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International Journal of Molecular Sciences 25 (7): 3771 (2024)
Children's Hospital (Vancouver, B.C.). Research Institute
Children's Hospital (Vancouver, B.C.)
Akbaba, Tayfun Hilmi
Toor, Kirandeep K.
Mann, Simranpreet K.
Gibson, Kristen M.
Alfaro, Gabriel Alejandro
Balci-Peynircioglu, Banu
Cabral, David A.
Morishita, Kimberly
Brown, Kelly L.
Chronic primary systemic vasculitis (PSV) comprises a group of heterogeneous diseases that are broadly classified by affected blood vessel size, clinical traits and the presence (or absence) of anti-neutrophil cytoplasmic antibodies (ANCA) against proteinase 3 (PR3) and myeloperoxidase (MPO). In small vessel vasculitis (SVV), ANCA are not present in all patients, and they are rarely detected in patients with vasculitis involving medium (MVV) and large (LVV) blood vessels. Some studies have demonstrated that lysosome-associated membrane protein-2 (LAMP-2/CD107b) is a target of ANCA in SVV, but its presence and prognostic value in childhood MVV and LVV is not known. This study utilized retrospective sera and clinical data obtained from 90 children and adolescents with chronic PSV affecting small (SVV, n = 53), medium (MVV, n = 16), and large (LVV, n = 21) blood vessels. LAMP-2-ANCA were measured in time-of-diagnosis sera using a custom electrochemiluminescence assay. The threshold for seropositivity was established in a comparator cohort of patients with systemic autoinflammatory disease. The proportion of LAMP-2-ANCA-seropositive individuals and sera concentrations of LAMP-2-ANCA were assessed for associations with overall and organ-specific disease activity at diagnosis and one-year follow up. This study demonstrated a greater time-of-diagnosis prevalence and sera concentration of LAMP-2-ANCA in MVV (52.9% seropositive) and LVV (76.2%) compared to SVV (45.3%). Further, LAMP-2-ANCA-seropositive individuals had significantly lower overall, but not organ-specific, disease activity at diagnosis. This did not, however, result in a greater reduction in disease activity or the likelihood of achieving inactive disease one-year after diagnosis. The results of this study demonstrate particularly high prevalence and concentration of LAMP-2-ANCA in chronic PSV that affects large blood vessels and is seronegative for traditional ANCA. Our findings invite reconsideration of roles for autoantigens other than MPO and PR3 in pediatric vasculitis, particularly in medium- and large-sized blood vessels.
Citation: Akbaba, T.H.; Toor, K.K.;Mann, S.K.; Gibson, K.M.; Alfaro,G.A.; Balci-Peynircioglu, B.; Cabral,D.A.; Morishita, K.A.; Brown, K.L., onbehalf of the PedVas Investigator’sNetwork. Anti-LAMP-2 AntibodySeropositivity in Children withPrimary Systemic Vasculitis AffectingMedium- and Large-Sized Vessels. Int.J. Mol. Sci. 2024, 25, 3771. https://doi.org/10.3390/ijms25073771Academic Editor: Hajime KonoReceived: 20 February 2024Revised: 18 March 2024Accepted: 21 March 2024Published: 28 March 2024Copyright: © 2024 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). International Journal of Molecular SciencesArticleAnti-LAMP-2 Antibody Seropositivity in Children with PrimarySystemic Vasculitis Affecting Medium- and Large-Sized VesselsTayfun Hilmi Akbaba 1,2,3,† , Kirandeep K. Toor 1,4,†, Simranpreet K. Mann 1,5 , Kristen M. Gibson 1,6,Gabriel Alejandro Alfaro 7, Banu Balci-Peynircioglu 3, David A. Cabral 1,2,8, Kimberly A. Morishita 1,2,8and Kelly L. Brown 1,2,8,*,‡ on behalf of the PedVas Investigator’s Network1 BC Children’s Hospital Research Institute, Vancouver, BC V5Z 4H4, Canada2 Division of Rheumatology, Department of Pediatrics, University of British Columbia,Vancouver, BC V6T 1Z4, Canada3 Department of Medical Biology, Faculty of Medicine, Hacettepe University, 06800 Ankara, Turkey4 Women+ and Children’s Health Sciences, University of British Columbia, Vancouver, BC V6T 1Z4, Canada5 Department of Microbiology and Immunology, University of British Columbia,Vancouver, BC V6T 1Z4, Canada6 Department of Medical Genetics, University of British Columbia, Vancouver, BC V6T 1Z4, Canada7 Meso Scale Diagnostics, LLC, Rockville, MD 20850, USA8 BC Children’s Hospital, Vancouver, BC V6H 3V4, Canada* Correspondence: kbrown@bcchr.ca; Tel.: +1-604-875-2000 (ext. 7984)† These authors contributed equally to this work.‡ Contributing Members of the PedVas Investigator’s Network are indicated in Acknowledgments.Abstract: Chronic primary systemic vasculitis (PSV) comprises a group of heterogeneous diseases thatare broadly classified by affected blood vessel size, clinical traits and the presence (or absence) of anti-neutrophil cytoplasmic antibodies (ANCA) against proteinase 3 (PR3) and myeloperoxidase (MPO).In small vessel vasculitis (SVV), ANCA are not present in all patients, and they are rarely detected inpatients with vasculitis involving medium (MVV) and large (LVV) blood vessels. Some studies havedemonstrated that lysosome-associated membrane protein-2 (LAMP-2/CD107b) is a target of ANCAin SVV, but its presence and prognostic value in childhood MVV and LVV is not known. This studyutilized retrospective sera and clinical data obtained from 90 children and adolescents with chronicPSV affecting small (SVV, n = 53), medium (MVV, n = 16), and large (LVV, n = 21) blood vessels.LAMP-2-ANCA were measured in time-of-diagnosis sera using a custom electrochemiluminescenceassay. The threshold for seropositivity was established in a comparator cohort of patients withsystemic autoinflammatory disease. The proportion of LAMP-2-ANCA-seropositive individualsand sera concentrations of LAMP-2-ANCA were assessed for associations with overall and organ-specific disease activity at diagnosis and one-year follow up. This study demonstrated a greatertime-of-diagnosis prevalence and sera concentration of LAMP-2-ANCA in MVV (52.9% seropositive)and LVV (76.2%) compared to SVV (45.3%). Further, LAMP-2-ANCA-seropositive individuals hadsignificantly lower overall, but not organ-specific, disease activity at diagnosis. This did not, however,result in a greater reduction in disease activity or the likelihood of achieving inactive disease one-yearafter diagnosis. The results of this study demonstrate particularly high prevalence and concentrationof LAMP-2-ANCA in chronic PSV that affects large blood vessels and is seronegative for traditionalANCA. Our findings invite reconsideration of roles for autoantigens other than MPO and PR3 inpediatric vasculitis, particularly in medium- and large-sized blood vessels.Keywords: lysosome-associated membrane protein-2; anti-neutrophil cytoplasmic antibodies; childhood-onset primary vasculitis; autoantibodies; Takayasu’s arteritis; polyarteritis nodosa1. IntroductionChronic primary systemic vasculitis (PSV) is an umbrella term for a family of hetero-geneous diseases that are commonly characterized by inflammation and damage in bloodInt. J. Mol. Sci. 2024, 25, 3771. https://doi.org/10.3390/ijms25073771 https://www.mdpi.com/journal/ijmsInt. J. Mol. Sci. 2024, 25, 3771 2 of 15vessel walls. PSV in children and