Insulin-like growth factor I and insulin down-regulate growth hormone (GH) receptors in rat osteoblasts: evidence for a peripheral feedback loop regulating GH action.

The anabolic actions of GH are mediated by the production of insulin-like growth factor I (IGF-I) from the liver and by local production of IGF-I in extrahepatic tissues. Insulin facilitates the hepatic production of IGF-I by up-regulating GH receptors (GHRs) in the liver and augmenting the IGF-I response to GH. Although GHRs have also been identified in extrahepatic tissues that produce IGF-I, the possibility that IGF-I and insulin might partake in GHR regulation, thereby modulating the effects of GH locally has not received detailed study. The aim of this study was to investigate whether IGF-I and insulin are involved in the local regulation of GHRs, using osteoblasts as a model of GH-responsive extrahepatic tissues. We have used UMR106.06, a well differentiated rat osteoblast-like cell line that expresses GHRs and exhibits a mitogenic response to GH. IGF-I and insulin (0-10 nM) increased cell number and reduced [125I]GH binding in a concentration-dependent manner, with ED50 values of 0.8 and 0.3 nM, respectively. Although IGF-I increased cell number maximally by 36.9 +/- 1.2% (mean +/- SE) above the control value and insulin by 104.8 +/- 5.7% (P < 0.001), they decreased GH binding to 47.0 +/- 9.3% (P < 0.01) and 29.8 +/- 8.7% of the control value (P < 0.001), respectively. Scatchard analysis revealed that the down-regulation of GH binding was attributed to reduced receptor numbers and not binding affinity. The effects of IGF-I and insulin at submaximal concentrations were additive, although the combined effects did not exceed the maximal effect of either growth factor alone. Addition of an anti-IGF-I receptor antibody (alpha IR3) reversed the inhibition of GH binding induced by IGF-I, but not that caused by insulin; similarly, an antiinsulin receptor antibody (29B4) attenuated the inhibitory effect of insulin only. Addition of alpha IR3 alone or an ant-IGF-I antibody (Sm1.2) decreased cell number and increased GH binding in a concentration-dependent mode. GH at 1.5 nM significant increased cell number by 19.3 +/- 2.4% above the control level (P < 0.01), an increase that was reversed by alpha IR3. GH increased GH binding by 32.4 +/- 7.2% (P < 0.05) in cells treated with alpha IR3 to remove the secondary effect of IGF-I. In summary, IGF-I and insulin acted via specific receptors to stimulate cell proliferation and down-regulate GHRs in osteoblasts. GH stimulated cell proliferation, an action mediated by local production of IGF-I, and GH enhanced its own binding. The collective data suggest the presence of a peripheral negative feedback loop that allows IGF-I to limit locally the response of extrahepatic tissues to circulating GH.

Direct quantitation of growth hormone binding protein in human serum by a ligand immunofunctional assay: comparison with immunoprecipitation and chromatographic methods.

Current methods for measuring GH-binding protein (GHBP) are laborious, require separation of GHBP complex, and may be affected by endogenous GH content of the sample. Such methods estimate binding activity and GHBP concentration can only be obtained by Scatchard analysis. We have developed and validated a 2-site immunofunctional assay for the direct quantitation of GHBP in human serum employing a capture monoclonal antibody against GHBP and a polyclonal antibody against hGH. Results were compared with binding activity data and Scatchard-derived capacity estimates obtained by immunoprecipitation and gel chromatography procedures. Serum samples were obtained from 21 subjects with GH deficiency, 24 patients with acromegaly, and 56 normal subjects; 12 of whom were postmenopausal women who were studied before and during oral estrogen treatment. Using the immunofunctional assay, serum GHBP concentrations in normal subjects ranged from 0.14-3.28 nmol/L, was positively related to body mass index (BMI, P = 0.0004) and negatively related to age (P = 0.015). Women had significantly higher levels (0.99 +/- 0.12 vs. 0.63 +/- 0.09 nmol/L; P = 0.0191) than age and BMI-matched men. GHBP levels were not different between normal, acromegalic, or GH-deficient subjects. Oral estrogen therapy in postmenopausal women increased serum GHBP concentrations up to 5-fold. There was a significant nonlinear relationship between the immunofunctional assay measurements and binding activity by either immunoprecipitation (r = 0.84) or chromatographic (r = 0.73) methods; increase in GHBP concentrations was not reflected in proportionate increase in both activity assays. Estrogen-induced changes in circulating GHBP levels were greatest with the immunofunctional assay followed by Scatchard-derived values from immunoprecipitation and chromatographic methods. We conclude that ligand immunofunctional assay measurements of GHBP are higher but show good agreement with binding activity or Scatchard derived estimates from immunoprecipitation and chromatographic methods. This assay provides a direct and practical tool for rapid, accurate and sensitive estimation of GHBP concentration in serum.

