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While most patients with metastatic prostatic adenocarcinoma respond initially, nearly all develop resistance to multiple lines of androgen deprivation therapies. This is related to overexpression of the androgen receptor (AR), which is frequently driven by AR amplification. In some patients, increased AR renders tumor cells sensitive to high dose androgens which paradoxically inhibits their growth. Bipolar Androgen Therapy (BAT) was introduced in which patients with castration resistant prostate cancer (CRPC) are treated intermittently with high dose testosterone. This produces biochemical and objective responses, and may re-sensitize prostate cancer to subsequent second generation AR inhibitors. This study utilizes samples from a recently performed clinical trial for patients with CRPC that included sequential biopsies of soft tissue metastases before BAT and after 3 cycles of BAT and before patients were treated with nivolumab.Tumor cells were present in FFPE blocks for both the pretreatment and C4D1 time points for 24 of the patients. These FFPE samples were stained using a recently validated iterative multiplex IHC assay containing 6 antibodies including: CD3 (T cells), CD4 (Helper T Cells), CD8 (Cytotoxic T Cells), PD1 (T Cell Checkpoint), FOXP3 (T-Reg cells), and Keratin 8 (epithelial cell marker). Biopsy slides were scanned after each round of staining and whole slide scans were imported into the HALO image analysis where they underwent color deconvolution, image registration and fusion. Regions of tumor were demarcated manually, and computerized automated image analysis was used to determine cell densities for 8 cell phenotypes (total CD3+, CD3+CD4+, CD3+CD8+, CD3+CD4+Foxp3+, CD3+CD4+PD1+, CD3+CD8+PD1+, CD3+CD4+PD1-, CD3+CD8+PD1-). There was a higher density of CD3+CD8+PD1+ cells in the pretreatment biopsies of men who responded (R) (to BAT) as compared with those who did not respond (NR)(p=0.015 Wilcoxon rank-sum test; mean 92.7 [R] vs 13.9 [NR]). The difference in R vs. NR was less and not significant in the CD3+CD8+PD1- population. There was a trend towards a higher density of CD3+CD4+PD1+ cells in the pretreatment samples in responders, but less so in the CD3+CD4+PD1- population. In the C4D1 biopsies, only CD3+ total cells were significantly higher in the R vs. NR (P=0.02), but the other phenotypes were not. We conclude that increased density of CD3+C8+PD1+, but not CD3+CD8+PD1- cells was present in pre-treatment biopsies in responders as compared with non-responders to BAT. These results indicate that men who are likely to respond to high dose testosterone treatment harbor tumors that are immunologically distinct prior to treatment than those men unlikely to respond. Additional spatial analysis and comparisons with RNAseq data will be presented. Citation Format: Carolina Gomes Alexandre, Jessica Hicks, Tracy Jones, John T Isaacs, Kiara Bowers, Alyza Skist, Laura Sena, Jennifer Meyers, Emmanuel Antonarakis, David Sanin, Hanfei Qi, Samuel Denmeade, Mark Markowski, Srinivasan Yegnasubramanian, Angelo De Marzo. Molecular pathology studies reveal PD1+ CD8 T cell density correlates with response to supraphysiological testosterone treatment in pre-treatment biopsies and MYC mRNA and protein correlate with response after treatment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4345.