adolescents has an average age of onset of 10–14 yearsand is particularly rare (<23/100,000 cases annually in North America) compared to thedisease in adults (onset > 50 years) [1–3]. In both pediatric- and adult-onset vasculitis, dif-ferent disease subtypes are broadly classified under the predominant size—small, medium,and large—of the affected blood vessels and consideration of differing clinical features,histologic analysis of affected tissues, and the presence (or absence) of anti-neutrophilcytoplasmic antibodies (ANCA) [4,5].ANCA are a family of autoantibodies that can target distinct autoantigens in thecytoplasmic (c-ANCA) and perinuclear (p-ANCA) region of neutrophils and, to a lesserextent, monocytes [6]. ANCA seropositivity is predominantly observed in chronic PSV thataffects small vessels, and in these cases, it targets one of two antigens: proteinase 3 (PR3)and myeloperoxidase (MPO). Seropositivity and specificity of ANCA for PR3 or MPO hassome proven utility in the diagnosis and differentiation of “ANCA-associated vasculitis(AAV)” subtypes and more recently were demonstrated to have power in predicting disease-associated risks in adult-onset disease [4]. Similar diagnostic and prognostic tools do notexist for “ANCA-negative” vasculitis [7], which in children includes ~10–30% of cases ofsmall-vessel vasculitis and the majority, if not all, forms of vasculitis affecting medium tolarge blood vessels [8].Beyond PR3 and MPO, some studies have demonstrated that lysosome-associatedmembrane protein-2 (LAMP-2/CD107b) is an antigenic target of ANCA. LAMP-2 is aheavily glycosylated lysosome and plasma membrane protein that, in contrast to MPOand PR3, is expressed in almost every cell and tissue type in the body [9]. One epitope ofLAMP-2 has 100% amino acid homology with type I fimbriated bacterial adhesion protein,FimH. Kain et al. [10] demonstrated that FimH-immunized rats produce ANCA againsthuman LAMP-2 and spontaneously develop microvascular injury, glomerulonephritis,and lung damage. Previous reports have demonstrated an increase in LAMP-2 protein insera from adults with a medium-sized vessel vasculitis subtype called polyarteritis nodosa(PAN) [11]. Elevated circulating concentrations of anti-LAMP-2 autoantibodies (LAMP-2-ANCA) have been observed in adults with small-vessel ANCA-associated vasculitis(AAV) and pauci-immune crescentic glomerulonephritis [12], as well as children withAAV [13]. Subsequent to this, elevated circulating concentrations of LAMP-2 protein [11,14]and LAMP-2-ANCA [13,15] were detected in adults with chronic PSV affecting medium-and large-sized blood vessels. Stemming from these collective reports, we hypothesizedthat LAMP-2-ANCA would be present in childhood-onset chronic PSV subtypes thataffect medium to large blood vessels and have no detectable concentrations of circulatingPR3-ANCA or MPO-ANCA.2. Results2.1. Greater Prevalence and Elevated Sera Concentration of LAMP-2-ANCA in PR3- and MPO-ANCA-Negative Vasculitis Subtypes Affecting Medium to Large Blood VesselsA total of 90 children and adolescents newly diagnosed with chronic primary systemicvasculitis (PSV) affecting small- (small-vessel vasculitis, SVV, n = 53), medium- (MVV,n = 16), and large- (LVV, n = 21) sized blood vessels were included in this study (Table 1).The median age at symptom onset (13.3 years, range 1.9–17.8 years) and biological sex atbirth (66.7% female) were comparable between individuals with vasculitis affecting vesselsof different size: SVV median onset 14.2 years, 66.0% female; MVV median onset 11.2 years,62.5% female; and LVV median onset 12.6 years, 71.4% female. Compared to individualswith a systemic inflammatory disease lacking autoimmune features (SAID cohort; seeMethods), a greater percentage of participants with SVV, MVV, and LVV were LAMP-2-ANCA-seropositive, with the greatest prevalence observed in chronic PSV subtypes,where larger vessels were involved: 45.3% (24/53) of SVV patients are LAMP-2-ANCA-seropositive compared to 52.9% (9/16) in MVV and 76.2% (16/21) in LVV (Table 2). Theconcentration of sera LAMP-2-ANCA followed a similar trend in that a significantly higherconcentration of LAMP-2-ANCA were detected in individuals with SVV (p = 0.0225),Int. J. Mol. Sci. 2024, 25, 3771 3 of 15MVV (p = 0.0056) and LVV (p = 0.0001) compared to the SAID cohort (Figure 1A). Amongparticipants with PSV, significantly higher concentrations of LAMP-2-ANCA were de-tected in individuals with MVV (2027.8 ng/mL, range 237.7–6210.9 ng/mL) comparedto those with SVV (1197.1 ng/mL, range 134.0–4281.9 ng/mL) (p = 0.0207). Likewise,significantly higher concentrations of sera LAMP-2-ANCA were present in individualswith LVV (3965.7 ng/mL, range 103.8–12,413.7 ng/mL) compared to both SVV (p < 0.0001)and MVV (p = 0.0418). Focusing on seropositive participants only, the mean concentrationsof LAMP-2-ANCA detected in our cohort were 1976.3 ng/mL in SVV, 3172.9 ng/mL inMVV and 5040.5 ng/mL in LVV.Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 3 of 15 subtypes, where larger vessels were involved: 45.3% (24/53) of SVV patients are LAMP-2-ANCA-seropositive compared to 52.9% (9/16) in MVV and 76.2% (16/21) in LVV (Table 2). The concentration of sera LAMP-2-ANCA followed a similar trend in that a significantly higher concentration of LAMP-2-ANCA were detected in individuals with SVV (p = 0.0225), MVV (p = 0.0056) and LVV (p = 0.0001) compared to the SAID cohort (Figure 1A). Among participants with PSV, significantly higher concentrations of LAMP-2-ANCA were detected in individuals with MVV (2027.8 ng/mL, range 237.7–6210.9 ng/mL) com-pared to those with SVV (1197.1 ng/mL, range 134.0–4281.9 ng/mL) (p = 0.0207). Likewise, significantly higher concentrations of sera LAMP-2-ANCA were present in individuals with LVV (3965.7 ng/mL, range 103.8–12,413.7 ng/mL) compared to both SVV (p < 0.0001) and MVV (p = 0.0418). Focusing on seropositive participants only, the mean concentrations of LAMP-2-ANCA detected in our cohort were 1976.3 ng/mL in SVV, 3172.9 ng/mL in MVV and 5040.5 ng/mL in LVV. Figure 1. Sera LAMP-2-ANCA in chronic primary systemic vasculitis of childhood. (A) LAMP-2-ANCA concentration (y-axis; μg/mL) in sera from children and adolescents (x-axis) with systemic autoinflammatory disease (n = 18, SAID) and chronic primary systemic vasculitis (PSV) affecting small- (SVV, n = 53), medium- (MVV, n = 16) and large- (LVV, n = 21) sized blood vessels. Horizontal dotted line marks the threshold for LAMP-2-ANCA seropositivity in samples with greater than 1 μg/mL LAMP-2-ANCA. (B,C) LAMP-2-ANCA concentration (y-axis; μg/mL) in the pediatric chronic PSV cohort grouped (x-axis) based on (B) positivity for MPO-ANCA (n = 18), PR3-ANCA (n = 30), or neither MPO- or PR3-ANCA (negative, n = 35), and (C) biological sex. (D) Concentration of LAMP-2-ANCA (y-axis; μg/mL) in pediatric chronic PSV participants plotted against age of symp-tom onset (x-axis; years). (E) Age of symptom onset (y-axis; years) in LAMP-2-ANCA-seropositive and -seronegative patients with MVV and LVV (x-axis). Bars show means in (A–C), and