Michelle Longmire

Systemic sclerosis (SSc) is a disease at the intersection of autoimmunity and fibrosis. However, the epigenetic regulation and the contributions of diverse cell types to SSc remain unclear. Here we survey, using ATAC-seq, the active DNA regulatory elements of eight types of primary cells in normal skin from healthy controls, as well as clinically affected and unaffected skin from SSc patients. We find that accessible DNA elements in skin-resident dendritic cells (DCs) exhibit the highest… Show more

Systemic sclerosis (SSc) is a disease at the intersection of autoimmunity and fibrosis. However, the epigenetic regulation and the contributions of diverse cell types to SSc remain unclear. Here we survey, using ATAC-seq, the active DNA regulatory elements of eight types of primary cells in normal skin from healthy controls, as well as clinically affected and unaffected skin from SSc patients. We find that accessible DNA elements in skin-resident dendritic cells (DCs) exhibit the highest enrichment of SSc-associated single-nucleotide polymorphisms (SNPs) and predict the degrees of skin fibrosis in patients. DCs also have the greatest disease-associated changes in chromatin accessibility and the strongest alteration of cell-cell interactions in SSc lesions. Lastly, data from an independent cohort of patients with SSc confirm a significant increase of DCs in lesioned skin. Thus, the DCs epigenome links inherited susceptibility and clinically apparent fibrosis in SSc skin, and can be an important driver of SSc pathogenesis. Show less

Human tumors consist of heterogeneous populations of cells with distinct marker expression and functional properties. In squamous cell carcinoma of the head and neck (SCCHN), CD44 is a well-characterized marker of a resilient subpopulation of cells associated with increased tumorigenesis, radioresistance, and chemoresistance. Evidence indicates that these cells have an immunosuppressive phenotype; however, mechanisms have been elusive.

Here we survey variation and dynamics of active regulatory elements genome-wide using longitudinal samples from human individuals. We applied Assay of Transposase Accessible Chromatin with sequencing (ATAC-seq) to map chromatin accessibility in primary CD4+ T cells isolated from standard blood draws of 12 healthy volunteers over time, from cancer patients, and during T cell activation. Over 4,000 predicted regulatory elements (7.2%) showed reproducible variation in accessibility between… Show more

Here we survey variation and dynamics of active regulatory elements genome-wide using longitudinal samples from human individuals. We applied Assay of Transposase Accessible Chromatin with sequencing (ATAC-seq) to map chromatin accessibility in primary CD4+ T cells isolated from standard blood draws of 12 healthy volunteers over time, from cancer patients, and during T cell activation. Over 4,000 predicted regulatory elements (7.2%) showed reproducible variation in accessibility between individuals. Gender was the most significant attributable source of variation. ATAC-seq revealed previously undescribed elements that escape X chromosome inactivation and predicted gender-specific gene regulatory networks across autosomes, which coordinately affect genes with immune function. Noisy regulatory elements with personal variation in accessibility are significantly enriched for autoimmune disease loci. Over one third of regulome variation lacked genetic variation in cis, suggesting contributions from environmental or epigenetic factors. These results refine concepts of human individuality and provide a foundational reference for comparing disease-associated regulomes. Show less

Drug-induced immune thrombocytopenia has been associated with hundreds of medications and can lead to devastating consequences for the patient. We present a case of a healthy 33-year-old female undergoing in vitro fertilization who developed a severe drug-induced thrombocytopenia, petechiae, and a large hemoperitoneum after receiving Cefazolin antibiotic prophylaxis for a transvaginal oocyte retrieval. The patient was admitted to the intensive care unit for resuscitation with blood products… Show more

Drug-induced immune thrombocytopenia has been associated with hundreds of medications and can lead to devastating consequences for the patient. We present a case of a healthy 33-year-old female undergoing in vitro fertilization who developed a severe drug-induced thrombocytopenia, petechiae, and a large hemoperitoneum after receiving Cefazolin antibiotic prophylaxis for a transvaginal oocyte retrieval. The patient was admitted to the intensive care unit for resuscitation with blood products. The presence of drug-dependent platelet antibodies to Cefazolin was confirmed serologically. Show less

