SARS-CoV-2 Vaccine-Elicited Immunity after B Cell Depletion in Multiple Sclerosis.

The impact of B cell deficiency on the humoral and cellular responses to SARS-CoV2 mRNA vaccination remains a challenging and significant clinical management question. We evaluated vaccine-elicited serological and cellular responses in 1) healthy individuals who were pre-exposed to SARS-CoV-2 (n = 21), 2) healthy individuals who received a homologous booster (mRNA, n = 19; or Novavax, n = 19), and 3) persons with multiple sclerosis on B cell depletion therapy (MS-αCD20) receiving mRNA homologous boosting (n = 36). Pre-exposure increased humoral and CD4 T cellular responses in immunocompetent individuals. Novavax homologous boosting induced a significantly more robust serological response than mRNA boosting. MS-α CD20 had an intact IgA mucosal response and an enhanced CD8 T cell response to mRNA boosting compared with immunocompetent individuals. This enhanced cellular response was characterized by the expansion of only effector, not memory, T cells. The enhancement of CD8 T cells in the setting of B cell depletion suggests a regulatory mechanism between B and CD8 T cell vaccine responses.

A partial human LCK defect causes a T cell immunodeficiency with intestinal inflammation.

Lymphocyte-specific protein tyrosine kinase (LCK) is essential for T cell antigen receptor (TCR)-mediated signal transduction. Here, we report two siblings homozygous for a novel LCK variant (c.1318C>T; P440S) characterized by T cell lymphopenia with skewed memory phenotype, infant-onset recurrent infections, failure to thrive, and protracted diarrhea. The patients' T cells show residual TCR signal transduction and proliferation following anti-CD3/CD28 and phytohemagglutinin (PHA) stimulation. We demonstrate in mouse models that complete (Lck-/-) versus partial (LckP440S/P440S) loss-of-function LCK causes disease with differing phenotypes. While both Lck-/- and LckP440S/P440S mice exhibit arrested thymic T cell development and profound T cell lymphopenia, only LckP440S/P440S mice show residual T cell proliferation, cytokine production, and intestinal inflammation. Furthermore, the intestinal disease in the LckP440S/P440S mice is prevented by CD4+ T cell depletion or regulatory T cell transfer. These findings demonstrate that P440S LCK spares sufficient T cell function to allow the maturation of some conventional T cells but not regulatory T cells-leading to intestinal inflammation.

Innate and Adaptive Cell-Mediated Immune Responses to a COVID-19 mRNA Vaccine in Young Children.

Background: There is little information on cell-mediated immunity (CMI) to COVID-19 mRNA vaccines in children. We studied adaptive and innate CMI in vaccinated children aged 6 to 60 months. Methods: Blood obtained from participants in a randomized placebo-controlled trial of an mRNA vaccine before and 1 month after the first dose was used for antibody measurements and CMI (flow cytometry). Results: We enrolled 29 children with a mean age of 28.5 months (SD, 15.7). Antibody studies revealed that 10 participants were infected with SARS-CoV-2 prevaccination. Ex vivo stimulation of peripheral blood mononuclear cells with SARS-CoV-2 spike peptides showed significant increases pre- to postimmunization of activated conventional CD4+ and γδ T cells, natural killer cells, monocytes, and conventional dendritic cells but not mucosa-associated innate T cells. Conventional T-cell, monocyte, and conventional dendritic cell responses in children were higher immediately after vaccination than after SARS-CoV-2 infection. The fold increase in CMI pre- to postvaccination did not differ between children previously infected with SARS-CoV-2 and those uninfected. Conclusions: Children aged 6 to 60 months who were vaccinated with a COVID-19 mRNA vaccine developed robust CMI responses, including adaptive and innate immunity.

Expansion of extrafollicular B and T cell subsets in childhood-onset systemic lupus erythematosus.

