Plasma Progerin in Patients With Hutchinson-Gilford Progeria Syndrome: Immunoassay Development and Clinical Evaluation.

BACKGROUND: Hutchinson-Gilford progeria syndrome (HGPS) is an ultrarare, fatal, premature aging disease caused by a toxic protein called progerin. Circulating progerin has not been previously detected, precluding research using readily available biological samples. This study aimed to develop a plasma progerin assay to evaluate progerin's quantity, response to progerin-targeted therapy, and relationship to patient survival. METHODS: Biological samples were collected by The Progeria Research Foundation Cell and Tissue Bank from a non-HGPS cohort cross-sectionally and a HGPS cohort longitudinally. HGPS donations occurred at baseline and intermittently while treated with farnesylation inhibitors lonafarnib±pravastatin and zoledronate, within 3 sequential open-label clinical trials at Boston Children's Hospital totaling >10 years of treatment. An ultrasensitive single-molecule counting progerin immunoassay was developed with prespecified performance parameters. Intra- and interpatient group statistics were descriptive. The relationship between progerin and survival was assessed by using joint modeling with time-dependent slopes parameterization. RESULTS: The assay's dynamic detection range was 59 to 30 000 pg/mL (R2=0.9987). There was no lamin A cross-reactivity. Mean plasma progerin in non-HGPS participants (n=69; 39 male, 30 female; age, 0.2-71.3 years) was 351±251 pg/mL, and in drug-naive participants with HGPS (n=74; 37 female, 37 male; age, 2.1-17.5 years) was 33 261±12 346 pg/mL, reflecting a 95-fold increase in affected children (P<0.0001). Progerin levels did not differ by sex (P=0.99). Lonafarnib treatment resulted in an average per-visit progerin decrease from baseline of between 35% to 62% (all P<0.005); effects were not augmented by adding pravastatin and zoledronate. Progerin levels fell within 4 months of therapy and remained lower for up to 10 years. The magnitude of progerin decrease positively associated with patient survival (P<0.0001; ie, 15 000 pg/mL decrease yields a 63.9% decreased risk of death). For any given decrease in progerin, life expectancy incrementally increased with longer treatment duration. CONCLUSIONS: A sensitive, quantitative immunoassay for progerin was developed and used to demonstrate high progerin levels in HGPS plasma that decreased with lonafarnib therapy. The extent of improved survival was associated with both the magnitude of progerin decrease and duration at lower levels. Thus, plasma progerin is a biomarker for HGPS whose reduction enables short- and long-term assessment of progerin-targeted treatment efficacy. REGISTRATION: URL: https://www. CLINICALTRIALS: gov. Unique identifiers: NCT00879034 and NCT00916747.

A novel somatic mutation achieves partial rescue in a child with Hutchinson-Gilford progeria syndrome.

BACKGROUND: Hutchinson-Gilford progeria syndrome (HGPS) is a fatal sporadic autosomal dominant premature ageing disease caused by single base mutations that optimise a cryptic splice site within exon 11 of the LMNA gene. The resultant disease-causing protein, progerin, acts as a dominant negative. Disease severity relies partly on progerin levels. METHODS AND RESULTS: We report a novel form of somatic mosaicism, where a child possessed two cell populations with different HGPS disease-producing mutations of the same nucleotide-one producing severe HGPS and one mild HGPS. The proband possessed an intermediate phenotype. The mosaicism was initially discovered when Sanger sequencing showed a c.1968+2T>A mutation in blood DNA and a c.1968+2T>C in DNA from cultured fibroblasts. Deep sequencing of DNA from the proband's blood revealed 4.7% c.1968+2T>C mutation, and 41.3% c.1968+2T>A mutation. CONCLUSIONS: We hypothesise that the germline mutation was c.1968+2T>A, but a rescue event occurred during early development, where the somatic mutation from A to C at 1968+2 provided a selective advantage. This type of mosaicism where a partial phenotypic rescue event results from a second but milder disease-causing mutation in the same nucleotide has not been previously characterised for any disease.

Transthyretin is dysregulated in preeclampsia, and its native form prevents the onset of disease in a preclinical mouse model.

