Michael Kirschfink

Membranoproliferative Glomerulonephritis Type II (Dense Deposit Disease): An Update

Membranoproliferative glomerulonephritis type II (MPGN II) is a rare disease characterized by the deposition of abnormal electron-dense material within the glomerular basement membrane of the kidney and often within Bruchs membrane in the eye. The diagnosis is made in most patients between the ages of 5 and 15 yr, and within 10 yr, approximately half progress to end-stage renal disease, occasionally with the late comorbidity of visual impairment. The pathophysiologic basis of MPGN II is associated with the uncontrolled systemic activation of the alternative pathway (AP) of the complement cascade. In most patients, loss of complement regulation is caused by C3 nephritic factor, an autoantibody directed against the C3 convertase of the AP, but in some patients, mutations in the factor H gene have been identified. For the latter patients, plasma replacement therapy prevents renal failure, but for the majority of patients, there is no proven effective treatment. The disease recurs in virtually all renal allografts, and a high percentage of these ultimately fail. The development of molecular diagnostic tools and new therapies directed at controlling the AP of the complement cascade either locally in the kidney or at the systemic level may lead to effective treatments for MPGN II.

Eculizumab for Atypical Hemolytic–Uremic Syndrome

To the Editor: Atypical hemolytic–uremic syndrome is a disease of uncontrolled complement activation associated with a high mortality rate, and most cases progress to end-stage renal disease.1 Abou...

Eculizumab in Severe Shiga-Toxin–Associated HUS

This letter reports treatment of three children with severe, dialysis-requiring Shiga-toxin–induced hemolytic–uremic syndrome (HUS) with eculizumab, a monoclonal anti-C5 antibody. The condition of all three children improved rapidly.

Obstacles to cancer immunotherapy: expression of membrane complement regulatory proteins (mCRPs) in tumors.

Monoclonal antibodies (mAbs) are being increasingly used in cancer therapy owing to their ability to recognize specifically cancer cells and to activate complement- and cell-mediated cytotoxicity and/or to induce growth arrest or apoptosis. The therapeutic potential of anticancer antibodies is significantly limited due to the ability of cancer cells to block killing by complement. Of the multiple resistance strategies exploited by cancer cells, the expression of membrane complement regulatory proteins (mCRPs), such as CD46 (membrane cofactor protein (MCP)), CD55 (decay-accelerating factor (DAF)), CD35 (complement receptor type-1 (CR1)) and CD59, has received most attention. CD46, CD55 and CD35 block the complement cascade at the C3 activation stage and CD59 prevents assembly of the membrane attack complex of complement (MAC). These proteins protect normal tissues from accidental injury by activated complement, but also confer resistance on cancer cells, thereby limiting the effect of complement-fixing monoclonal antibodies. Expression of mCRPs on malignant cells is highly variable, yet there is clear indication that certain tumors express higher mCRP levels than the normal tissue from which they have evolved. mCRP level of expression and cellular location may also vary during malignant transformation and between differentiated and undifferentiated tumors. Neutralizing anti-mCRP mAbs have been used in vitro to elucidate the significance of mCRP expression to the tumor complement resistance phenotype. In general, CD59 appears to be the most effective mCRP protecting tumor cells from complement-mediated lysis. Nevertheless, it acts additively, and in certain tumors even synergistically, with CD55 and CD46. It is envisaged that treatment of cancer patients with mCRP blocking antibodies targeted specifically to cancer cells in combination with anticancer complement-fixing antibodies will improve the therapeutic efficacy.

Immune evasion of Borrelia burgdorferi by acquisition of human complement regulators FHL-1/reconectin and Factor H.

