Clonal variation in interferon response determines the outcome of oncolytic virotherapy in mouse CT26 colon carcinoma model.

In our earlier studies, Semliki Forest virus vector VA7 completely eliminated type I interferon (IFN-I)-unresponsive human U87-luc glioma xenografts, whereas interferon-responsive mouse gliomas proved refractory. Here, we describe in two clones of CT26 murine colon carcinoma, opposed patterns of IFN-I responsiveness and sensitivity to VA7. Both CT26WT and CT26LacZ clones secreted biologically active interferon in vitro upon virus infection but only CT26WT cells were protected. Focal infection of CT26WT cultures was self-limiting but could be rescued using IFN-I pathway inhibitor Ruxolitinib or antibody against IFNß. Whole transcriptome sequencing (RNA-Seq) and protein expression analysis revealed that CT26WT cells constitutively expressed 56 different genes associated with pattern recognition and IFN-I signaling pathways, spanning two reported anti-RNA virus gene signatures and 22 genes with reported anti-alphaviral activity. Whereas CT26WT tumors were strictly virus-resistant in vivo, infection of CT26LacZ tumors resulted in complete tumor eradication in both immunocompetent and severe combined immune deficient mice. In double-flank transplantation experiments, CT26WT tumors grew despite successful eradication of CT26LacZ tumors from the contralateral flank. Tumor growth progressed uninhibited also when CT26LacZ inoculums contained only a small fraction of CT26WT cells, demonstrating dominance of IFN responsiveness when heterogeneous tumors are targeted with interferon-sensitive oncolytic viruses.

Sphingosine kinase as an oncogene: autocrine sphingosine 1-phosphate modulates ML-1 thyroid carcinoma cell migration by a mechanism dependent on protein kinase C-alpha and ERK1/2.

Sphingosine 1-phosphate (S1P) induces migration of the human thyroid follicular carcinoma cell line ML-1 by activation of S1P(1) and S1P(3) receptors, G(i) proteins, and the phosphatidylinositol 3-kinase-Akt pathway. Because sphingosine kinase isoform 1 (SK) recently has been implicated as an oncogene in various cancer cell systems, we investigated the functions of SK in the migration, proliferation and adhesion of the ML-1 cell line. SK overexpressing ML-1 cells show an enhanced secretion of S1P, which can be attenuated, by inhibiting SK activity and a multidrug-resistant transport protein (ATP-binding cassette transporter). Furthermore, overexpression of SK enhances serum-induced migration of ML-1 cells, which can be attenuated by blocking ATP-binding cassette transporter and SK, suggesting that the migration is mediated by autocrine signaling through secretion of S1P. Inhibition of protein kinase C alpha, with both small interfering RNA (siRNA) and small molecular inhibitors attenuates migration in SK overexpressing cells. In addition, SK-overexpressing cells show an impaired adhesion, slower cell growth, and an up-regulation of ERK1/2 phosphorylation, as compared with cells expressing a dominant-negative SK. Taken together, we present evidence suggesting that SK enhances migration of ML-1 cells by an autocrine mechanism and that the S1P-evoked migration is dependent on protein kinase C alpha, ERK1/2, and SK.

Antibody responses to deamidated gliadin peptide show high specificity and parallel antibodies to tissue transglutaminase in developing coeliac disease.

Coeliac disease (CD) is an enteropathy induced in genetically susceptible individuals by gluten components, gliadin, hordein and secalin, polypeptides present in cereals such as wheat, barley and rye, respectively. Although the disease starts as intolerance to gliadins, antibodies to tissue transglutaminase (tTG) in the gut epithelium are characteristic of the disease. Whereas serum autoantibodies against tTG (tTGA) are highly specific for CD, antibodies to gliadin are less informative as they can also be detected in other enteropathies, and even in healthy individuals. However, it was shown recently that antibodies to certain gliadin peptides occur with high specificity in CD patient sera. We developed a solid phase lanthanide-based immunofluorometric assay for simultaneous detection of serum IgA and IgG antibodies to a synthetic peptide derived from gamma gliadin of wheat comprising amino acids 86-103. Three glutamine residues of this native 18-mer peptide were replaced by glutamic acids and the peptide was biotinylated. Sera from 87 individuals who had undergone duodenal biopsy and were diagnosed with CD and from 81 healthy individuals were analysed for the presence of both IgA and IgG anti-gliadin peptide antibodies. The performance of the peptide AGA assay was excellent, showing a specificity and sensitivity of 90% and 92% for IgA, and 98% and 75% for IgG, respectively. The corresponding values for conventional anti-gliadin antibody (AGA) enzyme-linked immunosorbent assay (ELISA) tests were 72% specificity and 87% sensitivity for IgA, and 64% specificity and 78% sensitivity for IgG. In a prospective study, almost all the tTGA-positive sera drawn from children who later developed CD were also positive for gliadin peptide antibodies.

