Angiogenin is a member of the ribonuclease superfamily and promotes degradation of basement membrane and extracellular matrix. After stroke in Type one diabetes (T1DM) rats, Angiogenin is significantly increased and the Angiogenin is inversely correlated with functional outcome. Neamine, an aminoglycoside antibiotic, blocks nuclear translocation of Angiogenin, thereby abolishing the biological activity of Angiogenin. In this study, we therefore investigated the effect and underlying protective mechanisms of Neamine treatment of stroke in T1DM.
T1DM was induced in male Wistar rats by streptozotocin (60mg/kg, ip), and T1DM rats were subjected to embolic middle cerebral artery occlusion (MCAo). Neamine (10 mg/kg i.p.) was administered at 2h, 24h and 48h after induction of embolic MCAo. A battery of functional outcome tests was performed. Brain blood barrier (BBB) leakage, and lesion volume were evaluated and immunostaining, and Western blot were performed.
Neamine treatment of stroke in T1DM rats significantly decreased BBB leakage and lesion volume as well as improved functional outcome compared to T1DM-control. Neamine also significantly decreased apoptosis and cleaved caspase-3 in the ischemic brain. Using immunostaining, we found that Neamine treatment significantly decreased nuclear Angiogenin, nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) activity, advanced glycation endproducts receptor (RAGE) number, the positive area of toll-like receptor 4 (TLR4) and increased Angeopoietin-1 expression compared to T1DM-MCAo control rats. Western blot results are consistent with the immunostaining.
Neamine treatment of stroke is neuroprotective in T1DM rats. Inhibition of neuroinflammatory factor expression and decrease of BBB leakage may contribute to Neamine induced neuroprotective effects after stroke in T1DM rats.
Angiogenin; Neamine; neuroprotection; stroke; type one diabetes
We describe the case of a large pseudoaneurysm after transcatheter closure of patent ductus arteriosus in a 42-year old male who developed back pain and dyspnoea 6 months subsequent to the first procedure. The patient's pseudoaneurysm was successfully treated using Talent prostheses (Medtronic AVE, Santa Rosa, CA, USA), though the postoperative course was without incident.
Pseudo aneurysm; Patent ductus arteriosus; Endovascular techniques
T-cell factor (TCF) proteins represent key transcription factors that activate Wnt/β-catenin signaling. We have reported that a pair of TCF-4 isoforms (TCF-4C and TCF-4D) exhibits differential TCF transcriptional activity in hepatocellular carcinoma (HCC) cells, although their structure differs by only the presence (TCF-4D) or absence (TCF-4C) of exon 4.
To demonstrate a regulatory role of exon 4 in HCC development.
TCF-4C and TCF-4D expression profiles were examined in 27 pairs of human HCC and adjacent liver tissues. The functional role of the TCF-4 isoforms was evaluated in OUMS-29 (an immortalized hepatocyte-derived) and HAK-1A (a well differentiated HCC) cell lines using stable clones overexpressing the TCF-4 isoforms.
TCF-4C was significantly upregulated in HCC tissues compared to corresponding peritumor and normal liver tissues; in contrast, there was no difference of TCF-4D expression. TCF-4C clones derived from both cell lines exhibited increased TCF activity, Wnt-responsive target genes, cell proliferation, cell cycle progression, and resistance to chemotherapeutic drugs compared to TCF-4D clones. Capability of cell migration and colony formation was significantly higher in TCF-4C than TCF-4D clones. In a nude mice xenograft model, the HAK-1A-derived TCF-4C clone rapidly developed tumors compared to the TCF-4D clone. TCF-4C clone-derived tumors exhibited upregulation of Wnt-responsive target genes compared to the slow developing and small TCF-4D-derived tumors.
These results demonstrate that the TCF-4C isoform lacking exon 4 is associated with a malignant phenotype compared to the exon 4-harboring TCF-4D isoform, indicating that exon 4 of TCF-4 plays a prominent role in HCC development.
T-cell factor (TCF)-4; isoform; Wnt; hepatocellular carcinoma (HCC); exon 4
Although several studies suggest that stromal fibroblasts mediate treatment resistance in several cancer types, little is known about how tumor-associated astrocytes modulate the treatment response in brain tumors. Since traditionally used metabolic assays do not distinguish metabolic activity between stromal and tumor cells, and since 2-dimensional co-culture system does not recreate the formidable complexity of the microenvironment within 3-dimensional structures such as solid tumor tissue, we instead established a glioblastoma (GBM) cell-specific bioluminescent assay for direct measurements of tumor cell viability in the treatment of clinical relevant drugs.
