Accelerated Time Course Research Articles

Sex Differences in the Development of an Opioid Addiction–Like Phenotype: A Focus on the Telescoping Effect

BackgroundWomen develop addiction and drug-related health consequences after fewer years of drug use than men; this accelerated time course, or telescoping effect, has been observed clinically for multiple drugs, including opioids. Preclinical studies indicate that this is a biologically based phenomenon; however, these studies have focused exclusively on cocaine, and none have considered health effects. MethodsIn this study, we used a rat (Sprague Dawley) model to determine sex differences in the time course for the development of an opioid addiction–like phenotype, as defined by the development of physical dependence (withdrawal-induced weight loss) and an increase in motivation for fentanyl (under a progressive-ratio schedule). Effects were determined following either 10 days (optimized, experiment 1) or 3 days (threshold, experiment 2) of extended-access fentanyl self-administration (24 hours/day, fixed ratio 1, 2- to 5-minute trials/hour) or following short-access fentanyl self-administration (subthreshold, experiment 3; fixed ratio 1, up to 40 infusions/day). Opioid-related adverse health effects were also determined (experiment 4). ResultsMotivation for fentanyl was similarly increased in males and females following 10 days of extended-access self-administration (experiment 1), was transiently increased in females, but not males, following 3 days of extended-access self-administration (experiment 2) and was not increased in either sex following short-access self-administration (experiment 3). Females developed fentanyl-associated adverse health effects more readily than males (experiment 4), with particularly robust differences during extended-access self-administration and withdrawal. ConclusionsAs with findings in humans, female rats developed opioid addiction–like features and adverse health consequences more readily than male rats. These data provide support for a biologically based telescoping effect in females for opioids, particularly for opioid-related adverse health consequences.

Abstract P1028: Elucidating Differential Mechanisms That Underlie Phenotypic Variability In Two Independent Dcm Models

Mutations in components of the cardiac thin filament cause disease via complex, multi-faceted myocellular mechanisms, ultimately presenting as heterogeneous, symptomatic cardiomyopathies. The heterogeneity of both the myocellular mechanism(s) involved and the clinical outcome(s) represents a challenge in ongoing efforts to link genotype to phenotype. Two DCM-associated thin filament mutations (D230N-Tm and R173W-cTnT) with similar clinical phenotypes, were selected explore the molecular basis of disease trajectory. In patient populations, D230N-Tm exhibits an earlier time of onset compared to R173W-cTnT, with children (<1yr) presenting with an often-fatal DCM. We hypothesize that the difference in time of onset can be explained by distinct myocellular mechanisms. We utilized transgenic mice expressing D230N-Tm and R173W-cTnT and defined the progression of pathogenic remodeling across 3 independent timepoints via 2D-echo. At 2 months the %FS of D230N-Tm was significantly reduced (21.79 ± 1.19) compared to NTg littermates (33.45±1.45). R173W-cTnT, however, exhibited no reduction in %FS until 4 months (22.53±0.78), thus phenocopying the patient population. Using these timepoints to guide our myocellular mechanics, contractility measurements were then utilized to assess the myocellular mechanics associated with the mutations. At 2 months D230N-Tm exhibited a decrease in the rate of contraction (4.53±0.24μm/sec) and rate of relaxation (2.88±0.23) compared to NTg. R173W-cTnT did not diverge from NTg until 4 months, exhibiting a later onset of impaired contractility. By 4 months, both mutations displayed the opposite, an increase in rate of contraction and relaxation (rate of contraction, D230N-Tm 4.49±0.18 and R173W-cTnT 5.83±0.27; and rate of relaxation, D230N-Tm 3.14±0.19 and R173W-cTnT 3.70±0.24). Our results suggest that the accelerated time course for the development of D230N-Tm – linked DCM is caused by differential myocellular mechanism(s) that induce the early reduction in contractility and relaxation observed. Ongoing studies aim to further identify possible myocellular mechanism(s) by which these mutations exhibit different myocellular mechanics.

