{"paper_id":"03356f25-62ac-4e5c-8c65-04464ce6eee5","body_text":"1\n1 Diagnostic Value of Antibody Responses to \n2 Mycobacterium avium subsp. paratuberculosis-Derived \n3 Proteins PtpA and PtpB in Rheumatoid Arthritis\n4\n5 Jorge Hernández-Bello 1, Sergio Cerpa-Cruz 4, Gabriela A. Sánchez-Zuno 5, Ferdinando \n6 Nicoletti 6, Horacio Bach 2,*, José F. Muñoz-Valle1,* \n7\n8 1 Instituto de Investigación en Ciencias Biomédicas, Centro Universitario de Ciencias de la \n9 Salud Universidad de Guadalajara, 44340 Guadalajara, México \n10 2 Division of Infectious Diseases, Faculty of Medicine, The University of British Columbia, \n11 Vancouver, BC, V6H 3Z6, Canada\n12 3 Division of Rheumatology, Guadalajara Civil Hospital \"Fray Antonio Alcalde\", \n13 Guadalajara, Jalisco, México\n14 4 Department of Medicine, Yale School of Medicine, New Haven, CT, USA\n15 5 Department of Biomedical and Biotechnological Sciences, University of Catania, Catania, \n16 Italy\n17\n18 Corresponding authors\n19 * Horacio Bach, Division of Infectious Diseases, Faculty of Medicine, The University of \n20 British Columbia, 410-2660 Oak Street, Vancouver, BC V6H 3Z6, Canada. E-\n21 mail:horacio.bach@ubc.ca.\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.08.698458doi: bioRxiv preprint \n\n2\n22 * José F. Muñoz-Valle, Instituto de Investigación en Ciencias Biomédicas, Centro \n23 Universitario de Ciencias de la Salud, Universidad de Guadalajara, 44340 Guadalajara, \n24 México. E-mail: biologiamolecular@hotmail.com\n25\n26 Abstract\n27 Evidence suggests that Mycobacterium avium subspecies paratuberculosis (MAP) may \n28 contribute to autoimmune diseases such as rheumatoid arthritis (RA), partly through effector \n29 proteins—particularly the tyrosine phosphatases PtpA and PtpB—that modulate macrophage \n30 signaling and promote bacterial persistence. This study evaluated whether serum antibodies \n31 against these proteins serve as biomarkers of RA. Humoral responses to PtpA and PtpB were \n32 quantified in Mexican RA patients (n = 100) and healthy controls (n = 100) using in-house \n33 ELISAs. Associations with disease activity (DAS28), ROC performance, and logistic \n34 regression models were assessed. Results showed that anti-PtpB antibody levels were \n35 significantly higher in patients with RA than in healthy controls (median OD 0.185 vs. 0.080; \n36 p < 0.0001) and had moderate discriminative capacity (AUC = 0.762). Anti-PtpB reactivity \n37 increased with higher disease activity and showed a significant positive association with \n38 DAS28 (p < 0.05). In addition, there was a functional disability measured by HAQ (p < \n39 0.001), as well as moderate correlations with erythrocyte sedimentation rate and rheumatoid \n40 factor. A combined logistic regression model integrating both antibodies markedly improved \n41 diagnostic accuracy (AUC = 0.934), achieving high sensitivity (90%) and specificity (89%). \n42 These findings support a potential role of MAP in RA immunopathogenesis and indicate that \n43 combined quantification of anti-PtpA and anti-PtpB antibodies captures complementary and \n44 non-redundant immunological information. This combined serological approach may \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.08.698458doi: bioRxiv preprint \n\n3\n45 enhance RA diagnosis and provide clinically relevant insights into disease activity and \n46 severity.\n47\n48 Keywords: Rheumatoid arthritis; Mycobacterium avium subspecies paratuberculosis; PtpA; \n49 PtpB; DAS-28; Tyrosine phosphatases; ELISA;\n50\n51 Introduction\n52 RA is a multifactorial autoimmune disease in which genetic predisposition, \n53 dysregulated immune pathways, and microbial exposures interact to promote chronic \n54 synovial inflammation and joint destruction [1,2]. Growing interest has focused on \n55 microorganisms capable of persisting within host immune cells and generating antigenic \n56 stimuli that may shape autoantibody production or amplify inflammatory cascades. Among \n57 these, Mycobacterium avium subsp. paratuberculosis (MAP) has emerged as a plausible \n58 environmental trigger of autoimmunity due to its ability to survive within macrophages and \n59 modulate intracellular signaling via secreted virulence factors [3–5].