Using lentiviral transfection, we established a panel of human GBM cell lines constitutively expressing a fusion transgene encoding luciferase and the enhanced green fluorescence protein (eGFP). We then initiated co-cultures with immortalized astrocytes, TNC-1, and the eGFP/Luc GBM cell lines. Next, we treated all eGFP/Luc GBM cell lines with Temozolomide (TMZ) or Doxorubicin, comparing co-cultures of glioblastoma (GBM) cells and TNC-1 astrocytes with mono-cultures of eGFP/Luc GBM cells. Cell viability was quantitated by measuring the luciferase expression.
Titration experiments demonstrated that luciferase expression was proportional to the number of eGFP/Luc GBM cells, whereas it was not influenced by the number of TNC-1 cells present. Notably, the presence of TNC-1 astrocytes mediated significantly higher cell survival after TMZ treatment in the U251, C6, A172 cell lines as well as the in vivo propagated primary GBM tumor cell line (P3). Moreover, TNC-1 astrocytes mediated significantly higher survival after Doxorubicin treatment in the U251, and LN18 glioma cell lines.
Glioma cell-specific bioluminescent assay is a reliable tool for assessment of cell viability in the brain tumor cell compartment following drug treatment. Moreover, we have applied this assay to demonstrate that astrocytes can modulate chemo sensitivity of GBM tumor cells. These effects varied both with the cell line and cytotoxic drug that were used, suggesting that several mechanisms may be involved.
Co-culture; Bioluminescent assay; Drug resistance; Glioblastoma
Background and purpose
We investigated the neurorestorative effects and underlying mechanisms of stroke treatment with human umbilical cord blood cells (HUCBCs) in Type one diabetes mellitus (T1DM) rats.
Type one diabetes mellitus rats were subjected to middle cerebral artery occlusion (MCAo) and 24 h later were treated with: (1) phosphate-buffered-saline; (2) HUCBCs. Brain endothelial cells (MBECs) were cultured and capillary tube formation was measured.
Human umbilical cord blood cells treatment significantly improved functional outcome and promoted white matter (WM) remodeling, as identified by Bielschowsky silver, Luxol fast blue and SMI-31 expression, increased oligodendrocyte progenitor cell and oligodendrocyte density after stroke in T1DM rats. HUCBC also promoted vascular remodeling, evident from enhanced vascular and arterial density and increased artery diameter, and decreased blood-brain barrier leakage. HUCBC treatment also increased Angiopoietin-1 and decreased receptor for advanced glycation end-products (RAGE) expression compared to T1DM-MCAo control. In vitro analysis of MBECs demonstrated that Ang1 inversely regulated RAGE expression. HUCBC and Ang1 significantly increased capillary tube formation and decreased inflammatory factor expression, while anti-Ang1 attenuated HUCBC-induced tube formation and antiinflammatory effects.
Human umbilical cord blood cells is an effective neurorestorative therapy in T1DM-MCAo rats and the enhanced vascular and WM remodeling and associated functional recovery after stroke may be attributed to increasing Angiopoietin-1 and decreasing RAGE.
HUCBC; Neurorestorative therapy; Stroke; T1DM; Vascular remodeling; White matter remodeling
Although the abundance and diversity of natural organochlorines are well established, much is still unknown about the degradation of these compounds. Triplicate microcosms were used to determine whether, and which, bacterial communities could dechlorinate two chlorinated xanthones (2,7-dichloroxanthone and 5,7-dichloro-1,3-dihydroxylxanthone), analogues of a diverse class of natural organochlorines. According to quantitative-PCR (qPCR) results, several known dechlorinating genera were either not present or not enriched during dechlorination of the xanthones. Denaturing gradient gel electrophoresis, however, indicated that several Firmicutes were enriched in the dechlorinating cultures compared to triplicate controls amended with nonchlorinated xanthones. One such group, herein referred to as the Gopher group, was further studied with a novel qPCR method that confirmed enrichment of Gopher group 16S rRNA genes in the dechlorinating cultures. The enrichment of the Gopher group was again tested with two new sets of triplicate microcosms. Enrichment was observed during chlorinated xanthone dechlorination in one set of these triplicate microcosms. In the other set, two microcosms showed clear enrichment while a third did not. The Gopher group is a previously unidentified group of Firmicutes, distinct from but related to the Dehalobacter and Desulfitobacterium genera; this group also contains clones from at least four unique cultures capable of dechlorinating anthropogenic organochlorines that have been previously described in the literature. This study suggests that natural chlorinated xanthones may be effective biostimulants to enhance the remediation of pollutants and highlights the idea that novel genera of dechlorinators likely exist and may be active in bioremediation and the natural cycling of chlorine.