Background: Neoadjuvant anti-PD-1-based therapies have shown promise in several cancers, including Merkel cell carcinoma (MCC), a rare and aggressive skin cancer. We have shown that on-treatment specimens from patients responding to anti-PD-1 are characterized histologically by exuberant proliferative fibrosis, neovascularization, and robust inflammation including diverse immune cell types. Understanding how the stroma participates in the process of tumor regression may open therapeutic opportunities by redirecting stromal remodeling in treatment-refractory tumors. Methods: To evaluate stromal changes in neoadjuvant treated MCC, 13 on-treatment FFPE tumor specimens from patients with resectable MCC who received anti-PD-1 for ~4 weeks before surgery (NCT02488759) were subjected to laser capture microdissection to collect stromal tissue and isolate RNA. We conducted multiplex qRT-PCR targeting 125 unique mRNAs representing TME-relevant immune cell subsets, immune checkpoint pathways, and immune-modulating transcription factors and cytokines, followed by whole transcriptome RNA sequencing. Differential gene expression was considered significant if the magnitude of the signed fold-change was ≥2 and the p-value was ≤0.1 (Benjamini-Hochberg adjusted for RNAseq). Gene set enrichment analysis (GSEA) was performed with R Topper. Pathologic response was scored on H&E staining as pathologic complete response (pCR, n=6), major pathologic response (MPR, ≤10% residual viable tumor [RVT]; n=4), or non-MPR/pCR (>10% RVT; n=3). Results: Stratifying by response status (6 pCR vs 3 non-MPR/pCR) demonstrated that pCR stroma in MCC is distinguished by upregulation of molecules associated with neovascularization (VEGFA, EGR2) and M1 and M2 macrophages (IL1A, ARG1) via qRT-PCR, and mesenchymal cells (fibroblasts, myocytes, adipocytes) and extracellular matrix formation via RNAseq. GSEA demonstrated a strong association of pCR with both myocyte and fibroblast differentiation and functional pathways, suggesting a role for myofibroblasts in tissue remodeling during anti-PD-1-mediated tumor regression. In contrast, the stroma of non-MPR/pCR tumors showed evidence for ongoing IFNg-mediated inflammation, as evidenced by upregulation of IFNg, IL-27, STAT1, CXCL10 and LAG-3 via qRT-PCR; these findings were supported by RNAseq, which also showed upregulation of genes associated with inflammation (e.g., PLXNC1, TRPV2, LYZ, LAIR1, HLA-A, B2M, CD4). Similar results were obtained when MPRs were grouped with pCRs (n=10) vs non-MPR/pCR (n=3). Conclusion: These findings demonstrate that improved response to anti-PD-1 therapy is associated with a shift in the tumor stroma from an inflammatory to a proliferative myofibroblastic phenotype, recapitulating the stages of normal wound healing. This suggests a potential functional role for myofibroblasts in that critical transition. Citation Format: Joel C. Sunshine, Tracee L. McMiller, Alyza Skaist, Yan Zhang, Alan Berger, Kornel Schuebel, Jennifer Meyers, Julie S. Deutsch, Aleksandra Ogurtsova, Elizabeth L. Engle, Leslie Cope, Janis M. Taube, Suzanne L. Topalian. Characterizing tumor stromal evolution after neoadjuvant anti-PD-1 therapy in Merkel cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2260.

Testosterone is the canonical growth factor of prostate cancer but can paradoxically suppress its growth when present at supraphysiological levels. We have previously demonstrated that the cyclical administration of supraphysiological androgen (SPA), entitled Bipolar Androgen Therapy (BAT), can result in tumor regression and clinical benefit for patients with castration-resistant prostate cancer [1–5]. However, predictors and mechanisms of response and resistance have been ill-defined. Here we show that growth inhibition of prostate cancer models by SPA requires high androgen receptor (AR) abundance and activity and is driven in part by downregulation of MYC. Using matched sequential patient biopsies, we show that high pre-treatment AR activity predicts downregulation of MYC, clinical response, and prolonged progression-free and overall survival for patients on BAT. BAT induced strong downregulation of AR in all patients, which is shown to be a primary mechanism of acquired resistance to SPA. Acquired resistance can be overcome by alternating SPA with the AR inhibitor enzalutamide, which induces adaptive upregulation of AR and re-sensitizes prostate cancer to SPA. This work identifies a predictive biomarker of response to BAT and supports a new treatment paradigm for prostate cancer involving alternating between AR inhibition and activation.

