TCF1-LEF1 co-expression identifies a multipotent progenitor cell (TH2-MPP) across human allergic diseases.

Repetitive exposure to antigen in chronic infection and cancer drives T cell exhaustion, limiting adaptive immunity. In contrast, aberrant, sustained T cell responses can persist over decades in human allergic disease. To understand these divergent outcomes, we employed bioinformatic, immunophenotyping and functional approaches with human diseased tissues, identifying an abundant population of type 2 helper T (TH2) cells with co-expression of TCF7 and LEF1, and features of chronic activation. These cells, which we termed TH2-multipotent progenitors (TH2-MPP) could self-renew and differentiate into cytokine-producing effector cells, regulatory T (Treg) cells and follicular helper T (TFH) cells. Single-cell T-cell-receptor lineage tracing confirmed lineage relationships between TH2-MPP, TH2 effectors, Treg cells and TFH cells. TH2-MPP persisted despite in vivo IL-4 receptor blockade, while thymic stromal lymphopoietin (TSLP) drove selective expansion of progenitor cells and rendered them insensitive to glucocorticoid-induced apoptosis in vitro. Together, our data identify TH2-MPP as an aberrant T cell population with the potential to sustain type 2 inflammation and support the paradigm that chronic T cell responses can be coordinated over time by progenitor cells.

Lymphatic/blood vessel plasticity: motivation for a future research area based on present and past observations.

The lymphatic system plays a significant role in homeostasis and drainage of excess fluid back into venous circulation. Lymphatics are also associated with a number of diseases including lymphedema, tumor metastasis, and various lymphatic malformations. Emerging evidence suggests that lymphatics might have a bigger connection to the blood vascular system than originally presumed. As these two systems are often studied in isolation, several knowledge gaps exist surrounding what constitutes lymphatic vascular plasticity, under what conditions it arises, and where structures characteristic of plasticity can form. The objective of this review is to overview current structural, cell lineage-based, and cell identity-based evidence for lymphatic plasticity. These examples of plasticity will then be considered in the context of potential clinical and surgical implications of this evolving research area. This review details our current understanding of lymphatic plasticity, highlights key unanswered questions in the field, and motivates future research aimed at clarifying the role and therapeutic potential of lymphatic plasticity in disease.

Outcomes for Physiologic Microsurgical Treatment of Secondary Lymphedema Involving the Extremity.

OBJECTIVE: The aim of this study was to examine the long-term impact of physiologic surgical options, including VLNT and LVB, on patients with secondary lymphedema of the upper or lower extremity (UEL/LEL). SUMMARY BACKGROUND DATA: VLNT and LVB have become increasingly popular in the treatment of lymphedema. However, there is a paucity of long-term data on patient outcomes after use of these techniques to treat lymphedema. METHODS: An analysis of prospectively collected data on all patients who underwent physiologic surgical treatment of secondary lymphedema over a 5.5-year period was performed. Patient demographics, surgical details, subjective reported improvements, LLIS scores, and postoperative limb volume calculations were analyzed. RESULTS: Two hundred seventy-four patients with secondary lymphedema (197 upper, 77 lower) were included in the study. More than 87% of UEL patients and 60% of LEL patients had reduction in excess limb volume postoperatively. At 3 months postoperatively, patients with UEL had a 31.1% reduction in volume difference between limbs, 33.9% at 6 months, 25.7% at 12 months, 47.4% at 24 months and 47.7% at 4 years. The reduction in limb volume difference followed a similar pattern but was overall lower for LEL patients. Greater than 86% of UEL and 75% of LEL patients also had improvement in LLIS scores postoperatively. Fifty-nine complications occurred (12.9%); flap survival was >99%. CONCLUSIONS: Patients with secondary UEL/LEL who undergo VLNT/LVB demonstrate improved functional status and reduced affected limb volumes postoperatively. Patients with UEL seem to have a more substantial reduction in limb volume differential compared to LEL patients.

Lymphovenous shunts: from development to clinical applications.

