Production Of Functional Protein Research Articles

643. Prolonged Expression of Human Alpha-1-Antitrypsin in C3H/Hej Mice with Helper-Dependent Adenoviral Vectors Irrespective of Tissue Distribution of Gene Expression

We have recently constructed a novel HAC vector called 21ΔqHAC, generated by deletion of a distal portion of the long arm of human chromosome 21 and insertion of a loxP sequence to facilitate insertion of any desired gene construct. We intend to use this HAC vector to rescue T and B cell development and the radiosensitive phenotype in the DNA-PKcs-deficient SCID (severe combined immunodeficiency) mice. Our approach will involve introduction of 21ΔqHAC containing the DNA-PKcs gene into DNA-PKcs-deficient ES cells, differentiation of corrected ES cells into blood stem cells and transplantation of these blood stem cells into SCID mice. To evaluate the usefulness of 21ΔqHAC vector for this purpose, we first attempted in vitro studies using CHO cells (V3) exhibiting radiosensitive phenotype due to a DNA-PKcs deficiency. In this experiment, we inserted a ~21 kb doxycycline-inducible DNA-PKcs expression cassette into 21ΔqHAC vector by Cre-mediated recombination. The resulting 21ΔqHAC/DNA-PKcs construct was then introduced into the V3 cells. RT-PCR results from cells containing the 21ΔqHAC/DNA-PKcs vector showed doxycycline-dependent regulation of the DNA-PKcs gene. Furthermore, radiosensitivity assays showed significant phenotype rescue in these cells due to the production of functional DNA-PKcs protein. To our knowledge, our study is the first to report successful artificial control of gene expression by placing the multiple regulatory elements of the tetracycline system in a single expression cassette within a HAC vector. Our results also show that 21ΔqHAC is a very useful tool in the introduction of a therapeutic gene and the correction of a defective phenotype.

Enhanced production of functional proteins from defective genes.

Nonsense mutations are associated with a host of genetic disorders. The protein products encoded by genes containing these mutations are either truncated or completely missing. Recently, members of the aminoglycoside family of antibiotics have been found to induce the ribosome to read past these inappropriately inserted stop codons, albeit at extremely modest efficiencies. We report herein a strategy that enhances the production of functional proteins from the corresponding mutated genes.

Exploring and exploiting bacterial proteomes.

The plethora of data now available from bacterial genome sequencing has opened a wealth of new research opportunities. Many of these have been reviewed in preceding chapters. Genomics alone, however, cannot capture a biological snapshot from an organism at a given point in time. The genome itself is static, and it is the changes in expression of genes, leading to the production of functional proteins, which allows an organism to survive and adapt to a constantly changing environment. Proteomics is the term used to describe the global analysis of proteins involved in a particular biological process. Such processes may be analyzed via comparative studies that examine bacterial strain differences, both phenotypic and genetic, bacteria grown under nutrient limiting conditions, growth phase, temperature, or in the presence of chemical compounds, such as antibiotics. Proteomics also provides the researcher with a tool to begin characterizing the functions of the vast proportion of "hypothetical" or "unknown" proteins elucidated from genome sequencing and database comparisons. For example, study of protein-protein, protein-ligand, protein-substrate, and protein-nucleic acid interactions for a given target protein may all help to define the functions of previously unknown proteins. Furthermore, genetic manipulation combined with proteomics technologies can provide an understanding of how gene expression is regulated. This chapter examines the technologies used in proteome analysis and the applications of proteomics to microbiological research, with an emphasis on clinically-relevant bacteria.

Pathogenicity of the hereditary colorectal cancer mutation hMLH1 del616 linked to shortage of the functional protein

