ELISA Technology Research Articles

Attenuated brain derived neurotrophic factor and depression in type 2 diabetes mellitus patients: A case-control study

Problem consideredType 2 diabetes mellitus (T2DM) and depression are two of the most prevalent chronic and devastating diseases. Brain derived neurotrophic factor (BDNF) is postulated to modulate the secretion and actions of insulin. Furthermore, a strong association has been demonstrated between BDNF and depression. Although depression is a common neuropsychiatric comorbidity of T2DM, the association of BDNF levels with depression in T2DM remains unclear. Thus, the purpose of the present study was to assess the serum BDNF levels in healthy controls and T2DM patients and explore the association of serum BDNF levels with depression in T2DM patients. MethodsIn this study depression was assessed using Patient Health Questionnaire-9 (PHQ-9) and serum BDNF levels were estimated by ELISA technology. Resultsand conclusions: A total of 88 subjects were included in the study. The mean PHQ-9 score for T2DM patients (3.91 ± 5.88) was higher than in healthy subjects (1.02 ± 3.02), p = 0.031. Subsequently, the prevalence of depression was higher in T2DM patients 16 (36.36%) as compared to healthy subjects 5 (11.36%), p < 0.001. The serum level of BDNF was lower in T2DM patients (15.17 ± 2.81 ng/ml) than in healthy subjects (24.65 ± 13.81 ng/ml) than, p < 0.0001. Additionally, serum BDNF levels were lower in T2DM patients with depression (13.87 ± 2.62 ng/ml) than in those without depression (15.92 ± 2.77 ng/ml), p = 0.02. An inverse association was found between BDNF levels and PHQ-9 scores (r2 = 0.2003, p = 0.002). The results suggest that BDNF might influence the presence of depression in T2DM patients. However, further studies are warranted to elucidate the mechanism by which BDNF may influences depression in T2DM patients.

Study on the pro-inflammatory mechanism of the HuD antibody in promoting M1 polarization and paraneoplastic neurological syndrome occurrence

ABSTRACT Paraneoplastic neurological syndrome (PNS) is a nonmetastatic complication of malignant tumors that may lead to immune-mediated neuronal dysfunction or death. The occurrence of PNS results from the binding of anti-neuronal antibodies to neuronal cell surface antigens or intracellular antigens, which hinders the function of target proteins and promotes cell death. The aim of this study is to research the effect and immune mechanism of the neuronal ELAV-like protein (HuD antibody) on PNS-related syndrome. Neuronal cells were co-cultured with monocyte macrophages with or without HuD antibody. Next, we detected the apoptosis of neuronal cells by flow cytometry. Meantime, macrophage M1/M2 polarization factors and the secretion of inflammatory factors in the co-culture system were also detected by quantitative polymerase chain reaction (qPCR), Western blots and ELISA technologies. The results showed that after adding the HuD antibody in the co-culture system, the apoptosis level of the neuroma cells were significantly increased, and the apoptosis level were not significant changed when co-culture with monocytes without HuD antibody. In addition, the level of factors of M1 macrophages TNF-α, IL-12, TGF-β and IFN-γ increased, while the level of factors of M2 macrophages IL-10, IL-4, and Arg-1 decreased. The outcomes demonstrated that absorption of the HuD antibody by cerebellar neuronal cells could promote the proliferation of M1 macrophages and stimulates macrophages to secrete inflammatory factors, further damage the neuronal cells, eventually resulting in the occurrence of PNS. This finding provided a theoretical basis for the subsequent treatment and prevention of PNS.

Systemic levels of the soluble co-inhibitory immune checkpoints, CTLA-4, LAG-3, PD-1/PD-L1 and TIM-3 are markedly increased in basal cell carcinoma

Although co-inhibitory immune checkpoint proteins are primarily involved in promoting cell-cell interactions that suppress adaptive immunity, especially tumor immunity, the soluble cell-free variants of these molecules are also detectable in the circulation of cancer patients where they retain immunosuppressive activity. Nevertheless, little is known about the systemic levels of these soluble co-inhibitory immune checkpoints in patients with various subtypes of basal cell carcinoma (BCC), which is the most invasive and treatment-resistant type of this most commonly-occurring malignancy. In the current study, we have measured the systemic concentrations of five prominent co-inhibitory immune checkpoints, namely CTLA-4, LAG-3, PD-1/PD-L1 and TIM-3, as well as those of C-reactive protein (CRP) and vitamin D (VD), in a cohort of patients (n = 40) with BCC, relative to those of a group of control participants, using the combination of multiplex bead array, laser nephelometry and ELISA technologies, respectively. The median systemic concentrations of CRP and VD were comparable between the two groups; however, those of all five immune checkpoints were significantly elevated (P = 0.0184 - P = < 0.00001), with those of CTLA-4 and PD-1 being highly correlated (r = 0.87; P < 0.00001). This seemingly novel finding not only identifies the existence of significant systemic immunosuppression in BCC, but also underscores the therapeutic promise of immune checkpoint targeted therapy, as well as the potential of these proteins to serve as prognostic/predictive biomarkers in BCC.

