Feasibility of continuous glucose monitoring in patients with type 1 diabetes at two district hospitals in Neno, Malawi: a randomised controlled trial.

OBJECTIVES: To assess the feasibility and change in clinical outcomes associated with continuous glucose monitoring (CGM) use among a rural population in Malawi living with type 1 diabetes. DESIGN: A 2:1 open randomised controlled feasibility trial. SETTING: Two Partners In Health-supported Ministry of Health-run first-level district hospitals in Neno, Malawi. PARTICIPANTS: 45 people living with type 1 diabetes (PLWT1D). INTERVENTIONS: Participants were randomly assigned to Dexcom G6 CGM (n=30) use or usual care (UC) (n=15) consisting of Safe-Accu glucose monitors and strips. Both arms received diabetes education. OUTCOMES: Primary outcomes included fidelity, appropriateness and severe adverse events. Secondary outcomes included change in haemoglobin A1c (HbA1c), acceptability, time in range (CGM arm only) SD of HbA1c and quality of life. RESULTS: Participants tolerated CGM well but were unable to change their own sensors which resulted in increased clinic visits in the CGM arm. Despite the hot climate, skin rashes were uncommon but cut-out tape overpatches were needed to secure the sensors in place. Participants in the CGM arm had greater numbers of dose adjustments and lifestyle change suggestions than those in the UC arm. Participants in the CGM arm wore their CGM on average 63.8% of the time. Participants in the UC arm brought logbooks to clinic 75% of the time. There were three hospitalisations all in the CGM arm, but none were related to the intervention. CONCLUSIONS: This is the first randomised controlled trial conducted on CGM in a rural region of a low-income country. CGM was feasible and appropriate among PLWT1D and providers, but inability of participants to change their own sensors is a challenge. TRIAL REGISTRATION NUMBER: PACTR202102832069874.

Economics of Emergency Department Visits by Patients With Inflammatory Bowel Disease: A Real-World Analysis.

Background: Inflammatory bowel disease (IBD) is associated with significant psychosocial, economic, and physical burden on patients. IBD care in the United States results in significant healthcare expenditure with recurring emergency department (ED) care and hospital admissions. Despite advances in therapy and improved access to specialty care, there is still room for improvement in cost-efficient care. Specialty medical homes and interdisciplinary care models have emerged as ways to improve medical care, patient outcomes, and quality of life, as well as improve the impact of healthcare costs. There is limited real-world data on cost in the United States, with many articles citing cost estimates from models. Methods: We analyzed real-world data from our tertiary care center with a focus on recurrent ED visits by IBD patients. Descriptive statistics were used for a cost analysis of multiple ED visits by IBD patients. Patients with ≥4 visits to the ED in a 6-month period were described as SuperUsers and were included in a separate analysis. The cost of hospitalization was also included. Results: Total cost associated with all ED visits from SuperUsers were $72 999.57 with an average of $6636.32 per patient. When the patients were admitted, the total cost of ED visits and hospitalizations was $721 461.52, with an average of $65 587.41 per patient. Conclusions: ED utilization by IBD patients with or without hospitalization is expensive and is typically driven by a cohort of SuperUsers. More work needs to be done to improve cost-effectiveness in IBD care, including reducing the frequency of ED visits.

Identification of arginine-vasopressin receptor 1a (Avpr1a/AVPR1A) as a novel candidate gene for chronic visceral pain sheds light on the potential role of enteric neurons in the development of visceral hypersensitivity.