Background & Aims: Hereditary nonpolyposis colorectal cancer is associated with mismatch repair deficiency. Most predisposing mutations prevent the production of functional mismatch repair protein. Thus, when the wild-type copy is also inactivated, the cell becomes mismatch repair deficient, and this leads to a high degree of microsatellite instability in tumors. However, tumors linked to nontruncating mutations may display positive or partly positive immunohistochemical staining of the mutated protein and low or atypical microsatellite instability status, which suggests impaired functional activity but not a total lack of mismatch repair. We found human mutL homology ( hMLH) 1 del616, one of the most widespread recurring mutations in hereditary nonpolyposis colorectal cancer, segregating in a large hereditary nonpolyposis colorectal cancer family. Because the predicted coding change is a deletion of only 1 amino acid, the pathogenicity of the mutation was evaluated. Methods: Many analyses were performed to assess the pathogenicity of hMLH1 del616 and to study the expression and function of the mutated messenger RNA and protein. Results: Genetic and immunohistochemical evidence supported hMLH1-linked cancer predisposition in this family. Microsatellite instability varied from low to high, and the hMLH1 protein was lost in 2 tumors but was partly detectable in 1 tumor. Whereas similar optimal amounts of mutated hMLH1 del616 and wild-type hMLH1 proteins were equally functional in an in vitro mismatch repair assay, the amount of in vivo-expressed hMLH1 del616 was much lower than the amount of wild-type protein; this suggests that the deletion imparts instability to the mutant protein. Conclusions: Our results suggest that the pathogenicity of hMLH1 del616 is not linked to nonfunctionality, but to shortage of the functional protein.

DiscernArray™ technology: a cell-free method for the generation of protein arrays from PCR DNA

Protein array technology offers a powerful tool to bridge genomics and proteomics. Currently, the bottleneck in the generation of protein arrays is the comprehensive production of functional proteins. We have developed a rapid cell-free method, DiscernArray™, which creates functional protein arrays directly from PCR DNA by in vitro synthesis of individual tagged proteins on tag-binding surfaces, such that the tagged proteins are immobilized on a surface as they are synthesised. DiscernArray™ is particularly useful for arraying proteins and domains which cannot be functionally produced in heterologous expression systems or for which the cloned DNA is not available.

Uromodulin promoter directs high-level expression of biologically active human alpha1-antitrypsin into mouse urine.

We have recently shown that the regulatory sequence of the uromodulin gene, containing the 3.7 kb promoter, exon 1 and a part of exon 2, provided for kidney-specific expression of the reporter lacZ gene in transgenic mice [Zbikowska, Soukhareva, Behnam, Chang, Drews, Lubon, Hammond and Soukharev (2002) Transgenic Res., in the press]. In the present study, we generated transgenic mice harbouring the regulatory sequence of the uromodulin gene to direct the expression of human alpha1-antitrypsin (alpha1AT) into urine. Of the 13 founder mice that tested positive by PCR, seven showed the presence of the human protein in their urine. The concentration of the recombinant human (rh) alpha1AT in the urine, estimated by using ELISA, ranged from 0.5 to 14 microg/ml in the F(0)-generation mice, and reached up to 65 microg/ml in the F1 generation. The transgenically produced rh alpha1AT was found to be N-glycosylated and biologically active. The N-terminal sequence analysis confirmed the identity of the human protein and revealed that the recombinant alpha1AT was correctly processed with the signal peptide cleaved off. Our results demonstrate for the first time that the uromodulin regulatory sequence provides a very attractive option for the potential large-scale production of functional therapeutic proteins in livestock.

PASTICCINO1 (AtFKBP70) is a nuclear-localised immunophilin required during Arabidopsis thaliana embryogenesis

The PASTICCINO1 ( PAS1) gene of Arabidopsis thaliana encodes a protein with homology to the FK506-binding protein (FKBP) class of immunophilins. To begin to understand more about the possible function of PAS1, we tested some properties of recombinant PAS1 protein and analysed the expression of the gene in Arabidopsis embryos and cell cultures and in tobacco cells. In pas1-1/+ heterozygote embryos the pas1-1 allele is expressed at very low levels in all cells, but it is misexpressed in the pas1-1 homozygote mutant at the same stage. Anti-PAS1 affinity-purified antibodies recognise a 70 kDa protein from dividing cell cultures of Arabidopsis. In indirect immunofluorescence, the same antibodies label the nuclei of dividing tobacco BY-2 cells. In a protease-coupled assay, recombinant PAS1 protein has low peptidylprolyl cis–trans isomerase (PPIase) activity, which is inhibited by the immunosuppressive drugs FK506 and rapamycin, but not by cyclosporin. PAS1 also binds calmodulin in vitro. This data suggests the importance of the correctly regulated production of functional PAS1 protein, a likely nuclear-localised FKBP, for the correct development of the plant embryo.