To understand immune evasion mechanisms of Borrelia burgdorferi we compared serum‐resistant B. afzelii and serum‐sensitive B. garinii isolates for their capacity toacquire human complement regulators. Here we demonstrate that the two borrelial genospecies show different binding of the two important human complement regulators, FHL‐1/reconectin and Factor H. All serum‐resistant B. afzelii isolates bound FHL‐1/reconectin and also Factor H, and all analyzed serum‐sensitive B. garinii isolates showed no or a significantly lower binding activity. Using recombinant deletion mutants, the binding domains were localized to the C terminus of FHL‐1/reconectin to short consensus repeats 5–7. The borrelial binding proteins were located in the surface of the bacteria as demonstrated by immunofluorescence staining of intact, serum‐exposed bacteria and by enrichment of outer membrane proteins. The surface‐attached complement regulators maintained complement regulatory activity as demonstrated in a cofactor assay. By ligand blotting two different borrelial binding proteins were identified that were responsible for the surface attachment of FHL‐1/reconectin and Factor H. These borrelial complement regulators acquiring surface proteins (CRASP) were further characterized as either CRASP‐1, a 27.5‐kDa molecule which preferentially binds FHL‐1/reconectin and which was present in all serum‐resistant borreliae, or CRASP‐2, a 20/21‐kDa protein which interacts preferentially with Factor H and the expression of which was more restricted, being detected in four of the six isolates analyzed. In summary, we describe a new immune evasion mechanism of B. burgdorferi, as these bacteria acquire human complement regulators to control complement activation on their surface and to prevent formation of toxic activation products.

Complement resistance of Borrelia burgdorferi correlates with the expression of BbCRASP-1, a novel linear plasmid-encoded surface protein that interacts with human factor H and FHL-1 and is unrelated to Erp proteins.

The etiologic agent of Lyme disease, Borrelia burgdorferi, is capable of circumventing the immune defense of a variety of potential vertebrate hosts. Previous work has shown that interaction of host-derived complement regulators, factor H and factor H-like protein 1 (FHL-1), with up to five complement regulator-acquiring surface proteins (CRASPs) expressed by resistant B. burgdorferi sensu lato isolates conferred complement resistance. In addition expression of CRASP-1 is directly correlated with complement resistance of Borrelia species. This work describes the functional characterization of BbCRASP-1 as the dominant factor H and FHL-1-binding protein of B. burgdorferi. The corresponding gene, zs7.a68, is located on the linear plasmid lp54 and is different from factor H-binding Erp proteins that are encoded by genes localized on circular plasmids (cp32). Deletion mutants of BbCRASP-1 were generated, and a high affinity binding site for factor H and FHL-1 was mapped to the C terminus of BbCRASP-1. Similarly, the predominant binding site of factor H and FHL-1 was localized to the short consensus repeat 7. Factor H and FHL-1 maintain their cofactor activity for factor I-mediated C3b inactivation when bound to BbCRASP-1, and factor H is up to 6-fold more efficient in mediating C3b conversion than FHL-1. In conclusion, BbCRASP-1 (i) binds the host complement regulators factor H and FHL-1 with high affinity, (ii) is the key molecule of the complement resistance of spirochetes, and (iii) is distinct from the Erp protein family. Thus, BbCRASP-1 most likely contributes to persistence of B. burgdorferi and to pathogenesis of Lyme disease.

Complement resistance of tumor cells: basal and induced mechanisms

Clinical and experimental studies have suggested that complement may play a role in tumor cytotoxicity. However, the efficiency of complement-mediated tumor cell lysis is hampered by various protective mechanisms, which may be divided into two categories: basal and induced mechanisms. The basal mechanisms are spontaneously expressed in cells without a need for prior activation, whereas the induced mechanisms develop in cells subjected to stimulation with cytokines, hormones, drugs or with sublytic doses of complement and other pore-formers. Membrane-associated complement regulatory proteins, such as CD55 (DAF, Decay-Accelerating Factor), CD46 (MCP, Membrane Cofactor Protein), CD35 (CR1, Complement Receptor type 1) and CD59, which serve as an important mechanism of self protection and render autologous cells insensitive to the action of complement. appear to be over-expressed on certain tumors. Furthermore, tumor cells secrete several soluble complement inhibitors. Tumor cells may also express proteases that degrade complement proteins, such as C3, or ecto-protein kinases which can phosphorylate complement components, such as C9. Besides this basal resistance, nucleated cells resist, to some extent, complement damage by removing the membrane attack complexes (MAC) from their surface. Several biochemical pathways, including protein phosphorylation, activation of G-proteins and turnover of phosphoinositides have been implicated in resistance to complement. Calcium ion influx and activation of protein kinase C (PKC) and of mitogen-activated protein kinase (MAPK) have also been demonstrated to be associated with the complement-induced enhanced resistance to lysis. The complete elucidation of the molecular mechanisms involved in basal and induced tumor cell resistance will enable the development of strategies for interfering with these evasion mechanisms and the use of the cytotoxic complement system against tumor cells.

