An investigation of the molecular basis of interactions between human monoclonal antibodies and antigens that are clinically relevant in systemic lupus erythematosus and the Antiphospholipid Syndrome.

Autontibodies to a wide variety of antigens are associated with systemic lupus erythematosus (SLE) and the Antiphospholipid Syndrome (APS). Previous studies have demonstrated the importance of somatic mutations and arginine residues in the complementarity determining regions (CDRs) of pathogenic anti-dsDNA and antiphospholipid antibodies. This thesis describes the study of two human monoclonal IgG antibodies, B3 (anti-DNA) and IS4 (antiphospholipid) that were derived from a patient with active SLE and primary APS respectively. I have demonstrated in-vitro expression and mutagenesis of B3 and IS4 and used this expression system to investigate the importance of the arginine residues in B3VH and IS4VH. The mutant heavy chains, as well as the wild-type VH were expressed with different light chains and the resulting antibodies assessed for binding to nucleosomes, alpha-actinin, cardiolipin (CL), phosphatidylserine (PS), beta-2-glycoprotein I (foGPI), and the N-terminal domain of p2GPI (Domain I) using direct binding assays. The results obtained have shown that the presence of arginine at position 53 in B3VH was essential but not sufficient for binding to dsDNA and nucleosomes. Conversely, the presence of this arginine reduced binding to alpha-actinin, pzGPI and Domain I of P2GPI. The fact that the arginine to serine substitution at position 53 in B3VH significantly alters binding of B3 to different clinically relevant antigens, but in opposite directions implies that this arginine residue plays a critical role in the affinity maturation of the antibody B3. Furthermore, of four arginine residues in IS4VH CDR3 substituted to serine, two at positions 100 and 100g reduced binding to all antigens, while two at positions 96 and 97 reduced binding to fcGPI but increased or decreased binding to CL and PS. Only one H/L chain combination bound neutral phospholipid and none bound dsDNA hence, these effects are particularly relevant to antigens important in APS. Therefore, my findings suggest that these four arginine residues have developed as a result of somatic mutations driven by an antigen containing both phospholipid and frGPI. These results extend our knowledge of the structure-function relationship of human anti-DNA and anti phospholipid antibodies and aid in our understanding of how these antibodies lead to pathogenicity and what we need to target in the future for possible therapies

Evidence of different sensitivity of muscle and tendon to mechano-metabolic stimuli

This study aimed to examine the temporal dynamics of muscle-tendon adaptation and whether differences between their sensitivity to mechano-metabolic stimuli would lead to non-uniform changes within the triceps surae (TS) muscle-tendon unit (MTU). Twelve young adults completed a 12-week training intervention of unilateral isometric cyclic plantarflexion contractions at 80% of maximal voluntary contraction until failure to induce a high TS activity and hence metabolic stress. Each participant trained one limb at a short (plantarflexed position, 115°: PF) and the other at a long (dorsiflexed position, 85°: DF) MTU length to vary the mechanical load. MTU mechanical, morphological, and material properties were assessed biweekly via simultaneous ultrasonography-dynamometry and magnetic resonance imaging. Our hypothesis that tendon would be more sensitive to the operating magnitude of tendon strain but less to metabolic stress exercise was confirmed as tendon stiffness, Young's modulus, and tendon size were only increased in the DF condition following the intervention. The PF leg demonstrated a continuous increment in maximal AT strain (i.e., higher mechanical demand) over time along with lack of adaptation in its biomechanical properties. The premise that skeletal muscle adapts at a higher rate than tendon and does not require high mechanical load to hypertrophy or increase its force potential during exercise was verified as the adaptive changes in morphological and mechanical properties of the muscle did not differ between DF and PF. Such differences in muscle-tendon sensitivity to mechano-metabolic stimuli may temporarily increase MTU imbalances that could have implications for the risk of tendon overuse injury

Arginine mutation alters binding of a human monoclonal antibody to antigens linked to systemic lupus erythematosus and the antiphospholipid syndrome

