Comparative lifespan and healthspan of nonhuman primate species common to biomedical research.

There is a critical need to generate age- and sex-specific survival curves to characterize chronological aging consistently across nonhuman primates (NHP) used in biomedical research. Accurate measures of chronological aging are essential for inferences into genetic, demographic, and physiological variables driving differences in NHP lifespan within and between species. Understanding NHP lifespans is relevant to public health because unraveling the demographic, molecular, and clinical bases of health across the life course in translationally relevant NHP species is fundamentally important to the study of human aging. Data from more than 110,000 captive individual NHP were contributed by 15 major research institutions to generate sex-specific Kaplan-Meier survival curves using uniform methods in 12 translational aging models: Callithrix jacchus (common marmoset), Chlorocebus aethiops sabaeus (vervet/African green), Macaca fascicularis (cynomolgus macaque), M. fuscata (Japanese macaque), M. mulatta (rhesus macaque), M. nemestrina (pigtail macaque), M. radiata (bonnet macaque), Pan troglodytes spp. (chimpanzee), Papio hamadryas spp. (baboon), Plecturocebus cupreus (coppery titi monkey), Saguinus oedipus (cotton-top tamarin), and Saimiri spp. (squirrel monkey). After employing strict inclusion criteria, primary analysis results are based on 12,269 NHP that survived to adulthood and died of natural/health-related causes. A secondary analysis was completed for 32,616 NHP that died of any cause. For the primary analyses, we report ages of 25th, 50th, 75th, and 85th percentiles of survival, maximum observed ages, rates of survivorship, and sex-based differences captured by quantile regression models and Kolmogorov-Smirnov tests. Our findings show a pattern of reduced male survival among catarrhines (African and Asian primates), especially macaques, but not platyrrhines (Central and South American primates). For many species, median lifespans were lower than previously reported. An important consideration is that these analyses may offer a better reflection of healthspan than lifespan. Captive NHP used in research are typically euthanized for humane welfare reasons before their natural end of life, often after diagnosis of their first major disease requiring long-term treatment with reduced quality of life (e.g., endometriosis, cancer, osteoarthritis). Supporting the idea that these data are capturing healthspan, for several species typical age at onset of chronic disease is similar to the median lifespan estimates. This data resource represents the most comprehensive characterization of sex-specific lifespan and age-at-death distributions for 12 biomedically relevant species, to date. The results clarify the relationships among NHP ages and will provide a valuable resource for the aging research community, improving human-NHP age equivalencies, informing investigators of the expected survival rates of NHP assigned to studies, providing a metric for comparisons in future studies, and contributing to our understanding of the factors that drive lifespan differences within and among species.

Single-cell analysis reveals transcriptional dynamics in healthy primary parathyroid tissue.

Studies on human parathyroids are generally limited to hyperfunctioning glands owing to the difficulty in obtaining normal human tissue. We therefore obtained non-human primate (NHP) parathyroids to provide a suitable alternative for sequencing that would bear a close semblance to human organs. Single-cell RNA expression analysis of parathyroids from four healthy adult M. mulatta reveals a continuous trajectory of epithelial cell states. Pseudotime analysis based on transcriptomic signatures suggests a progression from GCM2 hi progenitors to mature parathyroid hormone (PTH)-expressing epithelial cells with increasing core mitochondrial transcript abundance along pseudotime. We sequenced, as a comparator, four histologically characterized hyperfunctioning human parathyroids with varying oxyphil and chief cell abundance and leveraged advanced computational techniques to highlight similarities and differences from non-human primate parathyroid expression dynamics. Predicted cell-cell communication analysis reveals abundant endothelial cell interactions in the parathyroid cell microenvironment in both human and NHP parathyroid glands. We show abundant RARRES2 transcripts in both human adenoma and normal primate parathyroid cells and use coimmunostaining to reveal high levels of RARRES2 protein (also known as chemerin) in PTH-expressing cells, which could indicate that RARRES2 plays an unrecognized role in parathyroid endocrine function. The data obtained are the first single-cell RNA transcriptome to characterize nondiseased parathyroid cell signatures and to show a transcriptomic progression of cell states within normal parathyroid glands, which can be used to better understand parathyroid cell biology.

