MU value of more than 277 died of DIC when a sufficient amount of heparin was not administered. On the other hand, no DIC developed in any of the patients with a MU value of less than 90.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of phosphoinositol 3 kinase contributes to nanoparticle-mediated exaggeration of endotoxin-induced leukocyte procoagulant activity.

Disseminated intravascular coagulation is an increasing concern for certain types of engineered nanomaterials. Recent studies have shed some light on the nanoparticle physicochemical properties contributing to this toxicity; however, the mechanisms are poorly understood. Leukocyte procoagulant activity (PCA) is a key factor contributing to the initiation of this toxicity. We have previously reported on the exaggeration of endotoxin-induced PCA by cationic dendrimers. Herein, we report an effort to discern the mechanism. Poly(amidoamine) dendrimers with various sizes and surface functionalities were studied in vitro by the recalcification test, flow cytometry and other relevant assays. Cationic dendrimers exaggerated endotoxin-induced PCA, but their anionic or neutral counterparts did not; the cationic charge prompts this phenomenon, but different cationic surface chemistries do not influence it. Cationic dendrimers and endotoxin differentially affect the PCA complex. The inhibition of phosphoinositol 3 kinase by dendrimers contributes to the exaggeration of the endotoxin-induced PCA.

Inhibition of PI3K contributes to nanoparticle-mediated exaggeration of endotoxin-induced leukocyte procoagulant activity (INC6P.340)

Abstract Nanoparticles are increasingly used in biomedical applications due to their ability to change biodistribution, increase safety and improve efficacy of drugs. However, application of engineered nanomaterials in medicine is often halted by safety concerns. Endotoxin contamination is very common in nanomaterials and there is an increasing number of studies reporting that certain nanoparticles can exaggerate endotoxin-mediated inflammation. We studied the relationship between physicochemical properties of polyamidoamine (PAMAM) dendrimers and their ability to exaggerate LPS-induced procoagulant activity (PCA) of human leukocytes in vitro. The PCA is phospholipid-protein complex composed of tissue factor (TF) and phosphotidylserine (PS). We found that cationic but not anionic or neutral PAMAM dendrimers, increase LPS-induced leukocyte PCA. Cationic PAMAM dendrimers enhanced both LPS-induced TF expression and PS exposure on the cell surface. The mechanism of LPS-induced PCA exaggeration by PAMAM dendrimers is not mediated by the electrostatic interaction between cationic dendrimers and anionic LPS, calcium influx and secondary messengers released by dendrimer activated platelets. We hypothesize that dendrimers exaggerate LPS-mediated PCA through the inhibition of negative regulation of inflammation. Specifically, we demonstrate that cationic PAMAM dendrimers inhibit phosphoinositol-3-kinase (PI3K), a negative regulator of TF expression in response to endotoxin.

Dendrimer-Induced Leukocyte Procoagulant Activity Depends on Particle Size and Surface Charge

Thrombogenicity associated with the induction of leukocyte procoagulant activity (PCA) is a common complication in sepsis and cancer. Since nanoparticles are increasingly used for drug delivery, their interaction with coagulation systems is an important part of the safety assessment. The purpose of this study was to investigate the effects of nanoparticle physicochemical properties on leukocyte PCA, and to get insight into the mechanism of PCA induction. A total of 12 formulations of polyamidoamine (PAMAM) dendrimers, varying in size and surface charge, were studied in vitro using recalcification time assay. Irrespective of their size, anionic and neutral dendrimers did not induce leukocyte PCA in vitro. Cationic particles induced PCA in a size- and charge-dependent manner. The mechanism of PCA induction was similar to that of doxorubicin. Cationic dendrimers were also found to exacerbate endotoxin-induced PCA. PAMAM dendrimer-induced leukocyte PCA depends on particle size, charge and density of surface groups.

