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Does dysregulated complement activation contribute to haemolytic uraemic syndrome secondary to Streptococcus pneumoniae?
Medical Hypotheses 81 (2013) 400–403
Contents lists available at SciVerse ScienceDirect
Medical Hypotheses journal homepage: www.elsevier.com/locate/mehy
Does dysregulated complement activation contribute to haemolytic uraemic syndrome secondary to Streptococcus pneumoniae? Rodney D. Gilbert ⇑, Arvind Nagra, Mushfequr R. Haq Regional Paediatric Nephro-Urology Unit, University Hospital Southampton, Tremona Road, Southampton SO16 6YD, United Kingdom
a r t i c l e
i n f o
Article history: Received 1 November 2012 Accepted 21 May 2013
a b s t r a c t We describe two patients with haemolytic uraemic syndrome (HUS) associated with invasive Streptococcus pneumoniae infection. Both patients had transiently reduced serum concentrations of complement C3. One had reduced expression of CD46 and never recovered renal function. No constitutive defect in regulation of the alternative pathway of complement activation was demonstrated in the second patient but there was an apparent improvement in her condition after administration of eculizumab. The most widely accepted mechanism for pneumococcal HUS is endothelial cell damage by pre-formed antibodies against the Thomsen–Friedenreich antigen. This explanation does not bear rigorous scrutiny. We postulate that transiently dysregulated complement activation may play a role in the pathogenesis of pneumococcal disease. We further postulate that the mechanism could be enhanced binding of factor H to the neuraminidase-altered surface of endothelial cells or reduced binding of factor H to the endothelial cell surface mediated by competitive binding of factor H by pneumococcal surface protein C (pspC). Ó 2013 Elsevier Ltd. All rights reserved.
Introduction
Case reports
Haemolytic uraemic syndrome (HUS) is a triad of microangiopathic haemolytic anaemia, thrombocytopenia and acute kidney injury. Pathologically it is characterised by vessel wall thickening with swelling and detachment of endothelial cells with accumulation of material in the subendothelial space. There are intraluminal platelet thrombi with partial or complete luminal obstruction [1]. The concept that the initiating process is damage to the microvascular endothelium was first proposed by Altschule in 1942 [2]. Seventy years later it is still believed that endothelial damage is the primary event [3]. In the case of infection related HUS, microbial toxins are believed to be the primary damaging mechanism. In many of the remaining cases, inherited or acquired disorders of the regulation of complement activation lead to endothelial damage [4,5]. It has recently been suggested that complement activation plays an important role in the pathogenesis of shigatoxin associated disease and other infection-related cases of HUS [6,7]. There may therefore not be a clear separation between shigatoxin induced cases, those associated with other infections or drugs and those with inherited disorders of complement regulation. We present two patients with Streptococcus pneumoniae associated HUS (pHUS) with transiently reduced serum C3 levels and suggest that complement-mediated injury may have played an important role in their disease and suggest possible mechanisms by which this may have occurred.
Case 1
⇑ Corresponding author. Tel.: +44 0 2380796254; fax: +44 0 2380795230. E-mail address: [email protected] (R.D. Gilbert). 0306-9877/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mehy.2013.05.030
A previously well 11 month old male infant presented to his local hospital with a 4 day history of upper respiratory tract infection associated with fever and vomiting. He then became unresponsive with twitching of the right leg and right side of his face with progression to a generalised seizure. Admission blood tests showed a plasma sodium concentration of 130 mmol/L, potassium 3.9 mmol/ L, urea 2.2 mmol/L, creatinine 48 lmol/L, haemoglobin 110 g/L and platelets of 427 109/L. Blood culture yielded a growth of S. pneumoniae serotype 19A sensitive to penicillin, amoxicillin and cefotaxime. A computed tomography scan of his head showed mild dilatation of both lateral ventricles and florid contrast enhancement of the extracerebral spaces compatible with meningitis. He was given intravenous cefotaxime, acyclovir and phenytoin. On the second hospital day the urine output had fallen to 0.37 ml/kg/hour, he had become oedematous and had increasing respiratory distress. He was transferred to the Paediatric Intensive Care Unit at Southampton. Blood tests showed renal failure (urea 17.1 mmol/L, creatinine 191 lmol/L), anaemia (Hb 67 g/L), thrombocytopaenia (12 109/L), complement C3 0.45 g/L (normal 0.75– 1.65), C4 0.20 g/L (normal 0.14–0.54), haptoglobin <0.06 g/L (normal 0.5–2.0) and lactate dehydrogenase (LDH) 2486 iu/L (normal 266–500). A blood film was positive for red cell fragments and the peanut lectin assay for T-cryptantigen activation was positive. These results were considered compatible with a diagnosis of pHUS.
