Isoniazid is still, 60 years after its introduction, a main front line drug for treating tuberculosis. Isoniazid, a hydrazine compound, is metabolized through N-acetylation by the arylamine N-acetyltransferase (NAT) enzyme in humans and its metabolism was important in establishing the early observations on pharmacogenetics since its metabolism to N-acetylisoniazid was identified as being genetically controlled. The incidence of adverse side effects to isoniazid is also linked to its metabolism. These side effects include liver toxicity, neuropathy and a condition resembling the autoimmune disorder Systemic Lupus Erythematosus (SLE). The latter side effect shares similarities with side effects to hydralazine, an anti-hypertensive, which is also a hydrazine and, like isoniazid, induces SLE-like symptoms in a sub group of patients who are almost exclusively slow NAT acetylators. The complement system in humans is essential for immune complex clearance and the chemical mechanism by which isoniazid and hydralazine interact with the activation of the complement cascade has been established, demonstrating their interference with the activation of the thiol ester in complement component C4 such that immune complexes become deposited at inappropriate tissue sites in the small blood vessels, kidneys and joints, thereby generating a SLE-like condition. The relevance of immunohistocompatibitility types relating to the polymorphic C4 type is also explored.

• Isoniazid 
• Complement 
• Arylamine N-acetyltransferase 
• Thiol ester 
• Lupus erythematosus 
• Immune complexes 
• Acetylation

Sim E, Laurieri N (2018) Isoniazid Induced Toxicity: Systemic Lupus Erythematosus. J Drug Des Res 5(1): 1065.

ADPR: Adenosine Diphosphate Ribose; HLA: Human Leukocyte Antigen; NAD: Nicotinamide Adenine Dinucleotide; INH: Isoniazid; InhA: enoyl-[acyl-carrier-protein]-reductase; SLE: Systemic Lupus Erythematosis; TB: Tuberculosis

Isoniazid (INH) was first introduced for the treatment of tuberculosis (TB) in 1952. It has revolutionized treatment of TB, usually in combination with other drugs [1]. Nevertheless there is a growing search for new anti-tubercular therapies following the availability of genomic information and identification of possible new anti-tubercular targets [2,3] with new treatments reaching the clinical trials stage as part of a growing pipeline of novel anti-tuberculars [4]. Although drug resistance is a growing and real problem, INH is still a front line treatment in combined therapies [5]. The mechanism of action of isoniazid is important as it is one way of identifying new drug treatments [6,7] and resulted in the identification of the agent ethionamide [8]. With INH, the drug is activated by oxidation by KatG (Figure 1), inside the mycobacterial cells and the resulting activated moiety then forms an adduct with NAD+ [8].

Figure 1: Mechanism of action of Isoniazid. Isoniazid is a prodrug which can be activated by the catalase-peroxidase KatG. The activated form (isonicotinoyl) reacts with NAD+ to form the adducts isonicotinoyl-NAD which inhibit the target NADH-dependent enoyl-ACP reductase involved in the fatty acid synthase type II system; this results in mycolic acid biosynthesis inhibition and mybacterial cell lysis. Based on [8,9]. ADPR= adenosine diphosphate ribose.

There is now a consensus that the adduct inhibits the enoyl-[acyl-carrier-protein]-reductase (InhA) [9] and thus inhibits synthesis of the mycolic acid component of the mycobacterial cell wall. There was an earlier controversy as to the nature of the inhibited enzyme and a more recent study has used computational methods to investigate the range of targets for the adduct [10]. Understanding of the molecular changes which lead to isoniazid resistance [11], have been important not only in understanding resistance but also in understanding the mechanism of action of this mainstay of anti-tubercular therapy and of identifying new possible treatments [6,7,12].

Whilst INH is still a front line treatment and is the drug of choice for latent TB, it is however a drug which has been associated with a wide range of adverse side effects. The most common of these is hepatotoxicity [13], followed by neuropathy [14], and also a condition resembling systemic lupus erythematosus (SLE) [15,16]. The mode of action of isoniazid oxidation results in the formation of a covalent bond with NAD+ . The effectiveness of isoniazid in combating TB and its mode of action being is the “yin” to the “yang” in relation to its side effects. This review focuses on one particular side effect induced by isoniazid, namely systemic lupus erythematosus (SLE). The condition of drug-induced lupus is shared with a wide range of other drugs [17], and has also been reviewed recently in an excellent online article which sets out the facts [18].

