Molecular Approaches for the Validation of the Baboon as a Nonhuman Primate Model for the Study of Zika Virus Infection.

Nonhuman primates (NHP) are particularly important for modeling infections with viruses that do not naturally replicate in rodent cells. Zika virus (ZIKV) has been responsible for sporadic epidemics, but in 2015 a disseminated outbreak of ZIKV resulted in the World Health Organization declaring it a global health emergency. Since the advent of this last epidemic, several NHP species, including the baboon, have been utilized for modeling and understanding the complications of ZIKV infection in humans; several health issues related to the outcome of infection have not been resolved yet and require further investigation. This study was designed to validate, in baboons, the molecular signatures that have previously been identified in ZIKV-infected humans and macaque models. We performed a comprehensive molecular analysis of baboons during acute ZIKV infection, including flow cytometry, cytokine, immunological, and transcriptomic analyses. We show here that, similar to most human cases, ZIKV infection of male baboons tends to be subclinical, but is associated with a rapid and transient antiviral interferon-based response signature that induces a detectable humoral and cell-mediated immune response. This immunity against the virus protects animals from challenge with a divergent ZIKV strain, as evidenced by undetectable viremia but clear anamnestic responses. These results provide additional support for the use of baboons as an alternative animal model to macaques and validate omic techniques that could help identify the molecular basis of complications associated with ZIKV infections in humans.

Multiplexed Simian Immunodeficiency Virus-Specific Paired RNA-Guided Cas9 Nickases Inactivate Proviral DNA.

Human and simian immunodeficiency virus (HIV and SIV) infections establish lifelong reservoirs of cells harboring an integrated proviral genome. Genome editing CRISPR-associated Cas9 nucleases, combined with SIV-specific guiding RNA (gRNA) molecules, inactivate integrated provirus DNA in vitro and in animal models. We generated RNA-guided Cas9 nucleases (RGNu) and nickases (RGNi) targeting conserved SIV regions with no homology in the human or rhesus macaque genome. Assays in cells cotransfected with SIV provirus and plasmids coding for RGNus identified SIV long terminal repeat (LTR), trans-activation response (TAR) element, and ribosome slip site (RSS) regions as the most effective at virus suppression; RGNi targeting these regions inhibited virus production significantly. Multiplex plasmids that coexpressed these three RGNu (Nu3), or six (three pairs) RGNi (Ni6), were more efficient at virus suppression than any combination of individual RGNu and RGNi plasmids. Both Nu3 and Ni6 plasmids were tested in lymphoid cells chronically infected with SIVmac239, and whole-genome sequencing was used to determine on- and off-target mutations. Treatment with these all-in-one plasmids resulted in similar levels of mutations of viral sequences from the cellular genome; Nu3 induced indels at the 3 SIV-specific sites, whereas for Ni6 indels were present at the LTR and TAR sites. Levels of off-target effects detected by two different algorithms were indistinguishable from background mutations. In summary, we demonstrate that Cas9 nickase in association with gRNA pairs can specifically eliminate parts of the integrated provirus DNA; also, we show that careful design of an all-in-one plasmid coding for 3 gRNAs and Cas9 nuclease inhibits SIV production with undetectable off-target mutations, making these tools a desirable prospect for moving into animal studies. IMPORTANCE Our approach to HIV cure, utilizing the translatable SIV/rhesus macaque model system, aims at provirus inactivation and its removal with the least possible off-target side effects. We developed single molecules that delivered either three truncated SIV-specific gRNAs along with Cas9 nuclease or three pairs of SIV-specific gRNAs (six individual gRNAs) along with Cas9 nickase to enhance efficacy of on-target mutagenesis. Whole-genome sequencing demonstrated effective SIV sequence mutation and inactivation and the absence of demonstrable off-target mutations. These results open the possibility to employ Cas9 variants that introduce single-strand DNA breaks to eliminate integrated proviral DNA.

Local immune responses to tuberculin skin challenge in Mycobacterium bovis BCG-vaccinated baboons: a pilot study of younger and older animals.

