Alternative Strategies to Provide Actionable Results When a Supply of Urinalysis Strips Is Unavailable.

CONTEXT.­: Urinalysis instrument-specific dip strips offer physicians qualitative results for actionable analytes (protein, glucose, leukocyte esterase, nitrates, hemoglobin, and ketones). OBJECTIVE.­: To explain a strategy implemented to support clinical decision-making by providing urine quantification of protein, glucose, white blood cells (WBCs), and red blood cells because of urine strip shortages. DESIGN.­: During shortages, we implemented an automated algorithm that triggered sending urine samples to the automation line for quantification of protein and glucose and ensured that urine microscopy was performed to obtain WBC and red blood cell counts. The algorithm printed 2 labels so nursing staff would collect 2 specimens. We monitored the turnaround time from the specimen being received in the laboratory to result verification, ensured that the culture reflex order was triggered, and tracked complaints by physicians regarding not having usual urinalysis results. Prior to implementation, correlation between sample types for protein and glucose measurement was found acceptable. RESULTS.­: The algorithm was put in place twice during 2022. The turnaround time of urine microscopic study was identical to that obtained when the urinalysis was done with the strips; however, the quantification of glucose and protein took approximately 30 minutes more. Urine reflex cultures were triggered correctly with the algorithm, as they were derived entirely from a WBC count higher than 10 per high-power field. During the shortage period we had only 1 complaint, by a physician wanting to have results of nitrates. CONCLUSIONS.­: During urine strip shortages, we successfully implemented a diversion algorithm that provided actionable urinalysis analytes in a timely manner with minimal provider complaints.

Educating for Children's Health: Lessons Learned on Facilitating Media Literacy & Food Marketing Programming.

Food marketing is currently a multi-billion dollar industry. High levels of child-targeted food marketing, including on food packaging, suggests the need for media literacy skills to navigate persuasive techniques on food products. Evidence-based educational content on the topic of Media Literacy & Food Marketing (MLFM) was developed for children in Grades 3 to 9. This MLFM content has been taught to thousands of Canadian children across Canada, both in-person and virtually. This Practice Note highlights key strategies and lessons from implementing the program, and provides valuable insights into effective methods for empowering children's critical thinking around food promotion.

Synergistic Immunostimulation through the Dual Activation of Toll-like Receptor 3/9 with Spherical Nucleic Acids.

Toll-like receptors (TLRs) are a family of proteins that modulate the innate immune system and control the initiation of downstream immune responses. Spherical nucleic acids (SNAs) designed to stimulate single members of the TLR family (e.g., TLR7 or TLR9) have shown utility in cancer immunotherapy. We hypothesized that SNAs synthesized with multiple TLR agonists would enable the simultaneous activation of multiple TLR pathways for maximally synergistic immune activation. Here, we describe the synthesis of SNAs that incorporate both a TLR3 agonist (polyinosinic:polycytidylic acid, poly(I:C)) and TLR9 agonist (CpG oligonucleotide) on the same liposomal scaffold. In this design, CpG comprises the SNA oligonucleotide shell, and poly(I:C) is encapsulated in the liposome core. These dual-TLR activating SNAs efficiently codeliver high quantities of both agonists to the same target cell, yielding enhanced immunostimulation in various murine and human antigen-presenting cells (APCs). Moreover, codelivery of TLR agonists using the SNA both synchronizes and prolongs the duration of costimulatory molecule and major histocompatibility complex class II expression in APCs, which has been shown to be important for efficient downstream immune responses. Taken together, this SNA design provides a strategy for potently activating immune cells and increasing the efficiency of their activation, which likely will inform the preparation of nanomaterials for highly potent immunotherapies.

Comparison of Antibody Class-Specific SARS-CoV-2 Serologies for the Diagnosis of Acute COVID-19.

