Primary immunodeficiency and autoimmunity: lessons from human diseases.

Primary immunodeficiency diseases (PID) are a genetically heterogenous group of >150 disorders that affect distinct components of the innate and adaptive immune system and are often associated with autoimmune diseases. We describe PID affecting T-regulatory cells, complement and B cells or their products and discuss the possibility of a cause-effect relationship. The high concordance of T-regulatory cell defects to organ-specific autoimmune disease implies an obligatory role of these cells in maintaining tolerance to epithelial and endocrine tissues; the absence of central nervous system involvement may reflect immunological privilege. Congenital defects in C1q, C1r/s and C4 are strongly associated with systemic lupus erythematosus (SLE), and this pattern along with laboratory evidence suggests a major importance of classical pathway activity in safe elimination of immune complexes and prevention of immune complex disease (ICD). It is debatable whether this ICD is to be regarded as an autoimmune disease (resulting from a breakdown of immunological ignorance to antigens that are normally hidden), as autoantibodies may be absent, and tissue damage because of deposition of immune complexes could account for all of the pathology observed. Evidence for a causative link between primary antibody deficiencies and autoimmune disease is much less compelling and may in fact involve a common genetic background. However, arguments have also been made in favour of the notion that an intense antigen load as a result of recurrent or persistent infections may affect either tolerance or ignorance, e.g. by molecular mimicry or the presence of superantigens. Similar immunological mechanisms might account for the vast majority of autoimmune diseases.

Profiling of cell cycle genes of breast cells exposed to etodolac.

Breast cancer represents the second leading cause of cancer-related deaths in the world. There is increasing evidence that perturbation of cell cycle regulation is an important contributing factor to various cancer progression stages. There are key checkpoints in the cell cycle involving various regulatory proteins. The relationship between these cell cycle regulatory proteins and cell cycle arrest by cyclo-oxygenase (COX) inhibitors during neoplastic progression remains largely unknown. Preclinical studies and epidemiological investigations have consistently shown that non-steroidal anti-inflammatory drugs have some anti-proliferative and anti-oxidative stress response on various tumors. In this study, the effect of etodolac, a 1,8-diethyl-1,3,4,9-tetrahydro-pyrano (3,4-beta) indole-1-acetic acid on signaling pathways was investigated by examining the differential expression of various cell cycle regulatory protein genes. A human cell cycle gene array was used to profile the expression of 96 genes involved in the cell cycle regulation. Differentially expressed genes were highly altered by etodolac treatment. Twenty-six genes were up- and 20 down-regulated with 0.5 and 2 mM etodolac treatment, respectively. Seven genes (ATM, BAX, CCNA2, CDC27, RAD50 and p21) were prominently altered, and six (ATM, CCND2, CCNF, CDC20, CDK1A and RAD50) were commonly altered with both concentrations. This finding indicated that etodolac could play a critical role on cancer cells by inducing cell death.

Smoking and a complement gene polymorphism interact in promoting cardiovascular disease morbidity and mortality.

We have demonstrated previously that carriers of a genotype called C4B*Q0 (silent allele of the C4B gene) have a substantially increased risk to suffer from myocardial infarction or stroke, and are selected out from the healthy elderly population. Because smoking carries a major risk for cardiovascular disease (CVD), it seemed worthwhile to study if these two factors interact. Study 1 involved 74 patients with angina pectoris (AP), 85 patients with recent acute myocardial infarction (AMI) and 112 survivors of a previous AMI and 382 controls from Iceland. Study 2 involved 233 patients with severe CVD and 274 controls from Hungary. Smoking habits were registered for each subject. The number of C4A and C4B genes was determined by phenotyping or genotyping. Compared to controls, C4B*Q0 carrier frequency was significantly higher at diagnosis in Icelandic smokers with AP (P = 0.005) and AMI (P = 0.0003) and Hungarian smokers with severe coronary artery disease (P = 0.023), while no such difference was observed in non-smoking subjects. Age-associated decrease in C4B*Q0 observed previously in two remote Caucasian populations was found, in the present study, to be associated strongly with smoking, and to already occur in smokers after age 50 years both in Iceland and Hungary. Our findings indicate that the C4B*Q0 genotype can be considered as a major covariate of smoking in precipitating the risk for AMI and associated deaths.

