Suspension Array Research Articles

A homogenous fluorescence immunoassay based on a DNA-grafted labeling strategy of magnetic microspheres for multiple neonicotinoids monitoring

Given the detrimental impact on non-target pollinators, there is a high demand for monitoring multiple neonicotinoid residues in the environment. In this study, we proposed a novel physical-biological coupled multiplexing system (PBCMS) to achieve a homogeneous and high-performance indirect competitive suspension array technology (ic-SAT) for multiple neonicotinoids monitoring. First, hapten-decorated microspheres (HDMs) were prepared by ligating the streptavidin-modified microspheres with the biotinylated hapten, according to a novel molecularly-defined single-stranded DNA (ssDNA)-grafted labeling approach. Taking thiamethoxam (THX) and imidacloprid (IMI) as models, the binding properties of two anti-neonicotinoid antibodies were profiled by establishing fluorescence monoplex ic-SAT assays. Subsequently, two HDMs (2.8 and 5.0 μm) immobilized with THX and IMI haptens were synthesized and then implemented to fabricate a dualplex ic-SAT assay, while they were distinguished via a facile diameter-resolved decoding process through flow cytometry. Excitingly, the screening capacity of the dualplex ic-SAT assay was significantly expanded by employing antibodies with distinctive binding profiles. It enables the simultaneous monitoring of 7 representative neonicotinoid insecticides (THX, IMI, acetamiprid, clothianidin, thiacloprid, nitenpyram and imidaclothiz) and 4 major metabolites in a single test sample, with IC50 values ranging from 0.02 to 572.70 ng/mL. Furthermore, the dualplex ic-SAT assay was applied to honey, pollen and bee samples, demonstrating satisfactory recovery rates (74.1 %∼106.3 %) and nice agreement with LC-MS/MS analysis. In summary, the PBCMS offers an easy-to-implement, robust and cost-effective way for establishing user-defined ic-SAT, providing a powerful analytical tool to simultaneously monitor multiple neonicotinoid residues in environmental samples.

Bead-based spontaneous Raman codes for multiplex immunoassay

In the immunoassay process, for fulfilling the need to identify multiple analytes in a small amount of complex sample matrix, it is desirable to develop highly efficient and specific multiplex suspension array technology. Raman coding strategy offers an attractive solution to code the suspension arrays by simply combing narrow spectral bands with stable signal intensities through solid-phase synthesis on the resin beads. Based on this strategy, we report the bead-based spontaneous Raman codes for multiplex immunoassay. The study resulted in superior selectivity of the Raman-encoded beads for binding with single and multiple analytes, respectively. With the use of mixed types of Raman-encoded immunoassay beads, multiple targets in small amounts of samples were identified rapidly and accurately. By confirming the feasibility of bead-based spontaneous Raman codes for multiplex immunoassay, we anticipate this novel technology to be widely applied in the near future.

Mildronate ameliorates kidney injury and reduces inflammation in Sickle Cell Disease mice