R.D. Gilbert et al. / Medical Hypotheses 81 (2013) 400–403
He was initially managed with continuous veno-venous haemofiltration but converted to peritoneal dialysis after 2 days. He recovered well from the meningitis without focal neurological deficit except for profound deafness for which he received a cochlear implant. He remained anuric and received a live donor renal transplant 14 months after first admission. Investigations for regulatory defects in the alternative pathway of complement activation (performed 3 days after admission) showed that the C3 concentration had returned to normal and was 1.2 g/L. The complement factor B concentration was 191 mg/ L (normal 90–320), factor H 648 mg/l (normal 345–590), factor I 131% (normal 70–130) and no factor H antibodies were detected. The von Willebrand cleaving protease (ADAMTS13) activity was 18% and there were no inhibitors. There was reduced expression of CD46 (membrane cofactor protein, MCP) on peripheral blood leukocytes (mean fluorescence intensity score 523, normal 600– 1400) compatible with heterozygous deficiency. Genetic analysis revealed no exonic mutations of CFH, CFI, CD46, CFB or C3. Case 2 A 21 month old female infant presented to her local hospital with a 12 day history of an upper respiratory tract infection. Seven days prior to admission she was reviewed in the Emergency Department because of cough, vomiting and pyrexia. A chest radiograph was reported as normal and she was discharged but represented 48 h later with respiratory distress and grunting. Examination revealed dullness to percussion and reduced breath sounds on the left side of the chest and a repeat radiograph showed left lower lobe consolidation. Blood tests on admission were as follows: haemoglobin concentration 111 g/L, platelet count 423 109/L, plasma sodium concentration 130 mmol/L, potassium 4.4 mmol/L, urea 5.1 mmol/L, creatinine 17 lmol/L and bicarbonate 18.9 mmol/L. She was given intravenous amoxicillin/clavulanic acid and her fluid intake was restricted. After 48 h she was mildly icteric, the haemoglobin concentration was 51 g/L, platelet count 5 x 109/L, bicarbonate 15.3 mmol/ L, urea 30.9 mmol/L and creatinine 151 lmol/L. A blood film showed red cell fragments and the Coombs test was positive. She was hypertensive with a systolic blood pressure of 130 mmHg. She was given amlodipine and a red cell transfusion. The antibiotic therapy was changed to cefotaxime and clarithromycin. She was then transferred to Southampton General Hospital for management of her HUS. On arrival she was pale and distressed. The respiratory rate was 58/min but transcutaneous oxygen saturations were 100% in air. There were reduced breath sounds at the left base. The pulse rate was 158/min, the heart was clinically normal and the blood pressure 100/56 mmHg. The liver was palpable 4 cm below the right costal margin. Laboratory investigations confirmed renal failure (urea 36.8 mmol/L and creatinine 198 lmol/L) and a there were numerous red cell fragments on a blood film. The LDH was 13,782 iu/L, plasma haptoglobin was 0.38 g/L, complement C3 0.58 g/L and complement C4 was 0.14 g/L. ADAMTS13 activity was 80%. A chest ultrasonogram revealed a sub-pulmonary effusion and a chest drain was inserted. Blood cultures were negative but a urine sample was positive for pneumococcal antigen. She became anuric and was commenced on peritoneal dialysis via a Tenckhoff catheter. The chest drain was removed 7 days after admission and the antibiotics were stopped after 10 days. Because of the low initial serum complement C3 concentration and the fact that she remained anuric and thrombocytopaenic, she was given eculizumab 300 mg intravenously on the seventh day of admission to Southampton and then fortnightly thereafter, according to the manufacturer’s dosing recommendations. Immediately prior to
401
administration of eculizumab the serum complement C3 had normalised to 1.54 g/L and the total haemolytic complement was high at 143%. The platelet count was 138 109/L and 12 h after the infusion it was 208 109/L and she became noticeably less irritable. She remained completely anuric for a further 2 weeks after the first dose of eculizumab (total of 21 days of anuria) and remained dialysis dependent for a total of 30 days. Three months after admission the patient had improving, but still abnormal, renal function with a plasma creatinine concentration of 82 lmol/L giving her an estimated GFR of 40 ml/min/1.73 m2. Her early morning urine protein/creatinine ratio was significantly elevated at 121 mg/ mmol. Investigations were performed to detect abnormalities of regulation of the alternative pathway of complement activation. No abnormalities were found in the C3, CFH, CFB, CFI or CD46 genes. CD46 expression on peripheral blood neutrophils was normal and antibodies to factor H were not detected. Multiplex ligationdependent probe amplification of CFHR1 exons 3, 5 and 6 and CFHR3 exons 1–4 and 6 showed 2 copies of each exon. Eculizumab was therefore stopped after 4 doses.
