Molecular cloning of murine ICA69: diabetes-prone mice recognize the human autoimmune-epitope, Tep69, conserved in splice variants from both species.

The islet cell antigen ICA69 is an autoimmune target in most patients with insulin-dependent diabetes. Understanding its role in diabetic autoimmunity would be facilitated by an animal model. We therefore cloned mouse ICA69. The different splice variants now identified conserve Tep69, the single T cell epitope recognized by patient T cells. We show that diabetes-prone NOD mice had Tep69-specific, autoreactive T cell repertoires and thus provide a relevant model for the study of ICA69's role in diabetic autoimmunity.

Genomic organization and transcript analysis of ICAp69, a target antigen in diabetic autoimmunity.

Islet cell antigen p69 (ICAp69) is a target self-antigen in autoimmune (insulin-dependent) diabetes mellitus. Distributed over more than 100 kb on chromosome 6 (6{A1-A2}), the single murine genomic locus contains 14 coding exons, 39-271 bp in length. The identified human and mouse intron-exon junctions are identical, with intron sizes ranging from 94 bp to 24 kb and with conserved flanking region intron sequences. cDNA cloning identified alternatively spliced ICAp69 mRNA transcripts. The predominating alpha-transcripts lack exon 4, while beta-transcripts include this exon, which codes translation termination in all reading frames and a truncated molecule following in vitro expression. gamma-Transcripts show splice removal of exons 8-12, while delta-transcripts exclude exon 11. Transcripts use alternative polyadenylation signals including a less frequent ATTAAA sequence. 5'-Untranslated cDNA and genomic sequencing and long PCR analysis suggest the presence of more noncoding exons. All splice variants encode the conserved T-cell epitope (in exon 2) recognized by autoreactive T cells in diabetic children and diabetes-prone NOD mice.

Role of Epstein-Barr virus in fine-needle aspirates of metastatic neck nodes in the diagnosis of nasopharyngeal carcinoma.

BACKGROUND: The patient with nasopharyngeal carcinoma (NPC) frequently is initially seen with regional node dissemination. Preliminary investigations suggest that the presence of Epstein-Barr virus (EBV) genomes in neck metastases from an occult primary may be diagnostic and predictive of NPC. The goal of this study was to test this proposition. METHODS: The polymerase chain reaction (PCR) was used to detect the presence of EBV DNA in fine-needle aspirate (FNA) samples obtained from malignant neck nodes. Control samples were obtained from other locations in the head and neck. PATIENTS: The patients in this study were evaluated at the Toronto Princess Margaret Hospital, a province-wide tertiary-care cancer treatment center. Of the 23 patients evaluated with malignant neck masses, 6 had NPC, 5 patients had metastatic squamous cell carcinoma of an unknown primary, and 12 patients served as controls with other known head and neck carcinomas. One of the patients initially diagnosed as an unknown primary later demonstrated NPC. FNA specimens were also obtained from 24 normal parotid, submandibular, or thyroid glands for comparison. RESULTS: In the samples with sufficient DNA for analysis, EBV was detected in 5 of 5 neck nodes from patients with known NPC. EBV was also detected in the neck node of a patient who went on to develop NPC and in a cervical node from 1 of 2 patients in whom the primary tumor remained unknown. None of the evaluable control neck nodes of FNA controls from other sites demonstrated EBV. CONCLUSIONS: These results demonstrate the utility of NPC-diagnostic EBV gene amplification in FNA samples of neck metastases and suggest that the presence of the EBV genome in FNA samples of neck nodes is predictive of the presence of NPC.

A majority of inverted sinonasal papillomas carries Epstein-Barr virus genomes.

BACKGROUND: The relationship between sinonasal inverted papilloma (IP) and various strains of human papilloma virus (HPV) has been examined previously. Yet there is little consensus regarding the incidence or role of HPV in IP. The possible role of Epstein-Barr virus (EBV), which, like HPV, is a DNA virus linked to human lymphoid and epithelial malignancies, was investigated. METHODS: The polymerase chain reaction (PCR) was used to detect EBV genomic sequences in surgical specimens of IP, in benign nasal polyps, and various control tissues. The IP specimens were similarly examined for the presence of HPV types 6, 11, 16, and 18. RESULTS: EBV DNA was found in 13 of 20 IP specimens (65%) and none of the 10 control tissues. Nine of the 20 specimens contained HPV DNA, and 5 of 20 specimens contained both EBV and HPV. CONCLUSIONS: These results imply a previously unsuspected role for Epstein-Barr virus in the pathogenesis of sinonasal inverted papilloma.

