Activation of Complement Following Total Hip Replacement.

The aim of this study was to investigate whether complement activation, via the classical and alternative pathways, occurs following a cemented total hip replacement (THR) surgery due to osteoarthritis. Blood samples were collected systematically from 12 patients - six male and six women, with a median age of 75 (range: 59-90 years) - preoperatively, 6 h post-operatively and on the first, second and third post-operative day. Total function of classical (CH50) and alternative pathways (AH50) was evaluated, along with the determination of serum concentrations of the complement proteins C3, C4, C3d, the soluble terminal complement complex (sTCC) sC5b-9, as well as C-reactive protein (CRP) and albumin. Measurements of CRP and albumin levels elucidated a marked inflammatory response following the operation. The CH50, AH50 and C3 and C4 levels were significantly lower 6 h after the surgery compared with the preoperative levels, but elevated above the preoperative levels during the following 3 days. The complement activation product C3d levels increased continually during the whole observation period, from 13.5 AU/ml (range: 8-19 AU/ml) preoperative to 20 AU/ml (range: 12-34 AU/ml) on the third post-operative day. Furthermore, we observed an increase in the sC5b-9 levels between the preoperative and the third post-operative day. These results demonstrate a significant activation of the complement system following cemented THR. Further studies are needed to elucidate the time frame and the pathogenic role of this observed complement activation.

Do non-steroidal anti-inflammatory drugs influence chronic inflammation? The effects of piroxicam on chronic antigen-induced arthritis in rats.

OBJECTIVE: The effects of non-steroidal anti-inflammatory drugs (NSAIDs) on acute inflammation have been thoroughly investigated. NSAIDs are, however, also prescribed for patients with chronic inflammation, such as rheumatoid arthritis (RA), and objective improvement suggestive of anti-inflammatory action from NSAIDs has not been convincingly shown in chronic RA. An antigen-induced arthritis (AIA) model was used to investigate the effects of piroxicam on chronic inflammation. METHODS: AIA was induced by injecting methylated bovine serum albumin (mBSA) into the knee joints of previously immunized rats that were treated orally with the NSAID piroxicam or with saline. This treatment was started either before AIA was induced or after it had reached a chronic phase. The findings were recorded by clinical and histological assessment of the joints. RESULTS: The piroxicam group developed significantly less acute and subsequent chronic knee joint inflammation but this was only evident if the drug was administered prior to the intra-articular mBSA injections. Piroxicam treatment that was initiated during the chronic inflammation did not have any clinical effect, whereas short-term corticosteroid treatment abolished the chronic inflammation. Moreover, histological analysis of the chronic inflammation revealed significantly more inflammatory changes in the piroxicam group compared with the control group. CONCLUSIONS: Piroxicam treatment had no beneficial effects on the chronic stable inflammation in this model and might even delay histological resolution. As the anti-inflammatory effect of piroxicam is restricted to acute inflammation, the use of NSAIDs during periods of chronic stable arthritis in humans, such as in RA, may need to be investigated.

The effects of tobacco smoking and rheumatoid factor seropositivity on disease activity and joint damage in early rheumatoid arthritis.

OBJECTIVE: To study the effect of tobacco smoking and rheumatoid factor (RF) isotypes on disease activity and joint damage in early rheumatoid arthritis (RA). METHODS: One hundred early RA patients were followed prospectively for 2 yr. They were evaluated at recruitment and at 6 and 24 months. Sociodemographic information included smoking history, and radiographs of hands and feet were obtained. RF was monitored by IgM- and IgA-specific RF enzyme-linked immunosorbent assay and by agglutination, and serial measurements were also obtained for C-reactive protein. The influence of tobacco smoking and RF positivity on disease outcome was evaluated using multivariate analysis. Covariates for the regression analysis included sex, age, coffee consumption and IgA-RF positivity. RESULTS: A gradient of increase in disease activity was observed from never smokers to former smokers to current smokers during the 2 yr of observation, defined by number of swollen joints (SJC), tender joints (TJC) and visual analogue scale for pain (P<0.001, P=0.02 and P=0.005, respectively), but smoking status did not influence radiological progression. Ever smokers were more often IgA RF positive (P<0.05). IgA RF-positive patients had more active disease (SJC P=0.002, TJC P=0.01) and showed more radiological progression (P<0.0001) compared with IgA RF-negative patients. Of the RF-positive patients 22% had elevated IgM RF without IgA RF and these patients showed similar disease activity and radiological joint progression to the RF-negative patients. None of these associations were explained by possible confounders. CONCLUSION: Tobacco smoking has an adverse effect on patients with early RA and this is possibly immunologically mediated. IgM RF does not predict poorer prognosis in RA unless it is associated with a concomitant elevation of IgA RF.