Discussion The terms ‘‘typical’’, ‘‘diarrhoea-associated’’ or ‘‘D + ’’ HUS are commonly used to describe HUS caused by shigatoxin producing organisms, most frequently Escherichia coli O157:H7. The terms ‘‘atypical’’, ‘‘non-diarrhoea-associated’’ or ‘‘D-’’ HUS have generally been applied to any form of HUS not due to shigatoxin producing organisms. In this classification, atypical HUS includes those cases secondary to other infections such as S. pneumoniae [8], human immunodeficiency virus (HIV) [9] and H1N1 Influenza A [10], a variety of drugs [11] or methyl malonic aciduria with homocystinuria, a rare inborn error of metabolism [12]. It also includes a group of patients with inherited or acquired disorders of complement regulation. It has been suggested that the term ‘‘atypical HUS’’ is therefore inadequate and the term ‘‘complement dysregulation-associated HUS’’ has been recommended to describe the latter group.[5]. There are at least two significant objections to this nomenclature, however. The first is that in around 40% of patients with non-shigatoxin associated HUS no cause can be found with currently available investigations. The second is that there is increasing evidence of at least transient dysregulation of complement activation in those cases due to shigatoxin producing organisms. To date there is little in the medical literature on the role of complement activation in cases due to S. pneumoniae. Our two patients both had low serum concentrations of C3 suggesting exuberant activation of the alternative pathway. This phenomenon has been previously observed [13] and a role for complement in the pathogenesis of pneumococcal HUS has been previously postulated (5). HUS following infection with S. pneumoniae has traditionally been explained as resulting from the action of bacterial neuraminidase which exposes the Thomsen–Friedenreich antigen (T-antigen) to pre-formed IgM antibodies which then damage the cells leading to HUS [14]. The pathogenicity of pre-formed anti-T IgM seems doubtful, however as it is a cold reactive antibody and at 37 °C causes neither red cell agglutination nor complement activation [15]. Furthermore, anti-T antibodies are not uniformly present during severe pHUS [16]. The alternative pathway of complement activation (APC) activates spontaneously in aqueous solution and C3b is deposited on surfaces. Host cell membranes are protected against combination of bound C3b with factor B to form the C3/C5 convertase C3bBb and the subsequent assembly of the membrane attack complex
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R.D. Gilbert et al. / Medical Hypotheses 81 (2013) 400–403
by a number of complement regulators, namely factor H, factor I, CD46 (Membrane cofactor Protein, MCP) and CD59 [17]. Factor H is an abundant serum glycoprotein composed of 20 complement control protein (CCP) modules [18]. It is the most important regulator of the APC in the fluid phase. The N-terminal four CCPs act as a cofactor for factor I-mediated proteolytic inactivation of C3b, compete with factor B for C3b binding and accelerate the decay of the C3bBb C3/C5 convertase into its components [19,20]. CCPs 19–20 contain two C3b binding sites [21] as well as a sialic acid binding site allowing Factor H to bind to sialic acid residues or glycosaminoglycans on endothelial cells and at the same time to engage the C3b molecule, allowing the N-terminal 4 CCPs to prevent further activation of the complement cascade, thus protecting host cells against complement mediated damage. The C3b and glycosaminoglycan binding sites on CCP 20 are partially overlapping [22–24]. Mutations in Factor H are the most frequent known cause of HUS secondary to inherited deficiencies of complement regulation. HUS-causing mutations are mostly found in the region of the gene coding for the C-terminal CCPs 19–20 [25]. Known aHUS mutations may have reduced or increased binding affinity to glomerular endothelial cells or reduced binding affinity to C3b [26]. Since pneumococcal neuraminidase removes sialic acid from endothelial cell surfaces, it has been proposed that factor H binding to these cells would be reduced, thus exposing them to complement-mediated damage. In an in vitro model of pHUS, Johnson et al. exposed cultured glomerular endothelial cells to neuraminidase to expose the T-antigen. The cells were then incubated with fresh human serum. As expected there was increased deposition of C3b and membrane attack complex but surprisingly there was also increased factor H deposition in neuraminidase treated cells compared with controls. (13) The enhanced binding of factor H to neuraminidase-treated endothelial cells may reduce binding to C3b and prevent further complement activation. Lehtinen et al. found four mutant factor H proteins associated with aHUS that had increased binding to glomerular endothelial cells. These mutants had unchanged binding to C3b when these functions were tested separately. Simultaneous binding to glomerular endothelial cell surfaces and C3b was not tested in this study [26]. Other pathogen virulence factors might also be involved in disrupting the normal function of factor H on host glomerular endothelial cells. Invading micro-organisms must have the capacity to avoid or ameliorate complement-mediated destruction or they would rapidly be coated with C3b and killed by a combination of cell wall perforation by the membrane attack complex and intracellular killing by phagocytes. One of the virulence factors expressed by pneumococci is pneumococcal surface protein C (pspC) [27]. PspC binds factor H and regulates complement activation on the pneumococcal surface. The pspC locus may include one or two copies of the gene [28]. In mice infected with the same strain of S. pneumoniae the expression of pspC varied depending on the route of infection and the cytokine profile produced by the host [29]. The amount of pspC produced therefore may depend on both the organism involved and on the nature of the immune response mounted by the host. It is possible that under certain circumstances very large amounts are produced. Some may be liberated into the fluid environment where it can bind to the C-terminal end of the factor H molecule and thereby inhibit binding to cell surfaces in a manner analogous to anti-factor H antibodies [30]. This would reduce host protection against complement-mediated damage. In conclusion we have presented two patients with pHUS who had low concentrations of complement component C3 at presentation. The first has reduced expression of CD46 which probably exacerbated the renal injury and he never recovered renal function. The second has no demonstrable defect in APC regulatory factors
but appeared to respond to treatment with the complement inhibitor eculizumab with an abrupt increase in platelet count and a subjective decrease in irritability. These cases suggest a possible role for abnormal regulation of complement activation in this form of HUS. We suggest two possible mechanisms for disordered regulation mediated by S. pneumoniae, namely increased binding of factor H to endothelial cell surfaces, possibly nullifying the effect of the partially overlapping C3b binding site on factor H CCP 20 and interference with cell surface and binding by soluble pspC. Further research is required to determine which, if any, of these postulated mechanisms is involved in the pathogenesis of pHUS. If our hypothesis is verified it would imply that the use of complement inhibiting drugs should be part of the treatment of pHUS.
Conflict of interest All three authors have received financial assistance for attendance at conferences from Alexion Pharma, manufacturer of eculizumab.
