Lck signaling inhibition causes improvement in clinical features of psoriatic inflammation through reduction in inflammatory cytokines in CD4+ T cells in imiquimod mouse model.

Psoriasis is a chronic dermal inflammatory disease with a world-wide prevalence in which different immune/non-immune cells, e.g. T cells, macrophages, neutrophils, and keratinocytes play a decisive role. These immune cells interact among themselves by releasing multiple mediators which eventually cause characteristic psoriatic plaques in the skin. T cells are reported to be significant contributors to psoriatic inflammation through release of multiple cytokines which are controlled by several kinases, one of them being Lymphocyte-specific protein tyrosine kinase (Lck). Lck has been reported to be crucial for expression/production of several key inflammatory cytokines though modulation of several other kinases/transcription factors in T cells. Therefore, in this investigation, effect of Lck inhibitor, A-770041 was examined on PLCγ, p38MAPK, NFATc1, NFkB and STAT3, TNF-α, IFN-γ, Foxp3, IL-17A, in CD4+ T cells in imiquimod (IMQ)-induced psoriatic inflammation in mice. Results from the present study exhibit that p-Lck expression is enhanced in CD4+ T cells of IMQ-treated mice which is concomitant with enhanced clinical features of psoriatic inflammation (ear/back skin thickness, MPO activity, acanthosis/leukocytic infiltration) and molecular parameters (enhanced expression of p-Lck, p-PLCγ, p-p38-MAPK, NFATc1, p-NFkB, TNF-α, IFN-γ, p-STAT3, and IL-17A in CD4+ T cells). Inhibition of Lck signaling led to amelioration of clinical features of psoriasis through attenuation of Th1/Th17 immune responses and upregulation of Treg cells in IMQ-treated mice. In summary, current investigations reveal that Lck signaling is a crucial executor of inflammatory signaling in CD4+ T cells in the context of psoriatic inflammation. Therefore, Lck inhibition may be pursued as an effective strategy to counteract psoriatic inflammation.

Hop-derived fraction rich in beta acids and prenylflavonoids regulates the inflammatory response in dendritic cells differently from quercetin: unveiling metabolic changes by mass spectrometry-based metabolomics.

Dendritic cells (DCs) represent a heterogeneous family of immune cells that link innate and adaptive immunity and their activation is linked to metabolic changes that are essential to support their activity and function. Hence, targeting the metabolism of DCs represents an opportunity to modify the inflammatory and immune response. Among the natural matrices, Humulus lupulus (Hop) compounds have recently been shown to exhibit immunomodulatory and anti-inflammatory activity. This study aimed to evaluate the ability of specific Hop fractions to modulate DCs metabolism after stimulation with lipopolysaccharide (LPS) by an untargeted metabolomics approach and compare their effect with flavonol quercetin. Following liquid chromatography-based fractionation, three fractions (A, B, and C) were obtained and tested. Cytokine and gene expression were evaluated using ELISA and qPCR, respectively, while the untargeted metabolomics analysis was performed using a combined HILIC-HRMS and DI-FT-ICR approach. The HOP C fraction and quercetin could both reduce the production of several inflammatory cytokines such as IL-6, IL-1α, IL-1ß, and TNF, but differently from quercetin, the HOP C mechanism is independent of extracellular iron-sequestration and showed significant upregulation of the Nrf2/Nqo1 pathway and Ap-1 compared to quercetin. The untargeted analysis revealed the modulation of several key pathways linked to pro-inflammatory and glycolytic phenotypes. In particular, HOP C treatment could modulate the oxidative step of the pentose phosphate pathway (PPP) and reduce the inflammatory mediator succinate, citrulline, and purine-pyrimidine metabolism, differently from quercetin. These results highlight the potential anti-inflammatory mechanism of specific Hop-derived compounds in restoring the dysregulated metabolism in DCs, which can be used in preventive or adjuvant therapies to suppress the undesirable inflammatory response.

Selecting disease-modifying medications in 5q spinal muscular atrophy.