Co-regulation of survival of motor neuron (SMN) protein and its interactor SIP1 during development and in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a neuromuscular disease characterized by the degeneration of motor neurons in the spinal cord. The disease is caused by mutations of the survival of motor neuron 1 gene (SMN1), resulting in a reduced production of functional SMN protein. A major question unanswered thus far is why reduced amounts of ubiquitously expressed SMN protein specifically cause the degeneration of motor neurons without affecting other somatic cell types. In a first attempt to address this issue we have investigated the Smn interacting protein 1 (Sip1), with an emphasis on its developmental expression and subcellular distribution in spinal motor neurons in relation to Smn. By confocal immunofluorescence studies we provide evidence that a significant amount of Smn does not co-localize with Sip1 in neurites of motor neurons, indicating that Smn may exert motor neuron-specific functions that are not dependent on Sip1. Sip1 is highly expressed in the spinal cord during early development and expression decreases in parallel with Smn during postnatal development. Strikingly, reduced production of Smn as observed in cell lines derived from SMA patients or in a mouse model for SMA coincides with a simultaneous reduction of Sip1. The finding that expression of Sip1 and Smn is tightly co-regulated, together with the unique localization of Smn in neurites, may help in understanding the motor neuron-specific defects observed in SMA patients.

Genetic Design of Stable Metal-Binding Biomolecules, Oligomeric Metallothioneins

Metallothionein (MT) is a suitable model for investigating molecular interactions relating to the handling of metals in cells. However, the production of functional MT proteins in microorganisms has been limited because of the instability of MT—the thiol group of cysteine is easily oxidized and proteolysis occurs. To increase the binding ability and to stabilize MT, we designed genes for dimeric and tetrameric MT and the genes were successfully overexpressed in Escherichia coli to generate functional oligomeric MTs. A human MT-II (hMT-II) synthesized with prokaryotic codons, a linker encoding a glycine tripeptide, and Met-deficient hMT-II was ligated to create a dimeric MT, from which a tetrameric MT was then constructed. The increased molecular size of the constructs resulted in improved stability and productivity in E. coli. Cells of E. coli carrying the oligomeric MT genes showed resistance toward Zn and Cd toxicity. The oligomeric proteins formed inclusion bodies, which were dissolved with dithiothreitol, and the purified apo-MTs were reconstituted with Cd or Zn ions under reducing conditions. The dimeric and tetrameric MT proteins exhibited both Cd and Zn binding activities that were, respectively, two and four times higher than those of the hMT-II monomer protein. These stable oligomeric MTs have potential as a biomaterial for uses such as detoxification and bioremediation for heavy metals.

Understanding the art of producing protein and nonprotein molecules in Escherichia coli.

The high-level production of functional proteins in E. coli is a very extense field of research in biotechnology. A number of important aspects to be considered in the initial design of an expression system and their interplay, were clear years ago. However, in recent times, strategies that go beyond transcription, translation, stability, vector, and strain choice, have been developed; so now expression of active peptides can be viewed as a more integrated process. Coexpression of protein subunits, foldases and chaperones, protein folding, location and purification schemes, metabolic engineering of the cell's central metabolism, and in vitro refolding strategies, are some of the novelties that are now available to aid in the success of an efficient expression system for active heterologous proteins. This review presents a compilation of the basic issues that influence the success in the production of protein and nonprotein products in Escherichia coli, as well as some general strategies designed to facilitate downstream process operations and improve biosynthesis yields.

Liver cirrhosis is reverted by urokinase-type plasminogen activator gene therapy.

Liver cirrhosis represents a worldwide health problem and is a major cause of mortality. Cirrhosis is the result of extensive hepatocyte death and fibrosis induced by chronic alcohol abuse and hepatitis B and C viruses. Successful gene therapy approaches to this disease may require both reversal of fibrosis and stimulation of hepatocyte growth. Urokinase-type plasminogen activator (uPA) may serve this function, as it is an initiator of the matrix proteolysis cascade and induces hepatocyte growth factor expression. In a rat cirrhosis model, a single iv administration of a replication-deficient adenoviral vector encoding a nonsecreted form of human uPA resulted in high production of functional uPA protein in the liver. This led to induction of collagenase expression and reversal of fibrosis with concomitant hepatocyte and improved liver function. Thus, uPA gene therapy may be an effective strategy for treating cirrhosis in humans.

