Correction: New insights into intestinal phages.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

New insights into intestinal phages.

The intestinal microbiota plays important roles in human health. This last decade, the viral fraction of the intestinal microbiota, composed essentially of phages that infect bacteria, received increasing attention. Numerous novel phage families have been discovered in parallel with the development of viral metagenomics. However, since the discovery of intestinal phages by d'Hérelle in 1917, our understanding of the impact of phages on gut microbiota structure remains scarce. Changes in viral community composition have been observed in several diseases. However, whether these changes reflect a direct involvement of phages in diseases etiology or simply result from modifications in bacterial composition is currently unknown. Here we present an overview of the current knowledge in intestinal phages, their identity, lifestyles, and their possible effects on the gut microbiota. We also gather the main data on phage interactions with the immune system, with a particular emphasis on recent findings.

Veno-venous anastomoses in twin-twin transfusion syndrome: A multicenter study.

INTRODUCTION: The aim of this study is to evaluate the prevalence of veno-venous (VV) anastomoses in a large cohort of monochorionic (MC) twin placentas with twin-twin transfusion syndrome (TTTS) compared to a control group of MC placentas without TTTS. METHODS: All TTTS placentas not treated with fetoscopic laser surgery (TTTS group) and examined at five international fetal therapy centers were included in this study and compared with a control group of MC placentas without TTTS (non-TTTS group). MC placentas were routinely injected with colored dye. We recorded the presence of VV and arterio-arterial (AA) anastomoses. RESULTS: A total of 414 MC placentas were included in this study (TTTS group, n = 106; non-TTTS group, n = 308). The prevalence of VV anastomoses was significantly higher in the TTTS group than in the non-TTTS group, 36% (38/106) and 25% (78/308), respectively (p = .04; odds ratio (OR) 1.65; 95% confidence interval (CI): 1.03-2.64). In the subgroup of MC placentas without AA anastomoses, the prevalence of VV anastomoses in the TTTS group and non-TTTS group was 32% (18/57) and 8% (2/25), respectively (p = .03; OR: 5.31; 95% CI: 1.13-24.98). DISCUSSION: VV anastomoses are detected more frequently in TTTS placentas than in MC placentas without TTTS and may thus play a role in the development of TTTS.

Placental weight, birth weight and fetal:placental weight ratio in dichorionic and monochorionic twin gestations in function of gestational age, cord insertion type and placental partition.

INTRODUCTION: We established reference values for placental weight, birth weight, and fetal:placental weight ratio (FPR) (a possible index of placental functional efficiency) in monochorionic and dichorionic twin gestations. METHODS: Placental weight, birth weight, and FPR in function of gestational age, cord insertion type and placental sharing were determined in 151 dye-injected diamniotic-monochorionic and 198 double-disc diamniotic-dichorionic twin placentas (25-39 weeks' gestation). RESULTS: As expected, FPR values increased with gestational age in both groups. Birth weights and placental weights of monochorionic twins >28 weeks' gestation were significantly lower than those of age-matched dichorionic twins. When stratified per placental weight, the birth weights and FPR values of monochorionic twins were overall lower than those of dichorionic twins within the same placental weight category. However, in the subset of monochorionic twins with small share in unevenly partitioned placentas, birth weights and FPR values per placental weight were similar to those of dichorionic twins, and significantly higher than those of monochorionic twins with larger share or even placental sharing. Cord insertion type did not correlate with birth weight or FPR values per placental weight in either twin type. DISCUSSION: Reference values were generated for placental weight, birth weight and FPR in monochorionic and double-disc dichorionic twins. The generally lower FPR per placental weight in monochorionic twins compared with dichorionic twins is suggestive of inherently lower placental functional efficiency in monochorionic gestations. The mechanisms and clinical implications of the apparent differential modulation of FPR/efficiency in monochorionic twins according to placental partitioning remain to be determined.

Placental endoglin levels in diamniotic-monochorionic twin gestations: correlation with clinical and placental characteristics.