Characterizing discrete subsets of polycystic ovary syndrome as defined by the Rotterdam criteria: the impact of weight on phenotype and metabolic features.

CONTEXT: The Rotterdam criteria for polycystic ovary syndrome (PCOS) defines discrete subgroups whose phenotypes are not yet clear. OBJECTIVE: The phenotypic characteristics of women in the PCOS subgroups defined by the Rotterdam criteria were compared. DESIGN: The study was observational. SETTING: Subjects were studied in an outpatient setting in Boston and Reykjavik. PATIENTS: Four subgroups of subjects with PCOS defined by 1) irregular menses (IM), hyperandrogenism (HA), and polycystic ovary morphology (PCOM, n = 298); 2) IM/HA (n = 7); 3) HA/PCOM (n = 77); and 4) IM/PCOM (n = 36) and a group of controls (n = 64), aged 18-45 yr, were examined. INTERVENTION: Subjects underwent a physical exam; fasting blood samples for androgens, gonadotropins, and metabolic parameters; and a transvaginal ultrasound. MAIN OUTCOME MEASURES: The phenotype was compared between groups. RESULTS: Ninety-seven percent of women with IM/HA had PCOM. Therefore, the groups with and without PCOM were combined. The Ferriman-Gallwey score and androgen levels were highest in the hyperandrogenic groups (IM/HA and HA/PCOM), whereas ovarian volume was higher in all PCOS subgroups compared with controls, as expected based on the definitions of the PCOS subgroups. Body mass index and insulin levels were highest in the IM/HA subgroup. CONCLUSIONS: Subjects with PCOS defined by IM/HA are the most severely affected women on the basis of androgen levels, ovarian volumes, and insulin levels. Their higher body mass index partially accounts for the increased insulin levels, suggesting that weight gain exacerbates the symptoms of PCOS.

Defining constant versus variable phenotypic features of women with polycystic ovary syndrome using different ethnic groups and populations.

CONTEXT: The phenotype of women with polycystic ovary syndrome (PCOS) is variable, depending on the ethnic background. OBJECTIVE: The phenotypes of women with PCOS in Iceland and Boston were compared. DESIGN: The study was observational with a parallel design. SETTING: Subjects were studied in an outpatient setting. PATIENTS: Women, aged 18-45 yr, with PCOS defined by hyperandrogenism and fewer than nine menses per year, were examined in Iceland (n = 105) and Boston (n = 262). INTERVENTION: PCOS subjects underwent a physical exam, fasting blood samples for androgens, gonadotropins, metabolic parameters, and a transvaginal ultrasound. MAIN OUTCOME MEASURES: The phenotype of women with PCOS was compared between Caucasian women in Iceland and Boston and among Caucasian, African-American, Hispanic, and Asian women in Boston. RESULTS: Androstenedione (4.0 +/- 1.3 vs. 3.5 +/- 1.2 ng/ml; P < 0.01) was higher and testosterone (54.0 +/- 25.7 vs. 66.2 +/- 35.6 ng/dl; P < 0.01), LH (23.1 +/- 15.8 vs. 27.6 +/- 16.2 IU/liter; P < 0.05), and Ferriman Gallwey score were lower (7.1 +/- 6.0 vs. 15.4 +/- 8.5; P < 0.001) in Caucasian Icelandic compared with Boston women with PCOS. There were no differences in fasting blood glucose, insulin, or homeostasis model assessment in body mass index-matched Caucasian subjects from Iceland or Boston or in different ethnic groups in Boston. Polycystic ovary morphology was demonstrated in 93-100% of women with PCOS in all ethnic groups. CONCLUSIONS: The data demonstrate differences in the reproductive features of PCOS without differences in glucose and insulin in body mass index-matched populations. These studies also suggest that measuring androstenedione is important for the documentation of hyperandrogenism in Icelandic women. Finally, polycystic ovary morphology by ultrasound is an almost universal finding in women with PCOS as defined by hyperandrogenism and irregular menses.

Defective prevention of immune precipitation in autoimmune diseases is independent of C4A*Q0.