The most common other drugs associated with SLE include the anti-hypertensive, hydralazine, the anti-arrythmic procainamide (which is still used in the USA but only in special circumstances in the UK) and also the anti-arthritic drug penicillamine.

Figure 2: Comparison of the chemical structures of isoniazid and hydralazine.

Isoniazid and hydralazine are chemically similar, both being hydrazine compounds (Figure 2), and this review focuses on a the nature of SLE induced by isoniazid, using examples derived from isoniazid’s interaction with the immune system in comparison with hydralazine also.

The emergence of HIV and concommitant increase in TB, including paediatric TB [19], has resulted in an increased interest in isoniazid toxicity and this has been particularly important in relation to understanding the presentation of instances where children have suffered adverse side effects [14].

Isoniazid is still the main front line drug against tuberculosis, despite the growing problem of resistance. It is usually used in combination with other anti-tuberculars for latent TB and in ongoing drug regimens [20-22]. In addition, isoniazid is being used prophylactically in latent TB [23], and it has been studied in relation to treatment of children who are not receiving anti-viral agents for HIV and appears to have a positive effect in reducing deaths from TB.

INH resistance in TB has been widely studied and the overwhelming evidence suggests that mutations in the InhA gene and the KatG gene account for the majority of the incidences of resistance in clinical isolates [11,12], but in addition mutations in the gene encoding for the mycobacterial pumps and in the arylamine N-acetyltransferase (nat) gene in mycobacteria have been implicated. The latter two appear to have a minor effect [11,24-26].

SLE is one of the less common side effects of isoniazid therapy. The diagnosis relies on the appearance of a combination of a range of indicators such as rash, joint involvement, and is particularly linked with the appearance of autoantibodies [27- 29], which have been noted in a similar fashion to hydralazine and procainamide induced SLE [30-32]. One of the key features of the diagnosis of INH-induced SLE has been the recovery and reversal of symptoms on removal of the drug and predictive assays have been reported relating to the induction of autoantibodies [15,28], such as the antibodies identified against the DNA H2AH2B complex [28]. A predictive test involves a popliteal lymph node activation test and has been reported to be useful in both isoniazid and procainamide adverse reactions [33].

It has been proposed that the oxidation or peroxidation of INH and also of hydralazine are important in the development of the idiopathic immune response [34], however there is overwhelming evidence that isoniazid is metabolized in humans by N-acetylation [35-37]. N-acetylation of INH was amongst the first examples of pharmacogenetic variation to be identified [35] and the molecular basis of the variation in acetylation now extends to over 90 alleles [38]. The original observation that SLE was associated with slow N-acetylation of hydralazine [31,39-42] and INH [43], has been substantiated by extensive genotyping studies in different populations [15,32]. The implication is that the difference in the clearance of the drug in slow NAT acetylators creates sufficient of a non-acetylated metabolite to be involved in the adverse reaction. The competition between NAT-acetylation and KatG-activation of INH in humans has also been described in mycobacterial cells themselves [44]. It was demonstrated that mycobacterial cells have an enzyme which N-acetylates INH [26,45], and this has also been demonstrated to contribute to sensitivity to INH in mycobacterial gene deletion and overexpression studies [44,45]. Genetic mutations in the nat gene in clinical isolates of Mycobacterium tuberculosis [11,24,25], have demonstrated that whilst the nat gene does show mutations it makes a minor contribution clinically to INH resistance with the mutations in InhA and KatG genes being of most importance [11,12]. Whilst genetic variation in the mechanisms for pumping INH from the mycobacterial cells has also been identified, but it has also been found to contribute only marginally to the overall INH resistance [11,12].

Structural studies on NAT enzymes from mycobacteria in which each NAT protein has a very similar amino acid sequence [46] have demonstrated INH in the binding pocket of the NAT enzyme from M. smegmatis [47]. In a separate study, hydralazine has been located in the binding site of the NAT enzyme from M. marinum [48], which has shed light on the reaction mechanism for N-acetylation. Interestingly the nat gene itself and the operon in which it is found is essential for mycobacterial survival inside cells [45] and has been explored as a target for antibacterial therapy [49-51].

Isoniazid is activated inside macrophage and the enzyme KatG which catalyses the activation is essential for the action of isoniazid (Figure 1).

Once it is activated, the moiety forms a covalent interaction with NAD+ and the adduct formed gives rise to a complex which stops InhA working in the formation of mycolic acids [9].