Consumption of plasma factor VII in a rabbit model of non-overt disseminated intravascular coagulation

Introduction: We have recently described an experimental animal model of non-overt disseminated intravascular coagulation (DIC) in the rabbit in which the induction of tissue factor (TF) mRNA and TF antigen expression in peripheral blood leukocytes (PBL) was demonstrated to occur within 2 h of administration of low-dose endotoxin [Hematol. J. 2 (2001) 188]. In the present study, we demonstrate that the leukocyte TF expressed has procoagulant activity leading to a rapid decline in the concentration of factor VII (FVII) in rabbit plasma. Methods: Total plasma FVII antigen and FVIIa were quantitated by rabbit FVII-specific immunoassay and FVIIa-specific clotting assays, respectively. Plasma samples from either saline-injected rabbits or rabbits administered a single bolus of 10 μg/kg Salmonella lipopolysaccharide were compared over a 24-h period. Results: Total plasma FVII antigen decreased progressively post-endotoxin injection, reaching 71% of the baseline concentration at 8 h ( p<0.001, n=18), and remained low (78%) at 24 h post-injection ( p<0.01, n=16), returning to normal by 48 h. Plasma FVIIa levels increased to 120% within 2 h of endotoxin injection, fell to 73% of the baseline concentration at 8 h ( p<0.05, n=18) and returned to normal by 24 h post-endotoxin administration. Procoagulant activity of rabbit peripheral blood leukocytes was enhanced at 2 h ( p<0.01, n=6) and 4 h ( p<0.05, n=6) post-endotoxin injection. The prothrombin time (PT) was increased by <3 s, and thrombin–antithrombin (TAT) complex formation was not significantly increased in the plasma of endotoxin-treated rabbits. No significant changes in total plasma FVII antigen, FVIIa or leukocyte procoagulant activity were observed in rabbits treated with saline. Conclusions: We conclude that the activation of FVII to FVIIa and rapid consumption of total FVII/FVIIa occur very early and likely are integral events linked to the initiation and propagation of non-overt DIC induced by endotoxin.

Procoagulant inflammatory responses of monocytes after direct balloon angioplasty in acute myocardial infarction

This study sought to investigate monocyte procoagulant activity and Mac-1 expression after successful percutaneous transluminal coronary angioplasty (PTCA) in acute myocardial infarction (AMI). An increased leukocyte count is an important risk factor for subsequent adverse cardiac events in AMI. Cellular procoagulant responses may contribute to the risk of thrombotic events after AMI. In 20 patients with AMI serial venous blood samples were obtained before, 4, 8 hours, and daily after direct PTCA. Twenty patients with elective PTCA served as a control group. We measured leukocyte procoagulant activity with a 1-stage clotting assay, Mac-1 expression of monocytes by flow cytometry, concentrations of tumor necrosis factor-α, interleukin (IL)-1β, IL-6, and IL-8 using immunoassays. Forty-eight hours after PTCA in patients with AMI, an increase in systemic IL-6 and C-reactive concentrations was found (p = 0.001, p = 0.008) associated with an increase in monocyte Mac-1 expression by 49 ± 18% (p = 0.04) and followed by an increase in monocyte procoagulant activity by 140 ± 63% 72 hours after PTCA (p = 0.01). None of these changes were detectable in the control group. No changes in the concentrations of the cytokines IL-1β, tumor necrosis factor-α, or IL-8 were found. The present study demonstrates an increase in procoagulant activity along with an increase in Mac-1 expression on circulating monocytes after successful PTCA in AMI associated with an increase in systemic IL-6. These cellular procoagulant responses may limit the clinical benefit from timely reperfusion.

Cellular Mechanisms of Atherogenesis

The study of vascular cell function and the interactions of endothelial cells (EC), smooth muscle cells (SMC), and monocyte-derived macrophages has expanded greatly during the past 20 years, and the resultant information has reformed our views on the genesis of atherosclerotic plaque. The concept of an activated or injured endothelium that exhibits properties distinct from healthy adult endothelium is now well accepted. Activated EC may exhibit proatherogenic behavior, including increased leukocyte adhesivity, procoagulant activity, and SMC mitogen production. Thrombin, a coagulation-system protease, may serve as a physiologic activator of EC. Thrombin at sites of vascular injury may stimulate diverse functions, including increased expression of monocyte adhesion proteins and platelet-derived growth factor (PDGF). The monocyte-derived macrophage has been implicated as a participant in several aspects of atherosclerotic plaque development. The attachment of monocytes to EC is the initial event in the interaction of these cells with the vessel wall. Distinctly focal adhesion of monocytes to EC of large vessels is one of the earliest documented events in experimentally induced atherosclerosis and, thus, regulation of this process may be critical to the development of the disease. Intimal proliferation of SMC is another hallmark of the atherosclerotic lesion. Platelet-derived growth factor is both a chemoattractant and mitogen for SMC. Therefore, if EC secrete PDGF abluminally, both the migration of SMC into the intima and subsequent proliferation will be stimulated. Immunocytochemistry and insitu hybridization have verified that vascular EC express PDGF mRNA and protein in vivo under certain conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced procoagulant activity of mononuclear leukocytes in patients with atopic dermatitis and psoriasis.

