In oncology, anti-drug antibody (ADA) development that significantly curtails response durability has not historically risen to a level of concern. The relevance and attention ascribed to ADAs in oncology clinical studies have therefore been limited, and the extant literature on this subject scarce. In recent years, T cell engagers have gained preeminence within the prolific field of cancer immunotherapy. These drugs whose mode of action is expected to potently stimulate anti-tumor immunity, may potentially induce ADAs as an unintended corollary due to an overall augmentation of the immune response. ADA formation is therefore emerging as an important determinant in the successful clinical development of such biologics. Here we describe the immunogenicity and its impact observed to pasotuxizumab (AMG 212), a prostate-specific membrane antigen (PSMA)-targeting bispecific T cell engager (BiTE®) molecule in NCT01723475, a first-in-human (FIH), multicenter, dose-escalation study in patients with metastatic castration-resistant prostate cancer (mCRPC). To explain the disparity in ADA incidence observed between the SC and CIV arms of the study, we interrogated other patient and product-specific factors that may have explained the difference beyond the route of administration. Treatment-emergent ADAs (TE-ADA) developed in all subjects treated with at least 1 cycle of AMG 212 in the subcutaneous (SC) arm. These ADAs were neutralizing and resulted in profound exposure loss that was associated with contemporaneous reversal of initial Prostate Surface Antigen (PSA) responses, curtailing durability of PSA response in patients. Pivoting from SC to a continuous intravenous (CIV) administration route remarkably yielded no subjects developing ADA to AMG 212. Through a series of stepwise functional assays, our investigation revealed that alongside a more historically immunogenic route of administration, non-tolerant T cell epitopes within the AMG 212 amino acid sequence were likely driving the high-titer, sustained ADA response observed in the SC arm. These mechanistic insights into the AMG 212 ADA response underscore the importance of performing preclinical immunogenicity risk evaluation as well as advocate for continuous iteration to better our biologics. Biologics such as monoclonal antibodies and their associated bispecific antibody constructs consist of large and complex structures. Some of these amino acid sequences and structural motifs, may induce humoral immune responses due to non-self recognition by the patient’s immune repertoire, resulting in the formation of specific anti-drug antibodies (ADAs).The ADA response is initiated by antigen-presenting cells (APCs) that phagocytose, internalize, and process the drug into smaller peptides. These peptides are loaded onto major histocompatibility complex (MHC) class II at the APC cell surface for presentation to CD4+ T cell clones that recognize the specific peptide-MHCII complex (pMHC) –3. At thADAs can cause unintended clinical consequences affecting exposure and safety, with effects ranging from none to life-threatening. ADAs may impact the pharmacokinetics (PK) of a drug –6, maintIn addition to treatment-induced ADAs, pre-existing reactivity has been detected in drug-naïve individuals . While tIn oncology, the risk of ADA development may be lower in patients due to their disease state itself, or in patients who have recently completed chemotherapy and whose immune systems may still be recovering from these myeloablative regimens. The suppressed ability to mount a robust antibody response may have accounted for the historically low rates of ADAs observed to tumor-associated antigen targeted, monoclonal antibody-based investigational drugs.Clinical immunogenicity has gained renewed interest of late, as immunotherapies exhibiting ability to potently trigger an immune response have brought the topic of drug-induced immunogenicity into active discourse. In a focused review consolidating immunogenicity data from 81 clinical trials with anti-cancer biologics, Van Brummelen and colleagues observed that 63% of these studies report ADA formation , suggestIn a similar assessment, Davda and colleagues reviewed the incidence of ADA and NAb across multiple, approved, anti-cancer antibody-based immunomodulatory agents and found that the data is suggestive of a higher likelihood of immunogenicity to antibodies with T cell or APC targets compared to B cell targets . Not sur®) molecule, a class of biologics whose mode of action is such that when the BiTE® molecule is bound on one end to target protein on the surface of a target cell and bound to CD3 on a T cell at the other end, proximity-induced, redirected T cell lysis of target cells can occur. In NCT01723475, a FIH study, AMG 212 was tested in mCRPC patients, who were refractory to novel anti-androgen therapy  and had failed at least one (but not more than two) taxane regimen.Pasotuxizumab (henceforth referred to as AMG 212) is a 55 kD protein with an anti-PSMA target binding domain linked to an anti-CD3 binding domain. It is a Bispecific T cell engager  response and adverse events observed on study. We also describe a measure implemented mid-study to mitigate the ADAs detected in the SC arm. Further, we performed a root cause analysis to explain the immunogenicity observed, by assessing potential contributing factors such as the baseline immune status of subjects and product quality attributes of the drug lots administered to subjects. Finally, through a series of This was an open-label, multicenter, Phase I, dose-escalation study  conducted at five clinical study centers in Germany and Austria and sponsored by Bayer AG, Leverkusen, Germany. It was designed to determine the safety and maximum tolerated dose (MTD) of AMG 212 (primary objectives) and to assess pharmacokinetics, PSA and tumor response (secondary objectives) and biomarkers (exploratory objective) of AMG 212 administered either by daily SC injection or CIV infusion. An independent data monitoring committee was established to regularly review safety data. The starting doses for the SC and CIV arms were 0.5 and 5 μg daily, respectively. A cycle for this study, in both the SC and CIV arms, was defined as 21 days (or 3 weeks).In the SC arm, AMG 212 was administered daily by SC injection, with no breaks between cycles. The SC dosing schema is shown in In the CIV arm, AMG 212 was administered as a continuous IV infusion, using an on-body portable infusion pump and central venous port system. In the first 4 cycles (first 12 weeks on study), patients received treatment on a “5 week on-1 week off” schedule, whereby AMG 212 was administered over 5 weeks, followed by a treatment-free interval of 1 week. From cycle 5 onwards, patients could continue treatment on the “5 week on-1 week off” schedule or switch to a “4 week on-2 week off” schedule, at the discretion of the investigator and the subject. The CIV dosing schema is shown in In both the CIV arm and the higher dose-level SC cohorts, prophylactic oral or IV dexamethasone was administered before the administration of AMG 212 to mitigate cytokine release syndrome (CRS) risk. At the discretion of the investigators, concomitant therapy was allowed. For each patient, treatment continued until tumor progression, unacceptable toxicity, consent withdrawal, or withdrawal from the study. Further details on study design can be found in Hummel et al., 2021 .Men aged ≥ 18 years old with histologically or cytologically confirmed advanced CRPC with treatment failure after ≥ 1 taxane regimen and who were refractory to abiraterone and/or enzalutamide or refused any other standard therapy were eligible for inclusion in the study. Eligible patients had undergone bilateral orchiectomy or received continuous androgen deprivation therapy and had evidence of progressive disease after discontinuation of anti-androgen therapy  before study drug treatment. Additional inclusion and exclusion criteria have been described in Hummel et al., 2021 .This was an open-label, multi-center, phase 1, dose-exploration/dose-expansion study in patients with mCRPC from North America, Europe, Asia, and Australia (NCT03792841) and sponsored by Amgen. The primary objectives of the study were to evaluate the safety and tolerability of AMG 160, to determine the MTD and/or recommended phase 2 dose (RP2D). The secondary objectives were to evaluate the preliminary antitumor activity and characterize the PK and pharmacodynamics of AMG 160.AMG 160 was administered intravenously in 28-day cycles. Once the target dose was reached, AMG 160 was administered by short-term IV infusion over 1 hr, every 2 weeks. In the dose exploration phase, to mitigate against CRS, step-dosing was implemented. Similar to the premedications used in the AMG 212 study, prophylactic oral and/or IV dexamethasone was administered before the administration of AMG 160 to mitigate CRS risk.Following the dose exploration phase, a dose expansion study was conducted to confirm the safety, PK, and pharmacodynamics of AMG 160 at the RP2D, and to obtain further safety and efficacy data and carry out correlative biomarker analysis.The AMG 160 patient population enrolled in NCT03792841 was comparable to that enrolled for AMG 212 in NCT01723475. In brief, men aged ≥18 years of age were included if they had histologically or cytologically confirmed mCRPC that was refractory to novel hormonal therapy, had failed 1–2 taxane regimens (or were unsuitable for or had refused treatment with taxanes), and had evidence of progressive disease as defined by the Prostate Cancer Working Group 3 (PCWG3) guidelines. Patients were excluded if they had active autoimmune disease or required immunosuppressive therapy, had received prior PSMA–targeted therapy (patients treated with PSMA radionuclide therapy were considered eligible), or had evidence of central nervous system metastases, leptomeningeal disease, or spinal cord compression.Both the AMG 212 and AMG 160 clinical studies were conducted in accordance with the ethical principles derived from international guidelines, including the Declaration of Helsinki, Council for International Organizations of Medical Sciences International Ethical Guidelines, and applicable International Conference on Harmonisation guidelines, laws, and regulations. The study protocols were approved by the Institutional Review Board/Independent Ethics Committee at each study site. All patients provided written informed consent.In the SC arm, blood samples for immunogenicity evaluation were collected predose on Cycle 1 Day 1, 8 and 15, on Day 1 of each cycle from Cycle 2 to 8, on Day 1 of every second cycle thereafter and at least 36 hr after the last dose of AMG 212. In the CIV arm, blood samples were collected predose on Cycle 1 Day 1, 8 and 15, on Day 1 of each subsequent cycle and at least 36 hr after the last dose of AMG 212. The ADA collection schedule for the SC and CIV arms is shown in Anti-AMG 212 antibodies were measured using a validated electrochemiluminescence-based bridging assay. This immunoassay method followed a two-tiered assay approach consisting of a screening assay and confirmatory assay. Samples were diluted 1:10 in D-PBS (Gibco Cat# 14190-094) or