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.

Enhanced complement resistance in drug-selected P-glycoprotein expressing multi-drug-resistant ovarian carcinoma cells.

Multi-drug resistance (MDR) is a major obstacle in cancer chemotherapy. There are contrasting data on a possible correlation between the level of expression of the drug transporter P-glycoprotein (P-gp) and susceptibility to complement-dependent cytotoxicity (CDC). We therefore investigated the sensitivity of human ovarian carcinoma cells and their P-gp expressing MDR variants to complement. Chemoselected P-gp expressing MDR cells showed increased resistance to CDC associated with overexpression of membrane-bound complement regulatory proteins (mCRP) and increased release of the soluble inhibitors C1 inhibitor and factor I. MDR1 gene transfection alone did not alter the susceptibility of P-gp expressing A2780-MDR and SKOV3-MDR cells to CDC. However, subsequent vincristine treatment conferred an even higher resistance to complement to these cells, again associated with increased expression of mCRP. Blocking the function of P-gp with verapamil, cyclosporine A or the anti-P-gp-antibody MRK16 had no impact on their complement resistance, whereas blocking of mCRP enhanced their susceptibility to complement. These results suggest that enhanced resistance of chemoselected MDR ovarian carcinoma cells to CDC is not conferred by P-gp, but is due at least partly to overexpression of mCRP, probably induced by treatment with the chemotherapeutic agents.

Down-regulation of CD55 and CD46 expression by anti-sense phosphorothioate oligonucleotides (S-ODNs) sensitizes tumour cells to complement attack.

Overexpression of one or more membrane-bound complement regulatory proteins (mCRPs) protects tumour cells against complement-mediated clearance by the autologous humoral immune response and is also considered as a barrier for successful immunotherapy with monoclonal anti-tumour antibodies. Neutralization of mCRPs by blocking antibodies, enzymatic removal or cytokine-mediated down-regulation has been shown to sensitize tumour cells to complement attack. In our study we applied, for the first time, anti-sense phosphorothioate oligonucleotides (S-ODNs) to knock down the expression of the mCRPs CD55 and CD46 with the aim of exploiting complement more effectively for tumour cell damage. Potent anti-sense oligonucleotides against CD55 and CD46 were identified by screening various target sequences (n = 10) for each regulator. S-ODN anti-CD55(687) reduced CD55 protein expression up to 84% and CD46 protein expression was inhibited up to 76% by S-ODN anti-CD46(85). Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed a similar reduction of the CD55 and CD46 mRNA levels, which argues for an RNAse H-dependent anti-sense mechanism. T47D, A549 and PC3 cells, representing breast, lung and prostate carcinoma, were used for functional studies. Dependent on the particular cell line, anti-sense-based inhibition of mCRP expression enhanced complement-dependent cytolysis (CDC) up to 42% for CD55 and up to 40% for CD46, and the combined inhibition of both regulators yielded further additive effects in T47D cells. C3 opsonization of CD55/CD46-deficient tumour cells was also clearly enhanced upon mCRP suppression. Due to the clinical applicability of S-ODNs, the anti-sense approach described in this study may offer an additional alternative to improve the efficacy of antibody- and complement-based cancer immunotherapy.

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 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.

Exposure to pesticide mixer/loaders and applicators in California.

Pesticide exposure occurs both when preparing (mixing/loading) the pesticide for application and when actually applying the pesticide. Equipment cleanup and repair may also contribute to exposure. These separate tasks may be done by different people or a single individual may combine them. Different formulations, handling methods, and application methods may affect levels of exposure. Good workplace hygiene dictates that the first priority in workplace safety is to put in place engineering and administrative controls to make the workplace safer rather than rely on the use of PPE to prevent exposure. Providing a physical barrier, such as closed systems for the mixer/loader and an enclosed cab or cockpit for applicators, is associated with reduced exposures of workers. The Worker Health and Safety Branch in the DPR of Cal/EPA has been monitoring various work task exposures. The results of these studies are summarized in Figures 1-4, where exposure is shown based on both time worked and amount of material handled. The studies were done using dosimeters, skin washes/wipes, and air pumps. Water-soluble packets, which are very popular with users, surpassed closed systems in reducing exposure to mixer/loaders in these studies. Hand spraying proved to present the greatest risk of exposure of the methods of application studied. It was found that the dermal route of exposure is most important, comprising 87-95% of a handler's exposure. Although this survey cannot be considered conclusive, since it leaves many formulations, systems, and methods incompletely studied or unstudied, it is clear that exposure is affected by different handling strategies. Manufacturers, regulators and users should work more closely to refine or develop new systems for safely handling pesticides.

