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Two functions have been assigned to properdin; stabilization of the alternative convertase, C3bBb, is well accepted, whereas the role of properdin as pattern recognition molecule is controversial. The presence of nonphysiological aggregates in purified properdin preparations and experimental models that do not allow discrimination between the initial binding of properdin and binding secondary to C3b deposition is a critical factor contributing to this controversy. In previous work, by inhibiting C3, we showed that properdin binding to zymosan and Escherichia coli is not a primary event, but rather is solely dependent on initial C3 deposition. In the present study, we found that properdin in human serum bound dose-dependently to solid-phase myeloperoxidase. This binding was dependent on C3 activation, as demonstrated by the lack of binding in human serum with the C3-inhibitor compstatin Cp40, in C3-depleted human serum, or when purified properdin is applied in buffer. Similarly, binding of properdin to the surface of human umbilical vein endothelial cells or Neisseria meningitidis after incubation with human serum was completely C3-dependent, as detected by flow cytometry. Properdin, which lacks the structural homology shared by other complement pattern recognition molecules and has its major function in stabilizing the C3bBb convertase, was found to bind both exogenous and endogenousmolecular patterns in a completely C3-dependent manner. We therefore challenge the view of properdin as a pattern recognition molecule, and argue that the experimental conditions used to test this hypothesis should be carefully considered, with emphasis on controlling initial C3 activation under physiological conditions. [ABSTRACT FROM AUTHOR]

Properdin is well known as an enhancer of the alternative complement amplification loop when C3 is activated, whereas its role as a recognition molecule of exogenous pathogen-associated molecular patterns and initiator of complement activation is less understood. We therefore studied the role of properdin in activation of complement in normal human serum by zymosan and various Escherichia coli strains. In ELISA, microtiter plates coated with zymosan induced efficient complement activation with deposition of C4b and terminal complement complex on the solid phase. Virtually no deposition of C4b or terminal complement complex was observed with mannose-binding lectin (MBL)-deficient serum. Reconstitution with purified MBL showed distinct activation in both readouts. In ELISA, normal human serum-induced deposition of properdin by zymosan was abolished by the C3-inhibiting peptide compstatin. Flow cytometry was used to further explore whether properdin acts as an initial recognition molecule reacting directly with zymosan and three E. coli strains. Experiments reported by other authors were made with EGTA Mg2+ buffer, permitting autoactivation of C3. We found inhibition by compstatin on these substrates, indicating that properdin deposition depended on initial C3b deposition followed by properdin in a second step. Properdin released from human polymorphonuclear cells stimulated with PMA did not bind to zymosan or E. coli, but when incubated in properdin-depleted serum this form of properdin bound efficiently to both substrates in a strictly C3-dependent manner, as the binding was abolished by compstatin. Collectively, these data indicate that properdin in serum as well as polymorphonuclear-released properdin is unable to bind and initiate direct alternative pathway activation on these substrates. [ABSTRACT FROM AUTHOR]

Abstract: The main function of the complement system is pattern recognition of danger. Typical exogenous danger signals are pathogen associated molecular patterns inducing a protective inflammatory response. Other examples are exposure to foreign surfaces of biomedical materials including nanoparticles, which principally induce the same inflammatory response. If a surface is “foreign” to the host, it induces complement activation. Development of monoclonal antibodies to neoepitopes on complement activation products introduced an entirely new set of methods for assay of complement activation. Activation of complement by a surface occurs by impairment of the fine balance of the control system, e.g. by preferred binding of factor B at the expense of factor H. Sensitive methods to detect complement activation on surfaces and in the fluid phase are a prerequisite for investigation of the biocompatibility of artificial materials. This information can be used to develop new materials with enhanced biocompatibility. Here we review available methods to study human and animal complement function and activation in vitro and in vivo. [Copyright &y& Elsevier]

Abstract: Complement activation plays an important role in human pathophysiology. The effect of classical pathway activation is largely dependent on alternative pathway (AP) amplification, whereas the role of AP for the down-stream effect of mannan-induced lectin pathway (LP) activation is poorly understood. In normal human serum specific activation of LP was obtained after exposure to a wide concentration range of mannan on the solid phase. Reaction mechanisms in this system were delineated in inhibition experiments with monoclonal antibodies. Direct mannose-binding lectin (MBL) independent activation of AP was not observed even at high mannan concentrations since addition of the inhibiting anti-MBL mAb 3F8 completely abolished generation of the terminal C5b-9 complex (TCC). However, selective blockade of AP by anti-factor D inhibited more than 80% of TCC release into the fluid phase after LP activation showing that AP amplification is quantitatively responsible for the final effect of initial specific LP activation. TCC generation on the solid phase was distinctly but less inhibited by anti-fD. C2 bypass of the LP pathway could be demonstrated, and AP amplification was also essential during C2 bypass in LP as shown by complete inhibition of TCC generation in C2-deficient serum by anti-fD and anti-properdin antibodies. In conclusion, the down-stream effect of LP activation depends strongly on AP amplification in normal human serum and in the C2 bypass pathway. [Copyright &y& Elsevier]