References [1] Ruggenenti P, Noris M, Remuzzi G. Thrombotic icroangiopathy, hemolytic uremic syndrome, and thrombotic thrombocytopenic purpura. Kidney Int 2001;60:831–46. [2] Altschule MD. A rare type of acute thrombocytopenic purpura: widespread formation of platelet thrombi in capillaries. N Engl J Med 1942;227:477–9. [3] Obeidat M, Obeidat M, Ballermann BJ. Glomerular endothelium: a porous sieve and formidable barrier. Exp Cell Res 2012;318:964–72. [4] Keir L, Coward RJM. Advances in our understanding of glomerular thrombotic microangiopathy. Pediatr Nephrol 2011;26:523–33. [5] Loirat C, Frémeaux-Bacchi V. Atypical hemolytic uremic syndrome. Orphanet J Rare Dis 2011;6:60. [6] Johnson S, Waters A. Is complement a culprit in infection-induced forms of haemolytic uraemic syndrome? Immunobiology 2012;217:235–43. [7] Ståhl A, Sartz L, Karpman D. Complement activation on platelet-leukocyte complexes and microparticles in enterohemorrhagic Escherichia coli – induced hemolytic uremic syndrome. Blood 2011;117:5503–13. [8] Waters AM, Kerecuk L, Luk D, et al. Hemolytic uremic syndrome associated with invasive pneumococcal disease: The United Kingdom experience. J Pediatr 2007;151:140–4. [9] Sutor GC, Schmidt RE, Albrecht H. Thrombotic microangiopathies and HIV infection: report of two typical cases, features of HUS and TTP, and review of the literature. Infection 1999;27:12–5. [10] Caltik A, Akyüz SG, Erdogan O, Demircin G. Hemolytic uremic syndrome triggered with a new pandemic virus: influenza A (H1N1). Pediatr Nephrol 2011;26:147–8. [11] Zakarija A, Bennett C. Drug induced thrombotic microangiopathy. Semin Thromb Hemost 2005;31:681–90. [12] Menni F, Testa S, Guez S, Chiarelli G, Alberti L, Esposito S. Neonatal atypical hemolytic uremic syndrome due to methylmalonic aciduria and homocystinuria. Pediatr Nephrol 2012;27:1401–5. [13] Johnson SA, Williams JM, Morgan B, Taylor C, Savage COS. Evidence for complement dysregulation in pneumococcal haemolytic uraemic syndrome (Abstract). Pediatr Nephrol 2009;24:1781. [14] Copelovitch L, Kaplan BS. Streptococcus pneumoniae-associated hemolytic uremic syndrome. Pediatr Nephrol 2008;11:1951–6. [15] Crookston KP, Reiner AP, Cooper LJ, Sacher RA, Blajchman MA, Heddle NM. RBC T activation and hemolysis: implications for pediatric transfusion management. Transfusion 2000;40:801–12. [16] Eber SW, Polster H, Quentin SH, Rumpf KW, Lynen R. Hemolytic uremic syndrome in pneumococcal meningitis and infection. Importance of Ttransformation. Monatsschr Kinderheilkd 1993;141:219–22. [17] Devine DV. The regulation of complement on cell surfaces. Transfus Med Rev 1991;5:123–31. [18] Rodriguez de Cordoba S, Esparza-Gordillo J, Goicoechea de Jorge E, LopezTrascasa M, Sanchez-Corral P. The human complement factor H: functional roles, genetic variations and disease associations. Mol Immunol 2004;41:355–67. [19] Richards A, Kavanagh D, Atkinson JP. Inherited complement regulatory protein deficiency predisposes to human disease in acute injury and chronic inflammatory states: the examples of vascular damage in atypical hemolytic uremic syndrome and debris accumulation in age-related macular degeneration. Adv Immunol 2007;96:141–77. [20] Hocking HG, Herbert AP, Kavanagh D, et al. Structure of the N-terminal region of complement factor H and conformational implications of disease-linked sequence variations. J Biol Chem 2008;283:9475–87.
R.D. Gilbert et al. / Medical Hypotheses 81 (2013) 400–403 [21] Kajander T, Lehtinen MJ, Hyvärinen S, et al. Dual interaction of factor H with C3d and glycosaminoglycan in host-nonhost discrimination by complement. Proc Natl Acad Sci USA 2011;108:2897–902. [22] Schmidt CQ, Herbert AP, Kavanagh D, et al. A new map of glycosaminoglycan and C3b binding sites on factor H. J Immunol 2008;181:2610–9. [23] Hellwage J, Jokiranta TS, Friese MA, et al. Complement C3b/C3d and cell surface polyanions are recognised by overlapping binding sites on the most carboxyl-terminal domain of complement factor H. J Immunol 2002;169:6935–44. [24] Morgan HP, Schmidt CQ, Guariento M, et al. Structural basis for engagement by complement factor H of C3b on a self surface. Nat Struct Mol Biol 2011;18:463–70. [25] Neumann HP, Salzmann M, Bohnert-Iwan B, et al. Haemolytic uraemic syndrome and mutations of the factor H gene: a registry-based study of German speaking countries. J Med Genet 2003;40:676–81.
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[26] Lehtinen MJ, Rops AL, Isenman DE, van der Vlag J, Jokiranta TS. Mutations of factor H Impair regulation of surface-bound C3b by three mechanisms in atypical hemolytic uremic syndrome. J Biol Chem 2009;284:15650–8. [27] Quin L, Carmicle S, Dave S, Pangburn MK, Evenhuis JP, McDaniel LS. In vivo binding of complement regulatory factor H by Streptococcus pneumoniae. J Infect Dis 2005;192:1996–2003. [28] Zipfel PF, Hallström T, Hammerschmidt S, Skerka C. The complement fitness factor H: role in human diseases and for immune escape of pathogens, like pneumococci. Vaccine 2008;30(26 Suppl. 8):167–74. [29] Quin L, Moore QC, Thornton JA, McDaniel LS. Peritoneal challenge modulates expression of pneumococcal surface protein C during bacteremia in mice. Infect Immun 2008;76:1122–7. [30] Dragon-Durey M-A, Loirat C, Cloarec S, et al. Anti-factor H autoantibodies associated with atypical haemolytic uremic syndrome. J Am Soc Nephrol 2005;16:555–63.