S1811 Cecal Ulceration as the Presenting Finding of Metastatic Breast Cancer: A Case Report and Review of Literature

Introduction: Breast cancer is the second most common malignancy worldwide and the second leading cause of cancer mortality in women. Despite routine screening, early detection, and evolving treatment modalities, recurrence and metastatic disease often occur. While breast cancer metastases are traditionally identified in the lung, liver, and bone, metastases to the gastrointestinal tract are rare. Metastases to the stomach are more commonly identified than to the colon and rectum. Prior cases of metastatic breast cancer to the gastrointestinal tract are reported in individuals with an established diagnosis of breast cancer. We describe a case of an ulcerated lesion in the cecum as the initial presenting sign of metastatic primary breast cancer. Case Description/Methods: A 64-year-old-woman with hypertension and chronic constipation was referred to our institution for evaluation of seven weeks of fatigue, one week of urinary symptoms, and lower extremity edema. The patient had undergone ten years of unremarkable screening mammograms. On admission, laboratory workup was notable for markedly elevated liver enzymes. Computerized tomography scan indicated evidence of an ileocecal valve mass and extensive diffuse hepatic metastases. Endoscopic evaluation revealed a benign appearing 30mm sessile polyp in the proximal rectum 15cm from the anal margin and a 3mm ulcer in the cecum (A, B, C, D). Pathologic evaluation and immunohistochemical analysis of the cecal ulceration tissue was suggestive of a diagnosis of primary breast cancer with metastasis to the colon (E, F, G, H).The polyp in the proximal rectum was benign. The patient elected to be discharged to home and had close follow-up. The rapidly progressive nature of the patient’s presentation prevented further workup and ultimately the patient expired less than a month after diagnosis. Discussion: We present an unusual case of metastatic carcinoma of the breast to the colon and liver in an individual without a previous diagnosis of breast cancer who is treatment naïve. The accelerated time course from presentation to patient death was noteworthy. Accurate identification and differentiation between primary gastrointestinal cancer and metastatic breast cancer is paramount to proper management and treatment of the disease. This can be achieved through engaging a multidisciplinary team. This case highlights the need to maintain a high index of suspicion for colon ulcers and lesions as potential sites of metastasis of other primary malignancies such as breast cancer.Figure 1.: A 30mm sessile polyp in the proximal rectum 15cm from the anal margin (A), rectal polyp resected and retrieved (B), and 3mm ulcer in the cecum (C, D). Medium power (40x) and high power (100x) views of a H&E stained slide shows a biopsied fragment of colonic mucosa with metastatic carcinoma involving the deep mucosa and superficial submucosa. The moderately to poorly differentiated malignant epithelial cells infiltrate in nests and cords without forming clear glandular structures (E, F). Nuclear hyperchromasia is seen on higher magnification (F). Immunohistochemical staining, essential in the characterization of this tumor, supports the diagnosis of a carcinoma metastatic from the breast. CK7 shows strong, diffuse positivity, highlighting tumor cells (G). GATA3 shows scattered but strong, nuclear positivity (H). Tumor cells are negative for CK20, CDX2, and S100.

Females develop features of an addiction-like phenotype sooner during withdrawal than males.

Women meet criteria for substance use disorder after fewer years of drug use than men; this accelerated time course, or telescoping effect, has been observed for multiple drugs, including cocaine. Preclinical findings similarly indicate an enhanced vulnerability in females to developing an addiction-like phenotype; however, it is not yet known if this phenotype develops faster in females versus males. The goal of this study was to determine using a rat model whether two key features of addiction in humans, an enhanced motivation for cocaine and compulsive use, emerge sooner during withdrawal from extended access cocaine self-administration in females versus males. Motivation for cocaine, as assessed under a progressive-ratio reinforcement schedule, was determined prior to and following extended access cocaine self-administration (24h/day, 96 infusions/day, 10days) and after 7, 14, or 60days of withdrawal. Compulsive use, or use despite punishment, was evaluated once progressive-ratio responding stabilized by adding histamine, an aversive stimulus, to the cocaine solutions. Motivation for cocaine increased from baseline sooner during withdrawal in females than males (at 7 versus 14days); motivation was also highest in the 60-day group. Histamine decreased progressive-ratio responding for cocaine in both sexes, although effects were greatest in males in the 7-day withdrawal group; males reached the female-level of resistance to histamine punishment by 14days of withdrawal. Female rats developed addition-like features sooner during withdrawal than male rats indicating that the telescoping effect observed in humans is biologically based. Additionally, like drug-seeking/craving, motivation for cocaine and measures of compulsive use incubate over withdrawal.

Editors' note: COVID-19 presenting with ophthalmoparesis from cranial nerve palsy.