D-PBS and soluble drug (confirmatory assay only) prior to analysis. The samples were then incubated with conjugate mixture consisting of biotinylated-AMG 212 and ruthenylated-AMG 212. During this incubation, the two antigen binding sites of anti-AMG 212 antibodies were able to form a bridge between the labeled AMG 212 molecules. The sample mixture was then added to a blocked streptavidin microtiter plate, washed, and analyzed on a plate reader. The result was a series of electrically induced oxidation-reduction reactions involving ruthenium (from the captured complex) and tripropylamine. In this immunogenicity screening assay, a subject-specific floating cut point was calculated by adding a specific normalization factor to the pre-dose subject sample. Samples with results equal to or greater than the assay cut point were then tested to confirm specificity of the response. Samples classified as positive in the confirmatory assay were further titrated in 10% human serum pool and reported at the highest dilution titer at which a positive response was determined. The normalization factor and the confirmatory assay cut point were calculated from 40 prostate cancer donor serum samples. The assay sensitivity was 8.6 ng/mL based on a goat polyclonal positive control antibody. At 12, 120 and 1,200 ng/mL of anti-AMG 212 antibody, the assay tolerated at least 10,000 pg/mL of excess AMG 212.in-vitro cell-based competitive ligand binding assay. Effector cells (CD3-positive MC15 cells) were incubated with target cells (human PSMA-positive C4-2 cells), serum samples and AMG 212. After overnight incubation, the cytotoxic activity was measured with the luminescent CytoTox-Glo™ Cytotoxicity Assay kit (Promega). The CytoTox-Glo™ Cytotoxicity Assay uses a luminogenic peptide substrate, the AAF-Glo™ Substrate, to measure dead-cell protease activity released from cells that have lost membrane integrity. When the serum sample contained AMG 212 neutralizing antibodies, cytotoxicity was reduced. A sample was considered positive for neutralizing antibodies if the decrease of the cytotoxicity was greater than the cut point compared to the maximal toxicity sample. The cut point was calculated from 45 healthy donor serum samples. The assay sensitivity was 780 ng/mL based on a goat polyclonal positive control antibody. At 13,500 ng/mL of anti-AMG 212 antibody, the assay tolerated at least 3,000 pg/mL of excess AMG 212.The resulting immunoassay-positive samples were analyzed using a cell-based bioassay to determine whether the detected binding antibodies have neutralizing properties. This AMG 212 neutralizing assay was based on an In the SC arm, blood samples for PK assessment were collected predose at Cycle 1 Day 1 and 15, and at 1, 2, 4, 6, 8, 12 and 24 hr post-infusion at these two timepoints. Samples were also collected predose on Cycle 1 Day 3, 4 and 8. From Cycle 2 to 8, and every second cycle thereafter, a blood sample was collected on Day 1 within 2 to 6 hr post-dose. In the CIV arm, blood samples were collected on Cycle 1 Day 1 predose, at the 2-3 hr and 4-6 hr post-start of infusion timepoints, and on Day 2, 8 and 15. From Cycle 2 onwards, a blood sample was collected on Day 1 and 15 with every subsequent even cycle, and collected on Day 1, 2 and 15 with every subsequent odd cycle.The assay to quantify AMG 212 was based on a sandwich immunoassay format in which capture antibodies  were coated on a plate. After sample incubation, a mouse anti-idiotype monoclonal antibody against the CD3 binding domain of AMG 212 was bound to the captured AMG 212 and detected with ruthenylated anti-mouse antibody. The assay range was 0.150 to 111 ng/mL.Efficacy was assessed according to the Prostate Cancer Clinical Trials Working Group 2 recommendations . RECIST 6 monocytes per flask in differentiation medium (Dendritic Cell (DC) medium containing 100 ng/mL IL-4, 50ng/mL GM-CSF) and incubated for 5 days at 37°C, 5% CO2 to differentiate into DC. The DC were then loaded with AMG 212  and matured with Lipoprotein polysaccharide (LPS) for 24 hr. After maturation, the DC were lysed and the membrane fraction containing the HLA:peptide complexes was solubilized and incubated with Protein A mag sepharose beads  coated with anti-HLA-DR antibody (Lonza) at 4°C overnight. The following morning the beads were washed in Tris Buffer Solution (TBS) and the peptides eluted from the HLA-DR complex with 0.1% Trifluoroacetic acid (TFA). Finally, the peptides were purified by passing through a 10kDa Molecular Weight Cut-Off (MWCO) spin column and stored at -80°C for mass spectrometry (MS) analysis. MS data analysis was carried out using the PEAKS® Studio Software package. The identified peptides are then mapped back to the full-length AMG 212 protein sequence and compared to an Epibase® in silico analysis to identify which HLA-DR alleles are responsible for the peptide binding. Albumin was also included as an internal control in the samples to verify assay performance. Comparable numbers of total (173 vs 171) and distinct (85 vs 77) albumin peptides were detected in the AMG 212 sample in the first and second round of MAPPS respectively, indicating that the assay was consistent and sensitive across repeats.The Epibase® MAPPS assay was performed at Lonza. Monocytes were isolated from frozen PBMC samples by positive magnetic bead selection (Miltenyi Biotec). Monocytes were seeded into T12.5 flasks with 5 X 10Along with the MAPPS-identified sequences , peptides spanning the rest of the Complementarity Determining Regions (CDR) regions of AMG 212 were proactively synthesized. These additional sequences were labeled Peptide #6, 7, 9, 10 and 12. A separate peptide, Peptide #13, was synthesized as a known self-tolerant peptide and acted as a negative control peptide that demonstrated MHC class II-binding but was not expected to confer T cell reactivity. A total of 14 peptides were synthesized.Pool 1 consisted of 5 peptides  and Pool 2 consisted of 8 peptides . Peptide #8.5 was inadvertently missed in Pool 2. CEFTA, a peptide pool consisting of 35 MHC class II-restricted peptides from human CMV, EBV, influenza virus, tetanus toxin, and adenovirus 5, and PADRE (Pan DR-binding epitope), were used as positive controls as these peptides are designed to stimulate T cells with a broad array of HLA types.Isolated CD4+ T cells were stimulated with multiple rounds of autologous monocyte-derived DCs (moDCs) pulsed with our test peptides in a 4-week co-culture. For the first 3 weeks, the T cells were stimulated weekly with freshly-derived moDCs pulsed with peptide pools in the first 3 stimulations, and then with individual peptides from a pool, for a fourth and final stimulation.6/mL into a 96 well plate (200 µL/well) in Cellgenix DC GMP Medium (Sartorius) supplemented with 100 ng/mL IL-4 (Peprotech) and 50 ng/mL GM-CSF (Peprotech). After 5 days, the immature DCs were separately loaded with 5 µg/mL CEFTA peptide (Mabtech) pool, 5 µM PADRE peptide (Mayflower Biosciences), or 5 µM test peptide pool and matured with 10 ng/mL TNF-α (R&D Systems) and 5 ng/mL IL-1β (Peprotech) for 48 hours. The quality of the matured DCs was assessed by labeling of markers HLA-DR, CD14, CD80, CD83, CD86, CD209, and CD11b. CD4+ T cells were isolated from PBMC using negative selection magnetic microbeads (Miltenyi Biotech). CD4+ T cells (2 X 105/well) were stimulated by peptide-loaded DC and cultured initially in AIM-V supplemented with 2% Human AB Serum (Sigma-Aldrich) for 21 days. Freshly loaded and matured DCs were added to the T cell culture every 7 days, and the culture medium was refreshed every 7 days with AIM-V supplemented with 2% Human AB Serum (Sigma-Aldrich), 10 U/mL IL-2 (Peprotech), and 5 ng/mL IL-7 (Peprotech).On Day 1, CD14+ cells were isolated from PBMC using positive selection magnetic microbeads (Miltenyi Biotech). The CD14+ cells were differentiated into immature DCs by seeding at 1 X 104) of CD4+ T cells were taken from each well and stimulated with peptide pool-loaded DCs in pre-coated Human Interferon-γ PVDF Plates (ImmunoSpot®). After 48 h incubation, the manufacturer’s plate development instructions were followed to detect secreted IFN-γ. Spots were counted using a CTL ImmunoSpo® S6 Ultra M2 Analyzer. Wells with unloaded DCs  and wells with T cells only , served as negative controls. T cell lines reactive to individual peptides were determined as wells that had spot counts at least two-fold higher in the presence of peptide compared to the unloaded DC negative control, with a minimal difference of 30 spots, as described previously  timepoint was collected per subject, according to the Schedule of Assessments in the AMG 160 FIH Study 20180101 (NCT03792841). The blood was sent ambient on the same day of collection to the central lab for PBMC processing using the CTL protocol (ications . However5/well into pre-coated Human Interferon-γ PVDF Plates (ImmunoSpot®) and incubated with individual test peptides at 5 µM each, in CTL-Test Medium supplemented with 2mM GlutaMAX and 10 ng/mL of GM-CSF (Peprotech). The peptides tested were #1, 2, 8, 8.5, 11, along with peptides #13 and #4 which we had established previously from the restimulated T cell line assays to be negative controls (did not demonstrate T cell reactivity). Phytohemagglutinin (PHA) at 2 µg/mL was used as a strong, non-specific stimulator for a technical positive control. PBMCs from an AMG 160-naive healthy donor, acted as an additional negative control for the assay. After 72 hr of incubation , manufacturer’s plate development instructions were followed to detect secreted IFN-γ. Spots were counted using a CTL Immunospot® Series 5 Macro Analyzer.Ultimately, to perform the clinical memory recall assay, freshly-thawed patient PBMCs were seeded at 2 X 10Analyses of AMG 212 immunogenicity, PK and signs of activity were descriptive in nature and presented using summary statistics and individual subject profiles. ADA status, exposure impact and PSA response correlation analyses were performed using a logistic regression model with SAS (version 9.4) software. Further details on sample sizes, dose-limiting toxicities (DLTs) and safety summary statistics to fulfill the study objectives can be found in a prior clinical report summarizing the overall results of the AMG 212 FIH study . PatientNCT01723475 was first initiated in a cohort of 31 subjects who were subcutaneously administered AMG 212 into four regions around the navel (other injection sites were permitted) . Doses wPre-existing reactivity was not observed to AMG 212. However, as an aggregate across all doses in the SC dose escalation study, treatment-emergent ADA (TE-ADA) developed in 30/31 subjects (96.7%) who had a post-baseline result  developed early, within the first 2 cycles of AMG 212 treatment, (ii) often progressed to high titers that neutralized AMG 212 activity and (iii) was sustained till end of study as none of the ADAs were transient.Exposure-ADA correlation analyses were performed to determine the impact of the TE-ADA on exposure and efficacy. Following subcutaneous administration, PK was not consistently detectable at lower dose levels. Therefore, it was not reasonable to make any PK-ADA associations at these lower dose levels.From