Current Status and Future Direction to Address Disparities in Diversity, Equity, and Inclusion in Prostate Cancer Care.

PURPOSE OF REVIEW: Disparities in prostate cancer care and outcomes have been well recognized for decades. The purpose of this review is to methodically highlight known racial disparities in the care of prostate cancer patients, and in doing so, recognize potential strategies for overcoming these disparities moving forward. RECENT FINDINGS: Over the past few years, there has been a growing recognition and push towards addressing disparities in cancer care. This has led to improvements in care delivery trends and a narrowing of racial outcome disparities, but as we highlight in the following review, there is more to be addressed before we can fully close the gap in prostate cancer care delivery. While disparities in prostate cancer care are well recognized in the literature, they are not insurmountable, and progress has been made in identifying areas for improvement and potential strategies for closing the care gap.

Disparities in Lung Cancer Treatment.

PURPOSE OF REVIEW: Despite an overall reduction in lung cancer incidence and mortality rates worldwide, Blacks still have higher mortality rates compared to Whites. There are many factors that contribute to this difference. This review seeks to highlight racial disparities in treatment and the possible reasons for these disparities. RECENT FINDINGS: Factors attributing to racial disparities in lung cancer treatment include social determinants of health, differences in the administration of guideline-concordant therapy as well as molecular testing that is essential for most NSCLC patients. One way to circumvent disparities in lung cancer survivorship is to ensure equal representation of race in research at all levels that will provide insight on interventions that will address social determinants of health, differences in treatment patterns, molecular testing, and clinical trial involvement.

Expanding the prostate cancer cell line repertoire with ACRJ-PC28, an AR-negative neuroendocrine cell line derived from an African-Caribbean patient.

Prostate cell lines from diverse backgrounds are important to addressing disparities in prostate cancer (PCa) incidence and mortality rates among Black men. ACRJ-PC28 was developed from a transrectal needle biopsy and established via inactivation of the CDKN2A locus and simultaneous expression of human telomerase. Characterization assays included growth curve analysis, immunoblots, IHC, 3D cultures, immunofluorescence imaging, confocal microscopy, flow cytometry, WGS, and RNA-Seq. ACRJ-PC28 has been passaged more than 40 times in vitro over 10 months with a doubling time of 45 hours. STR profiling confirmed the novelty and human origin of the cell line. RNA-Seq confirmed the expression of prostate specific genes alpha-methylacyl-CoA racemase (AMACR) and NKX3.1 and Neuroendocrine specific markers synaptophysin (SYP) and enolase 2 (ENO2) and IHC confirmed the presence of AMACR. Immunoblots indicated the cell line is of basal-luminal type; expresses p53 and pRB and is AR negative. WGS confirmed the absence of exonic mutations and the presence of intronic variants that appear to not affect function of AR, p53, and pRB. RNA-Seq data revealed numerous TP53 and RB1 mRNA splice variants and the lack of AR mRNA expression. This is consistent with retention of p53 function in response to DNA damage and pRB function in response to contact inhibition. Soft agar anchorage-independent analysis indicated that the cells are transformed, confirmed by principal component analysis (PCA) where ACRJ-PC28 cells cluster alongside other PCa tumor tissues, yet was distinct. The novel methodology described should advance prostate cell line development, addressing the disparity in PCa among Black men.

Heterogeneity in head and neck cancer incidence among black populations from Africa, the Caribbean and the USA: Analysis of cancer registry data by the AC3.

