Microbead Assay Research Articles

Prevalence of anti-HLA antibodies in COVID-19 convalescent plasma donors: an Indian experience

BackgroundCOVID-19 convalescent plasma is one of the experimental therapies used widely in moderately sick COVID-19 patients. However, there are a few risks involved in plasma transfusion; notably, transfusion-related acute lung injury (TRALI) caused by antibodies against human leukocyte antigens (HLA). This study was designed to assess the prevalence of anti-HLA antibodies in convalescent plasma donors using the single antigen bead method. Study design and methodsThis was a hospital-based observational study of consecutive plasma donors. A total of 252 samples were screened for anti-HLA Class I and Class II antibodies using the microbead assay with the identification of anti-HLA Ab in positive samples being performed using a single antigen bead assay. Luminex-based normalized background cutoff ratios of 10.8 for Class I and 6.9 for Class II and mean fluorescence intensity cutoffs of 2500 for Class I and 1500 for Class II were used for screening and the single bead assay, respectively. ResultsOf 252 screened samples, 28 (11.1 %) were positive for Class I, Class II or both Class I and Class II anti-HLA antibodies in donors with no history of a previous immunizing event. Moreover, 20/252 (7.9%) donors without any history of prior immunization had specific anti-HLA antibodies of Class I or Class II or both by the single bead assay. ConclusionsThe high prevalence of anti-HLA antibodies in our cohort of donors raises an urgent and immediate need for anti-HLA antibody screening in all convalescent plasma donors for safe therapy of COVID-19 patients.

A multiplex microchamber diffusion assay for the antibody-based detection of microRNAs on randomly ordered microbeads

MicroRNAs (miRNAs) are small non-coding RNA regulators linked to various human diseases incl. heart disease, a leading cause of death in Western countries. Their alterations signify the need for early detection methods. We devised a diffusion microbead assay, combining it with antibody-based miRNA detection. Diffusion involves co-diffusion of miRNAs and antibodies in a microchamber. Randomly ordered size and dye encoded microbeads loaded with specific capture probes target heart disease-associated miRNAs. MiRNA detection is time- and dose-dependent using an anti-DNA:RNA hybrid antibody. The miRNAs are successively exposed to randomly ordered microbeads, which leads to microbeads that become saturated with the target molecules first in front rows. Unbound miRNAs diffuse further and bind to microbeads with free binding sites. Our assay provides real-time multiplex detection of multiple miRNA within 2 h in an isothermal amplification-free environment, with low detection limits (miR-21-5p: 0.21 nM; miR-30a-3p: 0.03 nM; miR-93-5p: 0.43 nM). This study presents a miRNA detection principle that differs from other microbead assays where all microbeads are simultaneously mixed with the sample solution, so that all target molecules bind to microbeads equally, ultimately resulting in an averaged signal intensity.

FLASH Proton Radiation Therapy Mitigates Inflammatory and Fibrotic Pathways and Preserves Cardiac Function in a Preclinical Mouse Model of Radiation-Induced Heart Disease

PurposeStudies during the past 9 years suggest that delivering radiation at dose rates exceeding 40 Gy/s, known as “FLASH” radiotherapy, enhances the therapeutic index of radiation therapy (RT) by decreasing normal tissue damage while maintaining tumor response compared to conventional (or standard) RT. This study demonstrates the cardioprotective benefits of FLASH proton RT (F-PRT) compared to standard (conventional) proton RT (S-PRT), as evidenced by reduced acute and chronic cardiac toxicities. Methods and MaterialsMice were imaged using cone beam computed tomography to precisely determine the heart's apex as the beam isocenter. Irradiation was conducted utilizing a shoot-through technique with a 5mm diameter circular collimator. Bulk RNA-sequencing was performed on non-irradiated (NR) samples, as well as apexes treated with F-PRT or S-PRT, at 2 weeks following a single 40 Gy dose. Inflammatory responses were assessed through multiplex cytokine/chemokine microbead assay and immunofluorescence analyses. Levels of perivascular fibrosis were quantified using Masson's Trichrome and Picrosirius red staining. Additionally, cardiac tissue functionality was evaluated by 2D echocardiograms at 8 and 30 weeks post-PRT. ResultsRadiation damage was specifically localized to the heart's apex. RNA profiling of cardiac tissues treated with PRT revealed that S-PRT uniquely upregulated pathways associated with DNA damage response, induction of tumor necrosis factor superfamily and inflammatory response, while F-PRT primarily affected cytoplasmic translation, mitochondrion organization and ATP synthesis. Notably, F-PRT led to a milder inflammatory response, accompanied by significantly attenuated changes in TGF-β1 and αSMA levels. Critically, F-PRT decreased collagen deposition, and better preserved cardiac functionality compared to S-PRT. ConclusionsThis study demonstrated that F-PRT reduces the induction of an inflammatory environment with lower expression of inflammatory cytokines and profibrotic factors. Importantly, the results indicate that F-PRT better preserves cardiac functionality, as confirmed by echocardiography analysis, while also mitigating the development of long-term fibrosis.

