Characterization of novel mouse models to study the role of necroptosis in aging and age-related diseases.

To study the impact of necroptosis-induced chronic inflammation on age-related diseases and aging, two knockin mouse models (Ripk3-KI and Mlkl-KI) were generated that overexpress two genes involved in necroptosis (Ripk3 or Mlkl) when crossed to Cre transgenic mice. Crossing Ripk3-KI or Mlkl-KI mice to albumin-Cre transgenic mice produced hepatocyte specific hRipk3-KI or hMlkl-KI mice, which express the two transgenes only in the liver. Ripk3 and Mlkl proteins were overexpressed 10- and fourfold, respectively, in the livers of the hRipk3-KI or hMlkl-KI mice. Treating young (2-month) hRipk3-KI or hMlkl-KI mice with carbon tetrachloride (CCl4), a chemical inducer of oxidative stress, resulted in increased necroptosis (Mlkl-oligomers) and inflammation in the liver compared to control mice receiving CCl4. Mlkl-oligomerization also was significantly increased in old (18-month) hRipk3-KI and hMlkl-KI mice compared to old control (Cre negative, Ripk3-KI and Mlkl-KI) mice. The increase in necroptosis was associated with an increase in inflammation, e.g., inflammatory cytokines (TNFα, IL-6) and macrophage markers (F4/80, CD68). Importantly, steatosis (triglycerides) and fibrosis (e.g., picrosirius red staining, hydroxyproline levels, and transcripts for TGFß, Col1α1, and Col3α1) that increase with age were significantly higher in the livers of the old hRipk3-KI or hMlkl-KI mice compared to old control mice. In addition, markers of cellular senescence were significantly increased in the livers of the old hRipk3-KI and hMlkl-KI mice. Thus, the first mouse models have been developed that allow researchers to study the impact of inducing necroptosis in specific cells/tissues on chronic inflammation in aging and age-related diseases.

Polycaprolactone Electrospun Nanofiber Membrane with Skin Graft Containing Collagen and Bandage Containing MgO Nanoparticles for Wound Healing Applications.

The objective of this study was to create a nanofiber-based skin graft with an antimicrobial bandage that could accelerate the healing of an open wound while minimizing infection. To this end, we prepared a bi-layer construct where the top layer acts as bandage, and the bottom layer acts as a dermal equivalent graft. A collagen (CG) gel was combined without and with an electrospun polycaprolactone (PCL) membrane to prepare CG and CG-PCL dermal equivalent constructs. The antibacterial properties of PCL with and without an antibacterial agent (MgO nanoparticles) against Staphylococcus aureus (ATCC 6538) was also examined. Human dermal fibroblasts were cultured in each construct to make the dermal equivalent grafts. After culturing, keratinocytes were plated on top of the tissues to allow growth of an epidermis. Rheological and durability tests were conducted on in vitro dermal and skin equivalent cultures, and we found that PCL significantly affects CG-PCL graft biological and mechanical strength (rheology and durability). PCL presence in the dermal equivalent allowed sufficient tension generation to activate fibroblasts and myofibroblasts in the presence of transforming growth factor-beta. During culture of the skin equivalents, optical coherence tomography (OCT) showed layers corresponding to dermal and epidermal compartments in the presence or absence of PCL; this was confirmed after fixed specimens were histologically sectioned and stained. MgO added to PCL showed antibacterial activity against S. aureus. In vivo animal studies using a rat skin model showed that a polycaprolactone nanofiber bandage containing a type I collagen skin graft has potential for wound healing applications.

A Genetically Heterogeneous Rat Model with Divergent Mitochondrial Genomes.