Nanozyme enhanced paper-based biochip with a smartphone readout system for rapid detection of cyanotoxins in water

Cyanobacterial harmful algal blooms in freshwater systems can produce cyanotoxins, such as microcystins (MCs) and nodularins (NODs), presenting serious threats to human health and ecosystems. Required routine monitoring of cyanotoxins in water samples, as posed by U.S. EPA drinking water contaminant candidate list 5 (CCL5), demands for cost-effective, reliable and sensitive MCs/NODs detection methods. We report the development of a colorimetric paper-based immunochip assisted by nanozyme catalysis with a smartphone readout system for rapid detection of cyanotoxins in water. We show that the introduction of biorthogonal click reaction enables in situ facile self-assembly of multi-layers of peroxidase-like nanozyme onto the anti-MCs/NODs monoclonal antibody. We can detect 13 variants of MCs/NODs even in the sub-microgram per liter range with detection limit of below 0.7 μg/L and satisfactory recovery percentages between 88 and 120% in different water matrices. Our technology shows a good correlation with the well-developed ELISA technology, demonstrating its great potential applications in resource-limited or less-developed regions for on-site and large-scale screening of cyanotoxins in water environment.

The Expression of Inflammasomes NLRP1 and NLRP3, Toll-Like Receptors, and Vitamin D Receptor in Synovial Fibroblasts From Patients With Different Types of Knee Arthritis.

Activated rheumatoid arthritis (RA) synovial fibroblasts (SFs) are among the most important cells promoting RA pathogenesis. They are considered active contributors to the initiation, progression, and perpetuation of the disease; therefore, early detection of RASF activation could advance contemporary diagnosis and adequate treatment of undifferentiated early inflammatory arthritis (EA). In this study, we investigated the expression of nucleotide-binding, oligomerization domain (NOD)-like receptor family, pyrin domain containing (NLRP)1, NLRP3 inflammasomes, Toll-like receptor (TLR)1, TLR2, TLR4, vitamin D receptor (VDR), and secretion of matrix metalloproteinases (MMPs) in SFs isolated from patients with RA, osteoarthritis (OA), EA, and control individuals (CN) after knee surgical intervention. C-reactive protein, general blood test, anticyclic citrullinated peptide (anti-CCP), rheumatoid factor (RF), and vitamin D (vitD) in patients’ sera were performed. Cells were stimulated or not with 100 ng/ml tumor necrosis factor alpha (TNF-α) or/and 1 nM or/and 0.01 nM vitamin D3 for 72 h. The expression levels of NLRP1, NLRP3, TLR1, TLR2, TLR4, and VDR in all examined SFs were analyzed by quantitative real-time PCR (RT-qPCR). Additionally, the secretion of IL-1β by SFs and MMPs were determined by ELISA and Luminex technology. The expression of NLRP3 was correlated with the levels of CRP, RF, and anti-CCP, suggesting its implication in SF inflammatory activation. In the TNF-α-stimulated SFs, a significantly lower expression of NLRP3 and TLR4 was observed in the RA group, compared with the other tested forms of arthritis. Moreover, upregulation of NLRP3 expression by TNF-α alone or in combination with vitD3 was observed, further indicating involvement of NLRP3 in the inflammatory responses of SFs. Secretion of IL-1β was not detected in any sample, while TNF-α upregulated the levels of secreted MMP-1, MMP-7, MMP-8, MMP-12, and MMP-13 in all patient groups. Attenuating effects of vitD on the expression of NLRP3, TLR1, and TLR4 suggest potential protective effects of vitD on the inflammatory responses in SFs. However, longer studies may be needed to confirm or fully rule out the potential implication of vitD in SF activation in inflammatory arthritis. Both VDR and NLRP3 in the TNF-α-stimulated SFs negatively correlated with the age of patients, suggesting potential age-related changes in the local inflammatory responses.

