Publications

Objectives: Cisplatin-based chemotherapy is moderately active in malignant pleural mesothelioma (MPM) due to intrinsic drug resistance and to low immunogenicity of MPM cells. CAAT/enhancer binding protein (C/EBP)-beta LIP is a pro-apoptotic and chemosensitizing transcription factor activated in response to endoplasmic reticulum (ER) stress.Materials and methods: We investigated if LIP levels can predict the clinical response to cisplatin and survival of MPM patients receiving cisplatin-based chemotherapy. We studied the LIP-dependent mechanisms determining cisplatin-resistance and we identified pharmacological approaches targeting LIP, able to restore cisplatin sensitiveness, in patient-derived MPM cells and animal models. Results were analyzed by a one-way analysis of variance test.Results: We found that LIP was degraded by constitutive ubiquitination in primary MPM cells derived from patients poorly responsive to cisplatin. LIP ubiquitination was directly correlated with cisplatin chemosensitivity and was associated with patients' survival after chemotherapy. Overexpression of LIP restored cisplatin's proapoptotic effect by activating CHOP/TRB3/caspase 3 axis and up-regulating calreticulin, that triggered MPM cell phagocytosis by dendritic cells and expanded autologous anti-tumor CD8(+)CD107(+)T-cytotoxic lymphocytes. Proteasome inhibitor carfilzomib and lysosome inhibitor chloroquine prevented LIP degradation. The triple combination of carfilzomib, chloroquine and cisplatin increased ER stress-triggered apoptosis and immunogenic cell death in patients' samples, and reduced tumor growth in cisplatin-resistant MPM preclinical models.Conclusion: The loss of LIP mediates cisplatin resistance, rendering LIP a possible predictor of cisplatin response in MPM patients. The association of proteasome and lysosome inhibitors reverses cisplatin resistance by restoring LIP levels and may represent a new adjuvant strategy in MPM treatment.

Keywords: Biochemistry & Molecular Biology; Cell Biology; Immunology

In 1976-1977, I adapted reversed-phase HPLC (RP-HPLC) to peptide and protein purification, starting with pituitary proteins and continuing with the first successful purification to homogeneity of human leukocyte interferon (IFN-α). Using this technology, I isolated and characterized 6-8 different leukocyte interferon subtypes, which were later identified as products of the IFN-α gene family. Since then, RP-HPLC became a standard procedure for isolation and analysis of proteins. The successful purification of IFN-α led to the development of Roferon-A™, a drug used for the treatment of hairy cell leukemia, hepatitis C and a variety of other diseases. Later studies with my colleagues in Israel and abroad led to isolation and discovery of several cytokine receptors and binding proteins, including those of Type I IFNs, TNF and IL-18. The use of HPLC was indispensable in most of these studies.

Background: Crohn's disease and ulcerative colitis are inflammatory bowel diseases with an unknown etiology. Interleukin-18 is a pro-inflammatory cytokine that is up-regulated in Crohn's disease. IL-18 binding protein neutralizes IL-18. The relationship of IL-18 and IL-18BP and disease activity in these diseases is not fully understood. Objectives: To investigate the correlation of IL-18 and IL-18BP with disease activity and other disease parameters in inflammatory bowel disease. Methods: IL-18 and IL-18BP isoform α were measured in 129 patients and 10 healthy individuals. Patients' mean age was 40.5 (range 15-70 years) and 43 were women; 58 Crohn's and 28 colitis patients were in remission and 52 and 14, respectively, were in exacerbation. Twenty-three (19 and 4 respectively) were studied in both remission and exacerbation. Results: The mean level of free IL-18 was significantly different between healthy individuals and Crohn patients, and between Crohn patients during exacerbation and remission (167 ± 32 vs. 471 ± 88 and 325 ± 24 pg/ml, respectively, P

Keywords: Peripheral Vascular Disease

IFN-γ induces its immunoregulatory activities by activating genes mainly through the Jak-STAT signaling pathway. Here we show that what was considered to be intrinsic IFN-γ activities depend largely on the basal level of NF-κB, which is maintained by constitutively expressed IL-1α. The IL-1 receptor antagonist and antibodies to IL-1α, but not to IL-1β, inhibited the antiviral activity of IFN-γ by 90%, whereas no inhibition of type I IFN activity was observed. Similarly, the induction of many genes by IFN-γ, including HLA-DR, ICAM-1, IL-18BP, and genes mediating its antiviral activity, greatly depended on basal IL-1α. Furthermore, IFN-γ induced serum IL-18 binding protein in wild-type mice but not in IL-1α/β double-deficient mice. Thus, constitutively expressed IL-1α is critical for numerous IFN-γ activities.

