Broad and strong memory CD4+ and CD8+ T cells induced by SARS-CoV-2 in UK convalescent individuals following COVID-19.

The development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines and therapeutics will depend on understanding viral immunity. We studied T cell memory in 42 patients following recovery from COVID-19 (28 with mild disease and 14 with severe disease) and 16 unexposed donors, using interferon-γ-based assays with peptides spanning SARS-CoV-2 except ORF1. The breadth and magnitude of T cell responses were significantly higher in severe as compared with mild cases. Total and spike-specific T cell responses correlated with spike-specific antibody responses. We identified 41 peptides containing CD4+ and/or CD8+ epitopes, including six immunodominant regions. Six optimized CD8+ epitopes were defined, with peptide-MHC pentamer-positive cells displaying the central and effector memory phenotype. In mild cases, higher proportions of SARS-CoV-2-specific CD8+ T cells were observed. The identification of T cell responses associated with milder disease will support an understanding of protective immunity and highlights the potential of including non-spike proteins within future COVID-19 vaccine design.

HLA Allele-Restricted Immune-Mediated Adverse Drug Reactions: Framework for Genetic Prediction.

Human leukocyte antigen (HLA) is a hallmark genetic marker for the prediction of certain immune-mediated adverse drug reactions (ADRs). Numerous basic and clinical research studies have provided the evidence base to push forward the clinical implementation of HLA testing for the prevention of such ADRs in susceptible patients. This review explores current translational progress in using HLA as a key susceptibility factor for immune ADRs and highlights gaps in our knowledge. Furthermore, relevant findings of HLA-mediated drug-specific T cell activation are covered, focusing on cellular approaches to link genetic associations to drug-HLA binding as a complementary approach to understand disease pathogenesis.

Activation of Human CD8+ T Cells with Nitroso Dapsone-Modified HLA-B*13:01-Binding Peptides.

Previous studies have shown that cysteine-reactive drug metabolites bind covalently with protein to activate patient T cells. However, the nature of the antigenic determinants that interact with HLA and whether T cell stimulatory peptides contain the bound drug metabolite has not been defined. Because susceptibility to dapsone hypersensitivity is associated with the expression of HLA-B*13:01, we have designed and synthesized nitroso dapsone-modified, HLA-B*13:01 binding peptides and explored their immunogenicity using T cells from hypersensitive human patients. Cysteine-containing 9-mer peptides with high binding affinity to HLA-B*13:01 were designed (AQDCEAAAL [Pep1], AQDACEAAL [Pep2], and AQDAEACAL [Pep3]), and the cysteine residue was modified with nitroso dapsone. CD8+ T cell clones were generated and characterized in terms of phenotype, function, and cross-reactivity. Autologous APCs and C1R cells expressing HLA-B*13:01 were used to determine HLA restriction. Mass spectrometry confirmed that nitroso dapsone-peptides were modified at the appropriate site and were free of soluble dapsone and nitroso dapsone. APC HLA-B*13:01-restricted nitroso dapsone-modified Pep1- (n = 124) and Pep3-responsive (n = 48) CD8+ clones were generated. Clones proliferated and secreted effector molecules with graded concentrations of nitroso dapsone-modified Pep1 or Pep3. They also displayed reactivity against soluble nitroso dapsone, which forms adducts in situ, but not with the unmodified peptide or dapsone. Cross-reactivity was observed between nitroso dapsone-modified peptides with cysteine residues in different positions in the peptide sequence. These data characterize a drug metabolite hapten CD8+ T cell response in an HLA risk allele-restricted form of drug hypersensitivity and provide a framework for structural analysis of hapten HLA binding interactions.

Immunogenicity of COVID-19 vaccines in patients with follicular lymphoma receiving frontline chemoimmunotherapy.

