Proof Of Principle Study Research Articles

In Situ Imaging of Subcutaneous Drug Delivery Systems Using Microspatially Offset Low-Frequency Raman Spectroscopy.

The noninvasive in situ monitoring of the status of drug retention and implant integrity of subcutaneous implants would allow optimization of therapy and avoid periods of subtherapeutic delivery kinetics. A proof-of principle study was conducted to determine the use of microspatially offset low-frequency Raman spectroscopy (micro-SOLFRS) for nonintrusive in situ analysis of subcutaneous drug delivery systems. Caffeine was used as the model drug, and it was embedded in a circular-shape Soluplus matrix via vacuum compression molding. For the exploratory analysis, prototype implants were positioned underneath skin tissue samples, and various caffeine concentrations (1-50% w/w) and micro-SOLFRS displacement settings (Δz = 0-8 mm) were tested from the pseudo three-dimensional (3D)-imaging perspective. This format allowed the optimization of real-time micro-SOLFRS analysis of implants through skin tissue that was embedded in an agarose hydrogel. Notably, this analytical approach allowed the temporal and spatial erosion of the implant and solid-state transformations of caffeine to be distinguished. The spectrometric results correlated with complementary high-performance liquid chromatography (HPLC) determination of changes in drug concentration, illustrating drug dissipation/diffusion characteristics. The discovered capability of micro-SOLFRS for in situ measurements of drugs and implants makes it attractive for biomedical diagnostics that, ultimately, could result in development of a new point-of-care technology.

Characterization of CD34+ Cells from Patients with Acute Myeloid Leukemia (AML) and Myelodysplastic Syndromes (MDS) Using a t-Distributed Stochastic Neighbor Embedding (t-SNE) Protocol.

Using multi-color flow cytometry analysis, we studied the immunophenotypical differences between leukemic cells from patients with AML/MDS and hematopoietic stem and progenitor cells (HSPCs) from patients in complete remission (CR) following their successful treatment. The panel of markers included CD34, CD38, CD45RA, CD123 as representatives for a hierarchical hematopoietic stem and progenitor cell (HSPC) classification as well as programmed death ligand 1 (PD-L1). Rather than restricting the evaluation on a 2- or 3-dimensional analysis, we applied a t-distributed stochastic neighbor embedding (t-SNE) approach to obtain deeper insight and segregation between leukemic cells and normal HPSCs. For that purpose, we created a t-SNE map, which resulted in the visualization of 27 cell clusters based on their similarity concerning the composition and intensity of antigen expression. Two of these clusters were "leukemia-related" containing a great proportion of CD34+/CD38- hematopoietic stem cells (HSCs) or CD34+ cells with a strong co-expression of CD45RA/CD123, respectively. CD34+ cells within the latter cluster were also highly positive for PD-L1 reflecting their immunosuppressive capacity. Beyond this proof of principle study, the inclusion of additional markers will be helpful to refine the differentiation between normal HSPCs and leukemic cells, particularly in the context of minimal disease detection and antigen-targeted therapeutic interventions. Furthermore, we suggest a protocol for the assignment of new cell ensembles in quantitative terms, via a numerical value, the Pearson coefficient, based on a similarity comparison of the t-SNE pattern with a reference.

Stage Shifting by Modifying the Determinants of Breast Cancer Stage at Diagnosis: A Simulation Study.

Breast cancer is the most common cancer in Canadian women; nearly 25% of women diagnosed with cancer have breast cancer. The early detection of breast cancer is a major challenge because tumours often grow without causing symptom. The diagnosis of breast cancer at an early stage (stages I and II) improves survival outcomes because treatments are more effective and better tolerated. To better inform the prevention of and screening for breast cancer, simulations using modifiable rather than non-modifiable risk factors may be helpful in shifting the stage at diagnosis downward. Breast cancer stages were simulated using the data distributions from Alberta's Tomorrow Project participants who developed breast cancer. Using multivariable partial proportional odds regression models, modifiable lifestyle factors associated with the stage of cancer at diagnosis were evaluated. The proportions or mean levels of these lifestyle factors in the simulated population were systematically changed, then multiplied by their corresponding estimated odds ratios from the real data example. The effects of these changes were evaluated singly as well as cumulatively. Increasing total dietary protein (g/day) intake was the single most important lifestyle factor in shifting the breast cancer stage downwards followed by decreasing total dietary energy intake (kcal/day). Increasing the proportion of women who spend time in the sun between 11 am and 4 pm in the summer months, who have had a mammogram, who have been pregnant or reducing the proportion who are in stressful situations had much smaller effects. The percentage of Stage I diagnoses could be increased by approximately 12% with small modifications of these lifestyle factors. Shifting the breast cancer stage at diagnosis of a population may be achieved through changes to lifestyle factors. This proof of principle study that evaluated multiple factors associated with the stage at diagnosis in a population can be expanded to other cancers as well, providing opportunities for cancer prevention programs to target specific factors and identify populations at higher risk.

