Polarization of macrophages to an anti-cancer phenotype through in situ uncaging of a TLR 7/8 agonist using bioorthogonal nanozymes.

Macrophages are plastic cells of the immune system that can be broadly classified as having pro-inflammatory (M1-like) or anti-inflammatory (M2-like) phenotypes. M2-like macrophages are often associated with cancers and can promote cancer growth and create an immune-suppressive tumor microenvironment. Repolarizing macrophages from M2-like to M1-like phenotype provides a crucial strategy for anticancer immunotherapy. Imiquimod is an FDA-approved small molecule that can polarize macrophages by activating toll-like receptor 7/8 (TLR 7/8) located inside lysosomes. However, the non-specific inflammation that results from the drug has limited its systemic application. To overcome this issue, we report the use of gold nanoparticle-based bioorthogonal nanozymes for the conversion of an inactive, imiquimod-based prodrug to an active compound for macrophage re-education from anti- to pro-inflammatory phenotypes. The nanozymes were delivered to macrophages through endocytosis, where they uncaged pro-imiquimod in situ. The generation of imiquimod resulted in the expression of pro-inflammatory cytokines. The re-educated M1-like macrophages feature enhanced phagocytosis of cancer cells, leading to efficient macrophage-based tumor cell killing.

Paramedic-Performed Prehospital Tele-Ultrasound: A Powerful Technology or an Impractical Endeavor? A Scoping Review.

Ultrasound with remote assistance (tele-ultrasound) may have potential to improve accessibility of ultrasound for prehospital patients. A review of recent literature on this topic has not been done before, and the feasibility of prehospital tele-ultrasound performed by non-physician personnel is unclear. In an effort to address this, the literature was qualitatively analyzed from January 1, 2010 - December 31, 2021 in the MEDLINE, EMBASE, and Cochrane online databases on prehospital, paramedic-acquired tele-ultrasound, and ten articles were found. There was considerable heterogeneity in the study design, technologies used, and the amount of ultrasound training for the paramedics, preventing cross-comparisons of different studies. Tele-ultrasound has potential to improve ultrasound accessibility by leveraging skills of a remote ultrasound expert, but there are still technological barriers to overcome before determinations on feasibility can be made.

Diarylidene-N-Methyl-4-Piperidones and Spirobibenzopyrans as Antioxidant and Anti-Inflammatory Agents.

Curcumin has antioxidant properties resulting from its radical scavenging ability and inhibition of inflammation-associated factors. However, its lack of solubility, instability, and poor bioavailability are impediments to its therapeutic use. As potential alternatives, we synthesized and performed chemical analysis of thirty diarylidene-N-methyl-4-piperidone (DANMP), diheteroarylidene-N-methyl-4-piperidone (DHANMP), and spirobibenzopyran (SBP) derivatives, one of which was also characterized by single crystal X-ray diffraction. All compounds were evaluated for antioxidant activity via 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay and for drug-like properties in silico. A subset of five compounds was investigated in terms of aqueous solubilities, which were significantly improved compared to that of curcumin. In vitro assessments of cellular and anti-inflammatory effects were conducted via real time polymerase chain reaction (RT-PCR) and Griess assays to evaluate the presence of inflammatory/activated (M1) markers and production of nitric oxide (NO) species, which are associated with inflammation. The five compounds reduced levels of markers and NO to extents similar to or better than curcumin in inflamed cells, and showed no adverse effects on cell viability. We show that these compounds possess anti-inflammatory properties and may be used as curcumin-substitutes with improved characteristics.

Murine macrophage-based iNos reporter reveals polarization and reprogramming in the context of breast cancer.

