COMPLEMENT-MEDIATED ADIPOCYTE LYSIS BY NEPHRITIC FACTOR SERA

Recent data indicate a previously unsuspected link between the complement system and adipocyte biology. Murine adipocytes produce key components of the alternative pathway of complement and are able to activate this pathway. This suggested to us an explanation for adipose tissue loss in partial lipodystrophy, a rare human condition usually associated with the immunoglobulin G(IgG) autoantibody nephritic factor (NeF) which leads to enhanced alternative pathway activation in vivo. We hypothesized that in the presence of NeF, there is dysregulated complement activation at the membrane of the adipocyte, leading to adipocyte lysis. Here we show that adipocytes explanted from rat epididymal fat pads are lysed by NeF-containing sera but not by control sera. A similar pattern is seen with IgG fractions of these sera. Adipocyte lysis in the presence of NeF is associated with the generation of fluid-phase terminal complement complexes, the level of which correlates closely with the level of lactate dehydrogenase, a marker of cell lysis. Lysis is abolished by ethylenediaminetetraacetic acid, which chelates divalent cations and prevents complement activation, and reduced by an antibody to factor D, a key component of the alternative pathway. These data provide an explanation for the previously obscure link between NeF and fat cell damage

Complotype affects the extent of down-regulation by Factor I of the C3b feedback cycle in vitro.

Sera from a large panel of normal subjects were typed for three common polymorphisms, one in C3 (R102G) and two in Factor H (V62I and Y402H), that influence predisposition to age-related macular degeneration and to some forms of kidney disease. Three groups of sera were tested; those that were homozygous for the three risk alleles; those that were heterozygous for all three; and those homozygous for the low-risk alleles. These groups vary in their response to the addition of exogenous Factor I when the alternative complement pathway is activated by zymosan. Both the reduction in the maximum amount of iC3b formed and the rate at which the iC3b is converted to C3dg are affected. For both reactions the at-risk complotype requires higher doses of Factor I to produce similar down-regulation. Because iC3b reacting with the complement receptor CR3 is a major mechanism by which complement activation gives rise to inflammation, the breakdown of iC3b to C3dg can be seen to have major significance for reducing complement-induced inflammation. These findings demonstrate for the first time that sera from subjects with different complement alleles behave as predicted in an in-vitro assay of the down-regulation of the alternative complement pathway by increasing the concentration of Factor I. These results support the hypothesis that exogenous Factor I may be a valuable therapeutic aid for down-regulating hyperactivity of the C3b feedback cycle, thereby providing a treatment for age-related macular degeneration and other inflammatory diseases of later life.This is the author's accepted manuscript and will be under embargo until the 13th of August 2015. This final version is available from Wiley in Clinical & Experimental Immunology at http://onlinelibrary.wiley.com/doi/10.1111/cei.12437/abstract

Aerobic capacity and skeletal muscle characteristics in glycogen storage disease IIIa: an observational study

Background: Individuals with glycogen storage disease IIIa (GSD IIIa) (OMIM #232400) experience muscle weakness and exercise limitation that worsen through adulthood. However, normative data for markers of physical capacity, such as strength and cardiovascular fitness, are limited. Furthermore, the impact of the disease on muscle size and quality is unstudied in weight bearing skeletal muscle, a key predictor of physical function. We aim to produce normative reference values of aerobic capacity and strength in individuals with GSD IIIa, and to investigate the role of muscle size and quality on exercise impairment. Results: Peak oxygen uptake (V̇O2peak) was lower in the individuals with GSD IIIa than predicted based on demographic data (17.0 (9.0) ml/kg/min, 53 (24)% of predicted, p = 0.001). Knee extension maximum voluntary contraction (MVC) was also substantially lower than age matched predicted values (MVC: 146 (116) Nm, 57% predicted, p = 0.045), though no difference was found in MVC relative to body mass (1.88 (2.74) Nm/kg, 61% of predicted, p = 0.263). There was a strong association between aerobic capacity and maximal leg strength (r = 0.920; p = 0.003). Substantial inter-individual variation was present, with a high physical capacity group that had normal leg strength (MVC), and relatively high V̇O2peak, and a low physical capacity that display impaired strength and substantially lower V̇O2peak. The higher physical capacity sub-group were younger, had larger Vastus Lateralis (VL) muscles, greater muscle quality, undertook more physical activity (PA), and reported higher health-related quality of life. Conclusions: V̇O2peak and knee extension strength are lower in individuals with GSD IIIa than predicted based on their demographic data. Patients with higher physical capacity have superior muscle size and structure characteristics and higher health-related quality of life, than those with lower physical capacity. This study provides normative values of these important markers of physical capacity

Functional expression of complement factor I following AAV-mediated gene delivery in the retina of mice and human cells.

Funder: NIHR Oxford Biomedical Research CentreDry age-related macular degeneration (AMD) is characterised by loss of central vision and currently has no approved medical treatment. Dysregulation of the complement system is thought to play an important role in disease pathology and supplementation of Complement Factor I (CFI), a key regulator of the complement system, has the potential to provide a treatment option for AMD. In this study, we demonstrate the generation of AAV constructs carrying the human CFI sequence and expression of CFI in cell lines and in the retina of C57BL/6 J mice. Four codon optimised constructs were compared to the most common human CFI sequence. All constructs expressed CFI protein; however, most codon optimised sequences resulted in significantly reduced CFI secretion compared to the non-optimised CFI sequence. In vivo expression analysis showed that CFI was predominantly expressed in the RPE and photoreceptors. Secreted protein in vitreous humour was demonstrated to be functionally active. The findings presented here have led to the formulation of an AAV-vectored gene therapy product currently being tested in a first-in-human clinical trial in subjects with geographic atrophy secondary to dry AMD (NCT03846193)