OBJECTIVE: While endoglin has been implicated in the pathogenesis of various complications in singleton pregnancies, its potential contribution to complications of monochorionic twinning remains largely undetermined. The aim of this study was to determine the correlation between relevant clinical and pathological variables and placental endoglin levels in diamniotic-monochorionic twin pregnancies. METHODS: Endoglin expression was studied by immunohistochemistry and Western blot in a prospective cohort of 68 non-TTTS and 7 TTTS monochorionic twin placentas. Placental endoglin levels were correlated with clinical and placental characteristics associated with twin-to-twin transfusion syndrome (TTTS) and selective growth restriction, including birth weight discordance, uneven placental sharing, peripheral cord insertion and choriovascular anatomy. RESULTS: In non-TTTS gestations discordant for these criteria, placental endoglin levels were significantly higher for the twin with smaller birth weight, intrauterine growth restriction, and/or abnormal ultrasound Doppler studies than for the more normal co-twin. Similarly, placental endoglin levels were significantly higher in the placental territory with smaller share and/or peripheral cord insertion in cases discordant for these placental characteristics. In TTTS gestations, placental endoglin levels tended to be higher for donor twins than for recipients. There was no correlation between endoglin levels and superficial choriovascular anastomoses. CONCLUSIONS: While the exact functional implications remain to be determined, our findings suggest a strong correlation between unbalanced placental endoglin levels and intertwin growth discordance in monochorionic twins.

A case of HELLP syndrome: an immuno-"logical" approach.

We report on a 27-year-old woman who developed severe arterial hypertension on a background of general malaise within 48 hours after vaginal delivery, suggesting severe acute-onset pre-eclampsia. Concomitant biochemical observations of haemolysis, elevated liver tests and low platelets lead to the diagnosis of (post-partum) HELLP syndrome. Our patient was transferred immediately to the intensive care unit (ICU), where she underwent plasmapheresis in combination with intravenous glucocorticoids, nicardipine and labetalol. Our patient recovered fully after three plasmapheresis sessions. Genetic testing of mutations responsible for complement deficits was negative.

Correlation between cord insertion type and superficial choriovasculature in diamniotic-monochorionic twin placentas.

BACKGROUND: Non-central cord insertion has been associated with diminished chorionic vascular distribution in singleton placentas. The choriovascular correlates of peripheral cord insertion in diamniotic-monochorionic twin placentas remain undetermined. AIM: To study the association between type of cord insertion and choriovascular distribution of both twin territories in diamniotic-monochorionic twin placentas. DESIGN: A prospective cohort of 138 monochorionic placentas was examined at Women and Infants Hospital between 2009 and early 2011. Thirty-five cases (25%), including disrupted placentas, placentas from higher order multiples and placentas from pregnancies complicated by twin-to-twin transfusion syndrome, were excluded. The correlation between cord insertion type and superficial choriovascular distribution was studied in the remaining 103 dye-injected diamniotic-monochorionic placentas. Cord insertion was categorized as paracentral, marginal or velamentous. The choriovascular distribution of each individual twin territory was assessed by analysis of number and density (number per surface area) of perforating chorionic arteries (PCA). RESULTS: In contrast with singleton placentas, there was no correlation between cord insertion type and a twin's own choriovascular distribution in diamniotic-monochorionic placentas. However, a strong correlation was found between the choriovascular distribution of one twin and the cord insertion type of the opposite twin. For a twin with paracentral or marginal cord insertion, the PCA density was significantly higher if the co-twin had a velamentous cord insertion than if the co-twin had a paracentral cord insertion (P < 0.001 and P < 0.05, respectively). Similarly, the PCA density of a twin with velamentous cord insertion tended to be higher if the co-twin had a velamentous, rather than paracentral cord insertion (P = 0.09). CONCLUSIONS: This is the first study to suggest that in diamniotic-monochorionic twin gestations, the choriovascular architecture correlates with the cord insertion type of the co-twin. In general, velamentous cord insertion is associated with expanded choriovascular distribution in the opposite twin territory. Our observations may reflect novel manifestations of twin interdependence in monochorionic pregnancies.

