Perfluorooctane sulfonate (PFOS) poses significant health and environmental risks due to its persistence and widespread use and has been linked to various adverse outcomes, such as liver toxicity. Although the molecular responses and toxicity effects of PFOS exposure have been extensively studied, considerable uncertainty remains regarding the causal mechanisms leading to PFOS-associated adverse effects. To help bridge this gap, we conducted CRISPR screens in HepG2/C3A human liver cells exposed to IC25 (170 µM) of PFOS to identify genes and pathways influencing PFOS-induced cytotoxicity. Using a genome-wide CRISPR knockout library targeting 18,819 genes, we identified 340 candidate genes that modulate PFOS-induced cytotoxicity when genetically disrupted (189 gene disruptions increased sensitivity and 151 gene disruptions increased resistance). From these candidate genes, we individually disrupted two candidate genes, SLC6A9 which encodes the glycine transporter GlyT1, and CPSF2, and confirmed increased resistance to PFOS exposure. Further, molecular docking analysis predicts that PFOS directly binds to GlyT1 and functional inhibition of GlyT1 also increases resistance to PFOS exposure. Gene-Disease outcome association analysis using the Comparative Toxicogenomics Database (CTD) indicated an enrichment of candidate genes associated with cancer-related and liver disease phenotypes. KEGG and STRING enrichment analyses found over representation of several biological pathways including DNA damage response and cell cycle. Lastly, cross-species conservation analysis using the top two validated gene targets found that their pathways were highly conserved in several environmentally relevant species. These findings provide new mechanistic and functional insights into PFOS-induced cytotoxicity, highlight potential molecular targets for toxicity mitigation, and establish a foundation for cross-species toxicogenomic modeling of PFOS health effects.

F. tularensis is a highly infectious Gram-negative bacterial pathogen that causes tularemia, a re-emerging zoonosis of public health concern. Here we identify respiratory complex I as a selective vulnerability in Francisella and define the mechanism of action of a pyrazole compound, tolfenpyrad, with species-specific antibacterial activity. Using F. novicida as a surrogate model, we demonstrated that tolfenpyrad selectively inhibits growth with no measurable effect on E. coli or P. aeruginosa . Tolfenpyrad rapidly suppressed oxygen consumption, depleted ATP, collapsed proton motive force, and induced reactive oxygen species, indicating disruption of bacterial metabolism. Biochemical assays demonstrated selective inhibition of NADH-dependent respiration and membrane-associated NADH oxidation, whereas succinate-driven respiration was unaffected. Moreover, the alternative NADH dehydrogenase ( ndh ) was not required for tolfenpyrad activity. Structural docking identified a potential tolfenpyrad-binding pocket within the membrane subunit NuoM. These findings reveal species-specific inhibition of Francisella complex I and establish respiratory metabolism as a promising antimicrobial target in these bacteria.

Melanoma differentiation-associated protein 5 (MDA5), encoded by IFIH1, is a cytosolic double-stranded RNA (dsRNA) sensor. Mutation of IFIH1 resulting in MDA5 deficiency causes immune dysfunction and predisposition to specific respiratory viral pathogens due to the inability of innate immune system to detect viral dsRNA. Additionally, gene variants in IFIH1 have been linked to autoimmunity, including type 1 diabetes. To understand structure-function, we integrate structural biology and signaling principles to explain how MDA5 architecture governs interactions with dsRNA and type 1 interferon (T1-IFN) outputs. MDA5 binds dsRNA via its helicase core and C-terminal domain, uses ATP-dependent conformational cycling to assemble filaments, and exposes N-terminal CARDs that nucleate mitochondrial antiviral signaling protein polymerization, activating TBK1/IKKε-IRF and NF-κB programs that amplify T1-IFN production and inflammatory gene expression. Risk-associated IFIH1 alleles are predicted to increase T1-IFN production/activation thresholds, whereas rare loss-of-function variants attenuate T1-IFN outputs and confer protection. Finally, we outline therapeutic entry points that preserve antiviral defense while constraining chronic T1-IFN signaling to restrain MHC class I expression, chemokine production, and autoreactive T-cell recruitment. Targeting downstream pathways with small molecule inhibitors may delay early autoimmunity and target tissue functions in genetically defined subgroups.

Background: Sjögren's disease (SjD) is a chronic autoimmune disorder characterized by lymphocytic infiltration of exocrine glands, leading to secretory dysfunction. While conventional mouse models have advanced understanding of disease mechanisms, translational limitations exist due to differences in immune system components, particularly MHC molecules. The NOD-DR3 mouse strain, expressing human HLA-DR3, a prominent genetic risk factor for SjD, offers a humanized model that has not been previously characterized for SjD phenotypes. Objective: To comprehensively characterize NOD-DR3 mice as a novel humanized model for SjD, evaluating functional, serological, histological, and immunological disease features with particular focus on sex dimorphism. Methods: Aged NOD-DR3 mice (20-32 weeks) were assessed for salivary flow rates, autoantibody profiles (ANA, anti-Ro52, anti-Ro60, anti-La), glandular focal scores, and immune cell infiltration patterns. Comparisons were made with established SjD models (NOD-Aec1/2, NOD) and healthy B6 controls, with sex-stratified analyses. Results: NOD-DR3 mice exhibited significantly reduced salivary flow rates, comparable to those in established models. Seventy percent were ANA-positive, with pronounced sex-dependent patterns: males showed higher ANA frequency with nucleolar patterns, while females displayed elevated anti-Ro/La antibodies with speckled patterns. Notably, NOD-DR3 demonstrated the most severe lacrimal gland infiltration among all strains. Cellular analysis revealed striking sex dimorphism, with females showing 12-fold higher B cell infiltration and enhanced Th17 responses, while males exhibited elevated macrophage and Th1 profiles. Conclusions: NOD-DR3 mice faithfully recapitulate cardinal SjD features with robust sex dimorphism, establishing this humanized model as a valuable translational platform for investigating HLA-restricted disease mechanisms and evaluating therapeutic interventions.