Simple ELISA Methods to Estimate Neutralizing Antibody Titers to SARS-CoV-2: IgG Quantification, the Avidity Index, and the Surrogate Virus Neutralization Test

A total of 104 sera sampled from vaccinated (“Sputnik V”, Russia) volunteers were tested in parallel to determine different markers of humoral immunity against SARS-CoV-2. Testing was conducted using the neutralizing antibodies titer (NtAb) in the virus neutralization assay (VNA), the IgG to RBD in the quantitative ELISA (BAU/ml), the avidity index (AI) of the IgG to RBD (also in ELISA), and the titers of “neutralizing antibodies” which can block an interaction between human ACE2 and the viral RBD proteins in a competitive ELISA surrogate virus neutralization test (sVNT). The correlation coefficients between the quantitative results in the tried ELISA assays and the “true” NtAb titers in the VNA were high, with the following values: r=0.86 for BAU/ml (95% confidence interval, CI: 0.80-0.91, p<0.0001); 0.54 for the AI (95% CI: 0,38-0,67, p<0.0001); and 0.84 for the sVNT titer (95% CI:0.79-0.90). Additionally, it was found that the multiplicative index of BAU/ml × AI (which corresponds to the concentration of the high-affinity fraction of the total IgG to the RBD) gives a maximum correlation to the NtAb titers (r=0.89, 95% CI: 0.84-0.92, p<0.0001). The sensitivity and specificity, respectively, of the tested ELISA assays in recognizing the supposedly protective NtAb titer of ≥1:160 were the following: for the BAU/ml – 87.7±8% and 97.4±5%; for the (BAU/ml × AI) index – 92.3±6.5% and 97.4±5%; for the sVNT—95.4±5.1% and 89.7±9.5%. This confirms that the tested ELISA technologies have potential as a safe and cheap alternative to the classical VNA for assessing the protective force of patient immunity against SARS-CoV-2 infection in routine clinical practice.

Prevalence of Mycotoxins and Endotoxins in Total Mixed Rations and Different Types of Ensiled Forages for Dairy Cows in Lithuania

In this study, 119 samples of total mixed rations and different types of ensiled forage (maize and grass silage, and haylage) collected in 2019–2020 from dairy farms in Lithuania were analyzed to evaluate the quantitative occurrence of mycotoxins and endotoxins. Samples were analyzed using high-performance liquid chromatography (HPLC) with a fluorescent (FLD) and an ultraviolet detector (UV) of mycotoxins and a detection assay based on the ELISA technology for endotoxins. The study included toxins regulated within the European Union (aflatoxin B1 (AFB1), zearalenone (ZEA), deoxynivalenol (DON) and T-2 toxin) and nonregulated toxins (endotoxins). Mycotoxin analysis showed that 49.58% of the samples out of 119 were positive for AFB1, 52.11% for ZEA and DON, 55.47% for T-2 toxin and 84.04% for endotoxins. In the contaminated samples, the highest mean values of AFB1 and T-2 toxin were determined in the grass silage samples, while ZEA and DON–were determined in the maize silage samples. Maize silage samples had the highest ZEA and DON concentrations, exceeding the EU maximum permissible concentration limits. In the haylage samples, AFB1 mycotoxin exceeded the maximum concentration limits. The highest mean value of endotoxins was determined in the total mixed rations samples. This is the first study to provide information about the concentrations of mycotoxins and endotoxins in total mixed rations and different types of ensiled forages for dairy cows in Lithuania.

