Early procoagulant shift in the bronchoalveolar compartment of patients with secondary peritonitis

BACKGROUND: In acute respiratory distress syndrome or pneumonia, a procoagulant shift is observed in bronchoalveolar lavage fluid (BALF). The effect of a primarily extrapulmonary infection on coagulation and fibrinolysis in the pulmonary compartment is unclear. METHODS: In 35 patients, 87 bronchoalveolar lavages were performed on the day of operation for secondary peritonitis (day 0) and on days 2 and 3 after surgery. Two noninfectious control groups were included: subjects undergoing bronchoalveolar lavage after elective surgery (n=8) and those undergoing long-term mechanical ventilation (n=10). RESULTS: In BALF from patients with peritonitis, a tissue factor (TF)/factor VIIa-mediated activation of coagulation was shown (high levels of thrombin-antithrombin complexes). Levels of fibrinolysis activators decreased rapidly after day 0, whereas levels of inhibitors increased. The net effect was reduced fibrinolysis (plasminogen activator activity). The sequential comparison of plasma levels of TF pathway inhibitor showed higher levels in patients who died (28-day mortality; P<.001). Sequential levels of TF in BALF were higher in patients with low PaO2 : FiO2 ratios (<200; P=.039). Differences between patients and control subjects were more pronounced in BALF than in plasma. CONCLUSIONS: Secondary peritonitis induces an early activation of the coagulation and inhibition of fibrinolysis in the systemic and bronchoalveolar compartments, possibly via a compartmentalized response. This imbalance may be associated with reduced oxygen delivery and an adverse outcome in secondary peritonitis.     

Fibrinopeptide A reactive peptides and procoagulant activity in bronchoalveolar lavage: relationship to rheumatoid interstitial lung disease

Extravascular, primarily, alveolar fibrin deposition is commonly associated with the alveolitis of many interstitial lung diseases including the interstitial lung disease associated with rheumatoid arthritis (RA). We therefore hypothesized that coagulation pathways, which promote fibrin formation, would be activated in the alveolar lining fluids of patients with rheumatoid interstitial lung disease. To test this hypothesis, we studied the bronchoalveolar lavage (BAL) fluids from patients with rheumatoid interstitial lung disease (n = 7) and patients with RA unassociated with interstitial lung disease (n = 10) to characterize and quantitatively compare the BAL procoagulant material and levels of fibrinopeptide A (FPA), which is cleaved from fibrinogen by thrombin. FPA reactive peptide concentrations were significantly greater in rheumatoid interstitial lung disease than RA when normalized per ml of concentrated BAL fluid (p = 0.02), per mg BAL total protein (p = 0.01) or BAL albumin content (p = 0.03) and correlated with BAL antigenic neutrophil elastase concentrations (r = 0.87). Procoagulant activity was present in similar concentration of BAL of patients with RA and rheumatoid interstitial lung disease and was mainly attributable to tissue factor associated with factor VII (or VIIa). Our results demonstrate that tissue factor and factor VII are endogenous in the alveoli of subjects with RA and interstitial lung disease and could interact with distal coagulation substrates which may enter the alveoli in interstitial lung disease to locally promote fibrin deposition.(ABSTRACT TRUNCATED AT 250 WORDS)     

N-Acetylcysteine derivative inhibits procoagulant activity of human islet cells

Aims/hypothesis The early loss of beta cells after islet cell transplantation has been attributed in part to blood coagulation at the implant site. Tissue factor expressed by beta cells and contaminating duct cells is considered to activate this process. Here, we investigated the ability of N-acetyl-l-cysteine to suppress the in vitro procoagulant activity of duct cells and human islet cell preparations. Materials and methods The effects of Nacystelyn, a salt derivative of N-acetyl-l-cysteine, were first assessed on procoagulant activity induced in human plasma by recombinant tissue factor, human primary duct cells or human islet cell preparations. The influence of Nacystelyn on clot formation, platelet counts and d-dimers were measured in a whole blood tubing loop model. Human beta cell viability and insulin synthesis after Nacystelyn treatment were assessed to exclude cytotoxicity of Nacystelyn. Results Nacystelyn efficiently inhibited the procoagulant activity of human recombinant tissue factor, primary duct cells and human islet cell preparations at clinically relevant concentrations without cellular toxicity. Conclusions/interpretation Nacystelyn is a pharmaceutical candidate to reduce early beta cell loss related to tissue factor-dependent coagulation after islet transplantation. All the authors are partners of the JDRF Center for Beta Cell Therapy in Diabetes, Brussels, Belgium.     