medians in (E). LAMP-2-ANCA-seropositive (+) and -seronegative (−) samples indicated with closed and open circles, respectively. * Denotes statistical significance with p < 0.05, *** p < 0.001, **** p < 0.0001. Figure 1. Sera LAMP-2-ANCA in chronic primary ystemic vascu itis of childhood. (A) LAMP-2-ANCA concentration (y-axis; µg/ L) in se a from children and adolescents (x-axis) with systemicautoinflammatory disease (n = 18, SAID) and chronic pri ary systemic vasculitis (PSV) affectingsmall- (SVV, n = 53), medium- (MVV, n = 16) and large- (LVV, n = 21) sized blood vessels. Horizontaldotted line marks the threshold for LAMP-2-ANCA seropositivity in samples with greater than1 µg/mL LAMP-2-ANCA. (B, ) LAMP-2-ANCA concentration (y-axis; µg/mL) in the pediatricchronic PSV cohort grouped (x-axis) based on (B) positivity for MPO-ANCA (n = 18), PR3-ANCA(n = 30), or neither MPO- or PR3-ANCA (negative, n = 35), and (C) biological sex. (D) Concentration ofLAMP-2-ANCA (y-axis; µg/mL) in pediatric chronic PSV participants plotted against age of symptomonset (x-axis; years). (E) Age of symptom onset (y-axis; years) in LAMP-2-ANCA-seropositive and-seronegative patients with MVV and LVV (x-axis). Bars show means in (A–C), and medians in (E).LAMP-2-ANCA-seropositive (+) and -seronegative (−) samples indicated with closed and opencircles, respectively. * Denotes statistical significance with p < 0.05, *** p < 0.001, **** p < 0.0001.Int. J. Mol. Sci. 2024, 25, 3771 4 of 15Table 1. Pediatric chronic primary systemic vasculitis (PSV) cohort.Small Vessel(n = 53)Medium Vessel(n = 16)Large Vessel(n = 21)PSV subtype, n (%)GPA/limGPA 43 (81.1) - -MPA 5 (9.4) - -EGPA 2 (3.8) - -ANCA + GN 1 (1.9) - -PAN/cPAN - 14 (87.5) -TA - - 19 (90.5)UPV 2 (3.8) 2 (12.5) 2 (9.5)Sex, n (%)Female 35 (66.0) 10 (62.5) 15 (71.4)Male 18 (34.0) 6 (37.5) 6 (28.6)No significant difference in representation of males/females between groups (p = 0.8400) based on chi-squared testAge (years) of symptom onset—median (range) * 14.2 (1.9–17.3) 11.2 (2.7–15.8) 12.6 (3.7–17.1)1 ANCA Antigen Positivity, n (%)Proteinase 3 (PR3) 29 (55.8) 1 (6.7) -Myeloperoxidase (MPO) 18 (34.6) - -PR3 and MPO 2 (3.8) - -Neither 3 (5.8) 14 (93.3) 18 (100)Disease activity (pVAS) at diagnosisTotal pVAS, median (range) 19 (6–50) 8 (1–18) 16 (6–26)Subcomponent pVAS, median (range)Renal 10 (0–12) 0 (0–6) 4 (0–12)Cardiovascular 0 (0–4) 0 4 (0–6)Pulmonary 0 (0–6) 0 (0–4) 0 (0–6)Upper respiratory tract 4 (0–6) 0 (0–6) 0 (0–4)2 Induction Treatment, n (%)3 Immune-suppressing agents 38 (73.0) 4 (25.0) 2 (9.5)4 Disease-modifying agents 6 (11.5) 6 (37.5) 11 (52.4)5 Biologic agents - 1 (6.3) 6 (28.6)Corticosteroids 48 (92.3) 12 (75.0) 20 (95.2)2 Maintenance Treatment, n (%)3 Immune-suppressing agents 16 (37.2) 4 (28.6) 4 (20.0)4 Disease-modifying agents 14 (32.5) 9 (64.3) 10 (50.0)5 Biologic agents - 2 (14.3) 8 (40.0)Corticosteroids 41 (95.3) 10 (71.4) 19 (95.0)1 Percentage of patients with ANCA calculated based on available data for 52/53 SVV cases, 15/16 MVV casesand 18/21 LVV cases, 2 Percentage of patients on each induction treatment (for 3–6 months following diagnosis)is calculated based on available data within the SVV (52/53), MVV (16/16), and LVV (21/21) groups andlikewise for maintenance treatment (initiated after induction treatment is complete) within the SVV (43/53),MVV (14/16), and LVV (20/21) groups. 3 Immune-suppressing agents are most commonly cyclophosphamideand rituximab. 4 Disease-modifying agents are most commonly azathioprine, methotrexate, and mycophenolatemofetil. 5 Biologic agents are most commonly infliximab and tocilizumab. GPA, granulomatosis with polyangiitis;limGPA, limited GPA; MPA, microscopic polyangiitis; EGPA, eosinophilic GPA; GN, glomerulonephritis; PAN,polyarteritis nodosa; cPAN, cutaneous PAN; TA, Takayasu’s arteritis; UPV, unclassifiable primary vasculitis; pVAS,pediatric vasculitis activity score. * Significant difference in median age between SVV and MVV (p = 0.0209), butnot SVV and LVV (p = 0.0666) or MVV and LVV (p = 0.5357) based on Mann–Whitney tests.Consistent with a greater number of LAMP-2-ANCA-seropositive individuals withMVV and LVV compared to SVV, LAMP-2-ANCA seropositivity was observed in themajority of individuals (23/35; 65.7%) seronegative for MPO-ANCA and PR3-ANCA, andby comparison, in fewer individuals seropositive for PR3-ANCA (11/30; 36.7%) (Table 2)or MPO-ANCA (9/18; 50%). Similarly, the concentration of LAMP-2-ANCA in individualsseronegative for MPO- and PR3-ANCA was significantly higher compared to LAMP-2-ANCA titers in PR3-ANCA- (p = 0.0008, Figure 1B) and MPO-ANCA-positive patientsInt. J. Mol. Sci. 2024, 25, 3771 5 of 15(p = 0.0553, Figure 1B). These data contrast with several previous reports [16–18] in whichLAMP-2-ANCA titers, although not correlated with titers of MPO- or PR3-ANCA, werecommonly detected in individuals also seropositive for either MPO- or PR3-ANCA.Table 2. LAMP-2-ANCA seropositivity in pediatric chronic primary systemic vasculitis (PSV).PSV Total(n = 90)Small Vessel(n = 53)Medium Vessel(n = 16)Large Vessel(n = 21)a LAMP-2-ANCA − + − + − + − +n n = 41 n = 49 n = 29 n = 24 n = 7 n = 9 n = 5 n = 16% 45.6% 54.4% 54.7% 45.3% 41.2% 52.9% 23.8% * 76.2%b Sex, n (%)Female 26 (63.4) 34 (69.4) 18 (62.1) 17 (70.8) 4 (57.1) 6 (66.7) 4 (80.0) 11 (68.8)Male 15 (36.6) 15 (30.6) 11 (37.9) 7 (29.2) 3 (42.9) 3 (33.3) 1 (20.0) 5 (31.2)c Age of onset, yearsMedian 13.8 13.2 14.5 13.1 8.1 13.7 10.5 13.2Range 1.9–17.3 1.9–17.2 1.9–17.8 1.9–17.2 2.7–15.5 4.0–15.8 3.7–13.3 5.3–17.1d ANCA antigen positivity, n (%)PR3 19 (22.4) 11 (12.9) 18 (34.6) 11 (21.2) 1 (5.9) − − −MPO 9 (10.6) 9 (10.6) 9 (17.3) 9 (17.3) − − − −PR3 and MPO 1 (1.2) 1 (1.2) 1 (1.9) 1 (1.9) − − − −Neither 12 (14.1) 23 (27.1) 1 (1.9) 2 (3.8) 6 (35.3) 8 (47.1) 5 (27.8) 13 (72.2)a LAMP-2-ANCA-seronegative indicated by (−) and -seropositive indicated by (+). b Percentage calculated fromthe total seronegative or seropositive participants of the same vessel size; no statistically significant differences(chi-squared test) between seropositive and seronegative individuals in the PSV (0.5494), SVV (0.5024), MVV(0.6963), and LVV (0.6269) groups. c No statistically significant differences between seropositive and seronegativeindividuals in the PSV (0.7235, Mann–Whitney test), SVV (0.1479, Mann–Whitney test), MVV (0.1146, unpairedt-test), and LVV (0.1085, unpaired t-test) groups. d Percentage calculated based on available data for participantswithin the PSV (85/90), SVV (52/53), MVV (15/16), and LVV (18/21) groups. * Significant difference in theprevalence of seropositive individuals between SVV and LVV (p = 0.0162) based on chi-squared test. Across allgroups, p = 0.0545 for the prevalence of seropositivity; SVV vs. MVV, p = 0.4415; MVV vs. LVV, p = 0.1993.Together, these results demonstrate that a greater majority of cases of pediatric-onsetmedium- and large-vessel vasculitis compared to small-vessel vasculitis in our cohort werenot only seropositive for autoantibodies against LAMP-2 (exceeding 1 µg/mL) but hadsignificantly elevated concentrations of