INTRODUCTION: Sickle cell disease (SCD) is a hereditary hemoglobin disorder characterized by hemolytic anemia, vaso-occlusive crisis and chronic inflammation that affects multiple organs including kidney, liver, brain, spleen, and heart. Renal disease, the most common complication in SCD, is a progressive disease that begins in childhood as glomerular hyperfiltration, then advances to albuminuria, following by a fast decline in glomerular filtration. It may further progress to a renal failure in adults. More than 50% of SCD patients develop chronic kidney disease (CKD), and 14-18% progress to the end-stage kidney disease. Renal injury in SCD results from a cascade of events starting with chronic red blood cells sickling and hemolysis leading to increased blood viscosity, microvascular obstruction and dilation, hypoxia in the medulla, oxidative stress, and inflammation. Mildronate (meldonium) is an anti-ischemic and anti-inflammatory medication approved in Eastern Europe. HYPOTHESIS: We hypothesized that mildronate administration reduces renal medullary hypoxia and decreases oxidative stress and inflammation ameliorating renal injury in SCD mice. METHODS: The study was approved by the Howard University IACUC. Townes SCD mice, expressing human HbS, and control mice, expressing human HbA, were injected with mildronate (400 mg/kg in saline) intraperitoneally or saline as vehicle control over the course of ten days. Blood was collected and used for hematological parameters analysis. Cytokines were measured by Bio-Plex suspension array in plasma. Hypoxyprobe Biotin kit (Hypoxyprobe Inc) was used for visualization of organ hypoxia. The effect of Mildronate on systemic and renal oxidative stress was measured by malondialdehyde (MDA) using ELISA (Abcam). Urinary KIM-1 was measured by TIM-1/KIM-1 ELISA kit (R&D Systems). RESULTS: Mildornate injections did not affect hematological parameters of SCD mice including hematocrit, MCV, MCH, CMCH, erythrocytes and hemoglobin. Mildronate administration resulted in a significant reduction of the circulating pro-inflammatory cytokines IFN-γ, IL-1β and TNF-α and a trend toward reduction of IL-6, and IL-17 in SCD mice. We found higher level of hypoxia in the medulla of SCD mice compared to control mice. Mildronate injections did not reduced hypoxia in renal medulla of SCD mice. Also, mildronate injections did not change MDA levels in plasma or kidney of SCD mice. We found a significant reduction of proteinuria in SCD mice injected with mildronate compared to SCD mice injected with vehicle. Analysis of H&E-stained kidneys showed significant reduction of glomerular size and medullar congestion areas in the mildronate injected SCD mice. Urinary KIM-1 levels were reduced by mildronate administration. CONCLUSIONS: Our results demonstrate that mildronate ameliorates glomerular and tubular injury in SCD mice and reduces capillary congestion. Mildronate improves renal function, in part by reducing inflammation but not through the reduction of hypoxia or oxidative stress. In future, we plan to assess the mildronate’s impact on kidney function and vessel inflammation. This work was supported by NIH grants R01HL125005, U54MD007597, and SC1HL150685. We thank Xiaomei Niu for helping with the Bio-Plex assay. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sustained clinical benefit of malaria chemoprevention with sulfadoxine-pyrimethamine (SP) in pregnant women in a region with high SP resistance markers

The effectiveness of intermittent preventive treatment of malaria in pregnancy with sulfadoxine-pyrimethamine (IPTp-SP) is threatened by increasing SP-resistance in Africa. We assessed the level of SP-resistance markers, and the clinical and parasitological effectiveness of IPTp-SP in southern Mozambique. P. falciparum infection, antimalarial antibodies and dhfr/dhps SP-resistance mutants were detected by quantitative polymerase chain reaction (qPCR), suspension array technology and targeted deep sequencing, respectively, among 4016 HIV-negative women in Maputo province (2016-2019). Univariate and multivariate regression models were used to assess the association between taking the recommended three or more IPTp-SP doses (IPTp3+) and parasitological and clinical outcomes. 84.3% (3385/4016) women received three or more IPTp-SP doses. The prevalence of quintuple mutants at first antenatal care (ANC) visit was 94.2%. IPTp3+ was associated with a higher clearance rate of qPCR-detected infections from first ANC visit to delivery (adjusted odds ratio [aOR]=5.9, 95% CI: 1.5-33.3; p=0.012), lower seroprevalence at delivery of antibodies against the pregnancy-specific antigen VAR2CSADBL34 (aOR=0.72, 95% CI: 0.54-0.95; p=0.022), and lower prevalence of low birth weight deliveries (aOR: 0.61, 95% CI: 0.41-0.90; p=0.013). A sustained parasitological effect of IPTp-SP contributes to the clinical effectiveness of IPTp3+ in areas with high prevalence of SP-resistance markers.

The cytotoxic and immunomodulatory mechanisms of photodynamic therapy evaluated in in vitro studies on cultures of selected skin cell lines

SignificanceThe aim of the study is to evaluate the cytotoxic and immunomodulatory effects of HYP-PDT in vitro studies on cultures of selected skin cell lines, as a possibility of using PDT for patients with atopic dermatitis and psoriasis. ApproachPrimary Dermal Fibroblast and Primary Epidermal Keratinocytes was used in the study. After incubation the cells were treated with hypericin, exposed to VIS visible light. Then cell viability was determined by the MTT assay and cytokines markings were done with Bio-Plex Pro™ Assay kit and with Bio-Plex Suspension Array System. ResultsThe experiment showed a significant cytotoxic effect of HYP-PDT and in sublethal doses, an immunomodulatory effect manifested in changes in the concentrations of selected cytokines ConclusionsThe obtaining results confirming the destructive effect of HYP- PDT on the selected, skin cells lines show a possibility of extending the indication for HYP-PDT, for patients with inflammatory skin diseases.

