Iodixanol activation of mast cells: Implications in the pathogenesis of iodixanol-induced delayed cutaneous adverse reactions.

Iodinated contrast media (ICM) is widely used in radiological examination and interventional therapy. In the commonly used ICM, iodixanol is considered to be the safer one. However, compared with other ICMs, it has a higher incidence of delayed cutaneous adverse reactions. The underlying mechanisms are unclear. In this study, mice with positive allergic reactions were selected based on the mouse clinical allergy symptom score and skin and blood samples taken 1, 6, 24, 48, and 72 h after ICMs (6 g iodine/kg) injection for histological and blood analyses. ICMs-induced pseudo-allergic reactions were investigated through in vivo intravital vascular imaging and passive cutaneous anaphylaxis (PCA) not mediated by IgE and through, calcium imaging degranulation of mast cells (MCs), and western blot assays in vitro. Results shows iodixanol-induced systemic anaphylaxis caused severe extravasation of plasma proteins and degranulation of skin MCs, and increased levels of plasma histamine, cytokines and inflammatory chemokines. Mechanistically, iodixanol increases degranulation of MCs and promotes the synthesis of inflammatory factors by activating PLC-γ and PI3K-related pathways. Trigonelline inhibit iodixanol-induced MC-related pseudo-allergic reactions in vitro and in vivo. These results suggest that mice in the iodixanol group had a higher incidence of delayed cutaneous reactions, characterized by cytokine release over time and delayed cutaneous MC degranulation. Iodixanol's delayed cutaneous adverse reactions may be due to a delayed phase of MC-related pseudo-allergic reactions. Trigonelline revealed anti-allergic activity in iodixanol-induced MC-related pseudo-allergic reactions.

Skin Resident Memory T Cells May Play Critical Role in Delayed-Type Drug Hypersensitivity Reactions.

Delayed-type drug hypersensitivity reactions (dtDHR) are immune-mediated reactions with skin and visceral manifestations ranging from mild to severe. Clinical care is negatively impacted by a limited understanding of disease pathogenesis. Though T cells are believed to orchestrate disease, the type of T cell and the location and mechanism of T cell activation remain unknown. Resident memory T cells (TRM) are a unique T cell population potentially well situated to act as key mediators in disease pathogenesis, but significant obstacles to defining, identifying, and testing TRM in dtDHR preclude definitive conclusions at this time. Deeper mechanistic interrogation to address these unanswered questions is necessary, as involvement of TRM in disease has significant implications for prediction, diagnosis, and treatment of disease.

The Ability of AhR Ligands to Attenuate Delayed Type Hypersensitivity Reaction Is Associated With Alterations in the Gut Microbiota.

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates T cell function. The aim of this study was to investigate the effects of AhR ligands, 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), and 6-Formylindolo[3,2-b]carbazole (FICZ), on gut-associated microbiota and T cell responses during delayed-type hypersensitivity (DTH) reaction induced by methylated bovine serum albumin (mBSA) in a mouse model. Mice with DTH showed significant changes in gut microbiota including an increased abundance of Bacteroidetes and decreased Firmicutes at the phylum level. Also, there was a decrease in Clostridium cluster XIV and IV, which promote anti-inflammatory responses, and an increase in Prevotella copri that facilitates pro-inflammatory responses. Interestingly, treatment of mice with TCDD attenuated the DTH response, induced Tregs, suppressed Th17 cells in the mesenteric lymph nodes (MLNs), and reversed the gut microbiota composition toward normalcy. In contrast, FICZ exacerbated the DTH response, induced heightened Th17 cells, and failed to cause a major shift in gut microbiota. Furthermore, TCDD but not FICZ caused an increase in the levels of short-chain fatty acids (SCFA), n-butyric acid, and acetic acid. Administration of sodium butyrate into mice with DTH suppressed the response, increased Tregs, and reduced Th17 cells IL17. Butyrate also caused an increase in the abundance of Clostridium and a decrease in Prevotella. Lastly, TCDD, as well as butyrate but not FICZ, were able to inhibit proinflammatory Histone deacetylases (HDACs) class I and II. Together, our data suggest that AhR ligands, such as TCDD that suppress DTH response, may mediate this effect by reversing the gut dysbiosis induced during this inflammatory response, while FICZ may fail to suppress the DTH response because of its inability to overturn the dysbiosis.

