Biomedical subjects

H Rothberger

Publications and source records attributed to H Rothberger.

At least 19 recordsLinked to original sources

PubMed1992

Livedo vasculitis: vasculitis or thrombotic vasculopathy?

Elevated fibrinopeptide A levels, suggestive of a thrombogenic state, were observed in six patients with livedo vasculitis. Serological studies demonstrated normal complement levels, and circulating immune complexes were not identified by standard assays. Morphological studies showed no evidence of immune complex deposition or a neutrophilic vascular reaction. These results support classification of this disorder as a thrombogenic vasculopathy rather than as a small vessel vasculitis. Further investigations into the thrombotic abnormalities underlying this entity are warranted.

Adult↗

PubMed1989

Identification of mRNA coding for factor VII protein in human alveolar macrophages--coagulant expression may be limited due to deficient postribosomal processing.

Clotting factors synthesized by monocytes and macrophages may initiate coagulation reactions during inflammation. Functional vitamin K-dependent coagulation factors have been found to be associated with human monocytes/macrophages, but there are no reports identifying mRNA coding for vitamin K-dependent proteins in these cells. In the present studies, factor VII mRNA was found in total RNA extracted from freshly isolated human alveolar macrophages using hybridization with a complementary DNA probe. On the other hand, vitamin K-dependent carboxylase activity which is required for postribosomal modification of the protein, was not detectable in the macrophages before or after culture, and human blood mononuclear leukocytes also lacked this enzyme activity. Control human and rat hepatoma cells exhibited high levels of carboxylase activity within the same experiments. Using sensitive kinetic assays, no increase in factor VII activity was detected during culture of alveolar macrophages under conditions promoting 1.78 +/- .24 (n = 8) fold increases of tissue factor activity. These findings with freshly isolated cells demonstrate that alveolar macrophages synthesize factor VII mRNA in vivo. However, the mRNA was found in the absence of evidence for gamma-carboxylase activity or processing of the factor into a functional clotting enzyme. The results imply that functional expression of any synthesized coagulation factor VII in alveolar macrophages may be limited or prevented due to a cellular deficiency at the level of postribosomal processing.

Carbon-Carbon Ligases↗

PubMed1989

Clinical significance of combined antinuclear and anticytoplasmic antibodies detected by immunofluorescence.

Anti-nuclear antibodies (ANA) combined with anti-cytoplasmic antibodies (ACA) in 0.6% (n = 43) of 7,121 consecutive patients referred for ANA screening were observed. Homogeneous/cytoplasmic was the most frequently combined ANA/ACA pattern among 6 different indirect immunofluorescent (IF) combinations. Distribution of diseases was similar in patients with ANA/ACA and those controls with ANA alone. However, the ANA/ACA results were often transitory, especially in systemic lupus erythromatosus (SLE). The appearance of transitory ANA/ACA combinations on serial ANA testing may be useful as a serologic marker for SLE.

Adult↗

PubMed1989

Initiation of the extrinsic pathway of coagulation by human and rabbit alveolar macrophages: a kinetic study.

We examined assembly and expression of the factor X activating complex on human and rabbit alveolar macrophages. Kinetic parameters of the factor X activating reaction were determined by functional titrations of factors VII and X with macrophage tissue factor (TF) added. We found rapid activation of factor X to Xa on alveolar macrophage surfaces. Detection of rapid factor Xa formation on macrophages required addition of exogenous factors VII and X. At plasma concentrations of the purified factors, factor Xa was formed on freshly isolated macrophages at approximately 5.4 pmol/min/10(6) cells. After macrophage maturation in culture for 20 hours with LPS (endotoxin) added, the factor X activation rate was increased two- to sixfold. The km' (apparent km) of TF-factor VII enzymatic complexes assembled on alveolar macrophages for factor X were (258 +/- 55 and 475 +/- 264 nmol/L for human and rabbit cells, respectively). The km' did not change during macrophage maturation in culture, but V'max (apparent Vmax) was consistently increased. The K1/2 of human factor VII (concentrations giving half maximal rates of factor X activation) for the interaction with human and rabbit alveolar macrophage TF were 0.191 +/- 0.096 and 1.7 +/- 0.7 etamol/L, respectively. The K1/2 were not significantly changed after maturation, whereas rates of Xa formation at saturation with factor VII were increased. The fast rates of factor X activation observed at physiologic concentrations of plasma-derived factors VII and X indicate that TF on alveolar macrophages is likely to provide sites for binding of factor VII and activation of factor X in vivo during clotting reactions associated with alveolar edema and inflammation.

