Innate immune cell response to host-parasite interaction in a human intestinal tissue microphysiological system.

Protozoan parasites that infect humans are widespread and lead to varied clinical manifestations, including life-threatening illnesses in immunocompromised individuals. Animal models have provided insight into innate immunity against parasitic infections; however, species-specific differences and complexity of innate immune responses make translation to humans challenging. Thus, there is a need for in vitro systems that can elucidate mechanisms of immune control and parasite dissemination. We have developed a human microphysiological system of intestinal tissue to evaluate parasite-immune-specific interactions during infection, which integrates primary intestinal epithelial cells and immune cells to investigate the role of innate immune cells during epithelial infection by the protozoan parasite, Toxoplasma gondii, which affects billions of people worldwide. Our data indicate that epithelial infection by parasites stimulates a broad range of effector functions in neutrophils and natural killer cell-mediated cytokine production that play immunomodulatory roles, demonstrating the potential of our system for advancing the study of human-parasite interactions.

Under-Oil Autonomously Regulated Oxygen Microenvironments: A Goldilocks Principle-Based Approach for Microscale Cell Culture.

Oxygen levels in vivo are autonomously regulated by a supply-demand balance, which can be altered in disease states. However, the oxygen levels of in vitro cell culture systems, particularly microscale cell culture, are typically dominated by either supply or demand. Further, the oxygen microenvironment in these systems is rarely monitored or reported. Here, a method to establish and dynamically monitor autonomously regulated oxygen microenvironments (AROM) using an oil overlay in an open microscale cell culture system is presented. Using this method, the oxygen microenvironment is dynamically regulated via the supply-demand balance of the system. Numerical simulation and experimental validation of oxygen transport within multi-liquid-phase, microscale culture systems involving a variety of cell types, including mammalian, fungal, and bacterial cells are presented. Finally, AROM is applied to establish a coculture between cells with disparate oxygen demands-primary intestinal epithelial cells (oxygen consuming) and Bacteroides uniformis (an anaerobic species prevalent in the human gut).

Immune cell mediated cabozantinib resistance for patients with renal cell carcinoma.

Renal cell carcinoma (RCC) is the third most common genitourinary cancer in the USA. Despite recent advances in the treatment for advanced and metastatic clear cell RCC (ccRCC), the 5-year relative survival rate for the distant disease remains at 12%. Cabozantinib, a tyrosine kinase inhibitor (TKI), which is one of the first-line therapies approved to treat advanced ccRCC as a single agent, is now being investigated as a combination therapy with newer immunotherapeutic agents. However, not much is known about how cabozantinib modulates the immune system. Here, we present a high throughput tri-culture model that incorporates cancer cells, endothelial cells, and patient-derived immune cells to study the effect of immune cells from patients with ccRCC on angiogenesis and cabozantinib resistance. We show that circulating immune cells from patients with ccRCC induce cabozantinib resistance via increased secretion of a set of pro-angiogenic factors. Using multivariate partial least square regression modeling, we identified CD4+ T cell subsets that are correlated with cabozantinib resistance and report the changes in the frequency of these populations in ccRCC patients who are undergoing cabozantinib therapy. These findings provide a potential set of biomarkers that should be further investigated in the current TKI-immunotherapy combination clinical trials to improve personalized treatments for patients with ccRCC.

Driving Time to Trauma Centers for Children Living in Wisconsin.

INTRODUCTION: Trauma is the number 1 cause of death among children. Shorter distance to definitive trauma care has been correlated with better clinical outcomes. There are only a small number of pediatric trauma centers (PTC) designated by the American College of Surgeons, and the resources available to treat injured children at non-PTCs are limited. To guide resource allocation and advocacy efforts for pediatric trauma care in Wisconsin, we determined the precise distance to trauma centers for all children living in the state. METHODS: The 2010 US Census data was used to determine ZIP-centroid geolocation. The Wisconsin Department of Health Services trauma classification database was used to identify trauma facilities in Wisconsin. SAS routines invoking the Google Maps application programming interface were used to calculate the driving distance to each of the trauma facilities. We quantified the percentage of children living within 30- and 60-minute driving distances of level I-IV trauma centers. RESULTS: Just 31.3% of Wisconsin children live within a 30-minute drive of a level I PTC; 32.7% live within 30 minutes of a level II center; 81.3% within 30 minutes of a level III center; and 74.6% within 30 minutes of a level IV center. CONCLUSION: Two-thirds of children in Wisconsin live beyond a 30-minute driving distance of a level I PTC, but most children live within 30 minutes of level III and IV trauma centers. As the closest hospitals for most children, smaller trauma centers should be adequately resourced to provide pediatric trauma care.

