A fast intraoperative PTH point-of-care assay on the Philips handheld magnotech system.

BACKGROUND: Intraoperative parathyroid hormone (ioPTH) is used during minimally invasive parathyroidectomy (MIP) to predict the success of surgery and should be accurate with a short turnaround time. MATERIAL AND METHOD: We developed an ioPTH point-of-care (POC) assay on Philips handheld magnotech system. Magnotech technology is based on magnetically controlled movement of superparamagnetic nanoparticles in stationary sample fluid. During first phase, intact-PTH is captured by magnetic particles coated with anti-N-terminal-PTH antibodies. Subsequently, magnetic particles are collected by magnetic forces at sensor surface coated with anti-C-terminal-PTH antibodies. Unbound/nonspecifically bound particles are pulled away from detection surface, using a second magnetic force. Amount of specifically bound particles is measured using a surface-sensitive optical imaging technique. RESULTS: ioPTH test could be performed with a turnaround time of less than 10 min and could detect low intact-PTH concentrations (picomolar). Integrated cartridge contains a blood separation filter and dry reagents for the assay. CONCLUSION: The next magnotech ioPTH assay will be the only POC test able to give accurate results in less than 10 min, using 25 µL of whole blood. Thanks to the ease-of-use, magnotech ioPTH could be performed in the operating theater by any member of surgical staff.

GITR signaling potentiates airway hyperresponsiveness by enhancing Th2 cell activity in a mouse model of asthma.

BACKGROUND: Allergic asthma is characterized by airway hyperresponsiveness (AHR) and allergic inflammation of the airways, driven by allergen-specific Th2 cells. The asthma phenotypes and especially AHR are sensitive to the presence and activity of regulatory T (Treg) cells in the lung. Glucocorticoid-induced tumor necrosis factor receptor (GITR) is known to have a co-stimulatory function on effector CD4+ T cells, rendering these cells insensitive to Treg suppression. However, the effects of GITR signaling on polarized Th1 and Th2 cell effector functions are not well-established. We sought to evaluate the effect of GITR signaling on fully differentiated Th1 and Th2 cells and to determine the effects of GITR activation at the time of allergen provocation on AHR and airway inflammation in a Th2-driven mouse model of asthma. METHODS: CD4+CD25- cells were polarized in vitro into Th1 and Th2 effector cells, and re-stimulated in the presence of GITR agonistic antibodies to assess the effect on IFNgamma and IL-4 production. To evaluate the effects of GITR stimulation on AHR and allergic inflammation in a mouse asthma model, BALB/c mice were sensitized to OVA followed by airway challenges in the presence or absence of GITR agonist antibodies. RESULTS: GITR engagement potentiated cytokine release from CD3/CD28-stimulated Th2 but not Th1 cells in vitro. In the mouse asthma model, GITR triggering at the time of challenge induced enhanced airway hyperresponsiveness, serum IgE and ex vivo Th2 cytokine release, but did not increase BAL eosinophilia. CONCLUSION: GITR exerts a differential effect on cytokine release of fully differentiated Th1 and Th2 cells in vitro, potentiating Th2 but not Th1 cytokine production. This effect on Th2 effector functions was also observed in vivo in our mouse model of asthma, resulting in enhanced AHR, serum IgE responses and Th2 cytokine production. This is the first report showing the effects of GITR activation on cytokine production by polarized primary Th1 and Th2 populations and the relevance of this pathway for AHR in mouse models for asthma. Our data provides crucial information on the mode of action of the GITR signaling, a pathway which is currently being considered for therapeutic intervention.

Rapid immunoassay for the determination of glial fibrillary acidic protein (GFAP) in serum.

BACKGROUND: This study was aimed to develop a sensitive and rapid assay for the determination of glial fibrillary acidic protein (GFAP) in serum and to evaluate the clinical applicability in serum samples from patients with acute stroke. METHODS: The two-site chemiluminometric immunoassay, intended to use in a near-patient setting with a single incubation step (20 min), was used to measure serum samples from healthy blood donors and from patients with brain injury and correlated to serum S100B levels. RESULTS: The GFAP assay covered a concentration range up to 18 microg/L with an analytical sensitivity of 0.014 microg/L. The intra-assay precision was 3.5% at 1.55 microg/L (n=20) and 4.1% at 0.39 microg/L (n=20). The inter-assay precision was 3.8% at 9.1 microg/L (n=10) and 10.3 % at 0.21 microg/L (n=9). Normal controls (n=46) showed non-detectable GFAP with a 99% upper limit of <0.04 microg/L. GFAP values were associated with progression and severity of the illness in acute stroke patients. CONCLUSIONS: We have developed an improved assay for the measurement of GFAP levels in serum. Serum GFAP is a potential marker for prognosis and outcome in patients with central nervous system disorders.

Stimulation of allergen-loaded macrophages by TLR9-ligand potentiates IL-10-mediated suppression of allergic airway inflammation in mice.

