Proteomic identification of tumor- and metastasis-associated galectin-1 in claudin-low breast cancer.

BACKGROUND: Metastasis and mortality remain high among breast cancer patients with the claudin-low subtype because these tumors are aggressive, chemoresistant, and lack targeted therapies. Our objective was to utilize discovery-based proteomics to identify proteins associated with claudin-low primary and metastatic tumors to gain insight into pathways and mechanisms of tumor progression. METHODS: We used nano-LC-MS/MS proteomics to analyze orthotopic and metastatic tumors from the syngeneic murine T11 tumor model, which displays gene expression profiles mirroring human claudin-low tumors. Galectin-1 identity, expression and spatial distribution were investigated by biochemical and immunochemical methods and MALDI/IMS. RNA seq data from mouse and human tumors in our study and publicly available microarray data were analyzed for differential galectin-1 expression across breast cancer subtypes. RESULTS: Galectin-1, an N-acetyllactosamine-binding protein, exhibited the highest sequence coverage and high abundance rank order among nano-LC-MS/MS-identified proteins shared by T11 claudin-low tumors but not normal tissue. Label-free quantitation, Western immunoblot and ELISA confirmed galectin-1 identity and significant differential expression. MALDI/IMS spatial mapping and immunohistochemistry detected galectin-1 in T11 metastatic lung foci. Immunohistochemistry of human claudin-low tumors demonstrated intermediate-to-high intensity galectin-1 staining of tumor and stroma. Gene expression analysis of mouse and human tumors found the highest galectin-1 levels in the claudin-low breast cancer subtype. CONCLUSIONS: Proteomics and genomics reveal high expression of galectin-1 protein and RNA in primary and metastatic claudin-low breast cancer. GENERAL SIGNIFICANCE: This work endorses proteomic approaches in cancer research and supports further investigations of the function and significance of galectin-1 overexpression in claudin-low tumor progression.

Development of the Subdural Hematoma in the Elderly (SHE) score to predict mortality.

OBJECTIVE: The purpose of this study was to describe the development of a novel prognostic score, the Subdural Hematoma in the Elderly (SHE) score. The SHE score is intended to predict 30-day mortality in elderly patients (those > 65 years of age) with an acute, chronic, or mixed-density subdural hematoma (SDH) after minor, or no, prior trauma. METHODS: The authors used the Prognosis Research Strategy group methods to develop the clinical prediction model. The training data set included patients with acute, chronic, and mixed-density SDH. Based on multivariate analyses from a large data set, in addition to review of the extant literature, 3 components to the score were selected: age, admission Glasgow Coma Scale (GCS) score, and SDH volume. Patients are given 1 point if they are over 80 years old, 1 point for an admission GCS score of 5-12, 2 points for an admission GCS score of 3-4, and 1 point for SDH volume > 50 ml. The sum of points across all categories determines the SHE score. RESULTS: The 30-day mortality rate steadily increased as the SHE score increased for all SDH acuities. For patients with an acute SDH, the 30-day mortality rate was 3.2% for SHE score of 0, and the rate increased to 13.1%, 32.7%, 95.7%, and 100% for SHE scores of 1, 2, 3, and 4, respectively. The model was most accurate for acute SDH (area under the curve [AUC] = 0.94), although it still performed well for chronic (AUC = 0.80) and mixed-density (AUC = 0.87) SDH. CONCLUSIONS: The SHE score is a simple clinical grading scale that accurately stratifies patients' risk of mortality based on age, admission GCS score, and SDH volume. Use of the SHE score could improve counseling of patients and their families, allow for standardization of clinical treatment protocols, and facilitate clinical research studies in SDH.

Using bald eagles to track spatial (1999-2008) and temporal (1987-1992, 1999-2003, and 2004-2008) trends of contaminants in Michigan's aquatic ecosystems.