High-throughput microbead assay system with a portable, cost-effective Wi-Fi imaging module, and disposable multi-layered microfluidic cartridges for virus and microparticle detection, and tracking.

In recent years biomedical scientific community has been working towards the development of high-throughput devices that allow a reliable, rapid and parallel detection of several strains of virus or microparticles simultaneously. One of the complexities of this problem lies on the rapid prototyping of new devices and wireless rapid detection of small particles and virus alike. By reducing the complexity of microfluidics microfabrication and using economic materials along with makerspace tools (Kundu et al. 2018) it is possible to provide an affordable solution to both the problems of high-throughput devices and detection technologies. We present the development of a wireless, standalone device and disposable microfluidics chips that rapidly generate parallel readouts for selected, possible virus variants from a nasal or saliva sample, based on motorized and non-motorized microbeads detection, and imaging processing of the motion tracks of these beads in micrometers. Microbeads and SARS-CoV-2 COVID-19 Delta variant were tested as proof-of-concept for testing the microfluidic cartridges and wireless imaging module. The Microbead Assay (MA) system kit consists of a Wi-Fi readout module, a microfluidic chip, and a sample collection/processing sub-system. Here, we focus on the fabrication and characterization of the microfluidic chip to multiplex various micrometer-sized beads for economic, disposable, and simultaneous detection of up to six different viruses, microparticles or variants in a single test, and data collection using a commercially available, Wi-Fi-capable, and camera integrated device (Fig. 1).

MO947: Evaluation of Bone Metabolism Markers in Kidney Transplant Recipients

Abstract BACKGROUND AND AIMS Immunosuppressive therapies, pre-existing renal osteodystrophy, de novo hyperparathyroidism, and other traditional risk factors influence Mineral and Bone Disorder (MBD) after kidney transplantation. However, little is known about their effect on bone metabolism biomarkers. Therefore, we aimed to understand how kidney transplant affects bone metabolism markers in comparison to patients on hemodialysis and healthy individuals. METHOD This is a cross-sectional study with 57 kidney transplant patients, 36 patients on hemodialysis, and 31 healthy controls. Plasma concentrations of Dickkopf-related protein 1 (DKK1), osteoprotegerin (OPG), osteocalcin (OC), osteopontin (OPN), sclerostin (SOST) and fibroblast growth factor 23 (FGF-23) were measured using the Luminex-based microbead assay (HBNMAG-51K, Millipore, Billerica, MA) in these three groups. Associations between the measurements of these molecules with clinical, demographic, and laboratory data were evaluated. For variables without Gaussian distribution, Mann–Whitney test was used to compare two groups and the Kruskal–Wallis test for comparisons between more than two groups. For variables with normal distribution, comparisons between two groups were made by unpaired Student's t-test and for more than two groups by analysis of variance followed by Bartlett’s post-test. Pearson or Spearman tests were adopted to evaluate correlations according to the variables’ distribution. All statistical tests were two-tailed with a significance level of P < 0.05. RESULTS Transplant recipients had significantly lower levels of DKK1 (P < 0.001), OPG (P < 0.001), OC (P < 0.001), OPN (P = 0.001), OST (P < 0.001) and FGF-23 (P < 0.001) when compared with patients on hemodialysis. In comparison to healthy controls, transplant recipients also presented lower levels of DKK1 (P = 0.019), OPG (P < 0.001), OC (P = 0.027), SOST (P < 0.001) and FGF-23 (P = 0.043) (Figure 1). Regarding demographic data, women presented lower plasma SOST levels when compared with men in the hemodialysis group (P = 0.012). Different correlations between the human bone metabolism components were detected in transplant recipients and hemodialysis patients. In the transplant group, DKK1 was positively correlated with SOST (r = 0.484, P = 0.001), OPN (r = 0.420, P = 0.005) and OPG (r = 0.521, P < 0.001). In the same group, SOST levels were positively correlated with FGF-23 (r = 0.424, P = 0.027, Figure 2D) and OPG (r = 0.703, P < 0.001), while OC only positively correlated with OPN (r = 0.572, P < 0.001). By contrast, patients on hemodialysis only showed positive correlations between OPN and DKK1 (r = 0.420, P = 0.005) and between SOST and OPG (r = 0.757, P < 0.001). Concerning laboratory parameters and their correlations with human bone metabolism molecules, both transplant recipients and hemodialysis patients showed a negative correlation between 25(OH)vitamin D levels and FGF-23. On the other hand, ALP levels were positively correlated with different molecules in the two groups. In transplant patients, ALP levels positively correlated with OPN (r = 0.572, P < 0.001), while, in hemodialysis patients, the positive correlation was with OPG (r = 0.548, P = 0.012). CONCLUSION Our findings showed a reduction in bone metabolism markers, DKK1, OPG, OC, OPN and SOST after kidney transplantation. The first years after kidney transplantation modulate MBD markers, revealing a significant improvement of MBD associated with end-stage kidney disease.

