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Preterm deliveries are a major multifactorial public health problem in French Guiana. Desert dust episodes have been associated with preterm delivery in Guadeloupe, a territory with similarities to French Guiana. We thus tried to replicate this finding in the context of French Guiana. A retrospective ecological cohort study combined daily PM10 concentration measurements during pregnancy and term at delivery extracted from French Guiana’s computerized pregnancy delivery registry. Daily PM10 concentrations during the course of pregnancy were analyzed as mean concentrations and as the proportion of intense dust episodes (≥55 μg PM10/m3). These exposure variables were studied in relation to the outcome of preterm delivery. Overall, 3,321 pregnant women with complete daily PM10 measurements were included, of whom 374 (11.26%) delivered prematurely. Among preterm deliveries, 168 (44.9%) were spontaneous deliveries and 206 (55.1%) were induced. Rank-sum tests showed that, for spontaneous and induced spontaneous deliveries, both mean PM10 concentrations and proportions of intense desert dust episodes were significantly greater among preterm births than among term births. Although the proportion of intense desert dust episodes during pregnancy was significantly associated with spontaneous preterm deliveries, the relation was U-shaped, with an adjusted odds ratio (AOR) = 2 (95%CI = 1.2–3.1) for lowest values relative to median values and AOR = 5.4 (95%CI = 3.2–8.9) for the highest values relative to median values. Similarly, the proportion of intense desert dust episodes during pregnancy was also significantly associated with induced preterm deliveries in a U-shaped manner (AOR = 2.7 (95%CI = 1.6–4.5) for the lowest relative to median values and AOR = 6.8 (95%CI = 3.9–11.9) for the highest relative to median values). Although in our study the relation between PM10 concentrations appeared non-linear, the highest mean concentrations and intense desert dust episodes were indeed associated with both spontaneous and induced preterm delivery.

Objectives: Small for gestational age (SGA) newborns have a higher risk of poor outcomes. French Guiana (FG) is a territory in South America with poor living conditions. The objectives of this study were to describe risk factors associated with SGA newborns in FG.Methods: We used the birth cohort that compiles data from all pregnancies that ended in FG from 2013 to 2021. We analysed data of newborns born after 22 weeks of gestation and/or weighing more than 500 g and their mothers.Results: 67,962 newborns were included. SGA newborns represented 11.7% of all newborns. Lack of health insurance was associated with SGA newborns (p < 0.001) whereas no difference was found between different types of health insurance and the proportion of SGA newborns (p = 0.86). Mothers aged less than 20 years (aOR = 1.65 [1.55–1.77]), from Haiti (aOR = 1.24 [1.11–1.39]) or Guyana (aOR = 1.30 [1.01–1.68]) and lack of health insurance (aOR = 1.24 [1.10–1.40]) were associated with SGA newborns.Conclusion: Immigration and precariousness appear to be determinants of SGA newborns in FG. Other studies are needed to refine these results.

IntroductionIn a context of high HIV prevalence, poor pregnancy follow-up, frequent poverty, preeclampsia, and preterm delivery, we aimed to describe the characteristics and outcomes of pregnancies among women living with HIV in French Guiana.MethodsA retrospective cohort study was conducted on HIV-infected pregnancies enrolled between January 1st 1992 to 31st July 2022. Overall, there were 1,774 pregnancies in 881 women living with HIV.ResultsFor 75.1% of pregnancies, the HIV diagnosis was already known before pregnancy and in 67.6% of women, HIV follow-up predated pregnancy. Nearly half of women, 49.6%, only had one pregnancy since having been diagnosed with HIV. Although most women received antiretroviral therapy during pregnancy, for those with the available information we found only 48.5% had an undetectable viral load at delivery. Overall, 15.3% of pregnancies ended with an abortion. There were a total of 110 newborns infected with HIV representing an overall transmission rate of 6.2% (110/1,771). Between 1993 and 2002, the transmission rate was 34%, between 2003 and 2012 it was 1.3%, and between 2013 and 2022 it was 0.7%. Overall, in Cayenne, since 2008, 106 of 581 HIV–infected pregnancies (18.2%) with available information were premature before 37 weeks of pregnancy; of these, 33 (5.7%) were very preterm deliveries and 73 (13.3%) were late preterm deliveries. Over time, in Cayenne, preterm delivery declined significantly.ConclusionsThe present study emphasizes that, despite spectacular progress in reducing mother to child transmission, pregnancy outcomes among women living with HIV are still preoccupying with high incidence of preterm delivery and low birth weight. Teasing out what fraction is linked to HIV and what fraction is linked to social precariousness and poor follow-up was not possible in this study. Despite the high incidence of very preterm delivery recent progress suggests that coordination efforts to improve follow-up may also have improved obstetrical outcomes.