Chronic abdominal pain in the absence of ongoing disease is the hallmark of disorders of gut-brain interaction (DGBIs), including irritable bowel syndrome (IBS). While the etiology of DGBIs remains poorly understood, there is evidence that both genetic and environmental factors play a role. In this study, we report the identification and validation of Avpr1a as a novel candidate gene for visceral hypersensitivity (VH), a primary peripheral mechanism underlying abdominal pain in DGBI/IBS. Comparing two C57BL/6 (BL/6) substrains (C57BL/6NTac and C57BL/6J) revealed differential susceptibility to the development of chronic VH following intrarectal zymosan (ZYM) instillation, a validated preclinical model for post-inflammatory IBS. Using whole genome sequencing, we identified a SNP differentiating the two strains in the 5' intergenic region upstream of Avpr1a, encoding the protein arginine-vasopressin receptor 1A (AVPR1A). We used behavioral, histological, and molecular approaches to identify distal colon-specific gene expression and neuronal hyperresponsiveness covarying with Avpr1a genotype and VH susceptibility. While the two BL/6 substrains did not differ across other gastrointestinal (GI) phenotypes (e.g., fecal water retention), VH-susceptible BL/6NTac mice had higher colonic Avpr1a mRNA and protein expression. These results parallel findings that patients' colonic Avpr1a mRNA expression corresponded to higher pain ratings. Moreover, neurons of the enteric nervous system were hyperresponsive to the AVPR1A agonist AVP, suggesting a role for enteric neurons in the pathology underlying VH. Taken together, these findings implicate differential regulation of Avpr1a as a novel mechanism of VH-susceptibility as well as a potential therapeutic target specific to VH. PERSPECTIVE: This article presents evidence of Avpr1a as a novel candidate gene for visceral hypersensitivity in a mouse model of irritable bowel syndrome. Avpr1a genotype and/or tissue-specific expression represents a potential biomarker for chronic abdominal pain susceptibility.

Mapping Human Immunity and the Education of Waldeyer's Ring.

The development and deployment of single-cell genomic technologies have driven a resolution revolution in our understanding of the immune system, providing unprecedented insight into the diversity of immune cells present throughout the body and their function in health and disease. Waldeyer's ring is the collective name for the lymphoid tissue aggregations of the upper aerodigestive tract, comprising the palatine, pharyngeal (adenoids), lingual, and tubal tonsils. These tonsils are the first immune sentinels encountered by ingested and inhaled antigens and are responsible for mounting the first wave of adaptive immune response. An effective mucosal immune response is critical to neutralizing infection in the upper airway and preventing systemic spread, and dysfunctional immune responses can result in ear, nose, and throat pathologies. This review uses Waldeyer's ring to demonstrate how single-cell technologies are being applied to advance our understanding of the immune system and highlight directions for future research.

Heterologous expression of the human wild-type and variant NaV 1.8 (A1073V) in rat sensory neurons.

BACKGROUND: Silent inflammatory bowel disease (IBD) is a condition in which individuals with the active disease experience minor to no pain. Voltage-gated Na+ (NaV ) channels expressed in sensory neurons play a major role in pain perception. Previously, we reported that a NaV 1.8 genetic polymorphism (A1073V, rs6795970) was more common in a cohort of silent IBD patients. The expression of this variant (1073V) in rat sympathetic neurons activated at more depolarized potentials when compared to the more common variant (1073A). In this study, we investigated whether expression of either NaV 1.8 variant in rat sensory neurons would exhibit different biophysical characteristics than previously observed in sympathetic neurons. METHODS: Endogenous NaV 1.8 channels were first silenced in DRG neurons and then either 1073A or 1073V human NaV 1.8 cDNA constructs were transfected. NaV 1.8 currents were recorded with the whole-cell patch-clamp technique. KEY RESULTS: The results indicate that 1073A and 1073V NaV 1.8 channels exhibited similar activation values. However, the slope factor (k) for activation determined for this same group of neurons decreased by 5 mV, suggesting an increase in voltage sensitivity. Comparison of inactivation parameters indicated that 1073V channels were shifted to more depolarized potentials than 1073A-expressing neurons, imparting a proexcitatory characteristic. CONCLUSIONS AND INFERENCES: These findings differ from previous observations in other expression models and underscore the challenges with heterologous expression systems. Therefore, the use of human sensory neurons derived from induced pluripotent stem cells may help address these inconsistencies and better determine the effect of the polymorphism present in IBD patients.

Service readiness for the management of non-communicable diseases in publicly financed facilities in Malawi: findings from the 2019 Harmonised Health Facility Assessment census survey.