Accurate diagnosis of acute severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is critical for appropriate management of patients with this disease. We examined the possible complementary role of laboratory-developed class-specific clinical serology in assessing SARS-CoV-2 infection in hospitalized patients. Serological tests for immunoglobulin G (IgG), IgA, and IgM antibodies against the receptor binding domain (RBD) of SARS-CoV-2 were evaluated using samples from real-time reverse transcription-quantitative PCR (qRT-PCR)-confirmed inpatient coronavirus disease 2019 (COVID-19) cases. We analyzed the influence of timing and clinical severity on the diagnostic value of class-specific COVID-19 serology testing. Cross-sectional analysis revealed higher sensitivity and specificity at lower optical density cutoffs for IgA in hospitalized patients than for IgG and IgM serology (IgG area under the curve [AUC] of 0.91 [95% confidence interval {CI}, 0.89 to 0.93] versus IgA AUC of 0.97 [95% CI, 0.96 to 0.98] versus IgM AUC of 0.95 [95% CI, 0.92 to 0.97]). The enhanced performance of IgA serology was apparent in the first 2 weeks after symptom onset and the first week after PCR testing. In patients requiring intubation, all three tests exhibit enhanced sensitivity. Among PCR-negative patients under investigation for SARS-CoV-2 infection, 2 out of 61 showed clear evidence of seroconversion IgG, IgA, and IgM. Suspected false-positive results in the latter population were most frequently observed in IgG and IgM serology tests. Our findings suggest the potential utility of IgA serology in the acute setting and explore the benefits and limitations of class-specific serology as a complementary diagnostic tool to PCR for COVID-19 in the acute setting.

Tumor cell lysate-loaded immunostimulatory spherical nucleic acids as therapeutics for triple-negative breast cancer.

Highly heterogenous cancers, such as triple-negative breast cancer (TNBC), remain challenging immunotherapeutic targets. Herein, we describe the synthesis and evaluation of immunotherapeutic liposomal spherical nucleic acids (SNAs) for TNBC therapy. The SNAs comprise immunostimulatory oligonucleotides (CpG-1826) as adjuvants and encapsulate lysates derived from TNBC cell lines as antigens. The resulting nanostructures (Lys-SNAs) enhance the codelivery of adjuvant and antigen to immune cells when compared to simple mixtures of lysates with linear oligonucleotides both in vitro and in vivo, and reduce tumor growth relative to simple mixtures of lysate and CpG-1826 (Lys-Mix) in both Py230 and Py8119 orthotopic syngeneic mouse models of TNBC. Furthermore, oxidizing TNBC cells prior to lysis and incorporation into SNAs (OxLys-SNAs) significantly increases the activation of dendritic cells relative to their nonoxidized counterparts. When administered peritumorally in vivo in the EMT6 mouse mammary carcinoma model, OxLys-SNAs significantly increase the population of cytotoxic CD8+ T cells and simultaneously decrease the population of myeloid derived suppressor cells (MDSCs) within the tumor microenvironment, when compared with Lys-SNAs and simple mixtures of oxidized lysates with CpG-1826. Importantly, animals administered OxLys-SNAs exhibit significant antitumor activity and prolonged survival relative to all other treatment groups, and resist tumor rechallenge. Together, these results show that the way lysates are processed and packaged has a profound impact on their immunogenicity and therapeutic efficacy. Moreover, this work points toward the potential of oxidized tumor cell lysate-loaded SNAs as a potent class of immunotherapeutics for cancers lacking common therapeutic targets.

OX40+ plasmacytoid dendritic cells in the tumor microenvironment promote antitumor immunity.

Plasmacytoid DCs (pDCs), the major producers of type I interferon, are principally recognized as key mediators of antiviral immunity. However, their role in tumor immunity is less clear. Depending on the context, pDCs can promote or suppress antitumor immune responses. In this study, we identified a naturally occurring pDC subset expressing high levels of OX40 (OX40+ pDC) enriched in the tumor microenvironment (TME) of head and neck squamous cell carcinoma. OX40+ pDCs were distinguished by a distinct immunostimulatory phenotype, cytolytic function, and ability to synergize with conventional DCs (cDCs) in generating potent tumor antigen-specific CD8+ T cell responses. Transcriptomically, we found that they selectively utilized EIF2 signaling and oxidative phosphorylation pathways. Moreover, depletion of pDCs in the murine OX40+ pDC-rich tumor model accelerated tumor growth. Collectively, we present evidence of a pDC subset in the TME that favors antitumor immunity.

Sequence Multiplicity within Spherical Nucleic Acids.

