Optimizing Flow Cytometric Analysis of Immune Cells in Samples Requiring Cryopreservation from Tumor-Bearing Mice.

Most shared resource flow cytometry facilities do not permit analysis of radioactive samples. We are investigating low-dose molecular targeted radionuclide therapy (MTRT) as an immunomodulator in combination with in situ tumor vaccines and need to analyze radioactive samples from MTRT-treated mice using flow cytometry. Further, the sudden shutdown of core facilities in response to the COVID-19 pandemic has created an unprecedented work stoppage. In these and other research settings, a robust and reliable means of cryopreservation of immune samples is required. We evaluated different fixation and cryopreservation protocols of disaggregated tumor cells with the aim of identifying a protocol for subsequent flow cytometry of the thawed sample, which most accurately reflects the flow cytometric analysis of the tumor immune microenvironment of a freshly disaggregated and analyzed sample. Cohorts of C57BL/6 mice bearing B78 melanoma tumors were evaluated using dual lymphoid and myeloid immunophenotyping panels involving fixation and cryopreservation at three distinct points during the workflow. Results demonstrate that freezing samples after all staining and fixation are completed most accurately matches the results from noncryopreserved equivalent samples. We observed that cryopreservation of living, unfixed cells introduces a nonuniform alteration to PD1 expression. We confirm the utility of our cryopreservation protocol by comparing tumors treated with in situ tumor vaccines, analyzing both fresh and cryopreserved tumor samples with similar results. Last, we use this cryopreservation protocol with radioactive specimens to demonstrate potentially beneficial effector cell changes to the tumor immune microenvironment following administration of a novel MTRT in a dose- and time-dependent manner.

Mixed-Methods Evaluation of Father Participation in an Adolescent Obesity Prevention Program With Multiple Delivery Methods.

Padres Preparados, Jóvenes Saludables was a Latino family-based obesity prevention intervention implemented from 2017 to 2020 across eight programs in-person only, in a blended format (online/in-person), and online only. The intervention aimed to enhance father parenting skills to improve adolescent diet and activity behaviors. Mothers were encouraged to attend. Factors associated with participation were explored using a mixed-methods, qualitative (focus group/individual interviews by Zoom) and quantitative (process evaluation) design. Eleven focus group and 24 individual interviews were completed after participation with 24 fathers, 27 mothers, and 40 adolescents with responses not sorted by delivery method before analysis. Binomial logistic regression models examined associations between fathers' program completion and predictor variables of delivery characteristics, father demographic characteristics, and family attendance patterns. Parents were married (96% fathers, 76% mothers), had low income, a high school education or less (68% fathers, 81% mothers), and had lived in the United States a mean of 19 years. Parents were motivated to participate to improve health, and to be involved with and improve communication with their child. Common barriers to participation were work and life priorities and programmatic factors including scheduling conflicts and technological issues. Participation was greater for fathers attending sessions in-person compared with online only (OR = 11.6). Fathers were more likely to participate if they attended sessions with family members vs. not attending with family members (OR = 7.2). To maximize participation, findings suggest involving multiple parents/caregivers and adolescents, addressing contextual and programmatic barriers, and promoting benefits of better health and relations with family members.

Fashion fades, Chanel No.†5 remains: Epistemology between Style and Technology.

Perfumes embody a chemical record of style and technology. Blurring the boundary between what counts as natural and artificial in both a material and a perceptual sense, perfumery presents us with a domain of multiple disciplinary identities relevant to social studies: art, craft, and techno-science. Despite its profound impact as a cultural practice, perfume has seldom featured in historical scholarship. The reason for this neglect is its inherently qualitative dimension: perfume cannot be understood via codified representation but requires direct acquaintance with its sensory and material basis. The historical study of perfumery thus necessitates an experimental approach that comes not without challenge. This article looks at contemporary recreations of old perfumes to identify the difficulties involved in the experimental recreation of fragrances as sensory and performative artifacts. We highlight the need for a reconceptualization of methodology for inconcrete objects of study as part of the broader interest in experimental approaches to the humanities.

