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1.  Zika virus infection in pregnant rhesus macaques causes placental dysfunction and immunopathology 
Nature Communications  2018;9:263.
Zika virus (ZIKV) infection during pregnancy leads to an increased risk of fetal growth restriction and fetal central nervous system malformations, which are outcomes broadly referred to as the Congenital Zika Syndrome (CZS). Here we infect pregnant rhesus macaques and investigate the impact of persistent ZIKV infection on uteroplacental pathology, blood flow, and fetal growth and development. Despite seemingly normal fetal growth and persistent fetal-placenta-maternal infection, advanced non-invasive in vivo imaging studies reveal dramatic effects on placental oxygen reserve accompanied by significantly decreased oxygen permeability of the placental villi. The observation of abnormal oxygen transport within the placenta appears to be a consequence of uterine vasculitis and placental villous damage in ZIKV cases. In addition, we demonstrate a robust maternal-placental-fetal inflammatory response following ZIKV infection. This animal model reveals a potential relationship between ZIKV infection and uteroplacental pathology that appears to affect oxygen delivery to the fetus during development.
Zika virus infection during pregnancy can result in birth defects, but underlying pathogenesis at the maternal-fetal interface is unclear. Here, the authors use non-invasive in vivo imaging of Zika-infected rhesus macaques and show that infection results in abnormal oxygen transport across the placenta.
doi:10.1038/s41467-017-02499-9
PMCID: PMC5772047  PMID: 29343712
2.  HLA-DR Genotyping and Mitochondrial DNA Analysis Reveal the Presence of Family Burials in a Fourth Century Romano-British Christian Cemetery 
Frontiers in Genetics  2017;8:182.
In Colchester, Britain's oldest recorded town, during the Roman period there were areas which were clearly used solely as cemeteries. One of the most significant is at Butt Road, which includes a late Roman probable Christian cemetery with an associated building, apparently a church, that overlies and developed from a pagan inhumation cemetery. DNA was extracted from the long bones (femurs) of 29 individuals, mostly from a large complex of burials centered on two timber vaults. These were thought to comprise a number of family groupings, deduced from osteological analysis, stratigraphical and other considerations. The use of a modified version of the silica-based purification method recovered nanogram quantities of DNA/gram of bone. Two-stage amplification, incorporating primer-extension preamplification-polymerase chain reaction, permitted simultaneous amplification of both mitochondrial and nuclear DNA. Sequence-specific oligonucleotide probes yielded human leukocyte antigen (HLA)-DR typing of seven samples, with four revealing the infrequent HLA-DR10 genotype. Examination of the control region of mitochondrial DNA (mtDNA) by direct sequencing revealed polymorphisms yet to be reported in the modern population. HLA-DRB typing and mtDNA analysis affirmatively supported kinship among some, if not all, individuals in the “vault complex” and demonstrate a continental European origin of the individuals investigated.
doi:10.3389/fgene.2017.00182
PMCID: PMC5723391
extramural fourth century Romano-British cemetery; family groupings; HLA-DR; mtDNA; Colchester (Camulodunum)
3.  Therapeutic administration of a recombinant human monoclonal antibody reduces the severity of chikungunya virus disease in rhesus macaques 
PLoS Neglected Tropical Diseases  2017;11(6):e0005637.
Chikungunya virus (CHIKV) is a mosquito-borne virus that causes a febrile syndrome in humans associated with acute and chronic debilitating joint and muscle pain. Currently no licensed vaccines or therapeutics are available to prevent or treat CHIKV infections. We recently isolated a panel of potently neutralizing human monoclonal antibodies (mAbs), one (4N12) of which exhibited prophylactic and post-exposure therapeutic activity against CHIKV in immunocompromised mice. Here, we describe the development of an engineered CHIKV mAb, designated SVIR001, that has similar antigen binding and neutralization profiles to its parent, 4N12. Because therapeutic administration of SVIR001 in immunocompetent mice significantly reduced viral load in joint tissues, we evaluated its efficacy in a rhesus macaque model of CHIKV infection. Rhesus macaques that were treated after infection with SVIR001 showed rapid elimination of viremia and less severe joint infiltration and disease compared to animals treated with SVIR002, an isotype control mAb. SVIR001 reduced viral burden at the site of infection and at distant sites and also diminished the numbers of activated innate immune cells and levels of pro-inflammatory cytokines and chemokines. SVIR001 therapy; however, did not substantively reduce the induction of CHIKV-specific B or T cell responses. Collectively, these results show promising therapeutic activity of a human anti-CHIKV mAb in rhesus macaques and provide proof-of-principle for its possible use in humans to treat active CHIKV infections.
Author summary
Chikungunya virus (CHIKV) causes fever, rash, and acute and chronic arthralgia. Currently there are no approved therapies to treat or vaccines to prevent CHIKV infection in humans. In this study, we engineered SVIR001, a recombinant fully human monoclonal antibody (mAb) that eliminated viremia, reduced viral load at the site of infection, and diminished spread to distant target tissues in rhesus macaques when administered after infection. SVIR001 treatment reduced joint inflammation and disease without impairing the induction of the adaptive immune response. These results demonstrate the efficacy of mAb therapy to reduce the severity of CHIKV disease.
doi:10.1371/journal.pntd.0005637
PMCID: PMC5491320  PMID: 28628616
4.  Human Cytomegalovirus Induces Cellular and Humoral Virus-specific Immune Responses in Humanized BLT Mice 
Scientific Reports  2017;7:937.
