Murine model of chemotherapy-induced extraintestinal pathogenic Escherichia coli translocation.

Escherichia coli is a major cause of life-threatening infections in patients with neutropenia, particularly those receiving chemotherapy for the treatment of cancer. In most cases, these infections originate from opportunistic strains living within the patient's gastrointestinal tract which then translocate to major organ systems. There are no animal models that faithfully recapitulate these infections, and, as such, the host or bacterial factors that govern this process remain unidentified. We present here a novel model of chemotherapy-induced bacterial translocation of E. coli. Oral gavage of BALB/c mice with a clinical isolate of extraintestinal pathogenic E. coli (ExPEC) leads to stable and long-term colonization of the murine intestine. Following the induction of neutropenia with the chemotherapeutic drug cyclophosphamide, ExPEC translocates from the intestine to the lungs, liver, spleen, and kidneys with concomitant morbidity in infected animals. Translocation can also occur in mice bearing mammary tumors, even in the absence of chemotherapy. Translocation of ExPEC is also associated with an increase of the diversity of bacterial DNA detected in the blood. This is the first report of a chemotherapy-based animal model of ExPEC translocation in cancerous mice, a system that can be readily used to identify important virulence factors for this process.

Resistin deficiency in mice has no effect on pulmonary responses induced by acute ozone exposure.

Acute exposure to ozone (O3), an air pollutant, causes pulmonary inflammation, airway epithelial desquamation, and airway hyperresponsiveness (AHR). Pro-inflammatory cytokines-including IL-6 and ligands of chemokine (C-X-C motif) receptor 2 [keratinocyte chemoattractant (KC) and macrophage inflammatory protein (MIP)-2], TNF receptor 1 and 2 (TNF), and type I IL-1 receptor (IL-1α and IL-1ß)-promote these sequelae. Human resistin, a pleiotropic hormone and cytokine, induces expression of IL-1α, IL-1ß, IL-6, IL-8 (the human ortholog of murine KC and MIP-2), and TNF. Functional differences exist between human and murine resistin; yet given the aforementioned observations, we hypothesized that murine resistin promotes O3-induced lung pathology by inducing expression of the same inflammatory cytokines as human resistin. Consequently, we examined indexes of O3-induced lung pathology in wild-type and resistin-deficient mice following acute exposure to either filtered room air or O3. In wild-type mice, O3 increased bronchoalveolar lavage fluid (BALF) resistin. Furthermore, O3 increased lung tissue or BALF IL-1α, IL-6, KC, TNF, macrophages, neutrophils, and epithelial cells in wild-type and resistin-deficient mice. With the exception of KC, which was significantly greater in resistin-deficient compared with wild-type mice, no genotype-related differences in the other indexes existed following O3 exposure. O3 caused AHR to acetyl-ß-methylcholine chloride (methacholine) in wild-type and resistin-deficient mice. However, genotype-related differences in airway responsiveness to methacholine were nonexistent subsequent to O3 exposure. Taken together, these data demonstrate that murine resistin is increased in the lungs of wild-type mice following acute O3 exposure but does not promote O3-induced lung pathology.

Neurovirulence and immunogenicity of attenuated recombinant vesicular stomatitis viruses in nonhuman primates.