Diphtheria and tetanus seroepidemiology among children in Ukraine, 2017

BackgroundThe drastic decline of Ukraine’s immunization coverage since 2009 led to concerns about potential resurgence diphtheria and tetanus, along with other vaccine-preventable diseases. MethodsTo assess population immunity against diphtheria and tetanus, we tested specimens from the serosurvey conducted in 2017 among children born in 2006–2015, the birth cohorts targeted by the nationwide outbreak response immunization following a circulating vaccine-derived poliovirus type 1 outbreak in Zakarpattya province in 2015. We surveyed four regions of Ukraine, using cluster sampling in Zakarpattya, Sumy, and Odessa provinces and simple random sampling in Kyiv City. We tested serum specimens for IgG antibodies against diphtheria and tetanus, using microbead assays (MBA). We estimated seroprevalence and calculated 95% confidence intervals. We also obtained information on the immunization status of surveyed children. ResultsSeroprevalence of ≥0.1 IU/mL diphtheria antibodies was <80% in all survey sites (50.0%–79.2%). Seroprevalence of ≥0.1 IU/mL tetanus antibodies was ≥80% in Sumy, Kyiv City, and Odessa (80.2%–89.1%) and 61.6% in Zakarpattya. Across the sites, the proportion of children vaccinated age-appropriately with diphtheria-tetanus-containing vaccines (DTCV) was 28.5%–57.4% among children born in 2006–2010 and 34.1%–54.3% among children born in 2011–2015. The proportion of recipients of <3 DTCV doses increased from 7.1%–16.7% among children born in 2006–2010 to 19.8%–38.6% among children born in 2011–2015, as did the proportion of recipients of zero DTCV doses (2.6%–8.8% versus 8.0%–14.0%, respectively). ConclusionsProtection against diphtheria among children born in 2006–2015 was suboptimal (<80%), particularly in Zakarpattya. Protection against tetanus was adequate (≥80%) except in Zakarpattya. Diphtheria-tetanus immunization status was suboptimal across all sites. Catch-up vaccination of unvaccinated/under-vaccinated children and other efforts to increase immunization coverage would close these immunity gaps and prevent the resurgence of diphtheria and tetanus in Ukraine, particularly in Zakarpattya.

