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The anticoagulant agent heparin inhibits thrombosis by interacting with antithrombin III. There are three distinct chemical elements that make up the heparin precursor molecule: Mucopolysaccharides, amino acids, and sugars. Separate from the chemical elements are three unique domains of activity that have been defined based on the functional interaction of heparin with antithrombin III. Bleeding complications are potential side effects of heparin therapy and may be caused by heparin interactions with platelets. However, reducing the size of the molecule (low-molecular weight heparins) and varying the dosage may reduce the risk of excessive bleeding.

Little is known about the influence of cigarette smoking on the ability to smell; previous studies on this topic have led to contradictory findings and have failed to take into account smoking dose and duration. In the present study, the 40-odorant University of Pennsylvania Smell Identification Test was administered to 638 subjects for whom detailed smoking histories were available. Smoking was found to be adversely associated with odor identification ability in a dose-related manner in both current and previous cigarette smokers. Among previous smokers, improvement in olfactory function was related to the time elapsed since the cessation of smoking. Logistic regression analysis found current smokers to be nearly twice as likely to evidence an olfactory deficit than persons who have never smoked. Overall, the data suggest that (1) smoking causes long-term but reversible adverse effects on the ability to smell and (2) the failure of some studies to demonstrate smoking effects may be caused by the inclusion of persons with a history of smoking in the nonsmoking groups.

To assess the effects of low-level organic solvent exposure on olfactory function, a cross-sectional study in paint manufacturing workers was undertaken. Workers in two paint manufacturing facilities (N = 187) were tested using the University of Pennsylvania Smell Identification Test (UPSIT), a standardized, quantitative test of olfactory function. Industrial hygiene air samples over the past 13-15 years revealed that average solvent exposures in these plants were 2-40% of the existing threshold limit values for the three chemicals measured. Stratification by smoking status revealed evidence of dose-related decrements in olfactory function (p = .01) only in non-smokers. Furthermore, those nonsmoking workers in the highest exposure category had UPSIT scores below the fifth percentile for their age. These results suggest that solvents may cause nervous system dysfunction at lower levels than previously suspected, and that the olfactory system may be a critical target organ for the neurotoxic effects of solvents and other chemicals.

Murine lymphoid cells respond rapidly to bacterial lipopolysaccharide or antigen-antibody complexes to initiate or accelerate the blood coagulation pathways. The monocyte or macrophage has been identified as the cellular source, although lymphocyte collaboration is required for the rapid induction of the procoagulant response. This procoagulant activity is identified in the present study as a direct prothrombin activator, i.e., a prothrombinase. Studies with plasmas deficient in single coagulation factors demonstrate that the induced murine procoagulant activity effector molecule does not require factors XII, VIII, VII, X, or V, but does require prothrombin to transform fibrinogen to fibrin. This enzyme(s) produces limited proteolysis of prothrombin to yield thrombin or thrombinlike products that are functionally capable of converting fibrinogen to fibrin. The prothrombinase is undetectable in freshly isolated Murine lymphoid cells respond rapidly to bacterial lipopolysaccharide or antigen-antibody complexes to initiate or accelerate the blood coagulation pathways. The monocyte or macrophage has been identified as the cellular source, although lymphocyte collaboration is required for the rapid induction of the procoagulant response. This procoagulant activity is identified in the present study as a direct prothrombin activator, i.e., a prothrombinase. Studies with plasmas deficient in single coagulation factors demonstrate that the induced murine procoagulant activity effector molecule does not require factors XII, VIII, VII, X, or V, but does require prothrombin to transform fibrinogen to fibrin. This enzyme(s) produces limited proteolysis of prothrombin to yield thrombin or thrombinlike products that are functionally capable of converting fibrinogen to fibrin. The prothrombinase is undetectable in freshly isolated

A panel of human purified protein derivative of the tubercle bacillus (PPD)-reactive T cell clones was derived by cloning out of soft agar followed by cultivation on inactivated feeder cells in the presence of interleukin-2. 1 of 4 clones tested was able to mediate an increase in monocyte procoagulant activity (PCA) in response to PPD. All four clones had identical surface marker phenotypes (T4+, T8-) and proliferated in response to antigen. The reactive T cell clone possessed no PCA of its own, but upon being presented with PPD was able to instruct monocytes to increase their expression of PCA. Antigen presentation could be performed only by autologous monocytes; allogeneic monocytes from donors unrelated to the donor of the reactive clone could not present antigen to cells of the clone in a way that would initiate the procoagulant response. Cells of the reactive clone did not mediate increased monocyte PCA in response to Candida, even though peripheral blood mononuclear cells from the donor demonstrated increased PCA to both Candida and PPD. Thus, the PCA response to specific antigen can be mediated by a single clone of cells that shows specificity in the recognition of both antigen and antigen presenting cell.

