Collaborative study for the characterisation of the BINACLE Assay for in vitro detection of tetanus toxicity in toxoids - Part 1.

For several decades the European Pharmacopoeia monographs Tetanus vaccine (adsorbed) (0452) and Tetanus vaccine for veterinary use (0697) required that Specific toxicity and Absence of toxin and irreversibility of the toxoidof each bulk of tetanus toxoids had to be tested by an in vivo toxicity test in guinea pigs before it could be included in vaccines for human or veterinary use. In line with the 3Rs concept of replacing, reducing and refining animal experiments, an in vitro method for the detection of active tetanus neurotoxin (TeNT) has been developed at the Paul-Ehrlich-Institut (PEI, Germany). This method, the so-called BINACLE (binding and cleavage) assay, uses the receptor-binding and proteolytic properties of TeNT for the specific detection of active toxin molecules. Successful in-house validation studies as well as a small-scale transferability study had demonstrated that this method may represent a suitable alternative to the compendial in vivo toxicity test. As a follow up, an international collaborative study aimed at verifying the suitability of the BINACLE assay as a potential alternative to the guinea pig toxicity test for tetanus toxoids was organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM) under the aegis of its Biological Standardisation Programme (BSP). Within the framework of this study, coded BSP136, a feasibility phase - also referred to as Phase 1 - was run to select and qualify critical study reagents and samples and to assess the performance of the BINACLE Standard Operating Procedure developed by the project leaders. Then the international collaborative study aimed at evaluating the BINACLE, referred to as BSP136 Phase 2, was started. A total of 19 international laboratories (comprising vaccine manufacturers as well as national control laboratories) were supplied with a detailed assay protocol, critical reagents required for the assay, three samples consisting of three different bulk tetanus toxoids donated by major European vaccine manufacturers and one international standard toxoid. Each of the participants was asked to perform three independent BINACLE assays following the provided protocol. The statistical analysis of the results showed that most of the participating laboratories were able to perform the BINACLE assay according to the provided protocol. However, the results obtained by the participants varied widely, and not all the laboratories were able to achieve a sensitive detection of active TeNT. Multiple factors may have contributed to the elevated variability of the BSP136 study results. From an analysis of these factors, strategies were developed to help increase the standardisation of the BINACLE assay and obtain more consistent results in a follow-up validation study, BSP 136 Phase 3 (Part 2), for which the experimental phase took place in 2023. The present manuscript summarises the outcome of Phases 1 and 2, which constitute Part 1 of the BSP136 project.

Preparing Veterans Health Administration Psychologists to Meet the Complex Needs of Aging Veterans.

Background: There are significant workforce shortages for geriatric mental health care. The imbalance is particularly pronounced in the Veterans Health Administration (VHA) due to the large number of aging veterans receiving care. Workforce-based educational programs are needed to train existing clinicians to meet the mental health needs of aging veterans. Observations: This article describes an expansion of the Geriatric Scholars Program to train VHA psychologists to care for aging veterans. The multicomponent program includes an introductory course and opportunities to apply geriatric knowledge and skills through quality improvement initiatives. The Geriatric Scholars Program-Psychology Track evolved to incorporate ongoing specialized elective learning opportunities for scholars. A webinar series extends the educational programs to reach the entire VHA workforce. Conclusions: The Geriatric Scholars Program-Psychology Track represents a longitudinal educational approach to training VHA psychologists in clinical geropsychology. Other community-based organizations can use this model to construct and implement similar programs.

Impact of Bepanthen® and dexpanthenol on human nasal ciliary beat frequency in vitro.