Background: Hepatocellular carcinoma (HCC) is a common solid malignant tumor occurring worldwide that leads to the third largest cause of death compared to other cancers. Genetic and environmental factors are involved in the pathogenesis of HCC. Epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) can stimulate the proliferation of epidermal and epithelial cells. The EGF signal pathway has a relationship with the growth of the embryo, tissue repairing, and tumorigenesis. Methods: In this study, 416 patients with hepatitis B virus infection (HBV)-related HCC and 645 individuals who had never been infected with HBV of the Chinese Han population were enrolled. Eight single-nucleotide polymorphisms (SNPs), whose minor allele frequency >20% in the EGF and EGFR genes, were genotyped to examine their associations with hepatocarcinogenesis. Genotyping experiments were carried out using TaqMan. Results: There were significant differences in genotype distributions (p=0.005) and allele frequencies (p=0.001, odds ratio [OR]=1.43, 95% confidence interval [CI]=1.15–1.79) of rs11569017 in the EGF gene between the HCC and control groups. After binary logistic regression to determine independent factors for susceptibility to HCC under an additive model, rs11569017 was still independently associated with the susceptibility to HCC (p=0.021, OR=1.48, 95% CI=1.06–2.07), but no significant differences in other SNPs were found. Additionally, the haplotype T-G constructed by rs11569017 and rs4444903 of the EGF gene might increase the risk of HBV-related HCC (p=0.002, OR=1.44, 95% CI=1.15–1.82). Conclusion: The rs11569017 T allele was associated with susceptibility to HBV-related HCC.
Depression is a frequent mood disorder that affects around a third of stroke patients and has been associated with poorer outcomes. Our aim was to determine whether there was a relationship between inflammatory markers (leptin) and post-stroke depression (PSD).
One hundred and ninety-one ischemic stroke patients admitted to the hospital within the first 24 hours after stroke onset were consecutively recruited and followed up for 3 months. Enzyme-linked immunosorbent assay (ELISA) was used to measure serum levels of leptin at admission. Based on the symptoms, diagnoses of depression were made in accordance with DSM-IV criteria for post-stroke depression at 3 month.
Forty-four patients (23.0%) were diagnosed as having major depression at 3 month. Patients with depression showed higher serum leptin levels at 3 month after stroke (32.2 [IQR, 20.8–57.7] v. 9.9 [IQR, 4.6–13.1]ng/ml, respectively; P = 0.000). Serum levels of leptin ≥20 ng/ml were independently associated with PSD [odds ratio (OR) 20.23, 95% confidence interval (CI) 9.11–51.26, P = 0.000], after adjusting for possible confounders.
Serum leptin levels elevated at admission were found to be associated with PSD and may provide a new proposal for the treatment of PSD.
Presently, the understanding of bacterial enteric diseases in the community and their virulence factors relies almost exclusively on clinical disease reporting and examination of clinical pathogen isolates. This study aimed to investigate the feasibility of an alternative approach that monitors potential enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) prevalence and intimin gene (eae) diversity in a community by directly quantifying and characterizing target virulence genes in the sanitary sewage. The quantitative PCR (qPCR) quantification of the eae, stx1, and stx2 genes in sanitary sewage samples collected over a 13-month period detected eae in all 13 monthly sewage samples at significantly higher abundance (93 to 7,240 calibrator cell equivalents [CCE]/100 ml) than stx1 and stx2, which were detected sporadically. The prevalence level of potential EPEC in the sanitary sewage was estimated by calculating the ratio of eae to uidA, which averaged 1.0% (σ = 0.4%) over the 13-month period. Cloning and sequencing of the eae gene directly from the sewage samples covered the majority of the eae diversity in the sewage and detected 17 unique eae alleles belonging to 14 subtypes. Among them, eae-β2 was identified to be the most prevalent subtype in the sewage, with the highest detection frequency in the clone libraries (41.2%) and within the different sampling months (85.7%). Additionally, sewage and environmental E. coli isolates were also obtained and used to determine the detection frequencies of the virulence genes as well as eae genetic diversity for comparison.