Trends of HIV infections using the anonymous mandatory reporting system and other data sources in Switzerland and in the Federal Republic of Germany.

In Switzerland and in the Federal Republic of Germany the reporting of HIV infections is based on an anonymous mandatory reporting system for laboratories. In Switzerland, physicians have to complete a questionnaire on the patient's clinical status and risk behaviour. To make a rough estimate of the prevalence of HIV infections in the general population at low risk, data on blood donations are used in both countries. In Switzerland, additional data with information on positive and negative test results have been obtained since 1985 from anonymous test sites. In both countries, the data do not show evidence of an increasing incidence of HIV infection. The number of positive test results reported by the laboratories and physicians remains stable, and the rates of HIV-positive blood donations are declining. In the Federal Republic of Germany, homo-/bisexual men play the most important role in the epidemic, whilst in Switzerland, injecting drug users contribute most to the burden of HIV infection, and the proportion of persons probably heterosexually infected is increasing steadily. Possible selection biases need to be further discussed.

Quantitation of the anaphylatoxin C3a in the presence of C3 by a novel sandwich ELISA using monoclonal antibody to a C3a neoepitope.

C3a levels in plasma are usually measured by a competitive inhibition radioimmunoassay (RIA) using 125I-labelled C3a-desArg and antibodies to C3a capable of detecting C3a determinants which are also present on the native C3. Therefore, prior to the assay native, non-cleaved C3 has to be removed completely from the C3a-containing sample by precipitation. We developed a new rapid two-site sandwich ELISA system for the quantitation of C3a-desArg in plasma. This immunoassay uses a monoclonal antibody (mAb H466) reacting with C3a-desArg but not with C3. The reactivity of mAb H466 with a neoantigenic determinant of C3a-desArg permitted the direct quantitation of C3a-desArg without removal of C3 from the sample. The mAb H466 was used as a capture antibody and bound C3a-desArg was detected with a second peroxidase-labelled anti-C3a mAb. The lower limit of detection of C3a-desArg in this ELISA was 1 ng/ml. The C3a-desArg levels measured in the plasma samples of various patients were found to differ over a wide range. A good correlation was observed between the results obtained in the RIA and those obtained in the ELISA (r = 0.95). High levels of C3a-desArg were detected in plasma from patients with multiple trauma and patients undergoing haemodialysis. The C3a-desArg assay described should facilitate the routine quantitation of C3a in samples of plasma.

Spatial analysis of limb bud myogenesis: elaboration of the proximodistal gradient of myoblasts requires the continuing presence of apical ectodermal ridge.

Myogenic tissue from embryonic chick wing and leg buds is composed of several subpopulations of myoblasts. These clonally distinct subpopulations first appear at different developmental stages, and are distributed differently along the proximo-distal axis of the buds, giving the appearance of a gradient of myoblast cell types. This myoblast distribution pattern has been utilized to investigate the dependence of muscle tissue outgrowth and development on the presence of the apical ectodermal ridge (AER). Wing buds which have had the AER removed at stages 17-18 (2 days) subsequently develop normal proximal regions, but fail to elaborate skeletal structures distal to the humerus. The myoblast pattern of operated buds is also normal proximally, but distal portions of the pattern are not observed. Removal of the AER at stage 20 (3 days) results in buds which develop slightly more distal skeletal structures and the coinciding portions of the myoblast pattern, but in which the more distal portions of the normal myoblast gradient are truncated. These data suggest that elaboration of the myogenic pattern in early limb buds is dependent on the continuing presence of the AER, and that early removal of the AER leads to the subsequent cessation of myoblast pattern specification.

Functional characterization of an adhesive component from the embryonic chick neural retina.

We have developed a quantitative assay for tissue-specific adhesive components which is based on the agglutination of glutaraldehyde-fixed cells. At least 2 components are required for fixed-cell agglutination: a cell-surface ligand which is obtained from tissue culture-conditioned medium, and a soluble 'agglutinin' which accumulates in conditioned medium from monolayer cultures. Our results suggest that the surface-binding ligand and the agglutinin interact directly, resulting in tissue-specific agglutination of cells. The agglutination reaction exhibits divalent cation, temperature, and pH dependence. Several models of cell adhesion are described; the simplest of these which can account for the data is a multicomponent model in which the 2 adhesive components have structural roles.