\n60 MAP effector proteins involved in host–pathogen interactions have attracted \n61 particular attention for their immunogenic properties and potential relevance in RA. A pivotal \n62 study from Italy demonstrated that the MAP-derived protein tyrosine phosphatases PtpA and \n63 PknG are recognized at significantly higher frequencies in the sera of RA patients than in \n64 those of healthy controls, supporting the notion that MAP exposure may leave a detectable \n65 humoral footprint in RA [6]. Building on this idea, our group recently reported that PtpA-\n66 specific antibodies are also elevated in Mexican patients with RA and may serve as an \n67 informative immunological marker in this population [7]. These observations collectively \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.08.698458doi: bioRxiv preprint \n\n4\n68 suggest that MAP phosphatases and kinases are antigenic targets that can elicit differential \n69 immune responses in RA.\n70 Mechanistically, MAP-secreted proteins such as PtpA and PtpB may become relevant \n71 to RA pathogenesis because their intracellular effects converge on processes central to joint \n72 inflammation. PtpA inhibits phagolysosomal fusion by dephosphorylating the human \n73 VPS33B, a protein involved in phagolysosome fusion. This dephosphorylation allows \n74 bacteria to persist within macrophages [8], major producers of the pro-inflammatory \n75 cytokines TNF, IL-1β, and IL-6, which drive synovitis and structural damage [9]. Persistent \n76 MAP antigens could therefore act as chronic stimuli, maintaining macrophage activation, \n77 promoting continuous cytokine release, and enhancing Th1/Th17 polarization [10]. In \n78 genetically susceptible individuals, repeated exposure to these antigens may also increase \n79 autoantibody formation via molecular mimicry [11]. Furthermore, a higher antigenic load or \n80 stronger immune recognition of MAP phosphatases may reflect ongoing innate immune \n81 activation, potentially explaining an association between elevated antibody levels and greater \n82 clinical activity in RA.\n83 Among MAP-secreted effectors, the tyrosine phosphatases PtpA and PtpB have \n84 attracted attention for their ability to disrupt phagosomal maturation and phosphoinositide \n85 metabolism, thereby interfering with vesicular trafficking and promoting intracellular \n86 survival—mechanisms that have been extensively characterized in related mycobacterial \n87 pathogens. Although evidence has begun to accumulate for PtpA-specific responses in RA, \n88 whether PtpB elicits a similar or complementary humoral signature remains unknown. \n89 Furthermore, whether combining immune responses to both phosphatases can improve \n90 diagnostic discrimination has never been explored.\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.08.698458doi: bioRxiv preprint \n\n5\n91 In this study, we analyzed antibody reactivity to PtpA and PtpB in patients with RA \n92 and healthy controls and assessed the diagnostic utility of each marker individually and in \n93 combination. By integrating our previous findings [7] with new data on PtpB, we aimed to \n94 clarify the immunological relevance of MAP-secreted phosphatases in RA and determine \n95 whether their combined measurement enhances diagnostic accuracy.\n96\n97 Materials and methods\n98\n99 Subjects\n100 Archived serum samples from patients and healthy controls were used in this study. \n101 These samples were used to determine the level of anti-PtpA in our previous study [7]. \n102 Briefly, RA patients (23 males, 77 females; median age 58) who fulfilled the 2010 \n103 ACR/EULAR Classification Criteria for RA were enrolled at the Rheumatology Unit of the \n104 Civil Hospital of Guadalajara, Fray Antonio Alcalde, Guadalajara, Jalisco, Mexico, between \n105 January 1, 2018, and December 31, 2021. Clinical and demographic data were collected, \n106 including disease duration, rheumatoid factor (RF) and anti-cyclic citrullinated peptide (anti-\n107 CCP) status, treatment with steroids and disease-modifying anti-rheumatic drugs \n108 (DMARDs), C-reactive protein (CRP) levels, erythrocyte sedimentation rate (ESR), Disease \n109 Activity Score-28 (DAS-28), and Health Assessment Questionnaire (HAQ) scores.\n110 A group of 100 healthy controls (20 males, 80 females; median age 40 years) was \n111 recruited at the same hospital. Control participants verbally confirmed having no prior history \n112 of tuberculosis. Antibody reactivity to PtpA in this cohort has been previously described [7]. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.08.698458doi: bioRxiv preprint \n\n6\n113 For the present study, the same archived sera were additionally evaluated for reactivity to \n114 PtpB, and combined analyses of PtpA and PtpB were performed.