The lymphatic system is a vast network of vessels that functions to return excess fluid from the interstitial space to the blood stream. Lymphovenous shunts are anastomoses, either natural or surgical, that connect the lymphatic and venous systems. Connections between the thoracic duct and venous system or between the right lymphatic duct and venous system are prime examples of anatomic lymphovenous shunts. Lymphovenous shunts are also present peripherally in tissues such as lymph nodes. Furthermore, pathologic lymphovenous shunts are observed in conditions such as lymphedema, malignancy, and lymphovenous malformations. Surgically, lymphovenous shunts may be constructed as an approach to treat lymphedema. Here, we discuss anatomic and surgical lymphovenous shunts in the context of normal development and disease. This perspective is intended to give an understanding of the role of lymphovenous shunts in health and disease and to show how they can be leveraged to treat disease surgically.

Coordinating Tissue Regeneration Through Transforming Growth Factor-ß Activated Kinase 1 Inactivation and Reactivation.

Aberrant wound healing presents as inappropriate or insufficient tissue formation. Using a model of musculoskeletal injury, we demonstrate that loss of transforming growth factor-ß activated kinase 1 (TAK1) signaling reduces inappropriate tissue formation (heterotopic ossification) through reduced cellular differentiation. Upon identifying increased proliferation with loss of TAK1 signaling, we considered a regenerative approach to address insufficient tissue production through coordinated inactivation of TAK1 to promote cellular proliferation, followed by reactivation to elicit differentiation and extracellular matrix production. Although the current regenerative medicine paradigm is centered on the effects of drug treatment ("drug on"), the impact of drug withdrawal ("drug off") implicit in these regimens is unknown. Because current TAK1 inhibitors are unable to phenocopy genetic Tak1 loss, we introduce the dual-inducible COmbinational Sequential Inversion ENgineering (COSIEN) mouse model. The COSIEN mouse model, which allows us to study the response to targeted drug treatment ("drug on") and subsequent withdrawal ("drug off") through genetic modification, was used here to inactivate and reactivate Tak1 with the purpose of augmenting tissue regeneration in a calvarial defect model. Our study reveals the importance of both the "drug on" (Cre-mediated inactivation) and "drug off" (Flp-mediated reactivation) states during regenerative therapy using a mouse model with broad utility to study targeted therapies for disease. Stem Cells 2019;37:766-778.

Lymphatic Microsurgical Preventive Healing Approach (LYMPHA) for the prevention of secondary lymphedema.

Lymphedema is a chronic, morbid condition in which the upper or lower extremity experiences swelling and fibrosis due to impaired lymphatic clearance. Among breast cancer patients, this condition is primarily attributed to axillary lymph node dissection (ALND) performed for oncologic management. While nonoperative and operative approaches to lymphedema management may be implemented to "manage" this condition, they are typically not curative. Therefore, lymphedema prevention in patients who have undergone ALND is of critical importance. Here, we briefly describe lymphedema and available management strategies, and focus on prevention in patients undergoing ALND using the Lymphatic Microsurgical Preventive Healing Approach (LYMPHA). Currently available clinical and experimental evidence suggests that LYMPHA may provide protection against the development of lymphedema in carefully selected patients. This procedure can serve as an adjunct surgical option for patients at the time of ALND.

Effect of Adenylyl Cyclase Type 6 on Localized Production of cAMP by ß-2 Adrenoceptors in Human Airway Smooth-Muscle Cells.