UNLABELLED: In previous work, a prototypic recombinant vesicular stomatitis virus Indiana serotype (rVSIV) vector expressing simian immunodeficiency virus (SIV) gag and human immunodeficiency virus type 1 (HIV-1) env antigens protected nonhuman primates (NHPs) from disease following challenge with an HIV-1/SIV recombinant (SHIV). However, when tested in a stringent NHP neurovirulence (NV) model, this vector was not adequately attenuated for clinical evaluation. For the work described here, the prototypic rVSIV vector was attenuated by combining specific G protein truncations with either N gene translocations or mutations (M33A and M51A) that ablate expression of subgenic M polypeptides, by incorporation of temperature-sensitive mutations in the N and L genes, and by deletion of the VSIV G gene to generate a replicon that is dependent on trans expression of G protein for in vitro propagation. When evaluated in a series of NHP NV studies, these attenuated rVSIV variants caused no clinical disease and demonstrated a very significant reduction in neuropathology compared to wild-type VSIV and the prototypic rVSIV vaccine vector. In spite of greatly increased in vivo attenuation, some of the rVSIV vectors elicited cell-mediated immune responses that were similar in magnitude to those induced by the much more virulent prototypic vector. These data demonstrate novel approaches to the rational attenuation of VSIV NV while retaining vector immunogenicity and have led to identification of an rVSIV N4CT1gag1 vaccine vector that has now successfully completed phase I clinical evaluation. IMPORTANCE: The work described in this article demonstrates a rational approach to the attenuation of vesicular stomatitis virus neurovirulence. The major attenuation strategy described here will be most likely applicable to other members of the Rhabdoviridae and possibly other families of nonsegmented negative-strand RNA viruses. These studies have also enabled the identification of an attenuated, replication-competent rVSIV vector that has successfully undergone its first clinical evaluation in humans. Therefore, these studies represent a major milestone in the development of attenuated rVSIV, and likely other vesiculoviruses, as a new vaccine platform(s) for use in humans.

Effect of antigen sensitization and challenge on oscillatory mechanics of the lung and pulmonary inflammation in obese carboxypeptidase E-deficient mice.

Atopic, obese asthmatics exhibit airway obstruction with variable degrees of eosinophilic airway inflammation. We previously reported that mice obese as a result of a genetic deficiency in either leptin (ob/ob mice) or the long isoform of the leptin receptor (db/db mice) exhibit enhanced airway obstruction in the presence of decreased numbers of bronchoalveolar lavage fluid (BALF) eosinophils compared with lean, wild-type mice following antigen (ovalbumin; OVA) sensitization and challenge. To determine whether the genetic modality of obesity induction influences the development of OVA-induced airway obstruction and OVA-induced pulmonary inflammation, we examined indices of these sequelae in mice obese as a result of a genetic deficiency in carboxypeptidase E, an enzyme that processes prohormones and proneuropeptides involved in satiety and energy expenditure (Cpe(fat) mice). Accordingly, Cpe(fat) and lean, wild-type (C57BL/6) mice were sensitized to OVA and then challenged with either aerosolized PBS or OVA. Compared with genotype-matched, OVA-sensitized and PBS-challenged mice, OVA sensitization and challenge elicited airway obstruction and increased BALF eosinophils, macrophages, neutrophils, IL-4, IL-13, IL-18, and chemerin. However, OVA challenge enhanced airway obstruction and pulmonary inflammation in Cpe(fat) compared with wild-type mice. These results demonstrate that OVA sensitization and challenge enhance airway obstruction in obese mice regardless of the genetic basis of obesity, whereas the degree of OVA-induced pulmonary inflammation is dependent on the genetic modality of obesity induction. These results have important implications for animal models of asthma, as modeling the pulmonary phenotypes for subpopulations of atopic, obese asthmatics critically depends on selecting the appropriate mouse model.

Endogenous osteopontin promotes ozone-induced neutrophil recruitment to the lungs and airway hyperresponsiveness to methacholine.

