Integrating cross-modality hallucinated MRI with CT to aid mediastinal lung tumor segmentation.

Lung tumors, especially those located close to or surrounded by soft tissues like the mediastinum, are difficult to segment due to the low soft tissue contrast on computed tomography images. Magnetic resonance images contain superior soft-tissue contrast information that can be leveraged if both modalities were available for training. Therefore, we developed a cross-modality educed learning approach where MR information that is educed from CT is used to hallucinate MRI and improve CT segmentation. Our approach, called cross-modality educed deep learning segmentation (CMEDL) combines CT and pseudo MR produced from CT by aligning their features to obtain segmentation on CT. Features computed in the last two layers of parallelly trained CT and MR segmentation networks are aligned. We implemented this approach on U-net and dense fully convolutional networks (dense-FCN). Our networks were trained on unrelated cohorts from open-source the Cancer Imaging Archive CT images (N=377), an internal archive T2-weighted MR (N=81), and evaluated using separate validation (N=304) and testing (N=333) CT-delineated tumors. Our approach using both networks were significantly more accurate (U-net P < 0.001; denseFCN P < 0.001) than CT-only networks and achieved an accuracy (Dice similarity coefficient) of 0.71±0.15 (U-net), 0.74±0.12 (denseFCN) on validation and 0.72±0.14 (U-net), 0.73±0.12 (denseFCN) on the testing sets. Our novel approach demonstrated that educing cross-modality information through learned priors enhances CT segmentation performance.

The PI3K inhibitor GDC-0941 combines with existing clinical regimens for superior activity in multiple myeloma.

The phosphatidylinositol 3'-kinase (PI3K) pathway is dysregulated in multiple myeloma (MM); we therefore tested a highly selective class I PI3K inhibitor, GDC-0941, for anti-myeloma activity. Functional and mechanistic studies were first performed in MM cell lines, then extended to primary MM patient samples cultured in vitro. GDC-0941 was then assessed as a single agent and in various combinations in myeloma tumor xenograft models. We show p110 α and ß are the predominant PI3K catalytic subunits in MM and that a highly selective class I PI3K inhibitor, GDC-0941, has robust activity as a single agent to induce cell cycle arrest and apoptosis of both MM cell lines and patient myeloma cells. Mechanistic studies revealed an induction of cell cycle arrest at G0/G1, with decreased phospho-FoxO1/3a levels, decreased cyclin D1 and c-myc expression, and an increase in the cell cycle inhibitor, p27kip. Induction of apoptosis correlated with increased expression of the pro-apoptotic BH3-only protein BIM, cleaved caspase 3 and cleaved poly (ADP-ribose) polymerase (PARP). In vitro, GDC-0941 synergized with dexamethasone (Dex) and lenalidomide (combination index values of 0.3-0.4 and 0.4-0.8, respectively); in vivo GDC-0941 has anti-myeloma activity and significantly increases the activity of the standard of care agents in several murine xenograft tumor models (additional tumor growth inhibition of 37-53% (Dex) and 22-72% (lenalidomide)). These data provide a clear therapeutic hypothesis for the inhibition of PI3K and provide a rationale for clinical development of GDC-0941 in myeloma.

Towards a role of interleukin-32 in atherosclerosis.

