Dysfunction of complement receptors CR3 (CD11b/18) and CR4 (CD11c/18) in pre-eclampsia: a genetic and functional study.

OBJECTIVE: To study genetic variants and their function within genes coding for complement receptors in pre-eclampsia. DESIGN: A case-control study. SETTING: Pre-eclampsia is a common vascular disease of pregnancy. The clearance of placenta-derived material is one of the functions of the complement system in pregnancy. POPULATION: We genotyped 500 women with pre-eclamptic pregnancies and 190 pregnant women without pre-eclampsia, as controls, from the FINNPEC cohort, and 122 women with pre-eclamptic pregnancies and 1905 controls from the national FINRISK cohort. METHODS: The functional consequences of genotypes discovered by targeted exomic sequencing were explored by analysing the binding of the main ligand iC3b to mutated CR3 or CR4, which were transiently expressed on the surface of COS-1 cells. MAIN OUTCOME MEASURES: Allele frequencies were compared between pre-eclamptic pregnancies and controls in genetic studies. The functional consequences of selected variants were measured by binding assays. RESULTS: The most significantly pre-eclampsia-linked CR3 variant M441K (P = 4.27E-4, OR = 1.401, 95% CI = 1.167-1.682) displayed a trend of increased adhesion to iC3b (P = 0.051). The CR4 variant A251T was found to enhance the adhesion of CR4 to iC3b, whereas W48R resulted in a decrease of the binding of CR4 to iC3b. CONCLUSIONS: Results suggest that changes in complement-facilitated phagocytosis are associated with pre-eclampsia. Further studies are needed to ascertain whether aberrant CR3 and CR4 activity leads to altered pro- and anti-inflammatory cytokine responses in individuals carrying the associated variants, and the role of these receptors in pre-eclampsia pathogenesis. TWEETABLE ABSTRACT: Genetic variants of complement receptors CR3 and CR4 have functional consequences that are associated with pre-eclampsia.

Bleeding complications and antithrombotic treatment in 264 pregnancies in antiphospholipid syndrome.

Purpose The purpose of this study was to evaluate the safety of antithrombotic treatments prescribed during pregnancy in patients with antiphospholipid syndrome (APS). Methods This international, multicenter study included two cohorts of patients: a retrospective French cohort and a prospective US cohort (PROMISSE study). Inclusion criteria were (1) APS (Sydney criteria), (2) live pregnancy at 12 weeks of gestation (WG) with (3) follow-up data until six weeks post-partum. According to APS standard of care, patients were treated with aspirin and/or low-molecular weight heparin (LMWH) at prophylactic (pure obstetric APS) or therapeutic doses (history of thrombosis). Major bleeding was defined as abnormal blood loss during the pregnancy and/or post-partum period requiring intervention for hemostasis or transfusion, or during the peripartum period greater than 500 mL and/or requiring surgery or transfusion. Other bleeding events were classified as minor. Results Two hundred and sixty-four pregnancies (87 prospectively collected) in 204 patients were included (46% with history of thrombosis, 23% with associated systemic lupus). During pregnancy, treatment included LMWH ( n = 253; 96%) or low-dose aspirin ( n = 223; 84%), and 215 (81%) patients received both therapies. The live birth rate was 89% and 82% in the retrospective and prospective cohorts, respectively. Adverse pregnancy outcomes occurred in 28% of the retrospective cohort and in 40% of the prospective cohort. No maternal death was observed in either cohort. A combined total of 45 hemorrhagic events (25%) occurred in the retrospective cohort, but major bleeding was reported in only six pregnancies (3%). Neither heparin nor aspirin alone nor combined therapy increased the risk of hemorrhage. We also did not observe an increased rate of bleeding in the case of a short interval between last LMWH (less than 24 hours) or aspirin (less than five days) doses and delivery. Only emergency Caesarean section was significantly associated with an increased risk of bleeding (odds ratio (OR) 5.03 (1.41-17.96); p=.016). In the prospective cohort, only one minor bleeding event was reported (vaginal bleeding). Conclusion Our findings support the safety of antithrombotic therapy with aspirin and/or LMWH during pregnancy in high-risk women with APS, and highlight the need for better treatments to improve pregnancy outcomes in APS. PROMISSE Study ClinicalTrials.gov identifier: NCT00198068.

A pilot study of office-based spirometry in patients with systemic lupus erythematosus.