Preeclampsia is a major pregnancy complication with potential short- and long-term consequences for both mother and fetus. Understanding its pathogenesis and causative biomarkers is likely to yield insights for prediction and treatment. Herein, we provide evidence that transthyretin, a transporter of thyroxine and retinol, is aggregated in preeclampsia and is present at reduced levels in sera of preeclamptic women, as detected by proteomic screen. We demonstrate that transthyretin aggregates form deposits in preeclampsia placental tissue and cause apoptosis. By using in vitro approaches and a humanized mouse model, we provide evidence for a causal link between dysregulated transthyretin and preeclampsia. Native transthyretin inhibits all preeclampsia-like features in the humanized mouse model, including new-onset proteinuria, increased blood pressure, glomerular endotheliosis, and production of anti-angiogenic factors. Our findings suggest that a focus on transthyretin structure and function is a novel strategy to understand and combat preeclampsia.

Sera from preeclampsia patients elicit symptoms of human disease in mice and provide a basis for an in vitro predictive assay.

Early diagnosis and treatment of preeclampsia would significantly reduce maternal and fetal morbidity and mortality. However, its etiology and prediction have remained elusive. Based on the hypothesis that sera from patients with preeclampsia could function as a "blueprint" of causative factors, we describe a serum-based pregnancy-specific mouse model that closely mirrors the human condition as well as an in vitro predictive assay. We show that a single administration of human preeclampsia serum in pregnant IL-10-/- mice induced the full spectrum of preeclampsia-like symptoms, caused hypoxic injury in uteroplacental tissues, and elevated soluble fms-like tyrosine kinase 1 and soluble endoglin, markers thought to be related to the disease. The same serum sample(s) induced a partial preeclampsia phenotype in wild-type mice. Importantly, preeclampsia serum disrupted cross talk between trophoblasts and endothelial cells in an in vitro model of endovascular activity. Disruption of endovascular activity could be documented in serum samples as early as 12 to 14 weeks of gestation from patients who subsequently developed preeclampsia. These results indicate that preeclampsia patient sera can be used to understand the pregnancy-specific disease pathology in mice and can predict the disorder.

Vascular endothelial growth factor C facilitates immune tolerance and endovascular activity of human uterine NK cells at the maternal-fetal interface.

Although replete with cytotoxic machinery, uterine NK (uNK) cells remain tolerant at the maternal-fetal interface. The mechanisms that facilitate the uNK cell tolerance are largely unknown. In this study, we demonstrate that vascular endothelial growth factor (VEGF) C, a proangiogenic factor produced by uNK cells, is responsible for their noncytotoxic activity. VEGF C-producing uNK cells support endovascular processes as demonstrated in a three-dimensional coculture model of capillary tube formation on Matrigel. Peripheral blood NK cells fail to produce VEGF C and remain cytotoxic. This response can be reversed by exogenous VEGF C. We show that cytoprotection by VEGF C can be related to induction of the TAP-1 expression and MHC class I assembly in target cells. Small interfering RNA-mediated silencing of TAP-1 expression abolished the VEGF C-imparted protection. Overall, these results demonstrate that empowerment of uNK cells with angiogenic factors keeps them noncytotoxic. This phenotype is critical to their pregnancy-compatible immunovascular role during placentation and fetal development.

Ingestion of Steamed and Dehydrated Placenta Capsules Does Not Affect Postpartum Plasma Prolactin Levels or Neonatal Weight Gain: Results from a Randomized, Double-Bind, Placebo-Controlled Pilot Study.

INTRODUCTION: Clinical studies conducted in the early to mid-twentieth century, and recent self-reports by some maternal placentophagy practitioners, suggest that human maternal placentophagy improves breast milk quality and quantity, although little research has evaluated this claim. Some placentophagy providers and advocates suggest that increased prolactin levels after placenta ingestion could account for the purported lactation benefits. The current study was conducted to evaluate these claims by comparing plasma prolactin levels of women consuming steamed, dehydrated, and encapsulated placenta with those of women consuming a placebo. Neonatal weight gain was also compared between the 2 groups. METHODS: A randomized, double-blind, placebo-controlled pilot trial was conducted in which postpartum women (N = 27) were given a supplement containing their dehydrated placenta (n = 12) or placebo (n = 15). Plasma prolactin concentrations were measured 4 times across late pregnancy and early postpartum, and neonatal weights were recorded 3 times over the first 3 weeks postpartum. RESULTS: The results showed no statistically significant (P < .05) differences in either plasma prolactin levels or neonatal weight gain between groups. DISCUSSION: Maternal consumption of steamed, dehydrated, and encapsulated placenta postpartum does not appear to affect maternal postpartum prolactin or neonatal weight in the first 3 weeks postpartum. Further research is needed to investigate the possible effects of variation in placenta preparation methods or daily intake on human lactation.