BACKGROUND: IL-32 has been previously shown to promote inflammation in rheumatoid arthritis patients and to contribute to IL-1ß-induced ICAM-1 as well as other proinflammatory cytokines synthesis in human umbilical endothelial cells (HUVECs). Given the high rate of atherosclerosis in RA, these observations suggest that IL-32 may be involved in the inflammatory pathways of atherosclerosis. METHODS: mRNA and protein levels of IL-32 were determined in human atherosclerotic arterial vessel wall tissue by quantitative real-time PCR and immunohistochemistry. HUVEC and M1/M2 macrophages were stimulated with proinflammatory cytokines and TLR ligands to assess IL-32 mRNA induction. Human THP1 macrophages were transduced with AdIL-32γ, to investigate induction of several proatherosclerotic mediators. Finally, aortas from IL-32γ transgenic mice were studied and compared with aortas from age-matched wild-type mice. RESULTS: IL-32 expression was detectable in human atherosclerotic arterial vessel wall, with the expression of IL-32ß and IL-32γ mRNA significantly enhanced. TLR3-ligand Poly I:C in combination with IFNγ were the most potent inducers of IL-32 mRNA expression in both HUVEC and M1/M2 macrophages. Adenoviral overexpression of IL-32γ in human THP1 macrophages resulted in increased production of CCL2, sVCAM-1, MMP1, MMP9, and MMP13. The IL-32γ transgenic mice chow a normal fat diet exhibited vascular abnormalities resembling atherosclerosis. CONCLUSIONS: IL-32 acts as a proinflammatory factor and may be implicated in the inflammatory cascade contributing to atherosclerosis. By promoting the synthesis of matrix metalloproteinases, it may further contribute to plaque instability. Further studies are warranted to investigate whether IL-32 may serve as a potential therapeutic target in fighting atherosclerosis.

Generating CTLs against the subdominant EBV LMP antigens by transient expression of an A20 inhibitor with EBV LMP proteins in human DCs.

Epstein-Barr virus (EBV) infection leads to Hodgkin's disease (HD) in some immunocompetent hosts. The malignant Reed-Sternberg cells of HD only express a limited array of subdominant EBV antigens to evade pre-existing immune responses to EBV. The EBV-encoded latent membrane proteins (LMP1 and LMP2), which are expressed by HD and various EBV-associated malignancies, have been proposed as a potential target for cytotoxic T lymphocyte (CTL)-based therapy. However, the precursor frequency for LMP-specific CTL is generally low in healthy EBV-infected hosts, and immunotherapy based on these antigens is often compromised by the poor immunogenicity and the oncogenic potential. In the present study, we report that transiently expressing an inhibitor of A20, a key negative regulator of inflammatory signaling pathways, together with the LMP antigens (truncated LMP1 and full-length LMP2) greatly enhances maturation and cytokine production of human (h) monocyte-derived dendritic cells (DCs). As a consequence, LMP1/2-expressed, A20-silenced hDCs have an enhanced potency to prime LMP-specific T-cell response. When the in vitro primed T cells are adoptively transferred into tumor-xenografted, severe-combined immunodeficient mice, some of the xenografted tumors approach complete regression. Thus, the study may provide an available resource of LMP-specific T cells for T-cell immunotherapy.

In utero cigarette smoke exposure impairs somatic and lung growth in BALB/c mice.

The aim of this study was to assess whether in utero tobacco smoke exposure alone affects early-life lung growth and development. Pregnant BALB/c mice were exposed to cigarette smoke from six cigarettes per day, or air, from day 8 to 20 of gestation. At 2 weeks of age, pups were weighed and had their lung volumes and lung mechanics measured. Pups born from mothers exposed to cigarette smoke (CS pups; n=17) were significantly lighter (6.76 ± 0.76 versus 7.72 ± 0.68 g) and had lower lung volumes (0.123 ± 0.02 versus 0.149 ± 0.02 mL) than control pups (n=20). Respiratory mechanics were adversely impacted by cigarette smoke exposure. CS pups had higher baseline airway resistance, tissue damping and tissue elastance. These differences were largely due to lower lung volumes. Both tissue damping and elastance were increased excessively in CS pups at high transrespiratory pressures, while other parameters were not affected. There were no histological differences between groups. In utero tobacco smoke exposure significantly affects growth and development in BALB/c mice. These impacts may partially explain the susceptibility of infants born to smoking mothers to early respiratory disease and chronic respiratory disease as adults.

Cyanide in bronchoalveolar lavage is not diagnostic for Pseudomonas aeruginosa in children with cystic fibrosis.

