In vivo gene delivery to immune cells.

Immune cell therapies are an emerging class of living drugs that rely on the delivery of therapeutic transgenes to enhance, modulate, or restore cell function, such as those that encode for tumor-targeting receptors or replacement proteins. However, many cellular immunotherapies are autologous treatments that are limited by high manufacturing costs, typical vein-to-vein time of 3-4 weeks, and severe immune-related adverse effects. To address these issues, different classes of gene delivery vehicles are being developed to target specific immune cell subsets in vivo to address the limitations of ex vivo manufacturing, modulate therapeutic responses in situ, and reduce on- and off-target toxicity. The success of in vivo gene delivery to immune cells - which is being tested at the preclinical and clinical stages of development for the treatment of cancer, infectious diseases, and autoimmunity - is paramount for the democratization of cellular immunotherapies.

Optogenetic Microwell Array Screening System: A High-Throughput Engineering Platform for Genetically Encoded Fluorescent Indicators.

Genetically encoded fluorescent indicators (GEFIs) are protein-based optogenetic tools that change their fluorescence intensity when binding specific ligands in cells and tissues. GEFI encoding DNA can be expressed in cell subtypes while monitoring cellular physiological responses. However, engineering GEFIs with physiological sensitivity and pharmacological specificity often requires iterative optimization through trial-and-error mutagenesis while assessing their biophysical function in vitro one by one. Here, the vast mutational landscape of proteins constitutes a significant obstacle that slows GEFI development, particularly for sensors that rely on mammalian host systems for testing. To overcome these obstacles, we developed a multiplexed high-throughput engineering platform called the optogenetic microwell array screening system (Opto-MASS) that functionally tests thousands of GEFI variants in parallel in mammalian cells. Opto-MASS represents the next step for engineering optogenetic tools as it can screen large variant libraries orders of magnitude faster than current methods. We showcase this system by testing over 13,000 dopamine and 21,000 opioid sensor variants. We generated a new dopamine sensor, dMASS1, with a >6-fold signal increase to 100 nM dopamine exposure compared to its parent construct. Our new opioid sensor, µMASS1, has a ∼4.6-fold signal increase over its parent scaffold's response to 500 nM DAMGO. Thus, Opto-MASS can rapidly engineer new sensors while significantly shortening the optimization time for new sensors with distinct biophysical properties.

Review of Diet Protocols Following Orthognathic Surgery and Analysis of Postoperative Weight Loss.

INTRODUCTION: Orthognathic surgery is routinely practiced, yet little comparative data exists to evaluate post-orthognathic surgery diet protocols. OBJECTIVE: To evaluate which postoperative diet protocols are recommended and to quantify post-orthognathic surgery weight changes in our institutional cohort. METHODS: An internet search was carried out on Google for "orthognathic surgery diet" and the postoperative diet recommendations from centers worldwide were quantified. Additionally, a retrospective analysis of patients that underwent orthognathic surgery at our institution was performed, and their preoperative and postoperative weights were recorded. RESULTS: The internet search yielded 58 centers that met our inclusion criteria. Most centers were in the United States (n = 37, 63.8%) and were oral and maxillofacial surgeon (OMFS)-led centers (n = 39, 67.2%). Postoperative diets were categorized into 7 distinct protocols, ranging from most to least restrictive-the most popular was liquid diet for 2 to 4 weeks followed by soft diet for 2 to 6 weeks. There were no significant patterns observed across different geographical regions or specialties.In our institution, 135 patients were identified. Overall, there was an average maximum weight loss of 4.1 kg by week 4, followed by a gradual increase in weight. Linear regression analysis showed that patients with greater preoperative body mass index (BMI) lost more weight postoperatively than patients with lower BMI (R2 = 0.25, P < .001). CONCLUSION: There is a significant variability in recommended postoperative diets following orthognathic surgery. Following a moderately restrictive diet at our institution, patients returned to their preoperative weight after approximately 4 months.

Reflections on Nursing Ingenuity During the COVID-19 Pandemic.

INTRODUCTION: This reflections article provides insight toward nursing innovations to reduce the overuse of personal protective equipment while maintaining a safe environment for staff taking care of COVID-19 patients. The secondary aim of this article to capitalize on recent advances in mass electronic communication through social media to encourage nurses across the globe to share their knowledge and expertise during this pandemic. INNOVATIONS: The many innovations that have been implemented fall into 3 categories of reducing unnecessary use of personal protective equipment, promoting staff safety and readiness, and reducing foot traffic. SUMMARY: These strategies are being shared to promote dissemination of innovative nursing interventions that will save lives during the COVID-19 pandemic.

