Bioengineering of Fetal Skin: Differentiation of Amniotic Fluid Stem Cells into Keratinocytes

PURPOSE Open fetal spina bifida repair has become a novel clinical standard of care. In very large spina bifida lesions, the skin defect cannot be covered primarily, asking for alternative solutions. We hypothesize that amniotic fluid stem cells (AFSC) could be differentiated into keratinocytes that could then be used to bioengineer autologous skin usable for in utero back coverage. METHODS To obtain human AFSC, amniotic fluid samples obtained from fetal surgeries were subjected to immunoselection for c-kit. C-kit-positive samples and controls were cultured with the additives morphogenetic protein 4 and vitamin C to induce differentiation towards keratinocytes. This process was monitored by measuring the expression of K8 and K14 via immunohistochemical staining, flow cytometry, and polymerase chain reaction. RESULTS After immunoselection and expansion, most cells were positive for K8, but none for K14. After completion of the differentiation protocol, cell colonies with keratinocyte-like appearance could be observed, but cells remained positive for K8 and negative for K14, indicating failed differentiation into keratinocytes. CONCLUSIONS Culturing of keratinocyte-like cells from AFSC, harvested intraoperatively, was not feasible in this setting. The reasons for failure must be investigated and eliminated, as bioengineering of fetal skin for clinical use during fetal surgery for spina bifida remains an attractive goal

Urinary excretion of melatonin and association with breast cancer: meta-analysis and review of the literature

BACKGROUND Melatonin is an endocrine hormone secreted by the pineal gland during night hours that provides several biological functions in the circadian rhythm of humans. Due to anti-estrogenic properties, melatonin is considered to exhibit a protective role against the development of breast cancer (BC). Moreover, disruption of melatonin production through environmental influences, such as night work, is assumed to be a risk factor for BC. MATERIALS AND METHODS We reviewed recent findings concerning biological effects of melatonin on BC and conducted a meta-analysis to evaluate the association between melatonin and BC incidence. In random and fixed effects statistical models, concentrations (tertiles, quartiles) of the primary urinary metabolite of melatonin, 6-sulfatoxymelatonin (aMT6s), were tested for the assumption that women with the highest values would exhibit a lower risk of BC. RESULTS Statistical analysis of data from 5 prospective case-control studies indicates an inverse association between BC risk and the highest levels of urinary aMT6s. This effect seems to be influenced by lag intervals between aMT6s collection and the occurrence of BC, timing and methods of urine sampling, as well as genetic and environmental factors. CONCLUSION On the basis of the results of our meta-analysis, melatonin is likely to affect BC occurrence in women. However, methodological dissonances may require further studies

Co-inhibition of immunoproteasome subunits LMP2 and LMP7 is required to block autoimmunity

Cells of hematopoietic origin express high levels of the immunoproteasome, a cytokine-inducible proteasome variant comprising the proteolytic subunits LMP2 (β1i), MECL-1 (β2i), and LMP7 (β5i). Targeting the immunoproteasome in pre-clinical models of autoimmune diseases with the epoxyketone inhibitor ONX 0914 has proven to be effective. ONX 0914 was previously described as a selective LMP7 inhibitor. Here, we show that PRN1126, developed as an exclusively LMP7-specific inhibitor, has limited effects on IL-6 secretion, experimental colitis, and experimental autoimmune encephalomyelitis (EAE). We demonstrate that prolonged exposure of cells with ONX 0914 leads to inhibition of both LMP7 and LMP2. Co-inhibition of LMP7 and LMP2 with PRN1126 and LMP2 inhibitors LU-001i or ML604440 impairs MHC class I cell surface expression, IL-6 secretion, and differentiation of naïve T helper cells to T helper 17 cells, and strongly ameliorates disease in experimental colitis and EAE. Hence, co-inhibition of LMP2 and LMP7 appears to be synergistic and advantageous for the treatment of autoimmune diseases.publishe

Neoadjuvant rituximab modulates the tumor immune environment in patients with high risk prostate cancer.