Spinal muscular atrophy (SMA) is an inherited lower motor neuron disease. SMA occurs secondary to alterations in the survival motor neuron 1 gene (SMN1), which is the main driver of SMN protein production. The severity of the disease is determined by the number of copies of the SMN2 gene, which is a homolog to SMN1 but not as efficient in protein production. Three medications have recently been approved for the treatment of SMA. Nusinersen is an intrathecal antisense oligonucleotide that alters SMN2 pre-mRNA, onasemnogene abeparvovec-xioi is an intravenous SMN1 gene replacement therapy, and risdiplam is an oral small molecule splicing modifier of SMN2. No head-to-head studies have been conducted comparing these medications, so selection of one of these medications for an individual with SMA can be challenging. In this article we outline the efficacy, safety, and other pertinent factors to consider when selecting a therapy for an individual with SMA. The age of the individual and comorbidities, such as liver or kidney disease, help guide treatment choices. All three of these medications are efficacious, and early initiation is critical for obtaining the best outcomes.

Tofacitinib restores the balance of γδTreg/γδT17 cells in rheumatoid arthritis by inhibiting the NLRP3 inflammasome.

Objective: Tofacitinib (TOF) is a Janus kinase (JAK) inhibitor used in the treatment of rheumatoid arthritis (RA), but the mechanism of its action remains unclear. In this study, we investigated the influence of TOF on gamma delta regulatory T-cell (γδTreg)/γδT17 cell balance in RA and the role of the nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3) inflammasome in this process. Methods: We detected levels of inflammatory factors in the serum of RA patients before and after administration of TOF using an enzyme-linked immunosorbent assay (ELISA). A collagen-induced arthritis (CIA) model was constructed to investigate the effect of TOF on arthritis symptoms, γδTreg/γδT17 cell balance and the NLRP3 inflammasome. We used bone marrow-derived macrophages (BMDMs) to study the effect of TOF on NLRP3 inflammasome activation. Nlrp3-/- mice were introduced to assess the influence of NLRP3 on γδT17 cell activation in RA. Results: TOF treatment decreased levels of γδT17 cell-related cytokine interleukin-17 (IL-17) in RA patients. In addition, TOF intervention in the CIA model reduced joint inflammation and damage, rebalanced the γδTreg/γδT17 cell ratio and inhibited excessive NLRP3 inflammasome activation in draining lymph nodes and arthritic joints. BMDM intervention experiments demonstrated that TOF decreased the level of secreted IL-1ß via downregulation of NLRP3. Furthermore, experiments using Nlrp3-/- mice verified that the NLRP3 inflammasome mediated the effect of TOF on γδT17 cell activation. Conclusions: Recovery of γδTreg/γδT17 cell balance was a novel mechanism by which TOF alleviated RA. Meanwhile, NLRP3 played a pivotal role in the process of TOF-mediated γδT17 cell activation.

Nalmefene alleviates the neuroimmune response to repeated binge-like ethanol exposure: A TSPO PET imaging study in adolescent rats.

A large body of preclinical research has shown that neuroimmunity plays a key role in the deleterious effects of alcohol (ethanol) to the brain. Translational imaging techniques are needed to monitor the efficacy of strategies to prevent or mitigate neuroinflammation and alleviate ethanol-induced neurotoxicity. Opioid receptor antagonists such as nalmefene are antagonists of the toll-like receptor 4, which may block the proinflammatory signaling cascade induced by ethanol at this specific target. Male adolescent rats received a validated protocol of ethanol injection (i.p, 3 g/kg daily for two consecutive days followed by two resting days) during 14 days. Positron emission tomography (PET) imaging with the translocator protein 18 kDa (TSPO) radioligand [18 F]DPA-714 was performed at day-15. Toxicity induced by repeated binge-like ethanol exposure (71% mortality) was drastically reduced by nalmefene pretreatment (0.4 mg/kg, 14% mortality). No mortality was observed in animals that received vehicle (control) or nalmefene alone. Compared with control animals (n = 10), a significant 2.8-fold to 4.6-fold increase in the volume of distribution (VT ) of [18 F]DPA-714 was observed among brain regions in animals exposed to ethanol only (n = 9). Pretreatment with nalmefene significantly alleviated the neuroimmune response to ethanol exposure in all brain regions (1.2-fold to 2.5-fold increase in VT ; n = 5). Nalmefene alone (n = 6) did not impact [18 F]DPA-714 VT compared with the control group. Nalmefene may protect against the neuroinflammatory response and overall toxicity associated with binge drinking. [18 F]DPA-714 PET imaging can be used to noninvasively address the neuroimmune impact of ethanol exposure and its modulation by pharmacological strategies in vivo, with translational perspectives.

