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BACKGROUND: Typical gait is often considered to be highly symmetrical, with gait asymmetries typically associated with pathological gait. Whilst gait symmetry is often expressed in symmetry ratios, measures of symmetry do not provide insight into how these asymmetries affect gait variables. To fully understand changes caused by gait asymmetry, we must first develop a normative database for comparison. Therefore, the aim of this study was to describe normative reference values of regional plantar load and present comparisons with two pathological case studies. METHODS: A descriptive study of the load transfer of plantar pressures in typically developed children was conducted to develop a baseline for comparison of the effects of gait asymmetry in paediatric clinical populations. Plantar load and 3D kinematic data was collected for 17 typically developed participants with a mean age of 9.4 ± 4.0 years. Two case studies were also included; a 10-year-old male with clubfoot and an 8-year-old female with a flatfoot deformity. Data was analysed using a kinematics-pressure integration technique for anatomical masking into 5 regions of interest; medial and lateral forefoot, midfoot, and medial and lateral hindfoot. RESULTS: Clear differences between the two case studies and the typical dataset were seen for the load transfer phase of gait. For case study one, lateral bias was seen in the forefoot of the trailing foot across all variables, as well as increases in contact area, force and mean pressure in the lateral hindfoot of the leading foot. For case study two, the forefoot of the trailing foot produced results very similar to the typical dataset across all variables. In the hindfoot of the leading foot, medial bias presents most notably in the force and mean pressure graphs. CONCLUSIONS: This study highlights the clinical significance of the load transfer phase of gait, providing meaningful information for intervention planning.
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Pie Equinovaro , Pie , Adolescente , Fenómenos Biomecánicos , Niño , Preescolar , Femenino , Marcha , Humanos , Masculino , PresiónRESUMEN
PURPOSE: Circulating adipose stromal cells (CASC) are thought to be increased in obesity and facilitate angiogenesis, and tumor metastases. METHODS: CASC were identified from buffy coat peripheral blood mononuclear cells (PBMCs) by flow cytometry as CD34brightCD31- CD45- and CASC frequency was compared to adiposity measures in 33 women at increased risk for breast cancer. Feasibility of CASC as a response biomarker for a diet and exercise intervention in ten breast cancer survivors was then explored. RESULTS: For 33 high-risk women, median CASC frequency was 9.7 per million PBMCs and trended positively with body mass index, fat mass index (FMI), and percent android fat. Correlation was significant when BMI was dichotomized at > versus < 35 kg/m2 (p = 0.02). For ten breast cancer survivors with a median BMI of 37 kg/m2, median CASC frequency was 16.4 per million PBMCs. In univariate analyses, change in BMI, total fat and visceral fat were significantly correlated with change in CASC frequency. On multivariate analysis, change in visceral adipose had the strongest association with change in CASC frequency (p < 0.00078). CONCLUSIONS: The association between the reduction in visceral adipose tissue and the decrease in frequency of circulating adipose stromal cells suggests that the latter might be a useful biomarker in clinical trials of obese breast cancer survivors undergoing a weight loss intervention.
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Tejido Adiposo/inmunología , Biomarcadores/sangre , Neoplasias de la Mama/sangre , Obesidad/terapia , Tejido Adiposo/citología , Anciano , Antígenos CD34/metabolismo , Neoplasias de la Mama/inmunología , Supervivientes de Cáncer , Estudios Transversales , Dietoterapia , Terapia por Ejercicio , Femenino , Humanos , Antígenos Comunes de Leucocito/metabolismo , Persona de Mediana Edad , Obesidad/sangre , Obesidad/inmunología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Posmenopausia , Premenopausia , Células del Estroma/citología , Células del Estroma/inmunologíaRESUMEN
We conducted a phase II feasibility study of a 6-month behavioral weight loss intervention in postmenopausal overweight and obese women at increased risk for breast cancer and the effects of weight loss on anthropomorphic, blood, and benign breast tissue biomarkers. 67 women were screened by random peri-areolar fine-needle aspiration, 27 were registered and 24 participated in the interventional phase. The 24 biomarker evaluable women had a median baseline BMI of 34.2 kg/m(2) and lost a median of 11 % of their initial weight. Significant tissue biomarker modulation after the 6-month intervention was noted for Ki-67 (if restricted to the 15 women with any Ki-67 at baseline, p = 0.041), adiponectin to leptin ratio (p = 0.003); and cyclin B1 (p = 0.001), phosphorylated retinoblastoma (p = 0.005), and ribosomal S6 (p = 0.004) proteins. Favorable modulation for serum markers was observed for sex hormone-binding globulin (p < 0.001), bioavailable estradiol (p < 0.001), bioavailable testosterone (p = 0.033), insulin (p = 0.018), adiponectin (p = 0.001), leptin (p < 0.001), the adiponectin to leptin ratio (p < 0.001), C-reactive protein (p = 0.002), and hepatocyte growth factor (p = 0.011). When subdivided by <10 or >10 % weight loss, change in percent total body and android (visceral) fat, physical activity, and the majority of the serum and tissue biomarkers were significantly modulated only for women with >10 % weight loss from baseline. Some factors such as serum PAI-1 and breast tissue pS2 (estrogen-inducible gene) mRNA were not significantly modulated overall but were when considering only those with >10 % weight loss. In conclusion, a median weight loss of 11 % over 6 months resulted in favorable modulation of a number of anthropomorphic, breast tissue and serum risk and mechanistic markers. Weight loss of 10 % or more should likely be the goal for breast cancer risk reduction studies in obese women.