Cutaneous small vessel vasculitis (CSVV) is a nonspecific finding with an extensive differential diagnosis. It is critically important to distinguish skin-limited presentations of CSVV from severe life-threatening systemic vasculitides presenting with CSVV as an initial manifestation. It can be challenging to determine which patients presenting with CSVV are at risk for systemic disease. Standard histopathologic evaluation, direct immunofluorescence, and serologic evaluation is typically… Show more

Cutaneous small vessel vasculitis (CSVV) is a nonspecific finding with an extensive differential diagnosis. It is critically important to distinguish skin-limited presentations of CSVV from severe life-threatening systemic vasculitides presenting with CSVV as an initial manifestation. It can be challenging to determine which patients presenting with CSVV are at risk for systemic disease. Standard histopathologic evaluation, direct immunofluorescence, and serologic evaluation is typically required to exclude a systemic vasculitis. Type 1 cryoglobulinemia may rarely present with CSVV. Herein, we report a case of type 1 cryoglobulinemia in the setting of occult multiple myeloma. CSVV with prominent intravascular crystal formation was noted. The presence of intravascular crystals in the setting of CSVV may represent an important early clue to the diagnosis of type 1 cryoglobulinemic vasculitis. Show less

Service-based learning offered an efficient model for incorporating ophthalmic training into the medical school curriculum. A viable tool for quantitatively testing ophthalmoscope skills is presented.

Dendrimers are versatile macromolecules with tremendous potential as magnetic resonance imaging (MRI) contrast agents. Dendrimer-based agents provide distinct advantages over low-molecular-weight gadolinium chelates, including enhanced r1 relaxivity due to slow rotational dynamics, tunable pharmacokinetics that can be adapted for blood pool, liver, kidney, and lymphatic imaging, the ability to be a drug carrier, and flexibility for labeling due to their inherent multivalency. Clinical… Show more

Dendrimers are versatile macromolecules with tremendous potential as magnetic resonance imaging (MRI) contrast agents. Dendrimer-based agents provide distinct advantages over low-molecular-weight gadolinium chelates, including enhanced r1 relaxivity due to slow rotational dynamics, tunable pharmacokinetics that can be adapted for blood pool, liver, kidney, and lymphatic imaging, the ability to be a drug carrier, and flexibility for labeling due to their inherent multivalency. Clinical applications are increasingly being developed, particularly in lymphatic imaging. Herein we present a broad overview of dendrimer-based MRI contrast agents with attention to the unique chemistry and physical properties as well as emerging clinical applications. Show less

Conventional diagnostic imaging methods such as X-ray CT, MRI, and nuclear medicine are inherently monochromatic meaning that they can depict only one molecular target at a time. Optical imaging has the unique ability to be polychromatic and therefore multi-color imaging employing targeted agents conjugated to fluorophores of varying wavelength enables multiple simultaneous readouts thus providing greater multiplexed information. Numerous successful multicolor imaging techniques have recently… Show more

Conventional diagnostic imaging methods such as X-ray CT, MRI, and nuclear medicine are inherently monochromatic meaning that they can depict only one molecular target at a time. Optical imaging has the unique ability to be polychromatic and therefore multi-color imaging employing targeted agents conjugated to fluorophores of varying wavelength enables multiple simultaneous readouts thus providing greater multiplexed information. Numerous successful multicolor imaging techniques have recently been reported using optical imaging in in vivo animal disease models, thus adding to a growing body of research supporting the clinical viability and applicability of these technologies. Herein, we review multicolor optical imaging from the basic chemistry and physics perspective and then extend this to biological and medical applications. Show less

Perineural granulomatous inflammation resembling the perineural infiltrate of leprosy appears to be an uncommon characteristic of granuloma annulare. Clinical correlation and acid-fast stains can assist in establishing the correct diagnosis.