The article presents a study which examined the possible practical use of the Gm system in paternity cases. Gm is a pair allelic genes which was able to express itself in single and double dose. Results showed that out of the 320 sera from unrelated adults that were tested for anti-Gm sera, 194 of these persons were type Gm(a+), 126 were of type Gm(a-). Observations on a serum of non-rheumatoid type are presented.

Discusses recent developments in clinical immunology with regard to immunodeficiency, complement, treatment with intravenous immunoglobulin, and infection and vaccines. Communication between cells; Complement's regulatory mechanisms and organ transplants; Role of complement in inflammatory tissue destruction; Complement and kidney disease; Development of new vaccines.

The article presents the author's experience of studying the development of people's knowledge and understanding of the genetic control of antibody structure and function. He mentions the three human serum proteins wherein genetic variation had been demonstrated. He relates a study conducted by researcher Henry G. Kunkel which identified the 4 subclasses of human immunoglobulin G.

Abstract: Background: Meconium aspiration syndrome has a complex pathophysiology. Meconium activates the complement system and meconium-induced cytokine formation is differentially mediated by complement and CD14. C1-inhibitor (C1-INH) regulates complement and contact-system activation mainly by protease inhibition, but may reduce inflammation by other mechanisms as well. Objective: The aim of the study was to investigate the initial mechanisms of meconium-induced complement activation and to study the effect of C1-INH on the meconium-induced inflammatory reaction. Methods: Human serum from five donors was preincubated with an anti-MBL monoclonal antibody and then incubated with meconium for 30min at 37°C. Human cord whole blood, anticoagulated with lepirudin, from six donors was preincubated with C1-INH and then incubated with meconium for 30min and 4h at 37°C. Complement activation products specific for the different pathways were measured by ELISAs: classical pathway C1rs/C1-INH complexes, classical and lectin pathway C4d, alternative pathway C3bBbP, and terminal pathway sC5b-9 complex (TCC). A Bio-Plex Array Reader was used to measure 27 inflammatory mediators. Results: The anti-MBL monoclonal antibody significantly reduced meconium-induced formation of C4d by 63% (p =0.0159) and TCC by 27% (p =0.0079). C1-INH dose-dependently inhibited meconium-induced formation of C1rs/C1-INH complexes, C4d, C3bBbP, and TCC compared to albumin (p <0.002 for all). C1-INH induced a dose-dependent and substantial inhibition of meconium-induced formation of the proinflammatory cytokines TNFα, IL-1β, IL-6 and IFN-γ (p <0.01 for all), the chemokines IL-8, MCP-1, MIP-1α, MIP-1β, and eotaxin (p <0.02 for all), the growth factors G-CSF, GM-CSF, basic FGF, and PDGFbb (p <0.05 for all), and the anti-inflammatory cytokine IL-1ra (p <0.001). Conclusions: Meconium activated the lectin complement pathway as well as the alternative pathway. C1-INH efficiently reduced a broad spectrum of inflammatory mediators even at the lowest concentration. Administration of C1-INH may thus reduce the inflammatory response in MAS. [Copyright &y& Elsevier]

The proposed role of the mammalian cell entry protein 1A (Mce1A) of Mycobacterium tuberculosis is to facilitate invasion of host cells. The structure of Mce1A was modelled on the basis of the crystal structure of Colicin N of Escherichia coli by fold prediction and threading. Mce1A, as the model predicts, is an α/β protein consisting of two major (α and β) domains, connected by a long α helix. The model further revealed that the protein contains 12 helices, 9 strands, and 1 turn. The final model of Mce1A was verified through the program VERIFY 3D and more than 90% of the residues were in the favourable region. A mouse monoclonal antibody, TB1–5 76C, is directed to an epitope within a 60-mer peptide that has been shown to promote uptake of bacteria in mammalian cells. We show here that the epitope could be narrowed down to a core of 4 amino acids, TPKD. Upstream flanking residues, KRR also contributed to binding. Mce2A does not promote uptake in mammalian cells and sequence comparison of Mce1A and Mce2A indicates that the epitope mediates uptake. The epitope was located at the surface of the Mce1A model at the distal β strand-loop region in the β domain. The localization of this epitope in the model confirms its potential role in promoting uptake of M. tuberculosis in host cells. [Copyright &y& Elsevier]