BACKGROUND: Africa and the Caribbean are projected to have greater increases in Head and neck cancer (HNC) burden in comparison to North America and Europe. The knowledge needed to reinforce prevention in these populations is limited. We compared for the first time, incidence rates of HNC in black populations from African, the Caribbean and USA. METHODS: Annual age-standardized incidence rates (IR) and 95% confidence intervals (95%CI) per 100,000 were calculated for 2013-2015 using population-based cancer registry data for 14,911 HNC cases from the Caribbean (Barbados, Guadeloupe, Trinidad & Tobago, N = 443), Africa (Kenya, Nigeria, N = 772) and the United States (SEER, Florida, N = 13,696). We compared rates by sub-sites and sex among countries using data from registries with high quality and completeness. RESULTS: In 2013-2015, compared to other countries, HNC incidence was highest among SEER states (IR: 18.2, 95%CI = 17.6-18.8) among men, and highest in Kenya (IR: 7.5, 95%CI = 6.3-8.7) among women. Nasopharyngeal cancer IR was higher in Kenya for men (IR: 3.1, 95%CI = 2.5-3.7) and women (IR: 1.5, 95%CI = 1.0-1.9). Female oral cavity cancer was also notably higher in Kenya (IR = 3.9, 95%CI = 3.0-4.9). Blacks from SEER states had higher incidence of laryngeal cancer (IR: 5.5, 95%CI = 5.2-5.8) compared to other countries and even Florida blacks (IR: 4.4, 95%CI = 3.9-5.0). CONCLUSION: We found heterogeneity in IRs for HNC among these diverse black populations; notably, Kenya which had distinctively higher incidence of nasopharyngeal and female oral cavity cancer. Targeted etiological investigations are warranted considering the low consumption of tobacco and alcohol among Kenyan women. Overall, our findings suggest that behavioral and environmental factors are more important determinants of HNC than race.

Unintended Consequences: Ambiguity Neglect and Policy Ineffectiveness.

When a policymaker introduces a novel policy, she will not know what citizens' choices will be under the policy, and citizens themselves may have to construct new choice sets. This imparts inherent ambiguity to novel policy implementation: The policymaker does not know the probability that citizens will select actions that align with her policy. Assuming that citizens will follow a fixed approach may expose the policymaker to ambiguity neglect, which can result in unintended consequences. We provide examples and a simple formalization. Our results suggest that before implementing novel policies, policymakers should attempt to elicit preferences from citizens.

Modulatory Effect of Guinep (Melicoccus bijugatus Jacq) Fruit Pulp Extract on Isoproterenol-Induced Myocardial Damage in Rats. Identification of Major Metabolites Using High Resolution UHPLC Q-Orbitrap Mass Spectrometry.

Guinep is traditionally used in the management of cardiovascular ailments. This study aims to evaluate its medicinal constituents and effects in the management of myocardial injury in an experimental isoproterenol (ISO) rat model. Sprague-Dawley rats were randomly assigned to four groups: Group 1 was the control group; Group 2 received M. bijugatus extract (100 mg/Kg; MB) for six weeks; Group 3 was given ISO (85 mg/Kg) i.p. twice during a 24-hour period; and Group 4 was given ISO (85 mg/Kg) i.p. and MB extract (100 mg/Kg) for six weeks. The MB was administered orally by gavage, daily. The blood pressure of conscious animals was measured, while ECG was performed under anesthesia. Blood and serum were collected for biochemical and hematological analysis. The ISO group treated with MB showed a significant decrease (p < 0.001) in (SBP), diastolic (DBP), mean arterial (MAP) and heart rate (HR) compared to the ISO only group. Conversely, MB treated rats that were not induced with ISO displayed a significant decreases (p < 0.001) in SBP, DBP, MAP, and HR. ISO significantly elevated the ST segment (p < 0.001) and shortened the QTc interval (p < 0.05), which were recovered after treatment with 100 mg/Kg of MB. In addition, the results showed a significant decrease (p < 0.001) in the heart to body weight ratio of the ISO group treated with MB compared to the ISO only group. Furthermore, the extract normalized the hematological values depressed by the ISO while significantly elevating the platelet count. UHPLC high-resolution orbitrap mass spectrometry analysis results revealed the presence of several antioxidants like vitamin C and related compounds, phenolic acids, flavonoid, fatty acids (oxylipins), and terpene derivatives. The results of this study indicated that Melicoccus bijugatus did display some cardio-protective effects in relation to myocardial injury.

Transgenic swine: expression of human CD39 protects against myocardial injury.