Cytoskeletal protein mRNA expression in the chick utricle after treatment in vitro with aminoglycoside antibiotics: effects of insulin, iron chelators and cyclic nucleotides

In birds, spontaneous recovery of the hair cells of the inner ear can occur after damage induced by aminoglycoside antibiotics. The factors that influence this recovery and the process of hair cell regeneration itself have until recently been investigated largely by morphological and histological methods. The aim of this work was to use a molecular biological approach to the analysis of hair cell regeneration by measuring the changes that occur in expression of mRNA for hair cell-specific cytoskeletal proteins fimbrin and class III β-tubulin, along with that for β-actin, in the utricle of chicks after hair cell damage both in vitro and in vivo. Utricles were removed from 1-day-old chicks and incubated with the aminoglycoside antibiotics gentamicin or neomycin (both 1 mM), or chicks were injected intraperitoneally with 100 mg/kg gentamicin or neomycin for 4 consecutive days. At the end of the treatment periods, total RNA was extracted from single utricles, reverse transcribed to cDNA and the cDNA amplified by PCR with primers for β-actin, fimbrin and class III β-tubulin. Co-amplification allowed quantitative comparison of mRNA between fimbrin, or class III β-tubulin and β-actin from the same utricle. Both aminoglycosides, either after 48 h in vitro or immediately after treatment in vivo, caused a significant decrease in the expression of fimbrin mRNA and class III β-tubulin mRNA, relative to β-actin mRNA, which itself increased. Light and electron microscopy confirmed that this corresponded to loss of, and damage to, hair cells. The relative expression of fimbrin and class III β-tubulin mRNAs was partly restored almost to control levels 4 days after cessation of treatment in vivo and fully normalised by 4 weeks, by which time hair cells appeared normal. However, their relative expression remained depressed 4 days after removal of antibiotic in vitro. The iron chelator desferrioxamine (100 μM) in vitro prevented the aminoglycoside-induced reduction in relative expression of mRNA for both fimbrin and class III β-tubulin. Neither insulin (5 μM) nor a combination of dibutyryl cyclic AMP (5 mM) and the phosphodiesterase inhibitor IBMX (0.5 mM) prevented the decrease in relative expression of the mRNAs for the hair cell-specific proteins, but both treatments allowed their partial recovery in vitro during the 4-day-period after removal of aminoglycoside. It is concluded that the cells of the sensory epithelium of the chick utricle subjected to aminoglycoside-induced damage undergo a process in which mRNA expression is switched away from the production of functional proteins and towards proteins necessary for structural re-organisation. The restoration of mRNA expression to a normal pattern is promoted by the growth factor insulin and by cyclic AMP.

Sustained expression of human factor VIII in mice using a parvovirus-based vector

Persistent therapeutic levels of human factor VIII (hFVIII) would signify a major advance in the treatment of hemophilia A. Here we report sustained expression of hFVIII in immunocompetent mice using recombinant adeno-associated virus (rAAV) vectors. AAV can stably transduce liver cells, the target tissue for efficient hFVIII production. Because of rAAV packaging constraints, we tested 2 constructs using small regulatory elements designed for liver-specific transgene expression linked to B-domain-deleted hFVIII (BDD-hFVIII) cDNA. More than 10(12)/mL rAAV/BDD-hFVIII virion particles were generated using a transfection scheme that eliminates adenovirus. Coatest and APTT assays confirmed the production of functional BDD-hFVIII protein after transduction of 293 and HepG2 cells. In vivo experiments were performed in C57BL/6 and NOD/scid mice receiving 10(10-11) rAAV/hFVIII particles via portal vein injection. All C57BL/6 mice tested developed anti-hFVIII antibody. In contrast, NOD/scid mice expressed hFVIII reaching 27% of normal human plasma levels. As expected, we could not detect hFVIII antigen from plasma samples isolated from control animals receiving equivalent doses of rAAV expressing enhanced green fluorescent protein (EGFP). Transgene mRNA expression was detected primarily in the liver and histologic analysis of the liver revealed no pathologic abnormalities. These results demonstrate a promising approach for treatment of hemophilia A. (Blood. 2000;95:1594-1599)

Severity of phenotype in cystinosis varies with mutations in the CTNS gene: predicted effect on the model of cystinosin.