Frequency, reactivity and evolution of human leukocyte antigen and human platelet antigen antibodies in the setting of hematopoietic cell transplantation

Background and objectivesAntibodies (Ab) against HLA and HPA antigens play an important role in HCT. In this prospective study we evaluated prevalence and kinetics of HLA- and HPA-Ab after HCT, including a possible donor-recipient transfer and their clinical relevance in respect to platelet transfusion refractoriness (PTR). Materials and methodsPatients were consecutively recruited. Ab were determined by microbead assay technique and a mean fluorescence intensity cut-off of 1,000. ResultsAt baseline, 21 donors (42 %) and 27 patients (54 %) had HLA-Ab with a mean panel reactivity (cPRA) of 34.9 ± 29.4 % and 46.1 ± 36.5 %, respectively. We observed a significant higher number of HLA-Ab specificities in female donors and patients and a predominance of HLA-class I Ab. At day 0 we detected an increase of HLA-Ab (from 526 to 673) and cPRA (55.2 ± 31.9 %). Thirty-six patients (72 %) developed new HLA-Ab, mainly 3 weeks after HCT. In 7 patients an HLA-Ab with the same specificity as detected in the corresponding donor emerged, suggesting a possible transfer from the donor to the recipient. Overall, MFI showed a high variation. Type and number of transfusions were not associated with number and intensity of HLA-Ab (ρ: -0.05 – 0.02). Number of HLA-Ab, cPRA and intensity were not associated with PTR, which occurred in 9 patients (18 %) and none had bleeding WHO > 2. ConclusionsAlthough a considerable number of patients have and develop HLA-Ab before and early after HCT, we found no association with PTR and bleeding and management should be individualized.

High Intrapatient Variability in Tacrolimus Exposure Calculated Over a Long Period Is Associated With De Novo Donor-Specific Antibody Development and/or Late Rejection in Thai Kidney Transplant Patients Receiving Concomitant CYP3A4/5 Inhibitors.

High intrapatient variability in tacrolimus trough levels (Tac IPV) is associated with poor allograft outcomes. Tac IPV was previously calculated using trough levels 6-12 months after kidney transplantation (KT). Data on the accuracy of Tac IPV calculation over a longer period, the association between high Tac IPV and donor-specific antibody (DSA) development after KT in Asian patients, and the role of IPV in patients receiving concomitant cytochrome P450 (CYP)3A4/5 inhibitors (CYPinh) are limited. A retrospective review of patients who underwent KT at our center in 2005-2015, and who received Tac with mycophenolate during the first 2 years after KT was performed. IPV was calculated using Tac levels adjusted by dosage. DSA was monitored annually after KT using a Luminex microbead assay. In total, 236 patients were enrolled. CYPinh were prescribed to 189 patients (80.1%): 145 (61.4%), 31 (13.1%), and 13 (5.5%) received diltiazem, fluconazole, and ketoconazole, respectively. Mean IPV calculated from adjusted Tac levels for 6-12 months (IPV6-12) and 6-24 months (IPV6-24) after KT were 20.64% ± 11.68% and 23.53% ± 10.39%, respectively. Twenty-six patients (11%) showed late rejection and/or DSA occurrence, and had significantly higher IPV6-24 (29.42% ± 13.78%) than others (22.77% ± 9.64%; P = 0.02). There was no difference in IPV6-12 (24.31% ± 14.98% versus 20.17% ± 10.90%; P = 0.18). IPV6-12 and IPV6-24 were comparable in patients who did and did not receive CYPinh. When using mean IPV6-24 as a cutoff, patients with higher IPV6-24 had a higher probability of developing DSA and/or late rejection (P = 0.048). Tac IPV6-24 was higher and more significantly associated with DSA development and/or late rejection than Tac IPV6-12, independent of Tac trough level. This is the first study to demonstrate the impact of high IPV on DSA development in Asian patients, and that Tac IPV is comparable between patients with and without CYPinh.

Analysis of plasma KIM-1 as a biomarker for recurrence risk after resection for localized renal cell carcinoma.

