A serum factor that suppresses the cytotoxic function of cytokine-stimulated human eosinophils.

A human subject (NR) was identified whose eosinophils and neutrophils failed to respond to TNF in vitro in 29 of 33 experiments, using several biological assays. There was a response rate to TNF of 100% among 37 control subjects whose leukocytes were tested in parallel. NR serum contained an activity that inhibited the cytotoxic function of TNF- and GM-CSF-stimulated normal human eosinophils. A similar activity was detected in 4 of 122 control sera and in sera of two subjects with hypereosinophilia. This activity (ECI) had an apparent molecular weight of 80,000-100,000 and was sensitive to heating at 80 degrees C or to trypsin treatment. HPLC sizing chromatography increased the titer of ECI by a factor of 50 to 2,000 in experiments using NR serum or other sera with detectable inhibitory activity. In seven experiments using sera with no inhibitory activity, HPLC generated ECI of the same apparent molecular weight. The effect of HPLC on ECI activity required the separation of serum components and did not result from exposure to HPLC system components or other sample processing methods. This suggests that ECI in serum can be stabilized in an inactive or partially active form and that HPLC removes the stabilizing component. ECI suppressed TNF-stimulated eosinophil cytotoxic function when added to cultures up to 4 h after exposure of eosinophils to cytokine. However, ECI did not protect L929 cells from the toxic effects of TNF. Thus, ECI did not act by preventing the initial interaction of TNF with eosinophils or by interfering with the binding of TNF to its receptor on L929 cells. The results suggest that ECI is a component of a feedback mechanism that suppresses functions of cytokine-activated eosinophils in inflammation.

Molecular cloning of the complementary DNA for human tumor necrosis factor.

Tumor necrosis factor (TNF) is a soluble protein that causes damage to tumor cells but has no effect on normal cells. Human TNF was purified to apparent homogeneity as a 17.3-kilodalton protein from HL-60 leukemia cells and showed cytotoxic and cytostatic activities against various human tumor cell lines. The amino acid sequence was determined for the amino terminal end of the purified protein, and oligodeoxyribonucleotide probes were synthesized on the basis of this sequence. Complementary DNA (cDNA) encoding human TNF was cloned from induced HL-60 messenger RNA and was confirmed by hybrid-selection assay, direct expression in COS-7 cells, and nucleotide sequence analysis. The human TNF cDNA is 1585 base pairs in length and encodes a protein of 233 amino acids. The mature protein begins at residue 77, leaving a long leader sequence of 76 amino acids. Expression of high levels of human TNF in Escherichia coli was accomplished under control of the bacteriophage lambda PL promoter and gene N ribosome binding site.

Tissue factor pathway inhibitor reduces mortality from Escherichia coli septic shock.

This study was designed to test the hypothesis that tissue factor pathway inhibitor (TFPI) plays a significant role in vivo in regulating coagulation that results from exposure of blood to tissue factor after vascular injury as in the case of gram negative sepsis. Highly purified recombinant TFPI (6 mg/kg) was administered either 30 min or 4 h after the start of a lethal intravenous Escherichia coli infusion in baboons. Early posttreatment of TFPI resulted in (a) permanent seven-day survivors (5/5) with significant improvement in quality of life, while the mean survival time for the controls (5/5) was 39.9 h (no survivors); and (b) significant attenuations of the coagulation response and various measures of cell injury, with significant reductions in pathology observed in E. coli sepsis target organs, including kidneys, adrenals, and lungs. TFPI administration did not affect the reduction in mean systemic arterial pressure, the increases in respiration and heart rate, or temperature changes associated with the bacterial infusion. TFPI treated E. coli infected baboons had significantly lower IL-6 levels than their phosphate buffered saline-treated controls, however tumor necrosis factor levels were similarly elevated in both groups. In contrast to the earlier 30-min treatment, the administration of TFPI at 4 h, i.e., 240 min, after the start of bacterial infusion resulted in prolongation of survival time, with 40% survival rate (2/5) and some attenuation of the coagulopathic response, especially in animals in which fibrinogen levels were above 10% of normal at the time of TFPI administration. Results provide evidence for the significance of tissue factor and tissue factor pathway inhibitor in bacterial sepsis, and suggest a role for blood coagulation in the regulation of the inflammatory response.

Binding of 125I-labeled recombinant beta interferon (IFN-beta Ser17) to human cells.

