Effectiveness of Trained Community Lay Workers on Glycemic Control, Knowledge, and Self-Efficacy Among Agricultural Workers with Diabetes in the Texas Panhandle.

Systemic health barriers, economic challenges, and lack of follow-up care exacerbate self-management of chronic diseases like diabetes among Hispanic agricultural workers. The primary objective of this pilot project was to determine the benefit of using community coaches to decrease A1C levels and increase diabetes knowledge among agricultural workers with diagnosed diabetes in the Texas Panhandle. A longitudinal study design with two phases was used to create, deliver, and evaluate a diabetes coaching program. Phase 1 was the development of the program and community coaches training (n = 4). In Phase 2, the coaches then delivered the program over 12 weeks to thirteen clients. Phase 1: All coaches were Hispanic females, 28.3 (SD 3.8) years of age, half had at most a high school education level and the other half had a vocational certification (n = 4). Mean DKQ-24 score was 54.2% (SD = 29.7) at baseline and 75.0% (SD = 31.4) after training (t (4) = 4.6, P < 0.05). We observed a very large difference between mean baseline and exit DKQ-24 scores relative to the pooled standard deviation, resulting in an effect size estimate of 0.59 indicative of a medium to large learning effect. Phase 2: Clients were Hispanic Spanish-speaking, predominantly female (55%), 44.4 (SD 6.8) years of age with at most a high school level of education (88.9%) and occupations varied from dairy farm worker (33.3%), meat processing worker (33.3%), and other agriculture or manufacturing position (33.3%). The mean SKILLD score was 40.0% (SD = 28.7) at baseline, increasing to 72.2% (SD = 25.4) at 12 weeks upon completion of the coaching program (t (9) = 2.956, P < 0.05). We observed a very large difference between mean baseline and exit SKILLD scores relative to the pooled standard deviation, resulting in an effect size estimate of 1.13 indicative of a large learning effect. The mean A1C levels at baseline screening was 8.3% (SD = 3.0) and 7.6% (SD = 3.0) at exit screening, representing a 0.7% decrease (p = 0.4730). No statistically significant differences were observed between depression (p = 0.786) or anxiety (p = 1.000) measures at baseline compared to exit. Training and coaching programs for hard-to-reach agricultural and meat processing workers must be culturally, linguistically, and literacy appropriate for both coaches and clients. The program must be feasible and sustainable, focus on empowering community members, capitalize on technological advances and persisting new-normals from the COVID-19 pandemic as well as dismantle common systemic barriers to health and understanding lived-experiences of agricultural working populations in rural regions.

Adaptive Intervention to Prevent Respiratory Illness in Cerebral Palsy: Protocol for a Feasibility Pilot Randomized Controlled Trial.

BACKGROUND: This study will pilot-test an innovative just-in-time adaptive intervention to reduce severe respiratory illness among children with severe cerebral palsy (CP). Our intervention program, Respiratory Exacerbation-Plans for Action and Care Transitions (RE-PACT), delivers timely customized action planning and rapid clinical response when hospitalization risk is elevated. OBJECTIVE: This study aims to establish RE-PACT's feasibility, acceptability, and fidelity in up to 90 children with severe CP. An additional aim is to preliminarily estimate RE-PACT's effect size. METHODS: The study will recruit up to 90 caregivers of children with severe CP aged 0 to 17 years who are cared for by a respiratory specialist or are receiving daily respiratory treatments. Participants will be recruited from pediatric complex care programs at the University of Wisconsin-Madison (UW) and the University of California, Los Angeles (UCLA). Study participants will be randomly assigned to receive usual care through the complex care clinical program at UW or UCLA or the study intervention, RE-PACT. The intervention involves action planning, rapid clinical response to prevent and manage respiratory illness, and weekly SMS text messaging surveillance of caregiver confidence for their child to avoid hospitalization. RE-PACT will be run through 3 successively larger 6-month trial waves, allowing ongoing protocol refinement according to prespecified definitions of success for measures of feasibility, acceptability, and fidelity. The feasibility measures include recruitment and intervention time. The acceptability measures include recruitment and completion rates as well as intervention satisfaction. The fidelity measures include observed versus expected rates of intervention and data collection activities. The primary clinical outcome is a severe respiratory illness, defined as a respiratory diagnosis requiring hospitalization. The secondary clinical outcomes include hospital days and emergency department visits, systemic steroid courses, systemic antibiotic courses, and death from severe respiratory illness. RESULTS: The recruitment of the first wave began on April 27, 2022. To date, we have enrolled 30 (33%) out of 90 participants, as projected. The final wave of recruitment will end by October 31, 2023, and the final participant will complete the study by April 30, 2024. We will start analyzing the complete responses by April 30, 2024, and the publication of results is expected at the end of 2024. CONCLUSIONS: This pilot intervention, using adaptive just-in-time strategies, represents a novel approach to reducing the incidence of significant respiratory illness for children with severe CP. This protocol may be helpful to other researchers and health care providers caring for patients at high risk for acute severe illness exacerbations. TRIAL REGISTRATION: ClinicalTrials.gov NCT05292365; https://clinicaltrials.gov/study/NCT05292365. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/49705.