Effects of Human Maternal Placentophagy on Maternal Postpartum Iron Status: A Randomized, Double-Blind, Placebo-Controlled Pilot Study.

INTRODUCTION: Advocates of human maternal placentophagy report that encapsulated placenta is an excellent source of dietary iron. Our study compared the effect of ingested encapsulated placenta on maternal postpartum iron status versus that of a beef placebo. METHODS: A randomized, double-blind, placebo-controlled pilot study (N = 23) was conducted among healthy human research participants experiencing a normal pregnancy. Maternal iron status was measured via hemoglobin, transferrin, and ferritin taken from blood samples drawn in the participants' homes at 4 time points: the 36th week of pregnancy, within 96 hours of parturition, between days 5 and 7 postpartum, and during week 3 postpartum. Iron concentrations in the encapsulated placenta and encapsulated beef placebo were compared using inductively coupled plasma mass spectrometry. RESULTS: Seventy-eight percent (18/23) of study participants' hemoglobin concentrations were above the World Health Organization cutoff for gestational iron deficiency (≥ 11.0 g/dL) during the 36th week of pregnancy. Results revealed no statistically significant differences (hemoglobin, P = .603; ferritin, P = .852; transferrin, P = .936) in maternal iron status (including postpartum iron rebound in the first week postpartum) between women in the placenta supplement (n = 10) and placebo (n = 13) groups. Average iron concentrations were considerably higher in encapsulated placenta (0.664 mg/g) compared to the encapsulated beef placebo (0.093 mg/g) but provided only 24% of the recommended daily allowance (RDA) for iron among lactating women based on the study's maximum daily intake. DISCUSSION: The current study suggests that encapsulated placenta supplementation neither significantly improves nor impairs postpartum maternal iron status for women consuming the RDA of dietary iron during pregnancy and lactation, compared to a beef placebo. This may be an especially important finding for women who are iron deficient postpartum and whose only source of supplemental dietary iron is encapsulated placenta, as this may provide an inadequate source of supplemental iron in cases of deficiency.

Structure/function aspects of human 3beta-hydroxysteroid dehydrogenase.

Separate genes encode the human type 1 (placenta, breast tumors, other peripheral tissues) and type 2 (gonad, adrenal) isoforms of 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD1, 3 beta-HSD2). Mutagenesis of 3 beta-HSD1 produced the Y154F, H156Y and K158Q mutant enzymes in the probable Y(154)-P-H(156)-S-K(158) catalytic motif. The H(156)Y mutant of the 3 beta-HSD1 created a chimera of the 3 beta-HSD2 motif (Y(154)-P-Y(156)-S-K(158)) in 3 beta-HSD1. The D241N, D257L, D258L and D265N mutants are in the potential isomerase site of the 3 beta-HSD1 enzyme. Homology modeling with UDP-galactose-4-epimerase predicted that Asp(36) in the Rossmann-fold domain is responsible for the NAD(H) specificity of human 3 beta-HSD1, and our D36A/K37R mutant tested that assignment. The H(156)Y mutant of the 3 beta-HSD1 enzyme shifted the substrate (DHEA) kinetics to the 14-fold higher K(m) value measured for the 3 beta-HSD2 activity. From Dixon analysis, epostane inhibited the 3 beta-HSD1 activity with 17-fold greater affinity compared to 3 beta-HSD2 and H(156)Y. The mutants of Tyr(154) and Lys(158) exhibited no dehydrogenase activity and appear to be catalytic 3 beta-HSD residues. The D257L and D258L mutations eliminated isomerase activity, suggesting that Asp(257) or Asp(258) may be catalytic residues for isomerase activity. The D36A/K37R mutant shifted the cofactor preference of both 3 beta-HSD and isomerase from NAD(H) to NADP(H). In addition to characterizing catalytic residues, these studies have identified the structural basis (His(156)) for an exploitable difference in the substrate and inhibition kinetics of 3 beta-HSD1 and 3 beta-HSD2. Hence, it may be possible to selectively inhibit human 3 beta-HSD1 to slow the growth of hormone-sensitive breast tumor cells and control placental steroidogenesis near term to prevent premature labor.