An Unbiased Immunoaffinity-Based Strategy for Profiling Covalent Drug Targets In Vivo.

Activity-based chemical proteomics approaches used for identifying cellular targets of drugs are mainly dependent on the availability of probes derived from drugs. However, all chemical probes are structurally different from the drugs themselves and cannot fully mimic the real actions of drugs in cells. Here we present a concise and unbiased immunoaffinity-based strategy for identifying covalent drug targets in vivo. By using the specific antibody, we not only confirm the well-known ibrutinib-binding target BTK, but also identify some previously undescribed strongly binding proteins, such as CKAP4 in human cell lines and TAP1 in mouse organs. The observed target profiles between species may partially explain why certain drug candidates are very effective in mice but not in humans. This approach avoids the chemical modification of drugs, eliminates the nonspecific bindings of chemical probes, and allows to unbiasedly decode the underlying mechanisms of action of covalent drugs.

Is the small heat shock protein HspB1 (Hsp27) a real and predominant target of methylglyoxal modification?

This study analyzed the interaction of commercial monoclonal anti-methylglyoxal antibodies that predominantly recognize argpyrimidine with unmodified and modified model proteins and small heat shock proteins. These antibodies specifically recognize methylglyoxal (MG)-modified bovine serum albumin and lysozyme, but they react equally well with both unmodified and MG-modified HspB1. Mutation R188W decreased the interaction of these antibodies with unmodified HspB1, thus indicating that this residue participates in the formation of antigenic determinant. However, these antibodies did not recognize either short (ESRAQ) or long (IPVTFESRAQLGGP) peptides with primary structure identical to that at Arg188 of HspB1. Neither of the peptides obtained after the cleavage of HspB1 at Met or Cys residues were recognized by anti-argpyrimidine antibodies. This means that unmodified HspB1 contains a discontinuous epitope that includes the sequence around Arg188 and that this epitope is recognized by anti-argpyrimidine antibodies in unmodified HspB1. Incubation of HspB1 with MG is accompanied by the accumulation of hydroimidazolones, but not argpyrimidines. Therefore, conclusions based on utilization of anti-argpyrimidine antibodies and indicating that HspB1 is the predominant and preferential target of MG modification in the cell require revision.

Broad-spectrum monoclonal antibody and a sensitive multi-residue indirect competitive enzyme-linked immunosorbent assay for the antibacterial synergists in samples of animal origin.

To monitor the abuse of antibacterial synergists, a hapten, trimethoprim carboxylic derivative (TMPCOOH), was designed by using molecular modelling technology. A broad-spectrum monoclonal antibody (mAb) TMP/2G1 was prepared, for which the IC50 values of trimethoprim, diaveridine, aditoprim, baquiloprim, ormetoprim, and brodimoprim were 0.232, 0.527, 1.479, 4.354, 0.965, and 0.119 µg L-1, respectively. Based on the broad spectrum mAb, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed to determine the residues of antibacterial synergists. The limit of detection regarding the developed ic-ELISA for antibacterial synergists ranged from 0.025 to 1.126 µg L-1 in milk, honey and edible animal tissues. The recoveries ranged from 81.4% to 107.7%, with a coefficient of variation less than 20%. A good correlation (R2 = 0.994) between the ic-ELISA and HPLC-MS/MS showed the reliability of the developed ic-ELISA.

Pyrimidine synthesis inhibition enhances cutaneous defenses against antibiotic resistant bacteria through activation of NOD2 signaling.