BACKGROUND: Dexpanthenol-containing ointments/fluids are recommended to restore impaired nasal mucosa. To date, there are no data about the influence of dexpanthenol or formulations including dexpanthenol on ciliary beat frequency (CBF) of nasal epithelial cells. METHODS: We tested the ciliary beat frequency of human nasal epithelial cells in RPMI 1640 cell solution using in vitro high-frequency video microscopy every 60 s over a period of 15 min (min). Bepanthen® solution and dexpanthenol in two clinically relevant concentrations (1.67% and 3.33%) were added to the cells. Addition of sterile water served as control group. To get a better overview, the measurements after 1 min, 5 min and 15 min were combined. RESULTS: The CBF in the control group (n = 17) after 15 min was 7.3 ± 2.6 Hz. In comparison, the CBF after 15 min was 1.8 ± 1.0 Hz in the 3.33% Bepanthen® group (n = 17) and 3.2 ± 1.2 Hz in the 1.67% group, which was statistically significantly lower in both groups (p < 0.001). With regard to the dexpanthenol group (n = 17) a CBF of 6.0 ± 2.6 Hz with 3.33% and 6.1 ± 2.4 Hz with 1.67% dexpanthenol, was detected, which was again statistically significantly lower (p = 0.06) compared to the control group except CBF at 15 min with 1.57% (n = 17; p = 0.04). In general, the effect on CBF was less pronounced with dexpanthenol compared with Bepanthen® with a statistically significant difference between the two formulations. The results were verified by calculating an analysis of variance (ANOVA). CONCLUSIONS: Bepanthen® as an ointment, solution or inhalation is commonly used in ENT for mucosal care. Our results have shown that both substances reduce CBF in clinically relevant concentrations, although the effect was more pronounced with Bepanthen® compared to dexpanthenol solution, which could be related to additives or change of physical properties in the solution. Further research is needed to assess potential clinical relevance.

Neurovascular territories of the canine fossa: Analysis of a South African population.

BACKGROUND: The canine fossa, a depression on the surface of the maxillary bone, is important clinically due to the nexus of the neurovascular elements which occur in this region and supply the superficial and deep structures of the face. While it is known that there is much variation in the neurovascular structures of this region, little is known about sex differences. The aim of this study was to investigate and map the neurovascular branching within the region of the canine fossa of a South African population, with particular reference to any sex differences. METHODS: Sixty hemifaces (n=30 female; n=30 male) of individuals between the ages of 40 and 100years were dissected. The origin, number of branches, connections between branches, origin of connecting branches and the distribution of the neurovascular structures associated with the mid-facial and canine fossa regions were documented. The data obtained was qualitative and was statistically analysed with SPSS v26 statistical analysis software. Frequency and contingency tables, along with Chi-squared analysis and Fischer's Exact test, were used for quantitative data analysis. RESULTS: While high levels of variation in the neurovascular elements were documented, there was no statistically significant variation between the sexes. A sex variation was only observed for the terminal branches of CN VII with females displaying a lower number of buccal nerve terminal branches than males. CONCLUSION: While only one sex difference of significance was found in the highly variable neurovascular structures resident in the region of the canine fossa, the variability of the neurovascular elements is of importance to surgeons.

Infusing geriatrics in Indian Health Service general primary care clinics: Extending VA workforce development training.

As federal partners, the Veterans Health Administration (VA) and the Indian Health Service (IHS) agreed to share resources, such as education. The VA Geriatric Scholars Program, a workforce development program, provides one of its training programs on team-based primary care of elders to clinicians working in IHS and Tribal Health Programs. The practical impact of that training is described. A mixed methods approach was applied to the course's evaluation survey at five clinics in the Northwestern Plains, Southwest, Pacific Coast, and Alaska. Quantitative approaches assessed participants' self-reported intention to improve recognition and assessment of common geriatric syndromes. A qualitative approach applied to open-ended text responses revealed intensions to improve team-based care. Among the 51 respondents in our sample, we found significant improvements in self-reported ability to recognize previously unfamiliar potential risks to elders' health and safety, t(49) = 8.0233, p < .001, as well as increased comfort with conducting geriatric assessments and increased confidence in interprofessional team-based communication. Improvements to team-based care included enhanced clinical skills, organizational factors and the need to train additional employees. This evaluation demonstrates the value of sharing resources among federal partners and its value for participants in IHS and Tribal Health Programs.

Depression and anxiety in women with malignant ovarian germ cell (MOGCT) and sex cord stromal tumors (SCST): an analysis of the AGO-CORSETT database.

INTRODUCTION: The intention of this study was to evaluate the level of anxiety and depression of malignant ovarian germ cell (MOGCT) and sex cord stromal tumors (SCST) survivors and to identify possible alterable cofactors. METHODS: CORSETT was an observational, multicenter, mixed retrospective/prospective cohort study of the AGO Studygroup. Women who had been diagnosed with MOGCTs and SCSTs between 2001 and 2011 were asked to complete the Hospital Anxiety and Depression Scale (HADS) to evaluate distress. Predictors of distress (type of surgery, chemotherapy, time since diagnosis, recurrence, second tumor, pain) were investigated using multivariate linear regression analysis. RESULTS: 150 MOGCT and SCST patients with confirmed histological diagnosis completed the questionnaire median seven years after diagnosis. They had a HADS total score ≥ 13 indicating severe mental distress in 34% of cases. Patients after fertility-conserving surgery had lower probability of severe mental distress than those without fertility-conserving treatment (ß = - 3.1, p = 0.04). Pain was associated with the level of distress in uni- and multivariate analysis (coef 0.1, p < 0.01, coef. Beta 0.5). DISCUSSION: Severe mental distress was frequent in patients with MOGCT and SCST and the level of pain was associated with the level of distress. Fertility conserving therapy, however, was associated with less mental distress. Screening and treatment of pain and depression is required to improve mental well-being in survivors of MOGCT and SCST.