Effects of different oral oestrogen formulations on insulin-like growth factor-I, growth hormone and growth hormone binding protein in post-menopausal women.

OBJECTIVE: Insulin like growth factor-I (IGF-I) levels in post-menopausal women are reduced by oral administration of the synthetic oestrogen ethinyl oestradiol but increased by transdermal delivery of 17 beta-oestradiol. Since these oestrogen types are different, the aim of this study was to clarify whether reduction in IGF-I is a specific effect of ethinyl oestradiol or common to other oral oestrogen formulations. DESIGN: Randomized cross-over study comparing one month of treatment with ethinyl oestradiol (20 micrograms), conjugated equine oestrogen (1.25 mg Premarin) and oestradiol valerate (2 mg). SUBJECTS: Six healthy post-menopausal women, age 60.3 +/- 5.6 years. MEASUREMENTS: Mean 24 hour GH (from hourly sampling), IGF-I, GH binding protein (GHBP), pituitary (LH, FSH) and hepatic function (SHBG and angiotensinogen) were measured. RESULTS: All three oestrogen formulations resulted in a significant reduction in IGF-I levels compared to baseline and significant elevations of GH and GHBP (P < 0.05). The percentage increase in GH during oestrogen treatment was significantly related to the percentage decrease in IGF-I levels (P = 0.04). All three oestrogen formulations resulted in significant suppression of LH and FSH and induction of the hepatic proteins, SHBG and angiotensinogen (P < 0.05). GHBP increased in parallel with other hepatic proteins. CONCLUSIONS: Reduction in IGF-I levels is an intrinsic effect of oral oestrogen therapy and increased GH levels may occur as a result of reduced feedback inhibition by IGF-I. Since GHBP activity is not changed by transdermal oestrogen, we conclude that the liver is a major source of circulating GHBP and that GHBP is an oestrogen sensitive protein.

Potent effects of human galanin in man: growth hormone secretion and vagal blockade.

Human galanin (hGAL) is a 30-amino acid neurohormone that has recently been shown to differ significantly from porcine and rat GAL. We investigated the endocrine and cardiovascular effects of hGAL in eight male volunteers. On three separate occasions, each received a 90-min infusion of saline, low dose (33 pmol/kg.min) and high (132 pmol/kg.min) dose hGAL, combined with an iv glucose bolus (to assess effects on insulin and GH release). hGAL was undetectable, 1.4 +/- 0.2 nmol/L, and 3.7 +/- 0.5 nmol/L during control, low dose, and high dose studies, respectively. The half-life of hGAL was 3.5 +/- 0.5 min. GH levels rose significantly in both studies (vs. control) and were not suppressed by hyperglycemia [low dose area under the curve (AUC), 1827 +/- 348 micrograms/min.L (P < 0.05); peak, 19.5 +/- 5.3 micrograms/L; high dose AUC, 1896 +/- 401 micrograms/min.L (P < 0.005); peak, 28.0 +/- 7.5 micrograms/L]. PRL levels rose significantly with the high dose study only (AUC, 12.8 +/- 1.1 micrograms/min.L; P < 0.01). FSH, LH, and catecholamine levels were unchanged. Glucose-stimulated insulin release was not inhibited. There was a dose-dependent increase in pulse rate and a profound decrease in sinus arrhythmia, but no change in blood pressure. These cardiovascular effects have not been reported with studies in humans using GAL of other species. We conclude that hGAL may play an important role in man in modulating GH secretion and cardiac vagal tone, but not insulin release.