Linking at-risk South African girls to sexual violence and reproductive health services: A mixed-methods assessment of a soccer-based HIV prevention program and pilot SMS campaign.

Grassroot Soccer developed SKILLZ Street-a soccer-based life skills program with a supplementary SMS platform-to support adolescent girls at risk for HIV, violence, and sexual and reproductive health challenges. We conducted a mixed-methods assessment of preliminary outcomes and implementation processes in three primary schools in Soweto, South Africa, from August to December 2013. Quantitative methods included participant attendance and SMS platform usage tracking, pre/post questionnaires, and structured observation. Qualitative data were collected from program participants, parents, teachers, and a social worker during 6 focus group discussions and 4 in-depth interviews. Of 394 participants enrolled, 97% (n = 382) graduated, and 217 unique users accessed the SMS platform. Questionnaires completed by 213 participants (mean age: 11.9, SD: 3.02 years) alongside qualitative findings showed modest improvements in participants' perceptions of power in relationships and gender equity, self-esteem, self-efficacy to avoid unwanted sex, communication with others about HIV and sex, and HIV-related knowledge and stigma. The coach-participant relationship, safe space, and integration of soccer were raised as key intervention components. Implementation challenges were faced around delivery of soccer-based activities. Findings highlight the relevance and importance of programs like SKILLZ Street in addressing challenges facing adolescent girls in South African townships. Recommendations for future programs are provided.

Luteinizing hormone-releasing hormone enhances T cell recovery following allogeneic bone marrow transplantation.

Posttransplant immunodeficiency, specifically a lack of T cell reconstitution, is a major complication of allogeneic bone marrow transplantation. This immunosuppression results in an increase in morbidity and mortality from infections and very likely contributes to relapse. In this study, we demonstrate that sex steroid ablation using leuprolide acetate, a luteinizing hormone-releasing hormone agonist (LHRHa), increases the number of lymphoid and myeloid progenitor cells in the bone marrow and developing thymocytes in the thymus. Although few differences are observed in the peripheral myeloid compartments, the enhanced thymic reconstitution following LHRHa treatment and allogeneic bone marrow transplantation leads to enhanced peripheral T cell recovery, predominantly in the naive T cell compartment. This results in an increase in T cell function in vivo and in vitro. Graft-versus-host-disease is not exacerbated by LHRHa treatment and graft-versus-tumor activity is maintained. Because LHRHa allows for reversible (and temporary) sex steroid ablation, has a strong safety profile, and has been clinically approved for diseases such as prostate and breast cancer, this drug treatment represents a novel therapeutic approach to reversal of thymic atrophy and enhancement of immunity following immunosuppression.

Comparison of knowledge and attitudes toward cancer among African Americans.

BACKGROUND: It has been noted that the African American population in the U.S. bears disproportionately higher cancer morbidity and mortality rates than any racial and ethnic group for most major cancers. Many studies also document that decreased longevity is associated with low educational attainment and other markers of low socioeconomic status (SES), both of which are prevalent in African American communities across the nation. Evidence suggests that this phenomenon may be due to attitudes that reflect a lack of knowledge surrounding facts about cancer awareness and prevention. This study was designed to yield data concerning the general population's attitudes toward cancer, taking into consideration racial and/or socioeconomic differences in the population studied. RESULTS: Two hundred and fifteen subjects participated in the survey, of which 74% (159/215) defined themselves as African-American, 20% were White, and 6% were of other races. While only 38% of the study population was able to identify at least 5 risk factors associated with cancer, a lower proportion of African Americans identified at least 5 risk factors than whites (34% vs. 53%, p = 0.03). In addition, a slightly higher percentage of African Americans (10%) were not aware of the definition of a clinical trial when compared to whites (8%, p > 0.1). Of those aware of the definition of a clinical trial, African Americans were more reluctant to participate in clinical trials, with 53% answering no to participation compared to 15% of whites (p = 0.002). CONCLUSION: When comparing results to a similar study conducted in 1981, a slight increase in cancer knowledge in the African American population was observed. Our results suggest that while knowledge of cancer facts has increased over the years amongst the general population, African Americans and lower income populations are still behind. This may affect their risk profile and cancer early detection.

Phase II evaluation of interferon-alpha-2a for progressive or recurrent craniopharyngiomas.