We investigated the binding of 125I-labeled beta interferon (IFN-beta Ser17), a nonglycosylated recombinant human fibroblast interferon in which cysteine at position 17 is replaced by serine by site-specific mutagenesis. An optimized chloramine T radiolabeling method produced a highly labeled, fully active 125I-IFN suitable for these studies. Unlike the case with the chloramine T method, incorporation of a single mole of Bolton-Hunter reagent into a mole of IFN-beta Ser17 led to nearly complete loss of biological activity. 125I-IFN-beta Ser17, prepared by the chloramine T method, bound specifically to human lymphoblastoid cells (Daudi) with a dissociation constant of 0.24 nM. The number of binding sites per cell was 4,000. In competition assays, unlabeled beta interferons (native, recombinant IFN-beta Cys17, and various preparations of IFN-beta Ser17) equally displaced labeled IFN-beta Ser17 on Daudi cells. Recombinant IFN-alpha-1 displaced 125I-IFN-beta binding to Daudi cells less efficiently than did unlabeled native or recombinant beta interferon. However, at the concentrations tested, native gamma interferon showed no competition with 125I-IFN. Our results indicate that IFN-beta Ser17 and native IFN-beta posses similar binding properties.

Growth regulation of melanoma cells by interferon and (2'-5')oligoadenylate synthetase.

We report that endogenous, as well as exogenous, interferon (IFN) regulates the growth of human melanoma cells in culture. When antibodies directed against human fibroblast IFN were incorporated into the media of high-density cells stimulated to proliferate with serum, the cells entered the cell cycle earlier than did the controls. In investigating the biochemical basis for this finding, we have found that there is an inverse relationship between the (2'-5')oligoadenylate synthetase levels and the percentage of cells in S in untreated cultures. Upon IFN treatment, the relationship is obliterated and (2'-5')oligoadenylate synthetase levels increase throughout all phases of the cell cycle. This increase in enzyme levels correlates well with the decreased probability of the IFN-treated cells to cycle. These findings suggest a biological role for IFN as a negative growth factor for cells in culture.

Cures and partial regression of murine and human tumors by recombinant human tumor necrosis factor.

We tested the effect of recombinant human tumor necrosis factor (TNF) on the growth of the murine methylcholanthrene induced fibrosarcoma and the human ovarian carcinoma (NIH:OVCAR-3) in mice. The mice received multiple doses (25-250 micrograms/kg) of TNF starting 7-10 days after s.c. transplantation of tumors when they were easily palpable. TNF was administered i.v. every other day for a total of 6 injections per mouse, or i.p. daily for 7 days. Complete tumor regression was observed in the methylcholanthrene induced tumor bearing mice in 90% of the mice treated with TNF (100 micrograms/kg), 67% treated with TNF (50 micrograms/kg), and 34% treated with TNF (25 micrograms/kg). Tumors which did not completely regress were growth retarded during the course of TNF treatment. All mice given the highest TNF dose are still alive and tumor free (currently over 400 days), whereas the median survival of control mice was 28-39 days. Partial regression was observed in 100% of mice bearing the ovarian carcinoma treated i.p. with 250 micrograms/kg. Injections of TNF i.v. resulted in higher percentage of cures than i.p. injections at similar dose levels. These results suggest that tumor necrosis factor represents a likely potent drug against solid tumors and that the method of administration is critical in optimizing its use in cancer.

Biological effects of recombinant human tumor necrosis factor and its novel muteins on tumor and normal cell lines.

We investigated optimal conditions for cytotoxicity to tumor cell lines by recombinant human tumor necrosis factor (rhTNF) and the effect of amino-terminal deletions on the bioactivity of the rhTNF molecule. Two of four deletion muteins (-4 and -7) of rhTNF exhibit 2- to 3-fold enhancement of cytotoxicity/cytostasis against a variety of human carcinomas, a fibrosarcoma, and a melanoma cell line with no toxicity on normal fibroblastic and epithelial cultures. Of the two other muteins the -8 displayed equivalent and/or increased cytotoxicity/cytostasis while the -10 was consistently less cytotoxic than the parent on the same cell lines. Continuous exposure to TNF for greater than or equal to 96 h led to maximal cytotoxicity to tumor lines (99.99% with L929 cells) with no evidence of recovery. Pretreatment with actinomycin D (0.003-10 micrograms/ml for 1 h) rendered 82% of rhTNF-resistant cell lines (both tumor and normal) susceptible to its cytotoxic action within 24 h. However, the highest nontoxic concentrations of Actinomycin D necessary for rendering normal cell lines susceptible to TNF action were about 10-3000-fold higher than those necessary for converting resistant tumor cell lines. Similarly, preinfection of L929 cells with vesicular stomatitis virus (multiplicity of infection, 10(-2)-10(-4) for 1 h) rendered the cells 2-10-fold more susceptible to the cytotoxic action of rhTNF in 18 h. Our data suggest that rhTNF and its muteins represent potentially useful anticancer agents; however, adequate dosing and prolonged exposure may be critical in demonstrating cytotoxicity/cytostasis. The data also show that although normal and tumor cell lines became susceptible to cytotoxicity by rhTNF and actinomycin D, combination therapy of the two agents may be possible at defined concentrations.

