Isolation and Characterization of a Novel Alpha-Hemolytic Streptococcus spp. from the Oral Cavity and Blood of Septicemic Periparturient Immunodeficient Mice.

MISTRG is an immunodeficient mouse strain that expresses multiple human cytokines that support hematopoietic stem cell maintenance and myelopoiesis. While establishing a breeding colony of MISTRG mice in a dedicated barrier room, 6 cases of death or disease occurred in pregnant or postpartum mice. Clinically, this manifested as hunched posture, dyspnea, and 1 case of emaciation with ataxia. Pathologic analysis of 7 mice revealed multisystemic necrosuppurative inflammation variably affecting the uterus and placenta, joints, meninges, inner and middle ears, kidneys, and small intestine. Bacteria cultured from the blood of septic mice were identified with 89% probability by the Vitek 2 identification system as Streptococcus sanguinus with atypical biochemical parameters; the API 20E/NE system fully differentiated the isolates as a novel Streptococcus species. MALDI Biotyper-based mass spectrometry also indicated that the phenotype represented a novel Streptococcus spp. Sequencing revealed that the full-length 16S rRNA gene identity was below 97% with known Streptococcus species, including the 2 closest species Streptococcus acidominimus and Streptococcus azizii. We propose the name Streptococcus murisepticum spp. nov to our novel isolates. All male mice in this colony remained healthy despite their association with diseased female mice. Overall, 19% of the colony carried the novel Streptococcus in their oral cavity, but it could not be detected in feces. The organism was sensitive to amoxicillin, which was administered via drinking water throughout pregnancy and weaning to establish a colony of pathogen-negative future breeders. The colony remained disease-free and culture-negative for Streptococcus murisepticum spp. nov after treatment with amoxicillin. We suspect that oral colonization of MISTRG mice with the novel Streptococcus species and its associated unique pathology in periparturient mice is potentially the principal cause of loss of this strain at several institutions. Therefore, screening the oral cavity for α-hemolytic streptococci followed by targeted antibiotic treatment may be necessary when establishing MISTRG and allied immunodeficient mouse strains.

The Impact of Homelessness on Mortality of Individuals Living in the United States: A Systematic Review of the Literature.

This systematic review assessed peer-reviewed research studies on mortality rates of the homeless population within the United States. Extrapolated data included definitions of homelessness, mortality data sources, findings on mortality rates, and causes of premature mortality. Results demonstrate that individuals experiencing homelessness die earlier than comparison groups not experiencing homelessness. Methodology and findings varied across studies. Subpopulations included veterans, families, youth, and unsheltered. Causes of death varied across subpopulations and changed over time. Top causes of death, predominantly determined by ICD codes, stemmed from neoplasms, heart disease, and substance use disorder. Sources used for mortality data included the National Death Index (NDI), the Social Security Death Index (SSDI), state death occurrence files, and city vital statistics. Important research foci include standardization, subpopulation variations, policy implications, and the influence of mortality risk factors, such as poverty and racism.

A Novel Orthotopic Implantation Technique for Osteosarcoma Produces Spontaneous Metastases and Illustrates Dose-Dependent Efficacy of B7-H3-CAR T Cells.

The outcome for metastatic pediatric osteosarcoma (OS) remains poor. Thus, there is an urgent need to develop novel therapies, and immunotherapy with CAR T cells has the potential to meet this challenge. However, there is a lack of preclinical models that mimic salient features of human disease including reliable development of metastatic disease post orthotopic OS cell injection. To overcome this roadblock, and also enable real-time imaging of metastatic disease, we took advantage of LM7 OS cells expressing firefly luciferase (LM7.ffLuc). LM7.ffLuc were implanted in a collagen mesh into the tibia of mice, and mice reliably developed orthotopic tumors and lung metastases as judged by bioluminescence imaging and histopathological analysis. Intratibial implantation also enabled surgical removal by lower leg amputation and monitoring for metastases development post-surgery. We then used this model to evaluate the antitumor activity of CAR T cells targeting B7-H3, an antigen that is expressed in a broad range of solid tumors including OS. B7-H3-CAR T cells had potent antitumor activity in a dose-dependent manner and inhibited the development of pulmonary metastases resulting in a significant survival advantage. In contrast T cells expressing an inactive B7-H3-CAR had no antitumor activity. Using unmodified LM7 cells also enabled us to demonstrate that B7-H3-CAR T cells traffic to orthotopic tumor sites. Hence, we have developed an orthotopic, spontaneously metastasizing OS model. This model may improve our ability not only to predict the safety and efficacy of current and next generation CAR T cell therapies but also other treatment modalities for metastatic OS.