BACKGROUND: Although pulmonary involvement is common in systemic lupus erythematosus (SLE), its effects on healthy lifestyle physical activity and its association with fatigue have not been well characterized. The goals of this study were to describe pulmonary function measured by office-based spirometry in patients with SLE and to compare spirometry with physical activity and systemic fatigue. METHODS: During an office visit, 49 patients with SLE completed spirometry assessing: a) forced expiratory volume in 1 s (FEV(1), a measure of airway patency and responsiveness); b) forced vital capacity (FVC, a measure of lung volume); and c) maximum voluntary ventilation (MVV, a measure of volume of air moved during rapid breathing) which has been hypothesized to be decreased in SLE due to muscle fatigue. Patients also performed a 2-min corridor walking test and completed self-reported questionnaires measuring weekly physical activity and systemic fatigue. RESULTS: Mean age was 45 years, 45 (92%) were women, mean SLEDAI and SLICC scores were 2.8 and 1.0, respectively. Some 24 patients had a smoking history, and 15 had a history of SLE-related pleuritis, which was not active at enrollment. FEV(1) and FVC were 96% of predicted, but MVV was only 55% of predicted. The distance walked during the corridor test was similar to that of patients with other chronic diseases; however, self-reported physical activity was less than recommended by national guidelines. There were no associations between spirometry values and history of pleuritis, other pulmonary diagnoses, or smoking (p > .10 for all comparisons), however, better FEV(1) (p = .04) and better FVC (p = .04) were associated with more self-reported activity and better FEV(1) (p = .03) was associated with longer distance walked during the corridor test. Most patients reported marked systemic fatigue; however, there were no associations between spirometry values and fatigue scores (p > .10 for all comparisons). CONCLUSIONS: MVV was markedly diminished, which supports the hypothesis that SLE may be associated with respiratory muscle fatigue during rapid breathing. MVV was not associated with mild-to-moderate patient-directed physical activity; however, lower FEV(1) and FVC were associated with less self-reported and performance-based physical activity.

Perceptions and measurements of physical activity in patients with systemic lupus erythematosus.

Promoting physical activity should be a priority for patients with systemic lupus erythematosus (SLE) because a sedentary lifestyle compounds patients' already disproportionately high risk for cardiovascular events and other adverse health outcomes. The objectives of this pilot study were to assess physical activity in 50 patients with SLE and to compare activity levels with clinical and psychosocial variables, such as fatigue, depressive symptoms, and social support and stress. Patients were asked open-ended questions about physical activity, and responses were coded according to Grounded Theory. Patients then completed the Paffenbarger Physical Activity and Exercise Index, a survey of lifestyle energy expenditure reported in kilocalories/week, performed a 2-minute walk test according to a standard protocol, and completed questionnaires measuring fatigue, depressive symptoms and social support and stress. Most patients (92%) were women, had a mean age of 45 years, and did not have extensive SLE. In response to open-ended questions, patients reported they avoided physical activity because they did not want to exacerbate SLE in the short term. However, if they could overcome initial hurdles, 46 patients (92%) thought physical activity ultimately would improve SLE symptoms. Walking was the preferred activity and 45 (90%) thought they could walk more. According to the Paffenbarger Index, mean energy expenditure was 1466 ± 1366 kilocalories/week and mean time spent in moderate-intensity activity was 132 ± 222 min/week. In total, 18 patients (36%) and 14 patients (28%) met physical activity goals for these values, respectively. Mean distance walked during the 2-minute test was 149 ± 28 m, equivalent to two blocks, which is similar to reports for stable patients with other chronic diseases. Patients with more social stress and more fatigue reported less physical activity. We conclude that the proportion of patients meeting physical activity goals was low; however, patients performed well on a standard walking test. Most patients believed physical activity provided long-term benefits for SLE and that they could be more physically active.

Dysregulated complement activation as a common pathway of injury in preeclampsia and other pregnancy complications.

The complement system protects the host against invading organisms, initiates inflammation and dispose of immune complexes and the products of inflammatory injury. The complement system provides an important link between the innate and adaptive immune systems. Experimental observations suggest that increased complement activation causes and/or perpetuates inflammation during pregnancy. Recent studies suggest a link between complement activation and preeclampsia. Excessive activation or insufficient regulation of complement recruits leukocytes and unleashes potent inflammatory and anti-angiogenic mediators associated with placental insufficiency and maternal endothelial dysfunction characteristic of preeclampsia. We review the animal and human studies that link complement activation and pathogenic events in preeclampsia, present evidence that activation of the complement system is associated with the development of preeclampsia and provides new targets to prevent its complications.

The interrelationship of complement-activation fragments and angiogenesis-related factors in early pregnancy and their association with pre-eclampsia.