Functional properties of proteins coded by three human alpha-interferon genes and a pseudogene.

We have characterized the functional properties of four highly purified recombinant human class I alpha-interferon subtypes whose biological activities have not been described previously. We selected biological and biochemical activities that may discriminate between different functions of these molecules. We found that the alpha subtypes could be discriminated only by antiviral-host range specificity and natural killer cell activation. Differences in their antiproliferative activity were cell line dependent. Competitive binding, antiproliferative activity in agar, enhancement of expression of HLA-ABC, elevation of 2'-5'-oligoadenylate synthetase levels and enhancement of phosphorylation of the Mr 69,000 protein kinase did not allow discrimination among the alpha I subtypes on the tested cell lines.

Randomized, blinded, placebo-controlled trial of tissue factor pathway inhibitor in porcine septic shock.

This study tested the hypothesis that tissue factor pathway inhibitor (TFPI) would improve mortality and morbidity evoked by peritonitis-induced bacteremia in pigs. Secondarily, it sought to determine if TFPI treatment would attenuate cardiodynamic abnormalities produced by this septic model. 32 pigs were chronically instrumented with intracardiac transducers to measure left ventricular pressure and diameter, pulmonary and aortic pressures, and cardiac output. At least 5 days after surgery to implant transducers, basal cardiovascular readings and blood samples were obtained. Using a randomized, blinded study design, either purified, reconstituted TFPI (1 mg/kg bolus, 10 mg/kg/min for 48 h), placebo (arginine buffer), or saline was administered to pigs immediately after Escherichia coli 0111.B4 (3.0-11 x 10(9) colony-forming U/kg)-laden fibrin clots were implanted intraperitoneally, producing peritonitis and bacteremia. Pigs did not receive antibiotics or supportive therapy. No significant differences in primary or secondary endpoints were noted between the arginine and saline groups, so these data were combined into a control group (N = 20). 5 of 12 TFPI pigs survived (42%), while 5 of 20 control pigs survived (25%); this difference was not significant (p = .714, Fisher's exact test). TFPI treatment augmented cardiac output in surviving pigs, but did not affect any other cardiovascular performance variable (heart rate, % diameter shortening, or systemic and pulmonary vascular resistance). In controls, peritonitis induced rapid increase in plasma tumor necrosis factor-alpha (428 +/- 771 to 5,933 +/- 559 pg/mL at 2 h) and interleukin-8 (180 +/- 153 to 1,393 +/- 145 pg/mL at 2 h). TFPI treatment significantly attenuated cytokine responses to sepsis, reducing peak tumor necrosis factor-alpha to 2,103 +/- 813 pg/mL and reducing peak interleukin-8 levels to 534 +/- 211 pg/mL at 2 h (p < .05, Tukey test, two-way ANOVA). In conclusion, TFPI treatment attenuated important mediator components of the inflammatory response but did not provide significant survival benefit.

Extremely low doses of tissue factor pathway inhibitor decrease mortality in a rabbit model of septic shock.