Vascular IL-10: a protective role in preeclampsia.

IL-10 is a pregnancy compatible cytokine that plays a vital role in maintaining the balance of anti-inflammatory and pro-inflammatory milieu at the maternal-fetal interface. Recent evidence now suggests that IL-10 is a potent vascular cytokine that can blunt hypertension and inflammation-mediated vascular dysfunction. Thus, a re-evaluation of IL-10 as a cytokine supporting endovascular interactions and angiogenesis as well as blunting hypoxic-injury and preeclampsia-like features is warranted. In this review, we highlight these novel functions of IL-10 and propose that its immune-modulating and vascular functions are mutually inclusive, particularly in the context of normal gestation.

Beyond the threshold: an etiological bridge between hypoxia and immunity in preeclampsia.

Taking a cue from the recent workshop 'Preeclampsia--a Pressing Problem' sponsored by the National Institutes of Child Health and Human Development, this review article takes a fresh look at hypoxia and a dysfunctional immune system as the key contributors to the etiology of preeclampsia and the mechanisms involved therein. In the context of epidemiological research on the intricate and multifactorial nature of preeclampsia, we focus on hypoxia as an upstream regulator of preeclampsia and its consequences in a model compromised by a deficiency in key pregnancy compatible immune modulators. It has been proposed that placental hypoxia releases cytotoxic factors produced at the maternal-fetal interface into the circulation to manifest the maternal symptoms associated with preeclampsia. However, it is not clear how this mechanism is empowered in pregnant women. Does systemic hypoxia exert preeclampsia-like effects on pregnancy? Are these effects further manifested by intrinsic inflammation in the absence of key immune modulators such as IL-10? Thus, it is of paramount importance that in vivo models be developed wherein the role of systemic hypoxia can be evaluated for preeclampsia-causing events. We present a discussion on whether prolonged exposure to hypoxia can lead to a perpetual cycle of compartmentalized uteroplacental tissue damage, release of anti-angiogenic and vasoconstrictive factors that impair trophoblast invasion and promote systemic vascular resistance resulting in the maternal syndrome.

Novel approaches for mechanistic understanding and predicting preeclampsia.

Despite intense investigation, preeclampsia (PE) remains largely enigmatic. Relatively late onset of diagnostic signs and heterogeneous nature of the disease further contribute to poor understanding of its etiology and clinical management. There exist no concrete animal models that can provide mechanistic underpinnings for evaluating targeted therapeutic intervention. Poor cross-sectional findings with potential biochemical markers reported so far have proved counterintuitive and suggest a need for novel approaches to predict the early onset of disease. Because of the co-onset of local placental anomalies and systemic manifestation of symptoms, it is highly likely that serum from PE patients can provide a "blueprint" of causative factors. Proteomic and/or functional analysis of maternal serum are expected to predict the onset of disease ahead of manifestation of clinical symptoms. A serum-based predictive assay should overcome complexities resulting from the heterogeneous etiology of PE. This review attempts to address some of these issues and discuss the signature biochemical serum factors and propose new and better ways to predict PE.

Structure/function relationships responsible for the kinetic differences between human type 1 and type 2 3beta-hydroxysteroid dehydrogenase and for the catalysis of the type 1 activity.

