Faculty Bibliography

Tissue repair and regeneration are thought to involve resident cell proliferation as well as the selective recruitment of circulating stem and progenitor cell populations through complex signaling cascades. Many of these recruited cells originate from the bone marrow, and specific subpopulations of bone marrow cells have been isolated and used to augment adult tissue regeneration in preclinical models. Clinical studies of cell-based therapies have reported mixed results, however, and a variety of approaches to enhance the regenerative capacity of stem cell therapies are being developed based on emerging insights into the mechanisms of progenitor cell biology and recruitment following injury. This article discusses the function and mechanisms of recruitment of important bone marrow-derived stem and progenitor cell populations following injury, as well as the emerging therapeutic applications targeting these cells.

Pancreatic ductal adenocarcinoma is an aggressive cancer that interacts with stromal cells to produce a highly inflammatory tumor microenvironment that promotes tumor growth and invasiveness. The precise interplay between tumor and stroma remains poorly understood. TLRs mediate interactions between environmental stimuli and innate immunity and trigger proinflammatory signaling cascades. Our finding that TLR7 expression is upregulated in both epithelial and stromal compartments in human and murine pancreatic cancer led us to postulate that carcinogenesis is dependent on TLR7 signaling. In a mouse model of pancreatic cancer, TLR7 ligation vigorously accelerated tumor progression and induced loss of expression of PTEN, p16, and cyclin D1 and upregulation of p21, p27, p53, c-Myc, SHPTP1, TGF-beta, PPARgamma, and cyclin B1. Furthermore, TLR7 ligation induced STAT3 activation and interfaced with Notch as well as canonical NF-kappaB and MAP kinase pathways, but downregulated expression of Notch target genes. Moreover, blockade of TLR7 protected against carcinogenesis. Since pancreatic tumorigenesis requires stromal expansion, we proposed that TLR7 ligation modulates pancreatic cancer by driving stromal inflammation. Accordingly, we found that mice lacking TLR7 exclusively within their inflammatory cells were protected from neoplasia. These data suggest that targeting TLR7 holds promise for treatment of human pancreatic cancer.

A quantitative understanding of tissue morphogenesis requires description of the movements of individual cells in space and over time. In transparent embryos, such as C. elegans, fluorescently labeled nuclei can be imaged in three-dimensional time-lapse (4D) movies and automatically tracked through early cleavage divisions up to ~350 nuclei. A similar analysis of later stages of C. elegans development has been challenging owing to the increased error rates of automated tracking of large numbers of densely packed nuclei. We present Nucleitracker4D, a freely available software solution for tracking nuclei in complex embryos that integrates automated tracking of nuclei in local searches with manual curation. Using these methods, we have been able to track >99% of all nuclei generated in the C. elegans embryo. Our analysis reveals that ventral enclosure of the epidermis is accompanied by complex coordinated migration of the neuronal substrate. We can efficiently track large numbers of migrating nuclei in 4D movies of zebrafish cardiac morphogenesis, suggesting that this approach is generally useful in situations in which the number, packing or dynamics of nuclei present challenges for automated tracking.

Vitiligo is characterized by depigmented skin patches caused by loss of epidermal melanocytes. Oxidative stress may have a role in vitiligo onset, while autoimmunity contributes to disease progression. In this study, we sought to identify mechanisms that link disease triggers and spreading of lesions. A hallmark of melanocytes at the periphery of vitiligo lesions is dilation of the endoplasmic reticulum (ER). We hypothesized that oxidative stress results in redox disruptions that extend to the ER, causing accumulation of misfolded peptides, which activates the unfolded protein response (UPR). We used 4-tertiary butyl phenol and monobenzyl ether of hydroquinone, known triggers of vitiligo. We show that expression of key UPR components, including the transcription factor X-box-binding protein 1 (XBP1), is increased following exposure of melanocytes to phenols. XBP1 activation increases production of immune mediators IL6 and IL8. Co-treatment with XBP1 inhibitors reduced IL6 and IL8 production induced by phenols, while overexpression of XBP1 alone increased their expression. Thus, melanocytes themselves produce cytokines associated with activation of an immune response following exposure to chemical triggers of vitiligo. These results expand our understanding of the mechanisms underlying melanocyte loss in vitiligo and pathways linking environmental stressors and autoimmunity.Journal of Investigative Dermatology advance online publication, 14 June 2012; doi:10.1038/jid.2012.181.