Lymphocyte transformation test and cytokine detection assays: Determination of read out parameters for delayed-type drug hypersensitivity reactions.

Drug hypersensitivity reactions (DHRs) occur in certain people and are often not predictable. DHRs can be classified as immediate and delayed reactions regarding to onset of clinical manifestations. Both reactions are considered to be an important public health problem because they can lead to life-threatening conditions; however, this review article will focus on delayed DHRs. The most important points for diagnosis of delayed DHRs are the recognition of drug hypersensitivity characteristics and culprit drug identification. While it is usually difficult to identify a culprit drug; clinical evaluation using the causality assessment method, a non-invasive process, can identify the culprit drug without the need for intensive investigation. Delayed DHRs can cause life-threatening conditions; therefore, in vivo skin tests, as well as drug provocation tests, have to be cautiously performed by a drug allergist and have not been recommended in uncontrolled conditions. ENDA/EAACI has recommended that in vitro tests (if available) be performed prior to any in vivo tests. Therefore, in vitro diagnostic tests can be alternative methods to identify a culprit drug for delayed DHR diagnosis as there is no or very low risk for patients under investigation. There are many testing approaches to identify causative agents for delayed DHRs such as: the lymphocyte transformation test (LTT), cytokine/mediator detection assays (i.e. ELISA and flow cytometry-based bead assays), multiplex bead-based immunoassay and ELISpot. The LTT is the most standardized method whereas it has been available in medical schools affiliated with university hospitals. Other in vitro tests, like cytokine detection assays, have also been used, even though they are still being evaluated. They could supplement LTT results that would provide drug allergist's with documentary evidence and prevent risk to patients by avoiding in vivo or drug provocation testing. Hence, the in vitro tests have been promising tests contributing to the management of the delayed DHR work-up process in clinical practice.

miR-155 Contributes to the Immunoregulatory Function of Human Mesenchymal Stem Cells.

Objectives: Mesenchymal stem/stromal cells (MSCs) are widely investigated in regenerative medicine thanks to their immunomodulatory properties. They exert their anti-inflammatory function thanks to the secretion of a number of mediators, including proteins and miRNAs, which can be released in the extracellular environment or in the cargo of extracellular vesicles (EVs). However, the role of miRNAs in the suppressive function of MSCs is controversial. The aim of the study was to identify miRNAs that contribute to the immunomodulatory function of human bone marrow-derived MSCs (BM-MSCs). Methods: Human BM-MSCs were primed by coculture with activated peripheral blood mononuclear cells (aPBMCs). High throughput miRNA transcriptomic analysis was performed using Human MicroRNA TaqMan® Array Cards. The immunosuppressive function of miRNAs was investigated in mixed lymphocyte reactions and the delayed type hypersensitivity (DTH) murine model. Results: Upon priming, 21 out of 377 tested miRNAs were significantly modulated in primed MSCs. We validated the up-regulation of miR-29a, miR-146a, miR-155 and the down-regulation of miR-149, miR-221 and miR-361 in additional samples of primed MSCs. We showed that miR-155 significantly reduced the proliferation of aPBMCs in vitro and inflammation in vivo, using the DTH model. Analysis of miRNA-mRNA interactions revealed miR-221 as a potential target gene that is down-regulated by miR-155 both in primed MSCs and in aPBMCs. Conclusion: Here, we present evidence that miR-155 participates to the immunosuppressive function of human BM-MSCs and down-regulates the expression of miR-221 as a possible inflammatory mediator.

AhR Ligands Differentially Regulate miRNA-132 Which Targets HMGB1 and to Control the Differentiation of Tregs and Th-17 Cells During Delayed-Type Hypersensitivity Response.