Individuals over the age of 65 are highly susceptible to infectious diseases, which account for one-third of deaths in this age group. Vaccines are a primary tool to combat infection, yet they are less effective in the elderly population. While many groups have aimed to address this problem by studying vaccine-induced peripheral blood responses in the elderly, work from our lab and others demonstrate that immune responses to vaccination and infectious challenge may differ between tissue sites and the periphery. In this pilot study, we established an in vivo delayed-type hypersensitivity model of Mycobacterium bovis BCG vaccination and tuberculin skin test in two adult and two aged baboons. Vaccination generates BCG-specific immune cells that are recruited to the skin upon tuberculin challenge. We tested short term recall responses (8 weeks post-vaccination) and long term recall responses (25 weeks post-vaccination) by performing skin punch biopsies around the site of tuberculin injection. In short term recall responses, we found increased oxidation and decreased production of immune proteins in aged baboon skin at the site of TST challenge, in comparison to adult skin. Differences between adult and aged animals normalized in the long term response to tuberculin. In vitro, aged peripheral blood mononuclear cells had increased migration and functional responses to antigen-specific stimulation, suggesting that age-related changes in the tissue in vivo impairs aged immune recall responses to antigenic challenge. These findings highlight the impact of age-associated changes in the local tissue environment in memory recall responses, which may be more broadly applied to the study of other tissues. Moreover, these findings should be considered in future studies aimed at understanding and improving aging immune responses to vaccination and tissue challenge.

Silencing integrated SIV proviral DNA with TAR-specific CRISPR tools.

BACKGROUND: One approach for a functional HIV cure is to prevent transcription from integrated proviral DNA. A critical step in HIV transcription is the Tat protein interaction with the TAR element viral RNA. We tested the strategy of blocking this Tat-TAR interaction in the SIVmac model. METHODS: We designed five CRISPR short guiding RNAs (sgRNAs) targeting the SIVmac TAR element, along with inactive versions of Cas9 (dCas9). These sgRNA constructs were delivered as ribonucleoproteins or plasmid DNA, along with SIV DNA. The constructs were also tested in integrated viral DNA in a cell line chronically infected by SIV. RESULTS: The sgRNAs targeting the coding strand of the TAR element inhibited SIV RNA transcription in association with dCas9-KRAB, but not with dCas9. CONCLUSIONS: Induction of epigenetic modifications may be more effective in inactivating provirus than transcriptional interference and thus may be a better strategy to achieve a functional cure in vivo.

Posttraumatic stress as a contributor to behavioral health outcomes and healthcare utilization in adult survivors of childhood cancer: a report from the Childhood Cancer Survivor Study.

PURPOSE: To examine the association between posttraumatic stress symptoms (PTSS), neurocognitive and psychosocial late-effects, health behaviors, and healthcare utilization in long-term survivors of childhood cancer. METHODS: Participants included individuals (N = 6844; 52.5% female; mean [SD] age at diagnosis = 7.6 [5.8], at follow-up = 34.9 [7.5]) in the Childhood Cancer Survivor Study (CCSS). Follow-up included the Posttraumatic Stress Scale, Brief Symptom Inventory-18, Short-form 36 Health-related quality of life (HRQOL) survey, CCSS Neurocognitive Questionnaire, and questions about sociodemographics, physical health, health behaviors, and healthcare utilization. Modified Poisson regression and multinomial logistic regression models examined associations between posttraumatic stress symptoms (PTSS) and neurocognitive, HRQOL, health behavior, and healthcare outcomes when adjusting for sociodemographics, disease, and treatment. RESULTS: Long-term survivors with PTSS (N = 995, 14.5%) reported more impairment in mental (relative risk [RR] 3.42, 95% confidence interval [CI] 3.05-3.85), and physical (RR = 2.26, CI = 1.96-2.61) HRQOL. PTSS was also associated with increased impairment in task efficiency (RR = 3.09, CI = 2.72-3.51), working memory (RR = 2.55, CI = 2.30-2.83), organization (RR = 2.11, CI = 1.78-2.50), and emotional regulation (RR = 3.67, CI = 3.30-4.09). Survivors with PTSS were significantly more likely to attend cancer-specific health visits in the past 2 years (OR = 1.89, CI = 1.50-2.39), and showed greater likelihood of either high frequency (OR = 1.89, CI = 1.50-2.39) or complete lack of (OR = 1.63, CI = 1.32-2.01) primary care visits compared to survivors without PTSS. CONCLUSIONS: Survivors with PTSS reported significantly more psychosocial and neurocognitive late effects, and were more likely to engage in variable use of healthcare. IMPLICATIONS FOR CANCER SURVIVORS: PTSS is associated with additional challenges for a population vulnerable to adverse late effects. Inclusion of integrative services during follow-up visits may benefit functional outcomes.