Fibrin deposition is an important histopathological feature of inflammatory skin lesions and is mediated in part, by procoagulants generated by mononuclear leucocytes (MNL). We examined whether MNL from patients with atopic dermatitis or psoriasis generate enhanced procoagulant activity (PCA). MNL isolated from the peripheral blood of 15 healthy control individuals, 15 patients with atopic dermatitis and 15 patients with psoriasis were incubated for 24 h in the presence or absence of bacterial lipopolysaccharide (LPS). MNL or the cell culture supernatants were then added to recalcified human plasma to determine the clotting time. We found that in both atopic dermatitis and psoriasis MNL cultured in the presence or absence of LPS expressed greatly enhanced PCA (p < 0.01 to < 0.002). Supernatants from MNL cultures from patients with psoriasis, but not those from patients with atopic dermatitis, also generated augmented PCA (p < 0.002). In psoriasis, PCA normalized after successful topical treatment with anthralin. We conclude that enhanced PCA is a characteristic feature of MNL in both atopic dermatitis and psoriasis. In psoriasis the enhanced PCA is directly related to disease activity.

Cell-mediated immunity to orbital tissue antigens in thyroid-associated ophthalmopathy determined using the leukocyte procoagulant activity assay.

While it is generally accepted that thyroid-associated ophthalmopathy (TAO), a progressive inflammatory disorder of the extraocular muscle and orbital connective tissue (OCT), is immunologically mediated the nature of the underlying abnormalities is poorly understood. Although there is considerable evidence for antibody-mediated immunity against both eye muscle (EM) and OCT antigens in TAO a role of cell-mediated immunity (CMI) has not been studied in detail. We have used a new sensitive test for CMI, the leukocyte procoagulant activity (LPCA) assay and tested blood leukocytes from patients with TAO and autoimmune thyroid disease (ATD) without evident ophthalmopathy for reactivity against pig eye muscle (PEM) and human thyroid and OCT membrane and soluble fractions. In some cases human EM fractions were also tested. Preparations of PEM membrane (PEMM) and human thyroid membrane induced a significant LPCA response in both groups of patients. PEM cytosol, human OCT membrane and cytosol and human spleen membrane did not evoke a significant response in either group of patients. There were significant positive correlations in patients with TAO between (i) LPCA in response to PEMM and that to human thyroid membrane and (ii) LPCA in response to human thyroid membrane and that to human EM membrane. In patients with TAO there were no significant associations between LPCA response to PEMM and the detection of serum antibodies to a 64 kDa EM membrane protein in immunoblotting, or between LPCA response to PEMM and the duration or severity of the ophthalmopathy or clinical evidence for eye muscle involvement. These findings confirm a role of cell-mediated immunity against eye muscle antigens in thyroid-associated ophthalmopathy.(ABSTRACT TRUNCATED AT 250 WORDS)

Haemorheology in Neonates: Procoagulant Activity in Leukocytes

Haemorheology in Neonates: Procoagulant Activity in Leukocytes

Effects of simulated extracorporeal circulation on human leukocyte elastase release, superoxide generation, and procoagulant activity