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Neoplasias de la Mama/sangre , Mama/patología , Posmenopausia/sangre , Pérdida de Peso , Adipoquinas/genética , Adipoquinas/metabolismo , Anciano , Antropometría , Biomarcadores/sangre , Biopsia con Aguja Fina , Mama/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Dieta , Femenino , Expresión Génica , Humanos , Antígeno Ki-67/metabolismo , Persona de Mediana Edad , Actividad Motora , Proyectos Piloto , Posmenopausia/genética , Posmenopausia/metabolismo , Proteómica , Calidad de Vida , Factores de RiesgoRESUMEN
BACKGROUND: Auslan is used by the Australian deaf community and relies heavily on hand, wrist, and elbow movement. Upper limb injury or dysfunction may require surgical intervention to alleviate pain and provide a stable skeleton for function, leading to partial or complete reduction in motion. The aim of this study was to assess the wrist, forearm, and elbow motion required to communicate via Auslan, to tailor optimal interventions in this population. METHODS: A biomechanical analysis was conducted on two native Auslan communicators, who signed 28 pre-selected and common Auslan words and phrases. RESULTS: Sagittal plane wrist and elbow motion was found to be of greater importance than axial plane forearm rotation. Relative elbow flexion and generous wrist motion was common for many of the words and phrases, while end-range elbow extension was not recorded. CONCLUSION: The maintenance of wrist and elbow motion should be prioritized when selecting surgical interventions for patients who communicate using Auslan.
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Clinical motion analysis involves quantitative measurement of gait patterns to identify gait anomalies that currently or have the potential to impact function, activities of daily living and participation. Clinical motion analysis services are equipped with motion capture technology and comprise specialised staff who deliver 3-dimensional motion analysis services to children and adults who present with varying levels of gait impairment. Data is then used to inform intervention recommendations to clinicians with a view to maintaining independent, functional and pain free walking (or appropriate mobility). The ANZ-CMAG (established in 2013) identified a need to establish recommendations to assist in standardising practice guidelines for both current and new clinical motion analysis services within the region. The group serves to promote collaboration between services in quality assurance processes, clinical practices, data sets and research activities. The clinical practice recommendations described in this paper cover: i) requirements for a motion analysis service (including staffing, facilities and equipment), ii) patient assessments (requirements, clinical information and data gathered, reporting and interpretation of patient data), iii) quality assurance processes (including motion capture system / biomechanical models & limitations, marker placement, data storage / record keeping, creation of normative dataset); iv) helpful resources. Better outcomes for children and adults with gait deviations is dependent upon accurate measurement and evaluation of walking and requires input from multidisciplinary clinical teams with specialist knowledge and skills. The ANZ-CMAG hopes these clinical practice recommendations are beneficial to motion analysis services with an aim to improve clinical practices, patient outcomes, and support research collaboration.
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INTRODUCTION: There is currently no clear indication in the literature regarding a single or double hamstring tendon (single bundle) autograft for anterior cruciate ligament (ACL) reconstruction in the paediatric patient. The primary aim of this single blind randomised controlled trial is to determine whether a single or double hamstring tendon graft ACLR leads to superior clinical outcomes postsurgery in paediatric patients with ACL injury. METHODS AND ANALYSIS: Single site, prospective, single blind, randomised controlled trial with two parallel treatment arms. 100 patients aged 10-18 years who present with an isolated ACL tear±meniscal injury, verified on MRI, will be randomly allocated to one of the two surgical groups. The primary outcomes will be side-to-side difference in anterior tibial translation and graft failure incidence 12 months postsurgery. Primary and secondary outcomes will also be assessed at 2-year and 5-year postsurgery. ETHICS AND DISSEMINATION: Results will be presented in peer-reviewed journals and at international conferences and disseminated to participants and healthcare professionals via newsletters and hospital presentations. This study is approved by the Children's Health Queensland Hospital and Health Service Human Research Ethics committee. TRIAL REGISTRATION NUMBER: ACTRN12620001170910p; Australian New Zealand Clinical Trials Registry.