Dr. Dinkin et al. reported 2 cases with MRI confirmation of cranial nerve inflammation within several days of systemic symptoms of the novel coronavirus disease 2019 (COVID-19). Both patients tested positive for SARS-CoV-2 by PCR from a nasal swab, and both were treated with hydroxychloroquine (one also with intravenous immunoglobulin), with mild short-term improvement. The authors suggest the inflammatory cranial neuropathies could have been because of a postviral immune-related response. Dr. Machado wonders whether the cranial neuropathies could have resulted from direct viral invasion, given their onset shortly after the viral symptoms started. As noted by Gutiérrez-Ortiz et al. (Dinkin et al., reference 2 below) 2 patients developed evidence of cranial nerve inflammation and autoimmune neuropathy within 3–5 days of COVID-19 symptom onset. Each of these 4 cases highlight the accelerated time course of inflammatory cranial neuropathies after systemic manifestations of COVID-19, which seems to be more rapid than has been typically reported for variants of Guillain-Barré syndrome after other infectious illnesses. These observations may also support that there is an asymptomatic phase of this viral infection during which an immune response may be generated. Dr. Dinkin et al. reported 2 cases with MRI confirmation of cranial nerve inflammation within several days of systemic symptoms of the novel coronavirus disease 2019 (COVID-19). Both patients tested positive for SARS-CoV-2 by PCR from a nasal swab, and both were treated with hydroxychloroquine (one also with intravenous immunoglobulin), with mild short-term improvement. The authors suggest the inflammatory cranial neuropathies could have been because of a postviral immune-related response. Dr. Machado wonders whether the cranial neuropathies could have resulted from direct viral invasion, given their onset shortly after the viral symptoms started. As noted by Gutiérrez-Ortiz et al. (Dinkin et al., reference 2 below) 2 patients developed evidence of cranial nerve inflammation and autoimmune neuropathy within 3–5 days of COVID-19 symptom onset. Each of these 4 cases highlight the accelerated time course of inflammatory cranial neuropathies after systemic manifestations of COVID-19, which seems to be more rapid than has been typically reported for variants of Guillain-Barré syndrome after other infectious illnesses. These observations may also support that there is an asymptomatic phase of this viral infection during which an immune response may be generated.

Geranylgeranyl Diphosphate Synthase Inhibitor and Proteasome Inhibitor Combination Therapy in Multiple Myeloma

Introduction: Multiple myeloma (MM) is characterized by proliferation of malignant plasma cells that produce monoclonal protein (MP). MM cells, compared with non-secretory cells, have a lower threshold for induction of the pro-apoptotic components of the unfolded protein response pathway (UPR) as a consequence of near-maximal expression of the protective UPR elements. Proteasome inhibitors (PIs) are widely used in the treatment of MM and have been shown to disrupt the UPR. As an alternative strategy by which to target protein homeostasis in MM, we have focused on the development of geranylgeranyl diphosphate synthase (GGDPS) inhibitors. Inhibition of GGDPS results in disruption of Rab protein geranylgeranylation, which in turn results in disruption of MP trafficking, leading to accumulation of intracellular MP, induction of the UPR and apoptosis. We have previously reported preclinical studies with our lead GGDPS inhibitor (GGDPSi), RAM2061, which demonstrated the agent's metabolic stability, prolonged half-life (plasma elimination half-life of 29.2 (+6.0) hours (hrs)), systemic distribution, in vivo disruption of geranylgeranylation and anti-tumor efficacy in a mouse MM xenograft model (Haney et al., ASH 2018, abstract 215). We hypothesized that the combination of GGDPSi and PI therapy would result in enhanced disruption of the UPR and increase anti-MM efficacy. Methods: MTT assays were conducted to evaluate the cytotoxic effects of combining RAM2061 with the PI bortezomib (Bor) in human MM cell lines (RPMI-8226, MM.1S) and isobologram analysis was performed. The effects of RAM2061 and/or Bor on markers of the UPR were evaluated via qRT-PCR (ATF4, ATF6, CHOP, PERK, IRE1) and immunoblot analysis (ATF4, IRE1, phosphorylated eIF2α). Apoptotic markers (cleaved caspases 3, 8 and 9 and cleaved PARP) were evaluated by immunoblot analysis. Cell assays were performed using both concurrent (both drugs added at the start of the 48 hr incubation period) and RAM2061 pre-treatment (RAM2061 added at the start, Bor added after 24 hrs) approaches. To evaluate the in vivo activity of the novel combination therapy, a flank xenograft model was used. NOD/SCID mice were subcutaneously inoculated in the flank with MM.1S cells. Once tumors became palpable, mice were randomly divided into four treatment groups (n=8 per group): control (PBS), RAM2061 (0.08 mg/kg IV two times/week (wk)), Bor (0.3 mg/kg SQ two times/wk) and RAM2061/Bor combination. Tumor volume (TV) and animal body weight were recorded three times/wk. Mice were euthanized when tumors reached 2000 mm3. Blood samples from day 9 post-initiation of treatment were analyzed via ELISA for human λ light chain. Blood samples collected at time of euthanasia were analyzed for hematologic, renal and hepatic parameters. Results: Isobologram analysis revealed a primarily additive effect with concurrent GGDPSi/PI incubations and primarily synergistic effect with the RAM2061 pre-treatment approach. The effect of Bor on augmenting RAM2061-induced upregulation of UPR and apoptotic markers was both concentration- and time-dependent. While there was in general an enhanced induction of apoptosis with the combination, Bor partially abrogated the RAM2061-induced upregulation of UPR markers at the tested time points. Studies are ongoing to determine whether this effect is associated with an accelerated time course of UPR activation in the presence of Bor. Consistent with our previous in vivo studies, the primary toxicity associated with RAM2061 treatment was hepatic transaminase elevation. No additional toxicity was observed when Bor was combined with RAM2061 based on animal weight, hematological, renal or hepatic parameters. Tumor growth over time was significantly decreased in the RAM2061 (p=0.0009) and RAM2061/Bor (p=0.0002) treatment groups compared to control animals and this corresponded to prolonged time to sacrifice (p=0.09 for RAM2061 group and p=0.003 for RAM2061/Bor group). Consistent with TV, mean plasma human λ light chain levels were decreased in the RAM2061 and RAM2061/Bor treatment groups compared to the control mice (p<0.05). Conclusion: These studies support further investigation into the therapeutic potential of GGDPSi/PI-based combination therapy for MM. Further studies are needed to dissect the mechanisms underlying the interactions between the two classes of drugs on the UPR and apoptotic pathways. Disclosures Holstein: Takeda: Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy; Celgene: Consultancy; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Sorrento: Consultancy; Genentech: Membership on an entity's Board of Directors or advisory committees.