INTRODUCTION: Non-communicable diseases (NCDs) are rising in low-income and middle-income countries, including Malawi. To inform policy-makers and planners on the preparedness of the Malawian healthcare system to respond to NCDs, we estimated NCD service readiness in publicly financed healthcare facilities in Malawi. METHODS: We analysed data from 564 facilities surveyed in the 2019 Harmonised Health Facility Assessment, including 512 primary healthcare (PHC) and 52 secondary and tertiary care (STC) facilities. To characterise service readiness, applying the law of minimum, we estimated the percentage of facilities with functional equipment and unexpired medicines required to provide NCD services. Further, we estimated permanently unavailable items to identify service readiness bottlenecks. RESULTS: Fewer than 40% of PHC facilities were ready to deliver services for each of the 14 NCDs analysed. Insulin and beclomethasone inhalers had the lowest stock levels at PHC facilities (6% and 8%, respectively). Only 17% of rural and community hospitals (RCHs) have liver and kidney diagnostics. STC facilities had varying service readiness, ranging from 27% for managing acute diabetes complications to 94% for chronic type 2 diabetes management. Only 38% of STC facilities were ready to manage chronic heart failure. Oral pain medicines were widely available at all levels of health facilities; however, only 22% of RCHs and 29% of STCs had injectable morphine or pethidine. Beclomethasone was never available at 74% of PHC and 29% of STC facilities. CONCLUSION: Publicly financed facilities in Malawi are generally unprepared to provide NCD services, especially at the PHC level. Targeted investments in PHC can substantially improve service readiness for chronic NCD conditions in local communities and enable STC to respond to acute NCD complications and more complex NCD cases.

Clinical outcomes associated with antidepressant use in inflammatory bowel disease patients and a matched control cohort.

Antidepressant medications (AMs) are frequently used in inflammatory bowel disease (IBD). Many AMs enhance serotonin (5-HT) availability, but this phenomenon may actually worsen IBD. We hypothesized that use of 5-HT-enhancing AMs would be associated with poor clinical outcomes in these disorders. We performed a retrospective cohort study using the Merative Health Marketscan® commercial claims database between 1/1/05 and 12/31/14. Participants (18-63 years) were either controls or had ≥ 2 ICD-9 diagnoses for IBD with ≥ 1 year of continuous insurance enrollment before index diagnosis and 2 years after. We identified new AM prescriptions using the medication possession ratio. Primary outcomes were corticosteroid use (IBD-only), IBD-related complication (IBD-only), IBD-related surgery (IBD-only), hospitalization, and emergency department (ED) visit(s) within 2 years of diagnosis or starting AM. We calculated adjusted hazard ratios (aHRs) in IBD AM users (for each outcome). We also performed subgroup analyses considering IBD and AM subtype. In the IBD cohort (n = 29,393, 41.4% female; 42.2%CD), 5.2% used AMs. In IBD, AM use was independently associated with corticosteroid use, ED visits, and hospitalizations, but not IBD-related complications. AM use was associated with a decreased risk of surgery. In the control cohort (n = 29,393, 41.4% female), AM use was also independently associated with ED visits and hospitalizations, and there was an increased likelihood of these two outcomes compared to the IBD cohort. In conclusion, while AM use was independently associated with an increased risk of ED visits and hospitalization in IBD, these risks were statistically more common in a matched control cohort. Additionally, AM use was associated with reduced risk of surgery in IBD, demonstrating a potential protective role in this setting.

Protocol for an evaluation of the initiation of an integrated longitudinal outpatient care model for severe chronic non-communicable diseases (PEN-Plus) at secondary care facilities (district hospitals) in 10 lower-income countries.