Contents lists available at SciVerse ScienceDirect
Medical Hypotheses journal homepage: www.elsevier.com/locate/mehy
Does dysregulated complement activation contribute to haemolytic uraemic syndrome secondary to Streptococcus pneumoniae? Rodney D. Gilbert ⇑, Arvind Nagra, Mushfequr R. Haq Regional Paediatric Nephro-Urology Unit, University Hospital Southampton, Tremona Road, Southampton SO16 6YD, United Kingdom
a r t i c l e
i n f o
Article history: Received 1 November 2012 Accepted 21 May 2013
a b s t r a c t We describe two patients with haemolytic uraemic syndrome (HUS) associated with invasive Streptococcus pneumoniae infection. Both patients had transiently reduced serum concentrations of complement C3. One had reduced expression of CD46 and never recovered renal function. No constitutive defect in regulation of the alternative pathway of complement activation was demonstrated in the second patient but there was an apparent improvement in her condition after administration of eculizumab. The most widely accepted mechanism for pneumococcal HUS is endothelial cell damage by pre-formed antibodies against the Thomsen–Friedenreich antigen. This explanation does not bear rigorous scrutiny. We postulate that transiently dysregulated complement activation may play a role in the pathogenesis of pneumococcal disease. We further postulate that the mechanism could be enhanced binding of factor H to the neuraminidase-altered surface of endothelial cells or reduced binding of factor H to the endothelial cell surface mediated by competitive binding of factor H by pneumococcal surface protein C (pspC). Ó 2013 Elsevier Ltd. All rights reserved.
Introduction
Case reports
Haemolytic uraemic syndrome (HUS) is a triad of microangiopathic haemolytic anaemia, thrombocytopenia and acute kidney injury. Pathologically it is characterised by vessel wall thickening with swelling and detachment of endothelial cells with accumulation of material in the subendothelial space. There are intraluminal platelet thrombi with partial or complete luminal obstruction [1]. The concept that the initiating process is damage to the microvascular endothelium was first proposed by Altschule in 1942 [2]. Seventy years later it is still believed that endothelial damage is the primary event [3]. In the case of infection related HUS, microbial toxins are believed to be the primary damaging mechanism. In many of the remaining cases, inherited or acquired disorders of the regulation of complement activation lead to endothelial damage [4,5]. It has recently been suggested that complement activation plays an important role in the pathogenesis of shigatoxin associated disease and other infection-related cases of HUS [6,7]. There may therefore not be a clear separation between shigatoxin induced cases, those associated with other infections or drugs and those with inherited disorders of complement regulation. We present two patients with Streptococcus pneumoniae associated HUS (pHUS) with transiently reduced serum C3 levels and suggest that complement-mediated injury may have played an important role in their disease and suggest possible mechanisms by which this may have occurred.
Case 1
⇑ Corresponding author. Tel.: +44 0 2380796254; fax: +44 0 2380795230. E-mail address: [email protected] (R.D. Gilbert). 0306-9877/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mehy.2013.05.030
A previously well 11 month old male infant presented to his local hospital with a 4 day history of upper respiratory tract infection associated with fever and vomiting. He then became unresponsive with twitching of the right leg and right side of his face with progression to a generalised seizure. Admission blood tests showed a plasma sodium concentration of 130 mmol/L, potassium 3.9 mmol/ L, urea 2.2 mmol/L, creatinine 48 lmol/L, haemoglobin 110 g/L and platelets of 427 109/L. Blood culture yielded a growth of S. pneumoniae serotype 19A sensitive to penicillin, amoxicillin and cefotaxime. A computed tomography scan of his head showed mild dilatation of both lateral ventricles and florid contrast enhancement of the extracerebral spaces compatible with meningitis. He was given intravenous cefotaxime, acyclovir and phenytoin. On the second hospital day the urine output had fallen to 0.37 ml/kg/hour, he had become oedematous and had increasing respiratory distress. He was transferred to the Paediatric Intensive Care Unit at Southampton. Blood tests showed renal failure (urea 17.1 mmol/L, creatinine 191 lmol/L), anaemia (Hb 67 g/L), thrombocytopaenia (12 109/L), complement C3 0.45 g/L (normal 0.75– 1.65), C4 0.20 g/L (normal 0.14–0.54), haptoglobin <0.06 g/L (normal 0.5–2.0) and lactate dehydrogenase (LDH) 2486 iu/L (normal 266–500). A blood film was positive for red cell fragments and the peanut lectin assay for T-cryptantigen activation was positive. These results were considered compatible with a diagnosis of pHUS.