The strict species specificity of Human Cytomegalovirus (HCMV) has impeded our understanding of antiviral adaptive immune responses in the context of a human immune system. We have previously shown that HCMV infection of human hematopoietic progenitor cells engrafted in immune deficient mice (huNSG) results in viral latency that can be reactivated following G-CSF treatment. In this study, we characterized the functional human adaptive immune responses in HCMV latently-infected huBLT (humanized Bone marrow-Liver-Thymus) mice. Following infection, huBLT mice generate human effector and central memory CD4+ and CD8+ T-cell responses reactive to peptides corresponding to both IE and pp65 proteins. Additionally, both HCMV specific IgM and IgG B-cell responses with the ability to neutralize virus were detected. These results indicate that the HCMV huBLT mouse model may provide a valuable tool to study viral latency and reactivation as well as evaluate HCMV vaccines and immune responses in the context of a functional human immune system.
doi:10.1038/s41598-017-01051-5
PMCID: PMC5430540  PMID: 28428537
6.  Zika Virus infection of rhesus macaques leads to viral persistence in multiple tissues 
PLoS Pathogens  2017;13(3):e1006219.
Zika virus (ZIKV), an emerging flavivirus, has recently spread explosively through the Western hemisphere. In addition to symptoms including fever, rash, arthralgia, and conjunctivitis, ZIKV infection of pregnant women can cause microcephaly and other developmental abnormalities in the fetus. We report herein the results of ZIKV infection of adult rhesus macaques. Following subcutaneous infection, animals developed transient plasma viremia and viruria from 1–7 days post infection (dpi) that was accompanied by the development of a rash, fever and conjunctivitis. Animals produced a robust adaptive immune response to ZIKV, although systemic cytokine response was minimal. At 7 dpi, virus was detected in peripheral nervous tissue, multiple lymphoid tissues, joints, and the uterus of the necropsied animals. Notably, viral RNA persisted in neuronal, lymphoid and joint/muscle tissues and the male and female reproductive tissues through 28 to 35 dpi. The tropism and persistence of ZIKV in the peripheral nerves and reproductive tract may provide a mechanism of subsequent neuropathogenesis and sexual transmission.
Author summary
Although it was first identified almost 70 years ago, Zika virus had rarely been associated with pathology in humans until the 21st century. Recent outbreaks in the South Pacific and the Americas have been characterized by numerous confirmed cases, some involving neurologic sequelae and, of most concern, birth defects following infection of pregnant women. Here, we present the results of experimental infection of adult rhesus macaques with a strain of Zika virus isolated during the recent epidemic in the Western hemisphere. Following infection, Zika virus was detected in the sera and urine of all infected animals. Further, we detected virus in multiple tissues of infected animals as late as 35 days post infection, indicating viral persistence. The apparent tropism of the virus for tissues of the peripheral nervous system as well as the reproductive tracts of males and females has implications for the further characterization of the mechanism(s) of Zika virus pathogenesis. Additionally, this model provides a platform for development and testing of preventative or therapeutic interventions to combat the emergence of this virus.
doi:10.1371/journal.ppat.1006219
PMCID: PMC5344528  PMID: 28278237
7.  Improving National Data Systems for Surveillance of Suicide-related Events 
American journal of preventive medicine  2014;47(3 Suppl 2):S122-S129.
Background
Describing the characteristics and patterns of suicidal behavior is an essential component in developing successful prevention efforts. The Data and Surveillance Task Force (DSTF) of the National Action Alliance for Suicide Prevention was charged with making recommendations for improving national data systems for public health surveillance of suicide-related problems, including suicidal thoughts, suicide attempts and deaths due to suicide. Data from the national systems can be used to draw attention to the magnitude of the problem and are useful for establishing national health priorities. National data can also be used to examine differences in rates across groups (e.g., sex, racial/ethnic, and age groups) and geographic regions, and are useful in identifying patterns in the mechanism of suicide, including those that rarely occur.
Methods
Using evaluation criteria from the Centers for Disease Control and Prevention, the World Health Organization, and the U.S.-based Safe States Alliance, the DSTF reviewed 28 national data systems for feasibility of use in the surveillance of suicidal behavior, including deaths, non-fatal attempts and suicidal thoughts. The review criteria included such attributes as the aspects of the suicide-related spectrum (e.g., thoughts, attempts, deaths) covered by the system, how the data are collected (e.g., census, sample, survey, administrative data files, self-report, reporting by care providers), and the strengths and limitations of the survey or data system.
Results
The DSTF identified common strengths and challenges among the data systems based on the underlying data source (e.g., death records, health care provider records, population-based surveys, health insurance claims). From these findings, the DSTF proposed several recommendations for improving existing data systems, such as using standard language and definitions, adding new variables to existing surveys, expanding the geographic scope of surveys to include areas where data are not currently collected, oversampling of underrepresented groups, and improving the completeness and quality of information on death certificates.