289. Post COVID Syndrome Cohort Characterization

BackgroundPost COVID Syndrome (PCS) is significant morbidity following COVID-19. This study aims to identify biomarkers that predict PCS in a Gulf Coast cohort known for poor health outcomes.MethodsSince March 2020 the study Collection of Serum and Secretions for SARS CoV-2 Countermeasure Development (aka ClinSeqSer) has been enrolling subjects with confirmed acute COVID-19, with initial visit at 1 month and follow up every three months from symptom onset. At follow-up, subjects complete symptom questionnaire, physical examination, nasopharyngeal swab/saliva collection, blood draw. Subjects with >= one symptom new since COVID are PCS, remainder are Non-PCS experienced at initial one month visit and six months or longer. Univariate and bivariate analysis was carried out to study significant associations of currently available dataset (N=60).Figure 1. Post-COVID Symptoms Included if “new since covid”. For 60 subjects consented post-covid with completed questionnaire, results were analyzed. Most common symptoms reported were fatigue/tiredness or exhaustion (52%), muscle aches (38%), difficulty concentrating (33%) and headache (32%) as the most common symptoms during one month prior to their initial follow-up visit. The persistent symptoms experienced for six months or longer were fatigue/tiredness or exhaustion (25%), forgetfulness (22%), muscle aches (18%), and sleep difficulties (18%).ResultsCohort is 36 (60%) female, 24 (40%) male, age group of 49 (82%) 18-64 years, 11 (18%) 65+ years, 33 (55%) African American, 27 (45%) Caucasian. Median follow-up time after symptom onset: 290 days. Study cohort reported fatigue (52%), myalgias (38%), difficulty concentrating (33%), headache (32%) as most common symptoms during first month from initial symptom onset. Persistent symptoms ( >=6 months) are fatigue (25%), forgetfulness (22%), myalgias (18%), sleep difficulties (18%). Bivariate analysis shows that gender (female, P=0.04), past stroke/transient ischemic attack (P=0.04), deep venous thrombosis (P=0.02), abnormal kidney function (P=0.01) associate with PCS. Convalescent antibodies (ReSARS N IgG, S-RBD IgG) were measured and percentage inhibition of ACE2 spike interaction was recorded. Plasma inflammatory protein levels were measured using multiplex ELISA and Proximity Extension Assay technology during follow-up visit. Increased antibody ReSARS N IgG (2.91, 0.74-10.93; P=0.02) response and higher convalescent IL-10 (P=0.04) was associated with PCS. Percent inhibition of ACE2: spike interaction was not associated (P=0.79) with PCS. Nasal swab/saliva SARS-COV-2 sequencing has not identified a specific SARS-CoV-2 virus mutation predictive of PCS. Table 1. Demographic and Clinical Characteristics The bivariate analysis results showed that the gender (female, P=0.0354), history of stroke or transient ischemic attack (P=0.0382), chest pain from narrow heart vessels (P=0.0479), deep venous thrombosis (P=0.0241) and abnormal kidney function (P=0.0142) were associated with Post-COVID syndrome.Table 2. Antibodies and ACE2 spike inhibition. The convalescent antibodies, ReSARS N IgG and S-RBD IgG were measured in U/mL and percentage inhibition of ACE2 spike interaction was recorded during follow-up visit for PCS vs Non-PCS subjects. The increased antibody ReSARS N IgG (2.91, 0.74-10.93; P=0.0159) response was associated with Post-COVID syndrome. Percent inhibition of ACE2: spike interaction was not associated (P=0.7932) with PCS.Table 3. Plasma inflammatory protein levels. Plasma inflammatory protein levels were measured using multiplex ELISA (MSD) and Proximity Extension Assay technology (Olink) recorded during follow-up visit for PCS vs Non-PCS subjects, revealing IL-10 (P=0.0379) was associated with development of PCS.ConclusionThis study identifies initial clinical and biomarker predictors of PCS in a cohort that is 55% African American.Figure 2. Antibody ReSARS N IgG ReSARS N IgG measured in post-covid patients is significantly associated with post-COVID syndrome(P=0.0159). X axis: number of months from symptom onset to blood draw. Y axis: N IgG U/mL.Figure 3. Spike amino acid mutations Spike amino acid mutations detected in SARS-CoV-2 from acute-phase respiratory isolates. Nasal swab/saliva samples were collected from subjects with acute COVID-19 at time of enrollment into ClinSeqSer, stored at -80°C followed by RNA isolation and SARS-CoV-2 qRT-PCR. Samples with Ct value of ≤30 were then sequenced using NextSeq (Illumina). All sequences are deposited on GISAID and under BioProject (ID PRJNA681020). X axis: subject ID, with ID number increasing chronologically. Y axis: amino acid position of each mutation moving from N- to C-terminus.Disclosures Robert Garry, PhD, Zalgen Labs (Shareholder)

Immunological Estimation of Inflammatory Interleukins (IL-4, IL-6 &amp; IL-10) Among Patients with Bladder Cancer

The aim of this study was to determine Immunomolecular expression of inflammatory interleukins (IL-4, IL-6 &amp; IL-10) by using ELISA, conventional PCR and Sequencing technologies, to give acknowledge about the roles of these interleukins in patients with bladder cancer in Basrah province. A case-control study included 85 confirmed bladder cancer patients and 80 individual as a control group. Data about age, gender, smoking, alcohol drinking, family history, occupation, residency and clinical findings for all patients with urothelial carcinoma were collected. This study show the effect of demographical factors ( age, gender smoking, family history, alcohol drinking, occupation and residency) in patients with bladder cancer. The current study measure interleukins (IL-4, IL-6 &amp; IL-10) concentration (pg\ml) by using ELISA technology among various age groups, gender, tumor sages (T1-T4) and case status (newly diagnosed, advance &amp; recurrence). The results show significant increase of interleukins 4, 6 and 10 in comparison with control group (P-value= 0.008, 0.009, 0.001) respectively.

Impact of point-of-care testing and treatment of sexually transmitted infections and bacterial vaginosis on genital tract inflammatory cytokines in a cohort of young South African women

ObjectivesSTIs cause inflammation that is detrimental for both HIV risk and reproductive health. We assessed the impact of point-of-care (POC) STI testing, immediate treatment and expedited partner therapy (EPT) on...