Enhancing specialty training in geropsychology competencies: An evaluation of a VA Geriatric Scholars Program advanced topics workshop.

There is a growing need for psychologists with specialized training in geriatric mental health competencies. The Geriatric Scholars Program for Psychologists (GSP-P) was created to address this shortage within a large integrated healthcare system. In 2019, GSP-P piloted an advanced workshop designed to enhance expertise in geriatric mental health competencies among graduates of its foundational competencies core course. The workshop included 3.5 days of expert-led seminars regarding the biopsychosocial needs of older adults with chronic medical illness and was followed by completion of an individualized learning plan. This paper describes the evaluation of the course using a mixed methods with data collected prior to the workshop, immediately post-workshop, and six months post-workshop. Results indicated enthusiasm for the workshop, significant improvements in four geropsychology domains on the Pikes Peak Geropsychology Knowledge and Skill Assessment Tool, and benefit from completion of the independent learning plans. Our findings demonstrate that continued enhancement of geropsychology competencies through advanced coursework is feasible and improves knowledge and skill, particularly when combined with individualized learning plans.

Developing the VA Geriatric Scholars Programs' Clinical Dashboards Using the PDSA Framework for Quality Improvement.

BACKGROUND: Involving clinician end users in the development process of clinical dashboards is important to ensure that user needs are adequately met prior to releasing the dashboard for use. The challenge with following this approach is that clinician end users can undergo periodic turnover, meaning, the clinicians that played a role in the initial development process may not be the same individuals that use the dashboard in future. OBJECTIVES: Here, we summarize our Plan, Do, Study, Act (PDSA)-guided clinical dashboard development process for the VA Geriatric Scholars Program (GSP) and the value of continuous, iterative development. We summarize dashboard adaptations that resulted from two PDSA cycles of improvement for the potentially inappropriate medication dashboard (PIMD), one of many Geriatric Scholars clinical dashboards. We also present the evaluative performance of the PIMD. METHODS: Evaluation of the PIMD was performed using the system usability scale (SUS) and through review of user interaction logs. Routine end users that were Geriatric Scholars and had evidence of 5 or more dashboard views were invited to complete an electronic form that contained the 10-item SUS. RESULTS: The proportion of Geriatric Scholars that utilized the PIMD increased for each iterative dashboard version that was produced as a byproduct from feedback (31.0% in 2017 to 60.2% in 2019). The overall usability of the PIMD among routine users was found to be above average (SUS score: 75.2 [95% CI 70.5-79.8]) in comparison to the recommended standard of acceptability (SUS score: 68) CONCLUSION: The solicitation of feedback during dashboard orientations led to iterative adaptations of the PIMD that broadened its intended use. The presented PDSA-guided process to clinical dashboard development for the VA GSP can serve as a valuable framework for development teams seeking to produce well-adopted and usable health information technology (IT) innovations.

Acute endometriosis-related sigmoid perforation in pregnancy- case report.

BACKGROUND: An acute abdomen is an emergency that requires accurate diagnosis and prompt treatment. In pregnancy, the process is even more challenging and sometimes the radiological findings are unclear. Moreover, endometriosis- related complications are rare, especially in previously unknown endometriosis. CASE PRESENTATION: We report on a case of acute endometriosis-related sigmoid perforation during pregnancy (34 weeks of gestation) due to a previously unknown deep intestinal infiltrating endometriosis with focal ulceration of the affected colonic mucosa. CONCLUSIONS: Despite the low relative risk, clinicians should be aware of possible endometriosis-associated complications in pregnancy with potentially life-threatening events, even in previously unknown endometriosis. Further studies should evaluate intestinal complications during pregnancy in relation to previous treatment of intestinal endometriosis (conservative vs. surgical).