Inhalation of ozone (O3), a common environmental pollutant, causes pulmonary injury, pulmonary inflammation, and airway hyperresponsiveness (AHR) in healthy individuals and exacerbates many of these same sequelae in individuals with preexisting lung disease. However, the mechanisms underlying these phenomena are poorly understood. Consequently, we sought to determine the contribution of osteopontin (OPN), a hormone and a pleiotropic cytokine, to the development of O3-induced pulmonary injury, pulmonary inflammation, and AHR. To that end, we examined indices of these aforementioned sequelae in mice genetically deficient in OPN and in wild-type, C57BL/6 mice 24 h following the cessation of an acute (3 h) exposure to filtered room air (air) or O3 (2 parts/million). In wild-type mice, O3 exposure increased bronchoalveolar lavage fluid (BALF) OPN, whereas immunohistochemical analysis demonstrated that there were no differences in the number of OPN-positive alveolar macrophages between air- and O3-exposed wild-type mice. O3 exposure also increased BALF epithelial cells, protein, and neutrophils in wild-type and OPN-deficient mice compared with genotype-matched, air-exposed controls. However, following O3 exposure, BALF neutrophils were significantly reduced in OPN-deficient compared with wild-type mice. When airway responsiveness to inhaled acetyl-ß-methylcholine chloride (methacholine) was assessed using the forced oscillation technique, O3 exposure caused hyperresponsiveness to methacholine in the airways and lung parenchyma of wild-type mice, but not OPN-deficient mice. These results demonstrate that OPN is increased in the air spaces following acute exposure to O3 and functionally contributes to the development of O3-induced pulmonary inflammation and airway and lung parenchymal hyperresponsiveness to methacholine.

Heart-specific overexpression of CUGBP1 reproduces functional and molecular abnormalities of myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is caused by a CTG expansion within the 3'-untranslated region of the DMPK gene. The predominant mechanism of pathogenesis is a toxic gain of function of CUG repeat containing RNA transcribed from the expanded allele. The molecular mechanisms by which the RNA containing expanded repeats produce pathogenic effects include: sequestration of muscleblind-like 1 (MBNL1) protein and up-regulation of CUG binding protein 1 (CUGBP1). MBNL1 and CUGBP1 are RNA binding proteins that regulate alternative splicing transitions during development. Altered functions of these proteins in DM1 lead to misregulated splicing of their target genes, resulting in several features of the disease. The role of MBNL1 depletion in DM1 is well established through a mouse knock-out model that reproduces many disease features. Here we directly test the hypothesis that CUGBP1 up-regulation also contributes to manifestations of DM1. Using tetracycline-inducible CUGBP1 and heart-specific reverse tetracycline trans-activator transgenes, we expressed human CUGBP1 in adult mouse heart. Our results demonstrate that up-regulation of CUGBP1 is sufficient to reproduce molecular, histopathological and functional changes observed in a previously described DM1 mouse model that expresses expanded CUG RNA repeats as well as in individuals with DM1. These results strongly support a role for CUGBP1 up-regulation in DM1 pathogenesis.

Imaging prostate cancer lymph node metastases with a multimodality contrast agent.

BACKGROUND: Methods to detect lymph node (LN) metastases in prostate cancer (PCa) are limited. Pelvic LN dissection is commonly performed during prostatectomy, but often followed by morbid complications. More refined methods for detecting LN invasion are needed. METHODS: We developed a dual-labeled targeting agent having a near-infrared (NIR) fluorophore for intraoperative guidance, and a conventional radiotracer for detection of LN metastasis. Nu/Nu mice were orthotopically implanted with DsRed-expressing human PCa (PC3) cells. Antibody (Ab) specific for epithelial cell adhesion molecule was conjugated to DOTA, IRDye 800CW, and radiolabeled with (64) Cu. Dual-labeled Ab was administered intravenously at 10-12 weeks post-implantation, and positron emission tomography/computed tomography (PET/CT) and fluorescence imaging were performed within 18-24 hr. RESULTS: Metastasis to lumbar LNs was detected by DsRed fluorescence imaging, as well as pathology, in 75% of mice having pathology-confirmed primary prostate tumors. These metastases were also detected by NIR fluorescence imaging. In some cases, metastases to sciatic, medial, renal, and axillary nodes were also detected. For all LNs examined, no significant differences were found between the percentages of metastases detected by NIR imaging (63%) and µPET/CT (64%) (P = 0.93), or between those detected by DsRed imaging (25%) and pathological examination (19%) (P = 0.12). CONCLUSION: This study demonstrates that a multimodality contrast agent is useful for early detection of metastatic disease, and has applications for intraoperative PCa treatment. Further agent optimization is necessary to enhance specificity, and provide validation for prostate and other LN metastasizing epithelial cancers.

High-fat Western diet consumption exacerbates silica-induced pulmonary inflammation and fibrosis.