Résumé Contexte L’excès de poids et les troubles métaboliques ont un impact négatif sur les fonctions de reproduction masculine. Les mécanismes impliqués sont nombreux et complexes, et des mécanismes épigénétiques peuvent également intervenir, notamment par le biais des petits ARN non codants. Parmi eux, les microRNAs (miRNAs) présentent un intérêt particulier. Cette étude préliminaire visait à identifier les miRNAs différentiellement enrichis dans le plasma séminal en relation avec des troubles métaboliques et si certains étaient également associés à des altérations des paramètres spermatiques. Cent soixante hommes âgés de 18 à 45 ans, partenaires de couple infertile, ont été inclus dans cette cohorte. Les miRNAs associés au métabolisme ont été sélectionnés dans la littérature et analysés par PCR quantitative en temps réel à l’aide de tests d’expression génique TaqMan. Un sous-ensemble de ceux présentant un profil intéressant dans le plasma séminal ont été secondairement testés dans le sang. Résultats Parmi les 11 miRNAs sélectionnés, sept ont été détectés dans le plasma séminal (miR10b, miR19a, miR19b, miR34b, miR34c, miR133b, miRlet7c). Une corrélation négative a été observée entre les niveaux du miR19a séminal et le syndrome métabolique, la glycémie et le C-peptide. Les niveaux de miR19b séminaux étaient également corrélés négativement avec le syndrome métabolique. Les niveaux de miR34c séminaux étaient négativement corrélés avec l’IMC et le tour de taille. Les niveaux de miR133b séminaux étaient positivement corrélés avec l’IMC, le tour de taille et les niveaux de leptine. Il est intéressant de noter que les modifications des miRNA dans le plasma séminal semblent spécifiques puisque les corrélations mises en évidence ci-dessus n’ont pas été retrouvées dans le plasma sanguin pour les miR19a, 19b, 10b, 34c. Conclusion Quelques désordres métaboliques et anthropométriques ont été observés corrélés avec le niveau de certains miRNAs dans le plasma séminal. Des études complémentaires sont nécessaires pour déterminer si d’autres petits ARN non codants sont corrélés aux troubles métaboliques et anthropométriques et pour évaluer leur implication potentielle dans l’altération des fonctions de reproduction chez les hommes souffrant d’obésité ou de troubles métaboliques.

Background: Involved in physical and brain development, immunity and metabolism, micronutrients have profound health effects. The nutritional status of pregnant women is a major determinant of foetal health. French Guiana has a rapid population growth. Social inequalities, cultural practices and gastrointestinal nematode infections in French Guiana could affect the prevalence of these deficiencies. The main objective of the present study was to estimate the prevalence of micronutrient deficiency among pregnant women in French Guiana. The secondary objective was to identify socio-demographic, dietary, obstetrical and neonatal risk factors associated with deficiencies. Methods: Pregnant women over 22 weeks of pregnancy hospitalized for delivery at the Obstetrical Emergency Department of the Hospital Center in Cayenne from May 2018 to March 2019 were included. A socio-demographic and food questionnaire was administered. Medical data were collected from the medical records. Blood and urine samples were taken. The descriptive analysis used Student and chi-squared tests. Results: A total of 341 women were included. The majority were born in Haiti (39%) and French Guiana (34%). At least one micronutrient deficiency was observed in 81% of women. Precarious women had a significantly greater risk of micronutrient deficiency during pregnancy compared to those with both normal and complementary health insurance. Conclusions: Micronutrient deficiencies in pregnant women in French Guiana are a public health problem, a fact that was previously overlooked in the context of rising obesity. With over half the women overweight or obese, and 81% with at least 1 micronutrient deficiency, balanced nutrition should be a major focus.

Nutrition during the early postnatal period can program the growth trajectory and adult size. Nutritionally regulated hormones are strongly suspected to be involved in this physiological regulation. Linear growth during the postnatal period is regulated by the neuroendocrine somatotropic axis, whose development is first controlled by GHRH neurons of the hypothalamus. Leptin that is secreted by adipocytes in proportion to fat mass is one of the most widely studied nutritional factors, with a programming effect in the hypothalamus. However, it remains unclear whether leptin stimulates the development of GHRH neurons directly. Using a Ghrh-eGFP mouse model, we show here that leptin can directly stimulate the axonal growth of GHRH neurons in vitro in arcuate explant cultures. Moreover, GHRH neurons in arcuate explants harvested from underfed pups were insensitive to the induction of axonal growth by leptin, whereas AgRP neurons in these explants were responsive to leptin treatment. This insensitivity was associated with altered activating capacities of the three JAK2, AKT and ERK signaling pathways. These results suggest that leptin may be a direct effector of linear growth programming by nutrition, and that the GHRH neuronal subpopulation may display a specific response to leptin in cases of underfeeding.

