Citations

Abstract Background: Transthyretin-associated hereditary amyloidosis (ATTR) is an inherited disease in which variants in the primary structure of transthyretin (TTR; prealbumin) lead to the extracellular polymerization of insoluble protein fibrils, causing organ failure and ultimately death when major organs are involved. We have developed an integrated approach to molecular diagnosis with initial analysis of intact plasma TTR by electrospray ionization mass spectrometry (MS) and referral of positive samples for DNA sequence analysis and real-time PCR to confirm the common Gly6Ser polymorphism. Methods: Samples from 6 patients previously diagnosed with ATTR and from 25 controls with (n = 15) or without (n = 10) polyneuropathy were analyzed in a blinded fashion for the presence of variant TTR. TTR protein was extracted with an immunoaffinity resin from 20 μL of archived plasma samples. The purified TTR was reduced with tris(2-carboxyethyl)phosphine and analyzed by MS. The appearance of two peaks (or a single peak shifted in mass indicative of a homozygous variant), including the wild-type mass of 13 761 Da, was indicative of the presence of a variant, and the individual was referred for DNA sequence analysis. Results: MS analysis of intact reduced TTR correctly identified each of six samples known to contain variant TTR. These results were corroborated by subsequent DNA sequence analysis. Additionally, all Gly6Ser polymorphisms were correctly called based on the +30 mass shift and an equal relative abundance of the +30 polymorphism relative to wild-type TTR. No false-positive results were seen. Conclusions: This referral method eliminates the necessity of sequencing most samples and allows screening for the familial forms of amyloidosis in a broad patient population in a timely fashion. This method correctly identified all previously known variants and also identified a novel variant, Val94Ala.

Collagen-induced arthritis (CIA) is an animal model of autoimmune inflammatory polyarthritis that has features similar to rheumatoid arthritis (RA). Much like RA, susceptibility to mouse CIA is influenced by the major histocompatibility complex (MHC), H-2, and restricted to the H-2q and H-2r haplotypes. Whereas the role of the H-2A molecule in susceptibility to CIA is well established, little is known about the role of H-2E molecule in the disease. In this study, we analyzed the effect of a transgenic E beta d molecule on CIA susceptibility in a recombinant mouse B10.RQB3, which expresses the CIA susceptible Aq genes and an Eak gene, but does not produce an E molecule since Ebq is nonfunctional. In the presence of an Ebd transgene, a viable E molecule is generated. Whereas B10.RQB3 were susceptible to CIA, B10.RQB3-E beta d+ showed a dramatic reduction in the incidence of arthritis as well as a decrease in the level of anti-mouse and anti-bovine CII antibodies in their serum. No clear cut differences in the expression of T cell receptor (TCR) V beta was observed between E beta d+ and E beta d- transgenic mice. Mechanisms underlying the protective effect of E beta d transgenic molecule on CIA may shed light on how HLA-DR molecules influence human RA.

HLA-B27 transgenic mice in the context of various H-2 haplotypes were produced. A high expression of the HLA-B27 antigen was observed in mice homozygous for H-2b, H-2f, H-2s, H-2p, H-2r, and H-2k haplotypes. Mice of the H-2v haplotype expressed HLA-B27 at an intermediate level. Expression of HLA-B27 was minimal in mice of the H-2q and H-2d haplotypes. This was observed both on the B10 background and in DBA/2 or BALB/c mice. Only minimal expression of HLA-B27 could be detected in B10.PL (KuDd) or B10.RKDB (KkSkDdLb) mice, indicating that the low level of HLA-B27 expression maps to the H-2D gene or a very closely linked gene. Integration and transcription of the HLA-B27 gene does not appear to be different between high-expressing haplotypes and low-expressing haplotypes as determined by Southern and Northern blot analysis. However, expression of HLA-B27 on the cell surface correlated with the amount of HLA-B27 and beta 2M that could be immunoprecipitated with an anti-B27 antibody. Therefore, the association of the B27 heavy chain with endogenous beta 2M and subsequent expression on the cell surface are disrupted in mice with some class I H-2D genes. Possible mechanisms that might contribute to this defect in assembly, transport, and expression of class I molecules are discussed.