Introduction: Most childhood-onset SLE patients (cSLE) develop lupus nephritis (cLN), but only a small proportion achieve complete response to current therapies. The prognosis of children with LN and end-stage renal disease is particularly dire. Mortality rates within the first five years of renal replacement therapy may reach 22%. Thus, there is urgent need to decipher and target immune mechanisms that drive cLN. Despite the clear role of autoantibody production in SLE, targeted B cell therapies such as rituximab (anti-CD20) and belimumab (anti-BAFF) have shown only modest efficacy in cLN. While many studies have linked dysregulation of germinal center formation to SLE pathogenesis, other work supports a role for extrafollicular B cell activation in generation of pathogenic antibody secreting cells. However, whether extrafollicular B cell subsets and their T cell collaborators play a role in specific organ involvement in cLN and/or track with disease activity remains unknown. Methods: We analyzed high-dimensional mass cytometry and gene expression data from 24 treatment naïve cSLE patients at the time of diagnosis and longitudinally, applying novel computational tools to identify abnormalities associated with clinical manifestations (cLN) and disease activity (SLEDAI). Results: cSLE patients have an extrafollicular B cell expansion signature, with increased frequency of i) DN2, ii) Bnd2, iii) plasmablasts, and iv) peripheral T helper cells. Most importantly, we discovered that this extrafollicular signature correlates with disease activity in cLN, supporting extrafollicular T/B interactions as a mechanism underlying pediatric renal pathogenesis. Discussion: This study integrates established and emerging themes of extrafollicular B cell involvement in SLE by providing evidence for extrafollicular B and peripheral T helper cell expansion, along with elevated type 1 IFN activation, in a homogeneous cohort of treatment-naïve cSLE patients, a point at which they should display the most extreme state of their immune dysregulation.

Dysregulated Lymphocyte Antigen Receptor Signaling in Common Variable Immunodeficiency with Granulomatous Lymphocytic Interstitial Lung Disease.

PURPOSE: A subset of common variable immunodeficiency (CVID) patients either presents with or develops autoimmune and lymphoproliferative complications, such as granulomatous lymphocytic interstitial lung disease (GLILD), a major cause of morbidity and mortality in CVID. While a myriad of phenotypic lymphocyte derangements has been associated with and described in GLILD, defects in T and B cell antigen receptor (TCR/BCR) signaling in CVID and CVID with GLILD (CVID/GLILD) remain undefined, hindering discovery of biomarkers for disease monitoring, prognostic prediction, and personalized medicine approaches. METHODS: To identify perturbations of immune cell subsets and TCR/BCR signal transduction, we applied mass cytometry analysis to peripheral blood mononuclear cells (PBMCs) from healthy control participants (HC), CVID, and CVID/GLILD patients. RESULTS: Patients with CVID, regardless of GLILD status, had increased frequency of HLADR+CD4+ T cells, CD57+CD8+ T cells, and CD21lo B cells when compared to healthy controls. Within these cellular populations in CVID/GLILD patients only, engagement of T or B cell antigen receptors resulted in discordant downstream signaling responses compared to CVID. In CVID/GLILD patients, CD21lo B cells showed perturbed BCR-mediated phospholipase C gamma and extracellular signal-regulated kinase activation, while HLADR+CD4+ T cells and CD57+CD8+ T cells displayed disrupted TCR-mediated activation of kinases most proximal to the receptor. CONCLUSION: Both CVID and CVID/GLILD patients demonstrate an activated T and B cell phenotype compared to HC. However, only CVID/GLILD patients exhibit altered TCR/BCR signaling in the activated lymphocyte subsets. These findings contribute to our understanding of the mechanisms of immune dysregulation in CVID with GLILD.

Peripheral immunophenotyping of AITD subjects reveals alterations in immune cells in pediatric vs adult-onset AITD.

Autoimmune thyroid disease (AITD) is caused by aberrant activation of the immune system allowing autoreactive B and T cells to target the thyroid gland leading to disease. Although AITD is more frequently diagnosed in adults, children are also affected but rarely studied. Here, we performed phenotypic and functional characterization of peripheral blood immune cells from pediatric and adult-onset AITD patients and age-matched controls using mass cytometry. Major findings indicate that unlike adult-onset AITD patients, pediatric AITD patients exhibit a decrease in anergic B cells (BND) and DN2 B cells and an increase in immature B cells compared to age-matched controls. These results indicate alterations in peripheral blood immune cells seen in pediatric-onset AITD could lead to rapid progression of disease. Hence, this study demonstrates diversity of AITD by showing differences in immune cell phenotypes and function based on age of onset, and may inform future therapies.

T and B cell abnormalities, pneumocystis pneumonia, and chronic lymphocytic leukemia associated with an AIOLOS defect in patients.