Evaluating the complex interplay of cell types in the tissue microenvironment is critical to understanding the origin and progression of diseases in the prostate and potential opportunities for intervention. Mouse models are an essential tool to investigate the molecular and cell-type-specific contributions of prostate disease at an organismal level. While there are well-documented differences in the extent, timing, and nature of disease development in various genetically engineered and exposure-based mouse models in different mouse strains and prostate lobes within each mouse strain, the underlying molecular phenotypic differences in cell types across mouse strains and prostate lobes are incompletely understood.In this study, we used single-cell RNA-sequencing (scRNA-seq) methods to assess the single-cell transcriptomes of 6-month-old mouse prostates from two commonly used mouse strains, friend virus B/NIH jackson (FVB/NJ) (N = 2) and C57BL/6J (N = 3). For each mouse, the lobes of the prostate were dissected (anterior, dorsal, lateral, and ventral), and individual scRNA-seq libraries were generated. In situ and pathological analyses were used to explore the spatial and anatomical distributions of novel cell types and molecular markers defining these cell types.Data dimensionality reduction and clustering analysis of scRNA-seq data revealed that basal and luminal cells possessed strain-specific transcriptomic differences, with luminal cells also displaying marked lobe-specific differences. Gene set enrichment analysis comparing luminal cells by strain showed enrichment of proto-Oncogene targets in FVB/NJ mice. Additionally, three rare populations of epithelial cells clustered independently of strain and lobe: one population of luminal cells expressing Foxi1 and components of the vacuolar ATPase proton pump (Atp6v0d2 and Atp6v1g3), another population expressing Psca and other stem cell-associated genes (Ly6a/Sca-1, Tacstd2/Trop-2), and a neuroendocrine population expressing Chga, Chgb, and Syp. In contrast, stromal cell clusters, including fibroblasts, smooth muscle cells, endothelial cells, pericytes, and immune cell types, were conserved across strain and lobe, clustering largely by cell type and not by strain or lobe. One notable exception to this was the identification of two distinct fibroblast populations that we term subglandular fibroblasts and interstitial fibroblasts based on their strikingly distinct spatial distribution in the mouse prostate.Altogether, these data provide a practical reference of the transcriptional profiles of mouse prostate from two commonly used mouse strains and across all four prostate lobes.

Molecular analysis of tissue samples from metastatic cancer lesions can improve our understanding of drug response and resistance. Since prostate cancer cells initially require androgen signaling through the androgen receptor (AR) for proliferation, the treatment of metastatic prostate cancer involves surgical or chemical castration. While most patients respond initially, nearly all develop acquired resistance to combined androgen blockade using both first and second line agents. This resistance is often related to overexpression of AR, which is frequently driven by AR gene amplification. In some patients, this increased AR appears to render tumor cells sensitive to high dose androgens in a manner that paradoxically inhibits their growth. Bipolar Androgen Therapy (BAT) was introduced several years ago at our institution in which patients with castration resistant prostate cancer (CRPC) are treated intermittently with high dose testosterone. Prior clinical trials have shown that BAT can produce biochemical and objective responses, and may re-sensitize prostate cancer to subsequent second generation AR inhibitors. To define the mechanism(s) by which BAT results in tumor regression, we performed a clinical trial (NCT03554317) for patients with CRPC that included biopsies of soft tissue metastases before BAT and after 3 cycles of BAT (C4D1). Up to four tissue cores were taken per biopsy with two cores for formalin fixation and paraffin embedding (FFPE) and the others snap frozen. Histologically viable tumor cells were present for both the pretreatment and C4D1 time points for 24 of the 42 enrolled patients. The FFPE samples were stained for H&E and by IHC for AR, MYC and Ki67. MYC and Ki67 IHC stained slides were subjected to quantitative image analysis using HALO software (Indica Labs). Adequate frozen tumor tissue was available for laser capture microdissection and adequate amounts of RNA were obtained from 15 patients with paired pretreatment and C4D1 biopsies. RNAseq libraries were prepared using the NuGEN Ovation RNA-Seq System V2 and barcoded libraries were sequenced to an average depth of > 100 million reads per sample on an Illumina NovaSeq. Gene expression values were obtained with RSEM using Star aligner with GRCh38 (human). When comparing the C4D1 samples to the pretreatment samples, there were marked decreases in tumor MYC protein levels in approximately half of the treated patients, which was associated with decreased Ki67, decreased tumor volume, and prolonged progression-free survival on BAT. Using an image analysis computed H-SCORE for MYC, there was a strong correlation between MYC protein levels and MYC mRNA using RNAseq (r=0.81, p = 2.3 x 10-7). This indicates that MYC protein levels correlate with MYC mRNA and that reductions of MYC protein by BAT are likely related to reductions of MYC mRNA in clinical metastatic tissue samples. Citation Format: Carolina Gomes Alexandre, Tracy Jones, Jessica L. Hicks, John T. Isaacs, Anuj Gupta, Alyza Skaist, Laura Sena, Jennifer Meyers, Emmanuel Antonarakis, Mark Markowski, Samuel Denmeade, Srinivasan Yegnasubramanian, Angelo Michael De Marzo. Molecular pathology of metastatic prostatic adenocarcinoma treated with bipolar androgen therapy (BAT) reveals a correlation between MYC mRNA and protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 653.