The impact of substratum surface property change on biofilm community structure was investigated using laboratory biological aerated filter (BAF) reactors and molecular microbial community analysis. Two substratum surfaces that differed in surface properties were created via surface coating and used to develop biofilms in test (modified surface) and control (original surface) BAF reactors. Microbial community analysis by 16S rRNA gene-based PCR-denaturing gradient gel electrophoresis (DGGE) showed that the surface property change consistently resulted in distinct profiles of microbial populations during replicate reactor start-ups. Pyrosequencing of the bar-coded 16S rRNA gene amplicons surveyed more than 90% of the microbial diversity in the microbial communities and identified 72 unique bacterial species within 19 bacterial orders. Among the 19 orders of bacteria detected, Burkholderiales and Rhodocyclales of the Betaproteobacteria class were numerically dominant and accounted for 90.5 to 97.4% of the sequence reads, and their relative abundances in the test and control BAF reactors were different in consistent patterns during the two reactor start-ups. Three of the five dominant bacterial species also showed consistent relative abundance changes between the test and control BAF reactors. The different biofilm microbial communities led to different treatment efficiencies, with consistently higher total organic carbon (TOC) removal in the test reactor than in the control reactor. Further understanding of how surface properties affect biofilm microbial communities and functional performance would enable the rational design of new generations of substrata for the improvement of biofilm-based biological treatment processes.
It has become increasingly apparent that the pain threshold of females and males varies in an estrogen dependent manner. To investigate the modulation of pain by estrogen and the molecular mechanisms involved in this process. A total of 48 rats were ovariectomized (OVX). At 14 and 20 days after OVX, rats were divided into eight groups: groups 1–4 were administered drugs intravenously (IV); groups 5–8 were administered through intrathecal (IT) catheter. Hind paw incision was made in all animals to determine incisional pain. Paw withdraw threshold (PWT) was tested prior to and 24 h after incision. The test drugs were applied 24 h after the incision. Rats were either IV or IT administered with: 17-β-estradiol (E2), G protein-coupled estrogen receptor (GPER)-selective agonist (G1), GPER-selective antagonist (G15) and E2 (G15 + E2), or solvent. Before and 30 min after IV drug administration and 20 min during the IT catheter administration, PWT was tested and recorded. 24 h after incisional surgery, the PWT of all rats significantly decreased. Both in the IV group and IT group: administration of E2 and G1 significantly decreased PWT. Neither administration of G15 + E2 nor solvent significantly changed PWT. Estrogen causes rapid reduction in the mechanical pain threshold of OVX rats via GPER.
estrogen; estrogen receptors; G protein-coupled estrogen receptor (GPER); incisional pain
Cell therapy is emerging as a viable therapy to restore neurological function after stroke. Many types of stem/progenitor cells from different sources have been explored for their feasibility and efficacy for the treatment of stroke. Transplanted cells not only have the potential to replace the lost circuitry, but also produce growth and trophic factors, or stimulate the release of such factors from host brain cells, thereby enhancing endogenous brain repair processes. Although stem/progenitor cells have shown a promising role in ischemic stroke in experimental studies as well as initial clinical pilot studies, cellular therapy is still at an early stage in humans. Many critical issues need to be addressed including the therapeutic time window, cell type selection, delivery route, and in vivo monitoring of their migration pattern. This review attempts to provide a comprehensive synopsis of preclinical evidence and clinical experience of various donor cell types, their restorative mechanisms, delivery routes, imaging strategies, future prospects and challenges for translating cell therapies as a neurorestorative regimen in clinical applications.
Stem cells; Cell-based therapies; Ischemic stroke; Neurorestoration
AIM: To explore the feasibility and oncologic outcomes of segmental jejunal resection on the left side of the mesenteric vessels in patients with tumors of the angle of Treitz using data from a single center.
METHODS: Thirteen patients with tumors of the angle of Treitz who underwent surgery at our institution were prospectively followed. A segmental jejunal resection on the left side of the mesenteric vessels was performed in all patients. Formalin-fixed and paraffin-embedded tumor samples were examined. The primary end point of this analysis was disease-free survival.
RESULTS: In this study, there were 8 males and 5 females (mean age, 50.1 years; range, 36-74 years). The mean tumor size was 8.1 cm (range, 3.2-15 cm). Histologic examination showed 11 gastrointestinal stromal tumors (GISTs) and 2 adenocarcinomas. Five of the GIST patients presented with potential low risk, and 6 presented with intermediate and high risk, according to the National Institutes of Health criteria. One potentially high-risk patient showed tumor progression at 46 mo and died 52 mo after surgery. One patient with locally advanced adenocarcinoma received neoadjuvant chemotherapy and adjuvant radiotherapy, but the disease progressed, and the patient died 9 mo after surgery. One GIST patient without progression died 16 mo after surgery because of a postoperative intestinal obstruction. The median overall survival rate was 84.6 mo, and the median disease-free survival rate was 94.5 mo.
