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1.  BALB/c-congenic ANP32B-deficient mice reveal a modifying locus that determines viability 
Experimental Animals  2015;65(1):53-62.
We previously found that deletion of the multifunctional factor ANP32B (a.k.a. SSP29, APRIL, PAL31, PHAPI2) resulted in a severe but strain-specific defect resulting in perinatal lethality. The difficulty in generating an adult cohort of ANP32B-deficient animals limited our ability to examine adult phenotypes, particularly cancer-related phenotypes. We bred the Anp32b-null allele into the BALB/c and FVB/N genetic background. The BALB/c, but not the FVB/N, background provided sufficient frequency of adult Anp32b-null (Anp32b−/−) animals. From these, we found no apparent oncogenic role for this protein in mammary tumorigenesis contrary to what was predicted based on human data. We also found runtism, pathologies in various organ systems, and an unusual clinical chemistry signature in the adult Anp32b−/− mice. Intriguingly, genome-wide single-nucleotide polymorphism analysis suggested that our colony retained an unlinked C57BL/6J locus at high frequency. Breeding this locus to homozygosity demonstrated that it had a strong effect on Anp32b−/− viability indicating that this locus contains a modifier gene of Anp32b with respect to development. This suggests a functionally important genetic interaction with one of a limited number of candidate genes, foremost among them being the variant histone gene H2afv. Using congenic breeding strategies, we have generated a viable ANP32B-deficient animal in a mostly pure background. We have used this animal to reliably exclude mouse ANP32B as an important oncogene in mammary tumorigenesis. Our further phenotyping strengthens the evidence that ANP32B is a widespread regulator of gene expression. These studies may also impact the choice of subsequent groups with respect to congenic breeding versus de novo zygote targeting strategies for background analyses in mouse genetics.
PMCID: PMC4783651  PMID: 26558540
chromatin regulators; congenic breeding; modifier locus; strain-dependent SNPs
2.  Pyrvinium selectively targets blast phase-chronic myeloid leukemia through inhibition of mitochondrial respiration 
Oncotarget  2015;6(32):33769-33780.
The use of BCR-ABL1 tyrosine kinase inhibitors (TKI) has led to excellent clinical responses in patients with chronic phase chronic myeloid leukemia (CML). However these inhibitors have been less effective as single agents in the terminal blast phase (BP). We show that pyrvinium, a FDA-approved anthelminthic drug, selectively targets BP-CML CD34+ progenitor cells. Pyrvinium is effective in inducing apoptosis, inhibiting colony formation and self-renewal capacity of CD34+ cells from TKI-resistant BP-CML patients, while cord blood CD34+ are largely unaffected. The effects of pyrvinium are further enhanced upon combination with dasatinib, a second generation BCR-ABL1 TKI. In a CML xenograft model pyrvinium significantly inhibits tumor growth as a single agent, with complete inhibition in combination with dasatinib. While pyrvinium has been shown to inhibit the Wnt/β-catenin signalling pathway via activation of casein kinase 1α, we find its activity in CML is not dependent on this pathway. Instead, we show that pyrvinium localizes to mitochondria and induces apoptosis by inhibiting mitochondrial respiration. Our study suggests that pyrvinium is a useful addition to the treatment armamentarium for BP-CML and that targeting mitochondrial respiration may be a potential therapeutic strategy in aggressive leukemia.
PMCID: PMC4741801  PMID: 26378050
chronic myeloid leukemia; pyrvinium; mitochondrial respiration
3.  Helicobacter pylori infection can affect energy modulating hormones and body weight in germ free mice 
Scientific Reports  2015;5:8731.
Helicobacter pylori, is an invariably commensal resident of the gut microbiome associated with gastric ulcer in adults. In addition, these patients also suffered from a low grade inflammation that activates the immune system and thus increased shunting of energy to host defense mechanisms. To assess whether a H. pylori infection could affect growth in early life, we determined the expression levels of selected metabolic gut hormones in germ free (GF) and specific pathogen-free (SPF) mice with and without the presence of H. pylori. Despite H. pylori-infected (SPFH) mice display alteration in host metabolism (elevated levels of leptin, insulin and peptide YY) compared to non-infected SPF mice, their growth curves remained the same. SPFH mice also displayed increased level of eotaxin-1. Interestingly, GF mice infected with H. pylori (GFH) also displayed increased levels of ghrelin and PYY. However, in contrast to SPFH mice, GFH showed reduced weight gain and malnutrition. These preliminary findings show that exposure to H. pylori alters host metabolism early in life; but the commensal microbiota in SPF mice can attenuate the growth retarding effect from H. pylori observed in GF mice. Further investigations of possible additional side effects of H. pylori are highly warranted.