5064 Background: During BAT, intramuscular (IM) administration of testosterone (T) results in rapid cycling of serum T from supraphysiologic to near-castrate levels in men with mCRPC. In a retrospective study, clinical responses to immune checkpoint blockade (ICB) in mCRPC patients (pts) previously treated with BAT were observed. Here, we report the OS and biomarker results of a Phase 2 study in mCRPC pts treated with BAT in combination with nivolumab (COMBAT; NCT03554317). Methods: This was a multi-center, single arm, open label Phase 2 study in mCRPC pts who received T cypionate 400mg IM (BAT) every 28 days plus nivolumab 480mg IV every 28 days. Pts initially received BAT alone for a 12-week period, prior to the addition of nivolumab. Eligible pts were those with asymptomatic mCRPC who had soft tissue metastases amenable to biopsy, and who progressed on at least one prior novel AR targeted therapy (and up to one prior chemo for mCRPC). The primary endpoint was confirmed PSA50 response. OS and radiographic progression free survival (rPFS) were key secondary endpoints. All pts underwent baseline metastatic biopsies, and 24 had a second biopsy after 12 weeks of BAT. Semi-quantitative IHC (for AR, Ki67, MYC, PTEN, TP53, RB1) was performed on 24 paired biopsies, of which 15 pairs were also evaluable for RNA (whole transcriptome) sequencing. Results: 45 pts were enrolled. As previously reported, the PSA50 response was 40% (18/45, 95% CI: 26-56%, P=0.02 against the 25% null hypothesis), and median rPFS was 5.6 (95% CI: 4.4–6.0) months. After a median follow-up of 17.8 months, the median OS was 27.8 (95%% CI: 17.6–NR) months. In 24 pts with paired biopsies prior to administration of nivolumab, BAT significantly decreased median MYC (P=0.046) and Ki-67 (P=0.030) expression by IHC. 71% (17/24) of pts had any decrease in MYC following BAT, with 29% (7/24) having a >50% decrease. A >50% MYC protein decline was associated with longer rPFS (HR 0.33, 95%CI 0.14–0.78, P=0.005) and a nonsignificant association towards longer OS (HR 0.78, 95%CI 0.24–2.48, P=0.679). MYC protein and mRNA levels were tightly intercorrelated (r=0.65, P50% declines in MYC mRNA levels (P>0.1 for both). Conclusions: BAT combined with nivolumab led to a median overall survival of >2 years in heavily pretreated mCRPC pts. BAT attenuated MYC expression, correlating with better outcomes. Clinical trial information: NCT03554317.