Barriers to and facilitators of a just-in-time adaptive intervention for respiratory illness in cerebral palsy: a qualitative study.

OBJECTIVE: To understand caregiver, healthcare professional and national expert perspectives on implementation of a just-in-time adaptive intervention, RE-PACT (Respiratory Exacerbation-Plans for Action and Care Transitions) to prevent respiratory crises in severe cerebral palsy. DESIGN: Qualitative research study. SETTING: Paediatric complex care programmes at two academic medical institutions. PARTICIPANTS: A total of n=4 focus groups were conducted with caregivers of children with severe cerebral palsy and chronic respiratory illness, n=4 with healthcare professionals, and n=1 with national experts. METHODS: Participants viewed a video summarising RE-PACT, which includes action planning, mobile health surveillance of parent confidence to avoid hospitalisation and rapid clinical response at times of low confidence. Moderated discussion elicited challenges and benefits of RE-PACT's design, and inductive thematic analysis elicited implementation barriers and facilitators. RESULTS: Of the 19 caregivers recruited, nearly half reported at least one hospitalisation for their child in the prior year. Healthcare professionals and national experts (n=26) included physicians, nurses, respiratory therapists, social workers and researchers. Four overarching themes and their barriers/facilitators emphasised the importance of design and interpersonal relationships balanced against health system infrastructure constraints. Intervention usefulness in crisis scenarios relies on designing action plans for intuitiveness and accuracy, and mobile health surveillance tools for integration into daily life. Trust, knowledge, empathy and adequate clinician capacity are essential components of clinical responder-caregiver relationships. CONCLUSIONS: RE-PACT's identified barriers are addressable. Just-in-time adaptive interventions for cerebral palsy appear well-suited to address families' need to tailor intervention content to levels of experience, preference and competing demands.

30 Jahre Nanobodies: Neues von kleinen Helfern mit großem Potenzial.

2023 marks the 30th anniversary of the discovery of single-domain antibody fragments in camelids, better known as nanobodies. This was the starting point for their tremendous success story in biomedicine. Here we highlight recent advances in the development of nanobodies for the detection of neutralizing SARS-CoV-2 antibodies, as biosensors for monitoring extracellular metabolites and as tracer molecules for non-invasive imaging of immune cells.

Antibody Binding and Angiotensin-Converting Enzyme 2 Binding Inhibition Is Significantly Reduced for Both the BA.1 and BA.2 Omicron Variants.

BACKGROUND: The rapid emergence of the Omicron variant and its large number of mutations led to its classification as a variant of concern (VOC) by the World Health Organization. Subsequently, Omicron evolved into distinct sublineages (eg, BA.1 and BA.2), which currently represent the majority of global infections. Initial studies of the neutralizing response toward BA.1 in convalescent and vaccinated individuals showed a substantial reduction. METHODS: We assessed antibody (immunoglobulin G [IgG]) binding, ACE2 (angiotensin-converting enzyme 2) binding inhibition, and IgG binding dynamics for the Omicron BA.1 and BA.2 variants compared to a panel of VOCs/variants of interest, in a large cohort (N = 352) of convalescent, vaccinated, and infected and subsequently vaccinated individuals. RESULTS: While Omicron was capable of efficiently binding to ACE2, antibodies elicited by infection or immunization showed reduced binding capacities and ACE2 binding inhibition compared to wild type. Whereas BA.1 exhibited less IgG binding compared to BA.2, BA.2 showed reduced inhibition of ACE2 binding. Among vaccinated samples, antibody binding to Omicron only improved after administration of a third dose. CONCLUSIONS: Omicron BA.1 and BA.2 can still efficiently bind to ACE2, while vaccine/infection-derived antibodies can bind to Omicron. The extent of the mutations within both variants prevents a strong inhibitory binding response. As a result, both Omicron variants are able to evade control by preexisting antibodies.