As part of the first line of defense against pathogens, macrophages possess the ability to differentiate into divergent phenotypes with varying functions. The process by which these cells change their characteristics, commonly referred to as macrophage polarization, allows them to change into broadly pro-inflammatory (M1) or anti-inflammatory (M2) subtypes, and depends on the polarizing stimuli. Deregulation of macrophage phenotypes can result in different pathologies or affect the nature of some diseases, such as cancer and atherosclerosis. Therefore, a better understanding of macrophage phenotype conversion in relevant models is needed to elucidate its potential roles in disease. However, there are few existing probes to track macrophage changes in multicellular environments. In this study, we generated an eGFP reporter cell line based on inducible nitric oxide synthase (iNos) promoter activity in RAW264.7 cells (RAW:iNos-eGFP). iNos is associated with macrophage activation to pro-inflammatory states and decreases in immune-suppressing ones. We validated the fidelity of the reporter for iNos following cytokine-mediated polarization and confirmed that reporter and parental cells behaved similarly. RAW:iNos-eGFP cells were then used to track macrophage responses in different in vitro breast cancer models, and their re-education from anti- to pro-inflammatory phenotypes via a previously reported pyrimido(5,4-b)indole small molecule, PBI1. Using two mouse mammary carcinoma cell lines, 4T1 and EMT6, effects on macrophages were assessed via conditioned media, two-dimensional/monolayer co-culture, and three-dimensional spheroid models. While conditioned media derived from 4T1 or EMT6 cells and monolayer co-cultures of each cancer cell line with RAW:iNos-eGFP cells all resulted in decreased fluorescence, the trends and extents of effects differed. We also observed decreases in iNos-eGFP signal in the macrophages in co-culture assays with 4T1- or EMT6-based spheroids. We then showed that iNos production is enhanced in these cancer models using PBI1, tracking increased fluorescence. Collectively, this work demonstrates that this reporter-based approach provides a facile means to study macrophage responses in complex, multicomponent environments. Beyond the initial studies presented here, this platform can be used with a variety of in vitro models and extended to in vivo applications with intravital imaging.

Successful outcomes of surgery procedures in patient with Glanzmann´s Thrombasthenia

We report a 35-year-old female patient with Glanzmann's thrombasthenia (GT) and severe anemia due to abnormal uterine bleeding secondary to uterine myomatosis. She required several admissions of red blood cells and platelet transfusions. An elective subtotal hysterectomy with salpingo-oophorectomy was proposed and recombinant factor VII was required. Surgical and postoperative outcomes were successful, without surgical complications, bleeding, or hemogram alterations. 4 years later, she required tooth extraction because of periodontal disease and pulp necrosis. In Peru, reports of GT patients requiring major and minor surgical procedures are lacking, given the low disease prevalence and the difficulties related to surgery. The report of these successful cases becomes relevant to continue improving GT management.

Presentamos el caso de una paciente de 35 años con trombastenia de Glanzmann (GT) y anemia severa por sangrado uterino anormal secundario a miomatosis uterina. Requirió varias admisiones de transfusiones de glóbulos rojos y plaquetas. Se propuso histerectomía subtotal electiva con salpingo-ooforectomía y se requirió factor VII recombinante. Los resultados quirúrgicos y postoperatorios fueron exitosos, sin complicaciones quirúrgicas, sangrado ni alteraciones del hemograma. 4 años después, requirió extracción dental por enfermedad periodontal y necrosis pulpar. En Perú faltan reportes de pacientes con GT que requieran procedimientos quirúrgicos mayores y menores, dada la baja prevalencia de la enfermedad y las dificultades relacionadas con la cirugía. El reporte de estos casos de éxito cobra relevancia para seguir mejorando la gestión de GT

Engineered Polymer-siRNA Polyplexes Provide Effective Treatment of Lung Inflammation.

Uncontrolled inflammation is responsible for acute and chronic diseases in the lung. Regulating expression of pro-inflammatory genes in pulmonary tissue using small interfering RNA (siRNA) is a promising approach to combatting respiratory diseases. However, siRNA therapeutics are generally hindered at the cellular level by endosomal entrapment of delivered cargo and at the organismal level by inefficient localization in pulmonary tissue. Here we report efficient anti-inflammatory activity in vitro and in vivo using polyplexes of siRNA and an engineered cationic polymer (PONI-Guan). PONI-Guan/siRNA polyplexes efficiently deliver siRNA cargo to the cytosol for highly efficient gene knockdown. Significantly, these polyplexes exhibit inherent targeting to inflamed lung tissue following intravenous administration in vivo. This strategy achieved effective (>70%) knockdown of gene expression in vitro and efficient (>80%) silencing of TNF-α expression in lipopolysaccharide (LPS)-challenged mice using a low (0.28 mg/kg) siRNA dosage.

Macrophage circadian rhythms are differentially affected based on stimuli.