THE IMMUNE RESPONSE AGAINST RABIES VIRUS AMONG CANINE DONORS IN A ROMANIAN BLOOD BANK

INTRODUCTION: Seventy-six samples were collected from sixteen canine blood donors at every blood donation performed in accord with the canine donor selection guidelines. Statistical analysis of ELISA OD values revealed no significant variation of anti-rabies antibodies values during repeat donations. Our results support the statement that blood donation does not affect the immune status of donors.&#x0D; OBJECTIVES: The study aimed to assess the immune status against rabies among the canine blood donors in a Romanian canine blood bank&#x0D; METHODS: Serum samples (n=76) were collected from pet owned dogs (n=16) recorded in the blood bank register of a Romanian blood bank were performed with the immunological status of the investigated canine blood donors was evaluated using the indirect ELISA commercial kit PLATELIA RABIES II (BIO-RAD, France) as per the manufacturer's instructions&#x0D; RESULTS: The overall picture of ELISA OD results revealed non-significant variation between and within D1-D6 groups of values (p&gt;0.05, F&lt;Fcrit).&#x0D; CONCLUSION: The next studies topic would be to compare the results provided by the ELISA technology with FAVN.

Potent and Persistent Antibody Response in COVID-19 Recovered Patients.

SARS-CoV-2 has caused a global pandemic with millions infected and numerous fatalities. Virus-specific antibodies can be detected in infected patients approximately two weeks after symptom onset. In this study, we set up ELISA technology coating with purified SARS-CoV-2 S and N proteins to study the antibody response of 484 serum samples. We established a surrogate viral inhibition assay using SARS-CoV-2 S protein pseudovirus system to determine the neutralization potency of collected serum samples. Here, we report robust antibody responses to SARS-CoV-2 in 484 recovered patients varying from 154 to 193 days, with 92% of recovered patients displaying a positive virus-specific spike glycoprotein IgG (s-IgG) response, while the ratio of positive spike glycoprotein IgM (s-IgM) reached 63%. Furthermore, moderate to potent neutralization activities were also observed in 62% of patients, correlating significantly with s-IgG response. This study strongly supports the long-term presence of antibodies in recovered patients against SARS-CoV-2, although all serum samples were collected from individuals with mild or moderate symptoms.

Dysregulation of soluble immune checkpoint proteins in newly-diagnosed early breast cancer patients.

556 Background: Checkpoint proteins regulate the immune system. Breast cancer (BC) cells exploit the up-regulation or down-regulation of these proteins to evade anti-tumor immune responses. Soluble forms of immune checkpoint molecules (ICM) can be measured in human plasma, however their biological and clinical significance remains essentially unknown. The aim of the present analysis was to measure the pre-treatment ICM in newly- diagnosed BC patients (pts) and compare them to healthy controls. Methods: Soluble forms of ICM, as well as cytokines and chemokines, were measured using Multiplex bead array and ELISA technologies. Plasma samples from 98 BC pts and 45 healthy controls were analyzed for each protein. Data was prospectively obtained. Measured levels were compared between BC pts and healthy controls using a non-parametric test (Mann-Whitney). P-values below 0.05 were considered statistically significant. Results: Soluble stimulatory molecules GITR (p &lt; 0,000002), GITRL (p &lt; 0,007), CD27 (p &lt; 0,002), CD28 (p &lt; 0,003), CD40 (p &lt; 0,003), CD80 (p &lt; 0,009), ICOS (p &lt; 0,0006), as well as inhibitory molecules PD-L1 (p &lt; 0.0000001), CTLA-4 (p &lt; 0,005), TIM-3 (p &lt; 0,00006), HVEM (p &lt; 0,00002) TLR-2 (p &lt; 0,05), levels were significantly lower in early BC pts compared to healthy controls. When analyzed according to BC characteristics (TNBC vs. non-TNBC, tumor size, stage, nodal status and age) no significant difference was detected between the soluble levels of these ICM between the different subsets. Additionally, serum CXCL5 (p &lt; 0,000001), CCL23 (p &lt; 0,04), IL-16 (p &lt; 0,00005), interferon-α (p &lt; 0,03) and IL1-RA (p &lt; 0,03) were significantly lower compared to healthy controls. Serum CX3CL1 or fractalkine (p &lt; 0,024465) was significantly higher compared with healthy controls. Serum interferon-γ (p &lt; 0,2), IL-6 (p &lt; 0.6) and IL-2 (p &lt; 0.6) levels were not significantly different between the BC pts and the healthy controls. Conclusions: We identified low levels of both the stimulatory and inhibitory immune checkpoint molecules, in newly-diagnosed, non-metastatic BC pts compared to healthy controls. These results indicate that early BC is associated with a down-regulation of both soluble stimulatory and inhibitory immune-checkpoint pathways. Newly-diagnosed early BC pts have a generalized immune-suppression independent of subtype (TNBC vs non-TNBC) and stage, which, to our knowledge, is the first study to describe soluble immune checkpoints in early BC pts.

