CRISPR/Cas9: reflexiones bioéticas sobre las modificacion es genómicas / CRISPR/Cas9: bioethical reflections on genome modifications

CRISPR/Cas9 puede ser considerado el descubrimiento biotecnológico del siglo. Sin embargo, las reflexiones sobre la aceptabilidad y factibilidad de producir cambios permanentes en el ADN de gametos y embriones, ha arrojado nueva luz sobre CRISPR. Por ejemplo al notar que las alteraciones en la línea germinal, pueden ser heredadas y por lo tanto observarse a lo largo de las generaciones. Aunque inicialmente estas transformaciones puedan ser deseables, más allá del impacto tecnológico, la tecnología CRISPR parece tener varias implicancias éticas para la sociedad. Estas pueden ser analizadas a partir de las distintas reacciones que ha despertado CRISPR en todo el mundo. Por ejemplo, en el pedido de suspensión total de la aplicación clínica (es decir, la prohibición de transferir en el útero un embrión previamente modificado con la tecnología CRISPR) hasta tanto no se hayan alcanzado y aprobado ciertos requisitos indispensables para la investigación clínica. En general, esta tecnología ha sido prematuramente caracterizada como "disruptiva". Este texto analizará las implicancias éticas, políticas, sociales, médicas y subjetivas a la luz del fenómeno social - no tecnológico - creado por CRISPR.

CRISPR/Cas9 can be considered the biotechnogical discovery of the century. However, reflections on the plausibility and feasibility of producing permanent changes in the DNA of gametes and embryos throw new light on CRISPR. As in modifications in the germinal line can be inherited and hence, observed throughout generations. While this might be desirable for some, besides the technological impact, CRISPR also seems to have an ethical impact on society. These ethical impacts can be observed in the diverse reactions to CRISPR from across the globe. For instance, a request for the complete suspension of clinical application (that is, the prohibition of implanting an embryo with CRISPR modifications in the uterus) till certain basic research requirements were met and approved. Broadly, this technology has been prematurely also characterized as "disruptive" by some. This paper will analyze these ethical, political, social, medical and subjective reactions in light of the social - not technological - phenomenon created by CRISPR.

Treatment with Ilex paraguariensis (yerba mate) aqueous solution prevents hepatic redox imbalance, elevated triglycerides, and microsteatosis in overweight adult rats that were precociously weaned.

Early weaning (EW) leads to overweight, visceral obesity, hyperleptinemia, and insulin resistance in adulthood. Treatment with Ilex paraguariensis (yerba mate) improves obesity and insulin resistance in these animals. Here, we evaluated the effects of chronic treatment with yerba mate on the redox balance and liver morphology of overweight early-weaned rats. To induce EW, we wrapped the dams with bandages to interrupt milk access during the last 3 days of lactation. Control pups (C) had free access to maternal milk for the full 21 days of lactation. On postnatal day (PN) 150, EW offspring were subdivided into the EW+YM group, which received the aqueous extract of yerba mate (1 g/kg bw by gavage once a day for 30 days) and the EW group, which received water by gavage for the same period. All rats were euthanized on PN180. The EW group showed higher bound carbonyl (a marker of total protein oxidation), higher TBARS levels (a marker of lipid peroxidation), and lower superoxide dismutase (SOD) activity in liver tissue than the C group, as well as higher triglyceride content and microsteatosis. In plasma, the EW offspring showed higher TBARS levels. One month of yerba mate treatment normalized these parameters. Thus, we have shown evidence that yerba mate improved antioxidant defenses and mitigated liver dysfunction in overweight adult rats that were weaned prematurely.

Developmental plasticity of endocrine disorders in obesity model primed by early weaning in dams.