Near infrared fluorescence-guidance can be used for the detection of small cancer metastases and can aid in the endoscopic management of cancer. Indocyanine green (ICG) is a Food and Drug Administration (FDA)-approved fluorescence agent. Through non-specific interactions with serum proteins, ICG achieves enhanced permeability and retention (EPR) effects. Yet, ICG demonstrates rapid clearance from the circulation. Therefore, ICG may be an ideal contrast agent for real-time fluorescence imaging… Show more

Near infrared fluorescence-guidance can be used for the detection of small cancer metastases and can aid in the endoscopic management of cancer. Indocyanine green (ICG) is a Food and Drug Administration (FDA)-approved fluorescence agent. Through non-specific interactions with serum proteins, ICG achieves enhanced permeability and retention (EPR) effects. Yet, ICG demonstrates rapid clearance from the circulation. Therefore, ICG may be an ideal contrast agent for real-time fluorescence imaging of tumors. To evaluate the usefulness of real-time dual fluorescence and white light endoscopic optical imaging to detect tumor implants using the contrast agent ICG, fluorescence-guided laparoscopic procedures were performed in mouse models of peritoneally disseminated ovarian cancers. Animals were administered intravenous ICG or a control contrast agent, IR800-conjugated to albumin. The ability to detect small ovarian cancer implants was then compared. Using the dual view microendoscope, ICG clearly enabled visualization of peritoneal ovarian cancer metastatic nodules derived from SHIN3 and OVCAR5 cells at 6 and 24 hr after injection with significantly higher tumor-to-background ratio than the control agent, IR800-albumin (p < 0.001). In conclusion, ICG has the desirable properties of having both EPR effects and rapid clearance for the real-time endoscopic detection of tiny ovarian cancer peritoneal implants compared to a control macromolecular agent with theoretically better EPR effects but longer circulatory retention. Given that ICG is already FDA-approved and has a long track record of human use, this method could be easily translated to the clinic as a robust tool for fluorescence-guided endoscopic procedures for the management and treatment of cancer. Show less

In recent years, numerous in vivo molecular imaging probes have been developed. As a consequence, much has been published on the design and synthesis of molecular imaging probes focusing on each modality, each type of material, or each target disease. More recently, second generation molecular imaging probes with unique, multi-functional, or multiplexed characteristics have been designed. This critical review focuses on (i) molecular imaging using combinations of modalities and signals that… Show more

In recent years, numerous in vivo molecular imaging probes have been developed. As a consequence, much has been published on the design and synthesis of molecular imaging probes focusing on each modality, each type of material, or each target disease. More recently, second generation molecular imaging probes with unique, multi-functional, or multiplexed characteristics have been designed. This critical review focuses on (i) molecular imaging using combinations of modalities and signals that employ the full range of the electromagnetic spectra, (ii) optimized chemical design of molecular imaging probes for in vivo kinetics based on biology and physiology across a range of physical sizes, (iii) practical examples of second generation molecular imaging probes designed to extract complementary data from targets using multiple modalities, color, and comprehensive signals (277 references). Show less

The expanded biological and medical applications of nanomaterials place a premium on better understanding of the chemical and physical determinants of in vivo particles. Nanotechnology allows us to design a vast array of molecules with distinct chemical and biological characteristics, each with a specific size, charge, hydrophilicity, shape, and flexibility. To date, much research has focused on the role of particle size as a determinant of biodistribution and clearance. Additionally, much of… Show more

The expanded biological and medical applications of nanomaterials place a premium on better understanding of the chemical and physical determinants of in vivo particles. Nanotechnology allows us to design a vast array of molecules with distinct chemical and biological characteristics, each with a specific size, charge, hydrophilicity, shape, and flexibility. To date, much research has focused on the role of particle size as a determinant of biodistribution and clearance. Additionally, much of what we know about the relationship between nanoparticle traits and pharmacokinetics has involved research limited to the gross average hydrodynamic size. Yet, other features such as particle shape and flexibility affect in vivo behavior and become increasingly important for designing and synthesizing nanosized molecules. Herein, we discuss determinants of in vivo behavior of nanosized molecules used as imaging agents with a focus on dendrimer-based contrast agents. We aim to discuss often overlooked or, yet to be considered, factors that affect in vivo behavior of synthetic nanosized molecules, as well as aim to highlight important gaps in current understanding. Show less

A 9-year-old girl presented with a choroidal tumor 6 years after remission of Burkitt lymphoma with no evidence of systemic recurrence. The tumor regressed after plaque radiotherapy. The second tumor could have been related to previous chemotherapy, caused by Epstein-Barr virus infection, or the result of independent lymphoma cell growth.