the 72 µg to 172 µg dose level, PK was detectable in all subjects at first. However, of the 17 TE-ADA+ subjects, 14 subjects had PK samples measuring below the lower limit of quantitation (LLOQ) and 3 subjects had PK samples measuring close to LLOQ, either at the same timepoint as the first positive ADA sample, or at timepoints thereafter (A key pharmacodynamic (PD) marker in prostate cancer is Prostate Surface Antigen (PSA), which acts as a clinically validated marker of disease progression and therefore a surrogate marker for drug activity. From the 36 to 172 µg dose level, reductions in PSA > 50% relative to baseline were observed in nine patients . In thesGiven the high titers of ADAs observed in the SC arm, the ADAs were assessed for any association with immune-complex related safety events known to be associated with ADAs, such as infusion reactions or other hypersensitivity events . While iTaken together, TE-ADA observed in the SC cohort was not clearly associated with adverse events but did result in uniform exposure loss. This most likely accounted for the curtailment of the PSA response observed initially.In the SC cohort, due to the consistent formation of ADAs and the observation of localized injection site reactions in many ADA-positive subjects, it was hypothesized that ADA development was induced by the skin-resident DCs, which are well-established to be excellent APCs , 21. To The ability of the topical GC to eliminate or suppress skin APCs was not confirmed, as skin biopsies were not retrieved from GC-treated subjects. As a surrogate marker of whether the topical GC eliminated or suppressed skin APCs, peripheral blood monocyte counts were analyzed. There were no significant differences in this parameter, at the 144 and 172 µg/d dose levels, between the paired cohorts comparing subjects treated with or without GC  administration was initiated. In stark contrast to the SC cohort, 0/16 subjects (0% incidence) developed ADAs in the CIV cohort when administered AMG 212 at dose levels ranging from 5 µg/d to 80 µg/d ).From the 5 to 80 µg/d dose levels, confirmed PSA responses (PSA 30 or PSA 50) were recorded in 5/16 subjects . Out of Taken together, in contrast to the SC route, AMG 212 did not induce any TE-ADAs when administered by CIV infusion. This enabled maintenance of exposure, yielding an exceptional durability of response in two subjects at the higher dose levels.Several factors contribute to a therapeutic’s immunogenic risk and observed immunogenicity in the clinic. We sought to explain the polar difference in TE-ADA incidence observed between the SC (near 100%) and CIV (0%) cohorts by systematically interrogating both product-related and patient-specific factors, beyond the route of administration.hi) on monocytes were assessed by flow cytometry in peripheral blood at the time of screening (7 days before Cycle 1 day 1). At the screening timepoint, no significant differences in CD14+ HLA-DR+ counts (hi median fluorescence intensity (MFI)  on CD4+ T cells and MHC class II upregulation  (larger than dimer) is an attribute widely acknowledged as a primary risk to immunogenicity –27. The In addition, we examined stability assays that tested whether AMG 212 was stable over time after reconstitution. AMG 212 consisted of over 97% monomer species as measured by size-exclusion chromatography, after 7 days at 5 ± 3°C, and an additional 4 days at 37 ± 2°C with agitation (data not shown). This data suggests that it was unlikely that AMG 212 drug product could have developed HMWS in concerning amounts over time at human body temperature for at least 4 days. Taken together, an assessment of HMWS in AMG 212 drug substance and drug product ruled out this attribute as a potential cause for the immunogenicity observed in the SC arm.Given the above, patient baseline immune status , and drug product quality attributes related to immunogenicity risk, did not appear to contribute to ADA formation in SC-administered subjects.The immunogenicity observed to SC-dosed AMG 212 may be most evidently explained by the route of administration. However, relying on this factor alone would be an oversimplification, as not all SC-injected protein therapeutics above 20kD in size, which are known to first encounter the lymphatic system before entering the peripheral circulation , 29, eliTo determine where the AMG 212 SC ADAs were binding to on AMG 212, we performed exploratory work evaluating the domain specificity of these ADAs. Using 8 ADA-positive and 14 ADA-negative samples from 4 subjects  in an exploratory assay and with appropriate reagents, these results demonstrated that AMG 212 ADAs bound predominantly to the PSMA binder, and not to the CD3 binder or the linker (data not shown).However, sustained, clinically impactful ADA responses such as those observed in the AMG 212 SC arm are often driven not by structural epitopes recognized by B cells alone, but by CD4+ T cells recognizing sequence-based epitopes located within the drug’s amino acid sequence. We therefore focused our efforts on seeking out potential T cell epitope(s) in AMG 212. Here we hypothesized that the combination of the SC drug delivery regimen and the existence of potentially immunogenic sequences in AMG 212 were responsible for driving the robust clinical ADA response.in vitro experiments to determine whether AMG 212 contained potentially non-tolerant, sequence-based, T cell epitopes.To address the latter part of this hypothesis, we performed a series of We applied a tiered approach in seeking out potential T cell epitopes in AMG 212. Starting at the level of APC recognition and presentation, we narrowed down suspect sequences through their reactivity in healthy donor T cells, and eventually tested the peptides in clinical memory recall assays using patient samples.First, we sought to determine whether there were specific sequences in AMG 212 that were being presented on the APC surface to T cells by employing MHC class II-associated peptide proteomics (MAPPS) , 31. WhiMAPPS was performed on AMG 212 twice, evaluating a total of 20 donors that included a variety of HLA-DRB alleles representing the major subtypes in the human population  to those found in memory responses (~1:103-5)  . The assThe accrued assay results showed that of the 13 suspect peptides tested, 4 peptides showed T cell reactivity in more than 1 donor , 32. Howex vivo stimulation from the immunogenic peptide(s), this pool of peptide-specific memory T cell clones within ADA+ patient PBMCs can be recalled upon to secrete Interferon-γ, detectable by ELISPOT.With our top suspect sequence regions in hand, we sought to ascertain which of these sequences could be driving AMG 212 immunogenicity in a clinical memory recall assay  using pa® molecule), was being investigated in a FIH trial at that time .Ideally, we would have performed this recall assay using PBMCs from AMG 212 ADA+ patients. However, at this point in our investigation, the AMG 212 FIH clinical trial had already concluded, and we were unable to obtain PBMCs from AMG 212 ADA+ subjects retrospectively. However, a follow-on molecule to AMG 212, AMG 160 , as disclosed in the virtual ESMO 2020 presentation describing interim results of the AMG 160 FIH study, 6 of 30 (20.0%) patients evaluated developed ADAs which affected drug exposure between cycles 1 and 10  PBMC samples from patients in the AMG 160 FIH trial, to evaluate Peptide #1, 2, 8, 8.5 and 11 in the clinical memory recall assay. In total, PBMC samples from 9 ADA-positive and 8 ADA-negative patients from the AMG 160 FIH trial were assessed in this assay.Of the suspect sequences tested, Peptide #1, 8 and 11, but not #2 or 8.5, exhibited a recall response in a single AMG 160 ADA+ subject . Anecdotally, across several T cell engager trials, we have observed that intra-subject dose-escalation can sometimes result in This body of work, built upon many others, establishes a thought process and a systematic approach in addressing how a sponsor may identify culprit T cell epitopes driving clinical immunogenicity Figure 8When a biologic exhibits high sequence-based risk based on available prediction tools, downstream assays to confirm possible epitopes should be initiated. One can envision that the tiered approach we utilized to identify culprit epitopes retrospectively, can also be implemented prospectively, to de-risk molecules before they enter the clinic. Indeed, others have built mechanistic models that additionally account for the drug’s mode of action when attempting to predict a molecule’s clinical immunogenic risk . Clearly® (blinatumomab) (CD19-targeting), although approved as a CIV formulation, has since been tested as a SC formulation in both Relapsed/Refractory indolent Non-Hodgkin’s Lymphoma (NHL) and Acute Lymphoblastic Leukemia (ALL). In both of these trials, anti-blinatumomab antibodies were not detected , a first-in-class T cell engager for HLA-A 02:01-positive metastatic uveal melanoma patients may be a case-in-point. A 29-33% binding ADA incidence graces the label of KIMMTRAK®. High-titer ADA was shown to decrease exposure by 97%  Krankenhaus der Barmherzigen Schwestern Linz, Ethikkommission der Barmherzigen Schwestern Linz, Seilerstätte 4, 4010 Linz, Austria (2); Ethikkommission des Landes Oberösterreich, Kepler Universitätsklinikum Neuromed Campus, Wagner-Jauregg Weg 15, 4020 Linz, Austria (3); Universitätsklinikum Heidelberg, Ethikkommission der Med. Fakultät Heidelberg, Alte Glockengießerei 11/1, 69115 Heidelberg, Germany (4); Klinikum der Universität Würzburg, Ethik-Kommission bei der Medizinischen Fakultät, Institut für Pharmakologie und Toxikologie, Versbach Str. 9, 97078 Würzburg, Germany (5); Landesamt für Gesundheit und Soziales, Geschäftsstelle der Ethik-Kommission des Landes Berlin, Fehrbelliner Platz 1 (Dienstgebäude), 10707 Berlin, Germany. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.HP: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing. KH: Formal Analysis, Methodology, Writing – review & editing. NT: Formal Analysis, Methodology, Writing – review & editing. BW: Writing – review & editing, Methodology. CB: Formal Analysis, Methodology, Writing – review & editing. CW: Formal Analysis, Methodology, Writing – review & editing. MM: Formal Analysis, Methodology, Writing – review & editing. SW-R: Conceptualization, Formal Analysis, Funding acquisition, Writing – review & editing. GK: Conceptualization, Formal Analysis, Funding acquisition, Writing – review & editing. SS: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Project administration, Supervision, Writing – review & editing. RB: Conceptualization, Investigation, Writing – review & editing. H-DH: Conceptualization, Investigation, Writing – review & editing. WL: Conceptualization, Investigation, Writing – review & editing. CG: Conceptualization, Investigation, Writing – review & editing. TE: Data curation, Formal Analysis, Investigation, Methodology, Project administration, Supervision, Writing – review & editing. BT: Investigation, Writing – review & editing. DM: Formal Analysis, Funding acquisition, Methodology, Project administration, Supervision, Writing – original draft, Writing – review & editing, Conceptualization, Data curation.