Exploring cell-free assays for COVID-19 serosurvey

Serosurveys to monitor immunity toward COVID-19 in the population are primarily performed using an ELISA to screen samples for SARS-CoV-2 antibodies, followed by confirmation by a virus neutralization test, which is considered the Gold Standard. However, virus neutralization test may not be feasible for some laboratories because of the requirement for specific facilities and trained personnel. In an attempt to address this limitation, we evaluated three cell-free methods as potential alternatives for assessing SARS-CoV-2 seroprevalence in human population from plasma. We report the establishment of two inhibition ELISAs designed to detect anti-Spike RBD IgG antibodies and a microsphere quantitative suspension array technology assay, based on the Luminex xMAP platform, to measure the presence of antibodies against various SARS-CoV-2 antigens, including anti-RBD. These methods were also compared to a commercial chemiluminescent immunoassay designed for anti-RBD antibodies detection and to the combined ELISA + virus neutralization test strategy. These cell-free assays performed equally to estimate the percentage of positive and negative samples and could be used to determine the prevalence of SARS-CoV-2 antibodies in human population, at least in cohort with high-expected prevalence, without the use of seroneutralization assay.

Effect of aflibercept combined with triamcinolone acetonide on aqueous humor growth factor and inflammatory mediators in diabetic macular edema.

To investigate the efficacy of aflibercept combined with sub-tenon injection of triamcinolone acetonide (TA) in treating diabetic macular edema (DME) and to examine changes in growth factors and inflammatory mediator levels in aqueous humor after injection. Totally 67 DME patients (67 eyes) and 30 cataract patients (32 eyes) were enrolled as the DME group and the control group, respectively. The DME group was divided into the aflibercept group (34 cases) and the aflibercept combined with TA group (combined group, 33 cases). The aqueous humor of both groups was collected during the study period. The aqueous levels of vascular endothelial growth factor (VEGF), monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-1β (IL-1β) were detected using a microsphere suspension array technology (Luminex 200TM). Aqueous cytokines, best-corrected visual acuity (BCVA), central macular thickness (CMT), and complications before and after treatment were compared between the aflibercept group and combined group. The concentrations of VEGF, MCP-1, IL-6, and IL-8 in the aqueous humor were significantly higher in the DME group than those of the control group (all P<0.01). After 1mo of surgery, the concentrations of VEGF, MCP-1, IL-6, and IL-8 in the aqueous humor were significantly lower in the combined group than those of the aflibercept group (all P<0.01). The BCVA and CMT values of the two groups were statistically different after 1 and 2mo of treatment (P<0.01). However, the difference was not statistically significant after 3mo of treatment (P>0.05). The cytokines VEGF, MCP-1, IL-6, and IL-8 in the aqueous humor of DME patients are significantly increased. Aflibercept and aflibercept combined with TA have good efficacy in DME patients, can effectively reduce CMT, improve the patient's vision, and have high safety. Aflibercept combined with TA can quickly down-regulate the aqueous humor cytokines and help to relieve macular edema rapidly. However, the long-term efficacy is comparable to that of aflibercept alone.

Analysis of Cytokine and Chemokine Level in Tear Film in Keratoconus Patients before and after Corneal Cross-Linking (CXL) Treatment.

Keratoconus (KC) is a degenerative corneal disorder whose aetiology remains unknown. The aim of our study was to analyse the expressions of cytokines and chemokines in KC patients before and after specified time intervals after corneal cross-linking (CXL) treatment to better understand the molecular mechanisms occurring before and after CXL in KC patients process of corneal regeneration.; Tear samples were gathered from 52 participants immediately after the CXL procedure and during the 12-month follow-up period. All patients underwent a detailed ophthalmological examination and tear samples were collected before and after CXL at regular intervals: 1 day before and after the surgery, at the day 7 visit, and at 1, 3, 6, 9, and 12 months after CXL. The control group consisted of 20 healthy people. 10 patients were women (50%) and 10 were men (50%). The mean age was 30 ± 3 years of age. Tear analysis was performed using the Bio-Plex 3D Suspension Array System. Corneal topography parameters measured by Scheimpflug Camera included: keratometry values (Ks, Kf), PI-Apex, PI-Thinnest, Cylinder.; All the 12 months post-op values of the KC patients' topographic measurements were significantly lower than the pre-op. As for the tear cytokine levels comparison between the patient and control groups, cytokine levels of TNF-α, IL-6, and CXCL-10, among others, were detected in lower amounts in the KC group. The pre-op level of IL-6 exhibited a significant increase the day after CXL, whereas comparing the day after the procedure to 12 months after CXL, this showed a significant decrease. Both TNF-α and IL-1 showed a significant decrease compared to the day before and after CXL. We observed significantly higher levels of IL-1β, IL-10, IFN-γ and TNF-α in moderate and severe keratoconus than in mild keratoconus (p < 0.05). We also demonstrated a statistically significant positive correlation between both pre-op and 12 months after CXL TNF-α, IFN-γ, IL-6 and Ks and Kf values (p < 0.05, r > 0); Alterations of inflammatory mediators in tear fluid after CXL link with topographic changes and may contribute to the development and progression of KC.

