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.

Our approach of isolating proteins from a rich source of human proteins by ligand-affinity-chromatography enabled rapid and efficient isolation of not only soluble receptors corresponding to cell-associated receptors, but also independent binding-proteins and associated enzymes. No other approach would yield the latter. During the early 80's we prepared the tools and the infrastructure that enabled the subsequent 20 years of achievements. Thus we described eight soluble receptors (R) and binding proteins (BP) for various cytokines including the IL-6R, IFN-gamma R, TNFRI, TNFRII, LDLR, IFN-alpha/beta PR, IL-18BP and IL-32BP identified as Proteinase 3. The isolation of the soluble IFN-alpha/beta receptor led to the cloning of its long sought cell surface ligand binding counterpart. We have established the concept that soluble receptors and binding proteins are normal constituents of body fluids in healthy individuals and that the levels of these biomarkers are modulated in various pathological situations. Each of these proteins contributed to basic science, one of them serves as a basis for therapy and some others are in various stages of clinical development. (c) 2007 Elsevier Ltd. All rights reserved.

Catecholamines are important regulators of homeostasis, yet their functions in hematopoiesis are poorly understood. Here we report that immature human CD34(+) cells dynamically expressed dopamine and beta(2)-adrenergic receptors, with higher expression in the primitive CD34(+)CD38(Io) population. The myeloid cytokines G-CSF and GM-CSF upregulated neuronal receptor expression on immature CD34+ cells. Treatment with neurotransmitters increased the motility, proliferation and colony formation of human progenitor cells, correlating with increased polarity, expression of the metalloproteinase MT1-MMP and activity of the metalloproteinase MMP-2. Treatment with catecholamines enhanced human CD34+ cell engraftment of NOD-SCID mice through Wnt signaling activation and increased cell mobilization and bone marrow Sca-1(+)c-Kit(+)Lin(-) cell numbers. Our results identify new functions for neurotransmitters and myeloid cytokines in the direct regulation of human and mouse progenitor cell migration and development.

Keywords: Biochemistry & Molecular Biology; Cell Biology; Immunology

Leptin, an adipokine, a major regulator of food intake, was recently suggested to play a role in immune response. We previously showed that weight reduction following IFN alpha therapy is due, at least in part, to direct induction of adipose tissue apoptosis. We now studied the effect of leptin on IFN alpha treated adipocytes in vitro and in vivo. Diet induced obese C57/136 mice were treated continually with recombinant (r) IFM alpha/D + leptin (100 U/g body weight + 10 mu g/day, respectably) or leptin (10 mu g/day) alone for 8 days. Co-administration of IFN alpha A/D + leptin significantly reduced plasma cholesterol (P

Hemophagocytic syndrome (HIPS) is characterized by an uncontrolled and poorly understood activation of T-helper 1 (Th-1) lymphocytes and macrophages. We studied 20 patients with HIPS 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 gamma (IFN-gamma) production, and stimulation of macrophages and natural killer (NK) cells were highly increased in HIPS 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

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-gamma (IFNgamma) levels in supernatants were measured by enzyme-linked immunosorbent assay. Levels of IL-18BP and IFNgamma 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-gamma (IFNgamma) 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 IFNgamma 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 IFNgamma 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

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 nonsense 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. (C) 2003, Editrice Kurtis.

Although interleukin (IL)-18 is a member of the IL-1 family of ligands, IL-18 appears to have unique characteristics, particularly in the regulation of the T helper type 1 (Th1) response. Th1 responses are required for tumor surveillance, killing intracellular organisms, and to provide help for antibody production. In patients with chronic renal failure, the well-known immunosuppression contributes to a failure to respond to infectious challenges and vaccinations. The most salient biological property of IL-18, linking this cytokine to the Th1 response, is its ability to induce interferon gamma (IFN-gamma). In fact, IL-18 was originally identified as an IFN-gamma-inducing factor, and IFN-gamma production is the hallmark of the Th1 response. Dysregulation of IFN-gamma production resulting from reduced activity of IL-18 would explain one of the mechanisms of immunosuppression in patients with chronic renal failure. The activity of IL-18 can be regulated by the IL-18-binding protein (IL-18BP), a glycoprotein of 40,000 daltons, which is constitutively expressed and appears to be the natural inhibitor of IL-18 activity. Unlike soluble receptors for IL-18, IL-18BP does not have a transmembrane domain; IL-18BP is a secreted protein possessing a high-affinity binding and ability to neutralize IL-18. IL-18BP was discovered in human urine and is excreted in health following glomerular filtration. With decreasing renal function, the concentrations of IL-18BP in the circulation are elevated as compared with subjects with a normal renal function, and these elevated levels may result in a decreased IL-18 activity. Because of the importance of IL-18 and IFN-gamma in the Th1 response, the biology of IL-18 and IL-18BP is reviewed here in the context of the immunosuppression of chronic renal failure. Copyright (C) 2003 S. Karger AG, Basel.