Challenges in changing sun protection behaviors in children and adolescents.

Better communication between families, schools, communities, and clinicians is critical for improved skin cancer prevention initiatives for children and adolescents. Contributions from research in this area, as exemplified by the two studies in this special issue, will help shape priorities for future sun protection research and will be useful in generating evidence-based policy to support sun safety for children and reduce their future skin cancer risk.

Cross-sectional comparison of health-span phenotypes in young versus geriatric marmosets.

The development of the marmoset as a translational model for healthspan and lifespan studies relies on the characterization of health parameters in young and geriatric marmosets. This cross-sectional study examined health phenotypes in marmosets for five domains of interest for human health and aging: mobility, cognition, metabolism, homeostasis, and immune function. Geriatric marmosets were found to have significant executive function impairment when compared to young animals. While geriatric animals did not show gross abnormalities in mobility and measures of locomotion, their types of movement were altered from young animals. Geriatric marmosets had alterations in cardiac function, with significantly increased mean arterial pressures; metabolism, with significantly lower VO2 ; and suppressed immune function. Further, this study sought to characterize and describe histopathology for both young and geriatric healthy marmosets. Overall this study provides a characterization of health parameters for young and geriatric marmosets which will greatly enhance future aging and interventional testing in marmosets.

Publisher Correction: Experimental Zika Virus Infection in the Pregnant Common Marmoset Induces Spontaneous Fetal Loss and Neurodevelopmental Abnormalities.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Experimental Zika Virus Infection in the Pregnant Common Marmoset Induces Spontaneous Fetal Loss and Neurodevelopmental Abnormalities.

During its most recent outbreak across the Americas, Zika virus (ZIKV) was surprisingly shown to cause fetal loss and congenital malformations in acutely and chronically infected pregnant women. However, understanding the underlying pathogenesis of ZIKV congenital disease has been hampered by a lack of relevant in vivo experimental models. Here we present a candidate New World monkey model of ZIKV infection in pregnant marmosets that faithfully recapitulates human disease. ZIKV inoculation at the human-equivalent of early gestation caused an asymptomatic seroconversion, induction of type I/II interferon-associated genes and proinflammatory cytokines, and persistent viremia and viruria. Spontaneous pregnancy loss was observed 16-18 days post-infection, with extensive active placental viral replication and fetal neurocellular disorganization similar to that seen in humans. These findings underscore the key role of the placenta as a conduit for fetal infection, and demonstrate the utility of marmosets as a highly relevant model for studying congenital ZIKV disease and pregnancy loss.

Baboon CD8 T cells suppress SIVmac infection in CD4 T cells through contact-dependent production of MIP-1α, MIP-1ß, and RANTES.

Simian immunodeficiency virus (SIV) infection in rhesus macaques is often characterized by high viremia and CD4 T cell depletion. By contrast, SIV infection in African nonhuman primate natural hosts is typically nonpathogenic despite active viral replication. Baboons are abundant in Africa and have a geographical distribution that overlaps with natural hosts, but they do not harbor SIVs. Previous work has demonstrated baboons are resistant to chronic SIV infection and/or disease in vivo but the underlying mechanisms remain unknown. Using in vitro SIVmac infections, we sought to identify SIV restriction factors in baboons by comparing observations to the pathogenic rhesus macaque model. SIVmac replicated in baboon PBMC but had delayed kinetics compared to rhesus PBMC. However, SIVmac replication in baboon and rhesus isolated CD4 cells were similar to the kinetics seen for rhesus PBMC, demonstrating intracellular restriction factors do not play a strong role in baboon inhibition of SIVmac replication. Here, we show CD8 T cells contribute to the innate SIV-suppressive activity seen in naïve baboon PBMC. As one mechanism of restriction, we identified higher production of MIP-1α, MIP-1ß, and RANTES by baboon PBMC. Contact between CD4 and CD8 T cells resulted in maximum production of these chemokines and suppression of viral replication, whereas neutralization of CCR5-binding chemokines in baboon PBMC increased viral loads. Our studies indicate baboon natural restriction of SIVmac replication is largely dependent on CD4-extrinsinc mechanisms mediated, in part, by CD8 T cells.