Water-stable perovskite-silica nanocomposites for encoded microbeads construction and multiplexed detection

All-inorganic lead halide perovskite nanocrystals exhibiting bright luminescence have great potential as fluorescence elements for optical encoding. However, their limited stability in water hinders the application in biosensing. In this study, novel optical encoded microbeads based on CsPbX3 (X = Cl, Br) nanocrystals are developed and applied in bead-based suspension arrays for the first time. Through the in-situ crystallization of CsPbX3 nanocrystals within mesoporous silica nano-templates (MSNs), accompanied by mesopores collapse after sintering, CsPbX3@MSNs (X3M) nanocomposites with uniform morphology and stable fluorescence intensity in aqueous solutions for up to 50 days are obtained. By assembling X3M with microspheres to form a host–guest structure, an optical encoding microbead (MX3M) library is established by varying the X3M ratio, halide composition, and the size of host microspheres, which can be easily decoded under multi-channel flow cytometer. As a result, MX3M exhibits outstanding capacity for specific target capture and negligible nonspecific absorption performance in the multiplex nucleic acid detection of respiratory viruses, with a low limit of detection (10 copies/rxn). This result highlights the tremendous potential of MX3M encoded microbeads constructed based on CsPbX3 nanocrystals for multiplexed bioassays.

Multiplexed suspension array immunoassays for detection of antibodies to pneumococcal polysaccharide and conjugate vaccines.

Combination and polyvalent vaccines not only provide protection against several different pathogens at the same time but can also increase vaccine protection against pathogens that have closely related pathogenic strains or serotypes. Multiplexed serological testing is a preferred method for determining the efficacy of combination and polyvalent vaccines, as it reduces the need for conducting multiple individual assays to confirm immune responses and cross-reactivity, uses less sample, and can be faster, more reliable, and more cost-effective. Bead-based suspension array technologies, such as the Luminex® xMAP® Technology, are often used for development of multiplexed serological assays for various vaccine trials and for routine testing in clinical laboratories to determine immune status of vaccinated individuals. This article reviews publications describing the development and implementation of bead-based multiplexed serological assays for detection of immune responses to polyvalent polysaccharide and conjugate vaccines against Streptococcus pneumoniae. Many of these serological assays on the bead array platform have been further optimized and expanded over time and are still widely used today.

