Sulfated glycolipids are the platelet autoantigens for human platelet-binding monoclonal anti-DNA autoantibodies

The human monoclonal autoantibody HF2-1/17, produced by a human-human hybridoma derived from lymphocytes of a lupus patient with thrombocytopenia, reacts with single stranded DNA and platelets. To determine the chemical nature of the autoantigen against which this antibody is directed on platelets, this platelet antigen was purified by the lipid extraction of sonicated platelets, DEAE-Sephadex chromatography, and high performance liquid chromatography. The purified glycolipids, a trace component in platelets, demonstrated high reactivity with the HF2-1/17 antibody using a competition enzyme-linked immunosorbent assay system or immunostaining of thin layer chromatograms. The purified glycolipids co-migrated with bovine sulfatides by thin layer chromatography. The purified glycolipids contain sulfate and galactose but not sialic acid or phosphate. Fast atom bombardment-mass spectrometry revealed these sulfatides to be sulfated monohexyl ceramides. The dominant species has a molecular weight of 794 while a minor form has a molecular weight of 812 due to an extra hydroxyl group and loss of a double bond. These results indicate that the platelet autoantigen against which the human monoclonal anti-DNA antibody is directed represents a family of novel monogalactosyl sulfatides.     

Anti-immunoglobulin autoantibodies are not preferentially induced in polyclonal activation of human and mouse lymphocytes, and more anti-DNA and anti-erythrocyte autoantibodies are induced in polyclonal activation of mouse than human lymphocytes

Using a plaque assay with immunoglobulin (Ig)-coated SRBC, we and others have previously reported that the majority of polyclonally activated mouse lymphocytes secreted antibodies that appeared to be IgM anti-IgG autoantibodies. Careful reexamination of this assay, with application of several highly purified mouse serum and myeloma IgG and IgM preparations, revealed that IgM, which was a minor contaminant of Ig preparations, rather than IgG, was responsible for the formation of these plaques. High numbers of plaques could also be detected in assays with polyclonally activated human lymphocytes, Ig-coated SRBC, and anti-Ig developing sera. Of all IgG-, IgM- or IgA-secreting cells, 40 to 100% were detected with SRBC coated with gamma-globulin or Ig of the same isotype as the isotype to which the developing serum was specific; in general, low proportions of all PFC were detected with SRBC coated with Ig of a different isotype. Studies on the sequence of events leading to the formation of plaques with Ig-sensitized SRBC (both in humans and mice) revealed that antibodies detected in these assays were not able to bind to the Ig-coated SRBC (without the presence of developing serum), and therefore were not anti-Ig autoantibodies. It is our conclusion that the plaque assays with Ig-coated SRBC represent another type of a reverse hemolytic PFC assay that detects cells secreting antibodies regardless of their specificity, and these plaques are formed due to the cross-linking by the anti-Ig developing serum of the Ig coated on SRBC and the Ig secreted by lymphocytes. Our results confirmed preferential induction of anti-DNA antibody secreting cells in mice by showing that these antibodies indeed bind to DNA coated on SRBC. In cultures of polyclonally activated human lymphocytes, anti-DNA and anti-erythrocyte autoantibody-secreting cells were over 10 to 100 times less frequent than in mice. These results, therefore, disprove the concept of preferential induction of anti-Ig autoantibodies in the polyclonal activation of mouse and human lymphocytes, and show that anti-DNA and anti-erythrocyte autoantibodies are easily induced in the polyclonal activation of mouse, but not human, lymphocytes.     

Suppression of murine lupus autoantibodies to DNA by administration of muramyl dipeptide and syngeneic anti-DNA IgG

We evaluated the effect of repeated subcutaneous administration of syngeneic anti-DNA IgG and muramyl dipeptide, a synthetic immunoadjuvant, to 6-mo-old (NZB X NZW)F1 female mice. This treatment had profound effects on both idiotype expression and anti-DNA antibody levels of morbid mice. It was also associated with appearance of anti-idiotypic antibodies specific for the injected antibody. These findings suggest that this approach with the use of synthetic immunomodulators and syngeneic antibodies may be of potential use in the management of autoimmune diseases.     