The impact of transplant rejection on cutaneous squamous cell carcinoma in renal transplant recipients.

The incidence of cutaneous squamous cell carcinoma (cSCC) is markedly increased in renal transplant recipients compared with that of the nontransplant population. To investigate whether there is a relationship between transplant rejection and cSCC. The Duke Enterprise Data Unified Content Explorer historical database was used to identify patients who had undergone a renal transplant at Duke University Hospital during a 20-year period. Data on patient demographics, transplant dates, first rejection episodes, first cSCC development, medication, laboratory results and survival were recorded. In total, 1684 patients were identified, of whom 126 (7.5%) experienced an episode of rejection and 46 (4.0%) developed a cSCC after transplant. The incidence of cSCC was significantly greater in the rejection group, with 8.7% of patients developing cSCC compared with 2.2% in the no-rejection group (P<0.001). Median lag time to cSCC was shorter in the rejection group (2.5years; age 0.4-9.0years) than the no-rejection group (4.2years; range 1.3-20.4years) (P<0.03). Transplant rejection is associated with both a higher incidence and an accelerated time course for development of cSCC following renal transplantation. Close dermatological surveillance should be considered following an episode of rejection in this patient population.

In vivo imaging of emerging endocrine cells reveals a requirement for PI3K-regulated motility in pancreatic islet morphogenesis.

ABSTRACTThe three-dimensional architecture of the pancreatic islet is integral to beta cell function, but the process of islet formation remains poorly understood due to the difficulties of imaging internal organs with cellular resolution. Within transparent zebrafish larvae, the developing pancreas is relatively superficial and thus amenable to live imaging approaches. We performed in vivo time-lapse and longitudinal imaging studies to follow islet development, visualizing both naturally occurring islet cells and cells arising with an accelerated timecourse following an induction approach. These studies revealed previously unappreciated fine dynamic protrusions projecting between neighboring and distant endocrine cells. Using pharmacological compound and toxin interference approaches, and single-cell analysis of morphology and cell dynamics, we determined that endocrine cell motility is regulated by phosphoinositide 3-kinase (PI3K) and G-protein-coupled receptor (GPCR) signaling. Linking cell dynamics to islet formation, perturbation of protrusion formation disrupted endocrine cell coalescence, and correlated with decreased islet cell differentiation. These studies identified novel cell behaviors contributing to islet morphogenesis, and suggest a model in which dynamic exploratory filopodia establish cell-cell contacts that subsequently promote cell clustering.