ß 2-Adrenoceptors (ß 2ARs) are concentrated in caveolar lipid raft domains of the plasma membrane in airway smooth-muscle (ASM) cells, along with adenylyl cyclase type 6 (AC6). This is believed to contribute to how these receptors can selectively regulate certain types of cAMP-dependent responses in these cells. The goal of the present study was to test the hypothesis that ß 2AR production of cAMP is localized to specific subcellular compartments using fluorescence resonance energy transfer-based cAMP biosensors targeted to different microdomains in human ASM cells. Epac2-MyrPalm and Epac2-CAAX biosensors were used to measure responses associated with lipid raft and nonraft regions of the plasma membrane, respectively. Activation of ß 2ARs with isoproterenol produced cAMP responses that are most readily detected in lipid raft domains. Furthermore, overexpression of AC6 somewhat paradoxically inhibited ß 2AR production of cAMP in lipid raft domains without affecting ß 2AR responses detected in other subcellular locations or cAMP responses to EP2 prostaglandin receptor activation, which were confined primarily to nonraft domains of the plasma membrane. The inhibitory effect of overexpressing AC6 was blocked by inhibition of phosphodiesterase type 4 (PDE4) activity with rolipram, inhibition of protein kinase A (PKA) activity with H89, and inhibition of A kinase anchoring protein (AKAP) interactions with the peptide inhibitor Ht31. These results support the idea that overexpression of AC6 leads to enhanced feedback activation of PDE4 via phosphorylation by PKA that is part of an AKAP-dependent signaling complex. This provides insight into the molecular basis for localized regulation of cAMP signaling in human ASM cells.

Characterizing the Circulating Cell Populations in Traumatic Heterotopic Ossification.

Heterotopic ossification (HO) occurs secondary to trauma, causing pain and functional limitations. Identification of the cells that contribute to HO is critical to the development of therapies. Given that innate immune cells and mesenchymal stem cells are known contributors to HO, we sought to define the contribution of these populations to HO and to identify what, if any, contribution circulating populations have to HO. A shared circulation was obtained using a parabiosis model, established between an enhanced green fluorescent protein-positive/luciferase+ donor and a same-strain nonreporter recipient mouse. The nonreporter mouse received Achilles tendon transection and dorsal burn injury to induce HO formation. Bioluminescence imaging and immunostaining were performed to define the circulatory contribution of immune and mesenchymal cell populations. Histologic analysis showed circulating cells present throughout each stage of the developing HO anlagen. Circulating cells were present at the injury site during the inflammatory phase and proliferative period, with diminished contribution in mature HO. Immunostaining demonstrated that most early circulatory cells were from the innate immune system; only a small population of mesenchymal cells were present in the HO. We demonstrate the time course of the participation of circulatory cells in trauma-induced HO and identify populations of circulating cells present in different stages of HO. These findings further elucidate the relative contribution of local and systemic cell populations to HO.

Inhibition of Hif1α prevents both trauma-induced and genetic heterotopic ossification.

Pathologic extraskeletal bone formation, or heterotopic ossification (HO), occurs following mechanical trauma, burns, orthopedic operations, and in patients with hyperactivating mutations of the type I bone morphogenetic protein receptor ACVR1 (Activin type 1 receptor). Extraskeletal bone forms through an endochondral process with a cartilage intermediary prompting the hypothesis that hypoxic signaling present during cartilage formation drives HO development and that HO precursor cells derive from a mesenchymal lineage as defined by Paired related homeobox 1 (Prx). Here we demonstrate that Hypoxia inducible factor-1α (Hif1α), a key mediator of cellular adaptation to hypoxia, is highly expressed and active in three separate mouse models: trauma-induced, genetic, and a hybrid model of genetic and trauma-induced HO. In each of these models, Hif1α expression coincides with the expression of master transcription factor of cartilage, Sox9 [(sex determining region Y)-box 9]. Pharmacologic inhibition of Hif1α using PX-478 or rapamycin significantly decreased or inhibited extraskeletal bone formation. Importantly, de novo soft-tissue HO was eliminated or significantly diminished in treated mice. Lineage-tracing mice demonstrate that cells forming HO belong to the Prx lineage. Burn/tenotomy performed in lineage-specific Hif1α knockout mice (Prx-Cre/Hif1α(fl:fl)) resulted in substantially decreased HO, and again lack of de novo soft-tissue HO. Genetic loss of Hif1α in mesenchymal cells marked by Prx-cre prevents the formation of the mesenchymal condensations as shown by routine histology and immunostaining for Sox9 and PDGFRα. Pharmacologic inhibition of Hif1α had a similar effect on mesenchymal condensation development. Our findings indicate that Hif1α represents a promising target to prevent and treat pathologic extraskeletal bone.

cAMP Signaling Compartmentation: Adenylyl Cyclases as Anchors of Dynamic Signaling Complexes.