V region sequences of an idiotypically connected family of pathogenic anti-DNA autoantibodies

The H and L chain V region sequences of nine anti-DNA mAb that are representative of a pathogenic population of autoantibodies produced by the nephritis prone (SWR x NZB)F1 (SNF1) mice, were determined. These nine anti-DNA autoantibodies were idiotypically connected members of a cross-reactive Id family called the Id564 cluster. Moreover, these autoantibodies were all cationic in charge, IgG2b in isotype, and their H chain C regions had the normal SWR parent's allotype. Although derived from two different SNF1 animals, these pathogenic autoantibodies possessed highly homologous Leader-VH sequences that could account for their idiotypic cross-reactivity. Furthermore, the VH region sequences of these anti-DNA antibodies contained numerous basic residues that could impart their cationic charge. The Leader-VH sequences of these autoantibodies were also highly homologous to that of an anti-NP antibody-related germ-line gene of C57BL/6 mice, called VH-23. Among these nine pathogenic autoantibodies, three sets of clonally related anti-DNA antibodies could be identified. Thus the Id564 cluster of cationic anti-DNA autoantibodies of SNF1 mice are encoded by highly related VH genes, and this idiotypically connected population of pathogenic autoantibodies are selected to undergo an oligoclonal expansion in the lupus-prone SNF1 mice.     

Production of high affinity autoantibodies in autoimmune New Zealand Black/New Zealand white F1 mice targeted with an anti-DNA heavy chain

Lupus-prone, anti-DNA, heavy (H) chain "knock-in" mice were obtained by backcrossing C57BL/6 mice, targeted with a rearranged H chain from a VH11(S107)-encoded anti-DNA hybridoma (D42), onto the autoimmune genetic background of New Zealand Black/New Zealand White (NZB/NZW) F1 mice. The targeted female mice developed typical lupus serologic manifestations, with the appearance of transgenic IgM anti-DNA autoantibodies at a young age (2-3 mo) and high affinity, somatically mutated IgM and IgG anti-DNA Abs at a later age (6-7 mo). However, they did not develop clinical, lupus-associated glomerulonephritis and survived to at least 18 mo of age. L chain analysis of transgenic anti-DNA Abs derived from diseased NZB/NZW mouse hybridomas showed a very restricted repertoire of Vkappa utilization, different from that of nonautoimmune (C57BL/6 x BALB/c)F1 transgenic anti-DNA Abs. Strikingly, a single L chain was repetitively selected by most anti-DNA, transgenic NZB/NZW B cells to pair with the targeted H chain. This L chain had the same Vkappa-Jkappa rearrangement as that expressed by the original anti-DNA D42 hybridoma. These findings indicate that the kinetics of the autoimmune serologic manifestations are similar in wild-type and transgenic lupus-prone NZB/NZW F1 mice and suggest that the breakdown of immunologic tolerance in these mice is associated with the preferential expansion and activation of B cell clones expressing high affinity anti-DNA H/L receptor combinations.     

Genomic mapping within the albino-deletion complex using individual early postimplantation mouse embryos

Sensitive methods for analysis of DNA from limited amounts of tissue are often difficult, error prone, and time consuming. Here, we describe a procedure for molecular analysis of individual early post-implantation mouse embryos by Southern analysis. The procedure involves embedding single embryos in agarose before lysing and deproteinizing in situ. The embedded DNA can be digested with restriction enzymes and analyzed by standard Southern-blotting procedures. The procedure is sensitive enough to detect single-copy sequences in embryos as early as day 6.5 of development. We have used the technique to genotype embryos homozygous for an embryonic lethal deletion. Normally, the lethal phenotype associated with such mutations is identified by a retrospective statistical analysis of abnormal embryos produced from a heterozygous cross as compared to those produced from a control cross. Now, if associated with a detectable DNA abnormality, the mutant embryo can be genotyped directly. We also report the use of this method for mapping cloned markers relative to deletion breakpoints. This approach can save considerable time since mapping would conventionally be done using restriction fragment length polymorphisms (RFLPs) detected in Mus musculus/Mus spretus interspecies hybrids. Using this procedure, we have been able to redefine the distal limits of the region of Chromosome (Chr) 7 containing a gene (eed) needed for development of the embryonic ectoderm.     