Fecal amine metabolite analysis before onset of severe necrotizing enterocolitis in preterm infants: a prospective case-control study.

Infants developing necrotizing enterocolitis (NEC) have a different metabolomic profile compared to controls. The potential of specific metabolomics, i.e. amino acids and amino alcohols (AAA), as early diagnostic biomarkers for NEC is largely unexplored. In this multicenter prospective case-control study, longitudinally collected fecal samples from preterm infants (born <30 weeks of gestation) from 1-3 days before diagnosis of severe NEC (Bell's stage IIIA/IIIB), were analyzed by targeted high-performance liquid chromatography (HPLC). Control samples were collected from gestational and postnatal age-matched infants. Thirty-one NEC cases (15 NEC IIIA;16 NEC IIIB) with 1:1 matched controls were included. Preclinical samples of infants with NEC were characterized by five increased essential amino acids-isoleucine, leucine, methionine, phenylalanine and valine. Lysine and ethanolamine ratios were lower prior to NEC, compared to control samples. A multivariate model was rendered based on isoleucine, lysine, ethanolamine, tryptophan and ornithine, modestly discriminating cases from controls (AUC 0.67; p < 0.001). Targeted HPLC pointed to several specific AAA alterations in samples collected 1-3 days before NEC onset, compared to controls. Whether this reflects metabolic alterations and has a role in early biomarker development for NEC, has yet to be elucidated.

Experiences of Sleep Problems Among Older Korean Immigrants.

Despite poor sleep among older adults, little is known about the sleep habits of older immigrants living in the United States. The current pragmatic qualitative descriptive study explored sleep among older Korean immigrants, using a focus group with six participants and individual phone interviews with 22 Korean immigrants aged ≥60 years. Transcripts were coded to identify underlying themes. Several thematic categories were identified under six domains: daytime function, getting ready for bed, falling asleep, awakenings during sleep, going back to sleep, and seeking advice from peers. Unhealthy sleep behaviors were found during daytime and bedtime, particularly among those who were retired/unemployed or living alone. Seeking advice from peers was common but none of the advice helped participants sleep. Sleep education programs in Korean-speaking communities can be used to target those who are socially isolated and may benefit older Korean immigrants with sleep difficulties. [Research in Gerontological Nursing, 15(4), 193-202.].

[Reconstruction after facial trauma with dento-alveolar injury]. / Reconstructie na aangezichtstrauma met dentoalveolair letsel.

While abroad, a healthy 36-year-old woman slammed head-on into a rock wall at high speed, resulting in significant facial trauma. The initial trauma care and first aid took place abroad. In the Netherlands, the woman was referred to the Department of Oral and Maxillofacial Surgery (OMFS) for reconstruction of her face and alveolar processes, gingiva and dentition. In view of the seriousness of the injuries, a 3D treatment plan was drawn up in a multidisciplinary collaboration with an OMF surgeon, an implantologist, dentist and dental technician. By making a digital setup of both the top and bottom front in advance, it was possible to work predictably. The first step consisted of bone augmentation by means of an iliac crest graft to reconstruct the major bone defects of the superior and inferior alveolar processes. Implants were then placed in the upper and lower jaws in the ideal position for the suprastructure by means of drill jigs. Within 10 months after the trauma, the implant bridges could be placed on individual zirconia frameworks to optimally restore oral function and aesthetics, completing the reconstruction.

NT-proBNP and stem cell factor plasma concentrations are independently associated with cardiovascular outcomes in end-stage renal disease hemodialysis patients.

Aims: End-stage renal disease (ESRD) treated by chronic hemodialysis (HD) is associated with poor cardiovascular (CV) outcomes, with no available evidence-based therapeutics. A multiplexed proteomic approach may identify new pathophysiological pathways associated with CV outcomes, potentially actionable for precision medicine. Methods and results: The AURORA trial was an international, multicentre, randomized, double-blind trial involving 2776 patients undergoing maintenance HD. Rosuvastatin vs. placebo had no significant effect on the composite primary endpoint of death from CV causes, nonfatal myocardial infarction or nonfatal stroke. We first compared CV risk-matched cases and controls (n = 410) to identify novel biomarkers using a multiplex proximity extension immunoassay (276 proteomic biomarkers assessed with OlinkTM). We replicated our findings in 200 unmatched cases and 200 controls. External validation was conducted from a multicentre real-life Danish cohort [Aarhus-Aalborg (AA), n = 331 patients] in which 92 OlinkTM biomarkers were assessed. In AURORA, only N-terminal pro-brain natriuretic peptide (NT-proBNP, positive association) and stem cell factor (SCF) (negative association) were found consistently associated with the trial's primary outcome across exploration and replication phases, independently from the baseline characteristics. Stem cell factor displayed a lower added predictive ability compared with NT-ProBNP. In the AA cohort, in multivariable analyses, BNP was found significantly associated with major CV events, while higher SCF was associated with less frequent CV deaths. Conclusions: Our findings suggest that NT-proBNP and SCF may help identify ESRD patients with respectively high and low CV risk, beyond classical clinical predictors and also point at novel pathways for prevention and treatment.