Immune responses to primary COVID-19 vaccination were investigated in 58 patients with follicular lymphoma (FL) as part of the PETReA trial of frontline therapy (EudraCT 2016-004010-10). COVID-19 vaccines (BNT162b2 or ChAdOx1) were administered before, during or after cytoreductive treatment comprising rituximab (depletes B cells) and either bendamustine (depletes CD4+ T cells) or cyclophosphamide-based chemotherapy. Blood samples obtained after vaccine doses 1 and 2 (V1, V2) were analysed for antibodies and T cells reactive to the SARS-CoV-2 spike protein using the Abbott Architect and interferon-gamma ELISpot assays respectively. Compared to 149 healthy controls, patients with FL exhibited lower antibody but preserved T-cell responses. Within the FL cohort, multivariable analysis identified low pre-treatment serum IgA levels and V2 administration during induction or maintenance treatment as independent determinants of lower antibody and higher T-cell responses, and bendamustine and high/intermediate FLIPI-2 score as additional determinants of a lower antibody response. Several clinical scenarios were identified where dichotomous immune responses were estimated with >95% confidence based on combinations of predictive variables. In conclusion, the immunogenicity of COVID-19 vaccines in FL patients is influenced by multiple disease- and treatment-related factors, among which B-cell depletion showed differential effects on antibody and T-cell responses.

Patients with naproxen-induced liver injury display T-cell memory responses toward an oxidative (S)-O-desmethyl naproxen metabolite but not the acyl glucuronide.

BACKGROUND: Exposure to nonsteroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen (IBU) and naproxen (NAP) is associated with idiosyncratic drug-induced liver injury (DILI). Carboxylate bioactivation into reactive metabolites (e.g., acyl glucuronides, AG) and resulting T-cell activation is hypothesized as causal for this adverse event. However, conclusive evidence supporting this is lacking. METHODS: In this work, we identify CD4+ and CD8+ T-cell hepatic infiltration in a biopsy from an IBU DILI patient. Lymphocyte transformation test and IFN-γ ELIspot, conducted on peripheral blood mononuclear cells (PBMCs) of patients with NAP-DILI, were used to explore drug-specific T-cell activation. T-cell clones (TCC) were generated and tested for drug specificity, phenotype/function, and pathways of T-cell activation. Cells were exposed to NAP, its oxidative metabolite 6-O-desmethyl NAP (DM-NAP), its AG or synthesized NAP-AG human-serum albumin adducts (NAP-AG adduct). RESULTS: CD4+ and CD8+ T-cells from patients expressing a range of different Vß receptors were stimulated to proliferate and secrete IFN-γ and IL-22 when exposed to DM-NAP, but not NAP, NAP-AG or the NAP-AG adduct. Activation of the CD4+ TCC was HLA-DQ-restricted and dependent on antigen presenting cells (APC); most TCC were activated with DM-NAP-pulsed APC, while fixation of APC blocked the T-cell response. Cross-reactivity was not observed with structurally-related drugs. CONCLUSION: Our results confirm hepatic T-cell infiltrations in NSAID-induced DILI, and show a T-cell memory response toward DM-NAP indicating an immune-mediated basis for the adverse event. Whilst bioactivation at the carboxylate group is widely hypothesized to be pathogenic for NSAID associated DILI, we found no evidence of this with NAP.

Characterization of Drug-Specific CD4+ T-Cells Reveals Possible Roles of HLA Class II in the Pathogenesis of Carbamazepine Hypersensitivity Reactions.

Carbamazepine (CBZ) is an aromatic anticonvulsant known to cause drug hypersensitivity reactions, which range in severity from relatively mild maculopapular exanthema to potentially fatal Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS-TEN). These reactions are known to be associated with human leukocyte antigen (HLA) class I alleles, and CBZ interacts preferentially with the related HLA proteins to activate CD8+ T-cells. This study aimed to evaluate the contribution of HLA class II in the effector mechanism(s) of CBZ hypersensitivity. CBZ-specific T-cells clones were generated from two healthy donors and two hypersensitive patients with high-risk HLA class I markers. Phenotype, function, HLA allele restriction, response pathways, and cross-reactivity of CBZ-specific T-cells were assessed using flow cytometry, proliferation analysis, enzyme-linked immunosorbent spot, and enzyme-linked immunosorbent assay. The association between HLA class II allele restriction and CBZ hypersensitivity was reviewed using Allele Frequency Net Database. Forty-four polyclonal CD4+ CBZ-specific T-cell clones were generated and found to be restricted to HLA-DR, particularly HLA-DRB1*07:01. This CD4+-mediated response proceeded through a direct pharmacological interaction between CBZ and HLA-DR molecules. Similar to the CD8+ response, CBZ-stimulated CD4+ clones secreted granulysin, a key mediator of SJS-TEN. Our database review revealed an association between HLA-DRB1*07:01 and CBZ-induced SJS-TEN. These findings implicate HLA class II antigen presentation as an additional pathogenic factor for CBZ hypersensitivity reactions. Both HLA class II molecules and drug-responsive CD4+ T-cells should be evaluated further to gain better insights into the pathogenesis of drug hypersensitivity reactions.