It has been argued that the adverse reaction in humans is caused by an oxidation reaction perhaps in activated macrophages [34]. It is clear that there is a sub population of individuals who are susceptible to drug-induced SLE. Not all individuals get the adverse reaction. The incidence of INH-induced SLE is low (much less than 5%) although in hydralazine-induced lupus the incidence is higher with up to 12% in the early days when higher doses were used [31,41]. In order to understand the contribution of genetics, studies have been carried out to investigate the Human Leukocyte Antigen (HLA) type of patients who experience SLE-like symptoms. These studies have identified that individuals who carry the HLA DR4 type are more prevalent in the adverse reactors [52], along with those who are slow acetylators for NAT. In addition to the HLA DR4 type, it has been observed that there is an increased incidence of side effects on individuals carrying the C4A-null type - a class 3 HLA antigen [53]. It is well established that deletion of the genes for the early components of the classical pathway of complement are at increased risk of developing SLE and the C4A-null type is a particular risk feature [54]. These studies have been confirmed for hydralazine-induced SLE in the clinic [52-54]. In addition, noting that it is the drug rather than the N-acetylated metabolite which is the likely causative agent, a study showed that hydralazine but not its N-acetylated metabolite will bind to C4 when C4 is activated [55], in effect creating a chemical knock out of C4. In addition, the C4A type is more susceptible to this inhibition that C4B [56]. The inhibition reaction occurs on activation of the crucial thiolester in C4 [57], and results in the drug becoming bound to the complement component via the activated thiolester [56]. This in turn hinders the amplification of the complement cascade such that binding of the main component C3 does not occur. It has been demonstrated that immune complexes which are bound in joints and kidneys in drug-induced SLE have a reduced binding of C3 [58,59].

Figure 3: Isoniazid inhibition of complement component C4. When C4 is activated by immune complexes and also by subcellular debris via the classical or lectin pathways of complement the C4 is cleaved by either C1s or MASP2 and the thiolester which is within the C4 structure is activated through a conformational change. The exposed short lived thiol ester can bind to either hydroxyl or amine groups on the activating surface but this binding can be inhibited by the presence of a nucleophile and isoniazid itself can become bound to the active site via an amide bond [64-66].

The mechanism of INH inhibition of C4 activation is shown in Figure 3. Other polymorphisms in complement receptor type 1 affecting the handling of immune complexes have also been investigated in hydralazine-related SLE cases [60-63].

Isoniazid is still a front line drug of choice in tuberculosis treatment. It is metabolized by N-acetylation in humans. Adverse side effects are associated with a genetic sub group of individuals in particular the slow NAT acetylators, although no distinct NAT allele has been specifically identified. SLE is a rare side effect in INH treatment. The disruption of immune regulation which leads to SLE can be associated with the ability of INH and the chemically similar anti-hypertensive agent hydralazine, to form a covalent inhibitory reaction with complement component C4, which is likely to result in an inability to clear immune complexes.

1. Agrawal S, Kaur KJ, Singh I, Bhade SR, Kaul CL, Panchagnula R. Assessment of bioequivalence of rifampicin, isoniazid and pyrazinamide in a four drug fixed dose combination with separate formulations at the same dose levels. Int J Pharm. 2002; 233: 169-177.

2. Ryan A, Polycarpou E, Lack NA, Evangelopoulos D, Sieg C, Halman A, et al. Investigation of the mycobacterial enzyme hsad as a potential novel target for anti-tubercular agents using a fragment-based drug design approach. Br J Pharmacol. 2017; 174: 2209-2224.

3. Zumla A, Nahid P, Cole ST. Advances in the development of new tuberculosis drugs and treatment regimens. Nat Rev Drug Discov. 2013; 12: 388-404.

4. Tomioka H. Editorial: Current status and perspective on drug targets in tubercle bacilli and drug design of antituberculous agents based on structure-activity relationship. Curr Pharm Des. 2014; 20: 4305-4306.

5. Mitchison DA, Davies GR. Assessment of the efficacy of new anti-tuberculosis drugs. Open Infect Dis J. 2008; 2: 59-76.

6. AlMatar M, Makky EA, Var I, Kayar B, Koksal F. Novel compounds targeting InhA for TB therapy. Pharmacol Rep. 2017; 70: 217-226.