Morphologic and Immunohistochemical Characterization of Spontaneous Lymphoma/Leukemia in NSG Mice.

The NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ strain (NOD scid gamma, NSG) is a severely immunodeficient inbred laboratory mouse used for preclinical studies because it is amenable to engraftment with human cells. Combining scid and Il2rgnull mutations results in severe immunodeficiency by impairing the maturation, survival, and functionality of interleukin 2-dependent immune cells, including T, B, and natural killer lymphocytes. While NSG mice are reportedly resistant to developing spontaneous lymphomas/leukemias, there are reports of hematopoietic cancers developing. In this study, we characterized the immunophenotype of spontaneous lymphoma/leukemia in 12 NSG mice (20 to 38 weeks old). The mice had a combination of grossly enlarged thymus, spleen, or lymph nodes and variable histologic involvement of the bone marrow and other tissues. All 12 lymphomas were diffusely CD3, TDT, and CD4 positive, and 11 of 12 were also positive for CD8, which together was consistent with precursor T-cell lymphoblastic lymphoma/leukemia (pre-T-LBL). A subset of NSG tissues from all mice and neoplastic lymphocytes from 8 of 12 cases had strong immunoreactivity for retroviral p30 core protein, suggesting an association with a viral infection. These data highlight that NSG mice may develop T-cell lymphoma at low frequency, necessitating the recognition of this spontaneously arising disease when interpreting studies.

Combinatorial screening using orthotopic patient derived xenograft-expanded early phase cultures of osteosarcoma identify novel therapeutic drug combinations.

Lead discovery in osteosarcoma has been hampered by the lack of new agents, limited representative clinical samples and paucity of accurate preclinical models. We developed orthotopic patient-derived xenografts (PDXs) that recapitulated the molecular, cellular and histologic features of primary tumors, and screened PDX-expanded short-term cultures and commercial cell lines of osteosarcoma against focused drug libraries. Osteosarcoma cells were most sensitive to HDAC, proteasome, and combination PI3K/MEK and PI3K/mTOR inhibitors, and least sensitive to PARP, RAF, ERK and MEK inhibitors. Correspondingly, PI3K signaling pathway genes were up-regulated in metastatic tumors compared to primary tumors. In combinatorial screens, as a class, HDAC inhibitors showed additive effects when combined with standard-of-care agents gemcitabine and doxorubicin. This lead discovery strategy afforded a means to perform high-throughput drug screens of tumor cells that accurately recapitulated those from original human tumors, and identified classes of novel and repurposed drugs with activity against osteosarcoma.

CE: Original Research: Understanding the Hospital Experience of Older Adults with Hearing Impairment.

: Background: Older hospitalized adults with hearing impairment are vulnerable to adverse outcomes. These patients are at risk for being labeled confused, experiencing a loss of control, experiencing heightened fear and anxiety, and misunderstanding the plan of care. OBJECTIVE: This qualitative study sought to assess the hospital experience of older adults with hearing impairment in order to formulate suggestions for improving nursing care. METHODS: Open-ended interviews were conducted with eight participants, ages 70 to 95 years, who were identified as having a hearing impairment and were admitted as inpatients to a midwestern medical center. RESULTS: Through data analysis, three common themes emerged: health care communication difficulties, passivity and vulnerability, and frustration with family. CONCLUSIONS: Nurses will benefit from having a deeper understanding of the hospital experience of this vulnerable population. Efforts to address their needs can be accomplished through the following nursing actions: assess, accommodate, educate, empower, and advocate.

Advance Care Planning in an Accountable Care Organization Is Associated with Increased Advanced Directive Documentation and Decreased Costs.