It is widely accepted that cAMP signaling is compartmentalized within cells. However, our knowledge of how receptors, cAMP signaling enzymes, effectors, and other key proteins form specific signaling complexes to regulate specific cell responses is limited. The multicomponent nature of these systems and the spatiotemporal dynamics involved as proteins interact and move within a cell make cAMP responses highly complex. Adenylyl cyclases, the enzymatic source of cAMP production, are key starting points for understanding cAMP compartments and defining the functional signaling complexes. Three basic elements are required to form a signaling compartment. First, a localized signal is generated by a G protein-coupled receptor paired to one or more of the nine different transmembrane adenylyl cyclase isoforms that generate the cAMP signal in the cytosol. The diffusion of cAMP is subsequently limited by several factors, including expression of any number of phosphodiesterases (of which there are 24 genes plus spice variants). Finally, signal response elements are differentially localized to respond to cAMP produced within each locale. A-kinase-anchoring proteins, of which there are 43 different isoforms, facilitate this by targeting protein kinase A to specific substrates. Thousands of potential combinations of these three elements are possible in any given cell type, making the characterization of cAMP signaling compartments daunting. This review will focus on what is known about how cells organize cAMP signaling components as well as identify the unknowns. We make an argument for adenylyl cyclases being central to the formation and maintenance of these signaling complexes.

Scleraxis-Lineage Cells Contribute to Ectopic Bone Formation in Muscle and Tendon.

The pathologic development of heterotopic ossification (HO) is well described in patients with extensive trauma or with hyperactivating mutations of the bone morphogenetic protein (BMP) receptor ACVR1. However, identification of progenitor cells contributing to this process remains elusive. Here we show that connective tissue cells contribute to a substantial amount of HO anlagen caused by trauma using postnatal, tamoxifen-inducible, scleraxis-lineage restricted reporter mice (Scx-creERT2/tdTomatofl/fl ). When the scleraxis-lineage is restricted specifically to adults prior to injury marked cells contribute to each stage of the developing HO anlagen and coexpress markers of endochondral ossification (Osterix, SOX9). Furthermore, these adult preinjury restricted cells coexpressed mesenchymal stem cell markers including PDGFRα, Sca1, and S100A4 in HO. When constitutively active ACVR1 (caACVR1) was expressed in scx-cre cells in the absence of injury (Scx-cre/caACVR1fl/fl ), tendons and joints formed HO. Postnatal lineage-restricted, tamoxifen-inducible caACVR1 expression (Scx-creERT2/caACVR1fl/fl ) was sufficient to form HO after directed cardiotoxin-induced muscle injury. These findings suggest that cells expressing scleraxis within muscle or tendon contribute to HO in the setting of both trauma or hyperactive BMP receptor (e.g., caACVR1) activity. Stem Cells 2017;35:705-710.

Strategic Targeting of Multiple BMP Receptors Prevents Trauma-Induced Heterotopic Ossification.

Trauma-induced heterotopic ossification (tHO) is a condition of pathologic wound healing, defined by the progressive formation of ectopic bone in soft tissue following severe burns or trauma. Because previous studies have shown that genetic variants of HO, such as fibrodysplasia ossificans progressiva (FOP), are caused by hyperactivating mutations of the type I bone morphogenetic protein receptor (T1-BMPR) ACVR1/ALK2, studies evaluating therapies for HO have been directed primarily toward drugs for this specific receptor. However, patients with tHO do not carry known T1-BMPR mutations. Here we show that, although BMP signaling is required for tHO, no single T1-BMPR (ACVR1/ALK2, BMPR1a/ALK3, or BMPR1b/ALK6) alone is necessary for this disease, suggesting that these receptors have functional redundancy in the setting of tHO. By utilizing two different classes of BMP signaling inhibitors, we developed a translational approach to treatment, integrating treatment choice with existing diagnostic options. Our treatment paradigm balances either immediate therapy with reduced risk for adverse effects (Alk3-Fc) or delayed therapy with improved patient selection but greater risk for adverse effects (LDN-212854).