Conclusions
Some of the DSTF recommendations are potentially achievable in the short term (<1–3 years) within existing data systems, while others involve more extensive changes and will require longer term efforts (4–10 years). Implementing these recommendations would assist in the development of a national coordinated program of fatal and nonfatal suicide surveillance to facilitate evidence-based action to reduce the incidence of suicide and suicidal behavior in all populations.
doi:10.1016/j.amepre.2014.05.026
PMCID: PMC4959537  PMID: 25145729
8.  Factors Predictive of Corneal Graft Survival in the Cornea Donor Study 
JAMA ophthalmology  2015;133(3):246-254.
Importance
The Cornea Donor Study (CDS) showed that donor age is not a factor in survival of most penetrating keratoplasties for endothelial disease. Secondary analyses confirm the importance of surgical indication and presence of glaucoma in outcomes at 10 years.
Objective
To assess the relationship between donor and recipient factors and corneal graft survival in the CDS.
Design
Multi-center prospective, double-masked, controlled clinical trial
Setting
105 surgeons from eighty clinical sites enrolled participants and forty-three eye banks provided corneas.
Participants
1090 subjects undergoing corneal transplantation for a moderate risk condition, principally Fuchs’ dystrophy or pseudophakic/aphakic corneal edema (PACE)
Intervention(s) for Clinical Trials or Exposure(s) for observational studies
Corneas from donors <66 years or ≥ 66 years were assigned, double-masked to donor age. Surgery and post-operative care were performed according to surgeons’ usual routines. Subjects were followed for up to twelve years.
Main Outcome Measure(s)
Graft failure defined as a regraft or a cloudy cornea for 3 consecutive months.
Results
The 10-year graft failure rate was higher in cases with PACE than with Fuchs’ dystrophy (37% versus 20%, p < 0.001) and in cases with a history of glaucoma prior to penetrating keratoplasty, particularly with prior glaucoma surgery (58% with prior glaucoma surgery and medications at time of surgery versus 22% with no history of glaucoma, p<0.001). There were trends towards increased graft failure in recipients who were older (p=0.04), African-American (p=0.11), or had a smoking history (p=0.02). Lower endothelial cell density (ECD) and higher corneal thickness (CT) at 6 months, 1 year, and 5 years were associated with subsequent graft failure (p=0.04 to <0.001).
Conclusions and Relevance
Most penetrating corneal grafts for Fuchs’ dystrophy or PACE remain clear at 10 years. The risk of failure is greater for those with PACE and when there is a history of glaucoma. ECD and CT measurements during the course of post-keratoplasty follow up are associated with risk of failure. However, even with very low ECD and high CT at 5 years, most corneas remain clear at 10 years.
doi:10.1001/jamaophthalmol.2014.3923
PMCID: PMC4394864  PMID: 25322173
10.  Fluorescence-Based Laser Capture Microscopy Technology Facilitates Identification of Critical In Vivo Cytomegalovirus Transcriptional Programs 
Summary
Cytomegalovirus gene expression in highly permissive, cultured fibroblasts occurs in three kinetic classes known as immediate early, early and late. Infection of these cells results in a predictable transcriptional program leading to high levels of virus production. Infection of other, so called, “non-permissive cell types” results in a transcriptional program that either fails to produce virus particles or production is substantially reduced compared to fibroblasts. We have found that CMV gene expression profiles in tissues from infected hosts differ greatly from those observed in infected tissue culture cells. The number of viral genes expressed in tissues is much more limited, and the number of highly active genes does not correlate with viral DNA load. Additionally, viral gene expression in vivo is tissue selective with no two tissues expressing the exact same viral gene profile. Thus, in vivo CMV gene expression appears to be governed by mechanisms that are still uncharacterized. Cytomegalovirus remains in a persistent phase for the lifetime of the host. During this phase only a limited number of host cells are infected, and it is very difficult to detect CMV gene expression in whole tissues without sub-fractionating infected vs. uninfected cells. Herein, we describe the development of a fluorescence-based laser capture microscopy technique coupled with small sample size microarray analysis to determine the viral gene expression in 50–100 infected cells isolated from frozen RCMV-infected tissue sections.
doi:10.1007/978-1-62703-788-4_13
PMCID: PMC4347879  PMID: 24639226
Laser capture microscopy; cytomegalovirus; green fluorescence protein; microarray analysis
11.  Chikungunya Viruses That Escape Monoclonal Antibody Therapy Are Clinically Attenuated, Stable, and Not Purified in Mosquitoes 
Journal of Virology  2014;88(15):8213-8226.