Background and Objectives: Sjögren’s disease (SjD) is a chronic autoimmune disorder in which the immune system attacks the glands that produce tears and saliva, leading to symptoms such as dry eyes and dry mouth. If left untreated, SjD can also cause inflammation and damage to other parts of the body, including the skin, lungs, kidneys, and nervous system, and increase the risk of developing lymphoma. The human leukocyte antigen (HLA) class II molecule HLA-DR3 is strongly associated with SjD. Materials and Methods: To investigate how post-translational modifications (PTMs) influence the presentation of SjD-associated autoantigens by HLA-DR3, we employed a computational framework to determine the binding of PTM-mimic peptides to HLA-DR3. We further supported the in-silico results with in-vitro experiments. Results: Our analysis revealed that PTM-mimic substitutions at canonical anchor positions rarely improved predicted binding affinity using the Stabilized Matrix Method, with most modifications resulting in reduced affinity. However, a comprehensive analysis of full-length SjD-associated autoantigen sequences (Ro60, Ro52, La) identified discrete regions with high densities of PTM-eligible anchor sites, specifically, the Ro60 HEAT solenoid, Ro52 RING/B-box/PRY-SPRY modules, and the La motif-RRM1 region, suggesting that PTMs may alter epitope presentation in a sequence-dependent manner. Experimental validation of selected PTM-mimic peptides showed enhanced T cell responses, which were associated with increased binding affinity to HLA-DR3. Structural modeling of a representative complex revealed that PTM-mimic peptides adopt a slightly shifted backbone orientation and altered side-chain positioning, leading to a larger peptide–DR3 interaction interface. Conclusions: These findings provide new insights into the role of PTMs in shaping the immunogenicity of SjD-associated autoantigens and highlight the potential for PTM-mimic peptides to modulate T cell responses in SjD.

Importance:Drug reaction with eosinophilia and systemic symptoms (DRESS) is a severe, potentially fatal hypersensitivity syndrome with 3% to 10% mortality, and lamotrigine, a first-line treatment for bipolar and seizure disorders, is among the top 5 causative agents of DRESS in the United States and globally. Commercial panels test HLA-B*15:02 and HLA-A*31:01 for risk of carbamazepine-associated severe cutaneous adverse reactions; however, these are not validated for lamotrigine-induced DRESS, and current reports from these commercial assays could therefore provide false reassurance. No HLA associations with lamotrigine-induced DRESS have been established in US populations. Objective:To identify HLA genetic variants associated with lamotrigine-induced DRESS in a US population. Design, Setting, and Participants:This matched case-control study was conducted at 2 US academic medical centers (Vanderbilt University Medical Center and Mass General Brigham). Patients with lamotrigine-induced DRESS confirmed by Registry of Severe Cutaneous Adverse Reactions (RegiSCAR) criteria (score ≥4) were prospectively enrolled between April 2016 and June 2025. Lamotrigine-tolerant controls were matched 10:1 from the Vanderbilt BioVU biobank on sex, self-reported race, and age. HLA typing included class I (HLA-A, HLA-B, and HLA-C) and class II (HLA-DPB1, HLA-DQA1, HLA-DQB1, and HLA-DRB1) loci for case (high-resolution typing) and control (imputed from genotyping array data) participants. Main Outcomes and Measures:HLA class I and II alleles and haplotypes were tested using logistic regression with Bonferroni correction for their association with lamotrigine-induced DRESS. Results:This study included 29 patients with lamotrigine-induced DRESS (case participants; median [IQR] age, 33 [24-56] years; 25 female [86.2%]; 2 Asian [6.9%], 2 Black [6.9%], 23 White [79.3%], and 2 unknown race [6.9%]) and 290 matched control participants. HLA-A*32:01 was associated with lamotrigine-induced DRESS (41.4% [12 case participants] vs 4.1% [12 control participants]; odds ratio [OR], 16.4; 95% CI, 6.4-42.5; Bonferroni-corrected P < .001). No other alleles or class II loci showed significant associations after correction. The A*32:01 ~ B*44:02 haplotype was enriched (OR, 18.4; 95% CI, 4.6-83.8; Bonferroni-corrected P = .001) in lamotrigine-induced DRESS. Conclusions and Relevance:In this case-control study, HLA-A*32:01 was associated with lamotrigine-induced DRESS in a US population. Given that HLA-A*32:01 is not included in existing commercial pharmacogenomic panels, adding this marker could improve preprescription DRESS risk identification for lamotrigine.