Analysis of Bone-Cartilage-Stromal Progenitor Populations in Trauma Induced and Genetic Models of Heterotopic Ossification.

Heterotopic ossification (HO), the formation of extra-skeletal bone in soft tissues, is a pathologic process occurring after substantial burns or trauma, or in patients with type I bone morphogenetic protein (BMP) receptor hyperactivating mutations. Identifying the cells responsible for de novo bone formation during adulthood is of critical importance for therapeutic and regenerative purposes. Using a model of trauma-induced HO with hind limb Achilles' tenotomy and dorsal burn injury and a genetic nontrauma HO model (Nfatc1-Cre/caAcvr1(fl/wt) ), we demonstrate enrichment of previously defined bone-cartilage-stromal progenitor cells (BCSP: AlphaV+/CD105+/Tie2-/CD45-/Thy1-/6C3-) at the site of HO formation when compared with marrow isolated from the ipsilateral hind limb, or from tissue of the contralateral, uninjured hind limb. Upon transplantation into tenotomy sites soon after injury, BCSPs isolated from neonatal mice or developing HO incorporate into the developing lesion in cartilage and bone and express chondrogenic and osteogenic transcription factors. Additionally, BCSPs isolated from developing HO similarly incorporate into new HO lesions upon transplantation. Finally, adventitial cells, but not pericytes, appear to play a supportive role in HO formation. Our findings indicate that BCSPs contribute to de novo bone formation during adulthood and may hold substantial regenerative potential. Stem Cells 2016;34:1692-1701.

Combined reflectance and Raman spectroscopy to assess degree of in vivo angiogenesis after tissue injury.

BACKGROUND: Angiogenesis, the formation of blood vessels, is a critical aspect of wound healing. Disorders of wound healing are often characterized by lack of angiogenesis, a condition frequently observed in aging and diabetic patients. Current techniques for assessing blood at injury sites are limited to contrast-imaging, including angiography. However, these techniques do not directly observe oxygenation of blood and are not amenable to serial evaluation. A multimodal noninvasive reflectance and Raman spectrometer have been proposed to help clinicians as a point-of-care tool to interrogate local angiogenesis and tissue architecture, respectively. The spectrometer system is a rapid, noninvasive, and label-free technology well-suited for the clinical environment. MATERIALS AND METHODS: To demonstrate feasibility, the spectrometer system was used to interrogate angiogenesis serially over 9 wk as a result of heterotopic ossification (HO) development in a validated murine model. End-stage HO was confirmed by micro-computed tomography. RESULTS: Our preliminary results suggest that reflectance spectroscopy can be used to delineate vessel formation and that pathologic wounds may be characterized by unique spectra. In our model, HO formed at sites 1-3, whereas sites 4 and 5 did not have radiographic evidence of HO. CONCLUSIONS: A point-of-care system like that demonstrated here shows potential as a noninvasive tool to assess local angiogenesis and tissue architecture that may allow for timely intervention in a clinical setting.

Hair follicle specific ACVR1/ALK2 critically affects skin morphogenesis and attenuates wound healing.

The bone morphogenic protein signaling (BMP) is intricately involved in the quiescence and regulation of stem cells through activation of BMP receptors. Hair follicle stem cells play a critical role in cutaneous homeostasis and regeneration. Here, we utilize a novel mouse model with targeted overexpression of the BMP receptor ALK2/ACVR1 in hair follicle stem cells, to characterize its role in skin development and postnatal wound healing. Initial histologic evaluation demonstrated significant dysregulation in hair follicle morphogenesis in mutant mice. These demonstrated increased numbers of individual hair follicles with altered morphology and localization. Mutant follicles were found to exhibit elevated proliferative activity as well as increased prevalence of CD34 and ITGA6 positive follicle stem cells. Interestingly, constitutive overexpression of ALK2 resulted in attenuation of cutaneous wound healing. These findings demonstrate that hair follicle specific ALK2 is intricately involved in maintenance of the stem cell niche and wound healing.

BMP signaling mediated by constitutively active Activin type 1 receptor (ACVR1) results in ectopic bone formation localized to distal extremity joints.