7. Rožman K, Sosi? I, Fernandez R, Young RJ, Mendoza A, Gobec S, et al. A new ‘golden age’ for the antitubercular target inha. Drug Discov Today. 2017; 22: 492-502.

8. Banerjee A, Dubnau E, Quemard A, Balasubramanian V, Um KS, Wilson T, et al. InhA, a gene encoding a target for isoniazid and ethionamide in mycobacterium tuberculosis. Science. 1994; 263: 227-230.

9. Rawat R, Whitty A, Tonge PJ. The isoniazid-nad adduct is a slow, tight-binding inhibitor of InhA, the mycobacterium tuberculosis enoyl reductase: Adduct affinity and drug resistance. Proc Natl Acad Sci USA. 2003; 100: 13881-13886.

10. Jena L, Deshmukh S, Waghmare P, Kumar S, Harinath BC. Study of mechanism of interaction of truncated isoniazid-nicotinamide adenine dinucleotide adduct against multiple enzymes of mycobacterium tuberculosis by a computational approach. Int J Mycobacteriol. 2015; 4: 276-283.

11. Vilcheze C, Jacobs WR Jr. Resistance to isoniazid and Ethionamide in mycobacterium tuberculosis: Genes, Mutations, and Causalities. Microbiol Spectr. 2014; 2: MGM2-0014-2013.

12. Unissa AN, Dusthackeer VNA, Kumar MP, Nagarajan P, Sukumar S, Kumari VI, et al. Variants of katg, inha and nat genes are not associated with mutations in efflux pump genes (mmpl3 and mmpl7) in isoniazid-resistant clinical isolates of mycobacterium tuberculosis from India. Tuberculosis (Edinb). 2017; 107: 144-148.

13. Nolan CM, Goldberg SV, Buskin SE. Hepatotoxicity associated with isoniazid preventive therapy: A 7-year survey from a public health tuberculosis clinic. JAMA. 1999; 281: 1014-1018.

14. Shah BR, Santucci K, Sinert R, Steiner P. Acute isoniazid neurotoxicity in an urban hospital. Pediatrics. 1995; 95: 700-704.

15. Shah R, Ankale P, Sinha K, Iyer A, Jayalakshmi TK. Isoniazid induced lupus presenting as oral mucosal ulcers with pancytopenia. J Clin Diagn Res. 2016; 10: OD03-OD5.

16. Preziosi P. Isoniazid: Metabolic aspects and toxicological correlates. Curr Drug Metab. 2007; 8: 839-851.

17. Rubin RL. Drug-induced lupus. Expert Opin Drug Saf. 2015; 14: 361- 378.

18. Kauffman CL, Amin CO, Fredeking AE. Drug-induced lupus erythematosus. Dermatol. 2017.

19. Venturini E, Turkova A, Chiappini E, Galli L, de Martino M, Thorne C. Tuberculosis and HIV co-infection in children. BMC Infect Dis. 2014; 14: S5.

20. Nahid P, Dorman SE, Alipanah N, Barry PM, Brozek JL, Cattamanchi A, et al. Official American Thoracic Society/Centers for Disease Control and Prevention/Infectious Diseases Society of America Clinical Practice Guidelines: Treatment of Drug-Susceptible Tuberculosis. Clin Infect Dis. 2016; 63: e147-e195.

21. Denholm JT, McBryde ES, Eisen D, Street A, Matchett E, Chen C, et al. Sircle: A randomised controlled cost comparison of self-administered short-course isoniazid and rifapentine for cost-effective latent tuberculosis eradication. Intern Med J. 2017; 47: 1433-1436.

22. Tam CM, Chan SL, Kam KM, Sim E, Staples D, Sole KM, et al. Rifapentine and isoniazid in the continuation phase of a 6-month regimen. Interim report: No activity of isoniazid in the continuation phase. Int J Tuberc Lung Dis. 2000; 4: 262-267.

23. Zunza M, Gray DM, Young T, Cotton M, Zar HJ. Isoniazid for preventing tuberculosis in hiv-infected children. Cochrane Database Syst Rev. 2017; 8: CD006418.

24. Upton AM, Mushtaq A, Victor TC, Sampson SL, Sandy J, Smith DM, et al. Arylamine n-acetyltransferase of Mycobacterium tuberculosis is a polymorphic enzyme and a site of isoniazid metabolism. Mol Microbiol. 2001; 42: 309-317.