Regulation of growth hormone binding protein in man: comparison of gel chromatography and immunoprecipitation methods.

GH circulates in part bound to a high affinity binding protein (GHBP). Gel chromatography is the established method for measuring GH binding activity in plasma, but is slow and tedious. The separation of bound from free GH by immunoprecipitation using a monoclonal antibody to the GH receptor may be a more practical alternative. We have examined the effects of GH and estrogen status on GHBP measured in 24-h pool samples and compared results obtained from gel filtration and immunoprecipitation. GHBP activity (percent specific binding of [125I]GH) was measured in normal, GH-deficient, and acromegalic subjects; and in two groups of postmenopausal women before and after oral (ethinyl estradiol 20 micrograms daily) or transdermal (17 beta-estradiol 100 micrograms daily) estrogen therapy. GHBP activity was not significantly different between normal, GH-deficient, and acromegalic subjects matched for age and sex. Neither GH administration to GH-deficient and normal subjects, nor octreotide treatment of patients with acromegaly, significantly altered GHBP activity. Oral estrogen treatment significantly increased GHBP activity. This change was associated with an increase in binding capacity (P = 0.001) but not affinity, as revealed by Scatchard analysis. GHBP activity did not change significantly with transdermal estrogen therapy. GHBP activity obtained by the two methods was significantly correlated (n = 70, r = 0.92, P = 0.001) although values for the antibody method were 25% higher. Similarly, capacity estimates were highly correlated (r = 0.90, P = 0.0001), with values by immunoprecipitation being 40% higher. We conclude that GH secretory status is not a determinant of GHBP. Oral estrogens increase GHBP activity through an increase in capacity and not affinity. The estrogen effect is route dependent and the oral response is likely to reflect a first pass hepatic effect. The higher binding activity and capacity estimated from immunoprecipitation are likely to reflect a more rapid separation process thereby minimizing dissociation of GHBP complex. Immunoprecipitation offers the advantages of simplicity, convenience, and accuracy for the measurement of GH binding activity and GHBP concentrations in serum.

Different modes of growth hormone (GH) administration do not change GH binding protein activity in man.

OBJECTIVE: Studies in rodents have shown GH binding protein (GHBP) levels to be dependent on the mode of GH administration. The aim is to determine whether GHBP levels in man are also modulated by the pattern of GH administration. PATIENTS: Six GH deficient subjects participated in a randomized study in which 2 IU GH were administered either as (i) a continuous 24-hour infusion, (ii) two intravenous boluses or (iii) eight intravenous boluses every 3 hours. In a second study, six normal men received a single subcutaneous injection of 0.2 U/kg GH and GHBP activity was measured over 24 hours. Control data were obtained from an untreated group of six age-matched normal men. MEASUREMENTS: GHBP activity was measured by immunoprecipitation using a monoclonal antibody that recognizes the human GHBP, and expressed as percentage specific binding of 125I-GH in 50 microliters of serum. RESULTS: GHBP activity was not significantly different between the GH deficient and normal subjects. GHBP activity did not rise significantly during GH administration with each of the three intravenous patterns of delivery nor were there any significant differences between treatments. In the second study, GHBP activity did not change significantly following subcutaneous GH injection nor did results differ from untreated normal controls. CONCLUSIONS: The level of GHBP in man is not dependent on GH secretory status or altered by short-term GH treatment or the mode of administration. These findings stand in contrast to GH treatment effects in rodents and suggest that GH regulation of GHBP may be different between species.

Measurement of human phospholipase A2 in arthritis plasma using a newly developed sandwich ELISA.