The bald eagle (Haliaeetus leucocephalus) is an extensively researched tertiary predator. Studies have delineated information about its life history and the influences of various stressors on its reproduction. Due to the bald eagle's position at the top of the food web, it is susceptible to biomagnification of xenobiotics. The Michigan Department of Environmental Quality implemented a program in 1999 to monitor persistent chemicals including polychlorinated biphenols (PCBs) and dichlorodiphenyltrichloroethane (DDE). The objectives of the present study were to evaluate spatial and temporal trends of PCBs and organochlorine pesticides in nestling bald eagles of Michigan. The authors' study found that concentrations of PCBs and DDE were higher in Great Lakes areas with Lakes Michigan and Lake Huron having the highest concentrations of DDE and Lake Erie having the highest concentrations of PCBs. Temporally (1987-1992, 1999-2003, and 2004-2008) the present study found declines in PCB and DDE concentrations with a few exceptions. Continued monitoring of Michigan bald eagle populations is suggested for a couple of reasons. First, nestling blood contaminant levels are an appropriate method to monitor ecosystem contaminant levels. Second, from 1999 to 2008 PCB and DDE concentrations for 30% and 40%, respectively, of the nestling eagles sampled were above the no observable adverse effect level (NOAEL) for bald eagles. Lastly, with the continued development and deployment of new chemistries a continuous long term monitoring program is an invaluable resource. Environ Toxicol Chem 2016;35:1995-2002. © 2016 SETAC.

Impacts of Short-Rotation Early-Growing Season Prescribed Fire on a Ground Nesting Bird in the Central Hardwoods Region of North America.

Landscape-scale short-rotation early-growing season prescribed fire, hereafter prescribed fire, in upland hardwood forests represents a recent shift in management strategies across eastern upland forests. Not only does this strategy depart from dormant season to growing season prescriptions, but the strategy also moves from stand-scale to landscape-scale implementation (>1,000 ha). This being so, agencies are making considerable commitments in terms of time and resources to this management strategy, but the effects on wildlife in upland forests, especially those dominated by hardwood canopy species, are relatively unknown. We initiated our study to assess whether this management strategy affects eastern wild turkey reproductive ecology on the Ozark-St. Francis National Forest. We marked 67 wild turkey hens with Global Positioning System (GPS) Platform Transmitting Terminals in 2012 and 2013 to document exposure to prescribed fire, and estimate daily nest survival, nest success, and nest-site selection. We estimated these reproductive parameters in forest units managed with prescribed fire (treated) and units absent of prescribed fire (untreated). Of 60 initial nest attempts monitored, none were destroyed or exposed to prescribed fire because a majority of fires occurred early than a majority of the nesting activity. We found nest success was greater in untreated units than treated units (36.4% versus 14.6%). We did not find any habitat characteristic differences between successful and unsuccessful nest-sites. We found that nest-site selection criteria differed between treated and untreated units. Visual concealment and woody ground cover were common selection criteria in both treated and untreated units. However, in treated units wild turkey selected nest-sites with fewer small shrubs (<5 cm ground diameter) and large trees (>20 cm DBH) but not in untreated units. In untreated units wild turkey selected nest-sites with more large shrubs (≥5 cm ground diameter) but did not select for small shrubs or large trees. Our findings suggest that wild turkey have not benefited from the reintroduction of prescribed fire to the WRERA.

Using nestling plasma to assess long-term spatial and temporal concentrations of organochlorine compounds in bald eagles within Voyageurs National Park, Minnesota, USA.

The bald eagle (Haliaeetus leucocephalus) population at Voyageurs National Park (VNP) provides an opportunity to assess long-term temporal and spatial trends of persistent environmental contaminants. Nestling bald eagle plasma samples collected from 1997 to 2010 were analyzed for polychlorinated biphenyls (PCBs) and organochlorine pesticides. Trends of total PCBs, total DDTs, 4,4'-DDE, and Dieldrin were analyzed since >50% of nestling plasma samples had detectable concentrations. Total PCBs, total DDTs, and 4,4'-DDE concentrations have all decreased over time (26.09%, 24.09%, and 40.92% respectively). Concentrations of Dieldrin have increased by 50.25%. In this study, 61.1% of all nestlings sampled had detectable concentrations of Dieldrin from all time periods and all areas of VNP. Since Dieldrin is a banned pesticide in North America, the source of this increase is unknown. However, increases and fluctuations in Dieldrin concentration suggest contaminant levels in VNP may be linked to a new source or environmental process.