Developing Ethics and Equity Principles, Terms, and Engagement Tools to Advance Health Equity and Researcher Diversity in AI and Machine Learning: Modified Delphi Approach.

BACKGROUND: Artificial intelligence (AI) and machine learning (ML) technology design and development continues to be rapid, despite major limitations in its current form as a practice and discipline to address all sociohumanitarian issues and complexities. From these limitations emerges an imperative to strengthen AI and ML literacy in underserved communities and build a more diverse AI and ML design and development workforce engaged in health research. OBJECTIVE: AI and ML has the potential to account for and assess a variety of factors that contribute to health and disease and to improve prevention, diagnosis, and therapy. Here, we describe recent activities within the Artificial Intelligence/Machine Learning Consortium to Advance Health Equity and Researcher Diversity (AIM-AHEAD) Ethics and Equity Workgroup (EEWG) that led to the development of deliverables that will help put ethics and fairness at the forefront of AI and ML applications to build equity in biomedical research, education, and health care. METHODS: The AIM-AHEAD EEWG was created in 2021 with 3 cochairs and 51 members in year 1 and 2 cochairs and ~40 members in year 2. Members in both years included AIM-AHEAD principal investigators, coinvestigators, leadership fellows, and research fellows. The EEWG used a modified Delphi approach using polling, ranking, and other exercises to facilitate discussions around tangible steps, key terms, and definitions needed to ensure that ethics and fairness are at the forefront of AI and ML applications to build equity in biomedical research, education, and health care. RESULTS: The EEWG developed a set of ethics and equity principles, a glossary, and an interview guide. The ethics and equity principles comprise 5 core principles, each with subparts, which articulate best practices for working with stakeholders from historically and presently underrepresented communities. The glossary contains 12 terms and definitions, with particular emphasis on optimal development, refinement, and implementation of AI and ML in health equity research. To accompany the glossary, the EEWG developed a concept relationship diagram that describes the logical flow of and relationship between the definitional concepts. Lastly, the interview guide provides questions that can be used or adapted to garner stakeholder and community perspectives on the principles and glossary. CONCLUSIONS: Ongoing engagement is needed around our principles and glossary to identify and predict potential limitations in their uses in AI and ML research settings, especially for institutions with limited resources. This requires time, careful consideration, and honest discussions around what classifies an engagement incentive as meaningful to support and sustain their full engagement. By slowing down to meet historically and presently underresourced institutions and communities where they are and where they are capable of engaging and competing, there is higher potential to achieve needed diversity, ethics, and equity in AI and ML implementation in health research.

Two birds with one stone: human SIRPα nanobodies for functional modulation and in vivo imaging of myeloid cells.

Signal-regulatory protein α (SIRPα) expressed by myeloid cells is of particular interest for therapeutic strategies targeting the interaction between SIRPα and the "don't eat me" ligand CD47 and as a marker to monitor macrophage infiltration into tumor lesions. To address both approaches, we developed a set of novel human SIRPα (hSIRPα)-specific nanobodies (Nbs). We identified high-affinity Nbs targeting the hSIRPα/hCD47 interface, thereby enhancing antibody-dependent cellular phagocytosis. For non-invasive in vivo imaging, we chose S36 Nb as a non-modulating binder. By quantitative positron emission tomography in novel hSIRPα/hCD47 knock-in mice, we demonstrated the applicability of 64Cu-hSIRPα-S36 Nb to visualize tumor infiltration of myeloid cells. We envision that the hSIRPα-Nbs presented in this study have potential as versatile theranostic probes, including novel myeloid-specific checkpoint inhibitors for combinatorial treatment approaches and for in vivo stratification and monitoring of individual responses during cancer immunotherapies.