Molecular biomarkers associated with respiratory insufficiency in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating and fatal neurodegenerative disorder.Death typically occurs within 3–5 years after disease onset. The main cause of death in ALS is respiratory failure (RF). No effective treatment is available and no molecular biomarker related to respiratory outcome and to early ventilatory dysfunction was described so far. The club-cell protein (CC-16) is a biomarker associated with respiratory distress and lung inflammation. The aim of this work is to test if CC-16 and IL-6 could be new biomarkers of ALS for early signs of respiratory insufficiency and disease progression. Additionally, we intend to study morphological and viscoelastic changes of the erythrocytes’ membrane associating them with ALS patients’ clinical profile. Eighty-one ALS patients and 30 matched controls were included. Functional capacity and respiratory function (forced vital capacity) were evaluated. CC-16 and IL-6 were quantified by ELISA and multiplex technology, respectively. Morphological and viscoelastic properties of the erythrocytes were analysed by Atomic Force Microscopy (AFM). CC-16 levels were significantly raised in ALS patients. In 17% of them, CC-16 level was above the upper cut-off value. On these patients, the risk of non-invasive ventilation was greater in the following 6 months and they tend to have higher mortality in the following 30 months. IL-6 values were not different in ALS population as compared with controls. ALS patients have higher erythrocyte maximum height, area and volume, decreased erythrocyte membrane roughness and increased membrane stiffness than the control group. These results indicates abnormal erythrocyte structure and possible changes on membrane lipid composition on ALS patients. We propose that increased CC-16 levels could be a marker of lung inflammatory response, associated with ventilatory insufficiency and related to impending respiratory failure, which are not fully predicted by conventional respiratory tests. Moreover, abnormalities in erythrocyte morphology may enhance the risk of tissue hypoxia. Amyotrophic lateral sclerosis (ALS) is a devastating and fatal neurodegenerative disorder. Death typically occurs within 3–5 years after disease onset. Respiratory failure (RF) is the main cause of death. No effective treatment and no molecular biomarker related to respiratory outcome and to early ventilatory dysfunction are available. The club-cell protein (CC-16) is a biomarker associated with respiratory distress and lung inflammation. The aim of this work is to test if CC-16 and IL-6 could be new biomarkers of ALS for early signs of respiratory insufficiency and disease progression. Additionally, we intend to study morphological and viscoelastic changes of the erythrocytes’ membrane associating them with ALS patients’ clinical profile. Functional capacity and respiratory function (forced vital capacity) were evaluated in 81 ALS patients and 30 matched controls. CC-16 and IL-6 were quantified by ELISA and Multiplex-technology, respectively. Morphological and viscoelastic properties of the erythrocytes were analysed by Atomic Force Microscopy (AFM). A significant negative correlation was found between [IL-6] and PhrAMPL, but not with ALSFRS-R or disease duration. CC-16 levels were significantly raised in ALS patients. In 17% of them, CC-16 level was above the upper cut-off value. On these patients, the risk of non-invasive ventilation was greater in the following 6 months and they tend to have higher mortality. ALS patients have higher erythrocyte maximum height, area, volume, decreased erythrocyte membrane roughness and increased membrane stiffness. These results indicates that abnormal erythrocyte structure and possible changes on membrane lipid composition on ALS patients. Our results show that IL-6 levels are not dependent on the duration or severity of the disease, however, IL-6 may provide a marker of respiratory dysfunction in ALS. We propose that increased CC-16 levels could be a marker of lung inflammatory response, associated with ventilatory insufficiency and related to impending RF, which are not fully predicted by conventional respiratory tests. Moreover, abnormalities in erythrocyte morphology may enhance the risk of tissue hypoxia.

Evaluation of fecal immunoassays for canine Echinococcus infection in China.

Human echinococcosis is present worldwide but it is in China that disease prevalence is the highest. In western China, especially in the Tibetan Plateau, the burden of echinococcosis is the most important. Dogs are a major definitive host of Echinococcus and monitoring the presence of Echinococcus worms in dogs is therefore essential to efficiently control the disease. Detection kits based on three different technologies including sandwich ELISA, (indirect) ELISA, and gold immunodiffusion, are currently marketed and used in China. The objective of this work was to assess the efficacy of these kits, in particular with respect to sensitivity and specificity. Four fecal antigen detection kits for canine infection reflecting the three technologies were obtained from companies and tested in parallel on 220 fecal samples. The results indicate that the performance is lower than expected, in particular in terms of sensitivity. The best results were obtained with the sandwich ELISA technology. The gold immunofiltration yielded the poorest results. In all cases, further development is needed to improve the performance of these kits which are key components for the control of echinococcosis.