Early detection of the cyanobacterium Pseudomonas aeruginosa in the lungs of young children with cystic fibrosis (CF) is considered the key to delaying chronic pulmonary disease. We investigated whether cyanide in bronchoalveolar lavage (BAL) fluid could be used as an early diagnostic biomarker of infection. Cyanide was measured in 226 BAL samples (36 P. aeruginosa infected) obtained from 96 infants and young children with CF participating in an early surveillance programme involving annual BAL. Cyanide was detected in 97.2% of P. aeruginosa infected and 60.5% of uninfected samples. Cyanide concentrations were significantly higher in BALs infected with P. aeruginosa (median (25th-75th percentile) 27.3 (22.1-33.3) µM) than those which were not (17.2 (7.85-23.0) µM, p<0.001). The best sensitivity, specificity, positive and negative predictive values were obtained with a cut-off concentration of 20.6 µM, and were 83%, 66%, 32% and 96%, respectively. Neutrophil number in BAL was a significant predictor of cyanide concentration (p<0.001). Cyanide concentration can distinguish between P. aeruginosa infected and uninfected BALs as a group, but not individually; therefore, cyanide is a poor diagnostic biomarker of P. aeruginosa infection. Cyanide levels in BAL are related to the level of neutrophilic inflammation.

Adoptive immunotherapy for cancer: the next generation of gene-engineered immune cells.

Adoptive cellular immunotherapy involving transfer of tumor-reactive T cells has shown some notable antitumor responses in a minority of cancer patients. In particular, transfer of tumor-infiltrating lymphocytes has resulted in long-term objective responses in patients with advanced melanoma. However, the inability to isolate sufficient numbers of tumor-specific T cells from most malignancies has restricted the broad utility of this approach. An emerging approach to circumvent this limitation involves the genetic modification of effector cells with T cell receptor (TCR) transgenes or chimeric single-chain variable fragment (scFv) receptors that can specifically redirect T cells to tumor. There has been much progress in the design of TCR and scFv receptors to enhance the antigen-specific activation of effector cells and their trafficking and persistence in vivo. Considerable effort has been directed toward improving the safety of this approach and reducing the immunogenicity of the receptor. This review discusses the latest developments in the field of adoptive immunotherapy using genetically modified immune cells that have been transduced with either TCR or scFv receptor transgenes and used in preclinical and clinical settings as anticancer agents.

Acquisition and eradication of P. aeruginosa in young children with cystic fibrosis.

When do infants and young children with cystic fibrosis acquire infection with Pseudomonas aeruginosa? Can this be eradicated when first detected? Children <6 yrs of age participated in an annual bronchoalveolar lavage (BAL)-based microbiological surveillance programme in Perth, Australia. When P. aeruginosa was detected, an eradication programme using combination treatment with i.v., oral and nebulised antibiotics was undertaken. Repeat BAL was performed 3 months following treatment, to assess eradication success. P. aeruginosa was detected in 33 (28.4%) children; median (range) age at detection was 30.5 (3.3-71.4) months. P. aeruginosa was mucoid at detection in six (18.2%) out of 33 patients and associated with respiratory symptoms in 16 (48.5%) out of 33 children. In total, 26 children underwent eradication therapy, with P. aeruginosa eradicated in 20 (77%) out of 26 following one eradication cycle and in three (total 88%) additional children following a second cycle. Eradication was associated with a significant decrease in neutrophil elastase and interleukin-1beta in BAL fluid 12 months post eradication. Eradication of Pseudomonas aeruginosa infection is achievable in young children with cystic fibrosis for up to 5 yrs using combination i.v., oral and nebulised antibiotic therapy and is associated with reduced pulmonary inflammation 12 months post eradication.

In vitro and in vivo characterisation of anti-murine IL-13 antibodies recognising distinct functional epitopes.