Monitoring extracellular ion and metabolite dynamics with recombinant nanobody-fused biosensors.

Ion and analyte changes in the tumor microenvironment (TME) alter the metabolic activity of cancer cells, promote tumor cell growth, and impair anti-tumor immunity. Consequently, accurate determination and visualization of extracellular changes of analytes in real time is desired. In this study, we genetically combined FRET-based biosensors with nanobodies (Nbs) to specifically visualize and monitor extracellular changes in K+, pH, and glucose on cell surfaces. We demonstrated that these Nb-fused biosensors quantitatively visualized K+ alterations on cancer and non-cancer cell lines and primary neurons. By implementing a HER2-specific Nb, we generated functional K+ and pH sensors, which specifically stained HER2-positive breast cancer cells. Based on the successful development of several Nb-fused biosensor combinations, we anticipate that this approach can be readily extended to other biosensors and will open new opportunities for the study of extracellular analytes in advanced experimental settings.

The interaction between anti-PF4 antibodies and anticoagulants in vaccine-induced thrombotic thrombocytopenia.

Life-threatening thrombotic events at unusual sites have been reported after vector-based vaccinations against severe acute respiratory syndrome coronavirus 2. This phenomenon is now termed vaccine-induced immune thrombotic thrombocytopenia (VITT). The pathophysiology of VITT is similar to that of heparin-induced thrombocytopenia (HIT) and is associated with platelet-activating antibodies (Abs) against platelet factor 4 (PF4). Therefore, current guidelines suggest nonheparin anticoagulants to treat VITT patients. In this study, we investigated the interactions of heparin, danaparoid, fondaparinux, and argatroban with VITT-Ab/PF4 complexes using an ex vivo model for thrombus formation as well as in vitro assays to analyze Ab binding and platelet activation. We found that immunoglobulin Gs (IgGs) from VITT patients induce increased adherent platelets/thrombus formation in comparison with IgGs from healthy controls. In this ex vivo flow-based model, the procoagulant activity of VITT IgGs was effectively inhibited with danaparoid and argatroban but also by heparin. Interestingly, heparin and danaparoid not only inhibited IgG binding to PF4 but were also able to effectively dissociate the preformed PF4/IgG complexes. Fondaparinux reduced the in vitro generation of procoagulant platelets and thrombus formation; however, it did not affect platelet aggregation. In contrast, argatroban showed no effect on procoagulant platelets and aggregation but significantly inhibited VITT-mediated thrombus formation. Taken together, our data indicate that negatively charged anticoagulants can disrupt VITT-Ab/PF4 interactions, which might serve as an approach to reduce Ab-mediated complications in VITT. Our results should be confirmed, however, in a clinical setting before a recommendation regarding the selection of anticoagulants in VITT patients could be made.

Is Your WebLitLegit? Finding Safe and Good Health Information on the Internet.

BACKGROUND: There exists a paucity of literature about teenager health literacy in general and teenagers are likely to turn to the internet for health information. Therefore, they need good e-health literacy to properly understand and apply the information obtained. Yet, many have limited e-health literacy, lacking the knowledge and skills to filter and distinguish reliable from unreliable health information and searches return large amounts of information, making it difficult to recognize whether information is reputable and raising concerns regarding teenagers' safety. Brief Description of Activity: We developed a toolkit in collaboration with community-based organizations serving teenagers and teenagers themselves usable with brief training to present a 1-hour, interactive workshop. We transformed current adult information for locating and appraising online health information into a teenager friendly format using relevant health topics to engage participants. IMPLEMENTATION: We met teenagers in teenager-friendly settings where they already gather to engage them and leverage the relationship fostered within those settings to bridge positive and negative social determinant influences on health literacy and e-health literacy as well as cross potential cultural, economic, political, and demographic barriers. Using the "train the trainer" method to build sustainability, we trained teenagers and group leaders to use the toolkit to run workshops with teenagers and placed the components in an easily available online format. RESULTS: After completing the workshop, teenager participants expressed a high level of confidence in using Medline Plus, locating health information online, identifying Truth versus Trash and making health decisions. Most teenagers reported they would recommend the WebLitLegit workshop to their friends and it improved their ability to find credible online health information. LESSONS LEARNED: The workshop's practical application provided participants with real-life examples for evaluating online information using the "LEGIT" acronym. The integration of this community-based program fostered relationships between the teenager participants, community organizations, and university students and faculty. All of the organizations involved benefited through exposure to health literacy concepts and knowledge of evaluation criteria, which may help expand e-health literacy in the community because the students, teenagers, and community partners are able to sustainably share the toolkit within their social network. [HLRP: Health Literacy Research and Practice. 2022;6(2):e151-e158.] Plain Language Summary: Teenagers use the internet to find health information but have difficulty deciding if the information is correct and safe. WebLitLegit workshops help teenagers find correct and safe information to make health decisions. Teenagers completing the workshop thought their ability to find correct information and make good health decisions improved. This best practice adds to the literature by addressing needed teenager e-health literacy.