Placental intravascular organisms: a case report.

Ascending amniotic fluid bacterial infection is a cause of perinatal morbidity and mortality. A diagnosis of amniotic cavity infection can be inferred by documenting maternal (acute chorioamnionitis) and/or fetal (chorionic plate vasculitis; umbilical vasculitis/funisitis) inflammatory response. A definitive diagnosis of intrauterine/neonatal sepsis as a cause of stillbirth requires positive blood cultures obtained at postmortem examination. However, if postmortem examination is not performed, acute chorioamnionitis with/without fetal inflammatory response cannot be classified as a cause of demise. We present a case of intrauterine demise associated with acute chorioamnionitis, villitis, and intervillositis of the placenta. Although postmortem examination was denied, a conclusive diagnosis of intrauterine sepsis could be rendered by demonstration of gram-positive cocci within fetal vessels of umbilical cord, chorionic plate, and stem villi. This report highlights the importance of identification of placental intravascular organisms as unequivocal evidence of fetal sepsis, especially in cases where cultures cannot be obtained.

Placental characteristics of selective birth weight discordance in diamniotic-monochorionic twin gestations.

Up to 21% of diamniotic-monochorionic twin pregnancies are complicated by severe birth weight discordance in the absence of twin-to-twin transfusion syndrome, a serious condition termed 'selective' birth weight discordance. While its pathogenesis remains incompletely understood, the development of selective intertwin growth discordance, related to fetal growth restriction of one twin, is generally attributed to aberrant placental characteristics. The aim of this study was to characterize the placental markers of selective birth weight discordance, with special emphasis on the choriovascular architecture. A prospective cohort of 319 consecutive diamniotic/monochorionic twin placentas was examined at Women and Infants Hospital between 2001 and 2009. After exclusion of placentas from pregnancies complicated by twin-to-twin transfusion syndrome (TTTS), monoamniotic, multiple and disrupted placentas, 216 placentas (36 birth weight (BW)-discordant and 180 BW-concordant) formed the subject of this study. Following dye injection, the anatomic characteristics and choriovascular anastomotic patterns of BW-discordant and BW-concordant placentas were compared. The BW-discordant placentas showed significantly higher frequencies of velamentous cord insertion (22% versus 8%, P < 0.001) and uneven placental sharing (56% versus 19%, P < 0.0001) compared with BW-concordant placentas. The frequencies of intertwin AA, VV and AV anastomoses, the net number of AV anastomoses, and the net cross-sectional area of AV anastomoses were similar in both groups. There was no correlation between the frequency of velamentous cord insertion and degree of placental sharing or patterns of choriovascular anastomoses in either group. In conclusion, velamentous cord insertion and uneven placental sharing are the two major placental determinants of selective birth weight discordance in diamniotic-monochorionic twins. The role of the intertwin anastomoses, even when unbalanced, is likely negligible. Elucidation of the mechanisms whereby velamentous cord insertion affects fetal growth may lead to more focused and effective therapeutic strategies for twin and singleton pregnancies complicated by dysregulated fetal growth.

Placental markers of twin-to-twin transfusion syndrome in diamniotic-monochorionic twins: A morphometric analysis of deep artery-to-vein anastomoses.