Objective To increase the usage rate of mothers' own milk (MOM) among neonates with prenatal opioid exposure from a baseline average of 47% to an average of 75% over two years. Study design Between October 2018 and December 2020, we implemented various Plan-Do-Study-Act cycles that involved engaging providers in postpartum counseling for mothers with opioid dependence, using electronic medical records to track the rate of counseling, providing NAS educational materials to parents, and establishing a rooming-in unit. Our outcome measure was the provision of MOM to eligible neonates, while our process measure was the rate of postpartum counseling. Results During this initiative, we witnessed a special cause variation with an increase in the usage rate of MOM from a baseline of 47% to a 27-month average of 85% by December 2020. Conclusion A series of quality improvement efforts resulted in increased usage of MOM among infants at risk of NAS.

PD-1 blockade unleashes CD8 T cells1, including those specific for mutation-associated neoantigens (MANA), but factors in the tumour microenvironment can inhibit these T cell responses. Single-cell transcriptomics have revealed global T cell dysfunction programs in tumour-infiltrating lymphocytes (TIL). However, the majority of TIL do not recognize tumour antigens2, and little is known about transcriptional programs of MANA-specific TIL. Here, we identify MANA-specific T cell clones using the MANA functional expansion of specific T cells assay3 in neoadjuvant anti-PD-1-treated non-small cell lung cancers (NSCLC). We use their T cell receptors as a ‘barcode’ to track and analyse their transcriptional programs in the tumour microenvironment using coupled single-cell RNA sequencing and T cell receptor sequencing. We find both MANA- and virus-specific clones in TIL, regardless of response, and MANA-, influenza- and Epstein–Barr virus-specific TIL each have unique transcriptional programs. Despite exposure to cognate antigen, MANA-specific TIL express an incompletely activated cytolytic program. MANA-specific CD8 T cells have hallmark transcriptional programs of tissue-resident memory (TRM) cells, but low levels of interleukin-7 receptor (IL-7R) and are functionally less responsive to interleukin-7 (IL-7) compared with influenza-specific TRM cells. Compared with those from responding tumours, MANA-specific clones from non-responding tumours express T cell receptors with markedly lower ligand-dependent signalling, are largely confined to HOBIThigh TRM subsets, and coordinately upregulate checkpoints, killer inhibitory receptors and inhibitors of T cell activation. These findings provide important insights for overcoming resistance to PD-1 blockade., Single-cell RNA sequencing and T cell receptor sequencing are combined to identify transcriptional programs specific to mutation-associated neoantigen-specific T cells in non-small cell lung cancers treated with anti-PD-1, providing insights into resistance to PD-1 blockade.

Background In cancers, maintenance of telomeres often occurs through activation of the catalytic subunit of telomerase, encoded by TERT. Yet, most cancers show only modest levels of TERT gene expression, even in the context of activating hotspot promoter mutations (C228T and C250T). The role of epigenetic mechanisms, including DNA methylation, in regulating TERT gene expression in cancer cells is as yet not fully understood. Methods Here, we have carried out the most comprehensive characterization to date of TERT promoter methylation using ultra-deep bisulfite sequencing spanning the CpG island surrounding the core TERT promoter in 96 different human cell lines, including primary, immortalized and cancer cell types, as well as in control and reference samples. Results In general, we observed that immortalized and cancer cell lines were hypermethylated in a region upstream of the recurrent C228T and C250T TERT promoter mutations, while non-malignant primary cells were comparatively hypomethylated in this region. However, at the allele-level, we generally found that hypermethylation of promoter sequences in cancer cells is associated with repressed expression, and the remaining unmethylated alleles marked with open chromatin are largely responsible for the observed TERT expression in cancer cells. Conclusions Our findings suggest that hypermethylation of the TERT promoter alleles signals transcriptional repression of those alleles, leading to attenuation of TERT activation in cancer cells. This type of fine tuning of TERT expression may account for the modest activation of TERT expression in most cancers.