Fibrin is a hallmark of immune-mediated tissue lesions. The presence of fibrin in such lesions implies both the formation of fibrin via coagulation and the accompanying restriction of fibrinolysis, allowing fibrin to persist. Previous work has shown that human monocytes exposed to an inflammatory stimulus such as lipopolysaccharide (LPS) produce both tissue factor (TF) and plasminogen activator inhibitor--type 2 (PAI-2). These two proteins favor fibrin deposition, and evidence implies that cellular production of these two molecules may be linked. Another proinflammatory process pertinent to immune-mediated tissue damage and fibrin deposition is the response to alloantigen. Peripheral-blood mononuclear cells (PBM), consisting of lymphocytes and monocytes together, responded to alloantigen stimulation with differential expression of TF and PAI-2. PBM exposed to alloantigen developed high levels of TF activity, with no concomitant increase in PAI-2 activity or antigen. Alloantigen-stimulated PBM did not accumulate intracellular PAI-2, nor did they degrade PAI-2 added to cultures. This lack of PAI-2 production was not due to inadequate stimulation, as tritiated thymidine uptake and TF production demonstrated recognition of, and a vigorous reaction to, alloantigen. The divergent TF and PAI-2 responses of PBM exposed to alloantigen was maintained over 5 days and was reflected by mRNA profiles. These results imply that under specific physiologically relevant conditions, the procoagulant and antifibrinolytic effectors of inflammatory mononuclear cells can be independently regulated. This would imply more flexibility to monocyte mechanisms that favor fibrin deposition than previously thought.

Isolated human plasma very low density, intermediate density, and high density lipo-proteins at physiologic concentrations have been demonstrated in the preceding report to induce significant increases in the procoagulant activity of human peripheral blood mononuclear cells in vitro, whereas low density lipoprotein did not. The monocyte was identified in this study by cellular fractionation and by direct cytologic assays as the source of this inducible activity, thus identifying the procoagulant activity as a monokine. The generation of these lipoprotein-induced procoagulant monokines was entirely dependent upon the presence of lymphocytes. Isolated lymphocytes that had been exposed to the stimulatory lipoproteins could induce monocytes to produce the procoagulant activity, whereas neither the culture medium from lipoprotein-stimulated lymphocytes, homogenates of lymphocytes, nor other cells such as platelets could substitute for this requirement. The interaction of the stimulatory lipoproteins with lymphocytes was rapid, reaching completion within 30 min, and was equally effective at either 4 degrees or 37 degrees C. Low density lipoprotein did not stimulate lymphocytes to induce monocyte procoagulant activity, but did actively suppress the production of the procoagulant monokines induced by each of the stimulatory lipoproteins, as well as bacterial lipopolysaccharide. The monocyte was identified as the cell sensitive to low density lipoprotein suppression, and no suppression of lymphocyte triggering was observed. These observations on the interaction of plasma lipoproteins with lymphocytes and monocytes in vitro introduce two new regulatory events by which plasma lipoproteins influence the function of cells, and define a regulatory network by which certain lipoprotein classes trigger lymphocytes, which can in turn induce monocytes to express procoagulant activity. Only this latter phase is subject to lipoprotein suppression by physiologic concentrations of low density lipoprotein.

An investigation of the olfactory function of 731 workers at a chemical facility which manufacturers acrylates and methacrylates was undertaken using a standardized quantitative test. In a cross-sectional analysis of the data, no associations of chemical exposure with olfactory test scores were observed. A nested case-control study designed to evaluate the cumulative effects of exposure on olfactory function, however, revealed elevated crude exposure odds ratios (95% confidence interval) of 2.0 (1.1, 3.8) for all workers and 6.0 (1.7, 21.5) for workers who never smoked cigarettes. Logistic regression analysis, adjusting for multiple confounders, revealed exposure odds ratios of 2.8 (1.1, 7.0) and 13.5 (2.1, 87.6) in these same groups, respectively, and a dose-response relationship between olfactory dysfunction and cumulative exposure scores--semi-quantitative indices of lifetime exposure to the acrylates. The data also revealed decreasing exposure odds ratios with increasing duration since last exposure to these chemicals, suggesting that the effects may be reversible.

