Mark M. Hammer

IGF-I increases bone marrow contribution to adult skeletal muscle and enhances the fusion of myelomonocytic precursors

Muscle damage has been shown to enhance the contribution of bone marrow–derived cells (BMDCs) to regenerating skeletal muscle. One responsible cell type involved in this process is a hematopoietic stem cell derivative, the myelomonocytic precursor (MMC). However, the molecular components responsible for this injury-related response remain largely unknown. In this paper, we show that delivery of insulin-like growth factor I (IGF-I) to adult skeletal muscle by three different methods—plasmid electroporation, injection of genetically engineered myoblasts, and recombinant protein injection—increases the integration of BMDCs up to fourfold. To investigate the underlying mechanism, we developed an in vitro fusion assay in which co-cultures of MMCs and myotubes were exposed to IGF-I. The number of fusion events was substantially augmented by IGF-I, independent of its effect on cell survival. These results provide novel evidence that a single factor, IGF-I, is sufficient to enhance the fusion of bone marrow derivatives with adult skeletal muscle.

A Steering Model of Endothelial Sheet Migration Recapitulates Monolayer Integrity and Directed Collective Migration

ABSTRACT Cells in endothelial cell monolayers maintain a tight barrier between blood and tissue, but it is not well understood how endothelial cells move within monolayers, pass each other, migrate when stimulated with growth factor, and also retain monolayer integrity. Here, we develop a quantitative steering model based on functional classes of genes identified previously in a small interfering RNA (siRNA) screen to explain how cells locally coordinate their movement to maintain monolayer integrity and collectively migrate in response to growth factor. In the model, cells autonomously migrate within the monolayer and turn in response to mechanical cues resulting from adhesive, drag, repulsive, and directed steering interactions with neighboring cells. We show that lateral-drag steering explains the local coordination of cell movement and the maintenance of monolayer integrity by allowing closure of small lesions. We further demonstrate that directional steering of cells at monolayer boundaries, combined with adhesive steering of cells behind, can explain growth factor-triggered collective migration into open space. Together, this model provides a mechanistic explanation for the observed genetic modularity and a conceptual framework for how cells can dynamically maintain sheet integrity and undergo collective directed migration.

A novel enzyme complementation-based assay for monitoring G-protein-coupled receptor internalization

G‐protein‐coupled receptor (GPCR) signaling is involved in a wide range of physiological processes and diseases, and around one‐half of currently used drugs target GPCRs. Assays for the signaling of GPCRs have suffered from drawbacks, including low signal‐to‐noise, temporally transient signals, and difficulty in applying a single assay to a wide range of GPCRs. We have developed a set of assays for G‐protein‐coupled receptor signaling based on β‐galacto‐sidase enzyme complementation in live mammalian cells. We previously described an assay for GPCR activation by monitoring the binding of β‐arrestin to the receptor. Here we describe a second assay that monitors the internalization of GPCRs to endosomes, an event that follows receptor activation and is critical in desensitizing and resensitizing the receptor. We show that both assays display high signal‐to‐noise ratios with low variability and are quantitative for a wide range of GPCRs. EC50s obtained with these assays closely match results reported in the literature. Finally, we show that these assays are readily adapted to high‐throughput chemical screens. Thus, these two assays for monitoring G‐protein‐coupled receptor activation and internal‐ization should prove valuable in basic biological studies as well as in high‐throughput screens.— Hammer M. M., Wehrman, T. S., Blau H. M. A novel enzyme complementation‐based assay for monitoring G‐protein‐coupled receptor internalization. FASEB J. 21, 3827–3834 (2007)

A universal technology for monitoring G-protein-coupled receptor activation in vitro and noninvasively in live animals