Compartmentalized ocular lymphatic system mediates eye-brain immunity.

The eye, an anatomical extension of the central nervous system (CNS), exhibits many molecular and cellular parallels to the brain. Emerging research demonstrates that changes in the brain are often reflected in the eye, particularly in the retina1. Still, the possibility of an immunological nexus between the posterior eye and the rest of the CNS tissues remains unexplored. Here, studying immune responses to herpes simplex virus in the brain, we observed that intravitreal immunization protects mice against intracranial viral challenge. This protection extended to bacteria and even tumours, allowing therapeutic immune responses against glioblastoma through intravitreal immunization. We further show that the anterior and posterior compartments of the eye have distinct lymphatic drainage systems, with the latter draining to the deep cervical lymph nodes through lymphatic vasculature in the optic nerve sheath. This posterior lymphatic drainage, like that of meningeal lymphatics, could be modulated by the lymphatic stimulator VEGFC. Conversely, we show that inhibition of lymphatic signalling on the optic nerve could overcome a major limitation in gene therapy by diminishing the immune response to adeno-associated virus and ensuring continued efficacy after multiple doses. These results reveal a shared lymphatic circuit able to mount a unified immune response between the posterior eye and the brain, highlighting an understudied immunological feature of the eye and opening up the potential for new therapeutic strategies in ocular and CNS diseases.

Recruited monocytes/macrophages drive pulmonary neutrophilic inflammation and irreversible lung tissue remodeling in cystic fibrosis.

Persistent neutrophil-dominated lung inflammation contributes to lung damage in cystic fibrosis (CF). However, the mechanisms that drive persistent lung neutrophilia and tissue deterioration in CF are not well characterized. Starting from the observation that, in patients with CF, c-c motif chemokine receptor 2 (CCR2)+ monocytes/macrophages are abundant in the lungs, we investigate the interplay between monocytes/macrophages and neutrophils in perpetuating lung tissue damage in CF. Here we show that CCR2+ monocytes in murine CF lungs drive pathogenic transforming growth factor ß (TGF-ß) signaling and sustain a pro-inflammatory environment by facilitating neutrophil recruitment. Targeting CCR2 to lower the numbers of monocytes in CF lungs ameliorates neutrophil inflammation and pathogenic TGF-ß signaling and prevents lung tissue damage. This study identifies CCR2+ monocytes as a neglected contributor to the pathogenesis of CF lung disease and as a therapeutic target for patients with CF, for whom lung hyperinflammation and tissue damage remain an issue despite recent advances in CF transmembrane conductance regulator (CFTR)-specific therapeutic agents.

Age-related calcium dysregulation linked with tau pathology and impaired cognition in non-human primates.

INTRODUCTION: The etiology of sporadic Alzheimer's disease (AD) requires non-genetically modified animal models. METHODS: The relationship of tau phosphorylation to calcium-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) dysregulation was analyzed in aging rhesus macaque dorsolateral prefrontal cortex (dlPFC) and rat primary cortical neurons using biochemistry and immuno-electron microscopy. The influence of calcium leak from ryanodine receptors (RyRs) on neuronal firing and cognitive performance was examined in aged macaques. RESULTS: Aged monkeys naturally develop hyperphosphorylated tau, including AD biomarkers (AT8 (pS202/pT205) and pT217) and early tau pathology markers (pS214 and pS356) that correlated with evidence of increased calcium leak (pS2808-RyR2). Calcium also regulated early tau phosphorylation in vitro. Age-related reductions in the calcium-binding protein, calbindin, and in phosphodiesterase PDE4D were seen within dlPFC pyramidal cell dendrites. Blocking RyRs with S107 improved neuronal firing and cognitive performance in aged macaques. DISCUSSION: Dysregulated calcium signaling confers risk for tau pathology and provides a potential therapeutic target.