A sandwich enzyme-linked immunosorbent assay (ELISA) for human non-pancreatic phospholipase A2 (PLA2) was developed using monoclonal antibodies raised against purified recombinant PLA2. This assay was shown to be specific for human non-pancreatic PLA2, showing no cross-reactivity with human pancreatic PLA2 or with snake venom PLA2 (Crotalus durissus). The immunoassay showed no cross-reactivity with plasma components, and was reproducible and quantitative between 39 pmol and 2.7 nmol PLA2/1. The levels of non-pancreatic PLA2 in the plasma of patients with arthritis was measured using this immunoassay. There were significantly higher levels of PLA2 in patients with rheumatoid arthritis than in those with osteoarthritis or healthy controls. Plasma PLA2 was highest in those patients with active rheumatoid arthritis.

Elevated maternal plasma immunoreactive phospholipase A2 in human preterm and term labour.

Peripheral plasma concentrations of immunoreactive phospholipase A2 (irPLA2) (Type II, non-pancreatic) were determined in 110 women during pregnancy. The concentration of irPLA2 did not significantly change during pregnancy (5.7 +/- 0.6 ng/ml, n = 72) until the onset of labour. When compared with non-labouring women, irPLA2 concentrations were significantly elevated in association with both preterm labour (13.3 +/- 2.4 ng/ml, n = 15, p less than 0.02) and labour at term (10.4 +/- 1.7, n = 23, p less than 0.02). These data suggest that maternal plasma irPLA2 may be reflective of the mechanism(s) underlying the labour-associated increase in human gestational tissue eicosanoid formation.

Circulating phospholipase A2 activity associated with sepsis and septic shock is indistinguishable from that associated with rheumatoid arthritis.

Elevation of circulating phospholipase A2 (PLA2) activity is associated with sepsis and septic shock. Elevated levels of PLA2 activity also are seen in association with chronic inflammatory disorders such as rheumatoid arthritis. The relationship between these phospholipases is unclear. We have developed a highly specific enzyme-linked immunosorbent assay (ELISA) capable of measuring human synovial PLA2 in plasma, using monoclonal antibodies raised to recombinant synovial PLA2. This ELISA has been used to quantitate circulating PLA2 levels in patients clinically diagnosed with sepsis. These elevated levels positively correlated with the elevation seen in plasma PLA2 enzyme activity. The antibodies also have been used to purify immunoreactive PLA2 from plasma of patients with sepsis, thus enabling characterization of the purified protein by amino-terminal sequence analysis. We conclude from this study that the increase in PLA2 activity seen in association with sepsis and septic shock results from a dramatic elevation in levels of a circulating PLA2 enzyme. This inflammatory PLA2 is indistinguishable, both immunologically and chemically, from that associated with rheumatoid arthritis. Therapeutic agents directed towards inhibition of this inflammatory PLA2 enzyme may have utility in the treatment of both chronic and acute inflammatory disease.

Abnormalities of neutrophil phagocytosis, intracellular killing and metabolic activity in alcoholic cirrhosis and hepatitis.

Neutrophil functions of phagocytosis and intracellular killing of bacteria were examined in 40 patients with alcoholic cirrhosis of whom 18 had a superimposed acute alcoholic hepatitis. In 65% of these, defective neutrophil phagocytosis was demonstrable, and in 62.5% there was a defect of intracellular killing of either Staphylococcus aureus or Escherichia coli. Studies of the patients' serum failed to reveal inhibitors of neutrophil function. Additional assays of superoxide (O2-) and hydrogen peroxide production, hexose monophosphate shunt activity, degranulation and cellular levels of granule enzymes and glutathione revealed that these neutrophil defects are caused by both reduced production of superoxide and defects of degranulation. The hydrogen peroxide/superoxide molar ratio was raised in patients' neutrophils, and the strong inverse correlation found between the value of this ratio and intracellular levels of reduced glutathione would be consistent with the hypothesis that the neutrophils from patients with cirrhosis are unable to detoxify hydrogen peroxide effectively and that this is a result of reduced levels of glutathione in the cells. The consequent increase in oxidant stress, both intra- and extracellularly, may be the cause of phagocytic and degranulation defects. The reduced responses of patients' neutrophils may be caused by previous exposure of the cells to activating stimuli in circulation, as evidenced by depleted intracellular levels of granule enzymes and glutathione. Neutrophils from the patients with a superimposed acute alcoholic hepatitis had depressed phagocytosis in the early stages of incubation but, on the whole, neutrophils from these patients had a greater capacity for ingestion and killing of bacteria than neutrophils from patients with cirrhosis alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of neutrophil function by hydrogen peroxide. Effect of SH-group-containing compounds.