Comparison between lung parenchyma and bronchoalveolar lavage collagenolytic activity

We have evaluated, in an experimental model of silicosis in guinea pigs, if the presence of collagenolytic activity in bronchoalveolar lavage (BAL) fluid reflects the collagen catabolism in lung parenchyma. We measured simultaneously BAL collagenase activity, using as substrate [³H]type I collagen, and lung collagenolytic activity by the tissue pellet assay. Animals (n = 30) were instilled intratracheally with 50 mg of quartz DQ-12 and sacrificed 15, 30, and 60 days after silica administration. Guinea pigs instilled with saline solution were used as controls. Our results showed that lung parenchymal collagenolytic activity was present in all experimental and normal guinea pigs. There were no statistical differences between silicotic and normal animals at 15 and 30 days. At 60 days, however, a significant decrease in tissue collagenolytic activity was observed in silicotic animals (161 ± 100 vs. 400 ± 152 units of collagenase activity; p < 0.001). In contrast, BAL collagenolytic activity was revealed only in 7 of 10 silicotic animals at 15 days and 30 days, and in 4 of 10 at 60 days. Normal guinea pigs did not exhibit BAL collagenase activity. BAL and tissue collagenase activity from each experimental animal were analyzed by straight line regression and no significant relationship was observed (r = 0.082; p = 0.87). This suggests that BAL collagenolytic activity does not reflect lung tissue collagen turnover. Offprint requests to: M. Selman     

Regulation of monocyte procoagulant by chemoattractants

Various n-formylated peptides function as receptor-specific chemoattractants for both granulocytes and monocytes. Because these agents are important tools in the study of leukocyte function in vitro, we chose to examine their effects on leukocyte procoagulant activity. The synthetic chemotactic peptide N-formyl-methionyl-leucyl phenylalanine (FMLP) induces a fourfold increase in procoagulant activity (PCA) in cultured human monocytes at an optimal dose of 5 X 10(-9) mol/L, whereas higher doses inhibit PCA response. Although nonadherent lymphocytes are not absolutely required for PCA expression, their presence significantly amplifies monocyte PCA. Irradiation of nonadherent lymphocytes before mixing them with FMLP and adherent cells abolishes their ability to amplify PCA. Kinetic studies demonstrate an increase in optimal dose FMLP-stimulated PCA over time whereas high- dose inhibition of PCA generation occurs at various incubation times. Cell viability is unaffected by inhibitory concentrations of FMLP. Supernates from high-dose FMLP-stimulated cells fail to inhibit later expression of PCA by cells exposed to endotoxin. The cellular procoagulant remains cell-bound and exhibits characteristics of thromboplastin (tissue factor), including inhibition by concanavalin A and phospholipase C as well as the ability to shorten the clotting times of factor VIII but not factor VII-deficient substrate plasmas. These results suggest a complex system of lymphoid cell regulation of procoagulant generation by monocytes exposed to various chemotactic peptides in vitro.     

Effect of human recombinant cytokines on the induction of macrophage procoagulant activity

A panel of human recombinant cytokines was tested for induction of procoagulant activity (PCA) in human monocyte-derived macrophages. Nonadherent culture conditions were used, and PCA was determined with whole cells rather than cell lysates. It was assured by Limulus amebocyte lysate assay that tested cytokines displayed low levels of endotoxin activity within the range of biologic activity. Additional evidence to rule out an endotoxin effect was provided by heat- inactivation experiments. Interferon-gamma (IFN-gamma), interleukin-1 beta (IL-1 beta), and tumor necrosis factor-alpha (TNF-alpha) were strong macrophage PCA inducers. The low level of PCA induced by IL-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), M-CSF, IL-4, IL-6, IL-10, and IFN-alpha could not be distinguished from that induced by traces of endotoxin contaminating the preparations. Transforming growth factor-beta decreased constitutively expressed PCA within 24 hours of exposure. PCA induced by IFN-gamma, IL-1 beta, and TNF-alpha depended largely on tissue factor expression, as evidenced by experiments with factor X-deficient plasma and antitissue factor antibodies. In macrophages subcultured in adherence, IL-1 beta was a strong PCA inducer, whereas IFN-gamma and TNF-alpha promoted little PCA increase. This observation and different kinetics of PCA induction suggested that mechanisms of PCA induction are distinct for the three cytokines. Thus, we showed that well-characterized cytokines critically involved in the promotion of cell-mediated antimicrobial defense/delayed-type hypersensitivity and considered for clinical application promote local fibrin deposition by a direct effect on macrophages.     