Consumption of a high-fat Western diet (HFWD) contributes to obesity, disrupted adipose endocrine function, and development of metabolic dysfunction (MetDys). Impaired lung function, pulmonary hypertension, and asthma are all associated with MetDys. Over 35% of adults in the U.S. have MetDys, yet interactions between MetDys and hazardous occupational inhalation exposures are largely unknown. Occupational silica-inhalation leads to chronic lung inflammation, progressive fibrosis, and significant respiratory morbidity and mortality. In this study, we aim to determine the potential of HFWD-consumption to alter silica-induced inflammatory responses in the lung. Six-wk old male F344 rats fed a high fat Western diet (HFWD; 45 kcal % fat, sucrose 22.2% by weight) to induce MetDys, or standard rat chow (STD, controls) for 16 wk were subsequently exposed to silica (6 h/d, 5 d/wk, 39 d; Min-U-Sil 5®, 15 mg/m3) or filtered air; animals remained on their assigned diet for the study duration. Indices of lung inflammation and histopathologic assessment of lung tissue were quantified at 0, 4, and 8 wk after cessation of exposure. Combined HFWD+silica exposure increased bronchoalveolar lavage (BAL) total cells, leukocytes, and BAL lactate dehydrogenase compared to STD+silica exposure controls at all timepoints. HFWD+silica exposure increased BAL proinflammatory cytokines at 4 and 8 wk compared to STD+silica exposure. At 8 wk, histopathological analysis confirmed that alveolitis, epithelial cell hypertrophy and hyperplasia, lipoproteinosis, fibrosis, bronchoalveolar lymphoid hyperplasia and granulomas were exacerbated in the HFWD+silica-exposed group compared to STD+silica-exposed controls. Our results suggest an increased susceptibility to silica-induced lung disease caused by HFWD consumption.

Selective nanoparticle-directed ablation of the canine prostate.

BACKGROUND AND OBJECTIVES: Prostate cancer is the most frequent cancer type and the second most common cause of cancer death among US men. This study, adapted a previously reported nanoparticle-directed photothermal treatment of brain tumors to the treatment of prostate disease by using normal canine prostate in vivo, directly injected with a suspension of nanoparticles as a proxy for prostate tumor, and by developing laser dosimetry for prostate which is marginally ablative in native tissue, yet producing photothermal coagulation in prostate tissue containing nanoparticles. METHODS: Canine prostates were exposed by surgical laparotomy and directly injected with suspensions of nanoparticles (nanoshells) and irradiated by a NIR laser source delivered percutaneously by an optical fiber catheter and isotropic diffuser. The photothermal lesions were permitted to resolve for up to 8 days, at which time each animal was euthanized, necropsied, and the prostate taken for histopathological and elemental analysis. RESULTS: Nanoparticles were retained for up to 4 hours in prostate and served as a proxy for prostate tumor. A marginally ablative laser dose of 3.0 W for 3 minutes was developed which would yield 4 mm-radius coagulo-necrotic lesions if nanoparticles were present. CONCLUSION: We have shown that the addition of nanoshells to native tissue, combined with a marginally ablative laser dose can generate ablative thermal lesions, and that the radial extent of the thermal lesions is strictly confined to within ∼4 mm of the optical fiber with sub-millimeter uncertainty. This, in turn, suggests a means of precise tumor ablation with an ability to obviate damage to critical structures limited primarily by the precision with which the optical fiber applicator can be placed. In so doing, it should be possible to realize a precise, nerve bundle and urethra sparing prostate cancer treatment using a minimally invasive, percutaneous approach.

c-Jun N-terminal kinase contributes to aberrant retinoid signaling in lung cancer cells by phosphorylating and inducing proteasomal degradation of retinoic acid receptor alpha.