Stimulated neutrophils generate appreciable amounts of hydrogen peroxide (H2O2) which may be responsible for auto-oxidative injury and damage to adjacent cells. In the present study we describe inhibitory effects of H2O2 on neutrophil phagocytosis, bactericidal activity and associated metabolic processes as well as the effect of non-protein SH-compounds on H2O2-treated cells. Preincubation of neutrophils with low concentrations of H2O2 (1 mumoles/5 X 10(6) cell) results in delayed phagocytosis of Escherichia coli, which returns to normal levels in the later stages of incubation, while the activity of the HMPS and the production of O-2 and H2O2 remain unaffected. Bactericidal activity of the cells was more sensitive to peroxide treatment and even at low concentrations H2O2 induced some inhibition (12.2%) of neutrophils' capacity to kill E. coli. Increasing the concentrations of H2O2 in the preincubation mixtures resulted in a progressive decline in the neutrophils phagocytic and killing capacity for E. coli and was accompanied by inhibition of HMPS activity and the release of granule enzymes but not of O-2 or H2O2. The H2O2/O-2 molar ratio of peroxide-treated cells was elevated by up to 26.7% and this was followed closely by the reduction in the intracellular levels of reduced glutathione (GSH). Incubation of H2O2-treated neutrophils with all five SH-compounds used in the study resulted in the improvement of the phagocytic capacity of the cells. Improvement of the bactericidal capacity and degranulation responses of H2O2-treated neutrophils was achieved by incubation with cysteine, penicillamine, alpha-MPG and MMPC but not GSH. Stimulus-dependent H2O2 production by H2O2-treated cells, the H2O2/O-2 molar ratio and the intracellular levels of GSH remained unaltered after treatment with SH-compounds. The data shows that SH-compounds, in addition to their antiinflammatory properties, also have the ability to reverse the oxidant-induced inhibition of neutrophil function, a property of potential therapeutic significance.

Rapid microassays of phagocytosis, bacterial killing, superoxide and hydrogen peroxide production by human neutrophils in vitro.

Simple, rapid microassays for simultaneous measurement of phagocytosis, bacterial killing, superoxide and hydrogen peroxide production by human neutrophils in vitro are described. All assays employ 96-well flat bottom tissue culture plates which were incubated on a microtitre plate shaker at 37 degrees C. The separate evaluation of ingestion and intracellular killing of E. coli and S. aureus was based on the incorporation of [3H]uridine into viable extracellular bacteria. There was good correlation between plate counts of viable bacteria and amount of radiolabel incorporation. Phagocytosis and killing can be measured in a maximum of 100 microliter reaction mixture, requiring only 2.5 X 10(5) neutrophils per test and the assay is complete within 60 min. Assay of superoxide production by stimulated neutrophils was based on superoxide-dependent reduction of ferricytochrome c as measured spectrophotometrically at 550 nm in wells of tissue culture plates containing 150 microliter of reaction mixture. The assay requires only 1.25 X 10(5) neutrophils per test and is complete within 50 min. Quantitation of hydrogen peroxide was based on horseradish peroxidase-dependent oxidation of phenol red. The technique is as for superoxide detection except that the reaction must be terminated by the addition of 1 M NaOH at the desired time intervals. None of the assays require sampling during the incubation period. The principal advantages of the described techniques are increased simplicity and speed, requirement of low numbers of neutrophils and applicability to analysis of large number of samples in parallel.