AIOLOS/IKZF3 is a member of the IKAROS family of transcription factors. IKAROS/IKZF1 mutations have been previously associated with different forms of primary immunodeficiency. Here we describe a novel combined immunodeficiency due to an IKZF3 mutation in a family presenting with T and B cell involvement, Pneumocystis jirovecii pneumonia, and/or chronic lymphocytic leukemia. Patients carrying the AIOLOS p.N160S heterozygous variant displayed impaired humoral responses, abnormal B cell development (high percentage of CD21low B cells and negative CD23 expression), and abrogated CD40 responses. Naive T cells were increased, T cell differentiation was abnormal, and CD40L expression was dysregulated. In vitro studies demonstrated that the mutant protein failed DNA binding and pericentromeric targeting. The mutant was fully penetrant and had a dominant-negative effect over WT AIOLOS but not WT IKAROS. The human immunophenotype was recapitulated in a murine model carrying the corresponding human mutation. As demonstrated here, AIOLOS plays a key role in T and B cell development in humans, and the particular gene variant described is strongly associated with immunodeficiency and likely malignancy.

Minimizing Batch Effects in Mass Cytometry Data.

Cytometry by Time-Of-Flight (CyTOF) uses antibodies conjugated to isotopically pure metals to identify and quantify a large number of cellular features with single-cell resolution. A barcoding approach allows for 20 unique samples to be pooled and processed together in one tube, reducing the intra-barcode technical variability. However, with only 20 samples per barcode, multiple barcode sets (batches) are required to address questions in robustly powered study designs. A batch adjustment procedure is required to reduce variability across batches and to facilitate direct comparison of runs performed across multiple barcodes run over weeks, months, or years. We describe a method using technical replicates that are included in each run to determine and apply an appropriate adjustment per batch without manual intervention. The use of technical replicate samples (i.e., anchors or reference samples) avoids assumptions of sample homogeneity among batches, and allows direct estimation of batch effects and appropriate adjustment parameters applicable to all samples within a batch. Quantification of cell subpopulations and mean signal intensity pre- and post-adjustment using both manual gating and unsupervised clustering demonstrate substantial mitigation of batch effects in the anchor samples used for this adjustment calculation, and in a second validation set of technical replicates.

CTLA4 Message Reflects Pathway Disruption in Monogenic Disorders and Under Therapeutic Blockade.

CTLA-4 is essential for immune tolerance. Heterozygous CTLA4 mutations cause immune dysregulation evident in defective regulatory T cells with low levels of CTLA-4 expression. Biallelic mutations in LRBA also result in immune dysregulation with low levels of CTLA-4 and clinical presentation indistinguishable from CTLA-4 haploinsufficiency. CTLA-4 has become an immunotherapy target whereby its blockade with a monoclonal antibody has resulted in improved survival in advanced melanoma patients, amongst other malignancies. However, this therapeutic manipulation can result in autoimmune/inflammatory complications reminiscent of those seen in genetic defects affecting the CTLA-4 pathway. Despite efforts made to understand and establish disease genotype/phenotype correlations in CTLA-4-haploinsufficiency and LRBA-deficiency, such relationships remain elusive. There is currently no specific immunological marker to assess the degree of CTLA-4 pathway disruption or its relationship with clinical manifestations. Here we compare three different patient groups with disturbances in the CTLA-4 pathway-CTLA-4-haploinsufficiency, LRBA-deficiency, and ipilimumab-treated melanoma patients. Assessment of CTLA4 mRNA expression in these patient groups demonstrated an inverse correlation between the CTLA4 message and degree of CTLA-4 pathway disruption. CTLA4 mRNA levels from melanoma patients under therapeutic CTLA-4 blockade (ipilimumab) were increased compared to patients with either CTLA4 or LRBA mutations that were clinically stable with abatacept treatment. In summary, we show that increased CTLA4 mRNA levels correlate with the degree of CTLA-4 pathway disruption, suggesting that CTLA4 mRNA levels may be a quantifiable surrogate for altered CTLA-4 expression.

A novel human IL2RB mutation results in T and NK cell-driven immune dysregulation.

