Determination of transforming growth factor beta 2 in human blood samples by ELISA.

Transforming growth factors beta (TGF beta s) show pleotrophic functions in vitro and in vivo. Biological effects depend on the cell type, the TGF beta isoform, and the availability of active TGF beta. Bioassays for TGF beta have not proven to be reliable tools in the measurement of TGF beta in human blood samples. Previously described sandwich ELISAs for measuring TGF beta are limited to single TGF beta isoforms and have not been evaluated for use in human sera or plasma. We describe a simple procedure to quantitate not only TGF beta 1 but also TGF beta 2 in human blood samples using commercially available antibodies. The sensitivity of the TGF beta 2 ELISA was 11 pg/ml. There was no crossreactivity between the TGF beta 1 and TGF beta 3 isoforms. High concentrations of TGF beta 1 and TGF beta 3 in spiked samples did not interfere with TGF beta 2 determination. TGF beta 2 recovery was highest in EDTA plasma (> 88%), and the intra- and interassay variance was 6% and 8.5% respectively. Applying the ELISA to human plasma specimens a significant percentage of TGF beta 2 (3.7 +/- 0.8 ng/ml) was found (about 50% of the TGF beta 1 content).

Discontinuous and continuous stimulation of FRTL-5 thyroid cells with bTSH cause different cAMP and nuclear proliferation antigen responses.

Pulsatile TSH secretion has been described in man. We investigated the effect of discontinuous TSH stimulation on FRTL-5 thyroid cells. FRTL-5 monolayers were pulsed with TSH in 4 h incubation periods with alternate 4 h TSH-free intervals, or continuously incubated with TSH. The cAMP production of cells was measured in the supernatant of monolayers. Expression of a nuclear proliferation antigen in FRTL-5 monolayers was determined by a monoclonal antibody (Ki-67) using the alkaline phosphatase-anti-alkaline-phosphatase staining method. The TSH concentration in the stimulation series ranged from 0.01 to 1.0 U/l medium. Rhythmic cAMP production was observed in both discontinuous and continuous stimulation. With discontinuous stimulation cAMP production peaked after about 24 and 48 h, while in the continuous presence of TSH peaks were observed at 32-40 and 48 h. At all TSH concentrations the effect of discontinuous stimulation was higher than in continuously stimulated cultures. The discontinuous incubation stimulated nuclear proliferation antigen expression of FRTL-5 more intensely and there was a positive correlation with TSH concentration. We conclude that the rhythmic pattern of cAMP production after TSH stimulation is independent of the TSH pulse. The amplitude of stimulation and proliferation of FRTL-5, however, is increased by discontinuous TSH application.

Increased cellular growth of FRTL-5 thyroid cells mediated by discontinuous stimulation with bTSH.

Previous reports showed pulsatile secretion of TSH in man. Therefore, we investigated the effect of discontinuous versus continuous TSH stimulation on the cellular level of FRTL-5 thyroid cells, namely the expression of a nuclear proliferation antigen (NPAg). The expression of this antigen correlates linearly with the 3H-thymidine incorporation and is a marker for cellular growth. The FRTL-5 cells were stimulated for 4 hours with bTSH (0.01-1.0 U/l). Compared to continuously stimulated cell cultures the discontinuous stimulation of FRTL-5 cells with bTSH showed a significant higher rate of NPAg expression, i.e. cellular growth.

Immunoblot analysis of the eighth component of human complement. Demonstration of subunits and detection of C8 alpha-gamma double and triple bands.

Using SDS-PAGE/immunoblot analysis of the eighth component of human complement, C8, we have been able to demonstrate an 85 kDa C8 alpha-gamma and a 62 kDa C8 beta subunit in normal human serum. Serum from an undiagnosed patient who presented undetectable hemolytic C8 activity possessed only the 85 kDa subunit, suggesting a defect in the C8 beta subunit. Serum of a patient with known C8 alpha-gamma deficiency possessed only the complementary 62 kDa subunit. Both sera used together were able to lyse antibody-sensitized sheep erythrocytes, whereas individual sera could not. Optimum conditions for C8 immunoblotting were determined using small amounts of serum or plasma, during low voltage electrophoresis and a sensitive staining technique (nitrobluetetrazolium/bromochloroindoxylphosphate). Using these conditions, the C8 alpha-gamma subunit was found to be composed of up to three bands, termed C8 alpha-gamma 1, -2 and -3. All three bands were found in pooled normal sera. Individual sera had at least the C8 alpha-gamma 2 and C8 alpha-gamma 3 bands. Two C8 beta-deficient sera from two unrelated patients exhibited only the C8 alpha-gamma 2 and C8 alpha-gamma 3 bands. We conclude that immunoblotting of C8 permits a detailed analysis of the molecular composition of this component and helps to establish a precise diagnosis in inherited C8 deficiencies.