R.D. Gilbert et al. / Medical Hypotheses 81 (2013) 400–403
He was initially managed with continuous veno-venous haemofiltration but converted to peritoneal dialysis after 2 days. He recovered well from the meningitis without focal neurological deficit except for profound deafness for which he received a cochlear implant. He remained anuric and received a live donor renal transplant 14 months after first admission. Investigations for regulatory defects in the alternative pathway of complement activation (performed 3 days after admission) showed that the C3 concentration had returned to normal and was 1.2 g/L. The complement factor B concentration was 191 mg/ L (normal 90–320), factor H 648 mg/l (normal 345–590), factor I 131% (normal 70–130) and no factor H antibodies were detected. The von Willebrand cleaving protease (ADAMTS13) activity was 18% and there were no inhibitors. There was reduced expression of CD46 (membrane cofactor protein, MCP) on peripheral blood leukocytes (mean fluorescence intensity score 523, normal 600– 1400) compatible with heterozygous deficiency. Genetic analysis revealed no exonic mutations of CFH, CFI, CD46, CFB or C3. Case 2 A 21 month old female infant presented to her local hospital with a 12 day history of an upper respiratory tract infection. Seven days prior to admission she was reviewed in the Emergency Department because of cough, vomiting and pyrexia. A chest radiograph was reported as normal and she was discharged but represented 48 h later with respiratory distress and grunting. Examination revealed dullness to percussion and reduced breath sounds on the left side of the chest and a repeat radiograph showed left lower lobe consolidation. Blood tests on admission were as follows: haemoglobin concentration 111 g/L, platelet count 423 109/L, plasma sodium concentration 130 mmol/L, potassium 4.4 mmol/L, urea 5.1 mmol/L, creatinine 17 lmol/L and bicarbonate 18.9 mmol/L. She was given intravenous amoxicillin/clavulanic acid and her fluid intake was restricted. After 48 h she was mildly icteric, the haemoglobin concentration was 51 g/L, platelet count 5 x 109/L, bicarbonate 15.3 mmol/ L, urea 30.9 mmol/L and creatinine 151 lmol/L. A blood film showed red cell fragments and the Coombs test was positive. She was hypertensive with a systolic blood pressure of 130 mmHg. She was given amlodipine and a red cell transfusion. The antibiotic therapy was changed to cefotaxime and clarithromycin. She was then transferred to Southampton General Hospital for management of her HUS. On arrival she was pale and distressed. The respiratory rate was 58/min but transcutaneous oxygen saturations were 100% in air. There were reduced breath sounds at the left base. The pulse rate was 158/min, the heart was clinically normal and the blood pressure 100/56 mmHg. The liver was palpable 4 cm below the right costal margin. Laboratory investigations confirmed renal failure (urea 36.8 mmol/L and creatinine 198 lmol/L) and a there were numerous red cell fragments on a blood film. The LDH was 13,782 iu/L, plasma haptoglobin was 0.38 g/L, complement C3 0.58 g/L and complement C4 was 0.14 g/L. ADAMTS13 activity was 80%. A chest ultrasonogram revealed a sub-pulmonary effusion and a chest drain was inserted. Blood cultures were negative but a urine sample was positive for pneumococcal antigen. She became anuric and was commenced on peritoneal dialysis via a Tenckhoff catheter. The chest drain was removed 7 days after admission and the antibiotics were stopped after 10 days. Because of the low initial serum complement C3 concentration and the fact that she remained anuric and thrombocytopaenic, she was given eculizumab 300 mg intravenously on the seventh day of admission to Southampton and then fortnightly thereafter, according to the manufacturer’s dosing recommendations. Immediately prior to
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administration of eculizumab the serum complement C3 had normalised to 1.54 g/L and the total haemolytic complement was high at 143%. The platelet count was 138 109/L and 12 h after the infusion it was 208 109/L and she became noticeably less irritable. She remained completely anuric for a further 2 weeks after the first dose of eculizumab (total of 21 days of anuria) and remained dialysis dependent for a total of 30 days. Three months after admission the patient had improving, but still abnormal, renal function with a plasma creatinine concentration of 82 lmol/L giving her an estimated GFR of 40 ml/min/1.73 m2. Her early morning urine protein/creatinine ratio was significantly elevated at 121 mg/ mmol. Investigations were performed to detect abnormalities of regulation of the alternative pathway of complement activation. No abnormalities were found in the C3, CFH, CFB, CFI or CD46 genes. CD46 expression on peripheral blood neutrophils was normal and antibodies to factor H were not detected. Multiplex ligationdependent probe amplification of CFHR1 exons 3, 5 and 6 and CFHR3 exons 1–4 and 6 showed 2 copies of each exon. Eculizumab was therefore stopped after 4 doses.
Discussion The terms ‘‘typical’’, ‘‘diarrhoea-associated’’ or ‘‘D + ’’ HUS are commonly used to describe HUS caused by shigatoxin producing organisms, most frequently Escherichia coli O157:H7. The terms ‘‘atypical’’, ‘‘non-diarrhoea-associated’’ or ‘‘D-’’ HUS have generally been applied to any form of HUS not due to shigatoxin producing organisms. In this classification, atypical HUS includes those cases secondary to other infections such as S. pneumoniae [8], human immunodeficiency virus (HIV) [9] and H1N1 Influenza A [10], a variety of drugs [11] or methyl malonic aciduria with homocystinuria, a rare inborn error of metabolism [12]. It also includes a group of patients with inherited or acquired disorders of complement regulation. It has been suggested that the term ‘‘atypical HUS’’ is therefore inadequate and the term ‘‘complement dysregulation-associated HUS’’ has been recommended to describe the latter group.