Rehabilitation combined with dietary intervention improve urinary incontinence in women with obesity: A proof-of-principle study.

This study aimed to assess the impact of add-on pelvic floor exercises on a weight management rehabilitation program. This proof of principle study was conducted between July 2019 and December 2019. Ninety-three adult female inpatients with obesity and diagnosis of urinary incontinence (UI) were assessed for inclusion, and the suitable patients were randomly assigned to the experimental group and the control group. Both groups underwent a weight management rehabilitation program, while the experimental group also performed pelvic floor exercises. The primary outcome was UI severity, assessed by the 1-h pad test. Secondary outcomes were urinary symptoms, assessed by the International Consultation on Incontinence Questionnaire Short Form (ICIQ-SF), Patient Global Impression of Improvement (PGI-I), and Incontinence Quality of Life Questionnaire (I-QOL). Sixty female inpatients were randomly assigned to the experimental group [n=30; median age: 64.50 (51.25 to 70.50) years] or the control group [n=30; median age: 67.50 (58.50 to 74.75) years]. The experimental group showed a statistically significant reduction in UI severity [pad test: 2.08 (1.21 to 8.85) g vs. 0.54 (0.24 to 1.13) g, p<0.01; ICIQ-SF: 14.00 (10.25 to 17.00) vs. 8.00 (6.25 to 11.75), p<0.01; I-QOL: 56.37 (42.28 to 73.64) vs. 78.64 (64.32 to 90.68), p<0.01]. Statistically significant differences were found in the between-groups analysis [pad test: 0.54 (0.24 to 1.13) g vs. 1.08 (0.83 to 3.86) g, p<0.01; ICIQ-SF: 8.00 (6.25 to 11.75) vs. 12.00 (10.00 to 16.00), p<0.01; I-QOL: 78.64 (64.32 to 90.68) vs. 68.18 (60.00 to 84.32), p<0.01]. Including pelvic floor exercises might provide additional benefits compared to standard rehabilitation in reducing UI symptoms in obese women.

Polygenic liability for antipsychotic dosage and polypharmacy - a real-world registry and biobank study.

Genomic prediction of antipsychotic dose and polypharmacy has been difficult, mainly due to limited access to large cohorts with genetic and drug prescription data. In this proof of principle study, we investigated if genetic liability for schizophrenia is associated with high dose requirements of antipsychotics and antipsychotic polypharmacy, using real-world registry and biobank data from five independent Nordic cohorts of a total of N = 21,572 individuals with psychotic disorders (schizophrenia, bipolar disorder, and other psychosis). Within regression models, a polygenic risk score (PRS) for schizophrenia was studied in relation to standardized antipsychotic dose as well as antipsychotic polypharmacy, defined based on longitudinal prescription registry data as well as health records and self-reported data. Meta-analyses across the five cohorts showed that PRS for schizophrenia was significantly positively associated with prescribed (standardized) antipsychotic dose (beta(SE) = 0.0435(0.009), p = 0.0006) and antipsychotic polypharmacy defined as taking ≥2 antipsychotics (OR = 1.10, CI = 1.05-1.21, p = 0.0073). The direction of effect was similar in all five independent cohorts. These findings indicate that genotypes may aid clinically relevant decisions on individual patients´ antipsychotic treatment. Further, the findings illustrate how real-world data have the potential to generate results needed for future precision medicine approaches in psychiatry.

The cantilever contact-point resin bonded bridge; adhesion 2.0.