DHEA‐induced Voltage‐dependent Inhibition of ICa,L Is Augmented by Cholesterol Depletion and Down‐regulated by GPCR Blockade in Aortic Smooth Muscle Cells

Dehydroepiandrosterone (DHEA) is a steroid hormone synthesized from cholesterol and abundantly secreted as DHEA sulfate from the adrenal gland. DHEA produces relaxation of systemic and pulmonary arterial smooth muscles (ASM) and epiandrosterone, a metabolite of DHEA, causes voltage‐dependent inhibition (VDI) of ICa,L in ventricular myocytes. Here, we characterized DHEA‐induced VDI of ICa,L recorded with Ba2+ as a charge carrier by whole cell clamp and studied underlying mechanism and modulation by signal transduction in ASM cells (ASMCs) from bovine coronary artery and A7r5 cells from rat aorta. DHEA decreased peak amplitude of ICa,L rapidly, reversibly and dose‐dependently and accelerated time course of current decay decreasing the ratio of current amplitude at 500 ms (r500) to peak amplitude and shifted steady‐state inactivation curve (f∞‐V) to the left. DHEA‐induced inhibition of ICa,L became prominent by depolarization of holding potential; e.g. at −30 mV, the inhibition was 29% at 10 μM and 89% at 100 μM. Blockade of GPCR by dialysis of GDP βS shifted both f∞‐V and I–V relationships to the depolarizing direction, and decreased 10 μM‐DHEA‐induced VDI obtained at HP of −40 mV by 40%. Depletion of membrane cholesterol by methyl‐b‐cyclodextrin increased r500 and shifted f∞‐V to the right. Conversely, loading PEG‐cholesterol, a non‐ionic amphiphile, to the membrane decreased r500 and shifted f∞‐V to the left. In the cholesterol‐depleted cells, DHEA produced more potent decrease of r500 and larger negative shift of f∞‐V than in control. Therefore, DHEA‐induced VDI of ICa,L in ASMCs is due, at least partly, to cholesterol‐like action of DHEA on CaV1.2 in the membrane lipid bilayer. GPCR‐mediated signaling regulates voltage‐dependence of basal ICa,L and indirectly augments DHEA‐induced VDI of ICa,L in ASMCs.

Additional in-series compliance reduces muscle force summation and alters the time course of force relaxation during fixed-end contractions.

There are high mechanical demands placed on skeletal muscles in movements requiring rapid acceleration of the body or its limbs. Tendons are responsible for transmitting muscle forces, but, because of their elasticity, can manipulate the mechanics of the internal contractile apparatus. Shortening of the contractile apparatus against the stretch of tendon affects force generation according to known mechanical properties; however, the extent to which differences in tendon compliance alter force development in response to a burst of electrical impulses is unclear. To establish the influence of series compliance on force summation, we studied electrically evoked doublet contractions in the cane toad peroneus muscle in the presence and absence of a compliant artificial tendon. Additional series compliance reduced tetanic force by two-thirds, a finding predicted based on the force-length property of skeletal muscle. Doublet force and force-time integral expressed relative to the twitch were also reduced by additional series compliance. Active shortening over a larger range of the ascending limb of the force-length curve and at a higher velocity, leading to a progressive reduction in force-generating potential, could be responsible. Muscle-tendon interaction may also explain the accelerated time course of force relaxation in the presence of additional compliance. Our findings suggest that a compliant tendon limits force summation under constant-length conditions. However, high series compliance can be mechanically advantageous when a muscle-tendon unit is actively stretched, permitting muscle fibres to generate force almost isometrically, as shown during stretch-shorten cycles in locomotor activities. Restricting active shortening would likely favour rapid force development.

Accelerated time course of saccadic inhibition of return in individuals with autism spectrum disorders.

The inhibition of return (IOR) refers the observer's slower response time when the target stimulus appears on the previously attended location. In the present study, we examined the time course of saccadic IOR by using five stimuli onset asynchronies (SOAs) in a group of adults with autism spectrum disorders (ASDs) and a comparison group. The results showed that the IOR effect occurred earlier (300 ms SOA) in participants with ASDs, relative to the comparison participants (500 and 700 ms SOAs). The ASD group also committed a greater number of anticipatory saccades, which positively correlated with scores on restricted and repetitive behaviors, as assessed by the Autism Diagnostic Interview-Revised (ADI-R; Lord et al. in J Autism Dev Disord 24:659-685, 1994). These findings reveal an accelerated time course for saccadic IOR along with diminished volitional oculomotor control in participants with ASDs. We discussed these results with reference to the atypical and the superior visual search abilities often reported in this population.

Neuronal injury from cardiac arrest: aging years in minutes.