We generated a genetically heterogenous rat model by a 4-way cross strategy using 4 inbred strains (Brown Norway [BN], Fischer 344 [F344], Lewis [LEW], and Wistar Kyoto [KY]) to provide investigators with a highly genetically diverse rat model from commercially available inbred rats. We made reciprocal crosses between males and females from the 2 F1 hybrids to generate genetically heterogeneous rats with mitochondrial genomes from either the BN (OKC-HETB, a.k.a "B" genotype) or WKY (OKC-HETW a.k.a "W" genotype) parental strains. These two mitochondrial genomes differ at 94 nucleotides, more akin to human mitochondrial genome diversity than that available in classical laboratory mouse strains. Body weights of the B and W genotypes were similar. However, mitochondrial genotype antagonistically affected grip strength and treadmill endurance in females only. In addition, mitochondrial genotype significantly affected multiple responses to a high-fat diet (HFD) and treatment with 17α-estradiol. Contrary to findings in mice in which males only are affected by 17α-estradiol supplementation, female rats fed a HFD beneficially responded to 17α-estradiol treatment as evidenced by declines in body mass, adiposity, and liver mass. Male rats, by contrast, differed in a mitochondrial genotype-specific manner, with only B males responding to 17α-estradiol treatment. Mitochondrial genotype and sex differences were also observed in features of brain-specific antioxidant response to a HFD and 17α-estradiol as shown by hippocampal levels of Sod2 acetylation, JNK, and FoxO3a. These results emphasize the importance of mitochondrial genotype in assessing responses to putative interventions in aging processes.

Benserazide racemate and enantiomers induce fetal globin gene expression in vivo: Studies to guide clinical development for beta thalassemia and sickle cell disease.

Increased expression of developmentally silenced fetal globin (HBG) reduces the clinical severity of ß-hemoglobinopathies. Benserazide has a relatively benign safety profile having been approved for 50 years in Europe and Canada for Parkinson's disease treatment. Benserazide was shown to activate HBG gene transcription in a high throughput screen, and subsequent studies confirmed fetal hemoglobin (HbF) induction in erythroid progenitors from hemoglobinopathy patients, transgenic mice containing the entire human ß-globin gene (ß-YAC) and anemic baboons. The goal of this study is to evaluate efficacies and plasma exposure profiles of benserazide racemate and its enantiomers to select the chemical form for clinical development. Intermittent treatment with all forms of benserazide in ß-YAC mice significantly increased proportions of red blood cells expressing HbF and HbF protein per cell with similar pharmacokinetic profiles and with no cytopenia. These data contribute to the regulatory justification for development of the benserazide racemate. Additionally, dose ranges and frequencies required for HbF induction using racemic benserazide were explored. Orally administered escalating doses of benserazide in an anemic baboon induced γ-globin mRNA up to 13-fold and establish an intermittent dose regimen for clinical studies as a therapeutic candidate for potential treatment of ß-hemoglobinopathies.

Effects of Chronic Viral Infection on Lymphocyte Populations in Middle-aged Baboons (Papio anubis).

Aging of the immune system is characterized by the loss of naïve T-cells, increased inflammation, and immune function impairment. Chronic infection with cytomegalovirus is thought to play a role in age-related changes in immunity. Therefore, to assess the effect of pathogens such as cytomegalovirus on the immune system, we determined lymphocyte populations and inflammatory markers over a 3-y period in captive, middle-age baboons, with various exposure to pathogens and shedding pressure. Groups included SPF (i.e., pathogen-negative; n = 14); large-group, conventionally housed (CONV LG; pathogen- positive; n = 14), and small-group, conventionally housed (CONV SM; pathogen-positive; n = 7). All baboon groups showed a decrease in CD45RA+ CD28+ (i.e., naive) cells over time during middle age, but the rate of decline appeared faster in CONV LG baboons than in the other groups. In addition, the reduction in CD45RA+ CD28+ cells in the CONV LG baboons coincided with higher IgG levels against baboon cytomegalovirus, increased serum cortisol concentration, and a greater inflammatory phenotype. The results of this project support a role for cytomegalovirus infection in immune system alterations in middle-aged baboons.

Doublecortin-like kinase 1 promotes hepatocyte clonogenicity and oncogenic programming via non-canonical ß-catenin-dependent mechanism.