342 Background: Recurrence is common after nephrectomy for renal cell carcinoma (RCC), but no circulating biomarkers are available to identify patients at highest risk of recurrence who may benefit from adjuvant therapy. Kidney injury molecule-1 (KIM-1) is overexpressed in RCC and its ectodomain circulates in plasma. We investigated whether plasma KIM-1 is a prognostic biomarker in patients with localized RCC after nephrectomy. Methods: Banked plasma samples were analyzed from the ECOG-ACRIN 2805 (ASSURE) trial evaluating adjuvant sunitinib, sorafenib, and placebo in resected high-risk RCC. KIM-1 levels were measured at trial enrollment 4-12 weeks post-nephrectomy (baseline) and on cycle 2 day 1 (C2D1) using a previously validated microbead assay. A lognormal accelerated failure time model was used to test for association between circulating KIM-1 and disease-free survival (DFS). Results: Plasma samples from 418 patients were analyzed. In univariable and multivariable analyses, higher post-nephrectomy KIM-1 was associated with worse DFS across all study arms. This association remained independently significant after adjustment for Fuhrman grade, T-stage, N-stage, and tumor histology (survival time ratio 0.56 for 75th vs 25th percentile of KIM-1, 95% CI 0.42-0.73, p &lt; 0.001). The association between KIM-1 and DFS was stronger among patients with pathologic nodal involvement. The addition of baseline KIM-1 improved the concordance of both the SSIGN and UISS prognostic models (SSIGN concordance 0.57 vs 0.43, p = 0.05; UISS concordance 0.60 vs 0.40, p = 0.0005). C2D1 KIM-1 was not an independent predictor for DFS after adjusting for baseline KIM-1. Conclusions: Elevated plasma KIM-1 level at post-nephrectomy baseline is associated with worse DFS in RCC. This is consistent with the hypothesis that post-nephrectomy plasma KIM-1 may be a biomarker for microscopic residual disease. The model was additionally adjusted for papillary and chromophobe histology, sex, and ECOG performance status. [Table: see text]

The Association Between Breast Milk Group B Streptococcal Capsular Antibody Levels and Late-onset Disease in Young Infants.

Animal-model studies have demonstrated less group B streptococcal (GBS) invasive disease and gastrointestinal colonization after enteral administration of serotype-specific capsular antibodies. There is, however, a paucity of information on the association of breast milk GBS serotype-specific capsular antibodies and risks for invasive disease in infants. The aim of this study was to explore the association between natural secretory immunoglobulin A (sIgA) capsular antibodies in breast milk and the occurrence of late-onset disease (LOD) in young infants. A matched case-control study was undertaken in infants <3 months of age in Johannesburg, South Africa. Breast milk samples were collected on cases and controls matched for gestational age, maternal age, and human immunodeficiency virus status at time of enrollment. Capsular serotype Ia, Ib, III, and V sIgA antibody concentrations were measured using the fluorescence-based micro-bead immunosorbent assay. Breast milk samples were available for 31 LOD cases (8 serotype Ia and 23 serotype III), 21 recto-vaginally colonized matched controls (10 serotype Ia and 11 serotype III), and 84 serotype Ia and 105 serotype III noncolonized matched controls. Using a Bayesian model to estimate the probability of disease, there were 90% reductions in the risks of developing serotypes Ia and III LOD with sIgA concentrations ≥0.14 µg/mL and ≥2.52 µg/mL, respectively. Breast milk sIgA capsular antibodies were associated with lower risks for LOD in young infants. The ability of GBS polysaccharide-protein conjugate vaccines currently under development to induce sIgA responses warrant investigation as potential mediators of protection against LOD.

A new reporter design based on DNA origami nanostructures for quantification of short oligonucleotides using microbeads

The DNA origami technique has great potential for the development of brighter and more sensitive reporters for fluorescence based detection schemes such as a microbead-based assay in diagnostic applications. The nanostructures can be programmed to include multiple dye molecules to enhance the measured signal as well as multiple probe strands to increase the binding strength of the target oligonucleotide to these nanostructures. Here we present a proof-of-concept study to quantify short oligonucleotides by developing a novel DNA origami based reporter system, combined with planar microbead assays. Analysis of the assays using the VideoScan digital imaging platform showed DNA origami to be a more suitable reporter candidate for quantification of the target oligonucleotides at lower concentrations than a conventional reporter that consists of one dye molecule attached to a single stranded DNA. Efforts have been made to conduct multiplexed analysis of different targets as well as to enhance fluorescence signals obtained from the reporters. We therefore believe that the quantification of short oligonucleotides that exist in low copy numbers is achieved in a better way with the DNA origami nanostructures as reporters.

Spatial arrangement of proteins using scCro-tag: application for an in situ enzymatic microbead assay.