Abstract In modern medicine, prenatal diagnosis can no longer be sufficient by ultrasound examination. The genetic technical progress and its contribution may remain a challenge in isolated sites with the consequences that this implies in perinatal health.

Objective: Individuals born with intrauterine growth retardation (IUGR) are more prone to cardio-metabolic diseases as adults, and environmental changes during the perinatal period have been identified as potentially crucial factors. We have studied in a preclinical model early-onset molecular alterations present before the development of a clinical phenotype. Methods: We used a preclinical mouse model of induced IUGR, in which we modulated the nutrition of the pups during the suckling period, to modify their susceptibility to cardio-metabolic diseases in adulthood. Results: Mice born with IUGR that were overfed (IUGR-O) during lactation rapidly developed obesity, hepatic steatosis and insulin resistance, by three months of age, whereas those subjected to nutrition restriction during lactation (IUGR-R) remained permanently thin and highly sensitive to insulin. Mice born with IUGR and fed normally during lactation (IUGR-N) presented an intermediate phenotype and developed insulin resistance by 12 months of age. Molecular alterations to the insulin signaling pathway with an early onset were observed in the livers of adult IUGR-N mice, nine months before the appearance of insulin resistance. The implication of epigenetic changes was revealed by ChIP sequencing, with both posttranslational H3K4me3 histone modifications and microRNAs involved. Conclusions: These two changes lead to the coherent regulation of insulin signaling, with a decrease in Akt gene transcription associated with an increase in the translation of its inhibitor, Pten. Moreover, we found that the levels of the implicated miRNA19a-3p also decreased in the blood of young adult IUGR mice nine months before the appearance of insulin resistance, suggesting a possible role for this miRNA as an early circulating biomarker of metabolic fate of potential use for precision medicine.

Abstract Background Early preterm births are still represented as a major public health problem in French Guiana. The objective of the present study was to study factors associated with early preterm birth in French Guiana. Methods A monocentric age-matched case control study was conducted at the sole level 3 maternity in French Guiana. In utero fetal deaths and multiple pregnancies were not included. Cases were defined as giving birth prematurely between 22 and 32 weeks of pregnancy. Controls were defined as women delivering on term. For each case three controls were matched on age. In utero deaths, medical pregnancy interruptions and multiple pregnancies (a known major cause of preterm delivery) were excluded from the study. Sociodemographic variables, medical and obstetrical history, the complications of the current pregnancy, and the results of the last vaginal swab before delivery were recorded in the second or the third trimester. Thematic conditional logistic regression models were computed. Results Overall 94 cases and 282 matched controls were included. Preterm delivery was spontaneous in 47.9% (45/94) of the cases and induced in 52.1% (49/94).A history of preterm birth was associated with both spontaneous and induced preterm delivery. The absence of health insurance was associated with spontaneous early preterm delivery AOR (adjusted odd ratio) = 9.1 (2.2–38.3), p = 0.002 but not induced preterm delivery adjusted odd ratio (AOR) = 2.1 (0.6–6.7), p = 0.2. Gravidic hypertension, placenta praevia, intrauterine growth retardation and mostly preeclampsia (66%, 32/49) were linked to induced preterm delivery but not spontaneous delivery. Gardnerellavaginalis and group B Streptococcus infections were significantly associated with induced early preterm delivery but not spontaneous early preterm delivery. Conclusions Social factors were associated with spontaneous early preterm delivery, suggesting that efforts to reduce psychosocial stressors could lead to potential improvements. Vaginal infections were also associated with induced preterm labor suggesting that early diagnosis and treatment could reduce induced early preterm delivery. Preeclampsia was a major contributor to induced early preterm delivery. Reliable routine predictors of preeclampsia are still not available which makes its prevention impossible in first pregnancies.

Conditional gene targeting, based on Cre-lox or other systems, requires frequent genotyping of transgenic mouse populations and monitoring of tissue-specific Cre recombinatory efficiency. This is currently achieved by Southern analysis from tailand tissue-derived DNA. Multiplex PCR amplification of the floxed (flanked by loxP sites) genomic region, combined with the PCR detection of the Cre transgene, simplifies this task. Here, we show that complete genotyping of a floxed locus is possible with three appropriately placed primers and that this triplex PCR can be performed simultaneously with a universal PCR assay for the detection of Cre transgenes. Using this approach, we also determined the ratios of recombined versus non-recombined floxed genomic segments in genomic DNA samples. This allowed us to estimate the efficiency of in vivo conditional inactivation from biopsy material and tissue samples that were too small for Southern analysis. As many new conditional knockouts are spatiotemporally restricted, such assays will become increasingly useful. The proposed PCR strategy is flexible and may be adapted to the structural specificities of any target gene.