BMP signaling mediated by ACVR1 plays a critical role for development of multiple structures including the cardiovascular and skeletal systems. While deficient ACVR1 signaling impairs normal embryonic development, hyperactive ACVR1 function (R206H in humans and Q207D mutation in mice, ca-ACVR1) results in formation of heterotopic ossification (HO). We developed a mouse line, which conditionally expresses ca-ACVR1 with Nfatc1-Cre(+) transgene. Mutant mice developed ectopic cartilage and bone at the distal joints of the extremities including the interphalangeal joints and hind limb ankles as early as P4 in the absence of trauma or exogenous bone morphogenetic protein (BMP) administration. Micro-CT showed that even at later time points (up to P40), cartilage and bone development persisted at the affected joints most prominently in the ankle. Interestingly, this phenotype was not present in areas of bone outside of the joints - tibia are normal in mutants and littermate controls away from the ankle. These findings demonstrate that this model may allow for further studies of heterotopic ossification, which does not require the use of stem cells, direct trauma or activation with exogenous Cre gene administration.

Lymphatic Contribution to the Cellular Niche in Heterotopic Ossification.

OBJECTIVE: The objective of this study was to determine the contribution of lymphatic tissue to heterotopic ossification (HO). BACKGROUND: HO is the pathologic development of ectopic bone within soft tissues often following severe trauma. Characterization of the tissue niche supporting HO is critical to identifying therapies directed against this condition. Lymphangiogenesis is upregulated during incidents of trauma, thereby coincident with the niche supportive of HO. We hypothesized that lymphatic tissues play a critical role in HO formation. METHODS: Mice underwent hindlimb Achilles' tendon transection and dorsal burn injury (burn/tenotomy) to induce HO. The popliteal and inguinal lymph nodes were excised ipsilateral to the tenotomy site. Flow cytometry and immunostaining were used to quantify and localize lymphoendothelium. MicroCT was used to quantify HO. RESULTS: Enrichment of mature lymphatic tissues was noted 2 weeks after injury at the tendon transection sites when compared with the contralateral, intact tendon based on LYVE1+ tubules (10.9% vs 0.8%, P < 0.05). Excision of the inguinal and popliteal nodes with draining popliteal lymphatic vessel significantly decreased the presence of mature lymphoendothelium 2 weeks after injury (10.9% vs 3.3%, P < 0.05). Bone-cartilage-stromal progenitor cells (CD105+/AlphaV+/Tie2-/CD45-/CD90-/BP1-) were also significantly decreased after lymph node excision (10.2% vs 0.5%, P < 0.05). A significant decrease was noted in the volume of de novo HO present within the soft tissues (0.12 mm vs 0.02 mm). CONCLUSION: These findings suggest that lymphatic vessels are intimately linked with the de novo formation bone within soft tissues following trauma, and their presence may facilitate bone formation.

The role of the adaptive immune system in burn-induced heterotopic ossification and mesenchymal cell osteogenic differentiation.

BACKGROUND: Heterotopic ossification (HO) is the pathologic process of extraskeletal bone formation. Although the exact etiology remains unknown, inflammation appears to catalyze disease progression. The goal of this study is to determine the impact of the adaptive immune system on HO. METHODS: HO was induced in 8-wk-old control C57BL/6 and immunocompromised Rag1tm1Mom (Rag1 KO) male mice deficient in B- and T-lymphocytes via combined Achilles tenotomy and burn injury. Microcomputed tomography quantified the extent of HO formation at the tenotomy site. Adipose-derived mesenchymal stem cells were harvested to evaluate osteogenic differentiation potential. RESULTS: Areas of developing HO demonstrated substantial enrichment of CD45 + leukocytes at 3 wk after injury. HO from Rag1 KO mice was substantially less mature with foci of cartilage and disorganized trabecular bone present 12 wk after injury. Rag1 KO mice formed 60% less bone compared to immunocompetent controls (4.67 ± 1.5 mm versus 7.76 ± 0.65 mm; P = 0.001). Tartrate-resistant acid phosphatase staining and immunofluorescent analysis of osteoprotegerin and nuclear factor kappa-light-chain-enhancer of activated B cells demonstrated no appreciable difference in osteoclast number or activation. Alizarin red staining in vitro demonstrated a significant decrease in osteogenic potential in immunocompromised mice compared to controls (29.1 ± 0.54 mm versus 12.1 ± 0.14 mm; P < 0.001). CONCLUSIONS: We demonstrate a prominent role for the adaptive immune system in the development of HO. In the absence of mature B- and T-lymphocytes, HO growth and development are attenuated. Furthermore, we demonstrate that mesenchymal populations from B- and T-cell deficient mice are inherently less osteogenic. This study identifies a potential therapeutic role for modulation of the adaptive immune system in the treatment of HO.

