Complement profile in neonates of different gestational ages.

Blood levels of regulators of the complement system in preterm babies were reported in few studies only. The aim of this study was to set up a complement profile in premature and term babies focusing on the development of blood levels of MBL, key regulatory proteins and on classical pathway activity, which may allow an estimation of potential susceptibility to infection. Complement activity (CH50), levels of mannan-binding lectin (MBL), complement regulators (factors H and I, C1 inhibitor, properdin) and C3a as marker of complement activation were assessed in three groups of healthy newborns: (1) prematures (≤34 weeks); (2) late prematures (>34-<37 weeks) and (3) term neonates (≥37 weeks). CH50 increased with gestational age with lower titres in cord blood than in day 5 post-delivery venous blood. MBL concentrations were not significantly different among groups. Quantitative and functional C1 inhibitor were below adult normal range in prematures <34 weeks and lower in cord blood as compared to day 5. Factor I, factor H and properdin remained below adult values in all groups. Low C3a levels excluded that low complement titres were due to activation-induced consumption. These results demonstrate the relative immaturity of the complement system and its regulation, especially in premature infants.

Complement 4 phenotypes and genotypes in Brazilian patients with classical 21-hydroxylase deficiency.

The aim of this work was to analyse C4 genotypes, C4 protein levels, phenotypes and genotypes in patients with the classical form of 21-hydroxylase deficiency. Fifty-four patients from 46 families (36 female, 18 male; mean age 10.8 years) with different clinical manifestations (31 salt-wasting; 23 simple-virilizing) were studied. Taq I Southern blotting was used to perform molecular analysis of the C4/CYP21 gene cluster and the genotypes were defined according to gene organization within RCCX modules. Serum C4 isotypes were assayed by enzyme-linked immunosorbent assay. The results revealed 12 different haplotypes of the C4/CYP21 gene cluster. Total functional activity of the classical pathway (CH50) was reduced in individuals carrying different genotypes because of low C4 concentrations (43% of all patients) to complete or partial C4 allotype deficiency. Thirteen of 54 patients presented recurrent infections affecting the respiratory and/or the urinary tracts, none of them with severe infections. Low C4A or C4B correlated well with RCCX mono-modular gene organization, but no association between C4 haplotypes and recurrent infections or autoimmunity was observed. Considering this redundant gene cluster, C4 seems to be a well-protected gene segment along the evolutionary process.

Recurrent infections in partial complement factor I deficiency: evaluation of three generations of a Brazilian family.

We report here on the evaluation of a factor I-deficient Brazilian family (three generations, 39 members) with strong consanguinity. The complete factor I-deficient patients (n = 3) presented recurrent respiratory infections, skin infections and meningitis; one of them died after sepsis. They presented an impaired total haemolytic activity (CH50), low C3, low factor H and undetectable C3dg/C3d. Partial factor I deficiency was detected in 16 family members (normal low cut-off value was 25 microg/ml). Respiratory infections were the most common clinical occurrence among partial factor I-deficient relatives. Two of them were submitted to nephrectomy following recurrent urinary tract infections. An additional two heterozygous relatives presented with arthritis and rheumatic fever. Apparently, patients with partial factor I deficiency are also at higher risk for recurrent infections. Vaccination against capsulated bacteria and the eventual use of prophylactic antibiotics should be considered individually in this patient group.

Immunological analysis in paediatric HIV patients at different stages of the disease.

There are only few clinical studies on complement in well-defined (or characterized) paediatric HIV patients. Aim of this study was to evaluate the complement system and immunoglobulins in HIV-infected children and to correlate data to stage of disease. Blood samples of 127 HIV-infected children (11-134 months; 62 male : 65 female) were collected in order to evaluate humoral immunity. The patients were classified according to CDC clinical (N-asymptomatic; A-mild symptoms such as common recurrent infections; B-moderate symptoms such as Candidiasis and herpes infections, meningitis, sepsis and anaemia; C-severe symptoms such as opportunistic infections and neoplasia) and with respect to immunological criteria (T CD4(+) cell count). Analysis of complement system included the classical (CH50), alternative (APH50) pathway activities and plasma concentrations of mannan-binding lectin (MBL), of the C4 allotypic variants C4A and C4B. (ELISA), and of the C3 split product C3d (rocket immunoeletrophoresis). Immunodiagnosis also included CD4(+) and CD8(+) lymphocyte count and immunoglobulin concentrations. Complement activation and consumption was observed in all patients correlating with disease activity. Activated classical and alternative pathways and elevated C3d were significantly correlated with immunologic category 3. C3d levels were also significantly correlated with immunologic category 1. Undetectable CH50 and APH50 were found in two (group C) and 10 patients (n = 2, A = 2, B = 2, C = 4), respectively. Low MBL values were found in 13/127 but without correlation to disease severity. Undetectable C4B levels were observed in three patients, favouring the diagnosis of a complete deficiency. Although not related to clinical symptomatology, a strong ongoing complement activation can be observed in all stages of HIV infection. In contrast to earlier reports MBL could not be considered as a risk factor for HIV.