\n115\n116 Ethics approval statement\n117 This study was approved by the Ethics Committee of the University of Guadalajara \n118 (Approval No. 0122017) and conducted in accordance with the ethical principles outlined in \n119 the Declaration of Helsinki (64th World Medical Association General Assembly, Fortaleza, \n120 Brazil, 2013). Written informed consent was obtained from all participants prior to inclusion \n121 in the study.\n122\n123 ELISA assays \n124 Plate preparation\n125 The recombinant PtpB protein was expressed in Escherichia coli harboring the ptpB \n126 gene in the ampicillin-resistant pET-22 vector. Purification was performed via Ni-NTA \n127 affinity chromatography, and the protein was stored at –20°C until use. The preparation of \n128 recombinant PtpA and the corresponding assay conditions have been previously described \n129 [7].\n130 For ELISA, Maxisorp plates (ThermoFisher) were coated with 50 μg/mL of antigen \n131 in phosphate-buffered saline (PBS) and incubated overnight at 4°C. Plates were washed three \n132 times with PBS containing 0.05% Tween-20 (PBS-T) and blocked with 3% bovine serum \n133 albumin (BSA) in PBS at 4°C overnight. Plates were air-dried prior to use.\n134\n135 Assay procedure \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.08.698458doi: bioRxiv preprint \n\n7\n136 Sera from RA patients and controls were tested in triplicate. After incubation with \n137 sera, plates were washed with PBS-T and subsequently incubated with a peroxidase-\n138 conjugated anti-human IgG secondary antibody. Optical density (OD) was measured at 450 \n139 nm using an Epoch microplate reader (BioTek, USA). The baseline signal, defined as the \n140 secondary antibody alone, was subtracted from all readings. Positive controls were included \n141 in all assays. Cut-off values were determined by Receiver Operating Characteristic (ROC) \n142 analysis to ensure specificity above 90%, with sensitivity adjusted accordingly.\n143\n144 Statistical analysis\n145 Differences in antibody reactivity between RA patients and controls were assessed \n146 using the Mann–Whitney U test. Associations between clinical variables and antibody levels \n147 were examined by linear regression. ROC curves and areas under the curve (AUC) were \n148 generated using Python (version 3.14) with the scikit-learn library. A multivariable logistic \n149 regression model integrating anti-PtpA and anti-PtpB antibody levels was constructed to \n150 assess combined diagnostic performance. Pairwise comparisons between ROC curves were \n151 performed using DeLong’s test. Sensitivity, specificity, positive predictive value (PPV), and \n152 negative predictive value (NPV) were calculated at the optimal cutoff determined by the \n153 Youden index. Graphical representations of ROC curves and correlation plots were generated \n154 using GraphPad Prism version 8.0 (GraphPad Software, San Diego, CA). Statistical \n155 significance was defined as a two-sided p-value < 0.05.\n156\n157 Results\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.08.698458doi: bioRxiv preprint \n\n8\n158 Patients with RA were significantly older than controls (median 54 vs. 40 years, p < \n159 0.0001), while the proportion of females was similar between groups (81% vs. 80%, p > \n160 0.90). Among RA patients, disease activity and disability indices showed median DAS-28 \n161 and HAQ scores of 3.2 and 0.75, respectively. Inflammatory markers were elevated, with C-\n162 reactive protein (CRP) at 6 mg/dL and ESR at 20 mm/h. Most patients were receiving \n163 DMARDs (73%), and more than one-third reported NSAID use (36%), whereas only 1% \n164 were on corticosteroid therapy (Table 1).\n165 As shown in Fig 1A, antibody levels against PtpB were higher in the RA group than \n166 in controls (p < 0.0001, Mann–Whitney U test). The median optical density (OD) in RA \n167 patients was 0.1847 [25 th–75th percentile: 0.1120–0.2483], whereas the control group \n168 exhibited a median OD of 0.0801 [25 th–75th percentile: 0.03275–0.1434]. ROC analysis \n169 demonstrated that anti-PtpB antibodies effectively discriminated RA patients from healthy \n170 controls, with an AUC of 0.762 (p < 0.0001; Fig 1B).\n171\n172 Fig 1. Serum antibody reactivity to PtpB in RA subjects and CS. (A) Comparison of anti-\n173 PtpB antibody levels between groups. Bars represent the median and interquartile range; \n174 dashed lines indicate antibody positivity thresholds, and p-values are shown above. (B) \n175 Receiver operating characteristic (ROC) curve evaluating the discriminative capacity of anti-\n176 PtpB antibodies.