In cancers, maintenance of telomeres often occurs through activation of the catalytic subunit of telomerase, encoded byTERT. Yet, most cancers show only modest levels of telomerase gene expression, even in the context of activating hotspot promoter mutations (C228T and C250T). The role of epigenetic mechanisms, including DNA methylation, in regulating telomerase gene expression in cancer cells is not fully understood. Here, we have carried out the most comprehensive characterization to date ofTERTpromoter methylation using ultra-deep bisulfite sequencing spanning the CpG island surrounding the coreTERTpromoter in 96 different human cell lines. In general, we observed that immortalized and cancer cell lines were hypermethylated in a region upstream of the recurrent C228T and C250TTERTpromoter mutations, while non-malignant primary cells were comparatively hypomethylated in this region. However, at the allele-level, we generally observe hypermethylation of promoter sequences in cancer cells is associated with repressed expression, and the remaining unmethylated alleles marked with open chromatin are largely responsible for the observedTERTexpression in cancer cells. Our findings suggest that hypermethylation of theTERTpromoter alleles signals transcriptional repression of those alleles, leading to the attenuation ofTERTactivation in cancer cells.SIGNIFICANCEHypermethylation of theTERTpromoter alleles to attenuateTERTactivation in cancer cells may account for the modest activation ofTERTexpression in most cancers.

Background CT scan measures of emphysema and airway disease have been correlated with lung function in cohorts of subjects with a range of COPD severity. The contribution of CT scan-assessed airway disease to objective measures of lung function and respiratory symptoms such as dyspnea in severe emphysema is less clear. Methods Using data from 338 subjects in the National Emphysema Treatment Trial (NETT) Genetics Ancillary Study, densitometric measures of emphysema using a threshold of −950 Hounsfield units (%LAA-950) and airway wall phenotypes of the wall thickness (WT) and the square root of wall area (SRWA) of a 10-mm luminal perimeter airway were calculated for each subject. Linear regression analysis was performed for outcome variables FEV 1 and percent predicted value of FEV 1 with CT scan measures of emphysema and airway disease. Results In univariate analysis, there were significant negative correlations between %LAA-950 and both the WT ( r = −0.28, p = 0.0001) and SRWA ( r = −0.19, p = 0.0008). Airway wall thickness was weakly but significantly correlated with postbronchodilator FEV 1 % predicted (R = −0.12, p = 0.02). Multivariate analysis showed significant associations between either WT or SRWA (β = −5.2, p = 0.009; β = −2.6, p = 0.008, respectively) and %LAA-950 (β = −10.6, p = 0.03) with the postbronchodilator FEV 1 % predicted. Male subjects exhibited significantly thicker airway wall phenotypes (p = 0.007 for WT and p = 0.0006 for SRWA). Conclusions Airway disease and emphysema detected by CT scanning are inversely related in patients with severe COPD. Airway wall phenotypes were influenced by gender and associated with lung function in subjects with severe emphysema.

Butyric acid and trichostatin A (TSA) are anti-cancer compounds that cause the upregulation of genes involved in differentiation and cell cycle regulation by inhibiting histone deacetylase (HDAC) activity. In this study we have synthesized and evaluated compounds that combine the bioavailability of short-chain fatty acids, like butyric acid, with the bidentate binding ability of TSA. A series of analogs were made to examine the effects of chain length, simple aromatic cap groups, and substituted hydroxamates on the compounds' ability to inhibit rat-liver HDAC using a fluorometric assay. In keeping with previous structure-activity relationships, the most effective inhibitors consisted of longer chains and hydroxamic acid groups. It was found that 5-phenylvaleric hydroxamic acid and 4-benzoylbutyric hydroxamic acid were the most potent inhibitors with IC50's of 5 microM and 133 microM respectively.

This study investigates the complementary nature of face-to-face and computer-mediated social support and the development of a context through which hyperpersonal communication can develop within online communities. Optimal matching theory is used as a framework for explaining how hyperpersonal communication develops within online cancer support communities. We compared online participants' perceptions of illness support from the list with the support they received from a nonmediated relationship. Respondents participated more within the online community only when they perceived that the depth and support that they received from the online community was high, and when the depth and support they received from the specific person in their life was low.