We have found cell surface expression of an E alpha molecule in recombinant and transgenic mouse strains lacking an E beta molecule. Flow cytometry has shown low level expression of E alpha in B10.RQB3 (I-AqEk alpha) and B10.RFB2 (I-AfEk alpha) mice. We have also found that B10.Q (H-2q) mice can express the Ek alpha transgene. Since these strains do not have functional E beta chains, we propose that the E alpha A beta hybrid dimers are formed in low numbers and can be picked up by FACS analysis. So far we have not been able to identify these hybrid molecules by cytotoxicity or immunoprecipitation. The E alpha/A beta molecule can function in vivo during thymic selection in the clonal deletion of two V beta TCR subsets, V beta 11 and V beta 6, which have been shown to interact with the intact I-E molecule.

We have identified an inbred strain of mouse, RIII S/J (H-2r), that has the largest known deletion of the TCR V beta genes by screening with mAb and TCR V beta specific probes. Upon screening of PBL with mAb F23.1, which is specific for V beta 8 TCR, RIII S/J was found to be negative. On further screening with mAb KJ 23a, which is specific for V beta 17a TCR, RIII S/J was completely negative. We next tested RIII S/J with mAb 44-22-1, which is specific for V beta 6 TCR, and found it also to be negative. The (B10 X RIII)F1 mice showed a 50% expression of V beta 6 gene, indicating a genomic rather than a clonal deletion. mAb KJ25, detecting V beta 3, was positive in RIII S/J, denoting the downstream boundary for the deletion. Southern blot analysis of liver DNA using TCR V beta-specific probes confirmed the deletion of V beta 8 gene subfamily and V beta 5 gene subfamily, along with V beta 9, V beta 11, V beta 12, and V beta 13 genes similar to the known TCR V beta deletion mutants (SWR, SJL, C57L, and C57Br). In addition, RIII S/J is missing V beta 6, V beta 15, and V beta 17 genes. Our mapping of the deletion indicates that RIII S/J has lost approximately 130 kb of V beta chromosome and with it 13 V beta genes out of the known 21 V beta genes of the TCR. The deletion is marked by the presence of V beta 10 gene upstream and V beta 3 gene downstream.

Arthritis was induced by immunization of type II collagen in adjuvant in mice from H-2q-bearing crosses between SWR (H-2q/q) and B10 (H-2b/b mice), two strains known to be resistant to collagen-induced arthritis (CIA). The resistance of B10 is known to be due to its MHC haplotype, but it was postulated that the resistance of SWR mice which expresses the susceptible MHC haplotype could be due to the deletion of close to 50% of the V beta genes of the T cell receptor (TCR) in them. 17% of the F1 hybrids, 33% of the SWR backcrosses, 68% of the B10 backcrosses, and 52% of the F2 hybrids developed arthritis on follow-up to 5 mo after primary immunization with collagen. There was no significant difference in anti-type II collagen antibody titers between the arthritic and nonarthritic mice in each of these crosses. The segregation of the TCR genes with arthritis was determined in the F2 population by typing with F23.1 mAb that reacts with T cells using V beta 8 subfamily genes in their TCRs. SWR mice are F23.1- as V beta 8 genes are deleted in them. All six of arthritic mice homozygous for H-2q, and thus with an H-2 haplotype similar to SWR mice, expressed the F23.1 marker. These studies indicate that for complete susceptibility to collagen-induced arthritis, not only is a susceptible MHC haplotype (H-2q) important, but possibly also the presence of a subset of T cells using certain specific V beta genes in their TCRs. Other background genes may, however, modulate the severity of arthritis.

To identify which polymorphic residues determine the allospecific antibody binding sites on A beta polypeptides, mutant Ak beta genes were constructed encoding single or multiple amino acids of the d allele at 14 polymorphic positions in the beta 1 domain. Cell lines expressing these genes were analyzed by quantitative immunofluorescence using 16 mAbs reactive to Ak beta or Ad beta. Substitution of d allele residues at positions 63 and 65-67 in the Ak beta polypeptide resulted in the loss of binding of all Ak beta-reactive antibodies and the gain of binding of most Ad beta-reactive antibodies. Two Ad beta-reactive mAbs bound to the mutant Ak beta polypeptide containing d allele-characteristic residue at position 40. In contrast, substitution of the other polymorphic residues in the NH2-terminal and COOH-terminal regions of the beta 1 domain did not alter antibody binding.

