Preimplantation genetic diagnosis (PGD) is a genetic screening of embryos conceived with assisted reproduction technologies (ART). A single blastomere from an early-stage embryo is removed and molecular analyses follow to identify embryos carrying genetic defects. PGD is considered highly successful for detecting genetic anomalies, but the effects of blastomere biopsy on fetal development are understudied. We aimed to determine whether single blastomere removal affects steroid homeostasis in the maternal-placental-fetal unit during mouse pregnancy. Embryos generated by in vitro fertilization (IVF) were biopsied at the four-cell stage, cultured to morula/early blastocyst, and transplanted into the oviducts of surrogate mothers. Nonbiopsied embryos from the same IVF cohorts served as controls. Cesarean section was performed at term, and maternal and fetal tissues were collected. Embryo biopsy affected the levels of steroids (estradiol, estrone, and progesterone) in fetal and placental compartments but not in maternal tissues. Steroidogenic enzyme activities (3beta-hydroxysteroid dehydrogenase, cytochrome P450 17alpha-hydroxylase, and cytochrome P450 19) were unaffected but decreased activities of steroid clearance enzymes (uridine diphosphate-glucuronosyltransferase and sulfotransferase) were observed in placentas and fetal livers. Although maternal body, ovarian, and placental weights did not differ, the weights of fetuses derived from biopsied embryos were lower than those of their nonbiopsied counterparts. The data demonstrate that blastomere biopsy deregulates steroid metabolism during pregnancy. This may have profound effects on several aspects of fetal development, of which low birth weight is only one. If a similar phenomenon occurs in humans, it may explain low birth weights associated with PGD/ART and provide a plausible target for improving PGD outcomes.
Ultraviolet irradiation triggers a response in mammalian cells known as the UV response. Part of the UV response forms the enhanced synthesis of various extracellular proteins able to transmit the response to non-irradiated cells. Because several cancer-prone syndromes with enhanced genetic instability also have an enhanced synthesis of the same set of proteins without prior stimulation it is possible that induction of these stress responsive proteins may be involved in the process of carcinogenesis and genetic instability. To test this hypothesis mouse T lymphoma cells, GRSL13, were treated with the conditioned medium of UV-induced cells under various experimental conditions. Overall the mutation rate is enhanced 1.8-fold (P < 0.01). However, the degree of enhancement is strongly influenced by culture conditions. UV-induced factors only lead to an enhanced mutation rate when cells, both for the production and response to those factors, originate from a similar cell density. In addition, it was found that fresh medium interferes with this response. To eliminate the hindrance of these factors on the effect of the conditioned medium on the mutation rate, serum-starved cells at high density were treated with serum-free medium derived from high-density UV-irradiated cultures. Using these conditions a 2.8-fold (P < 0.002) enhancement of the mutation rate was found. Fluctuation analysis indicated that the enhancement is 10-fold during the first five generations after treatment. UV-induced factors have also been found to induce cell growth, and the degree of induction was linearly correlated with the enhancement in mutation rate. These experiments are in agreement with the hypothesis that induction of stress responses leads to genetic instability.
B cells and the antibodies they produce in solid tumours including malignant melanoma and the mechanisms by which these may be polarized by cancer cells, remain an unexplored component of patient immunity. Using patient-derived material, we explored the presence and potential functions of infiltrating B cells and investigated the IgG subclasses expressed in melanoma. We found class-switched mature B cells infiltrating cutaneous melanoma lesions and evidence for local active class-switching, B cell clonal expansion and antibody affinity maturation. Importantly, tumour-resident B cell compartments featured proportionally lower levels of the immunoactivatory IgG1 subclass and higher representation of chronic inflammation-associated IgG4, when compared to B cells from lymph nodes and blood. To gain insights into the mechanisms by which tumour microenvironments could modulate antibody production, we identified key Th2 cytokines IL-10, IL-4 and VEGF and demonstrated that these could skew antibody subclass expression in favour of IgG4. A tumour antigen-specific IgG4 antibody displayed lower anti-tumour functions when compared to the corresponding IgG1. IgG4 could reduce the anti-tumour potency of a cytotoxic IgG1 in vivo. Consistent with these findings, we found dysregulated IgG4 serum levels in a subset of patients with melanoma. Elevated serum IgG4 was associated with a higher risk of progression-free and overall survival. In summary, infiltration of B cells, evidence of an active humoral immune surveillance in situ in a proportion of cutaneous melanoma lesions and the contribution of mature memory B cells with distinct immunoglobulin isotype-biased profiles to skin cancer immune surveillance suggest that B cell and antibody responses display alternatively polarised phenotypes that may contribute to immune escape.
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