Twin-to-twin transfusion syndrome (TTTS) is a multifactorial disorder that develops in 9-15% of diamniotic-monochorionic twin gestations. While the pathogenesis of TTTS remains poorly understood, unbalanced deep artery-to-vein (AV) anastomoses have traditionally been implicated in the gradual shift of blood from donor to recipient. The aim of this study was to define the placental markers of twin-to-twin transfusion syndrome, with special emphasis on the deep AV anastomoses. A prospective cohort of 284 consecutive diamniotic/monochorionic twin placentas was examined at Women and Infants Hospital between 2001 and 2008. Following exclusion of monoamniotic, multiple, disrupted and laser-treated placentas, 218 twin placentas (21 TTTS and 197 non-TTTS controls) formed the subject of this study. Placentas were injected with color-coded dyes. Anatomic characteristics and choriovascular anastomotic patterns of TTTS placentas were compared with non-TTTS controls. The TTTS placentas showed significantly higher frequencies of velamentous cord insertion, magistral vascular distribution patterns, uneven placental sharing, absence of AA anastomoses and presence of VV anastomoses. Deep AV anastomoses were identified in >or=95% of TTTS and non-TTTS placentas and were overall more abundant than previously reported. The total and net numbers of AV anastomoses were similar in both groups. However, the net cross-sectional area of AV anastomoses, which also takes into account the caliber of the vessels, was significantly smaller in TTTS placentas. There was no correlation between the direction of the AV imbalance and the twin donor/recipient status. In conclusion, TTTS has distinct placental characteristics, warranting their routine inclusion in the diamniotic-monochorionic placental pathology report. Our findings suggest imbalance of AV anastomoses is not required for the development for TTTS, although their presence, whether balanced or unbalanced, may contribute to the creation or perpetuation of the syndrome. Elucidation of the role of the various placental determinants in diamniotic-monochorionic twin gestations may lead to further refinement of therapeutic strategies.

Vascular distribution patterns in monochorionic twin placentas.

Several recent publications have focused on the association between the occurrence of twin-to-twin transfusion syndrome (TTTS) in diamniotic-monochorionic twins and the presence of a number of selected anatomic placental characteristics (distribution of vascular territory, cord insertion, type and number of inter-twin anastomoses). In contrast, the potential importance of the vascular distribution patterns of the individual twins remains to be elucidated. Based on its gross architectural distribution pattern, chorionic vasculature is traditionally described as disperse, magistral or mixed. The aim of this study was (1) to determine the relative prevalence of these vascular distribution patterns in monochorionic twin placentas, and (2) to correlate these patterns with the presence of TTTS and known anatomic placental features linked to TTTS. The placentas of 89 consecutive diamniotic-monochorionic twins (15 with TTTS, 74 without TTTS), examined at Women and Infants Hospital, were studied. Disperse vascular patterns were seen in 53% of twins, and magistral or mixed patterns in 47%. The prevalence of magistral/mixed vascular patterns was significantly higher in TTTS gestations than in non-TTTS gestations (60% versus 44%, P<0.05) and, in TTTS gestations, much higher in donor twins than in recipient twins (87% versus 33%, P<0.005). A strong association was found between the presence of magistral/mixed patterns and marginal/velamentous cord insertion, low number of inter-twin anastomoses, and uneven distribution of the vascular territories. These findings suggest that the magistral/mixed vascular distribution pattern may represent an important placental architectural feature contributing to the complex pathophysiology of TTTS.

Effect of lung fluid composition on type II cellular activity after tracheal occlusion in the fetal lamb.

