Increased Expression of ATG10 in Colorectal Cancer Is Associated with Lymphovascular Invasion and Lymph Node Metastasis

Background

Autophagy has paradoxical and complex functions in cancer development, and autophagy-related genes (ATG) are key regulators in autophagy. Until now, more than 30 different ATG proteins have been identified in yeast, and their mammalian counterparts also have been reported. Although the roles of a few ATG proteins in cancer have been characterized, the role of ATG10 is almost completely unknown.

Methodology/Principal Findings

To investigate the clinicopathological role of ATG10 in colorectal cancer, we analyzed ATG10 expression in colorectal cancer tissues and cell lines. Protein expression analysis showed that ATG10 is highly increased in colorectal cancer (tissue - 18/37 cases, 48%; cell line –8/12 cell lines, 66%). Immunohistochemical analysis with clinicopathological features indicated a strong association of the up-regulation of ATG10 with tumor lymph node metastasis (p = 0.005) and invasion (p<0.001). Moreover, both 5-year disease free survival and overall survival rates of patients bearing tumors that did not express ATG10 were significantly higher than those of patients bearing ATG10-expressing tumors (p = 0.012).

Conclusion/Significance

Increased expression of ATG10 in colorectal cancer is associated with lymphovascular invasion and lymph node metastasis indicating that ATG10 may be a potential prognostic maker in colorectal cancer.     

Abundance and biomass estimates of the benthic fauna in Admiralty Bay,King George Island,South Shetland Islands

Summary Quantitative benthic samples were collected along three transects in Admiralty Bay, King George Island, South Shetlands. At each of a total of 18 stations, between 15 and 250 m depth, we took 3 replicate samples with a van Veen grab. Animals collected were classed into major groups. Abundance and biomass per m² was calculated for each sampling site. Considerable population densities and high biomass values were found. Most abundant groups were Bivalvia, Polychaeta and Amphipoda, whereas the largest part of the biomass was due to Ascidiacea, Ophiuroidea, Echinoidea, Polychaeta and Bivalvia. The maximum abundance recorded was 36,000 ind m^-2 while the average was approximately 6500 ind m^-2. Maximum biomass was over 2400 g m^-2 with an average of ca. 700 g m^-2. The contribution to the total biomass by groups such as the Oligochaeta, Cumacea and Tanaidacea was higher in the inner shallow part of Admiralty Bay (Ezcurra Inlet) than in the deeper areas of the bay. Our results confirm the reports on an unusually high density and biomass of the Antarctic sublittoral benthic fauna. Sessile suspension feeders belonging to the Bivalvia, Ascidiacea, sedentary Polychaeta, and vagile scavengers of the Ophiuroidea, Amphipoda and errant Polychaeta are the most significant groups in the Antarctic Ecosystem. The total benthic biomass in Admiralty Bay, based on the present preliminary quantitative data, was estimated to be over 600,000 t. This value is probably still an underestimate.     

GSK-3α Is a Novel Target of CREB and CREB-GSK-3α Signaling Participates in Cell Viability in Lung Cancer

Overexpression or activation of cyclic AMP-response element-binding protein (CREB) has been known to be involved in several human malignancies, including lung cancer. Genes regulated by CREB have been reported to suppress apoptosis, induce cell proliferation, inflammation, and tumor metastasis. However, the critical target genes of CREB in lung cancer have not been well understood. Here, we identified GSK-3α as one of the CREB target genes which is critical for the viability of lung cancer cells. The CREB knockdown significantly reduced the expression of GSK-3α and the direct binding of CREB on the promoter of GSK3A was identified. Kaplan-Meier analysis with a public database showed a prognostic significance of aberrant GSK-3α expression in lung cancer. Inhibition of GSK-3α suppressed cell viability, colony formation, and tumor growth. For the first time, we demonstrated that GSK-3α is regulated by CREB in lung cancer and is required for the cell viability. These findings implicate CREB-GSK-3α axis as a novel therapeutic target for lung cancer treatment.     