ABSTRACT
Chikungunya virus (CHIKV) is a reemerging mosquito-transmitted alphavirus that causes epidemics of debilitating polyarthritis in humans. A prior study identified two anti-CHIKV monoclonal antibodies ([MAbs] CHK-152 and CHK-166) against the E2 and E1 structural proteins, which had therapeutic efficacy in immunocompetent and immunocompromised mice. Combination MAb therapy was required as administration of a single MAb resulted in the rapid selection of neutralization escape variants and treatment failure in mice. Here, we initially evaluated the efficacy of combination MAb therapy in a nonhuman primate model of CHIKV infection. Treatment of rhesus macaques with CHK-152 and CHK-166 reduced viral spread and infection in distant tissue sites and also neutralized reservoirs of infectious virus. Escape viruses were not detected in the residual viral RNA present in tissues and organs of rhesus macaques. To evaluate the possible significance of MAb resistance, we engineered neutralization escape variant viruses (E1-K61T, E2-D59N, and the double mutant E1-K61T E2-D59N) that conferred resistance to CHK-152 and CHK-166 and tested them for fitness in mosquito cells, mammalian cells, mice, and Aedes albopictus mosquitoes. In both cell culture and mosquitoes, the mutant viruses grew equivalently and did not revert to wild-type (WT) sequence. All escape variants showed evidence of mild clinical attenuation, with decreased musculoskeletal disease at early times after infection in WT mice and a prolonged survival time in immunocompromised Ifnar1−/− mice. Unexpectedly, this was not associated with decreased infectivity, and consensus sequencing from tissues revealed no evidence of reversion or compensatory mutations. Competition studies with CHIKV WT also revealed no fitness compromise of the double mutant (E1-K61T E2-D59N) neutralization escape variant in WT mice. Collectively, our study suggests that neutralization escape viruses selected during combination MAb therapy with CHK-152 plus CHK-166 retain fitness, cause less severe clinical disease, and likely would not be purified during the enzootic cycle.
IMPORTANCE Chikungunya virus (CHIKV) causes explosive epidemics of acute and chronic arthritis in humans in Africa, the Indian subcontinent, and Southeast Asia and recently has spread to the New World. As there are no approved vaccines or therapies for human use, the possibility of CHIKV-induced debilitating disease is high in many parts of the world. To this end, our laboratory recently generated a combination monoclonal antibody therapy that aborted lethal and arthritogenic disease in wild-type and immunocompromised mice when administered as a single dose several days after infection. In this study, we show the efficacy of the antibody combination in nonhuman primates and also evaluate the significance of possible neutralization escape mutations in mosquito and mammalian cells, mice, and Aedes albopictus vector mosquitoes. Our experiments show that escape viruses from combination antibody therapy cause less severe CHIKV clinical disease, retain fitness, and likely would not be purified by mosquito vectors.
doi:10.1128/JVI.01032-14
PMCID: PMC4135940  PMID: 24829346
12.  Human Cytomegalovirus-Encoded pUL7 Is a Novel CEACAM1-Like Molecule Responsible for Promotion of Angiogenesis 
mBio  2014;5(6):e02035-14.
ABSTRACT
Persistent human cytomegalovirus (HCMV) infection has been linked to several diseases, including atherosclerosis, transplant vascular sclerosis (TVS), restenosis, and glioblastoma. We have previously shown that factors secreted from HCMV-infected cells induce angiogenesis and that this process is due, at least in part, to increased secretion of interleukin-6 (IL-6). In order to identify the HCMV gene(s) responsible for angiogenesis promotion, we constructed a large panel of replication-competent HCMV recombinants. One HCMV recombinant deleted for UL1 to UL10 was unable to induce secretion of factors necessary for angiogenesis. Fine mapping using additional HCMV recombinants identified UL7 as a viral gene required for production of angiogenic factors from HCMV-infected cells. Transient expression of pUL7 induced phosphorylation of STAT3 and ERK1/2 MAP kinases and production of proangiogenic factors, including IL-6. Addition of recombinant pUL7 to cells was sufficient for angiogenesis and was again associated with increased IL-6 expression. Analysis of the UL7 structure revealed a conserved domain similar to the immunoglobulin superfamily domain and related to the N-terminal V-like domain of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). Our report therefore identifies UL7 as a novel HCMV-encoded molecule that is both structurally and functionally related to cellular CEACAM1, a proangiogenic factor highly expressed during vasculogenesis.
IMPORTANCE
A hallmark of cytomegalovirus (CMV) infection is its ability to modulate the host cellular machinery, resulting in the secretion of factors associated with long-term diseases such as vascular disorders and cancer. We previously demonstrated that HCMV infection alters the types and quantities of bioactive proteins released from cells (designated the HCMV secretome) that are involved in the promotion of angiogenesis and wound healing. A key proangiogenic and antiapoptotic factor identified from a proteomic-based approach was IL-6. In the present report, we show for the first time that HCMV UL7 encodes a soluble molecule that is a structural and functional homologue of the CEACAM1 proangiogenic cellular factor. This report thereby identifies a critical component of the HCMV secretome that may be responsible, at least in part, for the vascular dysregulation associated with persistent HCMV infection.
doi:10.1128/mBio.02035-14
PMCID: PMC4217178  PMID: 25352622
13.  High-Dosage Ascorbic Acid Treatment in Charcot-Marie-Tooth Disease Type 1A: Results of a Randomized, Double-Masked, Controlled Trial 
JAMA neurology  2013;70(8):981-987.
