Placental proteins with predicted roles in fetal development decrease in premature infants.

BACKGROUND: Emerging evidence from animal experiments indicate that factors secreted by the placenta are critical for normal fetal organ development. Our objective was to characterize the umbilical vein and artery proteome in preterm infants and identify proteins that decrease in the neonatal circulation following delivery. METHODS: Cord blood at delivery and neonatal blood at 48-72 h of life was collected in 25 preterm infants. Plasma protein abundance was determined using the SomaLogic platform. RESULTS: When comparing protein levels of umbilical venous to arterial cord blood, 434 proteins were significantly higher indicating placental secretion into the fetal circulation. Moreover, when comparing neonatal blood to umbilical vein levels, 142 proteins were significantly lower. These proteins included Endoplasmic reticulum resident protein 29, CD59, Fibroblast growth factor 2 and Dynactin subunit 2, which are involved in brain development and prevention of brain damage as well as Fibroblast growth factor 1 which prevents lung fibrosis. CONCLUSIONS: The late second trimester human placenta secretes proteins into the fetal circulation which decrease following delivery. Many of these proteins are predicted to be important in the development of fetal organs. Further studies are needed to directly link placental proteins to organ development and poor outcomes in preterm infants. IMPACT: Prematurity remains a leading cause of morbidity and mortality requiring the development of novel treatments. Emerging evidence from animal studies suggest that factors secreted from the placenta may be critical in the development of the fetus. We report that the preterm human placenta secretes an array of proteins into the fetal circulation. Some of these proteins are predicted to be involved in the development of the brain and the lung. When born prematurely, infants are deprived of these placental proteins, which may contribute to their poor outcomes.

In Vitro Study on the Effect of Maraviroc or Dolutegravir on Bilirubin to Albumin Binding.

We performed an in vitro evaluation of the effect of maraviroc or dolutegravir on bilirubin to albumin binding. At typical treatment and low albumin concentrations, maraviroc had no impact, while dolutegravir affected bilirubin to albumin binding to an equivalent extent as sulfisoxazole. However in vivo, neither is likely to significantly impact bilirubin to albumin binding because of their low concentrations relative to albumin.

A warning: don't be stumped by stump appendicitis.

When an emergency medicine physician evaluates a child with abdominal pain and a history of appendectomy, appendicitis is often excluded from the differential. We present a case of a 16-year-old boy who developed stump appendicitis 3 years after laparoscopic appendectomy. Knowledge of this rare phenomenon in children can lead to timely diagnosis and avoid the significant additional morbidity associated with perforation of the stump.

Human papillomavirus type 16 E6 and E 7 proteins alter NF-kB in cultured cervical epithelial cells and inhibition of NF-kB promotes cell growth and immortalization.

The NF-kB family of transcription factors regulates important biological functions including cell growth, survival and the immune response. We found that Human Papillomavirus type 16 (HPV-16) E7 and E6/E7 proteins inhibited basal and TNF-alpha-inducible NF-kB activity in human epithelial cells cultured from the cervical transformation zone, the anatomic region where most cervical cancers develop. In contrast, HPV-16 E6 regulated NF-kB in a cell type- and cell growth-dependent manner. NF-kB influenced immortalization of cervical cells by HPV16. Inhibition of NF-kB by an IkB alpha repressor mutant increased colony formation and immortalization by HPV-16. In contrast, activation of NF-kB by constitutive expression of p65 inhibited proliferation and immortalization. Our results suggest that inhibition of NF-kB by HPV-16 E6/E7 contributes to immortalization of cells from the cervical transformation zone.

Fiber type conversion by PGC-1α activates lysosomal and autophagosomal biogenesis in both unaffected and Pompe skeletal muscle.

PGC-1α is a transcriptional co-activator that plays a central role in the regulation of energy metabolism. Our interest in this protein was driven by its ability to promote muscle remodeling. Conversion from fast glycolytic to slow oxidative fibers seemed a promising therapeutic approach in Pompe disease, a severe myopathy caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA) which is responsible for the degradation of glycogen. The recently approved enzyme replacement therapy (ERT) has only a partial effect in skeletal muscle. In our Pompe mouse model (KO), the poor muscle response is seen in fast but not in slow muscle and is associated with massive accumulation of autophagic debris and ineffective autophagy. In an attempt to turn the therapy-resistant fibers into fibers amenable to therapy, we made transgenic KO mice expressing PGC-1α in muscle (tgKO). The successful switch from fast to slow fibers prevented the formation of autophagic buildup in the converted fibers, but PGC-1α failed to improve the clearance of glycogen by ERT. This outcome is likely explained by an unexpected dramatic increase in muscle glycogen load to levels much closer to those observed in patients, in particular infants, with the disease. We have also found a remarkable rise in the number of lysosomes and autophagosomes in the tgKO compared to the KO. These data point to the role of PGC-1α in muscle glucose metabolism and its possible role as a master regulator for organelle biogenesis - not only for mitochondria but also for lysosomes and autophagosomes. These findings may have implications for therapy of lysosomal diseases and other disorders with altered autophagy.