\n177\n178 To assess associations between anti-PtpB levels and disease activity, RA patients \n179 were stratified into four groups according to their DAS28 scores: remission, low disease \n180 activity, moderate disease activity, and high disease activity. As shown in Fig 2, patients in \n181 remission had the lowest antibody levels (median = 0.1187; IQR = 0.0447–0.1887). Those \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.08.698458doi: bioRxiv preprint \n\n9\n182 with low disease activity showed slightly higher values (median = 0.1752; IQR = 0.1053–\n183 0.2296), with no significant difference relative to remission (p = 0.3209). The moderate \n184 activity group displayed intermediate values (median = 0.1948; IQR = 0.1574–0.2527), \n185 which overlapped with those of the low activity group (p = 0.9999). Conversely, patients \n186 with high disease activity exhibited the highest antibody levels (median = 0.2357; IQR = \n187 0.1682–0.3324) and differed significantly from the remission group (p = 0.0001). \n188\n189 Fig 2. Association between serum anti-PtpB antibody levels and DAS-28 categories. \n190 Bars represent median ± interquartile range for each disease activity group. Statistical \n191 analysis was performed using the Kruskal–Wallis test followed by Dunn’s post hoc \n192 correction. P-values are shown above the distributions.\n193\n194 Fig 3 displays the correlation heatmap between anti-PtpB antibody levels and clinical \n195 and laboratory variables in patients with RA. Anti-PtpB antibody levels showed statistically \n196 significant positive correlations with disease activity and functional impairment, including \n197 DAS28 (ρ = 0.45, p < 0.001) and HAQ (p = 0.40, p < 0.001). In addition, moderate positive \n198 associations were observed with ESR (p = 0.37, p = 0.003) and rheumatoid factor (RF; p = \n199 0.49, p < 0.001). No significant differences in anti-PtpB levels were observed by sex or \n200 treatment status, including use of non-steroidal anti-inflammatory drugs (NSAIDs), \n201 corticosteroids, sulfasalazine, chloroquine, or methotrexate. No other clinical, hematological, \n202 or demographic variables were significantly associated with anti-PtpB levels.\n203\n204 Fig 3. Correlation heatmap of anti-PtpB antibody levels with clinical and laboratory \n205 parameters in patients with RA. The heatmap displays Spearman correlation coefficients \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.08.698458doi: bioRxiv preprint \n\n10\n206 (ρ) between anti-PtpB antibody levels and clinical and laboratory variables, including \n207 hematological parameters, inflammatory markers, functional disability, and disease activity \n208 indices. Each cell shows the corresponding correlation coefficient and p-value. Warm colors \n209 indicate positive correlations, whereas cool colors indicate negative correlations. \n210 Abbreviations: WBC, white blood cells; RBC, red blood cells; Hb, hemoglobin; MCV, mean \n211 corpuscular volume; PLT, platelets; ESR, erythrocyte sedimentation rate; CRP, C-reactive \n212 protein; RF, rheumatoid factor; BMI, body mass index; HAQ, Health Assessment \n213 Questionnaire; DAS28, Disease Activity Score 28. \n214\n215 The relationship between humoral immune responses to the MAP-derived proteins \n216 PtpA and PtpB was assessed by comparing antibody levels in RA patients and controls. As \n217 shown in Fig 4A and 4B, no significant correlation was found between anti-PtpA and anti-\n218 PtpB levels in either group.\n219\n220 Fig 4. Correlation between anti-PtPA and anti-PtpB antibody levels in RA patients and \n221 control subjects. (A) RA patients. (B) Control subjects. Spearman’s rank correlation \n222 analysis was used to assess associations between markers. Blue and red lines denote the best-\n223 fit linear regressions for each group. \n224\n225 To evaluate the diagnostic utility of combining anti-PtpA and anti-PtpB antibodies, a \n226 multivariable logistic regression model was constructed and its performance assessed. As \n227 shown in Table 2, the combined model demonstrated excellent discriminative capacity, with \n228 an AUC of 0.934. At the optimal cut-off value of 0.3869, determined using the Youden index, \n229 the model achieved a sensitivity of 96% and a specificity of 87%. This threshold also yielded \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.08.698458doi: bioRxiv preprint \n\n11\n230 high predictive values, with a positive predictive value (PPV) of 86% and a negative \n231 predictive value (NPV) of 97%.