Increased prevalence of C4 null alleles is a common feature of autoimmune diseases. We have shown previously that complement-dependent prevention of immune precipitation (PIP) is defective in patients with systemic lupus erythematosus (SLE), and correlated this defect with C4A*Q0 and low levels of the C4A isotype. To further clarify the role of C4A in the aetiology of SLE, we now extend our studies to other diseases which have been associated with C4A*Q0. The frequency of C4A*Q0 was increased in Icelandic patients with coeliac disease (0.50; P < 0.001), Grave's disease (0.30; P = 0.002) and insulin-dependent diabetes mellitus (0.23; P = 0.04) and in British patients with dermatitis herpetiformis (0.42; P = 0.002) and this was reflected in low levels of C4A. In spite of this, PIP was normal in these patients, and in marked contrast to our previous observations on connective tissue diseases, PIP measurements in these patient groups correlated more strongly with levels of C4B (r = 0.51, P = 0.0000004) than C4A. Patients with increased levels of anti-C1q antibodies had significantly lower PIP than patients without such antibodies (P < 0.01) and a negative association of PIP with anti-C1q antibodies was also reflected in an increased prevalence (P = 0.006) and levels (P = 0.006) of anti-C1q antibodies in patients with subnormal PIP, as well as a negative correlation between PIP and anti-C1q antibodies (r = - 0.25, P = 0.02). These results show that the PIP defect cannot be explained by low levels of C4A alone and suggest that measurements of anti-C1q antibodies may be useful in future studies on the molecular cause of the PIP defect in autoimmune connective tissue 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.

Increased frequency of C4B*Q0 alleles in patients with Henoch-Schönlein purpura.

Henoch-Schonlein purpura (HSP) is a vasculitis of unknown aetiology, possibly involving immune complexes. The complement system is essential for the clearance of immune complexes. Our aim was to explore the hypothesis that patients with HSP have abnormal complements, contributing to the development of the disease. The study included 56 patients diagnosed with HSP at the Children's Hospital, Iceland between 1984 and 2000, and 98 blood donors as controls. Serum levels of immunoglobulin A, C4A, C4B and mannan-binding lectin were measured and compared between the two groups. C4 null alleles were significantly more common in HSP patients than in controls (P = 0.018) and were carried by 66.1% of the patients compared with 41.2% of the controls. This difference was due to an increased frequency of C4B*Q0 allele in the HSP group (0.25 versus 0.11 in the control group; P = 0.002). The fact that the majority of our patients carried a C4 null allele indicates that children with C4 deficiencies may have an increased risk of developing HSP. This may reflect inadequate complement activity and possibly present an opportunity to identify patients at risk of developing serious morbidity associated with HSP.

Allergic diseases and asthma in relation to serum immunoglobulins and salivary immunoglobulin A in pre-school children: a follow-up community-based study.

BACKGROUND: We have previously reported an association between low IgA and allergic manifestations in early childhood (0-2 years) and have now followed our cohort for an additional 2 years. OBJECTIVE: To evaluate in a longitudinal community-based cohort study the association between maturation of Ig production and allergic manifestations in the first 4 years of life. METHODS: A cohort of 161 randomly selected children was followed from birth to the age of 42-48 months and evaluated at 18-23 months (EV1; n = 179) and again at the age of 42-48 months (EV2; n = 161). Diagnoses were made with the help of a clinical questionnaire, physical examination and skin prick tests (SPTs) to 10 common allergens. Serum immunoglobulins were measured at EV1 and EV2, and salivary IgA (sal-IgA) at EV2. RESULTS: Serum IgA, IgE, IgG1, IgG2 and IgG4 increased from 2 to 4 years of age (P < 0.001) and their levels showed close correlations (P < or = 0.01 for most comparisons). Children with one or more positive SPTs had lower serum IgA (P = 0.004) and IgG4 (P = 0.05) at EV2 than those who did not respond, and children who developed allergic rhinitis between EV1 and EV2 had low sal-IgA (P = 0.006) and IgG3 (P < 0.05) at EV2. Atopic eczema was associated with low sal-IgA at EV2, and children who developed eczema between EV1 and EV2 had significantly lower sal-IgA than those who recovered after EV1 (P = 0.02). CONCLUSION: Allergic manifestations in predisposed children may be influenced by the rate of maturation of immunological components that counteract sensitization or inhibit effector mechanisms of allergy.