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Lesiones del Ligamento Cruzado Anterior , Reconstrucción del Ligamento Cruzado Anterior , Tendones Isquiotibiales , Traumatismos de la Rodilla , Lesiones del Ligamento Cruzado Anterior/cirugía , Reconstrucción del Ligamento Cruzado Anterior/efectos adversos , Australia , Niño , Tendones Isquiotibiales/trasplante , Humanos , Traumatismos de la Rodilla/cirugía , Articulación de la Rodilla/cirugía , Estudios Prospectivos , Ensayos Clínicos Controlados Aleatorios como Asunto , Método Simple CiegoRESUMEN
The incidence of pancreatic cancer is increasing significantly and will soon become the second leading cause of cancer-related deaths in the United States. We have previously shown that the gastrointestinal peptide gastrin, which is only expressed in the fetal pancreas and not in the adult pancreas, is activated during pancreatic carcinogenesis where it stimulates growth in an autocrine fashion. In this investigation, we used transgenic LSL-KrasG12D/+; P48-Cre mice that develop precancerous pancreatic intraepithelial neoplasia (PanIN) lesions and pancreatic cancer over time. Starting at 3 months of age, mice were either left untreated (control) or were treated with a gastrin-targeted vaccine, polyclonal antibody stimulator (PAS 250 µg) followed by a monthly booster until the mice reached 8 months of age when pancreata were excised, and analyzed by histology for PanIN grade in a blinded fashion. High-grade PanIN-3 lesions were significantly less in PAS-treated mice (P = 0.0077), and cancers developed in 33% of the control mice but only in 10% of the PAS-treated mice. Compared with the control mice, fibrosis was reduced by >50%, arginase positive M2 macrophages were reduced by 74%, and CD8+ T cells were increased by 73% in the pancreas extracellular matrix in PAS-treated mice. PREVENTION RELEVANCE: PAS vaccination significantly decreased high-grade PanIN lesions and altered the pancreas microenvironment, rendering it less carcinogenic.
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Vacunas contra el Cáncer/uso terapéutico , Neoplasias Pancreáticas/prevención & control , Animales , Anticuerpos/uso terapéutico , Carcinogénesis/genética , Carcinogénesis/inmunología , Carcinogénesis/patología , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Páncreas/inmunología , Páncreas/patología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas p21(ras)/genética , Microambiente Tumoral/genética , Microambiente Tumoral/inmunología , Vacunación/métodos , Neoplasias PancreáticasRESUMEN
Gastric cancer is a leading cause of cancer-related deaths worldwide. Recently, clinical studies have demonstrated that many of those with advanced gastric cancer are responsive to immune checkpoint antibody therapy, although the median survival even with these new agents is less than 12 months for advanced disease. The gastrointestinal peptide gastrin has been shown to stimulate growth of gastric cancer in a paracrine and autocrine fashion through the cholecystokinin-B receptor (CCK-BR), a receptor that is expressed in at least 56.6% of human gastric cancers. In the current investigation, we studied the role of the gastrin-CCK-BR pathway in vitro and in vivo as well as the expression of the CCK-BR in a human gastric cancer tissue array. CCK-BR and PD-L1 receptor expression and gastrin peptide was found in two murine gastric cancer cells (NCC-S1 and YTN-16) by qRT-PCR and immunocytochemistry. Treatment of NCC-S1 cells with gastrin resulted in increased growth. In vivo, the effects of a cancer vaccine that targets gastrin peptide (polyclonal antibody stimulator-PAS) alone or in combination with a Programed Death-1 antibody (PD-1 Ab) was evaluated in immune competent mice (N = 40) bearing YTN-16 gastric tumors. Mice were treated with PBS, PD-1 Ab (50 µg), PAS (250 µg), or the combination of PD-1 Ab with PAS. Tumor growth was significantly slower than controls in PAS-treated mice, and tumor growth was decreased even more in combination-treated mice. There were no metastases in any of the mice treated with PAS either alone or in combination with PD-1 Ab. Tumor proliferation by the Ki67 staining was significantly decreased in mice treated with PAS monotherapy or the combination therapy. PAS monotherapy or combined with PD-1 Ab increased tumor CD8+ T-lymphocytes and decreased the number of immunosuppressive M2-polarized tumor-associated macrophages. CCK-BR expression was identified in samples from a human tissue array by immunohistochemistry confirming the clinical relevance of this study. These results confirm the significance of the gastrin-CCK-BR signaling pathway in gastric cancer and suggest that the addition of a gastrin vaccine, PAS, to therapy with an immune checkpoint antibody may decrease growth and metastases of gastric cancer by altering the tumor microenvironment.