Importance
No current medications improve neuropathy in subjects with Charcot-Marie-Tooth disease type 1A (CMT1A). Ascorbic acid (AA) treatment improved the neuropathy of a transgenic mouse model of CMT1A and is a potential therapy. A lower dosage (1.5 g/d) did not cause improvement in humans. It is unknown whether a higher dosage would prove more effective.
Objective
To determine whether 4-g/d AA improves the neuropathy of subjects with CMT1A.
Design
A futility design to determine whether AA was unable to reduce worsening on the CMT Neuropathy Score (CMTNS) by at least 50% over a 2-year period relative to a natural history control group.
Setting
Three referral centers with peripheral nerve clinics (Wayne State University, Johns Hopkins University, and University of Rochester).
Participants
One hundred seventy-four subjects with CMT1A were assessed for eligibility; 48 did not meet eligibility criteria and 16 declined to participate. The remaining 110 subjects, aged 13 to 70 years, were randomly assigned in a double-masked fashion with 4:1 allocation to oral AA (87 subjects) or matching placebo (23 subjects). Sixty-nine subjects from the treatment group and 16 from the placebo group completed the study. Two subjects from the treatment group and 1 from the placebo group withdrew because of adverse effects.
Interventions
Oral AA (4 g/d) or matching placebo.
Main Outcomes and Measures
Change from baseline to year 2 in the CMTNS, a validated composite impairment score for CMT.
Results
The mean 2-year change in the CMTNS was −0.21 for the AA group and −0.92 for the placebo group, both better than natural history (+1.33). This was well below 50% reduction of CMTNS worsening from natural history, so futility could not be declared (P > .99).
Conclusions and Relevance
Both treated patients and those receiving placebo performed better than natural history. It seems unlikely that our results support undertaking a larger trial of 4-g/d AA treatment in subjects with CMT1A.
Trial Registration
clinicaltrials.gov Identifier: NCT00484510
doi:10.1001/jamaneurol.2013.3178
PMCID: PMC3752369  PMID: 23797954
15.  Wolf Motor Function Test for Characterizing Moderate to Severe Hemiparesis in Stroke Patients 
Objective
To extend the applicability of the Wolf Motor Function test (WMFT) to describe the residual functional abilities of moderate-to-severely affected stroke patients. The WMFT is a motor function test for mild to moderate upper extremity weakness in stroke patients, but it has not been routinely used for evaluation of more severe hemiparetic stroke patients due to its numerical characteristics.
Design
Data was collected as part of two double-blind sham controlled randomized interventional studies, the Transcranial Direct Current Stimulation (tDCS) in Chronic Stroke Recovery and tDCS Enhanced Stroke Recovery and Cortical Reorganization. Stroke patients were evaluated with the upper-extremity Fugl-Meyer (UFM) and the WMFT in the same setting prior to treatment.
Setting
University inpatient rehabilitation and outpatient clinic.
Participants
32 stroke patients with moderate-to-severe hemiparesis enrolled in the tDCS in Chronic Stroke Recovery and tDCS Enhanced Stroke Recovery and Cortical Reorganization studies.
Intervention
Not applicable.
Main Outcome Measures
WMFT scores were calculated using 1) median performance times, 2) new calculation using the mean rate of performance. We compared the distribution of values from the two methods and examined the WMFT-UFM correlation for the traditional and the new calculation.
Results
WMFT rate values were more evenly distributed across their range than median WMFT time scores. The association between the WMFT rate and UFM was as good as the association between the median WMFT time scores and UFM (Spearman rs 0.84 vs −0.79).
Conclusions
The new WMFT mean rate of performance is valid and a more sensitive measure in describing the functional activities of the moderate to severely affected upper extremity of stroke subjects and avoids the pitfalls of the median WMFT time calculations.
doi:10.1016/j.apmr.2012.05.002
PMCID: PMC3483403  PMID: 22579647
Hemiparesis; Rehabilitation; Assessment; Stroke
16.  It’s Not That Simple: Tobacco Use Identification and Documentation in Acute Care 
This environmental telephone interview scan was designed to identify: (1) how hospitals in one Canadian province incorporated tobacco use identification/documentation systems into practice; and, (2) challenges/issues with tobacco identification/documentation. Participants included 36/139 hospitals previously identified to offer cessation services. Results showed hospitals aided by researchers monitored and tracked tobacco use; those not aligned with researchers did not. The wording of tobacco items most commonly included use within the last 6-months (42%), 30-days (39%), or 7-days (33%), or use without reference to time (e.g., “Do you smoke?”; 39%); wording sometimes depended on admitting form space limitations. The admission process determined where the tobacco item appeared, which differed by hospital—75% included it on an admitting form (75%) and/or nursing assessment (56%); the item sometimes varied by unit. There were also different processes by which the item triggered delivery of cessation interventions; most frequently (69%), staff nurses were triggered to provide an intervention. The findings suggest that adding a tobacco use question to a hospital’s admitting process is potentially not that simple. Deciding on the purpose of the question, when it will be asked and by whom, space allotted on the form, and how it will trigger an intervention are important considerations that can affect the question wording, form/location, systems required, data extraction, and resources.