Animals↗

PubMed1986

Assembly of the prothrombin activator complex on rabbit alveolar macrophage high-affinity factor Xa receptors. A kinetic study.

Efficient prothrombin activation occurs after assembly of factors Va, Xa, and phospholipid surface cofactor as a multimolecular complex. These components are provided by platelets and plasma within the vascular space, but molecules and membranes for prothrombin activator assembly in extravascular spaces have not been identified. In the present study, purified alveolar macrophages were found to produce high-affinity factor Xa receptors that mediate formation of enzymatic prothrombinase complexes and rapid prothrombin to thrombin conversion in the absence of exogenous factor V/Va or platelets. Thus, in reaction mixtures with alveolar macrophages cultured for 20 h in serum-free medium, the thrombin formation rate was 152 nM/min/0.66 X 10(6) cells, after adding prothrombin (1.5 microM), Ca2+ (5 mM), and factor Xa (3.7 nM). The observed Kd of factor Xa interaction with macrophage receptors is 2.1 +/- 0.94 X 10(-10) M. Kinetic analysis and inhibition studies using isolated factor V and anti-factor V antibody show that macrophage Xa receptors are functionally and antigenically similar to plasma factor V. By contrast, freshly isolated cells lacked receptors promoting prothrombin conversion at high rates. Inhibitors of protein synthesis and glycosylation, puromycin and monensin, respectively, abrogated production of Xa receptors in culture. Additionally, subcellular fractionation and enzyme-marker studies (alkaline phosphodiesterase I) indicate that internal and external membranes of alveolar macrophages have phospholipid surface cofactor activity required for prothrombinase complexes. Pulmonary surfactant is also shown to express this cofactor activity. Alveolar macrophages and surfactant comprise an efficient prothrombin activator system that is independent of plasma factor V. This system may facilitate rapid extravascular alveolar thrombin formation even at very low concentrations of factor Xa during lung defense reactions to inflammation or edema.

Animals↗

PubMed1986

Enzyme-linked coagulation assay. II. A sensitive assay for tissue factor and factors II, VII, and X.

We have developed a solid-phase clotting assay which uses peroxidase-fibrinogen in solution and fibrinogen bound to microtiter plates as a substrate for the thrombin generated from the clotting cascade. We have developed this assay for measurement of the extrinsic pathway factors thromboplastin (tissue factor, factor III), VII and VIIa, X, and II. Using long incubation times (40-90 min), thromboplastin could be measured in extracts of human brain at very low concentrations. Specificity for thromboplastin was demonstrated by showing a requirement for factors II, V, X, and VII but not for VIII, IX, XI, or XII; both substrate plasmas monodeficient in single factors and mixtures of the pure factors were used in demonstrating this specificity. The assay was modified to measure factors II, VII, VIIa, and X using appropriate deficient plasmas. The limit of detection was 2-3 orders of magnitude lower than a one-stage clotting test for all factors assayed. This assay has the advantages of convenience, specificity comparable to standard clotting tests, and high sensitivity.

Blood Coagulation Factors↗

PubMed1985

Increased tissue factor activity generation in vitro by canine blood leukocytes associated with allogeneic kidney transplantation and rejection.

Using a canine model, leukocyte populations enriched for monocytes and lymphocytes were isolated from blood during three week periods after kidney allotransplantation corresponding to episodes of acute rejection. Relative to controls, these cells incubated in vitro for five hours were found to generate increased amounts of PCA (procoagulant activity) characterized as tissue factor, the extrinsic clotting pathway activator. Controls included comparable blood leukocyte populations isolated from kidney autograft recipients and healthy animals. Differences in results for these two control groups were insignificant. These contrasts observed between allografted animals and controls demonstrate that leukocyte PCA generation is stimulated by the allogeneicity of histoincompatible kidneys rather than by direct effects of organ transplantation or non-specific postoperative effects. Results of in vitro transfer experiments provide evidence that cellular stimulation or induction in vivo accounted for the PCA increases observed. Stimulation of leukocyte tissue factor generation as a consequence of allogeneic kidney transplantation may in part account for coagulopathies and fibrin deposition during kidney rejection.