Early weaning is associated with changes in the developmental plasticity. Here, we studied the adipocytes morphology, adipokines expression or content in adipose tissue as well as adrenal and thyroid function of neonate and adult offspring primed by early weaning. After birth, lactating rats were divided into 2 groups: EW (early weaning)--dams were wrapped with a bandage to block access to milk during the last 3 days of lactation, and Control--dams whose pups had free access to milk throughout lactation (21 days). At postnatal day (PN) 21, EW pups had lower visceral and subcutaneous adipocyte area (-67.7% and -62%, respectively), body fat mass (-26%), and leptin expression in visceral adipocyte (-64%) but higher leptin expression in subcutaneous adipocyte (2.9-fold increase). Adrenal evaluations were normal, but neonate EW pups presented lower serum T3 (-55%) and TSH (-44%). At PN 180, EW offspring showed higher food intake, higher body fat mass (+21.6%), visceral and subcutaneous adipocyte area (both 3-fold increase), higher leptin (+95%) and ADRß3 (2-fold increase) content in visceral adipose tissue, and higher adiponectin expression in subcutaneous adipose tissue (+47%) but lower in visceral adipose tissue (-40%). Adult EW offspring presented higher adrenal catecholamine content (+31%), but no changes in serum corticosterone or thyroid status. Thus, early weaning primed for hypothyroidism at weaning, which can be associated with the adipocyte hypertrophy at adulthood. The marked changes in catecholamine adrenal content and visceral adipocyte ADRB3 are generally found in obesity, contributing to the development of other cardiovascular and metabolic disturbances.

Calcium supplementation reverts central adiposity, leptin, and insulin resistance in adult offspring programed by neonatal nicotine exposure.

Obesity is a worldwide epidemic. Calcium influences energy metabolism regulation, causing body weight loss. Because maternal nicotine exposure during lactation programs for obesity, hyperleptinemia, insulin resistance (IR), and hypothyroidism, we decided to evaluate the possible effect of dietary calcium supplementation on these endocrine dysfunctions in this experimental model. Osmotic minipumps containing nicotine solution (N: 6 mg/kg per day for 14 days) or saline (C) were s.c. implanted in lactating rats 2 days after giving birth (P2). At P120, N and C offspring were subdivided into four groups: 1) C - standard diet; 2) C with calcium supplementation (CCa, 10 g calcium carbonate/kg rat chow); 3) N - standard diet; and 4) N with calcium supplementation (NCa). Rats were killed at P180. As expected, N offspring showed higher visceral and total body fat, hyperleptinemia, lower hypothalamus leptin receptor (OB-R) content, hyperinsulinemia, and higher IR index. Also, higher tyrosine hydroxylase (TH) expression (+51%), catecholamine content (+37%), and serum 25-hydroxyvitamin D(3) (+76%) were observed in N offspring. Dietary calcium supplementation reversed adiposity, hyperleptinemia, OB-R underexpression, IR, TH overexpression, and vitamin D. However, this supplementation did not reverse hypothyroidism. In NCa offspring, Sirt1 mRNA was lower in visceral fat (-37%) and higher in liver (+42%). In conclusion, dietary calcium supplementation seems to revert most of the metabolic syndrome parameters observed in adult offspring programed by maternal nicotine exposure during lactation. It is conceivable that the reduction in fat mass per se, induced by calcium therapy, is the main mechanism that leads to the increment of insulin action.

Blocking leptin action one week after weaning reverts most of the programming caused by neonatal hyperleptinemia in the adult rat.

Hyperleptinemia during lactation programs for higher serum leptin in 30-day-old and adult rats, associated with metabolic changes. Here we evaluated the inhibition of serum leptin at 29 and 30 days on the metabolic phenotype of rats programmed with leptin during lactation. Pups from Wistar rats were saline-injected or leptin-injected from postnatal day 1 to day 10. At 29 and 30 days old, animals were injected with anti-leptin antibody (LA and CA) or saline (LS and CS). In adult animals, higher visceral (+53%) and total fat mass (+33%), hyperleptinemia (+67%), hypertriglyceridemia (+47%), and hypoadiponectinemia (-44%) observed in LS group compared to CS were prevented by immunoneutralization of leptin, since LA group had those parameters values similar to CS group. However, immunoblockade of leptin in normal animals led to the same metabolic changes seen in leptin-treated animals, in addition to lower serum adiponectin (-77% vs. CS) and higher insulin resistance index (+37%). Liver sirtuin1 (SIRT1) was higher (+41%) only in LA group, suggesting a role for SIRT1 in the prevention of leptin programming. Hypothalamic OBR was lower and SOCS3 higher in LS group and these changes were normalized in LA group. In conclusion, blocking leptin action one week after weaning seems to revert most of the alterations observed in rats programmed by neonatal hyperleptinemia. Higher liver SIRT1 expression may be one of the mechanisms involved, leading to a better glucose and lipid metabolism. Our data suggest that the lack or the excess of leptin programs an adverse metabolic phenotype in adulthood.