OBJECT: Craniopharyngiomas originate from the same cells as squamous cell skin carcinoma, which can be treated successfully with interferon-alpha (IFNalpha)-2a. The authors evaluated the activity and toxicity of systemic IFN in young patients with craniopharyngiomas. METHODS: Fifteen patients between the ages of 4.2 and 19.8 years who had progressive or recurrent craniopharyngiomas were enrolled in this study. Nine of these patients had never received external-beam radiation therapy. Therapy consisted of 8,000,000 U/m2 IFNalpha-2a administered daily for 16 weeks (induction phase) followed by the same dose three times per week for an additional 32 weeks (maintenance phase). Of the 12 patients who could be evaluated, radiological studies demonstrated a response to treatment in three with predominantly cystic tumors (one minor response, one partial response, and one complete response); one of these patients also showed improvement in visual fields. The size of the cystic component of the tumors often increased temporarily during the first several months of therapy. Three patients met the criteria for progressive disease during therapy. The median time to progression was 25 months. The need for radiation therapy in patients treated with IFN was delayed for 18 to 35 months (median 25 months) in six patients. All patients developed transient flulike symptoms shortly after receiving the first dose of IFN. Other toxicities (predominantly hepatic, neurological, and cutaneous) were seen in nine (60%) of the 15 patients during the first 8 weeks of treatment but resolved after temporary discontinuation and/or dose reduction. CONCLUSIONS: Interferon-alpha-2a is active against some childhood craniopharyngiomas; its toxicity precludes administration of high daily doses, and the optimum dose level and schedule remain to be defined.

Dose-intensive, time-compressed procarbazine, CCNU, vincristine (PCV) with peripheral blood stem cell support and concurrent radiation in patients with newly diagnosed high-grade gliomas.

The dose intensity of the PCV regimen can be doubled using peripheral blood stem cell (PBSC) support. This study sought to determine the feasibility of giving dose-intensive PCV concurrently with radiation therapy. Twelve patients, age 3.2-22.7 years, median 7.5 years, with newly diagnosed high grade gliomas were enrolled. Diagnoses included diffuse intrinsic brainstem gliomas (BSG) (n = 6), glioblastoma (n = 4), anaplastic astrocytoma (n = 2). PBSCs were harvested prior to chemotherapy with G-CSF priming. Chemotherapy consisted of CCNU 130 mg/m2 and vincristine 1.5 mg/m2 on day 0, and procarbazine 150 mg/m2 on days 1-7. PBSCs were reinfused on day 9 of each course. Four courses of chemotherapy were administered every 28 days or when blood counts recovered. The first course was administered the week prior to RT, the second course began on week 3 of RT and the third and fourth course were given after RT. Hematologic toxicity was mild and the majority of courses were given on schedule. Five of six patients with diffuse BSG showed clinical improvement and three showed a radiographic response; however, only one remains alive 12+ months from diagnosis. All four patients with non-brainstem large-volume tumors showed clinical deterioration and radiographic progression during or shortly after RT. MRI scans showed massive edema and enhancement. Median time to radiographic progression was five months. Median overall survival was 11 months. We conclude that dose-intensive, time-compressed PCV given concurrently with large-volume RT appears to result in unacceptable toxicity in patients with large residual tumors.

Dose-intensification of procarbazine, CCNU (lomustine), vincristine (PCV) with peripheral blood stem cell support in young patients with gliomas.

BACKGROUND: The regimen of procarbazine, CCNU, and vincristine is active against gliomas. Previous attempts at dose-intensification have been unsuccessful because of delayed and cumulative myelosuppression. We sought to determine whether peripheral blood stem cell (PBSC) infusions would allow dose-escalation and time compression. PROCEDURE: Eleven patients, age 2.8-35.9 years, with newly diagnosed (n = 10) or recurrent (n = 1) gliomas underwent PBSC harvesting after mobilization with G-CSF. Chemotherapy consisted of CCNU 130 mg/m2 on day 0, vincristine 1.5 mg/m2 on days 0 and 7, and procarbazine 150 mg/m2 on days 1-7. PBSCs were reinfused on day 9 of each course. Four courses of chemotherapy were administered 28 days apart or when counts recovered. Involved field radiation was administered to newly diagnosed high-grade glioma patients following recovery from chemotherapy. RESULTS: Compared to the standard PCV regimen given every 6 weeks, dose intensity received was 1.7- and 1.8-fold greater for CCNU and procarbazine. Chemotherapy was delivered on time in 33/41 (80.5%) courses. Four courses (9.8%) were complicated by absolute neutrophil counts < 200/microL; platelet nadirs < 50,000/microL occurred in two courses (4.9%). Fever with neutropenia complicated three courses. Eight of 9 patients with measurable disease had an objective decrease in tumor size and/or decreased enhancement. Median survival for patients with high-grade gliomas (n = 6) was 13 months. CONCLUSIONS: Dose-intensification of PCV is possible using PBSCs.

