William C. Satterfield

Guided tissue fabrication from periosteum using preformed biodegradable polymer scaffolds

A successful tissue engineering method for bone replacement would imitate natural bone graft by providing the essential elements for new bone formation using synthetic scaffolds, osteogenic cell populations, and bone induction factors. This is a study of the suitability of various formulations of poly(DL-lactic-co-glycolic acid) (PLGA) foams to provide a tissue conducting scaffold in an ovine model for bone flap fabrication. Three formulations were used of different copolymer ratio and molecular weight. Porous wafers of PLGA were stacked into rectangular chambers (volume 4 cm3) enclosed on five sides. Some chambers also contained autologous morcellized bone graft (MBG). The chambers were inserted with the open face adjacent to the cambium layer of the periosteum in rib beds of seven sheep and harvested after 8 weeks in vivo. Gross and histologic examination of the resulting tissue specimens demonstrated molded units of vascularized tissue generally conforming to the shape of the chambers and firmly attached to the periosteum. Polymer degradation appeared to occur by varying degrees based on polymer formulation. New bone formation was observed only in areas containing MBG. There was no evidence of significant inflammatory reaction or local tissue damage at 8 weeks. We conclude that a PLGA foam scaffold is (1) an efficient conductor of new tissue growth but not osteoinductive, (2) contributes to the shape of molded tissue, and (3) biocompatible when used in this model. Further studies are warranted to develop practical methods to deliver bone induction factors to the system to promote osseous tissue generation throughout the synthetic scaffold.

Immunotherapy of chronic hepatitis C virus infection with antibodies against programmed cell death-1 (PD-1)

Hepatitis C virus (HCV) persistence is facilitated by exhaustion of CD8+ T cells that express the inhibitory receptor programmed cell death 1 (PD-1). Blockade of PD-1 signaling improves in vitro proliferation of HCV-specific T lymphocytes, but whether antiviral function can be restored in infected individuals is unknown. To address this question, chimpanzees with persistent HCV infection were treated with anti–PD-1 antibodies. A significant reduction in HCV viremia was observed in one of three treated animals without apparent hepatocellular injury. Viremia rebounded in the responder animal when antibody treatment was discontinued. Control of HCV replication was associated with restoration of intrahepatic CD4+ and CD8+ T-cell immunity against multiple HCV proteins. The responder animal had a history of broader T-cell immunity to multiple HCV proteins than the two chimpanzees that did not respond to PD-1 therapy. The results suggest that successful PD-1 blockade likely requires a critical threshold of preexisting virus-specific T cells in liver and warrants consideration of therapeutic vaccination strategies in combination with PD-1 blockade to broaden narrow responses. Anti–PD-1 immunotherapy may also facilitate control of other persistent viruses, notably the hepatitis B virus where options for long-term control of virus replication are limited.

Safety of chronic intrathecal morphine infusion in a sheep model

Background The safety of chronically administered intrathecal morphine has been questioned. Therefore, the authors examined the behavioral and neurologic effects and neurotoxicity of continuous intrathecal morphine administration in sheep. Methods Groups of three sheep were implanted with intrathecal infusion systems for the continuous administration of morphine (3, 6, 9, 12, or 18 mg/day) or saline at a fixed infusion rate of 1.92 ml/day beginning approximately 7 days after implantation. Sheep were examined daily for any changes in behavior or neurologic function. After 28–30 days, the animals were humanely killed. Cerebrospinal fluid samples were collected and analyzed for protein, erythrocytes and leukocytes, and morphine content. The spinal cord and meninges with the catheter in situ was removed en bloc and fixed in formalin for histologic analysis. Results Unilateral hind-leg gait deficits were observed in two of three animals in each of the 12- and 18-mg/day dose groups. Gross and microscopic evaluation of spinal cord tissue from these animals revealed intradural-extramedullary inflammatory masses that compressed the spinal cord at the catheter-tip and mid-catheter areas. This inflammation was ipsilateral to extremities that exhibited gait deficits and had acute and chronic cellular components. Conclusions The toxicity of intrathecal morphine seems to be dependent on the amount of morphine infused, although the effects of dose versus concentration cannot be clearly distinguished in this study. Intrathecal morphine doses of 12- 18 mg/day produced inflammatory masses extending from the catheter tip down the length of the catheter within the subarachnoid space. Doses of 6–9 mg/day produced mild-to-moderate inflammation 5 cm cranial to the catheter tip. A dose of 3 mg/day produced no neurotoxicity and spinal histopathologic changes that were equivalent to those observed in the saline-treated animals.