BACKGROUND: Advance care planning (ACP) documents patient wishes and increases awareness of palliative care options. OBJECTIVE: To study the association of outpatient ACP with advanced directive documentation, utilization, and costs of care. DESIGN: This was a case-control study of cases with ACP who died matched 1:1 with controls. We used 12 months of data pre-ACP/prematch and predeath. We compared rates of documentation with logit model regression and conducted a difference-in-difference analysis using generalized linear models for utilization and costs. SETTING/SUBJECTS: Medicare beneficiaries attributed to a large rural-suburban-small metro multisite accountable care organization from January 2013 to April 2016, with cross reference to ACP facilitator logs to find cases. MEASUREMENTS: The presence of advance directive forms was verified by chart review. Cost analysis included all utilization and costs billed to Medicare. RESULTS: We matched 325 cases and 325 controls (51.1% female and 48.9% male, mean age 81). 320/325 (98.5%) ACP versus 243/325 (74.8%) of controls had a Healthcare Power of Attorney (odds ratio [OR] 21.6, 95% CI 8.6-54.1) and 172/325(52.9%) ACP versus 145/325 (44.6%) controls had Practitioner Orders for Life Sustaining Treatment (OR 1.40, 95% CI 1.02-1.90) post-ACP/postmatch. Adjusted results showed ACP cases had fewer inpatient admissions (-0.37 admissions, 95% CI -0.66 to -0.08), and inpatient days (-3.66 days, 95% CI -6.23 to -1.09), with no differences in hospice, hospice days, skilled nursing facility use, home health use, 30-day readmissions, or emergency department visits. Adjusted costs were $9,500 lower in the ACP group (95% CI -$16,207 to -$2,793). CONCLUSIONS: ACP increases documentation and was associated with a reduction in overall costs driven primarily by a reduction in inpatient utilization. Our data set was limited by small numbers of minorities and cancer patients.

Deliberate Practice with Standardized Patient Actors and the Development of Formative Feedback for Advance Care Planning Facilitators.

OBJECTIVE: Multimodal curricular assessment after adding standardized patient (SP) actor-based simulation to an advance care planning (ACP) facilitator training course and development of a formative feedback tool. BACKGROUND: ACP represents a highly valued service requiring more and better trained facilitators. METHODS: Participants were primarily nurses and social workers in a large multisite health system. The course included a precourse video demonstration of ACP, traditional lectures, and four 30-minute simulations with SPs. Knowledge was tested with a multiple choice question (MCQ) test. In addition to standard postcourse/postsimulation evaluations, learners were surveyed pre/post/30-90 days delayed for self-perceived confidence. A linear mixed-effects model was used to analyze changes over time. Trained faculty rated performance in simulations with an observational mini-clinical examination (mini-CEX)-type rating form with a checklist, global competency, and global communication rating. Inter-rater reliability (IRR) was calculated on randomly selected paired ratings. RESULTS: Sixty-seven individuals consented to participate. MCQ scores improved from 83% ± 10% to 92% ± 8% (p < 0.001). Paired learner surveys of self-confidence across six domains were available for 65 pre, 65 post, and 40 delayed with a mean positive change on a 0 to 10 point scale from pre-post (2.32 ± 1.65; p < 0.001) and predelayed (2.34 ± 1.96; p < 0.001) time frames. For the faculty observation ratings of simulation performance, the average raw agreement for critical actions was 82% and IRR was 0.71. CONCLUSIONS: Learner feedback and self-assessment suggest that actor-based simulation contributed to improved confidence in conducting ACP. The mini-CEX observation form is adequate for formative feedback, with further testing needed to make judgments of competence.

Ultrasonic Instrumentation Instruction in Dental Hygiene Programs in the United States.