IA-2 autoantibodies restricted to the IgG4 subclass are associated with protection from type 1 diabetes.

The tyrosine phosphatase-like protein IA-2 is a major target antigen for autoantibodies in the preclinical period of type 1 diabetes. In this study, we examined whether immunoglobulin isotypes and IgG subclass specific autoantibodies directed at IA-2 discriminate between children at risk of type 1 diabetes who progressed to diabetes vs. those who remained diabetes-free. IgG1-4, IgA and the IgE-specific IA-2 antibody (IA-2A) were measured by radioligand assays in 50 patients with type 1 diabetes and 41 ICA-positive siblings of patients with type 1 diabetes who were followed for diabetes development. Of 41 siblings, 32 were positive for IA-2A; of these, 59 % had IA-2 IgG1, 59 % IgG4, 16 % IgG3, 9 % IgG2, 16 % IgA and 13 % IgE antibodies. IA-2 IgG1 was the dominant isotype in prediabetic children (n = 14, 86 % positive) and patients with type 1 diabetes (98 % positive) whereas only 7 of 18 (39 %) non-progressors had antibodies of this isotype. In subjects that remained diabetes-free, a significantly higher frequency of IA-2 IgG4 in the absence of IgG1 was observed (50 %) compared to progressors (7 %) and patients with type 1 diabetes (0 %). Life-table analysis revealed that IA-2A restricted to IgG4 correlated with protection from type 1 diabetes (p < 0.003). In contrast, IA-2 IgG2, IgG3, IgE and IgA did not differ significantly between study groups. Our findings suggest that the measurement of IA-2 IgG1 and IgG4 subclass antibodies can serve as surrogate marker to discriminate between antibody positive subjects at high or low risk for rapid development of diabetes.     

Serum IgG from an autoimmune prone mouse C3H/HeJ-gld/gld supports the interleukin-3-dependent cell line through an autocrine mechanism

C3H/HeJ-gld/gld(C3H/gld) mice have been shown to develop massive lymphadenopathy with autoimmunity. In this study, we tested whether C3H/gld-IgG supports the growth of the IL-3-dependent cell line, FDC-P2/185-4. Serum IgG from C3H/gld mice stimulated FDC-P2/185-4 cells to proliferate. On the other hand, IgG from C3H/HeJ-+/+ did not show such activity. This activity increased with age in both sexes of C3H/gld mice. It was suggested that a monomeric IgG component was responsible for the proliferative activity of C3H/gld mouse sera. The cell-induced growth required Fc gamma receptors on FDC-P2/185-4 cells. FDC-P2/185-4 cells stimulated with C3H/gld-IgG, secreted IL-3, and grew by themselves, indicating an autocrine mechanism. Thus, cytokines produced by serum IgG may play an important role in the development of disease in mice bearing the autosomal recessive mutation gld.     

Whole-canopy carbon gain as a result of selection on individual performance of ten genotypes of a clonal plant

Game theoretical models predict that plant competition for light leads to reduced productivity of vegetation stands through selection for traits that maximize carbon gains of individuals. Using empirical results from a 5-year competition experiment with 10 genotypes of the clonal plant Potentilla reptans, we tested this prediction by analyzing the effects of the existing leaf area values on the carbon gain of the different genotypes and the consequent whole canopy carbon gain. We focused on specific leaf area (SLA) due to its role in the trade-off between light capture area and photosynthetic capacity per unit area. By combining a canopy model based on measured leaf area and light profiles with a game theoretical approach, we analyzed how changes in the SLA affected genotypic and whole-stand carbon gain. This showed that all genotypes contributed to reduced stand productivity. The dominant genotype maximized its share of total carbon gain, resulting in lower than maximal absolute gain. Other genotypes did not maximize their share. Hypothetical mutants of the dominant genotype were not able to achieve a higher carbon gain. Conversely, in other genotypes, some mutations did result in increased carbon gain. Hence, genotypic differences in the ability to maximize performance may determine genotype frequency. It shows how genotypic selection may result in lower carbon gains of the whole vegetation, and of the individual genotypes it consists of, through similar mechanisms as those that lead to the tragedy of the commons.     