Radiation therapy has the potential to convert the tumor into an in situ individualized vaccine by inducing immunogenic cancer cell death and pro-inflammatory cytokines and chemokines; however this potential is rarely realized by irradiation alone. We hypothesized that radiation-induced immunosuppressive factors may hinder its pro-immunogenic effects. Transforming growth factor beta (TGFbeta) has immunosuppressive function for dendritic cells and T cells and is activated by radiation. Here we tested the hypothesis that inhibiting TGFbeta during radiation treatment would induce an immunogenic response. Poorly immunogenic, highly metastatic 4T1 carcinoma cells were injected s.c. in syngeneic BALB/c mice (day 0). TGFbeta neutralizing 1D11 or isotype control 13C4 monoclonal antibodies were given i.p. (200 mg/mouse) every other day from day 12 to 28. Tumors were irradiated with 6Gy on five consecutive days beginning on day 13. Tumor growth was measured consecutively. Mice were euthanized at day 21 for analysis, at day 28 for enumeration of lung metastases, or followed for survival. Gene expression profiles were obtained using Affymetrix mouse genome 430 2.0 array. Tumor growth rates and the frequency of lung metastases were similar in mice receiving control antibody or 1D11 alone. Radiation treatment caused significant (P=0.0065) tumor growth delay but did not inhibit lung metastases. In contrast, mice treated with both 1D11 and radiation exhibited significantly greater tumor growth control and reduced lung metastases (P<0.0001), and significantly prolonged survival (P<0.005). As expected, TGFbeta signalling was inhibited with 1D11 as measured in CD4+ and CD8+ T cells from tumor-draining lymph nodes at day 21. CD8+ T cells producing IFN in response to a tumor-specific antigen were detected only in mice treated with 1D11 and radiation. Expression profiles showed that genes associated with immune response and T cell activation were upregulated in irradiated tumors of mice treated with 1D11 compared to other treatment groups. In vivo depletion experiments demonstrated that T cells were essential for the improved tumor control and inhibition of lung metastases of mice treated with 1D11 and radiation. These data support a critical role for TGFbeta as a regulator of the pro-immunogenic effects of local tumor radiotherapy. Inhibition of TGFbeta during radiotherapy may promote self-immunization and achieve systemic control of metastatic disease. Supported by DOD BCRP grant BC100481P2

Persistent paraprotein production in plasma cells necessitates a highly developed rough endoplasmic reticulum (ER) that is unusually susceptible to perturbations in protein synthesis. This biology is believed to account for the exquisite sensitivity of multiple myeloma (MM) to the proteasomal inhibitor bortezomib (BTZ). Despite remarkable response rates to BTZ in MM, BTZ carries the potential for serious side-effects and development of resistance. We, therefore, sought to identify therapeutic combinations that effectively disrupt proteostasis in order to provide new potential treatments for MM. We found that sulforaphane, a dietary isothiocyanate found in cruciferous vegetables, inhibits TNFalpha-induced Ikappabeta proteasomal degradation in a manner similar to BTZ. Like BTZ, sulforaphane synergistically enhances the cytotoxicity of arsenic trioxide (ATO), an agent with clinical activity in MM. ATO and sulforaphane co-treatment augmented apoptotic induction as demonstrated by cleavage of caspase-3, -4 and PARP. The enhanced apoptotic response was dependent upon production of reactive oxygen species (ROS) as demonstrated by glutathione depletion and partial inhibition of the apoptotic cascade after pretreatment with the radical scavenger N-acetyl-cysteine (NAC). Combination treatment resulted in enhanced ER stress signaling and activation of the unfolded protein response (UPR), indicative of perturbation of proteostasis. Specifically, combination treatment caused elevated expression of the molecular chaperone HSP90 (heat shock protein 90) along with increased PERK (protein kinase RNA-like endoplasmic reticulum kinase) and eIF2alpha phosphorylation and XBP1 (X-box binding protein 1) splicing, key indicators of UPR activation. Moreover, increased splicing of XBP1 was apparent upon combination treatment compared to treatment with either agent alone. Sulforaphane in combination with ATO effectively disrupts protein homeostasis through ROS generation and induction of ER stress to culminate in inhibition of protein secretion and apoptotic induction in MM. Our results suggest that sulforaphane deserves further investigation in combination with ATO in the treatment of MM.

There are many examples of problems in pattern analysis for which it is often possible to obtain systematic characterizations, if in addition a small number of useful features or parameters of the image are known a priori or can be estimated reasonably well. Often the relevant features of a particular pattern analysis problem are easy to enumerate, as when statistical structures of the patterns are well understood from the knowledge of the domain. We study a problem from molecular image analysis, where such a domain-dependent understanding may be lacking to some degree and the features must be inferred via machine-learning techniques. In this paper, we propose a rigorous, fully-automated technique for this problem. We are motivated by an application of atomic force microscopy (AFM) image processing needed to solve a central problem in molecular biology, aimed at obtaining the complete transcription profile of a single cell, a snapshot that shows which genes are being expressed and to what degree. Reed et al (Single molecule transcription profiling with AFM, Nanotechnology, 18:4, 2007) showed the transcription profiling problem reduces to making high-precision measurements of biomolecule backbone lengths, correct to within 20-25 bp (6-7.5 nm). Here we present an image processing and length estimation pipeline using AFM that comes close to achieving these measurement tolerances. In particular, we develop a biased length estimator on trained coefficients of a simple linear regression model, biweighted by a Beaton-Tukey function, whose feature universe is constrained by James-Stein shrinkage to avoid overfitting. In terms of extensibility and addressing the model selection problem, this formulation subsumes the models we studied.