G‐protein coupled receptors (GPCRs) are a versatile and ubiquitous family of membrane receptors that transmit extracellular signals to mammalian cells and constitute the most important class of drug targets. Yet, sensitive and specific methods are lacking that would allow quantitative comparisons of pharmacologic properties of these receptors in physiological or pathological settings in live animals. We sought to overcome these limitations by employing low affinity, reversible β‐galactosidase complementation to quantify GPCR activation via interaction with β‐arres‐tin. A panel of cell lines was engineered expressing different GPCRs together with the reporter system. In vitro evaluation revealed highly sensitive, dynamic, and specific assessment of GPCR agonists and antagonists. Following implantation of the cells into mice, it was possible for the first time to monitor pharmacological GPCR activation and inhibition in their physiological context by noninvasive bioluminescence imaging in living animals. This technology has unique advantages that enable novel applications in the functional investigation of GPCR modulation in live animals in biological research and drug discovery.— von Degenfeld G., Wehrman T. S., Hammer, M. M., Blau H. M. A universal technology for monitoring G‐protein‐coupled receptor activation in vitro and noninvasively in live animals. FASEB J. 21, 3819–3826 (2007)

Pulmonary Tuberculosis: Role of Radiology in Diagnosis and Management.

Tuberculosis is a public health problem worldwide, including in the United States-particularly among immunocompromised patients and other high-risk groups. Tuberculosis manifests in active and latent forms. Active disease can occur as primary tuberculosis, developing shortly after infection, or postprimary tuberculosis, developing after a long period of latent infection. Primary tuberculosis occurs most commonly in children and immunocompromised patients, who present with lymphadenopathy, pulmonary consolidation, and pleural effusion. Postprimary tuberculosis may manifest with cavities, consolidations, and centrilobular nodules. Miliary tuberculosis refers to hematogenously disseminated disease that is more commonly seen in immunocompromised patients, who present with miliary lung nodules and multiorgan involvement. The principal means of testing for active tuberculosis is sputum analysis, including smear, culture, and nucleic acid amplification testing. Imaging findings, particularly the presence of cavitation, can affect treatment decisions, such as the duration of therapy. Latent tuberculosis is an asymptomatic infection that can lead to postprimary tuberculosis in the future. Patients who are suspected of having latent tuberculosis may undergo targeted testing with a tuberculin skin test or interferon-γ release assay. Chest radiographs are used to stratify for risk and to assess for asymptomatic active disease. Sequelae of previous tuberculosis that is now inactive manifest characteristically as fibronodular opacities in the apical and upper lung zones. Stability of radiographic findings for 6 months distinguishes inactive from active disease. Nontuberculous mycobacterial disease can sometimes mimic the findings of active tuberculosis, and laboratory confirmation is required to make the distinction. Familiarity with the imaging, clinical, and laboratory features of tuberculosis is important for diagnosis and management. ©RSNA, 2017.

WebFlow: A Software Package for High-Throughput Analysis of Flow Cytometry Data

Flow cytometry has emerged as a powerful tool for quantitative, single-cell analysis of both surface markers and intracellular antigens, including phosphoproteins and kinase signaling cascades, with the flexibility to process hundreds of samples in multiwell plate format. Quantitative flow cytometric analysis is being applied in many areas of biology, from the study of immunology in animal models or human patients to high-content drug screening of pharmacologically active compounds. However, these experiments generate thousands of data points per sample, each with multiple measured parameters, leading to data management and analysis challenges. We developed WebFlow (http://webflow.stanford.edu), a web server-based software package to manage, analyze, and visualize data from flow cytometry experiments. WebFlow is accessible via standard web browsers and does not require users to install software on their personal computers. The software enables plate-based annotation of large data sets, which provides the basis for exploratory data analysis tools and rapid visualization of multiple different parameters. These tools include custom user-defined statistics to normalize data to other wells or other channels, as well as interactive, user-selectable heat maps for viewing the underlying single-cell data. The web-based approach of WebFlow allows for sharing of data with collaborators or the general public. WebFlow provides a novel platform for quantitative analysis of flow cytometric data from high-throughput drug screening or disease profiling experiments.