Retinoic acid (RA) is the ligand for nuclear RA receptors (RARs and RXRs) and is crucial for normal epithelial cell growth and differentiation. During malignant transformation, human bronchial epithelial cells acquire a block in retinoid signaling caused in part by a transcriptional defect in RARs. Here, we show that activation of c-Jun N-terminal kinase (JNK) contributes to RAR dysfunction by phosphorylating RARalpha and inducing degradation through the ubiquitin-proteasomal pathway. Analysis of RARalpha mutants and phosphopeptide mapping revealed that RARalpha residues Thr181, Ser445, and Ser461 are phosphorylated by JNK. Mutation of these residues to alanines prevented efficient ubiquitination of RARalpha and increased the stability of the protein. We investigated the importance of RARalpha phosphorylation by JNK as a mediator of retinoid resistance in lung cancer. Mice that develop lung cancer from activation of a latent K-ras oncogene had high intratumoral JNK activity and low RARalpha levels and were resistant to treatment with an RAR ligand. JNK inhibition in a human lung cancer cell line enhanced RARalpha levels, ligand-induced activity of RXR-RAR dimers, and growth inhibition by RA. These findings point to JNK as a key mediator of aberrant retinoid signaling in lung cancer cells.

In vivo imaging of prostate cancer involving bone in a mouse model.

BACKGROUND: We compared the abilities of clinically relevant imaging modalities to quantify prostate cancer involving bone in a mouse model. Such non-invasive methods are needed pre-clinically to understand tumor biology and to evaluate therapy. METHODS: Human prostate cancer cells (MDA PCa 2b) or vehicle were injected into the right or left femur of SCID mice (n = 8). Radiography, computed tomography, and magnetic resonance imaging were performed 5 and 8 weeks later (n = 7). Bone scintigraphy (n = 6) was also performed at week 8. Imaging findings were compared with histology and correlated with contemporaneous serum prostate-specific antigen levels. RESULTS: Among the modalities evaluated, only MR imaging delineated prostate tumors involving bone. Tumor volume assessed by MR imaging correlated with PSA levels (R(2) = 0.87, P < 0.001). MR imaging of tumors corresponded with histology. Imaging of mineralized bone by CT corresponded with histology. CONCLUSION: In a mouse model, prostate tumors involving bone can be quantified using MR imaging.

Magnetic resonance imaging of therapy-induced necrosis using gadolinium-chelated polyglutamic acids.

PURPOSE: Necrosis is the most common morphologic alteration found in tumors and surrounding normal tissues after radiation therapy or chemotherapy. Accurate measurement of necrosis may provide an early indication of treatment efficacy or associated toxicity. The purpose of this report is to evaluate the selective accumulation of polymeric paramagnetic magnetic resonance (MR) contrast agents--gadolinium p-aminobenzyl-diethylenetriaminepentaacetic acid-poly(glutamic acid) (L-PG-DTPA-Gd and D-PG-DTPA-Gd)--in necrotic tissue. METHODS AND MATERIALS: Two different solid tumor models, human Colo-205 xenograft and syngeneic murine OCA-1 ovarian tumors, were used in this study. Necrotic response was induced by treatment with poly(L-glutamic acid)-paclitaxel conjugate (PG-TXL). T(1)-weighted spin-echo images were obtained immediately and up to 4 days after contrast injection and compared with corresponding histologic specimens. Two low-molecular-weight contrast agents, DTPA-Gd and oligomeric(L-glutamic acid)-DTPA-Gd, were used as nonspecific controls. RESULTS: Initially, there was minimal tumor enhancement after injection of either L-PG-DTPA-Gd or D-PG-DTPA-Gd, but rapid enhancement after injection of low-molecular-weight agents. However, polymeric contrast agents, but not low-molecular-weight contrast agents, caused sustained enhancement in regions of tumor necrosis in both tumors treated with PG-TXL and untreated tumors. These data indicate that high molecular weight, rather than in vivo biodegradation, is necessary for the specific localization of polymeric MR contrast agents to necrotic tissue. Moreover, biotinylated L-PG-DTPA-Gd colocalized with macrophages in the tumor necrotic areas, suggesting that selective accumulation of L- and D-PG-DTPA-Gd in necrotic tissue was mediated through residing macrophages. CONCLUSIONS: Our data suggest that MR imaging with PG-DTPA-Gd may be a useful technique for noninvasive characterization of treatment-induced necrosis.