Activated leukocytes are thought to contribute to respiratory dysfunction, alterations in microvascular permeability, disseminated intravascular coagulation, and thrombosis, all of which can complicate extracorporeal circulation. The purpose of this work was to determine the effects of extracorporeal circulation on leukocyte functions likely to mediate organ damage. White blood cell counts in the bubble circuits (n = 5) fell to 51% +/- 7% (mean +/- standard error of the mean; p less than 0.05) of initial levels within 2 hours of recirculation. In contrast, counts from both the spiral coil (n = 5) and hollow-fiber (n = 5) groups remained at 91% +/- 12% and 100%, respectively. Plasma levels of human neutrophil elastase rose from 0.28 +/- 0.06 micrograms/ml to 3.14 +/- 0.36 micrograms/ml (p less than 0.05) and 0.20 +/- 0.02 micrograms/ml to 1.61 +/- 0.35 micrograms/ml (p less than 0.05) in bubble and spiral coil circuits, respectively, but from only 0.20 +/- 0.03 micrograms/ml to 0.96 +/- 0.42 micrograms/ml in the hollow-fiber circuit despite 2 hours of recirculation. Consistently, in response to N-formyl-L-methionyl-L-leucyl-L-phenylalanine, a chemotactic peptide, cells from spiral coil and bubble circuits released and generated significantly less elastase and superoxide anion, respectively. In contrast, neutrophils from the hollow-fiber circuits demonstrated enhancement of N-formyl-L-methionyl-L-leucyl-L-phenylalanine-induced elastase release and superoxide generation. Finally, mixed leukocytes from all circuits expressed procoagulant activity reaching statistical significance in bubble circuits. In conclusion, extracorporeal circulation has pronounced effects on neutrophil elastase release, superoxide anion generation, and leukocyte procoagulant activity. Spiral coil and bubble oxygenators cause granule release and, subsequently, reduced sensitivity to soluble agonists. In contrast, hollow-fiber oxygenators "prime" cells, actually enhancing reactivity. Recirculation through all circuits induces leukocyte procoagulant activity that is likely to contribute to surface-induced thromboses and excessive bleeding.

Blood leukocytes bind platelet glycoprotein (lIb-Ilia)' but do not express the vitronectin receptor

Within the integrin family of Arg-Gly-Asp(RGD)-binding adhesion receptors, the subfamily defined by the beta chain known as beta-3 or glycoprotein (GP)IIIa is known to contain two individual receptors. These are the GPIIb-IIIa complex of platelets, where the alpha chain of the heterodimer is GPIIb, and the vitronectin receptor (VnR) containing the alpha V subunit. The presence of either GPIIb-IIIa and/or the VnR on blood leukocytes has been controversial. We have investigated this problem by performing immunoprecipitation and immunoblotting studies with rabbit and monoclonal antibodies (mAb) to each of the subunits of GPIIb-IIIa and the VnR. On cultured cells of different origin, it was established that almost all expressed the VnR but none had GPIIb-IIIa, and the only polypeptide associated with beta 3 was alpha V. Platelets expressed predominantly GPIIb-IIIa, and weakly, the VnR. Monocytes and neutrophils freshly isolated from blood did not express the VnR but bore on their surface a modified form of GPIIb-IIIa. This molecule appeared identical to GPIIb-IIIa but an epitope on GPIIb was masked on the intact cell and was only revealed after immunoblotting. We have termed this modified form of GPIIb-IIIa, GP(IIb-IIIa)'. With differentiation in culture, monocytes rapidly lost surface GP(IIb-IIIa)' and concurrently began to express the VnR. Evidence is presented that GP(IIb-IIIa)' is derived from particles released by activated platelets and is bound firmly to the leukocyte membrane. Its primary function does not seem to be to mediate attachment to matrix proteins; thus, although U937 cells bearing platelet-derived GP(IIb-IIIa)' bound fibrinogen in an RGD-dependent manner, isolated blood monocytes did not. It is suggested that this transfer of membrane proteins from platelets to monocytes and neutrophils may regulate the expression of the leukocyte VnR and also serve as a means of facilitating leukocyte procoagulant activity.