[5]. There are at least two significant objections to this nomenclature, however. The first is that in around 40% of patients with non-shigatoxin associated HUS no cause can be found with currently available investigations. The second is that there is increasing evidence of at least transient dysregulation of complement activation in those cases due to shigatoxin producing organisms. To date there is little in the medical literature on the role of complement activation in cases due to S. pneumoniae. Our two patients both had low serum concentrations of C3 suggesting exuberant activation of the alternative pathway. This phenomenon has been previously observed [13] and a role for complement in the pathogenesis of pneumococcal HUS has been previously postulated (5). HUS following infection with S. pneumoniae has traditionally been explained as resulting from the action of bacterial neuraminidase which exposes the Thomsen–Friedenreich antigen (T-antigen) to pre-formed IgM antibodies which then damage the cells leading to HUS [14]. The pathogenicity of pre-formed anti-T IgM seems doubtful, however as it is a cold reactive antibody and at 37 °C causes neither red cell agglutination nor complement activation [15]. Furthermore, anti-T antibodies are not uniformly present during severe pHUS [16]. The alternative pathway of complement activation (APC) activates spontaneously in aqueous solution and C3b is deposited on surfaces. Host cell membranes are protected against combination of bound C3b with factor B to form the C3/C5 convertase C3bBb and the subsequent assembly of the membrane attack complex
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by a number of complement regulators, namely factor H, factor I, CD46 (Membrane cofactor Protein, MCP) and CD59 [17]. Factor H is an abundant serum glycoprotein composed of 20 complement control protein (CCP) modules [18]. It is the most important regulator of the APC in the fluid phase. The N-terminal four CCPs act as a cofactor for factor I-mediated proteolytic inactivation of C3b, compete with factor B for C3b binding and accelerate the decay of the C3bBb C3/C5 convertase into its components [19,20]. CCPs 19–20 contain two C3b binding sites [21] as well as a sialic acid binding site allowing Factor H to bind to sialic acid residues or glycosaminoglycans on endothelial cells and at the same time to engage the C3b molecule, allowing the N-terminal 4 CCPs to prevent further activation of the complement cascade, thus protecting host cells against complement mediated damage. The C3b and glycosaminoglycan binding sites on CCP 20 are partially overlapping [22–24]. Mutations in Factor H are the most frequent known cause of HUS secondary to inherited deficiencies of complement regulation. HUS-causing mutations are mostly found in the region of the gene coding for the C-terminal CCPs 19–20 [25]. Known aHUS mutations may have reduced or increased binding affinity to glomerular endothelial cells or reduced binding affinity to C3b [26]. Since pneumococcal neuraminidase removes sialic acid from endothelial cell surfaces, it has been proposed that factor H binding to these cells would be reduced, thus exposing them to complement-mediated damage. In an in vitro model of pHUS, Johnson et al. exposed cultured glomerular endothelial cells to neuraminidase to expose the T-antigen. The cells were then incubated with fresh human serum. As expected there was increased deposition of C3b and membrane attack complex but surprisingly there was also increased factor H deposition in neuraminidase treated cells compared with controls. (13) The enhanced binding of factor H to neuraminidase-treated endothelial cells may reduce binding to C3b and prevent further complement activation. Lehtinen et al. found four mutant factor H proteins associated with aHUS that had increased binding to glomerular endothelial cells. These mutants had unchanged binding to C3b when these functions were tested separately. Simultaneous binding to glomerular endothelial cell surfaces and C3b was not tested in this study [26]. Other pathogen virulence factors might also be involved in disrupting the normal function of factor H on host glomerular endothelial cells. Invading micro-organisms must have the capacity to avoid or ameliorate complement-mediated destruction or they would rapidly be coated with C3b and killed by a combination of cell wall perforation by the membrane attack complex and intracellular killing by phagocytes. One of the virulence factors expressed by pneumococci is pneumococcal surface protein C (pspC) [27]. PspC binds factor H and regulates complement activation on the pneumococcal surface. The pspC locus may include one or two copies of the gene [28]. In mice infected with the same strain of S. pneumoniae the expression of pspC varied depending on the route of infection and the cytokine profile produced by the host [29]. The amount of pspC produced therefore may depend on both the organism involved and on the nature of the immune response mounted by the host. It is possible that under certain circumstances very large amounts are produced. Some may be liberated into the fluid environment where it can bind to the C-terminal end of the factor H molecule and thereby inhibit binding to cell surfaces in a manner analogous to anti-factor H antibodies [30]. This would reduce host protection against complement-mediated damage. In conclusion we have presented two patients with pHUS who had low concentrations of complement component C3 at presentation. The first has reduced expression of CD46 which probably exacerbated the renal injury and he never recovered renal function. The second has no demonstrable defect in APC regulatory factors
but appeared to respond to treatment with the complement inhibitor eculizumab with an abrupt increase in platelet count and a subjective decrease in irritability. These cases suggest a possible role for abnormal regulation of complement activation in this form of HUS. We suggest two possible mechanisms for disordered regulation mediated by S. pneumoniae, namely increased binding of factor H to endothelial cell surfaces, possibly nullifying the effect of the partially overlapping C3b binding site on factor H CCP 20 and interference with cell surface and binding by soluble pspC. Further research is required to determine which, if any, of these postulated mechanisms is involved in the pathogenesis of pHUS. If our hypothesis is verified it would imply that the use of complement inhibiting drugs should be part of the treatment of pHUS.
Conflict of interest All three authors have received financial assistance for attendance at conferences from Alexion Pharma, manufacturer of eculizumab.