CONCLUSION: The overall survival of patients with tumors of the angle of Treitz was encouraging even when the tumor size was relatively large. A segmental resection on the left side of the mesenteric vessels is considered to be a reliable and curative option for tumors of the angle of Treitz.
Gastrointestinal stromal tumor; Adenocarcinoma; Angle of Treitz; Surgical treatment; Prognosis
Biglycan (BGN) is an important member of small leucine-rich proteoglycans family, and has been implicated in oncogenesis and development of various human cancer types. Here we report that BGN promotes tumor invasion and metastasis of gastric cancer both in vitro and in vivo. BGN expression is significantly higher in gastric cancer tissues and associated with lymph node metastasis, depth of tumor invasion and TNM stage. BGN enhances gastric cancer cell wound healing, migration and invasion ability as well as the tube formation ability of endothelial cells in vitro. Animal experiments results in vivo are consistent with outcomes in vitro. BGN induces increased phosphorylation of FAK (Tyr576/577, Tyr925 and Tyr397) and Paxillin. These results indicate that BGN is upregulated, and plays an oncogenic role, in gastric cancer metastasis by activating the FAK signaling pathway.
BGN; Gastric cancer; Metastasis; FAK
Gastrointestinal stromal tumors (GISTs) are mesenchymal tumors that arise from the gastrointestinal tract. In rare cases, these tumors are found in intra-abdominal sites unrelated to the gastrointestinal tract, such as the mesentery, omentum and retroperitoneum. However, pancreatic extra-gastrointestinal stromal tumors are extremely rare, with only 14 previous cases reported. A 61-year-old man with no clinical symptoms had a routine check-up, during which an abdominal mass located in the pancreas tail was detected. Abdominal surgery was performed with resection of the pancreas tail and the spleen, and he was diagnosed with low-risk GISTs. Another 60-year-old man with no clinical symptoms underwent Computed tomography which revealed a well-demarcated tumor, 6 cm in diameter, in the head of the pancreas. He was diagnosed with pancreatic GISTs. Here, we describe two rare cases of pancreatic GISTs and review the cases previously reported in the literature.
Gastrointestinal Stromal tumors; Extra-gastrointestinal Stromal Tumors; Pancreatic gastrointestinal stromal tumors
Recent studies have reported high levels of fecal indicator enterococci in marine beach sand. This study aimed to determine the spatial and temporal variation of enterococcal abundance and to evaluate its relationships with microbial community parameters in Hawaii beach sand and water. Sampling at 23 beaches on the Island of Oahu detected higher levels of enterococci in beach foreshore sand than in beach water on a mass unit basis. Subsequent 8-week consecutive samplings at two selected beaches (Waialae and Kualoa) consistently detected significantly higher levels of enterococci in backshore sand than in foreshore/nearshore sand and beach water. Comparison between the abundance of enterococci and the microbial communities showed that enterococci correlated significantly with total Vibrio in all beach zones but less significantly with total bacterial density and Escherichia coli. Samples from the different zones of Waialae beach were sequenced by 16S rRNA gene pyrosequencing to determine the microbial community structure and diversity. The backshore sand had a significantly more diverse community and contained different major bacterial populations than the other beach zones, which corresponded to the spatial distribution pattern of enterococcal abundance. Taken together, multiple lines of evidence support the possibility of enterococci as autochthonous members of the microbial community in Hawaii beach sand.
Our previous studies have found that bone-marrow-stromal cells (BMSC) therapy improves functional recovery after stroke in non-diabetic rats while increases brain hemorrhage and induces arteriosclerosis-like changes in type-one-diabetic (T1DM) rats. Niaspan treatment of stroke increases vascular stabilization, decreases brain hemorrhage and blood-brain-barrier (BBB) leakage in T1DM rats. We therefore tested the hypothesis that combination therapy of BMSC with Niaspan attenuates the side effects of BMSC monotherapy in T1DM rats.
T1DM-rats induced by streptozotocin were subjected to 2 hours of middle-cerebral-artery occlusion (MCAo) and treated with: 1) PBS; 2) BMSC (5×106); 3) Niaspan (40 mg/kg) daily for 14 days; 4) BMSC (5×106) +Niaspan (40 mg/kg, daily for 14 days) combination starting at 24 hours after MCAo. All rats were monitored for 14 days.
Combination BMSC+Niaspan treatment of T1DM-MCAo rats did not increase brain hemorrhage, and significantly decreased BBB leakage and vascular arteriosclerosis-like changes as well as decreased Angiogenin, matrix metalloproteinase 9 (MMP9) and ED1 expression in ischemic brain and internal-carotid-artery compared to non-treatment control and BMSC monotherapy animals.