Affinity maturation of immunoglobulin A anti-tissue transglutaminase autoantibodies during development of coeliac disease.

Coeliac disease (CD) is an immune-mediated enteropathy triggered by ingestion of wheat gluten and related cereals in genetically predisposed individuals. Circulating immunoglobulin A (IgA) class autoantibodies against tissue transglutaminase (IgA-TGA) are highly specific and sensitive serological markers for CD, which is ultimately confirmed by duodenal biopsy. Although CD is considered a life-long disorder, transient or fluctuating IgA-TGA seropositivity has been observed in asymptomatic individuals on a gluten-containing diet. We set out to explore possible differences in the maturation of IgA-TGA avidity between individuals progressing to CD and subjects remaining healthy despite occasional expression of autoantibodies. We developed a time-resolved fluorometric IgA-TGA assay based on human recombinant tissue transglutaminase (tTG), and further modified the method to also measure urea-dependent avidity of the autoantibodies. We measured the autoantibody titres and avidities of sequential serum samples from 10 children developing CD and 10 children presenting transient or fluctuating autoantibodies. Both the initial titres at seroconversion and peak values of transient/fluctuating IgA-TGA were significantly lower than corresponding autoantibody titres in samples drawn from individuals with progressing CD (P = 0.004 and P = 0.0002, respectively). However, there were no statistically significant differences in the initial or peak avidity index values between the two groups of children. The avidity index values increased during the follow-up period (P = 0.013 for both groups) with no significant difference in the rate of avidity maturation between children with transient/fluctuating IgA-TGA and children developing CD. According to our results, high autoantibody titres have a higher predictive value than avidity maturation of TGA-IgA in screening for CD.

Treatment of experimental autoimmune encephalomyelitis with a neurotropic alphavirus vector expressing tissue inhibitor of metalloproteinase-2.

Prompted by our recent observations of increased MMP-8 and MMP-9 with simultaneous downregulation of tissue inhibitor of metalloproteinase-2 (TIMP-2) and TIMP-3 mRNA levels in the central nervous system (CNS) of mice with severe experimental autoimmune encephalomyelitis (EAE), we used Semliki Forest virus (SFV) to transfer and express recombinant murine TIMP-1-3 genes in the CNS. TIMP-1, TIMP-2 and TIMP-3 expression was confirmed in cultured cells and in the CNS of infected mice. Following intraperitoneal infection with 10(6) plaque-forming units (PFU) of SFV-TIMP, focal TIMP protein expression was achieved throughout the brain. Although already treatment with empty vector inhibited development of EAE to some extent, the expression of TIMP-2 by the virus significantly enhanced the inhibition. TIMP-3-administered mice also had lower disease grade, but the inhibition was not statistically significant. In contrast, SFV-TIMP-1 had no effect, similar to co-infection with TIMP-2 and TIMP-3. We found TIMP-2 expression also by non-infected CNS-resident cells surrounding the virus-positive areas, suggesting a bystander TIMP-2 induction. These data strengthen the view that matrix metalloproteinases are involved in the pathogenesis of EAE and provide clear evidence that virus-mediated delivery of their protein inhibitors can be effective in preventing the clinical disease. TIMPs might be candidates for novel treatment regimens in CNS autoimmune disorders, such as multiple sclerosis.

Recombination in uveitis-causing enterovirus strains.

The complete nucleotide sequences of three human echovirus (EV) 11 strains and one EV19 strain, all of which caused outbreaks of enterovirus uveitis (EU), a new infant disease first identified in 1980 in Siberia, were determined. One EV11 strain which caused an outbreak of sepsis-like disease in Hungary was also sequenced. All four EV11 strains were mosaic recombinants of the prototype EV11 strain Gregory, with their non-structural coding regions and 5' NTRs being more similar to other prototype enteroviruses (EV1, EV9). However, this finding is probably a feature of all circulating enterovirus strains and may not be related to their altered virulence. A full genome sequence comparison of the three subtypes of EU-causing strains excludes the role of recent recombination in their emergence, and points to their independent emergence.