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The inflammation-resolving and insulin-sensitizing properties of eicosapentaenoic (EPA) and docosahexaenoic (DHA) fatty acids have potential to augment effects of weight loss on breast cancer risk. In a feasibility study, 46 peri/postmenopausal women at increased risk for breast cancer with a body mass index (BMI) of 28 kg/m2 or greater were randomized to 3.25 g/day combined EPA and DHA (ω-3-FA) or placebo concomitantly with initiation of a weight-loss intervention. Forty-five women started the intervention. Study discontinuation for women randomized to ω-3-FA and initiating the weight-loss intervention was 9% at 6 months and thus satisfied our main endpoint, which was feasibility. Between baseline and 6 months significant change (P < 0.05) was observed in 12 of 25 serum metabolic markers associated with breast cancer risk for women randomized to ω-3-FA, but only four for those randomized to placebo. Weight loss (median of 10% for trial initiators and 12% for the 42 completing 6 months) had a significant impact on biomarker modulation. Median loss was similar for placebo (-11%) and ω-3-FA (-13%). No significant change between ω-3-FA and placebo was observed for individual biomarkers, likely due to sample size and effect of weight loss. Women randomized to ω-3-FA exhibiting more than 10% weight loss at 6 months showed greatest biomarker improvement including 6- and 12-month serum adiponectin, insulin, omentin, and C-reactive protein (CRP), and 12-month tissue adiponectin. Given the importance of a favorable adipokine profile in countering the prooncogenic effects of obesity, further evaluation of high-dose ω-3-FA during a weight-loss intervention in obese high-risk women should be considered. PREVENTION RELEVANCE: This study examines biomarkers of response that may be modulated by omega-3 fatty acids when combined with a weight-loss intervention. While focused on obese, postmenopausal women at high risk for development of breast cancer, the findings are applicable to other cancers studied in clinical prevention trials.
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Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/prevención & control , Ácidos Grasos Omega-3/administración & dosificación , Pérdida de Peso/fisiología , Programas de Reducción de Peso , Adulto , Anciano , Terapia Conductista , Biomarcadores de Tumor/sangre , Mama/metabolismo , Mama/patología , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Restricción Calórica , Citodiagnóstico , Suplementos Dietéticos , Ejercicio Físico/fisiología , Estudios de Factibilidad , Femenino , Humanos , Persona de Mediana Edad , Estadificación de Neoplasias , Obesidad/dietoterapia , Obesidad/metabolismo , Obesidad/terapia , Placebos , Lesiones Precancerosas/diagnóstico , Lesiones Precancerosas/metabolismo , Lesiones Precancerosas/patología , Programas de Reducción de Peso/métodosRESUMEN
Aerobic exercise reduces risk for breast cancer and recurrence and promotes visceral adipose tissue (VAT) loss in obesity. However, few breast cancer survivors achieve recommended levels of moderate to vigorous physical activity (MVPA) without supervision. In a two-cohort study, feasibility of 12 weeks of partially supervised exercise was started concomitantly with caloric restriction and effects on body composition and systemic risk biomarkers were explored. In total, 22 obese postmenopausal sedentary women (including 18 breast cancer survivors) with median age of 60 and BMI of 37 kg/m2 were enrolled. Using personal trainers twice weekly at area YMCAs, MVPA was escalated to ≥200 min/week over 9 weeks. For cohort 2, maintenance of effect was assessed when study provided trainer services were stopped but monitoring, group counseling sessions, and access to the exercise facility were continued. Median post-escalation MVPA was 219 min/week with median 12-week mass and VAT loss of 8 and 19%. MVPA was associated with VAT loss which was associated with improved adiponectin:leptin ratio. In total, 9/11 of cohort-2 women continued the behavioral intervention for another 12 weeks without trainers. High MVPA continued with median 24-week mass and VAT loss of 12 and 29%. This intervention should be further studied in obese sedentary women.