Setting Up for Success: Strategies to Foster Surgeons' Pursuit of Basic Science Research.

BACKGROUND: Surgeons make important contributions to basic science research and are in a unique position to innovate scientifically. The number of surgeons pursuing basic science research has been declining over the past two decades. We sought to describe perceived barriers to surgeons' pursuit of basic science research and identify interventions that mitigate these obstacles. MATERIALS & METHODS: An online survey was sent to chairs of academic surgery departments and practicing surgeons involved in basic science research. A subset of these participants were interviewed about their experiences. Interviews were audio-recorded, transcribed, and uploaded to NVivo. Two coders developed a codebook using inductive content analysis to identify relevant themes. RESULTS: 97 people responded to the survey, 27 (29%) were department chairs. Major barriers to basic science research for all respondents were lack of funding, clinical duties and lack of dedicated time for research. Nine surgeons and three departmental chairs were subsequently interviewed. The importance of having clear research goals and timetables with specific plans for attaining funding were mentioned by all. Chairs described the usefulness of embedding early surgeon scientists in their scientific mentors' labs in a post-doctoral model. Additionally, departmental leaders must actively work to protect surgeon scientists from encroaching clinical and administrative demands. CONCLUSIONS: While barriers to surgeons' pursuit of basic science research exist, the surgeon scientist is a phenotype that can be fostered with the dedication and commitment of surgeons to continue to pursue science research and active support of departmental leadership.

Case report: a step-wise management of concurrent presentation of congenital single lung and aberrant right subclavian artery in an infant girl.

INTRODUCTION: Congenital single lung (CSL) is a rare condition, and symptomatic patients often present with respiratory distress or recurrent respiratory infection due to mediastinal shift causing vascular or airway compression. Aberrant right subclavian artery (ARSA) is another rare congenital anomality that can lead to tracheal or esophageal compressions. There is only one other case of concurrent presentation of CSL and ARSA reported, which presented unique challenge in surgical management of our patient. Here we present a step-wise, multidisciplinary approach to manage symptomatic CSL and ARSA. CASE PRESENTATION: An infant girl with a prenatal diagnosis of CSL developed worsening stridor and several episodes of respiratory illnesses at 11 months old. Cross-sectional imaging and bronchoscopic evaluation showed moderate to severe distal tracheomalacia with anterior and posterior tracheal compression resulting from severe mediastinal rotation secondary to right-sided CSL. It was determined that her tracheal compression was mainly caused by her aortic arch wrapping around the trachea, with possible additional posterior compression of the esophagus by the ARSA. She first underwent intrathoracic tissue expander placement, which resulted in immediate improvement of tracheal compression. Two days later, she developed symptoms of dysphagia lusoria due to increased posterior compression of her esophagus by the ARSA. She underwent transposition of ARSA to the right common carotid with immediate resolution of dysphagia lusoria. As the patient grew, additional saline was added to the tissue expander due to recurrence in compressive symptoms. CONCLUSIONS: Concurrent presentation of CSL and ARSA is extremely rare. Asymptomatic CSL and ARSA do not require surgical interventions. However, if symptomatic, it is crucial to involve a multidisciplinary team for surgical planning and to take a step-wise approach as we were able to recognize and address both tracheomalacia and dysphagia lusoria in our patient promptly.

Toward improved in vitro models of human cancer.

Cancer is a leading cause of death across the world and continues to increase in incidence. Despite years of research, multiple tumors (e.g., glioblastoma, pancreatic cancer) still have limited treatment options in the clinic. Additionally, the attrition rate and cost of drug development have continued to increase. This trend is partly explained by the poor predictive power of traditional in vitro tools and animal models. Moreover, multiple studies have highlighted that cell culture in traditional Petri dishes commonly fail to predict drug sensitivity. Conversely, animal models present differences in tumor biology compared with human pathologies, explaining why promising therapies tested in animal models often fail when tested in humans. The surging complexity of patient management with the advent of cancer vaccines, immunotherapy, and precision medicine demands more robust and patient-specific tools to better inform our understanding and treatment of human cancer. Advances in stem cell biology, microfluidics, and cell culture have led to the development of sophisticated bioengineered microscale organotypic models (BMOMs) that could fill this gap. In this Perspective, we discuss the advantages and limitations of patient-specific BMOMs to improve our understanding of cancer and how these tools can help to confer insight into predicting patient response to therapy.

Reducing cost and waste in pediatric laparoscopic procedures.