Aryl hydrocarbon receptor (AhR), is a transcription factor and an environmental sensor that has been shown to regulate T cell differentiation. Interestingly, AhR ligands exert varying effects from suppression to exacerbation of inflammation through induction of Tregs and Th-17 cells, respectively. In the current study, we investigated whether the differential effects of AhR ligands on T cell differentiation are mediated by miRNA during delayed-type hypersensitivity (DTH) reaction against methylated Bovine Serum Albumin (mBSA). Treatment of C57BL/6 mice with TCDD attenuated mBSA-mediated DTH response, induced Tregs, decreased Th-17 cells, and caused upregulation of miRNA-132. TCDD caused an increase in several Treg subsets including inducible peripheral, natural thymic, and Th3 cells. Also, TCDD increased TGF-ß and Foxp3 expression. In contrast, treating mice with FICZ exacerbated the DTH response, induced inflammatory Th17 cells, induced IL-17, and RORγ. Analysis of miRNA profiles from draining lymph nodes showed that miR-132 was upregulated in the TCDD group and downregulated in the FICZ group. Transfection studies revealed that miRNA-132 targeted High Mobility Group Box 1 (HMGB1). Downregulation of HMGB1 caused an increase in FoxP3+ Treg differentiation and suppression of Th-17 cells while upregulation of HMGB1 caused opposite effects. Moreover, TCDD was less effective in suppressing DTH response and induction of Tregs in mice that were deficient in miR-132. In summary, this study demonstrates that TCDD and FICZ have divergent effects on DTH response and T cell differentiation, which is mediated through, at least in part, regulation of miRNA-132 that targets HMGB1.

Immunomodulatory role of reactive oxygen species and nitrogen species during T cell-driven neutrophil-enriched acute and chronic cutaneous delayed-type hypersensitivity reactions.

Rationale: Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are important regulators of inflammation. The exact impact of ROS/RNS on cutaneous delayed-type hypersensitivity reaction (DTHR) is controversial. The aim of our study was to identify the dominant sources of ROS/RNS during acute and chronic trinitrochlorobenzene (TNCB)-induced cutaneous DTHR in mice with differently impaired ROS/RNS production. Methods: TNCB-sensitized wild-type, NADPH oxidase 2 (NOX2)- deficient (gp91phox-/-), myeloperoxidase-deficient (MPO-/-), and inducible nitric oxide synthase-deficient (iNOS-/-) mice were challenged with TNCB on the right ear once to elicit acute DTHR and repetitively up to five times to induce chronic DTHR. We measured ear swelling responses and noninvasively assessed ROS/RNS production in vivo by employing the chemiluminescence optical imaging (OI) probe L-012. Additionally, we conducted extensive ex vivo analyses of inflamed ears focusing on ROS/RNS production and the biochemical and morphological consequences. Results: The in vivo L-012 OI of acute and chronic DTHR revealed completely abrogated ROS/RNS production in the ears of gp91phox-/- mice, up to 90 % decreased ROS/RNS production in the ears of MPO-/- mice and unaffected ROS/RNS production in the ears of iNOS-/- mice. The DHR flow cytometry analysis of leukocytes derived from the ears with acute DTHR confirmed our in vivo L-012 OI results. Nevertheless, we observed no significant differences in the ear swelling responses among all the experimental groups. The histopathological analysis of the ears of gp91phox-/- mice with acute DTHRs revealed slightly enhanced inflammation. In contrast, we observed a moderately reduced inflammatory immune response in the ears of gp91phox-/- mice with chronic DTHR, while the inflamed ears of MPO-/- mice exhibited the strongest inflammation. Analyses of lipid peroxidation, 8-hydroxy-2'deoxyguanosine levels, redox related metabolites and genomic expression of antioxidant proteins revealed similar oxidative stress in all experimental groups. Furthermore, inflamed ears of wild-type and gp91phox-/- mice displayed neutrophil extracellular trap (NET) formation exclusively in acute but not chronic DTHR. Conclusions: MPO and NOX2 are the dominant sources of ROS/RNS in acute and chronic DTHR. Nevertheless, depletion of one primary source of ROS/RNS exhibited only marginal but conflicting impact on acute and chronic cutaneous DTHR. Thus, ROS/RNS are not a single entity, and each species has different properties at certain stages of the disease, resulting in different outcomes.