However, as new single-cell technologies are introduced, as the community continues to adjust best practices for data processing, and as the alevin-fry ecosystem itself expands and grows, it is becoming increasingly important to manage the complexity of alevin-fry’s single-cell preprocessing workflows while retaining the performance and flexibility that make these tools enticing. We introduce simpleaf, a program that simplifies the processing of single-cell data using tools from the alevin-fry ecosystem, and adds new functionality and capabilities, while retaining the flexibility and performance of the underlying tools.The Simpleaf is written in Rust and released under a BSD 3-Clause license. It is freely available from its GitHub repository https://github.com/COMBINE-lab/simpleaf, and via bioconda. Documentation for simpleaf is available at https://simpleaf.readthedocs.io/en/latest/ and tutorials for simpleaf that have been developed can be accessed at https://combine-lab.github.io/alevin-fry-tutorials. Yet, the rapid arrival of new technologies and experimental modalities have led to data analysis pipelines that require increasingly complex and sophisticated workflows. For example, analyzing CITE-seq . Simpleaf  An example of streaming parser invocation can be found in Another useful feature provided by simpleaf “normalizes” these reads into an appropriate “simple” format (a format with only known position and fixed-length barcodes and UMIs) on the fly, and provides a modified (and simplified) geometry format description to the underlying mapper. Moreover, it streams the normalized reads directly to the mapper using FIFOs programmatically managed by simpleaf, thereby avoiding intermediate disk usage. This feature enables the existing mappers in the alevin-fry ecosystem, which are designed to process reads with simple geometry, to handle sophisticated geometries without modifying the underlying mapping tools, requiring extra preprocessing from the users, or taking the extra time and space to write and read the intermediate representations.When presented with a “complex” geometry specification, scg_lib_struct GitHub repository (https://github.com/Teichlab/scg_lib_structs). Furthermore, general parsers for such protocols have been developed  template, fill in minimal information about the current sample to instantiate it, and execute the instantiated template using the workflow executor. More details can be found in simpleaf workflows by taking advantage of the full functionality provided by the Jsonnet language. Furthermore, this design also makes it easy for users to create and share their workflows by simply sharing their workflow templates, without the need to understand and contribute to the codebase of simpleaf itself. To demonstrate the utility of the simpleaf workflow module, we have built such workflow templates for processing 10× Chromium 3′ v2 and v3 RNA-seq, CITE-seq (Supplementary Appendixsimpleaf workflow list program (The  program  for obtaSimpleaf provides a simple and flexible interface to access the state-of-the-art features provided by the alevin-fry ecosystem, tracks best practices using the underlying tools, enables users to transparently process data with complex fragment geometry, and to build and execute sophisticated workflows containing both simpleaf and external commands without the need to write code. Simpleaf has already seen adoption in community-led projects, for example, in the scrna analysis pipeline of the nf-core project (simpleaf can serve as an entry point and main interface to the alevin-fry ecosystem for most users. We also envision that our workflow feature can encourage those in the community to create and share their workflows and, thus, can help simpleaf to provide increasingly reusable building blocks to enable more varied and sophisticated single-cell data analysis pipelines.simpleaf provides a simple and flexible framework for single-cell data processing, the current implementation still has limitations, which motivate future work. For example, although the fragment geometry parser can parse barcodes with variable length and floating position, it currently lacks the ability to perform certain kinds of preprocessing, like the barcode substitution scheme required by the split-seq (simpleaf workflow is still under active development. Current efforts are underway to improve its generality, expand its library of standard functions, and develop more useful and sophisticated workflows for different purposes.While btad614_Supplementary_DataClick here for additional data file.


The survival of a species depends on its ability to reproduce . It has Puberty is the stage when fertility is achieved and maintained throughout all subsequent stages of life. This process is initiated by the pulsatile secretion of the gonadotropin-releasing hormone (GnRH) from the hypothalamus ,5. GnRH Factors such as genetic predispositions, under or overnutrition, and exposure to environmental endocrine disruptors can influence the timing of puberty and affect the integrity of the hypothalamic–pituitary–gonadal axis, which is essential for preserving fertility at all stages of life ,5. PatieAdolescence and young adulthood are critical stages when lifestyle factors such as weight, smoking, alcohol, caffeine consumption, diet, exercise, psychological stress, and environmental and occupational exposures can impact fertility in both sexes ,7,8. For example, obesity and being underweight are associated with gonad dysfunction and infertility, while certain vitamins  and food groups (including high-fat diets) have an important impact on reproductive health by influencing semen quality and ovulation ,11,12,13Additionally, excessive exercise can affect body energy balance and hypothalamic function, negatively impacting the reproductive system ,5. Finally, unhealthy lifestyle and environmental exposures can have transgenerational effects on endocrine disruptors in reproductive tissues, not only through oocytes, but also through sperm , demonstUnderstanding the impacts of environmental, nutritional, and lifestyle factors in regulating the reproductive system is essential for maintaining gonadal function, preserving fertility and protecting overall wellbeing. Preventative strategies that modify lifestyle factors can be useful in enhancing reproductive health and maximizing fertility outcomes, starting from childhood.


T cells are the essential effectors of the adaptive immunity. They use their unique receptor, the T cell receptor (TCR), to specifically recognize and respond to antigens associated with the major histocompatibility complex (MHC) molecules. Signal transduction of the TCR is initiated with the function of the CD3 subunits of the TCR complex. The very first event in TCR/CD3 complex activation is the phosphorylation of the cytoplasmic tails of CD3ζ by the tyrosine kinase Lck, allowing the recruitment of the kinase ZAP70 to phosphorylate a number of downstream mediators and effectors to amplify TCR/CD3 signaling.Adaptor molecules couple the TCR/CD3 complex to a variety of signaling molecules involved in T cell activation and as such play a key role as signal amplifiers. Adaptors include both transmembrane and cytosolic molecules, such as LAT, SLP-76, GRB2, SHC, and Gads. While some adaptor molecules, like TSAd and Nck, may participate in proximal TCR/CD3 signaling by recruiting Lck, other adaptors, such as PAG and SIT, may negatively regulate TCR signaling, thereby contributing to signal termination. Signaling by the TCR has been heavily investigated for exploitation in cancer immunotherapy through approaches involving CAR T cells or other engineered T cells. Adaptor proteins have fundamental roles in the fine-tuning the tightly controlled intricate process of TCR/CD3 signaling both in T cell homeostasis and immune responses. Harnessing their signal modulating properties may help develop better cancer immunotherapies. Additionally, adaptor molecules are among the components of the TCR signaling cascade being investigated for their implication in pathological conditions including autoimmunity and hematologic malignancies, and some adaptor molecules have been identified as therapeutic targets to treat or alleviate those conditions. This Research Topic focuses on adaptor proteins that participate in T cell signaling and thus modulate the adaptive immune response.Luff et al.  have docGartshteyn et al.  have rev+CD8+ double-positive but a decrease in that of CD4+ and CD8+ single-positive thymocytes. However, in the periphery these mice have reduced a percentage of CD8+ T cells and increased γδT cells. Additionally, Ruminski et al.  and the Src kinase associated protein 1 and 2 (SKAP1 and SKAP2) have been reviewed by Dadwal et al.  for theiu et al. , expandeVelnati et al.  have repO’Neill et al. have demonstrated the existence of a signalosome containing three interacting proteins, caspase recruitment domain family member 11 (CARD11), B-cell lymphoma/leukemia 10 (BCL10), and mucosa-associated lymphoid tissue 1 (MALT1), called the CBM complex. MALT1 is known to be a scaffold for the E3 ligase tumour necrosis factor (TNF) receptor-associated factor 6 (TRAF6). The authors showed in a mouse model that the CMB is crucial for balancing T cell activation and homeostasis. In addition, Oikawa et al. (Hornick and Bishop have reviewed the role of TRAF3 as a mediator of T cell differentiation and effector function. The essential function of TRAF3 has been demonstrated both in murine models and human disease caused by germline TRAF3 deficiency. The affected patients have autoimmunity, immunodeficiency, and a predisposition to B cell malignancies.Signaling transduction in T cells eventually leads to the NF-κB transcription factor activation. This process is a critical step for T cell survival, proliferation, differentiation and function. Collectively, this Research Topic contributes to provide new insights into T cell biology area involving adaptor proteins, including proximal and distal TCR signaling, thymocyte development and T cell differentiation, signalosomes relating to T cell signal transduction and regulation of cytokines and other signaling mediators. The knowledge obtained can set the foundation to novel approaches for treatment of autoimmunity and cancer.SP, AS, and CB drafted and edited the present editorial. All authors contributed to the article and approved the submitted version.


As evolutionarily conserved signaling cascades, AhR and Wnt signaling pathways play a critical role in the control over numerous vital embryonic and somatic processes. AhR performs many endogenous functions by integrating its signaling pathway into organ homeostasis and into the maintenance of crucial cellular functions and biological processes. The Wnt signaling pathway regulates cell proliferation, differentiation, and many other phenomena, and this regulation is important for embryonic development and the dynamic balance of adult tissues. AhR and Wnt are the main signaling pathways participating in the control of cell fate and function. They occupy a central position in a variety of processes linked with development and various pathological conditions. Given the importance of these two signaling cascades, it would be interesting to elucidate the biological implications of their interaction. Functional connections between AhR and Wnt signals take place in cases of crosstalk or interplay, about which quite a lot of information has been accumulated in recent years. This review is focused on recent studies about the mutual interactions of key mediators of AhR and Wnt/β-catenin signaling pathways and on the assessment of the complexity of the crosstalk between the AhR signaling cascade and the canonical Wnt pathway. AhR and pRb form a heterodimer that results in the blockade of cell cycle progression by downregulating S-phase genes , as well as leukotrienes and arachidonic-acid metabolites  ,76,77,78Pseudomonas aeruginosa) anthracene, Cd, CPF, indenopyrene, AS-IV, IAA, and βNF promote the activation of the Wnt/β-catenin signaling pathway ,138,143.CTNNB1, RSPO, Wnt5a, Rnf43, LGR, and DKK contain potential binding sites (XREs) for AhR [The enhancement of canonical Wnt signaling, as follows from the review of the literature, is likely due to the influence of AhR on such upstream components of the Wnt pathway as R-Spondin1 , Axin2,  for AhR ,163,189.Very recently, a novel AhR signaling mechanism underlying Wnt signal transduction activation was demonstrated , in whicAhR and CYP1 have been identified as target genes of β-catenin [CYP1A are located in close proximity [β-catenin plays the main part in the interaction with AhR signaling. Firstly, -catenin ,170; sec-catenin ,173,175;roximity ,178; androximity ,179,180.Lately, in chronic myeloid leukemia cell lines, a regulatory interaction between AhR and the Dvl protein, which plays a central role in β-catenin stabilization, has been demonstrated; this interaction manifests itself as a dependence of AhR protein expression on the silencing or overexpression of Dvl . There iIDO1 enzyme: after the triggering of IDO1 and the subsequent activation of AhR, β-catenin is activated [CCND1, RNF43, ZRNF3, LGR5, and ASCL2 [It is worth highlighting the involvement of key enzymes of the Kyn pathway of tryptophan metabolism—TDO2 and IDO1 —in the interactions between AhR and Wnt. The enhancement of the AhR and β-catenin pathways can proceed in a coordinated manner and is a consequence of the activation of the ctivated . The actnd ASCL2 .Thus, the presented evidence of the influence of AhR pathway signals on the Wnt/catenin signaling cascade paints a complicated picture where the outcome of such an influence is determined by the type of cells, tissues, or organs, as well as by the chemical structure of the AhR ligand. The Wnt/β-catenin pathway signals, in turn, are involved in interactions with AhR signaling. Taken together, the presented literature proves the important role of the interdependent regulation of AhR and Wnt/β-catenin signaling pathways in the maintenance of embryogenesis, organogenesis, and cell homeostasis under normal and pathophysiological conditions. In these interactions of the signaling pathways, AhR appears to act as a fine-tuner of Wnt signal transduction in a cellular, tissue, or organ context; this property indicates the high potential of AhR as a therapeutic target for Wnt pathway modulation in pathological conditions, including cancer and nonmalignant diseases.