The pleiotropic actions of interleukin-2 (IL-2) are essential for regulation of immune responses and maintenance of immune tolerance. The IL-2 receptor (IL-2R) is composed of IL-2Rα, IL-2Rß, and IL-2Rγ subunits, with defects in IL-2Rα and IL-2Rγ and their downstream signaling effectors resulting in known primary immunodeficiency disorders. Here, we report the first human defect in IL-2Rß, occurring in two infant siblings with a homozygous IL2RB mutation in the WSXWS motif, manifesting as multisystem autoimmunity and susceptibility to CMV infection. The hypomorphic mutation results in diminished IL-2Rß surface expression and dysregulated IL-2/15 signaling, with an anticipated reduction in regulatory T cells. However, in contrast to the IL-2Rß-/- animal model, which lacks NK cells, these siblings demonstrate an expansion of NK cells, particularly the CD56bright subset, and a lack of terminally differentiated NK cells. Thus, the early-onset autoimmunity and immunodeficiency are linked to functional deficits arising from altered IL-2Rß expression and signaling in T and NK cells.

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry.

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies in an attempt to understand the precise mechanisms that lead to the breakdown of self-tolerance and subsequent autoimmunity. Approaches thus far have been based on the study of one specific aspect of the immune system (a single or few cell types or cytokines), and do not offer a global assessment of complex autoimmune disease. While patient sera-based studies have afforded important insights into autoimmunity, they do not provide the specific cellular source of the dysregulated cytokines detected. A comprehensive single-cell approach to evaluate cytokine production in multiple immune cell subsets, within the context of "intrinsic" patient-specific plasma circulating factors, is described here. This approach enables monitoring of the patient-specific immune phenotype (surface markers) and function (cytokines), either in its native "intrinsic pathogenic" disease state, or in the presence of therapeutic agents (in vivo or ex vivo).

The Chlamydia trachomatis Inclusion Membrane Protein CpoS Counteracts STING-Mediated Cellular Surveillance and Suicide Programs.

Evading cell death is critical for Chlamydia to maintain a replicative niche, but the underlying mechanisms are unknown. We screened a library of Chlamydia mutants for modulators of cell death. Inactivation of the inclusion membrane protein CpoS (Chlamydia promoter of survival) induced rapid apoptotic and necrotic death in infected cells. The protection afforded by CpoS is limited to the inclusion in which it resides, indicating that it counteracts a spatially restricted pro-death signal. CpoS-deficient Chlamydia induced an exacerbated type I interferon response that required the host cGAS/STING/TBK1/IRF3 signaling pathway. Disruption of STING, but not cGAS or IRF3, attenuated cell death, suggesting that STING mediates Chlamydia-induced cell death independent of its role in regulating interferon responses. CpoS-deficient strains are attenuated in their ability to propagate in cell culture and are cleared faster from the murine genital tract, highlighting the importance of CpoS for Chlamydia pathogenesis.

Severe impingement of lumbar disc replacements increases the functional biological activity of polyethylene wear debris.

BACKGROUND: Wear, oxidation, and particularly rim impingement damage of ultra-high molecular weight polyethylene total disc replacement components have been observed following surgical revision. However, neither in vitro testing nor retrieval-based evidence has shown the effect(s) of impingement on the characteristics of polyethylene wear debris. Thus, we sought to determine (1) differences in polyethylene particle size, shape, number, or biological activity that correspond to mild or severe rim impingement and (2) in an analysis of all total disc replacements, regardless of impingement classification, whether there are correlations between the extent of regional damage and the characteristics of polyethylene wear debris. METHODS: The extent of dome and rim damage was characterized for eleven retrieved polyethylene cores obtained at revision surgery after an average duration of implantation of 9.7 years (range, 4.6 to 16.1 years). Polyethylene wear debris was isolated from periprosthetic tissues with use of nitric acid and was imaged with use of environmental scanning electron microscopy. Subsequently, particle size, shape, number, biological activity, and chronic inflammation scores were determined. RESULTS: Grouping of particles by size ranges that represented high biological relevance (<0.1 to 1-µm particles), intermediate biological relevance (1 to 10-µm particles), and low biological relevance (>10-µm particles) revealed an increased volume fraction of particles in the <0.1 to 1-µm and 1 to 10-µm size ranges in the mild-impingement cohort as compared with the severe-impingement cohort. The increased volume fractions resulted in a higher specific biological activity per unit particle volume in the mild-impingement cohort than in the severe-impingement cohort. However, functional biological activity, which is normalized by particle volume (mm3/g of tissue), was significantly higher in the severe-impingement cohort. This increase was due to a larger volume of particles in all three size ranges. In both cohorts, the functional biological activity correlated with the chronic inflammatory response, and the extent of rim penetration positively correlated with increasing particle size, number, and functional biological activity. CONCLUSIONS: The results of this study suggest that severe rim impingement increases the production of biologically relevant particles from motion-preserving lumbar total disc replacement components. LEVEL OF EVIDENCE: Prognostic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Characterization of wear debris in total elbow arthroplasty.

