Considerable evidence shows that the vestibular system contributes to adjusting sympathetic nervous system activity to maintain adequate blood pressure during movement and changes in posture. However, only a few prior experiments entailed recordings in conscious animals from brainstem neurons presumed to convey baroreceptor and vestibular inputs to neurons in the rostral ventrolateral medulla (RVLM) that provide inputs to sympathetic preganglionic neurons in the spinal cord. In this study, recordings were made in conscious felines from neurons in the medullary lateral tegmental field (LTF) and nucleus tractus solitarius (NTS) identified as regulating sympathetic nervous system activity by exhibiting changes in firing rate related to the cardiac cycle, or cardiac-related activity (CRA). Approximately 38% of LTF and NTS neurons responded to static 40° head up tilts with a change in firing rate (increase for 60% of the neurons, decrease for 40%) of ~50%. However, few of these neurons responded to 10° sinusoidal rotations in the pitch plane, in contrast to prior findings in decerebrate animals that the firing rates of both NTS and LTF neurons are modulated by small-amplitude body rotations. Thus, as previously demonstrated for RVLM neurons, in conscious animals NTS and LTF neurons only respond to large rotations that lead to changes in sympathetic nervous system activity. The similar responses to head-up rotations of LTF and NTS neurons with those documented for RVLM neurons suggest that LTF and NTS neurons are components of the vestibulo-sympathetic reflex pathway. However, a difference between NTS/LTF and RVLM neurons was variability in CRA over time. This variability was significantly greater for RVLM neurons, raising the hypothesis that the responsiveness of these neurons to baroreceptor input is adjusted based on the animal's vigilance and alertness.
Aldehyde dehydrogenase 1A1 (ALDH) is a cancer stem cell marker highly expressed in metastatic cells. Disulfiram (Dis) is an FDA-approved antialcoholism drug that inhibits ALDH and has been studied as a candidate for drug repurposing in multiple neoplasia. Dis cytotoxicity in cancer cells has been shown to be copper-dependent, in part due to Dis’s ability to function as a bivalent metal ion chelator of copper (Cu). The objectives of this research were to test ALDH expression levels and Cu concentrations in sarcoma patient tumors and human osteosarcoma (OS) cell lines with differing metastatic phenotypes. We also sought to evaluate Dis + Cu combination therapy in human OS cells. Intracellular Cu was inversely proportional to the metastatic phenotype in human OS cell lines (SaOS2 > LM2 > LM7). Nonmetastatic human sarcoma tumors demonstrated increased Cu concentrations compared with metastatic tumors. qPCR demonstrated that ALDH expression was significantly increased in highly metastatic LM2 and LM7 human OS cell lines compared with low metastatic SaOS2. Tumor cells from sarcoma patients with metastatic disease displayed significantly increased ALDH expression compared with tumor cells from patients without metastatic disease. Serum Cu concentration in canine OS versus normal canine patients demonstrated similar trends. Dis demonstrated selective cytotoxicity compared with human multipotential stromal cells (MSCs): Dis-treated OS cells demonstrated increased apoptosis, whereas MSCs did not. CuCl2 combined with Dis and low-dose doxorubicin resulted in a superior cytotoxic effect in both SaOS2 and LM7 cell lines. In summary, ALDH gene expression and Cu levels are altered between low and highly metastatic human OS cells, canine samples, and patient tumors. Our findings support the feasibility of a repurposed drug strategy for Dis and Cu in combination with low-dose anthracycline to specifically target metastatic OS cells.
Multiple Pearson's Chi-square of independence and Fisher's Exact tests evaluated comparisons of microaggressions by sex, race, Hispanic origin, sexual preference, and year in training. A multivariate regression analysis assessed associations between variables.Results: One hundred twenty-five participants responded to the survey (response rate: 10.2%). Of those who responded, 68.8% had experienced microaggressions in the past year. Female trainees experienced microaggressions more frequently than male trainees (p<0.05). Asians had higher odds to be a target of microaggressions compared to Caucasians (p=0.013). Non-heterosexual trainees were more likely to have experienced microaggressions compared to heterosexual trainees (p<0.05). Independent trainees were more likely to experience microaggressions than PGY 1-2 and 3-4 integrated residents (p<0.05). Conclusion:Approximately 7 in every 10 trainees stated that they experienced microaggressions in the past year. Females, racial minorities, sexual minorities, and independent trainees had higher odds of reporting that they experienced microaggressions. Further studies are needed to assess the implementation of strategies that address this problem in order to resolve inequities.