OBJECTIVE: We sought to determine the lowest dose of recombinant human tissue factor pathway inhibitor (TFPI) that can provide protection from lethality in a rabbit model of septic shock. METHODS: Sepsis was induced in New Zealand white rabbits by intraperitoneal implantation of 7.0 ml of a solution containing hemoglobin (4.8 g/dl), porcine mucin (6 g/dl), and 0.8-1.4 x 10(4) viable Escherichia coli (strain O:18 K+). Gentamicin (5 mg/kg) was administered 4 h following surgery, and this dose was repeated every 12 h for 3 days. Beginning 4 h following the induction of sepsis, animals were treated with a bolus (1 ml) plus a continuous infusion (100 ml over 24) of either TFPI (various doses) or its vehicle. Four different doses of TFPI were studied, and each experiment included a contemporaneous control group. The primary outcome parameter was survival time. Results were analyzed using the Wilcoxen log rank test. RESULTS: The average survival time for rabbits treated with the highest dose of TFPI tested (50 microg/kg bolus and 0.5 microg/kg per minute infusion) was 118 h, as compared to 81 h in vehicle-treated controls). The average survival time for septic rabbits treated with a much lower dose of TFPI (100 ng/kg bolus and 1.0 ng/kg per minute infusion) was 119 h as compared to 57 h in surviving vehicle-treated controls. Treatment with an even lower dose of TFPI (10 ng/kg bolus and 0.1 ng/kg per minute infusion) still produced a marginally significant prolongation of average survival time (80 h) relative to contemporaneously studied controls (47 h). When the dose of TFPI was decreased still further (1.0 ng/kg bolus and 0.01 ng/kg per minute infusion), average survival times were not significantly different between TFPI-treated and vehicle-treated rabbits (77 and 51 h, respectively). CONCLUSIONS: Delayed infusion with remarkably low doses of recombinant human TFPI prolongs survival in a rabbit model of antibiotic-treated Gram-negative bacterial sepsis. In planning human trials of TFPI as an adjuvant treatment for sepsis it may be reasonable to use much lower doses of the agent than were heretofore contemplated.

Murine monoclonal antibodies defining neutralizing epitopes on tumor necrosis factor.

A panel of four monoclonal antibodies (MAbs) was generated against recombinant human tumor necrosis factor-alpha (rTNF). These MAbs immunoprecipitate 125I-labeled rTNF, block binding of 125I-labeled rTNF to L929 mouse fibroblasts, and neutralize in vitro cytotoxicity of rTNF and native TNF (nTNF) in the L929 cytotoxicity assay. They define distinct epitopes closely associated with the receptor binding site of rTNF. In Western analysis they bind to both monomeric and dimeric rTNF. Two MAbs recognizing distinct epitopes were used to develop a 'sandwich' enzyme immunometric assay (EIMA) to measure rTNF levels in human serum and other fluids.

Tissue factor pathway inhibitor activity in severe sepsis.

OBJECTIVE: To review the preclinical and clinical evidence that provides the therapeutic rationale for recombinant human tissue factor pathway inhibitor (rTFPI) as a novel treatment for human sepsis. DATA SOURCES: A summary of published English-language literature regarding preclinical studies and limited information published about three phase II clinical studies for the evaluation of rTFPI safety in sepsis patients. DATA SUMMARY: Tissue factor pathway inhibitor, the physiologic inhibitor of the tissue factor pathway, interrupts activation of coagulation at multiple steps, including tissue factor VIIa activity, Xa activity, prothrombinase complex, and thrombin generation. Recombinant human TFPI exhibits anticoagulant and anti-inflammatory activities in animal models and humans with sepsis. These activities appear to have an important therapeutic role in protecting the microvasculature from injury and preventing multiple organ failure in sepsis. CONCLUSIONS: Tissue factor pathway inhibitor is a potent inhibitor of clotting in the microvasculature, which is thought to protect organs from injury. Recombinant TFPI improved survival of septic animals in multiple models. Recent phase II results suggest that rTFPI is well tolerated, and they show a trend toward reduction in 28-day all-cause mortality in rTFPI-treated patients; in addition, rTFPI demonstrated significant reduction in thrombin generation. These results suggest that a powered study is indicated to further evaluate rTFPI utility for the adjunctive management of severe sepsis.

Tissue factor pathway inhibitor blocks cellular effects of endotoxin by binding to endotoxin and interfering with transfer to CD14.