Pulmonary Immune Response of Dogs after Exposure to239PuO2

This study evaluated the cell-mediated (CMI) and humoral immune responses in four Beagle dogs five to six years after single inhalation exposures to different monodisperse 239PuO2 aerosols (0.72-1.4 microns activity median aerodynamic diameter). These exposures resulted in initial lung burdens ranging from 19 to 35 kBq. Four nonexposed dogs were used as age-matched controls. Anesthetized dogs were immunized by instillation of sheep red blood cells (SRBC) into selected lung lobes. Cells and fluids were obtained serially from blood samples and by bronchoalveolar lavage of the saline- and SRBC-treated lung lobes at 5-20 days after immunization. The CMI response evaluated by the leukocyte procoagulant activity test was similar in the saline- and SRBC-treated lobes of both groups of dogs. The humoral immune response was measured by the enzyme-linked immunosorbent assay. No differences were shown between the amount of antibody measured in the sera or lung lavages from control or Pu-exposed dogs. Histopathology of the tracheobronchial lymph nodes from the Pu-exposed dogs showed them to be fibrotic with no lymphoid cells, suggesting that these tissues could not respond to the antigen deposited in the lungs. However, both mediastinal and sternal lymph nodes did contain lymphoid tissue, and were likely to be the lymphoid tissues that produced the immunity to the antigen deposited in the lungs of the exposed dogs. Although both exposed and control dogs produced immune responses to the antigen instilled into their lungs, differences were observed in the number of neutrophils in lung lavages from the control and exposed animals. There was a dramatic influx of neutrophils into both the saline- and SRBC-treated lung lobes of the Pu-exposed dogs that was not seen in the age-matched controls. This suggests that the inhaled 239PuO2 produced chronically-active inflammation in the lung which may contribute to recruitment of lymphocytes to the lung following intrapulmonary deposition of antigen. In conclusion, the immune responses induced by lung immunization of dogs that had inhaled 239PuO2 were not suppressed by large doses of chronic alpha irradiation of the lungs and tracheobronchial lymph nodes, indicating that local pulmonary immune responses are preserved despite severe radiation-induced alteration of these tissues.

Induction of procoagulant activity in human leukocytes with lipoglycans from mycoplasmas.

Lipoglycans from mycoplasmas possess the ability to induce the elaboration of procoagulant activity. This represents a third endotoxic property for these polymers, in addition to the pyrogenicity and clotting of Limulus lysate reported previously. Lipoglycans from three species of Acholeplasma (A. axanthum, A. granularum, and A. oculi) stimulated the elaboration in vitro of procoagulant activity by human mononuclear cells to differing extents. Two serotypes, 3 and 4, of Ureaplasma urealyticum also exhibited this stimulatory activity. The activities of lipoglycans tested were, in decreasing order, A. oculi greater than U. urealyticum type 4 greater than A. axanthum greater than U. urealyticum type 3 greater than A. granularum.

Virus excretion and cell-mediated immunity during cytomegalovirus infection among healthy infants and children.

Specific cell-mediated immunity (CMI) and virus isolation were examined longitudinally to clarify the mechanism of the cessation of virus excretion in inapparent cytomegalovirus (CMV) infection among healthy infants and children. We measured leukocyte procoagulant activity (LPCA) responses to CMV antigen and to a purified protein derivative of tuberculin (PPD), with results expressed as a percentage reduction of recalcification (RC) time. Based on the results in seropositive and seronegative adult control subjects, reductions in RC time of more than 10% were considered indicative of a positive LPCA response. The CMV-specific LPCA response was negative in all infants shedding the virus, despite the presence of circulating antibodies, but were converted from negative to positive when the virus excretion ceased. This suggests that cessation of the virus excretion in inapparent CMV infection among healthy infants and children probably results from the specific CMI.

Procoagulant activity in renal transplant recipients.

Procoagulant activity (PCA) of leukocytes of renal transplant recipients was studied. This material, which activates coagulation, has previously been shown to be released from macrophages after they interact with mitogen-stimulated or antigen-stimulated T lymphocytes. Under endotoxin-free conditions, PCA of peripheral blood leukocytes, incubated for 90 min in tissue culture, was elevated in postoperative transplant recipients and in many transplant patients tested around the time of a rejection episode. The response to lipopolysaccharide added during culture was also increased in these populations. The PCA response was factor-VII-dependent when tested with washed peripheral blood mononuclear cells (PBMC), but was factor-VII-independent when tested with unwashed PBMC in their original culture medium. The results indicate a possible link between immunologic events and coagulation in transplant recipients.