INTRODUCTION: The Package of Essential Noncommunicable Disease Interventions-Plus (PEN-Plus) is a strategy decentralising care for severe non-communicable diseases (NCDs) including type 1 diabetes, rheumatic heart disease and sickle cell disease, to increase access to care. In the PEN-Plus model, mid-level clinicians in intermediary facilities in low and lower middle income countries are trained to provide integrated care for conditions where services traditionally were only available at tertiary referral facilities. For the upcoming phase of activities, 18 first-level hospitals in 9 countries and 1 state in India were selected for PEN-Plus expansion and will treat a variety of severe NCDs. Over 3 years, the countries and state are expected to: (1) establish PEN-Plus clinics in one or two district hospitals, (2) support these clinics to mature into training sites in preparation for national or state-level scale-up, and (3) work with the national or state-level stakeholders to describe, measure and advocate for PEN-Plus to support development of a national operational plan for scale-up. METHODS AND ANALYSIS: Guided by Proctor outcomes for implementation research, we are conducting a mixed-method evaluation consisting of 10 components to understand outcomes in clinical implementation, training and policy development. Data will be collected through a mix of quantitative surveys, routine reporting, routine clinical data and qualitative interviews. ETHICS AND DISSEMINATION: This protocol has been considered exempt or covered by central and local institutional review boards. Findings will be disseminated throughout the project's course, including through quarterly M&E discussions, semiannual formative assessments, dashboard mapping of progress, quarterly newsletters, regular feedback loops with national stakeholders and publication in peer-reviewed journals.

A distinct human cell type expressing MHCII and RORγt with dual characteristics of dendritic cells and type 3 innate lymphoid cells.

Recent studies have characterized various mouse antigen-presenting cells (APCs) expressing the lymphoid-lineage transcription factor RORγt (Retinoid-related orphan receptor gamma t), which exhibit distinct phenotypic features and are implicated in the induction of peripheral regulatory T cells (Tregs) and immune tolerance to microbiota and self-antigens. These APCs encompass Janus cells and Thetis cell subsets, some of which express the AutoImmune REgulator (AIRE). RORγt+ MHCII+ type 3 innate lymphoid cells (ILC3) have also been implicated in the instruction of microbiota-specific Tregs. While RORγt+ APCs have been actively investigated in mice, the identity and function of these cell subsets in humans remain elusive. Herein, we identify a rare subset of RORγt+ cells with dendritic cell (DC) features through integrated single-cell RNA sequencing and single-cell ATAC sequencing. These cells, which we term RORγt+ DC-like cells (R-DC-like), exhibit DC morphology, express the MHC class II machinery, and are distinct from all previously reported DC and ILC3 subsets, but share transcriptional and epigenetic similarities with DC2 and ILC3. We have developed procedures to isolate and expand them in vitro, enabling their functional characterization. R-DC-like cells proliferate in vitro, continue to express RORγt, and differentiate into CD1c+ DC2-like cells. They stimulate the proliferation of allogeneic T cells. The identification of human R-DC-like cells with proliferative potential and plasticity toward CD1c+ DC2-like cells will prompt further investigation into their impact on immune homeostasis, inflammation, and autoimmunity.

Lifestyle Factors and Silent Inflammatory Bowel Disease.

Introduction: Hypoalgesic or silent inflammatory bowel disease (IBD) is a poorly understood condition that has been associated with poor clinical outcomes. There is evidence that lifestyle factors, including diet, exercise, and substance use can influence inflammatory activity and symptoms in IBD. It is unclear, though, whether these issues impact pain experience in IBD. We performed this study to evaluate the potential relationship between several key lifestyle factors and silent IBD. Methods: We performed a retrospective analysis using an IBD natural history registry based in a single tertiary care referral center. We compared demographic and clinical features in 2 patient cohorts defined using data from simultaneous pain surveys and ileocolonoscopy: (a) active IBD without pain (silent IBD) and (b) active IBD with pain. We also evaluated the relative incidence of characteristics related to diet, exercise, sexual activity, and substance abuse. Results: One hundred and eighty IBD patients had active disease and 69 (38.3%) exhibited silent IBD. Silent IBD patients exhibited incidences of disease type, location, and severity as pain-perceiving IBD patients. Silent IBD patients were more likely to be male and less likely to exhibit anxiety and/or depression or to use cannabis, analgesic medication, or corticosteroids. There were no significant differences in dietary, exercise-related, or sexual activities between silent and pain-perceiving IBD patients. Conclusions: Silent IBD was associated with reduced incidence of substance and analgesic medication use. No relationships were found between silent IBD and diet, exercise, or sexual activity, though specific elements of each require further dedicated study.