Associations of Self-Reported Sleep Quality with Demographic and Other Characteristics in Older Korean Immigrants.

Poor sleep is common among older adults, affecting a wide range of health outcomes. However, little is known about sleep issues among older Korean immigrants, the fastest growing Asian American subgroup in the United States. We aimed to explore multiple factors associated with sleep among this group. We analyzed cross-sectional survey data from 43 older immigrants living in two large Korean communities in Southern California. Perceived sleep quality was significantly associated with gender, living arrangement, employment status, mental health, and sleep-related beliefs (all p-values < 0.05). Living with someone and being employed for wages were significantly uniquely associated with better sleep quality, accounting for demographic and health-related factors (R2 = 51.8%, adjusted R2 = 38.7%, p = 0.002). These findings suggest a potential role of sociocultural factors on sleep. Further studies are needed to confirm these findings and to inform a sleep intervention program tailored to the characteristics of older Korean immigrants.

Experiences of workplace bullying among academics in a health sciences faculty at a South African university.

BACKGROUND: Workplace bullying and other negative workplace behaviours are problems that need to be addressed across many work settings, including at universities. OBJECTIVES: To examine the prevalence of bullying among academics, and factors associated with bullying, in a faculty of health sciences (FHS) of a South African university. METHODS: All academic staff, except senior managers, were invited to participate by completing a self-administered, web-based questionnaire hosted on REDCap. In adition to sociodemographic information, the survey collected information on bullying, and the factors associated with experiences of workplace bullying. Survey data were exported to Stata 13 for analysis. The data were weighted to take account of the distribution of staff in the FHS. Chi-square tests and a multiple logistic regression model for bullying were utilised. RESULTS: The majority of study participants were white (52%), female (70%) and South African (85%). Bullying in the workplace was experienced by 58% of respondents, of whom 44% experienced bullying more than once, and 64% of participants had witnessed bullying. Being female (adjusted odds ratio (aOR) 1.83; 95% confidence interval (CI) 1.14 - 2.93; p<0.05) and being jointly appointed as both a clinician in a health facility and an academic in the university (aOR 1.73; 95% CI 1.29 - 2.32; p<0.001) increased the odds of experiencing workplace bullying. CONCLUSIONS: A combination of strategies is needed, including clear FHS policies to prevent bullying, training in bullying prevention and critical diversity, and positive practice environments.

Improving Geropsychology Competencies of Veterans Affairs Psychologists.

Older adults are more likely to seek mental health care through integrated care settings such as primary care. Currently, there exists a significant shortage of mental health providers trained in geropsychology and integrated care competencies. To address this need within the Veterans Health Administration, a national workforce development program was extended to include psychologists, which is called the Geriatric Scholars Program-Psychology Track (GSP-P). The GSP-P has two overarching educational program aims: (1) to improve geropsychology competencies of practicing VA psychologists, particularly those working within integrated settings (e.g., primary care) and (2) enrich psychologists' abilities to enact change in their clinical settings. Ninety-eight VA clinicians participated in the GSP-P, which includes a multi-day in-person course, from 2014 to 2018. Participants completed measures assessing confidence and self-reported knowledge in geropsychology and integrated care competencies pre-course and 3-months post-completion. Two-weeks post-course participants responded to open-ended survey questions regarding their perceptions of the course and potential applications of learning. Significant improvements in confidence in and knowledge of geropsychology and integrated care competencies emerged from pre-course to 3-months post-completion. Qualitative findings demonstrated that participants valued the face-to-face, integrated multimodal educational program. Findings provided insights regarding clinicians' planned application of the knowledge acquired, such as modifying treatments for older patients. Specialized workforce programs such as the GSP-P have a significant, positive impact on the care of older Veterans.