BACKGROUND: Co-trimoxazole is a leading global cause of severe cutaneous adverse drug reactions (SCAR) including Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/ TEN) and drug reaction with eosinophilia and systemic symptoms (DRESS). Co-trimoxazole-induced SCAR are associated with HLA class I alleles including HLA-B*13:01 and HLA-B*38:02 in Southeast Asian (SEA) populations. However, the global generalizability of these associations is unknown but critical for population-appropriate risk stratification and diagnosis. OBJECTIVE: To determine HLA risk factors associated with co-trimoxazole-induced SJS/TEN and DRESS in populations from the United States and South Africa. METHODS: We performed high-resolution HLA typing on dermatologist-adjudicated co-trimoxazole-induced patients with SCAR in the United States (n = 63) and South Africa (n = 26) compared with population controls. Peptide binding and docking analyses were performed using MHCcluster2.0 and CB-Dock2. RESULTS: In a multiple logistic regression model, HLA-B*44:03 (corrected P [Pc] < .001; odds ratio [OR] = 4.08), HLA-B*38:01 (Pc < .001; OR = 5.66), and HLA-C*04:01 (Pc = .003; OR = 2.50) were independently associated with co-trimoxazole-induced SJS/TEN in the United States. HLA-B*44:03 was also associated with co-trimoxazole-induced DRESS in South Africa (Pc = .019; OR = 10.69). Distinct HLA-B variants with shared peptide binding specificities (SPBS) and HLA-C*04:01 identified 94% and 78% of cotrimoxazole-induced SJS/TEN and DRESS in the United States, respectively. The SEA risk allele HLA-B*13:01, with SPBS to HLA-B*44:03, was identified in just one of 63 US patients with SCAR. CONCLUSIONS: HLA alleles with SPBS to SEA-related risk alleles, including HLA-B*44:03 (SPBS with HLA-B*13:01) and HLA-B*38:01 (SPBS with HLA-B*38:02) but also HLA-C*04:01, predisposed to co-trimoxazole-induced SCAR in the United States and South Africa. These findings provide biological plausibility and strategies for global risk prediction and diagnosis of co-trimoxazole-induced SCAR. (c) 2025 The Authors. Published by Elsevier Inc. on behalf of the American Academy of Allergy, Asthma & Immunology. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). (J Allergy Clin Immunol Pract 2025;13:3042-53)

BACKGROUND:Co-trimoxazole is a leading global cause of severe cutaneous adverse drug reactions (SCAR) including Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) and drug reaction with eosinophilia and systemic symptoms (DRESS). Co-trimoxazole-induced SCAR are associated with HLA class I alleles including HLA-B∗13:01 and HLA-B∗38:02 in Southeast Asian (SEA) populations. However, the global generalizability of these associations is unknown but critical for population-appropriate risk stratification and diagnosis. OBJECTIVE:To determine HLA risk factors associated with co-trimoxazole-induced SJS/TEN and DRESS in populations from the United States and South Africa. METHODS:We performed high-resolution HLA typing on dermatologist-adjudicated co-trimoxazole-induced patients with SCAR in the United States (n = 63) and South Africa (n = 26) compared with population controls. Peptide binding and docking analyses were performed using MHCcluster2.0 and CB-Dock2. RESULTS:In a multiple logistic regression model, HLA-B∗44:03 (corrected P [Pc] < .001; odds ratio [OR] = 4.08), HLA-B∗38:01 (Pc < .001; OR = 5.66), and HLA-C∗04:01 (Pc = .003; OR = 2.50) were independently associated with co-trimoxazole-induced SJS/TEN in the United States. HLA-B∗44:03 was also associated with co-trimoxazole-induced DRESS in South Africa (Pc = .019; OR = 10.69). Distinct HLA-B variants with shared peptide binding specificities (SPBS) and HLA-C∗04:01 identified 94% and 78% of co-trimoxazole-induced SJS/TEN and DRESS in the United States, respectively. The SEA risk allele HLA-B∗13:01, with SPBS to HLA-B∗44:03, was identified in just one of 63 US patients with SCAR. CONCLUSIONS:HLA alleles with SPBS to SEA-related risk alleles, including HLA-B∗44:03 (SPBS with HLA-B∗13:01) and HLA-B∗38:01 (SPBS with HLA-B∗38:02) but also HLA-C∗04:01, predisposed to co-trimoxazole-induced SCAR in the United States and South Africa. These findings provide biological plausibility and strategies for global risk prediction and diagnosis of co-trimoxazole-induced SCAR.