HRAS-Mutant Cardiomyocyte Model of Multifocal Atrial Tachycardia.

BACKGROUND: Germline HRAS gain-of-function pathogenic variants cause Costello syndrome (CS). During early childhood, 50% of patients develop multifocal atrial tachycardia, a treatment-resistant tachyarrhythmia of unknown pathogenesis. This study investigated how overactive HRAS activity triggers arrhythmogenesis in atrial-like cardiomyocytes (ACMs) derived from human-induced pluripotent stem cells bearing CS-associated HRAS variants. METHODS: HRAS Gly12 mutations were introduced into a human-induced pluripotent stem cells-ACM reporter line. Human-induced pluripotent stem cells were generated from patients with CS exhibiting tachyarrhythmia. Calcium transients and action potentials were assessed in induced pluripotent stem cell-derived ACMs. Automated patch clamping assessed funny currents. HCN inhibitors targeted pacemaker-like activity in mutant ACMs. Transcriptomic data were analyzed via differential gene expression and gene ontology. Immunoblotting evaluated protein expression associated with calcium handling and pacemaker-nodal expression. RESULTS: ACMs harboring HRAS variants displayed higher beating rates compared with healthy controls. The hyperpolarization activated cyclic nucleotide gated potassium channel inhibitor ivabradine and the Nav1.5 blocker flecainide significantly decreased beating rates in mutant ACMs, whereas voltage-gated calcium channel 1.2 blocker verapamil attenuated their irregularity. Electrophysiological assessment revealed an increased number of pacemaker-like cells with elevated funny current densities among mutant ACMs. Mutant ACMs demonstrated elevated gene expression (ie, ISL1, TBX3, TBX18) related to intracellular calcium homeostasis, heart rate, RAS signaling, and induction of pacemaker-nodal-like transcriptional programming. Immunoblotting confirmed increased protein levels for genes of interest and suppressed MAPK (mitogen-activated protein kinase) activity in mutant ACMs. CONCLUSIONS: CS-associated gain-of-function HRASG12 mutations in induced pluripotent stem cells-derived ACMs trigger transcriptional changes associated with enhanced automaticity and arrhythmic activity consistent with multifocal atrial tachycardia. This is the first human-induced pluripotent stem cell model establishing the mechanistic basis for multifocal atrial tachycardia in CS.

The Development and Pilot Evaluation of a Family-Based Education to Strengthen Latinx Adolescent Mental Health in the United States: The Familias Activas Experience.

Adolescent years are a time of joy and can represent a challenge for parents and youth, especially for immigrants to the US who are adjusting to their host country. Programs focusing on family skills and positive youth development (PYD) can contribute to youth wellbeing especially, however, few exist for low-income immigrant families. (1) Background: The major goals of this project are to strengthen both PYD and healthy parenting practices by implementing an evidence-informed program, Familias Activas. A theory of change guided the development of Familias Activas in which three factors: parent training, positive youth development, and youth physical activity sessions (soccer) aimed to improve Latinx youth mental health. Youth participated in weekly soccer practices led by trained soccer coaches while parents/caregivers attended parent education. Both sessions lasted eight weeks. (2) Method: We describe the formative stage of the research project as well as the pilot implementation of the Familias Activas program, which provides critical insights for the development of a PYD program. (3) Results: Evaluation surveys were administered to youth and their parents. Thirty youths and sixteen parents completed the survey. The Kidscreen scale had a mean for most items ranging from 3.6 to 4.2. Participating youth were 11 years old and most affirmed they were Latinx. The feasibility program quality mean was 4.2 indicating an overall positive result for the pilot program.. Implications of PYD programs for Latinx youth are discussed. (4) Conclusions: The current paper presents a model for positively influencing the physical and mental wellbeing of Latinx youth and their parents. The model is culturally responsive in its involvement of both parents and youth in programming.

Short-course neoadjuvant in situ vaccination for murine melanoma.

