Development and Evaluation of an In-House ELISA to Detect Anti-FcεR1α IgG Autoantibodies in Chronic Spontaneous Urticaria Patients.

BACKGROUND: Association between chronic spontaneous urticaria (CSU) and autoimmunity has been well documented. Autologous serum skin testing could support the autoimmune etiology of CSU, whereas it is difficult to interpret and could not be performed on antihistamine omitted patients. It was found that immunoglobulin G (IgG) autoantibodies (autoAbs) against high-affinity IgE receptor (FcεR1) were suggested as a potential trigger in the pathogenesis of CSU. Although many ELISA protocols have been developed to detect these autoAbs, they lacked validation or a reliable cut-off point. We, therefore, aimed to develop a validated ELISA with a reliable cut-off point to quantitate IgG anti-FcεR1α autoAbs for CSU. METHODS: We developed an in-house ELISA to quantitate IgG anti-FcεR1α autoAbs. Sera from 233 CSU patients and 25 healthy people were used to test with ELISA. The cut-off point was obtained from the results subjected to analyze with receiver operating characteristic (ROC) analysis. ELISA was validated with 116 CSU patients and 150 healthy donors. RESULTS: ELISA revealed that healthy people had a basal level of IgG anti-FcεR1α autoAbs, whereas their levels were significantly lower than autoAbs levels in CSU patients. ROC analysis of ELISA determined the cut-off point at 936.7 ng/ml. Our ELISA was validated and provided excellent sensitivity and specificity at 98.28% and 92.67%, respectively. CONCLUSION: Our ELISA could detect significant levels of IgG anti-FcεR1α autoAbs in CSU, supporting that these autoAbs were associated with CSU etiology. Our validated ELISA with the reliable cut-off point provided excellent accuracy at 95.11% (98.28% sensitivity and 92.67% specificity). Our ELISA could be an alternative test benefit for the patient who is unable to omit antihistamine treatment.

The effect of temperature on the stability of PCSK-9 monoclonal antibody: an experimental study.

BACKGROUND: PCSK9 monoclonal antibody lowers plasma PCSK9 and LDL-cholesterol levels. The manufacturers recommend drug storage at 2-8 °C, and not above 25 °C. This study aimed to investigate drug stability at various temperatures that this drug could be exposed to during medication handling and transportation in tropical countries. METHODS: Alirocumab and evolocumab were tested in 3 study conditions: room temperature (RT), cooler device with cold pack, and freeze-thaw for 9 and 18 h. Heated drugs were used as negative control. Free plasma PCSK9 levels from 9 hyperlipidemia subjects were measured with ELISA. RESULTS: Average subject age was 49.2 ± 18.4 years. Percent PCSK9 inhibition significantly declined in heated drugs compared to baseline. Average RT during the study period was 30.4 ±2.6 °C. Change in percent PCSK9 inhibition of PCSK9 mAb at RT from baseline was - 5.8 ± 4.4% (P = 0.005) and - 11.0 ± 8.9% (P = 0.006) for alirocumab at 9 h and 18 h, and - 9.7 ± 11.8% (P = 0.04) and - 15.1 ± 14.3% (P = 0.01) for evolocumab at 9 and 18 h, respectively. In contrast, there were no significant changes in percent PCSK9 inhibition from baseline when PCSK9 mAb was stored in a cooler. In freeze-thaw condition, changes in percent PCSK9 inhibition from baseline to 9 and 18 h were - 5.2 ± 2.9% (P = 0.001) and - 2.6 ± 4.9% (P = 0.16) for alirocumab, and - 1.8 ± 4.2% (P = 0.24) and 0.4 ± 6.1% (P = 0.83) for evolocumab. CONCLUSION: Proper drug storage according to manufacturer's recommendation is essential. Drug storage at RT in tropical climate for longer than 9 h significantly decreased drug efficacy; however, storage in a cooler device with cold pack for up to 18 h is safe.

Defective cytokine production from monocytes/macrophages of E-beta thalassemia patients in response to Pythium insidiosum infection.

BACKGROUND: Pythium insidiosum has been mainly reported to cause morbidity and mortality in thalassemia patients. P. insidiosum zoospores can germinate to be hyphae within a few hours; therefore, it is difficult to study the initial immune response that P. insidiosum zoospores induce. The present study aims to compare immune responses against P. insidiosum zoospore infection by comparing monocytes/macrophages from thalassemia patients with those from non-thalassemia controls. METHODS: In order to keepP. insidiosum in the zoospore stage in vitro for inoculation, the P. insidiosum zoospores were preserved without germination by treatment with inorganic hypochlorite solution. CD14+ cells were isolated from peripheral blood mononuclear cells of thalassemia and non-thalassemia donors and then left to transition to macrophages. Monocytes/macrophage culture was infected with P. insidiosum zoospores and culture supernatants were subjected to Th1/Th2 multiplex cytokine detection. RESULTS: Our study of cytokine production revealed that the basal level of GM-CSF produced by thalassemia monocytes/macrophages was lower than that observed in monocytes/macrophages of non-thalassemia individuals. Higher GM-CSF and IFN-γ response was also found when cells from non-thalassemia people were stimulated with P. insidiosum zoospores compared to thalassemia cells. It was also found that TNF-α, GM-CSF and IFN-γ productions from monocytes/macrophages of thalassemia patients who received iron chelator treatment were significantly higher than those produced from thalassemia patients without iron chelator treatment. CONCLUSION: For the first time, the present study demonstrates defective immune responses in monocytes/macrophages derived from thalassemia patients in response toP. insidiosum zoospore infection. The results also show an inverse correlation between iron overload and cytokine production in monocytes/macrophages of thalassemia patients. This finding could explain why thalassemia patients are susceptible to P. insidiosum infection.

Evaluation of a lymphocyte transformation test and cytokine detection assay to identify phenytoin and carbamazepine provoked DRESS or SJS/TEN in epilepsy patients.

Phenytoin (PHE) and carbamazepine (CBZ) are first rank causative drugs that can induce drug rash with eosinophilia and systemic symptoms (DRESS) and Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN). Identification of anti-epileptic drugs as a culprit drug has been problematic; hence, in vitro tests could be promising methods to define causative drugs without clinical risk. The aim of this study is to evaluate the efficacy of lymphocyte transformation tests (LTT) and cytokine detection assays in identifying PHE and CBZ as culprit drugs. Peripheral blood mononuclear cells were collected from normal, PHE/CBZ tolerance and PHE/CBZ hypersensitivity cohorts and utilized for cell-culture assays. LTT was performed and culture supernatants were subjected to multiple cytokine detection assays. Our study showed that LTT correlated with outcomes of Naranjo's assessment with statistical significance (r = 0.614). Various sensitivities of LTT and cytokine detection assays were demonstrated. Although both assays provided excellent specificity, LTT yielded higher sensitivity as compared to those of cytokine detection assays in both DRESS and SJS/TEN. Regardless whether specificity is, this is the first report to demonstrate that IL-4 detection assay could enhance sensitivity to identify culprit drug when it was combined with LTT for SJS/TEN patients or it was combined with IL-2/IFN-γ detection assays for DRESS ones.