Mildronate Ameliorates Kidney Function and Reduces Inflammation in SCD Mice

INTRODUCTION:Sickle cell disease (SCD) is a genetic hemoglobin disorder characterized by hemolytic anemia, vaso-occlusive crisis and inflammation that affects organs such as the kidney, liver, brain, spleen, and heart. SCD is driven by a mutation in the β-globin gene that causes sickling of red blood cells (RBCs). More than 50% of SCD patients are affected by chronic kidney diseases (CKD) and other comorbidities. Vasculopathy is caused by hemolysis-related endothelial dysfunction and inflammation, which collectively contribute to the development of renal disease. Mildronate (also known as Meldonium) is an anti-ischemic and cardio protecting medication approved in Eastern Europe. Mildronate facilitates a switch from fatty acid oxidation to less oxygen-intensive glycolysis that allows less oxygen consumption by peripheral tissues and higher oxygenation of heart. We hypothesized that mildronate administration would reduce tissue hypoxia and decrease inflammation in SCD mice. METHODS: The study was approved by the Institutional Animal Care and Use Committee at Howard University (IACUC). We employed the Townes SCD mouse model, which mimics SCD complications. SCD and control mice were injected with mildronate (400 mg/kg in saline) intraperitoneally or saline as vehicle control over the course of ten days. Blood and plasma were collected and used for hematological parameters analysis. Cytokines were measured by Bio-plex suspension array in plasma and real-time RT-PCR or ELISA in organs. Protein expression of HO-1, HIF-1α, NFκB and NRF2 was measured by Western blot. The effect of Mildronateon systemic and renal oxidative stress was measured by malondialdehyde (MDA) using ELISA (Abcam). RESULTS: We observed significant reduction in the cytokine's levels of IFN-γ, IL-1, and TNF-α in plasma of SCD mice after mildronate injection. We also observed reduction in IL-6, IL-10 and IL-17 levels but differences were not statistically significant. There were no significant changes in hematological parameters (Hematocrit, MCV, MCH, CMCH, Erythrocytes, Hemoglobin) after mildronate injection. Also, we did not observe changes in MDA levels in plasma or kidney in SCD mice compared to control mice, both non-injected and mildronate injected. We further evaluated HO-1 and NRF2 protein expression by western blot and observed no significant differences between SCD mice with or without mildronate injection. In H&amp;E staining, cortex glomerular area and medullar congestion area were significantly reduced after mildronate injection. We also observed reduction of proteinuria in SCD mice injected with mildronate. DISCUSSION: Our examination of the effect of mildronate showed decreases inflammation in the mildronate injected SCD mice evidenced by reduced levels of IFN-γ, IL-1, IL-17, IL-6, IL-10, and TNF-α in circulation. There was a slight decrease in the expression levels of Nrf2 and HO-1 after mildronate injection. HO-1 is crucial for the catabolism of hemoglobin and protects against tissue damage in hemolysis. The HO-1 promoter has binding sites for several transcription factors, including phosphorylated Nrf2. Previous studies from our lab showed increased HIF-1α expression in PBMCs obtained from SCD patients. Here we did not detect significant changes in NFκB-p65 or HIF-1α expression levels. While we expected mildronate to decrease the oxidative stress, we did not observe significant reduction in MDA levels in treated mice compared to control mice. CONCLUSION: Mildronate improved kidney function in SCD mice by reducing circulatory and renal inflammation. In future, we plan to assess in more details the mildronate's impact on kidney function and inflammation. ACKNOWLEDGMENTS: This work was supported by NIH grants 1R01HL125005, 5U54MD007597, and 1SC1HL150685.

Clinical significance of interleukin-6, total bilirubin, CD3 + CD4 + T cells counts in the acute exacerbation of connective tissue disease-associated interstitial lung disease: a cross-sectional study

ObjectiveInterstitial lung disease (ILD) is a severe complication of connective tissue disease (CTD) that can significantly impact patients' prognosis and quality of life. However, the current diagnostic arena lacks reliable biomarkers for detecting and monitoring the progression and exacerbation of CTD-ILD. This study aimed to investigate the clinical value of 12 serum cytokines in the diagnosis of CTD-ILD and prediction of the risk of acute exacerbation (AE) in this disease.MethodsThis study was a cross-sectional investigation. Ninety-one hospitalized CTD patients were allocated into two groups: CTD-ILD group (n = 61) and CTD-non-ILD group (n = 30), and 30 sex-age matched healthy volunteers were enrolled as controls. The serum concentrations of interferon (IFN)-α, IFN-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-17A, and IL-1β were measured by Luminex suspension arrays. Logistic regression was employed to determine the significance of variables in the occurrence of AE-CTD-ILD. A nomogram was constructed to visualize the independent variables.ResultsElevated levels of IL-6, IL-8, and TNF-α were observed and compared in the CTD-ILD group with CTD-non-ILD (all P < 0.05). Similarly, the levels of IL-6, IL-8 and TNF-α were higher in the acute exacerbation (AE-CTD-ILD) group compared with stable CTD-ILD (S-CTD-ILD) (P < 0.001, P < 0.001, and P = 0.022). Significant correlations between serum IL-6 and PaO2/FiO2 ratio (r = − 0.463, P < 0.001), percent predicted forced vital capacity (FVC%; r = − 0.362, P < 0.05), and total ground-glass opacity (GGO) score (r = 0.439, P < 0.001) were observed in CTD-ILD patients. Multivariate logistic regression analysis revealed that elevated IL-6 levels, total bilirubin (TBil), and decreased CD3 + CD4 + T cells counts were independent risk factors for the occurrence of AE-CTD-ILD (OR = 1.121, P = 0.024; OR = 1.865, P = 0.047; OR = 0.983, P = 0.037, respectively). Furthermore, by employing these three variables in combination for the prediction of AE status, their collective impact surpasses the independent effects of any single biomarker.ConclusionsElevated levels of serum IL-6, IL-8, and TNF-α were associated with the complication of ILD in CTD patients and the occurrence of AE in CTD-ILD patients. IL-6 could be a promising serum biomarker of severity and the occurrence of AE in CTD-ILD patients. The combination of the three variables (IL-6 level, TBil and CD3 + CD4 + T cells) predicted the AE-CTD-ILD better.