T cell activation and anergy to islet cell antigen in type I diabetes.

Early exposure to cow milk proteins was linked to the development of type I diabetes by consistent epidemiology, and by feeding and tolerization studies in diabetes-prone rodents. Dietary BSA was suggested as the culprit because patients and relevant rodents have elevated anti-BSA Abs that precipitate the recently cloned protein, p69, from beta cell lysates. A total of 68 of 78 children with recent onset diabetes had BSA-reactive T cells at the time of diagnosis. Here we 1) map the fine specificity of these T cells, 2) delineate a homologous peptide sequence near the N-terminus of p69, and 3) demonstrate T cell recognition of this p69 sequence (T cell epitope p69, Tep69) by patient T cells. The Tep69 sequence is conserved in p69 of patients and diabetes-prone rodents. Whereas BSA triggers T cell proliferation, recombinant p69 and a synthetic Tep69 peptide induce early stages of T cell activation (IL-2R transcription) but insufficient IL-2 production and thus anergy. Exogenous IL-2 overrides anergy and allows proliferative expansion of p69-responsive T cells. In mixing experiments, p69 and Tep69 peptide prevented proliferative responses to BSA even at 100-fold smaller concentrations. These findings imply that high-affinity self-peptide triggers anergy, whereas low-affinity mimicry Ag triggers proliferative expansion of these T cells. This implies a disease model in which mimicry Ag would rescue autoreactive cells from ablation by self-Ag.

Cloning of human and rat p69 cDNA, a candidate autoimmune target in type 1 diabetes.

Triggering of autoimmunity in insulin-dependent diabetes was linked to dietary bovine serum albumin (BSA). Anti-BSA antibodies from diabetes-prone rats precipitate a protein, p69, from islet cell lysates. We have used these antibodies to identify rat p69 cDNAs. Human p69 cDNA was identified by crosshybridization. The p69 coding regions show 87% nucleotide and 89% amino acid homology. Recombinant p69 is recognized by autoantibody and T cells from diabetic children.

Detection of viral sequences by internally calibrated gene amplification.

Inherent pitfalls of the polymerase chain reaction (PCR) can become serious difficulties when transferring research applications to high-volume routine procedures such as biofermentation process control and clinical diagnostics. Difficulties include 1) the danger of accidental sample contamination with positive control templates; 2) variable amplification due to positional effects in thermocycler blocks and unequal primer efficiency for sense/anti-sense strands; and 3) the need for reliable controls, which provide confidence for reporting negative reactions. Using the PCR detection system for Epstein-Barr virus as a model, we have developed a quick process to generate mutant internal co-amplification templates. These can be used for titration of amplification sensitivity. More importantly, single tube co-amplification without titrations allows determination of the minimum sensitivity achieved in each individual reaction; critical information when reporting negative diagnostic results. Mutant and native fragments are easy to distinguish by size, and sample cross contamination can be readily identified. The system should be easily adaptable to gene amplification procedures, which aim to routinely detect the presence of a given gene fragment in a controlled fashion.

Expression of IgE from a nonrearranged epsilon locus in cloned B-lymphoblastoid cells that also express IgM.

During development, B lymphocytes have the ability to switch from synthesis of IgM to immunoglobulins of another isotype such as IgG, IgA, or IgE. This class switching mechanism has been shown to involve DNA rearrangement and concomitant deletion of intervening CH genes. In our report, an EBV-transformed B lymphoblastoid cloned cell line is described that simultaneously expressed and secreted both IgM and IgE. DNA analysis showed the (nonproductive) rearrangement of one allele to gamma and (productive) rearrangement of the other allele to mu. Germ-line arrangement of the C epsilon gene was preserved on both alleles.

EBV utilizes a unique activation pathway for the transformation of human B cells.