Human class I major histocompatibility complex allele HLA-B27 is associated with a group of human diseases called "spondyloarthropathies." Studies on transgenic rats expressing HLA-B27 and human beta 2-microglobulin have confirmed the role of HLA-B27 in disease pathogenesis. Here we report spontaneous inflammatory arthritis in HLA-B27 transgenic mice lacking beta 2-microglobulin (B27+ beta 2m-/-). In the absence of beta 2-microglobulin, B27+ beta 2m-/- animals do not express the HLA-B27 transgene on the cell surface and have a very low level of CD8+ T cells. Most of the B27+ beta 2m-/- male mice showed nail changes, hair loss, and swelling in paws, which leads to ankylosis. The symptoms occur only after the B27+ beta 2m-/- mice are transferred from the specific pathogen-free mouse colony. These results suggest that aberrant assembly, transport, and expression of the HLA-B27 molecule may predispose an individual for development of the disease when exposed to an appropriate environmental trigger.

Guinea pig eosinophil granules are characterized by the presence of a basic protein of low molecular weight which accounts for greater than 50% of granule protein. This protein, termed the major basic protein (MBP), readily aggregates and becomes insoluble, and the formation of aggregates is dependent on the establishment of disulfide bonds. Analysis of concentrated preparations of MBP often revealed a series of bands which were multiples of a monomeric unit with a mol wt of approximately 11,000. Analysis of reduced and alkylated MBP on a 10% agarose column equilibrated with 6 M guanidinium chloride revealed a single polypeptide chain with a mol wt of 10,800. Amino acid analysis of the protein revealed the presence of 13% arginine, consistent with the basic character of the molecule. Four residues of tryptophan, were present, indicating that MBP is not a histone. The MBP did not increase vascular permeability when injected into the skin of guinea pigs, nor did it antagonize the effect of histamine and bradykinin in the skin. MBP also did not contract the isolated guinea pig ileum and when mixed with histamine or bradykinin did not inhibit their activity on the gut. MBP had only weak, if any, antihistaminic activity. MBP possessed weak bactericidal activity when compared to histone and then only with one strain of E. coli. MBP precipitated DNA, neutralized heparin, and activated papain. On a molar basis MBP was more active than cysteine in activating papain. These results do not point to any unique biological activity associated with MBP other than those expected of a protein as basic as it is and one which possesses reactive sulfhydryl groups. Possible functions of eosinophils based on the properties of the MBP are discussed.

AbstractBackground: One of the numerous proteins causing amyloidosis is transthyretin (TTR), a protein usually responsible for the transport of thyroxine and retinol-binding protein. Variants within TTR cause it to aggregate and form insoluble fibers that accumulate in tissue, leading to organ dysfunction.Methods: TTR was immunoprecipitated from serum by use of a polyclonal antibody and subsequently reduced with tris(2-carboxyethyl)phosphine. The purified TTR was then analyzed by fast-gradient liquid chromatography–dual-electrospray ionization Fourier-transform ion-cyclotron-resonance (FT-ICR) mass spectrometry. DNA sequencing was performed on all samples used in this study.Results: Because of the inherent limitations in achieving high mass measurement accuracy based on the most abundant isotopic mass, we applied a fitting procedure that allowed determination of monoisotopic mass. Wild-type TTR (mean molecular mass, 13 761 Da) and its associated variant forms could be distinguished because of the high molecular mass accuracy afforded by FT-ICR (≤3 ppm) except for instances involving isobaric species or when isotopic distributions overlapped significantly. The [M + 11 H+]11+ charge state for all samples was used to determine the mass accuracies for both wild-type and variant forms of the protein. We correctly assigned seven of seven TTR variants. Moreover, using a combination of proteomic and genomic technologies, we discovered and characterized a previously unreported cis double mutation with a mass only 2 Da different from wild-type TTR. Furthermore, DNA sequencing of the TTR gene for all individuals in this study completely agreed with the intact protein measurements.Conclusions: FT-ICR mass spectrometry has sufficient mass accuracy to identify genetic variants of immunoaffinity-purified TTR. We believe that 91% of known TTR variants could be detected by this technique.