Using nestling feathers to assess spatial and temporal concentrations of mercury in bald eagles at Voyageurs National Park, Minnesota, USA.

Bald eagles (Haliaeetus leucocephalus) have been utilized as a biosentinel of aquatic ecosystem health in the Great Lakes Region since the early 1960s. Bald eagle populations have been monitored at Voyageurs National Park (VNP), Minnesota, since 1973. For the past 20 years, researchers have collected feathers from nestling bald eagles to assess their dietary exposure to mercury (Hg) on Rainy, Kabetogama, and Namakan lakes in VNP. Mercury is an environmental pollutant with both natural and anthropogenic sources, and negatively affects many species of wildlife. In a previous study, geometric mean concentrations of Hg in feathers of nestling bald eagles were greater at VNP (20 mg/kg Dry Weight (DW)) than in nestling feathers from other Great Lakes subpopulations (~7 mg/kg DW), for the period 1985-1989. Current geometric mean concentrations have declined by 77.4% since 1989 at VNP. While all samples from 1985 to 1989 had detectable concentrations of Hg, 10% of current samples had concentrations below the reportable detection limit (0.001 mg/kg DW, n = 180). The major lakes at VNP are impounded, and Hg concentrations also declined greatly after the lake level stabilization order by the International Joint Commission was implemented in 1999. Mercury concentrations in feathers of nestling bald eagles from 1989 to 2010 ranged from ND (<0.001) to 34.97 mg/kg DW. The highest single concentration in a nestling was from Namakan Lake in 2010. The five-year geometric means for Rainy, Kabetogama, and Namakan lakes for 2006-2010 were 6.08, 1.07, and 5.56 mg/kg DW (n = 28, n = 32, n = 27) respectively. Although Hg concentrations in feathers of nestlings greatly declined after the change in water level management in 1999 and are lower than 1989 concentrations, recent samples suggest a gradual increase. Continued monitoring of nestling feather concentrations will be essential to assess this increase, to determine the source of Hg, to determine if there are changes to methylation potential, and to evaluate and optimize water level management.

American Society of Clinical Oncology-recommended surveillance and physician specialty among long-term breast cancer survivors.

BACKGROUND: It is unclear whether it is appropriate to transfer the follow-up care of breast cancer (BrCa) survivors from cancer specialists to primary care physicians (PCPs). This contemporary study compared physician specialty and documented the long-term surveillance of survivors who underwent surgery at an American academic center. METHODS: Women in this institutional review board-approved study underwent breast surgery between 1996 and 2006. Data were collected for 270 patients with stage I to III BrCa (mean follow-up, 6 years). Charts were reviewed based on American Society of Clinical Oncology (ASCO) guidelines for recommended surveillance frequency and care. RESULTS: The majority of patients (90%; n = 242) were followed by specialists with 10% (n = 28) followed by PCPs. Patients with advanced disease and a greater risk of disease recurrence more often received specialist care. Patients followed by specialists were more often seen at ASCO-recommended intervals (eg, 89% vs 69% of patients followed by a PCP at follow-up Year 6; P < .01); however, many patients were followed inconsistently. Breast disease was often not the focus of PCP visits or mentioned in clinic notes (18% patients). Women seen by specialists were more likely to have documented clinical examinations of the breast (93% vs 44% at Year 6), axilla (94% vs 52%), or annual mammograms (74% vs 48%; P = .001-.02). CONCLUSIONS: Consistent compliance with surveillance guidelines and chart documentation needs improvement among all providers; however, specialists more consistently met ASCO guidelines. If transfer of care to a PCP occurs, it should be formalized and include follow-up recommendations and defined physician responsibilities. Providers and patients should be educated regarding surveillance care and current guidelines incorporated into standard clinical practice.

Real-time PCR to determine transgene copy number and to quantitate the biolocalization of adoptively transferred cells from EGFP-transgenic mice.