PURPOSE: The purpose of this study was to determine the extent of ultrasonic scaling instrumentation instruction in dental hygiene programs in the U.S. Currently, there is no publication available defining a consensus of instruction for ultrasonic instrumentation. METHODS: Exempt status was received from the West Virginia University Institutional Review Board. A survey was developed with dental hygiene administrators and faculty, based on assumptions and a list of questions to be answered. The survey was tested for validity and revised after feedback from additional faculty. The instrument was 64 questions divided into demographics, curriculum and equipment. Most questions included a text box for additional comments. An email survey was sent to all directors of accredited dental hygiene programs in the U.S. (n=323). The final possible number of respondents was n=301. Results were collected in aggregate through the Secure Online Environment (SOLE). Results were transferred to an Excel spreadsheet for statistical analysis. RESULTS: After 3 emails, the response rate was 45% (n=136). No significant differences in methods of instruction were found between associate and baccalaureate degree granting programs. Eighty-nine percent of programs introduce hand scaling prior to ultrasonic scaling instruction. Students in 96% of the programs were required to administer pre-procedural mouth rinse intended to reduce the amount of bacteria. The magnetostrictive ultrasonic scaler is widely used in dental hygiene instruction. A variety of inserts/ tips were available although a universal or straight insert/tip was most common. Calculus, not inflammation, was the primary criterion for ultrasonic scaler use. CONCLUSION: The results of this study demonstrate that ultrasonic instrumentation is an integral component of the clinical curriculum and the majority of the dental hygiene programs prescribe to similar teaching methods. Programs could benefit from incorporating current scientific research findings of using site specific inserts to perform periodontal debridement based on thorough biofilm removal measured by resolution of inflammation.

Asparaginase Potentiates Glucocorticoid-Induced Osteonecrosis in a Mouse Model.

Osteonecrosis is a common dose-limiting toxicity of glucocorticoids. Data from clinical trials suggest that other medications can increase the risk of glucocorticoid-induced osteonecrosis. Here we utilized a mouse model to study the effect of asparaginase treatment on dexamethasone-induced osteonecrosis. Mice receiving asparaginase along with dexamethasone had a higher rate of osteonecrosis than those receiving only dexamethasone after 6 weeks of treatment (44% vs. 10%, P = 0.006). Similarly, epiphyseal arteriopathy, which we have shown to be an initiating event for osteonecrosis, was observed in 58% of mice receiving asparaginase and dexamethasone compared to 17% of mice receiving dexamethasone only (P = 0.007). As in the clinic, greater exposure to asparaginase was associated with greater plasma exposure to dexamethasone (P = 0.0001). This model also recapitulated other clinical risk factors for osteonecrosis, including age at start of treatment, and association with the systemic exposure to dexamethasone (P = 0.027) and asparaginase (P = 0.036). We conclude that asparaginase can potentiate the osteonecrotic effect of glucocorticoids.

Initial Evidence for the Reliability and Validity of the Educational Needs Assessment Questionnaire.

BACKGROUND AND PURPOSE: The purpose of this study was to evaluate the reliability and validity of the revised Educational Needs Assessment (ENA) questionnaire, a 32-item questionnaire designed to assess educational needs of nurses. METHODS: Data were obtained from 317 nursing home nurses in central Illinois and underwent testing for psychometric properties. RESULTS: The principal component analysis yielded a 6-factor solution that explained 65.9% of the variance and Cronbach's alpha for each factor was satisfactory. CONCLUSIONS: The revised ENA can be a useful tool to measure and identify in what areas of educational needs nursing home nurses need to develop their skills to help patients more effectively.

Promoting oral health as part of an interprofessional community-based women's health event.

Heart disease is the number one killer of women, and studies have shown connections between cardiovascular and oral health. However, interprofessional community-based participatory initiatives promoting women's oral health have received little research attention. This study evaluated the effectiveness of personalized oral health education (POHE) during a free one-day interprofessional women's health promotion event. The objectives were to 1) assess the participants' knowledge about the connection between oral health and heart disease; 2) disseminate information about oral-systemic linkages; 3) encourage comprehensive dental examinations; and 4) evaluate POHE outcomes. West Virginia University School of Dentistry faculty and students delivered POHE to the participants. These POHE instructors were calibrated with a standardized script regarding periodontal disease, health impact of tobacco, xerostomia-inducing medications, and oral hygiene instruction. Immediately prior to and following each POHE session, all the participants (N=165; 100 percent response rate) completed a number-coded questionnaire. The findings showed that the participants' knowledge of oral-systemic health linkages had increased following the POHE. The respondents received oral health kits and were offered discount vouchers toward the cost of a comprehensive oral examination at the dental school. This replicable model may prove useful to other dental schools in promoting women's oral health.