When young patients are congenitally missing anterior teeth, different treatment modalities can be used to complement the dental arch. This article proposes a new treatment modality for the replacement of anterior teeth, the cantilever contact-point resin bonded bridge(CCP-RBB). In this proof of principle study, CCP-RBB's delivered by one operator were clinically assessed. Patients who were missing maxillary incisors and had suitable intra-oral conditions for a contact-point cantilever RBB were included. Three cases are presented to describe all adhesive steps. This proof of principle clinical study is presented with up to 60 months follow-up of the cantilever contact resin bonded bridges. A total of 19 CCP-RBB's were evaluated after a mean period of 29.8 months. None of the restorations exhibited failure, carious lesions or fractures during the follow-up periods, demonstrating an absence of restoration debonding or the need for repair. The new cantilever contact-point resin bonded bridge exhibited an excellent treatment modality without failure or debonding up to 5 years. More and extended duration in vivo studies are needed to evaluate this new treatment modality. In this proof of principle the new cantilever contact-point resin bonded bridge obtained excellent results up to 5 years of clinical follow-up.

Genome-scale analysis of essential gene knockout mutants to identify an antibiotic target process.

We describe a genome-scale approach to identify the essential biological process targeted by a new antibiotic. The procedure is based on the identification of essential genes whose inactivation sensitizes a Gram-negative bacterium (Acinetobacter baylyi) to a drug and employs recently developed transposon mutant screening and single-mutant validation procedures. The approach, based on measuring the rates of loss of newly generated knockout mutants in the presence of antibiotic, provides an alternative to traditional procedures for studying essential functions using conditional expression or activity alleles. As a proof of principle study, we evaluated whether mutations enhancing sensitivity to the β-lactam antibiotic meropenem corresponded to the known essential target process of the antibiotic (septal peptidoglycan synthesis). We found that indeed mutations inactivating most genes needed for peptidoglycan synthesis and cell division strongly sensitized cells to meropenem. Additional classes of sensitizing mutations in essential genes were also identified, including those that inactivated capsule synthesis, DNA replication, or envelope stress response regulation. The essential capsule synthesis mutants appeared to enhance meropenem sensitivity by depleting a precursor needed for both capsule and peptidoglycan synthesis. The replication mutants may sensitize cells by impairing division. Nonessential gene mutations sensitizing cells to meropenem were also identified in the screen and largely corresponded to functions subordinately associated with the essential target process, such as in peptidoglycan recycling. Overall, these results help validate a new approach to identify the essential process targeted by an antibiotic and define the larger functional network determining sensitivity to it.

FRI614 Rescuing Aging-associated CD4+ T Cell Dysfunction Via Mitochondrial Transplantation

Abstract Disclosure: C.A. Headley: None. Mitochondrial dysfunction alters cellular metabolism, increases oxidative stress, and may be principal to the dysregulated signaling and function of CD4+ T lymphocytes in the elderly. In this proof of principle study, we investigated whether the transfer of functional mitochondria into CD4+ T cells that were isolated from old mice and elderly humans, could abrogate aging-associated mitochondrial dysfunction and improve the functionality of aged CD4+ T cells. Our results show that the delivery of exogenous mitochondria to aged T cells led to cellular and mitochondrial reprogramming. This was evidenced by significant mitochondrial proteome alterations that coincided with improved aerobic metabolism and decreased mitoROS in non-activated CD4+ T cells from old mice that received healthy mitochondria. Compared to non-manipulated CD4+ T cells, CD4+ T cells that received functional mitochondria (i.e. mito-transferred CD4+ T cells from old mice) showed improvements in activation-induced TCR-signaling kinetics that correlated with increased frequencies of CD4+ T cells that displayed markers of activation (CD25), increased IL-2 production, as well as enhanced proliferation ex vivo. Immunodeficient mouse models (RAG-KO and TCR-KO) showed that the adoptive transfer of mito-transferred naive CD4+ T cells, protected recipient mice from influenza A and M. tuberculosis infections and promoted effector memory T cell differentiation. Further, mito-transfer improved CD4+ T cell activation in human T cells by decreasing activation-induced senescence. These findings support mitochondria as targets of therapeutic intervention in aging. Presentation: Friday, June 16, 2023

A population-based study exploring phenotypic clusters and clinical outcomes in stroke using unsupervised machine learning approach.