Combination therapy using BMSC with Niaspan decreases BBB leakage and cerebral arteriosclerosis-like changes. These beneficial effects may be attributed to the decreased expression of Angiogenin, MMP9 and ED1.
White matter remodeling plays an important role in neurological recovery after stroke. Bone marrow stromal cells (BMSCs) and Niaspan, an agent which increases high density lipoprotein (HDL), each induces neurorestorative effects and promotes white matter remodeling after stroke in non-diabetic rats. In this study, we test whether combination of BMSCs with Niaspan induces an enhanced white matter remodeling in the ischemic brain of diabetic rats.
Research design and methods
Type-1 diabetes (T1DM) rats were subjected to transient middle cerebral artery occlusion (MCAo) and treated with or without BMSCs; Niaspan; and the combination of BMSCs + Niaspan daily for 14 days after MCAo. Immunostaining for white matter remodeling and synaptic protein expression including NG2; CNPase; BS (Bielschowsky silver); LFB (luxol fast blue); Synaptophysin and SMI-31 immunostaining were performed.
BMSC monotherapy did not regulate NG2 and CNPase expression compared to T1DM control rats. Both, combination of BMSCs + Niaspan treatment, and Niaspan monotherapy significantly increase NG2 and CNPase expression compared to T1DM control. While combination BMSC+Niaspan, BMSC monotherapy and Niaspan monotherapy groups all increase BS, LFB, synaptophysin, and SMI-31 expression in the ischemic brain compared to T1DM-MCAo control. In addition, the combination treatment significantly enhances LFB, SMI-31, and Synaptophysin expression compared to BMSC monotherapy.
Combination treatment of stroke with BMSCs and Niaspan in T1DM rats increases white matter remodeling and additively increases BMSC monotherapy induced myelination and synaptic plasticity after stroke in T1DM rats.
type-one diabetic rats; stroke; bone marrow stromal cells; Niaspan; white matter remodeling; synaptic plasticity
Glioblastoma (GBM) is the most malignant brain tumor where patients' survival is only 14.6 months, despite multimodal therapy with debulking surgery, concurrent chemotherapy and radiotherapy. There is an urgent, unmet need for novel, effective therapeutic strategies for this devastating disease. Although several immunotherapies are under development for the treatment of GBM patients, the use of natural killer (NK) cells is still marginal despite this being a promising approach to treat cancer. In regard of our knowledge on the role of NG2/CSPG4 in promoting GBM aggressiveness we investigated the potential of an innovative immunotherapeutic strategy combining mAb9.2.27 against NG2/CSPG4 and NK cells in preclinical animal models of GBM. Multiple immune escape mechanisms maintain the tumor microenvironment in an anti-inflammatory state to promote tumor growth, however, the distinct roles of resident microglia versus recruited macrophages is not elucidated. We hypothesized that exploiting the cytokine release capabilities of activated NK cells to reverse the anti-inflammatory axis combined with mAb9.2.27 targeting the NG2/CSPG4 may favor tumor destruction by editing pro-GBM immune responses. Combination treatment with NK+mAb9.2.27 diminished tumor growth that was associated with reduced tumor proliferation, increased cellular apoptosis and prolonged survival compared to vehicle and monotherapy controls. The therapeutic efficacy was mediated by recruitment of CCR2low macrophages into the tumor microenvironment, increased ED1 and MHC class II expression on microglia that might render them competent for GBM antigen presentation, as well as elevated IFN-γ and TNF-α levels in the cerebrospinal fluid compared to controls. Depletion of systemic macrophages by liposome-encapsulated clodronate decreased the CCR2low macrophages recruited to the brain and abolished the beneficial outcomes. Moreover, mAb9.2.27 reversed tumor-promoting effects of patient-derived tumor-associated macrophage/ microglia (TAM) ex vivo. Taken together, these findings indicate that NK+mAb9.2.27 treatment may be an amenable therapeutic strategy to treat NG2/CSPG4 expressing GBMs. We provide a novel conceptual approach of combination immunotherapy for glioblastoma. The results traverse beyond the elucidation of NG2/CSPG4 as a therapeutic target, but demonstrate a proof of concept that this antibody may hold potential for the treatment of GBM by activation of tumor infiltrated microglia/macrophages.
Microglia; NK cells; glioblastoma; immunotherapy; NG2/CSPG4
AIM: To investigate the therapeutic efficacy and mechanisms of action of oncolytic-herpes-simplex-virus encoding granulocyte-macrophage colony-stimulating factor (HSVGM-CSF) in pancreatic carcinoma.