Complement activation in infective endocarditis: correlation with extracardiac manifestations and prognosis.

In an infectious process complement activation is necessary for a proper immune and inflammatory response, but when exacerbated may cause tissue injuries. In infective endocarditis (IE) patients tend to develop high titres of circulating immune complexes (CIC) that activate complement. The aim of this study was to evaluate for the first time complement activation in IE for possible correlation with extracardiac manifestations and clinical prognosis. Twenty patients with IE, 14 healthy controls and 15 patients presenting mitral and aortic valve lesions (with no signs of either infection or other associated diseases), were studied. Plasma levels of C3adesArg, SC5b-9, C1rs-C1Inh and C3b(Bb)P were determined by ELISA and C3d by double decker immunoelectrophoresis. C3 and C4 levels were assayed by turbidimetry and CIC by ELISA. Elevation of plasma levels of all complement activation products, with the exception of C3b(Bb)P, indicated a significant classical pathway activation in IE patients when compared to controls (C3d: P < 0.00004; C3adesArg: P < 0.03, SC5b-9: P < 0.01, C1rs-C1Inh: P < 0.00007). CIC levels were significantly increased (P < 0.005) and C3 reduced in IE patients (P < 0.05). Elevated C3d (P < 0.02) and C3adesArg (P < 0.03) levels were associated with pulmonary manifestations. In addition, C3d was significantly elevated in the patients who died when compared to those who had a good recovery (P < 0.02). Our data demonstrate the activation of the complement classical pathway, most probably mediated by CIC, in IE and suggests C3d and C3adesArg as possible markers for extracardiac lesion and severity of the disease.

Avaliaçäo do sistema complemento em crianças, em diversos estádios da infecçäo pelo HIV: correlaçäo clínico-laboratorial / Evaluation of complement system in children in different phases of HIV infection: clinical and laboratorial correlation

Objetivo: A ativaçäo do complemento foi demonstrada em pacientes adultos infectados pelo HIV, no entanto, poucas informaçöes estäo disponíveis em crianças infectadas no período perinatal. O objetivo do presente estudo foi analisar a funçäo do sistema complemento em crianças infectads pelo HIV. Método: 127 crianças infectads pelo HIV no período neonatal (onze a 134 meses, 62 do sexo masculino: 65 do sexo feminino) foram incluídas e classificadas de acordo com os critérios clínicos e imunológicos do Centro de Controle de Doenças (Atlanta, EUA), de 1994. O diagnóstico da ativaçäo do sistema complemento foi realizado pelos seguintes ensaios: CH50 para via clássica, APH50 para via alternativa (APH50), ELISA para via das lectinas (MBL) e 'rocket' imunoeletroforese para o produto de C3,C3dg/C3d. Resultados: As infecçöes mais freqüentes foram: pneumonia bacteriana, otite, diarréia e infecçöes oportunistas como pneumonia por Pneumocystis carinii e tuberculose (31,5 por cento). A miocardiopatia foi a única apresentaçäo clínica que se relacionou com o estado imunológico (categoria 3). Nenhuma diferença estatisticamente significante nas funçöes da via clássica e alternativa foi observada entre os pacientes das diferentes categorias. Valores médios aumentados de CH50, APH50 e MBL foram verificados em pacientes nos estádios mais avançados da doença. Níveis elevados de C3d na maioria dos pacientes indicam que o complemento encontra-se ativado durante a infecçäo pelo HIV em crianças. CH50, APH50, e MBL estavam abaixo dos limites inferiores em duas, dez e duas crianças, respectivamente.