Individuals developing stroke have varying clinical characteristics, demographic, and biochemical profiles. This heterogeneity in phenotypic characteristics can impact on cardiovascular disease (CVD) morbidity and mortality outcomes. This study uses a novel clustering approach to stratify individuals with incident stroke into phenotypic clusters and evaluates the differential burden of recurrent stroke and other cardiovascular outcomes. We used linked clinical data from primary care, hospitalisations, and death records in the UK. A data-driven clustering analysis (kamila algorithm) was used in 48,114 patients aged ≥ 18 years with incident stroke, from 1-Jan-1998 to 31-Dec-2017 and no prior history of serious vascular events. Cox proportional hazards regression was used to estimate hazard ratios (HRs) for subsequent adverse outcomes, for each of the generated clusters. Adverse outcomes included coronary heart disease (CHD), recurrent stroke, peripheral vascular disease (PVD), heart failure, CVD-related and all-cause mortality. Four distinct phenotypes with varying underlying clinical characteristics were identified in patients with incident stroke. Compared with cluster 1 (n = 5,201, 10.8%), the risk of composite recurrent stroke and CVD-related mortality was higher in the other 3 clusters (cluster 2 [n = 18,655, 38.8%]: hazard ratio [HR], 1.07; 95% CI, 1.02-1.12; cluster 3 [n = 10,244, 21.3%]: HR, 1.20; 95% CI, 1.14-1.26; and cluster 4 [n = 14,014, 29.1%]: HR, 1.44; 95% CI: 1.37-1.50). Similar trends in risk were observed for composite recurrent stroke and all-cause mortality outcome, and subsequent recurrent stroke outcome. However, results were not consistent for subsequent risk in CHD, PVD, heart failure, CVD-related mortality, and all-cause mortality. In this proof of principle study, we demonstrated how a heterogenous population of patients with incident stroke can be stratified into four relatively homogenous phenotypes with differential risk of recurrent and major cardiovascular outcomes. This offers an opportunity to revisit the stratification of care for patients with incident stroke to improve patient outcomes.

Targeted NGS on sequential bone marrow biopsies aids in the evaluation of cytopenias and monocytosis and documents clonal evolution—a proof of principle study

Differential diagnosis of clonal versus reactive cytopenia and monocytosis, respectively, frequently presents a diagnostic challenge. With the two recent classifications of myeloid disorders, mutational analysis has gained importance as a diagnostic tool. However, reports on its utility on trephine bone marrow biopsies (BMB) are sparse. The aim of our proof of principle study was to determine the suitability of targeted sequencing for the longitudinal evaluation of cytopenia and monocytosis and demonstration of clonal evolution on sequential BMB. Seventy-seven EDTA-decalcified BMB of 33 patients with peripheral cytopenia and/or monocytosis, including at least one follow-up biopsy/patient, were included. Initial morphological diagnoses were idiopathic cytopenia of undetermined significance (ICUS, 8 cases), MDS (without blast increase, 7 cases), MDS with increased blasts/excess blasts (MDS-IB/EB) (11 cases), and CMML (7 cases). Thirty-one genes relevant for myeloid disorders were examined using two custom AmpliSeq NGS panels. Mutations were found in the initial BMB of 5/8 cases of ICUS, thus changing the diagnosis to clonal cytopenia of unknown significance (CCUS), 5/7 MDS, 10/11 MDS-IB/EB, and 7/7 CMML. Clonal evolution was observed in 14/33 (42%) cases, mostly associated with disease progression. None of the wild-type patients acquired mutations during follow-up. NGS-based mutation profiling is a robust diagnostic tool for BMB and provides valuable additional information, especially for cases with no/minimal dysplasia, and for better risk stratification of MDS. Tracking variant allele frequency and appearance of mutations over time allows for observing clonal evolution or relapse.

Automated diagnosis of 7 canine skin tumors using machine learning on H&E-stained whole slide images.