References [1] Ruggenenti P, Noris M, Remuzzi G. Thrombotic icroangiopathy, hemolytic uremic syndrome, and thrombotic thrombocytopenic purpura. Kidney Int 2001;60:831–46. [2] Altschule MD. A rare type of acute thrombocytopenic purpura: widespread formation of platelet thrombi in capillaries. N Engl J Med 1942;227:477–9. [3] Obeidat M, Obeidat M, Ballermann BJ. Glomerular endothelium: a porous sieve and formidable barrier. Exp Cell Res 2012;318:964–72. [4] Keir L, Coward RJM. Advances in our understanding of glomerular thrombotic microangiopathy. Pediatr Nephrol 2011;26:523–33. [5] Loirat C, Frémeaux-Bacchi V. Atypical hemolytic uremic syndrome. Orphanet J Rare Dis 2011;6:60. [6] Johnson S, Waters A. Is complement a culprit in infection-induced forms of haemolytic uraemic syndrome? Immunobiology 2012;217:235–43. [7] Ståhl A, Sartz L, Karpman D. Complement activation on platelet-leukocyte complexes and microparticles in enterohemorrhagic Escherichia coli – induced hemolytic uremic syndrome. Blood 2011;117:5503–13. [8] Waters AM, Kerecuk L, Luk D, et al. Hemolytic uremic syndrome associated with invasive pneumococcal disease: The United Kingdom experience. J Pediatr 2007;151:140–4. [9] Sutor GC, Schmidt RE, Albrecht H. Thrombotic microangiopathies and HIV infection: report of two typical cases, features of HUS and TTP, and review of the literature. Infection 1999;27:12–5. [10] Caltik A, Akyüz SG, Erdogan O, Demircin G. Hemolytic uremic syndrome triggered with a new pandemic virus: influenza A (H1N1). Pediatr Nephrol 2011;26:147–8. [11] Zakarija A, Bennett C. Drug induced thrombotic microangiopathy. Semin Thromb Hemost 2005;31:681–90. [12] Menni F, Testa S, Guez S, Chiarelli G, Alberti L, Esposito S. Neonatal atypical hemolytic uremic syndrome due to methylmalonic aciduria and homocystinuria. Pediatr Nephrol 2012;27:1401–5. [13] Johnson SA, Williams JM, Morgan B, Taylor C, Savage COS. Evidence for complement dysregulation in pneumococcal haemolytic uraemic syndrome (Abstract). Pediatr Nephrol 2009;24:1781. [14] Copelovitch L, Kaplan BS. Streptococcus pneumoniae-associated hemolytic uremic syndrome. Pediatr Nephrol 2008;11:1951–6. [15] Crookston KP, Reiner AP, Cooper LJ, Sacher RA, Blajchman MA, Heddle NM. RBC T activation and hemolysis: implications for pediatric transfusion management. Transfusion 2000;40:801–12. [16] Eber SW, Polster H, Quentin SH, Rumpf KW, Lynen R. Hemolytic uremic syndrome in pneumococcal meningitis and infection. Importance of Ttransformation. Monatsschr Kinderheilkd 1993;141:219–22. [17] Devine DV. The regulation of complement on cell surfaces. Transfus Med Rev 1991;5:123–31. [18] Rodriguez de Cordoba S, Esparza-Gordillo J, Goicoechea de Jorge E, LopezTrascasa M, Sanchez-Corral P. The human complement factor H: functional roles, genetic variations and disease associations. Mol Immunol 2004;41:355–67. [19] Richards A, Kavanagh D, Atkinson JP. Inherited complement regulatory protein deficiency predisposes to human disease in acute injury and chronic inflammatory states: the examples of vascular damage in atypical hemolytic uremic syndrome and debris accumulation in age-related macular degeneration. Adv Immunol 2007;96:141–77. [20] Hocking HG, Herbert AP, Kavanagh D, et al. Structure of the N-terminal region of complement factor H and conformational implications of disease-linked sequence variations. J Biol Chem 2008;283:9475–87.
R.D. Gilbert et al. / Medical Hypotheses 81 (2013) 400–403 [21] Kajander T, Lehtinen MJ, Hyvärinen S, et al. Dual interaction of factor H with C3d and glycosaminoglycan in host-nonhost discrimination by complement. Proc Natl Acad Sci USA 2011;108:2897–902. [22] Schmidt CQ, Herbert AP, Kavanagh D, et al. A new map of glycosaminoglycan and C3b binding sites on factor H. J Immunol 2008;181:2610–9. [23] Hellwage J, Jokiranta TS, Friese MA, et al. Complement C3b/C3d and cell surface polyanions are recognised by overlapping binding sites on the most carboxyl-terminal domain of complement factor H. J Immunol 2002;169:6935–44. [24] Morgan HP, Schmidt CQ, Guariento M, et al. Structural basis for engagement by complement factor H of C3b on a self surface. Nat Struct Mol Biol 2011;18:463–70. [25] Neumann HP, Salzmann M, Bohnert-Iwan B, et al. Haemolytic uraemic syndrome and mutations of the factor H gene: a registry-based study of German speaking countries. J Med Genet 2003;40:676–81.
403
[26] Lehtinen MJ, Rops AL, Isenman DE, van der Vlag J, Jokiranta TS. Mutations of factor H Impair regulation of surface-bound C3b by three mechanisms in atypical hemolytic uremic syndrome. J Biol Chem 2009;284:15650–8. [27] Quin L, Carmicle S, Dave S, Pangburn MK, Evenhuis JP, McDaniel LS. In vivo binding of complement regulatory factor H by Streptococcus pneumoniae. J Infect Dis 2005;192:1996–2003. [28] Zipfel PF, Hallström T, Hammerschmidt S, Skerka C. The complement fitness factor H: role in human diseases and for immune escape of pathogens, like pneumococci. Vaccine 2008;30(26 Suppl. 8):167–74. [29] Quin L, Moore QC, Thornton JA, McDaniel LS. Peritoneal challenge modulates expression of pneumococcal surface protein C during bacteremia in mice. Infect Immun 2008;76:1122–7. [30] Dragon-Durey M-A, Loirat C, Cloarec S, et al. Anti-factor H autoantibodies associated with atypical haemolytic uremic syndrome. J Am Soc Nephrol 2005;16:555–63.