BACKGROUND: Surgical resection remains an important component of multimodality treatment for most solid tumors. Neoadjuvant immunotherapy has several potential advantages, including in-situ tumor vaccination and pathologic assessment of response in the surgical specimen. We previously described an in-situ tumor vaccination strategy in melanoma using local radiation (RT) and an intratumoral injection of tumor-specific anti-GD2 immunocytokine (IT-IC). Here we tested whether neoadjuvant in-situ tumor vaccination using anti-GD2 immunocytokine and surgical resection, without RT, could generate immunologic memory capable of preventing recurrence or distant metastasis. METHODS: Mice bearing GD2 expressing B78 melanoma tumors were treated with neoadjuvant radiation, IT-IC, or combined RT + IT-IC. Surgical resection was performed following neoadjuvant immunotherapy. Immune infiltrate was assessed in the resection specimens. Mice were rechallenged with either B78 contralateral flank tumors or pulmonary seeding of non-GD2 expressing B16 melanoma metastasis induced experimentally. Rejection of rechallenge in mice treated with the various treatment regimens was considered evidence of immunologic memory. RESULTS: Neoadjuvant IT-IC and surgical resection resulted in increased CD8 T cell infiltration, a higher CD8:regulatory T cell ratio, and immunologic memory against contralateral flank rechallenge. The timing of resection did not significantly impact the development of memory, which was present as early as the day of surgery. There was less local wound toxicity with neoadjuvant IT-IC compared with neoadjuvant RT +IT IC. Neoadjuvant IT-IC and resection resulted in the rejection of B16 lung metastasis in a CD4 T cell dependent manner. CONCLUSIONS: Neoadjuvant IT-IC generates immunologic memory capable of preventing distant metastasis despite limited efficacy against large primary melanoma tumors. By combining neoadjuvant tumor vaccination and surgery, the toxicity of local RT was avoided. These preclinical data support further investigation regarding the use of neoadjuvant IT-IC in patients with melanoma at high risk for occult distant disease.

Mechanism of effective combination radio-immunotherapy against 9464D-GD2, an immunologically cold murine neuroblastoma.

BACKGROUND: Most pediatric cancers are considered immunologically cold with relatively few responding to immune checkpoint inhibition. We recently described an effective combination radio-immunotherapy treatment regimen ( c ombination a daptive- i nnate immunotherapy r egimen (CAIR)) targeting adaptive and innate immunity in 9464D-GD2, an immunologically cold model of neuroblastoma. Here, we characterize the mechanism of CAIR and the role of major histocompatibility complex class I (MHC-I) in the treatment response. METHODS: Mice bearing GD2-expressing 9464D-GD2 tumors were treated with CAIR (external beam radiotherapy, hu14.18-IL2 immunocytokine, CpG, anti-CD40, and anti-CTLA4) and tumor growth and survival were tracked. Depletion of specific immune cell lineages, as well as testing in immunodeficient R2G2 mice, were used to determine the populations necessary for treatment efficacy. Induction of MHC-I expression in 9464D-GD2 cells in response to interferon-γ (IFN-γ) and CAIR was measured in vitro and in vivo, respectively, by flow cytometry and quantitative real-time PCR. A cell line with IFN-γ-inducible MHC-I expression (9464D-GD2-I) was generated by transfecting a subclone of the parental cell line capable of expressing MHC-I with GD2 synthase and was used in vivo to assess the impact of MHC-I expression on responsiveness to CAIR. RESULTS: CAIR cures some mice bearing small (50 mm3) but not larger (100 mm3) 9464D-GD2 tumors and these cured mice develop weak memory responses against tumor rechallenge. Early suppression of 9464D-GD2 tumors by CAIR does not require T or natural killer (NK) cells, but eventual tumor cures are NK cell dependent. Unlike the parental 9464D cell line, 9464D-GD2 cells have uniformly very low MHC-I expression at baseline and fail to upregulate expression in response to IFN-γ. In contrast, 9464D-GD2-I upregulates MHC-I in response to IFN-γ and is less responsive to CAIR. CONCLUSION: Treatment with CAIR cures 9464D-GD2 tumors in a NK cell dependent manner and induction of MHC-I by tumors cells was associated with decreased efficacy. These results demonstrate that the early tumor response to this regimen is T and NK cell independent, but that NK cells have a role in generating lasting cures in the absence of MHC-I expression by tumor cells. Further strategies to better inhibit tumor outgrowth in this setting may require further NK activation or the ability to engage alternative immune effector cells.