A Psychometric Analysis of the Health Literate Health Care Organization-10 Item Questionnaire.

The concept of a Health Literate Healthcare Organization (HLHO) is a relatively new approach to health literacy that moves the focus from the individual patient to the overarching health care system. The HLHO-10 questionnaire was developed internationally to assess the 10 Attributes of HLHOs as described by participants of the Institute of Medicine Roundtable on Health Literacy. The purpose of this study was to establish reliability and validity of the HLHO-10 among a sample of United States hospitals. Reliability and validity were established through assessing the factor structure for the HLHO-10 and psychometric evaluation. The HLHO-10 was found to be reliable with a Cronbach's alpha of .855 and a two-factor structure was revealed through exploratory factor analysis. Additional research is needed to further validate use of the HLHO-10 in the U.S., but initial findings of this emerging tool are promising and timely as the issue of health literacy comes to the forefront of U.S. health care systems and associated regulatory agencies. [HLRP: Health Literacy Research and Practice. 2022;6(2):e137-e141.].

Feasibility Testing of Tubes@HOME: A Mobile Application to Support Family-Delivered Enteral Care.

BACKGROUND: Assistance from medical devices is common for children with medical complexity (CMC) but introduces caregiving challenges. We tested the feasibility of "Tubes@HOME," a mobile application supporting CMC family-delivered care using enteral care as a model. METHODS: Caregivers of CMC with enteral tubes participated in a 30-day feasibility study of Tubes@HOME November 2020 through January 2021. Tubes@HOME was available on mobile devices and designed to support collaborative care and tracking over time. Key features include child profile, caregiving network management, care routines, feedback loop, and action plans. Care routines delineated nutrition, medication, and procedural tasks needed for the child: frequencies, completions, and reminders. Metadata summarized feature use among users. Feasibility was evaluated with postuse questionnaires and interviews. Measures of Tubes@HOME's usability and usefulness included the NASA Task Load Index (TLX), System Usability Scale (SUS), and Acceptability and Use of Technology Questionnaire (AUTQ). RESULTS: Among n = 30 children, there were 30 primary (eg, parent) and n = 22 nonprimary caregivers using Tubes@HOME. Children had a median (IQR) 10 (5.5-13) care routines created. For care routines created, 93% were marked complete at least once during the study period, with participants engaging with routines throughout study weeks 2 to 4. Results (mean [SD]) indicated low mental workload (TLX) 30.9 (12.2), good usability (SUS) 75.4 (14.7), and above-average usefulness (AUTQ) 4.0 (0.7) associated with Tubes@HOME, respectively. Interviews contextualized usefulness and suggested improvements. CONCLUSIONS: Longitudinal use of Tubes@HOME among caregiving networks appeared feasible. Efficacy testing is needed, and outcomes could include reliability of care delivered in home and community.

A Novel PNGase Rc for Improved Protein N-Deglycosylation in Bioanalytics and Hydrogen-Deuterium Exchange Coupled With Mass Spectrometry Epitope Mapping under Challenging Conditions.

