Therapeutic strategies designed to treat HIV infection with combinations of antiviral drugs have proven to be the best approach for slowing the progression to AIDS. Despite this progress, there are problems with viral drug resistance and toxicity, necessitating new approaches to combating HIV-1 infection. We have therefore developed a different combination approach for the treatment of HIV infection in which an RNA aptamer, with high binding affinity to the HIV-1 envelope (gp120) protein and virus neutralization properties, is attached to and delivers a small interfering RNA (siRNA) that triggers sequence-specific degradation of HIV RNAs. We have tested the antiviral activities of these chimeric RNAs in a humanized Rag2−/−γc−/− (RAG-hu) mouse model with multilineage human hematopoiesis. In this animal model, HIV-1 replication and CD4+ T cell depletion mimic the situation seen in human HIV-infected patients. Our results show that treatment with either the anti-gp120 aptamer or the aptamer-siRNA chimera suppressed HIV-1 replication by several orders of magnitude and prevented the viral-induced helper CD4+ T cell decline. In comparison to the aptamer alone, the aptamer-siRNA combination provided more extensive inhibition, resulting in a significantly longer antiviral effect that extended several weeks beyond the last injected dose. The aptamer thus acts as a broad-spectrum HIV-neutralizing agent and an siRNA delivery vehicle. The combined aptamer-siRNA agent provides an attractive, nontoxic therapeutic approach for treatment of HIV infection.

Dengue viral (DENV) pathogenesis and vaccine studies are hampered by the lack of an ideal animal model mimicking human disease and eliciting an adaptive human immune response. Although currently available animal models have been very useful in dissecting some key aspects of disease pathogenesis, a major limitation with these is the lack of a human immune response. In this study, we sought to overcome this difficulty by utilizing a novel mouse model that permits multi-lineage human hematopoiesis and immune response following transplantation with human hematopoietic stem cells. To generate immunocompetent humanized mice, neonatal RAG2(-/-)gamma(c)(-/-) mice were xenografted with human CD34+ hematopoietic stem cells, resulting in de novo development of major functional cells of the human adaptive immune system. To evaluate susceptibility to dengue viral infection, humanized mice were challenged with DEN-2 serotype. Viremia lasting up to 3 weeks was detected in infected mice with viral titers reaching up to 10(6.3) RNA copies/ml. Fever characteristic of dengue was also noted in infected mice. Presence of human anti-dengue antibodies was evaluated using an antibody capture ELISA. Anti-dengue IgM was first detected by 2 weeks post-infection followed by IgG at 6 weeks. Sera from some of the infected mice were also found to be capable of dengue virus neutralization. Infected mouse sera showed reactivity with the viral envelope and capsid proteins in immunoprecipitation assay. These results demonstrate for the first time that humanized mice are capable of dengue viral primary human immune responses thus paving the way for new dengue immunopathogenesis and vaccine studies.

Several new immunodeficient mouse models for human cell engraftment have recently been introduced that include the Rag2−/−γc−/−, NOD/SCID, NOD/SCIDγc−/− and NOD/SCIDβ2m−/− strains. Transplantation of these mice with CD34+ human hematopoietic stem cells leads to prolonged engraftment, multilineage hematopoiesis and the capacity to generate human immune responses against a variety of antigens. However, the various mouse strains used and different methods of engrafting human cells are beginning to illustrate strain specific variations in engraftment levels, duration and longevity of mouse life span. In these proof-of-concept studies we evaluated the Balb/c-Rag1−/−γ−/− strain for engraftment by human fetal liver derived CD34+ hematopoietic cells using the same protocol found to be effective for Balb/c-Rag2−/−γc−/− mice. We demonstrate that these mice can be efficiently engrafted and show multilineage human hematopoiesis with human cells populating different lymphoid organs. Generation of human cells continues beyond a year and production of human immunoglobulins is noted. Infection with HIV-1 leads to chronic viremia with a resultant CD4 T cell loss. To mimic the predominant sexual viral transmission, we challenged humanized Rag1−/−γc−/− mice with HIV-1 via vaginal route which also resulted in chronic viremia and helper T cell loss. Thus these mice can be further exploited for studying human pathogens that infect the human hematopoietic system in an in vivo setting.

ObjectiveOverexpression of tissue-nonspecific alkaline phosphatase (TNAP) in endothelium leads to arterial calcification in mice. The purpose of this study was to examine the effect of elevated endothelial TNAP on coronary atherosclerosis. In addition, we aimed to examine endogenous TNAP activity in human myocardium.Approach and resultsA vascular pattern of TNAP activity was observed in human non-failing, ischemic, and idiopathic dilated hearts (5 per group); no differences were noted between groups in this study. Endothelial overexpression of TNAP was achieved in mice harboring a homozygous recessive mutation in the low density lipoprotein receptor (whc allele) utilizing a Tie2-cre recombinase (WHC-eTNAP mice). WHC-eTNAP developed significant coronary artery calcification at baseline compared WHC controls (4312 vs 0μm2 alizarin red area, p<0.001). Eight weeks after induction of atherosclerosis, lipid deposition in the coronary arteries of WHC-eTNAP was increased compared to WHC controls (121633 vs 9330μm2 oil red O area, p<0.05). Coronary lesions in WHC-eTNAP mice exhibited intimal thickening, calcifications, foam cells, and necrotic cores. This was accompanied by the reduction in body weight and left ventricular ejection fraction (19.5 vs. 23.6g, p<0.01; 35% vs. 47%, p<0.05). In a placebo-controlled experiment under atherogenic conditions, pharmacological inhibition of TNAP in WHC-eTNAP mice by a specific inhibitor SBI-425 (30mg*kg-1*d-1, for 5 weeks) reduced coronary calcium (78838 vs.144622μm2) and lipids (30754 vs. 77317μm2); improved body weight (22.4 vs.18.8g) and ejection fraction (59 vs. 47%). The effects of SBI-425 were significant in the direct comparisons with placebo but disappeared after TNAP-negative placebo-treated group was included in the models as healthy controls.ConclusionsEndogenous TNAP activity is present in human cardiac tissues. TNAP overexpression in vascular endothelium in mice leads to an unusual course of coronary atherosclerosis, in which calcification precedes lipid deposition. The prevalence and significance of this mechanism in human atherosclerosis requires further investigations.