OBJECTIVE: To determine the interrelationships during early pregnancy of complement-activation fragments Bb, C3a and sC5b-9, and angiogenesis-related factors placental growth factor (PiGF), soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng), and their associations with pre-eclampsia. DESIGN: Prospective cohort study. SETTING: Denver complement study (June 2005-June 2008). POPULATION: A total of 668 pregnant women with singleton gestations, recruited between 10 and 15 weeks of gestation. METHODS: Using univariable and multivariable logistic regression analysis, concentrations of complement-activation fragments and angiogenesis-related factors were compared between 10 and 15 weeks of gestation in women who subsequently did or did not develop pre-eclampsia. Interrelationships between these variables were tested using the non-parametric Spearman rank correlation coefficient. MAIN OUTCOME MEASURE: Pre-eclampsia. The association of complement-activation fragments and angiogenesis-related factors with obesity was also examined. RESULTS: The mean (+/-SD) levels of complement Bb in early pregnancy among women who did and did not develop pre-eclampsia were 0.84 (+/-0.26) microg/ml and 0.69 (+/-0.2) microg/ml, respectively (P = 0.001). Concentrations of PiGF were significantly (P = 0.01) lower (31 +/- 12 pg/ml) in early pregnancy in the pre-eclamptic group of women, as compared with the normotensive group (39 +/- 32 pg/ml). The adjusted odds ratio (AOR) of Bb and PiGF were 2.1 (CI = 1.4-3.1, P < 0.0003) and 0.2 (CI = 0.07-0.7, P = 0.01), respectively. There was no significant difference in the levels of C3a, sC5b-9, sFlt-1 and sEng in early pregnancy among women who developed pre-eclampsia, compared with women who remained normotensive during pregnancy. Higher levels of Bb (P = 0.0001) and C3a (P = 0.03), and lower levels of sFlt-1 (P = 0.0002) and sEng (P = 0.0001) were found among women with obesity, compared with non-obese controls. No meaningful relationships were found between the complement-activation fragments and the angiogenesis-related factors. CONCLUSIONS: In this cohort during early pregnancy, increased concentrations of complement-activation factor Bb and lower concentrations of PiGF were associated with the development of pre-eclampsia later in pregnancy.

Serum complement activation on heterologous platelets is associated with arterial thrombosis in patients with systemic lupus erythematosus and antiphospholipid antibodies.

Complement plays a major role in inflammation and thrombosis associated with systemic lupus erythematosus (SLE) and the antiphospholipid syndrome (APS). A cross-sectional retrospective analysis was performed to evaluate serum complement fixation on platelets and thrombotic incidence using banked sera and clinical data from patients with SLE (n = 91), SLE with antiphospholipid antibodies (aPL) or APS (n = 78) and primary aPL (n = 57) or APS (n = 96). In-situ complement fixation was measured as C1q and C4d deposition on heterologous platelets using an enzyme-linked immunosorbent assay approach. Platelet activation by patient serum in the fluid phase was assessed via serotonin release assay. Enhanced in-situ complement fixation was associated with the presence of IgG aPL and IgG anti-beta2 glycoprotein 1 antibodies (P < 0.05) and increased platelet activation (P < 0.005). Moreover, enhanced complement fixation, especially C4d deposition on heterologous platelets, was positively associated with arterial thrombotic events in patients with SLE and aPL (P = 0.039). Sera from patients with aPL possess an enhanced capacity for in-situ complement fixation on platelets. This capacity may influence arterial thrombosis risk in patients with SLE.

Pathogenic role of antiphospholipid antibodies.

The antiphospholipid antibody syndrome (APS) is characterized by recurrent arterial and venous thrombosis and/or pregnancy in association with antiphospholipid (aPL) antibodies. The pathogenic mechanisms in APS that lead to in vivo injury are incompletely understood. Recent evidence suggests that APL antibodies alter regulation of haemostasis and induce activation of complement. We will discuss the current knowledge on how aPL antibodies trigger increased inflammation and enhanced thrombotic tendency, and thereby lead to tissue damage.

Anti-heparin platelet factor 4 antibodies in systemic lupus erythematosus are associated with IgM antiphospholipid antibodies and the antiphospholipid syndrome.