N-linked glycosylation is a ubiquitous posttranslational modification of proteins. While it plays an important role in the biological function of proteins, it often poses a major challenge for their analytical characterization. Currently available peptide N-glycanases (PNGases) are often inefficient at deglycosylating proteins due to sterically inaccessible N-glycosylation sites. This usually leads to poor sequence coverage in bottom-up analysis using liquid chromatography with tandem mass spectrometry and makes it impossible to obtain an intact mass signal in top-down MS analysis. In addition, most PNGases operate optimally only in the neutral to slightly acidic pH range and are severely compromised in the presence of reducing and denaturing substances, which limits their use for advanced bioanalysis based on hydrogen-deuterium exchange in combination with mass spectrometry (HDX-MS). Here, we present a novel peptide N-glycanase from Rudaea cellulosilytica (PNGase Rc) for which we demonstrate broad substrate specificity for N-glycan hydrolysis from multiply occupied and natively folded proteins. Our results show that PNGase Rc is functional even under challenging, HDX quenching conditions (pH 2.5, 0 °C) and in the presence of 0.4 M tris(2-carboxyethyl)phosphine, 4 M urea, and 1 M guanidinium chloride. Most importantly, we successfully applied the PNGase Rc in an HDX-MS workflow to determine the epitope of a nanobody targeting the extracellular domain of human signal-regulating protein alpha (SIRPα).

COVID-19 patient serum less potently inhibits ACE2-RBD binding for various SARS-CoV-2 RBD mutants.

As global vaccination campaigns against SARS-CoV-2 proceed, there is particular interest in the longevity of immune protection, especially with regard to increasingly infectious virus variants. Neutralizing antibodies (Nabs) targeting the receptor binding domain (RBD) of SARS-CoV-2 are promising correlates of protective immunity and have been successfully used for prevention and therapy. As SARS-CoV-2 variants of concern (VOCs) are known to affect binding to the ACE2 receptor and by extension neutralizing activity, we developed a bead-based multiplex ACE2-RBD inhibition assay (RBDCoV-ACE2) as a highly scalable, time-, cost-, and material-saving alternative to infectious live-virus neutralization tests. By mimicking the interaction between ACE2 and the RBD, this serological multiplex assay allows the simultaneous analysis of ACE2 binding inhibition to the RBDs of all SARS-CoV-2 VOCs and variants of interest (VOIs) in a single well. Following validation against a classical virus neutralization test and comparison of performance against a commercially available assay, we analyzed 266 serum samples from 168 COVID-19 patients of varying severity. ACE2 binding inhibition was reduced for ten out of eleven variants examined compared to wild-type, especially for those displaying the E484K mutation such as VOCs beta and gamma. ACE2 binding inhibition, while highly individualistic, positively correlated with IgG levels. ACE2 binding inhibition also correlated with disease severity up to WHO grade 7, after which it reduced.

A Nanobody-Based Toolset to Monitor and Modify the Mitochondrial GTPase Miro1.

The mitochondrial outer membrane (MOM)-anchored GTPase Miro1, is a central player in mitochondrial transport and homeostasis. The dysregulation of Miro1 in amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD) suggests that Miro1 may be a potential biomarker or drug target in neuronal disorders. However, the molecular functionality of Miro1 under (patho-) physiological conditions is poorly known. For a more comprehensive understanding of the molecular functions of Miro1, we have developed Miro1-specific nanobodies (Nbs) as novel research tools. We identified seven Nbs that bind either the N- or C-terminal GTPase domain of Miro1 and demonstrate their application as research tools for proteomic and imaging approaches. To visualize the dynamics of Miro1 in real time, we selected intracellularly functional Nbs, which we reformatted into chromobodies (Cbs) for time-lapse imaging of Miro1. By genetic fusion to an Fbox domain, these Nbs were further converted into Miro1-specific degrons and applied for targeted degradation of Miro1 in live cells. In summary, this study presents a collection of novel Nbs that serve as a toolkit for advanced biochemical and intracellular studies and modulations of Miro1, thereby contributing to the understanding of the functional role of Miro1 in disease-derived model systems.

Willingness of a Multiethnic Immigrant Population to Donate Biospecimens for Research Purposes.