Infantile nephropathic cystinosis is a rare, autosomal recessive disease caused by a defect in the transport of cystine across the lysosomal membrane and characterized by early onset of renal proximal tubular dysfunction. Late-onset cystinosis, a rarer form of the disorder, is characterized by onset of symptoms between 12 and 15 years of age. We previously characterized the cystinosis gene, CTNS, and identified pathogenic mutations in patients with infantile nephropathic cystinosis, including a common, approximately 65 kb deletion which encompasses exons 1-10. Structure predictions suggested that the gene product, cystinosin, is a novel integral lysosomal membrane protein. We now examine the predicted effect of mutations on this model of cystinosin. In this study, we screened patients with infantile nephropathic cystinosis, those with late-onset cystinosis and patients whose phenotype does not fit the classical definitions. We found 23 different mutations in CTNS; 14 are novel mutations. Out of 25 patients with infantile nephropathic cystinosis, 12 have two severely truncating mutations, which is consistent with a loss of functional protein, and 13 have missense or in-frame deletions, which would result in disruption of transmembrane domains and loss of protein function. Mutations found in two late-onset patients affect functionally unimportant regions of cystinosin, which accounts for their milder phenotype. For three patients, the age of onset of cystinosis was <7 years but the course of the disease was milder than the infantile nephropathic form. This suggests that the missense mutations found in these individuals allow production of functional protein and may also indicate regions of cystinosin which are not functionally important.

Production of functional rat liver PSP protein in Escherichia coli.

An efficient Escherichia coli expression system for the production of a perchloric acid-soluble protein (PSP) has been constructed. Complementary DNA encoding PSP was inserted into an inducible bacterial expression vector pGEX-4T-1. After the plasmid introduced into E. coli was expressed by isopropyl 1-thio-beta-D-galaetopyranoside (IPTG), the recombinant product was purified by glutathione-Sepharose 4B affinity chromatography. The purified product showed the expected NH2-terminal sequence, but the translation inhibitory activity of this product was 10 times lower compared with that of authentic PSP isolated from rat liver.

Use of the Glyceraldehyde-3-phosphate Dehydrogenase Promoter for Production of Functional Mammalian Membrane Transport Proteins in the YeastPichia pastoris

The promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (PGAP) was employed to produce the mammalian peptide transporters hPEPT1 and rPEPT2 as models for polytopic transmembrane proteins in the methylotrophic yeastPrichia pastoris.Cells of a recombinant renal peptide transporter (rPEPT2) clone produced constitutively the functional carrier protein. The level of functional expression of rPEPT2 with PGAPvaried depending on the carbon source used for cell growth, but was up to five times higher than that obtained with the commonly employed inducible alcohol oxidase 1 promoter (PAOX1). Similar results were obtained for the expression level of the human intestinal peptide transporter hPEPT1 controlled by either PGAPor PAOX1. Therefore, the PGAPseems to be an attractive alternative to PAOX1for generation of transgenicP. pastoriscells expressing functional mammalian membrane transport proteins at high levels.

Collagen-binding growth factors: production and characterization of functional fusion proteins having a collagen-binding domain.

The autocrine/paracrine peptide signaling molecules such as growth factors have many promising biologic activities for clinical applications. However, one cannot expect specific therapeutic effects of the factors administered by ordinary drug delivery systems as they have limited target specificity and short half-lives in vivo. To overcome the difficulties in using growth factors as therapeutic agents, we have produced fusion proteins consisting of growth factor moieties and a collagen-binding domain (CBD) derived from Clostridium histolyticum collagenase. The fusion proteins carrying the epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF) at the N terminal of CBD (CBEGF/CBFGF) tightly bound to insoluble collagen and stimulated the growth of BALB/c 3T3 fibroblasts as much as the unfused counterparts. CBEGF, when injected subcutaneously into nude mice, remained at the sites of injection for up to 10 days, whereas EGF was not detectable 24 h after injection. Although CBEGF did not exert a growth-promoting effect in vivo, CBFGF, but not bFGF, strongly stimulated the DNA synthesis in stromal cells at 5 days and 7 days after injection. These results indicate that CBD may be used as an anchoring unit to produce fusion proteins nondiffusible and long-lasting in vivo.