Slc20a1/Pit1 and Slc20a2/Pit2 are essential for normal skeletal myofiber function and survival.

Low blood phosphate (Pi) reduces muscle function in hypophosphatemic disorders. Which Pi transporters are required and whether hormonal changes due to hypophosphatemia contribute to muscle function is unknown. To address these questions we generated a series of conditional knockout mice lacking one or both house-keeping Pi transporters Pit1 and Pit2 in skeletal muscle (sm), using the postnatally expressed human skeletal actin-cre. Simultaneous conditional deletion of both transporters caused skeletal muscle atrophy, resulting in death by postnatal day P13. smPit1-/-, smPit2-/- and three allele mutants are fertile and have normal body weights, suggesting a high degree of redundance for the two transporters in skeletal muscle. However, these mice show a gene-dose dependent reduction in running activity also seen in another hypophosphatemic model (Hyp mice). In contrast to Hyp mice, grip strength is preserved. Further evaluation of the mechanism shows reduced ERK1/2 activation and stimulation of AMP kinase in skeletal muscle from smPit1-/-; smPit2-/- mice consistent with energy-stress. Similarly, C2C12 myoblasts show a reduced oxygen consumption rate mediated by Pi transport-dependent and ERK1/2-dependent metabolic Pi sensing pathways. In conclusion, we here show that Pit1 and Pit2 are essential for normal myofiber function and survival, insights which may improve management of hypophosphatemic myopathy.

Transverse myelitis following measles vaccination in a rhesus macaque (Macaca mulatta).

A 16-year-old rhesus macaque presented with progressive, ascending quadriparesis following measles vaccination. He was diagnosed with transverse myelitis following MRI, gross necropsy, and histopathology. This is the first report of transverse myelitis in a rhesus macaque following measles vaccination.

Doxorubicin-Induced Cardiotoxicity in Collaborative Cross (CC) Mice Recapitulates Individual Cardiotoxicity in Humans.

Anthracyclines cause progressive cardiotoxicity whose ultimate severity is individual to the patient. Genetic determinants contributing to this variation are difficult to study using current mouse models. Our objective was to determine whether a spectrum of anthracycline induced cardiac disease can be elicited across 10 Collaborative Cross mouse strains given the same dose of doxorubicin. Mice from ten distinct strains were given 5 mg/kg of doxorubicin intravenously once weekly for 5 weeks (total 25 mg/kg). Mice were killed at acute or chronic timepoints. Body weight was assessed weekly, followed by terminal complete blood count, pathology and a panel of biomarkers. Linear models were fit to assess effects of treatment, sex, and sex-by-treatment interactions for each timepoint. Impaired growth and cardiac pathology occurred across all strains. Severity of these varied by strain and sex, with greater severity in males. Cardiac troponin I and myosin light chain 3 demonstrated strain- and sex-specific elevations in the acute phase with subsequent decline despite ongoing progression of cardiac disease. Acute phase cardiac troponin I levels predicted the ultimate severity of cardiac pathology poorly, whereas myosin light chain 3 levels predicted the extent of chronic cardiac injury in males. Strain- and sex-dependent renal toxicity was evident. Regenerative anemia manifested during the acute period. We confirm that variable susceptibility to doxorubicin-induced cardiotoxicity observed in humans can be modeled in a panel of CC strains. In addition, we identified a potential predictive biomarker in males. CC strains provide reproducible models to explore mechanisms contributing to individual susceptibility in humans.

Acute Abdominal Distension Due to Disseminated Peritoneal Neoplasia in a Rhesus Macaque (Macaca mulatta).