Molecular epidemiology of enteroviruses causing uveitis and multisystem hemorrhagic disease of infants.

We studied molecular epidemiology of highly virulent echovirus 11 and 19 strains that were isolated during five outbreaks of enterovirus uveitis (EU) in Siberia in 1980-1989, and three outbreaks of multisystem hemorrhagic disease of infants (MHD) in 1988-1991. Three genome regions, 5'NTR, VP1-2A junction, and a fragment of 3D polymerase, were analyzed. Phylogenetic grouping in the VP1-2A region correlated with serotyping results. All studied EV11 and EV19 strains, including the prototype EV11 and EV19, formed a major phylogenetic group in VP1-2A region. Within that group, several EV11 isolates from EU and MHD outbreaks formed a distinct cluster in VP1-2A and 5' NTR genome regions, designated EV11/B. All strains of this cluster possessed high virulence for monkeys compared with the prototype echoviruses. Subgrouping within this cluster correlated with year of virus isolation, not with the disease the viruses caused in infants (EU or MHD).

Up-regulation of MMP-8 and MMP-9 activity in the BALB/c mouse spinal cord correlates with the severity of experimental autoimmune encephalomyelitis.

Induction of EAE can be inhibited or repressed by administration of soluble metalloproteinase inhibitors. We studied the matrix metalloproteinase (MMP) and their tissue inhibitor (TIMP) expression pattern in experimental autoimmune encephalomyelitis (EAE) of the resistant Th2 prone BALB/c mouse, where the disease can be induced with ultrasound-emulsified antigen/adjuvant (son-ag), but not with conventional technique (syr-ag). We found highly elevated expression of MMP-8 (neutrophil collagenase) mRNA and protein in diseased son-ag challenged mice, colocalizing to neutrophil infiltrates found in brain and extensively in the spinal cord submeningeal space. MMP-8 expression has not been found previously in sensitive mouse strains. The infiltrates stained positive also for MMP-9 protein, and brain homogenates from corresponding mice showed MMP-9 activity during overt disease (days 12-16 post-immunization). TIMP-1 gene expression could be detected in CNS samples from diseased son-ag challenged mice but not in syr-ag or control mice, and the TIMP-1 protein colocalized with GFAP-staining. In contrast, in syr-ag mice both TIMP-2 and TIMP-3 gene expression in the spinal cords was elevated. The results show that sonication, but not extrusion, creates an adjuvant formula potent in activating the matrix metalloproteinase cascade similar to sensitive mouse strains, strongly implicating their role in EAE induction in this Th2 prone strain. The study provides the basis for establishment of MMP-specific therapy in this model.

Dual-colour imaging of membrane protein targeting directed by poa semilatent virus movement protein TGBp3 in plant and mammalian cells.

The movement function of poa semilatent hordeivirus (PSLV) is mediated by the triple gene block (TGB) proteins, of which two, TGBp2 and TGBp3, are membrane proteins. TGBp3 is localized to peripheral bodies in the vicinity of the plasma membrane and is able to re-direct TGBp2 from the endoplasmic reticulum (ER) to the peripheral bodies. For imaging of TGBp3-mediated protein targeting, PSLV TGBp3 tagged with a red fluorescent protein (DsRed) was used. Coexpression of DsRed-TGBp3 with GFP targeted to the ER lumen (ER-GFP) demonstrated that ER-GFP was contained in typical ER structures and peripheral bodies formed by TGBp3 protein, suggesting an ER origin for these bodies. In transient coexpression with viral membrane proteins tagged with GFP, DsRed-TGBp3 directed to the peripheral bodies the homologous TGBp2 protein and two unrelated membrane proteins, the 6 kDa movement protein of beet yellows closterovirus and the putative movement protein encoded by the genome component 4 of faba bean necrotic yellows nanovirus. However, coexpression of TGBp3 with GFP derivatives targeted to the ER membranes by artificial hydrophobic tail sequences suggested that targeting to the ER membranes per se was not sufficient for TGBp3-directed protein trafficking to peripheral bodies. TGBp3-induced targeting of TGBp2 also occurred in mammalian cells, indicating the universal nature of the protein trafficking signals and the cotargeting mechanism.

Coordinated induction of extracellular proteolysis systems during experimental autoimmune encephalomyelitis in mice.