Microscopic evaluation of hematoxylin and eosin-stained slides is still the diagnostic gold standard for a variety of diseases, including neoplasms. Nevertheless, intra- and interrater variability are well documented among pathologists. So far, computer assistance via automated image analysis has shown potential to support pathologists in improving accuracy and reproducibility of quantitative tasks. In this proof of principle study, we describe a machine-learning-based algorithm for the automated diagnosis of 7 of the most common canine skin tumors: trichoblastoma, squamous cell carcinoma, peripheral nerve sheath tumor, melanoma, histiocytoma, mast cell tumor, and plasmacytoma. We selected, digitized, and annotated 350 hematoxylin and eosin-stained slides (50 per tumor type) to create a database divided into training, n = 245 whole-slide images (WSIs), validation (n = 35 WSIs), and test sets (n = 70 WSIs). Full annotations included the 7 tumor classes and 6 normal skin structures. The data set was used to train a convolutional neural network (CNN) for the automatic segmentation of tumor and nontumor classes. Subsequently, the detected tumor regions were classified patch-wise into 1 of the 7 tumor classes. A majority of patches-approach led to a tumor classification accuracy of the network on the slide-level of 95% (133/140 WSIs), with a patch-level precision of 85%. The same 140 WSIs were provided to 6 experienced pathologists for diagnosis, who achieved a similar slide-level accuracy of 98% (137/140 correct majority votes). Our results highlight the feasibility of artificial intelligence-based methods as a support tool in diagnostic oncologic pathology with future applications in other species and tumor types.

Type IV pili facilitated natural competence in Fusobacterium nucleatum

ObjectivesMany bacterial species naturally take up DNA from their surroundings and recombine it into their chromosome through homologous gene transfer (HGT) to aid in survival and gain advantageous functions. Herein we present the first characterization of Type IV pili facilitated natural competence in Fusobacterium nucleatum, which is a Gram-negative, anaerobic bacterium that participates in a range of infections and diseases including periodontitis, preterm birth, and cancer. MethodsHere we used bioinformatics on multiple Fusobacterium species, as well as molecular genetics to characterize natural competence in strain F. nucleatum subsp. nucleatum ATCC 23726. ResultsWe bioinformatically identified components of the Type IV conjugal pilus machinery and show this is a conserved system within the Fusobacterium genus. We next validate Type IV pili in natural competence in F. nucleatum ATCC 23726 and show that gene deletions in key components of pilus deployment (pilQ) and cytoplasmic DNA import (comEC) abolish DNA uptake and chromosomal incorporation. We next show that natural competence may require native F. nucleatum DNA methylation to bypass restriction modification systems and allow subsequent genomic homologous recombination. ConclusionsIn summary, this proof of principle study provides the first characterization of natural competence in Fusobacterium nucleatum and highlights the potential to exploit this DNA import mechanism as a genetic tool to characterize virulence mechanisms of an opportunistic oral pathogen.

Prestrain in the eardrum investigated using laser-ablation perforation: A proof of principle study on the New Zealand white rabbit

While the presence of residual stress (also called prestress) in the tympanic membrane (TM) was hypothesized more than 150 years ago by von Helmholtz (1869), little experimental data exists to date. In this paper, a novel approach to study residual stress is presented. Using a pulsed laser, the New Zealand white rabbit TM is perforated at seven predefined locations. The subsequent retraction of the membrane around the holes is computed using digital image correlation (DIC). The amount of retraction is the so-called prestrain, which is caused by the release of prestress due to the perforation. By measuring the prestrain using DIC, we show that residual stress is clearly present over the entire rabbit TM surface. In total, fourteen TMs have been measured in this work. An automated approach allows tracking the holes’ deformation during the measurement process and enables a more robust analysis than was previously possible. We find similar strains (around 5%) as reported in previous work, in which slits were created manually using flattened surgical needles. However, the new approach greatly reduces measurement time, which minimizes dehydration artifacts. To investigate the effect of perforation location on the TM, the spatial decrease of the prestrain (α) around the perforation was quantified. Perforations inferior to the umbo showed the least negative α values, i.e., the most gradual decrease around the hole, and were the most consistent. Perforations on other locations showed more negative α values, i.e., steeper decrease in strain, but were less consistent across samples. We also investigated the effect of the holes’ creation sequence but did not observe a significant change in the results. Overall, the presented method allows for consistent residual stress measurements over the TM surface. The findings contribute to our fundamental knowledge of the mechanics of the rabbit TM and provide a basis for future work on human TMs.