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BACKGROUND: Obesity is associated with worse breast cancer prognosis, however little is known about the level of weight loss required to improve pathway biomarkers. The effects of weight regain on biomarkers are also largely unknown. METHODS: Overweight/obese breast cancer survivors enrolled in an 18-month behavioral weight loss trial provided weight and serum biomarkers [leptin, adiponectin, insulin, plasminogen activator inhibitor-1 (PAI-1), IL-6, TNFα, and hepatocyte growth factor HGF] at baseline, 6, and 18 months (n = 138). Change in biomarkers over time and by weight loss thresholds were examined. RESULTS: Mean weight loss at 6 months was 13.3 ± 5.0 kg; from 6 to 18 months, mean regain was 4.0 ± 5.2 kg. Favorable biomarker modulations were observed at 6 months for leptin, adiponectin, insulin, PAI-1, IL-6, and HGF (P < 0.006 to P < 0.0001). These changes remained significant overall at 18 months despite attenuation in some. Women who lost <10% of baseline weight showed significantly smaller modulation effects for leptin (P < 0.0001), adiponectin:leptin (A/L) ratio (P < 0.0001), PAI-1 (P < 0.001), and insulin (P = 0.003) compared with women who lost >10%. Women who lost >10% observed a significant increase in adiponectin (P < 0.0001), and these women continued to show improved adiponectin from 6 to 18 months despite weight regain. Physical activity contributed additional effects on biomarker change for leptin, A/L ratio, and PAI-1. CONCLUSIONS: These findings are consistent with a clinical target of 10% weight. IMPACT: Sustained increases in adiponectin likely confer benefits for breast cancer prognosis even with weight regain.
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Biomarcadores/metabolismo , Neoplasias de la Mama/complicaciones , Obesidad/fisiopatología , Sobrepeso/fisiopatología , Aumento de Peso/fisiología , Pérdida de Peso/fisiología , Anciano , Neoplasias de la Mama/mortalidad , Supervivientes de Cáncer , Femenino , Humanos , Población Rural , Análisis de SupervivenciaRESUMEN
We conducted a multiinstitutional, placebo-controlled phase IIB trial of the lignan secoisolariciresinol diglucoside (SDG) found in flaxseed. Benign breast tissue was acquired by random periareolar fine needle aspiration (RPFNA) from premenopausal women at increased risk for breast cancer. Those with hyperplasia and ≥2% Ki-67 positive cells were eligible for randomization 2:1 to 50 mg SDG/day (Brevail) versus placebo for 12 months with repeat bio-specimen acquisition. The primary endpoint was difference in change in Ki-67 between randomization groups. A total of 180 women were randomized, with 152 ultimately evaluable for the primary endpoint. Median baseline Ki-67 was 4.1% with no difference between arms. Median Ki-67 change was -1.8% in the SDG arm (P = 0.001) and -1.2% for placebo (P = 0.034); with no significant difference between arms. As menstrual cycle phase affects proliferation, secondary analysis was performed for 117 women who by progesterone levels were in the same phase of the menstrual cycle at baseline and off-study tissue sampling. The significant Ki-67 decrease persisted for SDG (median = -2.2%; P = 0.002) but not placebo (median = -1.0%). qRT-PCR was performed on 77 pairs of tissue specimens. Twenty-two had significant ERα gene expression changes (<0.5 or >2.0) with 7 of 10 increases in placebo and 10 of 12 decreases for SDG (P = 0.028), and a difference between arms (P = 0.017). Adverse event incidence was similar in both groups, with no evidence that 50 mg/day SDG is harmful. Although the proliferation biomarker analysis showed no difference between the treatment group and the placebo, the trial demonstrated use of SDG is tolerable and safe.
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Neoplasias de la Mama/tratamiento farmacológico , Butileno Glicoles/uso terapéutico , Glucósidos/uso terapéutico , Hiperplasia/tratamiento farmacológico , Lignanos/uso terapéutico , Premenopausia , Adulto , Neoplasias de la Mama/patología , Femenino , Lino/química , Estudios de Seguimiento , Humanos , Hiperplasia/patología , Persona de Mediana Edad , Proyectos Piloto , Pronóstico , Factores de Riesgo , Adulto JovenRESUMEN
Interventions that relieve vasomotor symptoms while reducing risk for breast cancer would likely improve uptake of chemoprevention for perimenopausal and postmenopausal women. We conducted a pilot study with 6 months of the tissue selective estrogen complex bazedoxifene (20 mg) and conjugated estrogen (0.45 mg; Duavee) to assess feasibility and effects on risk biomarkers for postmenopausal breast cancer. Risk biomarkers included fully automated mammographic volumetric density (Volpara), benign breast tissue Ki-67 (MIB-1 immunochemistry), and serum levels of progesterone, IGF-1, and IGFBP3, bioavailable estradiol and testosterone. Twenty-eight perimenopausal and postmenopausal women at increased risk for breast cancer were enrolled: 13 in cohort A with baseline Ki-67 < 1% and 15 in cohort B with baseline Ki-67 of 1% to 4%. All completed the study with > 85% drug adherence. Significant changes in biomarkers, uncorrected for multiple comparisons, were a decrease in mammographic fibroglandular volume (P = 0.043); decreases in serum progesterone, bioavailable testosterone, and IGF-1 (P < 0.01), an increase in serum bioavailable estradiol (P < 0.001), and for women from cohort B a reduction in Ki-67 (P = 0.017). An improvement in median hot flash score from 15 at baseline to 0 at 6 months, and menopause-specific quality-of-life total, vasomotor, and sexual domain scores were also observed (P < 0.001). Given the favorable effects on risk biomarkers and patient reported outcomes, a placebo-controlled phase IIB trial is warranted.