Light and scanning electron microscopic studies of blood from five patients with drug-induced oxidant hemolysis are presented. None of the patients had a previous history of hemolytic disease and laboratory studies indicated no evidence of either glucose-6-phosphate dehydrogenase (G6PD) deficiency or unstable hemoglobinopathy. Although the red cell deformities in our patients overlapped to some extent with those reported in patients with microangiopathic hemolytic anemia (MAHA) and in patients with G6PD deficiency undergoing oxidant hemolysis, striking differences were also observed. Cell fragments, commonly found in patients with MAHA, and eccentrocytes, frequently found in patients with G6PD deficiency undergoing oxidant hemolysis, were seldom found in blood samples from the five patients in this study. Bite cells were extremely common in our patients. They are rare in patients with either of the above disorders. An awareness of the morphologic abnormalities detailed in this report may help characterize the nature of a hemolytic process so that appropriate therapy can be initiated.

PRB was measured in standardized skin puncture wounds. Platelets in wound blood samples collected in EDTA-heparin-containing micropipettes were counted electronically following gravity separation of plasma. The relationship between the wound platelet count (expressed as percent of the venous blood platelet count) and time was found to be linear during the first 3 min of bleeding. The slope of the linear regression line for percent of venous blood platelet count vs. time was 20.8 +/- 8.9 (mean +/- S.D.) in 31 normal subjects. Nine of 10 patients with significant, nonpharmacologic platelet dysfunction were found to have markedly decreased PRB. The determination of PRB was found to be reproducible, and control studies excluded platelet clumping and sedimentation of platelets as factors which significantly influenced the results. In contrast to previous observations, the results of this study indicate that platelets are removed from circulating blood at an increasing rate during the first 3 min of bleeding in normal subjects. Furthermore, a significant degree of EDTA-reversible clumping of platelets normally occurs during bleeding. The method described permits an accurate assessment of the quantitative and dynamic aspects of platelet participation in the arrest of bleeding. The determination of PRB holds promise in the evaluation of the biological significance of in vitro evidence of platelet dysfunction, and it may be useful in the evaluation of patients with hemorrhagic and thromboembolic disorders.

The present study explores the interactions between lymphocytes and monocytes that are required for expression of procoagulant activity (PCA) by monocytes in response to purified protein derivative of the tubercle bacillus (PPD) or tularemia antigen. The PCA response was antigen specific: peripheral blood mononuclear cells (PBM) from donors sensitive to PPD or tularemia showed an increase in PCA only in response to the sensitizing antigen. The PCA was tissue factorlike in that Factors VII and X were required for expression of the activity, whereas Factor VIII was not. Maximum PCA developed only after 36 to 72 h. Fractionation of PBM into lymphocytes and monocytes after antigenic stimulation demonstrated that greater than 90% of the PCA was associated with monocytes. Isolated monocytes or lymphocytes incubated with sensitizing antigen had the same PCA as control cells. Purified lymphocytes that had been pulsed with antigen were unable to elicit a PCA response from monocytes to which they were added. However, adherent monocytes incubated with antigen, then washed free of unbound protein, were able to trigger lymphocytes to become stimulatory for PCA toward responding monocytes. The development of antigen-specific PCA in PBM could be blocked by including a monoclonal antibody to HLA-DR antigen in the incubation. The antibody had no effect on the clotting assay, on preformed PCA, cell viability, or on stimulatory antigen itself. These results indicate that elaboration of PCA by mononuclear cells may be an intrinsic part of the classical immune response to antigen, and may explain the presence of fibrin in immune lesions, as well as the occurrence of thrombotic complications in many immune disorders.

Both aggregated IgG and soluble immune complexes composed of human serum albumin and specific antibody at a 4:1 ratio were demonstrated to rapidly induce an increase in procoagulant activity (PCA) of murine peripheral blood mononuclear cells. Direct cytologic assays as well as fractionation of cells by adherence to plastic established the monocyte as the cell producing PCA, and demonstrated further that almost all monocytes, rather than a subpopulation, participated in the response. Monocytes that were depleted of lymphocytes did not respond to these stimuli with production of PCA; that is, induction of monocyte PCA was observed only when stimulation was performed in the presence of lymphocytes. Induction was equally efficient when only the lymphocyte was stimulated by aggregated IgG or soluble immune complexes, washed free of unbound stimulus, and added to monocytes. This suggests a unidirectional pathway by which the stimulus triggers lymphocytes after which there is direct lymphocyte-mediated induction of monocytes to produce PCA. Evidence for participation of soluble lymphocyte products was not obtained, and it is suggested that this series of events may be mediated by direct lymphocyte contact with responding monocytes. The role of this pathway in the genesis of immunologically initiated tissue lesions is discussed.