Plasminogen activators (PAs) and matrix metalloproteinases (MMPs) are considered to play an important role in the pathogenesis of multiple sclerosis. Experimental autoimmune encephalomyelitis (EAE) is widely used as an animal model of multiple sclerosis. Whereas several studies have addressed the expression of various MMPs and their inhibitors in the pathogenesis of EAE, the expression of the molecules of the PA system during EAE has not been reported previously. The present study was undertaken to investigate the expression of the molecules of the PA system (tPA, uPA, PAI-1, uPAR, LRP), as well as several members of the MMP family and their inhibitors in the course of actively induced EAE in BALB/c mice. During clinical EAE, the PA system was up-regulated in the central nervous system at several levels. Induction of expression of tPA and PAI-1 transcripts was detected in activated astrocytes in the white matter. Inflammatory cells expressed uPA receptor, uPAR. In situ zymography demonstrated the presence of increased tPA and uPA activities in the areas of the inflammatory damage. Accumulation of fibrin, fibronectin, and vitronectin immunoreactivity was seen in perivascular matrices of symptomatic animals. In addition, transcription of MT1-MMP and metalloelastase (in inflammatory cells), and TIMP-1 (in activated astrocytes) was induced during EAE. Increased gelatinolytic activity was detected at the sites of inflammatory cell accumulation by in situ zymography of fluorescently labeled gelatin; substrate gel zymography identified the up-regulated gelatinolytic activity as gelatinase B. Overall, our study demonstrates concurrent induction of PA and MMP systems during active EAE, supporting further the concept that the neuroinflammatory damage in EAE involves altered balance between multiple extracellular proteases and their inhibitors.

Responses of coxsackievirus B4-specific T-cell lines to 2C protein-characterization of epitopes with special reference to the GAD65 homology region.

Coxsackie B viruses (CBV) have been indicated as environmental triggers initiating autoimmune destruction of insulin-producing pancreatic beta-cells, and molecular mimicry might be the mechanism. A prime candidate for inducing cross-reactive immune responses is a homology sequence, PEVKEK, found both in CBV4 2C protein and in GAD65. To characterize the CBV4-specific T-cell epitopes, overlapping peptides covering the 2C protein were synthesized and CBV4-specific T-cell lines were established from healthy and diabetic subjects. The T-cell epitopes were dependent on the HLA-DR genotype of the T-cell donor, but no difference between diabetic and healthy subjects could be detected. Peptide p4, which included the PEVKEK sequence, contained an HLA-DR1-restricted T-cell epitope. Three randomly selected CBV4-specific T-cell lines, which responded to peptide p4, failed to recognize GAD65 protein or GAD65 peptides containing the PEVKEK sequence. We conclude that the CBV4 2C protein is strongly immunogenic for T-cells and PEVKEK is included in a T-cell epitope. However, presentation of this epitope in the context of neutral HLA-DR1 allele does not support its role in pathogenesis of type 1 diabetes.

Cell membrane-associated measles virus components inhibit antigen processing.

Measles virus (MV)-induced immune suppression is an important reason for MV-associated mortality and morbidity. Despite numerous studies, the mechanisms of immune suppression still remain poorly defined. In the present study we analyzed the effect of MV components on the T-cell recognition of specific non-MV antigens. We demonstrated that even inactivated MV could inhibit the presentation of unprocessed protein antigen to specific T cells, whereas MV did not affect the responses of specific T cells to representative synthetic peptide epitopes derived from complex antigens. The inhibition was induced by MV-infected cell membranes. The kinetics of the MV-dependent inhibition suggested an impaired antigen processing in mononuclear cells as addition of MV-infected cell debris 4 h after the beginning of cell cultures no longer inhibited T-cell responsiveness.

A time-resolved fluoroimmunometric assay for the detection of microcystins, cyanobacterial peptide hepatotoxins.

An immunoassay based on the time-resolved fluorometry (TR-FIA) was developed for microcystins, cyanobacterial peptide hepatotoxins. The assay was performed in a competitive mode and it utilised the monoclonal antibodies raised against microcystin-LR, and a europium chelate of microcystin-LR as a competitive antigen. The sensitivity of the assay was 0.1microg/l. The detection method of TR-FIA was compared to a commercially available kit based on the enzyme-linked immunosorbent assay (ELISA). The same level of sensitivity could be obtained with TR-FIA (in a non-optimised system). The simplified method of TR-FIA leads to a shorter analysis time.

Novel fusion proteins in the analysis of diabetes-associated autoantibodies to GAD65 and IA-2.