A predator's selection of an individual prey from a group

This paper examines the mechanisms by which a predator selects an individual target from a group of prey. In a predatory situation, both the group of prey and the predator move around in a two-dimensional space. The predator has to select one individual among these prey to catch. For the mathematical treatment of such a selection, this paper introduces priority functions. Three different priority functions, labeled Strategies N, P and S, are then defined to indicate selection of the Nearest victim, the Peripheral victim or the Split victim (an individual separated from the group), respectively. It is found that, from the predator's standpoint, Strategy P is the best of the three regardless of how the prey group moves in reaction to an attack. Such reaction motions are classified into three types: ordered, partially disordered and fully disordered motion. Of the three types of motion, partially disordered motion is the most difficult type, as it confuses the predator in selecting and tracking a target individual.     

Eleven MRL-lpr/lpr anti-DNA autoantibodies are encoded by genes from four VH gene families: a potentially biased usage of VH genes

The genes encoding 11 independently derived anti-DNA autoantibodies from the lupus-prone mouse strain, MRL-lpr/lpr, were examined with VH, D, and JH gene probes. These autoantibodies do not define new VH gene families, since all of the autoantibodies were encoded by VH genes from four of the nine known gene families. A minimum of nine different VH genes encoded this panel of 11 anti-DNA autoantibodies. These results are consistent with the stochastic use of the VH gene repertoire and the expression of multiple VH genes. However, the data is also consistent with a biased usage of the VH gene repertoire. First, two pairs of autoantibodies, one from the J558 family and one from the 7183 family, appear to express identical or closely related VH genes as determined by the position of two restriction enzyme sites 5' of the expressed VH genes. In addition, three autoantibodies that appear to be sister clones might define a third VH gene that is used repeatedly. Secondly, about 45% of the panel is encoded by the Q52 and 7183 families, which are the 3' most families. These families have been shown to be preferentially rearranged early in B cell ontogeny. This suggests that some anti-DNA autoantibodies might originate from a population of B cells that predominate early in ontogeny. An alternative hypothesis is that the potential bias in VH gene and gene family usage could be due to antigen selection. All four JH genes are expressed, although the JH1 gene appears to be underutilized in both expressed and unexpressed rearrangements. Two members of the panel that bind double-stranded DNA were encoded by two different VH gene families, the S107 family and the J558 family.     

Genome diversification within a clonal population of pandemic Vibrio parahaemolyticus seems to depend on the life circumstances of each individual bacteria

Background

New strains of Vibrio parahaemolyticus that cause diarrhea in humans by seafood ingestion periodically emerge through continuous evolution in the ocean. Influx and expansion in the Southern Chilean ocean of a highly clonal V. parahaemolyticus (serotype O3:K6) population from South East Asia caused one of the largest seafood-related diarrhea outbreaks in the world. Here, genomics analyses of isolates from this rapidly expanding clonal population offered an opportunity to observe the molecular evolutionary changes often obscured in more diverse populations.

Results

Whole genome sequence comparison of eight independent isolates of this population from mussels or clinical cases (from different years) was performed. Differences of 1366 to 217,729 bp genome length and 13 to 164 bp single nucleotide variants (SNVs) were found. Most genomic differences corresponded to the presence of regions unique to only one or two isolates, and were probably acquired by horizontal gene transfer (HGT). Some DNA gain was chromosomal but most was in plasmids. One isolate had a large region (8,644 bp) missing, which was probably caused by excision of a prophage. Genome innovation by the presence of unique DNA, attributable to HGT from related bacteria, varied greatly among the isolates, with values of 1,366 (ten times the number of highest number of SNVs) to 217,729 (a thousand times more than the number of highest number of SNVs).