Objective: Previous studies have shown the importance of somatic mutations and arginine residues in the complementarity-determining regions (CDRs) of pathogenic anti-double-stranded DNA (anti-dsDNA) antibodies in human and murine lupus, and in studies of murine antibodies, a role of mutations at position 53 in VH CDR2 has been demonstrated. We previously demonstrated in vitro expression and mutagenesis of the human IgG1 monoclonal antibody B3. The present study was undertaken to investigate, using this expression system, the importance of the arginine residue at position 53 (R53) in B3 VH. Methods: R53 was altered, by site-directed mutagenesis, to serine, asparagine, or lysine, to create 3 expressed variants of VH. In addition, the germline sequence of the VH3-23 gene (from which B3 VH is derived) was expressed either with or without arginine at position 53. These 5 new heavy chains, as well as wild-type B3 VH, were expressed with 4 different light chains, and the resulting antibodies were assessed for their ability to bind to nucleosomes, -actinin, cardiolipin, ovalbumin, 2-glycoprotein I (2GPI), and the N-terminal domain of 2GPI (domain I), using direct binding assays. Results: The presence of R53 was essential but not sufficient for binding to dsDNA and nucleosomes. Conversely, the presence of R53 reduced binding to -actinin, ovalbumin, 2GPI, and domain I of 2GPI. The combination B3 (R53S) VH/B3 VL bound human, but not bovine, 2GPI. Conclusion: The fact that the R53S substitution significantly alters binding of B3 to different clinically relevant antigens, but that the alteration is in opposite directions depending on the antigen, implies that this arginine residue plays a critical role in the affinity maturation of antibody B3

The association between IgG and IgM antibodies against cardiolipin, β2-glycoprotein I and Domain I of β2-glycoprotein I with disease profile in patients with multiple sclerosis

Antiphospholipid antibodies (aPL) occur in patients with multiple sclerosis (MS) with a number of studies reporting elevated levels; their exact prevalence and pathogenic role remain unclear. Epidemiological studies associate MS with an increased risk of deep venous thromboembolism and stroke; overlapping clinical features with APS. Antibodies against the first domain – Domain I (DI) – of β2glycoprotein I (β2GPI), show the most clinical significance and evidence for pathogenicity in the antiphospholipid syndrome (APS), but have not yet been investigated in MS. Serum from a well-defined cohort of 127 MS patients and 92 healthy controls were tested for IgM and IgG antibodies against cardiolipin (CL), β2GPI and DI. Higher frequency of IgM and IgG anti-CL were found in MS patients (18.1% and 21.3%), compared to controls (1.1% in both cases, p < 0.0001). We report that anti-DI antibodies were associated with MS patients, with 6.3% and 7.1% positive for IgM and IgG, respectively, compared to controls, 1.1% (p < 0.05). IgM anti-CL antibodies were elevated in secondary progressive MS and primary progressive MS compared to relapse-remitting MS, (p < 0.005). This study enrolled the largest number of patients with definite MS for studying the association with aPL. Although we confirmed IgM and IgG anti-CL antibodies occur in patients with MS, this is the first study that identified anti-DI antibodies in MS patients. This new finding may prove valuable and future studies are required to evaluate its role as a potential risk factor of thromboembolic phenomena in MS

Stable expression of a recombinant human antinucleosome antibody to investigate relationships between antibody sequence, binding properties, and pathogenicity

When purified under rigorous conditions, some murine anti-double-stranded-DNA (anti-dsDNA) antibodies actually bind chromatin rather than dsDNA. This suggests that they may actually be antinucleosome antibodies that only appear to bind dsDNA when they are incompletely dissociated from nucleosomes. Experiments in murine models suggest that antibody–nucleosome complexes may play a crucial role in the pathogenesis of glomerulonephritis in systemic lupus erythematosus. Some human monoclonal anti-DNA antibodies are pathogenic when administered to mice with severe combined immunodeficiency (SCID). Our objective was to achieve stable expression of sequence-altered variants of one such antibody, B3, in Chinese hamster ovary (CHO) cells. Purified antibodies secreted by these cells were tested to investigate whether B3 is actually an antinucleosome antibody. The pathogenic effects of the antibodies were tested by implanting CHO cells secreting them into SCID mice. Purified B3 does not bind to dsDNA unless supernatant from cultured cells is added, but does bind to nucleosomes. The strength of binding to dsDNA and nucleosomes is dependent on the sequence of the light chain. Mice that received CHO cells secreting wild-type B3 developed more proteinuria and died earlier than control mice that received nonsecreting CHO cells or mice that received B3 with a single light chain mutation. However, none of the mice had histological changes or deposition of human immunoglobulin G in the kidneys. Sequence changes may alter the pathogenicity of B3, but further studies using different techniques are needed to investigate this possibility

A novel expression system of domain I of human beta2 glycoprotein I in Escherichia coli