Chronic liver injury is a risk factor for cirrhosis and hepatocellular carcinoma (HCC). The molecular mechanisms that regulate the decision between normal injury repair and neoplastic initiation are unclear. Doublecortin-like kinase 1 (DCLK1), a tumor stem cell marker, is induced during cirrhosis and HCC. Here, we demonstrate that DCLK1-overexpressing primary human hepatocytes formed spheroids in suspension cultures. Spheroids derived from DCLK1-overexpressing hepatoma cells showed high level expression of active ß-catenin, α-fetoprotein, and SOX9, suggesting that DCLK1 overexpression induces clonogenicity and dedifferentiated phenotypes in hepatoma cells. DCLK1 overexpression in hepatoma cells also increased phosphorylation of GSK-3ß at Ser9. This was associated with an induction of a 48-kDa active ß-catenin with a preserved hypophosphorylated N-terminus that interacted with nuclear TCF-4 resulting in luciferase reporter activity and cyclin D1 expression. DCLK1 downregulation inhibited 48-kDa ß-catenin expression. The proteasome inhibitor bortezomib did not block the 48-kDa ß-catenin, instead, caused a threefold accumulation, suggesting a proteasome-independent mechanism. Liver tissues from patients with cirrhosis and HCC revealed epithelial co-staining of DCLK1 and active ß-catenin, and cleaved E-cadherin. Repopulated DCLK1-overexpressing primary human hepatocytes in humanized FRG mouse livers demonstrated active ß-catenin. In conclusion, DCLK1 regulates oncogenic signaling and clonogenicity of hepatocytes by a novel non-canonical/atypical ß-catenin-dependent mechanism.

Fifteen Years of Sm-p80-Based Vaccine Trials in Nonhuman Primates: Antibodies From Vaccinated Baboons Confer Protection in vivo and in vitro From Schistosoma mansoni and Identification of Putative Correlative Markers of Protection.

Recent advances in systems biology have shifted vaccine development from a largely trial-and-error approach to an approach that promote rational design through the search for immune signatures and predictive correlates of protection. These advances will doubtlessly accelerate the development of a vaccine for schistosomiasis, a neglected tropical disease that currently affects over 250 million people. For over 15 years and with contributions of over 120 people, we have endeavored to test and optimize Sm-p80-based vaccines in the non-human primate model of schistosomiasis. Using RNA-sequencing on eight different Sm-p80-based vaccine strategies, we sought to elucidate immune signatures correlated with experimental protective efficacy. Furthermore, we aimed to explore the role of antibodies through in vivo passive transfer of IgG obtained from immunized baboons and in vitro killing of schistosomula using Sm-p80-specific antibodies. We report that passive transfer of IgG from Sm-p80-immunized baboons led to significant worm burden reduction, egg reduction in liver, and reduced egg hatching percentages from tissues in mice compared to controls. In addition, we observed that sera from Sm-p80-immunized baboons were able to kill a significant percent of schistosomula and that this effect was complement-dependent. While we did not find a universal signature of immunity, the large datasets generated by this study will serve as a substantial resource for further efforts to develop vaccine or therapeutics for schistosomiasis.

Chronic whipworm infection exacerbates Schistosoma mansoni egg-induced hepatopathology in non-human primates.

BACKGROUND: Schistosomiasis continues to inflict significant morbidity and mortality in the tropical and subtropical regions of the world. The disease endemicity overlaps with the transmission of other parasitic diseases. Despite the ubiquity of polyparasitism in tropical regions, particularly in rural communities, little is known about the impact of multiple helminth infections on disease progression. In this pilot study, we describe the influence of chronic Trichuris trichiura infection on Schistosoma mansoni egg-induced hepatopathology in infected baboons. METHODS: Baboons with or without underlying whipworm infection were challenged with S. mansoni cercariae to establish schistosomiasis. Adult S. mansoni worms were recovered by perfusion and enumerated, hepatic granulomas were quantified via light microscopy, and transcriptional profiling of tissues were completed using RNA sequencing technologies. RESULTS: Co-infection with both S. mansoni and T. trichiura resulted in higher female schistosome worm burden and significantly larger liver granuloma sizes. Systems biology analyses of peripheral blood mononuclear cells (PBMC) revealed pathways associated with increased liver damage in co-infected baboons. CONCLUSIONS: Underlying chronic whipworm infection intensified schistosome egg-induced liver pathology in infected baboons. RNA-Seq analysis provided insight into pathways associated with increased liver damage, corroborating histological findings.