This report describes the clinical, radiographic, and pathologic findings in a female rhesus macaque that presented with acute abdominal distension and tympany. The macaque was euthanized after evidence of severe colonic distension on radiography and observation of widespread peritoneal adhesions on exploratory laparotomy. Gross and histopathologic evaluation revealed extensive entrapment of gastrointestinal and reproductive tracts by serosal fibrovascular proliferative tissue containing foci of endometriosis. The diagnosis of endometrial stromal sarcoma was supported by expression of CD10, Wilm tumor 1, estrogen receptor, and progesterone receptor and failure to express immunohistochemical markers characteristic of a range of differential diagnoses. In humans, this relatively uncommon neoplasm can arise from sites of endometriosis and often presents clinically as intestinal obstruction, similar to the presentation in this macaque.

Identification of a novel microRNA profile in pediatric patients with cancer treated with anthracycline chemotherapy.

Anthracycline chemotherapy (AC) is associated with decline in left ventricular ejection fraction (LVEF), yet the mechanisms remain unclear. Although changes in microRNAs (miRs) have been identified in adult cardiovascular disease, miR profiles in pediatric patients with AC have not been well studied. The goal of this study was to examine miR profiles (unbiased array) in pediatric patients with AC compared with age-matched referent normal patients. We hypothesize that pediatric patients with AC will express a unique miR profile at the initiation and completion of therapy and will be related to LVEF. Serum was collected in pediatric patients (10-22 yr, n = 12) with newly diagnosed malignancy requiring AC within 24-48 h after the initiation of therapy (30-60 mg/m2) and ~1 yr after completing therapy. A custom microarray of 84 miRs associated with cardiovascular disease was used (quantitative RT-PCR) and indexed to referent normal profiles (13-17 yr, n = 17). LVEF was computed by cardiac MRI. LVEF fell from AC initiation at ~1 yr after AC completion (64.28 ± 1.78% vs. 57.53 ± 0.95%, respectively, P = 0.004). Of the 84 miRs profiled, significant shifts in 17 miRs occurred relative to referent normal ( P ≤ 0.05). Moreover, the functional domain of miRs associated with myocardial differentiation and development fell over threefold at the completion of AC ( P ≤ 0.05). Moreover, eight miRs were significantly downregulated after AC completion in those patients with the greatest decline in LVEF (≥10%, P < 0.05). This study demonstrates, for the first time, that changes in miR expression occur in pediatric patients with AC. These findings suggest that miRs are a potential strategy for the early identification of patients with AC susceptible to left ventricular dysfunction. NEW & NOTEWORTHY Although anthracycline chemotherapy (AC) is effective for a number of pediatric cancers, an all too often consequence of AC is the development of left ventricular failure. The present study identified that specific shifts in the pattern of microRNAs, which regulate myocardial growth, function, and viability, occurred during and after AC in pediatric patients, whereby the magnitude of this shift was associated with the degree of left ventricular failure.

Use of integrated imaging and serum biomarker profiles to identify subclinical dysfunction in pediatric cancer patients treated with anthracyclines.

BACKGROUND: Anthracycline induced cardiomyopathy is a major cause of mortality and morbidity among pediatric cancer survivors. It has been postulated that oxidative stress induction and inflammation may play a role in the pathogenesis of this process. Accordingly, the present study performed an assessment of biomarker profiles and functional imaging parameters focused upon potential early determinants of anthracycline induced cardiomyopathy. METHODS: Patients (10-22 years) were prospectively enrolled between January 2013 and November 2014. Thirteen subjects completed the study and underwent serial cardiac magnetic resonance imaging and plasma biomarker profiling performed 24-48 h after the first anthracycline dose and at set dose intervals. In addition, we collected plasma samples from 62 healthy controls to examine normal plasma biomarker profiles. RESULTS: Left ventricular ejection fraction (LVEF) decreased from 64.3 ± 6.2 at the first visit to 57.5 ± 3.3 (p = 0.004) 1 year after chemotherapy. A decline in longitudinal strain magnitude occurred at lower cumulative doses. A differential inflammatory/matrix signature emerged in anthracycline induced cardiomyopathy patients compared to normal including increased interleukin-8 and MMP levels. With longer periods of anthracycline dosing, MMP-7, a marker of macrophage proteolytic activation, increased by 165 ± 54% whereas interleukin-10 an anti-inflammatory marker decreased by 75 ± 13% (both p < 0.05). MMP7 correlated with time dependent changes in EF. CONCLUSIONS: Asymptomatic pediatric patients exposed to anthracycline therapy develop abnormal strain parameters at lower cumulative doses when compared to changes in EF. A differential biomarker signature containing both inflammatory and matrix domains occur early in anthracycline treatment. Dynamic changes in these domains occur with increased anthracycline doses and progression to anthracycline induced cardiomyopathy. These findings provide potential prognostic and mechanistic insights into the natural history of anthracycline induced cardiomyopathy. TRIAL REGISTRATION NUMBER: NCT03211520 Date of Registration February 13, 2017, retrospectively registered.