Filamentous phage display has been extensively used to select proteins with binding properties of specific interest. Although many different display platforms using filamentous phage have been described, no comprehensive comparison of their abilities to display similar proteins has been conducted. This is particularly important for the display of cytoplasmic proteins, which are often poorly displayed with standard filamentous phage vectors. In this article, we have analyzed the ability of… Show more

Filamentous phage display has been extensively used to select proteins with binding properties of specific interest. Although many different display platforms using filamentous phage have been described, no comprehensive comparison of their abilities to display similar proteins has been conducted. This is particularly important for the display of cytoplasmic proteins, which are often poorly displayed with standard filamentous phage vectors. In this article, we have analyzed the ability of filamentous phage to display a stable form of green fluorescent protein and modified variants in nine different display vectors, a number of which have been previously proposed as being suitable for cytoplasmic protein display. Correct folding and display were assessed by phagemid particle fluorescence, and with anti-GFP antibodies. The poor correlation between phagemid particle fluorescence and recognition of GFP by antibodies, indicates that proteins may fold correctly without being accessible for display. The best vector used a twin arginine transporter leader to transport the displayed protein to the periplasm, and a coil-coil arrangement to link the displayed protein to g3p. This vector was able to display less robust forms of GFP, including ones with inserted epitopes, as well as fluorescent proteins of the Azami green series. It was also functional in mock selection experiments. Show less

One of the primary goals of oncological molecular imaging is to accurately identify and characterize malignant tissues in vivo. Currently, molecular imaging relies on targeting a single molecule that while overexpressed in malignancy, is often also expressed at lower levels in normal tissue, resulting in reduced tumor to background ratios. One approach to increasing the specificity of molecular imaging in cancer is to use multiple probes each with distinct fluorescence to target several surface… Show more

One of the primary goals of oncological molecular imaging is to accurately identify and characterize malignant tissues in vivo. Currently, molecular imaging relies on targeting a single molecule that while overexpressed in malignancy, is often also expressed at lower levels in normal tissue, resulting in reduced tumor to background ratios. One approach to increasing the specificity of molecular imaging in cancer is to use multiple probes each with distinct fluorescence to target several surface antigens simultaneously, in order to identify tissue expression profiles, rather than relying on the expression of a single target. This next step forward in molecular imaging will rely on characterization of tissue based on fluorescence and therefore will require the ability to simultaneously identify several optical probes each attached to different targeting ligands. We created a novel 'coincident' ovarian cancer mouse model by coinjecting each animal with two distinct cell lines, HER2+/red fluorescent protein (RFP)- SKOV3 and HER2-/RFP+ SHIN3-RFP, in order to establish a model of disease in which animals simultaneously bore tumors with two distinct phenotypes (HER2+/RFP-, HER2-/RFP+), which could be utilized for multicolor imaging. The HER2 receptor of the SKOV3 cell line was targeted with a trastuzumab-rhodamine green conjugate to create green tumor implants, whereas the RFP plasmid of the SHIN3 cells created red tumor implants. We demonstrate that real-time in vivo multicolor imaging is feasible and that fluorescence characteristics can then serve to guide the surgical removal of disease. Show less

In vivo molecularly targeted fluorescence imaging of tumors has been proposed as a strategy for improving cancer detection and management. Activatable fluorophores, which increased their fluorescence by 10-fold after binding tumor cells, result in much higher target to background ratios than conventional fluorophores. We developed an in vivo targeted activatable optical imaging probe based on a fluorophore-quencher pair, bound to a targeting moiety. With this system, fluorescence is quenched by… Show more

