Plasma Gc protein sequesters actin released into the circulation after massive hepatocyte necrosis, but is greatly depleted in the process. In fulminant hepatic failure (FHF), Gc is present in serum both as a complex with actin and as unbound protein, the latter becoming completely exhausted in those patients with the most severe FHF. In the present study, 47 consecutive patients with FHF, 39 of whom were the result of acetaminophen (AC) overdose, were evaluated to determine whether measurement of Gc protein levels could be used to predict survival. Using serum samples obtained shortly after admission as well as later samples, levels for total Gc protein, percentage of Gc complexed with actin, and calculated unbound Gc remaining in serum were compared for survivors and those who died of their illness. The most marked changes were present in unbound Gc levels in nonsurvivors, the mean of which for follow-up samples was 10% of normal mean values, as compared with 23% of normal mean values in those who survived (P < .01). Using a cutoff value for unbound Gc protein of ≥34 μg/mL to predict survival, outcome was correctly predicted in 32 of 47 (68%) patients using early samples, and in 24 of 27 (89%) patients using later sera. No differences were observed between values and/or outcome in AC and non-AC cases. Measurement of Gc protein level correctly predicted all patients dying of hepatic failure. This single measurement compares favorably with multifactorial predictive models, such as the King's College model, and might be a useful test for patients being considered for transplantation.

Studies were performed in Hep3B hepatocytes to better elucidate the mechanisms regulating circulating levels of human group-specific component (Gc). We measured changes in Gc messenger RNA (mRNA) synthesis and levels of secreted protein resulting fr om treatment of hepatocytes with cytokines and hormones known to influence synthesis of other proteins of hepatic origin, We particularly focused on compounds known to be prototypic stimulants during the acute phase response, interleukin-6 (IL-6) and dexamethasone were shown to increase Gc mRNA approximately twofold while transforming growth factor beta (TGF beta) decreased Gc mRNA in a dose-dependent fashion by up to fivefold. The effects on secreted Gc protein levels were similar. These results indicate that Gc protein appears to be regulated differently than the other members of this gene family, albumin and alpha-fetoprotein (AFP), which are negative acute phase reactants, in addition, these contrasting effects on Gc synthesis of IL-6 and dexamethasone and of TGF beta suggest that high basal levels of Gc synthesis may be maintained during the acute phase response.

The presence of Gc (vitamin D binding protein) has been consistently demonstrated on the membrane of B lymphocytes. This protein appears to be spatially associated with surface immunoglobulins. The origin of this surface protein has not yet been determined and the purpose of the present paper was to investigate if Gc may bind to human lymphocytes after immunoglobulin (Ig) capping. For this purpose the presence of Gc on B lymphocytes was examined by three different approaches. First, when cells were examined by immunofluorescence and quantified by flow cytometry, membrane Ig capping was followed by a dramatic decrease in positivity for Gc when compared to native cells. In addition, incubation of capped cells with purified Gc was followed by a significant increase in fluorescence, indicating that this protein had been able to bind again. Second, analysis of solubilized lymphocytes by Western blotting showed that native lymphocytes and capped cells incubated with purified Gc contained a large quantity of a 56kDa protein which was immunoreactive with anti Gc antibodies. This protein band was much weaker on blots from capped cells not treated with Gc. Third, radiobinding assays indicated that, following capping, cells were able to bind Gc in saturable fashion. These results suggest that membrane Gc could play a role in the entry of vitamin D metabolites into lymphocytes.

We have utilized enzyme-linked immunosorbent assay (ELISA) to quantitate PCR-amplified DNA. This method was used to measure mRNA for the vitamin D-binding protein (Gc), beta-actin and the transferrin receptor (TR) gene in the Hep3B cell line. Total RNA from Hep3B cells was reverse transcribed to obtain cDNA, which was amplified in the presence of digoxigenin-dUTP by PCR. The PCR products were then hybridized in liquid phase to a biotinylated, nested capture probe for the respective sequences. The hybridized products were bound to a streptavidin-coated ELISA plate and were detected by an alkaline-phosphatase-conjugated antibody to digoxigenin. ELISA standard curves for Gc and control genes, beta-actin and TR, were obtained after PCR amplification of serial dilutions of Hep3B total RNA. As an external standard, an ELISA standard curve for Gc was obtained after PCR amplification of serial dilutions of a full-length Gc cDNA insert obtained from a recombinant plasmid. Thus, we were able to develop a non-isotopic quantitation assay for PCR-amplified DNA that is highly sensitive and has the specificity of hybridization-based methods.

The expression of the DBP (vitamin D binding protein) gene was investigated in monocytes and in peripheral blood lymphocytes. DBP message was amplified through 35 cycles of PCR amplification using specific oligonucleotide primers. PCR products of the expected size were further identified by Southern blotting using a specific DBP probe. No expression of the DBP gene could be detected in peripheral blood lymphocytes, nor in the monocyte-derived U 937 cell line. In contrast, message for DBP was identified in monocytes activated with lipopolysaccharide when analyzed between 6 and 10 h following stimulation. These results suggest that the temporal expression of the DBP gene could play a major role in the activation of monocytes by 1-25(OH)2D3.

Vitamin D binding protein (Gc) is present on the surface of several blood cells and may interfere with the activity of 1,25(OH)2D3. It has previously been reported that Gc may bind to the U-937 line which is known to differentiate upon exposition to 1,25(OH)2D3. In the present paper, we evaluate the expression of Gc on the surface of U-937 and HL-60 lines. Both cell lines did not express Gc on their surface but U-937 cells were able to bind human purified Gc added to the medium whereas HL-60 were not. After culturing with 1,25(OH)2D3, HL-60 became able to bind Gc. This property seems to be related to the monocytic differentiation induced by 1,25(OH)2D3. Conversely, when present together, 1,25(OH)2D3 reduces binding on U-937.