Multidrug-resistant bacterial strains are a rapidly emerging healthcare threat; therefore it is critical to develop new therapies to combat these organisms. Prior antibacterial strategies directly target pathogen growth or viability. Host-directed strategies to increase antimicrobial defenses may be an effective alternative to antibiotics and reduce development of resistant strains. In this study, we demonstrated the efficacy of a pyrimidine synthesis inhibitor, N-phosphonacetyl-L-aspartate (PALA), to enhance clearance of methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Acinetobacter baumannii strains by primary human dermal fibroblasts in vitro. PALA did not have a direct bactericidal effect, but enhanced cellular secretion of the antimicrobial peptides human ß-defensin 2 (HBD2) and HBD3 from fibroblasts. When tested in porcine and human skin explant models, a topical PALA formulation was efficacious to enhance MRSA, P. aeruginosa, and A. baumannii clearance. Topical PALA treatment of human skin explants also resulted in increased HBD2 and cathelicidin (LL-37) production. The antimicrobial actions of PALA required expression of nucleotide-binding, oligomerization domain 2 (NOD2), receptor-interacting serine/threonine-protein kinase 2 (RIP2), and carbamoyl phosphatase synthase II/aspartate transcarbamylase/dihydroorotase (CAD). Our results indicate that PALA may be a new option to combat multidrug-resistant bacterial infections of the skin through enhancement of an integral pathway of the cutaneous innate immune defense system.

Imaging the neuroimmune response to alcohol exposure in adolescent baboons: a TSPO PET study using 18 F-DPA-714.

The effects of acute alcohol exposure to the central nervous system are hypothesized to involve the innate immune system. The neuroimmune response to an initial and acute alcohol exposure was investigated using translocator protein 18 kDa (TSPO) PET imaging, a non-invasive marker of glial activation, in adolescent baboons. Three different alcohol-naive adolescent baboons (3-4 years old, 9 to 14 kg) underwent 18 F-DPA-714 PET experiments before, during and 7-12 months after this initial alcohol exposure (0.7-1.0 g/l). The brain distribution of 18 F-DPA-714 (VT ; in ml/cm3 ) was estimated in several brain regions using the Logan plot analysis and the metabolite-corrected arterial input function. Compared with alcohol-naive animals (VTbrain  = 3.7 ± 0.7 ml/cm3 ), the regional VT s of 18 F-DPA-714 were significantly increased during alcohol exposure (VTbrain  = 7.2 ± 0.4 ml/cm3 ; p < 0.001). Regional VT s estimated several months after alcohol exposure (VTbrain  = 5.7 ± 1.4 ml/cm3 ) were lower (p < 0.001) than those measured during alcohol exposure, but remained significantly higher (p < 0.001) than in alcohol-naive animals. The acute and long-term effects of ethanol exposure were observed globally across all brain regions. Acute alcohol exposure increased the binding of 18 F-DPA-714 to the brain in a non-human primate model of alcohol exposure that reflects the 'binge drinking' situation in adolescent individuals. The effect persisted for several months, suggesting a 'priming' of glial cell function after initial alcohol exposure.

Ibrutinib Therapy Increases T Cell Repertoire Diversity in Patients with Chronic Lymphocytic Leukemia.

The Bruton's tyrosine kinase inhibitor ibrutinib is a highly effective, new targeted therapy for chronic lymphocytic leukemia (CLL) that thwarts leukemia cell survival, growth, and tissue homing. The effects of ibrutinib treatment on the T cell compartment, which is clonally expanded and thought to support the growth of malignant B cells in CLL, are not fully characterized. Using next-generation sequencing technology, we characterized the diversity of TCRß-chains in peripheral blood T cells from 15 CLL patients before and after 1 y of ibrutinib therapy. We noted elevated CD4+ and CD8+ T cell numbers and a restricted TCRß repertoire in all pretreatment samples. After 1 y of ibrutinib therapy, elevated peripheral blood T cell numbers and T cell-related cytokine levels had normalized, and T cell repertoire diversity increased significantly. Dominant TCRß clones in pretreatment samples declined or became undetectable, and the number of productive unique clones increased significantly during ibrutinib therapy, with the emergence of large numbers of low-frequency TCRß clones. Importantly, broader TCR repertoire diversity was associated with clinical efficacy and lower rates of infections during ibrutinib therapy. These data demonstrate that ibrutinib therapy increases diversification of the T cell compartment in CLL patients, which contributes to cellular immune reconstitution.

Pharmacokinetics of metronomic chemotherapy: a neglected but crucial aspect.