Pathology of drug hypersensitivity reactions and mechanisms of immune tolerance.

Immune-mediated type IV adverse drug reactions are idiosyncratic in nature, generally not related to the primary or secondary pharmacology of the drug. Due to their complex nature and rarity, these iatrogenic reactions are seldom predicted or encountered during preclinical/early clinical development stages, and often precipitate upon exposure to wider populations (i.e. phase III onwards). They confer a burden on the healthcare sector in both a clinical and financial sense presenting a severe impediment to the drug discovery and development process. Research over the past 50 years has improved our understanding of these reactions markedly as both in vitro and in vivo studies have placed the role of the immune system, in particular; drug-responsive T cells, firmly in the spotlight as the mediators of these reactions. Indeed, the role of different populations of T cells in adverse events and the interaction of drug molecules with HLA proteins expressed on the surface of antigen-presenting cells is of considerable interest. Herein, this review examines the pathways of immune-mediated adverse events including the various T cell subtypes implicated and the mechanisms of T cell activation. Additionally, we address the enigma of immunological tolerance and explore the role tolerance plays in determination of susceptibility to such adverse events even in individuals carrying immunogenic liabilities.

Drug-specific T-cell responses in patients with liver injury following treatment with the BACE inhibitor atabecestat.

BACKGROUND: Atabecestat is an orally administered BACE inhibitor developed to treat Alzheimer's disease. Elevations in hepatic enzymes were detected in a number of in trial patients, which resulted in termination of the drug development programme. Immunohistochemical characterization of liver tissue from an index case of atabecestat-mediated liver injury revealed an infiltration of T-lymphocytes in areas of hepatocellular damage. This coupled with the fact that liver injury had a delayed onset suggests that the adaptive immune system may be involved in the pathogenesis. The aim of this study was to generate and characterize atabecestat(metabolite)-responsive T-cell clones from patients with liver injury. METHODS: Peripheral blood mononuclear cells were cultured with atabecestat and its metabolites (diaminothiazine [DIAT], N-acetyl DIAT & epoxide) and cloning was attempted in a number of patients. Atabecestat(metabolite)-responsive clones were analysed in terms of T-cell phenotype, function, pathways of T-cell activation and cross-reactivity with structurally related compounds. RESULTS: CD4+ T-cell clones activated with the DIAT metabolite were detected in 5 out of 8 patients (up to 4.5% cloning efficiency). Lower numbers of CD4+ and CD8+ clones displayed reactivity against atabecestat. Clones proliferated and secreted IFN-γ, IL-13 and cytolytic molecules following atabecestat or DIAT stimulation. Certain atabecestat and DIAT-responsive clones cross-reacted with N-acetyl DIAT; however, no cross-reactivity was observed between atabecestat and DIAT. CD4+ clones were activated through a direct, reversible compound-HLA class II interaction with no requirement for protein processing. CONCLUSION: The detection of atabecestat metabolite-responsive T-cell clones activated via a pharmacological interactions pathway in patients with liver injury is indicative of an immune-based mechanism for the observed hepatic enzyme elevations.

Immune dysregulation increases the incidence of delayed-type drug hypersensitivity reactions.