Pigs genetically deficient in complement factor H all develop lethal membranoproliferative glomerulonephritis (MPGN) type II characterized by massive glomerular deposits of complement, intramembranous dense deposits, and mesangial hypercellularity. To elucidate the chronological relationship between these glomerular changes, and to precisely determine the localization of glomerular complement deposits, we studied kidney specimens from factor H-deficient piglets at different ages from fetal life until terminal kidney failure had developed. Deposits of C3 and the terminal complement complex localized within the glomerular basement membrane (GBM) were present already in factor H-deficient fetuses, without concurrent intramembranous dense deposits or mesangial hypercellularity. Incipient subendothelial dense deposits containing complement appeared no earlier than four days after birth, and intramembranous dense deposits in older piglets with established MPGN type II also contained large amounts of complement as detected by immune electron microscopy. Onset of kidney failure coincided with pronounced mesangial hypercellularity and expansion, compromising glomerular capillary patency. Formation of glomerular capillary wall double contours coincided with electron microscopic evidence of laminar disintegration of intramembranous dense deposits. Complement was also deposited in the mesangial matrix, but not on glomerular cells. We conclude that all components of the alternative and terminal pathways of complement have access into the GBM and the mesangial matrix. In the absence of factor H, complement is spontaneously activated and deposited in situ in these locations resulting in dense deposit formation. It is proposed that factor H dysfunction may play an essential role even in human MPGN type II. [ABSTRACT FROM AUTHOR]

Background: To investigate how the risk of active tuberculosis disease is influenced by time since original infection and to determine whether the risk of reactivation of tuberculosis increases or decreases with age. Methods: Cohort analysis of data for the separate ten year birth cohorts of 1876-1885 to 1959-1968 obtained from Statistics Norway and the National Tuberculosis Registry. These data were used to calculate the rates and the changes in the rates of bacillary (or active) tuberculosis. Data on bacillary tuberculosis for adult (20+) age groups were obtained from the National Tuberculosis Registry and Statistics Norway from 1946 to 1974. Most cases during this period arose due to reactivation of remote infection. Participants in this part of the analysis were all reported active tuberculosis cases in Norway from 1946 to 1974 as recorded in the National Tuberculosis Registry. Results: Tuberculosis decreased at a relatively steady rate when following individual birth cohorts, but with a tendency of slower decline as time passed since infection. A mean estimate of this rate of decline was 57% in a 10 year period. Conclusions: The risk of reactivation of latent tuberculosis decreases with age. This decline may reflect the rate at which latent tuberculosis is eliminated from a population with minimal transmission of tubercle bacilli. A model for risk of developing active tuberculosis as a function of time since infection shows that the rate at which tuberculosis can be eliminated from a society can be quite substantial if new infections are effectively prevented. The findings clearly indicate that preventative measures against transmission of tuberculosis will be the most effective. These results also suggest that the total population harbouring live tubercle bacilli and consequently the future projection for increased incidence of tuberculosis in the world is probably overestimated. [ABSTRACT FROM AUTHOR]

Complement component C5 is crucial for experimental animal inflammatory tissue damage; however, its involvement in human inflammation is incompletely understood. The responses to Gram-negative bacteria were here studied taking advantage of human genetic complement-deficiencies-nature's own knockouts-including a previously undescribed C5 defect. Such deficiencies provide a unique tool for investigating the biological role of proteins. The experimental conditions allowed cross-talk between the different inflammatory pathways using a whole blood model based on the anticoagulant lepirudin, which does not interfere with the complement system. Expression of tissue factor. cell adhesion molecules, and oxidative burst depended highly on C5, mediated through the activation product C5a, whereas granulocyte enzyme release relied mainly on C3 and was C5a-independent. Release of cytokines and chemokines was mediated to varying degrees by complement and CD14; for example, interleukin (IL)-1β and IL-8 were more dependent on complement than IFN-γ and IL-6, which were highly dependent on CD14. IL-1 receptor antagonist (IL-1ra) and IFN-γ inducible protein 10 (IP-10) were fully dependent on CD14 and inversely regulated by complement, that is, complement deficiency and complement inhibition enhanced their release. Granulocyte responses were mainly complement-dependent, whereas monocyte responses were more dependent on CD14. Notably, all responses were abolished by combined neutralization of complement and CD14. The present study provides important insight into the comprehensive role of complement in human inflammatory responses to Gram-negative bacteria. [ABSTRACT FROM AUTHOR]