Proof of principle study: synchrotron X-ray fluorescence microscopy for identification of previously radioactive microparticles and elemental mapping of FFPE tissues

Biobanks containing formalin-fixed, paraffin-embedded (FFPE) tissues from animals and human atomic-bomb survivors exposed to radioactive particulates remain a vital resource for understanding the molecular effects of radiation exposure. These samples are often decades old and prepared using harsh fixation processes which limit sample imaging options. Optical imaging of hematoxylin and eosin (H&E) stained tissues may be the only feasible processing option, however, H&E images provide no information about radioactive microparticles or radioactive history. Synchrotron X-ray fluorescence microscopy (XFM) is a robust, non-destructive, semi-quantitative technique for elemental mapping and identifying candidate chemical element biomarkers in FFPE tissues. Still, XFM has never been used to uncover distribution of formerly radioactive micro-particulates in FFPE canine specimens collected more than 30 years ago. In this work, we demonstrate the first use of low-, medium-, and high-resolution XFM to generate 2D elemental maps of ~ 35-year-old, canine FFPE lung and lymph node specimens stored in the Northwestern University Radiobiology Archive documenting distribution of formerly radioactive micro-particulates. Additionally, we use XFM to identify individual microparticles and detect daughter products of radioactive decay. The results of this proof-of-principle study support the use of XFM to map chemical element composition in historic FFPE specimens and conduct radioactive micro-particulate forensics.

The utilisation of digital droplet PCR to enhance the diagnosis of bladder and pancreaticobiliary tumours in cytology specimens.

Digital droplet PCR (ddPCR) is a relatively new technique used to detect molecular alterations with unprecedented precision and accuracy. It is particularly useful for detecting point mutations and copy number variation (CNV) in samples with small amounts of target DNA. This proof of principle study was conducted to see if ddPCR technology could be applied to cytology specimens to detect molecular alterations which may influence diagnostic decision making. A range of cytological specimens derived from bladder or pancreaticobiliary origin, with varying diagnoses but with an emphasis on abnormality, underwent ddPCR testing. DNA was manually extracted from Thinprep vials, cell blocks or direct fine needle aspiration smears. ddPCR was performed using two somatic point mutations TP53 R248W and TP53 R273H assays for both urine and pancreaticobiliary cytology specimens. Three CNV assays; CDKN2A, E2F3 and YWHAZ were applied to urine samples. SMAD4 and CDKN2A CNVs were applied to the pancreaticobiliary samples. 16 of 21 urine specimens showed molecular alterations using ddPCR testing. 12 of those 16 cases were associated with malignant outcomes. The pancreaticobiliary specimens showed 14 of 37 specimens with molecular alterations, all of which were associated with carcinoma. We demonstrated an increasing percentage of molecular aberrations associated with increasing severity of cytological results. Our results have shown that ddPCR can identify both mutations and CNVs in urine and pancreaticobiliary cytology derived samples. Being able to detect these molecular alterations may reduce the number of equivocal results leading to more timely and informed patient management decisions.

Evaluation of a compact composite sensor array for concentration monitoring of solutions and suspensions via multivariate analysis

Compact composite probes were identified as a priority to alleviate space constraints in miniaturized unit operations and pharmaceutical manufacturing platforms. Therefore, in this proof of principle study, a compact composite sensor array (CCSA) combining ultraviolet and near infrared features at four different wavelengths (280, 340, 600, 860 nm) in a 380 × 30 mm housing (length x diameter, 7 mm diameter at the probe head), was evaluated for its capabilities to monitor in situ concentration of solutions and suspensions via multivariate analysis using partial least squares (PLS) regression models. Four model active pharmaceutical ingredients (APIs): warfarin sodium isopropanol solvate (WS), lidocaine hydrochloride monohydrate (LID), 6-mercaptopurine monohydrate (6-MP), and acetaminophen (ACM) in their aqueous solution and suspension formulation were used for the assessment. The results demonstrate that PLS models can be applied for the CCSA prototype to measure the API concentrations with similar accuracy (validation samples within the United States Pharmacopeia (USP) limits), compared to univariate CCSA models and multivariate models for an established Raman spectrometer. Specifically, the multivariate CCSA models applied to the suspensions of 6-MP and ACM demonstrate improved accuracy of 63% and 31%, respectively, compared to the univariate CCSA models [1]. On the other hand, the PLS models for the solutions WS and LID showed a reduced accuracy compared to the univariate models [1].