Mannan-binding lectin and complement C4A in Icelandic multicase families with systemic lupus erythematosus.

OBJECTIVE: To determine whether low mannan-binding lectin (MBL) and C4A null alleles (C4AQ0) are associated with systemic lupus erythematosus (SLE) in multicase families with SLE. METHODS: Low MBL level was determined by measuring serum levels and by genotyping for mutant structural (B/C/D, designated as 0) and promoter (LX) alleles (by real-time polymerase chain reaction). C4AQ0 was detected by protein electrophoresis and corroborated with haplotype and genotype analysis. In nine Icelandic families, 24 patients with SLE were compared with 83 first-degree and 23 second-degree relatives without SLE. Twenty four unrelated family members and a population group of 330 Icelanders served as controls. RESULTS: Overall, the frequency of low MBL genotypes (0/0, LX/0 and wild-type/0) tended to be higher in patients with SLE than in their first-degree and second-degree relatives (p = 0.06), but the frequency was similar in the families and in the controls (p = 0.6). The frequency of C4AQ0 was, however, increased in patients and their relatives compared with that in the controls (p = 0.04). The combination of low MBL genotypes and C4AQ0 was found more often in the patients than in their relatives (p = 0.03) and controls (p = 0.02). However, low MBL level was observed only in patients and first-degree relatives in five of the nine multicase families. In these five families, patients with SLE had low MBL genotypes more often (64%) than their first-degree (38%) and second-degree (0%) relatives (p = 0.001), and the patients with SLE also had, accordingly, lower MBL levels than their relatives (p = 0.001). CONCLUSIONS: These findings indicate that low MBL levels can predispose people to SLE and highlight the genetic heterogeneity of this disease.

Childhood levels of immunoglobulins and mannan-binding lectin in relation to infections and allergy.

Respiratory tract infections, allergies and otitis media are common problems in early childhood. Our aim was to evaluate in a longitudinal community-based cohort study the association between maturation of immunoglobulin (Ig) and mannan-binding lectin (MBL) responses and disease manifestations in the first 4 years of life. Sustained low levels of IgA proved the strongest single indicator of susceptibility for recurrent otitis media (P = 0.008) and respiratory tract infections (P = 0.02), and this condition was also associated with low production of IgG subclasses. About 7% of the cohort had sustained low levels of MBL (<0.4 mg/l). Low MBL did not predispose to any ailments studied, but children with low IgA and recurrent otitis media had relatively low MBL at birth, which failed to increase during the study period and was significantly reduced at the age of 4 years (P = 0.04). MBL levels increased from birth to 2 years (P < 0.0001) and were higher in children than in adults (P = 0.001). The increase was 1.9-fold in children with no recorded clinical events and 1.7-fold in children with asthma or infections, but significantly lower, 1.2-fold, in children with recurrent otitis media. Low levels of IgA within the normal range may reveal disease susceptibility not detected by conventional criteria. Slow maturation of Ig appears to be the main factor of susceptibility during childhood, but a strong corollary role for MBL is indicated by the high levels produced during childhood as well as the precipitation of disease in children with low levels of MBL and Ig.

The potential role of mannan-binding lectin in the clearance of self-components including immune complexes.