Imaging in blunt cardiac injury: Computed tomographic findings in cardiac contusion and associated injuries

BACKGROUND Blunt cardiac injury (BCI) may manifest as cardiac contusion or, more rarely, as pericardial or myocardial rupture. Computed tomography (CT) is performed in the vast majority of blunt trauma patients, but the imaging features of cardiac contusion are not well described. PURPOSE To evaluate CT findings and associated injuries in patients with clinically diagnosed BCI. MATERIALS AND METHODS We identified 42 patients with blunt cardiac injury from our institutions electronic medical record. Clinical parameters, echocardiography results, and laboratory tests were recorded. Two blinded reviewers analyzed chest CTs performed in these patients for myocardial hypoenhancement and associated injuries. RESULTS CT findings of severe thoracic trauma are commonly present in patients with severe BCI; 82% of patients with ECG, cardiac enzyme, and echocardiographic evidence of BCI had abnormalities of the heart or pericardium on CT; 73% had anterior rib fractures, and 64% had pulmonary contusions. Sternal fractures were only seen in 36% of such patients. However, myocardial hypoenhancement on CT is poorly sensitive for those patients with cardiac contusion: 0% of right ventricular contusions and 22% of left ventricular contusions seen on echocardiography were identified on CT. CONCLUSION CT signs of severe thoracic trauma are frequently present in patients with severe BCI and should be regarded as indirect evidence of potential BCI. Direct CT findings of myocardial contusion, i.e. myocardial hypoenhancement, are poorly sensitive and should not be used as a screening tool. However, some left ventricular contusions can be seen on CT, and these patients could undergo echocardiography or cardiac MRI to evaluate for wall motion abnormalities.

Acute traumatic aortic injury: practical considerations for the diagnostic radiologist.

The diagnosis of acute traumatic aortic injury (ATAI) relies heavily on accurate and efficient imaging interpretation, thereby making the radiologist integral to the care of patients in whom these life-threatening lesions are suspected. Typically, this evaluation begins with the initial trauma radiograph, in which findings suggestive of mediastinal hematoma or ATAI can be detected. Definitive diagnosis of ATAI is made with the current gold standard, computed tomography, wherein indirect and direct signs of ATAI provide the means for sensitive and specific diagnosis. Although the diagnosis of ATAI on computed tomography can be straightforward, technical and anatomic pitfalls can complicate interpretation and must be understood. Once the diagnosis is made, the radiologist needs to provide a meaningful report that includes an appropriate description of the lesion location and characteristics. The purpose of this article is to review the key aspects of the imaging evaluation of ATAI with a focus on factors that affect the management of these patients.

Accuracy of computed tomography findings in acute pericarditis

Background: Acute pericarditis is a close clinical mimic of pulmonary embolism (PE) in the emergency department, and thus many of these patients are evaluated with chest computed tomography (CT). Purpose: To study whether CT findings can be diagnostic of acute pericarditis. Material and Methods: Using the electronic medical record, we retrospectively identified 46 cases of acute pericarditis and 46 control patients with pericardial effusions due to volume overload, all of whom underwent CT examination. Cases were reviewed by two blinded academic thoracic radiologists. Results: The majority, 67%, of the pericarditis cases were evaluated with PE-protocol CTs. Pericardial thickening/enhancement was the most accurate single parameter for pericarditis, with sensitivity of 54–59% and specificity of 91–96%. Conclusion: CT findings, while not sensitive for pericarditis, are diagnostic, with few false-positives. Radiologists should be attentive to pericardial thickening or enhancement on CT studies done for chest pain, as they may be able to suggest pericarditis as an alternative diagnosis for the chest pain.

Traumatic injuries of the diaphragm: overview of imaging findings and diagnosis

Injuries to the diaphragm muscle occur in penetrating and severe blunt trauma and can lead to delayed hernia formation. Computed tomography is the mainstay in the diagnosis of these injuries, which may be subtle at presentation. Imaging findings differ between blunt and penetrating trauma. Key features in blunt trauma include diaphragm fragment distraction and organ herniation because of increased intra-abdominal pressure. In penetrating trauma, herniation is uncommon, and the trajectory of the object is critical in making the diagnosis of diaphragm injury in these patients. Radiologists must keep a high index of suspicion for injury to the diaphragm in cases of trauma to the chest or abdomen.