SC5b-9 is the most sensitive marker in assessing disease activity in Brazilian SLE patients.

This study investigated whether increased plasma levels of terminal complement complex (SC5b-9) or split products correlate with disease activity and clinical manifestations in Brazilian systemic lupus erythematosus (SLE) patients. Comparisons with conventional measurements of complement and other inflammatory markers were also performed. Plasma levels of SC5b-9, C3a desArg, C1rs-C1Inhibitor, C3b(Bb)P, C3, C4, erythrocyte sedimentation rate (ESR) and mucoproteins (MP) were measured in 41 patients with SLE of different disease activity: 10 patients with none, 15 patients with mild, and 16 patients with moderate or severe activity. All parameters, with the exception of C3 and C3b(Bb)P, showed a statistically significant correlation with disease activity. Plasma levels of SC5b-9, C3a desArg, C4, CH50, ESR and MP revealed significant differences between the groups of patients without activity and those with moderate or severe disease. Although none of the variables were able to discriminate between patients without and those with mild activity, SC5b-9, C3a desArg, C4, ESR and mucoproteins showed significant differences between the patients with mild and those with moderate or severe disease. Among all the variables, SC5b-9 levels showed the most significant results and correlated well with the severity of the disease (p < 0.0005). Our data suggest that elevated levels of complement activation products, particularly of SC5b-9 are more sensitive markers in assessing disease activity than conventional laboratory diagnosis. Modern complement diagnosis is therefore recommended for monitoring disease progress in SLE patients.

[Hereditary angioedema: clinical and laboratory aspects of 7 cases]. / Angioedema hereditário: aspectos clínico-laboratoriais de sete casos.

Hereditary angioedema is caused by a defect in C1 inhibitor activity (C1INH). Its occurrence is rare and it is associated with an autosomal dominant mode of inheritance. We describe seven patients (4M:3F), age from 12 to 50 years old, who are affected by hereditary angioedema; four of them belong to the same family. The main clinical manifestations were: angioedema of face, hands and feet (6/7) and abdominal pain (2/7). No triggering factors were associated with symptoms in 4/7 patients and trauma (2/7) and menses (1/7) were reported in the other three ones. One patient was submitted to laparotomy for partial intestinal resection, before diagnosis. Laboratory complement analysis revealed the absence of hemolytic function of complement, reduced C4 (6/7) and low C1INH levels. All patients received Danazol (100 mg/day) with clinical control. Hereditary angioedema has to be considered in the differential diagnosis of angioedema, since an early diagnosis of this immunodeficiency, leading to specific treatment in order to decrease the complications.

Brazilian report on primary immunodeficiencies in children: 166 cases studied over a follow-up time of 15 years.

One hundred sixty-six cases of primary immunodeficiency diseases (PID) (95 males, 71 females), diagnosed according to WHO criteria, have been registered at the Children's Hospital, University of São Paulo, Brazil. The following frequencies were found: predominantly humoral defects, 60.8% (n = 101); T cell defects, 4.9% (n = 8); combined ID, 9.6% (n = 16); phagocyte disorders, 18.7% (n = 31); and complement deficiencies, 6% (n = 10). IgA deficiency was the most frequent disorder (n = 60), followed by transient hypogammaglobulinemia (n = 14), chronic granulomatous disease (n = 14), and X-linked agammaglobulinemia (n = 9). In comparison to other (national) reports, we observed higher relative frequencies of phagocyte and complement deficiencies. Recurrent infections were the cause of death in 12.7%. Allergic symptoms were observed in 41%, mainly in IgA-deficient, hypogammaglobulinemic, or hyper-IgE patients, and autoimmune disorders in 5%, predominantly in IgA and complement deficiencies. Five patients suffered from BCG dissemination; two of them died. This is the first Brazilian report on PID over an observation time of 15 years.

PIP: Over a 15-year observation period (1981-96), 166 cases of primary immunodeficiency disease (PID) were registered at the Department of Pediatrics, University of Sao Paulo, Brazil. PID was diagnosed according to World Health Organization criteria and only children with well-established deficiencies were included. The following frequencies were noted by PID classification: predominantly humoral defects (60.8%), T cell defects (4.9%), combined immunodeficiency (9.6%), phagocyte disorders (18.7%), and complement deficiencies (6%). The male to female ratio was 1.3 to 1. Immunoglobin A deficiency was the most frequent disorder (60 cases), followed by transient hypogammaglobulinemia (14 cases), chronic granulomatous disease (14 cases), and X-linked agammaglobulinemia (9 cases). Allergic symptoms occurred in 41% of cases. During the observation period, 23 children (13.8%) died, primarily of recurrent infections. Although improved diagnostic facilities have facilitated the recognition of immunodeficient children, the true incidence is likely to be higher than that detected in this study. Increased international collaboration is urged to improve the early detection of PID.

