Monica Casali

Apolipoprotein B–Dependent Hepatitis C Virus Secretion Is Inhibited by the Grapefruit Flavonoid Naringenin

Hepatitis C virus (HCV) infects over 3% of the world population and is the leading cause of chronic liver disease worldwide. HCV has long been known to associate with circulating lipoproteins, and its interactions with the cholesterol and lipid pathways have been recently described. In this work, we demonstrate that HCV is actively secreted by infected cells through a Golgi‐dependent mechanism while bound to very low density lipoprotein (vLDL). Silencing apolipoprotein B (ApoB) messenger RNA in infected cells causes a 70% reduction in the secretion of both ApoB‐100 and HCV. More importantly, we demonstrate that the grapefruit flavonoid naringenin, previously shown to inhibit vLDL secretion both in vivo and in vitro, inhibits the microsomal triglyceride transfer protein activity as well as the transcription of 3‐hydroxy‐3‐methyl‐glutaryl‐coenzyme A reductase and acyl‐coenzyme A:cholesterol acyltransferase 2 in infected cells. Stimulation with naringenin reduces HCV secretion in infected cells by 80%. Moreover, we find that naringenin is effective at concentrations that are an order of magnitude below the toxic threshold in primary human hepatocytes and in mice. Conclusion: These results suggest a novel therapeutic approach for the treatment of HCV infection. (HEPATOLOGY 2008.)

A novel formulation of oxygen-carrying matrix enhances liver-specific function of cultured hepatocytes

Oxygen is an important component of the cellular microenvironment, mediating cell survival, differentiation, and function. Oxygen supply is a limiting factor during culture of highly metabolic cells such as hepatocytes. Here we present a simple formulation of a fluorocarbon‐based oxygen carrier embedded in collagen gel that increases oxygen concentration in culture 6‐fold. Rat hepatocytes cultured on oxygen carrier‐collagen showed a significant increase in viability and function. Cytochrome P450IA1 activity was increased by 140% in serum‐free cultures and by 820% in serum‐containing cultures. The significantly higher hepatocellular function on oxygen carrier‐collagen matrix persisted and increased during long‐term culture. Long‐term albumin secretion was increased by 350% in serum‐free cultures and by 166% in serum‐containing culture. Long‐term urea secretion was increased by 79% in serum‐free cultures and by 76% in serum‐containing cultures. We conclude that oxygen supply may limit hepatocyte function in vitro. This limitation can be overcome by addition of an oxygen carrier to the extracellular matrix. Culture of hepatocytes on oxygen‐carrying matrix mimics the oxygen‐rich environment of the liver and provides a simple method for enhanced long‐term function.—Nahmias, Y., Kramvis, Y., Barbe, L., Casali, M., Berthiaume, F., Yarmush, M. L. A novel formulation of oxygen‐carrying matrix enhances liver‐specific function of cultured hepatocytes. FASEB J. 20, E1828–E1836 (2006)

Liver endothelial cells promote LDL-R expression and the uptake of HCV-like particles in primary rat and human hepatocytes†

Low‐density lipoprotein (LDL) is an important carrier of plasma cholesterol and triglycerides whose concentration is regulated by the liver parenchymal cells. Abnormal LDL regulation is thought to cause atherosclerosis, while viral binding to LDL has been suggested to facilitate hepatitis C infection. Primary hepatocytes quickly lose the ability to clear LDL during in vitro culture. Here we show that the coculture of hepatocytes with liver sinusoidal endothelial cells (LSEC) significantly increases the ability of hepatocytes to uptake LDL in vitro. LDL uptake does not increase when hepatocytes are cocultured with other cell types such as fibroblasts or umbilical vein endothelial cells. We find that LSECs induce the hepatic expression of the LDL receptor and the epidermal growth factor receptor. In addition, while hepatocytes in single culture did not take up hepatitis C virus (HCV)‐like particles, the hepatocytes cocultured with LSECs showed a high level of HCV‐like particle uptake. We suggest that coculture with LSECs induces the emergence of a sinusoidal surface in primary hepatocytes conducive to the uptake of HCV‐like particles. In conclusion, our findings describe a novel model of polarized hepatocytes in vitro that can be used for the study of LDL metabolism and hepatitis C infection. (HEPATOLOGY 2006;43:257–265.)