Hemophagocytic syndrome (HPS) is characterized by an uncontrolled and poorly understood activation of T-helper 1 (Th-1) lymphocytes and macrophages. We studied 20 patients with HPS secondary to infections, autoimmune disease, lymphoma, or cancer and observed that the concentrations of serum interleukin 18 (IL-18), a strong inducer of Th-1 responses, interferon γ (IFN-γ) production, and stimulation of macrophages and natural killer (NK) cells were highly increased in HPS but not in control patients. In contrast, concentrations of its natural inhibitor, the IL-18 binding protein (IL-18BP), were only moderately elevated, resulting in a high level of biologically active free IL-18 in HPS (4.6-fold increase compared with controls; P

Objectives. Interleukin 18 (IL-18) is a proinflammatory cytokine and a member of the IL-1 family. Animal models and investigations in humans point to an important role for this cytokine in inflammatory bowel diseases (IBD). IL-18 binding protein (IL-18BP) is a naturally occurring antagonist of IL-18. Methods. In this study, we measured IL-18 and IL-18BP plasma concentrations and spontaneous release in cultures of colonic explants from healthy subjects (n = 41), patients with Crohn's disease (CD, in = 135), and patients with ulcerative colitis (UC, n = 93). Results. Both CD and UC patients had higher IL-18BP plasma levels than controls. Plasma levels of free, unbound IL-18 were significantly elevated in CD patients compared to healthy controls, but not in UC patients. Colonic explant cultures from inflamed areas in IBD patients released significantly higher levels of IL-18 than non-inflamed areas and controls. IL-18BP levels from the same cultures were below the detection limit over a culture period of 24 h. Conclusions. Our results confirm the importance of IL-18 in the pathogenesis of IBD and suggest that especially in CD, IL-18BP might be produced in insufficient quantities to counteract the effects of endogenous IL-18.

Background: Laron Syndrome, first described in Israel, is a form of dwarfism similar to isolated growth hormone deficiency caused by molecular defects in the GH receptor gene. Objective: To characterize the molecular defects of the GH-R in Laron syndrome patients followed in our clinic. Methods: Of the 63 patients in the cohort, we investigated 31 patients and 32 relatives belonging to several ethnic origins. Molecular analysis of the GH-R gene was performed using the single strand conformation polymorphism and DNA sequencing techniques. Results: Eleven molecular defects including a novel mutation were found. Twenty-two patients carried mutations in the extracellular domain, one in the transmembrane domain, and 3 siblings with typical Laron syndrome presented a normal GH-R. Of interest are, on one hand, different mutations within the same ethnic groups: W-15X and 5, 6 exon deletion in Jewish-Iraqis, and E180 splice and 5, 6 exon deletion in Jewish-Moroccans; and on the other hand, identical findings in patients from distinct regions: the 785-1 G to T mutation in an Israeli-Druze and a Peruvian patient. A polymorphism in exon 6, Gly168Gly, was found in 15 probands. One typical Laron patient from Greece was heterozygous for R43X in exon 4 and heterozygous for Gly168Gly. In addition, a novel mutation in exon 5: substitution of T to G replacing tyrosine 86 for aspartic acid (Y86D) is described. Conclusions: This study demonstrates: a) an increased focal incidence of Laron syndrome in different ethnic groups from our area with a high incidence of consanguinity; and b) a relationship between molecular defects of the GH-R, ethnic group and geographic area.

Objective. To study the regulation of interleukin-18 binding protein (IL-18BP) production by rheumatoid arthritis (RA) or control peripheral blood mononuclear cells (PBMCs) and by RA synovial tissue cells, and to compare the levels of IL-18BP messenger RNA (mRNA) expression in whole blood from RA patients and controls. Methods. Unstimulated or phytohemagglutinin and phorbol myristate acetate (PHA/PMA)-stimulated PBMCs from 10 RA patients and 12 healthy controls and unstimulated or PHA-PMA-stimulated synovial tissue cells from 8 RA patients were cultured with or without IL-12 (1 ng/ml) and IL-18 (5 ng/ml) alone or in combination. IL-18BP and interferon-γ (IFNγ) levels in supernatants were measured by enzyme-linked immunosorbent assay. Levels of IL-18BP and IFNγ mRNA expression in whole blood samples from 22 RA patients and 12 healthy controls were determined by quantitative reverse transcriptase-polymerase chain reaction. Results. IL-12 decreased the basal levels of IL-18BP production by freshly isolated RA or control PBMCs, but increased those of synovial cells or PHA/PMA-stimulated PBMCs. IL-18 alone had no direct effect on IL-18BP production by PBMCs or RA synovial cells, with or without stimulation. Unstimulated whole blood samples from RA patients showed lower levels of IL-18BP mRNA expression than those from healthy controls. Conclusion. The production of IL-18BP in response to IL-12 and IL-18 was regulated differently in blood and synovial cells. The difference appears to be related to the level of cell activation.

