A new animal model of venous thrombosis in rats with low flow conditions in the venous blood stream.

The aims of the present investigation were to develop a new venous thrombosis animal model with low flow conditions in the venous blood stream and then evaluate this model for testing new anticoagulants. In this model, the vena cava of rats was narrowed with a Doppler flow probe, blood flow velocity continuously recorded and thrombus formation initiated by thromboplastin infusion. Sixty-five minutes following thromboplastin infusion the animals were sacrificed and the following parameters measured: thrombus wet weight, fibrinopeptide A (FpA), activated partial thromboplastin time and platelet number. The new model was evaluated with aspirin, a PGI2 mimetic, heparin and a soluble thrombomodulin analogue. Without thromboplastin infusion no thrombus formation or reduction of blood flow was observed. Controls receiving thromboplastin infusion developed a thrombus, blood flow was arrested, platelet number decreased and FpA was elevated. In contrast, animals pretreated with anticoagulants maintained a residual flow, while thrombus weight, thrombocytopenia and FpA elevation were reduced. The antiplatelet agents were not effective. This study demonstrates that, under low flow conditions, only a combination of blood flow reduction with a hypercoagulable state results in venous thrombus formation. This improved model of venous thrombosis more closely resembles the clinical situation and is applicable for testing anticoagulants.

Elevated plasma levels of the immunosuppressive complement fragment Ba in renal failure.

The complement fragment Ba is a 33 kD activation product of factor B which suppresses human B-lymphocyte functions in vitro. We report that plasma levels of Ba are highly elevated in patients with chronic renal failure (4.84 +/- 3.58 micrograms/ml) and in patients with end-stage renal disease undergoing regular hemodialysis (16.1 +/- 6.1 micrograms/ml) as compared to normals (1.01 +/- 0.30 micrograms/ml). Ba levels were strictly correlated with the creatinine clearance. The urinary excretion of Ba was 165-fold higher in patients with tubular proteinuria than in normals. These results indicate that the kidney is the major catabolic site for Ba. In addition, direct evidence was obtained for an enhanced turnover of the alternative pathway of complement in renal failure that, although it appears to be less important than the renal retention of Ba, contributes to elevated Ba plasma levels in these patients. Ba concentrations in dialysis patients who responded to hepatitis B vaccination were significantly lower than in non-responders. Furthermore, the in vitro IgM synthesis by purified mononuclear cells was negatively correlated with Ba concentrations determined in the plasma of these patients. These results suggest that the accumulation of Ba contributes to the defective immune response in patients with renal failure.

The role of C5a in interleukin-6 production induced by lipopolysaccharide or interleukin-1.

We investigated the effects of recombinant C5a on interleukin-6 (IL-6) production by peripheral blood mononuclear cells (PBMC) in vitro and compared them with the release of interleukin-1 (IL-1) and tumour necrosis factor (TNF). In a virtually lipopolysaccharide (LPS)-free culture system, C5a by itself did not induce any significant IL-6 translation. The IL-6 release in response to low amounts of LPS (500 pg/ml) or IL-1 beta, however, was markedly increased by the complement fragment. This enhancement of IL-6 synthesis was dose-dependent, reached its optimum at 5.8 x 10(-9)M rC5a and occurred regardless of the presence of serum components. At the level of transcription C5a by itself did not induce IL-6 gene expression, but in the presence of low amounts of LPS the stimulation of monocytes with C5a yielded an increase in IL-6 mRNA. The transcription of IL-1 beta, however, can be induced by C5a alone. These data are interesting, since they indicate a different regulation of IL-1 beta and IL-6 by the complement fragment C5a. Furthermore, we could show that the C5a-mediated IL-6 production influenced the synthesis of IgG rather than IgM in vitro. These results may be relevant for an understanding of the potentiating role of C5a in cytokine-dependent disease processes.

Determination of the epitope specificities of monoclonal antibodies using unprocessed supernatants of hybridoma cultures.

A double monoclonal antibody (mAb) ELISA has been developed to determine the epitope specificities of murine monoclonal (mAbs). It permits the mAbs which bind to the same or adjacent epitopes to be distinguished from those which bind to separate epitopes on soluble monomeric proteins. The assay is designed for the early evaluation of mAbs in hybridoma culture supernatants when purified or labeled mAbs are not available. It does not rely on chemical or enzymatic fragmentation of the antigen and does not generate results which may be due to differences in the affinities of the mAbs. Moreover the characteristic multiple binding of polyclonal antibodies to the same epitope is also avoided. A further advantage is the accessibility of epitopes on a given antigen since the antigen is presented by a solid-phase mAb, in contrast to assays in which the antigen itself is adsorbed onto the solid phase. The test was evaluated using culture supernatants from hybridomas which produced mAbs against the complement proteins C3a, C6 or C7.

Determination of the T3-3A1 antigen in PHA-induced human T-cells by standardized cell-ELISA.

The treatment of mononuclear leukocytes (MNL) with lectins induces marked changes in the cell's morphology, physiology and the composition of the cell surface. We used an immunoassay to monitor the PHA-induced expression of the T-lymphocyte-specific antigen T3-3A1 in fixed MNL with a monoclonal antibody (MoAb) specific for this antigen. This assay permits the detection and quantitation of the T3-3A1 antigen in a few thousand cells without the use of a FACS. The test was calibrated with isolated plasma membranes and, combined with a total protein determination, the relative content of T3-3A1 antigen in each sample could be calculated. Maximal T3-3A1 synthesis required a 10-fold lower concentration of PHA than was necessary for optimal DNA synthesis. The test may be used to screen for PHA stimulation.