Peripheral blood mononuclear cells (PBMs) produce both tissue factor and plasminogen activator inhibitor type 2 (PAI-2) in response to gram-negative bacterial lipopolysaccharide (LPS). The cellular roles in the tissue factor response have been previously elucidated, and we now report those roles in PAI-2 production. Monocytes are the only cells among LPS-stimulated PBMs that produce PAI-2 as assessed by measurement of PAI-2 activity and antigen. Concomitant immunohistochemistry demonstrated that monocytes contain PAI-2, with a greater number staining positively and more intensely after exposure to LPS. LPS-stimulated monocytes produced increased amounts of PAI-2 with or without addition of lymphocytes. Lymphocytes prestimulated with LPS and then washed did not induce PAI-2 production in monocytes to which they were added. Lipid X, a precursor in the biosynthetic pathway of lipid A and LPS, was able to inhibit LPS induction of monocyte PAI-2 in a dose-dependent manner. This inhibition was not due to cellular toxicity, the phospholipidlike nature of lipid X, interference with the PAI-2 assay, or monocyte production of a substance interfering with PAI-2. Lipid X was an effective inhibitor of PAI-2 production even when added up to 30 minutes after LPS.

To evaluate the interlaboratory and intralaboratory agreement in the performance of Lyme disease serological testing, we sent serum specimens from 132 outdoor workers in New Jersey to as many as four independent laboratories. These included one state department of health laboratory, one large commercial laboratory, and two research laboratories. The measurement of agreement employed, the kappa statistic, ranged from .45 to .53 among the four laboratories and from .50 to .54 within the commercial laboratory. These values represent low levels of agreement. The data suggest that Lyme disease serological testing procedures should be standardized so that Lyme disease test results are more comparable between laboratories.

Monocytes produce thrombospondin (TSP), a trimeric glycoprotein whose primary function is not yet clear. Platelet-poor monocytes (less than 1 platelet/50 monocytes) cultured with [35S]methionine produced [35S] TSP barely detectable by immunoprecipitation with either monoclonal or polyclonal antibody to TSP. Platelet-poor monocytes that had not been so thoroughly depleted of platelets (6-12 platelets/50 monocytes) synthesized readily detectable amounts of [35S] TSP. Addition of increasing numbers of washed platelets to platelet-poor monocytes resulted in increasing synthesis of [35S]TSP. This monocyte-platelet interaction was specific for cell type; neither neutrophils nor red cells could substitute for platelets. The induction of synthesis was specific for TSP; monocyte synthesis of three other proteins was not induced upon the addition of platelets. Platelet lysate or thrombin-induced platelet releasate could not substitute for intact platelets. In fact, platelet lysate inhibited [35S]TSP synthesis by monocyte-platelet cultures. This inhibition was not due to endotoxin contamination, interference with immunoprecipitation, or dilution of the [35S]methionine pool. Platelets required contact with monocytes to exert their effect, as culturing the cell populations with a filter between them prevented increased [35S]TSP synthesis. Monocyte-platelet interactions may serve to specifically increase monocyte synthesis of the adhesive protein, TSP.

The phenotypic and ultrastructural characterization of the blast cells from a T-cell acute lymphoblastic leukemia (T-ALL) that was associated with disseminated intravascular coagulation (DIC) is described. The bone marrow blasts were considered to represent neoplastic medullary thymocytes and were acid phosphatase (+), terminal deoxynucleotidyl transferase (-), acid alpha-naphthyl acetate esterase (-), E-rosette (+) at 4 degrees C and 37 degrees C, Fc- and C3-receptor (-), and cALLA-, Ia-, 9.6+, OKT3+, OKT4+, OKT6+/-, OKT8+, OKT10+. On transmission electron microscopic study, the predominant cell was 6 micron in diameter and possessed an irregular nucleus, moderate-sized nucleolus, marginated heterochromatin, abundant Golgi elements and granules, and prominent intermediate filaments. The cells were analyzed with normal and factor-deficient human plasmas and contained significant amounts of tissue factor-like procoagulant activity. This is the first report of a well-characterized medullary thymocyte T-ALL in which DIC was an accompanying feature, and the first demonstration of tissue factor-like procoagulant activity in acute lymphoblastic leukemia. In view of thrombohemorrhagic phenomena observed in association with other medullary thymocyte leukemias, these findings suggest that tissue factor-like procoagulant activity may be a characteristic of medullary thymocyte-derived lymphoid leukemias.