Uremia is associated with suppressed cellular immune responses, manifested, in part, by impaired interferon-γ (IFNγ) production. We investigated the influence of kidney function on plasma levels of interleukin-18 binding protein (IL-18BP), the naturally occurring inhibitor of IL-18. Methods: Plasma levels of IL-18, IL-18BP and IFNγ were measured by specific immunoassays in patients with normal kidney function (NKF, n = 29), in patients with chronic renal insufficiency (CRI, n = 29), and in patients on hemodialysis (HD, n = 40). In addition, Staphylococcus epidermidis-induced production of IFNγ and IL-18 in whole blood cultures was determined in 12 patients on HD and compared to production in 9 controls with NKF. Results: Plasma IL-18 (mean ± SEM) in NKF was 17.9 ± 3.6 pg/ml, in CRI 42.6 ± 7.0 pg/ml (p 6 mononuclear cells (PBMC) but suppressed in HD to 27.3 ± 16 pg/10⁶ PBMC (p

Deletions and mutations in the growth hormone receptor gene are the underlying etiology of Laron syndrome (LS). Most of the patients are distributed in and originating from the Mediterranean and Middle-Eastern countries. Thirty-nine mutations have been described so far. We hereby report 2 novel non-sense mutations, one in exon 2 found in three Jewish-Iraqi patients from Israel; and another in exon 6 found in an Italian girl. DNA sequencing of exon 2 revealed a G to A transition at nucleotide 83 in the fourth codon of the signal peptide (W-15X). In exon 6, a T to A transversion was found in amino acid 141 (L141X). Both mutations introduced a premature termination codon that led to a truncated non-functioning receptor. In addition we found in the Jewish-Iraqi patients, a mutation in exon 7 (R211H, previously described) and in the Italian family the polymorphism Gly168, in exon 6.

Interleukin 12 (IL-12) and IL-18 act synergistically to stimulate interferon gamma (IFN-γ) production; moreover, IL-1 and tumor necrosis factor (TNF) may also augment IFN-γ synthesis. We have investigated the relative contributions of these cytokines in the production of IFN-γ and TNF by the Gram-positive bacterium Staphylococcus epidermidis, using the specific cytokine inhibitors IL-18 binding protein (IL-18BP), IL-1 receptor antagonist (IL-1Ra), anti-IL-12 antibodies (anti-IL-12 Ab), and TNF binding protein. Inhibition of caspase-1 reduced IFN-γ and IL-1β levels (by 80 and 67%, respectively) when heat-killed S. epidermidis was added to whole human blood cultures. IL-18BP reduced S. epidermidis-induced IFN-γ (77% maximal suppression). In contrast, blocking IL-1 receptors by IL-1Ra had no effect on IFN-γ production. Blocking endogenous IL-12 and TNF reduced IFN-γ production by 69 and 36%. S. epidermidis-induced TNF-α was inhibited by IL-18BP and IL-1Ra, but not anti-IL-12 Ab, whereas IL-8 production was unaffected by any of the specific cytokine blocking agents. In conclusion, S. epidermidis stimulates IFN-γ which is IL-18, IL-12 and TNF-dependent, but IL-1 independent.

Type 1 (insulin-dependent) diabetes, T1DM, is the result of an immune-mediated destruction of the pancreatic β cells dependent mainly on T helper cells and macrophages. Interleukin-18 (IL-18) is a proinflammatory cytokine produced mainly by macrophages. IL-18 is capable of inducing T lymphocyte synthesis of IFNγ, thereby skewing the T helper response toward a T helper type 1 (Th1) profile. IL-18 binding protein (IL18BP) neutralizes IL-18 and leads to a reduced Th1 response. Polymorphisms in IL18BP may affect the activity of IL-18 and the magnitude of the Th1 response and may play a role in the pathogenesis of T1DM. The aim of the study was therefore to identify polymorphisms in IL18BP and to test these for association with T1DM. We evaluated the human IL18BP gene on chromosome 11q13 as a candidate susceptibility gene for T1DM and scanned the entire IL18BP (promoter, exons 1-6, and 3UTR) for polymorphisms using single-strand conformational polymorphism analysis and direct sequencing. We identified a total of 11 polymorphisms, all having allele frequencies ranging between 0.05 and 0.10. Four were in the 5UTR: -257G→T,-78C→T,-65G→A, and -59A→G. Three were in intron 3: IVS3+140A→C, IVS4-147G→T, and IVS4-59G→T. The last four, 38*A→T, 48*T→A, 388*C→G, and 440*_441*insG, were in the 3UTR of IL18BP. However, none of these were frequent enough to permit association studies in T1DM and we conclude that IL18BP does not contribute to the overall genetic susceptibility to type 1 diabetes.

Interleukin 18 (IL-18) is a recently described proinflammatory cytokine. In mouse models it has been shown to play a key role in the development of liver injury. IL-18 binding protein (IL-18BP) is a naturally occurring antagonist of IL-18. In this study we investigated whether IL-18/IL-18BP levels are altered in patients with chronic liver disease (CLD). We measured IL-18 and IL-18BP plasma levels in 153 patients with CLD and 41 healthy controls by a specific ELISA. Plasma levels of IL-18 were significantly higher in CLD patients than in healthy controls. Cirrhotics had higher levels than noncirrhotics. IL-18 levels increased with disease progression. IL-18BP plasma levels paralled the increase of IL-18 with disease progression, except in stage Child C cirrhosis. IL-18 and IL-18BP levels were elevated independent of the etiology of CLD. IL-18 and IL-18BP correlated with laboratory parameters of inflammation and liver injury. Plasma levels of IL-18 and its antagonist, IL-18BP, are elevated in CLD and correlate with severity of disease. IL-18BP may not be sufficient to counteract the overwhelming proinflammatory response in end stage liver disease.