Evaluation of the immunomodulatory and anti-inflammatory activity of Bakuchiol using RAW 264.7 macrophage cell lines and in animal models stimulated by lipopolysaccharide (LPS).

Bakuchiol (BAK) has been reported to have a diverse pharmacological property as an antibiotic, anti-cancer, anti-hypolipidemic, anti-inflammatory and anti-convulsant agent. This study aimed to elucidate the immunomodulation and anti-inflammatory mechanism of bakuchiol using lipopolysaccharide stimulated RAW 264.7 macrophages and various animal models. The present study has shown that BAK significantly suppressed the pro-inflammatory cytokine expression in a dose-dependent manner and its oral administration significantly decreased delayed hypersensitivity responses as compared to control group. The assessment of immunomodulatory activity was carried out by the testing Hemagglutinating antibody (HA) titer, delayed type hypersensitivity (DTH) responses and phagocytic index by carbon clearance test. On the other hand, it showed significant decrease in circulating antibody titer and carbon clearance assay in a concentration-dependent manner. BAK has significantly potentiated the cellular immunity as well as humoral immunity by facilitating the footpad thickness responses in sheep RBCs in sensitized mice by significantly decreasing circulating antibody titer. Molecular studies revealed that BAK inhibited the activation of upstream mediator nuclear factor-κB by suppressing the phosphorylation of IκBα and p65. The responses were statistically significant as compared with the control (*p < 0.05, **p < 0.01).

The effect of cytoplasmic crude extracts of Trichophyton verrucosum on cell mediated immunity.

OBJECTIVE: Trichophyton verrucosum is a slow growing dermatophyte responsible for a number of skin diseases such as ringworm, and is characterized by patches of hair loss and thick crusts on the host skin in domestic animals. In this study, we examined the immunomodulatory effects of crude extract of Trichophyton verrucosum (TV)cytoplasm in a mouse model. METHODS: The TV variate was cultured on Sabouraud dextrose agar and the mycelium was grinded by mechanical force. The purified protein was obtained from crude extract of the fungus, and protein concentration was measured by BradFord assay. Six to eight week-female BALB/c mice were divided into three groups: test group, receiving cytoplasmic crude extract plus defibrinated sheep blood; control group, receiving defibrinated sheep blood; and normal group, receiving normal saline. Injections were performed on days 0, 3, 5, 7 and 9 and the mice were sacrificed four days after the last injection. T lymphocyte metabolic activity was examined by methyl thiazol tetrazolium (MTT) assay, and also interleukin-4 (IL-4) and interferon-γ (IFNγ) levels were evaluated by enzyme-linked immunosorbent assay (ELISA). RESULTS: MTT assay showed that the TV extract stimulated lymphocyte metabolic activity. ELISA results showed that despite increase in the level of IFNγ, no changes were observed in IL-4 level. CONCLUSIONS: Results indicated that crude extract of TV cytoplasm may probably act as an immune modulator, which affects Th1 responses. The TV crude extract may be an appropriate agent to induce cellular immunity for combating dermatophytosis infection in animals; and therefore, TV extract may have some potential applications in vaccine/adjuvant technology.

Annexin V expression on CD4+ T cells with regulatory function.

Regulatory T (Treg) cells induce immunologic tolerance by suppressing effector functions of conventional lymphocytes in the periphery. On the other hand, immune silencing is mediated by recognition of phosphatidylserine (PS) on apoptotic cells by phagocytes. Here we describe expression of the PS-binding protein Annexin V (ANXA5) in CD4+  CD25hi Treg cells at the mRNA and protein levels. CD4+  ANXA5+ T cells constitute about 0·1%-0·6% of peripheral blood CD3+ T cells, exhibit co-expression of several Treg markers, such as Forkhead box P3, programmed cell death protein-1, cytotoxic T-lymphocyte antigen-4 and CD38. In vitro, ANXA5+ Treg cells showed enhanced adhesion to PS+ endothelial cells. Stimulated by anti-CD3 and PS+ syngeneic antigen-presenting cells CD4+  ANXA5+ T cells expanded in the absence of exogenous interleukin-2. CD4+  ANXA5+ T cells suppressed CD4+  ANXA5- T-cell proliferation and mammalian target of rapamycin phosphorylation, partially dependent on cell contact. CD4+  ANXA5+ T-cell-mediated suppression was allo-specific and accompanied by an increased production of anti-inflammatory mediators. In vivo, using a model of delayed type hypersensitivity, murine CD4+  ANXA5+ T cells inhibited T helper type 1 responses. In conclusion, we report for the first time expression of ANXA5 on a subset of Treg cells that might bridge classical regulatory Treg function with immune silencing.