Delayed-type, T cell-mediated, drug hypersensitivity reactions are a serious unwanted manifestation of drug exposure that develops in a small percentage of the human population. Drugs and drug metabolites are known to interact directly and indirectly (through irreversible protein binding and processing to the derived adducts) with HLA proteins that present the drug-peptide complex to T cells. Multiple forms of drug hypersensitivity are strongly linked to expression of a single HLA allele, and there is increasing evidence that drugs and peptides interact selectively with the protein encoded by the HLA allele. Despite this, many individuals expressing HLA risk alleles do not develop hypersensitivity when exposed to culprit drugs suggesting a nonlinear, multifactorial relationship in which HLA risk alleles are one factor. This has prompted a search for additional susceptibility factors. Herein, we argue that immune regulatory pathways are one key determinant of susceptibility. As expression and activity of these pathways are influenced by disease, environmental and patient factors, it is currently impossible to predict whether drug exposure will result in a health benefit, hypersensitivity or both. Thus, a concerted effort is required to investigate how immune dysregulation influences susceptibility towards drug hypersensitivity.

Modification of the cyclopropyl moiety of abacavir provides insight into the structure activity relationship between HLA-B*57:01 binding and T-cell activation.

BACKGROUND: Abacavir is associated with hypersensitivity reactions in individuals positive for the HLA-B*57:01 allele. The drug binds within the peptide binding groove of HLA-B*57:01 altering peptides displayed on the cell surface. Presentation of these HLA-abacavir-peptide complexes to T-cells is hypothesized to trigger a CD8+ T-cell response underpinning the hypersensitivity. Thus, the aim of this study was to explore the relationship between the structure of abacavir with HLA-B*57:01 binding and the CD8+ T-cell activation. METHODS: Seventeen abacavir analogues were synthesized and cytokine secretion from abacavir/abacavir analogue-responsive CD8+ T-cell clones was measured using IFN-γ ELIspot. In silico docking studies were undertaken to assess the predicted binding poses of the abacavir analogues within the HLA-B*57:01 peptide binding groove. In parallel, the effect of selected abacavir analogues on the repertoire of self-peptides presented by cellular HLA-B*57:01 was characterized using mass spectrometry. RESULTS: Abacavir and ten analogues stimulated CD8+ T-cell IFN-γ release. Molecular docking of analogues that retained antiviral activity demonstrated a relationship between predicted HLA-B*57:01 binding orientations and the ability to induce a T-cell response. Analogues that stimulated T-cells displayed a perturbation of the natural peptides displayed by HLA-B*57:01. The antigen-specific CD8+ T-cell response was dependent on the enantiomeric form of abacavir at both cyclopropyl and cyclopentyl regions. CONCLUSION: Alteration of the chemical constitution of abacavir generates analogues that retain a degree of pharmacological activity, but have variable ability to activate T-cells. Modelling and immunopeptidome analysis delineate how drug HLA-B*57:01 binding and peptide display by antigen presenting cells relate to the activation of CD8+ T-cells.

T-Cell Activation by Low Molecular Weight Drugs and Factors That Influence Susceptibility to Drug Hypersensitivity.

Drug hypersensitivity reactions adversely affect treatment outcome, increase the length of patients' hospitalization, and limit the prescription options available to physicians. In addition, late stage drug attrition and the withdrawal of licensed drugs cost the pharmaceutical industry billions of dollars. This significantly increases the overall cost of drug development and by extension the price of licensed drugs. Drug hypersensitivity reactions are characterized by a delayed onset, and reactions tend to be more serious upon re-exposure. The role of drug-specific T-cells in the pathogenesis of drug hypersensitivity reactions and definition of the nature of the binding interaction of drugs with HLA and T-cell receptors continues to be the focus of intensive research, primarily because susceptibility is associated with expression of one or a small number of HLA alleles. This review critically examines the mechanisms of T-cell activation by drugs. Specific examples of drugs that activate T-cells via the hapten, the pharmacological interaction with immune receptors and the altered self-peptide repertoire pathways, are discussed. Furthermore, the impacts of drug metabolism, drug-protein adduct formation, and immune regulation on the development of drug antigen-responsive T-cells are highlighted. The knowledge gained from understanding the pathways of T-cell activation and susceptibility factors for drug hypersensitivity will provide the building blocks for the development of predictive in vitro assays that will prevent or help to minimize the incidence of these reactions in clinic.