BACKGROUND: In 2017 the healthcare cost in the United States accounted for 17.9% of the Gross Domestic Product (GDP). Furthermore, healthcare facilities produce more than 4 billion pounds of waste annually. Interhospital and intersurgeon variabilities in surgical procedures are some of the drivers of high healthcare cost and waste. We sought to determine the effect of a monthly surgeon report card detailing the utilization and cost of disposable and reusable surgical supplies on cost and waste reduction for pediatric laparoscopic procedures. METHODS: Starting in July 2017, surgeons were provided with an individual report with supply cost per case, high cost, and disposable supply utilization, and clinical outcomes. Cost, utilization, and clinical outcomes six quarters before and after the intervention were compared. RESULTS: A total of 998 pediatric laparoscopic procedures were analyzed. We reduced the median supply cost per case by 43% after the intervention with total cost savings of $71,035 for the first four quarters. We also reduced the use of disposable trocars by 56% and the use of disposable harmonics and staplers by 33%. CONCLUSIONS: Using a periodic surgeon report card, we significantly reduced supply cost and utilization of disposable items for all pediatric laparoscopic procedures performed at the University of Wisconsin American Family Children's Hospital. TYPE OF STUDY: Cost effectiveness study. LEVEL OF EVIDENCE: Level III.

Intestinal Perforation in Children as an Important Differential Diagnosis of Vascular Ehlers-Danlos Syndrome.

BACKGROUND Ehlers-Danlos Syndrome (EDS) is a group of connective tissue disorders with heterogeneous clinical features associated with varying genetic mutations. EDS type IV, also known as vascular EDS (vEDS), is the rarest type but has fatal complications, including rupture of major vasculature and intestinal and uterine perforation. Intestinal perforation can be spontaneous or a consequence of long-standing constipation, a common symptom among patients with EDS. CASE REPORT We present a case of a 6-year-old boy with the previous diagnosis of vEDS who presented with colonic perforation from a stercoral ulcer. He underwent diagnostic laparoscopy and loop colostomy, with an uneventful postoperative course. Unfortunately, he developed a second colonic perforation 14 months after the initial episode and underwent total abdominal colectomy with end ileostomy. CONCLUSIONS Intestinal perforation is a well-documented and devastating complication of vEDS. However, spontaneous intestinal perforation is extremely rare in a young child. Therefore, the diagnosis of vEDS should be included in the differential diagnosis if a child presents with intestinal perforation. There is no clear guideline available for surgical management of colonic perforation in patients with vEDS, but total abdominal colectomy appears to provide the best chance of preventing recurrent perforation.

Monocyte-derived macrophage assisted breast cancer cell invasion as a personalized, predictive metric to score metastatic risk.

Patient-to-patient variability in breast cancer progression complicates clinical treatment decisions. Of women undergoing prophylactic mastectomies, many may not have progressed to indolent forms of disease and could have benefited from milder, localized therapy. Tumor associated macrophages contribute significantly to tumor invasion and metastasis, with cysteine cathepsin proteases as important contributors. Here, a method is demonstrated by which variability in macrophage expression of cysteine cathepsins, their inhibitor cystatin C, and kinase activation can be used to train a multivariate model and score patients for invasion risk. These enzymatic profiles were used to predict macrophage-assisted MCF-7 breast cancer cell invasion in the trained computational model. To test these predictions, a priori, signals from monocytes isolated from women undergoing mastectomies were input to score their cancer invasion potential in a patient-specific manner, and successfully predicted that patient monocytes with highest predicted invasion indices matched those with more invasive initial diagnoses of the nine patients tested. Together this establishes proof-of-principle that personalized information acquired from minimally invasive blood draws may provide useful information to inform oncologists and patients of invasive/metastatic risk, helping to make decisions regarding radical mastectomy or milder, conservative treatments to save patients from hardship and surgical recovery.

Multiple paracrine factors secreted by mesenchymal stem cells contribute to angiogenesis.

The therapeutic effects of stem cell transplantation in ischemic disease are mediated by the production of paracrine bioactive factors. However, the bioactive factors secreted by human mesenchymal stem cells (hMSCs) and their angiogenic activity are not clearly identified or determined. We here found that hMSC-derived conditioned media (hMSC-CdM) stimulated in vitro angiogenic activity of endothelial cells and contained significant levels of various growth factors and cytokines, such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), monocyte chemotactic protein-1 (MCP-1), interleukin-6 (IL-6), and transforming growth factor-beta1 (TGF-ß1). The angiogenic activity of hMSC-CdM was significantly inhibited by pretreatment with neutralizing antibodies against VEGF, MCP-1, and IL-6, but not against TGF-ß1 and HGF. A mixture of those inhibitory antibodies blocked CdM-mediated activation of angiogenic signals, as well as inhibited CdM-mediated in vivo angiogenesis. Moreover, local injection of CdM increased angiogenesis and promoted blood flow in mice with hindlimb ischemia, and these effects were inhibited by co-treatment with these inhibitory antibodies. These results indicate that hMSC-CdM represents a promising cell-free therapeutic strategy for neovascularization in ischemic diseases. These results suggest the combination of VEGF, MCP-1, and IL-6 as a commercial application for therapeutic angiogenesis.