Realigning noncommunicable disease monitoring with health systems priorities in the Africa region.

The African region of the World Health Organization (WHO) recently adopted a strategy aimed at more comprehensive care for noncommunicable diseases (NCDs) in the region. The WHO's World Health Assembly has also newly approved several ambitious disease-specific targets that raise the expectations of chronic care and plans to revise and update the NCD-Global Action Plan. These actions provide a critically needed opportunity for reflection and course correction in the global health response to NCDs. In this paper, we highlight the status of the indicators that are currently used to monitor progress towards global goals for chronic care. We argue that weak health systems and lack of access to basic NCD medicines and technologies have prevented many countries from achieving the level of progress required by the NCD epidemic, and current targets do little to address this reality. We identify gaps in existing metrics and explore opportunities to realign the targets with the pressing priorities facing today's health systems.

Polysubstance use in inflammatory bowel disease is associated with increased risk of emergency department visits: a longitudinal study.

Background: Polysubstance use (PSU), the simultaneous use of 2 or more substances of abuse, is common in inflammatory bowel disease (IBD). Preliminary studies suggest it may be associated with poor outcomes. This prospective study evaluated the impact of PSU on disease activity and healthcare resource utilization in IBD. Methods: This study was conducted in a tertiary IBD center between October 29, 2015, and December 31, 2019. Participants were assessed over 2 time points (index and follow-up outpatient appointments) separated by a minimum of 6 months. Demographics, endoscopic disease activity, and surveys assessing symptoms, healthcare resource utilization and substance use (tobacco, alcohol, marijuana, cocaine, methamphetamine, heroin, opioid, or benzodiazepine) were abstracted. We identified PSU during the index appointment and computed descriptive statistics and contingency table analyses, and multivariate logistic regression models at follow up to evaluate outcomes. Results: 162 consecutively enrolled IBD patients were included. Seventy-five patients (46%) were polysubstance users at the index appointment. The most common cohorts were utilizing tobacco and alcohol (n=40) or tobacco and opioids (n=13). On bivariate and multivariate analyses, PSU during the index visit was positively associated with emergency department (ED) visits (odds ratio [OR] 2.51, 95% confidence interval [CI] 1.24-5.07; P=0.01) and negatively associated with extraintestinal manifestations (OR 0.37, 95%CI 0.18-0.74; P=0.005). Age, sex, disease activity, disease subtype and IBD-related symptoms were not associated with PSU. Conclusions: IBD patients exhibiting PSU had increased risk of future ED visits. This study highlights the risks of PSU and reinforces the importance of appropriate substance use screening.

Abdominal Pain in Inflammatory Bowel Disease: An Evidence-Based, Multidisciplinary Review.

Abdominal pain is one of the most common and impactful symptoms associated with inflammatory bowel disease (IBD), including both Crohn's disease and ulcerative colitis. A great deal of research has been undertaken over the past several years to improve our understanding and to optimize management of this issue. Unfortunately, there is still significant confusion about the underlying pathophysiology of abdominal pain in these conditions and the evidence underlying treatment options in this context. There is also a relative paucity of comprehensive reviews on this topic, including those that simultaneously evaluate pharmacological and nonpharmacological therapeutic options. In this review, our multidisciplinary team examines evidence for various currently available medical, surgical, and other analgesic options to manage abdominal pain in IBD.

Lifestyle Factors Associated with Abdominal Pain in Quiescent Inflammatory Bowel Disease.