Interleukin-13 (IL-13) sequentially binds to IL-13Ralpha1 and IL-4Ralpha forming a high affinity signalling complex. This receptor complex is expressed on multiple cell types in the airway and signals through signal transducer and activator of transcription factor-6 (STAT-6) to stimulate the production of chemokines, cytokines and mucus. Antibodies have been generated, using the UCB Selected Lymphocyte Antibody Method (UCB SLAM), that block either binding of murine IL-13 (mIL-13) to mIL-13Ralpha1 and mIL-13Ralpha2, or block recruitment of mIL-4Ralpha to the mIL-13/mIL-13Ralpha1 complex. Monoclonal antibody (mAb) A was shown to bind to mIL-13 with high affinity (K(D) 11 pM) and prevent binding of mIL-13 to mIL-13Ralpha1. MAb B, that also bound mIL-13 with high affinity (K(D) 8 pM), was shown to prevent recruitment of mIL-4Ralpha to the mIL-13/mIL-13Ralpha1 complex. In vitro, mAbs A and B similarly neutralised mIL-13-stimulated STAT-6 activation and TF-1 cell proliferation. In vivo, mAbs A and B demonstrated equipotent, dose-dependent inhibition of eotaxin generation in mice stimulated by intraperitoneal administration of recombinant mIL-13. In an allergic lung inflammation model in mice, mAbs A and B equipotently inhibited muc5ac mucin mRNA upregulation in lung tissue measured two days after intranasal allergen challenge. These data support the design of therapeutics for the treatment of allergic airway disease that inhibits assembly of the high affinity IL-13 receptor signalling complex, by blocking the binding of IL-13 to IL-13Ralpha1 and IL-13Ralpha2, or the subsequent recruitment of IL-4Ralpha.

Results of an international round robin for the quantification of serum non-transferrin-bound iron: Need for defining standardization and a clinically relevant isoform.

Non-transferrin-bound iron (NTBI) appears in the circulation of patients with iron overload. Various methods to measure NTBI were comparatively assessed as part of an international interlaboratory study. Six laboratories participated in the study, using methods based on iron mobilization and detection with iron chelators or on reactivity with bleomycin. Serum samples of 12 patients with hereditary (n=11) and secondary (n=1) hemochromatosis were measured during a 3-day analysis using 4 determinations per sample per day, making a total of 144 measurements per laboratory. Bland-Altman plots for repeated measurements are presented. The methods differed widely in mean serum NTBI level (range 0.12-4.32mumol/L), between-sample variation (SD range 0.20-2.13mumol/L and CV range 49.3-391.3%), and within-sample variation (SD range 0.02-0.45mumol/L and CV range 4.4-193.2%). The results obtained with methods based on chelators correlated significantly (R(2) range 0.86-0.99). On the other hand, NTBI values obtained by the various methods related differently from those of serum transferrin saturation (TS) when expressed in terms of both regression coefficients and NTBI levels at TS of 50%. Recent studies underscore the clinical relevance of NTBI in the management of iron-overloaded patients. However, before measurement of NTBI can be introduced into clinical practice, there is a need for more reproducible protocols as well as information on which method best represents the pathophysiological phenomenon and is most pertinent for diagnostic and therapeutic purposes.

Management of post cholecystectomy Mirizzi's syndrome.

Various strategies have been proposed for the management of retained calculi within the biliary tree following cholecystectomy. We present a unique case of a cystic duct remnant calculus causing Mirizzi syndrome, only the fourth such case of its kind. An open procedure was planned, however the calculus was eventually extracted endoscopically. The pathophysiology and management of Mirizzi syndrome and retained calculi within the cystic duct remnant are discussed along with the merits of a minimally invasive approach.

Plasmodium falciparum signal sequences: simply sequences or special signals?

The malaria parasite, Plasmodium falciparum, synthesises and exports several proteins inducing morphological and biochemical modifications of erythrocytes during the erythrocytic cycle. The protein trafficking machinery of the parasite is similar to that of other eukaryotic cells in several ways. However, some unusual features are also observed. The secretion of various polypeptides was inhibited when P. falciparum-infected erythrocytes were incubated with Brefeldin A. Immunoelectron microscopy studies revealed substantial morphological changes in the endoplasmic reticulum following exposure of parasitised erythrocytes to the drug. Immunofluorescence studies of Brefeldin A-treated parasites suggest that polypeptide sorting to different intracellular destinations begins at the endoplasmic reticulum. The parasite also secretes polypeptides by a Brefeldin A-insensitive route that bypasses the classical endoplasmic reticulum-Golgi complex pathway.