Laparoscopic Nephrectomy: Initial Case Report.

A tumor-bearing right kidney was completely excised from an 85-year-old woman using a laparoscopic approach. A newly devised method for intra-abdominal organ entrapment and a recently developed laparoscopic tissue morcellator made it possible to deliver the 190 gm. kidney through an 11 mm. incision.

Modular, Metal-Catalyzed Cycloisomerization Approach to Angularly Fused Polycyclic Aromatic Hydrocarbons and Their Oxidized Derivatives.

Palladium-catalyzed cross-coupling reactions of 2-bromobenzaldehyde and 6-bromo-2,3-dimethoxybenzaldehyde with 4-methyl-1-naphthaleneboronic acid and acenaphthene-5-boronic acid gave corresponding o-naphthyl benzaldehydes. Corey-Fuchs olefination followed by reaction with n-BuLi led to various 1-(2-ethynylphenyl)naphthalenes. Cycloisomerization of individual 1-(2-ethynylphenyl)naphthalenes to various benzo[c]phenanthrene (BcPh) analogues was accomplished smoothly with catalytic PtCl2 in PhMe. In the case of 4,5-dihydrobenzo[l]acephenanthrylene, oxidation with DDQ gave benzo[l]acephenanthrylene. The dimethoxy-substituted benzo[c]phenanthrenes were demethylated with BBr3 and oxidized to the o-quinones with PDC. Reduction of these quinones with NaBH4 in THF/EtOH in an oxygen atmosphere gave the respective dihydrodiols. Exposure of the dihydrodiols to N-bromoacetamide in THF-H2O led to bromohydrins that were cyclized with Amberlite IRA 400 HO(-) to yield the series 1 diol epoxides. Epoxidation of the dihydrodiols with mCPBA gave the isomeric series 2 diol epoxides. All of the hydrocarbons as well as the methoxy-substituted ones were crystallized and analyzed by X-ray crystallography, and these data are compared to other previously studied BcPh derivatives. The methodology described is highly modular and can be utilized for the synthesis of a wide variety of angularly fused polycyclic aromatic hydrocarbons and their putative metabolites and/or other derivatives.

Links between the three-dimensional movements of whale sharks (Rhincodon typus) and the bio-physical environment off a coral reef.

BACKGROUND: Measuring coastal-pelagic prey fields at scales relevant to the movements of marine predators is challenging due to the dynamic and ephemeral nature of these environments. Whale sharks (Rhincodon typus) are thought to aggregate in nearshore tropical waters due to seasonally enhanced foraging opportunities. This implies that the three-dimensional movements of these animals may be associated with bio-physical properties that enhance prey availability. To date, few studies have tested this hypothesis. METHODS: Here, we conducted ship-based acoustic surveys, net tows and water column profiling (salinity, temperature, chlorophyll fluorescence) to determine the volumetric density, distribution and community composition of mesozooplankton (predominantly euphausiids and copepods) and oceanographic properties of the water column in the vicinity of whale sharks that were tracked simultaneously using satellite-linked tags at Ningaloo Reef, Western Australia. Generalised linear mixed effect models were used to explore relationships between the 3-dimensional movement behaviours of tracked sharks and surrounding prey fields at a spatial scale of ~ 1 km. RESULTS: We identified prey density as a significant driver of horizontal space use, with sharks occupying areas along the reef edge where densities were highest. These areas were characterised by complex bathymetry such as reef gutters and pinnacles. Temperature and salinity profiles revealed a well-mixed water column above the height of the bathymetry (top 40 m of the water column). Regions of stronger stratification were associated with reef gutters and pinnacles that concentrated prey near the seabed, and entrained productivity at local scales (~ 1 km). We found no quantitative relationship between the depth use of sharks and vertical distributions of horizontally averaged prey density. Whale sharks repeatedly dove to depths where spatially averaged prey concentration was highest but did not extend the time spent at these depth layers. CONCLUSIONS: Our work reveals previously unrecognized complexity in interactions between whale sharks and their zooplankton prey.