In natural systems, various metabolic reactions are often spatially organized to increase enzyme activity and specificity. Thus, by spatially arranging enzyme molecules in synthetic systems to imitate these natural systems, it is possible to promote a high rate of enzymatic turnover. In this present study, a normal and mutant form of the scCro DNA-binding protein were shown to bind orthogonally to specific recognition sequences under appropriate conditions. Furthermore, these DNA-binding tags were used to establish an enzyme assay system based on the spatial arrangement of transglutaminase and its substrate at the molecular level. Together, the results of the present study suggest that the scCro-tag may be a powerful tool to facilitate the synthetic spatial arrangement of proteins on a DNA ligand.

Longitudinal Study of Cellular and Systemic Cytokine Signatures to Define the Dynamics of a Balanced Immune Environment During Disease Manifestation in Zika Virus-Infected Patients.

Since its unexpected reemergence, Zika virus (ZIKV) has caused numerous outbreaks globally. This study characterized the host immune responses during ZIKV infection. Patient samples were collected longitudinally during the acute, convalescence and recovery phases of ZIKV infection over 6 months during the Singapore outbreak in late 2016. Plasma immune mediators were profiled via multiplex microbead assay, while changes in blood cell numbers were determined with immunophenotyping. Data showed the involvement of various immune mediators during acute ZIKV infection accompanied by a general reduction in blood cell numbers for all immune subsets except CD14+ monocytes. Importantly, viremic patients experiencing moderate symptoms had significantly higher quantities of interferon γ-induced protein 10, monocyte chemotactic protein 1, interleukin 1 receptor antagonist, interleukin 8, and placental growth factor 1, accompanied by reduced numbers of peripheral CD8+ T cells, CD4+ T cells, and double-negative T cells. Levels of T-cell associated mediators, including interferon γ-induced protein 10, interferon γ, and interleukin 10, were high in recovery phases of ZIKV infection, suggesting a functional role for T cells. The identification of different markers at specific disease phases emphasizes the dynamics of a balanced cytokine environment in disease progression. This is the first comprehensive study that highlights specific cellular changes and immune signatures during ZIKV disease progression, and it provides valuable insights into ZIKV immunopathogenesis.

Multiplex biomarker approach to cardiovascular diseases.

Personalized medicine is partly based on biomarker-guided diagnostics, therapy and prognosis, which is becoming an unavoidable concept in modern cardiology. However, the clinical significance of single biomarker studies is rather limited. A promising novel approach involves combining multiple markers into a multiplex panel, which could refine the management of a particular patient with cardiovascular pathology. Two principally different assay formats have been developed to facilitate simultaneous quantification of multiple antigens: planar array assays and microbead assays. These approaches may help to better evaluate the complexity and dynamic nature of pathologic processes and offer substantial cost and sample savings compared with traditional enzyme-linked immunosorbent assay (ELISA) measurements. However, a multiplex multimarker approach cannot become a generally disseminated method until analytical problems are solved and further studies confirming improved clinical outcomes are accomplished. These drawbacks underlie the fact that a limited number of systematic studies are available regarding the use of a multiplex biomarker approach in cardiovascular medicine to date. Our perspective underscores the significant potential of the use of the multiplex approach in a wider conceptual framework under the close cooperation of clinical and experimental cardiologists, pathophysiologists and biochemists so that the personalized approach based on standardized multimarker testing may improve the management of various cardiovascular pathologies and become a ubiquitous partner of population-derived evidence-based medicine.

Dynamics of Akt isoforms and role of Immune Evader (RCAS 1) in different grades of Breast Cancer tissues in Pakistani Women

Breast Cancer (BC) is the leading cause of death among females. It has been documented that Akt isoforms and kRaS control the proliferative pathways in most cancer including BC. To address the issue of breast cancer signaling through mRNA quantification along with proteins quantifications in Akt pathway by microbead assay. A total of 42 specimens were selected from a pool of samples representing nearly all Bloom’s Richardson’s grades tissues (Normal, Hyperplasia, Ductal in situ Carcinoma, Grade I, Grade II and Grade III). No Grade IV sample was available. Total of 8 genes (Akt 1, Akt 2, Akt 3, p53, RCAS 1, Bcl 2, BclxL, and Mcl 1) and 11 proteins in PI3K / Akt - mToR pathway were assayed simultaneously using microbead BioPlex™ assay. Results on quantification of RCAS 1 gene (Immune Evader) indicates its significance by increased quantities to help to evade the breast cancer cells to distant places to metastasize in the body. Data obtained on Akt isoforms (Akt 1, Akt 2, Akt 3) show that all three play significant role in the progression of breast cancer. Our data indicate that in advance grades (grade II and III) Akt 3 along with Akt 1 act as oncogenes. Here we propose that Akt1 and Akt 3 appear to be involved in cellular growth/proliferation and angiogenesis/invasion respectively. Bioinformatics modeling have shown that the conformation of Akt 3 differs from Akt 1 and Akt 2 in PH domain that interacts to surface membrane from inner side. Based on our data, we conceived that roles of all three Akt isoforms in growth, hypertrophy, epithelial mesenchymal transition (EMT), adhesion, and metastasis been highlighted. As reported in other cancers, Akt 3 appears to be involved in the phosphorylation of VEGF and Integrin family proteins, thereby facilitating invasion and metastasis in the aggressive stages of breast cancer. We propose a model with dynamics of Akt and RCAS 1 for metastasis.