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Biomarcadores de Tumor , Densidad de la Mama/efectos de los fármacos , Neoplasias de la Mama/etiología , Estrógenos Conjugados (USP)/farmacología , Indoles/farmacología , Sistema Vasomotor/efectos de los fármacos , Anciano , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/sangre , Mama/efectos de los fármacos , Mama/patología , Neoplasias de la Mama/sangre , Neoplasias de la Mama/diagnóstico , Estradiol/sangre , Terapia de Reemplazo de Estrógeno/métodos , Estrógenos Conjugados (USP)/uso terapéutico , Estudios de Factibilidad , Femenino , Humanos , Indoles/uso terapéutico , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/sangre , Factor I del Crecimiento Similar a la Insulina/análisis , Factor I del Crecimiento Similar a la Insulina/metabolismo , Antígeno Ki-67/análisis , Antígeno Ki-67/sangre , Mamografía , Menopausia/sangre , Menopausia/efectos de los fármacos , Menopausia/fisiología , Persona de Mediana Edad , Proyectos Piloto , Posmenopausia , Progesterona/sangre , Calidad de Vida , Factores de Riesgo , Testosterona/sangreRESUMEN
Human monocytes and macrophages, which are also called mononuclear phagocytes, represent a major arm of the innate immune system. These cells not only protect against infection but are also central to tissue remodeling and production of chemokines, cytokines, and growth factors. Tissue macrophages reside in the human placenta and uterine decidua throughout pregnancy, where they comprise part of the host defense network and facilitate placental and extraembryonic development. The purpose of this chapter is to describe methods for establishing useful models of human uteroplacental macrophages: (1) differentiated U937 myelomonocytic cells, (2) peripheral blood monocytes, (3) peripheral blood monocyte-derived macrophages, (4) decidual macrophages, and (5) placental macrophages.
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Técnicas de Cultivo de Célula/métodos , Separación Celular/métodos , Macrófagos/fisiología , Placenta/inmunología , Útero/inmunología , Medios de Cultivo , Endometrio/inmunología , Femenino , Humanos , Leucocitos Mononucleares , Macrófagos/citología , Monocitos/citología , Embarazo , Células U937/fisiologíaRESUMEN
The human major histocompatibility complex (MHC) contains genes encoding the Human Leukocyte Antigens (HLA). Of these antigens, placental immunologists need study only the HLA class I molecules, because HLA class II expression is repressed in the fetal placental cells that are in direct contact with maternal blood and tissues containing maternal immune cells. The class I antigens are subdivided into two general categories. The class Ia antigens are highly polymorphic and are typified by HLA-A, -B, and -C; these are expressed by nearly all somatic cells and stimulate graft rejection when foreign to the host. By contrast, the HLA class Ib antigens, HLA-E, -F, and -G, have restricted expression, few variants, and appear rarely to be immunostimulatory. One class Ia antigen, HLA-C, and the three class Ib antigens are differentially expressed by trophoblast cell subpopulations. In order to understand immune privilege in the pregnant uterus and placenta, it is essential to study the unique structural and functional features of these four genes and their glycoprotein products. In this chapter, we focus on the first class Ib gene identified in human placentas, HLA-G, with emphasis on its two soluble isoforms, HLA-G5 and HLA-G6. We describe methods developed in our laboratory to distinguish mRNAs encoding HLA-G5 and HLA-G6, and antibody-based protocols for identification of the soluble isoforms.