PMCID: PMC4348668  PMID: 25736205
4.  Hepatic Stellate Cell–Targeted Delivery of Hepatocyte Growth Factor Transgene via Bile Duct Infusion Enhances Its Expression at Fibrotic Foci to Regress Dimethylnitrosamine-Induced Liver Fibrosis 
Human Gene Therapy  2013;24(5):508-519.
Liver fibrosis generates fibrotic foci with abundant activated hepatic stellate cells and excessive collagen deposition juxtaposed with healthy regions. Targeted delivery of antifibrotic therapeutics to hepatic stellate cells (HSCs) might improve treatment outcomes and reduce adverse effects on healthy tissue. We delivered the hepatocyte growth factor (HGF) gene specifically to activated hepatic stellate cells in fibrotic liver using vitamin A–coupled liposomes by retrograde intrabiliary infusion to bypass capillarized hepatic sinusoids. The antifibrotic effects of DsRed2-HGF vector encapsulated within vitamin A–coupled liposomes were validated by decreases in fibrotic markers in vitro. Fibrotic cultures transfected with the targeted transgene showed a significant decrease in fibrotic markers such as transforming growth factor-β1. In rats, dimethylnitrosamine-induced liver fibrosis is manifested by an increase in collagen deposition and severe defenestration of sinusoidal endothelial cells. The HSC-targeted transgene, administered via retrograde intrabiliary infusion in fibrotic rats, successfully reduced liver fibrosis markers alpha-smooth muscle actin and collagen, accompanied by an increase in the expression of DsRed2-HGF near the fibrotic foci. Thus, targeted delivery of HGF gene to hepatic stellate cells increased the transgene expression at the fibrotic foci and strongly enhanced its antifibrotic effects.
Narmada and colleagues demonstrate that vitamin A–coupled liposomes can be used to deliver hepatocyte growth factor (HGF) specifically to activated human hepatic stellate cells (HSCs) in vitro. In vivo, they show that this approach leads to regression of liver fibrosis in a rat model.
PMCID: PMC3655631  PMID: 23527815
5.  Absence of Intestinal PPARγ Aggravates Acute Infectious Colitis in Mice through a Lipocalin-2–Dependent Pathway 
PLoS Pathogens  2014;10(1):e1003887.
To be able to colonize its host, invading Salmonella enterica serovar Typhimurium must disrupt and severely affect host-microbiome homeostasis. Here we report that S. Typhimurium induces acute infectious colitis by inhibiting peroxisome proliferator-activated receptor gamma (PPARγ) expression in intestinal epithelial cells. Interestingly, this PPARγ down-regulation by S. Typhimurium is independent of TLR-4 signaling but triggers a marked elevation of host innate immune response genes, including that encoding the antimicrobial peptide lipocalin-2 (Lcn2). Accumulation of Lcn2 stabilizes the metalloproteinase MMP-9 via extracellular binding, which further aggravates the colitis. Remarkably, when exposed to S. Typhimurium, Lcn2-null mice exhibited a drastic reduction of the colitis and remained protected even at later stages of infection. Our data suggest a mechanism in which S. Typhimurium hijacks the control of host immune response genes such as those encoding PPARγ and Lcn2 to acquire residence in a host, which by evolution has established a symbiotic relation with its microbiome community to prevent pathogen invasion.
Author Summary
Enteric pathogens like S. Typhimurium convert the host intestine into an inflamed environment in which they are well adapted to thrive. However, the precise strategy that this pathogen employs to achieve such favorable conditions for its survival remains unclear. Here, we uncovered a novel mechanism whereby S. Typhimurium inhibits the expression of the transcription factor PPARγ in the host intestine, surprisingly without TLR-4 involvement; this inhibition worsened the severity of the host's colitis. Subsequent detailed analysis revealed that colitis severity was coupled with elevated levels of antimicrobials like Lcn2, which stabilized the pro-inflammatory endopeptidase MMP-9 in the intestinal milieu. Combination of this escalated antimicrobial action together with enhanced protease activity disrupted the intestinal homeostasis, promoting an inflamed environment suitable for S. Typhimurium. Interestingly, using Lcn2 mutant mice we show that lack of Lcn2 effectively reduced tissue damage and the degree of inflammation, thus supporting a pivotal role of Lcn2 and MMP-9 in infectious colitis. Our data suggests a model whereby the pathogenesis of S. Typhimurium involves manipulation of the host innate immune and protease system, here illustrated by PPARγ, Lcn2 and MMP-9, to establish colonization and infection within the host.