Macrophages are white blood cells that play disparate roles in homeostasis and immune responses. They can reprogram their phenotypes to pro-inflammatory (M1) or anti-inflammatory (M2) states in response to their environment. About 8-15% of the macrophage transcriptome has circadian oscillations, including genes closely related to their functioning. As circadian rhythms are associated with cellular phenotypes, we hypothesized that polarization of macrophages to opposing subtypes might differently affect their circadian rhythms. We tracked circadian rhythms in RAW 264.7 macrophages using luminescent reporters. Cells were stably transfected with Bmal1:luc and Per2:luc reporters, representing positive and negative components of the molecular clock. Strength of rhythmicity, periods and amplitudes of time series were assessed using multiple approaches. M1 polarization decreased amplitudes and rhythmicities of Bmal1:luc and Per2:luc, but did not significantly affect periods, while M2 polarization increased periods but caused no substantial alterations to amplitudes or rhythmicity. As macrophage phenotypes are also altered in the presence of cancer cells, we tested circadian effects of conditioned media from mouse breast cancer cells. Media from highly aggressive 4T1 cells caused loss of rhythmicity, while media from less aggressive EMT6 cells yielded no changes. As macrophages play roles in tumors, and oncogenic features are associated with circadian rhythms, we tested whether conditioned media from macrophages could alter circadian rhythms of cancer cells. Conditioned media from RAW 264.7 cells resulted in lower rhythmicities and periods, but higher amplitudes in human osteosarcoma, U2OS-Per2:luc cells. We show that phenotypic changes in macrophages result in altered circadian characteristics and suggest that there is an association between circadian rhythms and macrophage polarization state. Additionally, our data demonstrate that macrophages treated with breast cancer-conditioned media have circadian phenotypes similar to those of the M1 subtype, and cancer cells treated with macrophage-conditioned media have circadian alterations, providing insight to another level of cross-talk between macrophages and cancer.

Delta Lactate (Three-hour Lactate Minus Initial Lactate) Prediction of In-hospital Death in Sepsis Patients.

This study examines the relationship between serial serum lactate levels and in-hospital mortality in an adult cohort of emergency department patients with severe sepsis or septic shock. Of the 164 patients in the cohort, 130 also got three-hour lactate in addition to the initial one. The median initial lactate was 3.01 (interquartile range [IQR]: 1.71-4.62). The median repeat lactate was 2.58 (IQR: 1.4-3.9). The in-hospital death rate was 23% for men and 29% for women. The delta lactate was significantly higher in women (P=0.0070), driven by a lower initial lactate (P=0.0277). In a multivariate regression model controlled for age and gender, a statistically significant correlation was noted between an increase in the delta lactate and in-hospital death (P=0.0323; R2=11.3%). The results of this single-center study suggest that an increase in serum lactic acid is significantly associated with higher in-hospital death.

Serum Procalcitonin Level Is Associated with Positive Blood Cultures, In-hospital Mortality, and Septic Shock in Emergency Department Sepsis Patients.

This study examines the accuracy of initial and subsequent serum procalcitonin (PCT) levels in predicting positive blood cultures, in-hospital mortality, and development of septic shock in emergency department (ED) patients with severe sepsis. This study includes all patients who presented to our ED with an admission diagnosis of severe sepsis over a period of nine months. The median initial PCT was 0.58 ng/mL, interquartile range (IQR) 0.16-5.39. The median subsequent serum PCT was 2.1 ng/mL, with an IQR of 0.3-11.1. The PCT trend over the initial three hours increased in 67% of the study population. Blood cultures were positive in 38% of the cohort. The median maximum PCT in those with a negative blood culture was 1.06 ng/mL compared to 4.19 ng/mL in those with a positive blood culture (p=0.0116). Serum PCT levels >2.0 ng/mL display significant correlation with positive blood cultures, in-hospital mortality, and development of septic shock and as such may serve as a biomarker for more serious infections.

Assembly Line Education: A Novel Educational Technique for Today's Learners.

Background Education is undergoing a transformation. The traditional passive lectures are failing to capture and inspire the new generation of learners who value more active and collaborative learning techniques. Objective We sought to create a novel educational technique to integrate into our curriculum that would be more personalized, employ more active learning and collaboration, and allow for an effective assessment of resident strengths and weaknesses. Discussion We created a monthly assembly line education academic half-day that evolved to replace one of the typical in-classroom didactics each month. Faculty run small-group simulation rooms, procedure workshops, competitive ultrasound, and wellness stations through which residents and medical students rotate. Conclusion This novel education technique resulted in a more personalized approach that increased resident interest, sparked the creation of a very popular MedEd-Simulation elective, and allowed the faculty to gain a better sense of resident strengths and deficiencies.

Pericardial effusion with tamponade: bedside ultrasonography saves another life.