In vitro and in vivo antimalarial activity of ferrochloroquine, a ferrocenyl analogue of chloroquine against chloroquine-resistant malaria parasites.

Previous studies have shown that ferrochloroquine (FQ) exhibited an antimalarial activity against Plasmodium spp. The present work confirmed this activity, described the curative effect on P. vinckei and investigated the FQ toxicity in vitro and in vivo. The in vitro and in vivo growth inhibition of P. falciparum and P. berghei N, respectively, showed that FQ antimalarial activity was 1.5-10 times more potent than chloroquine. FQ completely inhibited the in vivo development of both chloroquine-susceptible and resistant P. vinckei strains and protected mice from lethal infection at a dose of 8.4 mg kg(-1) day(-1) given for 4 days subcutaneously or orally. This curative effect was 5-20 times more potent than chloroquine, according to the strains' resistance to chloroquine. At this curative dose, no clinical changes were observed in mice up to 14 days after the last administration. Nevertheless, the acute toxicity and lethality of ferrochloroquine seemed to be dependent on gastric surfeit. The FQ security index determined in vitro confirmed that it might be a promising compound.

Intratracheal injection of adenovirus containing the human MnSOD transgene protects athymic nude mice from irradiation-induced organizing alveolitis.

PURPOSE: A dose and volume limiting factor in radiation treatment of thoracic cancer is the development of fibrosis in normal lung. The goal of the present study was to determine whether expression prior to irradiation of a transgene for human manganese superoxide dismutase (MnSOD) or human copper/zinc superoxide dismutase (Cu/ZnSOD) protects against irradiation-induced lung damage in mice. METHODS AND MATERIALS: Athymic Nude (Nu/J) mice were intratracheally injected with 10(9) plaque-forming units (PFU) of a replication-incompetent mutant adenovirus construct containing the gene for either human MnSOD, human copper/zinc superoxide dismutase (Cu/ZnSOD) or LacZ. Four days later the mice were irradiated to the pulmonary cavity to doses of 850, 900, or 950 cGy. To demonstrate adenoviral infection, nested reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out with primers specific for either human MnSOD or Cu/ZnSOD transgene on freshly explanted lung, trachea, or alveolar type II cells, and immunohistochemistry was used to measure LacZ expression. RNA was extracted on day 0, 1, 4, or 7 after 850 cGy of irradiation from lungs of mice that had previously received adenovirus or had no treatment. Slot blot analysis was performed to quantitate RNA expression for IL-1, tumor necrosis factor (TNF)-alpha, TGF-beta, MnSOD, or Cu/ZnSOD. Lung tissue was explanted and tested for biochemical activity of MnSOD or Cu/ZnSOD after adenovirus injection. Other mice were sacrificed 132 days after irradiation, lungs excised, frozen in OCT, (polyvinyl alcohol, polyethylene glycol mixture) sectioned, H&E stained, and evaluated for percent of the lung demonstrating organizing alveolitis. RESULTS: Mice injected intratracheally with adenovirus containing the gene for human MnSOD had significantly reduced chronic lung irradiation damage following 950 cGy, compared to control mice or mice injected with adenovirus containing the gene for human Cu/ZnSOD or LacZ. Immunohistochemistry for LacZ protein in adenovirus LacZ (Ad-LacZ)-injected mice demonstrated expression of LacZ in both the upper and lower airway. Nested RT-PCR showed lung expression of MnSOD and Cu/ZnSOD for at least 11 days following infection with each respective adenovirus construct. Nested RT-PCR using primers specific for human MnSOD demonstrated increased expression of the human MnSOD transgene in the trachea and alveolar type II cells 4 days after virus injection on the day of irradiation. At this time point, increased biochemical activity of MnSOD and Cu/ZnSOD respectively, was detected in lungs from these two adenovirus groups, compared to each other or to control or adenovirus LacZ mice. Slot blot analysis of RNA from lungs of mice in each group following 850 cGy irradiation demonstrated decreased expression of mRNA for interleukin-I (IL-1), TNF-alpha, and transforming growth factor-beta (TGF-beta) in the MnSOD adenovirus-injected mice, compared to irradiated control, LacZ, or Cu/ZnSOD adenovirus-injected, irradiated mice. Mice receiving adenovirus MnSOD showed decreased organizing alveolitis at 132 days in all three dose groups, compared to irradiated control or Ad-LacZ, or Ad-Cu/ZnSOD mice. CONCLUSIONS: Overexpression of MnSOD in the lungs of mice prior to irradiation prevents irradiation-induced acute and chronic damage quantitated as decreased levels of mRNA for IL-1, TNF-alpha, and TGF-beta in the days immediately following irradiation, and decrease in the percent of lung demonstrating fibrosis or organizing alveolitis at 132 days. These data provide a rational basis for development of gene therapy as a method of protection of the normal lung from acute and chronic sequelae of ionizing irradiation.