25. Sholto-Douglas-Vernon C, Sandy J, Victor TC, Sim E, Helden PD. Mutational and expression analysis of tbnat and its response to isoniazid. J Med Microbiol. 2005; 54: 1189-1197.

26. Sikora AL, Frankel BA, Blanchard JS. Kinetic and chemical mechanism of arylamine n-acetyltransferase from Mycobacterium tuberculosis. Biochemistry. 2008; 47: 10781-10789.

27. Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, Rothfield NF, et al. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 1982; 25: 1271-1277.

28. Salazar-Páramo M1, Rubin RL, García-De La Torre I. Systemic lupus erythematosus induced by isoniazid. Ann Rheum Dis. 1992; 51: 1085- 1087.

29. Rothfield NF, Bierer WF, Garfield JW. Isoniazid induction of antinuclear antibodies. A prospective study. Ann Intern Med. 1978; 88: 650-652.

30. Alarcon Segovia D, Worthington JW, Ward LE, Wakim KG. Lupus diathesis and the hydralazine syndrome. N Engl J Med. 1965; 272: 462-466.

31. Hess E. Drug-related lupus. NEJM. 1988; 318: 1460-1462. 32.Handler J. Hydralazine?induced lupus erythematosis. J Clin Hypertens. 2012; 14: 133-136.

33. Patriarca C, Verdier F, Brouland JP, Descotes J. Popliteal lymph node response to procainamide and isoniazid. Role of beta-naphthoflavone, phenobarbitone and S9-mix pretreatment. Toxicol Lett. 1993; 66: 21- 28.

34. Cho T, Uetrecht J. How reactive metabolites induce an immune response that sometimes leads to an idiosyncratic drug reaction. Chem Res Toxicol. 2017; 30: 295-314.

35. Evans DA, Manley KA, Mc KV. Genetic control of isoniazid metabolism in man. Br Med J. 1960; 2: 485-491.

36. Weber WW, Hein DW. Clinical pharmacokinetics of isoniazid. Clin Pharmacokinet. 1979; 4: 401-422.

37. Ellard GA. The potential clinical significance of the isoniazid acetylator phenotype in the treatment of pulmonary tuberculosis. Tubercle. 1984; 65: 211-227.

38. Boukouvala S. Nat nomenclature. In: Laurieri N, Sim E, editors. Arylamine N-acetyltransferases in health and disease. Singapore: World Scientific Publishing. 2018.

39. Evans DA, Bullen MF, Houston J, Hopkins CA, Vetters JM. Antinuclear factor in rapid and slow acetylator patients treated with isoniazid. J Med Genet. 1972; 9: 53-56.

40. Strandberg I, Boman G, Hassler L, Sjöqvist F. Acetylator phenotype in patients with hydralazine-induced lupoid syndrome. Acta Med Scand. 1976; 200: 367-371.

41. Perry HM Jr. Late toxicity to hydralazine resembling systemic lupus erythematosus or rheumatoid arthritis. Am J Med. 1973; 54: 58-72.

42. Mansilla-Tinoco R, Harland SJ, Ryan PJ, Bernstein RM, Dollery CT, Hughes GR, et al. Hydralazine, antinuclear antibodies, and the lupus syndrome. Br Med J (Clin Res Ed). 1982; 284: 936-939.

43. Weber WW, Hein DW, Litwin A, Lower GM Jr. Relationship of acetylator status to isoniazid toxicity, lupus erythematosus, and bladder cancer. Fed Proc. 1983; 42: 3086-3097.

44. Bhakta S, Besra GS, Upton AM, Parish T, Sholto-Douglas-Vernon C, Gibson KJ, et al. Arylamine n-acetyltransferase is required for synthesis of mycolic acids and complex lipids in Mycobacterium bovis BCG and represents a novel drug target. J Exp Med. 2004; 199: 1191- 1199.

45. Payton M, Auty R, Delgoda R, Everett M, Sim E. Cloning and characterization of arylamine n-acetyltransferase genes from Mycobacterium smegmatis and Mycobacterium tuberculosis: Increased expression results in isoniazid resistance. J Bacteriol. 1999; 181: 1343-1347.

46. Evangelopoulos D, Bhakta S. Arylamine n-acetyltransferases in mycobacteria. In: Laurieri N, Sim E, editors. Arylamine N-acetyltransferases in health and disease. Singapore: World Scientific Publishing; 2018.