Sjögren's disease (SjD) is a chronic autoimmune disorder predominantly affecting females, characterized by exocrine gland dysfunction. This study investigates the therapeutic potential of 2-chloro-1-(4-hydroxy-phenyl)-ethanone (CHPE) and metformin in the C57BL/6.NOD-Aec1Aec2 mouse model, which closely mirrors human SjD. Molecular docking identified CHPE and metformin as high-affinity binders to the MHC class II I-Ab antigen-binding groove, suggesting their ability to inhibit antigen presentation and modulate immune responses. In-vitro assays confirmed their effectiveness in reducing T cell activation. In-vivo studies demonstrated that both preventative and therapeutic regimens of CHPE and metformin significantly reduced lymphocytic infiltration in the lacrimal glands, with metformin showing a more pronounced effect in females. Salivary gland infiltration was less responsive, though some reduction in focal scores was observed in male mice treated preventatively with CHPE. Both drugs altered the composition of lymphocytic infiltrates, particularly by reducing B cell populations, with notable sex-specific differences in response to treatment. CHPE and metformin also reduced anti-nuclear antibody levels, with CHPE showing stronger effects in females. Additionally, both drugs improved saliva and tear secretion, with metformin being more effective in the preventative regimen, especially in females. T cell receptor transductant assays revealed that CHPE and metformin exert their therapeutic effects through antigen-specific pathways, inhibiting T cell responses to SjD-associated autoantigens. Overall, this study provides compelling evidence that CHPE and metformin can modulate immune responses and improve gland function, with effectiveness varying by sex and age. These findings support the potential of these compounds as personalized treatments for SjD tailored to individual patient characteristics.

Pancreatic ductal adenocarcinoma (PDAC) is projected to become the second leading cause of cancer-related deaths by 2030, with most patients presenting with advanced, unresectable disease. Despite advances in chemotherapy, the 5-year survival rate remains low, underscoring the need for novel therapies. This study builds on the discovery that palmatine, a natural compound inhibits pancreatic cancer growth and enhances gemcitabine efficacy. This was done by using computational docking to explore palmatine's interaction with the STAT3 linker domain. A screening of 139,735 compounds from the National Cancer Institute Developmental Therapeutics Program identified compounds with superior STAT3 binding, with Striatal B emerging as the lead. In vitro tests confirmed the ability of Striatal B to significantly inhibit pancreatic cancer cell growth and potentiate gemcitabine effects, while computational modeling indicated effective binding to STAT3. Striatal B reduced STAT3 activation and epithelial-mesenchymal transition markers and modulated mechanical properties, suggesting a mechanism of action that involves altering cell mechanics, potentially providing a promising new therapeutic avenue for PDAC treatment.

Sjögren’s Disease (SjD) is an autoimmune disease characterized by dry mouth and eyes, affecting approximately 4 million Americans. SjD is a complex condition with a multifactorial etiology and a diverse patient population in terms of disease activity, severity, and response to therapy. To better comprehend the etiologies and develop effective treatments, it is crucial to utilize appropriate animal models to unravel the different aspects of the autoimmune process. In this study, we have been examining the Non-Obese Diabetic (NOD)-DR3 mouse model, an NOD-congenic animal with human HLA class II expression, specifically the DR3(DRw17) transgene. The DR3 transgene expresses the HLA-DR3 genotype, comprising HLA-DRA0101 and HLA-DRB10301. Our research revealed that NOD-DR3 mice develop SjD phenotypes characterized by decreased salivary flow rates, elevated levels of anti-nuclear autoantibodies, changes in salivary gland proinflammatory immune cell populations and their cytokines, and increased focal scores. A notable finding is that NOD-DR3 mice exhibit sexually dimorphic disease phenotypes, mirroring patterns observed in human patients. Lastly, employing machine learning algorithms targeting DR3, we have identified and tested novel drug compounds. Some compounds alleviated the clinical signs of SjD in the mouse model. In summary, our research demonstrates the potential of this inaugural humanized mouse model of SjD and identifies DR3 as a target for treating the disease. Supported by the National Institute of Dental and Craniofacial Research (NIDCR) (DE028544, DE028544-02S1) Therapeutic Approaches to Autoimmunity (THER)

Sjögren's syndrome (SjD) is a chronic autoimmune disorder in which the immune system attacks the glands that produce tears and saliva, leading to symptoms such as dry eyes and dry mouth. If left untreated, SjD can also cause inflammation and damage to other parts of the body, including the skin, lungs, kidneys, and nervous system, and increase the risk of developing lymphoma. The human leukocyte antigen (HLA) class II molecule HLA-DR3 is strongly associated with SjD. To investigate how post-translational modifications (PTMs) influence the presentation of SjD-associated autoantigens by HLA-DR3, we employed a computational framework to analyze the binding of PTM-mimic peptides to HLA-DR3. Our analysis revealed that PTM-mimic substitutions at canonical anchor positions rarely improved predicted binding affinity using the Stabilized Matrix Method, with most modifications resulting in reduced affinity. However, a comprehensive analysis of full-length SjD-associated autoantigen sequences (Ro60, Ro52, La) identified discrete regions with high densities of PTM-eligible anchor sites, specifically, the Ro60 HEAT solenoid, Ro52 RING/B-box/PRY-SPRY modules, and the La motif-RRM1 region, suggesting that PTMs may alter epitope presentation in a sequence-dependent manner. Experimental validation of selected PTM-mimic peptides demonstrated enhanced T cell responses, which were associated with increased binding affinity to HLA-DR3. Structural modeling of a representative complex revealed that PTM-mimic peptides adopt a slightly shifted backbone orientation and altered side-chain positioning, leading to a larger peptide-DR3 interaction interface. These findings provide new insights into the role of PTMs in shaping the immunogenicity of SjD-associated autoantigens and highlight the potential for PTM-mimic peptides to modulate T cell responses in SjD.