Mutations that decrease insulin-like growth factor (IGF) and growth hormone signaling limit body size and prolong lifespan in mice. In vertebrates, these somatotropic hormones are controlled by the neuroendocrine brain. Hormone-like regulations discovered in nematodes and flies suggest that IGF signals in the nervous system can determine lifespan, but it is unknown whether this applies to higher organisms. Using conditional mutagenesis in the mouse, we show that brain IGF receptors (IGF-1R) efficiently regulate somatotropic development. Partial inactivation of IGF-1R in the embryonic brain selectively inhibited GH and IGF-I pathways after birth. This caused growth retardation, smaller adult size, and metabolic alterations, and led to delayed mortality and longer mean lifespan. Thus, early changes in neuroendocrine development can durably modify the life trajectory in mammals. The underlying mechanism appears to be an adaptive plasticity of somatotropic functions allowing individuals to decelerate growth and preserve resources, and thereby improve fitness in challenging environments. Our results also suggest that tonic somatotropic signaling entails the risk of shortened lifespan.

Individuals born with intrauterine growth retardation (IUGR) are more prone to cardio-metabolic diseases as adults, and environmental changes during the perinatal period have been identified as potentially crucial factors. We have studied in a preclinical model early-onset molecular alterations present before the development of a clinical phenotype. We used a preclinical mouse model of induced IUGR, in which we modulated the nutrition of the pups during the suckling period, to modify their susceptibility to cardio-metabolic diseases in adulthood. Mice born with IUGR that were overfed (IUGR-O) during lactation rapidly developed obesity, hepatic steatosis and insulin resistance, by three months of age, whereas those subjected to nutrition restriction during lactation (IUGR-R) remained permanently thin and highly sensitive to insulin. Mice born with IUGR and fed normally during lactation (IUGR-N) presented an intermediate phenotype and developed insulin resistance by 12 months of age. Molecular alterations to the insulin signaling pathway with an early onset were observed in the livers of adult IUGR-N mice, nine months before the appearance of insulin resistance. The implication of epigenetic changes was revealed by ChIP sequencing, with both posttranslational H3K4me3 histone modifications and microRNAs involved. These two changes lead to the coherent regulation of insulin signaling, with a decrease in Akt gene transcription associated with an increase in the translation of its inhibitor, Pten. Moreover, we found that the levels of the implicated miRNA19a-3p also decreased in the blood of young adult IUGR mice nine months before the appearance of insulin resistance, suggesting a possible role for this miRNA as an early circulating biomarker of metabolic fate of potential use for precision medicine. • Male mice are highly sensitive to changes in nutrition during the perinatal period. • Post-natal nutrition modulates metabolic diseases induced by IUGR in male mice. • Metabolic programming by perinatal nutrition involves epigenetic mechanisms. • Pre-symptomatic IUGR mice present molecular alterations of the insulin pathway. • Plasma miRNA19a-3p levels are associated with the development of insulin resistance. [ABSTRACT FROM AUTHOR]

Nutrition during the early postnatal period can program the growth trajectory and adult size. Nutritionally regulated hormones are strongly suspected to be involved in this physiological regulation. Linear growth during the postnatal period is regulated by the neuroendocrine somatotropic axis, whose development is first controlled by GHRH neurons of the hypothalamus. Leptin that is secreted by adipocytes in proportion to fat mass is one of the most widely studied nutritional factors, with a programming effect in the hypothalamus. However, it remains unclear whether leptin stimulates the development of GHRH neurons directly. Using a Ghrh-eGFP mouse model, we show here that leptin can directly stimulate the axonal growth of GHRH neurons in vitro in arcuate explant cultures. Moreover, GHRH neurons in arcuate explants harvested from underfed pups were insensitive to the induction of axonal growth by leptin, whereas AgRP neurons in these explants were responsive to leptin treatment. This insensitivity was associated with altered activating capacities of the three JAK2, AKT and ERK signaling pathways. These results suggest that leptin may be a direct effector of linear growth programming by nutrition, and that the GHRH neuronal subpopulation may display a specific response to leptin in cases of underfeeding.