The transport of the histidine-rich protein I from Plasmodium falciparum is insensitive to brefeldin A.

During its intraerythrocytic development, Plasmodium falciparum synthesizes several proteins that are exported beyond its membrane. Some of these secreted antigens are involved in the formation of protuberances or knobs, a major virulence factor, at the erythrocyte membrane. Various secreted malarial polypeptides, the transport of which is sensitive to brefeldin A, are translocated in vitro into dog pancreatic microsomes. We present evidence that the histidine-rich protein I (PfHRPI) is secreted by the parasite via a novel pathway, independent of the ER/Golgi apparatus. The secretion of PfHRPI was not blocked by incubation of parasite cultures at 15 degrees C and 20 degrees C or 37 degrees C in the presence of brefeldin A. PfHRPI was not translocated into microsomes in an in vitro translation-translocation cell-free system. Unlike other polypeptides from eukaryotic cells that bypass the ER/Golgi pathway and do not have a signal peptide, PfHRPI has an atypical signal sequence consisting of 21 amino acids, including eight positively charged residues followed by 11 hydrophobic residues. We also found that the unusually charged PfHRPI signal sequence diverts Exp-1, which is usually exported, away from the translocation machinery of microsomal membranes.

Influence of mechanical stretch on thrombin regulation by fetal mixed lung cells.

Respiratory distress syndrome (RDS) is characterized by intrapulmonary fibrin deposition, which can adversely affect surfactant function, and stimulate fibroblast proliferation, which may contribute to the development of bronchopulmonary dysplasia (BPD). We speculated that the premature lung may have impaired regulation of thrombin, thus making preterm infants susceptible to fibrin formation within the lung. Therefore, we studied the effect of stretch, which simulates fetal breathing movements (FBMs), on the generation and inhibition of a key hemostatic enzyme-thrombin-by rat fetal mixed lung cells (FMLCs). Our results showed that stretch induced glycosaminoglycan production with increased antithrombin activity due to an increase in the concentration of active chondroitin sulfate. Stretch downregulated secretion of tissue factor procoagulant activity, which may lead to decreased thrombin generation on the surface of FMLCs. Overall, stretch enhanced the local control of thrombin by FMLCs. These results suggest that premature infants, who will have experienced less FBM, may have impaired thrombin regulation. Impaired thrombin regulation likely contributes to increased fibrin deposition and, potentially, the development of BPD.

Polypeptide-polysaccharide conjugates produced by spontaneous non-enzymatic glycation.

A fundamental dogma has developed over the past 20 years that non-enzymatic glycation involving saccharide chains of greater than 3 to 4 residues is an extremely unlikely reaction. Our investigations using glycosaminoglycans have shown that, given sufficient time, polypeptide-polysaccharide conjugates form via the Schiff base-Amadori rearrangement mechanism. Further, even though these straight chain polysaccharides are relatively charged and sterically hindered, spontaneous glycation can also occur in vivo. A complete reinvestigation of all aldose terminating polysaccharides is required to elucidate this new class of macromolecules, which is likely to contain unusual polypeptide-polysaccharide combinations and functions.