Flucloxacillin Hypersensitivity: Patient Outcomes in a Multicenter Retrospective Study.

BACKGROUND: Flucloxacillin is a narrow-spectrum, beta-lactamase-resistant penicillin. Type I (IgE-mediated) and type IV (T-cell-mediated) reactions are less frequently reported than with other penicillins. OBJECTIVE: To undertake a detailed clinical characterization of a cohort of patients referred with suspected flucloxacillin hypersensitivity. METHODS: We retrospectively analyzed demographic characteristics, presentation, investigation, and management of 108 patients presenting to 4 UK centers. Patients underwent skin prick and intradermal testing with flucloxacillin, major (benzylpenicilloyl poly-l-lysine) and minor determinants, amoxicillin, and benzylpenicillin with immediate and, where appropriate, delayed reading of the test. In the immediate group, a further 14 patients were tested to ampicillin and 16 to Augmentin (co-amoxiclav-combination of clavulanic acid and amoxicillin). Skin test-negative patients underwent oral drug provocation. A multistep protocol was used, depending on risk assessment. RESULTS: Forty of 108 (37%) patients were diagnosed with hypersensitivity to flucloxacillin, of whom 33 (82.5%) showed immediate and 7 (17.5%) nonimmediate hypersensitivity, respectively. In the immediate group, most reactions were severe: 19 of 33 (58%). Intradermal testing had a higher negative predictive value (86%) in the immediate group than in the nonimmediate group (67%). Only a minority of patients (6 of 17 [35%]) with IgE-mediated allergy were cross-sensitized on intradermal testing with other penicillins, compared with 3 of 4 (75%) in the delayed group. CONCLUSIONS: Immediate hypersensitivity reactions to flucloxacillin are more common than delayed. Cross-sensitization to other penicillins appears higher in delayed reactions than in immediate. The negative predictive value of intradermal testing is higher in the immediate group than in the nonimmediate group. Drug provocation testing remains the diagnostic criterion standard.

Vps34/PI3KC3 deletion in kidney proximal tubules impairs apical trafficking and blocks autophagic flux, causing a Fanconi-like syndrome and renal insufficiency.

Kidney proximal tubular cells (PTCs) are highly specialized for ultrafiltrate reabsorption and serve as paradigm of apical epithelial differentiation. Vps34/PI3-kinase type III (PI3KC3) regulates endosomal dynamics, macroautophagy and lysosomal function. However, its in vivo role in PTCs has not been evaluated. Conditional deletion of Vps34/PI3KC3 in PTCs by Pax8-Cre resulted in early (P7) PTC dysfunction, manifested by Fanconi-like syndrome, followed by kidney failure (P14) and death. By confocal microscopy, Vps34∆/∆ PTCs showed preserved apico-basal specification (brush border, NHERF-1 versus Na+/K+-ATPase, ankyrin-G) but basal redistribution of late-endosomes/lysosomes (LAMP-1) and mis-localization to lysosomes of apical recycling endocytic receptors (megalin, cubilin) and apical non-recycling solute carriers (NaPi-IIa, SGLT-2). Defective endocytosis was confirmed by Texas-red-ovalbumin tracing and reduced albumin content. Disruption of Rab-11 and perinuclear galectin-3 compartments suggested mechanistic clues for defective receptor recycling and apical biosynthetic trafficking. p62-dependent autophagy was triggered yet abortive (p62 co-localization with LC3 but not LAMP-1) and PTCs became vacuolated. Impaired lysosomal positioning and blocked autophagy are known causes of cell stress. Thus, early trafficking defects show that Vps34 is a key in vivo component of molecular machineries governing apical vesicular trafficking, thus absorptive function in PTCs. Functional defects underline the essential role of Vps34 for PTC homeostasis and kidney survival.