Greenhouse Rearing Methods for Brown Marmorated Stink Bug (Hemiptera: Pentatomidae) on Live Cowpea Plants.

Brown marmorated stink bug (BMSB), Halyomorpha halys (Stål) (Hemiptera: Pentatomidae), is a pest of concern that must be controlled for market access of host material and regulated articles to certain countries. This work outlines a rearing system for BMSB on live cowpea plants, Vigna unguiculata (L.) Walp. (Fabales: Fabaceae), including methods to induce adults to both enter and exit diapause. This scalable system affords continuous access to >600 specimens per week of each life stage and/or age group, which is particularly advantageous when developing treatment efficacy data.

Identification of the transgene insertion site for an adipocyte-specific adiponectin-cre model and characterization of the functional consequences.

The Adipoq-Cre transgenic mouse is widely used in the development of adipocyte-specific genetic manipulations for the study of obesity and type 2 diabetes. In the process of developing a new mouse model utilizing the adipocyte selective Adipoq-Cre transgenic mouse, strong genetic linkage between a gene of interest, Adam10, and the Adipoq-Cre transgene was discovered. Whole-genome sequencing of the Adipoq-Cre transgenic mouse model identified the genomic insertion site within the Tbx18 gene locus on chromosome 9 and this insertion causes a significant decrease in Tbx18 gene expression in adipose tissue. Insertion of genes Kng2, Kng1, Eif4a2 and Rfc4 also occurred in the Adipoq-Cre transgenic mouse, and these passenger genes may have functional consequences in various tissues.

Depth of tumor implantation affects response to in situ vaccination in a syngeneic murine melanoma model.

An important component of research using animal models is ensuring rigor and reproducibility. This study was prompted after two experimenters performing virtually identical studies obtained different results when syngeneic B78 murine melanoma cells were implanted into the skin overlying the flank and treated with an in situ vaccine (ISV) immunotherapy. Although both experimenters thought they were using identical technique, we determined that one was implanting the tumors intradermally (ID) and the other was implanting them subcutaneously (SC). Though the baseline in vivo immunogenicity of tumors can depend on depth of their implantation, the response to immunotherapy as a function of tumor depth, particularly in immunologically 'cold' tumors, has not been well studied. The goal of this study was to evaluate the difference in growth kinetics and response to immunotherapy between identically sized melanoma tumors following ID versus SC implantation. We injected C57BL/6 mice with syngeneic B78 melanoma cells either ID or SC in the flank. When tumors reached 190-230 mm3, they were grouped into a 'wave' and treated with our previously published ISV regimen (12 Gy local external beam radiation and intratumoral hu14.18-IL2 immunocytokine). Physical examination demonstrated that ID-implanted tumors were mobile on palpation, while SC-implanted tumors became fixed to the underlying fascia. Histologic examination identified a critical fascial layer, the panniculus carnosus, which separated ID and SC tumors. SC tumors reached the target tumor volume significantly faster compared with ID tumors. Most ID tumors exhibited either partial or complete response to this immunotherapy, whereas most SC tumors did not. Further, the 'mobile' or 'fixed' phenotype of tumors predicted response to therapy, regardless of intended implantation depth. These findings were then extended to additional immunotherapy regimens in four separate tumor models. These data indicate that the physical 'fixed' versus 'mobile' characterization of the tumors may be one simple method of ensuring homogeneity among implanted tumors prior to initiation of treatment. Overall, this short report demonstrates that small differences in depth of tumor implantation can translate to differences in response to immunotherapy, and proposes a simple physical examination technique to ensure consistent tumor depth when conducting implantable tumor immunotherapy experiments.