Assays to detect autoantibodies to glutamic acid decarboxylase (GAD65) and the protein tyrosine phosphatase-like molecule IA-2, which are both present in pancreatic islets, have been used in the diagnosis and prediction of type 1 diabetes. In this study a novel fusion protein combining the entire GAD65 molecule with the 40 kDa intracellular domain of IA-2 (GAD-IA-2) was constructed to detect autoantibodies to both antigens by one single assay. For the same purpose a truncated version of this fusion protein which contained the entire GAD65 linked to the 203 carboxy-terminal amino acids of IA-2 (GAD-dIA-2) was made. A panel of 34 diabetic sera which represented unequivocally positive or negative antibody responses to GAD65 and/or IA-2 as well as 20 serum samples from healthy controls were tested in a radioligand binding assay with the constructed fusion proteins as antigens. Nine of the samples from patients with type 1 diabetes reacted with GAD65 while being negative for IA-2. Six sera were positive for IA-2 only, 11 were double positive, and 8 negative for both antibodies using the standard in vitro transcription translation assay with single antigens. The full-length, as well as the truncated fusion protein detected all samples positive for antibodies either to GAD65 or IA-2 or both, except for one GAD65 antibody positive sample. All samples from healthy controls tested negative in all assays. We conclude that the principle of a combinatorial molecule where a fusion protein expresses both GAD65 and IA-2 epitopes is feasible, and such a fusion protein can be used instead of the single antigens to reduce time and costs of large-scale screening for clinical purposes.

Chemokine expression by central nervous system resident cells and infiltrating neutrophils during experimental autoimmune encephalomyelitis in the BALB/c mouse.

The active role of chemokines in the central nervous system (CNS) during the pathogenesis of experimental autoimmune encephalomyelitis (EAE) has been clearly established. In this study the expression pattern of several chemokines and cytokines was elucidated using reverse transcription-PCR and immunohistochemistry in a recently established EAE model of the BALB/c mouse that is characterized by CNS infiltration of polymorphonuclear neutrophils. Elevated mRNA levels of the chemokines MIP-1alpha, MIP-2 and MCP-1 were detected in the CNS of diseased mice, whereas no chemokine expression could be measured in asymptomatic mice. Activated astrocytes were shown to be the main source of MIP-1alpha and MIP-2 before and during cellular CNS infiltration. Among the infiltrating immune cells the neutrophils secreted MIP-1alpha and MCP-1. These results suggest involvement of ordered chemokine expression during the process of neutrophil attraction into the CNS, which may play an important role in the initiation and perpetuation of autoimmune CNS inflammation in the BALB/c mouse. This is the first EAE model to describe CNS expression of the C-X-C chemokine MIP-2, corresponding to an observed neutrophil accumulation in the CNS.

Effects of palmitoylation of replicase protein nsP1 on alphavirus infection.

The membrane-associated alphavirus RNA replication complex contains four virus-encoded subunits, the nonstructural proteins nsP1 to nsP4. Semliki Forest virus (SFV) nsP1 is hydrophobically modified by palmitoylation of cysteines 418 to 420. Here we show that Sindbis virus nsP1 is also palmitoylated on the same site (cysteine 420). When mutations preventing nsP1 palmitoylation were introduced into the genomes of these two alphaviruses, the mutant viruses remained viable and replicated to high titers, although their growth was slightly delayed. The subcellular distribution of palmitoylation-defective nsP1 was altered in the mutant: it no longer localized to filopodial extensions, and a fraction of it was soluble. The ultrastructure of the alphavirus replication sites appeared normal, and the localization of the other nonstructural proteins was unaltered in the mutants. In both wild-type- and mutant-virus-infected cells, SFV nsP3 and nsP4 could be extracted from membranes only by alkaline solutions whereas the nsP2-membrane association was looser. Thus, the membrane binding properties of the alphavirus RNA replication complex were not determined by the palmitoylation of nsP1. The nsP1 palmitoylation-defective alphaviruses produced normal plaques in several cell types, but failed to give rise to plaques in HeLa cells, although they induced normal apoptosis of these cells. The SFV mutant was apathogenic in mice: it caused blood viremia, but no infectious virus was detected in the brain.

Replicase complex genes of Semliki Forest virus confer lethal neurovirulence.