Cardiac arrest is a leading cause of death and permanent disability. Most victims succumb to the oxidative and inflammatory damage sustained during cardiac arrest/resuscitation, but even survivors typically battle long-term neurocognitive impairment. Although extensive research has delineated the complex mechanisms that culminate in neuronal damage and death, no effective treatments have been developed to interrupt these mechanisms. Of importance, many of these injury cascades are also active in the aging brain, where neurons and other cells are under persistent oxidative and inflammatory stress which eventually damages or kills the cells. In light of these similarities, it is reasonable to propose that the brain essentially ages the equivalent of several years within the few minutes taken to resuscitate a patient from cardiac arrest. Accordingly, cardiac arrest-resuscitation models may afford an opportunity to study the deleterious mechanisms underlying the aging process, on an accelerated time course. The aging and resuscitation fields both stand to gain pivotal insights from one another regarding the mechanisms of injury sustained during resuscitation from cardiac arrest and during aging. This synergism between the two fields could be harnessed to foster development of treatments to not only save lives but also to enhance the quality of life for the elderly.

Osteoclast Generation and Cytokine Profile at Prosthetic Interfaces: A Study on Tissue of Patients with Aseptic Loosening or Implant-Associated Infections

Aseptic loosening of implants or loosening due to persistent bacterial infection remains a severe complication in orthopaedic surgery. To investigate underlying cellular and molecular mechanisms, particularly with regard to bone loss, tissue samples of patients requiring surgery were examined. By histological methods and by quantitative RT-PCR, respectively, infiltration of leukocytes, expression of osteoclast-typical genes and of proinflammatory cytokines was determined. Samples were taken directly from osteolytic sites and for comparison from adjacent sites, distant sites and from muscle. At osteolytic sites, cathepsin K and the metalloproteinases MMP1 and MMP9 were found, as was expression of inflammation-related cytokines, particularly of interleukin (IL)-1β, CXCL8, S100A9 and a very moderate expression of receptor activator of NfκB ligand (RANKL) and tumour necrosis factor (TNF) a. Of note, expression of these parameters gradually decreased from sites of osteolysis to adjacent tissue, to distant tissue to muscle. In patients with infection and osteolysis, expression of cytokines, notably of CXCL8, was markedly enhanced, especially in adjacent and distant tissues, where expression was 10- to 20-fold higher compared to tissue of aseptic patients. A possible source of CXCL8 could be infiltrated cells, particularly neutrophils, because they were found in infected tissue only. Histological examination of the biopsies revealed an additional CXCL8 source, namely endothelial cells of small blood vessels. In conclusion, aseptic loosening and implant-associated infection are associated with osteoclast generation and a local inflammatory response. The proinflammatory environment could promote the differentiation of precursor cells to osteoclasts, thereby linking inflammation to bone resorption. The higher expression of cytokines, particularly of CXCL8 in tissue of patients with bacterial infection, could explain the accelerated time course of bone resorption as it occurs in infection compared to aseptic loosening.

Abstract A48: Optical imaging model for monitoring dynamic interactions of breast cancer cells and bone tissues in ex vivo co-cultures

Abstract Purpose: Bone is the most common site of breast cancer metastasis and model systems are needed to study aspects of this process directly at the microenvironmental level. Our goal was to establish optical imaging approaches to monitor and quantitate the dynamics of cell growth, migration and colonization in viable ex vivo co-cultures of breast cancer cells and bone tissue explants. Experimental Procedures: Femur tissues harvested from CD-1 nude mice and discarded femoral heads from human hip replacement surgeries were cultivated in 6-well tissue culture plates adjacent to 4T1 mouse mammary carcinoma or MDA-MB-231 human breast cancer cells harboring luciferase reporters. Serial bioluminescence imaging (BLI) was performed on an IVIS 50 Imaging System to quantify breast cancer cell growth, track breast cell migration toward bones, and monitor breast cell colonization of bone tissue over a period of days and weeks. Bioluminescence imaging of single breast cancer cells in these cultures was also performed over time using a prototype LV200 Bioluminescent Imaging System from Olympus. Summary of Results: BLI revealed the directed migration of breast cancer cells toward and into adjacent bone tissues within days, and documented extended colonization of bone tissues for up to several weeks. Quantitative BLI demonstrated variable growth rates for breast cancer cells grown in the presence vs. absence of bone tissues. Growth, migration and colonization varied dramatically and consistently according to the region of femur (epiphysis vs. diaphysis) used for co-culture. The variable patterns of breast cancer cell growth and migration observed in association with different anatomic regions of the femur are consistent with the presence of distinct cell populations within specific microenvironmental compartments of the bone. Migration of bone cells toward adjacent colonies of breast cancer cells was also observed. Conclusions: Cell behaviors associated with breast cancer metastasis occur reproducibly in viable, dynamic ex vivo co-cultures and can be monitored and quantified using optical imaging approaches. Because of the accelerated time course of interactions, and the immediate quantitation afforded by BLI, this model will facilitate the direct, rapid study of breast cancer cell interactions within the metastatic niche of bone tissues. This approach circumvents the prolonged time course associated with in vivo metastasis models and offers the potential for high throughput perturbation to identify and evaluate therapeutic interventions. Citation Format: Bonnie L. King, Marie Bammer, Irfan Ali-Khan, Jonathan W. Hardy, Christopher H. Contag. Optical imaging model for monitoring dynamic interactions of breast cancer cells and bone tissues in ex vivo co-cultures. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A48.