UNLABELLED: CD39 (ectonucleoside triphosphate diphosphohydrolase-1; ENTPD-1) rapidly hydrolyzes ATP and ADP to AMP; AMP is hydrolyzed by ecto-5'-nucleotidase (CD73) to adenosine, an anti-thrombotic and cardiovascular protective mediator. While expression of human CD39 in a murine model of myocardial ischemia/reperfusion (I/R) injury confers cardiac protection, the translational therapeutic potential of these findings requires further testing in a large animal model. To determine if transgenic expression of CD39 reduces infarct size in a swine model of myocardial ischemia/reperfusion injury, transgenic pigs expressing human CD39 (hCD39) were generated via somatic cell nuclear transfer and characterized. Expression of hC39 in cardiac tissue was confirmed by immunoblot and immunohistochemistry. Myocardial I/R injury was induced by intracoronary balloon inflation in the left anterior descending (LAD) artery for 60 min followed by 3 hours of reperfusion. The ischemic area was delineated by perfusion with 5% phthalo blue and the myocardial infarct size was determined by triphenyl tetrazolium chloride (TTC) staining. During ischemia, the rate-pressure product was significantly lower in control versus hCD39-Tg swine. Following reperfusion, compared to littermate control swine, hCD39-Tg animals displayed a significant reduction in infarct size (hCD39-Tg: 17.2 ± 4.3% vs. CONTROL: 44.7 ± 5.2%, P=0.0025). Our findings demonstrate for the first time that the findings in transgenic mouse models translate to large animal transgenic models and validate the potential to translate CD39 into the clinical arena to attenuate human myocardial ischemia/reperfusion injury.

Isolation and in vitro characterization of putative porcine embryonic stem cells from cloned embryos treated with trichostatin A.

We report here the establishment and characterization of putative porcine embryonic stem cell (ESC) lines derived from somatic cell nuclear transfer embryos (NT-ESCs). These cells had a similar morphology to that described previously by us for ESCs derived from in vitro produced embryos, namely, a polygonal shape, a relatively small (10-15 µm) diameter, a small cytoplasmic/nuclear ratio, a single nucleus with multiple nucleoli and multiple lipid inclusions in the cytoplasm. NT-ESCs could be passaged at least 15 times and vitrified repeatedly without changes in their morphology, karyotype, or Oct-4 and Nanog expression. These cells formed embryoid bodies and could be directed to differentiate in vitro to cell types representative of all three germ layers. Following their injection into blastocysts, these cells preferentially localized in the inner cell mass. In conclusion, we have isolated putative porcine ESCs from cloned embryos that have the potential to be used for a variety of applications including as a model for human therapeutic cloning.

In vitro and in vivo characterization of putative porcine embryonic stem cells.

We have developed a new method for the isolation of porcine embryonic stem cells (ESCs) from in vivo-derived and in vitro-produced embryos. Here we describe the isolation and characterization of several ESC lines established using this method. Cells from these lines were passaged up to 14 times, during which they were repeatedly cryopreserved. During this time, ESCs maintained their morphology and continued to express Oct 4, Nanog, and SSEA1. These cells formed embryoid bodies in suspension culture, and could be directed to differentiate into various lineages representative of all three germ layers in vitro. When injected into blastocysts these cells localized in the inner cell mass of blastocysts. To examine their pluripotency further, cells were injected into host blastocysts and transferred to recipient animals. Of the six transfers undertaken, one recipient became pregnant and gave birth to a litter of one male and three female piglets. Microsatellite analysis of DNA extracted from the tail tissue of these piglets indicated that two female piglets were chimaeric.

Development of culture conditions for the isolation of pluripotent porcine embryonal outgrowths from in vitro produced and in vivo derived embryos.

In the present study we examined the effect of culture media and protein source on the formation of pluripotent primary outgrowths from in vitro produced and in vivo derived porcine embryos as the first step towards the isolation of embryonic stem cells (ESCs). To do this we compared high glucose Dulbeccos Modified Eagles Medium (DMEM) with Minimal Essential Alpha Medium (αMEM) both supplemented with fetal bovine serum (FBS) or serum replacement (SR) in a 2 × 2 factorial design. Culture in DMEM or αMEM supplemented with 10% SR resulted in the establishment of homogenous populations of cells which expressed Oct 4 and Nanog. In contrast culture in either media with FBS resulted in the formation of embryonal outgrowths composed entirely of differentiated cells or a mixture of differentiated cells and putative ESCs which grew poorly and could not be passaged. Using αMEM medium containing 10% SR and culturing in 5% oxygen, putative ESC lines were isolated from in vitro and in vivo derived embryos at efficiencies of 2 and 10% respectively.