Surfactant protein C dampens inflammation by decreasing JAK/STAT activation during lung repair.

Surfactant protein C (SPC), a key component of pulmonary surfactant, also plays a role in regulating inflammation. SPC deficiency in patients and mouse models is associated with increased inflammation and delayed repair, but the key drivers of SPC-regulated inflammation in response to injury are largely unknown. This study focuses on a new mechanism of SPC as an anti-inflammatory molecule using SPC-TK/SPC-KO (surfactant protein C-thymidine kinase/surfactant protein C knockout) mice, which represent a novel sterile injury model that mimics clinical acute respiratory distress syndrome (ARDS). SPC-TK mice express the inducible suicide gene thymidine kinase from by the SPC promoter, which targets alveolar type 2 (AT2) cells for depletion in response to ganciclovir (GCV). We compared GCV-induced injury and repair in SPC-TK mice that have normal endogenous SPC expression with SPC-TK/SPC-KO mice lacking SPC expression. In contrast to SPC-TK mice, SPC-TK/SPC-KO mice treated with GCV exhibited more severe inflammation, resulting in over 90% mortality; there was only 8% mortality of SPC-TK animals. SPC-TK/SPC-KO mice had highly elevated inflammatory cytokines and granulocyte infiltration in the bronchoalveolar lavage (BAL) fluid. Consistent with a proinflammatory phenotype, immunofluorescence revealed increased phosphorylated signal transduction and activation of transcription 3 (pSTAT3), suggesting enhanced Janus kinase (JAK)/STAT activation in inflammatory and AT2 cells of SPC-TK/SPC-KO mice. The level of suppressor of cytokine signaling 3, an anti-inflammatory mediator that decreases pSTAT3 signaling, was significantly decreased in the BAL fluid of SPC-TK/SPC-KO mice. Hyperactivation of pSTAT3 and inflammation were rescued by AZD1480, a JAK1/2 inhibitor. Our findings showing a novel role for SPC in regulating inflammation via JAK/STAT may have clinical applications.

Safety and Efficacy of AAV5 Vectors Expressing Human or Canine CNGB3 in CNGB3-Mutant Dogs.

Achromatopsia is an inherited retinal disorder of cone photoreceptors characterized by markedly reduced visual acuity, extreme light sensitivity, and absence of color discrimination. Approximately 50% of cases are caused by mutations in the cone photoreceptor-specific cyclic nucleotide gated channel beta subunit (CNGB3) gene. Studies in CNGB3-mutant dogs showed that subretinal injection of an AAV vector expressing human CNGB3, which has 76% amino acid identity with canine CNGB3, driven by a 2.1 kb human red cone opsin promoter (PR2.1) and packaged in AAV5 capsids (AAV5-PR2.1-hCNGB3) rescued cone photoreceptor function, but at high doses was associated with an inflammatory response (focal chorioretinitis) consistent with immune-mediated toxicity. AAV vectors containing the PR2.1 promoter packaged in AAV5 capsids and expressing either the native canine CNGB3 (AAV5-PR2.1-cCNGB3) or the human CNGB3 (AAV5-PR2.1-hCNGB3) were evaluated at different dose levels in CNGB3-mutant dogs. The vector expressing canine CNGB3 achieved somewhat better rescue of cone function but unexpectedly was associated with a greater degree of retinal toxicity than the vector expressing human CNGB3. Very low-level T-cell immune responses to some AAV or CNGB3 peptides were observed in animals that received the higher vector dose. There was a more than twofold increase in serum neutralizing antibodies to AAV in one of three animals in the low-dose group and in two of three animals in the high-dose group. No serum anti-hCNGB3 antibodies were detected in any animal. The results of this study do not support the hypothesis that the focal chorioretinitis seen with high doses of AAV5-PR2.1-hCNGB3 in the initial studies was due to an immune response to human CNGB3.