Alzheimer’s disease (AD) and Parkinson’s disease (PD) represent the most prevalent neurodegenerative disorders severely impacting life expectancy and quality of life of millions of people worldwide. AD and PD exhibit both a very distinct pathophysiological disease pattern. Intriguingly, recent researches, however, implicate that overlapping mechanisms may underlie AD and PD. In AD and PD, novel cell death mechanisms, encompassing parthanatos, netosis, lysosome-dependent cell death, senescence and ferroptosis, apparently rely on the production of reactive oxygen species, and seem to be modulated by the well-known, “old” second messenger cAMP. Signaling of cAMP via PKA and Epac promotes parthanatos and induces lysosomal cell death, while signaling of cAMP via PKA inhibits netosis and cellular senescence. Additionally, PKA protects against ferroptosis, whereas Epac1 promotes ferroptosis. Here we review the most recent insights into the overlapping mechanisms between AD and PD, with a special focus on cAMP signaling and the pharmacology of cAMP signaling pathways. Alzheimer’s disease (AD) and Parkinson’s disease (PD) represent two of the most prevalent neurodegenerative disorders worldwide, affecting over 50 million and 10 million people, respectively . AD prim2-), hydrogen peroxide (H2O2) and hydroxyl radical (-OH), are intermediates in the process of aerobic metabolism. Mitochondria are the main organelles to generate ROS within the cells, particularly during aerobic respiration involving electron transfer through respiratory chain complexes I and III. Low levels of ROS function as signaling molecules, regulating transcription, phosphorylation and other pathways , such as superoxide anion  is a key second messenger that mediates a variety of physiological processes, including memory and learning, myocardial relaxation and contraction, inflammation and immune function , and varevations . The durevations . cAMP sievations . As the evations  , indicating the role of compartmentalized cAMP/PKA signaling in mitochondrial pathology  is tightly and precisely modulated by various molecular machineries, and it has been suggested that RCD can be inhibited genetically and/or pharmacologically . The firMature neurons in a healthy CNS are generally resistant to cell death and can persist throughout the individual lifespan . Neurona+) and ATP depletion, which in turn contributes to parthanatotic cell death (2+ influx and activation of nitric oxide synthase (NOS) (2−) which is a common ROS in neurodegenerative disorders, and forms into peroxynitrite (ONOO−). NO and ONOO− can potently induce DNA damage and consequently lead to parthanatotic neuronal death (Parthanatos is a caspase-independent RCD that is triggered by hyperactivation of poly (ADP-ribose) polymerase (PARP-1) . Unlike ll death . Furtherll death . In AD ase (NOS) . Nitric al death  -induced neurodegeneration  and downstream Raf-MEK-ERK cascades, which phosphorylate NADPH oxidase (NOX) and enhance ROS production (Netosis is a type of RCD dependent on neutrophil extracellular traps (NETs) in response to infection or injury . Activat+ influx . This eloduction   is a highly semipermeable border that insulates brain parenchyma from circulating leukocytes. Neutrophils infiltrated the BBB and migrated into parenchyma via adhesion to blood vessels, and released NETs and IL-17 in transgenic AD models . FurtherInhibition of NET formation has already been used as a therapeutic approach against autoimmune and inflammatory diseases. NOX is a good target in netotic pathway and has entered several preclinical/clinical trials . NOX inhLysosome-dependent cell death is a type of RCD mediated by the lysosomal release of the proteolytic enzyme cathepsin. This cell death is induced by various stimuli including ROS, lysosomotropic detergents, dipeptide methyl esters and lipid metabolites  Figure . After l+)-induced PD cell model , which ultimately inhibited lysosomal acid sphingomyelinase (SMase)    ; Table 1INK4a can specifically inhibit CDK4 and CDK6 and thereby maintain the senescent suspension of cell cycle  were observed  . Senesceo models . In AD pobserved . Similarpatients . Studiespatients . Similarpatients . Furtherpatients . Taken tINK4a in P19 tau pathology mice, and demonstrated that genetic and pharmacological clearance of those cells could reduce gliosis, neurofibrillary tangles (NFTs), neuronal loss and cognitive impairment  was the only glia cell type showing elevated senescent markers in AD patients and an Aβ mouse model. Selective elimination of senescent OPCs improved cognitive function, reduced neuroinflammation, and decreased Aβ load . Anotherpairment . In contpairment . Additiopairment . Taken tSirtuin 1 (SIRT1) is commonly accepted as an anti-senescence protein, as it possesses deacetylase activity that can be activated through cAMP signaling . Sung etDysregulation of trace metals is one of the pathological features in AD and PD. In AD, zinc, copper and iron accumulate in senile plaques, with zinc and copper inducing a conformational change in Aβ and resulting in non-fibrillar Aβ oligomerization, while iron is likely to be associated with plaques in a ferritin-bound form . Similarc−  iron chelators, 2) antioxidants, 3) modulators of GSH synthesis . ExampleIn astrocytes, Nrf2 upregulates antioxidant proteins during mitochondrial activation, while PKA suppresses mitochondrial activation and ROS production via blocking protein import into mitochondria, and indirectly inhibits Nrf2 via outcompeting with Nrf2 regulator, casein kinase 2 (CK2) . BesidesBoth AD and PD severely impact life expectancy and quality of life of millions of people worldwide. Unfortunate, despite the global burden, the search after novel drugs able to diminish or even prevent the pathophysiological disease patterns is still characterized by a very limited progress in the last decades. Intriguingly, recent evidence indicates that cAMP-linked drugs - already approved by both FDA  and EMA –such as β-adrenoceptor agonists  and antagonists  seem to enjoy some clinical success . HoweverGlobal elevators and/or inhibitors of cAMP will have numerous (side) effects based on the widespread expression of the effectors of the cAMP signaling pathway in many cells and tissues including the brain. Therapeutics that specifically activate and/or inhibit effectors of cAMP signaling will likely enjoy greater clinical benefits with fewer side effects. In addition, gaining increasing knowledge about the impact of cAMP effectors distinct from β-adrenoceptors may open new avenues for combination therapies–allowing reduction in the concentration of each active drug component thereby most likely limiting unwanted treatment effects. Such combination therapies are already operational to treat chronic lung disorders  and cancer. Neurodegenerative disorders may alter also cAMP effector expression and/or localization, causing dysfunctioning of cAMP signaling. Such mechanisms may profoundly contribute to oxidative stress triggered-RCD mechanisms known to result in neuronal death in AD and PD. Therefore, more research into how specific cAMP signaling play key roles in distinct subcellular compartments is needed.For this purpose, we provided in this review a comprehensive overview about mechanisms underlying ROS-related novel cell death mechanisms in AD and PD. Notably, cAMP signaling appears to play differential roles in these RCDs. Signaling of cAMP via PKA and Epac promotes parthanatos and induces lysosomal cell death, while signaling of cAMP via PKA inhibits netosis and cellular senescence. Additionally, PKA protects against ferroptosis, whereas Epac1 promotes ferroptosis. Specifically, in parthanatos, PKA and Epac have been implicated in promoting cell death through phosphorylation of PARP-1 and inhibition of PARP cleavage, respectively . In lysoPharmacologically modulating PKA can result in unintended adverse effects due to its ubiquitous expression in various tissues and cells. To address this issue, alternative strategies that increase the specificity of PKA signaling have been proposed . RecentlModulating Epac signaling could be another potential therapeutic strategy against RCDs in neurodegenerative diseases. Indeed, it was reported that Epac1 gene was upregulated whereas Epac2 gene was downregulated in hippocampal and frontal cortex samples of AD patients compared to age-matched controls . LikewisIn addition to traditional low molecular weight modulators, PROteolysis TArgeting Chimeras (PROTACs) have shown promising effect in selective degradation of target proteins opening new avenues to specifically inhibit the cAMP pathway. PROTACs are bifunctional molecules with specific ligands for protein of interest and E3 ubiquitin ligase, connected by a linker . The tar


This paper investigates the effect of under-five mortality, child support grant (CSG) coverage and the rollout of antiretroviral therapy (ART) on fertility in South Africa. The study employs the quality-quantity trade-off framework to analyse the direct and indirect factors affecting fertility using the two stage least squares fixed effects instrumental variable approach. The analysis uses balanced panel data covering nine provinces from 2001–2016. This period was characterised by significant increases in the child support grant coverage and ART coverage. Furthermore, this period was characterised by a significant decline in the under-five mortality rate. We find no evidence to support the hypothesis that increases in the CSG coverage are associated with an increase in fertility. This finding aligns with previous literature suggesting that there are no perverse incentives for childbearing associated with the child support grant. On the other hand, results indicate that an increase in ART coverage is associated with an increase in fertility. Results also show that a decrease in under-five mortality is associated with a decline in fertility over the sample period. HIV prevalence, education, real GDP per capita, marriage prevalence and contraceptive prevalence are also important determinants of fertility in South Africa. Although the scale up of ART has improved health outcomes, it also appears to have increased fertility in HIV-positive women. The ART programme should therefore be linked with further family planning initiatives to minimise unintended pregnancies. The Sustainable Development Goals call for major reductions in under-five mortality, poverty, gender inequality, HIV/AIDS and unmet need for family planning–all of which are thought to be important determinants of fertility. Few studies have investigated the effect of progress towards these goals on fertility in the South African context. Over the period 2001 to 2016, there has been a decline in the under-five mortality rate, an increase in child support grant (CSG) coverage and increase in antiretroviral treatment coverage in South Africa. These changes occurred while low levels of fertility prevailed in South Africa compared to the rest of the African continent. A better understanding of the factors behind the prevailing fertility levels would be useful not only for South Africa, but also more generally. South Africa is a unique context in which to analyse the determinants of fertility given these prevailing factors that have received limited scrutiny in the economic literature.The effect of under-five mortality reductions on fertility is one of the key mechanisms emphasised in the economic growth literature explaining demographic transitions , 2. LoreSeveral studies have assessed the impact of under-five mortality on fertility and the results are mixed. Some studies find that a decrease in under-five mortality is associated with a decline in fertility , 3, 5–9.In this paper, we argue that the mixed results may be an indication that the relationship between under-five mortality and fertility is context-specific, and as such requires country-specific analysis. We also argue that the mixed results suggest the presence of omitted variables bias. This is in line with observations by Aksan and Chakraborty  and BurgStudies about the determinants of fertility in South Africa based on econometric analysis are scarce , 15. ThiSouth Africa has a well-functioning CSG system. The provision of the CSG in South Africa continues to incite debates in the public discourse about the perverse incentives for childbearing associated with the grant –19. SpecThere is also a lack of attention given to the effect of antiretroviral treatment (ART) on fertility in the economic literature when evaluating determinants of fertility. Despite epidemiological data showing the effect of ART on fertility at the individual level, no study, to the best of our knowledge, has examined the effects of ART coverage on population-level fertility in the economic literature. Several epidemiological studies support the hypothesis that an increase in the ART coverage is associated with an increase in fertility –24. AccoThe study employs the two stage least squares fixed effects instrumental variable (2SLS-FE-IV) approach to analyse balanced panel data covering nine provinces from 2001–2016. The 2SLS-FE-IV estimator is robust to various estimation problems that often affect empirical analysis, including endogeneity from omitted variables and reverse causality . As obseThis paper investigates the role of under-five mortality, child support grant uptake and ART rollout as drivers for change in fertility. In doing so, the paper tests the hypothesis that a decrease in under-five mortality leads to a decline in fertility. Furthermore, the paper tests the hypotheses that CSG coverage leads to increases in the total fertility rate and that ART rollout leads to increases in the total fertility