This cross-sectional study explores the willingness to donate biospecimens for research purposes among six refugee communities in North Texas (spanning Myanmar, Central Africa, Somalia, Nepal, Arabic speaking countries, and others). Participants were asked four questions about biospecimen donation: (1) previously asked to donate, (2) ever agreed to donate, (3) willingness to donate for future research, and (4) what samples they would be willing to donate. Most participants (77%) were willing to donate biosamples for medical research; 58% were willing to donate samples. Fewer refugees from Somalia were willing to donate compared to immigrants from Myanmar, Central Africa, and Nepal (p < 0.01). Participants in the older age group (40 + years) were 3.2 times more likely to be willing for donation of biospecimens than the younger ones (OR 3.22, 95% CI 1.22, 8.55). Findings suggest refugees' willingness to participate in biospecimen donation which support intentional inclusion of multicultural populations into medical research.

Biparatopic nanobodies protect mice from lethal challenge with SARS-CoV-2 variants of concern.

The ongoing COVID-19 pandemic and the emergence of new SARS-CoV-2 variants of concern (VOCs) requires continued development of effective therapeutics. Recently, we identified high-affinity neutralizing nanobodies (Nbs) specific for the receptor-binding domain (RBD) of SARS-CoV-2. Taking advantage of detailed epitope mapping, we generate two biparatopic Nbs (bipNbs) targeting a conserved epitope outside and two different epitopes inside the RBD:ACE2 interface. Both bipNbs bind all currently circulating VOCs with high affinities and are capable to neutralize cellular infection with VOC B.1.351 (Beta) and B.1.617.2 (Delta) in vitro. To assess if the bipNbs NM1267 and NM1268 confer protection against SARS-CoV-2 infection in vivo, human ACE2 transgenic mice are treated intranasally before infection with a lethal dose of SARS-CoV-2 B.1, B.1.351 (Beta) or B.1.617.2 (Delta). Nb-treated mice show significantly reduced disease progression and increased survival rates. Histopathological analyses further reveal a drastically reduced viral load and inflammatory response in lungs. These data suggest that both bipNbs are broadly active against a variety of emerging SARS-CoV-2 VOCs and represent easily applicable drug candidates.

Single-Domain Antibodies for Targeting, Detection, and In Vivo Imaging of Human CD4+ Cells.

The advancement of new immunotherapies necessitates appropriate probes to monitor the presence and distribution of distinct immune cell populations. Considering the key role of CD4+ cells in regulating immunological processes, we generated novel single-domain antibodies [nanobodies (Nbs)] that specifically recognize human CD4. After in-depth analysis of their binding properties, recognized epitopes, and effects on T-cell proliferation, activation, and cytokine release, we selected CD4-specific Nbs that did not interfere with crucial T-cell processes in vitro and converted them into immune tracers for noninvasive molecular imaging. By optical imaging, we demonstrated the ability of a high-affinity CD4-Nb to specifically visualize CD4+ cells in vivo using a xenograft model. Furthermore, quantitative high-resolution immune positron emission tomography (immunoPET)/MR of a human CD4 knock-in mouse model showed rapid accumulation of 64Cu-radiolabeled CD4-Nb1 in CD4+ T cell-rich tissues. We propose that the CD4-Nbs presented here could serve as versatile probes for stratifying patients and monitoring individual immune responses during personalized immunotherapy in both cancer and inflammatory diseases.

Nanobodies - Little helpers unravelling intracellular signaling.

The identification of diagnostic and therapeutic targets requires a comprehensive understanding of cellular processes, for which advanced technologies in biomedical research are needed. The emergence of nanobodies (Nbs) derived from antibody fragments of camelid heavy chain-only antibodies as intracellular research tools offers new possibilities to study and modulate target antigens in living cells. Here we summarize this rapidly changing field, beginning with a brief introduction of Nbs, followed by an overview of how target-specific Nbs can be generated, and introduce the selection of intrabodies as research tools. Intrabodies, by definition, are intracellular functional Nbs that target ectopic or endogenous intracellular antigens within living cells. Such binders can be applied in various formats, e.g. as chromobodies for live cell microscopy or as biosensors to decipher complex intracellular signaling pathways. In addition, protein knockouts can be achieved by target-specific Nbs, while modulating Nbs have the potential as future therapeutics. The development of fine-tunable and switchable Nb-based systems that simultaneously provide spatial and temporal control has recently taken the application of these binders to the next level.