A socioecological framework to understand weight-related issues in Aboriginal children in Canada

Obesity prevention efforts in Aboriginal (First Nations, Métis, or Inuit) communities in Canada should focus predominantly on children given their demographic significance and the accelerated time course of occurrence of type 2 diabetes mellitus in the Aboriginal population. A socioecological model to address childhood obesity in Aboriginal populations would focus on the numerous environments at different times in childhood that influence weight status, including prenatal, sociocultural, family, and community environments. Importantly, for Aboriginal children, obesity interventions need to also be situated within the context of a history of colonization and inequities in the social determinants of health. This review therefore advocates for the inclusion of a historical perspective and a life-course approach to obesity prevention in Aboriginal children in addition to developing interventions around the socioecological framework. We emphasize that childhood obesity prevention efforts should focus on promoting maternal health behaviours before and during pregnancy, and on breastfeeding and good infant and child nutrition in the postpartum and early childhood development periods. Ameliorating food insecurity by focusing on improving the sociodemographic risk factors for it, such as increasing income and educational attainment, are essential. More research is required to understand and measure obesogenic Aboriginal environments, to examine how altering specific environments modifies the foods that children eat and the activities that they do, and to examine how restoring and rebuilding cultural continuity in Aboriginal communities modifies the many determinants of obesity. This research needs to be done with the full participation of Aboriginal communities as partners in the research.

Heart Failure Transcriptome

The article by Barth et al1 in this issue points to a critical facet in heart failure investigation, namely, the molecular divergence observed between preclinical models of disease and clinical authenticity. These authors compared gene expression patterns from hearts of human, dogs, and rodents.1 Women and men appear to differ from mice in respect to the transcriptome dynamics of the failing heart. Not surprised? The implications of this finding may be potentially game changing for the future design of translational strategies to advance discovery science into patient care. Article see p 475 Heart failure is a significant health problem, affecting nearly 1 in 5 adults, with a cost of >$40 billion per year.2 Barth et al present data from a meta-analysis of several studies to suggest that the gene expression patterns in preclinical animal models of heart failure are significantly different than those in human heart failure.1 Specifically, Barth et al show that the significant downregulation of metabolic signaling pathways and reciprocal upregulation of cell signaling in rodent and dog models are not found in all human heart failure specimens.1 If dissimilar patterns in gene expression reflect different pathophysiological characteristics, the current course of testing new therapies for human heart failure in rodent and dog models may be questioned. Modeling heart failure in rodents and dogs is typically performed on an accelerated time course compared with natural disease progression in humans. The gene expression changes seen in pre-clinical models of heart failure in this study may more closely simulate acute heart failure syndrome3,4 versus chronic heart failure. Chronic human heart failure may present with changes referred to as remodeling and include dilatation of the ventricular …

Ih “run-up” in rat neocortical neurons and transiently rat or human HCN1-expressing HEK293 cells

Hyperpolarization-activated cyclic nucleotide-gated ion channels (HCN) are key determinants of CNS functions. Here we describe an increase in hyperpolarization-activated current (I(h)) at the beginning of whole-cell recordings in rat layer 5 cortical neurons. For a closer investigation of this I(h) increase, we overexpressed the predominant layer 5 rat subunit HCN1 in HEK293 cells. We characterized the resulting I(h) in the cell-attached and whole-cell configurations. Breaking into whole-cell configuration led to about a 30% enhancement of rat HCN1-mediated I(h) accompanied by a depolarizing shift in voltage dependence and an accelerated time course of activation. This current enhancement is not species specific; for human HCN1, the current similarly increases in amount and kinetics. Although the changes were bound to cytosolic solution exchange, they were independent of cAMP, ATP, GTP, and the phosphate group donor phosphocreatine. Together, these data provide a characterization of heterologous expression of rat HCN1 and suggest that cytosolic contents suppress I(h). Such a mechanism might constitute a reserve in h-channel function in vivo.