Two distinct genes encode the 93% homologous type 1 (placenta, peripheral tissues) and type 2 (adrenals, gonads) 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD/isomerase) in humans. Mutagenesis studies using the type 1 enzyme have produced the Y154F and K158Q mutant enzymes in the Y(154)-P-H(156)-S-K(158) motif as well as the Y269S and K273Q mutants from a second motif, Y(269)-T-L-S-K(273), both of which are present in the primary structure of the human type 1 3beta-HSD/isomerase. In addition, the H156Y mutant of the type 1 enzyme has created a chimera of the type 2 enzyme motif (Y(154)-P-Y(156)-S-K(158)) in the type 1 enzyme. The mutant and wild-type enzymes have been expressed and purified. The K(m) value of dehydroepiandrosterone is 13-fold greater, and the maximal turnover rate (K(cat)) is 2-fold greater for wild-type 2 3beta-HSD compared with the wild-type 1 3beta-HSD activity. The H156Y mutant of the type 1 enzyme has substrate kinetic constants for 3beta-HSD activity that are very similar to those of the wild-type 2 enzyme. Dixon analysis shows that epostane inhibits the 3beta-HSD activity of the wild-type 1 enzyme with 14-17-fold greater affinity compared with the wild-type 2 and H156Y enzymes. The Y154F and K158Q mutants exhibit no 3beta-HSD activity, have substantial isomerase activity, and utilize substrate with K(m) values similar to those of wild-type 1 isomerase. The Y269S and K273Q mutants have low, pH-dependent 3beta-HSD activity, exhibit only 5% of the maximal isomerase activity, and utilize the isomerase substrate very poorly. From these studies, a structural basis for the profound differences in the substrate and inhibition kinetics of the wild-type 1 and 2 3beta-HSD, plus a catalytic role for the Tyr(154) and Lys(158) residues in the 3beta-HSD reaction have been identified. These advances in our understanding of the structure/function of human type 1 and 2 3beta-HSD/isomerase may lead to the design of selective inhibitors of the type 1 enzyme not only in placenta to control the onset of labor but also in hormone-sensitive breast, prostate, and choriocarcinoma tumors to slow their growth.

Differences in substrate and inhibitor kinetics of human type 1 and type 2 3beta-hydroxysteroid dehydrogenase are explained by the type 1 mutant, H156Y.

Two distinct genes encode the human type 1 (placenta, mammary gland) and type 2 (adrenal, gonad) isoforms of 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD). We have produced the Y154F, H156Y, and K158Q mutant enzymes in the Y154-P-H156-S-K158 motif of the human type 1 3beta-HSD/isomerase. The H156Y mutant was created to produce a chimera of the type 2 enzyme motif (Y154-P-Y156-S-K158) in the type 1 enzyme. The wild-type (WT) 1 and 2 plus the mutant enzymes were expressed and purified. The Km for dehydroepiandrosterone and Ki for epostane measured with both the H156Y mutant and WT 2 are 13-fold to 17-fold greater than those values obtained with the WT 1 3beta-HSD. The Y154F and K158Q mutants exhibit no 3beta-HSD but have significant isomerase activity. Thus, H156 in WT 1 vs. Y156 in WT 2 accounts for the substantially higher affinity of WT 1 3beta-HSD activity for these substrate and inhibitor steroids relative to the WT 2 enzyme.

Structure/function relationships responsible for coenzyme specificity and the isomerase activity of human type 1 3 beta-hydroxysteroid dehydrogenase/isomerase.

Human type 1 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD/isomerase) catalyzes the two sequential enzyme reactions on a single protein that converts dehydroepiandrosterone or pregnenolone to androstenedione or progesterone, respectively, in placenta, mammary gland, breast tumors, prostate, prostate tumors, and other peripheral tissues. Our earlier studies show that the two enzyme reactions are linked by the coenzyme product, NADH, of the 3 beta-HSD activity. NADH activates the isomerase activity by inducing a time-dependent conformational change in the enzyme protein. The current study tested the hypothesis that the 3 beta-HSD and isomerase activities shared a common coenzyme domain, and it characterized key amino acids that participated in coenzyme binding and the isomerase reaction. Homology modeling with UDP-galactose-4-epimerase predicts that Asp36 is responsible for the NAD(H) specificity of human 3 beta-HSD/isomerase and identifies the Rossmann-fold coenzyme domain at the amino terminus. The D36A/K37R mutant in the potential coenzyme domain and the D241N, D257L, D258L, and D265N mutants in the potential isomerase domain (previously identified by affinity labeling) were created, expressed, and purified. The D36A/K37R mutant shifts the cofactor preference of both 3 beta-HSD and isomerase from NAD(H) to NADP(H), which shows that the two activities utilize a common coenzyme domain. The D257L and D258L mutations eliminate isomerase activity, whereas the D241N and D265N mutants have nearly full isomerase activity. Kinetic analyses and pH dependence studies showed that either Asp257 or Asp258 plays a catalytic role in the isomerization reaction. These observations further characterize the structure/function relationships of human 3 beta-HSD/isomerase and bring us closer to the goal of selectively inhibiting the type 1 enzyme in placenta (to control the timing of labor) or in hormone-sensitive breast tumors (to slow their growth).