circulating LAMP-2-ANCA; this was especially evi-dent in cases of LVV. The observed differences in both LAMP-2-ANCA positivity (Table 2)and concentration in small-, medium- and large-vessel vasculitis was not associated withbiological sex (Figure 1C, p = 0.8400) or the age of symptom onset (Figure 1D,E). Paradoxi-cally, MVV and LVV are considered “ANCA-negative” subtypes of chronic PSV as they arerarely associated with autoantibodies against PR3 or MPO that are common to SVV.2.2. LAMP-2-ANCA Seropositivity Is Associated with Lower Overall Disease Activity at DiagnosisTo gain insight into the potential for clinical utility of LAMP-2-ANCA measures inMVV and LVV, we asked if seropositivity or concentration of LAMP-2-ANCA at diagnosiscould inform the present state of vasculitis-specific (inflammatory) activity or the ability toattain inactive disease within the first year following diagnosis. Results indicate that LAMP-2-ANCA concentration was not associated with generalized inflammation, as indicatedby the concentration of C-reactive protein (CRP) or with disease-specific (inflammatory)activity at diagnosis measured by pVAS (Figure 2A). When considering seropositivityindependently of LAMP-2-ANCA concentration, however, we observed significantly loweroverall disease activity at diagnosis in LAMP-2-ANCA-seropositive (median pVAS = 15)versus -seronegative (median pVAS = 19) individuals (p = 0.0176), irrespective of the size ofaffected blood vessels (Figure 2B and Table 3).Int. J. Mol. Sci. 2024, 25, 3771 6 of 15Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 7 of 15 Figure 2. Measures of sera LAMP-2-ANCA positivity and concentration relative to disease activity at diagnosis and follow-up. (A) Concentration of LAMP-2-ANCA (y-axis; μg/mL) plotted against the pediatric vasculitis activity score (pVAS) (x-axis) at the time of diagnosis for all participants with chronic primary systemic vasculitis (PSV—all) and stratified according to the involvement of small (SVV) or medium- and large-sized (MVV + LVV) vessels. (B) Pediatric vasculitis activity score (pVAS) (y-axis, bar shows median) at diagnosis in LAMP-2-ANCA-seronegative (−) and -seroposi-tive (+) participants (x-axis). (C) Reduction in disease activity between all LAMP-2-ANCA-seroneg-ative (−) and -seropositive (+) PSV participants (x-axis) shown by absolute change in pediatric vas-culitis activity score (pVAS) (y-axis) from diagnosis to (left panel) the completion of 3–6 months of induction therapy (seropositive, n = 40; seronegative, n = 34) and (middle panel) one-year follow-up (seropositive, n = 32; seronegative, n = 36). Improvement in disease activity expressed as percentage of maximal improvement from diagnosis to one-year follow-up. (D) Reduction in disease activity between LAMP-2-ANCA-seronegative (−) and seropositive (+) MVV/LVV participants (x-axis) shown by absolute change in pediatric vasculitis activity score (pVAS) (y-axis) from diagnosis to (left panel) the completion of 3–6 months of induction therapy (seropositive, n = 22; seronegative, n = 10) and (middle panel) one-year follow-up (seropositive, n = 20; seronegative, n = 10). Improve-ment in disease activity expressed as percentage of maximal improvement from diagnosis to one-year follow-up. (E) Concentration of LAMP-2-ANCA (y-axis; μg/mL, bar shows mean) at diagnosis in participants with medium-/large-sized vessel vasculitis (MVV + LVV) with inactive (pVAS at one-year ≤ 1) or active (pVAS at one-year ≥ 2) disease one-year later (left panel). Similarly (right panel), percentage (x-axis) of seronegative (−) and seropositive (+) MVV/LVV participants with inactive (black) or active (gray) disease one-year post-diagnosis. Bars show medians in (B,C) and means in (D). LAMP-2-ANCA-seropositive (+) and -seronegative (−) samples indicated with closed and open circles, respectively. ns indicates non-significant. * Denotes statistical significance with p < 0.05 . 2.3. LAMP-2-ANCA Seropositivity and Concentration Are Not Significantly Associated with the Extent or Type of Organ Involvement Given the observed relationship between LAMP-2-ANCA seropositivity and overall lower disease activity (pVAS) at diagnosis, we asked if the involvement of multiple, single, or particular organ systems were driving this association. In our cohort at the time of i i foll - . ( ) once trati of LA P-2- (y-axis; µg/ l tt ithe pediatric vasculitis activity score (pVAS) (x-axis) at the time of diagnosis for all participantswith chronic primary systemic vasculitis (PSV—all) and stratified according to the involvementof small (SVV) or medium- and large-sized (MVV + LVV) vessels. (B) Pediatric vasculitis activityscore (pVAS) (y-axis, bar shows median) at diagnosis in LAMP-2-ANCA-seronegative (−) and-seropositive (+) participants (x-axis). (C) Reduction in disease activity between all LAMP-2-ANCA-seronegative (−) and -seropositive (+) PSV participants (x-axis) shown by absolute change in pediatricvasculitis activity score ( VAS) (y-axis) from diagnosis to (left panel) the completi of 3–6 months ofinduction th rapy (seropositive, n = 40; seronegative, n = 34) and (middle panel) on -year follow-up(seropositive, n = 32; seronegative, n = 36). Improvement in disease activity expresse as p rcentageof maximal improvement from diagnosis to one-year follow-up. (D) Reduction in disease activitybetween LAMP-2-ANCA-seronegative (−) and seropositive (+) MVV/LVV participants (x-axis)shown by absolute change in pediatric vasculitis activity score (pVAS) (y-axis) from diagnosis to (leftpanel) the completion of 3–6 months of induction therapy (seropositive, n = 22; seronegative, n = 10)and (middle panel) one-year follow-up (seropositive, n = 20; seronegative, n = 10). Improvementin disease activity expressed as percentage of maximal improvement from diagnosis to one-yearfollow-up. (E) Concentratio of LAMP-2-ANCA (y-axis; µg/mL, bar shows mean) at diagnosisin participants with medium-/large-sized vessel vasculitis ( + LVV) with i active (pVAS atone-year ≤ 1) or active (pVAS at one-year ≥ 2) disease one-year later (left panel). Similarly (rightpanel), percentage (x-axis) of seronegative (−) and seropositive (+) MVV/LVV participants withinactive (black) or active (gray) disease one-year post-diagnosis. Bars show medians in (B,C) andmeans in (D). LAMP-2-ANCA-seropositive (+) and -seronegative (−) samples indicated with closedand open circles, respectively. ns indicates non-significant. * Denotes statistical significance withp < 0.05.Int. J. Mol. Sci. 2024, 25, 3771 7 of 15Table 3. Disease activity at diagnosis and 1-year follow up stratified by LAMP-2-ANCA seropositivity.PSV Total Small Vessel Medium Vessel Large Vessela LAMP-2-ANCA − + − + − + − +Disease activity (total pVAS), median (range)b at diagnosis 19 (5–50) * 15 (1–36) 20 (7–50) 18 (6–36) 8 (5–31) 7 (1–18) 19 (6–20) 15 (6–26)c at 1-year 0 (0–4) 0 (0–12) 0 (0–6) 0 (0–12) 0 (0) 1 (0–4) 2 (3–4) 0 (0–12)d Inactive disease (total pVAS ≤ 1), n (%)at 1-year 26 (37.1) 25 (35.7) 19 (48.7) 12 (30.8) 5 (38.5) 4 (30.8) 2 (11.1) 9 (50.0)a LAMP-2-ANCA-seronegative indicated