TLR agonists enhance responsiveness of inflammatory innate immune cells in HLA-B*57-positive HIV patients.

HLA-B*57 affects the course of HIV infection. Under antiretroviral therapy, its effects cannot be explained by outstandingly efficient T cell responses alone but may also involve cells of innate immunity. Studying in vitro stimulation with Pam3CSK4, E. coli LPS-B5 and CpG-ODN-2216, we observed greater induction of IL-6/IL-1beta double-positive CD14+CD16++ monocytes as well as IFN-gamma-positive cytotoxic CD56highCD16neg NK cells in HLA-B*57- versus HLA-B*44-positive HIV patients, while TNF-alpha induction remained unchanged. Differences were not seen in the other monocyte and NK cell subsets or in HLA-matched healthy controls. Our findings show that, in virally suppressed HIV infection, HLA-B*57 is associated with enhanced responsiveness of inflammatory innate immune cells to TLR ligands, possibly contributing to increased vulnerability in sepsis. KEY MESSAGES: • HLA-B*57 is a host factor affecting clinical outcomes of HIV infection. • HLA-B*57 modifies inflammatory subsets of NK cells and monocytes in HIV infection. • In HLA-B*57-positive HIV patients TLR agonists induce enhanced IL-6/IL-1beta in monocytes. • NK cells from HLA-B*57 HIV patients release more IFN-gamma upon TLR costimulation. • HLA-B*57 is linked to enhanced inflammatory responsiveness to TLR ligands.

Probe-hosted large area silicon photomultiplier and high-throughput timing electronics for enhanced performance time-domain functional near-infrared spectroscopy.

Two main bottlenecks prevent time-domain diffuse optics instruments to reach their maximum performances, namely the limited light harvesting capability of the detection chain and the bounded data throughput of the timing electronics. In this work, for the first time to our knowledge, we overcome both those limitations using a probe-hosted large area silicon photomultiplier detector coupled to high-throughput timing electronics. The system performances were assessed based on international protocols for diffuse optical imagers showing better figures with respect to a state-of-the-art device. As a first step towards applications, proof-of-principle in-vivo brain activation measurements demonstrated superior signal-to-noise ratio as compared to current technologies.

[Stimulatory and inhibitory signaling pathways of the T cell-APC interaction and the effect of TLR agonists on APCs]. / Stimulierende und inhibierende Signalwege der APZ- und T-Zell-Interaktion sowie Einfluss von TLR-Agonisten auf APZ.

BACKGROUND: CD8+ cells are key players in the identification and elimination of cancer cells. Cancers can escape an effective T cell response by inducing an exhausted cell state, which limits the cytotoxic capacity of the effector cells. Among other mechanisms, new checkpoint inhibitors reactivate exhausted, dysfunctional T cells. CD8+ T cells can eliminate tumor cells after presentation of tumor-specific antigens via antigen-presenting cells (APCs). APC-mediated tumor recognition is mainly stimulated by Toll-like receptors (TLRs). OBJECTIVE: This study investigates the effect of TLR agonists on APCs as well as stimulatory and inhibitory signaling pathways of the T cell-APC interaction. MATERIALS AND METHODS: Gene expression of interleukin (IL)12 and programmed death ligand 1 (PD-L1) was analyzed by quantitative polymerase chain reaction (qPCR) after 0, 8, 24, and 48 h of CD14+ cell stimulation with CpG. Protein expression of inhibitor of nuclear factor kappa B (IκBα) after CpG stimulation was investigated by western blot. CD8+ T cells were stimulated for 72 h with or without programmed cell death protein 1 (PD-1) checkpoint blockade and analyzed for expression of PD­1, Tim­3, CTLA4, and Lag3 by flow cytometry. RESULTS: TLR stimulation (by unmethylated CpG DNA) of APCs upregulates immunostimulatory signals such as IL12 expression but also activates immunoinhibitory signaling pathways such as PD-L1 expression. This signaling is NF-κB dependent. After blockade of the PD-1/PD-L1 signaling pathway, overexpression of other immune checkpoint inhibitory receptors was observed-a potential explanation for lacking therapeutic responses after TLR stimulation with PD­1 checkpoint blockade. CONCLUSION: TLR stimulation causes APCs in the tumor microenvironment to upregulate PD-L1 in an NF-κB-mediated fashion, thereby contributing to CD8+ T cell exhaustion. The effect of PD­1 blockade after TLR stimulation might be impaired due to upregulation of other checkpoint inhibitors.