ß-Hydroxybutyrate Deactivates Neutrophil NLRP3 Inflammasome to Relieve Gout Flares.

Aging and lipotoxicity are two major risk factors for gout that are linked by the activation of the NLRP3 inflammasome. Neutrophil-mediated production of interleukin-1ß (IL-1ß) drives gouty flares that cause joint destruction, intense pain, and fever. However, metabolites that impact neutrophil inflammasome remain unknown. Here, we identified that ketogenic diet (KD) increases ß-hydroxybutyrate (BHB) and alleviates urate crystal-induced gout without impairing immune defense against bacterial infection. BHB inhibited NLRP3 inflammasome in S100A9 fibril-primed and urate crystal-activated macrophages, which serve to recruit inflammatory neutrophils in joints. Consistent with reduced gouty flares in rats fed a ketogenic diet, BHB blocked IL-1ß in neutrophils in a NLRP3-dependent manner in mice and humans irrespective of age. Mechanistically, BHB inhibited the NLRP3 inflammasome in neutrophils by reducing priming and assembly steps. Collectively, our studies show that BHB, a known alternate metabolic fuel, is also an anti-inflammatory molecule that may serve as a treatment for gout.

Study of Malformin C, a Fungal Source Cyclic Pentapeptide, as an Anti-Cancer Drug.

Malformin C, a fungal cyclic pentapeptide, has been claimed to have anti-cancer potential, but no in vivo study was available to substantiate this property. Therefore, we conducted in vitro and in vivo experiments to investigate its anti-cancer effects and toxicity. Our studies showed Malformin C inhibited Colon 38 and HCT 116 cell growth dose-dependently with an IC50 of 0.27±0.07µM and 0.18±0.023µM respectively. This inhibition was explicated by Malformin C's effect on G2/M arrest. Moreover, we observed up-regulated expression of phospho-histone H2A.X, p53, cleaved CASPASE 3 and LC3 after Malformin C treatment, while the apoptosis assay indicated an increased population of necrotic and late apoptotic cells. In vivo, the pathological study exhibited the acute toxicity of Malformin C at lethal dosage in BDF1 mice might be caused by an acute yet subtle inflammatory response, consistent with elevated IL-6 in the plasma cytokine assay. Further anti-tumor and toxicity experiments proved that 0.3mg/kg injected weekly was the best therapeutic dosage of Malformin C in Colon 38 xenografted BDF1 mice, whereas 0.1mg/kg every other day showed no effect with higher resistance, and 0.9mg/kg per week either led to fatal toxicity in seven-week old mice or displayed no advantage over 0.3mg/kg group in nine-week old mice. Overall, we conclude that Malformin C arrests Colon 38 cells in G2/M phase and induces multiple forms of cell death through necrosis, apoptosis and autophagy. Malformin C has potent cell growth inhibition activity, but the therapeutic index is too low to be an anti-cancer drug.

Loss of endogenous Nfatc1 reduces the rate of DMBA/TPA-induced skin tumorigenesis.