OBJECTIVE: To investigate the prevalence and clinical correlates of anti-heparin platelet factor 4 antibodies (anti-HPF4) in systemic lupus erythematosus (SLE) patients with and without antiphospholipid antibodies (aPL). METHODS: Sera and clinical data were obtained from the Hospital for Special Surgery Autoimmune Disease Registry for 78 aPL-positive and 91 aPL-negative SLE patients without heparin-induced thrombocytopenia (HIT). Controls were 90 blood donors of comparable age and sex. Sera were assayed for anti-HPF4, IgG/IgM antiphospholipid antibodies (APhL), and IgG/IgM anti-beta2-glycoprotein 1 antibodies (anti-beta 2GP1). Serotonin release assays (SRAs) were performed for subjects with positive anti-HPF4. RESULTS: Positive anti-HPF4 was seen in 9% of aPL-positive SLE patients, 4% of aPL-negative SLE patients and 1% of controls (p = 0.026, aPL-positive SLE vs controls). Two of 12 subjects with positive anti-HPF4 had reactive SRAs. In SLE patients, anti-HPF4 significantly correlated with IgM APhL, IgM anti-beta2GP1, and inversely with complement C4. In immunoabsorption experiments, there was partial cross-reactivity of IgM anti-HPF4 with IgM APhL, but not with IgM anti-beta 2GP1. SLE patients with positive anti-HPF4 had increased odds of the antiphospholipid syndrome (APS; odds ratio (OR) 4.5, p = 0.019), and APS with arterial thrombosis (OR 6.1, p = 0.007). In multivariate linear regression analyses, APS and IgM APhL were independently associated with anti-HPF4. CONCLUSIONS: Anti-HPF4 is detectable in SLE patients with and without aPL in the absence of HIT, and is most prevalent in aPL-positive SLE patients. In this SLE cohort, anti-HPF4 correlates with IgM APhL, IgM anti-beta 2GP1 and inversely with C4, and is associated with manifestations of APS.

The clinical and public health value of non-culture methods in the investigation of a cluster of unexplained pneumonia cases.

During 2003, a cluster of initially unexplained pneumonia cases (two fatal) occurred in patients aged <50 years in a British city. Routine culture tests were inconclusive, however, pneumococcal infection was suspected and the putative outbreak was investigated using non-culture methods. Clinical samples from ten patients were tested by pneumococcal polymerase chain reaction (PCR) and multi-locus sequence typing (MLST), or Binax NOW pneumococcal urine antigen test and serotype-specific enzyme-linked immunosorbent assay (ELISA). Lung samples from the deceased patients were PCR positive and yielded different MLST types. Two patients in one family group were serotype 1 pneumococcal antigen positive. Two further patients were serotype 1 antigen positive, and one serotype 4 positive. Two antigen-positive cases were also serum PCR positive. Non-culture methods confirmed the disease aetiology in six cases. Serotype and MLST results showed no single outbreak, but a family cluster of cases in a high background of pneumococcal pneumonia, providing important epidemiological data that would not otherwise have been available.

Activation of complement mediates antiphospholipid antibody-induced pregnancy loss.

Although it is clear that the specific antigenic reactivity of antiphospholipid (aPL) antibodies is critical to their effect, the pathogenic mechanisms that result in injury in vivo are incompletely understood. We hyphothesized that aPL antibodies targeted to the placenta activate complement locally, generating split products that mediate placental injury and lead to foetal loss and growth retardation. To test this hypothesis, we used a murine model of APS in which pregnant mice are injected with human IgG containing aPL antibodies. Mice treated with inhibitors of complement activation and mice deficient in complement components were protected from aPL antibody-induced foetal damage. Although the cause of tissue injury in this disease is probably multifactoral, we have shown that complement activation is an absolute requirement for foetal loss and growth restriction and, therefore, thatthis pathway acts upstream of other important effector mechanisms. Identification of complement activation as a mechanism that is necessary for aPL-induced tissue damage and definition ofthe complement components necessary to trigger such injury is likely to lead to a better understanding of the pathogenesis of vascular and tissue injury in SLE and to new and improved treatments.

An outbreak of serious illness and death among injecting drug users in England during 2000.

An outbreak of serious illness and death occurred in injecting drug users during 2000 in Scotland, Ireland and England. National and international collaboration was necessary for the investigation and management of this outbreak. In England and Wales active case-finding was initiated, coupled with standardised data collection and microbiological investigation of cases. Twenty-six definite or probable cases were identified in England between 1 April and 31 Aug. 2000; 17 of these occurred in the North. The overall case fatality was 50% (13/26). The principal apparent risk factor was a history of intramuscular or subcutaneous injection of heroin and the limited duration of the outbreak suggested that the problem might have been related to a particular supply of heroin. Clostridium novyi was isolated from two English cases. Taken in conjunction with contemporaneous microbiological and epidemiological results from Scottish and Irish cases, the probable aetiology for this outbreak was infection with C. novyi associated with both a particular supply of heroin and the method of preparation and injection used. A 'toolkit' was distributed in Sept. 2000 to all Consultants for Communicable Disease Control in England and Wales to assist them with the ongoing surveillance, investigation and management of this condition. Lessons learned have been used to produce guidance for the investigation and management of outbreaks of unexplained serious illness of possible infective aetiology.