Sm-p80-based schistosomiasis vaccine: double-blind preclinical trial in baboons demonstrates comprehensive prophylactic and parasite transmission-blocking efficacy.

Schistosomiasis is of public health importance to an estimated one billion people in 79 countries. A vaccine is urgently needed. Here, we report the results of four independent, double-blind studies of an Sm-p80-based vaccine in baboons. The vaccine exhibited potent prophylactic efficacy against transmission of Schistosoma mansoni infection and was associated with significantly less egg-induced pathology, compared with unvaccinated control animals. Specifically, the vaccine resulted in a 93.45% reduction of pathology-producing female worms and significantly resolved the major clinical manifestations of hepatic/intestinal schistosomiasis by reducing the tissue egg-load by 89.95%. A 35-fold decrease in fecal egg excretion in vaccinated animals, combined with an 81.51% reduction in hatching of eggs into the snail-infective stage (miracidia), demonstrates the parasite transmission-blocking potential of the vaccine. Substantially higher Sm-p80 expression in female worms and Sm-p80-specific antibodies in vaccinated baboons appear to play an important role in vaccine-mediated protection. Preliminary analyses of RNA sequencing revealed distinct molecular signatures of vaccine-induced effects in baboon immune effector cells. This study provides comprehensive evidence for the effectiveness of an Sm-p80-based vaccine for schistosomiasis.

Histopathology of Bordetella pertussis in the Baboon Model.

Pertussis is a severe respiratory disease caused by Bordetella pertussis The classic symptoms of pertussis include paroxysmal coughing with an inspiratory whoop, posttussive vomiting, cyanosis, and persistent coryzal symptoms. Infants under 2 months of age experience more severe disease, with most deaths occurring in this age group. Most of what is known about the pathology of pertussis in humans is from the evaluation of fatal human infant cases. The baboon model of pertussis provides the opportunity to evaluate the histopathology of severe but nonfatal pertussis. The baboon model recapitulates the characteristic clinical signs of pertussis observed in humans, including leukocytosis, paroxysmal coughing, mucus production, heavy colonization of the airway, and transmission of the bacteria between hosts. As in humans, baboons demonstrate age-related differences in clinical presentation, with younger animals experiencing more severe disease. We examined the histopathology of 5- to 6-week-old baboons, with the findings being similar to those reported for fatal human infant cases. In juvenile baboons, we found that the disease is highly inflammatory and concentrated to the lungs with signs of disease that would typically be diagnosed as acute respiratory distress syndrome (ARDS) and bronchopneumonia. In contrast, no significant pathology was observed in the trachea. Histopathological changes in the trachea were limited to cellular infiltrates and mucus production. Immunohistostaining revealed that the bacteria were localized to the surface of the ciliated epithelium in the conducting airways. Our observations provide important insights into the pathology of pertussis in typical, severe but nonfatal pertussis cases in a very relevant animal model.

Translational Model of Zika Virus Disease in Baboons.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus with devastating outcomes seen recently in the Americas due to the association of maternal ZIKV infection with fetal microcephaly and other fetal malformations not previously associated with flavivirus infections. Here, we have developed the olive baboon (Papio anubis) as a nonhuman primate (NHP) translational model for the study of ZIKV pathogenesis and associated disease outcomes to contrast and compare with humans and other major NHPs, such as macaques. Following subcutaneous inoculation of adult male and nonpregnant female baboons, viremia was detected at 3 and 4 days postinfection (dpi) with the concordant presentation of a visible rash and conjunctivitis, similar to human ZIKV infection. Furthermore, virus was detected in the mucosa and cerebrospinal fluid. A robust ZIKV-specific IgM and IgG antibody response was also observed in all the animals. These data show striking similarity between humans and the olive baboon following infection with ZIKV, suggesting our model is a suitable translational NHP model to study ZIKV pathogenesis and potential therapeutics.IMPORTANCE ZIKV was first identified in 1947 in a sentinel rhesus monkey in Uganda and subsequently spread to Southeast Asia. Until 2007, only a small number of cases were reported, and ZIKV infection was relatively minor until the South Pacific and Brazilian outbreaks, where more severe outcomes were reported. Here, we present the baboon as a nonhuman primate model for contrast and comparison with other published animal models of ZIKV, such as the mouse and macaque species. Baboons breed year round and are not currently a primary nonhuman primate species used in biomedical research, making them more readily available for studies other than human immunodeficiency virus studies, which many macaque species are designated for. This, taken together with the similarities baboons have with humans, such as immunology, reproduction, genetics, and size, makes the baboon an attractive NHP model for ZIKV studies in comparison to other nonhuman primates.