Abstract PS17-36: Dysregulation of soluble immune checkpoint proteins in newly - diagnosed early breast cancer patients

Abstract Background: Checkpoint proteins regulate the immune system. Breast cancer (BC) cells exploit the up-regulation or down-regulation of these proteins to evade anti-tumor immune responses. Soluble forms of immune checkpoint molecules (ICM) can be measured in human plasma. However, their biological and clinical significance remains mostly unknown. The aim of the present analysis was to measure the levels of pre-treatment ICM in newly diagnosed BC patients (pts) and compare them to healthy controls. Method: Soluble forms of ICM, as well as cytokines and chemokines, were measured using Multiplex® bead array and ELISA technologies. Plasma samples from 98 BC pts and 45 healthy controls were analyzed for each protein. Data was prospectively obtained. Measured levels were compared between BC pts and healthy controls using a non-parametric test (Mann-Whitney). Results: Soluble stimulatory molecules GITR (p &amp;lt; 0.000002), GITRL (p &amp;lt; 0.007), CD27 (p &amp;lt; 0.002), CD28 (p &amp;lt; 0.003), CD40 (p &amp;lt; 0.003), CD80 (p &amp;lt; 0.009), ICOS (p &amp;lt; 0.0006) as well as inhibitory molecules PD-L1 (p &amp;lt; 0.0000001), CTLA-4 (p &amp;lt; 0.005), TIM-3 (p &amp;lt; 0.00006), HVEM (p &amp;lt; 0.00002) and TLR-2 (p &amp;lt; 0.05) levels were significantly lower in early BC pts compared to healthy controls. When analyzed according to BC characteristics (TNBC vs. non-TNBC, tumor size, stage, nodal status and age) no significant difference was detected between the soluble levels of these ICM and between the different subsets. Additionally, serum levels of CXCL5 (p &amp;lt; 0.000001), CCL23 (p &amp;lt; 0.04), IL-16 (p &amp;lt; 0.00005), interferon-α (p &amp;lt; 0.03) and IL1-RA (p &amp;lt; 0.03) were significantly lower compared to healthy controls. Serum CX3CL1 or fractalkine (p &amp;lt; 0.024465) was significantly higher compared to healthy controls. Conclusion: In the current study, we identified low levels of both stimulatory and inhibitory soluble immune checkpoint molecules in newly diagnosed, non-metastatic BC pts compared to healthy controls. These results indicate that early BC is associated with a down-regulation of both soluble stimulatory and inhibitory immune-checkpoint pathways. Newly diagnosed early BC pts have a generalized immune-suppression independent of subtype and stage, which, to our knowledge, is the first study to describe soluble immune checkpoints in early BC pts. Citation Format: Bernardo Rapoport, Helen Steel, Teresa Smit, Liezl Heyman, Annette Theron, Nomsa Hlatswayo, Luyanda Kwofie, Lidia Jooste, Farhana Moosa, Carol Ann Benn, Simon Nayler, Ronald Anderson. Dysregulation of soluble immune checkpoint proteins in newly - diagnosed early breast cancer patients [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-36.

A Longitudinal Analysis Reveals Early Activation and Late Alterations in B Cells During Primary HIV Infection in Mozambican Adults.

Primary HIV infection (PHI) and subsequent chronic infection alter B-cell compartment. However, longitudinal analysis defining the dynamics of B-cell alterations are still limited. We longitudinally studied B-cell subsets in individuals followed for 1 year after PHI (n = 40). Treated and untreated chronic HIV infected (n = 56) and HIV-uninfected individuals (n = 58) were recruited as reference groups at the Manhiça District in Mozambique. B cells were analyzed by multicolor flow-cytometry. Anti-HIV humoral response and plasma cytokines were assessed by ELISA or Luminex-based technology. A generalized activation of B cells induced by HIV occurs early after infection and is characterized by increases in Activated and Tissue-like memory cells, decreases in IgM-IgD- (switched) and IgM-only B cells. These alterations remain mostly stable until chronic infection and are reverted in part by ART. In contrast, other parameters followed particular dynamics: PD-1 expression in memory cells decreases progressively during the first year of infection, Transitional B cells expand at month 3–4 after infection, and Marginal zone-like B cells show a late depletion. Plasmablasts expand 2 months after infection linked to plasma viral load and anti-p24 IgG3 responses. Most of well-defined changes induced by HIV in B-cell activation and memory subsets are readily observed after PHI, lasting until ART initiation. However, subsequent changes occur after sustained viral infection. These data indicate that HIV infection impacts B cells in several waves over time, and highlight that early treatment would result in beneficial effects on the B-cell compartment.

A fast and ultrasensitive ELISA based on rolling circle amplification.