by (−) and seropositive indicated by (+). b At diagnosis, pVAS availablefor n = 90 PSV patients. * Significant difference in disease activity (p = 0.0176) across all groups based on unpairedt-test. c At 1 year post-diagnosis, pVAS available for n = 70 PSV patients: n = 39 SVV (15 LAMP2 ANCA+), n = 13MVV (8 LAMP2 ANCA+), n = 18 LVV (13 LAMP2 ANCA+). d Inactive disease defined as total pVAS ≤ 1 atone-year post-diagnosis. Percentage is relative to the total number of seronegative or seropositive participants ofthe same vessel size.Using a subset of participants with follow-up data after induction therapy (3–6 monthspost-diagnosis, n = 74) and one-year post-diagnosis (n = 70), we comparatively analyzedLAMP-2-ANCA seropositivity and concentration as being informative for disease trajectorybased on two measures: overall improvement in disease activity (i.e., reduction in pVAS)and achievement of inactive disease. Chronic PSV patients showed marked improvementover the first 12 months of disease, with even the least improved patients achieving a mini-mum 33.3% decline in disease activity (pVAS). Individuals seronegative for LAMP-2-ANCAshowed a greater improvement compared to LAMP-2-ANCA-seropositive individuals afterinduction therapy (Figure 2C left panel, p = 0.023) and one-year post-diagnosis (Figure 2Cmiddle panel, p = 0.038). However, when the reduction in disease activity was calculatedrelative to pVAS at diagnosis (lower in LAMP-2-ANCA-seropositive patients, Figure 2B),no significant differences in improvement related to LAMP-2-ANCA status were observed(right panels in Figure 2C,D). Focusing on the achievement of inactive disease (pVAS ≤ 1),our results showed a comparable number of seropositive (13/21, 61.9%) and seronegativeparticipants (7/10, 70.0%) with inactive disease or sustained disease activity one-yearfollowing diagnosis (Figure 2E right panel and Table 3) and no significant difference inLAMP-2-ANCA concentration at diagnosis between individuals (p = 0.5472, MVV + LVV,Figure 2E, left panel) that went on to achieve inactive disease one -year post-diagnosis.Although our data show a relationship between LAMP-2-ANCA positivity and lowerdisease activity at diagnosis, neither diagnostic LAMP-2-ANCA seropositivity nor con-centration was informative for overall disease activity following therapy induction or atone-year after diagnosis.2.3. LAMP-2-ANCA Seropositivity and Concentration Are Not Significantly Associated with theExtent or Type of Organ InvolvementGiven the observed relationship between LAMP-2-ANCA seropositivity and overalllower disease activity (pVAS) at diagnosis, we asked if the involvement of multiple, single,or particular organ systems were driving this association. In our cohort at the time ofdiagnosis, we observed 71.1% of patients with kidney involvement (renal pVAS ≥ 2), 37.7%of patients with upper respiratory tract involvement (ear, nose, throat (ENT) pVAS ≥ 2),27.8% of patients with pulmonary system involvement (chest pVAS ≥ 2), and 23.4% ofpatients with cardiovascular involvement (cardio pVAS ≥ 2). A multivariate logisticregression model, adjusted for age at diagnosis and biological sex at birth, was used toevaluate the association between organ involvement and vessel size. As expected, weobserved a significant association between vessel size and organ involvement. Comparedto individuals with LVV, MVV less frequently affected the cardiovascular (OR 0.024, 95%CI 0.001–0.163, p = 0.001) and renal (OR 0.169, 95% CI 0.035–0.707, p = 0.019) systems; SVVhad less cardiovascular (OR 0.023, 95% CI 0.004–0.097, p < 0.0001) system involvement, buthigher involvement of the upper respiratory tract (URT) (OR 12.212, 95% CI 2.936–84.855,p = 0.003) and pulmonary system (OR 6.011, 95% CI 1.456–41.134, p = 0.027) involvement.Int. J. Mol. Sci. 2024, 25, 3771 8 of 15The results of a regression model, adjusted for vessel size, assessing the relationshipbetween the presence or absence of LAMP-2-ANCA and the involvement of more thanone of these organ systems (p = 0.173) or a single organ (p = 0.281) were inconclusive.Furthermore, the results did not provide clear evidence of an association between LAMP-2-ANCA seropositivity/seronegativity and type of organ system involvement (Table 4).Table 4. Association between organ system involvement and LAMP-2-ANCA seropositivity.Organ SystemLAMP-2-ANCA b Adjusted OR(95% CI)Standard Error p-ValueSeronegative (n = 36) a Seropositive (n = 41) aCardiovascular 7 (19.4%) 14 (34.1%) 0.82 (0.15, 3.73) 0.79 0.800Renal 29 (81.0%) 35 (85.4%) 1.70 (0.46, 6.91) 0.68 0.429Pulmonary 16 (44.4%) 9 (22.0%) 0.49 (0.16, 1.46) 0.56 0.198URT 19 (52.8%) 13 (31.7%) 0.58 (0.19, 1.79) 0.57 0.339a Number of PSV patients with involvement of URT, cardiovascular, renal, and pulmonary systems. b The referencecategory is LAMP-2-ANCA seronegativity. OR; odds ratio, CI; confidence interval, URT; upper respiratory tract.We next observed significantly lower LAMP-2-ANCA concentrations in individu-als with (mean LAMP-2-ANCA 1154.4 ng/mL) versus without (mean LAMP-2-ANCA2498.6 ng/mL; p = 0.0043) URT involvement (Figure 3A), but not the pulmonary system asa whole (Figure 3B). In contrast, and despite no association between ANCA titers and hyper-tension (Figure 3D) [19], we observed significantly elevated LAMP-2-ANCA concentrationsin individuals with cardiovascular involvement (mean LAMP-2-ANCA 3475.6 ng/mL)versus those individuals without this manifestation (mean LAMP-2-ANCA 1538.9 ng/mL;p = 0.0003) (Figure 3C). When using a multivariate linear regression model adjusted forvessel size, biological sex, and age at diagnosis, however, the results neither supported anassociation between the level of organ involvement (multi or single) and LAMP-2-ANCAconcentration nor significant differences in LAMP-2-ANCA concentration between individ-uals with and without cardiovascular (p = 0.392), renal (p = 0.541), pulmonary (p = 0.511)and URT (p = 0.901) involvement.Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 9 of 15 Figure 3. Organ system involvement and LAMP-2-ANCA titers in PSV. (A–C) Sera LAMP-2-ANCA concentration (y-axis; μg/mL) versus disease activity (component pVAS ≤ 1 is inactive and ≥2 is ac-tive) in (A) upper respiratory tract (ear–nose–throat component of pVAS), (B) pulmonary system, (C) cardiovascular system, or relative to (D) the presence of hypertension (yes/no) in children and adolescents with chronic primary systemic vasculitis (x-axis, total, n = 25) affecting small (SVV, n = 11) and medium/large (MVV + LVV, n = 14) vessels. LAMP-2-ANCA-seropositive and -seronegative samples indicated with closed and open circles, respectively. Bars show means. ** Denotes statistical significance with p < 0.01, *** p < 0.001. 