47. Sandy J, Holton S, Fullam E, Sim E, Noble M. Binding of the antitubercular drug isoniazid to the arylamine n-acetyltransferase protein from Mycobacterium smegmatis. Protein Sci. 2005; 14: 775-782.

48. Abuhammad AM, Lowe ED, Fullam E, Noble M, Garman EF, Sim E. Probing the architecture of the Mycobacterium marinum arylamine n-acetyltransferase active site. Protein Cell. 2010; 1: 384-392.

49. Anderton MC, Bhakta S, Besra GS, Jeavons P, Eltis LD, Sim E. Characterization of the putative operon containing arylamine n-acetyltransferase (nat) in Mycobacterium bovis BCG. Mol Microbiol. 2006; 59: 181-192.

50. Westwood IM, Bhakta S, Russell AJ, Fullam E, Anderton MC, Kawamura A, et al. Identification of arylamine n-acetyltransferase inhibitors as an approach towards novel anti-tuberculars. Protein Cell. 2010; 1: 82-95.

51. Yam KC, D’Angelo I, Kalscheuer R, Zhu H, Wang JX, Snieckus V, et al. Studies of a ring-cleaving dioxygenase illuminate the role of cholesterol metabolism in the pathogenesis of Mycobacterium tuberculosis. PLoS Pathog. 2009; 5: e1000344.

52. Batchelor JR, Welsh KI, Tinoco RM, Dollery CT, Hughes GR, Bernstein R, et al. Hydralazine-induced systemic lupus erythematosus: Influence of hla-dr and sex on susceptibility. Lancet. 1980; 1: 1107-1109.

53. Speirs C, Fielder AH, Chapel H, Davey NJ, Batchelor JR. Complement system protein c4 and susceptibility to hydralazine-induced systemic lupus erythematosus. Lancet. 1989; 1: 922-924.

54. Jüptner M, Flachsbart F, Caliebe A, Lieb W, Schreiber S, Zeuner R, et al. Low copy numbers of complement c4 and homozygous deficiency of C4a may predispose to severe disease and earlier disease onset in patients with systemic lupus erythematosus. Lupus. 2018; 27: 600- 609.

55. Sim E, Gill E, Sim R. Drugs that induce systemic lupus erythematosus inhibit complement component C4. Lancet. 1984; 2: 422-424.

56. Sim E, Law SK. Hydralazine binds covalently to complement component C4. Different reactivity of C4a and C4b gene products. FEBS Lett. 1985; 184: 323-327.

57. Campbell RD, Gagnon J, Porter RR. Amino acid sequence around the thiol and reactive acyl groups of human complement component C4. Biochem J. 1981; 199: 359-370.

58. Sim E, Dodds AW, Goldin A. Inhibition of the covalent binding reaction of complement component c4 by penicillamine, an anti-rheumatic agent. Biochem J. 1989; 259: 415-419.

59. Sim E. Drug-induced immune-complex disease. Complement Inflamm. 1989; 6: 119-126.

60. Mitchell JA, Gillam EM, Stanley LA, Sim E. Immunotoxic side-effects of drug therapy. Drug Saf. 1990; 5: 168-178.

61. Mitchell JA, Sim RB, Sim E. Cr1 polymorphism in hydralazine-induced systemic lupus erythematosus: DNA restriction fragment length polymorphism. Clin Exp Immunol. 1989; 78: 354-358.

62. Mitchell JA, Batchelor JR, Chapel H, Spiers CN, Sim E. Erythrocyte complement receptor type 1 (CR1) expression and circulating immune complex (CIC) levels in hydralazine-induced SLE. Clin Exp Immunol. 1987; 68: 446-456.

63. Mitchell JA, Sim E. Size polymorphism of the erythrocyte complement receptor type 1 (cr1) in systemic lupus erythematosus induced by hydralazine. Complement Inflamm. 1989; 6: 88-93.

64. Holm L, Ackland GL, Edwards MR, Breckenridge RA, Sim RB, Offer J. Chemical labelling of active serum thioester proteins for quantification. Immunobiol. 2012; 217: 256-264.

65. Mortensen S, Kidmose RT, Petersen SV, Szilágyi Á, Prohászka Z, Andersen GR. Structural basis for the function of complement component C4 within the classical and lectin pathways of complement. J Immunol. 2015; 194: 5488-5496.

66. Carroll ‎ MV, Sim RB. Complement in Health and Disease. Adv Drug Deliv Rev. 2011; 63: 965-975.