In the process of analyzing the effects of lipoproteins on functions of lymphoid cells, it was observed that physiological concentrations of isolated human plasma lipoproteins possess varying capacities to rapidly enhance the expression of procoagulant activity of human peripheral blood mononuclear cells in vitro. In a strict dose-dependent fashion, very low density lipoprotein, intermediate density lipoprotein, and high density lipoprotein enhanced both the surface expression by viable cells and the total cellular content of procoagulant activity during a 6-h incubation. Very low density lipoprotein induced a maximal 6.7-fold increase in the expression of a thromboplastin activity, which was consistent with tissue factor, in that it was dependent on Factors VII, X, and II. Both intermediate density lipoprotein and high density lipoprotein induced approximately a 12-fold increase of a different procoagulant activity which appears to be a direct prothrombin activator. This prothrombinase was calcium dependent and was inhibited by 2.5 mM diisopropylfluorophosphate, but was not neutralized by anti-Factor X antibodies or by inhibitors of Factor Xa. In contrast to the other lipoprotein density classes, low density lipoprotein did not stimulate procoagulant activity, but instead actively suppressed the generation of the two procoagulant activities induced by the stimulatory lipoproteins. Suppression by low density lipoprotein was clearly evident at molar ratios of low density lipoprotein to stimulatory lipoproteins of 1:3 or less. Reconstitution of all lipoproteins to physiological concentrations was not stimulatory as a consequence of the suppressive effects of low density lipoprotein. These data indicate that isolated plasma lipoproteins are capable of regulating the expression of two different procoagulant activities of peripheral blood mononuclear cells in vitro. The possibility that these interactions may be implicated in the association between certain types of hyperlipoproteinemias and thromboembolic disease merits study.

Platelet consumption is a prominent feature of disseminated intravascular coagulation. We investigated whether monocyte procoagulant activity (PCA) might play a role in platelet consumption associated with gram-negative septicemia. Human mononuclear cells exposed in vitro to lipopolysaccharide demonstrated parallel dose-dependent increases in PCA and ability to induce platelet aggregation. Induction of platelet aggregation required the generation of thrombin dependent on coagulation Factors VII, X, and II, and calcium. This is consistent with monocyte tissue factor initiating thrombin generation. A specific monoclonal antimonocyte antibody was used to identify monocytes via indirect immunofluorescence, and demonstrated that all monocytes were included in platelet aggregates. Mononuclear cells that did not express PCA did not induce platelet aggregation and monocytes were not surrounded by platelet clumps. These data suggest that monocytes induced to express tissue factor on their surface may be important mediators of endotoxin-induced platelet, as well as fibrinogen, consumption.

Patients with acute promyelocytic leukemia often develop bleeding diatheses during treatment. In seven patients who had this disease, the plasma level of alpha-2-plasmin inhibitor was the best predictor of severity of coagulopathy and bleeding. Clinical bleeding occurred when alpha-2-plasmin inhibitor levels measured less than 30% of normal levels. Patients with acute promyelocytic leukemia who had acquired deficiencies of alpha-2-plasmin inhibitor were considered to have deficits similar to those in persons congenitally deficient in alpha-2-plasmin inhibitor, and were assumed to be at increased risk for bleeding. Treatment with the fibrinolytic inhibitor, epsilon-aminocaproic acid, along with heparin resulted in prompt cessation of bleeding, reversal of laboratory evidence of fibrinolysis, and a decreased need for blood product support. The only thrombotic complication--thrombosis around a central venous catheter--resolved when treatment with epsilon-aminocaproic acid was discontinued. Epsilon-aminocaproic acid is a safe and effective therapy for those patients with acute promyelocytic leukemia who develop coagulopathy associated with low levels of alpha-2-plasmin inhibitor.

