In-hospital blood collection increases the rate of indeterminate results in interferon-gamma release assays.

BACKGROUND: The interferon (IFN)-γ release assay (IGRA) has recently been established as a method to evaluate the infection status of tuberculosis instead of the tuberculin skin test. However, indeterminate results can create challenges to interpretation. The IGRA has been available in Japan since 2005, including the recently launched QuantiFERON-TB Gold Plus (QFT-plus) assay. OBJECTIVES: The aim of this study was to investigate the clinical features and predictors of indeterminate results by the QFT-plus test in routine practice. METHODS: This was a cross-sectional study of 1258 patients. Multivariate logistic regression models were employed to investigate the clinical factors related to indeterminate results by the QFT-plus. RESULTS: Overall, 91.8% of results were found to be conclusive and 8.2% were indeterminate. The QFT-plus indeterminate results were predominantly due to a low level of IFN-γ production by mitogens. Multivariate analysis indicated that an indeterminate result was significantly associated with age, sex, corticosteroid use, autoimmune disease, and inpatient setting. CONCLUSION: Certain types of individuals are at higher risk of an indeterminate IGRA result. The QFT-plus test for hospitalized patients should be avoided as much as possible, and it is better to perform the test for those patients in outpatient settings.

Correlation Between Thymoma and Soluble Interleukin-2 Receptor Expression in a Patient with Good Syndrome.

Good syndrome is a rare condition characterized by the presence of thymoma in combination with adult-onset hypogammaglobulinemia. Immunological features of Good syndrome include various immunodeficiencies accompanied with hypogammaglobulinemia. In patients with thymoma, paraneoplastic syndromes including hypogammaglobulinemia worsen the prognosis. We herein describe a patient with advanced-stage type A thymoma who was effectively treated with chemotherapy and exhibited a parallel decrease in the serum level of soluble interleukin-2 receptor (sIL-2R), which depends on cellular immunity. The present case suggests the efficacy of sIL-2R as a potential prognostic biomarker in a subset of patients with Good syndrome.

Impact of the interferon-γ release assay and glomerular filtration rate on the estimation of active tuberculosis risk before bronchoscopic examinations: a retrospective pilot study.

BACKGROUND: Bronchoscopic examinations are vital to diagnose pulmonary diseases. However, as coughing is triggered during and after the procedure, it is imperative to take measures against nosocomial infections, especially for airborne infections like tuberculosis (TB). The interferon-γ release assay (IGRA) has recently been established as a method to evaluate the infection status of TB. We aimed to ascertain the efficacy of IGRA and clinical findings in estimating the prevalence of active TB before bronchoscopy. METHODS: We obtained IGRA results from 136 inpatients using a QuantiFERON-TB Gold In-Tube test. Bronchoscopy samples were cultured in Mycobacteria Growth indicator tubes and 2% Ogawa solid medium. We evaluated the adjusted effects of multiple clinical variables on active TB status using a logistic regression model. In addition, multiple variables were converted into a decision tree to predict active TB. RESULTS: Five (3.7%) patients were diagnosed with culture-positive TB, two of whom were simultaneously diagnosed with non-small-cell lung carcinoma or small-cell lung carcinoma. The multivariate analysis suggested the probability of predicting active TB using the IGRA [odds ratio (OR), 72.7; 95% confidence interval (CI), 3.169-1668; P=0.007] and decreased estimated glomerular filtration rate (eGFR) (OR, 0.937; 95% CI, 0.882-0.996; P=0.038) in patients undergoing bronchoscopy. A decision tree validated the use of these two variables to predict active TB. CONCLUSIONS: IGRA test results are useful for predicting active TB before bronchoscopy. This strategy could identify patients who require antibiotic therapy to prevent TB or who are in the active phase of TB.

Addition of UVA-absorber butyl methoxy dibenzoylmethane to topical ketoprofen formulation reduces ketoprofen-photoallergic reaction.