Long-term hemodialysis (HID) induces an inflammatory response and is associated with a suppressed cellular immune response manifested, in part, by impaired interferon (IFN-gamma) production. We investigated the effect of high-flux HID using the synthetic Helixone(R) membrane and ultrafiltered dialysate on plasma levels of inflammatory mediators and on the whole blood production of IFN-gamma. Methods: Twelve ESRD patients were dialyzed under low-flux HID (polysulfone F6) and again after 6 weeks of high-flux HID (Helixone(R) FX100). Ultrafiltered bicarbonate dialysate without bacterial growth and no detectable endotoxin was used throughout the study. Plasma levels of urea, albumin, beta(2)-microglobulin (beta(2)-M), interleukin (IL)-6, C-reactive protein (CRP), IL-1 receptor antagonist (IL-1Ra), IL-18, and IL-18-binding protein (IL-18BP) were measured. In addition, the Staphylococcus epidermidis-induced production of IFN-gamma and IL-18 was assessed in whole blood cultures of HID patients as well as in 9 healthy subjects. Results: Plasma levels of urea, albumin, IL-6, IL-1Ra and CRP were not significantly different between high-flux and low-flux HID. In contrast, beta(2)-M levels decreased significantly by 31% with high-flux Helixone(R) (p

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.

Proinflammatory cytokines are important mediators of neuroinflammation after traumatic brain injury. The role of interleukin (IL)-18. a new member of the IL-1 family, in brain trauma has not been reported to date. The authors investigated the posttraumatic release of IL-18 in murine brains following experimental closed head injury (CHI) and in CSF of CHI patients. In the mouse model, intracerebral IL-18 was induced within 24 hours by ether anesthesia and sham operation. Significantly elevated levels of IL-18 were detected at 7 days after CHI and in human CSF up to 10 days after trauma. Published data imply that IL-18 may play a pathophysiological role in inflammatory CNS diseases; therefore its inhibition may ameliorate outcome after CHI. To evaluate the functional aspects of IL-18 in the injured brain, mice were injected systemically with IL-18-binding protein (IL-18BP), a specific inhibitor of IL-18, I hour after trauma, IL-18BP-treated mice showed a significantly improved neurological recovery by 7 clays, accompanied by attenuated intracerebral IL-18 levels. This demonstrates that inhibition of IL-18 is associated with improved recovery. However, brain edema at 24 hours was not influenced by IL-18BP, suggesting that inflammatory mediators other than IL-18 induce the early detrimental effects of intracerebral inflammation.

Human and rat granulosa cells express receptors to leptin which synergies with glucocorticoid hormones in stimulation of ovarian steroidogenesis. To examine whether leptin affects follicular development and maturation, we injected recombinant ovine leptin (300 ng- 10 mug/animal) daily to immature 21 day-old female rats. Non-treated rats reached puberty at 44.5 +/-1.6 (n=9) days. In contrast, in leptin treated animals, puberty was reached at 34.5 +/-1.6 (n=9) days. Ovarian sections revealed hypertrophy of granulosa cells in leptin treated animals. Moreover, the number of ovulations was 2-fold higher in the treated animals compared to controls (3-4 ovulations versus 7-8 on the first three estrous cycles, P

IL-18 binding protein (IL-18BP) is a circulating antagonist of the proinflammatory Th1 cytokine IL-18. It effectively blocks IL-18 ky 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 (similar to 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. (C) 2001 Academic Press.

Adipose tissues consisting of adipocytes, microvasculature, and stroma are completely ablated upon overexpression of leptin in rats. This tissue regression is mediated by enhanced lipid beta-oxidation, adipocyte dedifferentiation, and apoptosis. To further characterize this phenomenon, we studied the possible effect of leptin on the adipose microvasculature. Tissue microvasculature is maintained by the interplay between positive and negative signals mediated by factors including vascular endothelial growth factor (VEGF), basic fibroblast growth factor, angiopoletin-1 (Ang-1), and Ang-2. Expression of the negative signal Ang-2 was reported in fetal tissues and in the adult ovary, which undergoes vascular remodeling or regression. We demonstrate that leptin induces the expression of Ang-a in adipose tissue without a concomitant increase in VEGF. Induction of Ang-2 occurred in an autocrine manner, as demonstrated in cultured adipocytes but not in several other cell types. This tissue-specific induction of Ang-2 coincided with initiation of apoptosis in adipose endothelial cells. We propose that induction of Ang-2 by leptin in adipose cells is one of the events leading to adipose tissue regression.