COVID-19 is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The severity of COVID-19 is highly variable and related to known (e.g., age, obesity, immune deficiency) and unknown risk factors. The widespread clinical symptoms encompass a large group of asymptomatic COVID-19 patients, raising a crucial question regarding genetic susceptibility, e.g., whether individual differences in immunity play a role in patient symptomatology and how much human leukocyte antigen (HLA) contributes to this. To reveal genetic determinants of susceptibility to COVID-19 severity in the population and further explore potential immune-related factors, we performed a genome-wide association study on 284 confirmed COVID-19 patients (cases) and 95 healthy individuals (controls). We compared cases and controls of European (EUR) ancestry and African American (AFR) ancestry separately. We identified two loci on chromosomes 5q32 and 11p12, which reach the significance threshold of suggestive association (p<1×10 -5 threshold adjusted for multiple trait testing) and are associated with the COVID-19 susceptibility in the European ancestry (index rs17448496: odds ratio [OR] = 0.173; 95% confidence interval [CI], 0.08–0.36 for G allele; p=5.15× 10 -5 and index rs768632395: OR = 0.166; 95% CI, 0.07–0.35 for A allele; p= 4.25×10 -6 , respectively), which were associated with two genes, PPP2R2B at 5q32, and LRRC4C at 11p12, respectively. To explore the linkage between HLA and COVID-19 severity, we applied fine-mapping analysis to dissect the HLA association with mild and severe cases. Using In-silico binding predictions to map the binding of risk/protective HLA to the viral structural proteins, we found the differential presentation of viral peptides in both ancestries. Lastly, extrapolation of the identified HLA from the cohort to the worldwide population revealed notable correlations. The study uncovers possible differences in susceptibility to COVID-19 in different ancestral origins in the genetic background, which may provide new insights into the pathogenesis and clinical treatment of the disease.

The 2.6 Å crystal structure of the apo form of Hip1 (hydrolase important for pathogenesis) has been previously reported. However, very little is known about the active site architecture of this M. tuberculosis (Mtb), serine hydrolase drug target. To begin mapping the active site of Hip1, we cocrystallized Hip1 with the irreversible serine protease inhibitor, 4-(2-aminoethyl)-benzenesulfonylfluoride (AEBSF). We chose AEBSF for cocrystallization with Hip1 since the similar inhibitor, phenylmethylsulfonyl fluoride (PMSF), interestingly exhibited no activity against Hip1. We obtained crystals that diffracted to 2.1 Å but to our bewilderment, we did not observe any electron density for the inhibitor in the omit map for the Hip1-AEBSF complex. Rather, in the active site, dehydroalanine (dAla) was found to occupy the expected position of the catalytic Ser228, thus yielding anhydrohip1. Here we present a comparative analysis of the crystal structures of anhydrohip1 and Hip1 and provide a mechanism for the conversion of the enzyme to the anhydro-form through reaction with AEBSF. With the aid of molecular docking, we propose an explanation for the differential inhibition of Hip1 by AEBSF and PMSF. We also present a preliminary definition of the S1 and S2 pockets of the protease's active site and propose a mechanism for a ligand-induced conformational change within the S2 pocket. Finally, we expand upon the previous demarcation of the putative lipid binding pocket in the α-domain of the enzyme. We believe that this detailed analysis of the structures of anhydrohip1 and Hip1 provides valuable information useful for the structure-based drug design of novel Hip1-directed Mtb therapeutics.

Sjögren’s syndrome (SjS) is characterized by lymphocytic infiltration and the dysfunction of the salivary and lacrimal glands. The autoimmune response is driven by the effector T cells and their cytokines. The activation of the effector helper T cells is mediated by autoantigen presentation by human leukocyte antigen (HLA) class II molecules of antigen-presenting cells. Studies using familial aggregation, animal models, and genome-wide association demonstrate a significant genetic correlation between specific risk HLAs and SjS. One of the key HLA alleles is HLA-DRB1*0301; it is one of the most influential associations with primary SjS, having the highest odds ratio and occurrence across different ethnic groups. The specific autoantigens attributed to SjS remain elusive, especially the specific antigenic epitopes presented by HLA-DRB1*0301. This study applied a high throughput in silico mapping technique to identify antigenic epitopes of known SjS autoantigens presented by high-risk HLAs. Furthermore, we identified specific binding HLA-DRB1*0301 epitopes using structural modeling tools such as Immune Epitope Database and Analysis Resource IEDB, AutoDock Vina, and COOT. By deciphering the critical epitopes of autoantigens presented by HLA-DRB1*0301, we gain a better understanding of the origin of the antigens, determine the T cell receptor function, learn the mechanism of disease progression, and develop therapeutic applications.