Semliki Forest virus (SFV) is a mosquito-transmitted pathogen of small rodents, and infection of adult mice with SFV4, a neurovirulent strain of SFV, leads to lethal encephalitis in a few days, whereas mice infected with the avirulent A7(74) strain remain asymptomatic. In adult neurons, A7(74) is unable to form virions and hence does not reach a critical threshold of neuronal damage. To elucidate the molecular mechanisms of neurovirulence, we have cloned and sequenced the entire 11,758-nucleotide genome of A7(74) and compared it to the highly neurovirulent SFV4 virus. We found several sequence differences and sought to localize determinants conferring the neuropathogenicity by using a panel of chimeras between SFV4 and a cloned recombinant, rA774. We first localized virulence determinants in the nonstructural region by showing that rA774 structural genes combined with the SFV4 nonstructural genome produced a highly virulent virus, while a reciprocal recombinant was asymptomatic. In addition to several amino acid mutations in the nonstructural region, the nsp3 gene of rA774 displayed an opal termination codon and an in-frame 21-nucleotide deletion close to the nsp4 junction. Replacement in rA774 of the entire nsp3 gene with that of SFV4 reconstituted the virulent phenotype, whereas an arginine at the opal position significantly increased virulence, leading to clinical symptoms in mice. Completion of the nsp3 deletion in rA774 did not increase virulence. We conclude that the opal codon and amino acid mutations other than the deleted residues are mainly responsible for the attenuation of A7(74) and that the attenuating determinants reside entirely in the nonstructural region.

Autoimmunity to glutamic acid decarboxylase in patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED).

Antibodies to glutamic acid decarboxylase (GAD) occur frequently in patients with APECED, although clinical insulin-dependent diabetes mellitus (IDDM) is seen only in a subgroup of the patients. We studied the cellular immunity to GAD, antibodies to GAD and their association with the HLA DQB1 risk alleles for IDDM in patients with APECED. Proliferation responses to GAD were enhanced in the patients with APECED when compared with the control subjects (P = 0.004), but autoimmunity to GAD was not associated with IDDM in APECED. The levels of interferon-gamma (IFN-gamma) secreted by GAD-stimulated T cells were higher in the patients than in control subjects (P = 0. 001). A negative correlation (r = - 0.436, P = 0.03) existed between the antibody levels and the stimulation indices (SIs) to GAD. In 14 non-diabetic patients no difference in insulin secretion was observed in intravenous glucose tolerance test (IVGTT) between the patients with and without T cell reactivity to GAD. We conclude that cellular immunity to GAD detected as T cell proliferation response to GAD or IFN-gamma secretion by GAD-stimulated T cells was frequent in patients with APECED (69%) and was not restricted to the patients with clinically detectable beta-cell damage.

Enhancement of experimental autoimmune encephalomyelitis severity by ultrasound emulsification of antigen/adjuvant in distinct strains of mice.

Susceptibility to experimental autoimmune encephalomyelitis (EAE) is associated with the major histocompatibility complex (MHC) haplotype. In this study EAE could be induced in six out of ten mice of the resistant DBA/2 (H-2d) strain by ultrasound emulsified antigen/adjuvant, whereas none of the mice immunized with the conventional adjuvant developed the disease. Similar results were previously obtained for the MHC identical BALB/c mice. Further, while only few T cells were present in the central nervous systems (CNS) of the diseased DBA/2 mice, macrophages formed the majority of the infiltrates. In congenic BALB.B (H-2b) and BALB.K (H-2 k) mice, EAE could be induced with both sonicated and extruded antigen/adjuvant emulsion. The results indicate that the EAE resistance in mice carrying the H-2d MHC haplotype is dependent on the physical structure of the immunogen.

Production of the islet cell antigen ICA69 (p69) with baculovirus expression system: analysis with a solid-phase time-resolved fluorescence method of sera from patients with IDDM and rheumatoid arthritis.

Islet cell antigen 69 (ICA69), previously implicated as an autoantigen in autoimmune insulin-dependent diabetes mellitus (IDDM), was produced using baculovirus-mediated expression in Spodopterafrugiperda (Sf9) insect cells. In these cells the protein was effectively expressed and ICA69 carrying C-terminal histidine-hexapeptide could be efficiently purified using immobilized metal chelate affinity chromatography. Screening of patient and control sera using this protein as an antigen in time-resolved fluoroimmunoassay (TR-FIA) identified 4/50 of patients with IDDM and 6/73 of patients with rheumatoid arthritis (RA) to be positive for ICA69 antibodies. The number of positives did not differ significantly between patients and control subjects but the level of binding was higher in sera from RA patients compared to that of control sera (P = 0.003). The results show that some subjects have specific autoreactive antibodies against the ICA69 protein produced with recombinant technology.