Quantitative real-time PCR (qPCR) is a sensitive technique for the detection and quantitation of specific DNA sequences. Here we describe a Taqman qPCR assay for quantification of tissue-localized, adoptively transferred enhanced green fluorescent protein (EGFP)-transgenic cells. A standard curve constructed from serial dilutions of a plasmid containing the EGFP transgene was (i) highly reproducible, (ii) detected as few as two copies, and (iii) was included in each qPCR assay. qPCR analysis of genomic DNA was used to determine transgene copy number in several mouse strains. Fluorescent microscopy of tissue sections showed that adoptively transferred vascular endothelial cells (VEC) from EGFP-transgenic mice specifically localized to tissue with metastatic tumors in syngeneic recipients. VEC microscopic enumeration of liver metastases strongly correlated with qPCR analysis of identical sections (Pearson correlation 0.81). EGFP was undetectable in tissue from control mice by qPCR. In another study using intra-tumor EGFP-VEC delivery to subcutaneous tumors, manual cell count and qPCR analysis of alternating sections also strongly correlated (Pearson correlation 0.82). Confocal microscopy of the subcutaneous tumor sections determined that visual fluorescent signals were frequently tissue artifacts. This qPCR methodology offers specific, objective, and rapid quantitation, uncomplicated by tissue autofluorescence, and should be readily transferable to other in vivo models to quantitate the biolocalization of transplanted cells.

Oriented immobilisation of engineered single-chain antibodies to develop biosensors for virus detection.

Single chain variable fragment (scFv) molecules were selected from a synthetic phage display library then cloned into a generic vector for expression of the scFv fused to the light chain constant domain of human immunoglobulin with a C-terminal cysteine residue (scFvC(L)cys). A heterobifunctional maleimide linker was synthesised and a strategy for functionalization of gold with the scFvC(L)cys fusion proteins elaborated. Successful covalent attachment of functional scFvC(L)cys was demonstrated using a surface plasmon resonance-based sensor. The results showed that the immobilised scFvC(L)cys molecules were functional and specific binding curves (with response relative to the concentration of virus antigen) were obtained over more than 25 cycles of binding and dissociation. ScFv molecules lacking the C-terminal cysteine performed poorly in similar experiments. The work demonstrates the feasibility of using simple scFv selection and cloning procedures combined with oriented immobilisation of scFvC(L)cys fusion proteins for robust antigen sensing surfaces in immunosensor or other biotechnological applications.

Characterization of Lewis lung clonal variants in a model of syngeneic pulmonary murine metastases.

Lung cancer is the leading cause of cancer-related mortality world-wide. Since the majority of cancer deaths result from metastatic complications, understanding cellular alterations contributing to organ specific metastases is a continuing cancer research goal. Desirable models involve easy, efficient methodologies for development of pulmonary metastases utilizing genetically related syngeneic tumor cell lines varying in clonogenic frequency and growth rate for comparative studies. This work focused on development and characterization of primary and metastatic Lewis lung subclones (LLCC3, LLC1, LLCab) in a histocompatible C57B1/6 model. Surgical resection of primary tumors utilizing these cell lines resulted in reliable development of pulmonary metastases (> 90% of injected mice), while tail-vein injection proved sporadic (20% of injected mice). The preliminary analysis of selected cell-surface molecules indicates potential genetic differences that may underlie phenotypic variations. The combination of subcutaneous resection methodology and variant cell lines results in robust metastatic lung cancer for testing potential therapeutic interventions.

Suppression of Lewis lung tumor development in alpha 1,3 galactosyltransferase knock-out mice.