Estrogen receptor variants in epithelial compartment of normal human breast

Estrogen receptor (ER) splice variants have previously been identified in normal human breast. Normal breast, however, comprises many cell types including fat cells, fibroblasts, endothelial cells, and a variety of inflammatory cells besides breast epithelial cells. The objective of our study was to demonstrate the existence of several variants in the epithelial compartment of the normal human breast. To this end, highly enriched breast epithelial cells from reduction nammoplasty specimens were isolated using Precoll gradient centrifugation. We analyzed the presence of both wild-type (WT) and variant receptors in human breast epithelial cells using the seminested polymerase chain reaction and direct automated sequencing of the amplified products. We demonstrated that a number of splicedvariants of the ER coexist with the WT receptor. Variants lacking exons 2, 4, 5, and 7 are detected in the breast epithelial compartment of the normal human breast.     

Regulation of procoagulant factors in mononuclear phagocytes

Mononuclear phagocytes can be activated to develop enhanced expression of a wide range of macromolecules and mediators. Amongst these are several procoagulants, most notably thromboplastin. A variety of stimuli (particles, ligands for cell surface receptors, and some pharmacological agents) enhance procoagulant expression. We indicate that such enhancement may contribute to intra-and extravascular coagulation in several types of infections. We also point to the paucity of knowledge on the biosynthesis and intracellular transport of these procoagulants and speculate on the nature of the mechanisms involved.     

Asbestos exposure results in increased lung procoagulant activity in vivo and in vitro

Enhanced fibrin deposition is a common histologic finding in fibrotic lung disorders including asbestosis and may be an important mechanism by which fibroblast proliferation is modulated. Asbestos-induced activation of lung interstitial cells may result in enhanced expression of procoagulant activity which contributes to the inflammatory response resulting in subsequent fibrin deposition. The current study examines procoagulant activity in bronchoalveolar lavage fluid from patients with clinically diagnosed asbestosis, patients with asbestos exposure without asbestosis, and normal, control subjects. Results indicated that asbestos exposure resulted in increased lung procoagulant expression in vivo, and furthermore, suggested that both endothelial cells and alveolar macrophages represented lung parenchymal cells which may contribute to this activity. This imbalance in coagulation homeostasis may be important in the regulation of fibrotic responses observed in asbestosis.     

Electrical activity of the normal human stomach

Using monopolar electrodes, the gastric electrical activity was recorded in waking human beings from both the mucosal and the serosal sides of the gastric wall. For each electrode location in a given subject, a mean curve of the regular fluctuations in potential (pacesetter potential) was obtained by an iteration process allowing measurement of characteristic amplitudes. A detailed study of the variations of the electrical period within and between subjects was undertaken. The frequency variations of the normal human gastric pacemaker appear in a slow and gradual fashion rather than in irregularities in the period; rhythm disorders rarely occur. Since frequent shifts of the baseline prevent the routine use of DC recordings, the use of a time constant of 4 seconds allows correct reproduction of the second component of the pacesetter potential. The morphology of the pacesetter potential is dependent on the gastric area and on the recording source—ie, mucosal or serosal. The amplitude and slope of the first component are greater in the antrum than in the corpus; the relative amplitudes of the first and the second component are dependent on the recording source.     

Delineation of T-progenitor cell activity within the CD34+ compartment of adult bone marrow

T-cell production is largely dependent on the presence of a thymus gland where CD34+ precursors mature into T lymphocytes. Prethymic stages of T-cell development are less defined. Therefore, this study aims to delineate T-progenitor cell potential within the CD34+ Lineage-- (Lin-) cell compartment of adult bone marrow (ABM). Fractionation of CD34+ Lin- ABM cells with CD45RA, Thy-1, CD38, and HLA-DR failed to absolutely segregate T-cell reconstituting ability, indicating broad distribution of T-progenitor cell potential. Titration experiments showed that low numbers of CD34+ Lin- CD45RA+ (RA+) cells had greater thymus repopulating ability than CD34+ Lin- CD45RA- cells (RA-). The great majority (> 95%) of RA+ cells expressed CD38, HLA-DR and 70% to 90% of RA+ cells lacked Thy-1 surface expression. RA+ cells contained colony-forming unit granulocyte-macrophage (CFU-GM) progenitor cells but were depleted of erythroid potential, did not provide hematopoietic reconstitution of human bone fragments implanted into SCID mice, and did not efficiently maintain CD34+ cells with secondary clonogenic potential in bone marrow cultures. Thus, RA+ cells are oligopotent (nonprimitive) CD34+ progenitors with T-cell reconstituting ability. In contrast, these same assays indicated that CD34+ Lin- CD45RA- cells (RA- cells) comprised hematopoietic stem cells (HSC) with primitive multilineage (T, B, myeloid, and erythroid) hematopoietic potential. It was confirmed that HSC-containing populations, such as CD34+ Lin- CD45RA- Thy-1+ cells had thymus repopulating ability. Culture of RA- cells on murine bone marrow stromal cells in the presence of interleukin (IL)-3, IL-6, and leukemia inhibitory factor (LIF) generated CD34+ CD45RA+ progeny engrafting in a secondary severe combined immunodeficiency (SCID)-hu thymus assay. Altogether, our results underscore the fact that T-cell reconstituting potential can be dissociated from HSC activity. Furthermore, we speculate that HSC might develop into the T lineage indirectly, via differentiation into an intermediate oligopotent CD34+ CD45RA+ stage. Finally, T-progenitor cells can be cultured in vitro.     