Eculizumab treatment: stochastic occurrence of C3 binding to individual PNH erythrocytes

C5 blockade by eculizumab prevents complement-mediated intravascular hemolysis in paroxysmal nocturnal hemoglobinuria (PNH). However, C3-bound PNH red blood cells (RBCs), arising in almost all treated patients, may undergo extravascular hemolysis reducing clinical benefits. Despite the uniform deficiency of CD55 and of CD59, there are always two distinct populations of PNH RBCs, with (C3+) and without (C3-) C3 binding

Molecular changes in the expression of human colonic nutrient transporters during the transition from normality to malignancy

Healthy colonocytes derive 60–70% of their energy supply from short-chain fatty acids, particularly butyrate. Butyrate has profound effects on differentiation, proliferation and apoptosis of colonic epithelial cells by regulating expression of various genes associated with these processes. We have previously shown that butyrate is transported across the luminal membrane of the colonic epithelium via a monocarboxylate transporter, MCT1. In this paper, using immunohistochemistry and in situ hybridisation histochemistry, we have determined the profile of MCT1 protein and mRNA expression along the crypt to surface axis of healthy human colonic tissue. There is a gradient of MCT1 protein expression in the apical membrane of the cells along the crypt-surface axis rising to a peak in the surface epithelial cells. MCT1 mRNA is expressed along the crypt-surface axis and is most abundant in cells lining the crypt. Analysis of healthy colonic tissues and carcinomas using immunohistochemistry and Western blotting revealed a significant decline in the expression of MCT1 protein during transition from normality to malignancy. This was reflected in a corresponding reduction in MCT1 mRNA expression, as measured by Northern analysis. Carcinoma samples displaying reduced levels of MCT1 were found to express the high affinity glucose transporter, GLUT1, suggesting that there is a switch from butyrate to glucose as an energy source in colonic epithelia during transition to malignancy. The expression levels of MCT1 in association with GLUT1 could potentially be used as determinants of the malignant state of colonic tissue

Beneficial and Detrimental Effects of Plasmin(ogen) during Infection and Sepsis in Mice

Plasmin has been proposed to be an important mediator during inflammation/infection. In this study, by using mice lacking genes for plasminogen, tissue-type plasminogen activator (tPA), and urokinase-type PA (uPA), we have investigated the functional roles of active plasmin in infection and sepsis. Two models were used: an infection model by intravenous injection of 1×107 CFU of S. aureus, and a sepsis model by intravenous injection of 1.6×108 CFU of S. aureus. We found that in the infection model, wild-type (WT) mice showed significantly higher survival rates than plasminogen-deficient (plg-/-) mice. However, in the sepsis model, plg-/- or tPA-/-/uPA-/- mice showed the highest survival rate whereas WT and tPA+/-/uPA+/- mice showed the lowest survival rate, and plg+/-, tPA-/-, and uPA-/- mice had an intermediate survival rate. These results indicate that the levels of active plasmin are critical in determining the survival rate in the sepsis, partly through high levels of inflammatory cytokines and enhanced STAT3 activation. We conclude that plasmin is beneficial in infection but promotes the production of inflammatory cytokines in sepsis that may cause tissue destruction, diminished neutrophil function, and an impaired capacity to kill bacteria which eventually causes death of these mice

'Care and Prevent': rationale for investigating skin and soft tissue infections and AA amyloidosis among people who inject drugs in London.

BACKGROUND: Skin and soft tissue infections (SSTIs) are a leading cause of morbidity and mortality among people who inject drugs (PWID). International data indicate up to one third of PWID have experienced an SSTI within the past month. Complications include sepsis, endocarditis and amyloid A (AA) amyloidosis. AA amyloidosis is a serious sequela of chronic SSTI among PWID. Though there is a paucity of literature reporting on AA amyloidosis among PWID, what has been published suggests there is likely a causal relationship between AA amyloidosis and injecting-related SSTI. If left untreated, AA amyloidosis can lead to renal failure; premature mortality among diagnosed PWID is high. Early intervention may reverse disease. Despite the high societal and individual burden of SSTI among PWID, empirical evidence on the barriers and facilitators to injecting-related SSTI prevention and care or the feasibility and acceptability of AA amyloidosis screening and treatment referral are limited. This study aims to fill these gaps and assess the prevalence of AA amyloidosis among PWID. METHODS: Care and Prevent is a UK National Institute for Health Research-funded mixed-methods study. In five phases (P1-P5), we aim to assess the evidence for AA amyloidosis among PWID (P1); assess the feasibility of AA amyloidosis screening, diagnostic and treatment referral among PWID in London (P2); investigate the barriers and facilitators to AA amyloidosis care (P3); explore SSTI protection and risk (P4); and co-create harm reduction resources with the affected community (P5). This paper describes the conceptual framework, methodological design and proposed analysis for the mixed-methods multi-phase study. RESULTS: We are implementing the Care and Prevent protocol in London. The systematic review component of the study has been completed and published. Care and Prevent will generate an estimate of AA amyloidosis prevalence among community recruited PWID in London, with implications for the development of screening recommendations and intervention implementation. We aim to recruit 400 PWID from drug treatment services in London, UK. CONCLUSIONS: Care and Prevent is the first study to assess screening feasibility and the prevalence of positive proteinuria, as a marker for AA amyloidosis, among PWID accessing drug treatment services. AA amyloidosis is a serious, yet under-recognised condition for which early intervention is available but not employed