Reduced expression of IGF-1R increases lifespan and resistance to oxidative stress in the mouse, raising the possibility that this also confers relative protection against the pro-parkinsonian neurotoxin MPTP, known to involve an oxidative stress component. We used heterozygous IGF-1R(+/-) mice and challenged them with MPTP. Interestingly, MPTP induced more severe lesions of dopaminergic neurons of the substantia nigra, in IGF-1R(+/-) mice than in wild-type animals. Using electron spin resonance, we found that free radicals were decreased in IGF-1R(+/-) mice in comparison with controls, both before and after MPTP exposure, suggesting that the increased vulnerability of dopamine neurons is not caused by oxidative stress. Importantly, we showed that IGF-1R(+/-) mice display a dramatically increased neuro-inflammatory response to MPTP that may ground the observed increase in neuronal death. Microarray analysis revealed that oxidative stress-associated genes, but also several anti-inflammatory signaling pathways were downregulated under control conditions in IGF-1R(+/-) mice compared to WT. Collectively, these data indicate that IGF signaling can reduce neuro-inflammation dependent sensitivity of neurons to MPTP.

Increasing evidence suggests a developmental origin for a number of human diseases, notably after intrauterine or postnatal nutrient deprivation. Nutritional changes readily translate into alterations of somatic growth. However, whereas intrauterine growth retardation often shows postnatal catch-up growth, recovery from food restriction immediately after birth is limited. Therefore, we investigated whether early postnatal nutrition (undernutrition and overfeeding) modifies plasticity of growth through developmental control of the somatotropic hormone axis. We used cross-fostering in mice to induce changes in early nutrition, and examined endocrine growth regulation and the development of specific disease phenotypes in adults. We showed that underfeeding during the early postnatal period delayed growth, whereas overfeeding accelerated it. In both cases, final body size was permanently altered. We found coordinated alterations in pituitary GH, plasma IGF-I and acid labile subunit, and gene expression of hypothalamic GHRH during postnatal development. These changes were consistent with the observed phenotypes. Alterations in the somatotropic axis persisted throughout adulthood. Although limited to the early postnatal period, both underfeeding and overfeeding led to reduced glucose tolerance later in life. These metabolic abnormalities were in line with defective insulin secretion in restricted mice and insulin resistance in overfed mice. Moreover, both restricted and overfed mice had increased arterial blood pressure, suggestive of vascular impairment. Our findings indicate a significant link between early postnatal diet, somatotropic development, and specific late onset diseases in mice. We suggest that, together with other hormones like leptin, IGF-I may play a role in modulating hypothalamic stimulation of the developing somatotropic function.

Mutations that decrease insulin-like growth factor (IGF) and growth hormone signaling limit body size and prolong lifespan in mice. In vertebrates, these somatotropic hormones are controlled by the neuroendocrine brain. Hormone-like regulations discovered in nematodes and flies suggest that IGF signals in the nervous system can determine lifespan, but it is unknown whether this applies to higher organisms. Using conditional mutagenesis in the mouse, we show that brain IGF receptors (IGF-1R) efficiently regulate somatotropic development. Partial inactivation of IGF-1R in the embryonic brain selectively inhibited GH and IGF-I pathways after birth. This caused growth retardation, smaller adult size, and metabolic alterations, and led to delayed mortality and longer mean lifespan. Thus, early changes in neuroendocrine development can durably modify the life trajectory in mammals. The underlying mechanism appears to be an adaptive plasticity of somatotropic functions allowing individuals to decelerate growth and preserve resources, and thereby improve fitness in challenging environments. Our results also suggest that tonic somatotropic signaling entails the risk of shortened lifespan.

Recent evidence indicates that growth hormone (GH) is involved in liver regeneration. To test whether insulin-like growth factor I (IGF-I) mediates this effect, we studied liver regeneration induced by partial hepatectomy in liver-specific IGF type 1 receptor knockout (LIGFREKO) mice. The absence of IGF-1R caused a significant decrease in hepatocyte proliferation in males (-52%), but not in females, as assessed by Ki67 immunohistochemistry. Cyclin D1 and cyclin A protein levels in the livers of LIGFREKO males were only half those in controls, indicating that cyclin induction during liver regeneration is dependent on IGF-1R signaling. Analyzing the signaling cascade initiated by IGF-1R, we observed a lack of IRS-1 induction in LIGFREKO livers. In contrast, the induction of IRS-2 synthesis was similar in LIGFREKO and control groups, suggesting the existence of differential regulation of IRS synthesis during liver regeneration. Regenerating livers from LIGFREKO animals also showed significantly less activated ERKs than controls. Our findings demonstrate that IGF-1R makes a significant contribution to liver regeneration. Using the LIGFREKO model, we provide new evidence that IGF-1R/IRS-1/ERK signaling may be the intracellular pathway controlling the cell cycle via cyclin D1 and cyclin A in the regenerating liver.