Increased macrophage procoagulant activity but normal endothelial thrombomodulin in rabbits fed an atherogenic diet

We have studied the procoagulant activity (PCA) of blood and spleen mononuclear phagocytes and the thrombomodulin activity of aortic segments in rabbits fed an atherogenic diet for 6 weeks as compared to rabbits fed a standard diet. Blood monocytes expressed negligible basal PCA (i.e., PCA measured immediately after cell isolation) both in treated and control rabbits. PCA induced by endotoxin in vitro was not different in the two groups. In contrast, dietary treatment resulted in a significant increase in the basal PCA of spleen cells (p less than 0.01). Moreover, the latter produced significantly more PCA than control cells (p less than 0.002) in response to endotoxin in vitro. The thrombomodulin activity associated with aortic endothelium was not different in the two groups of animals despite the presence of visible fatty streaks on the aortic endothelium of treated rabbits. When rabbits were given a single injection of endotoxin, spleen mononuclear phagocytes harvested 60 min after the injection from treated animals expressed three times more PCA (p less than 0.01) than did cells from controls. In all instances PCA was identified as tissue factor. Endotoxin injection did not affect the thrombomodulin activity of thoracic aorta from both control and diet groups. It is suggested that dietary fats may cause early functional changes in mononuclear phagocytes that lead to an increased PCA production both in vivo and in vitro. These data may be relevant to an understanding of the role of monocytes-macrophages in the pathogenesis of atherosclerosis.     

Measurement of macrophage cellular procoagulant activity

We developed a simple technique for the measurement of the procoagulant activity exposed on the surface of macrophages. The cells are isolated, adhered to plastic surfaces, and assayed in the same device. This approach allows us to study the microcoagulation on the surface of intact macrophages by sensitive and specific clotting tests.     

Isolated deficiency of platelet procoagulant activity

This is a study of a 34 year old woman with a moderate to severe bleeding disorder in whom impaired platelet procoagulant activity (PPA) was found by several methods, including tests of factor 3 availability (PF-3a), prothrombin consumption and contact activation. No deficiencies of platelet adhesion, aggregation, secretion, metabolism or granule-bound substances were detectable. Under adequate platelet coverage, this woman underwent two surgical procedures without difficulty. These findings demonstrate the role of PPA in hemostasis and indicate that a defect in PPA can be an isolated occurrence. The abnormalities in PF-3a found in this patient could be due to the diminished number of factor V binding sites, resulting in impaired factor Xa binding, found in separate studies by Majerus et al.     

支气管肺泡灌洗液淋巴细胞端粒酶活性检测对肺癌诊断意义

目的通过检测支气管肺泡灌洗液（BALF）中的淋巴细胞的端粒酶活性（TA）,探讨其对BALF端粒酶活性检测的影响,评价TA在肺癌中的诊断意义。方法将52例患者按最终临床诊断结果分为恶性（27例）、良性（25例）两组,用密度梯度离心法分离BALF标本获得淋巴细胞;应用TRAP—ELISA法检测淋巴细胞和同组对照标本的端粒酶活性,结果以OD值表示,计算该方法的敏感性、特异性、阳性预测值、阴性预测值、准确性等,结果用t检验和X^2检验。结果端粒酶活性检测（TA）：25例良性肺病组中只有1例TA检测阳性（1／24）,OD值为0．091±0．027,分离的18例淋巴细胞无1例阳性,OD值为0．064±0．013;27例肺癌中有24例阳性,OD值为0．630±0．350,分离的淋巴细胞有1例阳性,OD值为0．084±0．013,分离的肿瘤细胞全部阳性;TA检测的敏感性、特异性、阳性预测值、阴性预测值、准确性分别为88．9％、95．8％、96％、88．5％、92．2％,经统计分析：恶性和良性两组问TA差异有显著性（P〈0．01）,淋巴细胞的TA对总体对照无明显影响（P〉0．01）。结论BALF中淋巴细胞的端粒酶活性对TA检测结果无明显影响,TA检测可以作为肺癌的诊断方法。     