Cytokines and chemokines play an essential role in recruiting leukocytes from the circulation to the peripheral sites of inflammation by modulating cellular interactions with endothelial cell ligands and extracellular matrix (ECM). Herein, we examined regulation of T cell adhesion to ECM ligands by two major proinflammatory cytokines, interleukin (IL)-12 and IL-18. IL-12 and IL-18 induced T cell adhesion to fibronectin (FN) and hyaluronic acid at low (pM) concentrations that were mediated by specific adhesion molecules expressed on the T cell surface, namely, β ₁ integrins and CD44, respectively. The induction of adhesion by IL-12 and IL-18 was inhibited by extracellular signal-regulated kinase and p38 mitogen-activated protein kinase inhibitors (PD098059 and SB203580, respectively). In contrast, IL-12- and IL-18-induced interferon-γ (INF-γ) secretion from T cells was inhibited by SB203580, but not by PD098059. It is interesting that low concentrations of IL-12 and IL-18 induced T cell adhesion to FN in a synergistic manner. Thus, in addition to the regulation of late inflammatory functions such as INF-γ production, IL-12 and IL-18, alone or in combination, regulate early inflammatory events such as T cell adhesion to inflamed sites.

Interleukin-18 (IL-18), derived from macrophages and Kupffer cells, is the central pro-inflammatory cytokine leading to experimental liver failure. IL-18 binding protein (IL-18BP) is a circulating protein that binds IL-18 and neutralizes its activity. Since IL-18 production is increased in chronic HCV infection, we asked whether IFN-α might act on the IL-18/IL-18BP system in HCV patients. IL-18BP, total and free IL-18 plasma levels were determined in 13 HCV patients receiving 1 × 10⁷ IU IFN-α subcutaneously daily for 28 days. The in vitro effects of IFN-α on macrophage IL-18BP and IL-18 were studied by enzyme-linked immunosorbent assays and Northern analysis. IFN-α administration increased IL-18BP plasma levels 3.24 fold 24 h after institution of therapy, resulting in a 67.4% reduction of free IL-18. Total IL-18 levels decreased from day +24 on. In vitro, IFN-α diminished IL-18 release from macrophages of healthy volunteers and chronic HCV patients. On top of its inhibitory effects on IL-1 and TNF-α release, IFN-α also exerts its anti-inflammatory action in vivo by induction of IL-18BP. These anti-inflammatory properties might account - together with its antiviral action - for its clinical efficacy in chronic hepatitis C.

IL-18 binding protein (IL-18BP) is a circulating antagonist of the proinflammatory Th1 cytokine IL-18. It effectively blocks IL-18 by forming a 1:1 high affinity (K_d=400 pM) complex, exhibiting a very low dissociation rate. We have developed a sandwich ELISA for IL-18BPa and determined its limit of detection (62 pg/ml). Interference by IL-18 and related cytokines, as well as cross reactivity with other IL-18BP isoforms (b, c, and d) were determined. Using this ELISA, we measured serum IL-18BPa in large cohorts of healthy individuals and in septic patients. Serum IL-18BPa in healthy individuals was 2.15 ± 0.15 ng/ml (range 0.5-7 ng/ml). In sepsis, the level rose to 21.9 ± 1.44 ng/ml (range 4-132 ng/ml). Total IL-18 was measured in the same sera by an electrochemiluminescence assay and free IL-18 was calculated based on the mass action law. Total IL-18 was low in healthy individuals (64 ± 17 pg/ml) and most of it (∼85%) was in its free form. Total IL-18 and IL-18BPa were both elevated in sepsis patients upon admission (1.5 ± 0.4 ng/ml and 28.6 ± 4.5 ng/ml, respectively). At these levels, most of the IL-18 is bound to IL-18BPa, however the remaining free IL-18 is still higher than in healthy individuals. We conclude that IL-18BPa considerably inhibits circulating IL-18 in sepsis. Yet, exogenous administration of IL-18BPa may further reduce circulating IL-18 activity.

IL-18 can be considered a proinflammatory cytokine mediating disease as well as an immunostimulatory cytokine that is important for host defense against infection and cancer. The high-affinity, constitutively expressed, and circulating IL-18 binding protein (IL-18BP), which competes with cell surface receptors for IL-18 and neutralizes IL-18 activity, may act as a natural antiinflammatory as well as immunosuppressive molecule. In the present studies, the IL-18 precursor caspase-1 cleavage site was changed to a factor Xa site, and, after expression in Escherichia coli, mature IL-18 was generated by factor Xa cleavage. Mature IL-18 generated by factor Xa cleavage was fully active. Single point mutations in the mature IL-18 peptide were made, and the biological activities of the wild-type (WT) IL-18 were compared with those of the mutants. Mutants E42A and K89A exhibited 2-fold increased activity compared with WT IL-18. A double mutant, E42A plus K89A, exhibited 4-fold greater activity. Unexpectedly, IL-18BP failed to neutralize the double mutant E42A plus K89A compared with WT IL-18. The K89A mutant was intermediate in being neutralized by IL-18BP, whereas neutralization of the E42A mutant was comparable to that in the WT IL-18. The identification of E42 and K89 in the mature IL-18 peptide is consistent with previous modeling studies of IL-18 binding to IL-18BP and explains the unusually high affinity of IL-18BP for IL-18.