In these video clinical images, the authors present the cause for an elderly gentleman's shortness of breath. It was presumed to be an exacerbation of chronic obstructive pulmonary disease, a condition for which he was in the process of being evaluated. However, bedside ultrasonography revealed a large pericardial effusion with tamponade. This timely diagnosis resulted in the patient being taken expeditiously to the operating room and saving his life.

High-content and high-throughput identification of macrophage polarization phenotypes.

Macrophages are plastic cells of the innate immune system that perform a wide range of immune- and homeostasis-related functions. Due to their plasticity, macrophages can polarize into a spectrum of activated phenotypes. Rapid identification of macrophage polarization states provides valuable information for drug discovery, toxicological screening, and immunotherapy evaluation. The complexity associated with macrophage activation limits the ability of current biomarker-based methods to rapidly identify unique activation states. In this study, we demonstrate the ability of a 2-element sensor array that provides an information-rich 5-channel output to successfully determine macrophage polarization phenotypes in a matter of minutes. The simple and robust sensor generates a high dimensional data array which enables accurate macrophage evaluations in standard cell lines and primary cells after cytokine treatment, as well as following exposure to a model disease environment.

Emergency Department Approach to Testicular Torsion: Two Illustrative Cases.

We present two cases of young men with spontaneous nontraumatic testicular pain. While the differential diagnosis for scrotal or testicular pain can include less urgent causes, such as epididymitis, hydrocele, referred pain, idiopathic scrotal edema, and inguinal hernia, for example, the most feared etiology for acute scrotal pain is testicular torsion. The fact that a testicle can torse and detorse is also a confounding factor. In this case review, we explore factors affecting the timely diagnosis, management, and outcomes of acute testicular pain. Prompt diagnosis is imperative in order to salvage a torsed testicle.

Diagnosing Appendicitis in Pregnancy Via Ultrasonography.

The authors present a case of acute appendicitis during a first trimester pregnancy. Appendicitis in pregnancy is especially dangerous because perforation of the appendix increases the likelihood of maternal and fetal morbidity significantly. For this reason, it is important to diagnose and treat suspected appendicitis in pregnancy as soon as possible. The patient was diagnosed with appendicitis via a transabdominal ultrasound. She was provided antibiotics and underwent a laparoscopic appendectomy and recovered without complications.

Incidental Left Ventricular Aneurysm Discovered after Chest Pain Following a Motor Vehicle Collision.

We report the case of a female with a history of coronary artery disease who came in with chest pain after a motor vehicle collision. Imaging revealed an incidental left ventricular aneurysm. The presentation and management of left ventricular aneurysms are discussed, along with imaging findings on computed tomography, plain radiography, and ultrasonography.

Macrophage activation by a substituted pyrimido[5,4-b]indole increases anti-cancer activity.

Immunotherapy has become a promising new approach for cancer treatment due to the immune system's ability to remove tumors in a safe and specific manner. Many tumors express anti-inflammatory factors that deactivate the local immune response or recruit peripheral macrophages into pro-tumor roles. Because of this, effective and specific ways of activating macrophages into anti-tumor phenotypes is highly desirable for immunotherapy purposes. Here, the use of a small molecule TLR agonist as a macrophage activator for anti-cancer therapy is reported. This compound, referred to as PBI1, demonstrated unique activation characteristics and expression patterns compared to treatment with LPS, through activation of TLR4. Furthermore, PBI1 treatment resulted in anti-tumor immune behavior, enhancing macrophage phagocytic efficiency five-fold versus non-treated macrophages. Additive effects were observed via use of a complementary strategy (anti-CD47 antibody), resulting in ∼10-fold enhancement of phagocytosis, suggesting this small molecule approach could be used in conjunction with other therapeutics.

The Role of Serial Ultrasounds in Diagnosing Suspected Deep Venous Thrombosis.

Venous thromboembolisms (VTE), which include deep venous thrombosis (DVT) and pulmonary embolism (PE), are a major cause of morbidity and mortality in the United States and globally. This often underdiagnosed medical condition has many known risk factors including pregnancy, malignancy, immobility, exogenous estrogen use, and hereditary factors. A significant portion of emergency department visits involves ruling out these diseases. This case presents a woman with unilateral leg pain and swelling who initially had a negative emergency room workup including a negative lower extremity Doppler study. Upon a repeat visit, she was found to have extensive deep venous thrombosis and was diagnosed with B-cell lymphoma. This case highlights the importance of having patients return for repeat imaging if Dopplers are negative in the initial encounter.