Background and Aims Trimethoprim (TMP)–sulfamethoxazole (SMX) is an important cause of idiosyncratic drug‐induced liver injury (DILI), but its genetic risk factors are not well understood. This study investigated the relationship between variants in the human leukocyte antigen (HLA) class 1 and 2 genes and well‐characterized cases of TMP‐SMX DILI. Approach and Results European American and African American persons with TMP‐SMX DILI were compared with respective population controls. HLA sequencing was performed by Illumina MiSeq (Illumina, San Diego, CA) for cases. The HLA genotype imputation with attribute bagging program was used to impute HLA alleles for controls. The allele frequency difference between case patients and controls was tested by Fisher’s exact tests for each ethnic group. For European Americans, multivariable logistic regression with Firth penalization was used to test the HLA allelic effect after adjusting for age and the top two principal components. Molecular docking was performed to assess HLA binding with TMP and SMX. The European American subset had 51 case patients and 12,156 controls, whereas the African American subset had 10 case patients and 5,439 controls. Four HLA alleles were significantly associated in the European American subset, with HLA‐B*14:01 ranking at the top (odds ratio, 9.20; 95% confidence interval, 3.16, 22.35; P = 0.0003) after covariate adjustment. All carriers of HLA‐B*14:01 with TMP‐SMX DILI possessed HLA‐C*08:02 , another significant allele ( P = 0.0026). This pattern was supported by HLA‐B*14:01–HLA‐C*08:02 haplotype association ( P = 1.33 × 10 −5 ). For the African American patients, HLA‐B*35:01 had 2.8‐fold higher frequency in case patients than in controls, with 5 of 10 patients carrying this allele. Molecular docking showed cysteine at position 67 in HLA‐B*14:01 and phenylalanine at position 67 in HLA‐B*35:01 to be the predictive binding sites for SMX metabolites. Conclusions HLA‐B*14:01 is associated with TMP‐SMX DILI in European Americans, and HLA‐B*35:01 may be a potential genetic risk factor for African Americans.