doi:10.3390/ijerph10052069
PMCID: PMC3709365  PMID: 23698699
tobacco use and dependence guideline; tobacco use identification; tobacco use documentation
17.  Cytomegalovirus Latency Promotes Cardiac Lymphoid Neogenesis and Accelerated Allograft Rejection in CMV Naïve Recipients 
Human Cytomegalovirus (HCMV) infection is associated with the acceleration of transplant vascular sclerosis (TVS) and chronic allograft rejection (CR). HCMV-negative recipients of latently HCMV infected donor grafts are at highest risk for developing CMV-disease. Using a rat heart transplant CR model, we have previously shown that acute rat CMV (RCMV) infection following transplantation significantly accelerates both TVS and CR. Here, we report that RCMV-naïve recipients of heart allografts from latently RCMV-infected donors undergo acceleration of CR with similar kinetics as acutely infected recipients. In contrast to acutely infected recipients, treatment of recipients of latently infected donor hearts with ganciclovir did not prevent CR or TVS. We observed the formation of tertiary lymphoid structures (TLOs) containing macrophages and T-cells in latently infected hearts prior to transplantation but not in uninfected rats. Moreover, pathway analysis of gene expression data from allografts from latently infected donors, indicated an early and sustained production of TLO-associated genes compared to allografts from uninfected donors. We conclude that RCMV-induced TLO formation and alteration of donor tissue T-cell profiles prior to transplantation in part mediate the ganciclovir-insensitive rejection of latently infected donor allografts transplanted into naïve recipients by providing a scaffold for immune activation.
doi:10.1111/j.1600-6143.2010.03365.x
PMCID: PMC3454525  PMID: 21199347
Cytomegalovirus; Chronic Rejection; Transplant Vascular Sclerosis; Latency
18.  Physicians’ attitudes and behaviour toward screening mammography in women 40 to 49 years of age 
Canadian Family Physician  2012;58(9):e508-e513.
Abstract
Objective
To determine family physicians’ attitudes and behaviour toward screening mammography, breast self-examination, and breast awareness in women aged 40 to 49 at average risk of breast cancer.
Design
Cross-sectional survey.
Setting
Women’s College Hospital and Sunnybrook Health Sciences Centre, both in Toronto, Ont.
Participants
Family medicine residents, fellows, and staff physicians at 2 academic family practice health centres affiliated with the University of Toronto (n = 95).
Main outcome measures
Physicians’ answers to questions about offering screening mammography and promoting breast self-examination and breast awareness.
Results
Fifty-two completed surveys were returned (response rate 55%). Less than half of all surveyed family physicians (46%) routinely offered screening mammography to women aged 40 to 49 who were at average risk of breast cancer. Although 40% of physicians did not think breast cancer screening was necessary for women aged 40 to 49, 62% indicated that they would offer screening if their patients requested it. Physicians’ reasons not to offer screening included no evidence of decreasing breast cancer deaths (63%), grade A recommendation to screen women starting at age 50 and not at age 40 (25%), and the harms of screening outweighing the benefits (19%). Physicians’ reasons to offer screening included patient request (55%), personal clinical practice experience or mentors’ recommendations (27%), and guideline recommendations (18%). Breast self-examination was not recommended by most physicians (74%), yet most encouraged women to practise breast awareness (81%).
Conclusion
Many women at average risk of breast cancer are not being offered the opportunity to discuss and initiate mammographic screening before 50 years of age. While breast-self examination is not recommended, most physicians promote breast awareness.
PMCID: PMC3440292  PMID: 22972742
19.  Tobacco cessation Clinical Practice Guideline use by rural and urban hospital nurses: a pre-implementation needs assessment 
BMC Nursing  2012;11:6.
Background
This study was a pre-program evaluation of hospital-based nurses' tobacco intervention beliefs, confidence, training, practice, and perceived intervention barriers and facilitators. It was designed to identify relevant information prior to implementing tobacco cessation guidelines across a large northern rural region, home to 1 urban and 12 rural hospitals.
Methods
This cross-sectional survey was distributed by nurse managers to nurses in the 13 hospitals and returned by nurses (N = 269) via mail to the researchers.
Results
Nurses were somewhat confident providing cessation interventions, agreed they should educate patients about tobacco, and 94% perceived tobacco counselling as part of their role. Although only 11% had received cessation training, the majority reported intervening, even if seldom--91% asked about tobacco-use, 96% advised quitting, 89% assessed readiness to quit, 88% assisted with quitting, and 61% arranged post-discharge follow-up. Few performed any of these steps frequently, and among those who intervened, the majority spent < 10 minutes. The most frequently performed activities tended to take the least amount of time, while the more complex activities (e.g., teaching coping skills and pharmacotherapy education) were seldom performed. Patient-related factors (quitting benefits and motivation) encouraged nurses to intervene and work-related factors discouraged them (time and workloads). There were significant rural-urban differences--more rural nurses perceived intervening as part of their role, reported having more systems in place to support cessation, reported higher confidence for intervening, and more frequently assisted patients with quitting and arranged follow-up.