Animals↗

PubMed1985

Enzyme-linked coagulation assay: a clot-based, solid-phase assay for thrombin.

A new, solid-phase microtiter plate assay for thrombin has been developed, using fibrinogen bound to wells of a microtiter plate and peroxidase-fibrinogen in solution as an indicator system. When small amounts of thrombin are added to the mixture, peroxidase-fibrin and plate-bound fibrin are formed, and the peroxidase-fibrin binds to the plate-bound fibrin. The amount of peroxidase-fibrin binding is proportional to the thrombin concentration and time of incubation. Using this assay, thrombin was measured at concentrations as low as 0.25 ng/ml (0.006 nM) in 150 microliter of sample. In the presence of the specific inhibitors benzamidine and D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone, the thrombin activity is reduced, at relative concentrations of inhibitors consistent with their affinities and mechanisms of action. The enzyme-linked coagulation assay is generally useful as a highly sensitive and convenient alternative to conventional "clot-based" tests of coagulation.

Amino Acid Chloromethyl Ketones↗

PubMed1985

Tissue factor in bronchoalveolar lavage fluids. Evidence for an alveolar macrophage source.

Local and systemic coagulation and fibrin deposition occur in many types of alveolar injury and inflammation, but clotting factors capable of initiating the coagulation cascade in the alveolus have not been thoroughly identified and characterized. In the present studies, BAL (bronchoalveolar lavage) fluids obtained from rabbits were found to have procoagulant activity detectable in dilutions containing as little as 1.3 ng of protein. The specific activity of the procoagulant in these fluids was within 1 order of magnitude of that found in brain thromboplastin. The BAL procoagulant was shown to be associated with particles having a molecular weight greater than 15 X 10(6) daltons by gel filtration chromatography, and was characterized as tissue factor by showing specific requirements for factors VII, X, and II. Further experiments were performed using membranes purified from alveolar macrophages by sucrose density gradients and characterized by studies of alkaline phosphodiesterase I, a cytoplasmic membrane marker, and electron microscopy. These studies demonstrate that alveolar macrophages, especially low-density subpopulations, generate and release membrane material that is a source of tissue factor in BAL fluids.

Animals↗

PubMed1985

Immunoperoxidase assay for anti-dsDNA antibodies using Crithidia luciliae substrates.

In studies on 779 fresh, unselected ANA+ sera, 50 (6.4%) were found to have anti-dsDNA antibodies, by using CIP (C. luciliae immunoperoxidase), and 84% of the 50 patients met the criteria for definite SLE. After freezing 36 of these sera for 13 +/- 1 months, only 52.8% were again positive by CIP. Using CIF (C. luciliae immunofluorescence), 16.7% and 52.8% of the same stored sera were anti-dsDNA+, with transmitted and epi-illumination, respectively. We favour CIP to CIF because: slide files are permanent, overcoming any lability of antibodies in frozen sera; kinetoplast, flagellum and nucleus are easily distinguished; perikinetoplast staining is avoided; specialized microscopic equipment is not needed.

Antibodies, Antinuclear↗

PubMed1984

Generation of coagulation factor V activity by cultured rabbit alveolar macrophages.

Alveolar macrophages are cell's important in immune defense and inflammation in the lung, and the coagulation system participates in these reactions. In earlier experiments, it was found that alveolar macrophages contain and produce tissue factor, the extrinsic clotting pathway activator. The present experiments explore possible production by alveolar macrophages of the sequence of the clotting proteins that interact to form thrombin following initiation of coagulation by tissue factor. In studies using alveolar macrophages purified from rabbits, factor V activity was not detected in cell preparations assayed directly after isolation. However, after short-term culture, we found generation and release of factor V activity by these cells, which was predominantly from subpopulations with densities of 1.060-1.068 g/ml, corresponding to intermediate stages of alveolar macrophage maturation. Cell viability and protein synthesis were required for generation of the activity as shown by inhibitory effects of cell lysis before culture and by effects observed after including puromycin in cultures with viable cells. The activity generated was characterized as factor V by demonstrating specific functional requirements in one- and two-stage coagulation tests. There was no detectable generation in these cultures of factors II, VII, X, or the more recently described factor X-independent monocyte/macrophage prothrombinases. Factor V activity generated by alveolar macrophages may contribute to prothrominase assembly, activation of clotting, and fibrin formation within the alveolus.