Prolactin inhibition at mid-lactation influences adiposity and thyroid function in adult rats.

Maternal hypoprolactinemia at the end of lactation (a precocious weaning model) increases milk leptin transfer and results in overweight, leptin resistance, and secondary hypothyroidism at adulthood. We studied the effects of prolactin (PRL) inhibition during mid-lactation (a partial malnutrition model) on milk leptin transfer, leptinemia, body composition, and thyroid function. Lactating rats were treated with bromocryptine (BRO, 1 mg/twice daily) or saline on days 7, 8, and 9 of lactation. Offspring were sacrificed 10, 21, and 90 days after birth. After treatment, BRO-treated dams showed hypoprolactinemia and hyperleptinemia, and produced less milk with lower levels of lactose and higher milk triglycerides. Milk leptin levels were lower at weaning. Offspring of BRO-treated dams had lower body weight and length as well as less visceral fat during lactation and adulthood. Total fat was also lower at weaning and adult life, whereas total protein was higher at 90 days-old. BRO offspring presented lower serum T4 and TSH at 10 days-old and weaning, respectively. When adults, these rats exhibited hypoleptinemia, lower levels of thyroid hormones, and higher TSH. Early inhibition of PRL therefore leads to offspring malnutrition and affects subsequent growth. Also, inhibition of PRL during lactation predisposes offspring to hypothyroidism; however, when the inhibition occurs during late lactation, the hypothyroidism is secondary, whereas when it is restricted to mid-lactation, the thyroid hypofunction is primary. The programming effect of milk suppression thus depends on the developmental stage of offspring.

Treatment of South American blastomycosis (paracoccidioidomycosis) with miconazole by the oral route: an on-going study.

Results are presented from 12 patients of both sexes, whose ages range from 34 to 65 years, observed over a period of 16 months and receiving miconazole treatment for South-American blastomycosis. Ten of them were being treated for the first time and the remaining 2 presented relapses after treatment with other chemotherapeutic agents. The blastomycotic lesions were localized in skin, mucosa, lung and lymphatics. The drug was administered orally, at a dosage of 1 g three times daily. One relapsing patient had his first daily oral dose replaced by an intravenous dose of 200 mg of miconazole diluted in 250 ml of glucose solution during the initial four weeks. The criterion for diagnosis was finding of the fungus in sputum and/or in the material collected through scrapings or biopsy of the lesions. All patients were treated in hospital until the cure was apparent, clinical examinations being conducted weekly and bi-weekly. Bi-weekly laboratory examinations were performed to evaluate the patient's tolerance to the drug. All treated patients presented complete healing of cutaneomucous lesions (mean time: 4 weeks) and involution of lymphatic and pulmonary features (mean time: 4 months). Two of them still presented palpable lymphatics after 5 months of treatment, in spite of the cure of mucosal and pulmonary lesions. Monitoring the progress of the cured patients was and is being carried out quarterly. Currently, this observation period ranges from three to 15 months. Nine patients are still returning and none have shown the reappearance of mycotic lesions. Diarrhoea was the only side-effect observed after a period of therapy, but it was easily controlled by specific medication (kaolin, pectin, &c.). In a single case the treatment was interrupted because of this symptom.(ABSTRACT TRUNCATED AT 250 WORDS)