Maternal Vaccination With a Monocomponent Pertussis Toxoid Vaccine Is Sufficient to Protect Infants in a Baboon Model of Whooping Cough.

Background: Bordetella pertussis is a human pathogen responsible for serious respiratory illness. The disease is most severe in infants too young to be vaccinated with most hospitalizations and deaths occurring within this age group. The Advisory Committee on Immunization Practices recommended immunization of pregnant women to protect infants from birth until their first vaccination at 6-8 weeks of age. We previously demonstrated that maternal vaccination with licensed acellular pertussis vaccines protected newborn baboons from disease. We hypothesized that protection was due to toxin-neutralizing, maternal anti-pertussis toxin antibodies and predicted that maternal vaccination with a pertussis toxoid (PTx)-only vaccine would protect newborns from disease. Methods: Infant baboons born to unvaccinated mothers or mothers vaccinated with a PTx-only vaccine were challenged with B. pertussis at 5 weeks of age and followed for infection and signs of disease. Results: Although all challenged infants were heavily colonized, the infant baboons born to mothers vaccinated with PTx-only vaccine were free from clinical disease following exposure to B. pertussis. In contrast, disease was observed in infants born to unvaccinated mothers. Conclusions: Our results demonstrated that maternal vaccination with a PTx-only vaccine is sufficient to protect newborn baboons from disease following exposure to pertussis.

Apnea induction for invasive lung function testing in infant olive baboons: Comparison of intravenous propofol versus hyperventilation.

BACKGROUND: In various types of pulmonary research, pulmonary function testing (PFT) is performed to quantify the severity of lung disease. Induction of apnea and positive pressure ventilation are required for accurate PFT measurements in non-cooperative subjects. We compared two methods of apnea induction in infant olive baboons (Papio anubis). METHODS: Pulmonary function testing results were compared during apnea induced by hyperventilation (CO2 washout) vs. intravenous propofol (1 dose 10 mg/kg). PFT was evaluated using a hot-wire pneumotachometer incorporated within an Avea ventilator in nine 1-month-old baboons. RESULTS: Propofol induced apnea faster and more reliably. In both groups, PFT values passed the statistical equivalence test and were not significantly different (Student's t-test). There was a trend toward less data variability after propofol administration. CONCLUSIONS: Intravenous propofol was non-inferior to CO2 washout for apnea induction in infant olive baboons. Propofol induced apnea faster and more reliably and yielded less variable PFT results.

Pilot Study to Assess the Efficacy of Ivermectin and Fenbendazole for Treating Captive-Born Olive Baboons (Papio anubis) Coinfected with Strongyloides fülleborni and Trichuris trichiura.

In this study, we evaluated the efficacy of combined treatment with ivermectin and fenbendazole (IVM-FBZ) for treating captive olive baboons (Papio anubis) infected with Strongyloides fülleborni and Trichuris trichiura, 2 common nematode parasites of these NHP. Infected baboons were treated for a total of 9 wk with ivermectin (400 µg/kg IM twice weekly) and fenbendazole (50 mg/kg PO once daily for 3 d; 3 rounds of treatment, 21 d apart). Five baboons naturally infected with both S. fülleborni and T. trichiura (n = 4) or S. fülleborni alone (n = 1) received the combination therapy; an additional baboon infected with both parasites served as a nontreated control. The efficacy of IVM-FBZ was measured as the reduction in fecal egg counts of S. fülleborni and T. trichiura as determined by quantitative fecal flotation examination after treatment of baboons with IVM-FBZ. All baboons treated with IVM-FBZ stopped shedding S. fülleborni and T. trichiura eggs by 8 d after treatment and remained negative for at least 161 d. The nontreated control baboon shed S. fülleborni and T. trichiura eggs throughout the study period. Our results indicate that the IVM-FBZ regimen was efficacious for treating olive baboons infected with S. fülleborni and T. trichiura.