Experimental Zika Virus Inoculation in a New World Monkey Model Reproduces Key Features of the Human Infection.

A monkey model of Zika virus (ZIKV) infection is urgently needed to better understand transmission and pathogenesis, given its proven association with fetal brain defects in pregnant women and acute neurological illness. Here we experimentally infected 4 male marmosets with ZIKV (prototype 1947 African strain) and monitored them clinically with sampling of various body fluids and tissues for nearly 3 months. We show that the course of acute infection with ZIKV in these New World monkeys resembles the human illness in many respects, including (1) lack of apparent clinical symptoms in most cases, (2) persistence of the virus in body fluids such as semen and saliva for longer periods of time than in serum, and (3) generation of neutralizing antibodies as well as an antiviral immunological host response. Importantly, ZIKV-infected saliva samples (in addition to serum) were found to be infectious, suggesting potential capacity for viral transmission by the oral route. Re-challenge of a previously infected marmoset with a contemporary outbreak strain SPH2015 from Brazil resulted in continued protection against infection, no viral shedding, and boosting of the immune response. Given the key similarities to human infection, a marmoset model of ZIKV infection may be useful for testing of new drugs and vaccines.

A Self-Administered Stress Management Intervention for Hispanic Patients Undergoing Cancer Chemotherapy.

This study evaluated whether a self-administered stress management training (SSMT) could improve quality of life (QOL) and reduce distress among Hispanics receiving chemotherapy across multiple community clinical settings. Participants were randomized to receive SSMT (n = 106) or usual care (UCO) (n = 113). The primary outcome-QOL (SF-36) and secondary outcomes depression (CES-D), and anxiety (STAI) were assessed longitudinally over four chemotherapy cycles. Acculturation (BAS) and patients' intervention adherence were assessed. About 63% of participants reported distress after the initial chemotherapy cycle. Hispanics with lower acculturation reported greater STAI-Trait scores (p = .003). No significant treatment effects on outcomes measures were observed for participants receiving SSMT. SSMT intervention techniques were reported useful and improved mental health scores were observed with patients on a psychotropic agent (p = .04). Hispanics experience an elevated level of distress, yet SSMT did not significantly improve primary outcomes. SSMT may be potentially effective when combined with a psychotropic agent. SSMT enhancing strategies are discussed.

Dicephalous v. diprosopus sharks: record of a two-headed embryo of Galeus atlanticus and review of the literature.

As far as is known, this paper gives the first description of a two-headed shark embryo belonging to an oviparous species, Galeus atlanticus (Carcharhiniformes: Scyliorhinidae). The specimen was detected among 797 embryos intended for cardiovascular studies, which represents a defect incidence of 0·13%. Each head had a mouth, two eyes, a brain, a notochord and five gill openings on each side. The two heads fused behind the gills. On the single body, there were four anticipated dorsal fins, two anterior, right and left and two posterior, right and left. Ventrally, the specimen possessed two pairs of pectoral fins, a pair of pelvic fins and one anal fin. Two adjacent notochords, two neural tubes and two dorsal aortas ran along the body, which bent 180° at its posterior portion. There were two hearts, two oesophaguses, two stomachs, two livers, but a single intestine with a spiral valve. Previous reports of conjoined twins in sharks are scarce and only refer to oviparous and ovoviviparous species. Seven dicephalous sharks reported so far were similar to the specimen described here, namely, with two totally separated heads on one body. Instead, only one case of diprosopus shark has been reported; it had a single body and a single head with partial duplication of the face. Two further cases described in the literature as dicephalous or simply as abnormal sharks should be better regarded as diprosopus, while another three cases, also considered dicephalous, showed a mixture of characteristics of diprosopia and dicephalia.