Complement factor I deficiency in a family with recurrent infections.

Factor I deficiency causes a permanent, uncontrolled activation of the alternative pathway resulting in an increased turnover of C3 and consumption of factor B, factor H and properdin. Factor I deficiency is clinically associated with recurrent bacterial infections already in early infancy, mainly affecting the upper and lower respiratory tract, or presenting as meningitis or septicemia. We here report on a Brazilian family (n = 9) with known consanguinity, where in 3/7 children, suffering from chronic otitis, meningitis, and respiratory infections, a complete factor I deficiency was recognized. One of the patients died after fulminant sepsis. Hemolytic activity of the alternative pathway was not detectable in the patients' sera due to decreased plasma concentrations of C3, factor B and properdin. As a consequence of factor I deficiency, C3b could not be metabolized with the result that no C3-derived split products (C3dg/C3d) were detectable in the patients' sera. In vitro reconstitution with purified factor I restored the regulatory function in the patients' sera with the subsequent cleavage of C3b to C3c and C3dg. Factor H levels were decreased in all patients' sera and found to be tightly complexed with C3b resulting in a modified electrophoretic mobility. Upon factor I reconstitution, factor H was released from C3b regaining its beta 1 electrophoretic mobility. Complement-mediated biological functions like opsonization of bacteria, chemotactic activity and phagocytosis in these patients were impaired. The parents (cousins, 2nd degree) and 3/4 siblings had significantly reduced factor I plasma levels without further alteration in their complement profile. 3 of these obviously heterozygously deficient family members suffered from recurrent bacterial infections of different frequency and severity.

Complement and IgG subclasses in agammaglobulinemic patients.

Complement and IgG subclass analysis was performed in six agammaglobulinemic patients suffering from recurrent bacterial infections. Complement analysis included functional activity of the classical (CH50) and the alternative pathway (APH50) of complement, the complement proteins C3, C4, factor B, the regulators C1-inhibitor, factors H and I as well as the C3-derived split product C3dg/C3d. Plasma concentrations of total IgG ranged between < 0.01 and 297 mg/dl. IgM and IgA were almost undetectable. Levels of all IgG subclasses, which in most cases were measurable only by sensitive ELISA, were markedly reduced in all patients. A pronounced activation of the complement system was observed in all patients as revealed by reduced titers of hemolytic function and elevated levels of C3dg/C3d. Despite the fact that complement activation by itself was the major reason for decreased C4 levels, from depressed plasma concentrations of the common allotypic variants C4A and C4B, a heterozygous C4 deficiency could not be excluded in 2/6 patients. Our data clearly show that, by application of sensitive analytical methods, agammaglobulinemic patients vary considerably in their ability to synthesize immunoglobulins. This may, at least in part, explain the heterogeneous pattern of clinical symptoms. The increased susceptibility of agammaglobulinemic patients to bacterial infections is reflected by a highly activated, and thereby depleted complement system.

Evaluation of complement activation in premature newborn infants with hyaline membrane disease.

Fifteen premature newborns with hyaline membrane disease causing acute respiratory distress were evaluated for complement activation. A high intrapulmonary right-to-left shunt and marked arterial-alveolar oxygen difference indicated the severity of the respiratory failure. Twenty preterm healthy infants served as controls. Total haemolytic activity, plasma concentrations of complement components and regulatory proteins (C3, C4, C1-inhibitor, factors H and I) as well as activation products (C3a, C3dg, C1rsC1-inhibitor, C3b(Bb)P) gave no evidence of significant complement activation. Functional activity of the ubiquitous regulatory protein C1-inhibitor was significantly reduced without impact on classical pathway activation. These data suggest that, in contrast to the adult form of respiratory distress syndrome, the low-pressure pulmonary oedema characterising hyaline membrane disease is not mediated by activation of the complement system.