The synthesis and evaluation of spherical nucleic acids (SNAs) incorporating two physically and chemically distinct classes of oligonucleotides (ODNs) at programmed ratios are described. These SNAs are single entity agents that enter the same target cell at defined stoichiometries, and as such allow one to control important cell signaling and regulatory processes. To study the effect of sequence multiplicity within such structures, we synthesized SNAs consisting of a mixture of class A CpG and class B CpG, immunostimulatory ODNs that activate two different toll-like receptor 9 signaling pathways, each in a sequence-specific fashion. These dual-CpG SNAs exhibit high cellular uptake and codelivery of the two ODNs, relative to mixtures of the linear ODN counterparts, and remain highly associated inside the cell over time. Furthermore, the dual-CpG SNAs augment dendritic cell maturation, compared to the same amounts of oligonucleotides delivered in linear or SNA form but not conjugated to one another. Consequently, these structures constitute a platform for designing oligonucleotide-based combination therapeutics with highly tailorable activities.

Manipulating Immune Activation of Macrophages by Tuning the Oligonucleotide Composition of Gold Nanoparticles.

This paper describes how the ligand shell containing immunostimulatory oligonucleotides surrounding gold nanoparticles affects the in vitro activation of macrophages. Nanoconstructs with similar ligand densities but different oligonucleotide compositions (from 0% to 100% immune-active cytosine-phosphate-guanine, CpG) were compared. Maximum immunostimulation was achieved with CpG content as low as 5% (with total oligonucleotide surface coverage remaining constant), correlating to high levels of antitumor cytokine release and low levels of cancer-promoting ones. Independent of CpG content, gold nanoparticles with low oligonucleotide densities exhibit poor cellular uptake, leading to insignificant immunostimulation and cytokine release. By identifying effects of ligand shell composition on macrophage activation, we can inform the design rules of therapeutic nanoconstructs to achieve specific immune responses.

Effects of bisphosphonate ligands and PEGylation on targeted delivery of gold nanoparticles for contrast-enhanced radiographic detection of breast microcalcifications.

A preclinical murine model of hydroxyapatite (HA) breast microcalcifications (µcals), which are an important clinical biomarker for breast cancer detection, was used to investigate the independent effects of high affinity bisphosphonate (BP) ligands and a polyethylene glycol (PEG) spacer on targeted delivery of gold nanoparticles (Au NPs) for contrast-enhanced radiographic detection. The addition of BP ligands to PEGylated Au NPs (BP-PEG-Au NPs) resulted in five-fold greater binding affinity for targeting HA µcals, as expected, due to the strong binding affinity of BP ligands for calcium. Therefore, BP-PEG-Au NPs were able to target HA µcals in vivo after intramammary delivery, which enabled contrast-enhanced radiographic detection of µcals in both normal and radiographically dense mammary tissues similar to previous results for BP-Au NPs, while PEG-Au NPs did not. The addition of a PEG spacer between the BP targeting ligand and Au NP surface enabled improved in vivo clearance. PEG-Au NPs and BP-PEG-Au NPs were cleared from all mammary glands (MGs) and control MGs, respectively, within 24-48 h after intramammary delivery, while BP-Au NPs were not. PEGylated Au NPs were slowly cleared from MGs by lymphatic drainage and accumulated in the spleen. Histopathology revealed uptake of PEG-Au NPs and BP-PEG-Au NPs by macrophages in the spleen, liver, and MGs; there was no evidence of toxicity due to the accumulation of NPs in organs and tissues compared with untreated controls for up to 28 days after delivery. STATEMENT OF SIGNIFICANCE: Au NP imaging probes and therapeutics are commonly surface functionalized with PEG and/or high affinity targeting ligands for delivery. However, direct comparisons of PEGylated Au NPs with and without a targeting ligand, or ligand-targeted Au NPs with and without a PEG spacer, on in vivo targeting efficiency, biodistribution, and clearance are limited. Therefore, the results of this study are important for the rationale design of targeted NP imaging probes and therapeutics, including the translation of BP-PEG-Au NPs which enable improved sensitivity and specificity for the radiographic detection of abnormalities (e.g., µcals) in women with dense breast tissue.

RNA-Based Immunostimulatory Liposomal Spherical Nucleic Acids as Potent TLR7/8 Modulators.