Numerous tumours, including hepatocarcinomas, produce IGFs, and some depend on these growth factors in a paracrine or autocrine fashion. We have shown that c-myc-induced experimental hepatocarcinogenesis is associated with enhanced production of IGF-II. To assess the role of the IGF-I receptor (IGF-IR) in hepatocarcinogenesis, we generated conditional mutant mice that overexpressed c-myc and were knocked out for IGF-IR specifically in the liver. We compared these mice with littermate controls that also overexpressed c-myc but had wild-type IGF-IR alleles. We found that the pretumoral phase, induced by early c-myc expression and characterised by increased cell proliferation, was largely unaffected by the lack of IGF-IR. To our further surprise, hepatocellular carcinomas (HCCs) lacking IGF-IR readily developed and progressed at the same rate as control HCCs. At 9 months, all c-myc transgenic mice displayed well-differentiated multifocal tumours, regardless of whether their livers-and their tumours-were able to produce IGF-IR. Levels of IRS-1 and IRS-2 were elevated in all tumours in the presence or absence of IGF-IR, suggesting that the signalling pathway downstream of IGF-IR is activated via IGF-IR-independent mechanisms in HCC. In conclusion, the deregulation of IGF signalling pathways, which often occurs during liver tumorigenesis, does not necessarily require IGF-IRs, and hepatic IGF-IR alone may not play a determinant role in c-myc-induced hepatocarcinogenesis.

The binary Cre-lox conditional knockout system requires an essential part of the target gene to be flanked by loxP sites, enabling excision in vivo upon Cre expression. LoxP sites are introduced by homologous recombination, together with a selectable marker. However, this marker can disturb gene expression and should be removed. The marker is therefore often prepared with a third, flanking loxP site (tri-lox construct), facilitating its selective removal by partial Cre-lox recombination. We have shown that this excision can be achieved in vivo in the germline using EIIaCre transgenic mice, and have described the advantages of in vivo over in vitro removal. We show here that MeuCre40, a new transgenic mouse, more reliably and reproducibly generates an optimal partial mosaic Cre-lox recombination pattern in the early embryo. This mosaicism was transmitted to the germline and to many other tissues. Alleles with partial deletions, in particular floxed alleles from which the selectable marker was removed, were readily recovered in the next generation, after segregation from the transgene. Segregation via paternal or maternal transmission led to successful recovery of the alleles of interest. We also obtained total deletion of the floxed regions in the same experiment, making this transgene a polyvalent Cre-lox tool. We rigorously tested the ability of MeuCre40 to solve tri-lox problems, by using it for the in vivo removal of neo(R)- and hprt-expression cassettes from three different tri-lox mutants.

Studies in invertebrates have led to the identification of a number of genes that regulate lifespan, some of which encode components of the insulin or insulin-like signalling pathways. Examples include the related tyrosine kinase receptors InR (Drosophila melanogaster) and DAF-2 (Caenorhabditis elegans) that are homologues of the mammalian insulin-like growth factor type 1 receptor (IGF-1R). To investigate whether IGF-1R also controls longevity in mammals, we inactivated the IGF-1R gene in mice (Igf1r). Here, using heterozygous knockout mice because null mutants are not viable, we report that Igf1r(+/-) mice live on average 26% longer than their wild-type littermates (P < 0.02). Female Igf1r(+/-) mice live 33% longer than wild-type females (P < 0.001), whereas the equivalent male mice show an increase in lifespan of 16%, which is not statistically significant. Long-lived Igf1r(+/-) mice do not develop dwarfism, their energy metabolism is normal, and their nutrient uptake, physical activity, fertility and reproduction are unaffected. The Igf1r(+/-) mice display greater resistance to oxidative stress, a known determinant of ageing. These results indicate that the IGF-1 receptor may be a central regulator of mammalian lifespan.

Conditional gene targeting, based on Cre-lox or other systems, requires frequent genotyping of transgenic mouse populations and monitoring of tissue-specific Cre recombinatory efficiency. This is currently achieved by Southern analysis from tail- and tissue-derived DNA. Multiplex PCR amplification of the floxed (flanked by loxP sites) genomic region, combined with the PCR detection of the Cre transgene, simplifies this task. Here, we show that complete genotyping of a floxed locus is possible with three appropriately placed primers and that this triplex PCR can be performed simultaneously with a universal PCR assay for the detection of Cre transgenes. Using this approach, we also determined the ratios of recombined versus non-recombined floxed genomic segments in genomic DNA samples. This allowed us to estimate the efficiency of in vivo conditional inactivation from biopsy material and tissue samples that were too small for Southern analysis. As many new conditional knockouts are spatiotemporally restricted, such assays will become increasingly useful. The proposed PCR strategy is flexible and may be adapted to the structural specificities of any target gene.