AM. Krüppel-like factor 4 is a radioprotective factor for the intestine following ␥-radiation-induced gut injury in mice.

SummaryIn response to ionizing radiation-induced injury, the normally quiescent intestinal stem cells marked by BMI1 participate in the regenerative response. Previously, we established a protective role for Krüppel-like factor 4 (KLF4) in the intestinal epithelium where it reduces senescence, apoptosis, and crypt atrophy following γ-radiation-induced gut injury. We also described a pro-proliferative function for KLF4 during the regenerative phase post irradiation. In the current study, using a mouse model in which Klf4 is deleted from quiescent BMI1+ intestinal stem cells, we observed increased proliferation from the BMI1+ lineage during homeostasis. In contrast, following irradiation, Bmi1-specific Klf4 deletion leads to decreased expansion of the BMI1+ lineage due to a combination of reduced proliferation and increased apoptosis. Our results support a critical role for KLF4 in modulating BMI1+ intestinal stem cell fate in both homeostasis and the regenerative response to radiation injury.

Brush border membrane vesicles (BBMVs) were prepared from the 2nd instar larvae of Helicoverpa armigera. Binding of the activated Cry1Ac of Bacillus thuringiensis (Bt) toxin was shown by immunoblot. A 120-kDa protein was identified as a receptor for the Cry1Ac type delta-endotoxin. The aminopeptidase-N activity of BBMVs was measured as the hydrolysis of L-leucine p-nitroanilide. The specific activity was 35 units/mg protein. The BBMV preparation also showed low level of alkaline phosphatase activity. Zn++ chelating agents 2,2'-dipyridyl and 1,10-phenanthroline inhibited aminopeptidase activity at 10 mM concentration, indicating the presence of zinc-dependent aminopeptidase in the brush border of H. armigera. The aminopeptidase activity was increased with increasing concentration of delta-endotoxin. The purified 120-kDa binding protein was N-terminally sequenced. The first 10-amino-acid sequence showed 60-77% similarity with human cysteine-rich secretory protein-1 precursor, inhibin alpha chain precursor. Salmonella flagellar hook protein and yeast carboxypeptidase S.

Pre-clinical animal studies have shown that triiodothyronine (T3) replacement therapy improves cardiac contractile function after myocardial infarction (MI). We hypothesized that T3 treatment could prevent adverse post-MI cardiomyocyte remodeling by maintaining T-tubule (TT) organization, thus improving contractility. Methods: Coronary artery ligation (MI) or sham surgeries were performed on female Sprague-Dawley rats (aged 12 wks), followed by treatment with T3 (5ug/kg/d) or vehicle in drinking water for 16 wks (10/gp). After echocardiographic/hemodynamic analyses, ventricular myocytes were isolated by collagenase digestion and plated on laminin-coated glass coverslips for TT analysis or analyzed for contractility using IonOptix’s edge-detection method. Live cell staining with wheat germ agglutinin (WGA)-488 or di-8-ANNEPS labeled sarcolemma and TT, and multiple z-stack images were captured by scanning confocal microscopy and TT organization measured using TTorg program. Ventricular RNA was analyzed by qPCR. Results: Echocardiography and in vivo hemodynamic measurements showed significantly improved systolic and diastolic function in T3- vs vehicle-treated MI rats. IonOpix analysis of isolated ventricular myocytes showed significant dysfunction in measures of relaxation in MI myocytes compared to sham, and improvements with T3: max relaxation velocity (Vmax), 2.98±1.41 vs 1.59±0.33, p<0.05; time to Vmax (sec), 0.23±0.04 vs 0.31±0.02, p<0.001, MI+T3 vs MI). Similarly, time to peak contraction was shortened by T3 treatment (0.16±0.02 vs 0.20±0.01 sec., MI+T3 vs MI; p<0.01. TT power measured by TTorg analysis of WGA-488 or di-8-ANNEPS labeled myocytes showed significant disorganization of the TT system in MI myocytes that was normalized by T3-treatment (TTpower = 82±7 sham, 74±8 MI and 79±6 MI+T3; sham vs MI p<0.0001; MI vs MI+T3, p<0.0001). qPCR results indicated that reduced expression of Bin1, Jph2, RyR2 and LTCC in the MI myocardium was normalized by T3. In conclusion, T3 treatment prevented TT disorganization in the post-MI cardiomyocyte, normalized contractile and relaxation parameters and improved cardiac output, thus supporting the therapeutic potential of T3 in heart failure.