Associations between prenatal malaria exposure, maternal antibodies at birth, and malaria susceptibility during the first year of life in Burkina Faso.

In this study, we investigated how different categories of prenatal malaria exposure (PME) influence levels of maternal antibodies in cord blood samples and the subsequent risk of malaria in early childhood in a birth cohort study (N = 661) nested within the COSMIC clinical trial (NCT01941264) in Burkina Faso. Plasmodium falciparum infections during pregnancy and infants' clinical malaria episodes detected during the first year of life were recorded. The levels of maternal IgG and IgG1-4 to 15 P. falciparum antigens were measured in cord blood by quantitative suspension array technology. Results showed a significant variation in the magnitude of maternal antibody levels in cord blood, depending on the PME category, with past placental malaria (PM) more frequently associated with significant increases of IgG and/or subclass levels across three groups of antigens defined as pre-erythrocytic, erythrocytic, and markers of PM, as compared to those from the cord of non-exposed control infants. High levels of antibodies to certain erythrocytic antigens (i.e., IgG to EBA140 and EBA175, IgG1 to EBA175 and MSP142, and IgG3 to EBA140 and MSP5) were independent predictors of protection from clinical malaria during the first year of life. By contrast, high levels of IgG, IgG1, and IgG2 to the VAR2CSA DBL1-2 and IgG4 to DBL3-4 were significantly associated with an increased risk of clinical malaria. These findings indicate that PME categories have different effects on the levels of maternal-derived antibodies to malaria antigens in children at birth, and this might drive heterogeneity to clinical malaria susceptibility in early childhood.

Standardization of Food Allergen Measurements Using Multiplex Array Technology.

Multiplex suspension array technology enables multiple protein analytes to be measured in a single assay. In this chapter, we describe a polystyrene bead-based fluorescent multiplex array for quantitative measurements of specific food allergens. The array uses monoclonal antibodies for allergen detection and purified allergens as reference standards. Each component of the array can be standardized. The array measures up to 17 foods that are regulated in the United States, the European Union, and Japan.

Serum cytokine profiles in myasthenia gravis with anti-muscle-specific kinase antibodies

This study measured the serum levels of of 15 cytokines in 15 patients with anti-muscle-specific kinase antibody-positive MG (MuSK-MG) using a multiplex suspension array system. Fifteen patients with non-inflammatory neurological diseases served as controls. Compared with controls, patients with MuSK-MG showed higher levels of Th1- (IFN-γ), Th2- (IL-25, IL-31, and IL-33), Th17- (IL-22), Treg-related cytokines (IL-10), and soluble CD40 ligand (sCD40L). Higher serum Th2-related cytokines (IL-25 and IL-31) levels were correlated with less MG Foundation of America (MGFA) class. These suggest that Th2-related cytokines have protective effects, whereas sCD40L and others may facilitate the disease.