Topical application of ketoprofen (KP) clinically evokes the allergic type of photocontact dermatitis. To avoid this adverse reaction, we investigated the beneficial effect of each ultraviolet (UV) filter that was included in topical ketoprofen formulation. We first tested the inhibitory effects of four UVA filters by a modified local lymph node assay following KP application on the mouse skin and UVA irradiation on the same site. In this assessment, butyl methoxy dibenzoylmethane (BMDBM), when included in KP application, exerted the most effective inhibitory effect on stimulation with KP and UVA. We manufactured topical patch and gel KP applicants containing BMDBM, which retained KP penetration through the skin and KP stability toward UVA. The ability of BMDBM in these formulations to inhibit KP photosensitivity was evaluated by a modified adjuvant and strip method in guinea pigs, and the photoallergic reactions induced by the BMDBM-containing KP applicants were lower than the non-containing ones. It is known that KP has a cross-reactivity with benzophenone upon UVA exposure, but such a photocross-reactivity of BMDBM with KP was not observed in a mouse ear swelling model. The anti-inflammatory effect of the BMDBM-containing KP patch applicant was comparable to the non-containing one. These results suggest that the addition of BMDBM into KP topical formulations is efficacious for inhibition of KP photocontact dermatitis.

Skin application of ketoprofen systemically suppresses contact hypersensitivity by inducing CD4(+) CD25(+) regulatory T cells.

BACKGROUND: Ketoprofen (KP) is a widely used nonsteroidal anti-inflammatory drug that inhibits prostaglandin biosynthesis. We have previously shown that topical KP treatment at the sensitizing site inhibits the development of contact hypersensitivity (CHS) to picryl chloride (PCl). OBJECTIVE: We investigated the mechanism underlying the KP-induced immunosuppression of CHS by application of KP. METHODS: We analyzed the CHS responses to the non-sensitizing site and subsequent sensitization with PCl, and by transfer of the draining lymph node cells (LNCs) from KP-tolerated mice to recipient mice. Changes in the Foxp3 expression of LNCs from KP-phototreated skin were also examined by real-time PCR. RESULTS: Topical application of KP to not only the sensitizing but also non-sensitizing site suppressed CHS response. The immunosuppression was transferred with LNCs from mice treated with PCl plus KP, but not from mice treated oxazolone plus KP. In this transfer study, the CD4(+) CD25(+) subset of LNCs exerted the suppressive effect, while CD25(+) cell-depleted LNCs lost the inhibitory ability. CTLA-4 blocking with a specific antibody, but not IL-10 blocking, abrogated the activity of CD4(+) CD25(+) cells. Moreover, Foxp3 mRNA expression was remarkably increased in LNCs from PCl and KP-treated mice. CONCLUSION: The immunosuppression of CHS by topical application of KP is systemic and haptein-specific. Treg cells play an important role in the suppressive effect by KP.

IL-10-producing Langerhans cells and regulatory T cells are responsible for depressed contact hypersensitivity in grafted skin.

Although skin grafting is a common surgical technique, the immunological state of grafted skin remains unelucidated. An experimental model has shown that the development of murine contact hypersensitivity (CHS) is depressed when mice are sensitized with a hapten through full-thickness grafted skin. We explored the immunological mechanisms underlying this hyposensitization, focusing on the fate of Langerhans cells (LCs). When FITC was applied to grafted skin, FITC-bearing LCs were capable of migrating to the draining lymph nodes. Epidermal cell suspensions isolated from the grafted skin produced a high amount of IL-10 as assessed by real-time PCR. Adoptive transfer of immune lymph node cells from the sensitized mice suppressed the CHS response of recipients in an antigen-specific manner. CD4(+)CD25(+) but not CD4(+)CD25(-) T cells purified from lymph node cells were responsible for this suppression. Finally, we detected high expression of receptor activators of nuclear factor kappa-B ligand (RANKL) in the grafted skin, and found that recombinant RANKL stimulated LCs to produce IL-10. These findings suggest that the hyposensitization of CHS through the grafted skin is not attributable merely to the reduction of LC number but that IL-10-producing LCs exert a downmodulatory effect by inducing regulatory T cells.

Stimulation of Langerhans cells with ketoprofen plus UVA in murine photocontact dermatitis to ketoprofen.