Metronomic chemotherapy describes the close, regular administration of chemotherapy drugs at less-toxic doses over prolonged periods of time. In 2015, the results of randomized phase III clinical trials demonstrated encouraging, albeit limited, efficacy benefits of metronomic chemotherapy regimens administered as adjuvant maintenance therapy for the treatment of breast cancer, or as maintenance therapy in combination with an antiangiogenic agent for metastatic colorectal cancer. Owing to the investigational nature of this approach, metronomic chemotherapy regimens are highly empirical in terms of the optimal dose and schedule for the drugs administered; therefore, greater knowledge of the pharmacokinetics of metronomic chemotherapy is critical to the future success of this treatment strategy. Unfortunately, such preclinical and clinical pharmacokinetic studies are rare. Herein, we present situations in which active drug concentrations have been achieved with metronomic schedules, and discuss their associated pharmacokinetic parameters. We summarize examples from the limited number of clinical studies in order to illustrate the importance of assessing such pharmacokinetic parameters, and discuss the influence this information can have on improving efficacy and reducing toxicity.

Preparation of Antibodies and Development of an Enzyme-Linked Immunosorbent Assay for the Tyrosine Kinase Inhibitors Lapatinib and Nilotinib.

In this paper, we describe the production of the first specific antibodies against the tyrosine kinase inhibitors lapatinib and nilotinib. Anti-lapatinib antibody was obtained by immunizing rabbits with an antigen conjugated with bovine serum albumin using 3-chloro-4-((3-fluorobenzyl)oxy)aniline. Anti-nilotinib antibody was produced by immunizing mice with an antigen conjugated with bovine serum albumin using 2-(5-amino-2-methylanilino)-4-(3-pyridyl)pyrimidine. The generated antibodies were used to develop highly sensitive and specific enzyme-linked immunosorbent assays (ELISAs) for lapatinib and nilotinib in human serum. The assays were capable of detecting lapatinib and nilotinib at serum concentrations as low as 40 and 8 ng/mL, respectively. Using the two ELISAs, drugs levels were easily measured in the serum of rats after a single dose oral administration of lapatinib or nilotinib. The assays are therefore expected be valuable tools for therapeutic drug monitoring in the clinical setting and pharmacokinetic studies of lapatinib and nilotinib.

Macrophage depletion ameliorates nephritis induced by pathogenic antibodies.

Kidney involvement affects 40-60% of patients with lupus, and is responsible for significant morbidity and mortality. Using depletion approaches, several studies have suggested that macrophages may play a key role in the pathogenesis of lupus nephritis. However, "off target" effects of macrophage depletion, such as altered hematopoiesis or enhanced autoantibody production, impeded the determination of a conclusive relationship. In this study, we investigated the role of macrophages in mice receiving rabbit anti-glomerular antibodies, or nephrotoxic serum (NTS), an experimental model which closely mimics the immune complex mediated disease seen in murine and human lupus nephritis. GW2580, a selective inhibitor of the colony stimulating factor-1 (CSF-1) receptor kinase, was used for macrophage depletion. We found that GW2580-treated, NTS challenged mice did not develop the increased levels of proteinuria, serum creatinine, and BUN seen in control-treated, NTS challenged mice. NTS challenged mice exhibited significantly increased kidney expression of inflammatory cytokines including RANTES, IP-10, VCAM-1 and iNOS, whereas GW2580-treated mice were protected from the robust expression of these inflammatory cytokines that are associated with lupus nephritis. Quantification of macrophage related gene expression, flow cytometry analysis of kidney single cell suspensions, and immunofluorescence staining confirmed the depletion of macrophages in GW2580-treated mice, specifically within renal glomeruli. Our results strongly implicate a specific and necessary role for macrophages in the development of immune glomerulonephritis mediated by pathogenic antibodies, and support the development of macrophage targeting approaches for the treatment of lupus nephritis.

Inhibitors of SRC kinases impair antitumor activity of anti-CD20 monoclonal antibodies.