BACKGROUND: Lifestyle factors, including diet, exercise, substance use, and sexual activity, have been shown to influence risk of inflammation and complications in inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC). Little is known about their potential role in abdominal pain generation in IBD. AIMS: We performed this study to evaluate for relationships between lifestyle factors and abdominal pain in quiescent IBD (QP-IBD). METHODS: We performed a retrospective analysis utilizing data from our institution's IBD Natural History Registry (January 1, 2017-December 31, 2022). Endoscopic evaluation, concurrent laboratory studies and surveys were completed by participants. Demographic and clinical data were also abstracted. RESULTS: We identified 177 consecutive patients with quiescent disease (105 females:72 males; 121 with CD:56 with UC) for participation in this study, 93 (52.5%) had QP-IBD. Compared to patients with quiescent IBD without pain (QNP-IBD, patients with QP-IBD exhibited no significant differences in IBD type, location, severity or complication rate. Patients with QP-IBD were more likely to have anxiety/depression (55.9% vs. 32.1%, p = 0.002) and to use antidepressants/anxiolytics (49.5% vs. 21.4%, p < 0.001). They were also less likely to engage in exercise at least three times per week (39.8% vs. 54.8%, p = 0.05) or participate in sexual activity at least monthly (53.8% vs. 69.1%, p = 0.04). On logistic regression analysis, antidepressant and/or anxiolytic use was independently associated with QP-IBD [2.72(1.32-5.62)], while monthly sexual activity was inversely associated [0.48(0.24-0.96)]. CONCLUSION: Lifestyle factors, including the lack of sexual activity and exercise, are significantly associated with QP-IBD. Further study is warranted to clarify the relationships between these factors and the development of abdominal pain in quiescent IBD.

Abdominal pain is associated with an increased risk of future healthcare resource utilization in inflammatory bowel disease.

BACKGROUND: Numerous factors influence healthcare resource utilization (HRU) in inflammatory bowel disease (IBD). We previously demonstrated an association between the presence of certain IBD-related symptoms and HRU. We conducted a longitudinal study to identify the clinical variables and IBD-related symptoms predictive of HRU. METHODS: This investigation utilized clinical encounters at an IBD center within a tertiary care referral center between 10/29/2015-12/31/2019. Participants were assessed over two time points (index and follow-up office visits) separated by a minimum of 6 months. Demographics, endoscopic disease severity, totals and sub-scores of surveys assessing for IBD-related symptoms, HRU, and substance use, and IBD-related medications. HRU was defined as any IBD-related emergency room visit, hospitalization, or surgery during the 6 months prior to follow-up appointment. We identified patients exhibiting HRU (at follow-up) and computed descriptive statistics and contingency table analyses of index appointment clinical data to identify predictors of HRU. Multivariable logistic regression models were fit incorporating significant demographic and clinical factors. RESULTS: 162 consecutively enrolled IBD patients (mean age 44.0 years; 99f:63 m; 115 Crohn's disease [CD], 45 ulcerative colitis [UC], 2 indeterminate colitis) were included. 121 patients (74.7%) exhibited HRU (mean age 43.6 years; 73f:48 m; 84 CD, 36 UC, 1 IC) preceding follow-up appointment. Abdominal pain (OR = 2.18, 95% CI 1.04-4.35, p = 0.04) at the index appointment was the only study variable significantly associated with HRU on bivariate analysis (Table 1). However, none of the clinical factors evaluated in this study were independently associated with HRU in our multivariable logistic regression model. CONCLUSIONS: In this longitudinal study, abdominal pain was the only clinical variable that demonstrated an association with future HRU (even when considering other symptoms and key variables such as disease activity, IBD-medications, and psychiatric comorbidities (i.e., anxious or depressed state). These findings reinforce the importance of regularly screening for and effectively treating abdominal pain in IBD.

The Impact of Cannabis Use on Clinical Outcomes in Inflammatory Bowel Disease: A Population-based Longitudinal Cohort Study.