It has previously been described that soluble antigen:antibody complexes in antigen excess can induce an increase in the procoagulant activity of human peripheral blood mononuclear cells. It has been proposed that this response may explain the presence of fibrin in immune complex-mediated tissue lesions. In the present study we define cellular participants and their roles in the procoagulant response to soluble immune complexes. Monocytes were shown by cell fractionation and by a direct cytologic assay to be the cell of origin of the procoagulant activity; and virtually all monocytes were able to participate in the response. Monocytes, however, required the presence of lymphocytes to respond. The procoagulant response required cell cooperation, and this collaborative interaction between lymphocytes and monocytes appeared to be unidirectional. Lymphocytes once triggered by immune complexes induced monocytes to synthesize the procoagulant product. Intact viable lymphocytes were required to present instructions to monocytes; no soluble mediator could be found to subserve this function. Indeed, all that appeared necessary to induce monocytes to produce procoagulant activity was an encounter with lymphocytes that had previously been in contact with soluble immune complexes. The optimum cellular ratio for this interaction was four lymphocytes per monocyte, about half the ratio in peripheral blood. The procoagulant response was rapid, reaching a maximum within 6 h after exposure to antigen:antibody complexes. The procoagulant activity was consistent with tissue factor because Factors VII and X and prothrombin were required for clotting of fibrinogen. WE propose that this pathway differs from a number of others involving cells of the immune system. Elucidation of the pathway may clarify the role of this lymphocyte-instructed monocyte response in the Shwartzman phenomenon and other thrombohemorrhagic events associated with immune cell function and the formation of immune complexes.

The Shwartzman reaction is a classic biologic response in which the coagulation system is activated in vivo. Cellular initiation of the extrinsic coagulation protease cascade can be mediated by one or more limbs of the lymphoid response to diverse biological stimuli. The T cell-instructed monocyte and macrophage responses that have been implicated are mediated by a number of different cellular pathways and are elicited not only by antigens and allogeneic cells but also by other stimuli such as immune complexes and the lipid A moiety of bacterial lipopolysaccharide (LPS). The latter response has been implicated in the pathogenesis of the disseminated intravascular coagulation associated with bacterial infection. In the rapid collaborative cellular pathway response to LPS, we have described a relatively rigorous requirement for T helper cells in induction of the biosynthesis of tissue factor and Factor VII by monocytes. To elucidate potential regulatory aspects of this cellular procoagulant response, we provide the first evidence for the existence of T suppressor cells for the cellular procoagulant response to LPS by the rapid T cell-instructed pathway. Human peripheral blood lymphocytes were separated by cytoaffinity into Fc gamma-positive and Fc mu-positive cells and were characterized for their functional properties in the procoagulant response. T mu cells mediated the monocyte response, consistent with their identity with instructor cells. T gamma cells suppressed the response of monocytes to LPS in the presence of T mu cells, suggesting that they possess suppressor function for this response. The T gamma suppressor cells required stimulation by LPS to express their suppressor function and they exerted their suppressive effect directly on the monocyte. The existence and participation of LPS-responsive T suppressor cells on the cellular procoagulant response in vitro add a new dimension to the complexity of the rapid pathway of the collaborative cellular procoagulant response and may be important in the pathogenesis of disseminated intravascular coagulation.

Motivated by complaints of respiratory symptoms at a large state pheasant breeding farm, an evaluation of the health hazards of pheasant handling was undertaken. Pheasant workers (N = 24) were compared with age-, sex-, and smoking-status-matched controls (N = 23) without occupational or home exposure to birds by a respiratory questionnaire, pulmonary function testing (before and after the season and pre- and post-shift), a number of fungal and chlamydial serology tests, and hypersensitivity immunoprecipitins. Despite a number of important differences in the prevalence of symptoms in the pheasant workers, no differences were found in the results of pulmonary function testing or serology tests. These data suggest that the symptoms were likely because of local irritant effects rather than via immunologic or infectious mechanisms.

Lyme disease, a multisystem illness caused by a spirochete (Borrelia burgdorferi) and transmitted to humans by ticks, is increasing in incidence. Outdoor workers in areas of endemic disease are at increased risk of infection. Increased recognition of this fact will be necessary to prevent the disease in working populations.

To determine the possible mechanism(s) promoting alveolar fibrin deposition in the adult respiratory distress syndrome (ARDS), we investigated the initiation and regulation of both fibrinolysis and coagulation from patients with ARDS (n = 14), at risk for ARDS (n = 5), and with interstitial lung diseases (ILD) (n = 8), and normal healthy individuals (n = 13). Bronchoalveolar lavage (BAL) extrinsic pathway inhibitor activity was increased in ARDS BAL compared with patients at risk for ARDS (P = 0.0146) or normal controls (P = 0.0013) but tissue factor-factor VII procoagulant activity was significantly increased in ARDS BAL compared with all other groups (P less than 0.001). Fibrinolytic activity was not detectable in BAL of 10 of the 14 patients with ARDS and low levels of activity were found in BAL of the other four ARDS patients. Depressed fibrinolysis in ARDS BAL was not due to local insufficiency of plasminogen; rather, there was inhibition of both plasmin and plasminogen activator. Plasminogen activator inhibitor 1 was variably detected and low levels of plasminogen activator inhibitor 2 were found in two ARDS BAL samples, but plasminogen activator inhibitor 2 was otherwise undetectable. ARDS BAL antiplasmin activity was, in part, due to alpha 2-antiplasmin. We conclude that abnormalities that result in enhanced coagulation and depressed fibrinolysis, thereby predisposing to alveolar fibrin deposition, occur in the alveolar lining fluids from patients with ARDS.