\n232 ROC curves for anti-PtpA, anti-PtpB, and their combination are presented in Fig 5. \n233 The combined model outperformed both individual markers (PtpA AUC = 0.925; PtpB AUC \n234 = 0.762). Pairwise comparisons of ROC curves using DeLong’s test revealed no statistically \n235 significant differences between the combined model and anti-PtpA alone (ΔAUC = 0.009, p \n236 = 0.975), nor between the combined model and anti-PtpB alone (ΔAUC = 0.172, p = 0.495). \n237 Similarly, the difference between anti-PtpA and anti-PtpB was not statistically significant \n238 (ΔAUC = 0.163, p = 0.485).\n239\n240 Fig 5. ROC curve comparison of models using anti-PtpA, anti-PtpB, and their \n241 combination. The green solid line represents the combined PtpA+PtpB model, which \n242 achieved the highest diagnostic accuracy. The blue dashed line represents the model using \n243 only PtpA, while the orange dashed line represents the model using only PtpB. The black \n244 diagonal line indicates random classification (AUC = 0.5).\n245\n246 Discussion\n247 Emerging evidence suggests that bacterial exposures may contribute to the etiology \n248 of RA by disrupting immune tolerance and promoting chronic inflammation in genetically \n249 susceptible individuals [18]. Several microorganisms, particularly those capable of persisting \n250 within macrophages or modifying host proteins, have been implicated as potential triggers of \n251 autoimmunity [19]. Intracellular bacteria such as MAP can interfere with phagosome \n252 maturation and sustain pro-inflammatory cytokine production [20,21]. Meanwhile, mucosal \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.08.698458doi: bioRxiv preprint \n\n12\n253 pathogens, including Porphyromonas gingivalis and Aggregatibacter \n254 actinomycetemcomitans, have been associated with aberrant protein citrullination and the \n255 induction of anti-CCP antibodies in RA [22], supporting the concept that microbial factors \n256 may converge on shared immunopathogenic pathways.\n257 In this study, we evaluated humoral immune responses to the MAP-derived tyrosine \n258 phosphatases PtpA and PtpB in patients with RA and healthy controls. Four principal \n259 findings emerged: (i) anti-PtpB antibodies were significantly elevated in RA; (ii) anti-PtpB \n260 titers increased with higher disease activity; (iii) anti-PtpA and anti-PtpB responses were \n261 immunologically independent; and (iv) a combined logistic model incorporating both \n262 antibodies markedly improved diagnostic accuracy. Collectively, these observations \n263 strengthen the hypothesis that MAP antigens may contribute to the immunological landscape \n264 of RA and may serve as complementary biomarkers in this population.\n265 Our findings extend existing evidence suggesting MAP exposure in RA. Previous \n266 studies have shown that MAP-secreted proteins, such as PtpA and PknG, are more frequently \n267 recognized by RA sera than by healthy control sera [12]. We also previously reported \n268 increased anti-PtpA responses in Mexican RA patients [7]. Molecular investigations in the \n269 USA, Europe, and the Middle East have detected MAP DNA or MAP-reactive antibodies in \n270 RA populations, reinforcing the plausibility of MAP as an environmental contributor to \n271 autoimmunity [6,23–25].\n272 The present study adds the observation that PtpB, an established virulence \n273 phosphatase in mycobacteria, also elicits increased antibody responses in RA. Given that \n274 PtpB participates in immune evasion and intracellular persistence [16,26], heightened \n275 seroreactivity in RA patients is biologically compatible with chronic antigen exposure. \n276 Importantly, anti-PtpB antibody levels were positively associated with established measures \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.08.698458doi: bioRxiv preprint \n\n13\n277 of disease activity and functional impairment, including DAS28 and HAQ, providing a \n278 clinically meaningful link between MAP-related immune responses and disease expression.