METHODS: Tumor blocks were homogenized in a sterile grinder in saline. The homogenate was injected into the right armpit of each mouse. After vaccination, the mice were randomly assigned into four groups: a control group, a high dose HSVGM-CSF group [1 × 107 plaque forming units (pfu)/tumor], a medium dose HSVGM-CSF group (5 × 106 pfu/tumor) and a low dose HSVGM-CSF group (5 × 105 pfu/tumor). After initiation of drug administration, body weights and tumor diameters were measured every 3 d. Fifteen days later, after decapitation of the animal by cervical dislocation, each tumor was isolated, weighed and stored in 10% formaldehyde solution. The drug effectiveness was evaluated according to the weight, volume and relative volume change of each tumor. Furthermore, GM-CSF protein levels in serum were assayed by enzyme-linked immunosorbent assays at 1, 2, 3 and 4 d after injection of HSVGM-CSF.
RESULTS: Injection of the recombinant mouse HSV encoding GM-CSF resulted in a significant reduction in tumor growth compared to the control group, and dose-dependent effects were observed: the relative tumor proliferation rates of the low dose, medium dose and high dose groups on 15 d after injection were 45.5%, 55.2% and 65.5%, respectively. The inhibition rates of the tumor weights of the low, middle, and high dose groups were 41.4%, 46.7% and 50.5%, respectively. Furthermore, the production of GM-CSF was significantly increased in the mice infected with HSVGM-CSF. The increase in the GM-CSF level was more pronounced in the high dose group compared to the other two dose groups.
CONCLUSION: Our study provides the first evidence that HSVGM-CSF could inhibit the growth of pancreatic cancer. The enhanced GM-CSF expression might be responsible for the phenomenon.
Pancreatic carcinoma; Gene therapy; Animal test; Herpes-simplex-virus encoding granulocyte-macrophage colony-stimulating factor
Background & Objective
Diabetes mellitus (DM) plays an important role in the pathogenesis of vascular complications including arteriosclerosis and ischemic stroke. Whether DM impacts intracranial aneurysm (IA) formation has not been extensively investigated. In this study, we tested the underlying mechanism of type one DM (T1DM) induced IA formation in rats.
T1DM was induced by streptozotocin injection. Rats were euthanized at 0, 4 and 10 weeks after T1DM induction. To evaluate cerebral vascular perfusion, Fluorescein isothiocyanate - dye was injected at 5 min prior to euthanasia. Vascular perfusion was measured by laser scanning confocal microscopy. Trichrome, Elastica van Gieson, alpha-smooth muscle actin (a-SMA) and receptor of advanced glycation end-products (RAGE), toll-like receptor 4 (TLR4) and matrix metalloproteinase 9 (MMP9) immunostaining were performed. The IA formation was classified by 0–3 stages: 0: Normal; 1: Endothelial damage; 2: Moderate protrusion; and 3: Saccular aneurysm formation.
T1DM significantly increased IA formation identified by the classification of aneurysmal changes compared with non-DM rats (p<0.05). However, T1DM induced IA formations were classified as stage 1 and stage 2, but not stage 3. Cerebral vascular perfusion was significantly decreased in T1DM rats compared to non-DM rats (p<0.01). DM10W rats exhibited a significant decrease of cerebral vascular perfusion compared to DM4W rats (p<0.05). T1DM rats also significantly increased the internal carotid artery (ICA) intimae and media thickness, and decreased the internal carotid artery diameter compared to non-DM rats. RAGE, MMP9 and TLR4 expression were significantly increased in T1DM rats compared to non-DM rats. The increased RAGE, TLR4 and MMP9 significantly correlated with IA formation (p<0.05).
T1DM increases IA formation. The increased RAGE, MMP9 and TLR4 expressions might contribute to IA formation in T1DM rats.
Naturalized soil Escherichia coli populations need to resist common soil desiccation stress in order to inhabit soil environments. In this study, four representative soil E. coli strains and one lab strain, MG1655, were tested for desiccation resistance via die-off experiments in sterile quartz sand under a potassium acetate-induced desiccation condition. The desiccation stress caused significantly lower die-off rates of the four soil strains (0.17 to 0.40 day−1) than that of MG1655 (0.85 day−1). Cellular responses, including extracellular polymeric substance (EPS) production, exogenous glycine betaine (GB) uptake, and intracellular compatible organic solute synthesis, were quantified and compared under the desiccation and hydrated control conditions. GB uptake appeared not to be a specific desiccation response, while EPS production showed considerable variability among the E. coli strains. All E. coli strains produced more intracellular trehalose, proline, and glutamine under the desiccation condition than the hydrated control, and only the trehalose concentration exhibited a significant correlation with the desiccation-contributed die-off coefficients (Spearman's ρ = −1.0; P = 0.02). De novo trehalose synthesis was further determined for 15 E. coli strains from both soil and nonsoil sources to determine its prevalence as a specific desiccation response. Most E. coli strains (14/15) synthesized significantly more trehalose under the desiccation condition, and the soil E. coli strains produced more trehalose (106.5 ± 44.9 μmol/mg of protein [mean ± standard deviation]) than the nonsoil reference strains (32.5 ± 10.5 μmol/mg of protein).