Diazirine-containing photoactivatable isoprenoid: synthesis and application in studies with isoprenylcysteine carboxyl methyltransferase.

Photoaffinity labeling is a useful technique employed to identify protein-ligand and protein-protein noncovalent interactions. Photolabeling experiments have been particularly informative for probing membrane-bound proteins where structural information is difficult to obtain. The most widely used classes of photoactive functionalities include aryl azides, diazocarbonyls, diazirines, and benzophenones. Diazirines are intrinsically smaller than benzophenones and generate carbenes upon photolysis that react with a broader range of amino acid side chains compared with the benzophenone-derived diradical; this makes diazirines potentially more general photoaffinity-labeling agents. In this article, we describe the development and application of a new isoprenoid analogue containing a diazirine moiety that was prepared in six steps and incorporated into an a-factor-derived peptide produced via solid-phase synthesis. In addition to the diazirine moiety, fluorescein and biotin groups were also incorporated into the peptide to aid in the detection and enrichment of photo-cross-linked products. This multifuctional diazirine-containing peptide was a substrate for Ste14p, the yeast homologue of the potential anticancer target Icmt, with K(m) (6.6 µM) and V(max) (947 pmol min(-1) mg(-1)) values comparable or better than a-factor peptides functionalized with benzophenone-based isoprenoids. Photo-cross-linking experiments demonstrated that the diazirine probe photo-cross-linked to Ste14p with observably higher efficiency than benzophenone-containing a-factor peptides.

Primary epiphyseal arteriopathy in a mouse model of steroid-induced osteonecrosis.

Patients undergoing glucocorticoid therapy for a variety of disorders, including autoimmune diseases and hematological malignancies, are at risk of developing osteonecrosis. Despite extensive research in both patients and animal models, the underlying pathogenesis remains unclear. Proposed inciting mechanisms include intravascular thrombotic occlusion, marrow fat hypertrophy, osteocyte and/or endothelial cell apoptosis, hypercoagulability, and vasoconstriction of specific arteries and arterioles supplying bone. Our laboratory has developed a model of steroid-induced osteonecrosis in BALBcJ mice which reflects clinically relevant exposures to glucocorticoids in which treated mice develop osteonecrosis of the distal femoral epiphysis when administered 4 to 8 mg/L dexamethasone in drinking water for 6 weeks. We identified lesions in arterioles supplying this area, with the mildest occurring in knees without any evidence of osteonecrosis. However, arteriopathy was more common among mice that did versus did not develop osteonecrosis (P < 0.0001); in mice with osteonecrosis, the associated vessels showed transmural necrosis and thickening of the vessel wall progressing to the point of luminal obstruction. In the most severe cases of osteonecrosis, end-stage lesions consisted of fully occluded vessels with marrow and bone necrosis involving the entire epiphysis. We propose that a primary arteriopathy is the initiating event in the genesis of steroid-induced osteonecrosis and provides a basis for future investigation of this disease process.

Substrain-specific differences in survival and osteonecrosis incidence in a mouse model.

We previously reported strain-specific susceptibility to dexamethasone-induced osteonecrosis in mice. Here we report that BALB/cJ and BALB/cAnNHsd mice display substrain-specific differences in dexamethasone-induced adverse effects. As compared with BALB/cJ mice, BALB/cAnNHsd weighed more (16.6 g compared with 13.7 g) at the beginning of dexamethasone administration on postnatal day 28 and fewer died during the dexamethasone regimen (10% compared with 50%). Although the 2 substrains had similar plasma concentrations of dexamethasone, BALB/cJ mice were more susceptible to developing dexamethasone-induced osteonecrosis. A higher dose of dexamethasone (8 mg/L) throughout the treatment period compared with a lower dose (8 mg/L loading dose during week 1 followed by 4 mg/L for the remainder of the treatment period) and earlier start of treatment (postnatal day 24 compared with postnatal day 28) was required to induce osteonecrosis with a similar frequency in BALB/cAnNHsd mice as in BALB/cJ mice. Our results show, for the first time, substrain-specific differences in the development of osteonecrosis in mice.