BACKGROUND: The purpose of this study was to evaluate wear debris in periprosthetic tissues at the time of revision total elbow arthroplasty. Polyethylene, metallic, and bone cement debris were characterized, and the tissue response was quantified. MATERIALS AND METHODS: Capsular and medullary tissue samples were collected during revision surgery. Polyethylene debris was characterized by scanning electron microscopy after tissue digestion. The concentrations of metal and cement debris were quantified by inductively coupled plasma mass spectrometry. Tissue response was graded with a semiquantitative histologic method. RESULTS: Polyethylene particle size varied from the submicron range to over 100 µm. The mean diameter ranged from 0.6 µm to about 1 µm. Particles in the synovial tissues were larger and less abundant than those in tissues from the medullary canal. Cement, titanium alloy, and low levels of cobalt-chrome debris were also present, with cement predominating over metal debris. Histiocyte response was associated with small polyethylene particles (0.5-2 µm), and giant cells were associated with large polyethylene particles (>2 µm). Histiocyte scores positively correlated with the polyethylene particle number and the presence of metal. DISCUSSION: We have shown that periprosthetic tissues of total elbow patients who have undergone revision for loosening and osteolysis contain polyethylene, cement, and metal debris. Although the polyethylene particles were of a size and shape that have been previously shown to result in activation of phagocytic cells, osteolysis after total elbow arthroplasty is a multimodal process. Because of the presence of multiple wear particle sources, a cause-and-effect relationship between polyethylene debris and osteolysis cannot be established with certainty.

Characteristics of highly cross-linked polyethylene wear debris in vivo.

Despite the widespread implementation of highly cross-linked polyethylene (HXLPE) liners to reduce the clinical incidence of osteolysis, it is not known if the improved wear resistance will outweigh the inflammatory potential of HXLPE wear debris generated in vivo. Thus, we asked: What are the differences in size, shape, number, and biological activity of polyethylene wear particles obtained from primary total hip arthroplasty revision surgery of conventional polyethylene (CPE) versus remelted or annealed HXLPE liners? Pseudocapsular tissue samples were collected from revision surgery of CPE and HXLPE (annealed and remelted) liners, and digested using nitric acid. The isolated polyethylene wear particles were evaluated using scanning electron microscopy. Tissues from both HXLPE cohorts contained an increased percentage of submicron particles compared to the CPE cohort. However, the total number of particles was lower for both HXLPE cohorts, as a result there was no significant difference in the volume fraction distribution and specific biological activity (SBA; the relative biological activity per unit volume) between cohorts. In contrast, based on the decreased size and number of HXLPE wear debris there was a significant decrease in total particle volume (mm(3)/g of tissue). Accordingly, when the SBA was normalized by total particle volume (mm(3)/gm tissue) or by component wear volume rate (mm(3)/year), functional biological activity of the HXLPE wear debris was significantly decreased compared to the CPE cohort. Indications for this study are that the osteolytic potential of wear debris generated by HXLPE liners in vivo is significantly reduced by improvements in polyethylene wear resistance.

Do tissues from THA revision of highly crosslinked UHMWPE liners contain wear debris and associated inflammation?