BACKGROUND: The antiphospholipid syndrome (APS), characterised by recurrent miscarriage and thrombosis, is a significant cause of morbidity and mortality. Domain I (DI) of human beta 2 glycoprotein I (β(2)GPI) is thought to contain crucial antibody binding epitopes for antiphospholipid antibodies (aPL), which are critical to the pathogenesis of APS. Expressing this protein in bacteria could facilitate studies investigating how this molecule interacts with aPL. METHODS: Using a computer programme called Juniper, sequentially overlapping primers were designed to be used in a recursive polymerase chain reaction (PCR) to produce a synthetic DI gene. Specifically Juniper incorporates 'major' codons preferred by bacteria altering 41 codons out of 61. This was cloned into the expression plasmid pET(26b) and expressed in BL21(DE3) Escherichia coli (E. coli). By virtue of a pelB leader sequence, periplasmic localisation of DI aided disulphide bond formation and toxicity was addressed by tightly regulating expression through the high stringency T7lac promoter. RESULTS: Purified, soluble his-tagged DI in yields of 750 μg/L bacterial culture was obtained and confirmed on Western blot. Expression using the native human cDNA sequence of DI in the same construct under identical conditions yielded significantly less DI compared to the recombinant optimised sequence. This constitutes the first description of prokaryotic expression of soluble DI of β(2)GPI. Binding to murine monoclonal antibodies that recognise conformationally restricted epitopes on the surface of DI and pathogenic human monoclonal IgG aPL was confirmed by direct and indirect immunoassay. Recombinant DI also bound a series of 21 polyclonal IgG samples derived from patients with APS. CONCLUSION: By producing a synthetic gene globally optimised for expression in E. coli, tightly regulating expression and utilising periplasmic product translocation, efficient, soluble E. coli expression of the eukaryotic protein DI of β(2)GPI is possible. This novel platform of expression utilising pan-gene prokaryote codon optimisation for DI production will aid future antigenic studies. Furthermore if DI or peptide derivatives of DI are eventually used in the therapeutic setting either as toleragen or as a competitive inhibitor of pathogenic aPL, then an E. coli production system may aid cost-effective production

Somatic hypermutation as a generator of antinuclear antibodies in a murine model of systemic autoimmunity

Systemic lupus erythematosus (SLE) is characterized by high-avidity IgG antinuclear antibodies (ANAs) that are almost certainly products of T cell–dependent immune responses. Whether critical amino acids in the third complementarity-determining region (CDR3) of the ANA originate from V(D)J recombination or somatic hypermutation (SHM) is not known. We studied a mouse model of SLE in which all somatic mutations within ANA V regions, including those in CDR3, could be unequivocally identified. Mutation reversion analyses revealed that ANA arose predominantly from nonautoreactive B cells that diversified immunoglobulin genes via SHM. The resolution afforded by this model allowed us to demonstrate that one ANA clone was generated by SHM after a VH gene replacement event. Mutations producing arginine substitutions were frequent and arose largely (66%) from base changes in just two codons: AGC and AGT. These codons are abundant in the repertoires of mouse and human V genes. Our findings reveal the predominant role of SHM in the development of ANA and underscore the importance of self-tolerance checkpoints at the postmutational stage of B cell differentiation

Redating the formation of Lake Bafa, western Turkey: Integrative geoarchaeological methods and new environmental and dating evidence

The ancient Gulf of Latmos is an iconic example of a dynamic landscape and humankind's historical relationship with it. Using extensive new primary data and original models for calibrating radiocarbon dates in transitional lagoon environments, we demonstrate that Lake Bafa (or Bafa Gölü, in Turkish) formed at a much earlier date than previously thought. In questioning the logical process by which previous dates were achieved, we re‐examine the relationship between sedimentological data, archaeology and written history. We reassert the need to establish independently dated environmental data sets as the foundation of regional studies as distinct from archaeological and historical interpretive processes. We conclude that Lake Bafa slowly transitioned to become an isolated lagoon sometime between the end of the second millennium B.C. and end of the first millennium B.C.; becoming a fully closed brackish lake during the second millennium A.D. This marks a major shift in our understanding of the nature of human occupation and activity here during the last four millennia but also in the way we date ancient lagoons and integrate historical and environmental data in general

Holocene demographic fluctuations, climate and erosion in the Mediterranean: A meta data-analysis

As part of the Changing the Face of the Mediterranean Project, we consider how human pressure and concomitant erosion has affected a range of Mediterranean landscapes between the Neolithic and, in some cases, the post-medieval period. Part of this assessment comprises an investigation of relationships among palaeodemographic data, evidence for vegetation change and some consideration of rapid climate change events. The erosion data include recent or hitherto unpublished work from the authors. Where possible, we consider summed probabilities of 14C dates as well as the first published synthesis of all known optically stimulated luminescence dated sequences. The results suggest that while there were some periods when erosion took place contemporaneously across a number of regions, possibly induced by climate changes, more often than not, we see a complex and heterogeneous interplay of demographic and environmental changes that result in a mixed pattern of erosional activity across the Mediterranean