3. Discussion Chronic primary systemic vasculitis (PSV) is a heterogeneous group of diseases char-acterized by inflammation and damage to blood vessels that vary in size and location. Pediatric PSV affecting small blood vessels is commonly associated with autoantibodies (ANCA) against proteinase 3 (PR3) or myeloperoxidase (MPO). Herein, we demonstrate an increased prevalence and concentration of ANCA that are specific for LAMP-2 in chil-dren with PR3/MPO-ANCA-negative vasculitis affecting medium (MVV) and large (LVV) blood vessels. Our findings may prompt reconsideration of the presence and potential of monitoring autoantibody seropositivity against LAMP-2, or other autoantigens, in sub-types of MVV and LVV previously regarded as seronegative for ANCA. PR3 and MPO are the predominant antigens of interest in small-vessel vasculitis and autoantibodies (ANCA) to these aid with classification of AAV subtypes granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA). There is conflicting data on the utility of ANCA titers to inform disease activity, and an as yet unknown role for MPO- or PR3-ANCA in organ-specific disease processes. Only recently, more than 40 years after their presence in vasculitis was discovered [20], a role for ANCA specificity towards PR3 and MPO in predicting disease outcomes in ANCA-associated SVV is being recognized. The presence of PR3-ANCA is associated with a higher risk of severe inflammatory lung disease, multi-organ involvement, and disease relapse; whereas MPO-ANCA are associ-ated with more severe, renal-limited disease at presentation [21–23]. Not all patients, how-ever, follow such patterns of disease, and there is evidence that other factors, such as type of organ involvement (e.g., renal versus non-renal) and genetic associations also impact risk [24,25]. ANCA specificity for LAMP-2 or other autoantigens, particularly those Figure 3. Organ system involvement and LAMP-2-ANCA titers in PSV. (A–C) Sera LAMP-2-ANCAconcentration (y-axis; µg/mL) v sus disease activity (compone pVA ≤ 1 is in ctive and ≥2 isactive) in (A) upper respiratory tract (ear–nose–throat component of pVAS), (B) pulmonary system,(C) cardiovascular system, or relativ to (D) the pres nce of hyperten ion (yes/no) in children andadolescents with chronic primary systemic vasculitis (x-axis, total, n = 25) affecting small (SVV, n = 11)and medium/large (MVV + LVV, n = 14) vessels. LAMP-2-ANCA-seropositive and -seronegativesamples indicated with closed and open circles, respectively. Bars show means. ** Denotes statisticalsignificance with p < 0.01, *** p < 0.001.Int. J. Mol. Sci. 2024, 25, 3771 9 of 153. DiscussionChronic primary systemic vasculitis (PSV) is a heterogeneous group of diseases char-acterized by inflammation and damage to blood vessels that vary in size and location.Pediatric PSV affecting small blood vessels is commonly associated with autoantibodies(ANCA) against proteinase 3 (PR3) or myeloperoxidase (MPO). Herein, we demonstrate anincreased prevalence and concentration of ANCA that are specific for LAMP-2 in childrenwith PR3/MPO-ANCA-negative vasculitis affecting medium (MVV) and large (LVV) bloodvessels. Our findings may prompt reconsideration of the presence and potential of moni-toring autoantibody seropositivity against LAMP-2, or other autoantigens, in subtypes ofMVV and LVV previously regarded as seronegative for ANCA.PR3 and MPO are the predominant antigens of interest in small-vessel vasculitis andautoantibodies (ANCA) to these aid with classification of AAV subtypes granulomatosiswith polyangiitis (GPA) and microscopic polyangiitis (MPA). There is conflicting data onthe utility of ANCA titers to inform disease activity, and an as yet unknown role for MPO-or PR3-ANCA in organ-specific disease processes. Only recently, more than 40 years aftertheir presence in vasculitis was discovered [20], a role for ANCA specificity towards PR3and MPO in predicting disease outcomes in ANCA-associated SVV is being recognized.The presence of PR3-ANCA is associated with a higher risk of severe inflammatory lungdisease, multi-organ involvement, and disease relapse; whereas MPO-ANCA are asso-ciated with more severe, renal-limited disease at presentation [21–23]. Not all patients,however, follow such patterns of disease, and there is evidence that other factors, suchas type of organ involvement (e.g., renal versus non-renal) and genetic associations alsoimpact risk [24,25]. ANCA specificity for LAMP-2 or other autoantigens, particularly thoseexpressed in affected tissues, may have an additional prognostic role. Although the preva-lence of LAMP-2-ANCA has been debated in PR3/MPO-ANCA-associated vasculitis (AAV)and AAV-related kidney disease, overlapping seropositivity for LAMP-2-ANCA with MPO-or PR3-ANCA in SVV is consistently observed [10,13,16,18,26].The value of LAMP-2-ANCA seropositivity as a biomarker may be greatest amongAAV patients who have MPO/PR3-ANCA-negative SVV, but more so in individualswith middle vessel vasculitis (MVV), namely, polyarteritis nodosa (PAN) and large vesselvasculitis (LVV), namely Takayasu’s arteritis (TAK). Due to the rarity of pediatric PANand TAK combined with the absence of PR3- and MPO-ANCA, predictive biomarkersand specific disease activity markers are lacking. Determining levels of disease activity inPAN and TAK patients has proved challenging for several reasons: vascular imaging ofmedium- or large-sized vessels is frequently invasive and may not reliably differentiatebetween active inflammation versus damage; biopsy of affected vessels is often too riskyand not feasible on a repeated basis; and traditional markers of inflammation (C-reactiveprotein and erythrocyte sedimentation rate) are non-specific and may not be elevated inorgan-limited disease.Our findings of elevated LAMP-2-ANCA seropositivity in pediatric PAN and TAK areconsistent with reports in adults with PAN and TAK of elevated sera LAMP-2 protein [11,14],as well as elevated sera LAMP-2-ANCA [15,19]. In AAV, seropositivity for MPO or PR3-ANCA, rather than serial measures of titer, appear to have greater clinical value in pre-dicting disease course [23–25,27]. In adult-onset PAN, Li et al. demonstrated a positivecorrelation between sera LAMP-2 protein concentration, C-reactive protein concentration,and overall disease activity in adult PAN [11]. The observed absence of a correlationbetween LAMP-2-ANCA titers and disease activity in this study was consistent withseveral previous reports on adult SVV [16–18] and our own observations in pediatricSVV [13]. Further, our work revealed that neither seropositivity nor titer at diagnosis wasinformative to disease course, with similar improvements in disease activity observed inLAMP-2-ANCA-seronegative and -seropositive participants.In 2013, Kawakami et al. observed positive perinuclear (p) ANCA staining in adultcases of cutaneous polyarteritis nodosa and proposed that there are as yet undiscoveredautoantigens [15]. More recently, Mukherjee et al. provided strong evidence for the presenceInt. J. Mol. Sci. 2024, 25, 3771 10 of 15of ANCA towards an unidentified antigenic target in sputa from adults with MPO/PR3-ANCA-negative eosinophilic granulomatosis with polyangiitis (EGPA). Importantly, theirstudy demonstrated that ANCA reactivity was not observed in sera, emphasizing theimportance of investigations in the affected tissue [28]. Additional studies support theexistence of ANCA/autoantigens in