Retinal Disease in Usher Syndrome III Caused by Mutations in the Clarin-1 Gene

To determine the retinal phenotype of Usher syndrome type III (USH3A) caused by clarin-1 (CLRN1) gene mutations in a non-Finnish population. Patients with USH3A (n = 13; age range, 24-69) representing 11 different families were studied and the results compared with those from patients with USH2A (n = 24; age range, 17-66). The patients were evaluated by ocular examination, kinetic and static perimetry, near-infrared autofluorescence, and optical coherence tomography (OCT). Ten of 11 families had Ashkenazi Jewish origins and the N48K CLRN1 mutation. Rod function was lost in the peripheral field in the first two decades of life, but central rod function could be retained for another decade. Peripheral cone function was detectable into the third decade of life. Central cone function had a slower decline that extended for decades. Photoreceptor layer loss and features of retinal remodeling were present in retinal regions with severe visual dysfunction, even at the youngest ages tested. Central retinal structure could be normal in younger patients but structural integrity was lost in older patients. RPE disease generally paralleled photoreceptor degeneration. Comparisons between USH3A and USH2A suggested a common rod and cone phenotype but a more accelerated time course of rod loss in USH3A. USH3A and USH2A share patterns of rod and cone dysfunction and retinal structural abnormalities. Peripheral function measurements showed USH3A to be more rapidly progressive than USH2A.

Lipidomics of cellular and secreted phospholipids from differentiated human fetal type II alveolar epithelial cells

Maturation of fetal alveolar type II epithelial cells in utero is characterized by specific changes to lung surfactant phospholipids. Here, we quantified the effects of hormonal differentiation in vitro on the molecular specificity of cellular and secreted phospholipids from human fetal type II epithelial cells using electrospray ionization mass spectrometry. Differentiation, assessed by morphology and changes in gene expression, was accompanied by restricted and specific modifications to cell phospholipids, principally enrichments of shorter chain species of phosphatidylcholine (PC) and phosphatidylinositol, that were not observed in fetal lung fibroblasts. Treatment of differentiated epithelial cells with secretagogues stimulated the secretion of functional surfactant-containing surfactant proteins B and C (SP-B and SP-C). Secreted material was further enriched in this same set of phospholipid species but was characterized by increased contents of short-chain monounsaturated and disaturated species other than dipalmitoyl PC (PC16:0/16:0), principally palmitoylmyristoyl PC (PC16:0/14:0) and palmitoylpalmitoleoyl PC (PC16:0/16:1). Mixtures of these PC molecular species, phosphatidylglycerol, and SP-B and SP-C were functionally active and rapidly generated low surface tension on compression in a pulsating bubble surfactometer. These results suggest that hormonally differentiated human fetal type II cells do not select the molecular composition of surfactant phospholipid on the basis of saturation but, more likely, on the basis of acyl chain length.

Induction of cell death in activated hepatic stellate cells by targeted gene expression of the thymidine kinase/ganciclovir system

Liver fibrosis is the result from a relative imbalance between synthesis and degradation of matrix proteins. Following liver injury of any etiology, hepatic stellate cells undergo a response known as activation, which is the transition of quiescent cells into proliferative, fibrogenic, and contractile myofibroblasts. Upon this cellular transdifferentiation the effector cell becomes the major source of fibrillar and non-fibrillar matrix proteins resulting in excessive scar formation and cirrhosis, the end stage of fibrosis. Concomitant with progressive liver fibrosis, the tissue inhibitor of metalloproteinases-1 (TIMP-1) is strongly activated in hepatic stellate cells. We have developed a recombinant replication-defective adenovirus in which the TIMP-1 promoter is coupled to the herpes simplex virus thymidine kinase gene rendering activated hepatic stellate cells susceptible to ganciclovir. This novel targeted suicide gene approach was validated in a culture model considered to reflect an accelerated time course of the cellular and molecular events that occur during liver fibrosis. We demonstrate that transfer of the suicide gene to culture-activated hepatic stellate cells results in a strong expression of the respective transgene as assessed by Northern blot and Western blot analyses. The enzyme catalyzed the proper conversion of its prodrug subsequently initiating programmed cell death as estimated by caspase-3 assay and Annexin V-Fluos staining. Altogether, these results indicate that induction of programmed cell death is a promising approach to eliminate fibrogenic HSC.