Nest Making and Oxytocin Comparably Promote Wound Healing in Isolation Reared Rats

Background Environmental enrichment (EE) fosters attachment behavior through its effect on brain oxytocin levels in the hippocampus and other brain regions, which in turn modulate the hypothalamic-pituitary axis (HPA). Social isolation and other stressors negatively impact physical healing through their effect on the HPA. Therefore, we reasoned that: 1) provision of a rat EE (nest building with Nestlets®) would improve wound healing in rats undergoing stress due to isolation rearing and 2) that oxytocin would have a similar beneficial effect on wound healing. Methodology/Principal Findings In the first two experiments, we provided isolation reared rats with either EE or oxytocin and compared their wound healing to group reared rats and isolation reared rats that did not receive Nestlets or oxytocin. In the third experiment, we examined the effect of Nestlets on open field locomotion and immediate early gene (IEG) expression. We found that isolation reared rats treated with Nestlets a) healed significantly better than without Nestlets, 2) healed at a similar rate to rats treated with oxytocin, 3) had decreased hyperactivity in the open field test, and 4) had normalized IEG expression in brain hippocampus. Conclusions/Significance This study shows that when an EE strategy or oxytocin is given to isolation reared rats, the peripheral stress response, as measured by burn injury healing, is decreased. The findings indicate an association between the effect of nest making on wound healing and administration of the pro-bonding hormone oxytocin. Further elucidation of this animal model should lead to improved understanding of how EE strategies can ameliorate poor wound healing and other symptoms that result from isolation stress.

DNA-triggered innate immune responses are propagated by gap junction communication

Cells respond to infection by sensing pathogens and communicating danger signals to noninfected neighbors; however, little is known about this complex spatiotemporal process. Here we show that activation of the innate immune system by double-stranded DNA (dsDNA) triggers intercellular communication through a gap junction-dependent signaling pathway, recruiting colonies of cells to collectively secrete antiviral and inflammatory cytokines for the propagation of danger signals across the tissue at large. By using live-cell imaging of a stable IRF3-sensitive GFP reporter, we demonstrate that dsDNA sensing leads to multicellular colonies of IRF3-activated cells that express the majority of secreted cytokines, including IFNβ and TNFα. Inhibiting gap junctions decreases dsDNA-induced IRF3 activation, cytokine production, and the resulting tissue-wide antiviral state, indicating that this immune response propagation pathway lies upstream of the paracrine action of secreted cytokines and may represent a host-derived mechanism for evading viral antiinterferon strategies.

Site-directed mutagenesis of the hinge peptide from the hemagglutinin protein: enhancement of the pH-responsive conformational change

Environmentally responsive proteins and peptides are increasingly finding utility in various engineered systems due to their ability to respond to the presentation of external stimuli. A classic example of this behavior is the influenza hemagglutinin (HA) fusion protein. At neutral pH, HA exists in a non-fusogenic state, but upon exposure to low pH, the conformation of the structure changes to expose a fusogenic peptide. During this structural change, massive rearrangements occur in a subunit of HA (HA2). Crystallography data has shown that a loop of 28 amino acids (residues 54-81) undergoes a dramatic transition from a random coil to an alpha-helix. This segment connects to two flanking helical regions (short and long) to form a long, continuous helix. Here, we report the results of site-directed mutagenesis study on LOOP-36 to further understand the mechanism of this important stimulus-responsive peptide. The conformational transition of a bacterially expressed LOOP-36 was found to be less dramatic than has been previously reported. The systematic mutation of glutamate and histidine residues in the peptide to glutamines (glutamine scanning) did not impact the conformational behavior of the peptide, but the substitution of the glycine residue at position 22 with alanine resulted in significant pH-responsive behavior. Therefore this mutant stimulus-responsive peptide may be more valuable for future protein engineering and bionanotechnology efforts.

A microfluidic bioreactor for increased active retrovirus output

Retroviruses are one of the most commonly used vectors in ongoing gene therapy clinical trials. To evaluate and advance virus production on the microscale platform, we have created a novel microfluidic bioreactor for continuous retrovirus production. We investigated the growth kinetics of a retroviral packaging cell line in microfluidic bioreactors for several compartment sizes, and packaging cells perfused in the microdevices showed similar growth kinetics to those cultured in conventional static conditions. To evaluate the efficiency of retrovirus production, virus titers from the microdevices were compared to those obtained from static tissue culture. When retrovirus production and collection were maintained at 37 degrees C, virus production levels were comparable for the microdevices and static tissue culture conditions. However, immediate cold storage downstream of the packaging cells in the microdevices resulted in 1.4- to 3.7-fold greater active virus production levels with the microdevices compared to the conventional static conditions over a 5 day period. Lastly, the use of microfluidics for virus production provides a continuous supply of virus supernatant for immediate infection of target cells or for preservation and storage. Such devices will be valuable for the optimization of production and evaluation of retroviruses and other viral vectors for gene therapy applications.