A comparison of two in vitro maturation media for use with adult porcine oocytes for adult somatic cell nuclear transfer.

Two media used to mature adult porcine oocytes for somatic cell nuclear transfer were compared. In the first experiment, parthenogenetic embryos were produced using a maturation medium used by us previously to clone pigs (OMM199) and that described by Kühholzer et al. (2001) to transport oocytes overnight (BOMED). There was no difference in maturation rates between the two different media. However, BOMED medium increased the percentage of parthenogenetic embryos that developed to the blastocyst stage compared with OMM199 (49% vs. 29%, respectively). In a second experiment, BOMED medium increased the percentage of SCNT embryos that developed to the blastocyst stage compared with OMM199 (22% vs. 8%, respectively). The efficiency of our cloning protocol using adult oocytes matured in BOMED medium was then determined by transferring SCNT embryos reconstructed using adult fibroblasts to synchronized recipients. Primary cultures of adult fibroblasts were obtained from two adult male pigs and used for SCNT (passages 2-4). Between 82 and 146 fused couplets were transferred to seven recipients synchronized 1 day behind the embryos. Five recipients (71% pregnancy rate) subsequently farrowed a total of 23 piglets (4.4 average litter size). Overall efficiencies (liveborn/embryos transferred) were 3.2% for all transfers and 4.3% for animals that gave birth.

Production of homozygous alpha-1,3-galactosyltransferase knockout pigs by breeding and somatic cell nuclear transfer.

We report here our experience regarding the production of double or homozygous Gal knockout (Gal KO) pigs by breeding and somatic cell nuclear transfer (SCNT). Large White x Landrace female heterozygous Gal KO founders produced using SCNT were mated with Hampshire or Duroc males to produce a F1 generation. F1 heterozygous pigs were then bred to half-sibs to produce a F2 generation which contained Gal KO pigs. To determine the viability of mating Gal KO pigs with each other, one female F2 Gal KO pig was bred to a half-sib and subsequently a full-sib Gal KO. F1 and F2 heterozygous females were also mated to F2 Gal KO males. All three types of matings produced Gal KO pigs. To produce Gal KO pigs by SCNT, heterozygous F1s were bred together and F2 fetuses were harvested to establish primary cultures of Gal KO fetal fibroblasts. Gal KO embryos were transferred to five recipients, one of which became pregnant and had a litter of four piglets. Together our results demonstrate that Gal KO pigs can be produced by breeding with each other and by SCNT using Gal KO fetal fibroblasts.

Use of adult mesenchymal stem cells isolated from bone marrow and blood for somatic cell nuclear transfer in pigs.

Mesenchymal stem cells (MSCs) isolated from bone marrow were used to examine the hypothesis that a less differentiated cell type could increase adult somatic cell nuclear transfer (SCNT) efficiencies in the pig. SCNT embryos were produced using a fusion before activation protocol described previously and the rate at which these developed to the blastocyst stage compared with that using fibroblasts obtained from ear tissue from the same animal. The use of bone marrow MSCs did not increase cleavage rates compared with adult fibroblasts. However, the percentage of embryos that developed to the blastocyst stage was almost doubled, providing support for the hypothesis that a less differentiated cell can increase cloning efficiencies. As MSCs are relatively difficult to isolate from the bone marrow of live animals, a second experiment was undertaken to determine whether MSCs could be isolated from the peripheral circulation and used for SCNT. Blood MSCs were successfully isolated from four of the five pigs sampled. These cells had a similar differentiation capacity and marker profile to those isolated from bone marrow but did not result in increased rates of development. This is the first study to our knowledge, to report that MSCs can be derived from peripheral blood and used for SCNT for any species. These cells can be readily obtained under relatively sterile conditions compared with adult fibroblasts and as such, may provide an alternative cell type for cloning live animals.

An efficient method for producing alpha(1,3)-galactosyltransferase gene knockout pigs.