Abstract #1408256: In Search of New Biomarkers of Thyroid Function: The Association Between Thyroid Dysfunction and 11-beta Hydroxysteroid Dehydrogenase 2 Activity

The high prevalence of persistent hypothyroid symptoms in patients with normal thyrotropin and free thyroxine levels calls for research to identify alternative biomarkers of thyroid function. Animal studies have shown thyroid hormones regulate the renal 11-beta hydroxysteroid dehydrogenase 2 (11βHSD2), a key enzyme in glucocorticoid metabolism. This study examines the impact of hypothyroidism (Overt and subclinical) and hyperthyroidism (overt and subclinical) on 11βHSD2 activity. In this single center prospective study conducted between July 2022-Dec 2022, 24-hour urine samples were collected from 6 patients with newly diagnosed hyperthyroidism, 6 with newly diagnosed hypothyroidism, and 33 age and sex matched healthy reference subjects. A quantitative analysis of 26 steroid metabolites was done in the hydrolyzed urine samples. Given that 11bHSD2 is responsible for the conversion of active cortisol to inactive cortisone, the cortisol to cortisone (F/E) ratio was used to evaluate the enzymatic activity. Differences in total glucocorticoid production and F/E ratios were analyzed based on Z-score deviations from the controls’ reference intervals. The mean participants’ age was 50 years, and 73.3% were female. In patients with hypothyroidism, the mean total glucocorticoid production was 12316.8 ng/mL which was similar to the reference subjects (Z-score -0.03, p = 0.9, CI 95%: -4691.4 to 4666.3), and the mean F/E ratio was 0.5 which was lower than the reference group (Z-score 2.6, p<0.01, CI 95%: 0.06-0.25). In patients with hyperthyroidism, the mean total glucocorticoid production was 16933.7 ng/mL, which was non-statistically higher than the reference subjects (Z-score 1.2, p = 0.1, CI 95%: -794 to 10269), and the mean F/E ratio was 0.4 which was non-statistically higher than the reference group (Z-score 1.6, p = 0.09, CI 95%: 0.01-0.25). After adjustment analysis for total glucocorticoid production, only the differences in the F/E ratio of the hypothyroid patients remained statistically significant (p<0.01). In this proof of principle study, patients with hypothyroidism appeared to have normal total glucocorticoid production but impaired 11βHSD2 activity. Further studies are needed to confirm these findings, determine any clinical implications from cortisol excess in patients with thyroid dysfunction, and evaluate the utility of glucocorticoid metabolomics as a biomarker of peripheral thyroid function action.

Pulmonary Application of Novel Antigen-Loaded Chitosan Nano-Particles Co-Administered with the Mucosal Adjuvant C-Di-AMP Resulted in Enhanced Immune Stimulation and Dose Sparing Capacity.

The most successful medical intervention for preventing infectious diseases is still vaccination. This effective strategy has resulted in decreased mortality and extended life expectancy. However, there is still a critical need for novel vaccination strategies and vaccines. Antigen cargo delivery by nanoparticle-based carriers could promote superior protection against constantly emerging viruses and subsequent diseases. This should be sustained by the induction of vigorous cellular and humoral immunity, capable of acting both at the systemic and mucosal levels. Induction of antigen-specific responses at the portal of entry of pathogens is considered an important scientific challenge. Chitosan, which is widely regarded as a biodegradable, biocompatible and non-toxic material for functionalized nanocarriers, as well as having adjuvant activity, enables antigen administration via less-invasive mucosal routes such as sublingual or pulmonic application route. In this proof of principle study, we evaluate the efficacy of chitosan nanocarriers loaded with the model antigen Ovalbumin (OVA) co-administrated with the STING agonist bis-(3',5')-cyclic dimeric adenosine monophosphate (c-di-AMP) given by pulmonary route. Here, BALB/c mice were immunized with four doses of the formulation that stimulates enhanced antigen-specific IgG titers in sera. In addition, this vaccine formulation also promotes a strong Th1/Th17 response characterized by high secretion of IFN-γ, IL-2 and IL-17, as well as induction of CD8+ T cells. Furthermore, the novel formulation exhibited strong dose-sparing capacity, enabling a 90% reduction of the antigen concentration. Altogether, our results suggest that chitosan nanocarriers, in combination with the mucosal adjuvant c-di-AMP, are a promising technology platform for the development of innovative mucosal vaccines against respiratory pathogens (e.g., Influenza or RSV) or for therapeutic vaccines.