BACKGROUND & PURPOSE: : Lower limb amputation in older adults has a significant impact on balance, gait, and cardiovascular fitness, resulting in diminished community participation. The purpose of this case study was to describe the effects of a balance training program utilizing the Nintendo Wii™ Fit (Nintendo of America, Inc, Redmond, Washington) balance board and body- weight supported gait training on aerobic capacity, balance, gait, and fear of falling in two persons with transfemoral amputation. CASE DESCRIPTIONS: : Participant A, a 62 year-old male 32 months post traumatic transfemoral amputation, reported fear of falling and restrictions in community activity. Participant B, a 58 year-old male 9 years post transfemoral amputation, reported limited energy and balance deficits during advanced gait activities. INTERVENTION: : 6-weeks, 2 supervised sessions per week included 20 minutes of Nintendo™ Wii Fit Balance gaming and 20 minutes of gait training using Body Weight Support. OUTCOMES: : Measures included oxygen uptake efficiency slope (OUES), economy of movement, dynamic balance (Biodex platform system), Activities-Specific Balance Confidence (ABC) Scale, and spatial-temporal parameters of gait (GAITRite). Both participants demonstrated improvement in dynamic balance, balance confidence, economy of movement, and spatial-temporal parameters of gait. Participant A reduced the need for an assistive device during community ambulation. Participant B improved his aerobic capacity, indicated by an increase in OUES. DISCUSSION: : This case study illustrated that the use of Nintendo Wii™ Fit training and Body Weight Support were effective interventions to achieve functional goals for improving balance confidence, reducing use of assistive devices, and increasing energy efficiency when ambulating with a transfemoral prosthesis. Language: en

We examined virally transformed murine fibroblast clones as targets for cytotoxic T lymphocyte (CTL)-triggered lysis and genome digestion. Strikingly, while all clones were essentially equivalent in the ability to be lysed, one clone, SV3T3-B2.1, failed to exhibit genome digestion associated with CTL attack. Other aspects of the physiological cell death process, including loss of adhesion and nuclear envelope breakdown (lamin phosphorylation and solubilization), were not altered in this clone. The absence of genome digestion associated with CTL-induced cell death correlated with the absence of endodeoxyribonuclease activity in the nuclei of that clone. Characterization of the activity affected identifies a calcium-dependent, DNase I-like endonuclease of approximately 40 kDa, normally present constitutively in all cell nuclei, as the enzyme responsible for genome digestion associated with CTL-mediated cell death. These observations indicate that neither genome digestion per se nor its consequences [such as activation of poly(ADP-ribose) polymerase] are essential for cell death resulting from the triggering of this cell suicide process.

The failure of Thy-1 and Ly-6 to trigger interleukin-2 production in the absence of surface T-cell antigen receptor complex (TCR) expression has been interpreted to suggest that functional signalling via these phosphatidylinositol-linked alternative activation molecules is dependent on the TCR. We find, in contrast, that stimulation of T cells via Thy-1 or Ly-6 in the absence of TCR expression does trigger a biological response, the cell suicide process of activation-driven cell death. Activation-driven cell death is a process of physiological cell death that likely represents the mechanism of negative selection of T cells. The absence of the TCR further reveals that signalling leading to activation-driven cell death and to lymphokine production are distinct and dissociable. In turn, the ability of alternative activation molecules to function in the absence of the TCR raises another issue: why immature T cells, thymomas, and hybrids fail to undergo activation-driven cell death in response to stimulation via Thy-1 and Ly-6. One possibility is that these activation molecules on immature T cells are defective. Alternatively, susceptibility to activation-driven cell death may be developmentally regulated by TCR-independent factors. We have explored these possibilities with somatic cell hybrids between mature and immature T cells, in which Thy-1 and Ly-6 are contributed exclusively by the immature partner. The hybrid cells exhibit sensitivity to activation-driven cell death triggered via Thy-1 and Ly-6. Thus, the Thy-1 and Ly-6 molecules of the immature T cells can function in a permissive environment. Moreover, with regard to susceptibility to Thy-1 and Ly-6 molecules of the immature T cells can function in a permissive environment. Moreover, with regard to susceptibility to Thy-1 and Ly-6 triggering, the mature phenotype of sensitivity to cell death is genetically dominant.