OBJECTIVE To investigate the frequency, epidemiology, clinical features, and prognostic significance of inflamed molluscum contagiosum (MC) lesions, molluscum dermatitis, reactive papular eruptions resembling Gianotti-Crosti syndrome, and atopic dermatitis in patients with MC. DESIGN Retrospective medical chart review. SETTING University-based pediatric dermatology practice. PATIENTS A total of 696 patients (mean age, 5.5 years) with molluscum. MAIN OUTCOME MEASURES Frequencies, characteristics, and associated features of inflammatory reactions to MC in patients with and without atopic dermatitis. RESULTS Molluscum dermatitis, inflamed MC lesions, and Gianotti-Crosti syndrome-like reactions (GCLRs) occurred in 270 (38.8%), 155 (22.3%), and 34 (4.9%) of the patients, respectively. A total of 259 patients (37.2%) had a history of atopic dermatitis. Individuals with atopic dermatitis had higher numbers of MC lesions (P < .001) and an increased likelihood of molluscum dermatitis (50.6% vs 31.8%; P < .001). In patients with molluscum dermatitis, numbers of MC lesions increased during the next 3 months in 23.4% of those treated with a topical corticosteroid and 33.3% of those not treated with a topical corticosteroid, compared with 16.8% of patients without dermatitis. Patients with inflamed MC lesions were less likely to have an increased number of MC lesions over the next 3 months than patients without inflamed MC lesions or dermatitis (5.2% vs 18.4%; P < .03). The GCLRs were associated with inflamed MC lesion (P < .001), favored the elbows and knees, tended to be pruritic, and often heralded resolution of MC. Two patients developed unilateral laterothoracic exanthem-like eruptions. CONCLUSIONS Inflammatory reactions to MC, including the previously underrecognized GCLR, are common. Treatment of molluscum dermatitis can reduce spread of MC via autoinoculation from scratching, whereas inflamed MC lesions and GCLRs reflect cell-mediated immune responses that may lead to viral clearance.

The validation of human biological models for inhaled radionuclides is nearly impossible. Requirements for validation are: (1) the measurement of the relevant human tissue data and (2) valid exposure measurements over the interval known to apply to tissue uptake. Two lung models, ICRP 30(( 1)) and ICRP 66(( 2)), are widely used to estimate lung doses following acute occupational or environmental exposure. Both ICRP 30 and 66 lung models are structured to estimate acute rather than chronic exposure. Two sets of human tissue measurements are available: (210)Po accumulated in tissue from inhaled cigarettes and ingested in diet and airborne global fallout (239,240)Pu accumulated in the lungs from inhalation. The human tissue measurements include pulmonary and bronchial tissue in smokers, ex-smokers and non-smokers analysed radiochemically for (210)Po, and pulmonary, bronchial and lymph nodes analysed for (239,240)Pu in lung tissue collected by the New York City Medical Examiner from 1972 to 1974. Both ICRP 30 and 66 models were included in a programme to accommodate chronic uptake. Neither lung model accurately described the estimated tissue concentrations but was within a factor of 2 from measurements. ICRP 66 was the exception and consistently overestimated the bronchial concentrations probably because of its assumption of an overly long 23-d clearance half-time in the bronchi and bronchioles.

The discovery of broadly neutralizing antibodies that recognize highly conserved epitopes in the membrane-proximal region of influenza virus hemagglutinin (HA) has revitalized efforts to develop a universal influenza virus vaccine. This effort will likely require novel immunogens that contain these epitopes but lack the variable and immunodominant epitopes located in the globular head of HA. As a first step toward developing such an immunogen, we investigated whether the 20-residue A-helix of the HA2 chain that forms the major component of the epitope of broadly neutralizing antibodies CR6261, F10, and others is sufficient by itself to elicit antibodies with similarly broad antiviral activity. Here, we report the multivalent display of the A-helix on icosahedral virus-like particles (VLPs) derived from the capsid of Flock House virus. Mice immunized with VLPs displaying 180 copies/particle of the A-helix produced antibodies that recognized trimeric HA and the elicited antibodies had binding characteristics similar to those of CR6261 and F10: they recognized multiple HA subtypes from group 1 but not from group 2. However, the anti-A-helix antibodies did not neutralize influenza virus. These results indicate that further engineering of the transplanted peptide is required and that display of additional regions of the epitope may be necessary to achieve protection.