Adaptation of a microbead assay for the easy evaluation of traditional anti-sickling medicines: application to DREPANOSTAT and FACA

Context: Sickle cell disease is a common inherited blood disorder affecting millions of people worldwide. Due to lack of progress in drug discovery for a suitable treatment, sufferers often turn to traditional medicines that take advantage of the plant extracts activity used by traditional healers.Objective: This study optimizes an anti-sickling screening test to identify preparations capable of reverting sickle cells back to the morphology of normal red blood cells. We focused on the miniaturization and practicability of the assay, so that it can be adapted to the laboratory conditions commonly found in less developed countries.Materials and methods: We tested two traditional anti-sickling herbal medicines, FACA® and DREPANOSTAT®, composed of Zanthoxylum zanthoxyloides (Lam.) Zepern. & Timler (Rutaceae) and Calotropis procera (Aiton) Dryand. (Apocynaceae) at screening concentrations of hydroethanol extracts from 0.2 to 1 mg/mL. Potential bioactive molecules present in the extracts were profiled using Ultra High Performance Liquid Chromatography coupled with High Resolution Mass Spectrometry (UHPLC-HRMS/MS) method, identified through HRMS, MS/MS spectra and in silico fragmentation tools.Results: Hydroethanol extracts of FACA® and DREPANOSTAT® showed low anti-sickling activity, inhibiting less than 10% of the sickling process. The UHPLC-HRMS/MS profiles identified 28 compounds (18 in FACA® and 15 in DREPANOSTAT®, including common compounds) among which l-phenylalanine is already described as potential anti-sickling agent. When used as positive control, 7 mg/mL phenylalanine reduced the sickled RBC to 52%.Discussion and conclusions: This assay has been optimized for the easy screening of plant extracts or extracted compounds from bioassay guided fractionation, valuable to laboratories from less developed countries.

Nephrotoxicity-induced proteinuria increases biomarker diagnostic thresholds in acute kidney injury

BackgroundParaquat ingestion is frequently fatal. While biomarkers of kidney damage increase during paraquat-induced acute kidney injury (AKI), significant concurrent proteinuria may alter diagnostic thresholds for diagnosis and prognosis to an unknown extent. This study evaluated the effect of albuminuria on biomarker cutoffs for diagnosis and outcome prediction.MethodsThis was a multi-centre prospective clinical study of patients following acute paraquat self-poisoning in 5 Sri Lankan hospitals. Biomarker concentrations were quantified using ELISA and microbead assays and correlated with urinary albumin. Functional-AKI was defined by the Acute Kidney Injury Network serum creatinine definition and alternatively by a ≥50% increase in serum cystatin C. Albuminuria was defined as albumin-creatinine ratio >30 mg/g. The study outcomes were compared with a retrospective analysis of a pre-clinical study of paraquat-induced nephrotoxicity with appropriate controls.ResultsAlbuminuria was detected in 34 of 50 patients, and increased with functional-AKI severity. The concentrations of uNGAL, uCysC, uClusterin, uβ2M, and uKIM-1 were higher in albuminuric compared to non-albuminuric patients (p < 0.001). Albuminuria correlated with biomarker concentration (r > 0.6, p < 0.01) and was associated with death (p = 0.006). Optimal biomarker cutoffs for prediction of death were higher in the albuminuric group. Similar outcomes with more detailed analysis were obtained in experimental paraquat nephrotoxicity.ConclusionAlbuminuria was associated with paraquat-induced nephrotoxicity and increased excretion of low-molecular weight protein biomarkers. AKI biomarker cutoffs for diagnosis, outcome prediction and AKI stratification increased in the presence of albuminuria. This may lead to over-diagnosis of AKI in conditions independently associated with proteinuria.