Neonatal Hemolytic Anemia Due to Inherited Harderoporphyria: Clinical Characteristics and Molecular Basis

AbstractPorphyrias, a group of inborn errors of heme synthesis, are classified as hepatic or erythropoietic according to clinical data and the main site of expression of the specific enzymatic defect. Hereditary coproporphyria (HC) is an acute hepatic porphyria with autosomal dominant inheritance caused by deficient activity of coproporphyrinogen III oxidase (COX). Typical clinical manifestations of the disease are acute attacks of neurological dysfunction; skin photosensitivity may also be present. We report a variant form of HC characterized by a unifying syndrome in which hematologic disorders predominate: harderoporphyria. Harderoporphyric patients exhibit jaundice, severe chronic hemolytic anemia of early onset associated with hepatosplenomegaly, and skin photosensitivity. Neither abdominal pain nor neuropsychiatric symptoms are observed. COX activity is markedly decreased. In a first harderoporphyric family, with three affected siblings, a homozygous K404E mutation has been previously characterized. In the present study, molecular investigations in a second family with neonatal hemolytic anemia and harderoporphyria revealed two heterozygous point mutations in the COX gene. One allele bore the missense mutation K404E previously described. The second allele bore an A→G transition at the third position of the donor splice site in intron 6. This new COX gene mutation resulted in exon 6 skipping and the absence of functional protein production. In contrast with other COX gene defects that produce the classical hepatic porphyria presentation, our data suggest that the K404E substitution (either in the homozygous or compound heterozygous state associated with a mutation leading to the absence of functional mRNA or protein) is responsible for the specific hematologic clinical manifestations of harderoporphyria.

Translation of an Uncapped mRNA Involves Scanning

tat, an essential gene of human immunodeficiency virus, when placed under the control of the RNA polymerase III promoter from the adenovirus VA RNA1 gene, is transcribed into an uncapped and nonpolyadenylated mRNA. This VA-Tat RNA is translated to produce functional Tat protein in transfected mammalian cells (Gunnery, S., and Mathews, M. B. (1995) Mol. Cell. Biol. 15, 3597-3607). The presence of an upstream open reading frame (ORF) in VA-Tat RNA is inhibitory to the translation of the Tat ORF, suggesting that the RNA is scanned during translation even though it is uncapped. Because the effect of the upstream ORF is relatively small (about 2-fold), we sought more definitive evidence of scanning by introducing secondary structures of varying stabilities into the 5'-untranslated region of VA-Tat RNA. The results of transfection experiments showed that highly stable secondary structure was inhibitory to Tat synthesis, whereas structures of lower stability were not inhibitory, confirming that uncapped mRNA is subject to scanning. Furthermore, translation of the downstream ORF was reduced but not eliminated by mutations that caused the upstream ORF to overlap the Tat ORF. Extending the overlap of the two ORFs further decreased the translation of the downstream ORF. This observation implies that ribosomes reinitiate after termination, possibly after migrating in a 3' to 5' direction through the overlap region of the mRNA. Similar results were obtained with a capped polymerase II transcript, indicating that the translation of polymerase II and polymerase III transcripts occurs through similar mechanisms.

Production of IL-18 (IFN-gamma-inducing factor) messenger RNA and functional protein by murine keratinocytes.

Recently, the novel cytokine IL-18 (IFN-gamma-inducing factor) has been described as a growth and differentiation factor for Th1 cells. Epidermal keratinocytes (KC) are known to direct T cell education by production of cytokines. Therefore, expression of IL-18 was sought in KC. Epidermal RNA was analyzed following stimulation with contact sensitizers or controls for IL-18 mRNA expression by semiquantitative reverse transcription-PCR. Constitutive expression of IL-18 mRNA was low in untreated epidermal cells (EC), but early up-regulation of IL-18 mRNA signals was detected following application of a contact allergen in vivo. The peak strength of IL-18 signals was observed within 4 to 6 h following stimulation with an allergen. Application of an irritant (benzalconiumchloride) or solvents did not result in increased signal strength. To determine the cellular origin of IL-18 mRNA in EC, depletion experiments were performed. IL-18 signals were not affected by depletion with anti-CD3 (T cells) or anti-MHC class II mAb-coupled beads identifying KC as a major source of IL-18. These results were confirmed by analysis of mRNA derived from the KC cell line PAM 212. Strong IL-18 signals could be detected by reverse transcription-PCR. To delineate whether IL-18 protein was produced by EC/KC, a sandwich ELISA was used to assay for IL-18 production. Supernatants from allergen-stimulated EC and KC showed production of IL-18 protein. To confirm that IL-18 protein was functional, EC or KC supernatants were tested for their ability to induce IFN-gamma production. Significant amounts of IFN-gamma were detected in supernatants of allergen-treated cells. In aggregate, our data indicate that murine KC are a source of both IL-18 mRNA and functional protein.