Morten Harboe

Persistence of DNA from Mycobacterium tuberculosis in superficially normal lung tissue during latent infection

BACKGROUND A third of the worlds population has latent infection with Mycobacterium tuberculosis, and in areas of low endemicity, most cases of active tuberculosis arise as a result of reactivation of latent bacilli. We sought to establish the cellular location of these latent organisms to facilitate their elimination. METHODS We applied in-situ PCR to sections of macroscopically normal lung tissue from 13 individuals from Ethiopia and 34 from Mexico who had died from causes other than tuberculosis. Sections of lung tissue from six Norwegian individuals (ie, individuals from a non-endemic population) acted as negative controls, and six Ethiopian tuberculosis cases acted as positive controls. FINDINGS Control necropsy samples from the Norwegian individuals were all negative by in-situ PCR and conventional PCR, whereas all samples from known Ethiopian tuberculosis cases were positive by both methods. However, in macroscopically normal lung tissue from Ethiopian and Mexican individuals without tuberculous lesions, the in-situ PCR revealed five of 13 and ten of 34 positive individuals, respectively. These results were confirmed by conventional PCR with extracted DNA. Positive cells included alveolar and interstitial macrophages, type II pneumocytes, endothelial cells, and fibroblasts. INTERPRETATION M. tuberculosis can persist intracellularly in lung tissue without histological evidence of tuberculous lesions. M. tuberculosis DNA is situated not only in macrophages but also in other non-professional phagocytic cells. These findings contradict the dominant view that latent organisms exist in old classic tuberculous lesions, and have important implications for strategies aimed at the elimination of latent and persistent bacilli.

Quantification of the Terminal Complement Complex in Human Plasma by an Enzyme‐Linked Immunosorbent Assay Based on Monoclonal Antibodies against a Neoantigen of the Complex

The fluid‐phase terminal complement complex (TCC), consisting of the components C5b, C6, C7, C8, C9, and the S‐protein, has recently been detected in normal human plasma by using antibodies against native terminal complement components. Increased amounts of TCC were then found in several patients with in vivo activation of complement. We now describe a sensitive, specific, and reliable enzyme‐linked immunosorbent assay for quantification of the TCC, based on monoclonal antibodies against a neoantigen of the complex. The results indicate that the TCC is present in normal human plasma and in increased amounts in patients with complement activation in vivo, thus confirming previously obtained results. The assay is easy to perform and can be used for examination of large numbers of plasma samples.

The quantitative role of alternative pathway amplification in classical pathway induced terminal complement activation.

Complement activation with formation of biologically potent mediators like C5a and the terminal C5b‐9 complex (TCC) contributes essentially to development of inflammation and tissue damage in a number of autoimmune and inflammatory conditions. A particular role for complement in the ischaemia/reperfusion injury of the heart, skeletal muscle, central nervous system, intestine and kidney has been suggested from animal studies. Previous experiments in C3 and C4 knockout mice suggested an important role of the classical or lectin pathway in initiation of complement activation during intestinal ischaemia/reperfusion injury while later use of factor D knockout mice showed the alternative pathway to be critically involved. We hypothesized that alternative pathway amplification might play a more critical role in classical pathway‐induced C5 activation than previously recognized and used pathway‐selective inhibitory mAbs to further elucidate the role of the alternative pathway. Here we demonstrate that  selective  blockade  of  the  alternative  pathway  by  neutralizing  factor  D  in  human  serum  diluted 1 : 2 with mAb 166–32 inhibited more than 80% of C5a and TCC formation induced by solid phase IgM and solid‐ and fluid‐phase human aggregated IgG via the classical pathway. The findings emphasize the influence of alternative pathway amplification on the effect of initial classical pathway activation and the therapeutic potential of inhibiting the alternative pathway in clinical conditions with excessive and uncontrolled complement activation.

The alternative complement pathway revisited

•  Introduction •  Methods to study and modify the alternative pathway ‐  Inhibition by monoclonal antibodies ‐  Knockout models •  Influence of AP amplification on the effect of specific activation of the other initial pathways ‐  Direct activation versus amplification ‐  Mechanisms of amplification •  Properdin ‐  Control protein in the AP amplification loop ‐  Recognition molecule in AP •  Influence of AP in deficiency states ‐  Factor H deficiency and variants ‐  C2 bypass in CP and LP ‐  Properdin deficiency •  Complement in sepsis •  Concluding remarks

Innate Immune Responses to Danger Signals in Systemic Inflammatory Response Syndrome and Sepsis

The systemic immune response induced by non‐infectious agents is called systemic inflammatory response syndrome (SIRS) and infection‐induced systemic immune response is called sepsis. The host inflammatory response in SIRS and sepsis is similar and may lead to multiple organ dysfunction syndrome (MODS) and ultimately death. The mortality and morbidity in SIRS and sepsis (i.e. critical illness) remain high despite advances in diagnostic and organ supporting possibilities in intensive care units. In critical illness, the acute immune response is organized and executed by innate immunity influenced by the neuroendocrine system. This response starts with sensing of danger by pattern‐recognition receptors on the immune competent cells and endothelium. The sensed danger signals, through specific signalling pathways, activate nuclear transcription factor κB and other transcription factors and gene regulatory systems which up‐regulate the expression of pro‐inflammatory mediators. The plasma cascades are also activated which together with the produced pro‐inflammatory mediators stimulate further the production of inflammatory biomarkers. The acute inflammatory response underlies the pathophysiological mechanisms involved in the development of MODS. The inflammatory mediators directly affect organ function and cause a decline in remote organ function by mediating the production of nitric oxide leading to mitochondrial anergy and cytopathic hypoxia, a condition of cellular inability to use oxygen. Understanding the mechanisms of acute immune responses in critical illness is necessary for the development of urgently needed therapeutics. The aim of this review is to provide a description of the key components and mechanisms involved in the immune response in SIRS and sepsis.