Novel Infectious cDNA Clones of Hepatitis C Virus Genotype 3a (Strain S52) and 4a (Strain ED43): Genetic Analyses and In Vivo Pathogenesis Studies

ABSTRACT Previously, RNA transcripts of cDNA clones of hepatitis C virus (HCV) genotypes 1a (strains H77, HCV-1, and HC-TN), 1b (HC-J4, Con1, and HCV-N), and 2a (HC-J6 and JFH1) were found to be infectious in chimpanzees. However, only JFH1 was infectious in human hepatoma Huh7 cells. We performed genetic analysis of HCV genotype 3a (strain S52) and 4a (strain ED43) prototype strains and generated full-length consensus cDNA clones (pS52 and pED43). Transfection of Huh7.5 cells with RNA transcripts of these clones did not yield cells expressing HCV Core. However, intrahepatic transfection of chimpanzees resulted in robust infection with peak HCV RNA titers of ∼5.5 log10 international units (IU)/ml. Genomic consensus sequences recovered from serum at the times of peak viral titers were identical to the sequences of the parental plasmids. Both chimpanzees developed acute hepatitis with elevated liver enzymes and significant necroinflammatory liver changes coinciding with detection of gamma interferon-secreting, intrahepatic T cells. However, the onset and broadness of intrahepatic T-cell responses varied greatly in the two animals, with an early (week 4) multispecific response in the ED43-infected animal (3 weeks before the first evidence of viral control) and a late (week 11) response with limited breadth in the S52-infected animal (without evidence of viral control). Autologous serum neutralizing antibodies were not detected during the acute infection in either animal. Both animals became persistently infected. In conclusion, we generated fully functional infectious cDNA clones of HCV genotypes 3a and 4a. Proof of functionality of all genes might further the development of recombinant cell culture systems for these important genotypes.

Previously Infected Chimpanzees Are Not Consistently Protected against Reinfection or Persistent Infection after Reexposure to the Identical Hepatitis C Virus Strain

ABSTRACT Protective immunity after resolved hepatitis C virus (HCV) infection has been reported. However, the breadth of this immunity has remained controversial, and the role of neutralizing antibodies has not been well-defined. In the present study, two chimpanzees (CH96A008 and CH1494) with resolved monoclonal H77C (genotype 1a) infection were rechallenged with low-dose homologous H77C virus about 12 months after viral clearance; CH96A008 became persistently infected, and CH1494 had transient viremia lasting 2 weeks. CH1494 was subsequently either partially or completely protected following five homologous rechallenges with monoclonal H77C or polyclonal H77 and after six heterologous rechallenges with HC-J4 (genotype 1b) or HC-J6 (genotype 2a) viruses. Subsequently, a final challenge with H77C resulted in persistent HCV infection. In both chimpanzees, serum neutralizing antibodies against retroviral pseudoparticles bearing the H77C envelope proteins were not detected during the initial infection or during rechallenge. However, anamnestic cellular immune responses developed during the initial homologous rechallenge, in particular in CH96A008, which developed a persistent infection. Polyprotein sequences of viruses recovered from CH1494 after the two homologous rechallenges that resulted in transient viremia were identical with the H77C virus. In contrast, the polyprotein sequences of viruses recovered from both chimpanzees after homologous rechallenge resulting in persistent infection had numerous changes. These findings have important implications for our understanding of immunity against HCV; even in the best-case scenario with autologous rechallenge, low-level viral persistence was seen in the presence of primed T-cell responses.

Challenge Pools of Hepatitis C Virus Genotypes 1–6 Prototype Strains: Replication Fitness and Pathogenicity in Chimpanzees and Human Liver-Chimeric Mouse Models

Chimpanzees represent the only animal model for studies of the natural history of hepatitis C virus (HCV). To generate virus stocks of important HCV variants, we infected chimpanzees with HCV strains of genotypes 1-6 and determined the infectivity titer of acute-phase plasma pools in additional animals. The courses of first- and second-passage infections were similar, with early appearance of viremia, HCV RNA titers of >10(4.7) IU/mL, and development of acute hepatitis; the chronicity rate was 56%. The challenge pools had titers of 10(3)-10(5) chimpanzee infectious doses/mL. Human liver-chimeric mice developed high-titer infections after inoculation with the challenge viruses of genotypes 1-6. Inoculation studies with different doses of the genotype 1b pool suggested that a relatively high virus dose is required to consistently infect chimeric mice. The challenge pools represent a unique resource for studies of HCV molecular virology and for studies of pathogenesis, protective immunity, and vaccine efficacy in vivo.

Characterization of Chimpanzee/Human Monoclonal Antibodies to Vaccinia Virus A33 Glycoprotein and Its Variola Virus Homolog In Vitro and in a Vaccinia Virus Mouse Protection Model

ABSTRACT Three distinct chimpanzee Fabs against the A33 envelope glycoprotein of vaccinia virus were isolated and converted into complete monoclonal antibodies (MAbs) with human γ1 heavy-chain constant regions. The three MAbs (6C, 12C, and 12F) displayed high binding affinities to A33 (Kd of 0.14 nM to 20 nM) and may recognize the same epitope, which was determined to be conformational and located within amino acid residues 99 to 185 at the C terminus of A33. One or more of the MAbs were shown to reduce the spread of vaccinia virus as well as variola virus (the causative agent of smallpox) in vitro and to more effectively protect mice when administered before or 2 days after intranasal challenge with virulent vaccinia virus than a previously isolated mouse anti-A33 MAb (1G10) or vaccinia virus immunoglobulin. The protective efficacy afforded by anti-A33 MAb was comparable to that of a previously isolated chimpanzee/human anti-B5 MAb. The combination of anti-A33 MAb and anti-B5 MAb did not synergize the protective efficacy. These chimpanzee/human anti-A33 MAbs may be useful in the prevention and treatment of vaccinia virus-induced complications of vaccination against smallpox and may also be effective in the immunoprophylaxis and immunotherapy of smallpox and other orthopoxvirus diseases.