Suppression of autophagy permits successful enzyme replacement therapy in a lysosomal storage disorder--murine Pompe disease.

Autophagy, an intracellular system for delivering portions of cytoplasm and damaged organelles to lysosomes for degradation/recycling, plays a role in many physiological processes and is disturbed in many diseases. We recently provided evidence for the role of autophagy in Pompe disease, a lysosomal storage disorder in which acid alphaglucosidase, the enzyme involved in the breakdown of glycogen, is deficient or absent. Clinically the disease manifests as a cardiac and skeletal muscle myopathy. The current enzyme replacement therapy (ERT) clears lysosomal glycogen effectively from the heart but less so from skeletal muscle. In our Pompe model, the poor muscle response to therapy is associated with the presence of pools of autophagic debris. To clear the fibers of the autophagic debris, we have generated a Pompe model in which an autophagy gene, Atg7, is inactivated in muscle. Suppression of autophagy alone reduced the glycogen level by 50­60%. Following ERT, muscle glycogen was reduced to normal levels, an outcome not observed in Pompe mice with genetically intact autophagy. The suppression of autophagy, which has proven successful in the Pompe model, is a novel therapeutic approach that may be useful in other diseases with disturbed autophagy.

Differences in the predominance of lysosomal and autophagic pathologies between infants and adults with Pompe disease: implications for therapy.

Pompe disease is a lysosomal storage disorder caused by the deficiency of acid alpha-glucosidase, the enzyme that degrades glycogen in the lysosomes. The disease manifests as a fatal cardiomyopathy and skeletal muscle myopathy in infants; in milder late-onset forms skeletal muscle is the major tissue affected. We have previously demonstrated that autophagic inclusions in muscle are prominent in adult patients and the mouse model. In this study we have evaluated the contribution of the autophagic pathology in infants before and 6 months after enzyme replacement therapy. Single muscle fibers, isolated from muscle biopsies, were stained for autophagosomal and lysosomal markers and analyzed by confocal microscopy. In addition, unstained bundles of fixed muscles were analyzed by second harmonic imaging. Unexpectedly, the autophagic component which is so prominent in juvenile and adult patients was negligible in infants; instead, the overwhelming characteristic was the presence of hugely expanded lysosomes. After 6 months on therapy, however, the autophagic buildup becomes visible as if unmasked by the clearance of glycogen. In most fibers, the two pathologies did not seem to coexist. These data point to the possibility of differences in the pathogenesis of Pompe disease in infants and adults.

The values and limits of an in vitro model of Pompe disease: the best laid schemes o' mice an' men...

In Pompe disease, a lysosomal glycogen storage disorder, cardiac and skeletal muscle abnormalities are responsible for premature death and severe weakness. Swollen glycogen-filled lysosomes, the expected pathology, are accompanied in skeletal muscle by a secondary pathology-massive accumulation of autophagic debris-that appears to contribute greatly to the weakness. We have tried to reproduce these defects in murine, Pompe myotubes derived from either primary myoblasts or myoblasts with extended proliferative capacity. The cells accumulated large lysosomes filled with glycogen, but, to our disappointment, did not have autophagic buildup even though basal autophagy was intact. When we suppressed autophagy by knocking down Atg7, we found that glycogen uptake by lysosomes was not affected, suggesting that macroautophagy is not the major pathway for glycogen delivery to lysosomes. But two apparently incidental observations-a peculiar distribution of both microinjected dextran and of small acidic structures adjacent to the interior membrane of large alkalinized glycogen containing lysosomes-raised the possibility that glycogen traffics to the lysosomes by microautophagy or/and by the engulfment of small lysosomes by large ones. The cultured myotubes, therefore, appear to be a useful model for studying the mechanisms involved in glycogen accumulation in Pompe disease and to test substrate deprivation approaches.

When more is less: excess and deficiency of autophagy coexist in skeletal muscle in Pompe disease.

The role of autophagy, a catabolic lysosome-dependent pathway, has recently been recognized in a variety of disorders, including Pompe disease, which results from a deficiency of the glycogen-degrading lysosomal hydrolase acid-alpha glucosidase (GAA). Skeletal and cardiac muscle are most severely affected by the progressive expansion of glycogen-filled lysosomes. In both humans and an animal model of the disease (GAA KO), skeletal muscle pathology also involves massive accumulation of autophagic vesicles and autophagic buildup in the core of myofibers, suggesting an induction of autophagy. Only when we suppressed autophagy in the skeletal muscle of the GAA KO mice did we realize that the excess of autophagy manifests as a functional deficiency. This failure of productive autophagy is responsible for the accumulation of potentially toxic aggregate-prone ubiquitinated proteins, which likely cause profound muscle damage in Pompe mice. Also, by generating muscle-specific autophagy-deficient wild-type mice, we were able to analyze the role of autophagy in healthy skeletal muscle.