Polymorphonuclear leucocyte locomotion and aggregation in patients with alcoholic liver disease.

Defects in stimulated movement of polymorphonuclear (PMN) leucocytes was detected in 57% of patients with alcoholic liver disease. Serum from patients with the cellular defect had no effect on the function of normal PMN leucocytes. Aggregation responses of patients' PMN leucocytes suggest that the cellular defect may be related to specific abnormalities in the response to the C5a chemotactic factor. Defective serum attractant activity was found in 65% of the patients tested and the presence in the patients serum of humoral directed antagonists appeared to be responsible for the defect in majority of cases. Further analysis pointed to the presence of at least two distinct antagonists and the possible involvement of proteases in this serum abnormality. The activity of the serum antagonists or the severity of the cellular defect were unrelated to the presence of bacterial infection or elevations in serum IgA or IgG. The high frequency of cellular defects, possibly as a result of in vivo activation, in conjunction with serum abnormalities could account for the increased susceptibility of patients with alcoholic liver disease to bacterial infection.

Enhancement of neutrophil response by SH-containing compounds: modulation of superoxide and hydrogen peroxide production.

Anti-inflammatory effects of SH compounds in vivo and their effects on lymphocytes and macrophages in vitro have been described, but little is known about the mechanism of action or their effects on the neutrophil. In the present study the activity of seven low molecular weight non-protein SH compounds was compared. At a concentration of 3 X 10(-4)M all the compounds enhanced the activity of the HMP shunt of zymosan-stimulated neutrophils by 26-48% and that of PMA-stimulated cells by 6-44% above the control value (14.2 nmol CO2/2.5 X 10(6) neutrophils/30 min). Pretreatment of neutrophils with SH compounds for 15 min resulted in enhanced release of O-.2 by stimulated neutrophils in all cases, with the exception of GSH, by up to 87% above that of control. These effects were largely related to the ability of the compounds to modulate the release of O-.2 and H2O2 by stimulated neutrophils when present in the reaction mixture. Only the compounds alpha-MPG and cysteine had a mild preserving effect on the intracellular GSH concentration of stimulated neutrophils. None of the compounds tested had any adverse effect on phagocytosis or killing of opsonized bacteria by the neutrophils. SH compounds may protect sensitive SH groups of functional proteins by providing an easily accessible source of oxidizable SH groups in times of high oxidative stress, and their ability to interact with oxygen products could in part explain their anti-inflammatory properties.

Defects in serum attractant activity in different types of chronic liver disease.

Serum attractant activity, measured in 57 patients with chronic liver disease, was significantly reduced in 66% of the 27 patients with alcoholic liver disease and in 29% of the 17 patients with chronic active hepatitis, but was normal in 13 patients with primary biliary cirrhosis despite the presence of established cirrhosis in nearly half of them. In patients with alcoholic liver disease, but not in those with chronic active hepatitis, there was a correlation between the serum defect and severity of liver disease. The defect could not be related to the deficiency of key complement components, raised concentrations of IgA or G or the concurrent presence of bacterial infection. These findings suggest that the aetiology of liver disease may be an important factor in the development of serum attractant abnormalities.

Neutrophil adherence in chronic liver disease and fulminant hepatic failure.

Abnormal adherence of neutrophils to nylon fibre in vitro was found in blood from 17 of 51 (33.3%) patients with chronic or acute liver disease of different aetiologies. Patients with chronic liver disease had a much wider range of values than the controls and the sub-group with alcoholic cirrhosis had significantly higher adherence (72.4 +/- SD 6.2%) than that of controls (65.8 +/- SD 5.2%). The patients with chronic active hepatitis (68.2 +/- 12.7%) or primary biliary cirrhosis (69.2 +/- 6.6%) were not different from controls. Significantly reduced neutrophil adherence (56.2 +/- 8.7%) was found in blood from patients with fulminant hepatic failure. These abnormalities in neutrophil adherence may be due to the effects of the split components of serum complement and dependent on the degree and duration of exposure of the neutrophils. Defects in neutrophil adherence may in part contribute to the increased susceptibility to infection in patients with acute and chronic liver disease.