The roles of endogenous cytokines induced by either intact staphylococcal microorganisms or staphylococcal exotoxins were examined using human whole-blood cultures. To accomplish this, interleukin-18 binding protein (IL-18BP) and tumor necrosis factor binding protein (TNFbp) were used to neutralize IL-18 and TNF, respectively, whereas an anti-IL-12 monoclonal antibody was used to neutralize IL-12 and the IL-1 receptor antagonist (IL-1Ra) was used to block IL-1 receptors. Heat-killed Staphylococcus epidermidis and Staphylococcus aureus, as well as the staphylococcal superantigens toxic shock syndrome toxin-1 (TSST-1) and staphylococcus enterotoxin B (SEB) induced gamma interferon (IFN-γ) production. Staphylococcus spp.-induced production of IFN-γ required the presence of endogenous IL-18, IL-12, and TNF. In contrast, TSST-1-induced IFN-γ was not significantly reduced in the presence of IL-18BP, anti-IL-12 antibodies, IL-1Ra, or anti-TNFbp. SEB-induced IFN-γ was significantly inhibited only by anti-IL-12 antibodies, indicating that endogenous IL-18, IL-1, and TNF are not required for SEB-induced IFN-γ. In conclusion, the mechanisms of IFN-γ stimulation by intact staphylococcal microorganisms and by exotoxins differ, and this is likely due to the different receptors which are triggered on the cell membranes. In contrast to its role in the interactions between staphylococci and host cells, IL-18 does not appear to play a major role in superantigen-induced IFN-γ.

IL-18 shares with IL-1 the same family of receptors and several identical signal transduction pathways. Because of these similarities, IL-18 was investigated for its ability to induce prostaglandin E-2 (PGE(2)) synthesis in human peripheral blood mononuclear cells (PBMC), a prominent, proinflammatory property of IL-1. IL-18 was highly active in PBMC by inducing the synthesis of the chemokine IL-8; however, no induction of PGE(2) synthesis nor cyclooxygenase type-2 gene expression was observed in PBMC stimulated with IL-18. In the same cultures, IL-1 beta induced a 12-fold increase in PGE(2). Although IL-1 beta-induced IL-8 synthesis was augmented 3-fold by IL-18, IL-18 suppressed IL-1 beta-induced PGE(2) production by 40%. The suppressive effect of IL-18 on PGE(2) production was mediated by interferon (IFN)-gamma because anti-human IFN-gamma-antibody prevented IL-18-induced reduction in PGE(2). Consistent with these observations, IL-12, a known inducer of IFN-gamma augmented IL-1 beta-induced IFN-gamma but suppressed IL-1 beta-induced PGE(2) by 75%. IL-18 binding protein (IL-18BP) is a naturally occurring and specific inhibitor of IL-18. When recombinant IL-18BP was added to PBMC cultures, unexpectedly, spontaneous PGE(2) production increased. PGE(2) production was also increased by the addition of IL-18BP to PBMC stimulated with either IL-1 beta or IL-12 and also in whole blood cultures stimulated with Staphylococcus epidermidis. These studies demonstrate that IL-18BP decreases endogenous IL-18 activity by reducing IFN-gamma-mediated responses.

Interleukin (IL-)18 is an activator of NK cells and a co-inducer of Th₁ cytokines, sharing structural features with the IL-1 family of proteins. Unlike most other cytokines, IL-18 and IL-1β lack a signal peptide, have an all β-pleated sheet structure and are synthesized as biologically inactive precursors (pro-IL-18 and pro-IL-1β). These precursors are cleaved by caspase-1 (IL-1β-converting enzyme, ICE) to form the biologically active mature cytokines. Direct expression of mature recombinant human IL-18 in E. coli resulted in a partially active cytokine. We tested the possibility that correct folding of huIL-18 requires its prior synthesis as pro-IL-18. Because caspase-1 is not readily available, we constructed an expression vector encoding human pro-IL-18 in which the caspase-1 cleavage site was mutated into a factor Xa site. To facilitate purification, the mutated pro-IL-18 cDNA was fused in frame to a glutathione-S-transferase (GST) coding sequence. The GST-pro-IL-18 fusion protein was expressed in E. coli, captured on glutathione agarose and mature human IL-18, exhibiting high biological activity was released upon cleavage with factor Xa. This result indicates that correct folding of huIL-18 occurs at the level of pro-IL-18 and provides a practical way to produce biologically active huIL-18. (C) 2000 Academic Press.

Initially described in 1989 as interferon-γ (IFN-γ) inducing factor (IGIF), interleukin-18 (IL-18) is a novel pro-inflammatory cytokine that is clearly more than an inducer of IFN-γ. The cytokine possesses several biological properties such as activation of nuclear factor-κB (NF-κB), Fas ligand expression, the induction of both CC and CXC chemokines, and increased production of competent human immunodeficiency virus. Most activities are due to a receptor complex that recruits the IL-1 receptor-activating kinase (IRAK), leading to translocation of NF-κB. This property and others support the concept that IL-18 is related to the IL-1 family. Indeed, one of the IL- 18 receptor chains is the IL-1 receptor-related protein, a member of the IL- 1R family. In addition, IL-18 is structurally similar to IL-1β and like IL- 1β is first synthesized as a leaderless precursor requiring the IL-1β converting enzyme for cleavage into an active molecule. The biology of IL-18 is reviewed in the overview and the implication for a role for this cytokine in disease is presented.