BACKGROUND: Ketoprofen (KP) clinically evokes the allergic type of photocontact dermatitis when applied to the skin and irradiated with ultraviolet A (UVA). We have established a murine model of photocontact dermatitis to KP, which is a T cell-mediated delayed type hypersensitivity. OBJECTIVE: To further explore the mechanism underlying this sensitivity, we investigated whether KP plus UVA activates the antigen-presenting ability of Langerhans cells (LCs). METHODS: We analyzed the expression of surface molecules on LCs in the murine epidermis treated with KP plus UVA by immunohistochemistry and flow cytometry. Changes in the cytokine expression of epidermal cells from KP-phototreated skin were also examined by real-time PCR. RESULTS: LCs became larger after treatment with KP plus UVA. The number of LCs was significantly decreased 2-3 days after KP phototreatment and recovered on day 5. A flow cytometric analysis revealed that KP plus UVA increased the percentage of LCs that highly expressed MHC class II, CD86, CD80, CD54 and CD40, whereas neither KP nor UVA alone enhanced the expression. KP phototreatment augmented the expression of I-A and CD86 on LCs in KP and UVA dose-dependent manners. A real-time PCR analysis of KP-phototreated skin showed that the expression of mRNA for IL-1alpha and GM-CSF was immediately increased after treatment. CONCLUSION: A photosensitizing regimen of KP plus UVA activates LCs at least partly by stimulating keratinocytes to produce cytokines. Two strains of mice (BALB/c and AKR) differ in responsiveness to KP and the difference is not related to the activation of keratinocytes.

Induction of keratinocyte apoptosis by photosensitizing chemicals plus UVA.

BACKGROUND: The capacity of photosensitizing chemicals with ultraviolet A light (UVA) to induce apoptosis is one of the methods to assess their phototoxic and potentially photoallergic properties, since apoptotic cells may be easily presented by antigen-presenting cells. OBJECTIVES: We examined the photoaggravated ability to induce keratinocyte apoptosis of various chemicals that are known as causative agents of photocontact dermatitis and drug photosensitivity involving photoallergic and/or phototoxic mechanisms. METHODS: HaCaT keratinocytes were incubated with 3,3',4',5-tetrachlorosalicylanilide (TCSA), bithionol, diphenylhydramine, chlorpromazine, 6-methylcoumarin, sparfloxacin, and enoxacin at 10(-7) to 10(-4)M and irradiated with UVA at 4J/cm(2). As positive control, 8-methoxypsoralen (8-MOP) was also tested. Apoptosis and necrosis were evaluated by flow cytometric enumeration of annexin V(+) 7-AAD(-) and annexin V(+) 7-AAD(+) cells, respectively. The expression of apoptosis-related molecules, caspase-3 and poly (ADP-ribose) polymerase (PARP), was tested by flow cytometric and Western blotting analyses. RESULTS: In a comparison with non-irradiated cells, significant apoptosis was found in TCSA, bithionol, chlorpromazine, sparfloxacin and enoxacin at 10(-4) or 10(-5)M as well as 8-MOP as assessed by both annexin V and active caspase-3 stainings, while necrosis occurred in most of these chemicals at 10(-4)M. Neither apoptosis nor necrosis was seen in diphenylhydramine or 6-methylcoumarin. PARP were activated in HaCaT cells phototreated with TCSA, bithionol and chlorpromazine. CONCLUSIONS: We suggest that our method is useful for in vitro assessment of phototoxicity and potential photoallergenicity of chemicals.

Establishment of murine model of allergic photocontact dermatitis to ketoprofen and characterization of pathogenic T cells.

BACKGROUND: Ketoprofen is well known to evoke the allergic type of photocontact dermatitis when it is applied to the skin and irradiated with ultraviolet A (UVA) light. OBJECTIVE: We aimed to establish a murine model of this photosensitivity and to characterize pathogenic T cells concerned with the sensitivity. METHODS: Various strains of mice were sensitized on two consecutive days by application of ketoprofen to the shaved abdomen and irradiation of the skin with UVA. Five days later, they were elicited with ketoprofen plus UVA on the earlobes. Immune lymph node cells and epidermal cells from the challenged sites were analyzed by RT-PCR. RESULTS: Mice were successfully sensitized and challenged with 4% and 2% ketoprofen, respective, plus UVA at 20J/cm2. The responses in H-2k mice were higher than those in the other strains examined. Immune lymph node CD4+ or CD8+ cells from ketoprofen-photosensitized H-2k mice were transferred i.v. to naïve syngeneic recipients. Mice receiving CD4+ but not CD8+ cells exhibited ketoprofen photosensitivity, but transference of both CD4+ and CD8+ cell populations was more effective. Lymph node cells from photosensitized mice expressed high levels of mRNA for Th2 cytokine (IL-4) and Th2 chemokine receptor (CCR4) as well as Th1 cytokine (IFN-gamma) and Th1 chemokine receptor (CXCR3), as assessed by RT-PCR. In addition, epidermal cells from challenged earlobes expressed increased levels of both Th1 (TARC) and Th2 (Mig) chemokines. CONCLUSION: It is considered that not only Th1 but also Th2 cells participate in the pathogenesis of murine photocontact dermatitis to ketoprofen.