Feasibility of sequential high-dose chemotherapy and peripheral blood stem cell support for pediatric central nervous system malignancies.

BACKGROUND: The outlook for many brain tumors remains poor. Increased dose intensity has been correlated with response rate and survival in many solid tumors. PATIENTS AND METHODS: Ten children with recurrent or newly diagnosed brain tumors were treated with four sequential courses of high-dose single agent chemotherapy with peripheral blood stem cell (PBSC) support. PBSC harvesting was undertaken prior to chemotherapy and following the first course of chemotherapy (3.6 g/m2 etoposide). Each course of chemotherapy consisted of a single drug followed 48 hours later by PBSC reinfusion. Three patients were treated on Regimen A: etoposide, carboplatinum 1.95 g/m2, cyclophosphamide 5 g/m2, and thiotepa 300 mg/m2; three patients were treated on Regimen A' with carmustine 600 mg/m2 replacing cyclophosphamide; four patients received Regimen B: etoposide, carboplatinum 1.95g/m2, cyclophosphamide 7 g/m2, and thiotepa 900 mg/m2. RESULTS: No course of chemotherapy was complicated by >14 days of neutropenia. Platelet recovery was more prolonged, particularly in patients who had previously received craniospinal irradiation. Non-hematologic toxicity was severe with three toxic deaths including two patients who developed hemolytic-uremic syndrome and respiratory failure. Two of three patients with primitive neuroectodermal tumors had a partial response; no responses were observed in patients with high-grade gliomas. CONCLUSIONS: Administration of multiple courses of high-dose chemotherapy with PBSC support is feasible in this patient population and successfully mitigates hematologic toxicity. Non-hematologic toxicity becomes prohibitive as chemotherapy doses are escalated.

Decomposition-enhanced spike-triggered averaging: contraction level effects.

Decomposition-enhanced spike-triggered averaging (DE-STA) was applied to the vastus medialis muscle to examine size distributions of surface-detected motor-unit action potentials (S-MUAPs) at various force levels. Using DE-STA, 15-20 S-MUAPs were identified during 5%, 10%, 20%, and 30% of maximum voluntary contraction. Average S-MUAPs showed increase in peak to peak (and negative peak) amplitude with force (In microV): 5% = 37.9 +/- 6.1 (16.6 +/- 2.5), 10% = 44.0 +/- 4.0 (20.4 +/- 1.8), 20% = 80.7 +/- 9.3 (41.3 +/- 4.5), and 30% = 102.5 +/- 10.3 (53.6 +/- 5.0). Test-retest variability of peak to peak (and negative peak amplitude) between repeated trials was 0.10 (0.14), 0.14 (0.14), 0.17 (0.15), and 0.21 (0.20) at 5%, 10%, 20%, and 30% respectively. A relationship was found between the S-MUAP amplitude and force (r2 = 0.78, df = 90, F = 160, P < 0.001). Increase in average S-MUAP amplitude with force suggests that STA performed only at low levels of contraction may result in a biased sampling and small average S-MUAP amplitudes.

Multiple circadian-regulated elements contribute to cycling period gene expression in Drosophila.

A new regulatory element necessary for the correct temporal expression of the period (per) gene was identified by monitoring real-time per expression in living individual flies carrying two different period-luciferase transgenes. luciferase RNA driven from only the per promoter was not sufficient to replicate the normal pattern of per RNA cycling; however, a per-luc fusion RNA driven from a transgene containing additional per sequences cycled identically to endogenous per. The results indicate the existence of at least two circadian-regulated elements--one within the promoter and one within the transcribed portion of the per gene. Phase and amplitude analysis of both per-luc transgenes revealed that normal per expression requires the regulation of these elements at distinct phases and suggests a mechanism by which biological clocks sustain high-amplitude feedback oscillations.

Quantitative analysis of Drosophila period gene transcription in living animals.

To determine the in vivo regulatory pattern of the clock gene period (per), the authors recently developed transgenic Drosophila carrying a luciferase cDNA fused to the promoter region of per. They have now carried out noninvasive, high time-resolution experiments allowing high-throughput monitoring of circadian bioluminescence rhythms in individual living adults for several days. This immediately solved several problems (resulting directly from individual asynchrony within a population) that have accompanied previous biochemical experiments in which groups of animals were sacrificed at each time point. Furthermore, the authors have developed numerical analysis methods for automatically determining rhythmicity associated with bioluminescence records from single flies. This has revealed some features of per gene transcription that were previously unappreciated and provides a general strategy for the analysis of rhythmic time series in the study of molecular rhythms.