We have reported relatively efficient methods for somatic cell nuclear transfer and for knocking out the alpha(1,3)-galactosyltransferase (alpha1,3-GT) gene in porcine fetal fibroblasts using a nonisogenic promoterless construct approach. Here we report the production of alpha1,3-GT gene knockout pigs using these procedures. Seven alpha1,3-GT gene knockout cell clones were identified by long-range PCR from 108 neomycin resistant (neo(R)) colonies, giving a 6.5% targeting efficiency. Three cell clones were used for nuclear transfer. Nuclear transfer was performed using a fusion before activation protocol using in vitro-matured adult oocytes. Between 51 and 110 fused couplets were transferred to 10 recipients synchronized 1 day behind the embryos. Parturition was induced on day 115, and piglets were delivered by caesarean section. Four recipients gave birth to a total of 18 live piglets. All pigs were female, and all three clones resulted in the birth of live pigs. alpha1,3-GT gene knockout pigs were identified by long-range PCR and confirmed by Southern blot analysis. The efficiency (embryos transferred/piglets born) of our cloning protocol was 1.9% for all transfers and 4.6% for animals that gave birth.

Efficient generation of alpha(1,3) galactosyltransferase knockout porcine fetal fibroblasts for nuclear transfer.

Pigs are currently considered the most likely source of organs for human xenotransplantation because of anatomical and physiological similarities to humans, and the relative ease with which they can be bred in large numbers. A severe form of rejection known as hyperacute rejection has been the major barrier to the use of xenografts. Generating transgenic pigs for organ transplantation is likely to involve precise genetic manipulation to ablate the alpha(1,3) galactosyltransferase (galT) gene. In contrast to the mouse, homologous recombination in livestock species to ablate genes is hampered by the inability to isolate functional embryonic stem cells. However, nuclear transfer using genetically targeted cultured somatic cells provides an alternative means to producing pigs deficient for galT. In this study we successfully produced galT+/- somatic porcine fetal fibroblasts using two approaches; positive negative selection (PNS) using an isogenic targeting construct, and with a promoterless vector using non-isogenic DNA.

Production of cloned pigs from cultured fetal fibroblast cells.

Somatic cell nuclear transfer was used to produce live piglets from cultured fetal fibroblast cells. This was achieved by exposing donor cell nuclei to oocyte cytoplasm for approximately 3 h before activation by chemical means. Initially, an experiment was performed to optimize a cell fusion system that prevented concurrent activation in the majority of recipient cytoplasts. Cultured fibroblast cells were fused in medium with or without calcium into enucleated oocytes flushed from superovulated gilts. Cybrids fused in the presence of calcium cleaved at a significantly (P < 0.05) greater rate (69%, 37 out of 54) after 2 days of culture compared with those fused without calcium (10%, 7 out of 73), suggesting that calcium-free conditions are needed to avoid activation in the majority of recipient cytoplasts during fusion. In the second experiment, cybrids fused in calcium-free medium were activated approximately 3 h later with ionomycin, followed by incubation in 6-dimethylaminopurine to determine development in vitro. Following 2 days of culture, cleavage rates of chemically activated and unactivated cybrids (fusion without activation control) were 93% (100 out of 108) and 7% (2 out of 27), respectively. After an additional 5 days of culture, activated cloned embryos formed blastocysts at a rate of 23% (25 out of 108) with an average inner cell mass and trophectoderm cell number of 10 (range, 3 to 38) and 31 (range, 16 to 58), respectively. In the third experiment, activated nuclear transfer embryos were transferred to the uteri of synchronized recipients after 3 days of culture to assess their development in vivo. Of 10 recipients receiving an average of 80 cleaved embryos (range, 40 to 107), 5 became pregnant (50%) as determined by ultrasound between Day 25 and Day 35 of gestation. Of the five pregnant recipients, two subsequently farrowed one piglet per litter originating from two different cell culture lines. In this study, efficient reprogramming of porcine donor nuclei by fusing cells in the absence of calcium followed by chemical activation of recipient cytoplasts was reflected in high rates of development to blastocyst and pregnancy initiation leading to full term development.