A highly sensitive microsphere-based assay for early detection of Type I diabetes

Type 1 Diabetes Mellitus (T1DM) is a T-cell mediated autoimmune disease in which deterioration of insulin producing pancreatic β-cells leads to a state of insulin deficiency. It has been shown that the clinical symptoms of T1DM are preceded by presence of islet cell autoantibodies (ICA) in serum. Radioimmunoassay (RIA) based detection of ICA are the current gold standard for diagnosis of T1DM. While the onset of hyperglycemia is an indicator of onset of T1DM, detection of ICA within the serum is important to differentiate T1DM from ketogenic Type 2 Diabetes (T2D) and Maturity Onset Diabetes of the Young (MODY). Due to their limited range of sensitivity, however, RIA cannot detect ICA at low concentrations in serum which could lead to delay in proper diagnosis and treatment. In addition, the use of radioactive species presents major disadvantages including exposure, waste removal, need for specialized licensed facilities to conduct the tests and the time required for the test (> 24 hours). To overcome these limitations, we have developed a rapid, highly sensitive, fluorescent and microsphere-based assay technique using Rolling Circle Amplification (RCA), to profile T1DM marker antibodies in serum. This assay utilizes the ability of RCA to detect very small amounts of DNA coupled with microsphere-immobilization resulting in an assay which is at least 50 times more sensitive than RIA. Further, this assay method requires very low volume of sample (5 μL), and can be easily adapted to detect other autoantibodies at similar sensitivities while reducing the assay time to ~6 hours. This powerful technique could enable detection of T1DM markers much earlier than current methods and enable earlier intervention to deter the progression of disease. In addition, the modularity of this assay would have implications for enhancing the sensitivities of any standard ELISA technique.

Moloney murine leukemia virus decay mediated by retroviral reverse transcriptase degradation of genomic RNA

Retroviral vectors are powerful tools for the introduction of transgenes into mammalian cells and for long-term gene expression. However, their application is often limited by a rapid loss of bioactivity: retroviruses spontaneously loose activity at 37 degrees C, with a half-life of 4 to 9 h depending on the retrovirus type. We sought to determine which components of the retrovirus are responsible for this loss in bioactivity and to obtain a quantitative characterization of their stability. To this end, we focused on RNA and viral proteins, two major components that we hypothesized may undergo degradation and negatively influence viral infectivity. Reverse transcription PCR (RT-PCR) targeting RNA encoding portions of the viral genome clearly demonstrated time-dependent degradation of RNA which correlated with the loss in viral bioactivity. Circular dichroism spectroscopy, SDS-PAGE and two-dimensional SDS-PAGE analyses of viral proteins did not show any change in secondary structure or evidence of proteolysis. The mechanism underlying the degradation of viral RNA was investigated by site-directed mutagenesis of proteins encoded by the viral genome. Reverse transcriptase and protease mutants exhibited enhanced RNA stability in comparison to wild type recombinant virus, suggesting that the degradation of RNA, and the corresponding virus loss of activity, is mediated by the reverse transcriptase enzyme.

FMR1 Deficiency Alters Self-Renewal and Proliferation of Mouse Embryonic Cells

The regulation of embryonic stem (ES) cell self-renewal and pluripotency is based upon highly orchestrated transcription factor networks. RNA inhibition has been demonstrated to affect ES cell function by altering gene expression levels that are critical to the maintenance and differentiation of ES cells. Fragile X mental retardation protein (FMRP) is a selective RNA-binding protein that can act as a translational repressor for bound mRNA and regulates the expression of a variety of gene transcripts in numerous adult cells. The absence of FMRP results in the most common form of inherited intellectual disability, Fragile X syndrome. In an effort to determine the role of FMRP during development, we silenced the FMRP gene (FMR1) using short hairpin RNA (shRNA). Prior to differentiation induction, we analyzed the phenotype of FMR1 knock down (FMR1-kd) mouse ES cells in their undifferentiated state. Herein, we report that FMR1-kd ES cells proliferate at a greater rate than wild-type ES cells resulting in a 25% reduction in doubling time. FMR1-kd ES cells were found to have an increased expression of three self-renewal genes (OCT-4, Sox2, Nanog) in the undifferentiated state. Moreover, FMR1-kd ES cells failed to downregulate OCT-4 during differentiation programs resulting in abnormal fate decisions in vitro. These results demonstrate an unexpected correlation between FMR1 expression and OCT-4 regulation suggesting that FMRP is involved in the silencing of OCT-4 during the commencement of differentiation programs.