Immunosuppressive therapies using calcineurin inhibitors, such as cyclosporine A, are associated with a higher incidence of squamous cell carcinoma formation in mice and humans. Calcineurin is believed to suppress tumorigenesis in part through Nfatc1, a transcription factor expressed primarily in hair follicle bulge stem cells in mice. However, mice overexpressing a constitutively active Nfatc1 isoform in the skin epithelium developed increased spontaneous skin squamous cell carcinomas. Because follicular stem cells can contribute to skin tumorigenesis, whether the endogenous expression of Nfatc1 inhibits or enhances skin tumorigenesis is unclear. Here we show that loss of the endogenous expression of Nfatc1 suppresses the rate of DMBA/TPA-induced skin tumorigenesis. Inducible deletion of Nfatc1 in follicular stem cells before tumor initiation significantly reduces the rate of tumorigenesis and the contribution of follicular stem cells to skin tumors. We find that skin tumors from mice lacking Nfatc1 display reduced Hras codon 61 mutations. Furthermore, Nfatc1 enhances the expression of genes involved in DMBA metabolism and increases DMBA-induced DNA damage in keratinocytes. Together these data implicate Nfatc1 in the regulation of skin stem cell-initiated tumorigenesis via the regulation of DMBA metabolism.

Expression of the CTCFL Gene during Mouse Embryogenesis Causes Growth Retardation, Postnatal Lethality, and Dysregulation of the Transforming Growth Factor ß Pathway.

CTCFL, a paralog of CTCF, also known as BORIS (brother of regulator of imprinted sites), is a testis-expressed gene whose function is largely unknown. Its product is a cancer testis antigen (CTA), and it is often expressed in tumor cells and also seen in two benign human vascular malformations, juvenile angiofibromas and infantile hemangiomas. To understand the function of Ctcfl, we created tetracycline-inducible Ctcfl transgenic mice. We show that Ctcfl expression during embryogenesis results in growth retardation, eye malformations, multiorgan pathologies, vascular defects, and neonatal death. This phenotype resembles prior mouse models that perturb the transforming growth factor ß (TGFB) pathway. Embryonic stem (ES) cells with the Ctcfl transgene reproduce the phenotype in ES cell-tetraploid chimeras. Transcriptome sequencing of the Ctcfl ES cells revealed 14 genes deregulated by Ctcfl expression. Bioinformatic analysis revealed the TGFB pathway as most affected by embryonic Ctcfl expression. Understanding the consequence of Ctcfl expression in nontesticular cells and elucidating downstream targets of Ctcfl could explain the role of its product as a CTA and its involvement in two, if not more, human vascular malformations.

Clostridium perfringens enterotoxin C-terminal domain labeled to fluorescent dyes for in vivo visualization of micrometastatic chemotherapy-resistant ovarian cancer.

Identification of micrometastatic disease at the time of surgery remains extremely challenging in ovarian cancer patients. We used fluorescence microscopy, an in vivo imaging system and a fluorescence stereo microscope to evaluate fluorescence distribution in Claudin-3- and -4-overexpressing ovarian tumors, floating tumor clumps isolated from ascites and healthy organs. To do so, mice harboring chemotherapy-naïve and chemotherapy-resistant human ovarian cancer xenografts or patient-derived xenografts (PDXs) were treated with the carboxyl-terminal binding domain of the Clostridium perfringens enterotoxin (c-CPE) conjugated to FITC (FITC-c-CPE) or the near-infrared (NIR) fluorescent tag IRDye CW800 (CW800-c-CPE) either intraperitoneally (IP) or intravenously (IV). We found tumor fluorescence to plateau at 30 min after IP injection of both the FITC-c-CPE and the CW800-c-CPE peptides and to be significantly higher than in healthy organs (p < 0.01). After IV injection of CW800-c-CPE, tumor fluorescence plateaued at 6 hr while the most favorable tumor-to-background fluorescence ratio (TBR) was found at 48 hr in both mouse models. Importantly, fluorescent c-CPE was highly sensitive for the in vivo visualization of peritoneal micrometastatic tumor implants and the identification of ovarian tumor spheroids floating in malignant ascites that were otherwise not detectable by conventional visual observation. The use of the fluorescent c-CPE peptide may represent a novel and effective optical approach at the time of primary debulking surgery for the real-time detection of micrometastatic ovarian disease overexpressing the Claudin-3 and -4 receptors or the identification of residual disease at the time of interval debulking surgery after neoadjuvant chemotherapy treatment.