Pathology of T-cell-mediated drug hypersensitivity reactions and impact of tolerance mechanisms on patient susceptibility.

PURPOSE OF REVIEW: T-cell-mediated drug hypersensitivity is responsible for significant morbidity and mortality, and represents a substantial clinical concern. The purpose of this article is to focus on T-cell reactions and discuss recent advances in disease pathogenesis by exploring the impact of tolerance mechanisms in determining susceptibility in genetically predisposed patients. RECENT FINDINGS: Certain drugs preferentially activate pathogenic T cells that have defined pathways of effector function. Thus, a critical question is what extenuating factors influence the direction of immune activation. A large effort has been given towards identifying phenotypic (e.g., infection) or genotypic (e.g., human leukocyte antigen) associations which predispose individuals to drug hypersensitivity. However, many individuals expressing known risk factors safely tolerate drug administration. Thus, mechanistic insight is needed to determine what confers this tolerance. Herein, we discuss recent clinical/mechanistic findings which indicate that the direction in which the immune system is driven relies upon a complex interplay between co-stimulatory/co-regulatory pathways which themselves depend upon environmental inputs from the innate immune system. SUMMARY: It is becoming increasingly apparent that tolerance mechanisms impact on susceptibility to drug hypersensitivity. As the field moves forward it will be interesting to discover whether active tolerance is the primary response to drug exposure, with genetic factors such as HLA acting as a sliding scale, influencing the degree of regulation required to prevent clinical reactions in patients.

Reduction of amine N-oxides by diboron reagents.

Facile reduction of alkylamino-, anilino-, and pyridyl-N-oxides can be achieved via the use of diboron reagents, predominantly bis(pinacolato)- and in some cases bis(catecholato)diboron [(pinB)(2) and (catB)(2), respectively]. Reductions occur upon simply mixing the amine N-oxide and the diboron reagent in a suitable solvent, at a suitable temperature. Extremely fast reductions of alkylamino- and anilino-N-oxides occur, whereas pyridyl-N-oxides undergo slower reduction. The reaction is tolerant of a variety of functionalities such as hydroxyl, thiol, and cyano groups, as well as halogens. Notably, a sensitive nucleoside N-oxide has also been reduced efficiently. The different rates with which alkylamino- and pyridyl-N-oxides are reduced has been used to perform stepwise reduction of the N,N'-dioxide of (S)-(-)-nicotine. Because it was observed that (pinB)(2) was unaffected by the water of hydration in amine oxides, the feasibility of using water as solvent was evaluated. These reactions also proceeded exceptionally well, giving high product yields. In constrast to the reactions with (pinB)(2), triethylborane reduced alkylamino-N-oxides, but pyridine N-oxide did not undergo efficient reduction even at elevated temperature. Finally, the mechanism of the reductive process by (pinB)(2) has been probed by (1)H and (11)B NMR.

In-Vitro Approaches to Predict and Study T-Cell Mediated Hypersensitivity to Drugs.

Mitigating the risk of drug hypersensitivity reactions is an important facet of a given pharmaceutical, with poor performance in this area of safety often leading to warnings, restrictions and withdrawals. In the last 50 years, efforts to diagnose, manage, and circumvent these obscure, iatrogenic diseases have resulted in the development of assays at all stages of a drugs lifespan. Indeed, this begins with intelligent lead compound selection/design to minimize the existence of deleterious chemical reactivity through exclusion of ominous structural moieties. Preclinical studies then investigate how compounds interact with biological systems, with emphasis placed on modeling immunological/toxicological liabilities. During clinical use, competent and accurate diagnoses are sought to effectively manage patients with such ailments, and pharmacovigilance datasets can be used for stratification of patient populations in order to optimise safety profiles. Herein, an overview of some of the in-vitro approaches to predict intrinsic immunogenicity of drugs and diagnose culprit drugs in allergic patients after exposure is detailed, with current perspectives and opportunities provided.

T cell mediated hypersensitivity to previously tolerated iodinated contrast media precipitated by introduction of atezolizumab.