BACKGROUND: Humans lack the gene alpha 1,3 galactosyltransferase (GalT) and instead produce abundant cytolytic antibodies against cells bearing the antigen [gal alpha1,3 gal] (alphaGal). We have previously studied humoral anti-alphaGal responses in GalT knock-out (GalT KO) mice and shown that murine anti-alphaGal IgM, like human anti-alphaGal IgM, causes extensive complement-mediated cytolysis of GalT+ murine Lewis Lung carcinoma cells (LLCa) in vitro. Here we test the hypothesis that anti-alphaGal immune responses can inhibit the in vivo development of GalT+ tumors. MATERIALS AND METHODS: GalT KO mice orally immunized to produce anti-alphaGal antibodies (n =52) and naïve non-immunized KO mice (n=37) were challenged s.c. with 10(5) LLCa tumor cells. Anti-alphaGal antibody titers were measured before and after LLCa challenge. RESULTS: Anti-alphaGal IgM titers present at challenge correlated with protection from tumor development (p<0.04). Seventy-five percent of mice with titers > or = 1:1280 remained tumor-free versus 43% of naïve mice. Tumor onset was delayed in mice with circulating anti-alphaGal IgM versus naïve animals (p=0.02). LLCa challenge itself induced and augmented anti-alphaGal IgM and post-challenge titers correlated highly with protection from tumor development (p<0.001). No mice with post-challenge anti-alphaGal IgM titers > or = 1:1280 developed tumors, compared to 83% of mice lacking antibody. Inhibition studies showed that 30% of post-challenge IgM recognized LLCa antigens distinct from alphaGal. Anti-alphaGal IgG was low or undetectable both pre- and post challenge and did not affect tumor formation. CONCLUSION: The finding that anti-alphaGal IgM suppresses GaIT+ tumor development in vivo supports the premise that immunotherapy using GalT expression can utilize human anti-alphaGal responses and induce significant anti-tumor effects.

Induction of cytolytic anti-Gal antibodies in alpha-1,3-galactosyltransferase gene knockout mice by oral inoculation with Escherichia coli O86:B7 bacteria.

Naturally occurring antibodies against [Gal alpha-1,3-Gal] structures (anti-Gal antibodies) are the primary effectors of human hyperacute rejection (HAR) of nonhuman tissue. Unlike most mammals, humans lack a functional alpha-1,3-galactosyltransferase (GalT) gene and produce abundant anti-Gal antibodies, putatively in response to GalT(+) enteric bacteria. GalT knockout (KO) mice have been generated as a small animal model of HAR but inconsistently express anti-Gal antibodies. We hypothesized that enteric exposure of GalT KO mice to live GalT(+) bacteria would produce cytolytic anti-Gal antibodies. Naive mice lacking anti-Gal antibodies were orally immunized with 10(10) live GalT(+) Escherichia coli O86:B7 bacteria and assayed for anti-Gal antibody titer, isotype, and cytolytic activity. Fecal samples were tested for E. coli O86:B7 prior to and after inoculation. In two separate experiments, 77 to 100% (n = 31) of mice developed serum anti-Gal immunoglobulin G (IgG; titer, 1:5 to 1:80) and/or anti-Gal IgM antibodies (titer, 1:5 to 1:1,280) 14 days postinoculation. Induced anti-Gal antibodies caused complement-mediated cytolysis of GalT(+) target cells, with extensive cytolysis observed consistently at serum IgM titers of >/=1:320. Absorption with synthetic [Gal alpha-1,3-Gal] inhibited both antibody binding and cytolysis. E. coli O86:B7 was recovered from stool samples from 83 to 94% of inoculated mice but not from naive mice, thus confirming enteric exposure. These findings demonstrate that oral inoculation with E. coli O86:B7 is a novel and effective method to induce cytolytic anti-Gal antibodies in GalT KO mice and support the premise that enteric exposure to GalT(+) bacteria induces anti-Gal antibodies in humans. These studies also suggest a role for GalT KO mice in elucidating anti-Gal responses in microbial immunity.

Human T-cell-porcine endothelial cell interactions induce human Th1 cytokines and porcine activation markers.