EBV growth-transforms primate B lymphocytes and directly causes mono/multiclonal B cell lymphomas in vulnerable hosts. In this report we demonstrate that the degree of B cell transformability is not quantitatively determined at the level of either the saturable, transformation-prerequisite virus receptors or of the actual viral cell entry process. Instead, post-receptor binding events [Na+/H+ exchange, Ca2+ flux, tyrosine phosphorylation of two proteins (55-60/130-140 kd)] were identified as critical determinants of transformability. The presence of competent virus in transformable cells was per se insufficient for transformation: blockade of Ca2+ fluxes (or the antiport) generates virus-loaded cells that express viral genes but remain untransformed. Delayed induction by ionomycin of appropriately sized Ca2+ fluxes ([Ca2+]i greater than 180 less than 400 nM) re-starts transformation processes in EGTA-blocked, virus-loaded cells, perhaps providing a model for the study of virus re-activation. Overall, EBV induces unique cellular activation events different from non-oncogenic lymphocyte mitogens/activators, and, given the oncogenic potential of transformed cells in susceptible hosts, we hypothesize that these events describe a novel oncogenic transformation pathway.

Properties and heterogeneity of human fetal pre-B cells transformed by EBV.

A series of 75 EBV-transformed pre-B and B-cell lines from fetal bone marrow at 14 to 18 wk of gestation was cloned for phenotypic, functional and molecular genetic studies. B-cell type volume regulation in response to hypotonic stress, low level CD9 (BA2), and, perhaps biased by our use of EBV, functional EBV receptors with expression of CD21 (B2) determinants characterized the most immature cells detected. These "B-progenitors" contained EBV genomes, maintained Ig H and L chain (as well as TCR) constant region genes in germ-line configuration and did not express other B, T, or myeloid lineage-associated surface markers including CD20 and MHC class II determinants. Such cells may represent B progenitor cells preceding classical pre-B-lymphocytes in pathways of B cell differentiation. Reminiscent of Abelson virus-induced transformation of immature murine B cells, EBV transformability together with the above properties may be the earliest markers of B lineage commitment in man. The expression of MHC class II Ag, CD20, C mu-H and then L chain rearrangements and expression followed in less immature pre-B lymphocytes and permitted a classification of lines into discrete subgroups of increasing maturity. The physical organization of the H chain locus in a given line was a stable characteristic. However, fetal pre-B cell lines showed considerable intraclonal heterogeneity with respect to H chain gene expression and that of differentiation markers such as CD20. Subcloning experiments indicated that this heterogeneity reflected clonally stable expression patterns distributed among subclones in an all-or-none fashion. The induction, by IL-6, of L chain expression in some but not all of these clones was linked to the presence of C mu transcription products, consistent with a possible regulatory role of mu protein in L chain rearrangement and expression. Although pre-B cell differentiation likely follows inherent programming, external signals seem able to hasten development along prescribed, hierarchical differentiation pathways.

Concerted generation of Ig isotype diversity in human fetal bone marrow.

The human fetal bone marrow B cell compartment of 14- to 21-wk gestational age was examined phenotypically and with respect to Ig H chain commitment and diversity. A dramatic expansion of fetal marrow B cell pools at 16- to 18-wk gestational age characterizes a rapid and concerted chain of differentiation events. Transiently up to 1/4 of nucleated marrow cells are CD20+/CD21+ cells which begin to express surface Ig other than IgM. Limiting dilution analysis of EBV-infected marrow cells delineated a virtually exclusive commitment to IgM production until 15 wk and the absolute and relative number of these cells were small (approximately 5% of comparable adult values). In parallel to the rapid increase in total B cell pools size, cells committed and able to secrete any of the five Ig isotypes are generated by 16-wk gestational age and by 18 wk the frequencies of these cells rapidly reach levels typical for adult peripheral tissue such as blood or lymph node. Fetal L chain diversity always anticipated that observed in adult serum. In addition to rising pool sizes and diverse IgH expression, EBV transformability is a major variable during this period of B cell development with up to 2/3 of B lineage cells transformable, about half of which are pre-B cells. By 21-wk gestational age transformable pre-B cells have disappeared and (as in adult tissue) approximately 10 to 20% of CD20+ cells are transformable. The rapid, concerted expression of full H chain diversity during a narrow period in fetal development is unique to marrow and implies a lymphopoietic process in a privileged site rather than an immunologic differentiation event. During this event, the relative proportions between the different IgH classes expressed, resembled that found in adult tissue, perhaps suggesting that B cell inherent programming rather than only antigenic forces determine heavy chain choice. The staggered expression, early in postnatal life, of IgH regions 3' of the C mu locus may reflect regulatory functions rather than inherent immaturity of the B lineage.