Animals↗

PubMed1984

Selective increases of procoagulant activity in rabbit lymphoid populations in vitro following stimulation with endotoxin: dependence on anatomic source.

Rabbit mononuclear leukocytes isolated from a variety of anatomic sites were examined for ability to generate procoagulant activity in vitro. Marrow, blood and spleen mononuclear cell populations were found to differ functionally from lymph node, thymus and alveolar populations by having much greater ability to increase in tissue factor activity in response to an endotoxin stimulus. Thus, after incubation in the presence of endotoxin, leukocytes obtained from marrow, blood, and spleen were found to increase in procoagulant activity characterized as tissue factor by 832, 1942 and 12.6 fold, respectively. In contrast, pulmonary alveolar macrophages increased in tissue factor activity only by 2.8 fold, and lymph node and thymus mononuclear cells showed little or no increases. These functional differences, demonstrated by exposing the six cell populations to endotoxin under controlled conditions in vitro, likely explain the similar pattern of anatomic selectivity of leukocyte tissue factor increases reported to occur in vivo during endotoxemia and Shwartzman reactions (1).

Animals↗

PubMed1984

Tissue factor activity. A marker of alveolar macrophage maturation in rabbits. Effects of granulomatous pneumonitis.

Experiments were carried out to examine relationships between alveolar macrophage maturity and amounts of tissue factor (Clotting Factor III) in these cells under physiologic conditions and during immunologically induced pneumonitis. Using discontinuous density gradient centrifugation, alveolar macrophages from healthy rabbits were rapidly isolated into five subpopulations at different stages of maturation, as demonstrated by morphologic and morphometric evaluation. Very large amounts of tissue factor activity were found in fully mature cells that were purified in the lowest density subpopulation and assayed without preliminary in vitro stimulation or culture. In the remaining four subpopulations of increasing density, amounts of tissue factor were found to progressively diminish in direct correlation with declines of cell maturity. These differences at mean levels were as great as 35-fold. In addition, blood monocytes had less than 1/219 and less than 1/6 of the activity of the fully mature and the least mature subpopulations, respectively. After 16 h culture of the five isolated subpopulations in the absence of lymphokines or of significant numbers of lymphocytes, tissue factor activity increased in inverse correlation with the preincubation stage of cell maturity (2,387 and 109% in the least mature and most mature subpopulations, respectively). These increases required protein synthesis and were accompanied by morphologic and morphometric changes which indicated cellular maturation during the period of tissue factor activity generation in vitro, thus further demonstrating relationships between macrophage maturity and tissue factor content. In additional experiments, direct correlations between cell maturity and tissue factor activity content were also found in activated alveolar macrophage populations from rabbits with Bacillus Calmette Guering (BCG)-induced granulomatous pneumonitis. However, as compared with controls, the BCG populations had increased total amounts of tissue factor activity due to the presence of large numbers of mature alveolar macrophage forms that had high levels of the procoagulant. Thus, tissue factor activity in alveolar macrophages is a marker of cellular maturation in vivo and in vitro. Increased amounts of this initiator of the extrinsic clotting pathway, as found in alveolar macrophage populations from animals with granulomatous pneumonitis induced by BCG hypersensitivity, suggest that alveolar macrophage tissue factor may contribute to the pathology of immune lung diseases.

Animals↗

PubMed1984

Increased tissue factor activity of monocytes/macrophages isolated from canine renal allografts.