Immunologic detection of Giardia duodenalis in a specific pathogen-free captive olive baboon ( Papio cynocephalus anubis) colony.

Several commercial Giardia immunoassays were evaluated in baboons for sensitivity and specificity as well as ease of use in a large specific pathogen-free (SPF) colony. An additional objective was to identify the assemblage(s) of Giardia duodenalis present in this baboon colony. A direct immunofluorescent antibody test (IFAT) was used as the reference test. Tests evaluated were a patient-side rapid test for dogs and cats, a human rapid test, and a well-plate ELISA designed for use with humans. Test sensitivities and specificities were compared using the McNemar paired t-test and were further evaluated for agreement using an unweighted Cohen kappa statistic. When compared to the IFAT reference, both human tests were more sensitive than the veterinary test. Based on PCR and sequencing of the G. duodenalis small-subunit ribosomal RNA and glutamate dehydrogenase loci, assemblage AI was present in this baboon colony. We found that 10 of the 110 (9%) baboons in this SPF colony were infected with a zoonotic strain of G. duodenalis.

Prior exposure to Bordetella species as an exclusion criterion in the baboon model of pertussis.

The baboon model of Bordetella pertussis infection is the newest and most clinically accurate model of the human disease to date. However, among the 15 experimentally infected baboons in this study, a subset of baboons did not exhibit the expected high bacterial colonization levels or increase in white blood cell count. Moreover, cultures of nasopharyngeal wash samples from several baboons suggested B. bronchiseptica coinfection. Analysis of serum antibodies recognizing filamentous hemagglutinin, pertussis toxin and B. pertussis lipo-oligosaccharide indicated that several baboons had likely been previously exposed to Bordetella species and that prior exposure correlated with partial protection from B. pertussis infection. Notably, all animals with a baseline Fha titer of 5 IU/ml or below exhibited symptoms typical of the model, suggesting this value can be used as inclusion criteria for animals prior to study enrollment. While B. pertussis infection is endemic to human populations and B. bronchiseptica is common in wild small mammals, this study illustrates that baboons can readily harbor both organisms. Awareness of Bordetella species that share antigens capable of generating protective immune responses and tracking of prior exposure to those species is required for successful use of the baboon model of pertussis.

A cocktail of humanized anti-pertussis toxin antibodies limits disease in murine and baboon models of whooping cough.

Despite widespread vaccination, pertussis rates are rising in industrialized countries and remain high worldwide. With no specific therapeutics to treat disease, pertussis continues to cause considerable infant morbidity and mortality. The pertussis toxin is a major contributor to disease, responsible for local and systemic effects including leukocytosis and immunosuppression. We humanized two murine monoclonal antibodies that neutralize pertussis toxin and expressed them as human immunoglobulin G1 molecules with no loss of affinity or in vitro neutralization activity. When administered prophylactically to mice as a binary cocktail, antibody treatment completely mitigated the Bordetella pertussis-induced rise in white blood cell counts and decreased bacterial colonization. When administered therapeutically to baboons, antibody-treated, but not untreated control animals, experienced a blunted rise in white blood cell counts and accelerated bacterial clearance rates. These preliminary findings support further investigation into the use of these antibodies to treat human neonatal pertussis in conjunction with antibiotics and supportive care.

PBN (Phenyl-N-Tert-Butylnitrone)-Derivatives Are Effective in Slowing the Visual Cycle and Rhodopsin Regeneration and in Protecting the Retina from Light-Induced Damage.