MPO/PR3-ANCA-negative vasculitides includingidentification of elastase-ANCA in adult ANCA-negative glomerulonephritis [29] andthe presence of alpha-enolase, a potential cytosolic autoantigen, in 82% of adults withpredominantly ANCA-negative EGPA [30].Our study has certain limitations. Despite having the largest reported pediatric chronicPSV study cohort, it may not be reflective of all populations. Further, the sample size maybe underpowered for some analyses. Notably, it may not have been possible to observeany associations between organ system involvement and LAMP-2-ANCA, given that ourmultivariate logistic regression model did not reveal associations between MPO/PR3-ANCA status and particular organ systems as would be expected, given that a majority ofcases have renal and pulmonary/URT involvement at diagnosis [31–33].Distinguishing between the presence (or absence) and the antigenic target of autoim-mune processes has important implications for therapeutic decision-making. Our datasuggest that LAMP-2-ANCA may be important in childhood-onset PSV, where ANCAagainst classical targets, PR3 or MPO, are commonly absent. Although traditionally studiedin the context of renal involvement, our data highlight a high prevalence and concen-tration of LAMP-2-ANCA in subtypes of PSV with extrarenal manifestations. Althoughsubstantive gaps in the understanding of LAMP-2-ANCA utility and pathogenicity remain,this report and others argue for continued examination of LAMP-2-ANCA, particularlyin the context of medium- and large-vessel vasculitides that are commonly consideredseronegative for autoantibodies.4. Materials and Methods4.1. Study Cohorts and BiosamplesThe children and youth with chronic PSV that are described in this study (n = 90;Table 1) were enrolled in the Pediatric Vasculitis Initiative (PedVas), for which eligibilitycriteria have been described previously [32,34]. The Children’s and Women’s ResearchEthics Board of the University of British Columbia [H12-00894] and the respective ethicsreview boards at participating PedVas centers gave their approval to the research protocol.Participants contributed blood in serum separation tubes (BD Biosciences, Franklin Lakes,NJ, USA) at the time of diagnosis. These sample were processed to sera according to astandard protocol and stored at −80 ◦C.Individuals also included in the study were children and youth (n = 18; 38.9% fe-male, median age of symptom onset 8.3 years, range 1.2–16.1 years) receiving care fora systemic autoinflammatory disease (SAID) characterized by recurrent episodes of un-controlled inflammation in the absence of infection or autoimmunity (i.e., no detectableautoantibodies or autoreactive immune cells) [35]. Cohort characteristics are summarizedin Supplementary Table S1. Approval from the Children’s and Women’s Research EthicsBoard of the University of British Columbia was obtained to record diagnostic and demo-graphic data in a research database (H14-00272) [36] and to collect and store (as describedfor PSV patients) sera (H15-00351). Sera were used to establish a range of LAMP-2-ANCAconcentrations associated with systemic inflammation, as indicated by concentrations ofhuman C-reactive protein (CRP ELISA kit) according to the manufacturer’s instructions(ThermoFisher, Waltham, MA, USA).4.2. Clinical Data for Chronic PSV ParticipantsClinical data were entered by participating centers on a web-based clinical data reg-istry [31,32] and used to formally classify chronic PSV patients (n = 90) into disease subtypesunder the broader designation of small- (SVV, n = 53), medium- (MVV, n = 16), and large-(LVV, n = 21) vessel vasculitis. A subset of relevant registry data is summarized in Table 1.Int. J. Mol. Sci. 2024, 25, 3771 11 of 15The majority of cases of SVV met EULAR/PRINTO/PRES criteria for granulomatosis withpolyangiitis (GPA, 43/53, 81.1%) [37,38]. MPA patients fulfilled the pediatric modifiedalgorithm of the EMA [39] and EGPA patients met the ACR or Lanham criteria [37,40].MVV subtypes included: polyarteritis nodosa (PAN, n = 11; EULAR/PRINTO/PRES cri-teria [37]), cutaneous PAN (cPAN, n = 3) and two cases of unclassified medium vesselvasculitis (uMVV, n = 2). Patients with LVV were predominantly Takayasu’s arteritis (TA,n = 19/21, 90.5%; EULAR/PRINTO/PRES criteria [37]) with two cases of unclassifiedlarge-vessel vasculitis (uLVV, n = 2). ANCA positivity and specificity for proteinase 3 (PR3)and myeloperoxidase (MPO) were entered in the registry by participating centers. Asexpected, the majority of cases of SVV were positive for MPO- or PR3-ANCA (49/52 caseswith available data; 94.2%)4.3. Quantification of Active Disease in Chronic PSV ParticipantsDisease activity at the time of sample/data collection (diagnosis) was calculated fromthe registry data using the pediatric vasculitis activity score (pVAS; range 0–63, wherezero indicates inactive disease), which is a cumulative weighted score of disease activityacross nine organ systems [41]. For individuals with follow-up data, improvement atone-year post-diagnosis was calculated as: % improvement = 100% × ((pVAS at diagnosis)− (pVAS at 1-year))/(pVAS at diagnosis). All participants had active disease at the timeof diagnosis (and sample/data collection), with a median overall disease activity (mediantotal pVAS) score equal to 17 (range 1–50). Using subcomponent scores of pVAS, anal-yses were focused on four critical organs/organ systems that drive treatment decisionsearly in the disease course and are frequently involved in SVV, MVV, and LVV, namely,kidneys (max. pVAS = 12), upper respiratory tract (max. pVAS = 6), pulmonary system(max. pVAS = 6), and cardiovascular system (max. pVAS = 6), where a subcomponentpVAS ≥ 2 indicated organ involvement [41].4.4. Meso Scale Discovery (MSD) Electrochemiluminescence Assay to Detect SerumAnti-LAMP-2-AntibodiesRecombinant human (rh) LAMP-2 protein (R&D Systems, Minneapolis, MN, USA)was diluted in phosphate-buffered saline (PBS) and added (30 µL at 2.5 µg/mL) to 96-wellelectrochemiluminescence assay plates (Meso Scale Discovery, Rockville, MD, USA). Plateswere sealed and incubated at 4 ◦C overnight, blocked (150 µL blocker A solution; RT,1 h, 700 rpm) and washed (3 × 150 µL/well PBS + 0.05% Tween 20 (PBST)). A standardcurve was generated with anti-human-LAMP-2 (H4B4) monoclonal antibody (BioLegend,San Diego, CA, USA, cat #354302) serially diluted from 1–4000 ng/mL in blocker A so-lution. Standards and sera (50 µL/well of 1:10 dilution) were added (1 h, RT, shaking),then wells were washed (3 × 150 µL/well PBST). A biotin-conjugated anti-IgG Fc (multi-species) antibody (ThermoFisher Scientific, Waltham, MA, USA, cat #7102852100) wasadded (50 µL/well at 1 µg/mL in blocker A; RT, 1 h, shaking) followed by strep-SULFO-TAG (50 µL/well at 0.5 µg/mL in blocker A; RT, 1 h, shaking) and 150 µL/well of MSD GoldRead buffer B with washing between steps (3 × 150 µL/well TBST). Plates were read usingan MSD QuickPlex SQ 120 MM electrochemiluminescence instrument and a standard curvegenerated using a 4-parameter logistical curve fit algorithm within MSD Discovery