rate. The remainder of the paper is organised as follows. First, we provide an overview on under-five mortality, total fertility rate, ART coverage and CSG coverage in section 2. This is followed by the conceptual framework on the determinants of fertility in section 3. A review of the empirical literature is provided in section 4 and this is followed by a brief exposition of the empirical model and the estimation approach in section 5. Section 6 focuses on data selection and sources of data. Thereafter, a discussion of the empirical results is provided in section 7 and finally some discussion and concluding remarks in section 8.Since 2001, South Africa has experienced a small decrease in fertility with the total fertility rate declining from 2.50 in 2001 to 2.35 in 2016 . TFR at At the end of the 2016/17 financial year, there were 3.8 million people receiving ART in the public sector in South Africa . GloballThe CSG was introduced in 1998 with a starting amount of R100 per month (US$6.71 per month). It has become the largest social grant in the country accounting for 71% of all social grants disbursed in 2016 . The CSGThe primary focus of this study is to analyse the effect of CSG coverage and ART coverage variables within Becker’s  quality-Although the conceptual framework outlined thus far allows us to understand fertility determinants at the macro level, it must be stressed that this is purely an economic viewpoint. Amarante  points oWithin this context, the level of fertility in a given population is determined by both direct and indirect factors . The dirKalemli-Ozcan  points oSeveral studies , 14, 41 With regards to marriage as a proximate determinant of fertility, Kuhnt and Trappe  suggest Both Becker and Barro  and BongThe link between cash transfers in the form of a CSG and fertility is not explained by Becker and Barro’s  quantityThere are several theoretical and empirical studies that show that reductions in under-five mortality lead to declines in fertility , 35, 47.Studies by Duflo  and HansFew studies have accounted for social security protection for children when analysing determinants of fertility. Most of these studies –54 have In South Africa, few studies have evaluated the relationship between the CSG and teenage fertility, and these find no evidence of CSG impact on teenage fertility –18. UsinContrary to these studies, a recent study by Kollamparimbil  using thAlthough these studies allowed for the effect of social security transfers when assessing determinants of fertility, they suffer from methodological weaknesses. Apart from Kollamparambil , the focMakiwane  used treAlthough Udjo  applied priori expectation is that ART rollout will lead to an increase in fertility.To the best of our knowledge, there has been no study thus far that has controlled for ART coverage when evaluating the determinants of fertility in South Africa. However, several epidemiological studies have found significant increases in fertility after starting ART –24. In aTFRit is the total fertility rate for the province i at time t, U5_MRit is the under-five mortality rate, CSGit is the CSG coverage, ARTit is the ART coverage, X′it is a vector of other control variables , μi is the time-invariant province-specific fixed effects, λt is the time-variant fixed effects, and εit is a random error term.The aim of this paper is to examine how under-five mortality, the CSG and the provision of ART individually affect fertility in South Africa, while accounting for other determinants of fertility that are well established in the literature. In this paper, other direct and indirect determinants of fertility are controlled by HIV prevalence, real GDP per capita, mean years of schooling, marriage prevalence, contraception prevalence, urban ratio, and sex ratio at birth. The selection of independent variables is informed by the frameworks proposed by Becker and Barro , Becker The main empirical challenges when evaluating the impact of under-five mortality on fertility are the reverse causality and omitted variable biases , 40. SpeTo deal with the endogeneity problem of the under-five mortality rate, this study employs the 2SLS-FE-IV approach using the xtivreg2 estimator in Stata to evaluate the impact of under-five mortality, CSG and ART coverage on fertility in South Africa. The xtivreg2 estimator implements instrumental variable /generalised method of moments (GMM) estimation of the fixed-effects and first-differences panel data models with possibly endogenous regressors . An instTFRit is the total fertility rate, lnU5_mrit is the natural log of under-five mortality rate, lnCSGit is natural log of the CSG coverage, lnARTit is the natural log of ART coverage, Educit is the education proxied by mean years of schooling, lnRGDP_pcit is the natural log of real GDP per capita, lnHIV_Previt is the natural log of HIV/AIDS prevalence, CMRit represents marriage prevalence per thousand of the population, lnContraceptiveit is the natural log of contraception prevalence, lnUrban_ratioit is the natural log of urban ratio which is a proxy for level of urbanisation and SRBit is the sex ratio at birth.Although our study adopts the 2SLS-FE-IV approach, results of the pooled ordinary least squares (pooled OLS), Random Effects (RE), and Fixed Effects (FE) models are also reported as a robustness check. Pooled OLS is the simplest estimation method used for panel data but does not recognise the panel structure of the data leading to biased estimates due to the presence of time invariant fixed effects . ContrarFollowing the approach of Kalemli-Ozcan , Aksan  and OsteIn this study, the under-five mortality is assumed to be endogenous, in line with the previous empirical studies , 9, 35. For the dependent variable, this study uses, consistent with the previous empirical literature , 35, 47,Data on under-five mortality rates, total HIV prevalence, HIV prevalence in females aged 15–49 years, MTCT of HIV, ART coverage and ART coverage in females aged 15–49 years are sourced from the Thembisa model, version 4.3 . The CSGData on the education variable, which is proxied by the average (mean) years of schooling and the income variable  are sourced from the Subnational Human Development Database . The meaOwing to data limitations on key variables prior to 2001 and after 2016, the study covers only the period of 16 years from 2001 to 2016. Since there are 9 provinces, the study has a balanced panel of 144 observations.Before investigating the empirical impact of under-five mortality, CSG and ART on fertility, it is important to analyse the pairwise correlation between explanatory variables used in the regression. This helps to check if the results of the regression are affected by the presence of high levels of multicollinearity or not . ResultsFrom From For our chosen 2SLS-FE-IV model in the last column of As mentioned earlier in this paper, the theoretical prediction of the effect of the CSG coverage on fertility is dependent on whether or not the grant is big enough to encourage family expansion. In this paper, we find no significant relationship between the CSG coverage and the TFR in South Africa over the period 2001 to 2016.ART coverage is statistically significant in all regression models in Except for the urban ratio and sex ratio at birth, coefficients of the control variables have the expected signs and are statistically significant. Consistent with the results of Burger et al. , Hansen Lower marriage prevalence rates are associated with lower fertility, consistent with the South African study by Burger et al. . The incHoechle  points oWe observe from As discussed earlier in this section, we have observed a high correlation between our key variables of interest, CSG and ART coverage. In addition, a high correlation was also observed between contraception prevalence and ART coverage. To demonstrate robustness of our estimated coefficients, we re-estimate the baseline Comparing regression results in We also demonstrate that our results are not sensitive to the inclusion of squared terms on marriage prevalence rate, contraception prevalence and sex ratio at birth variables. Statistical significance of the squared terms for the marriage prevalence and contraception prevalence variables suggests that their relationship with TFR may be non-linear. The negative sign on the squared marriage prevalence variable suggests that at low levels, marriage prevalence is associated with an increase in TFR, but for high levels, marriage prevalence is associated with a decline in TFR. The opposite is observed in the case of contraception prevalence.In Determinants of fertility are frequently contested in the public discourse. In the African setting, there is little consensus on the effects of child support grants and expanding access to HIV treatment. This paper adds to the growing research to improve our knowledge of the determinants of fertility by investigating the effects of under-five mortality rate, CSG coverage and ART coverage on fertility in South Africa over the period 2001 to 2016. This period was characterised by significant increases in the CSG and ART coverage. Furthermore, this period was characterised by significant declines in the under-five mortality rate mainly due to PMTCT and ART. Despite epidemiological data showing the effect of ART on fertility at the individual level, no study has examined the effects of ART coverage on population-level fertility in the economic literature. Although there are a few studies in South Africa that have evaluated the effect of the CSG on teenage pregnancy, there are no studies that have assessed the effect on fertility over all ages.Using provincial-level data from a panel of nine South African provinces over the period 2001–2016, we find no evidence to support the hypothesis that the increase in coverage of the CSG is associated with an increase in fertility. In other words, we find no evidence that there are perverse incentives to have a child associated with the child support grant. This finding is consistent with the empirical results of Udjo , MakiwanOn the other hand, our results indicate that an increase in the coverage of ART is associated with an increase in the total fertility rate, which could explain why the fertility decline in South Africa over the period 2001 to 2016 has been relatively modest when compared to that in earlier decades. This finding suggests that studies that evaluate the determinants of fertility in countries with significant HIV epidemics should account for ART coverage in their analysis. Failure to account for ART coverage can lead to omitted variable bias. This has policy implications for South Africa. Although the scale up of ART has improved health outcomes, the rollout should also be linked with further family planning initiatives to minimise unintended pregnancies. Several studies –77 confiWe have also found that a decline in under-five mortality is associated with a decline in fertility. The finding that a decrease in under-five mortality is associated with a decline in fertility is consistent with theoretical and empirical literature , 35, 47.Education, real GDP per capita, HIV prevalence, marriage prevalence and contraception prevalence are also important determinants of fertility in South Africa. However, our analysis does not find a significant relationship between urbanisation or the sex ratio at birth, and fertility.A limitation of this study is that we did not extend the data set beyond 2016 because variables such as the under-five mortality rate and to a lesser extent the TFR were less reliably estimated in recent periods. This is mainly due to the lack of a census or community survey since 2016. Also, due to lack of data on important variables like CSG and ART coverage, the study could not cover the period prior to 2001. As such, our study starts two years before the peak in under-five mortality due to HIV/AIDS. Thus, the study does not capture the increase in under-five mortality, due to HIV/AIDS, prior to 2001. Despite these limitations, the finding that the decline in under-five mortality is associated with a decline in fertility holds over the sample period. Future studies might address the related problem of small observation numbers by using municipal or district level data when these become available. In addition, future studies should also consider investigating the long-term relationship between under-five mortality rate and fertility in South Africa.S1 Table(DOCX)Click here for additional data file.S2 TableNotes: Robust SEs and p-values are given in parentheses. *, ** and ***Denote significance at the 10%, 5% and 1% levels, respectively. MTCT rate of HIV and immunisation coverage are used as instruments for under-five mortality rate.(DOCX)Click here for additional data file.S3 TableNotes: Notes: Robust SEs and p-values are given in parentheses. *, ** and ***denote significance at the 10%, 5% and 1% levels, respectively. MTCT rate of HIV and Immunisation coverage are used as instruments for Under-five mortality rate.(DOCX)Click here for additional data file.S4 TableNotes: Robust SEs and p-values are given in parentheses. *, ** and ***Denote significance at the 10%, 5% and 1% levels, respectively. MTCT rate of HIV, immunisation coverage and access to piped water are used as instruments for under-five mortality rate.(DOCX)Click here for additional data file.S5 TableNotes: Robust SEs and p-values are given in parentheses. *, ** and ***denote significance at the 10%, 5% and 1% levels, respectively. MTCT rate of HIV and immunisation coverage are used as instruments for under-five mortality rate.(DOCX)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.S1 File(DO)Click here for additional data file.