Inhibition of mammalian target of rapamycin reverses alveolar epithelial neoplasia induced by oncogenic K-ras.

The serine/threonine kinase AKT and its downstream mediator mammalian target of rapamycin (mTOR) are activated in lung adenocarcinoma, and clinical trials are under way to test whether inhibition of mTOR is useful in treating lung cancer. Here, we report that mTOR inhibition blocked malignant progression in K-ras(LA1) mice, which undergo somatic activation of the K-ras oncogene and display morphologic changes in alveolar epithelial cells that recapitulate those of precursors of human lung adenocarcinoma. Levels of phospho-S6(Ser236/235), a downstream mediator of mTOR, increased with malignant progression (normal alveolar epithelial cells to adenocarcinoma) in K-ras(LA1) mice and in patients with lung adenocarcinoma. Atypical alveolar hyperplasia, an early neoplastic change, was prominently associated with macrophages and expressed high levels of phospho-S6(Ser236/235). mTOR inhibition in K-ras(LA1) mice by treatment with the rapamycin analogue CCI-779 reduced the size and number of early epithelial neoplastic lesions (atypical alveolar hyperplasia and adenomas) and induced apoptosis of intraepithelial macrophages. LKR-13, a lung adenocarcinoma cell line derived from K-ras(LA1) mice, was resistant to treatment with CCI-779 in vitro. However, LKR-13 cells grown as syngeneic tumors recruited macrophages, and those tumors regressed in response to treatment with CCI-779. Lastly, conditioned medium from primary cultures of alveolar macrophages stimulated the proliferation of LKR-13 cells. These findings provide evidence that the expansion of lung adenocarcinoma precursors induced by oncogenic K-ras requires mTOR-dependent signaling and that host factors derived from macrophages play a critical role in adenocarcinoma progression.

High expression of ErbB family members and their ligands in lung adenocarcinomas that are sensitive to inhibition of epidermal growth factor receptor.

Recent findings in tumor biopsies from lung adenocarcinoma patients suggest that somatic mutations in the genes encoding epidermal growth factor receptor (EGFR) and Kirsten ras (KRAS) confer sensitivity and resistance, respectively, to EGFR inhibition. Here, we provide evidence that these genetic mutations are not sufficient to modulate the biological response of lung adenocarcinoma cells to EGFR inhibition. We found high expression of ErbB family members, ErbB ligands, or both in three models that were sensitive to EGFR inhibition, including alveolar epithelial neoplastic lesions in mice that develop lung adenocarcinoma by oncogenic KRAS, human lung adenocarcinoma cell lines, and tumor biopsies from lung adenocarcinoma patients. Thus, lung adenocarcinoma cells that depend on EGFR for survival constitutively activate the receptor through a combination of genetic mutations and overexpression of EGFR dimeric partners and their ligands.

Chemokine (C-C Motif) Receptor-Like 2 is not essential for lung injury, lung inflammation, or airway hyperresponsiveness induced by acute exposure to ozone.

Inhalation of ozone (O3), a gaseous air pollutant, causes lung injury, lung inflammation, and airway hyperresponsiveness. Macrophages, mast cells, and neutrophils contribute to one or more of these sequelae induced by O3 Furthermore, each of these aforementioned cells express chemokine (C-C motif) receptor-like 2 (Ccrl2), an atypical chemokine receptor that facilitates leukocyte chemotaxis. Given that Ccrl2 is expressed by cells essential to the development of O3-induced lung pathology and that chemerin, a Ccrl2 ligand, is increased in bronchoalveolar lavage fluid (BALF) by O3, we hypothesized that Ccrl2 contributes to the development of lung injury, lung inflammation, and airway hyperresponsiveness induced by O3 To that end, we measured indices of lung injury (BALF protein, BALF epithelial cells, and bronchiolar epithelial injury), lung inflammation (BALF cytokines and BALF leukocytes), and airway responsiveness to acetyl-ß-methylcholine chloride (respiratory system resistance) in wild-type and mice genetically deficient in Ccrl2 (Ccrl2-deficient mice) 4 and/or 24 hours following cessation of acute exposure to either filtered room air (air) or O3 In air-exposed mice, BALF chemerin was greater in Ccrl2-deficient as compared to wild-type mice. O3 increased BALF chemerin in mice of both genotypes, yet following O3 exposure, BALF chemerin was greater in Ccrl2-deficient as compared to wild-type mice. O3 increased indices of lung injury, lung inflammation, and airway responsiveness. Nevertheless, no indices were different between genotypes following O3 exposure. In conclusion, we demonstrate that Ccrl2 modulates chemerin levels in the epithelial lining fluid of the lungs but does not contribute to the development of O3-induced lung pathology.