Reduced IGF type I receptor levels diminish postnatal growth rate and adult body weight in mice. Here, we studied the impact of experimental IGF receptor deficiency on tissue-specific growth by Cre-lox-mediated dosage of a floxed IGF-IR gene. We generated mice with a wide spectrum of receptor deficiency (5-82%), and separated them into two groups with either strong (> or =50%) IGF-IR deficiency (XS mice) or moderate deficiency (<50%, M mice). The growth of XS mice was significantly retarded from 3 wk after birth onward, with respect to M littermates. This effect was twice as strong in males as in females. Growth deficits persisted throughout adult life, and at 10-12 months, most organs and tissues showed specific weight defects. Skin, bone and connective tissue, muscle, spleen, heart, lung, and brain were the most severely affected organs in the XS males. With the exception of muscle and spleen, the same tissues were also significantly reduced in size in females, although to a lesser extent. The most severe growth defect, however, concerned adipose tissue. Fat pad size in XS males was only 29% (females, 44%) of M mice. The estimated number of adipocytes in XS male fat pads was only 21% that of M males (XS female, 27%). Lipid content per cell was significantly higher in XS adipocytes, whereas plasma glucose and insulin levels were low in XS males. Thus, IGF type I receptor deficiency produced mice with disproportionate postnatal organ growth, and these effects depended strongly on sex. A marked reduction in IGF-IR levels resulted in a major defect in adipose tissue.

Conditional gene targeting uses the insertion of expression cassettes for the selection of targeted embryonic stem cells. The presence of these cassettes in the final targeted chromosomal locus may affect the normal expression of the targeted gene and produce interesting knock down phenotypes. We show here that the selection cassette may then be selectively removed in vivo, using three appropriately positioned loxP sites in the targeted gene and the transgenic mouse EIIaCre. This strategy was applied to two different target genes and we demonstrated that it is reliable and reproducible. First, we generated double transgenic EIIaCre/loxP mice (F1) that showed variable degrees of mosaicism for partially CRE-recombined floxed alleles. Efficiency of EIIaCre at creating mosaicism was dependent on the target gene and on parental transmission of the transgene. The segregation of partially recombined alleles and EIIaCre transgene was obtained in the next generation using mosaic F1 males. Mosaic females were unsuitable for this purpose because they systematically generated complete excisions during oogenesis. Our strategy is applicable to other approaches based on three loxP sites. As this procedure allows generation of knock down (presence of neo), knockout (total exision of the loxP-flanked sequences) and floxed substrains (excision of the selection cassette) from a single, targeted germline mutation and in a single experiment, its use may become more widespread in conditional mutagenesis.

The insulin-like growth factor (IGF) system is a major regulator of somatic growth in vertebrates. Both ligands (IGF-I and IGF-II) signal via the same IGF receptor (IGF-IR). Classical IGF-IR invalidation is lethal at birth, so that conditional models are needed to study the postnatal role of this receptor. To establish a genetically inducible invalidation of IGF-IR, we targeted the IGF-IR gene using a construct that introduced a neomycin resistance cassette into intron 2, leaving the rest of the gene intact. This neomycin resistance cassette interfered with the processing of the primary transcript, resulting in there being 12% fewer IGF-binding sites at the cell surface in heterozygous mice and 41% fewer in homozygous mice. Hetero- and homozygous offspring grew more slowly than their wild-type littermates. This difference was noticeable from 4 weeks after birth and was significant from 5 weeks after birth in males. In females, the effect on postnatal growth of insertion of the neo cassette was not significant. In males, IGF-I levels increased moderately (+26%) but significantly, indicating effective feedback regulation of the IGF system. IGF-binding protein-4 (IGFBP-4) levels, estimated by Western ligand blotting, were low in homozygotes (-38%), whereas IGFBP-1, -2, and -3 levels were unaffected. In females, IGF-I and IGFBP-1, -2, -3, and -4 levels did not differ significantly among heterozygous, homozygous, and wild-type animals. We investigated the molecular mechanism involved and characterized two RNA-splicing events that could account for the decrease in IGF-IR. The phenotype of these mice developed exclusively postnatally, and body proportions were maintained. IGF-IRneo mice constitute a new model for human postnatal growth deficiency.