Impaired opsonization by serum from patients with chronic liver disease.

Serum opsonization of two organisms, E. coli and yeasts (S. cerivisiae), was examined in 68 patients with chronic liver disease (CLD). Impaired opsonization for yeasts was found in seven (29%) of 24 patients with chronic active hepatitis, six (27%) of 22 with alcoholic cirrhosis and five (23%) of 22 with primary biliary cirrhosis. Opsonization for E. coli was normal in patients with primary biliary cirrhosis but impaired in seven (29%) patients with chronic active hepatitis and three (14%) of those with alcoholic cirrhosis. The defect of opsonization in chronic active hepatitis was found mainly in patients with histological evidence of active disease. A deficiency, rather than antagonism or inhibition, of normal opsonization factors was responsible, but could not be related to reduced levels of serum complement factors of either the classical or the alternative pathway present in 45% of the patients with chronic active hepatitis, 71% with alcoholic cirrhosis and 18% of those with primary biliary cirrhosis. Serum from two of 11 patients with impaired opsonization antagonised the function of normal polymorphonuclear leucocytes, and polymorphonuclear leucocytes from six of seven patients had slightly reduced phagocytosis/killing of E. coli opsonized in normal serum. Defects of serum opsonization, complement activity and polymorphonuclear leucocyte function may be causes of the increased susceptibility to bacterial infection in patients with CLD.

Serum stimulatory activity and polymorphonuclear leucocyte movement in patients with fulminant hepatic failure.

Serum from 27 patients with fulminant hepatic failure and grade IV encephalopathy had reduced ability to stimulate the movement in vitro of normal polymorphonuclear leucocytes. All patients had a deficiency of serum complement factors C3 and C5 and there was a significant positive correlation between C5 and serum stimulatory activity. However, in addition to this complement defect, serum from 22% of patients contained an antagonist to normal serum stimulatory factors. This antagonism was attributed to at least two different substances in the serum on the basis of differences in heat lability, dialysability and action on complement factor C5a. Polymorphonuclear leucocytes from eight of 13 patients had reduced movement toward serum, but serum from only one patient contained an antagonist acting on the cells; this was probably related to an underlying carcinoma of the breast. During the early stages of clinical recovery, serum stimulatory and complement activity returned to normal. These serum and cellular defects have not been reported previously in patients with fulminant hepatic failure and represent major defects in the body's defenses against bacterial infection.

Defects of serum chemoattraction and polymorphonuclear leucocyte movement in patients with primary hepatocellular carcinoma.

Movement of polymorphonuclear leucocytes to the site of tumour cells may be an important stage in host defences against tumours in a variety of organs. In this study, sera from 29 of 30 patients with primary hepatocellular carcinoma had reduced ability to stimulate the movement in vitro of normal polymorphonuclear leucocytes. The serum defect was more severe in 11 patients with underlying cirrhosis but was not related to abnormalities of tests of liver function, levels of serum alphafetoprotein, or deficiency of complement factors C3 and C5. Serial studies showed that the defect was persistent and progressive in patients in whom the tumour did not respond to treatment. In 35% of patients, mainly those with cirrhosis, the sera contained antagonists to normal serum chemotactic factors which were heat stable and dialysable, but could be distinguished by their effect on complement factor C5a. A heat labile dialysable antagonist(s) was found in sera from 28% of the patients (mainly those without cirrhosis) which antagonized the movement of normal polymorphonuclear leucocytes (cell directed antagonism). In addition to these serum defects, polymorphonuclear leucocytes from two of seven patients studied had reduced movement which was not related to the presence in the serum of cell directed antagonists. These serum and cellular defects have not been reported previously in patients with primary hepatocellular carcinoma, and could compromise the body's defences against the tumour.