Association Analysis between Genotype and Phenotype of α,β-Thalassaemia Carriers in Huizhou Area of Guangdong Province

To analyze the prevalence, genotype distribution and hematological characteristics of α,β-thalassaemia carriers in Huizhou area of Guangdong Province. 10 809 carriers of simple β-thalassaemia and 1 757 carriers of α,β-thalassaemia were enrolled as our study cohort. The hematological parameters were detected by automated blood cell counters and automatic capillary electrophoresis. Suspension array technology, gap-polymerase chain reaction (gap-PCR) and PCR-reverse dot blot were used for the genotyping of thalassaemia carriers. The prevalence of α,β-thalassaemia in Huizhou area of Guangdong Province was 1.99%. A total of 62 genotypes were detected, and the most prevalent genotype was --SEA/ αα, βCD41-42/ βN (19.29%), the next was --SEA/ αα, βIVS-II-654/ βN (16.73%). Significant differences in mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) were found between different genotype groups for simple β-thalassaemia and α,β-thalassaemia. Violin plots showed that carriers with co-inheritance of β-thalassaemia and mild α-thalassaemia expressed the lightest anemia, and carriers with co-inheritance of β-thalassaemia and hemoglobin H (Hb H) disease expressed the most severe anemia. There is a high prevalence of α,β-thalassaemia in Huizhou area of Guangdong Province. Because of the lack of specific hematological makers for diagnosis of α,β-thalassaemia, it is necessary to distinguish it from simple β-thalassaemia by genotyping of α- and β-thalassaemia in order to correctly guide genetic counseling and prenatal disgnosis.

Anti-Vascular Endothelial Growth Factor Therapy with or Without Initial Steroid Therapy for Macular Edema in Branch Retinal Vein Occlusion.

In branch retinal vein occlusion (BRVO), administering steroid injections to inhibit expression of inflammatory factors in the first phase of macular edema may reduce recurrence of the edema. The purpose of our study was to investigate the functional and morphological prognosis and frequency of recurrence after injection of an anti-vascular endothelial growth factor (VEGF) with and without initial steroid therapy to treat macular edema with BRVO. Patients with BRVO and macular edema (41 eyes) received intravitreal ranibizumab injection (IRI) alone (IRI group, 21 eyes) or subtenon triamcinolone (STTA) injection and IRI (combination group, 20 eyes). Patients in both groups with recurrent macular edema received further IRI as appropriate. A laser flare meter was used to assess aqueous flare values, and an optical coherence tomography device was used to measure central macular thickness (CMT). Before the first treatment, we obtained samples of aqueous humor and analyzed them by the suspension array method to evaluate VEGF, placental growth factor (PlGF), platelet-derived growth factor (PDGF)-AA, soluble intercellular adhesion molecule (sICAM)-1, monocyte chemoattractant protein 1 (MCP-1), interleukin (IL)-6, IL-8, and interferon-inducible 10-kDa protein (IP-10). The two groups were not significantly different with regard to levels of VEGF, PlGF, PDGF-AA, sICAM-1, MCP-1, IL-6, IL-8, or IP-10. Best-corrected visual acuity, CMT, and aqueous flare value (IRI group, baseline 8.69 ± 4.55 photon counts/ms; combination group, baseline 9.21 ± 3.72 photon counts/ms) improved significantly in both groups without significant intergroup differences. Analyses showed no significant intergroup differences in the mean number of IRIs during the 12-month follow-up, but the proportion of patients without recurrence (ie, who received only one IRI) was significantly higher in the combination group than in the IRI group (P = 0.032). Furthermore, the time to initial recurrence was significantly longer in the combination group than in the IRI group (P = 0.003). These findings suggest that initial STTA injection and IRI may have a synergistic effect in patients with BRVO and macular edema in that they increase the time between anti-VEGF treatments.

VCAM-1 complements CA-125 in detecting recurrent ovarian cancer

BackgroundClose to three-quarters of ovarian cancer cases are frequently diagnosed at an advanced stage, with more than 70% of them failing to respond to primary therapy and relapsing within 5 years. There is an urgent need to identify strategies for early detection of ovarian cancer recurrence, which may lead to earlier intervention and better outcomes.MethodsA customized magnetic bead-based 8-plex immunoassay was evaluated using a Bio-Plex 200 Suspension Array System. Target protein levels were analyzed in sera from 58 patients diagnosed with advanced ovarian cancer (including 34 primary and 24 recurrent tumors) and 46 healthy controls. The clinical performance of these biomarkers was evaluated individually and in combination for their ability to detect recurrent ovarian cancer.ResultsAn 8-plex immunoassay was evaluated with high analytical performance suitable for biomarker validation studies. Logistic regression modeling selected a two-marker panel of CA-125 and VCAM-1 that improved the performance of CA-125 alone in detecting recurrent ovarian cancer (AUC: 0.813 versus 0.700). At a fixed specificity of 83%, the two-marker panel significantly improved sensitivity in separating primary from recurrent tumors (70.8% versus 37.5%, P = 0.004), demonstrating that VCAM-1 was significantly complementary to CA-125 in detecting recurrent ovarian cancer.ConclusionsA two-marker panel of CA-125 and VCAM-1 showed strong diagnostic performance and improvement over the use of CA-125 alone in detecting recurrent ovarian cancer. The experimental results warrant further clinical validation to determine their role in the early detection of recurrent ovarian cancer.