Increases in NKG2C Expression on T Cells and Higher Levels of Circulating CD8+ B Cells Are Associated with Sterilizing Immunity Provided by a Live Attenuated SIV Vaccine.

Vaccines based on live attenuated viruses are highly effective immunogens in the simian immunodeficiency virus (SIV)/rhesus macaque animal model and offer the possibility of studying correlates of protection against infection with virulent virus. We utilized a tether system for studying, in naive macaques and animals vaccinated with a live-attenuated vaccine, the acute events after challenge with pathogenic SIV. This approach allowed for the frequent sampling of small blood volumes without sedation or restraining of the animals, thus reducing the confounding effect of sampling stress. Before challenge, vaccinated animals presented significantly higher levels of proliferating and activated B cells than naive macaques, which were manifested by high expression of CD8 on B cells. After SIV challenge, the only changes observed in protected vaccinated macaques were significant increases in expression of the NK marker NKG2C on CD4 and CD8 T cells. We also identified that infection of naive macaques with SIV resulted in a transient peak of expression of CD20 on CD8 T cells and a constant rise in the number of B cells expressing CD8. Finally, analysis of a larger cohort of vaccinated animals identified that, even when circulating levels of vaccine virus are below the limit of detection, live attenuated vaccines induce systemic increases of IP-10 and perforin. These studies indicate that components of both the innate and adaptive immune systems of animals inoculated with a live-attenuated SIV vaccine respond to and control infection with virulent virus. Persistence of the vaccine virus in tissues may explain the elevated cytokine and B-cell activation levels. In addition, our report underpins the utility of the tether system for the intensive study of acute immune responses to viral infections.

Simian Immunodeficiency Virus Infection of Chimpanzees (Pan troglodytes) Shares Features of Both Pathogenic and Non-pathogenic Lentiviral Infections.

The virus-host relationship in simian immunodeficiency virus (SIV) infected chimpanzees is thought to be different from that found in other SIV infected African primates. However, studies of captive SIVcpz infected chimpanzees are limited. Previously, the natural SIVcpz infection of one chimpanzee, and the experimental infection of six chimpanzees was reported, with limited follow-up. Here, we present a long-term study of these seven animals, with a retrospective re-examination of the early stages of infection. The only clinical signs consistent with AIDS or AIDS associated disease was thrombocytopenia in two cases, associated with the development of anti-platelet antibodies. However, compared to uninfected and HIV-1 infected animals, SIVcpz infected animals had significantly lower levels of peripheral blood CD4+ T-cells. Despite this, levels of T-cell activation in chronic infection were not significantly elevated. In addition, while plasma levels of ß2 microglobulin, neopterin and soluble TNF-related apoptosis inducing ligand (sTRAIL) were elevated in acute infection, these markers returned to near-normal levels in chronic infection, reminiscent of immune activation patterns in 'natural host' species. Furthermore, plasma soluble CD14 was not elevated in chronic infection. However, examination of the secondary lymphoid environment revealed persistent changes to the lymphoid structure, including follicular hyperplasia in SIVcpz infected animals. In addition, both SIV and HIV-1 infected chimpanzees showed increased levels of deposition of collagen and increased levels of Mx1 expression in the T-cell zones of the lymph node. The outcome of SIVcpz infection of captive chimpanzees therefore shares features of both non-pathogenic and pathogenic lentivirus infections.

Correlates of Sun Safety Practices in a Racially and Ethnically Diverse Sample of Adolescents: Implications for Skin Cancer Prevention Interventions.

To guide skin cancer preventive interventions, this study examined correlates of sun safety behaviors in a racially and ethnically diverse sample of 407 adolescents completing a self-report survey at the time of their pediatric wellness visit. Adolescents regularly practiced few sun safety behaviors, and greater interest in cancer prevention was associated with more sun safety behaviors, ever smoking cigarettes was associated with fewer sun safety behaviors, and nonwhite minority adolescents practiced fewer sun safety behaviors than non-Hispanic whites. Clinical preventive interventions to increase sun safety practices among adolescents of all racial and ethnic backgrounds could be integrated into general cancer prevention education, including combining skin cancer prevention with antismoking counseling.