Synthesis of cinnamoyl ketoamides as hybrid structures of antioxidants and calpain inhibitors

The excessive calpain activation causes serious cellular damage or even cell death in neurological disorders such as stroke and Alzheimer’s disease. Oxidative stress has also been implicated in the initiation or progression of neurodegenerative diseases. In the present studies, a series of cinnamoyl ketoamides 4a-4j were synthesized as hybrid structures of antioxidants and calpain inhibitors. Cinnamoyl ketoamides, possessing an alkyl chain at the α-position, showed potent μ-calpain inhibitory activities indicating that the cinnamoyl skeleton can be regarded as an acyclic variant of calpain inhibitory chromone carboxamide 2. Among synthesized, compound 4e was the most potent inhibitor of μ-calpain (IC₅₀ = 0.13 μM) and also exhibited strong antioxidant activities in DPPH and superoxide anion radical scavenging and lipid peroxidation inhibition assay systems.     

Histocompatibility antigens (HLA) in children with lipoid nephrosis]

Actual knowledge on the HLA relationship with the primary glomerulopathies, with particular reference to steroid - sensitive nephrosis of childhood, is surveyed. Occurrence of HLA B-8 and B-35 in this nephropathy has been investigated. The studies involved 47 patients aged between 3 and 15 years and 117 healthy children from Lower Silesian region. It has been showed, that HLA B-8 is present more frequently in sick children, than in healthy controls. The situation is reverse in case of HLA B-35 antigen. However, the difference is statistically insignificant. A probability of the lipid nephrosis sensitivity to corticosteroids can not be predicted on the base of the presence of these HLA antigens.     

A case of acute gastric anisakiasis provoking severe clinical problems by multiple infection

Acute gastric anisakiasis with multiple anisakid larvae infection is reported. A 68-year-old woman residing in Busan, Korea, had epigastric pain with severe vomiting about 5 hours after eating raw anchovies. Four nematode larvae penetrating the gastric mucosae in the great curvature of the middle body and fundus were found and removed during gastro-endoscopic examination. Another one thread-like moving larva was found in the great curvature of upper body on the following day. On the basis of their morphology, the worms were identified as the 3rd stage larvae of Anisakis simplex. This case is acute gastric anisakiasis provoking severe clinical problems by the multiple infection and the greatest number of anisakid larvae found in a patient in Korea.     

Activated platelets rapidly up-regulate CD40L expression and can effectively mature and activate autologous ex vivo differentiated DC

Background DC are a promising immunotherapeutic for treatment of infectious/malignant disease. For clinical trials, immature DC generated from precursor cells such as monocytes, using serum-free media containing GM-CSF and IL-4, can be matured with specific cytokines/factors. CD40 ligand (CD40L) plays an important role in DC activation/maturation but is not available for clinical applications. These studies evaluated the feasibility of using activated platelets with elevated CD40L surface expression to stimulate autologous DC maturation. Methods Pilot and clinical scale studies were executed using magnetic/centrifugal separation. Monocyte precursors were differentiated to immature DC with GM-CSF and IL-4 and the ability of activated autologous platelets to mature these cells was evaluated on the basis of phenotype and function. Results In small-scale studies, DC cultures stimulated with activated autologous platelets (CD40L-AP), tumor necrosis factor-alpha (TNF-alpha) or soluble CD40L (sCD40L) up-regulated expression of phenotype markers indicative of activation and maturation. CD86 expression was significantly enhanced (P<0.05) by stimulation with either CD40L-AP (75.5+/-14.5%) or sCD40L (80.5%+/-5.3%) compared with immature DC (55.2+/-14.8%), as were CD80 and CD83. Similarly, in large-scale studies using Isolex 300I to enrich for monocytes and platelets for DC generation/maturation on a clinical scale, stimulation with CD40L-AP increased CD86 and CD80 expression as well as the ability to stimulate allogeneic lymphocytes compared with control cultures. Discussion These results demonstrate that thrombin-activated platelets express CD40L and are effective at inducing matured DC with potent immunogenic activity. Furthermore, these studies demonstrate the feasibility of this approach for clinical immunotherapeutic interventions.     