Many adverse reactions associated with immune checkpoint inhibitor (ICI) treatments are immunologically driven and may necessitate discontinuation of the ICI. Herein, we present a patient who had been administered the radio contrast media amidotrizoate multiple times without issue but who then developed a Stevens-Johnson syndrome reaction after coadministration of atezolizumab. Causality was confirmed by a positive re-challenge with amidotrizoate and laboratory investigations that implicated T cells. Importantly, the introduction of atezolizumab appears to have altered the immunologic response to amidotrizoate in terms of the tolerance-elicitation continuum. Proof of concept studies demonstrated enhancement of recall responses to a surrogate antigen panel following in-vitro (healthy donors) and in-vivo (ICI patients) administrations of ICIs. Our findings highlight the importance of considering all concomitant medications in patients on ICIs who develop immune-mediated adverse reactions. In the event of some immune-related adverse reactions, it may be critical to identify the culprit antigen-forming entity that the ICIs have altered the perception of rather than simply attribute causality to the ICI itself in order to optimize both patient safety and treatment of malignancies.

Characterization of T-Cell Responses to SMX and SMX-NO in Co-Trimoxazole Hypersensitivity Patients Expressing HLA-B*13:01.

HLA-B*13:01-positive patients in Thailand can develop frequent co-trimoxazole hypersensitivity reactions. This study aimed to characterize drug-specific T cells from three co-trimoxazole hypersensitive patients presenting with either Stevens-Johnson syndrome or drug reaction with eosinophilia and systemic symptoms. Two of the patients carried the HLA allele of interest, namely HLA-B*13:01. Sulfamethoxazole and nitroso sulfamethoxazole specific T cell clones were generated from T cell lines of co-trimoxazole hypersensitive HLA-B*13:01-positive patients. Clones were characterized for antigen specificity and cross-reactivity with structurally related compounds by measuring proliferation and cytokine release. Surface marker expression was characterized via flow cytometry. Mechanistic studies were conducted to assess pathways of T cell activation in response to antigen stimulation. Peripheral blood mononuclear cells from all patients were stimulated to proliferate and secrete IFN-γ with nitroso sulfamethoxazole. All sulfamethoxazole and nitroso sulfamethoxazole specific T cell clones expressed the CD4+ phenotype and strongly secreted IL-13 as well as IFN-γ, granzyme B and IL-22. No secretion of IL-17 was observed. A number of nitroso sulfamethoxazole-specific clones cross-reacted with nitroso dapsone but not sulfamethoxazole whereas sulfamethoxazole specific clones cross-reacted with nitroso sulfamethoxazole only. The nitroso sulfamethoxazole specific clones were activated in both antigen processing-dependent and -independent manner, while sulfamethoxazole activated T cell responses via direct HLA binding. Furthermore, activation of nitroso sulfamethoxazole-specific, but not sulfamethoxazole-specific, clones was blocked with glutathione. Sulfamethoxazole and nitroso sulfamethoxazole specific T cell clones from hypersensitive patients were CD4+ which suggests that HLA-B*13:01 is not directly involved in the iatrogenic disease observed in co-trimoxazole hypersensitivity patients.

Palladium-Catalyzed Aryl Amination Reactions of 6-Bromo- and 6-Chloropurine Nucleosides.

Palladium-catalyzed C-N bond forming reactions of 6-bromo- as well as 6-chloropurine ribonucleosides and the 2'-deoxy analogues with aryl amines are described. Efficient conversions were observed with Pd(OAc)(2)/Xantphos/Cs(2)CO(3), in PhMe at 100 °C. Reactions of the bromo nucleoside derivatives could be conducted at a lowered catalytic loading (5 mol % Pd(OAc)(2)/7.5 mol % Xantphos), whereas good product yields were obtained with a higher catalyst load (10 mol % Pd(OAc)(2)/15 mol % Xantphos) when the chloro analogue was employed. Among the examples evaluated, silyl protection for the hydroxyls appears better as compared to acetyl. The methodology has been evaluated via reactions with a variety of aryl amines and by synthesis of biologically relevant deoxyadenosine and adenosine dimers. This is the first detailed analysis of aryl amination reactions of 6-chloropurine nucleosides, and comparison of the two halogenated nucleoside substrates.