A2E and related toxic molecules are part of lipofuscin found in the retinal pigment epithelial (RPE) cells in eyes affected by Stargardt's disease, age-related macular degeneration (AMD), and other retinal degenerations. A novel therapeutic approach for treating such degenerations involves slowing down the visual cycle, which could reduce the amount of A2E in the RPE. This can be accomplished by inhibiting RPE65, which produces 11-cis-retinol from all-trans-retinyl esters. We recently showed that phenyl-N-tert-butylnitrone (PBN) inhibits RPE65 enzyme activity in RPE cells. In this study we show that like PBN, certain PBN-derivatives (PBNDs) such as 4-F-PBN, 4-CF3-PBN, 3,4-di-F-PBN, and 4-CH3-PBN can inhibit RPE65 and synthesis of 11-cis-retinol in in vitro assays using bovine RPE microsomes. We further demonstrate that systemic (intraperitoneal, IP) administration of these PBNDs protect the rat retina from light damage. Electroretinography (ERG) and histological analysis showed that rats treated with PBNDs retained ~90% of their photoreceptor cells compared to a complete loss of function and 90% loss of photoreceptors in the central retina in rats treated with vehicle/control injections. Topically applied PBN and PBNDs also significantly slowed the rate of the visual cycle in mouse and baboon eyes. One hour dark adaptation resulted in 75-80% recovery of bleachable rhodopsin in control/vehicle treated mice. Eye drops of 5% 4-CH3-PBN were most effective, inhibiting the regeneration of bleachable rhodopsin significantly (60% compared to vehicle control). In addition, a 10% concentration of PBN and 5% concentration of 4-CH3-PBN in baboon eyes inhibited the visual cycle by 60% and by 30%, respectively. We have identified a group of PBN related nitrones that can reach the target tissue (RPE) by systemic and topical application and slow the rate of rhodopsin regeneration and therefore the visual cycle in mouse and baboon eyes. PBNDs can also protect the rat retina from light damage. There is potential in developing these compounds as preventative therapeutics for the treatment of human retinal degenerations in which the accumulation of lipofuscin may be pathogenic.

Novel Inducers of Fetal Globin Identified through High Throughput Screening (HTS) Are Active In Vivo in Anemic Baboons and Transgenic Mice.

High-level fetal (γ) globin expression ameliorates clinical severity of the beta (ß) hemoglobinopathies, and safe, orally-bioavailable γ-globin inducing agents would benefit many patients. We adapted a LCR-γ-globin promoter-GFP reporter assay to a high-throughput robotic system to evaluate five diverse chemical libraries for this activity. Multiple structurally- and functionally-diverse compounds were identified which activate the γ-globin gene promoter at nanomolar concentrations, including some therapeutics approved for other conditions. Three candidates with established safety profiles were further evaluated in erythroid progenitors, anemic baboons and transgenic mice, with significant induction of γ-globin expression observed in vivo. A lead candidate, Benserazide, emerged which demonstrated > 20-fold induction of γ-globin mRNA expression in anemic baboons and increased F-cell proportions by 3.5-fold in transgenic mice. Benserazide has been used chronically to inhibit amino acid decarboxylase to enhance plasma levels of L-dopa. These studies confirm the utility of high-throughput screening and identify previously unrecognized fetal globin inducing candidates which can be developed expediently for treatment of hemoglobinopathies.

The effects of age and cytomegalovirus on markers of inflammation and lymphocyte populations in captive baboons.

The human immune system undergoes age-related changes that can lead to increased disease susceptibility. Using the baboon as a model for human immune system aging, we examined age-related changes in relative and absolute numbers of T cell subpopulations, cytomegalovirus (CMV) titer and markers of inflammation. In addition, the effect of gender, social status and peer group on lymphocyte subpopulations was determined. Relative and absolute numbers of total lymphocytes (CD3+), T helper cells (CD4+), and cytotoxic T cells (CD8+) increased with age. The proportion of naïve T cells (CD45RA+) decreased, while the total number of cells negative for the co-stimulatory receptor, CD28 (CD28-) increased in an age-dependent manner. Furthermore, CMV titers were negatively correlated with the number of naive CD4+ cells. IL-6 and cortisol concentration were also negatively associated with T cell subpopulations. Additionally, socially dominant baboons exhibited decreases in naïve CD4+ and CD8+ cells (by 65% and 52%, respectively) compared to subordinate animals. These results suggest that factors such as CMV exposure and inflammation may contribute to the age-related decline in immune health and indicate that factors like social status should be considered when studying immunosenescence in animal models.