Process intensification strategies toward cell culture-based high-yield production of a fusogenic oncolytic virus

We present a proof-of-concept study for production of a recombinant vesicular stomatitis virus (rVSV)-based fusogenic oncolytic virus (OV), rVSV-Newcastle disease virus (NDV), at high cell densities (HCD). Based on comprehensive experiments in 1 L stirred tank reactors (STRs) in batch mode, first optimization studies at HCD were carried out in semi-perfusion in small-scale cultivations using shake flasks. Further, a perfusion process was established using an acoustic settler for cell retention. Growth, production yields, and process-related impurities were evaluated for three candidate cell lines (AGE1.CR, BHK-21, HEK293SF)infected at densities ranging from 15 to 30 × 10⁶ cells/mL. The acoustic settler allowed continuous harvesting of rVSV-NDV with high cell retention efficiencies (above 97%) and infectious virus titers (up to 2.4 × 10⁹ TCID₅₀/mL), more than 4–100 times higher than for optimized batch processes. No decrease in cell-specific virus yield (CSVY) was observed at HCD, regardless of the cell substrate. Taking into account the accumulated number of virions both from the harvest and bioreactor, a 15–30 fold increased volumetric virus productivity for AGE1.CR and HEK293SF was obtained compared to batch processes performed at the same scale. In contrast to all previous findings, formation of syncytia was observed at HCD for the suspension cells BHK 21 and HEK293SF. Oncolytic potency was not affected compared to production in batch mode. Overall, our study describes promising options for the establishment of perfusion processes for efficient large-scale manufacturing of fusogenic rVSV-NDV at HCD for all three candidate cell lines.     

Comparison of responses elicited by immunization with a Legionella species common lipoprotein delivered as naked DNA or recombinant protein

To evaluate the peptidoglycan-associated lipoprotein (PAL) antigen of Legionella pneumophila as a vaccine candidate, mice were immunized intramuscularly with pcDNA3-PAL and intraperitoneally with recombinant PAL (t-rPAL), which were compared for their ability to induce PAL-specific immune responses. The t-rPAL protein induced PAL-specific IgG antibody production significantly more than did pcDNA3-PAL. The IgG2a and IgG1 production was predominant after pcDNA3-PAL and t-rPAL administration, respectively. In particular, pcDNA3-PAL induced much higher PAL-specific cytotoxic T-lymphocyte responses than did t-rPAL. Furthermore, in vivo, CD19+ B-cell populations were dramatically increased by t-rPAL vaccination, suggesting a B-cell immunomodulatory activity of the lipoprotein. The PAL antigen was also conserved among Legionella species, as determined by PCR and immunoblot analyses. These results support a potential use of the t-rPAL protein and in particular DNA vaccines against Legionella infections.     

Characterization of a quail suspension cell line for production of a fusogenic oncolytic virus

The development of efficient processes for the production of oncolytic viruses (OV) plays a crucial role regarding the clinical success of virotherapy. Although many different OV platforms are currently under investigation, manufacturing of such viruses still mainly relies on static adherent cell cultures, which bear many challenges, particularly for fusogenic OVs. Availability of GMP-compliant continuous cell lines is limited, further complicating the development of commercially viable products. BHK21, AGE1. CR and HEK293 cells were previously identified as possible cell substrates for the recombinant vesicular stomatitis virus (rVSV)-based fusogenic OV, rVSV-NDV. Now, another promising cell substrate was identified, the CCX.E10 cell line, developed by Nuvonis Technologies. This suspension cell line is considered non-GMO as no foreign genes or viral sequences were used for its development. The CCX.E10 cells were thus thoroughly investigated as a potential candidate for OV production. Cell growth in the chemically defined medium in suspension resulted in concentrations up to 8.9 × 10⁶ cells/mL with a doubling time of 26.6 h in batch mode. Cultivation and production of rVSV-NDV, was demonstrated successfully for various cultivation systems (ambr15, shake flask, stirred tank reactor, and orbitally shaken bioreactor) at vessel scales ranging from 15 mL to 10 L. High infectious virus titers of up to 4.2 × 10⁸ TCID₅₀/mL were reached in orbitally shaken bioreactors and stirred tank reactors in batch mode, respectively. Our results suggest that CCX.E10 cells are a very promising option for industrial production of OVs, particularly for fusogenic VSV-based constructs.