A highly sensitive ELISA is critical for early diagnosis and biomarker discovery of various diseases. Although various ELISA technologies have been developed with high sensitivity, they are limited by poor repeatability, high cost, the dependence on complex equipment and/or a prolonged reaction time. To this end, we developed a fast and ultrasensitive ELISA (termed RELISA) based on rolling circle amplification (RCA) and enzymatic signal amplification. The RELISA is established on the traditional ELISA, with only one more RCA step that can be accomplished within 10 minutes. The prolonged single strand DNA (ssDNA) from RCA is able to enrich abundant horseradish peroxidase conjugate (HRP) modified detection probes. Consequently, the intensive HRP is able to catalyze TMB-H2O2 to produce significantly enhanced colorimetric signals. With CEACAM-7 as a model biomarker, the RELISA achieves the limit of detection as low as 2.82 pg mL-1, which is ∼50 times higher than that of the traditional ELISA. Therefore, we envision that the developed RELISA would be a powerful tool for the early diagnosis of various major diseases.

Kinetics of peripheral blood neutrophils in severe coronavirus disease 2019.

ObjectivesEmerging evidence of dysregulation of the myeloid cell compartment urges investigations on neutrophil characteristics in coronavirus disease 2019 (COVID‐19). We isolated neutrophils from the blood of COVID‐19 patients receiving general ward care and from patients hospitalised at intensive care units (ICUs) to explore the kinetics of circulating neutrophils and factors important for neutrophil migration and activation.MethodsMulticolour flow cytometry was exploited for the analysis of neutrophil differentiation and activation markers. Multiplex and ELISA technologies were used for the quantification of protease, protease inhibitor, chemokine and cytokine concentrations in plasma. Neutrophil polarisation responses were evaluated microscopically. Gelatinolytic and metalloproteinase activity in plasma was determined using a fluorogenic substrate. Co‐culturing healthy donor neutrophils with severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) allowed us to investigate viral replication in neutrophils.ResultsUpon ICU admission, patients displayed high plasma concentrations of granulocyte–colony‐stimulating factor (G‐CSF) and the chemokine CXCL8, accompanied by emergency myelopoiesis as illustrated by high levels of circulating CD10−, immature neutrophils with reduced CXCR2 and C5aR expression. Neutrophil elastase and non‐metalloproteinase‐derived gelatinolytic activity were increased in plasma from ICU patients. Significantly higher levels of circulating tissue inhibitor of metalloproteinase 1 (TIMP‐1) in patients at ICU admission yielded decreased total MMP proteolytic activity in blood. COVID‐19 neutrophils were hyper‐responsive to CXCL8 and CXCL12 in shape change assays. Finally, SARS‐CoV‐2 failed to replicate inside human neutrophils.ConclusionOur study provides detailed insights into the kinetics of neutrophil phenotype and function in severe COVID‐19 patients, and supports the concept of an increased neutrophil activation state in the circulation.

Preeclamptic Women Have Decreased Circulating IL-10 (Interleukin-10) Values at the Time of Preeclampsia Diagnosis: Systematic Review and Meta-Analysis.

A key immunomodulatory cytokine, IL-10 (interleukin-10), has been shown to be dysregulated in preeclampsia, a pregnancy-specific hypertensive disorder, further characterized by multi-system involvement. However, studies have reported inconsistent findings about circulating IL-10 levels in preeclamptic versus normotensive pregnancies. The aim of the present systematic review and meta-analysis was to assess circulating IL-10 levels in preeclamptic and normotensive pregnancies at 2 time points: before, and at the time of preeclampsia diagnosis. PubMED, EMBASE, and Web of Science databases were searched to include all published studies examining circulating IL-10 levels in preeclamptic and normotensive pregnancies. Differences in IL-10 levels were evaluated by standardized mean differences. Of 876 abstracts screened, 56 studies were included in the meta-analysis. Circulating IL-10 levels were not different before the time of active disease (standardized mean differences, -0.01 [95% CI, -0.11 to 0.08]; P=0.76). At the time of active disease, women with preeclampsia (n=1599) had significantly lower IL-10 levels compared with normotensive controls (n=1998; standardized mean differences, -0.79 [95% CI, -1.22 to -0.35]; P=0.0004). IL-10 levels were lower in both early/severe and late/mild forms of preeclampsia. Subgroup analysis revealed that IL-10 measurement methodology (ELISA or multiplex bead array) and the sample type (plasma or serum) significantly influenced the observed differences, with the use of sera paired with ELISA technology providing the best distinction in IL-10 levels between preeclamptic and normotensive pregnancies. These findings support the role of decreased IL-10 levels in the pathophysiology of preeclampsia. Future studies should address the therapeutic potential of IL-10 in preeclampsia.