BACKGROUND/PURPOSE: Fetal tracheal occlusion (TO) causes accelerated lung growth. However, prolonged TO is associated with a decline in the type II cell number. Type II cell function after TO is unclear. Herein, the authors examine type II cell function after TO and the role of tracheal fluid. METHODS: Fetal lambs (term, 145 days) underwent TO at 122 days. Tracheal pressure was recorded daily. In one group of animals (TF; n = 6), lung fluid was aspirated, measured, and reinfused daily. In their respective twins, NS group, lung fluid was replaced milliliter per milliliter with normal saline (NS; n = 6). At death near term, lung weight was obtained, and tissues were processed for stereologic volumetry. Type II cells were quantitated using antisurfactant protein B immunohistochemistry. Surfactant protein B-mRNA expression was studied by Northern analysis. Wilcoxon signed rank test and single factor analysis of variance (ANOVA) were used for statistical analysis (P<.05 was significant). RESULTS: In both experimental groups, intratracheal pressure rose from 1.9+/-1.0 torr to 3.7 to 4.8 torr by day 1, and remained constant thereafter. Lung fluid volume increased from 11.9+/-4.2 on day 0 to 36.8+/-8.0 mL/kg in TF, and to 28.4+/-9.3 mL/kg in NS by day 1 (P<.05). At death, lung weight/body weight ratio was higher in TF (5.45% +/- 0.91%) than in NS (4.40% +/- 0. 67%) or control (3.83%+/-0.58%; P<.05). Type II numerical density was substantially reduced after TO: 57.7+/-12.8 x 10(6)/mL (TF) and 45.0 +/-25.9 x 10(6)/mL (NS), versus 82.3+/-13.6 x 10(6)/mL in controls. Ultrastructurally, remaining type II cells in TF were enlarged and engorged with lamellar bodies; in NS, they were smaller and contained fewer lamellar bodies. Surfactant protein B mRNA expression was significantly decreased in NS, but not in TF, compared with controls. CONCLUSIONS: Type II cell function as well as overall lung growth are stimulated by TO. Lung growth after TO is therefore not unavoidably detrimental to type II cells. After isobaric saline exchange of lung fluid, type II cell function is severely inhibited, confirming the role of tracheal fluid composition in type II stimulating type II cell function.

Study of the optimum ideal protein level for weaned piglets.

The amount of ideal protein, represented by the first limiting valine (Val), for optimising the growth performances of weaned hybrid piglets was studied. Feeds were formulated based on the ideal protein concept and on a constant essential/nonessential amino acid (AA) ratio and net energy (NE) level. The animal performance trial was composed of five dietary treatments ranging from 0.57 to 0.81% calculated apparent (app.) ileal (il.) digestible (dig.) Val. The piglets, male and female in equal numbers (11 replicates x 6 piglets/pen x 5 treatments) entered the trial at about 4 weeks old (average live weight 8.1 kg). The piglets were a cross product of Piétrain sire x hybrid dam. Feed intake and weight were recorded every two weeks until the end of the trial at 10 weeks of age (average live weight 20.6 kg). The requirement was expressed in st. il. dig. AA-units, as this unit approaches available AA better than app. il. dig. AA. The standardised (st.) il. AA digestibility coefficients (DCAA) were determined for two feeds, close to the animal performance optima, in a digestion trial with four T-cannulated piglets of approximately 6 weeks old. The feed independent endogenous nitrogen excretion was measured with a protein-free feed; although this technique underestimates the actual endogenous N-losses, it provides a reasonable estimate of basal endogenous N-losses. The determined st. il. DCAA were lower than the calculated st. il. DCAA, based on the CVB (2000); this might be linked to the higher than expected crude fibre content of the experimental feeds. The Val-requirement necessary to optimise ADG and FCR was similar and amounted 0.70% st. il. dig. Val, which corresponded with a st. il. dig. Lys-level of 1.03%.

Fas ligand expression coincides with alveolar cell apoptosis in late-gestation fetal lung development.

Apoptosis plays a central role in the cellular remodeling of the developing lung. We determined the spatiotemporal patterns of the cell death regulators Fas and Fas ligand (FasL) during rabbit lung development and correlated their expression with pulmonary and type II cell apoptosis. Fetal rabbit lungs (25-31 days gestation) were assayed for apoptotic activity by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) and DNA size analysis. Fas and FasL expression were analyzed by RT-PCR, immunoblot, and immunohistochemistry. Type II cell apoptosis increased significantly on gestational day 28; the type II cell apoptotic index increased from 0.54 +/- 0.34% on gestational day 27 to 3.34 +/- 1.24% on day 28, P < 0.01 (ANOVA). This corresponded with the transition from the canalicular to the terminal sac stage of development. The day 28 rise in epithelial apoptosis was synchronous with a robust if transient 20-fold increase in FasL mRNA and a threefold increase in FasL protein levels. In contrast, Fas mRNA levels remained constant, suggestive of constitutive expression. Fas and FasL proteins were immunolocalized to alveolar type II cells and bronchiolar Clara cells. The correlation of this highly specific pattern of FasL expression with alveolar epithelial apoptosis and remodeling implicates the Fas/FasL system as a potentially important regulatory pathway in the control of postcanalicular alveolar cytodifferentiation.