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type plasma protease inhibitor that inhibits factor Xa and the factor VIIa/tissue factor catalytic complex. It plays an important role in feedback inhibition of the coagulation cascade (Broze, Annu Rev Med 46:103, 1995). TFPI has also been used successfully to prevent lethality and attenuate coagulopathic responses in a baboon model of septic shock (Creasey et al, J Clin Invest 91:2850, 1993; and Carr et al, Circ Shock 44:126, 1995). However, the mechanism of reduced mortality in these animals could not be explained merely by the anticoagulant effect of TFPI, because TFPI-treated animals also had a significantly depressed interleukin-6 response. Moreover, inhibition of coagulopathic responses by other anticoagulants has failed to block the organ damage or lethal effect of endotoxic shock (Coalson et al, Circ Shock 5:423, 1978; Warr et al, Blood 75:1481, 1990; and Taylor et al, Blood 78:364, 1991). We show here that recombinant TFPI can bind to endotoxin in vitro. This binding prevents interaction of endotoxin with both lipopolysaccharide binding protein and CD14, thereby blocking cellular responses.

Role of G0-G1 arrest in the inhibition of tumor cell growth by interferon.

We report here that human leukocyte interferon preparations are capable of influencing the transition of human melanoma cells from the "A" state to the "B" phase. Human melanoma cells that enter a quiescent stage at high cell density are more sensitive to the cytostatic action of interferon than those that continue to proliferate under similar conditions. Cell cycle perturbations caused by interferon in these cells include a decreased transition rate out of G0-G1 ("A" state) into S ("B" phase) and a prolongation of S. These findings support the idea that some metabolic event is required for progress through the G0-G1 phase of the cell cycle and is susceptible to interferon action.

A high molecular weight component of the human tumor necrosis factor receptor is associated with cytotoxicity.

We compared the molecular structure of the receptor to human recombinant tumor necrosis factor (HurTNF) on cells of different tissue origin that differ in their response to one of the known activities of TNF. We studied tumor cell lines that respond to the cytotoxic action of TNF and resistant variants that bind TNF, normal cell lines that are stimulated to proliferate by TNF and those that are not affected by TNF, and peripheral blood granulocytes whose activation is also augmented by TNF. Using 125I-labeled HurTNF, we found that it bound mainly to four cellular polypeptides (138, 90, 75, and 54 kDa), three of which were found in every cell type examined and one (138 kDa) that was observed only in a human breast carcinoma cell line (MCF-7) that is highly responsive to the cytotoxic action of TNF. The 138-kDa polypeptide was not found in resistant variants of MCF-7 that bind TNF. In contrast to the other polypeptides, the 138-kDa protein was detected 30 min after incubation at 4 degrees C, as compared to 5 min. Scatchard analysis and cross-linking data suggest a model for the TNF receptor structure whereby the receptor is composed of noncovalently linked membrane-bound polypeptides that bind TNF with high affinity (Kd, 0.05-0.8 X 10(-9) M) with the 138-kDa protein being the least abundant and/or even absent in most cells.

Use of a sensitive receptor binding assay to discriminate between full-length and truncated human recombinant tumor necrosis factor proteins.

A radioreceptor assay (RRA) capable of detecting picomolar concentrations of human recombinant tumor necrosis factor (TNF) was used to compare the relative binding affinities of genetically engineered full-length and truncated TNF proteins. The specific cell-surface receptors for TNF present on the human cervical carcinoma cell line ME-180 were characterized as having a Kd of 0.2 nM and a density of 2700 sites/cell. Conditions were then defined for an RRA that maximized the specific binding of 125I-TNF to this adherent cell line. Incubation of ME-180 cells with 125I-TNF at 37 degrees C in the presence of 0.02% sodium azide resulted in a 4-fold increase in assay sensitivity and a doubling of specific counts bound, as compared to binding done at 4 degrees C with or without sodium azide. Inhibition of receptor-ligand internalization under these conditions was a likely reason for the increases. This system was utilized to compare low concentrations of the full-length TNF protein and a genetically altered TNF protein (mutein) which lacks the 10 N-terminal amino acids and contains an N-terminal methionine. Previous studies showing the truncated TNF to be 2- to 3-fold lower in cytotoxic activity on a variety of tumor cell lines were corroborated by our findings that the mutein was also three and one-half times lower in relative affinity for the TNF receptor on ME-180 cells. These results suggest a possible role for these residues in receptor binding and illustrate the use of a highly sensitive RRA for the evaluation of TNF molecules altered by recombinant DNA technology.

The activity of tissue factor pathway inhibitor in experimental models of superantigen-induced shock and polymicrobial intra-abdominal sepsis.