In vivo molecularly targeted fluorescence imaging of tumors has been proposed as a strategy for improving cancer detection and management. Activatable fluorophores, which increased their fluorescence by 10-fold after binding tumor cells, result in much higher target to background ratios than conventional fluorophores. We developed an in vivo targeted activatable optical imaging probe based on a fluorophore-quencher pair, bound to a targeting moiety. With this system, fluorescence is quenched by the fluorophore-quencher interaction outside cancer cells, but is activated within the target cells by dissociation of the fluorophore-quencher pair. We selected the TAMRA (fluorophore)-QSY7 (quencher) pair and conjugated it to either avidin (targeting the D-galactose receptor) or trastuzumab (a monoclonal antibody against the human epithelial growth factor receptor type2 (HER2/neu)) and evaluated their performance in mouse models of cancer. Two probes, TAMRA-QSY7 conjugated avidin (Av-TM-Q7) and trastuzumab (Traz-TM-Q7) were synthesized. Both demonstrated better than similar self-quenching probes. In vitro fluorescence microscopic studies of SHIN3 and NIH/3T3/HER2+ cells demonstrated that Av-TM-Q7 and Traz-TM-Q7 produced high intracellular fluorescent signal. In vivo imaging with Av-TM-Q7 and Traz-TM-Q7 in mice enabled the detection of small tumors. This molecular imaging probe, based on a fluorophore-quencher pair conjugated to a targeting ligand, successfully detected tumors in vivo due to its high activation ratio and low background signal. Thus, these activatable probes, based on the fluorophore-quencher system, hold promise clinically for "see and treat" strategies of cancer management. Show less

Commonly used in flow cytometry, multiplexed optical probes can diagnose multiple types of cell surface marker, potentially leading to improved diagnosis accuracy in vivo. Herein, we demonstrate the targeting of two different tumor markers in models of disseminated ovarian cancer. Two ovarian cancer cell lines (SKOV3 and SHIN3) were employed; both overexpress D-galactose receptor (D-galR), but only SKOV3 overexpresses HER2/neu. Additionally, fusion tumors composed of SKOV3 and SHIN3/RFP were… Show more

Commonly used in flow cytometry, multiplexed optical probes can diagnose multiple types of cell surface marker, potentially leading to improved diagnosis accuracy in vivo. Herein, we demonstrate the targeting of two different tumor markers in models of disseminated ovarian cancer. Two ovarian cancer cell lines (SKOV3 and SHIN3) were employed; both overexpress D-galactose receptor (D-galR), but only SKOV3 overexpresses HER2/neu. Additionally, fusion tumors composed of SKOV3 and SHIN3/RFP were evaluated. Both galactosyl serum albumin-rhodamine green (GSA-RhodG), which binds D-galR, and trastuzumab-Alexa680, which binds HER2/neu, were administered to tumor-bearing mice for in vivo fluorescence imaging and in situ fluorescence microscopy. In vivo fluorescence imaging depicted 64 of 69 SKOV3 tumors (94.2%) based on their dual spectra corresponding to both RhodG and Alexa680, while all 71 SHIN3 tumors (100%) were detected based on their single spectrum corresponding only to RhodG. All 59 SHIN3 and 36 SKOV3 tumors were correctly diagnosed with in situ microscopy. Additionally, in the mixed tumor model, all tumors could be depicted using the RhodG spectrum, but only SKOV3 components also showed the Alexa680 spectrum. In conclusion, multitargeted multicolor optical imaging enabled specific in vivo diagnosis of tumors expressing distinct patterns of receptors, leading to improved diagnostic accuracy. Show less

Nanoparticles possess enormous potential as diagnostic imaging agents and hold promise for the development of multimodality agents with both imaging and therapeutic capabilities. Yet, some of the most promising nanoparticles demonstrate prolonged tissue retention and contain heavy metals. This presents serious concerns for toxicity. The creation of nanoparticles with optimal clearance characteristics will minimize toxicity risks by reducing the duration of exposure to these agents. Given that… Show more