Conclusions

The evolutionary forces (SNVs, HGT) acting on each isolate of the same population were found to differ to an extent that probably depended on the ecological scenario and life circumstances of each bacterium.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1385-8) contains supplementary material, which is available to authorized users.     

Light chain determines the binding property of human anti-dsDNA IgG autoantibodies

We have previously prepared human anti-double-stranded (ds) DNA IgG Fab clones using phage-display technology. Nucleotide sequence analysis of genes of immunoglobulin (Ig) heavy and light chain variable regions in these Fab clones suggested that the DNA-binding activity of the clones depended on light chain usage. To confirm the role of the light chain in antibody binding to DNA, we constructed in the present study's new recombined Fab clones by heavy and light chain shuffling between the original anti-dsDNA Fab clones. Clones constructed by pairing Fdgamma fragments with the light chain from a high DNA-binding clone showed high DNA-binding activities, whereas other constructed clones using light chains from low DNA-binding clones showed low DNA-binding activities. Our results indicate that light chains in anti-dsDNA antibodies can determine the DNA-binding activity of the antibodies. Ig chain shuffling of phage-display antibodies may be useful for investigating the molecular mechanisms for antigen-antibody binding of human autoantibodies.     

Proliferative activity of autoimmune MRL mouse serum IgG to interleukin 3-dependent cell line through autocrine mechanism

Serum of an autoimmune MRL/Mp-lpr/lpr (MRL/l) mouse supported the proliferation of interleukin 3 (IL-3)-dependent cell line, FDC-P2. This IL-3-like activity initially appeared at 1 month of age and increased with age. Females showed higher titers than did males. MRL/Mp-+/+ mouse sera also exhibited such activity, though somewhat later in life only in female. Other autoimmune mice, NZB, NZB/NZW F1, and BXSB, demonstrated no such activity in either males or females, young and old. The active component of MRL/l sera was shown to be IgG. F(ab')2 or Fc fragments of MRL/l-IgG lost such activity. Not all IL-3-dependent cell lines, however, responded to MRL/l-IgG. We subcloned MRL-IgG responding and nonresponding clones from FDC-P2 cells and both were still dependent to IL-3. Such nonresponding IL-3-dependent cell lines, however, could be stimulated by the culture supernatant of the responding cell line, FDC-P2/185-4, after being stimulated with MRL/l-IgG. In this culture supernatant, IL-3 was found, thus the existence of an autocrine system was suggested in the IL-3-dependent MRL/l-IgG responding cell line.     

The alpha 5 chain of type IV collagen is the target of IgG autoantibodies in a novel autoimmune disease with subepidermal blisters and renal insufficiency

We describe a novel autoimmune disease characterized by severe subepidermal bullous eruptions and renal insufficiency with IgG autoantibodies directed against the NC1 domain of the alpha5(IV) collagen chain. In vivo deposits of IgG and C3 were found along the dermal-epidermal junction of skin lesions. The identity of the target antigen was determined by immunochemical analyses of candidate antigens using the patients' autoantibodies. The patients' IgG autoantibodies reacted with a 185-kDa polypeptide that was distinguished from the known autoantigens of the extracellular matrix including type XVII collagen, type VII collagen, or the alpha3, beta3, and gamma2 chains of laminin 5. Preincubation of the serum with recombinant alpha5(IV)NC1 domain of type IV collagen abolished immunoreactivity with the 185-kDa antigen. The serum reacted specifically with the alpha5(IV)NC1, among the six NC1 domains of type IV collagen, by Western blot and enzyme-linked immunosorbent assay analyses. The patients' autoantibodies reacted with normal skin and renal glomerulus but not with skin and glomerulus of a patient with Alport syndrome in which the basement membranes are devoid of the alpha5(IV) collagen chain. This study provided for the first time unambiguous evidence for the alpha5(IV) collagen chain as the target antigen in a novel autoimmune disease characterized by skin and renal involvement.     

Natural selection and the traits of individual organisms

It has recently been argued that origin essentialism regarding individual organisms entails that natural selection does not explain why individual organisms have the traits that they do. This paper defends this and related theses against Mohan Matthens recent objections.