Premature termination codons induce rapid transcript degradation in eukaryotic cells through nonsense-mediated mRNA decay (NMD). This pathway can modulate phenotypes arising from nonsense or frameshift mutations, but little is known about the physiologic role of NMD in higher eukaryotes. To address this issue, we examined expression profiles in mammalian cells depleted of Rent1 (also called hUpf1), a factor essential for NMD. Upregulated transcripts included those with upstream open reading frames in the 5' untranslated region, alternative splicing that introduces nonsense codons or frameshifts, introns in the 3' untranslated region or selenocysteine codons. Transcripts derived from ancient transposons and endogenous retroviruses were also upregulated. These RNAs are unified by the presence of a spliced intron at least 50 nucleotides downstream of a termination codon, a context sufficient to initiate NMD. Consistent with direct regulation by NMD, representative upregulated transcripts decayed more slowly in cells deficient in NMD. In addition, inhibition of NMD induced by amino acid starvation upregulated transcripts that promote amino acid homeostasis. These results document that nonsense surveillance is a crucial post-transcriptional regulatory event that influences the expression of broad classes of physiologic transcripts, has been functionally incorporated into essential homeostatic mechanisms and suppresses expression of evolutionary remnants.

Background . A novel therapeutic option for the treatment of acute myocardial infarction involves the use of mesenchymal stem cells (MSCs). The purpose of this study was to investigate whether implantation of autologous MSCs results in sustained engraftment, myogenic differentiation, and improved cardiac function in a swine myocardial infarct model. Methods . MSCs were isolated and expanded from bone marrow aspirates of 14 domestic swine. A 60-minute left anterior descending artery occlusion was used to produce anterior wall infarction. Piezoelectric crystals were placed within the ischemic region for measurement of regional wall thickness and contractile function. Two weeks later animals autologous, Di-I–labeled MSCs (6 × 10 7 ) were implanted into the infarct by direct injection. Hemodynamic and functional measurements were obtained weekly until the time of sacrifice. Immunohistochemistry was used to assess MSC engraftment and myogenic differentiation. Results . Microscopic analysis showed robust engraftment of MSCs in all treated animals. Expression of muscle-specific proteins was seen as early as 2 weeks and could be identified in all animals at sacrifice. The degree of contractile dysfunction was significantly attenuated at 4 weeks in animals implanted with MSCs (5.4% ± 2.2% versus −3.37% ± 2.7% in control). In addition, the extent of wall thinning after myocardial infarction was markedly reduced in treated animals. Conclusions . Mesenchymal stem cells are capable of engraftment in host myocardium, demonstrate expression of muscle specific proteins, and may attenuate contractile dysfunction and pathologic thinning in this model of left ventricular wall infarction. MSC cardiomyoplasty may have significant clinical potential in attenuating the pathology associated with myocardial infarction.

A quick and reliable method to measure a vapor concentration within porous substrate was developed. The technique consists of two steps, where a modified head space single drop microextraction (HS-SDME) sampling is used to entrap the vapor phase. In the second step, the entrapped vapor concentration is measured by gas chromatography (GC-FID). The technique is used to measure an effective diffusion coefficient of n-pentane in dry medium grain sand, with the sand partially saturated with water as an inert liquid. The measurements are carried out in a cylindrical sand holder on which the HS-SDME sampling ports are mounted. A linear vapor concentration profile along the bed thickness is found. From known concentration gradient and measuring the mass flux gravimetrically, the effective diffusion coefficient is determined. It turns out that the diffusion coefficient decreases from 8.49 × 10−6 for dry sand to 7.13 × 10−6 m2 s−1 as a function of water saturation. Additional hindrance to the vapor transport is observed from both the simple volumetric effect due to the porous medium void space reduction caused by the presence of water and increase of the tortuosity.