Patients with systemic lupus erythematosus are deficient in complement-dependent prevention of immune precipitation.

OBJECTIVE: A functional deficiency of complement has been implicated but not conclusively demonstrated in the pathogenesis of systemic lupus erythematosus (SLE). To test this, we studied several aspects of complement in 44 patients with SLE, 46 patients with rheumatoid arthritis and 102 blood donors. METHODS: Prevention of immune precipitation (PIP) was measured by an enzyme immunoassay, levels of C1q, C4 and C3 by rocket immunoelectrophoresis, C4A, C4B and C3d by enzyme-linked immunosorbent assay (ELISA), complement haemolysis (CH50) by standard methods and C4 allotypes by high-voltage agarose electrophoresis and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: PIP was significantly reduced in SLE (P<0.001); the defect was revealed by a sensitive assay measuring this function of complement but not by the other tests employed. The patients were clinically well at the time of study, and levels of C3d, which have been shown to correlate with disease activity, were normal. The defect was more common in patients with early disease (P = 0.009), supporting a role in aetiology or early pathophysiology. PIP was positively correlated with levels of C4 (P = 3 x 10(-5)) and in particular the C4A isotype (P = 9 x 10(-10)) whereas C4B was redundant. CONCLUSIONS: Our results reveal a defect in prevention of immune precipitation in SLE that is apparent at an early stage in the disease and correlates with low levels of C4A. These results indicate that subtle deficiencies of complement may predispose to SLE.

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.

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.

A study of the association of HLA DR, DQ, and complement C4 alleles with systemic lupus erythematosus in Iceland.

OBJECTIVE: To perform an exploratory analysis of the relative contribution of single MHC genes to the pathogenesis of systemic lupus erythematosus (SLE) in a homogenous white population. METHODS: MHC class II alleles and C4 allotypes were determined in 64 SLE patients and in ethnically matched controls. HLA-DR and DQ typing was performed by polymerase chain reaction amplification with sequence specific primers. C4 allotypes were determined by agarose gel electrophoresis. RESULTS: The frequency of C4A*Q0 was significantly higher in patients than in controls (46.9% v 25.3%, p = 0.002). HLA-DRB1, DQA1, and DQB1 alleles in the whole group of SLE patients were not significantly different from those of controls. On the other hand increase in DRB1*03 was observed in the group of patients with C4A*Q0, as compared with patients with other C4A allotypes (p = 0.047). There was no significant correlation between severe and mild disease, as judged by the SLEDAI, and HLADR, DQ alleles and comparing the patients with C4A*Q0 with those with other C4A allotypes there was no significant difference regarding clinical manifestations. CONCLUSION: The results are consistent with the argument that C4A deficiency contributes independently to susceptibility and the pathogenesis of SLE. C4A*Q0 in SLE patients in Iceland shows weaker linkage disequilibrium with DR3 genes than reported in most other white populations and emphasises the role of ethnicity.

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.

An enzyme based assay for the measurement of complement mediated binding of immune complexes to red blood cells.

A new in vitro method is presented for measuring directly the ability of sera to induce binding of immune complexes (ICs) to erythrocytes (ICRB assay). The assay measures the binding of alkaline phosphatase (AP)-anti-alkaline phosphatase (anti-AP) complexes formed in the presence of the test sera to the complement receptor 1 (CR1) on normal human red blood cells (RBCs). By using a standard serum source, the assay can also be used to measure the IC binding ability of RBCs from different donors. As compared to the traditional CH50 method, the ICRB assay generally showed more pronounced abnormality in 10 individuals tested, of whom 5 had primary deficiency of classical pathway components. Seven out of ten individuals had systemic lupus erythematosus (SLE) and 2/10 had other rheumatic diseases without primary complement deficiency. The ICRB measured in samples from 9 other patients with SLE was significantly decreased when compared to values from 80 normal individuals. ICRB in serum samples from a C2 deficient SLE patient collected during plasma infusion treatment reflected closely the rising amount of C2 in the serum. Using RBCs from different donors ICRB activity correlated well with the numbers of CR1 as measured by a flow cytometric assay (FCA). These methods should be valuable for measuring the overall IC clearance capacity of the blood and have the advantage that the use of radioactive isotopes is avoided.