Cross-matches in Transplantation: Each is Complementary to Other

Immune system, which is an obligatory component of body’s defense mechanism, is the main barrier for allogenic transplantation. Perfect selection of donor on the basis of human leucocyte antigen (HLA) compatibility and cross-matching are the key components of successful transplantation. Cross-match eliminates the chance of hyper-acute rejection and also ensures the long term graft survival. In 1960s, T-cell complement-dependent cytotoxicity cross-match was firstintroduced; since then it is mandatory component of transplantation workup process. With the advances of time different newer cross-match technics are evolved for determining the likelihood of donor-specific antibody-mediated responses, including flow cytometric cross-match and virtual cross-match using luminous microbead assay. Flow cytometric cross-match and luminous microbead assay are more sensitive and specific than the previous one, however all are complimentary to each other for final selection of donor recipient couple. This article builds an understanding of modern day cross-match interpretation using a complicated case-based approach.Birdem Med J 2016; 6(2): 118-126

Expression of TSLP and Downstream Molecules IL-4, IL-5, and IL-13 on the Eye Surface of Patients with Various Types of Allergic Conjunctivitis.

Background. The pathogenesis of allergic conjunctivitis has not been clearly established. Moreover, previous studies fail to consider human models of allergic conjunctivitis. This study investigated the expression of thymic stromal lymphopoiet in TSLP and its downstream molecules in conjunctival scrappings and tear. Methods. This cross-sectional study compares patients with vernal keratoconjunctivitis (VKC), seasonal allergic conjunctivitis (SAC), and perennial allergic conjunctivitis (PAC) with normal controls. There are 80 people recorded in Shanxi Eye Hospital. Increasingly, 20 are with VKC, 20 are with SAC, 20 are with PAC, and the remaining 20 are normal controls. Conjunctiva were harvested for total RNA extraction and gene expression by real-time polymerase chain reaction. Epithelial cells were collected to make pathological sections for immunohistochemical staining. Human tears were evaluated by Luminex microbead assay. A P value less than 0.05 from Dunnett's post hoc test in SPSS means a statistical significant distinction. Results. Positive expression in conjunctival cells of patients with allergic conjunctivitis. The expression of TSLP and IL-4, IL-5, and IL-13 mRNA shows a statistically significant difference (P < 0.05). TSLP and IL-4, IL-5, and IL-13 concentrations show a statistically significant difference (P < 0.01). Conclusions. This study suggests that TSLP and downstream molecules are expressed in patients with various types of allergic conjunctivitis.

Subclass analysis of donor HLA-specific IgG in antibody-incompatible renal transplantation reveals a significant association of IgG4 with rejection and graft failure.

SummaryDonor HLA‐specific antibodies (DSAs) can cause rejection and graft loss after renal transplantation, but their levels measured by the current assays are not fully predictive of outcomes. We investigated whether IgG subclasses of DSA were associated with early rejection and graft failure. DSA levels were determined pretreatment, at the day of peak pan‐IgG level and at 30 days post‐transplantation in eighty HLA antibody‐incompatible kidney transplant recipients using a modified microbead assay. Pretreatment IgG4 levels were predictive of acute antibody‐mediated rejection (P = 0.003) in the first 30 days post‐transplant. Pre‐treatment presence of IgG4 DSA (P = 0.008) and day 30 IgG3 DSA (P = 0.03) was associated with poor graft survival. Multivariate regression analysis showed that in addition to pan‐IgG levels, total IgG4 levels were an independent risk factor for early rejection when measured pretreatment, and the presence of pretreatment IgG4 DSA was also an independent risk factor for graft failure. Pretreatment IgG4 DSA levels correlated independently with higher risk of early rejection episodes and medium‐term death‐censored graft survival. Thus, pretreatment IgG4 DSA may be used as a biomarker to predict and risk stratify cases with higher levels of pan‐IgG DSA in HLA antibody‐incompatible transplantation. Further investigations are needed to confirm our results.