Interleukin (IL-)18 is an activator of NK cells and a co-inducer of Th-1 cytokines, sharing structural features with the IL-1 family of proteins. Unlike most other cytokines, IL-18 and IL-1 beta lack a signal peptide, have an all beta -pleated sheet structure and are synthesized as biologically inactive precursors (pro-IL-18 and pro-IL-1 beta), These precursors are cleaved by caspase-1 (IL-1 beta -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.

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.

The Fifth Annual Conference of the International Cytokine Society was held on November 9-13, 1997 at Lake Tahoe, Nevada. This meeting kept up with the tradition of exciting talks and posters, presenting significant advances in our understanding of the cytokine world, As we advance our knowledge, a complex network of interacting cellular communication pathways is revealed. Targeted disruption of genes coding for cytokines, their receptors and their cytoplasmic signaling molecules, became the standard method for a proper assessment of the role of a given cytokine in vivo, Yet, fundamental questions remain unresolved, For instance, it is not yet known how signal specificity is maintained when different cytokine receptors use the same cytoplasmic signaling pathways, The following summary is not comprehensive, rather, it is a collection of representative communications. Many more top quality studies were presented at the meeting and we apologize for not being able to review all of them here, (C) 1998 Elsevier Science Ltd. All rights reserved.

Two transmembrane polypeptides, IFNAR and IFN-alpha/beta R, 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-alpha/beta R is a 102-kDa protein but upon reduction only a 51-kDa protein is detected, In host murine cells human IFN-alpha/beta R was expressed as a 51-kDa protein, Host cells expressing IFN-alpha/beta R bound IFN-alpha(2) 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-alpha/beta R, the affinity for IFN-alpha(2) 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-alpha/beta R bind IFN-alpha(2) to form an intermediate product, while IFNAR associates with this product to form a ternary complex, Hence, IFNAR and IFN-alpha/beta R are components of a common type I IFN receptor, cooperating in ligand binding, Ligand-induced association of IFNAR and IFN-alpha/beta R 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-alpha receptor (sIFN-alphaR) were identified in human serum and urine by Western blotting with monoclonal antibodies (MAb) directed against IFN-alphaR, and by immunoprecipitation (Iptn) of a covalently cross-linked complex of IFN-alphaR and [I-125]IFN-alpha with anti IFN-alpha MAb. Elevated levels of sIFN-alphaR 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-alphaR 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 as high as 70,000 Da. It is therefore concluded that sIFN-alphaR molecules, corresponding to truncated forms of the extracellular domain of the cell surface IFN-alphaR, are present in human serum and in normal human urine.

Keywords: CHO CELLS; METHOTREXATE AMPLIFICATION; MICROCARRIER CULTURES; IMMUNOAFFINITY PURIFICATION

Keywords: Chemistry, Analytical

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

Unfractionated preparations of the proteins of human urine provided protection against the in vitro cytocidal effect of tumor necrosis factor (TNF). In certain cells, the proteins decreased expression of the receptors for TNF in a temperature-dependent way. In all cells examined, the proteins were found to interfere also with the binding of both TNF and interleukin-1 when applied directly into the binding assays. That effect could be observed in the cold, suggesting that it was independent of cellular metabolism. A protein which protects cells against the cytotoxicity of TNF was purified from human urine by chromatography on CM-Sepharose followed by high performance liquid chromatography on Mono Q and Mono S columns and reversed phase high performance liquid chromatography. This protein is a very minor constituent of normal urine, with an apparent molecular weight of about 27,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. Homogeneity of the purified protein was confirmed by microsequence analysis which revealed a single N-terminal sequence: Asp-Ser-Val-Cys-Pro-. The protein protected cells from TNF toxicity at concentrations of a few nanograms per ml and interfered with the binding of both TNF-alpha and TNF-beta to cells, when applied simultaneously with the cytokines. However, unlike crude preparations of the urinary proteins, the purified protein did not induce in cells a decrease in ability to bind TNF nor did it interfere with the binding of interleukin-1 to its receptor. Direct, specific binding to the protein of TNF-alpha and, to a lesser extent, also TNF-beta, but not of interleukin-1 nor interferon-gamma could be demonstrated. It is suggested that this protein blocks the function of TNF by competing for TNF with the TNF receptor and not by interacting with the target cell.

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.