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Antígenos HLA/análisis , Antígenos de Histocompatibilidad Clase I/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Citometría de Flujo/métodos , Antígenos HLA/química , Antígenos HLA-G , Antígenos de Histocompatibilidad Clase I/química , Humanos , Immunoblotting/métodos , Inmunohistoquímica/métodos , Inmunoprecipitación , Isoformas de Proteínas , ARN Mensajero/análisisRESUMEN
Experimentation with most human cell types is restricted to the use of cell lines, and this limits our ability to extrapolate interpretations to the in vivo condition. However, in studying human trophoblast cells, we have a unique opportunity to obtain large quantities of readily available human tissue. In this chapter, we outline the methodology for purification of human trophoblast cells from term placentas. The procedures are based on enzymatic dissociation of villous placental tissue, followed by gradient centrifugation and immunomagnetic bead purification. Purity may be assessed by immunocytochemistry or flow cytometry using a number of markers to identify both cytotrophoblast cells and cellular contaminants. The resulting cytotrophoblast cell populations have excellent viability and purity, and may be subjected to long-term culture.
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Técnicas de Cultivo de Célula/métodos , Separación Celular/métodos , Trofoblastos/citología , Supervivencia Celular , Células Cultivadas , Vellosidades Coriónicas , Femenino , Humanos , Separación Inmunomagnética , Placenta/citología , Embarazo , Nacimiento a TérminoRESUMEN
Apoptosis-inducing tumor necrosis factor (TNF) ligands and receptors have been reported in human placentas, but the expression patterns of family members lacking this function [a proliferation-inducing ligand (APRIL), B lymphocyte stimulator (BLyS), CD30L/CD153, CD40L/CD154, TNF-related activation-induced cytokine, CD27L/CD70, OX40L, activation-inducible TNF receptor ligand (AITRL)] are incompletely documented or unknown. We therefore investigated expression of these eight ligands and nine of their receptors (B cell maturation antigen, transmembrane activator and calcium-modulator and cyclophilin ligand-interactor, CD30, CD40, receptor activator of nuclear factor-kappaB, osteoprotegerin, CD27, OX40/CD134, AITR). Analysis by reverse transcriptase-polymerase chain reaction revealed mRNAs encoding only three of the ligands (APRIL, BLyS, CD30L/CD153). Immunoblots demonstrated all three proteins in first-trimester and term placentas, and immunohistochemical experiments showed that expression was cell-specific and gestation-related. Although mRNAs encoding receptors for the three expressed ligands were absent, those encoding receptors for all of the unexpressed ligands were detectable. Collectively, the results are consistent with the postulate that nonapoptosis-inducing, placenta-derived TNF superfamily cytokines contribute to the T helper cell type 2 bias required for successful pregnancy. Patterns of placental expression of receptors suggest bidirectional maternal-fetal cytokine communication.
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Linfocitos B/inmunología , Proteínas de la Membrana , Placenta/inmunología , Embarazo , Receptores del Factor de Necrosis Tumoral/análisis , Células Th2/inmunología , Receptor del Factor Activador de Células B , Ligando CD30 , Femenino , Humanos , Inmunohistoquímica , Ligandos , Glicoproteínas de Membrana/análisis , Neuropéptidos/análisis , Proteínas Nucleares/análisis , Placenta/química , Placenta/citología , Primer Trimestre del Embarazo , ARN Mensajero/análisis , Receptores del Factor de Necrosis Tumoral/genéticaRESUMEN
The purpose of this study was to assess the feasibility of using the selective estrogen receptor modulator (SERM) acolbifene as a breast cancer prevention agent in premenopausal women. To do so, we assessed change in proliferation in benign breast tissue sampled by random periareolar fine-needle aspiration (RPFNA) as a primary endpoint, along with changes in other risk biomarkers and objective and subjective side effects as secondary endpoints. Twenty-five women with cytologic hyperplasia ± atypia and ≥2% of breast epithelial cells staining positive for Ki-67, received 20 mg acolbifene daily for 6-8 months, and then had benign breast tissue and blood risk biomarkers reassessed. Ki-67 decreased from a median of 4.6% [interquartile range (IQR), 3.1%-8.5%] at baseline to 1.4% (IQR, 0.6%-3.5%) after acolbifene (P < 0.001; Wilcoxon signed-rank test), despite increases in bioavailable estradiol. There were also significant decreases in expression (RT-qPCR) of estrogen-inducible genes that code for pS2, ERα, and progesterone receptor (P ≤ 0.026). There was no significant change in serum IGF1, IGFBP3, IGF1:IGFBP3 ratio, or mammographic breast density. Subjective side effects were minimal with no significant increase in hot flashes, muscle cramps, arthralgias, or fatigue. Objective measures showed a clinically insignificant decrease in lumbar spine bone density (DEXA) and an increase in ovarian cysts but no change in endometrial thickness (sonography). In summary, acolbifene was associated with favorable changes in benign breast epithelial cell proliferation and estrogen-inducible gene expression but minimal side effects, suggesting a phase IIB placebo-controlled trial evaluating it further for breast cancer prevention.