Mannan-binding lectin (MBL) is a pattern recognition receptor in the innate immune system. It recognizes certain sugar residues arranged in a pattern that enables MBL to bind with sufficient strength. Such sugar patterns are common on the surface of many microorganisms, and MBL has therefore been considered to be an agent that can discriminate between self and nonself. There is, however, increasing evidence supporting that MBL, like many membrane-bound C-type lectin-like receptors, also helps to dispose of various outworn or abnormal body components. Most self-components are protected with sialic acid or galactose that disrupt the pattern of the sugars that MBL can bind, but MBL may be significantly involved in the elimination of self-components that have lost these protective terminal residues. The role of MBL in the clearance of invading pathogens has previously been thoroughly reviewed. Here, we review some findings that support the notion that MBL may contribute to noninflammatory removal of immune complexes and abnormal cells by the reticuloendothelial system. Defects in this clearance mechanism may cause an accumulation of potentially dangerous self-components, thereby increasing the likelihood of chronic inflammation and autoimmunity.

Human plasma-derived mannose-binding lectin: a phase I safety and pharmacokinetic study.

Mannose-binding lectin (MBL) is an important component of innate immunity that can bind to certain sugar residues on the surface of many types of pathogenic micro-organisms. On binding, MBL generates opsonic activity mainly through activation of the complement system. Genetically determined MBL deficiency is very common and can be associated with increased susceptibility to a variety of infections, especially in children and immunosuppressed individuals. The potential benefits of MBL reconstitution therapy therefore need to be evaluated. We have carried out a phase I safety and pharmacokinetic study on 20 MBL-deficient healthy adult volunteers. The MBL was prepared from plasma of nonremunerated, voluntary Danish donors tested and found negative for hepatitis B surface antigen, antibodies to human immunodeficiency virus (HIV) and hepatitis C virus. Each volunteer received a total of 18 mg of MBL in three 6 mg doses given intravenously, once weekly over a period of 3 weeks. The volunteers were closely monitored at the University Hospital in Reykjavik for 8 h after each infusion and daily thereafter for 5 days after each infusion. No adverse clinical or laboratory changes were observed in any of the 20 participants, and frequent measurements did not reveal any signs of infusion-associated complement activation. No antibodies to MBL, HIV or hepatitis viruses were observed 24 weeks after the last infusion. Serum MBL levels increased up to normal levels (1200-4500 ng/ml) immediately after each infusion, but the half-life of the infused MBL was highly variable, ranging from 18 to 115 h (mean 69.6). It is concluded that infusion of purified MBL as prepared by Statens Serum Institut (SSI) is safe. However, adults have to be given at least 6 mg twice or thrice weekly for maintaining protective MBL levels assumed to be about 1000 ng/ml.

Deficiency of complement-dependent prevention of immune precipitation in systemic sclerosis.

BACKGROUND: Previous studies have indicated that complement may be activated or inherently abnormal in systemic sclerosis (SSc), and it has been suggested that immune complex deposition plays a part in the microvascular damage of this disease. OBJECTIVE: To study several aspects of the complement system in 24 patients with SSc. METHODS: Complement dependent prevention of immune precipitation (PIP) was measured by a sensitive enzyme immunoassay, levels of C1q, C4, and C3 by rocket immunoelectrophoresis, C4 allotypes by high voltage agarose electrophoresis, and C4A, C4B, and C3d by an enzyme linked immunosorbent assay (ELISA). RESULTS: PIP was markedly decreased in the patients with SSc (p<0.001). Abnormal complement activation was detected in nine patients as raised levels of the complement split product C3d. However, a relation between low PIP and complement activation was not seen. PIP was significantly lower in patients who carried the C4A*Q0 allotype (p=0.03), and a strong correlation was found between PIP and C4A concentration (p<0.00001). The PIP defect may, at least in some patients, be associated with the initial phase of the disease. CONCLUSIONS: The results show a previously unrecognised functional defect of complement in SSc; the defect correlates with low levels of classical pathway components and, in particular, C4A.

Low mannose binding lectin predicts poor prognosis in patients with early rheumatoid arthritis. A prospective study.