Early Development of the Mouse Morphome.

INTRODUCTION: Analytical morphomics focuses on extracting objective and quantifiable data from clinical computed tomography (CT) scans to measure patients' frailty. Studies are currently retrospective in nature; therefore, it would be beneficial to develop animal models for well-controlled, prospective studies. The aim of this study is to develop an in vivo microCT protocol for the longitudinal acquisition of whole-body images suitable for morphomic analyses of bone. METHODS: The authors performed phantom studies on 2 microCT systems (Inveon and CT120) to study tissue radiodensity and further characterize system performance for collecting animal data. The authors also describe their design of a phantom-immobilization device using phantoms and an ovariectomized (OVX) mouse. RESULTS: The authors discovered increased consistency along the z-axis for scans acquired on the Inveon compared with CT120, and calibration by individual slice reduces variability. Objects in the field of view had more impact on measurement acquired using the CT120 compared with the Inveon. The authors also found that using the middle 80% of slices for data analysis further decreased variability, on both systems. Moreover, bone-mineral-density calibration using the QCT Pro Mini phantom improved bone-mineral-density estimates across energy spectra, which helped confirm our technique. Comparison of weekly body weights and terminal uterine mass between sham and OVX groups validated our model. DISCUSSION: The authors present a refined microCT protocol to collect reliable and objective data. This data will be used to establish a platform for research animal morphomics that can be used to test hypotheses developed from clinical human morphomics.

Engendering allograft ignorance in a mouse model of allogeneic skin transplantation to the distal hind limb.

OBJECTIVE: The aim of this study was to demonstrate lymphatic isolation in a model of hind limb lymph node (LN) excision, consisting of ipsilateral popliteal and inguinal LN excision and to evaluate the immunologic response to allogeneic skin transplanted onto this region of lymphatic isolation. METHODS: To study lymphatic flow, C57BL/6 mice underwent lymphadenectomy (n = 5), sham lymphadenectomy (n = 5), or no intervention (n = 5), followed by methylene blue injection. Mice were dissected to determine whether methylene blue traveled to the iliac LN. To study host response to skin transplantation, C57BL/6 mice underwent allogeneic skin transplantation with LN excision (n = 6), allogeneic skin transplantation alone (n = 6), or syngeneic skin transplantation (n = 4). Skin grafts were placed distal to the popliteal fossa and mice were euthanized at day 10. Grafts were stained for endothelial cell and proliferation markers (CD31 and Ki67, respectively). Secondary lymphoid tissues (spleen, ipsilateral axillary LN, and contralateral inguinal LN) were removed and rechallenged with BALB/c alloantigen in vitro with subsequent assay of interferon-γ and interleukin 4 cell expression using ELISPOT technique. RESULTS: Mice that underwent LN excision had no evidence of methylene blue in the iliac nodes; mice without surgical intervention or with sham LN excision consistently had methylene blue visible in the ipsilateral iliac nodes. Mice treated with allogeneic skin transplantation and LN excision had lower expression of interferon-γ and interleukin 4 in the secondary lymphoid tissues. CONCLUSIONS: Lymph node excision completely interrupts lymphatic flow of the hind limb. This model of lymphatic isolation impairs the ability of the transplant recipient to acutely mount a Th1 or Th2 response to allogeneic skin transplants.