Comparison of skin dose measurement using nanoDot® dosimeter and machine readings of radiation dose during cardiac catheterization in children.

OBJECTIVES: Direct measurement of skin dose of radiation for children using optically stimulated luminescence (OSL) technology using nanoDot® (Landauer, Glenwood, IL, USA). BACKGROUND: Radiation dose is estimated as cumulative air kerma (AK) and dosearea product based on standards established for adult size patients. Body size of pediatric patients who undergo cardiac catheterization for congenital heart disease vary widely from newborn to adolescence. Direct, skindose measurement applying OSL technology may eliminate errors in the estimate. MATERIALS AND METHODS: The nanoDot® (1 cm × 1 cm × flat plastic cassette) is applied to patient's skin using adhesive tape during cardiac catheterization and radiation skin doses were read within 24 hrs. nanoDot® values were compared to the currently available cumulative AK values estimated and displayed on fluoroscopy monitor. RESULTS: A total of 12 children were studied, aged 4 months to 18 years (median 1.1 years) and weight range 5.3-86 kg (median 8.4 kg). nanoDot® readings ranged from 2.58 mGy to 424.8 mGy (median 84.1 mGy). Cumulative AK ranged from 16.2 mGy to 571.2 mGy (median 171.1 mGy). Linear correlation was noted between nanoDot® values and AK values (R2 = 0.88, R = 0.94). nanoDot® readings were approximately 65% of the estimated cumulative AK estimated using the International Electrotechnical Commission standards. CONCLUSIONS: Application of OSL technology using nanoDot® provides an alternative to directly measure fluoroscopic skin dose in children during cardiac catheterization. Our data show that the actual skin dose for children is approximately one-third lower than the AK estimated using international standards for adult size patients.

Approximate-model closed-loop minimal sampling method for HIV viral-load minima detection.

We present a closed-loop method for determining the minimum of a viral rebound curve for an HIV patient undergoing a therapy transition. This method fits the parameters for a reduced approximate solution to the viral load measurements using a Simulated Annealing direct search algorithm. Gaussian white noise is added, and a family of fits is obtained. A safety tolerance measure is applied to the family of fits to obtain the next sample time. Using parameters identified from HIV data, we show that this method exhibits robust performance on noisy data generated from identified patient models, while greatly reducing the number of samples needed compared to a fixed-interval sampling approach.

Determination of average LET of therapeutic proton beams using Al2O3:C optically stimulated luminescence (OSL) detectors.

In this work we present a methodology and proof of concept to experimentally determine average linear energy transfer (LET) of therapeutic proton beams using the optically stimulated luminescence (OSL) of small Al(2)O(3):C detectors. Our methodology is based on the fact that the shape of the OSL decay curve of Al(2)O(3):C detectors depends on the LET of the radiation field. Thus, one can use the shape of the OSL decay curves to establish an LET calibration curve, which in turn permits measurements of LET. We performed irradiations at the M D Anderson Cancer Center Proton Therapy Center, Houston (PTCH), with passive scattering beams. We determined the average LET of the passive scattering beams using a validated Monte Carlo model of the PTCH passive scattering nozzle and correlated them with the shape of the OSL decay curve to obtain an LET calibration curve. Using this calibration curve and OSL measurements, we determined the averaged LET at various water-equivalent depths for therapeutic spread-out Bragg peaks and compared the results with averaged LETs determined using the Monte Carlo simulations. Agreement between measured and simulated fluence-averaged LET was within 24% for low energy spread-out Bragg peak (SOBP) fields and within 14% for high energy SOBP fields. Agreement between measured and simulated dose-averaged LET was within 12% for low energy SOBP fields and within 47% for high energy SOBP fields. The data presented in this work demonstrated the correlation between the OSL decay curve shapes and the average LET of the radiation fields, providing proof of concept of the feasibility of using OSL from Al(2)O(3):C detectors to measure average LET of therapeutic proton beams.