Work-bench Software (version 4.0.12). The assay was validated with sera, courtesy of Dr. RenateKain at the University of Vienna, from young-onset ANCA-associated vasculitis patientsknown to be seropositive for anti-LAMP-2 antibodies [10,13,42] and a subset of participantswith an SAID (n = 5) or a chronic PSV (n = 44; predominantly SVV) that were previouslyassessed for sera LAMP-2-ANCA concentrations using an indirect, in-house colorimetricELISA [13]. Compared to the traditional colorimetric ELISA, the MSD platform displayed10–60 times the linear range and improvement in the signal-to-noise ratio with a broad dy-namic range. Using this assay, sera LAMP-2-ANCA concentrations in the majority (15/18;83.3%) of individuals with an SAID were detected at less than or equal to 1000 ng/mL,Int. J. Mol. Sci. 2024, 25, 3771 12 of 15with an average concentration of 661.0 ng/mL (range 44.4–1311.4 ng/mL) (Figure 1A) andinformed the threshold of seropositivity for LAMP-2-ANCA at >1000 ng/mL in this study.4.5. Statistical AnalysisGraphPad Prism v9.0 statistical software (GraphPad Software, La Jolla, CA, USA) wasused to conduct the statistical analyses. Group differences were examined by one-wayANOVA and/or two-tailed t-tests. Pearson’s correlation coefficient was used to evaluatecorrelations. Where indicated, group differences were analyzed by Kruskal–Wallis, Mann–Whitney, or chi-squared statistical tests. R (R Foundation for Statistical Computing, version4.2.2, Vienna, Austria) was used for multivariate analyses. A 95% confidence interval wasused for all analyses, and a p-value equal to or less than 0.05 was regarded as significant.Supplementary Materials: The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/ijms25073771/s1.Author Contributions: All authors (T.H.A., K.K.T., S.K.M., K.M.G., G.A.A., B.B.-P., D.A.C., K.A.M.and K.L.B.) contributed to the conceptualization of the project, writing, and review of the manuscript.T.H.A., K.K.T., S.K.M. and K.M.G. were responsible for the acquisition and analysis of data. T.H.A.,K.K.T. and S.K.M. prepared figures and tables. G.A.A. was responsible for methodology implementa-tion. D.A.C., K.A.M., B.B.-P. and K.L.B. were responsible for funding acquisition and project oversight.All authors have read and agreed to the published version of the manuscript.Funding: This project has been supported by Canadian Institutes of Health Research grants (TR2-119188 to DC and PJT-180302 to KLB). THA is supported by funds from a Global Affairs CanadaGo-Global Study in Canada Scholarship (THA). KT is supported by a Centre for Blood Researchgraduate award and a Women+ and Children’s Health Sciences Diversity Award. SM is supported bya Canadian Institutes of Health Research Graduate Student Scholarship (CGS-M) and a BC Children’sHospital Graduate Student Award. KMG is supported by a University of British Columbia Four-YearFellowship for PhD students. DC is supported by the Arthritis Society Canada through the Ross PettyArthritis Society Chair. KLB is supported by a BC Children’s Hospital Salary Award and a MichaelSmith Foundation for Health Research Scholar Award.Institutional Review Board Statement: This study complies with the Declaration of Helsinki andreceived approval from the Children’s and Women’s Research Ethics Board of the University ofBritish Columbia (H12-00894; renewed annually since 2012 and most recently on 23 February 2024)and the respective ethics review boards at participating PedVas centers. Diagnostic and demographicdata were recorded in a research database with approval from the Children’s and Women’s ResearchEthics Board of the University of British Columbia (H14-00272) as described previously (18) withbiosamples collected under a linked (ethics board-approved) research study (H15-00351).Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.Data Availability Statement: The data that support the findings of this study are available from thecorresponding author upon reasonable request.Acknowledgments: The authors would like to thank Stephanie Hughes (UBC) for excellent technicalassistance and advice on assay design, implementation, and analysis. The authors thank RenateKain (University of Vienna) for providing anti-LAMP-2 antibody-positive sera for assay validation.The authors would like to acknowledge all participating patients and their families, without whomthis study would not be possible. The authors thank the PedVas site investigators and clinicalcoordinators for their dedicated work: Else Bosman, BC Children’s Hospital, University of BritishColumbia, Vancouver, BC, Canada; Rae Yeung and Emily Hickey, Hospital for Sick Children, TorontoON, Canada; Linda Wagner-Weiner, University of Chicago Comer Children’s Hospital, Chicago,IL, USA; Suzanne Li and Justine Griswold, Joseph M. Sanzari Children’s Hospital, HackensackUniversity Medical Center, Hackensack, NJ, USA; Alan Rosenberg and Joan Dietz, Royal UniversityHospital and University of Saskatchewan, SK, Canada; Melissa Elder and Heather Bell-Brunson,University of Florida, Gainesville, FL, USA; Susanne Benseler and Michaela Grice, Alberta Children’sHospital, Calgary, AB, Canada; Susan Shenoi and Nidhi Naik, Seattle Children’s Hospital, Seattle,WA, USA; Stacey Tarvin and Jessica Lee, Riley Children’s Hospital, Indianapolis, IN, USA; NeilMartin and Yvonne McCafferty, Royal Hospital for Children, Glasgow, UK; Kathryn Cook and JessicaInt. J. Mol. Sci. 2024, 25, 3771 13 of 15Kracker, Akron Children’s Hospital, Akron, OH, USA; Eslam Al-Abadi and Ruth Howman, IWKHealth Centre and Dalhousie University, Halifax, NS, Canada;, Birmingham Children’s Hospital NHSFoundation Trust, Birmingham, UK; Karen James and Suzy Richins, University of Utah’s PrimaryChildren’s Medical Center, Salt Lake City, UT, USA; Adam Huber and Victoria Dempsey, IWK HealthCentre, Halifax, NS, Canada; Flora McErlane and Caroline Miller, Great North Children’s Hospital,Newcastle, UK; Marek Bohm and Heather Rostron, Leed’s Children’s Hospital, Leeds, UK; DirkFoell and Antje Hellige, University Children’s Hospital Munster, Munster, Germany; Eileen Baildamand Michelle Andrews, Alder Hey Children’s NHS Foundation Trust Hospital, Liverpool, UK; PaulDancey and Rana Aslanova, Janeway Children’s Health, St. John’s, NL, Canada; Phil Riley andAnn McGovern, CMFT Manchester, Manchester, UK; Roberta Berard and Michelle Diebold, LondonHealth Sciences Centre, London, ON, Canada; Sirirat Charuvanij, Siriraj Hospital, Bangkok, Thailand;Vidya Sivaraman and Joanne Drew, Nationwide Children’s Hospital, Columbus, OH, USA; KathrynBailey and Danielle Miller, Oxford University Hospitals, Oxford, UK.Conflicts of Interest: The authors declare no conflicts of interest. 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lysosome-associated membrane protein-2
anti-neutrophil cytoplasmic antibodies
childhood-onset primary vasculitis
autoantibodies
Takayasu’s arteritis
polyarteritis nodosa
Anti-LAMP-2 Antibody Seropositivity in Children with Primary Systemic Vasculitis Affecting Medium- and Large-Sized Vessels
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