Quantification of bleomycin-induced murine lung damage in vivo with micro-computed tomography.

RATIONALE AND OBJECTIVES: We explored noninvasive, in vivo cone-beam microcomputed tomography (micro-CT) to visualize and quantify fibrotic and inflammatory damage over the entire lung volume of mice. MATERIALS AND METHODS: We used bleomycin to induce pulmonary damage in vivo and compared the results from micro-CT with histologic measurements. Ten C57BL/6 mice were given 5 U/kg bleomycin intratracheally. Seven surviving mice were scanned with micro-CT before administration of bleomycin, and again before sacrifice. The resulting images were analyzed for lung volume measurements. After the final scan, all lungs were examined histologically and pulmonary damage was quantified. Damaged lung tissue regions were matched between micro-CT images and histologic sections for each mouse. RESULTS: The percent lung damage calculated from micro-CT and histology were correlated (r(2) = 0.49, r = 0.64 with P = 0.12), and the means of their respective distributions were not different (P > 0.05). CONCLUSION: This study shows that micro-CT is a promising alternative to predicting lung damage caused by bleomycin. CT image volumes of the thorax allow for global tissue sampling, which may be useful when following nonuniform lung damage that can occur from intratracheal administration of bleomycin.

Many human medulloblastoma tumors overexpress repressor element-1 silencing transcription (REST)/neuron-restrictive silencer factor, which can be functionally countered by REST-VP16.

Medulloblastoma, one of the most malignant pediatric brain tumors, is believed to arise from the undifferentiated external granule-layer cells in the cerebellum. It is a heterogeneous cancer, and the mechanism of tumorigenesis for the majority of types is unknown. Repressor element-1 silencing transcription/neuron-restrictive silencer factor (REST/NRSF) is a transcriptional repressor that can block transcription of a battery of neuronal differentiation genes by binding to a specific consensus DNA sequence present in their regulatory region. Previously, we found that some medulloblastoma cell lines express REST/NRSF at high levels compared with either neuronal progenitor cells or fully differentiated neurons. However, it is not known if REST/NRSF is indeed overexpressed in human medulloblastoma tumor specimens and in what frequency. Here, we did an immunohistochemical analysis of such tumor specimens using an anti-REST antibody. We show that among 21 human medulloblastoma tumors, 17 expressed REST/NRSF (6 strongly and 11 weakly). In contrast, adjacent normal cerebellum tissue sections and four of the tumor specimens did not express REST/NRSF, indicating that abnormal expression of REST/NRSF is observed in the majority of human medulloblastoma tumors. To determine whether countering REST/NRSF activity blocks tumorigenicity of medulloblastoma cells, especially in the intracranial (i.c.) environment, we found that adenovirus-mediated expression of REST-VP16, a recombinant transcription factor that can compete with REST/NRSF and activate REST/NRSF target genes instead of repressing them, blocked the i.c. tumorigenic potential of medulloblastoma cells and inhibited growth of established tumors in nude mice, suggesting that REST/NRSF may serve as a therapeutic target for medulloblastoma and that forced expression of neuronal differentiation genes in medulloblastoma cells through agents, such as REST-VP16, can interfere with their tumorigenicity.