Safety of continuous intrathecal midazolam infusion in the sheep model

We investigated the safety of midazolam administered by continuous intrathecal infusion in relevant animal models. Preservative-free midazolam was delivered to sheep and pigs by using implanted infusion systems (SynchroMed® pumps plus silicone catheters). Sheep received midazolam 5 mg/d (n= 4) or 15 mg/d (n= 7) or saline (n= 2) for 43 days at 125 μL/h. One sheep received 10 mg/d. Infusion concentrations ranged from 1.7 to 2.5 mg/mL (5 mg/d) and from 2.5 to 5.0 mg/mL (15 mg/d). Pigs were evaluated for toxicity only and received 15 mg/d (n= 2) or saline (n= 1) for 43 days at 125 μL/h. Behavior, neurologic function, and vital signs were documented. Serum and cerebrospinal fluid chemistry and cytology were evaluated, and histology was performed on spinal cord tissue. Behavior and neurologic function remained normal in all subjects. Gross and microscopic evaluation of spinal tissue revealed mild inflammation surrounding the catheter tract in both the midazolam-treated and the saline-treated groups. This inflammation was likely attributable to the mechanical presence of the catheter. These data demonstrate that continuous intrathecal infusion of preservative-free midazolam at doses up to 15 mg/d were well tolerated.

T-cell immunity and hepatitis C virus reinfection after cure of chronic hepatitis C with an interferon-free antiviral regimen in a chimpanzee.

Memory CD8+ T cells generated by spontaneous resolution of hepatitis C virus (HCV) infection rapidly control secondary infections and reduce the risk of virus persistence. Here, CD8+ T‐cell immunity and response to reinfection were assessed in a chimpanzee cured of an earlier chronic infection with an interferon (IFN)‐free antiviral regimen. CD8+ T cells expanded from liver immediately before and 2 years after cure of chronic infection with two direct‐acting antivirals (DAAs) targeted epitopes in the E2, nonstructural (NS)5a, and NS5b proteins. A second infection to assess CD8+ T‐cell responsiveness resulted in rapid suppression of HCV replication by week 2, but viremia rebounded 3 weeks later and the infection persisted. The E2, NS5a, and NS5b proteins remained dominant CD8+ T‐cell targets after reinfection. Resurgent HCV replication was temporally associated with mutational escape of NS5a and NS5b class I epitopes that had also mutated during the first chronic infection. Two epitopes in E2 remained intact throughout both persistent infections. Intrahepatic CD8+ T cells targeting intact and escape‐prone epitopes differed in expression of phenotypic markers of functional exhaustion 2 years after successful DAA therapy and in the capacity to expand in liver upon reinfection. Conclusions: The intrahepatic HCV‐specific CD8+ T‐cell repertoire established during chronic infection was narrowly focused, but very stable, after cure with DAA. Existing intrahepatic CD8+ T cells targeting dominant epitopes of the challenge virus failed to prevent persistence. Vaccination after DAA cure may be necessary to broaden T‐cell responses and reduce the risk of a second persistent infection. (Hepatology 2014;60:1531–1540)

Fluorescence spectroscopy as a biomarker in a cell culture and in a nonhuman primate model for ovarian cancer chemopreventive agents

OBJECTIVE The objective of this study was to compare the effects of chemopreventive agents on natural fluorescence emission of ovarian cells in a cell culture and in a primate model as a feasibility trial to monitor drug activity. METHODS Fluorescence emission spectra were collected from normal (NOE) and immortalized ovarian surface epithelial cells at 290, 360, and 450 nm excitation. Redox potentials were calculated and compared to % apoptosis and cell survival. Fluorescence emission spectra were collected from 18 female rhesus macaques receiving fenretinide [N-(-hydroxyphenyl)retinamide (4-HPR)] orally and/or oral contraceptive pills (OCP) or no medication. Fluorescence intensities and redox ratios were compared using a two-tailed Students t test. RESULTS Apoptosis and cell survival correlated with fluorescence emission consistent with metabolically active proteins [flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NAD(P)H)] and the resulting redox ratio in cells grown with 4-HPR. The 4-HPR consistently inhibited cell survival in a dose dependent manner. Degree of correlation varied between different cell lines. In primates receiving 4-HPR, fluorescence emission was increased at 450 nm excitation, 550 nm emission consistent with FAD presence, whereas those receiving OCP showed decreased emission at 350 nm excitation, 450 nm emission consistent with decreased NAD(P)H presence. Redox ratios were increased by both drugs. CONCLUSIONS Fluorescence intensity and redox ratio appear to be altered by 4-HPR treatment in vivo and in cell culture and by OCP in vivo. Fluorescence intensity may be useful to monitor chemopreventive agents in clinical trials.