Isolation and identification of Clostridium spp. from infections associated with the injection of drugs: experiences of a microbiological investigation team.

Pathogenic species of the genus Clostridium may contaminate the materials used in the injection of drugs and under the right conditions may cause serious or life-threatening disease. C. novyi type A was implicated in an outbreak of severe infection with high mortality in injecting drug users who injected heroin extravascularly. The isolation of such highly oxygen-sensitive clostridia from clinical material may require adherence to enhanced methods and, once isolated, commercially available anaerobe identification kits alone may not give an accurate identification. Additional phenotypic tests that are useful in recognising the main pathogenic species are described. Differentiation of C. novyi type A from C. botulinum type C in reference laboratories was based on 16S rDNA sequence data and specific neutralisation of cytopathic effects in tissue culture.

Amplified fragment length polymorphism (AFLP) analysis of Clostridium novyi, C. perfringens and Bacillus cereus isolated from injecting drug users during 2000.

As part of the follow-up investigations associated with an outbreak of severe illness and death among illegal injecting drug users during 2000, 43 cultures of Clostridium novyi type A, 40 C. perfringens type A and 6 isolates of Bacillus cereus were characterised by amplified fragment length polymorphism (AFLP) analysis. Among the 43 C. novyi isolates, 23 different AFLP profiles were detected. The same AFLP profile was detected in isolates from 18 drug users investigated during 2000 from Scotland, England, the Republic of Ireland and Norway and a wound from a patient in 2000 who was not identified as a drug user. Unique AFLP profiles were obtained from four drug users from England and the Republic of Ireland, 10 historical isolates from culture collections, an isolate from food (1989) and three isolates from wounds (1995, 1991, 1988). The 40 C. perfringens isolates were from 13 drug users, the contents of one syringe and two samples of heroin. Sixteen AFLP types of C. perfringens were distinguished and there was little evidence for commonality among the isolates. The AFLP types of C. perfringens from heroin differed and were unique. Six isolates of B. cereus were from four drug users and two samples of heroin. Four different AFLP patterns were distinguished. Three AFLP types were isolated from four drug users. B. cereus isolates from an aspirate and a heroin sample collected from the same drug user were identical, and were also indistinguishable from an isolate from a groin infection in a second drug user. The AFLP type of the isolate from a second and unrelated heroin sample was unique. The AFLP results showed no or very limited evidence for commonality between the different isolates of B. cereus and C. perfringens. In marked contrast, the C. novyi isolates from the majority of the drug users during 2000 were homogeneous, suggesting a common source or clonal selection of a C. novyi type, or both, which either had an adaptive advantage in spore germination, survival or growth following the drug preparation and the injection procedure, or produced a more severe clinical presentation.

Characterization of the FcgammaRIIA promoter and 5'UTR sequences in patients with systemic lupus erythematosus.

FcgammaRIIA is a candidate gene involved in the predisposition to systemic lupus erythematosus (SLE). The presence of low binding alleles in patients with SLE is not sufficient to explain the lower phagocytic capacity observed in SLE patients. We considered the possibility that nucleotide polymorphisms in the FcgammaRIIA promoter that cause alterations in receptor expression might be present in SLE patients. In the present study, a 2.0 kb region of the human FcgammaRIIA 5'UTR from 20 normal donors and 53 SLE patients was examined. The results demonstrate that the sequence of the human FcgammaRIIA 5' region differs from the published sequence. Two novel SNPs have been identified in the distal region of the FcgammaRIIA promoter. The polymorphisms are present in both disease-free and SLE donors and do not associate with quantitative changes in FcgammaRIIa phagocytic function.

Accelerated atherosclerosis in systemic lupus erythematosus: implications for patient management.

One of the most compelling clinical challenges in the management of systemic lupus erythematosus (SLE) is the high incidence of atherosclerotic cardiovascular disease (ASCVD). Potential mechanisms for accelerated atherosclerosis in SLE include chronic inflammation, excess of traditional risk factors, and corticosteroid therapy. Given the high prevalence of atherosclerosis in SLE patients relative to young women in the general population, we propose that the presence of SLE constitutes a sufficiently potent risk factor for ASCVD to warrant more aggressive goals for risk factor reduction and strategies to reduce inflammation.