Vancomycin is a glycopeptide antibiotic used to treat resistant gram-positive infections. It is associated with a life-threatening, delayed T-cell–mediated reaction, drug reaction with eosinophilia and systemic symptoms (DRESS) presenting with fever, rash, hematologic abnormalities, lymphadenopathy, and organ involvement that occurs 2 to 6 weeks after initiation of vancomycin treatment.1Blumenthal K.G. Peter J.G. Trubiano J.A. Phillips E.J. Antibiotic allergy.Lancet. 2019; 393: 183-198Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar We demonstrated that HLA-A∗32:01 is strongly associated with vancomycin-induced DRESS in European populations.2Konvinse K.C. Trubiano J.A. Pavlos R. James I. Shaffer C.M. Bejan C.A. et al.HLA-A∗32:01 is strongly associated with vancomycin-induced drug reaction with eosinophilia and systemic symptoms.J Allergy Clin Immunol. 2019; 144: 183-192Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar All glycopeptide antibiotics contain a heptapeptide core structure, and cross-reactivity should be considered when treating patients who have had a previous hypersensitivity reaction to vancomycin (see Fig E1 in this article's Online Repository at www.jacionline.org).3Van Bambeke F. Lipoglycopeptide antibacterial agents in gram-positive infections: a comparative review.Drugs. 2015; 75: 2073-2095Crossref PubMed Scopus (44) Google Scholar Cross-reactivity remains controversial, as some patients presenting with teicoplanin-induced DRESS showed subsequent tolerability to vancomycin4Kwon H.S. Chang Y.S. Jeong Y.Y. Lee S.M. Song W.J. Kim H.B. et al.A case of hypersensitivity syndrome to both vancomycin and teicoplanin.J Korean Med Sci. 2006; 21: 1108-1110Crossref PubMed Scopus (40) Google Scholar, 5Lye D. Athan E. O'Brien D. Teicoplanin hypersensitivity syndrome.Int J Antimicrob Agents. 2007; 29: 476-478Crossref PubMed Scopus (8) Google Scholar, 6Hsiao S.H. Chen H.H. Chou C.H. Lin W.L. Liu Yeh P.Y. Wu T.J. Teicoplanin-induced hypersensitivity syndrome with a preceding vancomycin-induced neutropenia: a case report and literature review.J Clin Pharm Ther. 2010; 35: 729-732Crossref PubMed Scopus (14) Google Scholar, 7Miyazu D. Kodama N. Yamashita D. Tanaka H. Inoue S. Imakyure O. et al.DRESS syndrome caused by cross-reactivity between vancomycin and subsequent teicoplanin administration: a case report.Am J Case Rep. 2016; 17: 625-631Crossref PubMed Scopus (16) Google Scholar and patients with teicoplanin-induced DRESS confirmed by a positive intradermal skin test result had a negative result of a skin test to vancomycin.8Ben Romdhane H. Chadli Z. Ben Fredj N. Chaabane A. Boughattas N.A. Aouam K. Teicoplanin-induced DRESS syndrome: the importance of skin tests.Med Mal Infect. 2018; 48: 291-293Crossref PubMed Scopus (3) Google Scholar To examine the immunologic cross-reactivity among 4 glycopeptide antibiotics (ie, vancomycin, teicoplanin, dalbavancin, and telavancin), adults who were at least 18 years old with a probable diagnosis of vancomycin-induced DRESS defined as having a corresponding Naranjo adverse drug reaction score of 5 or higher (probable adverse drug reaction), having a Registry of Severe Cutaneous Adverse Reactions score of 4 or higher (probable DRESS), and carrying HLA-A∗32:01 (the recently described risk allele for vancomycin-induced DRESS) were recruited between January 2010 and September 2019 through drug allergy clinics and inpatient facilities at participating institutions (Vanderbilt University Medical Center in Nashville, Tennessee, and Austin Health, Peter MacCallum Cancer Centre, Fiona Stanley Hospital and Royal Perth Hospital in Perth, Western Australia, Australia). All patients provided informed consent for collection of saliva and blood to be stored as DNA and PBMCs. IFN-γ release in response to overnight incubation with implicated drugs was performed by ELISpot assay (3420-2H; Mabtech, Stockholm, Sweden) in triplicate from thawed PBMCs (rested overnight) and included negative (unstimulated) and positive (anti-CD3 Mabtech antibody, staphylococcal enterotoxin B, and/or cytomegalovirus pp65) controls. Control PBMCs from glycopeptide unexposed HLA-A∗32:01–positive and HLA-A∗32:01–negative individuals were also used. PBMCs plated at 200,000 cells per well were incubated with vancomycin, teicoplanin, dalbavancin, telavancin, and other implicated drugs at concentrations representative of maximum serum concentrations, as well as those 10-fold higher and 10-fold lower (Fig 1). A positive response was defined as more than 50 spot-forming units per million cells after background removal as per previous the definitions.9Trubiano J.A. Strautins K. Redwood A.J. Pavlos R. Konvinse K.C. Aung A.K. et al.The combined utility of ex vivo IFN-gamma release enzyme-linked ImmunoSpot assay and in vivo skin testing in patients with antibiotic-associated severe cutaneous adverse reactions.J Allergy Clin Immunol Pract. 2018; 6: 1287-1296.e1Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar High-resolution 4-digit HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DR, and HLA-DQ typing was performed by using sequence-based typing with previously published protocols.2Konvinse K.C. Trubiano J.A. Pavlos R. James I. Shaffer C.M. Bejan C.A. et al.HLA-A∗32:01 is strongly associated with vancomycin-induced drug reaction with eosinophilia and systemic symptoms.J Allergy Clin Immunol. 2019; 144: 183-192Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar A total of 15 patients who met the clinical inclusion criteria for vancomycin-induced DRESS syndrome were enrolled into this study. Their demographics, clinical characteristics, and DRESS history are described in Table E1 (in this article's Online Repository at www.jacionline.org), and full HLA typing of all patients is described in Table E2 (in this article's Online Repository at www.jacionline.org). All patients with vancomycin-induced DRESS exhibited a dose-dependent positive IFN-γ ELISpot response to vancomycin (Fig 1 and see Table E3 in this article's Online Repository at www.jacionline.org); all had a clear negative response to both concentrations of dalbavancin (Fig 1). Three patients overall showed cross-reactivity, with all 3 showing a positive response to telavancin and 2 of them also demonstrating a positive IFN-γ ELISpot response to teicoplanin. One of the 2 patients who had positive IFN-γ ELISpot responses to vancomycin, teicoplanin, and telavancin (patient 15 and see Table E3) was intradermally skin-tested to both vancomycin and teicoplanin and showed positive responses to both (see Fig E2 in this article's Online Repository at www.jacionline.org), which was not seen in glycopeptide-unexposed controls (n = 5) and 3 patients (patients 9, 10, and 11) with HLA-A∗32:01–positive vancomycin-induced DRESS who showed positive delayed intradermal testing and IFN-γ ELISpot results to vancomycin but negative IFN-γ ELISpot and intradermal testing results to teicoplanin. Patients 1, 3, and 5 also tolerated ingestion challenges with medications concurrently administered at the time of vancomycin-induced DRESS. In samples with sufficient cell numbers, PBMCs were tested against other concurrently administered medications potentially implicated in DRESS development (see Fig E3 in this article's Online Repository at www.jacionline.org). IFN-γ ELISpot was also performed on PBMCs from non–HLA-matched healthy donors (n = 5) and a HLA-A∗32:01–positive vancomycin naive control (n = 1), with all exhibiting a negative response to all 4 drugs (data not shown). Vancomycin is implicated in up to 40% of patients with antibiotic-related DRESS.1Blumenthal K.G. Peter J.G. Trubiano J.A. Phillips E.J. Antibiotic allergy.Lancet. 