OBJECTIVES: To study recombinant human tissue factor pathway inhibitor (rhTFPI) in a superantigen-induced shock model and in a cecal ligation and puncture (CLP) model of peritonitis in mice. DESIGN: Prospective, randomized, experimental study. SETTING: An experimental animal research laboratory. SUBJECTS: Eighty BALB/c mice for the superantigen model, and 56 BALB/c mice for the CLP model. INTERVENTIONS: In the superantigen-induced shock model, animals received rhTFPI (350 mg/kg) subcutaneously every 12 hrs (n = 30) or saline control (n = 30) for 60 hrs after staphylococcal enterotoxin B (SEB; 10 microg iv) and a sublethal dose of E. coli 0111:B4 lipopolysaccharide (LPS; 75 microg ip). Control groups received SEB alone (n = 10) and LPS alone (n = 10). In the CLP model, rhTFPI or saline was given every 8 hrs for 48 hrs by using a 21-gauge needle (n = 9) or 23-gauge needle (n = 14) for CLP. A sham surgery control group (n = 10) was also included. MEASUREMENTS AND MAIN RESULTS: There was 0% mortality in the SEB and LPS control groups. The mortality rate was 64% in the saline control group that received both SEB and LPS (19 of 30), whereas the rhTFPI- treated animals had a mortality rate of 20% (6 of 30; p < .01). The rhTFPI-treated group had significantly lower interleukin-6 levels (61.8 +/- 41 pg/mL vs. 285 +/- 63 pg/mL; p < .05) than the control group but no differences in tumor necrosis factor-alpha or interferon-gamma levels. In the CLP experiment, rhTFPI-treated animals did not have any survival advantage over the control group after the large-bore (21-gauge) needle puncture. The rhTFPI group had significantly improved 7-day mortality rate after CLP with the small-bore needle (23-gauge; 21.4% [rhTFPI] vs. 71.4% [control], p < .01). Plasma LPS, interleukin-6, interferon-gamma, and tumor necrosis factor-alpha levels were unchanged by rhTFPI treatment, but significantly reduced LPS (p = .006) and IFNgamma (p = .001) levels were found in the peritoneal fluid. CONCLUSIONS: Tissue factor pathway inhibitor significantly improves the mortality rate in models of superantigen-induced shock and polymicrobial intra-abdominal infection, supporting its potential use in clinical trials for septic shock.

Delayed treatment with recombinant human tissue factor pathway inhibitor improves survival in rabbits with gram-negative peritonitis.

To determine whether treatment with recombinant human tissue factor pathway inhibitor (TFPI), an inhibitor of the extrinsic coagulation pathway, can improve survival in a clinically relevant model of gram-negative sepsis, rabbits were given an intraperitoneal inoculation of a suspension containing hemoglobin (40 microg/mL), porcine mucin (150 microg/mL), and viable Escherichia coli O18:K1 (1.0 +/- 0.5 x 10(5) cfu/kg). Treatment with gentamicin (5 mg/kg every 12 h for five doses) was instituted 4 h after induction of peritonitis. At the same time point, rabbits were randomized to receive a 24-h infusion of vehicle or one of three different doses of TFPI. Treatment groups, 7-day survival rates, and significance versus control were as follows: control, 1 of 20; TFPI(LOW DOSE) (0.1 mg/kg, then 1 microg/kg/min), 3 of 12 (P = .14); TFPI(MID DOSE), (0.5 mg/kg, then 5 microg/kg/min), 7 of 12 (P = .002); TFPI(HIGH DOSE) (10 mg/kg, then 10 microg/kg/min), 4 of 13 (P = .04). Thus, delayed treatment with TFPI improves survival in septic rabbits.

Interferon increases HLA synthesis in melanoma cells: interferon-resistant and -sensitive cell lines.

We report that human leukocyte interferon preparations increase the expression of beta 2-microglobulin by 100-200% on the surface of normal fibroblast and melanoma cell lines sensitive to interferon. This increase in expression can be correlated with an increase in HLA synthesis as measured by incorporation of [35S]methionine in these antigens. This enhanced HLA synthesis, which is 5- to 17-fold, is time dependent and dose related. Synchronized cells in the G0/G1 phase of the cell cycle appear to be more sensitive to this interferon action. Neither an increase in surface expression nor in HLA synthesis is observed in a melanoma cell line resistant to the antiviral and antigrowth effects of interferon. Furthermore, there appears to be a stronger correlation between this increased HLA synthesis and the antiviral function than between it and the antiproliferative action of interferon.