Conclusions
The findings showed nurses' willingness to engage in tobacco interventions. What the majority were doing maps onto the recommended minimum of 1-3 minutes but intervention frequency and follow-up were suboptimal. The rural-urban differences suggest a need for more research to explore the strengths of rural practice which could potentially inform approaches to smoking cessation in urban hospitals.
doi:10.1186/1472-6955-11-6
PMCID: PMC3384473  PMID: 22545579
20.  Investigation of the Efficacy of Micafungin in the Treatment of Histoplasmosis Using Two North American Strains of Histoplasma capsulatum▿ 
Micafungin alone and combined with liposomal amphotericin B was evaluated against two strains of Histoplasma capsulatum. Micafungin was active in vitro against the mold but not the yeast form but was ineffective in vivo. Micafungin appears to be ineffective in treatment of histoplasmosis.
doi:10.1128/AAC.01681-10
PMCID: PMC3165286  PMID: 21670186
21.  Sphingolipid-mediated Inhibition of Apoptotic Cell Clearance by Alveolar Macrophages* 
The Journal of Biological Chemistry  2010;285(51):40322-40332.
A decreased clearance of apoptotic cells (efferocytosis) by alveolar macrophages (AM) may contribute to inflammation in emphysema. The up-regulation of ceramides in response to cigarette smoking (CS) has been linked to AM accumulation and increased detection of apoptotic alveolar epithelial and endothelial cells in lung parenchyma. We hypothesized that ceramides inhibit the AM phagocytosis of apoptotic cells. Release of endogenous ceramides via sphingomyelinase or exogenous ceramide treatments dose-dependently impaired apoptotic Jurkat cell phagocytosis by primary rat or human AM, irrespective of the molecular species of ceramide. Similarly, in vivo augmentation of lung ceramides via intratracheal instillation in rats significantly decreased the engulfment of instilled target apoptotic thymocytes by resident AM. The mechanism of ceramide-induced efferocytosis impairment was dependent on generation of sphingosine via ceramidase. Sphingosine treatment recapitulated the effects of ceramide, dose-dependently inhibiting apoptotic cell clearance. The effect of ceramide on efferocytosis was associated with decreased membrane ruffle formation and attenuated Rac1 plasma membrane recruitment. Constitutively active Rac1 overexpression rescued AM efferocytosis against the effects of ceramide. CS exposure significantly increased AM ceramides and recapitulated the effect of ceramides on Rac1 membrane recruitment in a sphingosine-dependent manner. Importantly, CS profoundly inhibited AM efferocytosis via ceramide-dependent sphingosine production. These results suggest that excessive lung ceramides may amplify lung injury in emphysema by causing both apoptosis of structural cells and inhibition of their clearance by AM.
doi:10.1074/jbc.M110.137604
PMCID: PMC3001012  PMID: 20956540
Apoptosis; Inflammation; Lung; Macrophage; Phagocytosis; Sphingolipid; Cigarette Smoke; Emphysema; Human; Rat
22.  An Insertion Mutation That Distorts Antibody Binding Site Architecture Enhances Function of a Human Antibody 
mBio  2011;2(1):e00345-10.
The structural and functional significance of somatic insertions and deletions in antibody chains is unclear. Here, we demonstrate that a naturally occurring three-amino-acid insertion within the influenza virus-specific human monoclonal antibody 2D1 heavy-chain variable region reconfigures the antibody-combining site and contributes to its high potency against the 1918 and 2009 pandemic H1N1 influenza viruses. The insertion arose through a series of events, including a somatic point mutation in a predicted hot-spot motif, introduction of a new hot-spot motif, a molecular duplication due to polymerase slippage, a deletion due to misalignment, and additional somatic point mutations. Atomic resolution structures of the wild-type antibody and a variant in which the insertion was removed revealed that the three-amino-acid insertion near the base of heavy-chain complementarity-determining region (CDR) H2 resulted in a bulge in that loop. This enlarged CDR H2 loop impinges on adjacent regions, causing distortion of the CDR H1 architecture and its displacement away from the antigen-combining site. Removal of the insertion restores the canonical structure of CDR H1 and CDR H2, but binding, neutralization activity, and in vivo activity were reduced markedly because of steric conflict of CDR H1 with the hemagglutinin antigen.
IMPORTANCE
Antibody diversification through VDJ gene recombination, junctional variation, and somatic hypermutation has clear importance for the generation of mature, high-affinity antibodies. Between 1.3 and 6.5% of antibody variable gene sequences have been reported to contain insertions or deletions, but their structural and functional significance remains less well defined. The pandemic influenza virus hemagglutinin antibody 2D1 data suggest that somatic insertions and deletions in antibody genes contribute important structural and functional features. We predict that such features can be critical for affinity and functional maturation of the human antibody repertoire.
doi:10.1128/mBio.00345-10
PMCID: PMC3037006  PMID: 21304166
23.  Implementation of the Tobacco Tactics Program in the Department of Veterans Affairs 
Journal of General Internal Medicine  2010;25(Suppl 1):3-10.
BACKGROUND
Smoking cessation services in the Department of Veterans Affairs (VA) are currently provided via outpatient groups, while inpatient cessation programs have not been widely implemented.
OBJECTIVE
The objective of this paper is to describe the implementation of the Tobacco Tactics program for inpatients in the VA.