Asunto(s)
Neoplasias de la Mama/prevención & control , Mama/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Piperidinas/uso terapéutico , Premenopausia , Moduladores Selectivos de los Receptores de Estrógeno/uso terapéutico , Adulto , Biopsia con Aguja Fina , Densidad Ósea/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Femenino , Humanos , Antígeno Ki-67/análisis , Antígeno Ki-67/biosíntesis , Persona de Mediana Edad , Quistes Ováricos/epidemiología , Proyectos Piloto , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Riesgo , Transcriptoma/efectos de los fármacosRESUMEN
Associational studies suggest higher intakes/blood levels of the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) relative to the omega-6 arachidonic acid (AA) are associated with reduced breast cancer risk. We performed a pilot study of high-dose EPA + DHA in postmenopausal women to assess feasibility before initiating a phase IIB prevention trial. Postmenopausal women with cytologic evidence of hyperplasia in their baseline random periareolar fine needle aspiration (RPFNA) took 1,860 mg EPA +1500 mg DHA ethyl esters daily for 6 months. Blood and breast tissue were sampled at baseline and study conclusion for exploratory biomarker assessment, with P values uncorrected for multiple comparisons. Feasibility was predefined as 50% uptake, 80% completion, and 70% compliance. Trial uptake by 35 study entrants from 54 eligible women was 65%, with 97% completion and 97% compliance. Favorable modulation was suggested for serum adiponectin (P = 0.0027), TNFα (P = 0.016), HOMA 2B measure of pancreatic ß cell function (P = 0.0048), and bioavailable estradiol (P = 0.039). Benign breast tissue Ki-67 (P = 0.036), macrophage chemoattractant protein-1 (P = 0.033), cytomorphology index score (P = 0.014), and percent mammographic density (P = 0.036) were decreased with favorable effects in a proteomics array for several proteins associated with mitogen signaling and cell-cycle arrest; but no obvious overall effect on proteins downstream of mTOR. Although favorable risk biomarker modulation will need to be confirmed in a placebo-controlled trial, we have demonstrated feasibility for development of high-dose EPA and DHA ethyl esters for primary prevention of breast cancer.
Asunto(s)
Biomarcadores de Tumor/sangre , Neoplasias de la Mama/prevención & control , Ácidos Docosahexaenoicos/uso terapéutico , Ácido Eicosapentaenoico/uso terapéutico , Ácidos Grasos Omega-3/sangre , Adulto , Anciano , Neoplasias de la Mama/sangre , Neoplasias de la Mama/patología , Cromatografía en Capa Delgada , Combinación de Medicamentos , Ácidos Grasos Omega-3/uso terapéutico , Estudios de Factibilidad , Femenino , Humanos , Hiperplasia/patología , Persona de Mediana Edad , Proyectos Piloto , Posmenopausia , Lesiones Precancerosas/patología , Reacción en Cadena en Tiempo Real de la Polimerasa , Proyectos de Investigación , Factores de RiesgoRESUMEN
Higher intakes of the omega-3 eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) relative to the omega-6 arachidonic acid (AA) have been variably associated with reduced risk of premenopausal breast cancer. The purpose of this pilot trial was to assess feasibility and explore the effects of high-dose EPA and DHA on blood and benign breast tissue risk biomarkers before design of a placebo-controlled phase IIB trial. Premenopausal women with evidence of hyperplasia ± atypia by baseline random periareolar fine needle aspiration were given 1860 mg of EPA + 1500 mg of DHA ethyl esters daily for 6 months. Blood and benign breast tissue were sampled during the same menstrual cycle phase prestudy and a median of 3 weeks after last dose. Additional blood was obtained within 24 hours of last dose. Feasibility, which was predefined as 50% uptake, 85% retention, and 70% compliance, was demonstrated with 46% uptake, 94% completion, and 85% compliance. Cytologic atypia decreased from 77% to 38% (P = 0.002), and Ki-67 from a median of 2.1% to 1.0% (P = 0.021) with an increase in the ratio of EPA + DHA to AA in erythrocyte phospholipids but no change in blood hormones, adipokines, or cytokines. Exploratory breast proteomics assessment showed decreases in several proteins involved in hormone and cytokine signaling with mixed effects on those in the AKT/mTOR pathways. Further investigation of EPA plus DHA for breast cancer prevention in a placebo-controlled trial in premenopausal women is warranted.