PMCID: PMC3900641  PMID: 24465207
6.  Potent therapeutic activity of folate receptor-targeted liposomal carboplatin in the localized treatment of intraperitoneally grown human ovarian tumor xenograft 
Intraperitoneal (IP) therapy with platinum (Pt)-based drugs has shown promising results clinically; however, high locoregional concentration of the drug could lead to adverse side effects. In this study, IP administration was coupled with a folate receptor-targeted (FRT) liposomal system, in an attempt to achieve intracellular delivery of the Pt-based drug carboplatin in order to increase therapeutic efficacy and to minimize toxicity. In vitro and in vivo activity of FRT carboplatin liposomes was compared with the activity of free drug and nontargeted (NT) carboplatin liposomes using FR-overexpressing IGROV-1 ovarian cancer cells as the model. Significant reduction in cell viability was observed with FRT liposomes, which, compared with the free drug, provided an approximately twofold increase in carboplatin potency. The increase in drug potency was correlated with significantly higher cellular accumulation of Pt resulting from FRT liposomal delivery. Further evaluation was conducted in mice bearing intraperitoneally inoculated IGROV-1 ovarian tumor xenografts. A superior survival rate (five out of six animals) was achieved in animals treated with FRT carboplatin liposomes, injected intraperitoneally with a dose of 15 mg/kg and following a schedule of twice-weekly administration for 3 weeks. In contrast, no survivors were observed in the free drug or NT carboplatin liposome groups. The presence of cancer cells in lung and liver tissues was observed in the saline, free carboplatin, and NT carboplatin liposome groups. However, there was no sign of cancer cells or drug-related toxicity detected in tissues from the animals treated with FRT carboplatin liposomes. The results of this study have demonstrated for the first time that the approach of coupling IP administration with FRT liposomal delivery could provide significantly improved therapeutic efficacy of carboplatin in the treatment of metastatic ovarian cancer.
PMCID: PMC3282613  PMID: 22359453
liposomes; ovarian cancer; targeted therapy; FRT carboplatin liposomes
7.  Increasing Damage to Tumor Blood Vessels during Motexafin Lutetium-PDT through Use of Low Fluence Rate 
Radiation research  2010;174(3):331-340.
Photodynamic therapy (PDT) with low light fluence rate has rarely been studied in protocols that use short drug–light intervals and thus deliver illumination while plasma concentrations of photosensitizer are high, creating a prominent vascular response. In this study, the effects of light fluence rate on PDT response were investigated using motexafin lutetium (10 mg/kg) in combination with 730 nm light and a 180-min drug–light interval. At 180 min, the plasma level of photosensitizer was 5.7 ng/μl compared to 3.1 ng/mg in RIF tumor, and PDT-mediated vascular effects were confirmed by a spasmodic decrease in blood flow during illumination. Light delivery at 25 mW/cm2 significantly improved long-term tumor responses over that at 75 mW/cm2. This effect could not be attributed to oxygen conservation at low fluence rate, because 25 mW/cm2 PDT provided little benefit to tumor hemoglobin oxygen saturation. However, 25 mW/cm2 PDT did prolong the duration of ischemic insult during illumination and was correspondingly associated with greater decreases in perfusion immediately after PDT, followed by smaller increases in total hemoglobin concentration in the hours after PDT. Increases in blood volume suggest blood pooling from suboptimal vascular damage; thus the smaller increases after 25 mW/cm2 PDT provide evidence of more widespread vascular damage, which was accompanied by greater decreases in clonogenic survival. Further study of low fluence rate as a means to improve responses to PDT under conditions designed to predominantly damage vasculature is warranted.
PMCID: PMC2995951  PMID: 20726728
Ultrasound in medicine & biology  2010;36(5):853-857.
The goal of this murine investigation was to evaluate the effect of an antivascular ultrasound treatment on the growth of an implanted melanoma and the consequent survival rate. Following the intravenous injection of 0.2 mL ultrasound contrast agent (Definity), therapy (n = 15) was performed on 1 mL tumors for 3 minutes with low intensity, continuous ultrasound (3 MHz; 2.4 ± 0.1−2 [ISATA]); control mice (n = 17) received a sham treatment. Mice were euthanized once the tumor had reached 3 mL and survival percentage versus time curves were plotted. The median survival time (time for tumor to reach 3 mL) for the treated group was 23 days and for the control group was 18 days; the difference was statistically significant (P ≤ 0.0001). Antivascular ultrasound therapy reduced the growth rate of an implanted melanoma and increased survival time. The ultrasound therapy provides a further example of tumor vascular disruption and its future clinical potential should be investigated.
PMCID: PMC2905813  PMID: 20381952
Low-intensity ultrasound therapy; Ultrasound contrast agent; Antivascular; Angiogenesis; Melanoma
9.  Mutation of tyrosine 145 of lymphocyte cytosolic protein 2 (Lcp2; SLP-76) protects mice from anaphylaxis and arthritis 
The Src homology 2 (SH2) domain-containing leukocyte phosphoprotein of 76-kilodaltons (SLP-76) is an essential adaptor molecule in myeloid cells, where it regulates FcεRI-induced mast cell (MC) and FcγR- and integrin-induced neutrophil (polymorphonuclear leukocyte; PMN) functions. SLP-76 contains three N-terminal tyrosines at residues 112, 128 and 145 that together are critical for its function.