BackgroundImmunotherapeutic regulation of the tumor microenvironment in prostate cancer patients is not understood. Most antibody immunotherapies have not succeeded in prostate cancer. We showed previously that high-risk PCa patients have a higher density of tumor infiltrating B-cells in prostatectomy specimens. In mouse models, anti-CD20 antibody ablation of B-cells delayed PCa regrowth post-treatment. We sought to determine whether neoadjuvant anti-CD20 immunotherapy with rituximab could reduce CD20+ B cell infiltration of prostate tumors in patients.MethodsAn open label, single arm clinical trial enrolled eight high-risk PCa patients to receive one cycle of neoadjuvant rituximab prior to prostatectomy. Eleven clinical specimens with similar characteristics were selected as controls. Treated and control samples were concurrently stained for CD20 and digitally scanned in a blinded fashion. A new method of digital image quantification of lymphocytes was applied to prostatectomy sections of treated and control cases. CD20 density was quantified by a deconvolution algorithm in pathologist-marked tumor and adjacent regions. Statistical significance was assessed by one sided Welch's t-test, at 0.05 level using a gatekeeper strategy. Secondary outcomes included CD3+ T-cell and PD-L1 densities.ResultsMean CD20 density in the tumor regions of the treated group was significantly lower than the control group (p = 0.02). Mean CD3 density in the tumors was significantly decreased in the treated group (p = 0.01). CD20, CD3 and PD-L1 staining primarily occurred in tertiary lymphoid structures (TLS). Neoadjuvant rituximab was well-tolerated and decreased B-cell and T-cell density within high-risk PCa tumors compared to controls.ConclusionsThis is the first study to treat patients prior to surgical prostate removal with an immunotherapy that targets B-cells. Rituximab treatment reduced tumor infiltrating B and T-cell density especially in TLSs, thus, demonstrating inter-dependence between B- and T-cells in prostate cancer and that Rituximab can modify the immune environment in prostate tumors. Future studies will determine who may benefit from using rituximab to improve their immune response against prostate cancer. Trial registration NCT01804712, March 5th, 2013 https://clinicaltrials.gov/ct2/show/NCT01804712?cond=NCT01804712&draw=2&rank=1

Neoadjuvant rituximab modulates the tumor immune environment in patients with high risk prostate cancer.

BackgroundImmunotherapeutic regulation of the tumor microenvironment in prostate cancer patients is not understood. Most antibody immunotherapies have not succeeded in prostate cancer. We showed previously that high-risk PCa patients have a higher density of tumor infiltrating B-cells in prostatectomy specimens. In mouse models, anti-CD20 antibody ablation of B-cells delayed PCa regrowth post-treatment. We sought to determine whether neoadjuvant anti-CD20 immunotherapy with rituximab could reduce CD20+ B cell infiltration of prostate tumors in patients.MethodsAn open label, single arm clinical trial enrolled eight high-risk PCa patients to receive one cycle of neoadjuvant rituximab prior to prostatectomy. Eleven clinical specimens with similar characteristics were selected as controls. Treated and control samples were concurrently stained for CD20 and digitally scanned in a blinded fashion. A new method of digital image quantification of lymphocytes was applied to prostatectomy sections of treated and control cases. CD20 density was quantified by a deconvolution algorithm in pathologist-marked tumor and adjacent regions. Statistical significance was assessed by one sided Welch's t-test, at 0.05 level using a gatekeeper strategy. Secondary outcomes included CD3+ T-cell and PD-L1 densities.ResultsMean CD20 density in the tumor regions of the treated group was significantly lower than the control group (p = 0.02). Mean CD3 density in the tumors was significantly decreased in the treated group (p = 0.01). CD20, CD3 and PD-L1 staining primarily occurred in tertiary lymphoid structures (TLS). Neoadjuvant rituximab was well-tolerated and decreased B-cell and T-cell density within high-risk PCa tumors compared to controls.ConclusionsThis is the first study to treat patients prior to surgical prostate removal with an immunotherapy that targets B-cells. Rituximab treatment reduced tumor infiltrating B and T-cell density especially in TLSs, thus, demonstrating inter-dependence between B- and T-cells in prostate cancer and that Rituximab can modify the immune environment in prostate tumors. Future studies will determine who may benefit from using rituximab to improve their immune response against prostate cancer. Trial registration NCT01804712, March 5th, 2013 https://clinicaltrials.gov/ct2/show/NCT01804712?cond=NCT01804712&draw=2&rank=1