Under optimized conditions, two oxidation peaks at potentials of 0.80 V (UA) and 1.4 V (CAF) were observed; the application of these potentials using multiple-pulse amperometry yielded concentration linear ranges of 5.0 × 10−8–2.2 × 10−5 mol L−1 (UA) and 5.0 × 10−8–1.9 × 10−5 mol L−1 (CAF) and limits of detection of 1.1 × 10−8 and 1.3 × 10−8 mol L−1 for UA and CAF, respectively. The proposed method exhibited good repeatability and stability, and no interference was detected in the electrochemical signals of UA and CAF in the presence of glucose, NaCl, KH2PO4, CaCl2, urea, Pb, Ni, and Cd. The application of the FIA-MPA method for the analysis of environmental samples resulted in recovery rates ranging between 98 and 104%. The results obtained showed that the BDD sensor exhibited a good analytical performance when applied for CAF and UA determination, especially when compared to other sensors reported in the literature.The present study reports the development and application of a flow injection analysis (FIA) system for the simultaneous determination of uric acid (UA) and caffeine (CAF) using cathodically pretreated boron-doped diamond electrode (CPT-BDD) and multiple-pulse amperometry (MPA). The electrochemical profiles of UA and CAF were analyzed via cyclic voltammetry in the potential range of 0.20–1.7 V using 0.10 mol L To maintain a healthy level of uric acid, it is highly recommended that one avoids the excess consumption of purine-based foods  [Uric acid (UA) is the final decomposition product generated from the metabolism of purine (constituent of nucleic acids). It is present in the blood and can be detected in urine. The monitoring of UA in human serum is found to be essentially important, as the amount of UA present therein can be used as a relevant parameter for the diagnosis of many diseases including heart disease, Lesch–Nyhan syndrome, gout, diabetes, arthritis, rheumatic pain, and hyperuricemia . The noralcohol) .Caffeine (CAF) is a xanthine alkaloid compound , which is a naturally occurring substance found in the leaves, seeds, and fruits of various plant species; this compound is widely applied in pharmacological formulations, including analgesics and flu medication, and acts as stimulant in the central nervous system, where it increases alertness in addition to reducing sleepiness and improving short-term memory ,5. CAF cUA and CAF can be detected simultaneously in the human body, and the presence of these analytes above the recommended threshold or in excess can cause, as mentioned above, different health problems. These analytes can be found in biological matrices through food consumption and production in our body and in environmental matrices when disposed in the domestic sewage. Several studies reported in the literature have employed a wide range of methods for the determination of uric acid and caffeine; some of the methods applied have included the following: fluorescence , colorim3 hybridization, and this is one of the reasons for their high chemical and physical stability [The FIA system has been employed in combination with multiple pulse amperometry (FIA-MPA) for the simultaneous detection of analytes with different redox potentials. These analyses were primarily conducted using the activation potential, cleaning potential and redox potential of the analytes. Usually, under this type of analysis, boron-doped diamond (BDD) electrodes are often employed; these electrodes offer numerous analytical advantages, which include the following: better repeatability; ability to conduct analysis with negligible capacitive current; ease of operation/operational simplicity; low adsorption; and wide potential window ,20. In atability ,22.In this work, we propose the use of a simple, highly sensitive and low-cost method for the simultaneous determination of uric acid (UA) and caffeine (CAF) in river water samples using a FIA-MPA system based on a cathodically pretreated BDD electrode. UA and CAF were chosen as matrices for the conduct of this study because these substances are commonly detected in the human body and are disposed of indiscriminately in the environment through domestic sewage.2PO4), potassium phosphate dibasic (K2HPO4), sodium hydroxide (NaOH), and potassium chloride (KCl) were acquired from Synth (Brazil). UA and CAF solutions were prepared in basic and acid media, respectively, using ultrapure water. A 0.10 mol L−1 concentration sulfuric acid solution was prepared from stock solution, and a 0.10 mol L−1 phosphate buffer solution was prepared using a mixture of KH2PO4 and K2HPO4. Aqueous solutions were prepared using deionized water from the Milli Q system (resistivity > 18.2 MΩ cm).Uric acid (UA) and caffeine (CAF) were purchased from Sigma Aldrich (Brazil). Potassium phosphate monobasic (KHThe working electrode was a boron-doped diamond (BDD) film (8000 ppm) on a silicon wafer obtained from the Center Suisse de Electronique et de Microtechnique SA (CSEM), Neuchatêl, Switzerland. Voltammetric and amperometric experiments were performed using potentiostat/galvanostat  controlled by the GPES 4.9 software (Eco Chemie). ® Model 20A liquid chromatograph coupled with a UV/Vis detector and C18 column . The analysis was conducted under the following conditions: mobile phase: 40% methanol and 60% water (v/v); total running time for each analyte: 6 min; and detection wavelength: 275 nm [HPLC-UV analysis was performed using Shimadzu: 275 nm ,24.−1) for the reference electrode, platinum wire auxiliary electrode, and BDD working electrode (with exposed area of 0.66 cm2), respectively. To render the electrode surface clean without impurities, the BDD electrode was washed with ultrapure water and ethanol mixture in an ultrasonic bath for 3 min. The anodic pretreatment (APT) and cathodic pretreatment (CPT) were performed using 0.50 mol L−1 H2SO4 solution and applied potentials of 0.04 A cm−2 and −0.04 A cm−2 consecutively for 30 s and 180 s, respectively. Subsequently, the BDD electrode was applied in the electrochemical cell for the amperometric and voltammetric analyses.Initially, the BDD electrode was subjected to pretreatment using a glass cell with three electrodes: Ag/AgCl/KCl  and stocked in amber flasks in a refrigerator at 0 °C. The samples were filtered, and 10 mL of the samples was enriched using two concentration levels of uric acid and caffeine. Synthetic urine samples were prepared using 0.040 mmol L−1 H2SO4 by applying 0.04 A cm−2 or −0.04 A cm−2 for 60 s or 180 s, respectively. The analytical response (electric current) of uric acid and caffeine was evaluated separately for the anodic and cathodic processes, as these analyzes were performed after optimizing the electrolyte solution, such as electrolyte and pH. On the other hand, the applied potentials for the oxidation of the analytes using the MPA were chosen through hydrodynamic voltammograms constructed from the threshold current data recorded at each applied potential pulse. Other variables involved in this method were systematically analyzed, such as flow rate, sample injection volume and pulsation time. In this sense, the calibration curves were obtained simultaneously from continuous injections of the support electrolyte containing the target analytes in the FIA-MPA system. The limit of detection (LOD) was obtained by processing experimental data of the signal-to-noise ratio in a 3-to-1 ratio. The accuracy of the analysis method proposed here was verified from several analysis repetitions (n = 15) for UA and CAF with two different concentrations. In addition, tests of the analytes in different matrices were performed to verify the efficiency of the detection method in real systems of environmental and biological complexity, such as river water and urine samples, in which such samples were enriched with several chemical compounds that are possible interferents in samples of this magnitude. Finally, river water and urine samples were added with known concentrations of UA and CAF and analyzed (n = 3) in terms of percentage recovery of the added concentration using the analytical curves constructed under optimized conditions.The BDD electrode was pre-treated anodically (APT–BDD) or cathodically (CPT–BDD) in 0.5 mol L−2 for 60 s) and a cathodically pretreated BDD electrode (−0.04 A cm−2 for 180 s); this was done in order to improve the performance of the BDD electrode in the redox reaction. The cathodic pretreatment (CPT) of the BDDE presents a predominantly hydrogen-terminated surface, while the anodic pretreatment (APT) of the BDDE presents a predominantly oxygen-terminated surface. Therefore, the detection can be strongly influenced by the pretreatment on the BDDE surface [The analysis of the electrochemical profiles of UA and CAF was performed using an anodically pretreated BDD electrode  and cathodically (CPT) pretreated BDD electrodes. Looking at −1 and step potential = 4 mV), which is typically characteristic of irreversible processes. The anodic peak current values obtained from the application of the APT-BDD and CPT-BDD electrodes for UA and CAF were 2.8 (UA) and 10.1 µA (CAF) for APT-BDD and 6.2 (UA) and 14.5 µA (CAF) for CPT-BDD. As can be seen, the CPT of the BDD electrode surface provided a significant increase in the magnitude of the oxidation peak current for both analytes, thus indicating that a predominantly hydrogen-terminated surface significantly improves the electrochemical activity of the BDD electrode for the processes of UA and CAF oxidation. In addition, the application of the BDD electrode modified with carbon materials (nanotubes and graphene) and metallic nanoparticles yielded no significant changes in the current signal. By virtue of that, the cathodically pretreated BDD electrode was selected and used for the conduct of further experiments.2SO4) solution. The results obtained from this analysis showed that the sulfuric acid solution exhibited better electrochemical response. 2SO4 solution evaluated. Based on the results obtained from the analysis, the 0.10 mol L−1 H2SO4 solution was chosen for further experiments.First, the cyclic voltammetry technique was used to evaluate the type of electrolyte and concentration that can be used for the conduct of electrochemical measurements. The electrolytes tested were as follows: phosphate buffer solution and sulfuric acid (H−1 H2SO4 as the electrolyte solution. The optimization of the FIA-MPA system was performed on the CPT-BDD electrode using 0.10 mol L−1)  C, flow r−1) D, and pu−1) E were op−5 mol L−1 of UA, 1.5 × 10−5 mol L−1 of CAF, or a mixture of 1.5 × 10−5 mol L−1 of UA and CAF. The analysis of the amperograms at the potentials of 0.80 V and 1.4 V showed that only UA was oxidized at 0.80 V (in the presence or absence of CAF), while both analytes (UA and CAF) were oxidized at the potential of 1.4 V, that is, there was a sum of the magnitude of the signal of the oxidation process of UA and CAF. However, based on the results obtained, the UA oxidation current intensities do not equally apply the two potential pulses (0.80 V and 1.4 V), therefore, in view of the simultaneous determination of UA (0.80 V) and CAF (1.4 V), the use of a correction factor (CF) to obtain the exact value of the AU current at potential 1.4 V is necessary. The correction factor (CF) was calculated by dividing the magnitude of the current obtained at 1.4 V by the magnitude of the current obtained at 0.8 V (Equation (1)). Thus, the CF value (of 1.2 ± 0.1) obtained was useful for calculating the oxidation current of UA (1.4 V).