Recognition of two distinct groups of human IgM and IgA based on different binding to staphylococci.

Labeled monoclonal IgM was bound to the Cowan I strain of Staphylococcus aureus in the absence of anti‐IgM antibody This binding was inhibited by 11 of 33 monoclonal IgM proteins; 22 failed to inhibit Agammaglobulinemic serum alto failed to inhibit High concentrations of IgG inhibited the binding of IgM Se to staphylococci, but IgG and IgM probably react with different receptors on tin bacterial surface. Polyclonal IgM from all of 17 individuals tested inhibited the binding of IgM Se to staphylococci, which indicates that the distinction corresponds to an IgM subclass rather than to an allotype. Two of seven monoclonal IgA proteins also inhibited the binding of IgM Se to staphylococci.

The Antigens of Mycobacterium bovis, Strain BCG, Studied by Crossed Immunoelectrophoresis: a Reference System

A reference system for the soluble antigen of Mycobacterium bovis, strain BCG is described. The system is based on crossed immunoelectrophoresis with intermediate gel. A Commercially available immunoglobulin preparation made from rabbit anti‐BCG hyperimmune serum was used as reference antibodies, while a concentrated BCG culture filtrate was used as reference antigen. The pattern obtained was highly reproducible, and most of the components were stable when the fiftyfold‐concentrated culture filtrate was stored in –20°C. About thirty different antigenic components were selected as reference antigens and numbered. The majority of the reference antigens was present in extracts prepared from BCG by ultrasonication or bacterial press extraction. Use of the system for studies of antigenic relationship between mycobacteria, identification and quantification of antigens, and characterization of antimycobacterial antibodies are illustrated by examples. The antigens of two preparations of tuberculin purified protein derivative (PPD) were identified. The antigen designated BCG60 was found to be a major constituent of tuberculin PPD. Evidence is presented that this antigen is cell wall associated.

Human genetic deficiencies reveal the roles of complement in the inflammatory network: Lessons from nature

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—natures 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.

Identification of the Causative Organism of Tuberculous Lymphadenitis in Ethiopia by PCR

ABSTRACT Tuberculous lymphadenitis (TBLN) is a common form of extrapulmonary tuberculosis with multiple differential diagnoses. Demonstration of the etiologic agent by smear microscopy or culture of fine needle aspirate (FNA) specimens is often unsuccessful. FNA specimens from 40 patients presenting at a rural health center in South Ethiopia and diagnosed as positive for TBLN on the basis of clinical and cytological criteria were analyzed for mycobacterial DNA by PCR. Thirty (75%) had cervical lymphadenitis and 11 (27.5%) were seropositive for human immunodeficiency virus (HIV). Three primer sets were initially used to identify the causative agent at the genus (antigen 85 complex), complex (IS6110 insertion sequence), and species (pncA gene and allelic variation) levels. Among the forty TBLN cases, 35 (87.5%) were positive by PCR at the genus and complex levels. Based on PCR for detection of allelic variation at position 169, 24 (68.6%) of the 35 were positive for Mycobacterium tuberculosis and 6 (17.1%) were positive for M. bovis. These six were positive in additional PCR assays using the JB21-JB22 primer set, which is highly specific for M. bovis. Five (14.1%) showed amplification for both M. tuberculosis and M. bovis with the allele-specific primer set. Cooccurrence of pyrazinamide (PZA)-sensitive and -resistant M. tuberculosis in those five cases was indicated, since all were negative in assays with the JB21-JB22 primer set. This feature was seen in 3 of 11 HIV-positive and 2 of 29 HIV-negative individuals (P < 0.001). Conclusion: among 35 PCR-positive cases of TBLN from southern Ethiopia, 29 (82.9%) were caused by M. tuberculosis and six (17.1%) were caused by M. bovis.

Purification, characterization and identification of a 32 kDa protein antigen of Mycobacterium bovis BCG☆

An immunogenic protein called P32 has been purified from Sauton zinc deficient culture filtrate of Mycobacterium bovis BCG using successively hydrophobic chromatography on Phenyl-Sepharose, ion exchange on DEAE-Sephacel and molecular sieving on Sephadex G-100. The final preparation was found to be homogeneous as based on several analyses. This P32 protein was a constituent of BCG cells grown in normal conditions. It represented about 3% of the soluble fraction of a cellular extract, and appeared as the major protein released in normal Sauton culture filtrate. This protein was found to have a molecular weight of 32,000 by SDS-polyacrylamide gel electrophoresis and in molecular sieving. Its amino acid composition showed an abundance of acidic amino acids (or their amides). The NH2-terminal amino acid sequence (6 amino acids) was determined. Purified P32 was tested by various crossed immunoelectrophoresis techniques, and was shown to belong to the antigen 85 complex in the reference system for BCG antigens. It was more precisely identified as antigen 85A. The protein antigen elicited a weak delayed hypersensitivity reaction in guinea pigs sensitized with heat-killed or living BCG. No delayed hypersensitivity reaction was observed in living BCG sensitized mice, however, it induced significant amounts of gamma interferon in cultured spleen cells from BCG-sensitized mice. Moreover, P32 either pure or as part of BCG soluble extract promoted substantial antibody levels when injected in rabbits.