Two transmembrane polypeptides, IFNAR and IFN-α/BR, were previously identified as essential components of the type I interferon (IFN) receptor, but their interrelationship and role in ligand binding were not clear. To study these issues, we stably expressed and characterized the two polypeptides in host murine cells. In human cells, native IFN-α/βR is a 102-kDa protein but upon reduction only a 51-kDa protein is detected. In host murine cells human IFN-α/βR was expressed as a 51-kDa protein. Host cells expressing IFN-α/βR bound IFN-α₂ with a high affinity (K(d) of 3.6 nM), whereas cells expressing IFNAR exhibited no ligand binding. Upon coexpression of IFNAR and the 51-kDa IFN-α/βR, the affinity for IFN-α₂ was increased 10-fold, approaching that of the native receptor. We show by cross-linking that both the cloned (51-kDa) and native (102-kDa) IFN-α/βR bind IFN-α₂ to form an intermediate product, while IFNAR associates with this product to form a ternary complex. Hence, IFNAR and IFN-α/DR are components of a common type I IFN receptor, cooperating in ligand binding. Ligand-induced association of IFNAR and IFN-α/βR probably triggers transmembrane signaling.

Immunization of mice with conditioned media from human breast cancer cells yielded the monoclonal antibody SP-2, which recognized an antigen of approximately 90-95 kDa. This protein, designated 90K, was found to be present in the serum of healthy individuals and at elevated levels in the serum of subpopulations of patients with various types of cancer and AIDS. Here we report the primary structure of the SP-2 antigen and demonstrate its relationship to a family of proteins which carry a scavenger receptor cysteine-rich domain. Northern blot analysis of normal tissues, primary tumors, and tumor-derived cell lines indicates a broad expression spectrum of the 90K gene at widely varying levels. Functional characterization reveals stimulatory effects of 90K on host defense systems, such as natural killer cell and lymphokine-activated killer cell activity, and indicates that its immunostimulatory effects may be mediated through the induction of interleukin-2 and possibly other cytokines.

Soluble forms of the interferon-α receptor (sIFN-αR) were identified in human serum and urine by Western blotting with monoclonal antibodies (MAb) directed against IFN-αR, and by immunoprecipitation (Iptn) of a covalently cross-linked complex of IFN-αR and [¹²⁵I]IFN-α with anti IFN-α MAb. Elevated levels or sIFN-αR were found in sera of hairy cell leukemia patients. The soluble receptor from serum migrated as a 55 kDa protein in SDS-PAGE, and, as expected, the cross-linked product migrated as a 75 kDa protein. The soluble receptor from urine was found to be a protein of mol. wt. 45 kDa and its cross-linked complex migrated as a 65 kDa protein. The calculated mol. wt. of the entire extracellular domain of the IFN-αR prior to post-translational modifications is 47,000. Since there are 12 potential glycosylation points in this extracellular domain, its actual mol. wt. may be its high as 70,000 Da. It is therefore concluded that sIFN-αR molecules, corresponding to truncated forms of the extracellular domain of the cell surface IFN-αR, are present in human serum and in normal human urine.

Recombinant interferon alpha-C (rIFNα-C, Interpharm), is a new type of alpha-interferon that has a specific activity of 1-2×10⁹ units/mg protein. The pharmacokinetics of rIFNα-C were studied in 11 patients with metastatic renal-cell carcinoma. A total of 10 million units IFNα-C were injected intramuscularly and the serum level of IFN was evaluated up to 72 h post-administration. Measurable IFN concentrations appeared in the serum as early as 0.5 h, and levels peaked at 4-6 h (C_max=53.2±4.6 units/ml). Relatively high levels persisted for 24 h and declined thereafter with an apparent half-life of 3-4 h. The mean area under the serum-concentration curve (AUC) was 1,259±145 units h ml^-1, indicating good bioavailability of the preparation from the intramuscular injection.

Cyclosporine, but not its nonimmunosuppressive analog cyclosporine H (CsH), caused in a variety of hematopoietic cell types a growth arrest in the G0/G1 phase of the cell cycle. This arrest was associated with a significant reduction in the c-myc mRNA levels, which could be observed already 1 hr following CsA treatment. Similarity between the antiproliferative effects of CsA and IFN-α was observed. Thus, the IFN- α sensitive human B-lymphoblastoid cell line Daudi was also sensitive to CsA while an IFN- α resistant variant of Daudi cells was found to be resistant to CsA as well. Inhibition of protein synthesis with cycloheximide during IFN- α or CsA treatment blocked their ability to reduce the expression of c-myc. Depletion of protein kinase C (PKC) activity from cells by pretreatment of Daudi cells with phorbol-12-myristate 13-acetate (PMA) abolished the G0/G1 arrest induced by both CsA and IFN- α. Combinations of low concentrations of CsA and IFN- α had synergistic effects on cell-cycle distribution and on c- myc mRNA level, suggesting that CsA and IFN- α differ in some features of their antiproliferative action. This conclusion was supported by the observation that a CsA- resistant variant of Daudi cells was found to retain its sensitivity to IFN- α. In addition, reduction of ornithine decarboxylase mRNA expression was obtained with IFN- α but not with CsA. Taken together, our results suggest that CsA and IFN- α share some common element(s) in the pathways of their antiproliferative activity. The possible mechanisms of their antigrowth effects and the clinical significance of our findings are discussed.