Potential of Molecular Culture in Early Onset Neonatal Sepsis Diagnosis: A Proof of Principle Study.

Delay in the time-to-positivity of a peripheral blood culture (PBC), the gold standard for early onset neonatal sepsis (EOS) diagnosis, has resulted in excessive use of antibiotics. In this study, we evaluate the potential of the rapid Molecular Culture (MC) assay for quick EOS diagnosis. In the first part of this study, known positive and spiked blood samples were used to assess the performance of MC. In the in vivo clinical study, the second part of this study, all infants receiving antibiotics for suspicion of EOS were included. At initial EOS suspicion, a blood sample was collected for PBC and MC. MC was able to detect bacteria present in the spiked samples even when the bacterial load was low. In the clinical study, MC was positive in one infant with clinical EOS (Enterococcus faecalis) that was not detected by PBC. Additionally, MC was positive in two infants without clinical sepsis (Streptococcus mitis and multiple species), referred to as contamination. The other 37 samples were negative both by MC and PBC. MC seems to be able to detect bacteria even when the bacterial load is low. The majority of MC and PBC results were comparable and the risk for contamination and false positive MC results seems to be limited. Since MC can generate results within 4 h following sampling compared with 36-72 h in PBC, MC may have the potential to replace conventional PBC in EOS diagnostics in order to guide clinicians on when to discontinue antibiotic therapy several hours after birth.

Abstract 229: Methylation as a surrogate for mutations to identify therapeutic targets

Abstract Background: Patients whose tumors exhibit an inflammatory "hot” phenotype tend to show initial efficacy toward immune checkpoint inhibitors but develop resistance over time, becoming “cold” tumors. Understanding the biology involved in this transition is of interest, especially for the identification of genes where a mutation may be facilitating the transition. As the occurrence of mutations are often rare, identifying these markers in an affected population of patients can be challenging. Here we describe a method using hyper- and hypomethylation, which can mimic the loss and gain of function caused by mutations, as a surrogate to identify mutations potentially involved in the transition of tumors from hot to cold. Methods: Breast cancer methylation and corresponding gene expression data were downloaded from TCGA. Samples were classified as immune hot or cold tumors, the latter being either an immunosuppressive state or immune inert state, by a previously defined gene algorithm [1]. Genes that contributed to immune state classification were compared between gene expression and hypermethylation status by creating a new logistic regression model for the latter. A candidate list of genes that were hypermethylated in cold tumors was created. As a validation step, Cancer Cell Line Encyclopedia (CCLE) expression and mutation data were downloaded from Depmap. Cell lines were classified as hot or cold by the same algorithm and were curated for damaging mutations within the derived gene list. For each candidate gene, the number of damaging mutations in cold cell lines was compared to the number of wild type “hot” cell lines (Chi-squared analysis) and significance was determined by comparing observed values to the distribution of 1000 randomly selected gene sets. Results: Of the final 1562 candidate genes, 420 genes had at least a 5% difference in hot and cold phenotypes between cell lines with and without a damaging mutation in that gene when compared to the methylation data (Z=10.24 , p&amp;lt;0.001). The candidate list of genes was enriched for transcription factors and a previously curated list of tumor suppressor genes. Conclusions: In this proof of principle study, we show that there is a correlation between methylation status in genes that classify tumor immune microenvironment in tumors and the presence of mutations in the same immune effector cell lines which can potentially be used to identify candidate genes involved in the immune status of tumors. While the role of these mutations is unknown, they are enriched for transcription factors and tumor suppressors genes.