Monokine gene expression in normal human liver: selective involvement of the portal compartment

Abstract: Monokines play a major role in the regulation of hepatocyte functions. To document a possible in situ production of these mediators under physiological conditions, expression of IL-1β and of IL-6 genes was analyzed by in situ hybridization in four histologically normal human livers. We detected cells containing IL-1β mRNA or IL-6 mRNA in all cases. Cells expressing either the IL-1β gene or the IL-6 gene were found with equal frequency and were similarly distributed. Although present in all liver compartments, they were selectively enriched in portal areas, in which they were detected both in endothelial positions and in perivascular connective tissues. Few positive cells were observed in hepatic lobules, most of them being located in the walls of centrolobular veins, in an endothelial position. Subcapsular cells were also shown to express monokine genes. The location of positive cells and their pattern of labelling suggested that macrophages, fibroblasts and endothelial cells were the main cell populations expressing monokine genes. In contrast, Kupffer cells, biliary epithelial cells and hepatocytes did not express monokine genes. No marker of immune activation other than monokine gene expression was detected in these histologically normal livers. The expression of the IL-1β gene and of the IL-6 gene may be induced by gut-derived LPS, and could play a role in the modulation of hepatocyte function in normal liver.     

Different expression of procoagulant activity in macrophages associated with experimental and human tumors

Mononuclear phagocytes, an integral part of the lymphoreticular infiltrate of human and experimental tumors, might contribute to fibrin deposition within malignant tissues through the production of procoagulant activity (PCA). Recent studies in different types of experimental tumors and in some cancer patients indicate that the functional status in terms of PCA of mononuclear phagocytes at 'peripheral' sites is not the same as that of macrophages at the tumor site (tumor-associated macrophages, TAM). Peripheral blood monocytes and/or peritoneal macrophages from V2-carcinoma-bearing rabbits and from mice bearing different types of tumors, like cells from normal animals, have low levels of basal PCA and are able to respond with a marked increase in PCA to in vitro stimulation. Likewise, in patients with primary lung cancer, pulmonary alveolar macrophages (PAM) obtained from the contralateral side of the neoplasm and blood monocytes behave essentially as cells from normal individuals. In contrast, profound changes in PCA have been found in TAM. In some experimental tumors and in a number of the patients, macrophages taken at the tumor site express high levels of basal PCA, which most probably reflect in vivo activation. Local activation of macrophages for PCA production might contribute to fibrin deposition at the tumor site. In the other experimental tumors studied and in the remaining patients, not only have macrophages low basal PCA but they also fail to respond to various stimulants in vitro. Although the mechanisms underlying these changes and their pathophysiological significance remain to be established, it is apparent that neoplastic growth may modulate the expression of macrophage PCA at the tumor site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased procoagulant activity of peripheral blood monocytes in human and experimental obstructive jaundice

We studied the procoagulant activity of peripheral blood monocytes in 41 patients with severe obstructive jaundice and in 27 nonjaundiced control patients using a one-stage clotting assay. Mononuclear cells from jaundiced patients, tested immediately after isolation, expressed low levels of procoagulant activity, which were, however, significantly higher than in cells from controls (p less than 0.01). In addition, after incubation in short-term cultures with and without endotoxin, these cells generated more procoagulant activity than did the control ones (p less than 0.001). No significant difference in procoagulant activity was found between patients with and without malignancy in either group. The relief of biliary obstruction resulted in the reduction of both serum bilirubin levels and monocyte procoagulant activity. Endotoxin-induced monocyte procoagulant activity was about threefold higher in the jaundiced patients who died than in the survivors (p less than 0.001). In rabbits made icteric by bile duct ligation and separation (15 days), the endotoxin-induced monocyte procoagulant activity was markedly increased as compared with sham-operated animals (p less than 0.005). In all instances, procoagulant activity was identified as tissue factor. The increased capacity of mononuclear phagocytes to produce procoagulant activity might help explain the activation of blood coagulation in severe obstructive jaundice.