Glycolytic and gluconeogenic growth of Escherichia coli O157:H7 (EDL933) and E. coli K-12 (MG1655) in the mouse intestine.

Escherichia coli EDL933, an O157:H7 strain, is known to colonize the streptomycin-treated CD-1 mouse intestine by growing in intestinal mucus (E. A. Wadolkowski, J. A. Burris, and A. D. O'Brien, Infect. Immun. 58:2438-2445, 1990), but what nutrients and metabolic pathways are employed during colonization has not been determined. In this study, when the wild-type EDL933 strain was fed to mice along with an EDL933 DeltappsA DeltapckA mutant, which is unable to utilize tricarboxylic acid cycle intermediates and gluconeogenic substrates for growth, both strains colonized the mouse intestine equally well. Therefore, EDL933 utilizes a glycolytic substrate(s) for both initial growth and maintenance when it is the only E. coli strain fed to the mice. However, in the presence of large numbers of MG1655, a K-12 strain, it is shown that EDL933 utilizes a glycolytic substrate(s) for initial growth in the mouse intestine but appears to utilize both glycolytic and gluconeogenic substrates in an attempt to maintain colonization. It is further shown that MG1655 predominantly utilizes glycolytic substrates for growth in the mouse intestine whether growing in the presence or absence of large numbers of EDL933. Data are presented showing that although small numbers of EDL933 grow to large numbers in the intestine in the presence of large numbers of MG1655 when both strains are fed to mice simultaneously, precolonization with MG1655 affords protection against subsequent colonization by EDL933. Moreover, in mice that are precolonized with EDL933, small numbers of MG1655 are able to grow rapidly in the intestine and EDL933 is eliminated. In situ hybridization experiments using E. coli-specific rRNA probes showed that while MG1655 is found only in mucus, EDL933 is found both in mucus and closely associated with intestinal epithelial cells. The data are discussed with respect to competition for nutrients and to the protection that some intestinal commensal E. coli strains might afford against infection by O157:H7 strains.

Carbon nutrition of Escherichia coli in the mouse intestine.

Whole-genome expression profiling revealed Escherichia coli MG1655 genes induced by growth on mucus, conditions designed to mimic nutrient availability in the mammalian intestine. Most were nutritional genes corresponding to catabolic pathways for nutrients found in mucus. We knocked out several pathways and tested the relative fitness of the mutants for colonization of the mouse intestine in competition with their wild-type parent. We found that only mutations in sugar pathways affected colonization, not phospholipid and amino acid catabolism, not gluconeogenesis, not the tricarboxylic acid cycle, and not the pentose phosphate pathway. Gluconate appeared to be a major carbon source used by E. coli MG1655 to colonize, having an impact on both the initiation and maintenance stages. N-acetylglucosamine and N-acetylneuraminic acid appeared to be involved in initiation, but not maintenance. Glucuronate, mannose, fucose, and ribose appeared to be involved in maintenance, but not initiation. The in vitro order of preference for these seven sugars paralleled the relative impact of the corresponding metabolic lesions on colonization: gluconate > N-acetylglucosamine > N-acetylneuraminic acid = glucuronate > mannose > fucose > ribose. The results of this systematic analysis of nutrients used by E. coli MG1655 to colonize the mouse intestine are intriguing in light of the nutrient-niche hypothesis, which states that the ecological niches within the intestine are defined by nutrient availability. Because humans are presumably colonized with different commensal strains, differences in nutrient availability may provide an open niche for infecting E. coli pathogens in some individuals and a barrier to infection in others.