Clinical trials with SRC family kinases (SFKs) inhibitors used alone or in a combination with anti-CD20 monoclonal antibodies (mAbs) are currently underway in the treatment of B-cell tumors. However, molecular interactions between these therapeutics have not been studied so far. A transcriptional profiling of tumor cells incubated with SFKs inhibitors revealed strong downregulation of MS4A1 gene encoding CD20 antigen. In a panel of primary and established B-cell tumors we observed that SFKs inhibitors strongly affect CD20 expression at the transcriptional level, leading to inhibition of anti-CD20 mAbs binding and increased resistance of tumor cells to complement-dependent cytotoxicity. Activation of the AKT signaling pathway significantly protected cells from dasatinib-triggered CD20 downregulation. Additionally, SFKs inhibitors suppressed antibody-dependent cell-mediated cytotoxicity by direct inhibition of natural killer cells. Abrogation of antitumor activity of rituximab was also observed in vivo in a mouse model. Noteworthy, the effects of SFKs inhibitors on NK cell function are largely reversible. The results of our studies indicate that development of optimal combinations of novel treatment modalities with anti-CD20 mAbs should be preceded by detailed preclinical evaluation of their effects on target cells.

T-cell activation by transitory neo-antigens derived from distinct microbial pathways.

T cells discriminate between foreign and host molecules by recognizing distinct microbial molecules, predominantly peptides and lipids. Riboflavin precursors found in many bacteria and yeast also selectively activate mucosal-associated invariant T (MAIT) cells, an abundant population of innate-like T cells in humans. However, the genesis of these small organic molecules and their mode of presentation to MAIT cells by the major histocompatibility complex (MHC)-related protein MR1 (ref. 8) are not well understood. Here we show that MAIT-cell activation requires key genes encoding enzymes that form 5-amino-6-d-ribitylaminouracil (5-A-RU), an early intermediate in bacterial riboflavin synthesis. Although 5-A-RU does not bind MR1 or activate MAIT cells directly, it does form potent MAIT-activating antigens via non-enzymatic reactions with small molecules, such as glyoxal and methylglyoxal, which are derived from other metabolic pathways. The MAIT antigens formed by the reactions between 5-A-RU and glyoxal/methylglyoxal were simple adducts, 5-(2-oxoethylideneamino)-6-D-ribitylaminouracil (5-OE-RU) and 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU), respectively, which bound to MR1 as shown by crystal structures of MAIT TCR ternary complexes. Although 5-OP-RU and 5-OE-RU are unstable intermediates, they became trapped by MR1 as reversible covalent Schiff base complexes. Mass spectra supported the capture by MR1 of 5-OP-RU and 5-OE-RU from bacterial cultures that activate MAIT cells, but not from non-activating bacteria, indicating that these MAIT antigens are present in a range of microbes. Thus, MR1 is able to capture, stabilize and present chemically unstable pyrimidine intermediates, which otherwise convert to lumazines, as potent antigens to MAIT cells. These pyrimidine adducts are microbial signatures for MAIT-cell immunosurveillance.

The second-generation mTOR kinase inhibitor INK128 exhibits anti-inflammatory activity in lipopolysaccharide-activated RAW 264.7 cells.

Cross-talk between the mTOR (mechanistic target of rapamycin) and NF-κB (nuclear factor kappa-B) pathways has been reported to regulate macrophage responses to lipopolysaccharide (LPS). In this study, we aimed to explore the effect of INK128, a second-generation inhibitor of mTOR, on the inflammatory cytokine production in LPS-stimulated RAW 264.7 cells. Our data showed that INK128 strikingly inhibited the phosphorylation of p70S6K, 4E-BP1 and AKTSer473 in both unstimulated and LPS-stimulated cells. Although it increased the phosphorylation levels of inhibitor kappa-B (IκB) in LPS-stimulated cells, INK128 did not significantly change the levels of NF-κB phosphorylation. In addition, LPS-induced expression of IL-1ß and IL-6 was markedly suppressed by INK128 at both mRNA and protein levels. However, the expression of Tumor necrosis factor-alpha (TNF-α protein), but not its mRNA level, was suppressed by this reagent. Our results suggest that the mTOR inhibitor INK128 not only regulates the NF-κB signaling but also influences the inflammatory cytokine expression at both transcriptional and translational levels.

Development of a specific and sensitive enzyme-linked immunosorbent assay for the quantification of imatinib.