Short-term tracheal occlusion corrects pulmonary vascular anomalies in the fetal lamb with diaphragmatic hernia.

BACKGROUND: Sustained fetal tracheal occlusion (TO) results in accelerated lung growth but causes severe type II cell depletion. Temporary TO fails to cause lung growth in a congenital diaphragmatic hernia (CDH) model but preserves type II cells and corrects pulmonary hypertension. Herein, we study the pulmonary vascular changes caused by temporary TO. METHODS: CDH was created in 12 fetal lambs (65-70 d; term, 145 days). In 6 lambs, the trachea was occluded for 2 weeks (CDH + TO; 108-122 d). Animals were killed at 136 days. The lungs were processed with elastin stains and anti-alpha-smooth muscle actin antibody. Partial or circumferential presence of inner and outer elastic lamina was used to determine muscularization of pulmonary arterioles. The percent of medial wall thickness was plotted against vessel diameter for each group. RESULTS: Lung weight/body weight was smaller in lambs with CDH (1. 35% +/- 0.56%) and CDH + TO (1.70% +/- 0.34%) than in control lambs (3.55% +/- 0.56%; P <.05, single-factor analysis of variance). The smallest muscularized vessel was 113 +/- 50 microm, and the largest nonmuscularized vessel was 138 +/- 49 microm in lambs with CDH, significantly different from control lambs (185 +/- 69 microm and 350 +/- 116 microm, respectively) and lambs with CDH + TO (185 +/- 97 microm and 245 +/- 100 microm, respectively; P <.05). In lambs with CDH, only 25% of vessels of less than 60 microm were nonmuscularized, compared with 81% in control lambs (P <.05) and 74% in lambs with CDH + TO.Conclusions. Temporary tracheal occlusion, from 108 to 122 days, corrects the abnormal muscularization of pulmonary arterioles seen in CDH. These morphometric findings parallel physiologic results at birth and further suggest that short-term occlusion, which preserves surfactant-producing type II pneumocytes without lung growth, may be sufficient to improve neonatal outcome of diaphragmatic hernia.

Late-gestation tracheal occlusion in the fetal lamb causes rapid lung growth with type II cell preservation.

BACKGROUND: Fetal tracheal occlusion (TO) results in varying degrees of lung growth. This study examines whether gestational age influences lung growth response following TO. MATERIALS AND METHODS: Fetal lambs (term = 145 days) underwent TO early (108 days, n = 6) or late (122 days, n = 6) in gestation. Aspirated lung fluid volume (LFV) and intratracheal pressure (ITP) were recorded daily. Two weeks after TO, the fetuses were sacrificed. Lung growth was assessed by lung weight and stereologic volumetry. Type II cellular density was assessed by computer-assisted morphometry using antisurfactant protein B antibody. RESULTS: After early TO, ITP remained below 2 mm Hg for all but one of the first 5 days. In late TO, ITP rose to 4.8 +/- 1.7 mm Hg by Day 1 and remained elevated. LFV remained lower after early than after late TO (P < 0.05) for 8 days. Thereafter, pressure and volume reached similar levels in both TO groups; both were significantly higher than their respective controls (P < 0.05). Parenchymal fraction (1 - air-space fraction) was significantly smaller after late TO (22.8 +/- 1.2%) than after early TO (31.3 +/- 0.5%). Type II density was 38.0 +/- 12.4 x 10(6)/mL after early TO and 84.0 +/- 24.3 x 10(6)/mL in control (P < 0.05); the difference between late TO and control was not significant. CONCLUSIONS: Late tracheal occlusion in fetal lambs caused more rapid lung growth than earlier TO, although ultimate lung size was similar in both groups. Late TO also resulted in greater air-space fraction and better preservation of the type II cell population than early TO. Late-gestation tracheal occlusion may therefore be preferable to prolonged occlusion initiated earlier.