(0.80 V) × CF from the total oxidation current at the potential of 1.4 V (UA + CAF), thus obtaining the value of the oxidation current only for CAF (Equation (2)). In this way, it is possible to quantify the two analytes simultaneously without interference.To obtain the actual concentration of CAF (1.4 V) in the simultaneous determination, we subtract the value of the oxidation current UA−8 to 2.2 × 10−5 mol L−1 for UA and 5.0 × 10−8 to 1.9 × 10−5 mol L−1 for CAF, with limits of detection (LOD) of 1.1 × 10−8 and 1.3 × 10−8 mol L−1, for UA and CAF, respectively , and the ability to perform 126 analytical determinations per hour.−6 and 1.0 × 10−5 mol L−1) (n = 15), as shown in The precision of the proposed FIA-MPA method was also evaluated through the repeatability technique using two different concentrations of UA and CAF  in a concentration ratio of 1:1 . The results obtained from these tests were found to be satisfactory; no significant effects were observed on the anodic peak current (Ipa) intensities of the analytes after amperometric measurements with the exception of ascorbic acid and dopamine, which presented current values above the reference values for UA and CAF. The RSD values obtained for UA and CAF were less than 4.0%.To further prove the suitability of the proposed method for the analysis of the analytes, electrochemical tests were performed in the presence of various interfering chemical compounds B which a−7 and 8.0 × 10−6) resulted in recovery percentages ranging from 98 to 104%; clearly, this outcome points to low matrix effects. A comparative analysis of the results obtained from the application of the proposed flow-injection method and those obtained using the high-performance liquid chromatography (HPLC) reference method showed that the two methods exhibited very similar results; essentially, this shows that the proposed CPT-BDD electrode-based FIA-MPA method can be successfully applied for the simultaneous determination of UA and CAF considering that the relative error lies in the range of −2.5 to 1.2%.The FIA-MPA method coupled with the CPT-BDD electrode was employed for the determination of UA and CAF in river water and urine samples, as shown in t-test (at a confidence level of 95%). Considering that the t values calculated for UA (0.33) and CAF (1.7) were smaller than the t value (3.2), we can conclude that the results obtained using the two analytical methods did not present significant statistical difference at 95% confidence level. Hence, the here-proposed results demonstrated the precision of the FIA-MPA method and its effectiveness for the simultaneous determination of UA and CAF in river water and urine samples.The results obtained by the two methods also were statistically compared by applying the paired Student The results obtained from the cyclic voltammetry analysis conducted in the batch system showed that the cathodically pretreated BDD electrode exhibited a higher degree of efficiency compared to the anodically pretreated BDD electrode. A comparative analysis of the analytical performance of the proposed electrochemical method with other methods reported in the literature showed that the proposed method exhibited better results in terms of linear concentration range and/or limit of detection, especially when the comparison is centered on methods based on BDD electrodes. The CPT-BDD electrode-based FIA-MPA system proposed in this study exhibited good stability, satisfactory repeatability, and operational simplicity; apart from that, the technique was found to be extremely reliable and can be used for the conduct of numerous analyses per hour. The proposed method is also of low cost and has proven to be a highly efficient alternative method for the determination of UA and CAF with recovery rates close to 100% when applied in synthetic urine and river water samples. The findings of this study show that the proposed methodology is highly advantageous when compared to other analytical methods reported in the literature.


In vitro diagnostics (IVDs) for primary detection test/screening of human T-cell leukemia virus (HTLV) have recently been updated to new-generation products in Japan. In this study, the performance of these products was evaluated and discussed in terms of the usability of HTLV diagnosis in Japan.The performance of 10 HTLV IVDs for primary detection test and confirmatory/discriminatory test was evaluated. Plasma specimens that had been declared ineligible for transfusion were provided by the Japanese Red Cross Blood Center.The diagnostic specificity of the IVDs was 100% (160/160). Six sandwich assays resulted in all HTLV-1/HTLV-positive specimens being positive (46/46). On the other hand, one sandwich assay, IVD under development 2 (UD2), resulted in one HTLV-1-positive and one HTLV-positive specimen being negative . One indirect assay, HISCL HTLV-1, could not detect one HTLV-positive specimen , but the updated product, UD1, correctly detected it . Serodia HTLV-I, based on a particle agglutination assay, resulted in 44 of the 46 positive specimens, but could not detect two specimens . ESPLINE HTLV-I/II, based on an immunochromatography assay (ICA), was able to diagnose all specimens as positive .Six sandwich assays and an ICA demonstrated high diagnostic sensitivity and specificity and are recommended for use in HTLV diagnosis in conjunction with confirmatory/discriminatory test using the INNO-LIA HTLV-I/II Score.The online version contains supplementary material available at 10.1186/s12879-023-08402-w. Retroviridae, genus Deltaretrovirus. Although most HTLV-1 infections are asymptomatic, some carriers develop adult T-cell leukemia (ATL) [Human T-cell leukemia virus (HTLV) belongs to the family ia (ATL)  after a ia (ATL) , 4. Baseia (ATL) , and theia (ATL) . HTLV-1 A diagnostic test algorithm for HTLV-1 infection was established in Japan . In shorLately, HTLV in vitro diagnostics (IVDs) for primary detection tests have been updated to new-generation products based on sandwich assay. The HIV sandwich assay shows high performance because using enzymatic or chemically labeled HIV antigen instead of labeled human IgG to detect antigen–antibody complex on a carrier following bound/free separation , 11.In this study, HTLV-positive reference panel, including a low signal-to-cutoff ratio (S/CO) or cutoff index (COI), and HTLV-negative reference panel were recently prepared in Japan. The performance of HTLV diagnostics for primary detection testing was evaluated using the HTLV-positive reference panel and the HTLV-negative reference panel.The performance of 10 HTLV IVDs for the primary detection test and one for confirmatory/discriminatory test was evaluated in this study. Two IVDs under development (UD1 and UD2) were included. This list is presented in Table To prepare HTLV-positive/negative reference panels, plasma specimens that had been declared ineligible for transfusion were provided by the Japanese Red Cross Blood Center. Additional testing after dispensing was performed according to the HTLV testing algorithm in Japan  at the NA total of 160 HTLV-negative plasma specimens were tested using 10 HTLV IVDs for primary detection test/screening. All IVDs were negative for all tests , were removed from this IVD evaluation. Forty-four HTLV-1-positive and two HTLV-positive untypable specimens (No. 16 and 26), based on INNO-LIA, were tested for evaluation . HISCL HTLV-I, indirect CLEIA, could not detect one HTLV-positive specimen, and the positive agreement rate was 97.8% (45/46). However, their updated product, UD1, detected all of the specimens, and the COI was widely distributed to that of HISCL HTLV-I. The distribution of the other five sandwich assays was similar to that of UD1. The performances of the HTLV CLEIA/CLIA/ECLIA IVDs were also examined and have been highly acclaimed in previous reports –18. ThesSpecimens no.16 and 24 showed negative results using some IVDs for primary detection test/screening. No.16 showed p19 I/II ( ±), gp21 I/II ( ±), and no.24 was p19 I/II ( ±), gp21 I/II (1 +), p19 I (1 +) using INNO-LIA. HISCL HTLV-I, that decided specimen no.16 to be negative, use HTLV-1 p19 and gp46 for detection of anti-HTLV-1 antibodies. Serodia HTLV-I use purified HTLV-1 viral antigen and UD2 use HTLV-1/2 gp21 and p24. Those IVDs tested negative both no.16 and 24. These results suggested that the kind of the antigen used in these kits is not necessarily related to the contradiction with the test result of INNO-LIA.Eight of the ten HTLV IVDs for primary detection test/screening used in this study are based on chemiluminescence detection and require a specific analyzer. These IVDs have the following advantages: (1) The process after setting the specimens is performed automatically, preventing human error and enabling tests to be performed with consistent quality. (2) More specimens can be tested with fewer personnel. (3) Use of the IVDs does not require special manual skills, reducing the burden of education and training. (4) They are compatible with the automation of examination reception and result output. The sensitivity and specificity of IVDs are high, particularly in sandwich assays; thus, the performance is considered to be adequate. Because a system for transporting clinical specimens is in place in Japan, consolidating laboratory operations in large-scale medical facilities and testing laboratories facilitates more efficient processing of multiple specimens.By contrast, using these IVDs in small- and medium-sized laboratories and areas with low HTLV prevalence is difficult because they are expensive to purchase and maintain. ESPLINE HTLV-I/II is based on ICA and may be suitable for HTLV primary detection test/screening in small- and medium-sized diagnostic laboratories. Its performance was superior to that of Serodia HTLV-I, the only manual-use IVD approved in Japan. Its performance was equivalent to the new-generation CLEIA/CLIA/ECLIA tests in this study. Incorporating ESPLINE HTLV-I/II into the HTLV testing algorithm will be a subject in the future.INNO-LIA can detect HTLV-1/2 structural protein-specific antibody responses and is suitable for confirmatory/discriminatory test. In previous studies, INNO-LIA has been reported to display superior sensitivity and specificity compared to the previously used WB-based diagnostics , 19–21 aWe have included an entry of IVDs under development in this project and provided its evaluation results. We believe this type of research project contributes to the performance confirmation of approved IVDs and to improving the performance of new IVDs. We are still collecting new specimens. We will plan to continue evaluating approved HTLV IVDs periodically.CLEIA/CLIA/ECLIA IVDs allow for automatic and rapid multi-specimen results without complex manipulation. New-generation HTLV IVDs, based on sandwich assay, demonstrated HTLV testing capabilities with high sensitivity and specificity. They are recommended for use in HTLV primary detection test/screening. However, these IVDs are expensive to purchase and maintain for small- and medium-sized laboratories. The use of ICA, which showed as same high performance as these IVDs in this study may overcome this issue.Additional file 1. Results of HTLV IVDs for primary detection test using HTLV-negative reference panel.