Reduction of Secondhand Smoke Exposure in the Cars of Children With Cancer.

This study examined whether an intervention designed to reduce secondhand smoke exposure (SHSe) among children being treated for cancer had effects in the specific setting of a motor vehicle. The parents or guardians (n = 71) of children being treated for cancer were randomized to either a behavioral secondhand smoke (SHS) reduction program or a standard care control group. Parental reports of SHSe were collected over the course of 12 months. Younger children were exposed at baseline more than their older counterparts. The greatest initial declines in car exposure were observed among children ≤5 years old in the intervention group compared with same-aged peers in the control group. After the 3-month time point, the control group showed greater reductions in car exposure in comparison with the intervention group. Interventions that teach parents strategies to manage their smoking while driving in their personal vehicles may produce even greater reductions in child exposure and should be developed. Based on the age-specific results reported here, future studies should account for effects of child age and use setting-specific measures of SHS.

Full and home smoking ban adoption after a randomized controlled trial targeting secondhand smoke exposure reduction.

INTRODUCTION: The current study examined home and full (i.e., home plus car) smoking ban adoption as secondary outcomes to a randomized controlled trial targeting reduced secondhand smoke exposure (SHSe) for children under treatment for cancer. METHODS: Families with at least 1 adult smoker who reported SHSe for their children (n = 119) were randomized to control or intervention conditions and followed for 1 year with 5 assessments. Both groups were advised of the negative health outcomes associated with SHSe; the intervention group provided more in-depth counseling from baseline to 3 months. Parents reported on household and car smoking behavior, demographic, psychosocial, and medical/treatment-related information. RESULTS: Regardless of group assignment, there was an increase in home (odds ration [OR] = 1.16, p = .074) and full (OR = 1.37, p = .001) smoking ban adoption across time. Families in the intervention group were more likely to adopt a full ban by 3 months, but this difference was nonsignificant by 12 months. Married parents (OR = 2.33, p = .006) and those with higher self-efficacy for controlling children's SHSe (OR = 1.11, p = .023) were more likely to have a home smoking ban; parents who reported smoking fewer cigarettes were more likely to adopt a home (OR = 1.62, p < .0001) or full (OR = 7.32, p = .038) ban. CONCLUSIONS: Smoking bans are in-line with Healthy People 2020's tobacco objectives and may be more feasible for parents with medically compromised children for immediate SHSe reduction. Furthermore, interventions targeting full smoking bans may be a more effective for comprehensive elimination of SHSe.

Tobacco use and exposure among youth undergoing cancer treatment.

INTRODUCTION: Adolescents with cancer are susceptible to the health consequences associated with secondhand smoke exposure (SHSE) and tobacco use. The present study compared tobacco use, exposure, and risk factors between patients and population peers. METHOD: Self-reported data on tobacco use, SHSE, and tobacco-related risk factors were drawn from a pediatric oncology hospital and the National Youth Tobacco Survey. Conditional logistic regression was used to estimate odds ratios for patients and control subjects. RESULTS: Patients were as likely to have tried tobacco and report home SHSE as control subjects. Patients were more likely to report car SHSE, less likely to report that SHSE is harmful, and less likely to report home smoking bans. DISCUSSION: Patients experienced SHSE, tobacco use, and tobacco-related risk factors at rates greater than or equal to control subjects. These results provide support for consideration of intervention targets, health status, and delivery mechanisms, particularly by health care providers, when developing comprehensive tobacco control strategies.

Tobacco and the pediatric chronic kidney disease population.

Tobacco use and exposure are preventable causes of morbidity and mortality. Whereas the impact of this public health issue is well described in adults with kidney disease, its role in the pediatric chronic kidney disease (CKD) population is largely unknown. This review discusses the prevalence of tobacco use and exposure in children with CKD, updates the reader on how tobacco affects the kidney, and presents intervention strategies relevant to this patient population.