Tuberculous lymphadenitis (TBLN) is a diagnostic challenge in sub-Saharan Africa, where there is a high rate of human immunodeficiency virus (HIV) infection. This study aimed to find ways to improve the diagnosis in Butajira, rural Ethiopia, where TBLN constitutes 40% of the total tuberculosis (TB) diagnosis. Among 147 clinically suspected cases, 107 (72.8%) were confirmed as TBLN by fine-needle aspiration (FNA) cytology and acid-fast bacillus (AFB) smear examination. Of the remaining 40 cases, denoted non-tuberculous lymphadenitis (NTBLN) after this smear examination, 37 (92.5%) showed a cytological pattern with neutrophil aggregates. The clinical manifestations were similar and cervical lymph nodes were the most affected in these 2 groups. 24 of the 107 TBLN cases (22.4%) and 9 (22.5%) of the other cases were seropositive for HIV infection (p > 0.5). FNA cytology combined with AFB smear examination is a good alternative to histology in rural Ethiopia where the expertise in taking biopsies is very limited. Polymerase chain reaction for Mycobacterium tuberculosis complex DNA was positive in 15 of 23 cases tested with NTBLN cytology, showing that an additional independent criterion for the presence of M. tuberculosis is needed for diagnosis in lymphadenitis cases of this kind. These findings could help to strengthen the diagnostic algorithm suggested by the National TB Control Program. [ABSTRACT FROM AUTHOR]

New delivery methods are needed to improve the efficiency of existing DNA vaccines. We have measured the immune response to Mycobacterium tuberculosis antigens following intramuscular DNA injection in combination with or without electroporation. Three to 6-fold increase in the number of antigen specific CD4+ and CD8+ T cells, measured by IFN-γ-producing cells in an ELISPOT assay, was found in mice DNA injected and electroporated compared with non-electroporated mice. Similarly, 5 to 10-fold increase in antigen specific IgG1, IgG2a and IgG2b antibodies were found in an immunoglobulin subclass specific ELISA. A 100-fold reduction in DNA dose could be used without loss of efficiency when immunisation was combined with electroporation. A single injection of 1 μg of antigen 85b (ag85b) plasmid DNA was sufficient to elicit a higher and long lasting level of IgG2a antibodies against antigen 85B (Ag85B) compared to standard BCG vaccination. We conclude that DNA immunisation in combination with electroporation can significantly improve the immunogenicity of plasmid-based DNA vaccines. [Copyright &y& Elsevier]

Antigen 85 (mol. wt 30,000) (30 kD), secreted by actively growing mycobacteria under axenic conditions, and mol. wt 65,000 (65 kD), a cytoplasmic antigen released during mycobacterial lysis, were used to monitor the efficacy of chemotherapy in previously untreated pulmonary tuberculosis (UPTB) patients using enzyme-linked immunosorbent assay. Sera from 125 UPTB patients were examined for each of the 2 antigens individually and for the ratio of secretory (30 kD) to cytoplasmic (65 kD) antigen (SCR), before commencement of treatment, after intensive phase (IP), completion of optimum period of treatment (COPT) and 6 months post-COPT. 116 controls (normals and contacts) were also checked for these antigens. The detection of 30 kD and 65 kD antigens in UPTB patients had a sensitivity ranging from 50-57% (mean 30 kD value: 0.64±1.24 ngs/ml) to 20-22% (mean 65 kD value: 0.51±1.87 ngs/ml), respectively, whereas in controls it ranged from 2-8% (0.05±0.28 ngs/ml) to 14-47% (0.09±0.22 ngs/ml), respectively. Although the decline in 30 kD positivity was more evident at COPT, computation of the SCR denoted efficacy of chemotherapy more readily at IP. Similarly, SCR resolved the ambiguity between individual antigen levels and the clinical status of a patient. Since significant numbers of patients demonstrated 30 kD at IP it may be computed that the lifespan of circulating 30 kD in serum could be at least 2 months after the start of treatment, declining gradually thereafter. Although seromonitoring for secretory antigen generally reflects the efficacy of chemotherapy, the interpretation of findings clearly requires further elucidation. [ABSTRACT FROM AUTHOR]