2019; 393: 183-198Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar The prevalence of vancomycin-induced DRESS appears to be increasing, and it is the second most common cause of DRESS overall reported to the US Food and Drug Administgration Adverse Event Reporting System between 1999 and 2019 (https://open.fda.gov/data/faers/ [accessed March 2, 2020]). HLA-A∗32:01 has recently been reported as a genetic risk factor for vancomycin-induced DRESS in the European population, within which the allelic prevalence is approximately 6.8%.2Konvinse K.C. Trubiano J.A. Pavlos R. James I. Shaffer C.M. Bejan C.A. et al.HLA-A∗32:01 is strongly associated with vancomycin-induced drug reaction with eosinophilia and systemic symptoms.J Allergy Clin Immunol. 2019; 144: 183-192Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar Because of the high prevalence of the risk allele, the high incidence of vancomycin-induced DRESS, and the potential cross-reactive risk with dalbavancin given its extremely long half-life of 14 days, the detection of cross-reactivity to alternative glycopeptide antibiotics is very important for reducing the risk of DRESS and providing patients with future therapeutic options. Our study is reassuring because it demonstrated that 100% of vancomycin-induced DRESS cases with IFN-γ ELISpot responses showed a negative IFN-γ ELISpot response to dalbavancin, suggesting no or very low cross-reactivity between vancomycin and dalbavancin. Dalbavancin differs from vancomycin through a structural modification of the lipophilic side chain, which enhances its binding affinity to the cell membrane and prolongs its half-life.3Van Bambeke F. Lipoglycopeptide antibacterial agents in gram-positive infections: a comparative review.Drugs. 2015; 75: 2073-2095Crossref PubMed Scopus (44) Google Scholar Approximately 87% (13 of 15) and 73% (8 of 11) of vancomycin-induced DRESS patients showed no cross-reactivity to teicoplanin and telavancin, respectively. Two patients with vancomycin-induced DRESS demonstrated immunologic cross-reactivity to teicoplanin, and to telavancin with use of ELISpot, which was supported by a positive intradermal skin test result to teicoplanin in the 1 patient in whom this testing was performed. Of note, 1 of these 2 patients had a short interval between the original reaction and IFN-γ ELISpot assay (patient 8 [see Table E1]). Telavancin is a semisynthetic derivative of vancomycin, and dalbavancin is a semisynthetic lipoglycopeptide derived from a glycopeptide structure more similar to that of teicoplanin. In addition to having a long lipophilic side chain that extends its half-life and improves affinity for the D-Ala-D-Ala target in the bacterial cell wall, dalbavancin lacks the acetylglucosamine group of teicoplanin. Intriguingly the 2 patients (patients 8 and 15 [see Tables E2 and E3]) with shared ex vivo cross-reactivity among vancomycin, teicoplanin, and telavancin shared the same class II HLA haplotype. To determine whether vancomycin, teicoplanin, and telavancin have the potential to bind class II HLA molecules in particular that are shared by the patients who exhibited cross-reactive specificities, we used molecular docking (AutoDock Vina software). Vancomycin, teicoplanin, and telavancin were predicted to bind HLA-DQ (DQA1∗01:01, DQB1∗05:03) with estimated ΔG values of –7.7, –7.4, and –7.2 kcal/mol, respectively, whereas dalbavancin was predicted to bind only weakly (Fig 2). Teicoplanin and telavancin may hence bind class II HLA as the molecular basis for cross-reactive T-cell responses in HLA-A∗32:01–positive patients who have experienced vancomycin-induced DRESS. This could suggest a new model for cross-reactivity that includes recognition of drug/class II HLA complexes by CD8+ T cells matured by positive selection by HLA-A∗32:01; alternatively, CD4+ T cells may recognize teicoplanin and telavancin in the context of class II HLA molecules such as DQ. A limitation of our study is the lack of in vivo and rechallenge cross-reactivity data. We cannot be certain that the clinical phenotype of patients with prior vancomycin-induced DRESS and ex vivo cross-reactivity to teicoplanin and telavancin and positive intradermal skin test result to teicoplanin in the 1 patient in whom this testing was performed would also be indicative of DRESS; however, given the structural similarity of these drugs and the half-life of dalbavancin of greater than 1 week, rechallenge of these individuals would not be ethical unless the clinical need outweighed any risk. This study provides the first evidence that elucidates a risk for a potential immunologic cross-reactivity pattern between vancomycin, teicoplanin, and newer glycopeptide antibiotics in patients with previous DRESS induced by vancomycin. The lack of apparent cross-reactivity is reassuring for dalbavancin, particularly given the long half-life of this lipoglycopeptide; however, clinicians should be aware of the low but detectable risk of cross-reactivity in particular among teicoplanin, telavancin, and vancomycin in the HLA-A∗32:01–restricted vancomycin-induced DRESS. Our study suggests that ex vivo IFN-γ ELISpot assay or skin tests in combination with HLA typing could be performed to risk-stratify patients with a history of previous vancomycin-induced DRESS for potential risk of cross-reactivity between vancomycin, teicoplanin, and telavancin to aid in making decisions for future treatment. The shared class II HLA haplotype among 2 patients with cross-reactivity between vancomycin, teicoplanin, and telavancin and virtual docking of these drugs to HLA class II suggest a potential novel mechanism for cross-reactivity following sensitization that deserves further exploration. Download .docx (.29 MB) Help with docx files Fig E1 Download .docx (2.01 MB) Help with docx files Fig E2 Download .docx (.15 MB) Help with docx files Fig E3 Download .docx (.03 MB) Help with docx files Tables E1-E3