Human peripheral blood mononuclear cells (PBM) respond to lipopolysaccharide (LPS) with increased release of a plasminogen activator (PA) inhibitor. This response is dose dependent and parallels the LPS-induced expression of PBM tissue factor activity. The PA inhibitors of control and LPS-stimulated PBMs appear identical as both are identified by antibodies to PA inhibitor type 2 of human placenta, but not by antibodies to type 1 inhibitor of bovine aortic endothelial cells. The PA inhibitor is specific for urokinase type PA as determined by the 125I-fibrin plate assay, and direct cleavage of 125I-plasminogen; it does not effectively inhibit tissue-type PA. The inhibitor forms an active site-dependent complex with 125I-urokinase, which then demonstrates an increase in mol wt from 33 kd to 68 kd on reduced sodium dodecyl sulfate (SDS) polyacrylamide gels. PBMs neither secrete nor express active PA. Hence, the exposure of PBMs to LPS results in conditions highly favorable to fibrin deposition and persistence: increased procoagulant and antifibrinolytic activities, accompanied by no measurable PA. Such modulation of these effectors may be important in the pathogenesis of fibrin characteristically found in tissue lesions of endotoxin-initiated processes.

PRB was studied in normal human subjects before and after aspirin ingestion. Aspirin ingestion resulted in a prolongation of individual bleeding times greater than 2.4 min (greater than 2 S.D. beyond the group mean before aspirin) in 62.5% of 48 paired studies. The relationship of platelets retained vs. time was linear during the first 3 min of bleeding before and after aspirin. The mean PRB decreased from 22.1 +/- 9.2 to 9.6 +/- 8.6 (p less than 0.001) after aspirin ingestion. Subjects whose bleeding time was prolonged greater than 2.4 min had a significantly higher mean PRB before aspirin and a significantly greater mean decrease in PRB after aspirin than those whose bleeding time was prolonged less than or equal to 2.4 min. Aspirin ingestion reduced the number of EDTA-irreversible clumped platelets present in wound blood approximately 50% during the second and third minute of bleeding, but large numbers of EDTA-reversible platelet clumps were observed in wound blood before and after aspirin. Although platelet retention was significantly decreased during the first 3 min of bleeding after aspirin, the percent of venous blood platelets present in wound blood just prior to the arrest of hemorrhage was equal before and after aspirin. These observations indicate that aspirin prolongs the bleeding time by decreasing platelet clumping and slowing the rate of platelet thrombus formation in severed blood vessels. The presence of platelet clumps in wound blood after aspirin ingestion indicates that alternative mechanisms of platelet aggregation, independent of the arachidonate pathway of prostaglandin synthesis, proceed in vivo unaltered by aspirin.

Fox, Alfred E. (Warner-Lambert Research Institute, Morris Plains, N.J.), George L. Evans, Frank J. Turner, Benjamin S. Schwartz, and Ansel Blaustein. Stimulation of nonspecific resistance to infection by a crude cell wall preparation from Myocobacterium phlei. J. Bacteriol. 92:1-5. 1966.-Exposure of large quantities of viable Mycobacterium phlei to attrition in a colloid mill resulted in 90 to 95% disruption of the organisms. Isolation of the crude cell wall preparation was accomplished by centrifugation of the broken cells at 10,000 x g, resuspension of the sediment, and repeated centrifugation at 1,000 x g to remove intact cells. Single oral or parenteral doses of the cell wall preparation increased the resistance of mice and guinea pigs to experimental infection with Salmonella enteritidis, and of mice to Staphylococcus aureus, for prolonged periods after administration. Histological examination of the organs of mice treated orally or intraperitoneally revealed a lymphoid hyperplasia of the spleen and a Kupffer cell proliferation of the liver. The preparation was nontoxic to mice by the oral route at doses up to 5,000 mg/kg, and the intraperitoneal ld(50) was approximately 680 mg/kg.