\n279 The association between anti-PtpB antibody levels and clinical disease activity \n280 provides a biologically plausible link between MAP-related immune responses and \n281 established pathogenic mechanisms in RA. Macrophages play a central role in RA synovitis, \n282 acting as key effector cells that produce pro-inflammatory cytokines such as TNF-α, IL-1β, \n283 and IL-6, which drive both joint inflammation and structural damage [27]. MAP \n284 phosphatases directly modulate macrophage biology: PtpA inhibits recruitment of the \n285 vacuolar H⁺-ATPase to the phagosome, blocking acidification [28], whereas PtpB in other \n286 mycobacteria modulates intracellular kinase signaling and promotes bacterial survival \n287 [29,30]. In this context, elevated anti-PtpB titers in RA may reflect sustained activation of \n288 innate immune pathways, consistent with their correlations with DAS28 and HAQ.\n289 Anti-PtpB levels were also moderately associated with ESR and RF, but not with CRP \n290 or BMI. This pattern suggests that anti-PtpB immunity does not simply mirror acute-phase \n291 inflammation or metabolic status. Rather, it supports the existence of a more specific \n292 immunological axis, potentially centered on macrophage activation and humoral \n293 autoimmunity, in which MAP-related antigens contribute to disease severity without acting \n294 as nonspecific inflammatory markers. The lack of association with CRP may further indicate \n295 that anti-PtpB antibodies capture chronic or cumulative immune activation rather than \n296 transient inflammatory fluctuations.\n297 The absence of significant differences in anti-PtpB levels according to sex or \n298 exposure to conventional antirheumatic therapies adds an important dimension to this \n299 interpretation. These findings suggest that anti-PtpB responses are relatively stable and not \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.08.698458doi: bioRxiv preprint \n\n14\n300 readily modulated by therapies that primarily target downstream inflammatory cascades, \n301 raising the possibility that anti-PtpB antibodies reflect upstream or parallel pathogenic \n302 processes that are not fully addressed by current therapeutic strategies.\n303 One of the most distinctive findings was the independence between anti-PtpA and \n304 anti-PtpB titers in both RA patients and controls. This observation is consistent with and \n305 provides mechanistic support for our previous findings that anti-PtpA antibody levels were \n306 not associated with DAS28, autoantibody status, or inflammatory markers in RA [7]. Such \n307 independence likely reflects the functional divergence between the two phosphatases. PtpA \n308 disrupts the VPS33B–V-ATPase axis, primarily affecting phagosomal maturation and \n309 intracellular trafficking [31]. In contrast, PtpB operates through distinct lipid-mediated and \n310 kinase-dependent signaling pathways that influence host immune activation [32,33]. Their \n311 non-overlapping virulence mechanisms provide a plausible biological basis for differential \n312 immune recognition, suggesting engagement of distinct antigen-processing routes and B-cell \n313 activation pathways rather than a shared humoral response. In this framework, anti-PtpA \n314 responses may reflect exposure-related or host–pathogen interactions, whereas anti-PtpB \n315 immunity appears more closely linked to clinically relevant inflammatory and disease-\n316 activity pathways.\n317 The strong diagnostic performance of the combined anti-PtpA/anti-PtpB logistic \n318 regression model further supports this complementary behavior. The model achieved \n319 excellent discriminative accuracy (AUC = 0.934), a level conventionally interpreted as \n320 indicative of high diagnostic utility [34]. Although pairwise ROC comparisons did not \n321 demonstrate statistically significant superiority over individual antibodies, the combined \n322 model consistently yielded numerically higher performance metrics. At the Youden-\n323 optimized cut-off, the model achieved very high sensitivity (96%) and negative predictive \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.08.698458doi: bioRxiv preprint \n\n15\n324 value (97%), while maintaining favorable specificity (87%) and positive predictive value \n325 (86%). This diagnostic profile is comparable to that reported for multiepitope serological \n326 tools used in early RA [35,36], underscoring the potential of MAP-derived antigens as \n327 clinically meaningful complementary biomarkers rather than standalone diagnostic \n328 replacements.