Soluble IL-6 receptor (IL-6-R) purified to homogeneity from normal human urine was used for immunization of mice and rabbits. Spleen cells derived from a mouse showing a high binding titer to IL-6-R in an inverted solid phase radioimmunoassay (IsRIA) and in a Western blotting analysis were fused to mouse myeloma cells. The hybridomas were screened by the IsRIA, and 30 positive clones were isolated and characterized. They were suitable for affinity purification of the IL-6-R and for its detection by Western blot analysis, by ELISA and by sandwich type sRIA. Most of them inhibited the binding of labeled IL-6-R to IL-6 in a solid phase RIA.

Two major transmembranal polypeptides of bovine olfactory epithelium were identified by SDS electrophoretic analysis of Triton X-114 solubilized membranes. Both polypeptides were present in large amounts in membranes of the olfactory epithelium but were barely detectable in membranes of the nasal respiratory epithelium. Both polypeptides are enriched in the deciliated epithelium as compared with isolated cilia. One of them is a glycoprotein with an apparent molecular mass of 56 kDa (gp56); the other is an unglycosylated protein with an apparent molecular mass of 52 kDa (p52). Sequence analysis of peptides obtained by CNBr cleavage of purified gp56 indicates that it is highly homologous to UDP-glucuronosyl transferase (UDPGT). Parallel analysis shows that p52 is highly homologous to cytochrome P-450 sequences of the IIA subfamily. This protein is assigned the name P-450olf2. Polyclonal antibodies were raised against synthetic peptides corresponding to gp56 and p52 peptide sequences. Immunoblots with these antibodies reveal the following properties of gp56 and p52: (1) they are enriched in the microsomal fraction of the bovine olfactory epithelium; (2) they are possibly specific to the olfactory epithelium, as we could not detect reactivity in microsomes derived from respiratory epithelium or lung, and only a very small amount of basal reactivity was seen with liver microsomes; (3) cross-reacting proteins exist in microsomes derived from the rat olfactory epithelium. These results are consistent with a mechanism whereby the microsomal enzymes are involved in odorant modification and clearance from the nasal tissue.

The protective effects of interferons (IFNs) against NK cell-mediated cytotoxicity (NK-CMC) is well established. We report here that both recombinant tumor necrosis factor-α (TNF-α) and recombinant interleukin-1α (IL-1α) can also protect some adherent target cells (e.g., the amniotic cells WISH and the cervical epithelial carcinoma cells HeLa-229) from NK-CMC in a dose-dependent manner. Like in the case of IFNs, the level of conjugate formation between target and effector cells (nonadherent peripheral blood lymphocytes) is not affected by pretreatment of the target cells with either TNF-α or IL-1α. However, while the main effect of IFNs is to reduce the ability of target cells to stimulate the release of NK cytotoxic factor (NKCF) from effector cells, TNF-α and IL-1α do not affect this process but rather reduce the target cell sensitivity to the lytic effect of NKCF. Therefore TNF-α and IL-1α induce resistance to NK-CMC by a mechanism that differs from the one attributed to IFNs. The protective effect of TNF-α and IL-1α is not mediated by the induction of IFN-β₂/IL-6.

Keywords: Immunology; Medicine, Research & Experimental; Pathology; Physiology

Keywords: Biochemistry & Molecular Biology; Immunology; Medicine, Research & Experimental

Natural-killer cell mediated cytotoxicity (NK-CMC) is modulated by interferons both at the effector cell and at the target cell levels. Pretreatment of effector cells with interferon increases their cytotoxicity while pretreatment of target cells with interferons decreases their sensitivity to NK-CMC. Interferons are inducers of several genes including those of the major histocompatibility complex (MHC) and several earlier studies have established a circumstantial correlation between induction of class I MHC gene products and induction of resistance to NK-CMC. In the present study we demonstrate that interferon-α renders Daudi cells resistant to NK-CMC without inducing the surface expression of class I MHC antigens. Therefore we suggest that these two phenomena are independent responses to interferon treatment.