Bifenthrin causes transcriptomic alterations in mTOR and ryanodine receptor-dependent signaling and delayed hyperactivity in developing zebrafish (Danio rerio).

Over the last few decades, the pyrethroid insecticide bifenthrin has been increasingly employed for pest control in urban and agricultural areas, putting humans and wildlife at increased risk of exposure. Exposures to nanomolar (nM) concentrations of bifenthrin have recently been reported to alter calcium oscillations in rodent neurons. Neuronal calcium oscillations are influenced by ryanodine receptor (RyR) activity, which modulates calcium-dependent signaling cascades, including the mechanistic target of rapamycin (mTOR) signaling pathway. RyR activity and mTOR signaling play critical roles in regulating neurodevelopmental processes. However, whether environmentally relevant levels of bifenthrin alter RyR or mTOR signaling pathways to influence neurodevelopment has not been addressed. Therefore, our main objectives in this study were to examine the transcriptomic responses of genes involved in RyR and mTOR signaling pathways in zebrafish (Danio rerio) exposed to low (ng/L) concentrations of bifenthrin, and to assess the potential functional consequences by measuring locomotor responses to external stimuli. Wildtype zebrafish were exposed for 1, 3 and 5 days to 1, 10 and 50 ng/L bifenthrin, followed by a 14 d recovery period. Bifenthrin elicited significant concentration-dependent transcriptional responses in the majority of genes examined in both signaling cascades, and at all time points examined during the acute exposure period (1, 3, and 5 days post fertilization; dpf), and at the post recovery assessment time point (19 dpf). Changes in locomotor behavior were not evident during the acute exposure period, but were observed at 19 dpf, with main effects (increased locomotor behavior) detected in fish exposed developmentally to bifenthrin at 1 or 10 ng/L, but not 50 ng/L. These findings illustrate significant influences of developmental exposures to low (ng/L) concentrations of bifenthrin on neurodevelopmental processes in zebrafish.

The immune response in trichloroethylene hypersensitivity syndrome: A review.

Trichloroethylene (TCE) has been used for a variety of industrial and consumer cleaning purposes because of its ability to dissolve organic substances. The multisystem injuries include those of skin, liver, and kidney, which are defined as TCE hypersensitivity syndrome (THS). THS is a serious occupational health issue. However, the mechanism of immune dysfunction leading to organ injury is poorly understood. Many studies reveal that skin lesions and organ injury caused by TCE are consistent with type IV hypersensitivity, also called delayed hypersensitivity, mediated by T cells. However, many researchers found T cell-mediated type IV hypersensitivity could not account for the pathogenesis of THS fully. Humoral immunity, including immunoglobulins and complement activation, may also play a possible role in THS pathogenesis. This review will describe the history, current understanding, and future research directions of the mechanism of THS.

Delayed hypersensitivity to nanosecond pulsed electric field in electroporated cells.

We demonstrate that conditioning of mammalian cells by electroporation with nanosecond pulsed electric field (nsPEF) facilitates their response to the next nsPEF treatment. The experiments were designed to unambiguously separate the electroporation-induced sensitization and desensitization effects. Electroporation was achieved by bursts of 300-ns, 9 kV/cm pulses (50 Hz, n = 3-100) and quantified by propidium dye uptake within 11 min after the nsPEF exposure. We observed either sensitization to nsPEF or no change (when the conditioning was either too weak or too intense, or when the wait time after conditioning was too short). Within studied limits, conditioning never caused desensitization. With settings optimal for sensitization, the second nsPEF treatment became 2.5 times (25 °C) or even 6 times (37 °C) more effective than the same nsPEF treatment delivered without conditioning. The minimum wait time required for sensitization development was 30 s, with still longer delays increasing the effect. We show that the delayed hypersensitivity was not mediated by either cell swelling or oxidative effect of the conditioning treatment; biological mechanisms underlying the delayed electrosensitization remain to be elucidated. Optimizing nsPEF delivery protocols to induce sensitization can reduce the dose and adverse side effects of diverse medical treatments which require multiple pulse applications.