BACKGROUND: Since the future of clinical transplantation will undoubtedly include xenotransplantation, there is a need to examine human anti-pig cellular reactions. The objective of this study is to use human anti-porcine mixed lymphocyte endothelial cell culture (MLEC) to investigate cell interactions, cross-species molecular compatibilities, and the induction of human cytokines and porcine activation markers. METHODS: Human peripheral blood mononuclear cells or enriched CD4+ T cells depleted of professional antigen-presenting cells were cultured with resting pig aortic endothelial cells in the absence of exogenous cytokines. T-cell proliferative responses were measured and PAEC were monitored for cell surface markers by flow cytometry. Culture supernatants were assayed for human TNF-alpha and IFN-gamma by ELISA. RESULTS: Human T cells proliferated strongly in response to PAEC (median stimulation index = 75), even in serum-free cultures. High levels of the human Th1 cytokines TNF-alpha (20-350 pg/ml) and IFN-gamma (200-3800 pg/ml) were detected only in cultures containing PAEC, with levels peaking on Day 4. CD4+ T-cell-enriched, APC-depleted responders maintained proliferative anti-PAEC responses and cytokine release. By Day 3, MHC Class II and VCAM expression was induced in 92-96% PAEC: mean fluorescence intensity (MFI) increased from 5 to 83 +/- 12 and 166 +/- 74, respectively, and MHC Class I was increased from MFI 31 to 965 +/- 269. CONCLUSIONS: These results indicate that MLEC is an excellent in vitro model in which to study human anti-porcine cellular responses. Human T cells are activated in response to direct antigen presentation by PAEC, which are also activated in this system. Specific cytokines, receptors, and adhesion molecules appear to cross the xenograft barrier and play a critical role in T-cell - PAEC interactions. Such interactions are likely to affect VEC activation and immune responses to porcine xenografts in vivo.

Chronic tonsillar herniation and Crouzon's syndrome.

Patients born with craniofacial syndromes such as Crouzon's syndrome will often develop hydrocephalus after their initial craniofacial reconstructive procedures. We have treated 10 patients with Crouzon's syndrome; 5 patients required a shunting procedure after cranial remodeling. Each of these 5 shunted patients later demonstrated chronic tonsillar herniation on magnetic resonance imaging studies. One of these patients exhibited signs of pseudotumor cerebri and 1 had a spastic quadriparesis. Of the 5 patients who did not require a shunt, none displayed chronic tonsillar herniation. Our evidence suggests that jugular foramen stenosis produces an increased cerebral venous turgor that leads to a cerebrospinal fluid absorption defect and hydrocephalus. After the hydrocephalus is treated the increased venous turgor remains and provides the driving force for the development of chronic tonsillar herniation.

Neonatal intracranial teratomas.

Two neonates with intracranial teratomas presented with cranial enlargements a few weeks after birth. Both cases underwent surgery: one died intraoperatively; the other is the longest known survivor, alive 7 years and 9 months after subtotal excision of a mature teratoma of the left sylvian fissure. Previous operations have been relatively few and nearly all have been unsuccessful. Size and favorable location may be the most important prognostic features regardless of the histologic classification as mature or immature. One of our cases demonstrates that even subtotal excision of a mature teratoma can result in long-term survival.

Gadolinium-DTPA enhancement of a recurrent intramedullary ependymoma: a case report.

Magnetic resonance images enhanced with the paramagnetic contrast agent gadolinium-DTPA accurately differentiated a recurrent intramedullary spinal cord ependymoma from surrounding postoperative and postirradiation spinal cord tissue changes, thereby facilitating total excision of the lesion. The illustrative case and the merits of enhanced magnetic resonance imaging are presented.

Tension hydrothorax from intrapleural migration of a ventriculoperitoneal shunt.

A male newborn underwent a myelomeningocele repair, with subsequent placement of a ventriculoperitoneal shunt for treatment of hydrocephalus. Five days after shunt surgery, the infant acutely developed a deeply sunken fontanel, pallor, tachypnea, bradycardia, and irritability. Chest radiographs revealed intrathoracic migration of the distal shunt tubing and a tension hydrothorax. Treatment consisted of tube thoracostomy and temporary externalization of the distal shunt tubing. The patient fully recovered. The acute onset of shock in association with a sunken fontanel in a neonate with a shunt should raise the suspicion of tension hydrothorax. For critically ill infants immediate needle aspiration or thoracostomy is suggested. In less severely ill children, exposure of the shunt tubing in the neck and withdrawal of the pleural effusion by the distal shunt tubing may be performed as an emergency measure. The early recognition and urgent management of this problem are emphasized.