METHODS
This is a pre-/post-non-randomized control study initially designed to teach inpatient staff nurses on general medical units in the Ann Arbor and Detroit VAs to deliver the Tobacco Tactics intervention using Indianapolis as a control group. Coupled with cessation medication sign-off, physicians are reminded to give patients brief advice to quit.
RESULTS
Approximately 96% (210/219) of inpatient nurses in the Ann Arbor, MI site and 57% (159/279) in the Detroit, MI site have been trained, with an additional 282 non-targeted personnel spontaneously attending. Nurses’ self-reported administration of cessation services increased from 57% pre-training to 86% post-training (p = 0.0002). Physician advice to quit smoking ranged between 73–85% in both the pre-intervention and post-intervention period in both the experimental and control group. Volunteers made follow-up telephone calls to 85% (n = 230) of participants in the Ann Arbor site. Hospitalized smokers (N = 294) in the intervention group are reporting an increase in receiving and satisfaction with the selected cessation services following implementation of the program, particularly in regards to medications (p < 0.05).
CONCLUSION
A large proportion of inpatient nursing staff can rapidly be trained to deliver tobacco cessation interventions to inpatients resulting in increased provision of services.
doi:10.1007/s11606-009-1075-9
PMCID: PMC2806966  PMID: 20077145
implementation research; smoking cessation; veterans
24.  Tobacco Use among Emergency Department Patients 
This is the first study to systematically track the tobacco use prevalence in an entire emergency department (ED) population and compare age-stratified rates to the general population using national, provincial, and regional comparisons. A tobacco use question was integrated into the ED electronic registration process from 2007 to 2010 in 11 northern hospitals (10 rural, 1 urban). Results showed that tobacco use documentation (85–89%) and tobacco use (26–27%) were consistent across years with the only discrepancy being higher tobacco prevalence in 2007 (32%) due to higher rates at the urban hospital. Age-stratified outcomes showed that tobacco use remained high up to 50 years old (36%); rates began to decrease for patients in their 50’s (26%) and 60’s (16%), and decreased substantially after age 70 (5%). The age-stratified ED tobacco rates were almost double those of the general population nationally and provincially for all but the oldest age groups but were virtually identical to regional rates. The tobacco use tracking and age-stratified general population comparisons in this study improves on previous attempts to document prevalence in the ED population, and at a more local level, provides a “big picture” overview that highlights the magnitude of the tobacco-use problem in these communities.
doi:10.3390/ijerph8010253
PMCID: PMC3037073  PMID: 21318027
tobacco prevalence; epidemiology; population surveillance; ED tobacco prevalence; hospital tobacco prevalence; tobacco use identification and documentation
25.  HCMV pUS28 initiates pro-migratory signaling via activation of Pyk2 kinase 
Herpesviridae  2010;1:2.
Background
Human Cytomegalovirus (HCMV) has been implicated in the acceleration of vascular disease and chronic allograft rejection. Recently, the virus has been associated with glioblastoma and other tumors. We have previously shown that the HCMV-encoded chemokine receptor pUS28 mediates smooth muscle cell (SMC) and macrophage motility and this activity has been implicated in the acceleration of vascular disease. pUS28 induced SMC migration involves the activation of the protein tyrosine kinases (PTKs) Src and Focal adhesion kinase as well as the small GTPase RhoA. The PTK Pyk2 has been shown to play a role in cellular migration and formation of cancer, especially glioblastoma. The role of Pyk2 in pUS28 signaling and migration are unknown.
Methods
In the current study, we examined the involvement of the PTK Pyk2 in pUS28-induced cellular motility. We utilized in vitro migration of SMC to determine the requirements for Pyk2 in pUS28 pro-migratory signaling. We performed biochemical analysis of Pyk2 signaling in response to pUS28 activation to determine the mechanisms involved in pUS28 migration. We performed mass spectrometric analysis of Pyk2 complexes to identify novel Pyk2 binding partners.
Results
Expression of a mutant form of Pyk2 lacking the autophosphorylation site (Tyr-402) blocks pUS28-mediated SMC migration in response to CCL5, while the kinase-inactive Pyk2 mutant failed to elicit the same negative effect on migration. pUS28 stimulation with CCL5 results in ligand-dependent and calcium-dependent phosphorylation of Pyk2 Tyr-402 and induced the formation of an active Pyk2 kinase complex containing several novel Pyk2 binding proteins. Expression of the autophosphorylation null mutant Pyk2 F402Y did not abrogate the formation of an active Pyk2 kinase complex, but instead prevented pUS28-mediated activation of RhoA. Additionally, pUS28 activated RhoA via Pyk2 in the U373 glioblastoma cells. Interestingly, the Pyk2 kinase complex in U373 contained several proteins known to participate in glioma tumorigenesis.
Conclusions
These findings represent the first demonstration that pUS28 signals through Pyk2 and that this PTK participates in pUS28-mediated cellular motility via activation of RhoA. Furthermore, these results provide a potential mechanistic link between HCMV-pUS28 and glioblastoma cell activation.
doi:10.1186/2042-4280-1-2
PMCID: PMC3050435  PMID: 21429240

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