Betulinic acid derivative BA5, a dual NF-kB/calcineurin inhibitor, alleviates experimental shock and delayed hypersensitivity.

Betulinic acid (BA) is a naturally occurring triterpenoid with several biological properties already described, including immunomodulatory activity. Here we investigated the immunomodulatory activity of eight semi-synthetic amide derivatives of betulinic acid. Screening of derivatives BA1-BA8 led to the identification of compounds with superior immunomodulatory activity than BA on activated macrophages and lymphocytes. BA5, the most potent derivative, inhibited nitric oxide and TNFα production in a concentration-dependent manner, and decreased NF-κB activation in Raw 264.7 cells. Additionally, BA5 inhibited the proliferation of activated lymphocytes and the secretion of IL-2, IL-4 IL-6, IL-10, IL-17A and IFNÉ£, in a concentration-dependent manner. Flow cytometry analysis in lymphocyte cultures showed that treatment with BA5 induces cell cycle arrest in pre-G1 phase followed by cell death by apoptosis. Moreover, BA5 also inhibited the activity of calcineurin, an enzyme that plays a critical role in the progression of cell cycle and T lymphocyte activation. BA5 has a synergistic inhibitory effect with dexamethasone on lymphoproliferation, showing a promising profile for drug combination. Finally, we observed immunosuppressive effects of BA5 in vivo in mouse models of lethal endotoxemia and delayed type hypersensitivity. Our results reinforce the potential use of betulinic acid and its derivatives in the search for potent immunomodulatory drugs.

The Value of In Vitro Tests to DiminishDrug Challenges.

Drug hypersensitivity reactions have multiple implications for patient safety and health system costs, thus it is important to perform an accurate diagnosis. The diagnostic procedure includes a detailed clinical history, often unreliable; followed by skin tests, sometimes with low sensitivity or unavailable; and drug provocation testing, which is not risk-free for the patient, especially in severe reactions. In vitro tests could help to identify correctly the responsible agent, thus improving the diagnosis of these reactions, helping the physician to find safe alternatives, and reducing the need to perform drug provocation testing. However, it is necessary to confirm the sensitivity, specificity, negative and positive predictive values for these in vitro tests to enable their implementation in clinical practice. In this review, we have analyzed these parameters from different studies that have used in vitro test for evaluating drug hypersensitivity reactions and estimated the added value of these tests to the in vivo diagnosis.

Inhibition of effector antigen-specific T cells by intradermal administration of heme oxygenase-1 inducers.

Developing protocols aimed at inhibiting effector T cells would be key for the treatment of T cell-dependent autoimmune diseases including type 1 autoimmune diabetes (T1D) and multiple sclerosis (MS). While heme oxygenase-1 (HO-1) inducers are clinically approved drugs for non-immune-related diseases, they do have immunosuppressive properties when administered systemically in rodents. Here we show that HO-1 inducers inhibit antigen-specific effector T cells when injected intradermally together with the T cell cognate antigens in mice. This phenomenon was observed in both a CD8+ T cell-mediated model of T1D and in a CD4+ T cell-dependent MS model. Intradermal injection of HO-1 inducers induced the recruitment of HO-1+ monocyte-derived dendritic cell (MoDCs) exclusively to the lymph nodes (LN) draining the site of intradermal injection. After encountering HO-1+MoDCs, effector T-cells exhibited a lower velocity and a reduced ability to migrate towards chemokine gradients resulting in impaired accumulation to the inflamed organ. Intradermal co-injection of a clinically approved HO-1 inducer and a specific antigen to non-human primates also induced HO-1+ MoDCs to accumulate in dermal draining LN and to suppress delayed-type hypersensitivity. Therefore, in both mice and non-human primates, HO-1 inducers delivered locally inhibited effector T-cells in an antigen-specific manner, paving the way for repositioning these drugs for the treatment of immune-mediated diseases.