Complement in human diseases: Lessons from complement deficiencies

Complement deficient cases reported in the second half of the last century have been of great help in defining the role of complement in host defence. Surveys of the deficient individuals have been instrumental in the recognition of the clinical consequences of the deficiencies. This review focuses on the analysis of the diseases associated with the deficiencies of the various components and regulators of the complement system and their therapeutic implications. The diagnostic approach leading to the identification of the deficiency is discussed here as a multistep process that starts with the screening assays and proceeds in specialized laboratories with the characterization of the defect at the molecular level. The organization of a registry of complement deficiencies is presented as a means to collect the cases identified in and outside Europe with the aim to promote joint projects on treatment and prevention of diseases associated with defective complement function.

Complement Inhibitor Eculizumab in Atypical Hemolytic Uremic Syndrome

BACKGROUND AND OBJECTIVES Atypical hemolytic uremic syndrome (aHUS) is associated with a congenital or acquired dysregulation of the complement alternative pathway that leads to continuous complement activation on host cells causing inflammation and damage. Eculizumab, a humanized mAb against complement protein C5, inhibits activation of the terminal complement pathway. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS We report an adolescent with relapsing unclassified aHUS. On admission, a high plasma creatinine level indicated a poor prognosis, and hemodialysis had to be started. Plasma exchanges were initially effective against the microangiopathic hemolytic activity and allowed a temporary improvement of renal function with termination of hemodialysis after 7 wk. Subsequently, plasma exchanges (three times per week) failed to prevent ongoing aHUS activity and progressive renal failure. After 12 wk, aHUS treatment was switched to eculizumab. RESULTS Eculizumab was effective in terminating the microangiopathic hemolytic process in two aHUS relapses; however, after normalization of complement activity, aHUS recurred and ultimately led to anuric end-stage renal failure. CONCLUSIONS In this patient, complement inhibition by eculizumab temporarily terminated the microangiopathic hemolytic activity. Nevertheless, renal damage as a result of preceding and subsequent aHUS activity resulted in end-stage renal failure; therefore, therapeutic success may depend on early administration of eculizumab. The optimal duration of treatment may be variable and remains to be determined.

Immune evasion of Borrelia burgdorferi: mapping of a complement-inhibitor factor H-binding site of BbCRASP-3, a novel member of the Erp protein family.

The causative agents of Lyme disease, Borrelia burgdorferi s.s., B. garinii, and B. afzelii, differ in their susceptibility to complement‐mediated lysis. This phenomenon apparently depends on the expression of proteins termed complement regulator‐acquiring surface proteins (CRASP) and their binding to the inhibitory plasma proteins factor H and FHL‐1. To characterize these bacterial proteins in more detail we have now isolated from a B. burgdorferi expression library a novel factor H‐binding protein. In accordance with our previous studies this protein was termed BbCRASP‐3 and represents a novel member of the polymorphic Erp (OspE/F‐related) protein family. On the basis of protease accessibility assays using intact spirochetes, BbCRASP‐3 isidentified as a surface‐exposed protein and binds the C‐terminal short consensus repeats of factor H. Applying deletion mutants of BbCRASP‐3, the factor H‐binding site was mapped to the nine‐amino‐acid motif LEVLKKNLK localized at the C‐terminal end of BbCRASP‐3. Factor H bound to BbCRASP‐3 maintains its cofactor activity in factor I‐mediated C3b inactivation. Binding of BbCRASP‐3 to factor H can be inhibited by heparin, a physiological ligand of the complement regulator factor H. Blocking of factor‐H‐binding by soluble BbCRASP‐3 leads to an increase of complement deposition on intermediate serum‐resistant strain ZS7. In conclusion, BbCRASP‐3 has been identified as a novel factor H‐binding protein on B. burgdorferi which by conferring complement resistance to the pathogen may contribute to its persistence in the mammalian host.