A monoclonal antibody, E12, to human Gc globulin was raised in murine somatic cell using purified Gc. The antibody was subtyped IgG2bκ and had a kd of 3.0β10−8 M for antigen Gc. Monospecificity for Gc was demonstrated by Western blotting of normal human serum using nondenaturing polyacrylamide gel electrophoresis. As judged by ELISA, actin inhibitted binding of E12 to Gc in dose-dependent fashion. Affinity chromatography studies further showed that ternary complexes of actin-Gc-E12 were not formed, and actin displaced Gc from Gc-E12 complexes. Proteolytic digestion of Gc with trypsin showed that the monoclonal antibody E12 reacted with the major 30-kDa tryptic fragment containing the amino terminal fragment of Gc, but actin did not react with this fragment. These results indicate that interaction of actin with Gc causes conformational changes which inhibit binding of E12.

ACTIN is the most abundant protein in mammalian cells. Cell motility and change in the size and shape of cells depend on the ability of monomers of actin to polymerize to actin filaments. When actin is released from cells, however, its strong tendency to polymerize can become a liability, and the presence of filaments of actin in blood vessels can be fatal. Cell necrosis is associated with the release of actin in a variety of clinical situations, including hepatic necrosis, septic shock, the adult respiratory distress syndrome, and certain disorders of pregnancy. A newly described homeostatic mechanism, termed the actin-scavenger . . .

The identification of Gc (vitamin D binding protein) with the anionic polypeptide cochemotaxin has recently been reported. In this paper we investigate its dose dependent cochemotactic activity and report the inhibition of Gc enhanced chemotaxis by vitamin D3. These results further support the role of immunomodulating hormone played by vitamin D.

Group-specific component (Gc) and total albumin concentrations in the breast secretions from 20 full-term infants were measured. The Gc concentrations as well as the albumin concentrations correlated significantly (p less than 0.02) with the total cell count and the absolute concentration of each white blood cell type in the breast fluid. The ratio of albumin in neonatal breast secretion to that in neonatal serum was similar to the comparable ratio for Gc. Since albumin and Gc are of similar molecular size, these observations suggest leakage of these two proteins from serum to breast secretion and a possible chemotactic relationship between these proteins and the mononuclear cells in neonatal milk.

Binding studies with [3H] 25-(OH) D3 were performed under a variety of conditions using Gc (Vitamin D-binding protein) purified from individuals displaying different phenotypes. No significant differences in affinity of binding were found between Gc1f, Gc1s and Gc2 allelle products in either homozygous or heterozygous individuals, nor between Gc1 anodal and Gc1 cathodal isotypes. Affinity was not significantly affected by different reaction temperatures (4, 22 or 37 degrees C), the presence or absence of Ca2+ ions, and binary and ternary interactions with G-actin and G-actin-DNase complexes respectively. However, reduction of pH caused a progressive decrease in binding with virtual abolition at pH less than or equal to 5.0. The latter might promote dissociation of D3 metabolites from Gc carrier protein in acidic compartments of cells.

Septic shock is known to involve increased metabolism of arachidonic acid and generation of certain eicosanoids. Recently, a new extracellular pool of unsaturated fatty acids including arachidonate has been found in relation to group-specific component (Gc), a vitamin D-binding plasma protein that sequesters monomeric G-actin. Since complexing with G-actin displaces fatty acids, possible alterations in plasma levels of Gc and extent of complexing were sought in serial samples obtained from rats with shock induced by Salmonella enteritidis endotoxin (12.5-15 mg kg-1). Gc levels in animals receiving endotoxin exhibited bimodal alterations, with a significant reduction (P less than 0.001) at 1 hour, followed by a progressive elevation to 160% of starting concentrations at 6 days in animals that survived, whereas in sham-injected animals the change observed was a continuous rise to 147% at 6 days. A statistically significant increase in the percentage of Gc complexed was observed in all endotoxemic rats from 2 hours onward (P less than 0.01), in contrast to sham-injected animals, in which the percentage of Gc complexed remained at less than 5%. Levels in survivors peaked at 30 +/- 5.2% at 8 hours and then decreased to normal (2 +/- 0.9%) by 6 days (n = 7), whereas in nonsurvivors complexed Gc continued to rise until time of death (66-80%) at 6-12 hours (n = 4). Correlation of these results with glucose, transaminases, and immunoreactive TXB2 and 6-keto-PGF1 alpha indicated that decreased absolute levels of Gc represent a consistent early change in endotoxic shock and that the percentage of Gc complexed is an accurate prognostic indicator of severity.

Septic shock involves increased generation of eicosanoids from arachidonic acid. Gc (vitamin D-binding protein) has been recently found to bind the parent molecule arachidonic acid but can also complex actin released as a result of tissue damage which causes displacement of bound arachidonic acid. Possible changes in serum levels of Gc and extent of complexing were therefore investigated in patients with gram-negative sepsis. As compared to healthy controls, serum levels of Gc were significantly decreased in patients with septic shock (P less than 0.01). Moreover, the percentages of Gc circulating in complexed form were significantly increased (P less than 0.01) and correlated strongly with disease severity, with levels often greater than 90% in patients who died (normal mean 8% +/- 3). These results suggest a hitherto unsuspected role for Gc in septic shock syndrome.