BACKGROUND: Cannabis use is common in inflammatory bowel disease (IBD). Recent studies demonstrated that use of cannabis may relieve symptoms; however, it is still unclear how safe cannabis and its derivatives are for IBD patients. We performed this study to evaluate the impact of cannabis use on several key clinical outcomes in IBD. METHODS: We performed a retrospective study using the TriNetX Diamond Network. Cannabis use and noncannabis use subcohorts were identified for 3 patient groups: (1) IBD, (2) Crohn's disease (CD), and (3) ulcerative colitis (UC). Baseline differences between subcohorts for each group were controlled by propensity score matching. In each group, we compared relative incidence of emergency department (ED) visits, hospitalization, corticosteroid use, opioid use, IBD-related surgery, and death between cannabis users and noncannabis users. RESULTS: Inflammatory bowel disease cannabis users demonstrated an increased risk for corticosteroid use (risk ratios [R],1.095; 95% CI, 1.021-1.174; P = .011), ED visits (RR, 2.143; 95% CI, 2.034-2.257; P < .001), hospitalizations (RR, 1.925; 95% CI, 1.783-2.079; P < .001) and opioid use (RR, 1.35; 95% CI, 1.14-1.6); P < .001), but not an increased risk of IBD-related surgery or death. The CD and UC groups exhibited similar outcomes, except only CD demonstrated an increased risk for corticosteroid and opioid use. CONCLUSIONS: Cannabis use in IBD patients is associated with several poor clinical outcomes, including increased risk of corticosteroid and opioid use, ED visits and hospitalization, though not IBD-related surgery or death. It is not clear what drives these risks or whether they are directly related to IBD-associated disease activity or other factors. Further prospective studies are warranted to more carefully investigate these relationships.

Serum albumin binding knob domains engineered within a VH framework III bispecific antibody format and as chimeric peptides.

Background: Serum albumin binding is an established mechanism to extend the serum half-life of antibody fragments and peptides. The cysteine rich knob domains, isolated from bovine antibody ultralong CDRH3, are the smallest single chain antibody fragments described to date and versatile tools for protein engineering. Methods: Here, we used phage display of bovine immune material to derive knob domains against human and rodent serum albumins. These were used to engineer bispecific Fab fragments, by using the framework III loop as a site for knob domain insertion. Results: By this route, neutralisation of the canonical antigen (TNFα) was retained but extended pharmacokinetics in-vivo were achieved through albumin binding. Structural characterisation revealed correct folding of the knob domain and identified broadly common but non-cross-reactive epitopes. Additionally, we show that these albumin binding knob domains can be chemically synthesised to achieve dual IL-17A neutralisation and albumin binding in a single chemical entity. Conclusions: This study enables antibody and chemical engineering from bovine immune material, via an accessible discovery platform.

An impact of age on respiratory syncytial virus infection in air-liquid-interface culture bronchial epithelium.

Background: Elderly people are known to be vulnerable to virus infection. However, this has not been appropriately tested in in vitro studies due to a lack of appropriate virus infection models. In this report, we investigated the impact of age on respiratory syncytial virus (RSV) in pseudostratified air-liquid-interface (ALI) culture bronchial epithelium, which more closely mimic human airway epithelium morphologically and physiologically, than submerged cancer cell line cultures. Methods: RSV A2 was inoculated apically to the bronchial epithelium obtained from 8 donors with different ages (28-72 years old), and time-profiles of viral load and inflammatory cytokines were analyzed. Results: RSV A2 replicated well in ALI-culture bronchial epithelium. The viral peak day and peak viral load were similar between donors at ≤60 years old (n = 4) and > 65 years old (n = 4; elderly group), but virus clearance was impaired in the elderly group. Furthermore, area under the curve (AUC) analysis, calculated from viral load peak to the end of sample collection (from Day 3 to 10 post inoculation), revealed statistically higher live viral load (PFU assay) and viral genome copies (PCR assay) in the elderly group, and a positive correlation between viral load and age was observed. In addition, the AUCs of RANTES, LDH, and dsDNA (cell damage marker) were statistically higher in the elderly group, and the elderly group showed a trend of higher AUC of CXCL8, CXCL10 and mucin production. The gene expression of p21CDKN1A (cellular senescence marker) at baseline was also higher in the elderly group, and there was a good positive correlation between basal p21 expression and viral load or RANTES (AUC). Conclusion: Age was found to be a key factor affecting viral kinetics and biomarkers post virus infection in an ALI-culture model. Currently, novel or innovative in vitro cell models are introduced for virus research, but when virus studies are conducted, similarly to working with other clinical samples, the age balance is important to obtain more accurate results.