A human genomic DNA segment of 5.6 kb containing the entire gene for immune interferon-γ was fused through its 5-untranslated region to the corresponding region of the simian virus 40 (SV40) T-antigen gene. The SV40 early promoter used contained a modified transcriptional enhancer element with a 93-bp repeat. Supercoiled plasmid DNA was used to transfect Chinese hamster ovary (CHO) cells, the selectable marker being a SV40-dihydrofolate gene construct. Constitutive expression of the IFN-γ gene in primary transformants was high, especially if a Harvey murine sarcoma virus long terminal repeat (LTR) was present in addition to the SV40 promoter. After gene amplification by methotrexate selection, CHO-γ cell lines were obtained that produce 1.52 million units of IFN-γ per million cells and per day (200,000 molecules per cell per minute). Metabolic labeling showed that over 90% of the protein secreted by such cells is human IFN-γ. A one-step immuno-affinity chromatography on monoclonal antibodies yielded pure IFN-γ with 12 × 10⁸ units/mg protein. Like IFN-γ from human white blood cells, the IFN-γ from CHO-γ cells is a mixture of two glycoproteins of 26,000 and 20,000 daltons with traces of the unglycosylated 17,000-dalton polypeptide. Large-scale cultures in 1% serum routinely yield over 600,000 units of human IFN-γ/ml culture per day.

Previous studies carried out in our laboratory have demonstrated that goldfish brain contains substances that promote neurite extension from regenerating retinae in culture. Fractionation of the brain extract by molecular sieving chromatography revealed the presence of several molecular species, including two peaks that have neurotrophic activity, representing low-molecular-weight substances. One peak was eluted (P-a) with an apparent molecular weight of about 13 kDa and was designated substratum neurite extension factor (SNEF) because it retained its neurotrophic activity when adsorbed onto the substratum. This recovered Sephadex fraction (P-a) when applied in vivo intraocularly caused an earlier capacity of the corresponding retinae to sprout in vitro. Thus, at 3 and 5 days after injury the neuritic growth indices from the factor-treated retinae were of 0.9 +/- 0.2 and 2.8 +/- 0.5, respectively, as compared with indices of 0.3 +/- 0.1 and 0.9 +/- 0.2, respectively, in retinae of injured but nontreated nerves. The factor was further purified by two steps of HPLC (ion exchange followed by reversed phase). The results showed that it is an acidic glycoprotein with an apparent molecular weight of 10 kDa.

A rapid procedure for isolation of two biologically active human interferon-γ subtypes was developed. Crude interferon-γ produced in a serum-free culture of peripheral blood mononuclear cells by mitogen stimulation was concentrated and partially purified by chromatography on controlled-pore glass. Following desalting and concentration by ultrafiltration, a step of cation-exchange high-performance liquid chromatography was performed. A linear NaCl gradient (0.01-0.4 m) at pH 7 was employed and four peaks of biological activity eluting at 0.17, 0.20, 0.26 (major peak), and 0.3 m were obtained. The major peak of biological activity coincided with two protein peaks. Analysis of one fraction from the major activity peak by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a protein band having an apparent molecular weight of 26.000, while an adjacent fraction of the same activity peak contained a protein band corresponding to a molecular weight of 21.000. The specific activity of both subtypes was 7-10 × 10⁶ units/mg.

A low-density subpopulation of normal human peripheral blood mononuclear leukocytes obtained by percoll gradients was found to produce significant levels (up to 10,000 units/ml) of interferon in the absence of an external inducer. The interferon (IFN) was characterized as a mixture of IFN-γ (immune) and a pH-sensitive IFN-α by neutralization with specific antibodies, by lability to low pH, and by cross-reactivity on bovine cells. Significant levels of IFN were obtained only at high (≥5 × 10⁶ cells/ml) cell densities. These levels were not affected by the presence of small T lymphocytes and sharply decreased with cell dilution in a nonlinear fashion. A release of IFN in the absence of an external inducer, indicates additional physiological roles besides its involvement in pathological conditions.

Studies with crude or partly purified interferon have provided a significant amount of structural information. However, complete biochemical characterization required purification to homogeneity. Earlier work on fractionation has met with many difficulties because interferon was available only in minute quantities. A scale-up of production, adaptation of multi-step purification schemes, use of high-resolution separation techniques and highly sensitive analytical methods have yielded pure interferons and hence many structural data. Specific activities, amino-acid compositions, partial sequences and structural homologies of many interferons were determined. Finally, cloned copy DNA (cDNA) fragments derived from specific interferon mRNA, as well as isolated interferon genes, have been sequenced and the data were used to elucidate complete sequences of many interferons with a high degree of confidence.

Unfractionated human leukocyte interferon, as well as highly purified subspecies of this interferon, and a purified recombinant of human leukocyte interferon produced in bacteria are active in suppressing multiplicability of tobacco mosaic virus in tobaco leaf discs. Human fibroblast interferon exhibits diverse levels of antiviral activity against tobaco mosaic virus but becomes as active as human leukocyte interferon upon incubation with glycosidases. The effect of interferon is reversible; normal multiplication of tobaco mosaic virus resumes upon removal of interferon.

The synthesis of phosphate diesters from various alcohols and nucleosides is described, using the N-methylpyridinium salt of dichlorophosphoric acid (1) as a phosphorylating agent. Internucleotide bonds are formed by stepwise addition of two suitably protected nucleosides to 1. Side products, usually formed during oligonucleotide synthesis were not observed using this new method. In addition, a nucleoside 2, 3-cyclic phosphate was prepared by one step phosphorylation of a ribonucleoside protected at the 5-hydroxyl. The products were isolated in relatively high yields by simple separation methods.