Short-term tracheal occlusion in fetal lambs with diaphragmatic hernia improves lung function, even in the absence of lung growth.

BACKGROUND/PURPOSE: Prolonged tracheal occlusion (TO) accelerates lung growth but impairs surfactant production. Short-term TO results in less lung growth but preserves type II cell function. The authors studied the effects of short-term TO on lung physiology in diaphragmatic hernia. METHODS: Diaphragmatic hernia was created in 9 fetal lambs at 90 to 95 days. Five were left uncorrected (CDH), 4 underwent 2-week TO (108 to 122 days; CDH + TO). Five unoperated lambs served as controls. Near-term (136 days) fetuses were ventilated for 90 to 150 minutes. Pulmonary arterial pressure, postductal blood gases, quasistatic compliance, total lung capacity (TLC), and lung weight to body weight (LW/BW) were measured. RESULTS: There was an overall survival rate of 89% at full term. Short-term occlusion did not induce lung growth (TLC and LW/BW, 6.07 +/- 2.92 mL/kg and 0.022 +/- 0.008 in CDH, 4.86 mL/kg and 0.019 +/- 0.005 in CDH + TO, 10.81 +/- 3.55 mL/kg and 0.036 +/- 0.006 in controls, respectively). However, pulmonary hypertension in CDH (47.4 +/- 12.32/35.8 +/- 12.19 torr) was corrected by short-term occlusion (20.2 +/- 4.0/16.0 +/- 4.8 torr in CDH + TO, P< .05, single-factor analysis of variance [ANOVA]; similar to control). Best pO2 and pCO2 improved after occlusion (CDH, 48.6 +/- 6.7 torr and 107.1 +/- 34.3 torr, respectively; CDH + TO, 101.5 +/- 16.3 torr and 81.9 +/- 2.4 torr; control, 291.4 +/- 4.7 torr and 37.7 +/- 17.3), as did oxygenation index (P < .05, CDH vCDH + TO; CDH, 97.2 +/- 23.0; CDH + TO, 28.7 +/- 3.1; control, 5.6 +/- 0.6). CONCLUSIONS: Short-term TO corrects pulmonary hypertension and improves gas exchange in fetal lambs with diaphragmatic hernia despite failure to produce accelerated lung growth. Inducing lung maturation and correcting the physiological derangement in diaphragmatic hernia may be more important than achieving lung growth alone.

Genetic correction of sickle cell disease: insights using transgenic mouse models.

Sickle cell disease is a hereditary disorder characterized by erythrocyte deformity due to hemoglobin polymerization. We assessed in vivo the potential curative threshold of fetal hemoglobin in the SAD transgenic mouse model of sickle cell disease using mating with mice expressing the human fetal Agamma-globin gene. With increasing levels of HbF, AgammaSAD mice showed considerable improvement in all hematologic parameters, morphopathologic features and life span/survival. We established the direct therapeutic effect of fetal hemoglobin on sickle cell disease and demonstrated correction by increasing fetal hemoglobin to about 9-16% in this mouse model. This in vivo study emphasizes the potential of the SAD mouse models for quantitative analysis of gene therapy approaches.

Altered hematopoiesis in murine sickle cell disease.