Imatinib is an oral tyrosine kinase inhibitor used for first-line treatment of chronic myeloid leukemia. Therapeutic drug monitoring targeting trough plasma levels of about 1000 ng/mL may help to optimize imantinib's therapeutic effect. This paper reports a specific and sensitive enzyme-linked immunosorbent assay (ELISA) for a pharmacokinetic evaluation of imatinib. Anti-imatinib antibody was obtained by immunizing rabbits with an antigen conjugated with bovine serum albumin and succinimidyl 4-{(4-methyl-1-piperazinyl)methyl}-benzoate. Enzyme labeling of imatinib with horseradish peroxidase was similarly performed using succinimidyl 4-{(4-methyl-1-piperazinyl)methyl}-benzoate. A simple ELISA for imatinib was developed using the principle of direct competition between imatinib and the enzyme marker for anti-imatinib antibody which had been adsorbed by the plastic surface of a microtiter plate. Serum imatinib concentrations lower than 40 pg/mL were reproducibly measurable using the ELISA. This ELISA was specific to imatinib and showed very slight cross-reactivity (1.2%) with a major metabolite, N-desmethyl imatinib. Using this assay, drug levels were easily measured in the blood of mice after their oral administration of imatinib at a single dose of 50 mg/kg. The specificity and sensitivity of the ELISA for imatinib should provide a valuable new tool for use in therapeutic drug monitoring and pharmacokinetic studies of imatinib.

Inhibition of pyrimidine biosynthesis pathway suppresses viral growth through innate immunity.

Searching for stimulators of the innate antiviral response is an appealing approach to develop novel therapeutics against viral infections. Here, we established a cell-based reporter assay to identify compounds stimulating expression of interferon-inducible antiviral genes. DD264 was selected out of 41,353 compounds for both its immuno-stimulatory and antiviral properties. While searching for its mode of action, we identified DD264 as an inhibitor of pyrimidine biosynthesis pathway. This metabolic pathway was recently identified as a prime target of broad-spectrum antiviral molecules, but our data unraveled a yet unsuspected link with innate immunity. Indeed, we showed that DD264 or brequinar, a well-known inhibitor of pyrimidine biosynthesis pathway, both enhanced the expression of antiviral genes in human cells. Furthermore, antiviral activity of DD264 or brequinar was found strictly dependent on cellular gene transcription, nuclear export machinery, and required IRF1 transcription factor. In conclusion, the antiviral property of pyrimidine biosynthesis inhibitors is not a direct consequence of pyrimidine deprivation on the virus machinery, but rather involves the induction of cellular immune response.

Carbon nanotube-protein carriers enhance size-dependent self-adjuvant antibody response to haptens.

Carbon nanotubes (CNTs) are nanomaterials with interesting emerging applications. Their properties make CNTs excellent candidates for use as new nanovehicles in drug delivery, immunization and diagnostics. In the current study, we assessed the immune-response-amplifying properties of CNTs to haptens by using azoxystrobin, the first developed strobilurin fungicide, as a model analyte. An azoxystrobin derivative bearing a carboxylated spacer arm (hapten AZc6) was covalently coupled to bovine serum albumin (BSA), and the resulting BSA-AZc6 conjugate was covalently linked to four functionalized CNTs of different shapes and sizes, varying in diameter and length. These four types of CNT-based constructs were obtained using efficient, fast, and easy functionalization procedures based on microwave-assisted chemistry. New Zealand rabbits and BALB/c mice were immunized with BSA-AZc6 alone and with the four CNT-BSA-AZc6 constructs, both with and without Freund's adjuvant. The IgG-type antibody responses were assessed in terms of the titer and affinity, paying special attention to the relationship between the immune response and the size and shape of the employed CNTs. Immunization with CNT-BSA-AZc6 resulted in enhanced titers and excellent affinities for azoxystrobin. More important, remarkable IgG responses were obtained even in the absence of an adjuvant, thus proving the self-adjuvanting capability of CNTs. Immunogens were able to produce strong anti-azoxystrobin immune responses in rabbits even when administered at a BSA-AZc6 conjugate dose as low as 0.05 µg. The short and thick CNT-BSA-AZc6 construct produced the best antibody response under all tested conditions.