Rhythms of Drosophila period gene expression in culture.

The Drosophila clock genes period (per) and timeless (tim) have been studied behaviorally and biochemically, but to date there has been no viable culture system for studying the cell biology of the Drosophila clock. We have cultured pupal ring glands attached to the central nervous system and observed rhythms of period gene expression in the prothoracic gland for 4-7 days. A daily rhythm of Per protein can be entrained by light in culture, even when neural activity is blocked by tetrodotoxin. In cultures maintained for a week in constant darkness, a per-luciferase reporter gene revealed circadian rhythms of bioluminescence. As the first circadian culture system from Drosophila, the prothoracic gland provides unique advantages for investigating the interactions between clock genes and cellular physiology.

Three-year follow-up of bibliotherapy for depression.

This study examined the durability of cognitive bibliotherapy for mild to moderately depressed adults by conducting a 3-year follow-up of participants from a previous study (C. Jamison & F. Scogin, 1995). The Hamilton Rating Scale for Depression, Beck Depression Inventory, and questions relating to participants' perceptions of the program were administered. Results indicated that treatment gains were maintained over the 3-year follow-up period and support the usefulness of cognitive bibliotherapy as an adjunct to traditional treatment modalities in a general adult population.

Negative outcomes: what is the evidence on self-administered treatments?

D.C. Mohr (1995) suggested that high deterioration rates may occur in self-directed treatments. The investigators examined data from 5 studies of self-administered treatment for depression and found in contrast much lower rates (9% vs. 19%) than those cited in Mohr's review. The negative response rates for the self-administered treatments compared favorably with the negative response rates in the therapist-administered treatments provided in the National Institute of Mental Health Treatment of Depression Collaborative Research Program. The findings indicate that it may be the manner in which participants are prepared for self-administered treatment that is critical.

Novel features of drosophila period Transcription revealed by real-time luciferase reporting.

The rapid turnover of luciferase and the sensitive, non-invasive nature of its assay make this reporter gene uniquely situated for temporal gene expression studies. To determine the in vivo regulatory pattern of the Drosophila clock gene period (per), we generated transgenic strains carrying a luciferase cDNA fused to the promoter region of the per gene. This has allowed us to monitor circadian rhythms of bioluminescence from pacemaker cells within the head for several days in individual living adults. These high time-resolution experiments permitted neuronal per transcription and opens the door to vastly simplified experiments in general chronobiology and studies of temporally regulated transcription in a wide range of experimental systems.

The outcome of cognitive bibliotherapy with depressed adults.

The effectiveness of minimal-contact cognitive bibliotherapy was examined with a group of 80 depressed adults who were recruited from the community. Minimal-contact cognitive bibliotherapy was found to be superior to a waiting-list control group. The results were both statistically and clinically significant, and the treatment group maintained their levels of improvement at 3-month follow-up. The results also indicated significant decreases in dysfunctional attitudes and automatic negative thoughts after treatment. It appeared that the treatment also served a psychoeducational function. The interventive and preventive implications of these results are discussed.

Developmental expression of protein C and protein S in the rat.

In order to better understand the expression of the Protein C/Protein S anticoagulant system, we have isolated and characterized cDNAs coding for rat Protein C and Protein S. These cDNAs were used in Northern analysis to determine tissue-specificity and developmental expression patterns for mRNAs coding for Proteins C and S. In rats, Protein C mRNA is expressed almost exclusively in liver with a small amount of expression in kidney, diaphragm, stomach, intestine, uterus and placenta. Protein C mRNA was not expressed in brain, heart, lung, spleen, small intestine, large intestine, ovary, or urinary bladder. In liver, Protein C mRNA is expressed at very low levels at prenatal day 18 and these levels increased to maximal levels by postnatal day 13. The size of the mRNA coding for rat Protein C is approximately 1.9 kb. Rat Protein S mRNA was expressed in all tissues examined: brain, heart, lung, diaphragm, liver, spleen, stomach, small intestine, large intestine, kidney, adrenal ovary, uterus, placenta, and urinary bladder. Interestingly, there were 4 bands hybridizing with the rat protein S cDNA that were evident in many of the tissues examined, corresponding to mRNA sizes of approximately 3.5, 2.6, 1.8, and 0.3 kb. There was a difference in tissue-specificity of each mRNA. The 1.8 kb band is generally the most prominent autoradiographic band in any tissue. From these results, it is evident that the expression of Protein C mRNA is similar to that of other vitamin K-dependent proteins. The expression of Protein S mRNA, however, is surprisingly complex and may include alternative splicing of mRNA to generate the various sizes evident on Northern analysis.