We sought to explore the relative importance of tyrosines 112, 128 and 145 of SLP-76 during MC and PMN activation.
We examined in vitro MC and PMN functions using cells isolated from knock-in mice harboring phenylalanine substitution mutations at tyrosines 112 and 128 (Y112/128F) or 145 (Y145F). We also examined the effects of these mutations on in vivo MC and PMN activation using models of anaphylaxis, dermal inflammation and serum-induced arthritis.
Mutations at Y112/Y128 and Y145 both interfered with SLP-76 activity; however, Y145F had a greater impact than Y112/128F on most in vitro FcR-induced functions. In vitro functional defects were recapitulated in vivo, where mice expressing Y145F exhibited greater attenuation of MC-dependent passive systemic anaphylaxis and PMN-mediated inflammatory responses. Notably, the Y145F mutation completely protected mice against development of joint-specific inflammation in the MC and PMN-dependent K/B×N model of arthritis.
Our data indicate Y145 is the most critical tyrosine supporting SLP-76 function in myeloid cells. Future efforts to dissect how Y145 mediates SLP-76-dependent signaling in MC and PMN will increase our understanding of these lineages and provide insights into the treatment of allergy and inflammation.
PMCID: PMC2778804  PMID: 19895996
Mast cells; neutrophils; Fc receptors; integrins; signal transduction; SLP-76; passive systemic anaphylaxis; localized Shwartzman reaction; arthritis
10.  A Replication-Competent, Neuronal Spread-Defective, Live Attenuated Herpes Simplex Virus Type 1 Vaccine▿  
Journal of Virology  2008;82(17):8431-8441.
Herpes simplex virus type 1 (HSV-1) produces oral lesions, encephalitis, keratitis, and severe infections in the immunocompromised host. HSV-1 is almost as common as HSV-2 in causing first episodes of genital herpes, a disease that is associated with an increased risk of human immunodeficiency virus acquisition and transmission. No approved vaccines are currently available to protect against HSV-1 or HSV-2 infection. We developed a novel HSV vaccine strategy that uses a replication-competent strain of HSV-1, NS-gEnull, which has a defect in anterograde and retrograde directional spread and cell-to-cell spread. Following scratch inoculation on the mouse flank, NS-gEnull replicated at the site of inoculation without causing disease. Importantly, the vaccine strain was not isolated from dorsal root ganglia (DRG). We used the flank model to challenge vaccinated mice and demonstrated that NS-gEnull was highly protective against wild-type HSV-1. The challenge virus replicated to low titers at the site of inoculation; therefore, the vaccine strain did not provide sterilizing immunity. Nevertheless, challenge by HSV-1 or HSV-2 resulted in less-severe disease at the inoculation site, and vaccinated mice were totally protected against zosteriform disease and death. After HSV-1 challenge, latent virus was recovered by DRG explant cocultures from <10% of vaccinated mice compared with 100% of mock-vaccinated mice. The vaccine provided protection against disease and death after intravaginal challenge and markedly lowered the titers of the challenge virus in the vagina. Therefore, the HSV-1 gEnull strain is an excellent candidate for further vaccine development.
PMCID: PMC2519657  PMID: 18562543
Ultrasound in medicine & biology  2007;33(12):1901-1910.
This study investigated whether a microbubble-containing ultrasound contrast agent had a role in the antivascular action of physiotherapy ultrasound on tumor neovasculature. Ultrasound images (B-mode and contrast-enhanced power Doppler [0.02mL Definity]) were made of 22 murine melanomas (K173522). The tumor was insonated (ISATA = 1.7 W cm−2, 1 MHz, continuous output) for 3 min and the power Doppler observations of the pre- and post-insonation tumor vascularities were analyzed. Significant reductions (p = 0.005 for analyses of color weighted fractional area) in vascularity occurred when a contrast-enhanced power Doppler study occurred prior to insonation. Vascularity was unchanged in tumors without a pre-therapy Doppler study. Histological studies revealed tissue structural changes that correlated with the ultrasound findings. The underlying etiology of the interaction between the physiotherapy ultrasound beam, the microbubble-containing contrast agent and the tumor neovasculature is unknown. It was concluded that contrast agents play an important role in the antivascular effects induced by physiotherapy ultrasound.
PMCID: PMC2423191  PMID: 17720299
Ultrasound imaging; Cancer therapy; Physiotherapy; Antivascular; Tumor angiogenesis; Power Doppler; Insonation; Ultrasound bioeffects; Microbubble contrast agent

Results 1-11 (11)