Considerable evidence shows that the vestibular system contributes to adjusting sympathetic nervous system activity to maintain adequate blood pressure during movement and changes in posture. However, only a few prior experiments entailed recordings in conscious animals from brainstem neurons presumed to convey baroreceptor and vestibular inputs to neurons in the rostral ventrolateral medulla (RVLM) that provide inputs to sympathetic preganglionic neurons in the spinal cord. In this study, recordings were made in conscious felines from neurons in the medullary lateral tegmental field (LTF) and nucleus tractus solitarius (NTS) identified as regulating sympathetic nervous system activity by exhibiting changes in firing rate related to the cardiac cycle, or cardiac-related activity (CRA). Approximately 38% of LTF and NTS neurons responded to static 40° head up tilts with a change in firing rate of approximately 50%. However, few of these neurons responded to 10° sinusoidal rotations in the pitch plane, in contrast to prior findings in decerebrate animals that the firing rates of both NTS and LTF neurons are modulated by small-amplitude body rotations. Thus, as previously demonstrated for RVLM neurons, in conscious animals NTS and LTF neurons only respond to large rotations that lead to changes in sympathetic nervous system activity. The similar responses to head-up rotations of LTF and NTS neurons with those documented for RVLM neurons suggest that LTF and NTS neurons are components of the vestibulo-sympathetic reflex pathway. However, a difference between NTS/LTF neurons and RVLM was variability in CRA over time. This variability was significantly greater for RVLM neurons, raising the hypothesis that the responsiveness of these neurons to baroreceptor input is adjusted based on the animal's vigilance and alertness.
Osteosarcoma (OS) is the most common primary bone cancer and disproportionately affects pediatric and adolescent patients. Survival rates for OS patients with metastatic disease is only 15-30% and has not improved in several decades. Novel treatments targeting specifically metastatic markers of OS are needed. ALDH1A1 is a known cancer stem marker and highly expressed in metastatic cancer cells. Disulfiram (Antabuse®) is an FDA-approved anti-alcoholism drug which acts as an ALDH inhibitor and has been widely studied for repurposing as an effective therapy in multiple types of cancer. Disulfiram cytotoxicity in cancer cells has been shown to be copper-dependent due to its ability to function as a bivalent metal ion chelator of copper (Cu). Cu concentrations of OS patient tumors, human OS cell lines, and OS patient plasma were determined using a Perkin Elmer AAnalyst 600 atomic absorption spectrophotometer. qPCR using custom primers for ALDH1A1 was performed on human OS cell lines and OS patient tumors differing in metastatic potential. OS cell cytotoxicity assays were performed with disulfiram and copper chloride after 24 hours of treatment. Balb/C mice were challenged intratibial with Luciferase+ K7M2, and treated with disulfiram and copper gluconate by oral gavage. Primary tumor growth and pulmonary metastases were monitored using the IVIS XR live imaging system. Metastatic OS patient tumors displayed significantly increased ALDH1A1 expression compared to non-metastatic OS patient tumors (n=8, P=0.02). Metastatic OS tumors displayed decreased Cu levels compared to non-metastatic OS tumors (n=18, P=0.05). Conversely, serum Cu levels from OS patients with metastases demonstrated increased blood Cu levels compared with non-metastatic patients (n=24, P=0.01). qPCR displayed ALDH1A1 expression was significantly increased in highly metastatic human OS cell lines LM2 and LM7 compared to low metastatic SaOS-2 (n=3, P=0.002). Intracellular Cu was inversely proportional to metastatic phenotype in human OS cell lines SaOS-2>LM2>LM7. Copper chloride displayed clear potentiation of disulfiram resulting in a strong cytotoxic effect in both SaOS-2 and LM7. Doxorubicin IC50 for SaOS-2 and LM7 was 1.2 µM and 2.5 µM respectively, while disulfiram IC50 with a 50 nM copper chloride supplement for SaOS-2 and LM7 was 1.2 µM and 0.6 µM respectively. In our preclinical mouse model, after primary tumor resection, differences in recurrent growth at amputation site were seen between treatment groups. Disulfiram and copper gluconate combination treated mice displayed 13% (1/8) recurrence rates at the primary tumor amputation site, while untreated animals displayed 60% (9/15) recurrence rates. This study demonstrates that both ALDH1A1 and Cu bioavailability are altered between low and highly metastatic OS. Since ALDH1A1 is a direct target and Cu is a potentiator of disulfiram, this supports the potential of using this repurposed drug in combination with surgical and chemotherapy interventions for metastatic OS. Citation Format: Jonathan B. Mandell, Nerone Douglas, Vrutika Ukani, Carolyn Anderson, Jan Beumer, Rebecca Watters, Kurt Weiss. Altered ALDH1A1 expression and cellular copper levels between low and highly metastatic osteosarcoma provides a case for novel repurposing of disulfiram [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 345.
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