OBJECTIVE: To determine whether low mannose binding lectin (MBL) is associated with poor prognosis in rheumatoid arthritis (RA) and whether patients with RA have increased frequency of MBL deficiency. METHODS: Patients with recent onset symmetric polyarthritis (< 1 year, median 3 mo) were recruited if they had not been treated longer than 2 weeks with disease modifying drugs. They were reevaluated after 6 months and their disease activity and progression were correlated with their MBL concentration, rheumatoid factor (RF) isotypes, and C-reactive protein (CRP). Sixty-three female patients with advanced RA were also analyzed. RESULTS: Sixty-five patients with early arthritis fulfilled American College of Rheumatology criteria for RA and 52 were followed for 6 months or longer. Low MBL was associated with raised RF, IgA RF in particular (p = 0.02). and also with a combined elevation of IgM and IgA RF (p = 0.035). Patients with low MBL (lowest 25th percentile) showed less improvement after 6 months of treatment than patients in the highest MBL quartile. This applied to the Thompson joint score (p = 0.03) and grip strength (p = 0.004). Low MBL was also significantly associated with radiological joint erosions at recruitment and at 6 month followup (p = 0.039); and the group with advanced RA also showed a significant association between low MBL concentration and radiological damage (p = 0.036). However. neither patient group had increased frequency of MBL deficiency compared to healthy controls. CONCLUSION: Low MBL predicts poor prognosis in patients with early RA.

Enzyme immunoassays for measuring complement-dependent prevention of immune precipitation (PIP) and solubilization of preformed antigen-antibody complexes (SOL).

We have developed simple and sensitive enzyme-based methods for evaluating the ability of serum complement to prevent immune complex precipitation (PIP) or to solubilize preformed immune complexes (SOL). Alkaline phosphatase, serving both as antigen and label, is added to goat IgG anti-alkaline phosphatase antibodies, with serum present throughout the assay (PIP), or added after immune complex formation (SOL). After incubation at 37 degrees C for 1 h followed by centrifugation, the enzyme activity of the supernatant, reflecting the amount of immune complexes in solution, is measured by colorimetry. Results are expressed with reference to a standard serum pool assigned 100 arbitrary units (AU). Intra- and inter-assay variabilities are within 10%. The normal ranges were 67-133 AU for PIP and 72-129 AU for SOL. These methods have been standardized for clinical use in relation to impaired complement function and immune complex disease, and adapted for measuring complement mediated binding of immune complexes to erythrocytes. They are sensitive, easy to perform and do not require expensive facilities. By measuring the interaction of complement with immune complexes, these methods may highlight aspects of the classical and the alternative pathway that are different from those detected using haemolysis as an endpoint.

Reconstitution of opsonizing activity by infusion of mannan-binding lectin (MBL) to MBL-deficient humans.

Mannan-binding lectin (MBL, previously named mannan-binding protein, MBP) is a serum collectin, which activates complement upon binding to microbial carbohydrates. This results in opsonization of the microorganisms as well as direct complement-mediated killing. Clinical evidence indicates that MBL has an important role in the innate immune defence against various pathogens. Genetically determined MBL deficiency is associated with increased susceptibility to infections. We have infused two MBL-deficient individuals with clinical grade MBL, purified from pooled donor plasma, in doses sufficient to attain normal concentration of MBL in serum. This resulted in normalization of complement-mediated opsonization. An initial rapid decrease in the serum concentration of MBL was followed by a slower decline with an estimated half-life of about 6 days. No adverse effects were observed and anti-MBL antibodies could not be detected following several MBL infusions. One of the two MBL recipient, a two-year-old girl, who had been suffering from repeated infections from the age of 4 months, was given a total of six MBL infusions. She has subsequently remained healthy for more than three years.

Rate-limiting components and reaction steps in complement-mediated haemolysis.

The aims of this study were to identify the rate-limiting components and reaction steps in the integrated activation sequence of the alternative (AP) and classical (CP) pathways of the complement (C) system. In an initial correlation analysis we found that the haemolysis rate in AP was correlated with the concentrations of C5 and IgM. In CP, the haemolysis rate was correlated with the concentrations of C2-C6, factors I and B, and IgM. In order to identify the rate-limiting components, we added single, purified C components and IgM to pooled, normal human serum and measured the resultant change in the haemolysis rate. We found that a large number of different components, rather than a single one, were rate-limiting in AP and CP. In reconstitution experiments we found that in CP the rate-limiting reaction steps are the activation of C4 and C2. In AP we cannot identify the rate-limiting step precisely, but can only state that it is at the C3 activation step or earlier.