We investigated the mechanisms of sickle cell disease (SCD) hematopoietic/erythropoietic defects using bone marrow, spleen, and/or peripheral blood from the transgenic SAD mouse model, which closely reproduces the biochemical and physiological disorders observed in human SCD. First, the erythropoietic lineage late precursors (polychromatophilic normoblasts to the intramedullary reticulocytes) of SAD mouse bone marrow were significantly altered morphologically. These anomalies resulted from high levels of hemoglobin polymers and were associated with increased cell fragmentation occurring during medullary endothelial migration of reticulocytes. Secondly, analysis of bone marrow erythropoiesis in earlier stages showed a marked depletion in SAD erythroid burst-forming units (BFU-E; of approximately 42%) and erythroid colony-forming units (CFU-E; of approximately 23%) progenitors, despite a significant increase in their proliferation, suggesting a compensatory mechanism. In contrast to the bone marrow progenitor depletion, we observed (1) a high mobilization/relocation of BFU-E early progenitors (approximately 4-fold increase) in peripheral blood of SAD mice as well as of colony-forming units-granulocyte-macrophage (CFU-GM) and (2) a 7-fold increase of SAD CFU-E in the spleen. Third, and most importantly, SAD bone marrow multipotent cells (spleen colony-forming units [CFU-S], granulocyte-erythroid-macrophage-megakaryocyte colony-forming units [CFU-GEMM], and Sca(+)Lin(-)) were highly mobilized to the peripheral blood (approximately 4-fold increase), suggesting that peripheral multipotent cells could serve as proliferative and autologous vehicles for gene therapy. Therefore, we conclude the following. (1) The abnormal differentiation and morphology of late nucleated erythroid precursors result in an ineffective sickle erythropoiesis and likely contribute to the pathophysiology of sickle cell disorders; this suggests that transfer of normal or modified SCD bone marrow cells may have a selective advantage in vivo. (2) A hematopoietic compensatory mechanism exists in SAD/SCD pathology and consists of mobilization of multipotent cells from the bone marrow to the peripheral blood and their subsequent uptake into the spleen, an extramedullary hematopoietic site for immediate differentiation. Altogether, these results corroborate the strong potential effectiveness of both autologous and allogeneic bone marrow transplantation for SCD hematopoietic therapy.

Lung growth response after tracheal occlusion in fetal rabbits is gestational age-dependent.

In utero tracheal occlusion (TO) is a potent stimulus of fetal lung growth, and is currently being applied in clinical trials to treat severe forms of pulmonary hypoplasia. The aim of this study was to examine the effect of timing of TO on pulmonary growth and maturation rates. Fetal rabbits (term = 31 d) were subjected to in utero tracheal clipping at 24 (late pseudoglandular stage) or 27 d of gestation (late canalicular/early terminal sac stage). Sham-operated littermates served as controls (C). Animals were killed at time intervals ranging from 1 to 6 d (early group) or 1 to 3 d (late group) after occlusion. Lung growth was measured by computerized stereologic volumetry and 5'-bromo-2'-deoxyuridine (BrdU) pulse labeling. Pneumocyte II population kinetics were analyzed using a combination of anti-surfactant protein-A and BrdU immunohistochemistry and computer-assisted morphometry. Statistical analysis was performed using unpaired Student's t test. Early TO was followed by an initial 3-d stagnation of growth and subsequently a dramatic acceleration of growth (BrdU-labeling index [LI] 10.1 +/- 0. 6% in TO versus 2.7 +/- 0.5% in C at 29 d, P < 0.001). In contrast, late TO induced an immediate and sustained moderate increase of lung growth (BrdU-LI 2.8 +/- 0.9% in TO versus 1.1 +/- 0.2% in C at 30 d, P < 0.05), associated with relatively more pronounced air-space distension. Whereas late TO caused no significant alterations in type II cell density or proliferation, early TO was followed by a marked increase in type II cell proliferation, paradoxically associated with dramatic reduction of type II cell density after 29 d. The effects of intrauterine TO on fetal lung growth and type II cell kinetics critically depend on the gestational age, and thus on the maturity of the lungs at the time of surgery. These findings have important clinical implications with respect to the timing of fetal interventions aimed at promoting lung growth. The fetal rabbit provides an invaluable model to study the mechanics and age dependency of TO-induced lung growth.