Effects of Intravitreal Ranibizumab Injection on Peripheral Retinal Microcirculation and Cytokines in Branch Retinal Vein Occlusion with Macular Edema.

Background and Objectives: To investigate peripheral blood flow in retinal vessels and vessel diameters after intravitreal ranibizumab injection (IRI) and the relationship between these parameters and cytokines in branch retinal vein occlusion (BRVO) with macular edema. Materials and Methods: We assessed relative flow volume (RFV) and the width of the main and branch retinal arteries and veins in the occluded and non-occluded regions before and after IRI in 37 patients with BRVO and macular edema. Measurements were made using laser speckle flowgraphy (LSFG). When performing IRI, we obtained samples of aqueous humor and analyzed them using the suspension array method to evaluate vascular endothelial growth factor (VEGF), placental growth factor (PlGF), platelet-derived growth factor (PDGF)-AA, soluble intercellular adhesion molecule (sICAM)-1, monocyte chemoattractant protein 1 (MCP-1), interleukin (IL)-6, IL-8, and interferon-inducible 10-kDa protein (IP-10). Results: In both retinal regions, before and after IRI, the RFV in the main artery and vein showed a significant correlation with the summed RFV in the respective branch vessels 1 and 2. In the occluded region, the RFV in the main vein was significantly negatively correlated with MCP-1, PDGF-AA, IL-6, and IL-8; the RFV in branch vein 1 was significantly negatively correlated with PlGF, MCP-1, IL-6, and IL-8; PDGF-AA was significantly negatively correlated with the width of the main and branch veins; and the RFVs of the main artery and vein decreased significantly from before to 1 month after IRI. Conclusions: Contrary to expectations, the study found that anti-VEGF therapy does not affect RFV in arteries and veins in patients with BRVO and macular edema. Furthermore, retinal blood flow is poor in patients with high MCP-1, IL-6, and IL-8. Finally, high PDGF-AA may result in smaller venous diameters and reduced retinal blood flow.

Identification of Inflammatory Endotypes by Clinical Characteristics and Nasal Secretion Biomarkers in Chronic Rhinosinusitis with Nasal Polyps

Introduction: The emergency of biologics and surgical techniques targeting the specific inflammatory endotype in chronic rhinosinusitis with nasal polyps (CRSwNP) asks for efficient identification of patients with different endotypes. Although mucosal IL-4, IL-5, IL-13, and IgE have been used to define type 2 (T2) inflammation, the optimal one remains unclear. In this study, we aimed to determine the optimal anchor for T2 inflammation and identify clinical characteristics and nasal secretion biomarkers predicting different endotypes in CRSwNP. Methods: Six mediators in sinonasal tissue and 36 mediators in nasal secretion samples were detected by the Bio-Plex suspension array system. Mucosal IFN-γ and IL-17A levels were used to define the T1 and T3 endotype, respectively. The efficacy of mucosal IL-4, IL-5, IL-13, and IgE to define the T2 endotype was compared. The power of clinical characteristics and nasal secretion biomarkers to predict the T1, T2, and T3 endotype was analyzed. Results: Among mucosal IL-4, IL-5, IL-13, and IgE, IL-13 was the best one to coincide with the expression of other T2 biomarkers. A combination of atopy, facial pain symptom score, ethmoid/maxillary computed tomography score ratio, and blood eosinophil percentage had a moderate predictive performance for T2 endotype (area under the receiver operating curve [AUC] = 0.815), comparable to that of nasal secretion IL-5 (AUC = 0.819). For the T3 endotype, nasal secretion IL-1Rα identified it with an AUC value of 0.756. No efficient marker for the T1 endotype was found. Conclusion: IL-13 is a primary anchor for the T2 endotype in CRSwNP. Clinical characteristics and nasal secretion biomarkers are helpful for identifying the T2 and T3 endotype of CRSwNP.