\n329 Accordingly, PtpA/PtpB serology is not intended to replace established RA \n330 biomarkers such as RF or anti-citrullinated protein antibodies, which remain central to the \n331 ACR/EULAR classification criteria. Instead, these MAP-derived immune markers may \n332 provide orthogonal information related to disease biology and immune activation. \n333 Significantly, future studies should extend the evaluation of these markers to other \n334 autoimmune and inflammatory diseases to determine their disease specificity. \n335 MAP has been implicated in autoimmune diseases through mechanisms of molecular \n336 mimicry, including shared epitopes between MAP Hsp65 and human GAD65 [37]. Although \n337 this study did not evaluate epitope overlap, cross-reactive immunity may contribute to the \n338 amplification of adaptive responses, including the formation of RA-associated autoantibodies \n339 [38].\n340 While this study was not designed to investigate transmission routes, previous work \n341 has demonstrated that MAP can be acquired through consumption of unpasteurized dairy \n342 products or contact with infected livestock, both of which have been associated with \n343 increased MAP positivity in humans. Given that MAP is shed in milk, feces, and aerosols \n344 from infected ruminants, these findings underscore the importance of zoonotic and foodborne \n345 exposure pathways in interpreting MAP-derived immune responses [39,40]. Such reservoirs \n346 may be particularly relevant in regions where traditional dairy practices persist or where rural \n347 populations have greater exposure to livestock. Future studies should therefore incorporate \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.08.698458doi: bioRxiv preprint \n\n16\n348 detailed exposure histories. In addition, the influence of long-term immunosuppressive \n349 therapies should be carefully considered, as disease-modifying antirheumatic drugs may alter \n350 host immune surveillance.\n351 Some limitations should be considered when interpreting these findings. The cross-\n352 sectional design does not allow causal relationships to be established, and direct detection of \n353 MAP in tissues was beyond the scope of the present study. Although age differences were \n354 observed between groups, age did not correlate with antibody titers, suggesting that \n355 cumulative environmental exposure is unlikely to account for the observed MAP-reactive \n356 immune responses. Together, these data provide a strong rationale for future longitudinal and \n357 mechanistic studies to define further the pathogenic relevance of MAP-derived immune \n358 signatures in RA and their potential utility for patient stratification.\n359 Conclusions\n360 This study demonstrates that anti-PtpB antibodies are elevated in RA, are associated \n361 with disease activity and functional impairment, and provide clinically relevant information \n362 that complements anti-PtpA responses. While anti-PtpA remains the strongest individual \n363 discriminator between RA patients and healthy controls, anti-PtpB antibodies appear to \n364 capture a distinct immunological dimension linked to inflammatory burden and disease \n365 severity. Accordingly, the combined assessment of both antibodies achieves excellent overall \n366 diagnostic performance, reflecting their complementary and non-redundant biological roles.\n367 Importantly, anti-PtpB antibody levels were independent of sex and conventional \n368 antirheumatic treatments, supporting the notion that MAP-related immune responses are not \n369 merely secondary to therapy or demographic factors. Together, these findings reinforce the \n370 hypothesis that exposure to MAP may represent a relevant environmental component in RA \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.08.698458doi: bioRxiv preprint \n\n17\n371 immunopathogenesis, with different MAP-derived antigens contributing heterogeneously to \n372 disease expression.\n373 Further studies incorporating longitudinal sampling, tissue-level MAP detection, \n374 treatment stratification, and host genetic factors are warranted to clarify the mechanistic and \n375 clinical significance of MAP-related immunity in RA. Such efforts will also be essential for \n376 determining the specificity and broader relevance of combined anti-PtpA/anti-PtpB profiling \n377 in other autoimmune and rheumatic diseases in which MAP has been proposed as a potential \n378 environmental trigger.