The synthesis of ferritin, the iron-storing molecule, is regulated at the translational level by iron through interaction between a cytoplasmic protein, iron regulatory protein (IRP), and a conserved nucleotide motif present in the 5' non-coding region of all ferritin mRNAs--the iron responsive element (IRE). This region forms a stem-loop structure and when the supply of iron to the cells is limited, the IRP is bound to IRE and represses ferritin synthesis. Ferritin is composed of a 24-subunit protein shell surrounding an iron core. The two types of subunit, H and L, are encoded by two genes located on chromosomes 11q13 and 19q13.1, respectively. Both genes are ubiquitously expressed but transcriptional regulation mediates tissue-specific changes in the H/L mRNA ratio and isoferritin profiles. We now report the identification of a single point mutation in the IRE of the L-ferritin mRNA in members from a family affected with dominantly inherited hyperferritinaemia and cataract. This mutation consists of an A to G change in the highly conserved CAGUGU motif that constitutes the IRE loop and mediates the high-affinity interaction with the IRP. We show that this mutation abolishes the binding of IRP in vitro and leads to a high constitutive, poorly regulated L-ferritin synthesis in cultured lymphoblastoid cells established from affected patients. This is, to our knowledge, the first mutation affecting the IRP-IRE interaction and the iron-mediated regulation of ferritin synthesis. We suggest that excess production of ferritin in tissues is responsible for the hyperferritinaemia and that intracellular accumulation of ferritin leads to cataract.

The spontaneous glucocorticoid production in control adrenal cells (N = 10) and in the adenoma cells (N = 15) exhibited comparable geometric mean values: 1.896 nmol/ml/4-5 x 10(5) cells per 2 h (confidence limits: 0.428-8.391) and 1.852 nmol/ml (0.326-12.241), respectively. The same results were obtained for the three samples of nodular hyperplasia cells. When cortisol and corticosterone were measured separately, there was no significant difference between the outputs for control cells and those for pathological cells. Baseline aldosterone production in control cells showed a geometric mean of 2.525 pmol/ml (0.236-27.192). In the 15 adenomas, spontaneous production was extremely important: 57.297 pmol/ml (3.357-976.692). The difference was highly significant (P less than 0.0005). Aldosterone levels in the 3 samples of nodular hyperplasia cells were not different from the control values. In 9 out of the 15 adenomas, aldosterone responses to 10(-10) mol/l ACTH, expressed as stimulated/basal production, were above normal: 3.58 +/- 0.86 (SEM) against 1.48 +/- 0.08 (P less than 0.025). In the remaining 6 and in the 3 samples of nodular hyperplasia cells, there was a slight or no response. Angiotensin II (AII) stimulated both adenoma and nodular hyperplasia cells to varying degrees, without any obvious difference between these two categories. A combination of ACTH (10(-12) mol/l) and AII (10(-12) mol/l) had a synergistic action on aldosterone production in cells classed in the adenoma group. These findings demonstrate that despite the abnormal rate of aldosterone formation in adenoma cells, the production rate of corticosterone and cortisol remains normal. They unmask two functional categories with regard to ACTH in the adenoma group. Finally, they underline the relative insensitivity of nodular hyperplasia cells to ACTH.

Human adrenocortical tissue obtained, on eight occasions, at the time of nephrectomy for renal carcinoma (outside the adrenal pole) was treated by collagenase to dissociate the cells. These were hen submitted to a short, 2-h, incubation with the N-terminal fragment (16 K) of POMC, its derivative, gamma 3-MSH, beta-lipotropin and beta-endorphin, in parallel with ACTH 1-24 (Synacthen Ciba) and angiotensin II (AII, Hypertensin Ciba). Under the influence of ACTH (10(-10) M), and AII (10(-10) M), basal glucocorticoid output, including more than 80% cortisol, was increased by factors of 3 +/- 0.51 (SEM) and 1.35 +/- 0.12 (SEM), respectively. The corresponding aldosterone responses were 1.60 +/- 0.13 for ACTH and 1.38 +/- 0.09 for AII. With the exception of gamma 3-MSH, the POMC peptides under study had no steroidogenic effect. gamma 3-MSH (10(-9) M) and AII (10(-10) M) stimulated aldosterone production to approximately similar levels of, respectively, 1.23 +/- 0.05 and 1.38 +/- 0.09 times the basal production. In contrast to AII however, gamma 3-MSH showed no apparent effect on glucocorticoid output. Steroidogenic response to ACTH was potentiated by gamma 3-MSH at a concentration of 10(-10) M which, when used alone, proved ineffective. This potentiating effect was pronounced for the aldosterone response, whereas the glucocorticoid production was hardly affected. This action ceased to be visible when the cells reached maximal stimulation by ACTH. These findings suggest that gamma 3-MSH--a portion of the 16 K fragment--may have a possible role in aldosterone secretion.