Immunostimulatory spherical nucleic acids (IS-LSNAs) comprised of RNA selective for toll-like receptors (TLRs) 7/8 are synthesized and characterized. These structures consist of liposomal cores functionalized with a dense shell of RNA inserted into the wall of the lipid core via hydrophobic cholesterol moieties. IS-LSNAs potently activate TLR7/8 via NF-κΒ signaling in reporter cell lines and in primary immune cells as evidenced by cytokine production and the upregulation of costimulatory receptors. Importantly, they are preferentially taken up by plasmacytoid dendritic cells, an observation that makes them potentially useful for immunotherapy. In addition, these structures contain a core that can be loaded with antigens and used to prime T cells. In this regard, it is shown that dendritic cells treated with IS-SNAs loaded with ovalbumin peptide can prime ova specific CD8+ T cells. In addition to introducing the first IS-LSNAs consisting of RNA, these experiments show that one can facilitate an antigen-specific T cell response greater than that of free or cationic lipid-transfected RNA of the same sequence selective for TLR7/8. This work points toward the promise of using IS-LSNAs comprised of RNA as potent and highly tunable TLR-specific agents for the development of vaccines and other pharmaceuticals that require selective immunomodulation.

Smaller CpG-Conjugated Gold Nanoconstructs Achieve Higher Targeting Specificity of Immune Activation.

This study describes a side-by-side comparison of the in vitro immunostimulatory activity of cytosine-phosphate-guanine (CpG)-conjugated gold nanoparticles. Three different gold nanoparticle cores (13 nm spheres, 50 nm spheres, and 40 nm stars) with the same CpG surface density were investigated for toll-like receptor 9 activation. For this parameter set, 13 nm spheres displayed significantly higher specificity for targeting immune receptors and larger nanoparticles (50 nm spheres and 40 nm stars) showed higher cellular uptake and higher immune activation because of off-target effects. Changes in nanoparticle size and presentation of activating ligands affect construct-induced immune responses at different levels, and care must be taken when considering practical and global design rules for CpG delivery.

Dual bioluminescence and near-infrared fluorescence monitoring to evaluate spherical nucleic acid nanoconjugate activity in vivo.

RNA interference (RNAi)-based gene regulation platforms have shown promise as a novel class of therapeutics for the precision treatment of cancer. Techniques in preclinical evaluation of RNAi-based nanoconjugates have yet to allow for optimization of their gene regulatory activity. We have developed spherical nucleic acids (SNAs) as a blood-brain barrier-/blood-tumor barrier-penetrating nanoconjugate to deliver small interfering (si) and micro (mi)RNAs to intracranial glioblastoma (GBM) tumor sites. To identify high-activity SNA conjugates and to determine optimal SNA treatment regimens, we developed a reporter xenograft model to evaluate SNA efficacy in vivo. Engrafted tumors stably coexpress optical reporters for luciferase and a near-infrared (NIR) fluorescent protein (iRFP670), with the latter fused to the DNA repair protein O6-methylguanine-DNA-methyltransferase (MGMT). Using noninvasive imaging of animal subjects bearing reporter-modified intracranial xenografts, we quantitatively assessed MGMT knockdown by SNAs composed of MGMT-targeting siRNA duplexes (siMGMT-SNAs). We show that systemic administration of siMGMT-SNAs via single tail vein injection is capable of robust intratumoral MGMT protein knockdown in vivo, with persistent and SNA dose-dependent MGMT silencing confirmed by Western blotting of tumor tissue ex vivo. Analyses of SNA biodistribution and pharmacokinetics revealed rapid intratumoral uptake and significant intratumoral retention that increased the antitumor activity of coadministered temozolomide (TMZ). Our study demonstrates that dual noninvasive bioluminescence and NIR fluorescence imaging of cancer xenograft models represents a powerful in vivo strategy to identify RNAi-based nanotherapeutics with potent gene silencing activity and will inform additional preclinical and clinical investigations of these constructs.

Improving Gram stain proficiency in hospital and satellite laboratories that do not have microbiology.

BACKGROUND: Consolidation of laboratories has left many hospitals and satellite laboratories with minimal microbiologic testing. In many hospitals and satellite laboratories, Gram stains on primary specimens are still performed despite difficultly in maintaining proficiency. METHODS: To maintain Gram stain proficiency at a community 450-bed hospital with an active emergency room we designed bimonthly challenges that require reporting Gram staining and morphology of different organisms. The challenges consist of five specimens prepared by the reference microbiology laboratory from cultures and primary specimens. Twenty to 23 medical laboratory scientists participate reading the challenges. Results from the challenges are discussed with each medical laboratory scientists. In addition, printed images from the challenges are presented at huddle to add microbiology knowledge. RESULTS: On the first three challenges, Gram staining was read correctly in 71%-77% of the time while morphology 53%-66%. In the last six challenges correct answers for Gram stain were 77%-99% while morphology 73%-96%. CONCLUSIONS: We observed statistically significant improvement when reading Gram stains by providing frequent challenges to medical laboratory scientists. The clinical importance of Gram stain results is emphasized during huddle presentations increasing knowledge and motivation to perform the test for patients.