Background and objective
We investigated axonal plasticity in the bilateral motor cortices and the long term therapeutic effect of Niaspan on axonal remodeling after stroke in type-1 diabetic (T1DM) rats.
T1DM was induced in young adult male Wistar rats via injection of streptozotocin. T1DM rats were subjected to 2 h transient middle cerebral artery occlusion (MCAo) and were treated with 40 mg/kg Niaspan or saline starting 24 h after MCAo and daily for 28 days. Anterograde tracing using biotinylated dextran amine (BDA) injected into the contralateral motor cortex was performed to assess axonal sprouting in the ipsilateral motor cortex area. Functional outcome, SMI-31 (a pan-axonal microfilament marker), Bielschowsky silver and synaptophysin expression were measured. In vitro studies using primary cortical neuron (PCN) cultures and in vivo BDA injection into the brain to anterogradely label axons and terminals were employed.
Niaspan treatment of stroke in T1DM–MCAo rats significantly improved functional outcome after stroke and increased SMI-31, Bielschowsky silver and synaptophysin expression in the ischemic brain compared to saline treated T1DM–MCAo rats (p<0.05). Using BDA to anterograde label axons and terminals, Niaspan treatment significantly increased axonal density in ipsilateral motor cortex in T1DM–MCAo rats (p<0.05, n=7/group). Niacin treatment of PCN significantly increased Ang1 expression under high glucose condition. Niacin and Ang1 significantly increased neurite outgrowth, and anti-Ang1 antibody marginally attenuated Niacin induced neurite outgrowth (p=0.06, n=6/group) in cultured PCN under high glucose condition.
Niaspan treatment increased ischemic brain Ang1 expression and promoted axonal remodeling in the ischemic brain as well as improved functional outcome after stroke. Ang1 may partially contribute to Niaspan-induced axonal remodeling after stroke in T1DM-rats.
Type-one diabetes rats; Stroke; Angiopoietin; Axonal remodeling; Niaspan
Background and Purpose
Cell therapy with bone marrow stromal cells (BMSCs) improves functional recovery after stroke in nondiabetic rats. However, its effect on diabetics with stroke is unknown. This study investigated the effect of BMSCs on stroke outcome in Type 1 diabetic (T1DM) rats.
T1DM was induced in adult male Wistar rats by injecting streptozotocin. Nondiabetic and T1DM rats were subjected to 2 hours of middle cerebral artery occlusion (MCAO), treated with or without BMSCs (3×106) at 24 hours after MCAO, and monitored for 14 days.
Functional benefit was not detected in T1DM-MCAO treated with BMSC rats compared with corresponding T1DM-MCAO controls. BMSC treatment in T1DM-MCAO rats had increased mortality, blood–brain barrier leakage, brain hemorrhage, and angiogenesis. Internal carotid artery neointimal formation and cerebral arteriole narrowing/occlusion were also observed in T1DM-MCAO+BMSCs rats compared with T1DM-MCAO controls (P<0.05), but not in nondiabetic stroke rats. We further studied the underlying mechanisms responsible for BMSC-induced blood–brain barrier leakage and accelerated vascular damage in T1DM-MCAO rats. We found that the expression of angiogenin (an angiogenic factor) and ED1 (a marker for macrophages) was significantly increased in the T1DM-MCAO+BMSC rats in the ischemic brain and internal carotid artery compared with nontreated T1DM-MCAO rats, but not in nondiabetic stroke rats.
BMSC therapy in T1DM-MCAO rats does not improve functional outcome. On the contrary, it increases blood–brain barrier leakage and cerebral artery neointimal formation, and arteriosclerosis, which possibly is due to increased expression of angiogenin. Thus, BMSC treatment starting 24 hours after MCAO may not be beneficial for diabetic subjects with stroke.
angiogenin; arteriosclerosis; bone marrow stromal cell; stroke; Type 1 diabetic