Kidney allografting was performed in a group of ten beagles, and viable leukocytes infiltrating the transplanted organs were isolated during episodes of acute rejection 5 or 6 days postoperatively. These infiltrate populations, consisting predominantly of lymphocytes and monocytes/macrophages, were found to have significantly increased amounts of procoagulant activity relative to control leukocytes isolated from circulating blood and lymph. Using nonspecific esterase staining in an agar microclot assay, procoagulant activity in the infiltrate leukocytes was found to reside in monocytes/macrophages rather than other coisolated cell types. By contrast, control monocytes from blood had no activity in this microclot assay. Procoagulant activity in the infiltrate cells was characterized as tissue factor. Increased amounts of this activator of the extrinsic pathway, as found in infiltrate monocytes/macrophages, may initiate clotting reactions and fibrin deposition within allografts.

Animals↗

PubMed1984

Primary lymphoma of bone presenting as monoarthritis.

We report an unusual case of primary non-Hodgkin's lymphoma or reticulum cell sarcoma of bone presenting as monoarthritis in a middle-aged woman without previous rheumatic disease. She underwent rheumatologic evaluation of plasma and synovial fluid, synovial cytology, arthroscopy, synovial and bone biopsies. Our evaluation may be the most thorough rheumatologic one yet reported.

Arthritis↗

PubMed1983

The clinical significance of anticytoplasmic antibodies found on fluorescent antinuclear antibody testing.

We evaluated anticytoplasmic antibodies (FACA) found on immunofluorescent antinuclear antibody tests (FANA). Of 1830 sera submitted to our laboratory for FANA testing, we found a 2.7% incidence of FACA as compared to a 21.5% incidence of FANA. Patients with FACA had rheumatologic and other systemic diseases closely resembling those present in controls with FANA, indicating that FACA provide a pathologic marker independent of FANA. Among FACA+ patients studied further, 59% were found to have serum antibodies to mitochondrial (Mit) and/or smooth muscle (SMus) antigens, largely in the absence of liver disease. By contrast, sera from FANA+ controls lacked anti-Mit and anti-SMus antibodies, but did contain antibodies to SSA, SSB, dsDNA, Sm, and RNP in much higher frequency than FACA+ sera.

Adolescent↗

PubMed1983

Procoagulant activity of lymphocyte-macrophage populations in rabbits: selective increases in marrow, blood, and spleen cells during Shwartzman reactions.

The present experiments examine leukocyte procoagulant activity using mononuclear cell populations purified or enriched from rabbit bone marrow, blood, spleen, lymph node, thymus, and pulmonary alveoli. Cells from these six sites, obtained from control and endotoxemic animals and assayed without an intermediate culture step, were found to have procoagulant activity identified as tissue factor. Under control conditions, tissue factor activity was found to be at low levels in marrow and blood populations compared to median activities 3- and 11-fold higher in populations from spleen and lymph node, and 33- and 45-fold higher in thymus and alveolar populations. By contrast to respective controls, significantly increased amounts of tissue factor (35-, 15-, and 12-fold at median levels) were found in marrow, blood, and spleen populations from endotoxemic animals. The types of leukocytes in these latter three populations were morphologically and histochemically indistinguishable from respective controls, indicating that endotoxin induced increases of activity in cells with relatively low amounts under control conditions. Activity did not change significantly in lymph node, thymus, or alveolar populations after endotoxemia. These studies show that tissue factor is present in a range of leukocyte populations not previously reported to have procoagulant activity. In addition, the finding of widespread gains of tissue factor in the marrow-blood-spleen pool due to endotoxemia provides new evidence supporting the importance of leukocyte procoagulants in Shwartzman-like reactions.

Animals↗

PubMed1982

Increases of leukocyte tissue factor activity stimulated by red cells sensitized with human blood group alloantibodies.

Human Rh+ and A+ red cells sensitized with human anti-Rh and anti-A blood group alloantibodies are shown to stimulate large increases of tissue factor activity as compared to controls after incubation with leukocytes for 3-18 hours. As little as a 1/1280 dilution of human anti-Rh serum was stimulatory. Immunospecific effects in experiments with 5 different antibodies exclude possible artifacts resulting from contaminants such as endotoxin. These results suggest that production of leukocyte tissue factor may be similarly stimulated by sensitized red cells during immune hemolysis and serve as an activator of coagulation.

ABO Blood-Group System↗