BACKGROUND: Polyethylene wear debris is a major contributor to inflammation and the development of implant loosening, a leading cause of THA revisions. To reduce wear debris, highly crosslinked ultrahigh-molecular-weight polyethylene (UHMWPE) was introduced to improve wear properties of bearing surfaces. As highly crosslinked UHMWPE revision tissues are only now becoming available, it is possible to examine the presence and association of wear debris with inflammation in early implant loosening. QUESTIONS/PURPOSES: We asked: (1) Does the presence of UHMWPE wear debris in THA revision tissues correlate with innate and/or adaptive immune cell numbers? (2) Does the immune cell response differ between conventional and highly crosslinked UHMWPE cohorts? METHODS: We collected tissue samples from revision surgery of nine conventional and nine highly crosslinked UHMWPE liners. Polarized light microscopy was used to determine 0.5- to 2-µm UHMWPE particle number/mm2, and immunohistochemistry was performed to determine macrophage, T cell, and neutrophil number/mm2. RESULTS: For the conventional cohort, correlations were observed between wear debris and the magnitude of individual patient macrophage (ρ=0.70) and T cell responses (ρ=0.71) and between numbers of macrophages and T cells (ρ=0.77) in periprosthetic tissues. In comparison, the highly crosslinked UHMWPE cohort showed a correlation between wear debris and the magnitude of macrophage responses (ρ=0.57) and between macrophage and T cell numbers (ρ=0.68). Although macrophages and T cells were present in both cohorts, the highly crosslinked UHMWPE cohort had lower numbers, which may be associated with shorter implantation times. CONCLUSIONS: The presence of wear debris and inflammation in highly crosslinked UHMWPE revision tissues may contribute to early implant loosening.

Distinct immunohistomorphologic changes in periprosthetic hip tissues from historical and highly crosslinked UHMWPE implant retrievals.

Assessment of immune response to implant wear debris in periprosthetic tissue following total hip arthroplasty suggests that multiple factors are involved in the loss implant function. The current study investigated wear debris and the associated immunohistomorphologic changes in tissues from nine patients with historical (gamma air-sterilized) and nine highly crosslinked UHMWPE implant components. Paraffin embedded tissue sections were evaluated for the presence of histiocytes, giant cells, fibrocartilage/bone, and necrosis. To determine the incidence, degree and co-localization of immunohistomorphologic changes and wear, overlapping full-field tissue arrays were collected in brightfield and polarized light. The historical cohort tissues predominantly showed histiocytes associated with significant accumulations of small wear (0.5-2 microm), and giant cells associated with large wear (> or =2 microm). Frequently, focal regions of necrosis were observed in association with wear debris. For the highly crosslinked cohort, inflammation and associated wear debris were limited, but in tissues from patients revised after implantation times of >2 years a response was observed. Whereas significant amounts of fibrocartilage/bone were observed in patients at earlier implantation times. In both cohorts, tissue responses were more extensive in the retroacetabular or proximal femoral regions. The current findings suggest that wear debris-induced inflammation may be a major contributor to the loss of implant function for both the historical and highly crosslinked cohorts, but it is not the primary cause of early implant loosening. This study highlights the importance of using a more quantitative and standardized assessment of immunohistomorphologic responses in periprosthetic tissues, and emphasizes differences in specific anatomical regions of individual patient tissues.

Comparison of periprosthetic tissue digestion methods for ultra-high molecular weight polyethylene wear debris extraction.

There is considerable interest in characterization of wear debris from polyethylene (UHMWPE) bearing components used in total joint replacement. To isolate UHMWPE wear debris, tissue samples must be excised from regions adjacent to revised UHMWPE implant components, followed by exposure to one of many available tissue digestion methods. Numerous studies demonstrate successful digestion, but the relative efficiency of each method is not clear. The purpose of this study was to evaluate a variety of conditions for tissue digestion to provide a quantitative comparison of methods. Porcine and human hip tissues were exposed for 24 h to basic, acidic or enzymatic agents, filtered and digestion efficiency calculated based on the percentage of initial to final tissue weight. Of the conditions tested, 5 M NaOH, 5 M KOH, 15 M KOH or 15.8 M HNO(3) yielded the most complete porcine hip tissue digestion (<1% residual tissue weight; p < 0.05). Proteinase K and Liberase Blendzyme 3 did not effectively digest tissue in a 24 h period. Similar to results from the porcine dataset, human tissues digestion was most efficient using 5 M NaOH, 5 M KOH or 15.8 M HNO(3) (<1% residual tissue weight; p < 0.05). To verify that particle surface modifications did not occur after prolonged reagent exposure, GUR415 and Ceridust 3715 particles were immersed in each solution for 24 h. Overall, this study provides a framework for thorough and efficient digestive methods for UHMWPE wear debris extraction.