Kronish, Donald P. (Warner-Lambert Research Institute, Morris Plains, N.J.), Raam R. Mohan, and Benjamin S. Schwartz. Distribution of radioactivity in autolyzed cell wall of Bacillus cereus during spheroplast formation. J. Bacteriol. 87:581-587. 1964.-Spheroplasts of Bacillus cereus strain T were produced from cells grown in the presence of uniformly labeled C(14)-glucose. At regular intervals during spheroplast formation, enzymatically degraded cell wall was isolated by a new procedure. Radioactivity of solubilized cell wall in cell-free material increased from 2.5 to 42% of the total incorporated label during spheroplast formation. The rate of cell-wall degradation as measured by increase in radioactivity was biphasic with relative slopes of 2.0 and 5.0. During autolytic depolymerization of B. cereus cell wall, two major components were solubilized at different rates. Chemical fractionation revealed these to be a peptide and a mucopeptide. The possibility of two enzymes being involved in spheroplast formation and cell-wall degradation is discussed.

Mohan, Raam R. (Warner-Lambert Research Institute, Morris Plains, N.J.), Donald P. Kronish, Roland S. Pianotti, Ray L. Epstein, and Benjamin S. Schwartz. Autolytic mechanism for spheroplast formation in Bacillus cereus and Escherichia coli. J. Bacteriol. 90:1355-1364. 1965.-Spheroplasts of Bacillus cereus strain T and Escherichia coli B were prepared by incubating early log-phase cells in appropriate buffers and stabilizers for 3 hr at 30 and 37 C, respectively. Upon incubation in 0.05 m tris(hydroxymethyl)aminomethane buffer osmotically stabilized with 16% polyethylene glycol at pH 7.5, 99% of the B. cereus cells formed spheroplasts; 90% of the E. coli cells were converted to spheroplasts in 0.4 m sodium acetate buffer osmotically stabilized with 1.6 m sucrose at pH 6.0. The extent of spheroplast formation was determined by phase-contrast microscopic examination, by measuring the rate of fall of optical density in the reaction mixture when subjected to osmotic shock, and by viable intact cell counts. The effect of a selected group of metabolic inhibitors on the autolytic system of B. cereus and E. coli has been examined. B. cereus and E. coli wall components comprising 26% of the dry weight of the original cellular material were recovered from dialyzed fractions by precipitation in 70% ethyl alcohol. Chemical and chromatographic analysis of cell-wall hydrolysates from B. cereus and E. coli indicated the presence of glucosamine, alanine, lysine, glycine, aspartic acid, diaminopimelic acid, glutamic acid, and muramic acid.

A Boivin preparation of Brucella abortus, unlike common enterobacterial endotoxins, failed to depress water intake or increase numbers of hemolysin-producing spleen cells in mice, or to cause delayed inflammatory reactions in rabbit skin. Reactivity to the B. abortus endotoxin was found only in animals which were previously given the endotoxin with, but not necessarily in, complete Freund's adjuvant. Previous treatment with the endotoxin in saline or with only the adjuvant was ineffective. Sensitization appeared within 10 days and waned after 5 weeks. Passive sensitization was obtained with sensitized donor spleen cells but not with serum. Serum antibody titers did not correlate with the appearance and disappearance of sensitization.

Fox, Alfred E. (Warner-Lambert Research Institute, Morris Plains, N.J.), Joachim Anschel, George L. Evans, Raam R. Mohan, and Benjamin S. Schwartz. Isolation of a soluble resistance-enhancing factor from Mycobacterium phlei. J. Bacteriol. 92:285-290. 1966.-Extraction of a crude cell wall preparation from Mycobacterium phlei with 20% urea yielded a fraction which induced a state of enhanced resistance to microbial challenge. The resulting soluble extract, after removal of the urea, represented a 15% yield of solids with the separation of the biologically active component(s) and elimination of toxicity. Single oral or subcutaneous submicrogram doses of this material induced a prolonged state of increased resistance to subsequent challenge with Salmonella enteritidis in mice. This effect appeared as early as 2 hr after oral administration and persisted for at least 30 days. Protection against experimental infection with Staphylococcus aureus was also demonstrated. Resistance to viral challenge with influenza type A was observed after intranasal administration of the M. phlei extract to mice. The isolated material was found to contain carbohydrate, protein, nucleic acids, and lipids. The lipids represented 60% of the total solids, and were all short-chain fatty acids. No toxic effects, including pyrogenicity, could be demonstrated after oral or parenteral administration of this preparation.