Targeted delivery to bone and mineral deposits using bisphosphonate ligands.

The high concentration of mineral present in bone and pathological calcifications is unique compared with all other tissues and thus provides opportunity for targeted delivery of pharmaceutical drugs, including radiosensitizers and imaging probes. Targeted delivery enables accumulation of a high local dose of a therapeutic or imaging contrast agent to diseased bone or pathological calcifications. Bisphosphonates (BPs) are the most widely utilized bone-targeting ligand due to exhibiting high binding affinity to hydroxyapatite mineral. BPs can be conjugated to an agent that would otherwise have little or no affinity for the sites of interest. This article summarizes the current state of knowledge and practice for the use of BPs as ligands for targeted delivery to bone and mineral deposits. The clinical history of BPs is briefly summarized to emphasize the success of these molecules as therapeutics for metabolic bone diseases. Mechanisms of binding and the relative binding affinity of various BPs to bone mineral are introduced, including common methods for measuring binding affinity in vitro and in vivo. Current research is highlighted for the use of BP ligands for targeted delivery of BP conjugates in various applications, including (1) therapeutic drug delivery for metabolic bone diseases, bone cancer, other bone diseases, and engineered drug delivery platforms; (2) imaging probes for scintigraphy, fluorescence, positron emission tomography, magnetic resonance imaging, and computed tomography; and (3) radiotherapy. Last, and perhaps most importantly, key structure-function relationships are considered for the design of drugs with BP ligands, including the tether length between the BP and drug, the size of the drug, the number of BP ligands per drug, cleavable tethers between the BP and drug, and conjugation schemes.

Contrast-Enhanced X-ray Detection of Microcalcifications in Radiographically Dense Mammary Tissue Using Targeted Gold Nanoparticles.

Breast density reduces the accuracy of mammography, motivating methods to improve sensitivity and specificity for detecting abnormalities within dense breast tissue, but preclinical animal models are lacking. Therefore, the objectives of this study were to investigate a murine model of radiographically dense mammary tissue and contrast-enhanced X-ray detection of microcalcifications in dense mammary tissue by targeted delivery of bisphosphonate-functionalized gold nanoparticles (BP-Au NPs). Mammary glands (MGs) in the mouse mammary tumor virus - polyomavirus middle T antigen (MMTV-PyMT or PyMT) model exhibited greater radiographic density with age and compared with strain- and age-matched wild-type (WT) controls at 6-10 weeks of age. The greater radiographic density of MGs in PyMT mice obscured radiographic detection of microcalcifications that were otherwise detectable in MGs of WT mice. However, BP-Au NPs provided enhanced contrast for the detection of microcalcifications in both radiographically dense (PyMT) and WT mammary tissues as measured by computed tomography after intramammary delivery. BP-Au NPs targeted microcalcifications to enhance X-ray contrast with surrounding mammary tissue, which resulted in improved sensitivity and specificity for detection in radiographically dense mammary tissues.

Gold nanoparticles as contrast agents in x-ray imaging and computed tomography.

Computed tomography enables 3D anatomic imaging at a high spatial resolution, but requires delivery of an x-ray contrast agent to distinguish tissues with similar or low x-ray attenuation. Gold nanoparticles (AuNPs) have gained recent attention as an x-ray contrast agent due to exhibiting a high x-ray attenuation, nontoxicity and facile synthesis and surface functionalization for colloidal stability and targeted delivery. Potential diagnostic applications include blood pool imaging, passive targeting and active targeting, where actively targeted AuNPs could enable molecular imaging by computed tomography. This article summarizes the current state of knowledge for AuNP x-ray contrast agents within a paradigm of key structure-property-function relationships in order to provide guidance for the design of AuNP contrast agents to meet the necessary functional requirements in a particular application. Functional requirements include delivery to the site of interest (e.g., blood, tumors or microcalcifications), nontoxicity during delivery and clearance, targeting or localization at the site of interest and contrast enhancement for the site of interest compared with surrounding tissues. Design is achieved by strategically controlling structural characteristics (composition, mass concentration, size, shape and surface functionalization) for optimized properties and functional performance. Examples from the literature are used to highlight current design trade-offs that exist between the different functional requirements.