Characterization of internodal collecting lymphatic vessel function after surgical removal of an axillary lymph node in mice.

Secondary lymphedema is an acquired lymphatic disorder, which occurs because of damage to the lymphatic system from surgery and/or radiation therapy for cancer treatment. However, it remains unknown how post-nodal collecting lymphatic vessels (CLVs) draining to the surgical wound area change in response to lymphadenectomy. We investigated functional and architectural changes of inguinal-to-axillary internodal CLVs (ICLVs) in mice after a single axillary LN (ALN) dissection using near-infrared fluorescence imaging. Our data showed no lymph flow in the ICLVs draining from the inguinal LN (ILN) at 2 days post-surgery. External compression enabled visualization of a small segment of contractile fluorescent ICLVs, but not all the way to the axillary region. At day 6, abnormal lymphatic drainage patterns, including lateral and retrograde lymph flow via vessels branching off the ICLVs were observed, which started to disappear beginning 9 days after surgery. The administration of vascular endothelial growth factor (VEGF)-C into the wound increased resolution of altered lymphatic drainage. Lymphatic drainage from the base of the tail to the ILN did not significantly change over time. These results demonstrate that lymph flow in the CLVs is dramatically affected by a LN dissection and long-term interruption of lymph flow might cause CLV dysfunction and thus contribute to chronic lymphatic disorders.

Plasminogen activator inhibitor-1 does not contribute to the pulmonary pathology induced by acute exposure to ozone.

Expression of plasminogen activator inhibitor (PAI)-1, the major physiological inhibitor of fibrinolysis, is increased in the lung following inhalation of ozone (O3), a gaseous air pollutant. PAI-1 regulates expression of interleukin (IL)-6, keratinocyte chemoattractant (KC), and macrophage inflammatory protein (MIP)-2, which are cytokines that promote lung injury, pulmonary inflammation, and/or airway hyperresponsiveness following acute exposure to O3 Given these observations, we hypothesized that PAI-1 contributes to the severity of the aforementioned sequelae by regulating expression of IL-6, KC, and MIP-2 following acute exposure to O3 To test our hypothesis, wild-type mice and mice genetically deficient in PAI-1 (PAI-1-deficient mice) were acutely exposed to either filtered room air or O3 (2 ppm) for 3 h. Four and/or twenty-four hours following cessation of exposure, indices of lung injury [bronchoalveolar lavage fluid (BALF) protein and epithelial cells], pulmonary inflammation (BALF IL-6, KC, MIP-2, macrophages, and neutrophils), and airway responsiveness to aerosolized acetyl-ß-methylcholine chloride (respiratory system resistance) were measured in wild-type and PAI-1-deficient mice. O3 significantly increased indices of lung injury, pulmonary inflammation, and airway responsiveness in wild-type and PAI-1-deficient mice. With the exception of MIP-2, which was significantly lower in PAI-1-deficient as compared to wild-type mice 24 h following cessation of exposure to O3, no other genotype-related differences occurred subsequent to O3 exposure. Thus, following acute exposure to O3, PAI-1 neither regulates pulmonary expression of IL-6 and KC nor functionally contributes to any of the pulmonary pathological sequelae that arise from the noxious effects of inhaled O3.

Potential role of growth factors in diminishing radiation therapy neural tissue injury.

Human growth factors are firmly established in treatment of cytopenias that are associated with cancer chemotherapy, and have been used successfully to reduce severe mucositis in patients receiving radiation therapy and chemotherapy in the setting of autologous bone marrow transplantation. The ability of growth factors that are involved in differentiation and proliferation of neural tissue cells to prevent or accelerate recovery from radiation injury currently is being evaluated in preclinical studies. Data from these studies indicate that brief therapeutic intervention with platelet-derived growth factor, insulin-like growth factor-1, vascular endothelial growth factor, and the combination of insulin-like growth factor-1 and basic fibroblast growth factor can prevent or delay radiation myelopathy after spinal cord irradiation. Additional investigation is required to define potentially clinically useful growth factor regimens in the clinic.