\n379\n380 Acknowledgments\n381 The authors would like to thank the patients who participated in this study. \n382\n383 Funding source\n384 The Universidad de Guadalajara supported the work performed in México through the \n385 Programa de Fortalecimiento de Institutos, Centros y Laboratorios de Investigación 2025. \n386 The work performed in Canada was supported by the Antibody Engineering and Proteomics \n387 Facility, Immunity and Infection Research Centre, Vancouver, Canada. The Universidad \n388 Politécnica del Centro, Tabasco, México supported LAB. \n389\n390 Author contributions\n391 JHB: Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – \n392 original draft, Writing – review and editing. HB: Conceptualization, Data curation, Formal \n393 analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.08.698458doi: bioRxiv preprint \n\n18\n394 Writing – review and editing. SCC: Formal analysis, Investigation, Validation, Writing – \n395 original draft, Writing – review and editing. GSZ: Formal analysis, Investigation, \n396 Methodology, Validation, Visualization, Writing – review and editing. FN: Formal analysis, \n397 Investigation, Methodology, Validation, Visualization, Writing – review & editing. FMV: \n398 Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, \n399 Visualization, Writing – original draft, Writing – review and editing.\n400\n401 Declaration of competing interest\n402 The authors declare that they have no competing interests.\n403\n404 Data availability\n405 Data will be made available upon reasonable request.\n406\n407 Supporting information\n408 S1 Data. (XLSX)\n409\n410 References \n411 1. Li J, Kuhn KA. Microbial threads in the tapestry of rheumatoid arthritis. J Clin Invest. \n412 2025;135: e195374. doi:10.1172/JCI195374\n413 2. Masoumi M, Solaymani M, Abbasifard M, Houshmandfar S, Iravani P, Saeedi-\n414 Boroujeni A, et al. The genetic puzzle of rheumatoid arthritis: Causes, progression, \n415 and treatment. Biochem Biophys Rep. 2025;43: 102148. \n416 doi:10.1016/j.bbrep.2025.102148\n417 3. 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Specific \n538 immunoassays confirm association of Mycobacterium avium Subsp. paratuberculosis \n539 with type-1 but not type-2 diabetes mellitus. PLoS One. 2009;4: e4386. \n540 doi:10.1371/journal.pone.0004386\n541 39. Falkinham JO. Mycobacterial aerosols and respiratory disease. Emerg Infect Dis. \n542 2003;9: 763–767. doi:10.3201/eid0907.020415\n543\n544 Table 1. Clinical and demographic features of RA patients and control subjects (CS).\nVariable RA (n = 100) Controls (n = 100) P-value\nAge, years (median, IQR) 54 (41–61) 40 (31–53) <0.0001\nFemale sex, n (%) 81 (81%) 80 (80%) >0.90\nHAQ score (0–3) 0.75 (0.25–1.25) – –\nDAS-28 score 3.2 (2.6–5.1) – –\nC-reactive protein (CRP), mg/dL 6 (2–12) – –\nErythrocyte sedimentation rate (ESR), mm/h 20 (12–44) – –\nWhite blood cells (WBC), ×10⁹/L 7.0 (5.8–8.5) – –\nRed blood cells (RBC), ×10¹²/L 4.5 (4.1–4.9) – –\nHemoglobin (Hb), g/dL 13.4 (12.1–14.8) – –\nMean corpuscular volume (MCV), fL 31.0 (29.0–33.0) – –\nPlatelets (PLT), ×10⁹/L 260 (210–320) – –\nRheumatoid factor (RF), IU/mL 45 (20–118) – –\nBody weight, kg 65 (56–74) – –\nHeight, cm 158 (152–165) – –\nBody mass index (BMI), kg/m² 27.5 (24.0–31.5) 26.3 (23.6–32 >0.90\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.08.698458doi: bioRxiv preprint \n\n23\nDisease evolution time, years 8 (2–20) – –\nCurrent steroid therapy, n (%) 1 (1%) – –\nDMARDs, n (%) 73 (73%) – –\nNSAIDs, n (%) 36 (36%) – –\n545\n546 Data are expressed as median (interquartile range, IQR) for continuous variables and as absolute numbers with \n547 percentages for categorical variables. Age was compared using the Mann–Whitney U test, and sex distribution \n548 was analyzed using the chi-square test. A p < 0.05 was considered statistically significant. Abbreviations: RA, \n549 rheumatoid arthritis; CS, control subjects; HAQ, Health Assessment Questionnaire; DAS-28, Disease Activity \n550 Score 28; DMARDs, disease-modifying antirheumatic drugs; NSAIDs, nonsteroidal anti-inflammatory drugs.\n551\n552 Table 2. Diagnostic performance of the logistic regression model combining anti-PtpA and \n553 anti-PtpB antibody levels.  \nMetric Value\nArea under the curve (AUC) 0.934\nOptimal cut-off point (Youden index) 0.3869\nSensitivity 0.9638\nSpecificity 0.87\nPositive predictive value (PPV) 0.8602\nNegative predictive value (NPV) 0.9666\n554\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.08.698458doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.08.698458doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.08.698458doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.08.698458doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.08.698458doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted January 8, 2026. ; https://doi.org/10.64898/2026.01.08.698458doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}