BACKGROUND: Previously, we demonstrated that OVA-loaded macrophages (OVA-Mphi) partially suppress OVA-induced airway manifestations of asthma in BALB/c mice. In vitro studies showed that OVA-Mphi start to produce IL-10 upon interaction with allergen-specific T cells, which might mediate their immunosuppressive effects. Herein, we examined whether IL-10 is essential for the immunosuppressive effects of OVA-Mphi in vivo, and whether ex vivo stimulation of the IL-10 production by OVA-Mphi could enhance these effects. METHODS: Peritoneal Mphi were loaded with OVA and stimulated with LPS or immunostimulatory sequence oligodeoxynucleotide (ISS-ODN) in vitro. The increase of IL-10 production was examined and, subsequently, ex vivo stimulated OVA-Mphi were used to treat (i.v.) OVA-sensitized mice. To further explore whether Mphi-derived IL-10 mediates the immunosuppressive effects, Mphi isolated from IL-10-/- mice were used for treatment. RESULTS: We found that stimulation with LPS or ISS-ODN highly increased the IL-10 production by OVA-Mphi (2.5-fold and 4.5-fold increase, respectively). ISS-ODN stimulation of OVA-Mphi significantly potentiated the suppressive effects on allergic airway inflammation. Compared to sham-treatment, ISS-ODN-stimulated OVA-Mphi suppressed the airway eosinophilia by 85% (vs. 30% by unstimulated OVA-Mphi), IL-5 levels in bronchoalveolar lavage fluid by 80% (vs. 50%) and serum OVA-specific IgE levels by 60% (vs. 30%). Importantly, IL-10-/-Mphi that were loaded with OVA and stimulated with ISS-ODN ex vivo, failed to suppress OVA-induced airway inflammation. CONCLUSIONS: These results demonstrate that Mphi-derived IL-10 mediates anti-inflammatory responses in a mouse model of allergic asthma, which both can be potentiated by stimulation with ISS-ODN.

Allergen immunotherapy induces a suppressive memory response mediated by IL-10 in a mouse asthma model.

BACKGROUND: Human studies have demonstrated that allergen immunotherapy induces memory suppressive responses and IL-10 production by allergen-specific T cells. Previously, we established a mouse model in which allergen immunotherapy was effective in the suppression of allergen-induced asthma manifestations. OBJECTIVE: In this study, we examined whether immunotherapy induces a long-lasting effect and investigated the role of IL-10 in successful immunotherapy. METHODS: Ovalbumin-sensitized BALB/c mice were treated with 3 injections of ovalbumin (1 mg, subcutaneous) on alternate days. After a short interval (1 week) and after a long interval (5 weeks), mice were challenged by ovalbumin inhalation, and subsequently, airway reactivity, airway eosinophilia, ovalbumin-specific IgE, and T(H)2 cytokine profile were measured. Flow cytometry and blocking of IL-10 receptors in vivo were used to gain insight in the role of IL-10 in the beneficial effects of allergen immunotherapy. RESULTS: After a long interval between ovalbumin immunotherapy and ovalbumin challenge, the development of airway eosinophilia and hyperresponsiveness to methacholine were as strongly suppressed as after a short interval. These suppressive effects coincided with significantly reduced serum ovalbumin-specific IgE levels and T(H)2 cytokine production. On immunotherapy, the IL-5:IL-10 ratio in the bronchoalveolar lavage fluid shifted toward IL-10. In ovalbumin-restimulated lung cell and thoracic lymph node cultures from these mice, IL-5 levels dramatically decreased, whereas the percentage of IL-10(+)CD4(+) T cells was not affected. Finally, in mice treated with mAb against IL-10 receptors, the beneficial effects of immunotherapy were largely abrogated. CONCLUSION: These data demonstrate that allergen immunotherapy induces a memory suppressive effect in which IL-10 is essential.

Phenotypical and functional characterization of clinical grade dendritic cells.

Dendritic cells (DC) are the professional antigen presenting cells of the immune system. Therefore, several clinical studies have been initiated in which tumor antigen-loaded DC are used as a vaccine to boost an immune response against malignant tumors in patients with cancer. A prerequisite for DC used in these vaccination studies is not only that they are grown under "Good Manufacturing Practice" but equally important that they retain their functional properties. In an extensive study, various conditions were tested to optimize the maturation and yield of DC grown for clinical use. DC grown in XVIVO-15 medium supplemented with 5% HS yielded the best results, morphologically and phenotypically. Mature DC expressed significant amounts of mature DC markers (CD83) and the costimulatory molecules CD80 and CD86. It was shown that mature and immature DC can be frozen and retain their phenotype and function after thawing. These clinical grade DC secreted high levels of the chemokines dendritic cell chemokine 1 (DC-CK1), interleukin-8 (IL-8), macrophage-derived chemokine (MDC), and thymus and activation-regulated chemokine (TARC). This implicates that these DC can attract naïve T and B cells as well as natural killer cells and memory T cells. Finally, to test their migratory capacity in vivo, (111)In-labeled DC were injected into tumor-free lymph nodes of patients with melanoma. Autoradiographic analysis of the dissected lymph nodes indicated that these DC could migrate into the T cell area of adjacent lymph nodes. In conclusion, a culture procedure was established to generate large numbers of monocyte-derived immature and mature DC that retain their morphologic, phenotypic, and functional characteristics in vitro and can be visualized in situ.

Renal cell carcinoma-associated antigen G250 encodes a naturally processed epitope presented by human leukocyte antigen-DR molecules to CD4(+) T lymphocytes.

We previously identified an HLA-A2.1-restricted epitope within the RCC-associated antigen G250 that is recognized by CTLs. Using DCs of healthy individuals, which were loaded with overlapping 20 mer G250-derived peptides, we here report the induction of CD4(+) T cells that recognize the G250 peptide of amino acids 249-268. Moreover, naturally processed G250 protein is readily recognized by these G250-specific CD4(+) T cells in the context of HLA-DR molecules. Interestingly, peptide G250:249-268 overlaps the previously identified HLA-A2.1-restricted G250 epitope recognized by CTLs. These data and the high prevalence of G250 in RCC patients make peptide G250:249-268 a potential target in peptide-based vaccines to induce both CD4(+) and CD8(+) T-cell responses in patients.