Investigation of a Multistate Outbreak of Listeria monocytogenes Infections Linked to Frozen Vegetables Produced at Individually Quick-Frozen Vegetable Manufacturing Facilities

In 2016, the U.S. Food and Drug Administration (FDA), the Centers for Disease Control and Prevention (CDC), and state partners investigated nine Listeria monocytogenes infections linked to frozen vegetables. The investigation began with two environmental L. monocytogenes isolates recovered from Manufacturer A, primarily a processor of frozen onions, that were a match by whole genome sequencing (WGS) to eight clinical isolates and historical onion isolates with limited collection details. Epidemiologic information, product distribution, and laboratory evidence linked suspect food items, including products sourced from Manufacturer B, also a manufacturer of frozen vegetable/fruit products, with an additional illness. The environmental isolates were obtained during investigations at Manufacturers A and B. State and federal partners interviewed ill people, analyzed shopper card data, and collected household and retail samples. Nine ill persons between 2013 and 2016 were reported in four states. Of four ill people with information available, frozen vegetable consumption was reported by three, with shopper cards confirming purchases of Manufacturer B brands. Two identified outbreak strains of L. monocytogenes (Outbreak Strain 1 and Outbreak Strain 2) were a match to environmental isolates from Manufacturer A and/or isolates from frozen vegetables recovered from open and unopened product samples sourced from Manufacturer B; the investigation resulted in extensive voluntary recalls. The close genetic relationship between isolates helped investigators determine the source of the outbreak and take steps to protect public health. This is the first known multistate outbreak of listeriosis in the United States linked to frozen vegetables and highlights the significance of sampling and WGS analyses when there is limited epidemiologic information. Additionally, this investigation emphasizes the need for further research regarding food safety risks associated with frozen foods

The Drosophila Copper Transporter Ctr1C Functions in Male Fertility*

Living organisms have evolved intricate systems to harvest trace elements from the environment, to control their intracellular levels, and to ensure adequate delivery to the various organs and cellular compartments. Copper is one of these trace elements. It is at the same time essential for life but also highly toxic, not least because it facilitates the generation of reactive oxygen species. In mammals, copper uptake in the intestine and copper delivery into other organs are mediated by the copper importer Ctr1. Drosophila has three Ctr1 homologs: Ctr1A, Ctr1B, and Ctr1C. Earlier work has shown that Ctr1A is an essential gene that is ubiquitously expressed throughout development, whereas Ctr1B is responsible for efficient copper uptake in the intestine. Here, we characterize the function of Ctr1C and show that it functions as a copper importer in the male germline, specifically in maturing spermatocytes and mature sperm. We further demonstrate that loss of Ctr1C in a Ctr1B mutant background results in progressive loss of male fertility that can be rescued by copper supplementation to the food. These findings hint at a link between copper and male fertility, which might also explain the high Ctr1 expression in mature mammalian spermatozoa. In both mammals and Drosophila, the X chromosome is known to be inactivated in the male germline. In accordance with such a scenario, we provide evidence that in Drosophila, the autosomal Ctr1C gene originated as a retrogene copy of the X-linked Ctr1A, thus maintaining copper delivery during male spermatogenesis