Invasive ductular carcinoma in 2 rhesus macaques (Macaca mulatta).

In the United States, breast cancer is the most common malignancy among women, with an estimated lifetime incidence of approximately 12% in American women. Invasive ductal carcinoma is the most common form of breast cancer in women, accounting for approximately 60% of all breast carcinomas. Prognostic markers are used to assess aggressiveness, invasiveness, and extent of spread of a neoplasm and thus may be correlated with patient survival. Immunohistochemistry is currently widely used for this purpose, with a variety of prognostication markers available. Classic markers for breast cancer in women include estrogen and progesterone receptor steroid hormone proteins and human epidermal growth factor receptor 2. Many additional markers have been used in diagnosis and prognostication, including p53, p63, and E-cadherin and cell proliferation markers such as Ki67. Despite an estimated lifetime incidence of approximately 6.1%, naturally occurring mammary neoplasms in nonhuman primates are uncommonly reported, with only sporadic references over the past 75 y. The majority of reported tumors occur in rhesus macaques, although this prevalence has been suggested to be a consequence of their high frequency of usage in biomedical research. Here we present 2 cases of mammary carcinoma in adult female intact rhesus macaques, with cytology, histopathology, and extensive immunohistochemical analysis. According to current classifications for human breast tumors, both tumors were classified as invasive ductal carcinoma. The prognostic value of immunohistochemical markers in human breast cancer and in reported cases in nonhuman primates is discussed.

Preclinical safety evaluation of a recombinant AAV8 vector for X-linked retinoschisis after intravitreal administration in rabbits.

X-linked retinoschisis (XLRS) is a retinal disease caused by mutations in the gene encoding the protein retinoschisin (RS1) and one of the most common causes of macular degeneration in young men. Currently, no FDA-approved treatments are available for XLRS and a replacement gene therapy could provide a promising strategy. We have developed a novel gene therapy approach for XLRS, based on the administration of AAV8-scRS/IRBPhRS, an adeno-associated viral vector coding the human RS1 protein, via the intravitreal route. On the basis of our prior study in an Rs1-KO mouse, this construct transduces efficiently all the retinal layers, resulting in an RS1 expression similar to that observed in the wild-type and improving retinal structure and function. In support of a clinical trial, we carried out a study to evaluate the ocular safety of intravitreal administration of AAV8-scRS/IRBPhRS into 39 New Zealand White rabbits. Two dose levels of vector, 2e(10) and 2e(11) vector genomes per eye (vg/eye), were tested and ocular inflammation was monitored over a 12-week period by serial ophthalmological and histopathological analysis. A mild ocular inflammatory reaction, consisting mainly of vitreous infiltrates, was observed within 4 weeks from injection, in both 2e(10) and 2e(11) vg/eye groups and was likely driven by the AAV8 capsid. At 12-week follow-up, ophthalmological examination revealed no clinical signs of vitreitis in either of the dose groups. However, while vitreous inflammatory infiltrate was significantly reduced in the 2e(10) vg/eye group at 12 weeks, some rabbits in the higher dose group still showed persistence of inflammatory cells, histologically. In conclusion, intravitreal administration of AAV8-scRS/IRBPhRS into the rabbit eye produces a mild and transient intraocular inflammation that resolves, at a 2e(10) vg/eye dose, within 3 months, and does not cause irreversible tissue damages. These data support the initiation of a clinical trial of intravitreal administration of AAV8-scRS/IRBPhRS in XLRS patients.