Nanoparticles possess enormous potential as diagnostic imaging agents and hold promise for the development of multimodality agents with both imaging and therapeutic capabilities. Yet, some of the most promising nanoparticles demonstrate prolonged tissue retention and contain heavy metals. This presents serious concerns for toxicity. The creation of nanoparticles with optimal clearance characteristics will minimize toxicity risks by reducing the duration of exposure to these agents. Given that many nanoparticles possess easily modifiable surface and interior chemistry, if nanoparticle characteristics associated with optimal clearance from the body were well established, it would be feasible to design and create agents with more favorable clearance properties. This article presents a thorough discussion of the physiologic aspects of nanoparticle clearance, focusing on renal mechanisms, and provides an overview of current research investigating clearance of specific types of nanoparticles and nano-sized macromolecules, including dendrimers, quantum dots, liposomes and carbon, gold and silica-based nanoparticles. Show less

Optical imaging has the potential to improve the efficacy of surgical and endoscopic approaches to cancer treatment; however, the optimal type of fluorescent probe has not yet been established. It is well-known that rhodamine-core-derived fluorophores offer a combination of desirable properties such as good photostability, high extinction coefficient, and high fluorescence quantum yield. However, despite the ubiquitous use of rhodamine fluorophores for in vivo optical imaging, it remains to be… Show more

Optical imaging has the potential to improve the efficacy of surgical and endoscopic approaches to cancer treatment; however, the optimal type of fluorescent probe has not yet been established. It is well-known that rhodamine-core-derived fluorophores offer a combination of desirable properties such as good photostability, high extinction coefficient, and high fluorescence quantum yield. However, despite the ubiquitous use of rhodamine fluorophores for in vivo optical imaging, it remains to be determined if unique chemical properties among individual rhodamine core family members affect fluorophore parameters critical to in vivo optical imaging applications. These parameters include preserved fluorescence intensity in low pH environments, similar to that of the endolysosome; efficient fluorescence signal despite conformational changes to targeting proteins as may occur in harsh subcellular environments; persistence of fluorescence after cellular internalization; and sufficient signal-to-background ratios to permit the identification of fluorophore-targeted tumors. In the present study, we conjugated 4 common rhodamine-core based fluorescent dyes to a clinically feasible and quickly internalizing D-galactose receptor targeting reagent, galactosamine serum albumin (GmSA), and conducted a series of in vitro and in vivo experiments using a metastatic ovarian cancer mouse model to determine if differences in optical imaging properties exist among rhodamine fluorophores and if so, which rhodamine core possesses optimal characteristics for in vivo imaging applications. Herein, we demonstrate that the rhodamine-fluorophore, TAMRA, is the most robust of the 4 common rhodamine fluorophores for in vivo optical imaging of ovarian cancer metastases to the peritoneum. Show less

The extensive adaptability of dendrimer-based contrast agents is ideal for the molecular imaging of organs and other target-specific locations. The ability of literally atom-by-atom modification on cores, interiors, and surface groups, permits the rational manipulation of dendrimer-based agents in order to optimize their physical characteristics, biodistribution, receptor-mediated targeting, and controlled release of the payload. Such modifications enable agents to localize preferentially to… Show more

The extensive adaptability of dendrimer-based contrast agents is ideal for the molecular imaging of organs and other target-specific locations. The ability of literally atom-by-atom modification on cores, interiors, and surface groups, permits the rational manipulation of dendrimer-based agents in order to optimize their physical characteristics, biodistribution, receptor-mediated targeting, and controlled release of the payload. Such modifications enable agents to localize preferentially to areas or organs of interest for facilitating target-specific imaging as well as assume excretion pathways that do not interfere with desired applications. Recent innovations in dendrimer research have increased agent directibility and new synthetic chemistry approaches have increased efficiency of production as well as led to the creation of novel dendrimer-based contrast agents. In addition, by taking advantage of the numerous attachment sites available on the surface of a single dendrimer molecule, new synthetic chemistry techniques have led to the development of multi-modality magnetic resonance, radionuclide, and fluorescence imaging agents for molecular imaging. Herein we discuss advances in dendrimer-based contrast agents for molecular imaging focusing mainly on the chemical design as applied to optical, magnetic resonance, computer tomography, radionuclide, and multi-modality imaging. Show less