Patient specific proteolytic activity of monocyte-derived macrophages and osteoclasts predicted with temporal kinase activation states during differentiation.

Patient-to-patient variability in disease progression continues to complicate clinical decisions of treatment regimens for cardiovascular diseases, metastatic cancers and osteoporosis. Here, we investigated if monocytes, circulating white blood cells that enter tissues and contribute to disease progression by differentiating into macrophages or osteoclasts, could be useful in understanding this variability. Monocyte-derived macrophages and osteoclasts produce cysteine cathepsins, powerful extracellular matrix proteases which have been mechanistically linked to accelerated atherosclerotic, osteoporotic, and tumor progression. We hypothesized that multivariate analysis of temporal kinase activation states during monocyte differentiation could predict cathepsin proteolytic responses of monocyte-derived macrophages and osteoclasts in a patient-specific manner. Freshly isolated primary monocytes were differentiated with M-CSF or RANKL into macrophages or osteoclasts, respectively, and phosphorylation of ERK1/2, Akt, p38 MAPK, JNK, c-jun, and IκB-α were measured at days 1, 3, 6, and 9. In parallel, cell diameters and numbers of nuclei were measured, and multiplex cathepsin zymography was used to quantify cathepsins K, L, S, and V activity from cell extracts and conditioned media. There was extensive patient-to-patient variability in temporal kinase activation states, cell morphologies, and cathepsin K, L, S, and V proteolytic activity. Partial least squares regression models trained with temporal kinase activation states successfully predicted patient-specific morphological characteristics (mean cell diameter and number of nuclei) and patient-specific cathepsin proteolytic activity with predictability as high as 95%, even with the challenge of incorporating the complex, unknown cues from individual patients' unique genetic and biochemical backgrounds. This personalized medicine approach considers patient variability in kinase signals to predict cathepsin activity. Such analyses may provide beneficial tools for personalized kinase and protease inhibitor therapies for tissue destructive diseases.

Manipulating substrate and pH in zymography protocols selectively distinguishes cathepsins K, L, S, and V activity in cells and tissues.

Cathepsins K, L, S, and V are cysteine proteases that have been implicated in tissue-destructive diseases such as atherosclerosis, tumor metastasis, and osteoporosis. Among these four cathepsins are the most powerful human collagenases and elastases, and they share 60% sequence homology. Proper quantification of mature, active cathepsins has been confounded by inhibitor and reporter substrate cross-reactivity, but is necessary to develop properly dosed therapeutic applications. Here, we detail a method of multiplex cathepsin zymography to detect and distinguish the activity of mature cathepsins K, L, S, and V by exploiting differences in individual cathepsin substrate preferences, pH effects, and electrophoretic mobility under non-reducing conditions. Specific identification of cathepsins K, L, S, and V in one cell/tissue extract was obtained with cathepsin K (37 kDa), V (35 kDa), S (25 kDa), and L (20 kDa) under non-reducing conditions. Cathepsin K activity disappeared and V remained when incubated at pH 4 instead of 6. Application of this antibody free, species independent, and medium-throughput method was demonstrated with primary human monocyte-derived macrophages and osteoclasts, endothelial cells stimulated with inflammatory cytokines, and normal and cancer lung tissues, which identified elevated cathepsin V in lung cancer.

Self-assembly of nucleoamphiphiles: investigating nucleosides effect and the mechanism of micrometric helix formation.

A new family of self-assembling systems based on nucleoamphiphiles is described. Nano to micrometric left-handed helix formation in aqueous solution was induced simply by complexing a GMP or an AMP with a nonchiral monocationic amphiphile. The assembling behavior such as micellar formation, monolayer at air-water interface, as well as the aggregates in solution of these nucleoamphiphiles are strongly influenced by the presence of nucleosides in solution. The observed effects depend on the properties of complexed nucleotides and nucleosides with a complex mixture of pi stacking, hydrophobicity of the bases, and hydrogen bonding.